PROG NUCLEIC ACID RES&MOLECULAR BIO V7, Volume 7 (v. 7)

PROGRESS IN Nucleic Acid Research and Molecular Biology Volume 7 Contributors to Volume 7 D. M. BROWN A. BURNY H. CH...

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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.

ACADEMIC PRESS, INC. 111 Fift,h Avenue, New York, New York 10003

United Kingdom Edition published by ACADEMIC PRESS INC. (LONDON) LTD. Berkeley Square House, London W.1

LIBRARY OF

CONGRESS

63-15847 CATALOG CARDNUMBER:

PRINTED IN THFi UNITED STATES OF AMERICA

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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. . . . . . . . . . . . PREF.ACE. . . . . . . . . . . . . . . . ABBREVIATIONSAND SYMBOLS . . . . . . . . . . . . CONTEXTS OF PREVIOUS VOLUMES .

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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

. . . . . . . . . . . . . . . 111. Stimulation of Amino Acid Incorporation by Reticulocyte RNA . . I V. Isolation of the mRNA Thread from Polyribosomes . . . . . V . Concluding Remarks . . . . . . . . . . . . References . . . . . . . . . . . . . . .

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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303 305

xi

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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369 370 376 380 383 390 392 408 414 419 420 427

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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

xiv


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


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

1 2 4

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7 7 14 16 16 20 22

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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,


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).


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


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 . . . . . . . . . .

. . . . .

.

Origin

. . . .

. . . .

. . .

. . .

. . .

. . .

.

.

25

.

. .

26 26 33 41 41 43 48 48 60

. . .

. . . . . . . . .

.

.

.

.

.

. .

. .

. .

.

.

.

. . .

. . . . . . .

. . .

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).


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


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


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).


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-


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


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).

48

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


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


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.


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


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


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


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


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


73

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


83

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).

REFERENCES 1. A. Kossel, “The Protamines and Histones.” Longmans-Green, London, 1928. 2. V. G . Allfrey, A. E. Mirsky, and H. Stern, Advan. Enzymol. 16, 411 (1955). 3. D. M. P. Phillips, Progr. Biophys. Biophys. Chem. 12, 211 (1961).

4. H. Busch and W. J. Steele, Aduaw. Cancer Res. 8, 41 (1964). 5. H. Busch, “Histones and Other Nuclrar Proteins.” Academic Press, New York, 1965. 6. H. Busch and W. C. Starbuck, Ann. l i c v . Biochem. 33, 519 (1964). 7 . I

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errors that do occur; AmG niny occur (139). Codons containing pyrimidines and purines show typical pyrimidine mistakes, though the pattern may not be so extensive as that shown by UUU ( 1 4 2 ) . Thus the error pattern for a given base in a given codon position may not be altogether independent of the overall composition of the codon, though it seems to be so to a first approximation.

TRANSLATION OF MRNA’s COKTAINING UNUSUALBASES Another class of phenomena related to the normal and streptomycin translation error patterns, but that could not in the strictest sense of the term be classed as errors, are the translations of mRNA’s containing abnormal bases. It has been known for some time that 5-fluorouracil causes phenotypic reversion of “amber” mutants. The most reasonable mechanism here is a translation mistake (7’9, 80). Similarly, mRNA’s containing inosine are given to rather high levels of translation errors in vitro (139). The most spectacular examples of this sort of “error,” however, are those involving the translation of mRNA’s containing the heavier halogenated pyrimidines. The best-studied cases arc those of the 5bromo pyrimidines. Poly BrU produces a polypeptide product that coiitains Phe, Ile, Leu, and Ser, Init little if any Tyr, Val, or Cys ( I @ ) . This is precisely what would bc expected on the basis of the above error pattern for poly U. If the mistake pattern for poly BrC were analogous to that for poly BrU (in that BrC were translated as C, U, and A, as BrU i s ) , then poly BrC should bring about synthesis of a peptide product containing Pro, Ser, Leu, and Thr. The polypeptide produced by poly BrC contains high amounts of Pro and Thr, but no Ser or LeuS (143). Thus BrC is not trailslated as U, though BrU is translated as C. (As seen above, there arc also cases where a translation error that would involve CmU does not occur under “high error’, conditions.) An understanding of the mechanisms behind translation errors and translation of mRNA’s containing unusual bases should yield an understanding of codon-anticodon recognition. The translation (‘error” data so far seem unexplainable in terms of simple base-pairing considerations, such as those used to explain the known codon-anticodon degeneracies (125, 11). It does not appear reasonable that such substituents as Br and I should manifest themselves mainly in terms of polar interactions. It is more likely that these atoms soniehow effect codon-anticodon interactions through their relatively high nonpolar reactivities. The exact mechanism remains obscure. R A scant amount of LCU is actually incorporated, but it is less than is incorporated with a poly C message ( 1 4 3 ~ ) .

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CARL R. WOESE

The Mechanisms of Polypeptide Chain Punctuation

Although i t is known that special nucleotide sequences are involved in both peptide chain initiation and termination, the mechanisms in both cases remain to be elucidated. A few relevant facts are available, however. Concerning initiation punctuation as stated above, two species of tRNAMeL are known to exist, each responding to the AUG codon, and one of these permits Met to be N-formylated subsequent to its attachspecies show different responses ment to tRNA ( 7 8 ) .These two tRNAMeL to the antibiotic puromycin, which seems to behave as a tRNA analog. The normal tRNAMet (not susceptible to formylation) binds to ribosomes in the presence of the AUG triplet with or without puromycin present (as would a typical tRNA) . However, the tRNAMet carrying N-formylMet (fMet-tRNAfMet)interacts with puromycin in the presence of AUG and the ribosome to form N-formylmethionyl puromycin. This is true whether the Met has been N-forinylated or not (1.44). We should consider these facts together with the following additional ones: ( a ) In general the first codon in (synthetic) mRNA has a very low probability of being translated. (The message AAAAACAAAAAA . . . yields the peptide Asn-Lys-Lys . . . ? but not Lys-AsnLys-Lys. . . .) However, the first codon is somehow “counted” (27). [AAAAACAAAAA . . . produces N-terminal Asn peptides; AAAACAAAAA . . . produces N-terminal Thr peptides; but neither produces a mixture of the two types ( 2 7 ) . ] (b) I n a low Mg2+ in vitro system, there is very little polypeptide synthesis brought about by poly (U, A, G ) unless N-formylMet-tRNA or certain peptidyl tRNA’s--e.g., PhePhe-tRNA-are present ( 7 6 ) . All these facts can be understood in terms of a model that postulates two kinds of sites on the ribosome: one site for interaction with the incoming tRNA, which holds the amino acid about to be placed into peptide link (the “decoding” site), and a second site to catalyze peptide bond formation and hold the tRNA that is covalently linked to the growing peptide chain (the “peptide” site). Clearly the two types of Met tRNA’s distinguish these two sites. The tRNA that permits hrformylation (tRNAfXlet)locates in (or possibly creates) a peptide site. This would also be true of tRNA’s carrying small peptides. The “normal” tRNAMet(which does not carry a formylated Met) and all other tRNA’s that carry amino acids locate in the decoding site. The failure to translate the initial codon in an RNA such as AAAACAAAA . . . (though it is nevertheless “counted”) could result from either of two mechanisms: (1) the initial codon somehow locates in the peptide site (and so is inaccessible to normal tRNA’s), or (2) since aminoacyl-tRNA bonds are

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n101’c labile than peptidyl-tRNA bonds (144a) , the amino acid attachcd to the tRNA reading the first codon in a synthetic mRNA is somehow prematurely split from its tRNA (in the framework of the ribosome) before i t can form a peptide link. At the present time there is some uncertainty as to how the reading frame is established for mRNA, and the role of the AUG codon in establishing it. It seems clear that the reading frame can be uniquely determined in the absence of the AUG codon, though the initial three nucleotides of the message do not seem to be translated into an amino acid residue with a measurable probability in this case (27, 145). The presence of the AUG codon a t or near the 5’ terminus of mRNA does appear to exert a directing effect on the choice of reading frame (146) and, as might be expected in view of the above, terminal AUG is translated into an amino acid residue (N-formyl-Met) (1.65). Several factors involved with reading-frame determination and/or peptide chain synthesis initiation by the AUG codon have been isolated (1.65). Little is known of the mechanism of peptide chain termination beyond that reported in the punctuation section above. The prevalent opinion is that a chain-terminating “tRNA” is involved. Recently a techniquc for assaying for “amber” suppressor tRNA’s has hecw clevclopcd, based upon the fact that the normal viral coat polypeptide producclcl by thc nucleic ncicl of :in R N A phagc in :In in z d r o system remains attachcd to the RNA, whereas the partial coat polypeptide produced from a mutant phage RNA containing an “amber” codon does not yield an RNA-bound peptide product ( 1 4 7 ) . This sort of assay could detect not only suppressor tRNA’s for the amber codon, but also any “terminator tRNA’s” responding to the UAG codon.

G. Suppressor tRNA’s The tRNA suppressors are of especial interest in that their properties should be particularly revealing not only with regard to immediate underlying molecular mechanisms, but also with regard to certain aspects of the code’s evolution. At present, our understanding of the phenomenon is rudimentary. We can record the following facts, (a) There are two kinds of tRNA suppressors, which, however, are not fundamentally different from one another-suppressors inserting an amino acid other than the normal one in response to a given codon, and suppressors inserting an amino acid in response to one of the chain-terminating codons, UAA and UAG. (b) The level of suppression, i.e., the probability that the suppressor response as opposed to the normal response will occur when the suppressed codon is translated ranges from less than 1% in some cases to as high as 60% in others (86, 148). One

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special case where suppression approaches the 100% level is discussed below. Let us note, too, that an E. coli Arg-Gly suppressor (Gly inserted in response to the Arg AGA codon) normally functions a t the 5% level, but introduction of the suppressor gene into a particular strain of E . coli (having an abnormally high level of Gly-activating enzyme) results in a 50% level of suppression (149). The obvious explanations for why suppression is not all-or-none need no discussion. (c) Suppressor tRNA’s, in all cases characterized, insert an amino acid one of whose codons is related (by a single base substitution) to the codon suppressed. Examples are Ser(UCG), Tyr (UAU,), and Gln (CAG) suppressors of the UAG codon (86, 150). These results suggest the suppressor tRNA’s to be created by modifications of the anticodon portion of the molecule. (Modification of either the anticodon, the enzyme recognition site, or possibly some other part of the tRNA molecule could conceivably create a suppressor tRNA.) More direct evidence for anticodon modification as the origin of suppression comes from the Tyr amber suppressor in E . coli. I n this case, it has been possible to incorporate the suppressor gene or the corresponding wild-type gene in a lysogenic phage genome. Cells infected with these phages can be caused to produce large amounts of the tRNA’s in question. The Tyr amber suppressor tRNA has thus been proved t o respond to the UAG but not the UAU codon (by the triplet binding assay of Nirenberg and Leder) , while the corresponding wild-type gene produces a Ty r tRNA, which does bind the UAU codon (151).Sequence analysis of these two tRNA’s shows that the change in primary structure in creating the suppressor is a G + C substitution in a segment whose sequence is GUA-i.e., precisely what would be expected for an anticodon alteration in a Tyr tRNA (151a). Several facts argue that not all suppressors are created by anticodon alteration. For one, yeast super-suppressors, which are believed to be analogous to E . coli amber suppressors, arise almost exclusively from addition-deletion mutations, not base substitutions (156).For another, a Cys-Gly suppressor tRNA (Gly in response to Cys codon) shows marked inhibition of Gly incorporation in the presence of excess Cys (15.2). [An Arg-Gly suppressor tRNA is not, however, inhibited by excess Arg (154).] This amino acid inhibition is more consistent with an ERS modification than an anticodon modification. The above-mentioned phenomenon of a shift in suppression level, from 5% to 50% upon introduction of the Arg-Gly suppressor gene into a high Gly activating enzyme environment, also suggests a modification in the enzyme recognition site. [The reasoning here is that in its original environment the suppressor tRNA must have existed largely in the uncharged state, which is atypical of normal tRNA’s (163); this in turn implies difficulty in

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charging this tRNA.1 It. should be recognized a t this point that were the anticodon a part of the enzyme recognition site, as the above evidence tends to suggest, an alteration of the former would automatically alter the latter.

VIII. The Fundamental Nature of the Genetic Code We have, in the above, surveyed the major facets of the coding problem-the cryptographic puzzle, the use of various encoded instructions, the decoding process. All this, however, gives an understanding of the code that is, for the most part, descriptive and relatively superficial. It should be clear that to know that UUU is assigned to Phe, or that mRNA is processed as if it were a tape, is one thing; to know why UUU is assigned to Phe, or why translation involves this sort of process (rather than a direct template mechanism, for example) is far more basic. I n other words, we still need to understand the principles upon which the structure of the genetic code rests. It seems obvious intuitively that this problem is practically inseparable from that of determining the way in which Nature actually built the code, i.e., the problem of the code’s evolution. A number of characteristics of the genetic code appear to reflect something of the code’s fundamental nature. I n this concluding section, we review these characteristics in detail.

A. The Problem of the Locked-in Code One of the more intriguing properties of the genetic code is th a t it is almost or perhaps entirely universal. All organisms appear to use the same set of codon assignments (with the exception of a few suppressor strains) (157-159). The mechanism of translation may well be universal also, in that parts of the translation machinery from one organism function well, if not perfectly, in the translation apparatus of another organism (112, 160). (There is a little doubt, as we have seen, just how universal Trans-I is, but it is nearly if not totally so.) Even if all life arose from a single cell line, how can universality of the code be maintained today in the face of mutational pressure? One has, of course, the old argument that once a universal code was established it could never change because an alterated codon assignment, etc., would be extremely deleterious for any cell line. Certainly a mutation that in one step completely changes the assignment of a major codon should be lethal, for it is equivalent to as many mutations in a genome as there are occurrences in that genome of the codon in question. (In a small genome the size of that of E . coli, this could easily mean 2 x lo4 simultaneous “mutations.”) But is this the correct, or the only, picture of what could happen? Suppressors do exist and the suppression level is some-

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times high, about 50%. These suppressors do not seem to put a n undue burden on the cell. The high-level suppressors seem, however, to involve codons whose occurrence in the genome is very restricted--e.g., AGA and UAG in E . coli. Since suppressor lines can be created in Nature, they must also be able to revert to nonsuppressed lines again. However, the reversion of a suppressor line t o the nonsuppressed condition need not recreate the original set of codon assignments. We may picture a mechanism whereby the UAG codon, for example, could be given a n unambiguous amino acid assignment, or in which one codon assignment for, say, Arg or Ser, might become eliminated, paving the way for a reassignment of such codons to different amino acids a t some later stage. (I mention here one case where a complete change of codon assignment does seem to have occurred: Two strains of mice differ in the sequence of their hemoglobin chains a t a particular site; one strain has an Asn residue a t a certain position, the other has an average of residue each of Ser and Thr at the same position. Using in vitro hemoglobin synthesizing systems, it has been possible to determine that the cause of the difference lies in the supernatant fractions-i.e., not in the message RNA’s (161).) This discussion does not prove anything definitely, but it does make one doubt whether processes that alter codon assignments are necessarily lethal, and leaves one wondering why suppressor mutations, etc., have remained “localized,” have not lead to a randomization, a destruction of the various sorts of universality in the genetic code. It has also been argued that universality is maintained because a certain set of codon assignments is somehow “optimal” for the organism, which would mean that a selective pressure exists to adjust assignments to this optimal set and keep them there. This seems a weak argument. It is hard to see why a single set of codon assignments should be optimal for an organism. Far more likely, many sets of assignments (though a very small percentage of the total possible sets) should be equally good. Even admitting that one set of assignments could bc optimal for an organism, it is still impossible to see why the same set then should be optimal for all organisms. Organisms exist under vastly differing conditions of pH, temperature, oxidation-reduction potential, etc. ; organisms have vastly differing compositions of their DNA-and (smaller) differences in overall protein composition-which could well reflect very different mutational pressures. A single code could not be “optimal” for all these fundamentally different situations ; perhaps not even a unique set of amino acids would be. Thus while there may exist codon assignments that optimize certain parameters (e.g., minimize deleterious effect of certain mutations) and although these may even

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have played some role in shaping the general form of the codon catalog long ago, it is impossible to see how these constraints could maintain the universality of the code, let alone create it. By now the difficulties in giving a satisfactory explanation for universality of the code should be apparent. There is a serious question as to how the genetic code became universal in the first place (which we have not gone into in any depth) and then how this characteristic became “locked in” in the second. Failure to explain these matters in any alternative way lcacls t o the idea that the answer must be in the nature of the code itself (rather than in the effect on the cell of changing codon assignments, etc.). There may be a clue to the solution in the possibility that the ERS contains the anticodon (discussed above). Were this so, changes in the latter would necessarily alter the former. Given a rather complex ERS to begin with, it might be impossible for an altered form of it to accommodate any activating enzyme “optimally.” Thus a change of anticodon, of codon assignment, would always bring with it a selective disadvantage. It is also possible that universality is a manifestation of some “recognition” of amino acids by oligonucleotides, an interaction that would make the evolutionary choice of codon assignments a predestined matter, and could function as the essence of R “locking-in” mechanism.

B. The Problem of the Indirect Template Much of the controversy over the nature of the genetic code during the early theoretical period in the code’s history centered about whether the association of amino acid and template is a direct one, involving “recognition” of an amino acid by its corresponding codon, or whether the burden of recognizing both amino acid and codon falls upon some intermediary molecule (s)-a so-called “adaptor” system. This same controversy is still with us, perhaps in a somewhat more sophisticated form. Do specific amino acid-oligonucleotide interactions (“recognitions”) somehow underlie and/or maintain the structure of the genetic code? If such interactions are important, such is not clearly manifested. The important experiment of Chapeville et al. (119) shows that the specificity of codon-anticodon interaction does not depend upon the nature of the amino acid carried by a tRNA. This indicates that ‘(intermediaries” (“adaptors”) are responsible for the association of amino acid with its codon in mRNA.O But we are still left with the possibilities: (a) t ha t aminoacyl-oligonucleotide interactions in some way fashioned *However, this experiment is not a proof, as is sometimes claimed, of the postulate from which the existence of “adaptors” was originally predicted-i.e., that it is impossible for an oligonucleotide to “recognize” (react preferentially with) a particular amino acid.

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codon assignments a t early stages of evolution; and/or (b) that these interactions still operate in Trans-I. Let us review the arguments, regarding “recognition” of amino acids by oligonucleotides. I n the 1940’s and early 1950’s the concept of “complementary” biological structures was developed and became a foundation of biological thought. This idea stemmed from the precise specificity demonstrated by antibodies and enzymes for their antigens and substrates ( 2 0 ) . It was then natural to extend the concept to cover the relationship of nucleic acid to histones, etc. (17)and, as Gamow did, to amino acids ( 5 ) . However, in formulating his adaptor idea, Crick took the iconoclastic position, as stated, of denying that nucleic acids can in any way show specificity toward amino acids. [The argument here in essence is that the functional groups on the nucleic acid bases do not appear “complementary” to the side chain of the amino acids, and in any case such interactions should be energetically unfavorable in solution (21, 162, ISS).] Such an argument is more a matter of intuitive prejudice than a product of thorough analysis, so it is not convincing in itself. However, all the initial experimental evidence that bore on this question tended to support this view. [For one example, equilibrium dialysis of amino acids against RNA showed no detectable binding of amino acids (164). For another, a polyuridylic acid column failed to retard amino acids (165).] Nevertheless, all that this proves is that under the conditions used, binding between amino acids and polynucleotides was not strong enough to be detected. If binding cannot be detected, one obviously cannot argue much about specificity of binding. It can be countered, however, that if this binding is so weak, it cannot play a role in determining the form of the genetic code. It must be admitted that interactions too weak t o be detected under these conditions certainly could not place specific amino acids on specific codons with high accuracy (and so, in a restricted sense, Crick’s original postulate must be correct). Yet this is far from saying that these weak interactions are not stronger under other conditions; or that weak interactions could not have played a “slow” but definitive role in guiding the course of the code’s evolution; or that in some such process as Trans-I the weak interactions cannot somehow be “summed” to give stronger and highly specific interactions. There is now rather good, if indirect, evidence to suggest that amino acid-heterocyclic base “recognition” interactions did play a dominant role in shaping the structure of the genetic code. An excellent way to detect and quantitate weak interactions is by means of chromatography. Thus it was very interesting to note that the classification of amino acids by chromatographic criteria bears a resemblance to amino acids grouped

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TABLE VIII CORRELATIONS BETWEEN AMINOACIDCODONASSIGNMEXTS AND POLAR REQUIREMENTS [Data from Woese et al., (167).] UCU UCC

cuu CUC

Leu 4.9

AUA AUG

Ile 4 . 9 Ile Met 5 . 3

GUU GUC GUA GUG

UGU c y s 4 . 8 UGC

UCA UCG

UAA UAG

UGA T r p 5 . 2 UGG

ccu ccc

CAC

ACU ACC

CAG

CGU CGA

Gln 8 . 6

CGA CGG

AAC AAG

GCU GCC

EtE

Asn 10.0 Lys 10.1

Asp13.0

GGU GGC

CIAA Glu 12.5 GAG

GGA GGG

Ala 7.0

GCA GCG

Arg 9 . 1

Thr 6 . 6

ACA ACG

Val 5 . 6

His8.4

Pro 6 . 6

CCA CCG

CUA CUG AUC

ITAU Tyr 5 . 4 UAC

Ser 7.5

Gly 7 . 9

according to codon assignment (166). Experiments indicate that this correlation is quite exact (Table VIII) . I n the table, the amino acids are arranged according to codon assignment (in the customary fashion), and for each amino acid its “polar requirement”lo is given ( 1 6 7 ) . The order in the codon catalog had already suggested something special about the I11 position in codons, in that changing a base in this position w r y often left the amino acid assignment of a particular codon unchanged. Now we see that in those cases where a base change in the I11 position of a codon does change an amino acid assignment, it is always ““Polar requirement” is defined as the slope of the straight line that results when amino acid RM’s are plotted us. log of the mole fraction of water in the chromatographic solvent. This quantity seems to be determined, loosely speaking, by the number of water molecules the amino acid will bind (expressed in energy units) minus the energy of interaction with the organic component of the chromatographic solvent. Thus, for example, Phe and Ala are expected to bind the same numhcr of water molecules, but Phe will interact more strongly in a nonpolar fashion with a molecule such a8 pyridinc, giving Plie the lower polar requirement. Similarly Asp will bind many more w a k r molecules than does Gly, and so has a higher polar requirement.

