PROGRESS IN
Nucleic Acid Research and Molecular Biology Volume 7
Contributors to Volume 7 D. M. BROWN A. BURNY H. CHANTRENNE FELICE GAVOSTO A. A. HADJIOLOV LUBOMIR S. HNlllCA AGNE LARSSON G. MARBAIX MARTIN NEMER J. H. PHILLIPS PETER REICHARD DAVID SHUGAR HALINA SIERAKOWSKA CARL R. WOESE
PROGRESS IN
NucIeic Acid Research and Molecular Biology edited by
J. N. DAVIDSON
WALDO E. COHN
Department of Biochemistry T h e University of Glasgow Glasgow, Scotland
Biology Division Oak Ridge National Laboratory Oak Ridge, Tennessee
Volume 7 7967
ACADEMIC PRESS New York and London
COPYRIGHT @ 1967,
BY AC.4DEMIC PRESS
INC.
ALL RIGHTS RESERVED. NO PART OF THIS BOOK MAY BE REPRODUCED I N ANY FORM, BY PHOTOSTAT, MICROFILM, OR ANY OTHER MEANS, WITHOUT WRITTEN PERMISSION FROM THE PUBLISBERS.
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List of Contributors Numbers in parentheses refer to the pages on which thc authors' contrihutions brgin.
D. M. BROWN(349), University Chemical LaboratoTy, Cambridge, England A. BURNY (173), Laboratory of Biological Chemistry, Faculty of Sciences, University of Brussels, Brussels, Belgium H. CHANTRENNE (173), Laboratory of Biological Chemistry, Faculty of Sciences, University of Brussels, Brussels, Belgium FELICEGAVOSTO (1), Hematology Laboratory, General Medical Clinic, University of Turin, Turin, Italy A. A. HADJIOLOV (195), Biochemical Research Laboratory, Bulgarian Academy of Sciences, Sofia, Bulgaria LUBOMIR S . HNILICA(25), Department of Biochemistry, T h e University of Texas, M . D . Anderson Hospital and Tumor Institute, Houston, Texas AGNELARSSON (303), Department of Chemistry I I , Karolinska Institutet, Stockholm, Sweden G. MARBAIX(173) Laboratory of Biological Chemistry, Faculty of Sciences, Cniversit y of Brussels, Brussels, Belgium MARTINNEMER(243), Division of Biochemistry, T h e Institute for Cancer Research, Fox Chase, Philadelphia, Pennsylvania J. H. PHIL LIPS^ (349) C'nivcrsity Chemical Laboratory, Cambridge, England PETERREICHARD (303) Department of Chemistry I I , Karolinska Institutet, Stockholm, Sweden DAVIDSHUGAR (369) Institute of Biochemistry and Biophysics, Acade m y of Sciences, and Department of Biophysics, University of Warsaw, Warsaw, Poland HALIKA SIERAKOWSKA (369) Institute of Biochemistry and Biophysics, Academy of Sciences, Warsaw, Poland CARLR. WOESE(107) Department of Microbiology, University of Ilknois, Urbana, Illinois
' Present address: Department of Riockrmistry, Makcrere IJni\-rrsity Collrge, Kampala, Uganda. \-
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Preface In introducing Volume 7 of Progress in Nucleic Acid Research and Molecular Biology we should once more point out that i t is not our intention to sponsor an annual or fixed-date publication in which literature appearing in a given period of time is summarized, as in the more customary type of bibliographic review or literature survey. As we have cmphasized on several previous occasions, our aim is to present “essays in circumscribed areas” in which recent developments in particular aspects of the field of nucleic acids and niolccular biology are discussed by workers provided with an opportunity for more personal expression than is normally met in review articles. T o this end i t is our policy to encourage discussion, argument, and speculation, and the expression of points of view that are individualistic and perhaps even controversial. It is, of course, to be expected that different authors will interpret this charge in different ways, some essaying a broad and philosophical vein, some developing or describing new theories or techniques, some taking the opportunity to assemble a number of fragmentary observations into a coherent pattern, and some reviewing a field in a more conventional manncr. We have not attempted t o 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 and 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 will welcome further suggestions from readers as to how this end may best be served. Indeed, we should likc to encourage readers to write to us with their comments. Abbreviations used for nucleic acids and their derivatives are now fairly well established by international authority. Those pertinent to our subject are not listed a t the beginning of each chapter, but will be found on the following page. J.N.D. W.E.C. August, 1967
vii
Abbreviations and Symbols Abbreviations used without definition are those recommended by the IUPAC-IUB Combined Commission on Biochemical Nomenclature, as printed in the J. Biol. Chem. 241, 527 (1966), Biochim. Biophys. Actu 108, 1 (1965), Biochemistry 5, 1445 (1966), Arch. Biochem. Biophys. 115, 1 (1966), Virology 29, 480 (1966), Biochem. J . 101, 1 (1966), European J. Biochem. 1, No. 3 (1967), and 2. Physiol. Chem. 348,245 (1967).
pu, PY AMP, CMP, GMP, IMP, UMP, q M P , TMP, XMP, etc. dAMP, etc. 2’-gMP, 3’-AMP, (5’-AMP), etc.
ribonucleoside residues in polymers (specific) ribonucleoside residues in polymers (general) purine, pyrimidine ribonucleoside (general) 5’-monophosphates of the above nucleosides
5’-monophosphate IJf 2’-deoxyribosyl adenine, etc. 2‘-, 3’-, (and 5’-, where nee-led for contrast) phosphate of adenosine, etc. 5’-(pyro)diphosphate of adenosine, etc. ADP, etc. 5‘-(pyro)triphosphate of adenlsine, etc. ATP, etc. inorganic orthophosphate and pyrophosphate Pi, PPi 3‘ + 5’ polymer of ribonucleotide N poly N, or (Wn,or (rN),, 3‘ + 5’ polymer of deoxyribonucleotide N poly dN, or (dN). 3’ + 5’ copolymer of N-N’-N-N’- in regular, poly (N-N’), or r(N-N’)” alteriiating, knorun sequence or (rN-rN’)n 3‘ --t 5’ copolymer of dN-dN’-dN-dN’- in poly d(N-N’), or d(N-N’),, regular, alternat,ing, known sequence or (dN-dN’), 3’ + 5’ copolyiner of N and N’ in random poly (N, N’) or (N, N ),, sequence two chains, geiierally or completely associated p o b (A) poly (B) or (A) . (B) two chains, asiociation unspecified or unknown P O ~ Y(A),poly (B) or (A),(B) P O ~ Y(A) POIY (B) or (A) (B) two chains, generally or completely urmssociated ribonucleic acid or ribonucleite RNA deoxyribonucleic acid or deoxyribmucleate DNA messenger RN.4; ribosomal RNA mRNA; rKNA nuclear RNA nItNA transfer ItNA (RNA that accepts and transfers tRNA amirio acids; amino acid-accepting RNA) “Charged” t l t N A (tltNA carrying amirioacyl Aminoacyl-tRN A residues) the transfer R S A molecule that normally Alanine tRNA or accepts alauine, e k . tRNAAl”,etc. the stme, with alanyl residue covalently linked Alanyl-tRNAA1a or Ala- t R N A iil)oiiuc*le:tse,deoxyril)oriurlease RNase, DNase
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I n naming cnzymes, the recommendations of the Commission on Enzymes of the International Union of Biochemistry (1965) are followed as far as possible.
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LIST OF CONTRIBUTORS .
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Autoradiographic Studies on DNA Replication in Normal and Leukemic Human Chromosomes
FELICE GAVOSTO I . Introduction . . . . . . . . . . . . . I1. Recognition of Chromosomes . . . . . . . . . I11. Autoradiographic Techniques . . . . . . . . . IV. The Chromosomal DNA of Normal Blood Cells . . . . . V . The Chromosomal DNA of Leukemic Cells . . . . . . VI . Conclusions : Some Tentative Hypotheses and Future Apprcnclips to the Problem . . . . . . . . . . . . . References . . . . . . . . . . . . . .
Proteins of the Cell Nucleus
LUBOMIRS. HNILICA
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I. Introduction . . . . . I1. Classification of Nuclear Proteins I11. Protamines . . . . . . IV. Histones . . . . . . V . Nonhistone Proteins . . . . VI . Conclusions and Sunimary . . References . . . . . .
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The Present Status of the Genetic Code
CARLR . WOESE I. Introduction . . . . . . I1. Historical . . . . . . . I11. The Cryptographic Problem . . . I V . Colinearity of Gene and Polypeptide . V . Codon Size . . . . . . .
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107 109 117 126 128
CONTENTS
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VI . Punctuation and Other Encoded Instructions . . . . VII . The Translation Tape-Reader and the Translation Process VIII . The Fundamental Nature of the Genetic Code . . . References . . . . . . . . . . . .
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130 138 155 167
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173 174 175 179 192 192
The Search for the Messenger RNA of Hemoglobin
H . CHANTRENNE. A . BURNY.AND G . MARBAIX I . Introduction
I1. Location of the Information for Hemoglobin in Reticulocyte Extracts
Ribonucleic Acids and Information Transfer in Animal Cells
A . A . HADJJOLOV I. General Considerations . . . . . . . I1. Remarks on the Methods of mRNA Identification 111. The Sequential Synthesis of RNA . . . . IV . Nuclei-the Site of mRNA Synthesis . . . . V . Cytoplasm-the Site of mRNA Expression . . VI . Synopsis . . . . . . . . . . References . . . . . . . . . .
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196 198 201 205 220 235 237
Transfer of Genetic Information During Embryogenesis
MARTINNEMER I . Introduction
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I1. The Egg as an Informational Structure .
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111. Schedules for the Activation of Protein Synthesis in Eggs IV . Activation of Protein Synthesis in the Sea Urchin Egg V. Transitions in Genic Activity . . . . . . VI . Developmental Regulation of Genetic Expression . . VII . Summary . . . . . . . . . . . References . . . . . . . . . . .
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243 245 255 259 266 278 290 293
Enzymatic Reduction of Ribonucleotides
AGNELARSSON AND PETERREICHARD I. Introduction
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11. Ribonucleotide Reduction in Escherichia coli
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CONTENTS
I11. Ribonucleotide Reduction in Lactobacillus Zeichmannii IV . Ribonucleotide Reduction in Animal Cells . . . V . Concluding Remarks . . . . . . . . References . . . . . . . . . . .
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324 338 342 345
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The Mutagenic Action of Hydroxylamine
J . H . PHILLIPS AND D . M . BROWN I . Introduction . . . . . . . . . . . I1. Genetic Background . . . . . . . . . . I11. General Chemistry of Hydroxylamine Action . . . . IV . Experimental Investigation of Hydroxylaminc Mutagenesis V. Conclusions . . . . . . . . . . . References . . . . . . . . . . . .
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349 350 353 358 364 366
Mammalian Nucleolytic Enzymes and Their Localization
DAVID SHUGAR AND HALINA SIERAICOWSKA I. Introduction . . . . . . . . . .
I1. Types of Nucleolytic Enzpmcs . . . I11. Methods of Assay . . . . . . IV . Substrate Preparations . . . . . V . Cellular Fractionation . . . . . VI . Histochrmical Methods . . . . . VII . Cytochemical Procedures . . . . VIII . Nucleolytic Enzymes in Pathological States I X . Possible Functions of Nucleolytic Enzymes Addendum . . . . . . . . References . . . . . . . . Note Added in Proof . . . . .
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AUTHOR INDEX
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SUBJECP INDEX
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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 Roie of DNA in RNA Synthesis
JERARD HURWITZ AND d . T. AVGVST Polynucleotide Phosphorylase
M. GRUNBERG-MANAGO Messenger Ribonucleic Acid
FRITZLIPMANN The Recent Excitement i n the Coding Problem
F. H. C. CRICK Some Thoughts on the Double-Stranded Model of Deoxyribonucleic Acid
AARONBEXDICHAND HERBERT S.ROSENKRANZ Denaturation and Renaturation of Deoxyribonucleic Acid
J. MARMUR, R. ROWND, AND C. L. SCHILDKRAUT Some Problems Concerning the Macromolecular Structure of Ribonucleic Acids
A. S. SPIRIN The Structure of DNA as Determined by X-Ray Scattering Techniques
VITTORIO LUZZATI Molecular Mechanisms of Radiation Effects
A. WACKER AUTHORINDEX-SUBJECTINDEX Volume 2 Nucleic Acids and Information Transfer
LIEBEF.CAVALIERX AND BARBARA H. ROSENBERG Nuclear Ribonucleic Acid
HENRYHARRIS xii
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CONTENTS OF PREVIOUS VOLUMES
Plant Virus Nucleic Acids
ROYMARKHAM The Nucleases of Escherichia coli
1. R. LEHMAN Specificity of Chemical Mutagenesis
DAVIDR. KRIEG Column Chromatography of Oligonucleotides and Polynucleotides
R~ATTHYS 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
AUTHOR INDEX-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
FUJIOEGAMI,KENJITAKAHASHI, AND TSUNEKO UCHIDA Chemical Effects of Ionizing Radiations on Nucleic Acids and Related Compounds
JOSEPH J. WEISS The Regulation of RNA Synthesis i n Bacteria
FREDERICK C. NEIDHARDT Actinomycin and Nucleic Acid Function
E. REICHAND I. H. GOLDBERG De Novo Protein Synthesis in Vifro
B. NISMANAND J. PELMONT
x111
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CONTENTS OF PREVIOUS VOLUMES
Free Nucleotides i n Animal Tissues
P. MANDEL AUTHORINDEX-SUBJECT INDEX Volume 4 Fluorinated Pyrimidines CHARLES KEIDELBERGER
Genetic Recombination in Bacteriophage
E. VOLKIN DNA Polymerases from Mammalian Cells
H. M. KEIR The Evolution of Base Sequences in Polynucleotides
B. J . MCCARTHY Biosynthesis of Ribosomes i n 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 b y Living Cells
L. L m u x AUTHORINDEX-SUBJECT INDEX 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 BOREK Hormones and the Synthesis and Utilization of Ribonucleic Acids
J. R. TATA
CONTENTS OF PREVIOUS VOLUMES
xv
Nucleoside Antibiotics
JACKJ. Fox, KYOICHIA. WATANABE, AND ALEXANDER BLOCH Recombination of DNA Molecules
JR. CHARLES A. THOMAS, Appendix I. Recombination of a Pool of DNA Fragments with Complementary Single-Chain Ends
G. S. WATSON, W. K. SMITH,A N D 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
ROBERTWARNER CHAMBERS The Biochemistry of Pseudouridine
EUGENE GOLDWASSER A N D ROBERT L. HEINRIKSON
AUTHOR INDEX-SUBJECTINDEX Volume 6 Nucleic Acids and Mutability
STEPHENZAMENHOF Specificity in the Structure o f Transfer RNA
KIN-ICHIRO MIURA Synthetic Polynucleotides
A. M. MICHELSON, J . MASSOULI~, AND W. GUSCHLBAUER The DNA of Chloroplasts, Mitochondria, and Centrioles
S. GRANICK AND AHARON GIBOR Behavior, Neural Function, and RNA
H. H Y D ~ N The Nucleolus and the Synthesis o f Ribosomes
ROBERT P. PERRY The Nature and Biosynthesis of Nuclear Ribonucleic Acids
G. P. GWRGIEV Replication of Phage RNA
CHARLES WEISSMANN AND SEVERO OCHOA AUTHOR INDEX-SUBJECT INDEX
Articles Planned for Future Volumes Purine N Oxides
G. B. BROWN “What Really Is DNA?”
E. CHARGAFF The Topography of sRNA
J. E. FRESCO Alterations of DNA Base Composition in Bacterial Cells
G. F. GAUSE Determination of the Nucleotide Sequences of large Oligonucleotides and Small Nucleic Acids
R. W. HOLLEY Photochemistry
R.B. SETLOW Chemistry of Guanine and I t s Biologically Significant Derivatives
R. SHAPIRO +X and 513
R. L. SINSHEIMER DNA and Protamine in the Salmon Testes
M. SMITHAND G. H. DIXON Oligonucleotide Separation
H. A. SOBER AND G. W. RUSHIZKY The Recognition Reaction in Protein Synthesis
P. C. ZAMECNIK
Autoradiographic Studies on DNA Replication in Normal and Leukemic Human Chromosom esl FELICE GAVOSTO Hematology Laboratory, General Medical Clinic, University of Turin, Turin, I t a ly
I. Introduction . . . . . . . . . . . . . 11. Recognition of Chromosomes . . . . . . . . . 111. Autoradiographic Techniques . . . . . . . . . A. Technique for i n Vitro Experiments . . . . . . . B. Technique for i n Vizlo Experiments . . . . . . . C. The Evaluation of Labeling-Approach to Quantitative Autoradiography . . . . . . . . . . . . IV. The Chromosomal DNA of Normal Blood Cells . . . . . V. The Chromosomal DNA of Leukemic Cells . . . . . . A. Acute Leukemia . . . . . . . . . . . . B. Chronic Myeloid Leukemia . . . . . . . . . VI. Conclusions : Some Tentative Hypotheses and Future Approachrs t o the Problem . . . . . . . . . . . . . References . . . . . . . . . . . . . .
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1. Introduction The leukemic transformation of hematopoietic cells involves an alteration in their genetic substance. This genetic event can occur in the genome a t different levels or to different extents. One or more chromosomes may be affected. It may take place a t the subchromosomal level and involve only a single chromosome segment, or i t may concern only one or few genes. I n the first case, the alteration can be observed morphologically. I n the lcukemic field, researches at this level bore fruit as long ago as 1960 [Philadelphia abnormality ( I ) ] , but since then, and in spite of intense study, no equally interesting finding has come t o light. Investigation a t the subchromosomal level and the analysis of chromosome segments The personal research referred to in the text was supported by EURATOM (contracts 016-62-1 BIOI and 061-66-3 BIOI) and by C.N.R.-Rome (contract 115/469/720). 1
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FELICE GAVOSM
as small as 2-3 microns are facilitated by the high-resolution autoradiographic technique. This technique also enables us to study the duplication process and to obtain a more functional view of the genetic substance. At the present time, research a t the molecular level or a t the level of the gene is impossible, a t least in mammalian cells. Autoradiographic studies of the duplication process of human chromosomes began a t almost the same time as morphological studies, and as early as 1961-1963 some important results were reported (2-16). These results included the discovery of a female X chromosome replicating a t a very late stage and thus easily differentiated from the other X chromosome (6, 7) and the finding of a clear asynchrony in human chromosome replication in the final phase of the S period (3-7, 15-17). Furthermore, in 1962, the basis for quantitative evaluation of the synthetic activity in single chromosomes and their segments was established (7). Further autoradiographic, biochemical, and biophysical studies clearly indicated that DNA replication in the nuclei of mammalian cells is an orderly process and involves, among other things, the existence of “early” and “late” synthesized DNA complexes in the genome (10, 18). There are now numerous reasons for supposing that DNA synthesized a t ari early stage represents the most active part of the genome and determines the synthesis of messenger RNA. DNA synthesized a t a later stage, on the other hand, is a repressed fraction of the genetic substance. In this article we discuss the methodological aspects of chromosome labeling in human blood cells and some results of our studies on the duplication of the genetic substance in the chromosomes of normal and leukcniic human blood cells.
II. Recognition of Chromosomes A standard method is necessary for arranging karyotypcs (Fig. 1 ) . The Denver system (19) has generally been used. Where this proved insufficient, the specifications and modifications added later (20) have been adopted. It is of paramount importance in autoradiographic experimcnts to establish well-defined criteria for recognizing single chromosomes in order t o compare their activity in synthesis in different tissues. It is in fact probable that many of the discrepancies in the data published by different authors are due to the different criteria used for arranging the karyotypes. No difficulties were encountered in group A. In group B, on the other hand, particularly in the case in which the chromosomes are supercontracted, i t proved difficult to distinguish between the two pairs 4 and 5. Chromosome 9 of group C is clearly recognizable by the heterochro-
DNA REPLICATION IN HUMAN CHROMOSOMES
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FIG.1. Human normal karyotypc (female subject).
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FELICE GAVOSTO
matic portion. This enables two groups, respectively to left and right of this chromosome, to be identified. Pair No. 6 is easily isolated because of the greater length of the chromosomes, as are also pairs No. 7 and No. 8 because the latter are more metacentric than the former. On the right of pair No. 9, the most metacentric pair is considered to be No. 11. The smaller of the other two is taken as No. 12. Group D is recognized on the basis of chromosome length in mitoses that are not too contracted, and in the supercontracted mitoses identification is based on the chromosome shape (long convex arms outward in chromosome 15, straight in the other two). Group E does not present any difficulty. Group F is not identified with certainty as the only criterion of recognition between the two pairs is a small band of heterochromatin in pair 19 close to the centromere. In group G, differentiation between pairs 21 and 22 is based on length, measuring only the long arms. It is always easy to isolate chromosome Y because of its different length in comparison with the chromosomes of group G. The only chromosome for which labeling is taken into account is heteropyknotic chromosome X ; morphological recognition of this chromosome is made impossible by the extreme variability of its dimensions.
111. Autoradiographic Techniques A preferred autoradiographic technique implies the possibility of removing silver grains from slides (21, 22) (Fig. 2 ) . This not only makes it possible to recognize chromosomes otherwise hidden by the heavy labeling, but also to arrange the karyotypes without being influenced by the distribution of grains on the chromosomes; only when the karyotype is completed can grains previously photographed be legitimately counted (Fig. 3).
A. Technique for in Vitro Experiments One milliliter of bone marrow, with 0.05 ml of heparin (30,000 units/liter) aspirated from hematopoietic regions, is incubated in a rotating system a t 37°C with 1 ml of TC199 and 0.02 ml of tritiated thymidine (100 aC/ml) for 4 hours; 1 ml of solution of colchicine (1:100,000) is added during the last 2 hours. After incubation, cells are centrifuged and the medium is replaced by 7 ml of hypotonic solution (1 part TC 199-3 parts water). After 10 minutes the material is fixed in a 3:l methanol-glacial acetic acid solution (changed three times). The cells are spread on slides by the air-drying method. For the autoradiographic process the darkroom is supplied with a lamp filter (Ilford S. No. 902). An iron thermostatic plate (40°C) is necessary for heating the slides. Fifteen milliliters of nuclear emulsion (Ilford Kz or La) is mixed with 10 ml of distilled water and dissolved by warming in a glass double container a t 45°C; the
DNA REPLICATION I N HUMAN CHROMOSOMES
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FIG.2. Mitotic spread of a human leukemic cell before (A) and after (B) removal of autoradiographic grains (male subject).
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FELICE GAVOSTO
FIG.3. Labeled and unlabeled karyotype from a normal bone marrow cell (male subject). solution is filtered through gauze to eliminate air bubbles and the emulsion is placed in a thermostatic container a t 45°C. Warm slides are twice dipped in the emulsion and the slides held vertically for a few seconds. The backs of the slides are cleaned with gauze and they are dried overnight in silica gel, then are packed tightly in boxes and left at 4°C for 5-15
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DKA REPLICATION IN HUMAN CHROMOSOMES
days. Finally they are processed with Ilford ID 19 developer for 5 minutes, washed in distilled water for 1 minute, fixcd in Kodak AL 4 for 6 minutes a t a uniform temperature of 15”C, and dried in a currcnt of cold air. The slides are stained through the emulsion for 4 minutes in a 2% solution of orcein (Gurr G T London) in acetic acid (30% acetic acid, 70% water), rinsed in water and dried in an air current. Acetic acid treatment can damage the emulsions and dissolve the silver grain, but w r ronsidrr the concentra.tion of acetic acid employed safe enough to avoid this. The silver grains can be removed by treatment of thc slides for 5 minutes in a 75% solution of acetic acid, for 5 minutes in a 30% solution of methyl alcohol, for 25 minutes in a 7.5% solution of potassium ferricyanide, and finally for 10 minutes in a 24% solution of sodium thiosulfate. The cells are stained again with acetic orcein. The technique is derived from that of Bianchi et al. (22). This method for in vitro experiments is particularly suitable for studying the terminal phase of the S period of normal and leukemic bone marrow as well iss spleen or lymph node cells.
B. Technique for in Vivo Experiments A &rect method for studying in vivo the incorporation of thymidine into human leukemia chromosomes was developed in 1963 (15). The following procedurc was adopted: about 2W300 pC/kg of thymidine were injected intravenously into leukemia patients and 3-5 mg of colceniid was injected, also intravenously, in order to arrest mitosis (this is less than a therapeutic dose). Osmotic shock was performed immediately after collecting the bone marrow specimen. The specimens were fixcd and the slides prepared. To obtain satisfactorily labeled mitotic cells under these conditions, the autoradiographic slides must be exposed for as long as 4-6 months, and sometimes even longer. As was demonstrated by Rubini e t al. (2.3, intravenous injection of thymidine in human subjects corresponds to a pulsc labrling procedure. Clearly, this rrpresents an optimum condition for studying DKA synthesis in chromosomes. Since injection of the precursor is a “pulse-labcling” process, one has only to take specimens of bone marrow a t different times after the injection to have selections of mitotic cells that, a t the time of labeling, are in different stages of the synthetic period (94). Figure 4 shows an example of a plan for an experiment of this type. For certain types of experiment, such as kinetic studies, i t may be preferable not to inject colchicine. I n such cases, mitoses of course, do not accumulate. The suggested in vivo technique has a number of attractive merits: (1) it provides pulse labeling of cells in the synthetic period; (2) i t obviates the need for in vitro culture; and (3) it makes it possible for different stages of the S period to be studied a t the same time in the same experiment.
C. The Evaluation of Labeling-Approach
to Quantitative
Autoradiography
After preparation of the karyotypes and transposition of the grains, the latter are counted and the synthetic activity in the chromosomes of each map is evaluated. The mean values of a t least 50 karyotypes must be considered for each evaluation. The number of grains on each chromosome must be corrected for the
8
FELICE GAVOSTO
relative length of that chromosome and for the total number of grains present in the whole mitosis (7,95). Values obtained in this way are related to the quantity of DNA of the single chromosomes and to the absolute quantity of DNA synthesized in each single mitosis. Thus expressions of the synthetic activity of the single chromosomes and of their segments can be compared one with the other. Furthermore, the correction in the total number of grains as obtained from this calculation enables us to compare mitoses from slides that differ in exposure time. It is now generally agreed that a more analytical and quantitative study of the synthetic process in the single chromosomes as offered by the autoradiographic technique is needed. Consequently, the main purpose of rec,ent studies is clearly to compare different experimental apThymidine pulse labeling
Bone marrow samples
Fro. 4. Design for an in vivo experiment.
proaches or, a t least, data obtained in different conditions (eg., normal and leukemic). It should be remembered that synthetic activity differs from one mitosis to another, albeit over a limited period of time. Many observations are necessary to obtain a reliable mean value, partly because of the random nature of radioactive decay but particularly because individual mitoses are not in synchrony and therefore have different contact times with the precursor. I n other words, even if the period of synthesis involved in the experiment is short in comparison with the duration of the entire S period, synthetic activity may appear quite different from one mitosis to another. For example, if we are studying the terminal phase of the S period, we have to deal with some mitoses corresponding to cells that have begun to synthesize DNA 30 minutes before the end of the S period and that have continued until the end,
DNA REPLICATION IN HUMAN CHROMOSOMES
9
others a few minutes later, and so on up to the end of the synthetic phase. On the other hand, in this period, the relative synthetic rate in some chromosomes and in many chromosome segments has already stopped completely, in others i t undergoes sudden reductions, in others again sharp acceleration as in heteropyknotic X. It is thus evident that any evaluation obtained simply by taking a mean value of the whole period observed will be only a n approximation. Consequently, to evaluate the rate of DNA synthesis, all the variables influencing the experimental conditions must be considered. We can easily evaluate the duration of the G2 period if we know the time of contact with colchicine and the ratio between the number of labeled and nonlabeled mitoses. I n this way the exact moment when the S period ends can be clearly established and the mitoses of different experiments can be compared by relating them to the end of S and by regression toward the earlier phases of S. Cell adaptability to cultures can be controlled by the number of mitoses formed per unit of time throughout the experiment (in our experiments we have observed that when culture times are limited to a few hours, the rate of mitosis is constant). Finally, it is necessary to know the precursor contact time for each cell. The fact that the different chromosomes have quite different rates of DNA synthesis in the final phase of the S period enables us t o place any single cell exactly in the S period and compare i t with another cell observed in another experiment. Two methods have been used to calculate the position of a cell in the S period. One (26) exploits the fact that the grains among the chromosomes of the same cell are distributed differently. Thus, by considering a group of labeled mitoses, we are able to obtain a parameter indicating the period of time in which each mitosis was in contact with the precursor. This index is given by the number of grains localized on the heteropyknotic X chromosome OT on another chromosome with delayed synthesis, if there is one. If, for example, the heteropyknotic, latereplicating X chromosome accounts for 20% of the labeling 10 minutes before the end, 5% 20 minutes before, and so on, the ratio of total grains on the mitosis to grains on this chromosome (X*) indicates the position of the cell in the S period (Fig. 5 ) . Actually, the increase in relative synthetic activity in this chromosome in the terminal phase of the S period is not linear and must be exprcssed as a more complex function. We are currently attempting to calculate this function from experimental data so as t o verify its general validity. I n any case, once the curve has been established empirically, or once the function is known, we can choose
10
FELICE GAVOSM
homologous classes for single cases, each corresponding to a particular synthetic activity of the chromosomes considered as a “pacemaker,” and compare them. The second method of placing each mitosis in the S period with a sufficient degree of accuracy involves an evaluation of grains over the whole cell. By using a “cumulative distribution” curve, the time during which a particular cell has been in contact with the precursor can be calculated (27,18).
.
.. . .
*. *
I Total groins on the mitoses
End of the S period
FIG.5. Behavior of DNA synthesis in the late-replicating X (X*) chrornosome during the terminal phase of the S period.
All the cells arrested in mitosis with colchicine and known to be uniformly distributed over the whole time of the experiment are considered. If we divide this time into a number of equal intervals, each containing an equal number of cells, we can suppose that each time class is characterized by a certain mean rate of synthesis. If we then construct a cumulative curve of cells with n or less grains, we will have an expression of the mean rate of each class (Fig. 6 ) . The number of labeled mitotic figures among the whole cell popula-
DNA REPLICATION I N HUMAN CHROMOSOiMES
11
tion considered and known to be uniformly distributed in the time interval has been calculated statistically by considering the grains on the chromosomes and comparing them with those in the background. This cumulative curve is made up of all the labeled mitoses, beginning, of course, with those with the smallest grain count. The abscissa of the cumulative curve corresponds to the time interval of the S period in which incorporation takes place; it is therefore pos-
L . LI60 200 Number of cells with N grains or less
' 2
A
'
'?f
s"
I 0 ' I2 ' I4 ' I6 ' I6 ' 2 0 '
Minutes before end of S
FIG.6. Cumulative curve of Inheld mitosrs in the terminal phase of the S period (grains on the autosornes).
sible to substitute the time units of this interval for the cell units. JT7e can therefore consider the curve as a cumulative curve in which the grains arc plotted as a function of the synthesis time under examination. By allocating to each mitosis a place in the synthetic period, we can obtain a kinetic picture of successive stages of the synthetic period up to the end of S. In investigations where a comparatively long period of S is being considered, it is possible to examine shorter periods by subdividing the mitoses of each case into different groups according to the total number of grains present on the single mitoses. We are thus able to explore different successive periods of 6-8 minutes each and to
12
FELICE GAVOSTO
FIG.7. Synthetic activity in four successive phases during the last 24 niinutes of t,he S period (normal bone marrow erythroblasts) (X* = late replicating 9). - - -, between 24‘-18’ before end of S ; -.-. , tictween 18’-12‘ before end of S ; . . . . . . . , between 12’4’ before end of S ; -, between 6‘4’ before end of S.
-
draw up an ideal karyotype for each of them, so as to indicate the synthetic activity in each chromosome (29, 50). This study of successive stages can tell us how the synthetic process behaves in each chromosome and how the asynchrony of the overall process is determined. At the conclusion of the S period it was clearly
lA
2A
3A
48
5B
FIG.8. Pattern of DNA synthesis in rcgions of chromosomes in groups A and B (terminal phase of S).
DNA REPLICATION IN HUMAN CHROMOSOMES
13
observed that the characteristics of the concluding pattern are foreshadowed a t least 20 minutes before the end. Another observation deriving from this study is that all the chromosomes complete their synthetic activity within a short period, of the order of 10 minutes (Fig. 7). Another line of development of the investigation carries the study up to the limits of resolution permitted by the autoradiographic technique with ordinary microscopy by studying the synthetic process a t subchromosome level. I n order to evaluate incorporation in chromosome segments, the following technique was adopted (29): for each pair of chromosomes, all the grains of an entire set of mitoses were collected and recorded graphically (as shown in Fig. 8) , noting of course, the exact localization of the grains during the transfer. Thus for each case examined we obtained an ideal karyotype (24 chromosomes, since the two homologs were indistinguishable) where each chromosome presented all the grains of the corresponding chromosomes of the karyotypes examined and where the most active zones are indicated with denser labeling (Fig. 8 ) . I n this way segments are obtained corresponding to regions where synthetic activity is more or less intense. This technique eliminates the choice of arbitrary segments and is therefore more physiological. Furthermore, it enables us to make a statistical comparison of the synthetic activity between different segments. This comparison should be done in two different ways: (1) a “qualitative” comparison of the localization within each chromosome of the segments identified with this technique; ( 2 ) a “quantitative” study of the incorporation rate in each segment.
Minutes bpfr’re imd rjf
S
FIG.9. Pattern of DNA synthesis in chromosome pair 3 during three successive terminal stages of S.
14
FELICE GAVOSTO
Figure 9 shows how the segments form in the terminal phase of S through successive, analytically investigated subperiods.
IV. The Chromosomal DNA of Normal Blood Cells For several interrelated reasons, a study was undertaken of the mitoses of hematopoietic cells of different type and origin. Up to now, studies have mostly covered the pattern of the final duplication phase of stimulated lymphocytes and after some days of in vitro culture (6,7' , 16, 16, 31, 32) ; moreover, it is not yet known with precision whether there is a single population of stem cells for the hematopoietic cells, or whether they ifor example lymphocytes and bone marrow cells) have different precursors. I n connection with this latter consideration, it seemed useful to compare the way DNA replicates in populations of hematopoietic cells with totally different functions, and to study the duplication process during spontaneous mitoses and not only in cells stimulated by phytohemoagglutinin (PHA) . A study was made on mitoses of normal lymphocytes stimulated with PHA, normal bone marrow (short-term culture), and mitoses of erythrobIasts (taken from a subjec,t with severe posthemolytic anemia) (29, 41). Experimental conditions were set up in such a way that the maximum period of contact between the different cells in the S period and thymidine was as similar as possible. All three tissues present a very similar pattern to that known to be Characteristic of the terminal phase of the S period for human cells. I n addition to a very active X, and in males to an active Y, we find very high values in both pairs of group B, particularly in one (considered as No. 4) and in a pair of group E (considered as No. 13) whereas particularly low values are to be seen in group F and in one of the pairs of group E. A second observation is that there are no significant differences between the means of the values for the single populations (lymphocytes, normal bone marrow, erythroblastic bone marrow) (Fig. 10). Actually, in the case of some pairs of chromosomes, a difference between the mean values of the three groups appears more evident than for others; however, not even in these cases are the differences statistically significant, a fact that can be established by calculating the variance within the groups. It can also be observed that in some cases, the differences between the values of the pairs in the same group tend to balance out. Thus, if we consider the mean values of the groups rather than the single pairs making them up, these are the same for the different types of cells. All
DNA REPLICATION IN HUMAN CHROMOSOMES
15
this means that, a t least within the resolution limits of the techniques employed, no significant differences can be found between different cell populations of hen~atopoietictibsue, that is, between cells like lymphocytes and erythroblasts whosc functions, properties, and fates are quite different. Apart from overall activity, the synthetic activity of the terminal phase of the S period was also evaluated analytically in four successive phases by the technique described in the previous section. The mean synthetic activities in the single pairs of chromosomes were evaluated for the mitoses belonging to each of these subperiods. The results show that synthetic activity falls progressively in all cases as they approach the end of the S period. But not even this analytical development of the investigation was able to show significant differences between the various populations of normal hematopoietic cells considered.
BIG.10. Late DNA synthesis in normal hematopoictic cells (terminal phase of tlic
S period) (X* = late-replicating XI. -, Peripheral blood lymphocytes; . . . . , whole bone marrow; - - - , erythroblastic bone marrow. The strong similarity in the patterns of duplication observed for lymphocytes and erythroblasts and in a mixed population of erythroblasts and granuloblasts of normal bone marrow are of considerable interest. It will be interesting eventually to have a complete picture of all the most important human physiological tissues. For the moment, it is clear that among the different cell types that make up hematopoietic tissue there are either no differences a t all or differences that cannot be revealed with the methodology a t hand.
16
FELICE GAVOSTO
V. The Chromosomal DNA of leukemic Cells I t is now known that tumors may contain cells in which the chromosome constitution differs from that of nonmalignant cells, but investigations have failed to dcmonstratc the existence of a specific exclusive chromosome alteration except in chronic myeloid leukemia. In all other neoplasms and hematological disorders, morphological anomalies in the chromosomes are absent or very variable (33-40). Consequently, the numerical and/or morphological abnormalities observed in most cases of human tumors or leukemias cannot be considered as an expression of a “first lesion,” related in some way to the onset of the neoplastic process. The chromosome alterations may be secondary phenomena resulting from the neoplasia and depending on abnormal mitoses in cells already damaged as a result of the neoplastic process. This is also the case in acute leukemias in which there is no constant or initial alteration: in almost 50% of the cases morphological alterations are absent; when they are present, they are extremely variable from one patient to another, often appearing late in the course of the disease. Unlike chronic myeloid leukemia, other forms of leukemia present no constant or initial alteration. It has therefore become necessary to extend the investigation to the subchromosomal level, tackling the problem from a functional rather than from a morphological point of view.
A. Acute leukemia DNA replication a t the end of the S period in chromosomes from cases of acute leukemia has been studied recently (24, 4 1 ) . Some of these cases were lymphoblastic forms and others myeloblastic or paramyeloblastic. For this study, all cases with a karyotype of 46 chromosomes and a near totality of blast cells were chosen. Synthetic activity was calculated first for the whole terminal phase of thc S period (about 30 minutes). Extremely similar values were observed within the same form of acute leukemia (namely lymphoblastic leukemia), and none of the differences are significant. The degree of homogeneity is actually higher than that observed between normal hematopoietic cells of different types. As in the normal cases, homogeneity is more evident for pairs of chromosomes of certain identity. I n the other pairs, differences due to uncertainty of classification can appear, as has already been observed in normal tissues. If we consider the mean values of a whole group rather than the single pairs making it up, homogeneity is even more evident.
DNA REPLICATION IN H U M A N CHROMOSOMES
17
I n the cases of myeloblastic lcukcmia too, expcriments were conducted so as to cover terminal phaseb of the S period varying from 26 to 30 minutes. Most pairs of chromosomes present homogeneous values ; in some pairs there is a fair hcterogeneity of values. Therefore, unlike the groups considered previously, :tcute lrukcmias classified as myeloblastic do not present a homogeneity of values among the single cases for all pairs of chtomosomes. For piiirs 16, 18, 21, arid 22 the variance within
FIG.11. Late-replicating T chromosome (arrow) in a leukrmic mitosis.
the individual cases is significant. The first consequence of this ohscrvation is t h a t we cannot consider a mean value of all the cases for these pairs. A first conclusion is that the pattern of synthetic activity in the various chromosomes in acute leukemias also reflects the classical latereplicating pattern of one X in females, of the Y chromosomes in males (Fig. 11) and, among autosomes, of one pair of group B (considered to be the 4th) and in one of group E (13th). If we consider, respectively,
18
FELICE GAVOSTO
the values for normal bone marrow cells, the values for lymphoblastic leukemias and those for the forms considered a s myeloblastic (except for the pairs where the difference of values within the group did not cnable us to reach a general mean), we observe similar patterns. The observation of a notable degree of homogeneity among the values of the cases considered enables us to evaluate the mean value of the cases for each pair of chromosomes and to use it for comparison with the values obtained from the mitoses of normal hematopoietic cells. Figure 12 shows the mean synthetic activity of the three cases of
d Grow G
10
5
FIG. 12. Late DNA synthesis in normal lymphocytes and in lymphoblastic , Normal lymphocytes; , lympholeukemia. (X* = late-replicating X). blastic leukemia.
-
---
lymphoblastic leukemia and the means of the activities of the normal lymphocytes. This comparison shows very few differences between the values of the mitoses of normal hematopoietic cells and those of the leukemic cells. It is, of course, necessary to establish whether these differences are statistically significant. The previously observed homogeneity of values in normal and leukemic cases makes possible a statistical comparison between the means. Furthermore, as we have already observed, the experimental conditions were such that even the durations of the terminal phases of the S period were very nearly the same in both normal and leukemic cases. The statistical comparison proved significant ( P < 1%) only for the pairs of group G. More specifically, data analysis shows that, for both pairs 21 and 22, synthetic activity in the final phase of
19
DNA REPLICATION I N HUMAN CHROMOSOMES
the S period Is significantly greater in leukemic pairs. For no othrr pair of autosomes or for chromosomes X were significant differences observed between normal and leukemic pairs. Even should such differences actually exist, it is very unlikely that present techniques are capable of revealing them. I n myeloblastic leukemia we observed higher values of synthetic activity rate in pair 16, in pair 18, and in pair 21 (in one case). The observed differences in the rate of synthetic activity between normal and leukemic chromosomes are most evident when the inveetigation is extended to segmentary level. Figure 13 refers to pair 21 of the
Leukemic
' .: . _ .'... . .. ;
% . :.
. . ..... .. . ,
.. .
/ L >
:: .
, ~
..
FIG.13. Patterns of terminal DNA synthesis in chromosomes 21 from normal and leukemic subjects.
chromosomes of group G and shows that the difference between normal and leukemic cases is almost exclusiveIy in the short arms of chromosomes 21. The kinetic study of the synthetic activity of the terminal phases of the S period, splitting i t into subperiods according to the technique described earlier, did not reveal any important differences between normal and acute leukemia chromosomes and proved that in acute leukemia cells the replication of DNA is completed in a similar way and falls progressively in all the chromosomes toward the end of the S period. I n acute leukemia it may be of some importance to include a study
20
FELICE GAVOSTO
of the synthetic activity of the extra chromosomes often found in such cases. This presents no problem where the extra chromosomes are morphologically recognizable. On the other hand, in the cases of trisomy or polysomy, it may be extremely important t o ascertain whether the distribution of grains on the three (or more) homologous chromosomes is homogeneous or not, and if not, whether the distribution in one of the three is different from that in the other two. It is possible t o count the total number of grains on the three or more chromosomes and correlate these with the grains on the mitotic figures. It may then be decided whether or not the grains on the three or more chromosomes may be considered to be equally distributed. Some mathcmatical models have been developed to evaluate whether or not the grains on the trisomy or polysomy under consideration are homogeneously distributed among the different chromosomes that form it. One of the simplest models is a statistical comparison of the actual distribution with the theoretical distribution, accepting the null hypothesis of equal grain distribution among the different chromosomes bclonging t o the same groups. Several cases of acute leukemias with extra chromosomes have been examined. One of the first questions to be answered was whether, as one would expect, DNA synthesis takes place regularly in these extra chromosomes. We found that thymidine is always incorporated into the extra chromosomes and that DNA synthesis retains its customary asynchrony in the various extra chromosome segments ( 4 2 ) . It was also found that DNA synthesis in these extra chromosomes is often independent of that in the analogous autosomes, in the sense that it is asynchronous with respect to the chromosomes belonging to the same group as the extra chromosomes in question. I n other words, incorporation is quite late in some instances. This is more clearly seen when the extra chromosomes are readily recognized by their morphological features. I n such cases, it there are might be supposed that, as in the late-replicating X (Xb), niany reprcsscd regions of little functional importance. In other instances, no appreciable differences in degree of synthesis are demonstrable between extra chromosomes and the corresponding autosomes. This seems to be especially so when the extra chromosomes are not easily or not a t all identifiable. I n this case, one might suppose that the extra chromosome is of major functional importance in the pathology of the malignant cell.
B.
Chronic Myeloid leukemia Autoradiographic studies on DNA synthesis in the chromosomes of chronic myeloid leukemia cells have been performed mainly with the
DNA REPLICATION IN HUMAN CHROMOSOXIES
21
aim of investigating DNA synthesis a t the end of the S period in the . specific abno-niality of the disease, i.e., the Ph' chromosome ( 4 )Delayed synthesis in this chromosome was demonstrated by Muldal in 1963 ( 4 4 ) .This finding was supported by statistical analysis and the Ph' chromosome was found to replicate later than either homologous 21 OT chromosome 21 from a normal case ( 4 4 ) . This result was con-
FIG.14. Mitotic figure of a leulicniic cell with three late-replicating Ph' chronioaomtss (arrows), in a case of chronic mycloid leukemia during terminal blastie crisis.
firmed by Schmid (31) in the same year but without quantitatiye analyyis. On t.he other hand, other investigators found no significant differences in the degree of DNA synthesis between the Ph' arid the normal counterpart of the pair. A quantitativc investigatioii has becri performed in one case of the disease and in a case of blaxtic. crisis developing a Ph' trisomy. In both cases, the Ph' chromosome presented a high degree of synthetic activity in the terminal phase of the S period (Fig. 14). A quantitative analysis
22
FELICE GAVOSTO
based on a total of 110 mitoses revealed that the incorporation of thymidine in the Phl chromosome is higher than in chromosomes 21 from normal cells and in the other chromosomes of the G group of the leukemic cells.
VI. ConcIusions: Some Tentative Hypotheses and Future Approaches to the Problem
The results obtained in the study of the replication of the genetic material in human leukemic cells suggest certain conclusions : 1. The duplication of DNA follows a particular time sequence in leukemic chromosomes as well as in normal cells. 2. I n the extra chromosomes, the synthetic process appears to retain its customary asynchrony and, in some instances, is completed later than in other autosomes. 3. The Phl chromosome has a great quantity of repressed DNA. 4. Finally, pushing the present autoradiographic technique to its limits and using it quantitatively, it is possible to reveal some differences between normal and leukemic chromosomes. One of the differences noted concerns chromosome 21 whose connections with other leukemia forms and mongolism are already established. A deletion does, in fact, exist in chronic myeloid leukemia involving a real loss of genetic material from a part of one chromosome 21, and this observation is now considered t o be of great importance in the pathogenesis of the disease. Mongolism is characterized by another type of alteration, an extra chromosome in the same pair; but of particular interest here is that the genetic alteration in chromosomes 21 of mongoloids represents an important predisposing cause of acute leukemia. Finally, an alteration in these chromosomes, characteristic of some familial forms of lymphatic leukemia (probably a predisposing cause) has been described (36)I n this series, a possible functional defect in chromosomes 21 of acute leukemia could provide another argument in favor of the hypothesis that many important genetic activities connected with control of leukopoiesis are situated in these chromosomes. The evidence of late replication in these chromosomes, might mean that many repressed regions have arisen in them under the influence of the leukemogenic factors. In this connection, it is also probable that some genetic mechanisms regulating differentiation of these cells, which are themselves controlled by the action of operator, regulator, and structural genes, have been repressed and that, in the phenotypic expression,
DNA REPLICATION Ih- H UMAN CHROMOSOMES
23
this event is seen in the loss of all differentiation potential by these blast cells. It might therefore be interesting to try to establish same correlation between the type of leukemia and the chromosome alteration with which it is connected. Chronic myeloid leukemia is characterized by a particular type of genetic lesion, involving only one chromosome 21 , the other being normal. In this form of leukemia, therc is a granulopoietic situation as a result of which the cells differentiate and mature almost normally although they have lost all control over their growth activity and therefore multiply without homeostatic regulation. The type of genetic lesion that is becoming evident in some cases of ac.ute leukemia, and which involves both chromosomes 21, might result in a double functional consequence: loss of control over cell growth as in chronic leukemia and loss of the system that controls cell differentiation. Finally, it should be observed that the unbalanced genetic situation determined by the presence of the three chromosomes 21 in mongolism, corresponds t o a predisposition to acute leukemia.
ACKNOWLEDGMENTS I wish to thank Professor G. C. Dogliotti and Drs. A. Pileri and L. Pegoraro for their advice and rritirism. Thanks are also due to Mr. Alan Nixon for his help in revising the English text.
REFERENCES P. C. Nowell and D. A. Hungerford, Science 132, 1497 (1960). J. H. Taylor, J . Biophys. Biochem. Cylol. 7, 455 (1960). J. L. German and A . G. Bearn, J . Ctin. Invest. 40, 1041 (1961). H. B. Painter, J . Biophys. Biochem. C y t o l . 11, 485 (1961). M. A. Bender and D. M. Prescott, Exptl. Cell. Res. 27, 221 (1962). J. L. German, Trans. N . Y . Acad. Sci. 24, 395 (1962). C. W. Gilbert, S. Muldal, L. G. Lajtha, and J. Rowley, Nature 195, 869 (1962). S. A . Lima-de-Faria and K. Borum, J . Cell Biol. 14, 381 (1962). 9. V. Monesi, Boll. Zoot. 29, 749 (1962). 20. E. Stubblefield and C. C. Mueller, Cancer Res. 22, 1091 (1962). 11. L. Atkins, J. A. Book, K. H. Gustavson, 0. Hansson, and M. Hjelrn, C y t o genetks 2, 208 (1963). 12. S. Bader, 0. J. Miller, and B. B. Mukherjee, Exptl. Cell Res. 31, I00 (1963). 13. F. Gavosto, A. Pileri, L. Pegoraro, and A. Momigliano, Nature 200, 807 (1963). 14. F. Giannelli, Lancet i, 863 (1963). 15. Y. Kikuchi and A. A . Sandberg, J . Clin. Invest. 42, 947 (1963). 16. P. S. Moorhead and V. Defmdi, J . Cell Biol. 16, 202 (1963). 27. M. M. Grumbach, A. Morishima, and J . H. Taylor, Proc. Natl. Acad. Sci. U . S. 49, 581 (1963). I S . G. C. Mueller and K. Kajiwara, Biochim. Biophys. Acta 114, 108 (1966). f. 2. 3. J. 5. 6. 7.
24
FELlCE GAVOSTO
19. A Proposed Standard System of Nomenclature of Human Mitotic Chronrosornes. Lancet i, 1063 (1960). 20. London Conference on “The Normal Human Karyotype,” Ann. Human Genet. 27, 295 (1964). 21. F. Gavosto, L. Pagoraro, and A. Pileri, Proc. 9th Congr. European Soc. Hematol., Lisbon, p. 1549. Kargrr, Basel, 1963. 22. N. 0. Bianchi, A. Lima-de-Faria, and H. Jaworska, Hereditas 51, 207 (1964). 23. J . R . Rubini, E. P. Cronkitr, V. P. Bond, and T. M. Fliedner, 1. Clin. Itirwst. 39, 909 (1960). 24. F. Gavosto, L. Pegoraro, and A. Pileri, “Current Research in Leukemia,” 11. 177. Cambridge Univ. Press, London and New York, 1965. 25. F. Gavosto, A. Pileri, and L. Pegoraro, A t t i Soc. Ital. Ematol., 19th t?o)tgy., Pavia, p. 67. Viscontea, Pavia, 1963. 26. F. Gavosto, L. Pegoraro, and A . Pileri, A t t i Conv. Farmital. “Citogenetica tlclle Leucemie,” Torino, p. 161. Mincrva Medica, Torino, 1965. 27. S. Muldal, Atti Conv. Furmifnl. “Cilogenetica delle Leucemie,” Toritio, 1). 130. Minerva Medica, Torino, 1965. 2s. C. W. Gilbrrt, S. Muldal, and I,. G. Lajtha, Nature 208, 159 (1965). 99. F. Ga\-osto, L. Pegoraro, P. Masera, and G. Rovera, unpublished data. 30. P. Masera, L. Prgoraro, G . Rovcra, and W. Gabutti, Boll. Soc. Ital. Biijl. Spcr. 43, 187 (1967). 31. W. Sclrmid, Cytogeiietics 2, 175 (1963). 32. J . I,. Gcrman, J. Cell B i d . 20, 37 (1964). 33. W. M. Court Brown and I. M. Tough, Aclvan. Cancer Rer. 7, 351 (1963). 34. P. H. Fitzgerald, A. Adams, and I”. W. Gunz, J. Natl. Cancer Inst. 32, 395 (1964). 35. F. Gavosto, A. Pileri, I,. Pegoraro, and R. Bernadelli, Abstr. 10th Congr. Inkrn. Soc. Haemntol., Stockholtn. 1.964. Sect. A , N o . 4. Ljunglijfs Litografiskn -415, Stoclillolm, 1964. 36. F. M:. Cunz, P. H. Fitzgflrald, and A. Adams, Brit. M e d . J. 11, 1097 (1962). Si. K. A . Kioesoglou, E. H. Rosenbaum, W. J. M. Dameshek, and W. D:trneslick, Blood 24, 114 (1964). 9s. A. A. Sandberg, T. Ishihara, T. Miwa, and T. S. Hauschka, Cancer It‘es. 21, 678 (1961). $9. A. A. Sandberg, T. Isliilrara, 1,. H. Crosswhite, and T. S. Hauschka, Cnticer Res. 22, 748 (1962). 40. A. A. Sandberg, T. Ishiliara, I . Kikuchi, and L. H. Crosswhite, Ann. N.1’. Acnil. Sci. 113, 663 (1964). 41. F. Gavosto, I,. Pegoraro, P. Masera, and G. Rovera, Proc. A m . Assoc. Cnticer Res. 8, 21 (1967). 42. P. Gavosto, I,. Pegoraro, and A. Pileri, Proc. 9th Congr. European SOC.flenmtol., Lisbon, p . 63. Karger, Basel, 1963. 4.9.A. Lima-de-Faria, N. 0. Bianclii, and P. Nowell, J. Cell Biol. 23, 5 4 1 (1964). 44. S. Muldal and C. H. Ockey, K e p t . Brit. K m p . Cancer Camp. 41, 517 (1963).
Proteins of the Cell Nucleus LUBOMIR S. HNILICA Department of Biochemistry, The University of Texas, M . D . Anderson Hospital and Tumor Institute, Houslmi, Terns
I. Introduction . . . . . . . . . 11. Classifiration of Nuclear Protrins . . . . A. Classification Based on Solubility . . . . B. Classification of Nuclear Proteins Based on Tllrir 111. Protamines . . . . . . . . . . A. Chemical Properties . . . . . . . B. Biological Properties . . . . . . . IV. Histones . . . . . . . . . . A . Molecular Properties . . . . . . . n. Molecular Functions . . . . . . . C. Cell and Species Spcc.ific.it.y . . . . . D. Histonr Biosynthesis . . . . . . E.Histonw as Enzymatic Inld>itcii.s . . . . F. Hist.ones as Gcne Heprrssors . . . . . G. Histones in Embryonir Dtw~1ol)mriit. . . V. Nonhistone Proteins . . . . . . . VI. Conclusions and Summary . . . . . . References . . . . . . . . . .
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26 26 33 41 41 43 48 48 60
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05
70 74 79 89 90 92 95
1. Introduction In the last decade, advances in biological and biochemical research have brought about new understanding and intensified interest in genetic information and replication. The mechanisms operating in transcription and translation of genetically coded information into protein sequence is onc of recent achievement. Concurrent with intensified interest in the cell nucleus and the genetic material is the interest in nuclear proteins. Kosscl's reports of the discovery and study of histones and protamines belong to the classics of chemical literature ( 1 ) . Recent reviews have dealt with certain RI'ORS or fuiictions of nuclear proteins. Allfrey, Mirsky, and Stern (6)discussed the biochemistry and physiology of the nucleus. Phillips (3) limited his review to the histones. Busrh and associates ( 4 4 ) discussed nuclear proteins in normal and neoplastic cells. 25
26
LUBOMIB S. HNILICA
Murray (7) reviewed the biochemistry of basic nuclear proteins. A collection of papers presented a t the First World Conference on Histone Chemistry and Biology (1963) was edited by Bonner and Ts'o (8) and nucleoproteins were discussed by Butler and Davison (9). A recent Ciba Foundation monograph discussed the biological properties of histones relating to their role in the transfer of genetic information (10). This review is intended as a brief summary of basic information with discussion of recent developments in the field of function and properties of nuclear proteins. An exhaustive listing of references is not possible, and some biochemical aspects, e.g., nuclear enzymes, are only briefly mentioned. Since histones are probably of most functional importance to the cell nucleus and its genetic role, they are the subject of the most active research and therefore occupy most of this review.
II. Classification of Nuclear Proteins A. Classification Based on Solubility The cell nucleus is a well-delineated functional structure containing mainly DNA, RNA, and proteins. Despitc intensive investigation, the biological function of most of the nuclear proteins remains unknown. Before the chemical and biological properties of a specific nuclear protein can be studied, the large number of proteins present in the nucleus must be fractionated either into protein species or into groups of proteins of similar chemical or biological properties. The classification of nuclear proteins according to their solubility in buffered salt solutions, dilute acids, bases, etc., is the conventional system, as it follows the initial steps in separation of fractions. I n this system, which was developed by several independent groups of investigators (11-17),there are five main classes of nuclear proteins: (1) nuclear globulins or globulin-like proteins soluble in 0.14 M NaCl (usually buffered with phosphate or citrate) ; (2) proteins of nuclear ribosomes that can be extracted into Tris buffer (pH 7.2-7.6); (3) basic nuclear proteins (histones and protamines) soluble in dilute acids; (4) the acidic nuclear proteins or residual nonhistone proteins, extracted together with some lipoproteins by 0.05-0.1N NaOH ; ( 5 ) the insoluble residue or the residual nuclear proteins. All five categories represent mixtures of proteins with a common feature, i.e., the solubility in the solvent used for their extraction. Further fractionation is based on differences in the chemical composition or on differences in the size of the constituents of each solubility group. Based on the solubility and sedimentation properties, a fractionation scheme, Fig. 1, can be compiled from the major procedures employed for fractionation of nuclear proteins.
27
PROTEINS OF THE CELL NUCLEUS
1. NUCLEAR GLOBULINS The proteins soluble in 0.14M NaC1, or nuclear globulins, first reported by Dounce et al. ( 2 1 ) and by Kirkham and Thomas ( l a ) , are actually mixtures of ribonucleoproteins, probably of ribosomal origin (15, 22-24). The amino acid composition shows an excess of TABLE I AMINOACIDCOMPOSITION OF THE MAJORNUCLEAR PROTEIN FRACTIONS IN RAT LIVER^ Soluble in
Soluble in 0.14111 NaCl Amino acidd Liysine Hrstidine Asginine Apartic acid Threonine Serines Glutamic acid Proline Glycine Alanine Half cystine Valine Methionine Isoleuicine Leurcne yheosine T P nylalanine
RiboWholec somesb 8.7 4.0 4.6 9.6 4.8 7.3 9.2 5.3 8.5 9.0 0.5 7.3 1.6 4.0 9.0 2.6 4.4
8.7 2.3 5.6 9.8 4.8 6.7 11.2 5.5 10.8 7.5 5.1 1.4 3.9 7.8 2.5 4.3
Microsomed 7.0 2.2 4.9 10.0 5.1 6.7 12.1 6.0 8.4 8.1 6.5
1.9 4.4 9.6 2.9 5.1
0.2 N
Sap6
Trisc
HC1
8.3 4.0 2.9 10.4 5.1 6.2 9.2 5.5 8.4 9.8 0.8 7.6 1.3 3.8 9.9 2.2 4.4
8.0 2.2 7.9 9.2 5.2 7.1 11.o 5.0 7.2 7.5 0.9 6.3 1.8 4.7 8.9 2.9 4.0
13.1 2.0 8.7 5.7 5.5 5.7 8.8 5.2 9.1 12.0 0.1 6.2 1.2 4.2 7.6 2.4 2.1
0.1 N NaOHc Residual 6.9 2.4 6.7 9.4 5.1 8.2 12.4 5.1 7.2 7.3 0.8 5.9 2.1 4.4 9.5 2.9 3.7
6.0 2.2 5.6 8.8 5.1 7.1 13.5 5.2 7.3 7.8 0.5 6.3 2.2 4.4 10.2 3.3 4.0
All values are expressed as percent of total moles of amino acids recovered. From Busch and Steele ( 4 ) . Nucleic acids were removed by extriictioii with hot trichloroacetic acid. Tryptophan was not determined. eAll serine values (except those from Buach and Steele) were corrected (10%) for hydrolytic losses. a
dicarboxylic over basic amino acids and does not differ substantially from the composition of other nuclear proteins, with the exception of histones (Table I ) .This is probably due to the complexity of the protein population present in the 0.14M NaCl nuclear extracts. Alanine, glycine, serine, aspartic and glutamic acids, the leucines and valine are the main NH,-terminal amino acids in nuclear proteins soluble in 0.14M NaCl from various tissues (4, 5 , 2 5 ) . Because of their hetero-
Nuclei
I
Extractionwith 0.14 MNaCl (buffered) (repeat if neceeeary)
T
S (nuclear globulins, nuclear sap) (12,13,16,18,19)
1
Extractionwith 0.1 M Trie
2-K
5% OOo x 0 07)
105,000 X g (20)
A A i g. T; 1 /way
Ppt
7
Riboeomal
proteins or nuclear sap If (16)
Nuclear microeomee
105’7\
9
-
P p t s
Fractions of the
nuclear 6-
Ppt
s
Ppt
DOC*- Nuclear Nuclear Nuclear Soluble ribosomes sap1 ultraribosomal mlcmsomee proteins
* Deoxycholate Ezo. la. For legend we opposite page.
y m t Cmde
deoxyribonucleopmtein
T)
Extraction with 2.0 M NaCl
8 Extraction with 0.25 N HC1 or 0.25 N H,SO, (16)
Precipitation with ethanol, extraction with 0.05 N NaOH or 0.25N H,SO, (histones, acidic nuclear, residual proteins) (17)
DNP I
Acidic nuclear and residual proteins (nucleolar residue)
supt
~
i
~ b Extraction with phenol-PAS**
Phenol phase Acid-insoluble proteins
/ \PPt
Supt
x
hPt
PPt
DOC*Ribosomal Soluble fraction nuclear proteins * Deoxycholate ** p -Aminosalicylic acid
Extraction with 0.05 N NaOH supt A Acidic nuclear proteins
P
~
~
~
A
m
zc!
d (contains RNA) (16)
Aqueous phase Phenol-insoluble protein 6
mr
9
x
Extraction kith 0. 05 N
Extraction with DOC* (17)
Centrifugation
I
Extraction with 2 M NaCl
P t Residual nuclear proteins
supt
Acidic nuclear proteins
Ppt Residual proteins
FIG.l a and b. Scheme for fractionation of nuclear proteins based on selective extraction
h3
W
30
LUBOMW S. HNILICA
geneity, the amino acid composition of these nuclear proteins from different tissues and species is quite similar (4, 6). As can be determined from the incorporation of labeled amino acids in vim, the nuclear proteins soluble in 0.14M NaCl are biosynthesized a t rates similar to those of the ribosomal proteins soluble in Tris buffer, thus indicating their similarity (4, 6 ) . Both the rate of biosynthesis and the amount of nuclear proteins soluble in 0.14 M NaCl are changed substantially after partial hepatectomy (26,f l ) .The amount of these proteins in spleen and thymus of rats is not affected by X-ray radiation (98) ; however, some qualitative changes are induced by radiation as determined by a 30% decrease in sulfhydryl content (29). During mitosis of HeLa cells, the isotonic saline-soluble proteins were found to form a halo surrounding the chromosomal plate and to return to the nucleus during telophase ( 3 0 ) . The nuclear globulin fraction forms insoluble complexes with histones (81). Barton (32) found that the nuclear isotonic saline-soluble proteins are not released from nuclei during isolation in isotonic sucrose media even if the nuclear envelopes are disrupted. When nuclei extracted by isotonic saline are washed with isotonic sucrose and exposed to the liver cell-supernatant fraction, an uptake of a soluble protein fraction is detected. These soluble proteins are again released if the nuclei are washed with isotonic saline solution. This indicates that a t least a part of the nuclear saline-soluble proteins is reversibly bound to the nuclear structures and is not retained in the nucleus by the nuclear membrane only. This behavior resembles to some extent that of cytonucleoproteins found in Amoeba ($336). 2. PROTEINS SOLUBLE IN TRISBUFFER
Proteins soluble in Tris buffer a t nearly neutral pH and proteins soluble in deoxycholate solutions represent another group of ribonucleoproteins derived mainly from nuclear ribosomes. Their amino acid composition and NH,-terminal amino acids are similar to those of the nuclear globulins (Table I). It is possible that the Tris buffer-soluble proteins are part of the complex soluble in isotonic saline and their different solubility is due to their association with species of RNA different from those associated with the saline-soluble proteins. Wang (3’7) fractionated the nuclear globulin fraction from calf thymus into several components each having a different rate of incorporation of ATP-C14 into their RNA in vitro. The in vivo biosynthetic rates of the Tris buffer-soluble proteins are quite similar to those. of the saline-soluble fraction (4, 6).
PROTEINS OF THE CELL NUCLEUS
31
3. ACID-SOLUBLE PROTEINS The acid-soluble proteins in most somatic cells are histones, a heterogeneous group of basic proteins characterized by their association with DNA in chromatin. In sperm cells and in lower multicellular organisms, histones are partially or completely replaced by more basic and structurally simple proteins, the protamines. I n addition to histones and protamines, nuclear ribosomes and nucleoli yield soluble proteins when extracted with strong acids (3840). This feature often complicates the extraction of histones, and unless precautions are taken, acid-soluble nuclear ribonucleoproteins contaminate histones, especially in acid extracts of nuclei from tissues other than calf thymus ( 7 , 39, 4 1 ) . If dilute sulfuric acid is used for extraction of the histones, contamination with acid-soluble ribosomal proteins is minimal. 4. ACIDICNUCLEAR PROTEINS
The alkali-soluble nuclear protein fraction is n mixture of DNA, RNA, lipoprotein (5, 42-47), and proteins containing sulfur and tryptophan (2,l.4,4 8 , 4 9 ) . This fraction, which is also referred to as the nuclear residual protein fraction (14, 20, 50-59) contains alkali-soluble proteins of nucleoli, chromatin, and nuclear membranes. The amino acid composition of acidic nuclear proteins differs slightly from that of the saline- and Tris-soluble proteins (Table I). The NH,-terminal amino acids of acidic nuclear proteins are alanine, glycine, serine, aspartic and glutamic acid, valine, and the leucincs ( 4 ) , again indicating the great heterogeneity of this protein mixture. The function of acidic nuclear proteins is not known. They probably contain part of the ribonucleoprotein network of the cell nucleus (53, 5 4 ) , part of the nuclear enzymes, and the residual chromosomal proteins. It has been suggested that a t least some of the acidic nuclear proteins participate in the chromosomal architecture (14, 51, 5 5 ) . Dounce and Hilgartner (51) reported that deoxyribonucleoprotein gels containing a considerable amount of acidic nuclear proteins can be dissociated by agents that disrupt disulfide bridges. When treated with sodium thioglycolate, the acidic nuclear protein yielded two fractions that could be resolved by elcctrophoresis on cellulose acetate strips a t p H 9.2. Lipoproteins (protein bound to cholesterol and phospholipid) are contained in the cell nucleus, presumably in the nuclear membrane. They are also present in the fraction soluble in diluted alkali (43, 56, 5 7 ) . I n bovine brain, lysolecithin, sphingomyelin, lecithin, phosphatidylethanol-
32
LUBOMIR 8. HNILICA
amine, and phosphatidylserine were detected in the nuclear lipoprotein fraction (67). Acidic nuclear proteins are rapidly labeled when labeled amino acids are administered to experimental animals, and their specific activity is the highest of all nuclear proteins (4,16, 58-62). In rapidly dividing tissues, such as malignant tumors, the specific activities of histones approach those of the acidic nuclear proteins, probably reflecting the increased demand for histones by perpetually dividing neoplastic cells (4,62, 6 3 ) . The biosynthesis of acidic nuclear proteins is significantly suppressed when alkylating agents and base analog inhibitors are injected into tumorbearing rats (64-66). Steele found that the acidic nuclear proteins are crosslinked to the DNA in Ehrlich ascites tumor. Furthermore, treatment with difunctional alkylating agents substantially increased the amount of crosslinked protein. No such increase was observed with monofunctional alkylating agents (67). The protein crosslinked to the DNA by means of difunctional mustards has a high content of dicarboxylic amino acids. Bendich and Rosenkranz suggested that this protein may link together DNA strands in chromatin (68). More recently, Balis et al. (69) proposed that the DNA-associated acidic nuclear proteins may function as derepressors of the regulatory genes. I n accordance with this proposal, Salser and Balis (70) found a significantly higher content (2.b8 times the content of spleen) of the DNA-associated proteins in different tumors. Holoubek et al. (7l),who studied the relationship of this protein fraction to the DNA, reported that the amino acid composition of the DNA-associated protein is similar to the composition of the bulk of alkali-soluble nuclear proteins. However, after 30 minutes of labeling with arginine-C'* of Ehrlich ascites cells maintained in a minimal medium, the specific activity of this fraction was significantly higher than the activities of other proteins in the nucleus; most of the arginine-C14 was bound to thymidine. The labeling of the DNA-associated protein was also found to parallel the incorporation of uridine-H3 into the nuclear RNA. Pate1 and Wang (72, 73) succeeded in solubilizing the acidic nuclear proteins in aqueous buffers, and such solubilized proteins effectively incorporate labeled amino acids in vitro (74).I n addition, Wang pretreated rat liver nuclei with MgCl, containing Tris buffer, p H 7.5, and then with 1M NaCl to obtain a residue of the acidic nuclear proteins (52). Extraction with 2% deoxycholate solubilized approximately 90% of the residue. The solution was then fractionated by adjusting the p H first to 6, removing the precipitate, adjusting the supernatant liquid to pH 5 , and precipitating the proteins soluble a t pH 5 with ammonium sulfate. The four protein fractions resulting from this method (i.e., the
PROTEINS OF THE CELL NUCLEUS
33
three precipitates and a residual protein labeled R-RNP) differed significantly in their amino acid composition. All four fractions contained RNA, approximately 1% in the pH 6 precipitate, 19% in the p H 5 precipitate, and approximately 33Fin the R-RNP and (NH,) ,SO4 precipitates. All fractions interacted strongly with calf thymus histones, forming insoluble complexes. It is difficult to decide whether such precipitation arises from selective interaction between the two species of proteins or whether it is a nonspecific association of basic histones with thc RNA moiety of the acidic ribonucleoproteins. Such interaction of histones with the nonhistone proteins in the cell nucleus may be the mechanism of the derepression of the genetic loci repressed by histone (52, 7 5 ) .
5 . THERESIDUAL NUCLEAR PROTEINS Thc last group of nuclear proteins resulting from the fractionation scheme in Fig. 1 are proteins that resist common solvents. The amount of such insoluble residue varies from tissue t o tissue and can represent more than 50% of the protein content in the nucleus (76-78). The ninount of residual proteins is increased in neoplasia (78, 79). The origin of this fraction is obscure, but the possibility must be considered that a t least a part of this fraction arises from cytoplasmic membranes contaminating the nuclei. Zbarsky and Dmitrieva (80) proposed that the residual nuclear proteins may be “inactivatcd” or denatured acidic nuclear proteins. Amino acid composition and 1 y ~ i n e - Cincorporation ~~ data support this possibility (4, 16, 6 2 ) . Steele and Busch (16) found a considerable amount of collagen in residual protein preparations from rat liver and Walker carcinosarcoma nuclei, but the origin of the collagen is unknown.
B. Classification of Nuclear Proteins Based on Their Origin A much more sophisticated but complex classification results from fractionation of morphological substructures of the cell nucleus such as chromatin, chromosomes, nucleoli, nuclear ribosomes, nuclear membrane, and nuclear sap, prior to the extraction of specific proteins from each structure. 1. PROTEINS OF THE CHROMOSOMES AND CHROMATIN
Most knowledge about the chemical composition of chromosomes comes from histochemical studies. Methods for isolation and fractionation of chromosomes have been developed only recently (see Gavosto in this volume). Early attempts to isolate chromosomes from mammalian tissues showed that chromosomal fragments are composed mainly of
34
LUBOMIR 5. HNILICA
DNA and histone with some acidic, tryptophan-containing protein (21, 81, 82). Recent methods for the isolation of metaphase chromosomes
permit a more detailed analysis, Metaphase chromosomes from L2 ascites tumor cells contain 13.5% RNA, 13.5% DNA, and 68.3% protein (83).Huberman and Attardi (84) analyzed isolated chromosomes from HeLa S-3 cells. Their chromosomes showed much more protein than expected relative to their DNA content (approximately 15.7% DNA, 10.4% RNA, 31.4% acid-soluble protein, and 42.5% acid-insoluble protein). The chemical nature of acid-soluble and acid-insoluble proteins in chromosomes was not determined, but it can be assumed that at least part are histones and acidic nuclear proteins. Direct chemical analyses of isolated chromosomes agree in general with cytochemical measurements whereby, in addition to the DNA and RNA, histones (fast green positive, acid-soluble proteins) and acidic proteins have been determined (85, 86). More knowledge is available about the proteins of nuclear chromatin. Nuclei of interphase cells contain two kinds of chromatin; dense (compact) chromatin that, in well-differentiated cells, represents a major portion of the chromatin content, and diffuse (extended) chromatin (87, 88), present in relatively small amount. Most of the RNA synthesis of the nucleus is associated with the extended chromatin (88, 89). Both the dense and diffuse chromatin can be isolated by mild sonication and sucrose gradient centrifugation (88,90).Chemical analysis indicates that chromatin, in addition to DNA and RNA, contains a considerable amount of histones, acidic nuclear proteins, and a small amount of residual nuclear proteins. The fractionation scheme presented in Fig. 1 can be applied to nuclear chromatin. 2. NUCLEOLAR PBOTEINS
One of the most interesting cellular organelles is the nucleolus. Up to 80% of its dry weight is protein. Nucleoli are difficult to obtain in purity sufficient for biochemical analysis because of their diffuse connection with nucleoplasm and chromatin and their lack of a delineating membrane. Proteins associated with nucleoli resemble the nuclear ribosomal proteins (soluble in 0.14 M NaCl and in Tris buffer), histones, acidic nuclear proteins, and the insoluble residue (40, 91, 92) and can be fractionated according to the scheme in Fig. 2. The amino acid composition of nucleolar proteins from mammalian tissues is similar to the composition of the corresponding fractions isolated from whole nuclei (40, 92,9S) (Table 11). No significant differences were observed in the distribution and composition of the five main
TABLE I1 AMIXO ACID COMPOSITION OF MAJORNUCLEOLAR PROTEIN FRACTIONS FROM RAT LIVER" Soluble in 0.14M NaCl Soluble in 0.2 N HCl
Amino acid*
Whole
Wholed
H20 Ppt"
Soluble in NaC1HCld
Soluble in HzOe
Soluble in NaCI, insoluble in HCld
Soluble in Tris
Histone
Acidsoluble
Soluble in 0.1 N NaOH
Lysine Histidine Arginine Aspartic acid Threonine Serinec Glutamic acid Proline Glycine Alanine Half-cystine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine
8.5 1.8 5.6 10.8 5.2 7.6 13.2
8.1 2.9 3.9 10.2 4.6 6.6 11.3
8.4 2.1 6.1 9.7 4.8 7.0 12.4
8.7 2.3 3.6 11.o 4.7 6.3 11.7
9.8 1.4 3.4 11.8 5.3 10.9 15.5
4.7 1.6 2.9 11.o 4.9 7.6 14.0
8.0 3.0 6.8 8.7 5.3 7.4 10.0
14.1 1.9 7.2 7.4 5.2 6.9 10.1
10.9 2.1 7.5 8.1 5.1 6.4 12.7
5.3 8.7 7.7 0.4 5.8 1.9 4.1 7.8 2.5 3.4
5.7 9.0 9.1 1.9 7.3 1.o 4.3 9.7 1.7 4.6
5.3 8.7 7.9 0.6 6.1 2.2 4.2 8.2 2.7 3.4
6.2 8.5 9.2 1.9 6.4 1.6 3.7 8.5 1.3 3.3
6.6
5.1
5.0
4.8
8.2 6.5 1.6 4.6 1.7 4.6 10.9 2.0 3.5
7.6 9.0 0.9 6.1 2.0 4.6 9.3 2.6 3.5
5.6 8.0 11.7 5.9 1.2 3.6 7.0 1.9 2.2
4.6
5.8 7.5 0.2 5.2 1.5 3.4 7.8 1.6 2.7
8.5 9.4 0.4 6.2 1.4 3.7 8.0 2.2 2.7
8.8 7.8
8.0
2.1 6.4 8.8 4.7 7.8 13.0
0.7 5.9 2.1 4.2 9.1 2.4 3.2
Residual 7.2 2.7 7.2 8.7 5.1 7.1 12.7 4.7 7.4 8.2 0.7 6.5 2.1 4.6 10.5 1.0 3.6
All values are expressed as percent of tot.al moles of amino acids recovered. Except for the histones, all proteins were treated to remove nucleic acids. Tryptophan was not determined. All serine values [except t.hose from Grogan et aZ. (4011 were corrected (10%) for hydrolytic losses. From Grogan et al. (40). The H20-soluble and HzO-insoluble fractions were obtained by dialysis of the 0.14M NaCl extract against distilled water.
Nucleali
Extraction with 0.14 M NaCl (buffered)
7Lwy
(92)
pPtA Extraction with 0.1 Af Tris,
4t
/"'"\
77
I
Extraction with
77
4t
supt
Residual proteins
17HistOnes" (acid-soluble nuclear proteins 1
Precipitation with 20%TCA* (nuclear globulins) Dialysis against 0.25 N HCl
:%i/
supt NaC1-solubleHC1-soluble fraction
Ppt
95% Ethanol
wrh
Nucleolar SUPt globulins and ribosomal fraction
supt Nucleolar ribosomal prOteiM
Extraction with 0.2 N HCl
/\*Pt
fraction
T
Histones and acid soluble proteins
Extraction with
YT
PPt
Nucleolar residual proteins
*Pt
Nucleolar acidic proteins
* Trichloroacetic acid FIG.2. Scheme for fractionation of nucleolar proteins.
3
0
p: Z
E
P
PROTEINS OF THE CELL NUCLEUS
37
histone fractions from isolated nuclei and nucleoli of Novikoff ascitic hepatoma ( 9 2 ) . Grogan et al. (40) analyzed the saline-soluble proteins, histones, and nucleolar acidic proteins from rat liver and Walker carcinosarcoma. I n addition to the similarity of the amino acid compositions of these proteins from both tissues, they were unable to detect any proteins specific for the nucleolus. Quantitatively, more saline-soluble protein, more histone, and less acidic nuclear protein (acid-insoluble residue) were found in liver than in Walker carciiiosarconia nucleoli. However, since the nucleolus forms a continuum with the nucleoplasm, it is very difficult to establish whether the observed differences in concentration of nucleolar proteins in these two tissues are meaningful. Birnstiel et al. (38)separated nucleolar proteins into three fractions; the protein soluble in 0 . 2 N HCl was similar in its amino acid composition t o the insoluble nucleolar residual protein, which in turn was similar to the protein of cytoplasmic ribosomes. This indicates that a large portion of nucleolar proteins are probably ribosomal and that only a part of the nucleolar protein soluble in 0 . 2 N HCl is histone. However, treatment of the nucleoli with 0.14 M NaCl and with 0.1 M Tris buffer (pH 7.6) removes most of the acid-soluble nucleolar nonhistone proteins (92). The incorporation of 1 y ~ in e-C'~ into nucleolar acidic proteins of liver and Walker carcinosarrorna decreases significantly after administration of uracil mustard to the experimental animals ( 9 3 ) . Grogan et al. (40) also confirmed data reported by Liau et al. (91) that in the nucleolus, the ratio of histone to DNA is almost twice that found in the whole nucleus. It has been reported, on the basis of pulse-labeling experiments, that the nucleolus in plants is relatively rich in histones and that these proteins may actually be biosynthesized in the nucleolus and then released into the nucleoplasm (94, 9 5 ) . I n accordance with such findings, nucleolar histones as compared with the whole nucleus in Novikoff ascitic hepatoma have a higher labeling in vivo ( 9 2 ) . The function of nucleolar proteins is not known. The presence of acidic proteins in the nucleolus has been related to the role of the nucleolus in the biosynthesis of ribosomes (91, 96-101). Also, the nucleolus may be the site of histone biosynthesis (92, 94, 9 5 ) . Liau et al. ( 9 1 ) observed that histones may actually participate in the regulation of RNA synthesis by the nucleolus. I n the presence of nucleolar histones or in nucleoli reconstituted with calf thymus histones, isolated nucleoli biosynthesize the ribosome-like RNA. Actinomycin D also affects the base composition of RNA synthesized in nucleoli in a similar manner ( 1 0 2 ) . Enzymes, such as RNA polymerase, RNase, S-adenosylmethionine-RNA methyltransferase, and, to a lesser extent, NAD pyrophosphorylase and ATPase A are present in isolated nucleoli (105, 104).
38
LUBOMIR S. HNILICA
Vincent [quoted in ( 7 5 ) ] found that starfish oocyte nucleoli contain a homogeneous protein comprising over 80% of the dry weight of the nucleoli. The molecular weight of this protein is in excess of 60,000 and it contains a small amount of RNA. Lysine comprises 16 of its 20 molepercent of basic amino acids. Dicarboxylic amino acids represent another 20 mole-percent of its total amino acid content.
3. NUCLEAR RIBOSOMAL PROTEINS Structures in nuclei similar to ribosomes have been noted by several workers (15, 22, 98, 105-107). Purified ribosomes are obtained from isolated nuclei by extensive homogenization in 0.1 M phosphate buffers, in buffered 0.14M NaCl, or in Tris buffer a t p H 7.1 (15, 37, 108, 109) followed by ultracentrifugation of the extract. Nuclear ribosomes consist almost entirely of RNA and protein in approximately equal ratios as do their cytoplasmic counterparts. Ribosomal proteins are highly heterogeneous ( 110-1 l 4 ) , and the amino acid compositions of cytoplasmic and nuclear ribosomal proteins from tissues as different as pea seedlings, rat liver, rabbit reticulocytes, calf thymus, rat Walker carcinosarcoma are very similar (4, 58, 114-117). As the content of basic amino acids exceeds slightly the content of dicarboxylic amino acids, the ribosomal proteins are moderately basic, especially since part of the aspartic and glutamic acid residues are probably in the form of their amides. Wang (22) and Frenster et al. (15, 107) reported rapid utpake of labeled amino acids by nucleoribosomal structures. Allfrey and co-workers (15, 118) using isolated nuclei, established that nuclear ribosomes participate actively in nuclear protein synthesis. Initially, the C14-leucine used for labeling was incorporated preferentially into the ribosomal “core” proteins. After a “chase” with nonradioactive leucine, the specific activity of the “core” proteins decreased with a simultaneous rise in the speciflc activity of ribosomal “coat” proteins ((‘coat’’proteins are readily extracted with 0.5% deoxycholate, whereas “core” proteins are resistant to such treatment). Later, the specific activity of the “coat” proteins also decreased. This situation was interpreted to reflect the migration of nuclear proteins during their biosynthesis by nuclear ribosomes. Similar results were reported by Wang ( 2 0 ) . Under proper conditions in the presence of an ATP-generating system, amino acid activating enzymes and GTP, isolated nuclear ribosomes are capable of independent protein synthesis. The uptake of labeled amino acids is sensitive to puromycin and RNase, indicating a similarity in the mechanisms operating in both the cytoplasmic and nuclear ribosomes (118-161). The in vitro protein synthesis in the nuclear ribosome system is a t least partially DNA-dependent (122).
PROTEINS O F THE CELL NUCLEUS
39
Ribosomal preparations from plant nuclei behave differently from those from calf thymus ribosomes. Nuclear ribosomes extracted from tobacco cells cultured and incubated with C’+-labeled amino acids incorporate the label in such n way as to suggest that nuclear ribosomes in plants are the product rather than the apparatus of nuclear protein synthesis (106,123, 124). In “pulse-chase” experiments, the newly biosynthesized ribonucleoprotein is lost from the nucleus, which may indicate migration of nuclear ribosomes to the cytoplasm. The differences between experiments on calf thymus nuclei and those in tobacco cells in culture can be interpreted on the basis of different rates of ribosomal production by thymocytes (low ribosomal turnover, high protein synthesis) and tobacco cells in culture (high output of new ribosomes).
4. PROTEINS OF THE MITOTICAPPARATUS The mitotic apparatus is an interesting functional structure. It represents approximately 10% of the total cellular protein content and, according to analyses performed on mitotic spindles isolated from sea urchin eggs undergoing cleavage, 90% or more of their mass is protein (125).Proteins isolated from the mitotic apparatus are relatively rich in dicarboxylic amino acids and leucine and their amino acid compositions resemble that of muscle actin (126). Miki and Osawa (127) demonstrated that mitotic apparatus protein interacts with rabbit myosin. The molecular weight of the digitonin-isolated mitotic apparatus protein is 315,000 k 20,000 (128). Two components, S, 3.7 (major) and S,, 8.6 (minor), were found by Zimmerman (128). On dialysis, the 3 . 7 s particles were converted to 3.2 S and the 8.6s particles disappeared. Sakai (129) more recently demonstrated the presence of three groups of protein particles in the mitotic apparatus of Strongylocentrotus purpuratus. The sedimentation coefficients of the three groups were 3.2-3.5, 11-13, and 21-22. The 3.5 S and most of the 13 S particles were cleaved by sulfitolysis or dithiothreitol to 2.5 S particles. Oxidation dimerized the 2 . 5 s particles to 3 . 5 s . The molecular weight of the monomer was determined to be 34,700 -C 200 and the 3 . 5 s particles contained 4% nucleotides. The nucleotide material is RNA of base composition similar to that of unfertilized eggs. The RNA of the mitotic apparatus also resembles the base composition of cytoplasmic RNA to some extent (128).
The origin of the proteins of the mitotic apparatus is believed to be cytoplasmic. It is not clear whether the nonchromosomal proteins of the mitotic apparatus are made de novo during each mitosis or whether they rearrange by polymerization from preexisting macromolecules. Stafford and Iverson (130) presented evidence that mitotic apparatus
40
LUBOMIR S . HNLIJCA
proteins actively incorporate leucine-14C during the metaphase. They also participate in the Rapkine cycle, i.e., an interesting rhythm of SHgroup fluctuation in cleaving eggs of sea urchins and other animals. This periodic fluctuation seems to be associated with the KC1-soluble proteins, and its possible biochemical mechanisms were recently reviewed by Dan (131). Actin and myosin-like proteins were reported by Ohnishi et al. in calf thymus nuclei (132).
5. PROTEINS OF THE NUCLEAR SAPAND NUCLEAR MEMBRANE The nuclear proteins comprising the chemically least well-defined group are those of the nuclear sap and membrane. Usually those proteins of the saline or Tris buffer extracts (pH 7.1-7.6) that do not sediment by ultracentrifugation fall into this category (4, 5, 32, 133-139). The amino acid composition of the nuclear sap proteins from rat liver and Walker carcinosarcoma resembles, with minor exceptions, the composition of nuclear ribosomal proteins (4, 5 ) . The most striking characteristic of the nuclear sap proteins is the low content of arginine (2.9-3.6%) and the relatively high content of histidine (approximately 4%). In this way, they resemble the globins in composition. Bakay and Soroff (19) divided nuclear sap proteins soluble in buffered isotonic saline into 18 hypothetical classes grouped into five charge types : basic, near-neutral, weakly acidic, highly acidic, and strongly polyanionic. Nuclear saline extracts of hepatomas produced by diets containing azo dyes were markedly depleted in the near neutral and basic proteins and enriched in the highly acidic components as compared with nuclear extracts from normal liver. It was also implied that nuclear sap proteins are capable of arresting the development of frog embryos (1.40). The nuclear membrane seems to be composed of several layers of lipoprotein membranes interlocked with more rigid protein structures. Electron microscopy demonstrates high porosity of the nuclear membrane with pores large enough to permit transport of ribosomes or similar particles. The nuclear membrane may form a continuum with the cytoplasmic reticulum and thereby provide a simple vehicle for the transport of ribosomes into the cytoplasm. This observation finds biochemical support in the findings that in certain cell types (thymocytes and neoplastic cells) the increased difficulty of obtaining cytoplasm-free nuclei by shearing in a mechanical homogenizer is associated with the increased content of nuclear residual protein in such nuclei. Lipoproteins and nuclear residual proteins are thought to be the main constituents of nuclear mcmbranes (18, 141-1.43).
PROTEINS OF THE CELL NUCLEUS
41
111. Protamines A. Chemical Properties Basic proteins associated with DNA in the cell nucleus that are extractable with acid are generally categorized as protamines and histones. It is very difficult to distinguish exactly between these two groups of proteins since basic proteins with an amino acid composition and properties permitting their classification in either of the two categories have been described in the literature. Protamines are frequently characterized as low molecular weight proteins (average mol. wt. 5000) of a strongly basic character, with arginine representing more than 50% of the total amino acid content. Protanlines contain a small amount of lysine and/or histidine. They also contain neutral amino acids, such as serine, proline, alanine, glycine, with a smaller amount of threonine, valine, the leucines, and occasionally methionine, tyrosine, aspartic and glutamic acids. Protamines are almost exclusively localized in sperm of fish and other lower animals ; however, a protamine-like protein (galline) has been described in rooster sperm (144, 1 4 5 ) . Protamines are classified into three categories according to their content of basic amino acids: monoprotarnines containing only arginine (clupeine, salmine, scombrine, fontinine, truttine, galline) ; diprotamines containing, in addition to arginine, either histidine or lysine (iridine, lacustrine) ; and triprotamines containing all three basic amino acids, arginine, lysine, and histidine (sturine) . Chemically, protamines behave like large peptides, and spermatozoa of one species usually contain a mixture of closely related proteins frequently categorized under one name derived from the species name (e.g., clupeine from Clupeus harengus, salmine from Salmo salar, scombrine from Scomber scombrus). Protamines, like any polypeptides, can be fractionated by paper chromatography, countercurrent distribution, column chromatography on activated alumina, or on ion-exchange resins (7, 146-1.48). The heterogeneity of clupeine increases upon chelation with copper ion (149). Divalent copper also increases the resistance of nucleoprotamines to thermal denaturation (nucleoprotamine is a complex of DNA with protamine) ( 1 5 0 ) , Typical protamines are composed of a relatively simple sequence of polyarginine peptides interrupted by residues of neutral amino acids. The NH,-terminal amino acids of protamines are almost exclusively proline and alanine ( 1 4 7 ) . The small quantities of serine, and other amino acids detected in some preparations may be attributed t o impurities or to partial proteolytic degradation of the samples during preparation or handling. The NH,-terminal amino acid of muguline is
42
LUBOMIR S. HNILICA
arginine while sturine has alanine and glutamic acid as NH2-terminals ( 1 4 7 ) .The presence of alanine and proline as NH,-terminal amino acids in protamines indicates their close relationship to somatic histones in which alanine and proline are the NH,-terminal amino acids of the fractions F3 and F2b, respectively. I n protamines, valine was found to be adjacent to the NH,-terminal proline; however, alanine is adjacent to proline in clupeine (151). More recently, Ando and Sawada (148) who fractionated clupeine from Pacific herring (Clupea pallasii) into fractions YI, YII, and Z reported the NH,-terminal sequences for fraction YI to be Ala-Arg-Arg-; for fraction YII, Pro-Arg-Arg-; and for fraction Z, Ala-Arg-Arg. The C-terminal amino acid sequences of most protamines investigated to date is Arg-Ala-Val- ; in salmine this is followed by proline (151). Though the complete amino acid sequence is difficult to determine because of protamine heterogeneity, it has been shown that polyarginine sequences alternate with neutral amino acids, although with no regularity. The polyarginine sequences are two, three, or five residues, thereby contradicting the earlier proposal that pairs of neutral amino acids alternate with four arginine residues ( 1 4 7 ) . The following amino acid sequence for the Z fraction of clupeine was suggested by Ando e t al. (156): H-Ala-Arg,-Ser-ArgrAla-Ser-~rg-Pro-Val-Arg~-Pr~,~r~~Val~~er-.~rg,-.~la-~rg*-OH
More recently, Ando and Suzuki (153) reported amino acid sequence of the YII fraction: H-Pro-A rg,-Thr-ArgpAla-Ser-Arg-Pro-Val-Arga-Pro-~~rg~-Val-Ser-.~rg~-~4la-Arg,-OH
Except for the NH,-terminal portion of the two fractions, the remainder of both clupeines is identical. The molecular weights for the polypeptides of these sequences are 4142 and 4777, respectively which compare favorably with the average minimum molecular weight for clupeine obtained by Zimmermann [quoted in ( 1 4 7 ) l .Molecular weights of other protamines range from 4100 (free base) for iridine (154) to 5150 (free base) for salmine ( 1 5 5 ) . However, since protamines are known to be heterogeneous, accurate molecular weight measurements have little meaning. The amino acid composition of protamines remains remarkably constant for the same species regardless of geographical location ( 1 5 6 ) , or age (157, 158). Iridine isolated from three individual fish of the same species has been found to be heterogeneous, thereby indicating that the heterogeneity of protamines is not introduced by mixing spermatozoa from several individuals of the same species (159). A similar obser-
PROTEINS OF THE CELL NUCLEUS
43
vation was reported for clupeine from the Pacific herring (Clupea pallasii) (160). Protamines found in fish spermatozoa are present in the form of protamine-DNA complex, the nucleoprotamine. It was assumed tlhat arginine residues oppose the nucleic acid phosphates with the neutral amino acid links bent outward. In nucleoprotamine, the polypeptide chain has an extended p-form and follows the shallow groove of the DNA helix (161).More recent studies indicate however, that the structure of nucleoprotamines is probably more complicated (147, 162). The number of basic amino acids in nucleoprotamines approximately equals the number of phosphate residues on DNA indicating almost complete saturation of the negatively charged DNA molecule (147). The bond between protamine and DNA is strong, much stronger than the binding of histones in nucleohistones. Measurements of the affinity of the protamines to DNA in solutions of high ionic strength suggest that forces other than the electrostatic interaction between arginine and phosphate contribute to the conformational stability of nucleoprotamines (163). Since protamines appear during the transformation of male reproductive cells into metabolically inert spermatozoa, the much higher content of arginine in protamines as compared with the somatic histones was interpreted as the basis for the mechanism by which the DNA in spermatozoa is kept tightly packed for its delivery during fertilization. In some viruses, the basic substances protecting the DNA are polyamines such as sperniine or spermidine (164, 165).
B. Biological Properties The appearance of protanlines during spermatogenesis in fish was studied by Miescher, who cliscovered these specialized proteins nearly one century ago. The Rhine salmon, after reaching sexual maturity in the Atlantic Ocean, starts its journey back to the breeding grounds upstream in the Rhine. During this period, the fish accepts no food and its originally small testes enlarge substantially and fill with ripe spermatozoa. During the &9 months of its upstream travel, the histones originally present in the testes are replaced almost completely by protamines. The histone-protamine transition during spermatogenesis occurs in many fish species. It was observed by Miescher and confirmed by others that protamines appear relatively late during spermatogenesis. In a detailed study, Felix et al. (166) analyzed basic proteins in testes of the brook char (Salmo fontinalis) at 10-day intervals during the maturation of spermatozoa, which begins about 90 days before spawning. The nucleoprotamine appeared suddenly, approximately 40 days
44
LUBOMIR S. HNILICA
before spawning. The testes of most animals also contained a more complex water-soluble nucleoprotein, probably nucleohistone, that was present a t all stages of maturation, even immediately before discharge of the semen. This indicates its origin in cells other than spermatozoa (147, 158). During sperm maturation, the amount of DNA in testes remains constant while the amount of nucleohistone decreases probably due to the replacement by protamines ( 1 6 7 ) . Alfert (168), utilizing the difference of solubility of histones and protarnines in 5% trichloroacetic acid (most histones are insoluble except for the very lysine-rich fraction 1) and the stainability of basic proteins with fast green, also found that protamines appear late in spermatogenesis. I n Chinook salmon, histones were replaced rapidly by protamines and the change occurred relatively late during spermatogenesis. One theory concerning the origin of protamines was that histones may be converted into protamines directly during spermatogenesis. Ingles et al. (169),in a series of experiments on the maturing testes of the Steelhead trout and the Pacific salmon, demonstrated clearly that protamines are biosynthesized de novo in the course of formation and ripening of the spermatozoa. At an early stage of sperm maturation, polyacrylamide gel electrophoretic patterns became more complex by addition of a broad band slower than the mobility of somatic histones originally present in the testes. Later, 52 days after the induction of spermatogenesis by injections of the Chinook salmon pituitary extract, an additional electrophoretically fast band corresponding to the mobility of protamines appeared in the electrophoretograms. Labeled arginine was actively incorporated into the protamines by cells isolated from maturing testes of several salmonide species. The incorporation was strongly inhibited by cycloheximide, puromycin, and to a lesser extent by chloramphenicol. Actinomycin D did not inhibit protamine synthesis a t all over a 4-hour period, possibly because of the long life of mRNA coding for protamine biosynthesis. It can be concluded that protamines arc indeed biosynthesized de novo during spermatogenesis and that the mechanisms governing their biosynthesis do not deviate essentially from the mechanisms of biosynthesis of other cellular proteins. The late appearance of protamines during spermatogenesis also occurs in the Pulmonate snail H e l k aspersa ( l 7 0 ) , squid Loligo opalescens ( 17 1 ), Drosophila melanogaster ( 1 72, 173) , in grasshopper Chortophaga viridifasciata (de Geer) ( 1 7 4 ) , in mouse ( l 7 5 ) , and possibly in many other species. However, all these transitions which were detected mainly by histochemical staining procedures and by autoradiography indicate
PROTEINS OF THE CELL NUCLEUS
45
the presence of a sperm-specific, arginine-rich histone in mature spermatozoa instead of a typical protamine. Recent biochemical studies lend support to this possibility. Hultin and Herne (176) analyzed proteins in spermatozoa from the mollusks Patella vulgata and Patella coerulea and from the sea urchin Arbacia lkula. The mollusks spermatozoa contained peculiar heterogeneous basic proteins, rich in arginine (25% and 46% of all amino acid residues), regarded as transitional between histones and protamines. The mollusk proteins also contain a considerable amount of lysine (20% and 8% of all amino acid residues), and their amino acid composition resembled the composition of histones. The sea urchin sperm contained basic proteins that could be classified as histones not dissimilar from the mammalian lysine-rich histone fraction. Recently, basic proteins prepared from the mollusk Mytilus, from the cytoplasm of frog eggs, and from the sea urchin Strongylocentrotus purpuratus were fractionated and analyzed ( 17 7 ) . Proteins similar to the arginine-rich group from Patella were present in fractions 3 and 4 of the Mytilus protein chromatographed on carboxymethyl cellulose. Frog oocytes contained proteins in which arginine, histidine, and lysine comprised approximately 576, 5%, and 12% of the total amino acid residues, respectively. Horn (178) described similar proteins in the oocytes of R a m pipiens. The basic proteins from the sperm of Strongylocentrotus purpuratus were interesting in that no protamine was found in the spermatozoa; all basic proteins were extractable with 0 . 2 N NCI or with an absolute ethanol-HC1 mixture (179, 180). All acid-soluble proteins were typical histones and could be fractionated by chromatography on Sephadex G75 into the arginine-rich histones of the F3 and F2a type and into the lysine and arginine-rich transitional histones similar to these reported by Hultin and Herne in the Patella (176). illammalian spermatic1 cells also accumulate arginine-rich proteins (175, 181). Since techniqucs of artificial insemination have been developed in the agricultural scienccs, spcrrnatozoa from domestic animals hecame available in quantities suitable for chemical investigation. Difficulties in extraction of the sperm proteins from ram (182) and bull (183) spermatozoa and the composition of the acidic and alkaline extracts indicate that mammalian spermatozoa are covered by a keratinous membrane which protects the nucleus against mechanical damage. Bril-Petersen and Westenbrink (184) analyzed isolated nuclei from bovine spermatozoa ; difficulties in obtaining acid-soluble proteins led the authors to conclude that the spermatoeoan nucleus may resemble a
46
LUBOMIR S. HNILICA
sponge, consisting of a keratinoid not unlike ordinary keratin in its high sulfur content, but containing much more arginine (approximately 35% in the sperm heads as compared with 10% in keratin) ; DNA and soluble proteins would be enclosed in the cavities of the keratinoid sponge. Fractionation of the arginine-rich keratinoid solubilized by oxidation with performic acid resulted in three fractions with varying proportions of arginine, lysine, and cysteine. The ratio of total basic amino acids to the DNA phosphoric acid groups was unity indicating that the arginine-rich keratinoid in bull spermatozoa serves a purpose similar to that of histone in the somatic cell or of protamine in fish spermatozoa. More detailed information of the arginine-rich keratinoid fractions was presented by Henricks and Mayer (185, 1867, who disrupted the keratinoid structure in bovine spermatozoa with mercaptoethanol. Increasing mercaptoethanol concentration solubilized several protein fractions differing from each other by their arginine and cysteine content. One fraction of the porcine spermatozoa contained up to 46.8% arginine. The protein soluble in 0 . 3 N HCI was not basic. The mercaptoethanol fractions were electrophoretically heterogeneous on polyacrylamide gels. The scheme by which the arginine-rich keratinoid is biosynthesized is not known. Increased histochemical staining for arginine before the termination of spermatogenesis in bovine testes indicates that the biosynthesis of the arginine-rich keratinoid proteins may follow a schedule similar to that demonstrated for protamines and transitional proteins in fish and lower animals. Therefore, the only apparent biochemical characteristic of spermatogenesis is the high arginine content of the participating proteins. Proteins incorporating the arginine may be as different as protamines in fish, arginine-rich histones in sea urchins, transitional proteins in mollusk, or arginine-rich keratinoids in mammalian spermatozoa. The amino acid composition of several such proteins is illustrated in Table 111. In addition to their natural role in spermatogenesis, protamines exhibit significant effects on living cells. They have been found to be bacteriostatic, bactericidal, and cytotoxic, probably by changing the permeability of cellular membranes. They can change the membrane potential in nerve cells. I n tissue cultures and in vivo, protamines enhance the infectivity of viral RNA, probably by protecting it against degradation by RNase ; they also prevent emergence of antibiotic-resistant bacteria if added to the culture media. Interaction with other proteins, such as insulin and &lipoproteins of serum is another of the many capabilities of protamines. However, most of these biological properties of protamines arise from their high polycationic character and are not biologically specific.
TABLE 111 AMINOACID COMPOSITION OF ARQININE-RICHSPERM PROTEINS" Amino acid Lysine Histidine Arginine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Half-cys tine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine
Clupeine
-
Galline
-
1.6 60.0 0.8 2.4 11.2 1.6
7.5 6.6
5.6 8.0 1.6
3.7
1.6
74.1
-
2.1 4.6
-
-
1.2
0.8
Patella vulgata
Patella coerulea
20.1 0.6 25.0
-
-
4.4 9.4 -
8.0
45.7 1.7 12.0
-
9.4 9.4 12.5 4.4 1.2 3.7
12.0 8.0 5.1
-
-
-
-
3.4 0.6 3.4 -
Porcine sperm F2
Bovine sperm F2
H-1
H-3
E-1
E-2
E-3
3.6 2.4 25.8 5.1 6.5 6.4 6.5
3.5 2.2 39.1 3.5 5.7 5.1 4.3
20.2 1.7 19.0 3.4 4.8 7.6 4.2
11.9 2.3 20.1 4.6 6.3 11.2 5.0
9.9 1.3 13.5 4.0 6.1 5.8 9.1
16.0 1.3 15.3 3.4 4.6 6.1 6.0
11.8 1.8 11.2 6.0 3.7 5.1 7.1
5.3
2.5 5.4 4.1 6.7 3.7
2.8 4.2 18.9
2.0 10.0 7.8 5.8 1.2 4.2 4.0 2.3 1.1
3.9 8.3 12.2 0.4 5.4 1.2 5.0 8.2 2.3 2.6
5.8 6.5 16.6
3.8 11.4 12.4
5.0 0.4 3.6 6.0
6.2 4.5 10.3 2.3 1.8
5.0
5.9 6.5 5.1 1.1 3.8 4.9 4.3 1.7
1.o
2.5 4.8 3.3 2.5
Strongylocenlrotus purpuratus sperm
-
4.5 1.3 2.5 2.7 1.1 0.8
-
1.4 1.5
-
All values are expressed as percent of total moles of amino acids recovered. The data were recalculated from following references: clupeine (151), galline (151, 144), Patella vulgata and PateZZa coerulea (151, 176), porcine and bovine spermatozoa (186). Fractions H-1 and H-3 and E-1, E 2 , and E-3 correspond to the protein peaks resulting from chromatography on Sephadex G-75. Fractions E comprise the ethanol-HC1 extract, fractions H comprise subsequent extraction with 0.2 N HCl (177).
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LUBOMIR S. HNILICA
IV. Histones A. Molecular Properties Ever since the discovery of histones nearly a century ago, their extractability by dilute mineral acids has been the principal procedure for their preparation. In a typical isolation of histones from calf thymus, the tissue is repeatedly extracted with isotonic saline, preferably buffered with phosphate or citrate. The resulting white mass consists mainly of nucleohistone (14, 187), from which histones can be extracted with dilute mineral acid. Strong acids, such as HC1 or H,SO,, are used for complete extraction (187, 188) as the use of weaker acids (H,PO,, oxalic acid, citric acid, HCIO,, etc.) achieves partial fractionation of histones. Unless high concentrations of weak acids are used, part of the histones (arginine-rich) remains associated with the DNA (189-193). Dilute HCl is often preferred to H,SO, because the higher aggregation of histone sulfates may complicate their later fractionation (194, 195). The procedure developed for calf thymus was successfully applied to spleen and Walker carcinosarcoma (62,180). For other tissues (liver, kidney, brain, etc.) it is necessary to prepare “clean” nuclei prior to the isolation of histones. Removal of proteins soluble in isotonic saline is essential even if clean nuclei are used, since part of the protein in nuclear sap and ribosomes is soluble in acid and will contaminate the histones. Additional extraction of the saline-washed nuclei with 0.1 M Tris buffer, pH 7.6, is recommended to further decrease contamination (16, 41). Because of the presence of proteolytic enzymes in most nuclear preparations (31, 196, 197), acid extraction of histones is preferred to the use of alternate procedures such as the dissociation of nucleohistones with salt solutions of high ionic strength (14, 187). Lacking a rigid tertiary structure, histones are very sensitive to degradation by proteolytic enzymes; therefore, use of protease inhibitors during the isolation of nucleohistone may be advantageous (31, 62). Since histones are protected from the action of proteolytic enzymes as long as they remain associated with DNA in nucleohistone, employment of proteolytic inhibitors is especially indicated in procedures involving the dissociation of nucleohistones during histone isolation. The extraction of the nuclei or tissues with isotonic saline removes most of the proteolytic enzymes (196). After solubilization in acid, the histones can be recovered by dialysis and lyophilization or by precipitation with ethanol or acetone (187, 188). 1. HETEROGENEITY OF HISTONES
Since the research of Kossel (1) and his associates, it has been known that histone is a heterogeneous protein. Later investigations revealed the
PROTEINS OF THE CELL NUCLEUS
49
presencc of several components sep:irnI)le by electrophoresis, ultracentrifugation, and chemical fractionation ( 3 ) .The first fractionation procedures distinguished two main histonc fractions, later designated as lysine-rich and arginine-rich histones from their amino acid compositioi (188, 190). Stedman and Stedman (188, 198), on the basis of theii experiments on chromosomal proteins, proposed that histones may participate in the regulation of biochemical expression of the genes. According to this theory, more than two kinds of histones would be expected in nuclei of mammalian cells. Indeed, after extensive investi~, S y , 1.6 Sy) were isolated and gation, six histone fractions ( ( Y , , L Y ~ , ( Y , ,0.8 :in:tlyzetl in their laboratory ( 189). The hetc~rogeneityof liihtoncs became fully appreciated when zone electrophoresis in starch gel was applied to histones from different tisbucs. Neelin and liis associates observed a t lcnst 16-1 8 distinct electrophoretic zones in preparations of unfractionated chicken erythrocyte, calf thymus, and other histones (200-202). Using urea to decrease the aggregation and interaction of histones a total of 22 zones was obscrved in calf thymus histone preparations (201). ,Johns et al. (205), with a simplified version of the horizontal starch gel elcctrophoresis, isolated the individual major bands, extracted protein from each and determined amino acid compositions. With minor exceptions, the compositions of the three main bands matched those of the individual fractions isolated by chemical fractionation or by column chromatography. This indicates that histone heterogeneity observed in gel electrophoresis comes a t least partially from true chemical heterogeneity and is not a n artifact produced by aggregation. Similar studies were performed more recently by MacPherson and Murray (204); chromatographic fractions of calf thymus histone were subfractionated hy preparative electrophoresis in starch gel. The amino acid composition of the electrophoretic components again indicated that histones are composite proteins. Zone electrophoresis in starch gel has been used successfully for the studies on fractionation, heterogeneity, and tissue specificity of histones (39, 41, 62, 92, 179, 180, 205-209). The resolution of the starch gel method was improved by including small amounts of AlCl, in the electrophoretic media (41). The improved procedure was used for quantitative studies on histones from different tissues. The introduction of zone electrophoresis in polyacrylamide gels by Cruft (210) further refined studies on histone heterogeneity. This method in various modifications has been used by many investigators (206, 211-217). The greatest advantage of the polyacrylamide gel is its high resolution. The resolution recently achieved by Shepherd and Gurley (218, 219) , who compared samples of unfractionated calf thymus histones from several investigators with the main histone fractions, showed that the high resolution of polyiwylamide electrophoresis actually may
50
LUBOMIB S. HNILICA
be too sensitive for comparing samples of unfrsctioiiated histones. However, this method is an excellent aid to the studies on fractionation of histones. As in starch gel electrophoresis, the resolution of histones in polyacrylamide gels can be improved to some extent by adding urea to the electrophoretic medium (211,220). Owing to the sensitivity of histones to proteolytic enzymes, it was considered that histone heterogeneity may arise a t least partially from degraded histone molecules derived from a few basic fractions. It is very probable that some claims of tissue specificity of histones based mainly on comparison of their electrophoretic profiles may come from such degradation (209). However, careful studies by Rasmussen e t al. (206) on fractions obtained from three different preparations of calf thymus histone revealed remarkable similarity of the chromatographic and starch gel electrophoretic patterns regardless of the method used for their preparation, thus discarding the possibility of major changes in histone heterogeneity during their isolation. Another consideration that histone heterogeneity may he produced by partial hydrolysis during the extraction with strong acid was made and received some support from ,Johns (221),who studied the effect of mild acid hydrolysis on the heterogeneity of the very lysine-rich fraction F1. However, by comparing the electrophoretic or chromatographic profiles of unfractionated histones obtained by direct acid extraction of the nucleohistone with samples prepared by dissociation of nucleohistone in salt solution of high ionic strength containing inhibitors of proteolytic enzymes, no significant hydrolysis could be shown. The dissociated histone can be separated from DNA by prolonged ultracentrifugation (14). When two such preparations were run in zone electrophoresis, identical patterns were obtained for both (222). Since there is no known enzyme with exactly the same Epecificity as hydrolysis by dilute HC1 (0.2 N ) , it can be concluded that the electrophoretic heterogeneity of histones as observed by many authors truly reflects many protein fractions. The exact number of histone fractions in various tissues is not known. In addition to several main histones that are relatively homogeneous, a large number of “minor components” are present in each histone preparation (212, 218, 219, 223). Whether the “minor components” are a reflection of true microheterogeneity of histones, or whether they are products of aggregation and/or partial degradation is not known. It is noteworthy that Reid and Cole (224), who studied the biosynthesis of very lysinerich histones in calf thymus, observed increased electrophoretic heterogeneity of samples prepared from incubated nuclei as compared with samples ohtained from freshly frozen tissues. The increased heterogeneity was attributed to proteolysis. Recent discoveries that histone
PROTEINS OF THE CELL NUCLEUS
51
fractions are partially acetylated, phosphorylated, thiolated, or methylated may also contribute to the observed microheterogeneity of histones. 2. HISTONEFRACTIONATION Differences in the amino acid composition between the arginine-rich and lysine-rich histones effect sufficient differences in the chemical properties to permit their separation. Isolation of a calf thymus histone fraction containing more than 25 mole-percent lysine and almost as much alanine was reported by several authors. Methods as different as prccipitation with ethanol (188, 194, 225), isoelectric precipitation with ammonia (190, 226), extraction with citric acid (189, I N ) , and gradual dissociation of the nucleohistone with salts (190, 227, 228) were employed to obtain the lysine-rich histones. The residue left aftef the removal of the lysine-rich fraction and extractable with strong acid was frequently referred to as the arginine-rich histone fraction. More sophisticated fractionation reflecting the heterogeneity of histones was introduced by the application of ion-exchange chromatography to the fractionation of histones. Fractions very rich in lysine, moderately rich in lysine, and moderately rich in arginine were obtained by chromatography of histones on Ambcrlite IRC 50 (229-232), or on carboxymethyl cellulose (62, 180, 206, 253-236). Several fractions resulting from these procedures are compared in Table IV. Gel filtration on Sephadex G-75 of calf thymus histone dissolved in 0.02N HC1 was introduced by Cruft (210, 237) and resulted in its resolution into four diffuse peaks corresponding approximately to the fractions 01, p , and y described previously by Cruft e t al. (199, 238). Johnson et al. (206) studied the effects of extraction of the calf thymus histones with various concentrations of acid, comparing the fractions by gel filtration on several different grades of Sephadex. Their results together with Cruft’s clata indicate that the whole histone cannot be successfully fractionated into its main fractions in a single-step procedure. Better separation was obtained by using selective extraction to fractionate the histones into the argininc-rich group, F2aF3, and the lysine-rich group, FlFPb, prior to their chromatography on Sephadex G-75. The two histone groups were then successfully fractionated into the fractions F3, F2a1, F2a2 and into Fl and F2b (63, 239, 240). Cross-contamination of the fractions could be largely prevented by discarding the overlapping portions of the protein peaks. Gel filtration on Sephadex and on Biogel was also successfully applied to the subfractionation of the arginine-rich histones F3 and F2a (240, .%$l)and to the arginine-rich /3 and the very lysinerich (Y histones from chicken erythrocytes (222, 2@-244). The main advantages of gel filtration of histones are the high recovery of the frac-
TABLE IV A COMPARISON OF AMINOACIDCOMPOSITION O F MAJORHJSTONE FRACTIONS* Very lysine-rich
Moderately lysine-rich F3
111-IVb
10.1 1.9 11.1 0.3
10.3 2.4 13.6 ND
9.4 0.9 13.3 1.8
5.7 5.2 3.2 9.2
4.7 7.0 4.9 9.2
4.5 6.5 3.9 10.6
4.3 6.4 3.8 9.8
4.0 8.1 11.3
3.4 11.1 11.9
3.9 9.9 11.1
4.4 6.0 13.4
6.5 1.6 4.9 8.9 3.2 1.3
5.8 0.4 4.4
6.4 1.1 5.2 8.5 2.7 2.0
4.1 7.9 11.5 5.6 1.1 5.2 9.5 2.5 2.8
Amino acid
F1
Ibb
a2c
F2b*
IIb*b
1.6 Syc
F2a*
Lysine Histidine Arginine r-N-methyllysine Aspartic acid Threonine Serine Glutamic acid
27.7
-
28.6 1.5 -
26.0 0.8 3.3
15.4 2.3 7.6
13.5 2.8 7.9
-
11.3 3.2 8.1 ND
10.7 2.6 11.5 ND
2.1 5.4 6.8 3.7
1.9 5.2 5.9 3.0
2.4 4.9 4.9 4.1
5.6 5.2 7.8 8.7
6.5 5.7 6.5 8.9
Proline
10.0 6.9 25.1 4.3
10 3
9.8 9.0 22.8
4.7 8.2 11.5 6.7 0.8 4.5 8.6 3.0 1.3
Glycine Alanine Half-cystine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine
-
1.8
-
0.9 4.3
0.5 0.5
6.7 26.3 4.9 0.8 3.9 0.5 0.4
I
-
4.9 I
1.6 4. I 0.8 0.8
-
6.3 4.3
7.9 7.4 5.0 7.1
11.2 7.5 1.4 5.0 6.4 3.7 1.6
Arginine-rich
-
-
10.8
2.3 1.3
L
0.1
4.8 1.5 4.9 9.2 2.1 2.0
pd
8.8 2.0
11.9
ND
5.2 6.2 4.1 9.6 4.5 9.3 11.8
-
5.7
1.o 4.9 9.8 2.7 2.6
All values are expressed as percent of total moles of amino acids recovered. Fractions marked with an asterisk (*) -can be further fractionated. From Murray (193). c From Cruft et al. (199). dFrom Mauritcen and Stedman (391). e ND, not determined.
PROTEINS OF THE CELL NUCLEUS
53
tioiis, mild conditions of the fractionation, and excellent reproducibility even for histones difficult to fractionate by ion exchange chromatography (those from liver, spleen, testes, nucleated erythrocytes, etc.) (63, 222, 2-42-245). Because of the limitations of ion-exchange chromatographic procedures in the separation of closely related histone fractions and because of their much lower efficiency in fractionating histones from tissues other than calf thymus (180), fractionation of histones by selective extraction and precipitation is being increasingly utilized. The very lysine-rich histones (Ia, Ib, A, a, F1) are held by much weaker bonds to the DNA than tlic rest of the histone fractions. This feature of lysine-rich histones n.liich lcd to their early recognition and isolation was recently utilized in aevcral isolation procedures [e.g., extraction with H,SO, in the pH interval 2.C1.75 (19S),extraction with 0.02M HCl (922),or with 0.2 M citric acid (189),and by salting-out procedures ( 2 4 6 ) l .The very lysinerich histone fraction is solublr in 5% trichloroacetic acid or 5% perchloric acid while other histones form precipitates (194, 180). This feature was employed by deNooij and Westenbrink (247) and by Johns and Butler (179, 648) for rapid and highly selective extraction of the very lysine-rich histones from calf thymus and other tissues. Selective dissociation of the very lysine-rich histones from the DNA in nucleohistone can be also achieved by gradual increase of the NaCl concentration in the extraction mixture (190, 998, 249-251a) ; a concentration of 0.5-0.6M NaCl is sufficient to remove most of the lysine-rich histones ( B l ) . Further increase in the ionic strength will release the moderately lysine-rich group, F2, and the last t o be dissociated are the arginine-rich histones, F3 (in 2.0 M NaC1). An interesting modification of the dissociation procedure was reported by Huang et al. [quoted in ( 7 ) ] ,who fractionated histones by centrifugation through a sodium perchlorate gradient. The histone fractions dissociate in the following sequence: (a) w r y lysinc-rich (Fl or Ih) , 0.24.25 M ; (b) modcrately lysinc-rich (F2sF21, or IIb), 0.4-0.5M; :md ( c ) argininc-rich (part of F2a and F3 or I11 and I V ) , O . M . 8 ill NnC10,. Similar rcsults were reported hy Olden buscli ef al. (951b) wlio sturlicd the dissociation of nucleohistones by NaCl and NaC10, in great dctail. It is assumed that the main factor determining the force by which histones are held to the DNA is the lysine and arginine content and possibly the size and shape of the fractions (3, 7, 163). A combination of differences in dissociation and solubility properties of histones, already utilized by Stedman and Stedman (188) and by Bijvoet ( 2 2 5 ) , was more recently vmployed by Johns e t al. (17.9, 180, 235, 948) in a method for a large-scale fractionation of histones into the five main fractions, F1, F2a, F2a1, F2a2, and F3. I n the original pro-
54
LUBOMIB S. HNILICA
cedure, the ethanol-washed nucleohistone was extracted with a mixture of absolute ethanol and 1.25N HCl (4 :l) to yield the arginine-rich fractions F2a and F3, which are solubiliaed by this procedure. The residue was then treated with 0.25 N HCl to obtain the lysine-rich group, F1 and F2b. The arginine-rich histones can be fractionated into the F3 and F2a components by dialysis against absolute ethanol (fraction F3 precipitates) (179, 235), by chromatography on carboxymethyl cellulose (62, 180), or by gel filtration on Sephadex G-75 (239, 240). The lysinerich histones F1 and F2b can be separated by precipitation with acetone (248) or with 5% trichloroacetic acid (62, 180), by chromatography on carboxymethyl cellulose (62, 252), or by gel filtration on Sephadex G-75 (63). The main advantages of the selective extraction procedure are its simplicity, speed, and, most importantly, its applicability to various mammalian tissues (62, 63, 180, 177, 252-255). However, this procedure requires close scrutiny of the fractions by gel electrophoresis or amino acid analysis due to the tendency of the fractions F2a and F2b to crosscontaminate each other (7, 4 1 ) . More recently, Johns and Butler (179, 248) further improved their method by extracting the very lysine-rich fraction F1 with 5% HC10, prior to the treatment with ethanol-HC1. Preparation and analysis of basic proteins by means of their complex formation with the Reinecke salt was recently reviewed by Lindh and Brantmark (256). The F2a histones were further fractionated into two components differing in their amino acid composition and in tryptic peptide maps (F2al and F2a2) by differential precipitation with acetone (257), by dialysis against n-propanol and n-propanol-ethanol mixtures (207), by chromatography on carboxymethyl cellulose (257), by gel filtration on Sephadex G-75or Biogel P60 (240, 257), by salting-out with NaCl (24O), and by preparative electrophoresis (204). The amino acid composition of histones is partially reflected in theii nomenclature. There are a t least five major histone fractions obtainable by the current fractionation procedures that can be sufficiently characterized by their amino acid composition, electrophoretic mobility, etc. The amino acid composition of the five major fractions from calf thymus is shown in Table V. A small amount of an unusual amino acid, C - N methyllysine, has been recovered from the arginine-rich histone fraction 111 and IV by Murray (205, 6 5 8 ).The presence of this amino acid seems to be a general feature of the arginine-rich histones in several tissues (2.39).
