Progress in Nucleic Acid Research and Molecular Biology: v. 13

PROGRESS IN

Nucleic Acid Research and Molecular Biology Volume

73

This Page Intentionally Left Blank

PROGRESS IN

NucIeic Acid Research and Molecular Biology edited by

J. N. DAVIDSON

WALDO E. COHN

Department of Biochemistry The University of Glasgow Glasgow, Scotland

Biology Division Oak Ridge National Laboratory Oak Ridge, Tennessee

Volume

73

7973

ACADEMIC PRESS New York and London

COPYRIGHT 0 1973, BY ACADEMIC PRESS, INC. ALL RIGHTS RESERVED. NO PART OF THIS PUBLICATION MAY BE REPRODUCED OR TRANSMITTED IN ANY FORM OR BY ANY MEANS, ELECTRONIC OR MECHANICAL, INCLUDING PHOTOCOPY, RECORDING, OR ANY INFORMATION STORAGE AND RETRIEVAL SYSTEM, WITHOUT PERMISSION IN WRITING FROM THE PUBLISHER.

ACADEMIC PRESS, INC.

111 Fifth Avenue,New York, New York 10003

United Kingdom Edition published by ACADEMIC PRESS, INC. (LONDON) LTD. 24/18 Oval Road, London NW1

LIEIRARY OF CONORESS CATALOO CARDNUMBER:63-15847

PRINTED IN THE UNITED STATES OF AMERICA

Contents

. . . . . . . . . . PREFACE. . . . . . . . . . . . . . . ABBREVIATIONS .4ND SYMBOLS . . . . . . . . . . SOMEARTICLESPLANNED FOR FUTURE VOLUMES . . . . . ERRATA . . . . . . . . . . . . . . . OBITUABYAAMES NORMAN DAVIDBON. . . . . . . .

.

.

ix

. .

. .

...

xi11

.

.

xvii

.

.

xix

.

.

xxi

I. Introduction . . , . . , . . . . . 11. Interaction of Formaldehyde with Bases, Nucleosides and Nucleotides 111. Interaction of Formaldehyde with Polynucleotides . . . . IV. Interaction of Formaldehyde with Nucleoproteins . . . . . V. Use of Reactions of Nucleic Acids and Nucleoproteins with Formaldehyde . . . . . . . . . . . . . . VI. Related Reactions and Their Effects (as Compared to Formaldehyde . . . . . . . . . . . . . Reactions) . VII. Conclusion . . . . . . . . . . . References . . . . . . . . . . .

1 3 15 30

LIST

OF

C~NTRIBUT~RS .

xi

Reactions of Nucleic Acids and Nucleoproteins with Formaldehyde

M. YA. FELDMAN

.

. .

.

.

.

.

.

. .

.

. .

35 40 44 44

Synthesis and Functions of the -C-C-A Terminus of Transfer RNA

MURRAY P. DEUTSCHER

.

. . .

. . .

I. Introduction . . . . . . . . . . 11. Location of the -C-C-A Terminus in the Three-Dimensional Structure of tRNA . . . . . . . . . . . . 111. Synthesis and Turnover of the -C-C-A Terminus in Vivo . . . IV. Enzymatic Synthesis of the -C-C-A Terminus in Vitro . . . . V. Role of the -C-C-A Terminus in tRNA Function . . . . . VI. Possible Control Functions of the -C-C-A Terminus of tRNA . . References . . . . . . . . . .

.

.

.

V

.

.

.

51

52 57

60 78 86 88

vi

CONTENTS

Mammalian RNA Polymerases

SAMSON T. JACOB I . Introduction . . . . . . . . . . . . . . 93 I1. Quantitative Extraction of RNA Polymerase from Mammalinn Cells 95 I11. Multiplicity. Nomenclature and Intranuclear Localixntion . . . 101 IV . Properties of RNA Polymerases . . . . . . . . . . 106 V . Subunit Structure and Molecular Weight . . . . . . . 112 VI . Regulation of RNA Polymerases . . . . . . . . . . 114 VII . Mitochondria1 RNA Polyrnerasc . . . . . . . . . . 117 VIII . Summary and Conclusions . . . . . . . . . . . 119 Notes Added in Proof . . . . . . . . . . . . 121 References . . . . . . . . . . . . . . . 121

Poly(adenosine diphosphate ribose)

TAKASHI SUGIMURA I . Introduction . . . . . . . . . I1. Chemical and Physical Properties of Poly(ADP-Rib) I11. Purification of Poly(ADP-Rib) . . . . . IV . Biosynthesis of Poly(ADP-Rib) . . . . . V . Biodegradation of Poly (ADP-Rib) . . . . VI . Natural Occurrence . . . . . . . . VII . Possible Biological Significance . . . . . VIII . Related Phenomena . . . . . . . . I X . Future Problems . . . . . . . . References . . . . . . . . . .

.

. .

.

. .

.

. .

. . . . . . . . . . . .

.

. .

. . . .

.

. .

. . . .

. . . . . . . . . . . . . . .

127 129 133 134 142 14.5 146 147 148 140

The Stereochemistry of Actinomycin Binding to D N A and Its Implications in Molecular Biology

HENRYM . SOBELL I . Introduction . . . . . . . . . . . . I1. Solution Studies of the Actinomycin-DNA Interaction . . I11. The Actinomycin-Deoxyguanosine Crystalline Complex . . IV . The ActinomycinaDNA Complex . . . . . . . V . A General Principle Governing Protein-Nucleic Acid Recognition VI . Possible Medical Implications . . . . . . . . VII . Summary . . . . . . . . . . . . . References . . . . . . . . . . . . .

. . . . .

.

. . . . . .

. . . .

153 155 158 165 178 187 158 189

vi i

CONTENTS

Resistance Factors and Their Ecological Importance to Bacteria and to M a n

M . H . RICHMOND I . Introduction . . . . . . . . I1. Resistance Factors and the Genes They Carry I11. Resistance Determinants . . . . . . IV . Other Plasmids Related to Resistance Factors V . The Mating Process . . . . . . . VI . Resistance Factor Transfer in Nature . . References . . . . . . . . .

.

.

.

.

.

.

. . . . . . . . . . . . . . . . . .

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

191 193 213 220 221 233 244

Lysogenic Induction

ERNESTBOREKAND ANN RYAN I . Introduction . . . . . . . . . . . I1. Historical . . . . . . . . . . . . I11. Dircct Methods for Inducing Lysogcns . . . . . IV. A Program Analysis of Early Phage Functions . . . V . Prophage Induction: A TwoStage Process . . . . VI . Indirect Modes of Induction . . . . . . . VII . Proposed Mechanisms for Lysogenic Induction in Bacteria VIII . Analogies in Mammalian Systems . . . . . . References . . . . . . . . . . . .

. . . . . . . . . . . .

.

.

.

. .

. .

. .

.

.

.

.

.

.

249 250 252 254 260 265 282 289 292

Recognition in Nucleic Acids and the Anticodon Families

.JACQUES NINIO I . Introduction . . . . I1. Remarks on Recognition . I11. The Wobble Hypothesis . . IV . The Missing Triplet Hypothesis V . The Experimental Evidence . VI . Discussion . . . . . References . . . . .

. . . . . .

. . . . . .

. . . . . .

. . . . . .

. . . . . .

. . . . . .

. . . . . .

. . . . . .

. . . . . .

. . . . . .

301 303 312 317 327 333 335

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

339 340 341 348

. . . . . . . . . .

Translation and Transcription of the Tryptophan Operon

FUMIOIMAMOTO I . Introduction . . . . I1. Historical Background . . I11. Translation of the t r p Operon IV . Transcription of the trp Operon

...

CONTENTS

V ll l

V . Translation and Transcription of the trp Operon in Nonsensr Mutants of E. coli . . . . . . . . . . . VI . Effect of n Block in Translation on Transcription . . . . References . . . . . . . . . . . . .

. .

. . . .

377 398 402

lymphoid Cell RNA’s and Immunity

A . ARTHURG~TTLIEB I . Introduction . . . . . . . . . . . . . . I1. Historical Perspective . . . . . . . . . . . I11. Biosynthesis of RNA in Immunized Systems . . . . . . IV . Transfer of Immune Phenomena by RNA . . . . . . V . Nonspecific Stimulators of Immune Responses . . . . . VI . Macrophage RNA’s and Immunity . . . . . . . . VII . Possible Mechanisms of Action of RNA’s in the Immune Response VIII . A Hypothesis Regarding the Mechanism of Action of Antigen. . . . . . . . . Ribonucleoprotein Complexes IX. Conclusion . . . . . . . . . . . . . . Referencrs . . . . . . . . . . . . . . SUBJECTINDEX

. .

. . . . .

409 410 412 425 440 442 454

. .

.

457 459 480

. . . . . . . . . . . . . . .

467

CONTENTS OF PREVIOUS

VOLUMES

.

.

.

.

.

.

.

.

.

.

.

470

List of Contributors Numbers in parentheses refer to the pages on which the authors' contributions begin.

ERNESTBOREK(249), Department of Microbiology, University of Colorado Medical Center, Denver, Colorado MURRAY P. DEUTSCHER (51) Department of Biochemistry, University of Connecticut Health Center, Farmington, Connecticut

M. YA. FELDMAN ( l ) ,Institute of Molecular Biology, Academy of Sciences, MOSCOW, U.S.S.R. A. ARTHUR GOTTLIEB (409) Institute of Microbiology, Rutgers University, New Brunswick, New Jersey FUMIOIMAMOTO (339) Department of Microbial Genetics, The Research Institute for Microbial Diseases, Osaka University, Yamada-kami, Suita, Osaka, Japan SAMSON T. JACOB(93) Physiological Chemistry Laboratories, Department of Nutrition and Food Sciences, Massachusetts Institute of Technology, Cambridge, Massachusetts

NINIO(301), Laboratoire de Biochimie du De'veloppement, Faculte' des Sciences de Paris VII, Paris, France

JACQUES

M. H. RICHMOND ( l g l ) , Department of Bacteriology, University Bristol, Bristol, England

of

ANN RYAN(249),t Department of Microbiology, University of Colorado Medical Center, Denver, Colorado HENRYM. SOBELL(153) ,$ Department of Chemistry, The University of Rochester, Rochester, New York; and Department of Radiation Biology and Biophysics, The University of Rochester, School of Medicine and Dentistry, Rochester, New York

* Present address: Department of Pharmacology, The Pennsylvania State University College of Medicine, Hershey Medical Center, Hershey, Pennsylvania. i Deceased. $ Present address: Department of Pharmacology, Stanford University School of Medicine, Palo Alto, California. ix

X

LIST OF CONTRIBUTORS

TAHASHI SUGIMURA ( 127), Biochemistry Division, National Cancer Center Research Institute, Chuo-ku, Tokyo, and Department of Molecular Oncology, The Institute of Medical Science, T o k y o Uwiwersity, Minato-ku, Tokyo, Japan

Preface Volume 13 of Progress in Nucleic Acid Research and Molecular Biology includes ten essays covering a wide spectrum in the nucleic acid field. We believe that our readers will find the contributions of topical interest and importance. They follow our usual pattern of attempting to present “essays in circumscribed areas” in which recent developments in particular aspects of the field of nucleic acids and molecular biology are discussed by workers provided with an opportunity for more personal expression of points of view that may be individualistic and perhaps even controversial. We have not attempted to define or restrict any author’s approach to his chosen subject, and have confined our editing to ensuring maximum clarity to the reader, whom we envisage to be a person himself active in or concerned with the general field of nucleic acids or molecular biology. Needless to say, we do not necessarily share all the opinions or concepts of all the authors and accept no responsibility for them. We seek rather to provide a forum for discussion and debate, and we welcome further suggestions from readers as to how this end may best be served. Indeed, we should like again to remind readers that we wish them to write to us with their comments. Abbreviations and symbols used for nucleic acids and their derivatives are now well established by the authority of the Combined Commission on Biochemical Nomenclature (CBN) of the International Union of Biochemistry (IUB) and the International Union of Pure and Applied Chemistry (IUPAC). Those pertinent to our subject are not usually listed a t the beginning of each chapter, but are listed on the following pages. I n this volume, in the interests of conserving space and reducing costs, we have made an innovation by simplifying the contractions for the titles of some of the most commonly cited journals. A list of these is appended to the section on abbreviations and symbols. We hope that this new usage will be acceptable to our authors and readers alike.

J. N. D. W. E. C.

xi

This Page Intentionally Left Blank

Abbreviations and Symbols All contributors to this Series are asked to use the terminology (abbreviations arid symbols) recommended by the IUPAC-IUB Commission on Biochemical Nomenclature (CBN) and approved by IUPAC and IUB, and the Editors endeavor to assure conformity. These Recommendations have been published in many journals (I) and compendia (5)in four languages and are available in reprint form from the NAS-NRC Ofice of Biochemical Nomenclature (OBN), as stated in each publication, and are therefore considered to be generally known. Those used in nucleic acid work, originally set out in section 5 of t,he above Recommendations ( 1 ) and recently revised and expanded (2-4), are given in condensed form (I-V) below for the convenience of the reader.

I. Bases, Nucleosides, Mononucleotides 1. Bases (in tables, figures, equations, or chromatograms) are symbolized by Ade, Gua, Hyp, Xan, Cyt, Thy, Oro, Ura; Pur = any purine, Pyr = any pyrimidine, Base = any base. The prefixes S-, H,, F-, Br, Me, etc., may be used for modifications of these. 2. Ribonueleosides (in tables, figures, equations, or chromatograms) are symbolized, in the same order, by Ado, Guo, Ino, Xao, Cyd, Thd, Ord, Urd (Urd), Puo, Pyd, Nuc. Modifications may be expressed as indicated in (1)above. Sugar residues may be specified by the prefixes r (optional), d ( = deoxyribo), a, x, 1, etc., to these, or by two threeletter symbols, as in Ara-Cyt (for aCyd) or dRib-Ade (for dAdo). 3. Mono-, di-, and triphosphutes of nucleosides (5‘) are designated by NMP, NDP, NTP. The N (for “nucleoside”) may be replaced by any one of the nucleoside symbols given in 11-1 below. 2’-, 3’-, and 5’- are used as prefixes when necessary. The prefix d signifies “deoxy.” [Alternatively, nucleotides may be expressed by attaching P to the symbols in (2) above. Thus: P-Ado = AMP; Ado-P = 3’-AMP.] cNMP = cyclic 3‘: 5‘-NMP.

11. Oligonucleotides and Polynucleotides 1. Ribonucleoside Residues (a) Common: A, G, I, X, C, T, 0, U, U, It, Y, N (in the order of 1-2 above). (b) Base-modified: sI or M for thioinosine = 6-mercaptopurine ribonucleoside; sU or S for thiouridine; brU or B for 5-bromouridine; hU or 1) for 5,Gdihydrouridine; i for isopentenyl; f for formyl. Other modifications are similarly indicated by appropriate lower-ease prefixes (in contrast to 1-1 above) (8, 3). (c) Sugar-modified: prefixes are d, a, x, or 1 as in 1-2 above; alternatively, by italics or boldface type (with definition) unless the entire chain is specified by an appropriate prefix. The 2’-O-methyl group is indicated by suflz m (e.g., -Am- for 2’-O-methyladenosine, but -mA- for N-methyladenosine). (d) Locants and multiplien, when necessary, are indicated by superscripts and subscripts, respectively, e.g., - 4 A - = Gdimethyladenosine; - s W - or -‘s-= 4-thiouridine; -a“= 2’-O-methyl-P-acetylcytidine. (e) When space is limited, 89 in two-dimensional arrays or in aligning homologous sequences, the prefixes may be placed over the capital letter, the suffixes over the phoaphodiester symbol. xiii

xiv

ABBREVIATIONS AND SYMBOLS

2. Phosphoric Acid Residues [left side

= 5’,

right side

= 3’

(or 2’)

I

.

(a) Terminal: p; e.g., pppN . . is a polynucleotide with a 5‘-triphosphate a t one end; Ap is adenosine 3’-phosphate; C > p is cytidine 2’:3‘-cyclic phosphate (1,6,3). (b) Internal: hyphen (for known sequence), comma (for unknown sequence); unknown sequences are enclosed in parentheses. E.g., pA-G-A-C(C2,A,U)A-U-G-C > p is a sequence with a (5’) phosphate at one end, a 2’: 3’-cyclic phosphate a t the other, and a tetranucleotide of unknown sequence in the middle. (Only codon triplets are written without some punctuation separating the residues.)

3. Polarity, or Direction of Chain The symbol for the phosphodiester group (whether hyphen or comma or parenthesis, as in 2b) representg a 3’-5’ link (i.e., a 5‘ . 3’ chain) unless otherwise indicated by appropriate numbers. “Reverse polarity” (a chain proceeding from a 3’ terminus at left to a 5’ terminus at right) may be shown by numerals or by right-to-left arrows. Polarity in any direction, as in a two-dimensionalarray, may be shown by appropriate rotation of the (capital) letters so that 5’ is a t left, 3’ a t right when the letter is viewed right-side-up.

..

4. Synthetic Polymers The complete name or the appropriate group of symbols (see 11-1 above) of the repeating unit, enclosed in parentheses if complex or a symbol, is either (a) preceded by ‘‘poly,” or (b) followed by a subscript “n” or appropriate number. No epace follows “poly” (8, 6). The conventions of 11-2b are used to specify known or unknown (random) sequence, e.g.1

polyadenylate = poly(A) or (A),,, a simple homopolymer; poly(3 adenylate, 2 cytidylate) = poly(AIC2) or (AI,CZ)., a random copolymer of A and C in 3:2 proportions; poly(deoxyadenylabdeoxythymidy1ate) = poly [d(A-T)]or poly (dA-dT)or (dA-dT), or d(A-T),, an allernuting copolymer of dA and dT; poly(adenylate, guanylate, cytidylate, uridylate) = poly(A,G,C,U) or (A,G,C,U)., a random assortment of A, G, C, and U residues, proportions unspecified. The prefix copoly or oligo may replace poly, if deaired. The subscript ‘WJ may be replaced by numerals indicating actual size.

111. Association of Polynucleotide Chains 1. Associated (e.g., H-bonded) chains, or bases within chains, are indicated by a center dot (not a hyphen or a plus sign) separating the comp2ete names or symbols, e.g. : P O ~ Y(A).poly (U1 or (A),. (U )m poly(A).2 poly(U) or (A)n+2(U)m poly(dA-dC).poly(dG-dT) or (dA-dC)..(dG-dT),; also, “the adeninethymidine base-pair” or “A-T base-pair” in text. 2. Nonasaociated chains are separated by the plus sign, e.g. : 2boly(A).poly(U)I 5 polyW.2 poly(U) poly(A) (1I-W or 2[An.Um]5 A..2U, A,, (114b). 3. Unspecified or unknown association is expressed by a comma (again meaning “unknown”) between the completely specified residues. Note: In all cases, each chain is completely specified in one or the other of the two systems described in 11-4 above.

+

+

xv

ABBREVIATIONS AND SYMBOLS

IV. Natural Nucleic Acids ribonucleic acid or ribonucleate deoxyribonucleicacid or deoxyribonucleate messenger RNA; ribosomal RNA; nuclear RNA “DNA-like” RNA; complementary RNA mitochondrial DNA transfer (or acceptor or amino acid-accepting) RNA; replaces sRNA, which is not to be used for any purpose aminoacyl-tRNA “charged” tRNA (i.e., tRNA’s carrying aminoacyl reaidues); may be abbreviated to AA-tRNA tRNA normally capable of accepting alanine, to form alanine tRNA or alanyl-tRN A tRNAA’a, etc. The same, with alanyl residue covalently attached. alanyl-tRNA or [Note: fMet = formylmethionyl; hence tRNA1M.t or alanyl-tRNAA1a tRNAP*l Isoacceptors are indicated by ‘appropriate subscripts, i.e., tRNA;”, tRNA:’&, etc. RNA DNA mRNA; rltNA; nRNA D-RNA; cRNA mtDNA tRNA

V. Miscellaneous Abbreviations Pi, PPi inorganic orthophosphate, pyrophosphate RNase, DNase ribonuclease, deoxyribonuclease Others listed in Table I1 of Reference 1 may also be used without definition. No others, with or without definition, are used unless, in the opinion of the editors, they increase the ease of reading.

Enzymes In naming enzymes, the recommendations of the IUB Commission on Enzymes, approved by IUB in 1964 (4), are followed as far as possible. At first mention, each enzyme is described either by its systematic name or by the equation for the reaction catalyzed, followed by its EC number in parentheses. Subsequent mention may use a trivial name. Enzyme names are not to be abbreviated except when the substrate has an approved abbreviation (e.g., ATPase, but not LDH, is acceptable). REFERENCES* 1. JBC 241, 527 (1966);Bchem 5, 1445 (1966);BJ 101, 1 (1966);ABB 115, 1 (1966), 129, 1 (1969);and elsewhere.t 8. EJB 15, 203 (1970);JBC 245, 5171 (1970);J M B 55, 299 (1971);and eh3ewhere.t

S. “Handbook of Biochemistry” (H. A. Sober, ed.), 2nd ed. Chemical Rubber Co., Cleveland, Ohio, 1970,Section A and pp. H130-133. 4. “Enzyme Nomenclature,” Elsevier Publ. Co., New York, 1965. [Revision under construction.] 6. “Nomenclature of Synthetic Polypeptides,” JBC 247, 323 (1972);Bwpolymem 11, 321 (1972);and e1sewhere.t

* Contractions for names of journals follow. t Reprints of all CBN Recommendations are available from the Office of Biochemical Nomenclature (W. E. Cohn, Director), Biology Division, Oak Ridge National Labors tory, Box Y, Oak Ridge, Tennessee 37830, USA.

xvi

ABBREVIATIONS AND SYMBOLS

Abbreviations of Journal Titles

Journals

Abbreviations w e d

Annu. RRv. Biochem. Arch. Biochem. Biophys. Biochem. Biophys. Res. Commun. Biochemistry Biochem. J. Biochim. Biophys. Acta Cold Spring Harbor Symp. Quant. Biol. Eur. J. Biochem. Fed. Proc. J. h e r . Chem. SOC. J. Bacteriol. J. Biol. Chem. J. Chem. SOC. J. Mol. Biol. Proc. Nat. Acad. Sci. U.S. Proc. SOC.Exp. Biol. Med.

ARB ABB BBRC Bchem BJ BBA CSHSQB EJB FP JACS J. Bact. JBC JCS JMB PNAS PSEBM

Some Articles Planned for Future Volumes DNA Modification and Restriction

W. ARBER Mechanisms in Polypeptide Chain Elongation on Ribosomes

E. BERMEKAND H. hfATTHAEI Primary Structure of Ribosomal RNA

P. FELLNER Bacterial Ribosomal Proteins

R. A. GARRETT,K. NIERHAUS, AND H. G. WITTMAN DNA Polymerases II and Ill

M. L. GEFTER RNA-Directed DNA Polymerases

M. GREENAND G. GERARD Initiation of Protein Synthesis

M. GRUNBERG-MANAGO AND F. GROS Immunogenic Polynucleotides

L. D. HAMILTON X-Ray Diffraction Studies of Nucleic Acids and Their Components

R. LANGRIDGE, E. SUBRAMANIAN, AND P. J. BOND Base Sequence Determination in DNA

K. MURRAY AND R. W. OLD Mechanism of Bacterial Transformation and Transfection

J. K. SETLOWAND N. K. NOTANI Chemistry of Alkylation and Its Relationship to Mutagenesis and Carcinogenesis

B. SINGER Structure and Function of Viral RNA

C. WEISSMAN Aliphatic Polyomines and the Regulation o f Macromolecular Biosynthetic Reaction in Eukaryotes

H. G. WILLIAMS-ASHMAN AND A. CORTI xvii

This Page Intentionally Left Blank

Errata Volume 12

Page 56. Thc structurcs for 11 and 111 should he cxchangcd with cach other.

Pages 63 arid 67. Structures of 2-mcthyladenosine and l-mcthylguanosine should appcar on pagc 67, and structures VIII and IX should appear on page 63. Page 75, Table V. Codon recognition of tRNAp’ should be as shown below.

-

xix

JAMES NORMAN DAVIDSON

James Norman Davidson 191 1-1972

Professor Norman Davidson, Gardiner Professor of Biochemistry in the University of Glasgow since 1948, died a t his home on September 11th at the age of 61. Dux of George Watson’s College in Edinburgh, he won a scholarship in medicine to the University of Edinburgh where he graduated B.Sc. with 1st Class Honours in Chemistry in 1934 and M.B. Ch.B. with Honours in 1937. Even by this time his interests had come to lie in the laboratory and in 1937 he was awarded a Carnegie Fellowship to work a t the Kaiser Wilhelm Institut fur Zellphysiologie in Berlin under the direction of Otto Warburg. On his return to Britain in 1938 he was appointed to a lectureship in Biochemistry a t the University of St. Andrews, a t University College Dundee, and in 1939 he received the degree of M.D., from Edinburgh University, for a thesis on the enzyme uricase. At the outbreak of war he became interested in the so-called “wound hormones.” This led him to learn the techniques of tissue culture and to his first investigations of the nucleic acids. In 1940 he moved to the University of Aberdeen where he continued his work on tissue culture and nucleic acids, and it was during the period 1940-1945 that he demonstrated that deoxyribonucleic acid and ribonucleic acid were normal constituents of both plant and animal cells. In 1945 he was awarded the degree of D.Sc. by Edinburgh University for work on cellular proliferation, and in the same year he joined the scientific staff of the Medical Research Council in London. After only one year he was appointed Professor of Biochemistry a t St. Thomas’ Hospital Medical School. Norman Davidson always felt very strongly about the effect on Scotland of the drain of trained personnel to the south and overseas and it is not surprising that in 1947 he accepted the offer of appointment to the Gardiner Chair of Physiological Chemistry in the University of Glasgow. Under his influence the department at Glasgow flourished and grew to an independent department of biochemistry second to none in Britain and with an international reputation for research, particularly in the fields of nucleic acids and cell culture. Work in Glasgow showed that deoxyribonucleic acid was largely confined to the cell nucleus while ribonucleic acid occurred in both the nucleus and the cytoplasm. Major contributions were also made to the demonstration of the conxxi

xxii

JAMES NORMAN DAVIDSON

stancy in the amount of deoxyribonucleic acid per avcrage cell in the somatic cells of any given species and to our present understanding of the mechanisms of biosynthesis of both deoxyribonucleic acid and ribonucleic acid in normal, malignant, and virus-infected cells. An outstanding teacher and research worker himself, Norman Davidson held very strong views on the importance of teaching and research in university departments and of the place of biochemistry in relation to medicine and science. As an administrator he had a remarkable capacity for grasping the main objectives clearly while dismissing the trivia and remaining aware of all the relevant matters of detail. He took a keen interest in other areas of science and medicine and was active in the affairs of many medical and scientific societies. In 1960 he was elected a Fellow of the Royal Society and he served two terms as President of the Royal Society of Edinburgh. He was for a time Secretary and later Chairman of the Committee of the Biochemical Society, and among other societies in which he took a particular interest were the Association of Clinical Biochemists, the European Molecular Biology Organization, the Institute of Biology, and the Nutrition Society. I n 1967 he was made a Commander of the British Empire for his services to science in the United Kingdom. I n addition to his own contributions in the field of nucleic acids, he was the author of a monograph (“The Biochemistry of the Nucleic Acids”), which has run to seven editions and is to be found on the bookshelves of all workers in this field throughout the world. With Erwin Chargaff he edited a three-volume series, “The Nucleic Acids,” which for many years served as one of the most important reference works in the field, and with Waldo Cohn he has been editor of the serial publication “Progress in Nucleic Acid Research and Molecular Biology.” His writings, however, were not restricted to the nucleic acid field and he was joint author of a textbook of physiology and biochemistry intended principally for medical students. He was at various times a guest lecturer a t many European and American universities and was well known in universities and research institutes throughout the world. His counsel and advice were widely sought by many official bodies and private individuals and he gave willingly of his time where he knew that he was able to make a useful contribution. The strain of all these activities took its toll on his health and a t the peak of his career he was beset by illness. He would not allow this, however, to prevent him from playing a full part in the affairs of his department and the university. Norman Davidson will be remembered by his friends not only for his energy, his powers of organization, his grasp of his subject, his clarity and incisiveness in the lecture theatre or in debate, but as a man who

xxiii

JAMES NORMAN DAVIDSON

was courteous, kindly, warm-hearted and generous. His death is a serious loss to the scientific community in general and more particularly to biochemistry, the field of nucleic x i d s , his university, and his department.

R. M. S. SMELLIE The U n i v e r d y Glasgow, Scotlnird

This Page Intentionally Left Blank

Reactions of Nucleic Acids and Nucleoproteins with Forma Idehyde'

I

M. YA. FELDMAN Institute of Molecular Biology, Acrtdemy of Scie)ices, iMoscoto, V.S.S.R.

I. Introduction . . . . . . . . . . . . . 11. Interaction of Formaldehyde with Bases, Nuclcosides and Nucleotides . . . . . . . . . . . . . . A. Primary Reactions : Formation of Methylol Derivatives, R--CH?OH . . . . . . . . . . . . . 13. Secondary Rciictions: Formntion of Metliylcne Dinucleotides, R-CHL-R' . . . . . . . . . . . . 111. Interaction of Formaldchydc with Polynuclrotidcs . . . . A. Synthetic Polynucleotidcs . . . . . . . . . B. Ribonucleic Acids . . . . . . . . . . C. Deoxyribonucleic Acids . . . . . . . . . . D. Effect of Secondary Structure . . . . . . . . E. Effect of Formaldehyde on thc Functional Activity of Nuclcic Acids . . . . . . . . . . . . . . IV. Interaction of Formaldcliyde with Nucleoproteins . . . . . A. Formation of Mcthylene Bridges in thc Reaction of Protein with . . . . . . . Formaldeh ydc B. Effect of Formaldehyde on Nuclcoprotcins . . . . . V. Use of Reactions of Nucleic Acids and Nucleoproteins with Formaldehydt . . . . . . . . . . . . A. Structural and Functional Studics of Nidcic Acids . . . B. Inactivation of Viruses by Formoldchydc in Vaccine Production. C. Effect of Formaldehyde on the Genetic Aplmratus of thc Cell . VI. Related Reactions and Thcir Effects (:I* Compsred to Formaldc. . . . . hyde Reactions) . . . A. Miscellanc~ousAldehydes . . . . . . . . . . B. Difunctional Alkylating Agents . . . . . . . . VII. Conclusion . . . . . . . . . . . . . . References . . . . . . . . . . . . .

.

.

. .

. . . .

.

. . . . .

1 3 3 10 15 15 16 19

m

28 30 30 33 35 35 36

38 40 40 42 44 44

1. Introduction Three main reiLsons for the chemical modification of nucleic acids can be singled out in a review of the information available on modifying agents. These agents are used for inactivations of various kinds (such as inactivation of viral RNA or of cytostatic action), for directed functional Translntcd by A. L. Pumpinnsky, Moscow. 1

2

M. YA. FELDMAN

changes of nucleic acids in vivo (mutagenesis, oncogenesis) and for the elucidation, through resulting modification, of structural and functional characteristics peculiar to synthetic or native polynucleotides. Some of these agents can be useful for one, two or all three of these purposes. One of the few agents that serves all three of them is formaldehyde. It is widely used as an inactivator of viruses to obtain vaccines ( I ) and is reported to exert a cytostatic (carcinostatic) effect (a). It is also one of the most promising mutagenic agents affecting multicellular organisms (3, 4 ) . Formaldehyde is used extensively in structural and functional studies of nucleic acids as an agent not so much for causing denaturation as for preventing renaturation, and as a fixator of nucleic acids and nucleoproteins in electron microscope and sedimentation investigations. The use of nucleic acid reactions with formaldehyde has outstripped our knowledge of their mode of action. In many cases a chemical mechanism was postulated for some biological or physical effect that could not plausibly be substantiated, and such diverse products as Schiff bases (5-7), monomethylol derivatives (RNH-CH,OH) (8,9), (R-N=CH,) mono- and dimethylol (R-N(CH,OH) *) derivatives ( l o ) , as well as methylol and methylene (RNH-CH,-NHR) compounds (11-13) were suggested., Recently some progress in the structural study of formaldehyde interaction products with nucleotides and nucleic acids has been made. Evidence has been presented that the reactions proceed according to the following scheme:

A '

0 -NH RNH, RNH-CHZOH

A '

+ CHZO F? 0 -N-CHzOH + CHzO + RNH-CHIOH + RNHZ + RNH-CHz-NHR

4-HzO

(14 (1b) (2)

Reaction ( l a ) proceeds with the participation of the -CO-NH- grouping of pyrimidine and purine heterocycles. In reaction ( l b ) , formaldehyde interacts with the exocyclic amino groups of AMP, GMP and CMP. Reaction (2) involves aminopurines only. This review is the first attempt to sum up the data available on the interaction of formaldehyde with nucleic acids and nucleoproteins with particular emphasis on the evidence for the formation of various structures and the molecular mechanisms of biological and other effects of formaldehyde. *Useful information on formaldehyde chemistry is provided by Walker in his book (14).

REACTIONS OF FORMALDEHYDE WITH NUCLEIC ACIDS

3

II. Interaction of Formaldehyde with Bases, Nucleosides and Nucleotides

A. Primary Reactions: Formation of Methylol Derivatives, R-CH OH The reactions described in this section were discovered by FraenkelConrat in 1954 ( 5 ) . They result in labile noncrystallizable compounds found only in solutions also containing starting compounds (formaldehyde and the nucleic base or its derivative). The reaction product is readily dissociated on simple dilution. However, it was later shown (15, 16) that these reactions are not the only ones to occur when formaldehyde acts on bases or nucleotides. When the reaction mixtures are allowed to stand for many days, the labile (hydroxymethyl or methylol) derivative reacts with the starting base present in the mixture [Eq. ( 2 ) ] to form a methylene bis-compound as the end product. The formation of methylene derivatives does not affect the investigation of primary (methylol) derivatives, the former appearing much later. Some methylene derivatives are precipitated quantitatively ( 1 5 ) . 1. FUNCTIONAL GROUPS

The question, what functional groups react with formaldehyde, was formerly settled by comparing different bases, nucleosides and nucleotides. The comparison was made by means of two tests involving the spectral changes of bases under the action of formaldehyde ( 5 ) and the quantitative estimation of the formaldehyde added (12). Reactions were essentially carried out in neutral buffered aqueous medium a t room temperature for 24-48 hours. I n this time, the primary reaction was almost completed whereas the products of the secondary reaction were still practically absent. The resulting data are presented in Table I. I n all cases studied, compounds containing amino groups reacted with formaldehyde with a marked change in the ultraviolet spectrum, the maximum shifting to longer wavelengths by 3-5 nm and its intensity rising by about 20%. Similar spectral changes occur when formaldehyde acts on deoxyribonucleotides containing amino groups (6). The data presented in the last column of Table I indicate the participation in the reaction not only of the exocyclic groups, but also of the NH-groups in position 9 (or 7) of purines and, possibly, in position 1 of pyrimidines (e.g., in hypoxanthine, 1,3-dimethylxanthine, 2,6,8-trichloropurine, uracil). No reaction takes place if the hydrogen atoms in these positions are replaced by methyl or ribosyl residues (inosine, 1,9-di- and 1,3,9-trimethylxanthine, uridine) .

4

M. YA. FELDMAN

TABLE IR INTERACTION OF PYRIMIDINE AND PURINE DERIVATIVES WITH FORMALDEHYDE IN NEUTRAL AQUEOUS SOLUTION AT ROOM TEMPERATURE AND RELATIVELY Low CONCENTRATION OF CHsO. PRIMARY REACTION (24-48 HOURS) Spectral changes in the presence of 1-2% CHzO (6) Increase of

Compound

emax

(%I

Formaldehyde bound (mole per Shift of XmRx to 100 moles of longer wave- purine or pyrimilength dine derivative)b (nm1 (18)

Pyrimidine derivatives

Uracil Thymine 1,3-Dirnethyluracil Uridine Uridylic acid Cytosine 2-Amino-4-ox yp yrimidine (isocytosine) 2-Aminop y rimidine Cytidylic acid

None None

-

None None -

1 3 None None

-

-

16

3

4 2 0 0

11

5 10

-

Purine derivatives

Adenine Adenosine Adenylic acid Guanylic acid Hypoxanthme 1,3-Dimethylxanthine 2,6,8-Trichloropurine Inosine 1,g-Dimethylxanthine 1,3,9-Trirnethylxanthine

23 19 22 5 -

5 5 5 5

-

-

20

10 9 13 10 9 24 0 1 0

Nucleic acida

RNA (TMV) DNA (thymus)

29 None

3 None

-

Reproduced by permission of Elsevier Publishing Co., Amsterdam. concentrations of reagents were 0.01 M. The free formaldehyde was determined (17,18) and the bonded aldehyde was calculated as the difference between the overall and free formaldehyde. 4

* The initial

REACTIONS OF FORMALDEHYDE WITH NUCLEIC ACIDS

5

It was later shown that the experiments listed in Table I failed to take into account the reaction of formaldehyde with ring NH-groups in position 3 of pyrimidines and 1 of purines, seemingly owing to the relatively low formaldehyde concentration used. At high CH,O concentrations, the absorption spectra of nucleosides with no NH,-group change slightly, thus indicating that they react with formaldehyde. The ultraviolet maxima for uridine and thymidine in 3.3M formaldehyde solution (pH 6.9) decrease by 2% and are shifted to longer wavelength by 2 nm (18). The spectra of uridylic and polyuridylic acids (18, 19) as well as that of inosine (20) reveal similar changes. These changes are believed to be caused by addition of formaldehyde to the N-3 of uridine and the N-1 of inosine. The respective N-1 and N-9 positions must be excluded as they are blocked by ribose residues. That the spectral changes cannot result from formaldehyde addition to C-5 was shown by experiments with thymidine (18), whose spectral changes with formaldehyde were similar to those for uridine, in spite of its blocked C-5 position. The reported spectral data (18-20) do not allow for possible formaldehyde interaction with hydroxyl groups of bases and ribose. The spectral analysis of pseudouridine with different formaldehyde concentrations suggested (18) that, in compounds containing two unsubstituted NH-groups in the ring (pseudouridine, uracil, thymine), both groups react. The participation of ‘
PH

FIG.1 . Titration of 0.00227 M adenine hydrochloride solution, with (B) and without (A) added formaldehyde (0.34%) at 19”C,by aqueous sodium hydroxide (21). By permission of the Chemical Society, London.

6

M. YA. FELDMAN

(9.8) is shifted upward ( 2 1 , 2 2 ) .I n the first case, the pH depression arises from the interaction of the free amino groups with formaldehyde, similar to the reaction of formaldehyde with other amines, in particular with amino acids. I n the region of the acidic dissociation constant (pKg), arising from the g-NH-group, the pH is raised by the following mechanism:

\NH

/

+F

\NHI:

/

[see (19-23)]. The pK values (ca. 4) of cytidylic ( 2 4 ) , adenylic and polyadenylic (25) acids are also lowered by formaldehyde, additional proof of the interaction of formaldehyde with the amino groups of nucleotides. A marked shift of the titration plot to higher p H is observed in the pI(, region of the NH-groups of inosine (20) and of uridylic and polyuridylic acids (19). This fact, together with spectral evidence, proved the interaction of formaldehyde and compounds containing the HN3-C40

I I

grouping in the pyrimidines or the HN1-CEOgrouping in the purines.

I I

A reaction of formaldehyde with the hydroxyl groups of uracil in the tautomeric form is excluded because the pH of solutions of compounds containing “acidic” hydroxyl groups, such as phenol, is not affected on formaldehyde addition whereas such a change is typical of compounds containing amino and imino groups ( 2 1 ) .Titration data do not indicate a participation of the N-1 of adenine in the formaldehyde reaction (22, 25), nor does spectral evidence (see Section 11, A, 2). Formaldehyde addition to the C-5 of uracil (26) (resulting in the formation of a crystalline product, 5-hydroxymethyluracil) takes place in the presence of 0.42 M KOH (73 hours a t 50°C) or 0.5 M HCl (reflux for 25 hours). To prepare 5-hydroxymethyl derivatives of uridylic and cytidylic acids, even more rigorous conditions were used (27). The results on the reaction of formaldehyde with sugars in neutral aqueous medium (14) do not unequivocally exclude the interaction of formaldehyde with the hydroxyl groups of ribose and deoxyribose to form labile semiacetals (R-0-CH,OH) . There is, however, no evidence that such an interaction does actually take place. In any case, low concentrations of CH,O (0.01 M ) do not react in a measurable amount with D-ribose and D-2-deoxyribose (12). Under ordinary conditions, if the nucleotides are not destroyed, their phosphate groups are unlikely to

7

REACTIONS OF FORMALDEHYDE WITH NUCLEIC ACIDS

react with formaldehyde [on formaldehyde reactions with phosphoric acid, see (14)1. It can thus be concluded that a t a pH close to neutral, formaldehyde reacts with the NH,- and NH-groups marked in formulas I-IV with asterisks.

Rib

Rib

(n)

(1)

NH;

0

I

Rib

(III)

Rib

(IV)

This scheme can be considered as proved for uridine ( I ) , cytidine (11) and adenosine (111) but not as conclusively for guanosine (IV). When the ribose residue is replaced by hydrogen, the glycosyl nitrogen a t position 1 of the pyrimidine bases and position 9 of the purine bases can also be attacked by CH,O. 2. SUBSTANTIATION OF THE FORMATION OF MONOMETHYLOL

DERIVATIVES The identification of the primary products of the reaction of formaldehyde with bases and nucleotides is made difficult by their lability. There exists only indirect, spectral and kinetic, proof that these products are monomethylol derivatives (>NCH,OH) . a. Spectral Evidence. Michelson and Grunberg-illanago (65) synthesized N6-hydroxyethyladenylic and poly ( No-hydroxyethyladenylic) acids and found their spectra to be similar to those of adenylic and polyadenylic acids treated with formaldehyde (1% CH,O, pH 6.8, 2.5 hours a t 37"). The similarity shows that the formaldehyde reaction with adenylic and polyadenylic acids most likely gives rise to hydroxymethyl (monomethylol, -NH-CH,OH) derivatives rather than to Schiff bases (-N=CH,) , As No-hydroxyethyladenylic and poly (Ns-hydroxyethyl-

8

M. YA. FELDMAN

adenylic) acids do not react with formaldehyde (the spectra remaining unaffected on addition of CH,O), formaldehyde is unlikely to react with adenylic and polyadenylic acids to give N6-dialkyl derivatives. Similar results were reported by Scheit (28). 2',3'-O-Isopropylidene cytidine reacted with formaldehyde (pH 7, 37°C) to give a product that could be separated chromatographically from the starting product on a thin silica gel layer. The ultraviolet spectrum of this product was found to be similar to that of isopropylidene N6-hydroxyethylcytidine. The latter compound failed to react with formaldehyde. It follows from this that formaldehyde reaction with isopropylidene cytidine leads to isopropylidene N6-hydroxymethylcytidine (V) rather than to a bis (N6hydroxymethyl) derivative. HN-C&OH

I

H,C0 x 0 CH,

The formaldehyde addition products with AMP (25) and isopropylidene adenosine (28) have spectra similar to those of No-methyladenosine (maximum shift to long wavelengths and increased absorption in the maximum as compared to adenosine in both acidic and alkali media), but different from those of N1-methyladenosine. This fact leads again to the conclusion that in the formaldehyde reaction alkylation proceeds a t the 6-amino group rather than N-1. b. Kinetic Evidence. The results from the kinetic study of the primary reaction only (at the early stage of the interaction) are in fair accord with the conception that one NH,-group reacts with one formaldehyde molecule (6, 8, 29). The same result is also always observed from the analysis of pH shifts and spectral changes due to formaldehyde interaction with ring NH-groups (18-23).In the latter case, the l-to-1 ratio points unambiguously to the formation of the structure >NCH,OH, because another derivative allowing for the same ratio, the Schiff base, cannot be produced in the reaction with NH-groups. The proof for the formation of monomethylol derivatives obtained

9

REACTIONS OF FORMALDEHYDE WITH NUCLEIC ACIDS

(SO) by the kinetic study of the overall conversion of adenosine in the primary and secondary reactions is presented in Section 11, B, 1.

3. PROPERTIES OF METHYLOL DERIVATIVES

It has been noted above that these compounds are extremely labile and dissociate into the starting components on simple dilution of the reaction mixtures. Since this dissociation takes place under chromatographic conditions, few workers (31, 28) have succeeded in observing the separation of hydroxymethyl derivatives from the starting compounds on chromatography or electrophoresis. When equimolar concentrations of reagents are used, the amount of formaldehyde bound to nucleotides and bases is much lower than would be expected from considerations of equivalency (see Table I). Spectral changes (8, 22, 19, 18) and the shift of titration plots (24,22,19) increase up to a certain limit as the formaldehyde concentration is raised. This dependence on concentration is used to estimate the equilibrium constants presented in Table 11. It will be seen that various authors have obtained almost the same results by different methods. The reaction constants for the ring NH-groups are much lower than those for the exocyclic amino TABLE I1 EQUILIBRIUM CONSTANTS (K = [RCH20H]/[RH].[CH20])OF PRIMARY FORMALDEHYDE REACTIONS WITH NUCLEOSIDES AND NUCLEOTIDES Compound reacting with CHzO

Reaction conditions

Adenosine

pH 4.8; room temp.

Adenosine

pH 7.3; room temp.

Adenylic acid 5'-AMP 5'-dAMP 5'-CMP 5'-dCMP Uridine 5'-UMP

pH 7.3; room temp.

Inosinic acid 4

pH 7.05; room temp. pH 7.05; room temp. pH 7.05; room temp. pH 7.05; room temp. pH 6.6; 23°C pH 4.7; 20°C 20°C 40°C pH 6.6; 23°C

K Method Quantitative analysis of the reaction products Quantitative analysis of the reaction products Quantitative analysis of the reaction products UV spectrophotometry UV spectrophotometry UV spectrophotometry UV spectrophotometry UV spectrophotometry UV spectrophotometry Titration Titration UV spectrophotometry

(1.mole-1)

Reference

15.8

30

12.3

a

10.9 11.4 11.3 16.6 15.5 2.5

2.6 2.42 1.35 1.7

K is calculated from data (18) listed in the last column of Table I.

(I

8 8 8 8 18 19

19 19

18

10

M. YA. FELDMAN

groups. No results for uracil (23) and guanylic acid (8) are given, for further studies have indicated that formaldehyde may add to each of these substances in two positions rather than one, as previously suggested (see Section 11, A, 1). In the reactions with the NH-groups of nucleosides and poly(U), the equilibrium is reached in less than 30 seconds (18).At room temperature and in a neutral medium, the primary reactions of CH,O with NH,-groups are close to equilibrium in 1-2 days, The adenosine reaction with formaldehyde a t pH 4.8 and 20°C attains equilibrium in 72 hours (SO). Kinetic analysis of spectrophotometric data reveals (69) that as the temperature is raised from 30" to 45°C the formaldehyde interaction with adenosine and poly(A) in a neutral medium is accelerated in both the direct and reverse reactions, the reaction rates increasing from 5 to 10 times. The values of the equilibrium constants for the primary formaldehyde reaction with bases and nucleotides fall with rising temperature as first observed in the titration of the NH,- and 9-NH-groups of adenine (22, 23) and the NH-group of uridylic acid (19).The decreased K for 5'-UMP when the temperature is raised from 20" to 40°C is exemplified in Table 11. This evidence was substantiated spectrophotometrically for mixtures of formaldehyde with AMP and CMP over a wide temperature range, from 15" to 85"C, a t p H 7.5 (32) as well as for CH,O and adenosine (30"-45") (29). Higher temperature never affects the extent of the reaction markedly, and in experiments with G M P (32) and poly (A) (29), the reaction is practically unaffected. There are insufficient results on the influence of hydrogen ion concentration to warrant any general conclusions. Lowering the pH to 2.4 to the acidic side or raising it to 10.8 to the alkaline side does not increase formaldehyde bonding to adenine, adenosine, adenylic acid, hypoxanthine, or thymine. On the contrary, even a t these pH values the amount of formaldehyde bound is lower than that in neutral solution (12). From spectrophotometric results (8), the primary CH,O reactions with nucleotides involving the NH,-group, equilibrium is attained a t the same rate over the pH range of 4 to 8, whereas at pH 10 it is sharply increased. It was shown spectrophotometrically that changing the concentration of the neutral phosphate buffer does not affect ( I I ) , or only weakly affects (8),the interaction of formaldehyde with nucleotides.

B. Secondary Reactions: Formation of Methylene Dinucleotides, R-CH,-R' Numerous stable products of the reaction of formaldehyde with purine and pyrimidine compounds have been isolated and identified, such as

REACTIONS O F FORMALDEHYDE WITH NUCLEIC ACIDS

11

5-hydroxymethyl derivatives of uracil (26), uridylic and cytidylic acids (27) (the reactions proceeding in the presence of alkali or acid with heating) , 7-hydroxymethyluric acid (condensation in the presence of KOH) (33), methylene bis (2-aminopyrimidine) ( 3 4 ) , methylene bis (6arninouracil) (12, 55) (synthesized in neutral medium with or without heating) (see reviews (36-38)) . The patent literature describes the preparation of resins through the action of formaldehyde on di-, tri- and tetraaminopyrimidines under different conditions (39-41). However, if the choice of bases is confined to those characteristic of nucleic acids and the reaction conditions are limited to those compatible with biological experiments, only those purine compounds will be of interest that react with formaldehyde to give stable condensation products, methylene derivThese compounds are dealt with in this atives of the type R-CH,-R’. section. Methylene bis-compounds are formed in the reactions of formaldehyde with adenine, adenosine and adenylic and guanylic acids (15,16). A reaction mixture containing AMP, GMP and formaldehyde gives, together with methylene bis-adenylic and methylene bis-guanylic acids, methylene adenine-guanine dinucleotide ( 1 6 ) . Pyrimidine components of nucleic acids fail to form stable condensation products with formaldehyde under similar conditions (16). 1. METHYLENE BIS-COMPOUNDS OF THE ADENINE SERIES

a. Methylene bis-Adenine ( V I ; R = H ) and Methylene bis-Adenosine ( V I ; R = Ribose Residue), These compounds are formed when adenine or adenosine and formaldehyde are kept a t room temperature and pH 4.5 for many days ( 1 5 ) . This pH is optimal. The product yield is diminished in a more acidic medium as well as in neutral and weakly alkaline media.

I

I

R

R

(VI)

The proof of the formation of methylene bis-compounds is as follows. Two moles of adenine or adenosine react with one mole of formaldehyde ( 1 5 ) . The dependence of the yield of reaction product (during time t ) upon the logarithm of the initial concentration of formaldehyde is graphically represented by a bell-shaped curve (Fig. 2) (SO). Both criteria are specific for methylene bis-compounds.

12

M. YA. FELDMAN

3.0 2.5 +

E

b 2.0 n 0

+

L

1.5

L11

I

1 " 1.0 V I

rn

0.5 0

-3

-2

-1

0

Logarithm of initial molar CH,O concentration

FIQ. 2. Influence of CH,O concentration on the formation of methylene bisadenosine (SO).pH 4.8, 20°C. Curve 1: initial adenosine concentration 0.01 M, incubation for 85 days; Curve 2: 0.015 M adenosine, 53 days.The experimentally found points are placed on the curves derived from theory, taking K N 16 l.mole-', kz 1.4 l.mole-'.day-', K being the equilibrium constant of reaction (3) and kl the rate constant of reaction (4). Reproduced by permission of Nauka, USSR.

The curves presented in Fig. 2 need special explanation. The experimental data are in good accord with theoretical plots corresponding to the reaction sequence RH R-CHIOH

+ CH20 @ R-CHIOH + R H R-CHZ-R + HzO +

(3) (4)

Raising the formaldehyde concentration gives an increased amount of methylol derivative (R--CHIOH) and, conversely, a decreased amount of adenosine (RH) in the reaction mixture. Ideally, the maximal rate of the secondary reaction is when [R-CH20H] = [RH] . With an initial adenosine concentration of 0.01-0.015 M, conversion of 50% of adenosine to its methylol derivative necessitates a formaldehyde 7 (the equilibrium constant for the primary concentration of ~ 0 . 0 M reaction being c 16). With this initial concentration of formaldehyde (0.07M), the secondary reaction runs a t the maximal rate (Fig. 2), whereas higher or lower formaldehyde concentrations lead to slower rates of methylene bis-adenosine formation.

13

REACTIONS O F FORMALDEHYDE WITH NUCLEIC ACIDS

Two important reaction characteristics should be borne in mind when constructing the ideal curve and in plotting the experimental results, 1. The primary reaction practically reaches equilibrium before the secondary reaction starts (prior to the appearance of a measurable amount of methylene bis-derivative) . With an initial adenosine concentration of 0.01-0.015 M and a formaldehyde concentration of 0.014.1 M, methylene bis-adenosine is reported (30) to appear (as a negligible precipitate) only after the solutions have been kept at room temperature (pH 4.8) for 6-8 days. This is in contrast to the primary reaction (Section 11, A, 3 ) , which reaches equilibrium in about 70 hours. On the other hand, if the methylene bis-derivative is readily formed, the reaction fails to give a bell-shaped curve for concentration dependence, as exemplified by the interaction of CH,O with 6-aminouracil (30). 2. No essential shift in equilibrium (3) is observed during reaction (4),which requires an equimolar amount of RH and R-CH,OH on the left and right sides of Eq. ( 3 ) , respectively. The superimposition of the theoretical and experimental plots (Fig. 2) is considered (30) as definitely proving the reaction to lead to methylene bis-adenosine, the monomethylol derivative being an intermediate product. Other possible reactions (see Section I) cannot give a symmetrical bell-shaped curve of concentration dependence. The ultraviolet absorption spectra of methylene bis-adenine and methylene bis-adenosine are characterized by the shift of the maximum to longer wavelengths and an increased absorption a t the maximum as compared with adenine and adenosine, respectively (Table 111). These spectral changes are typical of 6-N-alkyl substitution, being stronger for TABLE I11 ULTRAVIOLET MAXIMA OF METHYLENE BI&OMPOUNDS Compound Methylene bis-adenine Methylene bis-adenosine Methylene bis(adenosine 2’(3’)-phosphate) Methylene bis(guanosine 2’(3’)-phosphate)b

Reference

Xmax

fmnxa

Solvent

(nm)

(X10’)

0 . 0 5 M HCl 0 . 1 M NaOH 0.25 M KOH Acetate, pH 5

28 1 277 and 284 272 272

17.2 14.8 and 13.4 16.8 -

16 48

Acetate, pH 5

255

13.0

43

16 43

Absorbance at the maximum, calculated for 1 M monomer (or 0.5 M methylene bis-compound). Under the same conditions, Xmax = 252 nm and emnr = 12100 for guanosine-2‘(3’)phosphate. (1

14

M. YA. FELDMAN

methylene than 6-N-hydroxymethyl derivatives (see Section 11, A, 1 ) . The nuclear magnetic resonance spectrum of methylene bis-adenine (42) (100 Mc/sec, 10% solution of the compound in 1M NaOD) reveals three signals (singlets) due to protons bonded to carbons. Two signals common to adenine (44, 45) with a chemical shift of 8.46 and 8.27 ppm 6 are due to H-2 and H-8 (2 and 8 positions remaining thus unsubstituted). The third signal (5.51 ppm) has the same intensity as the first two (corrcsponding to two protons) and is to be accounted for by the inethylene group binding two adenine residues. The chemical shift of 5.51 ppm conforms to the position of the methylene group between two exocyclic nitrogens, HN-CH,-NH. (The methylene group bonded to two nitro-

I

I

gens involved in the heterocycles is expected to cause a chemical shift of about 7 ppm.) Methylene bis-adenine and methylene bis-adenosine share several common characteristics (15), such as insolubility in water and organic solvents, decomposition in mineral acids with liberation of formaldehyde and base, low chromatographic mobility and high melting points. b. Methylene bis-Adenylic Acid (VZ; R, Phosphoribosyl Residue). This acid may be isolated chromatographically from the mixture of AMP and CH,O ( 1 6 ) . The reaction goes to completion in 15 days (at room temperature and pH 4.8) and gives rise to a very low yield of C H , ( A P ) ~ not exceeding 6 4 % of theory. When chromatographed according to Cohn (46) on Dowex 1 in a formate system, methylene bis(adenosine 2'(3')phosphate) is separated from 2'- and 3'-AMP to give three isomers (16) with different positions of phosphate groups ( P-2'-Ado-CH2-Ado-2'-P, P-2'-Ado-CH2-Ado-3'-P, P-3'-Ado-CHz-Ado-3'-P) . Dephosphorylation of the three products results in methylene bis-adenosine. When chromatographed on DEAE-cellulose in a concentration gradient of NaCl (pH 5 ) , CH2(Ap) separates without dissociating into isomers (4s). 2. METHYLENE BIS-COMPOUNDS OF THE GUANINE SERIES These products have been less extensively studied than those of the adenine series. The low solubility of guanine and guanosine hinders the investigation of their interaction with formaldehyde. Dissolution of guanosine a t 70" (0.01 M, pH 4.8) and immediate addition of formaldehyde (0.1 M ) leads to a stable (at 36°C) solution that gives rise, in 10-15 days, to a slowly growing precipitate ( 4 7 ) .This precipitate has not yet been identified but its properties resemble those of the methylene bisderivatives of adenine and adenosine. It is insoluble in water, on paper chromatography in alcohol-acid mixtures it follows guanosine with an R, close to that of methylene bis-adenosine, and on hydrolysis in 1 M

15

REACTIONS O F FORMALDEHYDE WITH NUCLEIC ACIDS

HC1 a t 100" it gives off CH,O. It might thus be suggested that the precipitated product is methylenc bis-guanosine. The reaction product of GMP and CH,O, methylene bis-guanylic acid, is formed as slowly as is CH2(Ap)a(16).The effect of formaldehyde concentration is expressed by a symmetrical bell-shaped curve. On paper chromatography, CH, (Gp) , runs far behind GMP. Ion-exchange chromatography on Dowex 1 makes it possible to separate methylene bis(guanosine 2'(3') -phosphate) into isomers that are eluted over the same range of formic acid concentrations (approximately from 2.6 M to 4.0M ) as are C H , ( A P ) ~isomers, though the CH,(Ap), and CH,(Gp), isomers can be separated quite sharply.

3. MISCELLANEOUS DIMERS A reaction mixture containing CH,O and two nucleotides (AMP and GMP) yields a product not found in mixtures involving CH,O and only one of the two nucleotides. Thus the reaction product should have the structure P-Guo-CHa-Ado-P, which agrees with base composition (A and G in a 1-to-1 ratio) as well as by the bell-shaped curve for the concentration dependence. Other mixtures, such as UMP CMP + C H 2 0 and AMP G M P UMP CH,O have also been studied, but failed to show any additional condensation products (16, 48).

+

+

+

+

111. Interaction of Formaldehyde with Polynucleotides

A. Synthetic Polynucleotides The only results available on the interaction of biosynthetic polynucleotides with formaldehyde are concerned only with the labile primary products formed in the early stage of the reaction. On treatment of polynucleotides with [W]formaldehyde (0.1%) in neutral solutions a t room temperature for 16-20 hours followed by precipitation of polynucleotides with ethanol (11), the label is bound appreciably only in those polynucleotides containing amino groups: poly(A) and poly ( C ) , but not poly(U) and poly(1). When poly(A) (49, 29) and poly(C) (6, 50) are treated with formaldehyde, their spectra change just as do those of corresponding monomers. Poly (N6-hydroxyethyladenylic acid) does not react with formaldehyde ( 2 5 ) , nor does N6-hydroxyethyladenylic acid. The similarity in spectra indicates that the action of CH,O on polymers gives rise to the same products obtained on CH,O treatment of the corresponding monomers, that is, aminomethylol derivatives R-NH CH,OH. This

.

16

M. YA. FELDMAN

accords the decreased pk' of poly(A) in formaldehyde solutions, to 3.0 (25j.

The reactions of formaldehyde with poly(U) and UMP have many common characteristics (18, 19), such as identical spectral changes in solutions with a high formaldehyde concentration, increased pK, ready completion of the reactions that rcach equilibrium in less than 30 seconds, and similar equilibrium constants whose values are similarly lowered a t higher temperature (evidence for UMP is given in Section 11,A, 1).It is evident that formaldehyde is bound to the uracil residues in poly(U) just as it is in free uridylic acid. The resulting N3-hydroxymethyl derivatives are extremely labile, and it apparently suffices for their quantitative dissociation to remove the free formaldehyde by repeated polynucleotide precipitation. This, as well as the low initial formaldehyde concentration, seems to explain the almost complete absence of bound [14C]formaldehyde in the experiments (11) with poly(U) and poly(1).

B. Ribonucleic Acids As far back as 1901 the patent literature carried reports on the treatment of nucleic acid with formaldehyde (51). Yet it was only after Fraenkel-Conrat indicated a change in the ultraviolet absorption spectrum of TMV-RNA in the presence of formaldehyde ( 5 ) (see Table I) that biologists and biochemists directed their attention to the reaction of RNA with formaldehyde. In the same work (5)-that is, two years before it was discovered that TMV-RNA is infectious (56, 53)-he suggested that the formaldehyde inactivation of viruses is due to the action of formaldehyde on the nucleic component rather than on the protein. In 1958, Staehelin (11) suggested that the TMV-RNA reaction with formaldehyde gives rise both to labile products that dissociate on dialysis and to stable derivatives. He also suggested that by analogy with the formaldehyde interaction with proteins, this reaction can be considered as resulting in labile aminomethylol compounds and stable methylene compounds R-CH,-R'. However, Staehelin's results could be interpreted quite differently and did not necessarily point to the formation of two types of compounds (9). Yet, his suggestion about the successive formation of methylol and methylene derivatives was substantiated both by low molecular models (see Section 11, B) and by direct study of the reaction of RNA with formaldehyde (6.4,16). 1. METHYLOL DERIVATIVES

The main methods used to study the primary reaction products of RNA and formaldehyde are spectral analysis and quantitative estimation of bound [ I T ] formaldehyde. However, neither of them provides any

17

REACTIONS O F FORMALDEHYDE WITH NUCLEIC ACIDS

information about CH,O addition to cyclic nitrogen (N-3 of uracil and N-1 of guanine residues). Minute changes in the ultraviolet spectrum caused by this addition are obscured by the large spectral changes due to the reaction of exocyclic amino groups with formaldehyde. Quantitative cstiniation of the bound label requires the removal of the free [14C]forinaldehyde, which seems to be accompanied by the complete dissociation of extremely labile products of the addition of formaldehyde to NHgroups in the heterocycles. Therefore, it is only possible to discuss formaldehyde interaction with exocyclic amino groups of ribonucleic acids. The formation of aminomethylol derivatives (R-NHCH,OH) may be judged by characteristic spectral changes (see Table I ) .These spectral changes are almost completely reversible on prolonged (4 days) dialysis ( 5 5 ) . The dynamics of the formation of methylol derivatives in the formaldehyde reaction with amino groups of RNA a t room temperature and pH 4.6 is shown in Fig. 3 (curve 1 ) . To simplify the understanding of primary reactions, the equilibrium shift due to the very slow formation of methylene derivatives is not considered. The same time is required for equilibrium to be reached (about 100 hours) a t pH 7.6 (9) whereas the aminomethylol derivative of adenosine is formed in 72 hours a t p H 4.8 and 20°C (SO).

!

8

y I

100

-

200

300

3

I

4 I

400

I

I

500

600

Hours

FIG.3. Reaction of tRNA with "CH,O with time. Measurements were made by different workers (9, 56) under similar conditions. The nonfractionated yeast tRNA reacts with "CH20 in a weak acidic solution (pH 4.3-4.7) a t low ionic strength a t room temperature. Curve 1: Total number of nucleotides labeled in 0.1 M "CH,O without Mg" (tRNA concentration on nucleotides, 0.003 M) ; Curve 2: the same in the presence of Mg2+( 9 ) ; Curve 3: tRNA nucleotides participating in the formation of methylene cross-links L0.2 M "CH,O, 0.02 M (as nucleotides) tRNA1; Curve 4 : same as 3 in the presence of Mg2+( 6 6 ) . By permission of John Wiley & Sons, Inc., Nrw York, and Elsevier Publishing Co., Amsterdam.

18

M. YA. FELDMAN

Equilibrium is attained much more quickly in the primary RNA reaction with formaldehyde as the temperature is raised. Thus, a t 63" in 1 M formaldehyde (0.1 M phosphate, pH 8.5), the reaction with amino groups is nearly complete (about 85%) in 10 minutes (32). I n a 0.5% solution of CH,O (in 0.001 M phosphate buffer) the reaction is over in 10 hours a t 80°C (57). Such an acceleration of the reaction a t higher temperatures cannot be considered as due solely to the destruction of the secondary structure of RNA. In Section 11, A, 3 evidence was presented (29) on the increased rate of both the direct and reversed reactions when CHrO mixtures with adenosine or poly(A) are heated in neutral medium. Judging by the equilibrium constants, the NH,-groups of RNA react with formaldehyde just as do the amino groups of free nucleotides. According to Boedtker ( 3 2 ) , under denaturation conditions (at 63°C) the NH,-groups of RNA are responsible for the bonding of an amount of l'CH,O that corresponds to K N 11.0, i.e., to a value close to those of equilibrium constants for the formaldehyde reactions with the NH,groups of free nucleotides (see Section 11, A, 3 ) . Changes in pH over the range of 5-8 do not affect the amount of 14CH,0 bound to RNA (11). At pH 4.6, [14C]formaldehyde is bound to the amino groups of tRNA in a somewhat smaller amount than a t pH 7.6 (9), with 40% of the tRNA amino groups reacting with formaldehyde in the former case (see Fig. 3) and 48% in the latter. The difference is accounted for by a partial dissociation at pH 4.6 of the amino groups of cytidylic acid (pK,, = 4.5)'. 2. METHYLENE DERIVATIVES Methylene bis-adenylic acid, methylene bis-guanylic acid and methylene adenine-guanine dinucleotide have been isolated chromatographically (on Dowex 1, formate) from alkali hydrolyzates of formaldehyde-treated rRNA (rabbit liver) (16). Every methylene bis-nucleotide is in turn divided into isomers differing in the positions of phosphate groups whose exact location in the isomers is, however, as yet uncertain. No stable condensation products of formaldehyde with pyrimidine residues of RNA have been discovered (16). The dynamics of the formation of methylene his-derivatives in the tRNA reaction with formaldehyde is shown in Fig. 3. The same kinetic plots as in Fig. 3 are also obtained on '*CH,O action on the rRNA of rabbit liver and TMV-RNA (16, 56). In our experiments (16, 43) a t pH 3.5, from 27 to 30% of purine nucleotides of the rRNA are involved in the formation of methylene bridges; a t pH 4.8, the percentage is 12-17; a t p H 7 to 8.5, only 6-7 (15-24 days, room temperature, 0.2 M CH,O; ionic strength of buffer, 0.1). The reaction becomes faster as the formaldehyde

REACTIONS O F FORMALDEHYDE WITH NUCLEIC ACIDS

19

concentration is raised to 0.2 M (pH 4.8, room temperature, 0.02 M rRNA nucleotides) . A further increase in formaldehyde concentration does not affect the rate of formation of methylene bridges (16). The concentrations of hydrogen ion and CH,O do not affect the secondary RNA reaction with formaldehyde in the same way as they do the secondary reactions of adenosine and purine mononucleotides (see Section 11, B ) . The elucidation of this fact requires more detailed study allowing, in particular, for the role played by the macrostructure of RNA in the reaction with CH,O. At 37°C the amount of methylene cross-links formed is 2.5 times higher than a t 20” (tRNA, p H 4.8) ( 5 6 ) . Dialysis does not reduce the amount of the cross-links (16). The effect of ionic strength and Mg2+is dealt with in Section 111, D, 2.

C. Deoxyribonucleic Acids I n 1953 Zamenhof, Alexander, and Leidy (58) reported that formaldehyde a t high concentrations ( 4 M , p H 7.2, 30”) causes a sharp decrease in the transforming activity of DNA and a gradual drop of viscosity. This does not happen with 0.33M CH,O. It was suggested that formaldehyde reacts with the amino groups of nucleic acid and destroys hydrogen bonds. Later, direct proof of the interaction of DNA with formaldehyde was presented (11).I t was shown that denatured DNA was capable of adding [14C]f~rmaldehyde(for the role of the secondary structure of DNA in this reaction, see Section 111, D, 3 ) . The reaction of DNA with formaldehyde is much less well understood than are the reactions with mono- and polyribonucleotides. However, it can be expected that the bases in DNA react with C H 2 0 in principle just as do the free bases or bases in RNA. Early results (11) on the RNA-formaldehyde reaction, which gives not only labile derivatives but also derivatives remaining intact after prolonged dialysis, were also found t o be valid for denatured DNA ( 5 9 ) . The DNA reaction with 14CH20was carried out under denaturation conditions (10 minutes, 100°C). A small part of bound radioactivity was not removed even on prolonged dialysis (up to 5 days) ( 5 9 ) . According to spectral evidence, the primary formaldehyde reactions with ribo- and deoxyribonucleoside phosphates result in the same (monomethylol) derivatives (see Section 11, A). Similar changes of ultraviolet spectra are observed after formaldehyde treatment of single-stranded and denatured double-stranded bacteriophage DNA’s (60, 8). The evidence for methylene bridge formation between purines, reviewed in Sections 11, B and 111, B, 2, suggest that a t least some of the so-called “firmly bound formaldehyde” that is not detached from DNA

20

M. YA. FELDMAN

on dialysis is actually in methylene bonds. Some authors tried to determine by ultracentrifugation whether DNA chains separate on formaldehyde treatment and thermal denaturation or are prevented from doing so by the cross-links formed. Centrifuging in a density gradient (61, 62, 69) gave equivocal results. Analytical ultracentrifuging appears to indicate the formation of cross-links hindering the complete separation of chains (6% 64) *

D. Effect of Secondary Structure Numerous studies have been concerned with the effect of the secondary polynucleotide structure on the reaction of formaldehyde and with the reverse relation, that of the effect of the reaction on the structure of the polymer. The evidence available can be summed up as follows. 1. The base functional groups involved in the formation of hydrogen bonds do not react with formaldehyde, and, conversely, bound formaldehyde hinders the formation of the usual hydrogen bonds between complementary bases of nucleic acid. 2. The stacking interaction of bases does not hinder the reaction of amino groups with formaldehyde. In turn, the reaction with CH,O does not prevent base stacking. 3. Formaldehyde does not destroy the polymer chain, does not disrupt base stacking, and does not seem by itself to break hydrogen bonds. It does, however, make nucleic acid more sensitive to the action of denaturing agents, in particular to the action of heat (the “melting temperature” of DNA is lowered in the presence of formaldehyde). This may be explained by the fact that in the presence of formaldehyde denaturation is irreversible because the formaldehyde bound to the bases hinders rmaturation. The main experimental evidence presented below is principally obtained a t the first reaction stages when practically only methylol derivatives are formed. (The effect of the secondary structure on the formation of methylene bridges has been studied only for ribonucleic acids; see Section 111,D, 2.) 1. SYNTHETIC POLYNUCLEOTIDES

The stable complex of poly(U) and poly(A) (0.1M phosphate buffer, room temperature) is similar to DNA in not binding [14C]for-* maldehyde (in 0.05% solution). However, when the ionic strength is low, (0.001M phosphate), poly (A) reacts with formaldehyde as if there were no poly(U) in the solution a t all (11). Thus the DNA-like secondary structure fixed by hydrogen bonds hinders the formaldehyde reaction with bases.

REACTIONS OF FORMALDEHYDE WITH NUCLEIC ACIDS

21

The question how formaldehyde treatment affects the polynucleotide secondary structure has been discussed by Haselkorn and Doty (6). Having studied differential spectra, the authors chose a wavelength a t which changes in the intensity of absorption characterize either the extent of denaturation or only that of the reaction. At about 280 nm for poly(A,U) and 290 nm for poly(I,C), the changes in optical density are due to formaldehyde binding, whereas at about 260 and 250 nm, respectively, they are due only to denaturation. Kinetic studies during the short incubation period (up to 1.5 hours) at pH 6.8 (0.11 M phosphate) showed formaldehyde to cause denaturation of polynucleotide complexes a t a relatively high concentration (about 1 M) and somewhat elevated temperature (35”C), the rate of denaturation greatly exceeding that of the reaction and, thus, not limiting the latter. Poly (A,U) reacts with formaldehyde a t the same rate as does poly(A). Similarly, poly(1,C) reacts a t the same rate as does poly(C). However denaturation becomes markedly slower as the ionic strength is raised to 1.0. Under these conditions, it limits the reaction and lowers the formaldehyde interaction rate with amino groups. It is to be noted that what is termed by Haselkorn and Doty (6) as “denaturation” has been actually shown by further studies to be a much more complex process, apparently involving thermal denaturation and a fast formaldehyde addition to uridine and inosine residues that cannot be registered spectrophotometrically. The formaldehyde added to ring -NH-COgroups hinders renaturation and thus favors the less rapid formaldehyde reaction with exocyclic amino groups reported by the authors. Such an interpretation is in accord with experimental data on polydeoxynucleotides, poly (dA,dT) and poly (dA) apoly (dT) (65). The alternative concept of the “induction effect” (66) (see Section 111, D, 3), which completely ignores the very fast CHzO addition to the ring NH-groups, can hardly provide a better understanding of the process. The effect of formaldehyde concentration on denaturation of polynucleotides was studied with poly(1) (6). When the concentration is increased by 1% (ionic strength of about 0.9, pH 6 ) , the T, drops by about 18”. To minimize denaturation it is proposed (6) to make use of as low a CH,O concentration as possible (about 0.1 M ) , high ionic strength and low temperature (about 20°C). It has been shown by optical rotatory dispersion (50) that in neutral solution poly(C) has a highly ordered secondary structure. It is not destroyed in the presence of formaldehyde, i.e., it contains few or no hydrogen bonds, and it is completely disrupted by ethylene glycol as would be expected to be the case for structures maintained by hydro-

22

M. TA. FELDMAN

phobic interactions. It is thought (50) that poly(C) a t pH 7 has a single-stranded helical structure stabilized by intrastrand stacking of pyrimidine bases. (The pH is critical as poly(C) a t pH 4.1 has a hydrogen-bonded, double-stranded structure.) The reaction with CH,O plays an important role for two reasons. First, it helps to elucidate the nature of forces maintaining the secondary structure of poly(C) in neutral medium, and second, it shows that base stacking does not affect the formaldehyde reaction with amino groups and is not destroyed by it. Formaldehyde treatment of poly(C) was carried out by heating up to 90°C followed by slow cooling down to 20" (50), guaranteeing the irreversible destruction of hydrogen bonds (see Section 111, D, 3 ) . Yet, the Cotton effect, whose intensity can be considered as a measure of asymmetry (helicity) of poly (C) molecules, is the same whether poly(C) is treated with formaldehyde or not. It is thus seen that the secondary structure of poly(C) a t pH 7 has no relevance to hydrogen bonding and CH,O does not destroy the forces maintaining this structure. This was also proved in the same work (50) by the following facts. a. Rising temperature affects the intensity of the Cotton effects of poly (C) and formaldehyde-treated poly (C) similarly. In both cases, the changes are gradual and noncooperative as distinct from the temperature dependence for the DNA-like double-stranded structures supported by hydrogen bonds. b. On formaldehyde treatment, the absorption spectrum of poly (C) undergoes the usual changes: the intensity of the maximum rises by 14% and the maximum is shifted to longer wavelengths by 4-5 nm, showing that the reaction actually takes place. However, on heating treated and untreated poly (C) , the relative increases in optical density due to thermal denaturation were both gradual and identical. I n the experiments with formaldehyde-treated poly (C) , structures stabilized by hydrogen bonds were certainly absent before thermal denaturation started. The hyperchromic e k c t was caused in this study by the disruption of other forces maintaining the secondary structure of poly (C) in neutral medium, such as, possibly, the stacking interaction. Fasman et al. (50) proved that formaldehyde does not affect the stacking interaction. Later, Stevens and Rosenfeld ($9) showed unequivocally that single-stranded base-stacking does not affect the chemical affinity of bases for formaldehyde. Their work on poly(A) a t p H 7.5 and with 1-2% CH,O suggests that: (i) a t p H 7.5, poly(A) has few or no hydrogen bonds; (ii) the secondary structure of poly(A) can be judged by the form of the thermal denaturation plots, which resemble those of poly(C) and are apparently due to stacking interaction between adjacent bases, with formaldehyde leaving the Structure intact; and (iii) base

REACTIONS O F FORMALDEHYDE WITH NUCLEIC ACIDS

23

stacking does not hinder the interaction of bases and formaldehyde as evidenced both by an almost quantitative addition of ["C] formaldehyde without any disruption of the secondary structure and by the results of comparative kinetic studies of formaldehyde reactions with poly (A) and adenosine. The ratios of the rate constants for the direct and back reactions were practically identical for the interaction of formaldehyde with poly(A) and in the reaction with adenosine at 30", 35", 40", and 45°C. The absolute values of these constants were somewhat lower in the former than in the latter case. 2. RIBONUCLEIC ACIDS At a low ionic strength, all the amino groups of tRNA can react with formaldehyde at 25°C. The extent of the primary reaction is determined by the equilibrium a t the particular concentration of reagents chosen and is independent of the initial secondary structure (9).As the ionic strength is raised, the rate and extent of both the primary (11, 57, 66) and secondary (16) reactions fall. Mg2+ions are particularly effective in this respect (Fig. 3). RNA is generally much less stable to formaldehyde than is doublestranded DNA. The hydrogen-bonded regions of RNA are much smaller and less stable than those of DNA, the cooperative effects being accordingly weaker. It might be suggested that limited and short-lived unwinding of double helices is much more frequent in RNA than it is in DNA, thus providing more opportunities for base interaction with CH,O. Formaldehyde addition to the double-helical regions of RNA thus becomes possible a t room temperature and a t low concentrations of CH,O. Stabilization of RNA double helices by means of Mg2+ gives them almost the same stability as DNA. At room temperature and in the presence of Mg2+,a high percentage of tRNA bases is completely unavailable to the action of formaldehyde [Fig. 3; see also references (67, S S ) ] . Similar results were reported for tRNAPheat 35°C (66). The influence of formaldehyde treatment on the hyperchromic effect (57, 32, 69) deserves special consideration. Figure 4 shows typical "melting curves" of RNA. With untreated RNA, the sigmoid curve reflects the cooperative process of thermal denaturation. As the ionic strength is decreased, the midpoint of the ascending part of the curve, the T,, is shifted toward lower temperatures. The gradual and comparatively small increase in absorption on heating RNA treated with formaldehyde is independent of the ionic strength. The shape of the curve resembles those of poly (A) and poly(C) (either treated or untreated with formaldehyde) in neutral solutions (50, 2 9 ) . This resemblance, as well as the absence of

24

M. YA. FELDMAN

F---l

0.55

10

30

50

70

90 OC

FIQ.4. Absorbancc as a function of temperature of TMV-RNA bcfore and after reaction with formaldehyde (32).RNA was treated with 1.2 M CH20 for 15 minutes at 85' and rapidly cooled. Measurements were made at 260 nm. At this wavelength, the changes in optical density with rising temperature depend on thermal denaturation only, rather than on the reaction of bases with formaldehyde (heating formaldehyde mixtures with mononucleotides has practically no effect on optical density a t 260 nm). Control (without CH20) in 0.1M phosphate buffer, pH 7.5 ( 0 ) and 0.001M phosphate (0) ; RNA treated with formaldehyde in 0.1 M phosphate (A) and 0.Wl M phosphate ( A ) , Similar curves for thermal denaturation were obtained in experiments with other virus RNA's (32, 70) and with rRNA (71).

the ionic strength effect, shows that hypochromism and, accordingly, the secondary structure of RNA treated with formaldehyde are due to the interaction of stacked bases rather than to hydrogen bonds. I n such an RNA there are few or no double-stranded helical regions. The change in the absorbance of hydroxymethylated RNA on heating is reversible and is eliminated on cooling (32,68-71). A record of the dependence of optical density on temperature, not after RNA treatment with formaldehyde but from the beginning of this treatment, gives the usual melting curves showing a sharp increase in optical density over a limited temperature range. I n this case, cooling reveals two hypochromic fractions. The first, due to base stacking, is rather small and reversible, the second is large and irreversible (70,7 1 ) . Formaldehyde addition to RNA amino groups makes it impossible for

REACTIONS OF FORMALDEHYDE WITH NUCLEIC ACIDS

25

them to participate in the formation of hydrogen bonds (57) but does not hinder base stacking (68, 69, 32) under suitable temperature. Melting curves similar to those presented in Fig. 4 have been recorded by Hall and Doty [see Fig. 2 in reference ( 5 7 ) ] ,who used urea rather than formaldehyde as ti denaturant. It was later shown that urea fails to give such unequivocal effects as a denaturing agent as does CH,O (72, 7.3, because it appears to weaken rather than to completely abolish both hydrophobic interactions (74) and hydrogen bonding (72). Formaldehyde treatment increases the effective hydrodynamic volume of tRNA molecules. The sedimentation coefficient of tRNA falls by 0.40.7 Svedberg unit (69, 56). Thc intrinsic viscosity rises from 0.06 dl/g to 0 . 0 9 4 1 dl/g (69) and remains so even after free formaldehyde is removed (tRNA precipitation with ethanol and dialysis for 18 hours) (69). A more prolonged dialysis (for 48 hours), to remove not only the free but also the labile bound formaldehyde, results in almost complete restoration of the initial tRNA sedimentation rate. The presence of methylene bonds does not affect the sedimentation characteristics of tRNA ( 5 6 ) . The sedimentation and chromatographic results point to no chain rupture or dimerization (or polymerization) of tRNA on prolonged formaldehyde treatment (15 days, 20°C, pH 4.8). After limited guaniloribonuclease digestion of tRNA containing methylene bridges, large fragments (“halves”) are not separable from each other. Thc methylene cross-links formed both in the presence and the absence of Mg2+ are intramolecular, of the type

TCH7 . . R-

-It.

rather than intermolecular (66). The reversible decrease of Sedimentation coefficients without rupture of the chain on CH,O treatment is also observed in experiments with ribosomal and virus RNA (75-78). The aggregation of RNA a t pH <4.2 and low Mg2+ concentration leads, in the presence of 7.7% CH,O (20 minutes, 60°C), to a quantitative formation of stable specific dimers (78).The 28 S molecules combine with 28 S rRNA only. Similar specific complexes are formed by the 16 S rRNA of E. coli and by the RNA of bacteriophage MS2. Thus formaldehyde appears to fix the dimers by means of interchain cross-links (78, 79):

-RI

CHI

I

-R-

26

M. YA. FELDMAN

3. DEOXYRIBONUCLEIC ACIDS

Native double-stranded DNA fails to react with formaldehyde (6,11). For reaction to occur, it is necessary to destroy the hydrogen bonds of DNA by prolonged dialysis against watcr (11, 80),heating, or alkaline treatment (81). I n some cases, particularly in experiments with heating, the denaturant and formaldehyde are both used, but the mechanism remains the same as whcn the denaturant acts before the formaldehyde. After reaction with formaldehyde, DNA is not capable of renaturation (8, 61). The effect of formaldehyde on the secondary DNA structure is shown by thc melting curves in Fig. 5 (82). I n the absence of formaldehyde and on slow cooling, the DNA is almost completely renatured. On repeated heating, DNA “melts” as usual, the optical density increasing sharply a t about 82°C. The picture is different in the presence of formaldehyde. At first, denaturation proceeds as sharply as when no formaldehyde is present (this part of the curve is not shown in Fig. 5 ) , but a t a lower temperature. At 1% CH,O, the T,,,decreases by about 15°C. A stronger ‘Lhyperchromic” effect (Fig. 5 ) is due to the modification of chromophoric groups. When the temperature is lowered, no renaturation takes place. A small decrease in optical density does not account for even partial renaturation, as evidenced by the form of the curve, the optical density changing gradually both on cooling and repeated heating. However, the presence of some hypochromism points to a secondary structure of hydroxymethylated DNA. On repeated heating, the temperaturedependence curve resembles those for poly(A) and poly(C) in neutral

I

1

1

25”1Oo0 80

I

1

60

40

IIII

I

20”20 40 Temperature ( C ” )

1

I

I

60

80

100

FIG.5. Changes in absorbance at 260 nm on hcnling, slow cooling, and reheating T4 bacteriophage DNA with and without formaldehyde (6’2) (pH 8, 0.37 M CH20). 0,+HCHO; 0 , -HCHO. By permission of Academic Press, Inc., New York.

REACTIONS O F FORMALDEHYDE WITH NUCLEIC ACIDS

27

medium both in the presence and the absence of CH,O as well as that for hydroxymethylated RNA. This likeness suggests that hydroxymethylated DNA has a secondary structure maintained by base stacking only. Such a structure is not peculiar to native DNA, being due to formaldehyde treatment (this holds only partly for RNA, and the secondary structure of poly(C) and poly(A) at pH 7 is unaffected by formaldehyde; see Sections 111,D, 1 and 2 ) . There is enough evidence to justify the presumption that on partial denaturation caused, e.g., by moderate heating, only unwound sites of deoxyribonucleic acid react with formaldehyde. As formaldehyde prevents renaturation, the cooling of the solution leaves these sites open. It is possible to find by electron microscopy the DNA regions denatured by heating and fixed with formaldehyde (83,84). These are distributed in DNA in a strictly regular fashion rather than randomly. Their number, size and location in the molecules of a particular deoxyribonucleic acid are quite reproducible. Thus, these sites have a specific structure. I n the presence of formaldehyde, DNA undergoes denaturation just as it does in its absence, with the destruction of A - T pairs preceding that of G - C pairs. This fact was proved by two methods, differential spectrophotometry, which allows the melting of A . T and G - C pairs of DNA to be observed separately (85), and by electron microscopy (86). The latter is used to follow the partial denaturation of deoxyribonucleic acids with different base compositions. DNA is heated in 12% CH,O solution a t various temperatures (46"-52") for 10 minutes and then quickly cooled. I n the DNA molecules of papilloma virus with a high content of A - T pairs ( 5 8 % ) , the denatured sites arc evident a t lower temperatures than in those of polyoma virus with fewer A * T pairs (52%).The DNA molecules of Cancer pagurus with very high content of A - T pairs (over 90%) and the synthetic polymer, poly (dA,dT) , melt a t relatively low temperatures following the principle "total or no" change. No melted sites are to bc seen in these polynucleotides. Thus, in the presence of formaldehyde, DNA starts melting in regions enriched with A - T pairs, just as it docs in its absence. Therefore, formaldehyde acts to fix denaturation changes in the DNA structure caused by thermal fluctuations (85). This conclusion is not coiitradictcd by the facts that in the presencc of formaldehyde the melting temperature of DNA is lowered and its denaturation is dependent not only on tcmperaturc, but on CH,O concentration as well. The structure of DNA is dynamic rather than static, being determined by the cquilibrium between two processes: denaturation and renaturation (87, 65, 88). This equilibrium dcpcnds on temperature, with higher temperatures shifting it toward denaturation. I n the presence of formalde-

28

M. YA. FELDMAN

hyde, denaturation becomes irreversible and, therefore, the strand separation takes place a t a lower temperature, This seems to account for the mechanism of the T, lowering of DNA in the presence of formaldehyde (8, 63, 87). DNA denaturation is dependent on the concentration of CH,O. At 45”C, pH 7.2 and an ionic strength of about 0.1, 1% formaldehyde was practically unreactive with phage T4 DNA (no spectral changes observed during 48 hours), but reaction did take place in 15% CH,O solution (81). I n view of the above facts, the effect of high CH,O concentrations can be considered as due to an acceleration of formaldehyde reaction with opening sites produced by thermal fluctuations rather than to an active denaturation of DNA. Thus with respect to DNA, formaldehyde is not an “active,” but a “passive,” denaturing agent that opposes renaturation without causing denaturation. The reaction proceeds only with denatured DNA or with its denatured sites. A pn’ori, it is anticipated that the sites a t the ends of helices (where the chain is ruptured, a t the ends of molecules, or at sites adjacent to the already unwound DNA fragments in which the separation of chains is fixed by formaldehyde) should be less stable on heating than those in the middle of helices (89, 90, 65). Hence, formation of fixed centers of unwinding on moderate heating of DNA in CH,O solutions would induce the accelerated thermal denaturation of adjacent sites (“induction effect” (65)). Such a picture is in accordance with the results of detailed kinetic investigations (81, 89,90,65). However, the number of denatured nucleotide pairs of bacteriophage T 7 DNA containing free (with no CH,O added) amino groups is found to be very small (spectroscopy a t two wavelengths, 3.7% CH,O, pH 9.1, 58°C) ( 6 5 ) .It is doubtful that the induction effect plays an important part in the DNA interaction with formaldehyde. The cooperative character of the helix-coil transition is practically completely retained (the presence of formaldehyde in the solution does not affect the width of the DNA melting range). Electron microscopy (91) does not provide reliable evidence on accelerated melting of the ends of helices.

E. Effect of Formaldehyde on the Functional Activity of Nucleic Acids Some interesting results have been reported on the effects of CH,O modification of RNA’s on their functional properties. It was most surprising to find that under certain conditions modified RNA’s retain a considerable part of the original functional activity, sometimes even in greater degree.

REACTIONS OF FORMALDEHYDE WITH NUCLEIC ACIDS

29

The amino-acid accepting ability of transfer RNA is completely or almost completely suppressed by omission of Mg2’ from the reaction medium (92-94, 56). However, tRNA that has reacted with CH,O in the presencc of Mg2+ ions retains a considerable amount of its accepting activity. The activity of unfractionated tRNA with respect to four amino acids decreased by 30-50% on treatment with 3% CH,O (pH 7.6, 25°C) during the first 10 hours. Inactivation then stopped completely (92, 93) while the formaldehyde reaction continued. The primary reaction with amino groups is over in 100 hours when 15% of the NH,-groups has been converted to -NH-CH,OH-groups (9). Considerable retention of the acceptor activity in the presence of Mg2’ is also observed with a more intensive modification of tRNA leading to the formation of methylene bonds (1-4 per molecule) (56) whereas with no Mg2+the formation of 4 methylene bonds results in the total loss of acceptor activity (in this study hydroxymethyl groups were all or almost all removed by prolonged dialysis). Denaturation of tRNA in a formaldehyde solution not containing Mgz+(Section 111,D, 2) leads to a complete loss of the amino-acid accepting ability. However, an average of about 50% of the tRNA loses activity w e n with Mg2’ ions present in the solution (92, 93, 56). It is tempting to suggest (56) that intramolecular methylene bonds fix, in the presence of Mg“, not one, but at least two tRNA conformations, the “active” and “nonactive” ones, on the assumption that the two conformations were in equilibrium before formaldehyde fixation. This hypothesis is in accord with the results obtained when studying the acceptor activity of the yeast tRNAV”’ modified by CME-carbodiimide (95).3 Similar ideas are advanced with respect to chymotrypsin cross-linked with formaldehyde (96). The kinetics of CH,O inactivation of the “transfer” function of tRNA (as determined by the incorporation of [“C] phenylalanine into protein in the presence of poly(U) in a cell-free system) is rather similar t o that of the inhibition of acceptor activity. In this study (93), the formaldehyde treatment of the tRNA (an unfractionated preparation from Escherichia coli) was carried out over periods of time in which only methylol, but not methylene, derivatives would be expected to be formed. Some interesting results on the action of formaldehyde on mRNA were obtained for RNA bacteriophage f2 (97), whose messenger activity in the cell-free system is enhanced by mild treatment with 1 M CH,O a t 37°C for 11 minutes (followed by cooling and ethanol precipitation). Such a pretreatment of f2 RNA intensifies the synthesis of four proteins specific to this viral RNA and induces the synthesis of a t least three more CME-carbodiimide is N-cyclohexyl-N’-/3-(4-methylmorpholinium) ethylcarbodiimide p-toluenesulfonate.

30

M, YA. FELDMAN

polypeptides. All syntheses begin with N-formylmethionine, being thus initiated by AUG or GUG codons. By fixing the denaturation of the RNA a t limited sites, formaldehyde seems to make these codons more available for ribosomes thus leading either to increased synthesis of usual proteins or to the appearance of new polypeptides, depending on the localization of these codons. The CH,O modification has thus enabled us to obtain proof for the important functional role of the conformation of tRNA and mRNA. The same modification has also been used to prove the dominant role of the primary rather than secondary structure. Ribosomal RNA (from rabbit reticulocytes) was treated with 3% CHzO a t 63°C for 15 minutes (with subsequent cooling) to destroy completely the secondary structure (98). It was found that the resulting rRNA with disrupted secondary structure is hydrolyzed by ribonuclease T1 (in the same 3% CH,O solution) much more rapidly than is the normal rRNA. The intermediate products of partial ribonuclease (Tl) hydrolysis (10 minutes, 0°C) were discovered, however, to be similar as far as their molecular weight is concerned both for the native and formaldehyde-treated preparation of rRNA. Thus the disruption of the secondary structure does not affect the points of attack of the enzyme. Hence ribonuclease T1 recognizes definite nucleotide sequences. These experiments have excluded an alternative explanation. Formation of relatively stable intermediate products cannot result from the existence of “weak” sites (more available to the enzyme) in the RNA secondary and tertiary structures. This is because these products are also formed when the enzyme acts upon the fully unwound RNA molecules.

IV. Interaction of Formaldehyde with Nucleoproteins

This interaction involves formaldehyde reactions with nucleic acid and protein. It might well be that nucleoproteins undergo specific reactions with formaldehyde to form bridges between the nucleic and protein moieties (93-101). However, no strict chemical proof for this hypothesis has as yet been presented. Before proceeding to review the results available on the action of formaldehyde on nucleoproteins, a brief look a t the present state of the problem of formaldehyde interaction with proteins is pertinent.

A. Formation of Methylene Bridges in the Reaction of Protein with Formaldehyde The main events in the reaction of protein with formaldehyde (under conditions close to physiological ones) can be depicted by the following scheme:

31

REACTIONS OF FORMALDEHYDE WITH NUCLEIC ACIDS

RNH, RNH-CH2OH

+ CH2O RNH-CH?OH + R'H + RNH-CH2-R' + HzO

At high formaldehyde concentrations, dimethylol derivatives, RN(CH,OH),, are also formed. No labile mono- and dimethylol derivatives, formed in very fast reactions, have been isolated; their identification was made indirectly by physicochemical investigations of amino acids in formaldehydc solutions (102,103). The results of thcsc studies were generalized in the classical revicw by French and Edsall (104) and were subsequently substantiated kinetically (105).Spectral evidence was also provided for the addition of formaldehyde to imidazole protein groups (a-chymotrypsin) (106). Slow secondary reactions result in stable condensation products, methylene derivatives (RNH-CH,-R') . The first to present proof for condensation in reaction of protein with formaldehyde were Nitschmann and Hadorn (107),who showed that when gaseous formaldehyde binds to casein, water is eliminated. However, such a condensation could supposedly take place also on formation of compounds R=CH, and again on formation of methylene bridges . (R-CHZ-R') The first alternative was disproved. Various physical approaches showed that the action of formaldehyde on proteins, under conditions close to physiological ones, gives intermolecular cross-links causing increased molecular weight of proteins and/or intramolecular cross-links hindering denaturation of protein and favoring its renaturation (108112). It was also found that no cross-links are formed when formaldehyde acts on proteins with acetylated amino groups (113,11.6, 108). Methylene cross-links are most likely formed between amino groups and those of amide, guanidyl, phenol, imidazole or indole (114, 115). This conclusion was based on the study of a number of model systems such as alanine CH,O acetamide, proline + CH,O acetamide, threonine + CH,O + $,Cdimethylphenol, threonine CH,O + a-N-acetyl-L-histidine, polyglutamine C H 2 0 alanine, methylene tyrosine polymer CH,O methyl guanidine, etc. The first three reactions yield crystalline products (114, 116) and other products not isolated but. identified by analytical tests, in particular, by color reactions. I n the first stage, formaldehyde is added to the amino group to form the aminomethylol derivative (114) whose secondary reaction with the primary amide or another functional group givcs rise to compounds of the type

+

+

+

+

RNH-CH,-NHCOR',

+

+

RNH-CI&-NHC<

+

+

/m NHR'

As distinct from primary amides, secondary amides do not undergo

32

M. YA. FELDMAN

condensation with formaldehyde and amines under similar conditions (weak acid, room temperature) (114). The claim made a priOri by Nitschmann (107,113)about the formation of methylene bridges between c-amino groups of lysine residues and peptide bonds in adjacent protein chains was actually rejected in model investigations by Fraenkel-Conrat and Olcott. Special investigations were also made of the possible formation of methylene bridges between amide and guanidyl groups as well as between phenolic and amide or phenolic and guanidyl groups (114, 116). These alternatives could not be confirmed. Attempts to obtain methylene bridges between primary and secondary amide groups and between amides and aromatic rings failed even with acid used as catalyst (117). There appears to exist no convincing evidence for the formation of methylene cross-links between the amino groups of protein (114, 108). Direct proof was recently presented for methylene bridge formation by the action of formaldehyde on proteins (118, 119). Formaldehyde reacts with a-N-acetyllysine and a-N-acetyltyrosine to give, a t 37"C, a condensation product that, after deacetylation ( 1 M HC1, 37"C, 48 hours), leads to compound VII ( Lys-CHn-Tyr).

In the same studies, this coinpound was isolated from hydrolyzates of proteins treated with formaldehyde, that is, from serum albumin and toxoids of tetanus and diphtheria. Many authors have determined the amount of formaldehyde bound to proteins (120,121). Of greatest interest appear to be estimations obtained with labeled [ 14C]formaldehyde (122).Under conditions used to prepare toxoids (0.033 M CIZO, or 2200 moles of formaldehyde per mole of diphtheria toxin, a t 37°C) the protein reactions are complete in 15 days. The formaldehyde bound is distributed as follows: part is bound reversibly and can be removed by dialysis; 1 mole of toxin (MW 64,500) binds irreversibly 62 moles of formaldehyde, 14 of which are not liberated even by acid hydrolysis. Lys-CH2-Tyr, amounting to 4 moles per mole of anatoxin, is also stable to acid. Sedimentation constants of the diphtheria and

REACTIONS OF FORMALDEHYDE WITH NUCLEIC ACIDS

33

tetanus toxoids show little or no difference from those of the starting toxins (about 4.2 S for all cases studied) (123-125). A number of formaldehyde reactions with amino acids and proteins that lie outside the scope of this review have also been reported. Among these are reactions characteristic of free amino acids rather than of proteins, and protein reactions proceeding under conditions that are very far from physiological ones (in the presence of acid or alkali, or heating). Thesc reactions arc reviewed in special works on chemistry of amino acids, proteins and formaldehyde (10.4, 126-128, 1.4).

B. Effect of Formaldehyde on Nucleoproteins 5-Hydroxymethyluracil, obtained from the addition of formaldehyde to uracil (see Section 11, A, l ) , undergoes condensation with some amino acids (26) yielding products in which the residues of uracil and of amino acid are linked by methylenc group. The amino acid residue is connected to the mcthylene group through a sulfur atom (cysteine, homocysteine) or through an amino group (glycine). The reaction proceeds in the presence of acid or alkali and a t elevated temperature, that is, under conditions that are very far from those used with nucleoproteins. More adequate low molecular models do not appear in the literature. The results available on formaldehyde action on nucleoprotein particles such as ribosomes indicate that nucleoprotein fixation is the main result of this effect.

1. RIBONUCLEOPROTEINS There is a resemblance between the changes in ultraviolet spectra of ribosomes and in those of free RNA on formaldehyde treatment (129, 130). These spectral changes serve as proof of formaldehyde addition to the amino groups of rRNA bases in ribosomes. Just as with free rRNA, of ribosomes decreases in the presence of formaldehyde. The dethe T,,, crease is the greater, the stronger the formaldehyde concentration (130). The thermal denaturation of double helices of rRNA, both free and bound in the ribosome, is irreversible in the presence of formaldehyde (the starting double-helical structures are not reestablished on cooling) (130). Such a denaturation of RNA in the ribosome does not, surprisingly, change the hydrodynamic volume of the ribosome particle. Viscosity (130) and sedimentation coefficients of ribosomes (1.29) remain unaffected. It is to be noted that, in these viscosity and sedimentation studies, formaldehyde was not removed from the solutions (lSO),or the removal was effected only partially (129). Under such conditions, the free RNA has an increased effective hydrodynamic volume (Section 111, D, 2). Conversely, thermal denaturation of ribosomes in solution, with no

34

M. YA. FELDMAN

formaldehyde added, causes changes in ribosome conformations that are not fully reversible, judged from the same viscosimetric and sedimentation approaches (131). Ribosome fixation by formaldehyde is apparently due to methylene bonds formed essentially in the protein part of the nucleoprotein (129, 130). Neither high ionic strength (132) nor the presence of dodecyl sulfate (129) or urea (101) induces protein separation from RNA when ribosomes are given a preliminary treatment with formaldehyde. For ribosomes to be fixed, a very mild formaldehyde treatment suffices. Thus, for example, treatment of ribosomes with 0,05% CH,O a t 20°C and p H 7.5 for 75-120 minutes prevents their dissociation to proteins and RNA under the action of 1% dodecyl sulfate. The same treatment a t 0" is insufficient (129). I n other cases short incubation with 24% formaldehyde gives, a t a low temperature (0"4"C,1 hour, p H 6),the necessary fixation and proves useful in electron microscope studies (133, 134) and on ribosome centrifugation in the CsCl density gradient (132). The explanation of the fixing effect of formaldehyde under such mild conditions meets with considerable difficulties. Short treatment periods, low temperatures and neutral rather than weak acidic media do not favor the formation of methylene cross-links, as is known from the results of formaldehyde reactions with proteins and nucleic acids. It may be supposed that stabilization of nucleoproteins necessitates very few crosslinks. It is also likely that the nucleoprotein particle, having a rather rigid structure (e.g., the ribosome), involves functional groups held in a position sterically favorable to their linkage by means of methylene crosslinks in the reaction with CH,O. This steric factor may cause a sharp acceleration of the reaction. 2. DEOXYRIBONUCLEOPROTEINS The first to draw the attention to the interaction of nucleohistone with formaldehyde were Romakov and Bozhko (135). Later, the properties of the product of this reaction were studied in detail (100). Nucleohistone (of chromatin from pea buds) was treated with formaldehyde under extremely mild conditions (24 hours, O'C, p H 7.8), and formaldehyde was then removed by dialysis (100).Under these conditions, 0.5-1% CHIO concentration is enough to fix the nucleoprotein complex. I n contrast to native nucleoprotein, such a complex does not liberate protein in a saline solution of high ionic strength and can be studied in the CsCl gradient. It was found that its buoyant density is 1.411 g/ml, the same as the value calculated for the DNA-protein ratio in nucleohistone. Nucleohistone treated with formaldehyde is more stable to heating than is the native material, and the T, of the treated nucleohistone is

REACTIONS OF FORMALDEHYDE WlTH NUCLEIC ACIDS

35

higher than the T , of the untreated complex. With ribosomes, an opposite relation was observed (see above). Such a divergence in T , might be due to the different experimental conditions used: the T , of ribosomes was determined in the presence of formaldehyde, that of nucleohistone was estimated after CH,O was removed by dialysis. Even after the fifth treatment with Pronase, not all protein is removed from nucleohistone treated with formaldehyde; 4% of the protein remains firmly bound to DNA. I t is suggested (100) that formaldehyde reacts with nucleohistone to form methylene bridges between nucleic acid and protein.

V. Use of Reactions of Nucleic Acids and Nucleoproteins with Formaldehyde

No reaction of nucleic acids seems to have been as useful in experiment and practice as that with formaldehyde. The main fields of its application are the following: (1) study of nucleic acids and nucleoproteins, their structure and biochemical functions; (2) fixation of various biological structures, from ribosomes and bacteriophages to microscopic tissue sections; (3) inactivation of viruses for producing vaccines; and (4) chemical mutagenesis. The first three applications are essentially based on the fixing action of formaldehyde. This effect involves two mechanisms and, respectively, leads to two main results. Hydroxymethyl groups formed in the primary reaction of nucleic acid with formaldehyde fix denaturation by hindering renaturation. Methylene cross-links, formed in the secondary reaction under conditions not causing denaturation, fix the structure of the native RNA or of nucleoprotein. The type of fixation used depends on the particular aim pursued in the work. Of all applications of the reaction not concerned with the fixing action of formaldehyde, the most important is chemical mutagenesis. Aldehyde fixation in morphological studies does not receive special consideration in this review. The fixation of nucleoprotein particles and complexes of RNA’s has been discussed in Sections IV, B and 111, D, 2. The biochemical basis for the application of aldehydes (formaldehyde and acrolein) together with metachromatic dyes for histochemical investigations of RNA and DNA is dealt with in references (136-138).

A. Structural and Functional Studies of Nucleic Acids Formaldehyde is often used when it is desired to destroy double helices and to prevent their regeneration throughout the whole experiment.

36

M. YA. FELDMAN

Denaturation is usually attained by raising the temperature in the presence of formaldehyde followed by rapid cooling of the mixture, heating causing denaturation and formaldehyde preventing renaturation. Formaldehyde possesses a number of advantages over other denaturing agents: the necessary effect is produced by low concentrations of formaldehyde, nucleic acid can be investigated in an essentially aqueous medium, methylene glycol (hydrated formaldehyde) does not absorb ultraviolet light. Some important applications of “fixed” denaturation have already been considered above, namely, the use of formaldehyde reactions in functional studies of nucleic acids [Section 111, E; see also ( 1 3 9 ) ] , denaturation of polynucleotides in the presence of formaldehyde to study base stacking in poly(C), poly(A) and RNA (Section 111, D ) , location by means of electron microscopy of sites of DNA molecules enriched by A.T pairs [Section 111, D, 3; see also (91, 1.40,1 4 1 ) I . The primary formaldehyde reaction is of great value to achieve the separation of polynucleotide chains in order to elucidate their actual size. Thus, for example, heating the giant (>26 S) “DNA-like” RNA of yeasts in the presence of formaldehyde has showed that the preparation consisted actually of aggregates involving RNA molecules with molecular weight close to that of the 17 S component of rRNA (142). The same method has made it possible t o determine (81) that the polynucleotide DNA chain of phage T 4 is continuous and that its molecular weight is half that of the native DNA. This has provided one of the most convincing proofs that the DNA molecule consists of only two uninterrupted polynucleotide chains. Formaldehyde reactions also used for many other purposes, in particular when it is necessary to distinguish single- and double-stranded virus nucleic acids (60, 70, 143), to determine quantitatively the double-helical content of RNA (6, 32) and the number of secondary structure “defects” to eliminate specific features in the conformation of in DNA (89, a), some ribonucleic acids for a more exact estimation of their’ molecular weight (76, 77, 7 2 ) ,to separate the DNA chains in immunological experiments (82, 144), to study nucleic acids in situ in bacteriophages (146147) and in cells (136, 137).

B. Inactivation of Viruses by Formaldehyde in Vaccine Production

Formaldehyde action on virus nucleoproteins is of great practical value for preparation of viral vaccines for human subjects (14.8) and animals (149). Many investigations have dealt with virus “formol” in-

REACTIONS OF FORMALDEHYDE WITH NUCLEIC ACIDS

37

activation [see, e.g. ( 1 5 0 ) ] , but the chemical mechanism of this process has not been studied directly. The following four features of formaldehyde interaction with proteins, nucleic acids, and nucleoproteins can be considered as the most essential for the CH,O inactivation of viruses in vaccine preparation. 1. Formaldehyde acts both on the protein and nucleic virus component without causing any destruction of polypeptide or polynucleotide chains. 2. Modification of bases, formation of cross-links in nucleic acid and “hardening” of protein ensure that the virus is reliably rendered harmless. 3. Formaldehyde fixes the conformation of nucleoprotein. Recently evidence was produced concerning the special role of antigen conformation in inducing the synthesis of antibodies (151). Most antibodies induced by TMV are likely to be specific for the structure of the surface of the virus particle, for its conformation, rather than for the amino-acid sequence of virus subunits (152). It has also been shown (153) that the nucleoprotein and the free protein of the turnip yellow mosaic virus (crystallizable preparations isolated from the plant sap) are identically precipitated by antiserum to nucleoprotein but, when injected into rabbits, induce the formation of antibodies differently ; the antigenicity of the nucleoprotein is much higher than that of the free protein. Thus, in order for antigenicity to be stabilized it is desirable to fix the nucleoprotein as such, not its protein only. This condition is met by formaldehyde. 4. The small size of the formaldehyde molecule facilitates its passage through the protein shell of the virus. The importance of this fact can be exemplified as follows. Kethoxal (a-keto-P-ethoxybutyraldehyde) inactivates the isolated TMV-RNA much more readily than does formaldehyde. On the other hand, RNA in the virus is inactivated by kethoxal more slowly than by formaldehyde. It might be suggested that the relatively large size of the kethoxal molecule hinders its passage through the protein shell (154). All these peculiarities of formaldehyde action provide a post-facturn explanation of the choice of formaldehyde as an inactivator and fixative for vaccine preparation. The future will tell whether this delayed theoretical explanation will help to improve the preparation of vaccines and to prepare new vaccincs. It is to be noted that soiiie L‘anoinalics”of formaldehyde inactivation are also being explained. This is the ease for the reversible inactivation of TMV (155) and poliovirus (156) on short-term formaldehyde treatment (apparently due to the formation of only methylol derivatives) and the increased stability of the carcinogenic virus SV40 to the inactivating

38

M. T A . FELDMAN

action of formaldehyde [ thc virus contains a double-helical DNA (149)1. The question is now poscd whether all viruses retain their conformation in the formaldehyde reaction. This question, as well as many others cannot be answered a priori. If formaldehyde inactivation fails to give a satisfactory immunological effect, special physicochcmical studirs with purificd virus prcparations will clearly be nccdcd.

C. Effect of Formaldehyde on the Genetic Apparatus of the Cell The mutagenic action of formaldehyde was first described by Rapoport in 1946 ( 3 ) . Of mutagens acting on multicellular organisms, formaldchyde is one of the most interesting and well known (157, 4 ) . Formaldehyde causes mutation in viruses (158, 159), bacteria (1601, Neurospora (161) and Drosophila [on injection into adult flies (162) or introduction into culture medium for larvae (3, 163, 13,10,4) 1. The mechanism of mutagenesis induced by formaldehyde is best known for Drosophila larvae. Formaldehyde causes mutation in male larvae only (163, 4) and solely a t a definite stage of spermatogenesis (immediately preceding meiosis) (164). This shows formaldehyde to be a highly specific mutagen ( 4 ) . Its mutagenic effect has something to do with DNA rcplication, bccause after DNA synthesis is over formaldehyde loses its activity and cxcrts no mutagenic effect on late spermatocytes (165). Formaldehyde can cause mutation (recessive lethal) if the culture medium contains AMP (13). Adenylic acid can be replaced by adenosine or RNA, but not by adenine, cytosine or inosinic acid. Deoxyadenylic acid is less effective than adenylic acid (13, 166, 167, 10, 157). Mutations appear to be induced in Drosophila larvae not by formaldehyde as such, but by its reaction product with adenylic acid. It is suggested (13, 10) that mutation is causcd by the methylene or hydroxymethyl dcrivativcs cntcring one of the DNA (or RNA ( 1 5 7 ) ) chains during synthesis. It should bc noted that participation of a very labile hydroxymethyl product in formaldchydc-induced mutagenesis appears to be unlikely. In any case, the mutagenic action of formaldehyde (on Drosophila larvae) is not a dircct and primary one, but a much more complex phenomenon occurring probably during thc replication of the DNA. I n Drosophila larvac, formaldchydc induccs crossing-over either in the presencc or abscncc of AMP. The second alternative is of considerable interest in principlc as the agents inducing crossing-over act also as mutagens (157, 168). I n this case, formaldehyde is of great value because it allows crossing-over to be induced under conditions (absence of AMP) preventing mutagenesis (157).

REACTIONS O F FORMALDEHYDE WITH NUCLEIC ACIDS

39

Formaldehyde and number of its addition products, such as methylol derivatives of succinimide, glutarimide, piperidine, pyrrolidine, morpholine, dikctopipcrazine, 2-mercaptoiinidazolc, etc., arrest the growth of the mouse ascitic carcinoma in vivo ( 2 ) and lead to considerable changes in tumor metabolism. Expcrimcnts with mcthylene bis-guanylic acid give some indications as to the mechanism of formaldehyde effect (43). This compound, produced from the reaction of GMP and CH,O (see Section 11, B, 2), suppresses drastically the mitotic activity of human amniotic cells in continuous culture (strain FL). I n the presence of CH,(Gp),, DNA Synthesis is a t first enhanced for 1.5 hours (presumably owing to increased nucleotide pool in the cell) and then hindered. I n contrast, the synthesis of cell RNA is little affected. Reproduction of viruses containing RNA undergoes quite independent changes. With CH, (Gp), in the culture medium, reproduction of poliovirus in FL cells is first suppressed (for 8 hours) and then restored. A hypothesis has been advanced to account for these results ( 4 3 ) . After suitable enzymatic conversions in the ribosephosphate part, the methylene dinucleotide enters the newly synthesized DNA chain with one of its nucleotide residues in place of the appropriate normal nucleotide. During the next replication the free methylene dinucleotide residue can enter the complementary chain. Thus two DNA chains are bonded by a methylene cross-link -Piir1

-Piir-

and mitosis is inhibited. It is supposed that similar cross-links between the complementary chains of the replicative virus RNA inhibit the reproduction of the virus. The methylene bridge links two purine residues, i.e., a purine in one chain is opposed by a purine in the complementary chain, but not by pyrimidine as is usually the case. If the methylene cross-link is removed by hydrolysis or enzymatic reparation, chains will be separated. Purine substitution for pyrimidine thus causes mutation. This hypothesis would be substantially confirmed if methylene dinucleotide could be isolated from DNA. There are, however, as yet no suitable methods available to obtain such compounds from DNA. The highly specific mutagenesis induced by formaldehyde in Drosophila larvae cannot be even hypothetically accounted for (165, 4 ) . The results available (165,4 ) on localization of mutations (X chromosome and the second, but not the Y, chromosome) fail to explain why the formaldehyde reaction product with AMP affects the early spermatocytes

40

M. YA. FELDMAN

and why spermatogonia and all stages of oogenesis are not sensitive to the mutagenic activity of this reaction product. With [ ''C] formaldehyde in the culture medium, the label is discovered autoradiographically in the germ cells a t all stages (169).However, it is extremely doubtful whether this label always belongs to the product of the reaction between AMP and l'CH,O (tentatively CH, (Ap) ?). Just what cells this product permeates and how long it stays in different cells still remains unknown. It is possible that the carly spermatocyte has the most favorable conditions for accumulating methylene dinucleotide up to the concentration needed for its inclusion into the polynucleotide. The cytostatic action and cross-over induction do not necessarily require the intermediate formation of methylene dinucleotide. These effects may also be due to another mechanism such as the direct formaldehydc interaction with cell DNA.

VI. Related Reactions and Their Effects (as Compared to Formaldehyde Reactions) A detailed review of these reactions is not within the scope of this essay, particularly as they have already been dealt with in many reviews (38,170-17s). The purpose of this section is to show the place of formaldehyde as a modifying agent among other aldehydes and bifunctional compounds whose action on nucleic acids has been studied rather thoroughly. A. Miscellaneous Aldehydes A number of aldehydes other than formaldehyde are useful for modification of nucleic acids. These are almost all dicarbonyl compounds. Their interaction with nucleic acid or its component is likely to involve two consecutive reactions with two carbonyl groups. Yet, only the stable end products of these reactions have been well studied; these are discussed below. Particularly well known are the reactions with 1,2-dicarbonyl compounds (154, 174, 176), namely, with glyoxal (OHC-CHO), kethoxal (CH,-CH (OC2H,)-CO-CHO) and pyruvaldehyde (CH,-CO-CHO) . These modifying agents differ from formaldehyde in the following respects. 1. They are more specific than formaldehyde, and stable condensation products are found only in their reactions with guanine, guanosine and deoxyguanosine. At the nucleic acid level, this specificity for guanine rcsiducs of RNA has been unequivocally shown only for kethoxal (176).

REACTIONS O F FORMALDEHYDE WITH NUCLEIC ACIDS

41

(Vm) 2. l12-l)icarbonyl compounds do not form bridges between two niolccules (or residues) of the base. Two carbonyl groups react with two nitrogen atoms (N-1 and N-2) of guanine, forming a new cycle. I n formula VIII, R = H if guanine interacts with glyoxal and R = CH, or CH,CH (OC,H,) if it reacts with pyruvaldehyde or kethoxal, respectively (174,175).New five-membered rings are also formed in the reactions of guanosine and guanine with ninhydrin (174). 3. With glyoxal (177)and kethoxal (178,l79),the reactions proceed rather rapidly. Thus nonfractionated tRNA reacts with kethoxal a t 37°C and pH 5.0 to give a quantitative modification of guanine residues in about 15 hours with no MgZfand 30 hours in the presence of Mg2+(178). 4. I n principle, l12-dicarbonyl compounds are presumed to affect the secondary structure, just as does formaldehyde, by reacting only with such bases as are localized in single-stranded or unwound double-stranded RNA sites and preventing renaturation in the modified segments (178, 179). However, a very short-lived and limited unwinding, taking place in the fluctuating RNA structure, suffices to cause condensation with 1,2dicarbonyl compounds, which react readily to form a stable product. It appears that even stabilization by M g + ions does not provide for the retention of the secondary structure in the presence of kethoxal (178, 179).Only a t the start of the reaction (30 minutes, pH 7, 37"C, Mg2+) is it possible to locate those tRNAPh' sites that have reacted with [3H]kethoxal without destroying the tRNA conformation (179). 5. l12-Dicarbonyl compounds and formaldehyde are used in different fields. The former are never used for fixation, their mutagenic activity is unknown but kethoxal has been successfully used for labeling and identification of exposed guanine tRNA residues (179,180).On the other hand, the carcinostatic effect and the inactivation of DNA replication are observed under the action of both formaldehyde (or methylene dinucleotides) (2, 43) and l12-dicarbonyl compounds (181,182). Covalent cross-links, hindering the separation of complementary DNA chains on denaturation, are formed on DNA treatment with oxidized spermine [ dialdehyde, OHC (CH,) ,NH (CH,) ,NH (CH,) ,CHO] but are absent when DNA is treated with oxidized spermidinc [monoaldehyde, OHC(CH,),NH (CH,),NH,] (183,184). It follows that both carbonyl

42

M. YA. FELDMAN

groups of oxidized spermine are needed for cross-links. The reaction is nonspecific, for the carbonyl groups of oxidized spermine react with all bases containing amino groups and, though to a considerably smaller extent, with thymine and uracil (186, 184). Another dialdehyde, glutaric aldehyde (OHC (CH2);
B. Difunctional Alkylating Agents4 Formaldehyde is the simplest difunctional reagent. It is of interest to compare the modification of nucleic acids by formaldehyde with the effects produced by other conventional difuctional compounds, such as , aliphatic nitrogen mustard butadiene dioxide (CH,-CH-CH-CH,)

\ /

\/

0 0 HN2 [bis (2-chloroethyl)methylamine, CH,N (CH,CH,Cl) ,] and mustard gas [bis (2-chloroethyl)sulfide, S (CH,CH,Cl) 2]. Reacting with the N-7 of guanine residues in nucleic acids, these compounds give rise to products of monofunctional as well as bifunctional alkylation. Diguanyl derivatives of butadienc dioxide and HN2 are the most chemically studied compounds. The physicochemical and biochemical consequences of bifunctional alkylation of nucleic acids have been most thoroughly investigated in experiments with mustards (188-192). Difunctional alkylation proceeds with intermediate formation of the monoalkyl derivative (170,38, 193). The secondary structure does not Only those rcfercncrs nre given in this srihscction thnt, are not found in the reviews (170, 38).

REACTIONS OF FORMALDEHYDE WITH NUCLEIC ACIDS

43

prevent the action of alkylating agents. Reacting with the native doublestranded DNA, the difunctional agent forms both interchain and intrachain cross-links (192,194). Modification of nucleic acids by difunctional alkylating agents offers the following advantages over modification by formaldehyde. First, reactions with alkylating agents are very fast, less than 1 hour's treatment with mustard gas (37"C, pH 7) giving the necessary effect in many cases. Second, cross-links are specifically formed only between guanine residues [although the poly(A) reaction with mustard gas gives, together with monofunctional alkylation products, an additional product of difunctional alkylation, presumably bis (adenin-l-ylethyl) sulfide (198)1. Third, it is often possible to compare the difunctional alkylating agent and the corresponding monofunctional agent, for instance, mustard gas, bis (2-chloroethyl) sulfide, and hemisulfur mustard, 2-chloroethyl 2-hydroxyethyl sulfide. Compared to formaldehyde, difunctional alkylating compounds display a number of disadvantages, two of which are of particular importance. First, together with diguaninyl derivatives, a great number of stable by-products of monofunctional alkylation are formed. Thus, 20 minutes' treatment of salmon sperm DNA with mustard gas (37", pH 7.2) gives the following alkylation products (190) : bis (P-guanin-7-ylethyl) sulfide, 2270 of total alkylated bases ; 7- (2-hydroxyethylthioethyl) guanine, 59% ; 1- (2-hydroxyethylthioethyl) adenine, 4% ; 3- (2-hydroxyethylthioethyl) adenine, 15%. Second, alkylation reactions are complicated by hydrolytic release of the alkylated purines. Subsequent chain fission of nucleic acid should also not be ignored, however negligible. Comparison of the biological effects of mono- and difunctional alkylating agents in parallel experiments shows what effects are mainly or partly due to the formation of cross-links. The formation of cross-links in DNA results in the inactivation of bacteriophages containing doublestranded DNA (198, 195, 196),exerts a pronounced cytostatic (carcinostatic) effect, and inhibits DNA replication. The effects of difunctional alkylating agents in vivo are observed in both microorganisms (191, 197) and mammalian cells (198).The compounds exert only a weak action on the synthesis of RNA and protein, just as do corresponding monofunctional agents. The part played by each type of cross-linking, intra- and interstrand, is as yet unknown. There is not yet enough evidence to clarify the role of cross-links in mutagenesis induced by alkylating agents. The mechanism of reparation of defects caused by alkylating agents in vivo also requires further study (191, 198). Although many important problems await elucidation, the information available as to the action of difunctional alkylating agents on nucleic

44

M. YA. FELDMAN

acids in vitro and in vivo is very useful for a general biological interpretation of the nucleic acid modifications caused by difunctional agents.

VII. Conclusion Forrnaldehydc is not only one of thc most useful, but also one of the most extensively studied, agents modifying nucleic acids. It is with formaldehydc that the relationship between the modifying agent and the secondary polynucleotide structure appears to have been most thoroughly invcstigatcd. Thc main field of formaldehyde application is fixation. Formaldehyde fixes two opposite structural states, denatured and native. The denaturation of nucleic acid is fixed by the formation of primary products, methylol derivatives. Thc native macrostructure of the polymer is fixed by methylenc cross-links formed in the secondary reaction. For each fixation, suitable conditions must be carefully chosen. Otherwise, mixed effects arc produccd, which lead to incorrect intcrpretations of results. New and important applications of formaldehyde fixation are to be expected, such as fixation of different tRNA conformations and of oncogenic viruses for vaccine preparation (199). The possible biological effects of a new group of analogs of nueleic componcnts, methylene dinucleotides, are also of great interest.

ACKNOWLEDGMENT The author wishes to thank Dr. E. I. Budowsky for fruitful discussion of some sections of the review.

REFERENCES 1. J. E. Salk and J. 13. Gori, Ann. N . Y . Acnd. Sci. 83, 609 (1960). 2. G. Weitrel, F. Schneider, A.-M. Fretzdorff, K. Seynsche and H. Finger, HoppeSeyler’s Z . Physiol. Chem. 334, 1 (1963). 3. I. A. Rapoport, Dokl.Akad. Nnuk SSSR 54, 65 (1946). 4 . W. Ratnayake, Genet. Res. 12, 65 (1968). 6. H. Fraenkel-Conrat, BBA 15, 307 (1954). G. R. Hnselkorn and P. Doty, JBC 236, 2738 (1961). 7. G. Zuibay and R. Marciello, BBRC 11, 79 (1963). 8. L. Grossman, S. S. Levine and W. S. Allison, JMB 3, 47 (1961). 9. J. T. Penniston and P. Doty, Biopolymers 1, 145 (1963). 10. T. Alderson, Mutalion Res. 1, 77 (1964). 11. M. Staehelin, BBA 29, 410 (1958). 19. M. Ya. Feldmnn, Biokhimiyn 25, 563 (1960). 13. T.Alderson, Nature (London) 187, 485 (1960). 14. J. F. Walker, “Formaldehyde,” 3d ed. Reinhold, New York, 1964. 16. M. Ya. Feldmnn, Biokhimiya 27, 378 (1962). 16. M. Yn. Feldmnn, BBA 149, 20 (1967).

REACTIONS OF FORMALDEHYDE WITH NUCLEIC ACIDS

45

17. M. Ya. Feldman, Bwkhimiyn 23, 917 (1958). 18. E. J. Eyring and J. Ofengand, Bchem 6,2500 (1967). 19. N. N. Aylward, J. Chem. SOC.B 11. 627 (1966). 20. S. Lewin, Expeiientiu 20, 666 (1964). 21. S. Lewin, J . Chem. SOC. p. 1462 (1962). 22. S. Lewin, J . Chem. Soc. p. 792 (1964). 23. S. Lcwin and M. A. Barncs, J. Chem. Soc. B 13. 478 (1966). 24. D. E. Hoard, BBA 40, 62 (1960). 26. A. M. Michelson and M. Grunhcrg-Manago, BBA 91, 92 (1964). 26. R. E. Cline, R. Fink and K. Fink. J-4CS 81, 2521 (1959). 27. A. H. Alegria, BBA 149, 317 (1967). 2s. K. H. Scheit, I’etrrrhetlroii Lett. 11. 1031 (1965). 29. C. L. Stevens and A. Rosenfeld, Bchem 5, 2714 (1966). 30. M. Ya. Feldman, Biokhimiyn 29, 720 (1964). 31. D. L. Woodhouse, Nature (Loiitlon) 192, 336 (1961). 3.2. H. Boedtker, B c h m 6, 2718 (1967). 33. German Patent 106493 (1897). 34. B. Skowro6skaSerafinowa, Roczn. Chem. 29, 932 (1955). 36. W. Pfleiderer, F. Sigi and L. Grozinger, Chem. Ber. 99, 3530 (1966). 3G. G. W. Kenner and A. Todd, in “Heterocyclic Compounds” (R. C. Elderfield, ed.), Vol. 6, p. 234. Wiley, New York, 1957. 37. R. K. Robins, in “Heterocyclic Compounds” (R. C. Elderfield, ed.), Vol. 8, p. 162. Wiley, New York, 1967. 3s. N. K. Kochetkov, E. I. Budowsky, E. D. Sverdlov, N. A. Simukova, M. F. Turchinsky and V. N. Shibaev, “Organichcskayn khimiya nucleinovikh kislot.” Khimiya, Moskva, 1970. 89. German Patent 667542 (1938). 40. German Patent 669188 (1938). 41. US. Patent 2211710 (1940). 4.2. M. Ya. Feldmnn, unpublished data. 43. M. Ya. Fcldmnn, E. S. Zalmanson and L. N. Mikhailova, Mol. Biol. 5, 847 (1971). 44. C. D. Jardetzky and 0. Jardetzky, JACS 82, 222 (1960). 46. H. A. Szimansky and R. E. Yelin, “ N M R Band Handbook.” IFI/Plenum, New York, 1968. 4G. W. E. Cohn, in “The Nucleic Acids” (E. Chargaff and J. N. Davidson, cds.), Vol. 1, p. 211. Academic Press, New York, 1955. 47. M. Ya. Feldman, unpublished results. 4s. M. Yn. Fcldman and M. R. Shubinn. Dokl. Akud. Nnuk SSSR 174, 236 (1967). 49. R. F. Beers, Jr. and R. F. Steincr, Nature (London) 179, 1076 (1957). 60. G. D. Fasman, C. Lindblow and L. Grossman, Bchem 3, 1015 (1964). 61. German Patent 139907 (1901). 62. H. Fraenkel-Conrat, JACS 78, 882 (1956). 63. A. Gierer and G. Schramm, Nature (London) 177, 702 (1956). 64. M. YR. Feldman, Biokhimiyn 30, 203 (1966). 66. M. Ya. Feldman, Biokhimiyn 25, 937 (1960). 6G. V. D. Axelrod, M. Ya. Feldman, I. I. Chuguev and A. A. Baycv, BBA 186, 33 (1969). 67. B. D. Hall and P. Doty, JMB 1, 111 (1959). 6s. S. Zamenhof. H. E. Alexnnder and G. Leidy, J. Exp. Med. 98, 373 (1953).

46 69. C. GO. R. 61. C.

M. YA. FELDMAN

J. Collins and W. R. Guild, BBA 157, 107 (1968).

L.Sinsheimer, J M B 1, 43 (1959). A. Thomas, Jr. and K. I. Berns, J M B 4, 309 (1962).

62. P. Sheldrick and W. Szybalski, J M B 29, 217 (1967). 63. D.Freifelder and P. F. Davison, Biophys. J. 3, 49 (1963). G4. A. J. van tler Eh, I,. M '. van Kesteren nnd E. F. J. van Rruggm. BBA 182, 530 (1969). 06. H. Utiyama and P. Doty, Bchem 10, 1254 (1971). 66. A. Rosenfeld, C. L. Stevens and M. P. Printz, Bchem 9, 4971 (1970). 67. G.Zubay and R. Marciello. BBRC 11, 79 (1963). 6s. G. D. Fasman, C. Lindblow and E. Seaman, J M B 12, 630 (1965). 69. D. B. Millar and M. MacKrnzir, Bcliem 6, 2520 (1967). 70. R. A. Cox, K. Kanagalingam iind E. S. Sutherland, BJ 120, 549 (1970). T i . M. Edelman, I. M. Verma, D. Saya and U. Z. Littauer, BBRC 42, 208 (1971). 72. H.Boedtker, BBA 240, 448 (1971). 73. V. S. Mayo and S. R. de Kloet, BBA 247, 74 (1971). 74. P. L. Whitney and C. Tanford, JBC 237, 1735 (1962). 76. W. M.Stanley, Jr. and R. M. Bock, Bchem 4, 1302 (1965). 76. M.L.Fenwick, BJ 107, 851 (1968). 77. H.Boedtker, J M B 35, 61 (1968). 78. H. Slegers and W. Fiers, FEBS Lett. 7, 55 (1970). 79. H. Slegers and W. Fiers, Biopolymers 9, 1373 (1970). 80. N.K. Sarkar and A. L. Dounce, BBA 49, 160 (1961). Si. K.I. Berns and C. A. Thomas, Jr., J M B 3, 289 (1961). S9. D. Stollar and L. Grossman, J M B 4, 31 (1962). 83. M. Beer and C. A. Thomas, Jr., J M B 3, 699 (1961). 84. R. B. Inman, JMB 18, 464 (1%). S5. V. V. Simonov, N. I. Ryabchenko and A. M. Povcrenny, Mol. Biol. 1, 297 (1967). 86. E. A. C. Follet and L. V. Crawford, J M B 34, 565 (1968). 87. P. H. von Hippel and K.-Y. Wong, J M B 61, 587 (1971). 88. R.J. Cohen and D. M. Crothers, JMB 61, 525 (1971). 89. E. N. Trifonov, N. N. Shafranovskaya, M. D. Frank-Kamenetskii and Yu. S. Lazurkin, Mol. B i d . 2, 887 (1968). 90. Yu. S. Lazurkin, M. D. Frank-Knmenetskii and E. N. Trifonov, Biopolymers 9, 1253 (1970). 91. R. B. Inman and M. Sch~os,J M B 49, 93 (1970). 92. J. T.Penniston and P. Doty, Biopolymers 1, 209 (1963). 93. J. T. Penniston. M. Steward and M. D. Tucker, BBRC 15, 358 (1964). 94. M. K. Iiuklinnora, I,. I,. Kisrlcv and I,. Tu. Frolova, Biokliimiya 28, 1053 (1963). 96. A. S. Girshovich, M. A. Grachev, N. I. Komnrova and N. I. Mensorova, FEBS Lett. 14, 195 (1971). DG. L. J. Saidel, S. Leitzes and W. H. Elfring, Jr., BBRC 15, 409 (1964). 97. H. F. Lodish, J M B 50, 689 (1970). 98. J. C.Pinder and W. B. Gmtzer, Bchem 9, 4519 (1970). 99. M.Ya. Feldman, Vopr. &fed. Khim. 9, 232 (1963). 100. D.Brutlag, C. Schlehuber and J. Bonner, Bchem 8, 3214 (1969). 101. M.Roberts and I. 0. Walker, BBA 199, 184 (1970). iO2. M.Levy and D. Silberman, JBC 118, 723 (1937).

REACTIONS O F FORMALDEHYDE WITH NUCLEIC ACIDS

47

103. E. H. Frieden, M. S. Dunn and C. D. Coryell, J. Phys. Chem. 47, 10 (1943). 104. D. French and J. T. Edsall, Advan. Protein Chem. 2, 277 (1945). 105. B. P. Zhantalai and Ya. I. Tur’yan, Kinet. Kntal. 3, 325 (1962). 106. Ch. J. Martin and M. A. Marini, JBC 242, 5736 (1967). 107. H. Nitschmann and H. Hndorn, Helv. Chim. Acta 27, 299 (1944). 10s. H. Fraenkel-Conrat and D. Mcrham, JBC 177, 477 (1949). 109. Ii. I. Stracliitskii, iir “Voprosi medicinkoi lihimii” (8. R . Mardnslicv. cd.), Vol. 1, p. 292. Mcdgiz, Moskva, 1949. 110. M. P. Drake and A. Veis, Bchem 3, 135 (1964). 111. D. W. Bannister, BJ 114, 509 (1969). 112. P. V. Hauschka and W. F. Harrington, Bchem 9, 3734 (1970). 113. H. Nitschmnnn and H. Laucncr, Helv. Chim. A d a 29, 184 (1946). 114. H. Fraenkel-Conrat and H. Olcott, JACS 70, 2673 (1948). 115. H. Fraenkel-Conrat and H. Olcott. JBC 174, 827 (1948). llG. J. Blass, BuI1. SOC.Chim. p. 3120 (1966). 117. R. D. Haworth, D . Peacock, W. Smith and R. MacGillivry, J. Chem. SOC. p. 2972 (1952). 11s. J. Blass, B. Bizzini and M. Raynaud, C . R . Acad. Sci. 261, 1448 (1965). 119. J. Blass, B. Bizzini and M. Raynaud, Bull. SOC.Chim. p. 3957 (1967). 120. K. H. Gustavson, Advan. Protein Chem. 5, 353 (1949). 121. N. I. Egorkin, M. A. Mamedov and 0. D. Rokhwarger, “Formaldegidnoye dubleniye.” Gizlegprom, Moskva, 1957. 12.2. J. Blass, B. Bizzini and M. Raynaud, Ann. Znst. Pasteur IlG, 501 (1969). 123. M. L. Petterman and A. M. Pappenheimer, Jr., J. Phys. Chem. 45, 1 (1941). 124. J. F. Largier and F. J. Joubcrt, BBA 20, 407 (1956). 125. J. Blass, Biol. Me‘d. 53, 202 (1964). 126. H. Fraenkel-Conrat, in “Amino Acids and Proteins” (D. M. Greenberg, ed.), p. 532. Thomas, Springfield, Illinois, 1951. 127. P. Desnuclle, in “The Proteins. Chemistry, Biological Activity and Methods” (H. Neurath and K. Bailey, eds.), Vol. 1, Part A, p. 87. Academic Press, New York, 1953. 1%. F. W .Putnam, in “Thc Proteins. Chemistry, Biological Activity and Methods” (H. Neurath and K. Bailey, cds.), Vol. 1, Part B, p. 893. Academic Press, New York, 1953. 129. R. A. Cox, BJ 114, 743 (1969). 130. M.Tal, BBA 224, 470 (1970). 131. M. Tal, Bchem 8, 424 (1969). 132. A. S. Spirin, N. V. Belitsina and M. I. Lerman, J M B 14, 611 (1965). 133. H . E. Huxley and G. Zubay, JMB 2, 10 (1960). 134. S. T . Bayley, JMB 8, 231 (1964). 135. Yu. A. Rornakov and G. V. Bozhko, Biokhimiya 32, 471 (1967). 1SG. M. E. Lamm, L. Childers and M. K. Wolf, J. Cell Bio2. 27, 313 (1965). 137. N. Feder and M. K. Wolf, J . Cell Biol. 27, 327 (1965). 13S. A. V. Furano, D. F. Bradley and L. G. Childers, Bchem 5, 3044 (1966). 139. Yu. N. Kosaganov, M. I. Zarudnaya, Yu. S. Lazurkin, M. D. Frank-Kamenetskii, R. Sh. Beabenlashvili and L. P. Savochkina, Nature (London) New Biol. 231, 212 (1971).

140. W. Doerfler and A. K. Kleinschrnidt, JMB 50, 579 (1970). 141. A. S. Bayev, Yu. L. Lyubchenko, Yu. S. Lazurkin, E. N. Trifonov and M. D. Frank-Kamenetskii, Mo2. Biol. 6, 760 (1972).

