INTERNATIONAL SERIES OF MONOGRAPHS ON
PURE AND APPLIED BIOLOGY

Division: MODERN TRENDS IN PHYSIOLOGICAL SCIENCES

General Editors: P. Alexander and Z. M. Bacq

Volume 14

THE BIOSYNTHESIS
OF PROTEINS

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OTHER TITLES IN THE SERIES ON PURE AND APPLIED BIOLOGY

MODERN TRENDS IN PHYSIOLOGICAL SCIENCES DIVISION

FLORKIN — Unity and Diversity in Biochemistry

BRACKET — The Biochemistry of Development

GEREBTZOFF — Cholinesterases

BROUHA — Physiology in Industry

BACQ and ALEXANDER — Fundamentals of Radiobiology

FLORKIN (Ed.) — Aspects of the Origin of Life

HOLLAENDER (Ed.) — Radiation Protection and Recovery

KAYSER — The Physiology of Natural Hibernation

FRANQON — Progress in Microscopy

CHARLIER — Coronary Vasodilators

GROSS — Oncogenic Viruses

MERCER — Keratin and Keratinization

HEATH — Organophosphorus Poisons

RIVERA — Cilia, Ciliated Epithelium and Ciliary Activity

ENSELME — Unsaturated Fatty Acids in Atherosclerosis

BIOCHEMISTRY DIVISION

Vol. 1. PITT-RIVERS and TATA - The Thyroid Hormones

Vol. 2. BUSH — The Chromatography of Steroids

Vol. 3. ENGEL — Physical Properties of Steroid Hormones

BOTANY DIVISION

BOR — Grasses of Burma, Ceylon, India and Pakistan
TURRILL (Ed.) — Vistas in Botany
SCHULTES — Native Orchids of Trinidad and Tobago
COOKE — Cork and the Cork Tree

PLANT PHYSIOLOGY DIVISION

SUTCLIFFE — Mineral Salts Absorption in Plants
SIEGEL - The Plant Cell Wall

RAVEN — An Outline of Developmental Physiology

RAVEN — Morphogenesis : The Analysis of Molluscan Development

SAVORY — Instinctive Living

KERKUT — Implications of Evolution

TARTAR — The Biology of Stentor

JENKIN — Animal Hormones

CORLISS - The Ciliated Protozoa

GEORGE — The Brain as a Computer

ARTHUR— Ticks and Disease

RAVEN — Oogenesis

MANN — Leeches (Hirudinea)

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^

c

THE BIOSYNTHESIS
OF PROTEINS

by

H. CHANTRENNE

Free University of Brussels
Belgium

PERGAMON PRESS

NEW YORK • OXFORD • LONDON • PARIS
1961

PERGAMON PRESS INC.

122 East 55th Street, New York 22, N. Y.

1404 New York Avenue N.W.,

Washington 5, D.C.

PERGAMON PRESS LTD.

Headington Hill Hall, Oxford
4 & 5 Fitzroy Square, London W.l.

PERGAMON PRESS S.A.R.L.
24 Rue des Ecoles, Paris V^

PERGAMON PRESS G.m.b.H.
Kaiserstrasse 75, Frankfurt am Main

Copyright

©

1961

Pergamon Press Ltd

Library of Congress Card Number 61-14246

Set in Imprint 11 on 12 point and printed in Great Britain at
THE ALDEN PRESS. OXFORD

au Professeur Jean BRACKET,
mon maitre et mon ami

Contents

PAGE

Preface

Introduction 1

Chapter I. The Genetic Control of Protein S^tstthesis

A. The one gene — one enzyme hypothesis

1. Basic observations 5

2. Fine structure of the gene 12

(a) Evolution of the notion of gene 12

(b) Difficulties in the use of the cistron concept 19

3. On a few complications of the one gene-one protein relation 20

(a) Pleiotropy 20

(b) Suppression 21

(c) Controlling genetic units 22

B. Chemical nature of the genetic determinants of protein structure

1. Genetic material of bacteria and bacteriophages 23

2. Genetic material of ribonucleoprotein viruses 25

3. Genetic material of higher organisms 27

4. Structure of DNA 28

5. Structure of virus RNA 32

C. The colinearity hypothesis 35

1. Principle 35

2. Coding problems 36

Chapter II. The Sites of Protein Formation within the Living Cell

A. Early cytochemical data 40

B. Fractionation of cell organelles 41

1. Basic observations 41

2. General occurrence of ribosomes 46

C. Protein synthesis in cell fragments and organelles 48

1. Protein synthesis in enucleate cytoplasm 48

2. Protein synthesis in isolated mitochondria 53

3. Protein synthesis in chloroplasts 54

4. Protein synthesis in the cell nucleus 55

D. Summing up 56

Chapter III. Nucleic Acids and Protein Synthesis

A. The position of DNA in protein synthesis 57

1. Higher organisms 57

2. Bacteria 58

3. Conclusion 62

vii

79730

viii CONTENTS

B. Ribonucleic acids and protein synthesis 63

1 . Plurality and Metabolic Heterogeneity of cellular RNAs 63

2. Metabolism of RNA and protein synthesis 66

3. Importance of the structural integrity of RNAs 70

(a) Effects of ribonuclease 70

(b) Modification of RNAs by analogues of purines and

pyrimidines 73

(c) Summing up 78

4. RNA as the genetic messenger 79

(a) Do RNA molecules carry genetic information? 80

(b) Is cytoplasmic RNA made in close contact with DNA? 83

(c) Where is the genetic messenger? 89

5. Concluding remarks 90

Chapter IV. Chemical Pathways of Protein Biosynthesis

A. Energy requirement 92

B. The activation of amino acids 96

1. Amino acid activation enzymes 96

2. Transfer RNA 102

C. Other factors involved in protein synthesis 109

D. The sequential condensation of amino acids into polypeptides 112

1. Are there free peptide intermediates? 113

2. Are polypeptides made on a template? 117

E. Formation of the protein molecule

(a) Folding 122

(b) Disulphide bridges 123

(c) Association of polypeptides 123

(d) Further transformations 123

(e) Integration of proteins into structures 124

Chapter V. Regulation of Protein Synthesis

A. Enzymic adaptation 126

1. Induced synthesis of enzymes in micro-organisms 127

2. Repression of enzyme synthesis in micro-organisms 130

3. Mechanisms of repression and induction 131

4. Induced transformation of an enzyme 137

5. Permeases and enzyme synthesis 138

6. Enzymic adaptation in animal tissues 139

B. Non-mendelian hereditary factors in enzyme synthesis 141

C. Changes in protein synthesis during differentiation 150

D. The formation of antibodies 156

Subject Index 197

Author Index 207

Preface

Knowledge on the biosynthesis of proteins is increasing so rapidly, and
relevant data are obtained from such a variety of approaches that it is
more and more difficult to keep abreast with the progress of research.
Chemistry, crystallography, genetics, cytology, cellular physiology,
embryology, immunology, microbiology, enzymology all contribute to the
analysis of the process. It is obviously impossible to master all these
sciences, and each worker must go his own way, trying his best with what
he can do. But he must from time to time stop and look around to appre-
ciate the advances made along the other lines of research, lest he might
lose contact altogether with the other approaches. The author felt this
necessity for himself and for the students in his laboratory, and he tried
to outline a picture of the whole field of protein biosynthesis as he sees
it presently. The outcome of his attempt was this little book, in which
well established facts were merely summarized, and greater emphasis was
laid upon recent developments and new perspectives. It is realized that
the discussions and interpretations of recent data that this book contains
will soon become obsolete : this is unavoidable in a field of research which
is developing so rapidly. The picture presented here should be regarded
as a snapshot taken at the end of 1960; blurred spots on the picture are
due in part to the lens, and in part to the fog that still covers large regions
of the field.

The present book has profited very much by frequent conversations
with Professors R. Jeener, J. Brachet and M. Errera, and with Dr R.
Thomas and Dr M. De Deken. The author wishes to express his sincere
thanks to Dr G. Palade who kindly gave beautiful electronmicrographs, to
Mrs Bonnami and Mrs Hamers for their help in the publication of the
manuscript, and to Mrs Chantrenne who prepared the index.

H. Chantrenne

Introduction

Remarkable advances in the knowledge of protein structure have been
made during the last decade, and although many aspects of protein struc-
ture still raise some difficult problems, essential features are now clarified.
Protein molecules are made of one single polypeptide, or of a few poly-
peptidic chains associated together in a specific manner. The backbone of
polypeptides is a quite regular structure in which one nitrogen and two
carbon atoms alternate ; one of these carbons carries an oxygen, the other
one, which is asymmetrical, carries a hydrogen and a radical R.