The free NH,-terminal amino acids in most histones are alanine and proline (3, 62, 180, 231, 252, 269-261). I n addition to these two major NH,-terminals, small quantities of almost all other amino acids except methionine, arginine, and tyrosine are found in histone preparations from
55
PROTEINS O F T H E CELL NUCLEUS
TABLE V OF THE MAIN HISTONE FRACTIONS FROM CALF THYMUS^ AMINO ACID COMPOSITION Amino acid
Fl
F2aI
F2aII
F2bI
F2bII
F3
10.3 2.4 13.6 4.5 6.5 3.9 10.6 4.4 6.0 13.4 0.1 4.8 1.5 4.9 9.2 2.1 2.0
~
Lysine Histidine Arginine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Half-cystine Valine Methionine Isoleucine Leucine Tyrosine P henylalaniiie
27.7 -
1.8 2.1 5.4 6.8 3.7
10.0 6.9
26.1 4.3 0.9 4.3 0.5 0.5
10.0 1.9
13.9 5.1 6.7 2.5 6.4 1.4
12.6
16.6
14.3
2.8 9.5 5.6 4.9 5.0 8.9 4.2 9.1
2.6 6.9 4.8 6.2
2.4 8.7 5.7 4.8 3.7 8.8 4.4 9.9
16.9 7.6
13.1
-
-
8.0 1 .0 5.6 8.1 3.5 2.3
6.0 0.3 4.3 10.3 2.3 1.0
10.7 7.9 4.6 5.6 10.2
-
6.8 1.6
4.7 4.9 4.1 1.7
14.3 5.7 0.3 3.9 10.1 2.0 1.0
All values are expressed as percent of total moles of amino acids recovered. The serine values were corrected (10%) for hydrolytic losses. Fractions F2bI and FLbII were prepared by chromatography oil sulfoethyl cellulose. The main features distinguishing each fraction are printed i t i Idtlfare type.
various tissues (3, 62, 180, 231, 245, 261). Whether their presence indicates a microheterogeneity of histones similar to that observed in polyacrylaniide gel electrophoresis remains to be determined. It can be concluded from some early work by Phillips and Johns (197) that proteolytic enzymes are present in many histone preparations and that their action can easily increase the incidence of amino acids other than proline and alanine in NH,-terminal amino acid analysis of histones. Proline and alanine are the NH2-terminal amino acids of the histones F2b and F3, respectively. Few free NH,-terminal amino acids were demonstrated in fractions F1, F2a1, and F2a2. Phillips (260) demonstrated the presence of acetyl in all histone fractions, and it is assumed that in the fractions F1, F2a1, and F2a2 the NH,-terminals are acetylated; the minimum molecular weight calculated per acetyl residue for histones F1 and F2a was 15,000-18,000 and 10,00~14,000,respectively ( 2 6 0 ) . The proline and alanine NHr-terminal histones (F2b and F3) also contain a small amount of acetyl groups, but much less than the fractions F1 and F2a ( 2 6 0 ) . Serine bears the N-terminal acetyl in the histones F2al and F2a2 ( 2 6 2 ) . The carboxy-terminal amino acid in calf thymus histone fractions F1, F2a2, F2b is lysine, in fraction F2al it is glycine, and arginine and/or
56
LUBOMIR S. HNILICA
alanine is the C-terminal in fraction F3 (231, 252, 262, 2 6 3 ) . The 5% histone from chicken erythrocytes, rat spleen, rat thymus, and Novikoff hepatoma have lysine as the C-terminal amino acid ( 2 5 2 ) ; the Cterminal amino acid of the chicken erythrocyte specific histone fraction F2c is also lysine (263). Thc histone fraction nomenclature used by different investigators is rather confusing. Most authors rcfer to their fractions by letters or numbers indicating the scquence in which the histones emerge from chromatographic columns, e.g., A, B, C (229, %'SO), Ia, Ib, IIa, IIb, 111, IV, and V (231, 264), F1, F2, F3 (235). Others prefer to characterize histone fractions by their electrophoretic mobility, e.g., a l , cr2, as,P, 0.8 S y , 1.6 Sy ( 2 3 8 ) ,or E l , E2, E3 (203). Terminology based on the turnover rates (265) or the NH,-terminal amino acid composition ( 5 ) are among the more unusu:d. A need for a generally acceptcd nomenclature for histones that would sufficiently distinguish the existing histone fractions and accommodate :t11 future discoveries is obvious. Murray attcmptcd to introiluce &: sy&m 1):tscd on the ratios of basic amino acids, n:mely lysine :tnd arginirie (266). Unfortunately, his suggestions have not been gencrally ;tcwptrd, partially as a result of thc conservativc :tttitutlc of histonc I)ioclic*mists and because the lysioc-nrginine rntios alonc c:tnnot cliarnctcrizc the histone fractions sufficiently. Additional features, such as mobility in high-rcsolution zone elcctroplioresis and coiitcnt of amino acids most c1i:iracteristic for the fraction (e.g., alanine, scrine, leucine, isoleuciiie, glycine) may servc as the hasis of a general nomenclature for histones.
3. HISTONESTRUCTURE The heterogeneity of histones and formation of high molecular weight aggrcgates a t p H values near neutral and higher make accurate determination of molecular weights of histones very difficult. Davison e t al. (226, 26Y), Cruft et al. (195), and Ui (194, 268-271) investigated the aggregation of histones in detail; ionic strength, valency of the ions, pH, and temperature were all found to affect the aggregation substantially. When the molecular interaction was prevented or a t least decreased, e.g., by dissolving the histones in urea or guanidine hydrochloride solutions, the molecular weights of all histones decreased to a minimum between 10,000 and 16,000 (272, 273). More recent measurements of molecular weights of histone fractions are summarized in Table VI. Since a homogeneous protcin is required for meaningful sequential work, studies on the amino acid sequence of histones have obviously been limited t o preliminary studies on the size and nature of peptides present in enzymatic digests of several liistone fractions, chiefly of calf thymus
TABLE VI
MOLECULARWEIGHTSOF HISTONEFRACTIONS* Molecular weight determined from:
F1
XH2- and C-terminal
Sedimentation analysis
Fraction
amino acid content
F l : 13,000 (ref. 10, p. 36) F1: 21,100-24,600 (273) A: 8,000-12,000 (272) I: 8,400 (194) F l : 21,O0Oa
F2a
F2b
F3
F2a:
12,000"
F2bI: 13,O0Oa F2b: 22,000 (252)
.
F3III: 14,000°*b 8: 57,000 (195) B : 14,ooO-18,000 (272) 11: 37,000 (194)
+
Acetyl NHz-terminal amino acid content
63
F1: 37,oo(t61,000 (260) F1: 49,000-68,OOO (260) I-Ic: 7,60&11,000 (231) -
F1: 12,000-14,000 (260) -
-
F2a : 6 3 , 0 W l 4 9 ,000 (260) F2a: 85,000-113,000 (240) I : l5,00&17,500 (231)
F3a:
F2b: 15,oo(r17,000 (260) F2b : 23,ooO-25,000 (256) 11: 15,ooQ-17,500 (251)
F2b: 10,000-11,000 (260)
F3: 16,0W19,000 (260) F3 : 40,00(r55,000 (240) 111-IV: 18,000-21,000 (251)
9,OW12,500 (260) -
F3: 10,ooO (260)
* Numbers in parentheses are reference numben. Molecular weights estimated ,by Dr. A. Ansevin (Department of Physics, The University of Texas, &I. D. Anderson Hospital and Tumor Institute, Houston, Texas) on the basis of partial specific volume calculated from the amino acid composition and assuming a 2% reduction in partial specific volume on denaturation of the subunit in 5 711guanidine-HC1; the estimated error is f3,000g/mole. * Extrapolated value for monomer in a monomer-dimer system. 0
r r 2
58
LUBOMIR S. HNILICA
origin. Thc first to chcck tlic suspected regularity of amino acid sequences in histones (it was assumed that every fourth residue is a basic amino acid) was Satakc et al. (192), who digested several calf thymus histone fractions, obtained by chromatography on Amberlite IRC 50, with proteiriasc from S t r e p t o m y e s yriseus. The peptides were fractionated by chromatography ; analysis of their amino acid composition revealed tt whole spectrum of peptides containing arginine connected to either arginine, lysine, or another amino acid. Similar results were obtained for all the fractions (i.c., Ia, IIa, IIb, 111, and IV) indicating that there is no regularity in the distribution of basic amino acid residues in the histones. Phillips and Simson (27.4) confirmed the irregularity of amino acid sequences in liistonrs Ity isolating and analyzing tryptic peptides from calf thymus argiiiine-rich histone F3. In addition to the insoluble core, seven peptides were obtained. The spacing between the basic amino acids in that part of F3 histone from which the soluble peptides were prepared varied from adjacent (zero) to four nonbasic residues. In the insoluble core*, which was lieterogeneous, there wiis one basic :mino acid to every seveii residucs. Later, peptides with basic amino ncicl sparing as high as 7 :ind 8 residues were isolated from tlic tryptic tligclsts of F2a liistones of calf thymus and Walker carcinos;irconia ( 7 5 ) . Thc moticratcly lysinc-rich histone FZh, purified to :t consitlerablc extent, was studied by Hnilica ( 2 5 2 ) .A total of 25 major peptides was detected by paper c1iroin:ttogr:ipliy and electrophorrsis (“fingerprinting” or mapping) of thc tryptic digests. Preparations of this fraction from rat thymus, Walker carcinosarcoma, rat spleen, Novikoff hcpatoniu and chicken erythrocytes yielded identical peptide maps, indicating a high degree of homogeneity of this fraction (252, 2751. Out of these 25 major peptides, nine were isolated and analyzed, revealing the presence of peptides in length from 3 to 10 amino acid residues, several containing 2 to 4 residues of the same amino acid in one peptide ( 2 5 2 ) . Further studies on the homogeneity of F2b histone from calf thymus revealed the presence of two components both with the same C-terminal (-ThrSer-Ser-Lys) and differing in their NH,-terminal peptides [major fraction: Pro-Glu-Pro-Ala-Lys-, and minor fraction: Pro- (Asp,Glu,Gly,His, Ile,Ser,,Thr,Val) Lys] . Both components werc resolved by chromatogr:xphy on Fnlfoethyleellulo..c : m l the tryptic prptides of the major fraction were isolated by chromatography on Dowex 50-X-2 resin. The amino acid composition and squences of several of the isolated peptides :ire shown in Table VII. The composition and sequcwces of tryptic peptides obtained from the very lysine-rich histone fraction Ia were studied by Murray (7, 27G)
59
PROTEINS OF THE CELL NUCLEUS
TABLE VII AMINOACID COMPOSITION AND SEQUENCES OF TRYPTIC PEPTIDES FROM CALFTHYMUS HISTONEFZbI Peptide"
Composition
w c
Glu-Gl y-L y s (Glu,Serz,Tyr3,Valz) Lys Glu-Ile-Glu (Ala,Thr,Val)Arg (Ala,Glu,Gly,Leu4,Pro)Lys Ile-Ala-Gly-Glu(Ala,Ser)A g (Ile,Serz,Thrz)Arg Thr-Ser-Ser-Ly b: (Ala,Glu,Gly,Ser,Thr,Val)Lys Ma-Val-Thr-Lys Ala-Glu-Lys Val-Leu-L ys Lys- Gly-Ser-Lys Ser- Ala-Pro- Ala-Pro-L ys Pro (Ala,Glu,Pro)Lys Ser-Arg
Core
(Ala,Asp3,Arg,Gluz,Gly,Iles,Lys,Me~,Phe~,Ser~,Tyr,Val~)
1 2 3 4
5a 5b 7b 9 10 11 12
18 A
Peptides are numbered according to their position on a two-dimensional peptide map. Lettered peptides remain to be located. CORE is a precipitate resulting from tryptic digestion of the F2bI histone. * C-terminal peptide. c NHz-terminal peptide.
and his associates. A combination of ion-exchange chromatography, paper chromatography, and electrophoresis resulted in the isolation of more than 60 peptides, indicating considerable heterogeneity of this fraction. The isolated peptides varied in length with the longest being a pentadecapeptide. Accumulation of several identical amino acids in one peptide was common; the most peculiar peptide contained 4 prolyl residues ( Ala,,,Pro,,Glu2,Thr,Ser,Lys) .
Another histone suitable for scquential studies is the clcctrophoretically fast portion of the fraction F2a (component F2a2). This fraction nas recently isolatcd in a fairly homogeneous form by Phillips and Johns ( 2 5 7 ) ,hy Hnilica and Bess (207, 24O), and by MacPherson and Murray (204). The NH,-terminal sequence of the F2a2 histone is N-acetyl-SerGly-Arg (262) ; the same peptide was also isolated from the arginine-rich subfraction F2al. Analyses of the peptides from various histone fractions indicate that the histone molecules cannot follow the DNA helix very closely without parts of their sequences looping out and possibly crosslinking several strands of DNA.
60
LUBOMIR S. HNILICA
6. Molecular Functions The functional form of DNA in higher organisms is an association of histones, lipids, RNA, and nonhistone proteins in a complex called chromatin or nucleoprotein (DNP) . The physicochemical and biochemical properties of chromatin have been difficult to study because of the poor solubility in dilute solutions of salt, e.g., isotonic saline. Solubilisation of the D N P by exposure to solutions of high ionic strength produces dissociation of the primarily electrostatic bonding between DNA and histone and is therefore not suitable for physicochemical studies ( 14, 228). The complex in concentrated NaCl can be easily reassociated by diluting the NaCl to the physiological molarity ; the reassociated nucleohistone precipitates in the form of long fibers (14). However, physicochemical studies on the reassociated D N P indicate tbat it differs from “native” samples ( 7 , 927, 277). I n water, D N P forms a gel that progressively swells on dilution and ultimately dissolves into a viscous dispersion (278). The behavior of D N P in water was found to depend on its concentration, thereby indicating strong interaction of the macromolecules (279). The interaction caused by a negative charge on D N P can be decreased by addition of salts to eliminate the polyelectrolyte effect. Unfortunately, DN P precipitates even a t salt concentrations as low a s 0.01 M (280). The gel-forming properties of nucleoproteins suspended in water are regarded by many authors as a characteristic of enzymatically undegraded samples (51, 281-283). Since DNA a t similar concentrations does not form crosslinked gels, the contribution of protein to the gel-forming properties of D N P is obvious. Most likely the formation of gel is assisted by histones crosslinking DNA strands and interlocking the whole structure (284-286). Electron microscopy, reconstitution, and dissociation studies indicate that the histone fraction participating in the crosslinking of D N P is the lysine-rich histone F1 (298, 287-9899). However, according to Ilounce and Hilgartner ( 5 1 ) , the sulfur-containing acidic nuclear proteins may play a major role in the maintenance of the D N P gels. According to these workers, the acidic proteins are linked to the DNA by covalent bonds and the crosslinking is realized by the formation of -S-Sbridges. Zubay and Doty ( 9 7 8 ) , using EDTA and careful homogenization, prepared gel-free solutions of D N P from calf thymus. The DNP, dissolved in 0.7 mM phosphate buffer, consisted of highly assymetric molecules completely dispersed in the solution. The molecular weight of the particles was 18.5 million with the DNA accounting for approximately one-half of this weight. Later reports by other investigators
PR(YI'E1NS OF THE CELL NUCLEUS
61
confirmed the reproducibility of this procedure (290, 291). The DNA in aqueous solutions of D N P is double helical; most of the proteins conform to the a-helix (278). The gel-forming property of D N P is a t least partially dependent on the method of preparation and possibly on a partial enzymatic degradation of the D N P (282). Fredericq (283) prepared two nucleoprotein fractions from calf thymus, one :t gel and the other a solution. These two differed in their protein/DNA ratios (1.3 and 1.1); the relative proportions of the two fractions depended on the cxtraction procedure. The soluble fraction was similar to the soluble D N P prepared by Zubay and Doty (978).Comnierford e t a2. prepared the D N P from calf liver ( 2 5 1 ~, ) using a procedure similar to that of Zubay and Doty; however, gel formation was never observed during the extraction from isolated nuclei. The lack of gel formation by nucleoproteins from other tissues was also noted by others (292-294). It is noteworthy that histones isolated from water-soluble D N P preparations (calf thymus, r a t spleen, liver, etc.) showed considerable proteolytic damage when inspected by zone electrophoresis and other criteria (222). Electron microscopy of D N P preparations shows that the protein surrounds the DNA core in the form of an evenly distributed continuous envelope (86, 278, 295) . Electrometric and spectrophotometric titration reveal that, in the D N P of calf thymus, 80% of the lysine and arginine residues are inaccessible for titration a t p H 11.8, indicating that they are involved in ionic bonds to the phosphate groups on the DNA backbone. Carboxyl, imidazole, and tyrosyl groups are freely accessible to hydrogen and hydroxyl ions and therefore must be a t the surface of the D N P complex and in contact with the solvent (296). Optical rotary dispersion and infrared spectroscopy of the watersoluble calf thymus D N P indicate that about two-thirds of the protein is in an a-helical conformation. Zubay and Doty (278), assuming the partially a-helical structure of histone in DNP, proposed that the histone chain is accommodated in the large (10 A radius) groove of the DNA helix. I n order to achieve maximal packing, the histone a-helix must be interrupted by nonhelical regions spaced approximately 15-20 A apart. Such breaks in the a-helical structure of histone would permit the next a-helical region to make maximum contact. However, the highly irregular spacing of basic amino acids revealed by the analysis of tryptic peptides of several histones makes the maximum lengthwise contact of histones and DNA quite improbable (697). More recently, Zubay (284) has suggested that the histone molecules can change their direction relative to the DNA depending on the state of hydration. In the gel state, histones crosslink DNA strands by being
62
LUBOMIR S. HNILICA
parallel to the large groove of DNA, with their long axis a t an angle of 60 degrees to the long axis of DNA; in solution, the histones lie parallel to the DNA helix. The proposed model results in a sheetlike structure, with bridges in the plane of the sheet. I n oriented sheets of D N P films a t relative humidities over 80%, the DNA is in the B form (698). Itzhaki (27’7, 285), who used electric birefringence in combination with ultracentrifugation and enzymatic degradation, concluded that rat thymus D N P in very dilute phosphate buffer represents an array of laterally arranged molecules of the same range of length, up to 1.6 pm. In the gel, the molecules form an interlocked network that partially breaks up into clusters on dilution. I n very dilute solutions, the clusters disperse into individual moieties. Precipitation of the water-soluble gel in 0.15 M NaCl induces irreversible changes of the original structure. The protein (histone) does not associate with DNA lengthwise unless t o form frequent loops. Metal bonds, if present, do not affect the aggregation behavior of the DNP. An arrangement of D N P into micelles, with DNA helices running parallel and with water and histones in the interstitial spaces, was suggested by Luzzati and Nicolaieff (162). I n isolated form, histones are much less a-helical than in the association with DNA. When dissolved in water, all histone fractions are disordered (299-301). Infrared spectra of solid histone samples indicate the presence of an a-helix (298, 502) ; samples treated with ethanol or samples stored in the cold for some time show a decrease in their ahelical content with a simultaneous increase i r ~the @-type 1630 cm-’ absorption (698, 302). In films cast from water, the optical rotatory dispersion (309) indicates an a-helix content of calf thymus histone hetween 25 and 37%. Again, the aging of the samples decreases their ahelical conformation considcrahly. In 2-rhloroethitnol or in n-propanol, the a-helical content of calf thymus histone samples increases to 5367%, i.e., to the values observed in native DNP (278, 302). A similar, though not so dramatic, increase of the a-helical content of histones was achieved by anionic detergents (304, 305). The four main calf thymus histone fractions (Fl, F2a, F2b, F3) behave much as the unfractionated histone. When dissolved in water, all the fractions are disordered. However, the presence of small amounts of salts (0.02-0.1 M ) or DNA facilitates the formation of a-helix in all the fractions except in F1, which remains disordered even in 1.0 M NaCl (300, 301). I n 2-chloroethanol, all the fractions become highly a-helical (48-600/0). The ability of DNA, salts, and anionic detergents to facilitate the formation of an a-helix in the histones is probably due to their decrease of the polycationic effect of histones (electrostatic repulsion). It
PROTEINS OF THE CELL NUCLEUS
63
is of some interest that the very lysine-rich histone F1, which is also the least a-helical of all the fractions studied, is very weakly bound to the DNA in D N P and can be easily dissociated by dilute acids or by exposing the DNP to 0.6 M NaC1. Walker (296), by electrometric and spectrophotometric titration, observed that the unfractionated histone from calf thymus appears in fairly stiff coils a t neutral pH, When the pH was lowered, the coils unfolded giving a more flexible structure ; this process was reversible. The charge distribution along the polypeptide chains of histones appeared to be very nonuniform; the positive charges were grouped in clusters. Such an arrangement of basic residues in histones is basically in agreement with the results of the analysis of tryptic peptides of several histone fractions. Histoiies stabilize the double helix of DNA against thermal denaturation. The melting temperature of the DNA in association with histones increases with the increasing lysirie content of the haistones and with the rising A+T content of the DNA (288, 289, 306). Deoxyribonucleoprotein, partially heat-denatured, sedimented on ultracentrifugation as two distinct boundaries, one having a sedimentation coefficient of completely denatured material. This indicates that thermal denaturation of D N P is an all-or-none phenomenon (307). A similar stabilization effect on the DNA was also observed with various polyamines (308), proflavine (309),actinomycin D (310), cystamine (311), and other substances. In the D N P complex, the DNA is partially protected from radiation damage by the close association with proteins, mainly histones (312, 313). Exposure of th.c D N P to NaCl concentrations that partially dissociate the DNA-histone complex (0.4-0.8 M ) result in a substantial increase in the radiation damage to D N P as measured by the extent of the release of histone by y-radiation. The released histones are relatively rich in lysine, and the histone remaining with the D N P after irradiation is weakly attached to the DNA. This loose association probably arises from partial deamination of the basic amino acids ( 314) . Lloyd and Peacocke (315) observed that the dissociation of histone from DNA by y-radiation is probably the result of a one-hit process while the degradation of DNA is a two-hit process. A combination of these two processes could explain the radiation-induced decrease in the molecular weight in the radius of gyration of the DNP. The release of histones from irradiated D N P could then be a result of the breakage of one of the DNA chains with. consequent disorganization of the helix in the vicinity of the break. The partial degradation or dissociation of DNP by radiation can
64
LUBOMIB S. HNILJCA
explain the observed loss of proteins from the nuclei of liver, thymus, and spleen of irradiated rats (316). The possible function of thiol groups in the radiosensitivity of D N P was discussed by Jellum and Eldjarn (317),who separated rat liver and calf thymus D N P into fractions differing significantly in their thiol content. Part of the sulfur was bound to the histones, predominantly to the arginine-rich F3 fraction (318). During mitotic division, D N P present in the nucleus in the form of chromatin condenses into chromosomes. According to Ris, two DNA molecules may be bound together by histone side by side to form a fiber; further folding of the fibers into fibrils 100 or 200A thick is achieved by their association with histones and divalent cations (86, 319). The exact arrangement of such supcrcoilcd fibrils in the chromosome is not known ; electron microscopy of metaphase chromosomcs offers some evidence that the 200-230A thick fibrils are again supercoiled and then folded into a ropelike structure. Helices made of such ropelike supercoiled D N P form the individual chromosomes (320, 3 2 1 ) . While arginine-rich histones are thought to participate in the association of individual DNA molecules (322, 323), the very lysine-rich histones seem to maintain the chromosomal superstructure (287). The structure and composition of the giant polytene chromosomes and of the expanded looplike lampbrush chromosomes in oocytes are further complicated by their high metabolic activity. Both types contain regions of loosely packed DNA that, in the lampbrush chromosomes, extend into very thin fibers. The extended parts, puffs in polytene chromosomes or loops in lampbrush chromosomes, are the sites of active RNA and protein synthesis. The ratio of histone to DNA as determined cytophotometrically is rather constant throughout the entire length of the polytene chromosome (85). Histones are still present in puffs, but may be more diluted by the products of their biosynthetic activity. In the lampbrush chromosomes in which DNA loops occur a t almost all chromomeres, histochemical localization of histones has been much more difficult. The lampbrush chromosomes of the newt oocytes contain 200 times the amount of protein found in liver chromosomes of the same animal. Most of this protein is not histone (324). Arginine-rich histones (F3) when added to the lampbrush chromosomes produce retraction of the loops within 3 minutes. Actinomycin D and polylysine have a similar effect whereas the very lysine-rich histones F1 have only a minor effect on the chromosomal loops (3125). Cytophotometric studies also reveal quantitative constancy of histones in chromosomes, similar to the constancy of DNA (325).Similarly, the ratio of arginine to the DNA phosphorus in somatic nuclei of many species remains constant (326). Using tritiated arginine and Vi& faba
PROTEINS OF THE CELL NUCLEUS
65
root meristem cells, Prensky and Smith (327) demonstrated almost complete turnover of a major fraction of chromosomal protein during one cell division cycle. In human leukocyte chromosomes (see Gavosto in this volume) , the incorporation of lysine-H3 into chromosomal protein occurred throughout interphase. The incorporation rates differed during various periods of the interphase ; the incorporation was diminished during GI, increased in early S reaching the peak in late S. The high rate continued into G,. Lysine-H3 incorporated into chromosomal protein was not distributed to the chromosomes of daughter cells in the semiconservative manner described for the DNA. This indicates that the chromosome proteins are dissociated during interphase and reassociate for the next mitosis in a random fashion (328). The exact composition and distribution of proteins in the sheath surrounding the chromosome is not known, but histones contribute a significant part of sheath protein. The amount of histone in the chromosomes isolated and analyzed to date accounts for less than one-half of the total sheath. protein (83, 84). Chemical evidence indicates that the histones are in salt linkage with the phosphate groups of the DNA and that there is nearly one basic histone amino acid residue for every DNA phosphorus (3). In order to achieve linkages with all the DNA phosphorus, histone molecules would have to be rather extended, either following the same strand or crosslinking several strands of the DNA in the chromosome. If this is indeed the state of histone, it would not contribute for more than the inner layer of the sheath, being sandwiched between the DNA and the acidic coat protein. That the main bulk of the sheath protein is indeed acidic is supported by recent analytical findings on isolated metaphase chromosomes (83, 84) and by the fact that such chromosomes are stabilized in acid solution (S29).
C. Cell and Species Specificity The biological function of histones has remained a challenge to many investigators. Based on their early observations, Stedman and Stedman (188, 198) proposed that histones interact with DNA in a specific manner, thereby preventing the DNA from relaying its genetic information to the biosynthetic apparatus of the cell. T o carry out this function, histones were expected to manifest a considerable degree of species and cell specificity. To support this thcory, Stedman and his associates studied the properties of a number of histones from various sources. In addition to the already known difference between the somatic histones and sperm protamines, they were able to detect a significant specificity in the arginine content of histones from chicken erythrocytes and wheat germ (188, 198). Mauritzen and Stedman (330,331) observed tissue
66
LUBOMIR S. HNILICA
specificity in the amino acid composition of the arginine-rich p-histones from a number of tissues of domestic fowl and ox. However, these differences were only minor and later investigation showed that they might have arisen from contamination of the P-histones by other proteins (180). Crampton e t al. (230) prepared the A and B fractions of histones from calf thymus, kidney, liver, and from guinea pig testis. The amino acid compositions of all the fractions and the chromatographic profiles of 21-hour tryptic digests of histone fractions B from all the four tissues were remarkably similar. The question of tissue and species specificity of histones in normal rat spleen and liver, leukemic rat spleen and liver, and in calf thymus was more recently investigated by Hnilica et al. (180). Amino acid compositions, starch gel electrophoretic patterns, and the NH,-terminal amino acids of the very lysine-rich histones F1, moderately lysine-rich histones F2, and the arginine-rich histones F3 were very similar, if not identical, in all the tissues. A similar conclusion was reached when the fractions F1, F2a, F2b, and F3 were prepared from Walker carcinosarcoiiia and compared with those from calf thymus ( 8 2 ) .More detailed studies, including analysis of the distribution and composition of tryptic peptides from the calf thymus, rat thymus, r a t spleen, Walker tumor, and chicken erythrocytes F2b histones, further confirmed their essential identity (252, 275). Laurence et al. (255, 332), who studied histone fractions F1, F2a, F2b, and F3 from calf thymus, Crocker sarcoma, spontaneous mammary tumor of mice, and osteogenic rat sarcoma D 177, also confirmed the striking lack of specificity of the histones. Davis and Busch (236, 2/35) analyzed acid extracts from crude nuclear preparations of various tissues from rats injected with 1 y ~ i n e - C ~ ~ . They reported a radioactive chromatographic peak (RP2L) supposedly specific for neoplastic tissues. Isolation and partial purification of the proteins from the RP2L peak revealed the presence of histone fractions F1, F2a, and F2b (333). However, subsequent analyses of corresponding histone fractions from Walker carcinosarcoma and from calf thymus indicated no significant tissue specificity (52). It was concluded that the peculiar behavior of nuclear acid extracts from malignant tumors when chromatographed on carboxyrnethyl cellulose probably arises from contamination and is not from the histones (334). The lack of tissue and species specificity of histones is not a characteristic of the mammalian species. Histone fractions strikingly similar to the calf thymus histones F1, F2a, F2b, and F3 were found in chicken liver, chicken erythrocytes (239, 245, 2G4), trout liver (253), buds of pea seedlings (335), sea urchin embryos (336), and sea urchin sperm
PRCWJCINS OF THE CELL NUCLEUS
67
(177). The amino acid composition of several of these fractions is shown in Table VIII. Stedman and Stedman (188, 198) noticed a much higher content of argininc in one of the two histone fractions from the erythrocytes in domestic fowl. This finding was later confirmed by starch gel electrophoresis of the chicken erythrocyte histones, which showed a characteristic slowly moving band absent in other chicken tissues (202). A fraction corresponding to this electrophoretic zone was later isolated by Neelin et al. (d64), with the aid of chromatography on Amberlite IRC 50 (fraction V), and by Hnilica (2451, by gel filtration on Sephadex G-75 (fraction 2c). The amino :icid composition of this fraction is very characteristic (approximately 21 % lysine, 15% alanine, 12% serine, and 11% arginine). The NH,-terminal amino acid of the F2c histone is mainly threonine, but its low recovery indicates possible inaccessibility of the NH,-terminal to dinitrophenylation. The C-terminal amino acid in the F2c histone is lysine ; cartioxypeptidase digestion disclosed that the F2c fraction is homogeneous (263). The biological function of F2c histones is not known. Similar histones were recently isolated from the nucleated erythrocytes of other genera (frog, fish, turtle) and from the sperm of the sea urchin Strongylocentrotus purpuratus (1’77, 337). Their amino acid compositions are shown in Table IX. Even though these proteins are not identical, their close similarity indicates that this type of histone may be characteristic of specialized tissues with arrested RNA liiosynthesis. Proteins similar to mammalian histones are found in Allium cepa, I‘icia faba, Pisum sativum (338), Tetrahymena pyriformis (339, 340) , in wheat germ (208), in Chlorella ellzpsoidea, and in rice embryos (341). All are typical histones, in general containing more lysine-rich fractions than their mammalian counterparts. The arginine-rich fractions appear to bc comp!ctely absent from wheat germ (208). Histone-like proteins werc alko observed in Staphylococcus aureus (342) and Staphylococcus epidermis (,?-$3), in EscheTichicr. coli (344, 345), and Bncillws mtvpterium (345). Unfoi tunatcly, few analyticd data on tlic bacterial basic proteins are arailahle, and i t is possihlc that the “histone-likc” :tcid-soluhle proteins in hactciia originttc from the ribosonieb. The high resolving power of zone electrophoresis on starch or polyacrylamide gels was also employed for the study of histone specificity in various tissues and animals. Starch gel electrophoretic patterns of histones from various tissues of chicken indicate significant specificity for the erythrocyte and testis histone (902). This observation led directly to the isolation by Neelin et al. (264) of the specific histone fraction of chicken erythrocytes. Vendrely e t al. (346), also using starch
8
TABLE V I I I AMINOACIDCOMPOSITION OF HISTONESFROM TROUT LIVEX,PEABUDS,AND CALFLIVER" Amino acid
Trout liver Flb
Pea buds I&
Calf thymus Ibd
Trout liver F2bb
Calf thymus F2b
Pea buds IIbc
Calf thymus IIbd
Trout liver F3b
Calf thymus F3
23.8 1.o 3.6 4.3 4.0 6.4 5.3
22.9 0.9 2.7 3.0 4.6 5.6 7.8
26.2 0.2 2.6 2.5 5.4 6.5 4.3
13.3 2.1 8.3 5.8 6.3 6.9 8.7
15.4 2.3 7.6 6.3 4.3 7.9 7.4
14.1 1.1 7.6 6.2 4.8 6.2 7.7
13.5 2.8 7.9 5.6 5.2 7.0 8.7
8.5 1.6 12.1 5.0 6.5 4.5 11.2
10.3 2.4 13.6 4.5 6.5 3.9 10.6
9.7 1.9 10.8 6.1 6.1 4.4 8.8
9.7 1.9 11.9 5.0 6.7 4.6 10.4
7.3 5.8 19.9 0.4 7.2 0.8 2.5 4.4 1.8 1.4
10.0 3.7 22.9
9.1 7.3 24.2
5.3 10.0 11.8 6.9 0.6 5.0 8.4 1.8 2.4
4.7 8.2 11.5 6.7 0.8 4.5 8.6 3.0 1.3
4.4 6.0 13.4 0.1 4.8 1.5 4.9 9.2 2.1 2.0
3.9 9.8 9.7
4.2 8.6 11.6
4.1 0.1 1.2 5.0 0.7 0.6
5.0 7.1 11.2 7.5 1.4 5.0 6.4 3.7 1.6
4.6 6.4 12.6
6.2 Trace 2.9 4.6 0.9 1.3
3.7 8.7 10.7 0.6 6.7 1.4 5.0 6.7 3.3 2.0
6.7 0.4 5.9 10.5 2.4 3.1
5.9 1.3 5.3 8.9
~
Lysine Histidine Arginine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Half-cystine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine a
-
-
All values are expressed as percent of total moles of amino acids recovered. From Palau and Butler (253). From Fambrough and Bonner (335). From Rasmussen et al. (205).
.o
1
5.1 1.3 5.1 9.3 2.5 3.0
Pea buds 111-IV" ~
~~
-
Calf thymus 111-IVd ~~
-
:::
B
P
!l
E P
TABLE IX AMINO ACID COMPOSITION OF HISTONES FROM NUCLEATED ERYTHROCYTES"
F3 histones Amino acid Lysine Histidine Arginine Aspartic acid Threonine Serine Glutamie acid Proline Glycine Alanine Half-cystine Valiiie Methionine Isoleucine Leucine Tyrosine Pheny lalanine
F 2 c histones
F2a histones
Chick
Fish
Frog
CTh
Chick
Fish
Frog
CTh
Chick
Fish
Frog
Urchin
9.0 2.4 12.6 5.9 6.2 5.2 10.9 4.4 6.1 11.5 0.2 5.0 1.7 4.6 9.2 2.3 3.0
9.8 3.0 13.6 7.1 5.8 5.4 9.5 3.8 5.7 11.3 0.2 5.3 1.4 4.0 9.2 2.1 3.3
10.0 2.6 13.0 5.5 6.2 4.5 10.0 4.4 6.0 12.4 0.1 5.2 1.6 4.4 9.2 2.1 2.9
10.3 2.4 13.6 4.5 6.5 3.9 10.6 4.4 6.0 13.4 0.1 4.8 1.5 4.9 9.2 2.1 2.0
10.9 1.9 12.6 5.4 5.1 4.3 7.4 3.0 11.5 10.8 7.0 0.9 5.0 9.7 2.8 1.7
9.4 2.2 13.1 6.2 6.2 3.4 8.8 2.9 10.6 10.3 7.1 1.0 5.2 9.1 3.0 1.6
10.3 2.3 12.7 6.1 5.4 3.8 8.5 2.7 10.7 11.8
10.4 2.1 12.6 5.2 5.9 4.2 7.6 3.0 11.5 10.8
24.9 1.8 11.4 1.7 3.1 13.1 3.7 6.9 4.7 15.2
23.9 0.9 7.1 3.9 4.9 8.8 3.9 6.2 5.6 16.2
28.7 0.5 9.9 3.1 4.2 7.8 1.7 7.0 4.7 17.0
20.0 1.7 20.7 2.8 4.5 9.4 3.1 2.8 5.0 16.8
6.3 0.6
6.7 1.0 5.3 8.9 3.0 1.7
6.2 0.7 3.4 5.8 1.5 1.0
5.8 0.1 2.8 5.3 0.6 0.6
4.8 1.0 2.7 2.8 1.2 0.6
-
4.8
10.2 2.8 1.5
-
c
4.0 0.5 3.0 4.0 1.4 0.5
-
-
-
All values are expressed as percent of total moles of amino acids recovered. Serine values were corrected (10%) for hydrolytic losses. Fish = Caranx hippos (common jack). Frog = R a m catesbeiana (bullfrog). CTh = calf thymus. Urchin = Strongylocentrotus ~ U T ~ U T ~ 0
~ U S .
70
LUBOMIR S. HNILICA
gel electrophoresis, observed a remarkable similarity in the histone patterns from calf thymus, liver, lung and from rat thymus and liver. However, significant differences were observed in the histone patterns from nucleated erythrocytes of several genera (chicken, duck, guinea chick, viper, tortoise, trout, carp, pike, pleurodele, and frog). Only minor differences were observed when the histones of several chicken tissues were compared with each other and with the corresponding preparations of histones from chickens at ages ranging from 4 days to adult (209). Similarly, polyacrylamide gel electrophoresis of histones from rat spleen, thymus, liver, kidney, and testis ( 2 1 2 ) ,and from brain, liver, and kidneys of newborn and adult rats, of adult rabbits and guinea pigs (213) showed only minor variations from the general pattern of all mammalian histones. No detectable changes were observed in the composition and electrophoretic patterns of histones during early embryogenesis (df6 ), and during the organogenesis (347) of chickens. It can be assumed that the minor differences among histones from various tissues of higher animals detectable by electrophoresis depend mainly on the method by which the histones are isolated (218, 219). Mild proteolytic degradation of histones increases their heterogeneity in starch gel electrophoresis (348) ; therefore, claims of histone specificity based solely on comparison of electrophoretic patterns and not supported by chemical analysis should be considered with great caution. The lack of species and tissue specificity of histones observed among vertebrates and in some other animals poses an interesting problem. It is now well established, that most proteins, both functional and structural, show in the amino acid sequences phylogenetic differences detectable by various analytical techniques. Yet, the histones are strikingly similar in most species. The biological significance of such. similarity is not known. It can be speculated that histones are part of a rather general and essential biological mechanism. The template for biosynthesis of the histones must then be a part of the primordial genome carried through the generations during the evolution of species. The fact that histones are rather poor antigens supports this suggestion.
D. Histone Biosynthesis Early studies on the incorporation of labeled amino acids into nuclear proteins led to the discovery that histones readily incorporate the label and that the biosynthetic rates of nuclear proteins reflect the physiological state of the cells (2, 4, 5, ‘7, 349352). I n well-differentiated and slowly dividing tissues such as liver, the acidic nuclear proteins are much more active metabolically than histones. As the mitotic rate increases, histones incorporate mare actively until, in rapidly dividing tissues, the
PROTEINS OF THE CELL NUCLEUS
71
histones turn over at rates approximately equal to those of the acidic nuclear proteins (4-6, 16, 63). Improvement of the methods for fractionation of histones brought about an increasing interest in the metabolism of individual histone fractions. Differences in the incorporation of labeled amino acids into several major histone fractions indicated that histone biosynthesis is controlled by individual genetic loci and that the observed heterogeneity of histones is natural (4, 5, 62, 63, 349, 353). Chalkley and Maurer (353) recently investigated the incorporation of leucine-C14 into the chromatographic fractions Ia, Ib, IIb, 111, and IV of histones from rat liver, calf endometrium, pea cotyledon, and tobacco cells. Their work showed that the turnover of histone fractions depends to a considerable extent on the mitotic rate of the tissues. I n slowly dividing tissues, only the arginine-rich histone fractions 111 and IV became labeled; the lysine-rich histoncs I and I1 were actively labeled only in tissue or cells with extensive DNA replication. However, complete absencc of labeling of the lysinc-rich histones of rat liver in the presence of active uptake into the arginine-rich group is still controversial and has not been confirmed (63, 254, 349, 354). The labeling behavior of the four main histone fractions (Fl, F2a, F2b, and F3) in normal and regenerating rat liver and in Novikoff ascitic hepatoma was studied by Hnilica et al. (63). They also observed pronounced differences in the labcling of histone fractions in the liver. However, tlicre was significant uptakc of the labeled amino acids into all the histones in liver. The very-lysine-rich histone (F1) and the arginine-rich histone (F3) incorporated lysine-C" more readily than did fractions F2a and F2b. The differences in histone labeling decreased with increasing mitotic activity of the cells, arid in Novikoff hepatoma all the histone fractions had approximately similar turnover rates. Ord et al. (215, 5551, who studicd primarily the thiol content and the phosphorylation of nuclear proteins, also reported differences in the incorporation rates of labeled amino acids into histone fractions. Laurence and Butler (254) compared the incorporation of lysine-C1* or arginine-V into the histone fractions F1, F2a, F2b, and F3 from several experimental tumors and from liver of rats. I n the tumors, all thc fractions were labeled to approximately the same extent. I n liver, differences were observed that were not regarded as significant. It was also observed that histones, once labeled, turned over very slowly; in liver the whole histone lost about 50% of its activity in about a week. However, the activity of the arginine-rich group F2aF3 (soluble in ethanol-HC1) decreased only by 24% in this time. The more rapid disappearance of label in the lysine-rich histone from the DNP complex in liver does not seem
72
LUBOMIB S. HNILICA
to be compatible with the observation stated in the same paper indicating that the rates of the biosynthesis of all histone fractions in liver are practically equal. The turnover rates of histones in different tissues of the rat indicate a metabolic integrity of the DNA-histone complex (354) and do not confirm the faster turnover of the lysine-rich histones in liver observed by Laurence and Butler (254). Slow turnover rates of histones from brain and other tissues were also reported by Piha, Cuenod, and Waelsch (356). Incorporation of labeled amino acids into nuclear proteins during the regeneration of rat liver was studied by several workers (26, 27, $67, 358). Again, individual histone fractions differed from each other in their uptake of labeled amino acids; more importantly, the incorporation changed profoundly during regeneration. I n general, two maxima of incorporation, both preceding the peaks of mitotic activity, were observed, one a t about 20 hours, the other much broader a t approximately 28-43 hours after hepatectomy. As the peak of histone biosynthesis reached its maximum shortly before the peak of DNA synthesis, the histones are apparently biosynthesixed in cells preparing for mitosis, just prior to the biosynthesis of DNA. Maximum labeling of nuclear globulins and acidic nuclear proteins is similar to that of the cytoplasm and RNA and precedes slightly the histone maximum except for the histone fraction I which peaks simultaneously with other nuclear proteins (26, 27). Since the histone I is an extract with 0 . 2 N HClO, of the 0.05M citrate nuclear wash, its protein might actually be of ribosomal origin. Significant differences are also reported for the actual amounts of histones and other nuclear proteins during the regeneration of rat liver ($6, 27, 358). Experiments on regenerating rat liver indicate the close relationship of histone and DNA biosynthesis. Such data support numerous cytochemical observations of almost simultaneous biosynthesis of the DNA and acid-soluble nuclear protein in individual cells. Combining cytochemical methods with autoradiography, Gall (359) and Prescott (360) observed that in the Euplotes macronucleus, DNA synthesis proceeds a s a wave in the form of two narrow bands; the protein synthesis (incorporation of histidine-H3) is limited to this zone of DNA synthesis. Further support for the timing of DNA and histone synthcsis was obtained by Prescott from the study of the DNA, histone, and total protein synthesis during the cell cycle of a synchronized population of Euplotes eurystomus (361). As both the DNA and histone biosynthesis started a t 30% completion of the cell cycle and were terminated shortly before the onset of cell division, the histone and DNA synthesis are apparently
PROTEINS OF THE CELL NUCLEUS
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closely related events. Total cell protein was labeled through the entire cell cycle. During the mitosis, DNA, histone, and total protein were equally distributed between the daughter macronuclei. Spalding et al. (36%') who studied biosynthesis of acid-soluble nuclear proteins, presumably histones, in synchronised HeLa cells also observed the close association of DNA and histone biosynthesis. Labeled nuclear proteins were separated by electrophoresis in polyacrylamide gels and the specific activities of the three most prominent electrophoretic zones were determined a t different stages of cell synchrony. During the absence of DNA synthesis, only small turnover of the three proteins was found; their amounts remained constant. After the onset of DNA synthesis, the radioactivity of the three bands increased greatly and the amount of basic proteins doubled. The three electrophoretically isolated basic proteins displayed generally similar metabolic patterns; however, the small differences observed indicated that they were biosynthesized independently. A similar behavior of DNA and histones was observed in synchronized Tetrahymena pyriformis cultures (340). The increase of histone biosynthesis during DNA replication is most probably responsible for the observation of radioactive peaks once thought to be specific for ncoplastic tissues (RPZ-L). The significance and the properties of such peaks have been discussed in detail by Busch and his associates ( 4 4 ) . The conclusions derived from cytochemical observations of histone biosynthesis in nuclei are supported by studies on isolated nuclei in vitro. Active incorporation of labeled amino acids into histone in isolated nuclei has been observed by several investigators (109, 224, 363, 364). The sensitivity of such incorporation to puromycin and analysis of the radioactivity present in individual peptides of the tryptic digest from very lysine-rich histones isolated from thymocyte nuclei labeled in vitro indicate that histone biosynthesis takes place in the nucleus (224, 363). Recent investigations on the incorporation of lysine-H3 into nuclear proteins in situ indicates that the nucleolus and perinucleolar chromatin are the sites of most active incorporation (365). It is noteworthy that high incorporation rates of labeled amino acids into nuclear histones led Birnstiel and associates to conclude that the nucleolus may be the site of histone biosynthesis (94, 9 5 ) . High incorporation rates of nucleolar histones in Novikoff hepatoma cells were also observed by Hnilica et al. (92). Although DNA and histone biosynthesis proceed practically in parallel in normal cells, selective inhibition of DNA synthesis does not decrease the incorporation of amino acids into the histones (366, 367). On the other hand, histone synthesis may be required for DNA replication.
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I n Novikoff hepatoma cells, treatment with colchicine produces a major decrease in the lysine-C" incorporation into all histone fractions in 3-6 hours; thymidine incorporation into DNA decreases much more slowly, reaching a coinplcte arrest a t 8-24 hours after the addition of colchicine. By this time, histone biosynthesis has returned almost to the control rate. Other nuclear proteins were inhibited by the colchicine much less than any of the histone fractions. Lysine-rich histones Fl and F2b are inhibited more than the F2a, F3 fractions ; however, more substantial differences are observed in the recovery of individual histone fractions following colchicine arrest. Even 24 hours after the administration of colchicine, the F2a fractions are still strongly inhibited (368). These data indicate that histone biosynthesis may be a prerequisite for DNA replication. Independence of histone biosynthesis from DNA replication is also evident from studies on Ehrlich ascites cells infected with the ME (Maus-Elberfeld) virus (369). The biosynthesis of histones increases substantially following the infection while thymidine incorporation into the DNA remains unchanged until the virus-induced degeneration of the cells. Moreover, nucleated erythrocytes, which do not divide, still actively incorporate labeled amino acids into their histones. Puromycin (363, S70), chloramphenicol (364), actinomyein D (368, 371-3733), and 5,6-dichloro-l-~-~-ribofuranosylbenzimidazole (371) inhibit the biosynthesis of histones. This indicates that histones are biosynthesized by mechanisms similar to those for other proteins. The last two inhibitors selectively suppress RNA synthesis and seem to exhibit a selective effect on the inhibition of incorporation of labeled amino acids into individual histone fractions; e.g., in chicken reticulocytes (371) and in Novikoff hepatoma (368),the lysine-rich fractions F1, F2b are inhibited more than are other histones. Nuclear proteins other than histones are also inhibited, but to a lesser extent-than the histone fractions. Studies on the rates of biosynthesis of individual histone fractions in various tissues suggest the possibility that genetic regulation may be achieved by tissue-specific variations in the amounts of individual histones biosynthesized rather than by variations in their amino acid sequences. Quantitative studies on the content of major histone fractions separated by starch gel electrophoresis show that various tissues differ in the amount of their individual histone fractions (41).
E.
Histones as Enzymatic Inhibitors The possible function of histones as genetic regulators, as first suggested by Stedman and Stedman (188, 198), received substantial support
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by the findings that histones act as potent inhibitors of DNA-dependent RNA synthesis or of DNA replication. Huang and Bonner (374), working with chromatin from pea embryos, observed a substantial decrease in the ability of DNA to serve as a template for the RNA biosynthesis in vitro after the DNA became complexed with histones. The natural complex of DNA and histone, nucleochromatin, supported the i n vitro RNA biosynthesis very little. In the reconstituted mixtures of DNA and histone, the decrease was directly proportional to the amount of added histone. At a DNA: histone ratio of 1:1, the incorporation of C14-nucleotides into RNA practically ceased. Deproteinizatioii of the chromatin resulted in a spectacular increase of its template activity. Allfrey e t al. (375) removed histones from isolated thymocyte nuclei by a short trypsinization. Such treatment markedly increased the subsequent incorporation of radioactive nucleosides into the nuclear RNA. Even though the specificity of trypsin is not limited to the histones, it was assumed that the removal of histones by trypsinization was responsible for the increased incorporation. Addition of histones to the incubated nuclei substantially decreased their ability to biosynthesize RNA. The arginine-rich histones inhibited biosynthesis about three times as much as did the lysine-rich. fractions. The base composition of the RNA synthesized in nuclei after the removal of histones was similar to that of the DNA, indicating that most of the DNA repressed originally bccanie available for eiizynintic transcription. The differential inhibition of RNA biosynthesis in vitro by histone fractions was also observed by Huang e t al. (689) when both the chromosomal RNA polymerase and RNA polymerase from Escherichia coli were used in systems containing labeled nucleotides. However, in contrast to the reports of Allfrey e t al. (375), the lysine-rich histones Ib and I I b were more inhibitory than the arginine-rich fractions 111 and IV. The differences between individual calf thymus histone fractions in the system containing polymerase from Escherichia coli were more pronounced when the DNA and histone were reconstituted by a slow decrease of salt concentration during dialysis than when the DNA and histone were mixed directly in low ionic strength media. Histones also stabilize DNA toward thermal denaturation. I n complexes with, a constant DNA:histone ratio, the increase in the melting temperature of DNA associated with histones is proportional to the decrease of its template activity. Protamine reconstituted with DNA is almost as active in RNA biosynthesis as is the DNA alone. The high inhibitory effect of lysine-rich histones on the DNA-ticpendent biosynthesis of RNA was also confirmed by Barr and Butler (376),
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who used polymerase from Bacillus megatherium. Similarly, when isolated Novikoff hepatoma nucleoli were incubated with histone fractions F1, F2a, F2b, or F3, their RNA synthesizing system (aggregate enzyme) was most inhibited by the very lysine-rich fraction F1 (91). On the other hand, Hindley (377), using histones prepared according to the methods developed in Butler’s laboratory, found exactly the opposite. The arginine-rich histone F3 was more inhibitory than the fractions very rich in lysine. Skalka et al. (878), who used histones rich in lysine and arginine fractionated by gel filtration on Sephadex, also observed much higher inhibition of the DNA-primed RNA synthesis by arginine-rich histones than by lysine-rich fractions. This unfortunate controversy in the behavior of histones is not yet resolved. It does not depend on the origin of the enzyme; Huang et al. (289) and Skalka et al. (378) both used enzyme systems derived from Escherichia coli. The methods for the isolation and fractionation of histones also seem to contribute very little since Allfrey et al. (876),Barr and Butler (S76),Hindley (877),and Liau et al. (91) all used histones prepared essentially by the same procedure. Also with calf thymus nuclei, polylysine was found to be only a moderate inhibitor of the RNA synthesis (375) while other investigators found this polymer t o inhibit the in vitro reaction very efficiently (288, 348, 376, 378). Allfrey and Mirsky (979) attempted to resolve this controversy by careful comparison of the histone fractions prepared in their laboratory with samples supplied by Murray (289). I n a system containing calf thymus RNA polymerase, the F1 and I b histones behaved in an exactly opposite manner, thus indicating that the differences in their interaction with DNA as manifested by the inhibition of RNA synthesis may arise from changes brought about during their isolation. Whether the more “native” sample of very lysine-rich histone should be more inhibitory was not determined. The differences may be resolved by recent observations of the phosphorylation and thiolation of the very lysine-rich fraction F1 and the arginine-rich fraction F3. Partial oxidation of the arginine-rich fraction F3 drastically changed its effect on the in vitro synthesis of RNA (380). Histone function in RNA synthesis remains controversial. While some investigators regard the DNA-histone interaction to be random and nonspecific, others attribute the function of genetic regulation to the histones. Experimental evidence that histones alter the base composition (91, 375) and the nearest neighbor frequency (878) of the newly synthesized RNA seems to support the second possibility. The role of histones in the replication of DNA (DNA-dependent DNA synthesis) was investigated by Bazill and Philpot (381). Unfrac-
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tionated calf thymus histone inhibited the DNA replication by calf thymus DNA polymerase almost completely in the ratio of DNA: histone 1:1. Billen and Hnilica (348, 386) also observed that calf thymus histone effectively inhibits DNA synthesis by the DNA polymerase of E. coli in vitro. The four main histone fractions F1, F2a, FZb, and F3 were all inhibitory, with the very lysine-rich fraction F1 suppressing the incorporation of TTP-H3 into the DNA more effectively than did the other fractions. Mild trypsinization of histones abolished their inhibitory effect completely. The extent of inhibition of the in vitro biosynthesis of DNA by individual histone fractions seemed closely related to their stabilization effect on the DNA against thermal denaturation (288).The very lysine-rich histones F1 (which also were most inhibitory) are most effective in increasing the T,, of the DNA. These results agree with those of Huang et al. (289) and were confirmed by Sluyser et al. (383).On the basis of the solubility of the reconstituted DNA-histone complexes, it was concluded that the inhibition of the DNA-primed biosynthesis of DNA in vitro by histones may be caused by the removal of the DNA from solution. The possibility that histones affect the enzymatic activity of DNA polymerase directly was eliminated since a complete reversion of the inhibition could be obtained by adding excess of DNA to the histone-inhibited reaction mixture (348). Gurley et al. (38.4) investigated the inhibitory effect of histones on DNA biosynthesis in vitro. All the histone fractions inhibited the reaction and the very lysine-rich histones were more effective than the other fractions. The authors observed practically no effect of histones on the activity of thymidine kinase or of deoxyribonucleotide kinase. I n agreement with the in vitro data, Sluyser et al. (383) found that the in vivo synthesis of DNA in regenerating rat liver is significantly inhibited by injections of histones or polylysine. The latter was a much more powerful inhibitor than any of the histone fractions. Although the very lysine-rich histone stabilized the DNA against thermal denaturation inore than all other histone fractions, it did not differ from the other histones in its effect in vivo on DNA synthesis. However, the results may be complicated by proteolytic degradation of the histones after their administration to the animals. It is noteworthy that no discrepancies in the inhibition by very lysine-rich histones similar to those found in the systems synthesizing RNA seem t o exist in the in vitro biosynthesis of DNA. Although the absolute amount of inhibition varied to some extent, all investigators have reported much higher inhibition by the very lysine-rich histones. Should the arginine-rich versus lysine-rich histone controversy described for the RNA polymerase really depend on minor changes of the histones
>
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during their isolation, then the enzyme for DNA replication must be much less sensitive to such alterations. The biosyntheses of RNA and DNA are not, the only enzymatic processes affected by histones. McEwen et al. (385) reported that histones drastically inhibit mitochondrial respiration and completely uncouple phosphorylation in calf thymus. At concentrations inhibiting the mitochondria, histones had little effect on isolated nuclei. At higher histone concentrations, nuclear phosphorylation was also inhibited. Argininerich histones were more effective inhibitors than the lysine-rich fractions. However, this effect is not specific for histones since other basic proteins (e.g., protamines) are also known to uncouple oxidative phosphorylation in mitochondria. The effects of histones and other polycations on mitochondria were extensively studied by Schwartz and his associates (586-388) . Histone in low concentration was found markedly to stimulate mitochondrial respiration (386); in very low concentration (0.25-50 pg/ml) histones and other polycations stimulated mitochondrial ATPase activity. Concentrations of 100 pg/ml and higher were inhibitory; the effects on ATPase appeared to be consistent with the changes in oxygen consumption of the mitochondria. I n low concentrations, which stimulated the ATPase activity, histones caused a marked inhibition of the ADPATP exchange reaction (387). Stimulation of oxygen consumption was dependent on the composition of the histones, the F2a fraction being the most effective. The very lysine-rich fraction F1 in low concentration had practically no effect (388, 389). Histone fractions also affected the swelling-contraction cycle of mitochondria (388). It appears that specific histones may interact with the mitochondrial membranes by selectively altering their permeability and perhaps controlling the active transport process across the membranes (386490). The effect of histones on enzyme induction is of interest. If adrenalectomized mice are injected with adrenocorticotropic or adrenocortical hormones, the levels of tryptophan pyrrolase and of tyrosine-a-ketoglutarate transaminase increase significmtly. The hormone-induced increase of cnzymtttic artivity can be revrrscd by actministration of actinoniyrin D. This indicates that synthesis of RNA is a prerequisite for the induction of these two enzymes. Histones were found to act in a way similar to actinomycin D ; i.e., injection of 5-10 mg of histone per mouse inhibits substantially the cortisone induction of tryptophan pyrrolase and tyrosine-a-ketoglutarate transaminase in mice. Interestingly, the activity of these two enzymes increased if the adrenalectomized mice were injected with histone only (391). Lactic dehydrogenase activity in cultured embryonic chicken brain
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tissue is significantly increased after exposure to histones (392). The control over enzyme synthesis was explained by assuming that histones alter the synthesis of proteins. Acetylcholine esterase does not respond to the histone stimulus. Whether the increased enzymatic activity of lactic dehydrogenase is indeed the specific result oi the histone treatment or whether it arises from its effect on cellular energetics remains to be determined. Kischer and Hnilica (393), attempting to produce similar effects on different embryonic tissues of the chick, observed a significant toxicity of histones a t levels much lower than those reported by Goodwin and Sizer (392) (the LD,,, was approximately 25 pg/ml) and also showed that the toxicity of histone may be ascribed to its high positive charge.
F.
Histones as Gene Repressors Most primitive organisms, such as bacteria, when placed into a favorable medium continue to divide a t their maximum capacity until lack of food or accumulation of metabolic wastes gradually arrests the explosive growth of the colony. The appearance of a well-formed nucleus in the course of the evolution of species decreased the capability of unlimited growth. As the cells diffcrentiate, their capability of unlimited growth further decreases and in most instances completely disappears. The occurrence of histones in differentiated systems as compared with their absence in bacteria suggests some biological function in cell division. Assuming that all the DNA is functional in the fertilized egg and must be gradually inactivated as differentiation proceeds, a search was begun for substances capable of acting in this manner. Because of their proximity to the DNA, histones are the most likely candidates for the function of genetic repressors (188, 198). The discovery that histones are capable of the actual inhibition of the transcription process in vitro (374, 375) further supports this possibility. However, most of the evidence suggesting a repressor function for histones is only circumstantial. Histones decrease the template activity of DNA, and removal of them from nucleochromatin increases priming activity substantially. In artificial mixtures with DNA, different fractions of histones differ in their powcr t o inhibit DNA-primed RNA synthesis in vitro (289, 375, 376). A similar behavior has been reported for DNA replication in vitro (288, 382, 384) and in viva (383). Histoncs also stabilize the DNA helix toward thermal denaturation (288, 289, 374). All available evidence indicates that the mechanism by which histoncs inhibit the DNA-primed enzymatic reaction is essentially by the formation of a stable DNAhistone complex; DNA merely associated with histones is a poor primer.
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Neither of the enzymes involved in DNA-primed RNA synthesis or in DNA replication in vitro is known to dissociate the DNA-histone complex so as to initiate the biosynthesis. Histones may increase the energy required for the strand separation of the DNA. Amounts of histones smaller than what is required for complete inhibition of the enzymatic biosynthesis inhibit to a lesser extent. This dependence is linear. The base composition (91, 376) and the nearest-neighbor frequency (378) of the RNA synthesized in the presence of various fractions of histones depends on the fraction used for inhibition. Histones are the only nuclear proteins known to inhibit the enzymatic biosynthesis of RNA or DNA. Histones mimic to a great extent the behavior of actinomycin D, an antibiotic known to interfere specifically with genetic transcription (310). Histone biosynthesis runs almost parallel to DNA replication in cells, thus indicating that histone may be associated with the newly made DNA (361,362). In regenerating liver, histone biosynthesis actually precedes the peak of DNA replication, which is then followed by a mitotic wave (27, 28, 357, 358). Transition of spermatids into sperm is in many species accompanied by major changes in the composition of histones, indicating that drastic functional changes in cellular metabolism and function are therefore either the cause or the consequence of the changing pattern of basic nuclear proteins. Similar changes seem to operate during the differentiation of nucleated erythrocytes in some animals (177, 246, 264). The amount of histone fractions and their biosynthetic rates also change with increase of mitotic activity (41, 63, 363, 362). On the other hand, many other facts do not lend support to the theory that histones function as genetic repressors. Histones lack specificity in their primary structure. Although major differences exist between several main histone fractions, there is practically no specificity in the composition of similar fractions from different species. In the early theories, tissue specificity of histones was considered a prerequisite for their function as genetic repressors. The histone:DNA ratio in most somatic eelIs is close to unity ( S ) , and one might expect this ratio to vary with the degree of derepression of nuclear chromatin. The chemical composition and distribution of the histone fractions, and the histone: DNA ratio appear very similar, if not identical, in isolated repressed (condensed) and derepressed (extended) chromatin (394). The increase of template activity of chromatin after sonication also reinforces the old criticism that the changes in the priming activity of chromatin come from changes in the physical state (structure) of the nucleohistones rather than from a specific repression by individual histone molecules (396).
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The objection that the extended chromatin may be a sonication artifact must also be considered. The biosynthetic rates of individual histone fractions differ slightly in various tissues ; however, much greater differences might be expected if individual histone fractions were actively engaged in the process of repression. The turnover of histones closely follows that of DNA, indicating that histone, once associated with DNA, remains attached a t least until the next mitosis (254, 364). The distribution of labeled histones in postmitotic cells is random, not semiconservative as was found for the DNA (361).This indicates that the same histone does not remain with the DNA. Histones can be found in unfertilized sea urchin eggs. During cleavage and further developmcnt, the amount of histones in the embryos may change in quantity, but the general makeup of the fractions remains constant (336). A similar persistence of histones is seen during embryonic development of the chick (216). The inhibition of DNA-directed RNA synthesis or DNA synthesis may be a nonspecific artifact produced by the DNA-histone interaction (396, 397). The inhibition of in vitro RNA synthesis by individual histone fractions is a controversial issue (289,375377).Substances other than hSstones, e.g., polylysine, lysozyme, antimalarial drugs, acridines, actinomycin D, similarly inhibit the in vitro synthesis of RNA. Artificial reconstituted DNA-histone complexes show little selectivity in supporting DNA-dependent RNA synthesis or DNA synthesis; a t a given DNA: histone ratio, about the same inhibition occurs in both systems (288). However, the natural nucleohistone seems to have more selectivity. The suggestion has been made that histones may in some way regulate the extent of DNA replication while allowing RNA biosynthesis to proceed (398). Although, despite intense collective effort, the biological function of histones remains unknown, the evidence supporting an essential role of histones in the process of genetic transcription and/or genetic replication of the DNA in higher organisms is stronger than the evidence against such a function. Since in vitro experiments have demonstrated the importance of free or “naked” DNA for its template function, a mechanism for the removal of histones from thc repressed sites is essential to the normal function of the cell. Several such mechanisms have been suggested, all based on experimental evidence that modification of histones, either by direct chemical substitution or by interaction, can decrease their repressor activity (399). While investigating the inhibitory effect of histones on the RNA synthesis in isolated nuclei, Allfrey and Mirsky (379) noted that the
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inhibition of their system by the very lysine-rich histone can be substantially increased by making this fraction “arginine-rich” by partial guanidylation of the free NH, groups on the lysine residues. This first biologically active modification of histones opened a completely new chapter in histone biochemistry. The same group later observed the decreasing inhibition of histone fractions in proportion t o the increasing extent of acetylation (399). Currently, four types of chemical modifications of histones that decrease their repressor activity are being investigated. 1. HISTONE ACETYLATION Allfrey et al. (399) found that partial acetylation of histones decreases their inhibition of the DNA-primed biosynthesis of RNA. The inhibition is inversely proportional to the extent of acetylation and does not depend on the origin of the enzyme used for RNA biosynthesis. Acetylated arginine-rich histones also protect the DNA from thermal denaturation less effectively than do untreated histones, indicating weaker binding forces between the acetylated histones and DNA. When isolated nuclei are incubated in the presence of labeled acetate, there is a rapid incorporation of the isotope into the histones. The three mbin histone fractions (Fl, F2, and F3) differ in their uptake of the label; the F3 histones (arginine-rich) accept the acetyl most readily. Since the F3 histones have a much less total acetyl content than fractions F1 and F2a (260),this was unexpected. The acetylation of histones occurs after their biosynthesis, acetyl coenzyme A being the acetyl donor (400). Acetylation of isolated histone fractions in vitro can be achieved by using partially purified enzyme from pigeon liver. Such in vitro acetylation results in the attachment of labile 0-acetyl together with more stable N-acetyl to the arginine-rich fraction F3, while only the N-acetyl can be detected in the fractions F1 and F2 (401). To support the proposal that histone acetylation may be one of the possible mechanisms for derepression of histones, Allfrey et al. (394, 409) studied histone acetylation in various systems both in vivo and in vitro. The repressed (condensed) form of chromatin showed a much lower incorporation of acetate-2-C4 than did the extended (derepressed) part. RNA synthesis is chiefly associated with this extended form (88, 89). When a ~ e t y l - l - C ~ ~ - Cwas o A used, both forms of chromatin became labeled to the same extent. A similar situation was found when isolated nuclei were incubated with acetyl-l-Cl’-CoA directly. The interpretation was that the two states of chromatin differ, not with respect to their acetylase content, but with respect to their ability to convert acetate
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to acetyl-CoA. Diffuse chromatin probably represents a metabolically active part of the nucleus, and this is reflected in the increased acetylation of histoncs. Similar results were obtained when salivary gland chromosomes from Chironomus tentans were incubated with uridine-H3 and with acetate-Me-H3; intense uridine incorporation was observed mainly in the puffs. Similarly, the acetate was incorporated directly over the stained chromosomes (394). I n lymphocyte cultures induced with phytoheinngglutinin (403), the acetylation of histones increases markedly shortly heforc the onset of RNA synthesis (4O.Z). Phytohemagglutinin induces marked changes in the structure of the chromatin in treated lymphocytes as is shown by the increased binding of acridine orange to the chromatin of cells exposed to it (404). The timetable of such increase is closely related to the schedule of acetylation ($94). Based on the close relationship between histone acetylation and the RNA synthesis, Allfrey e t al. (394) concluded: “A change in the structure of the chromatin-brought about by, or coincident with, acetylation of the histones-is a necessary prerequisite for the synthesis of new RNA’s a t previously repressed gene loci.” The mechanism by which the histone derepression by acetylation could operate in viuo is difficult to explain. From the chemical studies on the distribution of acetyl residues in a number of histone fractions, it seems evident that the only amino acids acetylated in the histones are the NH,-terminals. Experiments in vitro demonstrated the feasibility of the enzymatic acetylation of the C-terminal amino acid in fraction F3 (4001). However, it is difficult to accept that such a relatively minor modification could significantly alter the interaction with DNA. Studies of the actual receptor sites of the acetyl by histone molecules are needed before the relationship between histone acetylation and RNA hiosynthcsis can be fully understood. 2. HISTONE PHOSPHORYLATION
Another mechanism that most likely participates in derepression by histones is their enzymatic p1ios~)hoiylation.Ord and Stocken (355) and Kleinsmith et al. (405) have shown incorporation of p h 0 ~ p h a t e - P ~ ~ into histones in rats in v i m or in calf thymus or rat thymus nuclei incubated in vitro. The main acceptor of the phosphate is the very lysinerich histone F1 ( 3 5 5 ) .Histonc phosphorylation is energy-dependent and takes place after the histone has been completely biosynthesizcd (405, 406). The acceptor of phosphate in the lysine-rich fraction F1 is serine (355).The enzyme responsible for histone phosphorylation was recently purified from liver by Langan and Smith (407). This histone phospho-
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kinase differs from the phosphoprotein kinase and is histone and protamine specific. It phosphorylates seryl residues in all histone fractions, but it shows a distinct preference for the very lysine-rich histone F1. Proteins other than histones are not phosphorylated. Stevely and Stocken (406) obtained evidence that phosphorylation of histones may modify the repressed sites of chromatin. F1 histone phosphorylated in vitro (34.2 v o l e P/mg protein) was less inhibitory in the in vitro RNA biosynthesiaing system than a sample obtained from animals directly (22.4 mpmole P/mg protein). Irradiation of the experimental animals markedly decreased the incorporation of the phosphate-P3* into the very lysine-rich fraction F1 (408). The doseresponse curve showed a remarkable similarity to a similar curve obtained by Ord and Stocken (409) for the incorporation of phosphate-PS2 into the thymus DNA. Significant differences in the incorporation of p h o ~ p h a t e - P into ~~ histone fractions isolated by a combination of selective extraction, gel filtration on Sephadex, and starch gel electrophoresis were reported by Gutierrea and Hnilica (410). The incorporation of the phosphate into corresponding histone fractions from normal liver, regenerating rat liver, and Novikoff hepatoma was tissue specific and indicated a substantial decrease in the phosphorylation of histones in the hepatoma. Trevithick et al. (411) studied phosphorylation of protamine during spermatogenesis in Steelhead trout testis. Nuclear ribosomes appeared to be the sites of protamine biosynthesis. Both the histones and protamines were phosphorylated in testes incubated with phosphate-Ps2, and the extent of phosphorylation decreased during the process of maturation. Again serine was the recipient of phosphate, and several P3*-containing peptides were located in tryptic digests. The phosphorylation of protamine appeared to take place during its biosynthesis; mature sperm contained little covalently bound phosphate. The phosphorylation of histones is not related as closely to the activation of RNA synthesis as acetylation appears to be. Agents inhibiting RNA synthesis do not affect phosphorylation. Allfrey et al. (394) proposed that histone phosphorylation may be associated with the spatial conformation of chromatin, i.e., the coiling and superstructure of chromosomes during transition between condensed and extended chromatin.
3. METHYLATION AND THIOLATION OF HISTONES Two other chemical modifications of histones that may change their biological properties are methylation of the r-amino group on lysine and oxidation and reduction of thiol groups present mainly in the argininerich histones, F3.
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The presence of c-N-methyllysine in histones was first reported by Murray who found a small amount (up to 1 mole percent) of this unusual amino acid in the arginine-rich fractions IIaa, IIa, 111, and IV of the calf thymus histone (258). Experiment with methyl-labeled methionine showed that methylation of histones occurs after their biosynthesis. Methionine functions as the donor of the methyl radical to lysine (268, 399). Methylation in isolated calf thymus nuclei proceeds through an 8-adenosylmethionine intermediate (412). Tidwell et al. (413) studied the methylation of histones during regeneration of rat liver. An increase of specific activity of the c-N-meth.yllysine in the basic proteins was not noticeable until 30 hours after hepatectomy. Since the first peak of RNA, histone, and DNA synthesis occurs much earlier [16-22 hours (26, 27, 357, 3 5 8 ) ] ,methylation of histones does not seem to correlate directly with the activation of chromatin during regeneration in liver. The presence of r-N-methyllysine in arginine-rich histones F2a and F3 is a general feature; varying amounts of this amino acid are found in the arginine-rich histones from various tissues (239). Phillips (414) found that the cysteine content occasionally observed in the arginine-rich fractions of histones is not due to contamination with acidic nuclear proteins as was previously believed. The thiolcontaining peptide of the F3 histone from calf thymus was isolated recently (262) and shown to have the probable composition (Ala, Asp, Cys, Gluz, Ser, Thr). In agreement with Phillips (414), Ord and Stocken (415) found that the F3 histone is the only fraction containing thiol groups. They also presented evidence that the histone fractions F1 and F3 exist in nuclei as families of proteins differing with respect to their sulfur and/or phosphorus content. I n addition to the F3 histone, fast electrophoretic components (polyacrylamide electrophoresis) obtained from the acid nuclear extracts fractionated on Sephadex G-75 contained significant amounts of thiol groups (355).The identity of this material has not been determined, but it is suggested that it may be a low-molecular nucleotide-peptide that perhaps can associate with certain hiatoiics (317,355). Hilton and Stocken (380,416) studied the effect of oxidation of the thiol-containing arginine-rich histone F3 on its repression of RNA biosynthesis in vitro. No differences were observed between the fully reduced histone and that in which the SH groups were completely masked by mercaptide formation. As oxidation increased the repressor activity of F3, the higher proportion of the total sulfur present as thiol (76 and 100%) found in the 50 m M HC1 extracts from extended chromatin as compared with the 5143% in the condensed form is consistent with the higher repression of RNA synthesis by dense chromatin.
LWOMIB S. HNILICA
4. DEEEPWSION BY INTERACTIONS a. Effect of Hormones. Another aspect of gene activation is the effect of hormones on cellular and tissue metabolism. The mode of action of hormones is considered to be a selective activation of the previously repressed sites on the DNA. Assuming that the repression of genes is achieved by the association of histones with DNA, activation by hormones must influence such association to permit synthesis of new RNA (mRNA) . Probably the clearest proof of this assumption is the activation of selective sites on polytene chromosomes by a steroid hormone, ecdysone (the molting hormone in Diptera) . The activation can be observed directly under the microscope by the appearance of extended zones of acute increase of RNA biosynthesis on the chromosomes-Balbiani rings and puffs. The mechanism of the ecdysone action is not known. Involvement of histones in the puffing process is indicated by unfolding of the tightly coiled chromosome fibers in the related chromomeres. It is known that such an unfolding is a prerequisite for the activation of genetic loci. According to more recent models of chromosome structure (319321), the successive supercoiling of the chromatin fibers in the chromosomes is achieved by association of the DNA with histone. Dissociation of such bonds will lead to the uncoiling of the chromosome. Robert and Kroeger (417) reported that histones in puffs are more sensitive to trypsin than are histones in the rest of the chromosome. Kroeger also proposed that the ecdysone effect on chromosomes may be based on a selective activation of ions, which would bring about partial dissociation of the DNP complex (418). An alternate mechanism for the appearance of puffs has been suggested by Allfrey et at. (S94), whereby partial derepression in the puffing region would be brought about by acetylation of the involved histone fractions. Also considered was the possibility of a direct interaction of the hormone with histones or with the DNA. Administration of corticosteroids to adrenalectomized animals will bring about a threefold increase in the activities of several liver enzymes. The induction of the enzymes can be prevented by administration of actinomycin D or by histones (391). I n accordance with the increased enzymatic activity, Dahmus and Bonner (419) observed an increase in the general template activity of the livers in hydrocortisone-treated adrenalectomized rats as compared with that in controls. Elimination of these differences by the deproteinization of the two chromatin samples indicates the involvement of proteins, most likely histones. Similar stimulation was achieved in vitro by treating the liver nuclei with cortisol (4.20). Since a significant increase in the RNA synthesis in isolated nuclei occurred with-
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in minutes after their cxposurc to cortisol, interaction of the hormone with the constituents of chromatin must have occurred. A similar protein-related increase in the template activity of chromatin was obtained by the administration of thyroxine t o tadpoles. Chromatin from the treated animals was much more efficient in supporting RNA synthesis th.an samples from the control animals (4.21). Similarly, injection of estradiol-17p into ovariectomized mature rats resulted in a tissue-specific increase of the template activity of uterine chromatin (422). Liao et al. (423, ,424) demonstrated that the increase of RNA synthesis in prostatic nuclei after androgen administration to castrated rats occurs at selective genetic sites. The activation of these sites was suppressed by actinomycin D, indicating androgen-induced changes in the template activity of involved parts of chromatin. The authors postulated that the sites of androgen action are the nucleolus and perinucleolar chromatin. The exact mechanism of the liormone-induced selective derepressioii of chromatin is not known. A rapid increase in the synthesis of nuclear proteins immediately prior to functional change (lactation) was observed in the mammary gland of pregnant rats (426). The changes in teniplatr activity of chromatin in hormone-treated animals may be the result of a direct interaction of the hormones with eithcr DNA (426, @7) or histone. M’hilr some studies indicate the binding of hydrocortisone (.@%), cortisone (@9), and testosterone (430) directly to the histones, other investigators observe no such binding, even though the main activity of labeled hormone administered to the animals was found in the chromatin (431). It is more likely that the hormones affect the repression by histones by chemical modification, such. as by acetylation of histones in cortisone-trested animals, recently described by Allfrcy et al. (394). b. Interactions of Histones with R N A . The capacity of RNA to function as a genetic template is well known from numerous studies with viruses, especially tobacco mosaic virus. Assuming an active complex of the RNA with a protein such as histone, RNA segments complementary to the specific “recognition sites” on DNA would easily provide all the specificity required for selective repression. This possibility attracted the attention of Allfrcy and hlir.-lw and their associates, who investigated the role of RNA in RNA synthesis. Various RNA fractions inhibited the RNA synthesis in isolated nuclei (394) or in isolated chromatin (432). Even though the RNA used in their experiments may have interacted with the RNA polymerase and therehy inhibited RNA synthesis by competing with DNA for the sites on the enzyme (433, 434), the idea appears feasible since functional DNARNA-protein complexes have been reported (435).
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Huang and Bonner (4.36) recently isolated a natural complex of RNA with nuclear proteins, presumably histones. Such protein-bound RNA can be separated from the bulk of chromatin by dissolving the complex in 2.09M CsCl and by equilibrium centrifugation of the solution a t 39,000 rpm (Spinco No. 39 rotor). The protein-bound RNA sediments as a zone separated from the dense DNA and from the light histone. Material recovered from this band contains about 10% RNA; the rest is protein, mainly histone. The RNA has an average length of 40 nucleotides and contains, in addition to the four common nucleotides, 27.5 moles percent of dihydrouridylic acid. The binding of this RNA to the protein is realized through the carboxyl groups of the acyclic form of dihydrouridylic acid (p-ureidopropionic acid) . The union is cleaved by acid. Since, in the pea-bud nucleohistone, all the histone was found associated with this new form of RNA (by CsCl centrifugation), and since only 10% of the mass in this complex is RNA, Bonner and Huang (437, 438) concluded that only a part of the protein present in the band can be associated with the RNA directly. Such RNA-protein (histone) is thought to serve as an aggregation center for the assembly of the remaining histone. The complex is held together by hydrogen bonds between the individual molecules since treatment with guanidine released most of the protein. Such an arrangement of histone molecules in clusters directed to specific parts of thc DNA molecule by their RNA would require a drastic revision of current views on the molecular structure of nucleoproteins and chromosomes. Chromatography of histones from the complex labeled with P32 on Amberlite GC 50 columns revealed the label to be associated only with the “run off” peak containing a n acidic protein and with the argininerich fractions I11 and IV. It is noteworthy that Hnilica and Bess (2.4O.0) also reported the presence of nucleotide material in the acidic protein fraction contaminating the arginine-rich histones F3. This acidic nuclear protein fraction is probably the actual carrier of the dih-ydrouridine RNA, as can be concluded from the amino acid composition of the “run off,’ peak analyzed by Bonner and Huang (437, 4.38). More recently, Huang (4.39) isolated the site of attachment of this RNA in chicken embryo chromatin. Pronase digestion, followed by alkaline hydrolysis of the RNA yielded, in addition to the nucleotides, a “linker” of a small peptide bound to the dihydropyrimidine base. The linker was both chromatographically and electrophoretically homogeneous. When hydrolyzed in 6 N HC1, its major ninhydrin-positive products were dihydropyrimidine, p-alanine, and serine. The bond between the RNA and the protein is thought to be a n amide linkage. The existence of RNA-histone complexes was confirmed by Benjamin
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et al. ( 4 4 0 ) ,who found a hist,oiic :issocintcd with RNA in rat liver. This RNA is heterogeneous in sucrose density gradient centrifugation and has a high adenine uracil content. The biological function of the RNA-histone complexes remains hypothetical. If the histones function as genetic repressors, a polynucleotide-protein unit could function :is u highly specific repressor. Sypherd and Strauss (441) postulated such ;I functional complex in which the RNA is supposed to hybridize with the complementary receptor segment of the DNA-gene and t.hus provide the necessary specificity for the protein effector. Another function for the RNA in genetic regulations was proposed by Frenster (43.2, 44.2). I n this scheme, RNA acts as a specific depressor by partially displacing histoncs from a DNA segment and allowing a specific depressor RNA to hybridize with one strand of the DNA, freeing the Complementary strand for the synthesis of mRNA. Only further work will determine whether the RNA-histone association is really functional and assess what significance it might have in the process of genetic transcription. It should be mentioned that Commerford and Delihas (449) wcre unable to confirm the high RNAhistone ratio complexes in nucleohistoncs from mousc liver and intestine. I n their preparations, the ratios were :it most 2 RNA-histone nuc.leotides/ 1000 DNA nucleotides as coin])arcd with Huaiig and Bonner’s findings of 100 RNA-histone nucleotides/1000 DNA iiucleotides (436, 437). The authors consider this ratio much too low to control effectively the genetic activity.
+
G. Histones
in Embryonic Development One of the fundamental problems in biology is differentiation, i.e., a spatiotemporal sequence of processes by which originally the omnipotent, unicellular zygote develops into a complex organism containing a variety of specialized cells. The process by which the fertilized ovum gives rise to the tissues so diversified in their metabolic and biochemical properties is not known. The most prevalent view is that cytoplasmic-nuclear interactions of a highly specialized nature bring about gradual specialization of cellular regions in the developing embryos. Since histones may act as gene repressors, the question whether histones change during clevelopincnt was investigat.ed by sevcral authors. It appears from numerous studies with actinomycin D that the zygote operates through many divisions on the already present reserve of ribosoinal and mRNA. Embryos trcated with actinornycin D will continue to develop until gradual arrest a t early gastrula stage (310).Since histones parallel in many ways the action of actinomycin D, it was no
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surprise when reports appeared that the development of embryos can be arrested by histones and polylysine (444-446) . The lysine-rich histones were found most effective. Similarly, Sherbet (447) reported developmental arrest and malformations (brain and somites) in chick embryos exposed to the bistones. Fractions F1, F2a, and F3 were found to have similar effect. Similar changes were also produced by polylysine. The mechanism of the histone action on embryos appears to be nonspecific (393, 448) probably because of plasmotropic interactions (447). An analytic approach to the studies of histones during embryogenesis has also been used. Dingman and Sporn (449) found practically no changes in the DNA:histone ratios and in the physicochemical behavior of the D N P over a large span of the developmental schedule in chickens. Similarly, histones prepared from tissues and cells at various stages of embryonic development in chick and other animals showed only minor variations in the main histone patterns as determined by zonal electrophoresis (209,215, ,216) or by analysis of the individual histone fractions (336, 347). Quantitative changes in the concentration of the three main histone fractions F1, F2, and F3 in chick embryos were reported during the first 5 days of incubation (450).Similarly, the amount of proteins soluble in 0.25 N HC1 was found t o decrease during gastrulation of the sea urchin (Paracentrotus lividus) embryos. However, such changes seem to come from the decreasing amount of acid-soluble cytoplasmic proteins since the content of histone proper remains relatively constant at all stages of the early development (461). Transition of the “cleavage” into the “adult” histones observed cytochemically (462) has not been confirmed by biochemical analysis. Even histochemically, such transition appears highly controversial (4~75).It can be concluded that embryogenesis is not accompanied by major changes in the composition of histones.
V. Nonhistone Proteins The acidic nuclear proteins represent a substantial part of the nuclear protein content. They appear to be firmly associated with nucleic acids, mainly RNA ( 6 2 ) , and attempts to dissociate such complexes result in the denaturation and precipitation of the protein component. Owing to difficulties in their solubilization, very little information is available about their biology and biochemistry. From electron microscopic observations it can be concluded that a large body of the nucleolochromosomal apparatus consists primarily of the RNA-acidic protein complex. Therefore, a part of the acidic nuclear proteins must participate in the metabolic functions of these structures (55,64). Another part of
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the acidic nuclear proteins can be found associated with nuclear ribosomes. A much snialler portion distinguished by the higher content of dicarboxylic amino acids, is closely associated with the DNA. It has been known for many years that a small fraction of such proteins resists attempts to remove them from their association with the DNA (454-456). The amount of this DNA-associated protein varies according to the procedure used for the isolation of DNA, and also with the metabolic and mitotic activity of the cells (69-7f). The function of the nuclear acidic proteins is not known and several suggestions have been made concerning its importance in the process of genetic regulations. Agrell and Christensson (457) reported a complex formation of histones and other basic proteins with acidic substances such as polysaccharides, and acidic proteins. It was this observation, probably together with the elusive behavior of a tumor-specific chromatographic histone peak (RP2L) , that prompted Busch and his associates (75) to postulate that a part of the acidic nuclear proteins may compete with the DNA for histones and thereby affect the DNA repression by the latter. Similar conclusions were also reached by Ursprung (458). Frcnster (4332, 4-62, 459, 460) further elaborated on this possibility and suggested a mechanism by which nuclear polyanions (acidic nuclear proteins, RNA, etc.) can produce a selective derepression of th.e genetic DNA coated by histones. Later, this scheme was further implemented by including other substances known to affect the DNA-directed synthesis of RNA (460). These theoretical considerations received experimental support from the findings that the acidic proteins, not the histones, become crosslinked to the DNA in cells exposed to bifunctional alkylating agents (67). No crosslinking was found in similar experiments with monofunctional alkylating agents; this may be interpreted to indicate a close proximity of acidic nuclear proteins to the DNA. A complex of acidic proteins, DNA, and lysine-rich. histones present in tumor cells was described by Frearson and Kirby (461). The RNA-linked protcin, which is supposed to serw as an aggregation nucleus for histones in pea cmbryo chromatin, is also acidic (436-439). The rapid incorporation of labeled amino acids into the DNA-associated protein parallels the rate of RNA synthesis in the nuclei of Ehrlich ascitic tumor. This was thought to indicate that this protein may be a part of a mechanism controlling th.e rate of RNA synthesis in response to physiological requirements ( 7 f ) . This interpretation was based on earlier studies by Balis e t al., who investigated the DNA-associated protein in bacteria (69) and in mammalian cells (7'0) and ascribed a function of the repressors of rcgulatory genes to these proteins. However,
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the parallel labeling of the DNA-associated acidic proteins and the RNA can also be interpreted as a biosynthesis of proteins required for the assembly and coating of the newly synthesized nuclear ribosomes. More information about how this protein is attached to the DNA [RNAprotein linkers were isolated and analyzed by Huang ( @ 9 ) ] , and which species of RNA in the nucleus is labeled concomitantly with the DNAassociated protein synthesis, is necessary before more definite functions can be ascribed to the DNA-associated acidic nuclear proteins. The possibility that the derepressor protein may be a phosphoprotein was investigated by Langan and Smith (462, 463). A phosphoprotein fraction isolated from nuclei, complexed easily with the histones; the inh.ibitory effects of histones on the in vitro biosynthesis of RNA was greatly reduced by such association. Phosphoproteins were found to be greatly accumulated in the derepressed (diffuse) chromatin as compared with the clumps of repressed (condensed) chromatin [quoted in (394)3. This may suggest a function of the phosphoproteins as genetic derepressors. The fact that eukaryotic (nucleated) organisms have much higher levels of phosphoproteins than the prokaryotic (bacteria, blue-green algae) lends further support to this possibility (463). Another group of nuclear proteins that may be involved in the coordination of nucleocytoplasrnic activities are the cytonucleoproteins, first found in the nuclei of amoebae. This group of proteins, which was observed by Goldstein (464) and further investigated by Prescott (35, 36) and Byers e t al. (33, 34), is lost to the cytoplasm during mitosis but rapidly reenters the nucleus after the division. This can be seen best when a nucleus from a heavily protein-labeled cell is transplanted into the cytoplasm of a nonradioactive host. I n Byers’ experiments, the label appeared in the host nucleus within 10 minutes and an equilibrium ratio 7:3 of the donor:host nucleus radioactivity was reached in 4-5 hours a t 25°C (34). Similar migration was observed for chromosomal proteins; Kroeger e t nl. (465) transplanted protein-labeled salivary gland chromosoines with their nucleoli into thc cytoplasm of nonr:idionctive salivary gland cells in Chi~otioinusthuuz7tii. The label, which again moved as a protein and not in form of free amino acids, rapidly appeared in the nucleus. The exact nature of the “shuttle” proteins in Amoeba and in Chironoinus is not known. It would be of considerable interest t o determine whether the occurrence of cytonucleoproteins is a feature specific to lower animals or whether similar shuttles operate in all species.
VI. Conclusions and Summary While research on the nonhistonc nuclear proteins has hardly begun, iiiformation available on the histones is plcntiful but their function re-
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mains controversial. The two niaiii functions ascribed to the histones are a specific one of genetic repressors and a completely nonspecific one of a chromosomal “glue.” Among the most favored arguments supporting the latter idea is the amazing lack of structural, sequential, and metabolic specificity of histones from various tissues and species. Histone can be visualized as binding the genetic units of DNA together as mortar binds stones into an architectural structure. The mortar of the cathedral in Reims may diffcr very little from that used in Cologne. Similar argumcnts seem to prevail in the interpretation of the function of ribosomal proteins, which are regarded as structural. After all, the helical structure of DNA seems important to its biological function. This fact of a macromolecular structure serving as a basis for its function may bring the histone function controversy to a compromise. It is possible that the lack of histone specificity is actually one of the main virtues of these proteins and may be related to their function. Histones obviously must be very old proteins phylogenetically. Only drastic changes in cellular functions lead t o the appearance of fundamentally new kinds of histones. The replacement of somatic histones by arginine-rich protamines does not seem to be, as was generally interpreted, a provision for the better metabolic immobilization of the DNA in sperm. Huang and Boiiner have shown that protamine in artificial complexes with DNA decreaws only slightly the DNA-primed biosynthesis of RNA in vitro (689); it would be very interesting to see how efficient a natural nucleoprotamine is as a primer. It might appear that the replacement of histones by protamines is to make the genetic material of the sperm more sensitive to the dcrepression mechanism operating during fertilization so i t rimy open up rapidly to initiate the first cleavage niech:tnism. Rccent findings on the acctylation, phosphorylation, tkiolation, and rnt4Jiylation of histones (394), all of which seem t o decrease the extent of the repression on chromatin, also indicate little need for a sequential hpecificity of histones in various tissucs. Probably the best arguments for the function of histones as genetic repressors are the observations that tlcproteinization of chromatin leads invariably to a substantial increase in its priming activity for the biosynthesis of RNA (689, 375, 419). Vice versa, addition of histones to the in vitro RNA-biosynthesizing systems causes a marked inhibition (289, 37’4379). A similar inhibition occurs in the replication of DNA both in vivo (383) and in vitro (288, 348, 381384). More complicated is the problem of spatiotemporal selectivity of the genetic repression by nuclear proteins, presumably histones. Studies on isolated chromatin (374, 4667, on chromosomal puffs in Diptera (467’),
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and from the molecular hydridisation of the newly synthesized RNA with its DNA template in partially repressed ch,romatin (468, 469), indicate that the restriction of template activity of DNA in the chromatin from various tissues is highly specific. I n most cells, only a small part of the DNA seems to be available for genetic transcription. The repression appears t o be quite permanent and much more difficult to reverse than in bacterial cells. Again, the absence of histones in bacteria is very suggestive for their function as “genetic brakes” in somatic cells of higher organisms. However, the histone type of repression must differ from the widely known feedback type of repression control described by Jacob and Monod (470).Only a few cases of this kind of genetic control have been observed in higher organisms, most of them in cultured cells and in liver, i.e., in tissues capable of an instantaneous regenerative response to injury. Assuming that genetic repressors exist in real life and are not “a logical construct rather than a chemical entity” (471), the existence of a t least two classes of genetic repressors operating in higher organisms appears possible (71, 472). Accepting the function of nuclear proteins as genetic repressors, one might visualize the acidic nuclear proteins (most of which exist in the form of ribonucleoproteins) as associated with the less permanent kind of repression. The RNA in such a complex would provide the site of interaction with the specific genetic locus on the DNA molecule; the protein part, because of its acidic character will, among other possible functions, protect the DNA in the neighborhood of the DNA-RNA interaction site from the association with histones and thereby make possible its genetic transcription. All remaining DNA loci not protected by the ribonucleoproteins will then associate with histones and remain genetically inactive. The specificity of such association need not be extensive and would depend 011 the local distribution of lysine and arginine in the histone chains as compared with the AT- or GC-rich sites on the DNA. This scheme is compatible with the obvious lack of cell and species specificity of histones. Derepression would be acbieved by the chemical modification of histones (394), thereby exposing the involved part of DNA to the interaction with ribonucleoprotein. Findings of discontinuous increases of RNA synthesis in response to the gradual removal of proteins, presumably histones, from chromatin in vitro (473) and the sudden change of the base composition of newly synthesized RNA toward the DNA-like RNA during such gradual derepression of chromatin support the possible function of histones as permanent repressors. The mechanism of the less permanent repression is almost completely unknown. Piecemeal information concerning the effect of certain hor-
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moiies on the DNA-dependent biosynthesis of RNA (474) and on the profound modification of RNA synthesis in chromatin-containing systems after exposure to low concentrations of mono- and divalent cations (418, 475) may prove to be the beginning of a new era of the investigation on the functions of the nonhistone nuclear proteins.
ACKNOWLEDGMENTS I wish to express my gratitude to Dr. Violctte S. Hnilica and Mrs. Louise C. Littlejohn for their invaluable assistance in the preparation of this review. Original studies by the author reported in t.liis paper were supported by grants from the IJnitcd Statcs Public Health Service (CA-07746), the American Cancer Society (E-388, IN43-F2, and IN43-Gll1, and The Robert A. Welch Foundation ((2-138).
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The Present Status of the Genetic Code
I
I
CARL
*OESE
DcparLment of Microbiology. L’iiiversity .of Illinois. lirbana. !l~inois
I . Introduction . . . . . . . . . . . . . I1. Historical . . . . . . . . . . . . . . A . Theoretical Attempts a t a Cryptographic Solution . . . B . The Nucleic Acid-Polypeptide Relationship and the Nature of Translation . . . . . . . . . . I11. The Cryptographic Problem . . . . . . . . . A . Codon Assignments Determined by Polypeptide Synthesis in Vitro . . . . . . . . . . . B. Other Approaches to Codon Assignments . . . . . IV . Colinearity of Gene and Polypeptide . . . . . . . V . Codon Size . . . . . . . . . . . . . VI . Punctuation and Other Encoded Instructions . . . . . A . Transcription Punctuation . . . . . . . . . B. Translation Punctuation . . . . . . . . . V I I . The Translation Tape-Reader and the Translation Process . . A . tRNA Structure . . . . . . . . . . . B. The Trans-I Proccss . . . . . . . . . . C . The Trans-I1 Process . . . . . . . . . . D . The Direction of Translation . . . . . . . . E . Errors in Trans-I1 . . . . . . . . . . . F . The Mechanisms of Polypeptide Chain Punctuation . . . G . Suppressor tRNA’s . . . . . . . . . . . VIII . The Fundamental Nature of the Genetic Code . . . . . A . The Problem of thr Locked& Code . . . . . . B . The Problem of the Indirect Templatc . . . . . . C . The Problem of Errors and Evolution . . . . . . D . The Problem of Constraints Governing the Code’s Evolution E . The Problem of thc Origin of the Components . . . . References . . . . . . . . . . . . .
107 109 110
113 117
117 121 126 128 130 132 134
138 139 142 146 149 149 152 153 155 155
157 161 162 163 167
.
1 Int.roduction Advances in elucidating the genetic code made over the past five years have proceeded a t a bewildering pace and have been sweeping in scope (see article by F. H . C . Crick in Volume I of this series) . With the recent completion of the catalog of codon assignments, the tempo has slackened somewhat . Thus it now seems a particularly appropriate time 107
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to review the subject. While there is a need to recount the various facts that have accumulated over this period, I feel th a t there is a far greater need to appreciate their impact on our concept of the genetic code. Therefore, the main purpose of this review is to reflect in broad terms upon what has been accomplished-to attempt to define its significance and to see what the still unsolved problems are and where they may lead us. Throughout its brief history, particularly of late, the coding field has heen plagued by a notoriety-an unfortunate consequence of the import of coding discoveries. The effect of this has been, in the net, rather negative. In the intense, narrow focusing of attention on a few intriguing aspects of the problem, there arises a tendency both to overlook and to distort the complete picture. Thus it is important to start our consideration of the genetic code with a tabula rasa, to strip the facts and concepts of any extrascientific trappings and view them merely for their own value. Perhaps the best place to begin this discussion is in inquiring how the genetic code came to be called just that. The very beginnings of the matter lie in the recognition, a halfcentury ago, of a phenotype as opposed to a genotype. However, the whole did not really begin to take form until the obtaining of evidence that DNA is the genetic material (1), the conceiving of the one geneone enzyme hypothesis ( 2 ), the realization that genes and polypeptides are basically linear polymers with unique primary structures [ a primary structure that, in the case of polypeptides, appears solely responsible for the overall geometry of the molecule in any given environment ( S ) ] , and the concept of genetic maps of single genes as linear arrays ( 4 ) . Thus the idea grew that the essence of gene expression lies in some special relationship between the primary structure of a gene and that of the corresponding polypeptide chain. Such a linear relationship bears obvious analogies to the one-dimensional information transfer in written language (as opposed, for example, to the two-dimensional display of information in a diagram, etc.), so that it became easy and natural to speak of the gene-polypeptide relationship in terms of “words,” “mesetc. I n particular, the analogy sages,” “dictionaries,” LLtranslations,)l between elucidating the gene-polypeptide relationship and cryptanalysis has been most appealing; thus the term “genetic code” received quick general acceptance, and decoding terminology and concepts were freely expropriated. There is a certain danger in taking this sort of analogy too literally -of treating i t too seriously, of failing to see where it is inapplicable. Such a mistake can lead (and has, I feel, led) to the popular misconception that a cryptographic solution-a “cracking” of the code-is all
PRESENT STATUS OF THE GENETIC CODE
109
there is to the genetic code. Certainly it can be argued that one is a t liberty to define the term “genetic code” in just such a way if he SO chooses. But then what term is to be used t o describe the other (and closely related) facets of the problem of the immediate molecular aspects of gene expression? It seems most reasonable to use the now accepted term “genetic code” to cover all these related facets of gene expression, and realize that the analogy between the genetic code, so-defined, and cryptography is not totally accurate. Two of the more serious conceptual errors that can result from a too-close adherence to this analogy are a failure to condsider any possible relationships between the nature of the bet of codon assignments and thc nature of the translation apparatus, and a failure to consider the sck of codon assignments in the light of its possible evolutionary origin. Defined in the above way, we see the genetic code to comprise a number of interrelated, but often technically separable, problems: (a) the cryptographic problem-the elucidation of the set of codon assignments (or “codon catalog” as I will call it) ; (b) the problem of instructions to the “decoding machinery” that are encoded in the genes; (c) the nature of the translation machinery, etc.; and (d) the evolution of the whole. On perhaps another level is the problem of what principles and/or mechanisms may underlie and so give rise to the structure of the whole-in short, the problem of the fundamental nature of the genetic code. The above, I think, adequately answers what the “genetic code” is and how it came to be called that. Let us turn then t o the details of the various facets of the coding problem. A useful way to begin elaborating a coding concept is through a brief review of the salient features of the code’s early history-covering that ten-year period preceding the discovery of the cxperimcntal appro:tch to codon assignments. It was during this time that treatment of the code was almost entirely theoretical; Imt, it was the time when the mattcr of the nature of the code was given wrious attention. The concepts developed then have been but slightly modified in the subsequent (five-ycar) period.
II. Historical The early theoretical attempts to characterize the genetic code were best known for their bold, spectacular conjectures as to the cryptographic solution, but their real value lay in their contributions to general properties of the code-to framing the questions that must be answered. As the initiated reader knows, all these theoretical solutions of the cryptographic problem have turned out to be totally incorrect, but many of the early
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surmises concerning more general features of the code have now been verified. We shall go into these latter contributions and their relationship to the more recent experimental findings in some detail. But first, let us examine the more famous of the attempts a t theoretical solutions to the cryptographic aspect of the problem. These more than anything convey the general intellectual climate in which ideas of the code arose.
A. Theoretical Attempts at a Cryptographic Solution Although Gamow was not the first to consider the coding problem, he was the first to treat seriously the cryptographic problem involved (6)J In fact, his overriding concern with this facet of the code not only set a definite tone for subsequent approaches to the code for years afterward, but seems to have been the weak point in his own approach. Gamow possessed a physicist’s “faith” in numerology. He felt that natural phenomena tend to manifest themselves in very simple numerical relationships. Therefore, to him, it was not incidental th a t precisely 20 kinds of amino acids are used in the synthesis of protein. For the reasons discussed below Gamow assumed the codon to be in essence a triplet of nucleotides, which meant that 43= 64 different kinds of codons were possible. The occurrence of only 20 kinds of amino acids in proteins then had to mean that some sort of biological or chemical “degeneracy” (equivalence of states) existed that reduced the distinguishable number of triplets to exactly 20. Certain triplets had to be identical in their coding role. Gamow devised two plausible schemes for placing the 64 possible triplet codons into exactly 20 equivalence categories, and each scheme had one or more tantalizing features that argued convincingly for its acceptance. Note that if proteins had contained some such number as 19 or 23 kinds of amino acids, it would have been impossible to devise a systematic rule for deriving that number-hence the special significance to the number 20. Gamow’s first code drew its raison d’dtre from the newly postulated Watson-Crick double-stranded structure for DNA. There appeared to be “diamond-shaped pockets” in this structure, each “diamond” being bounded and formed by a base in one DNA chain, the adjacent base pair (spanning both chains) and the next base adjacent to this in the opposite DNA chain (6). Gamow saw these “pockets’1 as specific templates for the amino acids, holding them in position until they could be polymerized into a polypeptide. By requiring that the symmetry operations of reflection and rotation (through 180 degrees) leave the amino acid (‘recognizing” properties of a diamond unchanged, the 64 different ‘The first to suggest that coding was related to various combinations of bases was Dounce (6).
111
PRESBNT STATUS OF THE GENETIC CODE
possible diamonds were divided into the requisite 20 distinguishable categories. (I leave the interested reader to deduce the details of such a scheme.) By requiring that the code be translated in a “fully overlapping” fashion (defined below) the spacing between adjacent diamonds c.ould be made to correspond very closely to the spacing between adjacent amino acid residues in a polypeptide chain. This last feature made Gamow’s code a particularly good one, in the sense that the theory was readily testable; fully overlapping codes place severe constraints on permissible amino acid sequences. Thus Gamow himself was TABLE I GAMOW’S TRIANGLE CODE (8) Class I Codons-Nondegenerate 1 AAA 2 ccc 3 GGG 4 uuu
1 AAA 2 AAG 3 AAU 4 CCA 5 CCG 6 CCU
Class I1 Codons-Triply Degenerate ACA CAA 7 GGA AGA GAA 8 GGC AUA UAA 9 GGU CAC ACC 10 UUA CGC GCC 11 UUC CUC UCC 12 UUG
1 ACG 2 ACU 3 AGU 4 CGU
Class I11 Codons-Sextuply Degenerate AGC CAG CGA GAC AUC CAU CUA UAC AUG GAU GUA UAG CUG GCU GUC UCG
GAG GCG GUG UAU UCU UGU
AGG CGG EGG AUU CUU GUU
GCA UCA UGA UGC
able subsequently to show that this code was inconsistent with known amino acid sequences in the insulin molecule ( 7 ) . Gamow’s second coding attempt, the “triangle code,” is shown in Table I ( 8 ) . The idea behind this derived in part from the growing realization that single-stranded RNA, not double-stranded nucleic acid, is probably responsible for the synthesis of protein. The triangle code is actually quite similar to its predecessor, the “diamond code,” in that essentially the same symmetry operations were used to produce the degeneracy that yielded the required 20 categories of codons. All triangles that were interconvertible by either a rotation or reflection operation were taken as equivalent in coding properties. As Table I shows, the constraint employed leads to four groups of codons that are nondegenerate, twelve groups that are triply degenerate, and four groups that
112
CARL R. WOESE
are sextuply degenerate. The main rationale for this code lay in the demonstration that the relative frequency of occurrence of the various codon categories, calculated according to the RNA composition for TMV-RNA, produced a distribution of just about the same shape as that yielded by the relative frequency of occurrence of the amino acids in TMV coat protein (9). The disproof of this coding scheme came with the demonstration that codons of the same composition, but different order of bases, are not assigned to the same amino acid. While Gamow’s specific codes were to be accepted for a short time only, the impact of his general thinking here was profound. Not only did his codes recognize, in one way or another (as we shall see more fully below) , most of the important facets of the coding problem, but it is fair to say that the comma-free codes (and the “adaptor hypothesis”), which supplanted Gamow’s schemes, arose in part as a reaction to certain of his ideas. The comma-free codes were designed to solve a special problem that arises when a message is translated in a nonoverlapping fashion. TO understand both problem and solution consider the following: Suppose we are given a segment of a (linear) message, taken from somewhere in its middle; how are we to tell which combinations of contiguous letters are the words and which are not? The structure of our (written) language is such that words in a sentence are clearly defined through the use of a special symbol, the “space.” However, suppose we are dealing with a form of writing in which the equivalent of a “space” symbol is not employed, so that all the words are run together. Then how does one distinguish which of the groupings of letters in the segment constitute words? There appear to be two ways to solve the problem. For one, it is possible to pick a known point, say the beginning of the message, and count off “words.” However, this method is possible only if there exists a definite counting rule--e.g., all words are of length three, so one counts in groups of three letters. The other solution is t o divide all possible words into two classes. The one class contains all and only those words that have i‘meaning,” the intended words. The words in the other class have no meaning. The classes must be constructed so that no two words in the “intended” class, when placed adjacent to one another, produce a group of contiguous letters that is itself a member of the intended class. For example, if ABC and DAC are two words from the intended class, then BCD, CDA, ACA, and CAB are not permitted to be in this same class. This rule then eliminates any confusion as to which groupings of letters in a message are supposed to be (meaningful) words, and so permits an unambiguous reading. When Crick and co-workers considered the ramifications of the
113
PRESENT STATUS OF THE GENETIC CODE
second alternative, they uncovered the remarkable coincidence that (if one deals with three-letter words and a four-letter alphabet) the maximum number of words that, can have “meaning” is 20-exactly the number of amino acids in protein (10) ! Table I1 shows one such grouping. Although this “comma-free code,” as it was called, had no facts to support it, the generation of the numbrr 20 from such simple and hasic assumptions was a t that time rrason enough for most scientists to accept the comma-free code as the probable solution t o the coding problem. In retrospect, it appears that Naturc has had a bit of a joke a t our expense, for there seem to be quite a few different logical oprrations that Can place 64 objects into 20 rl:isces, bnt none FO far produce an oldcring to tlic set of codons that rrsembles the cxperimcntally obTABLE I1 A “COMMA-FREE” TRIPLET (!ODE
OF
MAXIMUM SIZE GCA GCC GCG GCU
ACA ACC ACG ACU
AlT.1 AUl J
served one. Thus we are left wondering why the intriguing number 20 characterizes the encoded amino acids, but now also whether such a question really has a n answer a t all. A number of other cryptographic solutions for the code were proposed during the 1950’s, but these are of little or no interest now because they added nothing new to the overall concept of the genetic code dcvelopcd by Gamow, Crick, and, as we shall see, Dounce. Thus there is no reason to consider them in the present context. The interested reader will find discussion of these other attempts in references (7’) or (11).
B. The Nucleic Acid-Polypepfide
Relationship and the Nature
of Translation
1. COLINEARITY OF GENE AND POLYPEPTIDE
All coding theories implicitly or explicitly took for granted that the nucleic acid message and its corresponding polypeptide chain were “colinear.” That is to say, it was assumed that units (now called codons)
114
CARL
R. WOESE
in the nucleic acid chain correspond to units (amino acid residues) in the polypeptide chain, and the order of codons along the nucleic acid chain is exactly the same as the order of the corresponding amino acid residues along the polypeptide chain (1.2). The ease with which this assumption was made is certainly in part a function of the structure of language. However, the suggestion, as we have seen, is also strong in the nature of the biopolymers involved, and in the linearity of the genetic map of a gene. Although a good deal of indirect evidence long supported this assumption, it was not until rather recently, as we shall see, that a simple direct proof of this axiom could be accomplished. 2. CODONSIZE
On the matter of codon size it again turns out that the early theoretical assumptions (Dounce’s, Gamow’s, and Crick’s) are correct. Gamow appears to have assumed a triplet codon for two reasons: For one, three bases is the smallest grouping that provides sufficient information to code for 20 amino acids. For another, Gamow’s original scheme envisioned amino acids fitting into “diamond-shaped” pockets in the nucleic acid double helix, these pockets being basically triplet codons-i.e., two bases plus one base pair. This sort of model also led to the (correct) postulate that all codons are of uniform size. 3. OVERLAPPING vs. NONOVERLAPPING TRANSLATION
The structure of our language is such that no words in a sentence share common letters among them. However, it is possible to conceive of a manner of reading a message in which some of the letters that are a part of one word are also parts of the next word, etc. For example, consider the message ABCDEFGHIJKL, assumed to be composed of three-letter words only. Our experience with language would lead us t o assume that ABC, DEF, GHI, and JKL are the only words in the message. However, it is also possible to read the message in such a way that ABC, BCD, CDE, DEF, EFG, etc., are all words, read in that order, or such that ABC, CDE, EFG, GHI, etc., are the words. I n the first case the reading is called a “nonoverlapping” one; in the second case a “fully overlapping” one, and in the remaining case a “partially overlapping” one. As we have seen above, the issue of overlapping types of translation arose rather early in the code’s history, for in order to adjust the spacings between “diamonds” in DNA to the spacing between amino acid residues in a polypeptide chain, Gamow had to assume his code to be translatable in a fully overlapping fashion. I n contrast, the comma-free codes assumed the opposite, a rionoverlapping translation. The reason
PRESENT STATUS OF THE GENETIC CODE
115
for this latter assumption is inherent in the “adaptor” concept, discussed below, for if one “adaptor” is occupying a codon, no other adaptor can use bases common with that codon. Of course, experimental evidence has now shown that a nonoverlapping translation is the one actually involved in peptide synthesis (see Crick in Volume I of this series). I n any case, the impossibility of having a fully overlapping translation involving triplet codons was known for some time before this. The ronstraints that a fully overlapping triplet code places on peptide sequences require that only 4 x 4 x 4 X 4 = 256 dipeptide sequences are permissible, out of 20 x 20 = 400 possible sequences. By 1957 a sufficient number of peptide sequences had been experimentally deterniined that (assuming the genetic code to be universal) the impossibility of a fully overlapping translation could be proved (IS).
4. PUNCTUATION Again, the close analogy between the genetic code and the structure of language, particularly written language, readily suggests the possibility of various sorts of punctuation being employed by the cell. Actually the early theories were rather weak in their treatment of this facet of the genetic code. As seen, the comma-free code arose from a consideration of problems arising out of a message structure that does not use a “space” symbol to delimit lLwords.”However, beyond this, little or no attention seems to have been given to the other aspects of punctuation, such as the signals for beginning and ending of translation. I suppose it was assumed implicitly that the physical length of the nucleic acid message defined the length of the corresponding polypeptide, which could make initiation and termination punctuation a trivial matter. However, we iiow know that a single-message RNA in some instances is large enough to, and does, produce more than one kind of polypeptide chain (14). The details of how the cell accomplishes punctuation, the sequences involved, dx.lare postponed for the experimental discussion below.
5. THEMECHANISMS OF TRANSLATION AND TRANSCRIPTION Punctuation is of interest not only for what sequences are involved, but also in terms of the mechanisms by which the process operates. Thus punctuation is in a sense a transition point, between the purely formal considerations of what codons correspond to what amino acids and the consideration of the process of decoding. Although the details of the overall mechanism of translation were obviously too subtle to attempt treating them in the early coding theories, two general aspects of translation did receive attention a t that time, one of which was the subject of considerable controversy. I refer to the matters: (a) the order
116
CARL R. WOEBE
in which peptide links are made in synthesizing the polypeptide chain; and (b) the question of whether the relationship between an amino acid and its corresponding codon in the nucleic acid template is a direct (physical) interaction, which presumably then involves a “recognition” of the amino acid by the codon, or whether an intermediary moleculean “adaptor”-is somehow interposed between the amino acid and its codon. Concerning the matter of order of synthesis of peptide links, it appears that the two major theories were incorrect in their suppositions. Both the Gamow schemes and the comma-free code were predicted upon the assumption that the template, in one way or another, lines up all the amino acids prior to their being linked together. In particular, the formulation of the comma-free code rejected the idea that reading a message correctly and unambiguously is merely the result of starting a t a defined point and “counting off” the words. This sort of sequential counting mechanism in protein synthesis would imply, of course, that amino acids are aligned in relation to the template and that peptide links are made one a t a time, starting from the beginning of the message-exactly a s a sentence is spoken. As it turned out, sequential synthesis of a polypeptide is the mechanism used by the cell (15, 16). With regard to the matter of the necessity of intermediary molecules in the codon-amino acid relationship, the opposing views were well defined. On the one hand, Gamow, building on the earlier sperulations of Pauling and Delbriick and others (17-19), favored a direct interaction, a “recognition,” involving codon and amino acid. This concept had simplicity in its favor, which made it particularly appealing to the physicist. And too, a direct recognition had analogies in the recognition of substrates by enzyme, or antigens by antibodies ( 2 0 ) . On the other hand, Dounce had earlier postulated the existence of intermediaries between the amino acids and their codons-a set of enzymes that each “ ~ O S S C S S specificity for a certain limited segment of the polynucleotide Chain, as well as for a given amino acid . . .” (6),thus relegating the burden for recognition of both the codon and the corresponding amino acid to these enzymes. Dounce may have assumed implicitly what was stated later explicitly by Crick, th at the existence of these intermediaries is necessitated by the fact that oligonucleotides are incapable of “recognizing” amino acids-i.e., that oligonucleotides cannot show specificity for amino acids (21).Crick’s picture of the intermediary molecules was a bit more complex than Dounce’s. Crick postulated two classes of intermediary molecules: (a) the “adaptors,” l‘small molecules” carrying the amino acids and recognizing the codons (and lining up on the nucleic acid template) ; and (b) a set of enzymes that recognize and link together the individual amino acids and their corresponding adaptors. As
PRESENT STATUS OF THE GENETIC CODE
117
we shall see below, the question of whether the amino acid is related to the template nucleic acid directly or through intermediary molecules is now definitely settled in favor of the latter mechanism, but the related questions of whether specific oligonucleotide segments can in any sense “recognize” specific amino acids, and whether such interactions, if they exist, play any role in the translation process, remain unsettled. 6.
O R I G I N AND
EVOLUTION OF
T H E G E N E T I C CODE
The most notable shortcoming in the early, theoretical attempts to clinractcrize the code was the almost complete lack of attention to the cvolution of the whole. [Dounce alonc seems to have considered this niatter (22).] An Understanding of the code’s evolution is almost certainly ccntral to any understanding of the fundamental nature of the code itself. Paucity of fact cannot be the reason for this oversight, for a similar paucity of fact did not deter speculations concerning most other facets of the coding problem. I will grant that early evolution-the “pre-Darwinian era”-is a rather nebulous area a t present, but still there are reasonable bounds within which the code’s evolution must have occurred. And these should have been taken into account in framing any comprehensive coding scheme. For example, it seems to me that such a code as the comma-free code, which would have to come about through a process of trial and error ( i.e., reassignment of codons), could never evolve in actuality. The number of trials required to bring the code to this condition are, for all practical purposes, infinite. Perhaps the failure to consider the code’s evolution in these early theories can be nttributed to the language-genetic code analogy ; an understanding of the structure of language does not rest upon a knowledge of whence it originated. The problem of the code’s evolution is just now beginning to receive its proper attention. Hopefully this historical survey provides somc concept of the genetic code, :i framework in which the cipwimcntal details e m be presented, cvaluntcd, and assimilated. We follow below the swmc g e n e d pattern a s in the liihtorical bur~rcy-bc,ginriiilg with the rryptographic :tspect of t l i v problcni, through various jicnc~nlpropcrties of the gene-polyi)eI,tide relationship and ccrtain of details of translation, and finally on to consideration of the fundamental nature of the code and its evolution.