M. YA. FELDMAN

48

142. S. R. de H o e t , V. 8. Mayo and B. A. G . Andretin. BBRC 40, 454 (1970). 143. J. M. Bishop and G. Koch, JBC 242, 1736 (1967). 1 4 . A. M. Poverenny, T. L. Aleinikova and A. S. Saenko, Biokhimiya 31, 1150 (1966). 146. R. L. Sinsheimer, JMB 1, 37 (1959). 146. W. Sauerbier, Nature (Loiidon) 188, 327 (1960). 147. T. I. Tikhonenko and E. N. Dobrov, JMB 42, 119 (1969). 148. World Health Organization, “Human Viral and Rickettsia1 Vaccines,” Technical Report Series, No. 325. Geneva, 1966. 149. Ch. Andrewes, “Viruses of Vertebrates.” Bailliere, Tindall and Cox, London,

1964. 160. Chemical Inactivation of Viruses, Ann. N . Y . Acad. Sci. 83, No. 4, Part 11, 564653 (1960). 161. E. Maron, C. Shiozawa, R. Arnon and M. Scla, Bchem 10, 763 (1971). 162. F. A. Anderer, M. A. Koch and S. D. Hirschle, Eur. J . Immunol. 1, 81 (1971). 163. R. Markham, R. E. F. Matthews and K. M. Smith, Nnture (Londoti) 162, 88 (1948). 164. M. Staehelin, BBA 31, 448 (1959). 166. A. Ross and W. Stanley, J . Gen. Physiol. 22, 165 (1938). 166. R. Haas, R. Thomssen, V. Dostal and E. Ruschmann, Arch. Ges. Virusforsch. 9, 470 (1959). 167. T. Alderson, Nature (London) 215, 1281 (1967). 168. G. D. Zasukhina and I. A. Rapoport, Genetika 5, 33 (1965). 169. Yu. Z. Gendon, “Genetika virusov cheloveka i zhivotnikh.” Nauka, Moskva, 1967. 160. E. Englesberg, J. Bactenol. 63, 1 (1952). 161. F. H. Dickey, G. H. Cleland and C. Lotz, PNAS 35, 581 (1949). 162. F. H. Sobels, Radial. Res. SuppZ. 3, 171 (1963). 163. I. H. Herskowitz, Science 112, 302 (1950). 164. C. Auerbach and H. Moser, 2. Vererbungslehre 85, 479 (1953). 166. H. Nafei and C. Auerbach, 2. Vererbungdehre 95, 351 (1964). 16G. T. Alderson, Nature (London) 191, 251 (1961). lG7. A. F. E. Khishin, Mutation Res. 1, 202 (1964). 168. W. E. Ratnayake, Mutation Res. 9, 71 (1970). 169. W. D. Kaplan, Science 108, 43 (1948). 170. P. D. Lawley, This Series 5, 89 (1966). 171. R. Shapiro, This Series 8, 73 (1968). 172. A. Loveless, “Genetic and Allied Effects of Alkylating Agents.’’ Butterworths, London, 1966. 173. R. K. Lyakyavichus, Uspekhi Sour. Genet. 1, 165 (1967). 174. R. Shapiro and J. Hnchmann, Bchem 5,2799 (1966). 176. R. Shnpiro, B. I. Cohen, 8.5.Shiuey and H. Maurer, Bchem 8, 238 (1969). 176. R. Shapiro, B. I. Cohen and D. C. Clagett, JBC 245, 2633 (1970). 177. N. E. Broude, E. I. Budowsky and N. K. Kochetkov, MoZ. BWZ. 1, 214 (1967). 178. M. Litt and V. Hancock, Bchem 6, 1848 (1967). 179. M. Litt, Bchem 8, 3249 (1969). 180. M. Litt, Bchem 10, 2223 (1971). 181. F. A. French and B. L. Freedlander, Cancer Res. 18, 172 (1958). 18.3. 0. L. Klamerth, BBA 155, 271 (1968). 183. U. Bachrach and G. Eilon, BBA 145, 418 (1967).

REACTIONS OF FORMALDEHYDE WITH NUCLEIC ACIDS

49

l S 4 . U. Baclirarh and G. Eilon, BBA 179, 473 (1969). 186. G.Eilon and U. Buclirdi, BBA 179, 464 (1969). 186'. U.Bachrach and S. Pcrsky, BBA 179, 484 (1969). l S 7 . B. W. Kimes and D. It. Morris, BBA 228, 235 (1971). 188. K . W. Kohn, C. I,. Spears cind P. Dots, JMB 19, 266 (1966). 1259. K. W.Kohn and D. M. Green, J M B 19, 289 (1966). 190. P. D.Lawley and P. Brookcs, J M B 25, 143 (1967). 191. P. D.Lawley and P. Brookrs, BJ 109, 433 (1968). 192. P. D. Ltnvley, J. H. Lethbridgc, P. A. Edwards iind K.V. Shootcxr, J M B 39, 181 (1969). 193. R . J. Rutman, E. H. I,. Chun and J. Jones, BBA 174, 6f33 (1969). 104. W.G . Flnmm, N. J. Brrnlieim and I,. Fishbein, BBA 224, 657 (1970). 196. W.G. Verly and I,. Brnkirr, BBA 174, 674 (1969). 1911. L. Brakier and W-.G. Vrrly, BBA 213, 296 (1970). 197. S. Venitt. P. Brookes and P. D. La\vlcy, BBA 155, 521 (1968). 198. B. D.Reid and I. G.Walker, BBA 179, 179 (1969). 199. K.Takizawit and T. Yaniamoto, Gatin 61, 605 (1970).

This Page Intentionally Left Blank

Synthesis and Functions of the -C-C-A Terminus of Transfer RNAl

I

MURRAY P. DEUTSCHER Departmelit of Biochemistry, University of Connecticut Health Center, Farmingtoii, Connecticut

I. Introduction . . . . . . . . . . . 11. Location of the -C-C-A Terminus in the Three-Dimensional

Structure of tRNA . . . . . . . . . . 111. Synthesis and Turnover of the -C-C-A Terminus in Vivo . IV. Enzymatic Synthesis of the -C-C-A Terminus in Vitro . A. Historical . . . . . . . . . . . B. Purified tRNA Nucleotidyltransferases . . . C. tRNA Recognition . . . . . . . . . D. Catalytic Properties . . . . . . . . . E. Misincorporation of Nucleotides . . . . . . V. Role of the -C-C-A Terminus in tRNA Function . . A. Aminoacyl-tRNA Formation . . . . . . . B. Pept,ide Bond Formation . . . . . . . . VI. Possible Control Functions of the -C-C-A Terminus of tRNA References . . . . . . . . . . . .

. .

51

. . . . . .

52 57 60 60 62 67 71 76 78 78 82 86 88

. . . . . .

. .

. . . . . .

. . . . .

.

1. Introduction The central role of tRNA in the biosynthesis of proteins is now well established. Early studies of tRNA demonstrated the presence a t the 3’-terminus of a specific trinucleotide sequence, -C-C-A,’ which is required for its biological activities ( 1 ) . Numerous sequence analyses in the past several years have confirmed the existence of this terminal sequence in all tRNA molecules specific for most amino acids, independent of their biological sources (a). I n fact, the sequence determination of a tRNA involved exclusively in cell wall synthesis revealed

Studies from the author’s laboratory referred to here were supported by grants from the U.S. Public Health Service (GM-16317) and the American Cancer Society (P-523). Abbreviations used : tRNA-C-C-F, tRNA containing a terminal formycin residue ; P-cell, phosphocellulose ; DEAE, diethylaminoethyl cellulose ; tRNA-N, tRNA lacking the entire -C-C-A sequence. 51

52

MURRAY P. DEUTSCHER

that the -C-C-A terminus is present in molecules used for processes other than protein synthesis (S, Sa). Enzymes that incorporate nucleotides into the terminal positions of tRNA have been known since the earliest work describing aminoacyltRNA formation ( 4 ) . During the initial period of investigation (19581963), a substantial amount of information dealing with the function and turnover of the terminal nucleotides was accumulated. However, in the intervening years since those early studies, relatively little work has been devoted to the understanding of the role of this common trinucleotide in tRNA function or to its mode of synthesis. It is only in the past three years that there has been a rcncwed interest in this area, probably due to the increasing number of studies of tRNA biosynthesis ( 5 ) and to the observations that an identical -C-C-A sequence is present a t the 3’-end of many viral RNA molecules (6). It is the purpose of this article to review the studies dealing with the synthesis and function of the -C-C-A terminus of tRNA. A t the present time, other than the anticodon, the only part of the tRNA molecule assigned a specific function (i.e., carrying the amino acid) is the 3’-terminus. This region of tRNA is particularly amenable to study since it is possible to modify its structure without affecting the rest of the nucleic acid chain by use of the enzyme tRNA nucle~tidyltransferase.~ I n addition, this enzyme is itself of interest and is discussed, since its mode of action is particularly pertinent to an understanding of nucleic acid recognition and to thc mechanism of phosphodicster bond synthesis. Other reviews rclatcd to tRNA structure havc appeared recently (6a, 6b).

II. location of the -C-C-A Terminus in the Three-Dimensional Structure of tRNA Although all tRNA molecules sequenced to date can be arranged in a cloverleaf structure with an exposed 3’-terminus, the three-dimensional organization of these molecules is unknown. Nevertheless, some progress has been made and several models for tRNA structure have been proposed [sec article by Cramer in Vol. 11 of this series for a discussion of the problem ( 7 ) l . I n the present article we are particularly interelited in the location of the -C-C-A terminus and its possible interaction with other parts of thc tRNA molecule. Inasmuch as the 3’-terminus must be * The enzyme has also been called -C-C-A pyrophosphorylase or tRNA pyrophosphorylase. However, the trivial name tRNA nucleotidyltransferase (EC 2.7.7.25) is used throughout this article, esprcially since the enzymc undoubtedly works in the direction of synthesis in v i v a

-C-C-A

TERMINUS OF TRANSFER

RNA

53

available for reaction with aminoacyl-tRNA synthetases, peptidyltransferase and tRNA nucleotidyltransferase, it is likely that the -C-C-A sequence is present in an exposed region for a t least a fraction of the time. Several approaches have been employed in order to examine more directly whether the nucleotides in the -C-C-A terminus interact with other parts of thc molecule and are involved in the three-dimensional structure of tRNA, or whether they are completely exposed. These studies have included chemical modification by reagents specific for singlestranded regions of tRNA, degradation by specific enzymes, and physical measuremcnts of tRNA molecules containing fluorescent or spin-labeled moieties at their 3'-termini. The conclusions to be drawn from these various studies are contradictory. Studies involving modification of tRNA by reagents presumably specific for adenine and cytosine residues in exposed regions suggest that the terminal trinucleotide may be exposed under certain conditions. The terminal adenosine in yeast tRNAP'Ie is one of only four residues oxidized to the l-N-oxide by monoperphthalic acid (8). Similarly, 89% of the terminal adenylate moiety of bulk E. coli tRNA can be deaminated to inosinate by an algal adcnylatc deaminase (9). Exposed cytosine residues in tRNA can also be specifically modified by the reagents methoxyamine (o-methylhydroxylamine) or bisulfite. With both yeast tRNAVa' (10)and Escherichia coli SuIIl tRNATYr ( 1 1 ) , under conditions in which only a limited number of cytosine residues are derivatized by methoxyaminc, both cytosines in the -C-C-A terminus are reactive. Likewise, both terminal cytosine residues in yeast tRNATyr (12) are converted to uracil by the action of bisulfite. Only one of the two cytosine residues in E . coli tRNAolU reacts promptly with bisulfite (IS), suggesting that part of the -C-C-A terminus in this tRNA is less accessible under the conditions of the experiment. I n contrast to the chemical modification studies, digestion of tRNA by exonucleases suggests that the terminal trinucleotide is involved in the three-dimensional structure. The early work of Zubay and Takanami (14) indicated that only the terminal -C-A residues of tRNA are removed by snake venom phosphodicsterasc a t 20°C in the presence of Mg2+,whereas more extensive degradation takes place a t 37". These results were interpreted as evidence that only these two residues are exposed a t 20", but that a t higher temperatures the secondary structure of tRNA is sufficiently disrupted to allow further degradation. However, a recent study by Miller and co-workers (15) indicates that all three terminal nuclcotidcs arc, in fact, removed even a t 20°C, but the terminal AMP is rcmovcd about four timcs faster than the first C M P and about

54

MURRAY P. DEUTSCHER

seventy times faster than the second CMP moiety. These same relative rates of nucleotide removal are also found a t 37" indicating that the differences found a t the two temperatures are due only to the general effect of temperature on the rate of reaction, not to the weakening of tRNA structure. What is clear from these studies is that the terminal C M P residues, a t least, must be involved in the three-dimensional structure of tRNA since it is known that all four nucleotides in RNA can be removed a t similar rates if no secondary structure is present (16). The fact that the last four nucleotides of tRNA are not base-paired in the simple cloverleaf configuration suggests that the difference in rates of removal of the terminal residues is due to their interaction with other parts of the tRNA molecule. The action of polynucleotide phosphorylase on tRNA, which has been studied by Grunberg-Manago and her co-workers over the past several years (17-19), suggests that a certain fraction of tRNA molecules can assume a conformation in which they are so totally resistant to phosphorolysis that even the terminal AMP residue is not removed (17).This is true even in solutions containing only a single species of tRNA, although the proportion of resistant structures a t a given temperature varies from one tRNA to another (19).The resistant conformation can be converted to the sensitive one by heating the tRNA a t a temperature above 90°C or by including purine in the reaction mixture (18).Despite the fact that the -C-C-A termini of the resistant forms are inaccessible to polynucleotide phosphorylase, these molecules can be aminoacylated by aminoacyl-tRNA synthetases (It?), indicating that they are not denatured forms of tRNA (20, 2 1 ) . It is difficult to explain these observations unless it is assumed that the binding of tRNA to the synthetase alters its conformation in such a way that the 3-terminus becomes exposed, but that this change in tRNA structure does not occur upon binding to the phosphorylase. These ideas would fit very well with a model for tRNA structure recently proposed by Danchin (22) (see below). Additional evidence for the involvement of the %-terminal nucleotides in the tertiary structure of tRNA comes from physical studies of this nucleic acid. The fluorescent nucleotide, formycin monophosphate can be incorporated into tRNA in place of the terminal AMP residue by the action of tRNA nucleotidyltransferase (23). Examination of both the fluorescence intensity and pK of the formycin residue in tRNA-C-C-F* compared to those of the free nucleoside suggests that the terminal formycin moiety does interact with other parts of the tRNA molecule. I n addition, the T, of tRNA based on fluorescence of the formycin residue is 7" lower and considerably narrower than one based on absorbance (24).These results suggest that the terminal sequence is involved in a

-C-C-A

TERMINUS OF TRANSFER

RNA

55

part of the tertiary structure that melts independently of the whole tRNA molecule. The ordered structure of the -C-C-F terminus is probably identical to that in normal tRNA, since the former molecule also can accept amino acids ( 2 3 ) . Rich and co-workers (25, 26) obtained similar results using a nitroxide spin-label attached to the free amino group of valine in valyl-tRNA. Based on the rate of tumbling of the spin-label, these workers also observed a lower melting temperature for the 3’-terminus relative to the whole molecule, but only under certain conditions. For example, in dilute salt, the transition of spin-label tumbling occurs at the same temperature as the midpoint of the optical density melting curve, although the transition is sharper. Another study (27) in which the polarization of fluorescence of an acriflavin-tRNA conjugate was measured also showed that the nucleoside terminus becomes more mobile about 10°C below the T, based on absorbance when the measurements are performed in 0.02M Mg?’, but not in dilute salt ( 2 7 ) .These results suggest that the -C-C-A terminus may be involved in tertiary structure even when aminoacylated, although the ionic environment obviously plays an important role in the type of structure found. It has also been suggested that the low temperature form may represent a single-stranded stack of bases a t the 3’-terminus rather than an interaction of these residues with other parts of the tRNA molecule ( 2 6 ) . I n view of the suggestions that charged tRNA has a structure different from that of uncharged tRNA (28-29a), it would be of interest to reexamine these spin-label studies with a nitroxide-labeled nucleotide, such as “tempo-AMP” (SO),in the terminal position. It might be possible to incorporate this compound into tRNA with tRNA nucleotidyltransferase. Such studies would allow an investigation of the 3’4erminus without the complication introduced by amino acid loading. Additional, but less direct, evidence for interaction of the -C-C-A terminus with other regions of the tRNA molecule come from the following studies. Millar (31) has shown that cyanoethylation of pseudouridylate residues in bulk E. coli tRNA renders the terminal nucleotides more susceptible to degradation by snake venom phosphodiesterase a t 20°C compared to untreated tRNA, although no differences were found a t 0”. The data suggest that some interactions between 0” and 20” are disrupted, in cyanoethylated tRNA leading to greater exposure of the -C-C-A residues. Sen0 and co-workers demonstrated (32) that the 3’half of E . coli tRNAfMetdoes not interact with the 5’-half if the terminal six nucleotides are missing, even though in this particular tRNA the last five nucleotides are not based-paired in the cloverleaf structure. These workers suggested that the 3’-terminal nucleotides may play an important

56

MURRAY P. DEUTSCHER

role in forming the proper tertiary structure. On the other hand, it has also been shown that yeast tRNAsep and tRNAPhecan assume a denatured conformation in the absence of the last two nucleotides, -C-A (3s). Another piece of information favoring the involvement of the terminal cytosine residues in tRNA structure is that their conversion to uracils by bisulfite treatment renders them more susceptible to nuclease attack ( l a ) , suggesting that they are probably protected by pairing with guanine residues somewhere in the tRNA chain. In the past few years several models for the tertiary structure of tRNA have been proposed (7). I n most of these, the -C-C-A terminus is represented as being completely exposed, an idea not consistent with the available information, Cramer et al. (8) have suggested that the -C-C of the 3‘-terminus is hydrogen-bonded to the invariant -G-G sequence of the dihydrouridine loop and thereby held in a fixed position. Connors et al. (94) suggested that the C-C-A end “threaded through” the dihydrouridine loop, and Melcher (36) hypothesized that the 3‘terminus slipped through a ring made by all three major loops, thus leaving the -C-C-A residues near the anticodon. Upon aminoacylation the terminus would move out of the ring leading to a more exposed position which probably would be required for accepting the growing peptide chain. A main recent model suggested by Danchin (22) is related to the previous ones in that it visualizes the -C-C-A terminus passing through the dihydrouridine loop and pairing with the -G-G sequence in that loop to form a slipknot type of structure. The model further proposes that upon aminoacylation the hydrogen bonding between the terminal cytosine residues and these guanine residues would be disrupted and the loop would slip further down the aminoacyl stem exposing the -C-C-A end for peptide bond formation ( $ 8 ) . It is also suggested that a tRNA minus its -C-C-A would be folded in such a way that the aminoacyl stem would be positioned just under the center of the dihydrouridine loop and that tRNA nucleotidyltransferase could then add the -C-C-A sequence from above and complete the slipknot (29). Although some of the details of this last model might be open to question, the general outlines appear attractive and, furthermore, are consistent with recent data of Danchin and Gunberg-Manago (36, S7). These workers found that modification of the dihydrouridine loop of yeast tRNAPhe by reduction with sodium borohydride does not affect amino-acid charging (S6). However, if the tRNA is first heated to 70” to disrupt structure, the reduced tRNA exhibits greatly decreased aminoacylation compared to heated, unmodified tRNA. Incubating the tRNA a t 25°C leads to a recovery of activity and a conformation change as shown by NMR. The authors postulate that opening the dihydrouri-

-C-C-A

TERMINUS OF TRANSFER

RNA

57

cline residues by reduction decreases the space inside the loop and thereby decreases the rate a t which the -C-C-A terminus can reenter the loop after heat treatment, presumably to reform the slipknot structure ( 3 6 ) . Since yeast tRNAP1’“contains only eight residues in the dihydrouridine loop it would be of interest to repeat this experiment with a tRNA containing a larger loop to determine whether the charging ability of the tRNA returns after a shorter period of incubation. I n another study (37), these workers also found that more oligo (C) can be bound to acylated yeast tRNAP’le and E . coli tRNAVal than to the deacylated species. This would be consistent with the model described above, since, after charging, the -G-G (or -G-G-G) sequences in the dihydrouridine loop would no longer be paired with the -C-C residues of the terminus. Finally, it was observed (19) that tRNA’s with a long dihydrouridine loop are well phosphorolyzed by polynucleotide phosphorylase, possibly because their 3’-termini can move in and out of the loop more readily. It remains to be explained why chemical modification studies suggest a greater degree of exposure of -C-C-A terminus than other types of measurements. Undoubtedly, the conditions under which the modification reactions are performed (pH, temperature, ionic strength) play an important role. For example, all the chemical modifications were performed in high concentrations of salt, which tighten nucleic acid structure (38). In the context of Danchin’s model (ZZ), such conditions might tighten the structure in such a way that the flexible dihydrouridine arm slips down into the position normally found in aminoacylated tRNA, thereby exposing the -C-C-A terminus to chemical attack. The enzymatic and physical studies, in contrast, were carried out a t much lower salt concentrations. It is also possible to imagine that some enzymes, such as the aminoacyl-tRNA synthetases, could bind to the tRNA in a manner that pulls down the dihydrouridine loop, thereby exposing the -C-C-A end, whereas the various degradative enzymes studied might not induce such a change in conformation.

111. Synthesis and Turnover of the -C-C-A Terminus in Vivo Details of the mechanism of tRNA biosynthesis remain to be elucidated. However, it is clear from studies of both microorganisms and mammalian cells that the tRNA is first synthesized as a larger precursor molecule containing extra nucleotides a t both ends and is then specifically cleaved and modified to produce mature tRNA (39).Since all tRNA molecules contain the same terminal -C-C-A sequence, the question arises whether these nucleotides are synthesized during transcription or are added during the subsequent maturation process, especially since there

58

MURRAY P. DEUTSCHER

is an enzyme that can incorporate nucleotides into this terminal grouping. It has been known for some time that the nucleotides in the -C-C-A terminus are preferentially labeled by comparison with nucleotides in the interior of the tRNA chain ( 4 0 ) . This terminal incorporation is particularly pronounced in the presence of actinomycin D since relatively few nucleotides are incorporated into internal positions of the tRNA chain in the presence of the antibiotic while incorporation into terminal either residues is almost unaffected (41-43). I n labeling tRNA with s2Pp,, in the presence or absence of actinomycin, the radioactivity is found to be concentrated in the terminal AMP residue (40, 4.2, 4 4 ) . Such studies led to the postulate that the nucleotides of the -C-C-A terminus, and especially the AMP moiety, turn over independently of the rest of the tRNA molecule (40-43). However, in some of these studies it is not clear that only turnover was being measured. I n fact, the studies of Rosset and Monier (46) with yeast suggest that net synthesis of the -C-C-A terminus on tRNA molecules lacking these residues may also be taking place during the labeling period. These workers found that in a 15-minute pulse-label with 32Pi,the ratio of specific activities of terminal to internal AMP residues is 6.4, and that for CMP residues is 4.3,despite the fact that the C M P residues are not turning over ( 4 5 ) . That turnover of the terminal residues of tRNA also occurs was demonstrated by studies in E. coli (46, 4 7 ) , yeast ( 4 5 ) , rat liver ( 4 4 ) , and rabbit reticulocytes ( 4 8 ) . Cannon labeled tRNA in exponentially grown E. coli with ["Cladenine and showed that the radioactivity is removed from the terminal AMP residue independently of the internal AMP residues (46). This turnover amounts to about 0.5% of the total terminal AMP per minute, or about 20% per generation, and remains constant for four generations ( 4 6 ) .I n a similar study, Rosset and Monier demonstrated that the turnover rate increases from 0.27% to 0.63% of the total terminal AMP residues per minute as the growth rate increases fom 0.57 to 1.09 ( 4 7 ) .The turnover per generation remains constant a t about 20% ( 4 7 ) . Cannon showed further that turnover ceases when chloramphenicol is added to exponentially growing cells, or if cells with stringent control of RNA synthesis are deprived of a required amino acid ( 4 9 ) . I n cells with relaxed control, turnover of the terminal AMP moiety is observed, but only under certain conditions of amino acid starvation ( 4 9 ) . Studies with yeast (45) and rat liver (44) also indicated that turnover of the terminal CMP residues is relatively low compared to that of the AMP, although no quantitative values were assigned to the turnover rates. I n an examination of turnover in rabbit reticulocytes, Holt et al. (48) concluded that the terminal AMP of tRNA turns over com-

-C-C-A

TERMINUS OF TRANSFER

RNA

59

pletely in 4-8 hours, whereas the two CMP residues require an average of about 30 hours. I n addition, it was shown that these values represent actual turnover, not net synthesis. At present, the functional significance of turnover of the terminal nucleotides of tRNA is not known (see Section V I ) . Some of the labeling studies described above suggested that a portion of the radioactivity found in the terminal nucleotides might arise from net synthesis on tRNA molecules lacking the -C-C-A sequence. Such molecules are presumed to he intermediates in tRNA biosynthesis. Since an enzyme exists that can incorporate these nucleotides into tRNA, it has been assumed that the terminal residues are added in a posttranscriptional process, although no direct evidence for this idea has been described. This hypothesis was strengthened by the observations of Daniel et al. ( 5 0 ) , who found that the terminal AMP and penultimate CMP residues of tRNA are sensitive to pancreatic RNase digestion in tRNA. DNA hybrids, whereas these moieties are not removed by T1 RNase. These data suggest that the -C-C-A terminus does not have a complementary sequence on the DNA. However, it is also possible that the last few residues on tRNA do not hind tightly to DNA and, for that reason, are sensitive to the RNase. Despite this earlier evidence, the suggestion that the -C-C-A terminus may be synthesized independently of the rest of the tRNA molecule has been thrown into doubt recently by the results of Altman and Smith (50~1,5 1 ) . These workers sequenced a precursor tRNA molecule isolated from E. coli infected with $80 phage. The phage carried a mutant SuIII gene specifying tyrosine tRNA, which leads to high yields of the precursor. The precursor molecule of about 130 nucleotides contains the complete tRNA primary sequence as well as additional residues a t both the 3’- and 5’-ends (Fig. 1 ) . Since the -C-C-A sequence is already present and is followed by the three additional nucleotides, -U-C-U, it was concluded that the -C-C-A terminus is part of the transcriptional unit of this tRNA. In addition, incubation of the tRNA precursor with an E. coli extract leads to removal not only of the extra -U-C-U sequence, but also of the two terminal residues in the normal -C-C-A terminus ( 5 2 ) . If the C-C-A terminus of tRNA is, in fact, transcribed from every tRNA gene, one wonders what is the function of tRNA nucleotidyltransferase. This point is particularly perplexing since in many cases turnover of the terminal residues is limited to the AMP moiety, whereas the enzyme can accurately synthesize the complete -C-C-A sequence. I n view of the high turnover number and specificity of tRNA nucleotidyltransferase, it seems unlikely that the enzyme’s role would be limited to repairing the small number of tRNA molecules that lose part of their

60

MURRAY P. DEUTSCHER

A U G

A

C

A C-G

0,

C*G U -G U S A C-G pppG

*

C AG G C C AG U A A A AG C AU UA C C CG *C C A

c

U C

U

FIG.1 . Sequence of the SUIIItRNA precursor, adapted from Altman and Smith (61).The arrows indicate where cleavage must occur to generate the mature tRNA.

terminal sequence during turnover. The fact that tRNA nucleotidyltransferase is present in all sources examined, as well as in mitochondria ( 5 S ) , further suggests a significant function for this enzyme. Since incubation of the tRNA precursor leads to loss of part of the terminal sequence, the enzyme may, in fact, play a role during tRNA maturation. Isolation of mutants lacking tRNA nucleotidyltransferase would help to clarify its function in tRNA metabolism. Of course, the possibility also exists that tRNATyr is an exception, and in other tRNA molecules the -C-C-A sequence is not transcribed. It may be that the terminal -C-C-A sequence in this particular tRNA precursor actually is part of the 3’fragment removed during maturation, but fortuitously containing the same nucleotides reincorporated. Such an idea might be valid since tRNATYr contains three consecutive -C-C-A sequences a t its 3‘-end. This suggestion would also explain the unexpected removal of the terminal -C-A residues accompanying hydrolysis of the extra 3’-nucleotides. Otherwise, this process would appear to be quite wasteful.

IV. Enzymatic Synthesis of the -C-C-A Terminus in Vitro A. Historical The investigations into the nature of protein and RNA synthesis during the late 1950’s are an interesting example of how two completely separate lines of inquiry can ultimately lead to the same facts. At that

-C-C-A

TERMINUS OF TRANSFER

RNA

61

time, the importance of the RNA in the “soluble” portion of the cell (tRNA) was discovered and it was shown that a common trinucleotide sequence was present on these RNA molecules. Simultaneously, many investigators examining the incorporation of RNA precursors into RNA, reported that AMP is often incorporated into terminal positions and adjacent to CMP residues. As it turned out, in most cases, the RNA synthesis measured actually represented the limited incorporation of nucleotides into the terminus of tRNA. It was first shown by Heidelberger et al. (54),using [s2P]AMP and a liver fraction free of nuclei, that there is a specific site of nucleotide incorporation since close to 90% of the label was released as 2’- and 3’C M P after alkaline hydrolysis. I n contrast, Goldwasser (55) had demonstrated earlier, with a complete liver homogenate, that label from [3*P] AMP was distributed among all four nucleotides. It is clear now that in the earlier study (55) the incorporation probably represented net RNA synthesis taking place in nuclei, whereas the results of Heidelberger et al. (54) represented terminal addition to tRNA. I n a series of further studies from several laboratories, it became apparent that AMP incorporation is into the terminal position of the soluble RNA fraction, adjacent to a CMP residue (4, 56-60), and that ATP is the precursor (4, 58, 5 9 ) . I n detailed examinations of the terminal addition reaction, Hecht et al. (1, 4 ) showed that ATP, C T P and UTP are precursors for nucleotide incorporation into the RNA of the “pH 5 fraction,” but that G T P is inactive. A prior incubation of the pH 5 fraction decreased AMP incorporation unless CTP was also present. RNA’s from the soluble fraction of mammalian, bacterial or yeast cells were all active nucleotide acceptors, whereas microsoma1 and nuclear RNA had no activity. The reaction required Mg2+,and was inhibited by pyrophosphate, which reversed the reaction. The 2: 1 ratio of C M P and AMP incorporation, as well as the location of the incorporated residues, indicated that the -C-C-A sequence was synthesized by a terminal addition of nucleotides. This sequence of three nucleotides a t the end of tRNA is required for amino-acid charging (1, 6 1 ) , and furthermore, it has been demonstrated that the amino acid is actually esterified to either the 2‘ or 3‘ hydroxyl of the terminal adenosine (1,62, 63, 63a).

By 1960, the terminal addition of nucleotides to tRNA had been observed in extracts from rat liver (4, 58, 59, 64), Ehrlich ascites cells ( 4 ) , chick embryo (65) and E . coli ( 6 6 ) .Although much of the early data has been substantiated, several puzzling observations still remain to be explained. For instance, Harbers and Heidelberger (64) found that GMP was incorporated into a particular fraction of RNA and almost exclusively next to a UMP residue. These workers suggested the possibility that a

62

MURRAY P. DEUTSCHER

-U-U-G sequence might be synthesized. The incorporation of UMP into tRNA in vitro was also demonstrated in these carly studies ( 4 ) and shown to be in the terminal position, although the level of incorporation was relatively low. The significance of these labeling patterns is not clear since -U-U-G termini have not becn found in tRNA. UMP incorporation by tRNA nucleotidyltransferasc will be discussed further below. I n another puzzling observation, Hecht et al. (4) showed that the extent of AMP incorporation into pH 5 RNA is doubled by the presence of GTP, but not by UTP. CTP, as expected, also led to a large increase in AMP incorporation, but the effects of GTP and CTP were not additive. At present, there is no adequate explanation for this effect of GTP. Finally, Hurwitz and Bressler (67) isolated an enzyme from calf thymus glands that utilizes CTP and adds a limited number of CMP residues to the cnd of RNA chains. No other triphosphate was a substrate, and the only active RNA acceptor was a low molecular weight RNA isolated from the thymus. The relation of this CMP incorporating activity to tRNA nucleotidyltransferase is not understood. It may be related to the poly (C) polymerase activity found in thymus extracts (68).I n addition, the low molecular-weight thymus RNA used in these studies was a substrate only for CMP incorporation, even with an enzyme preparation from E . coli (69).The identity of this RNA is not known.

B. Purified tRNA Nucleotidyltransferases The first purification of the enzyme involved in the terminal addition of nucleotides to tRNA was reported by Canellakis and Herbert in 1960 (70, 71) . These workers isolated three presumably distinct ribonucleoproteins from rat liver, which incorporated AMP and CMP into the RNA present in the preparation (70, 7 1 ) .I n the next several years, the enzyme was purified several hundredfold from E . coli (69, 759, rat liver (73) and rabbit muscle (74) to specific activities in the range of 1-6 pmoles of AMP incorporated per hour per milligram of protein (see Table I).Each of these preparations also incorporated CMP, and the ratios of the two activities remained essentially constant throughout the purification, suggesting that a single enl;yme was probably involved. None of these enzymes was characterized with regard to either purity or physical and chemical properties of the protein. Further detailed studies of these interesting enzymes were neglected until the past two or three ycars when highly purified preparations from E . coli (75-78), yeast (79-80) and rabbit liver (81) became available. Some of the properties of these tRNA nucleotidyltransferase preparations are listed in Table I. I n these recent studies, the enzymes have been purified as much as several thousandfold. Although the actual extent of

TABLE I

PROPERTIES O F PURIFIED tRNA

Source

1. Escherichia coli B

Purification (-fold) -100

Specific activit.y (pmoles/mg/ hour) 4.9,37”C

2. E. eoli W 3. E. coli MRE 600 4. E . coli B

-200 -1000 -4000

5.1,37”C 0.2, 37°C 29, 37°C

5. E. coli B 6. E. coli MliE 600

-7000 -

36, 30°C 1.1, 37°C

7. Bakers’ yeast 8. Bakers’ yeast

-8000 -5000

1400, 32°C

-100 -400 -250 -25,000

0.0075, 25°C 6.3, 37°C 0.8, 37°C 2000, 37°C

9. Rat liver 10. Rat liver 11. Rabbit muscle 12. Rabbit liver

a

Sephadex.

c

Equilibrium ultracentrifugation.

-

Molecular weight

37, O W 45, oooa 54, OOob 42, OOOa 70,000’ 71,000” 44,000” 47, 000’ 48, 000”

NUCLEOT1DYLTRANSFE:RASES

Constant ratio Sedimentation CMP/AMP rate incorpora(S) tion?

3td z Comments Two peaks on IIEAEcellulose

-

60% pure Peak fraction, almost pure

Two peaks on IIEAEcellulose Homogeneous 3 Ribonucleoproteins Two peaks on phosphocellulose, homogeneous

Reference 72

2 cl a

u)

*

H

w

P

60 75 77

78 76

4

u)

9

m

s Y

5

70 80 71 73 74 81

* Dodecyl sulfate-acrylamide-gel electrophoresis. 0,

w

64

MURRAY P. DEUTSCHER

purification may be incorrect because of interfering activities in the crude extracts, it is obvious that these enzymes are present in cells in extremely small amounts. Despite the fact that several of these preparations were reported to be pure (80, 8 l ) , or nearly so (75, 7 7 ) ,their specific activities vary over an extremely wide range. Miller and Philipps (77) reported that their preparation from E. coli has a specific activity of about 30, whereas a 60%-pure enzyme from another strain of E. coli described by Carre et al. (75) has a specific activity of only 0.2 when expressed in the same units. Other purified E. coli preparations, which were not characterized for purity, also have specific activities in the same range (76, 7 8 ) . I n contrast, the enzymes purified from yeast by Sternbach et al. (80) and from rabbit liver by Deutscher (81) have specific activities of about 2000 pmoles per milligram of protein per hour. The turnover number calculated for the rabbit liver enzyme amounts to about 1500 molecules of AMP incorporated per molecule of enzyme per minute, which is probably greater than that for any enzyme synthesizing a phosphodiester bond. This high turnover number is even more surprising since the enzyme must dissociate from its tRNA substrate after the addition of every AMP residue, whereas DNA polymerase and RNA polymerase presumably remain attached to DNA during their action. It is curious that the eukaryote enzymes have such high turnover numbers compared to the prokaryote enzymes. Perhaps this may reflect different roles for tRNA nucleotidyltransferase in the two types of cells. On the other hand, the number of tRNA molecules per molecule of enzyme may vary considerably in prokaryotic and eukaryotic cells, but this has not been determined. In all cases in which they have been examined, the highly purified enzymes incorporate both nucleotides, AMP and CMP, into tRNA. It was demonstrated for the rabbit liver enzyme (82) that the two activities have the same relative rates of incorporation over a 7000-fold purification range, and that on the basis of acrylamide gel electrophoresis, isoelectric focusing and heat inactivation, AMP and CMP are incorporated by a single protein. The tRNA nucleotidyltransferases purified from the various sources generally fall into a relatively narrow molecular weight range (Table I). Miller and Philipps reported that the enzyme purified from E. coli B has a molecular weight of 54,000 based on dodecyl sulfate-acrylamide-gel electrophoresis, and 45,000 based on chromatography on Sephadex G-100 ( 7 7 ) .Similarly, enzymes purified from E. coli M R E 600 have a molecular weight determined on Sephadex of either 42,000 (76) or 37,000 ( 7 5 ) .The latter preparation also has a sedimentation coefficient of 2.9 S. Deutscher reported that the rabbit liver enzyme has essentially the same molecular weight of 44,000-48,000 when examined by equilibrium ultracentrifuga-

-C-C-A

TERMINUS OF TRANSFER

RNA

65

tion, dodecyl sulfate-acrylamide-gel electrophoresis or Sephadex G-100 chromatography (81). The sedimentation coefficient ( s ~ ~ of , ~ 3.6-4.0 ) for this protein is in good agreement with the molecular weight value, assuming a globular protein (81).I n contrast to these results, the tRNA nucleotidyltransferase purified from yeast by Sternbach et al. (80) has a molecular weight of about 70,000,both by equilibrium ultracentrifugation and of 4 . 2 s . Another purified yeast enzyme gel electrophoresis, and an szo,w also has a sedimentation coefficient of about 4 5 determined by sucrose gradient centrifugation (79). For the few cases in which it has been studied (77, 80,81),the molecular weight of tRNA nucleotidyltransferase determined by dodecyl sulfate-acrylamide-gel electrophoresis is essentially identical to that measured by nondisruptive methods, suggesting that these enzymes are single polypeptide chains. Furthermore, both the yeast (80) and rabbit liver enzymes (81) are devoid of any nucleic acid as determined by their ultraviolet absorption spectra. These results indicate that the synthesis of a specific -C-C-A sequence is not directed by a protein-bound template. Despite the fact that the yeast and rabbit liver enzymes have considerably different molecular weights, they have almost identical isoelectric points of about 7.5 (80, 82) (the liver enzyme actually has multiple forms with isoelectric points between 6.5 and 8, but the two main peaks are a t 7.2 and 7.5). The homogeneous rabbit liver enzyme contains no unusual amino acids and displays no unusual patterns in its amino acid composition (81).Both the rabbit liver (81) and rabbit muscle enzymes (82a) are relatively insensitive to sulfhydryl reagents and in some cases are actually stimulated. The enzyme from E . coli B, however, is inhibited 50% by 4-8 pM N-ethylmaleimide or p-mercuribenzoate (83).The rabbit liver (81) and some E. coli enzymes (75, 78) are stimulated by p-mercaptoethanol, but the rabbit liver tRNA nucleotidyltransferase is inhibited by dithiothreitol (81). The presence of multiple forms of tRNA nucleotidyltransferase has been reported by several workers. As discussed above, Canellakis and Herbert described the separation on hydroxyapatite of three ribonucleoproteins from rat liver, each of which is active for AMP and C M P incorporation (70). Furthermore, each fraction is eluted a t its original salt concentration when rechromatographed on hydroxyapatite. Multiple peaks for the E . coli enzyme were also reported by Preiss et al. (72) and by Gross et al. (76). I n the latter case, it was also shown that the molecular weight of the two forms are identical, but that one enzyme incorporates more AMP residues for a given tRNA preparation. Deutscher showed that the rabbit liver enzyme can be separated into two major forms and several minor ones by isoelectric focusing (82). Furthermore,

66

MURRAY P. DEUTSCHER

two forms of the enzyme also can be separated on phosphocellulose each of which incorporates AMP, C M P and UMP into tRNA, but a t different relative rates (81). These two proteins have very similar structural and catalytic properties, but some differences can be detected. For example, one protein appears to contain seven cysteine residues and the other four (81).I n addition, the response of the two enzymes to changes in reaction temperature or to the presence of high concentrations of salt in the assay are markedly different ( 8 4 ) .At present, the significance of multiple forms of tRNA nucleotidyltransferase is still not understood. Since, in the case of the liver enzymes, the two forms are so similar, it was suggested (81) that one of the proteins may have been damaged or modified during purification. Alternatively, if one of the proteins were modified specifically, this may indicate a control mechanism or physiological response for altering the enzymatic activity of the protein. Finally, it is possible that, the two forms of the enzyme represent proteins with different subcellular localizations. The last suggestion bears some further consideration. tRNA nucleotidyltransferases have generally been purified from high-speed supernatant fractions and were thought to be localized in the cytoplasmic fraction of cells. Mukerji and Deutscher (63),prompted by the finding of multiple forms of the liver enzyme, examined the subcellular localiza-

SUBCELLULAR DISTRIBUTION

TABLE I1 OF tRNA NUCLEOTIDYLTRANSFERASE IN RAT LIVER

Fraction Homogenate Crude nuclear Purified nuclei Crude mitochondrial Extracted mitochondria (freeze-thawing)* Extracted mitochondria (Triton X-loo)* Crude microsomal Purified microsomes High-speed supernatant

Total activitya (units)

8.8 0.5
Distributionc

(%) 69 4 <1 2

35 27 2 <1

65

0 Total activity is based on 10 g of liver. One unit equals 1 pmole of AMP incorporated per hour. * Recovery of purified mitochondria was only about 40%, so these values must be multiplied by 2.5 for total activity (values in parentheses). A value of 12.8 units was set equal to 100% of total cellular activity. This number was obtained by assuming that all the activity was localized in the high-speed supernatant (8.3units) plus the extracted mitochondria1 (4.5 units after correction) fractions.

-C-C-A

TERMINUS OF TRANSFER

RNA

67

tion of tRNA nucleotidyltransferase in rat liver (Table 11).It was observed that in addition to the cytoplasmic protein, extracted mitochondria also contain activity, amounting to about 35% of that in the whole cell. The other major subcellular fractions, nuclei and microsomes, are devoid of activity. The mitochondrial enzyme is localized in the matrix of the organelle and is not assayable unless the mitochondria are first ruptured by either freezing and thawing or by treatment with Triton X-100. The crude mitochondrial enzyme has similar catalytic properties to the one in the cytoplasm. However, the relation of the mitochondrial and cytoplasmic enzymes to the two forms of the enzyme purified from rabbit liver is still being explored.

C. tRNA Recognition The tRNA nucleotidyltransferases offer an interesting contrast t o the aminoacyl-tRNA synthetases with regard to their specificity for tRNA. Enzymes in the latter group utilize as substrates only a small number of tRNA chains, and, in many cases, even display species specificity such that tRNA molecules specific for the same amino acid may not be recognized in a heterologous system (85). In contrast, tRNA nucleotidyltransferases appear to recognize tRNA molecules specific for all amino acids and isolated from all organisms. Thus, aminoacyl-tRNA synthetases recognize the differences between tRNA molecules and tRNA nucleotidyltransferases recognize the similarities. An understanding of the structural properties required for the interaction of a nucleic acid with tRNA nucleotidyltransferase should give considerable information about the common features of tRNA structure. Although tRNA nucleotidyltransferases can utilize any type of tRNA as a substrate, these enzymes are almost completely inactive with other types of nucleic acid. Daniel and Littauer demonstrated that the enzyme isolated from rat liver cannot incorporate AMP into rRNA, TMV RNA, poly ( A ) , poly (C), poly (A,C) or poly (A,G,U,C) (73), and, in addition, these substrates are pyrophosphorolyzed very poorly, or not a t all. Similar results were also obtained with the enzyme from E . coli M R E 600 (75). The latter enzyme is also inactive with R17 and 5 S RNA, but it can incorporate an AMP residue into turnip yellow mosaic virus RNA (86). Likewise, under the usual conditions of assay, the rabbit liver enzyme is inactive with poly(A), poly(U), poly(C), poly(G), QB RNA and DNA (84, 87). However, a t high levels of enzyme, rabbit liver tRNA nucleotidyltransferase is able to incorporate nucleotides into liver rRNA (87). This anomalous incorporation occurs a t a very slow rate and very high concentrations of enzyme and rRNA are required in order to measure the reaction, So, although tRNA nucleotidyltransferases may have high

TABLE 111 CATALYTIC PROPERTIES OF PURIFIED tRNA NUCLEOTIDYLTRANSFERASES Apparent K , (triphosphates) (mM)

source 2. 3. 4. 5. 6.

7. 10. 11.

Escherichia coli B E . coli W E. coli MRE 600 E . coli B E. coli B E . coli MltE 600 Bakers’ yeast Ratliver Rabbit muscle

12. Rabbit livep

DH optimum 8.5 8.4 9.5 9.0-9.4 9.5

9.5 9.5 9.5 9.5

Apparent K , (tRNAp (pM)

ATP

CTP

UTP

tRNA-C-C

tRNA-C

tRNA-N

Reference

-

-

-

0.023 0.33 0.095 0.16 0.19

0.083 0.017 0.015 0.06

-

-

-

-

0.18 6.5

-

0.072 -

0.21 9 9.6

0.98 3.8

0.18 0.008

-

78 69 76 85 78 76 79 73 82

2

0.037 0.004

4

84

0.40

0.17

-

0.05

-

12

-

-

-

6

0.20

18

26d

a The apparent K, for tRNA-C-C was determined with ATP; the apparent K, for tRNA-C and tRNA-N was determined with CTP except as noted. Numbers of the various enzymes correspond to those in Table I. Constants for the rabbit liver enzyme were determined with phosphocellulose peak 11. Determined with ATP.

z 5 cd

-C-C-A

TERMINUS OF TRANSFER

RNA

69

specificity for tRNA molecules, other types of nucleic acid may act as acceptors if the conditions are forced (see Section IV, E). Relatively little work has been done to examine the binding of tRNA to tRNA nucleotidyltransferases. Kinetic studies of the E. coli (76, 7 8 ) and rabbit liver (84) enzymes have generally given K , values in the range of 5-20 pM for the various tRNA substrates (Table 111). However, Miller and Philipps (83)reported K , values of about 0.2 pM with the enzyme from E . coli B, which is about two orders of magnitude lower than that found by Best and Novelli (78) with an enzyme from the same source. The reason for this discrepancy is not apparent. Direct binding of the enzyme to tRNA has also been studied by the use of either sucrose gradient centrifugation, binding to nitrocellulose filters, or Sephadex chromatography. Honda, working with a relatively impure E . coli tRNA nucleotidyltransferase, showed that the enzyme forms a complex with yeast and E. coli tRNA’s that could be detected on sucrose gradients, but that rRNA and MS2 RNA are not bound ( 8 8 ) . His data also suggested that the enzyme has a lower molecular weight than tRNA, although such results have not been obtained with more highly purified preparations. Deutscher demonstrated that the enzyme isolated from rabbit liver forms a tight 1 : 1 complex with tRNA-C-C that can be isolated on Sephadex G-100 (81). Furthermore, this enzyme appears to be bound to tRNA during the early stages of purification, but the nucleic acid can be removed by chromatography on DEAE-cellulose. Morris and Herbert detected complex formation between the yeast enzyme and tRNA’s with a variety of termini, including those with 3’-phosphoryl groups ( 7 9 ) .However, tRNA with an intact -C-C-A terminus does not bind to nitrocellulose filters in the presence of the enzyme. Poor binding of tRNA-C-C-A to tRNA nucleotidyltransferases is also indicated by its high Ki as an inhibitor of the E . coli (83) and rabbit liver enzymes (891, and by its 10-fold higher K , (89) as a substrate for the poly(C) polymerase activity of the rabbit liver enzyme ( 8 7 ) . Studies of the recognition site on tRNA for tRNA nucleotidyltransferase have generally followed the same path as those for elucidating the aminoacyl-tRNA recognition region. These have included the use of chemically modified or degraded tRNA’s as AMP and C MP acceptors. The results suggest that the structural requirements for nucleotide acceptance are considerably less stringent than those for amino-acid loading. Thus, modification of tRNA by NH,OH, which destroys cytosine residues ( g o ) , or by cyanoethylation, which affects pseudouridine residues ( 9 1 ) , or by sodium borohydride reduction, which destroys dihydrouracil residues (92, 9 3 ) , has relatively little effect on the ability of tRNA to accept AMP. In addition, substitution of 60% of the uracil residues of yeast

70

MURRAY P. DEUTSCHER

tRNA by fluorouracil (94), which converts the common -G-T-$-C sequence to -G-flu-flu-C and also affects secondary structure, likewise does not impair AMP incorporation. On the other hand, treatment of rat liver (90) or E. coli (95) tRNA with nitrous acid or by UV irradiation (96) decreases AMP acceptance. Low levels of bromination or methylation of yeast tRNA actually stimulate AMP addition, but a t high extents of modification the tRNA is no longer an acceptor (97). tRNA can still be recognized by the tRNA nucleotidyltransferase even after removal of a considerable number of nucleotides. Thus, removal of five nucleotides from the anticodon region of yeast tRNA does not affect AMP incorporation (98, 99). Gross et al. (76) have shown that undefined yeast fragments containing 10 or 40 nucleotides are still able to accept AMP. However, Bernardi and Cantoni observed that removal of only 15% of the nucleotides from the 5' end of yeast tRNA with spleen phosphodiesterase decreased AMP incorporation by 50% , and 30% digestion decreased incorporation by 80% (100). Similarly, half-molecules of tRNA do not protect E . coli tRNA nucleotidyltransferase against heat inactivation unless they are first reannealed ( 1 0 a ) . I n the most complete examination to date of the AMP and CMP acceptor activity of tRNA fragments, Overath et al. (101) showed that half and three-quarter molecules and mixtures of fragments from tRNAPhe or tRNAser can accept AMP and CMP. The poorest acceptors are those in which the normally double-stranded aminoacyl stem is disrupted by the absence of the 5'-terminal nucleotides. However, the results from this work are very puzzling. It was claimed that all the fragments used in the study were pure. Yet, many fragments could accept AMP and CMP to only a small fraction of the theoretically expected value. If a homogeneous population of fragments can accept AMP or CMP a t all, it would be expected that these nucleotides should be incorporated completely. One possibility to explain these results is that the rate of incorporation of nucleotides into some of the fragments is so slow that the yeast tRNA nucleotidyltransferase is inactivated before the reaction is completed. A similar phenomenon was observed with the rabbit liver enzyme during studies of the misincorporation of nucleotides since these reactions also occur a t an extremely slow rate (87). From these results it is clear that only a part of the overall tRNA structure is required for recognitidn by tRNA nucleotidyltransferase. Nevertheless, something about the overall conformation may also be recognized, since denatured tRNALeUis not an AMP acceptor ( 1 0 2 ) . Possibly in the denatured conformation, the aminoacyl stem is buried and inaccessible to the enzyme. It appears from these observations that a likely candidate for the recognition site, and one that is common to all

-C-C-A

TERMINUS O F TRANSFER

RNA

71

tRNA’s, is the double-stranded aminoacyl stem with its seven base pairs. Consistent with this idea is the relatively poor activity of single-stranded fragments, and the loss of activity upon removal of only a few nucleotides from the 5‘-end of the molecule. Likewise, removal of nucleotides from the anticodon region would be expected to have no effect. If this hypothesis is correct, then synthetic stems such as oligo(G) *oligo(C) or oligo(A) oligo(U) might be acceptors of C M P and AMP.

D. Catalytic Properties The enzyme tRNA nucleotidyltransferase can incorporate AMP and CMP into tRNA molecules from which all, or part, of the terminal sequence has been removed, thus regenerating intact tRNA-C-C-A with the usual amino acid acceptor activity (1, 72, 7 3 ) . One wonders how the enzyme can limit nucleotide incorporation to two C M P residues and one AMP residue, and in the proper sequence. Since the purified tRNA nucleotidyltransferases contain no nucleotide material that could act as template, the mechanism whereby the specific sequence is synthesized must be determined by the proper relationship of active sites within the protein. At present, generalizations about the mechanism of nucleotide addition and the arrangement of triphosphate binding sites cannot be made since, in many respects, the bacterial and mammalian enzymes appear to be quite different. All the tRNA nucleotidyltransferases studied have alkaline pH optima, in the range of pH 9-10 for nucleotide incorporation (Table 111). Apparently, the enzyme does not work a t its p H optimum in vivo. Alternatively, the reaction in vitro may be quite different from that catalyzed in vivo. Despite the similarity of high pH optima and also that of being able to work on all tRNA’s, the enzymes purified from E. coli appear to differ from the mammalian enzymes, especially with respect to their triphosphate sites. Thus, the apparent K, values for ATP generally vary from about 0.1-0.3 mM for the E . coli enzymes (7578, 83) although one older report indicated a value of 0.02 mM (69) (Table 111).In contrast, the three purified mammalian enzymes have K , values one order of magnitude higher, approximately 1-4 m M ( 7 3 , 8 2 , 8 4 ) . Likewise, the K , values for C T P are also different. Those for the E . coli enzymes are in the range of 0.015-0.08 mM (69, 76-78, 83), whereas the rat liver enzyme has a K , value of 0.18 mM ( 7 3 ) .I n addition, the rabbit muscle (82a) and rabbit liver (84) enzymes give nonlinear double reciprocal plots for CTP, suggesting the presence of multiple C T P binding sites. Anthony et al. extrapolated the two linear portions of the l/v vs 1/S plot for C T P and calculated K , values of 0.008 mM and 0.037 mM ( 8 2 ~ ) . Deutscher, using a wider concentration range for CTP, was able to fit

72

MURRAY P. DEUTBCHER

the curved double reciprocal plot by computer to two K,, values of 0.004 mM and of 0.4 mM (84).The 10-fold difference in the high K,,, value for the two rabbit enzymes is probably due to the fact that the experiments of Anthony et al. (8%) extended only to 0.16 mM C T P and the K,, was calculated by an extrapolation of the three highest C T P concentrations even though theoretical considerations indicate that the line should be curved throughout. Nevertheless, these results suggest that the rabbit enzymes, a t least, probably contain two C T P binding sites. It should be pointed out, however, that all of these K,, values are really only apparent K,’s and may be greatly affected by the concentration of tRNA present. Further differences between the bacterial and mammalian enzymes have been observed in their response to the presence of a second triphosphate. Thus, with the bacterial enzymes, ATP inhibits CMP incorporation and C T P inhibits AMP incorporation (75-78, 83). This inhibition has been reported to be competitive (77, 78) in the case of two enzymes, and noncompetitive (75) with another one. I n the case of the rabbit enzymes, C T P also inhibits AMP incorporation. However, ATP stimulates, rather than inhibits, CMP incorporation (84, 103). The inhibition of AMP incorporation by C T P is competitive with the rabbit muscle enzyme (8%) and noncompetitive with the enzyme from rabbit liver (103). I n addition, AMP incorporation catalyzed by the rabbit muscle enzyme is inhibited (3040%) by equal concentrations of G T P or U T P (8Za), whereas the E. coli enzymes are inhibited only when U T P is present in large excess, and not a t all by G T P (75, 83). With the rabbit liver enzyme, it was shown that AMP incorporation in the presence of 1 mM ATP is 50% inhibited by 3 mM U T P and about 15-20 mM G T P (103). The different catalytic properties of the mammalian and bacterial tRNA nucleotidyltransferases, as well as the different specific activities discussed above, still remain to be explained. Several pieces of evidence suggest that, in the case of purified rabbit liver, tRNA nucleotidyltransferase, ATP and CTP bind a t separate sites (84,103).First of all, the apparent K , value for ATP is about one order of magnitude greater than even the higher K,, for C T P ( 8 4 ) .Second, high concentrations of salt inhibit AMP incorporation completely, whereas CMP incorporation catalyzed by the major form of the enzyme is stimulated as much as 2-fold (84). Third, CTP inhibits AMP incorporation noncompetitively (103), suggesting distinct sites. Finally, ATP stimulates CMP incorporation a t C T P concentrations of 0.05 to 4 mM (103), and it is difficult to explain how ATP could effect stimulation without binding a t a separate site. Anthony et al. (82a) also postulated the existence of separate sites for ATP and C T P based on the additivity of AMP and CMP incorporation in the presence of both substrates and appropriate

tRNA acceptors. Since the enzyme probably binds only a single tRNA acceptor at a time, it is not clear why these workers observed additivity of nucleotide incorporation. Possibly, their observations could be explained by the use of nucleoside triphosphates at concentrations considerably below saturating levels, so that the presence of both ATP and CTP enabled more enzyme molecules to participate in the reaction. Inasmuch as it appears that the rabbit liver enzyme contains two binding sites for CTP, and that ATP and C T P have separate sites, it has been suggested (84) that tRNA nucleotidyltransferase may contain three sites in tandem, two for CTP and one for ATP. The possibility also exists that the ATP site corresponds to one of the two CTP sites, but that model seems less attractive. In the model suggested, tRNA molecules lacking all, or some, of the terminal residues would bind opposite the appropriate triphosphate site and would, thereby, accept the proper nucleotides for generation of the complete -C-C-A sequence. If a tRNA molecule were bound adjacent to the wrong triphosphate site, it might explain the misincorporation of nucleotides observed with this enzyme (see Section IV, E) . However, the strength of tRNA binding a t the wrong site may be weaker; this is borne out by the finding that the K , for tRNA-C as an acceptor of AMP in the misincorporation reaction is 26 pM, whereas it is only 4-6 pM when CMP and UMP are incorporated (Table 111) ( 8 4 ) ,On the other hand, the K , for ATP for incorporation into tRNA-C is unaffected suggesting that the usual ATP binding site is also involved in this reaction. I n addition to incorporation of the expected nucleotides, A M P and CMP, purified tRNA nucleotidyltransferases also incorporate U M P into tRNA. Daniel and Littauer showed that the ratio of UMP to AMP incorporation remains constant during the 400-fold purification of the rat liver enzyme (73). Deutscher demonstrated UMP incorporation by a homogeneous rabbit liver protein (81) that is inactivated by heat a t the same rate as the AMP and CMP activities (81).UMP incorporation was also observed by Best and Novelli with a purified E. coli enzyme (i’O4), although Miller and Philipps reported that this activity is lost upon further purification (77). Under optimal conditions, UMP is incorporated into tRNA-C and tRNA-N by the rat and rabbit liver enzymes at about one-tenth the rate of CMP (73, 84). Since tRNA-C-C is not a substrate for UMP addition (73, 84, 105),it seems that UTP is acting as an analog of CTP. The K , for UTP with the mammalian enzymes was reported to be in the range of 0.05 to 0.26 mM (73, 84, 106),which is lower than that for ATP, but greater than the K , at the tight binding site for C T P (Table 111). Low concentrations of CTP completely abolish UMP incorporation (73, 103, 105, 106) and the inhibition is competitive (103,

74

MURRAY P. DEUTSCHER

106). I n addition, UTP inhibits CMP incorporation (103, 106), but this inhibition reaches a maximum of only 60% (103).It has been suggested (84) that UTP acts as an analog a t the low K , site for CTP, since U M P incorporation is completely abolished by concentrations of C T P that can saturate only the tight binding site, and because U T P can only partially inhibit CMP incorporation. I n addition, low concentrations of ATP stimulate UMP incorporation, in a manner similar to that found for CMP addition (103, 106). UMP is incorporated into tRNA molecules in the positions normally occupied by C M P residues, thereby leading to the synthesis of tRNA-U, tRNA-C-U and tRNA-U-C (73, 87, 105, 106). tRNA molecules containing two UMP residues are generally not made (73, 87, 106) although this reaction can be forced a t high levels of enzyme ( 8 7 ) .tRNA chains containing UMP residues accept AMP relatively poorly (73, 8 7 ) , in contrast to chains containing C M P residues. At high levels of the rabbit liver enzyme it has also been possible to incorporate UMP into the terminal position usually occupied by AMP, generating tRNA-C-C-U and tRNAC-U-U ( 8 7 ) . Other analogs of ATP and C T P can also be used as substrates by tRNA nucleotidyltransferase. Thus, bromo-CMP (107) and iodo-CMP ( 7 ) can be added to tRNA in place of CMP. The AMP base analogs tubercidin, toyocamycin, sangivamycin (108) and formycin monophosphate (23) (Fig. 2 ) , can also be incorporated into the terminal position of tRNA. Another ATP analog, adenosine 5’-0- (1-thiotriphosphate) is also used as a substrate and is incorporated into yeast tRNApheto generate a phosphorothioate linkage between the terminal adenosine and cytidine residues (109).In contrast, other analogs of AMP, such as G M P ( 8 4 ) , 2-aminopurine nucleotide ( d d ) , 2,6-diaminopurine nucleotide (24) and I M P (110), are not incorporated into tRNA. dATP is also not a substrate for either the E. coli (72, 7 5 ) , rabbit muscle (74) or rabbit liver (84) tRNA nucleotidyltransferases. With the rabbit liver enzyme, this is true even in the presence of Mn2+ rather than Mg2+ ( 8 9 ) . If nucleotide analogs such as dAMP, cordycepin phosphate or 2’-O-MeAMP were incorporated, they would generate tRNA molecules lacking the 2 or 3’-hydroxyl group. The amino acid acceptor activity of such tRNA molecules might elucidate the site of attachment of amino acids to tRNA. Nucleotide incorporation catalyzed by tRNA nucleotidyltransferases is accompanied by stoichiometric pyrophosphate release (72-74), and it has been known for some time that these enzymes can also catalyze the reverse reaction, the pyrophosphorolysis of tRNA (4, 69, 71-74). This reaction is dependent on the presence of RNA, and the same nucleotides

-C-C-A TERMINUS

HO

O F TRANSFER

OH

HO

Adenosine

75

RNA

OH

HO

Tubercidin

H

OH

Toyocamycin

HocQHH HO

OH

Sangivamycin

HO

OH

Formycin

FIG.2. Structure of adenosine and the adenosine analogs tubercidin. toyocamycin, sangivamycin. and form\.cin.

arc removed as are incorporated by the enzyme. Incorporation of label from ["PIPP, into G T P and UTP has also been reported (65, 7 1 ) , but not with any of the more purified enzymes (72, 7 3 ) . It has also been observed that greater incorporation of PP, into nucleotide mat,erial occurs if unlabeled triphosphates are present (69),and this finding may indicate that pyrophosphate exchange takes place in addition t o the pyrophosphorolysis of tRNA. Ribosomal RNA is not a substrate for pyrophosphorolysis (72, 7 3 ) , although a slow rate of reaction with poly(C) and poly (A,G,U,C) has been observed ( 7 3 ) .Blockage of the vicinal hydroxyls of tRNA by aminoacyl- or peptidyl-groups prevents pyrophosphorolysis (111). Surprisingly, the optimal p H for pyrophosphorolysis is 7.5 compared to 9.5 for nucleotide incorporation (73) although this may just indicate that the active form of pyrophosphate in the reaction is present in greater amounts a t the lower pH. Despite the more normal p H optimum for the reverse reaction, it is doubtful that pyrophosphorolysis plays a role in vivo since active pyrophosphatases would prevent the

76

MURRAY P. DEUTSCHER

accumulation of any significant amounts of PPi. The pyrophosphorolysis and pyrophosphate exchange reactions of tRNA nucleotidyltransferases have been very poorly examined, although such studies would be very valuable in elucidating any intermediate steps (i.e., an enzyme-nucleotide) that may occur during nucleotide incorporation.

E. Misincorporation of Nucleotides Under the appropriate conditions tRNA nucleotidyltransferases synthesize a perfect -C-C-A sequence on the 3'-terminus of tRNA. Nevertheless, with other conditions these enzymes catalyze the incorporation of incorrect nucleotides and utilize other RNA's as acceptors. It was first shown by Daniel and Littauer (112) that rat liver tRNA nucleotidyltransferase could add AMP to tRNA-C to generate tRNA-C-A. Klemperer and Haynes, using an enzyme from the same source, reported that in the presence of MnZ+,runs of CMP, as many as 10 nucleotides long, could be incorporated into 5 S and rRNA, and that -A-A and -U-U sequences could be made on phosphodiesterase-treated tRNA (113). These workers also observed that a small amount of CMP is incorporated into poly (A) epoly (U) and poly(1) epoly (C) , whereas the single stranded polymers are inactive. Miller and Philipps described the incorporation of three C M P residues into tRNA-N and two CMP residues into tRNA-C, but reported that this anomalous incorporation is lost upon further purification of the E . coli enzyme (77). I n contrast, Best and Novelli reported that a 7000-fold purified E. coli tRNA nucleotidyltransferase catalyzes the incorporation of excess CMP residues into tRNA-C and tRNA-C-C, but is inhibited by ATP (104). These workers also described the synthesis of tRNA-C-A, and the possible formation of tRNA-C-A-A. Deutscher, using a homogeneous rabbit liver tRNA nucleotidyltransferase, examined this problem in more detail (87). It was observed that a t low levels of enzyme the nucleotides incorporated into tRNA-C-C-A, tRNA-C-C, tRNA-C and tRNA-N are those expected for synthesis of the normal -C-C-A sequence except for the formation of tRNA-C-A (see Table IV) . At high levels of enzyme, tRNA molecules with a variety of unusual termini can be made. These include molecules with extra A*MP residues, tRNA-C-A-A, tRNA-A-A and tRNA-A-A-A, as well as molecules containing all lengths of CMP residues. Intact tRNA-C-C-A is not a substrate for AMP incorporation, even at high levels of enzyme, but CMP is incorporated. However, the presence of C T P stimulates AMP incorporation into this substrate. That C M P incorporation is actually into intact tRNA and not into a contaminating nucleic acid was shown by the release of the terminal AMP residue as 2'- and 3'-AMP

-C-C-A

TERMINUS O F TRANSFER

RNA

77

TABLE IV SUMMARY OF PRODUCTS SYNTHESIZED BY tRNA NUCLEOTIDYLTRANSFERASP tRNA substrate

Enzyme level

tRNA-C-C-A

Low

Triphosphate substrate

Product ~~~~

High tRNA-C-C

Low High

tRNA-C

Low High

tItNA-N

Low High

ATP CTP ATP CTP ATP CTP ATP CTP ATP ATP CTP ATP CTP ATP ATP CTP ATP CTP

+ CTP + CTP

None None None tRNA-C-C-A(pC), tRNA-C-C-A None tRNA-C-C-A tRNA(pC), tRNA-C-C-A tRN A-C-A tRNA-C-C tRNA-C-A-A tRNA(PC)n tRNA-C-C-A None tRNA-C-C tRNA-A, tRNA-A-A, tRNA-A-A-A tRNA(pC),

after alkaline hydrolysis. Increasing levels of enzyme and long times of incubation lead to the formation of runs of CMP on every tRNA substrate. When both ATP and CTP are present, the terminal position is usually occupied by AMP, even when many C MP residues have been incorporated. The excess C M P incorporation can be inhibited somewhat by ATP, but can be prevented completely by high concentrations of KC1 (87). These results indicate that purified tRNA nucleotidylfransferase contains poly (C) polymerase activity. Recent data (89) have demonstrated that the poly (C) polymerase activity actually is associated with tRNA nucleotidyltransferase. The activity copurifies during 5t 25,000-fold purification and is heat inactivated at a n essentially identical rate a t several different temperatures. The responsc of poly(C) polymerase to pH, Mg2+ concentration, C T P concentration and incubation temperature is almost identical to that of the normal CNP-incorporating activity. Poly (C) polymerase activity is most pronounced in the presence of Mn2+,although Mg2+ and Co2+are also active. Since the rate of poly(C) polymerase activity is less than 1% of that of normal CMP incorporation, it is not normally detected. The poly(C) polymerase can also utilize rRNA as a substrate, incorporating as many as 40 CMP residues per chain (87). With rRNA

78

MURRAY P. DEUTSCHER

as the substrate, it is also possible to incorporate a single AMP residue on each nucleic acid chain (89). The mechanism whereby tRNA nucleotidyltransferase synthesizes these various anomalous termini is still a matter of conjecture. Nevertheless, it is clear that these reactions occur a t very slow rates and can be detected only a t high levels of enzyme. It is curious that CTP, which appears to have two binding sites, is incorporated into tRNA and rRNA extensively, whereas ATP, which has only a single binding site, is incorporated only to a limited extent. Possibly slippage back and forth between the two CTP sites accounts for the poly (C) polymerase activity. The finding that homogeneous, mammalian tRNA nucleotidyltransferase preparations also contain poly (C) polymerase activity raises questions about the significance of the poly (C) polymerases described previously (68, 114, 116).

V. Role of the -C-C-A Terminus in tRNA Function It has been known since the earliest studies of tRNA function that the activated amino acid is attached to the 2'- or 3'-hydroxyl group of the terminal AMP moiety, and that removal of this nucleotide residue renders the tRNA inactive (1, 62, 63, 6%). Since the 3'-terminal residue carries the amino acid, it is obvious that this portion of the tRNA molecule must interact with the aminoacyl-tRNA synthetases during the charging reaction and also with the peptidyltransferase in the course of peptide bond formation. In trying to assess the role of the -C-C-A terminus in the charging and transfer reaction, it must be borne in mind that the action of tRNA in these processes requires proper recognition of the terminus as well as its ability to accept and transfer the amino acid. Thus, it is possible to imagine circumstances in which the terminal residues might be modified in such a way that tRNA could still interact with the enzymes, but could not accept or transfer amino acids. Generally, the recognition process has been examined by either direct binding studies or by testing modified tRNA as a competitive inhibitor. A. Aminoacyl-tRNA Formation Investigations into the function of the -C-C-A terminus in the aminoacylation reaction have generally employed two types of approach. I n both cases, tRNA is modified in its terminal residues and then tested either for its ability to interact with a synthetase or for its acceptor activity. The conclusion to be drawn from these studies is that modification of the terminal nucleotides has different effects depending on the synthetase under study. A number of workers have examined the effect

-C-C-A

TERMINUS OF TRANSFER

RNA

79

of oxidizing tRNA or of removing residues from the -C-C-A terminus on the interaction of tRNA with a synthetase. Since these modifications lead to tRNA molecules that cannot accept amino acids, interaction with the synthetase cannot be measured by the overall biological reaction, aminoacylation, but only by less direct methods. Converting the 2'- and 3'-hydroxyls of tRNA to a dialdehyde by periodate oxidation destroys acceptor activity ( 6 3 ) . Nevertheless, such modified tRNA molecules appear to bind to synthetases since they act as competitive inhibitors (116'-121). Moreover, since the binding constant of the tRNA does not appear to be greatly affected by the oxidation (116-121) , it appears that, although these oxidized molecules are inactive, they interact normally with the synthetases. Presumably i t is the presence of the aldehyde grouping that prevents the tRNA from being an acceptor. In fact, Cramer et al. have shown that reduction of periodateoxidized yeast tRNAPhe with NaBH, to generate a diol structure leads to an active tRNA with a normal K,, but about one-half the normal V,,, (122). However, in the case of yeast tRNALy8, reduction with borohydride decreases the inhibitory properties of oxidiaed tRNA (118, 123), possibly because the tRNA becomes a substrate. Although periodate-oxidized tRNA does bind to synthetases, i t cannot participate in some of the reactions of intact tRNA. Thus, oxidized tRNA does not stimulate the pyrophosphate exchange reaction observed with glutamyl- (124) or arginyl-tRNA synthetase (126). Similarly, oxidized tRNA does not induce the breakdown of valyl-AMP catalyzed by isoleucyl-tRNA synthetase (126'). However, oxidized tRNA can protect arginyl-tRNA synthetase against heat denaturation (127). tRNA's lacking all or part of the -C-C-A terminus are prepared either by treatment with snake venom phosphodiesterase (14) or by the successive actions of periodate, an amine and alkaline phosphatase (128). Using the latter method, it is possible to remove only the terminal nucleoside residue, leaving the phosphate group intact. I n contrast to oxidation of the terminal ribose moiety, which generally has little effect upon binding of tRNA to its synthetase, actual removal of terminal residues can lead to more drastic changes in tRNA-enzyme interactions, although again this varies with the system studied. Thus, E . coli tRNA-C inhibits valyl- and isoleucyl-tRNA formation (117, 126), and liver tRNA-C-Cp inhibits glutamyl-tRNA formation (124). I n each of these cases, the modified tRNA is a competitive inhibitor, but with a higher Ki than periodate-oxidized tRNA, suggesting that the terminal residues contribute to the binding of tRNA to the synthetase. A more extreme example was found with yeast serine tRNA-C-C in that this molecule, even in excess, does not inhibit serine incorporation (129). I n a more

80

MURRAY P. DEUTSCHER

detailed examination of the inhibitory properties of tRNA molecules lacking all, or part, of the -C-C-A terminus, Roy and Tener observed that the inhibition varied with the synthetase being examined (118). Phenylalanine incorporation is relatively little affected by any modified tRNA, whereas valyl-tRNA formation is sensitive to all the inhibitors. Direct binding studies of modified tRNA’s to synthetases indicate that tRNA-C-C and tRNA-C bind strongly to E . coli isoleucyl-tRNA synthetase (130).Similarly, valine tRNA-C-Cp forms a complex with yeast valyl-tRNA synthetase and inhibits formation of a complex with intact tRNAV*l (131). The affinity of the modified tRNA for the enzyme is within a factor of two of that for intact tRNA (131),although if Sephadex rather than sucrose gradients is used to detect complex formation, tRNA-C-Cp does not inhibit (132). Binding of modified tRNA’s to synthetases can also be studied indirectly by other methods. For example, intact yeast tRNASe’ labilizes the seryladenylate-enzyme complex, and a similar effect is observed with tRNA-C-C (133). However, 10-fold higher concentrations of the modified tRNA are required to effect the same degree of complex breakdown. E. coli arginyl-tRNA synthetase is protected against heat denaturation by tRNA (127).Removal of the terminal AMP and cytidine moieties does not decrease the ability of tRNA to protect, whereas removal of the phosphate between the two cytidine residues leads to a large decrease in protector activity. These studies with modified tRNA’s indicate that for some synthetases the nucleotides of the-C-C-A sequence contribute significantly to tRNA binding, although presumably they only reenforce the binding of the “recognition site.” On the other hand, if the “recognition site” is, in fact, the overall conformation of a particular tRNA molecule, including the location of the -C-C-A terminus, this distinction between conformation and “recognition site” becomes meaningless. Since all synthetases must bind to the terminal residues in order to effect aminoacid attachment, it may only be in those cases in which the -C-C-A terminus also contributes to the “recognition site” that removal of terminal residues abolishes binding of the tRNA. The second approach employed to examine the function of the -C-C-A terminus in aminoacylation is to alter one, or more, of these residues, leaving the terminal ribose intact and then determining whether these modified tRNA’s can still accept amino acids, The terminal residues can be altered either by chemical modification or by using tRNA nucleotidyltransferase to incorporate nucleotide analogs. Several modifications of the terminal adenine have been reported, and, in many cases, these altered tRNA’s still retain acceptor activity.

-C-C-A

TERMINUS O F TRANSFER

RNA

81

Deamination of the AMP moiety of E . coli tRNA to I M P by algal adenylate deaminase leads to a product that accepts glycine and an amino-acid mixture to the same extent as untreated tRNA (9). As discussed above, the terminal adenosine moiety of tRNA can be substituted by tubercidin, toyocamycin, sangivamycin (108) or formycin (23) using tRNA nucleotidyltransferase (Fig. 2). tRNA containing tubercidin a t the 3’4erminus accepts lysine arginine, valine, leucine, tyrosine, proline and serine a t close to normal levels, whereas phenylalanine is poorly incorporated (108). tRNA’s containing either toyocamycin or sangivamycin accept an amino-acid mixture at half normal levels in 15 minutes. However, it is not clear from these observations whether extents of incorporation are being measured (108). tRNA containing a formycin terminus also accepts a variety of amino acids a t close to normal levels (23). I n contrast to the data with nucleotide analogs, chemical modification of yeast tRNAPhe with monoperphthalic acid to yield the N-loxide of adenosine renders the tRNA inactive as an acceptor (8). It has also been possible to modify yeast tRNAPheso that a phosphorothioate linkage is introduced between cytidine 75 and adenosine 76 (109). This modified tRNA has a normal K,, but its V,,, for aminoacylation is reduced to one-half and the extent of charging is only 60% of normal. Several other studies have described modification of the terminal cytosine residues. Incorporation of 5-bromo-CMP into liver tRNA using tRNA nucleotidyltransferase and 5-bromo-CTP leads to a product that accepts several amino acids a t close to the levels found with a tRNA reconstituted with C T P (107). However, it is not apparent in this experiment whether both CMP residues or only the terminal one has been modified. Similarly, yeastPhe containing 5-iodocytidine in the penultimate position is still chargeable (7). I n contrast, modification of the two terminal cytosine residues of E . coli SuIII tRNATrrwith methoxyamine destroys the acceptor activity of this tRNA, whereas modification of only cytosine 84 does not inactivate the molecule (194).Likewise, modification of the two terminal cytosine residues of E . coli tRNAeMet by UV light inactivates this particular molecule (196). In these latter two cases it could be shown that the inactivation of the tRNA’s was due almost entirely to modification of the cytosine residues in the -C-C-A sequence. In contrast, conversion of both terminal cytosine residues in yeast tRNATyr to uracils by the action of bisulfite does not affect the extent of tyrosine charging ( 1 2 ) . The conclusion to be drawn from the foregoing chemical modification studies is that the -C-C-A terminus can tolerate considerable alteration and still remain active. The only change of the terminal adenosine moiety that leads to inactivation is N-1 oxidation, whereas modification of the

82

MURRAY P. DEUTSCHER

6-amino group or the imidazole ring has relatively little effect. Inactivation by N-1 oxidation may be due to steric effects caused by introduction of the oxygen atom or to removal of a potentially hydrogen-bonding nitrogen. On the other hand, a bulky bromine or iodine atom, or a methoxy group, can be introduced into a t least one of the cytosine residues without affecting charging. Modification of both cytosine residues, however, appears to inactivate the molecule, although conversion to uridines, in one case, has no effect. At the present time insufficient data are available to generalize whether certain residues within the -C-C-A terminus or certain functional groups on these residues are more important to tRNA function than others. A final approach to elucidating the function of the -C-C-A terminus in aminoacylation has been to change the length or sequence of residues within the terminus with tRNA nucleotidyltransferase. Thus, Daniel and Littauer synthesized liver tRNA-C-A, which lacks one of the CMP residues, and suggested that this molecule could accept leucine and phenylalanine to the usual extent, but a t considerably lower rates (112). Recent evidence, however, indicates that those results may have been due to some contamination by tRNA containing two CMP residues or to synthesis of intact tRNA during the aminoacylation reaction (136). Using rabbit liver tRNA nucleotidyltransferase, it has been possible to synthesize tRNA’s with a variety of 3’-termini (87) (Table IV). Examination of the amino-acid acceptor activity of these molecules should prove very useful for understanding the function of the terminal -C-C-A sequence in the reaction catalyzed by aminoacyl-tRNA synthetases.

B. Peptide Bond Formation The synthesis of peptide bonds takes place by transfer of the growing peptide chain from the -C-C-A terminus of one tRNA molecule to an amino acid attached to the -C-C-A terminus of a second tRNA. This reaction is catalyzed by the enzyme peptidyl transferase which is part of the larger ribosomal subunit (137). Raacke has recently suggested (138) that two peptidyltransferases may, in fact, be required. Inasmuch as the formation of peptide bonds involves amino acid moieties attached to the termini of tRNA molecules, it is appropriate to inquire how important the actual -C-C-A sequence is for this process. It was shown early, by measurement of the binding of modified tRNA’s to ribosomes, that the -C-C-A terminus plays a role in this interaction. Thus, tRNA molecules lacking the entire -C-C-A terminus do not bind to rat liver ribosomes in the presence of transfer enzymes (139, 140). Similarly, removal of the terminal adenosine moiety prevents the nonenzymatic binding of tRNA to E . co2i ribosomes (141). Cleavage

-C-C-A

TERMINUS OF TRANSFER

RNA

a3

of the C2’-C3’ bond of the terminal adenosine of tRNAPhe likewise prevents binding to ribosomes a t both T-factor-dependent and -independent sites (142). Interaction of modified tRNA’s with E . coli ribosomes has also been examined by measuring the inhibition of the nonenzymatic binding of aminoacyl-tRNA’s in the presence of mRNA (143-146). Since periodate-treated tRNA inhibits phenylalanyl-tRNA binding equally as well as deacylated tRNA (145), an intact terminal ribose is not required for interaction with ribosomes, although there is some question about this result (144). tRNA-C-Cp, tRNA-C-C and tRNA-C also inhibit phenylalanyl-tRNA binding to 70 S ribosomes, but not as well as intact, deacylated tRNA (149, 144). I n contrast, these modified tRNA’s are as effective as intact tRNA in inhibiting aminoacyl-tRNA binding to 3 0 s ribosomal subunits (143, 144). These results suggest that the terminal adenosine moiety plays a role in binding to 70 S, but not 30 S ribosomes. tRNA lacking the entire -C-C-A sequence also inhibits phenylalanyltRNA and lysyl-tRNA binding, but not as well as deacylated tRNA (144, 145). A significant advance in our understanding of the mechanism of peptide bond formation came with the discovery that substrate analogs containing only fragments of the tRNA molecule could act as peptide donors (146) and peptide acceptors (147-149). The use of these various analogs of the peptidyl-tRNA and aminoacyl-tRNA molecules has allowed a detailed examination of the structural requirements for peptide bond formation. From this information it has been possible to establish what features of the -C-C-A terminus are required for binding and reaction at the P-site and A-site on ribosomes (150). Structural requirements for the P-site were determined by measuring the reaction of various N-acylaminoacyl oligonucleotides with puromycin in the presence of ribosomes and methanol (161). Under these conditions, reaction with the hexanucleotide fragment, C-A-A-C-C-A-fMet is 5Q76 completed within 15 minutes a t 0”.The nucleotide fragments A-A-C-CA-fMet, A-C-C-A-fMet and C-C-A-fMet are equally active, whereas C-A-fMet and fMet-adenosine are without activity. The active fragments lead to an initial rate of reaction about one-half that of intact fMettRNA. Similarly, C-C-A-acetylleucine is active for transferring acetylleucine to puromycin. Although these studies do not distinguish between effects of structure on the affinity for peptidyltransferase or on the rates of reaction once bound, it is clear that the -C-C-A terminus by itself is sufficient for binding and reaction at the P-site of ribosomes. However, further removal of one 3’-CMP residue leads to an inactive substrate. Since intact tRNA’s have greater activities than the corresponding terminal fragments, other parts of the tRNA molecule must also interact

84

MURRAY P. DEUTSCHER

with the ribosome to increase the stability of binding. Inasmuch as the effect of concentration was not examined in these experiments, it is not possible to determine whether -C-C-A-fMet could attain the same initial rate as intact fMet-tRNA, given high enough levels of the fragment. Information is also available on the structural requirements for interaction a t the A-site. It has been known for some time that puromycin, an. analog of the aminoacyl-adenosine terminus of tRNA, can act as a peptide acceptor ( 1 4 7 ) , suggesting that only the terminal adenosine residue of tRNA is required for interaction a t the A-site. However, the only active analogs of puromycin are those in which the p-methoxyphenylalanine moiety is replaced by certain other aromatic amino acids (147, 152, 153). This result is surprising since the aminoacyl-tRNA’s that normally act as acceptors must include derivatives of all amino acids. It appears that the peptidyltransferase center on the ribosome may contain a hydrophobic binding site that helps to stabilize the binding of certain puromycin derivatives, but not others. More important for our purposes are the results, with puromycin analogs substituted on the 5‘-hydroxyl group. Addition of a 3’-CMP moiety to the 5’-hydroxyl of the glycyl analog of puromycin made this molecule almost as active as puromycin for inhibiting poly (U) -directed polyphenylalanine synthesis either in an E . coli (152) or a reticulocyte system (153).Thus, an analog of the aminoacyl-terminus of tRNA containing both the terminal cytidine and AMP residues is an inhibitor even if the aminoacyl moiety is the usually inactive residue, glycine. When the glycyl analog of puromycin is substituted a t the 5‘-hydroxyl with AMP, UMP or GMP, the resulting compounds are not inhibitors (152, 153), substantiating the specificity for the terminal C-A residues of tRNA a t the A-site. Further information about the specificity of the A-site comes from an examination of the ability of various aminoacyl-nucleoside or nucleotide derivatives to act as acceptors of the peptide chain. It was first demonstrated by Takanami (154) that the nascent polypeptide could be transferred to tRNA fragments prepared by digestion of aminoacyltRhTA’s with T1 RNase. Using more defined compounds, it was shown that leucyl-, phenylalanyl- and tyrosyladenosine are inhibitors of pro. tein synthesis in a cell-free system from E . coli, whereas adenosine, free amino acids, or other leucyl esters are inactive (148). Inhibitions of about 50% are obtained with 1 pM puromycin, but require about 100 pM leucyladenosine and 10 pM phenylalanyl-and tyrosyladenosine (148). Thus, as with the puromycin derivatives, aromatic amino acids appear to stabilize the interaction with the A site. Glycyladenosine or glycylAMP, even a t 10 mM, are almost totally inactive in releasing polylysine from E. coli ribosomes, whereas C-A-Gly is half as active as puromycin

-C-C-A

TERMINUS OF TRANSFER

RNA

85

(149). In contrast, U-A-Gly has a small effect, and U-U-Gly is inactive (149). In further studies of this phenomenon, Rychlik and co-workers (155157) showed that a variety of other aminoacyl-nucleosides could act as acceptors of the nascent peptide, with both Ac-Phe-tRNA and poly (Lys) tRNA as donors. The activity of the acceptors decreases in the sequence Phe-Ado > Phe-Ino > Phe-Cyd ; whereas Phe-Guo, Phe-Urd and PhedAdo are inactive (155).Furthermore, C-I-Gly releases the nascent polypeptide chain from poly (Lys)-tRNA, but dC-A-Gly has little activity (157). These results demonstrate that molecules containing only the terminal adenosine residue and an amino acid are sufficient for binding and reaction a t the A-site. The presence of a C-A sequence further stabilizes the binding in the case of certain aminoacyl-derivatives (particularly glycine). Since Phe-Cyd can act as an acceptor, base pairing between the terminal adenine of tRNA and rRNA probably does not play a role in the peptidyltransferase reaction. However, the 2’-hydroxyl groups on both the terminal adenosine and penultimate cytidine residues appear to be required. These 2‘-hydroxyl groups may be important for forming hydrogen bonds to the transferase or for forming intramolecular hydrogen bonds required for proper orientation of the tRNA terminus (155). Alternatively, the 2’-hydroxyl group of the terminal adenosine residue may be the initial acceptor of the nascent peptide prior t o reaction with the aminoacyl residue (158). Further evidence for the involvement of the -C-C-A terminus of tRNA in peptide bond formation was suggested by the finding that the trinucleotide, C-C-A stimulates the formation of fMet-OEt catalyzed by peptidyltransferase in the presence of fMet-tRNA and ethanol (159). The trinucleotide is only 10% as active as deacylated tRNA on a molar basis, but can attain the same maximal rate of ester formation. The stimulation of peptidyltransferase is specific for C-C-A, and C-A is inactive. Sequence isomers such as C-A-C and A-C-C as well as a variety of other trinucleotides, are inactive. Furthermore, periodate oxidation of the terminal adenosine of tRNA eliminates the stimulation of fMet-OEt formation. Aminoacylation of the tRNA leads to dipeptide formation rather than ethyl ester synthesis. The data suggest that the trinucleotide sits in the A-site, thereby stimulating peptidylransferase to catalyze a nucleophilic attack by ethanol. From the foregoing discussion it is clear that the -C-C-A terminus of tRNA plays a major role in peptide bond formation. At both the Asite and the P-sites, oligonucleotide fragments containing only a complete, or partial, -C-C-A sequence are sufficient to promote peptide bond formation. However, the structural requirements a t the two sites are different.

MURRAY P. DEUTSCHER

86

The complete C-C-A terminus is required for activity a t the P-site, whereas in some cases an aminoacyl-adenosine is all that is necessary to accept the nascent peptide, although addition of a CMP residue does increase activity. When the aminoacyl group is lacking, such as in the case of ethyl ester formation, a complete C-C-A sequence must be present for activity. If, in fact, the peptidyltransferase activities a t the putative A- and P-sites actually represent two different enzymes, as suggested by Raacke ( I % ) , the different structural requirements are not a t all surprising.

VI. Possible Control Functions of the -C-C-A Terminus of tRNA The turnover rate of the terminal nucleotides of tRNA is too slow to be coupled one-to-one with peptide bond formation. Both in E . coli (49) and rabbit reticulocytes (48) the slow turnover rate and the number of tRNA molecules involved also rules out any direct relationship between turnover and peptide chain release. Studies with in vitro protein synthesizing systems have also indicated that the adenylate moiety of aminoacyl-AMP is not incorporated into tRNA (160) and that there is no turnover of the terminal AMP of tRNA coupled to amino-acid transfer (161). Nevertheless, there does appear to be some relation between turnover and protein synthesis. For example, inhibition of protein synthesis in E. coli by chloramphenicol or amino-acid starvation leads to a cessation of turnover ( 4 9 ) . Similarly, injection of puromycin or cycloheximide into rats decreases the relative specific activity of the terminal adenylate residue of liver tRNA compared to internal residues ( l e g ) , suggesting an inhibition of turnover. It has been suggested (46, 47) that the turnover of the terminal residues corresponds simply to a limited action on tRNA by a nuclease such as the RNase I1 of E . coli. This reaction then would really have no metabolic significance, but would represent only the occasional damage of tRNA molecules that happen to be uncharged a t any given instant. This degradation could occur on the ribosome after the poptide chain has been transferred. On the other hand, the turnover of all, or part, of the terminal -C-C-A sequence in vivo raises the possibility that this process may serve a control function. A simple and rapid method for turning off protein synthesis would be to remove the terminal adenylate moiety from tRNA. It has been assumed by many workers that the enzyme that removes the terminal residues from tRNA is, in fa&, tRNA nucleotidyltransferase, and until recently this eneyme was actually designated “CCA pyrophos-

-C-C-A

TERMINUS O F TRANSFER

RNA

87

phorylase.” However, it is unlikely that this activity is responsible for nucleotide removal in cells since the pure enzyme contains no nuclease activity, even for terminal residues (81). Furthermore, the high levels of pyrophosphatase in cells would preclude the accumulation of pyrophosphate and, therefore, would prevent reversal of the synthetic reaction. These considerations suggested the existence of a nuclease that selectively removes only a limited number of residues from the 3’terminus of tRNA. Such an activity has been detected in rabbit liver and partially purified (163).Control of protein synthesis by removal of the 3’-terminal residue of tRNA, therefore, could be exercised either by tRNA nucleotidyltransferase or by this nuclease. Alternatively, the rate of protein synthesis may be controlled by addition of the -C-C-A sequence to a pool of inactive tRNA molecules lacking these residues. Several observations are consistent with a possible control function for the turnover of the terminal nucleotides of tRNA. Stationary phase and commercial yeast tRNA generally lack the terminal AMP moiety, whereas this residue is largely intact in growing cells (46). However, strain differences have been found (164). Another dormant system, unfertilized sea urchin eggs, may also contain deficient tRNA (165) possibly because of incomplete termini. Furthermore, upon fertilization, there is a dramatic increase in the incorporation of label into the nucleotides of the -C-C-A terminus (166, 167). It should be pointed out, however, that the lack of labeling in unfertilized eggs may be due to the inability of the 32Pilabel to be incorporated into ATP and CTP. In addition, completion of the -C-C-A terminus of tRNA cannot be the only mechanism for triggering the onset of protein synthesis following fertilization since ribosomes from unfertilized eggs seem also to be inactive [see Timourian (166) for references]. Yet another system in which protein synthesis may be controlled a t the level of the terminus of tRNA is the nonlactating bovine mammary gland (168). tRNA isolated from this tissue appears to lack the terminal trinucleotide sequence and thus cannot be charged. The terminal nucleotides can be replaced in the presence of rat liver tRNA nucleotidyltransferase, ATP and CTP to regenerate active molecules (168). It is curious that those systems that contain a defect in the terminal sequence (i.e., stationary phase yeast, unfertilized sea urchin eggs and nonlactating mammary gland) are all relatively dormant. Finally, Stent has suggested a model (169) whereby removal and addition of nucleotides from the -C-C-A termini of specific tRNA molecules could regulate the rate of synthesis of specific proteins. Removal of the terminus from tRNA molecules needed to translate certain codons could effectively shut off or decrease the rate of the synthesis of specific

88

MURRAY P. DEUTSCHER

proteins. Such a mechanism might explain thc slow rate of terminal turnover of tRNA that has been observed.

REFERENCES 1. L. I. Hrcht. M. 1,. Strplienson and P. C. Znniecnik, P N A S 45, 505 (1059). 2. M. Staehelin, Experie,ilin 21, 1 (1971). 3. G. G. Loringer, R. J. Roberts and J. L. Strominger, FP 30, 1217 (1971). Sn. H. A. Sober, ed., “Handbook of Biochemistry and Molecular Biology,” 2nd ed., pp. H-130-133. Chemical Rubber Co. 4 . L. I. Hecht, P. C. Zamecnik, M. L. Stephenson and J. F. Scott, JBC 233, 954 (1958).

5. D. So11, Science 173, 293 (1971). 6. U. Rensing and J. T. August, Nature (Londoii) 224, 853 (1969). 60. D. H. Cause, F. yon der Haw, A. Maelicke and F. Cramer, A R B 40, 1045 (1971).

Gb. S. Arnott, Progr. Biophys. Mol. B i d . 22, 181 (1971). 7. F. Cramer, This Series 11, 391 (1971). S. F. Cramer, H. Doepner, F. yon der Hnar, E. Schlimme and H. Seidel, PNAS 61, 1384 (1968). 9. C. Li and J. Sii, BBRC 28, 1068 (1967). 10. T. I. Jilyaeva and L. L. Kisselev, FEBS Lett. 10, 229 (1970). 11. A. R. Cashmore, D. M. Brown and J. D. Smith, J M B 59, 359 (1971). 12’. Z. K h a n , K. A. Freude, I. Kilrnn and R. W. Chambers, Nature (London) New Biol. 232, 177 (1971). 13. R. P. Singhal, JBC 246, 5848 (1971). 14. G. Zubay and M. Takanami, BBRC 15, 207 (1964). 15. J. P. Miller, M. E. Hirst-Bruns and G. R. Philipps, BBA 217, 176 (1970). 16. T. Nihei and G. L. Cantoni, JBC 238, 3991 (1963). 17. M. N. Thang, W. Gusehlbauer, H. G. Znchau and M. Grunberg-Mnnngo, J M B 26, 403 (1967). 18. M. N. Thang, B. Beltchev and M. Grunberg-Manago, EJB 19, 184 (1971). 19. B. Beltchev, M. N. Thang and C. Portier. EJB 19, 194 (1971). 20. T. Lindahl, A. Adams and J. R. Fresco, PNAS 55, 941 (1966). 21. W. J. Gartland and N. Sueoka, PNAS 55, 948 (1966). 32. A. Danchin, FEBS Lett. 13, 152 (1971). 23. D. C. Ward, A. Cernmi, E. Reich, G. Acs and L. Altwerger, JBC 244, 3243 (1969). 24. D. C. Ward, E. Reieh and L. Stryer, JBC 244, 1228 (1969). 35. B. M. Hoffman, P. Scliofield and A. Rich, P N A S 62, 1195 (1969). 26. P. Schofield, B. M. Hoffman and A. Rich, Bchem 9, 2525 (1970). 87. D. B. Millar and R. F. Steiner, Bchem 5, 2289 (1966). 25. P. S. Sarin and P. C. Zamernik, BBRC 20, 400 (1965). 29. R. Stern, L. E. Zulra and U. Z. Littauer, Bchem 8, 313 (1969). 29n. M. Colin, A. Danchin and M. Grunberg-Mnnago, J M B 39, 199 (1969). 30. T. R. Krugh, Bchem 10, 2594 (1971). 31. D. B. Millar, BBA 174, 32 (1969). 52. T. Seno, M. Kobayashi and S. Nishimura, BBA 190, 285 (1969). 33. R. E. Streeck and H. G . Zachau, FEBS Lett. 13, 329 (1971). 34. P. G. Connors, M. Labanauskas and W. W. Beemnn, Science 166, 1528 (1969).

89 56. G. Melcher, FEBS Lett. 3, 185 (1969).

SO. A. Danchin and M. Grunberg-Manago, BBRC 39, 683 (1970). S7. A. Danchin and M. Grunberg-Manago, FEBS Letl. 9, 327 (1970). 38. J. R. Fresco, in “Informational Macromolecules” (H. J. Vogel, V. Bryson and J. 0. Larnpen, eds.), p. 121. Academic Press, New York, 1963. 3.9. R. H. Burdon. This Scrics 11, 33 (1971). 40. C. Scholtissek, BBA 61, 499 (1962). 41. R. M. Franklin, BBA 72, 555 (1963). 4% I. Merits, BBA 108, 578 (1965). 43. Y. Moulk and R. M. Landin, BBRC 20, 491 (1965). 44. R. M. Landin and Y. Moulk, BBA 129, 249 (1966). 46. R. Rosset and R. Monier, BBA 108, 376 (1965). 4G. M. Cannon, BBA 87, 154 (1964). 47. R. Rosset and R. Monier, BBA 108, 385 (1965). 4s. C. E. Holt, P. B. Joel and E. Herbert, JBC 241, 1819 (1966). 49. M. Cannon, BBA 129, 221 (1966). 60. V. Daniel, S. Sarid and U. Z. Littauer, Science 167, 1682 (1970). 60a. S. Altman, Nature (London) New B i d . 229, 19 (1971). 61. S. Altman and J. D. Smith, Nature (London) N e w B i d . 233, 35 (1971). 62. S. Altman, personal communication. 63. S. K. Mukerji and M. P. Deutschrr, JBC 247, 481 (1972). 64. C. Heidelberger, E. Harbers, K. C. Leibman, Y. Takagi and V. R. Potter, BBA 20, 445 (1956). 66. E. Goldwasser, JACS 77, 6083 (1955). 6G. A. R. P. Paterson and G. A. LePage, Cnncer Res. 17, 409 (1957). 67. M. Edmonds and R. Abrams, BBA 26, 226 (1957). 6s. E. S. Canellakis, BBA 25, 217 (1957). 69. E. Herbert, JBC 231, 975 (1958). GO. C. Coutsogeorgopoulos, BBA 44, 189 (1960). GI. L. I. Hecht, M. 1,. Stephenson and P. C. Zamecnilr, BBA 29, 460 (1958). G2. H. G. Zachau. G. Acs and F. Lipmann, PNAS 44, 885 (1958). 6.9. J. Preiss, P. Berg, E. J. Ofengand, F. H. Bergmann and M. Dieclrmann, PNAS 45, 319 (1959). G3n. H. G. Zachau and H. Feldmann, This Series 4, (1965). G4. E. Harbers and C. Heidelberger, BBA 35, 381 (1959). 06. C. W. Chung, H. R. Mahler and M. Enrionc, JBC 232, 1448 (1960). 66. K. Moldave, BBA 43, 188 (1960). 67. J. Humitz and A. E. Bresler, JBC 236, 542 (1961). 68. M. Edmonds, JBC 240, 4621 (1965). G9. J. J. Furth, J. Hurwitz, R . Krug and M. Alexander. JBC 236, 3317 (1961). 70. E. S. Canellalris and E. Herbert, PNAS 46, 170 (1960). 71. E. S. Canellakis and E. Herbert, BBA 45, 133 (1960). 72. J. Preiss, M. Dieckmann and P. Berg, JBC 236, 1748 (1961). 7s. V. Daniel and U. Z. Littauer, JBC 238, 2102 (1963). 74. J. L. Starr and D. A. Goldthwait, JBC 238, 682 (1963). 76. D. S. Carrk, 8. Litvak and F. Chaperille, BBA 224, 371 (1970). 7 s . H. J. Gross, F. R. Duerinck and W. C. Fiers, EJB 17,116 (1970). 77. J. P. Miller and G. R. Philipps, JBC 246, 1274 (1971). 78. A. N. Best and G. D. Novelli, ABB 142, 527 (1971). 79. R. W. Morris and E. Herbert, Bchem 9, 4828 (1970).

90

MURRAY P. DEUTSCHER

80. H. Sternbach, F. von der Haar, E. Schlimme, E. Gaertner and F. Cramcr, EJB 22, 166 (1971). 81. M. P. Deutscher, JBC 247, 450 (1972). 82. M. P. Deutscher, JBC 245, 4225 (1970). SZa. D. D. Anthony, J. L. Starr, D. S. Kcrr and D. A. Goldthwait, JBC 238, 690 (1963). 83. J. P. Miller and G.

R. Philipps, JBC 246, 1280 (1971). 84. M. P. Deutscher, JBC 247, 459 (1972). 86. K. B. Jacobson, This Series 11, 461 (1971). 86. S. Litvak, D. S. Car& and F. Chapeville, FEBS Lett. 11, 316 (1970). 87. M. P. Deutscher, JBC 247, 469 (1972). 88. H. Honda, BBA 195, 587 (1969). 89. M. P. Deutscher, unpublished results. 90. M. Takanami and K. I. Miura, BBA 72, 237 (1963). 91. A. V. Rake and G. M. Tener, Bchem 5,3992 (1966). 92. P. Cerutti, BBRC 30, 434 (1968). 93. T. Igo-Kemenes and H. G. Zachau, EJB 10, 549 (1969). 94. R. Giege, J. Heinrich, J. H. Weil and J. P. Ebel, BBA 174, 53 (1969). 96. J. A. Carbon, BBA 95, 550 (1965). 96. P. D. Harriman and H. G. Zachau, JMB 16, 387 (1966). 97. B. Rether, J. H. Weil and J. P. Ebel, BUZZ. Sac. Chim. Biol. 47, 1591 (1965). 98. I. I. Chuguev, V. D. Axelrod and A. A. Bayev, BBRC 34, 348 (1969). 98. I. I. Chuguev, V. D. Axelrod and A. A. Bayev, BBRC 41, 1CB (1970). 100. A. Bernardi and G. L. Cantoni, JBC 244, 1468 (1969). 100a. J. P. Miller and G. R. Philipps, Bchem 10, 1001 (1971). 101. H. Overath, F. Fittler, K. Harbers, R. Thiebe and H. G. Zachau, FEBS Lett. 11, 289 (1970). 102. T. Lindahl, A. Adams, M. Geroch and J. R. Fresco, PNAS 57, 178 (1967). 103. S. K. Mukerji and M. P. Deutscher, unpublished results. 104. A. N. Best and G. D. Novelli, ABB 142, 539 (1971). 106. H. G. Klemperer and E. S. Canellakis, BBA 129, 157 (1966). 106. A. FernandezSorensen, D. D. Anthony, and D. A. Goldthwait, JBC 241, 5019

(1966). 107. R. L. Soffer, S. Uretsky, L. Altwerger and G. Acs, BBRC 24, 376 (1966).

105'. S. C. Uretsky, G. Acs, E. Reich, M. Mori and L. Altwerger, JBC 243, 306 (1968). 109. E. Schlimme, F. v. d. Haar, F. Eckstein and F. Cramer, EJB 14, 351 (1970). 110. A. Mehler and Y . Tal, personal communication. 111. P. Pulkrabek and I. Rychlik, BBA 179, 245 (1969). fiZ. V. Daniel and U. Z. Littauer, JMB 11, 692 (1965). 113. H. G. Klemperer and G. R. Haynes, BJ 104, 537 (1967). 114. M. G. Page, G. R. Haynes and H. G. Klemperer, BJ 102, 181 (1967). 116. J. G. Cory, A. W. Benson and A. J. Girgenti, BBRC 42, 778 (1971). 116. H. Hayashi and K. I. Miura, JMB 10, 345 (1964). 117. J. Torres-Gallardo and M. Kern, PNAS 52, 91 (1965). U S . K. L. Roy and G. M. Tener, Bchem 6, 2847 (1967). 119. M. P. Stulberg and K. R. Isham, PNAS 57, 1310 (1967). 120. M. P. Deutscher, ABB 125, 758 (1968). 121. V. A. Korzhov and L. S. Sandakhchiev, Biokhimiga 31, 71 (1966). 122. F. Crumer, F. von dcr Hnnr and Schlimme, FEBS Letl. 2, 136 (1968).

-C-C-A

TERMINUS OF TRANSFER

RNA

91

123. V. A. Korzhov, Mol. B i d . 4, 47 (1970). 124. M. P. Deutscher, JBC 242, 1132 (1967). 126. A. H. Mehler and S. K. Mitra, JBC 242, 5495 (1967). 126. A. N. Baldwin and P. Berg, JBC 241,839 (1966). 127. S. K. Mitra, K. Chakraburtty and A. H. Mehler, J M B 49, 139 (1970). 128. H. C. Neu and L. A. Heppel, JBC 239, 2927 (1964). 129. M. H. Makman and G. L. Cantoni, Bchem 5, 2246 (1966). 130. M. Yarus and P. Berg, J M B 42, 171 (1969). 131. U. Lagerkvist and 1,. Rymo, JBC 245, 435 (1970). i32. U. Lagerkvist, L. Rymo and J. Waldenstrijm, JBC 241, 5391 (1966).

1%. H. G. Bluestein, C. C. Allende, J. E. Allende and G. L. Cantoni, JBC 243, 4693 (1908). 134. A. R. Cashmore, FEBS Lelt. 12, 90 (1970). 136. L. H. Schulman, PNAS 66, 507 (1970). 136. Y. Tal and U. Z. Littauer, personal communication. 187. R. E. Monro, T. Staehelin, M. L. Gelma and D. Vazquez CSHSQB 34, 357 (1969). 13s'. I . R. Raacke, PNAS 68, 2357 (1971). 139, M. Takanami, BBA 55, 132 (1962).

140. W. S. Bont, F. Puizinga, H. Bloemendal, M. F. Van Weenen and L. Bosch, A B B 109, 207 (1965). iq. M. Cannon, R. Krug and W. Gilbert, J M B 7, 360 (1963). 149. C. M. Chen and J . Ofengand, BBRC 41, 190 (1970). 1.43. Y. Kuriki, I. Fukuma and A. Kaji, JBC 244, 1365 (1969). 144. S. Pestka, JBC 2-45, 1497 (1970). 146. N. Shimizu, H. Hayashi rind K. I . Miura, JBC 67, 373 (1970). 146. R. E. Monro and K. A. Marcker, J M B 24, 347 (1967). 147. D. Nathans and A. Neidle, Nature (London) 197, 1076 (1963). 148. 5. P. Waller, T. Erdos, F. Lemoine, S. Guttman and E. Sandrin, BBA 119, 566 (1966). 149. I. Rychlik, S. Cliludek and J . Zcmlicka, BBA 138, 642 (1967).

160. M. S. Bretscher, CSHSQB 31, 289 (1966). 161. R. E. Monro, J. Cernti and K. A. Marcker, PNAS 61, 1042 (1968). 162. R. H. Symons, R. J. Harris, L. P. Clarke, J. F. Wheldrake and W. H. Elliott, BBA 179, 248 (1969). 163. R. J. Harris, J. E. Hanlon and R. H. Symons, BBA 240, 244 (1971). 164. M. Takanami, PNAS 52, 1271 (1964). 166. I. Rychlik, J. Cernh, S. ChlBdek, J. Zemlicka and Z. Haladovk, J M B 43, 13 (1969). 16G. I. Rychlik, J. Cernli, S. ChlBdek, P. Pulkrabek and J. Zemlicka, EJB 16, 136 (1970). 167. J. Zemlicka and S. Chlkdek, Bchem 10, 1521 (1971). 168. H. Neumann, V. E. Shashova, J. C. Sheehan and A. Rich PNAS 68, 1207 (1968). 169. E. Scolnick, G. Milman, M. Rosman and T. Caskey, Nature (London) 225, 152 (1970). 100. K. K. Wong and K. Moldavc, JBC 235, 694 (1960). 161. D. Nathans and F. Lipmann, PNAS 47, 497 (1961). 162. Y. Moulk and R. M. Landin, FEBS Lett. 8, 189 (1970). 163. M. P. Deutscher, FP 29, 871 (1970). 104. R. Giege and J.-P. Ebel, BBA 161, 125 (1968).

92

MURRAY P. DEUTSCHER

f66. H. Timourian, Develop. Biol. 16, 594 (1967). 166. V. R. Glisin and M. V. Glisin, PNAS 52, 1548 (1964). f f l . P. R. Gross, K. Kraemer and L. I. Malkin, BBRC 18, 569 (1965). 168. M. D. Herrington and A. 0. Hawtrey, BJ 116, 405 (1970). 169. G. 5. Stent, Science 144, 816 (1964).

Mammalian RNA Polymerases SAMSON T. JACOB' Physiological Chemistry Laboratories, Department of Nutrition and Food Sciences, Massachusetts Institute of Technology, Cambridge, Massachusetts

1. Introduction . . . . . . . . . . . . . 11. Quantitative Extraction of RNA Polymerase from Mammalian Cells . . . . . . . . . . . . . . . A. Methods of Solubiliaation . . . . . . . . . B. Recovery of the Enzyme . . . . . . . . . 111. Multiplicity, Nomenclature and Intranuclear Localization . . A. Separation of Multiple Forms of RNA Polymerase by Column Chromatography . . , . . . . . . . . B. Nomenclature . . . . . . . . . . . . C. Nuclear Localization . . . . . . . . , . D. Relative Concentrations and Specific Activity . . . . IV. Properties of RNA Polymeraees . . . . . . . . A. Stability . . . . . . . . . . . . . B. Metal Ion and Ionic Strength Optima . . . . . . c. Sensitivity to a-Amanitin in vitl.0 . . . . . . . D. Effect of a-Amanitin in Bivo . . . . . . . . E. Template Specificity . . . . . . . . . . F. Factor Requirements . . . . . . . . . . V. Subunit Structure and Molecular Weight . . . . . . VI. Regulation of RNA Polymerases . . . . . . . . VII. Mitochondrial RNA Polymerase . . . . . . . . VIII. Summary and Conclusions . . . . . . . . . . Notes Added in Proof . . . . . . . . . . . References . . . . . . . . . . . . . .

93

95 95 101 101 101 101 103 103 106 106 106 107 108 110 111 112 114 117 119 121 121

1. Introduction Spectacular progress has recently been made in the elucidation of the structure of bacterial DNA-dependent RNA polymerase (EC 2.7.7.6) and of its role in the regulation of transcription [reviewed recently by Burgess ( I ) , Martin ( l a ) , and Sethi ( I b ) ] .The ease with which this enzyme is solubilized and purified from bacterial sources is mainly responsible for Present address : Department of Pharmacology, The Pennsylvania State University College of Medicine, Hershey Medical Center, Hershey, Pennsylvania. 93

94

SAMSON T. JACOB

these achievements. Unlike the prokaryotic RNA polymerase, the eukaryotic enzyme is tightly bound to a complex consisting of DNA, histones, acidic proteins and RNA. Consequently, studies on mammalian RNA synthesis have utilized either whole nuclei or an “aggregate enzyme” obtained as a crude, insoluble preparation from nuclei by precipitation with KC1 ( 2 ) . The quality of the RNA synthesized in isolated nuclei (3-10) or nucleoli (11-14) is significantly affected by divalent metal ions and salt concentration. I n the presence of Mg”, the RNA formed in the polymerization reaction is predominantly (G C)-rich rRNA, whereas the RNA synthesized with high salt and Mn2+is mostly (A U)-rich, DNA-like RNA ( 4 ) . These results are compatible with the following possibilities: (a) a single polymerase transcribes selective regions of the chromatin template under different ionic conditions, or (b) there are different species of RNA polymerase with specific cation and salt requirements. Although the first possibility has been widely accepted, there are instances where changes in ionic conditions have no effect on the template activity. For example, salt in bacterial systems stimulates the initiation of RNA synthesis without any significant modifications of the template (16).Moreover, prokaryotic cells with their simple chromosomal structure can exhibit remarkable specificity of transcription, with other factors (16) conferring initiator specificity on the “core” RNA polymerase. The second possibility seems attractive for the following reasons : (a) multiple forms of many enzymes have been described, particularly in eukaryotic cells; (b) the ribosomal genes are apparently regulated independently (17,18) and are usually localized within the nucleolus (19-22). Thus, the RNA polymerase responsible for the synthesis of rRNA probably functions within the nucleolar structure and the enz y m e ( ~ )involved in the synthesis of nonribosomal RNA species is active within the extranucleolar compartment (nucleoplasm) of the nucleus. Furthermore, several physiological factors such as hormones have been shown to stimulate specifically the Mg2+-dependentpolymerization reaction (1); it appears that a t least two polymerases are present in the nucleus. It is evident that the possibility of the existence of multiple forms of RNA polymerase in eukaryotes cannot be tested with crude aggregates of RNA polymerase systems in which the enzyme is associated with the deoxyribonucleoprotein template. Consequently, the role of RNA polymerase in the control of genetic expression in mammalian cells has not been clearly understood. Such a regulatory role of RNA polymerase can be adequately evaluated only by analyzing purified enzyme preparations with exogenous templates. The recent success of several investigators in obtaining RNA polym-

+

+

MAMMALIAN

RNA

POLYMERASES

95

erase in good yields with high degrees of purity from a variety of eukaryotic cells has resolved many such problems and has thus paved the way to a better understanding of the mechanism(s) of RNA synthesis in higher organisms. This article deals with the recent developments in the field of mammalian nuclear RNA polymerases. I have also attempted to speculate and advance my own interpretations of the available information on this subject.

II. Quantitative Extraction of RNA Polymerase from Mammalian Cells

A. Methods of Solubilization Attempts have been made to release RNA polymerase free of DNA from chick embryo (as), bovine lymphosarcoma ( d 4 ) , rat testes (d5), corn seedlings (96), HeLa cells (97)and rat liver (98-32). I n all these instances, the amounts of soluble enzyme recovered were either low or not reported a t all. The instability of the enzyme following solubilization was primarily responsible for failure in further purifications. Consequently, these procedures have not furnished the exact amount or all the types of RNA polymerase present in a tissue. Recently, several groups of investigators have succeeded in extracting the enzyme from a variety of animal tissues in quantities that are sufficient for further purification and analysis. Thus RNA polymerase has been solubilized quantitatively from liver tissue of the rat (33-.@), Xenopus Zuevis (47) and the monkey (@), from calf thymus (39, 49-51), sea urchin embryos (39,52),embryos (47) and ovaries (53) of Xenopus Zaevis, mouse embryo cells ( 5 4 ) ,HeLa cells (55), KB cells (48), bovine lymphoid tissue (56) and rat ventral prostate gland ( 5 7 ) . The various methods employed for the quantitative extraction of RNA polymerase from mammalian tissues are outlined in Table I and are described below with the references to the original procedure. They can be broadly classified into three categories: ( a ) mild treatment, (b) moderately drastic (strong) treatment, and (c) drastic treatment of the tissue or the cell nuclei. The solubilization of RNA polymerase by mild treatment can be achieved by one of the following procedures: (i) suspending the cells in buffer for various lengths of times a t 4' (54); (ii) homogenizing the tissue in a slightly alkaline buffer (41, 5 1 ) ; (iii) grinding the tissue in a Waring Blendor using a suitable buffer (56); and (iv) incubating the nuclei a t 37" in Tris buffer with pH in the range of 7.9-9.1 (33, 43). The moderately drastic treatment consists of brief sonication in a medium of low ionic strength (34, 35). The more drastic treatment in-

TABLE I SOLUBILIZATION AND CHROMATOGRAPHIC SEPARATION OF MAMMALIAN RNA POLYMERASES

Source of enzyme

Outline of the procedure for the extraction of enzyme(s)

Chromatographic elution properties of the enzymes"

Number of enzymes resolved

W

Q,

References

Rat Liver (a) Isolated nuclei

Incubation at 37"for 30 min in alkaline Fractionation on DEAESephadex with buffer (pH 8.9-9.1), followed by cf.; (NH&SO4 IA (0-0.03M) enzyme usually ppted with IB (0.12 M ) (NH4)eSOr and dialyzed.

4 (possibly 5)

SS, 56

2

S5,36

Fractionation on DEAESephadex with I (0.15 M) I1 (0.25 M) I11 (0.30 M)

3

58

Fractionation on DEAESephadex with (NHI)&OI I (0.15 M) Negligible I1 and I11 Fractionation on DEAESephadex with (N&)&O4 I1 (0.25 M) I11 (0.30M)

1

38

2

58

'IA (0.20-0.22 M)

(b) Isolated nucleoli Brief sonimtion, followed by incub% tion in alkaline buffer. Not ppted with salt.

IIB I11 (0.30 M) Fractionation on DEAESephadex or phosphocellulosewith (NH4)&04. Two enzymes were resolved IA (0-0.03M) IB (0.15 M)

Rut LioW (a) Isolated nuclei

Sonication for 1 min in presence of 0.3 M (NH4)&04, PH 7.9, followed by reduction of ionic strength and cf.; enzyme ppted with (NH4)&04 and dialyzed. (b) Isolated nucleoli Same as above

( c ) Isolated nucleoplasm

Same as above

4

P

c)

i?

Rat Liver Isolated nuclei

(a) Incubation a t 37" for 1 hour with Fractionation on DEAE-cellulose with KC1; I (0.23 M)-nucleolar enzyme. buffer containing 0.07 M KCl followed by cf. Enzyme not ppted with Bechromatography of I on phosphosalt. cellulose with KCl: IA (0.4-0.55 M) IB (0.6-0.70 M) (b) Sonication in presence of 0.3 M Fractionation on DEAESephadex with (NH4)&04, pH 7.9, followed by re(NHd804 B (0.35 M) duction of ionic strength and cf. EnRechromatography of B on phosphozyme ppted with (NH4)804and dialyzed. cellulose with (NH~)&OI B (0.25 M)

2

42

1

44

23

5

Total 3

Rut Liver Isolated nuclei

Sonication in presence of 0.75 M NaC1, Fractionation on DEAE-cellulose with stirring in the cold for 1.5 hours, reNH4Cl duction of ionic strength and cf. EnI (0.10M) I1 (0.28 M) zyme not ppted with salt.

2

40

Sonication for 7 min in presence of Fractionated on DEAE-cellulose with 0.48 M (NH,)804 followed by cf. ; (N&)&Ot followed by salt pption, reduction of A (0.12 M) ionic strength and removal of DNA B (0.30 M) with protamine sulfate. The enzyme finally ppted with (NH4)&04 and dialyzed.

2

46

Incubation in 0.32 M sucrose, pH 8.0 Fractionated on DEAE-Sephadex with at 0" for 8-15 hours, followed by cf., (N&)&O4 I (0.15 M) (NH&!% pption and dialysis I1 (0.25 M)

2

48

Rat liver Isolated nuclei

Rut lwer/African green monkeg liver Crude nuclei (700 g pellet)

CD

w

(Continued)

TABLE I (Continued)

Source of enzyme xenopus .?a& liver Whole tissue

Calf thymus Isolated nuclei

Calf thymus Isolated nuclei

Outline of the procedure for the extraction of enzyme(s)

Chromatographic elution properties of the enzymesa

Sonication in presence of 0.3 M (NH&~OI, pH 7.9, reduction of ionic strength and cf.; ppted with salt and dialyzed.

Fractionated on DEAESephadex with (NH~)&OI I (0.15 M) I1 (0.25M)

Fractionation on DEAE-cellulose with Sonication for 5-7 min in presence of 0.48 M (NH,)&o,, followed by d., (NH~)&OI,after the enzyme was initially adsorbed on the column with pption with salt and dialyzed. 0.04 M salt A (0.15 M) A (0.3 M) B (0.30M) Or B (0.25M) with phosphocellulose columns Rechromatography of B on D E A E cellulose, after the enzyme was initially absorbed on the column with 0.08 M salt: IB (0.22M) IIB (0.27 M) Sonication in presence of 0.3 M (NH4)2SO4, (PH 7.9), followed by reduction of ionic strength and cf.; ppted with salt and dialyzed.

Fractionation on DEAESephadex with (NHI)&OI I (0.15 M) I1 (0.27 M)

Number of enzymes resolved

2

References

47

Total 3

2

39 4

8

Calf thymus Whole tissue

Calf thymus Whole tissue

Sea urchin embryos Isolated nuclei

4 (possibly 5 )

Homogenizationof the tissue at pH 7.8, Fractionation on DEAEeellulose with KC1 addition of 0.1 M (NH4)&304 to reI (0.25 M ) move DNA and final pption with I1 (0.35 M) salt, followed by dialysis.

2

51

Sonication for 1 min in presence of Fractionation on DEAE-Sephadex.with 0.3 M (NHi)i?IO4, (PH 7.9),followed (NHi)i?IOd by reduction of ionic strength and I (0.15 M) cf.; ppted with (NH&?~OIand I1 (0.22 M) dialyzed. I11 (0.30M)

3

38

Fractionation on DEAE-Sephdex with (NHd804 I (0.12M) I1 (0.23 M) I11 (0.30M)

3

47

Sonication of crude chromatin prepara- Fractionation on DEAE-cellulose with tion in buffer containing 0.75 M KCl, KCl reduction of ionic strength and cf. I (0.15M) I1 (0.30M)

2

53

Xenopus lawis embryos Whole cells Same as above

z

xenspvs laavis ooytes

(a) Whole cella

56

Tissue ground with buffer in blender, Fractionation on DEAESephadex with cf. and ppted with protamine sulfate (NH4)SOt I (0.05 M) and ammonium sulfate. I1 (0.10M) I11 (0.13 M) IV (0.34 M)

(Continued)

TABLE I (Continued)

Source of enzyme

Outline of the procedure for the extraction of enzyme(s)

(b) Isolated nucleoli Same as above

HeLa cells Crude nuclei

Chromatographic elution properties of the enzymes" Fractionation on DEAE-cellulose with KC1 I (0.15 M)

+

Number of enzymes resolved 1

0 0

References 63

66

Incubation in hypotonic solution con- Fractionation on phosphocellulosewith taining 0.01 M (NH~)&OI and 0.5% (N&)801 Triton-X for 60 min at 4", cf. I (0.20 M), nucleolar enzyme

K B cells Whole cells

L ymphosarcoma Whole tissue

Incubation in 0.32 M sucrose (pH 7.9) Fractionation on DEAE-cellulose with a t 0" for 8-15 hours, followed by cf. KCl and (NH4)&04 pption and dialysis. I (0.25 M) I1 (0.35M)

2

Tissue ground with buffer in blender, cf., the enzyme ppted with (NH,)&O( and dialyzed.

3

Fractionation on DEAESephadex with (NHd80r I1 (0.10M) I11 (0.13 M)

J Y

(0.34

66

M) u,

+-

Rat ventral prostate gland Isolated nuclei

Sonication in presence of 0.3 M Fractionation on DEAE-cellulose with (NH4)&04, (PH 7.9), followed by KC1 reduction of ionic strength and cf.; I (0.3 M) ppted with (NH4)&04 and dialyzed. I1 (0.65 M)

2

67

a Nomenclature'as used by the investigators. The figures in parentheses indicate the molarity of the salt required for the elution of the enzymes.

!

Z

5 4

+-

8

MAMMALIAN

RNA

POLYMERASES

101

volves sonication in a medium of high ionic strength (37, 58, 60). The choice of a procedure depends largely on the type of tissue or cells from which the enzyme is extracted.

B. Recovery of the Enzyme The recovery of the enzyme in the initial extract is of primary importance. It is usually difficult to quantitate the yield of the enzyme after solubilization, since the solubilized enzyme is assayed with exogenous DNA as the template, whereas the aggregate enzyme uses its own chromosomal template in which much of the DNA is not available for transcription. If the polymerase is present in sufficient quantities in intact nuclei with DNA in the chromatin matrix as the limiting factor, addition of exogenous DNA to the solubilized enzyme preparations is likely to produce a dramatic rise in the enzyme activity. In most cases, this factor has been neglected in estimating the recovery of the enzyme after solubilization. Consequently, the yield of the enzyme in the initial extract has frequently been overestimated. This error can be considerably reduced if the difference in the activity of the nuclei before and after extraction of the enzyme is taken as an index of the recovery of the extracted enzyme (33). The recovery of the enzyme solubilized from rat liver nuclei by incubation in an alkaline medium, as measured by the latter procedure, is usually over 80%.

111. Multiplicity, Nomenclature and Intranuclear localization A. Separation of Multiple Forms of RNA Polymerase by Column Chromatography The chromatography of the enzyme solubilized from a variety of animal tissues on DEAE-cellulose or DEAE-Sephadex has revealed multiple forms of RNA polymerase (Table I).The multiplicity of RNA polymerase has also been observed in lower eukaryotes, such as an aquatic fungus (58), yeast (59)and maize (60).The enzymes are usually eluted from the column using a linear gradient of (NH,)*S04or KC1. Four discrete enzymes can be eluted from the column a t 0-0.05 M, 0.10-0.15 M, 0.200.30 M and 0.30 M-0.37 M ammonium sulfate (Table I). The enzymes of rat ventral prostate gland have been eluted from the column with higher than usual salt concentrations (67).

B. Nomenclature The nomenclature of mammalian RNA polymerases has not been uniform because different criteria have been adopted by various investi-

102

SAMSON T. JACOB

gators in classifying the enzymes. The three enzymes separated chromatographically between 0.10 and 0.37M salt concentrations are referred to as I, I1 and I11 in the order of their elution from the column (38, 6 1 ) . The enzymes I and 11,the major species of RNA polymerases detected in most tissues examined, are also designated by A and B, respectively (36, 50). A typical chromatographic profile of RNA polymerase solubilized from rat liver nuclei appears in Fig. 1. The recent suggestion by Chambon and his colleagues (45) to use the differential sensitivity of the RNA polymerases to a-amanitin as the only criterion for naming the different enzymes has neglected the large amount of other information available, such as cell localization and ionic requirements (see the section on the properties of RNA polymerases). On the other hand, a nomenclature based solely on the order of chromatographic elution of the enzymes presents some problems as it is possible to detect additional enzymes under proper conditions. Ideally, the enzymes within a given transcription system, such as nucleus, chloroplast and mitochondrion, should be grouped together: e.g., nuclear RNA polymerase I, 11, etc.; mitochondria1 polymerase I, 11, etc.; and so on. Since the isolation of enzymatically active nucleoli from many cells or tissues is extremely difficult, it is not always feasible to localize the nuclear polymerases further within nucleolar and nucleoplasmic regions, as has been achieved with the enzymes from rat liver. If the enzymes can be subsequently resolved into additional subspecies, the latter can be designated as A, B, m

'P

FRACTIONS (ML)

FIG. 1. Column chromatographic profile of RNA polymerase solubilized from rat liver nuclei (also see 36 and 61).

MAMMALIAN

RNA

POLYMERASES

103

C, etc., preceded by the number of the parent enzyme-for example, IA, IB, IC, etc. The subunits of the mammalian enzymes can be designated as a, p, y, etc. (subunits of I as Ia, IF, Iy, etc.; subunits of I1 as IIa, IIp, I I y , and so on), as in the case of bacterial RNA polymerase. The nucleolar enzyme eluted in the wash (0-0.05M salt) has been called V , (“void volume peak”) ( 3 6 ) .Since the only other enzyme found in the cell nucleolus a t this time was I, V , was redesignated as IA ( 6 1 ) . Recently, the enzyme I has been further resolved on a phosphocellulose column into two peaks of activity, IA and IB, eluted a t 0.48 and 0.65 M KCl, respectively (43). Consequently, two of the enzymes have the same terminology (IA) and hence the enzyme V,, separated from enzyme I as a distinct peak during the first fractionation on a DEAE-cellulose or DEAE-Sephadex column, is reclassified as enzyme IV. Thus the original nucleolar enzyme I is a mixture of enzymes IA and IB, and enzyme IV is an additional nucleolar RNA polymerase. Enzyme I1 has also been further resolved chromatographically into two additional enzymes, IIA and I I B (36, 45, 46).

C. Nuclear localization Enzyme I has been established to be of nucleolar origin (36, 38). Recently, it was demonstrated that enzyme IV is also of nucleolar origin (61). Enzymes IA and IB (43) are probably localized in the nucleolus since they are resolved from enzyme I of the nucleolus. Enzymes I1 (IIA I I B) and I11 (38) are of nucleoplasmic origin. Thus a t least in the case of rat liver, a total of six distinct forms of RNA polymerase have been separated by column chromatography, out of which three are localized in the nucleolus, the remaining three being in the nucleoplasm.

+

D. Relative Concentrations and Specific Activity It is not feasible to make an accurate estimate of the relative concentrations of different polymerases, as different procedures have been employed for the extraction of RNA polymerases from the same tissue. Furthermore, considerable variations in the stability of the enzymes, particularly the nucleolar polymerases, have been reported. Generally speaking, in almost all normal tissues, the amount of enzyme I appears to be less than that of enzyme 11. In rat liver, enzymes I11 and I V are present in equal proportions and are slightly lower than enzyme I ( 3 6 ) .Enzymes I11 and IV have not been reported to exist in tissues other than liver and sea urchin. The ratio of enzyme IIA to enzyme I I B is much higher in rat liver than in calf thymus (4.6). Whether this difference is a characteristic of the organs or results from selective loss of enzymes during the purification is presently unknown. The highest specific activity reported for a

TABLE I1 NUCLEAR LOCALIZATION, NOMENCLATURE AND PROPERTIES OF RNA POLYMERASES

Source of enzyme

Nuclear localization

Properties of the enzymes sugNomen- gested Sensitivity Optimal clature nomento Activity with Mn*+ (NH,)~o, used clature a-amanitin Activity with Mg*+ (M)

Template requirement

Rat liver Isolated nuclei and Nucleolus nucleoli Nucleolus

IA IB

IV

I

2.0

0.03 0.04

IIA IIB I11

8.0

0.06

Nucleoplasm

IIA IIB I11

Isolated nuclei and Nucleolus nucleoli Nucleoplasm Nucleoplasm

I I1 I11

I I1 I11

2

A IB IIB

I IIA IIB

IA IB B

IA

I I1

I I1

Nucleoplasm

-

Native nucleolar DNA most effective Denatured DNA

0.1-0.2

Rut liver

Rut liver Isolated nuclei

Rut liver Isolated nuclei

IB

5

1.1

0. 7

0.07 0.12

Native or denatured DNA Denatured DNA

0.025 0.025

I1

Rat liver Isolated nuclei

Native DNA Denatured DNA

References

Calf thymus Whole t.issue

A IB IIB

Xenopus laevis Liver Xenopus laevis Embryos Whole tissue

Native DNA Native DNA

Nucleolus

I I1 I11

I I1 I11

10 2.5

0-0.2

I

I1

I I1

3 7

0.W

I I1 I11

I I1 I11

I I1

I I1

HeLa cells Isolated nuclei Mouse embryos Whole cells

Rat ventral Nucleolus Prostate gland Isolated nuclei and Nucleoplasm nucleoli a

0.04O

0.10 0.10

46960

Pz

l-z

9

Sea urchin Embryos Isolated nuclei

Xenopus laevis oocytes Whole cells or nucleoli

I IIA IIB

0.016 in presence of M@+.

1

I I1

I I1

I I1

0.01-0. OF

47

c

5 cd 0

5

E

47

E

m

(I)

65

I I1 I I1

58, 76

0.04 0.09

Native DNA

66

54

+

2.4

57

3.3

* Determined in presence of Mnz+;in presence of M@+, the salt optima were 0.01 and 0.06 for enzymes I and 11, respectively.

F

0

cn

106

4

SAMSON T. JACOB

mammalian RNA polymerase is 300-600 units per milligram of protein [ l unit of activity is defined as nanomoles of nucleotide incorporated per 10 minutes a t 30°C (50)1. Recently, Weaver et al. (62)obtained polymerase I1 preparations from calf thymus and rat liver with specific activities ranging from 400 to 1000, which are similar to the specific activity reported by Burgess (63,64) for the E . coli polymerase. The selection of a suitable procedure for the estimation of enzyme protein is crucial in assessing the specific activity of RNA polymerase preparations. It is known that Lowry’s procedure for the estimation of proteins is liable to cause errors in the presence of a wide variety of compounds ( 6 5 ) . Glycerol and sucrose (66), tris (hydroxymethyl)aminomethane (67) and sulfhydryl compounds (for example, dithiothreitol and glutathione) interfere with the color produced in the Lowry reaction (66). I n general, protein precipitapts such as trichloroacetic or perchloric acids do not affect Lowry’s procedure, and consequently Bennett (68) has recommended a modification of Lowry’s procedure in which the interfering materials are first removed by acid precipitation. I n my laboratory, this procedure has been routinely and successfully used for the estimation of enzyme proteins. The large amount of tissue frequently used in the extraction of RNA polymerase yields enzyme in acid-precipitable quantities at the final stage of purification.

IV. Properties of RNA Polymerases The properties of the polymerases are summarized in Table 11.

A. Stability The nucleolar RNA polymerases appear to be less stable than the nucleoplasmic enzymes, particularly in the presence of salt (36, 4 3 ) . For this reason, it has been recommended that salt be avoided in the extraction and purification of the nucleolar enzymes. I n fact, the elimination of salt from the purification process results in higher yield of these enzymes (36, 42-44). Furthermore, salt has also been shown to induce alterations in the sedimentation properties of nucleolar enzyme I (50). The stability of all the enzymes can be considerably enhanced by the presence of large amounts of bovine serum albumin and 35% glycerol, and the nucleolar enzymes can even be stored for several weeks in this medium at -90°C without losing appreciable activity.

B. M e t a l ion a n d Ionic Strength Optima The polymerases exhibit different optima for divalent metal ions and (NH,) ?SO,. The nucleolar enzymes show their optimal activities a t low

MAMMALIAN

RNA

POLYMERASES

107

ionic strength and utilize MnZCand Mg2+equally well. On the other hand, higher Mn2+concentrations and ionic strengths are required for optimal activity of the nucleoplasmic enzymes. The enzyme from rat ventral prostate gland shows unusually large requirements of Mgz+and Mn2+for enzyme 11. Although the crude and purified nuclear enzymes have identical MgZf optima they differ markedly in their salt requirements. Thus, purified enzymes require considerably lower concentration of salt than the 0.4M (NH,),SO, that is usually employed for the analysis of the enzyme activities in isolated nuclei. Studies on the solubilized enzymes (33, 69, 70) indicate that the most significant effect of salt is on the enzyme. An identical observation is that KCl (0.2M) activates bacterial RNA polymcrasc in the presence of native DNA (1). Maitra and Barash (15) have shown that the function of salt in the bacterial RNA polymerase reaction is to detach the newly formed RNA from the enzyme and consequently to allow reinitiation of RNA chains. It is possible that salt can cause similar effects on mammalian polymerase(s) as well. The possibility that high concentrations of salt can, in addition, stimulate the chromntiii activity in isolated nuclei or nucleoli by detaching the proteins that mask the template DNA cannot be ruled out at present.

C. Sensitivity to a-Amanitin in Vifro Stirpe and Fiume (71)first observed that the activity of RNA polymerase in isolated nuclei under high ionic conditions (Mn2+and ammonium sulfate) can be inhibited by a-amanitin, a toxin isolated from the poisonous mushroom, Ainanita phalloides (72, 7 3 ) . Its selective effect on polymerase activity in the presence of Mn2+differs sharply from the effect of inhibitors that act on the DNA template, such as actinomycin ( 4 ) and aflatoxin ( 7 4 ) ,to which the Mg'f-activated enzyme is usually more sensitive. These observations suggested that a-amanitin might act on RNA polymerase itself. Experiments to test this possibility grove that this is indeed the case ( 7 5 ) . Subsequent studies in my laboratory (34) and elsewhere (50, 76, 77) have demonstrated that the toxin selectively inhibits the nucleoplasmic enzymes a t a concentration as low as 0.03 pg/ml (3 X 10-sM) whereas the nucleolar enzymes are not affected even a t inucli higher doses. In addition to the nucleolar RNA polymerases, the minor nucleoplasmic enzyme I11 does not appear to be inhibited by a-amanitin ( 7 6 ) . a-Amanitin is as specific for the eukaryotic enzyme (75) as rifamycin or rifampicin is specific for the prokaryotic enzyme (75, 78-80). Rifamycin has no significant effect on animal nuclear RNA polymerases (33, 39, 50, 7 8 ) . Unlike most inhibitors of RNA synthesis (81-90), a-amanitin inhibits RNA synthesis by binding to RNA polymerase (50, 75) rather

108

SAMSON T. JACOB

than to the DNA template. An analysis of the stoichiometry of the reaction by plotting initial velocity against enzyme concentration in the presence of different concentrations of the inhibitor ( 9 1 ) , shows that one molecule of amanitin inhibits one molecule of enzyme I1 ( 5 0 ) . Direct evidence for binding of the toxin to the enzyme has been obtained by cosedimentation of enzyme and [ methy2-14C]a-amanitin in glycerol gradients ( 9 2 ) . Further investigations on the mode of action of the toxin reveal that it inhibits RNA synthesis after the initiation step, presumably a t the level of chain elongation (75, 77, 9 3 ) . The mechanism of action of n-amanitin thus differs from that of rifampicin, which inhibits bacterial RNA synthesis by preventing chain initiation (80), but resembles that of another bacterial RNA polymerase inhibitor, streptolydigin that appears to inhibit chain elongation ( 9 4 ) . However, streptolydigin, in very high concentrations, also inhibits both the nucleolar and nucleoplasmic enzymes from calf thymus ( 5 0 ) . The overall conclusions from experiments using a-amanitin in vitro are (a) that a-amanitin reacts with the enzyme itself, not with the DNA template, (b) that it does not interfere with initiation of RNA synthesis and (c) that it is specific for eukaryotic RNA polymerase.