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from one amino acid to a “closely related” one (as defined by chromatographic criteria). Further, all amino acids possessing UII codons arc closely related, as are all amino acids possessing CII codons. There is also a suggestion in Table VIII that by some sort of “polar requirement” criterion, the bases rank U < C < A < G, as can be seen most clearly for the I position bases in the A,, column. Interestingly, the bases can also be ranked in the same order by their electronic properties (168).Finally, the close correlation between amino acid polar requirements and codon assignments can be obtained only when the organic component of the chromatographic solvent is a heterocyclic base, such as pyridine; aliphatic alcohols, phenols, etc., do not yield as exact a correspondence (101, 16’7). Thus the exact nature of the interaction of amino acid with the organic component of the solvent is important here. Even though the evidence is not direct, the good correlation observed is a strong argument for involvement of amino acid-nucleic acid base interactions in shaping thc codon ass;gnments. An objection often raised to the above argument is that one can also define “related” amino acid by the similarity of their function in the context of a protein molecule, and this definition of “relatcd” (having nothing to do with heterocyclic bases) would probably correlate just as well with codon assignments as does the above one. Unfortunately, “related” in a context of function in protein cannot yct be defined with any precision, and so an exact test of this alternative is not possible. The small amount of amino acid replacement data we now possess suggests that “related” by codon assignment is not necessarily “related” by function. For example, a large number of amino acid replacements have been recorded a t a certain locus in the tryptophan synthetase A protein. The protein is a t least partially functional when Ala, Ser, Thr, Val, Ile, or Asn replace Gly a t this position. It is not functional when Glu, Asp, or Arg are placed there. [Since a large number of revertants from Arg, -AGA-, to wild phenotype have not included Lys a t this position, Lys also presumably produces an inactive protein (&).I Thus there is no evidence for a similarity in function of Asn-Lys, or Ser-Arg, though both pairs of amino acids arc related by codon assignment. Further, one would not suspect that Cys and Trp, or His and Gln, or Ala and Pro would function similarly in protein, though each pair again is related by codon assignment as well as by polar requirement criteria. Thus, the best correlation so far between codon assignments and “related” amino acids is obtained when the “relatedness” is defined in terms of amino acid-heterocyclic base interactions. I think a reasonable conclusion to draw from the above is that the


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structure of the genetic code is determined by amino acid-nucleic acid “recognition” interactions (or their equivalent) , but being very weak interactions they could not have given rise directly to the genetic code as i t now exists. Instead they must have played the role of constraints operating on the evolutionary process, in this way gradually shaping the form of the code. Since the interactions are weak and therefore cannot manifest an all-or-none sort of specificity with regard to amino acidcodon pairings, it is reasonable to expect that they cannot align amino acids along a nucleic acid template directly, and so this “recognition” role has been filled by the evolution of an lLintermediary” system, the tRNA’s and activating enzymes, that recognize with vety high accuracy both an amino acid and its codons.

C. The Problem of Errors and Evolution The essence of the cell (if there be a single essence) lies in the capacity to construct a protein of unique primary structure from any given nucleic acid primary structure; i.e., the capacity to translate a gene essentially perfectly. It is hard to imagine what a cell would be like were it not capable of accurately creating protein from nucleic acid. Yet this is necessarily what must have been the case for the early cell, the cell of, say, 1 to 3)< lo9 years ago. Two considerations argue the point. ( a ) Biological specificity turns on weak interactions (ix., dispersion forces and the like). Singly (i.e., between two simple molecules) , these interactions manifest very little specificity (selectivity). When “summed” in the proper way, as in an enzyme site, they are not only rather strong, but highly specific and highly selective. (b) The great accuracy of the translation process is a product of extensive evolution and relies upon the accurate functioning of many enzymes-the set of activating enzymes, the set of enzymes that modify tRNA’s, etc. The presence of highly specific enzymes demands the existence of accurate translation and vice versa. Here is an apparent paradox, the solution of which probably lies in a simultaneous and gradual evolution to high-specificity enzyme functions and to accurate translation, starting from low-specificity enzymes and inaccurate translation. Since we have yet to uncover traces of a cell with an incompletely developed translation capacity, it is impossible to know in detail what such a cell was like and how it exactly evolved into the “modern” cell (an evolution that must have been complete a t least 500 x lo6 years ago). However, one can reason what such a cell and its evolution probably were like, a t least in general outline (169). (a) A highly inaccurate translation process meant that it was impossible to make two identical translations of any given nucleic acid sequence. This situation would

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severely limit both the kinds of enzyme functions available to a cell, and perhaps the size of the polypeptides translated (the smaller the polypeptide the greater the probability of a perfect translation). (b) The genetic differences between two species are often subtle. To be able to speak of “species,” etc., means that the states of a biological system must be definable within very narrow limits. This in turn demands precise enzyme functions and in particular precise control of the levels of enzyme function and changes in these levels. Thus complex interrelationships among various enzyme activities and a high degree of regulatory control are necessary. Certainly this level of regulatory sophistication was far beyond the capabilities of early cells. ( I n fact, it probably was impossible for multicellular organisms, i.e., differentiated cells, to exist.) Therefore, evolution as we have come to know i t s p e c i a tion, etc.-did not occur when cells possessed primitive translation systems. The general character of evolution in such cells was conceivably so different from what we have observed that it is probably useful to distinguish it as “pre-Darwinian” evolution. (c) The inaccuracies in translation also should have severely limited the genetic complexity of primitive cells; genomes even as large as phage genomes may have been impossible (or useless) to achieve. (d) Pre-Darwinian evolution, being completely limited by the accuracy of translation, was in a sense entirely concerned with evolution of the genetic code. This evolution perhaps occurred in discrete stages: the beginning of a stage would be some improvement in the translation process; the stage itself would be the working out of evolutionary ramifications of this improvement, which would then prepare the way for another improvement in translation, the beginning of the next stage, etc. (169).

D. The Problem of Constraints Governing the Code’s Evolution The genetic code must have evolved gradually, through many stages. There must be overall constraints and considerations that have shaped this evolution. As we have seen, there exists a reasonable case for amino acid-oligonucleotide recognition interactions supplying constraints. Since the point remains unproven, however, alternatives must also be considered. Could the high degree of order manifested by codon assignments, for example, possibly be accounted for without postulating some feature such as amino acid-codon recognition? I n other words, could the constraints governing the code’s evolution be external to the coding apparatus per se? Several attempts have been made to construct a model accounting for the highly ordered array of codon assignments without resort to constraints that are a part of the coding apparatus. The first detailed


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scheme of this sort, and the prototype for the others, was proposed by The starting point for this model is a cell with an Sonneborn (17 0). unordered codon catalog (but an otherwise fully developed translation system). Sonneborn argues that the codon catalog could have achieved its high degree of order in the following way. Practically all mutations are events deleterious to a cell or its descendants. Thus a cell that can reduce its burden of deleterious mutations is placed a t a selective advantage. Other things being equal, this can be achieved by reassigning codons until a particular optimal set (or sets) is obtained. An optimal set of codons is one in which all possible codons are assigned to amino acids (an unassigned codon, introduced by a mutation, would certainly be lethal to the cell), and all codons assigned to the same amino acid are “maximally connected”-i.e., the number of base changes necessary to convert any one of an amino acid’s codons to all others of its codons is a minimum. For example UUU, UUC, and UUA are a set of maximally connected codons; UUU, UUC, and UAU are not. The individual sets of maximally connected codons must then be further interrelated to one another by the condition that functionally related amino acids possess “connected” sets of codons wherever possible. These constraints would then serve to make as many mutations as possible lead to no change in the overlying amino acid, and when amino acid replacements did result from mutations, as large a fraction of these as possible would involve functionally related amino acids and so have little or no deleterious effect. The codon catalog resulting from such constraints could resemble the actual one in overall order. The ordering is a relative onei.e., there is no constraint that drives Phe to be associated particularly with UUU, etc. A number of criticisms can be directed against this model ( l l ) , and some of these we have encountered above. In any case, it seems possible to circumvent these by using a somewhat different model (169). The trouble with any basically stochastic scheme (as this one is) is that these schemes seem to have to start with a fairly well-developed translation system. Thus such a model never gives an indication of how translation could have begun initially. And that, after all, is the real question-an especially puzzling one too, if one is not permitted to invoke any “specificity” or selection in the interaction of nucleic acids and amino acids or their derivatives.

E. The Problem of the Origin of the Components The translation process involves a set of no less than twenty highly specific activating enzymes, a set of perhaps forty kinds of tRNA’s, a ribosome comprising two enoriiious RNA molecules and as many as

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35-40 kinds of protein molecules. Primitive translation could never have involved so many components. Not only must the number of different molecules in any class have been much smaller initially, but the number of classes of macromolecules in the translation apparatus initially must have been smaller, too; i.e., most of the components now a part of translation could not have been present in the earliest attempts. Since Occam’s razor demands that primitive translation be far simpler and perforce much different than its modern day counterpart, it is difficult to think of the former because of preconceived ideas derived from knowledge of the latter. I n fact it is wise even to question whether as a synthetic process translation began as a translation a t all-i.e., mapping the primary structure of nucleic acid into the primary structure of a polypeptide (albeit inaccurately). For several reasons, I take as axiomatic that some sort of “recognition” interactions involving amino acids and bases or their derivatives lies a t the root of the code’s origin. I n the first place, arguments brought against this sort of “specificity,” or “recognition,” etc. (as in the formulation of the adaptor hypothesis) are by no means compelling, as we have already seen. For another, there is evidence strongly suggesting these interactions (167). Finally, it seems difficult to evolve a relationship between nucleic acid and protein primary structures unless there exists some tendency for the two (or their components) to associate with some “specificity” to begin with. However, in light of the previous paragraph it may be wise to take a broad view of what kinds of interactions of amino acids and bases might contribute to the code’s origin and evolution. It seems useful to begin these considerations from the opposite end, as it were, by asking what components of the modern genetic coding apparatus (more properly, the progenitors of these components) might not have been present a t the earlier stages of the code’s evolution. Perhaps the least likely molecule to have been prescnt initially is one like an activating enzyme. This type of molecule could not have existed hzforc precise translation existed ; its major function is accurate discrimination among amino acids, which requires it to have a unique, fixed primary structure. On the other hand, tRNA progenitors may well have existed earlier, for several reasons. For one, tRNA is always covalently linked to the growing polypeptide chain, consistent with and suggesting a primeval association between the two macromolecules. For another, tRNA appears to be a “molecular anachronism”; it is a nucleic acid, yet the post-transcriptional modifications (methylations, etc.) it undergoes to be properly functional, as well as some of its properties, e.g.,

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readily reversible configurational changes (171, 17.2), give it more the characteristics of a protein. On the grounds that it is easier to adapt an existing function than to create a new one, the evolution of tRNA to a form so unlike other nucleic acids, argues that tRNA’s progenitors were indeed of relatively ancient origin (and may originally have had a quite different function). The ribosome, which seems so complex, could not have existed as such early in the evolutionary process, but this is not to say that a much simpler version of it was not a t one time a component of translation, Protein-RNA associations occur readily. Perhaps the key question of all is a t what stage an actual “tape-reading” process came into use in translation-i.e., an external tape (the message) passing through a tape-reading machine (the ribosome). Again the complexity of this operation, the number of separate parts participating, and the precision of their function, suggests that evolution of the tapereading feature was preceded by other developments. Since so little is known about possible progenitors of the present genetic coding apparatus, little would be gained by constructing detailed and untestable models for the code’s evolution. However, there is some virtue in cataloguing and briefly discussing types of interactions that could have contributed to the code’s origin, and in trying to formulate pertinent questions regarding this origin. It seems important to make some distinction as to whether initially the “unit of recognition” between the class of nucleic acid components and polypeptide components was the amino acid or some minimum size of peptide, on the one hand, and the individual base or some oligonucleotide, on the other. I n one case, we might expect primitive ‘%ranslation” t o involve addition of ainino acid subunits to growing peptide chains; in the other case, proteins could be synthesized from assembly of small peptide units, the small units having been made by ‘hondirected” synthesis and selected in the translation process. As we have seen, a major problem with amino acid-nucleic acid interactions is the apparent weakness of binding and consequent low specificity. Peptide-nucleic acid interactions would necessarily be much stronger and, in certain ways, might be more specific, We know that nucleic acid-peptide interactions do exist-in the ribosome, in histone-DNA complexes, in the activating enzyme-tRNA interaction, for example-and these show specificity to varying degrees. Recently it has been shown that poly Lys associates preferentially with DNA of high A T content while poly Arg prefers a high G C DNA, an encouraging sign (173). What we do not know in all cases is ( a ) whether the association of nucleic acid and peptide is a colinear one;

+

+

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(b) the number of bases with which an amino acid residue associates;ll and (c) the effect on strength of binding of changing one amino acid residue or base in the complex-i.e., the “specificity” of binding. Polypeptide-nucleic acid association might have been used in the primitive cell as a basis for feedback from protein primary structure to nucleic acid primary structure, a device for checking the accuracy of a translation, etc. There seems little point, in general, in having a direct feedback from polypeptide primary structure to synthesis of nucleic acid primary structure when a primitive translation process, in both directions, is a most inaccurate one. However, in certain special cases a direct influence of protein primary structure on nucleic acid primary structure could be advantageous. If the peptide product of a translation associates with the nucleic acid of whose primary structure it is a translation, there is a mechanism serving both to select the better translations from their less accurate counterparts and to influence nucleic acid primary structure, limiting the speed of “genetic drift,” etc.-e.g., by protecting from hydrolysis those nucleic acids associating most strongly with polypeptides. It is interesting to note one present-day system where protein primary structure might direct the synthesis of nucleic acid. A poly A synthetase has been isolated from Clostridiuin perfringens. The enzyme has an unusual cofactor requirement, poly Lys ( 1 7 6 ) . I n view of the coding relationship between AAA and lysine, one is tempted to speculate that perhaps in some rich primitive milieu poly Lys could have brought about the synthesis of poly A and vice versa, thus creating an autocatalytic system. The association of poly A with poly Lys would then serve to help eliminate (by not protecting adequately) ‘Ibad” translations in either direction. A phenomenon well known to the polymer chemist may have some bearing on origins of the code. I refer to the synthesis of stereo-regular polyhydrocarbons by various mineral catalysis-e.g., the Ziegler catalyst. Such interactions manifest a very rudimentary specificity. It is tempting to extrapolate from such a mechanism to one in which nucleic acid acts as a catalytic site for polypeptide synthesis. Perhaps some primeval RNA llThere is a controversy at present regarding whether or not rRNA can code for ribosomal proteins (174, 176). The answer to this point is particularly germane to the present discussion. It is argued that rRNA could not code for its own proteins because a n RNA optimally evolved to bind with protein (in ribosome formation) could not be optimally evolved to perform an unrelated function, coding for protein. This argument is reasonable if the two functions-coding us. binding-are unrelated to one another. However, if the two are indeed related, then i t might be expected that optimal binding of ribosomal protein is completely consistent with coding for the same protein.


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The Search for the Messenger RNA of Hemoglobin H. CHANTRENNE, A. BURNY, AND G. MARBAIX Laboratory of Biological Chemistry, Faculty of Sciences, University of Brussels, Brussels, Belgium

I. Introduction . . . . . . . . . . . . . 11. Location of the Information for Hemoglobin in Reticulocyte . . . . . . . . . . . . . . Extracts 111. Stimulation of Amino Acid Incorporation by Reticulocyte RNA . A. Cell-Free Preparations from E . coli . . . . . . . B. Cell-Free Preparations from Reticulocytes . . . . . IV. Isolation of the mRNA Thread from Polyribosomes . . . . A. Principles . . . . . . . . . . . . . B. Detection of a 9 s RNA with the Expected Properties . . . C. Direct Observation of 9 s RNA . . . . . . . . D. Purification of the 9 s Fraction . . . . . . . . E. Detachment of 9 s RNA from Ribosomal Particles . . . F. Properties of 9s RNA . . . . . . . . . . G. NaF-Resistant Association between 9 s RNA and Ribosomes . V. Concluding Remarks . . . . . . . . . . . References . . . . . . . . . . . . . .

173 174 175 175 176 179 179 179 183 185 186 189

191 192 192

1. Introduction Synthetic polynucleotides can certainly fulfill the main function of natural messenger RNA’s: they are accepted by ribosomes and translated into polypeptides. However, it is highly desirable to ohtain the messenger RNA corresponding to a known protein. Natural messengers must carry, besides the information for amino acid selection, signals of various kinds that control the machinery of translation: sequences for starting or interrupting translation, special properties that ensure fixation to the ribosome and correct phasing; they may possibly contain regulation sites. Messengers represent but a small fraction-only a few percent-of the total cellular RNA; they must make up a mixed population of individual molecules varying in size and half-life, and are subject to the constant risk of degradation by nucleases. This actually occurs in most cases when the cell is broken open and the delicate balance of its 173

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H . CHANTRENNE, A. BURNY, AND G. MARBAIX

constituents becomes disturbed. Nucleases are a major obstacle in attempts to isolate intact messenger RNA’s ; technical skill will eventually circumvent this difficulty. But to select the messenger of a known protein out of the whole messenger population of a cell raises another kind of a problem, for which there is as yet no obvious solution. For the time being, the best one can hope for is to find favorable cases in which the selection has been made for us by nature as, for example, in viruses. A viral RNA is a homogeneous molecular species that contains the information for several proteins, three in the simplest viruses and in some defective viruses perhaps only one. Some virus RNA’s are indeed excellent messengers ( I S ) . I n higher organisms, the messenger that seems the most accessible is that responsible for hemoglobin, owing to the exceptional properties of mammalian reticulocytes. Rabbit reticulocytes are easy to separate from other cells (apart from inactive mature erythrocytes); they can be washed free of blood plasma, they can be opened in a medium of controlled composition by a slight lowering of the osmotic pressure; moreover, they are free of ribonuclease. Mammalian reticulocytes do not make RNA (4, 5 ) ; their nucleus is lost in the process of differentiation. Nevertheless they retain for several days their ability to make hemoglobin. The genetic messengers for hemoglobin, therefore, are long-lived. Since rabbit reticulocytes make practically no other protein (6, 7 ) , it can reasonably be supposed that most of the messenger population of the cell is composed of hemoglobin messengers. As a first approximation, the problem of sorting out the messengers can be neglected; it has largely been solved by the physiological differentiation of the red cell. Several of the main technical difficulties encountered in the general case simply do not exist or are considerably alleviated in the case of hemoglobin messengers. Nevertheless, in order to isolate a messenger, one must be able to recognize it. The final criterion of a hemoglobin messenger is that it cause the synthesis of globin in a system that does not possess the information for that protein. It is therefore quite reasonable to attempt to identify this messenger on the basis of its informational properties.