111. The Cryptogruphic ProQIem A. Codon Assignments Determined by Polypeptide Synthesis in Vifro The vlegance of the coding thcories of the 1950’s had accustomed us to think in terms of a “cracking of the biological code”-of a sudden
118
CARL R. WOESE
realization of the structure of the whole stemming from brilliant theoretical analysis based upon the most meager factual clues. However, this was not t o be. Serendipity, not brilliant design nor esoteric analyses, was to produce the prize. And when facts did finally come, they came in superabundance. The whole character of our approach to the code was suddenly changed by the development of a satisfactory in vitro protein-synthesizing system. It is not my purpose to review the history or technology of these in vitro systems. Actually in the context of deciphering the codon assignments, the workings of an in vitro protein-synthesizing system are unimportant, for the whole can be treated merely as a black box; mRNA can be considered the input, in response to which a polypeptide chain, the output, is produced. The question of codon assignments-i.e., the relationship between the primary structures of input and output can be answered without knowing what happens in between. Nirenberg and Matthaei’s remarkable initial discovery was that addition of the simple synthetic RNA, poly U, to such a system, resulted in production of the simple polypeptide poly Phe (23). We need not know how it was produced in order to assign the codon UUU to the amino acid Phe. The essence of this in vitro approach to codon assignments is to use input RNA’s of either defined primary structure or of undefined primary structure simple enough so that the gross composition of the output polypeptide reveals either the exact codon assignments or very narrow limits for the codon assignment for an amino acid. In the beginning only the homopolymeric RNA’s (poly U, poly A, etc.) and copolymeric RNA’s of random primary structure (e.g., poly U,C) were available for these studies. More recently, it has been possible to synthesize RNA’s of known nontrivial primary structures, by combining the actions of synthesizing enzymes, degrading enzymes, and chemical polymerization (24-28) .
There is little point any longer in detailing the earliest experimental attempts a t fixing the codon assignments, which werc based upon the use of homopolymeric and random sequencc copolymeric RNA’s. [A more detailed discussion of these early experiments, their drawbacks, the accuracy of the assignments so determined, etc., can be found in previous reviews (e.g., 11, I,% ).] A hypothetical example will convey the essence of the approach. A random sequence RNA composed of two kinds of bases, A and B, contains eight different nucleotide triplet sequences: AAA, (AAB, ABA, BAA), (ABB, BAB, BBA), and BBB. If A # B , then the frequency of occurrence of the various triplets is not the same. Setting A:B = 1:3 would mean that if AAA is taken to occur with a frequency of 1.0, then the group of triplets of composition
PRESENT STATUS O F T H E GENETIC CODE
119
A,B will occur with relative frequciicy of 3, that of composition B2A with relative frequency 9, and BBB with relative frequency 27. Therefore, when an amino acid is incorporated into polypeptides in response to the introduction of such an RNA into an in vitro system, not only do we know that the amino acid i n question has a codon containing only A’s and/or B’s, but the level of that amino acid’s incorporation (relative to that of the other amino acids also incorporated) generally fixes the c,odon assignment a t one of no more than three possibilities. Similarly copolymeric RNA’s of three bases contain 27 possible codons, and fix assignments a t one of no more than six possibilities. Using such RNA’s, the gross compositions of about 40 codon assignments were determined. As is apparent, the use of random sequence RNA’s in this approach does not really fix codon assignments uniquely, for the order of bases within the codon is not determinable in this way. For a while this matter of the absolute order of bases in codons appeared to be a major stumbling block for the in vitro system approach, because RNA’s of known nontrivial sequence could not be obtained (or the few that could did not function correctly in the in vitro systems then employed) (29, 30). However, this iinpasse w:is short-lived. Nearly simultaneously three niethods were devised to overcome it: the development of methods for synthesizing either “honiopolymeric” RNA’s containing one or two codons of noiitrivial sequcnce or copolymerir RNA’s of repeating sequence (as we have scen above), and the devising of a new approach that did not require polypeptide synthesis a t all, the “triplet binding” method of Nirenberg and Leder. The last of these was first to yield an appreciable number of ordered codon assignments. The triplet binding method is based upon the observation by Kaji and Kaji ( 3 1 ) , by Nakanioto et ‘at. ( 3 2 ) , and others, that in the presence of poly U, ribosomes bind tRNAPh‘ specifically. The original observation relied upon sedimentation to separate bound tRNAP’” from the remaining tRNA’s. Nirenberg and Leder introduced an important change in technique, making use of the fact that ribosomes adhere to cellulose nitrate membrane filters under proper ionic conditions, whereas tRNA alone does not ( 3 3 ) . Thus tRNA bound t o ribosomes is readily separable froin tRNA not so bound. With this technique it was possible to ask and answer several important questions: (a) whether an oligonucleotide of the suspected size of a codon-i.e., three nucleoside units --brings about the binding of tRNA corresponding to a particular amino acid, and (b) whether such “triplet binding” bears a relationship to the codon assignments determined by peptide synthesis. It was found that trinucleotides do cause the binding of tRNA’s to ribosomes. This binding appears weaker than that caused by polynucleotides, but it is as
120
CARL R. WOESE
firm for a trinucleotide as for a tetra- or pcntanuclcotide, and most importantly, trinucleotide binding is highly specific for corresponding tRNA’s. Thus Nirenberg and Leder (33) could show that UUU, AAA, and CCC caused the binding to ribosomes of the tRNA’s for Phe, Lys, and Pro, respectively-in complete accord with the Phe, Lys, and Pro codon assignments, determined by polypeptide synthesis using homopolymeric RNA’s. Further, when a number of nucleotide triplets of nontrivial sequence were tested, it was found that they caused the binding of the tRNA’s for amino acids whose codon assignments were consistent with such binding (34). It appeared then that this “triplet binding” method was a valid one for determining codon assignments. It must be kept in mind, however, that triplet binding per se is not a determination of codon assignment; this must be done by actual peptide synthesis. This cautionary note is substantiated by the fact that triplet binding studies alone could not reveal the correct codon assignments in a number of cases-eithcr because negligible binding was caused by some triplets (34), or because some triplets caused significant binding of tRNA’s for more than one amino acid ( 3 5 ) . All in all, though, this method has proved most useful, and the final, unequivocal determination of codon assignments more recently by peptide synthesis has suhstantiatcd practically all the assignments that were proposed as “certain” on the basis of triplet binding. The use of RNA’s of nontrivial known primary structure to determine codon assignments, which followed the triplet binding studies, needs no further explanation. Table I11 lists the repeating sequence copolymers that have been synthesized and tested for directing polypeptide synthesis t o date. Table IV is a compilation of codon assignments determined by all the various methods. It can be seen that all codons but three have been assigned to amino acids. Two of these, UAA and UAG, as we shall sce, appear to serve as peptide chain termination punctuation. The remaining one, UGA, may also serve some similar function (40). This codon catalog shows a high degree of order. For the moment we shall be content with a superficial description of this order, dealing later with its details and possible significance: ( a ) All amino acids having codons of the form XYU also possess codons of the form XYC. (b) A similar rule holds for most, but not all, amino acids possessing codons of the form XYG us. XYA. (c) Amino acids generally possess either two or four codons; the exceptions are Trp and Met (each having a single codon, of the form XYG), Ile (having three codons of the form XYU, XYC, and XYA), and Ser and Arg (each having six codons). (d) Amino acids with similar chemical properties seem in some general way to possess similar codon assignments.
121
PRESENT STATUS O F THE GENETIC CODE
TBBLE: I11 THETRANSLATION o r COPOLYMERIC 13 NA’s Repeating sequence copolymer
OF
REPEATINGSEQIJENCE~
Codoiis involved
~~
~
~
Polypeptide into which translated
~~~~
Data from ~hCJrallael ul. (18). No polymer rorrespoiidiiig to the lr(;A C O ~ C J I Iis formed. No polymer corresponding to the IIA(; codoii is formed. The exact reason for failure to observe pept,ide synthesis in these cases is not clear. Each one contains one of the rodons implicated in peptide chain termination punctuation, but it, is also possible that, secondary structure of tjhe polymer may prevent translation.
B. Other Approaches to Codon Assignments There is no longer reason to discuss in detail the determination of codon assigniiients by methods other than those utilizing in vitro systems. The rapid success of the intter approaches has totally eclipsed the former. However, certain of these alternative approaches remain rclevant today, not as methods for detcimining the bulk of codon assignments, but as incans by which the few uncertain spots that remain in the codon catalog can be filled in, as tools by which the functional roles for the various coclons in thc cell can be elucidated, and as general probes for studying genes, gene expression, and aspects of evolution. Perhaps the most useful of thcse alternative approaches are the “amino acid replacement” am1 the related “reading-frame displacement” approaches. Both rely upon ineasuring the cliangcs in amino acid sequence caused by mutations in the underlying gene. Amino acid re-
Source of data
Source of data Assignments UUU UUC UUA UUG
Phe Phe Leu Leu
CUU
Leu
CUC
Leu
CUA CUG
Leu Leu
AUU ATJC AU.4 AUG
Ile Ile Ile Met
GUU GLTC GUA
Val Val Val Val
GUG
a
b
c
+ + + + + +
+ + + + + + +
+ + + + +
d
+
+ + + + + +
X
+ + + + x
+ + +
UCU UCC UCA UCG
Ser Ser Ser Ser
CCU CCA CCG
Pro Pro Pro Pro
ACU ACC ACA ACG
Thr Thr Thr Thr
GCU GCC GCA GCG
Ala Ala Ala Ala
CCC
+ + +
+ + + + +
Assig1iment.s a
X
b
c
d
Source of data Assignments
+ + + UAU + + UAC + + + LJAA + X UAG + c.iu + + CAC + f + CAA
+ + + + + + + + + + + + + + + + +
X
x
Tyr Tyr
* *
CAG
His His Gln Gln
AAU
Asn
AAC: AA.4 AAG
-4SIl
GAU GAC GAA GAG
Lys Lys Asp
Asp Glu
a
b
c
d
Assigrlments
X X
UGU UGC UGA UGG
Trp
CGU CGC CGA CGG
Arg Arg Arg Arg
AGU AGC ,4GA AGG
Ser Ser Arg Arg
GGU GGC
Gly Gly Gly Gly
+ + +
+ + + m
+ + + + + + + + + + + X + X + + + + + +
+ + +
X
f
X
+ + + + + + + + + + n
Source of data
X X
GGA GGG
cys Cys
#
a
b
+ + + + +
X
+ + +
+ +
+ + +
I1
I1
+
d
+
m
f
+
c
+
+ + + + +
x
+ X
X
Methods a-d: a : Composition deduced from peptide synthesis using random sequence RNA. b: Composition and order deduced by peptide synthesis using ordered sequence RNA. c: Composition and order deduced by triplet (or RNA) binding method. d : Composition and/or order deduced in part by some other method (amino acid replacements ( X ) or reading-frame shift (+) experiments). b Symbols and abbreviations: * : “Amino acid replacement” analysis indicates that these codons are involved in peptide chain termination punctuatioI1. #: Unassigned to an amino acid, also possibly involved in peptide chain termination punctuation. + : positive evidence for this assignment by method a, though there should have been. n: Positive result, but no better than binding caused by similar but “unacceptable” triplets. m: incorporation observed by method b. (L
PRESENT STATUS OF THE GENETIC CODE
123
placements measure the effect of base substitution mutations. Readingframc displacements deal with the effect of adding or deleting nucleotides. A single base substitution manifests itself as the replacement of one codon by another, which in turn means the replacement of one amino acid by another in the polypeptide chain. As is obvious, amino acids that can “replace” one another must possess codons related t o one another in a particular way, but a determination of the set of possible amino acid replacements does not tell anything about the composition of codons, or order of bases within codons. However, both these matters can be approached through refinements of the replacement approachby the use of mutagens producing (partially) defincd base substitutions (41) , and through genetic recombination analysis (recombination splitting the codon in question) ( 4 2 ) . As we shall see, amino acid replncement analysis has been used very effectively to elucidate the matter of chain termination, and more recently it has been applied to the matter of relative usage, in who, of the various codons assigned to particular amino acids (43). A considerable amount of amino acid replacement data has accumulated over the last seven to ten years, particularly from comparisons of normal and abnormal hemoglobins ( 4 4 ) , from spontaneous or chemically produced mutations in TMV coat protein (41, 45), and from the many mutations involving a very few codon sites in the gene controlling the synthesis of the A protein of tryptophan synthetase in E. coli (46).Table V gives thcsc amino acid replacement data. A comparison between these and the codon catalog (Table IV) reveals very few inconsistencies. I n the one case where an inconsistency has been investigated in detail (the Glu-Met replacement) it has been shown that the replacement undoubtedly results from a double base change in the underlying codon ( 4 6 ) . Addition or deletion of base pairs from a gene has, of coursc, a more far reaching and drastic cffect than does base substitution. If translation consists of a procc~sh wlic~el)y blocks of tlirce h s e s are countcd off, thcii deletion of oiiv 1 ) : ~ ) will have the same effect :is a inkcounting, in that the entire message distal to the point of the deletion (or miscounting) will be completely misread. For example, the mRNA AAGAAGAAG . . . would translate normally into poly Lys, but the deletion (or addition) of one base a t some point would result in the synthesis of poly Arg (or poly Glu) corresponding to the section of mRNA distal to the point of the alteration. This phenomenon is referred to as a “reading-frame displacement.” I n both the reading-frame displacenient and amino acid rcplaccment approaches amino acids are grouped by possessing codons which have two of three bases in cominon. (In thc amino acid rcplarcinent case only, however, arc the
124
CARL R. WOESE
AMINO
TABLE V ACIDREPLACEMENT PATTERNS
FOR VARIOUS PROTEINS”.b ~
I. Tryptophan Synthetase A Protein (46)
GUA
2b;A GAA
Ah Gly Asn GCU GGU AAU
y F $ + A
Ile Thr Ser Gly AUA ACA AGU GGA
Ala Gly Val GCA GGA GUA
L1
t?,kAla
Glu GAA
71 ,$
Gly GGA
b
Arg AGA
GCA
/ATL
Val Gln Met= UAG* GUG GAG AUG (amber)
Asn T h r Ser AGU AAU ACU
Gly Gly Ala GGU GCU GGU
Tyr +Cys UAU UGU
11. TMV Coat Protein (41,45) Spontaneous
Nitrous acid
Asp -+ Alan GAU GCU
Pro + Leu CCU cuu
ACU
UCU
Ile -+ Thr AUU ACU
Asn 4 Ser AAU AGU
Ser UCA
Leu UUA
Asp 4 Asn GAU AAU
Arg -+ Lys AGA BAA
Ile + Met AUA AUG
Thr --t Ile ACU AUU
Asn + Lys AAU AAA
Arg + Gly AGA GGA
Ile -+ Val AUU GUU
Thr + Met ACG AUG
Asn -+ Argx AAU AGA
Glu GAA
Ser -+ Phe UCU uuu
Tyr -+ Phe UAU UUU
Gly GGA
Asp + Gly GAU GGU
Leu CUU
--f
-
Phe UUIJ
Thr -+ Ser
125
PRESENT STATUS O F THE GENETIC CODE
TABLE V (Continued)
111. Hemoglobin Mutants (47) GluGAA
Ala GCA
Gln CAA
Glu rGln GAA CAA
AAU
Glu GAA
-
Arg
CGA
Asn +Lys
-
Val GUA
AA A
/2u C%\ TYr UAU Lys -Asp= AAA GAU
GlY GGA Gly -Asp GGU GAU
= Numbers refer to number of times the replacement is observed. b
Symbols: A: h c ~ e n s e dby 2-ami11opuriiie. * : Detected t)y “amber” suppressor gene. s: Not consistent wit,li single l m e chniige. 11: Not coii8istent with HKU2 inutageiiesis pattern.
common bases in the same positions in the codons.) Reading-frame displacements, of course, provide inore information. The power of reading-frainc displacement as a11 analytical tool is apparent. Given the codon assignments, this approach should permit the rktermination of the primary structure of a gene. To date the approach has been used in one instance only--a very restricted region of the lysozyiiie g c w of thc I)nrtriiol)liagt: 7‘4, shown in Table V I ( 4 8 ) . The TABLE VI CHANGEI N AMINOAcin SEQUENCE I N PHAGE T4 LYSOZOME CAUSEDBY READING-FRAME DISPLACEMENT (48)“ Wild-type sequence
.
. . Thr
Lys
Ser
Pro
Ser
Leu
Asn
Val
His
His
Leu
Met
Ala
...
Ala
...
Sequence in mutant
.
. . Thr
Lys
t -1
T +1
Arrows show points a t which deletion and addition mutations occurred.
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major limitation to this approach as now used, lies in the fact that unless the reading-frame displacement is confined to a relatively small portion of the gene, not only is the resulting protein functionless, but its physical and chemical properties are so changed that it may no longer be identified or isolated. However, it is clear that this technique will certainly become the method of choice for obtaining the sequences of structural genes, and should be useful as well for nonstructural genes, such as the ribosomal cistrons, if methods for producing reading-frame displacements in in vitro systems can be developed. At one time, nucleotide doublet frequencies (“nearest neighbor” analysis) in nucleic acids showed considerable promise as a means for determining codon assignments (49). It remains a useful technique for analyzing relative codon usage (in certain cases), for identification of nucleic acids, etc. The method relies upon the fact that nucleotides are introduced during nucleic acid synthesis as nucleoside 5‘- (tri) phosphate monomer units, and enzymatic digestion of nucleic acid in many cases produces 3’-phosphate monomer units, as does alkaline hydrolysis (of RNA). This, of course, means that a phosphate residue introduced into nucleic acid as a 5‘ attachment to one nucleoside can be recovered in 3’ linkage on the neighboring nucleotide residue. This allows one to determine the average frequency with which the four nucleotides lie adjacent (on the 5‘ side) to any single kind of nucleotide in nucleic acid. The technique has been extended (in principle) by Berg and co-workers, through the selective introduction of ribonucleotides into DNA during synthesis, and cleavage of the resulting “DNA” by a ribonuclease, yielding tri-, tetra-, etc., nucleotides in addition to dinucleotides ( 5 0 ) . This technique has been eclipsed as a method for determination of codon assignments by the in vitro system approach. It remains a rather powerful probe of the cell. T o give an example-calf thymus DNA reveals an extremely low frequency of the doublet CG, much lower than bacterial DNA’s of the same G C content (49). The CG frequency is so low that the codons for Arg of the form CGX must be used little if a t all in this organism. I n keeping with this suggestion, it has been shown that, upon infecting mammalian cells, herpes virus causes the synthesis (from its own genome) of several types of tRNA, one of which is a tRNAArg (61).
+
IV. Colinearity of Gene and Polypeptide As we have seen, the colinearity of the gene and its polypeptide product was both a natural assumption to make and one that was suggested by or consistent with many known facts. However, the direct proof of colinearity had to await the development of the necessary technical
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competence in both genetics and sequence analysis of proteins. Actually, two proofs appeared practically simultaneously, each by a different approach. The first proceeded along more familiar lines: The amino acid sequence of the A protein of tryptophan synthetase from E . coli was determined, making it possible to locate the position of any amino acid replacements. Mutations leading to such replacements could then be mapped by genetic recombination analysis. I n this way the order of a set of amino acid replacements in the polypeptide could be compared to the order of the corresponding mutations in the underlying gene. The two orderings agreed with one another, and distances in amino acid residue units along the polypeptide chain were roughly in a constant proportion to the corresponding distances on the genetic map between mutations ( 5 2 ) . The second proof of colinearity was elegant in its simplicity. By utilizing a particular property of the translation system, i t was possible to avoid the arduous task of sequencing a protein in order to test the colinearity assumption. A certain two codons serve as polypeptide chain terminators. When either of these are encountered (in the reading frame) in an RNA message, the synthesis of a polypeptide chain is there terminated and the “completed” polypeptide is released from its link to tRNA. Thus if such a codon (generally called an “amber” codon, unfortunately) is accidentally created through mutation in the middle of what was a structural gene, the translation of the gene then produces a shortened polypeptide, corresponding to the genetic segment proximal to the “amber” mutation only. (From the point of view of making the experiment feasible, it is also necessary to note that “amber” mutations can be suppressed in bacterial strains carrying the proper suppressor gene-the mechanisms involved in this are discussed below.) The actual experiment was as follows: A number of “amber” mutations involving the structural gene for the head protein of T4 were isolated (the mutant phage was maintained on E . coli strains carrying an “amber” suppressor gene) . The mutations were mapped-i.e. ordered-by genetic recombination analysis. Each amber mutant should permit the synthesis of a portion of the head protein polypeptide (in nonsuppressed host strains), the size of which should be characteristic of the mutation’s position in the head protein gene. The approximate proportion of the head protein sequence produced in each case could be measured by the simple device of digesting the polypeptide produced on phage infection with either trypsin or chymotrypsin2-proteolytic enzymes that hydrolyze polypeptides at specific amino acid residues only. The number of (small) ’At a certain period in the infectious q d c , head protein is the the major species produced. Therefore cells labeled with radioactive amino acids during this period yield mainly labeled peptide fragincnts derivcd from liead protein.
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peptides so produced would measure in each case approximately the length of the portion of the head protein produced (even though the order of these peptides in the head protein sequence was not necessarily known). If colinearity holds, then an “amber” mutant located near the proximal end of the head protein gene would produce only a few of the tryptic peptides found in the total head protein; an “amber” mutation located further along the gene would produce these same peptides plus others, and so on. In other words, the ordering of various “amber” mutations by the number of tryptic or chymotryptic peptide fragments they produce should be the same as that determined by genetic mapping of these mutations. This was indeed the case, so proving the thesis of colinearity ( 6 3 ) .
V. Codon Size It is generally impossible to separate experimentally, and so to discuss separately, codon size and the question of whether or not translation is of the overlapping type. Most determinations of codon size are actually determinations of the coding ratio. In all there have been four major experiments bearing upon codon size, cte., all utilizing rather different approaches. One is that of Crick et al., based upon the properties of addition and deletion mutations ( 5 4 ) . The second is that of Staehelin et al., in which the size of mRNA corresponding to a given size of polypeptide was determined (55).The third, by Nirenberg and co-workers, utilizes the triplet binding approach ( 3 3 ) . The fourth uses synthetic RNA’s of simple repeating sequence, as synthesized by the techniques of Khorana et al. (68).Only the first of these will be discussed in any detail. Crick et al. reasoned that it might be possible to deduce the coding ratio, if not the codon size, from genetic experiments, by utilizing the properties of addition or deletion mutations. The addition or deletion of 3n 1 or 3 n 2 base-pairs from a gene produces (as we have seen) a reading-frame displacement, which results in translating the gene into a completely different polypeptide sequence distal to the point of the mutation’s occurrence. They argued as follows: If deletion of a single base-pair displaces the reading frame, and so produces a mutant phenotype, the addition of a base-pair somewhere else in that gene will restore the reading frame to norinal, and perhaps yield something resembling a wild phenotype--provicted that the polypeptide segment cor1 esponding to the genetic scgnicnt hetwcen the addition and deletion mutations is not critical to the function of the protein. More importantly,
+
+
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however, if the coding ratio is n (i,e., the translation tape reader jumps n nucleotides for each amino acid placed into the polypeptide chain), then by combining in the same gene exactly n equivalent deletion (or addition) mutations, the reading frame should also be restored. I n this way the number n, the coding ratio, could be determined. I n practice the experiment was performed in the following manner: Acridine orange is known to produce mutations of the addition-deletion ( 5 6) type in recombining systems, such as the T-even bacteriophages. An initial reading-frame displacement mutation in the B cistron of the rII region of the T 4 phage genome was obtained by this means. Spontaneous revertants of this mutant phage were then isolated. I n almost all cases reversion results from the occurrence of a second mutation a t a locus near the original one. This second mutation is also of the addition-deletion type, compensating for the reading-frame displacement produced by the original mutation, i.e., restoring the original reading frame. I n isolation, the second mutation, of course, manifests the characteristics of a typical addition-deletion type of mutittion, and so it too reverts to a wild phenotype by the occurrence of still another mutation at a nearby locus within the same gene. Proceeding in this manner, a series of mutations can be isolated, where the second in the series arises to restore the displacement caused by the first, the third arises to restore the displacement produced by the second (when the latter is isolated), the fourth restores the displacement of the third, and so on. In such a series numbers 1, 3, 5, 7, etc. are all equivalent in that all produce the same displacement. Similarly numbers 2, 4, 6, 8, etc., are equivalent to one another. The reading-frame displacement produced by any member of the one group should be compensated by that produced by any member of the other group, a prediction that was experimentally verified. It then remained to determine the number of mutations from a single class that must be inserted into the B cistron of T 4 phage rII locus in order to restore the correct reading frame. This number turned out to be three. A number of points about this experiment should be emphasized. For one, the experiment measures the coding ratio, not the codon size. If, for cxarnple, the codon size were actually five nucleotides, and the code were translated in a partially overlapping fashion so that the coding ratio were three, the rcsult of Crick e t al. would still be obtained (57). For another, this rcsult indicates that the code is highly degenerate; the presence of a large fraction of unassigned codons would manifest itself in the areas of the cistron for which translation occurs in an abnormal reading frame-and so prevent completion of translation. For another, the result shows that the codon size (actually coding ratio) for all codons encountered must be uniform-i.e., three is not just an average number.
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The experiment of Crick et al. did yield one important anomalous result, which is best discussed in the context of punctuation. The determination of coding ratio by No11 and associates proceeds by the isolation of polysomes of various sizes and the determination of the size of mRNA associated with these. I n all cases, approximately a 90nucleotide segment of mRNA is associated with every ribosome in the polysome. By determining the size of the polypeptide synthesized per 90 nucleotides of mRNA, the coding ratio was estimated to be between 2 and 4. Since hemoglobin is known to be synthesized by a polysome that is a pentamer, and the hemoglobin chains are all approximately 150 amino acids in length, a closer estimate (55) of the coding ratio can be made: 5 x 90/150 = 3. Of course, the coding ratio determined by this approach is merely an average value. Triplet binding experiments provide a strong, though not certain, indication of codon size (but not of coding ratio). As seen above, specific nucleotide triplets bring about the binding of specific tRNA’s. Tetraand pentanucleotides do not cause any better binding, however. Dinucleotides do not bring about binding of tRNA’s to ribosomes. The simplest conclusion one can draw from these facts is that the codon is a triplet of nueleotides. The failure to determine whether changes in the 4 and 5 positions of tetra- or pentanucleotides produce changes in oligonucleotides specificity for tRNA’s, plus failure to explain why dinucleotides do not bring about tRNA binding t o ribosomes, do not permit us to state the conclusion as a certainty, however. The final experiments bearing on codon size essentially settle the matter, if it was not already so. By using repeating copolymers of the form poly AAB, Khorana et al. have shown that three homopolymeric polypeptides are produced (see Table III), the result of translation in three reading frames. This of course rules out doublet codons for all cases so tested. Further, the fact that a given nucleotide triplet can be preceded or succeeded by a number of bases without changing codon assignment-e.g., the triplet UCU, as it appears in the copolymer poly (U-C), in poly (U-U-C), or in poly (U-C-U-A)-argues strongly that the codon size is not larger than three. When we add to all the above experiments the probability that in translation two tRNA’s occupy adjacent triplet codons a t the same time, the conclusion becomes unavoidable that the size of the codon is three nucleotides and translation is of the nonoverlapping sort.
VI. Punctuation and Other Encoded Instructions As we have seen, the early theorists paid little or no attention to the matter of punctuation. Had the nuinher of ainino acids encoded turned
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out to be 19 rather than 20, perhaps the desire to use the number 20 would have forced such considerations. Be that as i t may, it is now useful, in approaching punctuation and related phenomena, to extend the analogy between the genetic code and language to a more specific analogy between the workings of the code and tape-reading processes. The cellular processes of nucleic acid replication, transcription, and translation work exactly like tape-reading processes. DNA or mRNA can be regarded as input tapes that feed through such tape readers as the replication polymerase, the transcription polymerase, and the translation tape-reading complex. Thc output of these tape readers is also a tape-DNA from replication, mRNA (or tRNA, rRNA, etc.) from transcription, and a polypeptide from translation. DNA is the ultimate reference tape of the cell, containing the information for making (almost) all the macromolecules in the cell. It is a supermolecule of molecular weight 2 to 3 x 10’ (for E . coli, that is) ( 5 8 ) ,arid thus contains sufficient information to make about 3000 different kinds of polypeptide chains (of average inolecular weight 40,000). Structurally, the DNA tape is one continuous unit ( 5 9 ) ,most probably without any double-strand breaks in it. Functionally, the DNA tape is a single tape with regard to the replication process, beginning at R single starting point and proceeding linearly to the opposite end (60, 61). In terms of transcription, however, DNA appears to be a collection of thousands of tapes, for selected segments (the cistrons or operons) can be transcribed independently of the remainder of the tape, a t characteristic and (sometimes) variable frequencies ( 6 2 ) .All RNA of the (normal bacterial) cell appears to be produced by this transcription process, from DNA, some of i t functioning as a part of the translation tapereader system, some of i t as the input tape for this t,ape reader. The tape-reader analogy makes apparent what sorts of punctuation and other encoded instructions the cell may utilize. I n some cases a t least it wenis necessary to control wtierc a tape reader attaches to and tlcti1Cllex froiii a tapr (sinrc :dl attnchiiient, :mtl tlrtachnient is not ;it entL of tnpeb). “Punctuation” wferh to thcse sorts of instructions, which control tape-reader attachniciit and detachment and the initiation and termination of output tapes. It is also apparent from the fact that some cistrons are transcribed thoussrids of times per cell cycle while others are transcribed but once that there must be control over the frequency with which a cistron is transcribed. This undoubtedly has nothing to do with the actual rate a t which the transcription tape reader travels along a cistron, but is instead controlled by the probability with which tape readers attach to or begin transcription of a cistron. This second type of control can be called “modulation.” The subject of modulation a t the
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transcriptional and translational levels has now become so extensive that it cannot be covered adequately in the present context. Therefore, we confine ourselves to a very brief summation of its highlights. Transcription modulation sequences (popularly called “operator” loci) are well known and documented in E . coli (63). They occur a t the “head” of operons ; they are probably reasonably extensive sequences in 0“ mutations-are that all mutations known t o affect them-i.e., deletions ( 6 4 ) . They occur in front of the sequence for transcription tapereader attachment (64). The presumed mechanism for their action-i.e., repressor attachment-is well known, and recent work indicates that repressor substances are proteins, or perhaps nucleoproteins (65, 65a) . Modulation a t the translational level is, however, not SO clear-cut a matter a t present. It appears that some sort of translational modulation may exist (66).Such modulation may be connected to transcriptional modulation, so that failure to translate causes a failure to transcribe. [This latter point is demonstrated by the fact that “amber” codons, i.e., translation blocks, also cause transcription blocks (67).]
A. Transcription Punctuation Very little conclusive evidence regarding punctuation for beginning or ending transcription now exists. We are essentially certain of the existence of such punctuation through experiments involving deletions of genetic regions presumably containing such punctuation. Champe and Benzer have shown that deletion of the portion of the T4 phage genome that spans the end of the A cistron and the beginning of the B cistron of the rII region, creates a single cistron of the remaining portions of these two (6%). Jacob et al. (64) showed that deletions that include the operator locus of the lac opcron sometimes place expression of the lac operon under control of a nearby purine locus. Further, a type of mutation with the characteristics expected for a mutation of a tape reader attnchmcnt site has recently been reported for the E . coli lac operon (69). Specifically, such a mutant shows a reduced rate of production of p-galactosidase both i n th(1 constitutive state and the inducible state. The mutation in qucstion is locatcd between thc existing Oc mutants and the z gene proper. While the existence of transcription punctuation is a certainty, its nature is not. It is reasonable to expect, however, that the sequences involved in transcription punctuation are repeated a great number of times throughout a genome the size of that of E . coli. It is then reasonable to expect such sequences to be universal, or nearly so. Thus, in searching for phenomena that are manifestations of transcription punc-
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tuation, we should look for sequences that are highly multiple and/or universal. A number of genetic phenomena show one or both characteristics. For one, the sex factor in E . coli is known to “integrate” a t any of a large number of loci throughout the bacterial genome (70). The integration process most certainly involves genetic homologies and crossing over. It is more reasonable to assume that the genetic segments in the bacterial genome responsible for this process are not SO placed specifically to facilitate integration, but rather are common for some other functional reason. The multiple integration sites for certain prophages argues similarly (‘71). The multiple sites discovered by Szybalski and co-workers are particularly intriguing in the present context ( 7 2 ) . The basic finding is that rRNA will “hybridize” with a fair fraction of denatured DNA (in Bacillus subtilis). Such hybridized DNA can be detected by its heavier buoyant density in a CsCl density gradient. If the DNA pieces are large enough (say 210 x loG)as much as half of the DNA strands will associate with rRNA. These DNA strands and their nonassociating counterparts have an interesting property: neither class of denatured DNA alone can be made to renature, but mixtures of equal amounts of both classes will re-form double stranded DNA when heated and cooled slowly. Thus this association with rRNA has separated the two complementary strands of DNA for every gene. It has also been determined that the primary structure of the rRNA segment associating with DNA is rich in G, if it is not actually poly G, for poly I,G will substitute perfectly well for rRNA in this interaction. Using other homopolymers, regions rich in A and T have also been detected in the genome. These “homopolymeric” stretches in DNA are estimated to be roughly 50 nucleotides long. Thus we have here highly multiple homopolymeric segments (of either G and C or A and T composition) throughout the genome of a bacterium. The only fact that might argue against a transcription punctuation function for them is that they are apparently homologous with a rRNA sequence-suggesting perhaps a role in translation. Yet our relatively extensive knowledge of translation has revealed no such role as yet. Finally, several cases have been recorded-eg., the (pX174 phageE . coli system, a Bacillus phage-host system-where the viral genome has a small homology with a particular segment(s) in the host genome (actually detected by hybridization with host RNA) (73, 7 4 ) . I n each case, the host RNA hybridizes with only one strand of the viral doublestranded DNA (in the case of +X174 this is the “replicative form”), always the opposite strand from the one hybridizing with viral mRNA. Again there seems to be a bias toward a strange composition of the
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segments involved-over 40% G, less than 10% C in host RNA homologous with the viral DNA.
B. Translation Punctuation Polycistronic mRNA’s are as much the rule as the exception. Thus there must be at least one kind of “signal” employed in the interior portions of such mRNA’s to determine the end of one polypeptide sequence and the beginning of the next. I t is important to recognize several points about translation punctuation a t the outset: For one, the same signal could conceivably serve to begin and to end polypeptide synthesis. Second, the signals need not be nucleotide triplets (or even employ only the major four bases). Third, punctuation sequences may have dual functions, being translated as well as serving as punctuation. I n vitro studies showing that almost any RNA sequence can be translated, regardless of what nucleotide sequence appears a t the beginning, indicate that no punctuation is required t o begin translation. However, this result seems to be an artifact of the Mg2+level in the in vitro system. When Mg2+concentration is held below 0.01 M , then a need for a particular peptide chain initiation punctuation manifests itself (25).3 Thus, while chain initiation punctuation may not be strictly essential to the onset of translation, it certainly seems to play some facilating role. The first indications of a chain initiation punctuation came from the studies of Waller showing that Met is very often the N-terminal amino acid in E . coli proteins ( 7 6 ) . Met, Ala, and Ser among them account for almost all N-terminal acids in this organism. Such a situation would occur if a chain initiator existed and were also translated. The definitive demonstration of this punctuation lay in two observationsone, that the coat protein of an RNA phage synthesized in vitro began with the peptide sequence Met-Ala-Ser-, whereas the same coat protein isolated from virus was identical with the exception that it no longer had the N-terminal Met. [Actually the Met introduced in vitro is N formylMet ( 7 7 ) . ] The other was that Met in E. coli is provided with two tRNA species, both responding to the sole Met codon, AUG; however, one of these permits the N-formylation of the Met subsequent to charging ( 7 8 ) .In in vitro systems low in Mg” the presence of N-formylMet-tRNA has a marked stimulatory effect upon peptide synthesis (when the AUG codon occurs in the mRNA) ( 7 5 ) . We shall return t o this phenomenon again in the discussion of translation mechanisms. ‘As the reader can see, this high-MgZe “artifact” probably has made the difference between solving the cryptographic aspect of the code by the in vitm approach and not solving it.
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The chain initiation punctuation discussed so far applies with certainty only t o that occurring c?t the beginnings of mRNA chains. Before we take up the matter of possible chain initiation punctuation in the interior of polycistronic InRNA’s it is best to consider chain termination, as some relationship between the two may exist. Unlike chain initiation, peptide chain termination sequences do not seem to be translated (i.e,, they don’t cause an amino acid residue or residues to be incorporated into polypeptide). Benzer and Champe were the first to observe this punctuation, though its full significance was not recognized a t the time ( 7 9 ) . Certain of the rII mutants of coliphage T4 were observed to grow in one strain of E. cola K12, but not in others. The subgroup of mutants so defined was also unique in being suppressed phenotypically by 5-fluorouracil. The explanation offered for these characteristics was that mutation produced a “nonsense” codon, one not assigned to an amino acid, and so translation is halted, except in strains carrying suppressors for the “nonsense,” or in the presence of 5-fluorouracil, which causes a mistranslntion of the “nonsense” (see discussion of translation errors below). Garen and Siddiqi (80) reported a similar phenomenon for a group of alkaline phosphatase negative mutants: the gene in question would produce phosphatase (at lower than wild-type levels) if transferred into a particular suppressed strain of E. coli, or when 5-fluorouracil was added to the growth mediuin. (The strain of E . coli used by Bcnzer and Champe also suppressed these mutants.) The so-called ‘Lambel”’mutants, which h a w been so useful in mapping the genome of the T-even coliphages (81), are, of course, the same “nonsense” mutations reported originally by Benzer and Champe and by Garen and Siddiqi. The complete elucidation of “amber” or “nonsense” codons has come about largely through the work of Brenner and co-workers and Garen and co-workers. By analysis of the polypeptides synthesized by a gene carrying an “amber” mutation (discussed above under colinearity) , it was shown that an amber codon does indeed halt translation distal to the point of its occurrence in mRNA (53). By collecting a sufficient number of revertants of a given amber mutation and determining the amino acid residue introduced in each instance-i.e., by (‘amino acid replacement” analysis-it has been possible to deduce the composition of the ‘(amber” codon from the known codon assignments (82-84). It is UAG. This assignment is also consistent with a characterization of this codon by various mutagens. Thcse studies have also revealed the existence of a second codon causing termination of peptide chain synthesis, UAA (85). Both UAG and UAA arc not normally assigned to amino acids, as
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Table IV shows. The main difference between the two seems to be in thc patterns of suppression they show. As we have seen, the UAG codon can be suppressed. Suppressors here are often “high level”-i.e., the translation process will pass the amber block and translate the UAG codon with a probability as high as 60% (86). Suppressors of the UAA codon, however, seem to be “low level” (less efficient) and, in addition to suppressing UAA, these also suppress UAG, though the reverse of this is not the case (85). Reasoning simply, one would feel that to suppress the true chain termination punctuation used by the cell a t a high level would cause severe damage to the cell. Thus, if UAG is used by the cell to terminate polypeptide synthesis, it is probably used sparingly. Since suppression of UAA occurs a t a lower level, this codon stands a better chance of having a major chain terminating function in the cell. One other nucleotide sequence could perhaps be involved in the process of chain termination. In determining the coding ratio by readingframe displacements, Crick et al. encountered an anomaly that they called “barriers” ( 5 4 ) . Although most coinbinations of an “addition” mutation with a “deletion” mutation in the rII B cistron produce a wild phage phenotype, a few cases were encountered where this was not the case. The failure to produce a wild phenotype could once again arise from a block of translation upon reaching some particular sequence in mRNA. The fact that base substitution mutations a t the locus in question eliminate the phenomenon is consistent with this interpretation 112). Subsequent investigation of these “barriers” has shown them to involve any of three sequences, UAA, UAG, or a sequence that does not respond to known amber (UAG) or ochre (UAA) suppressors. More recent studies show this sequence to be the remaining unassigned codon UGA. Bacterial strains containing suppressors of UGA can be isolated (4U). Thus the UGA codon seems to be functionally like UAA and UAG in all detectable respects. Thus, a t least three nucleotide sequences are known or suspected to cause peptide chain termination. We do not know for certain that any of them are the normal chain termination punctuation used by the cell. It is worth noting, however, that in the few examples that now exist where one has available amino acid replacements for the chain termination punctuation in normal proteins (e.g., in the cytochromes c (87), where the yeast sequence terminates before some of the others do) the predominant amino acids that “replace” the presumed termination punctuation are Glu and Lys-amino acids whose codon assignments are one base change removed from UAA or UAG. Returning to the matter of chain initiation punctuation for the interior cistrons in polycistronic mRNA’s two sorts are conceivable,
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punctuation permitting ribosomal attachment, or punctuation initiating translation of a new cistron utilizing previously attached ribosomes. In the second case, it is assumed that the only ribosomal attachment site is a t the head of the first cistron in the nicssage. These two types of punctuation should have rather different characteristics. For the first type of punctuation, the frequency of translation of any cistron in the mRNA would be independent, or almost so, of the frequency of translation of any other cistron in the same message. In the second instance, the frequency of translation of one cistron limits the frequency of translation of all cistrons distal to it. The following facts argue for the second alternative : the UAG (chain terminating) codon has a “polar” character, i.e., the occurrence of UAG in one cistron of an operon reduces the frequency of translation of cistrons distal to this one. This phenomenon has been demonstrated for typical operons such as the E. coli lac (88) and T r p (89) operons, and also for the case of an RNA phage. If there were separate chain initiation sequences a t the head of each cistron that allowed ribosomal attachment, i t is hard to see how this situation could occur. Another interesting characteristic of these chain terminator sequences is that the degree of polar character they ma n if e s ti.e ., the extent to which they suppress the expression of cistrons distal to the one in which they occur-seems to be correlated with the position of the sequence in the cistron (89). Mutations near the head of a cistron tend to be extremely polar, but as the position of the sequence approaches the other end of the cistron, the degree of polarity shown becomes so slight as to he undetectable. Again this holds for the RNA phage genome as well as more conventional operons (90). This is a somewhat puzzling observation, but might be rationalized by picturing the ribosome as ceasing translation but not detaching from mRNA when it encounters the UAG sequence. Instead, the ribosome may continue to travel along the mRNA in some nontranslating, or abortive translational mode, with, however, a nonnegligible probability of becoming detached per unit distance traveled. Thus if the UAG codon occurs far from a presumed chain initiation signal (at the head of the next cistron) the ribosome has a low probability of staying attached to mRNA long enough to encounter the interior chain initiation punctuation. However, if the two signals are relatively close to one another, the ribosome is almost certain to encounter the chain initiation signal. It has recently been demonstrated, through translation of a polycistronic phage RNA, that interior cistrons as well as the initial cistron in a message can use chain initiator punctuation (presumably the AUG sequence) resulting in the placement of N-formyl methionine as the N terminal amino acid ( 9 0 ~ ) .
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VII. The Translation Tage-Reader and the Translation Process Our present knowledge of the cell’s translating mechanism is rather fragmentary. This makes it difficult to relate much of what we know about translation to the context of the genetic code. Our present understanding of translation can best be given in biochemical terms. Translation appears to be separated into two distinct stages, “Trans-I,” and “Trans-11.” Trans-I comprises two steps, one in which the amino acid interacts with ATP under the influence of an “activating enzyme’’ specific for that amino acid, the product of the reaction, an aminoacyl adenylate, remaining enzyme bound (91) aa
+ ATP + E Ftaa-AMP-E + PP,
and a step in which the enzyme complex in turn interacts with a specific tRNA to “charge” (link) the tRNA with the amino acid, aa-AMP-E
+ tRNA + aa-tRNA + E + AMP
In Trans-I1 the charged tRNA interacts with one of its corresponding codons in mRNA when the latter is positioned properly in the framework of the ribosome, and in so doing positions the amino acid it carries so that it can be enzymatically added to the growing end of a polypeptide chain. Relating all this t o the context of the genetic code depends on defining such words and phrases as “specific,” “interacts,” “framework of the ribosome,’’ in molecular terms. Thus we shall now survey the pertinent facts about translation with this in mind. Perhaps one of the most potent methods a t our command for elucidating the mechanism of translation is through a study of the “errors,” “malfunctions,” “abnormal behavior,” etc., that the apparatus manifests. For sufficiently complex mechanisms, their “abnormal” modes of functioning are as characteristic of the mechanism as are their “normal” modes, and often far more revealing of the underlying processes. Thus we shall lay considerable emphasis upon such “abnormal” functioning, “error,” etc., in what follows. However, there is another, and perhaps more important, reason for emphasizing “errors” in the translation process: it seems that reduction of translation mistakes to an “acceptable” level was one of the dominant factors in the early stages of evolution, and so a study of the residual “errors” in the mechanism, in that they reflect in a qualitative way the presumed primeval errors, may provide some indication of the evolution of the genetic code. Before proceeding further, then, let us get an idea of what the overall translation error rate is. The average polypeptide chain has a molecular weight of approxi-
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mately 40,000,or 400 amino acid residues. Probably a t least 2/3 to 9/10 of each kind of polypeptide is a “perfect translation-i.e., every amino acid residue is exactly what is expected on the basis of the primary structure of the corresponding mRNA and the catalog of codon assignments. (This lower bound on the accuracy of translation is consistent with the fact that the thermal inactivation kinetics of most enzymes appears to be strictly first order for at least two decades. Since most if not all amino acid substitutions result in an altered temperature sensitivity of enzymes, this first order kinetics would not be expected to hold if an appreciable proportion of the enzyme population comprised “incorrect” translations of the corresponding mRNA’s.) To achieve this level of correct translations requires that the mistake rate per codon per translation be somewhere in the range 2 to 10 x lo-‘. The upper limit on the mistake rate for Ile-Val substitutions in egg albumin seems to be in this range (92). No single step in the translation process can then have an error frequency greater than 2 to 10 x unless there exists a subsequent step a t which the error is detected and corrected. It is worth noting that this level of mistakes is far lower than one expects from most if not all enzymes in the recognition of their substrates, and is far lower than theoretical calculations (for the recognition of a substrate by its enzyme) would place it (93).
A. tRNA Structure4 To date, the complete primary structures for four of the yeast tRNA’s have been determined (94-97‘). These are presented in Fig. 1. Perhaps the most striking features of these structures are (a) the relatively high fraction of rare (altered) nucleotides they contain, and (b) the fact that the overall geometry of the molecule does not seem to be determined solely by base-pairing considerations, which would lead to a fairly straightforward double helical structure. Although we do not now know the overall geometry of the tRNA molecule, it is clear it is rather coinpact, more so than the schematic representation in Fig. 1 would indicate. This has been inferred from tRNA’s relative resistance to endonuclease cleavage (98), resistance to formylation (99), and fairly high (25%) hyperchromicity upon heating (100). The most immediate problem is to relate the functional aspects of the tRNA moleeule to its structure. We are aided in this by the obvious homologies among the four known tRNA primary structures and possible secondary structures, but hindered by the failure to comprehend tRNA’s overall geometry and our lack of knowledge with regard to the details of tRNA’s function. ‘ S e e Miura in Volume 6 of this series.
140
CARL R. WOESE
‘cm
A- G-U,
. . .
C - C - U - G - C. - A -.G - U . - ~.- G - U - C - G - C - C - A ~ “ i;-&A-&G &C-A-&&(+&p
/
.IA)G
\
C-Y-T
/
/
\
u,
C m ;
G
h
G,
,,-G-A-G-U\
G-c-c C-G-6
F m
h
‘A-A-U-u
hAG
b --.A ..A $--+ /*“--A, A ....- ._ (J
t RNA
(* )
?u
i4 A
(b)
‘G’
i
FIG.1 . The primary structures of four yeast t R 5 . 4 ’ ~(94-97). The particular folded configuration shown is not to bc considered as necessarily representing the true geometry of the molecules. It is a device for revealing hydrogen bonding possibilities within each molecule and the similarities thus made possible. The homologies in sequences are independent of the configuration. Explanation of Symbols In11
=
1-methylinosine
mlG = 1-methylguanosine m2G = N2-methylguanosine miG = Nz-dimethylguanosine Gm = 2’-O-methylguanosine mA = methyladenosine .n:A = Nodimethyladenosine iA = NB-isopentenyladenosine N = unidentified nucleoside
T = ribosylthymine hU = 5,6-dihydrouridine = pseudouridine msC = 5-methylcytidine ac4C = N4-acetylcytidine Um = 2’-O-methyluridine Cm = 2’-0-methylcytidine Hyphen = -P(O)OH. . . , . , = hydrogen bonds
141
PhESENT STATUS OF THE GENETIC CODE
7A-U-C\$- A-F-A-G;A-A-U-U-C-G-C-A-C-C-
G
\
G - U-
6-
C-Y-T’ U‘
m7G:
-
c’
U -,
0-A-
G
A,”
-6-C- 6 ,
u\
A-G-
A\
m 2 G. C. U-. C.
c- &A
,
h U h
-6
YG
‘A-G-G’
FIGS.l c and d . For lcgend see opposite page.
Functionally, tRNA should have two recognizable sites specific for cacli kind of tRNA, the activating enzyme recognition site (ERS) and the site recognizing the codon (the “anticodon” or CRS). Over and above this, tRNA may possess :is many as three more nonspecific “sites”-onC for attachment of the amino acid, one for interaction with the ribosome, and perhaps one for interaction with another tRNA molecule, in the process of polypeptide chain formation. Each tRNA molecule can be drawn as having five sections, or “arms,” though the lower left-hand one (in Fig. 1) is often extremely “rudimentary.” With regard t o locating the various possible functions on the molecule, we can say the following. ( a ) The CCA terminus, common to all tRNA’s is the site of attachment of the amino acid (91). (b) The trinucleotide segment,
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CARL R. WOESE
that in all cases falls in the center of the “dependent arm” of the tRNA structure (as drawn in Fig. 1) is in all cases the base-pairing complement of one of the codons for the amino acid carried by the tRNA, and so is (c) The almost certainly the anticodon, or the essential core sequence TqGC, found in the upper left-hand “arm” as drawn, is common to most, if not all tRNA’s (94).’” It is therefore a candidate for the role of one of the functions common to all tRNA’s. (d) There is no definitive evidence regarding the location of the enzyme-recognizing site (ERS). The little suggestive evidence we have is discussed below. (e) The functions of the rare bases are still quite unknown. Roles for inosine and pseudouridine in anticodon function have been postulated (125, IOI), and the absence of methyl groups on tRNA seems to make some subtle, but important, changes in the translation process. More detailed speculation about the meaning of the similarities or differences in the various segments of the tRNA sequences is pointless until more experimental evidence is available.
B. The Trans-I Process The specific “recognition” of an amino acid by its activating enzyme is the key step in distinguishing among the amino acids in the translation process. However, this is not the whole story. While any given activating enzyme will activate its proper amino acid readily, there are a few cases where an activating enzyme activates the “incorrect” amino acid ( 1 02). In particular, it seems difficult to make a sharp distinction between Val and Ile-perhaps the two most closely related amino acids of all the naturally occurring ones. The Ile activating enzyme also activates Val, though the Val-activating enzyme does not activate detectable amounts of Ile. A number of years ago, Pauling calculated that the distinction between Val and Ile by an Ile-handling enzyme should be no better than 20:l ( 9 3 ) . Experiment seems to bear this out, for the K , for activation of Ile by the Ile enzyme is roughly 2% of the K , for inhibition of the reaction by Val (103). (The I<, for inhibition of Val activation by Ile, again using the Ile enzyme, is about the same as the Ile KM-i.e., of the order of 1P.) Several other such activation errors are known, the activation of a-amino-n-butyric acid, Thr and allo-Thr by the Val enzyme (IOS),and p-F-Phe by the Phe enzyme (104).I n this last case, the K , for the unnatural analog is only ten times that of its natural counterpart. It should be noted that homologies in the location of various sequences are independent of the arbitrary configuration chosen. They appear equally well in a simple linear representation.
PRESENT STATCS OF THE GENETIC’ C‘OIIE:
143
However, such high levels of activating errors as these seem not to incapacitate the cell. Berg and his co-workers have shown that when Val is activated by the Ile enzyme, the Val is not attached to tRNA in detectable amounts (103, 105). The Val-AMP-enz,,, complex reacts specifically with tRNA1le to bring about discharge of the enzyme, yielding free Val. This, then, is an error detection-correction device. However, it is unlikely that the cell actually makes a s high a level of mistakes in activating Ile. Loftfield and Eigner found that the presence of tRNA1Ie influences both the rate of Ile activation-increasing it-and the ability of the enzyme to distinguish Ile from Val-increasing that too (106). If we assume that the binding constant of tRNA for the activating enzyme is approximately lo6 (calculated from the data of Loftfield and Eigner), the concentrations of activating enzymes and tRNA’s that exist in the cell (roughly 10-5M) are such that most of the activating enzyme molecules may be complexed with tRNA molecules in v i m . We should also note that when the “wrong” amino acid is placed on tRNA (e.g., by reducing Cys-tRNACYs to Ala-tRNACYs), then it is not possible for an activating enzyme to remove it, as is the case normally (107).Finally, there is no evidence to indicate that an activating-enzyme complex tends to charge the wrong tRNA a t an appreciable level. These phenomena raise the interesting and important possibility that the tRNA molecule in some way “recognizes” (has a specificity for) the amino acid i t is to carry. Such a mechanism would account nicely for the added discrimination of Ile from Val during activation when tRNA is present, the failure of Val-enzIl, to charge tRNA, and the fact that the p-F-Phe-enz,,,, complex is about one-fourth as efficient in the charging as is its normal counterpart (104). Of course, i t is not necessary to postulate this sort of recognition in order t o account for these results, as the rather indefinite assumption of allosteric transitions in the enzyme could be invoked to account for the phenomena. However, I feel the allosteric explanation to be somewhat contrived in this instance. I n any case, let us realize that one of the central issues of the genetic code, the question of “recognition” of amino acids by oligonucleotides, is raised, but as yet unresolved, by these observations. 1. HETEROLOGOUS TRANS-I SYSTEMS
The study of heterologous Trans-I systems-where the activating enzyme comes from one source while the tRNA’s are derived from an “unrelated” one-might yield some degree of understanding of the nature of the sites involved in Trans-I. The idea is that if the ERS and the corresponding site on the activating enzyme have some special property,
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CARL R. WOESE
such as recognition of the activated amino acid by the ERS, then all heterologous Trans-I systems might be expected to function-i.e., TransI would be “universal.” Otherwise Trans-I sites might be expected to have diverged from one another during the course of evolution sufficiently so that heterologous Trans-I systems would not work. The results from heterologous Trans-I systems seem to defy interpretation. When heterologous Trans-I systems are derived from bacteria, then all such systems appear t o yield “correct” tRNA charging. When such systems are derived solely from the “higher forms” (among which I include yeast and Neurospora), correct charging is likewise the result. However, when one of the components in the heterologous Trans-I system comes from bacteria while the other comes from “higher forms,” then correct charging seems to result for only about half of the amino acids (108-110). The other half of the cases show mainly no appreciable charging, but a very few cases of “incorrect” charging (the “wrong” amino acid on a tRNA) also occur. The most clear-cut case of incorrect tRNA charging in a heterologous Trans-I system is the charging of the E. coli tRNA*’” and tRNAVa’ with Phe by Neurospora Phe-activating enzyme (110). This case has been investigated rather extensively. It is known that Neurospora possesses two Phe activating enzymes, one of which will charge E . coli tRNAPhe correctly, the other of which performs the incorrect charging (111). The puzzling feature of this incorrect charging is the loss of specificity-i.e., both tRNAAIaand tRNAVa’ are charged. It is hard to say what all these results mean. Certainly the universality of Trans-I among the bacteria or among the higher forms is suggestive of special features in the Trans-I sites, but this by no means proves the point. The negative results in some heterologous Trans-I systems are uninterpretable, and the very few tRNA chargings recognized as incorrect could possibly be written off in terms of some inconsequential mechanism. On the other hand, it is conceivable that Trans-I sites change but slowly during evolution but in time will evolve to become completely unlike one another in unrelated species ; we could be witnessing the beginnings of this slow evolution. The heterologous Trans-I systems presently tell us very little about the nature of the sites involved. It is worth recording in passing that undermethylated tRNA is, in some cases a t least, not charged in a heterologous Trans-I systemwhere the same tRNA is charged in a homologous Trans-I system, and its methylated counterpart is charged in the same heterologous TransI system (112).
145
PRESENT STATUS O F THE GENETIC CODE
2. THERELATIONSHIP OF
THE
ERS
M THE
ANTICOWN
There are a number of lines of evidence that suggest the ERS to have some relationship to the anticodon, perhaps including the anticodon as a part of the ERS. Unfortunately the matter cannot be decided definitely a t this stage. Hayashi and Miura (113) report that the charging of four tRNA species is inhibited by synthetic pentanucleotides according to the pattern shown in Table VII. The inhibition arises from TABLE VII CHARGING INHIBITION BY OLIGONUCLEOTIDES
(U),
(C).
((3 n
competition between the oligonucleotide and the tRNA for the activating enzyme (114). (An interaction between tRNA and oligonucleotide may also be seen in the slight inhibition shown by oligo U for tRNAP1”charging, etc.) The pattern reported is exactly what one would expect were the anticodon a part of the ERS. The failure of these workers to report data for other tRNA’s and to report the results for oligo G with any but one tRNA is puzzling. Attempts to repeat their results have failed (116). Thus we shall have to refrain from accepting thc implications of this study until such time as it can be verified and extended. Burton et al. (116) studied the inactivation of tRNA charging by osmium tetroxide, an agent that appears to destroy U residues readily (but not C, A, or G ) when these are in an “exposed” part of the nucleic acid structure. I n t,his case Tyr, His, Gln, Asn, Lys, Glu, Asp, Phe, Pro, and Ser tRNA’s are the ones readily destroyed by the reagent. From codon assignments, all except the last three should have U in the central position of the anticodon. Again, the composition of the ERS appears to correlate with the composition of the anticodon. Further evidence along these lines in support of this conclusion has been reviewed by Miura in Volume 6 of this series. Also bearing on this question is the number of kinds of activating enzymes an amino acid possesses. I n other words, does a n amino acid have one kind of activating enzyme for each kind of tRNA (differentiated by codon response) ? Here again the answer is not totally clear.
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CARL R. WOESE
I n somc cases an amino acid does seem to have one enzyme that charges all of its tRNA’s (109). However, consider the case of Leu. This amino acid has six codons and probably five tRNA’s-one recognizing the UUA and UUG codons, one the UUG codon, one the CUU and CUC codons, one the CUA and CUG codons, and one the CUG codon (see discussion of codon-anticodon recognition in Section VII, C) . Thermal inactivation of an activating enzyme preparation shows the Leu activity to decay according to first-order kinetics, suggesting a single (major) component (117). However, hydroxylapatite chromatography reveals a t least three kinds of Leu enzyme activity differing quantitatively in their response to the various Leu tRNA’s but apparently partially interconvertible (118). Further, heterologous Trans-I systems will .sometimes charge certain of an amino acid’s tRNA’s but not others. It seems likely from the above that each amino acid has fewer kinds of activating enzymes than it does kinds of tRNA’s-for some amino acids, only one enzyme. If the ERS contains the anticodon, it is then necessary that the enzyme recognize several different kinds of ERS’s.
C. The Trans-ll Process The essence of Trans-I1 lies in the accuracy of the recognition of a codon by its tRNA’s. It is reasonable to expect the interaction between codon and anticodon to occur through base pairing, as both constituents are nucleic acids. However, though a remote possibility, one must also consider any involvement of the amino acid carried by tRNA in the specificity of codon-anticodon recognition. This possibility has definitely been eliminated by the experiments of Chapeville et al., which demonstrated that an amino acid on its tRNA can be changed to another amino acid without altering grossly the codon recognition properties of that tRNA (119). Cys-tRNA”Y” was reduced to Ala-tRNACy8or oxidized to cysteic acid-tRNACY8.The resulting tRNA “chimeras” responded in translation to the Cys codons-both with synthetic messages and with a natural mRNA, such as that for hemoglobin (119, 120). More recently it has been possible to create other tRNA chimeras, such as tRNAAIaor tRNAva‘ carrying Phe (through use of heterologous Trans-I systems, as seen above). These too seem to manifest codon specificity that is a function of tRNA alone ( 1 2 1 ) .Thus the result with these chimeras seems quite general, and so the conclusion that there is no role for the amino acid in codon-anticodon recognition seems highly likely if not certain. Is the specificity of codon-anticodon recognition accounted for solely by base pairing? Initially, of course, it was felt that “adaptors” recognized their codons through typical Watson-Crick base-pairs. However, it was later suggested on theoretical grounds that perhaps a tRNA mole-
147
PRESENT STATUS O F THE GENETIC CODE
cule could recognize more than one kind of codon (belonging t o the same amino acid) (11).The initial experimental results on (partially) isolated tRNA’s for Leu suggested that this idea was wrong and that individual codons had separate tRNA’s (122). However, more recent results do show indeed that one tRNA may recognize a number of different codons. This is particularly clear for the case of tRNAAIafrom yeast. Here the tRNA appears to be of unique primary structure. Yet this tRNA will bind the Ala triplets GCU, GCC, and GCA (in the triplet binding method of Nirenberg and Leder) (123, 124). Such results do not eliminate base pairing as the mechanism for codonanticodon recognition, however. In fact, they reinforce it. Crick has pointed out that by permitting pairing (defined as two or more H bonds) between bases other than the usual ones, a pairing scheme can be constructed that accounts nicely for the codon-anticodon degeneracies observed (125).Assuming certain constraints, the following set of base-pairs is possible: U - A , U.G, C - G , A.U, and 1 - U , I . C , 1.A. (The difference between the pairing predicted for I and G is rationalized by the assumption that the 2-amino group of G cannot form a hydrogen bond to water if it pairs with A, while I, having no 2-amino group, should forin an energetically favorable pair with A.) If these alternate forms of pairing (the “wobble” hypothesis) w e arbitrarily restrictcd to the third position of the codon, the accompanying codon-anticodon recognition pattern is produced. Base in third anticodon position
Codoiis recognized
1-u 2-c
XYA, XYG XYG XYU XYU, XYC XYU, XYC, XYA
3-A 4-G 5-1
Codon binding studies with fractionated yeast tRNA’s show the existence of tRNA types 1,2,4, and 5 ( 1 2 3 ) .Among E . coli tRNA’s, types 1, 2, and 4 have been recognized (194). Classes of tRNA’s not conforming to this rule may also exist, however. For example tRNA’s responding to codons of the form XYU, XYC, XYA, and XYG seem to exist, as do thosc responding mainly to XYU, XYA, and XYG (123, 124). However, the incomplete separation of tRNA’s in each case makes it impossible to eliminate the possibility that these exceptions arise from mixtures of two tRNA species difficult to separate from one another. I n any case, many if not all tRNA’s appear to fall into one of four of the above five classes (tRNA’s responding solely to XYU are yet to be found).
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Perhaps the most puzzling thing about Trans-I1 is the accuracy of the process. As we have seen, Trans-I1 must occur with a mistake rate of no more than one part in about 3000. It is difficult to think that codonanticodon recognitions could be this accurate. As Loftfield has pointed Out, a 3000:1 discrimination should mean that the binding energy difference between correct codon-anticodon interactions and incorrect ones is about 5.0 kcal (92). Such a difference is certainly greater than expected for two hydrogen bonds of a correct base pair vs. one (or even no) hydrogen bond of an incorrect base pair. tRNA binding studies (including those using mRNA’s much larger than trinucleotides) give evidence that considerable “false” binding may occur, in keeping with this calculation. A good example is the binding of tRNAAspby poly (A-G) (in the presence of ribosomes) (126). Nevertheless, this sort of false binding, if it does occur in the cell, does not result in appreciable levels of incorrect amino acid incorporation. Does this mean that the overall Trans-TI process can somehow detect and correct false codon-anticodon recognitions? I n any case, it is useful to inquire whether factors other than base pairing (and the usual concomitant base-stacking interactions) go into codonanticodon recognition. tRNA does contain a rather high percentage of rare nucleotides, and these are sometimes found in the trinucleotide segment in tRNA presently considered to be the anticodon. We have just discussed one possible role for inosine in the third position of anticodons. O-MeG also occurs in the presumed anticodon, in one case so far ( 9 7 ) . Further evidence for the role of methylated bases comes from studies using so-called undermethylated tRNA’s, produced by growth of “relaxed” methioninerequiring auxotrophs of E . coli in the absence of methionine. The tRNA in such cells may contain less than 50% of its normal complement of methyl groups ( 1 2 7 ) . Rather surprisingly, the translation characteristics of such methyl-deficient tRNA preparations (in both Trans-I and Trans-11) are, to a first approximation, normal (128). However, closer inspection does reveal important differences. I n particular the codon recognition properties of tRNA’s seem altered by undermethylation. Littauer and co-workers report a striking increase in the frequency of translation errors involving Phc when demethylated tRNA’s are employed in Trans-I1 (1.29). Peterkofsky and co-workers report that the absence of methyl groups actually can change a codon response for the Leu tRNA’s, from a UUPu response to a CUX response (130). These initial reports are a strong indication that methylated bases play a direct role and/or a role in the codon-anticodon interaction. I n view of this, it may be wise not to settle on an oversimplified concept of codon-anticodon interaction a t this point; the anticodon may contain
PRESENT STATUS OF THE GENETIC CODE
149
more than three essential bases and/or codon recognition may not be solely a function of base pairing.
D. The Direction of Translation The direction of translation (relative to the mRNA) has been determined, though what relationship this bears to the mechanism of translation remains unknown. The first attempts to determine the direction of translation were unaccountably incorrect (29, 131). However, the matter has now been settled through the synthesis of mRNA’s that are homopolymers containing one nontrivial codon sequence a t or near one end of tlic molecule. A poly A ending with C, thus giving an AAC codon at the 3’ end of the chain, brings about Asn incorporation a t the Cterminal end of some of the polypeptides whose synthesis it directs. This same codon, placed as the second codon from the 5’ end of the message, permits the synthesis of polypeptides with amino-terminal Am5 (27). Thus translation starts from the 5-phosphate end of the RNA message, as protein synthesis is known to proceed from the aminoterminal to the carboxyl-terminal of the molecule (16). This makes the direction of translation the same as that for transcription, which may or may not be a coincidence. I n any case, the situation seems to permit translation of mRNA to begin before its transcription is complete. The demonstration that the DNA counterpart of the UAG (chain-terminating) codon in one of the cistrons of an operon halts transcription distal to this point but does not interfere with translation proximal to it is consistent with this notion ( 6 7 ) .
E. Errors i n Trans-ll The crror rate for in vivo protein synthesis is one part in 3000, or less. Measurement of in vitro translation errors are in agreement with this figure. Translation of a poly U mRNA in vitro under “optimal” conclitions shows incorporation errors involving Leu, Ilc, and Ser to occur a t frcqucncies of 1%, 0.01”/., and 0.02%, respectively ( 1 3 2 ) . Measurement of translation crrors would be a rather difficult task if one had always to detect low levels such as the above. However, under suboptimal conditions, the error rate for Trans-I1 increases dramatically. These conditions include a lower temperature (13S), a higher Mg2+level ( 132), the presence of alcohol ( l S 4 ) , of certain antibiotics (135), mutations affecting the ribosome (1%). Of especial interest are the effects of streptomycin and related antibiotics, for streptomycin, in particular, ‘ S e e discussion below regarding chain initiation and the role of the first codon in mRNA.
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CARL R. WOESE
appears to cause a great increase in the error frequency without changing grossly the overall pattern exhibited by an in vitro system translating under “optimal” conditions (132, 135). The site of streptomycin action appears to be the ribosome; mutation to streptomycin-resistance can be located on the 305 ribosomal subunit ( 1 3 7 ) ,within the group of proteins that remain tightly bound t o the 30 S particle in media of high ionic strength (138). The location of streptomycin action on the 30s ribosome is in keeping with the antibiotic’s role in increasing translation errors, for the 30 S particle appears to play the primary role in mRNA attachment and codon recognition by tRNA. The initial studies on errors produced by elevated Mg” levels showed that mistranslation of the UUU codon seemed to involve only those amino acids whose codons were related to UUU (132). The exact pattern for errors in UUU translation can be seen when these results, the streptomycin studies (135, 139), and work on fractionated tRNA’s in a high Mg2+in vitro system are combined (140). These errors are a function of (a) the position in the codon, and (b) the nature of the base for which U is mistaken. Of those amino acids possessing codons related to UUU, only Leu, Ile, and Ser are incorporated in appreciable amounts (Tyr is incorporated to a small extent). With regard to Leu, this applies to its tRNA’s that respond to UUA, UUG, and CUU. Together, the results yield the following pattern: I n order of susceptibility to error, the positions in the codon can be ranked 111> I > 11. I n each position, the ease with which U is mistaken for another base is UmC > UmA >> UmG,G a rule approximately independent of position in the codon.’ This particular error pattern is not unique for the codon UUU; it holds to a first approximation for all codons composed of pyrimidines (133, 139). An exception seems to be that CmU does not occur in codon position I, and not in I1 under certain error-producing conditions (139). With regard to the purine codons, the error pattern is rather different. In the first place thc level of errors in this instance is much lower than for the pyrimidine codons. Secondly, not much pattern is seen in the ‘UmG, etc., is read “U mistaken for G.” ‘With regard to the I and I1 positions this pattern is clear. With regard to the 111 position UmC is not now an “error” in the strict sense, in that tRNAPhe normally does not, distinguish W U from UUC; this might mean that the UmC error in 111 could have been a high level one at a more primitive stage in evolution. In this codon position, it does appear that UmA UmG, however, for even under “optimal” to poly U cannot be eliminated in vitro, conditions the response of tRNALeu(UU“) though that of tRNALeu‘UUG) responds to polg U a t appreciable levels only in the presence of increased Mg’+ levels (140, 1 4 1 ) .
>
PRESENT STATUS O F THE GENETIC CODE
151
errors that do occur; AmG niny occur (139). Codons containing pyrimidines and purines show typical pyrimidine mistakes, though the pattern may not be so extensive as that shown by UUU ( 1 4 2 ) . Thus the error pattern for a given base in a given codon position may not be altogether independent of the overall composition of the codon, though it seems to be so to a first approximation.
TRANSLATION OF MRNA’s COKTAINING UNUSUALBASES Another class of phenomena related to the normal and streptomycin translation error patterns, but that could not in the strictest sense of the term be classed as errors, are the translations of mRNA’s containing abnormal bases. It has been known for some time that 5-fluorouracil causes phenotypic reversion of “amber” mutants. The most reasonable mechanism here is a translation mistake (7’9, 80). Similarly, mRNA’s containing inosine are given to rather high levels of translation errors in vitro (139). The most spectacular examples of this sort of “error,” however, are those involving the translation of mRNA’s containing the heavier halogenated pyrimidines. The best-studied cases arc those of the 5bromo pyrimidines. Poly BrU produces a polypeptide product that coiitains Phe, Ile, Leu, and Ser, Init little if any Tyr, Val, or Cys ( I @ ) . This is precisely what would bc expected on the basis of the above error pattern for poly U. If the mistake pattern for poly BrC were analogous to that for poly BrU (in that BrC were translated as C, U, and A, as BrU i s ) , then poly BrC should bring about synthesis of a peptide product containing Pro, Ser, Leu, and Thr. The polypeptide produced by poly BrC contains high amounts of Pro and Thr, but no Ser or LeuS (143). Thus BrC is not trailslated as U, though BrU is translated as C. (As seen above, there arc also cases where a translation error that would involve CmU does not occur under “high error’, conditions.) An understanding of the mechanisms behind translation errors and translation of mRNA’s containing unusual bases should yield an understanding of codon-anticodon recognition. The translation (‘error” data so far seem unexplainable in terms of simple base-pairing considerations, such as those used to explain the known codon-anticodon degeneracies (125, 11). It does not appear reasonable that such substituents as Br and I should manifest themselves mainly in terms of polar interactions. It is more likely that these atoms soniehow effect codon-anticodon interactions through their relatively high nonpolar reactivities. The exact mechanism remains obscure. R A scant amount of LCU is actually incorporated, but it is less than is incorporated with a poly C message ( 1 4 3 ~ ) .
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F.
CARL R. WOESE
The Mechanisms of Polypeptide Chain Punctuation
Although i t is known that special nucleotide sequences are involved in both peptide chain initiation and termination, the mechanisms in both cases remain to be elucidated. A few relevant facts are available, however. Concerning initiation punctuation as stated above, two species of tRNAMeL are known to exist, each responding to the AUG codon, and one of these permits Met to be N-formylated subsequent to its attachspecies show different responses ment to tRNA ( 7 8 ) .These two tRNAMeL to the antibiotic puromycin, which seems to behave as a tRNA analog. The normal tRNAMet (not susceptible to formylation) binds to ribosomes in the presence of the AUG triplet with or without puromycin present (as would a typical tRNA) . However, the tRNAMet carrying N-formylMet (fMet-tRNAfMet)interacts with puromycin in the presence of AUG and the ribosome to form N-formylmethionyl puromycin. This is true whether the Met has been N-forinylated or not (1.44). We should consider these facts together with the following additional ones: ( a ) In general the first codon in (synthetic) mRNA has a very low probability of being translated. (The message AAAAACAAAAAA . . . yields the peptide Asn-Lys-Lys . . . ? but not Lys-AsnLys-Lys. . . .) However, the first codon is somehow “counted” (27). [AAAAACAAAAA . . . produces N-terminal Asn peptides; AAAACAAAAA . . . produces N-terminal Thr peptides; but neither produces a mixture of the two types ( 2 7 ) . ] (b) I n a low Mg2+ in vitro system, there is very little polypeptide synthesis brought about by poly (U, A, G ) unless N-formylMet-tRNA or certain peptidyl tRNA’s--e.g., PhePhe-tRNA-are present ( 7 6 ) . All these facts can be understood in terms of a model that postulates two kinds of sites on the ribosome: one site for interaction with the incoming tRNA, which holds the amino acid about to be placed into peptide link (the “decoding” site), and a second site to catalyze peptide bond formation and hold the tRNA that is covalently linked to the growing peptide chain (the “peptide” site). Clearly the two types of Met tRNA’s distinguish these two sites. The tRNA that permits hrformylation (tRNAfXlet)locates in (or possibly creates) a peptide site. This would also be true of tRNA’s carrying small peptides. The “normal” tRNAMet(which does not carry a formylated Met) and all other tRNA’s that carry amino acids locate in the decoding site. The failure to translate the initial codon in an RNA such as AAAACAAAA . . . (though it is nevertheless “counted”) could result from either of two mechanisms: (1) the initial codon somehow locates in the peptide site (and so is inaccessible to normal tRNA’s), or (2) since aminoacyl-tRNA bonds are
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n101’c labile than peptidyl-tRNA bonds (144a) , the amino acid attachcd to the tRNA reading the first codon in a synthetic mRNA is somehow prematurely split from its tRNA (in the framework of the ribosome) before i t can form a peptide link. At the present time there is some uncertainty as to how the reading frame is established for mRNA, and the role of the AUG codon in establishing it. It seems clear that the reading frame can be uniquely determined in the absence of the AUG codon, though the initial three nucleotides of the message do not seem to be translated into an amino acid residue with a measurable probability in this case (27, 145). The presence of the AUG codon a t or near the 5’ terminus of mRNA does appear to exert a directing effect on the choice of reading frame (146) and, as might be expected in view of the above, terminal AUG is translated into an amino acid residue (N-formyl-Met) (1.65). Several factors involved with reading-frame determination and/or peptide chain synthesis initiation by the AUG codon have been isolated (1.65). Little is known of the mechanism of peptide chain termination beyond that reported in the punctuation section above. The prevalent opinion is that a chain-terminating “tRNA” is involved. Recently a techniquc for assaying for “amber” suppressor tRNA’s has hecw clevclopcd, based upon the fact that the normal viral coat polypeptide producclcl by thc nucleic ncicl of :in R N A phagc in :In in z d r o system remains attachcd to the RNA, whereas the partial coat polypeptide produced from a mutant phage RNA containing an “amber” codon does not yield an RNA-bound peptide product ( 1 4 7 ) . This sort of assay could detect not only suppressor tRNA’s for the amber codon, but also any “terminator tRNA’s” responding to the UAG codon.
G. Suppressor tRNA’s The tRNA suppressors are of especial interest in that their properties should be particularly revealing not only with regard to immediate underlying molecular mechanisms, but also with regard to certain aspects of the code’s evolution. At present, our understanding of the phenomenon is rudimentary. We can record the following facts, (a) There are two kinds of tRNA suppressors, which, however, are not fundamentally different from one another-suppressors inserting an amino acid other than the normal one in response to a given codon, and suppressors inserting an amino acid in response to one of the chain-terminating codons, UAA and UAG. (b) The level of suppression, i.e., the probability that the suppressor response as opposed to the normal response will occur when the suppressed codon is translated ranges from less than 1% in some cases to as high as 60% in others (86, 148). One
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special case where suppression approaches the 100% level is discussed below. Let us note, too, that an E. coli Arg-Gly suppressor (Gly inserted in response to the Arg AGA codon) normally functions a t the 5% level, but introduction of the suppressor gene into a particular strain of E . coli (having an abnormally high level of Gly-activating enzyme) results in a 50% level of suppression (149). The obvious explanations for why suppression is not all-or-none need no discussion. (c) Suppressor tRNA’s, in all cases characterized, insert an amino acid one of whose codons is related (by a single base substitution) to the codon suppressed. Examples are Ser(UCG), Tyr (UAU,), and Gln (CAG) suppressors of the UAG codon (86, 150). These results suggest the suppressor tRNA’s to be created by modifications of the anticodon portion of the molecule. (Modification of either the anticodon, the enzyme recognition site, or possibly some other part of the tRNA molecule could conceivably create a suppressor tRNA.) More direct evidence for anticodon modification as the origin of suppression comes from the Tyr amber suppressor in E . coli. I n this case, it has been possible to incorporate the suppressor gene or the corresponding wild-type gene in a lysogenic phage genome. Cells infected with these phages can be caused to produce large amounts of the tRNA’s in question. The Tyr amber suppressor tRNA has thus been proved t o respond to the UAG but not the UAU codon (by the triplet binding assay of Nirenberg and Leder) , while the corresponding wild-type gene produces a Ty r tRNA, which does bind the UAU codon (151).Sequence analysis of these two tRNA’s shows that the change in primary structure in creating the suppressor is a G + C substitution in a segment whose sequence is GUA-i.e., precisely what would be expected for an anticodon alteration in a Tyr tRNA (151a). Several facts argue that not all suppressors are created by anticodon alteration. For one, yeast super-suppressors, which are believed to be analogous to E . coli amber suppressors, arise almost exclusively from addition-deletion mutations, not base substitutions (156).For another, a Cys-Gly suppressor tRNA (Gly in response to Cys codon) shows marked inhibition of Gly incorporation in the presence of excess Cys (15.2). [An Arg-Gly suppressor tRNA is not, however, inhibited by excess Arg (154).] This amino acid inhibition is more consistent with an ERS modification than an anticodon modification. The above-mentioned phenomenon of a shift in suppression level, from 5% to 50% upon introduction of the Arg-Gly suppressor gene into a high Gly activating enzyme environment, also suggests a modification in the enzyme recognition site. [The reasoning here is that in its original environment the suppressor tRNA must have existed largely in the uncharged state, which is atypical of normal tRNA’s (163); this in turn implies difficulty in
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charging this tRNA.1 It. should be recognized a t this point that were the anticodon a part of the enzyme recognition site, as the above evidence tends to suggest, an alteration of the former would automatically alter the latter.
VIII. The Fundamental Nature of the Genetic Code We have, in the above, surveyed the major facets of the coding problem-the cryptographic puzzle, the use of various encoded instructions, the decoding process. All this, however, gives an understanding of the code that is, for the most part, descriptive and relatively superficial. It should be clear that to know that UUU is assigned to Phe, or that mRNA is processed as if it were a tape, is one thing; to know why UUU is assigned to Phe, or why translation involves this sort of process (rather than a direct template mechanism, for example) is far more basic. I n other words, we still need to understand the principles upon which the structure of the genetic code rests. It seems obvious intuitively that this problem is practically inseparable from that of determining the way in which Nature actually built the code, i.e., the problem of the code’s evolution. A number of characteristics of the genetic code appear to reflect something of the code’s fundamental nature. I n this concluding section, we review these characteristics in detail.
A. The Problem of the Locked-in Code One of the more intriguing properties of the genetic code is th a t it is almost or perhaps entirely universal. All organisms appear to use the same set of codon assignments (with the exception of a few suppressor strains) (157-159). The mechanism of translation may well be universal also, in that parts of the translation machinery from one organism function well, if not perfectly, in the translation apparatus of another organism (112, 160). (There is a little doubt, as we have seen, just how universal Trans-I is, but it is nearly if not totally so.) Even if all life arose from a single cell line, how can universality of the code be maintained today in the face of mutational pressure? One has, of course, the old argument that once a universal code was established it could never change because an alterated codon assignment, etc., would be extremely deleterious for any cell line. Certainly a mutation that in one step completely changes the assignment of a major codon should be lethal, for it is equivalent to as many mutations in a genome as there are occurrences in that genome of the codon in question. (In a small genome the size of that of E . coli, this could easily mean 2 x lo4 simultaneous “mutations.”) But is this the correct, or the only, picture of what could happen? Suppressors do exist and the suppression level is some-
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times high, about 50%. These suppressors do not seem to put a n undue burden on the cell. The high-level suppressors seem, however, to involve codons whose occurrence in the genome is very restricted--e.g., AGA and UAG in E . coli. Since suppressor lines can be created in Nature, they must also be able to revert to nonsuppressed lines again. However, the reversion of a suppressor line t o the nonsuppressed condition need not recreate the original set of codon assignments. We may picture a mechanism whereby the UAG codon, for example, could be given a n unambiguous amino acid assignment, or in which one codon assignment for, say, Arg or Ser, might become eliminated, paving the way for a reassignment of such codons to different amino acids a t some later stage. (I mention here one case where a complete change of codon assignment does seem to have occurred: Two strains of mice differ in the sequence of their hemoglobin chains a t a particular site; one strain has an Asn residue a t a certain position, the other has an average of residue each of Ser and Thr at the same position. Using in vitro hemoglobin synthesizing systems, it has been possible to determine that the cause of the difference lies in the supernatant fractions-i.e., not in the message RNA’s (161).) This discussion does not prove anything definitely, but it does make one doubt whether processes that alter codon assignments are necessarily lethal, and leaves one wondering why suppressor mutations, etc., have remained “localized,” have not lead to a randomization, a destruction of the various sorts of universality in the genetic code. It has also been argued that universality is maintained because a certain set of codon assignments is somehow “optimal” for the organism, which would mean that a selective pressure exists to adjust assignments to this optimal set and keep them there. This seems a weak argument. It is hard to see why a single set of codon assignments should be optimal for an organism. Far more likely, many sets of assignments (though a very small percentage of the total possible sets) should be equally good. Even admitting that one set of assignments could bc optimal for an organism, it is still impossible to see why the same set then should be optimal for all organisms. Organisms exist under vastly differing conditions of pH, temperature, oxidation-reduction potential, etc. ; organisms have vastly differing compositions of their DNA-and (smaller) differences in overall protein composition-which could well reflect very different mutational pressures. A single code could not be “optimal” for all these fundamentally different situations ; perhaps not even a unique set of amino acids would be. Thus while there may exist codon assignments that optimize certain parameters (e.g., minimize deleterious effect of certain mutations) and although these may even
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have played some role in shaping the general form of the codon catalog long ago, it is impossible to see how these constraints could maintain the universality of the code, let alone create it. By now the difficulties in giving a satisfactory explanation for universality of the code should be apparent. There is a serious question as to how the genetic code became universal in the first place (which we have not gone into in any depth) and then how this characteristic became “locked in” in the second. Failure to explain these matters in any alternative way lcacls t o the idea that the answer must be in the nature of the code itself (rather than in the effect on the cell of changing codon assignments, etc.). There may be a clue to the solution in the possibility that the ERS contains the anticodon (discussed above). Were this so, changes in the latter would necessarily alter the former. Given a rather complex ERS to begin with, it might be impossible for an altered form of it to accommodate any activating enzyme “optimally.” Thus a change of anticodon, of codon assignment, would always bring with it a selective disadvantage. It is also possible that universality is a manifestation of some “recognition” of amino acids by oligonucleotides, an interaction that would make the evolutionary choice of codon assignments a predestined matter, and could function as the essence of R “locking-in” mechanism.
B. The Problem of the Indirect Template Much of the controversy over the nature of the genetic code during the early theoretical period in the code’s history centered about whether the association of amino acid and template is a direct one, involving “recognition” of an amino acid by its corresponding codon, or whether the burden of recognizing both amino acid and codon falls upon some intermediary molecule (s)-a so-called “adaptor” system. This same controversy is still with us, perhaps in a somewhat more sophisticated form. Do specific amino acid-oligonucleotide interactions (“recognitions”) somehow underlie and/or maintain the structure of the genetic code? If such interactions are important, such is not clearly manifested. The important experiment of Chapeville et al. (119) shows that the specificity of codon-anticodon interaction does not depend upon the nature of the amino acid carried by a tRNA. This indicates that ‘(intermediaries” (“adaptors”) are responsible for the association of amino acid with its codon in mRNA.O But we are still left with the possibilities: (a) t ha t aminoacyl-oligonucleotide interactions in some way fashioned *However, this experiment is not a proof, as is sometimes claimed, of the postulate from which the existence of “adaptors” was originally predicted-i.e., that it is impossible for an oligonucleotide to “recognize” (react preferentially with) a particular amino acid.
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codon assignments a t early stages of evolution; and/or (b) that these interactions still operate in Trans-I. Let us review the arguments, regarding “recognition” of amino acids by oligonucleotides. I n the 1940’s and early 1950’s the concept of “complementary” biological structures was developed and became a foundation of biological thought. This idea stemmed from the precise specificity demonstrated by antibodies and enzymes for their antigens and substrates ( 2 0 ) . It was then natural to extend the concept to cover the relationship of nucleic acid to histones, etc. (17)and, as Gamow did, to amino acids ( 5 ) . However, in formulating his adaptor idea, Crick took the iconoclastic position, as stated, of denying that nucleic acids can in any way show specificity toward amino acids. [The argument here in essence is that the functional groups on the nucleic acid bases do not appear “complementary” to the side chain of the amino acids, and in any case such interactions should be energetically unfavorable in solution (21, 162, ISS).] Such an argument is more a matter of intuitive prejudice than a product of thorough analysis, so it is not convincing in itself. However, all the initial experimental evidence that bore on this question tended to support this view. [For one example, equilibrium dialysis of amino acids against RNA showed no detectable binding of amino acids (164). For another, a polyuridylic acid column failed to retard amino acids (165).] Nevertheless, all that this proves is that under the conditions used, binding between amino acids and polynucleotides was not strong enough to be detected. If binding cannot be detected, one obviously cannot argue much about specificity of binding. It can be countered, however, that if this binding is so weak, it cannot play a role in determining the form of the genetic code. It must be admitted that interactions too weak t o be detected under these conditions certainly could not place specific amino acids on specific codons with high accuracy (and so, in a restricted sense, Crick’s original postulate must be correct). Yet this is far from saying that these weak interactions are not stronger under other conditions; or that weak interactions could not have played a “slow” but definitive role in guiding the course of the code’s evolution; or that in some such process as Trans-I the weak interactions cannot somehow be “summed” to give stronger and highly specific interactions. There is now rather good, if indirect, evidence to suggest that amino acid-heterocyclic base “recognition” interactions did play a dominant role in shaping the structure of the genetic code. An excellent way to detect and quantitate weak interactions is by means of chromatography. Thus it was very interesting to note that the classification of amino acids by chromatographic criteria bears a resemblance to amino acids grouped
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TABLE VIII CORRELATIONS BETWEEN AMINOACIDCODONASSIGNMEXTS AND POLAR REQUIREMENTS [Data from Woese et al., (167).] UCU UCC
cuu CUC
Leu 4.9
AUA AUG
Ile 4 . 9 Ile Met 5 . 3
GUU GUC GUA GUG
UGU c y s 4 . 8 UGC
UCA UCG
UAA UAG
UGA T r p 5 . 2 UGG
ccu ccc
CAC
ACU ACC
CAG
CGU CGA
Gln 8 . 6
CGA CGG
AAC AAG
GCU GCC
EtE
Asn 10.0 Lys 10.1
Asp13.0
GGU GGC
CIAA Glu 12.5 GAG
GGA GGG
Ala 7.0
GCA GCG
Arg 9 . 1
Thr 6 . 6
ACA ACG
Val 5 . 6
His8.4
Pro 6 . 6
CCA CCG
CUA CUG AUC
ITAU Tyr 5 . 4 UAC
Ser 7.5
Gly 7 . 9
according to codon assignment (166). Experiments indicate that this correlation is quite exact (Table VIII) . I n the table, the amino acids are arranged according to codon assignment (in the customary fashion), and for each amino acid its “polar requirement”lo is given ( 1 6 7 ) . The order in the codon catalog had already suggested something special about the I11 position in codons, in that changing a base in this position w r y often left the amino acid assignment of a particular codon unchanged. Now we see that in those cases where a base change in the I11 position of a codon does change an amino acid assignment, it is always ““Polar requirement” is defined as the slope of the straight line that results when amino acid RM’s are plotted us. log of the mole fraction of water in the chromatographic solvent. This quantity seems to be determined, loosely speaking, by the number of water molecules the amino acid will bind (expressed in energy units) minus the energy of interaction with the organic component of the chromatographic solvent. Thus, for example, Phe and Ala are expected to bind the same numhcr of water molecules, but Phe will interact more strongly in a nonpolar fashion with a molecule such a8 pyridinc, giving Plie the lower polar requirement. Similarly Asp will bind many more w a k r molecules than does Gly, and so has a higher polar requirement.
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from one amino acid to a “closely related” one (as defined by chromatographic criteria). Further, all amino acids possessing UII codons arc closely related, as are all amino acids possessing CII codons. There is also a suggestion in Table VIII that by some sort of “polar requirement” criterion, the bases rank U < C < A < G, as can be seen most clearly for the I position bases in the A,, column. Interestingly, the bases can also be ranked in the same order by their electronic properties (168).Finally, the close correlation between amino acid polar requirements and codon assignments can be obtained only when the organic component of the chromatographic solvent is a heterocyclic base, such as pyridine; aliphatic alcohols, phenols, etc., do not yield as exact a correspondence (101, 16’7). Thus the exact nature of the interaction of amino acid with the organic component of the solvent is important here. Even though the evidence is not direct, the good correlation observed is a strong argument for involvement of amino acid-nucleic acid base interactions in shaping thc codon ass;gnments. An objection often raised to the above argument is that one can also define “related” amino acid by the similarity of their function in the context of a protein molecule, and this definition of “relatcd” (having nothing to do with heterocyclic bases) would probably correlate just as well with codon assignments as does the above one. Unfortunately, “related” in a context of function in protein cannot yct be defined with any precision, and so an exact test of this alternative is not possible. The small amount of amino acid replacement data we now possess suggests that “related” by codon assignment is not necessarily “related” by function. For example, a large number of amino acid replacements have been recorded a t a certain locus in the tryptophan synthetase A protein. The protein is a t least partially functional when Ala, Ser, Thr, Val, Ile, or Asn replace Gly a t this position. It is not functional when Glu, Asp, or Arg are placed there. [Since a large number of revertants from Arg, -AGA-, to wild phenotype have not included Lys a t this position, Lys also presumably produces an inactive protein (&).I Thus there is no evidence for a similarity in function of Asn-Lys, or Ser-Arg, though both pairs of amino acids arc related by codon assignment. Further, one would not suspect that Cys and Trp, or His and Gln, or Ala and Pro would function similarly in protein, though each pair again is related by codon assignment as well as by polar requirement criteria. Thus, the best correlation so far between codon assignments and “related” amino acids is obtained when the “relatedness” is defined in terms of amino acid-heterocyclic base interactions. I think a reasonable conclusion to draw from the above is that the
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structure of the genetic code is determined by amino acid-nucleic acid “recognition” interactions (or their equivalent) , but being very weak interactions they could not have given rise directly to the genetic code as i t now exists. Instead they must have played the role of constraints operating on the evolutionary process, in this way gradually shaping the form of the code. Since the interactions are weak and therefore cannot manifest an all-or-none sort of specificity with regard to amino acidcodon pairings, it is reasonable to expect that they cannot align amino acids along a nucleic acid template directly, and so this “recognition” role has been filled by the evolution of an lLintermediary” system, the tRNA’s and activating enzymes, that recognize with vety high accuracy both an amino acid and its codons.
C. The Problem of Errors and Evolution The essence of the cell (if there be a single essence) lies in the capacity to construct a protein of unique primary structure from any given nucleic acid primary structure; i.e., the capacity to translate a gene essentially perfectly. It is hard to imagine what a cell would be like were it not capable of accurately creating protein from nucleic acid. Yet this is necessarily what must have been the case for the early cell, the cell of, say, 1 to 3)< lo9 years ago. Two considerations argue the point. ( a ) Biological specificity turns on weak interactions (ix., dispersion forces and the like). Singly (i.e., between two simple molecules) , these interactions manifest very little specificity (selectivity). When “summed” in the proper way, as in an enzyme site, they are not only rather strong, but highly specific and highly selective. (b) The great accuracy of the translation process is a product of extensive evolution and relies upon the accurate functioning of many enzymes-the set of activating enzymes, the set of enzymes that modify tRNA’s, etc. The presence of highly specific enzymes demands the existence of accurate translation and vice versa. Here is an apparent paradox, the solution of which probably lies in a simultaneous and gradual evolution to high-specificity enzyme functions and to accurate translation, starting from low-specificity enzymes and inaccurate translation. Since we have yet to uncover traces of a cell with an incompletely developed translation capacity, it is impossible to know in detail what such a cell was like and how it exactly evolved into the “modern” cell (an evolution that must have been complete a t least 500 x lo6 years ago). However, one can reason what such a cell and its evolution probably were like, a t least in general outline (169). (a) A highly inaccurate translation process meant that it was impossible to make two identical translations of any given nucleic acid sequence. This situation would
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severely limit both the kinds of enzyme functions available to a cell, and perhaps the size of the polypeptides translated (the smaller the polypeptide the greater the probability of a perfect translation). (b) The genetic differences between two species are often subtle. To be able to speak of “species,” etc., means that the states of a biological system must be definable within very narrow limits. This in turn demands precise enzyme functions and in particular precise control of the levels of enzyme function and changes in these levels. Thus complex interrelationships among various enzyme activities and a high degree of regulatory control are necessary. Certainly this level of regulatory sophistication was far beyond the capabilities of early cells. ( I n fact, it probably was impossible for multicellular organisms, i.e., differentiated cells, to exist.) Therefore, evolution as we have come to know i t s p e c i a tion, etc.-did not occur when cells possessed primitive translation systems. The general character of evolution in such cells was conceivably so different from what we have observed that it is probably useful to distinguish it as “pre-Darwinian” evolution. (c) The inaccuracies in translation also should have severely limited the genetic complexity of primitive cells; genomes even as large as phage genomes may have been impossible (or useless) to achieve. (d) Pre-Darwinian evolution, being completely limited by the accuracy of translation, was in a sense entirely concerned with evolution of the genetic code. This evolution perhaps occurred in discrete stages: the beginning of a stage would be some improvement in the translation process; the stage itself would be the working out of evolutionary ramifications of this improvement, which would then prepare the way for another improvement in translation, the beginning of the next stage, etc. (169).
D. The Problem of Constraints Governing the Code’s Evolution The genetic code must have evolved gradually, through many stages. There must be overall constraints and considerations that have shaped this evolution. As we have seen, there exists a reasonable case for amino acid-oligonucleotide recognition interactions supplying constraints. Since the point remains unproven, however, alternatives must also be considered. Could the high degree of order manifested by codon assignments, for example, possibly be accounted for without postulating some feature such as amino acid-codon recognition? I n other words, could the constraints governing the code’s evolution be external to the coding apparatus per se? Several attempts have been made to construct a model accounting for the highly ordered array of codon assignments without resort to constraints that are a part of the coding apparatus. The first detailed
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scheme of this sort, and the prototype for the others, was proposed by The starting point for this model is a cell with an Sonneborn (17 0). unordered codon catalog (but an otherwise fully developed translation system). Sonneborn argues that the codon catalog could have achieved its high degree of order in the following way. Practically all mutations are events deleterious to a cell or its descendants. Thus a cell that can reduce its burden of deleterious mutations is placed a t a selective advantage. Other things being equal, this can be achieved by reassigning codons until a particular optimal set (or sets) is obtained. An optimal set of codons is one in which all possible codons are assigned to amino acids (an unassigned codon, introduced by a mutation, would certainly be lethal to the cell), and all codons assigned to the same amino acid are “maximally connected”-i.e., the number of base changes necessary to convert any one of an amino acid’s codons to all others of its codons is a minimum. For example UUU, UUC, and UUA are a set of maximally connected codons; UUU, UUC, and UAU are not. The individual sets of maximally connected codons must then be further interrelated to one another by the condition that functionally related amino acids possess “connected” sets of codons wherever possible. These constraints would then serve to make as many mutations as possible lead to no change in the overlying amino acid, and when amino acid replacements did result from mutations, as large a fraction of these as possible would involve functionally related amino acids and so have little or no deleterious effect. The codon catalog resulting from such constraints could resemble the actual one in overall order. The ordering is a relative onei.e., there is no constraint that drives Phe to be associated particularly with UUU, etc. A number of criticisms can be directed against this model ( l l ) , and some of these we have encountered above. In any case, it seems possible to circumvent these by using a somewhat different model (169). The trouble with any basically stochastic scheme (as this one is) is that these schemes seem to have to start with a fairly well-developed translation system. Thus such a model never gives an indication of how translation could have begun initially. And that, after all, is the real question-an especially puzzling one too, if one is not permitted to invoke any “specificity” or selection in the interaction of nucleic acids and amino acids or their derivatives.
E. The Problem of the Origin of the Components The translation process involves a set of no less than twenty highly specific activating enzymes, a set of perhaps forty kinds of tRNA’s, a ribosome comprising two enoriiious RNA molecules and as many as
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35-40 kinds of protein molecules. Primitive translation could never have involved so many components. Not only must the number of different molecules in any class have been much smaller initially, but the number of classes of macromolecules in the translation apparatus initially must have been smaller, too; i.e., most of the components now a part of translation could not have been present in the earliest attempts. Since Occam’s razor demands that primitive translation be far simpler and perforce much different than its modern day counterpart, it is difficult to think of the former because of preconceived ideas derived from knowledge of the latter. I n fact it is wise even to question whether as a synthetic process translation began as a translation a t all-i.e., mapping the primary structure of nucleic acid into the primary structure of a polypeptide (albeit inaccurately). For several reasons, I take as axiomatic that some sort of “recognition” interactions involving amino acids and bases or their derivatives lies a t the root of the code’s origin. I n the first place, arguments brought against this sort of “specificity,” or “recognition,” etc. (as in the formulation of the adaptor hypothesis) are by no means compelling, as we have already seen. For another, there is evidence strongly suggesting these interactions (167). Finally, it seems difficult to evolve a relationship between nucleic acid and protein primary structures unless there exists some tendency for the two (or their components) to associate with some “specificity” to begin with. However, in light of the previous paragraph it may be wise to take a broad view of what kinds of interactions of amino acids and bases might contribute to the code’s origin and evolution. It seems useful to begin these considerations from the opposite end, as it were, by asking what components of the modern genetic coding apparatus (more properly, the progenitors of these components) might not have been present a t the earlier stages of the code’s evolution. Perhaps the least likely molecule to have been prescnt initially is one like an activating enzyme. This type of molecule could not have existed hzforc precise translation existed ; its major function is accurate discrimination among amino acids, which requires it to have a unique, fixed primary structure. On the other hand, tRNA progenitors may well have existed earlier, for several reasons. For one, tRNA is always covalently linked to the growing polypeptide chain, consistent with and suggesting a primeval association between the two macromolecules. For another, tRNA appears to be a “molecular anachronism”; it is a nucleic acid, yet the post-transcriptional modifications (methylations, etc.) it undergoes to be properly functional, as well as some of its properties, e.g.,
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readily reversible configurational changes (171, 17.2), give it more the characteristics of a protein. On the grounds that it is easier to adapt an existing function than to create a new one, the evolution of tRNA to a form so unlike other nucleic acids, argues that tRNA’s progenitors were indeed of relatively ancient origin (and may originally have had a quite different function). The ribosome, which seems so complex, could not have existed as such early in the evolutionary process, but this is not to say that a much simpler version of it was not a t one time a component of translation, Protein-RNA associations occur readily. Perhaps the key question of all is a t what stage an actual “tape-reading” process came into use in translation-i.e., an external tape (the message) passing through a tape-reading machine (the ribosome). Again the complexity of this operation, the number of separate parts participating, and the precision of their function, suggests that evolution of the tapereading feature was preceded by other developments. Since so little is known about possible progenitors of the present genetic coding apparatus, little would be gained by constructing detailed and untestable models for the code’s evolution. However, there is some virtue in cataloguing and briefly discussing types of interactions that could have contributed to the code’s origin, and in trying to formulate pertinent questions regarding this origin. It seems important to make some distinction as to whether initially the “unit of recognition” between the class of nucleic acid components and polypeptide components was the amino acid or some minimum size of peptide, on the one hand, and the individual base or some oligonucleotide, on the other. I n one case, we might expect primitive ‘%ranslation” t o involve addition of ainino acid subunits to growing peptide chains; in the other case, proteins could be synthesized from assembly of small peptide units, the small units having been made by ‘hondirected” synthesis and selected in the translation process. As we have seen, a major problem with amino acid-nucleic acid interactions is the apparent weakness of binding and consequent low specificity. Peptide-nucleic acid interactions would necessarily be much stronger and, in certain ways, might be more specific, We know that nucleic acid-peptide interactions do exist-in the ribosome, in histone-DNA complexes, in the activating enzyme-tRNA interaction, for example-and these show specificity to varying degrees. Recently it has been shown that poly Lys associates preferentially with DNA of high A T content while poly Arg prefers a high G C DNA, an encouraging sign (173). What we do not know in all cases is ( a ) whether the association of nucleic acid and peptide is a colinear one;
+
+
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(b) the number of bases with which an amino acid residue associates;ll and (c) the effect on strength of binding of changing one amino acid residue or base in the complex-i.e., the “specificity” of binding. Polypeptide-nucleic acid association might have been used in the primitive cell as a basis for feedback from protein primary structure to nucleic acid primary structure, a device for checking the accuracy of a translation, etc. There seems little point, in general, in having a direct feedback from polypeptide primary structure to synthesis of nucleic acid primary structure when a primitive translation process, in both directions, is a most inaccurate one. However, in certain special cases a direct influence of protein primary structure on nucleic acid primary structure could be advantageous. If the peptide product of a translation associates with the nucleic acid of whose primary structure it is a translation, there is a mechanism serving both to select the better translations from their less accurate counterparts and to influence nucleic acid primary structure, limiting the speed of “genetic drift,” etc.-e.g., by protecting from hydrolysis those nucleic acids associating most strongly with polypeptides. It is interesting to note one present-day system where protein primary structure might direct the synthesis of nucleic acid. A poly A synthetase has been isolated from Clostridiuin perfringens. The enzyme has an unusual cofactor requirement, poly Lys ( 1 7 6 ) . I n view of the coding relationship between AAA and lysine, one is tempted to speculate that perhaps in some rich primitive milieu poly Lys could have brought about the synthesis of poly A and vice versa, thus creating an autocatalytic system. The association of poly A with poly Lys would then serve to help eliminate (by not protecting adequately) ‘Ibad” translations in either direction. A phenomenon well known to the polymer chemist may have some bearing on origins of the code. I refer to the synthesis of stereo-regular polyhydrocarbons by various mineral catalysis-e.g., the Ziegler catalyst. Such interactions manifest a very rudimentary specificity. It is tempting to extrapolate from such a mechanism to one in which nucleic acid acts as a catalytic site for polypeptide synthesis. Perhaps some primeval RNA llThere is a controversy at present regarding whether or not rRNA can code for ribosomal proteins (174, 176). The answer to this point is particularly germane to the present discussion. It is argued that rRNA could not code for its own proteins because a n RNA optimally evolved to bind with protein (in ribosome formation) could not be optimally evolved to perform an unrelated function, coding for protein. This argument is reasonable if the two functions-coding us. binding-are unrelated to one another. However, if the two are indeed related, then i t might be expected that optimal binding of ribosomal protein is completely consistent with coding for the same protein.
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was a fixed template site that synthesized a polypeptide of crudely specified amino acid composition. Having only a single RNA site, this sort of synthesis could not be considered a translation, for there is no colinear relationship between nucleic acid and polypeptide primary structure, but i t could be considered the progenitor of the present day tRNA-peptide relationship.
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(1966). 166. G. E. Magni and P. P. Puglisi, Cold Spring Harbor Symp. Quunt. Biol. 31, 699 (1966). 166. D . Gillespie and S. Spiegelman, in preparation. 167. E. S. Maxwell, Proc. Natl. Acad. Sci. U S . 48, 1639 (1962). 168. J. F. Speyer, P. Lengyel, C. Basilio, A. J. Wahba, R. S. Gardner, and S. Ochoa, Cold Spring Harbor Symp. Quant. Biol. 28, 559 (1963). 169. R. Sager, I. B. Weinstein, and Y . Ashkenazi, Science 140, 304 (1963). 160. G. von Ehrenstein and F. Lipmann, Proc. Natl. Acad. Sci. US. 47, 941 (1961). ’61. D. Rifkin, D. Hirsh, M. R. Rifkin, and W. Konigsberg, Cold Spring Harbor Symp. Quant. Biol. 31, 715 (1966). IGI. F. H. C. Crick, Symp. SOC.E z p t l . BbE. 12, 138 (1958). 163. J. D. Watson, “Molecular Biology of the Gene.” Benjamin, New York, 1965. 164. G. Zubay and P. Doty, Biochim. Biophys. Acta 29, 47 (1958). 166. R. J. Britten and C. Woese, unpublished results. 166. C. Woese, Proc. Natl. Acad. Sci. U S . 54, 71 (1965). 167. C. Woese, D. H. Dugre, W. C. Saxinger, and S. A. Dugre, Proc. Natl. Acad. sci. us. 55, 966 (1966). 268. B. Pullman, J . Chem. Phys. 43, 5233 (1965); personal communication. 169. C. Woese, Proc. Natt. Acad. Sci. U S . 54, 1546 (1965). 270. T. M. Sonneborn, in “Evolving Genes and Proteins” (V. Bryson and H. J. Vogel, eds.), p. 377. Academic Press, New York 1965. 171. P. S. Sarin, P. C. Zamecnik, P. L. Bergquist, and J . F. Scott, Proc. Natl. Acad. Sci. U.S. 55, 579 (1966). 172. T. Lindahl, A. Adams, and J. R. Fresco, Proc. Natl. Acad. Sci. U.S. 55, 941 (1966); J. R. Fresco, A. Adams, R. Ascione, D. Henley, and T. Lindahl, Cold Spring Harbm Symp. Quant. Bwl. 31, 527 (1966). 173. M. Leng and G. Felsenfeld, Proc. Natl. Acad. Sci. US.56, 1325 (1966). 174. D. Nakada, Biochim. Biophys. Acta 103, 455 (1965). 176. P. Sypherd, J . Mol. Biol. 24, 329 (1967. 176. M. I . Dolin, Biochem. Biophys. l i es. Commun. 6, 11 (1961).
The Search for the Messenger RNA of Hemoglobin H. CHANTRENNE, A. BURNY, AND G. MARBAIX Laboratory of Biological Chemistry, Faculty of Sciences, University of Brussels, Brussels, Belgium
I. Introduction . . . . . . . . . . . . . 11. Location of the Information for Hemoglobin in Reticulocyte . . . . . . . . . . . . . . Extracts 111. Stimulation of Amino Acid Incorporation by Reticulocyte RNA . A. Cell-Free Preparations from E . coli . . . . . . . B. Cell-Free Preparations from Reticulocytes . . . . . IV. Isolation of the mRNA Thread from Polyribosomes . . . . A. Principles . . . . . . . . . . . . . B. Detection of a 9 s RNA with the Expected Properties . . . C. Direct Observation of 9 s RNA . . . . . . . . D. Purification of the 9 s Fraction . . . . . . . . E. Detachment of 9 s RNA from Ribosomal Particles . . . F. Properties of 9s RNA . . . . . . . . . . G. NaF-Resistant Association between 9 s RNA and Ribosomes . V. Concluding Remarks . . . . . . . . . . . References . . . . . . . . . . . . . .
173 174 175 175 176 179 179 179 183 185 186 189
191 192 192
1. Introduction Synthetic polynucleotides can certainly fulfill the main function of natural messenger RNA’s: they are accepted by ribosomes and translated into polypeptides. However, it is highly desirable to ohtain the messenger RNA corresponding to a known protein. Natural messengers must carry, besides the information for amino acid selection, signals of various kinds that control the machinery of translation: sequences for starting or interrupting translation, special properties that ensure fixation to the ribosome and correct phasing; they may possibly contain regulation sites. Messengers represent but a small fraction-only a few percent-of the total cellular RNA; they must make up a mixed population of individual molecules varying in size and half-life, and are subject to the constant risk of degradation by nucleases. This actually occurs in most cases when the cell is broken open and the delicate balance of its 173
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constituents becomes disturbed. Nucleases are a major obstacle in attempts to isolate intact messenger RNA’s ; technical skill will eventually circumvent this difficulty. But to select the messenger of a known protein out of the whole messenger population of a cell raises another kind of a problem, for which there is as yet no obvious solution. For the time being, the best one can hope for is to find favorable cases in which the selection has been made for us by nature as, for example, in viruses. A viral RNA is a homogeneous molecular species that contains the information for several proteins, three in the simplest viruses and in some defective viruses perhaps only one. Some virus RNA’s are indeed excellent messengers ( I S ) . I n higher organisms, the messenger that seems the most accessible is that responsible for hemoglobin, owing to the exceptional properties of mammalian reticulocytes. Rabbit reticulocytes are easy to separate from other cells (apart from inactive mature erythrocytes); they can be washed free of blood plasma, they can be opened in a medium of controlled composition by a slight lowering of the osmotic pressure; moreover, they are free of ribonuclease. Mammalian reticulocytes do not make RNA (4, 5 ) ; their nucleus is lost in the process of differentiation. Nevertheless they retain for several days their ability to make hemoglobin. The genetic messengers for hemoglobin, therefore, are long-lived. Since rabbit reticulocytes make practically no other protein (6, 7 ) , it can reasonably be supposed that most of the messenger population of the cell is composed of hemoglobin messengers. As a first approximation, the problem of sorting out the messengers can be neglected; it has largely been solved by the physiological differentiation of the red cell. Several of the main technical difficulties encountered in the general case simply do not exist or are considerably alleviated in the case of hemoglobin messengers. Nevertheless, in order to isolate a messenger, one must be able to recognize it. The final criterion of a hemoglobin messenger is that it cause the synthesis of globin in a system that does not possess the information for that protein. It is therefore quite reasonable to attempt to identify this messenger on the basis of its informational properties.
II. location of the Information for Hemoglobin in Reticulocyte Extracts Schweet et al. (8) demonstrated that an extract of rabbit reticulocytes can incorporate amino acids into hemoglobin in vitro. When the protein was chromatographed on ion-exchange resins, more than 80% of the
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incorporated amino acids were recovered with hemoglobin, and the relative rates of incorporation of three amino acids (leucine, isoleucine, and valine) corresponded to the ratios in which they are present in rabbit hemoglobin. Since a heterologous system made of rabbit reticulocyte ribosomes and a “pH 5” fraction from guinea pig liver produced hemoglobin just as well, it can be concluded that the information for hemoglobin synthesis is associated with the microsome fraction. Hemoglobin is even made by a simplified mixed system derived from reticulocyte ribosomes and Escherichia coli transfer RNA charged with amino acids ( 9 ) . That the protein synthesized has the primary structure of hemoglobin was proved by chromatographic analysis (“fingerprinting’,). Bishop et al. (10) made crossed systems with ribosomes from rabbit reticulocytes and a supernatant fraction from mouse reticulocytcs. The two hemoglobins were separated by chromatography ; the protein labeled in vitro belonged to the species contributing the ribosomes. However, two other groups reported experiments a t about the same time indicating that both the ribosomes and the supernatant fraction can supply information in crossed systems (11-13). As these results conflicted with previous reports and could not easily be confirmed (14, 1 5 ) , they were generally attributed to contamination of the supernatant fractions with remaining particles, which are difficult to eliminate completely. Quite recently, Schapira et al. (16, 17) presented additional evidence that a soluble fraction of the supernatant portion can provide information for hemoglobin synthesis in a crossed system. Whatever the reasons for these conflicting results, it is a t least clear and undisputed that the ribosome sediment (including polyribosomes) does contain the information that causes amino acids to condense in the correct sequences of hemoglobin chains. A large fraction of the genetic messengers of hemoglobin is associated with ribosomes and can be translated correctly in vitro.
111. Stimulation of Amino Acid lncorporution by Reticulocyte RNA A. Cell-Free Preparations from E. coli The success achieved with cell-free E. coli extracts (18) in unraveling the genetic code and the remarkable response of this system to a variety of synthetic polyribonucleotides with random (19, 20) or ordered (61-23) sequences of nucleotides made it the standard test for messenger properties of RNA. Many RNA preparations from all kinds of organisms indeed stimulate amino acid incorporation into polypeptides in this system. The RNA extracted from bacteriophage f2 or R17 causes the synthesis of recognizable viral proteins (1-3). The system embodies all
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the features required for initiation and termination of chains and for release of the protein, and it responds to the “punctuation marks” of the tricistronic messenger of R17; even suppression can be observed in vitro with this system, indicating a high degree of fidelity (3). Total reticulocyte RNA, as obtained by phenol extraction, stimulates amino acid incorporation in the cell-free E . coli system (24-27). When RNA is fractionated on sucrose gradients or by salt precipitation and the fractions are assayed for stimulatory activity, the most active fraction sediments together with 16s ribosomal RNA or on the lighter side of the 16s RNA peak (26-28). But the ratio of incorporation rates of two characteristic amino acids is very different from that expected for hemoglobin (24, 2 5 ) , and the labeled product does not behave as globin or hemoglobin in chromatography (24, 2 5 ) . Trypsin hydrolysis and peptide separation clearly show that the protein made does not resemble hemoglobin; the pattern of peptides is almost the same as that obtained from material made by the E . coli system alone (background incorporation) without addition of reticulocyte RNA ( 2 9 ) .Therefore, the RNA does not provide information to the system, it does not act as a messenger; i t merely stimulates in a n unknown manner n synthesis that was proceeding slowly in the E . coli extract. Clearly, one cannot equate stimulatory activity in amino acid incorporation and messenger properties. If RNA’s can stimulate the in vitro system without being translated, great caution must be exercised in using the in vitro E. coli system for identifying messenger RNA. In the present case, it cannot be concluded that the fraction that stimulates is messenger RNA, but neither is there any reason to conclude that the RNA preparation contains no hemoglobin messenger. Too little is known about the conditions required for a messenger to be accepted by a ribosome and translated. So far, only some viral RNA’s appear to have been translated correctly in vitro ( I S ) . This may possibly be related to their parasitic nature; they must indeed fulfill conditions that make them acceptable, even preferentially acceptable, by host ribosomes. It is quite conceivable that E. coli ribosomes cannot accept hemoglobin messengers or cannot bind them properly.
8. Cell-Free Preparations from Reticulocytes Although E . coli ribosomes may conceivably be unable to accommodate hemoglobin mRNA, ribosomes from reticulocytes can certainly translate it. RNA from reticulocytes can increase the synthesis of hemoglobin chains in vitro. Kruh e t al. (SO) obtained an increase in amino acid incorporation by adding the total RNA of reticulocytes to a cell-free
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reticulocyte extract. The ratio of incorporation of four different amino acids corresponded to their proportions in hemoglobin. Fingerprints of the labeled protein indicated that its amino acid sequence was that of hemoglobin. However, the N-terminal peptide was only very slightly labeled, as if most of the incorporation corresponded to completion of growing polypeptide chains rather than to a complete synthesis of the chains from end to end. The stimulating agent appears to be RNA (it is destroyed by RNase, not by DNase) and RNA’s from E. coli, yeast, or guinea pig inhibit the endogenous hemoglobin synthesis. Reticulocyte RNA was centrifuged on a sucrose gradient and separated into four fractions, which were assayed for stimulatory activity ( 3 1 ) . The fraction containing 23 S ribosomal RNA was slightly inhibitory, those containing either 16 S or 4 S RNA gave a slight stimulation, but fraction 111, which contains part of the light side of the 16s peak and the region of the gradient between 1 6 s and 4S, did stimulate hcmoglohin synthesis in the cell-free reticulocyte system. The average stimulation was 75% above the control, 150% in some experiments. It should be emphasized that fraction I11 comprises the region of the gradient in which an RNA of the size predicted for hemoglobin messenger would be expected, and part of the fraction that stimulates amino acid incorporation in the E . coli system (26, 27). Comparable studies by Arnstein et al. (3.2) showed that high molecular weight RNA extracted from “heavy” ribosomes (mostly polyribosomes) stimulated hemoglobin synthesis especially when the system was made of light ribosomes (mainly 80s) and the supernatant fraction. Considerable synthesis of new chains was observed in these experiments. The same group (33) later studied the stimulatory action of ribosomal subunits obtained by EDTA treatment of polyribosomes or by brief exposure to dilute alkali; the action of the RNA extracted from these particles and centrifuged on sucrose gradients was also examined. No RNA in the size range expected for free mRNA stimulated hemoglobin synthesis; all the activity was associated with ribosomal RNA. This led to the suggestion (33) that mRNA is covalently bound to ribosomal RNA, a concept that implies a departure from the classical model. Whatever the intrinsic interest of these results, they cannot avoid the fundamental objection that the system used for testing the RNA already contains, by itself, all the information necessary for making hemoglobin. It is, therefore, a priori, a poor test system for a hemoglobin messenger of the same species. It would be an excellent one if the endogenous messenger could be completely removed from the in vitro system to start with, but this has not been achieved so far. It is true that the nature of the polypeptides made can be influenced by the added RNA
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(27, 3 4 ) . But as long as endogenous messenger is present, stimulation of hemoglobin synthesis-just as much as inhibition-might arise from an action on any component of the system able to affect in one or the other direction the rate a t which the system operates. Certain facile criticisms were refuted and some obvious possibilities checked [see, for example, reference (SO)1. The system unfortunately is incompletely characterized, and so complex that discarding one more objection is not enough to establish a conclusion with certainty. The clear-cut results obtained by Drach and Lingrel (29) with the E. coli system prove that stimulation does not necessarily mean messenger activity. For instance, RNA’s from liver nuclei, and from the kidney or intestine of the rabbit stimulate hemoglobin synthesis by the in vitro reticulocyte system (35, 36). If the test system were devoid of information for hemoglobin, this would prove that liver, kidney, and intestine contain hemoglobin messenger RNA-a very important conclusion in relation to the problem of differentiation. But with the test system as it stands, the authors (36) are quite right in presenting this conclusion as tentative. Obviously, no definite conclusion can be based a t present on the stimulatory activity of an RNA in the reticulocyte system. When the RNA stimulates the completion of partly made hemoglobin chains, there is even reason to doubt that the stimulation of hemoglobin formation is due to messenger activity of the added RNA. Can one seriously consider the possibility that mRNA can bind on each ribosome with an unfinished chain, just a t the site where translation has been interrupted? The message must be read from the end, and mRNA must be able to bind t o a ribosome a t the starting end only; it cannot bind a t random. The fact that the polypeptides made in vitro correspond to hemoglobin shows that the system is correctly phased. Should the messenger bind a t random, two-thirds of the polypeptides made would not resemble hemoglobin a t all since two out of three messages would be out of phase. Therefore, in these cases where RNA stimulates the completion of hemoglobin chains, it is improbable that it works in the same way as informational RNA; effects of the type analyzed by Drach and Lingrel (29)are to be suspected. The present situation is indeed an embarrassing one : reticulocyte RNA stimulates amino acid incorporation into both E. coli and reticulocyte cell-free preparations. I n the E . coli system, the polypeptides made are characteristic of E . coli and do not resemble hemoglobin. I n the reticulocyte system, which does make hemoglobin by itself and therefore contains the corresponding genetic information, no definite conclusion
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can be based a t present on the stiniulatory action of RNA on true hemoglobin synthesis.