-NH-CH-CO-NH-CH-CO-NH-CH-CO-NH-CH-CO-

I I I I

Ri R2 R3 R4

Fig. 1

There are twenty common varieties of R radicals, and the sequence of
these confers their individuality to the polypeptides, which would other-
wise be all alike. Hydrolysis splits the peptide bonds — CO— NH— and the
chains thus break down into a mixture of some twenty a-amino acids. All
belong to the l series; this means that the relative positions of — NH2,—
COOH, — R and — H around the asymmetric carbon is the same whatever
the nature of the R radical. A very important consequence of the identical
steric configuration of all the amino acids is that certain regular types of
folding of the chains tend to form, whatever the nature of the R radicals.
Among the possible types of folding a few are privileged ; especially a cer-
tain type of helical folding, the a helix (Pauling et ah, 1951), which is found
in many proteins. In this structure, each —CO— of one spire is hydrogen
bonded with an — NH— in the next one ; this confers a great stability and
some rigidity to the framework.

Protein molecules, however, should not be visualized as straight and
perfectly regular helices. Some R side chains may interact and thus cause
deformations of the helices. Proline, one of the natural protein constituents,
is actually an a-imino acid, and its presence causes the helix to bend sharply.
Besides, extensive regions of polypeptides often are not in the form of
perfect helices. As a result, the folded polypeptide is not contained within
a straight cylinder as would be a perfect a helix; it is actually much dis-
torted, and it must look like an oddly contorted piece of tubing, as depicted

1

2 THE BIOSYNTHESIS OF PROTEINS

in the model of myoglobin (Kendrew, 1959) (Fig. 4). This picture also
shows the position of the haematin prosthetic group within the molecule.

The haemoglobin molecule is made of four chains, of two different
compositions, each of w^hich very much resemble myoglobin by the general
shape (Perutz et al, 1960). They are not very strongly bound with one
another and they can be made to separate or reassociate rather easily.

Sometimes, protein molecules associate into structures of a higher order
either with identical molecules or w^th others. For instance, many mole-

12 3 4 5 6 7 8 9 10 11 12 13 14

Acetyl-Ser-Tyr-Ser-Ileu-Thr-Pro-Thr-Ser-GluNH2-Phe-Val-Phe-Leu-Ser-

15 16 17 18 19 20 21 22 23 24 25 26, 27 28

Ser-Ala-Try-Ala-Asp-Pro-Ileu-Glu-Leu-Ileu-Asp*(CyS03H,Thr,Asp*)-

29 30 31 32 33 34 35 36 37 38 39 40

Leu-Ala-Leu-Gly-AspNH,-GluNH2-Phe-Glu*-Thr-Glu-GluNH2-Ala-

41 42 43 44 45 46 47 48 49 50 51 52 53
Arg-Thr-Val-Glu*-Val-Arg-GluNH2-Phe-Ser-GluNH2-Val-Try-Lys-

54 55 56 57 58 59 60 61 62 63 64 65 66 67
Pro-Pro-Ser-GluNH2-VaI-Thr-Val-Arg-Phe-Pro-Asp*-Ser-Asp-Phe-

68 69 70 71 72 73 74 75 76 77 78 79 80 81 82
Lys-Val-Tyr-Arg-Tyr-Asp*-Ala-Val-Asp-Pro-Leu-Val-Thr-AIa-Leu-

83 84 85 86 87 88 89 90 91 92 93 94 95 96 97
Leu-Leu-Gly-AIa-Phe-Asp*-Thr-Arg-AspNH2-Arg-Ileu-Glu*-Val-Glu*-Asp*-

98, 99, 100 101, 102 103 104 105 106 107 108 109 110 111
(Ala,GIu*,Thr,Asp*,Pro)-Thr-Ala-Glu*-Thr-Leu-Asp*-Ala-Thr-Arg-
112 113 114 115 116 117 118 119 120 121 122 123 124 125
Arg-Val-Asp*-Asp*-AIa-Thr-Val-AIa-Ileu-Arg-Ser-Ala-Asp*-Ileu-

126 127 128 129 130 131 132 133 134 135 136 137 138 139
AspNH2-Leu-Ileu-VaI-Glu-Leu-Ileu-Arg-Gly-Thr-Gly-Ser-Tyr-AspNH2-
140 141 142 143 144 145 146 147 148 149 150 151 152 153
Arg-Ser-Ser-Phe-Glu*-Ser-Ser-Ser-Gly-Leu-Val-Try-Thr-Ser-
154 155 156 157
Gly-Pro-Ala-Thr

Asp* = Asp or AspNH2 ; Glu* = Glu or GluNH2

Fig. 2. Amino acid sequence of the protein of tobacco mosaic virus
(Anderer et al., 1960).

cules of the tobacco mosaic virus protein unite in a tightly packed hollow
rod.

Several levels of complexity can thus be considered in proteins. The
polypeptidic backbone is made of amino acids which are firmly bound
together and arranged in a strictly determined sequence (Fig. 2); this is
the primary structure. Folding of the chains into a construction — helices
or otherwise — held together by hydrogen bonds, creates the secondary
structure. The tertiary structure results from the folding of the helices in
space as in Fig. 4. Higher orders of organization make the transition
between molecular and microscopic structures (Fig. 5).

Fig. 4. A three-dimensional model of myoglobin illustrating the tertiary
structure. The grey disc is the haem group (Kendrew, 1959).

INTRODUCTION

Each step of the synthesis of any cell constituent is controlled by a
protein, the specific enzyme. This confers on proteins a unique position in
the activity and in the formation of cell material. It is to be expected that

Fig. 3. A portion of an a-helix. The dashed lines figure hydrogen bonds
(Pauling and Corey, 1951).

the biosynthesis of proteins will raise questions which are not encountered
for the biosynthesis of other constituents, since it is the formation of the
tools themselves which is now being considered.

4 THE BIOSYNTHESIS OF PROTEINS

At the time of writing, no single problem of protein biosynthesis is com-
pletely solved, but the problems at least are clearly stated. The technology
of protein biosynthesis is partly understood ; the systems which supervise
and control the process are being eagerly investigated and some of the key
principles upon which they operate are known already. Nucleic acids are
all important in these systems. Here chemistry and genetics meet and a
new fascinating field opens: molecular biology.

Maximum radius

20A -^— Radius of hole

Nucleic acid

Fig. 5. Schematic representation of a tobacco mosaic virus. The protein

units form a hollow rod with the nucleic acid fibre embedded in the

protein material (Franklin et ah, 1957).

CHAPTER I

The Genetic Control of Protein Synthesis

A. THE ONE GENE-ONE ENZYME HYPOTHESIS

1. Basic Observations

At the beginning of this century, Garrod called attention to several
hereditary abnormalities of man. These abnormal conditions are character-
ized by obvious biochemical defects, for instance the excretion of unusual
substances in the urine, or changes in pigmentation. These traits are
transmitted from parent to progeny as if they were controlled by single
recessive alleles. A classical case of 'inborn error of metabolism' is alcap-
tonuria. Patients excrete in the urine homogentisic acid which gives rise
to a black pigment. When the acid is fed to an alcaptonuric, it can be re-
covered in the urine, whereas it disappears in a normal individual. It was
established as early as 1914 by Gross that normal human serum destroys
homogentisic acid whereas this substance is not changed in alcaptonuric
serum. Clearly, alcaptonurics suffer from a metabolic lesion, from the
failure of some enzymic processes. Since the disease is controlled by a
single gene, it looks as if genes could control enzymic processes in some
specific way.

Indications in the same direction were provided by studies on the in-
heritance of flower pigments, which were initiated by Onslow and Bassett
(1913, 1914) and brilHantly developed by Scott-Moncrieff (1930, 1939)
and Lawrence (1935, 1950). These researches are well illustrated by the
work on the Cape primrose Streptocarpus.