II. location of the Information for Hemoglobin in Reticulocyte Extracts Schweet et al. (8) demonstrated that an extract of rabbit reticulocytes can incorporate amino acids into hemoglobin in vitro. When the protein was chromatographed on ion-exchange resins, more than 80% of the

THE SEARCH FOR MESSENGER RNA OF HEMOGLOBIN

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incorporated amino acids were recovered with hemoglobin, and the relative rates of incorporation of three amino acids (leucine, isoleucine, and valine) corresponded to the ratios in which they are present in rabbit hemoglobin. Since a heterologous system made of rabbit reticulocyte ribosomes and a “pH 5” fraction from guinea pig liver produced hemoglobin just as well, it can be concluded that the information for hemoglobin synthesis is associated with the microsome fraction. Hemoglobin is even made by a simplified mixed system derived from reticulocyte ribosomes and Escherichia coli transfer RNA charged with amino acids ( 9 ) . That the protein synthesized has the primary structure of hemoglobin was proved by chromatographic analysis (“fingerprinting’,). Bishop et al. (10) made crossed systems with ribosomes from rabbit reticulocytes and a supernatant fraction from mouse reticulocytcs. The two hemoglobins were separated by chromatography ; the protein labeled in vitro belonged to the species contributing the ribosomes. However, two other groups reported experiments a t about the same time indicating that both the ribosomes and the supernatant fraction can supply information in crossed systems (11-13). As these results conflicted with previous reports and could not easily be confirmed (14, 1 5 ) , they were generally attributed to contamination of the supernatant fractions with remaining particles, which are difficult to eliminate completely. Quite recently, Schapira et al. (16, 17) presented additional evidence that a soluble fraction of the supernatant portion can provide information for hemoglobin synthesis in a crossed system. Whatever the reasons for these conflicting results, it is a t least clear and undisputed that the ribosome sediment (including polyribosomes) does contain the information that causes amino acids to condense in the correct sequences of hemoglobin chains. A large fraction of the genetic messengers of hemoglobin is associated with ribosomes and can be translated correctly in vitro.

111. Stimulation of Amino Acid lncorporution by Reticulocyte RNA A. Cell-Free Preparations from E. coli The success achieved with cell-free E. coli extracts (18) in unraveling the genetic code and the remarkable response of this system to a variety of synthetic polyribonucleotides with random (19, 20) or ordered (61-23) sequences of nucleotides made it the standard test for messenger properties of RNA. Many RNA preparations from all kinds of organisms indeed stimulate amino acid incorporation into polypeptides in this system. The RNA extracted from bacteriophage f2 or R17 causes the synthesis of recognizable viral proteins (1-3). The system embodies all

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H . CHANTRENNE, A. BURNY, AND G . MARBAIX

the features required for initiation and termination of chains and for release of the protein, and it responds to the “punctuation marks” of the tricistronic messenger of R17; even suppression can be observed in vitro with this system, indicating a high degree of fidelity (3). Total reticulocyte RNA, as obtained by phenol extraction, stimulates amino acid incorporation in the cell-free E . coli system (24-27). When RNA is fractionated on sucrose gradients or by salt precipitation and the fractions are assayed for stimulatory activity, the most active fraction sediments together with 16s ribosomal RNA or on the lighter side of the 16s RNA peak (26-28). But the ratio of incorporation rates of two characteristic amino acids is very different from that expected for hemoglobin (24, 2 5 ) , and the labeled product does not behave as globin or hemoglobin in chromatography (24, 2 5 ) . Trypsin hydrolysis and peptide separation clearly show that the protein made does not resemble hemoglobin; the pattern of peptides is almost the same as that obtained from material made by the E . coli system alone (background incorporation) without addition of reticulocyte RNA ( 2 9 ) .Therefore, the RNA does not provide information to the system, it does not act as a messenger; i t merely stimulates in a n unknown manner n synthesis that was proceeding slowly in the E . coli extract. Clearly, one cannot equate stimulatory activity in amino acid incorporation and messenger properties. If RNA’s can stimulate the in vitro system without being translated, great caution must be exercised in using the in vitro E. coli system for identifying messenger RNA. In the present case, it cannot be concluded that the fraction that stimulates is messenger RNA, but neither is there any reason to conclude that the RNA preparation contains no hemoglobin messenger. Too little is known about the conditions required for a messenger to be accepted by a ribosome and translated. So far, only some viral RNA’s appear to have been translated correctly in vitro ( I S ) . This may possibly be related to their parasitic nature; they must indeed fulfill conditions that make them acceptable, even preferentially acceptable, by host ribosomes. It is quite conceivable that E. coli ribosomes cannot accept hemoglobin messengers or cannot bind them properly.

8. Cell-Free Preparations from Reticulocytes Although E . coli ribosomes may conceivably be unable to accommodate hemoglobin mRNA, ribosomes from reticulocytes can certainly translate it. RNA from reticulocytes can increase the synthesis of hemoglobin chains in vitro. Kruh e t al. (SO) obtained an increase in amino acid incorporation by adding the total RNA of reticulocytes to a cell-free


177

reticulocyte extract. The ratio of incorporation of four different amino acids corresponded to their proportions in hemoglobin. Fingerprints of the labeled protein indicated that its amino acid sequence was that of hemoglobin. However, the N-terminal peptide was only very slightly labeled, as if most of the incorporation corresponded to completion of growing polypeptide chains rather than to a complete synthesis of the chains from end to end. The stimulating agent appears to be RNA (it is destroyed by RNase, not by DNase) and RNA’s from E. coli, yeast, or guinea pig inhibit the endogenous hemoglobin synthesis. Reticulocyte RNA was centrifuged on a sucrose gradient and separated into four fractions, which were assayed for stimulatory activity ( 3 1 ) . The fraction containing 23 S ribosomal RNA was slightly inhibitory, those containing either 16 S or 4 S RNA gave a slight stimulation, but fraction 111, which contains part of the light side of the 16s peak and the region of the gradient between 1 6 s and 4S, did stimulate hcmoglohin synthesis in the cell-free reticulocyte system. The average stimulation was 75% above the control, 150% in some experiments. It should be emphasized that fraction I11 comprises the region of the gradient in which an RNA of the size predicted for hemoglobin messenger would be expected, and part of the fraction that stimulates amino acid incorporation in the E . coli system (26, 27). Comparable studies by Arnstein et al. (3.2) showed that high molecular weight RNA extracted from “heavy” ribosomes (mostly polyribosomes) stimulated hemoglobin synthesis especially when the system was made of light ribosomes (mainly 80s) and the supernatant fraction. Considerable synthesis of new chains was observed in these experiments. The same group (33) later studied the stimulatory action of ribosomal subunits obtained by EDTA treatment of polyribosomes or by brief exposure to dilute alkali; the action of the RNA extracted from these particles and centrifuged on sucrose gradients was also examined. No RNA in the size range expected for free mRNA stimulated hemoglobin synthesis; all the activity was associated with ribosomal RNA. This led to the suggestion (33) that mRNA is covalently bound to ribosomal RNA, a concept that implies a departure from the classical model. Whatever the intrinsic interest of these results, they cannot avoid the fundamental objection that the system used for testing the RNA already contains, by itself, all the information necessary for making hemoglobin. It is, therefore, a priori, a poor test system for a hemoglobin messenger of the same species. It would be an excellent one if the endogenous messenger could be completely removed from the in vitro system to start with, but this has not been achieved so far. It is true that the nature of the polypeptides made can be influenced by the added RNA

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H . CHANTRENNE, A. BURNY, AND G. MARBAIX

(27, 3 4 ) . But as long as endogenous messenger is present, stimulation of hemoglobin synthesis-just as much as inhibition-might arise from an action on any component of the system able to affect in one or the other direction the rate a t which the system operates. Certain facile criticisms were refuted and some obvious possibilities checked [see, for example, reference (SO)1. The system unfortunately is incompletely characterized, and so complex that discarding one more objection is not enough to establish a conclusion with certainty. The clear-cut results obtained by Drach and Lingrel (29) with the E. coli system prove that stimulation does not necessarily mean messenger activity. For instance, RNA’s from liver nuclei, and from the kidney or intestine of the rabbit stimulate hemoglobin synthesis by the in vitro reticulocyte system (35, 36). If the test system were devoid of information for hemoglobin, this would prove that liver, kidney, and intestine contain hemoglobin messenger RNA-a very important conclusion in relation to the problem of differentiation. But with the test system as it stands, the authors (36) are quite right in presenting this conclusion as tentative. Obviously, no definite conclusion can be based a t present on the stimulatory activity of an RNA in the reticulocyte system. When the RNA stimulates the completion of partly made hemoglobin chains, there is even reason to doubt that the stimulation of hemoglobin formation is due to messenger activity of the added RNA. Can one seriously consider the possibility that mRNA can bind on each ribosome with an unfinished chain, just a t the site where translation has been interrupted? The message must be read from the end, and mRNA must be able to bind t o a ribosome a t the starting end only; it cannot bind a t random. The fact that the polypeptides made in vitro correspond to hemoglobin shows that the system is correctly phased. Should the messenger bind a t random, two-thirds of the polypeptides made would not resemble hemoglobin a t all since two out of three messages would be out of phase. Therefore, in these cases where RNA stimulates the completion of hemoglobin chains, it is improbable that it works in the same way as informational RNA; effects of the type analyzed by Drach and Lingrel (29)are to be suspected. The present situation is indeed an embarrassing one : reticulocyte RNA stimulates amino acid incorporation into both E. coli and reticulocyte cell-free preparations. I n the E . coli system, the polypeptides made are characteristic of E . coli and do not resemble hemoglobin. I n the reticulocyte system, which does make hemoglobin by itself and therefore contains the corresponding genetic information, no definite conclusion

THE SEARCH FOR MESSENGER RNA O F HEMOGLOBIN

179

can be based a t present on the stiniulatory action of RNA on true hemoglobin synthesis.

IV. Isolation of the mRNA Thread from Polyribosomes A. Principles In our laboratory, a completely different approach was chosen. After a few attempts with the in vitro systems, which gavc unrewarding (and unpublished) results, we thought that it should be possible to recognize and isolate reticulocyte messenger RNA on the basis of other properties. According to the classical model, the messenger is the RNA fiber that connects the ribosomes in the polyribosome structure (37-400) This fiber has been clearly observed in electron micrographs of reticulocytes, and the measured length (15OOA) is that expected for a messenger RNA coding for a single globin chain ( 4 1 ) . We thought that isolating this RNA fiber should not raise insurmountable difficulties. The size of this RNA thread having been established directly, it was possible to estimate its sedimentation constant to be in the range of 9s. As the RNA thread is much more sensitive to pancreatic ribonuclease than is ribosomal RNA in ribosomes (37-40), a very simple test is available for identifying any isolated RNA as the thread: i t should be destroyed when the polyribosomal structure is treated with just sufficient ribonuclease to disrupt the polyribosomes without attacking ribosomal RNA. Hemoglobin messenger RNA is long-lived compared to bacterial messengers. However, it was hoped that under favorable circumstances the messenger would be labeled more strongly than ribosomal RNA and this might help in its recognition. There was, of course, no hope of labeling the mRNA of rabbit reticulocytes in vitro; these cells have lost their nucleus and they can only incorporate precursors into the CA end of transfer RNA (4, 5 ) . Therefore, mRNA must be labeled during its formation in the bone marrow.

B. Detection of a 9 S RNA with the Expected Properties With these principles in mind, we isolated polyribosomes, extracted the RNA, and fractionated it by sucrose gradient centrifugation, looking for a new RNA fraction with the expected properties. a component When the RNA was obtained by LiCl precipitation (~$2)~ with a sedimentation coefficient of about 36s was consistently found, as had been observed before by Barlow e t al. (42). Treatment of the polyribosomes by RNase under conditions more than sufficient to destroy

180

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the RNA thread of the polyribosomes did not affect the 3 6 s peak; this fraction, therefore, could not be the RNA thread. The LiCl method, and most of the usual methods for obtaining RNA, involve a precipitation a t some stage in the preparation. As mRNA is a small fraction of total RNA, it is likely to be lost in precipitation steps. We looked for a method in which all losses could be avoided. The whole polyribosome pellet was dissolved in a 0.5% solution of dodecyl sulfate according to Kurland ( 4 3 ; see also @u). Ribosomal proteins and RNA are thus dissociated. Since the pellet dissolves completely, the RNA thread cannot be lost under these conditions. A rather concentrated solution of polyribosomes was simply layered on a 5-20% sucrose gradient and centrifuged in the SW 39 rotor of the Spinco for 7 hours a t 37,000 rpm (115,000 )( 8). The same experiment was repeated with reticulocytes from anemic rabbits to which radioactive phosphate had been injected a t various times. Reticulocyte RNA from rabbits injected with P325 hours before blood collection showed radioactivity in the 4 S peak only, as the result of chain-end turnover (4, 5 ) . With rabbits killed between 10 and 20 hours after a 10 m C injection of radioactive phosphate, the 23S, 16S, and 4 5 peaks were labeled, but a new peak of radioactivity was consistently observed between the 16 S and the 4 5 peak that was clearly distinct from both. This is the region where a single strand of RNA containing just enough nucleotides for coding one globin chain is to be expected. This fraction was hydrolyzed in KOH, together with unlabeled RNA as a carrier, and chromatographed on an ion-exchange column. The radioactivity distribution coincided with the nucleotides, showing that the original labeled substance was RNA. I n order t o find out whether this was the messenger RNA thread, a ribonuclease sensitivity test was applied as follows. FIG. 1 . ( A ) Sedimentation pattern of reticulocyte polyribosoinrs in a linear 18-36%> sucrose gradient equilibrated in 0.05 M Tris-HC1 (pH = 7,4), 0.025 M KC1, and 0.005 M magnesium acetate. Cent)rifugation was carricd out for 2 hours at 32,000 rpm (85,000 x g) in the S1)inc.o rotor SW 39 a t 4°C. (B) Scdinientation lmttern of retirriloc*yt,r polyribosomes after t.rr:ttmeut of thc polyribosomal suspension with 0.01 fig o f 1)anc.rratic ribonurleasr per 1111 for 5 niinut.es a t 37". Conditions of centrifugat,ion as in ( A ) . (C) Sedimentation pattern of (P3'-labeled) polyribosomal RNA extracted with sodium dodecyl sulfate in a linear 5-20'70 sucrose gradient equilibrated in 5 mM Tris-HC1, pH 7.4. Centrifugation wm earried out for 7 hours a t 37,500 rpm (115,000 x g) in the Spinco rotor SW 39 a t 4". (D) Sedimentation pattern of RNA extracted with sodium dodecyl sulfate from (P=-labeled) polyribosomes treated with ribonuclease (see B). Conditions of centrifugation as in (C). I n (C) and ( D ) : -optical density at 260 n m ; - - - radioactivity in counts per minute. [Taken from Marbaix and Burny (441.1

182

H. CHANTRENNE, A. BURNY, AND G. MARBAXX

[It should first be realized that polyribosomes isolated from washed reticulocytes by centrifugation through a sucrose cushion are quite stable. Their sedimentation profile in the analytical centrifuge does not change after a %-minute incubation at 37°C in neutral buffer containing Mg2+. In a systematic study of their sensitivity to pancreatic ribonuclease, it was found that a 5-minute treatment a t 37" with 0.01 pg of ribonuclease per ml was just sufficient to destroy most of the polyribosomes. Such a limited treatment has no effect on ribosomal RNA.] Polyribosomes were thus treated for 5 minutes a t 37" with 0.01 pg ribonuclease per ml and then chilled. Control polyribosomes were similarly treated without the enzyme. Part of the suspension was used to determine the sedimentation profile of the polyribosomes ; the rest was dissolved in sodium dodecyl sulfate and centrifuged on a sucrose gradient. The result of such an experiment is shown in Fig. 1. Clearly an RNase treatment just sufficient to disrupt the polyribosome structure destroys the RNA fraction sedimenting between the 4 s and 16s peaks, in the 9s region. The radioactivity is now found on the heavy side of the 4 S peak ( 4 4 ) .The treatment was very mild since the polyribosomal pattern did not completely disappear ; some small polyribosomes remained as if the thread had been cut in several places but not completely hydrolyzed. However, some doubt as to the origin of the new RNA remained. Was it really a reticulocyte RNA? Could it not originate from contaminating white cells? Although most of the leukocytes are eliminated by centrifugation and by differential lysis, some leukocytes might possibly be lysed a t the same time as reticulocytes so that their polyribosomes would contaminate the pellet from which the new RNA fraction is obtained. I n order to test this possibility, whole blood was deliberately incubated for 5 hours in vitro, the c,ells (including leukocytes) were lysed, and the RNA isolated from polyribosomes was centrifuged in a sucrose gradient. Under these conditions, all the radioactive RNA made should be derived from nucleated cells, i.e., leukocytes. The distribution of radioactivity (Fig. 2) shows a major peak a t 16S, slightly on its lighter side. This is quite different from the pattern observed with reticulocytes labeled in vivo and freed as far as possible of leukocytes. There is therefore good reason to presume that the 9 S RNA labeled in vivo, which has several of the properties expected of a messenger, is indeed coming from reticulocytes, rather than from white cells, and that it is essentially hemoglobin messenger RNA. Recent publications by Scherrer et al. (45-47) and by Attardi et al. (48) have described the occurrence, in the cytoplasm of immature duck erythrocytes, of 8-10s RNA, which is rapidly labeled when these


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nucleated cells are incubated with radioactive precursors. This is probably the analog of the 9 s RNA fraction we find in rabbit reticulocytes.

C. Direct Observation of 9 S RNA A rough estimate of the amount of messenger RNA in reticulocyte polyribosomes as compared with ribosomal RNA indicated that it should be possible to detect its ultraviolet light absorption by merely scaling up the operations by a factor of ten. Thus instead of centrifuging 200 pg of polyribosomal RNA in the Spinco SW 39 rotor, ten times as much was used. This required the adaptation of the method to the larger SW 25 rotor. After 40 hours of centrifugation a t 24,000 rpm (60,OOOX g), the 26 S component is a t the bottom of the tube, 16s is well resolved, and the new 9s fraction is clearly visible as a low peak, well separated from 4 S and 16 S, as shown in Fig. 3A. The limited RNase treatment described above was applied, giving the result shown in Fig. 3B, which confirmed our previous observations: the 9 s fraction, which is now observable by its optical density and its radioactivity, is destroyed when the polyribosomes are

184

H . CHANTRENNE, A. BURNT, AND G . MARBAIX

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disrupted by mild RNase treatment, as expected for the RNA fiber of the polyribosome. Quantitative comparisons of the spc'cific radioactivity of this fraction with that of ribosomal RNA, show that it is always higher. Depending on the time of injection of P,32,and on the individual rabbits, the new fraction is 2-8 times more radioactive than 16s RNA. A factor of 3-4 is most commonly observed when P3?is injected 15 hours before collecting the blood (44, 4 9 ) . I n summary, the new RNA fraction has the following properties predicted for the messenger RNA's of single hemoglobin chains: (1) it originates from reticulocytes; (2) it sediments in the region of the gradient where a single strand RNA of about 430 nucleotides (the number required to code for the 141-146 amino acid residues of each hemoglobin chain) should be expected. (3) its specific radioactivity is higher than that of ribosomal RNA; (4) the new RNA fraction is degraded when the polyribosomes are trcated briefly with an extremely small amount of RNase, just sufficient to disrupt the polyribosomal structure without causing any detectable damage to ribosomal RNA.

D. Purification of t h e 9 S Fraction The method used for detecting 9 S RNA was easily adapted to its isolation. A first procedure (49)' which was later improved by small technical modifications (50)' rests on repeated sucrose gradient centrifugations in the SW 25 rotor (Spinco). Three steps of the purification are illustrated in Fig. 4 [for details, see (50)1. Up to 250 pg of the 9 S fraction may be isolated from a 2 kg rabbit, but the yield is usually in the range of 150 pg per rabbit. The greatest care must be taken to avoid any trace of ribonuclease (from the hands for instance) especially for the operations in which the RNA is not protected by sodium dodecyl sulfate ; otherwise the isolation presents no difficulty. Quite recently, we have been able to scale up further and improve the preparations (51) by using Anderson's zonal rotor (52) and a new method for concentrating the RNA ( 5 3 ) .The resolution in Anderson's FIG.3. (A) Sedimentation pattern of (€'"-labeled) polyribosomal RNA extracted with sodium dodecyl sulfate in a linear sucrose gradient, &20C/o, equilibrated in 5 mM Tris-HC1 p H 7.4. Centrifugation was carried out for 40 hours a t 24,000 rpm (60,000 x g) in the Spinco rotor SW 25.1 a t 4". (B) Sedimentation pattern of RNA extracted wit.h sodium doderyl sulfate from a (Pa-labeled) polyribosornal suspension treatcd for 5 minutes at 37" with 0.01 $g of pancreatic ribonuclease per ml. Conditions of centrifugation as in ( A ) . Optical density a t 260 nm ; - - - - radioactivity in counts per minute. ~

186

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rotor is comparable with that achieved with the swinging bucket rotors; it is especially suitable for centrifuging the crude polyribosomal solution.