IV. Isolation of the mRNA Thread from Polyribosomes A. Principles In our laboratory, a completely different approach was chosen. After a few attempts with the in vitro systems, which gavc unrewarding (and unpublished) results, we thought that it should be possible to recognize and isolate reticulocyte messenger RNA on the basis of other properties. According to the classical model, the messenger is the RNA fiber that connects the ribosomes in the polyribosome structure (37-400) This fiber has been clearly observed in electron micrographs of reticulocytes, and the measured length (15OOA) is that expected for a messenger RNA coding for a single globin chain ( 4 1 ) . We thought that isolating this RNA fiber should not raise insurmountable difficulties. The size of this RNA thread having been established directly, it was possible to estimate its sedimentation constant to be in the range of 9s. As the RNA thread is much more sensitive to pancreatic ribonuclease than is ribosomal RNA in ribosomes (37-40), a very simple test is available for identifying any isolated RNA as the thread: i t should be destroyed when the polyribosomal structure is treated with just sufficient ribonuclease to disrupt the polyribosomes without attacking ribosomal RNA. Hemoglobin messenger RNA is long-lived compared to bacterial messengers. However, it was hoped that under favorable circumstances the messenger would be labeled more strongly than ribosomal RNA and this might help in its recognition. There was, of course, no hope of labeling the mRNA of rabbit reticulocytes in vitro; these cells have lost their nucleus and they can only incorporate precursors into the CA end of transfer RNA (4, 5 ) . Therefore, mRNA must be labeled during its formation in the bone marrow.
B. Detection of a 9 S RNA with the Expected Properties With these principles in mind, we isolated polyribosomes, extracted the RNA, and fractionated it by sucrose gradient centrifugation, looking for a new RNA fraction with the expected properties. a component When the RNA was obtained by LiCl precipitation (~$2)~ with a sedimentation coefficient of about 36s was consistently found, as had been observed before by Barlow e t al. (42). Treatment of the polyribosomes by RNase under conditions more than sufficient to destroy
180
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THE SEARCH FOR MESSENGER RNA OF HEMOGLOBIN
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the RNA thread of the polyribosomes did not affect the 3 6 s peak; this fraction, therefore, could not be the RNA thread. The LiCl method, and most of the usual methods for obtaining RNA, involve a precipitation a t some stage in the preparation. As mRNA is a small fraction of total RNA, it is likely to be lost in precipitation steps. We looked for a method in which all losses could be avoided. The whole polyribosome pellet was dissolved in a 0.5% solution of dodecyl sulfate according to Kurland ( 4 3 ; see also @u). Ribosomal proteins and RNA are thus dissociated. Since the pellet dissolves completely, the RNA thread cannot be lost under these conditions. A rather concentrated solution of polyribosomes was simply layered on a 5-20% sucrose gradient and centrifuged in the SW 39 rotor of the Spinco for 7 hours a t 37,000 rpm (115,000 )( 8). The same experiment was repeated with reticulocytes from anemic rabbits to which radioactive phosphate had been injected a t various times. Reticulocyte RNA from rabbits injected with P325 hours before blood collection showed radioactivity in the 4 S peak only, as the result of chain-end turnover (4, 5 ) . With rabbits killed between 10 and 20 hours after a 10 m C injection of radioactive phosphate, the 23S, 16S, and 4 5 peaks were labeled, but a new peak of radioactivity was consistently observed between the 16 S and the 4 5 peak that was clearly distinct from both. This is the region where a single strand of RNA containing just enough nucleotides for coding one globin chain is to be expected. This fraction was hydrolyzed in KOH, together with unlabeled RNA as a carrier, and chromatographed on an ion-exchange column. The radioactivity distribution coincided with the nucleotides, showing that the original labeled substance was RNA. I n order t o find out whether this was the messenger RNA thread, a ribonuclease sensitivity test was applied as follows. FIG. 1 . ( A ) Sedimentation pattern of reticulocyte polyribosoinrs in a linear 18-36%> sucrose gradient equilibrated in 0.05 M Tris-HC1 (pH = 7,4), 0.025 M KC1, and 0.005 M magnesium acetate. Cent)rifugation was carricd out for 2 hours at 32,000 rpm (85,000 x g) in the S1)inc.o rotor SW 39 a t 4°C. (B) Scdinientation lmttern of retirriloc*yt,r polyribosomes after t.rr:ttmeut of thc polyribosomal suspension with 0.01 fig o f 1)anc.rratic ribonurleasr per 1111 for 5 niinut.es a t 37". Conditions of centrifugat,ion as in ( A ) . (C) Sedimentation pattern of (P3'-labeled) polyribosomal RNA extracted with sodium dodecyl sulfate in a linear 5-20'70 sucrose gradient equilibrated in 5 mM Tris-HC1, pH 7.4. Centrifugation wm earried out for 7 hours a t 37,500 rpm (115,000 x g) in the Spinco rotor SW 39 a t 4". (D) Sedimentation pattern of RNA extracted with sodium dodecyl sulfate from (P=-labeled) polyribosomes treated with ribonuclease (see B). Conditions of centrifugation as in (C). I n (C) and ( D ) : -optical density at 260 n m ; - - - radioactivity in counts per minute. [Taken from Marbaix and Burny (441.1
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[It should first be realized that polyribosomes isolated from washed reticulocytes by centrifugation through a sucrose cushion are quite stable. Their sedimentation profile in the analytical centrifuge does not change after a %-minute incubation at 37°C in neutral buffer containing Mg2+. In a systematic study of their sensitivity to pancreatic ribonuclease, it was found that a 5-minute treatment a t 37" with 0.01 pg of ribonuclease per ml was just sufficient to destroy most of the polyribosomes. Such a limited treatment has no effect on ribosomal RNA.] Polyribosomes were thus treated for 5 minutes a t 37" with 0.01 pg ribonuclease per ml and then chilled. Control polyribosomes were similarly treated without the enzyme. Part of the suspension was used to determine the sedimentation profile of the polyribosomes ; the rest was dissolved in sodium dodecyl sulfate and centrifuged on a sucrose gradient. The result of such an experiment is shown in Fig. 1. Clearly an RNase treatment just sufficient to disrupt the polyribosome structure destroys the RNA fraction sedimenting between the 4 s and 16s peaks, in the 9s region. The radioactivity is now found on the heavy side of the 4 S peak ( 4 4 ) .The treatment was very mild since the polyribosomal pattern did not completely disappear ; some small polyribosomes remained as if the thread had been cut in several places but not completely hydrolyzed. However, some doubt as to the origin of the new RNA remained. Was it really a reticulocyte RNA? Could it not originate from contaminating white cells? Although most of the leukocytes are eliminated by centrifugation and by differential lysis, some leukocytes might possibly be lysed a t the same time as reticulocytes so that their polyribosomes would contaminate the pellet from which the new RNA fraction is obtained. I n order to test this possibility, whole blood was deliberately incubated for 5 hours in vitro, the c,ells (including leukocytes) were lysed, and the RNA isolated from polyribosomes was centrifuged in a sucrose gradient. Under these conditions, all the radioactive RNA made should be derived from nucleated cells, i.e., leukocytes. The distribution of radioactivity (Fig. 2) shows a major peak a t 16S, slightly on its lighter side. This is quite different from the pattern observed with reticulocytes labeled in vivo and freed as far as possible of leukocytes. There is therefore good reason to presume that the 9 S RNA labeled in vivo, which has several of the properties expected of a messenger, is indeed coming from reticulocytes, rather than from white cells, and that it is essentially hemoglobin messenger RNA. Recent publications by Scherrer et al. (45-47) and by Attardi et al. (48) have described the occurrence, in the cytoplasm of immature duck erythrocytes, of 8-10s RNA, which is rapidly labeled when these
THE SEARCH FOR MESSENGER RNA OF HEMOGLOBIN
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nucleated cells are incubated with radioactive precursors. This is probably the analog of the 9 s RNA fraction we find in rabbit reticulocytes.
C. Direct Observation of 9 S RNA A rough estimate of the amount of messenger RNA in reticulocyte polyribosomes as compared with ribosomal RNA indicated that it should be possible to detect its ultraviolet light absorption by merely scaling up the operations by a factor of ten. Thus instead of centrifuging 200 pg of polyribosomal RNA in the Spinco SW 39 rotor, ten times as much was used. This required the adaptation of the method to the larger SW 25 rotor. After 40 hours of centrifugation a t 24,000 rpm (60,OOOX g), the 26 S component is a t the bottom of the tube, 16s is well resolved, and the new 9s fraction is clearly visible as a low peak, well separated from 4 S and 16 S, as shown in Fig. 3A. The limited RNase treatment described above was applied, giving the result shown in Fig. 3B, which confirmed our previous observations: the 9 s fraction, which is now observable by its optical density and its radioactivity, is destroyed when the polyribosomes are
184
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disrupted by mild RNase treatment, as expected for the RNA fiber of the polyribosome. Quantitative comparisons of the spc'cific radioactivity of this fraction with that of ribosomal RNA, show that it is always higher. Depending on the time of injection of P,32,and on the individual rabbits, the new fraction is 2-8 times more radioactive than 16s RNA. A factor of 3-4 is most commonly observed when P3?is injected 15 hours before collecting the blood (44, 4 9 ) . I n summary, the new RNA fraction has the following properties predicted for the messenger RNA's of single hemoglobin chains: (1) it originates from reticulocytes; (2) it sediments in the region of the gradient where a single strand RNA of about 430 nucleotides (the number required to code for the 141-146 amino acid residues of each hemoglobin chain) should be expected. (3) its specific radioactivity is higher than that of ribosomal RNA; (4) the new RNA fraction is degraded when the polyribosomes are trcated briefly with an extremely small amount of RNase, just sufficient to disrupt the polyribosomal structure without causing any detectable damage to ribosomal RNA.
D. Purification of t h e 9 S Fraction The method used for detecting 9 S RNA was easily adapted to its isolation. A first procedure (49)' which was later improved by small technical modifications (50)' rests on repeated sucrose gradient centrifugations in the SW 25 rotor (Spinco). Three steps of the purification are illustrated in Fig. 4 [for details, see (50)1. Up to 250 pg of the 9 S fraction may be isolated from a 2 kg rabbit, but the yield is usually in the range of 150 pg per rabbit. The greatest care must be taken to avoid any trace of ribonuclease (from the hands for instance) especially for the operations in which the RNA is not protected by sodium dodecyl sulfate ; otherwise the isolation presents no difficulty. Quite recently, we have been able to scale up further and improve the preparations (51) by using Anderson's zonal rotor (52) and a new method for concentrating the RNA ( 5 3 ) .The resolution in Anderson's FIG.3. (A) Sedimentation pattern of (€'"-labeled) polyribosomal RNA extracted with sodium dodecyl sulfate in a linear sucrose gradient, &20C/o, equilibrated in 5 mM Tris-HC1 p H 7.4. Centrifugation was carried out for 40 hours a t 24,000 rpm (60,000 x g) in the Spinco rotor SW 25.1 a t 4". (B) Sedimentation pattern of RNA extracted wit.h sodium doderyl sulfate from a (Pa-labeled) polyribosornal suspension treatcd for 5 minutes at 37" with 0.01 $g of pancreatic ribonuclease per ml. Conditions of centrifugation as in ( A ) . Optical density a t 260 nm ; - - - - radioactivity in counts per minute. ~
186
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rotor is comparable with that achieved with the swinging bucket rotors; it is especially suitable for centrifuging the crude polyribosomal solution.
E. Detachment of 9 S RNA from Ribosomal Particles I n the method described above, the entire polyribosomal structure is dissolved in sodium dodecyl sulfate, which disrupts the protein-RNA associations and liberates all the RNA. A better principle for separating mRNA from palyribosomes would
THE SEARCH FOR MESSENGER RNA OF HEMOGLOBIN
187
be simply to detach the messenger from intact ribosomal particles. An obvious possibility is offered by decreasing the magnesium concentration, since the binding of artificial messengers and the stability of natural polyribosomes depend on these ions. Bacterial ribosomes dissociate easily and they do not, fix messenger in M Mg2+.Animal ribosomes are more resistant; it was necessary to add a chelating agent (EDTA) or to pass the polyribosomal suspension through a carboxymethylcellulose (CMC) column in order to disrupt the polyribosomes of reticulocytes. In the process, the ribosomes were split into subunits (54).The sedimentation constants of the two ribosomal subparticles obtained after EDTA treatment are about 36 and 2 6 s instead of the classical 60 and 40s. Apparently, the shape of the subparticle is changed, thus making the sedimentation slower. This has been noticed before ( 5 5 ) : but neither RNA (56, 57) nor proteins (58) seem to be degraded or lost by this trcatment. The EDTA- or CMC-treated polyribosomes were layered on a sucrose gradient and centrifuged. Figure 5 A and B show the result of such experiments for two different times of centrifugation. Clearly, a highly labeled fraction sediments between the light ribosome particle and 4s RNA. The ribonuclease test is more difficult to apply in the present case. When sodium dodecyl sulfate is used for extracting RNA, RNase is inactivated and does not interfere. I n the present case, the enzyme must be removed before the ribosomal suspension is freed of Mg, otherwise i t degrades the 16s RNA of the partly unfolded particles and the test is meaningless. Ribonuclease can be removed easily by passing the treated polyribosomc suspension through a short column of carboxymethylcellulose equilibrated with 5 mM Mg2+ (54) ; the ribosomal particles remain intact. Addition of EDTA then causes their dissociation. Again the fraction released by chelating Mg2+that sediments between the lighter particles and the 4s RNA disappears after ribonuclease treatment of the polyribosomes. The fraction released from the polyribosomes by EDTA was collected and recentrifuged together with a sodium dodecyl sulfate extract of nonradioactive reticulocyte polyribosomes as in the first isolation method. Figure 6 shows that most of its RNA sediments together with the small 9 s peak. It is therefore probable that both represent the same RNA. The fact that this RNA can be separated from the ribosomal particles without dissociating them into protein and nucleic acid is further evidence that the 9 S fraction is the RNA thread of polyribosomes, i.e., messenger RNA. The shoulder shown on the light side of 9 s peak in Fig. 6 is consistently observed. I t s properties are under investigation.
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A B FIG.5. (A) Sedimentation pattern of the ribosomal particles obtained by treating a suspension of P3*-labeled polyribosomes (8 mg/ml in 10 mM Tris-HC1, p H 7.4; 10 m M KCl) with half a volume of 0.1 M EDTA. Centrifugation was for 16 hours a t 24,000 rpm (60,OOO x g) in a linear 15-30% sucrose gradient equilibrated in 10 mM Tris-HC1, p H 7.4; 10 mM KCI. Spinco rotor SW 25.1; temperature = 4°C. (€3) Same preparation and conditions as in (A) except that the centrifugation was extended to 40 hours. I n (A) and (B): -optical density at 260 nm; radioactivity in counts per minute.
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189
THE SEARCH FOR MESSENGER RNA O F HEMOGLOBIN
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FIG.6. Sedimentation profile of unlabeled polyribosomal RNA extracted with sodium dodecyl sulfate supplemented with the high specific radioactivity RNA detached from labeled polyribosomes by EDTA treatment,. Centrifugation for 40 hours at 24,000 rpm in a linear 5-20% sucrose gradient rquilibrated in 5 nidl Tris-HC1, IIH 7.4. Spinco rotor SR' 25.1 ; temperature = 4°C. -Optiral drnsity at, 260 n m ; - - - - radioactivity in counts per minute.
F. Properties of 9 S RNA Figure 7 shows a sedimentation diagram of 9 s RNA isolated by the sodium dodecyl sulfate method. The sedimentation constant was measured for two different preparations, and the values found were respectively 9.0 and 9.3 S. These were measured in low ionic strength tnedium containing 8 p g of RNA per ml. Using the relation worked out by Gierer (59) for a single strand of RNA, this indicates a molecular weight in the range of 150,000. The synthesis of hemoglobin requires two messengers of that size, one for each chain; the corresponding genes are not linked (60). The 9 s fraction should therefore be a mixture of two molecular species. As their molecular weight should differ by less than 3.5%, it is not surprising that they are not resolved by the sedimentation method used. The observed homogeneity of the 9s fraction may seem unexpected for another reason. It is indeed isolated from a heterogeneous polyribosomal population comprising from 1 to 6 ribosomes. If the 9 S RNA is the messenger fiber, this indicates that the polyribosomes, whether they contain two or six ribosomes, all possess a full-length mes-
190
H . CHANTRENNE, A. BURNY, AND G . MARBAIX
FIG.7. Drnsitometer tracings of the sedimentation pattern of messenger RNA (8 pg/ml in water a t 20 C) in a Brckman Spinco ultracentrifuge, equipped with ultraviolet optics, a t 42,040 rpm. Tracings were taken a t &minute intervals; exposure 80 seconds; wavelength 2655d. Sedimentation from left to right. [Taken from Rurny and Marbaix (491.1
senger. This conclusion fits perfectly with observations on hemoglobin synthesis on polyribosomes in vitro: Lamfrom and Knopf (61) have shown that even monosomes (“programmed” single ribosomes) make complete hemoglobin chains in vitro. It was possible to determine the base composition of the 9s fraction by column chromatography of an alkaline hydrolyaate. The
10
20
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90
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FIG.8. Chromatographic separation of an alkaline hgdrolyzate of 9s RNA. Tlie neutralized solution was loaded on a 0.5 x 40 cm column of Dowex AG 1x 2-Cl- and chromatographed in a linear gradient ( 0 4 . 1 7 M ) of HCl. C = cytidine 2’(3’)-phosphate; A = adenosine 2’(3’)-phosphate ; G = guanosine 2’(3’)-phosphate ; U = uridine 2’(3’)-phosphate.
THE SEARCH FOR MESSENGER RNA OF HEMOGLOBIN
191
elution diagram (Fig. 8 ) , obtained on a 200 p g sample, shows no component absorbing a t 258 nm other than the usual nucleotides. If such components are present, they amount to a very small fraction of the total. The composition was also estimated from the distribution of radioactive phosphate among the nucleotides according to Volkin and Astrachan (6.2). This method, which confirmed the results for both ribosomal RNA’s in the same experiment, does not give identical compositions in the 9 s fraction, which shows an excess of radioactivity in the adenine nucleotides. Possible reasons for the discrepancy have been considered, but this point has not yet been clarified ( 5 0 ) . When chromatographed on a methylated serum albumin-kieselguhr column ( 4 9 ) , the 9s RNA is eluted in the same region of the gradient as 2 6 s ribosomal RNA, although the molecular weights of these two molecular species are quite different. This means that 9s RNA has a more open structure than ribosomal RNA’s (63, 64).
G. NaF-Resistant Association between 9 S RNA and Ribosomes Sodium fluoride inhibits the synthesis of hemoglobin by otherwise intact reticulocytes ; polyribosomes disappear and only 80 S particles persist after fluoride treatment (6 5 ). However, hemoglobin synthesis is restored when the reticulocytes are washed free of fluoride, while polyribosomes re-form slowly and sequentially ( 6 5 ) . Restoration of hemoglobin synthesis is perfect; complete chains are made, starting from the N-terminal amino acid (66). As no synthesis of mRNA can occur in the anucleate reticulocyte, one must consider that after fluoride action the messenger is released in the cytoplasm or that it remains bound to single 8 0 s particles. Our method for isolating 9 S RNA was applied t o the particles of fluoride-treated cells and to those of control cells that had received NaCl instead of NaF. It was confirmed that the polyribosomes completely disappear in NaF-treated cells. But just as much 9 S RNA was extracted from the 80 S particles of NaF-treated cells as from the polyribosomes of control cells (67). The 9 S fraction thus remains associated with single ribosomes. Recent results by Ravel et al. (68) and Lin et al. (69) demonstrate that fluoride blocks the initiation of new polypeptide chains and that the ribosome pellet still contains the information for hemoglobin synthesis. Considering these observations together with ours, we are led to conclude that a ribosome can attach to mRNA in presence of fluoride but is blocked a t or close to the starting end of the message. Fluoride may thus help to analyze the very first step of translation.
192
H. CHANTRENNE, A. BURNY, AND G . MARBAIX
V. Concluding Remarks Wc have describcd the isolation from rabbit reticulocytes of a new
RNA fraction with the following properties: it represents about 2% of the total RNA; its turnover rate in marrow cells is higher than that of ribosomal RNA ; it is preferentially degraded when polyribosomes are treated with minute quantities of pancreatic ribonuclease ; it is released from polyribosomes when the concentration of magnesium ions is drastically reduced; it sediments as a homogeneous substance with a sedimentation constant in the range expected for the messenger of individual hemoglobin chains. The new RNA has thus many properties expected for the messenger of hemoglobin. However, we have been unable so far to obtain stimulation of amino acid incorporation in a cell-free system with this RNA. The interaction of natural messenger RNA with ribosomes has not yet been well analyzed. Up to the present time, only a few viral RNA’s have been shown to direct the production of real proteins in vitro. The ease with which they are accommodated by the host ribosomes may be related to their parasitic nature. They may be able to circumvent a control that operates with cellular messengers. We consider that the 9 s RNA isolated from reticulocytes is hemoglobin messenger RNA although proof will be obtained only when conditions arc found in which it causes the synthesis of recognizable globin chains in a system demonstrably devoid of the corresponding information. ACKNOWLEDGMENTS The authors are grateful to Dr. P. Malpoix for hcr help in preparing the manuscript. The original work reported was carried out as part of the association contract Euratom-University of Brussels 016-61-10 ABIB. G. Marbaix is a Fellow of the Belgian Fonds National de la Recherche Scientifique.
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42. J. J. Barlow, A. P. Mathias, R. Williamson, and D. B. Gammack, Biochem. Biophys. Res. Commun. 13, 61 (1963). 43. C. G. Kurland, J. M o t . Biot. 2, 83 (1960). 4%. T. Staehelin, F. 0. Wettstein, H. Oura, and H. Noll, Nature 201, 264 (1964). 44. G. Marbaix and A. Burny, Biochem. Biophys. Res. Commun. 16, 522 (1964). 45. K. Scherrer and L. Marcaud, Bull. SOC.Chim. Biol. 47, 1697 (1965). 46. K. Scherrer, L. Marcaud, F. Zajdela, B. Breckenbridge, and F. Gros, Bull. SOC. Chim. Biol. 48, 1037 (1966). 47. K. Scherrer, L. Marcaud, F. Zajdrla, I. M. London, and F. Gros, Proc. Natl. Acad. Sci. U.S. 56, 1571 (1966). 48. G. Attardi, H. Parnas, M. I. Hwang, and B. Attardi, J. M o l . Biol. 20, 145 (1966). 49. A. Burny and G. Marbaix, Biochim. Biophys. Acta 103, 409 (1965). 50. G. Marbaix, A. Burny, G. Hucz, and H. Chantrenne, Biochim. Biophys. Acta 114, 404 (1966). 51. G. Huez, A. Burny, G . Marbaix, and E. Schram, Europcati 1. Biochem. 1, 179 ( 1967). 52. N. G. Anderson, J. Phys. Chem. 66, 1984 (1962). 53. R. Barber, Biochim. Biophys. Acta 114, 422 (1966). 54. G. Marbaix and A . Burny, Arch. Intern. Physiol. Biochim. 72, 689 (1964). 55. A. S. Spirin, N. A. Kisselev, R. S. Shukulov, and A. A. Bogdanov, Biokkimiya 28, 920 (1963). 56. R. F. Gesteland, J. Mol. Biol. 18, 356 (1966). 57. H. J. Gould, H. R. V. Arnstein, and R. A. Cox, J. M o l . Biol. 15, 600 (1966). 68. C. Godfroid, unpublished results. 59. A . Gierer, 2. Naturjorsch. 13b, 477 (1958). 60. C. Baglioni, i n “Molecular Genet.ics” (J. H. Taylor, ed.), 11. 405 Academic Press, New York, 1963. 61. H. Lamfrom and P. M. Knopf, J. Mol. B i d . 9, 558 ( 1 9 6 4 ) . G2. E. Volkin and L. Astrachan, Virology 2, 149 (1956). G3. K. Asano, J. M o l . Biol. 14, 71 (1965). G4. K. A. 0. Ellem, J. M o l . Biol. 20, 283 (1966). 65. P. A. Marks, E. R . Burka, F. Conconi, W. Perl, and R. A . Rifkind, Proe. NatE. Acad. Sci. U.S. 53, 1437 (1965). G6. F . M . Conconi, A. Bank, and P. A. Marks, J. M o l . Biol. 19, 525 (1966). 67. B. Lebleu, G. Huez, A. Burny, and G. Marbaix, Biochim. Biophys. Acta 138, 186 (1967). 68. J. M. Ravel, R. D. Mosteller, and B. Hardesty, Proc. N a t l . Acnd. Sci. U.S. 56, 701 (1966). 69. S. Y. Lin, R. D. Mosteller, and B. Hardesty, J. Mol. B i d . 21, 51 (1966).
Ribonucleic Acids and Information Transfer in Animal Cells A. A. HADJIOLOV Biochemicat Research Labora tory, Bulgarian Academy of Sciences, Sofia, Bulgaria I. General Considerations . . . . . . . . . 11. Remarks on the Methods of mRNA Identification . . A. Isolation of Native RNA . . . . . . . B. Molecular Characterization of RNA . . . . . C. Mononucleotide Composition and Base Sequences of RNA 111. The Sequential Synthesis of RNA . . . . . . IV. Nuclei-the Site of mRNA Synthesis . . . . . A. Molecular Characteristics of nRNA . . . . . B. Kinetics of nRNA Labeling-the First Products of Genetic Transcription . . . . . . . . C. Stable nRNA with Messenger Characteristics . . . D. Tracing of Messengers among Nuclear RNA’s . . V. Cytoplasm-the Site of mRNA Exprcwion . . . . A. Molecular Characteristics of Cytoplasmic RNA . . B. Labeling and Turnover of Cytoplasmic RNA . . . C. Mononucleotidr Composition. Cytoplasmic D-RXA’ . D. Stimulation of Cell-Free Polypeptide Synthesis . . E. Cytoplasmic Carriers of mRNA . . . . . . VI. Synopsis . . . . . . . . . . . . A. The Orthodox Interpretation . . . . . . B. One Plausible Unorthodox Interpretation . . . . References . . . . . . . . . . .
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196 198 198 199 201 201 205 205 206
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It is no longer a question that genetic information is mediated by specific base sequences of RNA chains. Research on the role of ribosomes in protein synthesis seemed for some time to indicate that rRNA is the carrier of genetic information (1). However, the unifying analysis by Jacob and Monod ( 2 ) of experiments with induced and phage-infected bacteria indicated that genetic information is transferred by a particular group of RNA molecules, termed messenger RNA. Consequently, a rather vague structural role was assigned to ribosomes and rRNA, the
’Abbreviafions used: RNP, ribonucleoprotein; D-RNA and R-RNA, RNA’s identical in their mononucleotide compositions to DNA or ribosomal RNA (see Georgiev in Volume 6 of this Series). 195
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base sequences in the latter being conceived as devoid of any genetic significance ( , 2 , 3 ) . Originally applied to bacteria, the messenger hypothesis was soon extended to animal cells, for which it was agreed that the same basic mechanisms operate. It was soon realized, however, that some of the postulated features of mRNA, i.e., metabolic instability, are not always observed in animal cells ( 4 ) . In the last few years, numerous studies have been carried out with animal cells, in which the assumed characteristics of mRNA were sought. Although significant progress was made, the isolation and chemical identification of the suspected mRNA has not yet been attsined (see chapter by Chantrenne e t al. in this volume). However, several enigmatic coincidences, which point to the close relationship between mRNA and rRNA, emerged from these investigations. I n this paper, the evidence on the physical and chemical characteristics, the biosynthesis, and the mode of operation of the RNA molecules likely to be the messengers peculiar to animal cells is reviewed. The author takes the liberty of proposing some unorthodox interpretations in the hope that they will stimulate new experimental approaches and further theoretical evaluations. The important findings concerning the genetic code and the basic mechanisms of protein synthesis obtained in studies with synthetic polynucleotides and viral RNA are not discussed.lB Their application to the course of events in the intact cell is considered precarious. The interaction of external messengers with ribosomes or endogenous messengers poses several problems beyond the scope of this essay. I n view of the extensive critical survey of the literature on mRNA made recently by Singer and Leder ( 5 ) ,we refer mainly to papers that appear to be of importance for the present approach.
1. General Considerations Animals belong to the group of eukaryotes, which implies the existence of basic differences from prokaryotes in the mechanisms of genetic information storage and transfer. The following features of animal cells appear to be of particular importance when RNA-mediated information transfer is envisaged. 1. The structure of the genetic material constituting the chromosomes of animal cells appears to be radically different from that of bacteria (6, 7 ) . Although the linear arrangement of genes along the chromosome is a classical fact, it does not seem that enough evidence has been " S e e article by Woese in this volume.
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presented for an “operon” type disposition. Moreover, it has been proved that closely related characters (for example, the two polypeptide chains of an enzyme molecule) may be situated in two distant chromosome loci and even on two different chromosomes [see ( g ) ] . If substantiated, these observations indicate that monocistronic messengers are more likely in animal cells. 2. Most animal cells are highly specialized in their structure and function. Accordingly, only a restricted part of the entire genome (probably about 2-5%) is transcribed during the life cycle of a differentiated animal cell. The possible role of histones as gene “repressors” has heen discussed (9, 10), but their specificity still remains to be substantiated experimentally.”) I n any case, histones stabilize the structure of chromosomes and the switching of genes “on” and “off” is likely to be more complicated than in bacteria. Even during embryogenesis, the expected interpolation of newly transcribed information is not easily observed. Instead, unmasking of preexisting stable “messages” seems to play a substantial role in development (11, 12). 3. Animal cells exist in a homeostatic milieu. Consequently, the needs for the synthesis of new types of mRNA are limited and a stable protein pattern is typical for most cells in the adult organism. Thus, regulation of mRNA synthesis is expected to be primarily directed toward quantitative rather than qualitative requirements. Only in a few cases has it been possible to connect the observed variations of enzymatic activity with an increased synthesis of enzyme molecules, and even in these cases (for example, tryptophan pyrrolase) only a five- to tenfold increase of enzyme level is recorded after induction. Accordingly, the relative role of regulatory mechanisms operating beyond the gene level would be increased. 4. The rigidity of the protein pattern in animal cells is correlated with the existence of stable messengers. Observations with enucleated cells indicate a stable protein synthesis persisting for several days and obviously independent of the continuous nuclear supply of short-lived mRNA (11, 1 3 ) . Experiments with actinomycin D (even neglecting thc general toxic effects of this drug) reveal that synthesis of total liver protein (14) and different liver enzymes (15) persists for hours and even days after inhibition of RNA synthesis. The stability of mRNA for specific proteins is well documented for reticulocyte hemoglobin ( 4 ) ’ ~ and silk gland fibroin (16, 1 7 ) . 5. Compartmentation of transcription and translation sites in the animal cell implies the existence of an irreversible flow of information ”See article by Hnilica in this volumr. “See article by Chantrenne eI (11. in this volume.
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from the nucleus into the cytoplasm. The flow of ribosomes required in the translation process is also irreversible (18).If ribosomes are actively engaged in the release of mRNA from the DNA template, as in bacteria (19), each ribosome can participate only once in this process. If not, unprotected mRNA would have to cross a long distance through the karyoplasm quite exposed to nuclease attack. It has been shown (20) that ribosomes of nucleated cells are markedly heavier (80 S) than those of bacteria and blue-green algae (70 S). The difference in size of rRNA’s in eukaryotes and prokaryotes is also well known. This enigmatic extra mass in animal cell ribosomes and rRNA suggests the existence of basic dissimilarities in the mechanisms of genetic information transfer.
II. Remarks on the Methods of mRNA ldentificution Work with biopolymers has made it necessary to depart from the chemical criteria of a pure compound, i.e., analysis of structure and reconstitution by synthesis. Instead, a substance found to be homogeneous by physicochemical methods is identified by its particular function. For mRNA, a single and chemically defined RNA molecule would direct, in a heterologous ribosomal system, the synthesis of a protein molecule with known structure. Since this functional identification of mRNA has not yet been achieved (see chapter by Chantrenne et al. in this volume), the term “messenger” RNA has been used to designate various RNA fractions possessing one or more traits presumed t o be inherent in this type of molecule ( 2 ) . However, it becomes increasingly clear that all the criteria used to identify mRNA, such as rapid labeling, nucleotide composition, hybridization with DNA, stimulatory activity in cell-free polypeptide synthesis, provide only indirect evidence ( 5 ). Moreover, with all these criteria, the burden of the proof is dependent on the methods of RNA isolation and characterization. Therefore, a brief discussion of some methodical imperfections seems to be of particular importance for our understanding of the genetic role of cellular RNA.
A. Isolation of Native RNA The introduction of the phenol technique (see Georgiev in Volume 6 of this series) has made possible the isolation of “native” RNA. However, it soon became evident that in several cases this native state is more apparent than real. I n fact, nuclease degradation is a very important factor when any one of the present methods of cell fractionation and subsequent RNA isolation is considered. A few phosphodiester bond breaks per molecule will result in shorter polynucleotide chains whose relation to the original RNA molecule is difficult to establish. This is
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illustrated by the controversy as to the intact nature of rRNA chains and, although convincing evidence has been presented ( 2 1 ) , some doubts still persist ( 2 2 ) . The isolation of “stable” rRNA in a truly native state requires direct homogenization of cells with phenol ( 2 1 ) . Obviously, such a requirement should apply even more rigorously to mRNA, which is thought not only to be more “unstable,” but to represent a small part of the total RNA. Thus, it is likely that in any procedure involving the previous fractionation of cell structures (nuclei, polysomes, etc.) , the isolated mRNA represents a more or less degraded product. Further, it is a general observation that preparations of RNA are unstable during storage. “Spontaneous” degradation is usually invoked, although there is no apparent chemical reason for such degradation. More probably, some nucleases resist phenol treatment ( 2 3 ) and contaminate the RNA preparations. Our own experience indicates that introduction of sodium dodecyl sulfate and polyvinyl sulfate (or bentonite) is helpful, but does not by itself remove all nuclease contaminants. Repeated deproteinization (involving several chloroform steps) yields the best RNA preparations. A routine test involves incubation of RNA solutions a t 37°C and subsequent analysis by agar gel electrophoresis. Our best preparations remain stable for 24 hours without marked changes in the electrophoretic pattern. Evaluation of nuclease contaminants in RNA samples is highly desirable, but appears lacking in most studies on mRNA. Yet, such control is critical with RNA samples suspected to contain mRNA, since methods of further characterization are often of long duration.
B. Molecular Characterization of RNA Density gradient centrifugation is the most widely used method for
RNA fractionation. However, important limitations exist when a minor fraction, like mRNA, is considered. These include: interaction with the major rRNA fractions, formation of aggregates with contaminants like DNA or polysaccharides, irregular influences of the medium (pH, ionic strength, Mg2+ etc.) , cross-contamination of RNA fractions on the gradient [cf. ( 5 ) ] .Sucrose is almost exclusively used as the gradient material, but trials with other materials and varying media are necessary prerequisites for the adequate identification of a minor RNA fraction. Thus, uncertain S values can be obtained with RNA’s of a higher molecular weight than rRNA under different conditions (24). These limitations justify the search for alternative methods of RNA fractionation. Gel electrophoresis may be such a method. Agar gel electrophoresis was originally used as a qualitative method of RNA fractionation, the
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separate bands being localized by pyronine staining (25, 2 6 ) . Recently, Tsanev and Staynov (27) developed a new technique for the direct UV densitometry of dried agar gel electrophoregrams. This technique allows the localization and quantitative estimation of the different RNA fractions (28). The RNA patterns are identical with those of density gradient centrifugation, and an almost linear relationship between S 30 -
TMV RNA
25 20 -
Liver [I8 S]
I5 10-
5-
I Mobility, centimeters from start
sRNA I
,
1 2 3 4 5 Mobility, centimeters from start
FIG.1. Comparison of RNA fractionation by agar gel electrophoresis and drnsity gradient centrifugation. Lefl : Agar gel electrophorcsis pattern of rat liver cytoplasmic RNA’s. The absorbance is recorded by direct UV densitometry of the dried electrophoregram by the method of Tsanev and Staynov ( 2 7 ) . Right: Corrrlation between sedimentation coefficients of different RNA fractions and their elcctrophoretic mobilities in agar gel. [From Hadjiolov et al. (29).1
electrophoretic mobility and S value is found (29) (Fig. 1 ) . Similar results were obtained by the use of polyacrylamide gels ( 3 0 ) .Although most of the pitfalls of density gradient centrifugation mentioned above are likely to intervene in gel electrophoresis, comparative studies permit a more precise identification of mRNA. Another method of RNA fractionation is chromatography on methylated albumin-kieselguhr columns [see (31)]. The results obtained by this technique are not directly comparable with those from the first two methods, and it seetns that its resolving power is more limited. On the other hand, the influence of base composition on the fractionation of nucleic acids has been found helpful in the isolation of RNA fractions likely to represent mRNA (see below). I n brief, the methods available for the molecular characterization of RNA are far from perfect. The adequate identification of a minor fraction like mRNA requires the Concomitant use of more tha,n one technique in order to minimize the artifacts.
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C. Mononucleotide Composition a n d Base Sequences of RNA The significance of nucleotide composition as a criterion of mRNA is limited because the composition of the active genes is unknown. The designation of an RNA fraction as “DNA-like” is often controversial. The artifacts that may arise when the mononucleotide composition of RNA is studied by P32distribution are discussed below. RNA-DNA hybridization (see McCarthy in Volume 4 of this series) has only recently been applied to the identification of RNA from animal cells. The use of different hybridization methods makes comparison difficult. Application of these techniques to genetically defined systems has yielded spectacular results for the DNA cistrons involved in rRNA synthesis [see Perry ( 3 2 ) ] .Unfortunately this is not yet the case with mRNA. Further, the chemical basis of the method has not been clarified and numerous controls are needed in order to allow a correct quantitative evaluation. The length of the RNA chain involved in specific base pairing is not known, but it seems to be much shorter than that of the added molecule. Thus, in the case of rRNA from HeLa cells, about 3050% of the hybridized material was recovered in the form of acidsoluble oligonucleotides ( 3 3 ) . It is evident that the shorter the chains involved, the higher the hazard of unspecific hybridization. This possibility is strengthened by the observation that DNA may form hybrids with poly U, poly G, or poly I,G, with only 10-50 residues engaged in base pairing (34). The presence of nuclease contaminants in RNA, discussed above, combined with a higher susceptibility of mRNA to enzyme attack, may thus become critical in the quantitative estimation of hybridization tests.
111. The Sequential Synthesis of RNA Labeling experiments of short duration have been, by definition, a crucial approach in the search for niRNA in animal cells. A theoretical evaluation of the mechanisms of RNA synthesis is consequently of particular importance for the adequate interpretation of these studies. The prohlem of the sequen tial synthesis of single-chain biopolymers arose from studies on protein biosynthesis. The theoretical grounds have been discussed in detail and experiments with different proteins have consistently shown that the growth of polypeptide chains is vectorial, proceeding from the N-terminal toward the C-terminal end [see (35) for references]. It is now firmly established that animal RNA molecules are composed of single polynucleotide chains (21, 2.2). Studies with the
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Escherichia coli phage 4x174 (36) and the Bacillus subtilis phage SP 8 (37) provide conclusive evidence that only one strand of DNA is copied in the transcription process in viva Extension of these results to enzymatic systems has shown that, when native phage DNA is used as template, only one of the two DNA strands is transcribed (38, 3 9 ) . General considerations indicate that asymmetry of transcription is a common mechanism operating in vivo (40). Further, experiments with enzyme systems (41, 4.2) and with E . coli cells (43) indicate that the synthesis of RNA is also vectorial and procecds by the stepwise addition of nucleotides from the 5’ phosphate end
-
Direction of chain growth
5’ c _
Labeling gradient
FIG.2. Vectorial synthesis of RNA chains. New nucleotides are attachrd a t the 3’-end of the polynucleotide chain. B = P u or P y base.
towards the 3’ hydroxyl end of the polynucleotide chain (Fig. 2 ) . The mechanisms determining the polarity of transcription are not clear, but they may involve a preferential binding of RNA polymerase to the 3’ hydroxyl of the template DNA (44). Asymmetry and polarity of the transcription process provide the necessary basis for considering the problem of sequential synthesis of RNA molecules. In the process of rRNA synthesis in animal cells, the following successive steps may be considered: assembly of free nucleotides on the DNA template; release of the rRNA precursor; transformation into 28 S and 18 S rRNA; nucleocytoplasmic transfer of the two rRNA’s [see (SZ)]. The distribution of the label expected along the polynucleotide chain of these RNA species after a short period of labeling is outlined in Fig. 3. It is evident that in any case of RNA synthesis studied with labeled precursors there must be a time period during which there is a nonrandom distribution of the label along the polynucleotide chain. This time period may be designated as the completion time of RNA. As a consequence, the following correlations may be expected in short-term labeling experiments. 1. Uneven labeling of a given nucleotide residue along the polymer chain will reflect the completion time of RNA molecules if the specific
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RNA AND INFORMATION TRANSFER I N ANIMAL CELLS
activity of the free nucleotidc in the precursor pool is kept constant. If this condition is not satisfied, then uneven labeling of RNA will also arise from variations in the labeling of the precursor nucleotide. However, this will hold true only if the completion time is comparable with the labeling time. Accordingly, in both cases, uneven labeling along the polynucleotide chain indicates that the completion time is commensurate with the labeling time. 2. Uneven labeling of a nucleotide residue along the polynucleotide chain would be detected not only during the actual completion timc of RNA molecules, but as long as the amounts of partly labeled and Nuclear precursor RNA P
3‘-end
5‘-end
m ‘vvv\
-
5L.iwww
3‘ 5‘
>--1 B
3‘
5b-3‘5;
P
3‘
___zMj
18 S RNA
28 S RNA
28 S RNA
FIG.3. Model for the sequential synthesis of nuclear precursor RNA and the expected distribution of label in short-term labeling experiments. The straight lines represent the unlabeled segment of the RNA chain; the zigzag lines, the labeled segment. A single split of the precursor is envisaged to give the product 285 and 18s RNA. Depending on whether the split occurs in A or B, the ratio of the specific radioactivities of lSS/ZSS RNA’s will be lower ( A ) or higher ( B ) than unity.
randomly labeled RNA molecules are comparable. As a consequence, the apparent completion time could be severalfold longer than the actual one. Obviously the time necessary for the precursor-product transformations and the nucleocytoplasmic transfer of RNA molecules only prolongs the time period during which uneven distribution of the label along the RNA chain may he detected. These considerations show that the completion time of RNA molecules is a very important paramcter to be determined in studies on RNA synthesis. Conceivably, its importance may become crucial in the shortterm (“pulse”) labeling experiments now widely used for mRNA identification. The labeling time used in such studies with animal cells
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varies from 5 minutes to a few hours. It is considercd that thr radioactive pulse should comprise 1/10 to 1/20 of the generation time of the cells studied. What then is the conipletion time of RNA molecules? Is it commensurate with the labeling time used in studies on niRNA? No direct answer to these questions is available. Some information may be derived from data on the sequential synthesis of proteins. Since any RNA molecule (including mRNA) is likely to participate in the production of several protein molecules, it seems reasonable to assume that the rate of protein synthesis will exceed the rate of RNA synthesis. Thus, studies on hemoglobin synthesis in intact reticulocytes show a definite labeling gradient after 7 minutes of incubation for the /3 chain and after 16 minutes for the LY chain (45).Experiments with perfused rat liver fixes the completion time of albumin molecules a t 2-5 minutes ( 4 6 ) . Similar figures were obtained for RNase synthesis in pancreas slices (4'7),lysozyme formation in minced hen's oviduct ( 4 8 ) ,etc. Where 100600 amino acid residues are involved, an average stcp-time of 0.5 to 1 second may be calculated. If the rate of RNA synthesis is taken as equal to that of proteins, then the completion time of a precursor: RNA molecule of 1.0 t o 2.0 X lo' nucleotides will be about 80-330 minutes. On the other hand, the experiments of Goldstein et al. (43) with E . coli cells labeled in vivo with uridine-C14 established a step-time of RNA synthesis of 13 seconds a t 0".Extrapolation of these results to 37"' taking into consideration the known temperature coefficients of enzyme reactions, gives a step-time of about 0.5 second and an estimated completion time of the precursor RNA envisaged of about 80-160 minutes. These figures for the completion time of RNA molecules in animal cells are unexpectedly high. Rough estimates of the actual completion time of RNA molecules have therefore been made on the basis of the doubling time of growing cells (with respect to their RNA) . Approximate values of 2-10 minutes were obtained. If we multiply these figures by a factor of 10 in order to evaluate the apparent completion time of RNA molecules, then it may be expected that uneven labeling of RNA molecules would be observed with growing cells a t labeling times shorter than 20-100 minutes. At present, we do not know exactly the size of the genome involved in RNA synthesis. We also do not know if RNA synthesis is a continuous or an intermittent process. Therefore, the above approximations may as well be erroneous. Nevertheless, there is little doubt that in any brief labeling experiment, the completion time of RNA molecules and the possibility of uneven labeling should be taken into account. A particular aspect of short-term labeling cxperiments is the estima-
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tion of the base composition of rapidly labeled RNA from the distribution of PASamong the four nucleotides obtained a t alkaline hydrolysis. Alkaline hydrolysis of RNA, by displacing P32to the nearest nucleotide on the left, eliminates differences in the specific activities of the nucleotides arising from nonrandom labeling of the precursors. Accordingly, it is deduced that distribution of P32among the four 2’(3’) mononucleotides represents the composition of labeled RNA molecules [see ( 4 9 )1. This interpretation is correct on1y if the distribution of nearest-neighbor nucleotides is random along the whole length of the RNA molecule in question. Although there is now general agreement on the general validity of this postulate, remarkably little ev-dcnce has been obtained to support it. I n fact, a direct comparison of the labeling of 2’(3’) and 5’ mononucleotides obtained from the same rapidly labeled RNA has been made only with yeasts ( 5 0 ) , T2-phage infected E . coli ( 5 1 ) , Ehrlich ascites tumor cells ( 5 2 ) , and rat liver ( 5 3 ) . Indications of statistically random distributions of nearest-neighbor nuclcotides were obtained in the first three cases only. Moreover, when the complction time of RNA molecules is considered, the following possibilities should be noted: 1. The Pd2distribution among RNA nucleotides yields information on the composition of the RNA segment labeled during the P,32pulse. Likewise, deductions about the random distribution of nearest-neighbor nucleotides apply to the labeled segment only. 2. Changes in the labeling of a-phosphates of precursor nucleoside 5’-triphosphatcs with time result in uneven labeling of the different segments of RNA moleculcs. As a consequence, the different segments of the molecule will contribute to varying extents to the estimated overall composition.
IV. Nuclei-the
Site of mRNA Synthesis
A. Molecular Characteristics of nRNA General agreement has not yet been attained even on the major RNA species present in the nucleus [see (54, 55) 1. The present discussion is limited to those aspects likely to have some bearing on nuclear mRNA. Cold phenol treatment of animal tissues and cells extracts “cytoplasmic” RNA, while nuclei remain entrapped in the water-phenol interphase layer. Furtlier bricf cxtraction with hot phenol-sodium dodecyl sulfate brings the total (more than 90%) interphasc RNA into solution ( 5 5 ) . This “nuclear” RNA is represented by well-shaped 28 S and 18s peaks. Our own experience with brain, livcr, hypophysis, and ascites tumor cells confirms the good reproducibility of this technique. However, this approach cannot exclude either loss of nRNA or contanii-
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nation with cytoplasmic RNA. Isolation of RNA from pure nuclei obtained by centrifugation in hypertonic sucrose reveals again the main 28 S and 18 S peaks (56-58), but in this case the relative amount of 18 S RNA is lower and the evidence suggests that degradation of RNA during isolation of nuclei by this technique cannot be avoided ( 5 9 ) . Isolation of HeLa cell nuclei by subsequent hypotonic and detergent treatments results in the complete absence of the 18s RNA component (60). Recently, methods have been developed that allow the rapid isolation of pure nuclei by the use of nonionic detergents in the cold (6f-63).The RNA extracted from such nuclei contains 28 S and 18 S components in a ratio analogous to that observed with interphase "nuclear" RNA (52, 5 9 ) . Thus, there is little doubt that the bulk of true nRNA is represented by the 28s and 18s molecular species. However, since variations in their ratios are commonly observed, the influence of the following factors should be considered. 1. Loss of nRNA components may occur in any extended procedure for isolation of nuclei. Preferential loss of 18s RNA would account for the smaller amounts of this RNA observed in some experiments (57, 58, 60). 2. On the other hand, incomplete extraction of some nRNA components is a possibility in any mild procedure ( 5 5 ) . The importance of factors, such as DNA-RNA binding, interaction of RNA with histones and other proteins, entrapping of RNA in lipoprotein structures, remains unknown. 3. Several authors have observed that hot phenol treatment of RNA results in the selective degradation of 28 S RNA (28, 59, 6 4 ) . Heating a t 65" of isolated nRNA (stable for several hours a t 40") results in the selective degradation of 28 S RNA ( 6 5 ) . The detailed studies of Applebaum e t al. (66) with RNA from Hyalophora cecropia wings demonstrate the nonenzymatic conversion of 28s into 18s RNA on heat treatment. All these findings clearly indicate that uncertainties concerning the major nRNA components are likely to have repercussions on all attempts to characterize nuclear mRNA.
B. Kinetics of nRNA Labeling-the
First Products of Genetic
Transcription
1. AUTORADIOGRAPHIC STUDIES
Experiments with various RNA precursors support the view that all types of RNA molecules are made in the nucleus [see (54, 67) for references]. Early attempts to localize the site of RNA synthesis within the nucleus showed that the label appears almost simultaneously in both
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the nucleolus and the karyoplasm [ cf. ( 6 7 )1. Such observations suggested an independent synthesis of these two topically distinct RNA types. Selective inhibition of nucleolur RNA labeling by low doses of actinomycin D and other independent evidence rule out a major RNA flow from karyoplasm to nucleolus ( 3 2 ) , but migration of RNA in the opposite direction is not excluded. Thus, UV microbeam inactivation of the nucleolus reveals that at least 30% of karyoplasm labeling is dependent on the nucleolus (68). Recently Amano et al. ( 6 9 ) , in a detailed kinetic study of nRNA in liver and pancreas pulse-labeled with cytidine-H7, found the decay of RNA labeling in both karyoplasm and nucleolus to be essentially parallel. Yet the low resolving power of the light microscope limits the quantitative evaluation of the results. Rapid labeling of nRNA followed by electron microscopic autoradiography reveal a more intense labeling of nucleolar RNA as compared to karyoplasmic RNA (70, 7 1 ) (Fig. 4). One plausible interpretation is that labeling of karyoplasmic RNA is a transitory event; i.e., there is no accumulation of labeled RNA in the karyoplasm, but since the identity of the RNA species involved is not established, a correlation with biochemical studies is difficult. 2. THEFREENUCLEOTIDE POOL IN NUCLEI
The interpretation of RNA labeling depends on the study of the free nucleotide precursor pool [see (6, 5 4 ) ] . Hence progress in this field is disappointingly slow. A major obstacle lies in the fact that free nucleotides are washed out during isolation of nuclei. There is no doubt that nuclei contain all the nucleotides required for RNA synthesis, but their exact level is unknown. Rapid equilibration of nuclear precursor pools is a common presumption, but in fact, the dilution factor may vary a t nucleolar and karyoplasmic sites of RNA labeling. The importance of the metabolisni of the precursor is likely to be increased when incorporation of a common precursor into different RNA nucleotides is studied. For example, by following adenine-C14 incorporation into adenine and guanine of nuclear and cytoplasmic RNA, Harris (72) reached the eonclusion that a rapid degradation of RNA takes place inside the nucleus. Yet, these results could be also explained by a delayed metabolism of guanine nucleotides a t the nuclear sites of RNA synthesis ( 7 3 ) . The existence of specific nucleotide pools in nuclei is indicated by the finding that P32labeling of GMP derived from nRNA of Ehrlich ascites tumor cells is twelvefold lower than that of the other three nucleoside 5'-monophosphates ( 5 2 ) . Another aspect of this problem is the possible rate-limiting role of the free nucleotide supply in RNA synthesis (7'4). Indeed we do not
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209
RNA AND INFORMATION TRANSFER I N ANIMAL CELLS
know whether nuclei are dependent on mitochondria for their supply of precursor nucleotides. Oxygen-dependent formation of ATP and other nucleotides was reported in thymus nuclei (9, 75), though the universal occurrcnce of this phenomenon remains to be proved. On the other hand, the studies of Reid e t al. (76) demonstrate in liver nuclei a high level of enzymes involved in the conversions orotate + UMP + UDP + UTP
Thus, it is possible that nuclei are self-sufficient in their supply of RNA precursor nucleotides. I n this casc, the supply of nucleotides would be even more limited since overall nuclear synthesis of nucleoside 5’-triphosphates proceeds a t a markedly lower rate than does cytoplasmic synthesis. The nucleotide level may bccome critical when we consider some nRNA’s, suspected as messengers, that are characterized by exceedingly high turnover rates.
3. RAPIDLYLABELED HIGHMOLECULAR WEIGHTnRNA The incorporation of a labeled precursor in a short time period (“pulse”) is widely used in the search for mRNA although the validity of this criterion is limited. The first ultracentrifugation studies on nRNA of animal cells revealed an initial appearance of the label in a broad peak with a maximum a t about 45s (56, 77, 78). Although suspected as mRNA, it became soon evident that 45 S nRNA is in fact a precursor of rRNA [see review by Perry ( 3 2 ) ] .Nevertheless, the picture of a simple precursor-product relationship is complicated by several observations that seem to indicate that molecules that are similar in size but “DNA-like” in composition are also rapidly labeled. Actually, since the pioneer work of Davidson et al. (79, 80) [extended to a variety of animal cells (cf. 52, 81-83)], it has been known that the nucleotide composition of rapidly labeled nRNA is more or less shifted toward that of DNA. More recently, several authors isolated the heavy rapidly labeled RNA fractions of density gradients and determined their nucleotide composition. These data are summarized in Table I. As can be seen, the rapidly labeled RNA in the 40-50 S zone displays wide variations in base composition. The G+C/A+U ratios oscillate between values of 0.8-0.9 (as in DNA) to 1.5-1.6 (as in rRNA). Accordingly, the rapidly labeled nRNA was considered by different authors ~
FIG.4. Distrihution of rapidly lahclrd R N A in the nucleolus and the knryoplasm of cultured kidney cells. Labeling with ~ r i d 1 n e - Hfor ~ 10 minutes. Localization of thc label by electron microscopic ;iulor:xdiography. [From Granboulan and Gram boulan (70).]
E
TABLE I MONONUCLEOTIDE COMPOSI~O OF N RAPIDLYLABELEDHIGH MOLECULAR WEIGHTNUCLEARRNA
Source HeLa cells
Ehrlich ascites tumor cells Immature erythroid cells
Mouse plasmocytic sarocma Rat liver
Nuclear fractionb Phenol 60" Phenol 60" Phenol 60" Phenol 60" Phenol 60" Phenol 60" Phenol 60" Phenol 60" Phenol 60" Nuclei Phenol 60" Phenol 60" Phenol 60" Phenol 60" Phenol 60" Phenol 60" Phenol 60" Phenol 60" Phenol 60" Phenol 43-63" Nuclei
RNA fraction (S va1ue)c 45 60-80 30-45 45-70 >70 41-54 72-84 84-100 45
45 Total 45 50 50 30-45 30-45 45-75 45-75 >75 >75 35-45 >45 Total
Time of labeling (minutes)
40 40 5 5 5 30 30 30 60 180 30 120 15 120 30 120 30 120 30 120 30 30 5
0 OF
ANIMALCELLS=
Molar ratio (%) A
U
G
C
18 24 19.8 20.6 21.3 17.6 23.1 23.4 16 14 20.1 21.2 22.4 25.0 20.6 19.9 21.7 23.2 25.1 23.1 18.1 18.0 23.0
22 29 26.9 26.5 31.0 22.2 28.2 29.5 27 24 25.6 26.4 27.8 29.1 24.8 22.5 28.7 28.6 31.1 29.9 22.3 21.5 30.9
33 24 25.1 25.4 21.1 30.9 23.8 21.4 32 32 28.0 25.9 25.7 22.7 28.9 29.9 27.9 24.6 24.1 23.3 30.3 31 . O 25.6
27 23 28.0 27.3 26.4 29.1 24.7 25.5 25 30 26.3 26.4 23.9 23.0 25.7 27.7 21.7 23.6 19.7 23.2 29.3 29.5 20.5
G A
++ C/U
1.50 0.89 1.14 1.12 0.91 1.50 0.95 0.89 1.33 1.63 1.19 1.10 0.99 0.85 1.20 1.36 0.98 0.93 0.78 0.87 1.47 1.49 0.86
Reference
Rat liver
Xuclei Nuclei Nuclei Nuclei Nuclei Nucleoli Nucleoli Nucleoli
35 45 45 4.5 55 28 35 45
30 30 30 10 10 Directd Direct Direct
24.7 25.1 25.7 24.9 26.1 15.0 14.3 14.5
22.1 22.1 23.7 21.2 22.6 19.9 19.8 20.8
29.1 28.5 26.3 29.5 26.5 34.9 35.1 35.3
24.1 24.3 24.3 24.4 24.8 30.2 30.8 29.4
1.14 1.12 1.03 1.17 1.05 1.87 1.93 1.84
(91) (91) (91) (92) (92) (91) (91) (91)
;P
Z
U n
z
r
0
w
52 8
The mononucleotide composition is determined by P3*distribution analyses after various periods of labeling with inorganic p h 0 ~ p h a t e - P ~ ~ . a The RNA fractions obtained by hot phenol treatment represent the total cellular RNA. It is known that the rapidly labeled, high molecular weight RNA’s are confined to the nucleus (see Section IV, A). u, c Fractions obtained from the appropriate regions of sucrose density gradients. The sedimentation coefficient values are approximately 2 s estimated by the position of the RNA fraction analyzed, with respect to the 28 S and 18 S rRNA4components. d The composition of the respective RNA fractions, concentrated and purified by sucrose density gradient centrifugation, is determined 2 + by direct analyses. a
E
Z
E Brl
212
A. A. HADJIOLOV
as either D-RNA (93, 94) or a pure rRNA precursor (95). Since intermediate values are usually found, most authors consider the rapidly labeled 40-50s nRNA as a mixture of these two RNA types. I n any case, it is now clear that the first D-RNA molecules labeled are also bigger than are the bulk of nRNA molecules. The basic conclusions from these studies may be summarized as follows. a. A direct correlation exists between the growth rate of the cells studied and the base composition of the rapidly-labeled nRNA. Thus, nongrowing cells show a preferential labeling of D-RNA (87, 88), while only R-RNA is usually detected in growing cells (32, 95). Transfer of ascites tumor cells in the stationary phase of growth to a mineral medium does not cause a shift of rapidly labeled RNA composition ( 6 2 ) , but exponentially growing cells respond to a similar change in the medium by an increased D-RNA labeling (96, 97). A shift toward preferential labeling of R-RNA is observed when the growth rate is increased, as in phytohemagglutinin stimulation of lymphocytes (98) and in regenerating rat liver (9.2). b. Similar changes are obtained when RNA synthesis is inhibited by actinomycin D treatment. Thus, inhibition of 9&95% of RNA synthesis results in the almost exclusive labeling of D-RNA (32, 96, 99). A favored interpretation is that D-RNA synthesis is more resistant to actinomycin D than is the synthesis of rRNA precursors [cf. (%)I. c. When the time course of RNA labeling is followed, a gradual shift in composition of rapidly labeled nRNA from D-RNA towards R-RNA is recorded. This transition was well documented in studies on total nRNA of Cave cells (100) and 4 5 s nRNA of Ehrlich ascites tumor cells (86). d. Embryos of the anucleolate mutant of Xenopus laevis in which RNA synthesis is exceedingly slow, display a “DNA-like” base composition of labeled RNA even with very prolonged labeling times (101).I n this case, too, the initially labeled D-RNA is situated in the heavy region of density gradients (10.2). These observations suggest that a slower rate of overall RNA synthesis is correlated with a higher ratio of D-RNA in the population of rapidly labeled RNA molecules. Moreover, the initially labeled RNA molecules are invariably “DNA-like” and represent a more or less heterogeneous population situated preferentially in the heavy region of density gradients. Since this D-RNA is a likely candidate for a messenger, several attempts a t its further characterization have been made. One approach involves the detailed analysis of density gradient fractions heavier than rRNA precursors. Thus, in immature duck erythrocytes (87, 88, 103, 104) and in HeLa cells (84, 85), the rapidly
RNA AND INFORMATION TRANSFER I N ANIMAL CELLS
213
labeled RNA of 60 to 100s displays a “DNA-like” composition (see Table I ) . I n fact, the content of U is markedly higher than that of A, while both G and C are low. Experiments with DNase,2 pronase, and trypsin do not lower the high S values of this RNA, while various treatments, including Mg2+chelation or heating a t 80’, seem to indicate that a continuous polynucleotide chain is involved (24, 88). Sedimentation studies, confirmed by electron microscopy (104), indicate a molecular weight of about 5 to 7 X loF,i.e., a polynucleotide chain constituted of 1.5 to 2.0 X lo4 monomers. Another approach involves the fractionation of nRNA in the presence of hot phenol [see ( 5 5 ) ] .The rapidly labeled RNA in the fraction extracted a t 55-65’ shows a mononucleotide composition matching exactly that of DNA in liver and ascites tumor cells (55, 106). Somewhat higher G+C/A+U ratios for a similar fraction from liver have been found by other authors (107,108), while in a mouse plasmocytic sarcoma, Kempf and Mandel (89) have reported a G+C/A+U ratio of 1.49 for the 4 5 s RNA of the 43-63” fraction. As indicated above, it is likely that the observed variations reflect peculiarities in the rate of RNA synthesis within the different cells studied. It is interesting that with this method of RNA fractionation, the rapidly labeled RNA displays lower S values with an upper limit of 40-50S, and a clear-cut distinction from the 45 S rRNA precursor is not possible by this criterion. The reason for the observed discrepancies between the two fractionation methods is not readily evident. In the case of hot phenol fractionation, degradation of some RNA fractions is more likely to occur (see Section IV, A). Several attempts have also been made to obtain a cytologically controlled fractionation of nucleoli and karyoplasm. Although RNA degradation is an almost unavoidable hazard, it seems that new techniques (109, 110) allow a preservation of RNA sufficient to justify further studies. With this approach, Busch e t al. (91, 111, 112) obtained evidence for the nucleolar location of the 45 S rRNA precursor, while tracer kinetics confirmed the nucleolar site of the 45 S +-35 S + 28 S RNA conversions. Yet, contrary to the commonly favored scheme [see ( 3 2 ) ] ,formation of 18 S nRNA was located in the karyoplasm and a particular 45-55 S precursor RNA was envisaged (92, 113). The G+C/ A+U ratio of this rapidly labeled RNA fraction in liver is 0.86, which relates it to the “DNA-like” RNA found by other workers. Finally, a note of warning seems appropriate: rapidly labeled nRNA is not invariably found as a high molecular weight fraction. For ex-
‘Experiments with thymidine-HY indicatc that some DNA is associated with rapidly labeled RNA in the heavy regions of density gradients (106).
214
A.
A. HADJIOLOV
ample, an nRNA fraction of liver extracted a t pH 8.3 showed a welldefined rapidly labeled “DNA-like” component of 8-16 S (114). Even if degradation has taken place, it must have occurred a t specific sites on the high molecular weight RNA molecule. I n summary, most of the results discussed in this section indicate that RNA molecules of high molecular weight and a “DNA-like” composition are labeled more rapidly in animal cell nuclei than are other types of RNA that are similar in size but t h a t have an “rRNA-like” composition. The site of labeling is likely t o be the karyoplasm, while kinetic studies reveal that the labeling of the “DNA-like” nRNA invariably precedes th at of the nuclear rRNA precursor. These findings are discussed in section IV, D. 4. HYBRIDIZATION OF RAPIDLY LABELED nRNA As shown by the evidence given above, karyoplasmic DNA is the most likely candidate for the site of mRNA transcription. Since the genes involved in mRNA transcription are presumably more numerous than those for rRNA, hybridization studies are expected to yield valuable information on rapidly labeled nRNA. Unfortunately, the technical difficulties inherent in this technique (see Section 11, C) have not been adequately controlled in most studies. On a comparative scale, the highest levels of RNA hybridization are obtained with rapidly labeled RNA fractions of animal cells. However, even with very short pulses, the amount of labeled RNA hybridized a t saturation is in the range of 1-5% of the input labeled RNA (104, 115). Such results seem to indicate that in animal cells a limited portion of the genome is involved in the production of numerous identical mRNA copies (see Section I). Yet, the quantitative estimation of hybridization experiments may be misleading. Thus, in the case of the anucleolate mutant of Xenopus Eaevis, annealing of labeled RNA with five successive portions of DNA reach a plateau a t about 12% hybridization of the input RNA (102).Since no rRNA is expected to be labeled in this organism, failure to obtain 100% hybridization is not easily understood. Further, a clear-cut distinction between rapidly labeled rRNA precursors and D-RNA seems difficult. For instance, it was reported that labeled 45-54s RNA from normal and actinomycin D-treated mouse L fibroblasts shows identical hybridization and competition by rRNA (116). In addition, in the only case in which the hybridized rapidly labeled RNA was analyzed further, large discrepancies in base composition were found, depending on whether direct estimation or P32distribution was used for the determination (104).
RNA AND INFORMATION TRANSFER IN AKIMAL CELLS
215
C. Stable nRNA with Messenger Characteristics The studies outlined above disclosc that neither of the main nRNA components (28 S and 18 S) may be ronsidcrcd to be rapidly labeled. However, several attempts were .made t o study the other traits expected from mRNA as displayed by different nRNA fractions. It has long been known that total RNA from purified nuclei has a lower G+C/A+U ratio than the bulk of cytoplasmic RNA [see (55) for refs.]. Values of 1.05 to 1.15 werc reported with nuclei purified by centrifugation in hypertonic sucrose (109, 117). Although wide variations in the molar ratio of individual nucleotides were reported, these results suggest the existence of stable D-RNA in nuclei. Recently, several workers have described the isolation of such D-RNA from nuclei. I n thymus nuclei, it was found t o represent about 6% of nRNA (118). Phenol extraction of nRNA with a gradual increase of either temperature (119) or pH (120) was reported to yield a D-RNA in substantially higher amounts (up t o 30% of total nRNA). A wide size distribution of this D-RNA in sucrose density gradients was correlated with the expected size heterogeneity of mRNA ( 1 2 0 ) , but the initially reported size heterogeneity of this D-RNA fraction is likely to be a degradation artifact. Further studies by the same workers (106, 114) indicate that the bulk of this D-RNA constitutes a broad peak with a maximum a t about 1 8 s . I n fact, when degradation is minimized by reduction of the extraction time a t 65" and the addition of Mg2+ and polyvinyl sulfate, a rather narrow 18s peak constitutes the bulk of nuclear DRNA (65, 108) (Fig. 5 ) . Thus, if this fraction is to be considered as mRNA, one should bear in mind that this mRNA is not heterogeneous in size and its peak coincides with the peak of 18s rRNA. Further, although this fraction is designated as D-RNA it is not yet clear how "DNA-like" this RNA fraction is.3 The G+C/A+U ratios reported by Georgiev e t al. [cf. ( 5 6 ) ] for rat liver and Ehrlich ascites tumor cells fall in the range of 0.754.85. However, markedly higher values are reported by others who attempted the isolation of this DRNA fraction. Thus, by the use of a temperature gradient extraction, Morrison and McCluer (122) obtained a small amount of a D-RNA with a G+C/A+U ratio of 1.07. In another approach, by the gradual extraction of nRNA with EDTA-sodium acetate, Kimura et al. (123) obtained five nRNA fractions, but again the D-RNA fractions had ratios of 1.00 and 1.15. I n the case of canine pancreas Yang and Dickman
+
+
3 T h e G C/A T ratios of DNA from most animal tissues and cells vary between the relatively narrow limits of 0.65 and 0.80 (121).
216
A. A. HADJIOLOV
(124) isolated two nRNA fractions extracted with phenol a t 50-65” and at 65-80’, respectively, but showing ratios of 1.47 and 1.38. Our own studies (65) with liver invariably showed ratios for this D-RNA fraction in the range of 1.05-1.20. Determination of the base composition of the 18s peak of D-RNA yielded similar results (65, 114). Finally, values of 1.02-1.30 were reported for different extranucleolar fractions of
Mobility. centimeters from start
FIG.5. Agar gel eleclroplioresis pattern of a rat liver “DSA-like” nRNA fraction. The RNA is extracted in the temperature interval of 6 M 5 in the presence of phenol, 0.01 M Mg“, and polyvinyl sulfate [see (5511. The G C/A U ratio of this fraction is 1.12. Note the good homogeneity of the 18s component. [From
+
+
Venkov and Hadjiolov (G5).I
rat liver (113). Thus, it would seem that fractions of nRNA with a directly determined G+C/A+U ratio lower than 1.00 are not easily obtained. It .may be argued that D-RNA should not be exactly “DNAlike,” but it is also clear that in several cases the base composition of nuclear D-RNA fractions is closer to that of 18s rRNA than of DNA. Stimulation of cell-free polypeptide synthesis (called “template activity” in some studies) should represent a most functional assay for mRNA. I n general, the stimulatory activity of nRNA is ten- to twentyfold higher than that of cytoplasmic RNA (125-12828).This activity is associated with 18s (126) or 8-16s (114, 127) nRNA components. The latt.er figures are more likely to be due to partial degradation of RNA during isolation. Nuclear D-RNA fractions have a higher stimulatory activity in the E . coli cell-free system than the respective RRNA fraction (127, 128). In fact, stimulation by D-RNA was only
RNA AND IXFORMATION TRANSFER I N A K I M A L CELLS
217
thrcc times higher than by R-RNA and the pattern of the stimulated incorporation of different amino acids was similar (12%). These results indicate an unspecific stiniulation of polypeptide synthesis rather than thc formation of a defined product (see also Section V, D ) . Evidence from recent experiments indicates that stable D-RNA is associated with proteins to constitute a homogeneous population of R N P particles of about 40 S [see ( 5 6 )] .4 Aside from the “DNA-like” composition of the constituent RNA, these particles appear to be identical with the smaller subunit of ribosomes. The existence and the identity of ribosomes and ribosomal subunits in nuclei is a most debated subject, the bulk of the evidence backing the vicw that they are true nuclear components [see (32, 67‘)]. Recently, McCarty e t al. (129) not only have confirmed their presence in liver nuclei, but have adduced evidence that nuclcar and cytoplasmic ribosomes and ribosomal subunits are itlcntical by all criteria tested, including the base composition of the constituent RNA. Thus, the distinction between 40 S R N P particles containing 18 S D-RNA and those containing 18 S R-RNA is not olivious. I n brief, these results show that when degradation is adequately minimized, the nRNA fractions assunicd to be stable mRNA represent n rather homogeneous species of 18s RNA molecules. It is perhaps helpful to add that even in the anucleolate mutant of Xenopus laevis the stable D-RNA appears as a well-shaped 18 S peak (102).
D. Tracing of Messengers among Nuclear RNA’s The correct interpretation of experimental results on nuclear mRNA should await the accumulation of further evidence. This is easily understood when one considers that, within the last year, one paper appeared in which i t is stated that 18 S RNA is not present a t all in nuclei (60), while in another, 185 RNA is considered as not only a major nuclear species but the true messenger ( 5 5 ) . Two nRNA species are the likely candidates for mRNA: the rapidly labeled high molecular weight RNA and the stable 18s D-RNA. The various possibilities are briefly considered and some alternative explanations put forward. The existence of a rapidly labeled high molecular weight RNA ’Throughout this article, the d u e of 40 S is assigned to the smaller ribosomal subunit (existing free or as a constituent of the ribosome) while the value of 60 S is used for the larger ribosomal subunit. The same approximation is applied to drsignate the 28 S and 18 S nuclear and cytoplasmic RNA’s. These designations arc operational, and minor variations in experimentally determined S values are ronsidcred as being due to configurational changes or to methodical modifications, the elucidation of which requires further studies [see reviews in (22, .%’, S2)l.
218
A. A. HADJIOLOV
fraction has been estahlished beyond reasonable doubt, but its fate and its relation to rRNA and mRNA are controversial. Two basic interpretations have beeen considered. 1. The high molecular weight RNA niolecules are marked by an exceedingly high turnover rate with a half-life of about 2-20 minutes (24, 88, 100). They are made and degraded inside the nucleus. Therefore, the function of this D-RNA remains obscure. This view was discussed extensively by Harris (64, ISO), and recent more direct evidence corroborates and specifies further this interpretation (24, 88). However, although rapid labeling of D-RNA is consistently observed, unequivocal proof of rapid degradation has not been presented. On the contrary, experiments with actinomycin D indicate that either there is not a preferential degradation of D-RNA (88) or i t is more stable than rRNA precursor (89). 2. The high molecular weight RNA is a precursor of cytoplasmic mRNA and because of its large size is suspected to represent a polycistronic mRNA (103, 104). However, tracer kinetic data rule out a direct precursor-product relationship (24, 88). I n addition, the operation of special mechanisms selecting only the mRNA segments needed for cytoplasmic translation must be evisaged (104). Why the cell would transcribe more genetic information than is needed remains a mystery. Since neither view alone embraces the facts, several authors (24, 84, 88) leave open the possibility for the parallel existence of these two pathways of high molecular weight D-RNA turnover. A third explanation arises if sequential growth of RNA chains is considered (see Section 111). It was shown that RNA synthesis in animal cells may be a rather slow process, the completion time being estimated in minutes or perhaps hours. Recently, Greenberg and Penman (131), following the methylation of the 45 S rRNA precursor in growing HeLa cells, derived a lower limit for its completion time of 2.3 minutes. On the other hand, a kinetic study of D-RNA labeling in growing Cave cells showed t h a t this RNA is degraded within 1 or 2 minutes (100). Thus, we seem t o confront the paradox that high molecular weight DRNA is degraded faster than i t is synthesized! The discrepancy must be larger in nongrowing cells where the overall rate of RNA synthesis is ten- to a hundredfold slower, not to mention that the high turnover rate of these giant RNA niolecules inust be the most uneconomical process in cellular energetics. A plausible solution to this paradox is to admit that high molecular weight nRNA is a precursor of other RNA molecules. Since it has been shown that i t cannot be the immediate precursor of any type of cytoplasmic RNA, conversion into stable nRNA’s is suggested. Transformation of high molecular weight D-RNA
RNA AND INFORMATION TRANSFER I N ANIMAL CELLS
219
into stable 18s D-RNA has been put forward by several workers (55, 92, 102, 11.9). Still, formation of stable nuclear D-RNA is a rather slow process, which is not compatible with the high rate of high molecular weight D-RNA labeling. Since 4 5 s RNA is the first product to accumulate in nuclei in sizable amount, conversion of high molecular weight D-RNA into 45 S RNA fits the most exacting kinetic requirements. This possibility is not considered merely because the base composition (determined by P32distribution) is different for these two RNA’s. The model of sequential RNA synthesis suggests that in rapid-labeling experiments only the composition of the labeled segment of the polynucleotide chain is measured. Thus, “DNA-like” composition of high molecular weight RNA might simply indicate that the last (i.e., the first-labeled) segment completing the RNA chain is “DNA-like.” This does not mean that the whole molecule is “DNA-like” ; it inay be “rRNA-like.” As a consequence, the following scheme may be outlined: high molecular weight RN.4 -+ 45 S RNA -+ 35 S RNA --t 28 S rRNA
I--+
18 S RNA
The consequences of the modcl are considered to reconcile some controversial findings delineated in the preceding sections. a. mRNA is transcribed on karyoplasm DNA as a polynucleotide segment completing preexisting RNA chains. The size heterogeneity of the initially labeled high molecular weight RNA reflects the liberation from the DNA template of unfinished RNA chains. The high molecular weight RNA is further transformed into more homogeneous 45 S RNA. Only moderate secondary structure transitions may be involved ( 2 4 ) . b. The precursor RNA molecules have a hybrid base composition. The apparent composition detected by P32distribution analyses would reflect the completion time, i t . , the overall rate of RNA synthesis in a given cell type. An increased labeling time or RNA synthesis rate would nccclerate the transition from “DNA-like” toward “rRNA-like” precursor molecules (see Section IV, B ) . Extremes are reached in the anucleolate S e n o p u s laevis (101, 102) and i n actinomycin D administration (96-100, 116) when only the “DNA-likc” segment is labeled. c. Further transformations follow the pathway established for 45 S RNA [see Perry ( 3 2 ) ] .It is proposed that the mRNA segment is transformed into the respective 18s nRNA. Therefore some features of mRNA are inherent to this molecular species of RNA. This interpretation of the experimental results avoids the implication of giant-size nRNA molecules with extremely high turnovers and unknown biological roles. It explains also the formation in nuclei of stable 18s RNA molecules with mRNA features. It is obvious that, in any
220
A. A. HADJIOLOV
case, 18s nRNA is a most controversial molecular species. Is it a messenger RNA or a ribosomal subunit RNA? Is there one molecular species with hybrid structural and functional features or are there two independent molecular types having by accident the same size? Since the precursor-product relationships observed in nuclei do not operate in the cytoplasm, it is likely that more clear-cut conclusions could be derived from studies on cytoplasmic rRNA and mRNA. These aspects are considered next with the i,mplicit understanding that both rRNA and mRNA are transferred from the nucleus into the cytoplasm without major modifications in the process of transfer [see (18, 3.2, 5 5 ) ] .
V. Cytqhsm-the
Site of mRNA Expression
It is proposed to review here some aspects of studies aimed a t the identification of cytoplasmic mRNA, with emphasis on the relation between mRNA and rRNA [see also (5) and Chantrenne in this volume].
A. Molecular Characteristics of Cytoplasmic RNA 1. RIBOSOMAL RNA
By definition, rRNA is a component of ribosomes. Actually, every ribosome is composed of two unequal subunits and we invariably find two rRNA’s. In animal cells, the large (60S) subunit contains 28 S RNA and the small (40s) subunit, 18s RNA. Most likely, the two rRNA’s constitute single polynucleotide chains with molecular weights of 1.2 x lo6 and 0.6 X loG,respectively [see (.21)]. When RNA is extracted from either ribosomes or total cytoplasm under conditions that minimize degradation, the 28 S and 18 S components are invariably obtained as the sole RNA species in a 1:l molar ratio, i.e., a 2 : l mass ratio. The two rRNA’s constitute about 90% of cytoplasmic RNA, most of the remainder being tRNA. Given the sensitivity of our techniques, any other type of RNA cttnnot account for more than 2-3% of the total cytoplasmic RNA. 2. EXTRARIBOSOMAL RNA Several attempts have been made to isolate and identify cytoplasmic RNA fractions other than rRNA. Some of these fractions are suspected of being mRNA and are therefore worth considering. Mitochondria1 RNA (if i t exists as an entity) is not likely t o be directly involved in nucleocytoplasmic transfcr of genetic information. With the advent of techniques for the isolation of ergastoplasmic
RNA AND INFORMATION TRANSFER IN APiIMilL CELLS
22 1
lipoprotein membranes, a variable amount of RNA was found associated with this fraction. Several fcaturcs, such as base composition and rate of Inbeling, seeined to indicate that thib RNA is distinct from rRNA. This view was strengthened by reports indicating the existence in membrane RNA of fractions, revealed in density gradients, other than the two rRNA’s (132). However, recent observations indicate t h a t the bulk of membrane RNA is 28 S and 18 S in size (133-135). Moreover, BergeronBouvet aucl Moulk (135) showccl t h a t the membrane RNA is enriched in thc 28 S species with a 28 S/18 S mtss ratio of ahout 2.5. On the other hand, in total cytoplasmic extracts or in postribosomal fractions of HeLa cells (136, 137) and liver (138), free 60 s and 40 S R N P particles were found, with a marked prevalence of the latter type. As discussed further, these particles were suspected to contain RNA species distinct from the two rRNA’s. Still, the bulk of the RNA extracted from these 40 S particles was found t o be 18 S in size (137, 138). The nature of this L ~ e ~ t r a r i t ) ~ ~R~NmAa lis” a disputed point, but recent observations of Sabatini e t al. (139) throw some light on this issue. They demonstrated t h a t ribosomes are attached to lipoprotein membranes hy their 60 S subunit. Under some contictions, the 40 S subunit is rcleasetl, leaving the 60 S subunit behind. Thus, it is plausible that disruption and further fmctionation of cells ni‘iy result in ribosome tlissociation, the two subunits heing recovered in diffcrcnt fractions. Thus, the membrane fraction, enriched iii attachcd 60 S subunits, yields a “membrane” RNA, identical with 28 S RNA, while the 40 S subunits and tlicir 18 S RNL4 are likely t o be found in postribosomal fractions. Of courw, the existence of free 60 S and 40 S R N P particles with different localization in the cytoplasm is not excluded. In anv case, it should be hti.c>sxl that all directly tlotc,c.ted “t.xti.aribosoiiin!” fractions contain only 28 S or 18 S RNA, or more often, varying proportions of hoth types. Atlrlitional eridcncc is needed to tlecidc whether thc 28 S and 18 S “estrnriI)o~on~nl” RNA’h arc itlentir:il witli rRNA.
B. labeling and Turnover of Cytoplasmic RNA 1. RIBOSOMAL RNA
Most of the evidence for the prcsence of mRNA in the cytoplasm is based on the property of rapid labeling with the assumption that ribosomes and rRNA are stable, but important limitations to this presumption exist with animal cells. On one hand, i t is likely that stable messengers direct the synthesis of most proteins (see Section I ) . On the other, although some evidence for the stability of rRNA has heen obtained with growing cells (I@), this is apparently not the case in non-
A. A. HADJIOLOV
222
"r 1.0
L
I
5
t
10
I
I
I
15
20
25
Tube number
(A)
I
I
7 ’
Hours
(B) FIG. 6. Labeling and turnover of RNA in the two ribosomal subunits of rat liver. [From Hadjiolov ( I % ) . ] (A) Sucrose density gradient sedimentation pattern of ribosomal subunits obtained from animals 90 minutes after introduction of orotic acid-6-CI4. 0-0, O.D.*,; 0- -0, radioactivity. (B) Decay of RNA labeling in the two ribosomal subunits obatined as in (A). Labpling for 60 minutes in Vzvo with orotic acid-6-C" followed by orotic acid-CB. Note the parallel decrease in the specific activity of the two subunits.
R N A A N D INFORMATION TRANSFER I N A N I M A L CELLS
223
growing cells. For example, it was found that the half-life of ribosomes and rRNA in liver is about 2-5 days (128, 141), which is a limited part of the life-span of liver cells. The following aspects of rRNA labeling and turnover may be of relevance here [see also (SZ)]. a. I n brief labeling experiments, the label appears a t first in the 18s rRNA and later in 28 S rRNA (136-138, 14%’). The same correlation is observed with the two ribosomal subunits obtained from either cytoplasmic extracts (137, 14%’) or after dissociation of isolated ribosomes (128). I n both cases, the 40 S subunit is initially more highly labeled than the 60s subunit (Fig. GA). Studies with HeLa cells show also an initial higher labeling of the proteins in the 40 S subunit (1.43). b. Uneven distribution of labeled 28 S and 18 S RNA among different cytoplasmic compartments is observed with brief labeling times. One popular interpretation depicts the flow: free (immature) 40 S and 60 S subunits + polysomes -+I ~ I O I I O I I ~ P I - ~ Crihosomes (32, 136, 137). Other observations indicate that “meinhrane” RNA is more highly labeled than the RNA of single ribosomes (133-135). One explanation of the initial higher labeling of the 40 S subunit RNA would be that i t possesses a higher turnover rate. However, when the decay of orotic acid-C14 pulse-laheled ribosomes of liver was studied (128), it was found that the RNA’s of the two subunits decay a t essentially the same rate (Fig. 6B). Consequently, the release of the two subunits and their further turnover should be synchronized. What then may be the meaning of the higher pulse-labeling of the smaller subunit and its RNA? This problem, connected with the search for rapidly labeled cytoplasmic RNA, is considcrcd next. 2. RAPIDLYLABELED RNA FRACTIONS
I n his first studies with liver, Hiatt ( 5 6 ) could not trace any rapidly labeled RNA fraction other than 28 S and 18 S RNA. However, further studies with liver (144-146), HeLa cells (147, 148), mammary adenocarcinoma (149), and immature erythroid cells (88, 104), revealed the presence of a heterogeneous rapidly labeled RNA fraction situated in the 4 1 6 S zone of density gradients. One particular aspect emerged from these studies: the heterogeneous RNA fraction was observed only when RNA was extracted from previously isolated polysomes. Because polysomes are involved in protein synthesis [cf. ( 5 )1, the rapidly labeled fraction was considered to be mRNA. Actually, this fraction may well be the product of partial RNA degradation taking place during isolation of polysomes (see Section 11, A ) . That this is the case is shown by studies with liver (128, 138, 150, 1511, Cave cells ( l o o ) , and a plasma cell tumor (151),which revealed that when RNA is isolated under conditions
224
A. A. HADJIOLOV
avoiding degradation, the radioactivity and UV profiles coincide almost exactly.' Thus, the rapidly labelcd fraction is either associated with the 18 S rRNA or it has the same sedimentation coefficient. Binding of Mg2+ with EDTA and treatments known to destroy hydrogen bonding, such as heating with urea (128) or formaldehyde (I%), failed to release
Urea concentration
FIG.7. Stability of label in 28s and 18s rat liver rRNA's on urea treatment. Labeling of RNA for 90 minutes with orotic acid-6-C" in wiwo. The 28 S and 18 S RNA's are isolated from sucrose density gradients. Treatment with 0.4 M urea a t 60" for 10 minutes and subsequent centrifugation in urea density gradients. L e f t : 2 8 5 RNA; Right: 18s RNA. -0, O.D.so; O---O, radioactivity. The arrows indicate the average specific radioactivity in counts/min/mg RNA (128).
any labeled material from 18 S RNA (Fig. 7 ) . These results suggest that the rapidly labeled RNA is either covalently bound to the 18s rRNA or is itself 18 S. The first possibility is made more likely by the observation that, in liver, a 6-14s highly labeled material is released from the 18 S RNA under the action of cytoplasmic RNase's (1%). In summary, the results discussed above indicate that rapidly labeled RNA of animal cells constitutes a homogeneous population of molecules that sediment with a coefficient of 18s. The rapidly labeled 18s RNA is preferentially degraded during isolation of polysomes to yield a heterogeneous 4-16 S material. The mRNA for serum albumin (mol. wt. 62,000) is expected to be 1618 S, while the mRNA for the secretory protein of the RPC-20 plasma cell tumor (mol. wt. 24,000) should be about 9-11 S (161).
RNA AND INFORMATION TRAXSFER I N A N I M A L CELLS
225
C. Mononucleotide Composition. Cytoplasmic D-RNA 1. DIRECTDETERMINATION
Since no major RNA species other than 2 8 s and 1 8 s is found in animal cell cytoplasm (Section V,A) only the base composition of these two RNA’s can be determined directly. Several workers have isolated these two RNA components and determined their mononucleotide composition (Table 11).I n every case the 18 S RNA has a much lower G+C/A+U ratio than 2 8 s RNA. As a rule this ratio falls in the range of 1.1Cb1.35 for 18s RNA and of 1.70-1.95 for 2 8 s RNA. Since the 2 8 s and 1 8 s RNA’s isolated from either total cytoplasm or purified ribosomes are identical in base composition (156), the existence of a major “extraribosomal” RNA fraction other than 28 S and 18 S rRNA is excluded. Likewise, the reported G+C/A+U ratios for thz “membrane” RNA vary from 1.65 to 1.95 [cf. ( 1 6 5 ) ] , thus reinforcing the proposed interpretation that the membrane fraction is enriched in 60 S subunits, i.e., 28 S RNA. The observed difference in base composition suggests that 28 S and 18 S RNA’s represent different species. Further, one should note the fact that the composition of 1 8 5 rRNA is definitely shifted toward that of DNA, i.e., 18 S rRNA is relatively “DNA-like.” If a G+C/A+U ratio of 1.80 is taken as typical for rRNA and a ratio of 0.75 for DNA, a rough estimate indicates that 1 8 s RNA contains 40% “DNA-like” and 60% “rRNA-like” components. Consequently, one may ask whether cytoplasmic 18s RNA is a mixture of D-RNA and R-RNA molecules of the same size or is a hybrid molecule with an R-RNA segment covalently bound t o a D-RNA segment? A definite answer must await more knowledge of the primary structure of rRNA, of 1 8 s RNA in particular. A rat liver fraction insoluble in 10% NaCl, and thus probably rRNA, yielded some preferred sequences among the dinucleotide and trinucleotide products of pancreatic RNase (166). On the other hand, complete digestion of separate 28 S and 18 S RNA’s from different sources with pancreatic or T1 RNase demonstrated definite differences in the nucleotide sequences of these two rRNA types (167, 168). These initial studies reveal that random distribution of nearest-neighbor nucleotides niay not be the rule in rRNA structure. We havc attempted to approach this problem by the stepwise degradation of liver rRNA with snake venom phosphodiesterase, an exonuclease known to release 5’ mononucleotides from the 3“OH end of polynucleotide chains. The RNA preparations studied were composed of homogeneous 28 S and 18 S RNA and virtually free from contaminating endonucleases (see Section 11, A). Moreover, endonuclease contami-
N
TABLE I1 MONONUCLEOTIDE COMPOSITION OF CYTOPLASMIC 28s
h3 0-4
AND
1 8 s RNA
OF
ANIMALCELLS"
~~~~
~
28 s
18 s
+
Molar ratio (%) Source
Rat liver Rib!
Rib. Rib. Reticulocyte Rib.
Hat kidney Rat spleen Rat brain Krebs I1 ascites cells HeLa cells BHK cells Mouse L fibroblasts Hen fibroblasts HLM cells
~
A
U
G
C
18.3 17.8 15.7 15.5 18.6 18.0 15.4 15.5 15.8 15.5 16.4 15.9 15.3 15.0 16.0 20.5 16.9 19.3 18.4 19.5 19.2 17.3
19.0 17.0 19.2 18.5 18.9 16.4 18.6 17.9 18.0 17.8 16.6 19.0 19.1 19.3 17.0 19.3 18.3 17.8 17.7 18.2 16.2 17.6
32.9 33.0 34.6 36.6 32.9 34.8 36.8 36.3 35.8 36.7 35.3 36.0 36.8 37.8 36.0 28.3 34.9 34.3 34.3 34.5 35.2 35.1
29.8 32.2 30.5 29.5 29.7 30.8 29.3 30.3 30.4 30.0 31.6 29.1 28.9 27.8 30.5 31.9 29.9 28.5 29.6 27.8 29.5
30.0
+ C/
G c/ A+U ratio
A
U
G
C
ratio
1.66 1.82 1.87 1.95 1.67 1.91 1.95 2.00 1.96 2.00 2.03 1.87 1.91 1.92 1.98 1.52 1.84 1.69 1.77 1.65 1.83 1.87
22.4 19.8 19.1 19.7 22.0 22.5 19.4 20.1 20.1 20.9 20.5 20.5 20.0 19.7 21.2 23.2 22.6 24.3 22.1 23.5 22.9 23.0
19.6 18.0 24.2 22.5 21.5 19.5 23.1 22.5 22.1 22.4 20.0 23.5 23.9 22.7 21.5 23.2 22.9 22.0 21.5 21.9 21.3 23.3
30.2 32.4 30.7 32.3 28.8 31.8 32.0 31.2 31.2 30.7 30.7 30.4 31.1 31.5 29.5 25.2 29.3 28.7 30.5 29.9 30.0 28.7
27.8 29.8 26.0 25.6 27.6 26.2 25.6 26.1 26.6 25.9 28.8 25.6 25.1 26.0 28.0 28.0 25.2 25.0 25.9 24.7 25.8 25.0
1.39 1.64 1.30 1.37 1.30 1.38 1.36 1.35 1.37 1.31 1.48 1.27 1.28 1.36 1.35
Molar ratio (yo)
G
A+U
1.14 1.20 1.16 1.29 1.20 1.26 1.16
Reference
Chick embryo 3 days 7 days Xenopus laevis Eggs Embryo Arbacia punctulata eggs Pot,ato tuber Pea seedlings
z
t
18 19
22
17 19 19.9 25.1 23.6
28
1.50 1.63
24 24
21 22
30 29
25 25
1.22 1.18
(161) (161)
30 30 26.0 22.0 22.6
2.03 1.86 1.56 1.16 1.21
22 22 23.1 25.4 23.7
18 18 23.2 25.2 25.1
31 32 29.9 27.2 31.1
29 28 23.8 22.2 20.1
1.50 1.50 1.16 0.98 1.03
(101) (162) (164) (163) (163)
24
18
36 34
16 16 19.3 21.2 21.6
37 35 34.9 31.7 32.1
Only values obtained by direct estimation of the mononucleotide composition are included. Data on some plant tissues are given for comparison. Rib.-The two RNA species have been obtained from previously isolated ribosomes. (1
t
Z
u
228
A. A. HADJIOLOV
-1
10
I
I
20
30
I
1
I
I
I
40 50 60 70 80 Hydrolysis, %
I
90
FIG. 8. Changes in the molar ratios of 5' mononucleotides released by stepwise hydrolysis of total rat liver rRNA with snake venom phosphodiesterase. Hydrolysis at constant pH of 8.5 in a pH-stat t,itration assembly. At a fixed percentage of hydrolysis, the undegraded RNA chains are precipitated with ethanol and the molar ratios of the released 5' mononucleotides remaining in solution is det,ermined. The undegradcd RNA is furt,her hydrolyzed in the same manner. Thus, the experimental points in the diagram represent the molar ratios of 5' mononucleotides in the rRNA segments degraded a t 0-10, 10-20, 20-40, and 40100% hydrolysis (see dotted line for pG) (176).
nants in the enzyme system could account for no more than 1 or 2 internal breaks per molecule RNA.6 The basic results obtained in these studies (169-172) are illustrat,ed in Fig. 8. They show that the molar ratio of the liberated mononucleotides varies with progress of rRNA ratio of the released monoenzyme degradation. The G+C/A+U nucleotides rises from 1.12 for the initial 10% of the RNA hydrolyzed to 2.75 for the last 60% (see Fig. 8 ) . Two explanations may be considered: ( a ) rRNA is R mixture of D-RNA and R-R.NA and the enzyme The possible presence of endonucle:tse contaminant,s in the enzyme system was controlled by the following tests: (a) the polynucleotide (Ap).Cp (average n = 15) was not hydrolyzed, while ( A p ) X was rapidly degraded to pA and pC; (b) the reaction proceeds to completion and yields PA, pU, pG, and pC as the only major products; (c) a t different stages of phosphodiesterase hydrolysis of rRNA, the reaction mas stopped by the addition of KOH and the liberated nucleosides (18 hours a t 37" and 0.5N KOH) were determined. The amounts of nucleosides liberated correspond to 1 or 2 internal breaks per molecule of rRNA; (d) when rapidly labeled rRNA is used as substrate, the specific radioactivity of the liberated nucleotides changes with the progress of phosphodiesterase action. Randomization of the label is expected under the act,ion of endonuclease contaminants [for details see ( l i l ) ] .
229
RNA AND INFORMATION TRANSFER I N A N I M A L CELLS
degrades preferentially D-RNA ; (b) rRNA is a hybrid molecule, “DNAlike” near the 3’-end and “rRNA-like” near the 5’-end of the polynucleotide chain. We consider the first possibility less likely since model experiments with double-stranded poly Aepoly U did not show a detectable influence of secondary structure on the action of this enzyme 1169, 171), and venom phosphoidesterase is devoid of base specificity [cf. ( l 7 3 ) ] .Further, the G C/A U ratio of 2.75 for the last 60% of rRNA is markedly higher than the values obtained with purified 2 8 s rRNA. Consequently, there should be other segments in rRNA compensatorily enriched in A and U, i.e., being more “DNA-like.” That these results reflect something more than a mere artifact is indicated by independent studies of Delihas and Bertman (174). As shown recently, partial digestion of rRNA with pancreatic (175) or T1 (176, 177) RNases yields several fragments of discrete size, but it has been demonstrated (174) that some of the high molecular weight fragments in partial T1 RNase digests, estimated t o represent 10-15% of the whole rRNA, have a G+C/A+U ratio of 3.1-3.6. Again, these results indicate that there are other segments of the rRNA chains more “DNA-like” than the average 2 8 s or 18s RNA’s. Thus, the scanty evidence available is consistent with thc possibility that 18 S RNA is a hybrid molecule of covalently bound D-RNA and R-RNA segments.
+
2. P
J
2
-
+
DSTUDIES ~ ~ ~
~
~
~
~
~
~
~
~
A “DNA-like” composition of rapidly labeled RNA is detected only by P32-distrib~tionanalyses. Thus, the heterogeneous &16 S RNA material obtained f i on1 previously isolated polysomes of HeLa cells (85, 147, 148), liver (145), and immature crythroid cells (88, 178) is enriched in D-RNA, although the G C/A U ratios obtained are in general significantly higher than the respective values for DNA. When undegradcd rat liver cytoplasmic RNA is isolated, the rapidly labeled D-RNA is found associated with both 2 8 s and 1 8 s RNA 1138, 154). On increasing the time of labeling, a gradual shift in the base composition of labeled RNA from D-RNA toward R-RNA is observed in liver (138, 154) and growing Cave cells (100). I n the case of HeLa cells, the heterogeneous 4-16 S rapidly labcled RNA fraction displays higher levels of hybridization with homologous DNA than the two rRNA peaks ( 1 7 9 ) . However, only a two- to threefold decrease in the amount of hybridized material from 1 8 s RNA is observed a t longer periods of labeling (179). All these studies are in good agreement with results of labeling experiments with other precursors (Section V, B, 2 ) . They show in addition that the rapidly labeled RNA fractions in the cytoplasm display a
+
+
230
A. A. HADJIOLOV
“DNA-like” base composition. When obtained from polysomes, this D-RNA is heterogeneous in size, while it coincides with 18s RNA (and 2 8 s RNA in some studies) when extracted under conditions chosen to minimize degradation. These results are interpreted as demonstrating the existence of independent D-RNA molecules identical in size with rRNA’s (100, 138, 154). The rapid labeling and the time-dependent shift in base composition are thought to reflect a high turnover rate of cytoplasmic D-RNA. However, it should be noted that no evidence whatsoever about the fast degradation of the suspected D-RNA molecules has been presented. An alternative interpretation of the findings on cytoplasmic D-RNA is given below.
D. Stimulation of Cell-Free Polypeptide Synthesis Several authors have determined the stimulatory activity of cytoplasmic RNA in E. coli or mammalian cell-free systems. In general, the most active material is found associated with the 18s RNA peak (114, 126, 128) obtained from either total cytoplasm, ribosomes or 4 0 s R N P particles. While the rapidly labeled components of 18s RNA are degraded to 6-14 S material, the stimulatory activity remains in 18 S RNA (138). The validity of this assay for mRNA is limited, since the secondary structure of a polynucleotide seems to determine its stimulatory activity. Thus, good stimulation of the E . coli system is obtained with denatured DNA (180) and partially degraded rRNA or tRNA (181), while double-stranded viral RNA is inactive (18%’). Many attempts have been made to isolate mRNA for a specific protein. Since most of this work has been concentrated on mRNA for hemoglobin i t need not be considered here (see article by Chantrenne et al. in this volume). Only two points will be mentioned. First, dissociation of hemoglobin mRNA from reticulocyte rRNA appears to be extremely difficult, if not impossible. The failure of several vigorous attempts led Gould e t at!. (183) to the conclusion that hemoglobin mRNA is covalently bound to rRNA. Secondly, various unrelated RNA fractions are likely to cause an unspecific stimulation of the synthesis of a specific protein (184). Therefore experiments with heterologous ribosomal systems are required for the adequate identification of a mRNA (see Section 11).
E. Cytoplasmic Carriers of mRNA 1. POLYSOMES
It was postulated ( 2 ) that mRNA bound to ribosomes would form the active complex for the translation of genetic information. With the
RNA A N D IKFORMATION TRANSFER I N ANIMAL CELLS
231
recognition of polysomes as such a complex, the presence of mRNA in these structures was assumed, and extensive supporting evidence has been collected [see (5, 35) for references]. Ribosomal aggregates (polysomes) connected by RNA threads are more active than single ribosomes in polypeptide synthesis both in vitro and in vivo. There is compelling evidence that the RNA linking the ribosomes is labeled more rapidly than rRNA, is heterogeneous in size, is “DNA-like” when analyzed by P32distribution, and is marked by a high stimulatory activity in cell-free systems. This indicates that the RNA threads in polysomes represent mRNA. However, all these facts do not show that the mRNA is external t o the ribosomes and provide no proof of the movement of ribosomes along an independent mRNA chain. On the contrary, several observations indicate that this model may not reflect the actual situation in vivo. It has been shown, for example, that polysomes are not an obligatory structure for protein synthesis. Single ribosomes, called monosomes [cf. ( 5 )], isolated from liver (185) or muscle (186‘) are active in cell-free polypeptide synthesis. The experiments of Lamfrom and Knopf (187, 188) showed that reticulocyte monosomes are self-sufficient in the synthesis of the separate chains of hemoglobin. Further, the experiments discussed in the preceding sections show that the heterogeneous messenger-like material isolated from polysomes is probably a degradation product. When degradation is avoided, the RNA molecules with several features of mRNA coincide exactly with 185 rRNA and cannot be dissociated by physical treatments to yield shorter and more heterogeneous RNA chains. I n brief, although we know for certain that mRNA is present in animal cell polysomes, the characteristics of this mRNA in the native state are controversial. When undegraded, the supposed mRNA coincides in size with rRNA, with 18s rRNA in particular. This was observed even in cases in which the mRNA for hemoglobin (183) and plas,ma cell tumor protein (151) should be much smaller in size than the rRNA. 2. RIBONUCLEOPROTEIN PARTICLES
The problem of cytoplasmic R N P particles as mRNA carriers is even more complicated. As mentioned before, several authors observed that a 40s R N P particle is labeled more rapidly than the related 60s particle. Kinetic and other evidence showed that the 40s particle is a precursor of (or is identical with) the smaller ribosomal subunit ( 3 2 , 1S‘i‘). However, the “excess” label in the 40s subunit found after short pulses and the identification of the constituent 18s RNA with some
232
A. A. HADJIOLOV
properties peculiar t o mRNA (“DNA-like” base composition, higher level of hybridization with DNA, stimulation of cell-free polypeptide synthesis) inclined others to think of the 40s particle as the carrier of mRNA in animal cells (136, 138, 179). The term “informosonies” was coined for similar R N P particles in order to distinguish them from the smaller ribosomal subunit (189).The close S values for these two particles, presumably distinct in function, remained unexplained. Then, unexpectedly it was found that the rapidly labeled R N P particles (assumed to be the 40s ribosomal subunit bearing mRNA) band a t CsCl densities that are slightly lower than those typical of the 40 S ribosomal subunit itself (190). These results rule out the possibility of an external mRNA being attached t o the 40s subunit, although the RNA of the 40 S R N P particle has been identified as mRNA by all criteria available a t present. Thus, we are left with the only possibility that the 18s RNA isolated from the 40s R N P particle is itself mRNA. Accordingly, we are obliged t o envisage the existence in the cytoplasm of two 40s R N P particles, the one containing 18s rRNA and the other, 18s mRNA. And again we end with the controversial 1 8 s RNA species.
3. 18 S RNA-MESSENGER OR RIBOSOMAL CONSTITUENT? Since in both nuclci and cytoplasm, the search for the RNA species involved in genetic information transfer has converged toward 18 S RNA, a general evaluation of its properties is appropriate here. a. In both nuclei and cytoplasm a species of RNA molecules is found that is homogeneous in size and constitutes a well-defined 18s peak. The molar ratio of 28 S/lS S RNA in total cellular RNA is close to unity. b. The 18s RNA is found as a constituent of 40s R N P particles. These particles are identical in physical properties to the smaller subunit of ribosomes. The R N P particles containing 18s RNA exist in three states: free; bound to the 60 S subunit in ribosomes; and involved in polysomal structures. No evidence for the existence of free 18s RNA in either nuclei or cytoplasm has been presented. c. The mononucleotide composition of 18s RNA is shifted toward that of DNA as compared to 28s rRNA. Approximate estimations indicate that 18s RNA is 40% “DNA-like” and 60% “rRNA-like.” d. In most cases studied, nuclear and cytoplasmic 18 S RNA’s show a higher stimulation of cell-free polypeptide synthesis compared to 2 8 s rRNA. e. Cytoplasmic 18s RNA is more rapidly labeled than 2 8 s RNA. When labeled with P3?,the 18 S RNA is found initially “DNA-like” in base composition. IVith extension of labeling time, the composition,
233
RNA AND IR’FORMATION TRANSFER I N ANlMAL CELLS
determined by P3?distribution, tends to reach the values obtained by direct determination. f. During isolation of cytoplasmic structures, the rapidly labeled 18 S RNA is preferentially degraded by endonucleases to yield a messengerlike material of heterogeneous size. This degradation is most likely to occur with 18 S RNA involved in the structure of polysomes. Thus, according to points ( a ) and (b) , 18 S RNA is a true rRNA. On the other hand, points (d), (e), and ( f ) suggest that 18s RNA may be the mRNA we seek. At present, it can hardly be decided whether 18 S RNA is a mixture of mRNA and rRNA molecules or whether it represents a hybrid molecule with characteristics of both RNA types. The “DNA-like” composition of rapidly labeled 18 S RNA is usually taken as evidence for the independent existence of 18s mRNA characterized by a high turnover rate (130, 138, 154). Further indications that an independent 18s mRNA exists are seen in the preferential degradation of 18s RNA moleculrs with messenger features, while 185 rRNA remains apparently unchanged (138, 191). Finally, in a recent study, Gazsrian and Schuppe (192) followed the changes in base composition of the rRNA of Cave cells after a prolonged “chase” of P32with nonradioactive phosphate. They ohserved that the G C/A U ratio of 18s RNA (determined by P33distribution) continues to rise during the chase to reach a value of 1.95, higher than the 1.45 obtained by direct determination. It was deduced that the composition of 18 S rRNA is identical with that of 2 8 s rHNA, while the lower values obtained by direct determination reflect contamination of 18 S rRNA by about 40% stable 18s D-RNA (see Table 11). The authors assumed that 18s D-RNA has a higher turnover rate than 18s R-RNA, although this is not the only possibility (see bclow). Besides the uncertainty in the experimental data, the view that an 18 S mRNA exists independently faces two basic obstacles. On one side, the expected size heterogeneity of total mRNA can hardly be reconciled with a homogeneous (18 S) mRNA.7 On the other, there is no apparent necessity for an mRNA t o be of the same size as rRNA, nor t o form R N P subunits identical with the smaller subunit of ribosomes. An alternative explanation may be found in the proposal that 18s RNA in both nuclei and cytoplasm represents a hybrid molecule consti-
+
+
‘ I n the case of liver, it is possible that thc bulk of mRNA codes for serum albumin (the major protein product of liver) and is expected to be 1618s in size. However, the synthesis of total liver proteins seems t o he little affected by actinomycin D ( 1 4 ) ; this suggests a stahle mRNA for serum albumin. On thc other hand, short-term labeling rsprriments indicate a half-lifa for tot,al mRNA in rat liver cytoplasm of about 4 hours (144, 146).
234
A. A. HADJIOLOV
tuted of covalently bound mRNA and rRNA segments. The following basic aspects .may be outlined. a. A heterogeneity in size of mRNA is shown by variations in the length of the mRNA segments within the limits determined by the “standard” size of 18 S RNA molecules (Fig. 9) .8 When functioning in F A v e r o g e size of 18 S RNA = 0.67 x lo6 doltons
L
1
Average size of mRNA segments =0.2?x doltons
lo6
f
FIG.9. Model illustrating the size heterogeneity of mRNA segments found within the limits of a standard 18s RNA molecule. The straight lines represent “ribosome-like” segments, and the zigzag lines designate the “messenger” segments (193).
polysomes, the inRNA segment is extended from one ribosome to another and is therefore more susceptible to endonuclease attack. As a result, heterogeneous 4-16 S mRNA material is easily obtained from previously isolated polysomes. b. A hybrid character for 18s RNA is compatible with the interpretation of 40 S R N P particles as being simultaneously a ribosomal subunit and a mRNA carrier, thus supplying a solution to the controversy concerning the nature of these particles. The existence of mRNA as a constituent of 40s R N P particles is compatible with the “protected” and “masked” forms of mRNA envisaged in several studies (cf. 12, 189). c. Shifts in the base composition of 18s RNA from D-RNA toward R-RNA with extension of labeling time are easily understood if we consider the model of sequential synthesis of RNA chains (see Section 111). Location of the mRNA segment a t the 3‘-end of the chain, which is the last synthesized (i.e., the first labeled), would explain both the higher pulse-labeling of 18 S RNA (and the 40 S subunit) and a “DNA‘The maximum sire of mRNA is thus limited to 185 RNA, ie., 0.67 x 10‘ molecular weight, or about 1900 nucleotides. This corresponds to a single protein chain of molecular weight about 76,000. Studies on the subunit structure of protein molecules reveal that no single polypeptide chains with molecular weights greater than 66,000 have been detected [see Reithel (194)l.
RNA AND INFORMATION TRANSFER IN ANIMAL CELLS
235
like” composition in the initial periods of labeling (see Fig. 3, case B). It is also evident t h a t a t very long periods after dilution with unlabeled P,, the reverse phenomenon is expected: P32 should disappear first from the mRNA segment, and P32 remaining in the rRNA segment would give a base composition similar to that of 28 S rRNA [see ( 1 9 2 ) l . d. Sequential labeling of 18s RNA would help t o reconcile the expected stability of mRNA derived from most studies on the synthesis of total or individual animal cell proteins (see Section I ) and the much faster “turnover” rate of mRNA deduced from short-term labeling experiments [cf. (24, 138, 144, 146, 1 4 8 ) l . The supporting evidence for this concept has been outlined throughout this article, together with the opposed findings. It seems likely that more conclusive evidence will soon be forthcoming.
VI. Synopsis The controversial experimental findings on the role of RNA in genetic information transfer in animal cells have been discussed. I n the current state of our knowledge we can hardly decide whether genetic information is mediated by independent messenger RNA molecules or by messenger segments in ribosomal RNA.
A. The Orthodox Interpretation This interpretation is endorsed in almost all studies on mRNA of animal cells. It is strengthened by the evidence in support of the messenger concept obtained in coding experiments with synthetic polynucleotides and in studies with bacteria and with virus-infected cells [see ( 5 )1 . The implicit assumption is that genetic information transfer in animal cells is governed by the same basic mechanisms which operate in bacteria. Consequently mRNA is considered as synthesized and transferred a t a rate and by a pathway independent of rRNA synthesis and of ribosome formation and turnovcr. The translation process involves only transitory association of the mRNA with genetically unspecific ribosomes. The evidence on information transfer in animal cells is thus fitted into the frame of two independent pathways (see p. 236). As discussed, the different steps of this hypothetical mRNA pathway are not equally well documented. Uncertainties exist on the functional significance of the rapidly turning over high molecular weight mRNA in nuclei; the existence of separate 40 S R N P particles as mRNA carriers; the native state of mRNA in polysomes; etc. The most striking aspect emerging from these studies is the close similarity between these two presumably independent pathways. It is clear that the almost iden-
236
A. A. HADJIOLOV
rRNA Pathway [see (32) ]
mRNA Pathway Nuclei
> 40 S mRNA -High
i
18 S mRNA (in 40 S RNP ?) Cytoplasm
45 S rRNA Precursor
turnover inside nuclei
j.
18 S mRNA "Protected," (in 40 S RNP) "masked" mRNA
18 S rRNA (in 40 S RNP)
t
18 S rRNA (in 40 S RNP)
t
18 S mRNA In polysomes, the active part being 4-16 S
28 S rRNA (in 60 S RNP)
1
28 S rRNA (in 60 S RNP)
\ /
PolyTes
Monomeric ribosomes (40 S + 60 S RNP)
tical physicochemical characteristics of the involved RNA molecules could hardly be due to a mere coincidence.
B. One Plausible Unorthodox Interpretation The proposed alternative interpretation (193)is an attempt to seek simplicity in the chemical organization of the living cell. It is based on the experimental results that point t o the close relationship between mRNA and rRNA in animal cells. The main feature of the advanced hypothesis is the consideration of mRNA as an integral part of some RNA's currently considered as pure rRNA's. The following tentative scheme may be considered : a. Messenger RNA is transcribed on karyoplasm DNA as a polynucleotide segment completing preexisting rRNA chains to constitute a hybrid precursor molecule. b. The precursor RNA is further transformed into 2 8 s and 18s molecules, which, combined with protein, constitute the 60 S and 40 S ribosomal subunits. I n this process the mRNA segment is found as a constituent of 18 S RNA. c. The 18s RNA is a hybrid molecule formed by covalently bound rRNA and mRNA segments. The size heterogeneity of .rnRNA is realized within the limits fixed by the standard size of the 18s RNA molecules (see Fig. 9 ) . d. The hybrid 18 S RNA integrated with proteins into 40 S R N P particles is transferred synchronously with the 60 S R N P particle into the cytoplasm. Thus, the 40 S R N P particle represents a "protected" form of mRNA. The hybrid character of 18s RNA results in the 40s RNP being simultaneously a mRNA carrier and a ribosomal subunit.
RNA AND INFORMATION TRANSFER I N ANIMAL CELLS
237
e. In the translation process the mRNA segment of the 18s RNA is extended between the 40 S subunits, forming the connection with other ribosomes to give polysomes. Every mRNA segment included in 18 S RNA codes the synthesis of a single protein chain. The experimental evidence in support of this scheme involves mainly: the attempts t o find a native mRNA in animal cells ended with a homogeneous 18s RNA (Sections IV, D and V, E ) ; the hybrid mononucleotide composition of 18 S R N A (40% “DNA-like” and 60% “rKNA-like”) ant1 indications that 18 S R N A consists of covalently bound mRNA and rRNA segments (Section V, C ) ; the possibility of explaining the controversial findings of short-term labeling experiments hy considering the sequeiitial synthesis of R N A (initiated with the “ribosomal” segment and finished with the ‘Lm~sseiiger”segment) correlated with a relatively extended time period necessary for the conipletion of RNA molecules (Section 111); the dual character of 40 S R N P particles identified as either a inRNA carrier or a ribosomal subunit (Section V, E). I n the p r e s ~ n state t of our kno~vletlgc,speculations on the consequences of this model are premature. Involvement of a ribosomal subunit in genetic information transfer appears to provide a more stable structure for this process as expected from the general considerations outlined in Section I. It is also self-evident that a distinct function for the tupo ribosomal subunits may be envisaged. Thus, the function of the large subunit seeins t o be concerned with the adaptation of the whole ribosome to the environmental conditions in the cell (interaction with membranes, with tRNA etc.). On the other hand, the small subunit is involved in the transfer of genetic information from the nucleus into the cytoplasm. Consequently, it seems appropriate to designate the large subunit as the ndaptosome, and to preserve the term znformosome for the small subunit (193), thus avoiding the often controversial designation by the respectire S mlueh. Perhaps other faccts of the process of genetic inforrii~ition traiisfer i n animal cells could t ~ calso givcn a more simple iiiterpretatioli.
ACKNOWLEDGMENTS Thr author \yould like to thank liis rollragues R. G. Tsanev, P. V. Venkov, and Rosrmary and Radoslav Bnchraroff for critical comments and help in the preparation of the manuscript.
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Transfer of Genetic Information During Embryogenesisl MARTINNEMER Division of Biochemistry. The Institute f o r Cancer Research. Fox Chase. Philadelphia . Pemsyluania I . Introduction . . . . . . . . . . . I1. The Egg as an Informational Structure . . . . . A . Ooplasmic Regionality . . . . . . . . . . . . . . . . B. Egg Messenger RNA C . Egg Ribosomes . . . . . . . . . . I11. Schedules for the Activation of Protein Synthesis in Eggs A . Fertilization neither Necessary nor Sufficient . . . B . Fertilization Necessary but Not Sufficient . . . C . Fertilization Necessary and Sufficient . . . . . IV . Activation of Protein Synthrsis in the Sea Urchin Egg . A . Amino Acid Transfer Activity . . . . . . B. Release of Ribosomal Activity . . . . . . C . Release of Messenger HNA Activity . . . . . D . Scheme for a Dual Activation . . . . . . . . . . . . . V . Transitions in Genic Activity A . Developmental Changes in RNA Transcription . . B . Changing Messenger RNA Populations . . . . C . Nucleocytoplasmic Interactions . . . . . . VI . Developmental Regulation of Genetic Expression . . . . . A . Functional Organization of Chromosomes B . Transmission of Genetic Information . . . . . C . Messenger Ribonucleoprotein . . . . . . . . . . . D . Regulation of Polysome Activity VII . Summary . . . . . . . . . . . . References . . . . . . . . . . . .
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i Introduction A direct approach for understanding the molecular basis of early embryonic development is to examine the informational macromolecules themselves. which are responsible for the transfer of genetic information and the synthesis of specific proteins . Such an approach has been taken 'This review was supported by Public Health Service Research Grants Nos . CA 05936 and CA 06927 from the National Cancer Institute and 1 SO1 F R 05539 from General Research Support . 243
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most extensively in studying the embryos of amphibians and echinoderms. Experiments on RNA and protein synthesis during early development of these embryos are reviewed here as a basis for understanding how the character of genetic regulation may be specified by the requirements of cellular differentiation. Insights drawn from various other approaches using a multitude of biological systems, such as those of molecular genetics ( I S ) , chromosome structure ( 4 4 , and nucleocytoplasmic interrelationships (7-10), are also used in an attempt to clarify the nature of thc problem. The aspects of embryogenesis are examined as a sequence of four problems that arise both in the course of development and apparently in a course of increasing complexity. The construction of the egg (oogenesis) involves the biosynthesis and laying down of macroinolecular structures, some of which contain genetic information or could be used to regulate the flow of genetic information. The determination of the origin, nature, and function of these macromolecular egg structures, then, is the first problem t o be encountered in the elucidation of the molecular basis of embryogenesis. The second problem of embryogenesis arises around the time of fertilization, when the flow of genetic information becomes activated from a nearly dormant state. This flow must be recognized to occur at two SUCcessive levels, transcription (RNA synthesis) and translation (protein synthesis), but it is at the level of translation that thc activation is vastly more prominent. This activation now appears to be a matter of mobilizing the preexisting components of the egg. Third is the problem of how genic activities change during development and what factors influence these activities. These influences may be viewed as being exerted on nuclear behavior or, in detail, a t the level of the genes and cliroinosomeb. The differential activities of genes are reflected at the level of transcription and appear to undergo significant changes as development gets under way. The function of transcription is to provide R flow of information in the form of templates for protein synthesis and to mediate translation by providing the other components of the protein synthesizing system. Thus in each aspect the nature of transcription may change to meet the requirements of development. The fourth problem is the regulation of genetic expression a t the level of translation. Beyond transcription, the flow of genetic information may not be readily or quickly consummated in protein synthesis. The process of regulation may involve a delay in the functioning of templates in protein synthesis. This apparent accumulation of templates may have a special role in cellular differentiation.