The chemical structure and the genetic control of the petal pigments
were analysed in a series of hybrids. Each strain was found to contain
essentially one single pigment. All the pigments are closely related sub-
stances belonging to the class of anthocyanidins ; they differ from each other
by the presence of a few glycosyl or methoxy groups on the hydroxyls of
the basic anthocyanin structure. The pigments are inherited as if they were
controlled by three pairs of alleles, with one dominant and one recessive in
each pair. Go, Rr, Dd. In the triple recessive genotype ord, the pigment is
mainly pelargonin-3-pentose-hexoside. Replacement of the recessive
allele r by the dominant R results in the substitution of a methoxy group
at position 3'. When the dominant allele O replaces the recessive o, two

6 THE BIOSYNTHESIS OF PROTEINS

methoxy groups appear, at positions 3' and 5', giving malvidin. When the
dominant allele D is present, a hexose is found at position 5, giving a 3,5-
dihexoside in place of the 3-pentosehexoside.

The individual genes thus correspond to certain chemical operations in
the synthesis of the pigments: methoxylation, glycosidation, at specified
positions of the anthocyanin nucleus. Obviously, the genes control well
defined steps in the enzymic processes involved in the synthesis of the
petal pigments.

Hexose-pentose

Hexose-pentose

ord
(Salmon)

oRd

(Rose)

— CHj
Hexose-pentose

Hexose

ORd

(Mouve]

ORD
(Blue)

Fig. 6. Chemical structure of pigments from four Streptocarpus varieties,
and corresponding genotypes (after Lawrence, 1950).

Brilliant studies by Beadle and Ephrussi on the development of the eye
pigments of Drosophila provided further information on the genetic control
of anabolic processes. The dark red colour of wild type eyes is due to the
presence of two types of coloured granules, red and brown, in the pigment
cells which surround the ommatidia. A series of mutants (vermilion
cinnabar, scarlet) which have various tinges of bright red eyes are deficient
in the brown pigment. Ephrussi and Beadle (1935, 1937) transplanted eye
disks of vcrmihon or cinnabar larvae into the abdomen of wild type larvae
and they observed that the mutant eye disks developed into adult struc-
tures with wild type pigmentation. The lymph of wild type flies therefore
proMdcd the mutant eye disks with substances which they did not contain

GENETIC CONTROL 7

and which allow them to make the normal pigment. Implantation of cinna-
bar into vermilion and vice versa showed that the transformations are due
to two substances : v+ which causes the change from vermilion to cinnabar,
and cn+ from cinnabar to wild.

A series of remarkable studies by the groups of Ephrussi, Tatum and
Butenandt led to the discovery that v+ is kynurenine, a product of the
oxidation of tryptophan ; cn+ was later identified as hydroxykynurenine, a
further step in tryptophan oxidation. Both substances are used up during
the transformation of the eye disks, they are intermediates in the bio-
synthesis of normal eye pigments (Kikkawa, 1941). It was clear that
mutation of the two genes each blocks one specified step in the oxidation
of tryptophan. Ephrussi suggested (1942) among other possibilities that the
genes involved control the formation of specific oxidation enzymes.

Further study of eye pigment synthesis from the enzymological point of
view looked very difficult, but it was clear that the type of approach was very
promising and that great developments both in genetics and in biochemistry
would follow once a material more amenable to enzymological studies
is found.

rf^

CO — CHg CH COOH /'%^/-'^^ ^^2 CH COOH

OH

Kynurenine Hydroxykynurenine

Fig. 7.

Neurospora crassa was such a material. The vegetative form of this
mould, the mycelium, which can be grown easily in fairly large batches,
produces asexual spores, the conidia. These make it possible to keep col-
lections of various strains and to grow unlimited amounts of a given
mycelium, the enzymes of which will easily be studied by classical bio-
chemical methods. On the other hand, Neurospora offers many features
which are of great interest for genetical studies. The mycelium is haploid ;
therefore, problems of dominance in the expression of the genetic char-
acters are avoided. Sexual reproduction is easily obtained by fusion of
mycelia of opposed mating types ; the zygote develops into an ascus con-
taining eight haploid spores from which the mycelium is obtained (Beadle,
1947).

Auxotroph mutants, that is mutants which require for growth a sub-
stance that the wild strain can dispense with, proved especially suitable for

B

8

THE BIOSYNTHESIS OF PROTEINS

biochemical studies. Let us consider one classical case, that of some trypto-
phan-less mutants oi Neurospora.

The wild type mould will grow readily on a very simple medium con-
taining salts, sucrose as the carbon source, and biotin. The mycelium forms
spores which, when transferred to fresh medium, will give a new
mycelium identical to the original one, and thus indefinitely. However,
from time to time a spore will not be able to grow on the basic medium,
unless tryptophan is added. Genetic analysis shows that this tryptophan
requirement is inherited as a single mendelian character ; it is the result of a
single mutation.

Conidia

Conidia

Mycelium (JV) of
mating type A

Trichogyne

Protoperithecimn
of mating type A
(maternal parent)

i.

Rsaalta of

first roeiotic

division

Ascoepore
germination

Zygote C2iVJ
nuclem

Results of

second meiotic

division

Mycelium (N) of
mating type a

Protoperithecium
of mating type a
(maternal parent)

Aflcoepore
germination

Ascoepores
from mitosis

Fig. 8. The life cycle of Neurospora crassa (from Wagner and Mitchell,

1955).

Tryptophan is a constituent of the proteins of the mould, and it is
normally produced in the wild type. If the mutant cannot grow in the
absence of the exogeneous tryptophan, this means that the mutant cannot
make tryptophan for itself as the wild type used to do, and that it must
depend on the medium for securing the amino acid. The mutation has
probably blocked biochemical processes involved in tryptophan bio-
synthesis.

Certain tryptophan-less mutants were found to excrete indol into the

GENETIC CONTROL 9

medium. Tryptophan is an indol derivative, and the production of indol
by a mutant which is unable to make tryptophan strongly suggests that
indol is an intermediate in tryptophan biosynthesis and that in the mutant
strain the further utilization of the indol nucleus is impaired.

Biochemical studies on the wild type mycelium indeed showed that
tryptophan is formed by the condensation of indol with serine (Tatum and
Bonner, 1944). This last step of tryptophan synthesis can be observed in
vitro ; it is catalysed by an enzyme present in extracts of wild type mycelium,
which was called tryptophan synthetase (Umbreit, 1946). The hereditary
defect is thus located at the level of one well defined chemical reaction,
catalysed by a known enzyme.

HOCHp CH COOH

+ I

^-. /CH2 — CH COOH

UL,

NHc

N^ NH2 ^^ ^N

H H

Indol Serine Tryptophan

Fig. 9.

It may be assumed that in tryptophan-less mutants which do not further
use indol, the mutation has resulted in the inhibition or destruction of the
enzyme, or else that it has prevented the formation of the enzyme. Mitchell
and Lein (1948) showed that the extracts obtained from the mycelium of
such mutants do not catalyse the condensation of indol with serine; if
tryptophan synthetase from a normal strain is added to the extract of the
mutant, it works perfectly. This indicates that no inhibitor of the enzyme
is contained in the extracts of the mutant. It is clear therefore that no
enzyme activity can be detected, simply because the enzyme is not present
in the extracts of the mutant mycelium.

It must be concluded that mutation of a mendelian gene has prevented
the synthesis of an enzyme which is normally formed in the wild strain.
The whole field of microbial genetics is an illustration of this strict control
of enzyme formation by mendelian determinants. A list of some 50
examples of well studied cases of genetic control of enzyme formation in
microorganisms will be found in a recent review by Fincham (1959).

In higher organisms, as well as in bacteria, enzymes depend on mende-
Uan genes. Inborn errors of metabolism in man, pigment heredity in flies
and flowers are explained by the genetic control of enzyme synthesis. Thus
the metabolic error in alcaptonuria results from the lack of homogentisic
acid oxidase (La Du et al, 1958) ; all the other enzymes of tyrosine oxida-
tion are present, only one is lacking as the result of one gene mutation.
Congenital galactosemia is due to absence of galactowaldenase (Kalckar

10 THE BIOSYNTHESIS OF PROTEINS

et al., 1956). Various forms of glycogen deposition diseases are due to lack
of glucose-6-phosphatase, amylo-l-6-glucosidase or muscle phosphorylase
(Hers and Malbrain, 1959; Hauk et al, 1959; Mommaerts et al, 1959;
Yi Yung Hsia and Kot, 1959; Larner and Villar Palasi, 1959; Gilles et al,
1960). Pentosuria may be due to hereditary deficiency in xylulose dehydro-
genase (Bozian and Touster, 1959). The eyes of vermilion mutants (v) of
Drosophila are devoid of tryptophan peroxidase, the enzyme which makes
kynurenine (Glass, 1957; Baglioni, 1959).