E. Detachment of 9 S RNA from Ribosomal Particles I n the method described above, the entire polyribosomal structure is dissolved in sodium dodecyl sulfate, which disrupts the protein-RNA associations and liberates all the RNA. A better principle for separating mRNA from palyribosomes would


187

be simply to detach the messenger from intact ribosomal particles. An obvious possibility is offered by decreasing the magnesium concentration, since the binding of artificial messengers and the stability of natural polyribosomes depend on these ions. Bacterial ribosomes dissociate easily and they do not, fix messenger in M Mg2+.Animal ribosomes are more resistant; it was necessary to add a chelating agent (EDTA) or to pass the polyribosomal suspension through a carboxymethylcellulose (CMC) column in order to disrupt the polyribosomes of reticulocytes. In the process, the ribosomes were split into subunits (54).The sedimentation constants of the two ribosomal subparticles obtained after EDTA treatment are about 36 and 2 6 s instead of the classical 60 and 40s. Apparently, the shape of the subparticle is changed, thus making the sedimentation slower. This has been noticed before ( 5 5 ) : but neither RNA (56, 57) nor proteins (58) seem to be degraded or lost by this trcatment. The EDTA- or CMC-treated polyribosomes were layered on a sucrose gradient and centrifuged. Figure 5 A and B show the result of such experiments for two different times of centrifugation. Clearly, a highly labeled fraction sediments between the light ribosome particle and 4s RNA. The ribonuclease test is more difficult to apply in the present case. When sodium dodecyl sulfate is used for extracting RNA, RNase is inactivated and does not interfere. I n the present case, the enzyme must be removed before the ribosomal suspension is freed of Mg, otherwise i t degrades the 16s RNA of the partly unfolded particles and the test is meaningless. Ribonuclease can be removed easily by passing the treated polyribosomc suspension through a short column of carboxymethylcellulose equilibrated with 5 mM Mg2+ (54) ; the ribosomal particles remain intact. Addition of EDTA then causes their dissociation. Again the fraction released by chelating Mg2+that sediments between the lighter particles and the 4s RNA disappears after ribonuclease treatment of the polyribosomes. The fraction released from the polyribosomes by EDTA was collected and recentrifuged together with a sodium dodecyl sulfate extract of nonradioactive reticulocyte polyribosomes as in the first isolation method. Figure 6 shows that most of its RNA sediments together with the small 9 s peak. It is therefore probable that both represent the same RNA. The fact that this RNA can be separated from the ribosomal particles without dissociating them into protein and nucleic acid is further evidence that the 9 S fraction is the RNA thread of polyribosomes, i.e., messenger RNA. The shoulder shown on the light side of 9 s peak in Fig. 6 is consistently observed. I t s properties are under investigation.

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A B FIG.5. (A) Sedimentation pattern of the ribosomal particles obtained by treating a suspension of P3*-labeled polyribosomes (8 mg/ml in 10 mM Tris-HC1, p H 7.4; 10 m M KCl) with half a volume of 0.1 M EDTA. Centrifugation was for 16 hours a t 24,000 rpm (60,OOO x g) in a linear 15-30% sucrose gradient equilibrated in 10 mM Tris-HC1, p H 7.4; 10 mM KCI. Spinco rotor SW 25.1; temperature = 4°C. (€3) Same preparation and conditions as in (A) except that the centrifugation was extended to 40 hours. I n (A) and (B): -optical density at 260 nm; radioactivity in counts per minute.

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FIG.6. Sedimentation profile of unlabeled polyribosomal RNA extracted with sodium dodecyl sulfate supplemented with the high specific radioactivity RNA detached from labeled polyribosomes by EDTA treatment,. Centrifugation for 40 hours at 24,000 rpm in a linear 5-20% sucrose gradient rquilibrated in 5 nidl Tris-HC1, IIH 7.4. Spinco rotor SR' 25.1 ; temperature = 4°C. -Optiral drnsity at, 260 n m ; - - - - radioactivity in counts per minute.

F. Properties of 9 S RNA Figure 7 shows a sedimentation diagram of 9 s RNA isolated by the sodium dodecyl sulfate method. The sedimentation constant was measured for two different preparations, and the values found were respectively 9.0 and 9.3 S. These were measured in low ionic strength tnedium containing 8 p g of RNA per ml. Using the relation worked out by Gierer (59) for a single strand of RNA, this indicates a molecular weight in the range of 150,000. The synthesis of hemoglobin requires two messengers of that size, one for each chain; the corresponding genes are not linked (60). The 9 s fraction should therefore be a mixture of two molecular species. As their molecular weight should differ by less than 3.5%, it is not surprising that they are not resolved by the sedimentation method used. The observed homogeneity of the 9s fraction may seem unexpected for another reason. It is indeed isolated from a heterogeneous polyribosomal population comprising from 1 to 6 ribosomes. If the 9 S RNA is the messenger fiber, this indicates that the polyribosomes, whether they contain two or six ribosomes, all possess a full-length mes-

190

H . CHANTRENNE, A. BURNY, AND G . MARBAIX

FIG.7. Drnsitometer tracings of the sedimentation pattern of messenger RNA (8 pg/ml in water a t 20 C) in a Brckman Spinco ultracentrifuge, equipped with ultraviolet optics, a t 42,040 rpm. Tracings were taken a t &minute intervals; exposure 80 seconds; wavelength 2655d. Sedimentation from left to right. [Taken from Rurny and Marbaix (491.1

senger. This conclusion fits perfectly with observations on hemoglobin synthesis on polyribosomes in vitro: Lamfrom and Knopf (61) have shown that even monosomes (“programmed” single ribosomes) make complete hemoglobin chains in vitro. It was possible to determine the base composition of the 9s fraction by column chromatography of an alkaline hydrolyaate. The

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loo

Fractions

FIG.8. Chromatographic separation of an alkaline hgdrolyzate of 9s RNA. Tlie neutralized solution was loaded on a 0.5 x 40 cm column of Dowex AG 1x 2-Cl- and chromatographed in a linear gradient ( 0 4 . 1 7 M ) of HCl. C = cytidine 2’(3’)-phosphate; A = adenosine 2’(3’)-phosphate ; G = guanosine 2’(3’)-phosphate ; U = uridine 2’(3’)-phosphate.


191

elution diagram (Fig. 8 ) , obtained on a 200 p g sample, shows no component absorbing a t 258 nm other than the usual nucleotides. If such components are present, they amount to a very small fraction of the total. The composition was also estimated from the distribution of radioactive phosphate among the nucleotides according to Volkin and Astrachan (6.2). This method, which confirmed the results for both ribosomal RNA’s in the same experiment, does not give identical compositions in the 9 s fraction, which shows an excess of radioactivity in the adenine nucleotides. Possible reasons for the discrepancy have been considered, but this point has not yet been clarified ( 5 0 ) . When chromatographed on a methylated serum albumin-kieselguhr column ( 4 9 ) , the 9s RNA is eluted in the same region of the gradient as 2 6 s ribosomal RNA, although the molecular weights of these two molecular species are quite different. This means that 9s RNA has a more open structure than ribosomal RNA’s (63, 64).

G. NaF-Resistant Association between 9 S RNA and Ribosomes Sodium fluoride inhibits the synthesis of hemoglobin by otherwise intact reticulocytes ; polyribosomes disappear and only 80 S particles persist after fluoride treatment (6 5 ). However, hemoglobin synthesis is restored when the reticulocytes are washed free of fluoride, while polyribosomes re-form slowly and sequentially ( 6 5 ) . Restoration of hemoglobin synthesis is perfect; complete chains are made, starting from the N-terminal amino acid (66). As no synthesis of mRNA can occur in the anucleate reticulocyte, one must consider that after fluoride action the messenger is released in the cytoplasm or that it remains bound to single 8 0 s particles. Our method for isolating 9 S RNA was applied t o the particles of fluoride-treated cells and to those of control cells that had received NaCl instead of NaF. It was confirmed that the polyribosomes completely disappear in NaF-treated cells. But just as much 9 S RNA was extracted from the 80 S particles of NaF-treated cells as from the polyribosomes of control cells (67). The 9 S fraction thus remains associated with single ribosomes. Recent results by Ravel et al. (68) and Lin et al. (69) demonstrate that fluoride blocks the initiation of new polypeptide chains and that the ribosome pellet still contains the information for hemoglobin synthesis. Considering these observations together with ours, we are led to conclude that a ribosome can attach to mRNA in presence of fluoride but is blocked a t or close to the starting end of the message. Fluoride may thus help to analyze the very first step of translation.

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V. Concluding Remarks Wc have describcd the isolation from rabbit reticulocytes of a new

RNA fraction with the following properties: it represents about 2% of the total RNA; its turnover rate in marrow cells is higher than that of ribosomal RNA ; it is preferentially degraded when polyribosomes are treated with minute quantities of pancreatic ribonuclease ; it is released from polyribosomes when the concentration of magnesium ions is drastically reduced; it sediments as a homogeneous substance with a sedimentation constant in the range expected for the messenger of individual hemoglobin chains. The new RNA has thus many properties expected for the messenger of hemoglobin. However, we have been unable so far to obtain stimulation of amino acid incorporation in a cell-free system with this RNA. The interaction of natural messenger RNA with ribosomes has not yet been well analyzed. Up to the present time, only a few viral RNA’s have been shown to direct the production of real proteins in vitro. The ease with which they are accommodated by the host ribosomes may be related to their parasitic nature. They may be able to circumvent a control that operates with cellular messengers. We consider that the 9 s RNA isolated from reticulocytes is hemoglobin messenger RNA although proof will be obtained only when conditions arc found in which it causes the synthesis of recognizable globin chains in a system demonstrably devoid of the corresponding information. ACKNOWLEDGMENTS The authors are grateful to Dr. P. Malpoix for hcr help in preparing the manuscript. The original work reported was carried out as part of the association contract Euratom-University of Brussels 016-61-10 ABIB. G. Marbaix is a Fellow of the Belgian Fonds National de la Recherche Scientifique.

REFERENCES 1 . D. Nathans, G. Notani, J. H. Schwartz, and N. D. Zinder, Proc. Natl. Acad. Sci. U.S. 48, 1424 (1962). 2. J. M. Clark, A. Y. Chang, S. Spiegelman, and M. E. Reichmann, Proc. Natl. Acad. Sci. U.S. 54, 1193 (1965). 3. M. R. Capecchi, J. M o l . B i d . 21, 173 (1966).

4. P. A . Marks, E. 13. Burkn, and D. Schlessingrr, PTOC.Natl. Acad. Sci. U S . 48, 2163 (1962). 6. A. Burny and H. Chantrennc, Riochim. Biophys. Actn 80, 31 (1964). 6. J. Kruh and H. Borsook, J. B i d . Chem. 220, 905 (1956).

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42. J. J. Barlow, A. P. Mathias, R. Williamson, and D. B. Gammack, Biochem. Biophys. Res. Commun. 13, 61 (1963). 43. C. G. Kurland, J. M o t . Biot. 2, 83 (1960). 4%. T. Staehelin, F. 0. Wettstein, H. Oura, and H. Noll, Nature 201, 264 (1964). 44. G. Marbaix and A. Burny, Biochem. Biophys. Res. Commun. 16, 522 (1964). 45. K. Scherrer and L. Marcaud, Bull. SOC.Chim. Biol. 47, 1697 (1965). 46. K. Scherrer, L. Marcaud, F. Zajdela, B. Breckenbridge, and F. Gros, Bull. SOC. Chim. Biol. 48, 1037 (1966). 47. K. Scherrer, L. Marcaud, F. Zajdrla, I. M. London, and F. Gros, Proc. Natl. Acad. Sci. U.S. 56, 1571 (1966). 48. G. Attardi, H. Parnas, M. I. Hwang, and B. Attardi, J. M o l . Biol. 20, 145 (1966). 49. A. Burny and G. Marbaix, Biochim. Biophys. Acta 103, 409 (1965). 50. G. Marbaix, A. Burny, G. Hucz, and H. Chantrenne, Biochim. Biophys. Acta 114, 404 (1966). 51. G. Huez, A. Burny, G . Marbaix, and E. Schram, Europcati 1. Biochem. 1, 179 ( 1967). 52. N. G. Anderson, J. Phys. Chem. 66, 1984 (1962). 53. R. Barber, Biochim. Biophys. Acta 114, 422 (1966). 54. G. Marbaix and A . Burny, Arch. Intern. Physiol. Biochim. 72, 689 (1964). 55. A. S. Spirin, N. A. Kisselev, R. S. Shukulov, and A. A. Bogdanov, Biokkimiya 28, 920 (1963). 56. R. F. Gesteland, J. Mol. Biol. 18, 356 (1966). 57. H. J. Gould, H. R. V. Arnstein, and R. A. Cox, J. M o l . Biol. 15, 600 (1966). 68. C. Godfroid, unpublished results. 59. A . Gierer, 2. Naturjorsch. 13b, 477 (1958). 60. C. Baglioni, i n “Molecular Genet.ics” (J. H. Taylor, ed.), 11. 405 Academic Press, New York, 1963. 61. H. Lamfrom and P. M. Knopf, J. Mol. B i d . 9, 558 ( 1 9 6 4 ) . G2. E. Volkin and L. Astrachan, Virology 2, 149 (1956). G3. K. Asano, J. M o l . Biol. 14, 71 (1965). G4. K. A. 0. Ellem, J. M o l . Biol. 20, 283 (1966). 65. P. A. Marks, E. R . Burka, F. Conconi, W. Perl, and R. A . Rifkind, Proe. NatE. Acad. Sci. U.S. 53, 1437 (1965). G6. F . M . Conconi, A. Bank, and P. A. Marks, J. M o l . Biol. 19, 525 (1966). 67. B. Lebleu, G. Huez, A. Burny, and G. Marbaix, Biochim. Biophys. Acta 138, 186 (1967). 68. J. M. Ravel, R. D. Mosteller, and B. Hardesty, Proc. N a t l . Acnd. Sci. U.S. 56, 701 (1966). 69. S. Y. Lin, R. D. Mosteller, and B. Hardesty, J. Mol. B i d . 21, 51 (1966).

Ribonucleic Acids and Information Transfer in Animal Cells A. A. HADJIOLOV Biochemicat Research Labora tory, Bulgarian Academy of Sciences, Sofia, Bulgaria I. General Considerations . . . . . . . . . 11. Remarks on the Methods of mRNA Identification . . A. Isolation of Native RNA . . . . . . . B. Molecular Characterization of RNA . . . . . C. Mononucleotide Composition and Base Sequences of RNA 111. The Sequential Synthesis of RNA . . . . . . IV. Nuclei-the Site of mRNA Synthesis . . . . . A. Molecular Characteristics of nRNA . . . . . B. Kinetics of nRNA Labeling-the First Products of Genetic Transcription . . . . . . . . C. Stable nRNA with Messenger Characteristics . . . D. Tracing of Messengers among Nuclear RNA’s . . V. Cytoplasm-the Site of mRNA Exprcwion . . . . A. Molecular Characteristics of Cytoplasmic RNA . . B. Labeling and Turnover of Cytoplasmic RNA . . . C. Mononucleotidr Composition. Cytoplasmic D-RXA’ . D. Stimulation of Cell-Free Polypeptide Synthesis . . E. Cytoplasmic Carriers of mRNA . . . . . . VI. Synopsis . . . . . . . . . . . . A. The Orthodox Interpretation . . . . . . B. One Plausible Unorthodox Interpretation . . . . References . . . . . . . . . . .

. . . . . . . .

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. .

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

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196 198 198 199 201 201 205 205 206

215 217 220 220 221 225 230 230 235 235 236

237

It is no longer a question that genetic information is mediated by specific base sequences of RNA chains. Research on the role of ribosomes in protein synthesis seemed for some time to indicate that rRNA is the carrier of genetic information (1). However, the unifying analysis by Jacob and Monod ( 2 ) of experiments with induced and phage-infected bacteria indicated that genetic information is transferred by a particular group of RNA molecules, termed messenger RNA. Consequently, a rather vague structural role was assigned to ribosomes and rRNA, the

’Abbreviafions used: RNP, ribonucleoprotein; D-RNA and R-RNA, RNA’s identical in their mononucleotide compositions to DNA or ribosomal RNA (see Georgiev in Volume 6 of this Series). 195

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base sequences in the latter being conceived as devoid of any genetic significance ( , 2 , 3 ) . Originally applied to bacteria, the messenger hypothesis was soon extended to animal cells, for which it was agreed that the same basic mechanisms operate. It was soon realized, however, that some of the postulated features of mRNA, i.e., metabolic instability, are not always observed in animal cells ( 4 ) . In the last few years, numerous studies have been carried out with animal cells, in which the assumed characteristics of mRNA were sought. Although significant progress was made, the isolation and chemical identification of the suspected mRNA has not yet been attsined (see chapter by Chantrenne e t al. in this volume). However, several enigmatic coincidences, which point to the close relationship between mRNA and rRNA, emerged from these investigations. I n this paper, the evidence on the physical and chemical characteristics, the biosynthesis, and the mode of operation of the RNA molecules likely to be the messengers peculiar to animal cells is reviewed. The author takes the liberty of proposing some unorthodox interpretations in the hope that they will stimulate new experimental approaches and further theoretical evaluations. The important findings concerning the genetic code and the basic mechanisms of protein synthesis obtained in studies with synthetic polynucleotides and viral RNA are not discussed.lB Their application to the course of events in the intact cell is considered precarious. The interaction of external messengers with ribosomes or endogenous messengers poses several problems beyond the scope of this essay. I n view of the extensive critical survey of the literature on mRNA made recently by Singer and Leder ( 5 ) ,we refer mainly to papers that appear to be of importance for the present approach.

1. General Considerations Animals belong to the group of eukaryotes, which implies the existence of basic differences from prokaryotes in the mechanisms of genetic information storage and transfer. The following features of animal cells appear to be of particular importance when RNA-mediated information transfer is envisaged. 1. The structure of the genetic material constituting the chromosomes of animal cells appears to be radically different from that of bacteria (6, 7 ) . Although the linear arrangement of genes along the chromosome is a classical fact, it does not seem that enough evidence has been " S e e article by Woese in this volume.

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presented for an “operon” type disposition. Moreover, it has been proved that closely related characters (for example, the two polypeptide chains of an enzyme molecule) may be situated in two distant chromosome loci and even on two different chromosomes [see ( g ) ] . If substantiated, these observations indicate that monocistronic messengers are more likely in animal cells. 2. Most animal cells are highly specialized in their structure and function. Accordingly, only a restricted part of the entire genome (probably about 2-5%) is transcribed during the life cycle of a differentiated animal cell. The possible role of histones as gene “repressors” has heen discussed (9, 10), but their specificity still remains to be substantiated experimentally.”) I n any case, histones stabilize the structure of chromosomes and the switching of genes “on” and “off” is likely to be more complicated than in bacteria. Even during embryogenesis, the expected interpolation of newly transcribed information is not easily observed. Instead, unmasking of preexisting stable “messages” seems to play a substantial role in development (11, 12). 3. Animal cells exist in a homeostatic milieu. Consequently, the needs for the synthesis of new types of mRNA are limited and a stable protein pattern is typical for most cells in the adult organism. Thus, regulation of mRNA synthesis is expected to be primarily directed toward quantitative rather than qualitative requirements. Only in a few cases has it been possible to connect the observed variations of enzymatic activity with an increased synthesis of enzyme molecules, and even in these cases (for example, tryptophan pyrrolase) only a five- to tenfold increase of enzyme level is recorded after induction. Accordingly, the relative role of regulatory mechanisms operating beyond the gene level would be increased. 4. The rigidity of the protein pattern in animal cells is correlated with the existence of stable messengers. Observations with enucleated cells indicate a stable protein synthesis persisting for several days and obviously independent of the continuous nuclear supply of short-lived mRNA (11, 1 3 ) . Experiments with actinomycin D (even neglecting thc general toxic effects of this drug) reveal that synthesis of total liver protein (14) and different liver enzymes (15) persists for hours and even days after inhibition of RNA synthesis. The stability of mRNA for specific proteins is well documented for reticulocyte hemoglobin ( 4 ) ’ ~ and silk gland fibroin (16, 1 7 ) . 5. Compartmentation of transcription and translation sites in the animal cell implies the existence of an irreversible flow of information ”See article by Hnilica in this volumr. “See article by Chantrenne eI (11. in this volume.

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from the nucleus into the cytoplasm. The flow of ribosomes required in the translation process is also irreversible (18).If ribosomes are actively engaged in the release of mRNA from the DNA template, as in bacteria (19), each ribosome can participate only once in this process. If not, unprotected mRNA would have to cross a long distance through the karyoplasm quite exposed to nuclease attack. It has been shown (20) that ribosomes of nucleated cells are markedly heavier (80 S) than those of bacteria and blue-green algae (70 S). The difference in size of rRNA’s in eukaryotes and prokaryotes is also well known. This enigmatic extra mass in animal cell ribosomes and rRNA suggests the existence of basic dissimilarities in the mechanisms of genetic information transfer.

II. Remarks on the Methods of mRNA ldentificution Work with biopolymers has made it necessary to depart from the chemical criteria of a pure compound, i.e., analysis of structure and reconstitution by synthesis. Instead, a substance found to be homogeneous by physicochemical methods is identified by its particular function. For mRNA, a single and chemically defined RNA molecule would direct, in a heterologous ribosomal system, the synthesis of a protein molecule with known structure. Since this functional identification of mRNA has not yet been achieved (see chapter by Chantrenne et al. in this volume), the term “messenger” RNA has been used to designate various RNA fractions possessing one or more traits presumed t o be inherent in this type of molecule ( 2 ) . However, it becomes increasingly clear that all the criteria used to identify mRNA, such as rapid labeling, nucleotide composition, hybridization with DNA, stimulatory activity in cell-free polypeptide synthesis, provide only indirect evidence ( 5 ). Moreover, with all these criteria, the burden of the proof is dependent on the methods of RNA isolation and characterization. Therefore, a brief discussion of some methodical imperfections seems to be of particular importance for our understanding of the genetic role of cellular RNA.