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II. The Egg as an Informational Structure A. Ooplasmic Regionality The egg is itself a highly specialized cell. The long and complicated processes of oogenesis produce a cell structure that has an important influence on the primary processes of differentiation. The informational architecture of the egg may he a conspicuous spatial arrangement of visible constituents or a n arrangement that may not be evidenced except by its apparent influence on early devclopnient itself. The main purpose of this cssay is to examine thc involvcmcnt of inforniational niacroiuoleculcs in the development of nn cin hryonic structure from a more or less specific egg structure. Thc primary process of development is, obviously, the construction of the egg. The directive influence in this construction may come cither from the oocyte nucleus or from the activities of surrounding nutritive cells (11-18) which can furnish it with materials. The oocyte may receive proteins from the serum, as in the case of insects (19-21), or directly from thc. accessory cells of various species among insects (26, 23) and amp1iibi:tns (24, 2 5 ) . RNA may also be supplied by the accessory cells ( 2 6 ) . On the other hand, the oocyte nucleus may be extremely active in tlie synthesis of RNA in amphibians (27-29) and in echinoderms (30-34). Whereas the architecture of the egg cell arises from numerous formative processes, the significance of the final structure lies in its relationship to development. This significance has been tested through classical studies directed toward tlie manipulation of ooplasmic regions directly, the transplantation of cells between different regions of the embryo, and the examination of the effects of different juxtapositions. Such studies have been on the animnl-vegetal polarities of amphibian ( 3 5 ) and echinoderm (see 36-40) c’ggs, tlie dorsally situated organizer of :tiiiphil)iiin gastrulae and it.: intlueiiig action on surrounding cells ( 14, 41,4 2 ) , antl tlic polar l o h witliout which ccrtain regions would not develop in rertnin snail ciiil)i,yos ( s c c 4 3 ) , cxtcnsively studied in Ilynncrssri (44-47). An cxaniplc of :i motlci~nattcinpt to probe tlie molecular conwquences of egg arcliitcrture might be cited in the studies of the animal-vegetal gradients in sea urchins and their involvement in RNA transcription (48, 4 9 ) . The animal and vegetal polar regions (anterior and posterior, respectively) of the sea urchin egg antl cmhryo appear to be centers for the elahoration of agents for thc support of two opposing developinental tentlenciw. Norin:il developnicnt rosiilt s from n controllctl balance I>e-
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tween these agents. If the 16-cell embryo is surgically separated into two equal fragments, consisting of animal and vegetal halves, independent development of these halves will result in the failure of mesenchyme and endoderm to form in the animal half (50). The vegetal half will form a great excess of endoderm compared to ectoderm. I n the whole embryo, the balance between these opposing gradients can be upset in one direction or the other by treatment with a large variety of chemicals ( 5 1 6 4 ) . Markman (55) observed that, after separation, the rate of incorporation in RNA increased during development more rapidly in the animal halves than in the whole embryos. Implantation of vegetal micromeres into animal halves moderated the developmental distortion fostering normal development (50) and prevented the increase in RNA incorporation in the developing animal halves ( 5 5 ) . The questions that arise here have a bearing on the distribution of preexisting information in the egg and the use of newly transcribed information. (a) Does the influence of the vegetal cells on the animal cells depend on agents that might arise via new RNA transcription? Alternatively, are such agents preexistent and prelocalized in the egg? Giudice and Horstadius ( 4 9 ) raised sea urchin embryos in the presence of actinomycin D and transplanted their vegetal micromeres into animal halves. They found that these micromeres had the normal inducing ability, insofar as they promoted normal development. Thus the specificity of vegetal influence was apparently derived from factors prelocalized in the vegetal region of the egg and did not depend on new RNA transcription. ( b ) Does the response evoked in the animal half by the agents of the vegetal half in whole embryos depend on the elicitation of the new RNA transcription? Corollary to this, does the specific animalized or vegetalized development of the separate halves depend on new transcription? Markman and Runnstrom (56) treated animal and vegetal halves with actinomycin for brief periods and found that each developmental tendency was diminished and was thus scemingly dependent on new transcription. Vegetalization can be effected in whole embryos by exposure to lithium ion, which upsets the developmental balance in favor of the vegetal tendency ( 5 1 ) . I n order to determine whether this vegetalization was mediated by RNA transcription, Runnstrom and Markman (48) exposed embryos to both lithium and actinomycin. They noted that actinomycin reduced the degree of vegetalization and thus ameliorated the effects of lithium. Therefore, the inducing action of the vegetalizing agent was blocked a t the level of transcription and the exaggerated formation of endoderm prevented. Lallier (57),using considerably longer exposures to lithium and actinomycin, obtained an enhancement of the effect of lithium rather than a diminution. A possible explanation
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for these contradictory results might be that the altered balance between the two gradients responded differently to short- and long-term incubations with actinomycin. It must also be pointed out that neither investigator tested the extent of inhibition of RNA synthesis. Thus only a tentative conclusion may be drawn that RNA transcription is involved in the process. Actinomycin apparently produced an effect in one direction or another. The nct result might well have depended upon the precise conditions. The prelocalization of cytoplasniic factors that may influence early gene activation has been pointed out by Davidson et al. (58) in studies on embryos of IZyanassa obsoleta. In these eggs and embryos, a region of egg cytoplasm known as the “polar lobe” is transiently extruded in the form of a spherical protuberance. The removal of this portion of cytoplasm results in the formation of an embryo lacking certain tissues. Ilavidson et aZ. (58) followed the incorporation in RNA in normal and “lobeless” embryos. They found that the extent of incorporation was eventually reduced i n the developing lobeless embryos, compared to the normal embryos. More germane to the role of egg cytoplasm in this niosaic embryo, however, would have been an examination of the possible qualitative differences in the RNA.
B. Egg Messenger RNA 1. EVIDENCE FOR EGGMESSENGER RNA
Messenger RNA (mRNA) has been postulated as the cytoplasmic representative of the genes, serving as template for the synthesis of specific protein (1) . In microorganisms inRNA has been characterized as “rapidly turning over” (5941), thus imparting the advantages of flexible regulation, sensitive to changes in milieu, and close coordination between transcription and translation of genetic information. The concept of mRNA stored in the egg (62, 63) is provocative in a different sense. First, we imagine that a meaningful ooplasmic segregation of genetic information in a stored forin of mRNA may underlie some of the observed influences of egg architecture on early development. Second, this inRNA may be peculiarly long-lived in order to survive long storage in the egg; but this unusually stable mRNA, if it proves to be relatively stable during development, iiiay have an important sustained influence and a t the same time allow a gradual transition toward a dependence on newly synthesized messengers. The evidence for the existence of mRNA in the egg might be divided into that deduced from the effects of various alterations on early development and that obtained by direct examination of the egg RNA.
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a. Developmental Evidence. Nuclear activity in the just-fertilized egg is not needed to supply any of the components of the protein synthesizing system, which in the sea urchin is activated following fertilization. This conclusion can be drawn from the couplcd observations that (i) parthenogenetic merogones can undergo early development, although to a limited extent (64, 65) and (ii) normal cell division in this development requires protein synthesis, as ascertained by puromycin inhibition of cleavage (66). This relationship has been substantiated by the direct observation that parthenogenetic merogones display the same enhanced rate of protein synthesis as fertilized eggs, compared to essentially dormant unfertilized eggs (67, 68). More specifically, new RNA synthesis does not contribute to the activation of protein synthesis following fertilization. Gross and co-workers (62, 69, 70) demonstrated that early incubation with actinomycin inhibits RNA synthesis in the just-fertilized egg, but fails to affect the activation of protein synthesis. They concluded that among the RNA components involved in protein synthesis, there must be messenger RNA stored in the mature egg, to be used after fertilization. Actinomycin has been used to demonstrate the lack of dependence of newly initiated protein synthesis on the continued production of RNA in amphibian eggs (72) and in germinating cotton seeds (72). b. Direct Characterizatio,t. (i) Echinoderm. RNA extracted from sea urchin eggs has been examined for properties of mRNA. Maggio et al. (73) reported a slight but appreciable stimulation of polypeptide synthesis by egg RNA in a system containing heterologous ribosomes and supernatant cofactors. A quantitative evaluation of this template activity by Slater and Spiegelman (74) was attempted through the use of the heterologous ribosomal system from Escherichiu coli. Relative to standard MS-2 RNA, regarded as a 1000/o-translatable message, the bulk RNA extracted from unfertilized eggs had approximately 6 5 % template activity. Unfortunately, the effect of the large excess of rRNA on template activity was not determined, as it might have been, for example, in a mixture of the MS-2 standard plus egg RNA. The RNA content of various species of sea urchin egg has been estimated at 2-6 iiig (75, 7 6 ) . Consequently, the niaturc sea urchin egg may contain 0.10.3 pg of template RNA. Another examination of the template activity of egg RNA was performed by Mano and Nagano (7'7), but with a homologous systcin eniploying egg ribosomes. The degree of stimulation was substantial, but no reference for template activity was employed. A simultaneous measurcnient of the teiiiplate activities of bulk egg RNA and that of a standard, such as MS-2 RNA, in both the lioinologous system from eggs and the hcterologous system from E . coli, might furnish a more reliable estimate of the amount of mRNA in the egg. A
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change in their relative incor1)orntions could then be attributable to a specific effect related t o the use of lieterologous and homologous test systems. Another approach has been to characterize the egg RNA labeled during oogenesis. Egg RNA labeled for 1 week in owo (78) was characterized by its sedimentation properties aiid as dRNA1" by virtue of its ability t o hybridize with DNA. The bulk of the label appeared in ribosomal RNA, but as the amount that might be nonribosomal and non-4 S (from sedimentation properties) and dRNA (from its ability to form DNA-RNA hybrids) was appreciable, an upper limit of 10-15% of the labeled RNA could be set by thesc studies. A corollary criterion for the dRNA character of the egg RNA is its ability to compete with labeled dRNA from developing embryos in hybridization with DNA. Egg dRNA has been demonstrated in this way in the sea urchin (79, 80). Half of the egg inRNA decays in approximately 10-20 hours of development, as estimated roughly from the eventual decline in the rate of incorporation in protein in the presence of actinomycin (69, 70). The rates of incorporation alone, of course, may not represent rates of synthesis. The calculation of thc longevity of mRNA in the absence of further RNA synthesis is basetl on t l i r assumption that the surviving or preexisting mRNA is rate-limiting in protein synthesis (81). A value of 1&12 hours can be estimated from the decline in the content of polyribosomes during development in the presence of actinomycin (Section VI, D). The longevity of this mRNA is considerably greater than that observed in bacteria (81), rapidly growing cells (82), and other differentiating tissues (85). However, there are many examples of long-lived mRNA's, possibly responsible for the synthesis of a limited number of proteins (84-88). I n the presence of actinomycin, the endogenous mRNA concentration will eventually become rate-limiting, as evidenced by the beginning of a decline in the rate of protein synthesis. This concentration is reached in Strongylocentrotus purpuratus a t 4-6 hours after fertilization (89). Considering that half of the egg mRNA decays in 10-20 hours, we can calculate that the egg stores 1.2-1.5 times the amount of mRNA required to sustain protein synthesis a t its maximal level during early cleavage. (ii) Amphibian. Davidson et al. (90) measured the template activity '"dRNA is the RNA class related operationally to DNA by having a high proportion of base sequences complementary to the total DNA genome. This complcmentarity is detected either by a high degree of DNA-RNA duplex ("hybrid") formation or a resemblance of their overall base compositions. Therefore, dRNA is a tentative category that includes all RNA that is not ribosomal, 4s or 5S, and thus includes mRNA as a subclass.
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of RNA extracted from oocytes of Xenopus lueuis by means of the heterologous ribosomal test system from E . coli (91). With the RNA of F2 coliphage as a messenger standard, they estimated that the mature oocyte contained 47 rqpg of template RNA. By the mid-lampbrush stage (stage 41, the oocyte contained 42 mpg of template RNA. These values correspond to approximately 27%of the egg RNA. Davidson et al. (90) concluded that the large store of mRNA accumulated in the stage 4 oocyte is still present in the completed stage 6 oocyte. An additional 16 mpg of RNA detectable as dRNA was synthesized during ovulation. This value was obtained by Brown and Littna (92) by measuring the specific activities of nucleotide precursors and the amount of incorporation in oocyte RNA approximately 12 hours after administration of isotope. Up till now, differences have not been sought between the dRNA synthesized by the lampbrush-stage oocyte and that of the oocyte induced t o ovulate by the administration of pituitary hormone. Dettlaff found that oocyte maturation leading to ovulation (see Table I) is inhibited by actinomycin (92a). The antibiotic was effective only during the period of hormone activity. Once the maturation process had begun, with the breakdown of the germinal vcsicle, the antibiotic had no effect. Thus it is possible that some of the dRNA synthesized during ovulation (92),but not that from the lampbrush oocyte ( g o ) , is involved in the support of terminal oocyte maturation. The dRNA synthesized by the lampbrush oocyte does not decrease detectably during development from the cleavage to the mid-blastula stage, as evidenced by equivalent capacities of ‘ and stage 7 to compete with labeled RNA preparations from stage 2 oocyte RNA in hybridization with DNA (92b). Between blastula stages 7 and 9, the dRNA population changes markedly, with the apparent elimination of over 28% of the genetic information derived from the lampbrush stage.
2.
MRNA The cellular fraction of the unfertilized sea urchin egg containing template RNA activity was found by Maggio et al. (73) in a high speed, unpurified ribosomal or microsomal pellet. The same fraction was treated with trypsin by Monroy et al. (93) to activate endogenous protein synthesis. The conclusion from these studies was that the stored egg mRNA is in the form of inactivated complexes with ribosomes. The existence of such “inactive polysomes” in other systems has been proposed. Stable and inactive polysomes appear to be stored in the undeveloped and early embryos of Ascaris (94) and in the developing chick feather (95). In both these cases, the question of whether or not mRNA is in fact involved in OOPLASMIC SEGREGATION OF
INFORMATION TRANSFER DURING EMBRYOGENESIS
251
the structure of the observed ribosomal aggregates must be raised (95a). Their insensitivity to low concentrations of RNase is difficult t o correlate with the polysomal structure proposed. The structures in the Ascaris embryos can be made sensitive to RNase, however, by prior treatment with trypsin (94). The concept of an inactive, storage polysome includes the participation of some sort of protein protector or repressor (93). Messenger RNA activity was assigned to a different cellular fraction of the unfertilized sea urchin egg by Mano and Nagano (77), who tested the template activity of RNA extracted from the 12,000 X g pellet, the microsomes, and the 105,000X g supernatant fractions. If the homogenization was performed in the presence of 2 m M CaCl,, template activity was considerably enriched in the RNA of large (L) particles sedimenting between 8,OOO and 15,000 x g in 10 minutes. Mano and Nagano noted that the omission of CaC1, or treatment of the homogenate with trypsin resulted in a redistribution with a considerable shift of template out of this low speed pellet. Although the low speed pellet had the highest concentration of template activity (per RNA content), the largest amount of this activity appeared to be associated wih the microsomal fraction. However, evidence was given that the fractionation easily resulted in cross contamination of components, most prominently the contamination of microsomes with the much larger L-particles of the 12,OOO x g pellet. The conclusion drawn by Mano and Nagano (77) was that the template activity attributed by Maggio e t al. (73) and Monroy e t al. (93) to a ribosomal or polysomal fraction was very likely due to contamination by their L-particles. The idea of a storage of egg mRNA in the L-particles of Mano and Nagano (77) supports a previous inference drawn by Baltus e t nl. (96) from the results of a comparison of the rates of protein synthesis observed in activated nucleate and anucleate halves of eggs. The anucleate halves were considerably the more active of the two, suggesting that the egg mRNA might be attached to “heavy” particles, such :is yolk platelets, pigment, cortical granules, which might he more prevalent in the anucleate halves. This asymmetric distribution of protein synthetic activity between nucleate and anucleate halves was not, however, noted by other investigators (68, 9 7 ) . The role of egg mRNA within the animal-vegetal gradient might be evaluated on the basis of the protein synthesis in the cells derived from these ooplasmic regions. Thc amount of incorporation in cells of the early blastula, examined by autoradiography, was the same in all regions of the embryo (98-101). Vegetal micromeres separated and purified from the other blastomeres of the 16-cell embryo incorporated amino acids into protein to the same extent per unit mass as did the other cells of the
252
MARTIN NEMER
embryo (97, 1 0 2 ) . Therefore, there was no segregation of mRNA in the egg that resulted in quantitative differences in protein synthesis of early blastomeres. However, these results should not discourage a n examination of the classes of newly synthesized proteins (103, 104) in these purified vegetal micromeres, to determine whether or not there may still be substantial qualitative differences in their respective new protein populations. 3. GENOMEREPRESENTATION OF THE MRNA POPULATION
Assays of the nature of the niRNA population of the mature egg have been perforined by studying the template activity and the hybridiaahility with DTSA, especially as compared with the RNA of the late stage embryo. By comprting RNA of the mature oocyte of Xenopus Zaevis against the RNA from lampbrush stage oocytes for hybridization with DNA, Davidson et al. (90) determined that at least 60% of the RNA of the mature oocyte was the product of genes active during the midlampbrush stage. This lampbrush stage dRNA was estimated t o be synthesized through the activity of about 2.7% of the genome. Considering the large amount of template RXA present in this preparation, and the small number of genes represented by the fraction of the DNA hybridized, they (90) concluded that the dRNA molecules may be present in multiple copies, as well as representing a specialized population. The lanipbrush chromosome loops of Triturus might comprise 2-3% of the genome at one time ( 1 0 5 ), thus being in close agreement with the percentage of the genome represented in RNA synthesis in X e n o p w oocytes. A further evaluation of the dRNA population of the mature egg and early cleaving embryo was made by Denis (106) by allowing this RNA to compete against labeled RNA from several late stage embryos for hybridization with DNA. I n all cases, the egg RNA was ineffective as a competitor and thus appeared t o contain no dRNA sequences in comnion with those byntlicsizcd later in development. These data all lead to the conclusion that only a. restricted population of mRNA’s are stored in the amphibian egg to w r v c the onrly tievelopiiirnt of the cnibryo. The situation seems quite different in the sea. urchin egg. The nature of the dRNA population of the sea urchin egg has been examined by using it to compete against labeled RNA from later stages in hybridizaiton with DN-4. This egg RNA was found to compete with non-4 S, pulse-labeled RNA from hatching blastulae to the same extent as unlabeled RNA from blastulae (79). It can be concluded that the unfertilized egg RTSA exhibits either the same or a greater genomic reprexiitation thnn the blastular RNA. It had been noted by Whiteley et at. (79) that the, RNA’s of tlie unfertilized egg and the blastula behave
253
INFORMATION TRANSFER DURING EMBRYOGENESIS
similarly in competition with labeled RNA from the prism stage, both depressing the binding of the latter to DNA by 50%. Thus both the unfertilized egg and the blastula lack a considerable portion of the population of dRNA molecules produced in the later stage. The observation (80) that only about 60% of the RNA preecnt in the blastula stage is generated in the later stages iinplieh that the dRNA population of the egg contains members not prewnt in the 1:itc stages.
C. Egg Ribosomes 1. RIBOSOMAL RNA RNA extracted from ribosomes of various species of eggs in various ways has been found generally to consist of the usual 2 8 s and 18s components. No change in sedimentation properties is detected in the bulk of this RNA after fertilization of the sea urchin egg ( 7 4 ) , thus excluding a shift in the sedimentation properties of ribosomal RNA as a developmental phenomenon. Whether or not ribosomal RNA, newly synthesized in the course of development, resembles the ribosomal RNA of the egg was tested by cosedirncntation of labeled ribosomal RNL4 of developing embryos with unlabeled egg RNA ( 1 0 7 ) . New and old ribosomal RNA’s had similar sedimentation properties. Other, inorc bubtle differences in the propertics of ribosoinal RNA from eggs and embryos, such as differences in ribosonial RNA nucleotide sequences being produced a t different stages, have not been sought. A heterogeneity of ribosomes may occur in bacteria (108). Certain females of the purple hea urchins produce mature eggs whose ribosomes can be extracted with sodium dodecyl sulfate phenol a t 60°C to yield an RNA product containing a n unusual component sedimenting a t approximately 13 S (107, 109 1. The same material extracted with phenol a t 20°C yields only tlic normal 18 S and 28 S ribo~omal RNA’s; however, the 18 S coniponc.nt can be transformed into 13 S material by heating at 60°C. A likely explnnation is that there are hreaks near the middle of tlic polymer chain of the 18s RNA that are not evident a t low temperatures because of the high degree of secondary structure. The transformation may involve the formation of two fragments from the original 18s molecule. This lesion can serve as a marker to distinguish egg ribosomes from those synthesized during development. A specific degradation of either the large or small ribosomal RNA has been noted in other material (1fO-119). I n certain batches of eggs, as much as 30% of the ribosomal RNA was 13s. Essentially all the 18s RNA in these eggs had the peculiar lesion, but development was normal despite this apparent defect. Thus the ribosomes containing a lesion were
+
254
MARTIN NEMER
still capable of supporting protein synthesis. The specific concentration of the 13 S RNA remained essentially constant during development to the mesenchyme blastula, but then began to decline with a loss of half the lesion in approximately 30 hours. Newly synthesized RNA did not yield a 13 S component, and the total amount of ribosomal RNA did not increase but remained constant or declined. One interpretation of the decreased concentration of the 13s RNA is that the lesion was mended during development. More likely, the original egg ribosomes were replaced by newly synthesized ribosomes, since the decline in 13s RNA marker begins a t the time of onset of suhstantial ribosomal RNA synthesis (109). The egg ribosomes are, therefore, consumed in the course of development. 2. SUPPORT OF DEVELOPMENT AND LONGEVITY
Besides their support of early protein synthesis, another possible use for egg ribosomes might be as a biosynthetic reservoir for nucleic acid synthesis. This use may prevail in the sea urchin (Section 11, C, 1) but apparently does not in amphibian eggs, in which the egg ribosomes seem to be conserved in early embryogenesis. In oogenesis, the oocytes pass through periods of variable RNA synthesis. It is during the early lampbrush stage in amphibia that the bulk of ribosomal RNA synthesis occurs (24, 28, 124). The next period, of several months, before ovulation is one of relative quiescence. Brown and Littna (124) labeled eggs of X e m p u s laeuis with radioactive precursors during the lanipbrush stage, waited 3 to 4 months for most of the eggs to mature, and studied the developmental rate of the prelabeled ribosomes. The amount of radioactive ribosomal RNA did not change appreciably, thus indicating a conservation of egg ribosomes. A control on the degree of recycling of RNA catabolites was obtained by noting the difference in the relative incorporations in 28 S and 4 S RNA in prelabeled embryos compared with embryos exposed to Iabelcd precursor during dcvelopment since considerably more 4 S RNA than 28 S RNA was synthesized. A breakdown and reutilization during development would have resulted in a shift of ribosomal RNA label into 4s RNA, but this did not appear to occur. The consumption of egg ribosomes could thus not be detected t o any extent. During oogenesis, the egg accumulates an amount of ribosomes that may be disproportionate for a single cell (113, 114). However, the stored ribosomes of several species (109, 115-118) are used in the early embryonic stages exclusively, without new ribosomal synthesis. I n the case of the teleost Misgournis fossilis (118), ribosomes are transported from external sources in the yolk, seemingly in preference to new syn-
INFORMATION TRANSFER DURING EMBRYOGENESIS
255
thesis. I n this embryo, the intraccllular ribosoiiial content is low enough to require importation, but these conditions do not elicit new ribosomal RNA synthesis. If ribosomal synthesis is evoked in some way by a developmental need, this need apparently does not stem simply from alterations in ribosome content. The processes of early embryogenesis of amphibia also appear not to require newly synthesized ribosomes. The “0-nu” (anucleolate) mutant of Xenopus Zaevis does not synthesize rRNA (119),yet its development appears to be normal ovrr a considerable period beyond the time a t which the normal embryo first begins to synthesize rRNA. Synthesis of rRNA is first detectable normally in the gastrula of ahout 10 hours, and the overall output of RNA does not contribute measurably to the total RNA content until about the 80-hour neurula stage (115). The 0-nu mutant develops normally through this period of a t least 70 hours apparently only with the support of egg ribosomes. One might conclude that since the ribosomal function is a general one in support of protein synthesis, the egg ribosomes serve this function as adequately as new ribosomes and that new ribosomes do not perform special developmental functions. The need for more ribosomes seems to be felt eventually, and the 0-nu mutants die as abnormal swimming tadpoles (119). At this late stage, numerous lesions might very likely be traced back to ribosomal dcficits. However, close examination reveals tissue-specific differences in RNA content (120). Present evidence (119, 121) discounts the likelihood that rRNA is actually synthesized in certain tissues of this mutant. Thus it is possible that the original egg ribosomes are retained to different extents in different tissues. In any event, early embryogenesis appears to be impervious to the absence both of nucleoli (122, 123) and the synthesis of new ribosomes (119).
111. Schedules for the Activation of Protein Synthesis in Eggs At some time during oogenesis, oocytes of various species experience a period of relative quiescence with respect to protein synthesis. However, after fertilization the early cleaving embryo is generally capable of substantial protein synthesis. The transition between the quiescent and active states may require a variety of cellular events, particularly those of maturation. I n the case of the frog R a m pipiens, protein synthesis is initiated during ovulation, which occurs after administration of pituitary hormone (125).The rate of this synthesis rises during the later stages of maturation of the oocyte, but does not change as a result of fertilization. On the other hand, in the sea urchin oocyte protein synthesis
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MARTIN NEMER
diminishes in the later states of oogcnesis ( 3 4 ) . Tlic mature sca urchin egg is engaged in protein synthesis to a negligible extent until it is activated by either fertilization or parthenogenesis. Intermediate between these cases, various species may undergo fertilization during the later stages of maturation of the egg, hut fail to exhibit an increased rate of protein synthesis until maturation has been completed. These include the starfish ( l a S ) , the mollusk Spisula sotidissima (128), and the nematode Ascaris Eumbricoides (94).
Protein synthesis
-e
.-6
Fertilization Breakdown of germinal vesicle Protein
c
E
Fertilization
First meiotic division
01 0 7
Fertilization 0
.-
5 -
Second meiotic division
Protein synthesis
Fertilization Protein synthesis
FIG.1. Schedules for fertilization and activation of protein synthesis.
The characteristics of these various activations of egg activity may or may not differ a t the molecular level. Our present discussion categorizes them according to their relationship to fertilization or, equivalently here, to parthenogenetic activation. Thus there seem to be three categories for the elicitation of protein synthesis under conditions in which (a) fertilization is neither sufficient nor necessary, (b) fertilization is necessary but not sufficient, and (c) fertilization is both necessary and sufficient (Fig. 1 ) .
A. Fertilization neither Necessary nor Sufficient The last stages of oogenesis of the frog ( R a n a pipiens) have been described by Subtelny and Bradt (129). After injections of pituitary extract into mature females, full-grown oocytes are released from the ovary. Their germinal vesicles (large nuclei) break down, and maturation continues to the second meiotic metaphase. Only at this point can the eggs be fertilized or parthenogenetically activated. They then complete the second meiotic division and continue their development. Smith e t al. (125) measured the extent of protein synthesis throughout this process by injecting labeled leucine into eggs and measuring incorporation into protein after 1 hour of incubation (Table I ) . Fully grown
257
INFORMATION TRANSFER DURING EhIBRYOGENESIS
oocytes of uninjectcd frogs show low but appreciable levels of protein synthesis. Protein synthesis begins to increase before the complete breakdown of the germinal vesicle. This synthesis continues to rise apparently until the eggs become mature (capable of being activated). Afterward, the same degree of activity pcrsist.s and the process, which in the absence of activation, it is suggested ( 1 2 5 ) , may lead to degeneration in the
INCORPORATION OF
Hours after pituitary injection
TAIILE I TRITIATEI) IAEUCINE I N T O ~ . ! I ~ I N E - ~ O L U B I X PROTEIN 1-HOUR EXI’OSURE TO T H E ISOTOPEn
Morphologicltl o1)servations s t 18°C
No pituitary 4 8
W5..\ 16
----. ,Breakdown
24
of germinal vesicles
AFTER A
Counts per Num- Total minute per ber number five-egg sample, of of mean 5 frogs five-egg standard used samples error
4 1 2 2
13 4 8 6
2
8
1575 f 125
3
22
1840 f 135
2
10
3156 f 296
1
6
3358 f 119
2
11
3923 f 113
1
4
5759
1 1 1
4 4 8
4836 f 91 4434 f 207 5643 f 118
406 536 687 1025
f 34 f 59
k 44 f 51
begins First dot appears
~
’ > (First polar body 32 /
40 36 46 49 a
Second dot appears
1
Eggs activatable or fertilizable
f 87
From Smith el al. (125).
uterus (overripe eggs), continues. Further studies are required to establish thc validity of these observations, both with respect to the contrihution of amino acid pools :tnd the nature of the kinetics of incorporation ( 1 5 0 ) . It appears, then, that pituitary hormones initiate protein synthesis in R a m pipiens eggs. This observation may apply broadly, where the other physiological functions of the female and the activities of her ovaries are coordinated through hormonal regulation. Protein synthesis in the frog egg continues without increase after
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MARTIN NEMER
fertilization. Smith and Ecker (131) demonstrated that the maintenance of this protein synthesis does not depend on the presence of a nucleus. They found that fertilized, activated, or enucleated eggs synthesize protein to the same extent. These results support the previous proposal by Brachet et al. (71) that protein synthesis after fertilization does not depend on the synthesis of new RNA. These workers based their hypothesis on the observation that the inhibition of RNA synthesis by actinoinycin injected into fertilized eggs has little effect on cleavage, whereas the injection of puroniycin, which inhibits protein synthesis blocks cleavage.
B. Fertilization Necessary but Not Sufficient Three species can be taken as examples to show that the activation of protein synthesis does not follow directly after fertilization or parthenogenesis, but must await maturation of the egg. Starfish eggs (Asterias) are shed with intact germinal vesicles that break down after contact with sea water. Fertilization may then occur, but, according to Monroy and Tolis ( I f % ) , protein synthesis remains a t a constant and negligible level until the sceond polar body is given off. Not only does the rate of incorporation in protein rise sharply a t this time, but so also does the uptake of precursor (amino acids or glucose). Thus incorporation may depend on uptake. Thesc authors noted that in the eggs of the surf clam (Spisula solidissima) , fertilization is followed by the breakdown of the germinal vesicle, which in turn is followed by an increase in the rates of both uptake of precursors and incorporation into protein. The eggs of the nematode Ascaris lumbricoides, isolated from the uterus, had been fertilized 12-24 hours previously. Both meiotic divisions have occurred and a shell has formed, but the pronuclei have not yet fused. Kaulenas and Fairbairn (94) found that most of the ribosomes of the zero-day, uneinbryonated eggs exist in “polyribosomal” aggregates rather than as monoribosomes. Only relatively high concentrations of RNase are able to convert these polysomes into monoribosomes. On the other hand, pretreatment with trypsin, which had no effect by itself on the polysornal sedimentation profile, allowed conversion to monoribosomes by subsequent treatment with low concentrations of RNase. Thc polysomes of thc developing embryo became progressively inore susceptible to attack by low concentrations of RNase itself. It was concluded that additional protein is attached to the egg polyribosomes, either for protection or inhibition of activity. Treatment of an unpurified preparation of ribosomes and polysomes from zero-day eggs with trypsin resulted in a severalfold activation of protein synthesis. A fraction sedimenting between lo00 X g and 15,000 x g was also
INFORMATION TRANSFER DURING EMBRYOGENESIS
259
activated by trypsin treatment. It was difficult to decide whether these very heavy particles simply contained polysomes or were another cellular fraction concerned with protein synthesis. It was concluded that stable and inactive polyribosomes become activated during development. The incorporation of newly synthesized RNA was observed to occur in these polysomes; however, the degree of involvement of the nucleus in the support of early protein synthesis could not be gauged here.
C. Fertilization Necessary and Sufficient I n the sea urchin, protein synthesis is activated after fertilization (132, 133) or parthenogenesis (134). This activation was demonstrated in cell-free ribosomal preparations (134). Considerable evidence has been
obtained in support of the original proposal (62, 63) that mRNA is present in the egg and that its activity accounts for the observed activation and sustained protein synthesis through early development (Section 11, B). The mature unfertilized sea urchin egg has no more than 5% of its ribosomal population in the form of aggregates or polysomes ( 1 3 5 ) . Monroy and Tyler (136) noted that after fertilization polysomes form and the amount relative to monoribosomes rises in the course of development. Nemer and Infante (135) estimated that in the first 3 hours after fertilization the polyribosome level rises to include approximately 50% of the total ribosomes. The time course of this change in the proportion of ribosomes present as polysomes should indicate the actual change in the rate of protein synthesis during this period. The rise in protein synthesis appears to extend over the first 2-3 hours after fertilization. Studies of incorporation into protein in vitro or in vivo indicate various time courses from abrupt to gradual increases after fertilization (114, 137-140). However, changes in rates of incorporation have all too frequently been interpreted as changes in rate of protein synthesis. Work directed toward the mechanism of activation has led to the view that a block is removed a t the level of translation. The present evidence supports the idea of a removal of repressor protein both from the ribosomes and from the mRNA stjored in the egg. We discuss below the release of ribosomal and mRNA activity as well as the involvement of other components, such as amino acid transfer activity.
IV. Activation of Protein Synthesis in the S e a Urchin Egg A. Amino Acid Transfer Activity The unfertilized sea urchin egg has substantial amino acid activating activity (141, 142) as gauged by the exchange reaction between ATP
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MARTIN NEMER
and pyrophosphate catalyzed by amino acids. Soon after fertilization and through the early cleavage stages, substantial addition of the terminal -pCpCpA sequences to tRNA is detected (143, 144). This incorporation is greater than that attributed to de novo RNA synthesis. It is possible that this activity is important in the activation of protein synthesis, but likely not rate-limiting. Whether or not this terminal addition increases upon fertilization cannot be ascertained from present evidence. An evaluation of the soluble components of the protein-synthesizing system in the unfertilized egg was made by Hultin (134), who found that the 105,000 x g supernatant from unfertilized eggs supports protein synthesis by ribosomes of fertilized eggs to the same extent as the TABLE I1 POLYPHENYLALANINE SYNTHESIS DIRECTED BY EXCESS POLYURIDYLIC ACIDIN THE PRESENCE AND ABSENCEOF ADDED YEASTsRXA BY POSTMI~TOCHONDRIAL SUPERNATANTS FROM EGGSA N D EMBRYOS OF Lytechinus petus' ~~
~
Phenylalanine incorporation (mpmole/45 min)
Stage
Unfertilized Zygote, 1-hour Blastula, 12-hour Gastrula, 24-hour
-tRNA (limiting)
+tRNA (in exces6)
Relative endogenous tRNA activity
3.9 4.3
42 53 96 90
1.0 1.3 3.8 5.7
14.7 22.2
From Nemer and Bard (114).
supernatant from the fertilized eggs. On the other hand, Candelas and Iverson (f 44a) obtained a considerably greater stimulation with supernatant from fertilized eggs than from unfertilized eggs. The nature of this soluble component has yet to be elucidated. Nemer and Bard (114) compared endogenous protein synthesis by postmitochondrial supernatants, prepared from eggs and embryos a t various stages, in the presence and absence of added heterologous, yoast tRNA (Table 11). In all cases, the rates of incorporation in protein were the same in the presence and absence of added tRNA. Thus i t appears that the content of functional tRNA is not rate-limiting in protein synthesis a t any stage, including the unfertilized egg. The heterologous preparation could well be lacking some species of tRNA ; however, adequate amounts of functional phenylalanine and leucine tRNA's were demonstrated. Although no response of endogenous protein synthesis was obtained by the addition of tRNA to the system derived from unfertilized eggs, a
INFORMATION TRANSFER DURING EMBRYOGENESIS
261
severalfold increase in phenylalanine incorporation was obtained by this addition in the presence of excess poly U. Thus the content of tRNA specific for phenylalanine was enough t o sustain endogenous protein synthesis, but not enough to support the maximum amount of poly U-directed polyphenylalanine synthesis of which the system was capable. That is, the capacity of the enzymatic and ribosomal components for polypeptide synthesis far outstripped the supply of tRNA available in the unfertilized egg. The same was true for embryonic stages up to gastrulation (114) ; however, the endogenous concentration of tRNA was able to support increasingly more polypeptide synthesis as development proceeded. The 24-hour gastrula had approximately 6 times the tRNA activity in the unfertilized egg with respect to phenylalanine and twice the amount in the egg with respect to leucine (114). As a corollary to these studies, a much greater content of ribosomal RNA than 4 5 RNA has been observed in the eggs of sea urchins (107, 109) and amphibians (113). I n each case, the content of 4 S RNA rises relative to that of the ribosomal RNA during development (107, 109, 1IS).
B. Release of Ribosomal Activity Hultin (134) suggested that the ribosomes of the egg might require activation, but his attempts to effect such an activation were only minimally successful. However, Rlonroy et al. (93) were able to activate the ribosomal system derived from the unfertilized egg by incubation with trypsin. The material they used was the resuspended unpurified ribosomal pellet. Two important results were obtained. The egg ribosomes, which were not able t o respond to “natural” mRNA derived from either eggs or blastulae, were made responsive to this RNA by the trypsin treatment and the response to the synthetic messenger poly U, which was low in these preparations, W U ~also considerably enhanced. The second result involved the elicitation by trypsin treatment of endogenous protein synthesis without further addition of inRNA. It was this response and the association of template RNA activity with egg microsomes (73) that led hlonroy et al. (93) t o the conclusion that the egg stores mRNA as inactive complexes with ribosomes and that these are activated by proteolytic removal of a protein coat. The previous observations by Lundblad (145-147) showing a pronounced increase in protease activity following fertilization, lend credence to the involvement in vivo of a proteolytic yelease of activity. Treatment with trypsin was also used successfully by Salb and Marcus (148) to activate poly U-directed polypeptide synthesis in ribosomes derived from mammalian tissue culture cells in metaphase, known to have a reduced rate of protein synthesis (14.9). Although this effect of trypsin treatment
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MARTIN NEMER
was similar to that obtained by Monroy et al. (93) with respect to enhanced receptivity to exogenous messenger polyribonucleotides, a n activation of endogenous messenger activity was not obtained with the metaphase ribosomes. It is therefore possible that the two activations are not entirely equivalent. The enhanced receptivity to exogenous mRNA in both cases indicate that ribosomes are being released from a repression of activity due to a protein. On the other hand, the increase in endogenous mRNA activity in the system derived from the egg may arise principally from an effect on the activity of the stored egg mRNA itself. Two laboratories have examined the rates of protein synthesis during the course of the highly synchronous cell divisions of the early cleaving embryo. Their results were in seeming conflict, Gross and Fry (139) reporting no discontinuity in the rate of protein synthesis, Sofer e t al. (140) noting a decline in this rate of metaphase. Timourian ( 1 5 0 ) , however, then revealed that in similar studies he had obtained a high degree of variability, thus not allowing a choice between the two alternatives. Gross and Fry (139) suggested that if nuclear proteins were responsible for the inhibition observed in tissue culture cells in metaphase, as proposed by Salb and Marcus (148), then their effect in the metaphase cells of the early embryo must be attenuated in the relatively great mass of cytoplasm. Thus, in the early embryo, metaphase ribosomes might be partially repressed. But a t the same time endogenous mRNA activity is being released by a proteolytic activity that also counters the ribosomal repression. This combination of events might well yield indeterminate experimental results. Furthermore, just as the metaphase ribosomes may be blocked by material released from the nucleus (148),the activity of the egg ribosomes may also be blocked (93) by the material released during the breakdown of the germinal vesicle in the last stages of oogenesis.
C. Release of
Messenger RNA Activity A composite of various experiments may be drawn upon to examine the mobilization of mRNA stored in the sea urchin egg (Section 11, B ) and its involvement in the activation of protein synthesis following fertilization. The observation that the addition of poly U to ribosomes of unfertilized and fertilized eggs elicits the same degrees of stimulation of polypeptide synthesis led to the proposal that the lack of endogenous mRNA activity, and not an absolute deficiency in ribosomal activity, is the basis of the unfertilized egg’s inactivity (151, 162). The egg ribosomes of four species of sea urchin-Arbacia punctulata (151), Psammechinus miliaris (152), Strongylocentrotw purpuratus ( 1 5 3 ) , and
INFORMATION TRANSFER DURING EMBRYOCENESIS
263
Lytechinus pictus (114, 153)-rcspond markedly to the addition of poly U or poly (U, C). The egg ribosomes of two other species, Hemicentrotus pulchem’mus and Pseudocentrotus depressus, are also stimulated by natural mRNA template derived from eggs and embryos ( 7 7 ) .A seventh species, Paracentrotus lividus, contains egg ribosomes that do not respond to natural mRNA (93) and only minimally to poly U (93, 154) unless they are first treated with trypsin (93). However, trypsin treatment of the purified ribosomes from H . pulcherrimus did not alter their already substantial capacity to respond to natural exogenous mRNA ( 7 7 ) . It is thus possible that only to a certain extent, depending on the species, the egg ribosomes may be repressed by adjunct protein in a manner similar to the depression of ribosomal activity in metaphase tissue culture cells. The major part of the activation of protein synthesis can be attributed to mRNA newly made available (11.4) from stores in the unfertilized egg (62, 63). During the first hour after fertilization of A . punctuzata, the stimulation of cell-free extracts by poly U increases less than 1.5-fold, whereas the rate of endogenous protein synthesis rises approximately twentyfold (114) (Fig. 2). There appear, then, to be two types of change: (a) A n increase in the activity of nonmessenger RNA components, either ribosomes or polypeptide polymerizing enzymes. This was the small 1.5-fold change observed with excess poly U and thus independent of endogenous mRNA activity. The use of the response to poly U as indicator of the activity of free or viable ribosomes may be considered valid, insofar as this synthetic polyribonucleotide, in spite of its high affinity for ribosomes, cannot react readily with ribosomes already occupied by mRNA. The need for a prior incubation, presumably to displace endogenous mRNA from ribosomes active in protein synthesis in order to render them responsive to poly U, has been shown in various systems (91, 93). (b) A n increase in endogenous activity, attributed to the release of egg m R N A . The relative extents of these two kinds of activation may well vary among species and indeed between batches of eggs ( l i d ) . The release of mRNA activity by the treatment of egg microsomes with trypsin led Monroy et al. (93) to propose that the egg mRNA is bound to ribosomes, which are themselves inactivated by adjunct protein. Further examination by Mano and Nagano (77) revealed that the template RNA is not predominantly associated with ribosomes, but is considerably enriched in much larger particles (Section 11, B, 2 ) . Mano and Nagano (77) furnished evidence that the microsome fraction of Monroy et al. (93) could have been contaminated with the more rapidly sedimenting particles (“L-particles”) that contain template
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activity. They were able to release template RNA from the L-particles by trypsin treatment. This release appears to have a counterpart in vioo, since a specific protease activity can be demonstrated to arise rapidly after fertilization, and this activity is associated with the cellular fraction containing the L-particles. This protease displays optimal
Minutes after fertilization
FIG.2. Polyphenylalanine and protein synthesis by the postmitochondrial supernatant fluids of Arbacia punctulata before and after fertilization. Rate of endogenous incorporation of phenylalanine in protein (filled circles), rate of incorporation in polyphenylalanine (open circles). The incorporations in protein were multiplied by 20, to give values of polypeptide bond formation. From Nemer and Bard (114).
activity a t pH 8 and is trypsin-like by virtue of its ability to hydrolyze benzoylarginine amide and to be inhibited by soybean trypsin inhibitor (155). Mano (155) suggested that a primary step in the activation of protein synthesis may be the removal of an inhibitor of the protease.
D. Scheme for a Dual Activation The evidence for a dual activation ( 1 1 4 ) of protein synthesis following fertilization can be ordered into a simple sequence of three steps
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(Fig. 3 ) . ( a ) The L-particles ( 7 7 ) , containing both protease and messcnger ribonucleoprotein (mRNP), may receive the primary signal of activation. The activation of the protease in these particles may be related to the changes, following fertilization, observed in cytoplasmic granules of the egg (156). Among these, the cortical granules, which apparently disappear after fertilization (156), might serve as repositories of template RNA (96). The protease may remove protein from mRNP, causing a release of niRNA from the L-particles. (b) Next, the released egg mRNA may react with those ribosomes that are free of any attached protein, that might serve as repressor (93) to form polyribosomes (136).(c) Finally, the protease of the L-particles may react with any protein attached to ribosomes, thus freeing the ribosomes for participation in polypeptide synthesis (93, 148). The proportion of egg ribosomes requiring this activation may depend on the species and be variable within each species. A speculative method for estimating the L- particles
Protein-ribosomes L F r e e ribosomes
3
FIG.3. Scheme for the dual artivation of protein synthesis in the egg.
proportions of egg ribosonies under repression might bc devised from the changes in their response to poly U. With trypsin treatment, the ribosomes of the eggs of P . lividus are activated 6-fold with respect to this response (93). Thus 84% of the ribosomal population may have been repressed. The activation due to fertilization of A . punctulata is 1.4-fold (114), suggesting that only 25% of the ribosomes are repressed. In the case of H . pulcherm'naus there is no activation of egg ribosomes with trypsin (77), and thus there are very few, if any, repressed ribosomes. The major focus of activation in this species is in the release of egg mRNA. The nature of the ooplasmic site of mRNA storage is not entirely settled by this scheme. An indeterminate portion of the mRNA may alternatively be bound to ribosomes, forming inactive polysomal structures (93). The trigger to this sequence of events has yet to be discovered. Although the initiation of protein synthesis occurs as early as 6 to 10 minutes after fertilization (156a),i t is comparatively late compared to several other processes that are activated by fertilization. There is thus no simultaneity hetween the initiation of protein synthesis and such events as the breakdown of cortical granules or the release of proteasc activity. On the other hand, we might ask whether or not the
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earlier responses to fertilization are prerequisite t o the activation of protein synthesis.
V. Transitions in Genic Activity A. Developmental Changes in RNA Transcription RNA transcription can be divided into two categories of developmental significance: (a) the apportionment of genic activity for the synthesis of the functional classes of rRNA, tRNA, and mRNA; and (b) the output of mRNA’s, each of which specifies a particular protein. The synthesis of rRNA occurs by an initial transcription of a 45 S RNA (157, 158), followed by conversion into 28 S and 18 S rRNA’s via several intermediate forms (159).Whether or not tRNA is transcribed directly as such from the DNA is not known. The synthesis of mRNA is now thought to occur a t least in part through the transcription of very high molecular weight precursors (160-163). These suspected precursors belong to a class of dRNA (DNA-like RNA) molecules that sediment heterogeneously from about 10s to 100s. The high molecular weight dRNA is to a large extent strictly nuclear (163), and the great preponderance of the RNA sequences in it are not represented in the cytoplasm (164). Whereas this cytoplasmic dRNA may be presumed to be identical to mRNA template for the synthesis of specific protein, the function of the bulk of the dRNA that is produced, never to leave the nucleus, is not known. These nuclear dRNA’s, if they are not merely expendable parts of mRNA precursors, may be surmised to have some regulatory role in nuclear activity. At any rate, we should be cautious about classifying mRNA as other than a subclass of dRNA’s. Another general class of RNA is the so-called “5 S RNA” (159, 160). I t s function is not known, although it has been associated both in its cellular location (165) and its production (166) with rRNA. 1. DNA-LIKE RNA (DRNA)
The intense activity of the early oocyte followed by a relative quiescence and later by a renewal of activity before or after fertilization describes a schedule for RNA synthesis resembling that for protein synthesis, and may be similarly characteristic for each species (Section 111). The immature oocyte of the starfish is active in RNA synthesis (SO), especially a t the site of its large nucleolus (167-173), which incorporates precursors into transfer RNA (164) and ribosomal RNA ( 1 7 2 ) . The structure of this nucleolus lends itself to a ready isolation and characterization (174).I n contrast to this single, large, echinoderm nucleolus, the nucleoli of amphibian oocytes, exemplified by Triturus
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( 175-1 7 7 ), consist of several hundred structures. Both extremes of nucleolar organization represent parts of the highly specialized nucleus of the immature oocyte, engaged in intense synthesis of egg ribosomes. The immature sea urchin oocytes, suspended in sea water, take up labeled uridine and incorporate i t extensively into RNA (34). Similarly incubated mature sea urchin eggs do not incorporate either labeled uridine or inorganic phosphate into RNA (34, 143). I n none of these measurements was it ascertained to what extent the incorporation into RNA might have been limited by the low permeability of the mature egg (178). Considerably higher concentrations of labeled uridine were used in other experiments ( l o g ) ,but only background bacterial contamination (143) could be detected as a result, However, fertilized eggs showed an increased incorporation and a pattern of sedimenting RNA components t ha t differed markedly, being heterogeneous with a prominent 4 S RNA label, from the background detected with unfertilized eggs (109). It appears, then, that the mature egg is essentially quiescent with respect to RNA synthesis. The reactivation of the genome of the sea urchin egg might be expected to occur some time after fertilization. The inability of various investigators (14.3, 179, 180) to detect RNA synthesis in the early cleaving embryo is not surprising considering the possibly large intermediary precursor pools (178, 181) and the low specific permeabilities of the unfertilized egg and recently fertilized egg (178, 189-185). The possibility that in the early cleaving embryo there is no de novo synthesis of RNA was raised by the observation that terminal addition t o tRNA seemed to account for the predominant portion of RNA incorporation at this time (I@). The failure to obtain other RNA species may have been due to incomplete extraction, since incorporation in heterogeneously sedimenting components of pulse-labeled RNA was indeed observed in other studies within the first hour after fertilization (109, 186-188). The RNA synthesized during middle and late cleavage stages was characterized as DNA-like in base composition (144). Furthermore, the rapidly labeled RNA of the 4-cell embryo was substantially of high molecular weight, heterogeneously sedimenting and specifically hybridizable with sea urchin DNA in high proportion (189).The successful detection of incorporation of pulse-labeled RNA in the early cleaving sea urchin embryo can be attributed to the use of very high concentrations of exogenous, labeled precursors. It is probable that de novo synthesis of RNA by the sea urchin embryo begins very soon after fertilization. This newly synthesized RNA, according to hybridization studies ( 18 9 ) , is almost exclusively dRNA. Mature amphibian eggs and embryos are fairly impermeable to the
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commonly employed RNA precursors. However, substantially labeled precursor pools could be built up in the oocyte by injection of isotope into gravid Xenopus females during the quiescent period following the vegetative stage of oogenesis, with little if any incorporation occurring in egg RNA. The administration of pituitary hormone (190) evokes further maturation of oocytes and appreciable incorporation in dRNA and 4 s RNA (115). This labeled RNA appears in cytoplasmic extracts associated with ribosomes (115, 116), possibly correlated with the observed protein synthesis activated by the hormone a t this time ( 1 2 5 ) . The same degree of incorporation persists after fertilization and through the cleavage stages. The labeling pattern noted in sedimentation analyses tends to obscure any other RNA incorporation that may take place during the early period of development. The observation that during development the prelabeled precursor pools of nucleotides change only slightly in specific activity allowed Brown and Littna (92) to calculate the amounts of each RNA class newly synthesized and accumulated. The proportion of newly synthesized, high molecular weight RNA that was dRNA could be estimated from the base composition, compared to the compositions of rRNA and DNA. Thus the accumulation of dRNA was measured during embryogenesis, and its steady-state amount was proportional to the increase in DNA content. An interpretation of this relationship might be that dRNA accumulation is quantitatively proportional to the number of cells; recent evidence indicates that it is qualitatively dissimilar in different parts of the embryo (191). Brown and Gurdon (119, 192) took advantage of the lack of rRNA synthesis in the O-nu mutant of Senopus in their characterization of dRNA. All the high molecular weight RNA synthesized by this mutant could be regarded as dRNA. Two general dRNA classes of apparent developmental significance were noted in the heterogcneously sedimenting components. The material greater than 20 S, which they called “heavy dRNA,” might be the same high molecular weight dRNA of the nucleus (163, 164) thought to be in part a precursor of cytoplasmic dRNA or mRNA. Indeed, Brown and Gurdon (192) presented evidence from pulse-chase experiments that the heavy dRNA is a t least partly a precursor of a “light” (less than 20 S) dRNA fraction. The heavy dRNA appeared in great preponderance after pulse incubations, but following an extensive chase incubation there was an accumulation of the light dRNA. I n any case, the light dRNA appeared to be considerably more stable than the heavy dRNA. This light dRNA might well contain a class of stable mRNA’s. Brown and Gurdon (192) also noted that the new RNA of the early embryos is predominantly heavy dRNA, whereas
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the late stage embryos tended to accumulate light dRNA. During development, the steady-state level of dRNA rises (92), and the dRNA population becomes represented in increasing proportion by previously synthesized members (106). There may then be a preferential accumulation of a portion of the immediate gene products, the heavy dRNA, in the form of light dRNA or mRNA. 2. RIBOSOMAL RNA
The onset of ribosomal RNA synthesis in the sea urchin appears to occur some time hetween the mrsenchymc blastula and gastrula stages (109, 180). The accumulation of labeled precursor in RNA has been followed after relatively long-trrm or chase incubations and is found not to be appreciably associated with rRNA before the mesenchyme blastula stage (about 20 hours in Strungylocentrotus pupuratus) (109). I n the course of development, the relative output of the different classes of RNA can be expected to change. The pulse-labeled RNA may be taken as the instantaneous gene product, whose composition reflects the composition of active gene sites. The high molecular weight (non4 s ) portion of the pulse-labeled RNA from various embryonic stages has bcen examined by hybridization with DNA in the presence of excess unlabeled ribosomal RNA (135, 189). Under these conditions, nonhybridizing, labeled RNA could bc considered rRNA, and the hybridizable labeled RNA could be considered dRNA. Relatively more rRNA compared to dRNA could be detected in the pulse-labeled RNA as development proceeded, since the extent of hybridization was observed to decline. There was thus a relative increase in the number of cistrons active in rRNA synthesis or the output of rRNA per cistron compared to dRNA synthesis. At about the mesenchyme blastula stage, this relative rate of rRNA output was c1i:tnging maximally. It is a t this time that nucleoli first appear (1I S ) . The synthesis of ribosoin:il HNA cannot be detected unequivocally in Xenopus laevis until the 10-hour gastrula dorsal lip stage (92, 1 1 5 ) . Thc total RNA content, niohtly rRNA, rcmains a t about 4 pg/emhryo froin fertilization to thc neuriiln Aagc (30-37 hours). New rRNA is accumulated a t increasing rates, SO that a t 20 hours, 50 mpg of the rRNA is new, and a t 50 hours, 500 m p g is new and contributing substantially to the total. The accumulation of rRNA is not a function of the rate of DNA synthesis and thus of cell number, as is the case with dRNA and tRNA (92, 166) (Fig. 4 1.Instead, the increase seems to reflect an increasing number of synthetic loci rclative to the production of DNA. Consequently, either ( i ) :in incrcasing number of genes for rRNA production are act1vatc.d in each cell or (ii) the rates of rRNA
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synthesis differed among cells. I n support of the first alternative is the observation that nucleoli, which have been extensively correlated with rRNA synthesis (see 19.3, 194), appear in possibly all the embryonic cells during a discrete period around gastrulation (195). Thus rRNA synthesis may be occurring in all cells. Also, Gurdon and Brown (196) concluded that the dorsal and ventral halves of tadpoles synthesize rRNA to the same extent relative to their RNA contents. However, although all cells have nucleoli, they may still synthesize ribosomes to
Time (hr) at 22 OC 1 1 1
1
I
I
I
9 I1 14 22 26 35 Niruwkoop-Faber stages
FIG.4. Amounts of dRNA-P" and rRNA-P3a present in the cmhryo of Xenopus laevi,v at, each stage of devrlopment. From Brown and Littna (98).
different extents, and these differences may not be detectable simply between arbitrary halves of the embryo. The problem is worth pursuing, since cellular differentiation may be expected to bring about conditions variably conducive to rRNA synthesis among cells. From the data of Esper and Barr (120), a differential sensitivity among tissues to the deprivation of new rRNA synthesis seems possible. They observed that the differences in RNA content between tissues from the homozygous anucleolate (0-nu) embryos and normal embryos of X'enopus laevis differ markedly from tissue to tissue and vary with stage.
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I n both the echinoid and ainphibian egg there is an amount of ribosomes apparently far beyond the immediate needs of the early embryo. The same situation appears to be true for the fish egg, which is supplied ribosomes from external yolk. The embryo of each seems then to face a biosynthetic problem, which may be directed toward a balanced distribution of the components of the protein synthesizing SYStem. The absence of ribosome synthesis in the early stages seems to satisfy the purpose of restoring this balance. The genes involved in rRNA synthesis can then be described as repressed, and so thus may be the forination of nucleoli. The signal to elicit new rRNA synthesis seems to tie linked to the process of development, rather than to an immediate requirement for new ribosomes. This signal is apparently given long before new ribosomes need to be used, and thus resembles other developniental signals that seem to anticipate the prospective course of development (see Section VI, B) .
3. LOWMOLECULAR WEIGHTRNA The content of 4s RNA in the egg of Xenopus laevis is only about 192 of the total RNA, and that of 5 S RNA 1.5 to 2% (166). Incorporation in tRNA during ovulation and early cleavage is all terminal addition. New synthesis of 4s RNA begins in the late blastula. From about 2 hours before gastrulation and the onset of rRNA synthesis, 4s RNA begins to accumulate in proportion to the accumulation of DNA and to the rate of cell division. Approximately 10% of the 4 S RNA is attached to ribosomes in the unfertilized egg. I n contrast all of the 5 s RNA is bound to ribosomes. The content of 5 s RNA eventually rises during development in parallel to the rise of new rRNA, indicating a possible coordination between the synthesis of 5 S RNA and rRNA. The initiation of tRNA synthesis appears to occur later than the cleavage stages in the sea urchin, since essentially all of the incorporation in 4 S RNA in the cleaving embryo can be accounted for by terminal addition (149, 144). The methylation of this RNA has been used as an indication of the extent of its de nouo synthesis (180, 197). Incorporation of the labeled methyl group of methionine in 4 s RNA was first detected in the mesenchyme blastula stage (at 14 hours in Lytechinus variegatus) .
6. Changing Messenger RNA Populations 1. EMBRYOGENESIS
Denis (106) has determined what portions of the genome are active in dRNA transcription and the composition of the dRNA population
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present at individual stages in the eiiibryogenesis of Senopus laevis. He used two different procedures involving the formation of duplexes between DNA and RNA: (a) the measurement of the levels of saturation of DNA by labeled RNA preparations and (b) the competition between labeled RNA and unlabeled RNA of the same or different stages. The dRNA populations examined a t each stage were of two origins. One was obtained by pulse incubation for 1-11 hours with C"O,, and represented the RNA specifically transcribed a t that stage. The other was derived by continuous incubation from the outset of development, utilizing P3?-inorganic phosphate originally introduced into the oocyte by injection of females (190).This labeled RNA represented the dRNA accumulated throughout development to and including the stage in question. These were not necessarily the same populations of molecules at each stage, but they did have sonic members in common. They differed extensively in the levels a t which they saturated DNA and in the degree of competition with unlabelcd RNA of the same and different stages. The results obtained by Denis (108) have been summarized in Fig. 5 . The amounts of dRNA either transcribed a t the gastrula, tail-bud, or tadpole stages or accumulated during development up to these stages have been represented by the designated areas. The content of dRNA rose during development from 12 nipg/embryo in the gastrula to 34 mpg in the tail-bud stage, and 78 mpg in the tadpole. An increasing portion of this population was represented by stable molecules. Of the total dRNA in the tail-bud embryo, 30% (10 mpg) was synthesized prior to that stage, and 46% (36 mpg) of the tadpole dRNA was synthesized before that stage. This population of stable dRNA molecules increased also in its genomic representation. There was no detectable stable dRNA in the gastrula. Some of the DNA sites active in the gastrula appear to be unique to that stage (stippled area in Fig. 5 ) and are no longer active in subsequent development; other sites continue to be active in later stages. These relationships were ascertained from the degree of competition by RNA preparations from the later stages, as well as from double saturation experiments involving these RNA combinations. A certain portion of these DNA sites persist in activity throughout development to the tadpole stage. The dRNA population of the tadpole consists not only of previously synthesized, relatively stablc molcdes, but also of a class of rapidly turning over moleculcs in multiple copies, that have becn transcribed a t that stage from a small segment of the genome (note the solid area of Fig. 5 ) . A portion of thc dRNA moleculcs transcribed by the gastrula are no longer detectable afterward. However, a certain portion as well as some that are synthesized by the tail-bud embryo (open area of Fig. 5 ) can be detected in
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1:itc.r stages, :ilthough tlicy arv 1 1 0 lo~igcrk i n g syntlicsizcd. One might enpcet that tlic average 1i:ilf-lifc of the tlRNA population should increase with development. Unfortunntcly, the persistence of highly radioactive precursor pools during pulse-chase experiments has interfered STAGE 12
A:
STAGE 26 TO 28
3.9
1<1(;.5. Diagram showing the relative nhundance of complementary RNA in ct:ul)ryos of Xenopus Zaeuis at different stages. The hatched areas correspond to stahlr RNA, i.e., RNA not labeled by C40,after an 11-hour pulse, but labeled by Pl when the latter is presrnt from the beginning of development. T h r solid areas rorrespond to the dRNA (mRNA), which can be labeled by an 11-hour pulse in stage 42 embryos. The dRNA (mRNA) transcribed on the same DNA sites in rarlier embryos is marked in the same way. In thcsc embryos, the pulse-labeled RNA is represented by the sum of solid and open areas. The length of the blocks is proportional to the percentage of DNA that, can be saturated by the R5.4 of each cIass and is a measure of the number of sitrs transcribed. R.NA present in the gastrula but not in later embryos is indicated by the separate, stippled block at the top left. The area of the blocks is proportional t o the quantity of hybridizahle RNA present. The height of the blocks is given in arbitrary units ohtained by dividing the amount of dRNA (mRNA), in mpglembryo, in stable and unstable forms by the percentage of DNA t o which rach class of dRNA is complementary. This height is a measure of the awrage number of copies of each class of dRNA (mRNA) . From Denis (106).
with a demonstration of surh R difference (106). On the other hand, the demonstration by Brown a n d Gnrdon (192) of a shift during development from a preponderancr of rapidly turning over hravy dRNA to a more stable light tlRNA docs woke the possibility of R correspond-
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ence between these respective classes and the transient and stable classes of dRNA, reported by Denis (106). Among the implications of these findings, according to Denis, is an indication of the manner in which the flow of genetic information might be regulated. Eliminated is the possibility that the complete release of genetic information occurs a t the onset of differentiation. It can be expected that the release of genetic information is controlled a t the level of transcription but also a t the level of translation, since the increasing prevalence of stable mRNA leads one to expect some regulation of its use. Since tissues do not differentiate synchronously in embryonic development, the differentiation of a specific tissue might be a predominant event a t a given time. Thus Denis suggests that the messengers involved with such specific tissue development might also predominate a t the same period in development. A closer correlation between specific tissue differentiations and specific messenger populations has been observed by Flickinger e t al. (191) in their comparison of the dRNA populations of the animal and vegetal halves of blastulae as well as the dorsal and ventral halves of tnil-bud cinbryos of Rana pipiens. The method was that of the double saturation plateau, whereby DNA was first saturated with the labeled RNA of one preparation, then tested for the availability of further complementary sites for hybridization with another RNA preparation. In each combination, DNA sites were discovered that were uniquely active in one half or the other. Thus as early in development as the stage 9 blastula, a differential transcription of dRNA could be observed among cells of different parts of the embryo. Their lengthy, 12-hour incubation of the embryo halves probably occurred with a fair amount of further development from their original stage 9 blastulae (191). We should like to know indeed just how much earlier in development such a differential genic activity occurs. I n contrast to the finding of Denis (106) that the dRNA of the egg and cleaving embryo has nothing in common with the dRNA synthesized by the late stage embryos, such as gastrula and tadpole, similar competition experiments indicate that the sea urchin egg contains RNA that can compete with approximately 40% of dRNA from prisms (79) and with essentially 100% of the dRNA of blastulae (80). However, as development proceeds to the gastrula and prism stages, dRNA is synthesized that is apparently characteristic of each stage. 2. ORCANOGENESIS
If the RNA’s from individual adult organs are used to compete in hybridization studies against labeled embryonic RNA, very low levels
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of homology are observed, indicating that in the differentiated tissue a restricted population of messengers exists. Whiteley et al. (7’9) found that the RNA of testes and gut from adult sea urchins had little effect on the hybridization of labeled RNA from prismoid embryos. Denis (106) found similarly that RNA from adult heart or adult liver competes poorly with labeled RNA from either gastrulae or swimming tadpoles. However, RNA from the whole adult competes strongly with the embryonic RNA. Thus a large fraction of the RNA synthesized by embryos may be present in adult cells. The process of individual organ formation, however, appears to involve n steady decline in the diversity of the dRNA population. Church and McCarthy (198) studied developmental changes in the synthesis of dRNA during both the organogenesis and the regeneration of mouse liver. RNA was extracted from embryonic livers a t the earliest feasible time, 14 days, and a t times subsequently to term (19 days). The RNA of the 14-day liver competed to a lower degree with labeled adult liver RNA than liver RNA preparations from any other embryonic stage. Thus a t its earliest point the embryonic liver has an RNA population that is the most divergent from the adult liver, in that it is the most lacking in characteristic adult liver RNA’s. Moreover, the 14-day liver contains the population of RNA molecules with the largest number of unique members among the embryonic liver RNA’s, since the RNA’s from the livers of the adult and the later embryonic stages compete the most poorly with the labeled 14-day liver RNA, as compared with other combinations used for competition. I n mutual competitions between embryonic and adult liver RNA’s with l-hour regenerating liver RNA from partially hepatectomized adults, the RNA population of the 14-day liver bore the closest resemblance to regenerating liver RNA. The similarity of these RNA populations indicates that genes specifically active in liver development have been repressed in the adult liver, but have been reactivated a t the outsct of regrneration. During organogenesis of the liver from day 14 to term the liver RNA became increasingly similar to that of the adult liver, but a t intermediate stages unique species arose and were present transiently.
C. Nucleocytoplasmic Interactions The blastula nuclei of several amphibian species are able to support the complete normal deveIopment of eggs into which they have been transplanted (7, 8, 199). However, in the course of further development, progressive changes in the nuclei restrict their ability to promote the normal development of test eggs (7, 200-204). Similarly, nuclear transplantations could be made between species of frogs (205) or toads (206).
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If the “nucleocytoplasmic hybrids’’ did not form early lethal hybrids, the transplanted nuclei a t an early stage before developmental arrest could be returned to homologous eggs to promote normal development (207). However, further development in the foreign cytoplasm eventually altered the nuclei in definite and consistent ways. Thus a simple exposure to foreign cytoplasm was not enough to cause irreversible nuclear alterations. An important physiological disparity between early and late embryos is in the durations of their division cycles. I n the cleaving amphibian embryo there is no G, phase and the G, phase is either very short or absent (208). I n going from late blastula t o early tail bud, the GI, S (DNA synthesizing), and G, phases increase, while the duration of metaphase remains relatively constant. Nuclei from late embryonic stages, which do not show appreciable DNA synthesis, do incorporate labeled thymidine within 90 minutes after transplantation into enucleated egg cytoplasm (209). This effect of recipient cytoplasm is analogous t o the effect in Am’oeba (10) of the cytoplasm of an S-phase (DNA synthesizing) cell on the transplanted nucleus of a G, cell. I n this case, the inactive nucleus begins to synthesize DNA. Since the period of DNA synthesis in the later embryonic stages is many times extended, the donor nuclei from these stages may not have completed the replication of their DNA before transplantation into the rapidly cleaving egg (204). Consequent errors in replication could lead to variable and indetermine chromosomal anomalies. A large proportion of nuclei transplanted into egg cytoplasm from late stage embryos thus experiences various degrees of chromosomal abnormalities ( 2 0 2 ) . The most severe abnormalities accompany arrest before apparent cellular differentiation occurs. The less pronounced abnormalities allow extended but abnormal development. A small group of transplanted nuclei has the standard karyotype, although undetectable lesions in chromosomes may be present. Within this group there are two classes. ( a ) There are nuclei that promote development in abnormal but characteristic ways. Nuclei of ectodermal origin afford good dcvelopmcnt of ertoclerrnal structures but arrest abnormally with deficient rnesodcrmal derivatives (202). Nuclei of endodermal origin give good development of endodermal structures but arrest abnormally with deficiencies in ectodermal and mesodermal derivatives (203, 204). (b) A very small number of nuclei of very late stage embryos are capable of promoting complete normal development (220a12 ) . The transplanted nuclei undergo structural and functional changes. The salient changes are the disappearance of the nucleolus (9) and a manyfold swelling of the whole nucleus (9, 613, 21.4). An intestinal
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epithelium cell nucleus increases from a volume of 160 p3 to 4500 p3 40 minutes after transplantation (9). Blastula nuclei of about 1900 ps increase to a similar size during the same period after transplantation (196). The nuclei become progressively smaller during the course of development. The nuclear swelling occurred during a period of 9Q minutes after transplantation, and this period was followed by the onset of DNA synthesis (209). Undoubtedly an accumulation of cytoplasmic material in the nucleus was in some way responsible for this biosynthetic activity. The nucleoli disappear froiii the transplanted nuclei in less than 40 minutes (196). Thus the situation a t the outset of normal development is restored, since the cleaving embryo lacks definitive nucleoli and these do not appear until the beginning of gastrulation (196, 215). The nucleolus has been implicated as the site of rRNA synthesis (193) and the appearance of nucleoli a t gastrulation in amphibians has been correlated with the initiation of substantial rRNA synthesis (113, 216). The changes in the nucleolus after transplantation are reminiscent of the dispersal of nucleoli that takes place in the oocyte long after the lampbrush period of intense rRNA synthesis and in the final stages of maturation. We might ask whether or not special cytoplasmic factors were operative in the disassembly of the large or multiple nucleoli of the oocyte, and if similar conditions prevailed and have some bearing on the response of the transplanted nucleus in the egg cytoplasm later. Concurrent with the disappearance of nucleoli is a change in the nature of the RNA transcribed by the transplanted nucleus (196). The nuclei from advanced embryonic stages, which have been engaged in rRNA synthesis, no longer synthesize this RNA species, but display an output of RNA seemingly characteristic of the cleaving embryo. The genic activities of these embryos apparently undergo a sequence of events programmed in a way similar to that of the normal donors, for if the embryos attain gastrulation, they begin (again) to synthesize rRNA. These experiments show directly that RNA transcription is regulated by the type of cytoplasm that provides the nuclear environment. These results might be extrapolated to :illow a consideration of the influences of the special regions of the egg cytoplasm. The animal and vegetal gradients of sea urchin eggs (48, 55) and the polar lobe of Ilyanassa (58), it might be expected, influence RNA transcription not only quantitatively, but also qualitatively. It is likely that the nature of dRNA synthesis also changes in response to cytoplasmic changes. The differences in the dRNA populations of the animal and vegetal regions of the late blastula and the dorsal and ventral halves of the tail-bud embryos of the frog (191) seem almost certainly to be basic to embryonic differentiation.
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They might be expected to arise in the course of development, promoted by either a transplanted or an endogenous nucleus. Thus a return to an earlier type of dRNA output should be expected in the transplanted nucleus.
VI. Developmental Regulation of Genetic Expression Genetic expression is the culmination of a stepwise process of chromosomal function and information transmission. The character of its regulation is determined by ( a ) the functional organization of chromosomes and (b) the interrelationship between gene transcription and translation. Both these processes leading to genetic expression may be significantly different in microorganisms and in the higher, developmental organisms.
A. Functional Organization of Chromosomes 1. THEOPERON
The achievement by an organism of a special physiological state, be it of growth, development, or response to the change of milieu, may depend on the coordinated functioning of several genes significantly grouped. Part of the flexibility of bacterial nutrition and of the efficiency of the growth of microorganisms may be attributed to a high degree of chromosomal organization. A large proportion of the genes of microorganisms may be arranged in clusters capable of acting in concert (3, 217). Such a cluster, designated an operon (1, Z I B ) , consists of genes specific for the synthesis of the enzymes of a single biochemical pathway. The coordinate control of the synthesis of these enzymes apparently follows from the activation or the inactivation of the cluster as a unit. Regulator genes produce a repressor substance that inhibits operon function (1). The original concept of the operon had the repressor action exerted directly on the DNA of the operator, the site for the initiation of transcription (1). Recent considerations have brought forth the possibility that the repressor action might also be exerted a t the level of translation. Control of polysome function has been suggested to occur either through inhibition by aminoacyl-tRN-4 (219-22.2) or by an influence of the inducer or corepressor on the folding of nascent polypeptide chains (223-225). Such a regulation during translation stipulates that the operator region is itself transcribed. The transcription of the operon proceeds from the operator end (226, 227) and results in the formation of a polycistronic mRNA the same length as the operon (228, 2 2 9 ) . The translation of mRNA also begins a t the operator end
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(219, 227, 230) and proceeds sequentially in the synthesis of proteins ( 2 3 1 ) , although the operator itself may not be translated ( 2 3 2 ) . An extrapolation of the basic concept of the operon as a unit of genetic regulation has been applied hy Jacob and Monod (233) to the regulation of differentiation. The overall relationship between a regulatory gene acting from a remote position upon a set of structural genes
could be used to describe controlling factors among complicated chromosomal structures. They (233) devised circuits to include several operons with metabolic products that themselves can serve as inducers or repressors. The same relationships as in microorganisms might hold, whether the repressor action is exerted at the level of transcription or translation, except that in eucaryotes cytoplasmic factors might act only after significant delays. 2. CHROMOSOMAL CHANGES I N DEVELOPMENT
The chromosomes of microorganisms are, of course, considerably smaller than those of higher organisms ; however, the apparent functional units in each may be fairly close in size. In Dmsophila the DNA content of bands (or chromomeres) range from 5,000 t o 50,000 nucleotide pairs per haploid strand, whereas the histidine operon of P22 phage is within this range a t a value of 13,000 iiucleotide pairs (2341. We might expect that the change in thc iiiagnitude of complexity of the chromosomes of higher organisms involves the introduction of new devices for genetic regulation. Processes of development have often been associated with visible changes in chromosomes, which may thus involve mechanisms operating on a large scale. These visible changes include the elimination of whole chromosomes (235-237) or the loss of parts or the diminution of chromosomes ( 2 3 7 ) . Also, we might include the process of endomitosis (which leads to polyploidy) , the division and separation of chromosomes without subsequent nuclear division, and polyteny, the multiplication of chromosomes with the adhesion of old and newly formed elements to form a morphological and physiological unit. The latter proccss is observed in most tissues of the larvae of Diptera, in which the cells grow but do not incrcasc in nuinber ( 2 3 8 ) . It occurs maximally in the salivary glands of the larvae. The selective rcplieation of certain genes would also fit into this category and might serve to illustrate an additional device for achieving an irreversible chromosomal change with differentiation. Such a device would bc opposite to the loss of genic activity during development, eithcr by elimination or repression. DNA is generally believed to hc constant in amount and equivalent in composition (239) among the diploid cells of an organism; however,
280
MARTIN NEMER
an exaggerated polyteny a t discrete loci might not be detectable except by direct cytological examination (240). Such an observation was made by Rudkin and Corlette (241) and Ficq and Pavan (242), who showed that certain bands of the polytene chromosomes of the salivary glands of Rhynchoscmra angelae have a disproportionate increase in DNA content compared to other bands. Thus whenever a cell must synthesize extraordinary amounts of a particular species of RNA, it is conceivable that its genes are first replicated to provide more templates. Certainly the formation of numerous nucleoli in the oocytes of amphibians allow an increase in the output of ribosomal RNA. Scarano et aZ. have proposed an interesting hypothesis for cellular differentiation through the modification of DNA base sequences ( 2 4 2 ~ ) . They point out that if DNA cytosine were methylated to 5-methylcytosine, and then converted to thymine by aminohydroxylation, an original CG base pair would thus be transformed into a T G pair. On subsequent replication of the DNA, the daughter chromosomes would become different, the one containing a T A pair and the other retaining the original (“germ line”) CG pair. The daughter cells would then differ in their chromosomal compositions. Although the methyltition of DNA is known to occur, the second step involving tliu conversion of 5-methylcytosine to thymine a t the DNA level has not been demonstrated. In support of this hypothesis, a heterogeneous origin of the thymine methyl group in DNA of sea urchin embryos has been indicated by virtue of differences in methionine and thymidine incorporation in fractions from DNase digestion. The data presented, however, are open to several alternative explanations. The differential gene activity observed in the polytene chromosomes of Diptera offers the broadest correlation between chromosomal and developmental events ( 4 ) . The bands of these chromosomes appear as diffuse (euchromatic) and compacted (heterochromatic) material. The euchromatic bands are active in RNA synthesis (243, 244) and may appear distended in the form of puffs or bulbs. A puff is an uncoiled band or chromomere (246, 2461, and the different patterns of puffing frequency may be characteristic of different tissues (244, 247-249) or different developmental periods (244-253) . Among the conditions that elicit characteristic puffing patterns (254-2601, the introduction of the molting hormone ecdysone (260, 261) results in the most precisely characterized series of events (261-263). The administration of ecdysone elicits puff formation in the salivary gland on chromosome I a t locus 18-C in 15-30 minutes and on chromosome I V a t locus 2-B in 30-60 minutes. Puffs arise as a consequence of RNA synthesis, since they fail to form when RNA synthesis is inhibited
INFORMATION TRANSFER DURING EhIBRl-OGENESIS
281
either by nctinomycin or high couccntl*ntionsof adenine (5). At least 20 hours after the appearance of thcsc primary puffs, further puffing occurs a t other loci. If protein synthesis is inhibited by puromycin (264) or cycloheximidc (265) before and after cwlysone injection into larvae, the primary puffs I-18-C and IV-2-I3 foim, but those that normally appear later than 15-20 hours after 110: mone :iclniinistration are missing. Clever (264) concluded that the primai~yaction of ecdysone was effectively a t the level of transcription and could not be mediated through protein synthesis. Furthermore, the carly-rcncting genes are involved in proc e w ~ bth:it lead to the sequcntial activation of later puffs. The intervcning proc(mes niay well involve protvin synthesis. The coordination of gciic activitics s c ~ n ihere to involvc :in interplay between transcriptional and tyanslational events.
B. Transmission of Genetic Information In bacteria, transcription, translation and mRNA degradation may occur in a tightly linked scquciicc. Hcforc the entire operon mRNA has h e n completely trmscribed, ri tiosonics may bccomc attached t o i t (2666), ~ ~ r c s u n i a l ~tol y facilitatc reniov;il frniii the DNA template (220, 267). Thc synthesis of p-galactosirl:w ran t:ikc place while its polycistronic 111HNA is still :itt:whul to thtb I)XX ( 2 2 7 ) .This closc linkage is possible Iwcwsc tmnslntion proccccls i n t h c s:iitic dirwtion as transcription, i n that it hegins a t thc ol)eratoi*I-cgion : m I goes froin the 5’-phosphate end to thc 3’-hydroxyl end (219. 227, 230, 268). Not only is there a rapid succession of transcription ancl translation, hut inactivation or degradation of the niRNA may follov7 close behind, according t o the brief life span of the iiiRNA coding for thc c’nzynies of the lactose operon (231, 269). The sequential translation of the cistrons of the operon mRNA may even bc followed by an orderly inactivation of template possibly by nurlense activity from the translatecl cnd ( 2 3 1 ) . In eucaryotes, the intervention of a nuclear membrane and a possible absence of nuclcar ribosomes, at least in certain cells (159), makes the t,ight linkage observed in prokaryotes unlikely. The conveyance of gene products into t,lie cytoplasin niay proceed through highly evolved mcchnnisms. I n the case of ribosome formation, the rRNA precursor molecules are elaborately processed in the nucleus to form subunits that do not give rise to whole rihosomes until they have entered the cytoplasm (see 1 9 3 ) . The possibility that inRNA may also undergo a special process of transport into thc cy top1:ism has recently become apparent. Particles of ribonuclcoprotcin with :I proposed transport function have been ohserved adjacent to puffs and Balbiani rings (270-27’4). I n the salivary glands of Chirono~noz~s tentans, the two large Balbiani rings
282
MARTIN NEMER
appear to produce characteristic round particles of 30 mp diameter (270). They are sensitive to RNase and to trypsin, and since the RNA elaborated by these puffs is probably messenger, by virtue of its base composition (675), the particles are probably mRNP. They float in the nuclear sap and can be followed to the nuclear envelope, through which their penetration seems to be accompanied by a change into rod and thread shapes. They have not been detected as such in the cytoplasm. The accumulation of RNA in puffs might be the result of an increased rate of synthesis or a decreased rate of removal. Experiments by Clever ( 6 ) indicate that an inhibition of protein synthesis may eventually lead to an accumulation of RNA in puffs that might be attributable to a decreased removal rate. After a 4-hour exposure to cycloheximide, intermolt larvae respond to the addition of ecdysone with the formation of larger puffs that are seen with ecdysone alone. The band IV-2-B does not form a puff in the presence of cycloheximide alone even after periods of 15-20 hours; however, ecdysone induces an oversized puff under these conditions. The deprivation of protein synthesis by cycloheximide cannot then be implicated as a condition for gene derepression and puff formation. Rather, the ecdysone is responsible for the puff induction, and the cycloheximide might then be viewed as leading to a diminished removal and transport of RNA. Ribonucleoprotein particles thought to contain messenger RNA have been extracted from nuclei of mammalian cells (276).These particles were of uniform shape and size, as determined by electron microscopy, as were those noted by Beerman and Bahr (270),but smaller and in the form of disks of 8 x 18 )( 18 mp. The RNA was characterized as messenger by DNA hybridization and base composition, and did not include a ribosomal RNA component. Hence the particles seemed to be complexes of mRNA and protein. The RNA with a Sedimentation coefficient of less than 10s may have been degraded in the purified particles. Removal of 75% of the RNA was possible with RNase treatment. The particles appeared to be largely protein and their sedimentation properties were not altered appreciably by RNase treatment. A consequence of the disengagement of transcription and translation is that a messenger RNA may be synthesized well in advance of the synthesis of its specifically coded protein. Spirin, in his review (277) of “masked” forms of messenger RNA, cites several examples of such a delayed sequence, besides the cases of stored mRNA in the eggs of sea urchins and frogs, presented here (Sections I1 and 111). (a) The synthesis of hemoglobin in the adult red blood cell occurs preponderantly after the cessation of RNA synthesis (678) (see Chantrenne et al. in this volume). The chick embryo can be shown to synthesize hemoglobin during
INFORMATION TRANSFER DURING EMBRYOGENESIS
283
a developmental period following the synthesis of specific messenger (279). (b) The inhibition of RNA synthesis by actinomycin D, before the onset of differentiation in the epithelial cells of the mouse pancreas, prevents differentiation. However, inhibition begun after differentiation has started does not prevent continued development (280). (c) The maximum synthesis of fibroin in silk glands takes place in the almost complete absence of RNA synthcsis (281). (d) Enucleated portions of Acetabularia experience an increase in phosphatase activity during regeneration of the cap to the same extent as nucleate portions (282). (e) In Bacillus cereus, the mRNA needed for spore formation is synthesized several hours in advance of the bulk of spore proteins (283). This case is of special interest, since i t involves a circumvention of the tight linkage between transcription and translation, suspected to be typical of prokaryotes. ( f ) Barros, Hand, and Monroy ( 2 8 3 ~ )have treated starfish embryos with actinomycin a t different periods during development, and concluded that the RNA transcription required for gastrulation occurs before the swimming blastula stage.
C. Messenger Ribonucleoprotein The emergence in the cytoplasm of a form of mRNA, functionally intermediate between transcription and translation, was proposed by Spirin and co-workers (284, 285). The suspected intermediates were thought to occur in the form of ribonucleoprotein particles that sediment more slowly than ribosomes (subribosomal) . The discovery of these subribosomal particles, however, led to two alternative formulations, concerning their structure: 1. mRNA-ribosomal subunit complex. Subribosomal particles extracted from mammalian liver and tissue culture cells were considered to contain mRNA attached to the m a l l , 40 S ribosomal subunits (286-289). The characterization of mRNA was based on rather low levels of template activity (288) and hybridization with DNA (289). I n all these studies only inaterial obtainccl from primary sucrose gradients was &died. Girard and Baltimore (2.90) found that free RNA can react with components of the cytoplasmic, high speed supernatant fluid, and cautioned against interpretations based on evidence obtained exclusively by means of sedimentation in sucrose gradients. Indeed, further examination by Perry and Kelley (291) revealed that the bulk of the labeled RNA in the subribosomal region, occurring principally a t about 45 S, is predominantly ribosomal. The particles containing this RNA had buoyant densities in CsCl gradients less than those of the ribosomal subunits. It was concluded that they are likely precursors to ribosomal subunits and not the postulated mRNA-subunit complexes.
284
MARTIN NEMER
2. Informosomes (mRNA-protein complexes). Subribosomal particles containing labeled RNA have been extracted from early embryos of the teleost Misgurnus fossilis (284, 285) and of sea urchins (189, 292). Since in the early embryonic stages no rRNA synthesis could be detected (109, 284), the particles were suspected of containing mRNA. The labeled RNA was characterized as heterogeneously sedimenting and thus nonribosomal (277, 292). This RNA hybridized extensively with homologous DNA (189, 292). I n addition to these criteria for mRNA content, Spirin et al. (284) noted a slight stimulatory effect on protein synthesis when the subribosomal material was added to a cell-free ribosomal system. This template activity was reminiscent of the demonstration by Hoagland and Askonas ($93) that the postribosomal supernatant fluid from r a t liver contains a component (their X-fraction) that stimulates protein synthesis. In both cases, the stimulatory component might have been either free mRNA or messenger ribonucleoprotein (mRNP). The mRNP component extracted from the polysomes of reticulocytes by Weisberger and Armentrout (294) that directs specific hemoglobin synthesis may be related to the X-fraction and to informosomes. I n the case of the hemoglobin synthesis, stimulation was obtained only with the mRNP and not with the mRNA extracted by deproteinization (see Chantrenne e t al. in this volume). The subribosomal mRNP particles obtained from the fish and the sea urchin embryos both displayed a heterogeneous sedimentation pattern. I n the case of the sea urchin, six sedimentation modes were distinguishable with values of 19 S, 30 S, 37 S, 44 S, 52 S, and 61 S (295) (Fig. 6 ) . The labeled RNA extracted from the entire subribosomal region of the sucrose gradient sedimented from about 10s to 40s with six distinguishable sedimentation modes. The six sedimentation modes could be classified as dRNA, by virtue of their extensive hybridization with DNA. If RNA was extracted from different portions of the subribosomal region of the sucrose gradient, the labeled RNA displayed a niean ncditnentation coefficient that was proportional to the average sedimentation coefficient of the particles extracted (89, 295). Therefore, the size of the RNA appeared to determinc the sedimentation behavior of the respective mRNY particles. Each sedimentation mode of mRNP probably reflected a sedimentation mode of the component mRNA. I n order to distinguish between a structure containing mRNA in complex with the ribosomal subunit and one with protein per se, the subribosomal particles were fixed with formaldehyde and their buoyant densities were measured in CsCl gradients (285, 296). A complex between mRNA and the ribosomal subunit would have a greater density than the subunit alone. This has been shown recently by Infante and
285
INFORMATION TRANSFER DURING EMBRYOGENESIS
Nemer (297) with artificial complexes. Spirin et al. (285) found that the subribosomal particles that had been concentrated by pelleting, then fixed with formaldehyde, had buoyant densities less than those of the ribosomes a t p = 1.55 gm/cm3. They concluded (285) that the labeled C
i 10
li
1000
ll 3000
CPM
2000 I IIII
~
1
0.5
\
I I
/
I000
/
FIG.G . Sedimentation diagram of suhibosornal part.icles containing labeled ItNA. Eight-hour morulae of Slmngylore ntrolus prirpu,ralus werr incubakd wit,li uridinrH.’ (100 pc/ml) for 15 minut,es. A 1)ostmit.oc.iiondrialsuliernat,nnt was prt~l~arrd and wntrifugrd in a 1&30% suerosc grndirrit :it. 39,000 rpm for. 4.5 hours. (dashed linc) was continuously nionitorcd through a recording spectrophotomcter ; ,O, radioactivity retained on membrane filters : x , acid-insoluble radioactivity that passed through membrane filters. Labeled material retained on niembranc filtcrs (10) was all RNA removable by RNase digestion and “bound RNA,” later shown bound to protein. From Infante and Nemer (297).
RNA could not be part of a complex with the ribosomal subunit, but with protein per se. They designated this complex the iLinformosome.’7 Spirin (277) has recently reported that an appreciable amount of labeled RNA of the subribosomal particles is associated with material of buoyant densities of 1.60-1.65 gm/cm3. This material, according to Spirin’s criterion, may or may not be informosomes.
u 1.65 160 155 1.50 145
1.40
DENSITY
- 1.60 P
- 1.55 - 1.50
I
I
30 90 150 210 DURATION OF HYDROLYSIS (SECONDS)
FIG.7. Buoyant densities of the subribosomal messenger ribonucleoprotein particles and the effects of RN‘ase digest,ion. Five-hour embryos of Lytechiniis pictus wrre incubated with uridinc-H” (100 pc/ml) for 30 minutes; the subribosomal components of 40s to 6 5 s were isolated from sucrose gradients (as in Fig. 61, and unlabeled 35s ribosomal subunit from the same embryos was added. Incubation a t 28°C with 0.2 pg/ml of boiled RNase was stopped at the indicated times by the addition of cold formaldehyde to 6%. In the lower figure the time course of the digestion was followed. The samples were immediately passed through membrane filters, and the filtrates were precipitated with cold trichloroacetic acid. 0, RNA-Hs retained on membrane filters, thus “bound RNA”; this bound RNA was not affected RNA-H3 in filtrates, in the course of the incubation if RNase was omitted. (NO), thus “free RNA”; x, mean densities of material in CsCl gradients, assayed by layering on preformed gradients. The buoyant densities of the untreated ( 0 )and material are represented after 30-second and the partially RNase-digested (10) 90-second incubations in the two upper figures. Solid line represents the A m in both the control and RNase-treated material. From Infante and Nemer (237).
INFORMATION TRANSFER DURING EMBRYOGENESIS
287
Infante and Nemer (297) recently detcrmincd that very little of the labeled RNA of the subribosomal particles extracted from sea urchin embryos is associated with material of buoyant density less than that of the ribosomal subunit. Their method was to fix the material with formaldehyde directly in the sucrose gradient and thus avoid the degradative effects of pelleting. Protein label never appeared in conjunction with the RNA label, even after a variety of conditions of incubation of the embryos. This failure of appearance of protein label in the dense region of the CsCl gradient was also noted by Spirin ( d r y ) , although i t did appear in the lighter regions in a n apparent correspondence with the RNA label of the light region. Infante and Nemer (697) were able to shift the particles into heavier regions by partial deproteinization with pronase. However, it was the response of the particles to mild ribonuclease treatment that enabled them to conclude that even the very dense m R N P particles are “informosomes” and not simple complexes of mRNA and ribosomal subunits (Fig. 7 ) . I n the course of this nucleolytic disassembly, the buoyant densities of the m R N P particles, which ranged from p = 1.50 to 1.75 gm/cm3, shifted into regions of the CsCl gradient less dense than the small ribosomal subunit a t p = 1.55. Such partial removal of mRNA from a complex with the ribosomal subunit would have caused the buoyant density to approach that of the subunit itself, since the subunit could be shown to be unaltered by the treatment. In conclusion, the labeled RNA that appears in the subribosomal particles of the cytoplasm is complexed with protein per se.
D. Regulation of Polysome Activity Monroy and Tyler (136) demonstrated that polysomes are formed after fertilization of the sea urchin egg and proposed that the poly,come activity is indicative of the activation of protein synthesis. Stafford e t al. (298) attributed the bulk of protein synthesis to the activity of a rapidly sedimenting class of polyribosomes. Spirin and Nemer (692) observed that newly synthesized RNA, characterized as dRNA (189), could be found associated with slowly sedimenting structures (20053 and less), but not with the rapidly sedimenting structures responsible for the major part of protein synthesis. They proposed that (a) the very active, rapidly sedimenting polysomes contain essentially only preexisting egg template RNA, and (b) the structurcs of 100S to 200 S containing the labeled RNA, are also polysomrs, but possibly blocked, a t least to some extent, in their ability to synthcsize protein. The newly synthesized RNA, according to various reports on the ability of the embryo to develop and synthesize protein in the absence of nuclei or RNA synthesis (see Section 11, B) would not be needed for the quantitative sup-
288
MARTIN NEMER
port of protein synthesis, nor would it be required for all aspects of development. Despite this apparent lack of essentiality, new dRNA is synthesized in the early cleaving embryo and becomes associated with cytoplasmic structures. I n these cytoplasmic forms the mRNA synthesized in the cleaving embryo might be either retained for translation at a later development stage or prevented from being used a t all. The utilization and expenditure of preexisting template RNA of the egg has been linked to the use of newly synthesized mRNA for polysome formation and activity (135, 699) : during the first 3 hours after fertilization of the eggs of Strongylocentrotus purpuratus, there is a continuous and rapid formation of polysomes with an average sedimentation coefficient of 300s (135, 2.99) (Fig. 8). The polysomes thus formed contain about 40% of the total egg ribosomes and are probably directed exclusively by preexisting egg template RNA. A second class of polysomes then begins to arise a t about 200s and gains prominence in development through the next 4 hours. Electron micrographs of tlicse polysomal classes reveal averages of 23 and 7 ribosomes per polysornc, respectively (299). I n the presence of actinomycin D, these more slowly sedimenting polysomes do not appear. Hence their formation seems to depend on the synthesis of RNA. I n the course of further normal development, the amount of 200s polysomes decreases and the two classes prevail in approximately equal amounts. If actinomycin D is present, the total content of polysomes begins to decline a t 4-6 hours of development and about half are lost during incubation of the embryos for thc next 10-12 hours (89). Therefore, a t the start of this decline, the preexisting egg mRNA becomes rate-limiting in the actinomycin-treated embryos, and polysomal activity in the control embryos becomes from then on increasingly dependent on newly synthesized RNA. The appearance of labeled RNA can be detected in the 2 0 0 s structures a t 23 hours after fertilization, even before the rise of the 200 S polysomes is detectable spectrophotometrically. Little if any labeled RNA is detectable in the 300s polysomes a t this time (292, 299). At least twothirds of the nascent protein is associated with the 300s structures. The rest of the new protein synthesis is associated with the 200 S polysomes. The specific activity (nascent protein per ribosome) of the 300s polysomes is 2-3 times that of the 200s polysomes. Hence the more slowly sedimenting class may be less active than the 300s polysomes. Thc possibility that the 200 S polysomes contain members that are inactive or minimally active is supported by the observation that the accumulation of these polysomes during the third to seventh hour of development occurs with a decrease in their specific activity (nascent protcin/ribosome) compared to the 300s polysomes. If the embryos are exposed to
289
INFORMATION TRANSFER DURING EMBRYOGENESIS ~
I
Control
I
300s
I
200
Actinomycin treated
I
s
74:
i
,
300s sedimentation constants
200s
74:
FIG.8. Sedimentation profiles of polyribosomes drrived from the embryos of Strongylocentrotus purpurnlus, developing in the presence and absence of actinomycin. Postmitochondrial supernatant fluids prepared from eggs and rmbryos were layered on 15-30"/, sucrose gradients, for crntrifugation a t 39,000 rpm for 45 minutes in the SW 39 rotor. Absorbance of gradient fractions was monitorrd continuously with a recording spectrophotometer. The unfertilized eggs in onr case wrrr incubated for 1 hour in actinomycin D a t 25 pglml. Thrse treated rggs were frrtilized and allowed to develop in the prrsrnw of actinomyrin a t 25 pg/nil From Infante and Nemer (89).
30-niinute incubations with labeled uridine during these early cleavage stages, the relative incorporation in RNA associated with the 200 S polysomes is much greater than that associated with the 300 S polysonies; however, the proportion of newly synthesized RNA in the 300s polysomes increases with development. The shift of the distribution of new RNA toward a greater amount in the 300s polysonial class may
290
MARTIN NEMER
represent an activation of the slowly sediinenting polysoines and conversion into the rapidly sedimenting structures. A series of “chase” incubations after a single pulse of labeled uridine, to incorporate RNA label into the 200s polysomes a t the 3-hour stage, resulted in a shift of labeled RNA into the 300 S polysomes. Therefore, the slowly sedimenting class seems to serve the function of holding the mRNA in abeyance for later translation. The interrelationship between informosomes and polysornes, especially the inactive 200 S polysomes, is not yet clear. I n preliminary experiments, Spiriii et al. (284) obtained results that indicate a reversible interconversion betwren inforiiiosoincs and polysomcs. Infante and Nemer (297) noted also that the kinetics of incorporation of labeled RNA allowed that the labeled RNA of the inforniosoines might be precursor to that of the polysomes. In experiments by Marks and co-workers (300, S o l ) , polysomes of the reticulocyte were caused to be disassembled by an inhibition of protein synthesis n-ith NaF. Functional polysomes could re-form after removal of the N a F and recovery of the cells. I n this case, the fate of the stable template RNA for hemoglobin synthesis was not studied. However, n complex hetween this RNA and protein (cf. 294) similar to the inforiiiosome is one conceivable structure that might account for its stability and survival during the period of polysome breakdown.
VII. Summary The flow of genetic information begins with the synthesis of RNA templates for specific proteins and mediators for the use of these templates. Variations in the output of each of these classes of RNA products can be detected throughout embryogenesis. The supply of the mediators (ribosomes and tRNA) in amphibian and echinoderm eggs is adequate to serve the needs of early development, so that new ribosomes are not synthesized until the gastrula and mesenchyme blastula stages, respectively. There is evidence to support the view that egg ribosomes are not consumed in the early amphibian embryo (124), but that they are consumed in the sea urchin ( 1 0 7 ) . The capacity of the enzymatic and ribosomal components for polypeptide synthesis far outstrips the supply of tRNA available in the unfertilized cgg. During the early period of development, considerable terminal addition to tRNA occurs, but de nova synthesis may not begin until the later stages when the tRNA content (11.3) and activity (11.4) rise relative to the content of ribosomes. The increase in tRNA is in proportion to the increase in cell number (166), whereas the accumulation of new ribosomes does not occur in parallel to this increase (92).
INFORMATION TRANSFER DURING EMBRYOGENESIS
291
Variations in genic activity have been detected as visible differences in the activities of chromosomal bands, in the form of puffing or selective RNA incorporation, as well as changes in the output of high molecular weight, nonribosomal or dRNA populations. Developmental, environmental, and hormonal influences on insect larvae have been related to chromosomal puffing, and the effects of possible regulatory agents on genic activity have been studied directly. I n certain cases the sequential puffing of chromosonial bands is linked by an intervening step involving protein synthesis (264, 265). An important influence on nuclear function is the physiological state of the cytoplasm. Its phase in the cell cycle (20,30%’)is involved in the promotion of DNA synthesis. The transplantation of nuclei into egg cytoplasm from later embryos promotes DNA synthesis (209), changes the chromosomal puffing patterns (%'54),and represses specifically the synthesis of rRNA (196). I n amphibians (108) and echinoderms (79, 8 0 ) , the dRNA populations themselves have been compared from stage to stage. I n each case, stage-specific species of dRNA are transcribed. I n the amphibian (106), these dRNA species may be either transient or stable. The stable members of the population may then persist through development to later stages. Consequently the late-stage embryos contain some species of dRNA that are no longer being transcribed, as well as species that are characteristically synthesized a t that stage. Furthermore, there is an increasing accumulation of stable dRNA molecules through the course of embryogenesis. Another class of dRNA molecules is one that is synthesized a t all stages. We expect that a portion of the dRNA molecules of all these classes represents template or mRNA. We do not know yet what the relationship is between the high molecular weight, nonribosomal RNA transcribed from the bulk of the genome, and thus characterized as DNA-like or dRNA, and the RNA that eventually serves as template in protein synthesis. The immediate transcription products may be very high molecular weight precursors that are subsequently coiiverted into maller molecules, as in the production of the 2 8 s and 18s rRNA from 4 5 s precursors. The immediate transcription products, reported u p to 100 S, may serve peculiarly nuclear or chromosomal functions, such as the regulation of DNA transcription (SOS), in addition to or alternatively to being precursor to template RNA. Since the dRNA of the cytoplasm is longer lived than that in the nucleus (164), the observed accumulation of stable dRNA molecules in the course of development (106) probably occurs in the cytoplasm, and represents stable templates. Many intervening steps may occur between the transcription and the use of template RNA. Part of the process involves the conveyance of this RNA into the cytoplasm to the sites of protein synthesis. Also involved
292
MARTIN NEMER
is the formation of a structure containing template RNA that will contribute to its stability and allow its specific accumulation. Cytoplasmic particles, designated “informosomes,” have been described in fish embryos (284, 285) and sea urchin embryos (189,292) and may serve these intermediate functions of transport and stabilization of templates. These particles consist of mRNA complexed with protein; they lack ribosomal components ( 9 9 7 ) .Whether or not the protein moiety is specific and involved also in the regulation of template activity remains to be seen. It is possible that informosomes react directly with ribosomes to form polyribosoiiial structures. The accumulation of stable RNA templates, it now appears, may be attributed to the existence of either inforinosomes or stable polyribosomes. The involvement of the accumulation of stable RNA templates in embryogcnesis might be elucidated by examining three early phases in template utilization: ( a ) Activation of e g g template R N A . The primary event in the activation of protein synthesis following fertilization may be the release of a specific proteolytic activity (145-147), which in turn is dircctcd a t the release of template activity (155). Two structures have been suggested for the storage of egg template RNA. The one consists of an informosome bound to a very large particle (77, 155); the other is an inactive, stable polyribosomnl structure (93).The bound informoboine may predominate as a specific storage form in the egg. A salient fcature in the activation of the egg is the formation of rapidly sedimenting, “r-polysomes” (containing 15-30 ribosomes), which account for the preponderant amount of the newly activated protein synthesis (292, 298, 299). The conversion of monoribosomes into r-polysomes occurs during the first 2-3 hours after fertilization (Fig. 9). (b) Accumulation of “s-polysomes.” During this early cleavage period, new dRNA can be detected. This new RNA appears in the cytoplasm in informosomcs and in a slowly sedimenting class of polysonies containing 5-10 ribosonws, tlic “s-polysoines” (292, 299). These s-polysomes, which n y t inactive or iiiinimally active in protein synthesis, accumulate to a maximum Concentration during tlcvelopinent from the 16-ccll stage to the 10-hour wrly blastula (2991, while the rontent of the active rpolysomes remains almost constant (Fig. 9 ) . The embryo may thus be thought to pass through a phase of pronounced accumulation of stable templates, seemingly made possible by a reliance on the support by preexisting egg template RNA. (c) Activation of ‘Is-polysomes.” I n further development from the 10-hour early blastula to the 20-hour mesenchyme blastula, the concentration of s-polysomes falls and, concomitantly, the content of r-polysomes increases (Fig. 9). It is proposed that the s-polysomes are activated, and thus take up additional ribosomes, to
INFORMATION TRANSFER DURIKG EMBRYOGENESIS
293
sediment rapidly as r-poIysomes. The period of development from cleavage to the early blastula has been termed one of “predifferentiation,” since there is little apparent morphological differentiation (304). However, the formation of the mesenchyme blastula that occurs during the next 10 hours represents the first period of massive cellular change.
TIME A F T E R F E R T I L I Z A T I O N I H O U R S I
FIG.9. Polysome content of sea urchin embryos in the course of development to the gastrula stage. The amounts of the total and constituent polysome classes were obtained by integration of areas under. the curves represented in Fig. 8. The ribosonial content as percentage of total ribosomes is given for the 200s or “spolysomes” ( x 1, the 300.3 or “r-polysomcs” (,O) and the total polysomes ( 0 ) . From Infante and Neiner (89).
These cellular changes may thus be brought about through the use of the accumulated polysonial templates. But we might wonder to what extent a specific distribution of these polysomes in the cytoplasm might be crucial to the effective use of template information in cellular differentiation.
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231. A. Kepes, personal communication, Information Exchange Group No. 7, Memo No. 623 (19s7). 256. E. Steers, Jr., G. R. Craven, and C. B. Anfinsen, Proc. N a t l . Acad. Sci. U S . 54, 1174 (1965). 2%. F. Jacob and J. Monod, in “Cytodifferentiation and Macromolecular Synthesis” (M. Locke, ed.), p. 30. Academic Press, New York, 1963. 234. G. T. Rudkin, Genetics 52, 665 (1965). 255. K. Mather, Nattire 161, 872 (1948). 956. M. J. D. White, J. Morphol. 79, 323 (1946). 23.57. C. Pavan, N a t l . Cancer Inst. Monograph 18, 309 (1965). B S . D. W. Bodenstein, in “Insect, Physiology” (K. D. Border, cd.), 11. 868. Wiley, New York, 1953. 2%. B. McCarthy and B. H. Hoyer, Proc. Natl. Acad. Sci. U.S. 52, 915 (1964). 2440. M. E. Breuer and C. Pavan, C h r o m o s a a 7, 371 (1955). 2.41. G. T. Rudkin and S. L. Corlette, Proc. Natl. Acad. Sci. U S . 43, 964 (1957). 242. A. Ficq and C. Pavan, Nature 180, 983 (1957). 2 4 2 ~ .E. Scarano, M. Iaccarino, P. Grippo, and E. Parisi, Proc. N a t l . Acad. Sci. U.S. 57, 1394 (1967). 243. J. H. Frenster, V. G. Allfrey, and A. E. Mirsky, Proc. N a t l . Acad. Sci. US. 50, 1026 (1963). S44. W. Beermann, Chromosoma 5, 139 (1952). 2446. W. Beermann, Naturwissenschnften 52, 365 (1965). 246. C. Pelling, Proc. R o y . SOC.B164, 279 (1966). $47. M. Breuer and C. Pavan, Caryologia 6, Suppl., 778 (1953). 24s. H. J. Becker, Chromosoma 10, 654 (1959). 249. U. Clever, Chromosoma 14, 651 (1963). 250. F. Mechelke, Chromosoma 5, 511 (1953). 261. U. Clever, Clrromosoma 13, 385 (1962). 266. H. D. Berendes, Chromosoma 17, 35 (1965). 253. N. Gabrusewgcz-Garcia, Chromosoma 15, 312 (1964). 254. H. Kroeger, Chromosoma 11, 129 (1960). 255. H. Kroeger, Chrmoso,ma 15, 36 (1964). 2556. F. M. Ritossa, Experientia 18, 571 (1962). 95‘57. F. M. Ritossa, Exptl. Cell Res. 35, 601 (1964). 25s. H. D. Rerendcs, F. M. A . van Rieugpl, and T. I<. H. Holt, Chr07n0soma 16, 35 (1965). 25:). IT. Clever, Clrromononia 17, 309 (1965). ?/ P.% &&on, I. Atigcw. C k e m . I n t c w . Ed. Engl. 2, 175 (1963). -061. U. Clever and P. Karleon, E z p t l . Cell Rea. 20, 623 (1961). 262. U. Clever, Chromosoma 12, 607 (1961). 263. U. Clever, Develop. Biol. 6, 73 (1963). 26‘4. U. Clever, Science 146, 794 ( 1 9 6 4 ) . 266. U. Clever, Develop. Biol. 14, 421 (1966). 266. R. Byrne, J. G. Levin, H. A. Bladen, and M. W. Nirenberg, Proc. N a t l . Acad. Sci. U.S. 52, 140 (1964). 26r. H. Bremer and M. W. Konrad, Proc. N a t l . Acad. Sci. U S . 51, 801 (1964). 2568. Y. Okada, E . Terzaghi, G. Streisinger, J. Emrich, M. Inouye, and A. Tsugita, Proc. Natl. Acad. Sci. U S . 56, 1692 (1966). 269. A. Kepes, Biochim. Biophys. Acta 76, 293 (1963). 2740. W. Beermann and G. F. Bahr, Eqcptl. Cell Res. 6, 195 (1954).
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271. W. Beermann, J. E z p t l . 2001.157, 49 (1964). 272. H . Swift, in “The Molecular Control of Cellular Activity” (J. M. Bllen, ed.), p. 73. McGraw-Hill, New York, 1962. 273. B. J. Stevens, J. Ultrastruct. Res. 11, 329 (1964). 274. V . I. Kalnins, H. F. Stich, and S.A. Bencosme, Can. J . 2001.42, 1147 (1964). 275. J . E . Edstrom and W. Beermann, J. Cell Biol. 14, 371 (1962). 276. 0.P. Samarina, H. A. Krichevskaya, J. Molnar, V. I. Bruskov, and G. P. Georgiev, Mol. Biol., USSR, in press (1966). AT. A. S. Spirin, in “Current Topics in Developmental Biology” (A. A. Moscona and A. Monroy, eds.), Vol. I, 11. 1. Academic Press, New York, 1966. 278. R. H . DeBellis, N. Gluck, and P. A. Marks, J . Cliii. I l i w s t . 43, 1329 (1964). f l 9 . F. H. Wilt, J. Mol. Biol. 12, 331 (1965). 280. N. I<. Wessells and F. H. Wilt, J . Mol. Biol. 13, 767 (1965).
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(1966). 285. M. Nemer and A. A. Infantc, Frdcrcition Proc. 24, 283 (1965). 296. A. S. Spirin, N. V. Brlitsina, nnd M. I. T,rrman, J . Mol. Biol. 14, 611 (1065). 297. A . A. Infante and M. Nemw, submittcd for publication (1967). 298. D. W. Stafford, W. € Sofcr, I. :mtl 11. M. T v ~ w o n ,Proc. N u l l . Acrrrl. Sci. t\.S. 52, 313 (1964). 299. M. Nrnirr, A . 4 . Infnntr, : c n t l hl. 14:. lhycr, un~~ulilishrtl (1967). ,300. 1’. A. Marks, E. R. Burkn, E’. M . Conc.oni, W. I ’ d , :mi R. A . Rifkind, Proc. N u t / . Acnd. Sci. U.S. 53, 1437 (1965). 501. F. M. Conconi, A. Bank, and 1’. A . Marks, J. Mol. B i d . 19, 525 (19666). 502. N. de Terra, Proc. N a t l . Acnd. Sci. U S . 57, 607 (1967). 305. J. H. Frenster, Nature 206, 1269 (1965). 504. J. Runnstriim, “Acid nucleiri e lor0 funaione biologica” (A. Baeelli, ed.), p. 342 1st. Lombardo, 1964.
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Enzymatic Reduction of Ribonucleotides' AGNELARSSON AND PETERREICHARD Department of Chemistry 11. Karolinska Institutet. Stockholm. Sweden
I . Introduction . . . . . . . . I1. Ribonucleotide Reduction in Escherichia coli .
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A General Remarks . . . . . . . B . Experiments with Crudc Extracts . . . C . Experiments with Purified Enzynirs . . . . . . D . The Thioredoxin System . . . . . . . . . E . Allosteric Regulation of Substrate Specificity . . . . F . Studies on the Reaction Mechanism . . . . . . 111. Ribonucleotide Reduction in Lacfobaczllus leichmanmi . . A . I n Viwo Experiments . . . . . . . . . . B. Experiments with Crude Extracts . . . . . . . C Experiments with Purificd Enzymcs . . . . . . D. Hydrogen Donor Specificity . . . . . . . . E . Substrate Specificity and Allosteric Effects . . . . F. Regulation of Enzyme Synthesis . . . . . . . G . Studies on the Reaction Mechanism . . . . . . IV . Ribonucleotide Reduction in Animal Cells . . . . . A . General Remarks . . . . . . . . . . . B . Experiments with Extracts from Chick Embryos . . . C . Experiments with Purificd Enzymes from Novikoff Hepatoma D . Evidence for Control Mechanisms in Intact Cells . . . V . Concluding Remarks . . . . . . . . . . References . . . . . . . . . . . . .
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1 Introduction By 1960 most of the metabolic pathways leading to the formation of the simpler body constituents had been discovered. and the relevant enzyme reactions in many CRSCS had been studied in considerable detail. 'Our studies on the subject undrr review wcre aided through grants from the Damon Runyon Fund. the U.S. Priblir Hralth Service. National Institutes of Health. U.S.A., thc Swedish Canrw Society. the Swedish Medical Rcsearch Council. Stiftelsen Therese och Johan Anderssons Miune. Magnus Bergvalls Stiftelse. Stiftclsen Gustaf och Tyra Svenssons Minne. and Knut och Alice Wallenbergs Stiftelse . It is a pleasure t o acknowledge the valuable support of these agencies . 303
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AGNE LARSSON AND PETER REICHARD
The biosynthesis of deoxyribose, however, formed a notable exception. In fact, the limited knowledge in this respect was based on isotope experiments with intact cells that suggested that many rapidly growing tissues contain a metabolic sequence in which ribose bound in a ribonucleoside or a ribonucleotide can be reduced to the corresponding deoxyribosyl compound (1-7) .2 At this time, however, attempts to demonstrate appropriate biosynthetic enzymes in extracts of these cells were unsuccessful, and considerable doubt therefore existed as to the quantitative significance of the in vivo experiments. Subsequently, studies in several laboratories, including our own, corroborated the results of the in vivo experiments and demonstrated, in several types of cells, the existence of specific enzyme systems capable of catalyzing the reduction of ribonucleotides (9-16). It now seems clear that the reactions catalyzed by these ribonucleotide reductases
RoH2cu RoH2 HO
OH
6e
401
-
HO
H
FIQ.1. Reduction of a ribonucleotidc. R stands for pyrophosphoryl in the cnzyme systems from Eschen'chia coli and Novikoff hepatomit, for triphosphoryl in the enzyme system from Lnclobacilliis leichntnnnii.
provide the major source of the precursors of DNA in the cells in question. The existence of alternative pathways of deoxyribose synthesis cannot, of course, be excluded in all instances. However, the only alternative enzyme reaction known-that catalyzed by deoxyribose aldolase (17)-appears to function in the degradation of deoxyribose 5phosphate, but not in its synthesis. Alternative synthetic enzymes therefore remain to be discovered. Figure 1 demonstrates schematically the chemistry of ribonucleotide reduction. The reaction involves the replacement of the hydroxyl group a t position 2' of the ribosyl moiety by a hydrogen atom. Two related, but in important details different, enzyme systems have been purified from Escherichiu coli B (9, 13, 18, 19) and Lactobacillus Zeichmannii (20-22), respcctively. The E . coli system utilizes ribonucleoside diphosphates as substrates, while that from L. leichmannii reduces ribonucleoside triphosphates. The most striking difference is the requirement of the enzyme from Lactobacilli for cobamide coenzyme, a deriva-
' For a detailed discussion of these tracer studies, the recent excellent review by Sable (8) should be consulted.
305
ENZYMATIC REDUCTION O F RIBONUCLEOTIDES
tive of vitamin B,?, which does not appear to be necessary for the activity of the system from E . coli. The mechanism of ribonucleotide reduction in animal cells is much less understood. Most knowledge in this area is derived from studies with partially purified enzymes from Kovikoff hepatoma (12, 23, 24).In many respects this system shows a close resemblance to the one from E . coli.
II. Ribonucleotide Reduction in
Escherichia coli
A. General Remarks Thc first attempts in our laboratory to demonstrate the formation of deoxyribonucleotides from ribonucleotides in a cell-free system were performed with extracts of 5-day-old chick embryos. Reduction of uridine nucleotides which occasionally could be stimulated by the addition of NADPH was noted in some experiments ( 2 5 ) , but these early results wcrc quite irreproducible. On the other hand, experiments with minced chick embryos, containing whole cells, consistently gave evidencc for the existence of a reductive pathway that could provide considerable amounts of the precursors necessary for DNA synthesis in these cells ( 2 6 ) . Thew experiments also showed that the reduction of ribose involved nucleotides and not nucleosides or polynuclcotides. The chick embryo system WHS the first example of many frustrating experiences that werc to follow. It seemed difficult or impossible to prepare in a reproducible way a proper soluble enzyme system from cells that were known, from in wivo or in vitro experiments, to carry out the reduction of ribonucleotides. This point is discussed further a t the end of this review. The difficulties with the chick embryo system prompted consideration of the use of bacteria, which appeared to be rich sources of the enzymes involved in DNA synthesis and, in addition, offered a means of preparing large amounts of starting material for enzyme purification. Accordingly, work with the chick embryo system was to a large extent terminated, and a serious effort was made to find the enzymc(s) responsible for reduction of ribonucleotides in extracts of E . coli B. Since that time, with a few exceptions, most of our work has been performed with this organism.
B. Experiments with Crude Extracts The initial studies (9, 27) involved the reduction of cytidine phosphates, since a relatively simple and reliable assay method for this nucleotide was available. In general, radioactive CMP (PS2or H3) was
306
AGNE LARSSON AND PETER REICHARD
incubated under varying conditions with a crude extract from E . coli B, and the amount of isotope appearing in the different deoxycytidine phosphates was used as a measure of the reduction. I n crude extracts, the isotope from CMP is rapidly distributed between the mono-, di-, and triphosphates of cytidine and, to a much lesser extent, also transferred to cytidine, uridine, and ~ r a c i l The . ~ isotope is distributed in a similar way between the different deoxycytidine phosphate compounds. These relationships are demonstrated in Fig. 2.
100"
HCIO,
Il:i *CTP
ATP, M g 2 + , TPNH crude extract
deoxrY
deoxy*CTP
FIG.2. Interconversion of nucleotides in extracts from Escherichia coli.
In the usual assay, the reaction is stopped by the addition of perchloric acid, and all nucleoside di- and triphosphates are transformed to monophosphates by acid hydrolysis (Fig. 2 ) . Subsequently, CMP is separated from dCMP by chromatography on Dowex 50 (28). A similar method, involving paper chromatography in place of the ion exchanger, can be used for assaying the reduction of uridine phosphates (29). Unfortunately, procedures involving acid hydrolysis of pyrophosphate bonds cannot be used to assay the reduction of purine ribonucleotides because of the acid lability of the purine sugar bond. Instead, dephosphorylation of all nucleotides with crude snake venom can be used and the resulting ribonucleosides and deoxyribonucleosides can then be separated by paper chromatography (28, SO). However, side reactions (deamination) reduce the reliability of this method when applied to crude extracts or partially purified enzymes. I n our opinion, the method outlined above for the assay of the reduction of cytidine phosphates is the method of choice when working with crude extracts containing low levels of ribonucleotide reductase activity. Extracts of E . co2i B4catalyze the reduction of C M P as measured by ’113 extracts from E . coli, only small amounts of nucleosides are formed. However, in other systems nucleoside formation may pose a serious problem. ' A much larger amount of CMP reduction occurs in extrarts from thyminerequiring E . coEi, the growth of which is limited by thymine starvation ( 3 1 ) . Such extracts were in recent years used for our enzyme purification.