Mutation of a mendelian gene thus suppresses the formation of an enzyme,
i.e. of a protein endowed with well specified catalytic properties. One may
wonder whether the enzyme protein does not form at all in the mutant, or
whether another protein lacking enzymic activity is made instead of the
normal enzyme.

Yanofsky studied 25 independent mutants of Neiirospora crassa all lack-
ing tryptophan synthetase activity. He found for many of them, that each
contains a protein which is closely related to the enzyme for it gives strong
cross reactions with an antiserum prepared against normal tryptophan
synthetase. Moreover, this cross reacting material behaves exactly like the
normal enzyme in biochemical fractionation procedures. A few mutants,
however, do not contain any such 'cross reacting material' (Suskind et al.,
1955; Yanofsky, 1956; Suskind, 1957).

In many cases, mutation thus results in the replacement of a normal
enzyme by some closely related protein devoid of normal catalytic proper-
ties. Cases are also known in which the abnormal protein formed still
possesses some enzymic activity, but it is more heat labile, or it is more
sensitive to an inhibitor, or it has an altered coenzyme requirement, or it
has an abnormal optimum of pH or temperature.

In the last case, the mutant may behave like an auxotroph at the usual
temperature and be able to grow like the wild strain at a higher tempera-
ture (Horowitz and Fling, 1953; Fincham, 1957; Yura, 1959; Lerner and
Yanofsky, 1957; Yanofsky and Stadler, 1958; Yanofsky, 1957; De Moss
and Bonner, 1959).

A shght change in the properties of the protein produced may thus be
brought about by gene mutation. The nature of the chemical change pro-
duced is not revealed by these studies : one may think of slight modifica-
tions in the folding of the polypeptide chains, or of breakages in the chains
or of changes in the amino acid sequence.

Extremely interesting and illuminating data were obtained in this respect
in studies on hereditary blood diseases of man. Genetic analysis, no doubt,
is at a great disadvantage here, but the ready availability of the proteins
involved and the ease of purification proved favourable to detailed chemical
investigation.

Sickle cell anaemia, a blood disease found rather frequently in popula-

^&::^^^ ^ ^ts?'

HaernogloDin A

Haemoglobin S

Fig. 10. Finger prints of trypsin digests of haemoglobins A and S

a: Haemoglobin A; b: Haemoglobin S.
Notice the identity of the two patterns, except for peptide 4 (Ingram, 1 958).

GENETIC CONTROL 11

tions originating from Nigeria, is characterized by an anomalous behaviour
of the red blood cells: when the oxygen tension is low, the blood cells
become twisted and under the microscope they look somewhat like sickles.
In 1949, Neel established that the disease is inherited in a mendelian
manner. Pauling et al. (1949) showed that the blood cells of individuals
carrying the sickle cell trait contain a haemoglobin which can be separated
from normal haemoglobin by electrophoresis. Sickle cell haemoglobin is a
little less negatively charged, and it is less soluble at low oxygen pressures
than normal haemoglobin; this lower solubility explains gelation and
sickling of the cells (Harris, 1950).

Mutant individuals containing the sickle cell gene make a slightly abnor-
mal haemoglobin, just as some tryptophan-less mutants of Neurospora
make a slightly abnormal protein which has lost the catalytic properties of
tryptophan synthetase. Sickle cell anaemia manifests itself in the individ-
uals who are homozygous for the sickle cell gene; these make only sickle
cell haemoglobin, no normal haemoglobin. The disease is the consequence
of an inborn defect in haemoglobin, one of the best known of all proteins.
The molecule of haemoglobin is made of two identical halves (Perutz et al.,
1951, 1960), each half containing two different polypeptide chains a and jS.
The complete amino acids sequence in haemoglobin is not known. Never-
theless, Ingram (1956, 1957) has been able to compare amino acid sequences
of haemoglobin S (sickle cell) and haemoglobin A (normal adult haemo-
globin). Haemoglobin is split by trypsin into 26 different peptides, and a
resistant core is left which can in turn be split into about as many peptides
by chymotrypsin. The peptides can be separated by paper chromatography
and the chromatograms display a pattern of spots which is sufficiently
constant and reproducible to be considered characteristic of the protein.
The 'finger prints' of haemoglobin A and S are nearly identical : no differ-
ence can be detected in the chymotrypsin digest of the trypsin resistant core
(Hunt and Ingram, 1958, 1959). Among the peptides produced by trypsin
hydrolysis, all except one are identical (Ingram, 1958) in both haemo-
globins. In the one peptide which differs between the two haemoglobins,
the only difference is the replacement of one glutamic residue by a valine
residue in S haemoglobin.

More than 20 varieties of abnormal haemoglobins are known at present
(Itano, 1957; Ager et al., 1958) and many of them have been shown to be
genetically determined (Benzer et al., 1958). The finger print procedure
again revealed a great similarity of structure between these variants and
normal haemoglobin; in all the cases analysed so far, each haemoglobin
differs from the others by the substitution of one amino acid for another
one, as shown in Fig. 11.

These remarkable studies clearly show that mutation of a gene can result
in the replacement of one amino acid by another in the protein, without any

12 THE BIOSYNTHESIS OF PROTEINS

Other change in the rest of the chains. Comparison of haemoglobin G, A
and S further indicates that two contiguous amino acids can be replaced
independently as the result of two independent mutations, and that
mutational changes can occur in different regions of the molecule (Hunt
and Ingram, 1959, 1960; Hill and Schwarz, 1959).

Haemoglobin A Val-His-Leu-Thr-Pro-Glu-Glu-Lys
Haemoglobin S Val-His-Leu-Thr-Pro-Fa/-Glu-Lys
Haemoglobin C Val-His-Leu-Thr-Pro-Lji-Glu-Lys

Haemoglobin G Val-His-Leu-Thr-Pro-Glu-GZj-Lys

Fig. 11. Amino acid sequence of the terminal part of the ^ chain in four
human haemoglobins (Hunt and Ingram, 1959; Hill and Schwartz, 1959).

This established that the genetic material controls the nature and
position of certain amino acids in the polypeptide chains and makes one
suspect that the position of each individual amino acid of a polypeptide
chain might indeed be controlled by the gene. It would seem that the gene
responsible for the production of a given protein contains a set of instruc-
tions concerning the nature and positions of the amino acids in the
polypeptide chain, perhaps a complete blue print of the polypeptide chains.
One must then examine closely the fine structure of the gene and try to
decipher the indications it contains.

2. Fine Structure of the Gene

(a) Evolution of the notion of gene. Early studies in genetics revealed
that the genes — i.e. the determinants of certain morphological characters
— are often inherited in groups, as if they were linked together in some way.
The physical basis of linkage in higher organisms is the chromosome. In
Drosophila, for instance, there are four chromosomes in a haploid set, and
the genes fall into four linkage groups. Linked genes are located on the
same chromosome. The linkage is not absolute, however, for separation
and recombination between usually linked genes occur in a certain fraction
of the progeny.

Crossing over can take place at many different points along a chromosome.
If the chances of crossing over were equal all along the chromosome, then
the frequency of recombination of two genes would be a simple function of
their distance on the chromosome : the further apart they would be located,
the more often would crossing over occur between them and the more fre-
quently would recombination be observed. It would be possible to compute
relative 'distances' between genes and the relative positions of the genes

GENETIC CONTROL 13

within a linkage group, from the frequency of recombinations. A most
remarkable outcome of studies based on such reasoning is that all the
experimental data could be accounted for by a linear arrangement of the
genes. Cytological observations revealed changes in the bands of giant
chromosomes of salivary glands of diptera at corresponding positions
(Bridges and Bridges, 1939; Demerec, 1941; Lewis, 1945).

In this outlook, crossing over between the members of a pair of homo-
logous chromosomes at meiosis involves breakage and reassociation of the
chromosome at the level of intergenic material; the genes are implicitly
considered as discrete units which are small compared to the distance
between them.

Developments of the genetics of micro-organisms made it possible to go
one step further in the analysis of the structure of the genome.