A. Isolation of Native RNA The introduction of the phenol technique (see Georgiev in Volume 6 of this series) has made possible the isolation of “native” RNA. However, it soon became evident that in several cases this native state is more apparent than real. I n fact, nuclease degradation is a very important factor when any one of the present methods of cell fractionation and subsequent RNA isolation is considered. A few phosphodiester bond breaks per molecule will result in shorter polynucleotide chains whose relation to the original RNA molecule is difficult to establish. This is

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illustrated by the controversy as to the intact nature of rRNA chains and, although convincing evidence has been presented ( 2 1 ) , some doubts still persist ( 2 2 ) . The isolation of “stable” rRNA in a truly native state requires direct homogenization of cells with phenol ( 2 1 ) . Obviously, such a requirement should apply even more rigorously to mRNA, which is thought not only to be more “unstable,” but to represent a small part of the total RNA. Thus, it is likely that in any procedure involving the previous fractionation of cell structures (nuclei, polysomes, etc.) , the isolated mRNA represents a more or less degraded product. Further, it is a general observation that preparations of RNA are unstable during storage. “Spontaneous” degradation is usually invoked, although there is no apparent chemical reason for such degradation. More probably, some nucleases resist phenol treatment ( 2 3 ) and contaminate the RNA preparations. Our own experience indicates that introduction of sodium dodecyl sulfate and polyvinyl sulfate (or bentonite) is helpful, but does not by itself remove all nuclease contaminants. Repeated deproteinization (involving several chloroform steps) yields the best RNA preparations. A routine test involves incubation of RNA solutions a t 37°C and subsequent analysis by agar gel electrophoresis. Our best preparations remain stable for 24 hours without marked changes in the electrophoretic pattern. Evaluation of nuclease contaminants in RNA samples is highly desirable, but appears lacking in most studies on mRNA. Yet, such control is critical with RNA samples suspected to contain mRNA, since methods of further characterization are often of long duration.

B. Molecular Characterization of RNA Density gradient centrifugation is the most widely used method for

RNA fractionation. However, important limitations exist when a minor fraction, like mRNA, is considered. These include: interaction with the major rRNA fractions, formation of aggregates with contaminants like DNA or polysaccharides, irregular influences of the medium (pH, ionic strength, Mg2+ etc.) , cross-contamination of RNA fractions on the gradient [cf. ( 5 ) ] .Sucrose is almost exclusively used as the gradient material, but trials with other materials and varying media are necessary prerequisites for the adequate identification of a minor RNA fraction. Thus, uncertain S values can be obtained with RNA’s of a higher molecular weight than rRNA under different conditions (24). These limitations justify the search for alternative methods of RNA fractionation. Gel electrophoresis may be such a method. Agar gel electrophoresis was originally used as a qualitative method of RNA fractionation, the

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separate bands being localized by pyronine staining (25, 2 6 ) . Recently, Tsanev and Staynov (27) developed a new technique for the direct UV densitometry of dried agar gel electrophoregrams. This technique allows the localization and quantitative estimation of the different RNA fractions (28). The RNA patterns are identical with those of density gradient centrifugation, and an almost linear relationship between S 30 -

TMV RNA

25 20 -

Liver [I8 S]

I5 10-

5-

I Mobility, centimeters from start

sRNA I

,

1 2 3 4 5 Mobility, centimeters from start

FIG.1. Comparison of RNA fractionation by agar gel electrophoresis and drnsity gradient centrifugation. Lefl : Agar gel electrophorcsis pattern of rat liver cytoplasmic RNA’s. The absorbance is recorded by direct UV densitometry of the dried electrophoregram by the method of Tsanev and Staynov ( 2 7 ) . Right: Corrrlation between sedimentation coefficients of different RNA fractions and their elcctrophoretic mobilities in agar gel. [From Hadjiolov et al. (29).1

electrophoretic mobility and S value is found (29) (Fig. 1 ) . Similar results were obtained by the use of polyacrylamide gels ( 3 0 ) .Although most of the pitfalls of density gradient centrifugation mentioned above are likely to intervene in gel electrophoresis, comparative studies permit a more precise identification of mRNA. Another method of RNA fractionation is chromatography on methylated albumin-kieselguhr columns [see (31)]. The results obtained by this technique are not directly comparable with those from the first two methods, and it seetns that its resolving power is more limited. On the other hand, the influence of base composition on the fractionation of nucleic acids has been found helpful in the isolation of RNA fractions likely to represent mRNA (see below). I n brief, the methods available for the molecular characterization of RNA are far from perfect. The adequate identification of a minor fraction like mRNA requires the Concomitant use of more tha,n one technique in order to minimize the artifacts.

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C. Mononucleotide Composition a n d Base Sequences of RNA The significance of nucleotide composition as a criterion of mRNA is limited because the composition of the active genes is unknown. The designation of an RNA fraction as “DNA-like” is often controversial. The artifacts that may arise when the mononucleotide composition of RNA is studied by P32distribution are discussed below. RNA-DNA hybridization (see McCarthy in Volume 4 of this series) has only recently been applied to the identification of RNA from animal cells. The use of different hybridization methods makes comparison difficult. Application of these techniques to genetically defined systems has yielded spectacular results for the DNA cistrons involved in rRNA synthesis [see Perry ( 3 2 ) ] .Unfortunately this is not yet the case with mRNA. Further, the chemical basis of the method has not been clarified and numerous controls are needed in order to allow a correct quantitative evaluation. The length of the RNA chain involved in specific base pairing is not known, but it seems to be much shorter than that of the added molecule. Thus, in the case of rRNA from HeLa cells, about 3050% of the hybridized material was recovered in the form of acidsoluble oligonucleotides ( 3 3 ) . It is evident that the shorter the chains involved, the higher the hazard of unspecific hybridization. This possibility is strengthened by the observation that DNA may form hybrids with poly U, poly G, or poly I,G, with only 10-50 residues engaged in base pairing (34). The presence of nuclease contaminants in RNA, discussed above, combined with a higher susceptibility of mRNA to enzyme attack, may thus become critical in the quantitative estimation of hybridization tests.

111. The Sequential Synthesis of RNA Labeling experiments of short duration have been, by definition, a crucial approach in the search for niRNA in animal cells. A theoretical evaluation of the mechanisms of RNA synthesis is consequently of particular importance for the adequate interpretation of these studies. The prohlem of the sequen tial synthesis of single-chain biopolymers arose from studies on protein biosynthesis. The theoretical grounds have been discussed in detail and experiments with different proteins have consistently shown that the growth of polypeptide chains is vectorial, proceeding from the N-terminal toward the C-terminal end [see (35) for references]. It is now firmly established that animal RNA molecules are composed of single polynucleotide chains (21, 2.2). Studies with the

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Escherichia coli phage 4x174 (36) and the Bacillus subtilis phage SP 8 (37) provide conclusive evidence that only one strand of DNA is copied in the transcription process in viva Extension of these results to enzymatic systems has shown that, when native phage DNA is used as template, only one of the two DNA strands is transcribed (38, 3 9 ) . General considerations indicate that asymmetry of transcription is a common mechanism operating in vivo (40). Further, experiments with enzyme systems (41, 4.2) and with E . coli cells (43) indicate that the synthesis of RNA is also vectorial and procecds by the stepwise addition of nucleotides from the 5’ phosphate end

-

Direction of chain growth

5’ c _

Labeling gradient

FIG.2. Vectorial synthesis of RNA chains. New nucleotides are attachrd a t the 3’-end of the polynucleotide chain. B = P u or P y base.

towards the 3’ hydroxyl end of the polynucleotide chain (Fig. 2 ) . The mechanisms determining the polarity of transcription are not clear, but they may involve a preferential binding of RNA polymerase to the 3’ hydroxyl of the template DNA (44). Asymmetry and polarity of the transcription process provide the necessary basis for considering the problem of sequential synthesis of RNA molecules. In the process of rRNA synthesis in animal cells, the following successive steps may be considered: assembly of free nucleotides on the DNA template; release of the rRNA precursor; transformation into 28 S and 18 S rRNA; nucleocytoplasmic transfer of the two rRNA’s [see (SZ)]. The distribution of the label expected along the polynucleotide chain of these RNA species after a short period of labeling is outlined in Fig. 3. It is evident that in any case of RNA synthesis studied with labeled precursors there must be a time period during which there is a nonrandom distribution of the label along the polynucleotide chain. This time period may be designated as the completion time of RNA. As a consequence, the following correlations may be expected in short-term labeling experiments. 1. Uneven labeling of a given nucleotide residue along the polymer chain will reflect the completion time of RNA molecules if the specific

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activity of the free nucleotidc in the precursor pool is kept constant. If this condition is not satisfied, then uneven labeling of RNA will also arise from variations in the labeling of the precursor nucleotide. However, this will hold true only if the completion time is comparable with the labeling time. Accordingly, in both cases, uneven labeling along the polynucleotide chain indicates that the completion time is commensurate with the labeling time. 2. Uneven labeling of a nucleotide residue along the polynucleotide chain would be detected not only during the actual completion timc of RNA molecules, but as long as the amounts of partly labeled and Nuclear precursor RNA P

3‘-end

5‘-end

m ‘vvv\

-

5L.iwww

3‘ 5‘

>--1 B

3‘

5b-3‘5;

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3‘

___zMj

18 S RNA

28 S RNA

28 S RNA

FIG.3. Model for the sequential synthesis of nuclear precursor RNA and the expected distribution of label in short-term labeling experiments. The straight lines represent the unlabeled segment of the RNA chain; the zigzag lines, the labeled segment. A single split of the precursor is envisaged to give the product 285 and 18s RNA. Depending on whether the split occurs in A or B, the ratio of the specific radioactivities of lSS/ZSS RNA’s will be lower ( A ) or higher ( B ) than unity.

randomly labeled RNA molecules are comparable. As a consequence, the apparent completion time could be severalfold longer than the actual one. Obviously the time necessary for the precursor-product transformations and the nucleocytoplasmic transfer of RNA molecules only prolongs the time period during which uneven distribution of the label along the RNA chain may he detected. These considerations show that the completion time of RNA molecules is a very important paramcter to be determined in studies on RNA synthesis. Conceivably, its importance may become crucial in the shortterm (“pulse”) labeling experiments now widely used for mRNA identification. The labeling time used in such studies with animal cells

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varies from 5 minutes to a few hours. It is considercd that thr radioactive pulse should comprise 1/10 to 1/20 of the generation time of the cells studied. What then is the conipletion time of RNA molecules? Is it commensurate with the labeling time used in studies on niRNA? No direct answer to these questions is available. Some information may be derived from data on the sequential synthesis of proteins. Since any RNA molecule (including mRNA) is likely to participate in the production of several protein molecules, it seems reasonable to assume that the rate of protein synthesis will exceed the rate of RNA synthesis. Thus, studies on hemoglobin synthesis in intact reticulocytes show a definite labeling gradient after 7 minutes of incubation for the /3 chain and after 16 minutes for the LY chain (45).Experiments with perfused rat liver fixes the completion time of albumin molecules a t 2-5 minutes ( 4 6 ) . Similar figures were obtained for RNase synthesis in pancreas slices (4'7),lysozyme formation in minced hen's oviduct ( 4 8 ) ,etc. Where 100600 amino acid residues are involved, an average stcp-time of 0.5 to 1 second may be calculated. If the rate of RNA synthesis is taken as equal to that of proteins, then the completion time of a precursor: RNA molecule of 1.0 t o 2.0 X lo' nucleotides will be about 80-330 minutes. On the other hand, the experiments of Goldstein et al. (43) with E . coli cells labeled in vivo with uridine-C14 established a step-time of RNA synthesis of 13 seconds a t 0".Extrapolation of these results to 37"' taking into consideration the known temperature coefficients of enzyme reactions, gives a step-time of about 0.5 second and an estimated completion time of the precursor RNA envisaged of about 80-160 minutes. These figures for the completion time of RNA molecules in animal cells are unexpectedly high. Rough estimates of the actual completion time of RNA molecules have therefore been made on the basis of the doubling time of growing cells (with respect to their RNA) . Approximate values of 2-10 minutes were obtained. If we multiply these figures by a factor of 10 in order to evaluate the apparent completion time of RNA molecules, then it may be expected that uneven labeling of RNA molecules would be observed with growing cells a t labeling times shorter than 20-100 minutes. At present, we do not know exactly the size of the genome involved in RNA synthesis. We also do not know if RNA synthesis is a continuous or an intermittent process. Therefore, the above approximations may as well be erroneous. Nevertheless, there is little doubt that in any brief labeling experiment, the completion time of RNA molecules and the possibility of uneven labeling should be taken into account. A particular aspect of short-term labeling cxperiments is the estima-

RNA AND IXFORMATION TRANSFER IN ANIMAL CELLS

205

tion of the base composition of rapidly labeled RNA from the distribution of PASamong the four nucleotides obtained a t alkaline hydrolysis. Alkaline hydrolysis of RNA, by displacing P32to the nearest nucleotide on the left, eliminates differences in the specific activities of the nucleotides arising from nonrandom labeling of the precursors. Accordingly, it is deduced that distribution of P32among the four 2’(3’) mononucleotides represents the composition of labeled RNA molecules [see ( 4 9 )1. This interpretation is correct on1y if the distribution of nearest-neighbor nucleotides is random along the whole length of the RNA molecule in question. Although there is now general agreement on the general validity of this postulate, remarkably little ev-dcnce has been obtained to support it. I n fact, a direct comparison of the labeling of 2’(3’) and 5’ mononucleotides obtained from the same rapidly labeled RNA has been made only with yeasts ( 5 0 ) , T2-phage infected E . coli ( 5 1 ) , Ehrlich ascites tumor cells ( 5 2 ) , and rat liver ( 5 3 ) . Indications of statistically random distributions of nearest-neighbor nuclcotides were obtained in the first three cases only. Moreover, when the complction time of RNA molecules is considered, the following possibilities should be noted: 1. The Pd2distribution among RNA nucleotides yields information on the composition of the RNA segment labeled during the P,32pulse. Likewise, deductions about the random distribution of nearest-neighbor nucleotides apply to the labeled segment only. 2. Changes in the labeling of a-phosphates of precursor nucleoside 5’-triphosphatcs with time result in uneven labeling of the different segments of RNA moleculcs. As a consequence, the different segments of the molecule will contribute to varying extents to the estimated overall composition.

IV. Nuclei-the

Site of mRNA Synthesis

A. Molecular Characteristics of nRNA General agreement has not yet been attained even on the major RNA species present in the nucleus [see (54, 55) 1. The present discussion is limited to those aspects likely to have some bearing on nuclear mRNA. Cold phenol treatment of animal tissues and cells extracts “cytoplasmic” RNA, while nuclei remain entrapped in the water-phenol interphase layer. Furtlier bricf cxtraction with hot phenol-sodium dodecyl sulfate brings the total (more than 90%) interphasc RNA into solution ( 5 5 ) . This “nuclear” RNA is represented by well-shaped 28 S and 18s peaks. Our own experience with brain, livcr, hypophysis, and ascites tumor cells confirms the good reproducibility of this technique. However, this approach cannot exclude either loss of nRNA or contanii-

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nation with cytoplasmic RNA. Isolation of RNA from pure nuclei obtained by centrifugation in hypertonic sucrose reveals again the main 28 S and 18 S peaks (56-58), but in this case the relative amount of 18 S RNA is lower and the evidence suggests that degradation of RNA during isolation of nuclei by this technique cannot be avoided ( 5 9 ) . Isolation of HeLa cell nuclei by subsequent hypotonic and detergent treatments results in the complete absence of the 18s RNA component (60). Recently, methods have been developed that allow the rapid isolation of pure nuclei by the use of nonionic detergents in the cold (6f-63).The RNA extracted from such nuclei contains 28 S and 18 S components in a ratio analogous to that observed with interphase "nuclear" RNA (52, 5 9 ) . Thus, there is little doubt that the bulk of true nRNA is represented by the 28s and 18s molecular species. However, since variations in their ratios are commonly observed, the influence of the following factors should be considered. 1. Loss of nRNA components may occur in any extended procedure for isolation of nuclei. Preferential loss of 18s RNA would account for the smaller amounts of this RNA observed in some experiments (57, 58, 60). 2. On the other hand, incomplete extraction of some nRNA components is a possibility in any mild procedure ( 5 5 ) . The importance of factors, such as DNA-RNA binding, interaction of RNA with histones and other proteins, entrapping of RNA in lipoprotein structures, remains unknown. 3. Several authors have observed that hot phenol treatment of RNA results in the selective degradation of 28 S RNA (28, 59, 6 4 ) . Heating a t 65" of isolated nRNA (stable for several hours a t 40") results in the selective degradation of 28 S RNA ( 6 5 ) . The detailed studies of Applebaum e t al. (66) with RNA from Hyalophora cecropia wings demonstrate the nonenzymatic conversion of 28s into 18s RNA on heat treatment. All these findings clearly indicate that uncertainties concerning the major nRNA components are likely to have repercussions on all attempts to characterize nuclear mRNA.

B. Kinetics of nRNA Labeling-the

First Products of Genetic

Transcription

1. AUTORADIOGRAPHIC STUDIES

Experiments with various RNA precursors support the view that all types of RNA molecules are made in the nucleus [see (54, 67) for references]. Early attempts to localize the site of RNA synthesis within the nucleus showed that the label appears almost simultaneously in both


207

the nucleolus and the karyoplasm [ cf. ( 6 7 )1. Such observations suggested an independent synthesis of these two topically distinct RNA types. Selective inhibition of nucleolur RNA labeling by low doses of actinomycin D and other independent evidence rule out a major RNA flow from karyoplasm to nucleolus ( 3 2 ) , but migration of RNA in the opposite direction is not excluded. Thus, UV microbeam inactivation of the nucleolus reveals that at least 30% of karyoplasm labeling is dependent on the nucleolus (68). Recently Amano et al. ( 6 9 ) , in a detailed kinetic study of nRNA in liver and pancreas pulse-labeled with cytidine-H7, found the decay of RNA labeling in both karyoplasm and nucleolus to be essentially parallel. Yet the low resolving power of the light microscope limits the quantitative evaluation of the results. Rapid labeling of nRNA followed by electron microscopic autoradiography reveal a more intense labeling of nucleolar RNA as compared to karyoplasmic RNA (70, 7 1 ) (Fig. 4). One plausible interpretation is that labeling of karyoplasmic RNA is a transitory event; i.e., there is no accumulation of labeled RNA in the karyoplasm, but since the identity of the RNA species involved is not established, a correlation with biochemical studies is difficult. 2. THEFREENUCLEOTIDE POOL IN NUCLEI

The interpretation of RNA labeling depends on the study of the free nucleotide precursor pool [see (6, 5 4 ) ] . Hence progress in this field is disappointingly slow. A major obstacle lies in the fact that free nucleotides are washed out during isolation of nuclei. There is no doubt that nuclei contain all the nucleotides required for RNA synthesis, but their exact level is unknown. Rapid equilibration of nuclear precursor pools is a common presumption, but in fact, the dilution factor may vary a t nucleolar and karyoplasmic sites of RNA labeling. The importance of the metabolisni of the precursor is likely to be increased when incorporation of a common precursor into different RNA nucleotides is studied. For example, by following adenine-C14 incorporation into adenine and guanine of nuclear and cytoplasmic RNA, Harris (72) reached the eonclusion that a rapid degradation of RNA takes place inside the nucleus. Yet, these results could be also explained by a delayed metabolism of guanine nucleotides a t the nuclear sites of RNA synthesis ( 7 3 ) . The existence of specific nucleotide pools in nuclei is indicated by the finding that P32labeling of GMP derived from nRNA of Ehrlich ascites tumor cells is twelvefold lower than that of the other three nucleoside 5'-monophosphates ( 5 2 ) . Another aspect of this problem is the possible rate-limiting role of the free nucleotide supply in RNA synthesis (7'4). Indeed we do not

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209


know whether nuclei are dependent on mitochondria for their supply of precursor nucleotides. Oxygen-dependent formation of ATP and other nucleotides was reported in thymus nuclei (9, 75), though the universal occurrcnce of this phenomenon remains to be proved. On the other hand, the studies of Reid e t al. (76) demonstrate in liver nuclei a high level of enzymes involved in the conversions orotate + UMP + UDP + UTP

Thus, it is possible that nuclei are self-sufficient in their supply of RNA precursor nucleotides. I n this casc, the supply of nucleotides would be even more limited since overall nuclear synthesis of nucleoside 5’-triphosphates proceeds a t a markedly lower rate than does cytoplasmic synthesis. The nucleotide level may bccome critical when we consider some nRNA’s, suspected as messengers, that are characterized by exceedingly high turnover rates.