D. Effect of a-Amanitin in Vivo Experiments using a-amanitin in vitro strongly indicate that the toxin can inhibit specifically the synthesis of mRNA in vivo without affecting ribosomal RNA synthesis in the nucleolus. If this is indeed the case, a-amanitin will then become a useful tool to study ( a ) the turnover of mRNA in higher organisms and (b) the transcriptional control of biochemical processes. In order to prove the possible differential effects in vivo, the incorporation of [14C]orotic acid into nucleolar and extranucleolar (nucleoplasmic) RNA following injection of a-amanitin to rats has been examined ( 9 5 ) . Contrary to expectations, the synthesis of ribosomal as well as nonribosomal RNA is significantly inhibited within an hour of amanitin treatment (95, 9 6 ) . The inhibition of rRNA synthesis has been further confirmed (95) by (a) the disappearance of the nucleolar 45 S RNA optical density peak from the sucrose density gradient profile of nuclear RNA and (b) a sharp decrease in the methylation of nuclear RNA with [~nethyl-'~C] methionine, which under these conditions methylates mainly rRNA, made in the nucleolus. The effect of amanitin on the rRNA synthesis is only transient and the inhibition disappears completely within 2 or 3 hours. However, the nonribosomal RNA synthesis remains severely depressed over long periods. The finding that rRNA synthesis is transiently inhibited after injection of the toxin into intact animals is interesting, However, it agrees

MAMMALIAN

RNA

POLYMERASES

109

with the early results obtained by electron microscopy (97), that. treatment of mice with amanitin in vivo causes rapid fragmentation of the nucleolus, implying that the nucleolus is susceptible to amanitin toxicity. Since rRNA synthesis is inhibited in vivo, it is important to know whether active nucleolar polymerase is lost from the nucleus after administration of the inhibitor. Treatment with a-amanitin in vivo usually results in extensive loss of activity of the nucleolar polymerases, and the recovery of the nucleolar enzyme activity is not as fast as that of the labeling of nucleolar RNA in vivo. One complicating factor involved in studies on the purified RNA polymerase obtained after amanitin treatment in vivo, is that the procedures for the solubilization and purification by chromatography may remove amanitin or an inhibitory metabolite from the enzyme and thus occasionally result in a higher activity than that in the intact cell ( 3 6 ) . Several explanations can be given for the inhibition of rRNA synthesis by amanitin in vivo. (a) It is possible that amanitin is converted to a toxic metabolite(s) that in turn inhibits the activity of nucleolar as well as extranucleolar polymerase. (b) It could be that the function of the nucleolar RNA polymerase is regulated by some extranucleolar factor sensitive to amanitin. (c) mRNA synthesized in the extranucleolar compartment may be needed for the synthesis of a rapidly renewed protein factor involved in the nucleolar polymerase function. Although all classes of RNA in rat liver are inhibited by treatment with amanitin in vivo there are reports that in some species, such as mouse (71) and Xenopus Eaevis embryos ( 5 3 ) , the ribosomal RNA-synthesizing machinery in the nucleolar compartment is unaffected by amanitin in vivo. The reason(s) for the difference in the response of the latter species is not very clear. It is possible that amanitin can be converted into a functional metabolitc (as suggested in the above paragraphs) by the active drug-metabolizing enzymes present in rat liver microsomes and that this derivative of a-amanitin affects the nucleolar RNA polymerase as well. Experiments to test this hypothesis have not been performed. It is known that actinomycin D, at doses commonly used for inhibition of mRNA synthesis in animal cells, not only produces cytotoxic effects in the liver, but also degrades newly synthesized RNA (98-100). It is thus evident that actinomycin D cannot be employed satisfactorily as a specific inhibitor of RNA synthesis, especially of mRNA synthesis. Unlike actinomycin D, a-amanitin can inhibit all RNA synthesis in vivo, when used a t very low doses. Consequently it should be possible to examine the requirement of de novo synthesis of RNA and specifically of mRNA for a biochemical process (if a proper time period such as 3 hours

110

SAMSON T. JACOB

after amanitin treatment is selected, when only extranucleolar RNA synthesis remains inhibited). Thus it has been demonstrated that the induction of tyrosine transaminase in rat liver by cortisol (101) or by cyclic AMP (109)is dependent on the de novo synthesis of RNA.

E. Template Specificity Most investigators have used calf-thymus DNA as template for purified enzymes. However, a t least for rat liver enzymes, purified liver nuclear DNA has been shown to be a better template than calf thymus DNA (35). Generally speaking, nucleolar polymerases act preferentially on native DNA whereas the nucleoplasmic enzymes utilize denatured DNA more efficiently. However, since neither polymerase I nor polymerase I1 of Xenopus laevis shows any striking preference for native DNA as opposed to denatured DNA, the specificity of transcription may require some other factor(s) that interacts with the enzyme or the DNA or both ( 4 7 ) .The nucleolar RNA polymerase of rat liver transcribes native nucleolar DNA fifteen times more efficiently than whole nuclear DNA. The activity of the nucleoplasmic enzymes is increased only slightly by substituting nuclear DNA with native or denatured nucleolar DNA. The higher activity of nucleolar DNA with the nucleolar enzyme is not due to fragmentation or single-strand breaks in the DNA (S. T. Jacob, unpublished observations). The nucleolar enzyme from Xenopus laevis embryos also acts preferentially on ribosomal DNA rather than on the bulk nuclear DNA ( 4 7 ) ,provided the salt concentration is above 0.05 M. However, no significant difference in the rates of transcription of the two templates has been observed at low salt concentrations. The only means to study the template specificity of purified RNA polymerase is to use DNA of defined structure in vitro. In studies performed so far, either polynucleotides or viral DNA have been used. Studies with synthetic polynucleotides have shown that templates composed of pyrimidine nucleotides are read far more readily by both nucleolar and nucleoplasmic enzymes than those made of purine nucleotides. This is true for both single and double-stranded templates (39). Polymerase I1 reads the dC, strand of dI,.dC, over thirteen times more rapidly than it does calf thymus DNA (39). These enzymes, however, differ in the rate of reading dT, which is transcribed by nucleolar enzyme I but not by nucleoplasmic enzyme I1 (39). Double-stranded DNA of adenovirus 2 with a mass of 23 X los daltons (103) and the circular, superhelical double-stranded DNA of SV40 virus (104) with a mass of 3 X lo6 daltons, have been used (48). It has been concluded that similar base sequences of these DNA’s are transcribed asymmetrically by both nucleolar and nucleoplasmic enzymes.

MAMMALIAN

RNA

POLYMERASES

111

The T 4 DNA is a poor template for all the enzyme fractions (39, 60) even in the presence of sigma factor, which dramatically stimulates the activity of the core E . coli polymerase (63). The enzymes from calf thymus transcribe twisted circular DNA (Py I) and open circular DNA (Py 11) from polyoma virus very poorly, whereas mouse DNA (Py 111) from the pseudovirions, at least a t nonsaturating levels, appears to be a better template than calf thymus DNA for the mammalian enzymes ( 5 0 ) . One possible explanation for the latter observation is that the relatively small Py I11 DNA offers more ends where initiation could occur than native calf-thymus DNA. However, the specificity of transcription, as evaluated by studies with synthetic or viral templates, may not be similar to that expressed in the cell. It should be pointed out that the RNA formed by “naked” DNA does not resemble qualitatively or quantitatively the natural RNA in the cell (106).The histones, specific chromosomal RNA, and acidic proteins of the chromatin possess some role in the regulation of transcription (105109). The experiments of Gilmour and Paul (106) clearly demonstrate that only the RNA transcribed from nucleoprotein that is reconstituted from the components of the chromatin is similar to natural RNA. It is thus clear that the specificity of RNA polymerase in the transcriptional process can be evaluated only by analyzing the product of the enzymes formed with native chromatin preparations as the template.

F. Factor Requirements Factors influencing the RNA polymerase of bacterial (63, 110, 111) and other microbial sources (112) play an important role in the positive control of gene transcription. It is tempting to assume that such a control mechanism exists also in higher organisms. Since bacterial sigma factor has no demonstrable stimulatory effect on the mammalian enzymes (50, 55) when added in vitro, it was deemed desirable to look for mammalianspecific factors that could regulate the transcript‘onel process. Two groups of investigators (40, 51, 113) have recently identified a factor that can stimulate the activity of nucleoplasmic enzymes obtained from rat liver and calf thymus. The factor in the enzyme preparations from liver appears to be present in the cytoplasm. Both the factors have been partially purified and a 100-fold purification of the one from liver has been achieved. The factor is a protein with a sedimentation coefficient of 3 3 . 5 s and a molecular weight of approximately 70,000. The specific stimulation of the nucleoplasmic enzyme I1 can be observed only in the presence of native double-stranded DNA. The preferential transcription of denatured DNA by the nucleoplasmic polymerase in the absence of the factor, implies that this enzyme is complete only with its associated

112

SAMSON T. JACOB

factor. The observed stimulation is not due to the action of a nuclease that can cause single-strand breaks in native DNA and consequently expose more initiation sites on the DNA. There seems to be a specific recognition between the enzyme and the factor as is suggested by the fact that the factor binds to the cnzyme. Since there is no simple stoichiometric relationship between factor bound to polymerase and its stimulatory effect on the enzyme, a catalytic action of the factor is suggested. These results are thus consistent with the effects of sigma factor on bacterial RNA polymerase. However, the question, whether or not the factor can promote initiation of RNA synthesis a t specific sites of DNA cannot be answered a t present. The possibility that the factor interacts with the enzyme or enzyme-template complex, changing its transcriptional properties, cannot be ruled out. The mechanism of action of the factor thus remains unanswered. It is interesting to note that no such factor for the nucleolar enzyme has yet been isolated. It should, however, be pointed out that microinjection of E . coli factor into Xenopus laevis oocytes results in a significant stimulation of both nucleolar and nucleoplasmic polymerases ( 5 3 ) . The absence of a stimulating effect when sigma is added in vitro may suggest that it interacts with endogenous “phosphocellulose-like” components (63) to initiate RNA synthesis.

V. Subunit Structure and Molecular Weight The most reliable information on the molecular structure of RNA polymerases has been obtained with the two a-amanitin-sensitive nucleoplasmic enzymes, arbitrarily named IIA and I I B (4446” 5 0 ) . Success in the elucidation of the structure of these enzymes came from their stability during the purification process, as compared to the relative instability of the nucleolar enzymes. I n the initial studies, polyacrylamide gel electrophoresis (114) of the polymerases suggested the presence of three major subunits with molecular weights of 215,000, 185,000 and 150,000 for calf thymus enzymes (50) and 200,000, 180,000 and 160,000 for the enzymes of rat liver (44).The molecular weights of the components present in the bands were determined by comparison of their mobilities with those of marker proteins by the procedure of Shapiro et al. (115). Recently Weaver et al. (66) succeeded in analyzing the minor components of the nucleoplasmic enzymes. These studies have revealed that the nucleoplasmic enzymes consist of four distinct subunits each, and thus the two forms of enzyme I1 of rat liver possess molecular structures of [ (190,OOO) (150,000) (35,000) (25,000) and [ (170,000) (150,OOO) (35,000) The corresponding enzymes from calf thymus have similar (25,000) subunit compositions; the enzyme with the subunit structure (190,000)

MAMMALIAN

RNA

113

POLYMERASES

(150,000) (35,000) (25,OOO) is the most predominant nucleoplasmic enzyme in this tissue. However, since the proportion of the enzyme containing the 170,000 subunit can be increased by addition of a proteolytic inhibitor during the isolation of the enzyme or “aging” the enzyme a t 4°C in the crude state, the 170,000 subunit may be derived from the 190,000 subunit (6.2). Whether both enzymes are present in rat liver nuclei or one is formed by degradation of the 190,000 subunit to the 170,000 subunit during the isolation is not yet clear. However, the production of two distinct polymerase species by specific proteolysis is a possibility ( 6 2 ) . It is interesting to point out that Leighton et al. (116) have recently observed a specific proteolytic modification of RNA polymerase during sporulation of Bacillus subtilk However, these studies do not rule out the possibility that the two subunits may be products of separate genes. Using highly purified preparations of enzymes IIA and IIB, Kedinger and Chhmbon (6%) established their subunit compositions as (214,000) (140,000)1 (34,000)1ol’ (25,000)~(16,500)a and (180,000)1 (140,000)1 (34,000) or (25,000), (165,000) 4, respectively. The subunit with molecular weight 16,500 has not been detected by Weaver et al. (62). There is also some discrepancy in the molar ratios of the smaller subunits. However, there is a general agreement on the size and molar proportion of the larger subunits. Kedinger and Chambon (62a) could not demonstrate any significant conversion of form IIA to form IIB by “aging” a t room temperature for a t least two hours. Whether such a conversion by a specific in vivo proteolysis is possible remains to be clarified. Sedimentation rates of 14.5-15.5 S have been obtained for calf thymus and rat liver enzyme 11. Minimum molecular weights estimated from these values, on the assumption of a globular shape, are about 350,000400,OOO (6.2), which compares well with the 495,000 reported for E . coli RNA polymerase (1).On the basis of densitometry analysis and sedimentation characteristics, a molecular weight of 500,000-700,OOO has been suggested by previous investigators (&, 5 0 ) . It is interesting to note that the large subunits of a t least one of the nucleoplasmic enzymes resemble the p and p‘ subunits of the E . coli polymerase in their electrophoretic mobility and molecular weight. Whether the functions of the large subunits of animal enzymes are identical to those ascribed to the p and p’ subunits of the E. coli enzyme is not known. I n view of the differential sensitivity of thc E . coli enzyme and the nucleoplasmic enzymes to inhibitors such as rifampicin and a-amanitin, it seems unlikely that the structures of their subunits are identical. This is further supported by the observation that rifampicin binds to the p subunit of the E. coli enzyme (117).Similarly, the smaller subunits of O1.

114

SAMSON T. JACOB

enzyme I1 do not exhibit any resemblance to either the subunits or the factors isolated from the E . coli enzyme. It is tempting to assume a structural relationship between nucleolar enzyme and bacterial RNA polymerase on the basis of their insensitivity to a-amanitin. However, the insensitivity of the nucleolar enzyme to rifampicin as well tends to rule out such a possibility. None of these observations indicate any structural similarity between the eukaryotic RNA polymerases and the prokaryotic “core” RNA polymerase. Since the large subunits of enzyme I differ from those of enzyme 11, an interconversion between these enzymes as proposed by Chesterton and Butterworth (@) is also highly improbable (&).

VI. Regulation af RNA Polymeruses The RNA-synthesizing ability can be regulated either by modifications in the chromatin template or by changes in the activity or levels of RNA polymerases. Qualitative changes in RNA synthesis are induced by hormones (118-1i?8), by partial hepatectomy (169-136) and by nutritional or dietary factors (153-159). I n certain cases, changes in RNA polymerase activity have also been measured. The most significant early changes are observed in the stimulation of ribosomal RNA synthesis in vivo and of the RNA polymerase activity assayed in a medium of low ionic strength. However, these investigations have been carried out with aggregate enzymes in which the polymerase is tightly bound to its chromatin template, Consequently, the actual mechanism (s) by which RNA synthesis is altered has not been elucidated. I n attempts to resolve this problem, animal chromatin preparations have been used with bacterial RNA polymerase to distinguish the effects on the template activity from those on the polymerase. The administration of appropriate hormones in vivo results in a stimulation of chromatin activity in rat liver (140,141), uterine tissue (I@), prostate gland (143) and skeletal muscle (144). Similar results, although less well established, have been reported after in vitro incubation of rat liver nuclei ( 1 4 ) and chromatin (146) with cortisol and uterine chromatin with estradiol (147).The reported in vivo or in vitro stimulation in the activity of isolated chromatin is usually far less significant than that of the whole nuclear preparations. However, it has been widely accepted that the activation of RNA synthesis induced by hormones and other factors is mostly due to the effect on the chromatin. No alternative suggestions have been given to explain the difference in the magnitude of response in the chromatin and in the whole nuclear preparations. Thc discovery of the multiple forms of RNA polymerase in

MAMMALIAN

RNA

POLYMERASES

115

higher organisms that are structurally diffcrciit from prokaryotic enzyme has cast serious doubt on the validity of the experiments involving bacterial polymerase and animal chromatin. Furthermore, since recent experiments (S. T. Jacob, unpublished observations) reveal that the mammalian chromatin is read much more efficiently by the nuclear enzymes than by the bacterial polymerase, a specificity in the recognition of the animal genome by the mammalian enzymes is indicated. Another factor that further complicates the interpretations of the results is the reported diurnal rhythmicity of rat liver chromatin in template activity ( I @ ) . To differentiate the alterations in the template from those in the polymerase, Yu and Fiegelson (149) used actinomycin D to inhibit the template function of endogenous nucleolar DNA and a synthetic template, poly(dC) (which is insensitive to actinomycin D ) , to assess RNA polymerase activity. Although these studies have suggested that the increased RNA synthesis is largely due to a stimulation of the activity of nucleolar RNA polymerase rather than an activation of chromatin, it is not possible to distinguish, under these conditions, the activity of homopolymer-forming enzymes from that of authentic RNA polymerase. From measurements of the number of RNA chains initiated by estradiol as an estimate of the RNA polymerase molecules, Barry and Gorski (150) concluded that this hormonc increases RNA polymerase activity in uterine nuclei by specifically enhancing the activity rather than the absolute amount of the polymerase. Blatti and co-workers (39) and Stirpe (151) have estimated (a) nucleolar polymerase activity by incubating the nuclei in presence of a-amanitin and media of low ionic strength, and (b) the nucleoplasmic enzyme I1 by subtracting the activity in the presence of Mn’+, high salt and amanitin from the activity under the same conditions in the absence of amanitin. Using this procedure, the former group of investigators showed that estradiol and glucocorticoids generally increase the activity of nucleolar enzyme I, whereas partial hepatectomy results in a stimulation of the activity of nucleoplasmic enzyme I1 as well. Experiments with inhibitors of protein synthesis such as cycloheximide and/or puromycin show that either de novo synthesis of a protein factor(s) or of RNA polymerase itself accounts for the enhanced synthesis of ribosomal RNA (a) in uteri after treatment of female rats with estradiol (152-154) ; (b) in liver following partial hepatectomy (155); (c) in liver after refeeding of starved rats with Stock diet (156) or with a meal of tryptophan-containing amino acid mixture (135, 138) ; and (d) in Ehrlich ascites tumor cells upon exposure to a medium enriched in amino acids (139). Since inhibitors such as cycloheximide can almost completely inhibit the RNA polymerase activity in control animals (157)

116

SAMSON T. JACOB

as well, the interpretation of the results obtained with these inhibitors may not be significant. Furthermore, cycloheximide has been shown to have a selective effect on ribosomal RNA synthesis in vivo (157-159) and on nucleolar RNA polymerase activity in vitro (157). All these investigations suggest a role for RNA polymerase(s) in the regulation of RNA synthesis. However, since these studies were performed using aggregate enzyme preparations, the results, although suggestive, are not conclusive. The use of purified RNA polymerases with exogenous templates has simplified this problem considerably. I n one such investigation (61), the effect of a single injection of hydrocortisone into adrenalectomized male animals has been to stimulate selectively the activities of purified nucleolar polymerases. The degree of stimulation of the purified enzymes accounts for the stimulation of the enzyme activity observed in the isolated nucleolar preparations where the enzyme is analyzed with its associated deoxynucleoprotein template. These observations thus clearly indicate a new type of cellular regulation, namely the regulation of RNA polymerase by hormones independently of the template activity. Similar results have been obtained with growth hormone and triiodothyronine (160) and with androgenic hormones ( 5 7 ) . Further studies (Sajdel and Jacob, unpublished observations) indicate that the increased secretion of corticosterone in response to stress can result in a stimulation of the activity of purified nucleolar RNA polymerases. These findings suggest that the enhanced hepatic RNA polymerase activity following in vivo administration of corticosteroids is due to a physiological rather than a pharmacological effect of the hormones. Recent studies ( 1 6 0 ~ show ) that RNA polymerase activities of forms IA, I B and I1 are approximately ninefold, twofold and twofold higher, respectively, in a minimal-deviation rat hepatoma cell line (Reuber H-35) in the liver, again suggesting the possible role of multiple RNA polymerases in the cellular regulation. At least two explanations can be given for the augmentation of the activities of purified RNA polymerases in response to various factors: (a) modification of the enzyme structure (allosteric changes) ; (b) increased de novo synthesis of the enzyme. Recently some attempts have been made to distinguish between these two effects. It has been demonstrated (61) that in order to saturate a fixed excess quantity of exogenous DNA, smaller amounts of RNA polymerase are required from adrenalectomized animals than from cortisol-treated animals and that a t the saturation point, the higher enzyme activity of the cortisol-treated animals still persists. These observations suggest that corticosteroids induce an activation of preexisting enzyme molecules rather than an increased synthesis of RNA polymerase. Using a different technique, Barry

MAMMALIAN

RNA

POLYMERASES

117

and Gorski (150) arrived a t similar conclusions on the action of estradiol. Recent findings (70), that histones can interact directly with the RNA polymerase, suggest that a stimulation of the RNA polymerase activity can be brought about by a dissociation of the polymerase from argininerich histones. It is possible that the complex containing histones and RNA polymerase can be disrupted by chemical modifications of histones such as acetylation (161) or phosphorylation (162). Following the treatment with hormones, the polymerase activity may also be modified by interaction with a hormone-receptor complex or a chemically modified hormone. The properties of the altered RNA polymerase have not yet been examined. The effects of other physiological and nutritional factors on purified polymerases have also not been explored. A de novo synthesis of RNA polymerase may be involved under certain conditions where an increased RNA synthesis has been observed. As mentioned earlier, the interpretations of experiments using inhibitors of protein synthesis are often misleading. The most satisfactory means to assess the synthesis of RNA polymerase is to measure the actual amount of the enzyme by immunoprecipitation, but this technique requires highly purified and homogeneous enzyme for the preparation of an authentic antiserum. Finally, the synthesis of the enzymes could be followed by labeling with a suitable amino acid.

VII. Mitochondria1 RNA Polymerase Only a concise summary of some relevant findings on mitochondrial RNA polymerase are presented in this section. Compared with nuclear RNA polymerase, relatively little is known about the properties and function of mitochondrial polymerase. Earlier studies on mitochondrial RNA polymerase have been conducted with intact organelles. Mitochondria isolated from fungi (163, 164), plants (165-168) and higher organisms (169-174) exhibit RNA polymerase activity; it is also much more active in embryonic rat tissue (176), germinating seeds (176) and in yeast cells in which glucose repression has been released (177).Recently, soluble enzyme has been obtained from Neurospora crassa (178), Blastocladiella emersonii (179), yeast (180, 181) and rat liver (182-184). The enzymes from N . crassa and yeast have been extensively purified. The requirement of sodium dodecyl sulfate for the extraction of rat liver mitochondrial polymerase (183) suggests a close association of the enzyme with the membrane. The partially purified rat liver enzyme has associated with i t almost saturating levels of DNA; consequently, it does not require additional exogenous DNA for its activity (182-184). The yeast mitochondria1 enzyme appears to occur

118

SAMSON T. JACOB

in at least two forms (180,181).Like the prokaryotic RNA polymerase, the enzymes from rat liver (182, 184) or heart (173)and N.crassa (178) are sensitive to rifampicin. However, there have been conflicting reports on the sensitivity of yeast mitochondrial enzyme to this drug. Scragg (181) has detected rifampicin-sensitive polymerase ( 8 ) in yeast, whereas Tsai et al. (180)have claimed that this enzyme is resistant to inhibition by the drug. The reason for this discrepancy is not clear. It is possible that a factor or factors needed for conferring sensitivity to rifampicin were lost from some enzyme preparations. I n contrast to rifampicin, a-amanitin does not inhibit the mitochondrial enzymes (178,180, 183, 184) although there are preliminary reports (184a, 184b) that show the inhibition of one form of yeast mitochondrial enzyme by a-amanitin. Each of the yeast mitochondrial enzyme differs from the corresponding nuclear polymerase with respect to response to divalent metal ions, ionic strength and templates ( 1 8 4 ~ )Compared . to thc mitochondrial enzymes of mammalian tissues, the yeast enzymes appear to have very high molecular weights ( 1 8 4 ~ )We . have recently observed that rat liver mitochondria contain very active homopolymer-synthesizing enzymes that are insensitive to pancreatic RNase, rifampicin and actinomycin D (Jacob and Schindler, unpublished observations). The varying degrees of response of some liver mitochondrial RNA polymerase preparations to actinomycin D (170,184), RNase (170), and rifampicin (184) may be the result of contamination of the polymerase with homopolymer-synthesizing enzymes. The enzyme from N . crassa transcribes poly (dA-dT) more efficiently than the mitochondrial DNA itself (178), which might reflect the presence of (A + T)-rich regions in mitochondrial DNA (186).Indeed, normal and mutant strains of yeast have usually a high content of A T(8&83%) (186). It is possible that the concentration of A T regions constitute a constraint on the coding capacity of mitochondrial DNA. Although the mitochondrial RNA polymerase is usually inhibited by rifampicin but not by a-amanitin, this enzyme resembles more the bacteriophage T7 specific enzyme (187)than the corresponding E. coli enzyme. The bacterial enzyme is composed of several subunits with other factors conferring specificity in the transcriptional process (1). Both phage and mitochondrial enzymes consist of a single subunit of relatively low molecular weight (T7 enzyme, MW 100,000; N . crassa enzyme, MW 64,000; rat liver enzyme, MW 64,000-68,000). This remarkable similarity between the phage and mitochondrial polymerases might' reflect the fact that both these enzymes transcribe a relatively small genome with only few cistrons or operons (188).The fact that rifampicin inhibits RNA synthesis by binding to the p subunit of E . coli polymerase

+

+

MAMMALIAN

RNA

POLYMERASES

119

(117)suggests the possibility that the mitochondria1 enzyme is similar to the

p subunit of prokaryotic enzyme. VIII. Summary and Conclusions

The objective of this rcview is to document the present status of RNA polymerases involvcd in RNA synthesis in mammalian cells, with emphasis on some recent developments in the field of nuclear RNA polymerase. The recent findings on this subject can be summarized as follows. (a) The RNA polymerases of eukaryotic nuclei exist in multiple forms. (b) These enzymes are compartmentalized within the nucleus. (c) They have different requirements for cations and salt. (d) The nucleolar enzymes are insensitive to a-amanitin in vitro whereas the nucleoplasmic enzymes arc generally inhibited by thc toxin, which binds to the enzyme and prevents RNA chain elongation. (e) The nucleolar enzymes are more activc with native DNA as the template, whereas the nucleoplasmic enzymes are more efficient with a denatured template; however, addition of a cytoplasmic protein factor can restore the ability of the latter enzyme to transcribe preferentially native DNA. ( f ) The subunit composition of nucleolar polymerase I appears to be different from that of the nucleoplasmic enzyme I1 ; both are structurally different from the bacterial RNA polymerase. (g) The RNA-synthesizing capacity of RNA polymerase can be modified by alterations in the activity and/or level of RNA polymerase, suggesting an independent role for RNA polymerase in the regulation of genetic expression in higher organisms. (h) The mitochondrial RNA polymerase of higher organisms has been partially solubilized and purified ; it is usually inhibited by rifampicin but is insensitive to a-amanitin. I n spite of the advances that have bcen made in our understanding of the transcriptional process in eukaryotic cells, there are several aspects of the problem that are not yet understood. For example, the role of the different mammalian RNA polymerascs in RNA synthesis remains to bc clarified. Since the subunit structures of the predominant nucleolar and nucleoplasmic polymerases appear to differ from one another, it is uiilikcly that these enzymes are derived from a common “corc.” Consequcntly, it is possible that thc nuclcolar enzymcs are involved in the synthcsis of ribosomal RNA precursor (45 S RNA) and of uridine-rich low-molecular-weight RNA of the nucleolus (189-191), whereas the nucleoplasmic enzymes make mRNA, tRNA, 5 S RNA, and possibly poly(A) involved in the transport of functional mRNA to the cytoplasm in some eukaryotes (192-196).A recent report (196~)shows that the

120

SAMSON T. JACOB

amanitin-sensitive enzyme from young maize seedlings can, under proper conditions, indeed make poly ( A ) . Only an analysis of the composition of RNA transcribed from native or reconstituted chromatin preparations will ultimately throw some light on these possibilities. Some attempts have recently been made in this direction (196%). A possible regulation of RNA polymerase activity by shifts in the intranuclear distribution of cations such as Mgz+ and Mn2+ and salts merits serious consideration. Since the polymerases show specific requirements for these ions, it is important (a) to examine the intranuclear levels of these metals and (b) to investigate changes in the polymerase activity in response to changes in their distribution. It may be pointed out here that polyanions such as spermine and spermidine, the concentrations of which are dramatically altered during growth and development (197203), stimulate RNA synthesis. The effect of these compounds on the activity of purified RNA polymerase should also be examined with a view to understanding their role in the transcriptional process and their specific function in the cellular regulation of RNA synthesis. The regulation of the activity of purified nucleolar RNA polymerase by hormones independent of any significant modification in the chromatin template is very interesting, considering the emphasis in the past on template modification as the most prominent means of gene regulation. Only the early effect of hormones on the purified RNA polymerase activity has been examined with purified RNA polymerases. A later and more permanent effect on chromatin activity exerted by hormones or by other physiological factors is a possibility that should be investigated. Qualitative changes in RNA synthesis are not always paralleled by changes in RNA polymerases. There are reports (47)showing no dramatic changes in the relative activities or amounts of polymerases I and I1 during the development of Xenopus laevis, while there are striking changes in the quantity and quality of nuclear RNA synthesis. The most surprising finding was the presence of normal amounts of polymerase I in the anucleolate mutant of Xenopus laevis, which is evidently not producing any ribosomal RNA (204).These observations tend to rule out any regulatory mechanism in which the amount of RNA polymerase is limiting within the cell. It is possible that, under certain conditions, RNA polymerases are present in excess, with other regulatory factors (needed for specific initiation) limiting. Future work to characterize any such factors in eukaryotic cells, such as u factor in prokaryotic cells, should be promising. Finally, the factors that are involved in initiation, termination and release of RNA chains in eukaryotic cells should be explored.

MAMMALIAN

RNA

POLYMERASES

121

NOTES ADDED IN PROOF 1. Recently, highly purified enzyme I has been obtained from calf thymus nuclei with a subunit composition of (20,000)1 (126,000)1 (51,000)1 (44,0W)1 (25,000)2 (16,!500), ( 1 ) . This subunit pattern accounts for the molecular weight of the native enzyme which is about 550,000 5 10%. 2. The inhibitory effect of various semi-synthetic derivatives of rifamycin SV on calf thymus DNAdependent RNA polymerases I and I1 has been examined ( 2 ) . Some of these derivatives having highly hydrophobic side-chains, like AF/013, completely inhibit both types of activities a t concentrations of 20 to 40 pglml. Unlike a-amanitin, initiation and not elongation is inhibited by these derivatives. 1. F. Gissinger and P. Chambon, EJB 28, 277 (1972). 2. M. Meilhac, Z. Tysper and P. Chambon, EJB 28, 291 (1972).

ACKNOWLEDGMENTS I wish to thank Dr. Elizabeth M. Sajdel for many useful discussions and for help in the preparation of the tables. The experimental work performed in the author’s laboratory was supported by a U.S. Public Health Service grant (GM 18534) and by the Damon Runyon Memorial Fund for Cancer Research (DRG 1062).

REFERENCES 1. R. R. Burgess, ARB 40, 711 (1971). l a . R. G. Martin, Annu. Rev. Genet. 3, 181 (1969). l b . V. S. Sethi, in “Progress in Biophysics and Molecular Biology” (J. A. V. Butler and D. Noble, eds.), p. 67. Pergamon, Oxford, 1971. 2. S. B. Weiss, PNAS 46, 1020 (1960). 3. C. C. Widnell and J. R. Tata, BBA 87, 531 (1964). 4 . C. C. Widnell and J. R. Tata, BBA 123, 478 (1966). 6. C. C. Widnell, T. H. Hamilton and J. R. Tata, JCB 32, 766 (1967). 6. X. J. Blackburn and H. G. Klemperer, BJ 102, 168 (1967). 7. P. Chambon, M. Ramue, P. Mandel and J. Doly, BBA 157, 504 (1968). 8. A. E. Pegg and A. Korner, ABB 118, 362 (1967). 9 . A. 0. Pogo, V. C. Littau, V. G. Allfrey and A. E. Mirsky, PNAS 57, 743 (1967). 10. J. D. Johnson, B. A. Jant, L. Sokoloff and S. Kaufman, BBA 179,526 (1969). 11. S. T. Jacob, E. M. Sajdel and H. N. Munro, BBA 157, 421 (1968). 12. T. 5. Ro and H. Busch, BBA 134, 184 (1967). 13. G . Siebert, J. Villalobos, Jr., T. S. Ro, W. J. Steele, G. Lindenmayer, H. Adxms and H. Busch, JBC 241, 71 (1966). 14. J. D. Johnson, B. A. Jant, S.Kaufman and L. Sokoloff, ABB 142, 489 (1971). 16. U. Maitra and F. Barash, PNAS 64, 779 (1969). 16. R. R. Burgess, JBC 244, 6168 (1969). 17. D. D. Brown and E. Littna, JMB 8, 669 (1964). 18. G. Guidice and V. Mutolo, BBA 138, 276 (1967). 19. F. M. Ritossa and S. Spiegelman, PNAS 53, 737 (1965). 20. H. Wallace and M. L. Birnstiel, BBA 114, 296 (1966). $1. D. D. Brown and I. B. Dawid, Science 160, 272 (1968). 2lu. M. L. Birnstiel, M. Chipchase and J. Spiers, This series 11, 351 (1971).

122

SAMSON T. JACOB

22. H. Busch and K. Smetana, in “The Nucleolus” (H. Busch and K. Smetana. eds.), p. 160. Academic Press, New York, 1970. 23. J. J. Furth and P. Loh, BBRC 13, 100 (1963). 24. J. J. Furth and P. Ho, JBC 240, 2602 (1965). 26. P. Ballard and H. G. Williams-Ashman, JBC 241, 1602 (1966). 26. E. R. Stout and R. J. Mans, BBA 134, 327 (1967). 27. A. Ishihama, BBA 145, 272 (1967). R8. M. Ramue, J. Doly, P. Mandel and P. Chambon, BBRC 19, 114 (1965). 29. D. D. Cunningham and D. F. Steiner, BBA 145, 834 (1967). SO. K. Seifart and C. E. Sekeris, Hoppe-Seyler’s 2.Physiol. Chem. 348, 1555 (1967). 31. M. L. Goldberg, H. D. Moon and W. Rosenau, BBA 171, 192 (1969). 32. S. Liao, D. Sagher and S. Fang, Nature (London) 220, 1336 (1968). 33. S. T. Jacob, E. M. Sajdel and H. N. Munro, BBRC 32, 831 (1968). 34. S. T. Jacob, E. M. Sajdel and H. N. Munro, BBRC 38, 765 (1970). 36. S. T. Jacob, E. M. Sajdel, W. Muecke and H. N. Munro, CSHSQB 35, 681 (1970). 36. S. T. Jacob, E. M. Sajdel and H. N. Munro, Advan. Enzyme Regul. 9, 169 (1971). 37. R. G . Roeder and W. J. Rutter, Nature (London) 224, 234 (1969). 38. R. G. Roeder and W. J. Rutter, PNAS 65, 675 (1970). 39. S . P. Blatti, C. J. Ingles, T. J. Lindell, P. W. Morris, R. F. Weaver, F. Weinbcrg and W. J. Rutter, CSHSQB 35, 649 (1970). 40. K. H. Seifart and C. E. Sekeris, EJB 7 , 408 (1969). 41. I<. H. Seifart, in “Lepetit Colloqium on RNA Polymerase and Transcription” (L. Silvestri, ed.), p. 233. Amer. Elsevier, New York, 1970. 42. C. J. Chesterton and P. H. W. Butterworth, FEBS Lett. 12, 301 (1971). 43. C. J. Chesterton and P. H. W. Butterworth, EJB 19, 232 (1971). 44. C. J. Chesterton and P. H. W. Butterworth, FEBS Lett. 15, 181 (1971). 46. C. Kedinger, P. Nuret and P. Chambon, FEBS Lett. 15, 169 (1971). 46. J. L. Mandel and P. Chambon, FEBS Lett. 15, 175 (1971). 47. R. G. Roeder, R. H. Reeder and D. D. Brown, CSHSQB 35, 727 (1970). 48. W. Keller and R. Goor, CSHEQB 35, 671 (1971). 49. M. Gniazdowski, J. L. Mandel, Jr., F. Gissinger, C. Kedinger and P. Chambon, BBRC 38, 1033 (1970). 60. P. Chambon, F. Gissinger, J. L. Mandel, Jr., C. Kedinger, M. Gniazdowski and M. Meihlac, CSHSQB 35, 693 (1970). 61. H. Stein and P. Hausen, CSHSQB 35, 709 (1971). 62. R. G. Roeder and W. J. Rutter, Bchem. 9, 2543 (1970). 63. G. P. Tocchini-Valentini and M. Crippa, CSHSQB 35, 737 (1970). 64. J. P. Monjardino and L. V. Crawford, CSHSQB 35, 659 (1970). 66. B. Sugden and J. Sambrook, CSHSQB 35,663 (1970). 66. J. J. Furth and G. E. Austin, CSHSQB 35,641 (1970). 67. W. I. P. Mainwaring, F. R. Mangan and B. M. Peterken, BJ 123, 619 (1971). 68. P. A. Horgen and D. H. Grifiin, PNAS 88,338 (1971). 69. H. Ponta, U.Ponta and E. M’intersberger, FEBS Lett. 18, 204 (1971). 60. E. C. Strain, K. P. Mullinix and L. Bogorad, €“AS 68, 2647 (1971). 61. E. M. Sajdel and S. T. Jacob, BBRC 45, 707 (1971). 62. R. F. Weaver, 5. P. Blatti and W. J. Rutter, PNAS 88, 2994 (1971). 6%. A. Kedinger and P. Chambon, EJB 28, 283 (1972).

MAMMALIAN

RNA

POLYMERASES

123

63. R. R. Burgess, A. A. Travers, J. J. Dunn and E. K. F. Bauta, Nature (London) 221, 43 (1969). 64. R. R. Burgess, JBC 244, 6160 (1969). 66. H. N. Munro and A. Fleck, in “Mammalian Protein Metabolism” (H. N. Munro, ed.), Vol. 3, p. 423. Academic Press, New York, 1969. 66. B. Gerhardt and H. Beavers, Anal. Biochem. 24, 337 (1968). 6’7.E . Layne, in “Methods in Enzymology” (S. P. Colowick and X. 0. Kaplan, eds.), Vol. 3, p. 447. Academic Press, New York, 1957. 65. T. P. Bennett, Nature (London) 213, 1131 (1967). 69. F. Stirpe and F. Novello, EJB 15, 505 (1970). 70. T. C. Spelsberg and L. S. Hnilica, BBA 195, 55 (1969). 71. F. Stirpe and L. Fiume, BJ 105, 779 (1967). 72. T. H. Wieland, Science 159, 946 (1968). 73. L. Fiume and T. H. Wieland, FEBS Lett. 8, 1 (1970). 74. H. V. Gelboin, J. S. Wortham, R. G. Wilson, M. Friedman and G. N. Wogan, Science 154, 1205 (1966). 76. S. T. Jacob, E. M, Sajdel and H. N. Munro, Nature (London) 225, 60 (1970). 76. T. J. Lindell, F. Weinberg, P. W. Morris, R. G. Roeder and W. J. Rutter, Scieiice 170, 447 (1970). 17. C. Kedinger, M. Gniaadowski, J. I,. Mandel, F. Gissinger and P. Chambon, BBRC 38, 165 (1970). ?S. W. Wehrli, J. Nuesch, F. Knusel and M. Staehelin, BBA 157, 215 (1968). 79. W. Wehrli, F. Knusel, K. Schmid and M. Staehelin, PNAS 61, 667 (1968). 50. A. Travers and R. R. Burgess, Nature (London) 222, 537 (1969). 81. E. Reich and I. H. Goldberg, This series 3, 183 (1964). 52. I. B. Weinstein, R. Carchman, E. Marner and E. Hirschberg, BBA 142, 440 (1965). 83. B. I(. Bhuyan and C. G. Smith, PNAS 54,566 (1965).

$4.

C;. Hartmann, H. Goller. K. Koschel, W. Kersten and H. Kersten, Biochem. Z . 341, 126 (1964). 56. A. M. DeRecondo, A. M. Frayssinet, C. Lafarge and E. LeBreton, BBA 119, 322 (1966). 86. C. D. Ward, E. Reich and I. H . Goldberg, Science 149, 1259 (1964). 57. M. J. Waring, J M B 13, 269 (1965). 55. B. Nicholson and A. Peacocke, BJ 100, 50 (1966). 59. R. W. Ruddon and J. M. Johnson, Mol. Pharmacol. 4, 258 (1968). 90. W. Troll, S. Belman, E. Berkowitz, Z. F. Chiemlewicz, J. L. Ambrus and T. J . Bardos, BBA 157, 16 (1968). 91. J. F. Morrison, BBA 185, 269 (1969). $9. M. Meihlac, C. Kedinger, P. Chambon, V. Govindan, H. Faulstich and T. Wieland, FEBS Lett. 9, 258 (1970). 93. F. Novello, L. Fiume and F. Stirpe, BJ 116, 177 (1970). 94. R. Schlief, Nature (London) 223, 1068 (1969). 06. S. T. Jacob, W. Muecke, E. M. Sajdel and H. N. Munro, BBRC 40, 334 (1970). 96. J. Niessing, B. Schnieders, W. Kuna, K . H. Seifart and C. E. Sekcris, X. Naturforsch. B 25, 119 (1970). 97. L. Fiume and L. Laschi, Sperimenlale 115, 228 (1965). 9s. H. S. Schwarta. J. E. Sodergren, M. Garofalo and S. S. Stenberg, Cancer nee. 25, 307 (1965).

124

SAMSON T. JACOB

H. S. Schwartz, S. S. Stcrnhrrg and F. S. Philips. Canner Ren. 26, 1873 (1966). H. S. Scliwarta and M. Garofalo. Mol. Phromncol. 3, 1 (1967). 101. C. E. Sekeris, J. Nirssing and K. H. S. Seifart, FEBS Lett. 9, 103 (1970). 102. P. Jolicoeur and F. Labric, FEBS Lett. 17, 141 (1971). 103. M. Green, M. Pina, R. Kimrs, P. Wcnsink, 1,. Maclinttic and C. A. Thomas, Jr.. PNAS 57, 1302 (1967). 104. L. V. Crawford and P. H. Black, Virology 24, 388 (1964). 106. J. Paul and R. S. Gilmour, JMB 34, 305 (1968). 106‘. R. S. Gilmour and J. Paul, JMB 40, 137 (1969). 107. I. Bekhor, G. Kung and J. Bonner, JMB 39, 351 (1969). 105. R. C. Hiiang and P. C. Hunng, JMB 39, 365 (1969). 109. J. E. Mayfield and J. Bonncr, PNAS 68,2652 (1971). 110. E. I(. F. Bnutz, F. A. Btiuta and J. J. Dunn, N n f w s (Lotdoti) 223, 1022 (1969). 111. W. C. Summers and 11. l3. Sirgel, Nature (Londoii) 223, 111 (1969). 112. R. Losick nnd A. L. Sonensheim, Nature (London) 204, 35 (1969). 113. I<. H. Seifart, CSHSQB 35, 719 (1970). 114. J. V. Maizcl, Jr., it& “Fiindamental Trcliniqucs in Virology” (K. Hahel and N. P. Salaman, eds.), 1). 334. Acadrmic Press, New York, 1969. 116. A. 1,. Shapiro, E. Vinuela and J. V. Maiael, Jr., BBRC 28, 815 (1967). 116. T. J. Leighton, R. K. Freese and R. H. Dol, FP 30, 1969 (1971). 117. W. Zillig, P. Palm, V. S. Sethi and D. Rabusstty, Ann. N . Y . Acad. Sci. 171, 910

99. 100.

(1970). 115. W. D. Wicks and F. T. Kenncy, Science 114, 1346 (1964). 11.9. J. Gorski, JBC 239, 889 (1964). 120. D. J. Begg and H. N. Munro, Nrrtnie (Lowdoit) 207, 483 (1965). 121. P. A. Dukes nnd C. E. Sekcris, Z. Physiol. Chem. 341, 149 (1965). 12.3. S. Liao, R. W. Barton and A. M. Lin, PNAS 55, 1593 (1966). 1.33. S. Liao and A. H. Lin, PNAS 57, 379 (1967). 124. 0. Barnabei and F. Scrcni, BBA 91, 230 (1967). 1%. F. L. Yu and P. Fcigelson, BBRC 35, 499 (1969). 126. S. T. Jacob, E. M. Sajdel and H. N. Munro, EJB 7, 449 (1969). 127. J. Hanoune and P. Feigelson, BBA 199, 214 (1970). 125. J. R. Tata, in “Biochemical Actions of Hormones” (G. Litwack, ed.), Vol. 1, p. 89. Academic Press, New York, 1970. 129. K. Tsukada and I. Lieberman, JBC 239, 2952 (1964). 130. M. Muramatsu and H. Busch, JBC 240, 3980 (1965). 131. S. T. Jacob, W. J. Stecle and H. Busch, Cancer Res. 27, 52 (1967). 132. A. 0. Pogo, V. C. Littau, V. G. Allfrey and A. E. Mirsky, PNAS 57, 743 (1967). 133. J. R. Warner, M. Girard, H. Latham and J. E. Darnell, JMB 19, 373 (1966). 134. J. R. Warner, JMB 19, 383 (1966). 136. J. Vesley and A. Cihak, BBA 904, 614 (1970). 136. M. E. Smulson and J. Thomas, JBC 244, 5309 (1969). 137. M. E. Smulson, BBA 199, 537 (1970). 13s. A. R. Henderson, BJ 120, 205 (1970). 139. M. T. Franze-Fernandez and A. 0. Pogo, PNAS 68, 3040 (1971). 140. M. E. Dahmus and J. Bonner, PNAS 54, 1370 (1965). 1.41. M. Beato, K. H. Seifart and C. F. Sekeris, ABB 138, 272 (1970). 148. K. Barker and L. Warren, PNAS 56, 1298 (1966). 143. F. R. Mangan, G. E. Nenl and D. C. Williams, ABB 124, 27 (1968). 1.44. C. B. Breuer and J. R. Florini, Bchem 5, 3857 (1966).

MAMMALIAN

RNA

POLYMERASES

125

146. M. Beato, J. Homoki, J. Lukiics and C. E. Sekcris, Hoppe-Seyler‘s 2. Physiol. Chem. 349, 1099 (1968). 140. H. L. Stackhousc and C. J. Chcstsanga, BBA 155, 159 (1968). 147. K. Barker and J. Warren, Endocrinology 80, 536 (1967). 14s. W. L. Steinhart, BBA 228, 301 (1971). 149. F. L. Yu and P. Feigelson. PNAS 68, 2177 (1971). 160. J. Barry and J. Gorski, Bchem 10, 2384 (1971). 161. F. Novello and F. Stirpe, FEBS Lett. 8, 57 (1970). 162. W. D. Noteboom and J. Gorski, PNAS 50, 250 (1963). 163. J. Gorski, JBC 239, 889 (1964). 164. J. A. Nicolette, M. A. Lemaliieu and G. C. Mueller, BBA 166, 403 (1968). 166. K. Tsukada and I. Lieberman, JBC 240, 1731 (1965). 166. T. Onishi, BBA 217, 384 (1970). 167. M. Maramatsu, N. Shimada and T. Higashinukagawa, J M B 53, 91 (1970). 168. M. Willems, M. Penman and S. Penman, JCB 41, 177 (1969). 169. F. Wanka and P. J. A. Schrauwen, BBA 254,237 (1971). 160. E. A. Smuckler and J. R. Tata, Nature (London) 234, 37 (1971). 1000. C. J. Cliesterton, S. M. Humphrey and P. H. W. Butterworth, BJ 126, 675 ( 1972). 1Gl. V. G. Allfrey, in “Regulatory Mechanisms for Protein Synthesis in Mammalian

Cells” (A. S. Pietro, M. R. Lamborg and F. T. Kenney, eds.), p. 65. Academic Press, New York, 1968. 1G2. T. A. Langan, in “Regulatory Mechanisms for Protein Synthesis in Mammalian Cells” (A. S. Pietro, M. R. Lsmborg and F. T. Kenney, eds.), p. 101. Academic Press, New York, 1968. 103. F. Herzfeld, Hoppe-Seyler’s Z. Physiol. Chem. 351, 658 (1970). lG4. D. J. K. Luck and E. Reich, PNAS 52, 931 (1964). 1/37. E. Wintersberger and U. Wintersberger, FEBS Lett. 6, 58 (1970). 1GG. E. Wintersberger, BBRC 40, 1179 (1970). 1G7. Y. Suyamn and J. Eyer, JBC 243,320 (1968). 168. S . Yang and R. S. Criddle, Bchem 9, 3063 (1970). 1GO. G. F. Kalf, Bchem 3, 1702 (1964). 170. D. Neubert and H. Helge, BBRC 18, 600 (1965). 171. G. Saccone, N. N. Cadaleta and E. Quagliariello, BBA 138, 474 (1967). 172. Z. G. Slimerling, BBRC 37, 965 (1969). 173. J. G. Gamble and R. H. McCluer, JMB 53, 557 (1970). 174. M. Ash!%-elland T. S. Work, A R B 39, 251 (1970). 176. D. Neubert, H. Helga and H. J. Merker, in “Biochemical Aspects of the Biogenesis of Mitochondria” (E. C. Slater, J. M. Papa and S. Quagliariello, eds.), p. 251. Adriatica Editrica. Bnri, 1968. 170. P. Lado and M. Schwendimann, Life Sci. 6, 16881 (1967). 177. D. J. South and H. R. Mahler, Nature ( L o d o n ) 218, 1226 (1968). 178. H. Kuntzel and K. P. Schafer, Nature (London) 231, 265 (1971). 179. P. A. Horgen and D. H. Griffi, Nature (London) 234, 18 (1971). 180. M. J. Tsai, G. Michaelis and R. S. Criddle, PNAS 68, 473 (1971). 181. A. H. Scragg, BBRC 45, 701 (1971). 182. M. N. Gadnleta, M. Greco and C. Saccone, FEBS Lett. 10, 54 (1970). 183. C. Saccone, R. Gallerani, M. N. Gadaieta and M. Greco, FEBS Lett. 18, 339 (1971). 184. B. D. Reid and P. Parsons, PNAS 68,2830 (1971).

126

SAMSON T. JACOB

184a. R. W . Benson, FASEB Abstr. 31, No. 1450 (1972). 184b. R. Eccleshall and R. S. Criddle, FASEB Abstr. 31, No. 1447 (1972). 186. G. Bernardi, F. Carnevali, A. Nicolaieff, G. Piperno and G. Tecce, J M B 37, 493 (1968). 186. L. I. Grossman, D. R. Cryer, E. S. Goldring and J. Murmur, JMB 62, 566 (1971).

187. M. Chamberlain, J. McGrath and L. Waskell, Nature (London) 228, 227 (1970). 188. M. M. K. Nass, Science 185, 25 (1969). 189. M. Muramatsu, J. L. Hodnett and H. Busch, JBC 241, 1544 (1966). 190. T. Nakamura, A. W. Prestayko and H. Busch, JBC 243, 1368 (1968). 191. H. Busch, in “The Nucleolus” (H. Busch and K. Smetana, eds.), p. 285. Academic Press, New York, 1970. 192. J. E. Darnell, R. Wall and R. J. Tushinski, PNAS 68, 1321 (1971). 193. S. Y. Lee, J. Mendecki and G. Brawerman, PNAS 68, 1331 (1971). 194. M. Edmonds, M. H. Vaughan, Jr. and H. Nakazato, PNAS 68, 1336 (1971). 196. L. Philipson, R. Wall, G. Glickman and J. E. Darnell, PNAS 68, 1336 (1971). 196. J. E. Darnell, L. Philipson, R. Wall and M. Adesnik, Science 174, 507 (1971). 196a. R. H. Benson and R. J. Mans, FASEB Abstr. 31, No. 1179 (1972). 196b. P. H. W. Butterworth, R. F. Cox and C. J. Chesterton, EJB 23, 229 (1971). 194. J. Janne, Acta Physiol. Scand. Suppl. 300, 1 (1967). 198. G. Moruzzi, B. Barbiroli and C. M. Caldarera, BJ 107, 609 (1968). 199. C. M. Caldarera, M. S. Moruzai, B. Barbiroli and G. Moruzri, BBRC 33, 266 (1968). 800. B. C. Monlton and S. L. Leonard, Endocrinology 84, 1461 (1969).

201. A. E. Pegg, D. H. Loekwood and H. G. Williams-Ashman, BJ 117, 17 (1970). 202. B. Barbiroli, A. Corti and C. M. Caldarera, FEBS Lett. 13, 169 (1971). 203. D. H. Russell, PNAS 68, 523 (1971). 204. D. D. Brown and J. B. Gurdon, PNAS 51, 139 (1964).

Poly(ad enosine diphosphate ribose) TARASHI SUGIMURA Biochemistry Division, National Cancer Center Research Institute, Chuo-ku, Tokyo, arid Department of Molecular Oncology, The Institute of Medical Science, Tokyo University, Minato-ku, Tokyo, Japan

I. Introduction . . . . . . . . . . . . . 11. Chemical and Physical Properties of Poly(ADP-Rib) . . . A. Characteristics of Poly(ADP-Rib) . . . . . . . B. Characteristics of Ado(P)-Rib-P . . . . . . . C. Derivatives of Ado(P)-Rib-P . . . . . . . . 111. Purification of Poly(ADP-Rib) . . . . . . . . . A. Incubation of Precursor with Enzyme Preparation . . . B. Separation of Poly(ADP-Rib) . . . . . . . . IV. Biosynthesis of Poly(ADP-Rib) . . . . . . . . A. General Character . . . . . . . . . . . B. Association of Polymerase with Chromatin . . . . . C. Association of Poly(ADP-Rib) with Nuclear Proteins . . D. Inhibitors . . . . . . . . . . . . . E. Relation between Enzyme Forming Poly (ADP-Rib) and . . . . . . . . . . . . . NADase F. Purification of Enzyme . . . . . . . . . . V. Biodegradation of Poly(ADP-Rib) . . . . . . . . A. Snake Venom Phosphodiesterase . . , . . . . B. Rat Liver Phosphodiesterase . . . . . . . . C. Enzymatic Cleavage of the Ribose-Ribose Bond . . . . VI. Natural Occurrence . . . . . . . . . . . VII. Possible Biological Significance . , . . . . . . . VIII. Related Phenomena . . . . . . . . . . IX. Future Problems . . . . . . . . . . References . . . . . . . , . . .

.

.

.

.

.

127 129 129 130 131 132 132 132 134 134 135 137 138 139 141 142 142 143 144 145 146 147 148 149

1. Introduction I n 1963, Chambon, Weill and Mandel made the observation that C ] into the acidincorporation of radioactivity of [~ d e n i n e - ~ATP insoluble material in a nuclear preparation from hen liver is enhanced a t least 1000-fold by the presence of NMN ( 1 ) . I n 1965, we confirmed this stimulation: using a nuclear preparation from rat liver (2).We found 127

128

TAKASHI SUGIMURA

that the acid-insolublc reaction product was not converted to an acidsoluble form by treatment in 0.5N NaOH a t 37" for 18 hours, and concluded that it was not poly (A) (2, 3). The reaction product was hydrolyzed by snake venom phosphodiesterase, and an unknown, radioactive substance was isolated from the hydrolyzate, which accounted for most of the initial reaction product and differed from 5'-AMP (3).The behaviors of this peak on paper and column chromatographies were very similar to those of adenosine 3',5'-bisphosphate and adenosine 2',5'-bisphosphate. This substance was later found to be 2'[5"-phosphoribosyl] -5'AMP, Ado (P)-Rib-P' (4-7,9, 11). I n Strasbourg, intensive studies were made on thc reaction product and the correct structure of Ado(P)-Rib-P was reported by Chambon et a2. (4) and Doly and Petek (5).The structure of Ado(P)-Rib-P was also elucidated by groups in the National Cancer Center, Tokyo (6-9) and Kyoto University (10,11). Knowing the structure of Ado(P)-Rib-P, the whole structure of the product was proved to be that shown in Fig. 1. The observations made in all three laboratories support this structure. The nuclear preparations contain NAD pyrophosphorylase, which produces [ adenine-"C] NAD from [adenine-W] ATP and NMN. These preparations also catalyze the polymerization of the ADP-Rib moiety of NAD. Thus, the reaction product found was [adenine-l'C]poly(ADP-Rib) (4,6,7,9,11). 12'-(5"-Phosphoribosyl)-5'-AMP (I) has been variously abbreviated as phosphoribosyl-AMP, PR-AMP, $-ADPR or Ado(P)-Rib-P. Ado(P)-Rib-P is recommended as the abbreviation. 2'-(Ribosyl)b'-AMP (11) has been abbreviated as ribosyl-AMP, R-AMP and Ado (P)-Rib ; 2'-(5"-phosphoribosyl) adenosine (111) as phosphoribosyladenosine, PR-AR and Ado-Rib-P ; fl'-ribosyladenosine (IV), as ribosyl adenosine, R-AR or Ado-Rib. Poly ADPR, poly(ADP-Rib), poly (ADPribose) and polymer of ADP-ribose have been used for the polymer; (ADP-Rib). is recommended as a short, unambiguous term. I Me I Rib-Rib I 1 P P Ado(P)-Rib-P P-L'-Ad0(2'-1')Rib-5"-P

m Me I Rib-Rib I P Ado-Rib-P Ad0(2'-l")Rib-5~-P

n Me I Rib-Rib I P Ado(P)-Rib P-5'-M0(2'-1")Rib

rv fde Rib-Rib Ado-Rib Mo(Z'-l")Rib

129

POLY (ADENOSINE DIPHOSPHATE RIBOSE)

Ade -Rib

I

P-P

kib-Rib

I

/Me

Ade

I

P-P

I

/Me

{ I :I Rib-Rib! Rib-Rib! RibI I l l I l l P+P

P+P

FIG.1. Structure of poly(ADP-Rib). Ade: adenine; Rib: n-ribose; P : phosphate. For the structure of the repeating unit (between broken lines), see Fig. 2.

This article surveys the information available on poly (ADP-Rib) and related subjects through December, 1971. The only previous reviews of studies on this unique biopolymer are two reviews in Japanese (12, IS) and a corcprehensivc rcvicw of the research in Strasbourg in a thesis (14).

II. Chemical and Physical Properties of Poly(ADP-Rib)

A. Characteristics of Poly(ADP-Rib) Incubation of nuclear preparations from hen or rat liver with N M N and ATP variously labeled showed that the 5'-phosphoribose moiety of NMN and the 5'-AMP moiety of ATP became incorporated into a n acidinsoluble product (4, 7, 8, 10).Studies with variously labeled NAD also revealed that all of the NAD except the nicotinamide moiety was incorporated into the product (4, 6-11). No incorporation of the nicotinamide moiety of NMN or NAD into the acid-insoluble material was detected even when NAD very highly labeled a t the nicotinamide moiety was used. The acid-insoluble product was highly purified, as described later, and completely freed from RNA and DNA (15,16),and was found to consist of polymerized ADP-Rib. Poly (ADP-Rib) shows the general characters of a polynucleotide. It is readily soluble in water in neutral and alkaline conditions. It precipitates in 5% trichloroacetic acid, 5% trichloroacetic acid 0.25% sodium tungstate (pH 2.0) or 66% ethanol 0.2M acetate buffer (pH 4.8). Poly(ADP-Rib) also precipitates in the presence of excess Mgz+ (14). (ADP-Rib),, has an s value of 2 4 s (4, 9, 11). Purified poly(ADP-Rib) has a buoyant density of about 1.57 on cesium sulfate equilibrium density gradient centrifugation ( 9 ) , being less dense than Fz virus RNA but heavier than T 4 DNA (17). On methylated albumin kieselguhr, the polymer moves as a broad band (11). On hydroxyapatite, poly(ADP-Rib) elutes after RNA and DNA and with a much higher concentration of buffer. This characteristic behavior was utilized in its purification (16). Purified poly(ADP-Rib) has an absorption maximum a t 258 nm a t pH 7.0, indicating no substitution in the adenine ring. It is deaminated by HNO,. Acid hydrolysis in 1M HCl a t 100°C for 10 minutes yields

+

+

130

TAKASHI SUGIMURA

hypoxanthine (11), Analysis indicates adenine, ribose and phosphate in the molar ratio of 1:2:2 (4,9, 11).The phosphate groups are all resistant to alkaline or acid monophosphatase (4, 9, 11). Poly(ADP-Rib) is relatively stable in alkali; little of the acidinsoluble material becomes acid-soluble on treatment with 0.5 M NaOH a t 37°C for 18 hours. However, boiling in 1M HC1 for 7 minutes results in total conversion to acid-soluble material (9). Acid treatment splits the ribose-ribose bond, yielding adenine and ribose 5-phosphate (11). Poly(ADP-Rib) is not hydrolyzed by DNase I, DNase 11, pancreatic RNase, RNase T1, micrococcal nuclease, spleen phosphodiesterase, potato phosphodiesterase, potato nucleotide pyrophosphatase, spleen NAD nucleosidase or trypsin (4, 9-11). It is readily hydrolyzed by phosphodiesterase from the venom of Crotalus adamanteus (4, 9-11). Snake venom phosphodiesterase, freed from 5‘-nucleotidase by the method of Keller (18) or of Sulkowski and Laskowski (19), also hydrolyEes poly (ADPRib) ( 2 0 ) . The main product of the hydrolyzate, separated by Dowex 1-formate column chromatography (6, 9-11) , is Ado(P)-Rib-P (4, 9-11, 13), a minor product is 5’-AMP (6,11, 15). The chain length of poly(ADP-Rib) can be determined from the molar ratio of Ado(P)-Rib-P to 5’-AMP plus 1, after complete hydrolysis of poly (ADP-Rib) by snake venom phosphodiesterase, since 5’-AMP should only be derived from a terminus (4, 9, 11). The A280:A2B0ratio of absorption of purified poly (ADP-Ribose) is 0.26. When poly (ADP-Rib) was prepared from an adenine-labeled precursor, either ATP or NAD, the specific radioactivity (counts per minute per OD?,,,) of polymer was about 1.27 times that of the precursor, indicating the presence of a tertiary structure due to interaction of adenine bases (15).Digestion of poly(ADP-Rib) with snake venom phosphodiesterase or heat treatment of poly (ADP-Rib) resulted in 20-25% increase in the optical density a t 260 nm (IS). Recently, a component of poly(ADP-Rib) was found with a much larger s value but with a chain length of 20-25 repetitions of ADP-Rib units. The heavier component could be completely separated from the lighter component (s values of 2-4 S) by sucrose-density gradient centrifugation or Sephadex G-200 gel filtration. The true molecular features of the heavier component are not yet known (91).

B. Characteristics of Ado(P)-Rib-P A major product isolated from a digest of poly(ADP-Rib) with snake venom phosphodiesterase is Ado (P)-Rib-P, 2’ [5”-phosphoribosyl] -5’AMP (4,9,11). Chambon e t al. (4) first designated this as p-ADPR. Ado(P)-Rib-P has absorption maxima a t 261, 259 and 258 nm under

131

POLY ( ADENOSINE DIPHOSPHATE RIBOSE)

HO

OH

FIG.2. Structure of Ado(P)-Rib-P.

alkaline, neutral and acidic conditions, respectively, showing that positions 1 and 3 of the adenine ring and the amino group of adenine are not substituted (4, 9, 11).Ado(P)-Rib-P contains 1 mole of adenine, 2 moles of ribose and 2 moles of phosphate (4, 9, 1 1 ) . The phosphate groups are all susceptible to Escherichiu coli alkaline phosphatase (4, 22), potato acid phosphatase (9) or prostate acid phosphatase ( 1 1 ) . Only 1 mole of periodate is consumed per mole of adenine of Ado(P)Rib-P, indicating that only one of the 2 moles of ribose in Ado(P)-Rib-P has free adjacent hydroxyl groups a t the 2' and 3' carbons (9). Doly and Pctek isolated N6-mcthyladeninc, 2,3,5-trimethylribose and 3,5-dimethylribose by two-dimensional paper chromatography from an acid hydrolyzate of the methylated Ado-Rib obtained from Ado (P)-Rib-P, confirming the presence of a 1' to 2' bond between two riboses ( 5 ) . Nonspecific adenosine deaminasc from Aspergillus oryzae deaminates adenosine but not adenosine substituted a t the 2' OH group (17).Ado(P)-Rib-P is not susceptible to this enzyme, providing additional support for the presence of a 1' to 2' bond between two ribose molecules ( 1 3 ) . Hydrolysis of Ado (P)-Rib-P by dilute mineral acid liberates ribosyl 5-phosphate and 5'-AMP ( 4 ) . All the above data are consistent with the structure of the main product, Ado (P)-Rib-P, shown in Fig. 2.

C. Derivatives of Ado(P)-Rib-P The two phosphates of Ado(P)-Rib-P are susceptible to alkaline phosphomonocstcrase. Partial digestion of Ado (P)-Rib-P with this enzyme yields 2'- (ribosyl)-5'-AMP, Ado (P)-Rib, and 2'- (5"-phosphoribosyl) adenosine, Ado-Rib-P. These two derivatives may be separated

132

TAKASHI SUGIMURA

from each other and from related compounds, including Ado-3‘,5‘-P2, Ado-2’,5’-Pz and ADP-Rib by column, paper and thin-layer chromatographies (22, 23). The separation of these dephosphorylated derivat.ives of Ado(P)-Rib-P was essential for determination of the terminal structure of poly(ADP-Rib) (24, 25). The phosphate group at the 5‘ position of the AMP moiety of Ado(P)-Rib-P is much more susceptible to alkaline phosphomonoesterase than the phosphate group at the 5“ position of the phosphoribose moiety of Ado(P) -Rib-P ( 2 2 ) . Complete hydrolysis of Ado (P)-Rib-P with phosphomonoesterase yields Ado-Rib. Ribosyladenosine has been reported to be present in a crude preparation of tRNA from Saccharomyces cerevisiae, although it is uncertain in this case whether the linkage between the two ribose molecules is 1’ to 2‘ or 1’to 3‘ (26).

111. Purification of Poly(ADP-Rib) A. Incubation of Precursor with Enzyme Preparation The nuclei of the livers of rats or hens contain very active NAD pyrophosphorylase for the polymerization of ATP and NMN. With NAD as a precursor, calf thymus is better because the nuclear preparation is easily separated. The following preparations are all suitable as enzyme sources: a crude preparation of nuclei obtained by means of 0.25M sucrose; a purified preparation of nuclei obtained by Chauveau’s method using 2.2 M sucrose (27) ; an aggregated enzyme preparation from nuclei ( 4 ); and chromatin prepared by Marushige and Bonner’s method (28,29).Addition of M ADP-Rib or M 3’:5’cyclic AMP effectively inhibits the enzyme degrading poly (ADP-Rib) and enhances the yield (dl). Incubation times of 10 minutes and 30 minutes are suitable with NAD and ATP + NMN, respectively, as substrates, under normal incubation conditions (8, 15, 16). With ATP plus NMN as substrates, addition of Mg2+is essential, since NAD pyrophosphorylase requires this ion.

B. Separation of PolytADP-Rib) 1.

GELFILTRATION

The procedure (15)involved (a) Pronase digestion and phenol extraction, (b) digestion with pancreatic RNase and DNase, (c) gel filtration through a Sephadex G-50 column, (d) digestion with micrococcal nuclease and spleen phosphodiesterase, and (e) a second gel filtration on Sephadex. With this procedure, the overall recovery of acid-insoluble radioactivity

POLY (ADENOSINE DIPHOSPHATE RIBOSE)

133

was more than 50% (15). Increase of the specific radioactivity (counts per minute per OD,,,) and decrease of the ratio of absorption a t 280 nm to that at 260 nm were used as criteria of purity of poly (ADP-Rib) . The ratio of the specific radioactivity per OD,,, unit of purified poly(ADPRib) to that of the [ C ~ - ~ ~ P ] A used T P as a precursor was 1.27:l. The A2Ra:A200 ratio of purified poly(ADP-Rib) a t the final step was 0.26. After the second gel filtration, poly(ADP-Rib) was free of RNA and DNA. The average chain length of the resulting poly (ADP-Rib) was 2030 ADP-Rib units. 2. HYDROXYAPATITE COLUMNCHROMATOGRAPHY

Hydroxyapatite column chromatography proved effective to separate poly(ADP-Rib) from DNA and RNA (16).Chromatin was incubated with NAD or ATP plus NMN and the product was precipitated. The acid-insoluble precipitate was digested with Pronase and the fraction containing DNA, RNA and poly (ADP-Rib) was prepared by phenol treatment. On chromatography of this fraction on hydroxyapatite, RNA, DNA and poly(ADP-Rib) with a chain length of 25 ADP-Rib units were eluted with 0.15 M, 0.25 M and 0.35 M phosphate buffer (pH 6.8), respectively. When the DNA, RNA and poly(ADP-Rib) fractions had previously been digested by pancreatic DNase and RNase, the separation of poly(ADP-Rib) from DNA was much better, and poly(ADP-Rib) of shorter chain length could be obtained in a highly purified state, the oligo ribo- and deoxyribonucleotides being eluted with more dilute phosphate buffer. A linear relationship was obtained between the chain length of the material eluted and the phosphate concentration of the elution buffer. Miwa et al. (21) recently developed a rapid method to prepare poly(ADP-Rib) in bulk. By incubating a crude nuclear preparation of calf thymus with NAD and subjecting the mixture of RNA, DNA and poly (ADP-Rib) to hydroxyapatite column chromatography, about 10 mg of poly(ADP-Rib) were obtained from 100 mg of NAD ( 2 1 ) . Separation of oligo (ADP-Rib) was also achieved on hydroxyapatite columns by elution with dilute phosphate buffer. 3. MAGNESIUM PRECIPITATION

Doly (14) purified poly (ADP-Rib) with magnesium precipitation from the aqueous solution after digestion with DNase and RNase, followed by Pronase and phenol extraction. Poly (ADP-Rib) was precipitated with 0.1 M MgCl, and precipitated poly (ADP-Rib) was dissolved in 50 mM EDTA. Ethanol precipitation was repeated several times, and

134

TAKASHI SUGIMURA

finally the poly (ADP-Rib) was dissolved in water and lyophilized. The chain length of the poly (ADP-Rib) thus obtained was 20 ADP-Rib units.

IV. Biosynthesis of Poly(ADP-Rib) A. General Character As described above, NMN-dependent incorporation of the radioactivity of [ ~ d e n i n e - ~ ~ C ] Awas T P first observed with chicken liver nuclei. Later the same reaction was observed with rat liver nuclei, and the reaction conditions wcrc reported in detail (4, 8, 10, 23, 29). Thc preparations of chicken and rat liver both contained NAD pyrophosphorylase, so NMN and ATP served as precursors of poly (ADP-Rib) . Incorporation of radioactivity from I adenine-14C]ATP in the presence of NMN was drastically decreased by addition of unlabeled NAD (4, 6, 10), suggesting that NAD was an obligatory intermediate. When NAD labeled in either part of its ADP-Rib moiety was used as a precursor, nuclei from various rat tissues all showed some activity to produce poly (ADP-Rib). Chambon et al. reported that incorporation of [32P]NADwas not inhibited by addition of unlabeled ADP-Rib ( 4 ) . Addition of NADasc from beef spleen abolished the incorporation ( 4 ) . NADH, NADP and ADP-Rib could not serve as substrates of poly (ADP-Rib) (8). Nuclei prepared from rat brain (SO), rat kidney (SO), regenerating rat liver (SO), fast-growing rat ascites hepatoma cells (30), slow-growing rat Morris hepatomas (SI), rat Novikoff hepatoma (S2), mouse liver ( S I ) , mousc Ehrlich ascites tumor ( 3 3 ) , hen erythrocytes (SO) and calf thymus (21) all form poly (ADP-Rib) from NAD. Nuclear preparations from trout and carp livers and a nuclear preparation from the protozoon Tetmhymenu pyrijormG, also have this ability (31).It seems quite probable that nuclear preparations from all species of animals may have an enzyme forming poly (ADP-Rib) from NAD. Nuclei from tissues with many mitotic cells, such as ascites hepatoma cells, or with few mitotic cells, such as normal hepatocytes, show similar abilities to form poly(ADP-Rib) from NAD, although the former showed slightly higher activity, indicating that the growth rate of cells is not directly related to the activity of poly(ADP-Rib) formation in the nuclei (34). Nuclei of Novikoff hepatoma cells show much higher activity. On the contrary, hepatoma cells do not form poly(ADP-Rib) from labeled ATP and NMN, because their nuclei have a very low NAD pyrophosphorylase activity ( 3 5 ) . Hilz and Kittler found no correlation between poly(ADP-Rib) polymerase activity and the rate of synthesis of DNA in neonatal and adult liver and hepatomas. Howevcr, they found a close

POLY ( ADENOSINE DIPHOSPHATE RIBOSE)

135

correlation between the activity and the DNA content of normal and malignant tissues that differed widely in proliferation rates (36‘).R a t liver nuclei were separated into various fractions by zonal centrifugation by Johnston et al. (37, 38). Nuclei of stromal cells (2nS) synthesized little RNA or DNA. The nuclei of both diploid and tetraploid parenchymal cells (2nP, 4nP) actively synthesized RNA, whereas the synthesis of DNA was concentrated in a fourth class of nuclei, intermediate in size between 2n and 4n. Haines et al. (39) found the highest activities of the enzyme catalyzing formation of poly (ADP-Rib) in nuclei involved in synthesis of RNA. Therc arc no obscrvations on thc ~ ~ r r s e n cofc this enzyiuc activity in the nuclear fraction of higher plants. However there is evidence that it is present in the nuclear fraction of the yeast Saccharomyces cerevisiae ( 4 0 ) . The enzymc was not found in a whole cell extract of Xeurospora crassa (30).No activity has yet been found in prokaryotic organisms, such as Escherichiu coli, Pseudo~nonasnerziyinosn and Lactobacillus casei (30). The formation of poly(ADP-Rib) seems to be restricted to eukaryocytic nuclei, having the chromatin structure. The most frequently used enzyme source has been the nuclear preparation obtained by Chauveau’s method using 2.2 M sucrose solution (23, 29). Nuclei may be disrupted mechanically or supersonically and a sediincntable fraction, called the aggregated enzyme preparation, was also used as an enzyme source (1, 4 ) . The apparent optimum pH for poly(ADP-Rib) formation was 8.6 (4) with glycine-NaOH buffer and p H 8.0 with Tris buffer (8, 41). The enzyme forming poly(ADP-Rib) from NAD was quite heat labile (4, 8). Nishizuka et al. (10) found that treatment of the crude enzyme preparation with DNase diminished formation of poly (ADP-Rib) from NAD, and Chamhon et al. ( 4 ) found that the reaction of A T P and N M N was inhibited by DNase digestion. DNase treatment was used by Shimizu et al. (4.2) and later Yamada et al. (43) to solubilize the polymerase activity from an aggregated enzyme preparation. Partially purified enzyme required DNA for the formation of poly(ADP-Rib) ( 4 3 ) and DNA seemed to be required for its elongation (50, 44).

B.

Association of Polymerase with Chromatin

On fractionation of nuclei, most of the enzyme activity is found in the chromatin fraction obtained by the method of Marushige and Bonner (28) while the soluble fraction, ribosomal fraction and nucleonemata from the nuclei have no activity (50). Thus the purified chromatin fraction is a good enzyme source (44,45). The chromatin contains 80-90% of the total

136

TAKASHI SCGIMTJRA

poly (ADP-Rib) polymerase activity in the rat liver nuclei, and about 20% of the protein. Ueda et al. (44, 46) treated chromatin with a high concentration of ammonium sulfate to dissociate DNA from protein and subjected the resulting preparation to gel filtration on Sephadex G-200. The activity to synthesize poly (ADP-Rib) was recovered exclusively in the void volume, which consisted mainly of DNA, while the bulk of protein was eluted later. Polymerase activity seemed to be bound relatively firmly to the DNA. They observed that low concentrations (0.1-0.5M ) of ammonium sulfate markedly depressed poly (ADP-Rib) formation with marked incrc'ase in RNA synthrsis. On increasing the concentration of ammonium sulfate, poly (ADP-Rib 1 synthesis increased again and reached a maximum a t a concentration of 1.7 M. The initial velocity in 1.7 M ammonium sulfate was 40% of that without salt. Only sulfate and phosphate ions a t high concentration increased poly (ADP-Rib) polymerase activity. Furthermore, it was noticed that there was no disappearance of poly(ADPRib) at high salt concentrations, which might inhibit degradation of the polymer by rat liver phosphodiesterase or by an enzyme splitting its ribose-ribose bonds. On centrifugation of the chromatin-enzyme complex in cesium sulfate solution, the activity to form poly (ADP-Rib) was found in the DNA peak at a density of 1.34, which carried less-dissociable protein. Gel filtration of this complex on Sephadex G-200 in the presence of 1.6 M ammonium sulfate yielded DNA in the void volume with a minute amount of protein and most of the polymer-forming activity (44, 4.5). Nishizuka et al. (44) reported that the average chain length of a polymer made by an intact chromatin fraction was 6.37. This chromatin was partially digested with increasing concentrations of DNase, resulting in a decrease in the average chain length and total acid-insoluble radioactivity. It was also found that a purified preparation of nucleoli from rat liver nuclei (46') contained no activity to synthesize poly (ADP-Rib) . Ueda et al. (46) also reported the association of the product, poly (ADP-Rib) , with DNA on gel filtration in the presence of salt as in the enzymatic reaction. However, the product dissociated from DNA on cesium sulfate density gradient equilibrium centrifugation, implying that the binding was not covalent (4.5). Proof that the bonds were not covalent was that treatment with sodium lauryl sulfate or proteinase resulted in release of the polymer from DNA (4.5). Hilz and Kittler incubated isolated nuclei of Ehrlich ascites tumor cells with [3H]adenine-labeled NAD, washed them with isotonic sucrose and subjected them to radioautographic analysis. Silver grains were evenly distributed over the whole nucleus, the nucleolus possibly being free (33).Oikawa et al. incubated cultured cells from dissected tissues of

POLY (ADENOSINE DIPHOSPHATE RIBOSE)

137

FIQ.3. Radioautographs of rat embryo cells. Cultured on a cover slip and incubated with I'HINAD. (A) X400; (B) X1000.

whole rat embryos with [~denine-~
C. Association of Poly(ADP-Rib) with Nuclear Proteins Poly (ADP-Rib) precipitates with aggregated enzyme or chromatin, and the addition of sodium dodecyl sulfate or proteinase promotes the release of poly (ADP-Rib) , hence poly (ADP-Rib) may be linked with protein (46). Nishizuka et al. (44) reported that the acid-insoluble radioactivity in the nuclei after incubation with [~denine-~'C]NAD is in various protein fractions. Tris-extractable proteins, phenol-soluble proteins, acidic proteins, residual proteins and especially histone (HC1-soluble) , all possessed

138

TAKASHI SUGIMURA

radioactivity. These histones were separated by CM-cellulose column chromatography (53),and all fractions contained radioactivity (53). The bond between ADP-Rib and histone was quite labile with a half-life of 5 minutes on incubation in 0.1N NaOH a t 0". The bond was broken on treatment with 2 M hydroxylamine a t pH 7.0 as rapidly as that of phenylalanine esterified to tRNA. These results are compatible with the supposition that the ADP-Rib moiety may be transferred to the carboxyl group in the protein molecule (4.4). Otake et a,?.(64) reported the binding of poly (ADP-Rib) with histone on CsCl equilibrium density gradient centrifugation in the presence of 1M guanidinium chloride, using the method of Huang and Bonner (55), providing additional support to the claim that the ADP-ribose polymer is covalently bound to histone. They incubated rat liver nuclei with [s2P]NADand extracted histone by the method of Steele and Busch (56). This crude histone preparation was separated into three distinct peaks of radioactive protein by CM-cellulose column chromatography. These fractions were then subjected to CsCl equilibrium density gradient centrifugation by the method described above. Miwa et a,?. (57) incubated rat liver nuclei with labeled NAD, disrupted the nuclei and extracted the histone fraction. They purified it by CM-cellulose and Amberlite IRC-50 column chromatographies. The purified histone fraction was then digested with Pronase, and the ADPribosylated histone fragment was isolated by Dowex 1 column chromatography. Meilhac (58) performed a similar experiment and isolated ADP-ribosylated peptides. These were digested with phosphodiesterase without and with alkaline phosphatasc, and phosphoribosyl peptides and ribosyl peptides were isolated. Their constituent amino acids have not yet been determined. Incidentally it was found that poly(ADP-Rib) bound to histone is not completely converted to an acid-soluble form by snake venom phosphodiesterase, possibly owing to steric hindrance to the action of phosphodiesterase on poly (ADP-Rib) combined with protein. This ADP-ribosylation or oligo ADP-ribosylation or poly ADPribosylation of nuclear proteins, including histones, might represent a new type of biochemical modification of histones in addition to the already known reactions such as methylation (59), pliosphorylation (60) and acetylation (61) of histones.

D.

Inhibitors This reaction is strongly inhibited by nicotinamide and less strongly by isonicotinic acid hydraside. The possible involvement of a certain type of NADase in this polymerization reaction has been suggested (8, 10, 40,

POLY(ADENOSINE DIPHOSPHATE RIBOSE)

139

62). Clark et al. reported that rat liver nuclear NADase and poly (ADPRib) polymerase are strongly inhibited both by nicotinamide and 5methylnicotinamide (63). The formation of poly(ADP-Rib) decreased 86% on addition of 2 mM parachloromercuribenzoate (4, 8). The incorporation was also inhibited by DNase, and this inhibition was reversed by DNA, heparin or dextran sulfate, but not by RNA, poly(A) or poly(U) ( 4 ) . Preiss et al. found that poly(ADP-Rib) polymerase from HeLa S3 cells is strongly inhibited by thymidine a t the concentration used to arrest cell cultures in the S-phase ( 3 2 ) . This inhibition is very specific and thymidine analogs like BrdIird and IdUrd have effects comparable to that of thymidine. However, FdUrd and other natural nucleosides do not inhibit poly (ADP-Rib) polymerase. They also found that nicotinainidc and 5-methyl nicotinamide inhibit poly (ADP-Rib) polymerasc from HeLa S3 cells (32). Many antibiotics, including actinomycin D, mitomycin C, chloramphcnicol, cycloheximide and chromomycin A, do not inhibit the rcaction (8).

E. Relation between Enzyme Forming Poly(ADP-Rib) and NADase Nakazawa et al. (SO,41) noted the reseiiiblance of the nuclear enzyme forming poly (ADP-Rib) to nuclear NAD glycohydrolase (NADase) . The nuclear NADase is known to carry out an exchange reaction between the nicotinamidc of NAD and certain pyridine derivatives (30,41). However, inicrosomal NADase, which can also catalyze the exchange reaction, failed to form poly(ADP-Rib). This might be related t o the fact that polymerization starts with ADY-ribosylation of nuclear proteins. Nishizuka et al. suggested the presence of threc different NADases: cytoplasmic NADase, nuclear NADase tightly bound to chromatin, and a third enzyme that is also bound to chromatin but that can catalyze poly(ADP-Rib) formation. The second and third enzyme might be a single enzyme (30). Furthermore the K,, values of cytoplasmic and nuclear NADases and the enzyme for poly (ADP-Rib) synthesis were 1.7 X 2.5 X lo-' and 2.5 X lO-"M, respectively. Shimizu et al. (42) digested chromatin with DNasc and obtained NADase activity specific for poly (ADP-Rib) formation in the supernatant. The insoluble fraction after DNase digestion still contained NADase and the properties of the latter were more closely related to those of a NAD hydrolase. Bock et al. also described the presence of NADase in Ehrlich ascites tumor cell nuclci ( 6 5 ) .They found somc differencc between thc NADases in nuclci and microsomes. The nuclear NADase was highly specific for

140

TAKASHI SUGIMURA

NAD, and was inhibited 50% by 0.2 mM nicotinamide, while the microsoma1 NADase could split NAD, NADP and NMN in an activity ratio of 1:0.5:0.2.They suspected that the former enzyme might be identical with an enzyme that polymerizes the ADP-Rib moiety of NAD. Romer et al. (62) also found two NADases in Ehrlich ascites tumor cells, one in the cytoplasm and the other in the nuclei. The nuclear NADase was inactive against NADP. Moreover it was not inhibited a t all by l e 3 M isonicotinic hydrazide, whereas the cytoplasmic NADase was inhibited 60%. The optimum pH values of the microsomal and nuclear NADases were pH 6.2 and 8.2, respectively ( 6 2 ) .The microsomal enzyme formed the acetylpyridine analog of NAD on incubation with NAD and acctylpyridine whereas the nuclear NADase did not ( 6 2 ) .Romer et al. found an almost stoichiometric relation between NAD disappearance, determined with cyanide, and [ a d e n i n e - W ]NAD incorporation into the acidinsoluble material, and suggested that all NADase activity measured by the cyanide method represented polymerizing activity in nuclei of Ehrlich ascites tumor cells. This was in contrast to the nuclear preparation of rat liver where degradation of NAD was usually greater than formation of poly (ADP-Rib) (41, 42). Nakazawa et al. (41) summarized comparative results on microsomal NADase, chromatin NADase and the poly (ADP-Rib) -forming enzyme as shown in Table I. NADase, which can catalyze the exchange reaction of the nicotinamide moiety of NAD with free nicotinamide, histamine or 4-aminoimidazole carboxamide, may be related to poly (ADP-Rib) polymerasc (63, 64) , since formation of poly (ADP-Rib 1 involves transglycosidation of thc nicotinamide of NAD with ribose of the AMP moiety of another molecule of NAD. Nishizuka et al. (SO) emphasized that the nicotinamide ribose linkage is a high-energy bond (8 kcal) as previously recognized by Kaplan and OF NAD.w: P~~OPEHTIIZS

Inhibitor Exchange reaction K , for NAD Optimum pH Heat stability Specificity DNase treatment

TABLE I POLY(ADP-RIH)-FORMING ENZYME(S)

AND

Microsomal NADase

Chromatin NADase

Pyridine derivative Positive 0.17mM 6.4 Heat stable NAD, NADP Resistaiit

Pyridine derivative Positive 0.25mM 7.6-8.0 Labile NAD Sensitive

Poly (ADP-Rib) formation Pyridine derivative 0.25mM 8.0 Labile NAD Semitivc

POLY ( ADENOSINE DIPHOSPHATE RIBOSE)

141

his co-workers (66). Zatman et al. reported that a single protein of mammalian NADase showed two different activities, NAD transglycosidase and NAD hydrolase activity. The common intermediate, ADPribosylated enzyme, might be formed with the concomitant release of nicotinamide. The ADP-ribose group of the intermediate would then be transferred to some other pyridine derivative in the NAD transglycosidase reaction or to water in the NAD hydrolase reaction (66).

F. Purification of Enzyme The cnzyme activity to form poly(ADP-Rib) is firmly bound to chromatin. The aggregated enzyme preparation from nuclei also has the activities of NAD pyrophosphatasc, NADase and phosphodiesterase. Attempts to purify the enzyme to form poly (ADP-Rib) by solubilization with DNase were partially successful (4%’).About one-third of the poly (ADP-Rib) polymerase activity was recovered in the soluble form, while only one-twentieth of the NAD hydrolase activity was recovered in the soluble fraction. The activity to hydrolyze poly(ADP-Rib) in the soluble fraction was also one-thirtieth of that in the aggregated fraction. It should be noticed that solubilized poly (ADP-Rib) polymerase activity represents the NADase activity determined using cyanide. The solubilized poly (ADP-Rib) polymerase was inhibited by nicotinamide, but not by isonicotinic acid hydrazide. Yamada et al. (43) partially purified the enzyme forming poly (ADPRib) from rat liver nuclei, by isolation of disrupted nuclei, solubilization of thc enzyme by DNase digestion and two fractionations with ammonium sulfate. The yield was about 15% with 10-fold purification. The enzyme required DNA, histone, MgCl,, and dithiothreitol for activity. DNA could not be replaced by polyanions, such as poly(U), poly(A), poly(C), RNA, polyvinyl sulfate, methyldextran sulfate or heparin. The enzyme was as active on native DNA as on heat-denatured DNA and on poly[d (A-T)], but less active on poly(dG) -poly(dC) and on acid-soluble oligodeoxyribonucleotide. Histones, lysine-rich histone and arginine-rich histone were equally effective in stimulating the reaction. The molecular weight of the enzyme was determined to be 78,000 by sucrose density gradient centrifugation. The product of the reaction was poly (ADP-Rib) with an average of 8 ADP-Rib units. Purification involving butanol treatment, acetone precipitation, DEAE-cellulose column chromatography and adsorption on and elution from calcium phosphate gel was also attempted, giving 20% recovery and 80-fold purification. The requirement of this preparation for DNA for activity was not very strict (67). Doly purified the enzyme to form poly(ADP-Rib) from the super-

142

TAKASHI SUGIMURA

natant fraction after disrupting chicken liver nuclei with homogenization in 0.2M NazHPOa (pH 8.5)-0.1 mM EDTA. Acetone precipitation and hydroxyapatite column chromatography were applied. Enzyme thus obtained with 20-fold purification contained histone in the preparation itself, but required the DNA for the reaction (14).

V. Biodegradation of Poly(ADP-Rib) A. Snake Venom Phosphodiesterase As described previously, the breakdown of poly (ADP-Rib) by snake venom (Crotalus adamanteus) phosphodiesterase was observed a t an It proved to be a early stage in studies on poly (ADP-Rib) (4,6,7,9,11). very useful tool in determining the structure of poly(ADP-Rib). Commercial snake venom phosphodiesterase was freed of 5’-nucleotidase by the method of Keller (18) or Sulkowski and Laskowski (19). This purified snake venom phosphodiesterase hydrolyzed poly (ADP-Rib) a t the pyrophosphate bond endonucleolytically, as illustrated in Fig. 4. An intermediate was obtained by gel filtration of a partial hydrolyzate of poly (ADP-Rib) with this enzyme (94). This intermediate, an oligomer of Ado (P)-Rib-P, was incubated with alkaline phosphomonoesterase, the phosphomonoesterase was removed and the product was completely hydrolyzed by snake venom phosphodiesterasc. Ado-Rib-P, Ado (P)-RibP, and Ado(P)-Rib were found in the hydrolyzate, supporting the conAde -Rib

I

P-P

t

-Rib

I

Ade

Ade

I I I Rib-Rib Rib-Rib Rib-Rib I P-P I I P--P I I I

Ade

Ade

kib-Rib

kib-Rib

kb-Rib

I

I

I

P-P

I

t

Ade

-Rib

l

P

I Rib-Rib l

P

l

P

Ade

I Rib-Rib l 1 P P

I

P

Ade

I I Rib-Rib Rib I P-P I I

partial hydrolysis

PTp!

Ade

I

Ade

Ade

Ade

kib-Rib

Rib

I

P

I

1

P--P

1 I

complete hydrolysis

Ade

I Rib-Rib I I P

P

Ade

I EBb-Rib I I P

P

Ade

I I P

Rib

FIQ.4. Hydrolysis of poly (ADP-Rib) by snake venom phosphodiesterase.

POLY (ADENOSINE DIPHOSPHATE RIBOSE)

Ade

Ade

I Rib-Rib +I I P

P-P

Ade

143 Ade

I I I Rib-Rib Rib-Rib Rib-Rib I I I I 1 IP-P P--P P

1

phosphomonoesterase

Ade

Ade

I Rib-Rib

I Rib-Rib I

P--P

I

t

Ade

I Rib-Rib I

P

Ade

I

P-P

I Rib-Rib I I

t

Ade

Ade

I Rib-Rib

I Rib-Rib

P

P

I

I

P

I

I

P

I I

Rib-Rib

P--P

t

Ade

complete hydrolysis by snake venom phosphodiesterase

1

Ade

I I

Rib-Rib

P

FIG.5. Studies of tcrminnl structure of oligo(ADP-Rib).

clusion that snake venom phosphodiesterase digested the polymer endonucleolytically, as illustrated in Fig. 5.

B. Rat liver Phosphodiesterase Futai and Mizuno found a new phosphodiesterase forming 5’-mOnOphosphate (68), and soon this enzyme was reported to be the first enzyme from animal tissues to hydrolyze poly (ADP-Rib) (49, 69). The enzyme was purified 200-fold from a rat liver homogenate by n-butanol treatment, streptomycin sulfate precipitation, ammonium sulfate fractionation, batchwise treatment with DEAE-cellulose and finally DEAE-Sephadex column chromatography. The optimal pH for hydrolysis of the polymer is around pH 10 (69). The products of the complete hydrolysis of poly (ADP-Rib) are Ado (P) Rib-P and 5’-AMP. This phosphodiesterase rapidly hydrolyzes p-nitrophenyl esters of nucleoside 5’-monophosphate and various oligonucleotides, while it hydrolyzes DNA and RNA slowly. The degradation product is 5’-monophosphate and the initial site of attack is the 3‘hydroxy end of the oligonucleotide. This enzyme hydrolyzes NAD, M NADH and ADP-Rib. Hydrolysis is completely inhibited by EDTA or HgCl,. For hydrolysis of p-nitrophenyl uridine 5’-monophosphate, NAD or A-U, the octimal pH is also 10. I n contrast to snake venom phosphodiesterase, this phosphodiesterase hydrolyzes poly (ADP-Rib) ezonucleolytically from a terminus from

-

TAKASHI SUGIMURA

144 Ade -Rib

I

P-P

Ade

Ade

Ade -Rib

I

P-P

Ade

Ade

Ade

I I I I I Rib-Rib Rib-Rib Rib-Rib Rib-Rib Wb I P-P I I P-P I I P-P I I P-P I I

Lb-Rib

I

I

P-P

I

4I

Ade

4

Ade

Ade

I I P

Rib

I

I t Ade

Ade

I I I Rib-Rib Rib-Rib Rib-Rib I P-P I I P-P I I PI

Ade

Ade

I I I I -Rib Rib-Rib Rib-Rib Rib-Rib Rib-Rib 1 1 P-P 1 l P-P 1 l P l Pl PI P-P

Ade

I I P

Rib

FIQ.6. Hydrolysis of poly(ADP-Rib) by rat liver phosphodiesterase.

which 5’-AMP should be released, as illustrated in Fig. 6. The actions of this enzyme on poly (ADP-Rib) and p-nitrophenyl uridine 5’-mOnOphosphate are inhibited by 5’-AMP. The direction of exonucleolytic hydrolysis was determined (26) by preparing separately poly (ADP-Rib) labeled with a t the ribose from the AMP phosphate derived from the NMN moiety or with moiety of NAD. It was concluded that this phosphodiesterase attacked poly (ADP-Rib) at the pyrophosphate bond a t the terminus, which should yield 5’-AMP. Kinetic studies suggest that both p-nitrophenyl uridine 5’-monophosphate and poly (ADP-Rib) are hydrolyzed by the same site or by overlapping active sites (49).

C. Enzymatic Cleavage of the Ribose-Ribose Bond Recently Miwa and Sugimura discovered a new enzyme in a calf thymus nuclear preparation that splits the ribose-ribose linkage of poly(ADP-Rib) as shown in Fig. 7 (60).This is a novel mechanism for degradation of poly (ADP-Rib) . This enzyme was mainly concentrated in the nuclear fraction and was easily extracted with 0.1 M sodium phosphate buffer (pH 7.0) and 1 mM 2-mercaptoethanol. The enzyme was purified about 100 times by streptomycin treatment, ammonium sulfate fractionation, DNase digestion, and phosphocellulose and hydroxyapatite column chromatographies. The optimum pH is around 7 and

145

POLY (ADENOSINE DIPHOSPHATE RIBOSE)

Ade -Rib

I

P-P

I Rib-Rib I I +

P-P

Ade -Rib

I

P-P

Ade

Ade

I

Rib-Rib

I

I

P-P

1

Ade

I I Rib Rib Rib-Rib I I I I P-P

P-P

I Rib-Rib I I +

P-P

Ade

Ade

Rib-Rib

Rib

I

I

I

+

P-P

I

I

Ade

Ade

Ade

Rib

Rib Rib

Rib Rib

P-P

P-P

I

I

I

I

I

I

I I

FIG.7. Hydrolysis of poly(ADP-Rib) at ribose-ribose bond by an enzyme from calf thymus.

activity is almost completely inhibited by M p-chloromercuribenzene sulfonate or M M HgCl,. Activity is also strongly inhibited by ADP-Rib. Partial hydrolysis of poly (ADP-Rib) with this enzyme yields the acid-soluble reaction products, ADP-Rib and oligo (ADP-Rib) . Ueda et al. found a similar enzyme in rat liver nuclei ( 5 1 ) .They found that the ribose-ribose linkage of Ado (P)-Rib-P and the linkage between protein and ADP-Rib are resistant to this enzyme. The activity to degrade poly (ADP-Rib) is inhibited by adenosine-3’: 5’-cyclic monophosphate, the apparent Ki being around 1.5 mM. The name poly(ADPRih) glycohyrlrolase was proposed for this cnzymc.

VI. Natural Occurrence Doly and Mandel (70) reported the natural occurrence of poly(ADPRib). They injected chickens with a dose of 300 pg of nicotinamide per 1OOg body weight and 4 hours later, 10 mCi of 32Piper 1OOg. The chickens were killed 6 hours later, and liver nuclei were separated. 3zPlabeled poly(ADP-Rib) was obtained by the method of Chambon et al. (4) in the presence of unlabeled poly (ADP-Rib) synthesized separately in vitro. They also showed that a hydrolyzate of this radioactive polymer contained radioactive Ado (P)-Rib-P. Hall reported the presence of 9- [2’ (3’)-O-ribosyl-~-~-ribofuranosyl] adenine in a crude preparation of tRNA of yeast ( 2 6 ) . Heated at 100” in 0.1 N HCl for 2 hours, this compound gave a single spot UV-absorbing corresponding to adenine. Only 1 mole of periodate was consumed per mole of ribosyladenosine, when determined a t pH 5.5. This indicated that one pair of vicinal hydroxyl groups in the molecule was blocked. Hall suggested that ribosyladenosine might arise from a fraction isolated fortuitously with tRNA (26).It is worth noting that the activity to

-

146

TAKASHI SUGIMURA

form poly(ADP-Rib) from NAD was found in a nuclear preparation isolated from yeast (40).

VII. Possible Biological Significance Since the discovery of the covalent bond between poly(ADP-Rib) or oligo(ADP-Rib) and nuclear proteins (&,64), it has been suspected that poly(ADP-Rib) may play a role in regulation of the function of chromatin. Burzio and Koide reported that a prior incubation of rat liver chrodTTP incorporation into acidmatin with NAD greatly reduces [ SH] insoluble material in the presence of three other kinds of dNTP. During this incubation, poly (ADP-Rib) is formed in chromatin. Incubation without NAD or with NAD plus nicotinamide did not produce this great decrease in the template capacity of chromatin for DNA synthesis ( W ) , as shown in Table 11. When chromatin was incubated with NAD and then with externally added DNA polymerase of Micrococcus luteus, its template activity was lost. Burzio and Koide concluded that the suppression of endogenous DNA polymerase activity in adult liver nuclei by a prior incubation with NAD is due to poly (ADP-Rib) , This observation was confirmed by Ueda et al. (72) and also be Hilz and Kittler ( 3 6 ) . Bureio and Koide (73) found that DNA obtained after complete removal of the protein from NAD-treated rat liver chromatin possesses template activity similar to that from untreated chromatin. Furthcrmorc stepwise removal of proteins from chromatin resulted in gradual release of the radioactivity of ADP-Rib and reduction of the inhibition. These results support the idea that the decrease in the template activity for DNA synthesis following incubation of chromatin with NAD is due to ADP-ribosylation of associated chromatin. Incubation of chromatin with NAD does not affect its capacity for RNA synthesis (73). TABLE I1 OF DNA SYNTHESIS INDUCED R Y PoLY(ADP-RIB) THEINHIBITION AND THE EFFECT OF NICOTINAMIDE

Prior incubation in the presence of -

NAD (4mM) NAD (4 mM) NAD (4 mM)

+ nicotinamide (10 mM) + nicotinamide (20 mM)

[JHIdTTP incorporation (cpm/100 mg protein) 2460 250 1500 2060

Inhibition

(%I 90 39 16

POLY (ADENOSINE DIPHOSPHATE RIBOSE)

147

Burzio and Koide (36)extended their work to other rapidly replicating tissues, regenerating liver and Novikoff hepatoma. They found again that DNA synthesis was greatly inhibited by incubation of nuclei from normal and regenerating liver with 4 mM NAD. On the contrary, treatment of nuclei from Novikoff hepatoma with 4 mM NAD did not alter thc template activity for DNA synthesis. They found that Novikoff hepatoma contained less of the total acid-insoluble radioactivity from labeled NAD in the histone fraction than other tissues. This suggests that ADPribosylation may be involved in regulation of DNA synthesis in normal and regenerating liver, but not in Novikoff hepatoma. Hilz and Kittler found that the endogeneous polymerase activity of the nuclei of Ehrlich carcinoma cells and HeLa 53 cells is suppressed by incubation with NAD ( 3 6 ) . This clear-cut difference between the effects of NAD on the nuclei of normal and malignant cells requires further studies. Smulson et al., using synchronized HeLa cells, reported that poly(ADP-Rib) polymerase showed maximal specific activity during the G1 phase and significantly lower specific activity during various periods of the S phase. This suggests that transfer of the ADP-Rib moiety from NAD to nuclear proteins is an important nuclear event at some time immediately before DNA replication ( 7 4 ) .Acetylation of histone showed a maximum in the late S phase in Chinese hamster cells (75) although Pogo et al. indicated that acetylation of histone occurs much earlier than the phytohemagglutinin-induced S phase (76). Poly(ADP-Rib) polymerase may be important in vivo in the control of the tissue concentration of the nicotinamide nucleotide (29). It was once thought that poly(ADP-Rib) might be a reservoir of NAD, but the reverse reaction, which should yield NAD from poly (ADP-Rib) and nicotinamide, was never observed (10). It is still possible that nicotinate can push the reverse reaction to produce deamido-NAD from poly(ADP-Rib) . Deamido-NAD could not be a substrate for polymerization ( l o ) , so that deamido-NAD could be reutilized to form NAD, if the reverse reaction with nicotinic acid was possible (39).The reversibility of formation of ADP-ribosylated nuclear proteins has not been studied extensively.

VIII. Related Phenomena A novel type of pyridine nucleotide, ADP-ribosyl NAD, was found in Azotobacter vinelandii by Imai et al. (77). ADP-ribose is linked glycosidically to NAD a t position 2' or position 3' of the nicotinamide mononucleotide moiety of NAD, giving a mixture of the a- and p-forms in

148

TAKASHI SUGIMURA

the configuration of the nicotinamide N-ribosyl linkage in the molar ratio of about 1 :1. However, ADP-ribosyl NAD did not serve as a coenzyme for yeast alcohol dehydrogenase and was not cleaved by Neurospora crmsa NAD glycohydrolase. Imai and Suzuki (78) also presented evidence of the occurrence of ATP-ribosyl NADP in the extract of Azoto-

bacter vinelandii. 5'-Phosphoribosyl pyrophosphate (P-Rib-PP) and ATP yielded phosphoribosyl-ATP, N-1- (5'-phosphoribosyl) adenosine triphosphate, with phosphoribosyl-adenosine triphosphate:pyrophosphate phosphoribosyltransferase, the enzyme involved in the first step of histidine biosynthesis. It was suggested that this enzymatic reaction proceeds through the formation of a covalent bond between the phosphoribosyl moiety and the enzyme (79). Ames described the spectra of N-1-ribosyladenosine 5'-triphosphate (80), which differ from those of adenosine-ribose. Honjo et al. (81) found that diphtheria toxin transferred the ADPRib portion of NAD to transferase 11,resulting in the inactivation of this enzyme and simultaneous release of a stoichiometric amount of nicotinamide. The covalent bond between ADP-Rib and transferase I1 is rather stable in alkaline conditions, unlike the bond formed on ADP-ribosylation of histone. A further difference of ADP-ribosylation of transferase I1 was that diphtheria toxin can catalyze the reverse reaction to form NAD from ADP-ribosylated transferase I1 and nicotinamide (82, 83). The ADP-ribosylation and the reverse reaction are most active a t p H 8.5 and 5.2, respectively. Diphtheria toxin does not catalyze either hydrolysis of NAD or the exchange reaction between NAD and nicotinamide (83). ADP-ribosylation of transferase I1 was also observed by Collier and Cole (841, Goor and Maxwell (85), Everse et al. (86) and Montanaro et al. (87).

IX. Future Problems Poly (ADP-Rib) was discovered in experiments in vitro on incubation of nuclear preparations of animal cells with NAD by groups of Mandel in Strasbourg ( 4 ) , of Sugimura in Tokyo (6-9) and of Nishizuka in Kyoto (10, 11).The natural occurrence of poly(ADP-Rib) was reported by Doly and Mandel (70).The most important outstanding problems at present are the demonstration of the existence of poly ADP-ribosylated, oligo ADP-ribosylated or ADP-ribosylated nuclear proteins in vivo. The chemical structure of the covalent bond between ADP-Rib and nuclear proteins should also be determined. Studies on phenomena related to chromatin function should also bc uscful. Thesc phcnomena include de-

POLY (ADENOSINE DIPHOSPHATE RIBOSE)

149

pression of template activity for DNA synthesis by poly (ADP-Rib) , inhibition of poly (ADP-Rib) synthesis by thyinidine and changes in the synthesis of poly (ADP-Rib) associated with the cell cycle. The physicochemical and biological properties of the highly purified polymer should also be investigated now that poly (ADP-Rib) can bc obtained in quantity. ACKNOWLEDGMENTS The author is grateful to Drs. S. Fujiniura, S. Haseyawa, Y. Shimizu, H. Okuyama, H. Matsubara, T. Shima, A. Oikawa, M. Yamada, M. Mina, N. Yosliimura, M. Nagao, H. Kagai and T. Matsusliima of this laboratory for their participation in experiments which made it possible to write this article. The work in the author’s laboratory was supported by grants from the Japanese Ministry of Education, the Japanese Ministry of Health and the Waksman Foundation.

REFERENCES 1. P . Chambon, J. D. Weill and P. Mandel, BBRC 11, 39 (1963). 2. S. Fujimura, T. Sugimura, K. Okabe and T. Yoshida, Seikngnki~( J . J a p . Biochem. Soc.) 37, 554 (1965). 3. S. Fujimura and T. Sugimura, SeikagaLzi ( J . Jap. Biochem. Soc.) 38, 691 (1966). Q. P. Cliambon, J. D. Weill, J. Doly, M. T. Strosser and P. Mandel, BBRC 25, 63s (1966). 5. J. Doly and F. Petek, C. R . Acntl. Sci. 263, 1341 (1966). 6 . S. Fujiniura, S. Hasegawa and T. Sugimura, BBA 134, 496 (1967). 7. T. Sugimura, S. Fujimura, S. Hasegawa and Y. Kawamura, BBA 138, 438 (1967). 8. S. Fujimura, S. Hasegawa, Y. Shimizu and T. Sugimura, BBA 145, 247 (1967). 9. S. Hasegawa, S. Fujimura, Y. Shimizu and T. Sugimura, BBA 149, 369 (1967). 10. Y. Nisliizuka, K. Ueda, K. Nakaznwn and 0. Hnyaishi, JBC 242, 3164 (1967). 11. R. H. Reeder, K. Ueda, T. Honjo, Y. Nishizuka and 0. Hayaishi, JBC 242, 3172 (1967). 12. T. Sugimura and S. Fujimura, Proteiir, Nucleic Acid and Enzyme 12, 1059 (1967). 13. T. Sugimura and Y. Shimizu, Seikagakii ( J . Jap. Biochem. Soc.) 40, 137 (1968). 14. J. Doly, P1i.D. Thesis, University of Strasbourg (1968). 16. T. Shima, S. Fujimura, S. Hasegawa, Y. Shimizu and T. Sugimura, JBC 245, 1327 (1970). 16. T. Sugimura, N. Yoshimura, M. Miwa, H. Nagai and M. Nagao, ABB 147, 660 (1971). 17. S. Hasegawa, S. Fujimura, Y. Shimizu, H. Okuyama and T. Suginiura, Seikagnku ( J . Jap. Biochem. Soc.) 39, 702 (1967). 18. E. B. Kellrr, BBlIC 17, 412 (1964). 19. E. Sulkonski and M. Laskowski, BBA 240, 443 (1971). 20. M. Yamada, unpublished. 91. M. Miwa, H. Nagni, T. Sugimura, M. Yamnda and N. Yoshimura, Seikagaku ( J . Jnp. Biochem. Soc.) 43, 685 (19771). 2.2. T. Shima. S. Hasegawa, S. Fujimura, H. Matsubara and T. Sugimura, JBC 244, 6632 (1969). 23. S. Fujimurn and T. Suyimura, in “Mctliods in Enzynioloyy,” Vol. 18U (D. B.

150

TAKASHI SUGIMURA

McCormick and L. D. Wright, vol. eds.), p. 223. Academic Press, New York, 1971. 34. H. Matsubara, S. Hasegan-a, S. Fujimura, T. Shima, T. Sugimuia and M. Futai, JBC 245, 3606 (1970). 26. H. Matsubara, S. Hascgtiwa, S. Fujimura, T. Shima, T. Sugimuia and M. Futai, JBC 245, 4317 (1970). 2G. R. H. Hall, Bchem 4, 661 (1965). ST. J. Chauveau, Y. MoulB and C. Rouiller, Exp. CeZl Res. 11, 317 (1956). 98. K.Marushige and J. Bonner, JMB 15, 160 (1966). 29. Y. Nishizuka, K . Ueda and 0. Hayaishi, in “Methods in Enzymology,” Vol. 18B (D. B. McCormick and L. D. Wright, vol. eds.), p. 230. Academic Press, New York, 1971. SO. Y. Nishizuka, K. Ueda, K. Nakazawa, R. H. Rccdcr, T. Honjo and 0. Hayaishi, J . Vitaminol. 14, Suppl. 1, 143 (1967). 31. T. Sugimura, unpublished. 32. J. Preiss, R. Schlaeger and H. Hilz, FEBS Lett. 19, 244 (1971). 33. H. Hilz and M. Kittler, Hoppe-Seyler’s Z.Physiol. Chem. 349, 1793 (1968). 34. S. Fujimura, Y. Shimizu, S. Hasegawa and T. Sugimura, Proc. Jap Cancer Ass. 26th Annu. Meeling 105 (1967). 36. L. Burzio and S. S. Koidr, FEBS Lett. 20, 29 (1972). 36. H. Hilz and M. Kittler, Hoppe-Seyler’s 2. Physiol. Chem. 352, 1693 (1971). 37. I. R. Johnston, A. P. Mathias, F. Prnnington and D. Ridge, Nature (London) 220, 668 (1968). 3s. I. R. Johnston, A . P. Mathias, F. Pennington and D. Ridge, BJ 109, 127 (1968). 39. M. E. Haines, I. R. Johnston, A. P. Mathias and D. Ridge, BJ 115, 881 (1969). 40. T. Sugimura, S. Fujimura, S. Hasegawa, Y. Shimizu and H. Okuyama, J . Vitamind. 14, 135 (1968). 41. K. Nakazawa, K. Ueda, T. Honjo, K. Yoshihara, Y. Nishizuka and 0. Hayaishi, BBRC 32, 143 (1968). 42. Y. Shimizu, S. Hasegawa, S. Fujimura and T. Sugimura, BBRC 29, 80 (1967). 43. M. Yamada, M. Miwa and T. Sugimura, ABB 146,579 (1971). 4.4. Y. Nishizuka, K. Ueda, K. Yoshihara, H. Yamamura, M. Takeda and 0. Hayaishi, CSHSQB 34, 781 (1969). 46. K. Ueda, R. H. Reeder, T. Honjo, Y. Nishizuka and 0. Hayaishi, BBRC 31, 379 (1968). 46. S. Hasegawa and T. Sugimura, unpublished. 47. A. Oikawa, Y. Itai, H. Okuyama, S. Hasegawa and T. Sugimura, Exp. Cell Res. 57, 154 (1969). 48. A. Oikawa and T. Sugimura, unpublished. 49. M. Futai, D. Mizuno and T. Sugimura, JBC 243, 6325 (1968). 60. M.Miwa and T. SugimurB, JBC 246, 6362 (1971). 61. K. Ueda, J. Oka, S. Narumiya, N. Miyakawa and 0. Hayaishi, BBRC 46, 516 (1972). 62. E. W. Johns, D. M. P. Phillips, P. Simson and J. A. V. Butler, BJ 77, 631 (1960). 63. Y.Nishizuka, K. Ueda, T. Honjo and 0. Hayaishi, JBC 243, 3765 (1968). 64. H. Otake, M. Miwa, S. Fujimura and T. Sugimura, J . Biochem. 65, 145 (1969). 66. R. C. Huang and J. Bonner, PNAS 54, 960 (1965). 66. W.J. Steele and H. Busch, Cancer Res. 23, 1153 (1963). 67. M.Miwa and T. Sugimura, unpublished. 68. M. Meilhnc, P1i.D. Thesis, Univewity of Strasbourg (1970).

POLY (ADENOSINE DIPHOSPHATE RIBOSE)

151

87, 258 (1963). GO. K. Murray, Bchem 3, 10 (1964). 61. M. G. Ord and L. A. Stocken, BJ 102, 631 (1967). G2. V. Riimer, J. Lambrecht, M. Kittler and H. Hilz, Hoppe-Seyler’s 2. Physiol. Chem. 349, 109 (1968). 03. J. B. Clark, G. M. Ferris and S. Pinder, BBA 238, 82 (1971). G4. S. G.A. Alivisatos, Federation Proc. 24, 769 (1965). G6. K. W.Bock, V. Gang, H. P. Beer, R. Kronau and H . Grunicke, EJB 4, 357 ( 1968). GO. L. J. Zatman, N. 0. Kaplan and S. P. Colowick, JBC 200, 197 (1953). G7. M. Yamada, M. Miwa and T. Sugimura, Seikagaku ( J . Jap. Biochem. Soc.) 42, 477 (1970). GS. M. Futai and D. Mizuno, JBC 242, 5301 (1967). GO. M. Futai, D. Miauno and T. Sugimura, BBRC 28, 395 (1967). 70. J. Doly and P. Mandel, C. R . Acad. Sci. 264, 2687 (1967). 71. L.Burzio and S. S. Koide, BBRC 40, 1013 (1970). 72. K. Ueda, Seikagaku ( J . Jap. Biochem. Soc.) 43, 438 (1971). 73. L.Burzio and S. S. Koide, BBRC 42, 1185 (1971). 74. M.Smulson, 0 . Henrilrsen and C. Rideau, BBRC 43, 1266 (1971). 76. G. R. Shephard, B. J. Norlond and J. M. Hardin, BBA 228, 544 (1971). 76. B. G. T. Pogo, V. G. Allfrey and A. E. Mirsky, PNAS 55, 805 (1966). 77. T.Imai, S . Okuda and S. Suzuki, JBC 244, 4547 (1969). 7s. T.Imai and S. Suauki, Protein, Nucleic Acid and Enzyme 15, 162 (1970). 78. R.M. Bell and D. E. Koshland, Jr., BBRC 38, 539 (1970). SO. B.N. Ames, R. G. Martin and B. J. Garry, JBC 236, 2019 (1961). 81. T.Honjo, Y. Nishizulca and 0. Hayaishi, CSHSQB 34, 603 (1969). 82. T.Honjo, Y. Nishiauka, 0. Hayaishi and I. Kato, JBC 243, 3553 (1968). 83. T. Honjo, Y. Nishizuka, I. Kato and 0. Hayaishi, JBC 246, 4251 (1971). 84. R. J. Collier and H. A. Cole, Science 164, 1179 (1969). 86. R.S. Goor and E. S. Maxwell, JBC 245, 616 (1970). 86. J. Eversc, D. A. Gardner, N. 0. Kaplan, W. Galasinski and K . Moldave, JBC 245, 899 (1970). 8Y. L. Montanaro, S. Sperti and A. Mattioli, BBA 238, 493 (1971). 69. D. M. P. Phillips, BJ

This Page Intentionally Left Blank

The Stereochemistry of Actinomycin Binding to DNA , and Its Implications in Molecular Biology HENRYM. SOBELL*

'

1

Department of Chemistry, The University of Rochester, Rochester, New York, and Department of Radiation Biology and Biophysics, The University of Rochester, School of Medicine and Dentistry, Rochester, New York

I. 1ntroduct.ion . . . . . . . . . . . . . 11. Solution Studies of the Actinomycin-DNA Interaction . . . A. Evidence Concerning the Polynucleotide Conformation . . B. Evidence Favoring Hydrogen-Bonded Recognition . . . C. Evidence Favoring Intercalation . . . . . . . . D. The Actinomycin-Deoxyguanosine Model Reaction . . . 111. The Actinomycin-DeoxyguanosineCrystalline Complex . . . A. Actinomycin Has 2-fold Symmetry . . . . . . . B. The Complex Has 2-fold Symmetry . . . . . . . C. The Water Structure . . . . . . . . . . IV. The Aetinomycin.DNA Complex . . . . . . . . A. Intercalation and Hydrogen Bonding to Base-Paired d G d C Sequences . . . . . . . . . . . . . B. Detailed Stereochemical Model . . . . . . . . C. Supporting Evidence . . . . . . . . . . D. Other Intercalating Agents . . . . . . . . . V. A General Principle Governing Protein-Nucleic Acid Recognition . A. Nuclease Specificity . . . . . . . . . . . B. Do Operators and Repressors Have 2-fold (or 4-fold) . . Symmetry? . . . . . . . . . . . C. A Model for Genetic Recombination . . . . . . . D. DNA Replication and Chromosome Structurc . . . . . VI. Possible Medical Implications . . . . . . . . . VII.Summary . . . . . . . . . . . . . . References . . . . . . . . . . . . . .

153 155 155 155 156 157 158 158 159 163 165 165 168 171 175 178 178 180 182 185 187 188 189

1. Introduction Actinomycin C1(D) is a cyclic-polypeptide-containing antibiotic widely used in the study of molecular and cell biology (Fig. 1). It is

* Present address : Department of Pharmncology, Stanford Universit.y School of Medicine, Pnlo Alto, California. 153

154

HENRY M. SOBELL

0

0

L-MeVal

Sar

o-Val

L - T ~ H,C/

MI

HN

L o I

I

CH,

o=c

/

CH,

I

I CHS

FIG. 1. Structure of actinomycin C,(D). Abbreviations: MeVal, methylvaline; Sar, sarcosine ; Pro, proline ; Val, valine ; Thr, threonine.

one of the most potent antitumor agents known, although, because of its extreme toxicity, has found only limited clinical use. The activity of the antibiotic stems from its ability to bind double-helical DNA and to inhibit specifically RNA synthesis (1-6).Deoxyguanosine residues are essential for the binding reaction as shown by extensive studies with naturally occurring and synthetic DNA duplexes (4, 6, 7 ) . The spectral perturbations that accompany actinomycin binding to DNA can be mimicked by the addition of deoxyguanosine to solutions of actinomycin (8),although similar spectral changes can be induced with a variety of other purines and organic aryl sulfonates (6, 9). Two models have been proposed to explain the binding of actinomycin to DNA. Thc first of these involves direct hydrogen-bonding recogniti’on between the guanine ring and the chromophore residue and is based on studies with a series of synthetic polymers possessing or lacking 2-amino functional groups on purine residues, as well as careful characterization of chcrnical groups on actinomycin essential for binding (10-12).The second model, based on detailed spectroscopic, hydrodynamic and kinetic measurements, and supported by observations that actinomycin promotes the unwinding of supercoilccl covalently circular DNA, involves inter-

STEREOCHEMISTRY OF ACTINOMYCIN BINDING TO

DNA

155

calation of the phenoxazone ring system between base pairs in the DNA helix, analogous to the binding of aminoacridines to DNA (9, 13, 14). This model, however, leaves unexplained the origin of guanine specificity in the binding reaction. We have already described a detailed molecular model for actinomycin-DNA binding which uses the geometry determined by X-ray crystallography of a n actinomycin. deoxyguanosine crystalline complex (15-19). Thc model is of broad interest for several reasons. I n the first place, it provides a structural explanation for a large mass of physical and biochemical data concerning the interaction of actinomycin with DNA and this, for the first time, correlates the three-dimensional structure of an antibiotic with its biological action. Second, the binding of actinomycin to DNA demonstrates two important structural features utilized by DNA in binding other types of molecules. The first of these, intercalation, is used in binding a large variety of drugs and antibiotics to the DNA helix (14), and the model provides detailed stereochemical information concerning this. The second feature concerns the demonstration of a general principle that several classes of proteins may utilize in recognizing symmetrically arranged nucleotide sequences on the DNA helix (,%-2S). The actinomycin-DNA stereochemical model presented here provides detailed information concerning this important principle, a principle central in our understanding of protein-nucleic acid recognition.

II. Solution Studies of the Actinornycin-DNA Interaction A large body of evidence is available concerning the interaction of actinomycin with DNA. This can be summarized as follows:

A. Evidence Concerning the Polynucleotide Conformation Actinomycin binds tightly to double helical DNA, but poorly, if a t all to double-helical RNA, an RNA-DNA hybrid helix, or single-stranded forms of DNA or RNA (24-26).

B. Evidence Favoring Hydrogen-Bonded Recognition The binding of actinomycin to DNA requires the presence of deoxyguanosine residues, the 2-amino group on guanine being essential for the binding reaction ( 4 ) . Thus, synthetic DNA-like polymers of the type, d(A-T),,, dA,,-dT,, d(1-C), and dI,.dC,, do not bind actinomycin, whereas polymcrs of the type, d (G-C) ,,, dG, dC,,, d (a2R-T)n1 and a?R = 2,6diaminopuriue nucleoside ; azR = 2-aminopurine nucleoside.

156

HENRY M. SOBELL

FIG.2. Unit structures of the synthetic polymers used in actinomycin binding studies. a2R = diaminopurine (nucleoside) . The associated homopolyrners, poly(dG* dC), poly (dI*dC), and poly(dA.dT) have also been synthesized and their binding properties studied. The 2-amino group on the purine residue appears to be essential for thc actinomycin binding reaction.

d(a2R-T),,l bind actinomycin tightly (7, 11, 24) (Fig. 2). The 2-amino group on the purine residue appears to be essential for the binding reaction. Substitution of the 2-amino group on the phenoxazone ring system with a hydroxyl-, chloro-, or a dimethylene amino group inhibits the actinomycin-DNA binding reaction (9,19).

C. Evidence Favoring Intercalation Addition of actinomycin to solutions containing low molecular weight (lo5) DNA causes an increase in the intrinsic viscosity and a decrease in the sedimentation constant for the complex ( 9 ) . Thc spectral changes observcd when actinomycin binds to DNA can be mimicked by the addition of a large number of different aryl sulfonates to solutions of actinomycin (9). The kinetics of association and of dissociation of actinomycin from DNA are sensitive to chemical substituents introduced on the 7-position of the phenoxazone ring system (9).

STEREOCHEMISTRY OF ACTINOMYCIN BINDIXC TO

DNA

157

The binding of actinomycin to covalently circular supercoiled DNA causes unwinding of the DNA, as evidenced by its sedimentation behavior a t increasing concentrations of actinomycin (IS).

D. The Actinomycin Deoxyguanosine Model Reaction It has been known for many years that a variety of purine nucleosides form complexes with actinomycin resulting in spectral changes similar to those observed in the actinomycin-DNA interaction (8). Of all the purine nucleosides tested, deoxyguanosine complexes most tightly with actinomycin, the association constant being 1.7 X lo3 M-l (26, 27). If ethanol-water solutions of actinomycin and deoxyguanosine are prepared

FIG.3. Single crystals of the actinomycin*deoxyguanosinecrystalline complex. Approximately X20.

158

HENRY M. SOBELL

and allowed to evaporate slowly at room temperature over several days, large single crystals develop containing actinomycin and deoxyguanosine cocrystallized as a 1:2 stoichiometric complex (Fig. 3 ) . The stoichiometry of the solid-state complex appears to be characteristic of the crystalline complex itself and does not reflect thc solution stoichiomctry from which the crystals arise.

111. The Actinomycin Deoxyguanosine Crystalline Complex Details of the X-ray analysis are not presented here, as they have already appeared (18). The crystalline complex consists of one actinomycin, two deoxyguanosine, and twelve water molecules, a total of 140 atoms (excluding hydrogens) in the asymmetric unit. The structure is observed a t atomic resolution.

A. Actinomycin Has 2-fold Symmetry The 2-fold symmetry is shown in Fig. 4, which shows a computerdrawn illustration of the actinomycin molecule as viewed down its approximate dyad axis (lying roughly along a vector connecting the 0-N bridging atoms in the phenoxazone ring). Although the symmetry is not exact (exact symmetry would require the amino group and the quinoid oxygen to be on both sides of the chromophore residue), both polypeptide chains closely obey this noncrystallographic 2-fold symmetry. The conformations of the peptide linkages are as follows: L-threonine-Dvaline, trans; D-valine-L-proline, cis; L-proline-sarcosine, cis; sarcosineL-methylvaline, trans; L-threonine-carboxamide carbonyl oxygen and carbon of chromophore, trans. A strong hydrogen bond exists between neighboring cyclic pentapeptide chains connecting the N-H of one Dvaline residue with the carbonyl oxygen of the other D-valine residue [2.94, 2.96 A]. No other hydrogen bonds stabilize the actinomycin structure either between chains or within chains. These findings are consistent with nuclear magnetic resonance data implicating the D-valine N-H groups as hydrogen bond donors, and also serve to confirm several of the assignments made regarding the conformation of the polypeptide chains (2830). It is evident from Fig. 4 that the quinoidal portion of the phenoxazone ring system is slightly twisted in a propeller-like fashion with respect to the aromatic portion of the chromophore. A marked deviation from planarity is observed for the amino group nitrogen atom, which lies 0.5 A from the least-squares plane of the phenoxazone ring system.

STEREOCHEMISTRY OF ACTINOMYCIN BINDING TO

DNA

159

FIG.4. Computcrdrnwn illustrniion of the nctinomycin molecule viewed down its approximate dyad axis. The phenoxazone ring is in the center of the figure and projects out toward the viewer. Hydrogen bonds connecting D-valine residues on neighboring chains are indicated by dashed lines. Note the twist of the quinoidal portion of t.he phenoxaaone ring system and the nonplanarity of the 2-amino group.

B. The Complex Has 2-fold Symmetry This symmetry is shown in Fig. 5. The two deoxyguanosine molecules interact with the two cyclic pentapeptide residues and stack on alternate sides of the phenoxazone ring system. A strong hydrogen bond [2.80, 2.82 A] connects the guanine %-amino group with the carbonyl oxygen of the L-threonine residue. A weaker hydrogen bond connects the guanine N-3 with the N-H group on this same L-threonine residue [3.15,3.25 A]. The conformation of both deoxyguanosine molecules is anti, this describing the relative orientation of the deoxyribose residues with the guanine

HENRY M. GOBELL

160

P

Q

F I ~5. . Same view as in Fig. 4, but with the two deoxyguanosine molecules in place. The approximate 2-fold symmetry of the complex is apparent. Notice a slight altcrat,ion in thc stacking of tlic deoxyguanosinc molecule immediately abovc the quinoidnl portion of the plienoxasone ring system, resulting from the nonplanarity of the chromophore in this region. Dotted lines indicate hydrogen bonds connecting thc actinomycin pcntnpeptidc chains with thc deoxygunnosine molccules.

rings ( 3 1 ) .One deoxyribose residue is puckered C3’endo-C2’exo, while the other is C2’endo-C3’em (32).The sugar residues of both deoxyguanosine molecules arc in closc stcric juxtaposition with the isopropyl groups of the L-methylvaline residues (see Fig. 6 ) , and hydrophobic contacts such as these, as well as the stacking of guanine and phenoxazone rings, imparts stabilization to thc complex. Hydrogen bonding, however, plays a key role in the association, and this explains the specificity that actinomycin dcmonstratcs for guanine residues in the modcl reaction ancl in the binding reaction with DNA. Related studies of associations bctwecn monomer

STEREOCHEMISTRY O F ACTINOMYCIN BINDING TO

DNA

161

FIG.6. Coniputcr-drawn illuxtration of tlic actinoni~cin.deoxyguanosine coinplcx viewed at an 80" angle to the phenoxazone ring system showing the sandwichlke stacked arrangement of guanine and phenoxazone rings. Dotted lines indicate hydrogen bonds connecting the nctinomycin pcntapeptide chains with the deoxyguanosine molecules.

purines and pyrimidines in solution and in thc crystalline state have demonstrated the importance of hydrogen bonding in base-pairing recognition (33-35), and, although the details of the stereochemistry differ, the same is truc in this examplc of protein-nucleic acid recognition. The 1 :Z stoichiotnetry of the complex is a direct consequence of the ,%fold symmetry of actinoinylcin and reflects the two chemically equivalent binding sites available to deoxyyuanosine for coinplex formation. It will bc scen that this has clccpcr mcaiiing with regard to actinomycin DNA binding, and, perhaps, to repressor-operator binding generally.

-

162

-...

HENRY M. SOBELL

-R

STEREOCHEMISTRY O F ACTINOMYCIN BINDING TO

DNA

163

C. The Water Structure The complete crystal structure is shown in Fig. 7. The structure contains 140 atoms in the asymmetric unit, of which 12 atoms are water oxygens (these and several symmetry-related water oxygens are shown as black balls). The water structure surrounding the complex is of particular interest in view of the large positive entropy change accompanying actinomycin -DNA binding, which probably reflects the rearrangement of water structure during the binding reaction ( 2 6 ) .Similar, but smaller, entropic changes accompany the actinomycin-deoxyguanosine solution association. The structure is stabilized by a total of 152 hydrogen bonds in each unit cell. Approximately 80% of these involve interactions with water structure. I n contrast with the symmetry observed in the complex, there appears to be little or no symmetry in the immediate water structure surrounding the complex. This reflects, in part, the amino and quinoid oxygen groups on the phenoxazone ring system, which are involved in water hydrogen bonding contacts, as well as a large amount of intermolecular hydrogen bonding between symmetry-related complexes that involve water bridges between various functional groups on the nucleosides and peptide chains. A significant number of water-water hydrogen bonding contacts are observcd, some of these resulting in “icelike” tetrahedrally coordinated structures. However, the majority act to bridge various functional groups on actinomycin and deoxyguanosine molecules, both within and between symmetry-related asymmetric units. The water structure appears to be well ordered and rigidly held in this structure (in contrast with thc situation in protein crystals), and this probably accounts for the hardness and excellent diffraction these crystals demonstrate. Almost all donor and acceptor groups are involved in hydrogen bonding. Exceptions are the nitrogen-7 on one deoxyguanosine and the methylFIG.7. The actinomycin*deoxyguanosine crystal structure as viewed down the axis. Black circles with highlights indicate water molecules. Deoxyguanosine molecules have been shaded with striped lines and are seen stacked above and below the plane of the cliromophore residue. The actinomycin molecule is indicated with dark open lincs and circlcs. Tlic surrounding crystal structurc is shown with lighter lines. Black dots indicate hydrogen bonds bctwecn atoms at neighboring levels. Daslicd lines indicate hydrogen bonds between atoms located at different levels in adjoining unit cells. Additional hydrogen bonding in tlic surrounding structure is indicated by small opcn circlcs. From Jain and Sobcll (IS). c

164

HENRY M. SOBELL

FIQ.8. Stepwise assembly of actinomycin.DNA complex using the space-filling Corey-Pauling-Koltun (CPK) molecular models. Top, left and right : actinomycin viewed down its dyad axis and perpendicular to the plane of thc chromophore, respectively. Middle, left and right: same views of act,inomycin.deoxygunnosine complex. Lower, left and right: same views of actinomycin. (dGdC) complex.

STEREOCHEMISTRY OF ACTINOMTCIK BINDING TO

DNA

165

valine carbonyl oxygen atom on one of the two pentapeptide chains. Although stacking of guanine and phenoxazone rings imparts energy of stabilization to the complex, it is clear that hydrogen bonding accounts for a far larger contribution to the total free energy of the lattice.

IV. The Actinomycin DNA Complex A. Intercalation and Hydrogen Bonding to BasePaired dG-dC Sequences This section provides a model for actinomycin- DNA binding that uses the geometry determined in the actinomycin. deoxyguanosine crystalline complex (Fig. 8 ) . Deoxycytidine 5'-phosphate is placed opposite deoxyguanosine to form a hydrogen-bonded guanine cytosine base pair. Adjacent deoxyguanosine and deoxycytidine residues immediately above and below the chromophore can then be connected by a phosphodiester bridge, giving rise to the sequence dG-dC. An immediate consequence of this is the close steric juxtaposition of the 2-amino group on the chromophore residue and a phosphate oxygen. A close steric contact is also noted between this amino group and the deoxycytidine furanose ring oxygen. These contacts suggest possible hydrogen bonding between these groups, although, in the case of the phosphate oxygen, hydrogen bonding may utilize a water bridge. It is of interest that, whereas this amino group appears to be important for actinomycin-DNA binding, it is not essential for the actinomycin .deoxyguanosine solution association (S6).This had previously suggested a geometric difference between these associations, but this must now be reexamined, Figures 9 and 10 show the complex with two base pairs added above and below the central intercalated base-paired dG-dC sequence. The 2-fold symmetry of the entire complex is apparent, this reflecting the 2-fold symmetry of the hexanucleotide duplex (both the sugar-phosphate chains as well as, in this case, the polynucleotide base sequence) coinciding with the 2-fold symmetry relating subunits on the actinomycin molecule. This important principle, first recognized by Bernardi in his studies of the nuclease enzyme, spleen acid deoxyribonuclease ( 2 0 ) , may be a general one used in protein-nucleic acid recognition and is discussed in detail below. The model predicts, therefore, that the chromophore on actinomycin intercalatcs between the base-paired dinucleotide sequence, dG-dC, while the peptide subunits lie in the narrow groove of the DNA helix and interact with deoxyguanosine residues on opposite chains through specific hydrogen bonds.

-

166

HENRY M. SOBELL

FIG.9. CPK space-filling model of actinomycin complexed with six base pairs, as viewed from the deep groove of the DNA helix. This model is obtained by adding two base pairs above, and two below, thc central intercalated d G d C sequence, shown in the lower portion of Fig. 8. The 2-fold symmetry of this complex is apparent.

STEREOCHEMISTRY OF ACTINOMYCIN BINDIX’G TO

DNA

167

FIG.10. CPK space-filling model of actinomycin complexed with six base pairs as viewed from the narrow groove of the DNA helix. The peptide subunits of actinomycin form numerous van der Waals contacts with atoms in both sugar-phosphate chains. The 2-fold symmetry of this complex is apparent,.

168

HENRY M. SOBELL

8. Detailed Stereochemical Model It is informative to review in detail the steps in assembly of the actinomycin- DNA hodel, beginning with the actinomycin molecule and the configuration obsetved in the actinomycin. deoxyguanosine crystalline complex. This is shown in Fig, 11 (A-D), and in Figs. 12 and 13 [these figures have been obtained from coordinates of the actinomycin. DNA model after careful model building studies using the Kendrew skeletal models ( i ' g ) ] . Figure 11A shows the actinomycin molecule viewed down its dyad axis. The two cyclic pentapeptide chains have been idealized to be related by exact 2-fold symmetry. The 7-carbon of the proline ring is puckered approximately 0.5A from the plane of the other four ring atoms and projects forward toward the viewer. This 2-fold symmetry means that the chemical environment above the plane of the chromophore is reproduced below the plane of the chromophore, neglecting the asymmetry of the phenoxazone ring system. It is not surprising, therefore, that the two deoxyguanosine molecules interact on opposite sides of the phenoxazone ring with the pentapeptide chains and that the complex has 2-fold symmetry (shown in Fig. 11B). The slight asymmetry in the complex reflects the twisting of one side of the phenoxazone ring system, which causes a small but significant stacking orientation change for the deoxyguanosine molecule located immediately above. Next, deoxycytidine is placed opposite deoxyguanosine using the Watson-Crick hydrogenbonded geometry. A phosphodiester bridge can then be formed connecting deoxyguanosine and deoxycytidine residues on either side of the phenoxazone ring, generating the unique dinucleotide sequence, dG-dC (Fig. 11C). Two base pairs are next added above and two below the central intercalated dG-dC sequence (Fig. 11D). The complete hexanucleotide sequence shown is $(A-T-G-C-A-T) . This DNA fragment (six base F i r s , or twelve nucleotides) interacts with. four hydrogen bonds and numerous van der Waals interactions with the actinomycin molecule. Figures 12 and 13 demonstrate steps in assembly of the actinomycin-DNA model as viewed from different angles in stereo pairs. Major structural features of the actinomycin * DNA stereochemical model therefore include: 1. Intercalation of the phenoxazone ring system between the baseFIG.11. Steps in assembly of detailed actinomycinaDNA stereochemical model. (A) Actinomycin, viewed down its approximate dyad axis. (B) the actinomycin. deoxyguanosine complex viewed down its dyad axis (compare with Fig. 5 ) . ( C ) The actinomycin. (dG-dC) complex. ( D ) The actinomycin- ( d A d T d G d C d A d T ) complex. Dotted lines indicate hydrogen bonds. Helix axis is shown by vertical dnsheddotted line. Arrows indicate 5' + 3' direction of DNA chains.