Moulds and yeasts form zygotes by fusion of haploid forms. In bacteria,
three processes have been discovered in which part of the genome of a bac-
terium can be introduced into another and give rise to recombinants. Thus
in 'conjugation', part of the genome of the 'male' Hfr bacterium enters the
F~ recipient cell (Lederberg and Tatum, 1946; Wollman et al., 1956). In
'transduction' (Zinder and Lederberg, 1952), a fragment of the bacterial
genome is carried over from one bacterium to another by a bacteriophage
produced in the donor bacterium. In 'transformation', a piece of DNA
extracted from a bacterium by the experimenter, is absorbed by a receptive
bacterium and thus a new piece of genetic material is introduced which may
eventually be integrated in the genome. In all these cases, the transfer is
unidirectional and only part of the genome is usually carried over from the
donor to the recipient bacterium. In bacteriophage, recombination of
genetic markers is observed when two related phages develop within the
same bacterium. (For a brief review, see Braun, 1953.)

The genome of bacteria, to say nothing of bacteriophage, is much smaller
than that of higher organisms. Bacteria and bacteriophages do not possess
chromosomal structures comparable to those found in animals or plants,
and recombination in these micro-organisms probably occurs by a process
which is quite different from crossing over as observed in higher organisms
(Hayes, 1960). Nevertheless, application of the same mapping principle as
used for higher organisms again led to results compatible with a congruent
linear arrangement of the genes. The linear order of the genes was more-
over confirmed by the observation of a progressive linear transfer of the
genetic markers during bacterial conjugation (Jacob et al, 1956; Jacob and
Wollman, 1958; Wollman and Jacob, 1958).

With moulds, yeast, bacteria and bacteriophages it was possible to in-
crease the resolving power of mapping procedures enormously. As many as
10^ individuals can be handled in one experiment. With well chosen selec-
tive media, extremely rare recombinants ca'n be recovered and isolated. It is

14 THE BIOSYNTHESIS OF PROTEINS

thus possible to determine rapidly the recombination frequency of series
of mutations, and to study quantitatively recombinations which occur with
a very low frequency, that is — according to the basic assumption —
recombinations between closely located sites.

When several mutants all lacking the same enzyme activity were isolated
and mapped, it was found that all the mutations are located within a narrow
region of the genome. This is the genetic region corresponding to the
enzyme in question. Crosses between these various mutants, however, may
give rise — with a very low frequency — to wild type organisms which
produce the normal enzyme, as if recombination had taken place between
different mutation sites within the genetic region corresponding to the
enzyme (locus).

Mapping of several such mutants on the basis of the frequency of
recombination again led to their arrangement in a linear order, this time
within the locus of a single enzyme. Although deviations from the pro-
portionality of frequency with distance are often observed for very short
distances, the linear order is maintained. The linear arrangement of the
mutation sites within a locus found strong confirmation in a recent analysis
of the topological relationships between markers in a bacteriophage, which
was based on a completely different principle (Benzer, 1959).

Each mutation thus corresponds to an alteration at one of many possible
sites within the linear array which makes up the genetic region correspond-
ing to a given enzyme protein (Pontecorvo, 1958; Demerec, 1956; Demerec
et aL, 1958; Hartman, 1958). The linear structure of this locus is of funda-
mental significance for the mechanism of protein synthesis.

Recombination after transduction in Salmonella showed that two
mutation sites corresponding to two different enzymes may not be more
distant from one another than two mutation sites within the same locus.
This indicates that two loci can be very close to one another, and most
probably contiguous (Hartman, 1957; Demerec et aL, 1958).

The elementary units of heredity cannot be visualized any more as well
separated beads on a string; they are part of a much more continuous
structure. It is obvious that the use of the term 'gene' to designate any
specific piece of genetic material would now lead to confusion, and there
has been a great deal of apparent conflict during the last few years, due to
the fact that the same words were being used with different meanings by
different authors. Benzer (1957) has greatly helped to clarify the field by
introducing new terms for the elementary units of heredity:

The muton is the smallest unit the alteration of which results in a muta-
tion. There is space for many mutons within the locus of an enzyme. The
muton is of great interest for protein synthesis, for it is directly related to
the smallest modification of the genetic material which causes a change in
the structure of the protein produced. It will be very interesting to estimate

GENETIC CONTROL 15

the size of a muton, how close together two different mutons can be located,
and finally to translate this genetic concept into chemical terms. This is
already partly accomplished, as we shall see later.

The recoil is the smallest element which can be interchanged, as a unit, by
genetic recombination. It is of interest for the analysis of recombination
processes and it is related to the limit of divisibility of the genetic material.
It has, so far, little bearing on studies of protein synthesis.

The cistron, which will be considered more closely in the following pages,
is the unit of function. It can be defined as a piece of genetic material which
must be present as a unit to accomplish its function. This function is the
control of a certain observable character or phenotype. The character con-
sidered may be e.g. a morphological feature, or the capacity of growing on
a defined medium, or the production of a pigment, etc. The observable
character may depend on the genetic material in a simple way or in a very
indirect way; it is clear therefore that the meaning of the 'genetic unit of
function' will depend on the character which is being considered. The
character we are interested in is the synthesis of a specific protein. The
cistron, in this particular case, is a piece of genetic material which specific-
ally controls the synthesis of this particular protein, and which ceases to
fulfil this function if it is fragmented. The size of this unit has deep implica-
tions for the mechanism of protein synthesis. Let us examine how the
cistron can be studied experimentally.

In the process of recombination, whatever the underlying mechanism, a
unique genetic structure is reconstituted or copied from two pieces of
genome which come each from one of the parents.

But it is also possible to confront within the same cell two genomes or
parts of genomes coming from two different cells, under such conditions
that they do not recombine or that recombination is negligible. This occurs
for instance in the formation of a zygote by fusion of two gametes. In
moulds, two haploid hyphae of the same mating type can fuse and form a
heterocaryon, in which each cell contains two types of nuclei, one type
coming from each organism (Beadle and Conradt, 1944). When a piece of
an Hfr genome enters a F" bacterium, a system resembling a zygote is
formed. Again when two different bacteriophages enter the same bacterium,
they multiply mostly independently, this is somewhat comparable to a
heterocaryon. Also in abortive transduction, part of the genome is intro-
duced into a bacterium and coexists with the bacterial genome without
recombining (Demerec and Ozeki, 1959).

The study of such heterozygotes or heterocaryons makes it possible to
find out whether two different mutations concern the same functionally
indivisible unit of genetic material or whether they affect two units which
can operate separately.

Let us consider two mutation sites A and B on the same linkage group

16 THE BIOSYNTHESIS OF PROTEINS

(chromosome or linear set of genes). Each site can exist in two alternative
forms: normal (+) or mutated (— ).

■//////. ■/ ■//.■'/// ,, 7/////////^

Recombination
A- B-'

A- B-

FiG. 12.

If the two mutants A+ B~ and A~ B+ are crossed and if recombination
occurs in between the two sites, a pair of recombinants will be obtained :
A+ B+ and A- B~. Obviously A+ B+, which possesses the two normal sites,
is normal ('wild type'). A" B- to the contrary is a double mutant.

Instead of looking for recombinants between the two strains A+ B~ and
A~ B+, let us introduce these genomes within the same cell under con-
ditions where there is no recombination (as in heterozygotes, heterocaryons,
etc.). Each cell contains a sample of the normal site A+ and a sample of the
normal site B+, but these are located on two separate pieces of genetic
material. If A and B belong to regions of the genome which can each per-
form their function independently of the other, the function of both A+ and B+
will be accomplished and the heterozygote or heterocaryon will behave as
the wild type, or in a very closely similar way. Complementation will be
observed.

For instance, if a heterocaryon is made between two mutants of Neuro-
spora, one tryptophan-less and one adenine-less, the heterocaryon will have
no requirement, because the nuclei which cannot control the synthesis of
tryptophan synthetase cause the formation of the enzyme of adenine meta-
bolism that the other type of nuclei cannot control, and vice versa.

But if the two mutation sites A and B both belong to the same functional
unit of genetic material, this unit is bad in both sets of genes: in one set it
does not work properly because site A is bad, in the other set, because site
B is bad. There is no complementation if the two mutation sites belong to
the same functional unit of genetic material.

Two mutations which do not show complementation when they are on
two separate pieces of genome (trans), although they can restore the wild
type when they are recombined within the same piece of genetic material
(cis) are said to be comprised within the same 'cistron' (Benzer, 1957).

If we are studying the genetic control of synthesis of a particular protein,
a cistron is thus a unit of nuclear genetic material which accomplishes one
indivisible function required for the synthesis of that specific protein.

GENETIC CONTROL 17

It could be for instance a segment of genetic material which controls the
structure of the smallest piece of a protein which can be made independ-
ently and which can later be integrated into the finished protein.