3. RAPIDLYLABELED HIGHMOLECULAR WEIGHTnRNA The incorporation of a labeled precursor in a short time period (“pulse”) is widely used in the search for mRNA although the validity of this criterion is limited. The first ultracentrifugation studies on nRNA of animal cells revealed an initial appearance of the label in a broad peak with a maximum a t about 45s (56, 77, 78). Although suspected as mRNA, it became soon evident that 45 S nRNA is in fact a precursor of rRNA [see review by Perry ( 3 2 ) ] .Nevertheless, the picture of a simple precursor-product relationship is complicated by several observations that seem to indicate that molecules that are similar in size but “DNA-like” in composition are also rapidly labeled. Actually, since the pioneer work of Davidson et al. (79, 80) [extended to a variety of animal cells (cf. 52, 81-83)], it has been known that the nucleotide composition of rapidly labeled nRNA is more or less shifted toward that of DNA. More recently, several authors isolated the heavy rapidly labeled RNA fractions of density gradients and determined their nucleotide composition. These data are summarized in Table I. As can be seen, the rapidly labeled RNA in the 40-50 S zone displays wide variations in base composition. The G+C/A+U ratios oscillate between values of 0.8-0.9 (as in DNA) to 1.5-1.6 (as in rRNA). Accordingly, the rapidly labeled nRNA was considered by different authors ~

FIG.4. Distrihution of rapidly lahclrd R N A in the nucleolus and the knryoplasm of cultured kidney cells. Labeling with ~ r i d 1 n e - Hfor ~ 10 minutes. Localization of thc label by electron microscopic ;iulor:xdiography. [From Granboulan and Gram boulan (70).]

E

TABLE I MONONUCLEOTIDE COMPOSI~O OF N RAPIDLYLABELEDHIGH MOLECULAR WEIGHTNUCLEARRNA

Source HeLa cells

Ehrlich ascites tumor cells Immature erythroid cells

Mouse plasmocytic sarocma Rat liver

Nuclear fractionb Phenol 60" Phenol 60" Phenol 60" Phenol 60" Phenol 60" Phenol 60" Phenol 60" Phenol 60" Phenol 60" Nuclei Phenol 60" Phenol 60" Phenol 60" Phenol 60" Phenol 60" Phenol 60" Phenol 60" Phenol 60" Phenol 60" Phenol 43-63" Nuclei

RNA fraction (S va1ue)c 45 60-80 30-45 45-70 >70 41-54 72-84 84-100 45

45 Total 45 50 50 30-45 30-45 45-75 45-75 >75 >75 35-45 >45 Total

Time of labeling (minutes)

40 40 5 5 5 30 30 30 60 180 30 120 15 120 30 120 30 120 30 120 30 30 5

0 OF

ANIMALCELLS=

Molar ratio (%) A

U

G

C

18 24 19.8 20.6 21.3 17.6 23.1 23.4 16 14 20.1 21.2 22.4 25.0 20.6 19.9 21.7 23.2 25.1 23.1 18.1 18.0 23.0

22 29 26.9 26.5 31.0 22.2 28.2 29.5 27 24 25.6 26.4 27.8 29.1 24.8 22.5 28.7 28.6 31.1 29.9 22.3 21.5 30.9

33 24 25.1 25.4 21.1 30.9 23.8 21.4 32 32 28.0 25.9 25.7 22.7 28.9 29.9 27.9 24.6 24.1 23.3 30.3 31 . O 25.6

27 23 28.0 27.3 26.4 29.1 24.7 25.5 25 30 26.3 26.4 23.9 23.0 25.7 27.7 21.7 23.6 19.7 23.2 29.3 29.5 20.5

G A

++ C/U

1.50 0.89 1.14 1.12 0.91 1.50 0.95 0.89 1.33 1.63 1.19 1.10 0.99 0.85 1.20 1.36 0.98 0.93 0.78 0.87 1.47 1.49 0.86

Reference

Rat liver

Xuclei Nuclei Nuclei Nuclei Nuclei Nucleoli Nucleoli Nucleoli

35 45 45 4.5 55 28 35 45

30 30 30 10 10 Directd Direct Direct

24.7 25.1 25.7 24.9 26.1 15.0 14.3 14.5

22.1 22.1 23.7 21.2 22.6 19.9 19.8 20.8

29.1 28.5 26.3 29.5 26.5 34.9 35.1 35.3

24.1 24.3 24.3 24.4 24.8 30.2 30.8 29.4

1.14 1.12 1.03 1.17 1.05 1.87 1.93 1.84

(91) (91) (91) (92) (92) (91) (91) (91)

;P

Z

U n

z

r

0

w

52 8

The mononucleotide composition is determined by P3*distribution analyses after various periods of labeling with inorganic p h 0 ~ p h a t e - P ~ ~ . a The RNA fractions obtained by hot phenol treatment represent the total cellular RNA. It is known that the rapidly labeled, high molecular weight RNA’s are confined to the nucleus (see Section IV, A). u, c Fractions obtained from the appropriate regions of sucrose density gradients. The sedimentation coefficient values are approximately 2 s estimated by the position of the RNA fraction analyzed, with respect to the 28 S and 18 S rRNA4components. d The composition of the respective RNA fractions, concentrated and purified by sucrose density gradient centrifugation, is determined 2 + by direct analyses. a

E

Z

E Brl

212

A. A. HADJIOLOV

as either D-RNA (93, 94) or a pure rRNA precursor (95). Since intermediate values are usually found, most authors consider the rapidly labeled 40-50s nRNA as a mixture of these two RNA types. I n any case, it is now clear that the first D-RNA molecules labeled are also bigger than are the bulk of nRNA molecules. The basic conclusions from these studies may be summarized as follows. a. A direct correlation exists between the growth rate of the cells studied and the base composition of the rapidly-labeled nRNA. Thus, nongrowing cells show a preferential labeling of D-RNA (87, 88), while only R-RNA is usually detected in growing cells (32, 95). Transfer of ascites tumor cells in the stationary phase of growth to a mineral medium does not cause a shift of rapidly labeled RNA composition ( 6 2 ) , but exponentially growing cells respond to a similar change in the medium by an increased D-RNA labeling (96, 97). A shift toward preferential labeling of R-RNA is observed when the growth rate is increased, as in phytohemagglutinin stimulation of lymphocytes (98) and in regenerating rat liver (9.2). b. Similar changes are obtained when RNA synthesis is inhibited by actinomycin D treatment. Thus, inhibition of 9&95% of RNA synthesis results in the almost exclusive labeling of D-RNA (32, 96, 99). A favored interpretation is that D-RNA synthesis is more resistant to actinomycin D than is the synthesis of rRNA precursors [cf. (%)I. c. When the time course of RNA labeling is followed, a gradual shift in composition of rapidly labeled nRNA from D-RNA towards R-RNA is recorded. This transition was well documented in studies on total nRNA of Cave cells (100) and 4 5 s nRNA of Ehrlich ascites tumor cells (86). d. Embryos of the anucleolate mutant of Xenopus laevis in which RNA synthesis is exceedingly slow, display a “DNA-like” base composition of labeled RNA even with very prolonged labeling times (101).I n this case, too, the initially labeled D-RNA is situated in the heavy region of density gradients (10.2). These observations suggest that a slower rate of overall RNA synthesis is correlated with a higher ratio of D-RNA in the population of rapidly labeled RNA molecules. Moreover, the initially labeled RNA molecules are invariably “DNA-like” and represent a more or less heterogeneous population situated preferentially in the heavy region of density gradients. Since this D-RNA is a likely candidate for a messenger, several attempts a t its further characterization have been made. One approach involves the detailed analysis of density gradient fractions heavier than rRNA precursors. Thus, in immature duck erythrocytes (87, 88, 103, 104) and in HeLa cells (84, 85), the rapidly


213

labeled RNA of 60 to 100s displays a “DNA-like” composition (see Table I ) . I n fact, the content of U is markedly higher than that of A, while both G and C are low. Experiments with DNase,2 pronase, and trypsin do not lower the high S values of this RNA, while various treatments, including Mg2+chelation or heating a t 80’, seem to indicate that a continuous polynucleotide chain is involved (24, 88). Sedimentation studies, confirmed by electron microscopy (104), indicate a molecular weight of about 5 to 7 X loF,i.e., a polynucleotide chain constituted of 1.5 to 2.0 X lo4 monomers. Another approach involves the fractionation of nRNA in the presence of hot phenol [see ( 5 5 ) ] .The rapidly labeled RNA in the fraction extracted a t 55-65’ shows a mononucleotide composition matching exactly that of DNA in liver and ascites tumor cells (55, 106). Somewhat higher G+C/A+U ratios for a similar fraction from liver have been found by other authors (107,108), while in a mouse plasmocytic sarcoma, Kempf and Mandel (89) have reported a G+C/A+U ratio of 1.49 for the 4 5 s RNA of the 43-63” fraction. As indicated above, it is likely that the observed variations reflect peculiarities in the rate of RNA synthesis within the different cells studied. It is interesting that with this method of RNA fractionation, the rapidly labeled RNA displays lower S values with an upper limit of 40-50S, and a clear-cut distinction from the 45 S rRNA precursor is not possible by this criterion. The reason for the observed discrepancies between the two fractionation methods is not readily evident. In the case of hot phenol fractionation, degradation of some RNA fractions is more likely to occur (see Section IV, A). Several attempts have also been made to obtain a cytologically controlled fractionation of nucleoli and karyoplasm. Although RNA degradation is an almost unavoidable hazard, it seems that new techniques (109, 110) allow a preservation of RNA sufficient to justify further studies. With this approach, Busch e t al. (91, 111, 112) obtained evidence for the nucleolar location of the 45 S rRNA precursor, while tracer kinetics confirmed the nucleolar site of the 45 S +-35 S + 28 S RNA conversions. Yet, contrary to the commonly favored scheme [see ( 3 2 ) ] ,formation of 18 S nRNA was located in the karyoplasm and a particular 45-55 S precursor RNA was envisaged (92, 113). The G+C/ A+U ratio of this rapidly labeled RNA fraction in liver is 0.86, which relates it to the “DNA-like” RNA found by other workers. Finally, a note of warning seems appropriate: rapidly labeled nRNA is not invariably found as a high molecular weight fraction. For ex-

‘Experiments with thymidine-HY indicatc that some DNA is associated with rapidly labeled RNA in the heavy regions of density gradients (106).

214

A.

A. HADJIOLOV

ample, an nRNA fraction of liver extracted a t pH 8.3 showed a welldefined rapidly labeled “DNA-like” component of 8-16 S (114). Even if degradation has taken place, it must have occurred a t specific sites on the high molecular weight RNA molecule. I n summary, most of the results discussed in this section indicate that RNA molecules of high molecular weight and a “DNA-like” composition are labeled more rapidly in animal cell nuclei than are other types of RNA that are similar in size but t h a t have an “rRNA-like” composition. The site of labeling is likely t o be the karyoplasm, while kinetic studies reveal that the labeling of the “DNA-like” nRNA invariably precedes th at of the nuclear rRNA precursor. These findings are discussed in section IV, D. 4. HYBRIDIZATION OF RAPIDLY LABELED nRNA As shown by the evidence given above, karyoplasmic DNA is the most likely candidate for the site of mRNA transcription. Since the genes involved in mRNA transcription are presumably more numerous than those for rRNA, hybridization studies are expected to yield valuable information on rapidly labeled nRNA. Unfortunately, the technical difficulties inherent in this technique (see Section 11, C) have not been adequately controlled in most studies. On a comparative scale, the highest levels of RNA hybridization are obtained with rapidly labeled RNA fractions of animal cells. However, even with very short pulses, the amount of labeled RNA hybridized a t saturation is in the range of 1-5% of the input labeled RNA (104, 115). Such results seem to indicate that in animal cells a limited portion of the genome is involved in the production of numerous identical mRNA copies (see Section I). Yet, the quantitative estimation of hybridization experiments may be misleading. Thus, in the case of the anucleolate mutant of Xenopus Eaevis, annealing of labeled RNA with five successive portions of DNA reach a plateau a t about 12% hybridization of the input RNA (102).Since no rRNA is expected to be labeled in this organism, failure to obtain 100% hybridization is not easily understood. Further, a clear-cut distinction between rapidly labeled rRNA precursors and D-RNA seems difficult. For instance, it was reported that labeled 45-54s RNA from normal and actinomycin D-treated mouse L fibroblasts shows identical hybridization and competition by rRNA (116). In addition, in the only case in which the hybridized rapidly labeled RNA was analyzed further, large discrepancies in base composition were found, depending on whether direct estimation or P32distribution was used for the determination (104).


215

C. Stable nRNA with Messenger Characteristics The studies outlined above disclosc that neither of the main nRNA components (28 S and 18 S) may be ronsidcrcd to be rapidly labeled. However, several attempts were .made t o study the other traits expected from mRNA as displayed by different nRNA fractions. It has long been known that total RNA from purified nuclei has a lower G+C/A+U ratio than the bulk of cytoplasmic RNA [see (55) for refs.]. Values of 1.05 to 1.15 werc reported with nuclei purified by centrifugation in hypertonic sucrose (109, 117). Although wide variations in the molar ratio of individual nucleotides were reported, these results suggest the existence of stable D-RNA in nuclei. Recently, several workers have described the isolation of such D-RNA from nuclei. I n thymus nuclei, it was found t o represent about 6% of nRNA (118). Phenol extraction of nRNA with a gradual increase of either temperature (119) or pH (120) was reported to yield a D-RNA in substantially higher amounts (up t o 30% of total nRNA). A wide size distribution of this D-RNA in sucrose density gradients was correlated with the expected size heterogeneity of mRNA ( 1 2 0 ) , but the initially reported size heterogeneity of this D-RNA fraction is likely to be a degradation artifact. Further studies by the same workers (106, 114) indicate that the bulk of this D-RNA constitutes a broad peak with a maximum a t about 1 8 s . I n fact, when degradation is minimized by reduction of the extraction time a t 65" and the addition of Mg2+ and polyvinyl sulfate, a rather narrow 18s peak constitutes the bulk of nuclear DRNA (65, 108) (Fig. 5 ) . Thus, if this fraction is to be considered as mRNA, one should bear in mind that this mRNA is not heterogeneous in size and its peak coincides with the peak of 18s rRNA. Further, although this fraction is designated as D-RNA it is not yet clear how "DNA-like" this RNA fraction is.3 The G+C/A+U ratios reported by Georgiev e t al. [cf. ( 5 6 ) ] for rat liver and Ehrlich ascites tumor cells fall in the range of 0.754.85. However, markedly higher values are reported by others who attempted the isolation of this DRNA fraction. Thus, by the use of a temperature gradient extraction, Morrison and McCluer (122) obtained a small amount of a D-RNA with a G+C/A+U ratio of 1.07. In another approach, by the gradual extraction of nRNA with EDTA-sodium acetate, Kimura et al. (123) obtained five nRNA fractions, but again the D-RNA fractions had ratios of 1.00 and 1.15. I n the case of canine pancreas Yang and Dickman

+

+

3 T h e G C/A T ratios of DNA from most animal tissues and cells vary between the relatively narrow limits of 0.65 and 0.80 (121).

216

A. A. HADJIOLOV

(124) isolated two nRNA fractions extracted with phenol a t 50-65” and at 65-80’, respectively, but showing ratios of 1.47 and 1.38. Our own studies (65) with liver invariably showed ratios for this D-RNA fraction in the range of 1.05-1.20. Determination of the base composition of the 18s peak of D-RNA yielded similar results (65, 114). Finally, values of 1.02-1.30 were reported for different extranucleolar fractions of

Mobility. centimeters from start

FIG.5. Agar gel eleclroplioresis pattern of a rat liver “DSA-like” nRNA fraction. The RNA is extracted in the temperature interval of 6 M 5 in the presence of phenol, 0.01 M Mg“, and polyvinyl sulfate [see (5511. The G C/A U ratio of this fraction is 1.12. Note the good homogeneity of the 18s component. [From

+

+

Venkov and Hadjiolov (G5).I

rat liver (113). Thus, it would seem that fractions of nRNA with a directly determined G+C/A+U ratio lower than 1.00 are not easily obtained. It .may be argued that D-RNA should not be exactly “DNAlike,” but it is also clear that in several cases the base composition of nuclear D-RNA fractions is closer to that of 18s rRNA than of DNA. Stimulation of cell-free polypeptide synthesis (called “template activity” in some studies) should represent a most functional assay for mRNA. I n general, the stimulatory activity of nRNA is ten- to twentyfold higher than that of cytoplasmic RNA (125-12828).This activity is associated with 18s (126) or 8-16s (114, 127) nRNA components. The latt.er figures are more likely to be due to partial degradation of RNA during isolation. Nuclear D-RNA fractions have a higher stimulatory activity in the E . coli cell-free system than the respective RRNA fraction (127, 128). In fact, stimulation by D-RNA was only

RNA AND IXFORMATION TRANSFER I N A K I M A L CELLS

217

thrcc times higher than by R-RNA and the pattern of the stimulated incorporation of different amino acids was similar (12%). These results indicate an unspecific stiniulation of polypeptide synthesis rather than thc formation of a defined product (see also Section V, D ) . Evidence from recent experiments indicates that stable D-RNA is associated with proteins to constitute a homogeneous population of R N P particles of about 40 S [see ( 5 6 )] .4 Aside from the “DNA-like” composition of the constituent RNA, these particles appear to be identical with the smaller subunit of ribosomes. The existence and the identity of ribosomes and ribosomal subunits in nuclei is a most debated subject, the bulk of the evidence backing the vicw that they are true nuclear components [see (32, 67‘)]. Recently, McCarty e t al. (129) not only have confirmed their presence in liver nuclei, but have adduced evidence that nuclcar and cytoplasmic ribosomes and ribosomal subunits are itlcntical by all criteria tested, including the base composition of the constituent RNA. Thus, the distinction between 40 S R N P particles containing 18 S D-RNA and those containing 18 S R-RNA is not olivious. I n brief, these results show that when degradation is adequately minimized, the nRNA fractions assunicd to be stable mRNA represent n rather homogeneous species of 18s RNA molecules. It is perhaps helpful to add that even in the anucleolate mutant of Xenopus laevis the stable D-RNA appears as a well-shaped 18 S peak (102).

D. Tracing of Messengers among Nuclear RNA’s The correct interpretation of experimental results on nuclear mRNA should await the accumulation of further evidence. This is easily understood when one considers that, within the last year, one paper appeared in which i t is stated that 18 S RNA is not present a t all in nuclei (60), while in another, 185 RNA is considered as not only a major nuclear species but the true messenger ( 5 5 ) . Two nRNA species are the likely candidates for mRNA: the rapidly labeled high molecular weight RNA and the stable 18s D-RNA. The various possibilities are briefly considered and some alternative explanations put forward. The existence of a rapidly labeled high molecular weight RNA ’Throughout this article, the d u e of 40 S is assigned to the smaller ribosomal subunit (existing free or as a constituent of the ribosome) while the value of 60 S is used for the larger ribosomal subunit. The same approximation is applied to drsignate the 28 S and 18 S nuclear and cytoplasmic RNA’s. These designations arc operational, and minor variations in experimentally determined S values are ronsidcred as being due to configurational changes or to methodical modifications, the elucidation of which requires further studies [see reviews in (22, .%’, S2)l.