This Page Intentionally Left Blank

STEREOCHEMISTRY OF ACTINOMYCIN BINDING TO

DNA

169

FIGS.12 ( l o p ) and 13 (bottom). Coinl)utrr-l$~~nerat~d stereo pairs showing the steps in assembly of the actinomycin DNA model. These were kindly supplied by Dr. Louis Katz, Graphic Facility for Interactive Displays, Department of Biological Sciences, Columbia University, New Yo&, New York.

170

HENRY &I. SOBELL

paired dinucleotide sequence, dG-dC. This results in movement of both guanine residues approximately 1.25 A toward the helix axis and unwinding the helix - 18”.Both the chromophore and the guanine-cytosine base pairs are tilted 10” to a plane normal to the helix axis. The nonplanarity of the quinoidal portion of the phenoxazone ring system alters the stacking of guanine and cytosine residues immediately above and below, and this results in a twist of 8” between guanine-cytosine rings. Local asymmctry therefore exists in the DNA structure a t the site of intercalation. 2. Conformational changes in the sugar-phosphate backbone. The sugar puckering for both deoxyguanosine residues can be described by C3‘endo-C2’ezo. The puckering for all other sugar residues is C2’endoC3‘exo. The sugar pucker in the I3 form of DNA is also C2’endo-C3’ezo, whereas in the A form i t is C3’endo-C2’exo (37). Deoxyribonucleotides have considerable flexibility in this regard ( 3 8 ) , and it is not entirely unexpected that both sugar conformations appear in the intercalation site. The stereochemistry of the polynucleotide chain in the immediate vicinity of intercalation is best described by the conformation about the C4’-C5’ bond for deoxycytidine residues, this being gauche-&. All other residues are gauche-gauche in this respect, with the exception of deoxyguanosine residues, which are cis-gauche. The gauche-cis and cis-gauche conformations are stereochemically acceptable conformations, although they would be expected to have somewhat higher energies than gauchegauche or gauche-trans conformations. We feel that these are not unreasonable, however, in view of the large number of van der Waals contacts in the actinomycin. DNA complex that stabilize this structure. 3. The pcptide portion of the actinomycin molecule lying in the narrow groove of the DNA helix, connected through hydrogen bonds to guanine residues. A strong hydrogen bond [2.90, 2.93 A] connects the guanine 2-amino group with the carbonyl oxygen of the L-threonine residue, while a weaker hydrogen bond connects the guanine N-3 with the N-H group on this same L-threonine residue [2.97,3.08 A]. The hydrogenbonded configuration closely resembles that found in the actinomycin deoxyguanosine crystalline complex (compare Figs. 5 and 11B).I n addition to these hydrogen bonds, numerous van der Waals contacts occur between hydrophobic groups on the actinomycin molecule and the DNA structure and these provide additional stabilization to the interaction. The two cyclic pentapeptide chains are related by 2-fold symmetry. The axis of symmetry relating subunits on actinomycin coincides with the axis of symmetry relating the sugar-phosphatc backbone and base sequence on the DNA helix. 4. Thymine-adenine base pairs immediately proximal to the intercalated dG-dC dinucleotide sequence undergo unwinding. The twist angle

-

STEREOCHEMISTRY OF ACTINOMTCIN BINDING TO

DNA

171

is reduced from 36" in the B form of DNA to 28". This partly reflects the C3'endo-C2'ezo sugar pucker for deoxyguanosine residues and the base tilt of 10" that the thymine-adenine base pairs make with a plane normal axis of symmetry relating subunits on actinomycin coincides with the proline residues on the actinomycin pentapeptide chains. The adeninethymine base pairs farthest from the intercalation site are also tilted 10" .to the plane normal to the helix axis; however, they are related by a twist of 36" from the preceding thymine-adenine base pairs. The total unwinding angle generated when actinomycin binds to DNA is, therefore, -18" (-lSo), or -34". 5. An additional hydrogen bond (not shown) possibly connecting the 2-amino group on actinomycin with a phosphate oxygen on the neighboring sugar-phosphate chain through a water bridge. Another possible hydrogen bond may involve this amino group and the furanose ring oxygen on the deoxycytidine residue [3.20 A].

+

C.

Supporting Evidence The stereochemical model we propose reconciles the data suggesting intercalation as well as hydrogen-bonded recognition, and, in a sense, is a synthesis of the two previous models advanced for actinomycin DNA binding. The specificity actinomycin demonstrates in binding doublehelical DNA, but not double-helical RNA or an RNA-DNA hybrid helix, most probably reflects the steric requirement imposed by the cyclic pentapeptides to recognize the B rather than the A form of a polynucleotide duplex. The rigidity that double-helical RNA and the RNA-DNA hybrid helix demonstrate with regard to their A conformation may be equally important in excluding actinomycin binding (39-41). The large number of favorable van der Waals contacts made between the cyclic peptides and atoms in the narrow groove of the DNA helix undoubtedly provide added stabilization to the complex and act to exclude water during complex formation. Amino-acid substitutions that interfere with this favorable fit would reduce the biological potency of the antibiotic, and this is the case, as seen in Table I. When proline is replaced by hydroxyproline (or hydroxyproline acetate) in one cyclic pentapeptide, the biological activity of the antibiotic is diminished some 20-fold. When, however, proline is replaced by L-allohydroxyproline (or L-allohydroxyproline acetate) , there is only a small change in biological activity. This position effect is readily interpretable from the actinomycin DNA stereochemical model presented hcre. An immediate prediction that this model makes is that poly[d(G-C)] should bind actinomycin most firmly and with highest efficiency (every twelve nucleotides, or every six base pairs) of all the synthetic DNA

-

172

HENRY M. SOBELL

TABLE I EFFECT OF SELECTED SINGLEAMINO-ACID SUBSTITUTIONS ON ANTIBACTERIAL ACTIVITYOF ACTINOMYCINO Amino-acid residue in position 3 of chain Actinomycin

c1

100

Xa

215

X08

57

Xo8 AC

xos

2.5

XoB Ac

0

200 50 25 5 1.5

100

x1.

A

B

L-Pro L-7-oxoPro L-aHYP L-aHypOAc L-HYP bHypOAc L-7-oxoPro

L-Pro LPro >Pro GPro L-Pro L-Pro Sar

Relative activity

From Reich and Goldberg (6).

duplexes, and this is verified in Table 11. Other sequences containing guanine can bind actinomycin, but with lower affinity and with less efficiency. This can be explained by assuming a complex with similar geometry, but forming fewer (less than four) hydrogen bonds between the cyclic pentapeptides and the dinucleotide base-paired sequence. There may be steric strain accompanying this altered interaction, and this could explain the diminished efficiency of binding observed with these polymers. Related data on the interaction between actinomycin and a large number of deoxyribo-dinucleotide monophosphates are now available (42). The dinucleotide, pdG-dC, interacts strongly with actinomycin and in a coTABLE I1 EQUILIBRIUM CONSTANTS AND NUMBER OF BINDING SITESFOR DNA-ACTINOMYCIN INTERACTIONS" DNA

%G+C

72 42 100 100 P~ly[d(T-G)]*p~ly[d(C-A)] 50 Poly[d(T-C)].poly[d(G-A)] 50 POI~[~(T-T-G)].~O~~[~(C-A-A)] 33 Po~~[~(T-T-C)I*~O~~[~(G-A-A)] 33 Poly[d(T-A-C)].poly[d(G-T-A)] 33 Poly(d1) Micrococcus luteus DNA Salmon sperm DNA Poly[d(G-C)] Poly(dG)*poly(dC)

a

From Wells and Lanon (7).

KaPp(M+) x 10-6

Nucleotides per site

2.5

9 20 12 91 25 37 45 67 50 111

2.0

3.2 2.0 1.3 0.6 1.2 0.7 0.8 1.3

STEREOCHEMISTRY OF ACTINOMYCIN BINDING TO

DNA

173

pdG-dA

d pdA-dT

/ 0.W

0.87

1.31

1.75

(NUCLEOT I DEI m M

FIG. 14. (A) Actinomycin.deoxyribodinuc1eotide binding studies, as monitored by difference spectra at 425 nm. N corresponds to thymidine, cytidine, adenosine or guanosine. Actinomycin concentration, 20 p M . (B) Actinomycin.pdG-dC and *pdCdG binding curves. Actinomycin conrentration, 36 p M . Redrawn from Krugh (U).

operative fashion (see Fig. 14A and B ) . Other sequences (either individually, or as complementary pairs) bind actinomycin less tightly and do not demonstrate cooperativity. It is of interest that the four dinucleotides, pdC-dG, pdT-dG, pdA-dG and pdG-dG (in the presence or the absence of the complementary sequence), resemble deoxyguanosine in

174

HENRY M. SOBELL

their binding properties, the reaction being distinctly first order in character. Dinucleotides of the type pdG-dT and pdG-dA bind actinomycin less tightly, the former, however, binding actinomycin more tightly than the latter. The reaction, again, appears to be first order in character. These observations can be explained in stereochemical terms as follows (@): in thc case of dinuclcotides with the general sequence pdN-dG (wherc N represents eitlicr T, C, A or G), binding occurs using the same hydrogen-bonded geometry found in the actinomycin.deoxyguanosine crystalline complex, this involving interaction with both cyclic peptide chains independently, without steric interference. These complexes would not involve intercalation. On the other hand, dinucleotides of the type pdG-dA and pdG-dT can use the same hydrogen-bonded configuration as in the crystalline complex ; however, these complexes would involve intercalation. (These observations follow immediately from an examination of CPK models of these complexes.) Here, complex formation between one dinucleotide and actinomycin would interfere with further complex formation with the second dinucleotide. This reflects the altered stereochemistry imposed by a noncomplementary d G - d T or dG-dA base pair. In the first case, (guaninesthymine base-pairing), the fit is more closely of the Watson-Crick type than in the second case (guanine-adeninebasepairing), and this interference effect almost certainly reflects stereochemical considerations such as these. Detailed kinetic studies on the binding of actinomycin with DNA demonstrate that several fast steps occur initially in the binding reaction, followed by a slow step extending over several minutes (9). This slow step in binding has also been observed in actinomycin binding studies with pdG-dC (46).Although it is not possible to relate these kinetic steps directly with specific stereochemical events accompanying binding, it has been suggested that the prolonged last step may reflect a conformational change affecting the peptide subunits in the actinomycin molecule (9). An alternative explanation, however,-is that an additional conformational change occurs in the polynucleotide backbone during the final stages of complex formation, this involving a change in the deoxyribose sugar puckering from C2’enndo-CS’ezo (as found in the B form of DNA) to C3’endo-CfL’ezo (as observed in the final model). The detailed actinomycin *DNA stereochemical model proposed involves no significant conformational change of the actinomycin pentapeptides (other than, perhaps, a change in puckering of one of two proline rings); for this reason, we favor the latter explanation at this time. Further information is necessary to establish this point. One can therefore envisage the stepwise association of actinomycin with DNA as follows: actinomycin first intercalates between a base-

STEREOCHEMISTRY OF ACTINOMTCIN BINDING TO

DNA

175

paired dG-dC sequence, recognition initially provided by the specific water structure surrounding actinomycin and the polynucleotide backbone and base sequence. As intercalation continues, this ordered water structure is “squeezed out” and specific hydrogen bonding with guanine residues takes place. The polynucleotide conformation a t this stage may cxist with all deoxyribose sugars C2‘endo-C3’ezo, helix unwinding reflecting thc altered stereochemistry in the immediate intercalation site (-18”). The final slow step is an alteration of the deoxyguanosine sugar puckering to C3’endo-C2’ezo, this providing better steric fit between the actinomycin cyclic peptides and the sugar-phosphate chain. This last step provides additional stabilization and specificity to the binding reaction and is accompanied by additional helix unwinding of base pairs immediately above and below the intercalated base-paired dG-dC sequence (-16’).

D. Other Intercalating Agents A decade has now passed since Lerman proposed his intercalation model to explain the strong binding of aminoacridines with DNA (14). It has now become increasingly clear that a large number of additional drugs and antibiotics utilize intercalation to bind DNA (Fig. 15). In addition to the aminoacridines, proflavin and acridine yellow, which act as frameshift mutagens, the list of intercalating agents includes the trypanocides, ethidium and propidium bromides, antitumor antibiotics such as daunomycin, nogalomycin and actinomycin, the antimalarial drug chloroquine, and hycanthone, a possible hydroxylated metabolite of the antischistosomite Miracil D. Important as diagnostic features of an intercalative process are two related effects on the hydrodynamic behavior of DNA: an increase in viscosity and a decrease in sedimentation coefficient (14, 9, 43). An additional sensitive indication concerning this is the alteration of the sedimentation behavior of covalently circular supercoiled DNA in the presence of increasing concentrations of the intercalating drug or antibiotic ( I S , 44) (Fig. 16). At low relative levels of drug bound per nucleotide, a decrease in the sedimentation coefficient is observed for supercoiled closed-circular DNA, but not for “nicked” circular DNA. This can be explained by assuming that right-handed supercoiled DNA exists as a compact structure (therefore having a large sedimentation coefficient) whose superhclix density depends on the duplex unwinding deficiency at the time of closure. Each intercalation event acts to unwind the helix approximately - 18” (this number arrived a t through the stereochemistry of intercalation provided through the actinomycin * DNA binding model). This means that the binding of twenty dye molecules results in the removal of one superhelical turn in the circular DNA duplex.

H

n

Acridtne yellow

Proflavtn

Hycanthone

OC% OH

CHsO

0

0 OH

HO

Meh

w+

OMe

Nogahmycin

Daunomycin

7%

HN-CH-(CH,),-N-H

gy3

c1

CH

+/'

k,H8

I

H Chloroqutne

IreMtamine A

I

DNA

STEREOCHEMISTRY OF ACTINOMYCIN BINDING TO

24

177

24

22

w

22

20 18

20

16

18 16

o

a05

0.10

Ethidium molecules bound/nucleotide

a15

o ao2004~06a08 F’ruflovin decules bound/nucleotide

o

a02 0.04 006 Actinomycin molecules bound/nucleotide

FIG.16. (A) Effect of ethidium bromide on the s9 of +X174 replicative form. The D N A preparation contained 72% closed circles. (B) Effect of proflavin on the 520 of +X174 replicative form. The D N A preparation contained 67% closed circles. ( C ) Effect of actinomycin on the s2,, of +X174 replicative form. The D N A preparaand ( A ) show results from tion contained 75% closed circles. The symbols (0) resolved boundaries of closed circles and nicked circles, respectively. From Waring (13).

Unwinding this tightly coiled, covalently circular DNA would decrease its sedimentation coefficient, a minimum obtained when all superhelical turns are lost. Continued binding after this results in formation of superhelical turns of opposite handedness, and, because of the coiled nature of this structure, the sedimentation coefficient increases. A detailed analytical theory for this process has been described (45, 46). It is of interest that the steroidal diamine, irehdiamine A, produces a clear rise and fall in the sedimentation coefficient of covalently circular supercoiled DNA; these findings are consistent with intercalation (13) (Fig. 17). This compound is, of course, a puckered ring structure and intercalation in the normal sense is not possible. The interaction with DNA appears to be biphasic as judged by optical thermal denaturation studies. At lower concentrations, irehdiamine A stabilizes DNA from thermal denaturation, whereas a t higher concentrations, the opposite is true (47). It has been suggested that these findings reflect two types of complexes, one involving electrostatic interactions between the protonated amino groups on irehdiamine A and the phosphate groups on opposite DNA chains, the other involving some nonspecific ordered micelle formation outside the helix ( 4 7 ) .It is possible that the first type of complex acts to unwind the helix by a nonintercalative mechanism, stabilizing the A FIG.15. Structural formulas for compounds thought to intercalate into the D N A helix. The steroidal diamine, irehdiamine A, has been included among these compounds, although its mode of binding is uncertain.

178

HENRY M. SOBELL

24

22

8 20

b

18

16 I

0 0.01

I

I

0.1 0.3 IDA/nucleotide ratio

0.03

I

1.0

3.0

FIG.17. Effect of irehdinminc A on the sill of +X174 re1)licwtivc form. Thc DNA preparntion contained 74% closed circles. The symbols (0) and ( A ) shorn results from resolved boundnries of closed circles and “nicked” cirrles. rrspcctirrly. From Waring ( I S ) .

form of the DNA duplex. An alternative explanation, however, is that the steroid nucleus fits between base pairs in the DNA helix, this accompanied by local denaturation of base pairs immediately above and below the intercalation site. Such an interaction is sterically feasible, and is immediately consistent with the sedimentation behavior of covalently circular supercoiled DNA in the presence of the steroid. Further data are necessary to elucidate the precise manner in which irehdiamine A binds to DNA.

V. A General Principle Governing ProteinNucleic Acid Recognition

A. Nuclease Specificity The binding of actinomycin to DNA demonstrates a general principle that several classes of proteins may utilize in recognizing symmetrically arranged nucleotide sequences on the DNA helix. This is shown schematically in Fig. 18A and B. Two nucleases that form double-strand scissions in DNA have been described (20, 2 2 ) . The first of these, a spleen acid deoxyribonuclease, is a dimer containing identical subunits. The enzyme is strongly inhibited by actinomycin, and was therefore thought to attack guanine-rich sequences. Bernardi (20) postulated that if the subunits of this enzyme were related by 2-fold symmetry, this would allow the enzyme to recognize the dyad axis on DNA, and, if each subunit had an active site, permit simultaneous double-strand scissions of the sugar-phosphate

STEREOCHEMISTRY OF ACTINOMYCIN BINDING TO

A

8

DNA

179

NUCLEASE SPECIFICITY ACTINOMYCIN SPECIFICITY

. P".

P"

GC

..

3' 5'

3' 5'

poly[d(G-C]]

M./uteus DNA

. .

BERNARD1 ( 2 0 )

KELLY 8 SMITH (22)

A

C

5' 3'

..

G C A T A T

... ...

DlMER RECOGNITION BERNARD1 (20) MONOD (21) KELLY (L SMITH ( 2 2 )

A T A T T A

3' 5'

..

TETRAMER RECOGNITION GIERER (23)

_ *_ _. _ * .

3' 5'

C

C

FIG.18. A schematic diagram illustrating the general principle governing proteinnucleic acid recognition, as exemplified by actinomycin. DNA binding specificity and endonuclease specificity (A and B). If a protein molecule has identical subunits related by 2-fold symmetry when it binds to DNA-the 2-fold axis coinciding with the dyad axis on DNA-then a necessary consequence is that the base sequence in the recognition site have 2-fold symmetry. (C) Extension of this general principle for dimer recognition to include tetramer recognition. One postulates a tandem genetic duplication of the DNA sequence involved in dimer recognition, followed by a hydrogen-bonding rearrangement. This nucleic acid structure can then be recognized by a tetrameric protein having identical subunits related by 4-fold symmetry. Patterns of recognition such as these may exist between operators and repressors.

180

HENRY M. SOBELL

backbone. A necessary consequence of this type of recognition would involve symmetrically arranged sequences, such as dC-dG or dG-dC, the latter being the sequence that actinomycin binds preferentially. The second nuclease is a restriction enzyme isolated from Hemophilus influenme that recognizes the symmetrically arranged hexanucleotide sequence shown in Fig. 18B and introduces a double-strand scission a t its central singularity. Although the subunit character of this enzyme is not yet established, Kelly and Smith (22) postulated a similar protein-nucleic acid recognition pattern for this restriction enzyme. It is of interest in this connection that Meselson and Yuan (48) have demonstrated that two classes of heteroduplex X ( 0 : K ) DNA produced by annealing isolated strands, one modified, the other not modified, are resistant to attack by E . co2i endonuclease. Thus, modification (presumably by methylation) of one strand imparts immunity from the restriction enzyme to the other strand, suggesting some type of 2-fold symmetry a t the recognition site. It remains to be seen whether these findings generally apply for restriction and modification systems in other bacteria [for recent reviews, see Arber and Linn (49) and Boyer (&I)].

B. Do Operators and Repressors Have 2-fold (or 4-fold) Symmetry? The binding of actinomycin to DNA (in particular, to dG-dC sequences) and its specificity in inhibiting the RNA polymerase reaction suggest a primitive repressor-operator character for this complex, which may prove to have more general meaning with regard to the recognition of naturally occurring operators by repressors. If a repressor molecule has identical subunits related by 2-fold symmetry when it binds to DNAthe 2-fold axis coinciding with the dyad axis on DNA-a necessary consequence is that the base sequence in the operator have 2-fold symmetry. This would be true regardless of precisely which groove (or grooves) the repressor binds. This geReral principle for dimer recognition can be extended to include tetramer recognition in the following way (see Fig. 18C).One postulates a tandem genetic duplication of the DNA sequence involved in dimer recognition, followed by a hydrogen-bonding rearrangement (23). This generates a cloverleaf-like structure that can be recognized by a tetrameric protein having identical subunits arranged in one of two ways. If the subunits of the protein are related by 4-fold symmetry, the operator must be a structure that itself possesses 4-fold symmetry, as indicated in Fig. 18C. Alternatively, if the subunits of the protein are related by only 2-fold symmetry (in particular, by one 2fold axis of symmetry), then the operator structure must also have this symmetry. Such a structure can be obtained from the cloverleaf structure

STEREOCHEMISTRY OF ACTINOMYCIN BINDING TO

DNA

181

A FIG.19. Alternative arrangements for a tetrameric protein containing identical subunits interacting with a Gierer-like operator site. (A) One 2-fold axis of symmetry replaces the 4-fold axis of symmetry. This figure is obtained from the right-hand figure shown in Fig. 18C by bending the horizontal leaves forward and the vertical leaves backward, giving rise to a roughly tetrahedral arrangement. It is noticed that the operation that takes the front dimer into the back dimer is a twist of go", a translation away from the viewer, and a change in the protein-protein subunit interaction necessary to sense the same nucleic acid chemical environment. One must thereforc postulate either different protein-protein subunit contacts to achieve identical environments for protein-nucleic recognition, or the same proteinprotein subunit contacts giving rise to different environments for protein-nucleic recognition. Both alternatives are unlikely. (B) 222 symmetry relating protein subunits to nucleic acid cloverleaf structure. Exact 222 symmetry is not possible without interrupting the continuity of the polynucleotide chain. However, pseudo-222 symmetry remains an nlternnte, although. for steric and energetic reasons, a less likely, possibility.

by bending the horizontal leaves forward and the vertical leaves backward, in a roughly tetrahedral arrangement (Fig. 19A). This gives rise to two distinct chemical environments involved in protein-nucleic acid recognition, however, and for this reason seems less likely. Higher symmetry, i.e., 222 symmetry, although possible for a tetrameric protein, is not (strictly speaking) possible for the operator structure shown without breaking the continuity of the polynucleotide backbone (Fig. 19B). While no structural evidence is yet available concerning the arrangement of subunits in the lac or the A repressors, the lac repressor is known to bind DNA as a tetramer (51, 5 2 ) , while the repressor may bind DNA in dimeric form (53, 54). The precise symmetry relating subunits of the 2ac repressor will eventually be revealed by X-ray crystallography; however, strong genetic evidence (55, 56) already points to the existence of %-fold symmetry in the lac operator genetic map (operator-constitutive mutants possessing similar levels of constitutive p-galactosidase activity) , with higher order subdivision possible. These findings, along

182

HENRY M. SOBELL

with the observations that lac repressor binds d (A-T) , selectively with high affinity (57), directly support the Gierer-like operator structure shown in Fig. 18C. However, the most definitive evidence regarding this will be provided by nucleotide sequence data of the lac operator and the X operator (s), and such studies are in progress (58,59).

C. A Model for Genetic Recombination The concept that regions of DNA possessing symmetrically arranged polynucleotide base sequences can exist either as Watson-Crick structures or Gierer-like structures (the latter being induced by a specific structural protein or proteins), leads to a simple but powerful molecular model to explain genetic recombination. This is shown schematically in Figs. 20 and 21. Any model for genetic recombination must explain: (i) the pairing of homologous chromosomes and events leading to reciprocal recombination ; (ii) gene conversion and polarity effects observed with gene conversion resulting in nonreciprocal genetic exchange [see Emerson (60) for an excellent recent review on linkage and recombination a t the chromosome level] ; (iii) genetic and physical chemical evidence regarding singlestrand exchange (the formation of insertion heteroduplexes, as, for example, in transformation), and double-strand exchange (with central heteroduplex formation, as occurs in breakage and reunion with flanking marker exchange) [see Bodmer and Darlington (61) for a review in linkage and recombination a t the molecular level] ; (iv) branched T 4 DNA molecules, which are observed in T 4 pol-lig- phage infection and interpreted as intermediates in T 4 recombination (62). The model presented explains these observations and involves the following features: (i) chromosomes pair due to the formation of Giererlike structures induced by a specific recombination structural protein (8) ; (ii) a Holliday (63) heteroduplex structure is formed, and this can migrate in either direction along the parental DNA molecules; (iii) reciprocal recombination results from the action of a nuclease or nucleases possessing 2-fold symmetry. Details of the model are as follows (see Figs. 20 and 21). Symmetrically arranged polynucleotide base sequences on homologous chromosomes (spaced every operon length or so along the chromosome) are fist converted to their Gierer structures in the presence of a specific structural protein(s), which, for simplicity, we shall call a recombination protein. Typically, a symmetrically arranged sequence contains central sequences [E,G] and [H,F] that do not possess symmetry. Therefore, the Gierer structure contains loops of single-stranded DNA susceptible (either randomly or with specificity) to nuclease attack. When complementary loops

STEREOCHEMISTRY OF ACTINOMTCIN BINDING TO

A

r’? _A.._ _.1._

A I‘

y

DNA

183

B

B

5’ 3,

.... ____

..

..

?’

A I 1 G C C A T G G

1A T A GC CG CG

tO Ei

C G A T A 1

AT A T

_; $-

I A --__ C G

GC

Ei E Z:G 2: -1g TA T A GC CG CG A T T A GC GC CG A T A T T I

_ _.._ _ j.5

1 A A C G G 1 A C C

A T A 1

H;:F GC A T 1 A

_ A_T _TA_ I T G C

A A C G

1 G G C A A T

A C C G T T A

:F

_ ._ _._

.. ..

.. .. I’ 5’

D

C

A T

FIG.20. A model for genetic recombination. Homologous chromosomes A-C and B-D possess specific regions (prrhaps placed every operon length or so along the chromosome) capable of forming Gierer-like structures in the presence of a specific recombination structural protein. Regions such as E-G and H-F form single-stranded denatured loops outside the immediate environment of the protein (which senses only the symmetry-related nucleic arid structure shown) and are therefore suscrptible to nuclease attack (shown by the arrows). When complementary loops G, H are “nicked” and opened, Watson-Crick base-pairing occurs, this followed by extensive propagation of the heteroduplex (shown in the lower two figures), The final structural intermediate is shown in the center of Fig. 21.

184 A

f. ?

..

..

HENRY M. SOBELL

B

I' I' ....

Ji

.... Y

C

D

5'

?

HOLLIDAY (1964)

BROKER & LEHMAN (1971)

FIG.21. A model for genetic recombination (continued). The central structural intermediate can give rise to the Broker-Lehman heteroduplex structure (62)in the absence of polynucleotide ligase, or to the Holliday heteroduplex (63) in the presence of polynucleotide ligase. The Holliday heteroduplex possesses 2-fold symmetry. It can therefore be recognized by a nuclease possessing 2-fold symmetry, which would be able to simultaneously nick strands of the same polarity at homologous sites. This could give rise to reciprocal recombination involving either single- or doublestrand exchange, depending on which strands were cut and joined. Another feature of this heteroduplex structure that results from its 2-fold symmetry is its ability to migrate (by rotary diffusion) in either direction along parental DNA molecules without unwinding difficulties. This would allow recombination to occur randomly throughout the genome and could give rise to polarity effects observed in gene conversion.

are "nicked," i.e., [G,H] , homologous Gierer structures can then come together through base pairing, this followed by propagation of the heteroduplex (the details of which are shown in the lower figures in Fig. 20). One then arrives a t the central intermediate shown in Fig. 21. I n the absence of ligase, this intermediate can become the BrokerLehman structure ( 6 2 ) . I n the presence of ligase, one can form the Holliday heteroduplex. This results from base pairing of "sticky ends" [H,G], followed by sealing of "nicks" in the polynucleotide chain by ligase. The Holliday heteroduplex is a particularly interesting structure

STEREOCHEMISTRY OF ACTINOMYCIN BINDING TO

DNA

185

in that it possesses 2-fold symmetry. It can therefore be recognized by n nuclease possessing 2-fold symmetry, which would simultaneously act to nick strands of the same polarity a t homologous sites. This would give rise to reciprocal recombination involving either single- or doublestrand exchange, depending on which strands were cut and joined. Another feature of the heteroduplex structure (which results from its 2fold symmetry) is its ability to migrate in either direction along parental DNA molecules without unwinding difficulties. This would allow genetic recombination to occur randomly throught the genome and, in addition, could explain polarity effects observed in gene conversion (64). For further details of the model and its genetic implications, see Sobell (65, 77). The model is attractive in its simplicity and logical design. It leads, furthermore, to models of DNA replication and of chromosome structure as well, and these are outlined below.

D. DNA Replication and Chromosome Structure The existence of Gierer-like loops induced by specific structural proteins and spaced every operon length I r so along the chromosome would provide single-stranded regions capablt of base-pairing with small (DNA or RNA) oligonucleotides, these possibly acting as primers for DNA polymerase activity. If these primers consisted of symmetrically arranged base sequences (for example, dA-dT or dA-dT-dA-dT) , DNA synthesis could proceed bidirectionally, resulting in newly synthesized DNA fragments of roughly operon size (see Fig. 22). These fragments could then be joined by polynucleotide ligase to form a continuous polynucleotide chain. The model is attractive in that it utilizes the known chemical properties of the DNA polymerase enzymes and their requirement for primer and template DNA (66-68). It also provides an explanation for the uniforrnity in size of newly replicated DNA fragments in alkaline sucrose gradients (69, 70). Finally, it is consistent with the replicon model for chromosomal replication (71), and these, as well as other aspects of the model will be discussed in further detail elsewhere (77). The ability of a specific protein to bind DNA a t specific sites leads to a model for DNA folding that may be relevant in understanding chromosome structure. One can imagine two states possible for a long DNA molecule, an extended linear state and a folded state (see Fig. 23A and B) , the latter reflecting protein-protein polymerization (either involving covalent or noncovalent bonding). Such a model can be used to explain the morphology of the lampbrush chromosomes [see, for example, a recent review by Taylor (%)I and the Drosophila and Chironomus giant chromosomes (73, 7 4 ) . A folded polymer state may

HENRY M. SOBELL

186

..

5' 3'

3' 5'

FIG.22. A model for DNA replication. Gierer-like loops induced by specific structural proteins and spaced every operon length or so along the chromosome could provide single-stranded regions capable of base-pairing with small (DNA or RNA) oligonucleotides (in this example, dAdT), these acting as primers for DNA polymerase activity. Synthesis could proceed bidirectionally and could simultaneously involve scveral continguoris Gierer-loop regions, this resulting in newly synthesized DNA fragments of roughly operon size. These fragments could then be joined by polynuclcotidc ligase to form a continuous polynucleotide chain.

explain the viscoelastic gel formation observed by Dounce and others for salt-extracted chromatin (75, 7 6 ) .The stability of this DNA-residual protein gel complex is very sensitive to disulfide reducing agents, such as mercaptoethanol, as well as to deoxyribonuclease and various pro-

STEREOCHEMISTRY OF ACTINOMTCIN BINDING TO

DNA

187

FIG.23. A model of DNA folding. If a protein binds to DNA at specific sites, it is possible for the polymer to exist in two states, an extended linear state (A) and n folded state (B). This simple model can be used to explain (in part) the morphology of the lampbrush chromosome and the Drosophila and Chironomus giant polytene chromosomes ( C ).

teinases. Observations such as these are consistent with a folded polymer state model forming the skeletal framework for mammalian chromosomes, and this as well as other aspects of chromosome structure will be discussed more completely elsewhere (77).

VI. Possible Medical Implications The detailed stereochemistry of actinornycin binding to DNA suggests a ratiorialc for thc synthesis of iicw variants of the actinomycin molecule that may find clinical use iii chemotherapy or neoplastic disease and viral infection. One of the major problems in using actinomycin

188

HENRY M. SOBELL

clinically is its extreme toxicity, since it passes through the cellular membrane of both normal and malignant cells without specificity. If it were possible to alter the peptide portion of the actinomycin molecule so as to change its permeability properties with respect to normal and malignant cells, the antibiotic could become a clinically useful drug. Thus, for example, D-valine could be replaced by a derivative of D-valine possessing a long alkyl chain, giving the actinomycin molecule a “tail.” A substitution such as this probably would not affect the ability of the molecule to bind to DNA, but may selectively prevent entrance of the antibiotic into a given cell type. One would hope that a proper tail could be devised that would prevent the antibiotic from passing through the normal cellular membrane while having little effect on transport across the malignant cellular membrane. Along the same lines, one might covalently attach the actinornycin molecule (through its “tail”) to serum albumin and hope that malignant cells that demonstrate pronounced pinocytosis will phagocytize these molecules and poison themselves with actinomycin. Other possible modifications may involve stereospecific peptide subunits designed to recognize other types of base sequences in DNA. These would serve as useful probes in the study of nucleic acid function, and may find chemotherapeutic use in the treatment of certain viral (DNA) infections. Finally, one wonders whether it may be possible to create an actinomycin-like antibiotic able to bind to doublehelical RNA. This would perhaps have use in the treatment of viral (RNA) infections as well as in the area of oncogenic research.

VII. Summary We have successfully cocrystallized actinomycin with its DNA substrate, deoxyguanosine, and have solved the three-dimensional structure of the complex by X-ray crystallography. The configuration observed in the crystalline complex explains in a natural way the stereochemistry of actinomycin binding to DNA. The phenoxazone ring system on actinomycin intercalates between the base-paired dinucleotide sequence, dGdC, while the peptide subunits lie in the narrow groove of the DNA helix and interact with deoxyguanosine residues on opposite chains through specific hydrogen bonds. The binding of actinomycin to DNA demonstrates a general symmetry principle that several classes of proteins may utilize in recognizing symmetrically arranged nucleotide sequences on the DNA helix. ACKNOWLEDGMENTS This work has been supported in part by grants from the National Institutes of Health, the National Science Foundation, the American Cancer Society, and the

STEREOCHEMISTRY OF ACTINOMYCIN BINDING TO

DNA

189

Atomic Energy Commission. H. M. S. was a recipient of a research career development award from the National Institutes of Health during part of the course of this work. This paper has been assigned report No. TTR-34!NK)-179nt, the Atomic Energy Project, the University of Rochester.

REFERENCES 1. J. Kirk, BBA 42, 167 (1960). 2. W. Kersten, H. Kersten, and H. M. Rauen, Nature (London) 187, 60 (1960). 3. E. Reich, R. M. Franklin, A. J. Shatkin, and E. L. Tatum, Science 134, 556 (1961). 4. I. H. Goldberg, M. Rabinowite. and E. Reich, PNAS 48, 2094 (1962). 6 . J. Hurwitz, J. J. Furth, M. Malamy, and M. Alexander, PNAS 48, 1222 (1962). 6. E. Reich and I. H. Goldberg, This series, 3, 183 (1964). 7. R. D. Wells and J. Larson, J M B 49, 319 (1970). S. W. Kersten, BBA 47, 610 (1961). 9. W. Miiller and D. M. Crothers, JiMB 35, 251 (1968). 10. L. Hamilton, W. Fuller, and E. Reich, Nature (Lotidon) 198, 538 (1963). 11. A. Cerami, E. Reich, D. C. Ward, and I. H. Goldberg, P N A S 57, 1036 (1967). 12. E. Reich, I. H. Goldberg, and M. Rabinowitz, Nature (London) 196, 743 (1962). 13. M. Waring, JMB 54, 247 (1970). 14. L. S. Lermm, J M B 3, 18 (1961). 16. H. M. Sobell, S. C. Jain, T. D. Sakore, and C. E. Nordman, Nature (Londoit) 231, 200 (1971). 16. H. M. Sobell, S. C. Jain, T. D. Sakore, G . Ponticello, and C. E. Nordman, CSHSQB 36, 263 (1971). 17. H. M. Sobell, Jerusalem Symp. Quaiit. Chem. Biochem. 4, 149 (1972). 1s. S. C. Jain and H. M. Sobell, J M B 68, 1 (1972). 19. H. M. Sobell and S. C. Jain, J M B 68, 21 (1972). 20. G. Bernardi, Aduan. Enzymol. 31, 1 (1968). 21. J. Monod, in “Symmetry and Function of Biological Systems a t the Macromolecular Level,” Nobel Symp. 11, p. 15. Wiley, New York, 1969. $2. T. J. Kelly and H. 0. Smith, J M B 51, 393 (1970). 23. A. Gierer, Nature (London) 212, 1480 (1966). 94. E. Reich, in “The Role of Chromosomes in Development” (M. Locke, ed.), pp. 73-81. Academic Press, New York, 1964. 26. R. Haselkorn, Science 143, 682 (1964). 26. M. Gellert, C. E. Smith, D. Neville, and G. Felsenfeld, J M B 11, 445 (1965). %’. M. Behme and E. H . Cordes, BBA 108, 313 (1965). 25. S. S. Danyluk and T. A. Victor, Jerusnlem Symp. Qunnt. Chem. Biochem. 2, 394 (1970). 29. F. Conti and P. De Santis, Nature (London) 227, 1239 (1970). 30. B. H. Arison and K. Hoogsteen, Bchem 9, 3976 (1970). 31. J. Donohue and K. N. Trueblood, J M B 2, 363 (1960). 32. M. Sundaralingam, JACS 87, 599 (1965). S3. H. M. Sobell, in “Genetic Organization” (E. Caspari and A. W. Rarin. eds.), Vol. 1, 11. 91. Academic Press, New York, 1969. 3g. H. M. Sobell, Jerusalem Symp. Qttnnt. Chem. Biochem. 4, 124 (1972). 36. D. Voet and A. Rich, This series, 10, 183 (1970). 36. E. Reich, Science 143, 684 (1964).

190

HENRY M. SOBELL

37. S. Arnott, P r o p . Bwphys. Mol. Biol. 21, 205 (1970). 38. M. Sundaralingam, Biopolymeis 7 , 821 (1969). 39. S. Arnott, M. H. F. Wilkins, W. Fuller, and R. Langridge, JMB 27, 525 (1967). 40. S. Arnott, M. H. F. Wilkins, W. Fuller, and R. Langridge, JMB 27, 535 (1967). 41. G. Milman, R. Langridge, and M. J. Chamberlin, PNAS 57, 1805 (1967). 42. T. R. Krugh, PNAS 69, 1911 (1972). 43. W. Kersten, H. Kersten, and W. Szybalski, Bchem 5, 236 (1966). 44. W. Bauer and J. Vinograd, JMB 33, 141 (1968). 46. W. Bauer and J. Vinograd, JMB 47, 419 (1970). 46. W. Bauer and J. Vinograd, JMB 54, 281 (1970). 47. H. R. Mahler, G. Green, R. Goutarel, and Q. Khuong-Huu, Bchem 7, 1568 (1968). 48. M. Meselson and R. Yuan, Nature (London) 217, 1110 (1968). 4.9. W. Arber and S. Linn, ARB 38, 467 (1969). 60. H. W. Boyer, Annu. Rev. Microbiol. 25, 153 (1971). 61. W. Gilbert and B. Muller-Hill, PNAS 58,2415 (1967). 62. A. D. Riggs, H. Suzuki, and S. Bourgeois, JMB 48, 67 (1970). 63. V. Pirrotta, P. Chadwick, and M. Ptashne. Nnture (London) 227, 41 (1970). 64. P. Chadwick, V. Pirrotta, R. Steinberg, X. Hopkins, and M. Ptashne, CSHSQB 35, 283 (1970). 66. T. F. Smith and J. R. Sadler, JMB 59, 273 (1971). 66. J. R. Sadler and T. F. Smith, JMB 82, 139 (1971). 67. S. Y. Lin and A. D. Riggs, Nature (London) 228, 1184 (1970). 68.

M. Ptashne, personal communication.

69. W. Gilbert, personal communication. 60. S. Emerson, in “Genetic Organization,” (E. Caspari and A. W. Ravin. eds.), Vol. 1, p. 267. Academic Press, New York, 1969. 61. W. F. Bodmer and A. J. Darlington, in “Genetic Organization” (E. Caspari and A. W. Ravin, eds.), Vol. 1, p. 223. Academic Press, New York, 1969. 62. T. R. Broker and I. R. Lehman, JMB 80, 131 (1971). 63. R. Holliday, Genet. Res. 5, 282 (1964). 64. S. Fogel, D. D. Hurst, and R. K. Mortimer, Stadler Sump. 1 and 2, 89 (1971). 66. H. M. Sobell, PNAS, 69, 2483 (1972). 66. M. J. Bessman, in “Molecular Genetics” (J. H. Taylor, ed.), Vol. 1, p. 1. Academic Press, New York, 1963. 67. M. Goulian, CSHSQB 23, (1968). 68. T. Kornberg and M. Gefter, PNAS 68, 761 (1971). 69. R. Okazaki, T. Okazaki, K. Sakabe, and K. Sugimoto. Jnp. J . Med. Sci. Biol. 20, 255 (1967). 70. E. K. Schandl and J. H. Taylor, BBRC 34,291 (1969). 71. F. Jacob, S. Brenner, and F. Cuzin, CSHSQB 28, 329 (1963). 72. J. H. Taylor, in “Genetic Organization” (E. Caspari and A. W. Ravin, eds.), Vol. 1, p. 163. Academic Press, New York, 1969. 73. C. B. Bridges, J . Hered. 28, Bo U935). 74. W. Beermann and G. F. Bahr, Exp. Cell Res. 8, 195 (1954). 76. A. L. Dounce, Amer. Scientist 59, 74 (1971). 76. A. E. Mirsky and H. Ris, J . Gen. Physiol. 34, 475 (1951). 77. H. M. Sobell, Advan. Genet. 17, in press.

Resistance Factors and Their Ecological Importance to Bacteria and to Man

I

I

M . H . RICHMOND Department of Bacteriology. Pniversity of Bristol. Bristol. England

I . Introduction . . . . . . . . . . . I1. Resistance Factors and the Genes They Carry . . . A . The Resistance Transfer Factor . . . . . . B . Plasmid Replication . . . . . . . . . C . The Maintennnce and Segregation of Plasmids . . . D. “Curing” . . . . . . . . . . . E . Practical Aspects of Maintenance . . . . . . F. The Gene Products Needed for Conjugation . . . G . DNA Transfer During Mating . . . . . . 111. Resistance Determinants . . . . . . . . A . Penicillin and Cephalosporin Resistance . . . . B. Chloramphenicol Resistance . . . . . . . C . Resistance to Aminoglycoside Antibiotics . . . . D . Tetracycline Resistance . . . . . . . . E . Sulfonamide Resistance . . . . . . . . F. Other Resistance Factor-Mediated Determinants . . I V . Other Plasmids Related to Resistance Factors . . . V . The Mating Process . . . . . . . . . . A . The Pilus Bridge and Its Formation . . . . . B. Gene Transfer . . . . . . . . . . C . The Properties of Newly Infected Recipients . . . D . The Transfer Process: A Summary . . . . . VI . Resistance Factor Transfer in Nature . . . . . A . The Early Observations . . . . . . . . B . Resistance Factors and Pseudomonas aeruginosa in Burns C . Resistance Factors and Resistance Transfer . . . References . . . . . . . . . . . .

. . . .

. . . .

. . . . . . . . . .

. . . . . . . . . .

. .

. .

. . . .

. . . . . .

. . . . . .

. . . . . .

191 193 194 195 203 206 208 209

212 213 213 215 216 219 219 220 220 221 221 223 230 231 233 233 235 238 244

.

1 Introduction The study of transferable drug resistance is a project very much in tune with the times: it has both a fundamental interest a t the molecular level and also the greatest relevance to the everyday life of all of US . At the molecular level. it throws light on how hereditary material introduced freshly into a bacterial cell may survive and become part. for a time a t least. of the informational content of that cell and of the bacterial popula191

192

M. H. RICHMOND

tion of which the cell forms part. I n practical tcrmns, it shows how bacterial cells may respond to a changing environment. Man has made the production of antibacterial substances into a multimillion dollar concern ; and the selection pressure that this cffort exerts on the bacterial population is immense-particularly in certain niches important to man. Everywhere one reads of the appearance and the spread of new or different patterns of bacterial resistance in the environment. Transferable antibiotic resistance is merely one example of a general phenomenon, but its study may show the nature of the steps involved in this process and thereby how to mitigate it. Spread certainly occurs: and it frequently goes a great way to undermine the value of new therapeutic agents. Too often, in the past, the study of gene transfer has interested two groups of people who had too little to say to one another. On the one hand, there were those who were concerned with elucidating the detailed steps of how transfer is effected, what form the transferred information takes, what steps are involved in the survival of the transferred piece in the recipient, and the like. The second group has compiled statistics about the incidence of resistance, charted the emergence of new resistant strains in thc bacterial population and tried to devise ways of reducing both the selection pressure of antibacterial therapy and the chance that resistant organisms would pass from one host to another. Too often these last people were busy hospital bacteriologists with a heavy routine professional commitment who often could not spare the time, and who frequently did not have the training to appreciate the molecular intricacies of the subject. Although the situation is rapidly improving with the worldwide increase in the number of groups making a concerted epidemiological and molecular attack on this problem, there are still only very few written accounts that attempt to deal with both aspects a t the same time [see, however, Anderson ( I ) ] : this article, therefore, tries to fill this gap and bring the story up-to-date. First we examine those aspects of bacterial genetics and biochemistry that are relevant to the survival and transmission of antibiotic resistance genes between bacterial cells. This involves a fairly detailed discussion of the nature and processes associated with the survival and transfer of R-factors' and other plasmids in bacteria. In the second part we examine certain examples claiming to show the importance of R-factor-mediated transfer of antibiotic resistance genes during the course of a clinical infection, and try to assess how widespread this

'Abbreviations: R, resistance (factor) ; RP, R-factors originally isolated in Pseudomonns aeruginosa; RTF, resistance transfer factor: F, fertilitv factor ,: I.. fertility factor specifying syntliesis of colicin I ; CCC, covklently closed circle (of DNA).

RESISTANCE FACTORS

193

process is and how serious in a clinical context. Since the discovery of transferable antibiotic resistance in Japan in the late 1950’s [see Watanabe ( 8 ) , for a review], bacterial mating has been held up as being of the greatest importance in the development of resistant bacterial populations. But there is real doubt as to whether the process is really important in giving rise to antibiotic resistant variants during the coursc of treatment or whether it has a more strategic role to play in building up a reservoir in thc so-called normal, healthy population. I n this review, we try to assess this situation-at least as it exists a t the time of writing. Before starting, it is important to stress that this review is in no way an attempt a t an exhaustive treatment of the molecular biology of R-factors, let alone bacterial plasmids. Throughout, emphasis is on those aspects of R-factor behavior that have most importance from the ecological point of view ; nevertheless, somc emphasis on thc molecular basis of thc process is important.

II. Resistance Factors and the Genes They Carry R-factors first emerged as elements that can promote transfer of genes conferring resistance to antibiotics from strains that carried the R-factors to strains that lacked them [see (1-3) for reviews]. Superficially, Rfactor transfer is analogous to transmission of the fertility factor; the act confers the ability to transfer in its turn on the recipient. There has been some doubt as to whether the donor loses the ability in the process ( 4 ) . Current evidence suggests that it does not [see ( 5 ) and Section V, B, 11. As studies on R-factors multiplied, it emerged that it is useful to divide the genes carried on the elements into two functional groups: The resistance transfer factor and the rcsistance determinants. The first group of genes is responsible for the replication and distribution of the plasmid on cell division and for the promotion of plasmid transfer to recipient cells. Nontransmissible R-factors (or a t least those that rely on methods other than conjugation for their transfer) seem to have the first two of these regions but not the third, and if transfer of this type of element is to take place, these genes must be provided by another plasmid ( I 1. The resistance determinants on an R-factor are thc genes that specify the molecules that confer the resistance. These are frequently biochemical products that destroy or inactivate the antibiotics (6-8), but sometimes they prevent the antibacterial agent from reaching its target within the cell (9). Certain R-factors also carry a number of genes apparently unrelated-or a t least only remotely related-to antibiotic resistance. These are discussed later (see Section 111,F.)

194

M. H. RICHMOND

A. The Resistance Transfer Factor Although it is easy enough to give a catalog of the overall functions of RTF,' the detailed genetic structure of the region, the exact nature of the biochemical products specified and the way in which they are COordinated to achieve plasmid survival and transfer are still largely unknown. It is not even certain how large the region is. Indirect estirhates obtained by coinparing the sizes of transmissible and nontransmissiblc variants of the same R-factor suggest a molecular weight in the region of 50 X 10" for R T F ; and this amount of DNA corresponds to about 70 genes (10-16).However, plasmids that are nontransmissible must still retain some genetic material characteristic of the RTF, and a transmissible fragment that has lost all its resistance determinants may yet carry information not properly part of RTF. Any indirect estimates of size must, therefore, be treated with extreme caution. Nevertheless, it does seem likely that a major part of the DNA of any plasmid is likely to be concerned with the survival and transfer of that plasmid, and this, in turn, stresses how important transmissible genes must be to bacterial populations, since there must be a considerable selection pressure tending to delete such large pieces of genetic information. Even when the genes and their products have no functional role, there must be a pressure tending to delete the genetic information from the cell. I n the last analysis, the energy required to synthesize the useless genes themselves will apply pressure toward their ultimate loss. Any cell that has lost the genes will be energetically more favorably placed than those that have not. The major functions specified by the RTF can be classified as follows: (1) initiation and regulation of plasmid DNA replication; (2) maintenance of plasmids in the cell and their segregation to daughter cells a t division ;

(3) specification of the gene products needed for conjugation; (4) transfer of DNA during conjugation.

Although such a list describes accurately enough the functions that depend primarily on the nature and the behavior of RTF, they are unlikely to reflect clearly the biochemical organization involved. For example, the DNA replication that seems to be an essential part of gene transfer during conjugation (IS) is unlikely to be rigidly independent, a t a biochemical level, from the DNA replication needed for plasmid duplication in growing cells. Furthermore, since each of the functions detailed above itself probably requires many gene products, it seems likely that some genes from the RTF have a relatively wide role to play in plasmid rnaintenancc and transfer, whereas others may be highly

RESISTANCE FACTORS

195

specific. Therefore, it is still premature to expect to get any clear, overall picture of how the genes comprising the RTF act. From the point of view of this article, we concentrate on those aspects of R T F structure and function that must play an important part in the practical aspects of gene transfer discussed later. Of course, all parts of the R T F are relevant to this topic, but some, such as the genes that decide on the number of plasmid copies per cell, those that specify the nature of the conjugation apparatus made by plasmid-carrying cells and those that allow autonomous replication of an incoming plasmid in a new host, must be of central importance. A number of recent detailed accounts of this subject are available (e.g., 14, 15).

B. Plasmid Replication With a number of important exceptions, the total amount of plasmid DNA forms a relatively constant proportion of the total DNA in plasmidcarrying bacterial strains. Between 0.5% and 5% of the DNA is extrachromosomal when a plasmid is present (16, 17).I n general, the carriage of bacterial plasmids seems much more widespread than originally anticipated, and plasmid DNA is often detected without any concomitant biochemical characters bcing evident (18). One can, therefore, think of “orphan” plasmids, much as one thinks of “orphan” viruses. The fact that plasmid DNA comprises a relatively fixed proportion of the cell’s DNA over relatively long periods implies that its rate of replication must be adjusted to be closely in step with that of the chromosome. Analysis often shows one plasmid copy pcr chromosome in the cell [e.g., RlOO in Escherichia coli (19-22)] ; but sometimes a number of plasmid copies exist for each set of chromosomal markers [e.g., the same plasmid in Proteus inirabilis (23, 2 4 ) l . Whichever is the case, a careful regulation of plasmid replication is necessary, since even a small difference between the two rates of DNA synthesis would lead rapidly either t o loss of the plasmid or to conversion of all the cell’s DNA to the plasmid state; this regulation of replication seems to be one important role played by the RTF. I n practice, plasmid replication must be considered in two parts: initiation and duplication. Since the plasmid is so small (usually only about 1% as long as the chromosome) the duplication of its base sequence will take a much shorter time than chromosomal duplication. It follows, therefore, that if the number of plasmid copies per chromosome is to remain constant, there must be strict control over some step in replication, since thc actual rate of duplication, base for base, is likely to be similar in both the chromosome and the plasmid. What evidence there is suggests

196

M. H. RICHMOND

that the control point is the initiation of new rounds of plasmid replieation, much as is the case for the chromosomal replicon (26-2’7). As far as duplication is concerned, it is still uncertain whether Rfactors specify their own DNA polymerase or use an enzyme provided by the host cell. Many workers who have tried to solve this problem have investigated the replicative behavior of plasmids following their transfer, either as a consequence of cell division or by mating, to “mini-cells” (28). These cells lack chromosomal DNA, and various authors reported various types of behavior with individual plasmids in such cells. The fertility factor, F, seems to be unable to segregate into a mini-cell ( 2 9 ) , although transfer of F to such a cell by mating leads to the formation of a DNA duplex on the template provided by the single DNA strand introduced, but no further replication ( 6 ) .Inselberg (30)claimed that the Col E l plasmid not only segregates to mini-cells but also replicates there. Both F-like and I-like’ R-factors may segregate to mini-cells on division, and some DNA synthesis may follow since the factors are present in the cells a t levels greater than expected from an incidence of one copy per cell (63).These experiments have the disadvantage, however, that it is always difficult to exclude the possibility that the necessary enzymes for replication, where it occurs, come from the parental cell when the minicell is budded off; and the whole question of whether the R-factors specify their own DNA polymerase is still wide open. Mutations that affect plasmid survival are common [see (31) for examples], but it is never easy to bc certain whether thcsc represent a failure in replication or in distribution (see Section 11, A). Mutation of a plasmid gene specifying a replicative step-probably regulatory rather than synthetic-does seem to be implicit in the strains studied by Nordstroin (32).Highly resistant mutants were obtained from Eschem’chia coli (R,+) after treatment with mutagens. These have now been analyzed and seem to contain multiple R-factor copies, whereas the parental strains carried one R-factor pcr set of chromosomal genes ( 3 2 ~ ) . The plasmid location of this lesion has been confirmed by conjugation and transduction experiments wherc the high level of resistance is maintained in recipients, unlike the situation found with the unmutated plasmid. Although Nordstrom’s experiments are the only well-documented cases where the regulation of plasmid replication is disturbed by mutation, variants in the number of R-factor copies pcr cell often occur under physiological conditions. Much work has been done with the R-factor R m (alias R222 and NR1). This phenomenon was originally reported by Rownd, Nakaya, and Nakamura (16),but the observation has been amply confirmed elsewhere (22, 24, 33).Although RlOO exists in Esche-

197

RESISTANCE FACTORS

richiu coZi a t a level of 1 copy per chromosome throughout growth (G + C = 51.5%, MW = 64 X loc) (19, 20, 22), the situation in Proteus inirabilis is much more complex (16, 17).Not only is the total number of R-factor copies greater than one per chromosome, but the R-factor gives rise to covalently closed circular plasmid fragments of different sizes, and these constitute different molecular proportions a t different phases of growth. One of the three molecular species is identical to that found in E . coZi, while the other two are smaller. The first of these has a molecular weight of 52 X 10" and a (G C) content of 48% while the other has a MW of 12 X 10" and a (G C) content of 56%. Most workers claim that the two smaller elements are related to the larger by the equation:

+ +

(52 X lo6)

+ (12 X lo6) = (64 X 10')

but there is no real evidence, apart from arithmetical plausibility, to support this view (11,34). During early exponential growth, the prcdominant form of RlOO in Proteus mirabilis is the 64 X loRdalton piece which is present a t a level of 5 copies per chromosomal copy. As the culture enters the stationary phase, there is a burst of R-factor synthesis that is accompanied by an increase in the cell content of p-lactamase and chloramphenicol transacetylase, two enzymcs whose genes arc carried on R100. This enzymatic change is accompanied by a relatively rapid increase in the incidence of the 56%-(G C) piece, which reaches ten times the level of the other two molecular forms in a period of 4-6 hours (11). So when the culture reaches the full stationary phase, the relative abundance of the various elements derived from RlOO is:

+

MW 64 X lo6: G MW 52 X lo6: G MW 12 X lo6: G

+ C = 51.5yo - -5 + C = 48.0% - -5 + C = 5S.0yo- --SO

copies/chromosome copics/chromosomc copies/chroniosomc

Restoration of exponential growth conditions by dilution of the culture into fresh medium leads to a return to a ratio of about 1: 1: 1 for these three molecular fragments ( 1 2 ) . Additional evidence suggests strongly that R T F is present on the two larger fragments but not on the smallest, since both the 51.5%(G C) and the 4&%-(G C) plasmids can be transferred to Rrecipients at the rate characteristic of RlOO and are, in all ways, typical plasmids. The 48oJo-(G C) piece does not, however, transfer any resistance determinants, although the 51.5%-(G C) piece does. All these data are consistent with thc arithmetic relationship quoted in equation form above. R-factor replication is therefore concerted with chromosomal replica-

+

+

+

+

198

M. H. RICHMOND

tion, but the rates of replication may be set to give a range of plasmid: chromosome ratios, and the actual ratio established may well reflect the relative number of plasmid and chromosomal initiations per cell cycle. The replication of many types of R-factor, therefore, represents an intermediate state between the presence of a single copy per chromosome (as is thc casc with some phages, but that may also be found with some R-factors) and the unrestrained replication of virulent phage. The fact that those parts of the R-factor that seem to carry the RTF are always present in low numbers in relation to the number of chromosomal copies suggests that this region has an important part to play in setting the rate of plasmid replication (11,36). There is much argument as to whether the smallest fragment from R100, which increases rapidly to about 50 copies per chromosome in the late exponential phase, and which seems to lack RTF, is under regulated or unrestrained replication. Certainly, for a short period, the differential rate of synthesis of this factor may be high and bears some superficial similarity to the replication of virulent phage. Furthermore, the cell does not escape this burst of replication unscathed since it is accompanied by abnormally high levels of cell lysis (24),much as is the case with lytic phage bursts. Watanabe (2) holds the view that R determinants niust be linked genetically to an R T F to replicate, while Anderson (I),since replication can be shown to occur in the absence of a known RTF, has claimed that R determinants can replicate autonomously. It is always difficult, however, to be sure that no “orphan” RTF exists in a strain used for test purposes. At present there is little to allow one to choose between these two models, a t least as far as RlOO in Proteus mirabilis is concerned. Since the 56%-(G C) fragment that bears the resistance determinants cannot be transferred by conjugation, it is not possible to examine its replicative properties in a cell known to be free of other replicons (11). And no studies have been published on derivatives of Proteus mirabilis (R&J that carry only the 56%-(G + C) piece. Perhaps this last point implies that such strains cannot ciccur, which would be indirect evidence that an RTF was indeed necessary for the replication of the 56%-(G C) fragment from R100. There has also been much speculation as to how a single CCC’ DNA linkage group-such as the 51.5%-(G + C) form of R100-can producc a disproportionate number of smaller CCC’ molecules. The fragmentation step can most readily be explained in terms of the “Rolling Circle” hypothesis for DNA replication (36) as modified for plasmid purposes by Novick (31)(see Fig. 1 ) . According to this model, complete replication would yield another plasmid of parental type. Alternatively a break could occur a t a point to give one 48%-(G + C) and one 56%-(G + C) piece.

+

+

199

RESISTANCE FACTORS

FIG.1. The “rolling-circle” model for DNA replication applied to the situation of a bacterial plasmid [after Novick ( 3 0 1 . The site on one strand that is attacked by a special endonuclease that cleaves one strand only prior to membrane attachment is shown by n. The location of a nucleotide sequence that serves to initiate DNA polymerase is shown by i. (A) A nonreplicating plasmid is carried in the bacterial cell unattached to the membrane. (B) One strand opens a t n and becomes attached to a special site on the membrane (V).(C) The unbroken circle starts to roll, and DNA replicntion commences. (D) The process continues and the new attachment site (V)is formed. (E, F) The two copies separate and become attached to their appropriate sites in the daughter cells.

To account for the disproportionate amounts of these two fragments, one would have to postulate autonomous replication of the 56%-(G C) fragment independently of the other two pieces-much as claimed by Anderson (1). It is important to stress, however, that Rownd’s labeling experiments on the replication of RPlOO in Proteus mirubilis (23) are incompatible with the Rolling Circle hypothesis applying to this plasmid. RlOO replicates under relaxed control in Proteus mirabiZis and R-factor molecules are withdrawn from the R-factor pool for replication without any discrimination against the molecules already replicated as would be the case with a Rolling Circle. An alternative explanation for the behavior of R1OO in Proteus mirabilis has been proposed by Rownd, Kasamatsu, and Mickel (12). Instead of a large number of CCC R-determinants containing about 56%(G + C) and of MW about 12 X lo6 in the stationary phase, Rownd and his colleagues detect a small number of very large linear R-factors each

+

M. H. RICHMOND

200

FIG.2. The length distribution of R-factor Rt DNA molecules having a density of 1.718 g/cm'. Ordinate: relative number of molecules; abscissa: length (am). Reprinted, with permimion, from Rownd et al. (@).

containing (they believe) multiple copies of the R determinants (see Fig. 2). If this model is correct [and Rownd supports his claims with electron

microscopy, in which he finds linear R-factor molecules up to 60 pm long, equivalent to MW = 120 X lo6, ( l a ) ] ,it would appear that replication of the R determinants without the R T F is impossible, but that the multiple copies are achieved by attaching them one after another on the RTF. At the moment one cannot easily reconcile the data provided by Falkow et al. (11) with those of Rownd et al. It should not be impossible to agree upon a molecular weight of the 56%-(G C) component that is so conspicuous in late exponential and stationary phase cultures of Proteus mirabilis carrying R100. If it were not for Rownd's electron microscope data, one would have to favor a low molecular weight for these 56%-(G C) plasmids since small CCC DNA molecules of MW about 10 X lo6 have been found by other workers on this system (34, 36). Perhaps the exact cultural conditions are important in these experiments and Rownd's large 5 6 % ~ ( G+ C) linear molecules are en route to becoming the small 56%-(G C) CCC molecule found by the other workers. More experiments are needed. From the point of view of R-factor behavior in naturally occurring strains, the replicative activity of R T F obviously has great importance.

+

+

+

201

RESISTANCE FACTORS

Quite apart from regulating DNA synthesis so that there are enough Rfactor copies for distribution a t cell division, the flexibility in the Rfactor:chromosome ratio and the fragmentation of the structure to give genetic diversity have a major importance as far as the survival of a hacterial population is concerned. Disproportionate replication of the R-determinants, as shown with RlOO in Proteus mirabilis, leads to an increase in the number of resistance genes per cell, and this, in turn, leads to an increase in the amount of antibiotic-destroying enzyme on a cell dry weight basis ( I S , 24). Certainly this is the case for the synthesis of p-lactamase and chloramphenicol transacetylase, although the gene dosage effect may not be so pronounced with resistance to tetracycline ( I S ) . I n Proteus fnirabilis, the increase in the incidence of the R-determinants in stationary phase or when chloramphenicol is used to select the highest possible level of R-factor per cell, gives an increase in the cell content of chloramphenicol transacetylase by 4- to 5-fold in the late exponential phase Table I ) . Since the amount of R-factor-mediated plactamase varies greatly in naturally occurring ampicillin-resistant strains that rely on the enzyme for their penicillin resistance (S?‘),it would be interesting to know how much of this variability is due to a modification in the rate of expression of the m p - r gene on the plasmid and how much to an alteration in the number of plasmid copies and hence 1. in the number of genes/cell [see also (3%’) The importance of plasmid fragmentation is more difficult to assess. Rownd would argue that it was an inescapable part of the regulatory mechanism whereby the number of resistance determinants was increased under conditions of selection pressure ( l a ) , but many would disagree that this is the primary objective. It is more likely that the fragmentation TABLE I THI.:1hL.\TIONSHIP IiIET!\I~;ICN CHLOll.AMPHENICOLTI~.\NS.\CI.:TYL.\S~.:; CONTKNT OF R+ Escherichia coli AND THE PHASEOF GROWTH OF THE ORGANISMSa

Chlorampheiiicol transacetylase Extinction

Growth phaseh

Units/ml

0.1 0.4

E E 1;; E/S S

0.02

0.6 0.8 0.85

0.07 0.17 0.38 0.49

Units/mg dry wt bacteria 0.13 0.17 0.34 0.58 0.7

Data from Falkow ct a / . (24). E, exponential phme; E/S, exponential to stationary transition; S, stationary phase. a

b

202

M. H. RICHMOND

pattern reflects the evolutionary origin of the R-factor. As time goes on, more and more plasmids are being detected in bacterial cells [see, for example (18)] , and it is certain that R T F regions are not confined to R-factors. The presence of such regions in F, for example, has been known for a long time (38) and plasmids conferring a number of biochemical properties unrelated to antibiotic resistance have recently been found in strains of Pseudomonas aeruginosa (39). It therefore seems that R-factors evolve under selection prcssurc by coupling R determinants to a general purpose RTF by genetic recombination (1, 40). Whenever a composite circular DNA molecule is formed in this way, there will always be a tendency for the structurc to return to the original statc by a break near the point a t which the original insertion was made. This is because the recombination events involved must give rise to two regions of homology in thc structurc; these two regions can act as pairing regions for later

TR

n

xd

e

r

Im

Xa/Xb

FIG.3. A diagram to show how recombinant plasmids may arise by the route proposed by Campbell (41). Such double-sized recombinant plasmids are frequently unstable because they contain regions of homology ( X a l X b ; X b / X a ) , which facilitatc reemergencc of the original ~ilnsmidsor dcriwtivcs froni thrm. RTF,rcsistancc transfer factor.

RESISTANCE FACTORS

203

“fragmentation” [see (41) and Fig. 31. Even if fragmentation of Rfactors does reflect their evolutionary origin, this is not to say that the process may not be turned to advantage by providing more copies of the resistant determinants under selection pressure if the possibility to do so exists. I n general, the formation and survival of R-factors in bacterial cells seem merely to be a special example of the genetic flexibility of bacterial cells-with all that implies for the response of bacterial populations to external selection pressures.

C. The Maintenance and Segregation of Plasmids Bacterial plasmids are replicons ; that is, their replication and segregation to daughter cells at division is independent of the bacterial chromosome. Chromosomal replication is necessary for plasmid survival only when the plasmid-linked maintenance systems are inactivated by mutation or are missing (38).The replicative part of this process is considered in the preceding section. In this section we are concerned primarily with the method by which plasmids are maintained in the cell and how they are passed to daughter cells at cell division. Novick (31) has pointed out that some specific plasmid maintenance system is necessary for plasmid survival in bacteria, particularly if the number of plasmid copies per cell is low. Even when this value is around 60 per cell, and replication is possible, thc absence of a specific maintenance system leads to ultimatc loss of thc clement from the culture (42, 43) [see discussion in Novick (31)1. One possible mechanism of plasmid survival might be a random process as appears to occur with mitochondria in eukaryotic cells. Cell division partitions approximately half the available organelles into each daughter cell randomly and the cell content of the daughters is completed during the ensuing growth cycle. With bacterial plasmids, however, not only are the number of copies often insufficient for such a process to work efficiently, but the regulatory aspects of plasmid replication and segregation are too well controlled for such a haphazard process to be possible. Jacob, Brenner and Cuzin (38) proposed that effective distribution of replicons-whether chromosomal or plasmid-at cell division requires contact between the replicon itself and some point on the surface of the bacterial cell ; subsequent electron microscopy has shown that there may indeed be such a point or region of contact (44-48). I n the original hypothesis, the single contact region was conceived to be highly specialized and concerned with two basic elements of replicon survival: the initiation of DNA duplication and the distribution of the duplicated copics to the daughter cells. Thus the division of R T F function into

204

M. H . RICHMOND

replication on the one hand and maintenance and segregation on the other, may indeed be artificial and even misleading. Many mutants that affect plasmid survival in bacterial cells have been isolated, but in no case is it certain whether the lesion represents a primary failure in plasmid duplication or a secondary failure following a disruption in the transfer of the duplicated plasmid to the daughter cells (31, 38, 49-51 ) . Most of the work on replicon distribution has been done with the chromosomal replicon and relatively little with plasmids-particularly R factors. Tremblay, Daniels, and Schaechter (52) have shown that between 60 and 80% of R-factor DNA may be recovered in association with the so-called “M-band’’ material derived from mini-cell membranes; this suggests that there is indeed a point of attachment between the R-factor replicons and the membrane, in intact mini-cells a t least. Levy (63) attempts to interpret his and others’ experiments on the segregation of R-factors and other plasmids to mini-cells by claiming that the point of R-factor attachment must be polar in the cell. Certainly the different attachment sites for F and F-like R-factors is consistent with many of the distinctions between these two classes of plasmid noted by Morrison and Malamy (51) ; but the polar location of the R-factor site in mini-cells must remain just an attractive possibility a t present. Attempts are often made to infer the properties of a plasmid’s attachment site from the cell’s sensitivity to infection by other plasmidsthough this gives information only about the identity of the site and tells little of its function and position. F+cells are immune to infection with F plasmids (54-56) and the same is true for pairs of F-like R-factors (57, 5 8 ) . This phenomenon is often referred to as “plasmid incompatibility” and is usually interpreted as being caused by competition for a common attachment site in the cell (31, 59). When the compatibility relationships of R-factors are examined i t emerges that there are a t least four compatibility classes if plasmid coexistence is used as a yardstick. The F-like and I-like classes have been known for some time (S),but two new classes can now be added. They are the RP1 plasmid class characterized by the plasmids originally isolated in carbenicillin-resistant strains of Pseudoinonus aeruginosa (60) but also able to exist in E . coli (61,63),and the class characterized by the R-factor Raa (62). There are two main difficulties about using compatibility studies to infer anything about the nature of a plasmid attachment site. The first is that onc must make allowances for the possibility of “entry exclusion.” A detailed description of this phenomenon is given by Novick ( 3 1 ) . As far as R-factor transfer is concerned, its principal effect is that it may

205

RESISTANCE FACTORS

exclude certain R-factors from cells that might otherwise be hospitable hosts. The other is the phenomenon of “restriction.” The fact that incoming phage DNA will not establish itself in certain strains is familiar enough (63) and the same process may affect R-factors that attempt to survive in strains also carrying restriction genes (64). Many plasmids restrict phage DNA and vice versa [see (3) for an account of this phenomenon], but the exact molecular processes that are involved are largely unknown. The net effect is that the incoming DNA does not establish itself but is degraded-probably by specific nucleases-within the restrictive host. This phenomenon most frequently manifests itself by a change in phage pattern in cells carrying plasmids, including R-factors (65). However, the reverse process may also be found (63, 66). Probably the best way to distinguish restriction and entry exclusion, on the one hand, from plasmid interaction at a maintenance site, on the other, is to examine the progeny of the cross carefully. If there is competition for a maintenance site there are always some colonies in which the incoming plasmid has (or a t least appcars to h a w ) displaced the resident, and probably there are also some recombinants. With entry exclusion and restriction there is unlikely to be any plasmid transfer even a t low frequency-particularly if normal multiplicities of infection are used. One of the major problems introduced by the “attachment site” hypothesis, a t least if attachment is necessary for replication as originally proposed, is the situation where a single plasmid (such as R100-see above) fragments and shows a different replication rate for the various pieces (as with RlOO in stationary phase Proteus inirabilis-see above). Either the parental replicon coiitains two RTF’s one of which is masked until fragmentation occurs, or one must allow the possibility of relaxed replication (replication unrelated to chromosomal replication) when a plasmid is in a cell but “unattached”-in violation of the replicon hypothesis. I n this connection, it is important to note that no plasmid seems to fragment into two or more parts all of which are found to be present as many copies per cell. This suggests, perhaps, that R-factors contain only one RTF, potential or active, and that on fragmentation this passes to one piece which replicates by attachment as suggested by Jacob, Brenner, and Cuzin (38).Such replication would bc regulated and the number of copies per cell could be set as required a t some low value. The other piece, lacking an RTF, would replicate in a relaxed manner because of its lack of the plasmid component of the attachment complex. An alternative possibility is that the small fragment is also attached but lacks many of the necessary regulatory regions so that its replication is unrestrained, and multiple copies are made in cvcry cell cycle. The fact that typical “small” fragments-like the 56%-(G C) piece from RlOO in Proteus

+

206

M. H. RICHMOND

mirabilis-are markedly less than 1% as large as the chromosome (94) would allow multiple copies to be run off without embarrassment to the cell as far as the total amount of extrachromosomal DNA to be synthesized was concerned. At present the situation is unclear, but on balance it does seem likely that membrane attachment is a prerequisite for “stringent” replication-replication that is tied to the replication of the chromosome.

D. “Curing” Before going on t o discuss the practical implications of maintenance, it is important to digress slightly to mention “curing” agents since they interfere with plasmid replication, or maintenance, or both. “Curing” occurs when the rate of plasmid replication and/or distribution fails to keep pace with chromosomal replication and cell division. All plasmidcarrying strains seems to throw off plasmid-less variants although the probability with which this occurs varies greatly from situation to situation. R-factors, as a class, seem relatively stable while other plasmidsnotably staphylococcal penicillinase plasmids-may be lost with a probper division in naturally occurring strains ( 6 7 ) . ability as high as Many chemical and physical agents can cause an abnormally high rate of plasmid loss, although there is some evidence that all enhance an inherently unstable situation rather than initiate loss by an entirely specific method. Such an enhancement is certainly found on raising the temperature of incubation (68) with ethidium bromide (69),with acridines (70), and with rifampicin (50, 71). The classic curing agent is acridine orange when acting on F+cells (72). In this case, the probability of loss in the presence of the dye is about 50% per division, and the dye rapidly converts an F+population to the plasmid-less state. Acridine dyes are not active against all plasmids, however; the compounds have little effect on the survival of many R-factors. I n general, molecules of this general structure (Fig. 4) are good intercalating agents (73), and ethidium bromide is another such agent that has curative properties-this time against staphylococcal plasmids ( 6 9 ) , but not against R-factors nor against F. Both agents become intercalatccl in the DNA helix of the plasmid and probably disrupt either polymerase action on the plasmid DNA or plasmid distribution, or both. Thc fact that no agent is a universal curing agent implies some selectivity of action, and the differentially affected site is likely to be on the plasmid. Perhaps there is a particular susceptible sequence in a critical point in some plasmids. Since intercalating agents frequently have some specificity for regions or base sequences in DNA (74),this is not impossible,

207

RESISTANCE FACTORS

Proflavine

Mepacrine

Ethidium

FIG.4. Chemical structures of

R

number of intercalating agents.

Sodium dodecyl sulfate is another effective curing agent that is active against most Gram-negative bacteria that harbor R-factors, including pseudomonads (75, 76). The exact mechanism of action of this compound is uncertain, although it seems likely that the compound selects those cells that have already lost their plasmids rather than acting primarily to cure. As far as is known, “curing” agents have little practical importance on the survival of R-factors in the environment. Most practical interest is centered around their potential value as therapeutic agents. However, it is not immediately apparent how important such agents might be. Of course, if a single compound could be developed that would instantly cure all the R-factors from the bacterial strains in the person under treatment, such a compound would certainly find a place in the therapeutic armament, provided always that it met strict requirements as to toxicity. However, it seems inherently rather unlikely that such a compound will be easy to find. As we have already discussed in this article, not only are the various plasmids that can confer resistance to antibiotics very variable in their nature, but they are also carried in a wide range of organisms that infect different localities in the host. Furthermore, it is doubtful whether a curing agent would ever be sufficiently active to work instantaneously and completely, and consequently prophylactic use is likely to be more successful than treatment of a full-blown infection. Any com-

208

M. H. RICHMOND

pound for prophylactic use should be particularly free from side effects. I n short, a curing agent is likely to be useful only as a preventive agent in people highly susceptihle to infections in which R-factors are likely to be critically important, This is not to say, of course, that research into “curing” agents may not be revealing for the light they throw on plasmid maintenance and replication, it is just that the widespread use of such agents for therapeutic purposcs seems unlikely-at least in the foreseeable future.

E.

Practical Aspects of Maintenance

It is difficult to overestimate the importance of the R T F maintenance systems-for they are, of course, specific to these structures rather than to whole R-factors-when considering the practical aspects of antibiotic resistance transfer. Not only is the maintenance system responsible for R-factor survival in the bacterial population, but it has a direct and important influence on the entry and survival of exogenous R-determinants. Furthermore, since a given R T F need not be coupled to resistance determinants but to other genes, apparently unrelated RTFs may restrict the uptake of R-factors because they are already using a common site. Morrison and Malamy (51)have shown that phage blocks the uptake of certain R-factors, and vice versa, so much so that some phage is regarded as being “female specific”; it will not survive in cells already carrying an incompatible RTF-coupled in this case to additional genes. Similarly, certain colicinogenic factors will exclude R-factors [for a review, see ( S ) ] . The existence of a number of compatibility groups among R-factors ensures that the restrictions against R-factor transfer to R+ cells are not as severe as they would have been if only one type of RTF were found coupled to R determinants. I n practice, the number of different maintenance sites that can maintain R-factors is probably greater than four, since workers have only recently started to investigate this problem systematically (61,62).The consequence of this multiplicity of sites is that some degree of plasmid-plasmid interaction between compatible R-factors is possible, quite independent of the chromosome and its replication. Furthermore, since there seems to be no very strong relationship between the nature of an RTF and the maintenance system used, on the one hand, and the linked resistance determinants on the other, the system seems to have evolved to a point where maximum genetic flexibility is possible within the limits imposed by the need for plasmid survival in the cells of a population. The redistribution of R determinants between R-factors that can occur by this route is of the greatest importance in practical terms.

RESISTANCE FACTORS

F.

209

The Gene Products Needed for Conjugation The most obvious gene products specified by the RTF region and needed for conjugation, as opposed to plasmid survival, are the sex-piliin some strains a t least. These structures are surface hairs very similar in appearance to the common pili or fimbriae (77, 78) that are found on many enteric bacteria and pseudomonads regardless of whether they carry R-factors or not. The first type of sex-pilus to be examined was that on cells carrying the fertility factor F (78,79). The F pilus may be detected on the surface of both F+and Hfr cells by the presence of the f antigen (80, 81) and by the reaction of the cells with the so-called male-specific phages. There are two main types of phage that bind to F-pili: isometric RNA phages (for example, MS2, f2, Qp and M12) which attach to the side of the pilus (79) and filamentous single-stranded DNA phages [for example f l (82)l which attach to the tip (83). With F+ and Hfr cells, every bacterium carries a sex-pilus, and these may be seen and characterized under the electron microscope, particularly after the preparation has been labeled along its length with tightly packed isometric phages [see illustrations in ( 3 ) ] . Sex-pili certainly have a role to play in bacterial conjugation since thcir removal, usually by shearing in a Waring Blendor (78), dramatically reduces the frequency of transfer, and if prolonged, abolishes it altogether (84). Furthermore, blanking off the tip of the pilus with filamentous phage also immediately blocks conjugation (85). With F+ bacteria, the frequency of gene transfcr is about 100% when measured in terms of the number of potential donor cells capable of transferring their F-factor a t any instant in time, and this is associated with the production of sex-pili in all the cells of the population. More than one pilus may be found on each cell, and the maximum length of the structure may be several times the length of the cell that bears them (3). While the frequency of the transfer of F and any associated genes approaches loo%, this is not so for R+ donors. Here the efficiency of transfer is commonly as low as per recipient when equal numbers of donors and recipients are present in the mating mixture for a n hour or so (2). However, R-factor mutants-the so-called derepressed, or drd, mutants (86) can be isolated in which the frequency of transfer is about 100%. Although the existence of drd mutants has proved very interesting and important, their detailed biochemistry and genetics are not dealt with a t length here, largely because such genotypically derepressed R-factors are inknown in naturc, even if some isolates carrying R-factors are some-

210

M. H. RICHMOND

times phenotypically derepressed when first encountered (87). The importance of drd R-factors carrying strains from our point of view, is that such cultures are extensively piliated and this allows the properties of these structures to be compared with those formed by F+or Hfr bacteria. Although R-factors do not share the same maintenance site as F in the cell, certain of them are related to F in that. they can repress the formation of F-like sex-pili in an F+/R+ hybrid (88, 89) ; that is, some character(s) specified by an R-factor can modify the expression of the F factor RTF. Such R-factors have been called jt+ factors (fertility inhibition positive factors) or F-like R-factors (90).When the sex-pili formed by the F-like R-factors were first examined, it was thought that they were identical with those specified by F itself. Certainly they react with the “male-specific phages” MS2, Qp and f l , and also specify the antigen f (3,78).However, it is now known that, although broadly similar, F-pili and F-like R-pili are distinct, both in their reaction with antiserum (91) and on density gradient centrifugation (92). Furthermore, hybrid cells that carry both an F factor and an F-like R-factor synthesize hybrid pili that have properties intermediate between true F-pili and F-like R-pili (91, 92). A further complication has arisen recently: there appear to be some minor immunological differences between different F-like R-pili (91). Armed with the knowledge that drd mutants of fi+ R-factors specify F-like pili with an incidence of about 1 pilus per cell or more, it became possible to look a t the properties of the naturally occurring repressed strains. Many of these were found to specify F-like R-pili, but only with 8 very low incidence, which almost certainly accounts for the very low level of gene transfer in mating mixtures involving naturally occurring R+ strains. Not all self-transmissible plasmids have been shown to specify F-like sex-pili, however. The plasmid specifying colicin I [the colI factor (93)] is somewhat repressed (transfer frequency about 1% per recipient), but immediately after transfer it exists for a period of up to three generations in a phenotypically derepressed state in the recipient (94). Under these conditions, no F-like pili can be detected either with antiserum or with F-specific phages. Examination under the electron microscope showed, however, the presence of a characteristic pilus on colI+ strains but not on COP(95). This type of pilus differs antigenically, morphologically and immunologically from F and F-like R-pili. As is the case with F-pili, Ipilus-specific phages have now been isolated (fil-1-filamentous, probably containing single-stranded DNA) that bind to the pilus tip (96). Not all R-factors repress pilus expression specificd by F (i.e., some are fi- (88), and among these R-factors are some that are found to specify

RESISTANCE FACTORS

211

a sex-pilus indistinguishable from that specified by the coZI factor (and also by coZEl and coZE2) (94). This has been demonstrated critically with rather few R-factors since unequivocal results are difficult to obtain without drd strains and these have proved harder to find with I-like than with F-like R-factors (86).Nevertheless, a few such mutants are available (e.g., R64 d r d l l ) , and it can be shown that the I-pili produced by the strains play the same role as the F-like pili do in strains carrying derepressed F-like R-factors (3). F+/R,+ hybrids and R(F-like)+JR (I-like) hybrids make both F-like and I-like pili, not intermediate forms as are found with F+/R(F-like)' hybrids (91). The great difficulty of working with I-like pili is that the only convenient method of detecting their presence is to use their specificity for I-specific phages (M), and since their phages plaque only poorly even on derepressed strains, one has to rely on an ability to propagate the phage using repressed strains for propagation. I n practice, a positive result is usually one where the I-specific phage titer maintains its value in the presence of the propagating bacteria over a period of some hours. Presumably the rather weak phage burst derived from the few sensitive cells present approximately balances the loss of phage by nonspecific absorption to the cells. Apart from the recent work showing that some R-factors are fi' but make I-like pili (97),a number of observations disturb the neat classification of R-factors into F-like and I-like ( 9 5 ) . The R-factors (RP factors) detected in strains of Pseudomonas aeruginosa (60, 98) apparently make neither F-like nor I-like pili; indeed, it is possible they specify no pili a t all (61). Similarly R46 is an R-factor that is in a compatibility group distinct from both F-like and I-like R-factors as well as R P factors (62). It seems, therefore, that the scheme suggested for the classification of R-factors (95) is too simple and will have to be extended. The chemical structure of the sex-pili specified by F+ and by Hfr strains has been examined to some extent by Brinton et al. (78, 9 9 ). Most of the structure is a single protein subunit, pilin, but this certainly cannot be the only component. Not only is the pilus differentiated to the extent that some phages bind to the tip of the structure and some to the side (83), but a number of genes located in the R T F of the F factor affect pilus structure, function and phage binding (100-105). Flike pili specified by F+ cells cross-react with similar pili synthesized by R(F-like)' cells but can be distinguished both physically (92) and by immunological cross-absorption experiments (91). Little is known about the chemical structure and composition of I-like pili. They are much shorter and finer than F-pili and consequently more difficult to purify. +

212

M. H. RICHMOND

Before leaving the topic of sex-pili to discuss other genes that may be involved in forming mating pairs, it is worth mentioning something about the formation of these structures. F-like sex-pili may be removed from F+ cells by blending (78),and this allows the rate of reappearance to be followed. Brinton and his colleagues showed that complete regeneration was possible within 5 minutes but that regeneration to halfmaximal length took only 30 seconds (84). This argues that pilus formation is due to extrusion of the structure from some preformed units already available within the cell. Furthermore, the fact that much pilus material can be recovered from the culture supernatant of F+ and R+ (92) suggests that F-like pili may have a short half-life in contact with the cell, being extruded and rapidly broken off. Certainly their length (up to 20 pm) (3) makes them rather unwieldy and likely to break a t the cell-pilus junction. I n summary, therefore, the RTF is responsible for synthesizing a specific surface appendage to facilitate transfer. As well as structural genes that are responsible for the formation of the sex-pilus itself. However, there are genes that influence the ability of the pilus to facilitate cell-to-cell contact, and genes that modify phage binding (102, 103). The R T F must also carry the genes needed to regulate the formation of sex-pili since the repressed state of pilus formation in most R+ strains, coupled with ability to isolate derepressed mutants (86) must argue for the presence of such regions [see the discussion in (103) and (104)]. Even though genetically derepressed RC strains have not been found to date in naturally occurring cells, and derepressed F factors only rarely (105), it is probably inadvisable to argue too strongly that the regulatory regions of the R T F have little practical importance. Naturally occurring R-factors vary greatly in the frequency of transfer they specify (1, a ) , and while some of this may be due to the state of the particular recipient (see the sections on restriction and entry exclusion, Section 11, C), the degree of repression of the R-factor in the donor is also likely to be important. This point is discussed further below.

G. DNA Transfer During Mating The precise role of the RTF and its products in DNA transfer during conjugation is still very obscure. Willetts (106) has detected ten distinct cistrons involved in conjugation when the mating ability of diploid F+ cells is examined, but only four of these seem to be concerned with pilus formation, structure or function. Some or all of the remaining six could specify products directly involved in DNA transfer, but no positive identifications have been made so far. All the direct evidence on this point has been obtained by using

213

RESISTANCE FACTORS

purified R+ mini-cells as donors of R-factors in mating experiments with appropriate normal cells as recipients (53, 107).Kass and Yarmolinsky (108) had already shown that mini-cells carrying F’.gal could mate with normal F- recipients but that the frequency of transfer was very poor, arguing pcrhaps more for the absence of a necessary cell-mediated product in the mini-cell than for any particular plasmid properties. Certainly all the expected plasmid markers wcrc prcscnt in the F’.gaZ minicells since the gal+ recipients derived from them by mating seemed completely normal. On the other hand, Levy and Norman (107) showed that R+mini-cells could mate with R- recipients and that the frequency of transfer was as high as when normal R+ donors were used. I n addition, the R+ mini-cells could act as donors in more than one successive cross, which argues that all the necessary genetic information for gene transfer is carried on the R-factor, since R- mini-cells are completely devoid of chromosomal genes ( 5 3 ) . The fact that the donors could act repeatedly also argued against the possible leak of essential gene products from the mother cell a t the time of mini-cell budding. Apart from this rather interesting, if preliminary work, there is little to decide how many and what type of genes concerned with DNA transfer are carried as part of the RTF. Furthermore, it is quite unknown to what extent the gene products involved in plasmid replication (see Section 11, B) may also play a part in gene transfer when that process takes place. It is always attractive to think of bacterial mating as the transfer of the duplicated copy of a replicon to the copulating partner rather than to thc daughter, the recipient normally provided by cell division. Whether this is a true representation of the facts is quite unclear a t present, and much work remains to hc done in this fascinating, if technically demanding, area.

111. Resistance Determinants The antibiotic resistance determinants carried on R-factors fall into two broad classes: (1) those that inactivate antibacterial agents, and (2) those that prevent the inhibitor reaching its target in the bacterial cell. Among the first class, some enzymes inactivate by destruction and some by substitution. These various products are described only briefly hcrc. Morc dctailcd accounts arc availahlc clscwhcre (e.g., 15, 109).

A. Penicillin and Cephalosporin Resistance R-factor-mediated resistance to pcnicillins and cephalosporins is caused by the opening of the p-lectam ring to yield antibiotically inactive products (Fig. 5 ) . Four distinct variants of this enzyme are R-factor-

M. H. RICHMOND

214

t CH,-COOH

O H H C

COOH

~

H

Penicillinase action

Cephalosporinase action

FIQ.5. Penicillinase and cephalosporinase action. Note that the opening of the p-lactam bond in cephalosporins leads to the expulsion of the substituent in the dihydrothiazine ring, if chemically possible.

mediated in enteric bacteria, although only two seem to be common (7, 110). All four are constitutive, and their properties are summarized in

Table 11. Although widely different in terms of the relative rate of substrate destruction that they catalyze, all probably represent relatively minor molecular variants of a common structure (111). One of the enzyme types (Type IIIa, Table 11) is R-factor mediated both in enteric bacteria and in Pseudoinonus aeruginosa (60, 98) where the gene concerned is carried on an RP factor (61). All the R-factor mediated p-lactamases in enteric bacteria and pseudomonads appear to be “periplasmic” enzymes; that is, they are located in such a position in the cell that they are readily released by osmotic shock (112, 113). The cell-bound location of the p-lactamase ensures that the enzyme effectively protects the single cell carrying the TABLE I1 PROPERTIE:S OP p - L r \ c ~ ~ ~ ~ MEDIATT,~D s ~ ; : s IIY R-FACTORS Sdbstrate profileb Enzyme type”

Pen G

Amp

Cephine

Ib IIIa Va Vb

100 100 100 100

-0 180 500 175

350

a

140 120 40

CeX 80

<10 -

Carb

Clox

MW

0 10

I I 200

-

200

29,000 25 ,000 25,000 4,0oo

-

The enzyme classification used here is based on the one in Richmond and Sykes

(110).

* Abbreviations: Pen G, benzyl penicillin; Cephine, cephaloridme; Carb, carbenicillin; Amp, ampicillin; CeX, cephalexin; Clox, cloxacillin; I, inhibition -, not available.

215

RESISTANCE FACTORS

enzyme, unlike the situation in Gram-positive bacteria where the plactamase is extracellular and acts widely t o give protection to all the cells in a population. This point has important implications when transfer of the p-lactamase gene occurs in a selective environment.

B. Chloramphenicol Resistance R-factor-mediated chloramphenicol rcsistancc is due to thc production of chloramphenicol acetyltransferase (114). Given time and appropriate substrate concentrations, the enzyme can acetylate both the free -OH groups in the chloramphenicol although the substitution a t the 3-position takes precedence and is rate limiting (Fig. 6). Shaw and his collaborators examined a number of R- strains of E . coli, Proteus inirabilis and Serratk marcescens and found low levels of an acetyl transferase that acetylated chloramphenicol (116, 116).This observation led to the suggestion that the R-factor in R+ strains may

Chloramphenicol

3-Acetoxychloramphenicol

Acetyl- CoA CoA

II

H, ~

z

~

-

~

~

,C-CHCI,

I

O H I O=C-CH,

-

I H

~

-

~

-

~

-

~

II 0

1,3-Diacetoxychloramphenicol

FIO.6. The action of chloramphenicol transacetylase.

-

~

~

3

216

M. H. RICHMOND

not carry the chloramphenicol acetyltransferase gene but a genetic region that modifies the expression of a chromosomal structural gene (67,117). However, point mutations in the acetyltransferase structural gene have been isolated and located genetically on the R-factor so the “modification” hypothesis is unlikely to be correct. Nevertheless, the presence of this transacetylase in R- bacteria, albeit a t very low levels, is an interesting observation that may have some bearing on the evolution of the R-factor-mediated chloramphenicol resistance. Recent work on this enzyme has been admirably reviewed by Shaw (6).

C.

Resistance to Aminoglycoside Antibiotics

A number of aminoglycoside antibiotics (Table 111) have been widely used against bacterial infections, some almost as long as the penicillins. Resistance to these antibiotics is not uncommon, and where encountered in hospitals is nearly always mediated by R-factors. This is in clear distinction to the kind of resistance that has been used so much in studies on microbial genetics, where resistance is due to the modification of the antibiotic target (118). R-factor mediated resistance to these antibiotics involves inactivation (119),and Umezawa and his colleagues have shown 0-phosphorylation and N-acetylation are commonly encountered in R+ strains (120-15’9). It is now known that a number of distinct enzymes are involved in R-factor-mediated resistance to aminoglycoside antibiotics. One enzyme adenylylates streptomycin and spectinomycin (Fig. 7) and is specific for the D-threo-methylamino alcohol residuc prescnt in both compounds. TABLE I11 AMINOQLYCOSIDE ANTIBIOTICS USED IN CLINICAL MEDICINE, THIS: NaTnnF. OF THE BACTERIAL RESISTANCE TO THEM, AND THEIR CltOS5Rl.:SIST.iNCE PATTERNS Antibiotic

Mechanism of inactivation

Streptomycin Streptomycin Spectinomycin Kanamycins Kanamycins Neomycins Neomycins Paromomycin Gentamicin A Gentamicin C,.

Adenylylation Phosphorylation Adenylylation Phosphorylation Acetylation Phosphorylation Acetylation Phosphorylation Phosphorylation Acetylation

Crowresistance pattern Spectinomycin

-

Streptomycin Neomycins, paromomycin, gentamicin A Neomycins, gentamicin CIa Kanamycins, paromomycin, gentamicin A Kanamycins, gentamicin CI. Neomycins, kanamycins, gentamicin A Neomycins, kanamycins, paromomycin Neomycins

217

RESISTANCE FACTORS

Streptidine I 0

n-threo-Nmethylamino alcohol H

H

NHCH,

I

1

H

Streptomycin O

H

I

HO-P- 0-Ribose-Adenine II 0

H

NHCH,

Spectinomycin

FIG.7. The adenylylation of various aminoglycoside antibiotics and related molecules.

Another distinct enzyme phosphorylates the 3'-hydroxyl in the glucosamine residue of streptomycin (Fig. 8). Spectinomycin is insensitive to this enzyme since no 3'-hydroxyl is available in this compound (8). Two distinct R-factor-mediated enzymes inactivate kanamycin and Streptidine t 0

Streptidine t

H

CHSN

O I

H

H

HO-P-OH 1 I 0

Fro. 8. The pliospl~orylationof streptomycin.

M. H. RICHMOND

218

2

NHZ

X -

Y -

NH,

OH

(B) Mi,

NH, NH,

Kanamycins (A)

(C) OH

FIG.9. The phosphorylation and acetylation of kanamycin.

a number of related antibiotics including neomycin; one phosphorylates (122) (Figs. 9 and 10) and the other acetylates (120-122) (Figs. 9 and 10). The detailed specificities of these two enzymes are given by Davies e t al. (8).

Neosamine: R = NH, Glucosamine: R = OH

0 I

Neamine: R = NH, Paromamine: R = OH

Deoxystreptamine

Ribose OH Neosamine Neomycin: R = NH, Paromomycin: R = OH

FIO. 10. The phosphorylation and acetylation of neomycin and related antibiotics.

219

RESISTANCE FACTORS

D. Tetracycline Resista nce Whereas all the examples of R determinants discussed so far modify the antibacterial agents chemically, tctracycline resistance depends upon an altered cell permeability to the antibiotic (9, 123). The primary target for tetracycline is the bacterial ribosome, and the compound is most unusual in that it is taken up into sensitive bacterial cells by an energydependent process. Resistance, in resistant cells, is inducible by tetracycline, and the net effect is that a much higher external concentration is needed to get the same amount of antibiotic into the cell (124) (see Fig. 11). Unfortunately, this system, interesting from so many points of view, is extremely difficult to investigate, partly because the primary mechanism of action of tetracycline is still obscure, and partly because changes in bacterial permeability are always hard to handle experimentally. Be that as it may, tetracycline resistance is one of the most commonly encountered and clinically important properties specified by R-factors in bacterial cells.

E. Sulfonamide Resistance Although sulfonamide resistance in pneumococci occurs primarily by mutation of the dihydropteric acid synthetase enzyme [the target for sulfonamide action (125)3, resistance in R+ enteric bacteria seems to involve restriction of access by the inhibitor rather than modification of loo[ I00

90 L"

k

c

r

0

60-

.+.

c

= 30i 207 10

. .NA 50

I I00

4 20

Tetracycline I p g /ml)

FIG.11. The resistance of Escherichia coli to tetracycline. Thc effect of various ; and in resistant E. coli external tetracycline concentrations in sensitive E. coli (0) either preincubated ( A ) or not preincubated (A)in a subinhibitory concentration of tetracycline. Ordinate: inhibition of protein synthesis by tetracycline (76); abscissa : tetracycline concentration (pg/ml). Redrawn from De Zeeuw (124).

220

M. H. RICHMOND

the target [ (126); see a discussion of Japanesc work in (127)l.Fcw details of Yokota’s work (128-130) are available in English; and, a t present, the precise nature of the permeability changes mediated by Rfactors are far from clear.

F. Other Resistance Factor-Mediated Determinants Smith (131) has claimed that R+ enteric strains carry markers conferring resistance to NiZ+,Co2+,and to Hg2+ions, although the difference in resistance between the R+ and R- variants is only about 4-fold when tested on plates. Such margins are markedly less than found with metalion-resistance markers carried by staphylococcal plasmids (132-134) and their importance has yet to be assessed critically.

IV. Other Plusmids Related to Resistance Factors As mentioned earlier, certain R-factors seem to specify sex-pili with a structure related to, if not identical with, those formed by cells carrying colicinogenic factors. The pili of I-like R-factors have obvious affinities with those specified by COZIand coZE plasmids, while the pili of F-like R-factors are related to those of COW.Table IV, taken from Meynell, Meynell and Datta’s review (3), summarizes these relationships. There seems to be some similarity between some colB factors and the F and F-like R-factor group, although in each case there are differences quite apart from those in the nature of the resistance determinant carried. ColB-K98 produces a repressor that can suppress F-pilus TABLE IV REACTIONS OF DIFFERENT SEX-PILI WITH DONOR SPECIFIC PHAGES AND WITH ANTISERUM (SP Antiserum to

Phage

Pilus F Col v F.lac Fo.lac Ri(fi+) Col l b R64(fi-) R144(fi-) a Symbols: done.

F

+ + + + +

I

F

Col v

Rl

+ + + + +

+ + NT

NT

NT

NT

NT

NT

NT NT NT

NT

+ + +

+

+

+ +-

R144

-

-, no antibody seen on pili; +, positive reaction; NT, experiment not

221

RESISTANCE FACTORS

formation by F+ strains, but this plasmid cannot repress all derepressed F-like R-factors (1%). I n general one gets the impression that all R-factors and many wl factors are a group of plasmids with much in common. It may even be that the R T F regions of some of these plasmids are identical despite wide differences in the other genes carried. The pactical importance of this point is that plasmids that do not carry antibiotic resistance determinants may nevertheless have a major effect in the efficiency of antibiotic resistance transfer under natural conditions (see Section VI, C) . Williams-Smith and Halls (136-138) have detected three distinct transferable plasmids among bacteria associated with enteric disease in the pig. These three plasmids Hly, Ent and K88 specify the production of a-hemolysin, enterotoxin and the K88 antigen, respectively, in enteric strains-all characters widely regarded as being characteristic of enteropathogenic E . coli. The relevance of these plasmids to R-factors proper is that while they never themselves mediate antibiotic resistance, they are often present together with an R-factor in enteropathogenic strains, and their presence undoubtedly enhances the ability of the strain to colonize the animal gut.

V. The Mating Process Up to this point we have been concerned primarily with the genetic constitution of R-factors, their survival in the bacterial strains that carry them and the way in which they modify their hosts so that they can act as donors in conjugation. This section concentrates on the mating process itself: how gene transfer occurs and how the event is adapted for the emergence and spread of antibiotic resistant bacterial populations. The mating process consists essentially of three parts: (1) the formation of the pilus bridge; (2) the t.ransfer of the resistance genes; (3) the survival of the genetic information in the recipient as the culture grows.

A. The Pilus Bridge and Its Formation I n the earliest hypothesis, the mating bacteria came into direct contact, cell surface to cell surface (139); this has now been superseded by the view that contact occurs between the tip of a sex pilus carried on the donor and the wall of the recipient (78). Thus the individual cells may be some distance apart. The tip of the pilus is differentiated both structurally (3,99) and in its phage binding properties ( M ) ,and it seems plausible (at least) that this region is responsible for making contact with the female cell. Certainly conjugating bacteria appear under

222

M. H. RICHMOND

the microscope to move in concert even though not in direct contact, and the behavior of the pairs under Brownian movement gives the impression of their being joined together by an invisible but flexible thread. Common pili are thought to stabilize pair formation even though they cannot themselves initiate it (78). Although the formation of the pilus bridge seems to be an essential prerequisite for DNA transfer (99), it is by no means proved that the nucleic acid passes to the female through the pilus. Certainly the DNA is insensitive to DNase during transfer (78), and the sex pili, a t least F and F-like pili, appear to have a hollow center of just the size necessary to allow single-stranded DNA to pass (3). Nevertheless, there is no proof that the DNA passes through, rather than over the surface of, the pilus, or even in some other protected form. A lot of rather inconclusive work has been carried out on the environmental conditions that affect conjugation. Pilus production-at least in F+ and Hfr bacteria-is maximal during exponential growth and tails off when the cultures become stationary (99). Cells grown in stationary culture routinely seem to carry more pili per cell than agitated cultures (I.@), but this may be because shearing forces are less pronounced in stationary conditions. Similarly, mating frequencies are commonly higher among stationary than in agitated cultures; again, the reason may be mechanical. Many workers imply that stationary cultures are “anaerobic” while agitated cultures are “aerobic,” but this is, of course, over simple: both are likely to have a relatively unrestricted oxygen supply unless the culture density is very great. There seems to be little laboratory information as to whether bridge formation and gene transfer can occur between facultative anaerobes when the oxygen supply is limited. However, R-factor transfer can undoubtedly occur in the gut of man and animals, and the site of transfer is likely to be the large intestine where the environment is highly anaerobic (141). This is not to say, of course, that the efficiency of transfer under these conditions is as great as when the cultures are growing aerobically under laboratory conditions. Nevertheless, the fact that it occurs at all in the anaerobic environment of the large intestine must have important practical consequences as far as gene transfer in the natural environment is concerned. The pilus bridge forms very rapidly when R+ and R- bacteria are mixed and approaches the maximum incidence possible (determined by the number of pilus-bearing cells) within a few minutes of mixing (11, 140).This step cannot, therefore, be rate limiting as far as gene transfer is concerned, a t least under laboratory conditions. Contact seems to occur by random collision, but the rate a t which pair formation takes place

RESISTANCE FACTORS

223

strongly suggests that R’ and R- cells once in contact do not readily part unless separated by mechanical forces.

B. Gene Transfer 1. THE PARTPLAYED BY DNA REPLICATION

The general metabolic activity of both donor and recipient has an important part to play in conjugation, particularly in DNA transfer. This has been investigated systematically only in crosses involving F+ and Hfr donors [see review by Curtiss e t al. ( 1 4 0 ) ] , but from these experiments it would seem that both the donor and the recipient in a bacterial conjugation require a metabolizable source of energy. Aminoacid starvation (by withholding an amino acid in auxotrophic strains) leads to an immediate loss in donor ability, but this can be restored by providing the missing amino acid [see also Levy (53)1. Therefore, protein synthesis seems to be needed to initiate transfer. Nalidixic acid, an inhibitor of DNA replication ( l & ) ,has a dramatic effect if the recipient is sensitive to the drug, but less when the donor is sensitive (140, 1 @ ) , an observation that fits well with the present views on the role of DNA replication during conjugation (see below). The model proposed by Jacob, Brenner, and Cuzin (38) to account for gene transfer by F+ and Hfr donors invoked DNA replication in the donor as the driving force for DNA transfer during mating. Certainly some replication must be involved in the process since the DNA found in the recipient immediately after transfer consists of one strand synthesized in the recipient during mating and one that existed in the donor before conjugation (1.64, 1.45). However, whether the DNA synthesis must occur in both participants, how this is related to the actual process of gene transfer, and where the driving force for DNA transfer lies, are still not absolutely clear. The most likely series of events-at least for F-is summarized as follows. A single DNA strand passes from the donor ( I - @ ) , and is replicated in the recipient (147),the work needed to get the DNA across the bridge being provided by the plasmid polynucleotide biosynthesis in the recipient, even though chromosomal replication is not needed (11). Thus the synthesis of one strand of plasmid DNA in the recipient is closely coupled to the actual act of nucleic acid transfer; moreover, the strand transferred is always the same (I@). When one DNA strand is passed to the recipient, an unbalanced situation must remain in the donor unless a preliminary round of replication has provided a “supernumerary” copy of one or both strands. DNA synthesis in the donor is not closely coupled to the actual act of nucleic acid transfer, and it is uncertain, a t present, when the replicative step

224

M. H. RICHMOND

needed to restore parity occurs in the donor. Since nalidixic acid has a much less dramatic effect on mating when the donor is sensitive than when the recipient can be inhibited, the single strand remaining in the donor after transfer is probably “repaired” later, but is not an obligate part of the actual transfer process. It would, of course, have to take place before the next round of bacterial growth occurred if the donor was not to lose the plasmid in the mating process. It is interesting to note in passing that the primary role of the recipient in transfer by conjugation puts this process on a par with transduction, where the exogenous DNA comes from a bacteriophage rather than another bacterial cell. I n both cases, the replicative activity is provided by the host. Falkow et al., have studied the transfer of R1 to Escherichia coli and the behavior observed accords well with the pattern observed with F (11) even though Bonhoeffer and Vielmetter (147)stress that the details of the transfer process observed with F+ bacteria may not apply universally. 2. THE KINETICSOF MATINGAND GENETRANSFER When the fertility factor F promotes chromosomal transfer by cellto-cell contact, mating may be interrupted a t any time with a direct effect on the amount of chromosomal material transferred (149) ; indeed, interrupted mating provides a convenient method of mapping (160). However, plasmid transfer-whether F’ or R-factor mediated-is either all or none: the genes either pass to the recipient or do not. Thus there is no possibility of “fragmenting” an R-factor by premature separation of mating pairs. This is because R-factor survival normally depends upon becoming established as an independent replicon in the recipient whereas chromosomal fragments, whether broken off by blending or not, must recombine with the resident chromosome for survival (see Section V, A ) . Transfer frequencies obtained by conjugation are easy to interpret only when all the donor cells are piliated and able to promote gene transfer. Figure 12 shows the results when derepressed plasmids are transferred to plasmid-less recipients: Fig. 12B shows an R+-drd/Rcross, and Fig. 12A an F+/F-. Little transfer can bc detected within 25 minutes, but thereafter the incidence increases to reach 80% in 80 minutes, in both examples. When repressed cultures are used, the interpretation of transfer experiments is greatly complicated by a number of factors. First, both the donor and recipient populations grow, and moreover, not necessarily a t the same rate. Second, the relative abundance of female cells over those that are male and piliated allows second and

225

RESISTANCE FACTORS

r

100I00 90 -

/-‘I

70 60

R+ a@

r-

5ot 40 40t 30 30t 20 10 -

/-

/-

/

-

50 -

R-

llJ

0 10 20 30 40 50 60 70 80 90 0 10 20 30 40 50 60 70 80 90 100 A ~

,

I

I

,

:A

B

I

I

I

,

I

,

FIG.12. Transfer kinetics of bacterial plasmids to plasmidless recipients. (A) Donor: Escherichia coii F’. lac; recipient: E. coii F-. (B) Donor: E. coli R1drdl9; recipient: E. coli R-. Culture densities: donor and recipient cells approximately 10’/ml in all experiments.

third cycles of plasmid transfer in which the newly infected “recipients” seem to be particularly effective donors (see below). Nevertheless, there is nothing so far to suggest that there is any difference in transfer kinetics a t the individual cell level between genotypically repressed but piliated donors and those that are both genotypically and phenotypically derepressed. The way in which these kinetic experiments are carried out gives a measure of the efficiency of the whole transfer process (time to mate, time for the DNA to pass to the recipient, time for the incoming DNA to establish itself and the time for the resistance determinants to express themselves a t a sufficient level to protect the cell when plated on selective agar), but gives little information about the individual parts of the process. The formation of mating pairs is extremely rapid when observed under the microscope [see Section V, A and (78) 1, so the majority of the time needed for effective gene transfer must be taken up by the later steps. Studies on the duration of DNA transfer from R+ donor to recipient, both with intact bacteria and with mini-cells, gives values between 30 and 60 minutes for the overall time needed to transfer the DNA from the donor and to establish the plasmid as covalently closed circular (CCC) DNA in the recipient (11,63). These experiments also give the times taken to reach some “intermediate” stages in transfer. About 15 minutes after mating, all the transferred DNA seems to be in a double-stranded linear form ( 4 3 s ) even though the material passed from the donor was probably singlestranded (148, 147). After 25 minutes, the total amount of 43 S DNA had decreased in absolute terms to be replaced by circular doubZe-

226

M. H. RICHMOND

2nd cycle of transfer possible

----0

10

20

30

40

50

60

70

00

FIO.13. Diagrammatic repreventation of the time-course of plasmid transfer to a plnemidless recipient.

stranded DNA, some of it covalently closed on both strands (50s) and the remainder closed on one strand only (or “nicked”) (75 S) ( ( 1 1 ) , but also see below). The time course of events following R1 transfer in E . coli a t least) seems therefore to be as shown in Fig. 13. I n general, the overall time scale of the process is similar to that taken by phage replication during a lytic cycle. 3. THE FATEOF TRANSFERRED DNA

IN THE

RECIPIENT

Once the single strand has passed from donor to recipient, the incoming piece of DNA must either establish itself as an autonomously replicating genetic unit or recombine with a resident replicon-another plasmid or the chromosome-to survive. An essential first step in this process seems to be the formation of a circular DNA molecule, presumably by ligase action ( 1 5 1 ) on the ends of the linear double-stranded molecule synthesized as part of the transfer process. This step has been studied in great detail with the bacteriophage P1 (IS,%?),where the phage DNA originally present as a linear double-stranded molecule 37 pm long circularizes to form a CCC molecule 32 pm in circumference. These values imply a region of homology (about P 5 pni a t each end) which is either single-stranded DNA with a complementary scquence or doublcstranded DNA with a common sequence. Falkow et al. ( 1 1 ) have shown that the scquence of events following thc appearance of liiicar double-stranded DNA in the recipient during

RESISTANCE FACTORS

227

R-factor transfer also involves the formation of circular molecules, some with one strand covalently closed (a so-called “nicked circle”: the 50 S molecule in their experiments) and others fully CCC DNA ( 7 5 s ) ; but whether there is any terminal redundancy is unknown. These experiments suggest strongly that membrane attachment plays a vital role in the formation of the circular molecules since R-factor DNA can be detected attached to the membrane within 5 minutes of the onset of mating and a t a point where no circular 50 S nor 75 S DNA has appeared in the cytoplasm. Linear 43 S double-stranded DNA can, however, be isolated on disruption of the cells. Thereafter, circular DNA does appear (both CCC and nicked circular), and the relative amount of plasmid DNA in the membrane fraction falls. The sequence of events suggested by this work is, therefore, as follows: the linear double-stranded DNA molecules synthesized immediately after the entry of the single-stranded DNA from the donor are made in contact with the membrane, immediately circularized and one strand sealed. This attached copy then acts as the replicative form to generate the multiple plasmid copies that are liberated free into the cytoplasm in the CCC form. These experiments certainly show that membrane attachment of the plasmid occurs early in the survival of the incoming DNA; indeed, it may be essential for replication. Yet a number of papers [notably De Haan and Stouthamer ( I S S ) ] claim that attachment may be delayed in some cases [see a review by Novick (Sl)].It could bc that the incoming DNA does not always attach to the maintenance site but sometimes circularizes free in the cytoplasm to form a CCC molecule. Although Falkow showed (11) that 43 S linear double-stranded DNA and membrane attachment precedes the appearance of any circular molecuIes, there are difficulties in having membrane attachment as the only course of action open to incoming DNA. I n some cases, recombination with the chromosome or another plasmid occurs, and there is strong circumstantial evidence that this requires the presence of nonattached DNA in the CCC form (41). Perhaps membrane attachment is the route of choice if the incoming R T F has the necessary genetic information, but that circularization in the cytoplasm occurs under other circumstances with subsequent integration into a resident replicon by recombination if the necessary sequence homology is available. Falkow’s experiments were all carried out with a recipient that lacked a resident incompatible R-factor. If the maintciiance site of the recipient is already occupied when a new incompatiblc plasmid arrives, there appears to be competition for the site. Genetic studies show that the frequency of transfer to such a recipient is much reduced when compnrcd with transfer to an “empty” cell, but a certain proportion of

228

M. H. RICHMOND

transcipients are. obtained in which the incoming element appears to have displaced the resident completely. This can probably occur by two routes: either recombination occurs outside the range of the known markers to give an apparent displacement (164) or a genuine interaction for the maintenance site occurs that results in the replacement of the resident by the incomer. This last possibility raises the question of whether a plasmid is continuously in contact with its membrane site. If not, displacement by competition would be easier to understand. If the resident plasmid is incompatible, recombination is not confined to a region outside the known markers but may occur either to produce a double-size plasmid or a recombinant that now carries a selection of markers derived from the two interacting elements (3,155). The exact molecular mechanism involved in plasmid-plasmid recombination is uncertain a t present, but it does seem likely that the interaction is between an attached copy of the resident and a CCC version of the incoming element [cf. Campbell ($I)]. If the resident plasmid is compatible, there is usually little interference with the uptake of the incoming element, but recombination between the two may occur. However, the incidence of such recombinants is normally very low, as it seems easier for an incoming plasmid to seek its own attachment site than to recombine with a plasmid maintained a t another site. Nevertheless, recombinants between F-like and I-like R-factors, and between R-factors on the one hand and col factors, or F, on thc other, have been reported (3,156-168). Such recombinants may have the compatibility relationships of either parent. Recombination between R-factors and the chromosome can occur but is extremely rare in naturally occurring isolates (159) ; with certain “laboratory” R-factors and recipients, it occurs more frequently (167). I n practice, integration of R-factors and the excision of chromosomal markers can be neglected as one of the attributes of R-factors that is important in the short term-probably because there is little sequence homology between most R-factors and the chromosome of enteric bacteria, unless steps have been taken to introduce some artificially. This,’ of course, is not the case with other plasmids (notably F) where integration occurs relatively frequently and has great practical importanceas a research tool, a t least [see Hayes (160) for a good general account]. Nor must integration and the mobilization of chromosomal markers be neglected when considering the role of R-factors in the long-term evolution of bacterial plasmids. There is some evidence that certain plasmids carry an identical, or a t least very similar, gene to one carried chromosomally (115, 116, 161), and this may well reflect a common evolutionary origin.

RESISTANCE FACTORS

229

4. TRANSFER OF PLASMID FRAGMENTS

Any fragment that retains those parts of RTF concerned with pilus expression and mating functions, but that lacks the regions necessary for the transferred DNA to survive autonomously, must rely entirely on recombination for survival. With R-factor fragments, the most likely candidate within the recipient is another R-factor, as the sequence homology between the resident and incoming pieces of DNA is likely to be greatest. I n this case, the compatibility relationships of the resident plasmid are less important since no maintenance affinities are expressed by the incoming piece. Recombination with other plasmids, or with the chromosome, may occur, but this is much less common, presumably for lack of sequence homology between the two stretches of DNA (157,159). When a resident plasmid provides a means of survival for incoming genes that would otherwise be lost, the resident plasmid is said to achieve “marker rescue.” This phenomenon is more commonly encountered with plasmids transferred by transduction (162)than by mating since, in the former case, the RTF region is less likely to be intact, particularly if the plasmid was originally a large one.

5. ABORTIVE TRANSFER Any transfer that fails to establish the incoming piece of DNA as part of the hereditary material of the recipient cell is known as “abortive transfer.” Failure can be due to a number of causes: sometimes the incoming DNA fails to form circular molecules [see Novick ( 3 1 ) ,quoting unpublished experiments by Dubnau and Maas], while in others circularization takes place but the resulting closed nicked circular molecule (whichever is appropriate) fails to take up its maintenance system (see Section V, B, 3 ) . The expression of genes carried on the abortive fragments gives valuable information about the state of that DNA in the cell since it must be double-stranded (though probably not necessarily circular) to be transcribed and translated (163). Any expression of plasmid markers following transfer implies, therefore, that synthesis of the strand complementary to that injected must have occurred even if the subsequent stages in DNA survival are blocked. Abortive transfer was first studied in relation to DNA transfer by transduction, but subsequently the phenomenon has been observed following transformation and conjugation (164). In all cases the result is the same: the transferred fragment is diluted out as the culture grows. I n practice, abortive transfer of extrachromosomal elements, as opposed to chromosomal fragments, is relatively uncommon since plasmids, ,by definition, are genetic elements capable of autonomous survival in a

230

M. H. RICHMOND

bacterial cell. Most can therefore establish themselves and survive in the recipient. It is only when a mutation in the recipient (31) or a resident plasmid (164) makes the host component of the plasmid maintenance system inactive, that, abortive transfer of a plasmid occurs. The fact that genes carried on abortive pieces of DNA may be expressed in the recipient has been mentioned above. The kinetics of expression are what would be expected from the abortive nature of the transfer: enzyme is formed a t a linear rate as the culture grows, and activity is therefore rapidly diluted out [see, for example, the expression of the lac genes transferred to E. coli by abortive transduction (165, 166)].

C. The Properties of Newly Infected Recipients In view of the nature of R-factors and other transmissible plasmids, one would expect newly infected recipients themselves to act as donors in subsequent crosses. When E. coli strains carrying col I are mixed with recipients lacking col I, a relatively low rate of transfer is obtained for about 2 hours but thereafter the spread of the plasmid in the recipient population becomes rapid (93).The low transfer rate obtained in the cultures initially is due to the low level of piliation among the col I+ donors, but analysis of the subsequent period shows that more than half the bacteria that recently obtained wl I are now donors in their own right. Indeed, about 10% of the newly infected cells donate their col plasmid within 2 minutes of mixing with a colI- recipient population, although the equivalent value for the initial donors is about 0.02%. Thus newly infected recombinants are derepressed with respect to col. I transfer, unlike the donors from which the plasmid was received. Examination of the duration of derepression following col. I transfer showed that a phenotypically derepressed donor can persist for up to three generations before falling back to the repressed state (94). Watanabe ( 2 ) repeated Stocker’s experiments with an R-factorcarrying strain and found that cells newly infected with the R-factor were also derepressed with respect to transfer to a further recipient. As with the cells carrying colI, about 50% of the newly infected R+ recipients would act, in turn, as donors within a few minutes of mixing with R- cells, even though the parental R+ culture would only promote R-factor transfer a t a frequency of about I n this case, the duration of the derepressed phase could not be measured, but it was long enough to ensure the “wildfire” spread of R-factors through a susceptible Rpopulation. The mechanism of this prolonged phenotypic derepression following transfer is quite unknown. Some have sought to explain this phenomenon

RESISTANCE FACTORS

231

by invoking a failure to produce a repressor in the newly infected cell (3), much as repression of P-galactosidase synthesis is delayed following the introduction of an F.i+z-genome into an i-z+ cytoplasm by conjugation in strains of E . coli (167,168). However, the phenotypic derepression in the experiments with the lac genes only persists about 45 minutes, not three generations as found with coZI transfer (94). At present, the molecular basis of the phenotypic derepression following transfer is unknown. Nevertheless, it has crucial importance for the transfer of Rfactors in natural populations since it is responsible for the rapid spread of cells carrying R-factor when a single piliated donor is mixed with a predominantly sensitive population. Since many R determinants may be expressed by R+ bacteria within minutes of receipt of the gene, this means that the very rapid conversion of an antibiotic-sensitive bacterial population to resistance can be achieved within a few hours of the initial transfer event.

D. The Transfer Process: A Summary Before going on t o discuss the possible role of R-factor mediated transfer in the emergence and spread of antibiotic resistant populations under natural conditions, it is worthwhile summarizing the steps in the process, to stress how well R-factors are adapted to facilitate gene transfer between bacteria. The steps in the process seem to be approximately as follows when the recipients lack a resident plasmid. 1. At any instant in time, a few of the cells carrying R-factor present in the population are piliated and capable of transferring their R-factors. 2. Collision of such a piliated cell with an appropriate recipient results in the formation of a pilus bridge between the bacteria. This bridge may be up to five times as long as the individual cell. 3. Some signal given at the time of bridge formation initiates the transfer of a single strand from the R-factor in the donor either through, over, or with the help of, the pilus bridge. 4. On arrival in the recipient, the single strand of DNA (which leads with its 5’ end) probably interacts with a point on the membrane of the recipient (the maintenance site) , and the synthesis of the complementary DNA strand begins immediately. This implies that the maintenance site must be near the point of entry of the donor strand since the energy for transferring the genetic information from the donor is thought to be provided by the replicative energy of polynucleotide synthesis in the recipient. No resident DNA need be in the recipient for this process to take place. ,5. The linear double-stranded DNA formed by the last step probably

232

M. H. RICHMOND

remains attached to the membrane but circularizes to form a molecule covalently sealed on one strand. 6. This replicative form of the plasmid may then either remain attached to the membrane in a quiescent state until the next round of cell division or start to run off copies of itself, which are then liberated into the cytoplasm in the form of covalently closed circular molecules. 7. Once in the double-stranded form (perhaps circularization is a necessary preliminary), the R determinants can be transcribed and translated, and the recipient then starts to express resistance to the appropriate antibiotics whose resistance genes are carried on the Rfactor. 8. The pilus-specifying genes on the R T F part of the R-factor also express themselves and the recipient cell now becomes a potential donorhence the possibility of “wildfire” or “epidemic” spread. 9. Before cell division, the membrane maintenance site is duplicated. The attached R-factor is also duplicated, and one copy is attached to each daughter site. As the sites move apart in the dividing bacteria, so plasmid copies are segregated. 10. Any cytoplasmic R-factor copies are probably partitioned randomly a t cell division. 11. After a period that varies from plasmid to plasmid, pilus synthesis specified by the RTF becomes repressed and the piliation of the cells carrying R-factor falls. 12. With low probability, replication of the plasmid fails to keep up with chromosomal replication and a plasmid-less variant arises “spontaneously.” When there is a resident plasmid in the cell, or when recombination is needed for survival, a variant of the above sequence seems to occur. Following entry, duplication and (probably) circularization, the incoming plasmid recombines either with another plasmid or with the chromosome, but the latter is rare with R-factors. If recombination is successful, the survival of the plasmid genes is then assured by the replicative mechanism of the replicon into which it has integrated. An R-factor is ideally adapted for these transfer sequences. Not only does it carry the necessary genes to maintain the R determinants in an autonomously replicating state in any enteric bacteria, it also stimulates the cell that carries it to form the necessary apparatus to promote its transfer to cells that lack it. In the process, however, the donor does not normally lose the plasmid. Once in the recipient, the replicon nature of the R-factor ensures that it has a good chance of surviving as an autonomously replicating genetic element. If this is not possible, recombination with a resident replicon may be an alternative means of survival in the

RESISTANCE FACTORS

233

recipient. Soon after transfer the newly infected cell becomes itself a potent donor; thus the derepressed synthesis of the sex-pili in the newly mated recipient ensures the rapid spread of the R-factor through the majority of cells of a susceptible population. Furthermore, the fact that the sex-pili can promote transfer to a wide range of enteric species and other Gram-negative bacteria ensures that R-factor is not confined to members of one strain or species. Wide taxonomic gaps can be jumped: for example, E . coli to Pasteurelln (leg),to Serratia ( 1 6 ) ,to Vibrio (170) and to Proteus ( 1 6 ) ,not to mention the narrower gap between E. coli and Salmonella and Shigella spp. (1).Some R-factors will even cross the gulf between enteric bacteria and pseudomonads (60, 171). R-factors, composed as they are of relatively all-purpose and highly specific R determinants, are therefore ideally evolved themselves to promote the evolution of antibiotic resistant populations. I n the next section we examine some practical examples of this process.

VI. Resistance Factor Transfer in Nature

A. The Early Observations Much of the early work on transferable drug resistance in clinical situations is published in Japanese, and most in the Western hemisphere are dependent on the early review by Watanabe ( 2 ) for a summary of this work. It is worth examining in detail, however, since the pattern revealed has become accepted, perhaps too readily, as typical of R-factors in action, and as such has greatly influenced people’s thinking. Bacillary dysentery has, for many years, been common in Japan, and one factor that seems to have played a part in the persistence of this picture is the high incidence of antibiotic-resistant strains. I n epidemiological studies, workers investigating large-scale outbreaks commonly found that antibiotic-sensitive shigellae could be isolated from some patients whereas resistant organisms with the same serotype occurred in others. Moreover, the resistant strains were often resistant to a range of antibiotics over and above the ones used for therapy. Some patients were even found to excrete both sensitive and resistant versions of the same Shigella serotype. Many attempts were made to interpret this pattern on the basis of mutation and selection, but it was Akiba (172) who proposed that gene transfer and selection rather than mutation and selection was occurring, and that the source of the resistance determinants might be the population of E. coli coexisting with ShigeUa in the alimentary tracts of the patients. Laboratory studies on the E. coli strains in question showed that they

234

M. H. RICHMOND

could act as donors in transfer experiments with shigellac as recipients (173-175), and this route of transfer was confirmed both in human volunteers (176) and in dogs (177). A number of features of the phenomenon observed by the Japanese should be stressed because they have had an important influence on subsequent thinking about this problem, and are relevant to our discussion here: (1) transfer was thought to occur from a reservoir of R-factors in common gut saprophytes; (2) the transfer was to a superinfecting pathogen that was predominantly sensitive at the beginning of an epidemic if not at the end; (3) the clinical conditions concerned were relatively long lasting, antibiotic therapy frequently being continued for some weeks; and (4) there was a high level of antibiotic resistance among E. coli strains in the environment at the outset, presumably related to the widespread h e of antibiotics in Japan, where the restriction of antibiotic usage has only recently been introduced. As a result of the Japanese work, it became widely assumed throughout the world that the emergence of antibiotic resistant strains during therapy, particularly in urinary tract infections, was often due to R-factor transfer from strains of E. coli and other enteric bacteria resident in the patient’s intestines to enteric bacteria present in the genitourinary tract and responsible for the clinical picture. Similarly, Anderson (1, 178) sought to blame R-factor transfer from nonpathogenic strains of E . coli, or some other saprophytic cnteric strain, to Salmonella typhimurium, type 29, as source of the highly resistant and R-factor-carrying type 29 strains that caused the massive epidemic of Salmonellosis in cattle in the years 1963-1968, which spilled over into the human population on morc than one occasion to cause some deaths among those handling the animals. Much has been made of the difficulty of proving that R-factor transfer occurs in natural conditions. For example, Walton (179) has statcd: “Since there is no positive means of labelling R-factors, infective drug rcsistance cannot be used as an epidemiological tool.” And indeed it is quite impossible, in practical terms, ever to observe a transfer event in nature. One can only infer its occurrence from the subsequent pattern of events, as was done by the Japanese. Even the elegant in vivo transfer experiments performed by Williams-Smith on himself (141) do not help greatly since they can easily be dismissed as “artificial.” Furthermore, it is certainly true that the identification of the R-factors by the markers they carry is quite inadequate. Plasmids with the markers (Su.Sm.Cm) , (Su.Sm.Tc), (Cm.Tcb, (Su.Sm) and (Cm) [the examples citcd by thc .Japanese (211 are far from uncommon, and the appearance both in E . coli and in a Shigella species of thc same sct of rnarkcrs can only bc suggestive cvidcncc for ta transfer event to liavc takcn placc wen if the

RESISTANCE FACTORS

235

strains concerned can act appropriately on donor and recipient. Could not a few R-factor-carrying shigellae among the sensitive majority have escaped detection until selected? Simple statements of the resistance pattern, particularly (as is often the case) determined by disc tests, is not enough. Furthermore, many R-factors have a molecular weight of about 50 X lo6 (S4), and such a molecule may accommodate about 100 genes. Therefore, the few known markers give little information about the structure as a whole. The difficulties of assessing the importance of transfer are compounded when the pathogen is itself a strain of E . coli, and one is faced with distinguishing pathogenic from saprophytic strains of the species. One is reminded of the definition that a weed is but a plant out of place; in this case, a pathogen may be a saprophyte out of place. Since many saprophytic E. coli strains carry R-factors, a much more detailed definition of the R-factor is needed if any really plausible inferences about transfer, as opposed to cross-infection, are to be drawn.

B. Resistance Factors and Pseudomonas aeruginosa in Burns To try to improve this situation, we have carried out detailed molecular studies on some R-factors implicated in a series of cases of systemic infection with carbenicillin-resistant Pseudoinonas aeruginosa following severe burning. The parameters of R-factor identity that have becn used are :

+

(1) the (G C)-content of the R-factor DNA, and the related parameter of buoyant density on centrifugation in CsCl gradients (see Section 111); (2) the inolecular weight of the R-factor as determined by its sedimentation characteristics (see Section 111) ; ( 3 ) the compatibility relationships of the plasmid (see Section 111) ; (4) the D N A-DNA hybridization relationships between the R-factors cncountered in epidemiologically related strains ; ( 5 ) the detailed enzymatic characters specified by the resistance determinants-for example, the type of p-lactamase specified and the nature of the aininoglycosidc inactivating enzyme involved (see Section 111).

The epidemiological sequence under investigation was divided into two main pliascs about 9 months apart. It followed a period of about thrcc ycars (April 1966 t o March 1969) during which carbenicillin therapy was uscd extensively and effectivcly in the MR C Industrial Injuries and Burns Unit a t the Birmingham Accident Hospital. I n the

236

M. H. RICHMOND

following two months, 116 isolates of highly carbenicillin-resistant P s e u d o m o m aeruginosa were made from burned patients (180). The resistance was not confined to one particular phage type and serotype of Pseudoinonas aeruginosa but was found in a t least three distinct strains within this period, all of which had been detected (but sensitive to carbenicillin) in the period immediately before the first appearance of resistance. I n one patient, both the sensitive and resistant versions of the “same” strain were isolated (sensitive from February 4, 1969 up to the isolation of the resistant variant on March 3, 1969), and other patients carried two resistant strains in the late phases of treatment whereas they originally had only one, and a sensitive variant a t that. The detection of an outburst of resistant strains all with similar properties [they were all resistant to about the same extent and all synthesized a carbenicillin-destroying enzyme (180)] in a range of strains that had hitherto been sensitive (there is, of course, no formal proof that the resistant and sensitive strains are linearly descended; it is only highly probable) suggested that R-factors and their transfer might be involved, particularly since the resistant lines gave rise to sensitive variants a t a relatively high frequency (180). Further examination in our laboratory showed unquestionably that an R-factor that mediated the synthesis of type IIIa p-lactamase and that could be transferred readily between strains of Pseudomonus aeruginosa and between pseudomonads and enteric bacteria, was involved (7, 60). This observation suggested that the onset of the “epidemic” of carbenicillin resistance in Pseudomonas aeruginosa in the Burns Units in March 1969 followed R-factor spread from enteric bacteria or other pseudomonads. Subsequent studies on the characteristics of the R-factor (s) involved in this outbreak supports this view. All the R-factors were indistinguishable regardless of the strain from which they were originally isolated. All ; the pen-r being responsible carried the markers (pen-r.neo/kana-r.tet-r) for type I I I a p-lactamase synthesis (7, 6 0 ) , and neo/kana-r to the presence of the aminoglycoside phosphorylating enzyme [ (8); J. Arrand, unpublished experiments]. The (G + C) content of the R-factor was about 58% (density = 1.719 g/ml) and the molecular weight about 40 X 10’ (181485). The R-factors were all members of the R P class, which can coexist with F-like and I-like factors without restriction in enteric bacteria (61, 6%‘). Unfortunately it was impossible to examine any other strains from this first period of occurrence of carbenicillin-resistant pseudomonads. Carbenicillin therapy was stopped in the Burns Unit, and resistance to the antibiotic ceased to be a problem. About 9 months later, a very serious case warranted the use of carbenicillin once more in view of the prolonged

237

RESISTANCE FACTORS

absence of carbenicillin-resistant strains. However, the step was followed by the immediate reappearance of resistance and with a pattern of events that allowed 9 detailed study of R-factors and their role in the emergence of resistant populations to be made. On November 17, 1969, the burned patient was carrying a strain of Pseudomonus aeruginosa sensitive to carbenicillin. On November 18, a strain of Klebsiella aerogenes also appeared in the burns, and this strain carried an R-factor that mediated resistance to penicillins and cephalosporins (pen-r), to neomycin and to kanamycin (neo/kanu-r), and to tetracycline (tet-r). Twelve days later, a strain of Pseudomonas aeruginosa of the same phage and serotype as the original sensitive strains, but now carrying an R-factor (pen-r.neo/ kana-r.tet-r), appeared in the burns, and by the end of the following week, an isolate of R+ Proteus mirabilis (also pen-r.neo/kana-r.tet-r) was obtained from the same burns. This sequence is summarized in Table V. Examination of the R-factors carried in these strains showed that, on isolation, all were indistinguishable in terms of the markers carried, molecular weights, buoyant density and (G + C) content and compatibility relationships with other factors.2 They could be transferred freely between pseudomonads and enteric bacteria and gave rise to carbenicillin resistance in the former and to carbenicillin and ampicillin resistance in the latter, by virtue of type I I I a p-lactamase production. Perhaps the most sensitive index of R-factor identity is DNA-DNA hybridization (24), and studies with selected R-factors from this series TABLE V SEQUENCE OF ISOL?LTION OF STRAINS FROM A SINGLE

BURNED PATIENT"

Date

Species

R-factor

17 Nov 1969 18 Nov 1969

Pseudmonas aerugimsab P . aeruginosa Klebsiella aerogenes

-

1 Dec 1969

P . aerugimsab K . aerogenes

Late Dec 1969

P . aeruginosab K . aetogenes Protews mirabilis

-

+ + + + + +

Carbenicillin

S S

R R

It

R R

R

a Data derived from Roe et al. (f83a)and Dr. E. J. L. Lowbury by personal communication. S, sensitivity; R, resistance. Typing pattern of the P . aeruginosa strains: 7/44/F8/FlO/lo9/352/1214/M6/~01 11.

'Unpublished experiments from this laboratory.

238

M. H. RICHMOND

showed them to be indistinguishable from one another while clearly different from R-factors of a different compatibility group2 I n view of the range of R-factor characteristics examined, it is difficult to escape the conclusion that the Pseudoinonas aeruginosa strains initially present but carbenicillin sensitive in the burns of this patient received the R-factor that conferred carbenicillin resistance from an enteric strain superinfecting the burn, probably by fecal contamination. I n seriously burned patients, this type of contaminant is difficult t o avoid, and burns often harbor enteric strains of fecal origin (184). The argument, that the R-factors in these strains are similar because it is the only type of R-factor capable of conferring carbenicillin resistance on strains of Pseudoinonas aeruginosa, can be refuted by examining the R-factors involved in a parallel but unconnected series of events involving burned patients in Glasgow (186). These strains carried Rfactors with the same markers as the Birmingham strains and belonged to the same plasmid compatibility group (6). However, the molecular weights of the Glasgow R-factors are about 76 X loe (against 40 X lo6 for the Birmingham plasmids) and the (G C) content is slightly lower [ (181) and J . Saunders, unpublished experiments]. Other types of Rfactor capable of causing carbenicillin resistance in strains of Pseudomonas aeruginosa do therefore exist.