In most cases, all the mutants affecting the synthesis of a specified
enzyme are found to be in the same cistron. This means that the genetic
information relative to these enzymes cannot be divided, it must be used in
one piece. It looks as if most enzymes could not he made piecemeal.

However, a few very interesting exceptions to this rule have been found
recently (Giles et al, 1957; Woodward et al., 1958, 1959; Case and Giles,
1958, 1960). Heterocaryons obtained from two mutants both lacking the
same enzyme, glutamic dehydrogenase, as the result of closely located
mutations showed complementation i.e. partial recovery of the enzyme
production (Fincham and Pateman, 1957; Pateman and Fincham, 1958;
Catcheside and Overton, 1958). This means that the two mutations are
located in two different cistrons. However, the enzyme level in the hetero-
caryon reached at most 25 per cent of that in the wild strain. The low level
of glutamic dehydrogenase could possibly be explained by assuming that
the enzyme is composed of two pieces, namely two polypeptide chains A
and B which are made independently, each under the control of one
cistron. In the heterocaryon, the nuclei of one type would provide for the
synthesis of normal A chains and abnormal B chains, whereas the other
type of nuclei would cause the formation of abnormal A and normal B
chains. Random association of A and B polypeptides would produce about
25 per cent of all good AB protein molecules and 75 per cent of protein
molecules in which at least one of the constituent chains is bad.

Association of polypeptides into a finished protein can indeed occur
spontaneously in vitro. Haemoglobin for instance can be made to dissociate
into two a and two j8 chains. Under suitable conditions, these polypeptides
will reassociate correctly and reconstitute normal haemoglobin molecules
(Itano and Singer, 1958, 1959; Itano and Robinson, 1959; Jones et al,
1959; Vinograd et al., 1959). Studies by Hunt (1959) on foetal haemo-
globin support the idea that the a and j8 chains might be made separately
under the control of two functionally independent pieces of genetic
material.

That complementation in certain heterocaryons occurs by association
of polypeptides has received a convincing demonstration in Woodward's
experiments (1959). By mixing extracts of two auxotroph mutants of
Neurospora both lacking adenylosuccinase, some enzyme activity was
restored. As the mixing took place at a low temperature under such conditions
that no metabolic process could take place, it must be admitted that an
active enzyme has been formed by spontaneous association of at least two
parts, one coming from each auxotroph, by some process akin to the
reassociation of a and ^ chains in the case of haemoglobin. The formation

18 THE BIOSYNTHESIS OF PROTEINS

of a hybrid enzyme in addition to the two parental types in a maize hetero-
zygote is most probably another example of the same phenomenon
(Schwartz, 1960).

To summarize, genetic evidence so far indicates that the information
necessary for the synthesis of a protein molecule is contained in one or
possibly in a very small number of cistrons.

It should be realized that the analysis has reached a point where the
definition of the genetic unit of function, that of protein molecule and that
of enzyme are at stake or raise difficulties. This is illustrated by recent
studies on tryptophan synthetase in Escherichia coli (Lerner and Yanofsky,
1957; Crawford and Yanofsky, 1958; Yanofsky and Stadler, 1958;
Yanofsky, 1959).

The normal enzyme as purified and isolated from the wild strain catalyses
three different reactions:

indole glycerophosphate > indole +triose phosphate (1)

indole + serine >■ tryptophan (2)

indole glycerophosphate + serine >■ tryptophan + triose phosphate (3)

Some mutants lose activity (1) and (2) but keep the other catalytic activity
(3). On the other hand, the mutants affecting tryptophan synthetase belong
to two contiguous pieces of genetic material. The normal protein trypto-
phan synthetase can be split into two inactive moieties which recombine in
vitro into a complex having all the activities of tryptophan synthetase.
Thus an enzyme having several related but clearly distinguishable catalytic
properties results in the present case from the association of two protein
structures the formation of which is controlled by two contiguous genetic
regions.

Going one step further in this direction, we will observe, as in Salmonella,
clusters of contiguous genetic loci arranged in the same relative order as the
steps in the pathway of biosynthesis they control (Demerec and Hartman,
1959; Yanofsky and Lennox, 1959; Hartman et al., 1960).

The names we give to pieces of genetic material and what we call
enzymes or an enzyme system is not very important. The fundamental facts
are that the smallest piece of protein material which can be made indepen-
dently is of the size of a protein molecule or of a large polypeptide chain, and
that all the specific information concerning such a major protein piece is
found within a unique and limited region of the genome.*

* An objection can be opposed to this conclusion (Atwood and Mukai, 1953). If
many proteins should share a common intermediate, for instance a quite small
peptide, a mutation which would prevent the synthesis of this small intermediate
would almost certainly be lethal and the mutant could not be detected. A selection
is therefore operated and the mutants recovered necessarily correspond to genes
controlling a restricted function such as the formation of one elaborate piece of
macromolecule. This is a purely theoretical objection, which it is important to keep
in mind, but there is no positive evidence that this situation exists.

GENETIC CONTROL 19

(b) Difficulties in the use of the cistron concept. Further studies on cases
of complementation in heterocaryons between mutants deficient in the
same enzyme showed that the apphcation of the cistron concept as the unit
of function of genetic material in enzyme synthesis can lead to absurdity.

Thus Giles et al. (1957) studied several mutants of the locus responsible
for adenylosuccinase synthesis. Certain associations of two such mutants in
a heterocaryon gave complementation, others did not. The puzzling situa-
tion which follows was observed: mutants A and B gave no complementa-
tion, which means that they were in the same cistron, by definition. In the
same way, mutants B and C were also found to be in the same cistron.
However, A and C gave complementation and — by definition — were not
in the same cistron. These three conclusions are obviously incompatible.

Similarly, Pateman and Fincham (1958) found that out of eleven
mutants in the glutamic dehydrogenase locus of Neurospora crassa, only
two pairwise combinations produced enzyme activity in heterocaryons,
whereas all the others gave no complementation. Again some pairs of
mutants were therefore in the same cistron, according to certain experi-
ments, and in different cistrons according to other experiments. In such
cases, the notion of cistron breaks down. The reason is most probably that
the operational definition of the cistron by the cis-trans test rests on the
common sense idea that adding two bad things together does not make a
good one. This assumption is not entirely justified. Fincham (1960) has
shown that the functional glutamic dehydrogenases produced by com-
plementation are not identical to the normal enzyme: they diff"er from the
wild type dehydrogenase and from each other in their thermostability, and
they have abnormal Michaelis constants. The co-operation of the genomes
of these mutants therefore has not reconstituted the normal protein, it has
produced doubly abnormal proteins. But these happen to have an enzymic
activity sufficiently similar to that of the normal enzyme to be functional.
The molecule of glutamic dehydrogenase results of the association of
several identical polypeptide chains. It is conceivable that polypeptides
with lesions in different places might be able to cover each other's deficien-
cies and to form an almost correctly folded protein structure with almost
normal enzyme activity (Fincham, 1960; Catcheside, 1960).

Fincham's observations are of great interest for protein synthesis. They
are compatible with the idea that the genetic locus strictly controls the
primary structure of the polypeptide chains (the amino acid sequence), and
they call attention to the fact that the appearance of the specific catalytic or
serological properties associated with the protein produced is an epiphe-
nomenon. This depends on eventual association of the polypeptide chains,
on their folding into helices, and on the manner parts of helices twist
around each other, thus bringing together the elements which constitute
the active centre of the enzyme, or the particular surface of the antigen.

20 THE BIOSYNTHESIS OF PROTEINS

Cases of complementation should be analysed very closely before being
taken as evidence for the resolution of the genetic locus of an enzyme into
several cistrons. The notion of cistron would recover its full value in such
cases if the 'function' considered was the production of a certain arrange-
ment of amino acids, but it is not an easy matter to study such a function.

The conclusions from the data examined in the preceding pages can be
summarized as follows : the genetic information which controls specifically
the arrangements of the amino acids of an enzyme is contained in a unique
segment of genetic material, the locus of the enzyme. This locus usually
acts as a unit; it may, in some cases, be composed of two or a quite small
number of functional units.

3. On a few Complications on the One Gene-One Protein Relation

The fact that two contiguous pieces of genetic material may be involved
in the control of the structure of one single enzyme does not contradict the
one gene-one protein hypothesis, provided one is ready to readjust the
definitions of gene and enzyme adequately. But a few categories of
experimental data seem to conflict with basic ideas contained in the
hypothesis.