218

A. A. HADJIOLOV

fraction has been estahlished beyond reasonable doubt, but its fate and its relation to rRNA and mRNA are controversial. Two basic interpretations have beeen considered. 1. The high molecular weight RNA niolecules are marked by an exceedingly high turnover rate with a half-life of about 2-20 minutes (24, 88, 100). They are made and degraded inside the nucleus. Therefore, the function of this D-RNA remains obscure. This view was discussed extensively by Harris (64, ISO), and recent more direct evidence corroborates and specifies further this interpretation (24, 88). However, although rapid labeling of D-RNA is consistently observed, unequivocal proof of rapid degradation has not been presented. On the contrary, experiments with actinomycin D indicate that either there is not a preferential degradation of D-RNA (88) or i t is more stable than rRNA precursor (89). 2. The high molecular weight RNA is a precursor of cytoplasmic mRNA and because of its large size is suspected to represent a polycistronic mRNA (103, 104). However, tracer kinetic data rule out a direct precursor-product relationship (24, 88). I n addition, the operation of special mechanisms selecting only the mRNA segments needed for cytoplasmic translation must be evisaged (104). Why the cell would transcribe more genetic information than is needed remains a mystery. Since neither view alone embraces the facts, several authors (24, 84, 88) leave open the possibility for the parallel existence of these two pathways of high molecular weight D-RNA turnover. A third explanation arises if sequential growth of RNA chains is considered (see Section 111). It was shown that RNA synthesis in animal cells may be a rather slow process, the completion time being estimated in minutes or perhaps hours. Recently, Greenberg and Penman (131), following the methylation of the 45 S rRNA precursor in growing HeLa cells, derived a lower limit for its completion time of 2.3 minutes. On the other hand, a kinetic study of D-RNA labeling in growing Cave cells showed t h a t this RNA is degraded within 1 or 2 minutes (100). Thus, we seem t o confront the paradox that high molecular weight DRNA is degraded faster than i t is synthesized! The discrepancy must be larger in nongrowing cells where the overall rate of RNA synthesis is ten- to a hundredfold slower, not to mention that the high turnover rate of these giant RNA niolecules inust be the most uneconomical process in cellular energetics. A plausible solution to this paradox is to admit that high molecular weight nRNA is a precursor of other RNA molecules. Since it has been shown that i t cannot be the immediate precursor of any type of cytoplasmic RNA, conversion into stable nRNA’s is suggested. Transformation of high molecular weight D-RNA


219

into stable 18s D-RNA has been put forward by several workers (55, 92, 102, 11.9). Still, formation of stable nuclear D-RNA is a rather slow process, which is not compatible with the high rate of high molecular weight D-RNA labeling. Since 4 5 s RNA is the first product to accumulate in nuclei in sizable amount, conversion of high molecular weight D-RNA into 45 S RNA fits the most exacting kinetic requirements. This possibility is not considered merely because the base composition (determined by P32distribution) is different for these two RNA’s. The model of sequential RNA synthesis suggests that in rapid-labeling experiments only the composition of the labeled segment of the polynucleotide chain is measured. Thus, “DNA-like” composition of high molecular weight RNA might simply indicate that the last (i.e., the first-labeled) segment completing the RNA chain is “DNA-like.” This does not mean that the whole molecule is “DNA-like” ; it inay be “rRNA-like.” As a consequence, the following scheme may be outlined: high molecular weight RN.4 -+ 45 S RNA -+ 35 S RNA --t 28 S rRNA

I--+

18 S RNA

The consequences of the modcl are considered to reconcile some controversial findings delineated in the preceding sections. a. mRNA is transcribed on karyoplasm DNA as a polynucleotide segment completing preexisting RNA chains. The size heterogeneity of the initially labeled high molecular weight RNA reflects the liberation from the DNA template of unfinished RNA chains. The high molecular weight RNA is further transformed into more homogeneous 45 S RNA. Only moderate secondary structure transitions may be involved ( 2 4 ) . b. The precursor RNA molecules have a hybrid base composition. The apparent composition detected by P32distribution analyses would reflect the completion time, i t . , the overall rate of RNA synthesis in a given cell type. An increased labeling time or RNA synthesis rate would nccclerate the transition from “DNA-like” toward “rRNA-like” precursor molecules (see Section IV, B ) . Extremes are reached in the anucleolate S e n o p u s laevis (101, 102) and i n actinomycin D administration (96-100, 116) when only the “DNA-likc” segment is labeled. c. Further transformations follow the pathway established for 45 S RNA [see Perry ( 3 2 ) ] .It is proposed that the mRNA segment is transformed into the respective 18s nRNA. Therefore some features of mRNA are inherent to this molecular species of RNA. This interpretation of the experimental results avoids the implication of giant-size nRNA molecules with extremely high turnovers and unknown biological roles. It explains also the formation in nuclei of stable 18s RNA molecules with mRNA features. It is obvious that, in any

220

A. A. HADJIOLOV

case, 18s nRNA is a most controversial molecular species. Is it a messenger RNA or a ribosomal subunit RNA? Is there one molecular species with hybrid structural and functional features or are there two independent molecular types having by accident the same size? Since the precursor-product relationships observed in nuclei do not operate in the cytoplasm, it is likely that more clear-cut conclusions could be derived from studies on cytoplasmic rRNA and mRNA. These aspects are considered next with the i,mplicit understanding that both rRNA and mRNA are transferred from the nucleus into the cytoplasm without major modifications in the process of transfer [see (18, 3.2, 5 5 ) ] .

V. Cytqhsm-the

Site of mRNA Expression

It is proposed to review here some aspects of studies aimed a t the identification of cytoplasmic mRNA, with emphasis on the relation between mRNA and rRNA [see also (5) and Chantrenne in this volume].

A. Molecular Characteristics of Cytoplasmic RNA 1. RIBOSOMAL RNA

By definition, rRNA is a component of ribosomes. Actually, every ribosome is composed of two unequal subunits and we invariably find two rRNA’s. In animal cells, the large (60S) subunit contains 28 S RNA and the small (40s) subunit, 18s RNA. Most likely, the two rRNA’s constitute single polynucleotide chains with molecular weights of 1.2 x lo6 and 0.6 X loG,respectively [see (.21)]. When RNA is extracted from either ribosomes or total cytoplasm under conditions that minimize degradation, the 28 S and 18 S components are invariably obtained as the sole RNA species in a 1:l molar ratio, i.e., a 2 : l mass ratio. The two rRNA’s constitute about 90% of cytoplasmic RNA, most of the remainder being tRNA. Given the sensitivity of our techniques, any other type of RNA cttnnot account for more than 2-3% of the total cytoplasmic RNA. 2. EXTRARIBOSOMAL RNA Several attempts have been made to isolate and identify cytoplasmic RNA fractions other than rRNA. Some of these fractions are suspected of being mRNA and are therefore worth considering. Mitochondria1 RNA (if i t exists as an entity) is not likely t o be directly involved in nucleocytoplasmic transfcr of genetic information. With the advent of techniques for the isolation of ergastoplasmic

RNA AND INFORMATION TRANSFER IN APiIMilL CELLS

22 1

lipoprotein membranes, a variable amount of RNA was found associated with this fraction. Several fcaturcs, such as base composition and rate of Inbeling, seeined to indicate that thib RNA is distinct from rRNA. This view was strengthened by reports indicating the existence in membrane RNA of fractions, revealed in density gradients, other than the two rRNA’s (132). However, recent observations indicate t h a t the bulk of membrane RNA is 28 S and 18 S in size (133-135). Moreover, BergeronBouvet aucl Moulk (135) showccl t h a t the membrane RNA is enriched in thc 28 S species with a 28 S/18 S mtss ratio of ahout 2.5. On the other hand, in total cytoplasmic extracts or in postribosomal fractions of HeLa cells (136, 137) and liver (138), free 60 s and 40 S R N P particles were found, with a marked prevalence of the latter type. As discussed further, these particles were suspected to contain RNA species distinct from the two rRNA’s. Still, the bulk of the RNA extracted from these 40 S particles was found t o be 18 S in size (137, 138). The nature of this L ~ e ~ t r a r i t ) ~ ~R~NmAa lis” a disputed point, but recent observations of Sabatini e t al. (139) throw some light on this issue. They demonstrated t h a t ribosomes are attached to lipoprotein membranes hy their 60 S subunit. Under some contictions, the 40 S subunit is rcleasetl, leaving the 60 S subunit behind. Thus, it is plausible that disruption and further fmctionation of cells ni‘iy result in ribosome tlissociation, the two subunits heing recovered in diffcrcnt fractions. Thus, the membrane fraction, enriched iii attachcd 60 S subunits, yields a “membrane” RNA, identical with 28 S RNA, while the 40 S subunits and tlicir 18 S RNL4 are likely t o be found in postribosomal fractions. Of courw, the existence of free 60 S and 40 S R N P particles with different localization in the cytoplasm is not excluded. In anv case, it should be hti.c>sxl that all directly tlotc,c.ted “t.xti.aribosoiiin!” fractions contain only 28 S or 18 S RNA, or more often, varying proportions of hoth types. Atlrlitional eridcncc is needed to tlecidc whether thc 28 S and 18 S “estrnriI)o~on~nl” RNA’h arc itlentir:il witli rRNA.

B. labeling and Turnover of Cytoplasmic RNA 1. RIBOSOMAL RNA

Most of the evidence for the prcsence of mRNA in the cytoplasm is based on the property of rapid labeling with the assumption that ribosomes and rRNA are stable, but important limitations to this presumption exist with animal cells. On one hand, i t is likely that stable messengers direct the synthesis of most proteins (see Section I ) . On the other, although some evidence for the stability of rRNA has heen obtained with growing cells (I@), this is apparently not the case in non-

A. A. HADJIOLOV

222

"r 1.0

L

I

5

t

10

I

I

I

15

20

25

Tube number

(A)

I

I

7 ’

Hours

(B) FIG. 6. Labeling and turnover of RNA in the two ribosomal subunits of rat liver. [From Hadjiolov ( I % ) . ] (A) Sucrose density gradient sedimentation pattern of ribosomal subunits obtained from animals 90 minutes after introduction of orotic acid-6-CI4. 0-0, O.D.*,; 0- -0, radioactivity. (B) Decay of RNA labeling in the two ribosomal subunits obatined as in (A). Labpling for 60 minutes in Vzvo with orotic acid-6-C" followed by orotic acid-CB. Note the parallel decrease in the specific activity of the two subunits.

R N A A N D INFORMATION TRANSFER I N A N I M A L CELLS

223

growing cells. For example, it was found that the half-life of ribosomes and rRNA in liver is about 2-5 days (128, 141), which is a limited part of the life-span of liver cells. The following aspects of rRNA labeling and turnover may be of relevance here [see also (SZ)]. a. I n brief labeling experiments, the label appears a t first in the 18s rRNA and later in 28 S rRNA (136-138, 14%’). The same correlation is observed with the two ribosomal subunits obtained from either cytoplasmic extracts (137, 14%’) or after dissociation of isolated ribosomes (128). I n both cases, the 40 S subunit is initially more highly labeled than the 60s subunit (Fig. GA). Studies with HeLa cells show also an initial higher labeling of the proteins in the 40 S subunit (1.43). b. Uneven distribution of labeled 28 S and 18 S RNA among different cytoplasmic compartments is observed with brief labeling times. One popular interpretation depicts the flow: free (immature) 40 S and 60 S subunits + polysomes -+I ~ I O I I O I I ~ P I - ~ Crihosomes (32, 136, 137). Other observations indicate that “meinhrane” RNA is more highly labeled than the RNA of single ribosomes (133-135). One explanation of the initial higher labeling of the 40 S subunit RNA would be that i t possesses a higher turnover rate. However, when the decay of orotic acid-C14 pulse-laheled ribosomes of liver was studied (128), it was found that the RNA’s of the two subunits decay a t essentially the same rate (Fig. 6B). Consequently, the release of the two subunits and their further turnover should be synchronized. What then may be the meaning of the higher pulse-labeling of the smaller subunit and its RNA? This problem, connected with the search for rapidly labeled cytoplasmic RNA, is considcrcd next. 2. RAPIDLYLABELED RNA FRACTIONS

I n his first studies with liver, Hiatt ( 5 6 ) could not trace any rapidly labeled RNA fraction other than 28 S and 18 S RNA. However, further studies with liver (144-146), HeLa cells (147, 148), mammary adenocarcinoma (149), and immature erythroid cells (88, 104), revealed the presence of a heterogeneous rapidly labeled RNA fraction situated in the 4 1 6 S zone of density gradients. One particular aspect emerged from these studies: the heterogeneous RNA fraction was observed only when RNA was extracted from previously isolated polysomes. Because polysomes are involved in protein synthesis [cf. ( 5 )1, the rapidly labeled fraction was considered to be mRNA. Actually, this fraction may well be the product of partial RNA degradation taking place during isolation of polysomes (see Section 11, A ) . That this is the case is shown by studies with liver (128, 138, 150, 1511, Cave cells ( l o o ) , and a plasma cell tumor (151),which revealed that when RNA is isolated under conditions

224

A. A. HADJIOLOV

avoiding degradation, the radioactivity and UV profiles coincide almost exactly.' Thus, the rapidly labelcd fraction is either associated with the 18 S rRNA or it has the same sedimentation coefficient. Binding of Mg2+ with EDTA and treatments known to destroy hydrogen bonding, such as heating with urea (128) or formaldehyde (I%), failed to release

Urea concentration

FIG.7. Stability of label in 28s and 18s rat liver rRNA's on urea treatment. Labeling of RNA for 90 minutes with orotic acid-6-C" in wiwo. The 28 S and 18 S RNA's are isolated from sucrose density gradients. Treatment with 0.4 M urea a t 60" for 10 minutes and subsequent centrifugation in urea density gradients. L e f t : 2 8 5 RNA; Right: 18s RNA. -0, O.D.so; O---O, radioactivity. The arrows indicate the average specific radioactivity in counts/min/mg RNA (128).

any labeled material from 18 S RNA (Fig. 7 ) . These results suggest that the rapidly labeled RNA is either covalently bound to the 18s rRNA or is itself 18 S. The first possibility is made more likely by the observation that, in liver, a 6-14s highly labeled material is released from the 18 S RNA under the action of cytoplasmic RNase's (1%). In summary, the results discussed above indicate that rapidly labeled RNA of animal cells constitutes a homogeneous population of molecules that sediment with a coefficient of 18s. The rapidly labeled 18s RNA is preferentially degraded during isolation of polysomes to yield a heterogeneous 4-16 S material. The mRNA for serum albumin (mol. wt. 62,000) is expected to be 1618 S, while the mRNA for the secretory protein of the RPC-20 plasma cell tumor (mol. wt. 24,000) should be about 9-11 S (161).

RNA AND INFORMATION TRAXSFER I N A N I M A L CELLS

225

C. Mononucleotide Composition. Cytoplasmic D-RNA 1. DIRECTDETERMINATION

Since no major RNA species other than 2 8 s and 1 8 s is found in animal cell cytoplasm (Section V,A) only the base composition of these two RNA’s can be determined directly. Several workers have isolated these two RNA components and determined their mononucleotide composition (Table 11).I n every case the 18 S RNA has a much lower G+C/A+U ratio than 2 8 s RNA. As a rule this ratio falls in the range of 1.1Cb1.35 for 18s RNA and of 1.70-1.95 for 2 8 s RNA. Since the 2 8 s and 1 8 s RNA’s isolated from either total cytoplasm or purified ribosomes are identical in base composition (156), the existence of a major “extraribosomal” RNA fraction other than 28 S and 18 S rRNA is excluded. Likewise, the reported G+C/A+U ratios for thz “membrane” RNA vary from 1.65 to 1.95 [cf. ( 1 6 5 ) ] , thus reinforcing the proposed interpretation that the membrane fraction is enriched in 60 S subunits, i.e., 28 S RNA. The observed difference in base composition suggests that 28 S and 18 S RNA’s represent different species. Further, one should note the fact that the composition of 1 8 5 rRNA is definitely shifted toward that of DNA, i.e., 18 S rRNA is relatively “DNA-like.” If a G+C/A+U ratio of 1.80 is taken as typical for rRNA and a ratio of 0.75 for DNA, a rough estimate indicates that 1 8 s RNA contains 40% “DNA-like” and 60% “rRNA-like” components. Consequently, one may ask whether cytoplasmic 18s RNA is a mixture of D-RNA and R-RNA molecules of the same size or is a hybrid molecule with an R-RNA segment covalently bound t o a D-RNA segment? A definite answer must await more knowledge of the primary structure of rRNA, of 1 8 s RNA in particular. A rat liver fraction insoluble in 10% NaCl, and thus probably rRNA, yielded some preferred sequences among the dinucleotide and trinucleotide products of pancreatic RNase (166). On the other hand, complete digestion of separate 28 S and 18 S RNA’s from different sources with pancreatic or T1 RNase demonstrated definite differences in the nucleotide sequences of these two rRNA types (167, 168). These initial studies reveal that random distribution of nearest-neighbor nucleotides niay not be the rule in rRNA structure. We havc attempted to approach this problem by the stepwise degradation of liver rRNA with snake venom phosphodiesterase, an exonuclease known to release 5’ mononucleotides from the 3“OH end of polynucleotide chains. The RNA preparations studied were composed of homogeneous 28 S and 18 S RNA and virtually free from contaminating endonucleases (see Section 11, A). Moreover, endonuclease contami-