+

C. Resistance Factors and Resistance Transfer A comparison of the Birmingham events in burns with the early Japanese work indicates a number of parallels. In both, the clinical conditions were relatively protracted with large-scale use of antibiotics; in both, the pathogenic strains were predominantly sensitive, a t least a t the outset; in both, the outbreak occurred against a background of a high level antibiotic resistance, much of it potentially transferable, in the saprophytes colonizing the patients. [Davis et a,?. (184) stated that, over the period 1965-1967, 93.7% of the isolates of E . coli made in the Burns Unit a t Birmingham were ampicillin resistant and other studies (186,187) suggest that a t least 40% of these will be carrying transmissible Rfactors.] Whenever this particular set of circumstances prevails, and provided always that there is an R-factor capable of making the necessary transfer, there can be little doubt that R-factor-mediated transfer can achieve “wildfire” spread of resistance determinants throughout a susceptible population. Once this has occurred, however, further spread is always more likely to be by cross-infection by the strains themselves than by R-factor transfer. Given, therefore, that R-factors can produce epidemic spread of antibiotic resistance among susceptible pathogens in nature, how widespread

239

RESISTANCE FACTORS

is the phenomenon? And what is its relevance to clinical medicine? Probably the most common medical condition that fulfills some, a t least, of the criteria proposed for transfer to be effective, is infection of the urinary tract. I n this case, however, the invading organisms are bacteria commonly carried saprophytically in the gut or elsewhere and that have escaped to set up an opportunistic infection. A survey of the types and resistance patterns of enteric bacteria isolated from urinary tract infections in the Bristol Royal Infirmary in the first six months of 1971 are shown in Table VI. Many of the causative organisms detected before the use of antibiotics were already resistant, and this is not surprising. A number of surveys on the incidence of R-factor-carrying bacteria in socalled normal, healthy individuals has shown that a few such bacteria are probably present a t all times in everyone, well or ill. Feeding tetracycline to healthy and hitherto untreated volunteers yields, tetracyclineresistant flora in the gut-often R-factor determined (see Fig. 14)within hours, and in a survey carried out in Bristol, at least one R+-

DRUQRESIST.4NCE

TABLE VI FECAL COLIFORMS ISOLATED FIRST4 MONTHSO F 1971"

PATTERNS O F

THE

Antibiotic resistance patterns

No. of strains

IN

BRISTOL IN

Rfactor (No. in parentheses)

~

A T S AT TS TSu

43 56 4 24 5 1

ssu

16

AsU

1 1

ANe Assu ACT TSC TSSu ATS ATSSu ACSSu TCSSU ATSSuNe STCSSu

12 1 1

1s

1 8 1 3 1

1

~~

Abbreviations: A, ampicillin resistance; T, tetracycline resistance; S, streptomycin resistance; Su, sulfonamide resistance; C, chloramphenicol resistance; Ne, neomycb resistance.

240

M. H. RICHMOND

100 r

,.-0-0-0,

ij so e 80 g 70

i

U 5,

c 0) c

c

6

c

.-UI

p

60-

5040-

Y0

2 cs . I -

0

30

-

20l0-

a-” -4 -2

\

/

0,

.-

\

0

lxxxxzl

I,i

0

2

4

6

8

10

12

14

Days before and after treatment

FIG.14. The response of fecal Escherichiu coli forms in volunteers to the feeding of tetracycline. The crosshatched area indicates the duratiot of medication.

colony-forming unit was found in the first million or so bacteria examined in fecal samples from about 40% of those surveyed-all of whom had been chosen because they had not had prescribed antibiotics in the last 6 months (188).The sewage arriving a t Bristol Sewage Works contains about 0.8% of R-factor-carrying tetracycline-resistant enteric bacteria: and R-factor carrying strains are abundant in many rivers and streams (189).In short, resistant enteric bacteria (many of them already carrying R-factors) are already extremely common, and the chances are high that an organism responsible for a urinary tract infection will be resistant in the first place. As Gardner and Smith (67) put it: “Environmental selection by antibiotics and colonization of patients with resistant organisms have far greater epidemiological significance than R-factor transfer in the gastrointestinal tract.” Under these circumstances, R-factor transfer must recede in importance as a source of resistance during therapy whenever the reservoir level of resistance in potential pathogens is already high, and transfer has probably rather less part to play in the common run of clinical cases than has been assumed by some. Where R-factor transfer is likely to be important, however, is in building up the reservoir level in a potential pathogen in the first place, but the probability is that this occurs in normally healthy individuals rather than in patients under treatment. A survey in Bristol shows that a t any instant there are likely to be more R+

RESISTANCE FACTORS

241

bacteria in the alimentary tracts of the hcalthy inhabitants of this city than in those that show clinical disease, and since the numbers of transfer events is likely to parallel the number of R+ strains, it follows that transfer outside hospitals is likely to be more common than within. This conclusion is sharply in contrast to what has been frequently written and discussed about the potential of R-factors transfer [see, for example, (15)1. The view expressed here implies that the possession and transfer of R-factors has a very much more subtle and long-term effect in shaping the antibiotic resistance patterns of therapeutically important bacteria than is perhaps immediately obvious. The identification of R-factors as the elements primarily responsible for the strategic rather than the tactical response of bacterial population to antibiotic therapy fits well with what we know of their molecular properties. I n their replicon nature they have all the necessary information to allow a group of adventitious genes to survive autonomously in bacterial cells. By virtue of their ability to form sex-pili and the associated transfer apparatus, they are able to promote their own transfer together with the genes they carry from cell to cell and from species to species. The detailed characteristics of the period immediately following transfer ensures that a newly infected recipient may act in turn as a donor and achieve epidemic spread of the plasmid. If, however, we think of transmissible R-factors as elements specifically evolved to transfer antibiotic-resistance genes, we will almost certainly be making a major mistake. R-factors and their prevalence in man and his domestic animals probably merely reflects the standard process of bacterial evolution-in this case, the problem of counteracting the enormous selection pressure exerted against the majority of bacteria by the combined efforts of the pharmaceutical industry and the medical profession. Plasmids have been found that can mediate resistance against camphors and phenols in pseudomonads (39).Presumably pine trees and their exudates may present pseudomonads with similar problems to those confronting E . coli in the gut of human beings in the 20th century, and the response is the same: to use the genetic flexibility available to bacterial cells to build up resistant populations. The fact that we know so much about R-factors and so little about the role of plasmids in other evolutionary situations derives from man’s well known preoccupation with his own health. But whether we like it or not, the response of bacteria to man’s antibacterial chemotherapy program gives us a wonderful chance of watching evolution in action; but for this to be profitable, studies must be pursued intensively a t both the poulational and the molecular level. So what can we do? At the purely molecular level the great unsolved problem is how replicons survive in bacterial cells. What is the nature of

242

M. H. RICHMOND

the connection between the DNA of the replicon itself and the cell? It seems that there may be a direct recognition of a DNA sequence (to give the necessary specificity) by a cell protein, and in many ways this is analogous to the recognition of an operator region by an i-gene product in an enzyme regulatory system, Is plasmid attachment then a sophisticated variant of enzyme regulation? Does the maintenance system play a part in regulating plasmid replication so that multiplication does not outstrip ccll growth with lethal consequences? Is there any limit to the number of different types of replicon that a cell can support? And indeed, do all replicons rely on the same underlying principles for their survival? The coining of such a useful word as replicon (38)may indeed have lulled us into believing that all self-replicating entities in the cell survive by the same mechanism. Perhaps some can survive in a controlled way without any point of contact with the host cell. To what extent are plasmids truly autonomous; that is, to what extent do they carry all the genetic information necessary to ensure their survival in the cell? Perhaps bare survival is possible with their own equipment, but some cell-specified characters (for example, recombination enzymes) are necessary for the full realization of all their genetic and evolutionary potential. All these questions are relatively precise, and apart from those that concern the details of the interaction of plasmid DNA with the bacterial cell in a detailed architectural context, all seem accessible by relatively straightforward techniques that have already been applied successfully to other systems, notably the study of bacteriophages. It is perhaps on the epidemiological side, and perhaps unexpectedly, that many of the problems lie if we are to use the epidemiology of plasmid infection of bacteria to study evolution. It is precise but simple molecular tests for plasmid identity that we lack. The flowering of the studies on bacterial epidemiology had to await the development of simple diagnostic tests-phage-typing, chemical tests, etc.-to allow epidemiological work to be carried out on the relatively massive scale needed for such studies. The Central Public Health Laboratory a t Colindale in London types about 16,000 strains of Staphylococcus aweus a year without making a particular fetish of the act. Who, a t this time, can identify 50 plasmids a year with any precision? Yet epidemiological studies on this small scale are likely to be unsatisfactory and may be downright misleading. So, from the point of view of understanding the role of plasmids in human affairs and thus of watching evolution a t work, the first attack must be on plasmid identification by simple and accurate methods. It seems to me unquestionable that the most fruitful approach is likely to be electron microscopy of plasmids after denaturation and hybridization with a reference plasmid DNA. This approach has already allowed com-

243

RESISTANCE FACTORS

parisons to be madc between various similar, but not identical, stretches of DNA, and it should be possible to adapt the process to the rapid investigation and identification of R-factors. The great advantage of the method is that not only does the method allow an assessment of the extent of homology between the DNA molecules being compared, it also allows the distribution of similarity and difference along the DNA chains to be established. The reason why such an approach is crucial is 2-fold: not only would it allow similar plasmids to be identified simply and accurately (rather like recognizing people by their faces), but it might also allow recombinant plasmids to be identified. It is already only too frustratingly clear to those working on plasmids-particularly if they ever study elements freshly isolated from “natural” sources-that R-factors are very flexible entities. By the time a culture carrying R-factor has grown from a single cell to a population of about los, a very large number of plasmid variants will be present in the population although not a t a level, normally, to disturb one’s investigation of “the plasmid” in the strain. But plasmid “fragmentation” and “recombination” do occur (see ref. 1, for example), and the flexibility of plasmid constitution in a given ecological niche is probably greatly underestimated at the present time. Although the plasmids in the burn cases considered earlier were “indistinguishable,” it is highly improbable that they were identical, just as two isolates of staphylococci related epidemiologically are unlikely to be identical even though they may give rise to colonies that are typical “Staphylococcus aureus” by Bergey’s classification and have the same typing-patterns. Part of the “flexibility” found with R-factors undoubtedly stems from interactions with plasmids that are in the same cell but remain undetected until they modify the R-factor behavior (1).Some attention must, therefore, be directed toward these “orphan” plasmids, replicons that may be present but without known function. So before we can use plasmid epidemiology to study evolution in action, we must adapt some of those sophisticated tools that made molecular biology in order to screen very large numbers of plasmid characters with precision. In this sense, we must retrace the steps of those medical bacteriologists of the late 19th and early 20th centuries who investigated the cause of bacterial infections in man if we are to understand plasmid infections in bacteria and their consequences for man. “Bigger fleas have lesser fleas. . . .” ACKNOWLEDGMENTS I would like to express my thanks to the members of this Departmcnt, and particularly to Dr. T. G. B. Howc and Mr. P. H. Beard, for many valuable discussions on various aspects of the work discussed in this review. I am also indebted to

244

M. H. RICHMOND

Dr. N. Datta for proof copies of papers on R-factor classification prior to publication. The work on bacterial plasmids carried out in this Department and mentioned in this review has been supported by Grants from the Medical Research Council, the Royal Society and I. C. I. Ltd.

REFERENCES 1 . E. S. Anderson, Annu. Rev. Microbiol. 22, 131 (1968). 2. T. Watanabe, Bacteriol. Rev. 27, 87 (1963). 3. E. M. Meynell, G. G. Meynell and N. Datta, Bacteriol. Rev. 32, 55 (1968). 4. N. Bouck and E. A. Adelberg, BBRC 11,24 (1963). 6. A. Cohen, W. D. Fisher, R. Curtiss and H. I. Adler, CSHSQB 33, 635 (1968). 6. W. Shaw, Ann. N.Y. Acad. Sci. 182, 234 (1971). 7. M. H. Richmond, G. Jack and R. B. Sykes, Ann. N.Y. Acad. Sci. 182, 243 (1971). 8. J. Davies, M. Brzerinska and R. Benveniste, Ann. N.Y. Acad. Sci. 182, 226 (1971). 9 . T.J. Franklin, BJ 105, 371 (1967). 10. P. Kontomichalou, M. Mitani and R. Clowes, J . Bacteriol. 104, 34 (1970). 11. S. Falkow, L. S. Tompkins, R. P. Silver, P. Guerry and D. J. Le Blanc, Ann. N.Y. Acad. Sci. 182, 153 (1971). 12. R. Rownd, H. Kasamatsu and S. Mickel, Ann. N.Y. Acad. Sci. 182, 188 (1971). IS. A. Cohen, W. D. Fisher, H. I. Adler and R. Curtiss, PNAS 61, 61 (1968). 14. CIBA Symposium, “Bacterial Episomes and Plasmids” (G. E. W. Wolstenholme

and M. O’Connor, eds.), Churchill, London. 16. New York Symposium, “The Problems of Drug-Resistant Pathogenic Bacteria.” Ann. N.Y. Acad. Sci. 182 (1971). 16. R. Rownd, R. Nakaya and A. Nakamura, JMB 17, 376 (1966). 17. S. Falkow, R. V. Citarella, J. A. Wohlheiter and T. Watanabe, JMB 17, 102 (1966). 18. B. C. Carlton and D.

R. Helsinki, PNAS 64, 592 (1968). 19. R. P. Silver and S. Falkow, J . Bacteriol. 104,331 (1970). 20. R. P. Silver and S. Falkow, J . Bacteriol. 104, 340 (1970). 21. S. N. Cohen and C. A. Miller, Nature (London) 224, 1273 (1969). 22. S. N. Cohen and C. A. Miller, JMB 50, 671 (1970). 23. R. Rownd, JMB 44, 387 (1969). 24. S. Falkow, D. K. HUpala and R. P. Silver, In “Bacterial Episomes and Plasmids,” CIBA Symposium (G. E . W. Wolstenholme and M. O’Connor, eds.), p. 136. Churchill, Landon. 26. C. E. Helmstetter and S. Cooper, JMB 31, 507 (1968). g6. C. E. Helmstetter, S. Cooper, 0. Pierucci and F. Revelas, CSHSQB 33, 809 (1968).

27. T. Watanabe, J. Bacteriol. 85, 788 (1963). 28. H. I. Adler, W. D. Fisher, A. Cohen and A. A. Hardigree, PNAS 57, 321 (1967). 29. A. Cohen, D. P. Allison, H. I. Adler and R. Curtiss, Jr., Genetics 56, 550 (1967). 30. J. Inselberg, J . Bacferiol. 102, 642 (1970). 31. R. P. Novick, Bacteriol. Rev. 33, 210 (1969). 32. K. Nordstrom, J . Gen. Microbiol. 66, 205 (1971). 32a. K. Nordstrom, L. C. Ingram and A. Lundback, J . Bacteriol. 110, 562 (1972). 33. T. Nisioka, M. Mitani and R. Clowes, J. Bacteriol. 97, 376 (1969).

RESISTANCE FACTORS

34. 36. 36. 37. 38. 39. 40. 41. 42. 43.

245

T. Nisioka, M. Mitani and R. Clowes, J. Bacteriol. 103, 166 (1970). D. J. Kopeck0 and J. D. Punch, Ann. N . Y . Acad. Sci. 182, 207 (1971). W. Gilbert and D. Dressler, CSHSQB 33, 473 (1968).

S.B. Levy, J. Bacteriol. 103, 836 (1970). F. Jacob, S. Brenner and F. Cuzin, CSHSQB 28, 329 (1963).

A. M. Chakrabarty and I. C. Gunsalus, Bacteriol. Proc. G 137 (1971). T. Watanabe, Ann. N . Y . Acad. Sn’. 182, 126 (1971). A. M. Campbell, Advun. Genet. 11, 101 (1962). M. Yarmolinsky and D. Korn, J M B 32, 475 (1968). K. Matsubara and A. D. Kaiser, CSHSQB 33, 769 (1968). 44. A. Ryter and F. Jacob, Ann. Inst. Pasteur 107, 384 (1964). 46. A. Ryter and F. Jacob, Ann. Inst. Pusteur 110 801 (1966). 46. A. Ryter and F. Jacob, C . R . Acad. Sci. 263, 1176 (1966). 47. A. Ryter, Y. Hirota and F. Jacob, CSHSQB 33, 669 (1968). 48. A. Ryter and 0. Landman, in “Microbial Protoplasts, Spheroplasts and LForms” (L. Guze, ed.), p. 110. Williams & Wilkins, Baltimore, Maryland, 1968. 4.9. H. Hashimoto, S. Iyobe and S. Mitsuhashi, Jap. J. Microbiol. 14, 343 (1969). 50. J. H. Johnston and M. H. Richmond, J. Gen. Microbiol. 60, 137 (1970). 51. T. G.Morrison and M. H. Malamy, J. Bacteriol. 103, 81 (1970). 52. G. Y. Tremblay, M. Daniels and M. Schaechter, J M B 40, 65 (1969). 53. S. B. Levy, Ann. N.Y. Acad. Sci. 182, 217 (1971). 54. F. Jacob, P. Schaeffer and E. Wollman, S y m p . Soc. Gen. Microbiol. 10, 67 (1960). 56. J. Scaife and J. D. Gross, BBRC 7, 403 (1962). 66. H. Echols, J. Bacteriol. 85, 262 (1963). 57. H. Hashimoto and Y . Hirota, J. Bacteriol. 91, 51 (1966). 58. K. Harada, M. Kameda, M. Suzuki and S. Mitsuhashi, J. Bacteriol. 88, 1257 (1967). 58. R. P. Novick and M. H. Richmond, J. Bactetiol. 90, 467 (1966). GO. R. B. Sykes and M. H. Richmond, Nature (London) 226, 952 (1970). G I . N. Datta, R. W. Hedges, E. Shaw, R. B. Sykes and M. H. Richmond, J. Bacteriol. 108, 1244 (1971). G2. N. Datta and R. W. Hedges Nnture (London) 234, 222 (1971). 63. W. Arber, Virology 11, 273 (1968). 64. T. Watanabe, T. Takano, T. Arai, H. Nishida and S. Sato, J. Bacteriol. 92, 477 (1966). 66. P. A. M. Guinke, R. T. Scholtens and H. M. Willems, Antonie van Leeuwenhoek J . Microbiol. SeroZ. 33, 30 (1967). 66. R. Nakaya, A. Nakamura and Y. Murata, BBRC 3, 654 (1960). 67. P. Gardner and D. H. Smith, N . Engl. J. M e d . 282, 161 (1969). GS. J. M. May, R. H. Houghton and C. J. Perret, J. Gen. Microbiol. 37, 157 (1964). 69. D. H. Bouanchaud, M R. Scavizzi and Y. A. Chabbert, J . Gen. Microbiol. 54, 417 (1969). 70. N. M. Schwartz, Genetics 57, 505 (1967). 71. F. Knusel, A. R o w l r t nnd W. Zimmrrinnn. Anti. N . Y . Acrtrl. Sci. 182, 329 (1971). 72. Y. Hirota, PNAS 46, 57 (1960) 73. M. Waring, S y m p . Soc. Gen. Microbiol. 16, 235 (1966). 74. E. F. Gale, E. Cundliffe, P. E. Reynolds, M. H. Richmond and M. Waring, “Molecular Basis of Antibiotic Action.” Wiley, New York, 1972.

246

M. H. RICHMOND

76. M. Tomoeda, M. Inuzukn, N. Kuho nnd S. Nnkaniurn, J. Bncteriol. 95, 1078 (1968). 76. N. Inuzuka, S. Nakamura, M. Inuzuka and M. Tomoeda, J. Bacteriol. 100, 827 (1969). 77. J. P.Duguid, I. W. Smith, G . Dcmpstcr and P. N.Edmunds. J. Pnthol. Bnctei’iol. 70, 335 (1955). 7s. C. C. Brinton, Trnils. N.Y. A d . Sci. 27, 1003 (1965). 70. C. C. Brinton, P. Gemski nnd J. Cnrnnhan, PNAS 52, 776 (1964). SO. F. 0rskov and I. @akov, Acta Pathol. Microbiol. Scniad. 48, 37 (1960). S1. M. Ishibashi, J . Bacteriol. 93, 379 (1967). S,?. L.Cnro and M. Sclinos, PNAS 56, 126 (1964). S3. H. Hoffman-Birling, H. C. Knerner and R. Knippers, Advan. Virus Res. 12, 329 ( 1966). S4. C. Novotny, J. Cnrnahan and C. C. Brinton, J. Bacteriol. 98, 1294 (1969). 56. K. A. Ippen and R. C. Valentine, BBRC 27, 674 (1967). SG. E. M. Meynell and N. Dntta, Nature (London) 214, 885 (1967). S7. P.A. Lee and K. B. Linton, Nnlilre (London) 222, 922 (1969). SS. T. Watanabe and T. Fuliusawa, J. Bacteriol. 83, 727 (1962). S9. T. Watanabe, T. Fukusnwa and T. Takano, Virology 17, 218 (1962). 90. T. Watnnnbe, H. Nishida, C. Ogata, T. Arai nnd S. Sato, J. Bncfeviol. 88, 716 (1964). 01. A. M. Lawn, E. Meynell and M. Cooke, Ann. h s t . Pasterir 120, 2 (1971). 92. P. Beard, T. G..B. Howe and M. H. Richmond, JMB 66, 311 (1972). 93. H. Ozeki, B. A. D. Stocker and S. M, Smith, J . Gen. Microbiol. 28, 671 (1962). 94. B. A. D. Stocker, S. M. Smith and H. Ozeki, J. Gen. Microbiol. 30, 201 (1963). 96. A. M. Lawn, E. M. Meynell, G. G. Maynell and N. Datta, Nature (London) 216, 343 (1967). 9G. G. G . Meynell and A. M. Lawn, Nature (London) 217, 1184 (1968). 97. J. N. Grindley and E. S. Anderson, Gem& Res. 17, 267 (1971). 0s. P. D. Fullbrook, S. W. Elson and B! Sloeombe, Nntwe (London) 226, 1054 (1970). 99. R. C. Valentine, P. M. Silverman, K. A. Ippan and H. Mobnch, Advan. Microbial. Physiol. 3, l(1969). 100. G. G . Meynell nnd E. Aufreiter, J. Gew. MicJrobioZ. 59, 429 (1969). 101. E. Ohtsubo, Y. Nishimura and Y. Hirota, Genetics 64, 173 (1970). 102. E. Ohtsubo, Geiietics 64, 189 (1970). 103. M. Achtman, N. S. Willets and A. J. Clark, J . Baderiol. 106, 520 (1971). 104. E.Meynell and M. Cooke, Genet. Re8. 14,’309 (1969). 106. 1,. S.Baron, W. F. Carey nnd W. M. Spilmm, PNAS 45, 1752 (1959). 106. N. S. Willetts, Nntiire (Lo~idon)New Biblr 230, 183 (1971). 107. S.B. Levy and P. Norman, Nature (London) 227,.606 (1970). 10s. L. R.Kass and M. B. YarmoJinsky, P N M . 6 8 , 815.(1970). 109. J. S. Kisser, G.,O. Gale and G. A. Kempi Advan. Appli Microbiol. 11, 77 (1969). 110. M. H. Richmond and R. B. Sykes, Advon. Microbid Physiol. 9, 31 (1973). 111. G. W. Jack and M. H. Richmond, FEBS Lett. 12, 301x1971). 112. H.C. Neu nnd A. L. Heppel, JBC 240, 36$5-(1965). 113. H.C. Neu and E. B. Winshell, ABB 139,2W (1970). 114. S. Okamoto and Y:Buzuki, Nature (Londo’n)1208,1301 (1965). 116. W. Shaw, JBC 242, 687 (1967). 1lG. W. Shnw, Awtinuio-ob. Ag. Chemother. 19G6’b~221 (19671.

HESISTANCE FACTORS

247

117. K. Mise and Y. Suruki, J. Bacteiiol. 95,2124 (1968). 11s. B. Weisblum and J. Davies, BacteiioI. R e v . 32, 493 (1968). 119. S. Okamoto and Y. Suzuki, Nature (Loiidon) 208, 1301 (1966). 120. M. Okanishi, S. Kondo, Y. Sueuki, S. Okamoto and H. Umerawa, J . Antibiot. Ser. A 20, 132 (1967). 121. H. Umcenwn, M. Oknnishi, R. Utnhnrn, I<. Mnrdn ant1 S. Kondo, J. Antibiot. Ser. A 20, 136 (1967). 122. H.Umerawa, M. Okanishi, S. Kondo, K. Hamnna, R. Utnhnrn. K. Mnrdn 2nd S. Mitsuhashi, Science 157, 1559 (1967). 123. T. J. Franklin, Symp. SOC.Gea. Microbiol. 16, 192 (1966). 124. J. R.De Zeeuw, J. Bacteriol. 95, 498 (1968). 126. B. Wolf and R. D. Hotchkiss, Bchem 2, 145 (1963). 126. M. L. Pato and G. M. Brown, ABB 103, 443 (1963). 157. S. Mitsuhashi, “Transferable Drug Resistance Factor R.” University Park Press, Tokyo, 1971. 128. T. Akiba and T. Yokota, Med. Biol. (Tokyo) 58, 161 (1961).(In Japanese.) 1 9 . T. Akiba and T. Yokota, Med. Biol. (Tokyo) 63, 155 (1962). (In Japanese.) 130. T. Yokota and T. Akiba, Med. Biol. (Tokyo) 63, 160 (1962).(In Japanese.) 131. D. H. Smith, Science 156, 1114 (1967). 132. B. Moore, Lancet 2, 453 (1960). 133. M.H.Richmond and M. John, Nature (London) 202, 1360 (1964). 134. G. Peyru, L. Wexler and R. P. Novick, J. Bacteiiol. 98;215 (1969). 136. A. Frydman and E. Meynell, Genet. Res. 14, 315 (1969). 136. H. WilliamsSmith and S. Halls, J. Gen. Microbiol. 47, 153 (1967). 137. H. Williams-Smith and S. Halls, J. Pathol. Bactem’ol. 93, 499 (1967). 138. H. Williams-Smith and S. Halls, J. Gen. Microbiol. 52, 319 (1968). 139. T. F. Anderson, E. Wollman and F. Jacob, Ann. Znst. Pasteur 93, 450 (1957). f4O. R. Curtis, Jr., L. J. Charamella, D. R. Stallions and J. A. Mays, Bactem’ol R e v . 32, 320 (1968). 141. H. WilliamsSmith, Ann. N . Y . A d . Sci. 182, 80 (1971). 142. W.A. Goss, W. H. Diete and T. M. Cook, J. Bacteriol. 89, 1068 (1965). 143. K. W. Fisher and M. B. Fisher, CSHSQB 33,629 (1968). 144. M. Ptashne, JMB 11, 829 (1965). 146. J. D. Gross and I,. Caro, JMB 16, 269 (1966). 146. W.D. Rupp and G. Ihler, CSHSQB 33,647 (1968). 147. R. Bonhoeffer and W. Vielmetter, CSHSQB 33, 635 (1968). 14s. D.Vapnek and W. D. Rupp, JMB 53, 287 (1870). 149. E. Wollman and F. Jacob, C. R . Acad. Sci. 240, 2449 (1955). 160. E. Wollman, F. Jacob and W. Hayes, CSHSQB 21, 141 (1956). 161. M. Gellert, PNAS 57, 148 (1967). 162. H. Ikeda and J. Tomieawa, CSHSQB 33, 791 (1968). 163. P. G. De Haan and A. H. Stonthamer, Genet. Res..4, 30 (1963). 164. M.H. Richmond, Advan. Microbial Physiol. 2, 43 YlWJ). 166. M.H. Richmond, BJ 113, 225 (1969). 166. T. Watanabe and C. Ogata, J. Bactem’ol. 91,43 i lm). 167. P. Fredericq, In “Bacterial Episomes Plasmids, Ciba Symposium (G. E. W. Wohtenholme and M. O’Connor, eds.), p. 163. Churchill, London, 1969. 168. E. S. Anderson, J. N. Mayhew and N. D. F. Glridtlfey, Nature (London) 222, 349 (1968). 169. Y. Sugino and Y. Hirota, J. Bauteriol. 84, 902 c1962).

248

M. H. RICHMOND

160. W. Hayes, “The Genetics of Bacteria & Their Viruses,” 2nd ed. Blackwell, Oxford, 1968. 161. M. H. Richmond, Geiiet. Res. 19, 187 (1972). 162. T.Watanabe and T. Fukusawa, J. Bactei-ioZ. 82, 202 (1961). 168. M. Hayashi, M. N. Hayashi and S. Spiegelman, PNAS 51, 351 (1964). 164. E. M. Dubnau and W. Maas, J. Bacteriol. 95, 531 (1968). 165. H. R. Revel, S. E. Luria and N. L. Young, PNAS 47, 1974 (1961). 166. H. R. Revel and S. E. Luria, CSHSQB 28, 403 (1963). 167. A. B. Pardee, F. Jacob and J. Monod, J M B 1, 165 (1958). 168. F. Jacob and J. Monod, J M B 3, 318 (1961). 169. H. S. Ginoza and T. S. Matney, J. Bacteriol. 85, 1177 (1963). 170. S. Kuwabara, T.Akiba, K. Koyama and T. Arai, J n p . J. Microbiol. 7, 61 (1963). 171. D.H. Smith and S. E. Armour, Lancet 2, 15 (19es). 179. T. Akiba, Proc. 15th Gen. Meeting Jap. Med. Ass. 5, 299 (1959). (In Japanese.) 178. K. Ochiai, T. Yamanaka, K. Kimura and 0. Sawada, Nippon Zji Shirnpo 1881, 34 (1959). (In Japanese.) 174. T. Akiba, K. Koyama, Y. Ishiki, S. Kimura and T. Fukushima, Jap. J. Microbiol. 4, 219 (1960). 175. T. Akiba, K. Koyama, Y. Ishika, S. Kimura and T. Fukushima, Nippon I j i Shimpo No. 1866, 46 (1960). (In Japanese.) 176. T. Akiba, K. Koyama, S. Kimura and T. Fukushima, M e d . BioZ. (Tokyo) 59, 185 (1961). (In Japanese.) 177. S. Mitsuhashi, K. Harada and H. Hashimoto, Jup. J. E q . Med. 30, 179 (1980). 178. E. S. Anderson and M. J. Lewis, Nnture (London) 206, 579 (1965). 179. J. R. Walton, Ann. N . Y . Acad. Sci. 182, 358 (1971). 180. E. J. L. Lowbury, A. Kidson, H. A. Lilly, G. A. J. Ayliffe and R. J. Jones, Lancet 2, 448 (1969). 181. M. H. Richmond, R. B. Sykes, J. Grinsted, L. C. Ingram and J. A. Saundera, Proc. 1st Int. Congv. R-Factors, Smoleiiice, p. 27 (1972). 182. L. C. Ingram, R. B. Sykes, J. Grinsted, J. R. Saunders and M. H. Richmond, J. Gen. Microbiol., 71, 289 (1972). 188. J. Grinsted, J. R. Saunders, L. C. Ingram, R. B. Sykes and M. H. Richmond, J . Bacteriol. 110, 529 (1972). 183a. E. Roe, R. J. Jones and E. J. L. Lowbury, Lancet 1, 149 (1971). 184. B. Davis, H. A. Lilly and E. J. L. Lowbury, J. C h i . Pathol. 22, 634 (1969). 186. W. A. Black and R. W. A. Girdwood, Brit. M e d . J. 2, 234 (1969). 186. N. Datta, Brit. Med. J. 2, 407 (1969). 187. G. W. Jack and M. H. Richmond, J . Gen. Microbiol. 61, 43 (1970). 188. K. B. Linton, P. A. Lee, M. H. Richmond, W. A. Gillespie, A. J. Rowland and V. N. Baker, J. Hyg. 70, 99 (1972). 189. H. WilliamsSmith, Nature (London) 228, 1286 (1970).

Lysoge nic Induction

I

ERNEST BOREK AND ANN R Y A N ~ Department of Microbiology, Uiiiversity oj Colorado Medical Center, Denver, Colorado

I. Introduction . . . . . . . . . . . . . 11. Historical . . . . . . . . . . . . . . 111. Direct Methods for Inducing Lysogens . . . . . . . A. Ultraviolet Irradiation . . . . . . . . . . B. Mitomycin C . . . . . . . . . . . . C. Thymine Starvation . . . . . . . . . . D. Fluoropyrimidines . . . . . . . . . . . E. Thermal Induction . . . . . . . . . . . F. Other Induceiv . . . . . . . . . . . . IV. A Program Analysis of Early Phage Functions . . . . . V. Prophage Induction: A Two-Stage Process . . . . . . A. The Derepression . . . . . . . . . . . B. The Excision and Cell Death . . . . . . . . VI. Indirect Modes of Induction . . . . . . . . . A. Indirect Ultraviolet Induction (Cross-induction or BorekRyan Effect) . . . . . . . . . . . . B. Other Indirect Modes of Induction . . . . . . . VII. Proposed Mechanisms of Lysogenic Induction in Bacteria . VIII. Analogies in Mammalian Systems . . . . . . . . References . . . . . . . . . . . . .

.

249 250 252 252 253 253 253 254 254 254 260 261 263 265 265 282 282 289 292

1. Introduction On this twenty-first anniversary of the discovery of lysogenic induction, the molecular mechanism still remains obscure. Yet, the intervening years have brought considerable understanding of “this Manichean situation in which . . . the good changed into the bad as a temperate phage was induced” ( I ) . The purely conceptual “prophage” is now well defined genetically and physically in several host-bacteriophage systems, although its transition to the vegetative state in most cases continues to be enigmatic. The demonstration that massive induction could follow the ultraviolet irradiation of lysogenic populations ( 2 ), whose propensity for phage production had been shown to be a heritable attribute (S),provided an operational handle for the intensive exploration of temperate 7 Deceased September 12, 1972. 249

250

ERNEST BORER AND ANN RYAN

phage development. Any retrospective of lysogeny must immediately designate these early observations in Lwoff’s laboratory as remarkably seminal findings, for the consequent suggestion that an early “decision” for lysogenization or lytic development occurred in infected cultures (4, 5 ) presaged the genetic control over evcnts later discerned in induced lysogens. The biochemical definition of lysogenic induction has demanded detailed understanding of two requisites for lysogeny : continuing repression of particular early phage-specific activities, and the integration of the phage genome with that of its host. These molecular mechanisms have such complexity that our comprchcnsion remains rudimentary. The multiplicity of inducing agents has also tcnded to compound the difficulty of clarifying thew processes a t the molecular level. Wc have selected from the voluminous phage literature of the past decade, with special emphasis on the last fivc years, experimental contributions with immediate relevance to lysogenic induction. Specific methods commonly used to induce bacterial lysogens are presented immediately. Bacterial lysogcny is then defined in the results of the intensive analyses of one temperate phage system. We consider indirect ultraviolet induction in a detailed rcvicw of the phenomenon. hlodels for the lysogenic induction mechanism suggested by these many observations are juxtaposed, and, finally, analogies with virus-transformed mammalian cell lines are indicated. The emphasis placed on the Escherichia coli K12 (A) system throughout the tcxt rcflects both the past bias of related research and our enduring confidence that this constitutes a representative model.

II. Historical Examination of the history of lysogcny over its first forty years reveals three eras of particular emphasis. Research for twenty years after the discovery of bacteriophage (6) focused on distinguishing carrier states from true lysogcny (7-11).By 1925, both Bail and Bordet had claimed the indepcndent discovery of strains in which each host cell produced a phage-yielding clone in continuity (8, 11). The genetic involvement that appeared in suggcstions that bactcriophages arc lethal gcncs ( l a ) was reinforced by Burnet and McKie’s isolation of a pcrmancntly lysogenic Salmonella strain within which phage development was considered to follow the activation of a specific “anlagc” (13). Observations that bactcriophagcs enter a noninfectious or “eclipse” stage within infected hosts (14, 15) and were

LYSOGENIC INDUCTION

251

absent in several purported lysogens (16)antedated by seventeen years the definition of lysogeny. Two reports ushered in a new era. The pedigree studies of Lwoff and Gutmann in which single-ccll isolates of Bacillus megateriwn remained lysogenic through nineteen generations (S), and the linkage of the capability for X phage production to the gal IV locus of the E . coli K12 chromosome (17,18) showed lysogeny to bc the “hereditary pcrpetuation of the phage integrated with the genome of the bacterium” ( 4 ) . Emphasis subsequently shifted to the temperate phages of the Enterobacteriaceae through which the new subdiscipline of Iiactcrial genetics rapidly evolved. Lysogeny appeared to be controlled by the prophage itself and by the bacterial site of attachment, for newly isolated coliphages, with the exception of P1,always associated with the host chromosome a t specific sites; only some were UV inducible or exhibited cross-immunity to X (19,20). An asymmetry in the recovery of this prophage in reciprocal bacterial crosses led t o the hypothesis that induction could occur during conjugation (21), a proposal subsequently confirrncd experimentally ( 2 2 ) . Each prophage transferred by double lysogens was induced a t a characteristic, independent rate that reflected its situation on the bacterial chromosome undergoing oriented transfer (93,24),Development within the cytoplasm of nonimmune recipients was limited to those prophages that were inducible; suitable crosses soon confirmed that immunity is dominant to nonimmunity. This suggested the presence within an immune cell of a diffusible repressor whose interaction with the phage genome prevented the induction of prophage or the multiplication of superinfecting homoimmune phages. Formulation of a repressor model analogous to that proposed for the lac operon (26) required that three mutational types be distinguished; all were found among the determinants in the C region of the phage genome (26).The capacity of the temperate P22 phage of Salmonella to lysogenize, similarly, is controlled by thc short C region of its genome (27).Other nonlysogenizing X mutants escape repressor control and were described as virulent (V) types (28).Since these involved polygenic changes, they sccmcd to result from operator mutations in genes that determine early repressible functions. By the decade’s end, a mutation, Xind-, which maps within the C1 region and renders lysogens insensitive to UV induction, was recognized (29).Its dominance over ind’ prophages in double lysogens and its zygotic induction on transfer to nonimmune recipients implied that the rcprcssor could be qualitntivcly or quantitatively altered. The swift pace of genetic analysis in the period 1951-1961 provided only a vague image of the complexity that was to be found in the sequen-

252

ERNEST BORER AND ANN RYAN

tial expression of temperate phage functions after lysogenic induction. That induction itself would be a multistep process with early lability had been suggested by the observations that extraneous factors such as temperature (SO, S I ) , dose of inducer (S2),and, particularly, inhibition of protein synthesis ( S 3 ) alter the decision for stable lysogenization or lytic development. The regulatory system appeared to be so delicately poised that inducing agents might exert their effect through the arrest or decrease of repressor synthesis or through the bypass or enhancement of particular early gene functions. Research in the ensuing decade, we shall show, clarified the gene activation sequence in induction but left unanswered questions about the modus operandi of diverse inducing agents.

111. Direct Methods for Inducing Lysogens Inducibility, which is determined by the genetic constitution of the provirus in all lysogenic systems, responds to specific alterations in the physiological state of the host cell. The diverse agents and treatments initiating bacteriophage syntheses arc most effective when cultures are in logarithmic growth. I n general, minimal inducibility exists when lysogens have reached stationary growth phasc or when they have experienced an amino acid starvation, an energy deprivation or exposure to inhibitors of protein synthesis (4, 34, 56). Inducing conditions either directly inactivate the repressor or prematurely terminate host DNA synthesis while other biosyntheses proceed. Any model proposed for an inductive mechanism must accommodate both circumstances. Common methods for inducing bacterial lysogens are outlined below. Full analysis of these factors and the known biological impairments so imposed can be found in the cited references. Unless otherwise noted, these methods can be applied to bacterial lysogens grown to mid-logarithmic growth phase in enriched medium or in any of several buffered synthetic media supplemented with essential growth factors and a utilizable energy source. In all cases, induced complexes or free phages are assayed by the soft agar ovcrlay nicthod (38).

A. Ultraviolet Irradiation (37-@b) Cclls, washed and resuspended in cold 0.9% saline or buffer, are irradiated by a low-voltage mercury resonance lamp with a maximal energy output at wavelength 2537 A, e.g., a 15-W germicidal lamp. This is most effective when a population approximating 2 X loR bacteria per milliliter is exposed in thin layers (1-2 mm) ; corrections for screening must be made when either parameter is increased ( 4 1 ) . The total incident

LYSWrENIC INDUCTION

253

dose for maximal induction should be empirically determined, for even substrains can vary in UV sensitivity. Samples can be removed immediately for infcetious centers assays or restored to growth medium for additional incubation. All operations following the irradiation are performed in subdued or yellow light to prevent photorestoration. [See Rupert and Harm (4a.l

B. Mitomycin C (43) Cultures are supplemented directly or resuspended in fresh growth medium containing thc antibiotic for continued incubation with aeration at the optimal growth temperature. Responsiveness is somewhat variable, but, in general, cell populations (2 X loxbacteria per milliliter) are fully and irreversibly induced after 10 minutes' growth with 5-10 pg of mitomycin C per milliliter or after 15 minutes with one-tenth these concentrations. The supplementation of synthetic media with Casamino acids enhances the phage yield, which parallels the residual growth. An increase in optical density occurs without cell division for 30 and 60 minutes a t the respective concentrations. Treatment with 1 pg/ml usually initiates lysis 90-100 minutes after the addition of the antibiotic and culminates in complete lysis 60-90 minutes later (44-46').

C. Thymine Starvation (47) Thymine auxotrophs, grown in synthetic medium, are washed and resuspended in the same medium without thymine. Incubation a t the optimum growth temperature is continued for several hours before the readdition of thymine. Both the burst size and latent period depend on the duration of the thymine starvation. Maximal yields are obtained usually after 150-210 minutes of prior starvation. Lysis starts 30-40 minutes after the addition of thymine and is complete in 60 minutes (45,4840)*

D. Fluoropyrimidines (51, 59) Lysogcns, grown in minimal salts glucosc medium supplemented with Casamino acids, are incubated in the presence of 5-fluorouracil (1-100 pg/ml) for 90 minutes. Thymine and uracil or their nucleosides are then added (50 pg/ml), and incubation is continued for an additional 120-150 minutes. The inducing activity of the deoxyribonucleoside fluorodeoxyuridine (FdUrd) depends on its concentration: a 5-hour incubation with 0.5 pg/ml induces only 5% of the X lysogens whereas 3 hours with ten times more FdUrd increases induction to 30%. When this higher concentration

254

ERNEST BOREK AND ANN RYAN

is supplemented with uracil (200 pg/ml), complete induction can be obtained within 90 minutes a t 37°C (6566).

E. Thermal Induction Genetically susceptible lysogens, grown in tryptone broth or synthetic medium to a population density approximating 2 X los cells per milliliter, are diluted 1:loO in fresh culture medium previously warmed to the desired temperature. The appropriate exposure time and temperature, as well as a possible requirement for concurrent protein synthesis, are genetically determined. I n the only system analyzed to date, E . coli K12 ( A ) , one group of clts mutants is induced irreversibly after a 30-second heat pulse; another group requires a minimal heating time of 2-5 minutes accompanied by protein synthesis. Some heterogeneity appears in thermal sensitivities, yet all mutants are fully induced a t 45°C (66).

F. Other Inducers Both X-rays and gamma rays are variably effective in inducible systems owing to their high lethal activity (67,68). A large group of mutagens and/or carcinogens are inducing agents in particular systems (4,691*

IV. A Program Analysis of Early Phage Functions The complex model proposed for the regulation of temperate phage development includes both positive and negative controls presumed, most often, to act a t the level of transcription. Impressive supporting evidence is available only for lambda, yet analogous results obtained with other temperate phages, while fragmentary, indicate that composite regulatory systems occur generally (60). Genetic maps of the vegetative coliphages, h (Fig. 1) and P22 designate both essential and dispensable genes, the known control sites and, where determined, their transcriptional direction (61-67).Cistrons of related function are clustered here, but, as detailed in Calendar's review (60), this feature is not universal in temperate phage genomes. These lysogenizing phages provide the most current information with relevance to lysogenic induction. This orientation might well restrict our interest to the negative control that prevents the initiation of phage syntheses, but earlier descriptive studies so often have equated induction with cell viability losses or repression with the absence of plaque formation that a cursory survey of an entire intricate regulatory scheme seems justified. The interdependency of phage syntheses was defined functionally by

255

LYSOGENIC INDUCTION

31% C+C

t

I

f

2 a

F l

4

57 % G+C

5’G 48%

Gtt

3‘

42 ‘1L C+C

FIG. 1. Genetic and molccular map of Escherichiu coli bacteriophage lambda. For definition of symbols scc W. Saybalaki, in “Handbook of Microbiology” (H. Lechevdier and A. I. Imkin. cda.), Chemical Rubber Co., Cleveland, Ohio, 1972.

complcrnentation studies utilizing the many phage mutations identified as conditional lethals or recovered in marker rcscue experiments (64, 757 9 ) . In cssencc, mutations in hctcroimmunc superinfecting phages whose functions were not rescued by wild prophages are postulated to be under dircct negative control by immunity substance. Other cistrons that bypass this regression in the tram configuration are controlled positively by a diffusible inducer provided by an earlier gene activity (80). Homoimmune phages infecting h lysogens can express only genes C1 (66)and Rex (63, 81) whose rcspcctive products arc the h repressor and T4rII cxclusion factor.

256

ERNEST BOREK AND ANN RYAN

Particular pleiotropic phage mutations, assigned to cistron N , result in slow replication of phage DNA (82, 83), the synthesis of little early mRNA (66, 84, 86),and the absence of the earliest enzyme detectable after induction or infection, the h exonuclease, and the late-appearing endolysin and serum-blocking power (86-90).Thomas suggested that this early N product induces early functions and alters later phage syntheses by virtue of its influence of h exonuclease synthesis (80). The direct relationship is confirmed by observations that exonuclease production is temperature sensitive in the presence of temperature-sensitive N mutations and is restored by ochre suppression of amber N mutations (89, 90). Since the “functional half-life” of the N product is only 3-5 minutes (91-93), its synthesis may be necessary throughout infection (94). Crosses between the deletion mutant hb, and seven independently isolated mutants devoid of integrating ability revealed two genetic sites, intA and intB, active in phage-specific recombinations (96). The intA product, “integrase,” acts in trans and appears to be a site-specific recombinase whose locus maps between b, and the cf 11 gene (96, 97). Recombination mediated by this enzyme is limited to the attachment site itself (98-100). Similar int mutants have been found among the P22 temperate phages (69),and, in several mutants of h and P22, the quality of the diffusible product is altered. IntB is necessary for stable lysogenization, but its role remains undefined. The simple possibility that prophage excision could entail only the interaction of integrase with a recombinant recognition site has succumbed to recent experimental attack. Other A mutants, xis, are able to integrate efficiently but cannot excise when repression is released (101).The gene involved maps between int and e m (102, 103), and, like them (10.4, 106), acts in tram under the positive control of gene N . Two additional genes, 0 and P , whose products are required for phage DNA replication (82, 83, 106, 107) are also under direct immunity control. Each mutant type synthesizes early mRNA a t a normal rate, but little late protein appears (92), possibly because no template multiplication occurs; both produce the large mRNA that is essential for their later transcription of mRNA from the region A J (108).Cistron P is assumed to direct the synthesis of the h endonuclease that creates the single-strand break required for the initiation and/or continuation of X DNA synthesis (109). However, both genes 0 and P must be intact for the appearance of the endonuclease (11 0 , f f 1).Neither gene function absolutely depends on N activity since N defectives replicate slowly (82, 112414) and accumulate the 0 product after UV induction. Both cistrons appear to be in the same operon; their simultaneous transcription is enhanced by the

LYSOGENIC INDUCTION

257

N gene product (116), responds uniformly to noncomplementing polar which mutations in region J: (117, 118) and becomes constitutive in hl,, has mutated in the y region (119, 120). If this last mutation is combined with certain c1 changes, the DNA constitutively synthesized overtitrates the available c1 product producing virulence ( 1 2 0 4 2 2 ) . An immediate response to gene dosage becomes evident in the activity of gene Q whose full expression follows completion of DNA replication (83, 86, 74). Its product stimulates transcriptioil of late genes, for Qmutants synthesize normal quantities of phage DNA, albeit noninfectious in transformations, but only reduced amounts of lysozyme and serumblocking power (86). Weak suppressors fail to correct Q mutations so the gene’s product is assumed to act stoichiometrically (123) a t a n “essential site” in cis located between genes Q and S (124-126). Later syntheses respond to this interaction, but replication of the template is required for the adequate production of lysozyme, head and tail components to permit infectious phage formation and release. The delineation of transcriptional units followed the physical separation of DNA half-molecules produced by transverse shear (127) or by the selection in CsCl gradients (128) of that DNA strand, rich in cytosine clusters, that preferentially binds poly (G) (63). Changes in nomenclature reflect the altered methodology; in short, the C strand, binding poly(G) and transcribed from left to right, was redesignated the r-strand while the W strand, oppositely oriented in the 5’G3’direction and, consequently, read in reverse, became the Z-strand (63, 129). Gene orientations were determined subsequently by comparing the genetic activity of selected markers in reciprocal DNA heteroduplexes formed in vitro (1%). The transcription pattern obtained by annealing to specific DNA segments that mRNA produced during the development of particular mutant phages permitted the development of transcription maps (131) within which the gene c1 lies 0.22 k 0.08 fractional molecular lengths from the right end of the A DNA molecule (132). General regions for initiation and termination can be inferred from the convergence of these transcriptions, the placement of two known promoters a t either side of the cl gene, and from the single switch point. The genetic segments whose template activities have been distinguished readily in hybridization analyses are cl-rex, N , c111-att on one strand and x-P, Q, SR, and A-J on the other DNA strand. Their transcription from the Z and r strands, respectively, reveal a convergence of RNA polymerase action in the genetically unmapped b, region from which only a small amount of mRNA is transcribed on each strand after induction (66, 133). Late transcription of the left segment of bz on the r-strand may terminate what appears to be a sequenced reading of the “late”

258

ERNEST BORER AND ANN RYAN

phage genes (134). Some dependence in v i v o on the physical contour of the phage chromosome does exist, for, should excision or circularization be blocked, neither early nor late l-mRNA from this region is synthesized (133). Control a t the transcriptional level is definite. If a thermolabile repressor is heat-inactivated, both the Z and r viral mRNA's are coordinately induced in a weight ratio of 5 : l with a decrease in 1 synthesis about 10 minutes after induction (135).An active N gene, whose transcription from the Z-strand occurs 30 seconds after induction (135,136), is required for the synthesis of this large 1 mRNA from genes c l l l , exo, B, xis, and int and of the large r MRNA from the region that includcs genes Q and R (66, 74, 84, 85, 136).Transcription of all genes between int and rex occurs a t a constant rate while that from gene N falls with time. Nsus mutations limit transcription to the N cistron itself and to regions immediately proximal to the cl gene (85, 136). Preferential transcription from the r strand to the right of cl starts in E . coli K12 (A) about 25-30 minutes after UV induction (63). The direction of mRNA transcription clearly changes between the segment cl-N and region x - P (63,133). A polar mutation between N and rex creates sex which is c&-defective in the expression of genes N , c l l l , e m , B, and xis (137, 138) and shows a 98% decrease in the 1 mRNA synthesized after derepression (139, 1400).This can be demonstrated both in vivo (93) and in vitro (ldla, l 4 l b ) . Sufficient N gene product is made to induce late essential syntheses, yet these inadequacies indicate that the entire region to the left of gene cl depends on one promoter fop leftward transcription. Accordingly, genes N , exo, and B are expressed independently of the transcription of the 2-0 segment (1.42).It appears that direct control by gene cl of x - P transcriptions may indirectly control phage replication (143), as superinfecting X phages cannot replicate under immune conditions even when a replicating helper phage Aimm 434 is present (144). Back-mutation of X sex restores to normal levels all syntheses from N through int (139, l @ ) , so it is likely that the product of the N gene extends the leftward transcription initiated at the promoter. Luzzati did observe, however, that supcrinfccting, heteroimmune N+ezo phages elicit no exonuclease synthesis from thermally inducible Nsus prophages before derepression (145). While the N product can function in trans, c1 control of the exo gene, as well as int, must controvert that of gene N (93, 104, 1.46). Regulation of the segment to the right of the repressor locus, cl, involves a similar interplay; synthesis from the right operon depends on N , yet cl is epistatic (82, 147-150). Genes c l l , 0 and P comprise a single operon while the Q gene is a t Ieasf partially independent of the y-P

LYSOGENIC INDUCTION

259

segment. Polar mutants in the x region fail to complement genes c l l , 0, P , Q, or Qsus (117, 118), and a particular mutation, xl3, can be designated a promoter mutation (141b).Several mutations to the right of the pR promoter further define the relation of these genes to the remainder of the phage genome. Significant production of the 0 and P products occurs in mutant cl7 without stimulation by the N gene as gene Q remains inducible in these partially virulent mutants (119, 120). An added mutation, b y p , mapping between P and Q, renders the Q gene constitutive, thus conferring a clear phenotype on these double mutants whose need for the N product to stimulate lytic growth disappears (123, 125, 151). In particular deletion mutants, Qin, lacking both the Q gene and the “essential site” to which it usually binds, the Q gene product is completely dispensable (152, 153). At least two genes are active in turning off the early transcriptions of individual operons. Although the T11 mutations in the x region block expression of e l l , 0, P , and Q (84, 117, 118), it resuts also in continuing and accelerated synthesis of exonuclease and the B protein beyond the 8-10 minute cutoff time after induction or infection (118,154).Normally, both proteins and their mRNA’s disappear simultaneously, so transcriptional control by a trans dominant signal from a cistron to the right of the x region fails in these mutants (142, 154). An immunity-specific, diffusible product (155) is encoded in the gene, tof, which maps in the same region as does cro whose product shuts off immunity itself late in the lytic cycle (156-159). The repressor regulator is immunity specific, and the immunity loss is dominant (156).If this regulation could be induced prematurely in lysogens, it might be assumed to effect the early cutoff of cl mRNA synthesis that occurs about 1 minute after X inductions (135, 136, 160). However, this normal shutoff has already been shown to follow the heat induction of A c1857cro- lysogens (116,160-162). The transcription of the operator mutants reflects the activity of the regulatory genes. Constitutive synthesis following the vlvS and v2 mutaand 0, operators, respectively, confers complete virulence tions in the 0,. only in the triple mutant Xvir (28, 164). Partially virulent mutants in the left operator preferentially synthcsize 2 mRNA and fail to transcribe the segments QR and A-J; those in the right operator produce more r mRNA than 2 mRNA with no transcription from the segment c-111-b2 (163). The insensitivity of each mutant type to repression is evident in vitro in the decreased binding of purified X repressor to each altered operator (164).The mechanism for regulation was studied in h. clts lysogens infected with these partial virulents (163) and in cells lysogenized by the autonomously replicating h dv deletion mutants described by Matsubara and Kaiser (106),which exhibit low expression of the cl-rex

260

ERNEST BOREK AND ANN RYAN

region and produce no rex product (166a, 166b). It appeared that the toj gene, under the influence of a constitutive 0,. gene may control the leftward transcription from the pL promoter or simultaneously enhance N gene transcription and repress synthesis from the cl-rex segment. More current interpretation holds that the gene tof negatively regulates transcription of two “scriptons,” p,-cl-rex and p,-clll-int regions; the more plausible modes of action of the N gene product and that of t o j have been considered in a recent review (166).

V. Prophage Induction: A Two-Stage Process Repression exerted in the immunity region assures the transcriptional and, consequently, the functional silence of prophages (71, 167-170). Stable lysogeny often depends, however, on both the transcendent negative control by specific immunity substance and on subsidiary, poorly defined controls operating in the same region. Among the lambdoid phages, point mutations within any of three distinct genetic units, cI, cII,or cIIIproduce variants characterized by the clear plaques formed on sensitive strains. These mutants complement each other in mixed infections but fail to grow in immune hosts; all are subject to immunity, yet none is self-repressing ( 2 6 ) . Studies on Aimm434,a recombinant containing mainly A genes together with the immunity region of the closely related phage 434, proved specific immunity to be encoded in the c l gene (171). Its activity alone maintains lysogeny and superinfection immunity, yet c l l and c l l l mutants lysogenize with a greatly reduced efficiency ( 2 6 ) .Synthesis from the c l l cistron is directly controlled by the immunity repressor without absolute dependence on gene N (147),but the stability of the repressed state is greater in both wild A and c l l mutants than in Aclll- lysogens (172). The repressor produced by infecting A phages usually reaches its maximum a t 15 minutes and falls off 10-15 minutes later as the cro protein antagonizes its synthesis (161). Mutations in either c l l or c l l l advance the onset and increase the quantity of late mRNA production (173). This seems to be independent of any effect of immunity, for the double mutants el-11- and c1-c111- exhibit changes identical to those of their repressing counterparts. The complex control circuit operating within the immunity region has been partially described since Eisen et al. first presented evidence that regulation in the synthesis of the A repressor appeared to depend on self-activation (119). Separate pathways of repressor synthesis act in establishing and maintaining the repression on the operators, 0, and 0, (161). Transcription of the cl gene can initiate at two different

LYSOGENIC INDUCTION

261

promoters, pre and prm, located, respectively, a t DNA sites cY and another close to the right operator. The c l l and c l l l products activate pre during lysogenization while the repressor itself acts a t the p r o site to sustain repressor synthesis within established lysogens. Consequently, the repression of c l l and c l l l mutants requires the presence of both an active repressor and a nonmutant right operator to which it can bind. This was inferred also from evidence that less repressor is made by hcll mutants infecting nonlysogens rather than h lysogens ( 1 7 4 . Since the repressor, bound at Or, prevents CTO transcription from the r strand as it promotes leftward transcription of the cl structural gene, a bifunctional cl product must act to maintain lysogeny. I n the temperate Salmonella phage P22, four genetic sites control lysogenization: the clustered genes, cl, c2, and c3 (176-l76b) and mnt which complements all c mutants (70).Those in group c1 and c2 behave as virulent phages, producing lysis and little if any lysogenizing. The latter is equivalent to c1 in lambda in that it encodes the immunity repressor while the other gene, cl, exerts a subsidiary repression on phage DNA synthesis during the first 11 minutes after infection (68). The combination of two mutations, k , and vx, respectively, within and to the left of the c2 gene, produces virulence (177). In crosses between P22 and the related temperate phage L, the immunity specificity of each appeared to be determined by at least two factors, one in the c region and the other in the I region; the c systems seem to be very different while the I systems are similar ( 178).

A. The Derepression The recognition that particular c1 mutations qualify the inducibility of prophages, intensified interest in lysogenic induction. Several h lysogens, when elevated temporarily or grown a t temperatures above 40°C, release phages capable of conferring thermal sensitivity on new hosts (179-181). The suppressibility o f one of these mutations, ~1857,and the failure of mutant strains carrying cl tl to develop superinfection immunity when held in chloramphenicol implied that the repressor was proteinaceous (181-183). Amost simultaneously, three groups distinguished two categories of thermolabile h lysogens: one class in which exposure to an elevated temperature was necessary but insufficient for induction without concomitant protein synthesis, the other inducible by high temperature alone (56, 184, 185). Twelve mutated sites distributed over the entire cl cistron fall in two genetic regions which correspond to the functional distinction between these X lysogens. Mutants in region A to the left of center belong to the second group while those in region B on the right

262

ERNEST BOREK AND ANN RYAN

side of the cistron require concurrcnt protein synthesis. Thc thermal sensitivity varies within each group, but all mutants are fully induced a t 45” (56). I n these studies, the “irreversible” killing specific to the lysogcnic condition operationally redefined induction, since this correlates well with the phages released at permissive temperatures when growth accompanies the thermal stress. So defined, lysogenic induction appears to be a twostep process, as suggested by Gingery and Echols, in which derepression precedes the lethal event (95). Derepression without subsequent phage release or killing occurs when a class B mutant is heated in chloramphenicol; immunity disappears and the consequent 40-fold increase in X mRNA production persists for about 30 minutes after their return to permissive temperatures and growth (f85).A certain amount of protein synthesis is, therefore, mandatory in their complete induction (56, 185), yet the full induction of class A mutants in the absence of protein synthesis follows the irreversible thermal inactivation of the repressor (186). From this disparity, it can be inferred that an essential “inducing protein,” if specified by the phage genome, must be an integral part of the repressor or be encoded in the cl-rex segment. Several explanations were considered theoretically possible, for intragenic complementation between mutations in the different regions had suggested that each involved a separate function (56). The cl product could then be: a single polypeptide with one or two active sites, a dimer of unique polypeptides, or a multimer whose disaggregation rather than conformational change would produce derepression (187, 188). Lieb indicated, however, that simple dominance between repressors within complexes reflects the sensitivity of the mutant genome whose resistance to induction from the prophage state is greatest ( 5 6 ) . Some restriction on these possibilities had become evident in the response of the thermolabile lysogens to ultraviolet irradiation. Three levels of UV sensitivity exist: several mutants in region B are more UVsensitive than A+, three on the left side are extremely sensitive, and another in the same region is noninducible by UV (189).The UV induction of all thermosensitive mutants can be dark-repaired and photorepaired, but their usual latent period is reduced relative to that of Xcl+, indicating more effective derepression of accelerated vegetative syntheses (56). The noninducibility of the ind- mutant currently is attributed to an increase in the repressor stability (192).Ogawa and Tomieawa have observed that the residual immunity of nonlysogenic segregants from cells abortively lysogenized by Xb, persists for four generations. The repressor was growth stable whether specified by c1+, inds or ind- phages, and the immunity within cells UV irradiated and incubated further in

LYSOGENIC INDUCTION

263

broth falls a t a rate that corresponds to tlic sensitivity of the Ab, phage originally elaborating the c, product i t . ind” > cl+>>ind- (190). The dispersion of the mutations altering the UV sensitivity with some, located in the A region, conferring extreme opposite phenotypes would preclude a simple niodel for repression. The isolations of the A+, X ind and, more recently, the 434 repressors in high chemical purity now make direct studies possible (191-195). Each binds specifically to native DNA, and the dissociation constants approximatc 10-lo M, which compare favorably with the value found for the lac repressor (194). When DNA from the mutant, X vir, was tested for binding, a dccreasctl affinity for the repressor was evident. The X repressors are single polypeptide chains of molecular weight 30,000, acidic and chromatographically distinguishable on DEAE-cellulose (191, 192). Unlike these, thc rcpressor of phage 434 proved to be a small basic protein (195). Several partially purified mutant c1 products have been tested in vitro for inhibitory action on the transcription of X DNA. Specific repression was inferred from a 50% reduction in the radioactivity incorporated by an E . coli RNA polymerase system in the presence of the DNA-specific repressor (169).

B. The Excision and Cell Death The induction sequence, now readily followed in the E . coli K12-A+ system, involves the same sequence of viral syntheses found after infection. However, prolonged thermal derepression even of XNsus prophages results in cell death, yet all N mutants kill much less efficiently than do 0, P , &, or R mutants. The last group immediately arrests host DNA synthesis but kills, as demonstrated with 0 and P mutants, only after lysogens carrying both a time lag (114). Studies of thermosensitive N and c1 mutations clearly indicated that killing was subject to immunity control. When heat-induced, the double mutants varied markedly in their killing potential, but the N activity of the prophage was dispensable. Some N mutations combined with c1tB reduce thermal sensitivity while others permit cell growth and division. If N mdtants carry c1tA mutations instead, their hosts die after heating and often exhibit incrcased sensitivity. Although Xcl tB-Nsus and several Xcl tA-Nsus mutants survive at 43°C and retain immunity to X superinfection, some among the lattcr die if the temperature is lowered before c1 activity is introduced. An accelerated DNA synthesis, observed when the temperature is lowered, correlates with a loss in the rescue of the cell through immunity (188). The coincidence between rapid DNA synthesis and an increased probability of killing implied that excision preceded or accompanied the

264

ERNEST BOREK AND ANN RYAN

lethal cvcnt. The time required for excision was determined by measuring the rescue of the X prophage in the matings of transiently heat-induced Hfr Xc1857 lysogens. Cured chromosomes began to appear in recomhinants after 2-3 minutes of heating, yet only after 6 minutes did infectious centers later appear. The excision disrupted the gal-bio linkage and responded to phage mutations that normally influence excision, e.g., thc presence of an “induecd” attached iV mutant prophage reduced the gal-bio linkage while Xint, mutants produced high excision. Thus, transient heat induction produced prophage excision and chromosomal healing hefore the cells were committed to phage production (196). Since repression is restored when Xind-cl857 is returned to permissive temperatures (186,196’), the observation was confirmed by chilling transiently induced cells in ice before superinfecting them and selecting clones capable of growth a t 40°C. The presence of both cured and substitution lysogens among these proved the point (197). The potential lethality of excision had already become manifest in UV and zygotic inductions. When the linkage between gal and bio was examined after the zygotic induction of various X mutants, it was apparent that linkage had been completely destroyed by the induction of N mutants and greatly reduced in the ease of 0 mutants. When x or P mutant lysogens are induced, a complete restoration of linkage is reflected in high survival and curing (198). If irradiated cells carry instead a defective FgalXd episome, mutant in x, N , 0 or P, no host death appears, but with the exception of the 2 mutant, the episome is lost. The zygotic induction of various X prophages, defective in any of the same genes carried on the sex factor, again resulted in thc same loss. Among the “sexductants” 75-9576 of the cells in which 0 and P mutants had been induced were A sensitive while only 211% of those carrying the N mutation on the episome became sensitive. The induction of these defective prophages thus established that lethality was confined to the genetic element from which the prophage had been excised (199). Excision, then, precedes phage replication and commits to death only bacterial hosts carrying X chromosomally. Curing, it should be noted, is usually a rare event, but even a short heat exposure, inadequate for the complete induction of temperaturesensitive mutants, can result in high survival of cured cells (2U0, 201). Nevertheless, Lieb found that the number of nonlysogens per milliliter of heated culture increased a t a rate to be expected from the division of preexisting immune cells (56).High W irradiation (2W) or superinfection of lysogens with weakly virulent phages or heteroimmune related phages (103, 202-204) produce curing without attachment of the superinfecting phages (205). Three requirements must be met for X curing:

LYSOGENIC INDUCTION

265

an intact int gene must be present either in the prophage or superinfecting phage, an xis gene must be active, and the a t t regions must be capable of undergoing int promoted recombination (204). The last implies that both ends of the prophage must be intact (97, 206, 207). However the derepression that permits the lethal event is attained, UV induction is clearly produced indirectly. At least one bacterial function, recA (208) controls the response, for immunity is not lifted in irradiated recA lysogens (209-212). The thermosensitive Salmonella typhimurium phage, P22c2ts, is not induced by UV or mitomycin C, although the excision mechanism remains undamaged, as their subsequent thermal induction indicates (213).Since immune r e d - segregants derived from cells abortively lysogenized by the deletion mutant Xb,clts2 also lose immunity by heat but not by UV irradiation, recA- cells fail to form a “UV inducer” both in the presence and absence of the phage genome (190). The difference in UV sensitivity of rec+ cells lysogenized by Xb,ind- did not seem to reflect an increase in the amount of residual immunity substance present in such cells (214).

VI. Indirect Modes of Induction Crucial questions concerning the induction of bacterial lysogens whose answers could well have applicability to other biosystems remain unresolved. We still ask: How is derepression of the well-programmed phage system initiated? Must inducing agents attack the control mechanisms directly or can thcy act through a metabolic intermediate, the hypothetical “inducer”? The wealth of information already documented has brought us only to the edge of understanding molecular mechanisms, so at this point, we will turn to observations that definitively answer the second query.

A. Indirect Ultraviolet Induction (Cross-induction or Borek-Ryan Effect)

Our demonstration that the inductive activity of ultraviolet light can be transferred unidirectionally in a mating bacterial system clearly establishes the participation of a highly efficient “inducer” (215, 216). As originally described, UV-irradiated E. coli auxotrophs, carrying F episomally, were mated to auxotrophic females in growth medium nutritionally adequate for the recipient bacteria alone. Free phages in excess of the spontaneous yield, released after an appropriate incubation period, were attributed to a transferred inductive process. Since major radiation damage to the productive lysogen is precluded, the system seemed ideal

ERNEST BOREK AND ANN RYAN

266

for the study of prophage induction and the possible identification of a biologically active UV-damaged intermediate. To avoid an a priori judgment about specificity, we descriptively referred to this phenomenon, mediated by bacterial conjugation, as “cross-induction.” On subsequent analysis, we found W irradiation to be uniquely effective (917, 918), and, in confirmation of this, Devoret, the foremost contributor to the field, applied the more specific term, “Indirect Ultraviolet Induction,” by which it has become more widely known (919). Several parameters give complexity to the system : radiation sensitivity, conjugation kinetics and stability, for, as could be anticipated, each limits the overall induction efficiency. Little disparity appears in these studies performed in Devoret’s laboratory and in ours, and indeed, the concordance of results obtained under differing nutritional conditions &nd based on somewhat different analytical criteria is noteworthy. We refer the reader to the cited literature for methodology but would point out now that the reproducibility and efficiency of “cross-induction” accomplished in minimal-glucose medium are entirely comparable t o those reported for experiments performed in enriched medium. 1. BACTERIAL CONJUGATION IN THE MEDIATION OF INDIRECT

ULTRAVIOLET INDUCTION

Most critical to any argument that cross-induction entails the neutralization or bypass of undamaged repression systems is that evidence which excludes generalized indirect radiation damage to recipient lysogens. The oriented transfer of inductive activity (Table I) implied an essential role for bacterial conjugation, yet only the simultaneous elirhination of both mating and indirect UV induction could discount the

POLARITY AND

TABLE I NONSPECIFICITY IN INDIRECT

~ _ _ _ _ _ _ _ ~

ULTRAVIOLET INDUCTION4

~ _ _ _ _ _ _

Representative bacterial matinga Irradiated donors

W 6 F+ (A)+ W1177 F- (A)+ Hfr H (A)+ W1485 F+ (A)58 F+Col Ir (A)HfC 49 Col 11 (A)a

8447.

Recipients

W1177 F- (A)+ W 6 F+ (A)+ W1177 F- (A)+ W1177 F- (A)+ 48 P C o l Ir (&)550 F-COP (A)+

~_______

a/c (free phages or lacunae*)

57 2 3 20 30* 211

These crosses and their efficiencies have been extracted from references 816, 816,

LYSOGENIC INDUCTION

267

involvement of some solubilized, radiation-activated intermediary. Interrupted mating experiments proved unequivocally that the induction of recipients is limited by the kinetics of conjugation itself as was inferred also from the observation that prior destruction of the conjugal capability of F+ donors by 0.2 mM sodium metaperiodate prevented inductive transfer (218). The patterns obtained occasionally reflect the distortion expected when gentle aeration of mating populations causes premature rupture of specific pairs (22U). Where linearity occurs, the curve extrapolates back to about 2 minutes, the minimal time required for the formation of specific pairs (Fig. 2 ) . Obviously, inductive activity enters the F- population almost immediately, increases as specific unions increase, and reaches its maximum when all effective unions have been accomplished. If minimal delays appear, the entry time coincides with that expected for the transfer of the F episome itself (218). A randomized transfer of particular chromosomal markers is excluded from causation since the levels of cross-induction observed implicate a major portion of the donor population. The donor chromosome itself does not appear to be the relevant target. Various Hfr strains tested as donors exhibit low efficiency, and others, such as AB313 in which F undergoes exceptionally low reversion to autonomy, are completely ineffective (215, 218, 221, 222). Devoret confirms that several HfrH stocks crossed to different females yield indirect UV induction levels that average about five times the spontaneous phage yields (222, 223) ; our stocks of HfrH are less than twice as effective when matings in mineral salts medium are uninterrupted (215, 216,224).

Confirmation that no specific chromosomal site contributed t o the minimal activity of these Hfr donors is found in the time course of inductive transfer (Fig. 2B). The curvilinear kinetics, exaggerated here in a semilogarithmic plot (218), would permit the view that the activity of Hfr strains in cross-induction can be attributed to a subpopulation of autonomous F+ revertants (223). Supporting data indicate that the frequency of F in surviving recipients increases with the time from an extrapolated original level of to lo-* when Hfr donors are killed by streptomycin after 40 minutes’ mating (225). From such initial frequencies, indirect UV induction would appear unlikely to reach 20% of the maximum attained by pure F+populations. Since Rosner detects no viable F in recipients cured of their prophage by indirect UV induction, we should reconsider this observation (226). Devoret has shown more recently that inactivated bacterial markers carried on the irradiated F-merogenote can be rescued in the recipients (227). Any sweeping conclusion that irradiated Hfr strains possess no

ERNEST BOREK AND ANN RI‘AN

268

8

46

24

32

‘ -4

40

140

TIME in MINUTES (post mating)

-

100 8 -

4

8

12 i6 20 24 28 32 36 4 0 4 4 48 52 TIME (MINUTES POST MATING)

FIG.2. The kinetics of transfer in cross-induction. Samples were removed a t the indicated times, diluted lo-’ in the growth medium of the recipient strain, vortexed in a Virtis ‘‘45” homogenizer for 1 minute and returned to incubation a t 37°C. The increment in the free phage yield relative to the background titer after 160 minutes is represented. The recipient in all experiments was Escheyichin coli K12W1177, F-, thr-, leu-, f h i - ( h ) + .

intrinsic capacity for cross-induction could be premature, for positive responses, comparable to those of F+stocks, can be obtained with strains that usually arc minimally act.ive. The anomalous pattern obtained with Hfr CSlOl presented in Fig. 2B exhibited an unusual acceleration a t 12 minutes. However, the same stock and clonal isolates, tested immediately before or after the experiment cited, produced indirect UV induction a t levels that were 9, 8 and 12 times the spontaneous yield. Enhanced act.ivity also occurs occasionally in HfrH, but, in b0t.h cases, only matings

LYSOGENIC INDUCTION

269

that are interrupted within 30-32 minutes permit its detection (218,224). Until these anomalies can be adequately explained, any postulated specific role for episomes as exclusive mediators of indirect UV induction, is suspect. 2. THEEFFICIENCY OF THE PHENOMENON

The unquestionablc involvement of bacterial conjugation prompted analysis of the quantitative response of indirect UV induction to variations in the participating bacterial populations. Two different approaches, independently pursued, lead to the same conclusion: under optimal conditions, both donors and recipients contribute equally to the crossinduction phenomenon, which fails to reach its theoretical maximum. T o compare the efficiencies of cultures grown in broth and synthetic medium, we must consider the details of these computations. Our own data are based on the relationship between the spontaneous and indirect inductions of recipient lysogens ; others compare indirect t o direct induction. The bacterial crosses required can be generalized as follows: a. Irradiated F+X F- A+ (indirect) b. F+X irradiated F- A+ (direct) c. F+X F- A+ (spontaneous)l Devoret estimated an average theoretical yield of 55.9% in broth from the relationship: a-c - X lOO/p = % ' yield b-c where p , the probability of mating, is drawn from a Poisson distribution about the mean F+/F- ratio (range 0.59-5.4). This efficiency was never attained experimentally even under optimal conditions, i.e., 800 ergs/mrn2 radiation dose; unit population ratio ( p = 0.67). Average experimental values indicated that only 41% or 46% of the recipients were induced indirectly when yields were calculated from free phages and infectious centers, respectively (223). While the results did not support the view that determinations of infectious centers arc more reproducible, they confirm the implicit assumption in these calculations that a comparable burst size exists in direct, indirect and spontaneous inductions. Given it the fact that spontaneous induction ( c ) occurs a t a frequency of is also evident that the correction of induction yields for this contribution is superfluous at all meaningful levels of indirect induction.

' When cultures were grown and mated in synthetic medium, the spontaneous burst of the female was determined in most experiments with unmated cells; this value differed little from that obtained with mated recipients.

270

ERNEST BOREK AND A N N RYAN

Fluctuations of this parameter when populations are mated in broth, nevertheless, render the more simple a/c ratio a deceptive indicator of the level of indirect induction. While this ratio ranged from 12 to 454 with a mean of 125 in the same series of experiments cited, no problem exists when cross-induction is effected in synthetic medium. The same relationship between phage yields in 22 randomly selected experiments, performed with the same recipient used by Devoret and another donor, varied from 20 to 82 with a mean yield that was 43 times the spontaneous bursts (224). Since the irradiation doses used in both experimental series would directly induce the entire F- population, the value of b is lo2times larger than c. We conclude, therefore, that the efficiencies of indirect ultraviolet induction are the same whether cells are mated in broth (41/100F-) or in synthetic medium (43/100F-).

-

I. -

k h r i c h l o coll Klbb fno./ml) A....Z.Vx If

-

c....7.4

B....l.S

I

10

107

-

-

.,P

d

1

2

3

DW, P*utotisNno./ml)in-e

Y

n

W

107

--

--

I

1

1

I

I

I

I

I

2

3

4

5

6

7

POPULATION R A T I O F+/F-

FIO.3. The free phage titer in excess of the control after 140 minutes' mating is presented. The recipient strain was Eyeherichin coli K-12-W1177, P,leu-, t h r , thi-, (A)+. Curve A, 2.9 X 108; B, 1.5 X 10'; C,7.4 X lo'.

LYSOGENIC INDUCTION

271

This statistical average confirms the value previously determined from a single experiment designed to provide information about the population dependence, maximal contribution of irradiated males and the absence of inductive transfer through the medium. I n Fig. 3, the relationship of free phage yields from indirect inductions in synthetic medium to the F+/F- ratio is indicated for several absolute donor concentrations. Proportionality between the curves obtained is obvious as is the lowered yield at F+/F- ratios above 2 (618). The latter could reflect triparental matings, reduction in the effective donor concentration through F + X F+ interactions or suppression of phage yields as a result of multiple matings. This suggests that calculations of theoretical maximal yields based on a Poisson distribution may be erroneous. An empirical approach to this question was satisfactory. The free phage yields obtained when the population ratio was 1 vary directly with the absolute donor population level (Fig. 3 inset). An extrapolate from the higher values passes through the origin, although the actual yield at the lowest donor population level is somewhat depressed. We know that actual yields fall off a t F+/F-ratios below 1, yet the maximal theoretical contribution of each irradiated population when the Fpopulation is infinitely large can be assumed to be the extrapolated value of the ordinate. Thus, the effective fraction of the male population can be calculated from the relationship: loo = % of males effecting indirect UV induction x total F+ population

Ordinate

burst size

Fortunately, the highest donor population is the same as that routinely placed in mixture with recipients by Devoret, so the calculated maxima can be compared. The theoretical maximum fraction contributing, as calculated from the upper curve, accounts for only 43% of the absolute donor population (&18). The concordance between this value and those obtained in both experimental series that define female productivity proves that donors and recipients contribute equally to indirect UV induction and implies that the males limit inductive transfer. We shall see later that the metabolic instability of inducing activity in the male population in conjunction with conjugation kinetics amply account for the insufficiency. 3. THEUNIQUE EFFECTIVENESS' OF ULTRAVIOLET IRRADIATION

The unique effectiveness of ultraviolet light in evoking a transferable inducing activity became evident in an extended series of experiments in which other inducing agents failed to impart cross-inducing activity to

ERNEST BOREK AND ANN RYAN

272

donors. Neither mitomycin C, X-rays, sulfur mustard, aeaserine nor

H,O, were effective in indirect induction (217,218). Since the last four produced low direct induction levels, i.e., ’< 40% and marked inactivations, the negative results could have reflected damage to the mating mechanism. Nevertheless, data obtained with X-rays and mitomycin C clearly show the absence of transferable inducing activity over a wide dose range that included levels a t which donor populations experience a complete loss of colony-forming ability and/or maximal direct induction. Our early studies of the cross-induction phenomenon indicated that the activity involved was sufficiently stable to permit a demonstration that inductive processes need not possess completely overlapping mechanisms. Telling experiments in which ultraviolet irradiation superimposed cross-inducing activity on both ineffective X-irradiated and mitomycin C-treated donors clearly proved the point (Table 11). Reproductive death, that is, the loss of colony-forming ability, appeared to be an appropriate and sensitive indicator of prior damage to the donors whose cross-inducing potential was reassessed after superimposing UV irradiation. I n neither case, did the secondary inducer act upon a fraction of the population that had escaped the primary inducing agent, for had this been true, an increase in the free phage yield from the doubly induced donors might be anticipated. Moreover, the surviving fraction present in the male cultures prior to ultraviolet irradiation was too low to account for the cross-inducing activity subsequently generated in the population. Results such as these compelled the conclusion that ultraviolet light TABLE I1 THEEFFECTIVENESS OF ULTRAVIOLET IRRADIATION, X-RAYS,AND MITOMYCIN

c IN

CROSS-INDUCTION

Expt.

Mated populations (No. cells/ml)

Inducer

Total dose

Percent survivors

a/@

A

F+ 8.7 x 107

Mitomycin

F- 8.2X 107

3d m l 900 ergs/mm*

0.10 4.30

3.5 47

Mitomycin

z

0.01

25

F+ 1.0x 108

uv X-ray uv

26/100 r 900 ergs/mm*

0.40 9.70

38

z

-

B

F- 1.5 x lo8

uv

+

X-ray

+

w ..As defined by Devoret. See page 269.

1.6 8.1

LYSOGENIC INDUCTION

273

differs from other inducers in its mode of action but did not imply any diversity in the terminal inductive event (217, 218). Our statement implicitly accepts that indirect and direct UV inductions share a common mechanism. Support for this view is found in comparisons of their dose-effect curves in which the differences observed can be attributed to the necessary intervention of conjugation in indirect UV induction. Direct, supraoptimal UV irradiation causes a rapid decline in infectious centers (or free phage yields) which parallels the survival curves of both the lysogenic strain and its cured counterpart. In indirect UV induction, however, neither free phages nor infectious centers decrease over a dose-range which extends to a t least 1400 or 2000 ergs/mm2 whether donors are grown in synthetic medium or in broth. The plateau reflects the absence of general radiation damage to the productive lysogen; maximal yields persist until the conjugation mechanism of the donor is directly impaired (222-224, 228). The intervening conjugation diminishes the optimal yield to about 40-50% of that obtained by direct induction and introduces an apparent 3-fold dose reduction in the ascending portion or the curve ( 2 2 s ) . A careful determination of indirect UV induction values obtained with small UV doses ranging from 32 to 400 ergs/mm2 provided data whose plot on logarithmic coordinates produced an ascending slope of 1.8 (223, 228), which closely approximates the value of 2 calculated by Marcovich for direct UV induction (229). The applicability of target theory to biological systems is now highly suspect, but in its terms, such data would indicate that two radiological events of equal probability occur in each inductive response. If the events themselves precede conjugation, the data would suggest that the direct, inducing UV damage to bacterial cells that is later transferred is independent of both host chromosomal loci and the presence of prophage itself. An identical induction mechanism was inferred also from the synergism found between low level direct irradiation and superimposed indirect UV induction (223, 228). Recipients exposed directly to minimal UV irradiation were mated subsequently to donors that had received optimal UV doses; the free phages released were compared to the sum of the indirect and direct contributions. With three male strains tested, the sum was inferior to the mixed production. However, some ambiguity exists in experiments which assume that the increased phage yield reflects only the sensitization of the induction mechanism. The possibility must be critically eliminated that F- cells directly irradiated a t noninductive UV levels are more proficient in phage multiplication or more delayed in lysis than are nonirradiated F- recipients. To this point, we have defined-uantitatively and qualitatively-

274

ERNEST BOREX AND ANN RYAN

the operational limitations on indirect W induction without particular concern for the specificities of the biological process itself. Experiments designed to this purpose confirm the predictive value of certain facts already presented. Bacterial conjugation may limit the efficiency of inductive transfer, but it restricts as well our view of the probable determinant. The immediacy and high efficiency of transfer and the marginal yield in Hfr matings all suggest that an episomal entity, or plasmid, mediates the effect. Nothing discussed above excludes a cytoplasmic, i.e., nonchromosomal, constituent except the obvious requirement for localization a t the site of cell contact and the minimal transfer in Hfr matings. Yet, a priori explanations for these are more readily found than are proposals that define the pertinence of irradiated episomes themselves to the inductive event. Their immediate participation in induction, as biological entities, could be inferred with assurance only if specificity exists in the UV response of either the donor or lysogenic recipient. That nonlysogenic donors effect the indirect induction of prophages suggested that no specificity would be found. 4. THEABSENCE OF EPISOMAL SPECIFICITY IN INDIRECT

ULTRAVIOLET INDUCTION Clues to the nature of the episomal involvement in indirect UV induction, which biological specificity could provide, are unfortunately few. Only a minimal distinction between the participating strains need exist: donor status demands an appropriate mating surface to facilitate transfer, while lysogenic recipients require only UV inducibility to respond positively. An additional restriction, the autonomy of the episome or plasmid that promotes its own transfer in mating, may reflect only a requirement for early transferability imposed by the metabolic instability of the inducing activity. Cellular interactions in mating are so vaguely understood that controversy over the molecular mode of DNA transfer continues (83U-234) as models for its physical passage to recipients multiply (836-839). However transfer occurs, we know that the most proficient donors of inducing potential in an F-determined mating system are males whose material contribution to females is limited to DNA, apparently equivalent in amount to the F genome (240,841). Integration of this episome into the bacterial chromosome confers Hfr status on the male by subsequently promoting the transfer in mating of variable leading segments of the chromosome with very infrequent transfer of the terminally located F factor. The marginal activity of these Hfr donors in cross-induction did suggest that the episome was the pertinent radiation target. Monk

LYSOGENIC INDUCTION

275

has presented data in which the entry time of inducing activity correlated well with a delay in F'lac transfer attributable to its increased replication time (2.42). A more detailed study which utilized F merogenotes of several sizes : F14, carrying both methionine (met+) and isoleucinevaline (iZva+) markers, and F16, carrying only the latter, produced similar correlations. In experiments performed with F14, the leading episomal marker, met+, first appeared in recombinants about 8 minutes after mixing; the second marker, ilua+, then entered a t 12 minutes in coincidence with the appearance of inducing activity. Without exception, all iZva+ recombinants in interrupted matings were sensitive to the malespecific f2 bacteriophage (243, 244). Thus, cross-inducing activity does not appear until most, if not all, of the episomal DNA unit has been transferred. While these results tend to identify the inducing activity with the irradiated episome, they do not exclude the possibility that the completion of episomal transfer permits entry of an unspecified inducing activity. Several autonomous genetic elements, including F, Col I (245) and R T F (Drug Resistance Transfer Factor) (246) have already been found to mediate indirect ultraviolet induction without specificity. When donor strains carry an unintegrated F, neither the presence of a free atth site, A+ (215-218) or hind- (223) alters the efficiency of the male in the indirect induction of X lysogens. The h repressor, obviously, plays no role in the generation of inducing activity and, more significantly, fails to inactivate it within the donor strain. Nevertheless, irradiated males will not induce hind- recipients, although they act efficiently with females carrying the prophage 434 or inducible Col I plasmids (223, 245, 247). Reciprocal crosses performed with HfC donors that are capable of producing an epidemic spread of Col I within 15 minutes after mating substantiate the nonselectivity of indirect UV induction. While stains bearing Col I plasmids have about twice the radiation resistance of F+strains, they are somewhat more efficient in the indirect induction of h lysogens. I n these HfC matings, induction exceeded 50%, yet the Col I plasmid was transferred at the 39% level; the inductive response did not correlate with the transfer and/or survival of the irradiated factors (247). The validity of this conclusion can be judged from the results of a most revealing three-strain cross in which direct irradiation of the mediating plasmid was avoided. In this instance, an HfC donor was mated for 10 minutes with an F-Col- strain previously irradiated. Donors were then destroyed by streptomycin, and the newly infected F-Col' intermediate strain was mated to an F-A+ recipient. Although Col I transfer itself was less than l%,the level of the indirect ultraviolet induction of the h lysogen was proportional to the UV dose over an extended range

276

ERNEST BOREK AND ANN RYAN

(247).Unless one presumes indirect irradiation damage to Col I in the intermediate strain, the plasmids, and, by analogy the sex factors, would not seem to be the relevant radiation targets. An alternate, but inadequately explored, possibility that the nonirradiated Col I may have excluded X development in control matings has been recognized (248). Controverting evidence also appears in experiments that describe thc exclusion of inductive activity when an autonomous or integrated F factor already resides in the recipient lysogcn. Here, irradiated F+strains were mated to either F+ of Hfr strains that had been converted to Fphenocopies. The absence of induction lead these workers to ascribe a “dominance” of the normal F over the entering episome (243). This would constitute the first instance in which the inducing agent was apparently subject to biological control. However, the usual techniques for producing phenocopies (220) are those classically used to create “inaptitude” for direct induction ( 4 ) . Low temperature cultivation employed in this instance may not present this problem. These workers report also that irradiated P1 phage upon infecting X lysogens induces indirectly with a maximum efficiency of about 2076 (243). The response appears to be dose-dependent and fails a t higher radiation doses possibly due to damage to the injection mechanism. This low level induction does suggest that entering irradiated DNA, comparable in size to the episomes, may trigger induction. I n this case also, exclusion appears, for irradiated P1 fails to induce double lysogens (X,Pl) alone or single X lysogens infected simultaneously with unirradiated P1. I n contrast, coinfection with F does not prevent a low level indirect induction by the irradiated P1 phages. This uncommon capability of P1 underscores the autonomy of all the genetic factors mediating indirect ultraviolet induction, since it too replicates extrachromosomally in the prophage condition (g4-69).From these studies, it would appear that the mediators of induction can be excluded from spccific membrane sites a t which induction would bc initiated. Until a detailed analysis establishes the identity to indirect UV induction of the low level induction effected by irradiated P1, we need not assume a coilinion initiation.

5. CLUESTO THE NATURE rn THE INDUCER Several independent evidential lincs prejudice our ideas about the chemical nature of the active inducer, yet none irrefutably proves its identity. Episomal mcdirttion without spccificity, photoprotection and photoreactivation, and the responses of thcsc cross-inducing systems to mutations that alter the radiation sensitivity or DNA restriction implicate DNA.

277

LYSOGENIC INDUCTION

That a radiation-damaged substrate for the photoreactivation enzyme might be causal in indirect UV induction found support in early experiments. These provided the additional information that preillumination a t wavelength 375 nm of either donor or recipient strains is equally effective in eliminating the indirect response (Table 111). The presence of a transferable inhibitory activity in X-irradiated donors after illumination was suggested by a reduction in the free phage yield from the mated system below that attained in the spontaneous induction of the female population alone. A similar diminution in phage production subsequent to photorcactivating treatment was detected by Latarjet, but no photoreactivation of lethal X-ray damages occurs (229).Significantly, when UV irradiation was superimposed on X-irradiation, postillumination reduced the donor efficiency in indirect UV induction, yet caused no greater reduction in its phage yield. The photoreversibility of the UV effect within males that had sustained irreparable X-irradiation damage answered in the negative the then moot question as to the identity of these radiation lesions (217). Experiments utilizing bacterial strains with an altered ability to enzymatically repair UV damage reinforce claims for DNA mediation. Mutant strains, phenotypically described as Hcr- on the basis of their decreased dark repair of lethal UV lesions or of UV-inactivated phages, fall into four distinct groups: uvrA, uvrB, uvrC, and uvrD, whose defective loci are widely separated on the E . coli chromosome. The first three genetic sites specify enzymes that effect the excision of pyrimidine dimers from UV-irradiated DNA (250), while the last determines repair

Strain illuminated Expt. Inducer

A

UV

Pr@

Post

a/c

-

-

F+

-

F+

F+

30 3.1 2.2 1.0

-

22

F-

-

-

B

UV

-

F+

1.9

Strain illuminated Expt. Inducer post only

c

uv X-ray

-

F+ -

F+ Both

-

F+

a/c 10.9 0.16 3.0 0.18 4.6 0.58

278

ERNEST BOREK AND A N N RYAN

synthesis (261). E . coli K12 ( X ) H c r mutants are ten times more sensitive to W induction (262) than is wild K12 ( A ) , and nonlysogenic mutant types exhibit different sensitivities to UV, killing, i.e., uvrB > A > C Y D , where each increment approximates one order of magnitude (261).

The efficiencies of strains with differing Hcr status in both F and Col I-mediated indirect ultraviolet induction reflect the repair capability only of the donors. While F+Hcr+males respond optimally to UV doses of 600 ergs/mm2, wild Col I donors are effective only a t doses above 600 ergs/mm2, apparently owing to a 5-fold increase in their radiation resistance (247).A logarithmic increase in the indirect W induction efficiency, extending a t least to a total UV dose of 2100 ergs/mm2, presumably indicates the insensitivity to UV inactivation of the conjugation mechanism in HfC donors. Nevertheless, nonrepairing Col I carriers irradiated a t 600 ergs/mm2 have an efficiency in indirect W induction approaching that of direct irradiation (247,248, 252). The absence of dimer-excision within these donors, apparently, offsets the increased UV resistance of recipients receiving the colicinogenic factor, for Col I+ strains are known to have a lowered spontaneous induction rate. Although the Hcr mutation in Col I donors increases their cross-inducing efficiency, nonrepairing recipients are slightly less responsive to transferred inducing activity than are their Hcr+ counterparts. Since Col I transfer from Hcr’ donors to nonrepairing females also falls below that found with wild recipients, the Hcr status influences the entry or survival of the presumed Col I mediator itself. Correlations between the restriction of transferred episomal DNA in heterospecific crosses and the disappearance of indirect induction similarly implied an inducing role for this macromolecule. I n these experiments, male E . coli K12 strains whose DNA either lacked modification or carried the E. coli B modification pattern failed to produce indirect UV induction in K12 (A) recipients (263). One could ask whether degradation of UV-damaged DNA “swamps out” an inducing small molecule or DNA fragment. The stability of inducing activity within donors responds markedly to variations in metabolism. Simultaneous alterations in the conjugal capability of the participants can complicate analyses that involve metabolic inhibitors or “holding,” but appropriately monitored experiments indicated that the active agent within irradiated males varies with the availability of an energy source, the oxygen tension and the nature of the mediating episome. In synthetic medium, a linear biphasic decay a t 37°C or 25°C occurs both in the presence and the absence of a carbon source (218,B64) yet appears to be logarithmically continuous when

LYSOGENIC INDUCTION

279

irradiated donors are held in enriched medium (223).If the carbon source is removed, the usual S 9 minute half-life is extended to 14 minutes. An energy dependence in this decline could also be inferred from the disappearance of the secondary phase of decline in unaerated cultures. Several observations raised suspicions that continuing synthesis or activation of the inducing agent might occur simultaneously with decay. First, the initial “decay” within donors irradiated in synthetic medium a t 37°C appeared to be more rapid when cells were shifted to 25°C. Second, the temperature dependence of the activity decline within irradiated males differed markedly in synthetic and broth medium: 50% of the activity in cells irradiated at 25°C or 37°C remained a t the lower temperature only 4 minutes in minimal glucose medium (254) while a comparable level persisted for at least 4.0 minutes when donors were held in broth (223). Either pattern could indicate a requirement for the immediate temperature-dependent conversion of the initial UV damage into a transferable state or the concomitant formation of inducing activity during the holding period. The shorter initial half-life occurs also in EDTA-treated males a t 37°C (255),even in the presence of actinomycin, and the decay curve exhibits the same plateau found when untreated irradiated donors are held in Synthetic medium without active aeration (254). Similar studies on the stability of inducing activity in broth-held colicinogenic donors weaken the evidence these strains provided that DNA mediates indirect UV induction. Thc exponential decline, which in this case is limited to the first 10 minutes after irradiation, does not reflect the ability of these strains to excise pyrimidine dimers; a 4-minute half-life in wild donors is further reduced to 2-3 minutes in Hcr- males (247) Thus, the decay within the irradiated participants varies markedly with metabolism but fails to correlate with the DNA repair that rapidly occurs in the donor within the period required for conjugation. Specific metabolic inhibitions imposed on the male strain prior to irradiation revealed that continuing macromolecular syntheses are dispensable in the formation of cross-inducing activity (218, 254). These studies employed the highly efficient auxotroph, E . coli K12-W6 (A+) met-, rel-1, which continues to synthesize stable RNA, DNA, and mRNA species when deprived of its essential amino acid. Additional restrictions that included inhibitions by chloramphenicol, puromycin, azauracil, hydroxyuridine, fluorouracil, or actinomycin were imposed individually on the donors before their exposure to UV in incomplete medium. Among these inhibitors, only chloramphenicol and fluorouracil markedly reduced the subsequent efficiency of donors in indirect UV induction. The

-

280

ERNEST BOREK AND A N N RYAN

TABLE I V

THEEFFECTOF

Expt. No.

CROSS-1NDUCINQ ACTIVITY OF ULTRAVIOLEFIRRADIATED DONORSO

PRIOR INHIBITION ON THE

Inhibitor Chloramphenicol Puromy cin 5-Methyltryptophan 6Azauracil 5-H ydrox yuridine

Aotinomycin Actinomycin 5-Fluorouracil

Concentrationb (rglml)

Time bin)

(set)

a/c

0 200 0 1000 0 100 0 100 0 10 0 10 0 10 0 2 10

0 30 0 30 0 20 0 15 0 10 0 Post= 0 Pred 0 20 20

400 400 200 200 180 180 200 200 400 400 200 200 200 200 240 240 240

26 5 42 27 43 75 11 9.5 23 24 60 57 174 81 100 94 46

uv

Bacterial strains used: Escherichia wli K12-W6 (F+, Met-, A+) a8 donon and E . coli K12W1177 (P,thr-, leu-, BI-, A+) aa recipients. *The unusually high concentrations of inhibitors were necessary to prevent net protein synthesis in the defined medium used. Added immediately after irradiation and present throughout the mating period. Added immediately before irradiation and present throughout the mating period. 0

0

dose-dependent decrease found in the latter case, accompanied also by a diminished phage production by the irradiated male lysogens themselves in complete medium, was apparent only when glucose had been present throughout the preparative washing and irradiation. I n contrast to the complete loss of inducing potential, the frequency of prototrophic recombinants in control matings fell from 3.0 X to 1.5 X after donors had been exposed to the higher fluorouracil concentration (10 pg/ml), which inhibits DNA synthesis and reduces RNA synthesis in E . coli. The negative findings with other inhibitors having similar effects on nucleic acid synthesis permitted the conclusion that the fluoropyrimidine had acted in some specific manner. Later results obtained with actinomycin argue against suggestions that defective protein synthesis during or immediately subsequent to irradiation eliminated indirect UV induction. Donors, rendered accessible to the antibiotic by pretreatment with EDTA, were effective in cross-

LYSOGENIC INDUCTION

281

induction whether irradiated in the presence or absence of the inhibitor. No efficiency loss appeared when the addition immediately followed irradiation. The 50% reduction in both the indirect UV induction efficiency and free phage yield of lysogenic donors irradiated and mated in the presence of the antibiotic reflected only extracellular screening by actinomycin. Escape synthesis of new proteins was discounted, therefore, even though prior exposure of the irradiated donors to chloramphenicol instead did severely depress their cross-inducing potential. Again, the 30% decrease in the number of prototrophic recombinants formed when males had been inhibited by chloramphenicol (200 &ml) for 30 minutes would not explain the 84% loss of cross-inducing activity. The validity of this control, in each experimental series, obviously depends on the assumption that the major I? population has no greater sensitivity to inhibition of specific pairing or transfer than have the rare Hfr males present which effect chromosomal transfer. There is no compelling reason to assume that chloramphenicol and 5-fluorouracil act through the same intermediate, but the specificity displayed in both inhibition studies must be considered. That deficiencies established by puromycin, 5-methyltryptophan and actinomycin failed to eliminate consistently the inducing activity of donors suggests that the response to chloramphenicol was not simply referable to its inhibitory action on general protein synthesis. The composite data indicated, rather, that such synthesis is unnecessary to the fixation of UV irradiation damage into a transferable intermediate. The unique effectiveness of fluorouracil would be most readily understood if it is considered to antagonize the function of uracil-containing cofactors or precursors to bacterial cell wall; defective wall polymers or unassembled constituents could alter radiation responsiveness. Since Versene-treated males that have lost approximately 50% of their cell wall lipopolysaccharides in the treatment period (266) are effective in cross-induction, such polymers, while not excluded as potential radiation targets, are much less suspect in theory than other wall constituents, particularly TDP sugars. Alternatively, structural impairment of the wall complex in both fluorouracil and chloramphenicol-treated donors may prevent the localization which is essential for efficient transfer of an active inducer. From these studies, it was concluded that only a preexisting macromolecule, altered by UV irradiation to a metabolically unstable form, or some radiation-damaged small molecule could be responsible for the indirect UV induction of bacterial lysogens. I n summary, most observations indicate that irradiated DNA entering

282

ERNEST BORER AND ANN RYAN

recipient lysogens in mating initiates phage production. Nevertheless, the temporal coincidence between episomal transfer and indirect UV induction, which we discerned quite early, could be misleading if some cellular component depends on the episome for passage. The inhibition studies suggested that the labile activity might be associated with the cell wallmembrane complex.

B. Other Indirect Modes of Induction Two unrelated series of experiments reinforce the above conclusion. The indirect induction of E . coli lysogens of opposite sex can be effected by strains exposed to high fluorouracil concentrations (100 pg/ml) for 60 minutes in hypertonic or glucose-deficient media. I n contrast with direct fluorouracil induction (requiring only 1 pg/ml) , thymidine rather than uracil enhances the effect (256).Thus, an inductive condition is produced whose efficiency increases when fluorouracil inhibition of DNA synthesis is reduced. It was shown subsequently that protoplasts of opposite mating type, maintained under rigorously controlled conditions, initiate induction on membrane contact. Polarity effects remain, so i t must be assumed that episomal elements produce important membrane alterations (257). Indirect ultraviolet induction and contact induction may not be independent phenomena. An interruption of episomal DNA synthesis in UV-irradiated cells may permit a persistent binding of the newly synthesized DNA strand to a membrane-bound component that initiates induction on entering recipient lysogens. If these are distinct responses instead, membrane contact may only release an activity essential to the induction response. In this view, two aspects of the induction mechanism would be reflected in the several modes of indirect induction described.

VII. Proposed Mechanisms for Lysogenic Induction in Bacteria The mechanisms proposed for lysogenic induction must now accommodate myriad new facts, Experimentally contrived inductions have shown that none of the inducing agents in general use bypasses dispensable early phage syntheses. Whether spontaneous derepression involves an indirect or immediate inactivation of the specific repressor or the subversion of its activity by inhibition and/or residual growth remains an open question. Before considering the suggested models, we might note some physiological impairments accompanying inducing treatments. The prejudicial observations that most inducing agents severely restrict host DNA synthesis (43, 51, 258, 2 9 ) now seem less informative. Ultraviolet irradiation not only inhibits this synthesis and that of RNA

LYSOCENIC INDUCTION

283

(26W262) but also decreases the rate of polypeptide chain initiation a t increasing doses and produces abnormal short polypeptides (263).Moreover, in both thermal and mitomycin C inductions of X prophages, the shutoff in DNA synthesis is lcss prompt than a major inhibition of bacterial RNA and protein syntheses (264). The latter is attributed to the action in trans of a phage-cneoded protcin whose gene maps near the ex0 and B protein loci. Prophage induction can be initiated, therefore, during DNA replication, but it is no less complete in irradiated lysogens that have already terminated a replication cycle, without reinitiation, a t nonpermissive temperatures (2666). Most bacterial strains that are sensitive to UV light have been found to be sensitive to mitomycin C (266), but several exceptions are now recognized. Recently isolated mitomycin-sensitive E . coli mutants, maximally inhibited by one-tenth the usual concentration of the antibiotic, retain the UV sensitivity of the wild strain, their ability to reactivate Wirradiated A phages and their susceptibility to UV induction (267, 268). The sensitivity to the antibiotic has been attributed to an alteration in the permeability barrier or to a mitomycin-inactivating system.

Induction Models Conjecture now focuses on derepression as the initiating event in prophage induction, yet this would be effective only if critical levels of the repressor are not regenerated. Most inducing agents do, in fact, derange DNA, RNA, and protein synthescs, and the exceptional cases of thermal inactivation of mutant repressors, presumably through conformational changes, seem straightforwardly analogous to the denaturations of several other mutationally altered proteins. Rcsidual growth might assure that repression is not rcncwed, but this complication could be circumvented if the derepressed phage gcnomc became inaccessible to newly synthesized repressor through a change of state or through the irreversible binding of an “inducer” that is prcsent in excess. Two models evolved from comprehensive studies of thermolabile X repression assume the active repressor to be an aggregate; the first proposes an oligomer consisting of the c l and N polypeptide chains while the second assumes a multimer of c1 polypeptides alone (56,188, 269, 270).In either case, a mutational change in the c l product would permit tlic same stcric alteration in the polypcptidc in response to heat (or an unspecified “inducer”) producing a partial or complete disaggregation of the complex. The characterization of repressing c l products as single polypeptide chains tends to negate this view, yet Ptashne’s isolations entailed heavy irradiation of the host before X infection to suppress bacterial syntheses (191,192).If the model were correct, the conditions

284

ERNEST BOREK AND ANN RYAN

generated would be expected to prevent aggregation. A theoretical model for the radiation induction of the X prophage similarly imputes an oligomeric structure to the repressor (271). Arguments for multiple binding sites on the cl product emphasize the functional differences between mutations in the A and B regions expressed in thermal inductions, the wide dispersion of cl mutant sites differentially affecting sensitivity to UV induction, and the binding of the repressor to both DNA strands at the pro promoter. Since mutations eliminating UV inducibility map in region A , it was inferred that this portion of the repressor binds UVgenerated inducers ; region B had been thought to combine specifically with another polypeptide chain. In the first model, some transcription of the N gene in uninduced N + or Nsus lysogens is assumed. The charge, size and secondary structure of this product would contribute to the ability of the complex to dissociate. Disaggregation would unblock the synthesis from the N gene, among others, but a delayed reassociation would occur in B mutants on their return to permissive temperatures. The alternate model assumed that mutations in the A region permit a major structural alteration in the cl product a t elevated temperatures while those in region B allow only minor distortions. The extent of disaggregation and derepression within each mutant group could vary, but mutations in the N gene would affect not repression itself but rather the expression of other operons. Allosteric properties ascribed to the repressor would accommodate either model to an indirect mode of repressor inactivation (187). Normally, XcltB mutants exhibit a survival curve after varied periods at nonpermissive temperatures that reflects their complete resistance to killing after very short heating intervals. In contrast, survival Xc,tA lysogens decrease precipitously following short exposures to inducing temperatures. If strains carrying c,tB prophages are UV irradiated a t sublethal doses and incubated for 15-20 minutes in chloramphenicol before the temperature shift, their survival curve duplicates that of c,tA lysogens. This was presumed to indicate the prior alteration of the repressor by bound, UVgenerated inducers ; disaggregation on heating would then follow the same time course observed for unirradiated Xc,tA lysogens. Complete derepression in UV (or nalidixic acid) induction(s) may require 20 minutes (W72), but this synergism implies that both agents modify the repressor itself. Devoret has shown that low, noninducing UV doses also enhance the phage yield of recipients in indirect UV induction (223, 2.98)) which introduces an alternate possibility that some trigger for derepression can be preset during the 15-minute postirradiation interval. Moreover, should the maximal rate of thermal induction possible be attained in hcl t A lysogens, whose requirements for derepression are minimal (273), any reduc-

LYSOGENIC INDUCTION

285

tion in competing host reactions after low UV irradiation could accelerate only the killing specific to the induction of heated hcltB lysogens. Another model places emphasis on the possibility that induction may follow the inactivation of the cl product through the presumed binding of adenine derivatives (274). Significantly, a highly pertinent host mutation permitting the thermal induction of E . coli T44 (A) appears t o involve the regulation system for nucleic acid precursors (274,275).These lysogens are spontaneously induced a t 30°C a t ten times the normal rate and can be fully induced if the temperature is shifted to 42°C. All phages normally induced by UV can be induced in this mutant by heat alone, but the addition of guanosine or cytidine a t the time of the thermal shift protects against induction. These same supplements also restore cured mutant cells to normal growth a t 40”C, although adenine counteracts them in each system. On the basis of these results, an induction model was proposed in which an adenine derivative, possibly activated during growth a t the nonpermissive temperature, inactivates the heat-stable repressor. The parallel response of septum formation and prophage induction is striking, particularly since these lysogens are induced only when they can grow a t the elevated temperature (274). I n this context, the hypothetical “inducing protein,” required by cl tB mutants a t high temperature and present in cltA mutants, could be a preexisting host protein essential for the binding of the active adenine derivative. Prophage induction following UV irradiation has been considered a t various times to result from: direct damage to the repressor (276, 277), the intervention of a UV-generated “inducerlJ1the activity of an induced host protein also involved in septa formation (278) or dilution of the repressor to subcritical levels by residual growth (279). Growth without division does occur during the latent periods of UV-irradiated E . coli and Pseudoinonus aerugimsa (280, 281 ) lysogens, among others. Induced B. megateriuin ( 2 ) and Staphylococcus uureus strains (279) undergo two divisions before phages are released. Nevertheless, this view is seriously undermined by observations that the derepression of the h prophage and the commitment of the bacterial cell to death precedes the usual division time in synthetic medium. Immediate UV damage to the repressor now seems an unlikely possibility since the quantum yield for proteins is two orders of magnitude smaller than that calculated for DNA by Smith and Hanawalt (282). More importantly, the cross-induction phenomenon (indirect UV induction) demonstrates unequivocally that UV irradiation may induce indirectly via donors that lack repressor. The following proposals may amplify specific aspects of the same inductive process. Consider the implications of indirect UV induction. This response proved that UV induction is mediated by a radiation-damaged,

286

EBNEST BOREK AND ANN RYAN

metabolically unstable product of nonviral origin that, if it is not a metabolite or “small” molecule, appears to be synthesized before UV irradiation. The transfer of the “inducer,” together with several episomes, indicates that the irradiated episomal material, to some element of which it may be attached or some UV-altered cell constituent dependent upon limited DNA transfer for passage, effects the induction of recipient lysogens. Usually, only the denser DNA strand in a CsC1-poly(U,G) gradient is transferred in F infection (283).This is synthesized in the donor prior to conjugation (241, 284, 285) and, as measured after a 60-90 minutes mating in broth, it constitutes from 0.3 to 1.0% of the DNA present in the male (240, 2.41, 286, 287). After transfer, it appears in doublestranded DNA molecules together with newly synthesized complementary strands (283, 285, 287). This must be compared with the negligible transfer of donor proteins and RNA whose upper limit is set a t 0.1% of the total complements of both Hfr and F+males ( 2 @ , 887). DNA was immediately implicated as the probable inducing agent by the photoreactivation and photoprotection of the activity and the immediate transfer by F+donors, as well as by the prevailing emphasis on this macromolecule as the most likely UV target. The chemical evidence cited above and later biological results obtained with Hcr+ donors or DNArestricting females further strengthened this view. Irradiated episomal DNA, if unrepaired prior to transfer, should then contribute UV damaged oligonucleotides or monomers to the soluble pool of the recipient cell. However, it has been reported that restricted, episomal, irradiated DNA does not induce the recipient (253). A general model offered by Hertman and Luria (811) assumes that exposure to an inducing agent causes the production of an unknown substance, possibly an adenine derivative by analogy with the T44 response, that could inactivate the repressor only in rec+ cells. In recA mutants, which retain heat inducibility if they carry a clts mutation but fail to respond to UV irradiation or thymine starvation in any case, an excessive degradation of DNA would permit accumulation of another unknown compound, Y, capable of counteracting the production or activity of X (211).Studies performed with the double mutant, E. coli K12 recAuvrB, fail to substantiate this in a mitomycin induction; little DNA breakdown and no induction occurred, yet the prophage was induced on the entry of recA+ activity. The cellular response to mitomycin may not correspond with its behavior after UV irradiation, of course, since both single urv B and double recA uvrB lysogens have different sensitivities to these agents. Moreover, the uwB+ gene has some unknown role in promoting cell survival during normal growth, although it is detectable only in the absence of recA activity (212). This last function also appears to act pleio-

LTSOGENIC INDUCTION

287

tropically on UV reactivation, UV mutagenesis and UV induction, implying some common pathway (288, 289). Tomizawa and Ogawa proposed that recA, however, is involved in the production or activation of the inducer (190). Yet additional bacterial mutations present in some recA+ survivors from a thymine deprivation prevent their induction by this treatment and reduce the frequency of their spontaneous induction (890). For this reason, several biochemical steps have been suggested to precede the formation of an inducer in the thymine-starved cell. Nevertheless, a defective TecA gene permits limited cell division when DNA synthesis is stopped by thymine starvation or nalidixic acid treatment. Specific differences were detected in the membrane proteins of recA+ and recA- cells; these may be associated with cell division and DNA synthesis. Inouye suggests, therefore, that septum formation is negatively controlled by the recA+ gene (291). The parallelism between the UV induction of prophages and filament formation by irradiated E . coli B, independently noted by Witkin, led her to conclude that this strain, whose radiation sensitivity results from filament formation a t low UV doses, may also contain a repressor that is inactivated when DNA synthesis is blocked (278).This failure to produce cross-septa, like prophage induction, is prevented when cells are preilluminated or treated with chloramphenicol before or after irradiation. Both filamentation and induction can occur after the formation of only 10-20 dimers per bacterium, which block DNA synthesis. She proposed, therefore, that an operon B, producing an inhibitor of cell division or leading directly to filament formation, is induced under these conditions. When DNA is repaired, repression is restored, and the subsequent recovery and cell division depend on the quantity of the inhibitor accumulated (278). A composite image of events reconcile direct and indirect induction models. If it is assumed that the inhibition of DNA synthesis by the usual inducing treatments leads to the production of substance X, a possible substrate (or product) of recA activity, the reaction of this component with a membrane protein, Z, would create an inducer, XZ, of both the prophage, if present, and the B operon. Alternatively, the conversion of Z might lead to the inhibition of cell division only as a result of the decreased available concentration of Z. The nature of the mutational change in Z or its availability would determine whether prophage induction and filament formation were correlated. Since the bacterial property altered in T44 confers thermolability only on UV inducible prophages, i.e., not on Xind-, the functional change seemed to affect the repressor. However, the increased concentration of the ind- repressor could render the available inducer inadequate, particularly if binding is altered by the mutational change. The proposed “inducer” would prevent

288

ERNEBT BORER AND ANN RYAN

this binding of the repressor to its DNA by serving as a preferred substrate. If analogous, rather than overlapping, mechanisms are involved in filament formation and prophage induction, the presumed receptor could be a phage-encoded protein transcribed prior to induction. In the h system, this would have to map in the cl-rex or N gene regions, although the position of the pL promoter to the right of N makes the last choice improbable. The activated inducer could then be a modified repressor, the rex product or another specified by an unrepressed phage gene. Green proposed that the inducer is an altered repressor on the basis of his analysis of the functional differences between thermosensitive mutants in the cl gene (186).Yet, we already know that nonlysogens can crossinduce. Immunity might be expected to fluctuate in growth in response to physiological and mutational changes. Calef and Neubauer have reported a phase variation in immunity a t different growth temperatures, which they attribute to quantitative changes in the “immunity substance” (292). An additional variation as a result of the rim mutation in the immunity region produces both normal large colonies and small clones due to frequent spontaneous induction. The mutation, detected first in hind- mutants, does not change the ind- character but does cause a partial breakdown in superinfection immunity. I n addition, cT protects hc1857 lysogens from thermal induction to a lesser extent than ind- and produces a rex- phenotype (693).The impact of mutational changes in the immunity region on cl repression is now under intensive study. That some phage or episome-specified protein could serve as an inactive inducer finds a precedent in the inducing activity of an exceptional new type of colicine, F,, produced by a noninducible colicinogenic E . co2i strain. After 10 minutes’ exposure to this product, several E . co2i K12 (A) strains subsequently exhibited a 100- to 1000-fold increase in phage titer (294). Moreover, the bacteriocins produced by an inducible Pseudoinonas aeruginosa lysogen include a group consisting of bacterial protoplasmic and cell wall proteins in addition to Pyocine 15 (295,296‘),which resembles the “tail-like” structures released by the defective lysogens of Bacillus subtilis (297) and E . coli 15 (298). Surface alterations due to episomal activities (2993Ul) are common, but the polysaccharides or proteins involved appeared to be unlikely UV irradiation targets. Since an inducing intermediary can be demonstrated for this agent alone, arguments for DNA mediation in an indirect process gain strength. How lysogenic induction could be effected indirectly remains to be resolved. It is now apparent that an inducer need only antagonize the binding of the phage-specific repressor (in the X system) to the T strand

LYSOGENIC INDUCTION

289

of the prophage at pro, for Szybalski finds that the induction of tofmutants does not turn off cl-rex transcription (165b).A role for irradiated DNA in indirect induction as an alternate binding substrate, while singlestranded, would seem less conjectural than other proposals, but an involvement of some membrane constituent can not be excluded.

VIII. Analogies in Mammalian Systems Lwoff was the first to analogize the spontaneous induction of bacterial lysogens with oncogenesis in higher organisms. Recent observations remove all doubt that lysogenization by oncogenic viruses can occur in infected animal cells. The rescue of defective viral genomes by helper viruses (3024U4) or in cocultivation experiments (306-510) , the recovery of infectious viruses from nonproducing cell lines after cell fusions with indicator strains (306, SlOSl4, 321), and an enhanced virus production following conventional inducing treatments (314, 317-320) constitute impeccable biological evidence for the persistence of latent viral genomes in virus-free transformed cells or in the tumors they initiate. DNA-RNA hybridhation studies indicate that multiple copies of viral DNA within these cells may number between 5-60 or 22-85 viral equivalents per cell transformed by the polyoma and simian virus-40 (SV40) (323-325) or by adenoviruses (326), respectively. Portions of each genome are transcribed preferentially, yet some early and late genes are not usually active (326).Recent reviews have presented the progress of relevant research (326-330). We note here only selected evidence that lysogenic induction also occurs in transformed mammalian cells. That cell lines transformed by SV40, Rous sarcoma virus (RSV), polyoma and adenoviruses often harbor intact viral genomes was demonstrated with variable difficulty (308,313, 322, 331). Nevertheless, the cocultivation of SV40-induced hamster tumor or transformed cells with permissive indicator strains did induce viral syntheses (308-310). Cell fusions promoted by UV-inactivated Sendai viruses so increased the effectiveness of parabiotic culturing that positive responses appeared in systems otherwise found to yield no viruses (510-314). The data accumulated indicate that more than ten transformed cell lines representing four species contain a t least one complete SV40 genome per cell (315).Intact adenovirus 12 and polyoma genomes were not detected, however, in hamster or mouse kidney tumor cells by cocultivation, cell fusions, chemical inductions or combined treatments (315).Polyoma transformed lines, in contrast, have yielded viruses in cell fusions with permissive cells (321), and 3-20% of the heterokaryons formed between cells from either of two established polyoma tumor lines and mouse embryo or

290

ERNEST BOREK AND ANN RYAN

kidney cells have specific fluorescence after 8 days (316). Similarly latent RNA viruses are recoverable when transformed cells are cocultivated or fused with natural host cclls in the presence of helper viruses (539). The fusion of clones from two different nonproducing classes of RSV transformed chick cells results in RSV production, SO complerncntation or recombination between latent proviruscs or defectivc viral genomes also restores infectivity (306). Conventional inducing agents including UV irradiation, X-rays and mitomycin C initiate virus producton in polyoma-transformed rat embryo muscle and SV40 transformed hamster kidney cclls (314, 320). I n general, mitomycin C induces clones that are positive by the cocultivation overlay assay. The spontaneous frequency of polyoma production, 1/104 cells, was increased 200, 100 and 3000-fold by UV irradiation, X-rays and mitomycin C, respectively. All clones exhibiting spontaneous inducibility and 50% of those remaining are chemically inducible but differ in yield per culture and degree of inducibility (320). Several observations suggest that repression again controls some essential viral synthesis when cells are lysogcniced by genetically competent viruses. A repressor-antirepressor model for control is proposed, since a protein, present only in permissive cells (?IS@, counteracts the 30-50% inhibition of phage production causcd by crude extracts from SV40 transformed (554), productively infected or abortively infected cells (535). However, heterogeneity exists in virus activation within hctcrokaryons from effective ccll fusions. Although 50% of those formed between SV40 Gwen monkcy kidney (GMK) transformants and permissive GMK cells yield viruses, only 1% of the heterokaryons resulting from fusions of transformed human sublines to GMK cells are productive (312). While all clones of an SV40 transformed mouse cell line were positive after cell fusions, again, in each of these clones, less than 10% of thc heterokaryons between any of six indcpcndent transformed lines and a permissive GMK linc formed plaques (313).Metabolic inhibitions imposed on transformed murine cells before fusion indicate that iododeoxyuridine and 8-azaguanine considerably increase the percentage of positive hctcrokaryons and the virus yicld. In contrast, 2-thiouracil and fluorophenylalanine are without effect, while mitomycin C and actinomycin D incrcasc the fraction of positive hctcrokaryons as they reduce tlic virus yicld. An intcrfcrciicc with rcprcssor syiitlicsis or thc formation of a less active mutant protein was suggested (336). Thc “induction of detectability” in heterokaryons could entail the overtitration of thc repressor, the introduction of an inducing or activating componcnt prcsent only in pcrmissivc strains, or the dilution and/or inhibition of repressor. Superficially, the cell fusion response

LYSOGENIC INDUCTION

291

resembles indirect UV induction or contact induction in bacteria. The role of the irradiated Sendai viruses may be complex, for several “fusing” viruses disrupt lysosomes and activate lysosomal enzymes (337).Nevertheless, cocultivation alone often induces, so membrane contact or particular pinocytic inclusions seem to be sufficient. Speculation on the many possibilities is limited by several additional facts obtained with SV40-transformed systems. No correlation exists between the number of viral equivalents in transformed cells and their ability to yield infectious viruses after cell fusions (325).Physiological conditions do not alter this number, and all clones in any cell line produce the same amount of T antigen (324). The heterogeneous productivity of lieterokaryons should not reflect a gene dosage effect on the amount of repressor specified by the viral genome, for thc nuclear ratios within hetcrokaryons did not affect the percentage of productive hctcrokaryons (313). Spontaneous induction may contribute to “natural” neoplastic transformations, since malignancy has been shown to correlate with inducibility in a t least one transformed linc (314).Huebner suggests that the vertical transmission of an inducible oncogenic (c-type) RNA viral genome, known to be integrated in cells of nine spccies and three classes of vertebrates, could account for the stochastic distribution of cancer (338,339). Infectious leukemia and sarcoma viruses appear in radiation and chemically induced earcomas of low-incidence mice (340) and group specific antigen occurs in 10% of the sarcomas induced by 3-methylcholanthrcnc in virus-free wild mice (341). Moreover, the association between the b-type RNA viral particlcs and murine mammary carcinomas (3.42) or heterokaryons formed by malignant cells and normal cells (34.9) seems to be paralleled in the human disease (3.4.4-346’).Direct cell-to-cell transfer of these particlcs in pinocytic vesicles, as has already been detected with Bittner factors (347),help establish latent proviruses whose later expression would be tumorigenic in an altered physiological context or after viral superinfcctions. Extrinsic infections during latency by unrelated viruses would promote both viral diversity and multiple lysogeny as a consequence of recombination, complementation and phenotypic mixing. Obviously, defective polylysogeny would increase the probability of a malignant transformation without correlation with persistent recognizahle virions. Low, reproducible levels of murine leukemia group-spccific antigrn appear in soiiic classes of SV3T3 transformants in the absence of virus production, so infecting with oncogenic DNA viruses, as well as chemical and physical inducing agents or hormonal stimulation, may activate the presumed latent “oncogene.” Viral gcnctics assumcs ovcrwhclming importance, for the biochemical characterization of identifiable mutants of the suspected etiological

292

ERNEST BOREK AND ANN RYAN

agents could take us beyond the oncogene theory. Bacterial geneticists have long recognized that certain episomal elements, lacking specificity in integration, have high mutagenic potential for the host genome (3.48). As more analogies between phage and viral lysogeny emerge, lysogenic induction may well become of paramount concern to all.

ACKNOWLEDGMENTS We are grateful to Drs. Seymour S. Cohen, Dale Kaiser and Arthur Weissbach for critical readings of this manuscript. We are also indebted to Dr. Waclaw Szybalski for providing Figure 1. The work reported from our laboratory was supported by contracts from the U.S. Atomic Energy Commission. It is a pleasure to express our indebtedness to the scientist-administrators of that agency for their support when both the reality nnd the significance of our work was questioned by many radiation biologists.

REFERENCES 1. Luria, S., Personal communication. 2. A. Lwoff, L. Siminovitch and N. Kjeldgnnrd, Ann. Inst. Pasteztr 79, 815 (1950). 3. A. Lwoff nnd A. Gutmnnn, Ann. Znst. Pnstenr 78, 711 (1950). 4. A. Lwoff, Bncteiz’ol. Hev. 17, 269 (1953). 6. G. Bertani, Advnn. Virus Res. 5, 151 (1958). 6. F. Twort, Lancet 2, 1241 (1915). 7. F. d’Herelle, “Le Bncteriophnge.” Mnsson, Paris. 8. 0. Bail, Med. Klin. (Munich) 21, 127 (1925). 9. E. Gildmeister and K. Herzberg, Zentr. Bnkteiiol. Parasifenk. Znfektwmkr. Hug. Abt. 1: Oiig. 93, 402 (1924). 10. E. McKinley, C. R. SOC.Biol. 93, 1050 (1925). 11. J. Bordet, Ann. Zitst. Pasteur 39, 711 (1925). 12. E. Wollmnn, Bull. Inst. Pastew- 26, 1 (1928). 13. F. Burnet and M. McKie, Aust. J . Exp. Biol. Med. Sci. 6, 277 (1929). 1.6.E. Wollman, Bull. Znst. Pnsteur 32, 945 (1934). 16. A. Gratia, C. R. SOC.Biol. 122, 812 (1936). 10. A. Doermann, Cnrnegie Inst. Wnsh. Y e a h 47, 176 (1948). 17. E. Lederberg and J. Lcdcrbcrg. Genetics 38, 51 (1953). 1s. E. Wollman, A m . Znst. Pasfezir 84, 281 (1953). 19. F. Jncob nnd E. Wollman, Ann. Znst. Pastew 91, 386 (1956). 90. F. Jncob nnd E. Wollmnn, “Sexuality and thc Genetics of Bacteria,” p. 87. Acntlemic Preas, New York, 1961. 91. F. Jacob and E. Wollman, C . R. Acad. Sci. 239, 455 (1954). 22. F. Jncob and E. Wollmnn, Ann. Znst. Pasteitr 91, 489 (1956). 23. F. Jacob and E. Wollman, in “The Chemical Bnsis of Heredity,” (W. D. , p. 468. Johns Hopkins Press, Baltimore, Maryland, MrElroy and B. C l a ~cds.), 1957.

24. F. Jacob and E. Wollman, Symp. SOC.Ezp. Biol. 12, 75 (1958). 96. F. Jncob and J. Monod, JMB 3, 318 (1%1). 26. A. Kaiser, Virology 3, 42 (1957). 27. M. Levine, Virology 3, 22 (1957). 1. F. Jncob and E. Wollman, Ann. Inst. Pasteur 87, 653 (1954).

LYSWzENIC INDUCTION

293

29. F. Jacob nnd A. Cnmpbell, C. R. Acad. Sci. 248, 3219 (1959). 30. G. Bertani, Ann. Zmt. Pnstew 84, 273 (1953). 31. M. I Ab , J. Bneteriol. 65, 642 (1953). 32. J. Boyd, J . Pathol. Bacteriol. 63, 445 (1951). 83. 13. Fry, J . Geti. Microbial. 21, 676 (1959). 34. E. Bore%, BBA 8, 211 (1952). 36. F. Jacob, A m . Ziisl. Pnsteiir 82, 433 (1952). 30. M. Adams, Methods Mecl. Res. 2, 7 (1950). 37. H. Marcovich, Ann. hist. Pastefir 91, 511 (1956). 3s. P. Hnnnnalt nnd R. Setlow, BBA 41, 283 (1960). 89. R. Srtlow, P. Swmson and W. Carrier, Science 142, (1963). 400. P. Swrnscn and R. Setlow, J M B 15, 201 (1966). 40b. P. Swrnsen and R. Setlow, JMB 17, 237 (1966). 41. H. Morowitz, Science 111, 229 (1950). 42. C . Rupert and Harm, Advnn. Radial. Biol. 2, 2 (1966). 43. W. Ssybalski and V. Iyer, Antibiotics 1, 211 (1967). 44. N. Otsuji, M. Sekiguchi, T. Iijima and T. Tnkngi, Nature (London) 184, 1079 (1959). 46. D. Korn and A. Weissbach, BBA 61, 775 (1962). 4G. M. Levine and M. Borthwick, Virology 21, 568 (1963). 47. S. Cohen and H. Barner, PNAS 40, 885 (1954). 4s. M. Levine, Virology 13, 493 (1961). 49. N. Meleclicn and P. Skaar, Virology 16, 21 (1962). 60. N. Sicard and R. Devoret, C. R. Acnd. Sci. 253, 1417 (1962). 61. S. Cohcn, J. Flaku, H. Barner, M. Locb and J. Lichtenstein, PNAS 44, 1004 ( 1958). 62. J. Horowitz, Saukkonen and E. Chargaff, JBC 235,3266 (196(1). 63. H. Marcovich and H. Kaplan, Natitre (London) 200, 487 (1963). 64. E. Geisslcr, Actn Biol. Med. Ger. 11, 286 (1963). 66. L. Bertani, BBA 87, 631 (1964). 6G. M. Leib, JMB IS, 149 (1966). 67. R. Lntarjet, Ann. Inst. Pastew 81, 389 (1951). 65. H. Marcovich, Ann. Zirsl. Pasteiir 90, 303 (1956). 69. S. Epstein and I. Saporoschets, Ezperientk 24, 1245 (1968). 60. R. Calendar, Aanzi. Rev. Microbiol. 24, 241 (1970). GI. E. Jordan, J d f B 10, 341 ( 1 M ) . 62. D. S. Hogness, W. Docrfler, J. B. Egan and L. W. Black, CSHSQB 31, 129 (1966). G3. K. Taylor, Z. Hradnecna and W. Szybnlski, PNAS 57, 1618 (1967). 04. J. S. Pnrkinson, Genetics 59, 311 (1968). (16.B. C. Westmoreland, W. Szybnlski and H. Ris, Science 163, 1343 (1969). GO. S. Kumar, K. Bovre, A. Guha, Z. Hradnccna, S. V. M. Malier and W. Szybalski, Nature (London) 221, 823 (1969). 07. M. Gough and M. Levinc, Ge,ielics 58, 161 (1968). GS. H. Smith and M. Lcvine, Virology 25, 585 (1965). 69. R. Kolstad and H. Prell, Mol. Gen. Genet. 104, 339 (1969). 70. M. Gough, J. Virol. 2, 992 (1968). 71. G. Attardi, S. Naono, J. Rouvierc, F. Jacob, and F. Gros, CSHSQB 28, 363 (1963). 72. W. S. Sly, H. Echols and J. Adler, PNAS 53,378 (1965).

294

ERNEST MREK AND ANN RYAN

73. S. Naono and F. Gros, CSHSQB 31, 363 (1966). 74. A. Skalka, B. Butler and H. Echols, PNAS 58, 576 (1967). 76. A. Campbell, Virology 14, 22 (1961). 76. R. Thomas, C. Leun, C. Dambly, D. Parmentier, L. Lambert, P. Brachet, N. Lefebre, S. Mousset, J. Porcheret, J. Szpirer and D. Wauten, Mutation Res.

4, 735 (1967).

77. A. Brown and W. Arber, Virology 24, 237 (1964). 7s. F. Jacob, C. R. Fuerst and E. L. Wollman, Ann. Znst. Pasteur 93, 724 (1957). 79. C. R. Fuerst and D. W. Mount, Can. Cancer Con!. 6, 293 (1966). 80. R. Thomas, JMB 22, 79 (1966). 81. W.Dove, Annu. Rev. Genet. 2, 305 (1968). 82. T. Ogawa and J. Tomizawa, JMB 38, 217 (1968). 83. A. Joyner, L. N. Isaacs, H. Echols and W. S. Sly, JMB 19, 174 (1966). 84. S. N. Cohen and J. Hunvite, JMB 37, 387 (1968). 86. P. Kourilsky, L. Marcaud, P. Sheldrick, D. Luzzati and F. Gros, PNAS 61,

1013 (1968).

86. W. Dove, JMB 19, 187 (1966). S7. J. J. Protass and D. Korn, PNAS 55, 1089 (1W).

88. C. M. Radding, BBRC 15, 8 (1964). 89. C.M. Radding and H. Echols, PNAS 60, 707 (1968). 90. C. M. Radding, Annu. Rev. Genet. 3, 383 (1969). 91. C. Babinet and H. Condamine, C. R. Acad. Sci. 231, 2670 (1968). 92. M. Konrad, PNAS 59, 171 (1968). 93. M. Schwartz, Virology 40, 23 (1970). 94. D. Rabovsky and M. Konrad, Virology 40, 10 (1970). 96. R. Gingery and H. Echols, PNAS 58,1507 (1967). 96. J. Zissler, Thesis, University of Rochester (1967).

97. J. Ziasler, Virology 31, 189 (1967). 9s.

H. Echols, R. Gingery and L. Moore, JMB 32,251 (l!M).

99. E. Signer and J. Weil, JMB 32, 261 (1968).

100. J. Weil and E. Signer, JMB 32, 273 (1968). 101. G.Guarneros and H. Echols, JMB 47, 565 (1970). 109. A. Kaiser and T. Masuda, JMB 47, 557 (1970). 103. A. Kaiser and T. Masuda, Virology 40, 522 (1970). 104. E. Signer, Virology 40, 624 (1970). 106. E. Signer, Nature (London) 223, 158 (1969). 106. K. Matsubara and A. Kaiser, CSHSQB 33, 769 (1968). 107. K.Brooks, Virology 26, 489 (1965). 10s. K. Oda, Y. Sakakibara and J. Tomizawa, Virology 39, 901 (1969). 109. R.Shuster and A. Weissbach, BBRC 33,514 (1968). 110. R. Shuster and A. Weissbach, Nature (London) 223, 852 (1969). 111. D. Freifelder and I. Kirschner, Virology 44, 223 (1971). 112. H.Eisen, L. Pereira da Silva and F. Jacob, C . R . Acad. Sci. 266, 1176 (1968). 118. L. Pereirn dn Silvn, H. Eisen nnd F. Jncob, C. R . Acnd. Sci. 266, 926 (1968). 114. W. Sly, H. Eisen and L. Siminovitch, Virology 34, 11% (1968). 116. L.Pereira da Silva and F. Jacob, Virology 33, 618 (1967). 116. S.Heineman and W. Spiegelman, PNAS 67, 1122 (1970). 117. P. Brachet and B. Green, Virology 40, 792 (1970). 118. H. Eisen, C. Fuerst, L. Siminovitch, R. Thomas, L. Lambert, L. Pereira da Silva nnd F. Jncob, Virology 30, 224 (19eS).

LYSOGENIC INDUCTION

119. 120. 121. 122.

295

S . Packman and W. Sly, Virology 34,778 (1968). L. Pereira da Silva and F. Jacob, Ann. Inst. Pnsleltr 115, 145 (1968). W. Sly and K. Rabideau, JMB 42, 385 (1969). G. Streisinger, J. Emrich, Y. Okada, A. Tsugita and M. Inouyc, JMB 31, 607 (1968).

123. 124. 126. 186. 127. lgS. 189. 130. 131. 132. 133. 134. 136. 138.

C. Dambly, M. Couturier and R. Thomas, JMB 32, 67 (1968). I. Hcrskowits and E. Signer, JMB 47, 545 (1970). N. Hopkins, Virology 40, 223 (1970). S . Tonegawa and M. Haynishi, JMB 40, 219 (1970). A. Skalka, CSHSQB 31, 377 (1986). W. Doerfler and D. Hognees, JMB 33, 635 (1968). Z. Hradneca and W. Szybalski, Virology 32, 633 (1967). W. Doerfler and D. Hogness, JMB 33, 661 (1968). A. Skalka, E. Burgi and A. Hershey, JMB 34,l (1968). P. Bear and A. Skalka, PNAS 62, 385 (1969). K. Bovre and W. Saybalski, Virology 38, 614 (1969). S. Tonegawa and M. Hayashi, PNAS 61, 1320 (1968). M. Konrad, JMB 53, 389 (1970). F. Gros, P. Kourilsky and L. Marcaud, Ciba Found. Symp. Homeostatic Regul.

107 (1969). 137. R. A. Weisberg and M. E. Gottesman, in “The Bacteriophage Lambda” (A. Hershey, ed.), p. 489. Cold Spring Harbor, New York. 138. R. Hendrix and M. Schwarta, cited by R. Calendar (GO). 139. P. Kourilsky, M. F. Bourguignon, M. Bouquet and F. Gros, CSHSQB 35, 305 (1970). 140. H. Nijkamp, K. Bovre and W. Szybalski, JMB 54, 599 (1970). 1 4 a . J. Roberts, Nature (London) 223, 480 (1969). l 4 lb. J. Roberts, Nature (London) 224, 1188 (1969). 142. C. Radding and D. Schreffler, JMB 18, 251 (1966). 143. W. Dove, E. Hargrovc, M. Ohashi, F. Haugli and A. Guha, Jap. J . Genet. 44, 11 (1969). 144. R. Thomas and L. Bertani, Virology 23, 241 (1964). 146. D. Luaaati, JMB 49, 515 (1970). 146. J. Zissler and A. Campbell, Virology 37, 318 (1969). 147. V. Bode and A. Kaiser, Virology 25, 111 (1965). 148. R. Thomas, JMB 49, 393 (1970). 14.9. R. Thomas and S. Moussct, JMB 47, 179 (1970). 160. R. Boyce, E. Kraiselburd, S. Ryan and H. Chesein, Virology 37, 679 (1969). 161. B. Butler and H. Echols, Virology 40,212 (1970). 162. D. Court, and K.Sato, Virology 39, 348 (1969). 163. K.Sato and A. Campbell, Virology 41, 474 (1970). 164. C. Radding, PNAS 52, 965 (1964). 166. J. Pero, Virology 40, 65 (1970). 166. H. Eiscn, P. Brachct, L. Pcreirn da Silva and F. Jacob, PNA8 66, 855 (1970). 167. E. Calcf and Z. Ncubaucr, CSIISQB 33, 765 (1968). 168. H. Eisen, L. Prrcira da Silra and F. Jacob, CSHSQB 33, 755 (1968). 169. Z. Neubauer and E. Calef, JMB 51, 1 (1970). 160. W. Spiegelman, Virology 43, 16 (1971). 161. L. Reichardt and A. Kaiser, PNAS 68, 2185 (1971). 162. P. Kourilsky, L. Marcaud, M. M. Portier, M. Zamansky and F. Gros, B d l . SOC.Chem. B i d . 51, 1429 (1969).