(a) Pleiotropy. In higher organisms, it is not exceptional that a single
mutation changes several morphological or physiological characters. This
has little impact on the hypothesis for it is highly probable that one single
biochemical deficiency in the zygote will have several eflFects in the course
of development and may in the end change the properties of the differenti-
ated cells in various ways. But pleiotropy has been observed also for bio-
chemical characters in micro-organisms. A single mutation can result in a
double requirement.

In well analysed cases, however, the double requirement was shown to
result from the lack of one single enzyme. For instance, the double require-
ment for methionine and threonine in a Neurospora strain results from a
single genetic block in the pathway of synthesis of homoserine, which is a
common precursor of threonine and methionine (Teas et al., 1948; Horo-
witz, 1956). The double requirement for valine and isoleucine is due to the
inhibition by a precursor of isoleucine of the transaminase which makes
valine. The precursor accumulates when the transaminase which makes
isoleucine is absent (Bonner, 1946). A mutant lacking the latter enzyme
therefore requires both valine and isoleucine for growth. A comparable case
has also been described for Escherichia coli (Myers and Adelberg, 1954;
Rudman and Meister, 1953).

(b) Suppression. A more puzzling phenomenon is the suppression of
a nutritional requirement by a mutation which occurs in a region of the
genome completely unrelated to the locus in which the first mutation has
occurred.

GENETIC CONTROL 21

In certain cases, the suppressor mutation was shown to open a pathway
of biosynthesis alternative to the one which was blocked in the auxotroph
(Lein and Lein, 1952), e.g. by relieving an inhibition (Strauss and Pierog,
1954; Howarth, 1958).

But all suppressors do not act in this way. Auxotroph behaviour in
mutants of the td locus (responsible for tryptophan synthetase) in Netiro-
spora can be 'suppressed' by mutations occurring at a great distance from the
td locus. With a mutant strain which produces a temperature sensitive
enzyme, it was observed that the 'suppressed' mutant still displayed a
temperature sensitivity which does not exist in the wild strain (Bonner,
1946; Yanofsky, 1956; Suskind, 1957). This suggested that the td locus
still controlled the structure of the enzyme and that the suppressor prob-
ably affected the operation of the abnormal protein in some indirect way.

Again, for a tryptophan synthetase mutant of Neurospora, such a mechan-
ism has been partly cleared up recently (Suskind and Kurek, 1959). The
mutant considered contained an enzyme protein which was much more
sensitive to inhibition by zinc ions than the normal enzyme. In a 'sup-
pressed' mutant, in which wild type behaviour was more or less restored,
the enzyme produced was actually the zinc-sensitive enzyme, but the sup-
pressor gene in some way controlled the adjustment of zinc ions concen-
tration in the mycelium. As a result, the abnormal protein could fulfil an
almost normal enzymatic function in the 'suppressed' mutant, whereas it
could not (due to zinc inhibition) in the non-suppressed auxotroph.

In all the cases considered above, suppressors affect the operation of
enzymes or of enzyme systems, and their action is irrelevant to the control
of the enzyme structure. These facts do not conflict with the idea that the
information relative to the primary structure of an enzyme is located within
one restricted piece of genetic material.

There are cases where the difficulties raised by suppression cannot be
completely dismissed at present. For instance, a strain of Escherichia coli
which lacks tryptophan synthetase and does not produce any corresponding
cross reacting material as judged by the usual immunological criteria, can
recover the capacity of making a protein having the immunological and
enzymic properties of the normal enzyme, as the result of a 'suppressor'
mutation in another region of the genome (Yanofsky, 1958; Yanofsky and
Crawford, 1959).

In another mutant, which does produce an enzymically inactive cross
reacting material, a suppressor mutation caused the production of a new
enzymically active protein, in addition to the altered one. It was shown,
besides, that the locus of the suppressor mutation in itself does not carry
the information for making the normal protein.

These facts indicate that suppressor mutations can indeed change some-
how the mode of expression of a gene. This could possibly mean that the

22 THE BIOSYNTHESIS OF PROTEINS

final shape of a protein is not completely determined by the corresponding
locus and that factors which are independent of this locus may play a part
in shaping the protein molecule. This calls for the same remarks as the cases
of complementation between non-identical alleles (cf. p. 20). The genetic
locus provides information — perhaps all the information — concerning the
linear arrangement of the amino acids in the polypeptide chain (the so-
called 'primary structure of the protein'). But the activity of an enzyme or
the immunological properties of a protein depend on their tertiary struc-
ture. The folding and association of polypeptide chains are conditioned by
the amino acid sequence, but they might also depend on contingent factors
like the concentration of various ions and possibly on somewhat more
specific actions. Certain suppressors might act by modifying such factors.

(c) Controlling genetic units. Another example of complication to the
gene-enzyme relationship might be seen in the genetic control of ^-galacto-
sidase synthesis in Escherichia coli. The production of the enzyme is con-
trolled by three independent and separable loci z, i, y. Only one of those,
the z locus, provides structural information for /S-galactosidase synthesis.
The / locus controls the actual production of the enzyme, it is involved in
the regulation of the synthesis. This locus does not carry any information
as to the structure of ^-galactosidase since mutation of this gene does not
alter the structure of the protein produced. The third gene y also regulates
the synthesis of ^-galactosidase indirectly, by controlling the formation of
a system which concentrates into the bacteria inducers of jS-galactosidase
synthesis (Jacob and Monod, 1959). A similar situation exists for tyrosinase
in Neurospora (Horowitz et al., 1960) and most probably for other inducible
and repressible enzymes (see Chapter V).

Actually, these facts are in perfect agreement with the concept that all
or at least an essential part of the structural information relevant to a
specified protein is located in a restricted part of the genome, in the present
case the z locus.

(d) Concluding remark. In discussing the value of the one gene-one
enzyme hypothesis, it should be clearly realized that two difi^erent mean-
ings can be attributed to this relation depending on the direction in which
it is expressed. This relation can be taken to mean that the structure of a
protein is specifically controlled by a restricted section of genetic material;
we have seen that this statement is supported by numerous experimental
data and contradicted by none, if we reserve a few cases that have not been
thoroughly analysed yet.

The other acception of the gene-enzyme relationship concerns the mode
of action of the genetic material ; this side of the matter has not been dis-
cussed here. It should be made clear that there are reasons to doubt that
the genetic material exerts its action on the cell exclusively by controlling
the structure of specific proteins. The genetic control of differentiation in

GENETIC CONTROL 23

higher organisms might involve other modes of action of the genes.
Moreover, there is evidence that the i gene which controls the inducible
or constitutive character of j8-galactosidase can be expressed under such
conditions that no protein formation can occur (Pardee et al., 1959); it
would seem therefore that the / gene does not act by controlling the
structure of any protein (Jacob and Monod, 1959).

B. CHEMICAL NATURE OF THE GENETIC
DETERMINANTS OF PROTEIN STRUCTURE

1 . Genetic Material of Bacteria and Bacteriophages

Direct evidence that DNA is the genetic material of bacteria was pro-
vided by studies on bacterial transformation. Griffith (1928) had observed
that material obtained from encapsulated Pneumococci can confer upon
non-encapsulated strains the ability to make a capsule com.posed of specific
polysaccharide. Avery et al. (1944) found that transforming activity is
associated with DNA. The transforming properties of the DNA prepara-
tions are not affected by proteolytic enzymes or by ribonuclease, but they
are extremely sensitive to deoxy ribonuclease. The transforming properties
also disappear under conditions which cause denaturation of DNA
(Zamenhof et al. 1953; Zamenhof, 1956; Lerman and Tolmach, 1959;
Lacks and Hotchkiss, 1960).

Many hereditary characters, including the capacity to make well defined
enzymes, can be transferred from one bacterial strain to another by highly
purified DNA. This DNA was shown to transfer for instance the capacity
to oxidize glucose (Ephrussi-Taylor, 1954) or to make mannitolphosphate
dehydrogenase (Marmur and Hotchkiss, 1955). In the transformed bacteria,
this capacity is thereafter inherited like any other character and DNA
extracted from the transformed bacteria is able to cause transformation.
DNA thus performs the two functions which are characteristic of the
genes : it brings to the bacterium the capacity of making a specific enzyme
and it is duplicated and transmitted to the progeny together with the
genetic determinants. The genetic characters introduced by the transform-
ing DNA are also capable of recombination (Ephrussi-Taylor, 1951, 1954,
1955 ; Hotchkiss and Marmur, 1954).