N

TABLE I1 MONONUCLEOTIDE COMPOSITION OF CYTOPLASMIC 28s

h3 0-4

AND

1 8 s RNA

OF

ANIMALCELLS"

~~~~

~

28 s

18 s

+

Molar ratio (%) Source

Rat liver Rib!

Rib. Rib. Reticulocyte Rib.

Hat kidney Rat spleen Rat brain Krebs I1 ascites cells HeLa cells BHK cells Mouse L fibroblasts Hen fibroblasts HLM cells

~

A

U

G

C

18.3 17.8 15.7 15.5 18.6 18.0 15.4 15.5 15.8 15.5 16.4 15.9 15.3 15.0 16.0 20.5 16.9 19.3 18.4 19.5 19.2 17.3

19.0 17.0 19.2 18.5 18.9 16.4 18.6 17.9 18.0 17.8 16.6 19.0 19.1 19.3 17.0 19.3 18.3 17.8 17.7 18.2 16.2 17.6

32.9 33.0 34.6 36.6 32.9 34.8 36.8 36.3 35.8 36.7 35.3 36.0 36.8 37.8 36.0 28.3 34.9 34.3 34.3 34.5 35.2 35.1

29.8 32.2 30.5 29.5 29.7 30.8 29.3 30.3 30.4 30.0 31.6 29.1 28.9 27.8 30.5 31.9 29.9 28.5 29.6 27.8 29.5

30.0

+ C/

G c/ A+U ratio

A

U

G

C

ratio

1.66 1.82 1.87 1.95 1.67 1.91 1.95 2.00 1.96 2.00 2.03 1.87 1.91 1.92 1.98 1.52 1.84 1.69 1.77 1.65 1.83 1.87

22.4 19.8 19.1 19.7 22.0 22.5 19.4 20.1 20.1 20.9 20.5 20.5 20.0 19.7 21.2 23.2 22.6 24.3 22.1 23.5 22.9 23.0

19.6 18.0 24.2 22.5 21.5 19.5 23.1 22.5 22.1 22.4 20.0 23.5 23.9 22.7 21.5 23.2 22.9 22.0 21.5 21.9 21.3 23.3

30.2 32.4 30.7 32.3 28.8 31.8 32.0 31.2 31.2 30.7 30.7 30.4 31.1 31.5 29.5 25.2 29.3 28.7 30.5 29.9 30.0 28.7

27.8 29.8 26.0 25.6 27.6 26.2 25.6 26.1 26.6 25.9 28.8 25.6 25.1 26.0 28.0 28.0 25.2 25.0 25.9 24.7 25.8 25.0

1.39 1.64 1.30 1.37 1.30 1.38 1.36 1.35 1.37 1.31 1.48 1.27 1.28 1.36 1.35

Molar ratio (yo)

G

A+U

1.14 1.20 1.16 1.29 1.20 1.26 1.16

Reference

Chick embryo 3 days 7 days Xenopus laevis Eggs Embryo Arbacia punctulata eggs Pot,ato tuber Pea seedlings

z

t

18 19

22

17 19 19.9 25.1 23.6

28

1.50 1.63

24 24

21 22

30 29

25 25

1.22 1.18

(161) (161)

30 30 26.0 22.0 22.6

2.03 1.86 1.56 1.16 1.21

22 22 23.1 25.4 23.7

18 18 23.2 25.2 25.1

31 32 29.9 27.2 31.1

29 28 23.8 22.2 20.1

1.50 1.50 1.16 0.98 1.03

(101) (162) (164) (163) (163)

24

18

36 34

16 16 19.3 21.2 21.6

37 35 34.9 31.7 32.1

Only values obtained by direct estimation of the mononucleotide composition are included. Data on some plant tissues are given for comparison. Rib.-The two RNA species have been obtained from previously isolated ribosomes. (1

t

Z

u

228

A. A. HADJIOLOV

-1

10

I

I

20

30

I

1

I

I

I

40 50 60 70 80 Hydrolysis, %

I

90

FIG. 8. Changes in the molar ratios of 5' mononucleotides released by stepwise hydrolysis of total rat liver rRNA with snake venom phosphodiesterase. Hydrolysis at constant pH of 8.5 in a pH-stat t,itration assembly. At a fixed percentage of hydrolysis, the undegraded RNA chains are precipitated with ethanol and the molar ratios of the released 5' mononucleotides remaining in solution is det,ermined. The undegradcd RNA is furt,her hydrolyzed in the same manner. Thus, the experimental points in the diagram represent the molar ratios of 5' mononucleotides in the rRNA segments degraded a t 0-10, 10-20, 20-40, and 40100% hydrolysis (see dotted line for pG) (176).

nants in the enzyme system could account for no more than 1 or 2 internal breaks per molecule RNA.6 The basic results obtained in these studies (169-172) are illustrat,ed in Fig. 8. They show that the molar ratio of the liberated mononucleotides varies with progress of rRNA ratio of the released monoenzyme degradation. The G+C/A+U nucleotides rises from 1.12 for the initial 10% of the RNA hydrolyzed to 2.75 for the last 60% (see Fig. 8 ) . Two explanations may be considered: ( a ) rRNA is R mixture of D-RNA and R-R.NA and the enzyme The possible presence of endonucle:tse contaminant,s in the enzyme system was controlled by the following tests: (a) the polynucleotide (Ap).Cp (average n = 15) was not hydrolyzed, while ( A p ) X was rapidly degraded to pA and pC; (b) the reaction proceeds to completion and yields PA, pU, pG, and pC as the only major products; (c) a t different stages of phosphodiesterase hydrolysis of rRNA, the reaction mas stopped by the addition of KOH and the liberated nucleosides (18 hours a t 37" and 0.5N KOH) were determined. The amounts of nucleosides liberated correspond to 1 or 2 internal breaks per molecule of rRNA; (d) when rapidly labeled rRNA is used as substrate, the specific radioactivity of the liberated nucleotides changes with the progress of phosphodiesterase action. Randomization of the label is expected under the act,ion of endonuclease contaminants [for details see ( l i l ) ] .

229

RNA AND INFORMATION TRANSFER I N A N I M A L CELLS

degrades preferentially D-RNA ; (b) rRNA is a hybrid molecule, “DNAlike” near the 3’-end and “rRNA-like” near the 5’-end of the polynucleotide chain. We consider the first possibility less likely since model experiments with double-stranded poly Aepoly U did not show a detectable influence of secondary structure on the action of this enzyme 1169, 171), and venom phosphoidesterase is devoid of base specificity [cf. ( l 7 3 ) ] .Further, the G C/A U ratio of 2.75 for the last 60% of rRNA is markedly higher than the values obtained with purified 2 8 s rRNA. Consequently, there should be other segments in rRNA compensatorily enriched in A and U, i.e., being more “DNA-like.” That these results reflect something more than a mere artifact is indicated by independent studies of Delihas and Bertman (174). As shown recently, partial digestion of rRNA with pancreatic (175) or T1 (176, 177) RNases yields several fragments of discrete size, but it has been demonstrated (174) that some of the high molecular weight fragments in partial T1 RNase digests, estimated t o represent 10-15% of the whole rRNA, have a G+C/A+U ratio of 3.1-3.6. Again, these results indicate that there are other segments of the rRNA chains more “DNA-like” than the average 2 8 s or 18s RNA’s. Thus, the scanty evidence available is consistent with thc possibility that 18 S RNA is a hybrid molecule of covalently bound D-RNA and R-RNA segments.

+

2. P

J

2

-

+

DSTUDIES ~ ~ ~

~

~

~

~

~

~

~

~

A “DNA-like” composition of rapidly labeled RNA is detected only by P32-distrib~tionanalyses. Thus, the heterogeneous &16 S RNA material obtained f i on1 previously isolated polysomes of HeLa cells (85, 147, 148), liver (145), and immature crythroid cells (88, 178) is enriched in D-RNA, although the G C/A U ratios obtained are in general significantly higher than the respective values for DNA. When undegradcd rat liver cytoplasmic RNA is isolated, the rapidly labeled D-RNA is found associated with both 2 8 s and 1 8 s RNA 1138, 154). On increasing the time of labeling, a gradual shift in the base composition of labeled RNA from D-RNA toward R-RNA is observed in liver (138, 154) and growing Cave cells (100). I n the case of HeLa cells, the heterogeneous 4-16 S rapidly labcled RNA fraction displays higher levels of hybridization with homologous DNA than the two rRNA peaks ( 1 7 9 ) . However, only a two- to threefold decrease in the amount of hybridized material from 1 8 s RNA is observed a t longer periods of labeling (179). All these studies are in good agreement with results of labeling experiments with other precursors (Section V, B, 2 ) . They show in addition that the rapidly labeled RNA fractions in the cytoplasm display a

+

+

230

A. A. HADJIOLOV

“DNA-like” base composition. When obtained from polysomes, this D-RNA is heterogeneous in size, while it coincides with 18s RNA (and 2 8 s RNA in some studies) when extracted under conditions chosen to minimize degradation. These results are interpreted as demonstrating the existence of independent D-RNA molecules identical in size with rRNA’s (100, 138, 154). The rapid labeling and the time-dependent shift in base composition are thought to reflect a high turnover rate of cytoplasmic D-RNA. However, it should be noted that no evidence whatsoever about the fast degradation of the suspected D-RNA molecules has been presented. An alternative interpretation of the findings on cytoplasmic D-RNA is given below.

D. Stimulation of Cell-Free Polypeptide Synthesis Several authors have determined the stimulatory activity of cytoplasmic RNA in E. coli or mammalian cell-free systems. In general, the most active material is found associated with the 18s RNA peak (114, 126, 128) obtained from either total cytoplasm, ribosomes or 4 0 s R N P particles. While the rapidly labeled components of 18s RNA are degraded to 6-14 S material, the stimulatory activity remains in 18 S RNA (138). The validity of this assay for mRNA is limited, since the secondary structure of a polynucleotide seems to determine its stimulatory activity. Thus, good stimulation of the E . coli system is obtained with denatured DNA (180) and partially degraded rRNA or tRNA (181), while double-stranded viral RNA is inactive (18%’). Many attempts have been made to isolate mRNA for a specific protein. Since most of this work has been concentrated on mRNA for hemoglobin i t need not be considered here (see article by Chantrenne et al. in this volume). Only two points will be mentioned. First, dissociation of hemoglobin mRNA from reticulocyte rRNA appears to be extremely difficult, if not impossible. The failure of several vigorous attempts led Gould e t at!. (183) to the conclusion that hemoglobin mRNA is covalently bound to rRNA. Secondly, various unrelated RNA fractions are likely to cause an unspecific stimulation of the synthesis of a specific protein (184). Therefore experiments with heterologous ribosomal systems are required for the adequate identification of a mRNA (see Section 11).

E. Cytoplasmic Carriers of mRNA 1. POLYSOMES

It was postulated ( 2 ) that mRNA bound to ribosomes would form the active complex for the translation of genetic information. With the

RNA A N D IKFORMATION TRANSFER I N ANIMAL CELLS

231

recognition of polysomes as such a complex, the presence of mRNA in these structures was assumed, and extensive supporting evidence has been collected [see (5, 35) for references]. Ribosomal aggregates (polysomes) connected by RNA threads are more active than single ribosomes in polypeptide synthesis both in vitro and in vivo. There is compelling evidence that the RNA linking the ribosomes is labeled more rapidly than rRNA, is heterogeneous in size, is “DNA-like” when analyzed by P32distribution, and is marked by a high stimulatory activity in cell-free systems. This indicates that the RNA threads in polysomes represent mRNA. However, all these facts do not show that the mRNA is external t o the ribosomes and provide no proof of the movement of ribosomes along an independent mRNA chain. On the contrary, several observations indicate that this model may not reflect the actual situation in vivo. It has been shown, for example, that polysomes are not an obligatory structure for protein synthesis. Single ribosomes, called monosomes [cf. ( 5 )], isolated from liver (185) or muscle (186‘) are active in cell-free polypeptide synthesis. The experiments of Lamfrom and Knopf (187, 188) showed that reticulocyte monosomes are self-sufficient in the synthesis of the separate chains of hemoglobin. Further, the experiments discussed in the preceding sections show that the heterogeneous messenger-like material isolated from polysomes is probably a degradation product. When degradation is avoided, the RNA molecules with several features of mRNA coincide exactly with 185 rRNA and cannot be dissociated by physical treatments to yield shorter and more heterogeneous RNA chains. I n brief, although we know for certain that mRNA is present in animal cell polysomes, the characteristics of this mRNA in the native state are controversial. When undegraded, the supposed mRNA coincides in size with rRNA, with 18s rRNA in particular. This was observed even in cases in which the mRNA for hemoglobin (183) and plas,ma cell tumor protein (151) should be much smaller in size than the rRNA. 2. RIBONUCLEOPROTEIN PARTICLES

The problem of cytoplasmic R N P particles as mRNA carriers is even more complicated. As mentioned before, several authors observed that a 40s R N P particle is labeled more rapidly than the related 60s particle. Kinetic and other evidence showed that the 40s particle is a precursor of (or is identical with) the smaller ribosomal subunit ( 3 2 , 1S‘i‘). However, the “excess” label in the 40s subunit found after short pulses and the identification of the constituent 18s RNA with some

232

A. A. HADJIOLOV

properties peculiar t o mRNA (“DNA-like” base composition, higher level of hybridization with DNA, stimulation of cell-free polypeptide synthesis) inclined others to think of the 40s particle as the carrier of mRNA in animal cells (136, 138, 179). The term “informosonies” was coined for similar R N P particles in order to distinguish them from the smaller ribosomal subunit (189).The close S values for these two particles, presumably distinct in function, remained unexplained. Then, unexpectedly it was found that the rapidly labeled R N P particles (assumed to be the 40s ribosomal subunit bearing mRNA) band a t CsCl densities that are slightly lower than those typical of the 40 S ribosomal subunit itself (190). These results rule out the possibility of an external mRNA being attached t o the 40s subunit, although the RNA of the 40 S R N P particle has been identified as mRNA by all criteria available a t present. Thus, we are left with the only possibility that the 18s RNA isolated from the 40s R N P particle is itself mRNA. Accordingly, we are obliged t o envisage the existence in the cytoplasm of two 40s R N P particles, the one containing 18s rRNA and the other, 18s mRNA. And again we end with the controversial 1 8 s RNA species.

3. 18 S RNA-MESSENGER OR RIBOSOMAL CONSTITUENT? Since in both nuclci and cytoplasm, the search for the RNA species involved in genetic information transfer has converged toward 18 S RNA, a general evaluation of its properties is appropriate here. a. In both nuclei and cytoplasm a species of RNA molecules is found that is homogeneous in size and constitutes a well-defined 18s peak. The molar ratio of 28 S/lS S RNA in total cellular RNA is close to unity. b. The 18s RNA is found as a constituent of 40s R N P particles. These particles are identical in physical properties to the smaller subunit of ribosomes. The R N P particles containing 18s RNA exist in three states: free; bound to the 60 S subunit in ribosomes; and involved in polysomal structures. No evidence for the existence of free 18s RNA in either nuclei or cytoplasm has been presented. c. The mononucleotide composition of 18s RNA is shifted toward that of DNA as compared to 28s rRNA. Approximate estimations indicate that 18s RNA is 40% “DNA-like” and 60% “rRNA-like.” d. In most cases studied, nuclear and cytoplasmic 18 S RNA’s show a higher stimulation of cell-free polypeptide synthesis compared to 2 8 s rRNA. e. Cytoplasmic 18s RNA is more rapidly labeled than 2 8 s RNA. When labeled with P3?,the 18 S RNA is found initially “DNA-like” in base composition. IVith extension of labeling time, the composition,

233

RNA AND IR’FORMATION TRANSFER I N ANlMAL CELLS

determined by P3?distribution, tends to reach the values obtained by direct determination. f. During isolation of cytoplasmic structures, the rapidly labeled 18 S RNA is preferentially degraded by endonucleases to yield a messengerlike material of heterogeneous size. This degradation is most likely to occur with 18 S RNA involved in the structure of polysomes. Thus, according to points ( a ) and (b) , 18 S RNA is a true rRNA. On the other hand, points (d), (e), and ( f ) suggest that 18s RNA may be the mRNA we seek. At present, it can hardly be decided whether 18 S RNA is a mixture of mRNA and rRNA molecules or whether it represents a hybrid molecule with characteristics of both RNA types. The “DNA-like” composition of rapidly labeled 18 S RNA is usually taken as evidence for the independent existence of 18s mRNA characterized by a high turnover rate (130, 138, 154). Further indications that an independent 18s mRNA exists are seen in the preferential degradation of 18s RNA moleculrs with messenger features, while 185 rRNA remains apparently unchanged (138, 191). Finally, in a recent study, Gazsrian and Schuppe (192) followed the changes in base composition of the rRNA of Cave cells after a prolonged “chase” of P32with nonradioactive phosphate. They ohserved that the G C/A U ratio of 18s RNA (determined by P33distribution) continues to rise during the chase to reach a value of 1.95, higher than the 1.45 obtained by direct determination. It was deduced that the composition of 18 S rRNA is identical with that of 2 8 s rHNA, while the lower values obtained by direct determination reflect contamination of 18 S rRNA by about 40% stable 18s D-RNA (see Table 11). The authors assumed that 18s D-RNA has a higher turnover rate than 18s R-RNA, although this is not the only possibility (see bclow). Besides the uncertainty in the experimental data, the view that an 18 S mRNA exists independently faces two basic obstacles. On one side, the expected size heterogeneity of total mRNA can hardly be reconciled with a homogeneous (18 S) mRNA.7 On the other, there is no apparent necessity for an mRNA t o be of the same size as rRNA, nor t o form R N P subunits identical with the smaller subunit of ribosomes. An alternative explanation may be found in the proposal that 18s RNA in both nuclei and cytoplasm represents a hybrid molecule consti-

+

+

‘ I n the case of liver, it is possible that thc bulk of mRNA codes for serum albumin (the major protein product of liver) and is expected to be 1618s in size. However, the synthesis of total liver proteins seems t o he little affected by actinomycin D ( 1 4 ) ; this suggests a stahle mRNA for serum albumin. On thc other hand, short-term labeling rsprriments indicate a half-lifa for tot,al mRNA in rat liver cytoplasm of about 4 hours (144, 146).

234

A. A. HADJIOLOV

tuted of covalently bound mRNA and rRNA segments. The following basic aspects .may be outlined. a. A heterogeneity in size of mRNA is shown by variations in the length of the mRNA segments within the limits determined by the “standard” size of 18 S RNA molecules (Fig. 9) .8 When functioning in F A v e r o g e size of 18 S RNA = 0.67 x lo6 doltons

L

1

Average size of mRNA segments =0.2?x doltons

lo6

f

FIG.9. Model illustrating the size heterogeneity of mRNA segments found within the limits of a standard 18s RNA molecule. The straight lines represent “ribosome-like” segments, and the zigzag lines designate the “messenger” segments (193).

polysomes, the inRNA segment is extended from one ribosome to another and is therefore more susceptible to endonuclease attack. As a result, heterogeneous 4-16 S mRNA material is easily obtained from previously isolated polysomes. b. A hybrid character for 18s RNA is compatible with the interpretation of 40 S R N P particles as being simultaneously a ribosomal subunit and a mRNA carrier, thus supplying a solution to the controversy concerning the nature of these particles. The existence of mRNA as a constituent of 40s R N P particles is compatible with the “protected” and “masked” forms of mRNA envisaged in several studies (cf. 12, 189). c. Shifts in the base composition of 18s RNA from D-RNA toward R-RNA with extension of labeling time are easily understood if we consider the model of sequential synthesis of RNA chains (see Section 111). Location of the mRNA segment a t the 3‘-end of the chain, which is the last synthesized (i.e., the first labeled), would explain both the higher pulse-labeling of 18 S RNA (and the 40 S subunit) and a “DNA‘The maximum sire of mRNA is thus limited to 185 RNA, ie., 0.67 x 10‘ molecular weight, or about 1900 nucleotides. This corresponds to a single protein chain of molecular weight about 76,000. Studies on the subunit structure of protein molecules reveal that no single polypeptide chains with molecular weights greater than 66,000 have been detected [see Reithel (194)l.

RNA AND INFORMATION TRANSFER IN ANIMAL CELLS

235

like” composition in the initial periods of labeling (see Fig. 3, case B). It is also evident t h a t a t very long periods after dilution with unlabeled P,, the reverse phenomenon is expected: P32 should disappear first from the mRNA segment, and P32 remaining in the rRNA segment would give a base composition similar to that of 28 S rRNA [see ( 1 9 2 ) l . d. Sequential labeling of 18s RNA would help t o reconcile the expected stability of mRNA derived from most studies on the synthesis of total or individual animal cell proteins (see Section I ) and the much faster “turnover” rate of mRNA deduced from short-term labeling experiments [cf. (24, 138, 144, 146, 1 4 8 ) l . The supporting evidence for this concept has been outlined throughout this article, together with the opposed findings. It seems likely that more conclusive evidence will soon be forthcoming.

VI. Synopsis The controversial experimental findings on the role of RNA in genetic information transfer in animal cells have been discussed. I n the current state of our knowledge we can hardly decide whether genetic information is mediated by independent messenger RNA molecules or by messenger segments in ribosomal RNA.

A. The Orthodox Interpretation This interpretation is endorsed in almost all studies on mRNA of animal cells. It is strengthened by the evidence in support of the messenger concept obtained in coding experiments with synthetic polynucleotides and in studies with bacteria and with virus-infected cells [see ( 5 )1 . The implicit assumption is that genetic information transfer in animal cells is governed by the same basic mechanisms which operate in bacteria. Consequently mRNA is considered as synthesized and transferred a t a rate and by a pathway independent of rRNA synthesis and of ribosome formation and turnovcr. The translation process involves only transitory association of the mRNA with genetically unspecific ribosomes. The evidence on information transfer in animal cells is thus fitted into the frame of two independent pathways (see p. 236). As discussed, the different steps of this hypothetical mRNA pathway are not equally well documented. Uncertainties exist on the functional significance of the rapidly turning over high molecular weight mRNA in nuclei; the existence of separate 40 S R N P particles as mRNA carriers; the native state of mRNA in polysomes; etc. The most striking aspect emerging from these studies is the close similarity between these two presumably independent pathways. It is clear that the almost iden-

236

A. A. HADJIOLOV

rRNA Pathway [see (32) ]

mRNA Pathway Nuclei

> 40 S mRNA -High

i

18 S mRNA (in 40 S RNP ?) Cytoplasm

45 S rRNA Precursor

turnover inside nuclei

j.

18 S mRNA "Protected," (in 40 S RNP) "masked" mRNA

18 S rRNA (in 40 S RNP)

t


t

18 S mRNA In polysomes, the active part being 4-16 S


1


\ /

PolyTes

Monomeric ribosomes (40 S + 60 S RNP)

tical physicochemical characteristics of the involved RNA molecules could hardly be due to a mere coincidence.

B. One Plausible Unorthodox Interpretation The proposed alternative interpretation (193)is an attempt to seek simplicity in the chemical organization of the living cell. It is based on the experimental results that point t o the close relationship between mRNA and rRNA in animal cells. The main feature of the advanced hypothesis is the consideration of mRNA as an integral part of some RNA's currently considered as pure rRNA's. The following tentative scheme may be considered : a. Messenger RNA is transcribed on karyoplasm DNA as a polynucleotide segment completing preexisting rRNA chains to constitute a hybrid precursor molecule. b. The precursor RNA is further transformed into 2 8 s and 18s molecules, which, combined with protein, constitute the 60 S and 40 S ribosomal subunits. I n this process the mRNA segment is found as a constituent of 18 S RNA. c. The 18s RNA is a hybrid molecule formed by covalently bound rRNA and mRNA segments. The size heterogeneity of .rnRNA is realized within the limits fixed by the standard size of the 18s RNA molecules (see Fig. 9 ) . d. The hybrid 18 S RNA integrated with proteins into 40 S R N P particles is transferred synchronously with the 60 S R N P particle into the cytoplasm. Thus, the 40 S R N P particle represents a "protected" form of mRNA. The hybrid character of 18s RNA results in the 40s RNP being simultaneously a mRNA carrier and a ribosomal subunit.


237

e. In the translation process the mRNA segment of the 18s RNA is extended between the 40 S subunits, forming the connection with other ribosomes to give polysomes. Every mRNA segment included in 18 S RNA codes the synthesis of a single protein chain. The experimental evidence in support of this scheme involves mainly: the attempts t o find a native mRNA in animal cells ended with a homogeneous 18s RNA (Sections IV, D and V, E ) ; the hybrid mononucleotide composition of 18 S R N A (40% “DNA-like” and 60% “rKNA-like”) ant1 indications that 18 S R N A consists of covalently bound mRNA and rRNA segments (Section V, C ) ; the possibility of explaining the controversial findings of short-term labeling experiments hy considering the sequeiitial synthesis of R N A (initiated with the “ribosomal” segment and finished with the ‘Lm~sseiiger”segment) correlated with a relatively extended time period necessary for the conipletion of RNA molecules (Section 111); the dual character of 40 S R N P particles identified as either a inRNA carrier or a ribosomal subunit (Section V, E). I n the p r e s ~ n state t of our kno~vletlgc,speculations on the consequences of this model are premature. Involvement of a ribosomal subunit in genetic information transfer appears to provide a more stable structure for this process as expected from the general considerations outlined in Section I. It is also self-evident that a distinct function for the tupo ribosomal subunits may be envisaged. Thus, the function of the large subunit seeins t o be concerned with the adaptation of the whole ribosome to the environmental conditions in the cell (interaction with membranes, with tRNA etc.). On the other hand, the small subunit is involved in the transfer of genetic information from the nucleus into the cytoplasm. Consequently, it seems appropriate to designate the large subunit as the ndaptosome, and to preserve the term znformosome for the small subunit (193), thus avoiding the often controversial designation by the respectire S mlueh. Perhaps other faccts of the process of genetic inforrii~ition traiisfer i n animal cells could t ~ calso givcn a more simple iiiterpretatioli.

ACKNOWLEDGMENTS Thr author \yould like to thank liis rollragues R. G. Tsanev, P. V. Venkov, and Rosrmary and Radoslav Bnchraroff for critical comments and help in the preparation of the manuscript.

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PROG NUCLEIC ACID RES&MOLECULAR BIO V7, Volume 7 (v. 7)

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