307
ENZYMATIC REDUCTION O F RIBONUCLEOTIDES
this assay ( 2 7 ) .A considerable stiniulation was observed when the incubation mixture was fortified with ATP and Mg ions, and-after dialysis NADPH. Even under or treatment with Dowex 2 or charcoal-with optimum conditions, the amount of synthesis observed was far below that required to account for DNA formation in bacteria growing logarithmically. This point is discussed later. It was important to establish that the observed transfer of isotope from C M P to dCMP occurred only by direct reduction of the ribonucleotide and not by a complcs series of enzyme reactions involving transfer of cytosine-H3 t o a preexisting deoxyribosyl moiety. The direct reduction mechanism was established in experiments demonstrating that corresponding amounts of isotopc are transferred to dCMP from C M P labeled with either P32or H3. The stimulation of the reduction of C M P by ATP and Mg2+turned out to be a complex phenomenon. Part of the explanation came from the finding that the reduction occurs a t the ribonucleoside diphosphate level and that ATP and Mgz+thus are required for the phosphorylation of C M P to C D P by C M P kinase. I n the presence of the side reactions summarized in Fig. 2, one cannot demonstrate directly that CDP is the preferred substrate of the reaction. However, by adding a large excess of a nonlabeled deoxycytidine phosphate to the incubation mixture a t the beginning of the experiment, most of thc isotope incorporated into the deI
I
mtn
FIG.3. Time curves for specific activities of different deoxycytidine phosphates formed from H3-CMP in the presence of nonlabeled dCDP (87).
308
AGNE LARSSON AND PETER REICHARD
oxycytidine phosphate compounds from the cytidine phosphate precursors can be trapped a t the original level of phosphorylation a t early timepoints before equilibration through kinase and pyrophosphatase reactions occurs. I n this way, it was found that dCDP has the highest specific activity during the first 10 minutes of the experiment, and thus is the most direct product of the reaction (Fig. 3). This finding strongly indicates that C D P is the substrate of ribonucleoticle reductase. When C D P was used as substrate in place of CMP, the reaction still required ATP and Mg2+,indicating still another function for these substances in ribonucleotide reduction. An attractive hypothesis a t that time appeared to be the intermediate formation of an “activated” ribonucleotide that required an ATP-dependent step for its synthesis (18). Experiments described later excluded such a reaction mechanism and now suggest that both ATP and Mgz+ are required for maintaining the proper active conformation of the enzyme.
C. Experiments with Purified Enzymes From our experiments with extracts from E. coli, i t became apparent that a rather complex enzyme system is involved in the reduction of CDP. The activities of different extracts varied widely, the results of kinetic experiments usually were very peculiar, and several lines of evidence suggested the presence of inhibitors in the crude extract. It seemed reasonable to assume that studies of the ribonucleotide reductase system from E. coli might give interesting insights into a complex enzyme system that could be expected to participate in a control mechanism of DNA synthesis. Obviously, work with purified enzymes was required. For some time, fractionation of the extract resulted in loss of enzyme activity and, while reinforcing the concept of the complexity of the system, attempts a t enzyme purification were fruitless. In retrospect, it seems clear that losses of enzyme activity resulted not so much from the instability of the protein catalysts, but rather from the partial separation of the five proteins required for the reduction of CMP, an occurrence that made adequate reconstruction experiments difficult. A turning point came when hydrogen donors other than NADPH were tried in the system. Of great importance in this connection was a paper by Grossman and Hawkins (32) reporting a reduction of ribonucleosides in extracts from Salmonella typhimurium that depended on the addition of a dithiol (dithiopropanol). In the crude E. coli system, dithiols, such as reduced lipoic acid (lip- (SH)z) , had no stimulatory effect. Very minor purification, however, yielded an enzyme preparation that was considerably more active with lip-(SH), than with NADPH
309
ENZYMATIC REDUCTION O F RIBONUCLEOTIDES
(18). Using lip- (SH),as the hydrogen donor during the assay, a further substantial purification of this enzymc, provisionally called fraction B, was obtained. The final preparation of the enzyme reduced C D P to dCDP in the presence of lip-(SH),, ATP, and Mg2+. It showed little activity with C M P or CTP as substrates (18). C M P could, however, be used as a substrate by fraction B, when a second protein fraction (fraction A ) was added. Fraction A was a side fraction obtained in an early step during the purification of fraction B (cf. Fig. 4) and, after extensive purification, was found to be identical Crude extract streptomycin
,
Streptomycin supernatant
Thioredoxin
\
Thioredoxin reductase
f
precip.
1 1 Crude
,
Crude fraction B
fraction A
I
J
Purified fraction A =
= [ C M P kinase
Purified fraction B
1 supernat.,,/
\eluate
FIG.4. Purification scheme for different proteins involved in t h e transformation of CMP t o dCDP in Escherichiu coli.
with C M P kinase. The results clearly established that the activity of CMP in thc crude extract dependcd on the presence of CMP kinase and that C D P is the actual substrate of the ribonucleotide reductase system of E . coli B. On the basis of this result, the purified enzyme
310
AGNE LARSSON AND PETER REICHARD
system was named the “CDP reductase system.” Fig. 5 summarizes our understanding of the reaction a t that time. Purified fraction B was separated further into two subfractions (enzymes B1 and B2), both of which were required for the reduction of CD P according to Fig. 5. Later (19), these two proteins were extensively purified from an extract of a thymine-requiring mutant of E . coli B, the CD P reductase of which had been derepressed by depriving the bacteria of thymine ( 3 1 ) . The final preparations of enzymes B1 and B2 are considered to be relatively pure. I n the conversion of C D P to dCDP,
ATP C M P Mgz+ kinase
.I
CDP $ Iip(SH),
fraction B
dCDP
ATP, Mg2+
+
lips,
FIG.5. The reduction of CDP by fraction B with reduced lipoate.
they are completely inactive by themselves and do not appear to catalyze any known partial reaction. Experiments now in progress indicate that the two fractions are nonidentical subunits of C D P reductase. This enzyme thus shows some similarity to tryptophan synthetase (33) and aspartate carbamyl transferase ($4). Recent unpublished experiments indicate that the binding of the two subunits requires Mg ions and that the absolute requirement of the ribonucleotide reductase for Mgz+ may be explained on this basis.
D. The Thioredoxin System The dithiol requirement is very specific; with purified fraction B, neither dithiopropanol nor any of several monothiols tested could substitute for lip- (SH)z.B In crude extracts, with NADPH as hydrogen donor, arscnite strongly inhibited the reduction of CDP. This finding indicated that, even in the absence of added lip-(SH), a dithiol participates in the reaction in the unfractionated system. Nevertheless, it seemed unlikely that free, reduced lipoate is the physiological hydrogen donor for the reduction of CDP. Lipoic acid functions in quite different types of oxidative enzyme reactions, and, more important, the crude bacterial extracts did not contain any free reduced lipoate, nor did they require added lip- (SH) for the reduction of CDP. The requirement for the addition of lip- (SH) appeared after the removal of fraction A, and it was therefore suggested that this fraction contained a protein-bound “Later it was found that dithioerythrol or dithiothrritol can substitute for lip-(SH)z.
311
ENZY M A T l C REDUCTION OF RIBONUCLEOTIDES
ditliiol (or a disulfide, reducible with NADPH) for which lip- (SH), could substitute. Fraction A thus yielded a protein that, together with NADPH, completely replaced lip-(SH)? when the reduction of C D P was performed with fraction B (35, 36). This protein was first called factor-& (35); later-when more was known about its structure and function-it was renamed thioredoxin (36). After nearly 6000-fold purification, it was obtained in an essentially pure form.
- Peptide B
-Peptide
A
__t
.COOH
CNBr
FIG.6. Strwturr of thiorcdoxin.
Thioredoxin is a heat-stable, acidic protein with a molecular weight of approximately 12,000 as determined by ultracentrifugation, amino acid composition, and enzyme activity (36). The protcin contains all the common amino acids, and a t the present time no metals or other nonprotein constitucnts have bcen detected. Thioredoxin differs in this respect from other ‘Lredoxins,”which contain nonheme iron and inorganic sulfur (ferrodoxin), nonheme iron (ruhredoxin), or a flavin (flavodoxin) (37-39) . Thioredoxin functions as a hydrogen carrier. It is isolated in its oxidized form. The functional group of the molecule consists of a disulfide bridge formed by two half-cystine residues (Fig. 6 ) . The molecule consists of a single polypeptide chain with an N-terminal serine. The S-S hridge is therefore of the intrachain type. The exact distance between the two half-cystines is not yet known but must be relatively short, since a decapeptidc recently isolated contains both half-cystine residues (40). As only one methionine residue is found among the 109 amino acids of thioredoxin, it is possible to use cyanogen bromide to cleave the molccule into two large peptides, scparable by chromatography on Sephadex-C* 50 ( 4 1 ) . The smaller fragment (peptide B) contains the active group of the molecule and consists of 37 amino acids, while the larger pcptide A contains 72 amino acids (cf. Fig. 6 ) . Since isolated thioredoxin contains no thiol groups, it cannot by itself replace lip-(SH), as the hydrogen donor in the reduction of C D P by fraction B. On addition of NADPH, however, fraction B reduces C D P in the presence of catalytic amounts of thioredoxin ( 3 6 ) .
312
AGNE LARSSON AND PETER REICHARD
Subsequently ( 4 2 ) , an enzyine was found in fraction B that catalyzes a partial reaction, in which the S-S bridge of the oxidized form of thioredoxin (thioredoxin-S2) is transformed by NADPH to a dithiol in the reduccd form of thioredoxin (thioredoxin- (SH ) L' J : thioredoxin-Sz
+ NADPH + H+
+ NAUP
thioredoxin-(SH)z
This enzyme (thioredoxin reductase) was purified extensively from crude fraction B (cf. Fig. 4) and has been obtained in pure form (43). It has a molecular weight of about 66,000 and contains two molecules of FAD, but apparently no metal. It is similar in a number of respects to glutathione reductase and lipoyl dehydrogcnase, both of which also catalyze the reduction of S-S bonds (4-46). Thioredoxin reductase is highly specific for the reduction of the S-S bond of thioredoxin and does not reduce any other disulfides (42, 4 3 ) . Both NADPH and NADH are used by the enzyme, but thc apparent K,, value for NADH is about 400 times higher than that for NADPH. At neutral pH the equilibrium is far in the direction of the dithiol, the equilibrium constant Keq
=
(thioredoxin-(SH)z)(NADP+) (thioredoxin-$1) (NADPH)(H+)
being around 5 x lo8. Marked inhibition of thioredoxin reductase by low conccntrntions of paramercuribenzoate or cadmium chloride (>go% inhibition a t 5 X lO-'M) indicates the involvement of SH groups of the enzyme in the reaction. Evidence has been reported for a direct participation of SH groups in the oxidoreduction mechanism of the two related enzymes, glutathione reductase, and lipoyl dehydrogenase (44). The stereospecificity of the hydrogen transfer from NADPH was found t o involve the B position of thc nicotinamide ring (46). This stereospecificity was also found for glutathione reductase (4'7), whereas the stereospecificity of lipoyl dehydrogenase apparently is not known. Thioredoxin and thioredoxin reductase together function as an electron and hydrogen transport system making available the reducing power of NADPH for the transformation of C D P to dCDP (Fig. 7 ) . The components, NADPH, thioredoxin, and thioredoxin reductase, can be called the thioredozin system. Table I gives a few typical experiments demonstrating the coupling of the highly purified enzymes B1 and B2 to the thioredoxin system. With catalytic amounts of oxidized thioredoxin and substrate amounts of NADPH, the formation of d C D P is completely dependent on all the components of the thioredoxin system. When chemically reduced thioredoxin is used, the requirement for
313
ENZYMATIC REDUCTION O F RIBONUCLEOTIDES ATC Mg2’ CDP
NADP’
Enzymes B , + B,
-
dCDP
Thioredoxin~-
reductose
NADPH +
H
FIG. 7. Hcdiiction of CDP hy i h p tliiorrdosin system ( I ! ? ) .
NADPH and thioredoxin reductase disappears and thioredoxin- (SH) functions in an almost stoichiomctrir niannrr. Thcsc results strongly support the scheme given in Fig. 7. The thioredoxin system also c:ttalyzes a nonspecific rcduction of “exposed” S-S bonds (42).Thus both the inter- and intrachain S-S bonds of insulin are conipletely reduced, and the A-peptide can be isolated in good yield after blocking the SH-groups with p-mercuribenzoate. I n these reactions reduced thioredoxin- (SH) acts as a “chemical” reducing agent. Enzyme systems, similar in function to that of the thioredoxin system, have been described for the reduction of methionine sulfone (48) TABLE I THE REQUIREMENT O F (:DP H E D U C T I O N
FOR
THIOREDOXIN dCDP formed (mpmoles)
Thioredoxin-Sz (0.16 mrmole) Thinredoxin-St (0.16 mpmole) ttiiorecloxiti reductase (1.2 pg) NADPH (82 mprnoles) Thioredoxin-(SH)z 0 . 5 mpmole 4 . 0 mpmole 8.0 mpmole
+
a
B1
+
0.0 7.2 0 3 2.5 5.1
The reaction mixture contained CDP, XTY, Mg*+,Tris buffer pH 8.0, and enzymes B2. The conditions used have been described by Holmgren et al. (19).
+
314
AGNE LARSSON AND PETER REICHARD
and sulfate (49) and possibly for the reduction of hydroxocobalamine (50) and glycine ( 5 1 ) . Howcver, these systems have not yet been characterizcd sufficiently to allow a more definite conclusion as to their possible relationship to the thioredoxin system.
E.
Allosteric Regulation of Substrate Specificity Crude extracts of E . coli B catalyze the reduction of all four ribonucleotides (CMP, UMP, AMP, and GMP) with approximately equal efficiency. Under the assay conditions used for determination of CDP reduction, purified fraction B (cf. Fig. 4) showed good activity toward UDP (69) but much weaker activity toward G D P and in particular ADP ( 1 3 ) . Furthermore, different preparations of fraction B showed wide variations in the capacity to reduce the four ribonucleotides ( 1 3 ) . These results were thought originally to indicate that different enzymes were involved in the reduction of purine and pyrimidine ribonucleotides, although the general requirements (e.g., a dithiol and Mg2+) were the same for both types of reactions. It was never possible, however, to establish the existence of a purine-specific enzyme, and when the CDP reductase was purified further, a small activity toward purine ribonuclcotides always remained throughout the purification. The situation remained confused until it was discovered that C D P reductase also catalyzes the reduction of purine ribonucleotides, provided that certain deoxyribonucleoside triphosphates are substituted for ATP. This was discovered during investigations on the ATP requirement in the reduction of CDP, and this latter point must first be discussed before we return to the reduction of purine ribonucleotides. When highly purified preparations of enzymes BS and B2, free from pyrophosphatases and kinases became available, the stoichiometry of the ATP requirement could be investigated ( 1 9 ) . No ATP was consumed during the reduction of C D P ; consequently the previous hypothesis involving an “activation” of the ribose moiety of C D P through consumption of stoichiometric amounts of ATP became untenable. Further studies (19, 52) indicated that the ATP requirement is not absolute and that the extent of stimulation observed with ATP depends on the concentration of C D P in the incubation mixture, being more pronounced a t low concentrations of the substrate. Under appropriate r.onditions, ATP increases the affinity of the enzyme for C D P (as measured by the K,, value for C D P ) nearly tenfold, and also increases the maximum velocity (V,,,,) of dCDP formation more than twofold. These results strongly suggest a role for ATP as a positive allosteric effector as defined by Monod e t al. (63). This type of stimulation is not specific for ATP; with dTTP the
315
ENZYMATIC REDUCTION O F RIBONUCLEOTIDES
same effects on K , and V,,, are observed. Since maximum stimulation 10-3M ATP is is found a t approximately 10-5M dTTP-whereas required-it was easy to demonstrate that the effect of dTTP is catalytic. When ATP and d?TP are present simultaneously, rather complicated effects are obtained depending on the relative concentrations of the two nucleotides. Although these experiments are not discussed in detail here, it was apparent that a t high concentrations of dTTP, relative to that of ATP, an inhibition of CDP reduction can occur. dATP has a specific and pronounced inhibitory effect and is considered to be a negative allosteric effector. The inhibition by dATP is counteracted by ATP, but not by dTTP. The latter, in fact, seems to potentiate the inhibition. As shown in Fig. 8, a series of S-shaped curves
10
20
ATP ( M Xlo4)
FIG.8. Inhibition of CDP reduction by dATP and reversal of inhibition by ,4TP (ID).
is obtained when increasing amounts of ATP are added to the enzyme system inhibited by different amounts of dATP. I n all cases, the inhibition is released a t high concentrations of ATP, but it does not appear to involve a simple competition between ATP and dATP for one site of the enzyme. Essentially identical effects of stimulatory and inhibitory nucleoside triphosphates were observed in the reduction of UDP. When the reduction of purine ribonucleoside diphosphates was reinvestigated with highly purified enzymes in the presence of different
316
AGNE LARSSON AND PETER REICHARD
allosteric effectors ( 5 4 ) ,it was soon discovered that the previous failures resulted from the use of the incorrect allosteric effector (ATP). Figure 9 shows that the reduction of G D P is stimulated strongly by low concentrations of d T T P and dGTP, while ATP has essentially no effect. As in the case of CDP, the reduction of G D P is strongly inhibited by dATP both in the presence and absence of dTTP. Also, in this case ATP, but not dTTP, counteracts the inhibition by dATP.
3
I
M x
5
lo5
FIG.9. Allosteric effects in the reduction of GDP.
Similar results were obtained in the reduction of ADP. Both d T T P and dGTP act as positive effectors, while dATP (and to a lesser degree dADP) are strong inhibitors. Studies of the reduction of ADP are therefore complicated by the fact that the product of the enzyme reaction is a quite strong inhibitor. Again, ATP, which by itself has no effect on the reduction of ADP, counteracts the inhibition by dATP and dADP. Thus, to obtain linear kinetics of ADP reduction over an extcnded time period, the incubation mixture must contain both a positive effector (dGTP or dTTP) and ATP (as “anti-inhibitor”). An important general point emerges from these studies: I n all instances the nucleoside triphosphate showed the most pronounced allosteric effects, although the diphosphates had some activity in several cases. On the other hand, the ribonucleoside diphosphates were the substrates of the enzyme system. The reduction of all four ribonucleoside diphosphates appears to be catalyzed by the same enzyme system. Two lines of evidence support
317
ENZYMATIC REDUCTION O F RIBONUCLEOTIDES
such a conclusion. First, during the later stages of purification of enzymes B1 and B2, the activities toward the different ribonucleotides run in parallel (64). Secondly, under the proper conditions, a definite competition is observed betwecn the different substrates (5d)-e.g., in the presence of dTTP as the positive allosteric effector, increasing amounts of G D P inhibit the reduction of CDP. This inhibition can in turn be reversed by increasing the concentration of CDP, and Lineweaver-Burk plots indicate a typical competitive situation. In one exIwiment, the K , value for G D P was 0.7 x M (Sd), which compares quite favorably with a K , value of 2.5 x 10-5AM obtained for the reduction of G D P with dTTP as effector ( 5 4 ) . On the other hand, when the same experiment was carried out with A T P as effector, G D P showed almost no effect on the reduction of CDP. As described earlier, ATP stimulates the reduction of CDP, but not that of GDP. The general conclusion is that the substrate specificity of the ribonucleoside diphosphate reductase is governed by the prescnce of different allosteric effectors. The results also suggest the presence of a common catalytic site for the binding of all four substrates. Pyrimidine ribonucleotides fit the site when ATP is bound t o the allosteric site of the enzyme; purine ribonucleotides fit it when dGTP is the allosteric effcctor; and all four substrates interact with the site with d T T P as effector. Within the framework of thc theory of hlonod et nl. (55) this means that the allosteric effectors stabilize different conformational states of the enzyme (Fig. 10) suitable for the reduction of the different groups of substrates. It is then ilssunied that dATP stabilizes an inactive state of the enzyme.
1
ATP Pyrimidine and purine specific state
=
dTTP
dGTP
dATP
Y
-
Purine specific state
318
AGNE LARSSON AND PETER REICHARD
The discussion of allosteric properties and substrate specificity of ribonucleoside diphosphate reductase6 has thus far not included a consideration of the requirements of the reaction for two proteins (enzymes B1 and B2).7There are few facts to offer on this point and only speculation is possible at the present time. Both proteins are relatively large, ~ , in ~sucrose gradients of around 7.8 ( B l ) and 5.5 (B2). with s ~ values Preliminary experiments suggest that both are subunits of the active enzyme and that Mgz+ is necessary for their mutual binding. It thus seems to be more appropriate to talk of the B1 and B2 proteins instead of enzymes B1 and B2. It is not a t all clear to what extent the two proteins of the ribonucleotide reductase resemble the catalytic and regulatory subunits found for aspartate carbamyl transferase by Gerhart and Schachman (S4). One dissimilarity is immediately apparent: neither of the B1 or B2 proteins is active in the absence of the other. The catalytic subunit of aspartate carbamyl transferase, on the other hand, is more active in the absence of the regulatory subunit. The availability of highly purified preparations of the B1 and B2 proteins should soon make possible a better understanding of the molecular mechanism of ribonucleoside diphosphate reductase. Ribonucleotide reduction is the first enzymatic step on a metabolic pathway leading specifically to the synthesis of DNA. It is a wellestablished fact that branch-points of metabolic pathways are targets of regulatory mechanisms. The allosteric control of the substrate specificity of the enzyme probably functions in the cell to furnish a balanced supply of the four deoxyribonucleoside triphosphates required for the synthesis of DNA. Figure 11 represents an attempt to formulate a reasonable model concerning the situation in the living cell, based on the findings in vitro with purified enzymes. I n the presence of ATP, the two pyrimidine ribonucleoside diphosphates are reduced to the corresponding deoxyribonucleotides. One of these, dUDP, is then transformed to d TTP in a series of reactions. The increasing concentration of d TTP initiates the reduction of purine ribonucleotides, and, as well, influences the reduction of pyrimidine ribonucleotides. The latter effect, depends on the relative concentrations of “Originally the enzyme was called CDP reductase. The enzyme is not specific for the reduction of CDP, however, and the name ribonucleoside diphosphate reductase is therefore more appropriate. ‘Thioredoxin does not enter the discurnion at this level, since it is looked upon w a substrate in the reaction. We have not found allosteric effects influencing the binding of thioredoxin.
319
ENZYMATIC REDUCTION O F RIBONUCLEOTIDES
GDP
AOP
FIG.11. Possible physiological interpretation of the allosteric effects involvcd in the regulation of the substrate specificity of the ribonucleoside diphosphate reduetase from Escheriehiu coli ( 6 4 ) .
ATP m d clTTP, so that either an inhibition or a stimulation of the reaction rcsults. A further stimulation of purine ribonucleotide reduction is caused by dGTP. While d T T P is most effective in the reduction of GDP, the greatest effect of dGTP is on the reduction of ADP. Finally, dATP, formed by phosphorylation of dADP, acts as a general feedback inhibitor and depresscs the reduction of all four substrates. Thus, in the absence of DNA synthesis, dATP is the key effector that prohibits the accumulation of excessive amounts of deoxyribonucleotides in the cell. This model is an attempt to integrate the findings with purified enzymes into a rather detailed physiological control mechanism and of UMP
It UDP
ribonucleotide
-GzziiF
dUDP
,,
It
CI
dUTP
dUMP
1
d T M P synthetase
dTMP
FIG.12. Enzyme reactions involved in the formation of dTMP from UMP.
320
AGND LABSSON AND PETER REICHARD
course represents only an approximation to the situation in vim. It should not be regarded in any other light. Similar types of regulatory mechanisms also appear to function in animal cells and are described below together with a discussion of experiments with intact cells that appear to substantiate the general picture given above. One final aspect of regulation involves the formation of deoxyuridine phosphates. The dUDP formed by the reduction of UDP is in the cell rapidly phosphorylated to dUTP, which can substitute for dTTP in the DNA polymerase reaction (56).However, with rare exceptions (57) , uracil does not occur in DNA. The explanation for this appears to be the existence of a powerful pyrophosphatase (deoxyuridine triphosphatasc) , which specifically cleaves dUTP to dUMP and pyrophosphate (29,5 8 ) . dUMP is then methylated to dTMP by thymidylate synthetase (59). These interrelationships are summarized in Fig. 12. The physiological function of deoxyuridine triphosphatase thus appears to be to prevent the accumulation of dUTP in the cell and involves the same principle as that underlying the action of the phage-induced deoxycytidine triphosphatase discovered previously (60,61).
F.
Studies on the Reaction Mechanism In principle, ribonucleotide reduction involves the replacement of a hydroxyl group by a hydrogen atom (see Fig. 1). In E. coli, the most immediate hydrogen donor identified is the dithiol group of reduced thioredoxin. I n order to understand the reaction mechanism, one thus must answer the question how a dithiol can reduce a secondary alcohol group. Some degree of understanding may be attained by localizing the position (s) in the deoxyribosyl moiety into which hydrogen is introduced during the enzyme reaction. This can be done by carrying out the reaction in water labeled with either H3 or H2. A rapid equilibration of isotope occurs with the functional groups of the hydrogen donor, since hydrogen atoms of SH groups exchange with the protons from water ( 6 2 ) . The isotope introduced into deoxyribose via the dithiol can then be localized after a chemical degradation, in the case of tritium, or by nuclear magnetic resonance (NMR) spectroscopy, in the case of deuterium. For the experiments with tritium, the degradation procedure outlined in Fig. 13 was used (63).dCMP was transformed to deoxyribose 5-phosphate by acid hydrolysis after labilixation of the bond between the base and the sugar by reduction. Deoxyribose 5-phosphate was then further degraded in the two following ways.
32 1
ENZYMATIC REDUCTION O F RIBONUCLEOTIDES
1. By treatment with deoxyribose aldolase in the presence of alcohol dehydrogenase and NADH, carbon atoms 1 and 2 were obtained as ethanol, and carbon atoms 3, 4, and 5 as glyceraldehyde phosphate. I t was necessary to use a large excess of alcohol dehydrogenase in order to remove the acetaldehyde as rapidly as it is formed. Otherwise loss of isotope from the original position 2 of deoxyribose would occur. 2. Deoxyribose 5-phosphate was oxidized with bromine to deoxyribonic acid 5-phosphate. I n this way the hydrogen attached to position 1 is recovered in water. Glyreraldehyde 3-P + ethanol
reduction
dCMP
+ acid hydrolysis*
Deoxyribose 5-P
/
\ Deoxy r i boni c acid 5-P
FIG.13. Degradation of dCMP for localization of tritium.
I n two separate experiments, 0.26 and 0.29 atom of tritium was incorporated into dCMP. The occurrence of an isotope effect explains these low values. By combining the two degradation procedures it could be clearly established that all isotope was localized a t position 2 of deoxyribose (Table 11). TABLE I1 DEGRADATION OF DEOXYRIBOSE 5-PHOSPHATE ENZYMATICALLY SYNTHESIZED H3-CDP
FROM
Specific radioactivity
(%I dCMP
100
1
deoxyribose 5-P
I
99
1 deoxyribonic acid 5-P
glyceraldehyde 3-P
+
ethanol
102
<1
94
322
AGNE LARSSON AND PETER REICHARI)
@@-o-cvy
w
P
HO
OH
HO
HO
@
FIG.14. Different possible reaction mechanisms for the reduction of CDP. The circled H denotes the incorporation of tritium expected from the different mechanisms.
The results exclude several of the possible reaction mechanisms shown in Fig. 14. For example, the formation of intermediates containing a double bond (either between carbon atoms 2‘ and 3‘ or between carbon atoms 1’ and 2’) is not possible since reduction in this manner would result in the introduction of tritium into positions 2’ and 3 ,or I’ and 2’, respectively. Another possible intermediate, involving a 3‘-keto-deoxyribosyl moiety, is also excluded, since in this case isotope would have been introduced into position 3’.
ENZYMATIC REDUCTION O F RIBONUCLEOTIDES
323
Similar experiments with D,O substantiated and extended the results obtained with tritiated water ( 64) . The NMR spectrum of enzymatically synthesized, deuterated deoxycytidine conclusively showed the presence of one atom of deuterium a t position 2‘. In this case the isotope effect did not obscure the stoichiometry of the introduction of hydrogen since the enzyme reaction was carried out in 99.776 D,O. The NMR spectrum also demonstrated that the deuterium is introduced stereospecifically into position 2‘. By combining the data from the N M R spectrum with earlier calculations of the coupling constants for the two hydrogens a t position 2’ with the hydrogen a t position 3‘ ( 6 5 ) , it appeared that the hydrogen is introduced trans to the base with no inversion occurring during the reaction. The stereochemistry of the reduction a t position 2’ seeins to exclude the replacement of the OH group by a hydride ion through a simple SN 2 reaction, unless we wish to invoke a double inversion during the reaction. It seems more likely that the reaction proceeds via an SN 1 reaction involving the formation of a carbonium ion. Many nonenzymatic SN1 reactions involve racemization, since the attack by the nucleophilic agent can occur with about equal probability on either side of a planar carbonium ion. I n enzyme reactions, racemizations usually do not occur, and one can easily visualize a situation during which a “hydride ion” is created by a part of the enzyme in a given steric position with respect to the plane of the carbonium ion, resulting in the insertion of the hydrogen a t only one side of the ring. Spectroscopic evidence (66) indicates a relatively low pK, value for the dissociation of the OH group a t position 2’ in ribonucleosides as compared to pK, values of OH groups a t other positions. The increased acidity of the OH group a t position 2’ is also apparent from the behavior of different nucleosides during chromatography on Dowex 1-OH- ( 6 7 ) . These results indicate a polarization of the C-0 bond 2t position 2’ in a ribonucleotide, which would favor the formation of a carbonium ion. It is difficult to visualize how the postulated “hydride ion” can be generated from a dithiol. As discussed in the next section, the ribonucleotide reductase from Lactobacillus leichmannii requires the participation of a cobamide coenzyme, which appears to function as a hydrogen carrier between reduced thioredoxin and the ribonucleotide. I n this system hydrogen is introduced into position 2’ of the deoxyribosyl moiety with the same stereospecificity as in the E . coli system. The participation of a cobamide coenzyme in the ribonucleotide reductase from L. leichmannii makes it easier to accept a hydride ion mechanism in this system. Although the participation of a cobamide derivative now appears to be firmly excluded from the mechanism of the E. coli reductase, the
324
AGNE LARSSON AND PETER REICHARD
possibility exists that some other unknown group (firmly bound to either the B1 or B2 proteins) acts as a carrier of hydrogen between reduced thioredoxin and the ribonucleotide.
111. Ribonucleotide Reduction in Lactobacillus leichmannii A. In Vivo Experiments I n several species of Lactobacillus, including L. leichmannii, the substitution by deoxyribonucleosides of the growth requirement for vitamin B,, (68) suggested the possible involvement of this vitamin in the biosynthesis of deoxyribosyl compounds in these organisms. Strong support for this idea was first obtained by Downing and Schweigert (69),who demonstrated that addition of vitamin BIZto growing cultures of L. leichmannii results in considerable dilution of isotope in the deoxyribosyl moiety of uniformly labeled thymidine-C14 incorporated into the bacterial DNA. Subsequently, more direct evidence for the involvement of vitamin B,, in the reduction of ribosyl compounds came from experiments demonstrating that L. leichmannii converts the ribosyl residue of guanosine to deoxyribose of DNA when growth is supported by vitamin B,,, but not by deoxyribonucleoside (70, 71). Furthermore, the size of the acid-soluble deoxyribosyl pools of L. leichmannii depend on the supply of vitamin B,, in the medium (72). Although the whole-cell experiments strongly suggested a function of vitamin B,, in the transformation of ribosyl to deoxyribosyl derivatives, definite proof was lacking a t this time. It could be argued that many of the results reflected a feedback type of inhibition of deoxyribose synthesis caused by the addition of deoxyribonucleosides (15). The existence of a cobamide-dependent ribonucleotide reductase in L. leichmannii was definitely established by the work of Blakley and Barker ( l 4 ) ,who demonstrated that a cobamide coenzyme was required for the transformation of C M P to dCMP in extracts of this organism.
B. Experiments with Crude Extracts Several investigators, including ourselves, unsuccessfully tried to demonstrate a conversion of ribonucleotides to deoxyribonucleotides in cell-free extracts of L. Zeichmannii. I n 1964 Blaklcy and Barker ( 1 4 ) presented the first evidence for the enzymatic reduction of ribonucleotides by showing that extracts of L. leichmannii, treated with Dowex 1C1-, catalyzed the conversion of CMP to dCMP without cleavage of the glyrosyl linkage. The reduction possessed an almost absolute dependence on the addition of dimethylbenzimidazolyl cobamide coenzyme. I n ad-
ENZYMATIC REDUCTION O F RIBONUCLEOTIDEY
325
dition, the reaction required ATP, Mg", 2-mercaptoethanol, and a NADPH-generating system (glucose &phosphate and glucose 6-ph0~phate dehydrogenase) . The pioneering work of Blakley and Barker was subsequently confirmed by Abrams and Duraiswami (15)and Beck and Hardy (16). I n a n extension of the initial work, all three groups examined the requirements of the reaction for cobamide coenzyme. Among the compounds tested, Blakley (73) found that the cobamide coenzyme was the most active derivative with a K , of 9 x lO-'M. Only low activities wcw found with hydroxycobalamin and cyanocobalamin. Similarly Beck and Hardy (16) found a higher activity with the coenzyme than with cyanorobalamin and demonstrated an interesting effect of ribosomes in the system: when these particles were removed from the extract, a nearly fivefold increase in the reduction of cytidine phosphates was observed with the cobamide coenzyme, and the activity with cyanocobalamin disappeared. These effects were explained by the discovery that ribosomes not only transform cyanocobalamin t o the cobamide coenzyme (in a reaction requiring ATP and a mono- or dithiol) , but also bind cxogenous cobamide coenzyme and render it unavailable for the enzymatic reduction of CMP. I n the early experiments with crude extracts of L. Zeichrnannii, the reduction of C M P required the simultaneous presence of a NADPHregenerating system and mercaptoethanol (14). Under these conditions, it may be assumed that NADPH is the ultimate hydrogen donor in the reaction. This work was followed by the discovery that reduced lipoate or ciithiothreitol-dithiols active as reductants in ribonucleotide reduction in E . coli-not only abolished the requirement for the NADPH-regenerating system and mercaptoethanol, but stimulated the reduction of cytidine ribonucleotides several hundredfold (16, 20, 7 3 ) . As in the E . roli system, monothiols and 2,3-dimercaptopropanol were inactiw. Optimum formation of dCMP required very high concentrations of reduced lipoate (3 x M or higher). Addition of nicotinamide nucleotides or flavins gave no further stimulation of the reaction. I n the crude extract, the conversion of C M P to d C M P was absolutely dependent on the addition of ATP and strongly stimulated by the addition of MgZ+.All three groups of workers found that in the crude extract optimum concentrations of ATP and Mg2+ were approximately 1 x 10-2M. It was noted that C D P and C T P have approximately equal activity as substrates, C M P less and cytidine very little ( 7 3 ) .It was suggested that C D P or CTP is the actual substrate. The activity observed with
326
AGNE LARSSON AND PETER REICHARD
CMP and cytidine was thought to be caused by the presence of kinases in the crude extract. Under conditions optimal for the reduction of cytidine phosphates, very little reaction was observed with UMP, UDP, AMP, or ADP. On the other hand, GMP was reduced a t about the same rate as CMP (7.3).
C. Experiments with Purified Enzymes The purification of the enzyme from lactobacilli was carried out simultaneously in the laboratories of Abrams, of Beck, and of Blakley. Many of the results also appear to have been obtained independently by the three groups. Abrams (20) purified the enzyme activity of the crude extract approximately 25-fold with respect to CTP reduction (specific activity: 20-30 v o l e s per minute and per mg) and successfully removed CMP and C D P kinases. The preparation lost the ability to reduce CMP and CDP, reducing only C T P to dCTP. A more extensive purification was achieved by Goulian and Beck ( 2 2 ) . The reductase was again purified with respect to C T P reduction to a final specific activity of 550-600. In the analytical ultracentrifuge, the enzyme appeared monodisperse on sedimentation velocity analysis, although a slight heterogeneity was found during a sediment.ation equilibrium analysis. Electrophoresis on cellulose acetate gave a single band, whereas polyacrylamide electrophoresis separated two major and two minor bands. It is possible that the latter method separated several subunits and that the enzyme preparation was, in fact, quite pure. This enzyme preparation was specific for the reduction of ribonucleoside triphosphates and was used for most of the experiments by Beck’s group. In Blakley’s laboratory, the reductase was purified with respect to the reduction of purine ribonucleoside triphosphates (21, ‘74).As yet only brief statements of the general principles of the purification procedures have been published, and no studies on the homogeneity of the most purified enzyme preparations have been reported. The best preparations show a specific activity of 1200-1700 with ATP as substrate. Again the enzyme is highly specific for the triphosphate. The choice of different substrates prevents a precise comparison of the activities of Beck’s and Blakley’s enzyme preparations. Nevertheless it seems probable that both groups have purified the reductase to about the same extent. It is clear that the same enzyme is involved in the reduction of C T P and ATP, as is discussed later. The absolute requirement of the purified preparations for the addition of the cobamide coenzyme indicates that the apoenzyme was purified independently of the naturally occurring form of its coenzyme.
ENZYMATIC REDUCTION O F RIBONUCLEOTIDES
327
Moreover, the reduction of CTP in crude extracts also showed an almost absolute requirement for the addition of the cobamide coenzyme. These observations suggest two possible explanations. Either the binding of the coenzyme is weak-an unlikely possibility in view of the low K , value for the cobamide coenzyme-or a large excess of apoenzyme exists in the bacterial extracts. The activity of the coenzyme in ribonucleotide reduction in vitro does not necessarily prove that this cobamide derivative is the naturally occurring coenzyme. However, since the major part of the cobalamin derivatives in lactobacilli can be isolated as dimethylbenzimidazolyl cobamide coenzyme, such an assumption is probably valid. The final answer will come when the holoenzyme is isolated from L. leichmannii and the identity of the bound coenzyme is established. A puzzling discrepancy exists between the values determined for the molecular weight of the Lactobacillus enzyme. From gel filtration experiments, Blakley et al. ( 2 1 ) estimated the molecular weight of the enzyme t o be approximately 25,000, whereas Goulian and Beck (2?2), from ultracentrifugation data, calculated a value of 110,000. The possibility remains that the lower value corresponds to the molecular weight of a subunit.
D. Hydrogen Donor Specificity All the different purified ribonucleoside triphosphate reductase preparations show a n absolute dependence on the addition of a dithiol. The specificity of the dithiol requirement was investigated in detail by Vitols and Blakley ( 7 4 ) , who found that dithiols containing either 6,8-, 1,3-, or 1,4-dithiol groups are active, whereas such compounds as 2,3dithiopropanol are inactive. It was suggested that activity depends on the ability to undergo intramolecular cyclization upon oxidation. The most efficient reductant was reduced lipoate, which was oxidized in stoichiometric amounts during the reduction of G T P to dGTP, thereby establishing the substrate nature of reduced lipoate. Furthermore, reduced lipoate in the reduction of ATP can be replaced thioredoxin reductase from E. coli ( 7 4 ) . by NADPH and thioredoxin All three components are required for activity and the requirement for the coenzyme remains. Thus the same naturally occurring dithiol can be used as hydrogen donor in both the cobamide-dependent system and the system from E. coli, which operates in the absence of a cobamide derivative. The hydrogen donor activity of reduced thioredoxin from E. coli with L. leichmannii reductase was confirmed and extended to the reductions of CTP, GTP, and U T P by Beck e t al. (75). Half-saturation
+
328
AGNE LARSSON A N D PETER REICHARD
with E. coli thioredoxin in the L. leichmannii system occurs a t 2 x 1M and the saturation curve is sigmoidal. With E . coli ribonucleotide reductase, however, the thioredoxin saturation curve is hyperbolic with an apparent K , value of about 1.5 x M (42). The affinity of the E . coli enzyme for E . coli thioredoxin therefore seems to be considerably higher than that of the L. leichmannii ribonucleotide reductase. With reduced lipoate, half-saturation in the L. leichmannii reductase system occurs a t concentrations in excess of 2 X M (16, 6 7 ) , indicating a much lower affinity for reduced lipoate than for thioredoxin. However, maximum rates of ribonucleotide reduction were slightly lower with thioredoxin than with reduced lipoate (75). The very high concentrations of reduced lipoate required for optimum activity made it quite unlikely that this compound was the naturally occurring active reductant. I n fact, the physiological role of reduced lipoate was effectively excluded when Orr and Vitols (76) isolated from lactobacilli a thioredoxin system that appeared to be the naturally occurring hydrogen donor. With the NADPH-dependent reduction of 5,5’-dithio-bis-2-nitrobenzoic acid as assay and by procedures patterned after those used for the purification of thioredoxin and thioredoxin reductase from E. coli (36, &), Orr and Vitols (76) partially purified two protein fractions that corresponded functionally to the two E. coli proteins. Their molecular weights, estimated by gel filtration, were 12,000 and 55,000, respectively, as compared to 12,000 for E . coli thioredoxin (36) and 66,000 for E . coli thioredoxin reductase ( 4 3 ) . A limited amount of cross reaction was found between the fractions from the two types of microorganisms. Thus E. coli thioredoxin was reduced by the reductase from L. leichmannii, while the combination of E. coli reductase and L. leichmannii thioredoxin was essentially inactive. The thioredoxin system from L. leichmannii replaced reduced lipoate in the reduction of GTP. Ribonucleotide reduction exhibited simple Michaelis-Menten kinetics with thioredoxin from either source in the presence of homologous thioredoxin reductase. The apparent K , value for L. leichmannii thioredoxin was 3 X M and for that of E . coli M . The same VmaXwas reached with both. 4x As mentioned earlier, Beck et al. (75) observed a sigmoidal saturation curve for E. coli thioredoxin with half-saturation a t about 2 x 1 @ 5 M . The conditions of incubation might explain the contradictory results, since Beck et al. measured dCTP synthesis in a system containing both Mg” and dATP, while Orr and Vitols assayed for the reduction of GTP in the absence of either Mg2+or a second nucleotide. An alternative explanation derives from differences in molecular weights reported
ENZYMATIC REDUCTION OF RIBONUCLEOTIDES
329
for the two reductases: the one used by Beck et al. had a molecular weight of 110,000 and that by Orr and Vitols 25,000. If the smaller protein were a subunit, it is entirely possible that a cooperative effect or control mechanism existing with the larger molecule was lost. Such a loss may well explain the difference between hyperbolic and sigmoidal saturation curves. This tentative explanation rests on the assumption that the observed molecular weights are valid under the conditions of incubation.
E. Substrate Specificity and Allosferic Effects The different groups working with the L. leichmannii reductase eventually concluded that the substrates for this enzyme are nucleoside triphosphates. From the early work, however, the specificity of the enzyme for the base constituents of the nucleotides remained obscure until i t was discovered that this problem was related to the requirement of ATP and Mgz+for C T P reduction. This latter point is discussed first. Blakley et al. (21) reported that their partially purified enzyme preparation was 10 times more active with G T P than with C T P or ATP, and that the reduction of U T P was only 2 4 % of the reaction with GTP. Furthermore, they stated that A T P and Mg2+ had no effects on any of the reactions. It was Concluded that the stimulation previously demonstrated in cruder systems was related to the transformation of CMP to CTP. This observation was in contrast to that reported by Abrams (go), who found that ATP and Mg2+ stimulated the reduction of C T P more than tenfold. The reduction of the other ribonucleoside triphosphates was not tested. Similar results were obtained by Beck (77). At about this time, experiments with the ribonucleotide reductase from E . coli indicated that ATP functions in the reduction of C D P as an allosteric activator (19). As described above, this finding led to the demonstration that the substrate specificity of the enzyme is regulated by different allosteric effectors (52, 5 4 ) . It seemed possible that the L. leichmannii system was regulated in a similar way and that some of the discrepancies observed could be explained on this basis. A joint investigation by Beck's group and ourselves indicated that the ribonucleoside triphosphate reductase from I,. leichmannii indeed appeared to be subject to allosteric regulation (75). I n the presence of Mg2+,the reduction of C T P is stimulated about twentyfold by ATP (or dATP), and that of U T P about sevenfold by dCTP. With the purine nucleotides, only about twofold stimula t'ions ( G T P reduction by d T T P and ATP reduction by dGTP) are observed (Table 111).Approximately the same maximum rates were observed with
330
AGXE LARSSON A N D PETER REICHARD
all four ribonucleoside triphosphates in the presence of the proper activator (Table 111). Since the enzyme preparation was considered to be essentially pure, it appeared likely that all four substrates are reduced by the same enzyme. A more detailed study of the effect of ATP by Goulian and Beck (22) revealed a complicated interplay between the effects of Mg2+and those of the allosteric effector. I n the presence of Mg2+,the reduction of CTP is almost completely dependent on the addition of ATP, whereas TABLE I11 SUBSTRATE SPECIFICITY OF RIBONUCLEOTIDE REDUCTASE FROM
Lactobacillus leichmannii Effector"
Substrate
None
ATP
dATP
dGTP
dCTP
dTTP
CTP GTP ATP UTP
0.23 2.91 2.61 0.31
6.67 0.80 2.93 0.42
6.44 3.50 0.80 0.42
0.85 1.62 7.30 0.40
0.54 3.37 0.70 2.02
0.77 6.02 0.75 0.54
All results were obtained in the presence of 1.6 X
M MgCl,. They are expressed
as millimicromoles of product formed. The condit.ions of the experimeiit have been
described (75).
in the absence of Mg2+the ATP requirement is less pronounced. With the four different substrates tested, the reduction of C T P is the only reaction stimulated by ATP Mgz+.All four reactions are inhibited by Mg2+alone. dATP replaces ATP as a stimulator of C T P reduction and is effective a t much lower concentrations. Since the enzyme also reduces ATP to dATP, i t was suggested that dATP-and not ATP-is the positive effector for CTP reduction. If this hypothesis were correct, a time study would reveal an initial lag in the rate of dCTP synthesis. Since such a lag was not found, we feel that the results more likely indicate that both ATP and dATP can stimulate the reduction of CTP. The studies of Goulian and Beck (22) demonstrated two further fundamental differences between the ribonucleotide reductases from E . coli and L. leichrnannii. All the reactions catalyzed by the former enzyme show an absolute requirement for Mg2+ and are strongly inhibited by dATP. The enzyme from L. leichmannii is in many instances actually inhibited by Mg2+,and dATP is a positive effector in the reduction of CTP. Recently Beck (78) made a very thorough and extensive study of the regulatory effects involved in the reductions of the four different ribo-
+
331
ENZYMATIC REDUCTION OF RIBONUCLEOTIDES
nucleoside triphosphates by the L. leichmannii enzyme. A major part of the results concerned the profound influence of the coneentration of Mg2+ on the results obtained with different deoxyribonucleoside triphosphates as effectors. Also, the order in which Mg2+was added to the incubation mixture-relative to the addition of substrate and/or effector-was very important. These latter effects are not easily understood. A few of the results from Beck’s paper on the influence of effector and Mg2+on the four different reductions are summarized in Table IV. TABLE I V INFLUENCE OF RIg2+ A N D EFFECTOR ON ACTIVITYOF RIBONUCLEOTIDE REDUCTASE FROM Lactobacillus leichmannii Specific activity of enzyme (mpmoles deoxyribonucleotide formed/mg/min) Minus
f3ubstrat.e
Effector
Complete”
Minus Mgz+
dATP dCTP dGTP dTTP
490 180 490 480
290 180 500 200
m2+,
Minus effector
minus effector
20 30 180 300
100 100 270 830
~~~
CTP UTP ATP GTP ~
The complete system contained both Mg’f arid the effector. The data are compiled from the paper by Beck (78).
In general, the results substantiate the stimulatory effects of the different ‘Lprime’’effectors on the reduction of the four substrates already demonstrated in Table 111. In addition, the inhibitory effect of Mg2+in the absence of the effectors are brought out. In the presence of the effectors, Mg2+ showed no effect on the reduction of UTP and ATP, but gave a slight stimulation of the reductions of both C T P and GTP. The reduction of GTP, however, proceeded at its highest value in the absence of Mg’+ and effector. These results are valid only a t this single concentration of Mg”. The influence of effector and Mg2+ on the substrate saturation curve for the reaction is exemplified by the reduction of C T P (Fig. 15). I n the presence of Mg2+,but without effector (dATP), a sigmoidal saturation curve was obtained. Addition of dATP straightened the curve and increased the affinity for CTP as well as the maximum velocity. With dATP alone, or in the absence of both dATP and Mg2+,nonsigmoidal curves with low maximum velocities were obtained. Similar but not identical effects were found with ATP, GTP, or U T P as substrates.
332
AGNE LARSSON AND PETER REXCHARD
The substrate specificity of the enEyme was influenced by changing the relative proportions of the effectors. Thus, in the presence of both CT P and GTP as substrates, and of dATP and d l T P as effectors, the enzyme reduced primarily C T P when the dATP/dTTP ratio was 1:9. I
I
I
I
CTP ( ~ ~ 1 0 ~ )
FIG.15. Effects of dATP and Mg” on the CTP saturation curve of the ribonucleoside triphosphatc reductase from Lactobacillus kichmunnii ( 7 8 ) .
When this ratio was changed to 9:1, G T P was reduced in preference to CTP. This result was observed only in the presence of Mg2’. I n the absence of Mg?’, GTP was always the preferred substrate, even though the absolute reaction rates decreased for G T P and increased for CTP when the dATP/dTTP ratio increased (Fig. 16). The analysis of most of the kinetic data is complicated by the fact that a given nucleotide can be both a substrate (or product) and an effector. In the E . coli system, the substrates and products are nucleoside diphosphates. There it is possible to obtain evidence for a single “substrate” site by the proper competition experiments. Corresponding experiments with the L. Zeichmannii system are not convincing, and a decision on this point must await direct binding studies. Similarly, it is not yet possible to decide with any degree of confidence whether one or several “allosteric” sites are involved in the binding of the different effectors and what the role of Mg2’ is in this connection. Earlier we discussed the difficulties involved in any attempt to relate the in vitro results with the E. coli system to a physiological control mechanism in vivo. These difficulties are even more apparent with the L. Zeichmannii system. First, any hypothesis must necessarily depend on
333
ENZYMATIC REDUCTION OF RIBONUCLEOTIDES
whether or not the effects of Mgz+are taken into consideration. Secondly, the L. Zeichmannii system appears to lack a strong, physiologically useful “feedback” inhibitor. Therefore, from an experiment such as that shown in Fig. 16, one can visualize a control mechanism that prevents the enzyme from reducing a single substrate, but not a mechanism that prevents the enzyme from catalyzing overproduction of all four deoxyribonucleotides. I n a purely speculative vein, however, we should like
LI -I
0
I
+I
log [dATP]/ [dTTP]
FIG. 16. Dependence of substrate specificity (toward C T P and GTP) of Lactobacillus leichmannii reductme on the relative concentrations of two effectors (dATP and dTTP) (78).
to suggest the possible existence of another type of control mechanism iiivolving cobamide coenzyme. L. leichmannii (whether grown in the presence of vitamin B,, or deoxyribonucleoside) contains relatively large amounts of apoenzyme relative to coenzyme. It is conceivable that a mechanism exists that regulates the binding of the coenzyme to the apoenzyme and that depends on the supply of deoxyribonucleotides in the cell.
F.
Regulation of Enzyme Synthesis I n addition to vitamin B,, or a deoxyribonucleoside, L. leichmannii also requires either folic acid or thymine for growth in an enriched medium. Beck and Hardy (16) observed that the ribonucleotide reductase activity of crude extracts of organisms previously depleted of folate by extended growth on thymine increases after exposure of these organ-
334
AGNE LARSSON AND PETER REICHARD
isms to media lacking thymine. I n fact, the capacity of extracts to reduce cytidine nucleotides increases nearly tenfold during a 5-hour period after transfer to a medium lacking thymine. After the discovery that the increase of the enzyme activity is blocked by chloramphenicol, Beck and Hardy concluded that the synthesis of the ribonucleotide reductase is subject to repression by an intracellular derivative of thymine, possibly dTTP. A similar conclusion was reached earlier for the enzyme from E . coZi15T-. Ghambeer and Blakley (79, 80) also studied the factors influencing the levels of ribonucleotide reductase in L. leichmannii, and observed that the capacity of a crude bacterial extract to reduce ATP is greatest a t the end of the linear phase of growth, and nearly absent during the stationary phase. When the concentrations of either cyanocobalamin or the different deoxyribonucleosides in the growth medium were increased, the reductase activities decreased. Assuming that the activity of dialyzed extracts accurately reflects the amount of reductase in the cells, Ghambeer and Blakley concluded that addition of cyanocobalamin or deoxyribonucleosides to the growth medium causes repression of enzyme synthesis. Furthermore, they suggested that cyanocobalamin is not an actual corepressor, but acts indirectly by increasing the cellular content of deoxyribonucleotides, considered to be the functional corepressors in the synthesis of the enzyme. Ghambecr and Blakley criticized the conclusion of Beck and Hardy, that an intracellular thymine derivative was a corepressor, on two points. First, they found that addition of thymine t o the medium does not influence the level of the reductase in the crude extracts, while addition of thymidine has a pronounced effect. Secondly, they were unable t o observe an increase in enzyme activity under conditions designed t o achieve a thymineless state, as described by Beck and Hardy. As far as we can see, the two groups have demonstrated that ribonucleotide reductase in L. leichmannii is a repressible enzyme. Furthermore, it appears that both succeeded in derepressing the synthesis of the enzyme and have exploited this success by starting enzyme purifications with extracts containing the derepressed enzyme. The major remaining point of controversy seems to be the manner in which derepression is effected. Does it require withdrawal of deoxyribonucleotides in general-as Ghambeer and Blakley s u g g e s t o r can i t be achieved by depletion of thymidine nucleotides alone? The different answers to this question may depend on differences between the two strains (ATCC 7830 and a mutant of ATCC 4797) used by the two groups. Thus the strains could differ in some genuine aspect of their control mechanism, or in the ability to store folic acid derivatives. A more likely explanation
ENZYMATIC REDUCTION OF RIBONUCLEOTIDES
335
derives from the possibility that the strain used by Blakley may not have the capacity to transform extracellular thymine to intracellular dTTP. If this assumption were correct, it would explain the repression by thymidine but not thymine. We do not think that the experiments of Ghambeer and Blakley rule out dTTP as corepressor in the synthesis of the L. leichmannii reductase. Besides, the observation that the enzyme level in different strains of E. coli increases markedly during thymine starvation (31, 19) makes it tempting to extrapolate this result t o L. Zeichmannii. However, the experiments of Beck and Hardy do not exclude the possible role(s) of other deoxyribonucleotides as corepressors as well as the possibility that “full derepression” may require the presence of all four deoxyribonucleotides.
G.
Studies on the Reaction Mechanism
One of the major distinctions between the two ribonucleotide reductases concerns the involvement of cobamide coenzyme in the reaction catalyzed by the L. leichmannii enzyme, and the first question regarding the reaction mechanism of this enzyme concerns the mechanism by which the coenzyme participates in the reaction. I n this respect, the recent work of Abeles and co-workers (81, 82) on the mechanism of action of
Dioldehydrase DBC roenzyme
H H I 1 H-C-C-H I 1 HO OH
FIG.17. Reactions catalyzed by dioldehydras?.
the cobamide coenzyme in the dioldehydrase reaction is extremely relevant. This enzyme catalyzes the conversions of propanediol to propionaldehyde, and of ethylene glycol to acetaldehyde (Fig. 1 7 ) . These reactions resemble the conversion of ribonucleotides to deoxyribonucleotides since they involve the replacement of a hydroxyl group by hydrogen. They differ, however, in one basic respect. I n the dioldehydrase reaction, the reduction a t one carbon occurs simultaneously with oxidation a t the adjacent carbon, whereas in the reaction catalyzed by ribonucleo-
336
AGNE LARSSON A N D PETER REICHARD
tide reductase the reduction of carbon 2' is coupled to the oxidation of an external dithiol. The formation of propionaldehyde from propanediol proceeds without incorporation of hydrogen from the medium. However, the hydrogen transfer is not strictly intramolecular, since when propanediol-1-H3 and nonlabeled ethylene glycol were incubated with the cobamide coenzyme and dioldehydrase, acetaldehyde with tritium in its a-position was formed in addition to labeled propionaldehyde. It was postulated (81) that the formation of an aldehyde from a diol occurred in a stepwise fashion. First, a hydrogen equivalent was transferred from the substrate to the coenzyme and, secondly, from the coenzyme to the product. T o account for the transfer of tritium from propanediol to acetaldehyde, the coenzyme must contain a t least two equivalent hydrogens, such that the one donated by the substrate is not necessarily the one inserted in the product. This was established by further experiments by Frey and Abeles (82) in the following way. Propanediol-1-H3 was incubated with dioldehydrase and the coenzyme. The coenzyme was isolated and found t o contain 2 atoms of tritium per molecule. When this labeled coenzyme was reincubated with nonlabeled propanediol and dioldehydrase, tritium was transferred to the aldehyde formed. To establish the group of the coenzyme that participates during the hydrogen transfer, Frey and Abeles (82) synthesized the coenzyme, specifically tritiated in the 5' positions of the adenosine moiety. When the labeled coenzyme was incubated with propanediol and dioldehydrase, the isotope was quantitatively recovered in the propionaldehyde formed. These studies conclusively show that in the dioldehydrase reaction the coenzyme participates as a hydrogen-transferring agent with apparent involvement of the 5'-methylene residue. I n order to find a similar function of the coenzyme in the reaction catalyzed by ribonucleoside triphosphate reductase, Beck et al. (83) investigated the fate of tritium from 5'-cobamide-H3 coenzyme during the reduction of CTP with reduced lipoic acid. Essentially all the tritium was recovered in the water of the incubation mixture and none was found in the dCTP formed. The release of isotope was absolutely dependent on the presence of enzyme, CTP, and reduced lipoate. Omission of the positive allosteric effector (dATP) markedly decreased the formation of dCTP but only slightly diminished the transfer of tritium to the water. The model depicted in Fig. 18 was suggested t o explain thesc results. I n reaction ( a ) , a complex consisting of enzyme, oxidized lipoate, and a reduced cobamide coenzyme is formed. During reaction (b) , this complex reduces CTP. Assuming that reaction ( a ) is much more
337
ENZYMATIC REDUCTIOK OF RIBONUCLEOTIDES
rapid than reaction (b), no tritium would necessarily be transferred to dCTP. Instead, tritium from position 5’ of the cobamide coenzyme would first equilibrate with the SH groups of reduced lipoate and then with the protons of water. The participation of sulfhydryl groups in the reduction of ribonucleotides thus leads to a complete loss of tritium from
[i *
E.DBC. H
+
CTP
t E
+ DBC
+. dCTP
(b)
FIG. 18. Scheme for the involvement of the cobamide coenzyme its hytlrogen carrier in the reduction of CTP by Lnctobacilkts Zeichmannii reductasc (M).
position 5’ of the coenzyme to the water. This does not occur during the dioldehydrase reaction in which the transferred hydrogen never occupies an exchangeable position. The model shown in Fig. 18 docs not, however, explain why CTP is required for the transfer of isotope from H?-coenzyme t o water. The mechanism of hydrogen transfer was also studied in tritiated (84) or deuterated (85) water as described earlier for the E . coli system. The results in both cases demonstrated the exclusive incorporation of isotope into position 2‘ of either dCTP or dATP by the L. leichmannii enzyme. The stereochemistry of the reaction was studied very thoroughly with NMR spectroscopy by Batterham et al. (86). During the reduction of ATP with L. leichmannii reductase, isotope was introduced into position 2‘ of dATP in a position trans to the base. The enzyme reaction thus occurred with retention of the configuration a t position 2’. I n these experiments, the resolution of the signals for the two protons a t positions 2’ of deoxyadenosine was better than in the corresponding experiments with deoxycytidine (64), probably in part depending on the choice of deuterated dirnethylsulfoxide as a solvent for the NMR spectroscopy. This made the assignments for the signals much safer in the experiment of Batterham et al. (86). It is clear, however, that in both experiments the same signal was lost in the deuterated deoxynucleoside and that the same stereochemistry is involved in both the L. leichmannii and E . coli systems despite the fact that cobamide coenzyme participates only with the L. leichmannii enzyme.
338
A G N E LARSSON A N D PETER REICHARD
IV. Ribonucleotide Reduction in Animal Cells A. General Remarks Most of our knowledge of ribonucleotide reduction with enzymes from animal cells comes from the work of Moore and collaborators with Novikoff rat hepatoma. Some work, in particular that on the regulation of the enzyme reaction, has also been performed with crude extracts from other tissues, notably chick embryos. These latter experiments are first discussed briefly before returning in more detail to the work with purified enzymes from Novikoff hepatoma.
B.
Experiments with Extracts from Chick Embryos
Crude extracts from 5-day-old chick embryos catalyze the reduction of CMP and GMP to the corresponding deoxyribosyl compounds (30). The reactions show the same general requirements for Mg2+and ATP as described earlier for the E . coli system. With the chick-embryo system, a regulatory mechanism of DNA synthesis operates a t the level of ribonucleotide reduction. This mechanism was discovered (87) in studies of the synthesis of DNA from ribonucleotides when ribonucleotide reductase was coupled to DNA polymerase. Conditions were first established for the optimum rate of incorporation of isotope from labeled CMP or GMP into DNA. In the coupled system, the ribonucleotides were transformed into deoxyribonucleotides before incorporation into DNA. When different deoxyribonucleoside triphosphates were added to the incubation mixture, the incorporation of isotope from CMP-P3Zwas strongly inhibited by dTTP, dATP, and dLGTP. The incorporation of label from GMP-C14 was inhibited by dATP, but stimulated by d TTP and dCTP. These effects were also found directly with the ribonucleotide reductase reaction, without coupling to the polymerase reaction. It then became apparent that the reductions of both CMP and GMP are inhibited by very low concentrations of dATP and dGTP (5OooJo inhibition a t a nucleotide concentration of M ) . These results suggested that this feedback type of inhibition may be a part of a general control mechanism participating in the regulation of DNA synthesis. The stimulation of the reduction of GMP by pyrimidine deoxyribonucleoside triphosphates, particularly dTTP, was not understood a t that and lCP4M, we regularly time. At concentrations of d TTP between observed a two- to threefold stimulation of the reduction. As discussed in the section on the ribonucleotide reductase from E . coli, this finding
339
ENZYMATIC REDUCTION OF RIBONUCLEOTIDES
eventually provided a key to an understanding of the substrate specificity of that enzyme.
C. Experiments with Purified Enzymes from Novikoff Hepatoma The ribonucleotide reductase system from Novikoff hepatoma was separated by Moore and Hurlbert (1.2) into three different components (Fig. 19) : (a) The actual reductase system, enzyme P ; (b) an auxiliary Dialyzed extract acetic acid to pH 5. 2 k c i p .
\ Crude Enzyme S
Crude Enzyme P
IDEA"
t
FIG.19. Separation of components of the ribonucleotidr rt,ductase system froni Novikoff hepatoma.
enzyme S; and (c) an additional heat-stable component, factor S,. The separation of enzyme P and of enzyme S factor S, was quite clear, but the separation of enzyme s from factor sb was not, and no preparation of enzyme S known to be free of factor Sb has been obtained. Enzyme S (probably together with factor S,,) could be replaced by thioredoxin reductase, and is probably funcbacterial thioredoxin tionally equivalent to these proteins (23). Under both conditions, NADPH functioned as a hydrogen donor. As with the bacterial system, dithiols such as reduced lipoic acid, dithioerythrol or reduced thioredoxin could substitute for enzyme S (+factor sb) and NADPH. At a glance, one may be tempted to equate factor S, with the bacterial thioredoxin and enzyme S with bacterial thioredoxin reductase. However, other explanations must be considered, particularly the possibility that enzyme S may be the functional counterpart of both thioredoxin and thioredoxin reductase. Further work in this area should prove very interesting. I n the absence of reduced lipoic acid, the formation of dCDP from C D P with an enzyme preparation purified approximately tenfold showed
+
+
340
AGNE LARSSON AND PETER REICHARD
an absolute requirement for the reducing system and Mg2+, and was stimulated about tenfold by ATP (23).CMP gave about 30% and C T P about 60% of the activity of CDP. The diphosphate was considered to be the preferred substrate and the presence of kinases and phosphatases in the enzyme preparation was thought to account for the activities observed with CMP and C T P ( 2 3 ) . No requirement for cobamide coenzyme could be demonstrated for this system. It was discovered that the reduction catalyzed by enzyme P was stimulated more than twofold by FeCl, (12,2 3 ) . No such stimulation was seen with the bacterial enzymes. Under appropriate conditions, enzyme P catalyzed the reduction of all four ribonucleotides (88), and, in addition, responded to additions of certain nucleoside triphosphates in a manner closely resembling the response of the E . coli system to the same allosteric effectors. Thus the reductions of both C D P and U D P were stimulated by ATP and inhibited by dATP. I n the presence of ATP, both dTTP and dGTP inhibited. I n the absence of ATP, only a minor stimulation of the reduction of C D P was observed with dTTP. The reduction of purine ribonucleotides was very little affected by ATP. Instead, the reduction of ADP was stimulated by dGTP (and to a smaller extent by d TTP), and that of GDP by dTTP. Both reactions were strongly inhibited by dATP, and the reduction of GDP was also inhibited by dGTP. The interpretation of the results with nucleoside triphosphates as effectors is complicated somewhat by the presence of contaminating enzymes in enzyme P. Nevertheless, it seems clear th a t the activators cause a considerable decrease of the apparent K , values for the different substrates, and in several cases also increase V,,,, for the enzyme reactions. Both activations and inhibitions were usually observed a t deoxyribonucleotide conc.cntrations of approximately M . It seems likely therefore, that the results were caused by allosteric effects, and, by analogy with the two previously described bacterial systems, it is tempting to suggest that one enzyme catalyzes all four reactions. However, as emphasized by Moore and Hurlbert, there is no evidence a t all on this point. On the basis of their experiments, Moore and Hurlbert (88) suggested a regulatory mechanism very similar to the one depicted earlier in Fig. 11 for the enzyme system from E . coli. There are actually only two rather minor differences between the proposed models (Fig. 20). In the mammalian system, d TTP is not considered as a possible stimulator of pyrimidine ribonucleotide reduction, and dGTP is procosed as an inhibitor rather than a stimulator of the reduction of GDP. Considering
34 1
ENZYMATIC REDUCTION OF RIBONUCLEOTIDES
the rather large differences often apparent between regulatory mechanisms of closely related bacterial systems, and also considering the fact that the two schemes were arrived a t independently by Moore and Hurlbert and ourselves, the similarity is quite remarkable.
CDP
dCDP-dCTP
UDP
GDP
dGDP
-
dGTP
ADP
FIG.20. Possible physiological interpretation of the allosteric effects involved in the regulation of ribonucleotide reduction in Novikoff hepatoma (88).
In most respects, the mammalian system is obviously very much like the ribonucleoside diphosphate reductase from E . coli. It should be realized, however, that only a very moderate purification of enzyme P has been achieved so far and that the specific activity of the mammalian enzyme is around 1% of that of the two microbial systems. Many of the conclusions with respect to the properties of the animal system must therefore be tentative.
D. Evidence for Control Mechanisms in Intact Cells Evidcnce for the operation of control niechanisms of the type discussed here has also been obtained in sevcral whole-cell systems. For cxaniple, inhibition of the synthesis of DNA in Ehrlich ascites cells by deoxyadcnosine was described before the effects of dATP on enzymatic ribonuclcotide reduction were known (89, 90). Further investigation of this observation indicated that deoxyadenosine is transformed to dATP in these cells, and that in all probability this nucleotide is responsihlc for the observed inhibition (91). Of significance is the fact that the inhibition by deoxyadenosine was reversed only by the simultaneous addition of both deoxyguanosine and deoxycytidine ( 9 2 ) . Under such circumstances, some deamination of deoxycytidine [or more likely
342
AGNE LARSSON A N D PETER REICHARD
dCMP (93, 9 4 ) ] can occur and thus allow the inhibition of ribonucleotide reductase to be bypassed completely. Several other whole-cell systems are known in which DNA synthesis is blocked by deoxyadenosine. Of particular interest is the demonstration that deoxyadenosine-as well as other inhibitors of DNA synthesis-causes chromosome breakage in bean roots (95). Under certain conditions, thymidine also inhibits DNA synthesis in different types of animal cells (96-98). From thymidine-sensitive P815Y cells in tissue culture, a variant cell line was isolated that is resistant to the inhibition by the nucleoside and is deficient in thymidine kinase (99). This finding suggested that a phosphorylated derivative of thymidine is the actual inhibitor. I n the sensitive line, the inhibition is reversed by deoxycytidine alone. These results fit the model proposed in Fig. 20 in that dTTP inhibits only the reduction of pyrimidine ribonucleotides. Not all cells, however, are inhibited by deoxyribonucleosides. This may indicate that such cells do not contain a ribonucleotide reductase subject to the type of control outlined in Fig. 20. Alternatively, lack of inhibition by deoxynucleosides may be explained by the fact that many cells have very powerful degradative enzymes, such as phosphorylases, nucleosidases, and deaminases, which rapidly remove the added deoxynucleosidcs before an appreciable pool of deoxynucleotides can accumulate within the cell. It seems significant to us that in most normal cells the amounts of purine deoxyribonucleoside triphosphates-the most poverful inhibitors according to Figs. 11 and 20-are either very small or cannot be detected a t all (100).
V. Concluding Remarks We have summarized some of the properties of the three ribonucleotide reductase systems described in this review in Table V. Several features are common to all three: 1. In crude extracts, NADPH appears to be the ultimate hydrogen donor and hydrogen transport occurs via the thioredoxin system or enzyme s. 2. Dithiols, such as reduced lipoic acid, dithioerythrol, or reduced thioredoxin, can function as reducing agents with the purified enzymes. 3. All three systems are subject to some sort of allosteric regulation by nucleoside triphosphates, which influence the substrate specificity of the enzymes. On the other hand, ribonucleotide reductases from E. coli and from
343
ENZYMATIC REDUCTION OF RIBONUCLEOTIDES
TABLE V COMPARISON OF HIBONUCLEOTIDE REDUCTASES E . coli
L. leichmannii
NovikoH hepatoma
Diphosphate
Triphosphate
Diphosphate
No
Yes
No(?)
Thioredoxin Yes
Thioredoxiii Yes
Enzyme S Yes
Yes Absolute No
Relative No
Parameter ~
_ _ _ _ _ ~
Level of phosphorylation of substrate Requirement for cobamide coenzyme Physiological hydrogen donor Allosteric regulation of substrate specificity dATP as general negative effector Requirement, for Mg2+ Additional metal effect
~~
NO
Yes Absolute Yes, FeC13
Novikoff hepatoma share several properties that distinguish them from the L. Eeichmannii reductase: 1. The reduction occurs with ribonucleoside diphosphates, not with triphosphates. 2. No derivative of vitamin B,, appears to be involved. This conclusion is tentative for thc maininaliaii system. 3. There is an absolute requirement for Mg", which, a t least in the E . coli reductase, appears to bc related to the presence of two subunits in thc active enzyme. 4. dATP is a strong inhibitor of the reduction of all four ribonuclcotides, and in general the pattern of allosteric effccts is very similar for the enzymes from E. coli and Novikoff hepatoma and different from that of the L. leichmannii enzyme. There are also certain differences between the E . coli system and that from Novikoff hepatoma. The latter is stimulated by FeC1, while the E . coli rcductase is not. Furthermore, the hydrogen transport system (enzyme S) in Novikoff hepatoma appears to differ in several respects from the thioredoxin system. More work is needed on this point, however. These differences should not conceal the basic similarities between the two systems. What is then the status of ribonucleotide reduction in other animaI tissues? It is tempting to use the system from Novikoff hepatoma as an example and to assume that all animal ribonucleotide reductases are patterned after the E. coli reductasc. However, to makc such a. comparison, we must first answer a more basic question. Is there a ribonucleotide reductase in all rapidly growing animal tissues, or is deoxyribose synthesized via a different pathway?
344
AGNE LARSSON A N D PETER REICHARD
Attempts to demonstrate ribonucleotide reductase activity in extracts from, e.g., thymus or regenerating rat liver have so far been rather inconclusive (10, 1.2, 101). On the other hand, incorporation studies with labeled ribonucleosides established the occurrence of the reductive pathway (e.g., 1-3, 98). Of particular interest are recent experiments demonstrating that isotope from inorganic Pszand from uniformly C14-labeled cytidine is incorporated with the same ratio into C M P from RNA and into dCMP from DNA (102)of regenerating rat liver. This experiment not only demonstrates the existence of ribonucleotide reduction in the intact rat but also strongly suggests that all deoxyribose synthesis occurs via this pathway in regenerating rat liver. Another type of evidence from whole cell experiments comes from thr observation that DNA synthesis is inhibited by certain deoxyribonucleosides as discussed previously in Section IV, D. These effects, observed with different types of tumors and thymocytes, clearly involve an inhibition of ribonucleotide reductases. Since this inhibition results in a block of DNA synthesis, the results again support the conclusion that ribonucleotide reduction in these cells provides the major source of DNA precursors. Furthermore, the inhibition pattern in whole cells agrees with the results obtained with the E . coli cnzyme, but not with those obtained with thc L. leichmannii enzyme. I n our opinion, the evidence for the ubiquitous nature and importance of ribonucleotide reductase is very strong. Not long ago, it was difficult or impossible to find rcductases in extracts from E . coli or L. Zeichmannii. The major difficulty in this respect involved several technical problems, such as maintenance of the proper level of substrate phosphorylation and the choice of the correct concentrations of ATP and MgZ+ (in E . coli), or the maintenance of NADPH (in L. leichmannii) . Furthermore, the presence of multienzyme systems complicated the experiments, and inhibitors in the E . coli extracts strongly influenced the activities observed. All of these complications still make it impossible t o measure enzyme activities quantitatively in crude extracts and may well explain the apparent low activities observed. Similar complications probably exist in extracts from most animal cells. The concentrations of ribonucleotide reductase in these extracts probably are much lower than in bacterial extracts since DNA synthesis occurs a t a much lower rate. It is not surprising, therefore, that in many cases no enzyme activity can be found. We believe that preliminary enzyme purification, patterned after that used for the Novikoff hepatoma system, may be required to remove inhibitors or other interfering enzyme systems before ribonucleotide reductase activity can be demonstrated.
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The Mutagenic Action of Hydroxylamine J. H. PHILLIPS' AND
D. M. BROWN University Chemical Laboratory, Cambridge, England I. Introduction . . . . . . . . . . . 11. Genetic Background . . . . . . . . . . 111. General Chemistry of Hydroxylamine Action . . . . A. Reaction with Nucleic Acids . . . . . . . B. Reaction with Cytosine Derivatives . . . . . C. Reaction with Uracil Derivatives . . . . . . IV. Experimental Investigation of Hydroxylamine Mutagrnesis A. The Model . . . . . . . . . . . B. Reaction of Hydroxylamine with Pols C . . . . C. Experiments with RNA Polymerase . . . . . D. Mechanism of Erroneous Replication . . . . . V. Conclusions . . . . . . . . . . . . References . . . . . . . . . . . .
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349 350 353 353 354 356 358 358 359 361 362 364 366
1. Introduction I n the field of chemical mutagenesis, uncertainty holds sway. Despite the efforts of many investigators, success in clarifying the chemical events leading to mutation has, in general, been very limited. In the absence of means for making direct observations on the genomic alteration that gives rise to the mutant progeny, a compromise position has to be taken, the degree of certainty regarding the mutagenic mechanism in question being judged by the convergence of a number of lines of evidence. The reasons for the difficulties inherent in the subject are not far to seek and have been discussed by others (I,,%?). From the chemical standpoint, the problems posed by reactions involving macromolecules are greater than might have been anticipated, and the difficulties in extending the details of apparently well-defined chemical reactions have proved formidable. One of the main stumbling blocks has been the 1 Preseiit address: Depart.mertt. o f Biochemistry, M:ikerere L1tiiversit.y College, Kampala, Uganda. 349
350
J. H. PHILLIPS AND D. M . BROWN
question of specificity. Specific reagents producing single base changes in DNA are difficult t o find and, indeed, only one possible candidate, methoxyamine, has been investigated a t all. Use of this reagent as a mutagen has been very limited although the chemically related hydroxylamine, which appears to be highly specific for the base transition C + T under the conditions normally employed for its use, is one of the more commonly used mutagens and has been investigated in detail. Indeed, its mutational specificity is such that it is the only mutagen to have been used confidently in codon assignment (see chapter by Woese in this volume) (3, 4, 4a). Hydroxylamine therefore, is a good choice for detailed chemical investigation. The objective of the work covered in this review has been to elucidate completely the mechanism by which hydroxylainine induces mutations upon interacting with the nucleic acid of a bacteriophage particle or with transforming DNA. In brief, our own and other work began with base analyses of treated DNA and examination of the reaction with the individual bases and nucleosides. It was then extended to polynucleotides and an in vitro polymerase system was used as a model for replication-this, in effect, providing a relay between the mutational studies themselves and the gross chemistry exhibited by the reagent. The latter phase is as yet incomplete, nor have the techniques developcd been extended to biological systems. Incomplete, too, are the physicochemical studies of the hydroxylamine reaction products, on which a detailed understanding of the error-induction in replication must depend.
II. Genetic Background Chemical mutagenesis of the rII region of bacteriophage T4 has been reviewed by Krieg ( 5 ) . However, a number of points of general importance with regard to hydroxylamine mutagenesis emerge also from studies of other bacteriophages and of transforming DNA. It causes mutations in bacteria (6),a yeast ( s a ) , and the fungus Neurospora crassa ( 7 ) . The mechanism whereby hydroxylamine induces abnormal chromosome patterns in mammalian (8, 9) and plant (10)cells is quite unknown and is not discussed in this review. Hydroxylainine has both strong inactivating and strong mutagenic effects on infectious nucleic acids. For the most part, these have been investigated separately, and the characteristics of the latter are known much more completely than those of the former. Freese and his coworkers have carried out systematic studies of the effects of hydroxylamine on T4 (11, fa) and Bacillus subtilis transforming DNA (1315) [being, in fact, the first to give an account of the mutagenic effect of the reagent ( f l ) ] , and other detailed analyses have been made by
MUTAGENIC ACTION OF HYDROXYLAMINE
351
Schuster (16, 1 7 ) , Benzer (18), and Tessman (19), and their collaborators. Detailed analyses of relatively few hydroxylamine-induced T4 mutants have been published. However, the data available from both forward mutation (19) and hydroxylamine-induced reversion (11, 18) clearly indicate that the reagent induces a single class of base-pair transition ( 5 ) ; the same is found in the case of induction of mutations in transforming DNA ( 1 3 ) . Study of chemical mutagenesis was greatly refined by Tessman, however, by the use of the single-stranded coliphage S13 (19).The phage is exceedingly small (it is closely related t o bacteriophage pX174, which has only about 4500 nucleotides) , and consequently, unlike T4, it presumably has no dispensable genetic regions. Forward and reverse mutants were selected by host range. Systems were developed that gave unambiguous results and were probably free from the difficulty of distinguishing suppressor mutations, a difficulty that is inherent (although surmountable) in studies with T4. It could be clearly demonstrated from the mutation data that hydroxylamine induces a single class of base transition (out of the four possible classes) ; from chemical data (vide infra) this change is taken to be C + T (i.e., G - C+ A - T for a double-stranded genome). [We may note that, in contrast t o the clcarcut results with hydroxylamine, a number of other mutagens appeared to induce all the possible transition classes (19, 20).It seems very probable that to some extent this must be a reflection of secondary effects (for example, a replication error during repair of the lesion) , necessarily obscuring the site and nature of the primary event (Z).] There is good evidence from an analysis ( 6 ) of the reversion data of Champe and Benzer (18) that in T4 there is a close correlation between mutagenesis by hydroxylamine and by 5-bromodeoxyuridine, although we should note that an opposite conclusion might be drawn from the results of a clean-growth mutagenesis experiment performed by Terzaghi e t al. ( 2 0 ~ )However, . the situation seems to be quite the opposite for 513 mutants (21),even when the same host strain of Escherichia coli is used. This may reflect some special response of the phage-induced DNA polymerase to the presence of base analogs. Mutants induced by hydroxylamine in T4 appear to be widely distributed over both rIx cistrons (12, 18). Although no particular evidence for “hot spots” emerges from the mapping data [as found also by Tess, rates of hydroxylamine-induced reversion a t difman for S13 ( 1 9 ) ] the ferent sites are in fact found to vary considerably (18). Drake, in his study of ultraviolet light-induced T4 mutants ( 2 2 ) ,found that many mutants reverting under the influence of base-analogs do so by suppressor mutations ; hydroxylamine, however, rarely induces such suppressors : this suggests the possibility that while very many G - C sites are suscep-
352
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PHILLIPS AND
D. M. BROWN
tible to mutation by a base-analog such as 5-bromodeoxyuridine, very many sites may in fact be refractory to hydroxylamine mutagenesis. The degree of secondary structure of the genetic material appears to be of critical importance. Freese pointed out that T 4 is a thousand times more mutable by hydroxylamine than is transforming DNA (13, 2 3 ) ; denatured DNA is even more subject to mutagenesis. Native transforming DNA can be made more susceptible by changing the solvent, especially by adding ethylene glycol, which reduces the degree of secondary structure of the polymer. This difference of susceptibility has recently been utilized by E. S. Tessman to provide a precise method of estimating the amount of the double-stranded replicative form from S13 DNA in a mixture with single-stranded infectious DNA, based on the fact that hydroxylamine may be used to inactivate the latter essentially selectively ( 2 4 ) . It seems clear that effective hydroxylamine mutagenesis requires that the genome contain rather exposed bases. Studies of the p H dependence of the mutagenic change are of considerable importance in understanding its mechanism (see Section IV, D ) . Induction of mutants by hydroxylamine at pH values between 6 and 9 has been tested with T4 (16, 18) 513 (19),and tobacco mosaic virus RNA (17 ) . The very much reduced mutation rate a t higher pH values has provided the main evidence to support the idea that hydroxylamine interacts with cytosine rather than thymine in the genome. Especially interesting, however, are the few experiments examining the effect of p H values lower than 6. Two separate effects seem to be indicated. Schuster and Vielmetter, in experiments with T 4 in which the pH was reduced as low as 4.5, found a definite maximum a t p H 6 (16). On the other hand, with transforming DNA a t high temperature, Freese and Strack found that mutagenesis was much more effective a t p H 5.5 than a t 6.2 (13) and, although it is difficult to combine the results in their two papers, the later data of Freese and Freese (15)suggest that mutagenesis a t as low pH as 4.2 may be considerably more effective than a t pH 5.5. More complete data on these effects is very desirable, and it would be most interesting to have comparable data for S13. Schuster and Vielmetter made a further point with regard to T4 mutagenesis, namely, that treatment of the phage particles a t several p H values (no values were given) after removal of hydroxylamine had no effect on the mutation rate (16). They concluded that the primary action of the reagent with the hydroxymethylcytosine residues is responsible for the mutagenic change. The possible implications of these observations are discussed in Section V. Hydroxylamine is a convenient mutagen for study in the sense that, despite the complications in determining its mechanism, it seems to produce rather specific mutagenic changes, acting as a reagent for naked
MUTAGENIC ACTION OF HYDROXYLAMINE
353
nucleic acid or phage particle in vitro. In vitro mutagens have considerable advantages over in vivo mutagens as is seen immediateIy when the effect of hydroxylamine on phage-bacterium complexes is investigated. The specificity of the reagent seems to be entirely lost (.25),presumably because of the many effects of hydroxylamine on the metabolism of the host cell and the possible further effects this may have on the bacteriophage replication. The possible complication in the interpretation of mutagenic data due to the presence of repair mechanisms in the host cell that reduce damage to the chromosome has already been mentioned, although more recent experiments (25a) suggest that this may not apply to hydroxylamine-induced mutagenic alterations in T4. The inactivating effects of hydroxylamine are not understood in any detail. The effect decreases a t relatively high hydroxylamine concentrations (above 1M ) (12, 15) , a phenomenon found both with bacteriophages and transforming DNA; there is little inactivation a t low pH ( 1 7 ) . N-Methylhydroxylamine, CH,NHOH, is completely similar to hydroxylamine in both mutagenic and inactivating effects on transforming DNA (15) although it does not seem to be mutagenic for T4 (1.2). Methoxyamine, NH,OCH,, on the other hand, is mutagenic in both systems (15, 26) but does not have an inactivating effect on transforming DNA. Light has been shed on the whole process of inactivation by the recent experiments of Freese and Freese (27), who observed that inactivation is decreased under anaerobic conditions and correspondingly increased in the presence of oxygen. Free radicals are generated under aerobic conditions, and doubtless these are involved in the inactivation process, since this, but not the mutagenic action, is diminished by radical inhibitors, such as pyrophosphatc.
111. General Chemistry of Hydroxylamine Action A. Reaction with Nucleic Acid,s Hydroxylamine, although not very basic (pK, 5.96) (28), is a powerful nucleophilic agent, for reasons still not entirely clear. Although the purine bases are not readily attacked by nucleophiles, cytosine and uraciI derivatives are quite susceptible (29, SO). Extensive treatment of salmon sperm DNA with hydroxylamine followed by base analysis reveals a progressive loss of cytosine and a very much smaller loss of thymine; other bases are unaffected (31, 3 2 ) . I n place of cytosine, another base, later identified as N4-hydroxycytosine, is detected. Investigation of the action of hydroxylamine on individual bases, nucleosides, and nucleotides using chromatographic and spectrophotometric methods to follow the course of the reaction confirms that the pyrimidine, but not the purine, bases are attacked (11, 31-35).
354
J. H. PHILLIPS AND D. M. BROWN
Furthermore, these studies reveal a marked p H dependence, cytosine derivatives reacting rapidly a t pH 6, uracil derivatives a t p H 10 ( 3 5 ) . Thymine derivatives react slowly but detectably under very forcing conditions (32), for example, heating in anhydrous hydroxylamine. By comparison with the free base itself, cytosine residues in native DNA react extremely slowly (32, 3 6 ) .
B. Reaction with Cytosine Derivatives We discuss here the chemistry of the reaction of cytosine and its derivatives (I) with hydroxylamine in strong aqueous solution. The p H optimum appears to be around 6.5, i.e., near the pK, of the reagent.
N5-
0A
N R
0
R
I
II
NOH
NHOH
IV
I
III
With cytosine (I; R = H) itself, essentially complete disappearance of ultraviolet absorption in the 270 mp region occurs and a product having A,,, = 220-225 mp is formed with the structure 111. Although this compound may decompose in a number of ways a t pH values away from neutrality, it is stable for some hours in neutral aqueous solution a t room temperature (37). Strong acid converts it rapidly and quantitatively to N4-hydroxycytosine (IV), as does heating the dry cornpound. It is significant that IV is converted to 111 by aqueous hydroxylamine, but a t a rate that is a t most a fifth of its rate of formation from I. It follows that the major pathway from I to I11 does not include I V but instead must involve the intermediate I1 (46'). This intermediate cannot be detected in the reaction with cytosine or cytidine 2'(3')-phosphate so that clearly I + I I is the rate-limiting step. However, when polycytidylic acid is used, the intermediate I1 is stabilized to a small extent and its presence in the polymer can be demonstrated ( 3 8 ) .
355
MUTAGENIC ACTION O F HYDROXfLAMINE
The mechanism of the niajor reaction of hydroxylamine with cytosine, and, indeed, with all of the A']-substituted derivatives studied, (e.g., I in which R = Me, ribosyl, 2'-deoxyribosyl, ribosyl 2'(3') -phosphate) (3.2, 38) is thus a conjugate addition of hydroxylamine leading to saturation of the 5:6 double bond followed by a rapid exchange of the amino group by hydroxylamine. The second step is an example of a very general displacement reaction: 5,6-dihydrocytosines (V) are very readily hydrolyzed to dihydrouracils (39) and they react extremely rapidly with hydrosylamine to yield i~'-liytlrosy-5,6-dihydl.oeytosincs (VI) (40) and with other nitrogen nuclcophiles to yield analogous products (40, 41 ) .
V
VI
At this point we should note briefly that methoxyamine reacts with cytosinc derivatives analogously to hydroxylamine although with a lower pH optimum ( 3 2 ) .No reaction has been demonstrated with uracil derivatives (4%'). The adduct formed between N-methylhydroxylamine and cytosine, viz. that corresponding to 111, is very unstable, and the exchange product, N'-methyl-N4-hydroxycytosine, is isolated from the reaction mixture (@). There are two positions on the cytosine ring a t which nucleophilic attack can occur. The C-6 position has already been discussed. Displacement reactions a t (3-4 are also known, exemplified by the very slow base-catalyzed hydrolysis to uracil derivatives (44). [The deamination observed in mild acid solution may depend on reversible addition of a nucleophiIe to the 5,6-double bond and hydrolytic displacement of the amino group in the internietliate (45j.l Janion and Shugar (46a) were the first to point out that the reaction between hydroxylamine and 5-hydroxymethylcytosine (VII) leads to the exchange product VIII. NOH
VII
VIII
They found no evidence for the presence of an intermediate corresponding to 111 and suggested that a direct displacement occurred. Present
356
J. H. PHILLIPS AND D. M. BROWN
kinetic evidence (do), however, does not distinguish between such a displacement (VII + VIII) and the formation of a 5,6-adduct that undergoes the displacement reaction followed by a rapid elimination from the 5,6-positions. The same is true for !j-methylcytosine, and in each case the reaction proceeds more slowly than with cytosine. More recently, Lawley TABLE I RATECONSTANTS FOR REACTIONS BETWEEN HYDROXYLAMINE A N D CYTOSINE DERIVATIVES
k Compound Cytosine (I; R
=
H)
2'-Deoxycytidine (I; R
=
2'-deoxyribosyl)
5-Hydroxymethylcytosine (VII) a In 3.5 M hydroxylamine, pH 6.5, 35°C. Data from Brown and Hewlins (40).
b
Reaction
(hr-I)
I-+ I1 I+IV IV -+ I11 I11 --+ Iv I --+ I1 I + N VII + VIII
4.5" 0.9"
0.2 0.02b
1.25O 0 . 25a 0.3"
In 4.0 M hydroxylamine, pH 6.5, 35°C.
(47') has obtained kinetic evidence that some N'-hydroxycytosine (IV) is formed simultaneously with I11 in the reaction of hydroxylamine with cytosine, and this has been confirmed in other work (40) that shows, in addition, that the back reaction (111 + IV) also contributes to its formation. Constants are given in Table I by which the rates of the various reactions can be compared. C. Reaction with Uracil Derivatives As mentioned above, hydroxylamine reacts with uracil derivatives, albeit to a much smaller extent than with cytosine derivatives, and the gross chemistry of this reaction has been elucidated by two groups (33, 3 5 ) . Examination of reaction profiles as followed by absorbance decrease (Fig. 1) shows that the initial rate of reaction with uracil derivatives is in fact fairly high, even a t pH values below neutrality, although the reaction does not proceed so far toward completion a s in the cytosine case. Thymine, by this criterion, appears to be essentially unreactive (11, 33). However, we detail here briefly the course of reaction with uracil, in view of its relevance t o the question of hydroxylamine reactivity and to its possible usefulness for modifying nucleic acids before enzymatic cleavage, (34) or as a probe for studying the function of specific bases, as applied recently to transfer RNA [e.g., (48-60)] and to synthetic messenger RNA's (61).
357
MUTAGENIC ACTION OF HYDROXYLAMINE
I
_--I
FIG. 1. Reaction with 2.5 M hydroxylamine hydrochloride, p H 6.7, 22"C, followed by decrease of optical density a t A.,
..
The shape of the reaction profile is indicative of a relatively rapidly attained equilibrium (IX X) . The reaction probably then proceeds by a mechanism analogous to hydrazinolysis (52). Following addition to the a,P-unsaturated carbonyl system (IX + X) ring closure occurs with formation of the 3,4-dihydro-3-ureidoisoxazo1-5-one(XI). Analogous pyrazolone derivatives have been isolated as hydrazinolysis intermediates ( 5 3 ) . The isoxazolone (XII) is liberated by elimination of urea. It may be assumed that adducts of type X are rather labile with respect to elimination of hydroxylamine ; under basic conditions the ring
Ix
X
1
358
J. H. PHILLIPS AND D. M. BROWN
closure is facilitated. This explains the base-catalysis observed. The rate decreases above p H 10, which is above the pK, of the uracil derivative, so that the initial nucleophilic addition is hindered. It thus seems that the equilibrium (IX e X) is rapidly established and is slowly displaced by irreversible isoxazolone formation as evidenced by the slow decrease in optical density that is observed. The adduct is unstable with respect both to reversal to the parent uracil and to isoxaxolone formation and presumably has a relatively short-lived existence. 5-Substituted uracil derivatives are generally unreactive, and this applies even to the electrophilic 5-nitrouracil, although this may reflect, in the latter case, simpIy an unfavorable equilibrium position in the addition reaction. The rapid reaction observed between hydroxylamine and 5-bromouridine (Fig. 1) (11, 4.3) is therefore unexpected, the more so as 5-bromocytidine reacts very slowly (4.3). I n the reaction with 5-bromo-l-methyluracil, bromide ion is liberated, the non-ultraviolet-absorbing component of the equilibrium mixture being, in fact, the adduct (X; R = Me) and 3-methyluracil is one of the products of the reaction. The reaction has a sharp pH-rate profile with a maximum a t pH 7.2 ( 4 3 ) . The high rate of the reaction, even faster than that of cytosine a t comparable pH values, suggests that the decomposition of the bromouracil residues causes the rapid inactivation of bacteriophages observed when thymine is replaced by 5-bromouracil (If).
IV. Experimental Investigation of Hydroxylamine Mutagenesis
A. The Model Modern biology is clearly a science of models. Whether conceived or built, they are connected to actual biological proccsses by reason and rarely by experirncnt ( 5 4 ) . Bacteriophage mutagcnesis, itself of interest as a model for studying genetic processes in higher organisms, must be imitated by a simpler model if a full understanding of the process on the molecular level is to be gained. The primc requirement of the model is that every mutational event be scored. Mutagenesis of even the well-defined rII region is impossibly ill-defined: not only is the genome a complex DNA helix but the phenotypic expression of mutations is inevitably largely prevented ( 2 ) . For obvious reasons, we, in our own work, decided to study the action of hydroxylamine on the readily available homopolymers, especially poly C. It is not immediately obvious that this polynucleotide, forming :L single-stranded structure with stacked bases a t p H values near neutrality (55, 5 6 ) , is a good model for the DNA molecule, the main contrast
MUTAGENIC ACTION OF HYDROXYLAMINE
359
being the nature of its secondary structure which seems to result mainly from the hydrophobic interactions of the bases (56, 57). A t first sight, a double-stranded structure such as poly I.poly C would appear to have greater relevance, for it is by no means certain that patterns of reactivity are the same when a particular base is situated in such differing environments. This problem has been studied principally with reference to photochemistry. For example, cytosine hydrate formation occurs on irradiation of poly C but not of the complex poly 1-poly C (58) or of poly dI-poly dC ( 5 9 ) , and hydrate formation is much reduced in poly Aepoly U compared with poly U (60). I n this connection, we may refer back t o the work of Freese et al. (12, 1.3, 15) on hydroxylamine-induced mutagenesis in bacteriophages and B. subtilis transforming DNA. From a consideration of mutation rates in various solvents and especially in aqueous glycol solutions it was concluded that the mutagenic reaction was strongly dependent on the exact structure of the DNA (IS). The contribution of the hydrophobic forces to the secondary structure of DNA is a matter of present dispute (61), but ethylene glycol presumably produces an unwinding effect. Bacteriophages were found to be one thousand times more mutable than transforming DNA under similar conditions (1.0M hydroxylamine in 1.3 M NaCl a t pH 7.5 and 37") ( I S ) ; the rate of induction of mutations in phage is intermediate between those of native and denatured transforming DNA, but the activation energy is similar to that for denatured DNA (14). A possible conclusion from these experiments was that the phage DNA was distorted by packing, rendering some regions more accessible to the mutagen (cf. also 61a,b). [One may recall Drake's suggestion from genetic evidence that not all C - G sites are equally accessible to hydroxylamine (Section 11).] It would thus appear that the comparatively exposed poly C molecule may in fact be a better model for in vitro mutagenesis than a highly structured complex such as poly I.poly C. Poly 5-methylcytidylic acid has recently become available (62). It has a greater degree of secondary structure than poly C and is clearly a better model for bacteriophages containing 5-hydroxymethylcytosine, such as T4 (46).
B. Reaction of Hydroxylamine with Poly C Reaction of poly C with hydroxylamine is considerably slower than that of cytidylic acid. I n excess reagent, the reaction proceeds with first-order kinetics, and analysis of the partly reacted polymer shows that the appearance of the N4-exchange product (111) is considerably delayed (38).Depending on whether this is consequent on the polyanionic nature of the substrate or steric hindrance, which affects C-4 and
360
J. H. PHILLIPS AND D. M. BROWN
C-6 differentially, denatured DNA may or may not show the same effect. There is a certain limit to the amount of useful information that may be derived from large extrapolations from relatively insensitive absorbance measurements. Using methoxyamine-C1* it is possible to measure the product content of the polymer a t very small extents of reaction ( 6 3 ) . Figure 2' shows the formation of products I1 and 111 in
2.o
I .5 D
e
c
0
u)
3
U .-
; 1.0
c
W
cn
0 * c 0
E
n
0.5
Hours of reaction with NH,OCH,
FIG.2. Reaction between poly C and 1.0M methoxyamine hydrochloride, pH 5.5, 37°C. Curve A : percentage of cytosinr residues rcarted, curve B: percentage residues converted to N*-methoxy-5,6-dihydro-6-m~thoxyaniinocytosine residues (111, sre text) ; curve C : percentagc. of residues converted to 5,6-dihydro-6-methoxyaminorytosine residues (11). [From the data of Phillips et al. (G3.1
the polymer for up to 2% of reaction. The intermediate adduct (11) reaches a low steady state concentration, although how long this is maintained cannot be deduced from the data. The product (111) may contain a small proportion of IV, possibly up to one-fifth ( 4 7 ) , since this is not distinguished by the assay. Concomitant with the reaction, there is a loss of secondary structure of the poly C. This presumably results directly from the saturation of
MUTAGENIC ACTION OF HYDROXYLZAMINE
361
the 5,6-double bond and the consequent lack of interaction of the affected base with its neighbors. An analogous situation is found when uracil residues in poly U undergo reduction (64).
C. Experiments with RNA Polymerase Grossman e t al. first used highly purified RNA polymerase from Micrococcus Iysodeikticus to investigate mutagenic effects by examining the effect of ultraviolet irradiation on the template properties of poly C (65). The same system has been used to investigate the effects of hydroxglamine. The enzyme used is thought to be that involved in the synthesis of RNA from a DNA template in vivo (66) ; as it can also utilize homoribopolymers as templates for the synthesis of complementary polynucleotides ( 6 7 ) ,it is a convenient model for the replication process. The clear advantage of this system as a model is that the “mutagenic” events in the template poly C should be detected by and relatable to adenylate incorporation in the poly G synthesized (if the theory of transitions is correct) without the difficulties due to code degeneracy in scoring methods relying on translation (such as identifying mutant proteins or use of an in vitro protein synthesizing system). The RNA polymerase appears t o “read” the template accurately and sequentially (68). When the template is poly C, one equivalent of poly G is formed in the presence of M . Eysodeikticus RNA polymerase and G T P (69).This is firmly complexed with the poly C, and the product does not act as a template for further guanylate incorporation. Treatment of poly C with hydroxylamine reduces its capacity to act as a template for poly G synthesis. This capacity is partially restored if ATP, but not CTP or UTP, is added to the reaction mixture: adenylate is incorporated into the newly synthesized poly G, primarily as single residues flanked on both sides by guanylate residues, as shown by nearest-neighbor analysis (70, 7 1 ) . Treatment of poly A, however, has relatively little effect on its ability to act a s a template for poly U synthesis. Treatment of poly IJ at pH 6.5 leads to some decrease of its template activity; however, this cannot he recovered by addition of a second nucleoside triphosphate. These experiments fend strong support to the original contention that hydroxylamine may act as a mutagen by altering a cytosine residue so that it is replicated as if it were thymine. The next problem is the determination of which possible product of hydroxylamine action directs adenylate incorporation and how this erroneous incorporation occurs. The kinetics both of guanylate incorporation in the presence of ATP and of the adenylate incorporation itself as a function of time of hydroxylamine treatment of the poly C strongly suggests that residues of 11,
362
J. H. PHILLIPS AND D. M. BROWN
the mono-adduct, rather than 111 in the polymer are responsible for the “mutagenic” error (70). As noted before, methoxyamine is a mutagen and in its reactions with cytosine derivatives is similar to hydroxylamine. Use of the CI4-labeled compound has two advantages; first it is possible to determine the proportions of each reaction product in the poly C after very small extents of reaction (Fig. 2) and, secondly, the number of alterations in the template necessary for each adenylate incorporated can be derived. These experiments (63) strengthen the case that, in this model system, 5,6-dihydro-6-hydroxylaminocytosineresidues (11) are replicated like uracil residues and indicate that each residue of this base in the template is replicated in this way.
D. Mechanism of Erroneous Replication The various theories that have been entertained for the explanation of hydroxylamine mutagenesis are indicated in Fig. 3. At present, mechanisms A and C are favored and we present here the evidence in their favor and against the others. Deamination to uracil or its adduct (D and E) seemed attractive hypotheses a t first (33) in view of the known lability of dihydrocytosines toward hydrolysis. However, analyses of even extensively hydroxylamine-treated poly C have failed to reveal any uridylate residues in the polymer (38),and the recent studies of Johns et al. on the deamination of 5,6-dihydro-6-hydroxycytosine (7%’) render it most unlikely that deamination could occur under mutagenesis conditions. I n the case of the poly C-RNA polymerase model system, both C and F are excluded because of the scarcity of these species in the poly C template while adenylate residues are being incorporated (63). They could be involved only if each altered base directed the incorporation of a t least two or three adenylate residues. The experimcnts with methoxyamine quoted above also excluded B as a possibility. From the estimated pK, of 11, taken to be close to 5.6, as found for the structurally similar cytosine hydrate (72), it is clear that not more than one in ten of the residues of I1 in the poly C could be protonated. Yet every residue is, in fact, replicated by adenylate incorporation. One is therefore brought to a consideration of mechanism A. The induction of errors in the replication process leading t o transition mutations has, until now, been discussed solely in terms of changes in the ionization level or tautomeric state of the altered base (73) although, doubtless, a more sophisticated view will be developed ( 7 4 ) . We have rejected mechanism B, based on a change in ionic state. The tautomeric constant of I1 is unknown. The related 1-methyl-5,6-
363
MUTAGENIC ACTION OF HYDROXYLAMINE
H
I
HO.NH
m FIG.3. Possible mechanisms for base-pairing with adenine in hydroxylamine mutagenesis.
364
J. H. PHILLIPS AND D. M. BROWN
dihydrocytosine (V: R = Me) has a tautomeric constant of ca. 25 in favor of the amino form in aqueous solution (40)~compared with cytosine in which KT N lo5 (75). The less polar imino form predominates in a medium of low dielectric constant, a situation that may correspond to the conditions a t the point of replication. Indeed, a tautomeric constant of 0.2 or less (i.e. KT N 5 in favor of the imino form), if it applied to 11, would adequately account for the incorporation results and support mechanism A. However, it must be emphasized that extrapolation from such results to the case in point is a matter of considerable uncertainty. We are compelled to reconsider the question of how far the poly C model represents the case of mutagenesis itself. A good case may be constructed for supposing that for bacteriophage T4, a t least, mutagenesis is mediated by mechanism C. As noted before, reaction of aqueous hydroxylamine with 5-h.ydroxymethy1cytosine leads to N4-hydroxy-5-hydroxymethylcytosine as the only detectable product (46), although we have no information on the degree to which the addition and exchange reactions are affected by secondary structure in DNA, relative to one another. Moreover Janion and Shugar (46) have pointed out that if hydroxylamine mutagenesis of transforming DNA is as strongly dependent on low pH as suggested by Freese e t al. (15),an explanation may be found in the acid-catalyzed elimination from residues of type I11 to yield a n N4-hydroxycytosine (IV) [cf. ($?)I. It is in any case clear that the poly C model is inappropriate as a test for mechanism C. However, copolymers of cytidylic and N4-hydroxycytidylic acids are available and act as effective templates for poly (A,G) synthesis in the presence of RNA polymerase (76). Furthermore the kinetic evidence indicates that N4-hydroxycytosine residues direct the incorporation of adenylate residues with high efficiency (>50%). Of some significance, too, is the finding that N1-substituted derivatives of N4-hydroxy- and N4-methoxycytosine are essentially completely in the oximino tautomeric form (IV) with KT of about 10 ( 7 7 ) .I n addition they show strong interaction with a 9-alkyladenine, but not with a 9-alkylguanine (40), in the system used by Rich e t al. (78). The specificity found in the hydrogen bond interactions of the normal base-pairs ( i e , G with C, A with U) (78-81) and the demonstration of bonding between N4-hydroxycytosine and adenine derivatives is strong presumptive evidence for error induction by mechanism C.
V. Conclusions Although a conclusive and unified theory for hydroxylamine mutagenesis cannot yet be presented, the two alternative possible mech-
365
MUTAGENIC ACTION OF HYDROXYLAMINE
anisms (A and C) seem to be well defined and to lead to a number of clear predictions. We favor addition of hydroxylamine to cytosine residues to yield 5,6-dihydro-6-hydroxylaminocytosineresidues as the active error-promoting species in the case of single-stranded cytosine-containing bacteriophages such as S13, and possibly transforming DNA. At the moment the mechanism for T-even bacteriophages remains open although the chemical and enzymatic evidence strongly favors that in which N4hydroxy-5-hydroxymethylcytosine residues lead to replication error. It may be very pertinent in this connection that N-niethylhydroxylamine is mutagenic for transforming DNA but not for T4 (Section 11); the exchange product (XIII) cannot take up the oximino form nor is it likely to base-pair effectively, while the 5,g-adduct (XIV) should be insig-
XIII
XIV
nificantly different from the hydroxylamine adduct. It is, in any case, clearly possible that no single unique mechanism covers all cases of hydroxylamine mutagenesis. Further experimental work is clearly needed ; for instance, chemical and enzymatic investigations using other model systems such as poly5MeC and poly I =poly C or appropriate deoxyribonucleotide polymers. Chemical investigation of bacteriophages or DNA treated with labeled methoxyamine, further biological experiments on the effect of acid treatment on mutagen-treated material and mutagenesis under conditions producing defined chemical products arc still required. In conclusion, we draw attention to the possible beginnings of a rationale for cytosine-based mutagenesis. As we have indicated, there is residues (XV) strong evidence that 5,6-dihydro-6-hydroxylaminocytosine may be responsible for C + T transitions in some systems; furthermore, it has been suggested (82) that hydrazine-induced mutations, thought to be primarily transitions in the case of T4 (83),result from the reducing action of di-imide to yield the 5,6-dihydrocytosine derivative (XVI ; R’ = CH,OH) . Irradiation of bacteriophages yields a high proportion of transition mutants; in the case of T4 these are mainly G - C + A - T transitions ( 2 2 ) ,and the majority of mutants induced in 513 have definitely been identified as C + T (20). Taken in conjunction with experiments utilizing the RNA polymerase model system described above (66) and
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J. H. PHILLIPS AND D. M. BROWN
with Johns's experiments showing cytosine hydrate to be a primary product of thc irradiation ( 8 4 ) ,there seems good evidence that 5,6-dihydro-6hydroxycytosine residues (XVII) are responsible for the transition. Com-
xv
XVI
XVIII
XVII
plementary experiments have now been performed with R.NA polymerase in which it has been shown that 5,B-dihydroCTP (XVIII; R = ribosyl Y-triphosphate) may act as a substrate for the DNA-dependent enzyme, being recognized as either CTP or UTP ( 8 5 ) . It therefore seems reasonable to expect that some unity in this area of chemical mutagenesis will be established within the fairly near future. REFERENCES 1. L.
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Mammalian Nucleolytic Enzymes and Their Localization DAVIDSHUGAR AND HALINASIERAKOWSKA Institute of Biochemistry and Biophysics, Academy of Sciences, and Department of Biophysics, University of Warsaw, Warsaw, Poland
I. Introduction . . . . . . . . . 11. Types of Nucleolytic Enzymes . . . . A. Endonucleases . . . . . . . . B. Exonucleases . . . . . . . . C. Cyclic Nucleotide Phosphodiesterases . . 111. Methods of Assay . . . . . . . . A. Endonucleases . . . . . . . . B. Natural Inhibitors . . . . . . . C. Exonucleases . . . . . . . . D. Cyclic Nucleotide Phosphodiesterases . . IV. Substrate Preparations . . . . . . . V. Cellular Fractionation . . . . . . . VI. Histochemical Methods . . . . . . VII. Cytochemical Procedures . . . . . . A. Immunofluorescence Techniques . . . B. Precipitate-Forming Techniques . . . C. Comparison of Cytochemical and Fractionation VIII. Nuclealytic Enzymes in Pathological States . Nucleases in Tumors . . . . . . . IX. Possible Functions of Nucleolytic Enzymes . Addendum . . . . . . . . . . References . . . . . . . . Note Added in Proof . . . . . . .
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1. Introduction A multitude of enzymes of varying degrees of specificity exhibit hydrolytic activity against RNA, DNA, and nucleoside cyclic phosphates. Relatively little is known about the biological function of these enzymes and, in particular, of their intracellular localization, both of these being, of course, interrelated. Even if we discount the difficulties normally encountered in investigations on enzyme localization, those involving nucleolyt.ic enzymes have been particularly arduous because of the wide range of enzymes with, frequently, overlapping specificities. 369
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The following brief review makes no claim to provide any clear-cut solutions to these problems. Its preparation was prompted rather by the fact that, during the past five years, the preparation of some specific substrates, and the development of methods for their application to cell fractionation techniques and histo- and cytochemical procedures, has a t least laid a reasonably solid foundation for future work. Furthermore, it is felt that no apology is required for the inclusion of brief references to some studies now known to be of questionable validity, but the principles of which may nonetheless prove of value in an important field still undergoing birth pangs.
II. Types of Nucleolytic Enzymes Attempts a t the direct intracellular localization of nucleolytic enzymes have been confined largely t o the differentiated materials of higher organisms. The following outline is therefore limited to mammalian enzymes, although reference is made below to some bacterial enzymes (Section IX) in connection with a discussion of their function. Only essential properties are listed, such as substrate specificity, type of attack, and nature of products; additional aspects are included when they appear to be of value in distinguishing between different enzymes in localization studies. Further details may be found in several review articles (1-9).
A. Endonucleases 1. RIBONUCLEODEPOLYMERASES
Pancreatic R N m e (EC 2.7.7.16). This enzyme is most abundant in the pancreas (10, I l ) , but similar activity is found in other tissues and body fluids (12-16). It is stable to acid and heat, and is optimally active a t pH 7-8. Its activity may be “masked” by natural inhibitors in mammalian tissues (16-20). Its action is biphasic and involves first the rapid hydrolysis of the internucleotide linkage between a pyrimidine nucleoside 3’-phosphate and the adjacent nucleoside to give a pyrimidine nucleoside 2’:3’-cyclic phosphate; this is followed by the much slower hydrolysis of the cyclic phosphate ring to give a pyrimidine nucleoside 3’-phosphate [for review, see reference (21)1. Exhaustive hydrolysis of RNA results in formation of a resistant “core” composed of purine oligonucleotides terminated by a pyrimidine nucleoside 3‘phosphate. High concentrations of RNase slowly degrade poly A, but normally
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the enzyme does not release free purine nucleotides, nor does it hydrolyze purine nucleoside 2’: 3‘-cyeIic phosphates ( 2 2 ) . RNase hydrolyzes esters of pyrimidine nucleoside 3’-phosphates, e.g., the benzyl, methyl, ethyl, or 0-naphthyl esters (23, 24), but not the esters of pyrimidine 2’-phosphates or of purine nucleoside 3’-phosphates. Under suitable conditions, the enzyme exhibits synthetic activity, catalyzing the formation of alkyl esters from nucleoside 2‘: 3’-cyclic phosphates and primary alcohols, or of oligonucleotides from nucleoside cyclic phosphates and nucleosides (25, as), and this synthetic activity can be enhanced relative to the hydrolytic activity by suitable modification of the structure ( 2 7 ) . Goldstein (28) has shown that a specific alkylated derivative of RNase I, ~-carboxymethyllysine-41RNase, is devoid of synthetic activity and inert toward 2’:3’-CMP, but retains its endonucleolytic activity and specificity. A ribonuclease with a specificity similar t o that for the pancreatic enzyme has been isolated and purified 260-fold from KB cultured mammalian epithelial cells. However, the enzyme differs in two respects from that of the pancreatic type: its pH optimum is lower, p H 6 ; and it is somewhat less heat-stable ( 2 8 ~ )It. was not tested in high concentrations against poly A, the results of which would have been valuable for further characterization of the enzyme. AEkaZine RNases. Several ohservers (2953) have isolated from rat liver supernatant a relatively thcnnostahle enzyme, optimally active a t pH 7.5-8.0, that hydrolyzes RNA to resistant oligonucleotides with a terminal pyrimidine 2’: 3’-nurleotide, and that is inhibited by a substance found in the supernatant of liver and other tissues (19, 34, 3 5 ) . The properties and specificity of the inhibitor have been extensively reviewed (9). Roth (31) also reports the isolation from rat liver mitochondria of another alkaline RNase active toward RNA and pyrimidine nucleoside cyclic phosphates, but inert toward poly A, poly U, and adenosine 2’: 3’-phosphate, and inhibited by a supernatant inhibitor but not by the antiserum to crystalline RNase; the meaning of this is uncertain, since it has been shown (36) that the mitochondria1 and supernatant enzymes exhibit identical chromatographic behavior, pH optimum, and specificity. A chromatographically purified enzyme preparation from beef liver was found by Maver and Greco (37) to hydrolyze RNA to cyclic nucleotides, leaving a small core; exhaustive digestion led to the appearance of 3’-phosphates from the cyclic nucleotides. Beard and Razzell (38) purified alkaline RNase from pig liver mitochondria and supernatant 3000-fold and found the enzyme from both fractions to be identical. The enzyme hydrolyzes RNA more rapidly
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DAVID SHUGAR AND HALINA SIERAKOWSKA
than synthetic polyribonucleotides, with poly A completely resistant, to liberate oligonucleotides with terminal 2’: 3’-phosphates. Activity against the cyclic phosphate ring could not be tested because of the presence of an inhibitor. The enzyme is not inhibited by antiserum to pancreatic RNase. RNase activity optimal a t alkaline p H has been found in many organs (39). Acid RNases (EC 2.7.7.17). Maver et al. (40) originally isolated from bovine spleen a heat- and acid-labile enzyme, with an optimum at pH 5.8, that degrades RNA to purine and pyrimidine nucleoside2’: 3’-phosphates. Purification showed the final products to be 3‘-phosphates (37), the 2’-phosphates previously reported being due to a very active contaminating 2 :3’-nucleotide phosphodiesterase (see below). Further purification gave an enzyme th at hydrolyzes tRNA and rRNA at almost equal rates, and poly U much more rapidly than other synthetic polynucleotides ( 4 1 ) . A similar enzyme ( S I - S S ) , subsequently shown to bc contaminated with a 2’: 3’-nucleotide phosphodiesterase (37), has been isolated from rat liver. However, Nodes et al. (36) used DEAE chromatography to isolate from rat liver mitochondria an RNase fraction inactive toward cyclic phosphates. This discovery was confirmed by Beard and Razzell (38) during the course of a study on acid RNase contamination of a hog liver alkaline RNase; after removal of a contaminating 2’:3’nucleotide phosphodiesterase, the acid RNasc exhibited little activity against nucleoside cyclic phosphates. Acid RNase-like enzymes are found in nearly all tissues, and are unaffected by natural (supernatant) inhibitors of alkaline RNases (9, 20, 51,SS).
RNases releasing 5‘-phosphate-temninated oligonucleotides (6’RNase) . Two such enzymes, both endonucleases, have been reported. One, purified from guinea pig (4.2) and pig (4.3) liver nuclei, hydrolyzes poly A to small oligonucleotides. It is optimally active a t pH 7, is activated by Mg2+, and hydrolyzes all polyribonucleotides except poly G (42). The other, with no marked base specificity, and isolated from the endoplasmic reticulum membranes of rat liver and Ehrlich tumor cells, is optimally active a t p H 7-8 and is insensitive to Mg” and N a F (44-46). 2. DEOXYRIBONUCLEODEPOLYMERASES DNase I (EC 3.1.4.5). This enzyme was first crystallized from the pancreas ( 4 7 ) , but similar activity occurs in other organs (16, 48-50), although no struetural identity between these has been established ( 9 ) .
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The enzyme is optimally active a t about p H 7 and is usually activated by Mg2+(49, 5 1 ) . Native DNA is depolymerized with a rapid decrease in viscosity; denatured DNA is attacked more slowly. The enzyme is inactive toward small oligonucleotides, esters of mononucleotides, apurinic acid, and deaminated single-stranded DNA ( 2 , 52, 5 3 ) . Many tissues contain natural inhibitors toward DNasc I (16, 54-57) ; two such protein inhibitors have been prepared in crystalline form from calf spleen (58, 5 9 ) . One of these, referred to as inhibitor 11, has now been identified as a labile protein with a molecular weight of approximately 60,000. It inhibits specifically DNase I and exhibits no adverse activity against DNase 11, snake venom phosphodiesterase, pancreatic RNase, or E . coli endonuclease ( 5 9 a ) . Under a variety of experimental conditions, in the p H range 6-9, it interacts directly with DNase I to form only one type of stable complex, with a ratio of inhibitor I1 to DNase I of 1:1 ( 5 9 b ) . Above pH 9.5 the complex dissociates with simultaneous denaturation of the inhibitor. The specificity, cation requirements, and nature of the products of DNase I action are still far from clear ( 8 ) , but studies by Bollum (60) with the aid of synthetic polydeoxyribonucleotides may lead to a resolution of these problems. DNase I I (EC 3.1.4.6). This enzyme, isolated from spleen (61) and thynius (62, 6 3 ) , occurs in nearly all mammalian cells (48, 50, 64, 6 5 ) , with an optimum a t p H 4.2-5.5 and maximum activity in rapidly multiplying tissues (66). The enzyme initially rapidly hydrolyzes about 10% of the internucleotide linkages, followed by a slow release of 3’-phosphate-terminated mono- and oligonucleotides ( 6 7 ) . Of considerable interest is the finding that the enzyme may split both strands in native DNA simultaneously to give two duplex strands, and the correlation of this behavior with its dimeric structure (68, 6 9 ) . DNase I1 is inhibited by Mg2+concentrations in excess of 1 mM and is activated by monovalent cations (49, 67); it is inhibited by denatured DNA ( 7 0 ) . It has been reported to exhibit some activity against bis-p-nitrophenyl phosphate and p-nitrophenyl esters of 3’-deoxynucleotides (71) . Tissue and urine inhibitors of the enzyme have been reportcd (2, 7 2 ) . DNase I1 from HeLa cells has now been purificd 700-fold ( 7 0 ) . Treatment of this enzyme with mercaptoethanol led to the production of two modified active enzyme species. Both of these, like the native enzyme, degraded native DNA by “single hit” kinetics. Kinetic data for both HeLa cell and calf spleen DNasc I1 suggested a “twin site’’ model capable of both “single hit” and “double hit” degradations.