ERNEST BOREK AND ANN RYAN

296

163. Y. Sakakibara and J. Tomizawa, Virology 44, 471 (1971). 164. M. Ptashne and N. Hopkins, PNAS 80, 1282 (1968). 166a. S. Kumar and W. Saybalski, Virology 41,665 (1970). 166b. S. Kumar, E. Calef and W. Szybalski, CSHSQB 35, 331 (1970). 166. W. Szyba.lski, K. Bovre, M. Fiandt, 5. Hayes, Z. Hradnecna, S. Kumar, H. Lozeron, H. Nijkamp and W. Stevens, CSHSQB 35, 341 (1970). 167. L. Isaacs, H. Echols and W. Sly, JMB 13,963 (1965). 1G8. W. Sly, H. Echols and J. Adler, PNAS 53, 378 (1965). 169. H. Echols, L. Pilanki and P. Cheng, PNAS 59, 1016 (1968). 170. W. Haywood and M. Green, PNAS 54, 1675 (1965). 171. A. Kaiser and F. Jacob, Virology 4, 509 (1957). 172. C. Malva, G. Razzino and E. Calef, Virology 38, 358 (1969). 173. R. McMacken, N. Mantel, B. Butler, A. Joyner and H. Echols, JMB 49, 639 (1970).

174. H. Echols and L. Green, PNAS 68, 2190 (1971). 176. M. Levine, Genetics 40, 582 (1955). 176a. M. Levine and R. Curtis, Genetics 42, 383 (1957). 176b. M. Levine and R. Curtim, Genetics 46, 1573 (1961). 177. M. Bronson and M. Levine, Bacteriol. Proc. 201 (1970). 178. P. Amati, Virology 36, 701 (1968). 179. R. Sussman and F. Jacob, C. R . Acad. Sci. 254, 1517 (1962). 180. R , Thomas and L. Lambert, Bull. Acad. Roy. Belg. 48, 688 (1962). 181. M. Lieb, Science 145, 175 (1964). 182. F. Jacob, R. Sussman and J. Monod, C. R . Acad. Sci. 254, 4214 (1962). 183. R. Thomas and L. Lambert, JMB 5, 373 (1962). 184. T. Horiuchi and H. Inokuchi, JMB 15, 674 (1966). 186. M. Green, JMB 16, 134 (1966). 186. S. Naono and F. Gros, JMB 25, 517 (1967). 187. M. Lieb, JMB 39, 379 (1969). 188. R. Cross and M. Lieb, J . Virology 6, 33 (1970). 189. M. Lieb, Nature (London) 214, 175 (1964). 190. J. Tomizawa and T. Ogawa, JMB 23,247 (1967). 191. M. Ptashne, PNAS 57, 308 (1967). 192. M. Ptashne, Nature (London) 214, 232 (1967). 193. V. Pirotta and M. Ptashne, Nature (London) 222, 541 (1969). 194. W. Gilbert and Miiller-Hill, PNAS 56, 1891 (1966). 196. A. Oppenheim, Virology 39, 832 (1969). 196. R. Weisberg, and J. Gallant, JMB 25, 537 (1967). 197. A. Oppenheim, Mol. Gen. Genet. 105, 21 (1969). 198. H. Eisen, I. Tallan and L. Siminovitch, Virology 32, 104 (1968). 199. H. Eisen, L. Siminovitch and P. Mohide. Virology 34, 97 (1968). 200. A. del Campillo Campbell, cited by Campbell, in “The Episomes,” p. 19.

Harper, New York, 1969. 201. R. Weisberg and J. Gallant, CSHSQB 31, 374 (1966). 802. D. Cohen, Virology 7, 112 (1959). 803. M. Gottesman and M. Yarmolinky, JMB 31, 487 (1968). 804. R. Weisberg, Virology 41, 195 (1970). 206. E. Signer, Annu. Rev. Microbiol. 22, 451 (1968). 806. E. Signer, J. Weil and P. Kimball, JMB 46, 543 (1969). 807. N. Franklin, W . Dove and C. Yanofsky, BBRC 18, 910 (1965).

LYSOGENIC INDUCTION

297

208. A. Clark and A. Margulies, PNAS 53, 451 (1965). 209. K. Brooks and A. Clark, J . Virol. 1, 283 (1967). 210. N. Willets, A. Clark and B. Low, J . Bacten'ol. 97, 244 (1969). 211. I. Hertman and S. Luria, JMB 23, 117 (1967). 212. I. Hertman, Genet. Res. 14, 291 (1969). 213. J. Wing, J . Virol. 2, 702 (1968). 214. T. Ogawa and J. Tomizawa, JMB 23, 225 (1967). 216. E. Borek and A. Ryan, PNAS 44,374 (1958). 216. E. Borek and A. Ryan, BBA 41, 67 (1960). 217. A. Ryan, BBA 60, 455 (1962). 218. A. Ryan, Ph.D. Thesis, Columbia University, 1963. 819. R. Devoret and J. George, C. R . Acad. Sci. 258, 2227 (1964). 220. A. Clarke and E. Adelberg, Atznu. Rev. Microbiol. 16, 15 (1962). 221. E. Calef, G . Giardino and G. Modiano, Atti Gen. Italy 5, 247 (1960). 222. R. Devoret and J. George, C . R . Acad. Sci. 258, 5287 (1964). 223. R. Devoret and J. George, Mutat. Res. 4, 713 (1967). 224. A. Ryan, unpublished data. 226. J. George, unpublished data. 226. J. Rosner, FP 26, 527 (1967). 227. J. George and R. Devoret, Mol. Gen. Genet. 111, 103 (1971). 228. R. Devoret, Intern. J . Rad. Biol. 6, 385 (1963). 229. H. Marcovich and R. Latarjet, Advan. Bwl. Med. Phus. 6, 75 (1958). 230. F. Jacob, S. Brenner and F. Cuzin, CSHSQB 28, 329 (1963). 231. N. Bouck and E. Adelberg, J . Bacteriol. 102, 688 (1970). 232. F. Bonhoeffer, 2. Vererbungslehw 98, 141 (1966). 233. W. Vielmetter, F. Bonhoeffer and A. Schutte, JMB 37, 8 (1968). 234. S. Barbour, JMB 28, 373 (1967). 236. T. Anderson, E. Wollman and F. Jacob, Ann. Inst. Pasteur 93, 450 (1957). 236'. J. Ou and T. Anderson, J . Bacteriol. 102, 648 (1970). 237. C. Brinton, Trans. N . Y . Acad. Sci. 27, 1003 (1965). 238. C. Brinton, P. Gemski and J. Carnahan, PNAS 52, 776 (1964). 239. R. Curtis and L. Charamella, Genetics 54, 329 (1968). $40. S. D. Silver, JMB 6, 349 (1963). 241. R. Herman and F. Forro, J. Biophys. 4, 335 (1964). 24% M. Monk, unpublished data. 2.43. J. Rosner, L. Kass and M. Yarmolinsky, CSHSQB 33, 785 (1968). 244. J. Rosner, Ph.D. Thesis, Yale University (1967). 246. M. Monk and R. Devoret, Ann. Inst. Pasteur 107, Suppl. 5, 163 (1964). 246. J. I. Tomizawa, cited by Rosner et al. (243). 247, R. Devoret, M. Monk and J. George, Zentralbl. Bakteriol. Parasitenk., Injeklionskr. Hyg. A b t . 1 : Orig. 196, 193 (1965). 248. M. Monk, Mol. Gen. Genet. 100, 264 (1967). 249. H. Ikeda and J. Tomizawa, CSHSQB 33, 791 (1968). 260. R. Howard-Flanders, R. Boyce and L. Theriot, Genetics 55, 1119 (1966). 261. K. Shimada, H. Ogawa and J. Tomizawa, Mol. Gen. Genet. 101, 245 (1968). 262. M. Monk, Molec. Gen. Genet. 106, 14 (1969). 263. J. George, C. R . Acad. Sci. 262, 1805 (1966). 264. A. Ryan, BBA 138, 140 (1967). 866. L. Leive, BBRC 21, 290 (1965). 266. V. Zgaga and D. Novak, JMB 29,125 (1967).

298

ERNEST BOREK AND ANN RYAN

D. Novak, S. Tkalac and V. Zgaga, J M B 29, 527 (1967). A. Kelner, J. Bacterial. 65, 252 (1953). L. Siminovitch, Ann. Znst. Pasteui 84, 265 (1953). M. Draculic, BBA 36, 172 (1959). E. Wainfan and E. Borek, I d . J. Radiat. Biol. 4, 327 (1962). E. Wainfan, L. Mandel and E. Borek, BBRC 10, 315 (1963). H. Michalke and H. Bremer, J M B 41, 1 (1969). S. Cohen and A. Chang, J M B 49, 557 (1970). 206. M. Monk and J. Gross, Mol. Gen. Genet. 110, 299 (1971). 266. R. Boyce and R. Howard-Flanders, 2. Veierbmgslehie 95, 345 (1964). 267. N. Otsuji, J. Bacterial. 95, 540 (1968). 268. Y. Imae, J . Bacterial. 95, 1191 (1968). 269. R. Cross and M. Lieb, Genetics 57, 531 (1966). 270. R. Cross and M. Lieb, Genetics 57, 549 (1966). 271. D. Noack, Biophysik 3, 118 (1966). 272. J. Cowlishaw and W. Ginosa, Virology 41, 244 (1970). 273. M. Butcher and M. Green, J M B 45, 433 (1969). 274. D. Goldthwait and F. Jacob, C . R . Acad. Sci. 259, 661 (1964). 276. E. Kirby, F. Jacob and D. Goldthwait, PNAS 58, 1903 (1967). 270. G. Stent, in “The Molecular Biology of the Viruses,” p. 206. Freeman, San Francisco, 1963. C77. W. Braun, “Bacterial Genetics,” p. 16. Saunders, Philadelphia, Pennsylvania, 267. 268. 269. 260. 261. 262. 263. 264.

1965. 278. E. Witkin, PNAS 57, 1275 (1967).

279. D. Sompolinsky, Y . Yiflah and M. Aboud, J. Gen. Viral. 2, 347 (1968). 280. F. Jacob and C. Fuerst, J. Gen. Microbial. 18, 518 (1958). 281. F. Jacob and E. Wollman, CSHSQB 18, 101 (1953). 282. K. Smith and P. Hanawalt, in “Molecular Photobiology,” p. 165. Academic Press, New York, 1969. 283. D. Vapnek and W. Rupp, J M B 53, 287 (1970). 284. J. Gross and L. Caro, Science 150, 1679 (1965). 286. D. Freifelder, J. Bacterial. 94, 396 (1967). 286. M. Ohki and J . Tomisawn, CSHSQB 33,651 (1968). 287. S. D. Silver, E. Moody and R. Clowes, J M B 12, 283 (1965). 288. J. Donch, J. Greenberg and M. Green, Genet. Res. 15, 87 (1970). 289. E. Witkin, Mutation Res. 8, 9 (1969). 290. R. Devoret and M. Blanco, Mol. Gen. Genet. 107, 272 (1970). 291. M. Inouye, J . Bacterial. 106, 539 (1971). 292. E. Calef and Z. Neubauer, CSHSQB 33, 765 (1968). 293. H. Strack, M. Kayser and S. Holder, Virology 42, 707 (1970). 294. Y. Hamon and Y. Peron, C . R . Acad. Sci. 265, 1433 (1967). 296. J. Homma and H. Shionoya, Jap. J. Exp. Med. 37, 395 (1967). 296. H. Shionoya, S. Goto and M. Tsukamoto, Jap. J. Exp. Med. 37, 359 (1967). 297. E. Siege1 and J. Marmur, J. Viral. 4, 610 (1969). 298. G. Medoff and S. Overholt, J. BacterioZ. 102, 213 (1970). 299. I. Orskov and F. Orskov, Acta Pathol. Microbial. Scand. 48, 37 (1960). 300. H. Uetake and S. Hagiwara, Virology 13, 500 (1961). 301. Y. Nishimura, M. Ishibashi, E. Meynell and Y. Hirota, J. Gen. Microbial. 49, 89 (1967).

LYSOGENIC INDUCTION

299

302. S. Chang, R. Toni, R. Gilden. M. Hatanaka and R. Huebner, J. Gen. Virol. 5, 443 (1969). 303. S. Chang, R. Gilden and R. Huebner, J. Gen. Virol. 10, 107 (1970). 304. F. Rapp, Annu. R e v . Microbiol. 23, 293 (1969). 305. N. Yamaguchi, M. Takeuchi and T. Yamamoto, Int. J. Cancer 4, 678 (1969). 306. T. Yamamoto, Jap. J . E x p . M e d . 40, 243 (1970). 307. D. Simkovic, J. Smida and V. Thurzo, Neoplasma 9, 9 (1962). 308. A. Sabin and M. Koch, PNAS 49, 304 (1963). 309. P. Black, W. Rowe and H. Cooper, PNAS 50,847 (1963). 310. P. Gerber, Virology 28, 501 (1966). 311. H. Koprowski and F. Jensen, Federation Proc. 26, 313 (1967). 312. H. Koprowski, F. Jensen and Z. Steplewski, PNAS 58, 127 (1967). 313. J. Watkins and R. Dulbecco, PNAS 58, 1396 (1967). 314. W. Burns and P. Black, J. Virol. 2, 606 (1968). 316. W. Burns and P. Black, Int. J. Cancer 4, 204 (1969). 316. M. Saito. T. Taguchi, K. Hasegawa, Y. Yoshida and D. Nagaki, Jup. J. Microbiol. 14, 512 (1970). 317. C. Monti-Bragadin, L. Conventi and C. Meloni, Boll. Soc. Ital. Biol. Sper. 46, 565 (1970). 318. L. Payne, Enuiron. Health 21, 366 (1970). 319. P. Gerber, Science 145, 833 (1964). 320. M. Fogel and L. Sachs, Virology 40, 1174 (1970). 321. M. Fogel and L. Sachs, Virology 37, 327 (1969). 322. P. Black, J. N a t . Cancer. Inst. 37, 487 (1966). 323. R. Dulbecco and M. Vogt, PNAS 50, 236 (1963). 324. J. Sambrook, H. Westphal, P. R. Srinivasan and R. Dulbecco, PNAS 60, 1288 (1968). 326. H. Westphal and R. Dulbecco, PNAS 59, 1158 (1968). 326. M. Green, A R B 39, 731 (1970). 327. P. Black, Annu. R e v . Mkrobiol. 22, 391 (1968). 328. W. Eckhart, Physiol. R e v . 48, 513 (1968). 329. R . Schlessinger, Advan. Virus Res. 14, 1 (1969). 330. E. Winocour, Advan. Virus Res. 14, 153 (1969). 331. P. Vigier, B~11.Cancer 57, 3 (1970). 332. P. Sarma, W. Vass and R. Huebner, PNAS 55, 1435 (1966). 333. R. Cassingena, P. Tournier, S. Estrade and M. Bourali, C. R . Acad. Sci. 269, 261 (1969). 334. R. Cassingena, P. Tournier, C. R . Acad. Sci. 267, 2251 (1988). 336. R. Cassingena, P. Tournier, M. Map, S. Estrade and M. Bourali, C. R . Acnd. Sci. 268, 2834 (1969). 336. J. Watkins, J. Cell. Sci. 6, 721 (1970). 337. L. Greenham and G. Poste, Microbios 3, 97 (1971). 338. R. Huebner, G. Todaro, P. Sarma, J. Hartley, J. Freeman, A. Peters, R.

Whitmore and C. Meier, Proc. 2nd Int. Symp. Tumor Viruses, Royaumont (1969), p. 33. 339. R. Huebner and C. Todaro, PNAS 64, 1067 (1969). 340. M. Gardner, J. Estes, H. Turner, R. Rongey and R. Huebner, unpublished observations, cited in Huebner and Todaro (839). 341. R . Huebner, G. Kelloff and W. Lane, unpublished observations, cited in Huebner and Todaro (339).

300

ERNEST BOREK AND ANN RYAN

34% B. Kramarsky, E. Lasfargues and D. Moore, Cancer Res. 30, 1102 (1970). 343. E. Lasfargues, B. Kramarsky and D. Moore, Proc. SOC.Exp. Biol. Med. 136, 777 (1971). 344. J. Charney and D. Moore, Natnrc (London) 229, 627 (1971). 346. D. Moore, J. Charnry, B. Kramarsky, E. Lasfarguca, N. Sarktlr, M. Brcnnan, J. Burrows, S. Sirsat, J. Paymaster and A. Vaidya, Nature (Londou) 229, 611 (1971). 340. R. Axel, J. Schlom and S. Spiegelman, Nature (London) 235, 32 (1972). 347. F. Gay, J. Clarke and E. Dermott, J . Gem Virol. 7 , 75 (1970). 348. A: L. Taylor, PNAS SO, 1043 (1963).

Recognition in Nucleic Acids and the Anticodon Families JACQUESNINIO Laboratoire de Biochimie du De'veloppement. Faculte' des Sciences de Park V I l . Paris. France

I. Introduction . . . . . . . . . . . I1 Rcmnrks on Rccognition . . . . . . . . A . Recognition from the Point of View of Thermocheniistry B. Oligonucleotide Associations in Water . . . . C The Replication of Nucleic Acids . . . . . D. Thc Formation of tRNA Secondary Structurc . . E . Structure of the Anticodon Loop . . . . . . I11 The Wobble Hypothesis . . . . . . . . . A The Approach . . . . . . . . . . B Subsidiary Hypothesis . . . . . . . . C . The Conclusions . . . . . . . . . D Limitations of the Wobble Hypothesis . . . . E . Evolutionary Implications . . . . . . . IV . The Missing Triplet Hypothesis . . . . . . . A . The Approach . . . . . . . . . . B . Hidden Dissymmetries in thc Coding Process . . . C . Further Conimcnts on Dissymmetry . . . . . D Diversity in the Binding Patterns . . . . . E The Sources of Ambiguity . . . . . . . F Why the Anticodons, and Not the Codons? . . . G Limitations of the Missing Triplet Hypothesis . . H Evolutionary Implications . . . . . . . V . The Experimental Evidence . . . . . . . . A . Suppression . . . . . . . . . . .

.

.

.

. . .

. . . . .

. . . .

. .

. . . .

. . . .

.

.

.

.

. . . . . . .

. . . . . . .

. . . .

. . . .

. .

. . . . . .

301 303 303 305 308 308 310 312 312 314 315 316 316 317 317 318 320 321 322 323 323 324 327 327 328 330

. . . . . . . .

E . Initiator and Noninitiator tRNA's F. A Fourth Base in the Anticodon? . G. Anticodons in the Third Column . VI . Discussion . . . . . . . References . . . . . . .

. . . . . . . . .

. .

. .

. .

. .

. . . .

. . . . . . . . . . . . . .

331 331 332 332 333 335

.

1 Introduction Fundamental ideas in molecular biology are easy to express. The existence of a genetic code. of regulatory mechanisms. of replication a t 301

302

JACQUES NINIO

the molecular level, once discovered gave the illusion of being selfexplanatory. In general, these ideas depict the functioning of the cell as that of a complex machine, describing how one element acts upon the others. Crick’s wobble hypothesis ( I ) departed somewhat from mechanistic explanations. It related some of the known and fundamental features of the genetic code to the imperfections of the translation machinery. A picture of codon-anticodon pairing was reached that seemed in agreement with most of the experimental results. Although the picture was clear, the reasoning through which it came into focus remained opaque for most of the readers of Crick’s article. The wobble hypothesis attempted to explain the general character of the degeneracy of the code. Although it put limitations on the anticodons that could be used, it did not say which anticodon should be used, and which should not. In each organism, there are fewer anticodons actually used than the maximum number allowed by the wobble. This restriction phenomenon cannot be explained by the wobble hypothesis alone. In general, the implicit additional assumption has been made that the cell is saving on tRNA genes. Thus, one needed two hypotheses, one to explain the degeneracy of the code, and the other to explain the restriction phenomenon. By logical arguments, one can show that this is unnecessary. Using premises less constraining than those of the wobble hypothesis, one can build a theory wherein the restriction phenomenon and the degeneracy of the code are two related consequences of a same phenomenon : the physical ambiguity of codon-anticodon recognition ( 2 ) . Several proposals have been made concerning the details of codonanticodon interaction (34).The present article is mainly concerned with the wobble hypothesis and the missing triplet hypothesis that deal with the presence or the absence of the various potential anticodons. Emphasis is put on the reasoning whereby a theory is constructed, and fitted with the existing data. The core of the article is constituted by Sections I11 and IV, in which the ideas and the logic of the approaches are exposed with as few interferences by technical discussions as possible. Section I1 presents part of the background knowledge required for the formulation of the physical assumptions that underlie the theories. Section V gives the experimental evidence through which the predictions of the theories can be checked.l I n o r d e r to facilitate the reading, the anticodons are written with an orerbar. t.hus XYZ, and the regions of the tRNA molecule are denoted as in Fig. 5.

303

RECOGNITION IN NUCLEIC ACIDS

II. Remarks on Recognition A. Recognition from the Point of View of Thermochemistry Let us consider two molecular species, A and B, that can form an association AB. At constant temperature and pressure, an equilibrium is reached for which one particular thermodynamic function, Gibbs free energy, is minimum. Writing this condition leads to the equation

where C(A)’s are concentrations in moles/liter and u is the free energy liberated when 1 mole of AB is formed from 1 mole of A and 1 mole of B. Writing the second member of Eq. (1) in the form of a constant K gives the law of mass action, where K is the equilibrium constant. Now, imagine that A can associate with B, and B,, with corresponding free energies u1and tk. Applying Eq. (1) twice yields the ratio of C(AB,) to C(AB,) a t equilibrium:

If c ( B , ) and E(B,) are the total concentrations of B, and B, (C(B1) = C(Bl) + C ( A B 1 ) ) ,and if K , K,,one can deduce that

>

In terms of recognition processes, the molecule A can recognize both B, and B?, but it displays some specificity toward the substrates B, and B?. If initially, both B, and B, are present in large excess, and in equal amounts, C(AB,)/C(AB,) will be close to exp - [ ( u , - u , ) / k T ] . A difference uI - u2 of 3 kcal/mole at 37°C will correspond to a specificity of 0.5%, and a difference of 4 kcal will mean that AB, is present 670 times as often as AB,. As shown by Eq. (3), the apparent specificity in a real process can be ) . trivial is masked or enhanced by changing the ratio ~ ( B l ) / ~ ( B ,Less the remark that specificity decreases when the absolute values of the concentrations of the substrates B, and B, decrease simultaneously, C (A) remaining large. When the concentrations of substrate become vanishingly small, and if I<, and K , are large enough, A may become “saturating.” Then, C (AB,)/C(AB,) approaches (B,)/c(Bz) whatever the difference u1 - u2.Provided the associations are possible, they are formed proportionally to the concentrations of the substrates. Recognition becomes an all-or-none process. Thus, if equilibrium equations can be

304

JACQUES NINIO

applied to codon-anticodon recognition, a factor that would reduce the number of tRNA molecules per active ribosome may increase all the errors of translation. Note that in poly (U)-directed polypeptide synthesis the ratio of isoleucine/phenylalanine incorporation increases when the total concentration of tRNA decreases (6, 7 ) . At equilibrium, the rate of the reaction expressed as the net production of AB per unit time is zero. On a microscopic scale, both the formation of AB and its dissociation into A and B continue a t equal rates. Thus, an association AB has, on the average, a certain lifetime T . There is no thermodynamic relation between u and T . Knowing (8) the rate of protein elongation on the bacterial ribosome (10-50 amino acids per second) and the thermodynamic parameters of tRNA binding in the presence of mRNA will not help in answering the following question: During the 1/50th of a second that a codon has to choose the tRNA, how many associations are tried? A thousand, a hundred, or a single one? Eigen has stated that four G C pairs in codon-anticodon associations would make translation too "sticky" (9),implying that the lifetime T of the codon-tRNA complex is rate limiting, but this is far from being demonstrated. At equilibrium, the lifetime I is also equal to the average time during which random collisions between A and B successfully lead to the formation of AB. Why only a certain fraction of the collisions gives rise to the association is a problem in reaction kinetics. When the underlying mechanism is simplest, i.e., when one molecule of AB is formed during an encounter between one molecule of A and one molecule of B, the rate of formation of AB in the microscopic process takes the form

-

vf

=

kr exp

- (uf/lcT)C(A)C(B)

(4)

where u' is the activation energy corresponding to the pathway of the reaction. In the simplest case, the rate of dissociation takes the form: yd =

kr exp - (ud/LT)C(AB)

(5) uf and ud are related by u = uf - ud. Hence, the lifetime of AB does not depend upon the advancement of the reaction. It is the same initially and at equilibrium. NOW,if A can associate with B, and B, and no association is initially present, the initial ratio of AB, to AB, is

In the case of codon-anticodon recognition, we can assume that the recognition site on the ribosome is equally accessible to all tRNA's and

RECOGNITION IN NUCLEIC ACIDS

305

that both the codon and the anticodon are initially in a configuration suitable for the association. Thus, we may neglect the activation energies and the influence of the k,.’s. If the formation of the association is ratelimiting in protein synthesis, recognition has the character of an all-ornone process. In general, one thinks of polypeptide elongation in terms of Eq. (4). As soon as a codon-an ticodon association is formed, elongation proceeds. However, it is far from obvious that the ribosome should function in this way. One can imagine instead an oscillatory mechanism by which the ribosome checks at intervals of time 0 the occupancy of the A site. Elongation proceeds only if the check is positive. I n this case, the important parameter controlling reliability in translation is 0/7. When this parameter is large, the discrimination is described by Eq. (2), and the rate of polypeptide elongation is related to the equilibrium constants. When the parameter is small, the rate of polypeptide elongation is related to the rate constant of Eq. (4) , and the fidelity in translation is described by Eq. ( 6 ) . Intermediate situations are conceivable. A factor that would modify ribosomal kinetics by changing the ratio 0 / T would increase or decrease accordingly all the errors of translation. A study of the rate of polypeptide elongation as a function of tRNA concentration should provide invaluable information on the mode of action of the ribosome. Unfortunately, the answer is not available. All the kinetic studies are made under conditions where chain initiation is the rate-limiting step so that “pure elongation kinetics” have never been determined.

B. Oligonucleotide Associations in Water I n stating that the two strands of a DNA molecule are complementary to each other, geometric and energetic criteria are mixed. The symmetry and the superposability of Watson-Crick base pairs (Fig. 1) make possible the construction of a double-helical model in which the geometry of the sugar-phosphate backbone can, in principle, be extremely regular, whatever the sequence of the bases on one strand. This geometrical observation does not imply that the base pairs are, by themselves, energetically favored. While the Watson-Crick base pairs can be found in a number of situations (hence, they do correspond to some energetic minimum) , other types of pairing between nucleotides have been observed, for instance, in the physical studies on homopolynucleotides and their associations. In this case, the geometry of the base pair may have nothing to do with Watson-Crick geometry, since parallel pairing can occur. Observations of a different kind are related to associations between

306

JACQUES NINIO

A

0

FIG.1. Geometry of a nucleotide plateau in a double-stranded nucleic acid. The two paired bases are chemically variable. In a purine, the &membered cycle is numbered in the clockwise direction. The interrupted lines symbolize hydrogen bonds, while the thick lines indicate the glycosyl bonds between the C-1 of the sugar and the base. In the double-helical structure of DNA, the chain carrying the base has the 5’ end below the plane of the figure. The 3’ OH is above this plane. The Watson-Crick pairings are symmetric. This means that when the tri-igle 0,AB is constructed for a pair A - U or G-C, it turns out to be isosceles with OAB = OBA. Furthermore, the Watson-Crick pairs are superposable, i.e.. the triangle OAB constructed with A . U is equal to the triangle OAB constructed with G.C. From the properties of symmetry and superposability, it follows that two polynucleotides of arbitrary sequence, but complementary, can associate to form an extremely regular double helix. More precisely, the sugar-phosphate backbone may form a strictly periodic structure, while the base pairs can he interchanged. This is of course approximate, and the double helix may be distorted owing to the action of several factors. I n particular, the interactions of stacking between consecutive plateaux may be reflected in deviations from the standard three-dimensional structure. If one wishes to know whether the geometry of a base pair is not too different from Watson-Crick geometry, one must look a t its triangle OAB. A first indication is given by the distance AB. I n some of the pairs considered by Crick, such as U . U or U.C. this distance is smaller than the standard distance of 11A.

+

+

oligo or polynucleotides in which the sequences of the bases are nearly complementary. The studies bear on whether the presence of noncomplementary pairs is tolerated by an otherwise complementary structure and on whether it leads to a measurable “defect” by energetic or spectroscopic criteria. A general conclusion is that, provided noncomplementary

RECOGNITION IN NUCLDIC ACIDS

307

G . U or 1.U or 1.A pairs are not clustered, the destabilizing effects are rather small (10-14). Let us try to describe the association of two oligonucleotides, A and B. I n the free state, A may adopt several configurations in various amounts. In some cases, one of these may be largely predominant. Most of the free rotations are located in the phosphate backbone -CH2-0-PO2-O-, which contains five degrees of freedom (however, at both ends, the rotation angles take discrete values). Thus, it is believed that the configuration of a small oligonucleotide will be dominated by the interactions of stacking between the bases. However, a few dinucleotides may adopt an unstacked structure in solution (15).As the oligonucleotide gets longer, the backbone assumes greater importance, for if the position of stacking of the seventh base over the sixth is optimized, this will lead to a rearrangement of the position of the sixth sugar, and, as a consequence, all the other sugars have to rotate. There is no reason why the configuration of A in the associated state should be the same as in the free state. On the contrary, the structure of the associated state depends upon the exchange of hydrogen bonds between the bases of A and the bases of B. This energetic contribution plays no role in determining the structure of the reactants in the free state. The energetic balance between the two states may comprise several terms: e.g., the “horizontal” energy corresponding to the exchange of hydrogen bonds; the ‘(vertical” interactions of stacking between bases on the same molecule (which will he less favorable than in the free state) ; the energy of deformation of the sugar-phosphate backbones ; the “diagonal” energies of interaction between bases of A with bases of B that are not in register with them. These additional contributions may be favorable or unfavorable. Furthermore, an entropy term must be considered, especially if one of the oligonucleotides, being small, has no definite structure. [For a quantitative discussion of the various terms in codon-anticodon recognition, see Eisinger et al. (16, 17) .] The process of association itself is complex. The base pairs do not form simultaneously. The lifetime of the first base pair formed is about to lo-” second. In general, it dissociates before a second pair can be formed. But once a “nucleus” of at least two or three base pairs is formed, the association proceeds like zippering. The kinetics of association of oligonucleotides of 10 to 20 bases is well documented (18, 19). Relaxation-kinetic experiments using the temperature-jump (T-jump) method, applied to tRNA, provide information on the kinetic behavior of associated regions containing 4-7 base pairs (20, d l ) . Trinucleotides do not associate in water (t?2),but their binding to macromolecules is currently being studied (23-28) .

308

JACQUES NINIO

C. The Replication of Nucleic Acids Replication makes use of a double recognition process. Let us consider a T residue on a template being replicated as an A or G residue. Elongation of the primer occurs only if the reactive phosphorus on the A or the G is in a suitable position. I n fact, there seem to be many positions allowing nonenzymatic elongation. Thus, in addition to 3'-5' bonds, a large majority of 5'-5' or 2'-5' phosphodiester linkages is found ( $ 9 ) . The statistical distribution of the various types of bonds need not be the same when T is read as an A or as a G. However, if one particular pathway (for instance the 3'-5' pathway) is predominant, owing to the presence of a replicase, the way the new base pair is superimposed over the preceding one becomes crucial for elongation. Thus, even if recognition a t the level of hydrogen-bonding between bases is inaccurate, replication can be reliable when an enzyme checks the external geometry. Similarly, one may think that an incorrect codon-anticodon pairing will shift the amino acid from the position required for the formation of the peptide bond. However, I would be surprised if the tRNA structure is rigid enough to transmit accurately a small deformation in the anticodon region up to the amino-acid region.

D. The Formation of tRNA Secondary Structure This is a recognition process of still another type. There are several ways in which small portions of a tRNA sequence can associate. Once an association is formed, the possibilities of base-pairing between other parts of the molecule are restricted. The free energy of the first association is partially compensated by the loss of entropy in the whole molecule. Even if the first association is correct, the final state may be inaccessible. For instance, if nucleation starts by the pairing of the 3' and 5' ends, the molecule acquires the topological properties of a closed curve and the final tertiary structure may not be accessible even if all the correct base-pairings are realized (SO). More generally, when one or two associations are realized, the final state may be accessible only across a high energy barrier. Thus, the formation of the structure must be considered dynamically. The final structure is generally stable ; i.e., when one portion opens, the reassociation occurs rapidly, before a similar' event happens in some other part of the molecule. Since it is the whole structure that counts, the formation of the base-pairing pattern is not the simple result of adding elementary recognition steps. This remark may explain why the complementarity rule is not always observed in the four stems of the cloverleaf structure. The amino acid stem of wheat germ tRNAphecontains the most unusual section:

RECOGNITION IN NUCLEIC ACIDS

69

309

-

67

C-A-C G-G-G 4-6

It turns out that this “bad” association might be necessary for the realization of the cloverleaf pattern, for if CAC was replaced by CCC or CUC, the following associations, incompatible with the cloverleaf: 69

-

65

c-c-c-u-c

-

or

G-G-G -A-G

18

22

70 + 62 C-C-U-C-U-C-G-C-A G-G-A-G-A-G-C-G-$ 19 e 27

could be formed, leading to an incorrect structure. There are well-known cases of dimorphism in tRNA’s. The replacement of a non-complementary base pair by a complementary one in E. coli tRNATPp(31) suppresses the dimorphism. Yeast tRNALeUexhibits a well-known case of dimorphism. Two subspecies have been sequenced. The main difference is in the last base pair of the anticodon stem, A * $ in one case and A . C in the other (38). Comparative studies of their dimorphic properties are not yet available. Whatever the reasons for the presence of noncomplementary base pairs in tRNA, their proportion in the helical regions is relatively high, about 6%. I n 28 tRNA’s of known sequence, involving 579 base pairs, there are 30 GeU’s, 4 C-A’s, one G * A and one U.U. These pairs are scattered in 14 out of the 21 positions available for base-pairing in the cloverleaf structure. The most favorable environments for G. U pairs are: G C IU GI, C G

G C IU GI U A

and

G C IG U G CI

In two cases (yeast tRNAVa’and T,-coded tRNALeU),the hU stem consists of an association of the three base pairs drawn on the left. The three associations represented start with

1: I:

In 60% of the cases, a U - G is preceded by a G - C (Table I). I n this situation, the guanines are diagonally stacked, which goes against the observed tendency to stack vertically, as shown by the relative occurrence of each of the three possibilities:

310

JACQUES NINIO

TABLE I ENVIRONMENT OF U . G PAIRSIN tRNAo

1"

U G '117

1;

3

6

1: 1:

3

7

3

9

1; 1::

y

3

1;

a The figures indicate the number of occurrences of each association in a statistical set comprising 28 tRNA sequences. The difference between the sums of the figures of the first and second row indicates that when a U G pair is terminating a stem, the U is at a 3' end.

One of the reasons for finding the

1: I:

association could be a bad stacking of the dinucleotide A-C as suggested by the comparison:

1"

U G '117

1;

:I25

These statistical observations suggest that during the formation of tRNA secondary structure, a dinucleotide G-U will recognize almost as easily the dinucleotide G-C as the complementary dinucleotide A-C. It should bc stressed that thc rules on the environment of U * G in tRNA are in good agreement with the observations on the terminal stems of 5 S RNA. On the other hand, all the models proposed so far for 5 S RNA disagree with thcsc rules in the base-paired regions except in thc terminal stems.

E. Structure of the Anticodon Loop Model-building studies (33) suggest that a hairpin structure with a three-membered loop can exist in nucleic acids. Indeed, one finds in the cxtra-loop region of tRNA such situations as: U C C C G U G G G C

U

(Very similar hairpins are likely to exist in Escherichiu coli and Pseudom o m 5 S RNA.) A segment of this kind, isolated from yeast tRNAse",

has been studied by T-jump experiments ( 3 4 ) . Its stability is high, although one cannot be sure that the G - C next to the loop is not

311

RECOGNITION I N NUCLEIC ACIDS

“breathing” more than usual ( 3 5 ) . Thus, thc constraints on a looped structure due to the sugar-phosphate backbone are so loose that they allow the presence of a three-membered loop. The anticodon loop contains four more nucleotides; with each of these, five degrees of freedom are added to the sugar-phosphate backbone. Thus the backbone of the anticodon loop has in principle 20 more degrces of freedom than that of a three-membered loop, which, physically, can exist. Hence, it would be difficult to gct some idea of thc structure of tlic anticodon loop without looking at the bases, and trying to figure out their interactions. Unfortunately, the base sequences in these loops can be widely different (Fig. 2), the only constancy being that of the second residue, a uracil (or a y?). Our ideas on how the dinucleotides U-U, A-C, etc. stack are rather limited (15,36, 37). Besides, it would require a good knowledge of the nucleic acid-water interactions to understand the rules of formation of the structure and propose a model. There is no way to decide whether the bases of the anticodon point inward or outward. Arguments based on what the anticodon loop geometry should be for translation to proceed did not result in any definite progress, and the best we can say a t present comes from direct experimental evidence. The binding of oligonucleotidcs to the anticodon loops of several purified tRNA’s has been studied by equilibrium dialysis (24, 25) and by spectroscopic techniques (IS). Thc binding constants for triplets complementary to the anticodon arc about lo00 or 10,000. Since the association of two complcmcntary triplets in water is not observed, the anticodon loop must have a special, rclativcly fixed configuration, allowing the anticodon to be in a geometry suitablc for recognition. Moreover, two tRNA’s with nearly complcmentary anticodons can form a very stable association, the binding constant being 200 times larger than in the previous case (17).If all the anticodons have a similar geometry,

G * C Cm A U A CAU

A-JI C A U A’

E coli

E coli tRNAMet

tRNANet

CA !U

C . 0 J I U U G* GAG

A - C

J I

c

U

m’G

~‘CAA

E coli K12 “renaturable” tRNAp yeast N A P

FIG.2. Primary structure of four nntirodon loops. It is often assumed that all tRNA’s have a uniform anticodon loop structure, except initiating tRNA’s. An inspection of the primary sequences of the four anticodon loops represented here (32, 72-74) provides little support for such a dichotomy.

312

JACQUES NINIO

TABLE I1

BINDING OF OLIGONUCLEOTIDES TO THE ANTICODON LOOPOF Eschcrichia wli tRNAI*e (37).

AN

Approximate association Oligonucleotide A-U-C A-U-A A-U-U A-U-G A-U-C-A A-U-A-C A-U-A-A A-U-C-C A-U-C-G A-U-G-C A-U-C-U U-C-A-G

constant 7,100

-

120,000

-

20,000 130,000

-

50,000 6,400

a The anticodon loop has the sequence C-U-G-A-U-A*-A. A dash means that the corresponding association constant is <2500. Reprinted by permission of the American Chemical Society.

the result suggests that this geometry is invariant upon a transformation from thc anticodon to its oomplcmentary structure. Then, its sugarphosphate backbone may be approximated by a geometrical curve that can be the transform of a helix by an orthogonal affinity (a straight or a “leaning” helix). Another piece of information comes from the study of the binding of tetranucleotides to the anticodon loop. The results show, for both initiator and noninitiator tRNA’s, that one base adjacent to the anticodon can participatc in cooperative binding of the anticodon loop to complementary or nearly complementary tetranucleotides. In all the cases studied so far, this base is on the 5’ side of the anticodon. The one on the 3’ side (sometimes considered as the “fourth base” of the anticodon, e.g., Section V, F) does not contribute to tetranuclcotide binding. Some of tlic rcsults arc displayed in Table 11.

111. The Wobble Hypothesis

A. The Approach N and M being any of the four bases A,U,G,C, the following rules are observed in the codon-amino-acid catalog: a. NMU and NMC always code for the same amino acid. b. NMA and NMG generally code for the same amino acid; UAA

RECOGNITION IN NUCLEIC ACIDS

313

and UAG are both termination triplets; AUA codes for Ile, while AUG codes for Met; UGA is a termination triplet, while UGG codes for Trp. c. NMU, NMC, NMA and NMG code for the same amino acid when N is G or C and M is G or C. d. NMU and NMG code for different amino acids when N is U or A and M is U or A. Knowing from physical studies on double-helical nucleic acids in solution that two G - C pairs are not less stable than three A-U’s, one can explain the features c and cl. One may suggest that two A - U pairs are not stable enough to allow a codon-anticodon pairing, hence rule d showing that the third base of the codon has to be specified; two G - C pairs are stable enough, hence the type of degeneracy observed in c, where the third base need not be specified. Even if unnecessary, the third base actually exists, and one is led to suggest that it has a somewhat smaller influence on codon-anticodon pairing than the others. The wobble hypothesis attempted to give a description of codonanticodon pairing that could show up this type of feature, and explicitly explain the rules a and b. Hence, i t was shown how U and C on one side, and A and G on the other could be equivalent in the third position of the codons. Equivalences of this kind were previously suggested by Woese (38) and by Eck (39). Woese proposed that A with C and G with U could be equivalent in the second position, and U equivalent to C in the third position. Rules a and b were first suggested by Eck. The wobble hypothesis was more than a proposal. It used a careful, deductive logical argumentation of a kind not uncommon in physics. Starting with a number of facts and a general theoretical framework, one looks for the special theory that remains within the framework and is simultaneously compatible with the facts. I n the general framework, codon-anticodon pairing is undcrstood as an all-or-none process. There are rules for pairing applying to each position of the codon-anticodon association. Pairing in one position is not influenced by the bases in the other positions. The associations in the first two positions are restricted to Watson-Crick pairs. Finding the pairing rules in the third position is the object of the special theory. Let us choose an arbitrary rulc. G in the first position of the anticodon pairs with A or U in the third position of the codon; C pairs with G or C. If such were the case, the glutamine codon CAG would be read by a CUG anticodon that would also be able to read the histidine codon CAC. The glutamine codon CAA and the histidine codon CAU would be read by GUG. Then, the decoding process would not permit the gluta-

-

314

JACQUES NINIO

mine codons to be distinguished from the histidine codons, which is contrary to the evidence. Thus, the arbitrary rule is wrong and one can try another arbitrary rule. It turns out that without additional assumptions, many such rules are compatible with the code. A subsidiary criterion must be used to restrict the number of solutions.

B. Subsidiary Hypothesis 1. The pairing in the third position makes use of two antiparallel bases exchanging a t least two hydrogen bonds. The acceptor and donor groups are the same as those considered in the Watson-Crick pairs, in their usual tautomeric form. I n addition to A,U,G,C, the base-pairing possibilities of inosine (I) wcre examined. From physical arguments, Crick showed that A-U, G - C , Anticodon

Codon

J FIO.3. The "positions" considered in the wobble hypothesis. Here, the geometry clements of a plateau are reduced to their simplest expression. On thc left, the base is represented by the corresponding glycosyl bond. The cross indicates the C-1 carbon of the sugar; i t is the same as point A in Fig. 1. Then, the position of the + base is unequivocally determined. The position of thc glycosyl bond for the minus base is a guanine, forming base depends upon the base pair considered. When the a pair with U- or C-, the glycosyl bond comes at the place indicated on the right by G+.U- and G+.C-, respectively. Since the pair G * U is not symmetric, G+*U-is not found a t the same place as G-.U+. Physically it is the same pair. It is not cquivalent, for several reasons, to have G on the codon, or on the anticodon; if the codon has a rigid structure on the ribosome, the anticodon deformation required for the use of U+*G-differs from that required by the use of G+*U-. The wobble theory admits that the four positions on the right of thc figure arc effectively used in codonanticodon association, and not the others. Note that the bases are considered to be in their usual tautomeric form. This assumption can be criticized (3,.@a). On the other hand, whatever the tautomeric configuration of I and A, the 1.A pair cannot bc in the standard geometry.

+

+

315

RECOGNITION IN NUCLEIC ACIDS

G * U , C- U , U - U , I.U, I.C, 1.A could bc taken into consideration, while A*A, G - G , C.C, A - C , A.G and I - G should be left out. 2. When two base pairs have the same “geometry,” they are either both used, or both excluded. Thc geoinctry of base pairs is outlined in Figs. 1 and 3. Thus, either all the Watson-Crick pairs are used, or none of them is used. Conversely, thc use of U - G with U on the codon does not imply tlic use of G - U with U on the anticodon. The thirtccn associations taken into consideration are spread over seven positions.

C. The Conclusions The rules relative to the pairings that occur in the third position now state which positions of Fig. 3 can be used, and which positions cannot. C; * C: = Sincc there arc scvcn positions, wc have to consider C: 127 possible systems of rules. Most of thesc can be easily rejected if one admits that (a) all four bases A,U,G,C on the codons must sometimes be read; (b) the code must in certain cases distinguish between U and C on the one hand, and A and G on the othcr. Thcii, only sevcn systcms of rulcs remain, thc most interesting of which is given in Table 111. In other systcms, U - G or G.U.may be forbidden. Thus, although compatible with the code, they do not provide an explanation for thc degcneracy rulcs a and b of Section 111, A. On the other hand, if Table I11 is correct, predictions can be made concerning the codons not yet assigned to amino acids. Table I11 can be interpreted geometrically. The positions allowed by the wobble hypothesis are clustered in a certain portion of Fig. 3

+ + -

TABLE I11 PAIBING AT THE THIRD POSITION OF Base on the anticodon

U C A G

I From Crick (1).

THE

CODON’

Bases recognized on the codon

316

JACQUES NINIO

around the standard position. Hence, the formulation that the general features of the degeneracy of the genetic code can be explained if there is a certain amount of play, or “wobble,” in the third position of the codon-anticodon association.

D. limitations of the Wobble Hypothesis One specific tRNA can bind to more than one codon, and this observation has contributed to the formulation of the wobble hypothesis. It was known that total E. coli tRNAPhecould respond to UUU ( 4 0 ) ,thus at least one isoaccepting tRNAPhe responded to both UUU and UUC. IGC was considered to be a possible anticodon of yeast tRNAAIn (41). This suggested an experimental way of verifying the wobble. After the isolation of purified tRNA species, the binding patterns could be studied, and Table I11 could be checked. It turned out that most of the anticodons sequenced initially started with G, U or I, as if the cell was always using the minimum number of anticodons that the wobble would require. Various assumptions can be added to the wobble hypothesis in order to try to explain the restriction phenomenon. None of them appears convincing. I n particular, economy arguments do not hold, since several genes may correspond to the same anticodon [there are at least seven genes for tRNATYrin yeast (42) ] , since one activating enzyme can charge several isoaccepting tRNA’s with various anticodons (although not with the same efficiency), and since a complex enzymatic system is required to insert inosine into the anticodons. I will try to show in Section IV that one can build a theory of codonanticodon recognition in which the anticodon restriction phenomenon can be deduced without further assumptions. I n doing so, the premises and the approach of the wobble hypothesis need to be reconsidered.

E. Evolutionary Implicat ions In considering the relationship of the wobble hypothesis to the origin of the code, two attitudes can be adopted. In the first, the ordering in the codon-aminoacid catalog is not due to the peculiarities of codonanticodon recognition. For instance, it has been suggested that this ordering reveals a stereochemical correspondence between amino acids and anticodons in prebiotic conditions (&a). Then, the wobble mechanism would be a late invention of the cell providing a better adaptation of the translation apparatus to the general features of the .code’s degeneracy. In the second attitude, the degeneracy of the code is a consequence of the wobble. A wobble in the third position would be a necessary feature of translation because the structure of the anticodon in a seven-

RECOGNITION IN NUCLEXC ACIDS

317

membered loop is necessarily, for physical reasons, more flexible in the wobble position than in the others (43). Crick’s attitude falls in this category. H e admits that if the wobble theory of codon-anticodon interaction is correct, the maximum number of things that can be coded in a positive way is 32 (say 31 amino acids and a chain terminator), not 64. Therefore, the wobble is some kind of property intrinsic to the threedimensional structure of interacting nucleic acids. Also implicit in this attitude is the fact that the reliability in the reading of the first two positions needs no explanation. Thus again, accurate recognition and replication of nucleic acids may have preceded the existence of a genetic apparatus ( 4 4 ) .

IV. The Missing Triplet Hypothesis A. The Approach I n 1967, P. Claverie made some energy calculations on DNA in which he attempted to determine the preferential position of stacking of every base pair upon another, respecting the continuity of the sugarphosphate backbone. The position of minimum energy appeared dependent upon the chemical nature of the bases. However, the turn angle of 36” per base pair found in the B form of DNA came out as an average over the various stacking positions (46). This result led me to reconsider the wobble hypothesis. If Claverie’s computations were qualitatively correct, it would be difficult to understand how a n accurate recognition could be achieved in the first two positions of the codon-anticodon association. Let us assume that there is an enzymatic system acting as a stereochemical filter that checks the geometry of the association. In several cases, two complementary triplets should have a tendency to adopt a nonstandard configuration (since the standard configuration is described as only an average). On the other hand, some noncomplementary associations should be able to pass through the filter, mainly the associations containing the two pyrimidines C and U in register. From a model-building point of view, it is quite possible to insert a C opposite a U in a Watson-Crick double-helix without distorting the sugar-phosphate backbone. In that case, C and U are in register, but do not exchange hydrogen bonds. Hence, a discrimination of “good” and “bad” associations based only on the geometry of the complex would lead to errors in translation. In addition, the energy of interaction between the triplets is not restricted to the horizontal contributions (e.g., Section 11, B ) . Since the other contributions may not be negligible, there will be a number of cases where the complementarity rule might be blurred. This

318

JACQUES NINIO

is consistent with the observations on base-pairing rules in tRNA (e.g., Section 11, D). If ambiguous reading is physically possible even in the first two positions, let us work out the consequences of an arbitrary misreading in which, for instance, the association

:I I!

is formed instead of

1: %I

Such a potential misreading implies, in practice, that when -the valine codon GUU appears on the ribosome, a tRNAG”Ywith an ACC anticodon may compete efficiently with the tRNAVn’species for codon binding. But if that particular tRNAG’y does not exist, the potential misreading will not show up. Hence one can attempt to put forward a theory that tries to explain the presence or the absence of the various anticodons by combining two ideas: (a) ambiguity is not restricted to the third position; (b) ambiguity not compatible with degeneracy is not normally visible because there is a restriction on the anticodons. (Why the anticodons, and not the codons? Section IV, F.)

B. Hidden Dissymmetries in the Coding Process The codon and the anticodon do not play symmetrical parts. Some important dissymmetries are related to the mechanism of translation. Let us compare two translation processes, one real and the other imaginary, I n the imaginary process, messenger RNA is in large excess and one tRNA is introduced at a time. This tRNA binds on the codon for which it has the largest affinity. Then, an induced-fit mechanism strengthens the binding. When another tRNA is introduced and chooses an adjacent codon, a peptide bond is formed. In the actual process, one codon a t a time appears a t the proper position on the ribosome, and we want to know which tRNA among the various acceptors will bind to that codon. The same “ambiguity” will manifest itself differently in the two processes. Taking the example of the preceding section, misreading means that the asparagine tRNA with a GUU anticodon will bind either to the asparagine codon AAC, or to the threonine codon ACC, if we are in the situation of the first process. The same misreading in the second process means that in the presence of the valine codon GUU, a tRNAG’y with an ACC anticodon may bind as well as a tRNAVal with anticodon AAC. From this kind of dissymmetry, a practical rule can be deduced. A G - U pair (where the first letter indicates the base on the codon) is not equivalent to a U - G pair. When U is on the codon, the error consists in

319

RECOGNITION IN NUCLEIC ACIDS

a substitution of U.G to U * A pairing. When G is on the codon, the alternatives are G * Uand G'C. Since G - C is stronger than A-U, misreading will more frequently involve U - G pairs than G-U's. Thus, if the isoleucine codon AUA has a corresponding anticodon beginning with U, such an anticodon will not necessarily perturbate the translation of AUG by a CAU anticodon. This, perhaps, is the reason why rule b of Section 111,A is less general than rule a. Now, let us take again our arbitrary - example - of misreading where the GUU codon is read by both AAC and ACC. For some reason, the - anticodons AAC and ACC do not correspond to the same amino acid. Then, the following argument can be made: -

1. The anticodon ACC does not exist. 2. The codon complementary to the missing anticodon is GGU. 3. GGU will be read by a noncomplementary anticodon. 4. Among the various possibilities, the best candidates -correspond to the formation of a pair G - U or U.G, thus GCC, AUC and ACU (momentarily, we suppose that we do not know the code). 5. Since U -G and G - U are not equivalent, the first solution is the best. Suppose ACU is chosen. This implies that AGU and GGU code for the same amino acid. The ambiguity relative to the reading of GUU is transformed into a degeneracy where AGU and GGU are synonyms. With the choice of the anticodon ACU, a new ambiguity is created

G IG U

U GI A

with

G C IG C l U G

for the association on the right contains one more G - C pair than the association on the left. Thus, by suppressing in this way the misreading of GUU, a misreading of GGU is created, in addition to the GGU-AGU degeneracy. 6 . The anticodon GCC exists. 7. The codon complementary to GCC is GGC. 8. Thus, GGC and GGU code for the same amino acid. This example shows how an ambiguity in the second position that is not transformed into a degeneracy in this positon can give rise to a degeneracy in another position that is not arbitrary. I n this particular example, we had ambiguity in the second position + degeneracy in the third position. We could have constructed a second example in which an ambiguity in the second position was revealed as a degeneracy in the first position. However, there is no rule by which the second example can be con-

320

JACQUES NINIO

structed as an obligatory counterpart of the first, even if the codonanticodon association is perfectly symmetric. The arguments presented in this section give a picture of the relationship between ambiguity in translation and degeneracy in the code. Physical ambiguities in the translation process ultimately correspond to degeneracies in the genetic language, but not necessarily in the same positions. Hence, the observation that degeneracy is concentrated on the third position does not imply that the ambiguity is concentrated exclusively on that position. The study of the positional shifts between the two factors should reveal the constraints in the genetic language that are not purely due to codon-anticodon recognition.

C. Further Comments on Dissymmetry 1. FULLER-HODGSON MODEL

Although the Fuller-Hodgson model for the anticodon loop (49) does not appear physically justified, there is a very interesting aspect in the author’s argumentation. When a loop is added a t one end of a perfectly regular double-helical section of a nucleic acid, one side is connected to a 5’-phosphate, and the other to a 3’-phosphate. This dissymmetry may show itself in the three-dimensional structure of the loop, which in turn may imply that translation must be ambiguous on precisely the third position. The wide appraisal the model received owed much to its being in line with Crick’s hypothcsis and to giving a very mechanistic answer to the question: Why is the wobble on thc third position? It was incorporated in most, but not all, subsequent models of tRNA. Yet, anyone who tries to build a tRNA model with atomic components has the opportunity to appreciate how little we can say on the precise structure of loops. 2. BINDING EXPERIMENTS

In most of the recent experiments on codon-anticodon recognition, a purified tRNA species is isolated, and its binding to ribosomes in the presence of a few triplets is examined. This type of in vitro experiment gives information on the reliability of translation in the imaginary process of Section IV, B. Several ambiguities are observed that are not in agreement with the wobble hypothesis. I n order to explain thcsc ambiguities, the authors suggest that there are special suppressor tRNA’s with definite functions in the normal cell (46). This interpretation is unnecessary. I n order to measure the reliability of translation in the real process one must see how various tRNA species compete for the same codon. Fortunately, the deciphering of the code was performed that way.

RECOGNITION IN NUCLEIC ACIDS

321

3. PRIMITIVE RECOGNITION I n order to test the predictions of the hypothesis that the amino acids were recognized by the anticodon loop region in primitive tRNA, computations were made (47)on the relative affinities of glycine for various dinucleotides. Actually, the “specificity” that can be deduced from the comparison corresponds to a proccss of revcrse translation, from amino acid to codon. 4. REPRESSOR SPECIFICITY

After the isolation of thc repressor for the lactose operon in E . coli, its affinity for about 20 synthetic DNA’s was studied (47a). Although none of these DNA’s binds repressor as tightly as does lac operator, most do bind to a measurable extent, especially poly (dA-dT) .poly(dAdT) and poly (dT-dT-dG) apoly (dC-dA-dA) . Thus, the lac repressor may appear rather unspecific, and onc may see a contradiction between the in vitro data and the genetic evidence. However, before discussing spccificity, the recognition process should be analyzed. The question that counts, for genetic expression is whether the operator is free, or whether it forms a complex with the repressor. Thus, efficiency in the regulatory mechanism depends on how the various proteins of the cell (including the repressor) compete for binding to the operator site. Once this point is answered, one can go next to the question: Will the repressor be present in sufficient amounts in the cell? If the number of repressor moleculcs is small, and the number of sites (other than the operators) to which they can bind is large, no repressor may be left for binding to the operator. However, the binding of the repressor to any other site must be analyzed similarly; i.c., one must find the protein that, among the other proteins of the cell, binds effectively to the site considered.

D. Diversity in the Binding Patterns Most of the binding experiments described after the proposal of the wobble hypothcsis wcrc considered to be in excellent agreement with it. From the bulk of the data, two new generalizations could be made, which were pertinently underlined by Caskey (48). a. The nonscnse codons arc actively recognized by the so-called release factors. Moreover, recognition of the termination codons by these proteins is partially degenerate. Caskey made the parallel between ambiguous reading by RNA and by proteins. If this was not the case, there would have been a serious objection to the missing anticodon hypothesis. One might have expected translation to

322

JACQUES NINIO

FIG.4. Patterns of recognition of codons by tRNA’s from different species. coli; A, yeast; 0, mammals. The points representative of codons “recognized” by the same tRNA species are joined by a line. Most of the information was presented in three articles (46, 63, 76). The binding experiments are not very reliable for several reasons. High magnesium concentrations are used. Some of the tRNA’s may not be entirely pure. Moreover, competition experiments are required to establish the effective binding patterns (Sections IV, B, and C). The “strength” of binding remains an ambiguous concept as long as the kinetics of polypeptide elongation (Section 11. A) are not yet understood.

0, Escherichia

proceed through the formation of a noncomplementary association between the iinonsense codon” and a tRNA species. b. Although the genetic code is universal, the type of binding patterns observed with isolated isoaccepting tRNA’s can vary widely with the organism from which the tRNA is extracted and with the particular amino acid considered (Fig. 4 ) . It is clear from Section IV, B that all the ambiguities cannot be resolved in a uniform manner. The observation that the binding patterns are not uniform and that every amino acid appears as a particular case removes another major difficulty of the theory.

E. The Sources of Ambiguity Since polypeptide elongation is an extremely complex process, several factors can affect its reliability: magnesium concentration, temperature, etc. Here, we focus on the slightly different question: What type of ambiguity is expected? I n other words, what are the potential codonanticodon mispairings which need to be resolved through anticodon suppression or modification We can make certain guesses.

323

RECOGNITION IN NUCLEIC ACIDS

a. From the study of tRNA structure, we guess that

1” “1

needs to

be resolved (e.g., Section 11,D). b. From Claverie’s computations showing that the diagonal interactions of stacking are much more favorable in

1’ ‘1

16

GI than in IG

61,

we guess that needs to be resolved. c. From the experimental observation that tRNAfMetfrom E . w2i responds to GUG, we are led to examine the consequences of

I# I:

d. A number of suggestions come from Claverie’s computations in the “rigid approximation.” In Sections V, B to D, we consider these various potential ambiguities, and try to see how they can be resolved. Then, we check the predictions against the experimental evidence, and this tells us whether the postulated ambiguity is physically possible in the decoding process.

F. Why the Anticodons, and Not the Codons? Let us assume that all the codons C,, C,, . . . CB1and all the anti-

codons A,, A, . . . A,, are used. Suppose a misreading in which C1*A2is formed instead of C,.A,. We have assumed that the ambiguity was resolved by suppressing or modifying A,. Let us work out the consequences of keeping A, and acting upon C,. Modifying all the GI's on mRNA appears difficult but, using the codon C, less and less, it is feasible. Once C, has disappeared, A, becomes useless and may disappear. Then, the codon C, can be used again and read A,. Now, it codes for the same amino acid as Cz.The pathway for transferring the ambiguity to a degeneracy is different from that described in Section IV, B, where A2 is suppressed, and C, acquires the meaning of a codon C, different from C1. Most of the codons have already been observed in vivo (49, & but I), one cannot say the same of the anticodons.

G. limitations of the Missing Triplet Hypothesis That recognition is not an all-or-none process, at least in the case of chain termination codons, was made clear in Hirsh’s experiments (29). He sequenced a UGA suppressor tRNA possessing a CCA anticodon and found that the suppressor tRNA differed from the normal tRNAT”p by a single-base substitution outside the anticodon loop. The normal tRNATrPalso acts as a weak UGA suppressor.

324

JACQUES NINIO

I n this case, the potential ambiguity involving an association of UGA with CCA was resolved without the use of an anticodon suppression or modification. The existence of solutions of that type puts limitations on the predictive value of the theory. On the other hand, the fact that the presence or the absence of the various anticodons appear to obey very special laws demands a theory, and the missing triplet hypothesis provides the only available framework for the understanding of the phenomenon.

H. Evolutionary Implications The idea that recognition in nucleic acids, without the help of complex devices, is rather ambiguous, leads to the question: How can a system making errors in translation evolve and lead to a reliably reproducing system? In the preceding sections, we have shown some of the pathways through which an ambiguity can be suppressed and converted into a degeneracy. However, this is not an answer, the problem being much deeper, as shown by Orgel’s argument on aging (51).Suppose that errors of translation can occur in a cell, and affect the synthesis of protein components on which the reliability of translation depends (such as aminoacyl synthetases) . Translation errors will then increase exponentially, and if the rate a t which this exponential increases is more rapid than that of cell division, all the progeny of the cell will die before reaching an Nth generation. In other words, if the level of errors in translation is too high, Darwinian selection cannot operate. What is that level above which (through replication and selection) a species cannot survive? It depends upon the complexity of the cell and that of the genetic apparatus. A crude guess can be made about the level of errors in replication that cannot be tolerated. A 1% error per nucleotide and per generation will change 3% of the anticodons on tRNA by one base. This makes an average change in the code of one codon-amino acid assignation per generation, which is enormous. Taking into account the fact that codon-anticodon recognition is affected by the structure of the whole tRNA molecule and by other factors as well, and taking into account Orgel’s effect, lo-* error per nucleotide and per generation would appear lethal. Unfortunately, it is not easy to work out the figures in the reverse direction, i.e., if translation errors of loL4 per amino acid inserted are assumed, what is the expected level of errors in replication? Let us assume as a crude estimate, that error in translation do not allow less than error in replication. Then, error in translation is lethal for any system in which the translation apparatus works in the way it actually does.

RECOGNITION IN NUCLEIC ACIDS

325

Let us call T the tolerable amount of errors in a given species of bacteria. T is the average number of errors in translation above which the propagation of errors makes impossible the survival of the species. A long time ago the ancestors of that cell were making errors in translation a t the level T AT larger than T. How is this possible? One possibility is that the ancestor made use of shorter proteins. For one error occurring in a protein 50 amino acids long will, on the average and with respect to function, be less harmful than ten errors occurring in a protein of 500 amino acids. In the larger protein of 500 amino acids, we will still find in the active site an average error of one per 50 amino acids so that we do not see how it could be less affected than the shorter protein. Besides, the probability of finding a protein with no error a t all is 36.4% for the short protein, and 0.004% for the large one. The mathematical argument necessitates a physical assumption: the same function can be carried by proteins of widely different lengths. The best example we have of this situation comes from the study of RNA polymerases in halophilic bacteria, the molecular weight of which is 20 times lower than in E. coli ( 5 6 ) . How far can we go with this evolutionary descent? As long as the genetic apparatus is working in the same fashion, T cannot be larger than a certain value depending on the logic of the translation B y decreasing apparatus, which we crudely estimated to be around the lengths of the proteins, we are approaching the oligopeptide level. Then, the situation looks qualitatively different, for we do not need a code. Several oligopeptides of defined sequences are synthesized by a noncoded mechanism on complex enzymes. Lipmann has discussed the chemical similarity between coded and noncoded peptide synthesis ( 5 3 ) . This leads to the question: Can we consider the translation apparatus as an evolved form of a particle performing noncoded peptide synthesis? First, we notice that a number of intermediate stages can be imagined between noncoded and coded peptide synthesis. To fix the ideas, let us start with a small enzyme catalyzing the synthesis of Met-His. In a second stage, an evolved form of the enzyme catalyzes the synthesis of Met-His-Val. The first two amino acids are linked as before, but the addition of the third is dependent upon the presence of a coenzyme, which might be an oligonucleotide; thus, we have a coupling between an oligonucleotide and one amino acid that is not a coding relationship. In a third stage, an evolved form of the complex synthesizes two different tetrapeptides, Met-His-Val-Tyr and MetHis-Val-Gln depending upon the coenzyme (a larger oligonucleotide) that is added. Now we have more than a coupling, we have a correspondence ‘(at the fourth position” between a portion of a variable oligonucleotide and the two amino acids, Tyr and Gln. If this is the

+

JACQUES NINIO

326 hU

acid

(d)

FIG. 5. A very speculative scheme for the evolution of transfer RNA threedimensional structure, consistent with the ideas exprerrsed in Section IV, H. It is proposed that diagram (a) represents the minimal requirement for the recognition of an amino acid by nucleic acids. According to our knowledge of tRNA threedimensional structure (70-78),a portion of the “extra-loop” may be accommodated as a third strand in the large groove of the hU stem. This suggests the following steps in tRNA evolution. (a) At the beginning, tRNA is a combination of two oligonucleotides allowing recognition of amino acids. Then, the hU stem and loop region would be the oldest part of the structure. (b) A better positioning of the amino acid is reached. (c) The adaptor function appears, not necessarily in connection with a process of linear translation. (d) Binding to protoribosomes is improved through the use of the G-T+-C stem and loop region. This is the point where molecular models of tRNA cease to agree, since this region is often believed to extend toward the anticodon region. On the other hand, the important point is that such a major change can be accomplished without displacing too much the amino acid position, whatever the solution adopted. (el Specificity in the acylation of tRNA becoming more and more controlled by activating enzymes, the amino acid no longer needs to be close to the hU loop. The amino acid stem grows. In this context, the nucleotidy1 transferases (CCA-adding enzymes) would sit on the G-T+-C stem and loop region, and control the length of the amino acid stem.

direction of the evolutionary process, it is clear that now very little is needed to reach a situation whcrc thcrc is a coclc. For having a code simply means that the process of addition of amino acids is becoming recurrent, i.e., the correspondence a t the Nth position is exactly of the same type as the correspondence at the ( N 1)th position. Again, we must try to find out whether each of the stages is consistent: Is the logic of the system such that selection can go faster than

+

RECOGNITION IN NUCLEIC ACIDS

327

propagation of errors? The system is certainly inconsistent if a complex enzyme is required to synthetise the di-or tripeptides. On the other hand, if small oligopeptides and oligonucleotides can get involved in selfsustaining catalytic cycles or supercycles, the difficulty can be removed. Prigoginc (54) and later Eigen ( 9 ) havc discussed the relevance to the origin of life of such cycles, which are currently studied in the thermodynamics of irreversible processes. However, the behavior of a mixture of molecules in which several reactions take place depends very much on the actual values of the coefficients of the rate equations. Thus, we cannot say that the picture proposed for the cvolution of the code is consistent. These ideas were found by elimination of other solutions. From the knowledge that recognition in nucleic acids is ambiguous, we cannot accept schcmes in which first RC h a w replicating nuclcic acids, then a code, and finally proteins. We cannot either accept schemes in which the logic of thc translation apparatus exists from thc beginning, with evolution bringing the codon-amino acid catalog progressively into focus. The examination of thc conscquences of translation errors has suggested a new way of considering the origin of the code, in which the important phenomenon is the progressive increase in the length of the synthesized oligopeptides. Thus, a gradual cvolution from noncoded to coded protein synthesis is proposed. The appearance of tRNA, a multifunctional molecule, would be easier to understand if its various functions were not required simultaneously from thc bcginning. It was proposed that tRNA ancestors may have h e n involved in noncoded polypeptide (54n) or dipeptide (54b) synthcsis. With such a starting point thc thrcc-dimensional structurc of tRNA may appcar less mysterious (Fig. 5 ) . Whcthcr this view is corrcct or not, i t shows how important is a good knowledge of the reliability in rccognition processes: it is one of thc kcy positions t o thc undcrstanding of prc-Darwinian evolution.

V. The Experimental Evidence A. Suppression I n thc normal cell, when a particular codon in a gcne mutates to ITAA, UAG or UGA, tlic synthcsis of thc cncodcd polypcpticle does not go beyond thc nonsense codon, and a shortcr chain is rcleascd. Release is an activc process, due not to the absence of tRNA’s with anticodons complcmcntary to the nonsensc codon, but to a recognition of the termination codons by rclcasc factors (55,56). The nonsensc mutations mentioned can he suppressed by other mutations altering the primary struc-

328

JACQUES NINIO

ture of a tRNA. I n thc prcscncc of the nonscnse codon, thcre is now a competition bctwccn thc rclcasc factors and the suppressor tRNA. Thus, the efficiency of the supprcssor can bc variablc and depends upon the location of the nonsense codon inside the gene. More precisely, a nonsense codon can producc 5% chain tcrmination when located a t onc place, and 70% chain tcrmination wlicn located at mothcr, in thc prescnce of the sanw supprcssor tRNA gcnc, as if the “rcading context” exerts an influence upon translation. This phenomenon had already been discussed and intcrprctctl in 1969 (57). Thc niiiiimal intcrpretation is that the release factor is actually able to recognizc a little more than a triplet. Thus, chain tcrmination would bc extrcmcly efficient whcn UAG is followed by U, but suppressors would compctc with relcase factors when thc ncxt basc is A, G or perhaps C. As we have secn in Scction 11, A, it is easy to find conditions in which rccogaition is altcrctl in such a way that the rcsolution between two competitors is always reduced. Streptomycin is known to alter the ribosomes in such a way that translation errors increase. Now, nonsense suppression is a translation error in which the chain termination codon is read by a suppressor tRNA instead of being read by the release factor. Thus, as expected, streptomycin increases the level of nonsense suppression. Here, the word “error” is taken in the more general sense of substitution of the molecule giving the highest binding, by a competing molecule. One must beware of a confusion in reasoning that goes as follows. In a strain that carries an ambcr or an ochre suppressor, the nonsense codon is read by the suppressor. If that reading was of the same type as the reading of normal codons, streptomycin should induce errors in that rcading, and thus it should decrease the level of suppression. Since suppression increases, one must assume that there is a misreading process essentially different from the process of normal reading. Reasoning of this type has been extended to missense suppression (58). I view the changes in thc levels of misreading, of nonsense suppression, and of missensc suppression as thrcc aspects of the same phenomenon-an increascd or dccrcascd capacity to discrimiaatc between two compctitors. Once this is takcn into account, one finds nothing in the experiments on phenotypic suppression in support of the view that “mistranslation is kept under control by thc wild-typc ribosome without intcrfcring with normal translation” (59).

B. Consequences of

I”

The occurrence of the above association in codon-anticodon pairs would lead to the ambiguities

RECOGNITION IN NUCLEIC ACIDS

1;

U

A

1;

U

with

G ( 3 1

or

1:

with

1:

329

In the first case, we are concerned with the translation of thc four codons beginning with U-G. UGA is a nonscrisc codon. Thc competition would be between a release factor and an arginine anticodon UCG. There is nothing against such a possibility (e.g., Section V, A). UGC is a cysteine codon. Is it read by an arginine anticodon GCG? ICG would be preferable to GCG since 1.C is weaker than G - C . Here, we can reasonably explain the following feature. Although inosine is practically never found in E . coli tRNA’s, it occurs in an arginine anticodon ICG (60). UGU is no problem since anticodons beginning with A do not exist, and to a first approximation we can neglect the associations containing two U - G pairs. The major problem would be the translation of UGG since the binding experiments suggest the presence of CCG as an arginine anticodon. In the cases in which U - G occurs in the second position, we are concerned with the translation of thc codons UUG, CUG, AUG and GUG. As for the leucine codon UUG, let us compare the three associations:

-

-

:I 4 :I $1 :I I: U A

U A

U A

-

and

We know that serine has a CGA anticodon - in rat liver tRNA. On the other hand, leucine has an anticodon CAA, which may appear unnecessary if the wobble is correct. Thus, the consideration of the consequences of

1” ‘1

occurring in the second and third positions lead to an inG C correct prehiction (CGA should not exist). However, the potential ambiguity may appear as a second-order phenomenon, thereby explaining the usc of the “unneccssary” anticodon CAA. Can the valine codon GUG be read by an alanine anticodon CGC? The major isoaccepting tRNA*’” in yeast has K C . A second subspecies apparently has UGC rather than CGC in its anticodon (61). If the preceding situations can be generalized to the translation - of CUG and AUG, one can expect two things. The presence of CGG for proline is dependent upon that of CAG for lcucine. The presence of CGU for threonine is made possible by the presence of CAU for methionine (with a restriction discussed in Section V, E) .

-

7

-

-

-

330

JACQUES NINIO

C. Consequences of

I“

The occurrence of the above association would lead to the ambiguities

1:

N

M with

;1

1;

G

and

C with

;1

G

C (31

The first of these, being coincident with the degeneracy of the code does not need to be resolved. Ambiguities of the second type would concern the reading of valine codons by alanine anticodons. Since both IAC and IGC are found in yeast tRNA’s, taking into account the above ambiguity leads to incorrect predictions. Once more, U * Gis in the second position of the codon-anticodon association. On the other hand, the examination of the consequences of the very special association of GUC with GGC (e.g., Section 11, D) may explain another strange observation. In yeast t.RNA’s, inosine is found whenever allowed by the wobble, with a possible exception: glycine (Fig. 4 ) . The binding patterns provided by et al. (63) do not exclude entirely the presence of a tRNAolY with So11an ICC anticodon. Their peak I1 could bind to ribosomes in response to GGC, GGU and GGA (Table IV). However, in their interpretation of the data, they considered tRNA,olY to be specific for GGU and GGC, as seems to be the case for their tRNA,o’Y. The proposed explanation - for the presence of an isoaccepting tRNAG1rwith a postulated GCC anticodon was that the aspartic acid anticodon GUC would efficiently compete against ICC for binding to the glycine codon GGC, hence the need for at least one glycine anticodon that binds to GGC more strongly than does Ice (2). TABLE IV OF GLYcYL-tRNAQIY SPECIES TO RIBOSOMES IN BINDINQ OF VARIOUS TRINUCLEOTIDES (63, 79)”

G-G-U G-G-C G-G-A G-G-G

PRESENCE

Escherichia wli

Yeast Species

THE

Peak 1 Peak 2 Peak 3 Peak 4 Peak 1 Peak 2 Peak 3 Peak 3‘ 0 0 0.21 0.06

1.60 0.80 0.20 0

0.06 0.12 0 0.24

0.23 0.60 0.02 0

1.5 1.1 0 6.9

0.6 0 2.8 3.9

6.5 6.0 0.3 0

0.18 0.12 0.65 0.53

0 The binding i s expressed in picomoles of glycyl-tRNAG’Y bound to ribosomes in the presence of 1 nmole of trinucleotides. The incubation mixtures contained 10-30 pmoles of glycyl-tRNAQIy.

RECOGNITION IN NUCLEIC ACIDS

331

D. The Rigid Approximation According to the energy-calculations applied to the recognition of triplets in a double-helical RNA configuration (66),all the potential glycine anticodons should produce ambiguities. However, because the release factor, this makes termination codon UGA -is recognized by apossible the use of UCC (6). The case of CCC is interesting. One of the tRNAG'y fractions in yeast (63) responded to GGG, and to a lesser extent, to GGC and GGU. Similarly, one of the tRNAG'y fractions in E. coli (79) responded predominantly to GGG, but also to GGU and GGC (Table IV). In both cases, the authors interpreted their binding patterns as revealing the existence of a GGG-specific tRNAG'Y. It turns out that the anticodon CCC is present in the corresponding E. coli tRNAG'y (6%). If the binding of that tRNA to GGU and GGC is confirmed, we would be in a situation somewhat similar to that of E. coli tRNAT"*: an ambiguity would be resolved by the production of a "strange" tRNA. If not, we may remember that the potential ambiguity concerning CCC would be due to the formation of a U .C pair in the second position of the codon-anticodon association. Once more, the recognition process would -appear rather strict on the second position. The requirements for GCC are contradictory. That E C instead of K C appears to be found in yeast tRNA was explained by the possible - association of GUC with GGC. In E. coli, a tRNAG'Y with a GCC anticodon has -been sequenced ( 6 4 ) . Thus, the prediction that a tRNAG'y with GCC should be able to read the cysteine codon UGC is incorrect. However, the prediction may be true as a second-order effect. The well-known missense suppressor, suf-78, in which a cysteine codon in the tryptophan synthetase gene is read as glycine (65) appears to be a tRNAG'Y differing from wild type by a post-transcriptional modification outside the anticodon GCC ( 6 3 b ) .

-

-

E. initiator and Noninitiator tRNA's Knowing that E. coli tRNAcMetcan bind to the ribosomes in the presence of GUG, we may wonder about the fate of an internal GUG codon. What will prevent tRNAMptfrom reading GUG, which is a valine codon? Actually, the requirement is very simple, tRNAMetmay bind to GUG provided that the affinity of GUG for a tRNAV"' is much larger than its affinity for tRNAMet.Noting that E. coli tRNAfMetcontains CAU, whereas in tRNAMetthe first C is modified, we are tempted to attribute to this modification the effect of reducing the affinity of the G - C pair, thus decreasing at the same time the affinities of tRNAMetfor its own codon AUG and for the valine codon GUG (6). The modified base C+

-

332

JACQUES NINIO

TABLE V BINDING CONBTANTS OF OLIGONUCLEOTIDES TO INITIATOR AND NONINITIATOR tRNA’s (80) Oligonucleotide

E. wli tRNA$t Yeast tRNAPt 4. wli ,,NAPt E . wli tRNAp’

A-U-G

G-U-G

6,100

910 950

11,100 1,700 500

U-G 270

<100

a

b

2,300

b

a There is a definite binding of G-U-G to E . wli tRNAFt ( K be suppressed by a 100-fold excess of unlabeled A-U-G. b Not determined.

= 1,900) that

cannot

was recently identified as N’-acetylcytidine (66a). It was shown earlier that poly (I)’ poly (C’) has a slightly higher melting temperature than poly(1) Vpoly (C) (66b). Several experiments, performed by Dr. Hogenauer’s group, tend to invalidate the idea of important qualitative differences between the anticodon loops of initiator and noninitiator tRNA’s (Table V ) . On the other hand, if one sticks to the Fuller-Hodgson interpretation of the wobble hypothesis (@), one must admit that the anticodon loop of E. Cali tRNArMethas a special structure, a t variance with the structure of noninitiator anticodon loops. Furthermore, since translation brings a noninitiator tRNA from the acceptor site to the peptidyl site, its anticodon should undergo a conformational change from a “noninitiator” to an “initiator” type.

F. A Fourth Base in the Anticodon? It has often been suggested that the base following the third position of the anticodon has a direct influence on codon-anticodon recognition (66). Correlations are found between the chemical nature of the third and the “fourth” base of the anticodon loop (see Nishimura’s review, Vol. 12 in this series). The fact that frameshift mutations can be suppressed (66a, 66%) by a tRNA with an altered anticodon loop (66c) stresses the importance of the whole loop structure and position during translation. I n the present article, the question is left aside. Before theorizing on the function of the fourth base, it is not unreasonable to pay attention to the anticodons themselves.

G. Anticodons in the Third Column The binding patterns observed with yeast tRNAQ’“appeared a t first sight inconsistent with the wobble hypothesis. One of the isoaccepting

333

RECOGNITION IN NUCLEIC ACIDS

-

species responded to GAA but not to GAG (67). A tRNAol" with UUC should be able, according to the wobble hypothesis, to bind to GAG. A sequence analysis revealed that the first base of the anticodon was a 2-thiouracil residue. The authors proposed that such a modification - made impossible the association with GAG. They suggested that UUC could bind to the aspartic acid codon GAU, while the modified anticodon could not (68). This idea is very close to that developed in the missing triplet hypothesis: a potential ambiguity is resolved by an anticodon modification. However, in several aspects, the authors remained within the framework of the wobble hypothesis, considering recognition as an all-or-none process (they did not consider competition of tRNA's for the same codon), ambiguity as being restricted to the third position, and the base pairs as defined in Section 111,B. The two glutamic isoacceptors insert glutamic acid a t different positions during in vitro translation of hemoglobin messenger (67). A similar observation is made with lysine isoacceptors (69), but in this case thiouracil does not seem to be present. Thus, the authors ask whether their tRNA has not been modified during the purification steps, reconverting SU to U. Clearly, once it is admitted that recognition is not an all-or-none process, the hypothesis of alteration during purification becomes unnecessary. (For further details, see Nishimura in Vol. 12 of this series.)

VI.

Discussion

The missing anticodon hypothesis differs from other theories encountered in molecular biology, in being nonmechanistic. However, it does not deny the usefulness of knowledge on ribosomal mechanics for a deeper understanding of the coding process. The argumentation is drawn in such a manner that the validity of the conclusions does not depend on a stereochemical model of the codon-anticodon association, or on a dynamic model of protein elongation. After analysis the recognition process, several conclusions have been reached. Reliability in translation depends on such an interplay of different factors that a strong doubt is raised about the possibility of achieving a reliable recognition of 64 codons by their 64 complementary anticodons (e.g., Sections 11, B, D, E, and IV, A). The relationship between the formation of noncomplementary codon-anticodon pairs and the occurrence of errors in translation is complex. The way translation proceeds in time enhances some of the physical ambiguities and disguises others. The degeneracy of the code allows physical ambiguities to be disguised in the form of degeneracy. The position of degeneracy need not be coincident with the position of ambiguity (e.g., Section IV, B). The resolution of an ambiguity by anticodon suppres-

334

JACQUES NINIO

sion or modification may generate an ambiguity elsewhere. Thus, within one species, the anticodons form a strongly connected family. The presence of guanine in one particular isoaccepting species demands the presence of inosine on some other anticodon. A reliable translation requires the adjustment of each tRNA to the set formed by the others. Thus, we have a general theoretical framework with which to understand why in one case (phenylalanine) there is one anticodon GAA for the two codons UUU and UUC, while in another (leucine) there are two different anticodons for the two codons UUA and UUG. It deals with problems that must arise whatever the precise mechanism of protein elongation. Even if the general framework is true, its practical value may be rather limited if it is describing second-order or third-order phenomena, or, in other words, if the physical process of recognition is accurate enough to allow the use of many more anticodons than the anticodons actually found. I n this case, the anticodon restriction phenomenon would have been the result of selection upon some other feature than the lowering of ambiguities in translation. Thus, after all, the hypothesis may be a failure. On the other hand, the discussion of the details of anticodon sequences and recognition patterns (e.g., Sections V, B-E) shows that the hypothesis can be used as a practical tool for understanding apparently unrelated observations, leading to experimental verification. In any case, the wobble hypothesis remains true as a first approximation. The anticodons that cannot exist, according to the theory, are never found. Its validity owes much to the logical character of the argumentation, as it has been exposed in Section 111,A. One can say of any process occurring in the cell that it is a recognition process. Does the study of eodon-anticodon recognition help to understand other phenomena? I would bc tempted to answer yes. The set of the molecules used in any particular cell form a coherent family, with quite unpredictable relationships between some of their features. For instance, within one cell, the set of the aminoacyl synthetases is adapted to the recognition of the set of the tRNA species of that cell. When one takes tRNA from one organism, and enzymes from another, mischarging occurs (70). Through thcsc experiments, some investigators are attempting to delineate a “recognition site” for the synthetase on tRNA. We confidently predict that such a reductionist hope will lead to inconsistent results. A connection between two apparently unrelated molecules (a tRNATyr and an enzyme, tryptophan pyrrolase) has recently been disclosed in Drosophila ( 7 1 ) . The informational content of a protein must include the compatibility of that protein with the various processes that take place in the cell. Part

RECOGNITION IN NUCLEIC ACIDS

335

of this information can be described in the form of negative instructions: do not go across the membrane, do not bind to thc active site of that protein, etc. The ability of one enzyme to pcrturb the functioning of another can be suppressed if it forms an oligomeric structure in which a portion of the monomer surface is now buried in the quaternary structure. The cvolutionary implications of thc missing triplet hypothesis were discussed in Section IV, H. We can add a few words about the present evolution. Since the molecules of a cell form a mutually consistent family, a mutation in one molecule may have effects that are unrelated t o its function, and that may not be revealed in its enzymatic properties. One may also expect that any modification in a component of the translation apparatus may result in a series of subsequcnt adjustments. ACKNOWLEDGMENTS I am grateful to F. Chapeville and A. M. Michelson for the warm hospitality of their laboratories. F. Gros’ advice and P. Clavwie’s comments helped me much in the expression of these ideas. Thanks are also due to A. L. Haenni and D. Lawrence for their constructive remarks.

REFERENCES 1. F. H. C. Crick, J M E IS, 548 (1966). 2. J. Ninio, J M E 56, 63 (1971). 3. S. I. Chan, G. C. Y. Lee, C. F. Schmidt and G. P. Kreishman EERC 46, 1536 (1972). 4. C. R. Woese, Nature (London) 226, 817 (1970). 5. F. M. Unger, in “First European Biophysics Congress” (E. Broda, A. Locker and H. Springer-Lederer, eds.), p. 13. Verlag Wien. Med. Akad., Vienna, 1971. 6‘. M. Gninberg-Manago and J. Dondon, BBRC 18,517 (1965). 7. S. Pestka, R. Marshnll and M. Nirenberg, PNAS 53, 639 (1965). S. 0. Maal@e and N. 0. Kjeldgaard, i i ~“Control of Macromolecular Synthesis” (B. D. Davis, ed.), 1). 70. Benjamin, New York, 1966. 9. M. Eigen, NaticrurLse,zschnfteii 58, 465 (1971). 10. E. K. F. Bautz and F. A. Bautz, PNAS 52, 1476 (1964). 11. M. Tsuboi, K. Matsuo and M. Nakanishi, Biopolymers 6, 123 (1968). 12. A. C. Wang and N. R. Kallenbach, J M B 62, 591 (1971). IS. 0. C. Uhlenbeck, F. H. Martin and P. Doty, J M B 57, 217 (1971). 14. S. K. Podder, Nature New Eiol. (London) 232, 115 (1971). 15. C. D. Barry, J. A. Glasel, A. C. T. North, R. J. P. Williams and A. V. Xavier, BEA 262, 101 (1972). 16. J. Eisinger, B. Feurr and T. Yamnnc, Nature (London) New Biol. 231, 126 (1971). 17. J. Eisinger, BBRC 43, 854 (1971). IS. D. Porschke, Biopolymers 10, 1989 (1971). 19. M. E. Craig, D. M. Crothers and P. Doty, J M E 62, 383 (1971). 20. R. Rome,, D. Riesner, G. Maasu, W. Wintermeyer, R. Thiebe and H. G. Zachau, FEES L e t t . 5, 15 (1969).

336

JACQUES NINIO

21. M. Dourlent, M. Yanir and C. H86ne, EJB 19, 108 (1971).

28. S. R. Jaskumas, C. R. Cantor and I. Tinoco, Jr., Biochemistry 7 , 3164 (1968). 83. G. Hogenauer, Hoppe-Seyler’s Z . Physiol. Chem. 348, 227 (1967). 24. G. Hogenauer, EJB 12, 527 (1970). 26. 0. C. Uhlenbeck, J. Baller and P. Doty, Nature (London) 225, 508 (1970). 36. J. B. Lewis and P. Doty, Nature (London) 225, 510 (1970). 87. P. R. Schimmel, 0. C. Uhlenbeck, J. B. Lcwis, 1,. A. Dirkson, E. W. Eldred niitl A. A. Schreier, Biochemisfry 11, 642 (1972). 28. 0. C. Uhlenbeck, JMB 65, 25 (1972). 20. J. E. Sulston, R. Lolirmann, L. E. Orgel, H. Schneider-Bernloehr, B. J. Weiman and H. T. Miles, JMB 40, 227 (1969). 30. J. Ninio, Biochimie 53, 485 (1971). 31. D. Hirsh, JMB 58, 439 (1971). 38. S. H. Chang, N. R. Miller and C. W. Hnrmon, FEBS Lett. 17, 265 (1971). 33. J. R. Fresco and B. M. Alberts, PNAS 46, 311 (1960). 34. S. M. Coutts, BBA 232, 94 (1971). 36. A. R. Cashmore, D. M. Brown and J. D. Smith, JMB 59, 359 (1971). 36. N. C. Seeman, J. L. Sussman, H. M. Bermnn and S. H. Kim, Nntzae ( L o d o n ) New Biol. 233, 90 (1971). 37. W. C. Johnson, M. S. Itzkowitz and I. Tinoco, Jr., Biopolymers 11, 225 (1972). 38. C. R. Woese, Natnire (London) 194, 1114 (1963). 39. R. Eck, Science 140, 477 (1963). 40. M. R. Bernfield and M. W. Nirenberg, Science 147, 479 (1965). 41. R. W. Holley, J. Apgar, G. A. Everett, J. T. Madison, M. Marquisee, S. H. Merrill, J. R. Penswick and A. Zamir, Science 147, 1462 (1965). 4.2. R. A. Gilmore, J. W. Stewart and F. Sherman, JMB 61, 157 (1971). 4%. C. R. Woese, D. H. Dugre, S. A. Dugre, M. Kondo and W. C. Saxinger, CSHSQB 31, 723 (1966). 43. W. Fuller and A. Hodgson, Nature (London) 215, 817 (1967). 44. F. H. C. Crick, JMB 38, 367 (1968). 46. P. Claverie, J . Chim. Phys. 65, 57 (1968). 46. C. T. Caskey, A. Beaudet and M. Nirenberg, JMB 37, 99 (1968). 47. M. S. Rendell, J. P. Harlos and R. Rein, Biopolymers 10, 2083 (1971). 47a. A. D. Riggs, S. Lin nnd R. D. Wells, PNAS 69, 761 (1972). 48. C. T. Caskey, Quart. Eev. Biophys. 3, 295 (1970). 48a. R. M. Bock, J. Theoret Biol. 16, 438 (1967).

R. Contrcras, R. De Wachter, G. Haegcman, J. Mcrrcgacrt, W. Min Jou and A. Vandenberghe, Bwchimie 53, 495 (1971). 60. Y. Ocada, S. Amagase and A. Tsugita, JMB 54, 219 (1970). 61. L. E. Orgel, PNAS 49, 517 (1963). 68. B. G. Louis, P. I. Peterkin nnd P. S. Fitt, BJ 121, 635 (1971). 63. F. Lipmann, Science 173, 876 (1971). 64. P. Glansdorff and I. Prigogine, “Thermodynamic Theory of Structure, Stnbilit>y and Fluctuations.” Wiley (Interscicnec), New York, 1971. 64n. C. R. Woese, “The Genetic Code.” Harper, New York, 1967. 64b. L. E. Orgel and J. E. Sulston, in “Prebiotic and Biochemical Evolution” (A. P. Kimball and J. Oro, eds.), p. 89. North-Holland Publ., Amsterdam, 1971. 66. J. L. Fox and M. C. Ganosa, BBRC 32, 1064 (1968). 66. M. S. Bretscher, JMB 34, 131 (1968). 67. W. Salser, M. Fluck and R. Epstein, CSHSQB 34, 513 (1969).

40. W. Fiers,

RECOGNITION IN NUCLEIC ACIDS

337

68. D. K. Biswas and L. Gorini, JMB 64, 119 (1972). 69. L. Gorini, Nature (London) New Biol. 234, 261 (1971). 60. K. Murao, T. Tanabe, F. Ishii, M. Namiki and S. Nishimura, BBRC, 47, 1332 (1972).

W. Chambers, PNAS 60, 1450 (1968). 6.2. P. Clnverie, JMB 56, 75 (1971). 63. D. Rill, D. S. Jones, E. Ohtsukn, R. D. Faulkncr, R. Lohrmnnn, H. Hayatsu, H. G. Khorana, J. D. Cherayil, A. Hampel and R. M. Bock, JMB 19,556 (1966). 6Sa. C. W. Hill, unpublished results, quoted by D. L. Riddle and J. Carbon (66~). 63b. J. Carbon, communication given at IUPAC tRNA meeting, Princeton (1971). 64. C. Squires and J. Carbon, Nature (London) New Biol. 233, 274 (1971). 66. J. Carbon, P. Berg and C. Yanofsky, CBHSQB 31, 487 (1966). 66a. Z. Ohashi, K. Murao, T. Yahagi, D. L. Von Minden, J. A. McCloskey and S. Nishimura, BBA 262, 209 (1972). G6b. F. Pochon, M. Leng and A. M. Michelson, BBA 169,350 (1968). 66. F. Kimura-Harada, F. Harada and S. Nishimura, FEBS Lett. 21, 71 (1972). GGa. D. L. Riddle and J. R. Roth, JMB 54, 131 (1970). 66%. J. Yourno and S. Tanemura, Nalure (London) 225, 422 (1971). 66c. D. L. Riddle and J. Carbon, Nature (London) New Biol., in press. 67. T. Sekiya, K. Takeishi and T. Ukita, BBA 182, 411 (1989). 68. Z. Ohashi, N. Saneyoshi, F. Harada, H. Harada and S. Nishimura, BBRC 40, 866 (1970). 69. E. Rudloff and K. Hilse, EJB 24, 313 (1971). 70. B. Dudock, C. DiPeri, K. Scileppi and R. Resrelbach, PNAS 68, 681 (1971). 71. K. B. Jacobson, Nature (London) New Biol. 231, 17 (1971). 7.8. S. K. Dube, K. A. Marclcer, B. F. C. Clark and S. Cory, Nature (London) 61. R. H. Reeves, N. Imura, H. Schwam, G. B. Weiss, L. H. Schulman and R.

218, 232 (1968). 73. S. Cory, K. A. Marckrr, S. I(. Dubc and B. F. C. Clark, Nature (London) 220, 1039 (1968). 74. H. U. Blank and D. SOU, BBRC 43, 1192 (1971). 76. S. Nishimura and I. B. Weinstein, Biochemistry 8, 832 (1969). 76. J. Ninio, A. Favre and M. Yaniv, Nnture (London) 223, 1333 (1969). 77. P. G. Connors, M. Labanauskas and W. W. Beeman, Science 166, 1528 (1969). 78. D. E. Bergstrom and N. J. Leonard, Biochemistry 11, 1 (1972). 79. J. Carbon, C. Squires and C. W. Hill, JMB 52, 571 (1970). 80. G. Hogenauer, F. Turnowsky and I?. M. Ungcr, BBRC 46, 2100 (1972).

This Page Intentionally Left Blank

Translation and Transcription of the Tryptophan Operon FUMIO IMAMOTO Depnrtment of Microbiut Geuetics, The Research Institute for Microbial Diseases, Osakn Universit y, Yomada-knmi, Suita, Osuka, Japan

I. Introductioii . . . . . . . . . . . 11. Historical Background . . . . . . . . . 111. Translation of the tip Operon . . . . . . . A. Mode of Regulation of Enzymc Synthesis . . . B. Sequential Initiation of Translation . . . . . c. Simultaneous Initiation of Translation . . . . D. Quantitative Aspects of Translation . . . . . IV. Transcription of the trp Operon . . . . . . . A. Detection of tip mRNA . . . . . . . . B. Characterization of trp mRNA . . . . . . C. Initiation of Transcription . . . . . . . D. Rate of Transcription Propagation . . . . . E. Degradation of trp mRNA . . . . . . . F. Repression of Transcription . . . . . . . G. Trp mRNA Synthesis during Amino Acid Starvation . H. Synthesis of trp mRNA in Vitro . . . . . . V. Translation and Transcription of the trp Operon in Nonsense Mutants of E. coli . . . . . . . . . . A. Translation Studies . . . . . . . . . B. Transcription Studies . . . . . . . . VI. Effect of a Block in Translation on Transcription . . . References . . . . . . . . . . . .

. . . . .

. . . . .

. . . . . . . . . .

. . . .

. . . . . .

.

.

. . . . . . . . .

.

339 340 341 341 344 346 347 348 348 352 354 366 367 369 374 376 377 378 381 398 402

1. Introduction I n this chapter I suminarise scveral years of studies on the regulation of translation and transcription of the tryptophan ( t r p ) operon of the enteric bacteria, especially Escherichia coli, with reference to Salmonella typhimurium where appropriate. I have chosen to limit topics and to refer to others only very briefly. Related topics have been discussed in previous reviews (1-4). The early history of genetic and biochemical studies on the biosynthetic pathway of tryptophan in E. coli was reviewed by 339

340

FUMIO IMAMOTO

Yanofsky (5).Recently Margolin ( 6 ) rcvicwed the regulation of tryptophan biosynthesis, covering in detail various facets of the protein chcmistry, biochemistry and genetics of this system.

II. Historical Background The trp operon is presently understood to consist of five genes, each spccifying a different polypeptide chain (Fig. 1). The genes arc arranged in the same sequence relative to the metabolic pathway in E . coli (7) and S. typhiinuriuin (8-lo), although the letter designations of the genes are in thc order E, D, C, B and A from thc operator end in E . coli while in S. typhirnuriurn, they are in the order A, B, E, D, and C. The first gene code6 for anthranilate synthetase component I (11, 1 2 ). The sccond gene codcs for a singlc polypcptidc chain, which serves as anthranilate synthetase component I1 and phosphoribosyl transferasc ( I S - 1 6 ) . The polypeptide products of the first two genes form a complex that catalyzes reactions 1 and 2 of the pathway in both E . coli (11) and S. typhimurium (12).Although both component I and component I1 are required for reaction 1, uncomplexcd component I1 efficiently catalyzes reaction 2. A singlc polypcptidc product of thc third gene, phosphoribosylanthranilate isomerase-indoleglycerol-phosphate synthetase, catalyzes reactions 3 and 4 of the pathway (17,18). The polypeptide products of the fourth and fifth genes form the tryptophan synthetase complex, a tetramer consisting of two polypcptidc chains of each type (19, 20). Anthranilate synthetase component I has been obtained in pure form from E . coli (21) and from S. typhiinuriunz ( 2 2 ) , as has anthranilatc Operatw

E

D

C

0

A

Gene sequence

Reactions

I I

2

Phosphoribosyl Indoleglycerol Tryptophan anthranilate' phosphate svnthetase isomerase synthetase I

I

I I

Chorisrnic; Anthranilic Anth-Rib~~Anth-dRbl~~lndGro&Tryptophan acid (0 acid (2) ( 3) (4) (5)

FIO.1. Tryptophan operon of Exherichin coli and tlie enaynies and reactions involved in tryptophan biosynthesis. Abbreviations used are : Anth-Rib-P, N-5'phosphoribosylanthranilate; AnthdRbl-P, antliranilic dcoxyrih~ilonucleotide; IndGroP, Indoleglycerol pliosphate.

TRANSLATION AND TRANSCRIPTION OF THE

trp

OPERON

341

synthetase component I1 of S . typhimurium ( 2 3 ) . The proteins determined by the distal three genes of E . coli, namely trp C ( 1 7 ) ,trp B (19) and trp A ( 2 4 ) ,have also been purified to homogeneity. The anthranilate synthetase complex from E . co2i (25) and from S. typhimurium (26) has been partially purified. The MW of the protein products of the third, fourth, and fifth genes of the trp operon in E . coli are 45,000 ( 1 7 ) , 45,000 (27) and 29,000 ( 2 8 ) , respectively. The comparable values for the first and the second gene products are not known with certainty, but the first gene product of E . coli appears to have a subunit M W in the vicinity of 60,000 (21).Since the second gene product has the same sedimentation coefficient as the first (11), it can be assumed that its subunit M W is also about 60,000. The MW of the anthranilate synthetase complex is S. typhimuriwn has been reported to 1)c 290,000 (26) and 260,000 ( 2 9 ) .Since comparative studies of hybrid anthranilate synthetase complexes from E . coli, 8. typhimurium and A . aerogenes gave about the same MW’s for anthranilate synthetase component I and component I1 (SO),the M W of each component could be somewhat higher than that estimated previously. An operator region for the trp operon exists adjacent to the gene that cncodes anthranilate synthetase componcnt I. This location is assigned from the finding that deletion of trp E in E . coli eliminates tryptophan regulation of the operon (31, 32). Polarity studies also support the above conclusion for E . coli (33, 34) and S. typhimurium (35). Operatorconstitutive mutants have been isolated and mapped in E . coli (36) and S . typhimuriu?n ( 3 7 ) . A tryptophan regulator (trp R ) gene has been located near thr on the chromosome of E. coli, far removed from the clustered tryptophan structural genes (38). Amber mutants of trp R of E . coli have recently been isolated, thereby indicating that its product is a protein ( 3 9 ) . Nondefective transducing phages ($30 pt’s), carrying all or part of the tryptophan operon, are available ( 4 0 ) .With the DNA of this phage, hybridization procedures have been used to detect intact tryptophan messenger RNA (trp mRNA) and messenger fragments corresponding to different regions of the operon ( 4 1 , & ) .

111. Translation of the trp Operon A. Mode of Regulation of Enzyme Synthesis An operon is defined as a segment of genetic material that bears the functionally coordinate multigene complex of operator and structural genes ( 4 3 ) .Apparent coordinate expression of the genes in an operon can

342

FUMIO IMAMOTO

be explained by a central feature of operon expression: all the proteins coded by a given operon are synthesized from a single polycistronic messenger whose synthesis is under the control of the regulator gene product. The enzymes of the tryptophan biosynthetic pathway are subject to repression during growth in the presence of tryptophan (44). Upon derepression, in E. coli, synthesis of the polycistronio messenger occurs sequentially in the direction from the E gene to the A gene (4.9, 45-47) ; upon repression by the addition of tryptophan, initiation of synthesis at trp E is terminated (4.9, 46, 47-49). In experiments with wildtype cells, a coordinate variation in the activities of the enzymes specified by the four operator-proximal genes with respect to trp A protein was found under a variety of derepressing conditions (33). The productivity of the trp genes for these enzymes was also subject to control by the trp R gene. In tryptophan auxotrophs of S. typhimurium grown with limiting amounts of tryptophan in a chemostat, a disparity exists between the multiplicity of derepression of the two operator-proximal genes and of the three distal genes of the operon (35). Such a semicoordinate production of the trp enzymes upon derepression also appears in the constitutive enzyme levels of a 5-methyltryptophan-resistant (repressor-deficient) mutant: comparison of the levels of enzymes synthesized constitutively with the activities of fully repressed control bacteria (5-methyltryptophan-sensitive parental strain) showed that in the mutant the enzymes for the two operator-proximal genes were present a t about 40 times the control level while the enzymes for the three distal genes were derepressed only about 25-fold. Additional studies (50, 51) led to the hypothesis that the basis for the semicoordinate production is the presence of a constitutive promoterlike initiator element (P2) near the boundary between the second and the third genes of this operon. A homologous internal initiator element was later recognized in the trp operon of E . coli ( 5 2 ) . A comparison of the enzyme levels in a maximally repressed culture with those in a culture of regulator-constitutive ( t r p R-) bacteria, showed the same disparity as seen in S. typhimurium. The enzymes encoded by the two operator-proximal genes showed constitutive levels higher than the repressed levels by an average factor of 300, while the enzymes encoded by the three distal genes increased by only an average factor of 60. Thus, since synthesis of five enzymes in the regulator-constitutive strain is equimolar (21, 52) , it must be concluded that the maximally repressed level of expression of the three distal genes is approximately five times that of the two operatorproximal genes. The apparent discrepancy in the findings of the mode of enzyme

TRANSLATION AND TRANSCRIPTION OF THE

trp

OPERON

343

production during derepression can be attributed to the different manners in which the extent of derepression was measured. Some (35, 52) expressed i t as a multiplicity factor, determined by dividing the basal repressed level of each enzyme into the steady-state level achieved in the derepressed culture. Others (33) determined the increase in the relative specific activity of the enzymes synthesized during the initial stage (50 minutes a t 37°C) after derepression. Moreover, the latter subtracted the basal repressed levels before plotting the relative specific activities (Ito, quoted by Martin, 3 ) . Similar studies with S. typhimurium yielded the conclusion that the rates of expression of trp genes are coordinate throughout derepression ( 5 3 ) . The internal initiators in S. typhimurium and E . coli are estimated to contribute only about 0.5-1% (35) and 28% ( 5 2 ) , respectively, of the maximum rate of synthesis of enzymes specified by the three operatordistal genes. This low-efficiency initiation, therefore, is insignificant under conditions of maximum operon expression. Accordingly, equimolar production of the trp E , trp C, trp B and trp A proteins produced under derepression conditions has been directly demonstrated (17,19, 54).This equality in the final derepressed level of the enzymes supports the idea that the translational capacity of each cistronic region on the trp polycistronic messenger is the same. An inequality in the amount of trp enzymes synthesized can occur when a tryptophan auxotroph is starved for tryptophan (33, 34, 52, 5 5 ) . The cumulative amount of trp mRNA produced in under starvation conditions has been determined by equating the enzyme-forming capacity that develops upon derepression by tryptophan starvation to the amount of specific messenger ( 5 6 ) .The capacity for synthesis of the enzymes for the first (trp E ) , the third (trp C ) and the fifth (trp A ) genes of the operon increases at an almost linear rate starting immediately after such derepression, indicating that an equal amount of messenger is made for each enzyme. However, measurement of the differential rate of synthesis of these three enzymes during starvation showed that the number of molecules of the trp E gene product is almost twice those of the trp C and trp A gene products. There is, therefore, an uncoupling of transcription and translation when auxotrophic cells are starved for tryptophan. On the other hand, the number of molecules of the trp A gene product is about 1.6 times that of the trp E gene product in prototrophic cells undergoing tryptophan starvation ( 5 2 ) . The difference in these results could have arisen from differences in the bacterial strains used in the experiments. However, the experimental results of the earlier studies of tryptophan auxotrophs (3.3,34) are consistent with the former finding (56) when the specific activities reported are converted into numbers of molecules of the

344

FVMIO IMAMOTO

enzymes. The relatively high production of the trp E gene product upon tryptophan starvation could be due to either a preferential attachment of ribosomes at a site at trp E or a preferential distribution of a limited amount of tryptophan supplied endogenously to the synthesis of the polypeptides specified by the trp E (56).

B. Sequential Initiation of Translation Evidence that the appearance of the enzymes of an operon after induction or derepression is sequential, beginning with the product of the most operator-proximal gene, appears in the lactose operon of E . coli (57, 58) , the histidine operon of S. typhimurium (59) and the galactose operon of E . coli (60), as well as in the trp operon of E . coli (61, 45). Anthranilate synthetase, the enzyme complex composed of the products of the two most operator-proximal genes, starts to increase within a few minutes after derepression effected by the addition of indole-3-propionic acid to wild-type cells growing in minimal medium a t 37"C,and translation of the most operator-distal gene commences a few minutes later (61). The kinetics of messenger and enzyme appearance after derepression of the trp operon in wild-type E . coli that transcription and translation take place virtually simultaneously (45). Upon derepression effected by the depleting of tryptophan, the trp enzymes appear sequentially in the same order as the structural genes of the operon (Fig. 2, A ) . Comparison of the results of both transcription and translation experiments carried out under the same conditions permit an estimate of the times of completion of transcription and translation a t four points along the operon. At 30°C,the tryptophan enzymes (all but the trp B enzyme were tested), begin to increase about 0.5 minute after transcription of thcir respective genes. Under these conditions, it takes about 8 minutes to transcribe the entire trp operon. Accurate analysis of indoleglycerol-phosphate synthetasc (trp C ) activity was not possiblc duc to the rclativc insensitivity of the assay for this enzyme. In a study of the kinetics of repression of transcription and translation of thc trp operon, the operon was first dcrcpresscd by dcplcting wild-type bacteria of tryptophan and then repressed by thc addition of excess tryptophan (45). All the tryptophan biosynthetic enzymes were repressed sequentially, again in the same order as the sequence of corresponding structural genes in thc opcron. Thc timing of cnzymc rcpression was significantly different from that of the appearance of enzyme after derepression. At 30"C, synthesis of anthranilatc synthctase component I ( t r p E gene product) terminated after 3 minutes, while the synthesis of the trp D and trp C gene products stopped approximately 6 and 8

trp

TRANSLATION AND TRANSCRIPTION OF THE 1

345

OPERON 1

1

1

B

Time of incubation (min)

FIG.2. (A) Sequential derepression of cnzymes for tryptophan biosynthesis. Escherichin eoli K12,wild-type strain (W3110)grown in rich medium was washed with cold minimal medium and then transferred into warmed minimal medium (30°C) containing glucose and nineteen amino acids, omitting tryptophan. Enzymes were assayed a t the indicated time after derepression. Data were taken from Ito and Imamoto (46). A, E enzyme; A,D enzyme; 0 , C enzyme; and 0,A enzyme. (B) Simultaneous appearance of enzymes synthesized during one round of transcription commencing at internal sites in the Irp operon. E. coli K12,wild-type strain (W3110) was derepressed for 1 minute at 30°C and then treated with 5 mM dinitrophenol for 29 minutes. The culture was then cooled rapidly and centrifuged and the cells were suspended in prewarmed minimal medium containing glucose and amino acids including tryptophan. The cell suspensions were shaken vigorously a t 3O"C, and enzymes were assayed a t indicated time of incudation. A ; E enzyme, 0 ; C enzyme, and 0; A enzyme. Data were taken from Imamoto and Ito (66).

minutes, respectively, after the initiation of repression. Under the same conditions, synthesis of trp mRNA for trp E, trp D and trp C was found to stop 1, 3 and 4.5 minutes after repression, respectively. Unexpectedly, synthesis of the trp A product proceeded for an even longer time (12 minutes) than that of the operator-proximal gene products. Synthesis of the trp E product ceased within 3 minutes after repression, in spite of the fact that synthesis of trp E mRNA continued for 1 minute after repression. Since synthesis of the trp D and trp C products stopped 3-3.5 minutes after cessation of synthesis of their respective mRNA's, halt in synthesis of the trp E product was unusually rapid. The authors suggested the possibility that repressor somehow blocks the translation of the operator-proximal portion of the tryptophan mcssenger without interfering with the translation of more operator-distal regions because of a close coupling of transcription and translation in the proximal region. These investigators of the kinetics of derepression and repression of the trp operon in E . coli indicate that translation of the tryptophan messenger is in progress before transcription of the operon is complete,

346

FUMIO IMAMOTO

and point to the possibility that, a t the operator-proximal end of the operon, transcription and translation are closely linked.

C. Simultaneous Initiation of Translation An apparent exception to the sequential mode of expression of the operon was observed with histidine auxotrophs of S. typhiinurium unable to form the intermediate, phosphoribosyl-aminoimidazolecarboxamide. These mutants produce the enzymes of the histidine pathway simultaneously rather than sequentially following derepression (6.2).Subsequent studies showed this result to be due to an unusual manner of translating the polycistronic histidine mRNA (63, 64) and hence can be considered a specific abnormality of translation in these mutants. Recently, an apparent exception to sequential transcription in the trp operon of E . coli was reported (6548).Following treatment of cells with 2,klinitrophenol (a reversible inhibitor of RNA synthesis) in the absence of tryptophan, transcription was initiated a t internal sites in the operon. Addition of the drug to derepressed cells causes a rapid decrease in the rate of trp mRNA synthesis as well as detachment from the operon and breakdown of any preformed mRNA molecules (68). The removal of the dinitrophenol results in immediate resumption of transcription with initiations occurring both a t the t r p promoter located a t the beginning of the operon and internally a t sites within the operon. If the inhibitor is removed in the presence of excess tryptophan, normal initiation a t the promoter is blocked and all newly synthesized trp mRNA molecules made from internal sites of the operon are small in size and contab only operator-distal information. To explain the internal initiation, it was suggested that the RNA polymerase molecules attach a t the beginning of the operon move along the operon without transcribing in the presence of the inhibitor and, upon its removal, resume synthesis of messenger a t their new positions in the operon (66,68). Translation studies were performed to inquire if the drug-induced trp mRNA fragments that lack the operator-proximal portion of the operon and the specific sites for ribosome attachment and initiation of polypeptide synthesis, can initiate translation (66). In contrast with the sequential mode of enzyme appearance after ordinary derepression of the operon, simultaneous initiation of synthesis of the enzymes for trp C and trp A was observed after dinitrophenol treatment (Fig. 2, B) . Initially the rate of synthesis of both proteins was nearly equivalent, but after the fifth minute, synthesis of the trp A product continued while that of the trp C product stopped. This is consistent with sequential termination of the synthesis of trp mRNA corresponding to their respective genes. The lack of significant synthesis of the trp E product verified the occurrence of

TRANSLATION AND TRANSCRIPTION OF THE

trp

OPERON

347

initiation of transcription exclusively a t internal sites within the operon under these conditions. The relative amounts of the trp mRNA and enzymes synthesized during one round of transcription after repression in normal and dinitrophenol-conditioned cells indicate that the t r p mRNA fragments lacking the operator-proximal segment can be translated with almost normal efficiency. Also, the number of trp C and trp A protein molecules accumulated after the completion of one round of transcription initiated a t internal sites along the operon is about proportional to the number of their respective mRNA copies synthesized during this period. It was concluded (65) that not only trp mRNA fragments carrying information for the t r p C , trp B and trp A genes but also those carrying information for the trp B and t r p A genes and for the trp A gene alone can be translated efficiently. In the t r p operon, translation immediately follows transcription. This makes it likely that the first ribosome attaches to the principal ribosome binding site of the nascent mRNA molecules soon after this region has been synthesized. Ribosomes attach to and then apparently move along the mRNA chain as its synthesis proceeds. This demonstrates seemingly only one attachment site for ribosomes a t the beginning of the polycistronic trp mRNA molecule. When transcription begins a t internal sites, however, translation appears to be initiated a t other sites on the trp mRNA fragments. This could occur at or near the translational initiation site in each cistron accessible to ribosomes.

D. Quantitative Aspects of Translation Morse e t al. (61) studied translation of the t r p operon of E . coli using the tryptophan analog indolc-3-propionic acid to derepress the operon in cells growing in minimal medium. By a combined use of this derepression and tryptophan repression, they obtained synchronous and limited transcription, and thus obtained a measure of the total yield of the anthranilate synthetase (products of the t r p E and trp D genes) and tryptophan synthetase SY (product of the trp A gene) molecules per trp mRNA molecule. A stepwisc increase of the amount of product of trp A was obtained by the addition of tryptophan a t various times after the derepression. They assumed that this one-step increase in capacity results from the synthesis of to synthesize tryptophan synthetase onc copy of trp mRNA; i.c., a fixed number of ribosomes translates this messcngcr. The number of enzyme molecules synthesized by translation of each trp mRNA molecule was estimated to be about 100. An equal number of molecules of anthranilate synthetase were synthesized in the same cells. Subsequently, it appeared that about 16 RNA poly(Y

348

FUMIO IMAMOTO

merase molecules can transcribe simultaneously one trp operon, and that the estimate of 100 ribosomes translating each mRNA should be revised to about 20 (69).It has been repeatedly observed that translation of the derepressed trp operon proceeds with a fixed periodicity (61, 7 0 ) . However, from studies on the kinetics of sequential appearance of the tryptophan-synthesizing enzymes in E. coli (&, 56) and of histidinedegrading enzymes in B. subtilis (71),and on the kinetics of appearance of /3-galactosidase in E. coli ( 7 9 ) , some question has arisen concerning the reproducibility of a definite periodicity of initial translation after derepression and induction. More recently, it was reported that, in growing culture of the trp Rstrain, about 30 ribosomes translate the tryptophan synthetase region of each trp mRNA molecule (73).Comparable estimates for derepressed cultures of a trp R+ strain suggest that severalfold more ribosomes than in log phase cultures of the trp R- strain translate each mRNA; i.e., transcription initiations occur with a frequency of 5.1 initiations per operon per minute during the first minute of derepression and 1.1 initiation per operon per minute at steady state, comparing with a frequency of ea. 2.6 initiations per operon per minute in exponentially growing trp R- cells, and the rate of synthesis of tryptophan synthetase .a! protein is ca. 460 molecules per cell per minute (or 250 molecules per genome per minute) in derepressed trp R+ cultures, a value approximately three times that of the trp R- culture (73).The interpretation was that there is a longer functional as well as chemical mRNA half-life under derepression conditions than during logarithmic growth of the trp R- bacterium (73).If degradation of trp mRNA is randomly initiated on the growing mRNA chain ( l o r ) ,any estimate of the number of ribosomes translating each mRNA would be subject to fluctuations. Measurement of the rate of ribosome movement along the trp mRNA molecule by measuring times for the commencement of appearance of anthranilate synthetase and tryptophan synthetase in E. coli yield an estimate of about 1300 nucleotides per minute a t 37°C ( 6 1 ) .The kinetics of sequential translation of the trp operon in E. coli indicate that the rate of ribosome movement along the messenger is ahout 900 nurleotidcs per minute a t 30°C (45). .(Y

IV. Transcription of the trp Operon A. Detection of trp mRNA The transfer of genetic information from gene to protein is mediated by messenger RNA. A specific mRNA coded by a defined genetic region

349

TRANSLATION AND TRANSCRIPTION OF THE tTp OPERON

has a base sequence complementary to one of the two complementary DNA strands of this region. The isolation of specific mRNA's for bacterial operons has become possible by DNA.RNA hybridization, using DNA from various temperate phages in which genes of the operon adjacent to the phage attachment site have been incorporated into the phage genome by apparent mispairing during excision of the prophage (41, 42, 74-76). By mixing and annealing bacterial RNA with singleTryptophan operon

E

Operator end Gene sequence

ki i i :...

i i i i

D

:'

'

'

."

v C

B

A

Polar mutants

.. :. .. ::

Deletion mutants

-

A E ~ ii 0AEl4 4 AE5 OAE2 KAE44 OAE 5 AE9 AE 15 BE40 OAE8 AEI4 AD28 AD5 Dt E

. . . ::: ..... .. .. ..:. i : .; :. .. : . ;iii ii :: .:: . .. i $ : - i ... . .:: .. . i: ,. ::: ; ... .. .. . .i ii . :. ,.. .' . . ... : .i . ::i:..i i .. ..:: i.. :

:: ::

i r

7

-

i

*

i:: iii

:

1

i : !ii ; : ;': J:;

' : i*

:

r i :

b

+ 8 0 phages pt-A-C pt A

-

pt E-D

FIG.3. Tryptophan operon segments in deletion mutants and 980 pt's. The order of mutationally altered sitcs were based on previous studies (34, 81) and personal communication from C. Yanofsky. T r p operon segments carried by the deletion mutant or the $80 pt are indicated by a solid line. The position of the deletion terminus is based on previous studies (4S, 1OS, 116). The relative size of the irp genes and distances between the mutant sites arc only approximate. Phages were isolated by A. Matsushiro ($80 and pt E distal). S. Deeb and B. Hall (pt C-A), K. Sat0 ( p t E-D), J. P. Gratia (pt. E), C. Ynnofsky (pt D) and M. Taylor (pt A). Type AE deletion mutants and the various polar mutants were isolated by C. Ynnofsky ; type OAE deletion mutants wcre isolated by J. Ito. Deletion mutant, KAE44, was isolated by Y. Kano.

350

FUMIO IMAMOTO

stranded DNA formed by heating the double-helical DNA of the phage, artificial DNA-RNA hybrids are specifically formed, which can be collected and assayed. The availability of various nondefective derivatives of phage $80, ($80 pt’s) (do), carrying all or part of the trp operon (Fig. 3) has made possible a detailed study of the regulatory mechanisms governing transcription of the trp operon. With the DNA from these phages, the hybridization reaction effectively distinguishes between the different states of regulation of the bacterium (i.e., repression and derepression) since significant production of trp mRNA (i.e., mRNA specifically hybridizable with DNA from pt phages) is detected only under derepression conditions (41, 42). Pulse-labeled RNA prepared from a derepressed culture of wild-type cells (m3110) makes varying amounts of DNAsRNA hybrid with DNA from various pt phages (Table I), reflecting presumably the relative length of the segment of the trp operon carried by each transducing phage. With this assortment of phage DNA’s, it was possible to demonstrate that the trp mRNA extracted from various deletion mutants lacks those mRNA segments corresponding to those genes missing in the deletion mutant. For example, RNA extracted from trp AE1, a deletion mutant TABLE I DETECTION OF THE trp mRNAa

DNA source 480

PtE ptE distal ptED ptC-A

Radioactive material tixed on the filter (CPd

89f 2 1,357 f 39 381 f 18 2,252 f 41 1.123 f 2

Percentage of added radioactive material fixed

(%) 0.023 f 0.001 0.355 f 0.010 0.099 f 0.005 0.590 f 0.011 0.294 f 0.001

trp mRNA value’

(%I 0.332 0.076 0.567 0.271

a RNA was prepared from derepressed or repressed culture of Escherichiu wli K12, wild-type strain (W3110),pulse-labeled with [3H]uridine for 20 seconds a t minute 18 of incubation; 58 gg of [aH]RNA (specific activity, 6630 cpm/#g) was annealed with 5 pg of heat-denatured phage DNA in a 0.25 ml reaction mixture. The values reported are the averages of duplicate determinations from derepressed cultures and the variation in each assay is represented as f . The background values (pt DNA value - 480 DNA value) found with [PHIRNA prepared from repressed cultures were 0.005%, O.OOO%, 0.012%, and 0.033% with DNA from ptE, ptE distal, ptE-D and ptC-A, respectively. Data were taken from Imamoto (49). a The background values with 480 DNA were subtracted from each hybrid value with pt DNA.

TRANSLATION AND TRANSCRIPTION OF THE

trp

35 1

OPERON

TABLE I1 DETECTION OF THE SPECIFIC trp E mRNA0 Trp mRNA hybridizable with DNA from ~

RNA source

ptE

ptE-D

ptC-A

trp A E l trp AE6

0 81

0 63

2 4

a RNA was prepared from derepressed cultures pulse-labeled for 2 minutes a t the tenth minute of incubation at 30°C (Imamoto, unpublished data). Other conditions were essentially as in Table I. Values are expressed as % x 108.

lacking all the genes of the trp operon, contains no significant amount of RNA hybridizable with the transducing phage DNA’s (Table 11).A deletion mutant, t r p AE5, which retains the operator-proximal portion of the trp E but lacks the other genes of the t r p operon, produces RNA hybridizable only with DNA from phages carrying the trp E (Table 11). A phage, ptEdiBtnl,which carries only the operator-distal segment of the trp E has been used to examine the possibility of the detection of trp mRNA corresponding to a part of the trp E . Hybridization experiments with RNA from deletion mutants t r p AE5, trp AE8, t r p AE11 and trp AD%, (see Fig. 3 for deletion termini) demonstrated that only those mutants whose deletion termini leave intact all or part of the distal trp E region carried by the ptEdistalphage produce mRNA hybridizable with DNA of this phage (Table 111). This unequivocal correspondence between the genetic characters of the deletion strains and the pt phage DNA’s in the hybridization reaction shows that the RNA hybridizable with the phage DNA containing the t r p TABLE I11 DETECTION OF THE SPECIFIC mRNA FOR THE OPERATOR-DISTAL PORTION OF THE trp E GENE^ trp mRNA hybridizable with DNA from

RNA source

PtE

ptE distal

trp AE6 trp AE8 trp A E l l trp AD28

129 335 456 495

0 8 72 100

a RNA was prepared from derepressed cultures pulse-labeled for 2 minutes at the twentieth minute of incubation a t 30°C (Imamoto, unpublished data). Values are expressed as yo x 103.

352

FUMIO IMAMOTO

genc segments is truly trp-specific mRNA carrying genetic information for the structures of the proteins of the tryptophan system. This direct method of hybridization using total pulse-labeled RNA and excess concentrations of DNA is in general appropriate to test the dynamics of trp inRNA metabolism. Quantitative measurement of the intracellular level of trp mRNA a t a steady state of trp inRNA mctabolism has been obtained by an indirect competition assay using unlabeled trp mRNA from the strain in question in competition with purified labeled trp inRNA for the trp sequences on $80 pt DNA (77).

B. Characterization of trp mRNA The coordinate control of expression of the genes in an operon is explicable by a model for operon expression, the central features of which arc (a) the transcription of the entire operon into a polycistronic mRNA molecule, and (b) the specific regulation of operon expression a t the level of synthesis of this mRNA. The possibility that operon mRNA is polycistronic is strongly supported by results of studies on the size of galactose ( 6 0 ) , the specific mRNA for the lactose (78, 79), histidine (80), and tryptophan (42) operons, and verified by the demonstration of the polycistronic nature of the trp operon mRNA ( 4 9 ) . Knowing the molecular size of the largest mRNA specific for a particular operon was considered significant in testing the possibility of polycistronic mRNA. The trp mRNA prepared from wild-type bacteria pulse-labeled for a short period under conditions of steady-state transcription of the operon exhibits a heterogeneous distribution when sedimented in a linear sucrose density-gradient (Fig. 4, a ) . This heterogeneity was assumed to be due to the labeling of a population of trp mRNA molecules of varying length caught in the process of synthesis a t different stages of completion a t the time of the pulse (@). Resedimentation of the trp mRNA molecules taken from several faster sedimenting regions of the first gradient resulted in profiles in which the distribution of the molecules was always shifted back to a more slowly sedimenting region with peaks in the region between 31 S and 35 S (Fig. 4, b-d). The total molecular weight of the polypeptides specified by the operon is estimated to be 244,000, equivalent to about 2400 amino acids and 7200 nucleotides. This value corresponds to a mRNA molecular weight of the order of 2 X loe, or about 33s. The size of the largest mRNA molecules in the pulse experiment is therefore about that necessary to carry the information from all of the genes of the trp operon. Another way used to verify the one-operon-one-messenger hypothesis is to examine if the molecular size of intact trp mRNA is larger than that of mRNA from bacteria having deleted regions in the trp operon. I n this study, it was hoped that the sedimentation profiles of the trp

TRANSLATION AND TRANSCRIPTION OF THE

20,000

trp

OPERON

353

400

i0,ooo .-C

\InE f

o

1

500 (cl

{ 0.75

400

300 200 100

0 Froction no.

FIG.4. Determination of molecular sire of trp mRNA. (a) '2P-Labeled W3102 ( E . coli K12, wild-typc strain) RNA pulse-labeled for 3 minutes 7.5 minutes after derepression was sedimented in a 520% linear sucrose gradient. (b-d) To A, B and C fractions shown by the arrows in (a), cold carrier RNA was added and precipitated with ethanol. Each precipitated RNA was sedimented. Data were taken from Iniamoto et nl. (41). O---O, hybridization with DNA from phage ptl (carrying frp D-lrp A genes) ; A---A, hybridization with DNA from phage $80 (background difference between these two counts (trp mRNA) ; ---, optical value) ; .----@, density at 260 nm.

mRNA from various deletion mutants would reflect the locations of the deletion termini in the operon, although logically this need not be the case. A number of deletion mutants with deletion termini in the vicinity of the end of the trp E and the beginning of the trp D were examined (42, 68, 81). The trp mRNA from these mutants sedimented more slowly than 19S, showing that most of the molecules are very much smaller than normal. From these and other observations, the size of the trp mRNA segment corresponding to the intact trp E gene was estimated to be 16.5 to 19.5 S with a calculated molecular weight of 5.8 to 7.6 X lo" (68). The trp E product has a subunit molecular weight of 60,000 (21) equivalent to 600 amino acids and thus to 1800 nucleotides. This value corresponds to a mRNA of molecular weight of 5.4 to 5.7 X lo5. The experimental observation is therefore in agreement with this predicted value. The trp mRNA carrying information for trp C , trp B and trp A was also shown to bc smaller than intact trp mRNA (4.2). Further evidence for the polycistronic trp mRNA comes from the

354

FUMIO IMAMOTO 1

1 X c

- 200 $.-

.-

h

<s

U v

Y

1

P

b 2 500 -

CT

P

- 100 5

.E

.b ._ 1

3 :4 Y

Y U

0

.o 2 0

20

10

0 30

f

%

Fraction number

FIG.5. Sedimentation profile of trp mRNA synthesized under steadystate conditions following derepression. RNA was prepared from Escherichia coli K12, wildtype strain (w3110) pulse-labeled with C'HIuridine for 1 minute at the fifteenth minute after derepression and sedimented in a linear 5-3070 sucrose gradient. Data were taken from Imamoto (66). --, Optical density at 260 nm; -, radioactivity of C'HIRNA; O---O, trp E-D mRNA; 0-0, t r , C-A mRNA.

observation that the trp mRNA species including the mRNA segment that carries information for the operator-distal genes (trp C , B and A ) are larger than those species including the mRNA segment for the operator-proximal genes (trp E and D ) (&), When t r p mRNA prepared from bacteria pulse-labeled for a very short period during steady-state derepression and collected immediately was sedimented, most trp mRNA molecules for the trp C , trp B and trp A sedimented faster than those for the trp D and trp E (Fig. 5 ) . This indicates that the C-A region of the mRNA occupies the operator-distal portion of the polycistronic trp mRNA molecule, and thereby also indicates that messenger synthesis is initiated a t the trp E end and proceeds to the trp A end of the operon

(@I

-

The existence of a polycistronic trp mRNA is further supported by the findings that the majority of the trp mRNA molecules from nonsense mutants of the trp operon are deficient in the mRNA segments corresponding to the genes of the operon on the operator-distal side of the mutated gene and that the sine of these molecules decreases as the distance from the beginning of the operon to the site of the mutational alteration decreases (68,81).

C. Initiation of Transcription The first step in operon expression is the initiation of transcription of the genes into mRNA. The current concept of transcription initiation

TRANSLATION AND TRANSCRIPTION OF THE

trp

OPERON

355

is that transcription begins a t the promoter, an element closely associated with the operator (82, 83), and regulates the rate of initiation of transcription of the operon (82,84,85). Deletion of the operator-proximal region of the histidine operon results in a marked reduction in the level of expression of the remaining genes of the operon, due to the loss of the initiating region (86). Most of the lac mutations were found later to be not deletions, but double mutants. In the trp operon of S. typhiinurium, strains having deletion mutations extending from outside into the most operator-proximal gene (trp A ) of the operon lack expression of the next gene (trp B ) , which is still physically intact, yet retain a low constitutive level of function of the last three genes, trp El D, and C , thereby indicating the existence of a relatively low efficiency, internal initiator element (Pa) controlling Several lines of evidence expression of the last three genes (60,51,87-89). indicate that the site of the principal initiator of expression of the trp operon is in the vicinity of the operator-proximal, first-gene terminus. 1. INITIATION AT THE PRINCIPAL trp PROMOTER

Synthesis of the polycistronic trp mRNA initiates virtually a t a single site (promoter) located at the operator-proximal end of the operon (42, 45-47). Following derepression effected by transferring bacteria grown in a rich medium to a minimal medium lacking tryptophan, the synthesis of trp E mRNA begins without delay and the synthesis of trp D, trp C-A and trp A mRNA lags 2, 3 and 6 minutes behind that of trp E mRNA (Fig. 6 ) . This indicates that transcription initiated at the trp E end progresses sequentially to the trp A end, taking about 8 minutes, under these conditions, for completion of one full-length trp mRNA molecule. The largest moleculc of trp mRNA detected to date has a sedimentation value estimated at about 33 S, which, as indicated earlier, agrees with the total molecular weight of the polypeptides specified by the operon. This consideration supports the view that there is but one principal initiation site for transcription at or near the extremity of the operon. It was also indicated that, following derepression, the trp mRNA molecules exhibit a gradual increase in size during the first round of transcription to the maximum size exhibited by the trp mRNA synthesized during the steady-state period (46). The orientation of the operon rclativc to the regions specifying the amino- and carboxy-terminal ends of the tryptophan synthetase A protein has been established (90,91): the region of trp A corresponding to the amino-terminal end of thc protein is closest to the operator end. This orientation is consistent with the well established facts that (a) synthesis of the polypeptide chain progresses from the amino- to carboxy-

356

FUMIO IMAMOTO

-a

$0.3

z K

E

Q

h

0.2

r

0

c

c

3

g

0.t

a

0

0

5 Time after depression

FIQ.6. Sequential transcription of the trp-operon after derepression. RNA was prepared from E . coli K12, wild-type strain (W3110) pulse-labeled with C'Hluridine for successive 1 minute periods after derepression at 30°C. The horizontal bars represent the pulse periods. Data from Imamoto (66). 0, trp E mRNA; trp D mRNA; 0 , trp C-A mRNA; 8,trp A mRNA.

a,

terminal end (92), (b) translation of mRNA is from the 5' to the 3' end (93-95) and (c) synthesis of mRNA is from the 5' to the 3' end (96-98). 2. QUANTITATIVE ASPECTOF INITIATION

Although repressor regulation of the extent of transcription of bacterial operons is at present reasonably well understood (43, 82, 83, 99101),relatively little is known about the mechanism that determines the proper frequency a t which the RNA polymerase is to transcribe the operon. It has been suggested that the promoter determines the maximum rate of expression of the operon, since mutational alterations of the promoter region of the lac operon markedly reduce the maximum level of expression of the operon (82, 86, 102). It is possible that some unknown factors interact a t the promoter, thereby determining the rate of attachment of RNA polymerase molecules to the binding region of an induced or derepressed operon and/or determining the rate of the initiation of transcription of the operon. Knowing the absolute number of trp mRNA molecules present in a growing bacterial cell is necessary for a quantitative appraisal of the rounds of trp transcription. For trp mRNA in bacteria, one minimum estimate is eight polycistronic trp mRNA molecules per cell for wild-

TRANSLATION AND TRANSCRIPTION OF THE

trp

OPERON

357

type cells grown under derepressed conditions, as compared to two per cell for wild-type cells repressed by tryptophan and 25 per cell for a trp R- (repressor-deficient) strain (77). By direct hybridization of pulselabeled RNA with $80 pt DNA, Edlin et al. (103)estimated that the steady-state number of t r p mRNA molecules per cell during derepression of the trp operon is about 4 4 . The level of trp mRNA in bacteria growing in minimal medium at 37"C, estimated by measuring competitive binding of labeled and unlabeled mRNA molecules to sites on 980 pt DNA, is 14 molecules of messenger in the derepressed trp R+ strain and 6 molecules in a trp R- strain (104). Improved methods for determining the t r p mRNA content of a given preparation of RNA labeled under conditions where the specific activity of the pyrimidine pool can be estimated gave steady-state levels of 6.5 trp E mRNA equivalents1 per cell for the trp R- strain grown in minimal medium a t 37°C (69). The variation in these results might arise from differences between strains, growth conditions, and techniques. A rough calculation of the number of mRNA molecules being synthesized on any one trp operon at any one time is possible. The half-life of trp mRNA is estimated to be approximately 1 and 2 minutes under the conditions employed by Baker and Yanofsky (69) and by Lava116 and DBHauwer (104), respectively. A tentative conclusion to be drawn from these two estimates of the level of trp mRNA is, therefore, that a trp R- cell must synthesize three trp E mRNA (69) or 1.5 polycistronic trp mRNA (104) equivalents every minute, and a derepressed trp R+ cell must synthesize 3.5 polycistronic trp mRNA equivalents every minute. If one assumes that each cell contains on the average two copies of the trp operon (106)and 3 minutes are required for a round of transcription of the operon under their conditions (69),the number of polycistronic trp mRNA molecules attached to any one trp operon is calculated to be 2 4 . 5 (for the trp Rstrain) or 5 (for the derepressed trp R+ strain) equivalents. A determination of the absolute amount of trp E mRNA synthesized in a cell derepressed for 1 minute, during which degradation of the trp mRNA synthesized is assumed to be insignificant, gives an estimate of 5.4 equivalents of trp E mRNA in a cell (69). This value corresponds to eight equivalents of polycistronic trp mRNA attached to any one trp operon, considering the foregoing assumptions. If polymerase molecules are spaced evenly on the t r p operon, then, on the average, a polymerase molecule would have one half of an intact polycistronic trp mRNA attached to it. Thus, 16 polymerase molecules could simultaneously transcribe one trp operon. The initiation frequency is reportedly higher One equivalent is defined as one or more pieces of trp mRNA whose combined radioactivity is equivalent to that of a single full-length trp E mRNA (69).

