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, y