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Lamb brain phosphodiesterase. This endonuclease preferentially hydrolyzes thermally denatured DNA. It has a broad p H optimum, 7-9, and leads to formation of 5’-phosphate-terminated oligonucleotides (73). 3. NONSPECIFIC ENDONUCLEASES
A thermolabile enzyme from chicken pancreas hydrolyzes DNA, RNA, and, a t somewhat slower rates, synthetic polynucleotides, with the release of small 5’-phosphate-terminated oligonucleotides. NO activity is manifested against nucleoside cyclic phosphates or esters of nucleoside phosphates. The enzyme is optimally active a t highly alkaline pH, is activated by divalent cations and inhibited by EDTA ( 7 4 ) . Rat liver mitochondria contain a nonspecific heat-labile endonuclease, optimally active a t pH 6.8; it hydrolyzes both RNA and DNA, with a marked preference toward single-stranded chains, to yield 5’-phosphateterminated oligonucleotides. It exhibits no base specificity and is inactive against p-nitrophenyl 3’- and 5’-TMP. The enzyme possesses an absolute requirement for Mg2+or Mn2+ (75, 7 6 ) . The relationship, if any, between this enzyme and another previously isolated from liver and purified fifteenfold in the same laboratory (77) is not clear. The latter endonuclease exhibits similar cation requirements and preference for denatured DNA. A similar heat labile endonuclease, with no base specificity or preference for the sugar moiety, optimal activity a t pH 7.0-7.5, and with a requirement for Mg2+,has been isolated from sheep kidney. The enzyme exhibits rigorous specificity with regard to secondary structure, attacking only nonstructured regions with formation of 5’-phosphate terminated oligonucleotides (77a). An additional enzyme hydrolyzing RNA a t alkaline pH occurs in the particulate fraction of rat liver ( 7 8 ) , but its specificity toward other substrates has not been examined. It is labile to heat and acid, with an optimum a t pH 9.CL9.5. It is not affected by rat liver supernatant inhibitor, is strongly inhibited by monovalent cations, and is activated by non-ionic detergents. 8. Exonucleases Phosphodiesterase I (EC 3.1.4.1). This enzyme, which resembles snake venom phosphodiesterase, is heat labile, optimally active a t p H 9.2, and is found in nearly all tissues (79-82). It is most active against chains with a 5’-phosphate terminal group, and it releases 5’-mononucleotides stepwise from the 3’-hydroxyl tertninal end. Acetylation of the terminal 3’-hydroxyl does not significantly affect enzyme activity, but chains terminating in a 3’-phosphate are relatively resistant. The enzyme is
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active against dinucleotides and nucleotide esters such as p-nitrophenyl or a-naphthyl thymidine S’-phosphate. It rather reluctantly hydrolyzes bis-p-nitrophenyl phosphate and dinucleoside monophosphates (83). Leukemic cell phosphodiesterase (EC 3.1.4.1). Isolated from mouse leukemic cells, this enzyme, optimally active a t p H 7-8, hydrolyzes oligo- and poly-, ribo-, and deoxyribonucleotides, with the stepwise release of 5’-mononucleotides. It is active against dinucleotides, but not nucleotide esters. It belongs to it newly discovered class of phosphodiesterases requiring the presence of two nucleoside moieties in ester linkage to a phosphate group ( 8 4 ) . Polynucleotide phosphorylase (EC 2.7.7.8). Although this enzyme is fairly widespread among bacteria, there are only several reports of its existence in mammalian cells (e.g., in rat liver nuclei and nucleoli) and exhibiting phosphorylytic activity ( 8 5 ) . Hilmoe and Heppel (86) isolated from guinea pig nuclei an enzyme converting poly A to ADP and catalyzing the exchange of P3‘0, with ADP; it is optimally active a t pH 7, requires Mg“, and is inhibited by fluoride. The discovery of such an enzyme in human sperm (87) has not been confirmed and is highly suspect. Phosphodiesterase IZ (EC 3.1.4.1). This enzyme is widely distributed in animal tissues (79, 88) and has been isolated from the spleen (89, 90). It is heat-labile, has an optimum a t pH 6, and releases 3’-mononucleotides from the 5’-hydroxyl end of the chain ; 5’-phosphate-terminated chains are resistant (90, 91). Phosphodiesterase I1 is active against some esters, like p-nitrophenyl thymidine 3’-phosphate, and may also catalyze formation of internucleotide linkages (26, 91). Mammary tumors of C3H mice contain an enzyme that produces 5’-mononucleotides from DNA, with a preference for denatured DNA (92, 9 3 ) . It is optimally active a t pH 8.5, with an absolute requirement for Mg2+,and is inhibited by Na+. Activity against p-nitrophenyl thymidine 5’-phosphate and RNA, to further define the specificity, was not reported.
C. Cyclic Nucleotide Phosphodiesterases 3’:5‘-iVucleotide phosphodiesterases. An enzyme found in various tissues, and most abundant in the brain, hydrolyzes 3’: 5’-ribonucleotides (3’: 5’-AMP) most rapidly, to 5’-phosphates, with optimum activity at pH 7.5-8.0 (94, 9 5 ) . It is activated by Mg?+ or Mn2+ ions, inhibited by methylxanthines (94, 95, 95a), nucleoside triphosphates, and inorganic pyrophosphate (95a, 95b), and is inert toward internucleotide linkages and nucleoside 2’:3’-cyclic phosphates ( 9 4 ) . Purified 170-fold from dog heart, it hydrolyzes 3‘: 5’-dAMP more rapidly than 3‘: 5’-AMP (96).
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DAVID SHUGAR AND HALINA SIERAKOWSKA
Subsequently a second, analogous, enzyme was partially purified from dog heart. It hydrolyzes 3’:B-UMP a t a rate fourfold that of 3’:5’-AMP and 20 times that of 3’:5’-CMP, with a n optimum a t p H 8. By the use of ammonium sulfate, this enzyme was partially separated from the one described in the previous paragraph, and was found to be inhibited by 100-fold lower concentrations of methylxanthines. On the basis of this evidence, it is considered to be a different enzyme than that hydrolyzing 3’: 5’-AMP (97). It is quite conceivable that additional enzymes of this class may yct be found. 2’:s’-Nucleotide phosphodiesterase. Only one such enzyme has hitherto been reported. It is most abundant in nervous tissue ($7, 98-100), has been isolated from beef brain, and hydrolyzes purine and pyrimidine nucleoside 2’: 3’-cyclic phosphates to the 2’-phosphates, with a pH optimum of 6-7. It is inert toward internucleotide linkages, 3‘: 5‘-nucleotides, and nucleotide esters.
111. Methods of Assay The assay methods for nucleolytic c’nzymes outlined here are usudly applied in fractionation studies. However, they may and should be employed in determining the validity of histo- or cytochemical procedures, as is shown below.
A. Endonucleases I n the past, tissue RNases have been assayed almost exclusively against RNA substrates. However, the oligonucleotides initially released by RNases are subject to further degradation by tissue phosphodiesterases. A similar situation prevails for DNases. In some instances, a suitable pH in the incubation medium may selectively inhibit phosphodiesterases. However, the usual assay of the acid-soluble products of what is believed to be RNase or DNase activity is a measure of both the endoand exonucleolytic activities of a tissue sample. It has been pointed out by Razzell (79) that the discovery of phosphodiesterases I and I1 in nearly all animal tissues renders dubious many of the earlier quantitative estimations of tissue RNase and DNase. 1. RNASES
Methods of measuring endonucleolytic RNase activity are generally unsuitable for tissue homogenates; this applies to the Kunitz (101) method and others, reviewed by Josefsson and Lagerstedt ( 5 ) and by Roth (9). The turbidimetric method has been claimed to be the only
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one generally applicable to crudc enzyme preparations (102). Assay of acid-soluble products formcrl in later stages of the reaction has been widely employed, although tliis is not always too accurate. The relative merits of various precipitating agents have been evaluated by Rot11 ( 9 ) . Furthermore, coniinercial RNA preparations, widely used in the past as the substrate, contain polyvalent cationic contaminants that may affect results by modifying enzyme activity directly (38) or by inactivating the inhibitors (34, 1031. Roth ( 9 ) circumvents this by addition of E D T A to the assay nicdium or by the use of purified, highly polymerized yeast RNA (104 ) , relatively insensitive to exonucleases. But it is important t o note that E D T A may unmask ribosome-bound enzyme activity ( 1 0 5 ) . Polyeytidylic acid, now commercially available, is a fairly sensitive substrate for RNases of the pancreatic type (106). Some increase in specificity may be attained by control of pH and ionic strength, or the use of activators and inhibitors, etc. Neu and Heppel (105) estimated the latcnt RNase I of Escherichia coli with tRNA in the presence of EDTA, i.e., under conditions mfavorable for E . coli phosphodiesterase and polynuclcotide phosphorylase. Free RNase was estimated using tRNA as the substrate in the presence of Mg“. Estimations may also be based on the relative rates of hydrolysis of various types of RNA or synthetic polynucleotides, and on the nature of the products formed (107, 108). It is rather difficult to obtain specific substrates for endonucleases. Pancreatic RNase, because of its biphasic mode of action, is an exception in view of its activity against pyrimidine nucleoside 2‘: 3’cyclic phosphates, but even it is difficult to assay specifically bccausc of susceptibility of the cyclic products t o 2‘: 3‘-nucleotide phosphodiesterases, which, as a result of their wide distribution and broad pH optima ( 9 9 ) , may interfere with RNase assays. Such interference can be detected by the formation of nucleoside 2’-phosphate products ( 3 8 ) . Hydrolysis of nucleoside cyclic phosphates may be followed by electrophoresis or by paper or thin-layer chromatography (5, 109, 110), but the rates of hydrolysis are low (Section VII). Titrimetric (5, 111) and spectrophotometric (5, 112) methods for following hydrolysis have been described but are difficult t o apply t o homogenates. Butcher and Sutherland (94) followed the hydrolysis of 3’: 5’-nucleoside phosphates by addition of a 5’-nurIeotidase, followed by colorimetric dctermination of the P, liberated. This procedure (cf. Scheme 31, originally suggested for RNase assay by Heppel [cf. ( l o g ) ] ,is applicable to any phosphodiesterase assay and niay be used with tissue extracts if the 5’-nucleotidase is replaced by a nonspecific phosphatase. Other useful substrates are nucleoside 3’-alkyl or aryl phosphates,
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although their susceptibility to phosphodiesterase I1 limits their application to alkaline RNases. After earlier attempts (11S), a diester, a-napthyl uridine 3’-phosphate was synthesized for this purpose. This suhstance yields a color-forming product on hydrolysis and, even more important, it is resistant to phosphodiesterase 11. The high rate of hydrolysis of this substrate jcf. Section VII, B) and its specificity make it very suitable for both colorimetric and histocheinical assays of RNase (114). Since alkaline RNases often occur in an inhibited form, the total, as contrasted with the free, activity is assayed in homogenates subjected to freezing and thawing in the presence of p-chloromercuribenzoate (17, 103). Acid RNases may be assayed with RNA “core” as substrate (115); the core is resistant to alkaline RNases, but not to phosphodiesterases. It is possible that a-naphthyl esters of purine nucleoside 3’-phosphates, resistant to phosphodiesterase 11, may be more suitable, and attempts are under way in our laboratory to prepare such a compound. RNases that lead to formation of 5’-phosphate-terminated products, like the K+-activated RNase or the liver nuclear enzymes, are generally assayed with poly A as substrate in the presence of the proper activating ions (42, 43, 46, 116). Free and latent acid RNase may be assayed together, in the presence of low concentrations of p-chloromercuribenzoate and with repeated freezing and thawing of the tissue extracts (10s). 2. DNASES
Methods of DNase activity assays and their applications have been reviewed by Kurnick ( 4 ) and Laskowski (8). The viscosimetric method, as well as that based on the UV hyperchromicity accompanying hydrolysis, both reflect initial cleavage by the enzyme, but are difficult to apply to homogenates ( 4 ) . A procedure that estimates exclusively endonucleolytic activity makes use of the loss of activity of transforming DNA ( 1 6 ) , or of A-phage infectivity ( 6 8 ) , but these are too complex and time-consuming for routine work. The methyl green method, which probably measures more than just endonuclease activity, is claimed to be applicable to alkaline and acid DNases of tissue homogenates ( 4 ) but has not gained many proponents. Assay of acid-soluble products, either by spectrophotometry or the diphenylamine reaction, has been most widely used in studies with crude systems. I n assaying DNase I, Mg2+ and pH control are used to discriminate against DNase 11, and conversely for DNase 11. A sensitive method for acid-
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soluble products, based on fluorimetry of thymine-containing oligonucleotides, has been proposed for acid and alkaline DNases ( 1 1 7 ) . Antiserum to DNase I has been employed for estimations of DNase activity in salivary glands of Chironomus thummi (lit?), but the specificity is questionable as the immune serum employed also precipitates DNase I1 and streptococcal DNase. Immunochemical assay of DNase preferentially attacking singlestranded DNA is highly specific, extremely sensitive, and free from exonuclease interference, since the serological activity depends on the size of the DNA molecule and hence responds to endonucleolytic breakage ( I 19). A rapid and sensitive method for endonucleolytic DNase activity (120), applicable to tissue extracts ( l d l ) , is based on the ability of nitrocellulose filters to retain only large fragments of denatured DNA. Labeled denatured DNA is incubated with the enzyme and then filtered; the decrease in retention of radioactivity is correlated with enzyme activity. For DNases with a preference for native DNA, the latter is denatured after incubation and then placed on the filter. Total DNase activity is assayed after dcstruction of inhibitors by aging or heat treatment (55, 56).
B. Natural Inhibitors Many tissue endonucleases are known to be associated with natural protein inhibitors, which may interfere with assay of enzyme activity. Methods have been elaborated for assay of the inhibited enzyme (see preceding sections) and of the inhibitor activity directly. Alkaline RNase inhibitor is estimated from the amount of tissue extract which gives 50% inhibition of crystalline RNase (9, 103), and similarly for DNase I (55, 57) and DNasc I1 (72).
C. Exonucleases Phosphodiesterase I . Earlier methods of assay of phospliodiesterase I were based on the use of bis-p-nitrophenyl phosphate, but this subbtratc is also susceptible to I)liospliodiesterase I1 and DNase I1 (71) and is hydrolyzed a t only 1% of the rate for the natural substrates, which are 5'-phosphate-terminated dinucleotides. Razzell and Khorana ( 8 3 ) introduced p-nitrophenyl thymidine 5'-phosphate, specific for 5'mononucleotide-forming exonucleases and hydrolyzed a t a rate six times that of the best natural substrates; the liberated p-nitrophenol is then easily estimated colorimetrically a t alkaline pH (6). Alternatively, a-naphthyl thymidine 5'-phosphate may be used but, apart from histo-
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DAVID SHUGAR AND HALINA SIERAKOWSRA
chemical applications (122), it presents no special advantages over the p-nitrophcnyl analog. Finally, both a-naphthyl and 3-pyridyl thymidine 5’-phosphates undergo small spectral changes a t 3101 mp when liydrolyzcd ( I d s ) , but these changes are too small to be of practical usc except with purified enzymes and difference spectrophotometry. Phasphodiesterase II. Early assay methods made use of bis-p-nitrophenyl phosphate, but this proved a poor substrate; it is also subject to some interference from phosphodiesterase I (83) and, to a lesser extent, from DNase I1 ( 7 1 ) . One may use RNA “core,” with spectral estimation of acid-soluble products (115),but interference from acid RNases is to be expected. Razzell (79) introduced p-nitrophenyl thymidine 3’phosphate for enzyme assay of tissue extracts; it is hydrolyzed a t about the same rate as the natural substrates and the rate can be estimated colorimetrically ( 9 1 ) . Although very convenient, its trustworthiness is somewhat liinited by a slight susceptibility to splenic acid DNase ( 7 1 ) . Bernardi and Bernardi (41) consider 3’-phosphate-terminated oligodeoxyribonucleotides (resulting from exhaustive digestion of DNA by splenic acid DNase) more specific substrates for phosphodiesterase 11; these undoubtedly merit further study.
D. Cyclic Nucleotide Phosphodiesterases 2’: 3‘-Nucleotide phosphodiesterase. Hydrolysis of 2 :3’-AMP to 2’AMP is followed by measuring the Pi released from the latter by an excess of phosphomonoesterase (6). The use of purine nucleoside cyclic phosphates eliminates the interference of alkaline RNases. 3’:5’-Nucleotide phosphodiesterases. Specific substrates are the 3’ :5’cyclic nucleotides. Here again the method of choice is to follow the release of P, in the presence of added phosphomonoesterase ( 9 4 ) .
IV. Substrate Preparations I n view of the multitude of nucleolytic enzymes found in rnammalian cells (and there are even more in lower organisms) and the overlapping specificities frequently encountered among these, it is perhaps somewhat of an anachronism to speak of specific substrates, unless it is with reference to a given enzyme. However, even a substrate with limited specificity is still indispensable in any method designed to estimate or localize a given enzyme, or class of enzymes. The following paragraphs list some of the techniques for the preparation of substrates with absolute or relative specificities. Ribonucleoside 2’: 3’-cyclic phosphates have been synthesized by a variety of procedures, but we limit ourselves here only to those that do
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not require supplementary purification techniques. The method used in our laboratories for several years is based on the reaction of the commercially available nuclcoside 2’ and 3’-phospliatt~s with dicyclohexylcarbodiiinide (DCC) in anhydrous medium, to give the cyclic phosphates in quantitative yield in the form of the sodium or ammonium salts (124, 125). The method of Smith et al. (126‘) involves the reaction of the ammonium salts of the nucleotides with DCC in aqueous tertbutyl alcohol and formamide. Tha t of Michelson ( l 2 7 ) , based on treatment of the nucleotide in the presence of a base with ethylchloroforinate, has been made quantitative with the aid of a slight inodification by Taylor and Hall (128) for uridine 2’:Y-cyclic phosphate. Wigler (129) has described a procedure for obtaining cytidine 2’: 3‘-cyclic phosphate in crystalline form. Ribonucleoside 3’:5’-cyclic phosphates are conveniently prepared by reacting the ribonucleoside 5’-phosphoromorpholidate with DCC as described by Smith and Khorana (130). Additional procedures for all the natural ribo and deoxyribo analogs, including the widely eiiiployed 3’: 5’-AMP (now available commercially) have been described in detail (131). p-Xitrophenyl thymidine 3’-phosphate is synthesized by phospliorylation of 5’-O-tritylthymidine with p-nitrophenyl phosphorodichloridate (132).
a-Naphthyl phosphoryldichloride ,I
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DAVID SHUGAR AND HALINA SIERAKOWSKA
p-ATitrophenyl thymidine 5’-phosphate may be prepared by esterification of thymidine 5’-phosphate with p-nitrophenol in the presence of
DCC (133). The synthetic procedures for the p-nitrophenyl esters of all the ribo- and deoxyribo-nucleoside 5’-phosphates and 3’-TMP have recently been slightly improved upon and described in extensive detail (1S4). a-Naphthyl thymidine 5’-phosphate has been prepared by treatment of 3’-acctylthymidine with a-naphthylphosphoi-yldichloride according to Scheme 1 (122). The 3’ isomer may be prepared in an analogous manner froiii .Y-O-tritylthymidine. a-Naphthyl uridine 5’-phosphate is obtained by treatment of 2‘,5‘di-0-tetrahydropyranyluridine with a-naphthylphosphate in the presence of DCC (Scheme 2) (114).
4
OH ANN 3
OAN pyranylation ____c
d
O
I
ICSI
HOCH,
0
-80% CH,COOH
rG O-e>
OH
U-3’-p-Naphthyl
SCHEME 2
A modification of this procedure (114) makes possible the preparation of a mixture of the 2’ and 3’ isomers, which may then be separated from each other by several methods. Dinucleoside monophosphates, dinucleotides, and higher oligonucleotides. A variety of these is now available commercially, and the number
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383
of procedures for their chemical synthesis is too numerous to present here (see, e.g., 155-1373. Some of the ribo compounds can be very conveniently obtained on a preparative scale with the help of pancreatic or T 1 RNases by techniques described by Bernfield (138) and Egami (139).
V. Cellular Fractionation Endonuclease determinations have, with relatively few exceptions (see below), been based on assays of acid-soluble products released from RNA and DNA. Substrates with better specificity have been employed for exonucleases. Reference has already been made (Section 111) t o the question of substrate specificity and to the use of unpurified commercial substrates affecting cellular inhibitors. An additional problem, which has received little attention in fractionation studies, is the concomitant liberation of cellular inhibitors and their effects on the activity of various fractions. Control of enzyme diffusion has usually been limited by the use of suitable thermal and tonicity conditions to prevent disruption of cytoplasmic structures; sucrose solutions have been widely applied for this purpose, since they also limit aggregation of the particles. In some investigations, the validity of the findings has been cxamined by testing for adsorption of the isolated enzyme to the various cellular fractions, low adsorption being regarded as evidence for the reality of the activity of a given fraction. The initial, erroneous localization of DNA polymerase almost exclusively in the cytoplasm [cited in reference ( l 4 O ) l illustrates the types of errors arising from enzyme diffusion in aqueous fractionation studies ; subsequent nonaqueous fractionation revealed the high specific activity of the enzyme in nuclei (56,141) and its extractability with buffer explained the aforementioned erroneous results. Another example is that of LLribosomalRNase,” discussed in Section IX. The presently available wide variety of cell fractionation techniques and their applicability to intracellular enzyme distribution studies are discussed in several reviews (1&-145). The isolation of nuclei with the aid of nonaqueous media by Allfrey and Mirsky (1.46) represented probably the earliest attempt to avoid artifacts due to enzyme diffusion and concomitant rcadsorption in studies on localization of nuclease enzymes. Their procedure has been widely adopted and its grneral aspects were recently rrviewed (147). The use of nonaqueous media has also found application in the isolation of defined fragments from individual cells by microdissection, fol-
384
DAVID SHUGAR AND HALINA SIERAKOWSKA
lowed by incubation of the fragments with a nucleoside 2’:3‘-cyclic phosphate for RNase estimations, the extent of hydrolysis being determined by microelcctrophoresis ( 1 4 8 ) . Difficulties encountered in microdissection of small mammalian cells limit the procedure to relatively large invertebrate ooeytes, but improvements in microdisPection techniques could considerably broaden the potentialities of this technique. It is not without interest that microdissection of cells for such purposes was attempted as early as 1941 by Bundling (149)) who separated salivary gland nuclei from the cytoplasm in studies on catalasc distribution. Localization data from isolation procedures. In a number of instances, specific enzymes have been isolated from psrticular cellular fractions, e.g., the endonucleolytic RNase from guinea pig liver nuclei, which releases oligonucleotides with terminal 5’-phosphate groups (42). Since the emphasis in such studies has been on isolation and/or purification, and not on intracellular localization, these examples are cited in Section 11, and their possible significance is referred t o in Section IX. Brid RNases. Intracellular localization of nucleases by cell fractionation techniques was initiated in 1952 by Schneider and Hogeboom (150), who observed that the specific activities of acid RNA and DNA depolymerases of rat liver mitochondria markedly exceed those of the homogenate. Ultrasonic disruption of the isolated mitochondria liberates their depolymerase activity into the medium. The liberated enzymes exhibit little tendency to adsorption on mitochondrial structures, testifying to the authenticity of the mitochondrial localization. Subsequent investigations (33, 63, 151, 152) of acid RNase revealed that the specific activity of particulate fractions exceeds those of the nuclei and supernatant. I n liver the activity has been found mainly in the mitochondrial fraction (33,151).D e Duve e t al. (153-156) attempted further subfractionation of the particulate fraction by various methods, including sucrosc density gradient equilibration, and found the acid RNase to be located, together with other acid hydrolases, in a distinct class of cytoplasmic particles, the lysosomes. These were shown to be enclosed by a membrane that could be disrupted by osmotic shock, sonification, or non-ionic detergents, with concomitant liberation and activation of acid RNase (cf. Schneider and Hogeboom, above). Reid and Nodes (3%’)attempted the localization of enzymes of known specificity by comparing activities of particular fractions against RNA and nucleoside 2.’ :3’-phosphates, and distinguished two acid RNases in the liver. They concluded that the acid RNase of lysosomes, which hydrolyzes RNA and also adcnosinc 2’: 3’-phosphate to 2’-nucleotides, is probably different from the acid RNaee that hydrolyzes cyti-
LOCALIZATION OF NUCLEASES
385
dine 2’:3’-phosphate to the 3’ isomer, the localization of which was difficult to establish. This finding warrants reexamination, since liver acid RNase has now been shown to tw inert toward cyclic nucleotides (Section 11, A, 1 ) . Roth (157) found no acid RNase associated with rat liver microsomes or ribosomes. In the kidney, acid RNase has been located in droplets together with other acid hydrolases (158, 159). I n mouse pancreas, the enzyme was found mainly in the zymogen granule and microsoinal fractions (160, 161), the microsomal enzyme being subject to activation by EDTA (161). The kidney droplets and the zymogen granule fraction are considered to be examples of polymorphism of lysosomes (144). Spleen acid RNase was reported to be concentrated in the mitochondria, but this must be regarded with reserve since no separation of mitochondria from lysosomes was undertaken (15.2). Smith and Winkler (162) lysed the purified bovine adrenal gland chromaffin granule fraction by exposure to hypotonic media, thus releasing acid RNase and DNase activities. Subsequent sucrose density gradient distribution studies showed that the acid RNase and other acid hydrolases of bovine adrenal medulla occur in the lysosoinee (163). Using the fractionation procedure of Schneider and Hogehoom (150), Girij a and Sreenivasan (2020) localized acid RNase in the mitochondria1 fraction of rat adrenal gland and liver. Freezing and thawing or detergent treatment appreciably increased the activity without affecting that of alkaline RNase. Acid RNase of rat brain was recovered from mitochondria, microsomes, and supernatant, with a somewhat higher specific activity in the supernatant fraction. Density gradient subfractionation of the mitochondria and microsomes showed the enzyme to be concentrated in the most dense fractions (164). Acid RNase of liver nuclei has been reported to range from negligible (33, 35) to 1-5% of the total (150, 153, 154). It has also been found in ‘honaqueous” nuclei (165). In other tissues, such as kidney (33) or thymus and spleen (18, 6 3 ) , the nuclear content of enzyme is higher. Atkatine RNase has been localized in the mitochondria1 (20, 33, 35, 151) and microsomal fractions (3.2, 33, 35, 151), and in the supernatant fraction (20, 32, 33, 35, 151) of liver cells. Roth (9) suggests that the supernatant activity arises not from alkaline RNase, which is rendered inactive by excess inhibitor in the supernatant, but rather from other RNases unaffected by the inhibitor. Inactivation of the RNase inhibitor in the supernatant increased appreciably the percent activity in the latter fraction (20, 3 5 ) . Roth (157) found some alkaline RNase as-
386
DAVID SHUGAR AND HALINA SIERAKOWSKA
sociated with rat liver microsomes and ribosomes, but subsequent studies (166) showed the latter finding to be an artifact (Section IX). Belousova (167) has claimed predominantly mitochondrial localization of spleen alkaline RNase, whereas in the pancreas the enzyme was concentrated in the microsoIncs, zyniogen granules, and supernatant (160, 168). In rat adrenals the enzyme was present in the mitochondria as well as in the supernatant (20). Studies dealing with the occurrence and inactivation of alkaline RNase inhibitors (20, 34, 35, 103) have altered our notions as to the ratio of alkaline to acid RNases in most tissues. Destruction of RNase inhibitor, which is localized in the supernatant, increases the level of the alkaline enzyme severalfold. Since the mitochondrial alkaline RNase occurs in an inhibited form to a lesser degree, removal of the inhibitor modifies the intracellular distribution pattern of the alkaline enzyme. Roth (35) compared the activities of acid and alkaline RNases in various fractions of rat liver cells. By inactivating the alkaline enzyme inhibitor, he estimated the total alkaline activity of a given fraction. The content of acid RNase was then calculated from the fractional activity resistant to inactivation. The relation between activities of individual fractions and the assay pH suggests that, in contrast to other fractions, the nuclei contain small amounts of alkaline RNase and no acid enzyme (169). Siebert et aZ. (85) found alkaline RNase in rat liver nuclei and nucleoli, partially in latent form, the nucleolar enzyme exhibiting a higher specific activity than the nuclear. The alkaline RNase activity of “nonaqueous” nuclei of rat liver is appreciably higher than that of nuclei isolated in sucrose ( 1 6 5 ) . Alkaline RNase has also been found in “aqueous” nuclei of kidney cells (33). 5’-Ribonuclease (cf. Section 11, A, 1) has been recently assayed in rat liver fractions, with poly A as substrate (Table I ) . About 65% of total activity was associated with the mitochondria. Nearly 25% appeared to be localized in the nuclear fraction, but its low specific activity makes this finding suspect and due, possibly, to contamination from the mitochondrial fraction ( 1 0 8 ) . DNase I distribution studies on liver fractionated by differential (170) and density gradient (155, 171) centrifugation point to the occurrence of this enzyme in mitochondria, the specific activity of the nuclear fraction being much less (170) and that of the nucleolar lower still ( 8 5 ) . Predominantly mitochondrial localization was likewise found in mouse pancreas ( 1 7 2 ) . DNase I activity has also been studied in “nonaqueous” nuclei of rat regenerating liver ( 5 6 ) and of calf and rabbit thymus (173).Specific activity of such nuclei was lower than that of the “nonaqueous” cyto-
387
LOCALIZATION OF NUCLEASES
plasm and was largely resistant to extraction by buffer. Tlic DNasc I of regenerating rat liver nuclei was associated to a larger extent than the cytoplasmic activity with a powerful inhibitor, the removal of which resulted in a twelvefold increase in enzyme activity ( 5 6 ) .Calf and rabbit thymus nuclei also contain DNase I inhibitors (173). TABLE I DISTRIBUTION OF PHOSPHODIESTERASE I A N D 5’-ltX.is~ INTRACELLULAR IN
RAT LIVER,WITH
(iLlrCOSE
6-PHOSPHATASE
AND
ACID
PHOSPHATASE AS REFEREN(IE ENZYMES (108)
Enzymes Proteins
Numher of Ahsoexperi- lute merits valuesa 4
Percentage distribution*
W
4
Phosphodiesterase I Glucose Bphosphatase Acid phosphatase
4
3
3
L
blc
S
Recovery
10.5 f2.1 3.7 f2.5 6.8 f6.2
17.0 k3.1 2.8 k0.8 35.9 f5.9
43.1 k2.8 5.7 k1.6 9.7 k6.7
101.7 k7.6 100.0 f22.9 99.9 k0.2
2.17 100 k0.72 3.01 100 k0.76
16 f 1 23 f 9 40 f 6
2
14 4 f0.7 64.2 k13.0 6.7 k2.6
20.49 100 k1.28
10.9 k6.4
9.0 +4.6
6.8 f4.0
69.9 flO.l
3.5 +1.6
100.1 +0.2
1.92 100
8.1 f2.5
14.8 k4.7
45.7 f10.5
20.3 k8.1
9.3 f1.8
98.2 k4.5
167
100
k 19 5’-RNase
nt
N
+0.27
7 7 G 1
x
Absolute values are in milligrams per gram of homogenate for proteins; in micromoles of AMP produced per rni1iut.e per gram of homogenate for 5‘-HNase using an extinction coefficient of 142 X lo8;i n mitw,moles of p-nitrophenyl5’-TMP hydrolyzed per minute per gram of homogenate; and in micromoles of phosphate liberated per minute per gram of homogenate for glurose 6-phosphatase and acid phosphatase. * To express the percentage distriliut,ion, the absolute values for the homogellate were taken as 100. H, homogenate; N, nuclei; M, mitochoiitlrin; L, lysosomes; Mc, microsomes; 6, superiiatant.
DiVase II was initially localized by Schneider and Hogeboom (150) and subsequently by others (6‘5, 174) predominantly in mitochondria of liver and other organs. Beaufay et al. (170) found the enzyme in the lysosomal fraction of liver cells, confirming this by sucrose density gradient centrifugation (155, l 7 1 ) , which has also been employed to localize acid DNase in the lysosomal fraction of bovine adrenal medulla (163). In the kidney it occurs in droplets, which are probably the biochemical equivalent of liver lysosornes (144) and are characterized by
388
DAVID SHUGAR AND HALINA SIERAKOWSKA
high specific activities of other acid hydrolases (158, 159). I n rat brain the enzyme is concentrated in mitochondria and microsomes, with the highest specific activity in the densest subfractions (164). Allfrey and Mirsky (146) localized DNase I1 in ‘Lnonaqueous” nuclei of various organs and found the ratio of nuclear to cytoplasmic activities to vary appreciably for different tissues, being highest with liver and heart. The higher values obtained, as compared to those from nuclei isolated in an aqueous medium, are probably due to enzyme diffusion from the latter into the aqueous medium. The enzyme content of nuclei isolated in an aqueous medium also varies with different organs (63, 65, 154, 170, 174). R a t liver nuclei isolated in the presence of Ca2+ ions (175) contained 14% of total cellular acid DNase, and this activity was released from nuclei on transfer to a calcium-free medium, but not by treatment that solubilized the lysosomes. The specific activity of rat liver nuclei was reported to exceed that of nucleoli (85). Phosphodiesterase I. An extremely useful study of the distribution of this enzyme in liver and kidney has been made by Razzell ( 7 9 ) , using p-nitrophenyl 5’-TMP as substrate. The enzyme was localized largely in the microsomes, with a relative specific activity manyfold in excess of that in other fractions (Table 11).Its occurrence in nuclei, as contrasted to adsorption to the nuclear membrane, could not be unequivocally established. De Lamirande et al. (108) reported 36% and 41% of total rat liver cell phosphodiestcrase I in the microsomal and nuclear fractions, respectively, with similar relative specific activities in both fractions, but negligible in others (Table I ) . Within microsomes, phosphodiesterase I was located in microsomal membranes but not in ribosomes ( 4 6 ) . The high nuclear activity is in sharp contrast with the results of Razzell (79) and those obtained by cytochemical methods (122). The enzyme has also been found in goat brain ribosomes (176). A phosphodiesterase active against bis-p-nitrophenyl phosphate a t pH 9 has been reported as an authentic component of the plasma membrane of liver cells. Its specific activity in the membranes was %fold that of the microsomes, the major contaminant of the membrane preparations. The membrane phosphodiesterase resisted extraction procedures that . the activity a t removed 75% of the membrane proteins ( 1 7 6 ~ )While pH 9 points to the foregoing enzyme as phosphodiesterase I, final classification must await tests against more specific substrates. Phosphodiesterase I I distribution in differentially fractionated liver and kidney cells of the rat has been studied with the aid of p-nitrophenyl thymidine 3’-phosphate, and was found predominantly in the supernatant (Table 11).In the case of hog kidney homogenates, density gradient centrifugation techniques pointed to the mitochondria1 localiza-
389
1,OCALIZATION OF NUCLEASES
tion of this enzyme, suggesting that the supernatant enzyme had been released preferentially from the mitochondria ( 7 9 ) . 3’:5‘-Nucleotide phosphodiesterases. With 3’: 5’-AMP as substrate, this enzyme has been assayed in tissues of the dog fractionated into a 2000 X g particulate fraction and supernatant, the latter being more active ( 9 7 ) . The corresponding particulate fraction from beef heart contained most of the activity, which was resistant to extraction in isotonic and hypotonic media ( 9 4 ) .Nair ( 9 6 ) found the total activity in dog heart to be equally divided between the 600 x g particulate fraction TABLE I1 INTRACELLULAR DISTRIBUTION OF PHOSPHODIESTERASE I A N D PHOSPHODIESTERASE I1 IN RAT LIVERA N D KIDNEY,USINGAS SUBSTRATES ~NITROPHEN 5’-TMP YL AND 3’-TMP, RESPECTIVELY (79) ~
~
~
~
Phosphodiesterase Sperific activitiesa Fraction
A. Liver fractions 1. Homogenate 2. Nuclei 3. Mitochondria 4. Microsomes 5. Supernatant solution B. Kidney fractions 1. Homogenate 2. Nuclei 3. Mitochondria 4. Microsomes 5. Supernatant a
b
Total unitsb
I
I1
I
3.2 2.1 4.8 18.2 1.9
0.30 0.10 0.21 0.29 0.67
820 198 180 2 40 123
70.0 8.5 7.8 3.7 44.0
7.5 3.4 4.6 38.0 2.9
0.29 0.19 0.28 0.32 0.65
1120 91 123 670 150
59.0 4.1 8.8 7.2 34.0
I1
In pmoles substrate/hr/mg prot.ein. In equal volumes of 10% homogeiiate in 0.25 M sucrose of each tissue.
and the 100,OOOx g supernatant; liberation of almost all activity from frozen tissue into the supernatant ( 9 6 ) probably excludes localization in nuclei. I n r a t brain the enzyme was found predominantly in mitochondria, followed by supernatant, microsomes, and nuclei in decreasing order of activity. Subfractionation of the mitochondria showed the enzyme in the cholinergic nerve endings; subjection of mitochondria to osmotic shock indicated association of the enzyme with the soluble synaptic neuroplasm ( 1 7 7 ) . Subsequent investigations (95a, I77a) , showed the major port,ion of the microsomal enzyme to be in the latent form, and to become un-
390
DAVID S H U G A R A N D HALINA SIERAKOWSKA
masked on addition of Triton X-100. The unmasked microsomal phosphodiesterase constituted more than one-half the total activity, the remainder being distributed almost equally between the synaptoplasm and the 100,000 x g supernatant ( 1 7 7 ~ ) . The 3': 5'-UMP phosphodiesterase of dog heart has been found largely associated with the 2,000 x g particulate fraction and, to a lesser extent, with the supernatant. The fractional activity in the 2000 X g supernatant from other tissues was higher (9’7).
VI. Histochemical Methods These are based on what has become known as the film-substrate technique, originally proposed by Daoust (178) for the localization of DNase, and subsequently RNase (179, 180). A tissue section is placed in contact with a film of gelatin containing RNA or DNA. After incubation, the film is stained with a basic dye, toluidine, to give reduced staining in those areas where enzymatic hydrolysis had occurred. I n essence, the method is an extension of one developed earlier for the histochemical localization of proteases with commercial photographic emulsions. The film-substrate method has since been successfully applied to other enzymes, e.g., hyaluronidase (181) and amylase (181, 182), which require niacromolecular substrates. The substrate film is prepared by dissolving the substrate in aqueous gelatin, spreading a few drops of this on a slide, and placing the slide on a level surface until gelation occurs. Thinner films may be obtained, with accompanying higher resolution, by allowing excess solution to drain from a vertically supported slide (179). The films are then fixed in formaldehyde to make them water-insoluble and resistant to proteases. I n the original procedure, an unfixed frozen section of the desired tissue is mounted on a glycerol-gelatin supporting pad on a second glass slide, and placed in contact with the substrate film. After incubation, the slides are separated, stained, and examined under the microscope. While useful for some purposes (see below), it is more advantageous to mount the freshly frozen sections directly on the substrate film, incubate in covered petri dishes saturated with water vapor, and flush the sections off with a stream of water; adjacent serial sections, fixed and stained, are used as controls (183, 184). Mayner and Ackerman (185) modified this further by coating the section on the film with glycerol-gelatin to prevent drying during incubation, but Daoust (184) found that this led to masking of some reactive sites. Since incubation techniques of 4-24 hours were required with this coating technique, it must clearly affect
LOCALIZATION O F N U C L E A S E S
391
enzyme activity adversely. Furthcrmore, some RNase sites identified by Mayner and Ackerman (185) have not been confirmed by others. The technique is obviously simple and readily reproduced, while the viscosity of the “incubation medium” helps to reduce enzyme diffusion. However, enzyme diffusion is of lesser significance here because of the low resolution which, despite several improvements, does not permit localization a t the intnacellular level. This could conceivably be improved by the use of thinner adhering films such as those employed in autoradiography ; and Daoust (184) has proposed that superior films might be attainable by means of Kohler’s centrifugal spreading technique. The specificity of the method is, of course, limited, since it is of necessity confined to the use of polymer substrates that will not diffuse out of the fixed film-substrate matrix (184). I n addition, since even short oligonucleotides are capable of binding basic dyes [see reference ( 1 3 5 ) , p. 4521, the resulting localization is usually that of a mixture of endo- and exonucleases. Some extension of specificity is feasible by the use of additional natural and synthetic oligo- and polynucleotides as substrates (183). The original method suffers from the disadvantage that it pcrniits of no pH control during incubation, nor of the use of activators or inhibitors. This difficulty may he partially circumvented by modification of the pH of the substrate film, or of thr glycerol-gelatin supporting pad, discussed in detail elsewhere (185).
FIG.1. RNA-gelatin film exposed t o a section of rat kidney. Activity in 1~os1111al convoluted tubules (ISO). X 30.
392
DAVID SHUGAR AND HALINA SIERAKOWSKA
FIG. 2. DNA-gelatin film exposed to a section of large inrestine of the rat. Activity in epithelial cells wit.h little activity in the lamina propria, submucosa, and the muscle layers (184). ~ 3 0 .
While substrate films are rendered resistant to proteases by formaldehyde fixation ( I & ) , it is nonetheless advisable to make use of substratefree films as controls, followed by staining with alkaline (pH 10) toluidine, which is a sensitive test for protease activity (186). Nonspecific changes in staining of substrate films occasionally require additional controls. Films made from alkaline gelatin solutions, free of substrate, stain with toluidine blue; on contact with a tissue section, the degree of staining decreases considerably with prolonged incubation (186). Some nucleases have been localized with substrate-film made alkaline to increase the solubility of the polynucleotide substrate (183) ; these obviously require appropriate controls. Ignorance of the foregoing has resulted in what is probably false positive localization of RNase (180) and other nucleases in the kidney outer medullary zone (183).
Representative examples of nuclease localization by the film-substrate technique are illustrated by Figs. 1 and 2. Additional data may be found in a review by Daoust (184).
VII. Cytochemical Procedures The general requirements for cytocheinical localization of enzymes have been reviewed in detail by several authorities in the field (187189). A few points of interest relating to nucleasc enzymes, reviewed in part elsewhere (190), are outlined below.
LOCALIZATION O F NUCLEASES
393
Substrates having the specificities discussed in Section 111 should be susceptible to the least possible number of enzymes. Specificity may occasionally be further limited by control of pH, ionic strength, and inhibitors or activators. “Substrate” specificity is not, of course, of importance in immunofluorescence techniques. Enzyme diffusion is less of a problem than in aqueous fractionation techniques because of the possibility of introducing fixation. Fixation may often effectively prevent enzyme diffusion, but it is rare indeed for a fixative to induce translocation of a n enzyme from one site to another. However, fixation may have a deleterious effect on a localized portion, or all, of the activity in a section; it is consequently desirable to examine staining intensity and localization with more than one fixation method. Absence of enzyme diffusion, absolutely essential for a fully satisfactory cytochemical technique, should be tested for by ( a ) prior incubation of sections, with no subsequent change in activity or localization on incubation with substrate medium, or ( b) incubation of a highly active section in contact with one exhibiting low activity; if there is no diffusion, each section will exhibit normal activity and localization. It is equally essential to test for nonspecific adsorption of the final precipitate, particularly a t sites of enzyme activity. This may be done by adding to the full incubation medium an exogenous source of the enzyme under study, and should result in formation of a uniform precipitate over the section area. If, however, it leads to an intensification of the reaction at sonie given site, localization of endogenous enzyme a t this site, while not disproved, is at least suspect.
A. Irnrnu nofl uorescence Techniques As early as 1954 attempts were made by Marshall (191) to localize RNase and DNase by means of this technique, which is based on the antigenic properties of the enzymes, so that they will readily precipitate fluorescein-labeled antibodies. Tissue sections were fixed in a buffered solution of formaldehyde in aqueous dioxane to avoid enzyme diffusion from sections. Both enzymes were localized in the zyniogeii granules and apical cytoplasm of bovine pancreatic acini ; there was some diffuse staining of the cytqplasm of certain acinar cells, while thc nuclei and mitochondria were negative. The validity of Marshall’s results for RNase were subsequently questioned by Ehinger (192) on the grounds that no tests had been made of the homogeneity of the antibody preparations and that this was responsible for the diffuse staining in Marshall’s preparations.
394
DAVID SHUGAR AND HALINA SIERAKOWSKA
Full details of the immunofluorescence technique in cytochemistry are given in several reviews (193-196). The technique is undoubtedly capable of high specificity, but this may be both an advantage and a hindrance. Proper purification of the antibody will lead to localization only of its antigen. Furthermore, antigenically identical enzymes exhibit narrow ranges of organ and species specificities. The antigenic specificity of antiserum to rat pancreatic and liver RNases has been examined by Gordon (197), who found the anti-pancreatic RNase serum to inhibit the activity of pancreatic and spleen RNases, and only partially those of kidney and serum, but not that of the liver. The anti-liver RNase serum was inert against the pancreatic enzyme and marginally inhibited the spleen enzyme but was highly inhibitory against liver and kidney alkaline RNases. No inhibition of acid RNases was noted. This apparently high specificity circumscribes the applicability of the method to highly purified enzymes. It is to be expected that, with further developments in purification techniques for enzymes, the high specificity of the immunofluorescence technique will make i t a highly refined research tool. It should be capable of differentiating between nucleases of different molecular structures and, possibly, of varying functional significance and should be applicable, for instance, to following the transfer of an enzyme within the secretory cells. It cannot, on the other hand, portray the overall enzyme activity in a cell; but this is a small price to pay for the high specificity attainable, the more so in that this gap can be filled with the use of less refined techniques of more general applicability (114). Fluorescent anti-liver RNase serum was used by Gordon and Myers (198) to localize RNase in the liver and kidney of the rat. The enzyme was found in the cytoplasm of liver parenchymal cells, with nuclei negative, and in the cytoplasm of proximal convoluted tubules of the kidney. No activity was noted in other normal tissues, nor in four liver hepatomas and one cholangioma. Intracellular details were rather poorly defined; this could have arisen from the use of ethanol fixation, which is known to be accompanied, during incubation, by enzyme diffusion (190). The same technique was applied by Ehinger (192) to localize RNase in Carnoy-fixed frozen sections of rat and ox pancreas. Two types of localization were reported, but not both in the same section: ( a ) positive nucleoli and basal ergastoplasmic zone of acinar cells (Fig. 3) with nuclei and remaining cytoplasm negative; (b) positive apical zymogenic areas, together with nucleoli, and the remaining cytoplasm negative. Islets of Langerhans were negative. This study included rigorous tests
LOCALIZATION O F NUCLEASES
395
for specificity. Oddly enough, however, a prior incubation of sections in buffer solutions led to subsequent decreased staining with antibody. The author suggested that this may have arisen from enzyme diffusion, w11ic.h normaIIy is prevented by blocking and fixation of the enzyme by anti-
FIG 3 Immunofluoresccnce localization of RNase in rat pancreas Actikity in I.)asal ergastoplasmic zone of acinar ccllls and tlmr nucleoli, w ~ t hnuclei and reinamIng cytoplasm ncgative (192). ~ 2 2 5
body. However, this cannot bc tlie full explanation for, with rapid enzyme diffusion, fixation by antibody should have been equally rapid ; since this should have led to staining, I t IS curious that 3 hours of incubation were required. I n attempting to explain the predominantly basal ergastoplasmic localization, as contrasted with the zymogen granule localization principally found by previous obstwers, it was proposed by Ehinger (192) that this might be due to looser binding of the zymogen enzyme after Carnoy fixation. If so, this should have been shown by other methods. Such discrepancies between immunofluorescence and other cytochemical
396
DAVID SHUGAR AND HALINA SIERAKOWSKA
methods are not limited to nuclease enzymes; the localization of aamylase in the basal portion of acinar cells by immunofluorescence (199) is in disagreement with results obtained by other procedures (182). Pilocarpine stimulation was found to lead to a pronounced decrease in RNase content of rat pancreas, but only to a given plateau level beyond which higher doses were ineffective. The inference was drawn that there must be two types of binding of the enzyme, sedentary and “exportable.” This was correlated with the finding of a dual localization, shown by histoiminunofluorescence and changes of localization during the diurnal cycle ( 2 0 0 ) . Finally, attention should be drawn to Ehinger’s (192) observation that formalin fixation of pancreas abolishes the antigenic activity of the tissue enzyme. This was not so for the crystalline enzyme, the antigenic activity of which was only partially modified by formalin treatment, nor did i t appear to be the case with the formalin-based fixative employed by Marshall (191), referred to above. I n any event the discrepancies between these results and those of Marshall (191) and ZanKowalczewska et al. (114) will require further study.
B. Precipitate-Forming Techniques 1. ALKALINE RIBONUCLEASES
Probably the first attempt a t localization of nucleases by precipitateforming techniques was that of Zugury e t al. (201), based on the observation that lead salts of RNA and oligonucleotides are more soluble than the corresponding mononucleotide salts. Formol-saline fixed sections were incubated in a medium containing RNA and lead nitrate (for RNase) , or oligonucleotides and lead nitrate (for phosphodiesterase) , the resulting enzymatically liberated mononucleotides being precipitated as the lead salts. Appropriate controls were used to assess possible interference from endogenous phosphomonoesterases. Localization patterns were identical for both substrates, mainly in cells of the Islets of Langerhans and a narrow band in the apical region of the secretory acini. Apart from the lack of specificity of this method, it was subsequently shown (183) that mononucleotide lead salts are far from insoluble; a t the lead concentrations used, precipitate formation a t pH 8 requires a mononucleotide concentration of about 2 mM, and even more a t lower pH. Concurrently with the above, RNase localization was attempted with the use of what a t that time was considered a specific substrate, a pyrimidine nucleoside 2’: 3’-cyclic phosphate (190). The specificity of this has since been shown to be only relative by the discovery of 2’:3J-
397
LOCALIZATION O F NUCLEASES
nucleotide phosphodiesterase (Section 11, C) . The scheme devised is essentially a two-step enzymatic reaction, the second step of which is based on the Gomori techniquc for alkaline phosphatase, as shown in Scheme 3. 4
PY tissue RNase
*P:O
HO
i I
OH
HO
OH
0
0
exogenous alkaline phosphatase
i
HO-P-OH II 0
Po:-
Calcium phosphate
SCHEME 3
The incubation medium coiitained 2’:3’-UMP, purified alkaline phosphatase and a soluble calcium salt. Hydrolysis of the cyclic phosphate a t RNasc sites liberated uridine 3’-phosphate, which was dephosphorylated by the exogenous phosphatase ; the liberated phosphate ions were trapped by calcium to form a calcium phosphate precipitate, which was then revealed by the usual Gomori procedure. A rather serious disadvantage of the foregoing is the slow rate of hydrolysis of cyclic phosphates by RNases [Section I1 and reference ( 2 0 6 ) ] ,so that unduly long incubation times are required. Equally disadvantageous was the use of ethanol- and acetone-fixed sections, from which RNase diffuses rapidly [cf. reference (203)1, leading to artifactual nuclear and nucleolar localization. Enzyme diffusion probably could have been reduced to permissible limits by the use of formalin fixation (see below), but this was not attempted a t the time because of the claim (204, 205) that RNase in pancreatic sections was totally inactivated by formalin. Further work on this method was subsequently abandoned when nucleoside 3/-naphthyl phosphates were found to be more suitable substrates (see below) , but the above procedure undoubtedly warrants reexamination with the use of formalin and other methods of fixation, and reference should be made to the original paper (190) for a dctailed discussion of the problems involved in nuclease localization.
398
DAVID SHUGAR AND HALINA SIERAKOWSKA
A technique similar to the above utilizes RNA as substrate (206). Blood smears fixed in formalin vapor were incubated in a medium containing RNA, acid phosphatase, and lead nitrate. Activity was observed in all neutrophilic cells, mainly in the perinuclear region, and in lymphocytic cells, while controls without substrate and with addition of sodium fluoride were negative. However, no suitable controls were run for endogenous phosphatase, or for tissue affinity for the final lead phosphate precipitate. This procedure was subsequently applied to a study of the changes in RNase activity in leukocytes of rabies-susceptible and refractory mice; rather surprisingly, no RNase could be detected in the latter (207). Much more promising, and widespread in scope, was the development of the nucleoside naphthyl phosphates: a-naphthyl uridine 3‘phosphate (see Section I V and ref. 114) is completely hydrolyzed by RNase t o 2’:3’-UMP and free naphthol (see Scheme 4) ; its relative specificity for RNase is enhanced by the resistance of a-naphthyl nucleoside 3’-phosphates to spleen phosphodiesterase (122), which hydrolyzes other esters of nucleoside 3’-phosphates (91). The liberation of 0
y$
HOCH,
0
H
0 OH I O=P-OH I
RNase
+
-
4 o, Ofl.-OH
+Naphthol
0
Insoluble red dye
5-Chloro-atoluidine
SCHEME 4
LOCALIZATION O F NUCLEASES
399
free naphthol accompanying hydrolysis by RNase logically suggested a one-step procedure for RNaee, based on coupling of the enzymatically liberated naphthol with a suitatilc djazotatc according to standard ~ Z O dye coupling techniques, as sliown i n Scheme 4. The utility of this substrate was further indicated by the fact that it is hydrolyzed by RNase a t w rate about 100-fold greater than that for 2’: 3’-UMP. The rapid rate of hydrolysis of a-naphthyl uridine 3’phosphate was further confirmed in collaboration with Witzel (unpublished rcsults), who found that I-,,,,, for this substrate was 80 times that for the corresponding benzyl ester, with the K , for the naphthyl derivative higher than that for 2’: 3’-UMP and uridine 3’-benzyl phosphate. Such a high rate of hydrolysis of the naphthyl derivative is probably caused by the high leaving tendency of the naphthyl group, which is reflected by the slow spontaneous hydrolysis of uridine 3’-naphthyl phosphate in aqueous medium. This slow rate of decomposition, leading to formation of free naphthol, would normally prove rather awkward in cytochemical work, but it is more than offset in this case by the rapid rate of enzymatic hydrolysis, making possible very short incubation periods. The slight, diffuse, nonspecific precipitate from spontaneous hydrolysis is readily removed during the normal rinsing procedure that follows incubation. The availability of this substrate led to a reexamination of the problem of enzyme diffusion, with the finding that cold formol-calcium fixation yields tissue sections with high RNase activity and m signs of enzyme diffusion (114). Previous reports (203-205) on the deleterious effects of formalin fixation a t 24” were confirmed, but fixation in Baker’s fonnol-calcium a t 4” led to little loss in activity, with either uridine 3’-naphthylphosphate or 2’: 3’-UMP as substrates. Activity against RNA was affected to a variable degree, depending on the effectiveness of foriiialin removal prior to incubation ; decrease in activity against RNA apparently resulted from interaction of residual formalin with RNA, rendering the latter more resistant to RNase. Cytochemical reactions were conducted at pH 9 to ensure a rapid coupling reaction and minimum diffusion of the reaction product. Frozen sections fixed in formol-calcium were routinely employed, with incubation times of 2-30 minutes. Extensive tests, including assays of incubation media for enzyme activity, established the absence of enzyme diffusion. The type of localization obtained is illustrated in Figs. 4 and 5. It is perhaps worth mentioning that with the incubation times employed no nuclear or nucleolar localization was ever observed (114). By contrast, and in agreement with previous observations (190), preparations of tissues fixed in acetone, alcohol, or Carnoy reagent ex-
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DAVID S H U G A R A N D H A L I N A SIERAROWSKA
hibited rapid and appreciable diffusion of enzyme into the incubation medium, which could be revealed by a variety of techniques. Such sections exhibited desmoactivity, which persisted following preincubation in water, and varied in different tissues from nil in the pancreas to a sizable percentage in the kidney. I n all cases investigated, activity of formalin-fixed material was far greater than the desmoactivity in alcohol or acetone fixed material, with the sole exception of the
FIG.4. Alkaline RNaae in rat pancreas by azo-dyc coupling technique. Intense activity in the suprannclear portion of acinar cells with lower activity in the remaining cytoplasmic area. Intense activity in the lumen of excrrtory ducts and slight activity in the epithelium of the excretory ducts adjoining the lumen (114). x 375.
vascular connective tissue enzyme. It has not been established whether the tissue-bound desmoactivity (located, for instance, in the kidney tubules) comes from pancreatic RNase differently bound to some tissue component(s) or, perhaps more likely, is due to a different RNase. It should be recalled that RNases differing immunologically from the pancreatic have indeed been detected in several organs, including the kidney (197).It would be desirable to determine whether the ratio of pancreatic to other alkaline RNases in a given organ can be correlated with the ratio of lyo- to desrnocomponent following acetone fixation. Should this prove
LOCALIZATION O F NUCLEASES
40 1
FIG.5. Alkaline RKase in rat kidney by aso-dye coupling technique. Intense activity in the brush border zone of thr proximal convoluted tubules (11 4 ) . X 1OOO.
to be so, it would imply a different functional significance for the two enzymes. This problem could Irofitably be tackled with the aid of iiiiniunohistochemical techniques. 2. DEOXTRIBONUCLEASES
Localization of DNase was first reported by Marshall (191), who used the histoinirnunofluorescence technique t o localize DNase I in the pancreas. The enzyme was found to occur mainly in the apical portion of acinar cell cytoplasm. No further work along these lines has since been done, possibly because of the antigenic nonhomogeneity of the enzyme. Precipitate-forming techniques have formed the basis of other methods, hit these h a w been somewhat limited in scope because of the absence of known simple substrates for these enzymes (see Section 11, A, 2 ) . A method for DNase I1 has been described (208) using D N A as substrate, with exogenous phosphatase in the incubation medium to liberate terminal phosphate groups exposed by tissue DNase. The libcrated phosphate was trappecl with lead ions and visualized by the Gomori technique. Substrate specificity is similar to that in other biochemical methods for DNasc I1 with DNA as substrate and elimination of othcr activities by pH control and a magnesium-free medium.
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DAVID SHUGAR AND HALINA SIEBAKOWSKA
DNase I1 activity, in frozen sections fixed in acetone-formalin-water and incubated for several hours, was reported in the nuclei and cytoplasm of splenic red pulp, in Kupffer cells, in nuclei of liver parenchymal cells, and in nuclei of the epithelium of kidney tubules and duodenum. These results must, however, be accepted with caution because of the following considerations. DNase localization was observed only in cells containing acid phosphatase. Furthermore, when exogenous phosphatase was omitted from the incubation medium, the same localization, but with decreased intensity, was observed ; while the exogenous conimercial phosphatase employed almost certainly contained phosphodiesterase activity. The long incubation periods required may have been accompanied by enzyme diffusion into the incubation medium [ cf. reference (190) 1. Finally, the nuclear localization observed was likely due to affinity of the nuclei for the lead phosphate precipitate released by the diffusing enzyme [cf. reference (189), p. 433, and reference (209)1. I n a subsequent application of this technique to tissues of irradiated animals (210) the fixation procedure was altered to include neutral formol-saline fixation of tissue blocks, washing in acetate buffer, dehydration with acetone, and paraffin embedding. This procedure was again modified by Vorbrodt ( a l l ) , using cold formol-calcium fixation, and a lower concentration of DNA and Pb2+in the incubation medium. The resulting localization was found to be pH dependent. It was mainly nuclear a t about pH 5, and largely in cytoplasmic granules a t about pH 6. The activity of cytoplasmic granules was inhibited by sulfate ions, which are known to inhibit DNase 11. It was also noted by Vorbrodt (211) that substitution of a commercial low molecular weight DNA preparation for the high molecular weight substrate resulted in an increase in activity. This raises some doubts as to the nature of the enzyme localized. Highly polymerized, native, DNA is subject to hydrolysis primarily by endonucleolytic DNases, but a degraded preparation is more susceptible to exonucleases. The Vorbrodt modification was subsequently applied by Coimbra and Tavares (212) to localization of DNase 11, as well as acid RNase (with an RNA substrate) in rabbit nerve tissue, with essentially similar pH-dependent results. Samorajski et al. (213) applied the foregoing to light and electron microscope localization of DNase I1 in dorsal root ganglia and the spinal cord of aged aniiiials, using 4-hOur incubation pcriods. The presence of DNase 11, acid phosphatase, and eathepsin-type-C estcrase reaction products in lysosomes and lipofuscin pigment masses was regarded as indicative of the role of lysosomes in formation of lipofuscin pigments.
LOCALIZATION OF NUCLEASES
403
Nuclear DNase I1 activity was also reported in neurons. It is clear that this entire procedure requires critical reexamination. 3. PHOSPHODIESTERASES Phosphodiesterase I . Apart from the trials carried out by Zugury et al. (20f),referred to above, cytochemical procedures for phosphodiesterase have been proposed and applied with success (82, 122). One enzyme localized was phosphodicstcrase I, using as substrate the synthetic a-naphthyl thymidine 5’-phospliate (Section IV) . The enzymatically liberated naphthol is couplcd with a diazotate to form an insoluble colored precipitate, as described above for RNase (see Scheme 5 ) . The substrate is relatively specific for phosphodicsterase I and is hydrolyzed by this enzyme a t tlic same rate as the corresponding p-nitrophenyl ester (2f4), i.e., about 5 tiriics as rapidly as the natural substrate, a 5’-phosphoryldinuclcotide. This high rate of hydrolysis made possible relatively short incubation periods. Cold formol-calcium fixation of tissue sections reduced enzyme activity t o about 20%, with no subsequent diffusion, as estimated from both cytochemical and paper chromatography tmts. Paraffinembedded, acetone- or ethanol-fixed sections retained only 5% of the original activity, but with unchanged localization patterns. Enzyme activity was optimal a t about pH 9, but some activity persisted even at pH 5.2. Phosphodiesterase I activity obtained with formol-fixed sections is illustrated in Figs. 6 and 7. A consistent lack of nuclear and nucleolar activity is apparent in all the tissues examined. Attempts to apply the above procedure to localization of phosphodiesterase I1 with a-naphthyl thymidine 3’-phosphate (Section IV) , an analog of p-nitrophenyl thymidine 3’-phosphate ( 8 3 ) , were abandoned when the former was found to be totally resistant t o this enzyme ( 1 2 2 ) . 3’:5‘-Sucleotide phosphodiesterase. In an extension of the method described above for the localization of RNase by means of ribonucleoside 2’: 3’-cyclic phosphates, Shanta et al. (215) developed a two-step reaction for localization of 3’: 5’-nucleotide phosphodiesterase. In addition to the substrate, the incubation medium contained snake venom as a source of 5’-nucleotidase, and Pb2+ions to precipitate the liberated phosphate by the Gomori procedure as shown in Scheme 6. Appropriate controls appear to satisfy the normally accepted criteria for cytochemical localization (Figs. 8 and 9 ) . Omission of snake venom (i.e., 5’-nucleotidase) was accompanied by some activity, in rabbit liver, obviously from endogenous phosphatase, but with different localization, mainly in nuclei arid nucleoli. I n the presence of snake venom, no
404
d
i-
bZ
0
I
o=Pl-0
I x I
0
I
DAVID SHUGAR AND HALINA SIEXAKOWSK.4
sA38
\
V d
I
i x o=b-o
405
LOCALIZATION OF NUCLEASES
FIG.6. Phosphodiesterase I in rat liver by azo-dye coupling techniqae. Intensc activity in proximity of bile canaliculi. Cytoplasm of hepatic cells weakly artivc with somewhat grcater activity a t thc lumen of sinusoids; activity in capillaries ( 1 2 2 ) . x300
-
0
~ 0 -IIp - 0 - c ~ ~0
tissue
nucleotide-
I
OH
PDase
%$ HO
OH
O=P-O I HO
+.PI
p0,S-
+
5’-nucleotidase exogenous L-
lead acetate
+
Lead phosphate
SCHEME 6
HOC@*
HO
OH
OH
406
DAVID SHUGAR AND HALINA SIERAKOWSKA
FIG.7. Phosphodiesterase I in rat tongue hy azo-dye coupling technique. Iiitenec activity in t,he cytoplasm of mast rells, in the intima of small blood vesels, and in capillaries, with less activity in the perineurium (122). x 100.
FIG.8. htlcnosinc 3': 5'-~yelic phosphatc d k t e r a s e in rabbit liver by technique of Shanta ef nl. (215). Moderately positive activity in the cytoplasm of parenchymal cells with nuclei and nucleoli negative. x260.
LOCALIZATION O F NUCLEASES
407
FIG.9. Control to Fig. 8 incubatcti in medium containing no snake venom. Thc localization corresponds to endogenous p1ioPy)honionoesterase and sites of high affinity for lead phosphate precipitiite. Not? the darkly stained nuclei and nucleoli (arrows) ~ 4 1 6 .
nuclear or nucleolar localization was observed, suggesting (a) that in the absence of snake venom, dcl)liosl,liorylation of AMP came from endogenous phosphatase, which diffused into the incubation medium, as occasionally happens, and that tlic rcsulting lead phosphate, liberated in the incubation medium, was adsorbed by the nuclei and nucleoli in accordance with their known affinity for these structurcs (189, 209) ; and (b) that in thc presence of snake venom, the resulting 5’-nucleotidase activity was high enough to override the effect of diffused endogenous phosphatase. Supplementary tests indicated no diffusion of cyclic nucleotidc phosphodicstcrase from the unfixed tissue sections employed in this study. Snake venom is known to hydrolyze slowly nucleoside 3’ :5’-cyclic phosphates; e.g., 3’: 5’-AMP is hydrolyzed by high concentrations of Crotalus adamanteus venom to give about 50% 3’-ARIIP and 50% adenosine, the latter resulting from dephosphorylation of 5’-AMP by the venom 5’-nucleotidase (216). This apparently did not interfere in the investigation discussed above since inactivated control sections were completely blank. Nonetheless, the replacement of snake venoiii by purified 5’-nucleotidase or highly purified nonspecific monophosphoesterase would be a desirable improvement.
C. Comparison of Cytochemical and Fractionation Findings A comparison of cytocheniical results with those of cell fractionation techniques is not a simple matter. Cytochemical procedures at present
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DAVID SHUGAR AND HALIN.4 SIERAKOWSKA
differentiate bctween specific regions within the cytoplasm or the nucleus, with the resolution of thc light microscope. Cell fractionation techniques locate enzymes a t an intraeellular level, i.e., in mitochondria, microsomes, lysosomes, ribosomes, more recently membranes, etc. With the current use of density gradient centrifugation and the perspectives opened up by zonal centrifugation ( 2 1 7 ) , the resolution begins to approach that of the electron microscope. The present availability of several suitable cytocheniical substrates emphasizes the need for application of electron microscopy to the cytochemical localization of nucleolytic enzymes. Efforts are already being made in the preparation and screening of coupling agents yielding electron-dense precipitates, by incorporation into diazotates of heavy metals (218) or osmiophilic radicals that, upon reaction with osmium, become electron dense (219). Either of these would be useful with the existing naphthyl derivatives. There is also the possibility of introducing electron-dense moieties into the precipitable component of a substrate. Alternatively, labeled naphthyl, combined with autoradiography, might serve for quantitative electron microscope localization. However, until these possibilities become realities, there are several lines of approach for improvement of existing methods. One of these is the search for additional specific substrates, as well as increasing the specificities of existing substrates with the aid of natural and synthetic inhibitors. Localization of natural inhibitors, which has hitherto received little attention, should be of value in elucidating their physiological functions (and that of the enzyme that each specifically inhibits). It should be perfectly feasible to localize such inhibitors by immunofluorescence techniques or, perhaps more simply, by fluorescein labeling of the appropriate enzyme. Quantitative evaluation of enzymatic activity is feasible with the use of appropriately labeled substrates or trapping reagents, in conjunction with autoradiography ( 2 2 0 ) . There remains also considerable room for improvements in trapping reagents, along the lines so extensively exploited, e.g., by Holt (221) for localization of esterases with the use of variously substituted indoxyl radicals.
VIII. Nucleolytic Enzymes in Pathological States Possible changes in levels of nucleolytic activities in tissues and body fluids in a variety of pathological states have been intensively investigated. There are, however, few, if any, instances where the results are sufficiently clear-cut t o permit definite conclusions. Some typical findings have been summarized ( 4 , 2 2 2 ) .
LOCALIZATION OF NUCLEASES
409
The major difficulty in interpretetion stems from the fact that the variations encountered usually lie within the range of activities exhibited by the subjects in their noriuztl physiological state. It is possible that closer attention to the problem of cnzyiiie specificity in such studies may prove more profitable. Similar extensive investigations have been devoted to the influence of partial or whole-body X-irradiation on nuclease activities in diverse organs, occasionally in conjunction with estimations of other enzymes. The overall findings are too complex to sumniarize here and reference should be made to several reviews (223-225). The influence of drugs on nuclease activities has likewise been examined. For example, corticostcroids are known to provoke involution of lymphoid tissue, and this is associated with an increase in nuclease activities. Wiernick and MacLeod (266) noted significant increases in thymus DNase I1 and alkaline and acid RNases following injection of 9-a-fluoroprednisolone. The effect of the steroid on alkaline RNase disappeared aftcr removal of alkaline RNase inhibitor, implying that the initial action is on the inhibitor. This is, however, to be contrasted with the observation that the increase of adrenal gland RNA resulting from ACTH treatment can be attributed at least in part to elevated levels of RNase inhibitor ( 6 2 7 ) . We return to this subject below. It is worth adding, in this context, that streptococcal DNase (streptodornase) and, to a lesser extent, pancreatic DNase I (because of its commercial availability) have themselves found applications in clinical practice, vie. via local infusion or intravenous injection for the purpose of liquefying insoluble DNA-protein in purulent exudates in local infections. These applications have bcen reviewed in detail by Tillett (628). On theoretical grounds, oiie might rlxpect that a study of nuclease activities following virus infection would contribute more to an understanding of the possible regulatory role of these enzymes. The finding that, as with bacteriophages, virus infection is associated with the appearance of induced polymerascs has recently led to a search for other induced enzymes involved in the metabolism of nucleic acids. An alkaline DNase active against denatured DNA in HeLa cells has been reported to double in activity following vaccinia virus infection (629), the induced character of the increased activity being inferred from the arrest of the latter on addition of puromycin or infection with ultravioletirradiated virus. Induced DNase activity has also been claimed in cultures of baby hamster kidney cells infected with herpes simplex virus (250) ; the criteria for the induced character of the enzyme activity were based on heat stability, behavior toward native and denatured DNA, and response t o cations. Somewhat more convincing than the above is the
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DAVID SHUGAR AND HALINA SIERAKOWSKA
demonstration that pox virus infection of HeLa cells is accompanied by the appearance of several new enzymes active against DNA (231, 239). Since thcse were not detected in the prcsence of puromycin or fluorodeoxyuridine, their induced character, as coritrastcd to activation of latent activity, was presumed. The induced enzymes, two of which are exonucleases, differ from the corresponding enzymes in the normal cell either in type of attack or preference for native or denatured DNA. It is clear that further evidence for the induced nature of these enzymes, which should be directed more to such factors as specificity and type of attack, is highly desirable from the point of view of establishing their role in the infective process. Nucleases in Tumors This is a field that has attracted widespread attention. The results of earlier studies have been summarized in several reviews (9, 233, 234), from which it appears that there is a general tendency toward a lower activity of these enzymes, as compared to those in normal cells, for such tumors as ascites, hepatoma, and carcinoma of human cervix. However these findings are by no means uniform, one of the difficulties in comparing results of different observers being the lack of any uniform baseline for quantitative measurements of activity. Histochemical (film-substrate) methods have been applied in this field by Daoust et al. (235,256), who showed that azo-dye carcinogenesis in the rat leads to a progressive decrease in liver RNase before the parenchymal cells become cancerous; by contrast the loss in DNase activity is abrupt and appears to be closely associated with the neoplastic transformation of parenchymal cells (Figs. 10 and 11).A further study on 32 different types of 65 experimental and human tumors corroborated the earlier results for neoplastic cells, whereas the connective tissue stroma and necrotic regions exhibited high levels of activity (237), in accord with the generally observed increase in nuclease activity during tissue regression and necrosis. The foregoing results are somewhat a t variance with those of Roth e t al. (238) on a series of transplantable rat hepatomas and normal rat liver, using fractionation techniques. The original paper should be consulted for full details, but it is worth noting here that the hepatomas exhibited a doubling of the microsomal alkaline RNase activity and an appreciable increase in supernatant acid RNase. The discrepancy betweeen the results of Daoust et al. (235-237) and Roth et al. (238) may be more apparent than real, in view of the difference in techniques. Daoust e t al., using the film-substrate method, with an unbuffered gelatin supporting pad for the sections, had no control
LOCALIZATION OF NUCLEASES
41 1
over the pH, and hence detected probably a mixture of different RNases. Thc state of the enzyme in a tumor cell might also be such that its activity in a section is masked. It is indeed curious that this should be so for both RNase and DNase, but the findings of Daoust et al. (635-237) cannot be readily dismissed.
FIG.10. RNA-gelatin film exposed to a section of cirrhotic liver. Note t,he high ItNase activity in trabeculae of bile ducts and connective tissue. The nodules of pwenchymal cells are relatively i n d i v e (184). X 30. FIG. 11. R N A - g e l a h film eqiosed to a section of primary hepatoma. Note RNase activity in bands of connective tissue and the relatively inactive neoplastic cclls (T)(18.4). x30.
Phosphodiesterase I localization in unfixed frozen sections of human breast carcinomas and fibroadenomas has been studied by Michalowski and Jasinska (unpublished results), using a-naphthyl 5’-TMP. No activity was observed in the cancer cells, apart from occasional traces of activity in the necrotic regions. There was, on the other hand, intense activity in bands of connective tissue immediately surrounding, as if cncapsulating, the nodules of neoplastic tissue. The enzyme appeared t o be localized extracellularly, but did not show up uniformly in all connective tissue adjacent to caiicer nodules. Connective tissue bands further removed from neoplastic nodules exhibited little activity (Fig. 12) . The epithelial cells in fibroadenomas behaved erratically, some being negative, other exhibiting very intense activity in some of the cells. Vorbrodt (239) examined the cytochcmical localization of DNase I1 in various animal and human tumors and found the lysosomal activity in neoplastic cells diminished by comparison with that in normal cells.
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DAVID SHUGAR AND HALINA SIERAKOWSKA
Daoust (236) reported low activity against DNA films in neoplastic nodules with high activity in necrotic regions. Linked to the foregoing was the observation of Roth et al. (238) of a decrease in alkaline RNase inhibitor in rat hepatomas. More important still is the finding that mouse ascites tumor cells contain a potent RNase inhibitor most active a t neutral pH, leading to apparent pH optima of enzyme activity at acid and alkaline p H values, where it dissociates
FIQ.12. Phosphodiesterase I in human breast carcinoma by azo-dye coupling technique. Note the ncgativc cancer cells and the intense activity in bands of connective tissue adjacent to cancer nodules. x 100. (A. Michalowski and J. Jasinska, unpublished.)
from the enzyme (240). Of no less significance was the demonstration by Gordon (197) that RNases extracted from 5 tumors derived from liver are not inhibited by the anti-liver RNase serum. Since fluorescent antibodies t o rat liver RNase that stain normal liver intensely did not stain the liver tumors, the tumor RNases must have been derived from cells of other than parenchymal origin; this indirectly supports the results of the histochemical studies referred to above. Because of the critical role of the nucleic acids in transmission of genetic information and in protein (and enzyme) synthesis, and the
LOCALIZATION OF NUCLEASES
413
value of the enzymatic approach in studies on the biochemistry of cancer ( 2 4 1 ) , it is t o be expected that further investigations on nucleolytic enzymes in carcinogenesis will be actively pursued. Undoubtedly there is an important requirement for more standardized quantitative methods. But i t may be profitable to place increased emphasis on the specificities of the enzymes involved. It would be of interest to know, e.g., whether leukemic cell phosphodiesterase (Section 11) is a specific tumor enzyme. The problems involved in such studies have been clearly set forth by Bergel (233) and Potter (241),among others. Various attempts have been made to control tumor growth by direct hydrolysis of nucleic acids, e.g., hy daily injections of high doses of RNase, apparently causing a transient regression of solid tumors in mice (24.2).Similar transient regressions provoked by RNase and DNase have been reviewed by Bergel (233), but there is no real evidence to support the claims that hydrolysis of tumor nucleic acids is really achieved. Both RNase and DNase have been shown t o be capable of penetrating membranes of mammalian cells (243, 244), with widely varying effects. I n any event, the foregoing has in part stimulated studies on intracellular levels of nucleolytic activity accompanying tumor regression induced by various agents, and led to the discovery of elevated RNase activity associated in some cases with regression of tumor growth. For example, regression of lymphosarcoma under corticosteroid treatment is accompaiiicd by a twofold increase in cellular acid RNase activity; this increased activity was shown to arise from tumor cells and not from the macrophages associated with them (245). Treatment of lymphosarcoma with 9-a-fluoroprednisolone also leads t o increases in acid RNase activity, but this occurs prior to tumor regression, with no modification of the intracellular distribution of the enzyme (246). It should be recalled, in this connection, that fluoroprednisolone similarly affects the nuclease activities of normal lymphoid tissues (see above). I n tumor strains resistant to steroid treatment but sensitive to 5-fluorouracil, regression caused by the latter is not accompanied by an increase in RNase activity. Mashburn and Wriston (247) have similarly reported on changes in RNase activity preceding regression of lymphosarcoma in strains sensitive to a-asparaginase. Treatment with N,N'-diethylene-N'-phencthylphosphoramide of Walker 256 rat tumors reduces the rate of tumor growth, and this is accompanied by a reduction in intracellular RNA and an increase in alkaline RNase (248). A variety of cytostatic agents that limit the proliferation of ascites tumor cells provoke a correlated increase in free and latent alkaline RNase activity (249). These observations, together with additional findings (226), are interpreted as indicating that some agents, provoking regression, inactivate
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DAVID SHUGAR AND HALINA SIERAKOWSKA
tissue inhibitors, leading to increased nucleasc activity. I n the case of acid RNases this may be the result of rupture of lysosomal membranes, the liberated acid RNase then participating in autolytic processes. It nonetheless remains to be established whether nuclease activation provokes regression, or whether it is one of the side effects accompanying such regression.
IX. Possible Functions of Nucleolytic Enzymes It is generally agrecd that, in mammalian organisms, nucleases fulfill a variety of functions. Some of them are known to be involved in the digestive processes, e.g., the intracellular digestion of food in the alimentary tract by nucleases of the pancreatic juice and saliva, which is reasonably well understood. The localization of RNases, DNases and phosphodiesterases in the serous cells of digestive glands (114, 122, 184, 191, 192) is in accord with this. Intracellular digestive functions are carried out by acid nucleases localized, along with other acid hydrolases, in the lysosomes ( 1 4 4 ) . Under normal conditions, the lysosomal enzymes hydrolyze intravessicularly materials rejected by, or foreign to, the cells. It is believed that thc compartmentalization of these enzymes by the lysosomal membrane serves to regulate their activity, thus rendering them innocuous toward cellular nucleic acids. I n agreement with this is the absence of any known natural specific inhibitors of acid RNases and the lack of activity toward these enzymes of alkaline RNase inhibitors. The structure-linked latency of acid nucleases is modified as a result of cellular injury or tissue regression. Rupture of lysosomes is accompanied by the appearance of elevated levels of acid nucleases in the supernatant. These nucleases are generally presumed to be irivolvcd in autolytic processes. The occurrence of RNase (250-253) and DNase (251-253) activities on the surface of mammalian skin in somewhat baffling from the point of view of possible function. Studies on interaction with viral RNA have also shown that HeLa (254) and normal mammalian (255) cells exhibit surface-associated RNase activity, the significance of which remains to be clarified (see below). It is also generally accepted that nucleases participate in some manner in the intracellular metabolism of genetically and informationally active nucleic acids. This subject has received a good deal of attention in recent years, particularly with regard to enzymes active against RNA. Studies in this field have been confined mainly to bacteria; but, from the general similarities in basic metabolic pathways, and the results of some
LOCALIZATION OF NUCLEASES
415
studies on higher organisms, it may be inferred that the results obtained with microorganisms are also applicable to higher organisms. A tentative hypothesis for the intracellular role of ribonucleases was advanced by Elson (256, 257) following the discovery of latent ribosomal nuclease activity in E . coli. The enzyme initially released 2’:3’phosphate-terminated oligonucleotides and then hydrolyzed the cyclic phosphate rings with a preference toward cytidine and adenosine linkages ( 2 5 8 ) . Elson postulated that the latent ribosomal enzyme, which constitutes the total RNase activity of the cells, might be involved in removal of mRNA from the ribosomal surfaces. This hypothesis received a major set-back following the discovery by Neu and Heppel (105, 259, 260) that latent ribosomal RNase could be released from E . coli cells without destruction of their viability or the integrity of their ribosomal population. Formation of E . coli spheroplasts Icd to the re!ease of a large fraction of the RNase activity, with the simultaneous disappearance of a corresponding amount of latent RNase (with nearly identical properties) from the ribosomes. It was further shown that free E . coli RNase (or pancreatic RNase) was readily adsorbed and “masked” by ribosomes, but was released on breakdown of the latter. E. coli ribosomes were able to bind a 26-fold excess of the RNase activity normally associated with them upon isolation (261). RNase activity similar to that released during formation of spheroplasts was recently found in the debris of E . coli ( 2 6 2 ) . This has raised serious doubts as to whether LL1atent’’RNase is localized on the ribosomes, rather than in the vicinity of the cell wall along with other degradative enzymes, being adsorbed and masked by the ribosomes during disruption of the cell structure. I n conjunction with supernatant inhibitors, this would account for the stability of polysomal structures in the intact cell (263, 264). Reservations have also been expressed as to whether the RNase of E . coli is responsible for inactivation or degradation of mRNA. Hydrolysis of the latter by ribosome-associated enzymes leads to formation of nucleoside 5’-mono- and -pyrophosphates; no fragments with terminal 3’-phosphate groups are found among the products (107, 265). Formation of 5’phosphates is caused by a K+-activated phosphodiesterase (265, 266) ; 5’-pyrophosphate products, and the activating effect of phosphate ions, are indicative of the action of polynucleotide phosphorylase (265, 267, 268). Only when the Mg” conrtntration is lowered, leading to ribosome dissociation, do nucleosi(1e 3’-phosphatcs appear, because of activated RNasc (269). Furthermore, E . coli spheroplasts, devoid of RNase, have bccn shown readily to degrade rapidly labeled RNA ( 2 7 0 ) . Escherichia coli RNase-
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DAVID SHUGAR AND HALINA SIERAKOWSKA
deficient mutants, with less than 1% of the RNase activity of the wild type, produce rapidly labeled RNA with a half-life identical with that of tlic wild type. Extracts from these cells support mRNA- or poly Ustimulated in vitro protein synthesis as effectively as the RNase-positive strain ( 2 6 1 ) . E . coli mutants deficient in RNase I and in both RNasc I and polynucleotide phosphorylase were shown to inactivate mRNA of induced P-galactosidase a t a rate equal to that for the wild strain (270a). Degradation of mRNA in RNase-deficient mutants of E . coli yields nucleoside 5’-mono- and -pyrophosphates ( 2 7 1 ) .Alcaligenes faecalis with no detectable RNase activity degrades both mRNA and rRNA via polynucleotide phosphorylase and K-activated phosphodiestcrasc (272).Singer and Tolbert (273) found no change in the level of 5‘-phosphate-forming, K+-activated RNase in strains dcficient in latent RNase ; in particular, they established that the enzyme, partially bound to ribosomes, is specific for polyribonucleotides in the random coil configuration. It consequently appears that the in vivo inactivation and degradation of mRNA is not due to formation of products with terminal 3‘-phosphatcs by RNase action, but is regulated in lower organisms by phosphodiesterases forming nucleoside 5’-phosphates and by polynucleotide phosphorylase, albeit the evidence regarding the role of the latter enzyme is still somewhat contradictory. These ribosome-associated enzymes degrade mRNA niorc readily than either tRNA or rRNA (107, 274). Latent RNase, on the other hand, hydrolyzes tRNA morc readily than other types of RNA ( 2 6 0 ) . Intact E . coli ribosomes are relatively resistant to endogenous latent and exogenous pancrcatic RNase a t Mg‘+ concentrations above 5 m M ; lower Mg2+ concentrations lead to their dissociation to smaller units, which are susceptible to the RNases ( 2 7 5 ) . I n intact E . coli cells subjected to phosphorus deprivation, initial degradation of rRNA to 3’-phosphate-terminated oligonucleotides is due largely to latent ribosomal RNase ( 2 7 6 ) . The probable localization of latent E . coli RNase in the region adjoining the cell membrane, together with its nonindispensibility for cell viability, imply some extracellular function. The surface localization of a number of E . coli degradative enzymes (phosphatases and nucleases) (277) is in agreement with such a concept. It is of interest in this connection that metazoan (255) and HeLa (254) cells contain a surface-associated RNase activity that appreciably decreases the infectivity, and leads to fragmentation, of infectious viral RNA. Thc existence of inicroorganims deficient in polynucleotide phosphorylwe points to the nonindispensibility of this enzyme as well. Kimhi and Littauer (278) found less than 10% of polynucleotide phos-
LOCALIZATION OF NUCLEASES
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phorylase firmly bound to washed ribosomes of E . coli. The specific activity of polysomes was low i n the heavy polysome region, and increased toward the smaller polysomes; such a distribution is in accord with that expected for activity bound to mRNA. Elson’s original work stimulated a series of investigations on latent ribosomal RNase in mammalian cells (46, 1/31),particularly with respect to the stability of mRNA (279, 280). Calf pancreas ribosomes were found to contain considerable RNase that differed from that of liver ribosomes in that it was precipitated by serum antibodies to crystalline pancreatic RNase. The ribosome-bound RNase was shown to exchange readily with added labeled paiicreatic RNase (281). Recently Utsonoiniya and Roth (166) found the alkaline RNase of rat liver polysomal preparations to be associated with subunits or degradation products of the ribosomes; intact ribosomes or polysomes were devoid of latent RNase and exhibited no masking of small quantities of exogenous pancreatic RNase or supernatant RNase, which associated with the smaller subunits. I n the presence of spermine or Mg”, these subunits re-form larger units, with concomitant masking of their RNase (9). On the other hand, treatment of guinea pig pancreatic ribosomes with spermine provoked a release of RNase (282). Of particular interest was the discovery of 5’-phosphate-forming endoribonuclease and phosphodiesterase I in the membranes of the endoplasmic reticulum of rat liver cells (46) and Ehrlicli tumor cells (44). Since protein synthesis was found to be more active in ribosomes attached to membranes (683),the inference was drawn that these enzymes may play some significant role in the protein synthesis machinery ( 4 6 ) . However, the possible role, if any, of polynucleotide phosphorylase in the degradation of mRNA of higher organisms is not clear. Hymer and Kuff (284) found the initial breakdown of high-molecular-weight rapidly labeled RNA of plasma cell tumor nuclei to 4-6 S components to be mediated by an enzyme resembling the supernatant ribonuclease. Harris (285) observed that the rapidly labeled RNA of HeLa cell nuclei is degraded to acid-soluble fragments by polynucleotide phosphorylase. The degradation of rRNA in rat liver microsomes has been claimed to be due to EDTA-stimulated RNase and a phosphodiesterase (286). The potential role of RNase in the regulation of mRNA-dependent protein synthesis has comparatively recently been advanced as a conceivable interpretation for the finding that maize endosperm, homozygous for the opaque-2-gene, exhibits a multifold increase in RNase activity paralleling a reduction in zein synthesis and modification of the amino acid composition of the endosperm (286a, 286b). The increase in RNase activity is apparently not due to removal of an inhibitor, and it was
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proposed that the opaque-2 locus may be a rcgulator of RNase synthesis (286~). Insofar as the DNases are conccrncd, there is now reasonable evidence pointing to some regulatory role for this class of enzymes in DNA synthesis. An increase in thc priming effectiveness of DNA by endonucleases releasing 3’-hydroxyl-terminal products has been observed with DNase I (287) and E. coli endonuclease I (288). While as a rule, DNA priming activity is reduced by endonucleases that produce 3‘phosphate-terminal products (289), at least one instance is known where the simultaneous presence of a specific DNA 3‘-phosphatase exonuclease not only removes this inhibition but results in additional enhancement of priming activity (288). These findings are further substantiated by the more recent observations of Buttin and Kornberg (290), who showed that E . coli endonuclease may play a key role in priming in vivo DNA synthesis under specific conditions. Attempts have been made to correlate activation of DNA polymerase activity by DNase I with high activity of the latter in young animals engaged in active DNA synthesis (291), but these findings are untenable in the light of further evidence (251, 291). Nucleases in Cellular Repair Mechanisms. Various microorganisms and mammalian cells are known to undergo recovery in thc dark following ultraviolet and ionizing radiation damage (292, 293). This process is, in the case of UV, accompanied by the release of small oligonucleotides containing pyrimidine dimers. The removal of these damaged fragments, which are a block to DNA synthesis and cell division, must be by means of some endonuclease(s) and is followed by repair (or “patching”) of the damaged (or “excised”) region by cellular polymerases. The significance of these “excision” endonucleases is further enhanced by the fact that other types of lesions in DNA may also be excised by them [see, e.g., reference ( 2 9 4 ) ] .I n a t least one instance, a crude cellular extract, from Micrococcus lysodeilcticus, was shown to act as an endonuclease relatively specific for DNA containing radiationinduced lesions (295, 296). It is, of course, conceivable that some nucleases involved in the excision of radiation-induced or other lesions may be known enzymes, for example, a DNase active preferentially against denatured DNA, which would be capable of excising the lesions because of the accompanying loss of secondary structure in the damaged region. But there now remains little doubt as to the existence of specific excision endonucleases, which form an integral part of some cellular machinery whose function it is to reverse or repair damage to essential genetic elements (293). An additional example of this class of enzymes is a nuclease in Bacillus subtilis that inserts breaks in DNA treated with the mono-
LOCALIZATION OF NUCLEASES
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functional alkylating agent, methyl methanesulfonate (MMS) . The enzyme is quite inactive against DNA extracted from cells that have recovered from MMS damage. Since the recovery process is accompanied by thymidine incorporation, it probably proceeds via excision and repair in a manner similar to, but not necessarily identical with, that for UV repair ( 2 9 7 ) . In a t least one instance, the excision process has been demonstrated in witro, the substrate being UV-irradiated DNA (i.e., DNA containing pyrimidine dimers) and the source of enzyme an extract from MicroCOCCUS lysodeikticus (298). It is to be anticipated that the purification of some such enzyme and a delineation of its properties will enhance our understanding of the mechanisms of mutagenesis and the capacity of the living cell for dealing with them. ADDENDUM The a-naphthyl derivatives of pyrimidine nucleotides have been applied to the cytochemical localization of alkaline RNase and PDase I in the central nervous system of the rat ( N I ) . l The localization patterns for both enzymes were similar, with most intense activity in the endothelium of blood vessels, the leptomeninges and membrana limitans superficialis of the brain, and the ependymal cells lining the surface of the ventricules, and the canalis centralis of the spinal cord. No activity was observed in the neuronal and glial cells. Initial trials in our laboratories have led to the preparation of the a-naphthyl esters of two purine nucleotides, 3’-IMP and 3’-AMP. The former was obtained by treatment of 2 ,5 -di-O-tetrahydropyranylinosine with a-naphthylphosphate in the presence of DCC; the latter by treatment of NG-acetyl-2 ,5 -di-O-tetrahydropyranyladenosine(obtained by enzymatic dephosphorylation of NG-acetyl-2 ,5 -di-O-tetrahydropyranyladenosine 3’-phosphate) with a-naphthylphosphate in the presence of DCC. Both of them were found to be rapidly hydrolyzed by RNase T,, while preliminary trials demonstrated their relative resistance to RNase T, and, of course, to pancreatic RNase. The stability of both substrates was sufficiently satisfactory for use in both section cytochemistry and colorimetric assays of the appropriate enzymes. Preparative methods for these substrates are therefore being elaborated. These findings considerably extend the range of application of the azo-dye coupling techniques to localization of nucleolytic enzymes. [The reader is referred to the Note Added in Proof, p. 427.1 ACKNOWLEDGMENTS Ke are indebted to the following for making available photographs and manuscripts prior to publication: Drs. R. Daoust, G . delamirande, B. Ehinger, U. Z. ‘See reference list on p. 429.
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Littaucr, J. S. Roth, T. R. Shanta., M. Laskowski, Sr., D. Elson, A. Michalowski, and J. Jasinska; and to the Wellcome Trust and the World Health Organization for support.
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NOTEADDEDIN PROOF Several new and rather interesting nucleolytic enzymes have been described since the completion of this review. An enzyme that specifically hydrolyzes phosphodiester linkages in RNA and synthetic polynucleotides containing a 2’-O-methyl in the ribose moiety has been isolated from Amcystis nidulans ( N 2 ) . The enzyme, referred to by the authors as 2’-O-methyl RNase, gives rise to products with 5’-phosphate terminal groups. Its specificity is testified to by the fact that it does not hydrolyze DNA and is not appreciably active against normal RNA or bis-p-nitrophenyl phosphate or p-nitrophenyl 5’-TMP. It is fairly heat stable and optimally active a t about pH 7.5. No such activity has been reported in higher organisms, but the remarkable specificity of this enzyme, and its potential importance in sequence studies, will undoubtedly stimulate further studies on its distribution and properties. Ehrlich ascites tumor cell nuclei have been found to contain an exoribonuclease active a t pH 7.69.2, with a preference for single-stranded, random coil RNA which is hydrolyzed to 5’-mononucleotides ( N S ) . The enzyme is inactive against DNA and p-nitrophenyl 5’-TMP. A similar enzyme is localized in rat liver cell nuclei. An acid DNase has been isolated from malignant tumors of C3H mice and partially purified ( N 4 ) .It is an endonuclease with a preference for double-stranded DNA, which is hydrolyzed to products with 3’phosphate terminal groups. It differs from splenic DNase I1 in its preference for a d(ApT) linkage.
428
DAVID SHUGAR AND HALINA SIERAKOWSKA
Further progress has been reported in the purification of DNase I inhibitor from rat serum ( N 5 ) . The inhibitor is protein-like in nature and acts by stoichiometric binding to the enzyme. It is inactive against DNase I1 and DNA polymerase. It is also of interest that rat bone marrow, which contains mainly mid RNase and relatively little of the alkaline enzyme, has been shown to be devoid of alkaline RNase inhibitor ( N 6 ) . Additional useful reviews have appeared on DNases ( N 7 ) , on ribosomal enzymes, including RNases ( N 8 ) , and on the properties and assay of tissue nucleases ( N 9 ) . The latter adduces further data on the specificity of acid RNase, which is postulated as being specific for the internucleotide linkage between a pyrimidine nucleoside 3’-phosphate and the adjacent nucleoside. Previously reported hydrolysis of other bonds is ascribed to contamination with alkaline RNase and/or spleen PDase.2 The author also proposes a survey procedure for nuclease assays in homogenates, essentially as follows: One half the homogenate is treated with acid to inactivate RNase inhibitors, 5’-RNase and PDases I and 11, and then assayed for acid and alkaline 3’-phosphate forming RNases; the other half is used to assay 5’-RNase against poly-A as substrate, and PDases I and I1 against p-nitrophenyl 5’-TMP and 3 TMP, respectively. It should be noted that the use of the p-nitrophenyl nucleotide esters for assay of PDases is to he strongly recommended, in place of the still widely employed bis-p-nitrophenylphosphate, for reasons other than merely greater specificity and higher rates of hydrolysis. The latter compound has been found to be an active inhibitor of carboxylesterases ( N 1 0 ) . It turns out that the inhibitory effect is due to phosphorylation of the carboxylesterase molecule with concomitant release of two moles of p-nitrophenol. Consequently, release of p-nitrophenol on incubation of the substrate with tissue homogenates may, in some instances, be due in whole or in part to the presence of carboxylesterase rather than PDases, particularly since the inhibition effect is rapid a t p H 8. An extremely informative and succinct review ( N 1 1 ) discusses the selective release of E. coli degradative enzymes by successive EDTA and “shock” treatment, and summarizes the available evidence for the presumed surface localization of these enzymes, i.e., in a region between the cell wall and the cytoplasmic membrane. However, the same article contains an addendum which points to the weakness of some of the arguments and emphasizes the need for additional evidence. As regards the still controversial problem of RNases in tumor cells, it has been reported that Ehrlich ascites tumor cells contain acid PDases = phoephodiesterases.
429
LOCALIZATION O F NUCLEASES
RNase but exhibit no alkaline activity, either in the presence or absence of p-chloromercuribenzoate ( N l d ) . Alkaline RNase was found only in cell suspensions contaminated with blood, and it therefore appears that it is derived from the host tissues, via the blood, in the later stages of tumor growth. Further progress has been achieved in isolation of the repair enzyme found in the wild type strain of Micrococcus lysodeikticus (see p. 419). Column chromatography led to the clear separation of an enzymatic coniponent with endonucleolytic activity against UV-irradiated D N A and with no activity against normal DNA (N1.9). The enzyme exhibited no requiremcnt for Mg2+. Somewhat surprisingly, an eytract from a mutant strain with similar UV sensitivity was only slightly active against DNA. Numerous attempts have been made to determine whether the enzymatic genetic repair mechanisms t h a t exist in microorganisms are also to be found in mammalian cclls. No such direct evidence has yet been forthcoming. But Rauth ( N l 4 ) has noted some striking similarities between dark reactivation of UV damage in mouse L cells and that in hacteria. It is to be expected that this subject will continue to elicit considerable interest, the more so in t h a t it had been previously shown, as might be expected, t h a t thymine dimers are induced by UV irradiation in the chromosomal D N A of Chinese hamster cells ( N 1 4 ) .