358

FUMIO IMAMOTO

during the first minute of derepression than in a steady state-derepressed trp R+ or a rapidly growing trp R- culture (73). Comparable estimates for derepressed cultures of a trp R+ strain gave 5.1 initiations per operon per minute during the first minute of derepression, 1.1 initiation per operon per minute at steady state and 2.6 initiations per operon per minute in exponentially growing trp R- cells. Other attempts have been made to determine the frequency of transcription initiation. Using techniques of pulse-labeling for very short successive periods during the initial stage after derepression (46) or of labeling for successive periods during a single round of transcription initiated by addition of the tryptophan analog indole-3-propionic acid ( 4 7 ) ,periodic changes of transcription rates of the operon were observed during the first round of transcription after derepression. An essentially similar phenomenon occurs in the lac operon of E . coEi (I&?). The simplest interpretation of these observations is that transcription initiations occur regularly every 3.5 to 4 minutes (46') or a t 2.5 minutes (47) depending on the conditions used, taking 8-10 minutes (at 30°C) or 6.5 minutes (at 37"C), respectively, for a round of transcription of the operon. It must be concluded from such periodicity that three mRNA molecules are being synthesized on any one trp operon at any one moment. The discrepancy in the conclusions concerning the number of spaced polymerase molecules on the trp operon from these experiments and those just described (69), might be due to different physiological conditions of the bacteria used in the experiments. For example, cells treated under the experimental conditions employed by Imamoto, in which bacteria were derepressed by transferring from rich medium to minimal medium with an intermediate washing of the cells with cold minimal medium (free of tryptophan), grew very slowly during the initial period after derepression. Such a temperature shock for cells for even a short, time causes a marked slowdown in both synthesis and degradation of lac mRNA, especially in the initial stages of incubation after transfer of cells to 35°C (IOSa). This slowdown might also affect the frequency of transcription initiations of the operon. It should be mentioned that some difficulty in the reproducibility of the defined periodicity has been noted (69). Mosteller et al. (107')measured the relative amounts of trp mRNA in trp R- bacteria growing with a 6- to 7-fold variation in cell growth rate, which was effected by changing the carbon source. The amount of trp mRNA relative to genome was estimated to be decreased 8- to 11-fold in proline-grown cells compared to glucose-grown cells. The reduction in the amount of trp mRNA was ascribed to a decreased transcription initiation frequency because trp mRNA chain elongation and degradation

TRANSLATION AND TRANSCRIPTION OF THE

t?'p

OPERON

359

rates were nearly constant, regardless of the large variation in the cell growth rate (107). Some information is available concerning the effect of deletions of large segments of the operator-distal portion of the operon on the level of transcription of the trp operon. This is particularly important in determining whether the rate of transcription of a given operon is governed solely by the nature of interactions occurring a t a specific restricted region of the operon at the operator end. The production of trp mRNA in deletion mutants that retain more than one-tenth of the operatorproximal region of the trp operon increases in proportion to the length of the operon segments present in these mutants (46, 108). This indicates that these deletions do not affect the rate of initiation of transcription. However, deletion mutants that, retain less than one-tenth of the operatordistal region of the operon produce trp mRNA fragments that are present a t a significantly higher level than normal and that are remarkably unstable (67, 6 8 ) . A similar phenomenon is also observable with nonsense mutants, which may cause premature termination of transcription a t or near the site of the mutational alteration (67, 6 8 ) . Such transcriptional events are interesting in view of the possibility that the opening up of the DNA duplex corresponding to the initial segment of the transcription unit might be involved in the mechanism determining the rate of transcription of the unit. 3. THE INTERNAL PROMOTEM OF

THE

trp OPERON

Investigations with the trp operon of S. typhimurium and E . coli reveal the existence of a relatively low-efficiency promoter element (P2) a t or near the boundary between the second and the third genes of the operon, in addition to the principal promoter (Pl) in the vicinity of the operator region. The trp operon of S. typhimurium appears to be composed of two independent units (trp A-trp B and trp E-trp D-trp C), each possessing a promoterlike initiator element, but the entire operon appears to function as a unit with respect to regulation by tryptophan, possessing a single operator region at the trp A end (35, 5 1 ) . Polarity mutations in the most operator-proximal gene (trp A ) affect not only the function of the next gene ( t r p B ) , but also of the last three genes (trp E , trp D and trp C ) . However, the effect on trp B is more severe and in even the strongest trp A polarity mutations an appreciable basal level of activity for the distal three genes persists. Deletion mutations extending into the operon from the operator end (trp A side of the operon) and ending in trp A eliminate expression of trp B but retain a low constitutive level of function of the distal three genes (50, 51). Deletions ending in the second

360

FUMIO IMAMOTO

gene (trp B ) yield essentially an identical result. However, expression of the distal three genes is completely lost in strains where the deletion extends past the boundary between the second and the third genes, terminating in the third or the fourth genes. Also, the basal repressed level of the distal genes is decreased in strains with internal deletions, eliminating the boundary between the second (trp B ) and the third (trp E ) genes. These findings suggest that the apparent noncoordinate synthesis of the enzymes of the trp operon results from the presence of the second promoterlike initiator element (P2) at or near the trp B-trp E boundary (60, 61). The level of constitutive synthesis of enzymes of the distal three genes is 2 4 % of the level of those enzymes synthesized by a partially constitutive, 5-methyltryptophan-resistant (repressor-deficient) mutant grown in the presence of tryptophan (36).The constitutive enzyme levels present in this mutant are three to four times lower than those obtained in derepressed (chemostat-grown) tryptophan auxotrophic bacteria. A homologous, low-efficiency promoter-like element exists between the second and the third genes of the trp operon of E . coli ( 6 2 ) . As in S. typhimurium, polarity mutations in the two most operator-proximal genes fail to reduce the low-level constitutive expression initiated by the internal promoter, while the polarity mutants in the third and the fourth genes produce the appreciable negative pleiotropy on the expression of the most operator-distal gene of the operon. Comparison of the enzyme levels of a maximally repressed culture with those of a culture of fully derepressed trp R- constitutive cells indicates that constitutive P2 function is approximately 2% of the maximum rate. P2 is located within trp D ; P2 maps near the operator-distal end of trp D on the operatorproximal side of two trp D point mutants (109). Since low-level constitutive expression of the operator-distal three genes is apparently not regulated by the tryptophan repressor, the internal initiator (P2) seems to serve as a transcriptional initiator (i.e., a promoter) rather than as a special translational initiator (61, 6 2 ) . The existence of two analogous internal initiators in the his operon of S. typhimurium has been observed (110). A homologous internal initiator can be created by mutation within the trp operon of S. typhimunum. Mutants with deletions terminating inside trp A lack expression of trp B yet retain expression of the operator-distal three genes a t a low constitutive level. Phenotypically trp B+ derivatives have been obtained by the induction of secondary mutations (called h i ) from such deletion mutants and it has been proposed that these strains had acquired new initiator elements that restore expression of the intact but promoterless trp B (60). Further study of the genetic

TRANSLATION AND TRANSCRIPTION OF THE

trp

OPERON

361

and physiological characteristics of these mutagen-induced initiator elements indicates that the Ini mutations can be created by a single DNA base-pair change in several regions of the nucleotide sequence of the trp A resulting in constitutive functioning of the distal four genes (111).Initiation at the Ini sites is a t the same low level as the naturally occurring internal initiator (P2), and is additive with P2 for the last three genes. Since expression of h i mutations is constitutive, being independent of derepression of the operon, and since the mutations do not require the proximity of a translation-terminating mutation for function, they differ from the translational “restart” mutations described in the rII region of bacteriophage T 4 (112) and in the lactose operon of E. coli (113). It is concluded that Ini mutations are probably low-efficiency promoter elements serving as initiators of transcription (111). Ini strains in which the trp operon is normal except for the retention of the Ini mutation in the trp A have been constructed (111). Since these recombinants are able to translate the Ini mutation as an apparent missense sequence, determination of the amino-acid replacement and the sequences neighboring the change might offer some idea of the base sequence and chemical properties of a t least one type of promoter (111). Similar initiator mutations have been observed in the histidine operon of S. typhimurium (114).

There is a promoterlike element, arising by spontaneous mutation, within the first structural trp E gene of the trp operon of E. coli (116). Unlike the Ini mutations, this mutation (trp E R ) inactivates the t r p E enzyme and simultaneously causes a partially constitutive functioning of the four operator-distal genes a t about 30-50% of the maximal level observed for repressor-deficient (trp R-) constitutive mutants. The loss of trp E function caused by the trp E R mutation is not suppressed by nonsense suppressors. The constitutivity of the trp ER mutation is not influenced by the introduction of a strong polar mutation preceding it in trp E. It was concluded that the trp ER mutation apparently creates a high efficiency initiator in the first gene of the trp operon which functions at the level of transcription (116). The internal initiation of transcription near the trp E R mutational site was observed by hybridization studies of trp mRNA synthesized in this mutant, using DNA’s from $80 pt phages carrying different segments of the trp operon. Since the t r p E R mutant has the same constitutive level of tryptophan synthetase when grown in rich medium (L broth) as when grown in minimal medium containing tryptophan alone, it was assumed that the promoterlike element of trp E R does not include a “foreign” operator controlled by a corepressor other than tryptophan (116). It is possible, however, that the mutation involves insertion of a foreign “promoter” internal to the trp operon

362

FUMIO IMAMOTO

analogous to the insertion in the absolute polar mutants of the lac and gal systems (116-118). Callaham and Balbinder (119) isolated a mutant that is capable of utilizing anthranilic acid (substrate for the second enzyme of the tryptophan pathway) as a growth factor only in the presence of the analog 5-methyltryptophan, normally a potent growth inhibitor and corepressor of synthesis of tryptophan biosynthetic enzymes. The mutation maps in the “unusual” region between trp A and trp B in 8. typhimurizlm. The reason for this peculiar phenotype is explained by the hypothesis that the mutation creates a promoter of transcription for constitutive expression of the four operator-distal genes when the principal trp promoter (Pl) is inactive due to repression caused by tryptophan or 5-methyltryptophan. When P1 is available for transcription in derepressed conditions, the mutation is read as a structural gene mutation in the “unusual” region and also acts as a terminator of transcriptions originating a t P1,resulting in an extreme polarity effect for the four distal genes. Since mutagenesis by agents presumed to cause a single base change in the nucleotide sequence of DNA can apparently create functional promoter sites, it would appear that initiation signals for transcription are extremely short. This is not unreasonable since a single base change in the lac promoter can drastically reduce its function (81, 86, 101).The significance of naturally occurring, low-efficiency internal promoters might be important with respect to the evolutionary development of mechanics of gene organization in bacteria (6).Alternatively, the production of a rclatively small number of messengers initiated a t specific sites within the operon might reflect an obligatory “leakiness” a t a distinct stage of the bacterial cell and replication cycles (36). 4. INITIATION AT SITESWITHIN

THE

OPERON

I n bacterial operons, the cistron nearest the operator is the first to be transcribed following induction or derepression, followed by the others in order until the transcript of the most operator-distal cistrbn appears. An apparent exception to this sequential mode of transcription has been observed in the trp operon of E. coli (66-68). When bacteria (E. coZi wild-type strain, w3110 K12, or CRM producing mutant trp A9952, K12) growing in the absence of tryptophan are treated with 2,4-dinitrophenol a t a concentration of 5 mM, thc transcription of the derepressed trp operon begins to slow immediately and stops altogether after a short time. The inhibition by dinitrophenol of the synthesis of RNA by RNA polymerase was first reported by Gros et al. (I&?). The possibility that the apparent decrease in the rate of transcription might be caused by rapid degradation of trp mRNA

TRANSLATION AND TRANSCRIPTION OF THE

trp

OPERON

363

molecules in the presence of the drug has been excluded (66,68).Following 20 or more minutes of exposure to dinitrophenol a t 30°C, when trp mRNA synthesis has ceased altogether, removal of the inhibitor results in immediate restoration of trp mRNA synthesis (attaining an almost normal rate within 1 minute) with initiations occurring a t the normal site (Pl) and at sites throughout the operon. I n the following, two typical experimental conditions are introduced as examples for the demonstration of apparent internal initiation of trp mRNA synthesis. a. Treatment of the Operon during Steady-State ,Transcription (66, 68). Dinitrophenol a t 5 mM, was added to a derepressed culture (to assure steady-state transcription of the operon) , and the culture was then incubated for 45 minutes a t 30°C. The addition promptly inhibited synthesis of both bulk RNA and trp mRNA. After 5 minutes, more than 96% inhibition was observed, and by 10 minutes, the synthesis of RNA had ceased altogether. Although degradation of trp mRNA slows down under these conditions, the bulk of the trp mRNA molecules synthesized before the addition of D N P detach from the operon and are broken down during incubation with the inhibitor for longer than 30 minutes. However, if a shorter period of incubation is used (15-30 minutes), some incomplete trp mRNA molecules remain attached to the operon after relief of inhibition. After the removal of dinitrophenol, the bacteria were quickly transferred into prewarmed standard medium including tryptophan and were pulsc-labclcd with [ R H ] ~ r i d i nduring c the first minute of incubation at 30°C. Tryptophan blocks ordinary initiation of transcription at promoter P1 but does not interfere with transcription in progress along the operon. The constitution of the trp mRNA synthesized during the first minute after the removal of D N P was then compared with that of the trp mRNA synthesized during the dcrepressed steady state by hybridization using the various $30 pt DNA's (Table I V ) . It was found that transcription subsequent to drug removal commences a t sites throughout the entire operon in contrast to the ordinary initiation of transcription a t the site of the trp E extremity (Fig. 6 ) . Initiation of transcription a t internal sites is remarkably efficient in the operator-distal region trp C-A and trp A mRNA, though somewhat less so in the operator-proximal region (trp E and trp D mRNA) . If internal initiation occurs, as thc results presented above imply, it would be expected that the trp mRNA molecules carrying the operatordistal information, synthesized during the first minute after relief of D N P inhibition, would be smaller than thosc synthesized in the steady state. This expectation was verified. When trp mRNA was !abeled for 1 minute at steady-state transcription of the operon, trp C-A mRNA

364

FUMIO IMAMOTO

TABLE I V COMPARISON OF trp mRNA LEVELS DURINQ STEADY-STATE DEREPRESSION AND AT THE INITIAL STAGEAFTER INTERNAL INITIATION OF TRANSCRIPTIONO Amount of trp mRNA synthesized (% x 108) Condition

trp E

trp D

Steady state Internal initiation

442 25

161 60

tTp

C-A

322 248

trp A

88 91

0 RNA was prepared from a CRM producing mutant (C 9941) of E . coli K12 pulselabeled with [3H]uridine for 1 minute at the twentieth minute after derepression (steady state), or pulse-labeled under conditions of repression during the first minute after the removal of dinitrophenol (internal initiation). Bacteria were incubated in the presence of the drug, added at the tenth minute after dereprwion, for 45 minutes. Repression W&B effected at the time of removal of the drug by the addition of tryptophan. (Imamoto, unpublished data.)

sediments faster than trp E - D mRNA indicating that the trp C-A region occupies the operator-distal portion of the polycistronic mRNA (Fig. 5 ) . On the contrary, trp C-A mRNA labeled in the inhibitor-conditioned state was extraordinarily small, sedimenting in the same region as trp E-D mRNA (Fig. 7).These trp mRNA molecules synthesized during 1 minute after relief of the inhibition were estimated to correspond in length to one-half to two-thirds of the trp E, the transcription of which would require 1 minute or so under the conditions used in these experiments. The conclusion from the foregoing is that after treatment with dinitrophenol the synthesis of trp mRNA is initiated simultaneously a t various locations within the operon. b. Treatment of the Operon Derepressed for 1 Minute (66). It is possible to demonstrate internal initiation of transcription in a restricted region of the trp operon. Addition of dinitrophenol 1 minute after derepression (30°C) promptly inhibited trp mRNA synthesis by more than 90%; by the fourteenth minute after the addition of the inhibitor, more than 98% inhibition was observed. The residual synthesis (less than 2%) seen after 14 minutes occurred in the operator-distal portion of the trp D and ceased altogether by the nineteenth minute. Thus, transcription already in progress over the operator-proximal region of the trp E before the addition slows to several percent of the normal level immediately after the addition, and thereafter proceeds along the operon at a gradually decreasing rate until it ceases in the middle of the operon. When the time of exposure to dinitrophenol is 4 minutes, then the trp mRNA synthesized during the first minute after relief of inhibition by DNP corresponded only to the trp E and trp D.When the exposure is for

TRANSLATION AND TRANSCRIPTION OF THE

trp

OPJCRON

365

Fraction number

FIG.7. Sedimentation profile of t r p mRNA synthesized a t the initial stage after internal initiation of transcription. RNA was prepared from a CRM-producing mutant (A9952) of E . coli K12 pulse-labeled with 'H-uridine under conditions of repression during the first minute after the removal of dinitrophenol, added at the tenth minute after derepression and removed 45 minutes later. Repression was initiated at the time of removal by the addition of tryptophan. RNA was sedimented in a 5 3 0 % linear sucrose gradient. Data were taken from Imamoto et al. (68). 0-0,trp C-A mRNA; .--a, trp E-D mRNA; -, hybridization with $80 DNA (background value.) ; - - - - -, optical density at 260 nm.

nine minutes, synthesis corresponded to the trp E , t r p D and t r p C. If exposure is extended to more than 12 minutes, the mRNA synthesized during the first minute after the removal corresponded to the whole region between t r p E and t r p A . The trp mRNA molecules synthesized in these experiments during the first minute immediately after the removal were unusually small. Thus, when dinitrophenol was present continuously, a small number of rounds of transcriptions that had started before the addition of the drug continued at a diminishing rate and reached as far as the operator-distal region of the t r p D before terminating, but when the operon was relieved from the inhibition a t various times, transcription was found to be initiated randomly a t sites throughout the region between the t r p E and trp A . This indicates that the transcribable area of the operon apparently spreads from the trp E t o the trp A in the absence of normal transcription, and that, following the treatment, t r p mRNA synthesis can be initiated within this region. When tryptophan was present throughout the period of inhibition, internal initiation of transcription did not occur after the relief of inhibition. When tryptophan was added 10 minutes prior to the removal of dinitrophenol, t r p mRNA could be synthesized over the operator-distal

366

FUMIO IMAMOTO

but not the operator-proximal portion of the operon. Thus, an area of nontranscribability appears to progress along the operon in the trp E to trp A direction when tryptophan is present while the cells are in the inhibited state. The rates of propagation of both the transcribable and nontranscribable states are similar under any one set of conditions, and are about half as fast as the normal rate of transcription propagation after derepression. The simplest interpretation of internal initiation of trp mRNA synthesis is that RNA polymerase molecules move along the operon without making RNA in the presence of dinitrophenol and, upon removal of the inhibitor, resume synthesis of mRNA whenever they happen to be in the operon. An alternative interpretation is that the drug alters the secondary structure of the DNA so as to create internal sites at which polymerase molecules may become attached. However, the results of experiments employing nonsense mutants of the trp operon argue against the latter possibility since it was found that the internal initiation of transcription on the operator-distal genes is less frequent with strong polar mutants than with weak polar mutants or with wild-type strains (67, 68). The former possibility is also supported by recent observation that internal initiation of trp mRNA synthesis is not inhibited by rifampicin, which blocks the ordinary initiation of transcription (121) (Table V, second row).

D. Rate of Transcription Propagation The rate of transcription in a given operon is a function of two parameters: the frequency of transcription initiation and the rate of transcription propagation. It has been possible to test if the RNA synthesis along the trp operon proceeds a t the same or perhaps a t a quite different rate than that of bulk RNA synthesis. Independent investigations have indicated that the propagation rate of trp mRNA synthesis during synchronous derepression is 1 6 1 9 nucleotides per second a t 30°C (46, 4.9, 66),17 nucleotides per second a t 30°C (70) , 17-20 nucleotides per sec a t 37°C (47) and 28 nucleotides per second a t 37°C (70).The experiments of the first group were performed under conditions of derepression effected by depleting tryptophan; the other experiments were done using either indole-3-acrylic acid or indole-3-propionic acid to derepress the trp operon. Additional evidence bearing on the rate of transcription propagation has been obtained by measuring the time required for the completion of transcription of the operon following addition of an inhibitor of transcription initiation, rifampicin, to repressor-negative (trp R-) cultures (122). The estimated rates of transcription propagation were 16-17 nucleotides

TRANSLATION AND TRANSCRIPTION OF THE

trp

OPERON

367

per second a t 25"C, 25-28 nucleotides per second at 30"C, and 3 7 4 5 nucleotides per second at 37"C, somewhat higher values than those estimated previously. It was suggested that the lower estimates in the previous studies (45, 47,49,66,70) might result from asynchrony of transcription initiation during derepression combined with the insensitivity associated with the measurement by hybridization of the precise time of appearance and/or disappearance of mRNA complementary to specific regions of the operon. The propagation rate was not altered over a 3-fold range of growth rates effected by changing the carbon source or supplementing the medium with casein hydrolyzate. Recent estimates of the average growth rates of bulk RNA chains in E. coli are 26 nucleotides per second a t 29°C (123),55 nucleotides per second at 37°C (l24),and 43 nucleotides per second a t 37.5"C (123). These rates are nearly independent of the bacterial growth rate over a 2-fold to 3-fold range (123,124).The average growth rate for T 4 "early" mRNA in E. coli has been estimated to be nucleotides per second a t 37°C (126). Unequivocal concurrence is generally observed between rates of polypeptide elongation and mRNA elongation (2). I n Salmonella typhimurizim, an average chain growth rate of 15 amino acids per second, which corresponds to 45 nucleotides per second for mRNA synthesis, has been estimated for in vivo polypeptide synthesis a t 37°C (106).Indirect estimates of the mRNA chain growth rate for the lac operon have been obtained from measurements of the time required for appearance of enzymes following induction of their synthesis (107,126). Such measurements indicate mRNA elongation rates of 29 nucleotides per second a t 30°C and of 38 nucleotides per second a t 37°C. Another estimate of lac mRNA chain growth calculated from the polypeptide elongation rate for the P-galactosidase of E. coli is 45 nucleotides per second a t 37°C (127). Rates of t r , mRNA elongation appear to be in agreement with the bulk RNA elongation rates, thereby suggesting that, in bacteria, a common mechanism of RNA synthesis operates and determines the rate of polynucleotide assembly. The observation that in the trp operon the rate of transcription propagation is not affected by variation in the cell growth rate supports the hypothesis that bacterial RNA polynucleotide chain growth is independent of DNA replication (123,128).

E. Degradation of trp mRNA I n spite of the attention that has been paid to the short-lived mRNA fractions in bacteria, very little is known at present of the mechanisms by which individual mRNA molecules are broken down or of the factors regulating the turnover of mRNA in vivo.

368

FUMIO IMAMOTO

By means of uridine “chase” experiments, following derepression effected by depleting tryptophan, it appears that t r p mRNA in E . coli is degraded sequentially from the t r p E (5’ end of the molecule) to the t r p A (3’ end) (199).The uridine “pulse-chase” technique, during a single round of transcription generated by the addition of tryptophan shortly after the synchronous initiation of derepression by the addition of indole3-acrylic acid, yielded similar results (130). These two experiments corrected a previous report (131) that proposed mRNA degradation in the reverse direction (i.e., 3’ + 5 ’ ) . The kinetics of degradation of t r p mRNA segments corresponding to different regions of the operon (129, 130) and the sedimentation analysis of t r p mRNA being degraded (129) suggest that trp mRNA is degraded exonucleolytically, starting from the 5’ end. The possibility has not been excluded that t r p mRNA is first segmented in a sequential fashion from the trp E to the t r p A by endonllcleolytic action, the resulting small fragments then being digested exonucleolytically from either or both ends. A new ribonuclease, RNase V, in E . coli, which is associated with and might possibly be an integral part of ribosomes and which degrades mRNA exonucleolytically in the 5’ to 3’ direction, has been discovered recently (132). There is ample support for an inactivating event occurring a t the 5‘ end of the t r p mRNA. After repression, synthesis of the tryptophan biosynthetic enzymes ceases sequentially in the same order as the sequence of the structural genes of the operon ( 4 6 ) .If functional inactivation of t r p mRNA werc duc simply to chcmical degradation, this finding would indicate a temporal sequence for the degradation of t r p mRNA. Under the conditions where repression follows derepression, appreciable degradation of trp mRNA occurs at the 5’ end before transcription of the operon is completed (10’7, 129, 130). Consistent with this is the finding that the synthesis of the first enzyme, anthranilate synthetase CoI, comes to an unusually rapid halt after the onset of repression by addition of tryptophan ( 4 6 ) . Under the conditions where repression has been initiated by the addition of either tryptophan or 4-methyltryptophan, degradation of trp mRNA begins randomly in time following initiation of synthesis (107).These findings suggest that very few “full length” molecules exist under repressed conditions. In the absence of repression, the degradation of trp mRNA has been observed to proceed somewhat more slowly than when tryptophan is present (70, 129). During an initial period after derepression effected by depletion of tryptophan, degradation of t r p mRNA takes about twice as long as its synthesis (129). Consistently, under these conditions, the existence of trp mRNA molecules large enough to be a transcript of the whole t r p operon has been observed as a significant fraction of the RNA (44&, 199).

TRANSLATION AND TRANSCRIPTION OF THE

trp

OPERON

369

Thus the possibility exists that the degradation of trp mRNA is triggered by a factor or factors acting in1 response to an increase in the rate of translation.

F.

Repression of Transcription

1. MODEOF REPRESSION

I n 1961, Jacob and Monod (43) proposed their classic operon model of the regulation of gene expression. According to this model, repression (operon shutoff) involves the blockage of initiation of transcription by the association of the regular gene product with the operator located a t one extremity of the operon. This assumes that repression is not primarily concerned with transcription already in progress along the operon. Hybridization experiments in the trp operon of E . wli have verified this assumption (4.2, 46,47-49,133). The trp operator region is located a t the trp E end of the operon (32, 36) and the trp mRNA appears in a temporal sequence corresponding to the positional sequence of the structural genes in the operon. When the trp operon is derepressed by transferring cells from rich medium to prewarmed minimal medium (30°C) containing glucose and a mixture of nineteen amino acids lacking tryptophan, the synthesis of trp E mRNA commences after a very brief lag while the synthesis of trp D mRNA lags a t least 2 minutes behind that of trp E mRNA (Fig. 8). Under these conditions, 7-8 minutes are required for completion of a round of transcription of the operon. If, under these conditions, repression is established by the addition of tryptophan the second minute after derepression (essentially a single round of transcription is in progress), both derepression and repression proceed simultaneously along the operon (Fig. 8). The synthesis of different regions of trp mRNA was repressed sequentially in the same order as the structural genes: the synthesis of trp E mRNA, trp D mRNA, trp C-B mRNA and trp A mRNA ceases, 1, 3, 5 and 6 minutes, respectively, after onset of repression. When repression is started at the sixteenth minute after derepression to assure a steady state of transcription of the operon, the synthesis of the trp E, trp D and trp C-B mRNA stops after 1, 3 and 5 minutes, respectively (46, 66). These experiments demonstrate that the trp repressor blocks initiation of mRNA synthesis on the operon but does not interfere with transcription already in progress. In principle, this could occur in one of three ways: (a) Repressor could prevent the binding of the RNA polymerase to the promoter of the operon. (b) RepreFsor could prevent the initiation of transcription by RNA polymerase molecules still able to bind to the promoter. (c) Repressor could inhibit mRNA synthesis in progress over a short but limited operator-proximal region of the operon, thereby in-

370

FUMIO IMAMOTO 1

I

I

1

1

1

1

I

T

2500 fmin/pg

Tryptophan

4 '0000

9a 7500

-!2la 0

.-

.>

B 'g

5000 g

m

2500

Time after derepression (or repredon)

FIG.8. Sequential progress of a single round of transcription after repression. RNA was prepared from wild-type Escherichia coli K12 (strain W3110) pulselabeled with ['Hluridine for successive 1 minute periods after repression which was initiated by the addition of tryptophan at the end of the second minute after derepression. Data were taken from F. Imamoto, Mol. Gen. Genet.. 106, 123 (1970), by permission of Springer-Verlag, Berlin and New York. 0,trp E mRNA ; 0,t r p D mRNA; 0 , t i p C-B mRNA; 8, t r p A mRNA; Q, specific activity (cpmlpp) of ['HIRNA.

directly preventing further binding and/or initiation of transcription by additional RNA polymerase. The following observations support possibility (c). In Fig. 8, it can be seen that, after the onset of repression, the time-course of transcription of trp E is apparently shortened; in the first round of transcription of the operon, the first 2 minutes after derepression wcre required for transcription of the trp E , although it stopped altogether l minute after repression. Transcription initiated a t trp E progresses somewhat sluggishly during the first 30 seconds after derepression and thereafter becomes faster: under steady-state conditions, approximately 1.5 minutes are required for transcription of the trp E (49).The shortening appears to correlate with the abrupt reduction in the amount of trp E mRNA synthesis following repression. The experiment depicted in Fig. 9 shows that such reduction is in part

TRANSLATION AND TRANSCRIPTION OF THE

U

trp

371

OPERON

0.2

E

a0.1

+ 0

5 0 0.3 U

k-.--%-.-+-JI

0.1

0

4

t

0.2

I

2

3min 0 I Time a f t e r r e p r e s s i o n

0.2

2

3min

0

FIG.9. Sequential cessation of transcription during repression of the t i p operon. RNA was prepared from wild-type Eschei-ichin coli K12 (strain W3110) pulselabeled with ['Hliiridine for 2O-second periods a t the time indicated during deRepression was initiated by the addition of repression ( 0 )and repression (0). tryptophan a t the fifteenth minute after derepression. Data were taken from F. Imamoto, Mol. Gen. Genet. 106, 123 (1970), by permission of Springer-Verlag, Berlin and New York. (a) Specific activity (cpm/pg) of ['HIRNA; (b) trp E mRNA; ( c ) trp D mRNA; (d) trp C-A mRNA; (e) trp E distal mRNA.

caused by a prompt blocking of the transcription in progress over the most operator-proximal segment of the operon. Here repression was started a t the fifteenth minute after derepression (steady-state transcription of the trp operon) and thereafter pulse-labeling was performed for successive 20-second periods. Although the specific activities of the total labeled RNA extracted from the repressed culture were approximately 70% higher than those of labeled RNA from the derepressed culture (Fig. 9, a ) this difference does not affect the estimation of the amount of trp mRNA, expressed as percent of total labeled RNA: i.e., the amount of trp C - A mRNA from repressed cultures was essentially the same as from derepressed cultures, when pulse-labeling was carried out within 1.5 minutes after repression (Fig. 9, d) (during which the reduction in

372

FUMIO IMAMOTO

the rate of synthesis of mRNA for the operator-distal region of the operon was reasonably free from the effect of repression). After repression, the rate of t r p E mRNA synthesis was abruptly reduced by 46% of that in the steady-state dereprcssed control during the first 20-second period of pulse-labeling ; thereafter the rate decreased gradually until the end of the first minute after repression (Fig. 9, b). The theoretical reduction of trp E mRNA synthesis caused by interference of initiation of transcription at thc promoter sitc, during thc first 20-scrond pcriod aftcr rcpression, was estimated to bc 13% of thc rate of t r p E mRNA synthesis under steady-state derepressed conditions (4.9). Thus the 33% difference must have arisen from a prompt cessation of the transcription in progress over the t r p E region. Such abrupt changes in the rate of synthesis are not observed in the synthesis of trp D mRNA and t r p C-A mRNA during the initial periods subsequent to the onset of repression (Fig. 9, c and d). The rate of synthesis of the t r p mRNA corresponding to the operator-distal region of the t r p E [Edistal mRNA (Fig. 3 ) ] , did not begin to decrease until 40 seconds had elapsed; thereafter the rate did begin to decrease, and eventually it ceased at the end of the first minute after repression (Fig. 9, e). The evidence prescntcd in Fig. 9 indicates that thc decrease in the rate of trp E mRNA synthesis caused by the onset of repression must arise from two sources: the inhibition of transcription already in progress over the most operator-proximal segment of the operon; and the failure of further initiation of transcription at the promoter site. The possibility has been excluded that the apparent block of the progress of transcription is caused by a selective and rapid degradation of the region of trp mRNA involved in this effect (49). The length of the transcription region blocked immediately by the onset of repression was estimated on the basis of more extensive quantitative determinations to be approximately the first 4% of the operon (49). The following evidence is also consistent with the foregoing. Employing the conditions of internal initiation of trp mRNA synthesis described in a previous section (IV, C, 4), it was observed that t r p mRNA synthesis initiated at internal sites in theregion between t r p D and t r p A is not affected by tryptophan added at zero time of internal initiation, while the synthesis of mRNA for t r p E is decreased by tryptophan to about half the rate observed in the presence of rifampicin (Table V) , A similar effect was observed with the tryptophan analogs, such as 4-methyltryptophan, 5-methyltryptophan or 6-methyltryptophan, each of which represses the t r p operon (l%$), but not with 7-azatryptophan which does not repress (134) (Table V ) . The decrease in the rate of synthesis of t r p E mRNA caused by the addition of tryptophan corepressors must arise from two

TRANSLATION AND TRANSCRIPTION OF THE tTp OPERON

373

TABLE V EFFRCT OF RIF.\MPICIN, TRYPTOPHAN AND VARIOUS TRYPTOPHAN ANALOGS ON INTERNAL INITIATION OF TRANSCRIPTION OF THE DINITROPHENOGCONDITIONED trp OPERON (121)”

Amount (%) of trp mRNA synthesized Addition a t 0 time of internal initiation None Rifampicin, 120 pg/ml &Tryptophan, 50pglml D&Methyltryptophan, 100 pgg/ml Db5-Methyltryptophan, 100 pgg/ml DL-6-Methyltryptophan, 100 pg/ml nb7-Azatryptophar1, 100 fig/ml

Relative ratio E d i d

E

Rdiatnl

D

C-A

E/C-A

C-A

D/C-A

100 95 40 40

100 95 78 79

100 78 80 89

100 79 92 92

1.00 1.20 0.44 0.46

1.00 1.20 0.85 0.86

1.00 0.99 0.87 0.97

52

96

90

100

0.52

0.96

0.90

42

76

87

93

0.45

0.82

0.94

100

101

80

86

1.16

1.17

0.93

0 RNA was prepared from cultures of rifampicin-sensitive strain RFS522 (donated by R. Schleif, to whom we are indebted) pulse-labeled with [aH]uridinefor the first 90 seconds after the removal of dinitrophenol (5 mM). Bacteria, were incubated in the presence of dinitrophenol, added a t the tenth minute after derepreasion, for 45 minutes.

sources: the failure of initiation of transcription a t the usual site ( P l ) , and the inhibition of transcription of an operator-proximal segment of

trp E . These findings provide a hypothesis for the mechanism by which the repressor blocks mRNA synthesis on the trp operon: the repressor, when it binds to the operator, directly blocks the progress of the RNA polymerase into the structural genes of the operon. An alternative explanation, namely that an RNA polymerase transcribing the operator-proximal region of the operon travels more rapidly following the addition of tryptophan, is unlikely, for the rate of transcription of the operator-proximal region would increase greatly upon the addition of tryptophan. Also consistent with this hypothesis is the recent demonstration that. transcription of the trp E ceases more rapidly after tryptophan addition than it does after the addition of rifampicin, which blocks initiation of transcription of the operon (133). 2. POSITIONAL SEQUENCEOF PROMOTER AND OPERATOR The promoter region preceding the operator region in the lactose operon of E . coli has been mapped (8483),and this has led to the proposition that

374

FUMIO IMAMOTO

transcription of the operon is initiated before (or at) the operator region, and can be directly blocked by the combination of the repressor with the operator (82).In the trp operon of Salmonella typhimurium, genetic evidence suggests that the promoter (Pl) and the operator region are arranged in the same order with respect to the first structural gene (trp A ) as that found for the promoter and operator in the lac system (89).The findings described in the foregoing section seem to favor the hypothesis that the synthesis of mRNA on the trp operon is initiated before the operator region and that repressor, by binding to the operator region, directly blocks the progress of transcription. It should be mentioned that the trp operon segment of phage +80 pt El used as the DNA source for the hybridization reaction, possesses the intact trp operator region (Imamoto and Ito, unpublished). If the above hypothesis is correct and if the operator is transcribed, the operator region of the trp operon in E. coli must be smaller than 4% of the operon (49). Although genetic evidence reported previously suggested that the trp operator region is 2040% of the size of the entire operon ( 3 6 ) , it could not be excluded that marker effects were responsible for the high estimate of genetic map distances (83,90). I n the lactose (83,136) and histidine [cited in (S)] operons, the operator region, measured by genetic mapping, has been estimated to be much smaller than the average gene. Additional evidence has been obtained in E . coli suggesting that, if transcription begins before the operator region, it must be smaller than about 13% the length of the trp E or than about 3% the length of the entire operon (108).Transcription in delction mutant trp OAE14 (Fig. 3 ) , which retains only the operator-proximal eighth (about 13% the length) of the trp E, is completely repressible, indicating that an intact functional operator region is present.

G. Trp mRNA Synthesis during Amino Acid Starvation Abundant evidence demonstrates an involvement of amino acids in the regulation of RNA synthesis (136).Synthesis of RNA in auxotrophic strains of E . coli carrying the wild-type RCatr*allele is severely inhibited during starvation for a required amino acid, while strains carrying the mutant RCre' allele of that gene are able to continue RNA synthesis in the absence of a required amino acid (137).It was first suggested that this stringent control of RNA synthesis operates a t the level of general transcription and that all RNA classes (rRNA, tRNA and mRNA) are coordinately controlled (128,137-1393. An alternative hypothesis is that * RC"", the allele that confers stringent control of RNA synthesis; RC'", the allele that confers relaxed control of RNA synthesis.

TRANSLATION AND TRANSCRIPTION OF THE tTp OPERON

375

the reduction in RNA synthesis during amino acid starvation of RCEtr strains concerns mainly rRNA and tRNA (140). In these studies, it was very difficult to obtain sound information concerning mRNA synthesis mainly due t o technical problems in recognizing the mRNA fraction as well as to the relatively instability of this fraction. One approach was to examine the rate of p-galactosidase mRNA synthesis following induction of an amino acid-starved stringent culture (141). Using the capacity for p-galactosidase formation after the establishment of enzyme repression by dilution of the inducer (lor),it was shown that the initial rate of P-galactosidase inRNA synthesis is independent of the presence of the required amino acid (arginine), thereby supporting the hypothesis that the synthesis of all classes of bacterial RNA’s are not coordinately regulated. In agreement, in a stringent strain of E . coli, the synthesis of total mRNA, estimated by measuring the initial rate of stimulation of amino-acid incorporation into protein in vitro by an extract from a stringent strain of E. coli, was the same in the presence as in the absence of required amino acids (141a). The synthesis of trp mRNA is not subject to RC““ amino-acid control. Using amino-acid starvation by depleting tryptophan from reprcsscd tryptophan auxotrophic strains and thereby simultaneously imposing derepression conditions, it was showa that pulse-labeled trp niRNA, detected by direct hybridization with $80 pt DNA is ten times RS great in the stringent strain as in the relaxed strain. Also that the forination of the tryptophan synthctasc LY and ,8 proteins following dercprcssion of thc trp operon is higher in the tryptophan-starved stringent strain than in the relaxccl strain (103).I n similar experiments, the amount of trp mRNA was three times as high in a dcreprcssed strain ( t r p R’) as that in exponential growth of a constitutive (trp R-) strain in the steady state, in both RCntr and Rere’combinations (104). It was also observed that synthesis of tryptophan synthetase LY protein, which contains no tryptophan, procccds during starvation of the stringent strain and not during starvation of the isogenic relaxed strain, although trp mRNA is also present in the latter case, thereby raising an argument for an obligatory connection between transcription and translation as originally proposed by Stent (142). A somewhat different result has becn presented with trp R- derivatives of RCRtrand RCret strains requiring arginine. The competitive binding assay between unlabeled and purified labeled trp mRNA for binding sites 011 $30 pt DNA, indicated (143) that both stringent and relaxed strains have &fold t o 7-fold lower levels of trp mRNA upon starvation for arginine. Direct hybridization of pulse-labeled RNA with 480 pt DNA showcd a great decreasc in the rate of trp mRNA upon starvation for

376

FUMIO IMAMOTO

arginine in both stringent and relaxed strains. However, the rate of synthesis of total mRNA hyhridizable with E . coli DNA was depressed 4.3fold in an arginine-starved RCSt”strain, but only 1.6-fold in the RCre’ strain, in agreement with findings obtained previously (139).Quantitative variations in the stringent response depending upon the amino acid withheld (104, 137, 140) mizht be responsible for the seemingly contradictory observations (143). Since the possibility has been ruled out that changes in the levels of endogenous nucleoside triphosphate pools serve to regulate RNA synthesis in stringent bacteria during amino-acid starvation (144), stringent control of RNA synthesis could operate a t the level of initiation and/or propagation of RNA synthesis. An increased rate of RNA degradation in bacteria under stringent control is an alternative possibility. It is possible that the apparent noncoordinate regulation of synthesis of trp mRNA and ribosomal and transfer RNA’s occurs only during tryptophan starvation. Obviously, the stringent response of trp mRNA synthesis requires further clarification, especially with regard to the specificity of the amino acid used in the starvation.

H. Synthesis of trp mRNA in Vitro A workable in vitro system could be invaluable in furthering our understanding of the mechanisms of gene expression of the trp operon. With RNA polymcrasc purificd from E. coli strain Y-me1 by the usual procedures, RNA hybridizablc with transducing $80 pt phage DNA can be synthesized from an E . coli DNA template at high ionic strength (0.18 M KC1) (145). Such synthesis is dependent on DNA as well as the enzyme and is sensitive to inhibition by deoxyribonuclease, ribonuclease or actinomycin D. The incorporation of labeled precursors into RNA proceeds almost linearly for 15 to 20 minutes at 37”C,and thereafter at a gradually decreasing rate for 60 minutes; it occurs with various template DNA’s from deletion mutants of the trp operon. The amount of RNA that formed specific ribonuclease-resistant hybrids with phage DNA carrying the entire trp operon has been estimated to be approximately 0.276 of the RNA synthesized in vitro (146). The synthesis of P-galactosidase under the control of lac repressor in a cell-free system with $180d h c DNA has been observed (145a, 146). Recently, the in vitro transcription of the lac operon has been demonstrated, with DNA’s from 480 plac, Aplac, A-480 dlac or F’lac as either a template and/or a hybridization complement for greater specificity in the detection of lac mRNA (147-149). With this set of DNA’s, essentially only the lac mRNA may be isolated. Asymmetric transcription on the correct strand of the lac region was apparent. Since both 480 ptrp and

TRANSLATION AND TRANSCRIPTION OF THE

trp

OPERON

377

X ptrp phages are now available; an analogous in vitro system for trp mRNA synthesis is a possibility and could be used to inquire into the regulatory mechanisms involved in the expression of a repressible system.

V. Translation and Transcription of the frp Operon in Nonsense Mutants of E. coli Genetic polarity is the phenomenon in which a nonsense mutation in one gene of an operon, in addition to resulting in inactivation of the product of that gene, also decreases the expression of all the operatordistal genes of the opcron (99, 150). This phenomenon appears to be a general characteristic of all operons transcribed into polycistronic mRNA. Mutational changes that produce polar effects appear to have as a common consequence the introduction, either directly or indirectly, of a polypeptide-chain-terminating codon internal to the gene (34, 151-153). This results in the production of a polypeptide fragment of the normal chain corresponding to the nucleotide sequence up to the chain-terminating codon (154-157), in addition to causing the polar effect. Mutations of the nonsense and fraineshift types, but not missense mutations, exert polarity (34, 151-153). Since suppressor genes whose products act a t the translational level relieve polarity (34, 151, 152, 165, 158-162) , it appears that polarity is due to a translational defect. On the other hand, transcription studies with extreme polar mutants of thc lac operon indicated that such strains do not produce significant levels of the lac mRNA in response to the addition of inducer ( 7 4 ) .One such lac mutant has later been shown to be a nonsense mutant of the type (163).Similarly, a polar mutant of the gal operon did not produce increased levels of specific mRNA upon the addition of inducer (164). On the other hand, there is normal formation of r l l mRNA in cells infected with an amber mutant of bacteriophage T4 containing a mutational alteration located well within the rll A cistron (166).In view of the earlier findings, it was not clear whether a nonsense codon causes polarity by reducing translation of mRNA regions past the nonsense codon, or by affecting the relative representation of different regions of the operon in the specific mRNA population. As a result of the development of mRNA assay techniques, polarity in the lac and trp operons was found to involve two associated phenomena. First, polar mutants produced a decreased amount of mRNA for the genes operator-distal to the mutation (76, 81). The observed decrease was sufficient to explain the reduced expression of the distal genes (81). Second, the mRNA molecules for the genes operator-proximal to the mutation werc of reduced sizc. The shortened mRNA segments werc apparently terminated near the site of the nonsense mutation (68, 81).

378

FUMIO IMAMOTO

The extent of both effects correlates with the position of the mutation in the gene and the severity of the polar effect exerted by thc mutation. Polarity phenomena also have been observed in the in vitro translation of the RNA of RNA phages possessing nonsense mutations, during which no RNA degradation occurs (166-169).I n this case, polarity is not due to a preferential destruction of the messenger from regions distal to the nonsense codon. Translation can normally be initiated a t internal sites on the phage RNA. In vitro polarity with RNA phage messenger is therefore believed to result from a conformational masking of the internal initiation signals of the RNA (157,167, 170, 171).I n contrast, polarity in the translation of bacterial operons results from a decrease of mRNA distal to the mutated gene, a consequence of either the premature termination of transcription at or near the site of mutation (67,68,172-175), or of an accelerated breakdown of the section of the messenger distal to the mutation (175-177). The degree to which a nonsense alteration is polar is a function of the distance of its site from the nearest operator-distal cistron boundary (178,179).The closer the alteration is to this boundary, the less polar it is (12,34, 35, 53, 68,151, 180-184).There is evidence suggesting that the degree of polarity depends, a t least in part, on the distance from the site of the nonsense alteration to the next translational initiation site (113,185). However, little is yet known about the mechanisms that determine the degree of polarity. In this section, the results of studies of translation and transcription with polar mutants of the trp operon are summarized.

A. Translation Studies 1. PLEIOTROPIC EFFECTS AND

POLARITY GRADIENT Polarity of the trp operon in E. coli was first reported by Ito and Crawford (33), and Matsushiro et al. (39). I n a study of coordinate THE

enzyme production employing mutants of the tryptophan operon of E . coli, the former observed that the trp C or trp B mutants lacking cross-reacting material (CRM-) and grown under conditions of derepression (a limiting amount of tryptophan) exhibit a reduction in the levels of enzymes formed by the genes on the side of the mutation distal to' the operator, while mutants with cross-reacting material (CRM+) showed no such polarity effect. Matsushiro et al. (32) reported one instance of a polarity effect involving a mutant of trp B. Subsequently, translational polarity of the trp operon in E. coli was more extensively studied by Yanofsky and Ito ( 3 4 ) .I n their experiments, tryptophan nonsense mutants combined with a trp R mutation were grown to log phase in thc presence of tryptophan. Since excess tryptophan

TRANSLATION AND TRANSCRIPTION OF THE

trp

379

OPERON

was present, the functioning of the tryptophan biosynthetic enzymes was not essential for growth. Thus the relative production of the different tryptophan biosynthetic enzymes in each auxotroph was a reflection of the effect of the mutation on the ability of the strain to make and translate mRNA. All amber and ochre mutants exhibited a reduction in the relative rates of synthesis of the enzymes specified by genes operatordistal to the gene harboring the nonsense mutation (Table VI) . Missense mutants producing cross-rcacting material (CRM") did not show polarity. Two frameshift mutants also wcrc polar. This is consistent with tlic interpretation (180) that frameshift mutatioiis result in polarity by generating a nonsense codon further along in the mutated gene. It was also verified in this study that amber and/or ochre suppressor mutations partially relieve the polarity effect approximately to the same extent as their efficiency of suppression of the nonsense codon ( 3 4 ) . The genetic locations and the relative t q ~(A protein) polarity values TABLE VI COORDIN.4TlC ENZYMIC PItODUCTION nY CRM+, Abxnm, FRAMI'.SHIFT MUTANTS"

OCHRl~?AND

Percent of wild-type valnd Tryptophan synthetase Mutant

Ant,h P-Rib Ind-Gro-P synthetase transferase synthetase

A

B

R-trp A9963 CRM+ R-trp A9796oc

100 100

104 95

114 100

100 49

90 0

R-trp B9681 CRM+ R-trp B40c R-trp B9679oc

100 100 100

64 111 90

105 83 76

0 0 0

112 3 5

R-trp C99qi CRM+ R-trp C.9870am

100 100

78 75

0 0

90 33

98 36

R-trp D9939 CRM+ R-lrp D9778oc

100 O(1OO)c

0 0

80 14

98 13

113 16

R-lrp It-trp It-kp It-trp R-lrp

O(100) O(1OO)c O(lo0)c O(1OO)E

94 6 25 24 5

98 7 26 24

E10330 CRM+ E9829am E9903fs E9887fs E9803am

O(1OO)c

7

-

27 6

5

Data from Yanofsky and Ito (34) and Imamoto et al. (181). Repressed R- cultures, anthranilate synthetase value set at 100% in each case. Anth = anthranilate; Ind = indole; Cro = glycerol. c No activity detected, wild-type value taken a s 100 for calculation of coordinate levels.

380

FUMIO IMAMOTO Tryptophan operon Gene sequence

B

l

a

D

E

Operator end b

I

1

1

1

I

l

l

l

l

B

C .1

A

i

FIG.10. Genetic? map of tlic / r p oprion and rrlatire polarity mlues in mutants. Thr order of mutntiontilly nltered sites and the trp A protein polarity values (in parentlieses) were bnsrd on previous studies (34, 81) and personal communication from C. Yanofsky. The tip A protein polarity value is the ratio, given as percent, of trp A protein production by the mutant to t i p A protein production by the wildtype control.

for polar mutations of trp E are represented in Fig. 10. It is evident that within trp E there is a gradient in the degree of polarity with the most extrcmc polar effects occurring with alterations closest to thc operator end. A gradient of polarity was observed in the trp R (S4). Bauerle and Margolin (1.2, 36) and Blume et al. (53) investigated polarity in the trp operon of Salinonella typhiinzsium and found a gradient of polarity in the trp A , the first gene of the operon. By comparing the genetic location of each mutation on the linear map of trp A to the degree of its polarity effect, it was observed, as in E . coli, that those mutations most proximal to the operator end of the gene exert the most severe polarity effect. The slope of the polarity gradient characteristic of each gene appears to differ from one gene to another, within an operon (16).In contrast with the observation that in trp E all chain termination mutations in the operator-proximal half of the genetic map reduce distal gene expression by 9576, there is a near-linear polarity gradient in trp D and a slightly concave gradient in trp C. The most extreme polar mutations a t the operator-proximal ends of both genes reduced synthesis of enzymes specified by distal genes of the operon by 80-90%, while the most operator-distal chain termination mutations had little or no polar effect. By contrast, in trp E many chain termination mutations reduced distal gene expression by 95% and nonpolar chain termination mutations were not detected (34).

MULTIPLE-NONSENSE MUTANTS The extent of polarity in several trp R- strains of E . coli in which two or more nonsense codons were present in the trp operon have been assessed (34).When nonsense codons were present in two different genes in the operon, each exerted its individual polar effect on enzyme forma2.

POLARITS IN

TRANSLATION AND TRANSCRIPTION OF THE

trp

OPERON

381

tion; i.e., the levels of the enzymes specified by genes past the ones with the polar mutations werc much lower than in the singlc mutant parents. In a trp E-D-C-B quadruple mutant, the trp A protein level was reduced to less than 0.2% of the wild-type control. When two nonsense codons were introduced into the same gene, the cxtcnt of polarity was the same as that obtained with the single-mutant parent whose nonsense codon was closer to the operator end. 3. ANTIPOLARITY

In coordinate cnzymc production studies with polar and dclction mutants of the trp operon in E . coli, Ito and Crawford (33) found that certain mutants caused what they termed a (short range) antipolar effect; i.e., the enzyme specified by the nearest unmutated gene on the operator side of the mutation is formed in reduced amounts. Yanofsky and Ito (34) also observed that a nonsense alteration in the C, B, or A gene often leads to a slight reduction in the synthesis of the enzyme specified by the gene immediately preceding (Table VI). The effect. was most scvcre in trp A mutants. I n thosc cases in which the extent of polarity and antipolarity in a nonsense mutant could be compared, the polarity effect was found to be the more extreme. Examination of a set of well-mapped trp A nonsense mutants of the ochre typc indicates that the extent of antipolarity is a function of the map location of the nonsense codon (186). A gradient of degree of antipolarity exists, with the orientation running in the same direction as the gradient of polarity seen in the trp E and trp B, i.e., nonsense mutations on the operator side of the gene have the most extreme polar and antipolar effects. In addition, the antipolar effect of an amber mutation mapping in the most operator-proximal region of trp A on trp B expression is partially relieved by introduction of an amber suppressor gene. From the similar orientation of the gradient of polarity and antipolarity in this operon, it is assumed that the polarity caused by nonsense alterations of trp A is primarily responsible for the antipolarity effect on trp B. Recent results (16) show that therc is no appreciable antipolar effect with several strong polar mutants in trp D , trp C and trp B except for the trp A mutants. The basis of the contradiction of these with previous results (33, 34) 187) is not yet clear.

B. Transcription Studies 1. PRODUCTION OF mRNA DEFICIENT IN DISTAL SEGMENTS

Amber, ochre and frame shift polarity mutants of the trp operon of E . Goli have been examined in transcriptional studies to determine whether translational polarity is associated with an alteration of the trp

382

FUMIO IMAMOTO

mRNA population (81, 181). In these experiments, trp mRNA was isolated from bacterial cultures pulse-labeled with [ 3H]uridine at a steady-state transcription of the operon, following a shift from repression to derepression conditions. Total trp mRNA and trp mRNA regions corresponding to different segments of the operon were measured by hybridizing with DNA from $80 transducing phages carrying different segments of the operon. The results obtained indicate that strong polarity mutants generally contain less total trp mRNA than the identically treated wild-type strain or missense mutant. This result was most evident with the six nonsense mutants with alterations located in the initial segment of the trp E . The relative amount of trp mRNA observed in these mutants ranges from about 10 to 30% of that of the wild-type controls

mutants

I

9851

9802

9758

1

9029

10203

1

42242

9887

5972

1

I

1

9903

5984

FIG.11. Trp mRNA levels in trp E polar mutants. The trp E mutants are aligned in the order as their respective sites on the genetic map. RNA was prepared from derepressed cultures pulse-labeled for 3 minutes at the steady state of transcription. The ordinate is the relative trp mRNA value compared to the value with trp mRNA from a wild-type culture determined in each experiment. The vertical lines joining points were added to avoid confusion with neighboring points. Data were taken from Imamoto and Yanofsky (81).

TRANSLATION AND TRANSCRIPTION OF THE

tTp

383

OPERON

(Fig. 11).The two trp E frame-shift mutants (trp E9903 and trp 39887) are moderately polar (Fig. 10) and produce higher levels of trp mRNA than the six strongly polar nonseiise mutants. The weak trp E polar mutants, E 5984 and E 5972, produce even higher levels of trp mRNA. Specific hybridization studies demonstrated that the trp mRNA of strong polar mutants is deficient in the mRNA regions corresponding to the genes of the operon on the operator-distal side of the mutated gene (81, 181).I n strong or moderately strong polar mutants of trp E, trp D or trp C , relatively little trp mRNA corresponding to the three distal genes was detected, while nearly wild-type amounts were found in the weak polar mutants of trp E and trp C .

0.3 0.2

E

0

% 0.1

.L

u)

0

5

0 .c

a

0.5

Froction number

FIQ.12. Sedimentation profifes of trp mRNA from various trp E polar mutants. RNA was prepared from derepression culture pulse-labeled with ['Hluridine for 2 minutes a t the 10th minute (steady state) of derepression and sedimented in a 630% linear sucrose gradient. The arrows indicate the peak position of each RNA species. Data were taken from Imamoto et al. (68). 0-0, trp E mRNA; -, hybridization with $80 DNA (background value) ; - - - -, optical density at 260 nm.

384

FUMIO IMAMOTO

In agreement with these findings, sucrose gradient sedimentation profiles of the trp mRNA of strong and moderately strong polar mutants demonstrated that the majority of the trp mRNA molecules of each mutant are smaller in size than normal (68,81, 181).Figure 12 shows the sedimentation profiles of trp mRNA from five strong or moderately strong trp E polar mutants, pulse-labeled for 2 minutes during steadystate transcription of the operon. The trp mRNA from these mutants peaked in the region between 1 6 s RNA and 4s RNA, showing that most of the mo!ecules were very much smaller than normal. The s values of the trp mRNA of mutants trp E9829, trp E10203, trp E122@, trp 39758 and trp E9903, were 7.0,7.5,11.5, 12.5 and 14.0 S, respectively (68). Comparison of the order of these trp mRNA sizes with the map order of the mutational alterations (Figs. 3 and 10) indicates that trp mRNA size increases in relation to the distance from the beginning of the operon to the site of the mutational alteration. To examine the possibility that the short trp mRNA molecules in relatively strong polar mutants lack the mRNA region corresponding to the portion of the operon beyond the site of the nonsense mutation, the size of these trp mRNA molecules was compared with those from various deletion mutants in which only a portion of the trp E is retained (68,81).It had been shown that trp deletion strains produce, as expected, trp mRNA molecules of reduced size, directly reflecting the locations of the deletion termini in the operon (42, 68, 81, 108). Sedimentation analysis of trp mRNA from various trp delction mutants was carried out and the molecular weight of thc trp mRNA of thcse mutants was calculated (68). Figure 13 depicts thc relative values of the molecular weight of. thc shortened trp mRNA molecules produced by various polar and deletion mutants. The values were aligned on a relative scale for the trp E of D gene -

Operator end

Deletion mutants

I

i

+

-

I

5

w a 0

%

I

I

I

I

I

I

II

2

3

4

5

I

I

im

N OD

a a

4

I

0

I

I +

82 IaQI I

I

6

7,

n

-

9 3

$5 a I

I

8

a

a1 I

9

10

FIG.13. Coniparison of relntive size of Irp niRNA segment from various polar and delction mutants. Explanntions arc in the text. Data wcrc takcn from Imamoto e t al. (68).

TRANSLATION AND TRANSCRIPTION OF THE

trp

OPERON

385

10 units, assuming that the trp E-trp D intercistronic punctuation point is located a t a site between deletion mutants trp AEll and trp AD28, whose termini lie in the vicinity of the trp E-trp D punctuation point. Comparison of the order of the trp mRNA size with the genetic order of the nonsense alterations and deletion termini (Fig. 3 ) shows that trp mRNA size is unambiguously correlated with the position of the mutational alteration or the deletion terminus in trp E , and thereby indicates that synthesis of trp mRNA molecules in the polar mutants (and also in the deletion mutants) had apparently terminated a t or near the site of their mutational alterations (or deletion termini). All the deletion mutants investigated so far are known to have their other terminus a t sites between ton B and tdK. Thus it can be concluded that the genetic region adjacent to this end of the deletion is oriented in the opposite direction or that the region fails to transcribe in some presently unexplained way (68). I n conclusion, polarity mutations lead to a marked alteration of the trp mRNA population. I n each polarity mutant, particularly when polarity is severe, there is a reduction in the relative representation of the trp mRNA region corresponding to the operon region beyond the site of the nonsense mutation. This reduction is accompanied by the appearance of truncated trp mRNA molecules corresponding to those genes operator-proximal to the polar mutation. Estimates of the relative numbers of normal length and smaller-thannormal trp mRNA molecules in strong and weak polar mutants give values consistent with the conclusion that the extent of translational polarity in any mutant approximates the relative extent of representation of different operon regions in the trp mRNA population (81). 2. PARAMETERS DETERMINING THE DEGREE OF POLARITY

The degree of polarity created by trp E nonsense mutations is a function of their location in trp E. Mutations in the operator-proximal region are strongly polar while mutations in the operator-distal region are weakly polar. Of the trp E polar mutants, five (trp B 9829, trp E 9851, trp E 10203, trp E 9802 and trp E 1224.2) with nonsense alterations at the beginning of trp E are strongly polar, while four mutants ( t r p E 9903, trp E 9887, trp E 598.4 and trp E 5972) with the alterations closer to the operator-distal end of trp E show weak polarity (Fig. 10). A nonsense mutation, trp E 9758, which maps between the locations of these two groups of mutants, exhibits moderately strong polarity by producing relatively little but detectable amounts of the distal gene products (Fig. 10). The location of the mutational site in strain trp E 9758 can be approximated from the experimental results presented in

386

FUMIO lMAMOTO

Fig. 13. On the basis of the assumption that the molecular size of the truncated trp mRNA in polar mutants corresponds to the distance from the beginning of the operon to the site of the nonsense alteration, the location of the nonsense alteration in trp E 9768 is estimated to be a t 40-540/0 of the trp E from the trp E-trp D intercistronic punctuation point, which is considered to be located a t a site lying between the end points of deletion mutants trp A E l l and trp AD28. It is currently believed that the degree of polarity is determined by the absolute distance between the nonsense codon and the next intercistronic punctuation point (113, 178, 179, 186). The trp E product has a subunit molecular weight of 60,000 (Section IV, B) ( 2 1 ) .The length of the region of trp E operator-distal to trp E 9768 can therefore be assumed to correspond to a polypeptide molecular weight between 24,000 and 35,000.In the 2 gene of the lac operon in E. coli, polarity tends to diminish along the omega region (142, 186) which is the most operator-distal section of the Z gene. p-Galactosidase is a tetramer of 135,000molecular weight monomer units (4.3, 188) composed of a single polypeptide chain (189-191).The omega peptide has been purified from several deletion mutants; i t appears to correspond to one-third to one-fourth of the Z gene product, that is, to have a molecular weight of 34,000 or 45,000.A molecular weight of 39,000 for the omega peptide (64) has been deduced from more recent work. The difference between the values for the trp E and for the Z gene of the lac operon might not be significant, considering the uncertainties in the estimates for either system. Further investigations of the quantitative differences in polarity between both systems might clarify further the factors determining the degree of polarity. The polarity of a nonsense mutation decreases when a deletion mutation brings the nonsense mutation closer to the subsequent intercistronic punctuation point but polarity is not affected when a deletion precedes the nonsense mutation (178,179).Also, the degree of polarity is reduced by the introduction, by mutation, of a new polypeptide reinitiation site close to the operator-distal side of a nonsense mutation (112, 113). These findings suggest that it is the distance from the site of a nonsense mutation to the next polypeptide reinitiation site that dctcrinines the degrec of polarity. In the trp operon in E . coli, the extent of antipolarity (i.e., the enzyme specified by the gene of the opcron immediatcly prcceding the mutated gcne is formed in reduced amounts) appears to bc a function of the genetic map location of the nonsense alterations in the trp A (186).From the similar orientation of thc gradient of polarity and antipolarity in this operon, it is assumed that the polarity created by nonsense alterations in trp A is primarily responsible for antipolarity. The gradient of

TRANSLATION AND TRANSCRIPTION OF THE

trp

OPERON

387

polarity in trp A might be determined, a t least in part, by the distance from the mutation to the operator-distal end (probably a terminator for either translation or transcription) of the operon. Recently, the effect of deletion of the operator-distal genes of the trp operon, including the trp E-trp D intercistronic punctuation point, on the degree of polarity created by nonsense mutations lying in the remaining intact region operator-proximal to the deletion terminus was determined (68). The double mutants employed contained a nonsense mutant such as trp E 9829, trp E 9758 or trp E 9905 and deletion trp AEl1 (Fig. 1 3 ) . As already mentioned, transcription of the trp operon segment in deletion mutants of trp E terminates near the site of their deletion termini. A transcription unit beginning from the trp gene segment in these deletion mutants is therefore assumed not to contain a foreign polypeptide initiation site arising by fusion of the remaining trp operon segment with a foreign gene lying a t the other end of the deletion. Thus the double mutants provide the possibility to examine how the gradient of polarity (in the sense of transcriptional events) is affected by deletion of the usually adjacent polypeptide initiation site or of the region between the site of the nonsense mutation and the operator-distal end of the transcription unit. The results of these experiments indicate that a gradient of polarity is conserved in such strains. It seems unlikely that the trp gene segment of trp AE 11 is fused to a very small genetic segment that contains a site functioning as a reinitiator for polypeptide synthesis, since in more extensive studies (192) a similar conclusion was obtained with double mutants containing such deletion mutants as trp AE 9 and trp AE 10. Thus it is tentatively proposed that the distance from the site of the nonsense alteration to either the next reinitiator for polypeptide synthesis (based on the evidence from the lac operon) or to the operatordistal end of the transcription unit, which includes a terminator for transcription, can determine, a t least in part, the degree of polarity (68).

3. EXAMINATION OF THE MOLECULAR MECHANISMS OF THE POLARITY EFFECT Polarity of bacterial operons is associated with a decrease in the amount of mRNA for the operator-distal genes; although some of the mRNA synthesized by the operon corresponds to its whole length, much of it is shorter and corresponds to genes operator-proximal to the mutation site. The loss of mRNA from beyond the mutation site could result either from premature termination of transcription a t or near the mutation or from an accelerated degradation of the messenger corresponding to the operator-distal genes.

388

FUMIO IMAMOTO

a. Kinetic Analysis of the Initial Stages of Transcription in Polar Mutants. During synthesis of the first round of trp mRNA molecules after the shift of a bacterial culture from repressing to derepressing conditions, the growing mRNA chain is presumably attached a t the site of synthesis on thc opcron. Such mRNA rnoleculcs might be expected to bc more resistant to dccay than thosc synthesized under steady-state conditions; i.e., there should bc a period of mRNA accumulation prior to the time a t which steady-state production and degradation of trp mRNA is established. I n fact, under the experimental conditions generally used to derepress the trp operon by depleting tryptophan, relatively slow degradation of trp mRNA is observed a t the initial stage of derepression (129).Degradation of the trp mRNA molecules takes about twice as long as their synthesis during the first 10 minutes after derepression, where 7-8 minutes are required to complete the first set of fulllength trp mRNA molecules. If complete polycistronic trp mRNA molecules are produced by polar mutants, and subsequently the portion of the molecule corresponding to the operator-distal region of the operon is selectively and rapidly degraded, these rcgions of the trp mRNA might be detected during the initial period of trp mRNA accumulation. I n experiments in which pulse-labeling was performed a t different times prior to the appearance of the first complete trp mRNA molecules, reduced levels of trp mRNA were detected in the strong polar mutants and the mRNA region corresponding to the operon region beyond the nonsense codon was conspicuously absent (173).I n addition, sucrose gradient sedimentation studies showed that, with the wild-type control, the size of trp mRNA continued to elongate for the initial 6 minutes after derepression, while the mRNA of a strong polar mutant (trp E 9758) ceased elongating by the end of the second minute after derepression and ended up smaller (173). Although the possibility of fast breakdown during synthesis of the section of the mRNA distal to the nonsense mutation could not have been ruled out, these findings suggestcd that polarity mutations may cause premature termination of transcription of an operon in the vicinity of an introduced nonsense codon. b. Production of Labile naRNA in Polar Mutant. In strongly polar trp E (trp E 9802) and trp D (trp D 9778) mutants, much of the trp mRNA corresponding to the gene carrying the nonsense mutation is apparently very rapidly degraded (176).When a very brief pulse-labeling was performed a t steady-state transcription of the operon derepressed by the addition of indole-3-propionic acid to bacteria growing in minimal medium, the trp mRNA for the mutated gene was detected a t a relatively high level, one that compared with the level of normal production of the mRNA by a missense mutant or wild-type strain. As the length of pulse-

TRANSLATION AND TRANSCRIPTION OF THE

trp

OPERON

389

labeling was increased, however, the detectable portion of the mRNA segments for the mutated gene decreased in the polar mutants. Similar observations were also made on a strong polar trp D amber mutant ( t r p D 9838) (177). Since the ochre mutation in trp D9778 is believed to be located a t thc beginning of trp D and close to thc trp E-trp D intercistronic punctuation point, Morse and Yanofsky (176)assumed that the bulk of the labile trp D mRNA detectable only in the very short period of labeling originated from the trp D region distal to the site of the nonsense mutation. With the strongly polar mutant t r p E 9802, they concluded that the disappearance of the bulk of the trp E mRNA as the length of pulselabeling was increased reflected the rapid degradation of the trp E mRNA segments distal to the nonsense mutation site. This conclusion was based on the determination of the operator-distal trp E mRNA segment by calculating the difference between total t r p E mRNA and that corresponding to only the operator-proximal part of the trp E, using the results of mRNA hybridization with DNA's from $80 pt ED,,, containing all of trp D and trp E, and $80 pt ED, (or $80 pt D2), containing all of trp D and only the distal part of trp E. The t r p operon segment of $80 pt ED, is known to include the trp E region distal to the trp E 9851 site (see Fig. 3) (115,176;pcrsonal communication from C. Yanofsky). An explanation proposed for the above findings is that the trp mRNA region that is untranslatable (and thereby prematurely exposed) due to the discharge of ribosomes at the nonsense codon may rapidly be attacked by endonuclease and degraded (176). The production of labile mRNA in strongly polar mutants can be attributed to premature termination of transcription near the nonsense codon rather than to selective degradation of the messenger distal to the nonsense codon (67,68). Rapid degradation of messenger occurs only in strong polar mutants with nonsense alterations in the initial region of trp E and the messenger, which is highly labile, does not correspond to the region operator-distal to the nonsense mutation but rather to the region that is operator-proximal. During steady-state transcription of the operon derepressed by depleting tryptophan, the most pronounced effect of varying the period of pulse-labeling on the detectable level of trp mRNA was seen in the case of the trp E mRNA of strongly polar trp E mutants (trp E 9829, trp E 9861, trp E 10203, trp E 9802, trp E 1224.2 and trp E 9758) (Table VII) , With other less polar trp E mutants, with the strongly polar mutant trp D 9778, and with the non-polar missense mutant trp C 9941,similar changes in the levels of trp mRNA corresponding to the gene carrying the mutation were not observed (67, 68). The labile mRNA in such strongly polar trp E mutants did not

390

FUMIO IMAMOTO

TABLE VII CHANQES IN trp mRNA LEVELIN POLAR MUTANTS AS A FUNCTION OF THE PERIOD OF PULSE-LABELING'

RNA source, strain trp E9899

trp E9861

trp ElON.3

trp E9809

trp ~ 1 9 8 4 9

trp E9768 trp E990.9

trp E9887 trp E6984

trp E697g trp (79941

Period of pulselabeling (seconds) 10 20 120 10 20 120 10 20 120 10 20 120 10 20 120 10 20 120 10 20 120 10 20 120 10 20 120 10 20 120 10 20 120

Amount of trp-mRNA synthesized (yox 108) trp E

trp E distal

252 275 140 286 270 147 333 431 154 362 296 96 413 404 212 454 440 224 402 435 386 448 434 352

0 9 8 9 6 4 15 14 6 18 10 0 13 12 15 25 24 18 54 60 62 56 51 51 68 65 64 43 99 82 130 125 151

44.4 403 376 641 600 605 530 562 550

labile trp P 112 135 139 123 179 277 266 200 201 192 230 216 16 49 96 82

68 27 36 (-5)

-

( 20) 12

RNA was prepared from derepressed cultures pulsslabeled with ['Hluridine for variousperiods at the tenth minute (steady state) after derepression.Data from Imamoto el al. (68). The trp mRNA values of RNA from cultures pulse-labeled for 2 minutes were subtracted in each case.

TRANSLATION AND TRANSCRIPTION OF THE

trp

OPERON

391

hybridize with DNA from +80 pt E distal phage, the trp gene segment of which includes the region distal to the trp E 9903 site (Fig. 3). The truncated trp mRNA molecules produced by strains trp E 122&, tip E 9758 and trp E 9903 were estimated to correspond to the operatorproximal 45, 53 and 63% of the intact trp E (Fig. 13). The molecular size of these truncated mRNA molecules has been assumed to correspond to the distance from the beginning of the operon to the site of the mutation (68, 8 1 ) . Thus the distance from the mutational sites in trp E 12242 and trp E 9758 to that in trp E 990s should correspond t o only 18 and lo%, respectively, of the trp E. As shown in Table VII, twice as much trp E mRNA was detected in strains trp E 12249 and trp E 9768 using very short pulse-labelings as when the labeling period was 2 minutes. Thus, the bulk of the labile mRNA produced by strongly polar mutants such as trp E 122.42 and trp E 9758 must correspond to the operatorproximal region before the nonsense mutation in trp E. The possibility that the trp mRNA synthesized during very short labeling periods (10 or 20 seconds) consists of molecules larger than those detected during a 60-second labeling period due to selective degradation of the messenger segment distal to the nonsense mutation site was tested using strain trp E 98U2 (67). The sedimentation profiles of the trp mRNA isolated following the lo-, 20-, and 60-second labeling periods were essentially the same (Fig. 14), negating this possibility. If transcription of the trp operon in strong polar mutants is frequently terminated near the site of the nonsense codon, the messenger detached prematurely from the operon might be rapidly attacked by nuclease and degraded. When a constant period of pulse-labeling is employed, the probability of survival of the premature messenger might decrease as the length of the transcribable region is shortened. This possibility was demonstrated in experiments measuring detectable trp mRNA as a function of the length of pulse-labeling with various deletion mutants of the trp E (68). Comparison of the order of the molecular size of the trp mRNA from various polar and deletion mutants of the trp E with the order of the mutational alterations and deletion termini on the genetic map indicates that trp mRNA size increases in relation to the distance from the beginning of the operon to the site of the deletion termini (Fig. 13), which indicates that the trp mRNA molecules of these deletion mutants have apparently terminated near the site of their deletion termini (68). All these deletion mutants have another deletion terminus at sites between the t o n B and td K genes (192). It is conceivable that these mutants have out-of-phase fusions or the genetic region adjacent to the end of the deletion is oriented in the opposite direction (192u).

392

FUMIO IMAMOTO

I500 -1.0

4000 500

0

-f 3000

0

5000 4000

3000

2000 I000 0 Fraction number

FIG.14. Sedimentation profiles of l r p mRNA of l ) p E 9802 pulse-labeled for various periods during steady-state transcription. Pulse-labeling was carried out with ['Hluridine for 10 (A), 20 (B) and 60 (C)seconds 20 minutes after derepression (30°C). RNA was sedimented in a 5-3076 linear sucrose gradient. Data were taken from Imamoto (67). 0-0, trp E mRNA; , hybridization with +80 DNA (background value) ; -, optical density at 260 nm.

---

-

As the deletion termini (trp operon side) of trp AE5 and trp OAE2 are located within the region where six strongly polar trp E mutants that produce labile mRNA are located (to the site of trp E ,9768) (Fig. 13), these two deletion mutants might also be expected to produce labile messenger. These expectations were realized (Table VIII) . The analogous production of labile mRNA in the strongly polar and deletion mutants of trp E suggests that the segment of the trp messenger detached prematurely from the operon in the strong polar mutants as a result of termination of transcription a t or near the site of nonsense

TRANSLATION AND TRANSCRIPTION OF THE

tTp

393

OPERON

TABLE VIII CHANGES IN frp-mRNA LEVELIN DELETION MUTANTS AS PEltIOD O F PULSE-LABELING'

A

FUNCTION OF THI.:

RNA source

Strain

Period of pulse-labeling (seconds)

Amount of trp-mRNA synthesized (% x 10-3) labile trp Eb

trp E ~~

trp AE6

trp OAE2 trp AE8

trp OAE6 trp A E l l

5 10 20 120 10 20 120 10 20 120 10 20 120 10 120

288 220 157 96 248 248 138 343 343 366 368 373 342 700 663

~

192 124 61 110 110 (-23) (-23) 26 31 37

a RNA was prepared from bacterial cultures pulse-labeled with [aHIuridine for the various periods indicated at the tenth minute after derepression. Data were taken from Imamoto el al. (68). bThe trp-mRNA values of RNA from cultures pulse-labeled for 2 minutes were subtracted in each case.

alteration is critically susceptible to nucleolytic attack. In this regard, it is of interest that the stability of messengers detached from the DNA template in E . coli was lower than that of molecules attached to the template (193). Recent observation indicate that two strongly polar mutants of t r p E ( t r p E 9914) and t r p D ( t r p D 159),derepressed by depleting tryptophan, produce labile E mRNA and D mRNA, respectively, which correspond to the region distal to the mutant nonsense codon (193~). Labile mRNA corresponding to genes distal to the gene with the nonsense codon could not be demonstrated. The labile mRNA in t r p E 9914 was not hybridizable with DNA from 480 ptEaiXtHl phage, the trp gene segment of which includes the operator-distal third of the trp E (cf. Figs. 3 and 13) (S. Hiraga, personal communication). The basis for the contradictory findings between the two groups of experiment described is not yet clear, but there are the following possibilities.

394

FUMIO IMAMOTO

The different techniques of derepressing the t r p operon might affect the degree of polarity. Polar trp D mutants show rapid degradation of trp D messenger when derepressed by indole-3-propionic acid or indole-3acrylic acid (176,177, 194).These tryptophan analogs decrease remarkably the severity of polarity. The preferential degradation of the exposed, untranslatable trp mRNA may be retarded by tryptophan starvation effected by adding a tryptophan analog (194).Alternatively, tryptophan analogs may cause artificial events in the “coupling” machinery by which transcription is allowed to continue independently of the ‘arrested translational machinery, thereby resulting in the production of untranslatable trp mRNA molecules that are somewhat susceptible to endonucleases. I n a constitutive strain, trp R- trp D 9778, grown in an excess of tryptophan, neither production of hyperlabile mRNA nor reduction in the extent of polarity was observed under the conditions used by Morse and Guertin (194) (also personal communication from D. E. Morse). An interpretation is that, when cells face an excess of intracellular tryptophan, degradation of untranslatable mRNA is so rapid that this mRNA cannot be detected (194).However, stimulation in the rate of trp mRNA degradation after the addition of tryptophan to tryptophan-starved cells is 2- to 3-fold the rate of degradation in starved cells (70;unpublished data of Morikawa and Imamoto). Differences in growth conditions of the bacteria might be responsible for the discrepancy in the findings. As the mechanisms by which transcription is prematurely arrested when translation is coupled, it may be that endonucleolytic cleavage of the nascent mRNA chain occurs (195) upon the arrest of transcription that occurs as a result of the event, whatever it may be, that terminates translation. The physiological conditions of the bacteria might determine how quickly all this might occur. The fact that detectable hyperlabile mRNA corresponds to the gene with the nonsense alteration but not to more distal genes (176)suggests that degradation catches up with RNA polymerase near the operatordistal end of the untranslatable region of mRNA, rendering distal mRNA regions undetectable (176,177,194),and that transcription terminates in genes distal to the one with the nonsense alteration site (67,68). c. Effect of Polarity Suppressors. The suppressor A (su A ) allele suppresses the polarity effect of nonsense mutations of the lac operon of E. coli but not the mutant phenotype itself (168,85).Relief of polarity by su A is associated with almost normal detectability of trp D mRNA with two strongly polar t r p D mutants (177).It was therefore suggested that the su A strain suppresses polarity since it lacks endonuclease, the normal product of the wild-type su A allele. The su A mutation is a lesion

TRANSLATION AND TRANSCRIPTION OF THE

trp

OPERON

395

in the gene coding for an endonuclease, but it appears that its activity is relatively weak, producing only a few breaks in the substrates in 10 to 20 minutes at 37°C (196). Subsequent observations indicate that the amber su A mutation relieves by only 7% the reduction in the amount of distal trp mRNA in the constitutive trp R- trp D polar mutant, whereas addition of a tryptophan analog such as indolepropionic acid or indoleacrylic acid leads to a greater extent (more than 70%) of relief by su A of the diminution in the level of presumed untranslatable trp mRNA in trp R+ or trp R- strains of trp D polar mutants. This might result from a peculiar ability of the tryptophan analogs to reduce remarkably the severity of transcriptional polarity (176, 177, 194; C. Yanofsky quoted in ref. 16). Two new polarity suppressors in E . coli, su 27 and su 78, relieve polarity in the lac and trp operons, and neither suppresses the mutant phenotype itself (196).The mapping indicates that su 78, like su A (177), is cotransduced with ilv, but that su 27 is genetically different from either of these suppressors. The effect of these suppressors on the production of mRNA for the y and a genes of the lac operon in a strain with the extreme polar mutation of the z gene was partial relief: the suppressors relieved by 2331% the reduction in the amount of distal lac mRNA. The rapid degradation of both lac mRNA and bulk mRNA induced by puromycin was partially reversed by su 78 and completely reversed by su 29. It was suggested that these suppressors act by stabilizing mRNA after premature termination of protein synthesis. d. Intactness of Full-Size Messenger in Polar Mutants. The relatively few messenger molecules produced by polar mutants has been assumed to consist of full size mRNA’s corresponding to the whole operon (81, 175). However, no critical test had been made as to whether large messenger molecules containing the untranslable region created as a result of the termination of translation in polarity mutants are resistant to endonucleolytic attack in this region. This would be particularly important in view of the results of in vitro translation studies of bacteriophage RNA where no RNA degradation occurs. Here the polarity of nonsense mutants results from the complete and rapid release of the ribosome from the messenger-ribosome complex a t strongly or weakly polar amber codons (157).It is known that, unless a reinitiator of polypeptide synthesis exists near by, polypeptide synthesis does not efficiently restart in the region between the nonsense codon and the subsequent intercistronic punctuation, i.e., the C-terminal polypeptide fragment is not efficiently made (154-157). If polarity of bacterial operons results from the rapid degradation of the segment of the messenger distal to the nonsense alteration leading to nontranslation of this region of the polycistronic messenger

396

FUMIO IMAMOTO

(76,176, 177, 194, 196), it might be expected that the small number of large messenger molecules made by the polar mutant might also be fragmented by endonucleolytic attack in the exposed internal untranslatable region. To test the above possibility, a double mutant consisting of the moderately strong polar mutation trp E 9903 and the deletion mutation trp AD5 whose terminus is located in the middle of trp D (Fig. 3) was used to examine the molecular size distribution of mRNA molecules containing trp D information (68).The degree of transcriptional polarity was observed to remain as strong in the double mutant as in the parental strain trp E 9903. I n the double mutant, the trp mRNA species including the trp D mRNA segment was obviously larger, sedimenting like the trp mRNA of trp AD5 (20S), than the predominant trp mRNA species, sedimented at the position of the short trp mRNA of trp E 9903 (14 S). If endonucleolytic cleavage of the mRNA had occurred near the site of the trp E 9903 nonsense codon, much smaller molecules of trp D mRNA would have been observed. The failure to observe such molecules suggests the large trp mRNA molecules in the double mutant are not subject to such an endonucleolytic cleavage and that the trp D mRNA synthesized is still covalently attached to the trp E mRNA of the polycistronic messenger. A similar finding was obtained with the large trp mRNA molecules produced by strain trp E 9903 (68).Thus the intactness of the large messenger molecules in polar mutants has been verified, constituting evidence unfavorable to the “degradation” hypothesis. e. Lower Frequency of Internal Initiation in Polar Mutants. An attempt was made to determine the distribution of RNA polymerase molecules over the region of the trp operon distal to the mutated gene (67,68). Use was made of the effect of 2-4-dinitrophenol during derepression (described previously in Section IV, C, 4), which severely but reversibly inhibits transcription, but which upon removal, leads to the synthesis of incomplete trp messenger molecules containing only operator-distal information as a result of random internal reinitiation of synthesis within the operon (65, 66). Those trp mRNA fragments synthesized as a result of internal initiation are translated with almost normal efficiency ( 6 5 ) . If polarity results from a rapid degradation of the segment of the messenger distal to the nonsense alteration due to its nontranslation, then after intragenic initiation of transcription in the presence of tryptophan (added to block ordinary initiation a t the trp promoter), normal numbers of functional messenger would be synthesized from the region distal to the nonsense alteration. On the other hand, if polarity is attributable to premature termination of transcription and the dissociation of the polymerase from the template, a scanty distribution of polymerase molecules over

TRANSLATION AND TRANSCRIPTION OF THE

trp

397

OPERON

the distal genes of the opcron would result, leading to a greatly reduced frequency of intragenic initiation. These predictions were tested in the following experiment: 5 mM dinitrophenol was added to a derepressed culture 10 minutes after derepression effected by depleting tryptophan (to ensure a steady state distribution of polymerase molecules over the operon), and the culture was further incubated for 45 minutes a t 30°C. During this incubation, t r p mRNA molecules which were in the process of synthesis before the addition become detached from the template and are broken down (68). After the removal of the dinitrophenol, the bacteria were quickly transferred into warm standard medium containing tryptophan and were pulselabeled with [3H]~ridinefor the first minute of incubation. The results (Table IX) show that the frequency of internal initiations in the operator-distal genes ( t r p C, t r p B and t r p A ) correlates closely with the extent of polarity. A similar experiment in which only trp A mRNA was measured (with $80 pt A DNA) yielded a similar result (68). Thus transcription of the operator-distal genes of the trp operon is limited in strong polar mutants. It appears likely that molecules of RNA polymerase wander along the operon without transcribing in the presence of dinitrophenol. Upon removal of the inhibitor, these polymerase molecules resume synthesis of mRNA a t their resident position on the template. TABLE IX COMPARISON OF trp-mRNA (trp C-A mRNA) LEVELSOF VARIOUSPOLAR MUTANTSIN TEE STEADYSTATE AND AT THE INITIAL STAGEAFTER INTERNAL INITIATION OF TRANSCRIPTION’ Amount of trp C-A mRNA synthesized (% X loa) Strain trp trp trp trp trp trp trp trp

E9829 E9802 39768 E6984 D9778 C10377

C9771 C9941

In steady state

After internal initiation

77 79 67 182 77 178 277 332

64 68 62 210 95 117 210 248

RNA was prepared from bacterial cultures pulselabeled with [aH]uridine for 1 minute at the twentieth minute after derepression (steady state), or from cultures pulse-labeled with [SHluridine under conditions of repression during the first minute after the removal of dinitrophenol (internal initiation). In internal initiation, the cultures were incubated in 5 mM dinitrophenol, added a t 10 minutes after derepression, for 45 minutes. Data from Imamoto et al. (68).

398

FUMIO IMAMOTO

The reduced frequency of intragenic initiation observed with the strong polar mutants is therefore best explained by a reduced number of RNA polymerase molecules on the operator-distal genes of the operon.

VI. Effect of a Block in Translation on Transcription In attempting to picture the mechanism of gcnc expression and of genetic polarity exerted by nonsense mutation in bactcrial operons, a hypothesis was proposed (14.2) based on the idea of a feedback mechanism existing between transcription and translation. I n spite of a great volume of suggestive experimentation, it is still not certain whether a direct inhibition of transcription is caused by an arrest of the translational machinery (2,197). From observations on the temperature-sensitive mutant of the E . coli 30s ribosomal P10 protein, the product of the str gene (198), it appears that under nonpermissive conditions thc overall ratc of trp mRNA synthesis, but not bulk mRNA synthesis, closely parallels the decreased rate of bulk protein synthesis (199, 2 0 ) . The P10 protein is known to be required for natural mRNA-directed protein synthesis and for the binding of N-formylmethionyl tRNA, but not for poly (U) -directed polyphenylalanine synthesis, and thereforc is believed to be involved in thc initiation of protein synthesis (201,202). Thus it was suggested that continued transcription of some species of gcncs (possibly induciblc-rcpressiblc opcrons including thc trp operon), but not necessarily all gcncs, is facilitated by initiation of protein synthesis and/or movement of ribosomcs along mRNA. A similar conclusion has been drawn from the results of more recent experiments (.$!03) using the temperature-sensitive mutant of the E . coli ribosome translocation G factor (.$!04),whcrc it was found that arrest of ribosome translocation is causally related to stabilization of nascent and unloaded mRNA (203) and also to inhibition of transcription of some species of genes, including the trp operon (203;D. E. Morse cited in ref. 203). The general significance of a model for “coupling” of transcription and translation in some operons has recently been reviewed (197). During the past fcw years the primary sitcs of action of many antibiotics that block protein synthesis have been elucidated. Antibiotics that act a t specific steps in the process of translation are valuable tools in testing the hypothesis of an obligatory “coupling” between transcription and translation. Transcription of the lac operon in chloramphenicolor puromycin-treated E. coli (205) and of the trp operon in chloramphenicol-treated E . coli (206) is diminished, an event attributable to

TRANSLATION AKD TRANSCRIPTION OF THE

trp

OPERON

399

either an accelerated degradation of nascciit inessciiger or to an arrest of transcription. In E . coli trcatcd with tctracycliiie, cliloraniphenicol, puromycin, crythroiiiycin or fusidic acid, tlic overall ratc of t r p mRNA synthesis is rctluccd coordinately with tlic tlccrcasc in the ratc of polypcptidc synthesis caused by the action of these antibiotics, wliilc the rate of syiitlicsis of bulk mRNA is only partially reduced, thcreby suggesting that the synthesis of sonic spccics of mRNA, including trp inRNA, dcpends on a functional translational machinery while that of other species of mRNA does not (207,208). Transcription of tlic “immcdiatc-carly” class of gencs of is not inhibited by chloramphenicol (209-912). It can be visualized that if the trp operon were translocated into the early region of A, certain orientations of the trp genes would be possible wherein trp transcription would now be controlled by both the X repressor and the trp repressor (Fig. 15). I n fact, it has been obscrvcd in translational experiments that the expression of trp genes fused into the X genome is affected by A immunity repressor as well as by trp repressor (213; personal communication from I. P. Crawford). Transcription of such a translocated trp opcron is also under this dual control (207,$ 1 4 ) . Synthesis of trp mRNA specific for the translocated trp operon of Apt was assayed after infecting deletion mutant trpAE1, which lacks the whole trp operon, with the Apt phage. Trp mRNA synthesis by XptE-A, which possesses the trp operon intact, was found to bc only partially rcpresscd by fully activated trp rcpressor in strain trpAE1, nonlysogcnic for A. On the other hand, trp inRNA synthcsis by AptE-A in strain trpAEl ( A ) (i.e., lysogenic for A and therefore possessing A repressor) is completely repressed when the trp repressor is fully activated (excess tryptophan). Phage AptBA which possesses intact trpA and trpB but does not possess thc trp promoter and operator exhibits transcription of trp mRNA that is not repressed a t all

FIG. 15. Diilgriiininutic rrprc~ccnlutionof dual control of transcript.ion of thc translocated trp operon of Apt phages.

400

FUMIO IMAMOTO

by trp repressor, but is regulated completely by X repressor (207,214) (Fig. 15). These results indicate that trp mRNA synthesis from XptE-A consists of two types. One is initiated at the trp promoter, controlled by trp repressor, another is initiated a t the X promoter, controlled by h repressor. If the indispensability of “coupling” of transcription to the translational machinery is determined by the nature of the promoter, then of the dual transcription of the trp genes in XptE-A, only that initiated a t the trp promoter should be inhibited by antibiotics. On the other hand, if indispensability of “coupling” is determined by signals located inside the operon, then both types of trp transcription should be initiated by antibiotics. Transcription of the trp genes in XptE-A after infection of trp AEl (nonlysogenic) is only partially inhibited by chloramphenicol in the absence of tryptophan, while in the presence of tryptophan, trp transcription originating a t the X promoter of XptE-A is not inhibited a t all (207). In contrast, synthesis of X early mRNA remains constant in the presence of chloramphenicol under both sets of conditions (207).Transcription of the trp genes of XptE-A after infection of strain trp AE1 (A) is totally sensitive to the action of chloramphenicol (207,215). The shutdown effect is the same as that seen with trp transcription originating on the bacterial chromosome. From these observations, it can be concluded that transcription initiated a t the trp promoter is sensitive to the uncoupling of translation by chloramphenicol, while trp transcription originated a t the A promoter is not (Fig. 16). An essentially similar effect was observed in experiments with tetracycline, puromycin, erythromycin and fusidic acid (215). Functional coupling between the translational machinery and transcribing RNA polymerase molecules is obligatory for continued transcription of some species of genes (possibly inducible-repressible operons, including the trp operon), but is dispensable for the expression of other species of genes. The promoter or the very beginning region of the gene or operon appears to play the most important role in determining the CM uensitivo

J

CM inuensltiva

\ I 1

I

3 f 9 A B C D E

N

EL

I

Apt € - A

Fro. 16. Diagrammatic representation of the antibiotic sensitivity of the transcription of translocation trp operon of Apt.

TRANSLATION AND TRANSCRIPTION OF THE

EFFECTOF trp-

Phage

Host cell

None

AE1 AEUX) AE1 AE1 AE1(X) AE1(X) AE1 AEI AEl(X) AEl(X)

XptE-A

XptBA

AND

tv OPERON

401

TABLE X X-REPRESSORS ON mRNA SYNTHESIS OF Apt“

Tryptophan, 50 pg/ml -

-

++++

Active repressor existing None X None trP X X trp None trP

+

X

X

+ trp

trp mRNA (% x 109

X early mRNA

14 0 1531 614 1428 36 651 492 6 29

21 44 856 705 248 139 649 740 288 172

(% x 109

a Bacteria were suspended in T1 medium (41) containing 2mM KCN and infected with Apt at a multiplicity of 5. After incubation for 15 minutes a t 30”C, unabsorbed phages were removed by centrifugation. The cells were transferred into warm (30°C) minimal medium supplemented with the 19 protein amino acids (66),except htryptophan, or with all 20 amino acids. The cells were shaken vigorously a t 30°C and pulse labeled with [aH]uridinefor 2 minutes at the eighteenth minute of incubation. Trp mRNA was assayed by employing &OptE-D and &OptC-A DNA’s. The sum of these hybrids is listed as XptE-A transcription. The hybrid with &OptC-A DNA is presented as XptBA transcription. Assay of early mRNA was carried out employing DNA from +80-X hybrid phage, iAbPh80,kindly supplied by E. R. Signer. Values for trp mRNA and X early mRNA are expressed aa the percent of total [‘HIRNA hybridized. Data were taken from Imamoto and Tani (207).

requirement for “coupling” of RNA polymerase to the translational machinery. The inability to detect a normal level of mRNA accumulation could result either from an arrest of synthesis or from the accelerated degradation of exposed mRNA left unoccupied by ribosomes as a result of blockage of translation. The finding that trp mRNA synthesis originating a t the trp promoter ceases upon blockage of translation by antibiotics, while trp mRNA synthesis originating a t the X promoter does not do so, seems to be unfavorable to the view that the reduction in the level of t r p mRNA found during blockage of translation is attributable to simple degradation of the nascent and unloaded mRNA. The event appears to be related to the eventual arrest of transcription in the beginning region of the trp operon. Mechanisms by which transcription is prematurely arrested when translation is uncoupled could involve the loss of some factor(s) structurally associated with ribosomes required to stimulate mRNA synthesis (Sl6-220), the entanglement of RNA polymerase with nascent mRNA chains unoccupied by ribosomes (S),or the prevention of the unfolding

402

FUMIO IMAMOTO

of a DNA-nascent mRNA hybrid transcriptional intermediate in the absence of ribosome movement. Alternatively, endonucleolytic cleavage of the nascent mRNA chitin may occur (196) upon the arrest of transcription, which occurs as a result of the event, whatever it might be, that terminates translation. The physiological conditions of the bacteria might determine how quickly all this would occur. More information is obviously necessary to gain a better understanding a t the molecular level of the principal mechanisms of the transcriptional events caused by an arrest of the translational machinery. AIthough opposing hypotheses have been proposed invoking either premature termination of transcription or premature degradation of the messenger, a further elucidation of the characteristics of normal and abnormal transcription termination and of messenger degradation is warranted. Only then will we arrive a t a complete understanding of the mechanism of polarity and of transcription in general. ACKNOWLEDGMENTS I am greatly indebted to Dr. R. H. Bauerle for his critical reading of the manuscript. I thank Drs. C. Yanofsky, P. Margolin and I. P. Crawford for the opportunity of reading their manuscripts before publication. I also thank Dr. J. Ito for helpful discussions.

REFERENCES 1. W.Epstein and J. R. Beckwith, ARB 37, 411 (1968). 2. E.P. Geiduschek and R. Haselkorn, ARB 38, 647 (1969). 3. R. G. Martin, Annu. Rev. Genet. 3, 181 (1909). 4. H.E.Umbarger, ARB 38, 323370 (1969). 6. C. Yanofsky, Bacteriol. Rev. 24, (1960). G . P. Margolin, in “Metabolic Pathways,” Vol. 5 (H. J. Vogel, ed.), p. 389. Academic Prew, New York, 1971. 7. C. Yanofsky and E. S. Lennox, Virology 8,425 (1959). 9. M. Demerec and Z. Hartman, Carnegie Znst. Wash. Publ. SlZ, 5 (1956). 9. E. Balbinder, Genetics 47, 469 (1962). 10. A. J. Blume and E. Balbinder, Genetics 53, 577 (1966). 11. J. It0 and C. Yanofsky, JBC 241, 4112 (1966). 12. R. H. Bauerle and P. Margolin, CSHSQB 31, 203 (1906). 13. R. H. Bauerle, J. B. Secor and M. Grieshaber, FP 30, Abstr., 1058 (1971). 14. L. H. Hwang and H. Zalkin, JBC 246, 2338 (1971). 16. M.Grieshaber and R. H. Bauerle, Nature (London) New Biol. 236, 232 (1972). 16. C. Yanofsky, V. Horn, M. Bonner and S. Stasiowski, Genetics 69, 409 (1971). 17. T.E.Creighton and C. Yanofsky, JBC 241,4614 (1966). 18. 0.H. Smith, Genetics 57, 95 (1967). 19. D.Wilson and I. P. Crawford, JBC 240, 4801 (1965). 20. M. E. Goldberg, T. E. Creighton, R. L. Baldwin and C. Yanofsky, JMB 21, 71 (1966). 81. J. Ito, E.Cox and C. Yanofsky, J. Bact. 97, 725 (1969).

.

TRANSLATION AND TRANSCRIPTION OF THE

trp

OPERON

403

12. H. Zalkin and D. Kling, Bchem 7, 3566 (1966). $3. E. J. Henderson, H. Zalkin and L. H. Hwang, JBC 245, 1424 (1970). 94. B. C. Carlton and C. Yanofsky, JBC 237, 1531 (1962). 26. T. I. Baker and I. P. Crawford, JBC 241, 5577 (1966). 20. D. Smith and R. H.Bauerle, Bchem 8, 1451 (1969). 27. G. M. Hathaway, S. Kida and I. P. Crawford, Bchem 8, 989 (1969). 28. U. Henning, D. R. Helinski, F. C. Chao and C. Yanofsky, JMB 237, 1523 (1962). 29. E. J. Henderson, H. Nagano, H. Zalkin and L. H. Hwang, JBC 245, 1416 (1970). SO. J. Ito, Nature (London) 223, 57 (1969). 31. A. Matsushiro, S. Kida, J. Ito, K. Sat0 asd F. Imamoto, BBRC 9, 204 (1962). 32. A. Matsushiro, K. Sato, J. Ito, S. Kida and F. Imamoto, JMB 11, 54 (1965). 33. J. Ito and I. P. Crawford, Genetics 52, 1303 (1965). 34. C. Yanofsky and J. Ito, JMB 2, 313 (1966). 36. R. H. Bauerle and P. Margolin, PNAS 56, 111 (1966). 36. S. Hiraga, JMB 39, 159 (1969). 37. E. Balbinder, R. Callahan 111, P. P. McCann, J. C. Cordaro, A. R. Weber, A. M. Smith and F. Angelosanto, Genetics 66, 31 (1970). 38. G. N. Cohen and F. Jacob, C. R. Acad. Sci. 248, 3490 (1959). 39. D. E. Morse and C. Yanofsky, JMB 44, 185 (1969). 40. A. Matsushiro, K. Sat0 and S. Kida, Virology 23, 299 (1964). 41. F. Imamoto, N. Morikawa, K. Sato, 5. Mishima and T. Nishimura, JMB 13, 157 (1965). 42. F. Imamoto, N. Morikawa and K. Sato, JMB 13, 169 (1965). 43. F. Jacob and J. Monod, JMB 3, 318 (1961). 44. J. Monod and Cohen-Bazire, C. R.Acad. Sci. 236,530 (1963). 4. J. Ito and F. Imamoto, Nature (London) 220, 441 (1968). 40. F. Imamoto, PNAS 60, 305 (1988). 47. R. F. Baker and C. Yanofsky, PNAS 60, 313 (1968). 48. F. Imamoto, Nature (London) 220, 31 (1968). 49. F. Imamoto, Mol. Gen. Genet. 106, 123 (1970). 60. P. Margolin and R.H. Bauerle, CSHSQB 31, 311 (1966). 61. R. H. Bauerle and P. Margolin, JMB 26, 423 (1967). 62. D. E. Morse and C. Yanofsky, JMB 38, 447 (1968). 63. A. J. Blume, A. Weber and E. Balbinder, J . Buct. 95, 2230 (1968). 64. M. E. Goldberg and S. I. Edelstein, JMB 46, 431 (1969). 66. R. L. Somerville and C. Yanofsky, JMB 8, 616 (1964). 66. J. Ito, BBA 204, 624 (1970). 67. D. H. Alpers and G. M. Tomkins, PNAS 53, 797 (1965). 68. A. Kepes, BBA 138, 107 (1967). 69. R. F. Goldberg and M. A. Berberich, PNAS 54, 279 (1965). 00. G. Michaelis and P. Starlinger, Mol. Gen. Genet. 100, 210 (1967). 61. D. E. Morse, R. F. Baker and C. Yanofsky, PNAS 60, 1428 (1968). 62. M. A. Berberich, P. Venetianer and R. F. Goldberger, JBC 241, 4426 (1966). 63. M. A. Berberich, J. S. Kovach and R. F. Goldberger, PNAS 57, 1857 (1967). 64. P. Venetianer, M. A. Berberich and R. F. Goldberger, BBA 166, 124 (1968). 66. F. Imamoto and J. Ito, Nature (London) 220,27 (1968). 86. F. Imamoto, JMB 43, 51 (1969). 67'. F. Imamoto, Nature (London) 228, 232 (1970).

404

FUMIO IMAMOTO

08. F. Imamoto, Y. Kano and S. Tani, CSHSQB 35, 471 (1970). 69. R.Baker and C. Yanofsky, CSHSQB 35, 467 (1970). 70. D. E. Morse, R. D. Mosteller and C. Yanofsky, CSHSQB 34, 725 (1969). 71. E. Kaminskas and B. Magasanik, JBC 245, 3549 (1970). 7%. M. Jacquet and A. Kepes, J M B 60,453 (1971). ?3.R. Baker and C. Yanofsky, J M B 69, 89 (1972). ?4. G. Attardi, S. Naono, J. Rouviere, F. Jacob and F. Gros, CSHSQB 28, 363

(1963).

76. M. Hayashi, S. Spiegelman, N. Franklin and S. E. Larin, PNAS 49, 729 (1963). 7G. G. Contesse, S. Naono and F. Gros, C. R. Acad. Sci. 263, 1007 (1966). 77. J. D. Stubbs and B. D. Hall, J M B 37, 289 (1968). 78. B. S. Guttman and A. Novick, CSHSQB 28, 373 (1963). 79. Y. Kiho and A. Rich, PNAS 54, 1751 (1965). 80. R. G. Martin, CSHSQB 28, 357 (1963). 81. F. Imamoto and C. Yanofsky, J M B 28, 1 (1967). 82. K. Ippen, J. H. Miller, J. G. Scaife and J. R. Beckwith, Nature (London)

217, 825 (1968). 83. J. H. Miller, K. Ippen, J. G. Scaife and J. R. Beckwith, J M B 38, 413 (1968). 84. F. Jacob, A. Ullman and J. Monod, C. R. Acad. Sci. 258, 3125 (1964). 86. J. G. Scaife and J. R. Beckwith, CSHSQB 31, 403 (1966).

80. B. N. Ames, P. E. Hartman and F. Jacob, J M B 7 , 23 (1963). 87. P.Margolin and F. H. Mukai, Bacteriol. Proc. p. 87 (1964). 88. R. H. Bauerle and P. Margolin, Bacleiiol. Proc. p. 94 (1965). 89. R. Callaham, A. J. Blume and E. Balbinder, J M B 51, 709 (1970). 90. C. Yanofsky, B. C. Carlton, J. R. Guest, D. R. Helinski and U. Henning, PNAS 51, 266 (1964). 91. J. R. Guest and C. Yanofsky, JBC 241, 1 (1966). 92. H.M. Dintzis, PNAS 47, 247 (1961). 93. R. E. Thach, M. A. Cecere, T. A. Sundarajan and P. Doty, PNAS 54, 1167 (1965). 94. M. Salas, M. A. Smith, W. M. Stanley, A. J. Wahba and S. Ochoa, JBC 240, 3988 (1965). 96. E. Terzaghi, Y. Okada, G. Streisinger, J. Emrich, M. Inoue and A. Tsugita, PNAS 56, 500 (1966). 96. A. Goldstein, J. Kirschbaum and A. Roman, PNAS 54, 1669 (1965). 97. U. Maitra and J. Hurwith, PNAS 54, 815 (1965). 98. H.Bremer, M. W. Konrad, K . Gaines and G. S. Stent, J M B 13, 540 (1965). 99. F. Jacob and J. Monod, CSHSQB 26, 193 (1961). 100. W. Gilbert and B. Muller-Hill, PNAS 56, 1891 (1966). 101. J. R.Beckwith, Science 156, 597 (1967). 101. R. R. Arditti, J. G. Scaifeand J. R. Beckwith, J M B 38, 421 (1968). 103. G. Edlin, G. S. Stent, R. F. Baker and C. Yanofsky, J M B 37, 257 (1968). 104. R. Lave114 and G. DBHauwer, J M B 37, 269 (1968). 106. 0. Maalfle and N. 0. Kjeldgaard, “Control of Macromolecular Synthesis.” Benjamin, New York, 1966. 106. G. Contesse, M. C&pin and F. Gros, Bull. SOC.Chim. Biol. 51, 1445 (1969). 106a. A. Kepes, BBA 76, 293 (1963). 107. R. D. Mosteller, J. K. Rose and C. Yanofsky, CSHSQB 35, 461 (1970). 108. F. Imamoto, MoZ. Gen. Genet. 105, 298 (1969). 109. Jackson and C. Yanofsky, J M B 69, 307 (1972).

TRANSLATION AND TRANSCRIPTION OF THE

trp

OPERON

405

110. J. F. Atkins and J. C. Loper, PNAS 65, 925 (1970). 111. G. Wuesthoff and R. H. Bauerle, J M B 49, 171 (1970). 112. A. Sarabhai and S,Brenncr, J M B 27, 145 (1967). 113. T. Grodzicker and D. Zipser, J M B 38, 305 (1968). 114. M. L. St. Pierre, J M B 35, 71 (1968). 115. D. E. Morse and C. Yanofsky, JMB 41, 317 (1969). 116. J. A. Shapiro, J M B 40, 93 (1969). 117. E. Jordan, H. Saidlrr and P. Starlinger, Mol. Gen. Genet. 100, 296 (1967). 118. E. Jordan, H. Saidler and P. Starlinger, Mol. Gen. Genet. 102, 353 (1968). 110. R. Callaham and E. Balbinder, Science 168, 1586 (1970). 180. F. Gros, J. M. Dubert, A. Tissikres, 8. Bourgeois, M. Michelson, R. Soffer and L. Legault, CSHSQB 28, 299 (1963). 121. F. Imamoto, in preparation. 122. J. K. Rose, R. D. Mosteller and C. Yanowsky, J M B 51, 541 (1970). 123. H. Manor, D. Goodman and G. S. Stent, J M B 39, 1 (1969). 1.84. H. Bremer and D. Yuan, J M B 38, 163 (1968). 125. H. Bremer and D. Yuan, J M B 34, 527 (1968). 126. R. 0. Kaempfer and B. Magasanik, J M B 27,475 (1967). 1xi. F. Lacroute and G. S. Stent, JMB 35, 165 (1968). 128. G. S. Stent, Proc. Roy. Soc. Ser. B 164, 181-197 (1966). 129. N. Morikawa and F. Imamoto, Nature (London) 223, 37, (1969). 150. D. E. Morse, R. Mosteller, R. F. Baker and C. Yanofsky, Nature (London) 223, 40 (1969). 131. R. F. Baker and C. Yanofsky, Nature (London) 219,26 (1968). 132. M. Kuwano, C. N. Kwan, D. Apirion and D. Schlessinger, in “First Depetit

Symposium on RNA-Polymerase a d Transcription,” North-Holland Publ., Amsterdam, pp. 222-232. 1969. 133. R. D. Mosteller and C. Yanofsky, J M B 48,525 (1970). 134. R. D. Mosteller and C. Yanofsky, J. Bact. 105, 268 (1971). 135. J. Davies and F. Jacob, J M B 36, 413 (1968). 136. F. C. Neidhardt, This series 3, 145 (1964). 137. G . S. Stent and S. Brenner, P N A S 47, 2005 (1961). 138. C. G. Kurland and 0. Maalee, J M B 4, 193 (1962). 139. J. D. Friesen, J M B 20, 559 (1966). 140. D. W. Morris and J. A. DeMoss, P N A S 56, 262 (1966). 141. P. W. Morris and N. D. Kjeldgaard, J M B 31, 145 (1968). 1 4 1 ~ J. . Forchhammer and N. 0. Kjeldgaard, J M B 37,245 (1968). 142. G. S.Stent, Science 144, 816 (1964). 143. J. D. Stubbs and B. D. Hall, J M B 37, 305 (1968). 144. G. Edlin and J. Neuhard, J M B 24, 225 (1967). 146. T. Yura, M. Imai, T. Okamoto and S. Hiraga, BBA 169, 496 (1968). 1450. G. Zubay and D. A. Chambers, CSHSQB 34, 753 (1969). 146. G. Zubay and M. Lederman, PNAS 62, 550 (1969). 147. R. Arditti, L. Eron, G . Zubay, G. Tocchini-Valentini, S. Connaway and J. Beckwith, CSHSQB 35, 437 (1970). 148. B. De Crombrugghe, B. Chen, M. Gottesman, I. Pastan, H. E. Varmus, M. Emmer and R. L. Perlman, Nature (London) New Biol. 230, 37 (1971). 140. Y. Ohshima, T. Horiushi, Y. Iida and T. Kameyama, MoZ. Gen. Genet. 106, 307 (1970). 150. N. C. Franklin and

S.E. Luria, Virology 15,

299 (1961).

406

FUMIO IMAMOTO

161. W. A. Newton, J. R. Beckwith, D. Zipser end S. Brenner, JMB 14, 290 (1965). 162. U. Henning, G. Dennert, K. Szolyvag and C. Deppe, JMB 13, 592 (1965). 163. R. G. Martin, N. F. Silbert, D. W. E. Smith and H. J. Whitfield, JMB 21, 357 (1966). 164. A. S. Sarabhai, A. 0. W. Stretton, S. Brenner and A. Bolle, Nature (Londo~i) 201, 13 (1964). 166. N. D. Zinder, D. L. Engelhardt and R. E. Webster, CSHSQB 31, 251 (1966). 166. T. Sugiyama, H. 0. Stone and D. Nakada, JMB 42, 97 (1969). 167. R. E. Webster and N. D. Zinder, JMB 42,425 (1969). 168. J. Beckwith, BBA 76, 162 (1963). 169. M. R. Capecchi and G. N. Gussin, Science 140,417 (1965). 160. S. Brenner, A. 0. W. Stretton and S. Kaplan, Nature (London) 206, 996 (1965). 161. D. L. Engelhardt, R. E. Webster, R. C. Wilhelm and N. D. Zinder, PNAS 54, 1791 (1965). 162. A. 0. W. Stretton, S. Kaplan and S. Brenner, CSHSQB 31, 173 (1966). 163. S. Brenner and J. R. Beckwith, JMB 13, 629 (1965). 164. C. W. Hill and H. Echols, JMB 10, 38 (1966). 166. E. K. F. Bantz, JMB 17, 298 (1966). 166. H. F. Lodish, JMB 32, 47 (1968). 167. H. F. Lodish, JMB 32, 681 (1968). 168. J. W. Robert and G. N. Gussin, JMB 30, 565 (1967). 169. D. L. Engelhardt, R. F. Webster and N. D. Zinder, JMB 29, 45 (1967). 170. H. F. Lodish, Nature (London) 220, 345 (1968). 171. P. Spahr and R. Gesteland, PNAS 50, 876 (1968). 172. R. K. Herman and A. B. Pardee, BBA 108, 513 (1965). 173. F. Imamoto and C. Yanofsky, JMB 28, 25 (1967). 174. T. Carter and A. Newton, Nature (Lonlon) 223, 707 (1969). 176. D. Zipser, Nature (London) 221, 21 (1969). 176. D. E. Morse and C. Yanofsky, Nature (London) 224, 329 (1969). 177. D. E. Morse and P. Primakoff, Nature (London) 226, 28 (1970). 178. A. Newton, CSHSQB 31, 181 (1966). 179. D. Zipser and A. Newton, JMB 25, 567 (1967). 180. R. G. Martin, H. J. Whitfield, Jr., D. B. Berkowitz and M. J. Voll, CSHSQB 31, 215 (19ea). 181. F. Imamoto, J. Ito and C. Yanofsky, CSHSQB 31, 235 (1966). 182. E. Jordan and H. Saidler, MoZ. Gen. Genet. 100, 283 (1967). 183. G. R. Fink and R. G. Martin, JMB 30, 97 (1967). 184. R . G. Martin and N. Talal, JMB 36, 219 (1968). 186. D. Zipser, S. Zabell, J. Rothman, T. Grodzicker, M. Wemk and M. Novitski, JMB 49, 251 (1970). 186. C. Yanofsky and J. Ito, JMB 24, 143 (1967). 187. E. Balbinder, A. J. Blume, A. Weber and H. Tamaki, J. Bact. 05, 2217 (1968). 188. E. Steers, Jr., G. R. Craven and C. B. Anfinsen, JMB 240, 2478 (1975). 189. P. Perrin, CSHSQB 28, 529 (1963). 190. S. Koorajiam and I. Zabin, BBRC 18, 384 (1965). 191. 9. G. Korenman, G. R. Craven and C. B. Anfinsen, BBA 124, 160 (1966). 192. Y. Kano and F. Imamoto, in preparation. 192a. M. Levinthal and H. Nikaido, JMB 42, 511 (1969). 193. L. Lindahl and J. Forchhammer, JMB 43, 693 (1969). 193a. S. Hiraga and C. Yanofsky, in preparation.

TRANSLATION AND TRANSCRIPTION OF THE i!Tp OPERON

407

194. D. E. Morse and M. Guertin, Nature (London) New Biol. 232, 165 (1971). 196. M. Kuwano, D. Schlessinger and D. E. Morse, Nature (London) New Biol. 231, 214 (1971). 196. T. Carter and A. Newton, PNAS 68, 2962 (1971). 197. D. Schlessinger, Stadler Symp., Univ. Missouri, Columbia, Missouri p. 3 (1971). 19s. S. Kang, PNAS 65, 544 (1970). 199. F. Imamoto, FP 30, Abstr., 490 (1971). 200. F. Imamoto and Y. Kano, Nature (London) New Biol. 232, 169 (1971). 201. M. Ozaki, S. Mizushima and M. Nomura, Nature (London) 222, 333 (1969).

202. M. Nomura, S. Mizushima, M. Ozaki, P. Traub and C. V. Lowry, CSHSQB 34, 49 (1969). 203. E. Craig, Genetics 70, 331 (1972). ,904. G. P. Tocchini-Vdentini and E. Mattochia, PNAS 61, 146 (1968). 206. H. E. Varmus, R. L. Perlman and L. Pastan, Nature (London) New Biol. 230, 41 (1971). 206. D. E. Morse, JMB 55, 113 (1971). 207. F. Imamoto and S. Tani, Nature (London) New Bio. in press. 908. F. Imamoto, JMB in press. 209. W. Sly, H. Echols and J. Adler, PNAS 53, 378 (1965). 210. A. Skalka, CSHSQB 31, 377 (1966). 211. K. Taylor, Z. Hradecna nnd W. Szybalski, PNAS 57, 1618 (1967). 212. W. Szybalski, K. Bovre, M. Fiamdt, A. Guha, Z. Hradecna, S. Kumar,

H. A. Lozeron, Sr., V. M. Maher, H. J. J. Nijkamp, W. C. Summers and K. Taylor, J. Cell Physiol. 74, Suppl. 1, 33 (1969). 213. N. C. Franklin, “The Bacteriophage Lambda,” p. 621. Cold Spring Harbor Lab., Cold Spring Harbor, Long Island, 1971. 214. S. Tani and F. Imamoto, in preparation. 216. F. Imamoto and 6. Tani, in preparation. 216‘. D. H. Shin and K. Moldave, JMB 21, 231 (1966). 217. M. Revel, M. Herzberg, A. Becarevic and F. Gros, JMB 33, 231 (1968). 218. J. C. Brown and P. Doty, BBRC 30, 284 (1968). 219. J. Davidson, K. Brookman, L. Pilarski and H. Echols, CSHSQB 35, 95 (1970). 220. J. C. Leavitt, R. H. Hayashi and D. Nakada, Amer. SOC.Biol. Chem., 62nd Ann. Meeting p. 838 (1971).

This Page Intentionally Left Blank

Lymphoid Cell RNA’s and Immunity A. ARTHTJR GOTTLIEB Institute of Microbiology, Rutgers University, New Brunsurick, New Jersey

I. Introduction . . . . . . . . . . . . . 11. Historical Pcrspective . . . . . . . . . . . 111. Biosynthesis of RNA in Immunized Systems . . . . . A. Changes in RNA Synthesis after Exposure to Antigen . . B. Evidence for the Formation of Unique Species of RNA in Response to Antigen . . . . . . . . . . C. Progress in Isolation of mRNA‘s Directing y-Globulin Synthesis . . . . . . . . . . . . . D. Effect of Actinomycin D on RNA Metabolism of Lymphoid Cells and Antibody Formation . . . . . . . . IV. Transfer of Immune Phenomena by RNA . . . . . . A. Transfer of Specific Antibody Synthesis . . . . . . B. Transfer of Cell-Mediated Immune Responses . . . . C. Transfer of Allotypic Specificity . . . . . . . V. Nonspecific Stimulators of Immune Responses . . . . . VI. Macrophage RNA’s and Immunity . . . . . . . . A. Peritoneal Exudate Cell Populations and Macrophages . . B. Some Effects of Macrophages on Immune Responses . . . C. Antigen-RNA Interactions . . . . . . . . . VII. Possible Mechanisms of Action of RNA’s in the Immune Respome . . . . . . . . . . . . VIII. A Hypothesis Regarding the Mechanism of Action of Antigen-Ribonucleoprotein Complexes . . . . . . . IS. Conclusion . . . . . . . . . . . . . References . . . . . . . . . . . . .

409 410 412 412 416 422 423 425 425 434 439 440 442 442 443 445 454 457 459 460

1. Introduction The subject of “RNA’s and Immunity” is a confusing one to both the biochemist and the immunologist. The latter, oriented in general t o look at interactions between cells, prefers to think in cellular terms. The biochemist is puzzled by the apparently crude nature of the systems with which the immunologist works, the failure to define precisely the components and products of the system, and the semiquantitative nature of many immunologic assays. I n this respect, the interactions of “RNA” with thc “lymphoid system” lacks form. For these reasons, I have attempted to look at this problem from both viewpoints, and to call atten409

410

A. ARTHUR GOTTLIEB

tion to certain phenomena that justifiably stimulate further investigation. Many of these phenomena should be better defined, and the reasons for conflicting results from one laboratory to another require clarification. Nevertheless, it does appear that this peculiar and as yet poorly defined area has already produced some important insights that will, I am confident, lead to a more clear understanding of immunity and control of immune phenomena.

II. Historical Perspective One of the first observations concerning the ability of ribonucleoprotein extracts to transfer immune phenomena was described by Sterzl and Hrubesova (1).They showed that the spleens of adult rabbits, previously immunized with Salmonella paratyphi B contain a ribonucleoprotein preparation that induces the production of specific antibody in 5-day-old rabbits, which normally fail to respond to salmonella antigens. I n some as yet unknown fashion, the RNA-containing material conferred immunocompetence on the immunologically immature rabbits. Another dimension to these phenomena was indicated by the classic studies carried out by Campbell and Garvey on the nature and fate of retained antigen in vivo ( 2 ) . Rabbits injected with S5S-labeledsulfanilate-azo-bovine serum albumin retained a small but significant amount of antigen in the liver for extended periods of time. Much of this material was associated with RNA, but the nature of the antigen was never carefully defined although it appeared in most cases to be degraded to fragments (3). These complexes of antigen and RNA were very strong immunogens in guinea pigs, assaying for in vitro anaphylaxis by the Schultz-Dale technique. Indeed, the complexes from the liver were up to 200 times more effective in sensitization to the modified albumin than the original antigen. Moreover, when the complexes of antigen and RNA were dissociated, neither the antigenic fragment nor the RNA itself were found to be capable of inducing antibody formation. These observations pointed to an important role for nucleate-protein complexes in the retention of antigen and development of immunity. Studies similar to those of Campbell and Garvey, carried out by Friedman ( 4 ), showed that a nucleoprotein preparation from lymph nodes, derived from rabbits immunized with Shigella paradysenteriae, was also capable of inducing the formation of specific agglutinins in normal and partially X-irradiated adult recipients after injection of the nucleoprotein extract. A substantial advance in these studies was the development by Fishman ( 5 ) of an in vitro system for this transfer. He exposed peritoneal exudate cells (consisting of 70% macrophages and 30% lymphocytes) to T2 bacteriophage and prepared a cell-free homogenate from

LYMPHOID CELL RNA’S AND IMMUNITY

411

these cells. These homogenates contained a material sensitive to ribonuclease and capable of inducing the formation of specific anti-T2 antibody in cultures of rat lymph nodes. The addition of T2 phage particles alone to such culturcs yielded no antibody formation. This indicated that peritoneal exudate cells produce a factor highly immunogenic for the phage antigen to which they are exposed. Later studies (6) showed that RNA alonc, purified by thc cold phenol technique from antigen-exposed macrophages, is also capable of producing specific neutralizing antibody to the antigen to which these cells are exposed. These studies were the first to show that ribonucleic acid might play a critical role in the transfer of immunity. By gross criteria, the RNA preparations employed were free of “antigen,” but more sensitive assays on this point by Askonas and Rhodes (7) and Friedman et al. (8) indicated that antigenic fragments were indeed present. Askonas and Rhodes studied the ability of RNA extracted from mouse peritoneal exudate cells to stimulate secondary responses in syngeneic’ recipients primed with Maio squinado hemocyanin. Remarkably, the antigen-RNA complex was about 20 times more potent as an immunogen than the equivalent amount of antigen in free form. As a result of such studies, the somewhat mystical and unsatisfactory term “superantigen” was applied to these complexes. Yet another aspect of the role of RNA in immunity was presented in 1962 by Mannick and Egdahl ( 9 ) , who showed that RNA preparations can transfer immunity to homografts. Exposure of normal rabbit lymph node cells in vitro to RNA extracted from lymph nodes of rabbits immunized with skin homografts conferred specific sensitivity on the normal cells, shown by their ability to elicit a positive skin reaction when injected into donors of the skin homografts but not into other rabbits. Again, this activity could be destroyed by treatment with ribonuclease. Fong et al. (10) demonstrated that cellular resistance of rabbit peritoneal exudate cells to the intracellular parasitism and degeneration caused by infection with virulent tubercle bacilli can also be transferred by ribosomal RNA preparations from the peritoneal exudate cells of BCG2-immunized rabbits. Thor (11) showed that RNA extracted from the lymph nodes of tuberculin- or histoplasmin-sensitive humans can transfer delayed hypersensitivity to nonsensitive cells, as manifested by the ability of these cells to inhibit migration of macrophages (a phenomenon correlated with the appearance of delayed hypersensitivity) upon subsequent incubation with the specific antigen. Again, the activity conferred by the RNA preparaSyngeneic refers to an interaction between cells or cellular materials derived from genetically identical members of the same species. ’BCG: Bacillus Calmette-GuCrin is an avirulent mutant of the tubercle bacillus. It is commonly employed in experimental studies on tuberculosis, and as a vaccine agninst tuberculosis.

412

A. ARTHUR GOTTLIEB

tion was inhibited by ribonuclease. These studies have been criticized by Bluestein et al. ( I d ) , who claim not to be able to reproduce this phenomenon. It is not certain, a t this point, how seriously the latter objection should be taken since in all biological systems a negative result can have many irrelevant explanations. Lately, Alexander (13) and Deckers and Pilch (14) have described the ability of RNA preparations, from animals immunized against certain tumors, to transfer tumor immunity to nonimmune recipients. In view of these studies, one cannot help but be impressed by the variety of immune phenomenon that can be transferred or induced with ribonucleic acids derived from specifically immunized systems. Nevertheless, the nature of the RNA’s involved, and we emphasize the plural, is not necessarily the same for all systems described, and the preparations employed are of varying degrees of purity. Further detailed characterization of the RNA’s and their modes of action is required.

111. Biosynthesis of RNA in Immunized Systems A. Changes in RNA Synthesis after Exposure to Antigen A number of investigations have dealt with changes in the RNA’s of lymphoid cells, as a result of exposure of these cells to antigen (s) . Early studies on the cellular changes occurring in lymphocytes during the production of antibody were carried out by Harris and Harris (16).On the second day following antigen stimulation, pyrinophilic material was found in the nuclei and cytoplasm of reticulum cells, lymphoblasts, and finally mature lymphocytes. This pyrinophilic material was shown to be RNA, as demonstrated by specific digestion with protease-free RNase. Antigens, in contrast to nonantigenic materials, give rise to increased RNA in the lymph node. Later, Dutton et al. (16) demonstrated that preparations of spleen cells making antibody in vitro following anamnestic3 stimulation in vivo exhibit markedly increased uptake of s2Pinto RNA and DNA compared with normal cells. More detailed radiographic studies were carried out by Mitchell and Nossal (17), who studied lymph node cells in the presence of [SH]uridine after exposure to a flagellin preparation from Salmonella’ adelaide. Plasmablasts incorporated the label rapidly, whereas the mature plasma cells exhibited little RNA turnover. It is interesting to note that, despite their low uptake of labeled uridine, the mature plasma cells exhibited active protein synthesis, consistent with an active production of antibody (18). Thus, the low level of uridine incorporation suggests that the mRNA’s coding for these pro-

’ The response following a second exposure to an antigen.

LYMPHOID CELL RNA'S AND IMMUNITY

413

teins are relatively stable. Schooley et al. (19) also concluded that mature plasma cells do not actively synthesize mRNA coding for proteins produced by these cells. In later studies, Parkhouse (20) incubated spleen cell suspensions derived from nonimmune or previously immunized rabbits in the presence or absence of inhibitors plus the labeled precursors ['HI thymidine on [ 'HI cytidine. He concluded that high concentrations of puromycin and mitomycin inhibited both DNA and RNA synthesis in these cells. The concentration of drug required to effect inhibition was similar in antigen-stimulated and control cultures. A detailed study of the biosynthesis of RNA in antibody-producing tissues has been carried out by Mach and Vassalli (81). Employing Hemophilus pertussis in the rat, immunization resulted in an unusually rapid rate of synthesis of ribosomal RNA and precursors of ribosomal RNA. Whereas the sedimentation profile of rapidly labeled RNA from the lymphoid cells of nonimmunized animals is widely scattered throughout the gradient, with a predominance of low molecular-weight components, the sedimentation profile of the newly synthesized RNA from immunized animals is characterized by an RNA with an sz0 > 30 S and a ribosomal base composition. The first newly synthesized RNA to reach the microsomes was a fraction of relatively low molecular weight, having many of the characteristics expected for mRNA. I n immunized cells, the ability of these RNA fractions to stimulate amino-acid incorporation in homogenates of Escherichia w l i was scattered over a wide range of szo values, although the highest activity in the cell-free systems appeared in RNA from the 6-12s region of the gradient. Thus, after immunization, the most prominent fraction of rapidly synthesized RNA is unexpectedly not of the messenger type, but of the ribosomal type. This unusually rapid rate of synthesis of ribosomal RNA is further manifested by the rapid accumulation of labeled ribosomal RNA on the microsomes. Nevertheless, the first labeled RNA's to reach the ribosomes were distinct from ribosomal RNA but had the characteristics of messenger RNA. These RNA's which had szo values of 3-12 S could theoretically direct the synthesis of proteins of 2000-30,OOO daltons and thus are large enough to specify fragments of the y-globulin molecule. I n another article (22), Mach indicates how to isolate an RNA fraction sedimenting between 10 and 35 S and having a high template activity. However, antibody has not been shown to be formed in vitro in response to addition of this RNA. Mach and Vassalli suggest that mRNA may exist in two different forms in antibody-forming systems, one of relatively high molecular weight as it is synthesized on DNA (possibly polycistronic) and one of lower size representing RNA, as it functions at the actual site of protein synthesis. I n an attempt to demonstrate the

414

A. ARTHUR GOTTLIEB

synthesis of RNA specific for antibody, the spleens and lungs of animals injected with Salmonella typhi or sheep erythrocytes were examined for quantitative changes in the rate of RNA synthesis ( 2 3 ) .An increase in the specific activity of RNA in the spleen, as contrasted with lung, in response to S. typhi at 72 hours was observed. In agreement with Mach and Vassalli, Church et al. (23) also noted that the RNA from spleens of animals immunized 18 hours is heterogeneous but contains a higher proportion of material larger than 28S, as compared with normal RNA. Changes in RNA synthesis following antigenic stimulation have also been described by Cohen (64) and Ortiz-Ortiz and Jarosiow ( 2 5 ) . The rate of incorporation of labeled uridine into RNA is accelerated in the spleens of mice after injection of antigen (sheep erythrocytes). This is accompanied by a progressive increase in the rate of labeling of the 8-16 S fraction of RNA ( @ ) , overall changes in base composition of lymph node RNA (principally in mRNA fractions) and an increase in the rate of ribosomal RNA synthesis, and number of ribosomes per lymphocyte (26, 27).Such variations in ribosomal RNA composition make it difficult to precisely determine the degree of labeling of specific mRNA’s. A high basal level of ribosomal RNA synthesis could account for the failure of Donahm et al. (28) to find significant differences in the synthesis of RNA in immune as compared with nonimmune lymph-node cells. An additional distinction in the labeling patterns of lymphoid cells following exposure to antigen has been observed by Lazda et al. ( 3 4 , 3 5 ) . Immunization of rats with Salmonella flagella induces an increased rate of incorporation of S2P-labelednucleotides into the RNA associated with the ribosomal fractions of these cells. A large portion of the newly synthesized RNA extracted from the ribosomal aggregates sediments as 29 S and 1 8 s ribosomal RNA, but the RNA having the greatest specific activity is polydisperse between 6 and 18s with a sharp peak of high specific activity in the 18s region. RNA’s in this size range could code for proteins of 7800 to 33,000 daltons. It was noted (34, 35) that this broad range of sedimentation values of the RNA is peculiar to RNA extracted from immune spleen. With increased time of incubation, the 29 S fraction becomes much more heavily labeled, whereas the amount of label in the light RNA fractions increases more slowly (35).This indicates that the 6-12 S RNA, although synthesized rapidly, does not accumulate, whereas the 29 S fraction appears to be more slowly synthesized but of greater stability. Moreover, the base composition of these two fractions differed in that the 6-12 S RNA resembled DNA more closely. On the basis of these observations, Lazda et al. (35) suggest that mRNA in immune spleen cells consists of two species that differ in stability. Such changes in RNA are not unique to spleen or lymph node, but

LYMPHOID CELL RNA'S AND IMMUNITY

415

have been observed by Cohen et al. in thymus gland as well, following exposure of mice to sheep red cells (33).Two principal peaks of RNA radioactivity at %12 S and 28 S were observed and the specific activities of these RNA fractions were increased 16-fold on day 3 after immunization. I n view of the ability of steroid hormones to suppress antibody production, it is of interest to observe that lymphoid cells treated with cortisol in vitro exhibit a diminished degree of incorporation of labeled precursor into RNA, DNA and protein (36). These phenomena parallel the changes in RNA that occur when lymphocytes are exposed to phytohemagglutinin. I n such a system, RNA production increases abruptly and progresses exponentially for 24 hours (29). Moreover, the prompt increase in RNA synthesis observed immediately after exposure to phytohemagglutinin occurs in nonribosomal components whereas ribosomal RNA synthesis does not occur for several hours. Rubin and Cooper (29) suggested that the unstable lymphocyte RNA is a messenger RNA of short half-life produced in increasing proportions and possibly concerned with successive stages of activity as the lymphocyte progresses from a resting stage to mitosis. A net increase in total cellular RNA 45-50 hours after addition of phytohemagglutinin to lymphocyte cultures was also seen by Forsdyke, who observed that the increase in amount of RNA was dependent on the concentration of the agent up to 0.3 mg/ml. Further addition did not lead to additional RNA synthesis (30). RNA synthesis following phytohemagglutinin stimulation of lymphocytes is blocked by the addition of actinomycin D, which also blocks the increase in labeling of protein commonly seen in cells responding to this agent (31). Both normal and stimulated cells incorporate acetate into their histones, but there is a striking increase in the acetylation of histones in the stimulated as compared with normal cells. Histone synthesis is not affected, but the increase in acetylation of basic proteins appears to precede the increase in nuclear RNA synthesis. I n further studies on this phenomenon, Rubin et a,?. (32) exposed nuclei isolated from resting lymphocytes to phytohemagglutinin for 1 hour. Under these conditions, the rate of DNA-directed RNA synthesis doubled. Moreover, treatment of resting nuclei resulted in increased binding of actinomycin D suggesting the liberation of additional segments of free chromatin. A parallel increase in incorporation of [ "C] acetate into acid-insoluble material was observed, and this effect was independent of protein synthesis. This suggested that there may be an augmentation in histone acetylation under these conditions. These authors suggest that phytohemagglutinin exerts a direct influence on nuclear histones, which

416

A. ARTHUR GOTTLIEB

in turn might free additional segments of extended DNA template for RNA synthesis.

8. Evidence for the Formation of Unique Species of RNA in Response to Antigen Several attempts have been made to show that there are unique RNA’s formed in lymphoid cells in response to antigen. The most extensive studies of this type have been carried out by Cohen e t al. (37)’ who immunized mice with sheep erythrocytes and labeled the RNA in vivo by subsequent injection of szP,.Significant changes in base composition were detected. Specifically, more molecular hybrids could be formed between mouse RNA and single-stranded DNA when both polynucleotides were isolated from homologous species. Moreover, RNA extracted 2 days after immunization formed more hybrids than did RNA from nonimmunized controls. This was particularly marked with respect to RNA having an s value of 9-12 S. Although such an increase is presumptive of mRNA, i t is not clear that the new species of RNA formed after immunization is directly or indirectly related to antibody formation. I n a subsequent report (38)’RNA’s from immunocompetent cells exposed to sheep erythrocytes hybridized with DNA immobilized on a nitrocellulose column. The RNA’s that failed to form hybrids and passed through the column presumably did so because they found no sites available for hybridization, possibly because these species of RNA represent RNA’s synthesized in relative excess from a few genes. The net specific activities of the RNA’s that failed to find sites for hybridization were higher with RNA from the normal mouse spleen than with RNA from the spleens of mice injected 2 days previously with the erythrocytes. Cohen suggests that this indicates that RNA’s from the spleens of immunized animals found more sites in the DNA for hybridization than did RNA from the controls. Since the DNA used in these studies was from nonimmunized mice, these experiments partially support the concept that part of the potential to produce antibodies of varying specificities arises from germ-line mechanisms of inheritance rather than entirely by somatic mutation. Raska and Cohen (39) exposed peritoneal exudate cells to antigen and annealed the RNA from these cells against DNA from unstimulated cells. The amount of RNA so annealed was higher when the RNA was isolated from such cells exposed to sheep erythrocytes. Unlabeled RNA from stimulated cells more efficiently inhibited hybridization than did RNA from nonstimulated control cells, but unlabeled RNA from cells exposed to a different antigen also competed. Thus, some similar and some distinct species of RNA appear after such cells are exposed to different

LYMPHOID CELL RNA’S AND IMMUNITY

417

antigens. New species of RNA were found in both the adherent and nonadherent cell population but different species of RNA were not found in the nonadherent cells. Raska and Cohen suggest that the information for the synthesis of these RNA’s is present in a large proportion of normal mouse DNA. This is, of course, consistent with the hypothesis that the potential to produce antibody of varying specificity is inherited and does not arise from somatic mutation. As indicated above, Church et al. carried out similar studies (23) using the same kind of approach. They compared the amounts of hybridizable RNA formed in the spleens of mice that had received prior injections of sheep erythrocytes or S. typhi, finding that the kinetics of RNA synthesis following immunization with different antigens differed. The production of hybridizable RNA differing from control RNA in response to S. typhi was more rapid than when sheep erythrocytes were employed as antigen. One of their studies compared the annealing to DNA of labeled RNA from spleens of mice injected 24 hours previously with S. typhi in the presence or the absence of unlabeled RNA from similar groups of animals injected with the same antigen, homologous mouse or sheep erythrocytes. Unlabeled RNA from the spleens of normal animals significantly suppressed the binding of the labeled RNA, but this effect was less than that exerted by the unlabeled RNA extracted from the mice immunized with S. typhi. The RNA’s from both sets of treated animals displayed more effective competition than did normal RNA, with that from the sheep-treated set exhibiting somewhat greater suppression of binding. This result suggests that the suppression observed in the mouse case reflects nonspecific changes in the RNA population of mouse spleen after exposure to mouse erythrocytes whereas the differential effects seen with unlabeled RNA’s from the S. typhi and mousetreated animals on the binding of labeled RNA from S. typhi immunized mice indicate a qualitative difference between RNA’s transcribed in the spleen in response to different antigens. Similar studies using labeled RNA from mice immunized with sheep erythrocytes demonstrated that, at 24 hours, only relatively minor differences in competitive ability were noted between spleen RNA derived from animals injected with this antigen or with S. typhi. Analogous studies conducted 60 hours after immunization revealed a greater response in the sheep-treated animals than in the S. typhi treated set. The simplest explanation of these results is that the relevant changes in the RNA synthesized in the spleen occur within 24 hours after S. typhi injection but only later in the case of sheep erythrocyte immunization. I n these studies of Cohen and Church, therefore, we have evidence for changes in these RNA fractions of spleen following immunization,

418

A. ARTHUR GOTTLIEB

but there is no evidence that any of these changes are in fact changes in the specific messenger RNA's coding for the specific antibodies to the antigens employed. An interesting piece of indirect evidence on this point is given by Cohen and Raska (@), who exposed mouse spleen cells in vitro to N,ph,,(Lys), (a poor antigen) and N , p h ( L y ~ ' O A l a ~(a ~ ) good ~ antigen) .' The latter compound stimulated the appearance of newly synthesized RNA's distinguishable from similar RNA's in control cells. The poor antigen failed to elicit new species of RNA. By molecular hybridization, Cohen and Raska indicated that there was no experimentally detectable difference between the DNA's of the nonimmune mouse as compared with the DNA of immune mice. These experiments support the ideas that different species of RNA appear in cells exposed to different antigens, and that the information for the synthesis of a t least part of these species of RNA exist in normal genome. This, of course, argues against somatic mutation as a mechanism for the generation of antibody diversity. Hybridization experiments (41) suggest that differences exist between the DNA's from liver and spleen of the same animal (rabbit) and between the RNA's from lymph nodes stimulated by different antigens. A larger proportion of spleen DNA than of liver DNA is complementary to the RNA isolated from antigen-stimulated lymph nodes, and there is more extensive hybridization by RNA from antigen-stimulated cells than from nonimmune cells. No difference between liver and spleen DNA is observed in hybrids formed with RNA from lymph nodes of nonimmune animals. These observations are consistent with the stimulation of cell proliferation observed in lymph nodes after antigen stimulation and implies that there should be a general increase in total cellular RNA in the spleen. Such an increase in the RNA content of spleen cells after immunization was found by Juhasz et al. (4.8). However, Little and Donahue observed only a minor difference in the overall rate of incorporation of [SH]uridine into RNA in antigen-stimulated or nonstimulated cells (41). The difference in the amount of RNA from antigenstimulated cells hybridizing with liver and spleen DNA suggests that there may be unique or redundant gene sequences in spleen coding for 41, 4.8) are representative of antibody protein. These experiments (B, attempts to determine whether the DNA's of lymphoid cells forming different antibodies are indeed different. Unfortunately, the methods employed are limited in sensitivity, and it is not possible to specify individual values of specific activity to singular RNA species. Moreover,

' These are conjugates prepared by reaction of dinitrofluorobenzene with poly(L-lysine) or poly(Lys"Ala"O), respectively. The products each contain 15 moles of dinitrophenyl residues per mole of backbone polypeptide carrier.