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Author Index Numbers in parentheses are rrfrrence numbers and indicate that an author’s work is referred to although his name is not cited in the text. Numbers in italic show the page on which the complete referrnce is listed. 413), 86(394), 87(394), 92(394), 93 A (375, 379, 3941, 94(394), 95(475), 05, Abeles, R. H., 335(81, 82), 336(81, 82, 96, 97, 08, 102, 103, 104, 105, 106, a),337(83), 346, 347 197(9), 209(9, 75), 215(118), 238,259, Abelson, N. N., 154(151), 171 240, 247(58), 277(58, 216), 280(243), Abrams, R., 304(10, 15, 20), 324(15), 325 295, 299, 300, 383(143, 146), 388(146), (15, 20), 326(20), 329(20), 344(10), 423 345 Ackerman, G. A., 390(185), 391(185), 424 Allison, J. B., 413(248), 426 Alpers, D. H., 278(227), 279(227), 281 Ackerman, W. W., 74(370), 104 (227), 299 Adams, A., 11334, 36), 22(36), 24, 165 Altman, K. I., 373(72), 379(72), 421 (172), 172 Adams, H., 375(85), 386(85), 388(85), rlmano, H., 410(236, 237), 411(236, 237), 412(236), 414(251), 418(251), 425, 422 426 Adams, H. R., 37(104), 66(334), 97, 103 Ammo, M., 207(69), “ 9 , 390(179), 4-74 Adams, J. M., 137(90a), 170 Ambellan, E., 413(245), 426 Adler, J., 320(56), 346 .4melunxen, F., 122(38), 16s Adman, R., 361(70), 362(70), 36s Amer, S. M., 32164, 6 5 ) , 06 Agrell, I. P. S., 90(450), 91(451), 106 278(219), I, 279 Ajtkhozin, M. A,, 232(189), 234(189), Ames, B. N., 43(164), +% (219), 281(219), 299 246, 254(118), 283(284, 285), 284 Ananieva, L., 94(473), 106, 213(106), 215 (284, 285), 285(285), 292(284, 2851, (106), 240 297, 301 Anderson, D. C., 70(350), I03 Akinrimisi, E. O., 43(163), 53(163), 99, L4nrlerson,E. P., 375(84), 422 358(55), 36s Andcrson, J., 134(78), 152(78), 169 Aldridge, W. S., 402(210), 426 Anderson, N. G., 185(52), 194, 408(217), Alexander, D. E., 417(286b), 427 413(244), 425, 420 Alexander, P., 409(225), 426 Ando, T., 41(148), 42(148, 152, 153, 157, Alfert, M., 44(168), 64(325), 99, 103 159), 43(160, 44(167), 67(339), 91 Allard, C., 371(33), 372(33), 384(33, 151), (339), 9s, 99, 103 385(33, 151), 3%(33), 420, 423 Andoh, T., 415(268), 426 Allen, E., 174(8), 193 Andrcev, V. M., 359(61a), 368 Allen, F. W., 376(98), 377(104), 422 Anfinsen, C. B., 108(3), 167, 204(48), Allende, J. E., 119(32), 168 259, 279(232), 300, 370(1, I l ) , 420 Allfrey, V. G., 25(2), 26(15), 27(15), 31 (2, SO), 32(58), 34(88, 891, 38(15, h g e l o r , E. Z., 213(108), 215(108, 117). 940 107, 108, 109, 118), 60(287), 64(287, 323, 3‘24), 70(2), 73(109, 363), 74 Anraku, Y., 415(262, 2691, 4% (363),75(375), 76(375, 379), 78(385), Antokolskaya, J. A,, 30(29), 96 79(375), 80(375, 3941, 81(375, 379, Apgar, J., 139(94), 140(94), 142(94), 170, 414(250),. 420 399), 82(88, 89, 394, 399, 400, 402), . 83(394, 402, 405), 84(394), 85(399, Aposhian, H. V., 418(289), 427
431
AUTHOR INDEX
432 Appelmans, F., 384(153), 423 Applebami, S., 206(66), 239, 253(110), 296
Agvist, S.E. G., 370(11), 420 Argetsinger, J. E., 137(90a), 170 Arlinghaus, R., 176(24), 19s Armentrout, S. A., 284(294), 290(294), so1
Arms, K., 276(209), 277(2O9), 291(209), 299
Arnstein, H. R. V., 175(14), 177(32, 33), 178(34), 187(57), 199, 194, 230(183), 231(183), 242 Aronson, A. I., 253(108), 283(283), 296, 301
Aronson, J., 401(208), 425 Artman, M., 415(270), 426 Asahi, T., 314(49), 346 Asano, K., 191(63), 194 Ascione, R., 165(172), 172 Ashkenazi, Y., 155(159), 172 Askonas, B. A., 284(293), 301 Astrachan, L., 191(62), 194, 205(51), 239, 247(60), 295 Atchley, W. A,, 61(292, 293), 87(427), 102, 105
Atkins, L., 2(11), 23 Attardi, B., 182(48), 194, 210(88), 212 (88), 213(88), 218(88), 223(88), 229 (88), 240, 266(161), 298 Attardi, G., 34(84), 65(84), 97, 182(48), 194, 201(33), 210(85,88), 212(85, 88), 213(88), 218(88), 223(88), 229(85, 88), 23S, 240, 266(161), 298 Atwal, 0. S., 398(206, 207), 425 Aurrbach, C., 350(6a), 366 August, J. T., 118(25), 134(25), 167 Avery, T. O., 108(1), 167 Avramerts, E., 200(25), 238 Axelrod, A. E., 223(146, 149), 233(146), 235(146), 241 Azegami, M., 42(152), 99
B Bachvaroff, R., 200(26), 288 Bachvarova, R., 277(216), 299 Bader, S., 2(12), I 3 BackstrGm, S., 90(451), f06 Bagatell, F. K., 304(7), 346
Baglioni, C., 189(60), 194 Bahr, G. P., 281(270), 282(270), SO0 Bakay, B., 31(49), 40(19), 96, 96 Baldesten, A,, 305(27), 307(27), 945 Baldwin, A. N., 143(105), 170 Balis, M. E., 32(69, 70), 91(69, 70), 96, 355(44), 367 Baltimore, D., 283(290), 301 Baltus, E., 251(96), 265(96), 266(169, 170, 171, 1731, 296, 298 Bandurski, R. S., 314(49), 346 Bank, A., 148(128), f71, 191(p6), 194, 290(301), 301 Banks, G. R., 364(76), 368 Barber, R., 185(53), 194 Bard, S. G., 254(114), 259(114), 260(114), 261(114), 263(114), 264(114), 265 (1141, 290(114), 297 Barker, G. R., 429(N12), 42.9 Barker, H. A., 304(14), 324(14), 325(14), 34 Barker, K. L., 87(422), 106 Barlow, J. J., 179(42), 194 Barnard, E. A,, 397(202), 425 Barner, H. D., 304(11), 346 Barnett, L., 120(40), 122(40), 128(54), 129(56), 136(40, 541, 168, 169, 350 (4a), 366 Barnett, S. R., 27(21), 34(21), 95 Barnett, W. E., 144(110, 1111, 170 Baron, F., 357(52), 367 Barondrs, S. H., 216(125), 241 Barr, G. C., 76(376), 79(376), 81(376), 93(376), la4 Barr, H. J., 255(120), 270(120), 297 Barrcll, B. G., 225(168), 842 Barrnett, R. J., 408(218), 425 Barros, C., 283(283a), 301 Barton, A. D., 30(32), 40(32, 136), 96, 9s
Barton, R. W., 87(423, 4241, 106 Bases, R. E., 414(255), 416(255), 426 Basilio, C., 119(29, 30), 149(29), 155 (1581, 168, 172 Batterham, T. J., 337(85, 86), 347 Bandhuin, P., 384(154, 155, 156), 385 (1541, 386(155, 170, 1711, 387(155, 170, 1711, 388(54, 1701, 423, 424 Bauer, R. D., 50(220), 100
433
AUTHOR INDEX
Bautz, E., 350(11, 12), 351(11, 121, 353 (11, 12), 356(11), 358(11), 359(12), 366
Bautz-Frcese, E., 350(11,12),351(11,12), 352(23), 353(11, 12), 356(11), 358 ( l l ) , 359(12), 366, 367 Bayer, M. E., 288(299), 292(299), 301 Bayler, S. T., 38(114), 98 B:tylry, P. M., 61(291), 102 Bazill, G. W., 76(381), 93(381), 104 Beadle, G. W., 108(2), 167 Beams, H. W., 245(16), 294 Beard, J. R., 371(38), 372(38), 377(38), 421 Bcarn, A. G., 2(3), 23 Bcaufay, H., 384(155, 1561, 386(155, 170), 387(155, 170), 388(170), 423, 424 Beaver, D. I,., 402(209), 407(209), 425 BeixrrviC, A,, 221(133), 223(133), 225 (165), 241, 242 Beck, J. S., 30(30), 96 Bwk, W. S., 304(16, 22), 306(31), 324 (72), 325(16), 326(22), 327(22, 75), 328(16, 75), 329(75, 77), 330(22, 75, 78), 331(78), 332(78), 33306, 78), 335(31), 336(83), 337(83, 84), 346, $46, 347 Bwkrr, E. D., 364(80), 968 Beclter, H. J., 280(248), 500 Reckrr, Y., 221(136), 223(136, 147), 229 (147), 232(136), 241, 249(82), 283 (286), 296, 301 Beckwith, J. R., 131(62), 132(69), 135 (85), 136(85), 169 Brermiinn, W., 244(5), 280(244, 245), 281 (5, 270, 271), 282(270, 2751, 294, 300, 301 Brrrs, R . F., 371(22), 420 Bclitsina, S. V., 232(189), 234(189), 249, 254(118), 283(284, 285), 284(284, 285, 296), 285(285), 292(284, 285), 297, 301
Belkhode, M. I,., 388(176a), 424 Bell, E., 227(161), 241, 249(83), 250(95), 296 Brllair, J. T., 51(242, 243, 2441, 53(242, 243, 244), 101 Rellamv, A . D., 226(158), S4l Belman, S., 354(36), 3 6’7
Belousova, A. K., 386(167), 424 Bencosme, S. A., 281(274), 301 Bendall, D. S., 384(155), 386(155, 170), 387(155, 170), 388(170), 423, 424 Bender, M. A., 2(5), 23, 30(35), 92(35), 96, 261(149), 297 Bendich, A., 32(68), 65(329), 96, 103, 350 (91, 366 Benjamin, W., 89(440), 106 Bcnsam, A,, 197(16), 238 Brnzcr, S., 108(4), 132(68), 135(79), 146 (119, 120), 147(122), 151(79), 157 (119), 167, 169, 170, 171, 350(W, 351(18), 367 Berberich, M. A., 279(230), 281(230), 299 Berendes, H. D., 280(252, 2581, 300 Berg, P., 87(433), 106, 126(50), 138(91), 141(91), 142(102, 1031, 143(103, 1051, ~ ~ ( 1 4 i9w, , 168, 170, 171, 172, 202 (44), 239 Brrg, W. E., 252(102), 296 Bergel, F., 410(233), 413(233), 425 Brrgrr, G , 428(N5), 423 Bergeron-Bouvet, C., 221(135), 223(135), 24.41 Bergh, A. K., 67(343), 103 Bergmann, F. H., 142(102, 103), 143 (1031, 170 Brrgquist,, P. I,., 165(171), 179 Berleur, A., 384(154), 385(154), 388(54), &3 Bernardelli, R., 16(35), S4 Rmmrdi, A., 372(41), 380(41), 421 Bwnardi, G., 372(41), 373(66, 69, 71), 379 (71), 380(41, 71), 421 Bernficld, M. R., 120(34, 35), 122(35, 361, 168, 371(27), 383(138), /,SO. 424 Brrry, J. M., 204(47), 239 Bertani, I,. E., 306(29), 314(29), 320(21), 545 Berthct, J , 384(156), 424 Bertman, J., 229(174), S4Z B ~ r,. ~G., ~51(239, , x o ) , 5 4 ~ ~ 240), 9 , 57(240), 59(240), 66(239), 88(240), 101
Bessmnn, M. J.. 320(56), 346 Brtheil, J. J.. 178(34), 1BS Bhagavan, N. V., 61(292, 2931, 102
434
AUTHOR INDEX
Bianchi, N. O., 4(22), 7(22), 21(43), 94 Bianchi, P. A., 91(456), 106 Bier, K., 245(23, 26), 294 Biezunski, N., 176(27), 177(27), 178(27), 19s Bijvoet, P., 51(225), 53(225), 100 Billen, D., 60(288), 63(288), 70(348), 76 (288, 348), 77(288, 348, 3821, 79 (288, 382), 81(288), 93(288, 348, 382), 102, 103, 104 Birnboim, H. C., 199(24), 210(84), 212 (84), 213(24), 218(24, 841, 219(24), 234(24), 238, 240, 266(162, 1631, 268 (163), 298 Birnstiel, M. L., 31(38), 37(38, 94, 95, 98, 1031, 38(38, 98), 39(123, 1241, 73 (94, 95, 3661, 96, 97, 98, 1U4, 255 (1211, 297 Biserte, G., 51(241), 54(261), 55(261), 101
Bishop, J., 116(15), 149(15), 167, 175 (10, 151, 193 Bissot, T . C., 353(28), 367 Biswm, B. B., 73(364), 74(364), lo4 Biswas, C., 306(31), 335(31), 3& Black, S., 312(45), 313(48), 346 Blackstein, M., 377(108), 386(108), 387 (1081, 388(108), 422 Bladen, H. A., 198(19), 238, 281(266), so0 Blakley, R. L., 304(14, 211, 324(14), 325 (14,73), 326(21, 73, 74), 327(21, 741, 329(21), 334179, 80), 337(85, 86), 545, 346, 3-47 Blaszek, V. A., 90(448), 106 Blobel, G., 415(264), 426 Bloch, D. P., 44(170, 171, 174), 99 Bock, R. M., 132(66), 147(123), 169, 171, 278(224), 299 Bodenstein, D. W., 279(238), SO0 Bodmer, W., 418(289), 487 Book, J. A., 2(11), 23 Bogdanov, A. A., 187(55), 194 Boileau, S., 372(46), 378(46), 388(46), 417(46), 421 Boiron, M., 266(165), 298 Bojadjieva-Mikhailova, A. G., 206(63), 939
Bolle, A,, 128(53), 135(53, 811, 168, 169 Bollum, F. J., 373(60), 418(287), 4.21, 4fl
Bond, H. E., 253(111), 296 Bond, V. P., 7(23), 24 Bonner, J., 26181, 34(87, 901, 43(163), 49 (217, 53(163), 6012891, 63(289), 66 (3351, 68(335), 75(289, 3741, 76(289), 77(289), 79(289, 374), 81(289, 398), 86(419), 88(436, 437, 438), 89(436, 437), 93(289, 374, 419, 466), 95, 97, 99, 100, 102, 103, 104, 105, 106, 197 (101, 238 Borden, R. K., 381(131), 382(134), 423 Borek, E., 37(103), 97, 148(127), 171 Borenfreund, E., 65(329), 103, 350(9), 366 Borsook, H., 174(6), 192 Borum, K., 2(8), 23 Bosco, M., 251(101), 296 Boulanger, P., 209(81), 239 Bourne, G. H., 403(215), 406(215), 426 Boyde la Tour, E., 135(81), 169 Brachet, J., 90(444), 206, 197(11), 238, 245(14), 248(67, 711, 251(96), 258 (71), 265(96), 267(179), 294, 295, 296, 298 Brack, 5. D., 44(174), 99 Bradbury, E. M., 62(298, 300, 302, 303), 102
Bradt, C., 256(129), 276(213), 297, 299 Branda, R., 267(180), 269(180), 271(180), 298 Brantmark, B. L., 54(256), 101 Braun, H., 40(133), 95 Braunitzer, G., 168 Brawerman, G., 176(27), 177(27), 178 (27), 193, 214(114), 215(114, 1201, 216(114), 226(114), 230(114), 240, 241 Breckenbridge, B., 182(46), 194,212(104), 213(104), 214(104), 218(104), 223 (104), 240 Bremer, H., 202(41), 239, 281(267), SO0 Brenncr, S., 115(13), 120(40), 122(40), 128(53, 54), 129(56), 135(53, 82, 85), 136(40, 54, 85, 86), 153(86), 154(86, 150, 151, 151a), 167, 168, 169, 172, 196(3), 9.97, 350(4, 4a), 966
435
AUTHOR INDEX
Bretcher, M., 52(144), 171 Breuer, C . B., 231(186), 648 Breuer, M. E., 280(240, 2471, YOU Briggs, R., 244(7), 275(7, 199, 200), $94, 299 Bril-Peterscn, E., 45(184), 99 Brimacombe, R., 120(35), 122(35, 361, 168
Brinton, C. C., 179(40), 195 13ritten, R. J., 158(165), 172 Urody, S., 153(148), 171 Brookes, P., 362(73), 368 Broquist, H. P., 344(101), 347 Brown, D. D , 37(101), 97, 206(64), 212 (101, 102), 214(102), 217(102), 219 (101, 102), 227(101, 162), 239, 240, 241, 250(92), 254(113, 115, 116, 117, 124), 255(115, 119), 261(113), 266 (166), 268(92, 115, 116, 119, 192), 269 (92, 113, 115, 166), 270(92, 194, 196), 271(166), 273(192), 277(113, 196), 290 (92, 113, 124, 1661, 291(196), 290, 2.97, 39s, 299
Brown, D. J., 353(30), 567 Brown, D. M., 353(29, 31, 32), 354(32, 381, 355(32, 38, 40, 43), 356(40), 357 (52), 358(43), 359(38), 360(63), 361 (70), 362(38, 63, 70), 364(32, 40, 76, 77), 365(82), 567, SGS, 371(23), 375 (88), 420, 426 Brown, F , 226(160), 241 Brown, G. 13, 304(3), 344(3), 345, 355 (44), 367 Brown, G. L , 139(100), 170 Brown, I(. D., 373(63), 384(63), 385(63), 388(63), 421 Brownlec, G. G., 225(168), 848 Brownson, C., 337(85, 86), 347 Brunish, R., 32(61), 96 Bruskov, V. I., 282( 2761, SO1 Buclianan, J. M , 320(60), 346 Buck, C. A,, 230(181), 242 Burhi, H., 118(28), 121(28), 122(28), 128(28), 167 Budovsky, E. I , 355(42), 3G7 Biirger, M., 199(23), 238 Buffa, F., 408(222), 425 Bundling, I. M., 384(149), 423 Burdick, C. J., 60(287), 64(287), 102
Burdon, M. G., 374(75, 771, 421, 486 Burka, E. It., 174(4), 179(4), 181(4), 191 (65), 192, 194, 290(300), SO1 Burny, A,, 174(5), 179(5), 181(5, 441, 182 (44), 185(44, 49, 50, 511, 186(50), 187 (54), 190(49), 191(50, 671, 192, 194, 229(178), 648 Burton, K., 145(116), 170 Busch, H., 25(4, 5, 61, 26(16), 27(4, 5, 25), 30(4, 5), 31(5, 40, 53, 54), 32(4, 16, 62, 64, 65, 66), 33(4, 16, 62, 75), 34(40, 931, 35(40), 36(40), 37(40, 93, 100, 102, 104), 38(4, 751, 40(4, 5, 18), 48(16, 621, 49(62), 51(62, 236), 54 (62), 55(62), 56(5, 2651, 58(75, 275), 66(62, 236, 265, 275, 333, 3341, 70(4, 5, 350, 3 5 0 , 71(4, 5, 6, 16, 621, 73 (4, 5, 6), 90(53, 541, 91(75), 95, 96, 97, 101, 102, 103, 206(58), 211(91, 92), 212(92), 213(91, 92, 110, 111, 112, 113), 216(113), 219(92, 113), 639, 240, 375(85), 386(85), 388(85), 422 Butcher, R. W., 375(94), 377(94), 380 (941, 389(94), 422 Butler, G. C., 49(202), 51(232), 53(246), 67(202), 100, 101 Butler, J. A. V., 26(9), 45(179, N O ) , 48 (180, 187, 189), 49(179, 180, 203,2081, 50(223), 51(180, 2351, 53(179, 180, 189, 235, 249), 54(179, 180, 235, 253, 254, 255), 55(180), 56(203, 235, 267), 60(279, 280), 66(180, 253, 265, 332), 67(208), 68(253), 70(352), 71(254), 72(254, 357), 76(376), 79(376), 80 (357), 81(254, 376, 396, 397), 85 (357), 91(454), 93(376), 95, 99, 100, 101, 102, 105, 104, 105, 106 Ruttin, G., 418(290), 4Z7 Byers, T. J., 30(33, 341, 92(33, 34), 96 Ryrne, R., 198(19), 238, 281(266), SOU Rpvoet, P., 37(100), 66(334), 71(354), 72(354), 81(254), 97, 103, lo4
C Caffery, J. M., 78(391), 86(391), lo4 Caicuts, M. J., 361(71), 568 Cairns, J., 131(58), 169 Callahan, P. X., 56(264), 66(264), 67 (2641, 80(264), 101
436
AUTHOR INDEX
Callan, H. G., 40(143), DS, 245(27, 29), 267(175), 294, 298 Cammack, K. A., 416(275), 486 Campagne, R. N., 278(223), 299 Campbell, D. H., 353(28), 367 Campbell, L. L., 133(74), 169 Campbell, P. N., 201(35), 231(35), 238 Candelas, G. C., 260(144a), 297 Canellakis, E. S., 338(87), 347 Canellakis, Z. N., 338(87), 347 Canfield, R. E., 204(48), 239 Cantero, A., 384(151), 385(151), 410 (2351, 411(235), 423, 4.25 Cantoni, G. L., 229(173), 24.2 Cantor, K. P., 34(83), 65(83), 97 Capecchi, M. R., 137(90a), 153(147), 170, 171, 174(3), 175(3), 176(3), 192 Capra, J. D., 148(130), 171 Carasso, K.,206(61), BYS Carbon, J., 154(149, 154), 171, 172 Carlton, B. C., 127(52), 168 Carnahan, J. E., 311(37), 3.46 Carrier, W. L., 219(298), 427, 429(N15),
4% Carroll, E., 304(3), 344(3), 3.46 Carter, W., 217(129), $41 Carver, M. J., 31(45), 96 Caston, J. D., 254(117), 297 Catalano, C., 259(142) 297 Cave, M. D., 65(328), 103 Cerna, J., 356(50), 367 Cerutti, P., 361(64), 368 Chakravorty, A. K., 73(364), 74(364), 104 Chalkley, G. R., 49(217), 71(353), 80 (353), 100, 104 Chamberlin, M., 126(50), 168 Chambers, E. L., 267(183), 298 Champagne, M., 61(294), 102 Champe, S. P., 132(68), 135(79), 151(79), 169, 350(18), 351(18), 350(18), 367 Chandler, B., 196(7), 238 Chang, A. Y., 174(2), 175(2), 176(2), 192 Chang, S. H., 139(97), 140(97), 148(97), 170 Changeux, J.-P., 314(53), 317(55), 346 Chantrenne, H., 174(5), 179(5), 181(5), 185(50), 186(50), 191(50), 191, 194
Chapeville, F., 143(107), 146(119), 151 (119), 170 Chargaff, E., 205(53), 215(121), 225(166), 23S, 251, 242, 248(76), 295, 373(52), 421
Chentsov, J. S., 40(134), 98 Cherayil, J. O., 147(123), 171 Chernick, W. S., 370(15), 420 Cheung, W. Y., 375(95a,b), 389(95a, 177, 177a), 390(177a), 422, 484 Chrvalley, R., 135(81), 169 Chevallier, M. R., 67(344), 103 Chien, S. C., 66(333), 103 Chipchase, M. I. H., 31(38), 37(38, 98), 38(38, 981, 96, 97 Chorazy, M., 65(329), 103 Christensson, E. G., 90(450), 91(457), 106 Chu, D., 413(248), @G Cliu, E. H. Y., 429(N15), 429 Chubukov, V. F., 353(26), 36Y Church, R. B., 275(198), 299 Clark, B. F. C., 134(78), 152(78), 154 (1511, 169, 171 Clark, J. M., 174(2), 175(2), 176(2), 192 Clark, R. E., 227(163), 242 Clement, A. C., 245(44, 45, 46, 47), 295 Clever, U., 244(6), 280(249, 251, 259, 261, 262, 263), 281(264, 265), 282(6), 291 (264, 265), 294, 900 Cline, A. L., 132(66), 169, 278(224), 299 Cohen, P. P., 87(421), 105 Cohen, S. S., 304(5, l l ) , 545, 355(39), 367, 415(265), 426 Cohn, N. S., 67(338), 91(438), 103, 350 (lo), 366 Cohn, P., 38(112), 72(357), 80(357), 85 (357), 98, 104 Coimbra, A., 402(211), 425 Coirault, Y., 67(346), 103 Cole, A,, 64(319), 86(319), 103 Cole, L. J., 87(435), 106, 388(175), 424 Cole, R. D., 50(224), 56(273), 57(273), 73(224), 87(425), 100, 102, 105 Collier, J. R., 245(43), 195 Colman, R. F., 312(45), 346 Colter, J. S., 372(39), 412(240), 421, 426
437
AUTHOR INDEX
Comb, D. G., 267(180), 269(180), 271 (180, 1971, 298, 299 Commerford, 8. L., 53(251a), 61 (251a), 89(443), 101, 106 Conconi, F. M., 191(65, 1661, 194, 290 (300, 3011, 301 Conger, N., 370(15), 420 Conn, E. E., 320(62), 346 Connel, G. E., 49(200), 100 Conway, B. E., 60(280), 102 Conway, T. W., 119(32), 168 Coons, A. H., 394(193), 396(199), 424, 425 Cooper, E. J., 373(54), 421 Cooper, H. L., 212(98), 219(98), 240 Cordonnier, C., 373(66), 421 Corlette, S. L., 280(241), 300 Coulson, E. J., 370(14), 420 Counts, W. B., 253(111), 296 Court Brown, W. M., 16(33), 24 Cousineau, G. H., 247(62), 248(62), 259 (62), 263(62), 295 Cox, D. C., 74(370), lo4 Cox, R. A., 175(14), 177(32, 33), 187 (57), 195, 194, 230(183), 231(183),
$42 Coszone, A., 74(373), 104 Crampton, C. F., 51(227, 229, 2301, 56 (229, 230, 272), 57(272), 60(227), 66 (2301, 100, 101 Crane-Robinson, C., 62(300, 302), 102 Craven, G. R., 279(232), 300 Crestfield, A. M., 377(104), 422 Crick, F. H. C., 113(10), 114(12), 116 (21), 118(12), 120(40), 122(40), 128 (54), 129(56), 136(12, 40, 541, 142 (125), 147(125), 151(125), 158(21, 1621, 167, 168, 169, 171, 172, 350(4a), 866
Crippa, M., 249(90), 250(90, 92b), 252 (go), 296 Crocco, R. M., 249(88), 296 Crocker, T. T., 32(71), 91(71), 94(71),
97 Cronkite, E. P., 7(23), i?4 Crossley, M. L., 413(248), 426' Crosswhite, L. H., 16(39, 401, 24 Cruft, H. J., 48(195, 199), 49(210), 51 (199, 210, 237, 238), 52(199), 54(238),
56(195, 238), 57(195), 67(342, 345), 100, 101, 103 Cuenod, M., 72(356), lo4 Cummins, J. E., 267(178), 298 Cunningham, L., 372(48), 373(48), 421 Curtis, P. J., 374(75, 761, 421 Cusimano-Carollo, T., 90(446), 106
D Dahcra, M. D., 206(59), 239 da Costa, H. C., 384(151), 385(151), 425 Dahmus, M., 49(217), 86(419), 93(419), 100, 105 Dalby, A,, 417(286a), 427 Dalcq, A., 245(35), 294 Dallam, R. D., 26(13), 31(42, 44), 95, 96 Daly, M. M., 31(50), 32(58), 41(144), 47 (144), 48(190), 51(190), 53(190), 96, 98, 99 Dameshek, W. J. M., 16(37), 24 Dameshek, W., 16(37), 24 Dan, K., 40(131), 98 Daniels, A,, 379(120), 423 Daoust, R., 390(178, 179, 180, 184), 391 (180, 184), 392(180, 184), 410(235, 236, 237), 411(184, 235, 236, 237), 412 (236), 414(164, 251), 418(251), 424, 425, 426 Darnell, J. E., 199(24), 209(77), 210(84), 212(84, 95), 213(24), 218(24, 84), 219 (24), 221(137), 223(137, 147, 148), 226(159), 227(161), 229(147, 148),231 (137) 235(24, 148), 258, 259, 240, 241, 249(82), 266(158, 162, 163), 268(163), 283(287), 296, 298, SO1 Das, D., 44(172, 1731, 99 Datta, R. K., 388(176), 424 Dautrevaux, M., 51(241), 101 Davern, C. I., 138(59), 169 David, B. D., 150(139), 151(139), 171 Davidson, E. H., 247(58), 249(90), 250 (90,92b), 252(90), 277(58, 216), 296, 296, 299 Dnvidson, J. N., 32(60), 96, 207(74), 209 (79, 801, 239, 374(77), 422 Davie, E. W., 149(134), 171 Davies, I. A. I., 417(286a), 427 Davies, J., 149(135), 150(135, 137, 1391, 151(139, 142), 171
438 Davies, M. C., 231(186), 2.42 Davis, F. F., 49(214), 100, 376(98), 422 Davis, J. R., 32(66), 40(18), 51(236), 56 (265), 66(236, 265, 3331, 70(350), 95, 96, 101, 103 Davison, N., 53(251b), 101 Davison, P. F., 26(9), 48(187, 189), 51 (226, 233, 2341, 53(189), 56(226, 2671, 60(279, 2801, 95, 99, 100, 101, 102 Deb, D., 408(219), 426 DcBellis, R. H., 89(440), 10G, 226(157), 241, 282(2781, 901 Debov, S. S., 31(47), 33(79), 96, 99 Decroly, M., 267(179), 298 de Duve, C., 383(144), 384(153, 154, 155, 1561, 385(154), 386(155, 1701, 387 (144, 155, 170), 388(54, 170), 414 (140, 42% 424 Defendi, V., 2(16), 14(16), 83 de Garilhe, M. P., 370(7), 396(201), 403 (201), 420, 486 Dekker, C. A., 323(67), 328(67), 346 de Kloet, S. R., 215(118), 840 de Lamirande, G., 371(33), 372(33, 44, 45, 46), 377(108), 378(46), 384(33, 1511, 385(33, 151), 386(33, log), 387 (108), 388(46, 417(44, 46, 286), 420, 421, 422, 42% 427 Delaney, R., 370(13), 420 Delbruck, M., 116(17), 158(17), 167 Delihas, N., 89(443), 106, 225(167), 229 (1741, 242 Demerec, M., 244(3), 278(3, 2171, 293, 299 Denhardt, G., 135(81), 169 Denis, H., 248(71), 252(106), 258(71), 269(106), 271(106), 272(106), 273 (106), 274(106), 275(106), 291 (1061, 296, 296 Denny, P. C., 248(68), 251(68), 296 deNooij, E. H., 53(247), 101 de Reuck, A. V. S., 26(10), 57(10), 96 Derumez, P., 210(90), 240 Desjardins, R., 31(40), 34(40, 931, 35(40), 36(40), 37(40, 93), 96, 97 Dessev, G. N., 223(150), 226(154), 229 (1541, 230(154), 233(191), 241, 242 de Terra, N., 291(302), 501 Dettlaff, T. A,, 250(92a), 896'
AUTHOR INDEX
Devi, A., 418(291), 427 de Vitrx, F., 248(71), 258(71), 295 Dim, D., 150(141), 171 DiBerardino, M. A,, 275(204), 276(204), 299 Dickman, S. R., 216(124), 941, 385(160, 161), 386(160), 417(161), 424 Dieckmann, M., 87(433), 106, 142(102, 103), 143(103), 170, 202(44), 239 DiGirolamo, A., 216(126), 230(126), 841 Dingman, W., 90(449), 10G, 206(57), 216 (125), 239, 241 Dinka, S., 74(370), 104 Dintzis, H. M., 116(16), 167, 174(7), 19.2, 204(45), 239 Dixon, G. H., 44(169), 84(411), 99, 106 Dmitrieva, N. P., 33(80), 40(142), 97, 98 Doctor, B. P., 147(124), 171 Dolapchiev, L. B., 205(52), 206(52), 207 (521, 209(52), 210(52), 212(52), 228 (169, 170, 171, 172), 229(169, 1711, 239, 242 D o h , M. I., 166(176), 172 Domagk, G., 304(6), 345 Doty, P., 60(278), 61(278), 62(278, 299), 102, 139(99), 158(164), 170, 172, 249 (80), 253(80), 2740301, 291(80), 296 Dounce, A. L., 27(21), 30(31), 31(51, 56), 34(21), 48(31, 1961, 60(51), 72(358), 80(358), 85(358), 96, 96, 100, 104, 110(6), 116(6), 117(22), 167, 383 (143, 423 Downing, M., 324(69), 346 Drach, J. C., 176(26, 28, 291, 177(26), 178(29), 195 Drake, J. W., 351(22), 367 Dreyfus, J. C., 175(12, 13, 16, 171, 176 (30), 177(31), 178(30, 35, 361, I93 Driedger, A., 49(206, 2121, 50(212), 51 (206), 70(212), 100 Drummond, G. I., 376(99, loo), 377(99), 407(216), @2, 426 Dubin, D. T., 43(164), 99 Diirwald, H., 416(271), 426' Dugre, D. H., 142(101), 159(167), 160 (101, 1671, 164(167), 170, 172 Dugre, 5. A., 142(101), 159(167), 160 (101, 1671, 164(167), 170, I78
439
AUTHOR INDEX
DuPraw, E. J., 64(320, 321), 86(320, 3211, 103 Duraiswami, S., 304(15), 324(15), 325 (151, 346 Dure, L., 248(72), 295 Durham, L. J., 323(64), 337(64), 846 nutting, D., 139(95), 140(95), 170 E Ebstein, R., 206(66), 239, 253(110), 296 Erker, R. E., 255(125), 256(125), 257 (125, 130), 258(131), 268(125), 297 Edger, R. S., 135(81), 169 Edmonds, M., 304(10), 344(10), 346 Edstrom, J. E., 37(96, 97), 97, 282(275), 301, 384(148), 423 Edwards, L. J., 31(41), 45(177), 47(177), 49(41), 54(41, 1771, 67(177), 74(41), 80(41, 177), 96, 99 Egami, F., 377(111), 383(139), 422, 423 Ehinger, B., 393(192), 394(192), 395 (192), 396(192, 200), 414(192), 424,
Engelberg, H., 415(270), 426 Englehardt, D. L., 134(77), 137(90), 169, 170 Enright, J. B., 398(206, 207), 425 Epel, D., 265(156a), 298 Epler, J. L., 144(111), 170 Epstein, R. H., 135(81), 169 Erbe, W., 413(249), 426 Erkama, J., 373(51), 421 Ernst, H., 30(28), 40(133, 137), 64(316), 80(28), 95, 98, 103 Errera, M., 207(68), 2S9, 245(33), 294 Esper, H., 255(120), 270(120), 297 Evans, J. H., 30(26, 27), 72(26, 27), 80 (271, 85(26, 27), 96 Everett, G. A., 139(94, 96, 96a), 140(94, 96, 96a), 142(94), 170 Evon, L. J., 379(121), 410(231), 423, 426
F
Fairbairn, D., 250(94), 251(94), 256(94), 258(94), 296 Fambrough, D., 49(217), 66(335), 68 426 (3351, 100, 103 Eichel, H. J., 370(15, 18), 371(34), 377 (34), 384(152), 385(18, 1521, 386 Fancher, H., 87(433), 106, 126(50), 168, 202(44), 239 (34), 409(223), 420, 421, 42% 4% Fangman, W. L., 142(104), 143(104), 170 Eidlic, L., 278(222), 299 Fanshier, L., 32(71), 91(71), 94(71), 97 Eigner, E. A., 143(106), 170 Farina, B., 374(81), 422 Eikenberry, E. F., 149(131), 171 Eisenstadt, J., 176(27), 177(27), 178(27), Farr, D. P., 120(40), 122(40), 136(40), 168 193, 215(120), 2.41 El-Aaaer, A,, 209(76), 239, 385(165), Fasman, G. D., 358(56), 359(56), 368 Faulkner, R., 81(399), 82(399), 85(399), 386(165), 424 106 Elder, A., 32(69), 91(69), 96 Faulkner, R. D., 139(97), 140(97), 147 Eldjarn, L., 64(317), 85(317), 103 (123), 148(97), 170, 171 Ellem, K. A. O., 191(64), 194, 372(39), Favard, P., 206(61), 239 412(240), 421, 426 Favelukes, G., 175(10), 176(25), 19s Elley, J., 374(74), 421 Feinstein, R. N., 373(55), 379(55), 421 Elliott, A., 62(303), 102 Feldman, H., 139(95), 140(95), 170 Elsdale, T. R., 255(122), 697 Elson, D., 248(76), 296, 415(256, 257), Feldman, M., 417(280), 426 416(270a), 417(279, 280), 426, 428 Felix, K., 41(147, 149), 42(147, 151, 156), 43(147, 166), 44(147), 47(151), 98, (NS), 429 99 Emerson, T. R., 359(61), 368 Felsenfeld, G., 165(173), 172 Emmel, V. M., 402(210), 426 Emmelot, P., 77(383), 79(383), 93(383), Ficq, A,, 245(24, 30, 331, 248(67), 251 (96), 254(24), 265(96), 266(30), 267 104 (1791, 280(242), 294, 296, 296, 898, Emrich, J., 122(48), 125(48), 168, 281 300 (2681, SO0
440
AUTHOR INDEX
Finney, R. J., 247(58), 277(58), 295 Firszt, D. C., 321651, 96 Ir'ischberg, M., 255(121, 122), 297 Fischer, G. A., 342(96, 991, 347 Fischer, H., 41(145), 42(151), 47(151), 98
Fisher, H. F., 320(62), 346 Fitsgerald, P. H., 16(34, 36), 22(36), 24 Flamm, W. G., 311381, 37(38, 95), 38 (381, 39(123, 1241, 73(95, 366), 96, 97, 98, 104, 253(111), 996 Flanagan, J. F., 371(28a), 420 Fleissner, E., 37(103), 9Y ' Flickinger, R. A., 268(191), 274(191), 277 (191), 299 Fliedner, T. M., 7(23), 24 Florini, J. R., 231(186), 242 Floyd, L., 211(92), 212(94), 213(92), 219 ( 9 3 , 240 Flyesher, M. H., 383(136), 423 Fowler, A. V., 76(378), 80(378), 93(378), 104
Friedrich-Freksa, H., 116(18), 167 Frolova, L. Y., 356(49), 367' Fry, B. J., 259(139), 262(139), 297 Frye, F. L., 398(206, 207), 425 Fujimura, F., 49(217), 100 Fukada, T., 212(93), 840 Futai, M., 415(269), 426
G Gabrusewyez-Garcia, N., 280(253), 300 Gabutti, W., 12(30), 24 Gaines, K., 202(41), 239 Galibert, F., 266(165), 298 Gall, J. G., 61(295), 7213591, 102, 10.4, 245(28, 291, 252(105), 254(28), 294, 296
Gallucci, E., 123(43), 135(83), 168, 169, Gammack, D. B., 179(42), 194 Gamow, G., 110(5), 111(7, 8), 112(9), 113(7), 158(5), 167 Gardner, R. S., 119(30), 155(158), 168,
172 Fox, C. F., 87(434), 106, 361(66, 67, 68, Garen, A., 123(43), 135(80, 83, 84), 151 691, 368 (So), 1G8, 169, 350(3), 366 Fox, J. J., 323(66), 346 Garren, L. D., 249(88), 296 Frajola, W. J., 379(117), 415(117), 4% Gavosto, F., 2(13), 4(21), 7(13, 24), 8 Frazier, J., 361(64), 368 (25), 9(26), 12(29), 13(29), 14(29, Frearson, P. M., 91(461), 106 41), 16(24, 35, 41), 20(42), 25, 24, Frcdericq, E., 60(281, 282, 283, 286), 61 408(222), 425 (282, 283), 106 Gavrilova, L. P., 283(284), 284(284), 292 Freedman, M., 74(371), 104 (284), 501 Freeman, K. B., 362(72), 3661841, 368 Frccse, E., 350(11, 12, 13, 14, 15), 351 Gay, H., 44(172, 173), 99 (11, 12, 13), 352(13, 15, 23), 353(11, Gazarian, K. G., 212(100), 218(100), 219 (loo), 223(loo), 229(1OO), 230(100), 12, 15, 25a, 27), 356(11), 358(11), 233(192), 235(196), 240, 24.2 359(12, 13, 151, 364(15), 366, 567 Freese, E. B. (see also Bautz-Freese, E.), Geiduschek, E. P., 202(39), 235, 379 350(14, 15), 352(15), 353(15, 25a, 27), (1201, 4.23 Qchatia, M., 42(154), 99 359(15), 364(15), 366, 567 Gellhorn, A., 89(440), 106 Freer, R. M., 27(21), 34(21), 95 Frenster, J. H., 26(15), 27(15), 34(88,89), Genchev, D. D., 228(171), 242 38(15, lo"), 82(88, 89), 87(432), 89 Gentry, G. A., 342(97), 347 (432, 442), 91(432, 442, 459, 4601, 95, Gentry, N., 67(346), 105 97, 105, 106, 280(243), 291(303), 300, Grorgatsos, J. G., 375(92, 93), 4.22, 427 301 (N4), 429 George, J. F., 259(140), 262(140), 297 Fresco, J. R., 165(172), 172 Frey, P. A,, 335(81, 82), 336181, 82), 346 Georgier, G . P., 33(76, 77), 40(134), 94 (473), 97, 98, 106, 205 ( 5 5 ) , 206(55), Friedkin, M., 320(59), 346 212(99), 213(55, 1061, 215(55, 106, Friedman, S. M., 149(133), 150(133), 171
441
AUTHOR INDEX
119), 216(55), 217(55), 219(55, 991, 220(55), 239, 240, 282(276), SO1 Gerhart, J. C., 310(34), 346 German, J. L., 2(3, 6), 14(6, 32), $3, 24 Geschwind, I. I., 64(325), 103 Gesteland, R. F., 187(56), 194, 415(261), 416(261), 4.26 Geuskens, M., 245(31, 3 3 , 294 Ghambeer, R. K., 304(21), 326(21), 327 (21), 329(21), 334(79, 801, 337(85, 86), 346, 346, 347 Ghosh, H., 118(28), 121(28), 122(28), 128(28), 167 Gliosh, J. J., 388(176), 424 Gianetto, R., 384(153), 423 Giannelli, F., 2(14), 23 Giannoni, G., 51(228), 53(228), 60(228), 63(306), 100, 102 Gierrr, A., 179(38), 189(59), 193, 294 Gilbert, C. W., 2(7), 8(7), 10(28), 14(7), 23, 2.4 Gilbert, W., 132(65), 149(135), 150(135), 153(144a), lG9, 171 Grldm, R . V., 34(90), D7 Gillespie, D., 172 Gilrnour, R. S., 94(468, 4691, 106 Girard, M., 199(24), 213(24), 218(24), 219(24), 221(137), 223(137), 231 (137), 235(24), 238, 841, 266(162), 283(290), 298, 301 Girija, N. S., 370(20), 385(20), 386(20), 420 Giudice, G., 245(49), 246(49), 249(85), 259(138), 296, 296, 297 Glisin, M. V., 249(80), 253(80), 260(143), 267(143), 271( 143), 274(80), 291 (SO), 296, 297 Glisin, V. R., 249(80), 253(80), 260(143), 267(143), 271(143), 274(80), 291(SO), 295, 297 Gluck, N., 226(157), 241, 282(278), 301 Godfroid, C., 187(58), 194 Gold, L., 215(120), 241 Goldberg, I. H., 63(310), 80(310), 89 (310), 102, 249(84), 296 Goldberg, M. L., 87(427), 106 Goldbergh, R. F., 279(230), 281(230), 299 Goldstein, A., 202(43), 204(43), 239
Goldstein, J., 30(33, 341, 92(33, 341, 96, 371(28), 4.20 Goldstein, L., 92(464), 106, 244(10), 276 (101,291(10), 294 Gonano, F., 147(122), 150(140), 171 Goodman, H. M., 154(151), 171 Goodwin, B. C., 79(392), 105 Goppold-Krekels, A., 41(146), 43(166), 98, 99 Gordon, J., 394(197, 198), 400(197), 412 (1971, 4.26 Gorin, A. I., 359(61b), 368 Gorini, L., 149(135, 136), 150(135, 139), 151(139), 171 Gottesman, M. M., 337(84), 347 Could, H., 229(177), 242 Gould, H. J., 177(33), 187(57), 193, 194, 229(177), 230(183), 231(183), 242 Gorilian, M., 304(22), 324(72), 326(22), 327(22, 75), 328(75), 329(75), 330 (22, 751, 345, 346 Goiitier, R., 409(224), 426 C:outier-Pirotte, M., 372(40), 49f C:rahain, A. F., 221(140), 241 Grnlrnm, C. F., 276(208, 209), 277(209), 291(2091, 29.9 Qrnmpp, W., 37(97), 97 C:ranboulan, N., 207(70), 209(70), 239 Granboulan, P., 207(70), 209(70), 8.59 Grant, P., 245(18), 294 Grataer, W. B., 229(176), 242 Grwo, A. E., 371(37), 372(37, 40), 373 (64), 376(37), 421 Green, M., 355(39), 367 Green, W. W., 45(182), 99 Greenberg, G. R., 320(58), 3.46 Greenberg, H., 218(131), 241 Greene, R., 268(191), 274(191), 277(191), 299
Greenman, D. L., 213(107), 240 Greenspan, C., 202(37), 238 Griffe, M., 373(71), 379(71), 380(71), $21 Griffith, J., 113(10), 167 Grifiths, W. M., 267(184), 298 Grippo, P., 280(242a), 300 Grobstein, C., 249(87), 296 Grogan, D. E., 31(40), 34(40), 35(40), 36(40), 37(40), 96 Gros, F., 182(46, 47), 194, 210(87), 212
442
AUTHOR INDEX
(87, I@$), 213(104), 214(104), 218 (m), 223(104), 240, 266(160), 298 Gross, P. R., 197(12), 234(12), 238, 247 (62), 248(62, 69, 70), 249(69, 70, 78), 252(104), 259(62, 1391, 260(144), 262 (139), 263(62), 267(144), 271(144), 296, 296, 29r Grossman, L., 308(32), 346, 358(56), 359 (56), 360(63), 361(65, 701, 3W63, 70), 364(76), 365(65), 366(85), 368 Gruber, M., 278(223), 299 Grumbach, M. M., 2(17), 23 Grunberg-Manago, M., 118(24), 145(115), 151(143, 143a), 167, 170, 171, 356 (511, 36’7, 374(77a), 422 Guest, J. R., 127(52), 168 Gulick, A., 26(11), 96 Gumport, R. I., 87(434), 106, 361(66), 388 Gunz, F. W., 16(34, 36), 22(36), $4 Gupta, N. K., 118(28), 121(28), 122(28), 128(28), 154(152), 16'7, 172 Giipta, S., 370(16), 372(16), 373(16), 378
(W, GO Gurdon, J. B., 37(101), 97,212(101, 102), 214(102), 217(102), 219(101, 102), 227 (101), 240, 244(9), 255(119), 268(119, 192), 270(196), 273(192), 275(206), 276(9, 209, 210, 212, 214), 277(9, 209), 291(196, 2091, 294, 297, 299 Gurley, L. R., 49(216, 218, 219), 50(218, 219), 70(216, 218, 219), 77(384), 79 (384), 81(216), 90(216), 93(384), 100, 104 Gussin, G. N., 137(90a), 153(147), 170, 171 Gustafson, T., 245(36), 246(54), 248(76), 294, 296 Gustavmn, K. H., 2(11), 23 Gutermmn, H., 42(158), 44(158), 99 Gutierrez, R. M., 84(410), 106 Guttman, B. S., 278(229), 299 Gyergyay, F., 90(448), 106
H Hackett, D. P., 227(163), 242 Hadjiolov, A. A., 200(29), 205(52), 206 (52, 63, 65), 207(52), 209(52, 831,
210(52), 212(52), 213(108), 215(65, 108, 117), 216(65, 128), 217(128), 222 (128), 223(128), 224(128), 228(169, 170, 171, 1721, 229(169, 1711, 230 (128), 234( 1931, 236(193), 237( 193), 238, 239, 240, 241, 242 Hadjivassiliou, A., 214(114), 215(114), 216(114), 226(114), 230(114), 240 Hiiggmark, A., 306(29), 314(29), 320(21), 345 Emmerling, J., 197(13), 238 Hagen, U., 40(137), 98 Hakim, A. A., 375(87), 422 Hall, C. E., 179(37, 411, 193 Hall, R. H., 381(128), 383(136), 423 Hallinan, T., 221(134), 223(134), 241 Hamilton, M. G., 38(108), 97 Hammarsten, E., 304(1), 344(1), 346 Hand, G. S., Jr., 283(283a), 301 Hanker, J. S., 408(219), 426 Hannibal, M. J., 392(188), 484 Hansson, O., 2(11), 23 Hardesty, B., 176(24), 191(68, 69), 193, 194 Hnrdman, J. G., 376(97), 389(97), 390 (971, 422 Hardy, J., 304(16), 306(31), 325(16), 328 (161, 333(16), 335(31), 345 Hardy, R. W. F., 311(39), 346 Harel, J., 226(155), 241 Harel, L., 226(155), 241 Hariharan, P. V., 366(84), 368 Harris, H., 205(54), 206(54), 207(54, 721, 218(54, 130), 233(130), 239, 241, 283 n (282), 301, 417(285), 4 Harris, J. I., 38(113), 98 Harte, E. M., 313(48), 346 Hartman, P. E., 244(3), 278(3, 219, 2251, 279(219), 281(219), 293, 299 Harvey, E. B., 248(64, 651, 296 Haselkorn, R., 361(67, 691, 368 Hashimoto, C., 42(154, 156, 157), 44 (167), 99 Haslett, G. W., 247(58), 277(58), 296 Hathaway, J. A,, 379(117), 415(117), 422 Hauschka, T. S., 16(38, 39), 24 Hawkins, G. R., 308(32), 346 Hay, J., 126(51), 168 Hayashi, H., 145(113, 114), I70
443
AUTHOR INDEX
Hayashi, M., 133(73), 169, 202(36, 381, 238, 247(59), 296 Hayashi, M. N., 133(73), 169, 202(36, 381, $38 Hayashi, Y., 253(112), E97 Hayatsu, H., 122(37), 147(123), 148(126), 168, 171
Hayes, W., 133(70), 169 Healy, J. W., 374(73), 379(119), 421, 422 Hearst, J. E., 34(83), 65(83),97 Heeter, M., 226(159), $41 Heider, K., 246(12), 294 Helinski, D. R., 127(52), 168, 310(33), 346
Hell, A., 207(68), 239 Heller, G., 122(38), 168 Hempel, A., 147(123), 171 Hempelmann, L. H., 401(208), 402(210), 426
Henley, D., 165(172), 172 Hennen, S., 276(207), 299 Henning, V., 127(52), 168 Henricks, D. M., 46(185, 186), 99 Henshaw, E. C., 216(126), 221(138), 223 (1381, 2241138, 226(138), 229(138), 230(126, 138), 232(138), 233(138), 235(138), 241, 283(288), 301 Heppel, L. A., 370(12), 371(25, 26), 372 (42, 43), 375(26, 84, 86, 88, 89), 377 (105), 378(42, 43), 384(42), 415(105, 259, 260), 416(260, 2771, 420, 421, 422, @6', 42S(N11), 429 Herbst, C., 246(51), 296 Herne, R., 45(176), 47(176), 99 Herre, G., 143(107), 170 Herriot, R. M., 370(16), 372(16), 373 (161, 378(16), 420 HPW,H. Y. C., 44(170), 90(452), $9, IOG Hewlins, M. J., 355(40), 356(40), 364 (40, 70), 367, 366 Hey, A. E., 31(41), 49(41), 54(41), 74 (41), 80(41), 96 Heymann, E., 428(N10), 429 Hiatt, H. H., 197(14), 2oS(56), 209(56), 216(126), 221(138), 223(56, 1381, 224 (138), 226(138), 229(138), 230(126, 1381, 232(138), 233(138), 235(138), 238, 239, 9.41, 283(288), 301, 304(6), 346
Higa, A., 249(81), 996 Hilgartner, C. A., 31(51), 60(51), 96 Hille, M. B., 153(145), 171 Hilmoe, R. J., 375186, 88, 89, 901, 378 (115), 4.92 Hilschmann, N., 168 Hilse, K., 168 Hilton, J., 76(380), 85(380), 104 Hilton, S., 387(174), 388(174), 410(238), 412(238), 424, 4.96 Hilz, H., 413(249), 496 Hindley, J., 49( 1991, 51( 199), 52(199), 76(377), 81(377), 93(377), 94(472), 100, 104, 106 Hirsch, C. A., 225(156), 226(156), 241 Hirsh, D., 156(161), 172 Hjelm, M., 2(11), 23 Hnilica, L. S., 31(4I), 32162, 63, 71), 33 (62, 75), 34(91, 9 3 , 36(91, 921, 38 (751, 45(177, 180), 47(177), 48(62, 180, 191), 49(41, 62, '32, 180, 207), 50(222), 51(62, 63, 180, 222, 239, 240), 53(63, 180, 222, 2451, 54(41, 62, 63, 177, 180, 207, 239, 240, 252), 55 (62, l80), 56(252, 263), 57(240, 2521, 58(75, 252, 275), 59(207, 240), 60 (288), 61(222), 62(301, 304), 63(288), 66(62, 180, 239, 245, 252, 333, 3361, 67(177, 245, 267, 275), 70(347, 3481, 71(62, 631, 73(92), 74(41, 3681, 76 (91, 288, 348), 77(288, 348, 3821, 79 (288, 382, 393), 80(41, 63, 91, 177, 245), 81(288), 84(410), 88(241), 90 (336, 347, 397), 91(71, 75, 4361, 93 (288, 348, 3821, 94(71), 96, 97, 99, 100, 101, 102, 103, 104, 106
Hnilica, V. S., 32(63), 51(63), 53(63), 71(63), 80(63), 96 Hoagland, M. B., 195(l), 237, 284(293), 301
Hodnett, J. L., 211(91), 213(91, 1121, 240
Hijrstadius, S., 245(38, 39, 40, 49), 246 (49, 50, 54), 294, 296 Hogeboom, G. H., 384(150), 385(150), 387(150), 423 Hokin, L. E., 209(82), ,840 Hokin, M. R., 209(82), 940 Holbrook, D. J., 30(26, 271, 70(349), 71
444
AUTHOR INDEX
(349), 72(26, 27), 77(384), 79(%4)1 80(27), W26, 271, 93(3&1), 96, 10% 104 Holland, J. J., 230(180, 181). 2-@ Hollander, V. P., 413(245, 243, 496 Holley, R. W., 139(94), 140(94), 142(94), 147(122), 170, 171, 414(250), 4-96 Hollingworth, J., 415(258), @6 Holmgren, A., 304(19), 310(19), 311(40, 41), 313(19), 314(19), 315(19), 329 (191, 335(19), 946, 346 Holoubek, V., 32(71), 74(369), 91(71), 94 (71), 97, 104 Holoweczyk, M., 253( 108), 296 Holt, S. J., 392(187), 408(221), 424, 496 Holt, T. K. H., 280(258), 300 Holtfreter, J., 245(42), 296 Holtzer, R. L., 386(172), 424 Honig, G. R., 74(371, 3721, 104 Hook, S., 324(72), 346 Hopkins, J. W.,229(179), 232(179), %.4& 283(289), 301 Horecker, B. L., 304(6), 346, 378(116),
4% Horiuchi, T., 416(272), 436 Horn, E. C., 45(178), 99 Horn, V., 122(46), 123(46), 160(46), 168 Horowitz, N. H., 94(4711, 106, 197(8), 238
Hosokawa, K., 150(138), 171 Houck, J. C., 377(102), 4.22 Hounaell, J., 229(176), 2& Houssais, J.-F., 210(85), 212(85), 229(85), 940
Houssier, C., 60(281, 286), I02 Howard, B. D., 351(20, 21), 367 Howell, R. R., 223(141), 241, 249(88), 296 Hoycr, B. H., 279(239), 900 Hsu, T. C., 350(8), 366 HSU,W.-T., 361(68), 368 Huang, P. C., 201(33), 238 Huang, R.-C. C., 34(87, 901, 49(21?), 60 (289), 63(289), 75(289, 374), 76(289), 77(289), 79(289, 374), 81(289), 88 (436, 437, 438, 4391, 89(436, 437), 92(439), 93(289, 3741, 97, 100, 109, 104, 106 Hubbard, M., 249(78), 996
Huberman, J. A., 34(84), 49(217), 65(84), 97, 100 Hudson, B., 313(48), 346 Huennekens, F. M., 314(50), 3-46 Huez, G., 185(50, 51), 186(50), 191(50, 67), 194 Hultin, T., 45(176), 47(176), 99, 248(66), 259(132, 1341, 260(134), 261(134), 262(152), 263(154), 296, 257 Humphrey, R. R., 275(200), 299 Humphrcys, T., 250(95), 296 Humprey, G. B., 383(147), 483 Hungcrford, D. A., 1(1), 2s hunt^, J. A., 175(14), 177(32), 1.3.9, 230 (184), S@
Hunter, M. J., 53(521a), 61(251a), 101 Huppert, J., 229(175), 242, 414(255), 416 (255)) 426
Hurlbert, R. B., 34(91, 921, 36(91, 92), 49(92), 73(92), 76(91), 80(91), 97, 304(12), 305(12, 24), 339(12), 340 (12, 88), 341(88), 344(12), 846, 347 Hurwitz, J., 76(3?8), 80(378), 93(378), 104, 118(25), 134(25), l f l , 202(42), 239
Hutchinson, W. C., 409(227), 426 Hutchison, D. J., 65(329), 103 Hyde, B. B., 37(94, 98), 38(98), 73(94), 97 Hymer, W. C., 206(62), 223(151), 224 (151), 231(151), 239, 241, 417(284), 496 Rwang, M. I. R.,182(48), 194, 210(88), 212(88), 213(88), 218(88), 223(88), 229(88), S/,O. 266(161), 6118
I Iaccsrino, M., 280(242a), 300 mar, A. T., 2 0 9 ( w , 240 Imamoto, F., 132(67), 137(89), 149(67), 169, lY0, 278(226), 2.99 Imhenotte, J., 226(155), 241 Tmmers, J., 251(98, 99), 296 Imrie, R. C., 409(227), 425 Infante, A. A., 249(89), 253(107), 259 (135), 261(107), 2670891, 269(135, 189), 284(89, 189, 295), 285(297), 286 (297), 287(189, 2971, Z88(S9, 135,
445
AUTHOR INDEX
299), 289(89), 290(107, 2971, 292(189, 297, 299), 293(89), 296, 297, 298, 301 Ingles, C. J., 44(169), 84(411), 99, 106 Inouye, M., 122(48), 125(48), 168, 281 (268), 300 Irvin, E. M., 70(349), 71(349), 103, 373 (65), 387(65), 388(65), 4.91 Irvin, J. I,., 30(26, 27), 49(211), 50(211), 70(349), 71(349), 72(26,27), 77(384), 78(391), 79(384), 80(27), 85(26, 27), 86(391), 93(384), 96, 100, 10% 104, 373(65), 387(65), 388(65), 49 .1 Ishihara, T., 16(38, 39, 40), 24 Ishii, S . I., 42(152), 99 Iso, K., 42(155), 99 Jto, J., 122(46), 123(46), 132(67, 891, 149 (67), 160(46), 168, 169, 170 It,zhaki, R. F., 60(277, 285), 62(277, 2851, 102 Iverson, R. M., 39(130), 98, 259(140), 260(144a), 262(140), 287(298), 292 (298), 297, 301 Iwai, K., 42(152), 67(339, 341), 91(439), 99, 103 Iwanaga, M., 31(57), 32(51), 96 Iyer, N. T., 376(100), 4.99 Iyer, T. K., 376(99), 377(99), 422 Izawa, M., 64(323, 324), 103
J Jackson, R. J., 231(185), 242 Jacob, F., 94(470), 106, 132(63, 64), 133 (70), 169, 195(2), 196(2), 198(2), 237, 244(1), 278(1, 218), 279(233), 293, 299, 300, 314(53), 346' Jarob, J., 92(465), 106 Jacob, T. M., 118(28), 121(28), 122(28), 128(28), 148(126), lU, 171, 175(21), 193 Jacobs, G., 373(63), 384(63), 385(63), 388 (631, 421 Jacobson, K. B., 144(110), 146(121), 170, 171 Jacques, P., 384(156), 494 James, D. W. F., 48(187), 56(267), 99, 101 Jancovi6, V., 225(165), %?$I Janion, C., 354(46), 355(41, 46a), 359 (46), 364(46), 367
Jnouni, T., 122(36), 168 Jaworska, H., 4(22), 7(22), 24 Jellum, E., 63(311), 64(317, 318), 85 (317), 102, 103 Jensen, R., 49(217), 100 Jesensky, C., 148(128, 130), 171 Jirgensons, B., 62(301, 302, 305), 102 Johns, E. W., 45(179, 180), 48(180, 197), 49(179, 180, 203, 208), 50(221), 51 (180, 235), 53(179, 180, 235, 248), 54 (179, 180, 235, 248, 257), 55(180, 197), 56(203, 235), 62(300), 66(180), 67(208), 81(396), 9.9, 100, 101, 102, 106 Johns, H. E., 359(60), 362(72), 366(84), 368 Johnson, C. L., 78(388, 389), 104 Johnson, L. D., 49(206, 2121, 50(212), 51(206), 70(212), 100 Johnsson, N., 397(204, 205), 399(204, 2051, 426 Joklik, W. K., 221(136), 223(136), 232 (136), 241, 283(286), 301, 409(229), 426 Jonrs, D. S., 122(37), 147(123), 148(126), 165, 171, 175(22), 193 Jonrs, R. N., 364(81), 36'8 Josrfsson, J,., 370(5), 376(5), 377(5, 110), 420, 422 Jossr, J., 126(49), 16'8 ,Jiingblut, P. W., 204(46), 239 .Jungwirth, G., 409(229), 426
K Kahat, S., 201(33), 238 Kaiser, A. D., 126(49), 168 Kaji, A,, 119(31), 168 Kaji, H., 119(31), 168 Kajiwara, K., 2(18), 23, 73(362), 80(362), 104 Kalnins, V. I., 281(274), 301 Kaplan, H. S., 370(12), 420 Kaplan, S., 135(82), 136(86), 153(86), 158(86), 169, 350(4), 366 Kappler, H. A., 32(63), 51(63), 53(63), 54(63), 71(63), 80(63), 96 Karasaki, S., 207(71), 239, 270(195), 999 Karlson, P., 280(260, 261), 300
446 Kmai, K., 374(77a), 48.8 Kataja, E., 149(136), 171 Kates, J. R., 373(70), 410(232), 4 2 f , 425 Kato, K., 366(85), 368 Katz, E. R., 120(40), 122(40), 136(40), 168, 350(4a), 366 Katz, K., 364(79), 368 Katz, S., 267(180), 269(180), 271(180), 298 Katzoff, L., 408(219), 426 Kaufmann, B. P., 44(172, 1731, 99 Kaulenas, M. S., 250(94), 251(94), 256 (941, 258(94), 296 Kawade, Y., 212(93), 240 Kawamura, H., 40(132), 98 Kay, D. T., 43(165), 99 Kedrova, E. M., 30(29), 96 Keefe, J. R., 402(213), 486 Keir, H. M., 373(56), 379(56), 383(56), 386(56, 173), 387(56, 173), 409(230), 481, 494, 486 Keith, J., 376(100), 422 Kellenberger, E., 135(81), 169 Kelley, D. E., 214(116), 232(190), 240, 242, 283(291), 301 Kellogg, D. A., 147(124), 171 Kempf, J., 210(89), 213(89), 218(89), $40 Kennan, A. L., 197(15), 238 Kenner, G. W., 364(75), 368 Kenney, F. T., 213(107), 840 Kepes, A., 279(231), 281(231, 269), 300 Keynan, A., 249(81), 296 Khorana, H. G.,118(28), 121(28), 122 (28, 37), 128(28), 139(97), 140(97), 147(123), 148(97, 1261, 154(152), 167, 168, 170, 171, lY2, 175(21, 221, 193, 370(3), 375(83, 91), 370(83), 380(83, 91), 381(126, 130, 132), 383(137), 398 (91), 403(83), 407(216), 420, 422, 423, 4g5 Kickhofen, B., 199(23), 238 Kihlman, B. A., 342(95), 347 Kikuchi, Y., 2(15), 14(15), 16(37), 23, 24 Killander, D.,83(404), 106 Kim, K. H., 87(421), 106 Kim, S., 85(412), 106 Kimhi, Y., 416(278), 486 Kimmel, D. L., 90(445), 106 Kimura, K., 215(123), 84f
AUTHOR INDEX
King, C., 413(246), 4.26 King, T. J., 244(7, 81, 275(7, 8, 199, 204), 276(204), 894, 899 Kinkade, J. M., 56(273), 57(273), 108 Kirby, K. S., 91(455, 461), 106, 213(105), 226(153), 240, 84l Kirkham, W.R., 26(21), 27(12), 31(42), 96, 96 Kinchbaum, J., 202(43), 204(43), 239 Kirschfeld, S., 324(70), 346 Kischer, C. W., 49(216), 70(216, 347), 79(393), 81(216), 90(216, 347, 393), 100, 103, 106 Kiselev, L. L., 356(49), 367 Kisselev, N. A., 187(55), 194 Kissoglou, K. A., 16(37), 24 Kitamura, T., 42(155), 99 Kitay, E., 324(68), 346 Kitazume, Y., 197(16), 238 Kivity-Vogel, T., 416(270a), 486 Klein, R. S , 355(45), 567 Kleinsmith, L. J., 80(394), 82(394), 83 (394, 4051, 84(394), 86(394), 87(394), 92(394), 93(394), 94(394), 106 Klenow, H., 341(89, 92), 347 Klimenko, S. M., 351(61a), 368 Knight, E., Jr., 311(39), 346 Knight, J., 26(10), 57(10), 96 Knobloch, A., 67(344), 103 Knobloch-Mazen, A., 64(326), 103 Knopf, P. M., 179(39), 190(61), 193, 194, 204(45), 231(186, 1871, 839, 242 Koch, A. L., 362(74), 368 Koch, G., 212(96), 219(96), 240 Kochrtkov, N. K., 355(42), 367 Koehn, P. V., 249(86), 296 Koenig, H., 385(164), 388( 1641, 424 Koerner, J. F., 320(60), 346, 373(61, 67), 421 Kohen, E., 32(65), 96 Kohl, D. M., 268(191), 274(191), 277 (191), 299 Kohlhage, H., 353(34, 351, 354(35), 356 (34, 351, 367 Kolakofsky, D., 134(75), 152(75), 169 Kolb, J. J., 31(49), 96 Kondo, M., 142(101), 160(101), 170 Kondo, N., 247(61), 296 Konigsberg, W., 156(161), 178
447
AUTHOR INDEX
Konrad, M. W., 202(41), 939, 281(267), 300 Koppelman, K., 207(73), 259 Kornberg, A., 118(26), 126(49), 167, 168, 320(56, 611, 346, 4181288, 289, 2901,
4n Kornberg, R. D., 87(433), 106, 202(44), 239
Korner, A., 223(145), 229(145), 231(185), 241,
su
Korschelt, E., 245(12), 294 Kossel, A., 25(1), 48(1), 96 Kowl, H., 118(28), 121(28), 122(28), 128(28), 167 Kowlessar, 0. D., 373(72), 379(72), 421 Kozlov, J., 94(473), 106 Kraemer, K., 260(144), 266(144), 271 (1441, 297 Kramer, F. R., 249(90), 250(90), 252 (go), 296 Krekels, A., 42(151), 47(151), 98 Kreuaer, L., 41(145), 08 Krichevskaya, H. A., 282(276), SO1 Krieg, D. R., 350(5), 351(5), 566 Krim, M., 350(9), 366 Krisch, K., 428(N10), 429 Kroeger, H., 86(417, 4181, 92(465), 93 (467), 95(418), 106, 106, 280(254, 255), 291(254), 300 Kruh, J . , 174(6), 175(12, 13, 16, 17), 176 (30), 177(31), 178(30, 35, 361, 192, 193 Kubacki-Enbring, V., 31(46), 96 Kubinski, H., 133(72), 169, 201(34), 212 (961, 219(96), 238, $40 Kubler, H., 122(38), 168 Kuff, E. L., 206(62), 223(151), 224(151), 231(151), 239, $41, 417(284), 426 Kuhn, J., 372(39), 412(240), 421, 426 Kullipev, P., 197(17), 238, 283(281), 301 Kumar, S., 350(19), 351(19), 352(19), 367 Kung, H., 139(96, 96a), 140(96, 96a), 170 Kunitz, M., 370(10), 372(47), 376(101), 4200, 421, &2 Kurland, C. G., 181(43), 194 Kurnick, N. B., 370(4), 378(4), 308(4),
4m
Kutsky, P., 268(190), 272(190), 298 Kyoguku, Y., 364(78), 368
L Lagerstedt, S., 370(5), 376(5), 377(5, IlO), 397(203, 204, 2051, 3M203, 204, 205), 420, 422, 426 Lajtha, L. G., 2(7), 8(7), 10(28), 14(7), 2% 24 Lallier, R., 246(57), 295 Lamar, C., 197(15), 238 Lamb, D. C., 56(264), 66(264), 67(264), 80(264), 101 Lnmfrom, H., 174(8), 175(11), 190(61), 193, 194, 231(186, 1871, 242 Litndsteiner, K., 116(20), 158(20), 167 Lang, N., 87(429), 105, 216(127), 241 I,angan, T. A., 83(407), 91(462), 92(462, 463), 106, 106 I,angford, P., 415(263), 42G I,anka, E., 123(43), 168 Lanning, M. C., 304(5), 545 I,ansing, A. I., 388(176a), 424 I,areau, J., 177(31), 193 Lark, K. G., 342(100), 347 Larsen, C. J., 266(165), 298 Larsson, A,, 304(13), 312(46), 314(13, 52), 316(54), 317(52, 54), 320(63), 323 (641, 327(75), 3281751, 329(52, 54, 75), 330(75), 337(64), 344(101, 1021, 346, 346, 347
Laskowski, M., 31(46), 96, 372(48), 373 (48, 54, 63), 384(63), 385(63), 388 (63), 421 Laskowski, M., Sr., 370(2, 8), 372(2), 373(2, 8), 378(8), 420, 428(N7), 429 Lwt, J. A., 153(145), 171 Laszlo, J., 217(129), 241 Latham, H., 212(95), 221(137), 223(137, 148), 229(148), 231(137), 235(148), 840, 241, 266(158), 283(287), 298, SO1 Laufer, H., 379(118), 422 Laurence, D. J. R., 54(254, 255), 66(255, 332), 70(352), 71(254), 72(254), 81 (254), 101, 103 Laurent, T. C., 311(36), 328(36), 346 Lavrin, D. H., 87(425), 106 Lawford, G. R., 415(263), 426
aa Lawley, P. D., 356(47), 360(47), 362 (731, 367 Lazarus, H. M., 427(N3), 4.29 Leahy, J., 116(15), 149(15), 167 Leaver, J. L., 67(342, 345), 103 LeBlanc, J. C., 362(72), 568 Lebleu, B., 191(67), 194 Leblond, C. P., 207(69), 239 Leder, P., 119(33), 120(33, 34, 351, 122 (35, 36), 128(33), 168, 196(5), 198(5), 199(5), 207(5), 220(5), 223(5), 231 (5), 235(5), 238 Lederberg, J., 418(289), 4%’’ Ledoux, L., 413(242, 2431, 4.96 Lec, H. A., Jr., 335(81), 336(81), 346 Lee, Y. C., 67(340), 73(340), 103 Lehman, I. R., 320(56), 346 Lehmann, H., 41(146), 43(166), 98, 99 Leilausis, A., 135(81), 169 I,elong, J. C., 266(165), 298 Lemieux, R. U., 323(65), 346 Leng, M., 165(173), 172 Lengyel, P., 119(29, 301, 149(29), 155 (158), 168, 172 Leone, E., 374(81), 422 I’erman, M. I., 212(99), 213(106), 215 (106), 219(99), 240, 284(296), SO1 Lerner, A. M., 227(161), 241 Letendre, C., 145(115), 170 Leuchtenberger, C., 40(135), 98 Levander, 0. A., 89(440), 106 Levin, J. G., 198(19), 838, 281(266), 300 Levine, E., 354(36), 367 Levine, L., 374(73), 379(119), 421, 422 Lrvinthal, C., 249(81), 296 Leezi, M., 93(467), 106 Liao, S., 87(423, 424), 106 Liau, M. C., 34(91, 92), 36(92), 37(91, 921, 49(92), 73(92), 76(91), 80(91), 97 Libcnson, L., 304(10), 344(10), 346 Lihonati, M., 374(81), 422 Lirhtenstein, J., 304(11), 345 Lie, S., 350(6), 366 Thberman, I., 388(176a), 424 Lima-dc-Fnria, A., 2(8), 4(22), 7(22), 21 (43), 23, 24 I i n , A . H., 87(423, 4241, I06 r h , S. Y., 191(69), 194
AUTHOR lNDEX
Lindahl, P. E., 245(37), 246(52), 294, 996 Lindahl, T., 165(172), 172 Lindberg, M. U., 373(58, 59), 421 Lindberg, U., 373(59a, 59b), 421 Lindblow, C., 358(56), 359(56), $68 Lindenmayer, G., 37(104), 97, 375(85), 386(85), 388(85), 422 Lingrel, J. B., 176(26, 28, 291, 177(26), 178(29), 193 Lingens, F., 357(53), 368 Lindh, N. O., 54(256), 101 Lindsay, D. T., 31(39), 49(39, 2091, 50 (209), 70(209), 90(209), 96, 100 Lipmann, F., 119(32), 146(119), 155(160), 157(119), 168, 170, 172, 175(9), 193, 196(4), 238 Lipsey, A., 32(65), 9G Lipshitz-Wiesner, R., 225(166), 642 Lison, L., 45(181), 99 Litchfield, J. B., 267(182), 898 Littau, V. C., 34(89), 39(108), 60(287), 64(287), 73(363), 74(363), 75(375), 76(375), 79(375), 80(375), 81(375), 82(89), 93(375), 97, 102, 104, 106 Littauer, U. Z., 148(129), 171, 416(278), &6 Littna, E., 206(64), 239, 250(92), 254 (115, 124), 255(115), 266(166), 268 (92, 115), 269(92, 115, 166), 270(92), 271(166), 290(92, 124, 166), 296, 297, 298 Lloyd, L., 267(175), 298 Lloyd, P. H., 63(315), 103 Loeb, J. N., 223(141), 241 Loeb, P. M., 87(431), 105 Loebel, J. E., 147(124), 171 Loenig, U. E., 200(30), 238 L&lie, A,, 266(173), 298 Loftfield, R. B., 139(92), 143(106), 148 (92), 170 Logan, R., 209(80), 839 Lohrmann, R., 122(37), 147(123), 168, 171 London, I. M., 182(47), 194, 210(87), 212 (87), 2.40 Lord, R., 364(78), 368 Lovcnbrrg, W., 311(38), 346 Lowett, S., 415(267), 426
449
AUTHOR INDEX
Lu, P., 87(426), 106 Luck, D. N., 204(47), 239 Luck, J. M., 32(61), 48(192), 49(205), 50(205), 51(231), 54(205, 2311, 55 (231), 56(231), 57(231), 58(192), 68 (205), 96, 99, 100 Lucy, J. A., 53(249), 101 Lukacs, I., 86(420), 106 Lundblad, G., 261(145, 146, 147), 292 (145, 146, 1471, 997 Luzzati, V., 43(162), 62(162), 99 Lynch, W. E., 388(176a), 424
Malamy, M. N., 378(116), 422 Maleknia, N., 175(16, 171, 193 Maley, F., 342(94), 347 Maley, G. F., 342(94), 347 Maling, B. D., 310(33), 346 Malkin, L. I., 248(69, 701, 249(69, 70, 781, 260(144), 267(144), 271(144), 296, 857 Malling, H. V., 350(7), 366 Mandel, L. R., 148(127), 171 Mandel, P., 210(89), 213(89), 218(89), 840
Mano, Y., 248(77), 251(77), 263(77), 264 (155), 265(77), 292(77, 1551, 296, 898 M Manson, L. A., 324(71), 3.46 McAllister, H. C., 49(211), 50(211), 100 Mantieva, V. L., 215(119), 240 Maor, D., 409(225), 426 McArthur, C. S., 67(343), 103 McAuslan, B. R., 373(70), 379(121), 410 Maraini, G., 408(222), 426 Marbaix, G., 181(44), 182(44), 185(44, (231, 232), 421, 423, 425 49, 50, 51), 186(50), 187(54), 190(49), McCarthy, €3. J., 214(115), 230(180, 181), 191(50, 67), 194, 229(178), 242 ,940, i),jY), 249(79), 252(79), 266(164), 268(164), 274(79), 275(79, 279 Marcaud, L., 182(45, 46, 471, 194, 210 (87), 212(87, 103, 104), 213(104), 214 (239), 291(79, 164), 295, 298, 29!1, :I011 (104), 218(103, 104), 223(104), 240, McCarty, K., 217(129), 241 266(160), 298 McCarty, M., 108(1), 167 Marchis-Mouren, G., 74(373), 104 McCluer, R., 215(122), 241 McConkey, E. H., 229( 179), 232 (1791, Marcker, K., 134(78), 152(78), 169 Marcus, P. I., 261(148), 262(148), 265 242, 283(289), 301 (148), 297 McEwen, B. S., 78(385), 104, 209(75), Margoliash, E., 136(87), 169 239 Marquisee, M., 139(94), 140(94), 142 McIndoe, W. M., 32(60), 96 (941, 170 MacLeod, C. M., 108(1), 167 MacLeod, R. M., 409(226), 413(226, 2461, Markert, C. L., 40(140), 98 Markham, R., 371(26), 375(26), 420 4.26, 4.26 Markman, B., 245(48), 246(48, 55, 561, McMaster, P. R. B., 200(26), 238 251(100), 277(48, 551, 296, 296 McNaught, A. D., 365(82), 368 Marko, A. M., 49(206, 212), 50(212), 51 McNutt, W. S., 324(68), 346 (206), 70(212), 100 MacPherson, A., 49(204), 54(204), 59 Markov, G. G., 223(150), 226(154), 229 (204), 100 (1541, 230(154), 233(191), 241, 242 McPhie, P., 229(176), 242 Madison, J. T., 139(94, 96, 96a), 140(94, Marks, P. A., 174(4), 179(4), 181(4), 191 (65, 66), 192, 194, 226(157), 241, 282 96, 96a), 142(94), 170 (278, 299(300, 3011, 301 Magasanik, B., 278(221), 299 Maggio, R., 213(109), 215(109), 240, 248 Marmur, J., 202(37), 238, 320(57), 346 (73), 250(73, 93), 251(73, 93), 259 Marone, R., 219(297), 4.27 (141, 142), 261(73, 931, 262(93), 263 Marrian, D. H., 355(44), 367 Marshall, J. M., 393(191), 396(191), 401 (93), 265(93), 292(93), 296, 296, 297 (191), 414(191), 424 Magni, G. E., 154(155), I79 Martin, E. M., 154(153), 172 Maitra, U., 202(42), ,939
AUTHOR INDEX
450
Miles, H. T., 361(64), 364(80), 368 Miller, C., 362(74), 368 Miller, 0. J., 2(12), 23 Martin, S. J., 226(160), 241 Marushige, K., 49(217), 93(466), 100, 166 Miller, 0. L., 267(176, 1771, 298 Miller, R. S., 119(29), 149(29), 168 Maruyama, H., 416(276), 426 Masera, P., 12(29, 30), 13(29), 14(29, Mingueil, J., 428(N6), 4% Mirsky, A. E., 25(2), 26(14, 15), 27(15), 411, 16(41), 23, 24 31(2, 14, 48, 50, 55), 32(58), 34(81, Mashburn, L. T., 413(247), 426 82, 88, 89, 107, 108, log), 38(15), 41 Massey, V., 312(44), 346 (1441, 47(144), 48114, 190), 50(14), Mather, K., 279(235), SO0 51(190), 53(190), 60(14, 2871, 64 Mathias, A. P., 179(42), 194 (287, 323, 324), 70(2), 73(109, 3631, Matsuda, M., 373(53), 421 74(363), 75(375, 76(375, 3791, 78 Matsushiro, A., 278(226), 299 (385), 79(375), 80(375, 394), Sl(375, Matthaei, J. H., 118(23), 122(38), 167, 379, 3991, 82 (88, 89, 394, 399, 4021, 168, 175(18, 19), 193, 250(91), 263 83(394, 402, 405), 84(394), 85(399, (911, 296 413), 86(394), 8713941, 92(394), 93 Mathingly, S. A., 731365), 104 (375, 379, 3941, 94(394), 95(475), 96, Maurer, H. R., 71(353), 80(353), 104 96, 97, 98, 99, 10.2, 103, 104, 106, 106, Mauritzen, C. M., 48(195), 49(199), 51 197(9), 209(9, 75),215(118), 258, 239, (199, 238, 242, 243, 2441, 52(199, 331), 240, 247(58), 249(90), 250(90, 92b), 53(242, 243, 2441, 54(238), 56(196, 252(90), 277158, 216), 280(243), 296, 238), 57(195), 65(330, 331), 100, 101, 2'8, %99, 300, 383(146), 388(146), 103 423 Maver, M. E., 371(37), 372(37, 40), 373 Mishima, S., 278(226), 299 (641, 376(37), 421 Mavioglu, H., 33(75), 38(75), 58(75), 91 Mita, T., 67(339), 91(439), 103 Mitrhison, J. M., 267(178), 298 ( 7 3 , 97 Miura, K., 145(113, 114), 170, 230(182), Maxwell, E. S., 155(157), 172 242, 356(48), 567 May, P., 428(N5), 429 Mayer, D. T., 26(11), 31(42, 43), 46(185, Miwa, T., 16(38), 24 Miyagi, M., 268(191), 274(191), 277(191), 186), 96, 96, 99 299 Mayersbach, H. v., 394(195), 424 Mizuno, D., 415(262, 268, 2691, 416(272, Mayner, D. A., 390(185), 395(185), 484 276), 426 Mazen, A,, 61(294), 102 Moffatt, J. G., 381(126), 382(133), 4-03 Mazia, D., 39(125, 126), 98 Molinaro, M., 90(446), 106 Mechelke, F., 280(250), 300 Molnar, J., 282(276), 301 Mehler, A. H., 148(128), 171 Momigliano, A,, 2(13), 7(13), 25 Mellors, R. C., 394(196), @d Moncsi, V., 2(9), 23, 44(175), 45(175), Meriii, S. H., 139(94), 140(94), 142(94), 99 170, 414(250), 426 Monny, C., 359(57), 368 Mmelson, M., 131(60), 150(38), 169, 171 Monod, J., 94(470), 106, 132(63, 641,169, Metzenberg, R. L., 94(471), 106, lWi'(8), 195(2), 196(2), 198(2), 237, 244(1), ,238 278(1, 218, 233), 293, 299, 300, 314 ( 5 3 , 317(55), 546 Michclson, A. M., 145(115), 151(143), 170, 171, 356(51), 359(57, 61), 367, Monroy, A., 197(12), 234(12), 238, 248 (73), 250(73, 931, 251U3, 93, IOl), 381(127), 383(135), 391(135), 423 256(128), 258(128), 259(133, 136, Miki, T., 39(127), 98 138), 261(73, 93), 262(93), 263(93), Milchev, G. I., 228(169), 229(169), 2.&
Martin, R. G., 115(14), 167, 278(228), 299
AUTHOR INDEX
265(93, 1361, 283(283a), 287(136), 292(93), 296, 296, 297, 301 Montagnier, L., 226(158), 241 Montjar, M., 223(146, 1491, 233(146), 235(146), 241 Montreuil, J., 209(81), 839 Monty, K. J., 31(56), 96, 117(22), 167’ Moon, M. W., 175(21), 193 Moore, B. C., 90(453), 106 Moore, E. C., 304(12), 305(12, 23, 241, 311(35, 361, 312(42), 313(42), 328'36, 42), 339(12, 23), 340(12, 23, 88). 341 (88), 344(12), 346, 346, 347 Moore, J. A., 275(205), 299 Moore, S., 51(229, 230), 56(229, 2301, 66 (230), 100 Moorehead, W. R., 73(367), 104 Moorhead, P. S., 2(16), 14(16), 23 Morais, R., 372(44, 45, 46), 377(108), 378(46), 386(108), 387(108), 388(46, 108), 417(44, 46, 2861, 4-91, 4-92, 427 Morgan, R., 118(28), 121(28), 122(28), 128(28), 157' Morgan, R. W., 276(208), 299 Moriguchi, E., 418(295), 497' Morikawa, N., 278(226), 299 Morishima, A., 2(17), 23 Morrill, G. A., 385(160), 386(160), 424 Morris, H. P., 410(238), 412(238), 426 Morris, N. R., 342(96, 991, 347 Morrison, J. M., 409(230), 496 Morrison, M., 117(22), 167 Morrison, W., 215(122), 241 Morse, P. A,, 342(97), 347 Mortenson, L. E., 311(37), 3.46 Moses, H. L., 402(209), 407(209), 4% Mosteller, R. D., 191(68, 691, 194 Moulk, Y., 221(135), 223(135), 941 Moyer, W. A,, 248(69), 249(69), 296 Mueller, G. C., 2(10,18), 23, 73(362), SO (363, 104 Miiller-Hill, B., 132(65), 169 Mukherjee, B. B., 2(12), 23 Mukundan, M. A., 418(291), 497' Muldal, S., 2(7), 8(7), lO(27, 28), 14 (71, 21(44), 23, 94 Munch-Petersen, A., 341(91), 347 Mundell, R. E., 266(173), 298
Munro, A. J., 223(145), 226(152), 229 (145), 231(185), 241, 242 Munro, H. N., 221(134), 223(134), 241 Muramatsu, M., 211(91), 213(91, 110, 111, 1121, 240 Murray, K., 26(7), 31(7), 41(7), 48(193), 49(204, 205), 50(205), 52(193), 53 (71, 54(7, 204, 205, 2581, 56(264, 2661, 58(7, 2761, 59(204), 60(7, 289), 61(290), 62(300), 63(289), 66(264), 67(264), 68(205), 70(7), 75(289), 76 (289), 77(289), 79(289), 80(264), 81 (2891, 85(258), 93(289), 96, 99, 100, 101, 102
Muto, A., 230(182), 248 Myers, J., 394(198), 426
N Nachlas, M. N., 392(188), 424 Nace, G. W., 245(25), 294 Nadler, N. J., 207(69), $39 Nagano, H., 248(77), 251(77), 263(77), 265(77), 292(77), 296 Nagata, T., 131(61), 169 Nair, K. G., 375(96), 389(96), 4% Nakada, D., 166(174), 172 Nakahara, C., 42(152), 99 Nakamoto, T., 119(32), 134(75), 152(75), 168, 169
Nakano, E., 259(133), 297 Nakase, Y., 379(118), 422 Nakayama, H., 429(N13), 429 Naora, H., 38(122), 98 Narang, S. A,, 118(28), 121(28), 122(28), 128(28), 167, 175(21), 193 Narayan, K. S., 31(54), 37(102), 90(54), 96, 97 Nasim, A., 350(6a), 366 Nast, H. P., 41(149), 98 Nathans, D., 174(1), 175(1), 178(1), 19.2 Natori, S., 415(268), 416(272), 4.26 Neelin, E. M., 38(114), 49(201), 98, 100 Neelin, J. M., 38(114), 49(200, 201, 202), 51(232), 56(264), 66(264), 67(202, 337), 80(264), 91(437), 98, 100, 101, 103
Neidhardt, F. C., 14211041, 143(104), 170, 244(2), 278(222), 393, 299
AUTHOR INDEX
Neidle, A., 49(213), 70(213), 90(213), 100 Neilands, J. B., 344(102), 347 Nemer, M., 249(89), 253(107, la),254 (109, 1141, 259(114, 1351, 260(114), 261(107, 109, 1141, 262(151), 263 (114), 264(114), 265(114), 267(109, 181, 188, 1891, 269(109, 135, 189, 284 (89, 109, 189, 292, 295), 285(297), 286 (297), 287(189, 292, 297), 288(89, 135, 292, 299), 289(89), 290(107, 114, 297), 292(189, 292, 297, 299), 293(89), 296, $97, 298, 301 Neth, R., 122(38), 168 Neu, H. C., 377(105), 415(105, 259, 260), 416(260, 277), 422, 426 Newton, A., 122(48), 125(48), 137(88), 168, 169 Nicolaieff, A., 43(162), 62(162), 99 Nihei, T., 229(173), ,942 Nikitina, L. A,, 275(201), Z99 Nirenberg, M. W., 118(23), 119(33), 120 (33, 34, 351, 122(35, 361, 128(33, 147 (1241, 167,168,iri, 175(18, ig), 193, 198(19), 938, 250(91), 263(91), 281 (266), 996, 300 Nishimura, S., 139(98), 148(126), 170, 171, 175(22), 193 Nishimura, T., 278(226), 999 Nixon, R. F., 304(21), 326(21), 327(21), 329(21), 5.46 Nodes, J. T., 371(30, 32, 361, 372(32, 361, 377(109), 384(32), 385(32), 420, 491, 429 Nohara, H., 82(401), 83(401), 106 Noll, H., 1281551, 130155), 169, 179(40), 181(43a), 193, 194, 223(144), 233 (1441, 235(144), 94f Nomura, M., 150(138), I71 Norman, A., 414(254), 416(254), 496 Norton, J., 427(N2), 429 Notani, G., 174(1), 175(1), 176(1), 193 Novelli, G. D., 139(98), 170, 249(85), 296 Novick, A., 278(229), ,999 Nowell, P., 21(43), 24 Nowell, P. C.,1(1), 93 Nghan, W. L., 32(64), 96
0 Ochoa, S., 118(27), 119(29, 301, 149(27, 29, 1321, 150(132), 152(27), 153(27, i55(158), 167,168, i n , ird, 175 (20, 23), 193, 372(42), 378(42), 384 (421, Ockey, C. H., 21(44), d4 O’Dell, R. A., 45(183), 99 Odmark, G., 342(95), 347 Ofengand, E., 142(102), 170 Ogata, K., 82(401), 83(401), 106 Ogoshi, H., 373(53), 421 Ohba, Y., 63(306), 64(322), 109, 103 Ohlenbusch, H., 49(217), 53, 100, 101 Ohnishi, T., 40(132), 98 Ohtsuka, E., 118(28), 121(28), 122(28, 371, 128(28), 147(123), 148(126), 167, 168, 171, 175(21), 193 Okada, S., 373(72), 379(’12), 401(208), 491, 426 Okada, Y., 122(48), 125(48), 168, 281 (268), 300 Okamura, N., 206(58), 211(92), 212(94), 213(92), 219(92), 939, 240 Okuba, S., 429(N13), 429 Olivera, B., 49(217), 53(251b), 100, 101 Oncley, J. L., 53(251a), 61(251a), 101 O’Neal, C.,120(35), 122(35), 168 Ono, J., 361(65), 365(65), 568 Ontko, J. A., 73(367), 104 Opara-Kubinska, Z., 133(72), 169 Orce, L., 366(85), 368 Ord, M. G., 49(215), 71(215, 3551, 83 (3551, 84(408, 409, 85(355, 4151, 100, 104, 106 Ordy, J. M., 402(213), 4.26 Orengo, A., 66(336), 90(336), 91(436), 103, 248(75), 996 Orgel, A., 129(56), 169, 365(83), 368 Orgel, L. E., 113(10), 167, 349(1), 366 Orr, M. D., 328(76), 346 Ortiz, P. J., 372(42), 378(42), 384(42), 421 Osawa, H., 39(127), 98 Osawa, S., 38(109), 73(109), 98, 247(61),
iw,
296
O’Sullivan, D. G., 392(187), 424 Oth, A., 372(49), 373(49), 373(49), 421
453
AUTHOR INDEX
Oura, H., 128(55), 130(55), 169, 181(43a), 194, 223(144), 233(144), 235(144), 241 Ozaki, H., 245(34), 252( 1031, 256(34), 267(34, 1851, 294, 296, 298
P Padieu, P., 175(16, 17), 193 Pailr, W. K., 85(412), 106 Painter, R. B., 2(4), I Palade, G. E., 213(109), 215(109), 221 (139), 240, 241, 386(168), 417(282), 424, &6 Palau, J., 54(253), 66(253), 68(253), 101 Palmade, C., 67(344), 103 Papaconstantinou, J., 249(86), 296 Pardee, A., 131(62), 169 Parisi, E., 280(242a), 300 Parmeggiani, A., 122(38), 168 Parnas, H., 182(48), 194, 210(88), 212 (88), 213(88), 2lS(SS), 223(88), 229 (88), 240, 266(161), 998 Parry, R. W., 353(28), 367 Parsons, J., 217(129), 841 Pasteels, J., 245(35), 294 Patel, G., 32(72, 73), 38(111), 97, 98 Paul, J., 94(468, 4691, 106 Pauling, L., 116(17), 123(44), 139(93), 142(93), 158(17), 167, 168, 170 Pavan, C., 279(237), 280(240, 242, 247), 300 Pavlik, J. G., 429(N12), 49.9 Peacocke, A. R., 51(228), 53(228), 60 (228), 61(290, 291), 63(306, 312, 315), 100, 102, 103 Pearse, A. G. E., 390(182), 392(189), 396 (182), 402(189), 407(189), 424 Pearson, M., 359(60), 368 Pegoraro, L., 2(13), 4(21), 7(13, 24), 8 (251, 9(26), 12(29, 301, 13(29), 14 (29, 41), 16(24, 35, 41), 20(42), 23, 94 Pelling, C., 280(246), SO0 Pelmont, J., 229(175), 242 Penman, S., 206(60), 217(60), 218(131), 221(137), 223(137, 147), 229(147), 231(137), 239, 2.41, 249(82), 250(95), 266(159), 281(159), 296, 298, 364(79), 368
Penniston, J. T., 139(99), 170 Penwick, J. R., 139(94), 140(94), 142 (941, 170 Peraino, C., 197(15), 238 Perl, W., 191(65), 194, 290(300), 301 Perretta, M., 428(N6), 429 Perrin, D., 278(218), 999 Perry, R. P., 201(32), 202(32), 207(32, 68), 209(32, 781, 212(32), 213(32), 216(116), 219(32), 220(32), 223(32, 142, 1901, 231(32), 236(32), 238, 239, 240, 241, 2&, 266(157), 270(193), 277 (193), 281(193), 283(291), 298, 299, 301 Peterkofsky, A., 144(112), 148(128, 1301, 155(112), 170, 171 Petermann, M. L., 199(22), 201(22), 217 (22), 238 Peterson, E. A., 372(40), &I PetroviE, J., 221 (1331, 223033) , 225(165), $41, PetroviE, S., 221(133), 223(133), 225(165), 241, 243 Phillips, D. M. P., 25(3), 48(197), 49(3, 203), 51(235), 53(3, 2351, 54(3, 235, 255, 257, 259, 260), 55(3, 197, 260, 262), 56(203, 235, 262), 57(260), 58 (274), 59(262), 61(297), 62(300), 65 (3), 66(265), 80(3), 82(260), 85(262, 414), 91(454), 95, 100, 101, 102, 105, 106 Phillips, J. H., 354(37, 38), 355(38, 43), 358(43), 359(38), 360(63), 361(70), 362(38, 63, 701, 367, 368 Philpot, J. S. I,., 76(381), 93(381), 104 Piatigorsky, J., 245(34), 256(34), 267(34, 185), 294, 298 Picheral, B., 275(202), 276(202), 299 Piha, R. S., 72(356), lo4 Pileri, A., 2(13), 4(21), 7(13, 241, 8(25), 9(26), 16(24, 35), 20(42), 23, 94 Pinzino, C. J., 267(180), 269(180), 271 (180), 898 Pitha, J., 364(81), 368 Pithova, P., 364(81), 368 Pitot, H. C., 197(15), 221(132), 238, 241 Plashna, M., 132(65a), 169 Platt, D. B., 30(33, 341, 92(33, 34), 96 Platt, J. R., 358(54), 368
AUTHOR INDEX
454 Pochon, F., 396(201), 403(201), 426 Poddar, R. K., 350(19), 351(19), 352(19),
367 Pogo, A. O., 38(108), 80(394), 82(394), 83(394), 84(394), 86(394), 87(394), 92(394), 93(394), 94(394), 95(475), 97, 106, 106 Pogo, B. G. T., 38(108), 80(394), 82(394, 402), 83(394, 4021, 84(394), 86(394), 87(394), 92(394), 93(394), 94(394), 97, 106 Pollister, A. W., 26(14), 31(14, 55), 40 (1351, 48(14), 50(14), 60(14), 96, 96, 98 Poort, C., 40(138, 139), 98 Porter, K. R., 245(17), 294 Potter, J. L., 373(72), 379(72), @ l Potter, V. R., 342(%)', 347, 413(241), 415 (264), 426 Preiss, J., 413(249), 4% Prensky, W., 65(327), 103 Prescott, D. M., 2(5), 23, 30(35, 361, 72 (360, 361), 80(361), 81(361), 92(35, 36), 96, 104, 198(18), 220(18), 3-38, 244(10), 261(149), 276(10), 291(10), 294, 297 Pressman, B. C., 384(153), 423 Preston, B. N., 61(291), 63(312), 102 Price, W. C., 62(298), 102 Prokoshin, B. D., 212(100), 218(100), 219 (loo), 223(100), 229(100), 230(100), 240 Prusoff, W. H., 341(90), 347 Puglisi, P. P., 154(155), 172 Pullman, B., 160(168), 172 Purkayastha, R., 67(337), 91(437), 103
Q Quatrano, R. S., 67(338), 91(438), 103 Quertier, J., 251(96), 265(96), 267(179), 296, 298
R Raab, W., 414(252), 4.96 Raaf, J. H., 49(215), 71(215), 100 Rabinovits, M., 74(371, 3721, 104 Racker, E., 304(17), 346 Rahman, Y. E., 374(78), 42.9
Rajbhandary, U. L., 139(97), 140(97), 148(97), 170 Rake, A. V., 221(140), 241 Rall, T. W., 375(95), 42.9 Ralph, R., 212(97), 219(97), 240 Ramenskaya, G., 197(17), 238 Randall, J., 72(358), 80(358), 85(358), 104 Rasmussen, P. S., 48(192), 49(205), 50 (205), 51(231), 54(205, 2311, 55(231), 56(231), 57(231), 58(192), 68(205), 99, 100 Rauth, A. M., 429(N14), 429 Ravel, J. M., 191(68), 194 Raven, C. P., 245(15), 294 Ray, W., 146(119), 157(119), 170 Razzell, W. E., 370(6), 371(38), 372(38), 374(79, 80),375(79, 83, 91), 376(79), 377(38), 379(6, 831, 380(6, 79, 83, 91), 388(79), 389(79), 398(91), 402 (214), 403(83), 420, 421, 422, 426, 428 (N9), 429 Reese, C. B., 364(75), 368 Reich, E., 63(310), 80(310), 89(310), 102 Reichard, P., 304(1, 4, 9, 18, 19), 305(9, 23, 25, 26, 27), 306(28, 29, 301, 307 (27), 308(18), 308(18), 310(19), 311 (35, 36, 41), 312(42), 313(19, 421, 314(19, 29, 52), 315(19), 316(54), 317 (52, 54), 320(29), 323(64), 327(75), 328(36, 42, 751, 329(19, 52, 54, 751, 330(75), 335(19), 337(64), 338(30, 87), 339(23), 340(23), 344(1), 345, 346, 347 Reichmann, M. E., 174(2), 175(2), 176 (21, 192 Reid, B. R., 50(224), 73(224), 100 Reid, E., 209(76), 289, 371(30, 32, 36), 372(32, 36), 384(32), 385(32, 165), 386(165), 420, U1,424 Reiter, H., 418(294), 419(297), 4.87 Reithel, F. J., 234(194), 242 Rendi, R., 38(106), 39(106), 97 Revel, M., 148(129), 171, 197(14), 221 (138), 223(138), 224(138), 226(138), 229(138), 230(138), 232(138), 233 (138), 235(138), Z38, 241, 283(288), 301
455
AUTHOR INDEX
Rich, A., 111(7), 113(7), 149(131), 16’7, 171, 179(37, 39, 41), 193, 364(78), 368 Richards, F. M., 377(112), 42.2 Richardson, C. C., 418(288, 289), 427 Rifkin, D., 156(161), 172 Rifkin, M. R., 156(161), 172 Rifkind, R. A., 191(65), 194, 290(300),
sot Rigler, R., 83(404), 105 Rinaldi, A. M., 248(73), 250(73, 931, 251 (73, 93), 261(73, 93), 262(93), 263 (93), 265(93), 292(93), 296, 2996 Ris, H., 31(48), 34(81, 82, 86), 41(144), 47(144), 61(86), 64(86), 96, 97, 98, 196(7), 238 Ritossa, F. M., 280(256, 2571, 300 Ro, T. S., 37(102, 1041, 97, 375(85), 386 (85), 388(85), 422 Robbins, J. H., 83(403), 105 Robbins, M., 418(296), 419(297), 427 Robert, M., 86(417), 105 Roberts, W. K., 210(86), 212(86), 240 Robinson, M. G., 63(313, 3141, 103 Robinson, W. G., 336(83), 337(83), 347 Robinson, W. S., 61(67, 681, 368 Rodinov, V. M., 30(29), 96 Roll, P. M., 304(3), 344(3), 946 Roman, A,, 202(43), 204(43), 239 Romanoff, P., 248(70), 249(70), 295 Rondoni, P., 116(19), 167 Roodyn, D. B., 383(145), 417(283), 423, @6
Rosa, J., 175(12, 131, 193 Rosas del Valle, M., 283(283), 301 Rose, I. A., 304(2), 344(2), 346 Rosenbaum, E. H., 16(37), 24 Rosenberg, M., 40(141), 98 Rosencwajg, R., 206(61), 239 Rosenkranz, H. S., 32(68), 96 Rosenthal, A. S., 402(209), 407(209), 425 Roth, J. S., 370(9, 17, 18), 371(9, 29, 31, 35), 372(9, 31), 373(57), 374(74), 376 (9), 377(9), 378(17), 379(9, 57), 384 (152), 385(9, 18, 35, 152, 157), 386 (35, 166, 169), 387(174), 388(174), 409(223), 410(9, 234, 238), 412(238), 417( 166), .$30. @ f , @3, @.$, $35, .j%, 427(N2), 62.9 Roth, T. F., 245(17), 294
Rotherham, J., 70(349), 71(349), 103, 373(65), 387(65), 388(65), Rottman, F., 120(35), 122(35), 168 Rovera, G., 12(29, 301, 13(29), 14(29, 411, 16(41), 24 Rowley, J., 2(7), 8(7), 14(7), 2.9 Rubin, A. D., 212(98), 219(98), 240 Rubini, J. R., 7(23), 94 Rudkin, G. T., 279(234), 280(241), 300 Rudolff, V., 168 Rucckert, R. R., 74(369), 104 Runnstrom, J., 245(48), 246(48, 53, 56), 265(156), 277(48), 293(304), 295, 298, 301
Russel, E., 175(10), 193 Rutberg, L., 304(9), 305(9, 27), 307(27), 346
Rutter, W. J., 249(87), 296 Ryclilek, I., 356(50), 367
S Sabatini, D., 221(139), 241 Sable, H. Z., 304(7, 8),345 Safer, B., 78(389), 104 Sager, R., 155(159), 172 Saidov, C. M., 33(78), 97 Sakai, H., 39(129), 98 Salas, M., 118(27), 122(39), 149(27), 152 (27), 153(27, 1451, 16Y, lF8, 171, 175 (231, 193 Salb, J. M., 261(148), 262(148), 265(148), 297 Salganicoff, L., 389(177, 177a), 390(177a), 424
Salser, J. S., 32(69, 701, 91(69, 701, G6 Snluste, E., 304(1), 344(1), 346 Samarina, 0.P., 26(17), 32(59), 95, 96, 197(17), 212(99), 213(106), 215(106), 219(99), 238, 240, 282(276), 501 Sambrook, J. F., 120(40), 122(40), 136 (40), 168 Samorajski, T., 402(213), 426 Sanchez, C., 278(218), 299 Sandberg, A. A., 2(15), 14051, W38, 39, 40), 23, 24 Sandeen, G., 377(106), 4% Smdm, C., 358(55), 568 Sander, G., 122(38), 268
456 Sanger, F., 225(168), 242 Sarabhai, A. S., 128(53), 135(53), 168 Sarin, P. S., 165(171), 172 Sarkar, N. K., 416(271), 418(291), 496, @7 Sarnat, M. T.,202(39), 238 Satake, K.,48(192), 51(231), 54(231), 55 (2311, 56(231), 57(231), 58(192), 99, 100 Sato, C. S., 38(105), 97 Sato, K.,278(226), ,999 Sato, S., 377(111), 428 Saunders, G. F., 133(74), 169 Sautiere, P.,51(241), 54(261), 55(261), 101 Sawada, F., 42(159), 43(160), 159, 160, 99 Sawada, S., 41(148), 42(148), 98 Saxingcr, W.C., 142(101), 159(167), 160 (101, 167), 164(167), 170, 178 Scaife, J. G., 132(69), 169 Srarano, E.,248(75), 259(141), 280(242a), 295, 297, 300, 342(93), 347 Schachman, H. K., 310(34), 546 S-hachter, H.,415(263), 436 Srhaeffer, J., 176(24, 251, 195 Srhapira, G.,175(12, 13, 16,171, 176(30), 177(31), 178(30, 35, 361, 193 Srhell, P., 353(31, 32), 354(32), 355(32), 364(32, 771, 365(82), 367, 368 Scherbaum, 0. H., 67(340), 73(340), 10s Srherrer, K.,182(45, 46, 47), 194, 209 (771, 210(87), 212(87, 95, 103, 104), 213(104), 214(104), 218(103, 104), 223(104, 1471, 229(147), 939, 840, 241, 249(82), 266(158, 1601, $96, 298 Schildkraut, C. L., 418(288, 289), 48'7 Schlesinger, S., 278(221), $99 Schlessinger, D.,174(4), 179(4), 181(4), 192, 377(107), 416(107), 428 Srhmid, W., 14(31), 21(31), t!4 S-hncidcr, W.C., 384(150), 385(150), 387 (1501, 423 Schneider-Bernlohr, H.,357(53), 368 Schoch, G.,122(38), 168 Schor, N.,37(97), 97 Schottelius, D.D., 373(65), 387(65), 388 (651, 4-91 Schram, E., 185(51), fQ4
AUTHOR INDEX
Schubert, G., 394(195), 424 Schuffman, S. S.,402(209), 407(209), 426 Schulta, J., 244(4), 280(4), 294 Schuster, H.,351, 352(16, 17), 353(17, 33), 356(33), 362(33), 367 Schwartz, A,, 78(386, 387, 388, 389, 3901, 104 Schwartz, J. H.,174(1), 175(1), 176(1), 192
Schweet, R., 116(15), 149(15), 167, 174 (8), 175(10, 151, 176(24, 251, 193 Schweigert, B. S., 204(2), 324(69), 344 (21, 346, 346 Schwimmer, S., 81(3981, 105 Scott, J. F., 165(171), 172 Scott, R. B., 249(83), 296 Searaski, T.,418(296), 427 Sccd, R. W., 249(84), 296 Seifert, R.,413(249), 426 Sckcris, C. E.,86(420), 87(429), 105, 216 (127), 241 Sekignchi, M., 415(265), 426, 429(N13), 429 Seligman, A. M., 408(219), 425 Sellingcr, 0.Z.,384(156), 424 Setlow, R. B., 359(59), 3G8, 418(292), 419(298), 4fl Setterfield, G., 38(114), 98 Shack, J., 372(50), 373(50), 4 1 Shanta, T. R., 403(215), 406(215), 426 Shapiro, H.S., 373(52), 4Zl Shapiro, R.,355(45), 367 Shapot, V.,221(132), 241 Shear, M.,390(182), 396(182), 424 Shearer, R. W., 214(115), 940, 266(164), 268(164), 291(164), 295 Sheldrick, P.,133(72), 169, 201(34), 238 Shepard, W. M.,126(51), 168 Shepherd, G. R.,49(216, 218, 219), 50 (218,219), 70(216, 218,219), 81(216), 90(216), 100 Sherbert, G. V., 90(447), 106 Shibaevn, R. P., 355(42), 367 Shigeura, H. T., 205(53), 23.9 Shimada, K.,429(N13), 4% Shiomi, H.,67(339), 91(439), 103 Shooter, K. V.,48(187), 51(226), 561226, 267), 91(454, 456), 99, 100, 101, 106 Shortman, K., 370(19), 371(19), 377
457
AUTHOR INDEX
(1031, 378(103), 379(103), 386(103), 420, 422
Shoup, R. R., 364(80), 368 Shugar, D., 323(66), 346, 354(46), 355 (41, 46a), 359(46, 58), 364(46), 567, 368, 371(24), 374(82), 378(114), 380 (122), 381(124, 125), 382(114, 1221, 384(148), 388(122), 390(181, 1831, 391(183), 392(183, 186, 1901, 394 (114, 1901, 396(114, 183, 1901, 397 (190), 398(114, 122), 399(114, 1901, 400(114), 402(190), 403032, 1221, 406 (1221, 406(122), 408(220), 414(114, 1221, 418(293), 4.203 4.22, 4.239 4241 4.356,
w
Shukulov, R. S., 187(55), 194 Shuppe, N. G., 212(100), 218(100), 219 (loo), 223(100), 229(100), 230(100), 233(192), 235(192), 240, 242 Sibatani, A,, 215(118, 1231, 240, 2.41 Siddigi, O., 135(80), 151(80), 169 Siebert, G., 37(104), 97, 209(76), 239, 373(56), 375(85), 379(56), 383(56, 140, 147), 385(165), 386(56, 85, 165), 387(56), 388(85), 4.21, 422, 423, 4-94 Siekevitz, P., 213(109), 215(109), 940, 386(168), 417(281, 282), 424, 496 Sierakowska, H., 371(24), 374(82), 378 (114), 380(122), 382(114, 122), 384 (148), 388(122), 390(181, 1831, 391 (183), 392(183, 186, 190), 394(114, 190), 396(114, 183,190,,397(190), 398 (114, 122), 399(114, 190), 400(114), 402(190), 403(82, 122), 405(122), 406 (1221, 414014, 1221, 480, 493, 423,
4-0,; Signoret, J., 275(200), 999 Simms, E. S., 320(56), 346 Simnett, J. D., 275(203), 276(203), 1 9 Simon, M. I., 374(73), 4 1 Simson, P., 38(112), 49(203), 51(235), 63 (235), 56(203, 2351, 58(274), 66(332), 98, 100, 101, 102, 103
Singer, M. F., 196(5), 198(5), 199(5), 207(5), 220(5), 223(5), 231(5), 235 (5), !B8, 416(273, 2741, 4.26 Singh, U. N., 207(73), 939 Sinsheimer, R. I,., 373(61, 67, 681, 378 (681, 421
Sirlin, J. L., 37(99), 92(465), 97, 106, 206(67), 207(67), 217(67), 239, 255 (121), ,997 Sizer, I. W., 79(392), 106 Skalka, A., 76(378), 80(378), 93(378), 104 Slater, D. W., 227(164), 248, 248(74), 253 (741, 296 Slayter, H. S., 179(41), 193 Sluyser, M., 77(383), 79(383), 87(428, 4301, 93(383), 104, 106 Smellie, R. M. S., 32(60), 96, 373(56), 374(75, 76, 77), 379(56), 383(56, 141), 386(56), 387(56), 421, 422, 423 Smetana, K., 31(53, 541, 37(100), 90(53, 54), 96, 97, 213(110), 240 Smillie, I,. B., 53(246), 101 Smirnov, M. N., 212(99), 219(99), 240 Smirnov, V. N., 283(281), 301 Smit, J. A,, 49(215), 71(215), 100 Smith, A. D., 385(162, 163), 387(163),
4.24 Smith, D. B., 53(246), 101 Smith, E. L., 136(87), 169 Smith, H. H., 65(327), 103 Smith, I., 266(159), 281(159), 298 Smith, J., 386(173), 387(173), 4 2 i Smith, J. D., 154(151), 171 Smith, K. C., 377(104), 422 Smith, L. D., 255(125), 256(125), 257 (125, 130), 258(131), 268(125), 276 (2111, 297, 299 Smith, L. K., 83(407), 91(462), 92(462), 106, 106
Smith, M., 44(169), 99, 381(126, 130, 131), 382(134), 407(216), 423, 425 Smith, M. A,, 118(27), 149(27), 152(27), 153(27), 167, 175(23), 193 Smith, M. S., 320(60), 346 Smith, N., 245(25), 294 Smith, S., 255(122), 297 Snell, E. E., 324(68), 346 So, A. G., 149(134), 171 Sobel, B. E., 311(38), 346 Sober, H. A., 372(40), 421 Soeiro, R., 199(24), 210(84), 212(84), 213 (24), 218(24, 84), 219(24), 235(24), 258, 240, 266(162, 163), 268(163), 298 So11, D., 122(37), 147(123), 168, 171
458 Sofer, W. H., 259(140), 262(140), 287 (298), 292(298), 297, 301 Somers, C. E., 350(8), 366 Somerville, R. L., 320(58), 346 Sonnenberg, B. P., 80(395), 106 Sonneborn, T. M., 163(170), 172 Sorm, F., 356(50), 367 Soroff, S., 40(19), 96 Soudek, D., 40(141), 98 Spahr, P. F., 38(105), 98, 377(107), 415 (258, 266), 416(107), 422, 426 Spalding, J., 73(362), 80(362), lo4 Spemann, H., 245(41), 296 Spencer, T., 283(282), 301 Speyer, J. F., 119(29, 301, 149(29), 155 (158), 168, 172 Spicer, V. L., 355(44), 86'7 Spiegel, M., 251(97), 252(97, 1031, 296 Spiegelman, S., 133(73), 169, 172, 174(2), 175(2), 176(2), 192, 202(36, 381, 227 (164), 238, 242, 247(59), 248(74), 253 (74), 296 Spirin, A. S., 187(55), 194, 199(21), 201 (21), 2171211, 220(21), 232(189), 234 (189), 238, 242, 254(118), 282(277), 283(281, 284, 285), 284(277, 284, 285, 292, 2%), 285(277, 2851, 287(277), 288(292), 292(284, 285, 2921, 297, 301 Spitkovsky, D. M., 359(61b), 368 Sporn, M. B., 90(449), 106, 206(57), 216 (1251, 239, 241, 427(N3), 429 Spyrides, G. T., 119(32), 168 Sreenivasan, A., 370(20), 385(20), 386 (201, 420 Srinivasan, P. R., 214(116), 24O Stadtrnan, T. C., 314(51), 346 Staehelin, M., 200(31), 238 Staehelin, T., 128(55), 130(55), 150(138), 169, 171, 179(40), 181(43a), 193, 194, 223(144), 233(144), 235(144) $41 Stafford, D. W., 39(130), 98, 287(298), 292(298), 801 Stahl, F. W., 131(60), 169, 350(20a), 367 Stanley, W. M., Jr., 118(27), 122(39), 149(27), 152(27), 153(27, 145), 167, 168, lY1, 175(23), 193 Starhuck, W. C., 25(6), 70(351), 71(6), 73(6), 78(3881, 96, lU3, 104 Stavy, L.,417(280), 426
AUTHOR INDEX
Staynov, D. Z., 200(27), 238 Stedman, Edgar, 48(188, 1951, 49(188, 198, 199), 51(188, 199, 2381, 52(199, 231), 56(195, 2381, 57(195), 6 5 ( W 198, 330, 3311, 67(188, 1981, 74(188, 198), 79(188, 1981, 99, 100, 101, 10s Stedman, Ellen, 48(188), 49(188, 1981, 51 (188), 65(188, 198), 67(188, 1981, 74 (188, 1981, 79(188, 198), 99, 100 Steele, W. J., 25(4), 26(16), 27(4, 2519 30(4), 31(53), 32(4, 16, 671, 33(4, 16, 75), 37(104), 38(4,75), 40(4),48(16), 58(75), 70(4), 71(4, 16), 73(4), 90 ( 5 3 , 91(67, 751, 96, 96, 97, 206(58), 213(113), 216(113), 219(113), 239, 240, 375(85), 386(85), 388(85), 422 Steers, E., Jr., 279(232), 800 Steigleder, G. K., 414(252), 426 Stein, W. H., 51(229, 230), 56(229, 2301, 66(230), 100 Steinberg, C. M., 135(81), 169 Stent, G. S., 154(153), 172, 202(40, 41), 23.9, 278(220), 289(220), 299 Stern, B. K., 312(47), 346 Stern, H., 25(2), 31(2), 70(2), 96 Sternberger, L. A., 394(194), 424 Stevely, W., 83(406), 84(406), 106 Stevens, B. J., 281(173), 301 Stevens, H., 370(14), 420 Stewart, J. A., 249(86), 296 Stich, H. F., 281(274), 301 Stocken, L. A., 49(215), 71(215, 355), 76 (380), 83(355, 406), 84(406, 408, 4091, 85(355, 380, 415, 416), 100, 104, 106 Stockx, J., 378(113), 422 Stollar, D., 374(73), 379(119), 421, 422 Storck, R., 198(20), 238 Strack, H. B., 350(13, 14), 351(13), 352 (13), 359(13), 366 Straus, W., 385(158, 1591, 388(158, 159),
494
Strauss, B., 418(294, 296), 419(297), 4 f l Strauss, N., 89(441), 106 Streisinger, G., 122(48), 125(48), 168, 281(268), 300, 351 (20a), 367 Stretton, A. 0. W., 128(53), 135(53, 82), 136(86), 153(86), 154(86, 150), 168, 16.9, 171, 350(4), 366 Stuart, A,, 139(97), 140(97), 148(97), 170
AUTHOR INDEX
459
Tavares, A. S., 402(211), 425 Taylor, A. L., 133(71), 169 Taylor, C. W., 33(75), 38(75), 58(75, 275), 66(275, 333), 91(75), 97, 102, 103 Taylor, J. H., 2(2, 171, 23, 196(6), 2358 Taylor, M. M., 198(20), 238 Taylor, P. R., 381(128), 423 Telfer, W. H., 245(19, 20, 21), 294 Teller, D. C., 56(273), 57(273), 102 Tencer, R., 248(67), 295 Tencheva, Z. S., 206(63), 23.9, 419(liU), 429 Terman, S. A., 252(104), 296 Terzaghi, E., 122(48), 125(48), 168, 281 (268), 300, 351(20a), 367 Tessman, E. S., 352(24), 367 Tessman, I., 351(19, 20, 21), 352(19), 353 (251, 367 R. E., 153(146), 171 Thach, 494 Thedford, R., 383(136), 423 Symeonidis, A., 375(93), 4.92 Sypherd, P. S., 89(449), 106, 166(175), Thelander, L., 304(19), 310(19), 312(42, 43, 46),313(19, 421, 314(19), 315(19), 178 328(42, 43), 329(19), 335(19), $45, Szempliiiska, H., 380(122), 382(122), 388 $46 (122), 390(1811, 398( 122), 403( 1221, 405(122), 406(122), 414(122), 423, Thomas, L. E., 26(12), 27(12), 31(42, 43, 44, 45), 95, 96 4.94 Thomasson, W. A., 50(220), 100 Szer, W., 149(132), 150(132), 171, 359 Thornblom, D., 246(53), 295 (62), 368, 381(125), 423 Szybalski, W., 133(72), 169, 201(34), 238 Thung, P. J., 77(383), 79(383), 93(383), 104 T Tice, L. W., 408(218), 425 Tidwell, T., 85(413), 105 Tabachnick, J., 414(253), 426 Tikhonenko, T . I., 359(61a,b), 368 Tabor, C. W., 63(308), 102 Tillett, W. S., 409(228), 425 Tabor, H., 63(308), 102 Timourian, H., 262(150), 297 Takagi, Y., 429(N13), 429 Tishoff, G. H., 27(21), 34(21), 96 Takahashi, I., 320(57), 346 Tocchini-Valentini, G. P., 202(39), 238 Takahashi, K., 383(139), 423 Tocco, G., 248(75), 296 Takahashi, T., 82(401), 83(401), 105 Todd, A. R., 364(75), 368, 371(23), 420 Takai, M., 247(61), 236 Toennies, G., 31(49), 96 Takanami, M., 356(48), 367 Tolbert, G., 416(273, 274), 426 Tal, M., 417(279), 426 Tolis, H., 256(128), 258(128), ,297 Tamm, C., 373(52), 421 Tomkins, G. M., 223(141), 241, 249(88), Tamoda, J., 215(123), 241 296 Tanaka, Y., 40(132), 98 TomIin, S. G., 40(143), 98 Tashiro, Y., 221(139), 241 Tompkins, G., 278(227), 279(227), 281 Tata, J. R., 95(474), 106 (2271, 299 Tatarinova, S. G., 353(26), 367 Tooze, J., 137(90a), 170 Tatum, E. L., 108(2), 167
Stubblefield, E., 2(la), 23 Subak-Sharpe, H., 126(51), 168 Subtelny, S., 255(125), 256(125, 129), 257 (125), 268(125), 276(213), 2 g , 299 Sueoka, N., 144(108, 1091, 146(109, 117), 170 Sundararajan, T. A., 153(146), 171 Susman, M., 135(81), 169 Sutherland, E. W., 375(94, 95), 376(97), 377(94), 380(94), 389(94, 971, 390 (971, 422 Suttie, J. W., 417(283), 426 Suutarinen, P., 373(51), 421 Suyama, T., 245(25), 894 Suzuki, K., 42(153), 99, 418(295), 427 Swan, R. J., 359(61), 368 Swift, H., 34(85), 64(85), 97, 281(272), 301 Swingle, K. F., 87(435), 106, 388(175),
460
AUTHOR INDEX
Tough, I. M., 16(33), 84 Trager, L., 324(70), 346 Trakatellis, A., 223(146, 149), 233(146), 235(146) , 841 Traub, P., 150(138), 171 Trautmann, M.L., 373(54), 4 1 Trautman, R., 56(272), 57(272), 101 Trevithick, J. R., 44(169), 84(411), 99, 106
Trojanova, J., 74(368), 104 Trosko, J. E., 429(N15), 429 Troll, W., 354(36), 367 Trupin, J., 120(35), 122(35, 36), 168 Trupin, K. M., 385(161), 417(161), 4B4 Tsanev, R. G.,200(27, 28, 291, 206(28, 591, 2230501, 226(154), 229(154), 230(154), 233(191), 238, $39, ,941, 2&
Tseytlin, P. I., 359(61b), 368 Ts’o, P. 0.P., 26(8), 38(105), 43(183), 53(163), 87(426), 96, 97, 99, 106, 358 (55), 368 Tsugita, A., 122(48), 123(45), 125(48), 168, 281(268), 300 Tsumita, T., 31(57), 32(57), 96 Tsvetikov, A. N., 51(231), 54(231), 55 (231), 56(231), 57(231), 100 Tuan, D., 53(251b), 1Of Tumanian, V. D., 213(106), 215(106),8@ Turner, A. F.,381(132), 423 Turner, M. K., 209(76), 239, 385(165), 386(165), 424 Tyler, A,, 245(13, 34), 247(63), 248(68), 251(68, 971, 252(97, 103), 256(34), 259(63, 136, 137), 262(153), 263(63, 153), 265(136), 267(34, 1851, 287 (1361,294, 296, 296, 297, 298
U Uchida, T., 377(111), 383(139), 482, @3 Ueda, K., 253(112), 997 Ui, N., 48(194), 51(194), 53(194, 250), 56(194, 268, 269, 270, 2711, 57(194), 99, 101 Ulbricht, T. L. V., 353(29), 359(61), 367, 368
Ullman, A., 132(64), 169 Umafia, R.,30(31), 48(31, 1961, 72(358), 80(358), 85(358), 96, 100, lo4
Updike, S., 72(358), 80(358),85(358), 104 Uriel, J., 200(25), ,988 Urnes, P.,62(299), 102 Ursprung, H.,40(140), 91(458), 98, 106 Utsunomiya, T., 386(166), 417(166), 494
V Valentine, R. C., 311(37), 346 Valeva, L. V., 213(108), 215(108), g4U van Breugel, F. M. A., 280(258), 300 Van de Woude, G. F.,49(214), 100 Van Lancker, J. L., 386(172), 484 Vamey, N.,145(116), 170 Venatanier, P., 279(230), 281(230), 890 Vandrely, C.,64(326), 103 Vendrely, R., 64(326), 67(344, 3461, 103 Venkov, P. V., 200(29), 205(52), 206(52, 65), 207(52), 209(52), 210(52), 212 (52), 213(108), 215(65, lOS), 216(65), 228(170, 171, 172), 229(171), 838, 839, 940, 848 Vennesland, B., 312(47), 320(62), 346 Veomett, R. C., 414(254), 416(254), 4% Verwoerd, D. W., 353(34, 35), 354(35), 356(34, 351, 367 Villalobos, J., 37(104), 97, 375(85), 386 (851,388(85), 4% Vincent, W. S., 205(50), 239, 266(167, 168, 169, 170, 172, 173, 1741, ,998 Vitols, E.,304(21), 314(50), 326(21, 74), 327(21, 74), 328(76), 329(21), 346, 346 Vittorelli, M. L., 248(73), 250(73), 251 (73), 259(138), 261(73), 296, 297 Voigt, H. P., 122(38), 168 Volkin, E., 191(62), 194, 205(49), 239, 247(60), 996 von Ehrenstein, G., 146(119, 1201, 147 (122), 150(140, 1411, 155(160), 157 (119), 170, 171, 172, 175(9), I93 von Vielmetter, W., 350(16), 35206), 567 von Zachau, H. G.,139(95), 140(95), 170 Vorbrodt, A,, 402(211), 411(239), 426, 426
W Wacker, A., 324(70), ,946 Wade, H.E., 415(267), 416(275), 4,96
461
AUTHOR INDEX
Waelsch, H., 49(213), 70(213), 72(356), 90(213), 100, lo4 Wagner, 0. W., 335(81), 336(81), 946 Wahba, A. J., 118(27), 119(29, 301, 122 (391, 149(27, 291, 152(27), 153(27, 145), 155(158), 167, 168, 171, 172, 175(23), 193, 320(59), 946 Waitzman, M. B., 403(215), 406(215), 495 Waldschmidt-Leitz, E., 42(158), 44(158), 99 Walker, G., 314(50), 346 Walker, I. O., 61(296), 63(296, 306, 3091, 102 Wall, R., 127(57), 169 Wallace, H., 255(121, 123), 277(215), 297, 299 Waller, J.-P., 38(108, 113), 98, 134(76), 1 0 Wan, Y. C., 49(211), 50(211), 100 Wang, K. M., 27(24), 96 Wang, T. Y., 27(22, 23, 24), 30(37), 31 (20, 42, 43, 521, 32(52, 72, 73, 741, 33(52), 38(20, 22, 37, 110, 111, 117, 119, 120, 121), 90(52), 95, 96, 97, 98 Wannemacher, R. W., 413(248), 426 Warner, J. R., 179(37, 39, 411, 193, 199 (24), 213(24), 218(24), 219(24), 223 (143), 235(24), 238, 841, 266(162), 2.98
Warren, J. C., 87(422), 106 Wartofsky, L., 313(48), 346 Wasserkrug, H., 408(219), 426 Watanabe, I., 42(155), 99 Waters, L., 248(72), 295 Watson, J. D., 137(90a), 158(163), 170, 172, 212(97), 219(97), 240 Wattiaux, R., 384(153, 154, 1551, 385 (154), 386(155), 387(155), 388(155), &$
Watts-Tobin, R. J., 128(54), 136(54), 168 Webb, M., 373(62), 491 Webb, S. J., 67(343), 103 Weber, K., 137(90a), 170 Webster, R. E., 134(77), 170 Weigert, M. G., 123(43), 169, 350(3), 366
Weinfeld, H., 304(3), 344(3), 346 Weinstein, I. B., 149(133), 150(133), 155 (159), 171, 172 Weisberger, A. S., 284(294), 290(294), 301 Weisblum, B., 1461119, 120), 147(122), 157(119), 170, 171 Weiss, J. J., 63(313, 3141, 103 Weiss, S. B., 87(434), 106, 361(66, 67, 681, 368 Wells, R. D., 118(28), 121(28), 122(28), 128(28), 167 Wessells, N. K., 249(87), 283(280), 296, 301
Westenbrink, H. G. K., 45(184), 53(247), 99, 101 Westheimer, F. H., 320(62), 346 Wettstein, F. O., 128(55), 130(55), 169, 179(40), 181(43a), 193, 194, 223(144), 233(144), 235(144), 9.41 Wheeler, C. M., 63(313, 3141, 103 Whichard, L., 78(391), 86(391), 104 Whitcut, J. M., 371(36), 372(36), 421 White, F. H., 370(1), 420 White, M. J. D., 279(236), 300 Whiteley, A. H., 249(79), 252(79), 267 (182, 183, 184), 274(79), 275(79), 291 (79), 296, 998 Whiteley, H. R., 249(79), 252(79), 274 (79), 275(79), 291(79), 295 Whitfield, P. R., 371(25, 26), 375(26), 420 Whittle, E. D., 342(98), 344(98), 347 Wicks, W. D., 213(107), .Z4O Widholm, J., 49(217), 100 Wiernik, P. H., 409(226), 413(226), 425 Wierzchowski, K. L., 359(58), 368, 381 (1241, 423 Wigler, P. W., 380(123), 381(129), 423 Wilkins, M. H. F., 43(161), 99 Wilkinson, B., 213(105), 230(184), 240, 242 Wilkinson, G. R., 62(298), 102 Williams, C. H., Jr., 312(44), 346 Williams, J., 245(11), 294 Williamson, R., 179(42), 194 Wilson, C. M., 417(286b), 427 Wilson, L. G., 314(49), 946 Wilson, J. D., 87(431), 106
AUTHOR INDEX
462 Wilson, R. G., 361(65, 711, 365(65), 368 Wilt, F. H., 262(152), 267(186, 1871, 283 (279, 2801, 297, 298, 901 Winkler, H.,385(162, 163, 387(1W, -494 Wittmann, H.G., 123(41), 168, 350(17), 352(17), 353(17), 367 Wittmann-Liebold, B., 123(41), 168 Witzel, H., 370(21), 397(202), 420, &5 Woese, C., 113(11), 118(11), 142(101), 147(11), 151(11), 158(165), 159(166, 167), 160(101, 1671, 161(169), 162 (169), 163 (11, 1691, 164(167), Ifl,
Z Zajdela, F., 182(46, 47), 194, 210(87), 212(87, 104), 213(104), 214(104), 218 (104), 223(104), 240 Zalite, B. R., 373(57), 379(57), 491 Zalokar, M., 245(22), 294 Zalta, J. P., 206(61), 239 Zamecnik, P. C., 145(116), 165(171), 170, 172
Zamenhof, S., 349(2), 351(2), 358(2), 366
Zamir, A., 139(94), 140(94), 142(94), 170 Zan-Kowalczewska, M., 371(24), 378 Wollman, E. L., 133(70), 169 (114), 382(114), 394(114), 396(114), Woods, W. D., 403(215), 406(215), 4% 398(114), 399(114), 400(114), 414 Work, T.S.,417(283), 426 (114), 419(N1), 420, 422, 429 Wright, E. M.,304(7), 346 Zbarsky, I. B., 26(17), 31(47), 33(76, 77, Wriston, J. C., 413(247), 426 80), 40(142), 95, 96, 97, 98, 197(17), Wyatt, G.,206(66), 299, 253(110), 296 255, 283(281), SO1 Wyrnan, J., 317(55), 346 Zillig, W., 353(34, 351, 354(35), 356(34, Y 351, 367 Yamane, T.,144(109), 146(109, 1171, 170 Zimmerman, A. M., 39(128), 98 Yang, J., 216(124), 241 Zimmerrnan, E.,41(149, 150). 98 Yanofsky, C.,122(46), 123(42, 461, 127 Zimmerman, E. F., 226(159), 242 (52), 132(67), l37(89), 1491671, 153 Zimmerman, S. B., 320(56, 611, 346, 377 (148), 154(149, 154), 168, 169, 170, (1061, 422 171, 172, 310(33), 346 Zinder, N. D., 134(77), 137(90), 169, 170, Yasuda, K., 396(199), 426 174(1), 175(1), 176(1), 192 Y&s, M., 111(7), 112(9), 16'7, 197(16), Zittle, C. A., 45(183), 99 205(50), %38, 239 Zubay, G., 60(278, 284), 61(278, 284), Yegian, C. D., 154(153), 172 62(278, 303), 80(395), 102, 106, 158 Yermolayeva, L. P., 40(142), 98 (1641,172 Yoshikawa-Fukada, M.,212(93, 941, 240 Zirckerkandl, E., 123(44), 168 Young, E. T., 11, 373(68), 378(68), 421 Zrigiiry, D.,396(201), 403(201), 426 Yu, C.-T., 146(118), 170 170, 172
Subject Index A
D
Allosteric regulation, ribonucleotide reduction and, 314320, 32%333 Amino acid(s), incorporation, rcticulocyte nucleic acid and, 175-179 Amino acid transfer activity, sea urchin egg, 259-261 Autoradiographic techniques, evaluation, approach to quantitation, 7-14 in vitro, 4-7 in vivo, 7
Deoxyribonucleases, methods of assay, 378-379 Deoxyribonucleic acid, chromosomal, leukemic cells, 16-22 normal blood cells, 14-15 replication, autora&ographic techniqucs, 4-14 conclusions and hypotheses, 22-23 general considerations, 1-2 Deoxyribonucleodepolymerases,types of, 372-374
B
E
Blood cells, normal, chramosomal deoxyribonucleic acid of, 14-15
C Cells, fractionation, nucleases and, 383390 Chick embryo, ribonucleotide reduction in, 33-9 Chromosomes, functional organization of, 278-283 recognition of, 2-4 Codon(s), assignments, in vitro polypeptide synthesis and, 117-121 other approaches, 121-126 size of, 12S130 Control mechanisms, ribonucleotide reduction and, 341-342 Crude extracts, ribonucleotide reduction and, 305-308, 32G326 Cyclic nucleoside phosphodiesterases, method of assay, 380 types of, 375-376 Cytochemistry, nucleases and, 392-408 Cytoplasm, messenger ribonucleic acid in, 220-235 Cytosine derivatives, hydroxylamine and, 354-356 463
Egg, activation of protein synthesis, schedules for, 255-259 as informational structure, 245-255 messenger ribonucleic acid in, 247-253 ribosomes of, 25S255 sea urchin, activation of protein synthesis in, 259-266 Embryogenesis, general considerations, 24S244 histones and, 89-90 nucleocytoplasmic interactions and, 27&278 regulation of genetic expression in, 278-290 summary of, 29C293 transitions in genic activity in, 266278 Endonucleases, methods of assay, 376-378 types of, 370-372 Enzymes, inhibition, histones and, 7 6 7 9 purified, ribonucleotide reduction and, 308-310, 326-327
Escherichia coli, cell-free preparations, reticulocyte nucleic acid and, 17&176 ribonucleotide reduction in, 305-324 Evolution, genetic code and, 161-163
464
SUBJECT INDEX
Exonucleaaes, methods of assay, 379-380 types of, 374-375
Genetic expression, developmental regulation of, 278-290 Genetic activity, transitions, embryogenesis and, 266267
F
H Fertilization, protein synthesis and, 256-259
G Gene ( s ), colinearity with polypeptides, 126128 repression, histones and, 79-89 Genetic background, hydroxylamine mutagenesis, 350-353 Genetic code, codon asignmenta, in vitro polypeptide synthesis and, 117-121 other approaches, 121-126 codon size and, 12&130 components, origin of, 163-167 the cryptographic problem, 117-126 direction of translation, 149 errors in translation-11 process, 149151 evolution, constraints and, 162-163 fundamental nature of, 155-167 general considerations, 107-109 historical, 109-117 indirect template and, 157-161 locked-in code and, 155-157 nucleic acid-polypeptide relationship and, 113-117 polypeptide chain punctuation, mechanism of, 152-153 problem of errors and evolution, 161162 punctuation and other encoded information, 130-132 theoretical attempts a t a cryptographic solution, 110-113 transcription punctuation and, 132-134 translation-I process, 142-146 translation-I1 process, 146-151 translation punctuation, 134-137 translation tape-reader and translation process, 138-165
Hemoglobin, information for, reticulocytes and, 174-175 Histochemistry, nucleases and, 390392 Histones, biosynthesis of, 70-74 cell and species specificity of, 65-70 embryonic development and, 89-90 enzyme inhibition and, 74-79 gene repression and, 7 W 9 moIecular function of, 6045 molecular properties of, 48-59 Hydrogen donor, ribonucleotide reduction and, 327-329 Hydroxylamine, cytosine derivatives and, 354-356 mutagenesis, conclusions, 364-366 experimental investigations of, 358364 general chemistry and, 353-358 general considerations, 349450 genetic background, 350-313 nuclei acids and, 353-364 polycytidylate and, 359-361 uracil derivatives and, 356-358
I Immunofluorescence, nucleases and, 393-396 Information transfer, orthodox interpretation, 235-236 a plausible unorthodox interpretation, 236-237 1 Lactobacillus kichmannii, ribonucleotide reduction in, 324-337 Leukemia, acute, chromosomal deoxyribonucIeic acid in, 1620
465
SUBJECT INDEX
chronic myeloid, chromosomal deoxyribonucleic acid in, 20-22
substrate preparations and, 380-383 types of, 370-376
M Mu tagenesis, hydroxylamine and, conclusions, 364-366 experimental inveatigations of, 358364 genetic background, 350-353 general chemistry and, 353-358 general considerations, 349-350 model of, 358-359 ribonucleic acid polymerase and, 361362
N Novikoff hepatoma, ribonucleotide reduction in, 339-341 Nuclear proteins, classification, origin and, 33-40 solubility and, 26-33 historical, 25-26 nonhistone, 90-92 summary and conclusions, 92-95 Nuclei, messenger ribonucleic acid synthesis in, 205-220 Nurleic acid, polypeptide relationship, genetic code and, 113-117 hydroxylamine and, 353354 Nucleocytoplasmic interaction, embryogenesis and, 275-278 Nucleolytic enzymes, cellular fractionation and, 383-390 comparison of cytochemical and fractionation findings, 407-408 cytochemical procedures and, 392-408 general considerations, 36S370 histochemical methods and, 3 W 9 2 immunofluorescence technique and, 393-396 methods of assay, 376380 natural inhibitors of, 319 pathological statcs and, 4 0 W 1 4 possible functions of, 414-419 precipitate forming techniques and, 396-407
0 Ooplasm, regionality of, 245-247
P Pathological states, nucleases and, 408414
Polycytidylate, hydroxylamine and, 35% 361
Polypeptide (s), colinearity with genes, 126-128 in vitro synthesis, codon assignments and, 117-121 nucleic acid relationship, genetic code and, 113-117 punctuation, mechanism of, 152-153 Polyribosomes, activity, regulation of, 287-290 isolation of messenger ribonucleic acid from, 17s191 Protamines, biological properties of, 43-47 chemical properties of, 41-43 Proteins, nonhistone, 90-92 synthesis, activation in sea urchin egg, 259-266 schedules for activation in eggs, 255259 scheme for dual activation of, 264266 Punctuation, genetic code and, 130-132 transcription and, 132-134 translation and, 134-137
R Reaction mechanism, ribonucleotide reduction and, 32M24, 335337 Rqdication, erroneous, mechanism of, 362-364 Reticulocytes, cell-free preparations, amino arid incorporation by, 176-179 information for hemoglobin in, 174175
466 ribonucleic acid, amino acid incorporation by, 175-179 Ribonucleic arid, cytoplasmic, carriers of, 230-235 cell-free polypeptide synthesis and, 230 labeling and turnover of, 221-224 molecular characteristics of, 220-221 mononucleotide composition of, 225230 information transfer by, general considerations, 196-198 mesaenger, changing populations of, 271-275 composition and base sequence of, 201 egg and, 247-253 isolation from polyribosomes, 179191 methods of identification, 198-201 molecular characterization of, 199200 problems of isolation, 173-174 release of activity in sea urchin egg, 262-264 native, isolation of, 198-199 nuclear, kinetics of labeling, 206-214 molecular characteristics of, 205-206 stability and messenger characteristics of, 215-217 tracing of, 217-220 reticulocyte, amino acid incorporation by, 175-179 9% detachment from ribosomes, 186-189 detection of, 179-183 direct observation of, 183-185 fluoride-resistant association with ribosomes, 191 properties of, 189-191 purification of, 185-186 sequential synthesis of, 201-205 transcription, developmental changes in, 366-371 transfer, structure of, 139-142
SUBJECT INDEX
suppressor, 153-155 Ribonucleic acid polymerase, hydroxylamine mutagenesis and, 361-362 Ribonucleoprotein, messenger, 283-287 Ribonucleotides, reduction, conclusions, 342-344 general considerations, 303-305 reduction in animal cells, chick embryo and, 33W39 evidence for control mechanism, 341-342 general remarks, 338 Novikoff hepatoma and, 339-341 reduction i n Escherichia coli, allosteric regulation, 314320 crude extracts and, 30E408 general remarks, 305 purified enzymes and, 308-310 reaction mechanism, 320-324 thioredoxin system, 310-314 reduction in Lactobacillus leichmannii, crude extracts and, 324-326 hydrogen donor specificity and, 327329 in vivo, 324 purified enzymes and, 326-327 reaction mechanism, 335-337 regulation of enzyme synthesis, 333335 substrate specificity and allosteric effects, 329-333 Ribosomes, activity, release in sea urchin egg, 261-262 detachment of 9 s ribonucleic acid from, 186189 egg and, 253-255
S Sea urchin, egg, activation of protein synthesis in, 25S266 Sodium fluoride, association of 9 s ribonucleic acid with ribosomes and, 191 Suppressork), transfer ribonucleic acid, 153-155
467
SUBJECT INDEX
T Thioredoxin system, ribonucleotide reduction and, 310-314 Translation, direction of, 149
errors in, 149-151 Tumors, nucleases in, 410-414
U Uracil derivatives, hydroxylamine and, 356-358
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