LYMPHOID CELL RNA’S AND IMMUNITY

419

the reactions are not locus specific owing to the reiteration of nucleotide sequences in the mammalian genome. A somewhat similar approach has been used by Greenberg and Uhr (4.3) to examine the DNA’s and rapidly labeled RNA populations of three different myeloma tumors derived from one strain of mice. Each of the DNA’s was obtained from a myeloma line synthesizing a different globulin (the lines were MPC,,, MPC, and BJ, which produce IgG, IgA and L-chain proteins, respectively). The RNA’s were labeled by incubation with 32Piand separated on sucrose gradients. DNA from the MPC,, line was annealed with fractions of RNA from the same line. 28 S, 18 S and 1 0 s RNA’s annealed to DNA a t levels of 0.095%, 0.050% and 0.26%’ respectively. This indicates that the 1 0 s fraction might be expected to be relatively enriched in messenger RNA. Each RNA preferred DNA from its own line. Kreuger and McCarthy (4.4) have also studied the annealing characteristics of pulse-labeled RNA’s to DNA’s from four different myeloma tumors (one of IgG type, two of the IgG type and one producing L chains only). They suggested that differences exist among the DNA’s of each tumor in those segments of the genome from which some of the pulse-labeled RNA is transcribed. They concluded that the pulse-labeled RNA species annealing must be represented in multiple reiterated copies of the genome and these copies must exist in greater numbers in homologous tumor DNA than in heterologous tumor DNA. I n a recent study ( 4 5 ) , my laboratory has generated RNA complementary to each of two distinct myeloma DNA’s through the use of E. coli DNA-dependent RNA polymerase. The tumors employed both produce globulins of the IgG class, but each of these globulins is antigenically distinct. Annealing of these cRNA’s to each of the tumor DNA templates a t temperatures 12 degrees below the T,,, of the DNA, under which conditions the formation of hybrids containing large amounts of adenine. thymine base pairs is limited, revealed no difference in the number of cRNA copies represented in each of the two tumor DNA’s. Nevertheless, normal mouse liver DNA could readily be distinguished by this technique. We conclude that the genes of high guanine and cytosine content in both tumors are not distinguishable. This is consistent with the idea that all the information for every globulin is contained in each antibody-forming cell but that restriction to produce one globulin involves a limiting translational or transcriptional mechanism. Another approach to the characterization of mRNA in antibodyforming systems is that of Norton et al. (&) and is based on the effect of progressive immunization on polyribosomal size in lymphoid cells. For all antigens used, a large proportion of the polyribosomes from animals

420

A. ARTHUR GOTTLIEB

receiving the more prolonged course of immunization were heavier. This result could be interpreted either as an enrichment of the lymph node with a distinct cell population containing larger polyribosomes, presumably plasma cells, or a progressive increase in polyribosomal size within the existing population. These investigators also observed that the ratio of mRNA to rRNA is reduced during immunization, so that the number of ribosomes associated with messenger RNA increases. A further observation was that the polyribosomal population on which there was evidence of antigenic determinants of y-globulin was composed of aggregates containing 5-10 ribosomes. This indicates the presence of an RNA large enough to code for a polypeptide of 20,000-40,000daltons. This result is in contrast to the work of Stenzel et a2. (47),which suggested that the component polypeptide chains of y-globulin were synthesized on single ribosomes or dimers of monoribosomes, although degradation of polyribosomes may have occurred in these studies. Scharff and Uhr (48) used the cytoplasm of HeLa cells to minimize the breakdown of polyribosomes and demonstrated that protein synthesis by lymph node cells obtained from hyperimmune rabbits occurs on polyribosomes and that the treatment of such cells in vitro with actinomycin D inhibits protein synthesis. Becker and Rich (49) described methods for isolating polyribosomes from antibody-forming tissues, finding a biphasic distribution with one cluster of polyribosomes containing 7 or 8 ribosomes and the other 16-29 ribosomes. In agreement with Shapiro et al. (60),they inferred that the heavy and light chains of antibody molecules were synthesized on separate messenger RNA molecules. When rabbits were immunized with bovine albumin, approximately 30% of the ribosomes were recovered as polysomes whereas only 15% were recovered as polysomes in nonimmunized rabbits. I n association with this change in ribosomal profile, there was a 3- to 4-fold increase of protein synthesis following stimulation of antibody formation. Subsequently, Shapiro et al. found heavy and light chains of y-globulin on different polyribosomesthe light chains on 190s and the heavy chains on 270s polyribosomes. “Messenger” RNA’s of sizes compatible with the known molecular weight of L and H chains were demonstrated in the appropriate polyribosome class. Using this information, Kuechler and Rich (51, 52) labeled lymph and extracted the RNA from isolated node cells in vivo with [3H]~ridine polysomes and monoribosomes. On polyacrylamide gels, two distinct peaks of labeled RNA were observed between the 18s ribosomal and the 45 transfer RNA. These species, which were found in the RNA extracted from polysomes, were absent from single ribosomes, and had molecular weights of 2.2 X los and 3.7 X lo5. This is consistent with the molecular weights expected for monocistionic mRNA’s coding for the

LYMPHOID CELL RNA’S AND IMMUNITY

421

light and heavy chains of antibody. Both these species occurred a t elevated levels in lymph nodes stimulated by antigen to synthesize specific antibody. Specific antibody is produced by the lymph nodes containing these unique RNA species, and as much as 60% of the total protein synthesis of the nodc is directed toward IgG production (53).I n a cell-free system prepared from rabbit lymph node that synthesizes H and L chains of immunoglobulins, the synthesis of complete H and L chains is directed by two different size classes of polysomes ( 5 4 ) . These polysome classes cach contain the number of ribosomes expected for mRNA coding for individual complete polypeptide chain of either the H or L variety. Synthesis of H or L chains also occurs if lymph node polysomes are incubated with supernatant enzymes from rabbit reticulocytes, rabbit or rat liver. These observations appear to be applicable to other antigens. Lymph nodes from rabbits immunized with bacterial a-amylase contain in the ribosomal fraction specific antibody against this antigen (56). Using a general method for the reproducible isolation of polyribosomes from rat spleen, Wust (57) has demonstrated an increased rate of synthesis of RNA associated with the ribosomes after immunization. This was accompanied by an increase in the capacity of these subcellular particles to promote protein synthesis. A recent observation that may be of considerable importance is that RNA-DNA complexes may exist in hemolytic plaque-forming cells; the RNA in this complex appears to be in a hybrid form with DNA, since it is resistant to ribonuclease (58). Most interesting is that this hybrid appears to be more abundant in antibody-producing cells than in nonantibody-producing cells, Although this material will clearly require further characterization, it may be that it. represents a process of gene amplification either through increased synthesis of RNA on a particular DNA or by DNA synthesis on a select RNA template. We have recently observed the presence in peritoneal exudate cells of a ribopolymer-transcribing enzyme that appears to be increased in activity (or perhaps absolute amount) on stimulation of these cells with antigen. It is conceivable that such an enzyme functions in lymphoid cells to replicate selected mRNA templates for antibody production. Of interest in this regard, is the discovery by Stein and Hausen (59) of an enzyme in extracts of calf thymus that specifically degrades the RNA moiety of DNA-RNA hybrids, but does not affect single-stranded RNA’s. Such an enzyme (ribonuclease H ) has also been found by us in extracts of myeloma cells (60). In the antibody-forming cell, such an enzyme could conceivably serve an “editing” function, perhaps by generating

422

A. ARTHUR GOTTLIEB

small ribonucleotide chains that then serve as initiators for DNA synthesis.

C. Progress in Isolation of mRNA's Directing y-Globulin Synthesis

Myeloma cell lines are of considerable interest to immunologists as they produce homogeneous populations of 7-globulin molecules and are thought to be functionally well-differentiated neoplastic variants of antibody-forming cells. By precipitation with antisera against various myeloma protein determinants, substantial fractions of the radioactive peptide chains on polyribosomes have been identified as nascent myeloma protein chains. Nascent H-chain determinants were located by precipitation with anti-Fc antibody, which precipitated about 30% of the labeled material sedimenting on 3 0 0 s polyribosomes while L chains could be detected in the 120-180 S region (55). In the past year, dramatic progress has been made in isolating specific mRNA molecules that direct the synthesis of 7-globulins in cell-free systems. An RNA fraction of the myeloma line MOPC 41A (which produces a light chain product of the kappa type known as K41) has been isolated from the 9-13 S region of a sucrose gradient. This fraction directs the synthesis of the K41 product in a cell-free reticulocyte system, as judged by tryptic peptide analysis and by the ability of authentic antiK41 antibody to precipitate specifically the product generated in the cellfree system (65a). A similar preparation of an mRNA from the microsomes of the MOPC-21 myeloma directs the synthesis (in a Krebs I1 ascites cell-free system) of light chains characteristic of the MOPC-21 tumor (55b).Very recently, Leder and co-workers (55c),using the Krebs I1 ascites system, purified a biologically active mRNA from the MOPC41 tumor line by chromatography on oligothymidylate cellulose. The RNA recovered in this way directs the synthesis of a product that yields tryptic peptides corresponding to those derived from the myeloma protein. The technique of oligothymidylic acid-cellulose chromatography has been successfully employed to isolate the mRNA for rabbit globin (564, which contains stretches of polyadenylate. This suggests that the myeloma mRNA also contains a region rich in adenylate residues. Based on the observed s20,w of 13S, corresponding to about 850 nucleotides, it appears that the mRNA for the light chain of the MOPC-41 myeloma globulin is monocistronic and longer than necessary to encode the variable and constant regions of the molecule. This indicates that both variable and constant regions are encoded in a single mRNA molecule and that assembly of the light chain does not occur via posttranscriptional linkage of these regions.

LYMPHOID CELL RNA'S AND IMMUNITY

423

D. Effect of Actinomycin D on RNA Metabolism of Lymphoid Cells and Antibody Formation

Actinomycin binds to deoxyguanosine residues in DNA and prevents DNA-dependent RNA polymerase from transcribing DNA (61).The effects of this drug on immune responses have been variable. At 0.05 pM, it completely inhibits the continuation of the primary response and the secondary response induced against T2 bacteriophage in rabbit lympli node in vitro, suggesting that messenger RNA for antibody formation has a half-life of less than several days, since responses programmed by longer-lived mcssengcr would be prolonged beyond this point (66). I n contrast, the same drug in rats, in conjunction with sheep erythrocytes, causes a delay in the immune response but has no effect on the rate or maximum amount of hemagglutinating antibody produced (63). The delay implies that at some time after administration of actinomycin the drug is no longer available for binding to DNA, at which point messenger RNA production may resume. Wust et al. interpret their results as indicating that mRNA is synthesized during the inductive phase of antibody formation. Such a conclusion is compatible with the observation of Svehag e t al. (64) that the formation of 1 9 s antibody to poliovirus by rabbit spleen cells in vitro is interrupted by adding actinomycin D (1-10 pg/ml) for 30 minutes or longer. Antibody formation was slowly renewed upon removal of the drug. This indicates that 1 9 s antibody formation is dependent on DNA-directed RNA synthesis with a messenger lifetime of less than 12 hours. Studies of the effect of actinomycin D on RNA synthesis and antibody formation on the secondary response in vitro suggest that the mRNA is stable for not more than 2-5 hours (66). The formation of all RNA fractions was inhibited by concentrations of the drug that produced no decrease in antibody synthesis. This implies that the level of mRNA may not be the limiting factor in the series of reactions involved in formation of antibody and that antibody production is only inhibited when a relatively stable mRNA fraction is reduced in concentration below a critical level by total inhibition of new synthesis. The inhibition by actinomycin D could not be reversed by removal of cells to a medium free of inhibitor. These results are somewhat in disagreement with the work of Lazda and Starr (66) in that they show that continued synthesis of antibody in vitro in the anamnestic response of rabbit spleen cells to dinitrophenylated-bovine y-globulin is independent of the inhibition of RNA synthesis by actinomycin D. The in vitro synthesis of antibody in spleen cells 3 days after secondary immunization Continued for at least 18 hours after treatment with actinomycin D, in-

424

A. ARTHUR GOTTLIEB

dicating that the mcsscngcr RNA for antibody production has a long half -1if e. The effects of thc drug in vivo are more variable. Actinomycin partially inhibits the secondary in vivo response to sheep erythrocytes in mice (68) and delays the appearance of antibody in rat serum in the primary response, as noted by Wust et al. (63).Nevertheless, Geller and Spiers (691 demonstrated persistence of the secondary response to tetanus toxoid in the presencc of toxic amounts of the drug (up to 2.5 mg per mouse) ; large doses of actinomycin D did not prevent previously sensitized recipient mice from responding to an injection of antigen. This points to the existence of a persisting inactivated mRNA activated by reinjection of antigen. These authors also showed that giving 0.6 mg/kg of actinomycin D following intraperitoneal injection of pertussis vaccine or tetanus toxoid led to an inhibition of primary immunization as dhown by low antitoxin titers 4 weeks after priming and a failure to achieve a normal secondary response to challenging antigen (70).Previously, Spiers (71) found that administration of actinomycin D concomitant with initial injection of tetanus toxoid reduced the antitoxin titers obtained during the secondary response. Stavitsky and Gusdon (72)pointed out that all these studies on the effect of actinomycin D on antibody formation depend on the assay for antibody in serum or culture medium. Hence, the precise time of cessation of antibody synthesis in vivo or in vitro is unknown. Antibody synthesis in rabbit lymphoid cells in response to hemocyanin and human serum albumin is somewhat independent of continued nucleic acid synthesis and is reduced to 50% of the control value 6 5 hours after addition of 1-10 pg/ml of actinomycin, a level adequate to cause immediate cessation of all RNA synthesis. However, i t continued a t one-third to one-half the initial rate for a t least one day, under these conditions. This implies that there may be two classes of RNA for antibody synthesis with halflives of 4-5 hours and 24 hours, respectively. I n this respect it is worth emphasizing that if antigen persists beyond the initial period of actinomycin D toxicity, the regeneration of competent cells may occur in its presence and these cells could then become sensitized. The effect(s) of actinomycin D would then vary depending on thc nature of the antigen employed. Thus, priming with soluble antigens such as fluid toxoids might be inhibited by an appropriately timed injection of actinomycin D. On the other hand, particulate antigens, such as alum precipitatedtoxoids, might persist for periods longer than the suppressive action of the drug. The available evidence suggests that an event sensitive to actinomycin D occurs during the establishment of immunologic memory, but that the expression of immunologic memory in the form of antibody

LYMPHOID CELL

RNA’SAND

IMMUNITY

425

production during thc secondary response is not sensitive to the action of the drug.

IV. Transfer of Immune Phenomena by RNA Many of the studies that have interested the immunologist in this area relate to the transfer of immune phenomena by RNA. I n many of these situations, the nature of the RNA is unclear, and, as we shall point out, many of these “immunologically active” RNA preparations contain impurities. The areas of greatest interest have been: (a) transfer of specific antibody synthesis, (b) transfer of delayed hypersensitivity responses, transplantation and tumor immunity, (c) transfer of allotypic markers

A. Transfer of Specific Antibody Synthesis These phenomena can be divided into two major areas: phenomena caused by RNA supposedly free of antigen and those not. I attempt in this review, to group experiments in these categories, though it should be recognized that the proof that an RNA is free of antigen is much more difficult than is the clear demonstration of antigen in an RNA preparation. I n some cases, it is not possible to place an RNA definitively in cither class. I n such cases, to preserve the author’s viewpoint, these RNA’s have been classed as antigen free with reservation. The effects of antigen-free RNA’s are discussed below, and studies dealing with antigen-containing RNA’s are covered in Section VI, C.

1. RNA’s FREEOF ANTIGEN As indicated in the introduction to this review, Sterzl and Hrubesova ( I ) were among the first to show that detectable antibody formation could be induced by RNA. I n their studies, antibody to Salmonella paratyphi B appeared in the blood 3-5 days after intraperitoneal injection into 5-day-old rabbits of a “ribonucleoprotein” from the spleens of normal adult rabbits that had themselves received a dose of heat-killed Sal?nonelZa 2 days before. This is of interest since these rabbits were incapable of mounting an immune response at this age. The possible association of antigen with RNA in this case was not studied in detail. Friedman (73, 74) did a similar experiment with ribonucleoprotein from lymphoid cells of rabbits exposed to Shigellu. Mice were injected with a digest of alcohol-killed Shigella paradysenteriae. Groups of mice were killed a t various times following injection, and cell-free lysates were pre-

426

A. ARTHUR GOTTLIEB

pared. Fractions rich in ribonucleoprotein and deoxyribonucleoprotein were obtained by salt extraction of the cellular supernatant. These fractions, as well as nuclei and mitochondria, were incubated with normal spleen cells from nonimmune mice, and these mixtures were transferred to other groups of nonimmune mice. The latter were tested for agglutinin formation, and it was found that the major immunogenic activity of the donor spleen cells appeared to be associated with the fractions enriched in ribonucleoprotein. In subsequent studies (76), suspensions of normal spleen cells from nonimmune mice were treated in vitro with RNA from spleen cells of donor mice previously immunized with sheep erythrocytes. Incubation of the RNA-treated cells in tissue culture a t 37°C resulted in a marked increase in the number of Jerne (76) plaques formed. Nonimmune cells did not form such plaques after incubation with RNA from immune mice, or RNase-treated RNA from immune cells, or RNA from donors immunized with different antigens. Treatment with DNase did not suppress the immunologic activity of the RNA preparation. Cultivation of the “recipient” cells with actinomycin D did not affect the plaquereduction activity of the RNA, but pretreatment of prospective donor mice with actinomycin D prevented plaque formation by donor spleen cells and resulted in the failure of RNA extracted subsequently to induce plaque formation by normal spleen cells. This suggests that a newly formed RNA was responsible for the immunologic activity of the RNA prepared from immunized mice (77). This general type of experiment has been the basis of a number of studies showing, with varying degrees of success, that RNA can transfer the capability to induce antibody formation. A number of studies of this type have been carried out by Cohen and his associates. I n an early study from this group, Cohen and Parks (78) extracted RNA from the spleens of mice that had been previously immunized with sheep erythrocytes and incubated it with spleen cells from nonimmune mice. The number of antibody-forming cells formed was measured by the Jerne plaque technique and was higher among the cells incubated with the RNA from immune mice than among cells incubated without RNA or with RNA from nonimmune animals. The response was specific for the antigen employed and sensitive to RNase, but not to trypsin, pronase or DNase. Addition of RNA from nonimmune mice to the active preparation reduced the number of antibody-forming cells. Most of the “active” RNA appeared in the 8-12s region on sucrose gradients (79). Somewhat similar results were described by Hashem (go), who noted that RNA extracted from antigen-stimulated peripheral lymphocytes promoted transformation and proliferation of unstimulated lymphocytes in a specific fashion, while RNA from unstimulated cells was not effective.

LYMPHOID CELL RNA’S AND IMMUNITY

427

In Hashem’s system, however, it was the heavy (ribosomal) RNA’s that were active. Cohen comments (81) that in the latter study, the sedimentation took so long that the 8-12s RNA had sedimented and appeared associated with ribosomal RNA, which in fact it was not. Cohen and hlosier (82) induced formation of antibody to sheep erythrocytes in vitro with extracts of RNA from lymphoid cells exposed to antigen for 30 minutes. Again, RNA extracts prepared from lymphoid cells exposed to heterologous RNA, or RNA from cells not incubated with antigen, failed to stimulate an immune response to the blood cells. Two relevant points made in this particular study were that there was considerable variation in the number of normal cells converted to PFC (plaque-forming cells) by “immunogenic RNA,” and that small numbers of peritoneal exudate cells were induced to form antibody. As noted in Section 111, B, Raska and Cohen (38, 39) carried out annealing studies on the new species of RNA formed after antigen stimulation. If the cell population was separated by means of glass adherence into adherent populations that were macrophage-enriched and nonadherent populations that were lymphocyte-enriched, both populations formed new RNA’s on exposure to antigen. The lymphocyte-enriched population contained some new species of RNA that were antigenspecific, as judged by comparative hybridization, whereas the macrophage population did not. Thus, two different populations of RNA from different cell sources were identified. The function of each of these cell groups and their relationship to each other in the transfer of immunity by RNA is discussed below. Other studies of this type, using peritoneal exudate cells, have been carried out by several other groups. When such cells from mice were exposed to bovine 7-globulin in vitro, the RNA prepared by phenol extraction of these cells induced hemagglutinating antibody in syngeneic mice (85). If the globulin was initially labeled with lS1I,no radioactivity was detected in the RNA fraction used for transfer. Treatment of the RNA preparation with Pronase, a crude proteolytic enzyme preparation, seemed to enhance rather than diminish the immunogenicity of the RNA preparation. On this basis, it was concluded that the immunologic activity of the RNA is attributable to the RNA itself, not to contaminating antigen. Mitsuhashi and co-workers (84) explored extensively a system in which transfers of this type appear to be dependent only on ribonucleic acid free of antigen. This system involves RNA derived from peritoneal exudate cells of mice immunized either with live Salmonella or with Salmonella flagellin preparations. These workers considered their population of cells to be nearly 99% macrophages based on the ability of the

428

A. ARTHUR GOTTLIEB

cells to phagocytose foreign material. The basic observation of this group was that mononuclear phagocytes immunized with S. enteritidis contain microsomal fractions capable of transferring the ability to inhibit intracellular multiplication of Salmonella organisms to nonimmune cells (85, 86). Subsequently, it was found that an RNA extracted from mononuclear cells exposed to $. enteritidis could transfer this ability in vitro (87). Antimicrobial cellular immunity was conferred by these RNA preparations not only on nonimmune peritoneal exudate cells but on pure populations of normal glass-adherent macrophages as well. Injection of this RNA into nonimmune mice led t o the production in 5 days of a peritoneal exudate population that had acquired antimicrobial cellular immunity as well as containing increased numbers of immunocytoadherent cells specific for the strain of Salmonella originally employed (88). Addition of antiflagellar antibody to the RNA preparation had no effect on the activity of the RNA. These experiments provide evidence for RNA from peritoneal exudate cells capable of transferring a type of immune response, but free of antigen. An interesting claim made in the preceding paper, as well as in subsequent reports from the same laboratory, is that the macrophage manufactures antibody. This is based on the appearance of cell-bound antibody against 5. enteriditis in mononuclear phagocytes of immune mice and those converted to immunity by RNA. The antibody was stated to be 19 S and was not detectable in serum (89, 90). Most immunologists do not accept the view that a macrophage can produce antibody under any circumstances, and it is not clear in Mitsuhashi’s studies whether the cell that contains antibody is a macrophage or a lymphocyte with phagocytic properties. Subsequently, antibody formation in vitro was studied using immunocytoadhesion as the assay technique. The development of cellular immunity by immune RNA was inhibited by puromycin but not by actinomycin D. However, the serial passive transfer of cellular immunity by “immune” RNA was inhibited by treatment of the recipient mouse with actinomycin D. This suggests that DNA-dependent RNA polymerase is involved in replicating the “immune” RNA in recipient cells (91). Saito et al. also pointed out (92) that mouse exudate cells that had received an RNA fraction extracted from the spleens of mice immunized with a live vaccine of S. enteriditis were resistant to infection with heterologous species of Salmonella as well. Mikami et al. (93) demonstrated that immunity conveyed by RNA is unaffected by DNase, proteases or antiserum to the specific antigen, and that the immunity can be serially transferred to nonimmune cells by RNA fractions. Moreover, in sequential administration of RNA and subpriming doses of antigen to an animal, only immune RNA followed by

LYMPHOID CELL RNA’S AND IMMUNITY

429

antigen was effective in causing an increase in serum antibody. The of 8-12 S and was eluted from a MAK column active RNA had an s20,w at 0.8-0.9 M NaCl, which is similar to the elution characteristics of 18 S ribosomal RNA (93). Another intriguing observation is that spleen cells of nonimmune mice gain the ability to produce antibody after treatment with RNA extracted from allogeneic mice immunized with xenogenic or allogeneic red cells (94). A small number of spleen cells from individual mice of certain strains form antibody against autologous red cells when the cells are treated in vitro with RNA obtained from the spleen of allogeneic mice immunized with red cells of that individual. I n nonimmune mice, there is no production of RNA (which the authors designate as “transfer agent”) capable of inducing autoantibody formation even though the nonimmune animal is continuously exposed to autologous substance. However, once this RNA (“transfer agent”) is produced by sllogeneic animals, it is accepted by nonimmune sheep cells to induce autoantibody formation. The response is ribonuclease-sensitive. To resolve the question of whether these effects of RNA were due to contaminating antigen, “immune” RNA was extracted from the spleens of guinea pigs immunized with diphtheria toxoid and given to nonimmune guinea pigs or mice. This led to the production of rosette-forming cells in host peritoneal exudate cells or spleen cells, but serum antibody could not be detected. By contrast, a high titer of serum antibody was demonstrated, and the number of rosette-formers increased after diphtheria toxoid was given to guinea pigs previously receiving immune RNA. Moreover, this secondary response did not take place after injection of immune RNA into animals primed with antigen. Secondary stimulation with antigen gave a secondary response in animals previously primed with a corresponding immune RNA preparation. These authors suggested that this implies that the RNA preparation is free of demonstrable antigen. Furthermore, serially transferring the same amount of RNA from one mouse to another through 4-6 transfers did not affect the enhancement of the number of immunocytoadherent or rosette-forming peritoneal cells in the last recipient. The calculated fraction of the original RNA present in the final passage was lower than the amount necessary to achieve a positive transfer on the first test (96).Thus, unless the antigenic component had some means of self-replication, i t is clear that it must have been diluted out and could not account for the immunologic activity of the RNA preparations. Also dealing with the identification of a transfer agent of the RNA type, Michelazzi et al. (96-99) suggested that after introduction of antigen, cells of the reticuloendothelial system synthesize a permanent

430

A. ARTHUR GOTTLIEB

template that can produce an RNA responsible for antibody-globulin synthesis in the lymphoid system. They designated this RNA as “RNAimmunocarrier” and predicted that such a material would be present in the sera of immunized animals. Indeed, they found a significant increase of RNA content in immune sera (chiefly in the y-globulin fraction). This RNA was capable of stimulating the production of antibodies against the same antigen used to immunize the animals from which the RNA immunocarrier was taken (96). Antibodies (to red cells) begin to appear in the serum of rabbits receiving RNA immunocarrier derived from rabbits given rat red cells. Moreover, RNA-immunocarrier extracted from the serum of immunized animals of one species could induce antibody production in animals of a different species (9”).Hemolytic plaque formation was also induced, as shown by experiments which tested the influence of RNA-immunocarrier from serum of rabbits immunized with guinea pig erythrocytes to stimulate thymus and spleen cells of rats injected with this material. Hemolytic plaques were formed in this system but none were formed in similar experiments using normal rats or rats injected with ribonuclease-treated RNA (98). It was also possible to obtain, from hyperimmune antidiphtheria rabbit serum, an RNA immunocarrier capable of inducing a precocious antidiphtheria response in normal rabbits (99). Esposito and ChBrie-Lignikre (100) have confirmed the presence of a Michelaszi-type “immunocarrier” in serum. They immunized rabbits with the “H” antigen of S. typhi. After 20 days, a t which time the serum contained a higher titer of anti-H antibody, RNA was extracted from the spleens and then injected into nonimmune newborn rabbits. Undegraded RNA from the immune rat conferred a specific antibody response on these neonatal recipients. Degraded and dialyzed RNA did not work. This was taken as evidence that the response was not attributable to contaminating protein and/or DNA. 2. “INFORMATIONAL” RNA AND REPLICASE

In a set of very unusual experiments, Jachertz et al. (101-109) claim to have identified several RNA’s containing information for the synthesis of antibody, and an enzyme that replicates these RNA’s in a cellfree system. A very striking point about these studies is that the RNA’s involved are active only at extremely dilute concentrations. This group employs a cell-free in vitro system, prepared from the peritoneal exudate cells of mice, that synthesizes an “antibody-analog product” after stimulation with phage receptor particles from E. wli. (It is worth noting that this is another example of antibody synthesized by peritoneal exudate cells.) The product appears to be a 19s globulin,

LYMPHOID CELL RNA’S AND IMMUNITY

431

and its activity can be demonstrated by its specific blocking effect upon the phage-inactivating potency of homologous receptor particles. The synthesis of the product requires ribosomes and 100,000 X g supernatant, and is abolished by treatment of the system with DNase or RNase, and by actinomycin or puromycin (101). A second point raised by Jachertz et al. concerns the in vitro stimulation of mouse macrophages by selected antigens notably bacteriophage, namely that exposure of macrophages to bacteriophage leads to the formation of two kinds of “informational” (messenger) RNA5 (102, 103). One of these acts as a messenger RNA in the same cell and leads to formation of antibody-analog product. The other type of RNA is not effective in the macrophage but acts on transfer to a culture of normal spleen cells where it initiates the production of antibody. Both of these RNA’s can be separately identified on sucrose gradients, and both contain complete information for the synthesis of immunospecific proteins, that is, they could induce the synthesis of the product or antibody in a cell-free system in the presence of actinomycin or in a cell-free system previously treated with DNase. Jachertz and Noltenius calculate that the synthesis takes place only in certain antigen-absorbing cells present in peritoneal exudate cells at a frequency of These cells not only synthesize the antibody-analog but also produce a special type of “informational” RNA that can be transferred to spleen cells and there lead to a synthesis of immune-specific 7-globulins. Furthermore, in contradistinction to clonal selection, one product-forming cell is capable of simultaneously synthesizing several kinds of products with different specificity. These informational RNA’s have not been extensively characterized but appear to be stable to heating and to exhibit no thermal hyperchromicity (104), and Jachertz noted that, as the spleen cells synthesize antibody, the informational RNA can be found in these cells. This phase of antibody production is followed by a latency phase that can be abrogated by irradiation with ultraviolet light. At such a point, vigorous RNA synthesis that is not inhibited by actinomycin begins. This suggests that, during latency, at least one copy of informational RNA is stored in spleen cells, and this, on activation, serves as template for additional informational RNA synthesis. I n later studies (105) Jachertz showed that informational RNA consists of four fractions (I-IV) , all of which are apparently free of antigen. The de novo synthesis of fractions I, I1 and I11 is induced by a protein synthesized in the spleen cell by fraction IV. This regulatory mechanism can be separated from the effect of antigen. These RNA’s have bouyant

’Jachertz

commonly refers to “informational” RNA(s) as “I-RNA(s).”

432

A. ARTHUR GOTTLIEB

densities of 1.68-1.69 g/ml in cesium sulfate and sedimentation coefficients I = 258; I1 = 190; I11 = 109 and IV = 33 S. Jachertz suggested that after the initial contact with antigen, only the macrophage responds with analog-product synthesis and production of “informational” RNA, which is transferred to other cells, as a result of which antibody is formed. On secondary stimulation, not only the macrophages respond, but numerous other lymphocytes in the latency phase can respond to antigenic stimulation with synthesis of such RNA. Fractions I, 11, I11 lead to 19 S antibody formation in spleen cell cultures, whereas fraction IV leads to production of protein(s) of MW 30,000-40,000 in cell-free systems but to 19 S antibody in spleen cell cultures. Apparently, I, I1 and I11 are synthesized de novo as a result of action of a protein produced by fraction IV. These studies have been extended to the use of influenza virus as antigen (106). Cell-free systems derived from monkey or guinea pig spleens were incubated with the hemagglutinating subunits of influenza virus. The RNA derived therefrom, designated informational RNA, induced specific immunologic responses in recipient nonimmune monkey and guinea pigs. The immunogenic activity of this RNA was speciesspecific and was abolished by ribonuclease. Curiously, very small amounts of RNA injected per animal were effective (5 X pg to 0.73 pg) and the number of responding animals and the actual antibody levels obtained were not correlated with the dose of RNA. According to Jachertz, the “informational RNA” has a density of 1.65-1.67 in cesium sulfate (compatible with RNA) is free of protein, is not inactivated by heat but is sensitive to RNase a t a concentration of pg/ml, losing 7576 of the activity within 5 minutes. Biologically, this RNA (a) is capable of initiating antibody synthesis in spleen cell cultures; (b) in combination with ribosomes and p H 5 fraction, can stimulate a cell-free system to synthesize antibody; (c) when inoculated into animal hosts, can induce antibody synthesis and immunity (106, 107).Being synthesized on DNA, it is sensitive to the action of actinomycin D. Two pieces of evidence (107)indicate that replication of informational RNA occurs by means of an RNA replicase: a. There exists a productive phase of antibody synthesis when spleen cell cultures are incubated with “informational RNA.” This lasts 6-8 days during which time this RNA and antibody are detectable. A latent period follows, during which neither antibody nor the RNA can be detected. Irradiation of such a culture with UV produces a rapid synthesis of “informational RNA” resistant to the action of actinomycin. This newly synthesized RNA carries immunologically specific information for

LYMPHOID CELL RNA’S AND IMMUNITY

433

the original antigen and can induce an immune response to this antigen. It is concluded that a few molecules of informational RNA are stored in the cells during the latent period and these molecules represent the starting point for subsequent RNA-directed RNA synthesis. b. As little as pg of “informational RNA” per animal leads to antibody synthesis and immunity, suggesting that an amplification mechanism for RNA synthesis is present in these cells (107). The ‘(rephase” enzyme was obtained by fractionation of a pH 5 fraction over Sephadex G-200. Three fractions containing activity capable of synthesizing informational RNA using informational RNA as template are obtained (108). By contrast, no RNA synthesis was observed using RNA from nonstimulated cell-free systems or nonstimulated macrophages as templates. It is suggested (108) that these fractions represent monomer, dimer and polymeric structures of a basic unit molecule having replicase activity, and also that, in vivo, the production of informational RNA is amplified by antigenic stimulation or nonspecific stimuli such as UV irradiation. In contrast, the synthesis of this RNA after primary antigenic stimulation of cell cultures and cell-free systems is sensitive to actinomycin. It is also claimed (109)that an RNA-dependent DNA polymerase exists in the cell-free system from spleen. This enzyme exhibits a marked preference for “informational RNA” as a template and the DNA product can initiate specific antibody formation (in the cell-free system) without reexposure to antigen. A somewhat similar claim has also been made by Mitsuhashi (110). While the implications of these studies appear to be dramatic, we must wait independent confirmation, particularly in view of the peculiar characteristics of the cell-free system employed (109). 3. EFFECTS OF RNA

ON

MYELOMA CELLS

Related to these studies are attempts to characterize the nature of RNA’s in immunoglobulin cells, using the myeloma cell as prototype. In a rather novel experiment to determine the effectiveness of RNA from a myeloma line to induce antibody formation in lymphoid cells, lymphoid cells were exposed in vitro to RNA from a C,H plasma cell tumor, then injected into sublethally irradiated isogenic recipients or cultured in vitro. Both methods supported protein synthesis by the RNA-treated cells. Although de novo synthesis of globulins by the RNA-treated cells was observed, these globulins did not react with antiserum directed against the C,H myeloma globulin. It was hypothesized that the failure to demonstrate induced myeloma globulin synthesis might arise from failure by a particular lymphoid cell to pick up the two separate RNA molecules required for complete synthesis, or to degradation of the exogenously

434

A. AFtTHUR GOTTLIEB

administered RNA. Success in generating a “modified” myeloma globulin in response to CsH RNA is claimed. In BalbJc mice bearing developing myeloma tumors, the surface immunoglobulin of circulating lymphocytes lose their reactivity with anti -1g antibody (118). This loss is accompanied by the appearance of an immunoglobulin that reacts only with anti-idiotypic antisera directed against the plasmacytoma-specific monoclonal globulin. Moreover, an RNA bound to globulin is found in the plasma of such mice. Incubation of this RNA with normal lymphocytes led to the development of Ig receptors with idiotypic characteristics of the particular myeloma of the mouse from which the RNA was derived. In other studies of the RNA in myeloma cells producing immunoglobulin, i t was noted that labeling of these cells leads to the rapid appearance of label associated with the ribosomes (113).Some of this synthesis could be reduced by incubating the cells with 0.05 pg/ml of actinomycin D. More of the ribosomes were associated with the endoplasmic reticulum in secreting cells than in nonsecreting myeloma cells.

B.

Transfer of Cell-Mediated Immune Responses

1. TRANSFER OF DELAYED HYPERSENSITIVITY Immune responses transferable by cells but not by serum are said to be cell-mediated, or to be manifestations of delayed hypersensitivity. Such responses can be transferred by RNA. I n a classic demonstration of this kind of phenomenon, Mannick and Egdahl (114) observed that nonimmune lymph-node cells from normal rabbits are converted to a state of transplantation immunity by incubation with RNA from the lymph nodes of rabbits bearing skin homografts. These results have been confirmed by other groups (116, 117-119) and the transfer reaction is inhibited by RNase to varying degrees. RNA by itself (i.e., without prior incubation with spleen cells) is not effective and the activity of the RNA is abolished by exposure to RNase. I n at least one case (118), the active RNA appears localized in the 8-12s region of a sucrose gradient. A novel extension of these studies is provided by the work of Guttman et al. (116). RNA was extracted from the liver of allogenics or syngeneicl mice and added to donor isogenic* skin grafts. These grafts. were then sutured to a host. The combination of Balb/c skin and Balb/c Allogeneic refers to an interaction between cells or cellular materials derived from genetically different members of the same species. ‘Isogenic refers to an interaction between cells or cellular materials derived from the same animal.

LYMPHOID CELL RNA’S AND IMMUNITY

435

RNA was not rejected, but a combination of BalbJc skin and C,H liver RNA when given to a Balb/c recipient led to increased rejection of the Balb/c graft. The effect of RNA was abolished by acid hydrolysis of the extract. Other correlates of delayed hypersensitivity have also been transferred. It is well established that the migration of sensitive human inonocyte cells from lymph nodes is regularly and specifically inhibited by purified protein derivative of tuberculoprotein or histoplasmin. When nonsensitive cells are incubated with an RNA extract from lymph nodes of donors sensitized to either or both of these proteins, the migration of these cells is inhibited by these antigens. The action of the RNA is inactivated by ribonuclease but is not affected by DNase and/or trypsin (120,121). Paque and Dray subsequently showed that such transfers of immunologic reactivity by RNA of specifically sensitized donors can cross species barriers (122). A substance capable of inducing cellular resistance to the tubercle bacillis is present in the microsomal fraction of histocytes-more specifically, in the ribosomes of these cells (10). Isolated ribosomal RNA can initiate the development of cellular resistance to the tubercle bacillus in normal animals. This effect is abolished by ribonuclease, but not by DNase or trypsin. This suggests that immunization may result in activation of a mechanism that originally derived its information from the genome but that is capable of self-replication after activation. It is thought that antigen is not involved since successful serial transfer of resistance may be achieved (10). Histocytes from an animal immunized with BCG2, and subcellular components of these histocytes (ribosomes and ribosomal RNA) are effective in inducing cellular resistance against other mycobacteria and brucella (123). A form of transplantation immunity can also be transferred by lymphoid cell ribosomes. When Balb/c mice are sensitized to cells of C,H mice, the Balb/c ribosomes can confer the ability to kill C,H cells on other nonsensitieed Balb/c lymphoid cells (124). This effect could be abolished by ribonuclease, but the authors noted that such RNAmediated transfers did not occur consistently. These studies dealing with the transfer of cellular immunity by lymphoid cell RNA should be distinguished from other studies in which the transfer of delayed hypersensitivity to bacterial antigens is mediated by ribosomal preparations from the bacteria themselves. Such transfers have been described for antigens from Salmonella (126, 126) and Mycobacterium tuberculosis (127, 128). These transfers can also be mediated by rRNA and are unaffected by trypsin. Upon treatment of the rRNA with ribonuclease, only about 50% of the immunogenic activity of the

436

A. ARTHUR GOTTLIEB

preparation is destroyed. This could reflect the presence of an immunologically active RNA core. The mechanism of action of this unusual ribosomal RNA is unclear, but four possibilities exist: (a) the RNA is a mRNA for antibody production, (b) the RNA contains fragments of mycobacterial antigens, (c) the action of RNA might be similar to the nonspecific amplification of immune responses produced by oligonucleotides [see Braun and Firshein ( I S O ) ] . (d) The mycobacterial RNA might be a potent stimulator of the specific immune system involved in acquired immunity to tuberculosis. This latter possibility as well as possibility (c) seem to be unlikely in view of subsequent work by Youmans and Youmans (131) which showed that poly (A), poly (U) poly (C) or poly (I), whether single- or double-stranded, could not replace mycobacterial RNA in the production of an immune response against tuberculous disease. Nor is possibility (a) very likely since it would be curious indeed to find an mRNA for antibody production in the tubercle bacillus. In view of this, it is possible that the RNA in question contains antigenic fragments derived from the mycobacterium. 2. TRANSFER OF IMMUNITY TO TUMOR GROWTH

An area of great interest and potential practical impact relates to the demonstration that RNA prepared from donors immune to specified tumors can transfer that immunity to a tumor-vulnerable host. The concept that an antitumor effect might be mediated by administration of a cell-free extract was suggested by the finding that lymphocytes from a sheep that had been previously stimulated with rat tumor had a pronounced and specific antitumor action. It seemed clear that the cells responsible for this antitumor effect are those large RNA-rich immunoblasts that leave the node 3-7 days after antigen stimulation. Extracts containing this RNA (along with a variable amount of DNA and protein), injected into nontumor bearing recipients, protected the latter from tumor challenge and brought about temporary regression of primary rat sarcomata (132-136). However, established grafted sarcomata failed to respond in any significant way to treatment with intact immune cells or RNA. Significantly, if purer RNA (containing only 10% DNA) was employed, such preparations failed to arrest primary sarcomata, but this relatively pure RNA was active in an in vitro test in which nonimmune spleen cells received cytotoxicity for L5178Y cells by 30-minute exposure to nucleic acids (100 pg/ml) extracted from lymphocytes of sheep that had been immunized with the L5178Y neoplastic line. Possibly, the presence of DNA facilitated the uptake of RNA by the lymphoid cells,

LYMPHOID CELL RNA’S AND IMMUNITY

437

or protected it from degradation. Apparently, therefore, RNA from immune large lymphoblasts or plasmablasts can enter uncommitted lymphoid cells and bestow upon them the ability to reach specific immunity against specific tumor antigens. It is clear, and this is perhaps the most interesting point of all, that the transfer is tumor specific and not due to some nonspecific interaction of RNA with the cell. Variations of this general approach have been presented by other groups including the demonstration of the transfer of tumor immunity in mouse (136,137) and syngeneic guinea pig systems (1.6s). Immune cytolysis of tumor could be transferred by RNA and while this effect was sensitive to ribonuclease, it could not be mimicked by exposure of lymphoid cells to tumor extracts rich in solubilized tumor-specific antigens (142). The work of Ramming and Pilch (140-143) points out the pronounced specificity of these transfers. Spleen cells incubated with RNA extracted from strain 2 guinea pigs immunized with the MCA-A liposarcoma did not cause significant immune cytolysis when applied to monolayers of MCA-25 (whose tumor specific antigens do not cross react with MCA-A). It was noted that there was a background of nonspecific lysis of cells by RNA. I n contradistinction to the suppressive effects of certain RNA molecules as noted above, a curious effect was observed if tumor cells were mixed with RNA-exposed spleen cells prior to injection into a host. Using RNA extracted following immunization with the BP-8 tumor, there was a consistently increased tumor incidence in treated mice over untreated controls. No increase in tumor incidence was seen when tumor cells were mixed with spleen cells incubated with RNA derived from guinea pigs immunized with normal C,H tissues or with BP-8 “immune” RNA treated with RNase. This observation parallels that of Morini et al. (138, 139). Rats that received normal RNA had larger tumors than untreated tumor-bearing rats suggesting that some type of tolerance to the tumor may have been induced. T o test this possibility, Balb/c mice were immunized with rat erythrocytes. “Immune” and “normal” RNA extracted from spleens were given intraperitoneally to other mice; 5 days later these recipients received rat erythrocytes. During the 5-day interval, no circulating hemagglutinins were detected. Such antibodies appeared only after injection of the rat red cells and the peak of the antibody response was observed in normal controls 6-8 days after this antigenic stimulus. At that time, the mice injected with RNA (both normal and immune) had striking depressions in immunologic response, the effect being greater in those animals receiving normal RNA. This indicates that such RNA preparations, in particular those derived from tumor-free animals may exert a strong immunosuppressive effect.

438

A. ARTHUR GOTTLIEB

3. “TRANSFER FACTOR” We have indicated above the relationship of certain RNA’s to manifestations of delayed hypersensitivity and the transfer of tumor immunity, immunologic functions thought to be cell- rather than serummediated. About twenty years ago, Lawrence noted that lysates of white blood cells derived from tuberculin-sensitive donors contains a factor that can transfer this sensitivity to insensitive individuals. This material is dialyzable, and its characteristics are reviewed by Valentine and Lawrence (144) and Lawrence (14.5).The active principle of “transfer factor” has not been isolated, but besides its small size, it contains material that reacts with orcinol and its ability to transfer sensitivity is not affected by DNase. Trypsin and RNase have no effect on the biological activity of “transfer factor.” These properties are consistent with those of a double-stranded RNA or double-stranded RNA joined to a protein moiety, thus conceivably resistant to proteolytic enzymes and ribonuclease. Baram et al. (146) have also called attention to a nondialyzable component of extracts of rhesus monkey leukocytes sensitized to keyhole limpet hemocyanin, which also transfers sensitivity to nonimmune lymphocytes and contains ribose, indicating that RNA was present. They also analyzed the dialyzable transfer factor of Lawrence and found i t to be free of uracil and antigen (147).A recent report by Burger et al. (148) also states that dialyzable “transfer factor” (transferring sensitivity to dinitrophenyl-antibody) is free of antigen as judged by failure to adsorb to columns of anti-dinitrophenyl-antibody. This latter study is also of interest since it describes the ability to transfer delayed hypersensitivity with transfer factor from peritoneal exudate cells or lymph node cells of a guinea pig. I do not believe that the observations of Baram or of Burger exclude the presence of minute amounts of antigen that may be necessary for the activity of “transfer factor.” Fireman et al. (14.9) estimated the maximum molecular weight of the active principle of “transfer factor” to be 4000 or less. They claimed that when leukocytes from tuberculin-negative individuals are cultured in the presence of “transfer factor,” the subsequent addition of tuberculin results in an increased transformation of the cells into large lymphoid cells. Human “transfer factor” can stimulate nonsensitive human cells to produce migration-inhibition factor in the presence of specific antigen (150).The relationship of “transfer factor” activity to oligoribonucleotides in these preparations remains to be explored, as does the explanation at a molecular level of this unusual type of immunologic transfer activity. It is interesting to speculate that both the phenomenon of transfer of tumor immunity by RNA and “transfer factor” activity can be ac-

LYMPHOID CELL RNA’S AND IMMUNITY

439

counted for by the observation of macrophage arming. Normal macrophagcs incubated with hyperimmune spleen cells, or with the supernatant of cultures of lymphocytes exposed to specific antigen, are converted to an “armed” state in which the macrophages are cytotoxic to specific target cells (151).It is conceivable that both RNA from the spleens of animals rendered tumor immune and “transfer factor” from appropriately sensitized donors can “arm” macrophages in this manner.

C. Transfer of Allotypic Specificity RNA extracted from peritoneal exudate cells exposed to T 2 phage leads t.0 the production of two phases of antibody production in nonimmune lymph node fragments (6, 6). The first phase consists mainly of 19s antibody and is maximal at about 5 days. The formation of the RNA responsible for this response was inhibited by actinomycin D, which indicates that the RNA is apparently synthesized after exposure of the cells to antigen. The immunogenic activity of the RNA is abolished by very low concentrations of RNase but was unaffected by treatment of Pronase or a specific antiserum. In contrast, the second phase consisted mainly of 7 s antibody and is maximal on day 12. The RNA responsible for this phase is not newly synthesized after exposure to antigen and its immunogenic activity is abolished by Pronase, specific antisera and high concentrations of ribonuclease A (enzyme to substrate ratio of 1 t o 5 ) . I n addition, Fishman and co-workers argued that both of these RNA’s come from macrophages because they failed to find them in cells pretreated with silica, a procedure that normally kills macrophages. They also found that RNA extracted from glass-adherent peritoneal cells is capable of giving rise to this biphasic antibody response. In other studies, Adler et al. (152) made the startling observation that the anti-T2 19 S molecules formed in this system contain light-chain markers of the donor allotype and do not contain allotype markers of the animal from which the lymph node fragments, which produce the antibody in question, were derived. In contrast, the IgG antibody formed later in the same culture have the light chain allotypic markers of the immune globulins of the donor of the lymph node fragments. It is important to recognize that the allotypic determinants are specified by a set of amino acids in the variable region of the L chain close to the antibody-combining site. Bell and Dray (153) extended their observation by using the technique of Cohen and Parks (78)in which plaque-forming cells specific for sheep erythrocytes are obtained after incubation of rabbit lymphoid cells with RNA from peritoneal exudate or lymph node cells obtained 5 days after injection of the erythrocytes. By use of antibodies directed

440

A. ARTHUR GOTTLIEB

against b4 and b5 allotypic markers of the IgG light chain, the allotypic specificity of the y h l produced by the plaque-forming cells was identified by direct precipitation of the y M in the plaque or by inhibition of plaque formation. The y M antibody produced by the converted spleen cells invariably possessed the allotypic specificity characteristic for immunoglobulin of the donor of the immune RNA. Such transfer of allotypic specificity could be inactivated by RNase but not by DNase or trypsin. I n a subsequent study (154),spleen cells from nonimmune rabbits were converted t o antibody-producing cells of the “indirect” type by incubation with the RNA of lymph node cells obtained from rabbits 18-24 days after immunization with sheep erythrocytes. The antibody produced was of the IgG class and was specific for the cells injected. Most of the indirect plaque-forming cells of the corrected spleen cells possessed IgG antibody with the allotypic character of the donor of the RNA extract, but up to 30% had recipient allotype. One notes with interest the fact that rather large amounts of RNA were required to elicit this response. Bell and Dray could also show that this effect was not due to carry over of anti-sheep antibody since such antibody could not be detected in any of the RNA extracts by “indirect” hemolysis in gel. These studies have been extended and show that such transfer can occur on both L and H chains (155). To date, these very interesting results have not been confirmed in other laboratories, and it is hoped that they will be in the near future. There are several possibilities to account for them. (a) There could be transfer of genuine “mRNA” for antibody synthesis. (b) It is possible that the DNA for b4 and b5 polypeptide chains are indeed present in all rabbits and that the allelic genes regulating the synthesis of these polypeptide chains are control rather than structural genes. In that case, RNA might act on the control genes. (c) The RNA could be a messenger or suppressor RNA capable of altering the transcription of information coded in the genome of the nonimmune cell so as to cause substitution of a few amino acids in the polypeptide sequence specifying allotypy. (d) The RNA’s preparation might contain unique tRNA molecules capable of specifying the specific amino acids in the allotypic locus. Such tRNA molecules have not, however, been demonstrated.

V. Nonspecific Stimulators of Immune Responses A number of RNA and RNA-like molecules are capable of stimulating immune responses in a nonspecific manner. I n the course of their studies on the ability to form hemolysin antibody in X-irradiated rats (400 r) , Taliaferro and Jaroslaw injected these animals intravenously with sheep erythrocytes together with various preparations of nucleic

LYMPHOID CELL RNA'S AND IMMUNITY

441

acids (156, 157). They found that enzymatically degraded DNA or RNA partially restored the ability of irradiated rats to form antibody. Neither intact nucleic acids nor nucleotides could do this, hence the restorative effect was attributable to partially degraded nucleic acids. Moreover, they found that a mixture of oligoribonucleotides restores the secondary response to polymerized flagellin previously inhibited by actinomycin D. Deoxyribonucleotides were not effective and there was no enhancement by oligoribonucleotides in the noninhibited culture. These findings suggest that the restorative activity of the oligonucleotides is related to enhanced RNA polymerase activity. Thus, the stimulation of nucleic acid synthesis by oligonucleotides at the time of antigen injection may result in the development of more cells into a state responsive to antigen. Braun and Nakano (159) confirmed that enzymatic digests of DNA can significantly increase the number of hemolysin-forming cells in spleens removed from AKR mice 48 hours after immunization with heterologous red cells. The number of antibody-forming cells produced was higher when the antigen was given with an enzymatic digest of calf-thymus DNA. Stimulation by an enzymatic digest of DNA was not matched by an enzymatic digest of RNA, nor were oligodeoxyribonucleotides capable of stimulating an immune response unless specific antigen was given concurrently. Actinomycin D interfered with the immune response in the absence or presence of oligodeoxyribonucleotides. I n later studies (160, 161) this group observed a pronounced stimulation of antibody production to sheep erythrocytes if associated homopolymers, such as poly (A-U), poly (G-C), or poly (I .C), were used. The polynucleotides appear not to affect the final number of antibody-forming cells but rather to stimulate the performance of the cells initially responding to antibody. These combinations of homopolymers were not effective as singlestranded polymers in vivo, but single-stranded homopolymers could be used in vitro. The duplexes could stimulate primary as well as secondary responses when given with antigen. Moreover, poly (A-U), given without specific antigen but with a modifier of lymphocyte permeability such as antilymphocyte serum, could evoke a significant booster type response to previously experienced antigenic stimuli. Antigen in combination with gave rise to premature initiation of antibody poly(A.U) or poly (I-C) formation in newborn mice, analogous to some effects of bacterial endotoxin (162) and in at least one case (I&?), polynucleotides could stimulate the production of normal responses in a genetically low-responder strain of mice. Studies that extend these observations demonstrate that peritoneal exudate cells exposed to BCG' reinjected into syngeneic mice led to a shortened induction period, an increased uptake of j3H]uridine, and increased antibody titers as compared to mice directly injected with

442

A. ARTHUR GOTTLIEB

antigen. Actinomycin D inhibits these effects and establishes that synthesis of mRNA is necessary for the adjuvant action of these adherent cells. The inhibition by actinomycin D can be reversed by poly(A.U) when it was given directly before or after the drug. Newly synthesized RNA could not induce an antibody response by itself, when injected into mice, unless poly (A-U) was given concomitantly. However, RNA by itself induced the formation of rosettes in numbers comparable to those induced by BCG alone. Again, when the RNA was given with poly(A.U), the number of rosette-forming cells was increased relative to RNA alone (164). It had been suggested that the adjuvant effect of endotoxin could be mediated through cell destruction and release of nucleic acids, which then stimulated division of antibody-forming cells. If the action of endotoxin was so mediated, endotoxins should affect the proliferation of antibody-forming cells in v i m and the postulated nucleic acid intermediates should amplify the antibody response to protein antigens. Merritt and Johnson (166) found that endotoxin accentuated antibody formation in the presence of fluorodoxyuridine (FdUrd) (which suppresses cell division by its action on thymidylate synthetase). FdUrd given 1 hour before or up to 14 hours after antigen injection exerted an adjuvant action resulting in higher than normal levels of antibody. When FdUrd was delayed until 18 hours or longer after injection of antigen, the antibody response was abolished unless endotoxin was given with the antigen. The postulated intermediates, nucleic acids, were capable of shortening the induction period and enhancing the level of anti-BCG when given with antigen. From these studies, it was clear that the nucleic acids were capable of acting as adjuvants, and that they exhibited effects similar to those of endotoxin. It is interesting to note that digestion of the nucleic acids with nuclease did not affect their action as adjuvants, unless low molecular weight products were removed by hydrolysis. Poly(A.U) or poly(1.C) is effective in reducing the incubation period and increasing antibody titers to several antigens in mice (160,166). Homopolymers are not effective and, curiously, poly (A-U) is immunosuppressive when injected 12-24 hours before antigen. The action of nucleic acids, in this nonspecific manner, may relate to stimulation of polynucleotide-related enzyme activities in rapidly proliferating cells.

VI. Macrophage RNA's and Immunity A. Peritoneal Exudate Cell Populations and Macrophages While a number of the studies described to this point relate to RNA's present in lymphoid tissue, and since macrophages (or histiocytic cells)

LYMPHOID CELL RNA’S AND IMMUNITY

443

makc up a substantial fraction of such tissues, some of the RNA species with which we have been dealing may in fact come from macrophages. These cells characteristically adhere to glass and/or plastic surfaces and a population of cells from lymph node or spleen can be rendered relatively, but not completely, macrophage-free by allowing the total cell culture to incubate on such surfaces for 16-20 hours. The nonadherent cells can then be recovered. Not every investigator who has studied lymph node or spleen cclls has taken pains to remove the adherent macrophages. There is, however, a fairly extensive literature on the characteristics of RNA from macrophages. Two types of cell populations have been cmployed: (a) cclls present in the peritoneal cavity either with or without induction with a macrophage-inducing substance, such as mineral oil (after recovery, these cells are plated on a glass or plastic surface and the monolayer of adherent cells is employed) ; or (b) lymphoid cells from lymph node or spleen similarly treated to yield the adherent cell population. Fewer macrophages are obtained from lymph node or spleen than from the peritoneal cavity. Moreover, it is well to emphasize the fact that not every cell that adheres to glass or plastic surfaces is necessarily a macrophage; “sticky” lymphocytes can lead to confusion and misinterpretation of studies on “macrophage” populations. Changes in RNA populations of peritoneal exudate cells have been described. The addition of bovine serum albumin to peritoneal exudate cells (60% monocytes and 33% lymphocytes) leads to significant changes in the base composition of RNA extracted from this mixture of cells, specifically, to a reduction in adenine and cytosine but an increase in guanine and uracil (167).However, it is not clear whether these changes occur in the lymphocytes or the macrophages. Adherent peritoneal exudate cells from the mouse do not synthesize much DNA (as judged by uptake of [3H]thymidine), but actively synthesize RNA (as scored by uptake of [SH]uridine) (168). During phagocytosis the incorporation of [”Iuridine into RNA is increased. Thus, we have the impression that the macrophage is a cell that will synthesize (and degrade) RNA upon exposure to foreign substances, but is reluctant to synthesize DNA and divide (169).

B.

Some Effects of Macrophages on Immune Responses A great deal has been written concerning the role of macrophages in immunity (17&172), and only selected aspects of this area are here covered. It is important to emphasize that there are two general schools of thought on the role of macrophages in immunity, one school holding that macrophages do nothing more than deliver antigen bound on their surface to antigen-reactive cells, the other that while surface-bound

444

A. ARTHUR GOTTLIEB

antigen may be effective and indeed necessary in the induction of certain forms of immunity, certain RNA species in the macrophage can become associated with fragments of antigen and it is these antigen-RNA complexes that are important and useful in initiating immunity. Levine and Benacerraf (173)were among the first to suggest that degradation of antigen is important in the immune response. They observed that those hapten-poly (L-lysine) conjugates that are degraded by macrophages are antigenic in certain guinea pigs, whereas those conjugates that are not degraded are not antigenic. Uhr and Weissman (174)injected phage into guinea pigs and noted the association of the phage with the lysosomes. With time, there was a sequential decrease in plaque-forming units, owning to degradation of the phage, but this was associated with increased immunogenicity of the phage residue. A classic study by Frei et al. (175)suggested that phagocytosis is “critical” for immunity. I n that study, particulate or aggregated antigens, which undergo extensive phagocytosis, gave rise to better immune responses than did soluble or poorly metabolized substances. Presently, many immunologists do not regard phagocytosis as an essential prerequisite for the immune response, since there is much evidence to indicate that antigens are capable of direct interaction with lymphocytes (176).I n particular, the point has been made that conformational determinants would undergo degradation by macrophages; the loss of native structure in such a process would lead to an abrogation of the immune response. I n my opinion, this is too severe a judgment. Sela has reviewed the evidence (177)that preservation of total native antigenic structure is required for immunogenicity. While this may be true for certain antigens, it leaves no room for a large number of observations that indicate that fragments of antigen play a very real role in the induction of certain forms of immunity. The work of Lapresle et al. on human serum albumin (178), Ada et al. on fragments of flagellin (179)and that of my own laboratory (180) speak to this point. It is well established that not only are foreign Substances catabolized and degraded by macrophages, but small amounts of antigens and/or antigenic fragments are retained in these cells for relatively long periods of time. For example, there is the preservation of antigen in lysosomes and RNA fractions of the spleens and livers of rabbits during antibody formation (181). Lysosomal fractions isolated from spleen and liver of animals a t various periods after administration of sheep erythrocytes contain fragments of antigen that retain immunogenicity and are capable of provoking the formation of specific antibodies in immunized and nonimmunized recipients. Immunogenicity is not associated with intact microsomal fractions. Antigen present in the lysosomes (and presumably

LYMPHOID CELL RNA’S AND IMMUNITY

445

degraded by lysosomal enzymes) is a more effective immunogcn than intact antigen (174). Moreover, a number of studies carried out by Mitchison and others on the effect of macrophages on the immunogenicity of macrophages (188) show that in general, “weak” antigens exhibit enhanced immunogenicity when administered enclosed in macrophages, whereas the immunogenicity of “strong” antigens is not affected by this process. Another view of the role of macrophages in immunity is given by Spitsnagel and Allison (183, 184), who have shown that several agents known to labilise lysosomal membranes, thus allowing for increased degradation of antigens, have adjuvant effects on antigens such as bovine serum albumin. In an accompanying paper (184), these workers studied the primary and secondary antibody responses measured in CBA mice after injection of different doses of free albumin or albumin taken up by syngeneic macrophages. The response to macrophage-associated albumin was higher than to free albumin and they suggested that free albumin elicits two competitive responses in immunocompetent cells, immunization and the induction of tolerance. When the antigen is associated with macrophages, the balance is strongly shifted in the direction of immunization. Bovine serum albumin associated with macrophages treated with E . wli lipopolysaccharide (endotoxin) induced consistently higher antibody responses in normal mice than did albumin bound by untreated macrophages. The lipopolysaccharide had no effect on uptake or degradation of antigen by macrophages. The authors suggested that macrophages contribute to augmented immunogenicity by presenting antigen to immunocompetent cells in a way that strongly favors immunity, through a reduction in the amount of albumin freely available for direct interaction with lymphocytes.

C. Antigen-RNA

Interactions

1. ANTIGENFRAGMENTATION

A considerable amount of interest in the function of macrophages has centered on the issue of association of antigens with RNA’s from the macrophage. For this article, it is perhaps appropriate to summarize some of the salient points concerning such interactions. Garvey and Campbell (8, 3) pointed out that tissue-retained antigen was, in some cases, attached to RNA. These papers make the important points that immunogenicity of an antigen is often associated with incomplete fragmentation of antigen and that small amounts of these fragments persist in tissues for long periods of time. Using 35S-labeled bovine serum albumin and ‘%-labeled keyhole limpet hemocyanin intravenously

446

A. ARTHUR GOTTLIEB

injected into rabbits, they found over 10% of the labeled antigen in the urine during the next 24 hours, and another 25% in the next 6 days. Curiously, the labeled antigen material found in the urine was quite heterogeneous and much of it was associated with low-molecular-weight RNA. The principal difference between the labeled material released following secondary injection, as compared with primary injection, was the high degree of association of antigen with nucleotide material. The nature of the RNA-bearing antigen was not fully characterized, and it is not clear whether the RNA was isolated in degraded form. Subsequent studies indicate that antigen can be released from RNA by heating to 80-90" and that the antigen-RNA complex has a molecular weight of about 30,000 (3).It is important to note that Garvey and Campbell consider that their antigen fragments are derived from hepatic cells. This view is in contrast to that of other investigators who suggest that such fragments are produced in the macrophage-like cells that line the hepatic sinusoids (Kupff er cells) . 2. MACROPHAGE RIBONUCLEOPROTEIN AND ANTIGEN-RIBONUCLEOPROTEIN COMPLEXES Certain antigens, when complexed to RNA, display augmented immunogenicity. This was first shown by Fishman and Adler, who demonstrated that RNA derived from macrophages exposed to T2 phage has the capacity to induce antibody formation in nonimmune lymph node and/or spleen fragments (186, 186). Subsequent investigation confirmed these observations (187)but also clearly demonstrated the presence of trace amounts of antigen in the RNA preparations (7,8). My laboratory demonstrated that antigen bound to a unique ribonucleoprotein of the macrophage contains all the immunogenic activity of the FishmanAdler RNA preparations. RNA extracted from adherent peritoneal exudate cells and banded in cesium sulfate displays a minor light-density species (1.58 g/ml), which we have referred to as macrophage r i b nucleoprotein. This species constitutes about 5% of the total RNA of the cell, has an approximate molecular weight of 12,000, and contains 28% protein. The species is found in comparable amounts in all peritoneal exudate cells, whether experimentally exposed to antigens or not. However, in the case of cells exposed to various antigens, fragments of the antigen are found associated with the RNA-protein complex, and phage T 2 antigenic fragments associated with this complex are demonstrably more immunogenic than the same amount of antigen in free form. Moreover, all the immunologic activity of the Fishman-Adler system is exclusively resident in the complex (188).It is of interest to us that Saha et al. (188a) observed a small molecular weight ribonucleate-antigen

LYMPHOID CELL RNA'S AND IMMUNITY

447

complex in the liver of immunized rabbits. This complex exhibits elution characteristics on anion-exchange columns similar to those of ribonucleoprotein. The complex is not an artifact of the technique used to extract RNA. Bishop and Gottlieb (189) demonstrated that RNA. protein is distributed in a 2 : l ratio between cellular supernatant and ribosomes and is clearly present in the cell in situ. Moreover, the complex appears to be unique to the adherent peritoneal exudate cell (macrophage) (18%). In examining the characteristics of a variety of 12sI-labeled antigens in regard to their ability to bind to ribonucleoprotein, the following facts were well established. 1. Fragmentation of antigen is a requirement for binding to ribonucleoprotein. Based on the estimated molecular weight of the complex and the protein content, the maximum size of the antigenic determinant is 3600 (190). For phage T2,the antigenic fragment in the complex is highly immunogenic and capable of absorbing out all neutralizing antibodies directed against the native T2 bacteriophage. 2. Molecules such as linear random copolymers of D amino acids, for example, poly ( D G ~ UnAla3' ~ " DTyrlO) are not degraded by the macrophage and are not found associated with the RNASprotein species to any large extent. Similar copolymers of L amino acids are bound very well. This observation indicates that the binding of such polymers (which bear substantial negative charges a t physiological pH) cannot simply be due to charge interactions with RNA through Mg2+bridges, as suggested by others, since such a process would not be expected to be stereospecific (191).

3. Poly(~Glu~O ~ A l a ~DTyrlO) ' prevents the binding of other synthetic copolymers of L amino acids to the complex. Since poly(~G1u~O ~ A l a DTyrlO) ~' does not bind to the complex itself, this suggests that there may be an enzyme in adherent peritoneal exudate cells that is required for linkage of the fragment of the L polymer and that this enzyme appears to be blocked by D polymers of large molecular size. It is clear that this suppression of binding of the L polymer by the D polymer is not due to an effect of D polymer on the catabolism of the L polymer (19B). 4. There is no direct relationship between the amount of antigen recovered in the complex and the immunogenicity or ((strength" of the antigen (191). Since these measurements depend on the availability of a labeled tyrosine residue in the fragment bound, it is certainly possible that unlabeled fragments of antigen might be associated with the complex but would not be seen in this system. 5. Cortisone suppresses the linkage of labeled antigen to macrophage ribonucleoprotein without strikingly affecting polymer catabolism. It is

448

A. ARTHUR GOTTLIEB

possible that the well-known effects of cortisone on immune responses may be related to this effect (193). Fishman and Adler (194) distinguish two types of R.NA that are extractable from antigen-exposed peritoneal exudate cells. One of these RNA’s is clearly associated with antigenic material, causes the production of 7 S antibody, has a sedimentation coefficient (on preparative sucrose gradients) of 4 4 S and elutes only with high salt from a methylated albumin-kieselguhr column. Employing a method for fractionating peritoneal exudate cells on discontinuous gradients, Walker ( 1 9 4 ~ and ) Rice (195) identified a fraction of macrophages that bands a t the interface between 8 and 11% albumin solutions and is the only subpopulation that gives rise to anti-T2 antibody. Curiously, this subpopulation was not as phagocytic as were the other fractions. These studies are of potential value in identifying the cell type involved in these responses. The second type of RNA, extractable from peritoneal exudate cells, is free of antigen, leads to the production of 19s globulin, and migrates in the 8-12s region of a sucrose gradient. It is analogous and perhaps identical to the RNA described, by Adler et al. (152) and by Bell and Dray (153-155), which is capable of transferring allotypic specificity. The precise cell from which this RNA is derived has not been determined. A summary of the distinctions between these two types of “immunologically active RNA” is given in Table I. Our studies lead us to believe that the antigen-RNA complex in the Fishman-Adler studies is identical to the antigen ribonucleoprotein complex we identified in peritoneal exudate cells (188). Our results indicate that this complex is smaller than indicated by Fishman, having an s20,w of 1.8. This difference may relate to the fact that the studies of Fishman were carried out on preparative sucrose gradients, while our studies were TABLE I OF IMMUNOLOQICILLY ACTIVIB R.NA’s CH.~R.WTERISTICS Characteristic Sedimentation coeffi cient Protein content Size of antigen moiety Ability to transfer allot,ypy Globulin produced Sensitivity to RNarte A Sensitivity to Pronase Guanine cytosine content Messenger RNA activity

+

Ribonucleoprotein, antigen-RNA complex 1.8 28%

5 36OO No 7 S, possibly 19 S Resistant Sensit.ive 58.9% None

“Informational” RNA’s 8-12 0 None present YeS 19 S only Very sensitive Resistant Undetermined Presumed to be mRNA

LYMPHOID CELL RNA'S AND IMMUNITY

449

performed by sedimentation velocity in the analytical centrifuge. Furthermore, the behavior of the Fishman antigen-RNP complex on column chromatography and its elution a t high concentrations of salt may be attributable to the protein component of the complex, since proteins are known to adhere firmly to such a matrix. I n our initial studies, we had noted that RNA extracted from peritoneal exudate cells exposed to T2 bacteriophage contained immunologic activity, for the production of anti-T2 antibody, only in the nucleoprotein band and that this activity migrated with the 2 8 s fraction of the RNA on sucrose gradients. Yet the complex is small. In view of the studies of Bishop and Gottlieb (189) which indicate that ribonucleoprotein is distributed in a 2:1 ratio between cellular supernatant and ribosomal fractions, it is conceivable that the antigenic fragment of T 2 bacteriophage may have resulted in preferential binding of the nucleoprotein bearing this particular antigenic fragment to the ribosome. This would lead to the recovery of the complex in the 28s fraction of the RNA. It should be noted that it is quite difficult to prepare undegraded RNA's from macrophages, and that ribonucleoprotein is most frequently recovered from macrophages in free form. Roelants and Goodman (196,1.97') have called attention to the fact that a large number of antigens (especially those bearing negative charges on their surfaces) will bind to various RNA's from macrophages. Their evidence indicates that this association involves a Mgz+ion bridge between negative groups on the antigen and phosphate groups on the backbone of the polynucleotide. They conclude that all interactions of RNA with antigen are, therefore, artifactual. Table I1 indicates the major differences between antigen-nucleoprotein interactions and nonspecific interactions of antigen with RNA. The sweeping conclusion of Roelants and Goodman seems to be unwarranted, and we would reTABLE I1 CHARACTERISTICS OF ANTIGEN-RIBONUCLEOPROTEIN AND ANTIGEN-RNA INTER.4CTIONS Criteria

Antigen-ribonucleopro tein

Source of RNA Unique to macrophages Favored by Mga+ ion No Abolished by EDTA No Abolished by hot phenol No Location in RNA profile" Only on nucleoprotein molecule Always fragmented Antjigenin complex Q

On polyacrylamide gel electrophoresis.

Ant,igen-R.NA Any cell homogenate Yes Yes Yes Broadly dispersed Native

450

A. ARTHUR GOTTLIEB

emphasize the point that it is necessary to distinguish critically between interactions of antigen with nucleoprotein and with other species of RNA. Although both types of interaction may lead to enhanced immunogenicity of the attached antigen or antigenic fragment, only interactions of antigen with nucleoprotein have been shown to exist in situ. The interaction of antigen with other non-nucleoprotein species is most likely without physiological relevance. It is the sweeping nature of the Roelants and Goodman conclusion that has led to a good deal of confusion and misunderstanding. Another series of studies on antigen fragments associated with small ribonucleotides demonstrated the presence of a low-molecular-weight antigen fragment in association with an oligonucleopeptide in lymph node and spleen (198). Using SB-labeled bovine serum albumin as antigen, Yuan and Campbell examined the soluble extracts of these cells 5 hours after adding antigen. Although retained antigenic material could be separated into four fractions on Sephadex, the bulk of the soluble antigen was present in material of small molecular weight. This component contained not only nucleotides and antigen, but also an oligonucleopeptide to which antigen was attached and which stimulated antibody formation to the albumin when injected into rabbits. The complex of a6S-labeled antigen and oligonucleopeptide did not elute in the void volume of Sephadex G-25, indicating a molecular weight of about 5000 to 10,000, and appeared to contain a basic protein, since it migrated toward the cathode, even with the sulfanilate residue attached. This indicates that this moiety is different from the nucleoprotein molecule we described, and perhaps is a more positively charged derivative of ribonucleoprotein. This SsS-labeled antigen-oligoribonucleotide conjugate produced a secondary immune response 4 days after injection into rabbits previously immunized with bovine serum albumin (199),and following incubation with specific anti-albumin, formed radioactive precipitin areas when subjected to immunoelectrophoresis against goat antirabbit IgG. This indicates that the antigen fragments that conjugate with the oligoribonucleotides are indeed antigenic determinants derived from the albumin molecule. The immunogenicity of the conjugates was also demonstrated in normal rabbits by detecting antibodies to the albumin in'the IgG fraction by radioimmunodiffusion and by scanning for antibody-forming cells by the Jerne plaque technique (76).An important point raised by these studies is that since the conjugates induce the formation of antibodies that react with the native antigen, bovine serum albumin, the fragment of antigen in the conjugate must have retained a configuration specific for thc native albumin molecule. I n this regard, it is worth

LYMPHOID CELL RNA'S AND IMMUNITY

451

recalling tlic studies of Cohen (84), who showed that partially degraded RNA from mice immunized with sheep erythrocytes inhibits the conversion of normal spleen cells into antibody-forming cells by undegraded RNA. This is a specific reaction and it is not inhibited by degraded or nondcgraded RNA from mice immunized with unrelated antigens. It was not shown whether the RNA contained sheep cell fragments, but if the antigen fragments that associated with the RNA are not detectable without a radioactive assay, the inhibitory effect could be explained as due t o the competitive binding of these partially digested antigen-oligoribonucleotide complexes to the lymphocytes, thus preventing the interaction of these antigenic dctcrminants with the lyniphocytcs. The same laboratory has partially characterized the nucleopeptide fraction obtained from livers of rabbits injected with 3zS-labeled sulfanilate-azoalbumin (bovine serum) (200).Fractionation of this material on Sephadex G-50 followed by electrophoresis led to the resolution of several nucleopeptide components, containing antigen and capable of triggering an anamnestic response. These studies suggest a covalent linkage between nucleotide and antigen fragment. Perhaps the fact that there appear to be several labeled nucleopeptide conjugates indicates that individual antigenic fraginents carrying different charges are linked to a common oligoribonucleotide matrix. The association of antigen and other proteins with small oligoribonucleotides is a feature common to this study as well as previous studies on macrophage ribonucleoprotein. Yadayatty e t al. (201) has suggested that such protein (or polypeptide components) may protect RNA molecules from degradation by nucleases. Conversely, the association of distinct antigenic determinants with RNA or oligoribonucleotides may be a way of conserving these determinants from further enzymatic degradation. The work of Abramoff and Brien (202) on the transfer in chickens of the primary immune response to sheep erythrocytes by RNA from immune chickens is best accounted for by an erythrocyte-RNA complex. Examination of the spleen cell populations of chickens receiving a single intravenous injection of sheep red cells indicated that a heightened amount of RNA accumulated in immune spleen cells relative t o nonimmune populations. RNA extracted from spleen 2 days after injection of the erythrocytes stimulated antibody formation in nonimmune recipient cells within 6 hours. The immunogenic activity was abolished upon incubation with RNase a t not less than a 1:7 weight ratio of bovine pancreatic RNase to RNA for 30-60 minutes a t 37".This is a relatively large amount of RNase and recalls the relative insensitivity of ribonucleoprotein to bovine pancreatic RNase. Specific anti-sheep-erythrocytc serum also inhibited thc immunogenic activity of this RNA. Addi-

452

A. ARTHUR GOTTLIEB

tional characterization of the active RNA in that system was not reported, but the results are consistent with linkage of sheep erythrocyte antigen to ribonucleoprotcin. Indeed, subsequent studies indicate that fragments of sheep red cells are associated with a ribonucleoprotein-like material in a cesiuni sulfate density gradient (203). 3. MIXEDEFFECTS:8-12 S RNA PREPARATIONS CONTAINING ANTIGEN

There are a number of observations dealing with preparations of antigen-containing RNA capable of transferring immune responses in which the iinmunologically active RNA appears to be considerably larger than the macrophage R N P species. It is well to point out that the source of these RNA’s (k.,lymphocyte or macrophage) has not been determined. Rat spleen cells immunized with sulfanilic acid conjugated to bovine albumin produce an RNA species that induces sulfanilic-acid-specific antibodies in nonimmune isologous cells in vitro (20.4). The effective RNA was localized in the 4-12s region of a sucrose gradient. RNase abolished the effect, and on the basis of tracer studies it was concluded that the imniunogenicity of the lymphoid cell RNA is not due to an antigenic moiety associated with the RNA. This conclusion may not be warranted since the specific activity of the sulfanilate-albumin conjugate was only 20.6 pCi/mg so that the ability to detect labeled material in the RNA would have been limited. Pronase treatment of the RNA preparation led to a reduction in the number of plaques to 55% of control values. Since a greater effcct was noted when the lymphoid cells wcre first treated with RNA and then with Pronase, Juras and Abramoff suggested that this again supports the view that antigen does not contribute to the immunogenicity of the RNA preparation. Similarly, RNA derived from the spleens of mice immunized with streptococcal phage induced the formation of specific phage-neutralizing antibodies when added to nonimmune mouse spleen fragments (206,206). It has been suggested that many of the RNA transfer effects seen with phages from gram-negative hosts are attributable to contaminating endotoxin (207). This study argues against this suggestion since phage 81a grows on a gram-positive host. The immunologically active RNA displayed a sedimentation coefficient of 8.2 on sucrose gradients, indicating an approximate molecular weight of 1OO,OOO, so that it would be capable of coding for a chain of l0,OOO molecular weight. The activity of the RNA in an immunological sense could be inactivated by RNase, but not by RNase or trypsin. Phage antigens were definitely present in the RNA as judged by the detection of labeled phage antigens, and sntiphage antibody preventcd the ability of the RNA to induce neutralizing anti-

LTMPHOID CELL RNA'S AND IMMUNITY

453

body in cultures of normal spleen fragments. Injection of immunogenic RNA into previously immunized mice resulted in an anamnestic response, whereas challenge with nonimmune RNA did not. Thus, in this case, it is not clear whether the immunogenicity of this RNA preparation was intrinsic to the RNA itself or to antigen linked, specifically or nonspecifically, to this RNA. Studies of the fate of labeled dinitrophenylated bovine serum albumin or to ribonuclease in spleen and lymph node showed that there is a n increase of the antigen retained in ribosomes of these tissues and in the cytoplasm as well. I n particular, one of these soluble forms of antigen was associated with RNA in a cesium chloride density gradient, and it was immunogenic as tested by the induction of antibody synthesis in ' normal spleen cells (208). This form of antigen closely resembles the antigen-nucleoprotein complex we have described (188). One major difference was noted in that the immunological effectiveness of this nuclcoprotein-like material was not affected by Pronase in the hands of these workers, but was abolished in ours. Perhaps this relates to the ' type of antigen employed in that the product of Pronase digestion of Npph-albumin or N,ph-RNase might retain effective immunogenicity, whereas T 2 phagc antigens might not. Howcver, it is more likely that these immunologic events are due to an antigen-free RNA and that the activity of the antigen-nucleoprotein complex is not reflected in these studies. It is of interest to note that Duke and Harshman (209) found no association of antigen with RNA when labeled NJph-albumin or N,phRNase were added to liver homogenates and subsequently extracted with phenol. This is an important observation that indicates that the coupling of antigen to RNA is not an artifactual process but requires the intact cell. They were able to show that RNA from liver containing antigen-RNA complexes induces specific de novo antibody formation in normal spleen cells in vitro. Antibody appeared as early as 2 hours after addition of RNA. Again in this study, Pronase did not affect the induction of antibody synthesis even though all detectable antigen was removed. However, the assertion th at this indicates complete removal of all antigenic fragments can only be evaluated in terms of the specific activities of the antigens employed. Thus it is not resolved whether the labeled N,ph-antigen fragment associated with the RNA plays a direct role in inducing normal rabbit spleen cells to produce antibody. However, since the antigenic fragment does not appear to be necessary for immunogenicity, it may well be that immunologic activity of their RNA preparations is exclusively attributable to RNA free of antigen. Duke et aE. (210) extended these observations to sheep erythrocytes.

454

A. ARTHUR GOTTLIEB

RNA from liver or spleen of erythrocyte-primed mice is capable of inducing the formation of plaque-forming cells, specific for these erythrocytes, in normal spleen cells. The active RNA was excluded by Sephadex G-100 but not by Sephadex G-200.This suggests that the active RNA had a molecular weight of approximately 100,000 and could therefore code for a protein of 10,000. The molecular weight of the RNA active in the Duke and Harshman system is severalfold greater than the antigen-nucleoprotein complex we have described. It is likely that Duke and Harshman are studying the immunologic activity of an RNA of molecular weight approximately 100,000 that is contaminated with trace amounts of antigen. This antigen causes a density shift of the RNA in CsCl, but the antigenic fragment itself, in contrast to the activity of the nucleoprotein moiety, does not play a role in the response. An important question regarding the role of these antigen-RNA complexes in immunity is whether these complexes are actually transmitted to lymphocytes (which are the precursor cells for antibody production). A direct cytoplasmic connection between macrophages and lymphocytcs has been described by Schoenberg (611). Miller and Avrameas (212) have described antibody-producing cells clustered around macrophages in lymph nodes. A distribution of labeled RNA between macrophages and lymphocytes suggesting a “feeding” of RNA from macrophages to lymphocytes was noted by Fishman et al. (213). Bona (214) incubated peritoneal exudate cells with [3H]uridine in the presence of endotoxin and observed that lymphocytes in contact with the labeled macrophages became labeled, while lymphocytcs not in contact with the macrophages remained unlabeled. These bits of evidence support the idea that RNA (and presumably antigen-RNA) complexes can be transmitted from macrophages to lymphocytes.

VII. Possible Mechanisms of Action of RNA’s in the Immune Response As we have noted in this review, there appear to be a myriad of immunologic phenomenon related to or caused by various RNA’s from lymphoid cells. In several respects many of the studies are clearly a t an early stage of development, and much more rigorous study of these systems is required to assess thc genuine nature of the phenomena and their physiological relevance. Better definition of the heterogeneous cell populations involved, more precise characterization of reaction components and products, more rigorous application of stoichiometric principles and development of more quantitative assays are all indicated. It is important to recognize that apart from the theoretical impact

LYMPHOID CELL RNA’R AND IMMUNITY

455

of these studies, the practical applications of some of these phenomena as in the possible treatment of tumors with RNA or the imaginative application of “transfer factor” to the solution of intractable clinical problems, indicate that we would be remiss in not pursuing these studies. Moreover, there are several observations in this area that suggest that something important is going on. These include: (a ) the ability to transfer allotypic markers; (b) the specificity of transfer and induction of dclsyed hypersensitivity and tumor immunity; (c) the dramatic “transfer factor” phenomenon; (d) the fact that macrophage ribonucleoprotein is unique to the adherent peritoneal exudate cell (macrophages) and comprises 5% of the total RNA of that cell type; (e) the demonstration (107)of a replicase system in cell free extracts of spleen and the important observation (if confirmed) that “informational RNA” is a preferred templatc for a reverse transcriptase-like enayme in spleen cxtracts; ( f ) thc demonstration of a high proportion of R N A - D N A hybrids in antibody-forming cells. A basic distinction can be madc between RNA or nucleoproteins that tlcpcnd on the prescnce of antigen for iminunologic activity and those that do not. Such traces of antigen could be randomly distributed as contaminating molecules in an RNA preparation or they may be linked to vcry few specific RNA species in the mixture and these molecules bearing antigen may be responsible for the activity of the preparation. The resolution of this question, particularly as regards the properties of “transfer factor,” is not an idle question. With respect to the “informational” RNA’s, most of these appear to be 8-12s in size and to be free of contaminating antigen, protein, and DNA, and are capable of coding for synthesis of an L chain. It is clear that these “informational” RNA’s need not necessarily be complete “messenger” RNA’s specifying the entire chain (or variable region thereof). Fusion of an RNA molecule coding for the variable chain with another RNA molecule coding for the constant region would be a way in which the information contained in a small RNA, which by itself might be too small to code for a complete chain, could be integrated into a larger RNA molecule. This kind of mechanism could explain many of the effects of 8-12 RNA’s on immune systems. It is well to remember that while a good deal of current-day interest centers on somatic rccombination of gcnrs and/or linkage of genes for the common and variable regions of the globulin molecule, such processes may reflect changes in the haw sequences of RNA molecules rather than changes in the genome. Fusion of RNA’s coding for different parts of the y-globulin molecule could account for the peculiar structure of antibody molecules.

456

A. ARTHUR GOTTLIEB

As for the phenomenon of allotype transfer, the RNA’s involved need only be large enough to specify the several amino acid substitutions involved. It is conceivable, if the number of substitutions is not large, that this could be accomplished by a few tRNA molecules that may conceivably exist in antibody-forming cells for the sole purpose of specifying allotypy (of course, they would be found in the 4 s region). Gyenes (816)has suggested a novel mechanism by which this, as well as some other patterns of constrained variation of amino-acid interchanges seen in y-globulin, could be explained. He suggests that the presence of inosine in the mRNA coding for y-globulin could lead to a special translational mechanism in which inosine-containing triplets would be decoded by different tRNA’s hypothesized to exist in lymphoid cells. Since inosine can pair with uridine and cytidine, different tRNA’s could be matched with an inosine-containing triplet leading to the inserU tion of different amino acids. Thus, Gyenes suggests that IUC could A A A be matched with tRNA’s containing the anticodons UAG, CAG or AAG

u

u

U

which correspond to the “conventional” codons AUC, GUC and UUC, which in turn code for isoleucine, valine and phenylalanine. In this way, U IUC could be translated as these amino acids. Thus, the single substitution, a t position 191 of the L chain, of leucine for valine in the human Inv+/Inv- allotypic system, could be explained by the presence of an IUG triplet a t the proper location in the mRNA of the light chain. I n addition, one must hypothesize that suppression of the inappropriate tRNA, corresponding to the amino acid that is not incorporated. in the particular L chain, takes place. Allotype transfer by RNA in the rabbit could be accounted for by such a mechanhm, and if such suppression were generalized and occurred in a unique fashion in every different clone, the tRNA’s of each clone could read the unique message in only one distinct way, which would be different for every clone. Gyenes states that direct support for this theory would be the demonstration of inosine in the specific mRNA molecules for antibody production. As I indicate, one might suggest that allotype transfer might be accomplished by a few unique tRNA molecules that may conceivably exist in antibodyforming cells for the purpose of specifying allotypy. The latter possibility is somewhat unlikely in view of the observation that the RNA’s active in allotype transfer sediment as 8-12s species. With regard to the ability of RNA’s to transfer delayed hypersensitivity and/or tumor immunity, there are several ways in which such RNA’s could operate in vivo.

LYMPHOID CELL RNA’S

AND IMMUNITY

457

1. This might be a “transfer factor” effect in the Lawrence sense, attributable to dialyzable “transfer factor.” This is probably ruled out on the basis of the size of the RNA’s involved and the fact that the RNA’s capable of transferring delayed hypersensitivity and/or tumor immunity are quite sensitive to pancreatic ribonuclease, whereas “transfer factor” is not. 2. This might be due to a (tumor) antigen-RNA or (tumor) antigenribonucleoprotein complex. Such a complex would not be expected to be so highly sensitive to ribonuclease. However, assuming that a complex of tumor antigen with RNA or RNA-protein were stable, one should be able to isolate such a complex by the techniques our laboratory has developed. 3. A nonspecific stimulatory effect of RNA in association with antigen carry-over. This would be similar to the phenomenon of macrophage “arming.” Such a mechanism cannot be excluded on the basis of currently available evidence. 4. A set of mechanisms involving replication of RNA: (a) leading to the production of messenger RNA’s or components of mRNA for specific antitumor antibodies, presumably of the cell-bound type; (b) as template for enzymes of the reverse transcriptase type leading to production of specific sets of DNA molecules complementary to RNA. Recent studies with the messenger RNA for globin, clearly indicate that if poly (A) sequences exist in the messenger RNA for globulin, short oligomers of d T could serve as efficient initiators for the synthesis of DNA complementary to the myeloma globulin genes; (c) as initiator for replication of single-stranded DNA. Brutlag et al. have suggested that short lengths of RNA serve as initiators for DNA in the M13 phage system (216). A similar type of phenomenon has been observed in our laboratory using the myeloma line MOPC-21. Perhaps “transfer factor” plays such a role at the molecular level.

VIII. A Hypothesis Regarding the Mechanism of Action of Antigen-Ribonucleoprotein Complexes One of the perplexing questions regarding the binding of antigenic fragments to ribonucleoprotein is the fact that such a fragment cannot retain the configuration of the native antigen. However, it is clear that excision of antigenic determinants occurs in the macrophage so that some, but by no means all, of these fragments are capable of recognizing antibody directed against the native antigen. Nevertheless, certain antigens lose certain determinants upon alteration of the native molecule, and such antigens no longer behave immunologically in the same

458

A. ARTHUR GOTTLIEB a

I.

Carrier

ASC (TorB)

/

-(3 Carrier

Tolerant B-cell

0

\a

-Tolerant T-cell

Carrier

I

/s“?;r\ 1

2d

Carrier derepression by R N P m

MacraDhaae

J

Carrier

I

a

I

Carrier

Antibody Mocraphaqe forming (odhcrent PE cell)

Memory cell for ontibody farmatian

Effector cell far D.H.

Memwy cell for D.H.

FIQ.1. A model for the ribonucleoprotein molecule in the immune response. I n this scheme, the first step in the immune response is the interaction of antigen with an antigen-sensitive cell (ASC) . Antigen (consisting of a hapten designated “a” and the balance of the molecule designated as carrier) can react with such a cell through either of these groups, depending on which receptor is available on the cell surface (i.e., whether the antigen-sensitive cell is a T (thymus-derived) or B (bone-marrow-derived) cell precursor). This initial interaction leads to the production of a state of “tolerance.” Immunity requires a second contact with antigen, and it is suggested that this occurs through presentation of fragments of antigen linked to a ribonucleoprotein (RNP) molecule. Carrier fragments in this form would go to “T” cells while haptenic determinants would be transmitted to “B” cells. Derepression of the selected clone then occurs, followed by proliferation. I n this process, nucleoprotein molecules bearing hapten or carrier determinants would be diluted out among the progeny lymphocytes. The cells retaining these molecules bearing haptenic or carrier determinants would serve as memory cells whereas those cells that lacked them (i.e., the majority of the cells) would be responsible for humorhl antibody production (if they were “B” cells) or for effecting delayed responses (if they were “T” cells). PE = peritoneal exudate, D.H. delayed hypersensitivity. From A. A. Gottlieb and R. H. Schwartz, Cell. Immunol. 5, 341 (1972).

-

LYMPHOID CELL RNA’S AND IMMUNITY

459

way as their native counterparts. It is therefore desirable to construct a model that will allow us to have native antigen and digest it as well. I n this model (Fig. l ) , antigen consisting of a hapten “a” and a carrier is seen to interact with an antigen-sensitive cell. This cell could be a “T” cell or “B” cell precursor, the distinction relating to the type of receptor that the cell bears on its surface. I n general, “T” cells are thought to have carrier-like receptors on their surfaces, while “B” cells are considered to have hapten receptors on their surfaces. Antigen will react either through its hapten with the “B” cell or through its carrier with the “T” cell. I n this model, this primary interaction leads to the production of a “tolerant” state. Immunity depends on a secondary contact with antigen, and we would suggest that this occurs through presentation of fragments of antigen linked to ribonucleoprotein. The introduction of carrier determinants attached to ribonucleoprotein into the “T” cell, or the introduction of haptenic determinants attached to it into the ‘iB”cell, leads to derepression of the appropriate cell, commitment to production of antibody (“B” cell) or delayed hypersensitivity (“T” cell) followed by proliferation of the committed clone of “T”or “B” cells. Cooperative interaction between “T” and “B” cells could also take place at this point. As a result of such proliferation, ribonucleoprotein molecules bearing hapten or carrier determinants would be diluted out among the progeny of the clone. The cells retaining ribonucleoprotein molecules bearing carrier or haptenic determinants would serve as memory cells. Those cells which lacked these molecules (i.e., the majority of the cells) would be responsible for the effector functions of the immune response either as producers of humoral antibody (“B” cells) or for effecting delayed responses (“T” cells). A principal virtue of this model is that it leaves antigen intact for selection of the clone and thus does not require that nonnative or altered antigen select the clone. Nevertheless, it is possible that such altered antigens or antigenic fragments might be capable of selecting the appropriate clone of antigenic-sensitive cells in certain cases. The model predicts that a critical role for fragments of antigen occurs after selection of the clone if immunity is to be produced.

IX. Conclusion Many observations indicate that RNA’s from immunized lymphoid systems display special properties with respect to the transfer of immune phenomena. I have attempted to summarize much of what is known concerning the nature of these RNA’s and the phenomena they control, induce or modify. Regretably, little is known concerning the mode of

460

A. ARTHUR GOTTLIEB

action of these various RNA species, whose effects are not easily explained in terms of conventional functions of RNA. This indicates that we may have underestimated the mechanisms by which RNA may serve in the differentiation of antibody-forming cells, and suggests that RNA’s may operate by as yet undetermined mechanisms in such systems, in addition to serving conventional roles as tRNA’s, mRNA’s or as components of the ribosome. It is likely that the future will yield a much clearer picture of many of the phenomena described herein, but if this is to be so I believe it will be necessary to maintain a receptivity for new ideas tempered by a healthy skepticism.

REFERENCES 1 . J. Sterzl and M. Hrubesova, Folk Biol. 2, 21 (1956). 3. D. H. Campbell and J. S.Garvey, J. Exp. Med. 105, 361 (1957). 3. D. H. Campbell and J. S. Garvey, Aduan. Immunol. 3, 261 (1963). 4 . H. Friedman, FP 18, 567 (1959). 6. M. Fishman, J . Exp. Med. 114, 837 (1961). 6. M. Fishman and F. Adler, J. Exp. Med. 117, 595 (1963). 7 . B. A. Askonas and J. M. Rhodes, Nature (London) 205, 470 (1965). 8. H. P. Friedman, A. B. Stavitsky and J. M. Solomon, Science 149, 1106 (1965). 9. J. A. Mannick and R. H. Egdahl, Science 137, 976 (1962). 10. J. Fong, D. Chin and S.S. Elberg, J. Exp. Med. 118, 371 (1963). 1 1 . D. E. Thor, Science 157, 1567 (1967). 12. H. G. Bluestein, L. Green and B. Benacerraf, PSEBM 135, 146 (1971). 13. P. Alexander, in “Progress in Experimental Tumor Research” (F. Hamburger,

ed.), pp. 22-71. Karger, Basel, 1968. 1.4. P. J. Deckers and Y. H. Pilch, Nature (London) New Biol. 231, 181 (1971). 16. T. N. Harris and S. Harris, J. Exp. Med. 90, 169 (1949). 16. R. W. Dutton, A. H. Dutton, M. George, R. Q . Marston and J. H. Vaughan, J . Immunol. 84, 268 (1960). 17. J. Mitchell and G. J. V. Nossal, Nature (London) 197, 1121 (1963). 18. J. Michell, Aust. J. Exp. Biol. Med. 42, 347 (1964). 19. J. C. Schooley, J. Zmmunol. 86, 331 (1961). 20. R. M. E. Parkhouse, Nature (London) 215,394 (1967). 51. B. Mach and P. Vassalli, PNAS 54, 975 (1965). 22. B. Mach and P. Vassalli, Science 150, 622 (1965). 23. R. B. Church, U. Storb, B. J. McCarthy and R. S. Weiser, J . Zmmunol. 101, 399 (1968). 24. E. P. Cohen, Nature (London) 213, 462 (1967). 26. L. Ortiz-Ortie and B. N. Jarosloy, Nature (London) 221, 1163 (1969). 26. U. Rife, Int. Arch. Allergy 36, 538 (1969). 27. B. Moav and T. N. Harris, J. Zmmunol. 104, 950 (1970). 28. H. A. Donahue, V. G. Moore and J. R. Little, J . Immunol. 105, 592 (1970). 29. A. Rubin and H. Cooper, PNAS 54, 469 (1965). SO. D. R. Forsdyke, Biochem. J. 105, 679 (1967). 31. B. G. T. Pogo, V. G. Allfrey and A. E. Mirsky, PNAS 55, 805 (1966). 38. A. D. Rubin, S. Davis and E. Schults, BBRC 46,2067 (1972).

461

LYMPHOID CELL RNA’S AND IMMUNITY

E. P. Cohen, J. Majer and K. Friedman, J . Exp. Med. 130, 1161 (1969). V . A. Lazda, J. L. Starr and M. Rachmeler, J. Immwtol. 101, 349 (1968). V . A. Lazda, J. L. Starr and M. Rachmeler, J . Immunol. 101, 359 (1968). M. H. Makman, B. Dvorkin and A. White, JBC 243, 1485 (1968). E. P. Cohen, PNAS 57, 673 (1967). K. Raska and E. P. Cohen, Nature (London) 221, 685 (1969). K. Raska and E. P. Cohen, Nature (London) 217, 720 (1968). E . P . Cohen and K. Raska, in “Nucleic Acids in Immunology” (0. J. Plescia and W. Braun, eds.), p. 573. Springer-Verlag, Berlin and New York, 1968. 41. J. R. Little and H . A. Donahue, PNAS 67, 1299 (1970). 48. A. Juhasz, B. Rose and M. B. Maude, Can. J . Biochem. Physiol. 41, 179 (1963). 43. L. J. Greenberg and J. Uhr, PNAS 58, 1878 (1967). 44. R. G. Kreuger and B. J. McCarthy, BBRC 41, 944 (1970). 46. A. A. Gottlieb and D. Tenney, J . Nut. Cancer Inst. 48, 1457 (1972). 46. W. 1,. Norton, D. Lewis and M. Ziff, PNAS 54, 851 (1965). 47. K. H. Stenzel, W. D. Phillips, D. D. Thompson and A. L. Rubin, PNAS 51, 33. 34. 36. 36. 3;. 38. 39. 40.

636 (1964). 48. M. D. Scharff and J. W. Uhr, Science 148,648 (1965). 49. M. T. Becker and A. Rich, Nature (London) 212, 142 (1966). 60. A. 1,. Shapiro, M. D. SchariT,

J. V. Maizel, Jr. and J. W. Uhr, PNAS 56, 216

(1966). 61. E . Kuechler and A. Rich, PNAS 63, 520 (1969). 62. E. Kuechler and A. Rich, Nature (London) 222, 544 (1969). 63. M. Becker, P. Ralph and A. Rich, BBA 199, 224 (1970). 64. P . Ralph and A. Rich, Biochemistry 10, 4717 (1971). 66. A. R. Williamson and B. A. Askonas, J M B 23,201 (1967). 66n. J. Stavnezer and R. C. C. Huang, Natuve (London) New BioZ. 230, 172 (1971).

56b. C. G. Brownlee, T. M. Harrison, M. B. Mathews and C. Milstein, FEBS Lett. 23, 244 (1972). 66c. D. Swan, H. Aviv and P. Leder, PNAS 69, 1967 (1972). 65d. H. Aviv and P. Leder, PNAS 69, 1408 (1972). 66. S. Nakashima, S. Ohi, K. Tsukada and H. Oura, BBA 145, 671 (1967). 67. C. J . Wust, ABB 118, 568 (1967). 68. A. M. C. Koros, L. H. Koster and M. J. Mowery, Nature (London) New Biol. 232, 239 (1971). 59. H. Stein and P. Hausen, Science 166, 393 (1969). 60. F. J. Penico, G. O’Cuinn and A. A. Gottlieb, in preparation. Gl. E. Reich and I. H. Goldberg, This series 3, 1837 (1964). 62. J. W. Uhr, Science 142, 1476 (1963). 63. C. J. Wust, C. L. Gall and G. D. Novelli, Science 143, 1041 (1964). 64. S. E. Svehag, Science 146, 659 (1964). 66. D. J. Smiley, J. G . Heard and M. Ziff, J . Exp. Med. 119, 881 (1964). 66. V. Lazda and J. L. Starr, J. Immunol. 95, 254 (1965). G7. G. Harris, J . Exp. Med. 127, 675 (1968). (is. H. C. Nathan, 6. Bieber, G. B. Elion and G. H. Hitchings, PSEBM 107, 796 (1961). 69. B. D. Geller and R. S. Spiers, PSEBM 117, 782 (1964). 70. B. D. Geller and R. S. Spiers, Immunology 15, 707 (1968). ? l . R . S . Spiers, Nature (London) 207, 371 (1965).

72. A. Stavitsky and J. P. Gusdon, Bacteriol.

Rev. 30,

418 (1966).

A. ARTHUR GOTTLIEB

73. H. Friedman, Ezperientiu 19, 537 (1963). 7.4. H. Friedman, Nature (London) 199, 502 (1963). 75. H. Friedman, Science 146, 934 (1964). 76. N. K. Jerne, A. A. Nordin and C. Henry, i ? ~“Cell-Bound Antibodies” (B. Amos and H. Koprowski, eds.), p. 109. Wistrtr Institute Press, Philadelphia, Pennsylvnnia, 1983. 77. H. Friedman, BBRC 17, 272 (1964). 7s. E. P. Cohen and J. J. Parks, Science 144, 1012 (1964). 79. E. P. Cohen, R. W. Newcomb and L. K. Crosby, J . Immzinol. 95, 583 (1965). SO. N. Hashem, Science 150, 1460 (1965). 81. E. P. Cohen, Science 152, 231 (1966). S2. E. P. Cohen and D. E. Mosier, Nature (London) 219, 969 (1968). 83. H. G. Johnson and A. G. Johnson, J . Ezp. Med. 133, 649 (1971). S4. K. Saito, I. Sato, T. Tnnaka and S. Mitsuhashi, Proc. Jap. Acnd. 39, 691 (1963). S5. I. Sat0 and S. Mitsuhashi, Proc. Jap. Acud. 39, 697 (1963). SO. I. Sato and S. Mitsuhashi, J. Bucl. 90, 1194 (1965).

ST. S. Mitsuhashi, S. Kurashige, M. Kawakami and T. Nojimn, Jop. J. Microbiol. 12, 261 (1968). SS. N. Osawa, S. Kurashige, M. Kawakami and S. Mitsuhashi, Jap. J . Microbiol. 12, 479 (1988). S9. 5. Mitsuhashi, Proc. Jap. Acud. 42, 280 (1966). 90. S. Kurashige, N. Osawa, M. Kawahnmi and S. Mitsuhashi, J. Bact. 94, 902 (1967). 91. K. Snito, S. Kurashige and S. Mitsuhnshi, Jup. J . Microbiol. 13, 122 (1969). 92. K. Saito, S. Kurashige, N. O~awa,E. Kato, M. Kawakami and S. Mitsuhashi, Jap. J . Microbiol. 13, 277 (1969). 93. H. Mikami, M. Kawakami and S. Mitsuhashi, Jap. J . Microbiol. 15, 169 (1971). 94.

M. Kawnknmi, K. Kitamura, H. Miknmi 2nd 8. Mitsuhnshi, Jap. J . Microbiol.

13, 247 (1969). 95. S. Kurashige, K. Kitamura, K. Akama and S. Mitsuhashi, Jap. J . Microbiol. 14, 41 (1970). 9G. L. Michelazai, G. Nanni, I. Baldini and A. Novelli, Ezpeiientiu 20, 447 (1964). 97. L. Michelaaai, I. Baldini, A. Novelli and G. Nanni, Nature (London) 205, 194 (1965). 9s. L. Michelaaai, A. Novelli, I. Baldini, U. M. Marinari and G. Nanni, Ezperientiu

21, 585 (1965). 99. L. Michelaaai, G. Nanni, I. Baldini and A. Novelli, Nature (London) 207, 1406 (1965). 100. S. Esposito and E. L. ChBrie-LigniBre, Ezpeiientiu 22, 603 (1966). 101. D. Jacherta, 2. Med. Mikrobiol. Immzinol. 152, 20 (1966). 102. D. Jachertz and H. Noltenius, Z . Med. Mikrobiol. Immunol. 152, 112 (1966). 103. D. Jacherta, 2. Med. Mikrobiol. Immunol. 152, 262 (1966). 104. D. Jacherta, 2.Med. Mikrobiol. Immunol. 153, 250 (1967). 106. D. Jachertz, 2. Med. Mikrobiol. Immimol. 154, 1 (1968). 100. D. Jacherta and J. Dreacher, J . Immunol. 104, 746 (1970). 107. D. Jacherta, Sonderduck aus Behringwerk Mitteilungen 49, 147 (1969). 10s. V. Neuhoff, W. B. Schell and D. Jachertz, Z . Physiol. Chem. 351, 167 (1970). 109. D. Jacherta, Ann. N.Y. Acad. Sci., in press.

110. 5. Mitsuhashi, Ann. N.Y. Acad. Sci., in prees. 111. P. R. Chin and M. S. Silverman, J . Immunol. 99, 489 (1967).

LYMPHOID CELL RNA’S AND IMMUNITY

463

112. P. Heller and V. Yakulis, Ann. N.Y. Acntl. Sci., in press. 113. C. B. Kimmel, BBA 182, 361 (1969). 114. J. A. Mannick and R. H. Egdnhl, Sciettce 137, 976 (1962). 116. B. D. Jankovic and H. F. Dvorak, J. Imniimol. 89, 571 (1962). 116. R. D. Guttmann, E. D. Kraiis and M. F. D o h , Nnfzire (London) 203, 196 (1964).

117. E. Sabbadini and A. Sehon, I t i t . Arch. Allergy Appl. Aficrobiol. 32, 55 (1967). 115. H. Bondevik and J. A. Mannick, PSEBM 129, 264 (1968). 119. K. P. Ramming and Y. Pilch, Trnnsplnnkttioii 7, 296 (1969). 120. D. Thor and S. Dray, J. Zmmunol. 101, 469 (1968). 121. R. E. Jureziz, D. E. Thor and S. Dray, J. Immunol. 101, 823 (1968). 122. R. E. Paque and S. Dray, J. Zmmiinol. 105, 1334 (1970). 123. J. Fong, D. Chin and S. S. Elberg, J. Exp. Meil. 120, 885 (1964). 124. R. M. Genighty, W. Rosenau and H. D. Moon, J. Zmmritiol. 97, 700 (1966). 136. M. R. Venneman and N. J. Bigley, J . Bncl. 100, 140 (1969). 1.26. R. A. Smith and N. J. Bigley, Infection Zmmuaity 6, 384 (1972). 137. A. S. Youmans and G. P. Youmnns, J. Bnct. 89, 1291 (1965). 125. A. S. Youmans and G. P. Youmrlns, J . Bmt. 91, 2146 (1966). 1.29. A. S. Youmans and G. P. Youmnns. J. Bnct. 91, 2139 (1966). 130. W. Braun and W’. Firshein, Bncteriol. lieu. 31, 83 (1967). 1Sf. A. S. Youmans and G. P. Youmnns, Zttfecf. Zmmzinity 3, 149 (1971). 133. P. Alexander, E. J. Delorme, L. D. Hamilton and J. G. Hall, in “Nucleic Acids J. Plescia and W. Brniin, cds.), p. 527. Springer-Verlag, in Immunology” (0. Berlin and New York, 1968. 133. P. Alexander, E. J. Delorme, L. D. Hamilton and J. G. Hall, Nntrire (London) 213, 569 (1967). 134. P. Alexander, Proc. Roy. SOC.60, 1181 (1967). 136. Z. B. Mikulska, C. Smith and P. .4lexnndrr, J. Nnt. Cnncer Znst. 36, 29 (1966). 130. C. T. C. Kennedy, D. B. Cater and F. Hnrtrcit. Actn Pnthol. Microbiol. S c a d 77, 196 (1969). 137. D. B. Cater and H. Waldmann, Brit. J. Cancer 21, 124 (1967). 13s’. M. V. Londner. J. C. Morini, M. T. Font and S. L. Rnbosa, Ezpeiientin 24, 598 (1968).

139. J. C. Morini, M. V. Londncr, M. T. Font and S. 1,. Rabasa, Ezperientia 25, 640 (1969).

140. Y. H. Pilch and K. P. Ramming, Cancer 26, 630 (1970). 141. K.P. Ramming and Y. H. Pilch, Science 168, 492 (1970). I&?. K. P. Ramming and Y. H. Pileh, Sio’g. Forum 21, 267 (1970). 143. K. P. Ramming nnd Y. H. Pilch, J. Nnt. Cancer Znst. 45, 543 (1970). 144. F. Valentine and H. S. Lawrence, Amer. J. Pothol. 60, 437 (1970). 146. H. S. Lawrence, AtEvan. Immzcnol. 11, 195 (1969). 146. P. Bnram, I,. Yuan and M. M. Mosko, J. Immrinol. 97, 407 (1966). 147. P. Bnram nnd W. Condoulis, J. Immnnol. 104, 769 (1970). 145. D. R. Burger, R. M. Vetto and A. Mallry, Science 175, 1473 (1972). 149. P. Fireman. M. Borsman, Z. H. Hnddad and D. Gitlin, Science 155, 338 (1967). 150. R. E. Paque, P. J. Kniskcrn, S. Drny and P. Bnram, J. Zntmrinol. 103, 1014 (1969).

161. R. Evans and P. Alexander, Nolure (London) 236, 168 (1972). 162. F. Adler, M. Fiehman and S. Dray, J . Zmmunol. 97, 554 (1966). 163. C. Bell nnd S. Dray, J . Zmmiinol. 103, 1196 (1969).

464

A. ARTHUR GOTTLIEB

164. C. Bell and S. Dray, J . Zmmunol. 105, 541 (1970). 166. C. Bell and S. Dray, J . Immunol. 107, 83 (1971). 160. W. H. Taliaferro and B. N. Jaroslow, J . Infect. D k . 107, 341 (1960). 167. B. N. Jaroslow, in “Nuclelc Acids in Immunology” (0.J. Plescia and W. Braun, eds.), p. 404. Springer-Verlag, Berlin and New York, 1988. 168. F. Gros, J. Dubert, A. Tissiires, S. Bourgeois, M. Michelson, R. Soffer and L. Legault, CSHSQB 28, 299 (1963). 169. W. Braun and M. Nakano, PSEBM 119, 701 (1965). 160. W. Braun and M. Nakano, Science 157, 819 (1967). lG1. W. Braun, M. Ishizuka, Y. Yajima, D. Webb and R. Winchurch, in “Biological Effects of Polynucleotides” (R. F. Beers and W. Braun, eds.), p. 139. SpringerVerlag, Berlin and New York, 1972. lG2. R. Winchurch and W. Braun, Nature (London) 223, 843 (1969). 163. E. Mows, G. M. Shearer, M. Sela and W. Braun, in “Biological Effects of Polynucleotides” (R. F. Beers and W. Braun, eds.), p. 197. Springer-Verlag, Berlin and New York, 1972. 164. H. G. Johnson and A. G. Johnson, J . Exp. Med. 133, 649 (1971). dG6. I(.Merritt and A. G. Johnson, J . Immunol. 94, 416 (1965). 166. J. R. Schmidtke and A. G. Johnson, J . Immunol. 106, 1191 (1971). lG7. E. Halac, U. Rife and L. Rinaldi, Nature (London) 202, 558 (1964). 168. D. Keast and G. D. Birnie, Exp. Cell Res. 58, 253 (1969). 169. K. U. Hartmann, 2.Naturforsch. B 24, 1317 (1969). 170. A. A. Gottlieb and S. R. Waldman, Crit. Rev. Microbiol. 1, 321 (1972). 171. R. S. Schwartz, D. Ryter and A. A. Gottlieb, Progr. Allergy 14, 81 (1970). 172. D. S. Nelson, “Macrophages and Immunity.” North-Holland Publ., Amsterdam, 1969.

173. B. B. Levine and B. Benacerraf, J . Exp. Med. 120, 955 (1964). 174. J. W. Uhr and G. Weissman, J . Immunol. 94,544 (1965). 176. P. C. Frei, B. Benacenaf and G. Thorbecke, PNAS 53, 20 (1965). 176. D. W. Dresser, Immunology 6, 345 (1965). 177. M. Sela, Science 166, 1365 (1969). 178. C. Lapresle, M. Kaminski and C. E. Tanner, J . Immunol. 82, 94 (1959). 179. A. C. Ada, P. G. Lang and G. Plymin, Immunology 14, 825 (1968). 180. A. A. Gottlieb, Science 165, 592 (1969). 181. I. Y. Uchitel, E. L. Khasman, L. G. Zaitseva and G. V. Shumakova, 2. Mikrobiol., Epidemiol., Immunobiol. 46, 17 (1969). 182. N. A. Mitchison, Immunology 16, 1 (1969). 183. J. K. Spitznagel and A. C. Allison, J . Zmmunol. 104, 119 (1970). 184. J. K. Spitznagel and A. C. Allison, J . Zmmunol. 104, 128 (1970). 186. J. S. Garvey and D. H. Campbell, Lab. Invest. 10, 1126 (1961). 186. J. S. Garvey and D. H. Campbell, J . Exp. Med. 125, 111 (1967). 187. D.S. Friend, W. Rosenau, J. S. Winfield and H. D. Moon, Lab. Invest. 20, 275 (1969).

188. A. A. Gottlieb, V. R. Glisin and P. Doty, PNAS 57, 1849 (1967). 188a. A. Saha, J. S. Garvey and D. H. Campbell, ABB 105, 179 (1964). 189. D. C. Bishop and A. A. Gottlieb, J. Immunol. 107, 269 (1971). 189a. A. A. Gottlieb, in “Nucleic Acids in Immunology” (0. Plescia and W. Braun, eds.), p. 471. Springer-Verlag, Berlin and New York, 1988. 190. A. A. Gottlieb and D. S. Straus, JBC 244, 3324 (1969).

LYMPHOID CELL RNA’S AND IMMUNITY

465

191. R. H. Schwartz, S. Leskowitz and A. A. Gottlieb, Immunochemistry 9, 601 (1972).

193. A. A. Gottlieb, R. H. Schwartz and S. R. Waldman, J. Immunol. 108, 719 (1972). 193. A. A. Gottlieb, R. H. Schwartz, S. A. Kudva and S. R. Waldman, Ann. N.Y. Acad. Sci., in press. 194. M. Fishman and F. L. Adler, CSHSQB 32, 343 (1967). 19.4~.W. S. Walker, Nature (London) New Bwl. 229, 211 (1971). 196. S. G. Rice,FP 31, 3177 (1972). 196. G. E. Roelants and J. W. Goodman, Bchem. 3, 1432 (1968). 197. G. E. Roelants and J. W. Goodman, J. E q . Med. 130, 557 (1969). 198. L.Yuan and D. H. Campbell, Immunochemistry 8, 185 (1971). 199. L. Yuan and D. H. Campbell, Immunochemitry 9, 1 (1972). 200. J. S. Garvey, H. Rinderknecht, B. G. Weliky and D. H. Campbell, Immunochemistry 9, 187 (1972). 201. J. D. Padayatty, M. D. Hensley and H. Van Kley, BBA 161, 51 (1968). 202. P. Abramoff and N. B. Brien, J. Immunol. 100, 1210 (1970). 203. D. C. Bishop, Ph.D. Thesis, Marquette Univ. (1968). 204. D. S. Juras and P. Abramoff, J. Immunol. 105, 1244 (1970). 206. H.B. Herscowitz and P. Stelos, J . Immunol. 105, 771 (1970). 306. H. B. Herscowitz and P. Stelos, J. Immunol. 105, 779 (1970). 207. J. A. Michael and C. J. Kuwatch, Nature (London) 222, 684 (1969). 208. S. Harshman, L. J. Duke and H. Six, Immunochemistry 6, 175 (1969). 209. L. J. Duke and S. Harshman, Zmmunochemistry 8,431 (1971). 210. L. J. Duke, C . Miller and S. Harshman, Nature (London) New Biol. 235, 180 (1972).

211. M. D. Schoenberg, V. R. Miuvaw, R. D. Moore and A. S. Weisberger, Science 143, 964 (1964).

212. H.R. P. Miller and S. Avrameas, Nature (London) New Biol. 229, 1% (1971). 213. M. Fishman, R. A. Hammerstrom and V. P. Bond, Nature (London) 198, 549 (1963).

214, C. Bona, R. Robineaux, A. Antennis and G. Chauvet, C . R . Acad. Sci. 269, 1359 (1969).

816. L. Gyenes, Rev. Can. Biol. 28, 179 (1969). 216. D. Brutlag, R. Schekman and A. Kornberg, PNAS 88, 2826 (1971).

This Page Intentionally Left Blank

SUBJECT INDEX A Actinomycin - deoxyguanosine crystalline complex, actinomycin symmetry, 158-159 symmetry of, 159-163 water structure, 163-165 Actinomycin .deoxyribonucleic acid complex, intercalation and hydrogen-bonding,

G Genetic apparatus, formaldehyde and, 38-40

Genetic recombination, model for, 1821 185

I Immune responses, nonspecific stimulators, 4-42

165-167

1

medical implications, 187-188 solution studies, deoxyguanosine model reaction, 157-

Lysogenic induction, analogies in mammalian systems, 28%

158

292

hydrogen-bonded

recognition, 155-

156

intercalation and, 156-157 polynucleotide conformation and, 155 stereochemical model, 16%171 supporting evidence, 171-175 Aldehydes, nucleic acids and, 40-42 Alkylating agents, nucleic acids and, 4244

Antigen-ribonucleoprotein complexes, mechanism of action, 457-459 B Bases, formaldehyde and, 3-15

direct methods, fluoropyrimidines, 253-254 mitomycin C, 253 other inducers, 254 thermal induction, 254 thymine starvation, 253 ultraviolet irradiation, 252-253 historical aspeck, 250-252 indirect methods, other modes, 282 ultraviolet cross-induction, 265-282 mechanisms, 282-289 phage functions, early, 254-280

M D Deoxyribonucleic acid, formaldehyde and, 19-20 intercalating agents other than actinomycin, 175-178 replication, chromosome structure and, 185-187

F Formaldehyde, interactions, bases, nucleosides and nucleotides,

Mating process, gene transfer and, 223-230 newly infected recipients and, 230-231 pilus bridge and, 221-223 transfer process and, 231-233 Missing triplet hypothesis, nucleic acid recognition and, approach and, 317-318 diversity in binding patterns, 321322 evolutionary implications, 324327 further comments on dissymmetry, 320321

hidden

3-15

nucleoprokins, 30-35 polynucleotides, 15-20 related reactions, 4044 uses of, 35

dissymmetries

in

coding,

318320

limitations of, 323-324 sources of ambiguity, 322-323 why anticodons, not codons?, 323 467

468

SUBJECT INDEX

N Nuclease(s), specificity, 17b180 Nucleic acids, experimental evidence for recognition in, anticodons in third column, 332-333 consequences of

1:3 ,]:I

330

consequences of 328329 fourth base in the anticodon, 332 initiator and noninitiator transfer ribonucleic acids, 331-332 rigid approximation and, 331 suppression and, 327-328 protein recognition, general principles, 178-187 recognition in, anticodon loop structure, 310-312 oligonucleotide associations in water, 305-307 replication and, 308 thermochemistry and, 303-305 transfer ribonucleic acid secondary structure and, 308-310 structure and function, formaldehyde and, 20-30, 35-36 Nucleoprotein(s), formaldehyde and, 3035 Nucleosides, formaldehyde and, 3-15 Nucleotides, formaldehyde and, 3-15 misincorporation, transfer ribonucleic acid, 76-78 Nucleo tidyltransferase, transfer ribonucleic acid, catalytic properties, 71-76 purified, 62-87

0 Operators, symmetry, 180-183

P Peptide bond, formation of, 82-86 Poly (adenosinediphosphate ribose), biodegradation, enzymatic cleavage of ribose-ribose bond, 144-145 rat liver phosphodiesterase, 143-144 snake venom phosphodiesteraoe, 142143

biological significance, 146-147 biosynthesis, chromatin and, 135c137 general character, 134-135 inhibitors, 138-139 nuclear proteins and, 137-138 purification of enzyme, 141-142 relation to nicotinamide adenine dinucleotidase, 139-141 chemical and physical properties, 129132 future problems, 148-149 natural occurrence, 145-146 purification, incubation of precursor with enzyme. 132 weparation of, 132-134 related phenomena, 147-148 Polynucleotides, synthetic, formaldehyde and, 15-16 Prophage induction, 260-261 derepression and, 261-263 excision and cell death, 263-265 Protein(s), formaldehyde and, 30-33 nucleic acid recognition, general principles, 178-187

R Repressors, symmetry, 180-183 Resistance determinants, aminoglycoside antibiotics, 216-218 chloramphenicol, 215-216 other factor-mediated, 220 penicillin and cephalosporin, 213-215 sulfonamide, 219-220 tetracycline, 219 Resistance factors, 193 “curing” and, 206-208 deoxyribonucleic acid transfer during mating, 212-213 gene products needed for conjugation, 209-212 maintenance, practical aspects, 208 plasmids and, maintenance and segregation, 203-206 others related, 220-221 replication, 195-203 resistance transfer factor and, 194-195 transfer in nature,

SUBJECT INDEX

469

early observations, 233-235 T Pseudomonas aeruginosa in burns, Transfer ribonucleic acid, 235-238 aminoacyl, formation of, 78-82 resistance transfer and, 238-243 -C-C-A terminus, Ribonucleic acid, control functions, 86-88 biosynthesis in immunized systems, enzymatic synthesis in vitro, 60-78 actinomycin D and, 4 2 W 5 location in three dimensional strucexposure to antigen and, 412416 ture, 5247 messenger isolation, 422 role in function, 78-86 unique species and, 416-422 synthesis and turnover in vivo, 57formaldehyde and, 16-19 60 immune phenomena transfer by, recognition of, 67-71 allotypic specificity, 439-440 Tryptophan operon, cell-mediated responses, 434-439 historical background, 340-341 specific antibody synthesis, 425434 nonsense mutants, 377-378 immunity and, transcription in, 381-398 historical perspective, 410-412 translation in, 378-381 mechanism of action, 454-457 transcription, of messenger, macrophage, amino acid starvation and, 374-376 antigen interactions, 445-454 block in, 398-402 immune responses and, 443-445 characterization, 352-354 peritoneal cell populations and, 442degradation, 367-369 443 detection, 348-352 Ribonucleic acid polymerase, initiation of, 354-366 localization, 103 rate of, 36-67 mitochondrial, 117-119 repression of, 369-374 multiple forms, separation of, 101 synthesis in vitro, 376-377 nomenclature, 101-103 translation, properties, block in, 3-02 a-amanitin effects, mode of regulation of enzyme synin vitro, 107-108 thesis, 341-344 in v i m , 108-110 quantitative aspects, 347-348 factor requirements, 111-112 sequential initiation, 344-346 metal ions and ionic strength, 106simultaneous initiation, 346-347 107 stability, 106 V template specificity, 110-111 Viruses, formaldehyde and, 36-38 quantitative extraction, recovery of enzyme, 101 W solubilization methods, 95-101 Wobble hypothesis, regulation of, 114-117 approach and, 312-314 relative concentration and specific conclusions, 315-316 activity, 103-108 evolutionary implications, 316-317 subunit structure and molecular weight, limitations, 316 112-114 subsidiary hypothesis, 314-315

Contents of Previous Volumes Volume 1 "Primer" in DNA Polymerase Reactions

F. J. BOLLUM The Biosynthesis of Ribonucleic Acid in Animal Systems

R. M. S. SMELLIE The Role of DNA in RNA Synthesis

JERARD HURWITZ AND J. T. AUGUST

Polynucleotide Phosphorylase

M. GRUNBERG-MANAGO Messenger Ribonucleic Acid

FRITZLIPMANN The Recent Excitement in the Coding Problem

F. H. C. CRICK Some Thoughts on the Double-Stranded Model of Deoxyribonucleic Acid

AARON BENDICH AND HERBERT S. ROSENKRANZ Denaturation and Renaturatian of Deoxyribonucleic Acid

J. MARMUR, R. ROWND, AND C. L. SCHILDKRAUT Some Problems Concerning the Macromolecular Structure of Ribonucleic Acids

A. 5. SPIRIN The Structure of DNA as Determined by X-Ray Scattering Techniques

VITTORIO LUZZATI Molecular Mechanisms of Radiation Effects

A. WACKER INDEX AUTHOR INDEX-SUBJECT Volume 2 Nucleic Acids and Information Transfer

LIEBEF. CAVALIERI AND BARBARA H. ROSENBERG Nuclear Ribonucleic Acid

HENRYHARRIS 470

CONTENTS OF PREVIOUS VOLUMES

Plant Virus Nucleic Acids

ROYMARKHAM

The Nucleases of Escherichia coli

I. R. LEHMAN Specificity of Chemical Mutagenesis

DAVID R. KRIEG Column Chromatography of Oligonucleotides and Polynucleotides

MATTHYS STAEHELIN Mechanism of Action and Application of Azapyrimidines

J. SKODA The Function of the Pyrimidine Base in the Ribonuclease Reaction

HERBERT WITZEL Preparation, Fractionation, and Properties of sRNA

G. L. BROWN AUTHORINDEX-SUBJECT INDEX

Volume 3 Isolation and Fractionation of Nucleic Acids

K. S. KIRBY Cellular Sites of RNA Synthesis

DAVID M. PRESCOTT Ribonucleases in Taka-Diastase: Properties, Chemical Nature, and Applications

FUJIO EGAMI, KENJITAKAHASHI, AND TSUNEKO UCHIDA Chemical Effects of Ionizing Radiations on Nucleic Acids and Related Compounds

JOSEPHJ. WEISS The Regulation of RNA Synthesis in Bacteria

C. NEIDHARM’ FREDERICK Actinomycin and Nucleic Acid Function

E. REICHAND I. H. GOLDBERG De Novo Protein Synthesis in Vitro

B. NISMAN AND J. PELMONT

471

472

CONTENTS OF PREVIOUS VOLUMES

Free Nucleotides in Animal Tissues

P. MANDEL

AUTHOR INDEX-SUBJECT INDEX Volume 4 Fluorinated Pyrimidines

CHARLES HEIDELBERGER Genetic Recombination in Bacteriophage

E. VOLICIN DNA Polymerases from Mammalian Cells

H. M. KEnt The Evolution of Base Sequences in Polynucleotides

B. J. MCCARTHY Biosynthesis of Ribosomes in Bacterial Cells

SYOZO OSAWA 5-Hydroxymethylpyrimidines and Their Derivatives

T. L. V. ULBRICHT Amino Acid Esters of RNA, Nucleosides, and Related Compounds

H. G . ZACHAUAND H. FELDMANN Uptake of DNA by living Cells

L. LEDOUX AUTHOR I N D E X ~ U B J EINDEX CT Volume 5 Introduction to the Biochemistry of D-Arabinosyl Nucleosides

SEYMOUR S. COHEN Effects of Some Chemical Mutagens and Carcinogens on Nucleic Acids

P. D. LAWLEY Nucleic Acids in Chloroplasts and Metabolic DNA TATSUICHI

IWAMURA

Enzymatic Alteration of Macromolecular Structure

P. R. SRINIVASAN AND ERNEST BORER Hormones and the Synthesis and Utilization of Ribonucleic Acids

J. R. TATA

CONTENTS O F PREVIOUS VOLUMES

473

Nucleoside Antibiotics

JACK J. Fox, KYOICHI A. WATANABE, AND ALEXANDER BLOCH Recombination of DNA Molecules

CHARLESA. THOMAS, JR. Appendix 1. Recombination of a Pool of DNA Fragments with Complementary Single-Chain Ends

G. S. WATSON, W. K. SMITH, AND CHARLES A. THOMAS, JR. Appendix II. Proof That Sequences of A, C, G, and T Can Be Assembled to Produce Chains of Ultimate Length, Avoiding Repetitions Everywhere

A. S. FRAENKEL AND J. GILLIS The Chemistry of Pseudouridine

ROBERT WARNER CHAMBERS The Biochemistry of Pseudouridine

EUGENE GOLDWASSER AND ROBERT L. HEINRIKSON AUTHORINDEXSUBJECT INDEX

Volume 6 Nucleic Acids and Mutability

STEPHEN ZAMENHOF Specificity in the Structure of Transfer RNA

KIN-ICHIRO MIURA Synthetic Polynucleotides

A. M. MICHELSON, J. MASSOULII~, AND W. GUSCHLBAUER The DNA of Chloroplasts, Mitochondria, and Centrioles

S. GRANICK AND AHARONGIBOR Behavior, Neural Function, and RNA

H. HYDEN The Nucleolus and the Synthesis of Ribosomes

ROBERT P. PERRY The Nature and Biosynthesis of Nuclear Ribonucleic Acids

G. P. GEORGIEV Replication of Phage RNA

CHARLES WEISSMANN AND SEVERO OCHOA AUTHORINDEXSUBJECT INDEX

474

CONTENTS OF PREVIOUS VOLUMES

Volume 7 Autoradiographic Studies on DNA Replication in Normal and leukemic Human Chromosomes

FELICE GAVOSTO Proteins of the Cell Nucleus

LUBOMIR S. HNILICA The Present Status of the Genetic Code

CARLR. WOESE The Search for the Messenger RNA of Hemoglobin

A. BURNY, AND G. MARBAIX H. CHANTRENNE, Ribonucleic Acids and Information Transfer in Animal Cells

A. A. HADJIOLOV Transfer ofGenetic Information during Embryogenesis

MARTINNEMER

Enzymatic Reduction of Ribonucleotides

AGNELARSSON AND PETER REICHARD The Mutagenic Action of Hydroxylamine

J. H. PHILLIPS AND D. M. BROWN

Mammalian Nucleolytic Enzymes and Their Localization

DAVID SHUGAR AND HALINA SIERAKOWSKA AUTHORI N D E X ~ U BINDEX JECT Volume 8 Nucleic Acids-The

First Hundred Years

J. N. DAVIDSON Nucleic Acids and Protamine in Salmon Testes

GORDON H. DIXONAND MICHAEL SMITH Experimental Approaches to the Determination of the Nucleotide Sequences of large Oligonucleotides and Small Nucleic Acids

ROBERT W. HOLLEY Alterations of DNA Base Composition in Bacteria

G. F. GAUSE Chemistry of Guanine and Its Biologically Significant Derivatives

ROBERT SHAPIRO Bacteriophage 4x1 74 and Related Viruses ROBERT L. SINSHEIMEB

CONTENTS OF PREVIOUS VOLUMES

The Preparation and Characterization of large Oligonucleotides

GEORGE W. RUSHIZKY AND HERBEW A. SOBER Purine N-Oxides and Cancer

GEORGE BOSWORTH BROWN The Photochemistry, Photobiology, and Repair of Polynucleotides

R. B. SETLOW What Really Is DNA? Remarks on the Changing Aspects of a Scientific Concept

ERWIN CHARGAFF Recent Nucleic Acid Research in China

TIEN-HSICHENGAND ROYH. Do1

AUTHORI N D E X ~ U B JINDEX ECT

Volume 9 The Role of Conformation in Chemical Mutagenesis

B. SINGER AND H. FRAENKEL-CONRAT Polarographic Techniques in Nucleic Acid Research

E. PALEEEK RNA Polymerase and the Control of RNA Synthesis

JOHNP. RICHARDSON Radiation-Induced Alterations in the Structure of Deoxyribonucleic Acid and Their Biological Consequences

D. T. KANAZIR Optical Rotatory Dispersion and Circular Dichroism of Nucleic Acids

JENTSIYANGAND TATSWASAMEJIMA The Specificity of Molecular Hybridization in Relation to Studies on Higher Organisms

P. M. B . WALKER Quantum-Mechanical Investigations of the Electronic Structure of Nucleic Acids and Their Constituents

BERNARD PULLMAN AND ALBERTE PULLMAN The Chemical Modification of Nucleic Acids

N. K. KOCHETKOV AND E. I. BUD~WSKY AUTHORINDEX-SUBJECTINDEX

475

476

CONTENTS OF PREVIOUS VOLUMES

Volume 10 Induced Activation of Amino Acid Activating Enzymes by Amino Acids and tRNA

ALANH. MEHLER Transfer RNA and Cell Differentiation

NOBORU SUEOKA AND TAMIKO KANO-SUEOKA NO- ( A2-lsopentenyl)adenosine: Chemical Reactions, Biosynthesis, Metabolism, and Significance to the Structure and Function of tRNA

Ross H. HALL Nucleotide Biosynthesis from Preformed Purines in Mammalian Cells: Regulatory Mechanisms and Biological Significance

A. W. MURRAY, DAPHNE C. ELLIOTT, AND M. R. ATKINSON

Ribosome Specificity of Protein Synthesis in Vifro

ORIOCIFERRI AND BRUNO PARISI Synthetic Nucleotide-peptides

ZOE A. SHABAROVA The Crystal Structures of Purines, Pyrimidines and Their Intermolecular Complexes

DONALD VOETAND ALEXANDER RICH AUTHORINDEX-SUBJECTINDEX

Volume 11 The Induction of Interferon by Natural and Synthetic Polynucleotides

CLARENCE COLBY, JR. Ribonucleic Acid Maturation in Animal Cells

R. H. BURDON Liporibonucleoprotein as an Integral Part of Animal Cell Membranes

V. S. SHAPOT AND S. YA.DAVIDOVA Uptake of Nonviral Nucleic Acids by Mammalian Cells

PUSHPA M. BHARGAVA AND G. SHANMUGAM The Relaxed Control Phenomenon

ANN M. RYANAND ERNEST BORER Molecular Aspects of Genetic Recombination

CEIDRIC I. DAVERN

CONTENTS O F PREVIOUS VOLUMES

477

Principles and Practices of Nucleic Acid Hybridization

DAVIDE. KENNELL Recent Studies Concerning the Coding Mechanism

THOMAS H. JUKES AND LILAGATLIN The Ribosomal RNA Cistrons

M. L. BIRNSTIEL, M. CHIPCHASE, AND J. SPEIRS Three-Dimensional Structure of tRNA

FRIEDRICH CRAMER Current Thoughts on the Replication of DNA

ANDREW BECKER AND JERARD HURWITZ Reaction of Aminoacyl-tRNA Synthetases with Heterologous tRNA's

K. BRUCEJACOBSON On the Recognition of tRNA by Its Aminoacyl-tRNA Ligase

ROBERT W. CHAMBERS

AUTHOR INDEX-SUBJECTINDEX Volume 12 Ultraviolet Photochemistry as a Probe of Polyribonucleotide Conformation

A. J. LOMANT AND JACQUESR. FRESCO Some Recent Developments in DNA Enzymology

MEHRAN GOULIAN Minor Components in Transfer RNA: Their Characterization, Location, and Function

SUSUMU NISHIMURA The Mechanism of Aminoacylation of Transfer RNA

ROBERT B. LOFTFIELD Regulation of RNA Synthesis

EKKEHARD K. F. BAUTZ The Poly(dA-dT) of Crab

M. LASKOWSKI, SR. The Chemical Synthesis and the Biochemical Properties Peptidyl-tRNA

YEHUDA LAPIDOTAND NATHAN DE GROOT SUBJECTINDEX

This Page Intentionally Left Blank