Treatment of DNA by nitrous acid is known to deaminate guanine,
adenine and cytosine ; when transforming DNA is treated in vitro by this
agent and used thereafter as transforming agent, many transformed
bacteria have acquired mutated characters. Modifications of DNA brought
about in vitro by a chemical agent can thus be expressed as mutations when

c

24

THE BIOSYNTHESIS OF PROTEINS

the DNA is introduced into a bacterium (Litman and Ephrussi-Taylor,
1959).

Experiments by Hershey on the genetic material of bacteriophage are
almost as convincing. Bacteriophages are complex organisms made of
packed DNA enclosed in an elaborate protein structure. Hershey and

Fig. 13. Schematic representation of the experiment of Hershey and
Chase (1952) (Reproduced from Kozloff, 1959).

Chase (1952) grew bacteriophage in a medium containing either 32PO4 or
35SO4; the collected phage contained ^^p in DNA exclusively or ^^S in
protein constituents only. When non-labelled bacteria were infected with
labelled phages, it was found that ^^p (therefore DNA) enters the bacteria
and is retained in the progeny whereas most of the ^^S stays outside (Her-
shey, 1955). Phage particles may be compared to small syringes which inject
the DNA they contain into a bacterium. The injected DNA confers to the
infected bacteria the capacity of making the phage proteins and several
enzymes involved in the synthesis of phage DNA precursors (Flaks and
Cohen, 1959; Flaks et ah, 1959; Romberg et al., 1959; Somerville et ah,
1959; Earner and Cohen, 1959; Bessman, 1959; Keck et al, 1960).

Phage DNA multiplies within the bacterium, it is later enwrapped into
its protein container. When two related phages infect the same bacterium,
that is when two phages inject their DNA into the same bacterium, re-
combination of hereditary characters may be observed. Phage DNA con-
tains all the information required for orienting the cell metabolism towards
the synthesis of a set of specific foreign proteins. It is clear that phage DNA
has all the features of the genetic material.

There is also good reason to believe that during bacterial conjugation
it is essentially a DNA fibre which passes into the recipient bacterium, for
decay of ^^p^ which breaks the DNA molecule, causes breakages in the
chain of genetic markers (Jacob and Wollman, 1958).

GENETIC CONTROL 25

2. Genetic Material of Ribonucleoprotein Viruses

Many viruses of plants and animals contain no DNA; they are made of
RNA enclosed in protein. Viruses certainly bring to the cell a set of instruc-
tions as how to assemble building blocks of proteins and nucleic acids into
virus RNA and protein. Extensive modifications of the protein moiety of
tobacco mosaic virus can be brought about by chemical treatment without
adverse effect on virus infectivity. Substitution of 70 per cent of the protein
NH2 groups by chlorobenzoyl groups (Miller and Stanley, 1942), addition
of leucyl residues (Fraenkel-Conrat, 1953), removal of the terminal
threonine of the protein by carboxypeptidase (Harris and Knight, 1952)
have no effect on infectivity, nor on the nature of the virus produced. A
large part of the protein can be removed without killing the virus (Schramm
etal, \955).

On the other hand, slight modifications to the virus RNA result in virus
inactivation. Structural analogues of purines and pyrimidines (e.g. 8-
azaguanine, 2-thiouracil or 5-fluorouracil) drastically reduce multiplication
(Commoner and Mercer, 1951, 1952; Matthews, 1952; Gordon and
Staehelin, 1959); this must be due to alterations of the virus RNA by the
abnormal purines or pyrimidines, for a close correlation was found between
incorporation of thiouracil into the virus RNA and the inhibition of virus
multiplication (Jeener and Rosseels, 1953). If ribonuclease is introduced
into the cells of a tobacco plant at the beginning of infection, virus multi-
plication is blocked (Hamers-Casterman and Jeener, 1957; Benda, 1958).
Multiplication of influenza virus is also inhibited by ribonuclease (Le
Clerc, 1957).

Such experimental data indicated that the integrity of RNA is more
essential to virus multiplication than integrity of the protein sheath (Jeener,
1956). Fraenkel-Conrat and Williams (1955) were able to separate the RNA
from the protein and to reassociate them into virulent particles. Virus
reconstituted by mixing RNA and protein from different strains always
caused lesions which resembled very much those corresponding to the
virus from which the RNA had been obtained (Fraenkel-Conrat, 1956;
Fraenkel-Conrat et al, 1957). Numerous mutants were formed, however,
as if the genetic material of the virus had been slightly damaged during the
process (Commoner et al, 1956; Fraenkel-Conrat and Singer, 1957;
Bawden and Pirie, 1957). Ribonuclease does not kill the intact virus, but
the slightest action of the enzyme upon the naked RNA leads to complete
inactivation of the reconstituted nucleoprotein (Fraenkel-Conrat and
Williams, 1955).

Finally, Gierer and Schramm (1956) were able to isolate from tobacco
mosaic virus, by phenol extraction, RNA which could transmit infection.
This fundamental observation has now been confirmed by work from many
laboratories and extended to other viruses. Control experiments of various

26 THE BIOSYNTHESIS OF PROTEINS

kinds make it quite clear that the infectivity of the RNA preparations is not
due to traces of contaminant virus (Fraenkel-Conrat et ah, 1958; Bawden
and Pirie, 1957; StaeheHn, 1959; Engler and Schramm, 1959).

Infectious RNA has been isolated by phenol or detergent extraction from
turnip yellow mosiac virus (Fraenkel-Conrat et ah, 1957; Cohen and
Schachman, 1957), from tobacco Ring Spot (Kaper and Steere, 1959),
from the virus of necrotic Ring Spot of cucumber (Diener and Weaver,
1959) and tomato Bushy Stunt (Rushizky and Knight, 1959). Ribonucleo-
protein viruses which infect animal cells also provide infectious RNA.
This was estabhshed for poliomyelitis virus. Encephalitis viruses and
other neurotropic viruses by numerous workers: Alexander et al, 1958;
Huppert and Sanders, 1958; Ada and Anderson, 1959; Franklin et al,
1959; Cheng, 1958; Wecker, 1959; Mountain et al, 1959; Gerber and
Kirschstein, 1960. Infectious RNA was also isolated from animal cells
infected with neurotropic viruses (Colter et al., 1957; Wecker and Schaffer,
1957; Sokol et al, 1959), with Influenza virus (Maassab, 1959) or with Foot
and Mouth disease (Brow and Stewart, 1959; Mussgay et al., 1959) and
also from an insect virus (Krieg, 1959).

It is now possible to study directly the effects of chemical or enzymic
modifications of the isolated RNA upon its infectivity. Treatment of the
isolated RNA by nitrous acid under very mild conditions rapidly inactivates
the RNA. Nitrous acid acts upon adenine, guanine and cytosine and
replaces their amino groups by a hydroxyl. It has been estimated that the
modification of one nucleotide out of some three thousand can inactivate a
virus particle (Schuster and Schramm, 1958); this amounts to one or two
changes per virus particle. It was also observed (Gierer and Mundry, 1958)
that the fraction of mutants recovered is increased very much after limited
treatment of the RNA by nitrous acid. This suggests that replacement of,
for example, a cytosine residue by its product of deamination uracil might
lead to viable mutated forms of the virus (Gierer and Mundry, 1958).

RNA of the ribonucleoprotein viruses must carry the hereditary charac-
teristics of the virus, since it is able to cause the complete process of in-
fection, including the synthesis of the specific protein coating. It would
seem that the protein moiety is a sheath which protects (Siegel et al., 1956)
RNA in the resting extracellular form of the virus. In normal infection, the
RNA is indeed liberated into the cell where it reproduces first (Engler and
Schramm, 1959); the unprotected RNA is then very sensitive to ribonu-
clease action within the cell (Hamers-Casterman and Jeener, 1957; Benda,
1958). The specific virus protein is made later and it associates into the
typical rods in which one RNA fibre is enwrapped.

The protein moiety plays, nevertheless, an important part in virus
infection. The host range of viruses is often rather restricted. Recent
observations indicate that the susceptibility or resistance of animal or plant

GENETIC CONTROL 27

cells to certain viruses depend on specific interactions between the surface
of the cell and the virus protein (De Somer, et al. 1959 ; Holland et al., 1959 ;
Gordon and Smith, 1960). Only primate cells are susceptible to polio-
myelitis virus. But cells of rabbit, pig, mouse and chicken can be infected
by RNA extracted from the virus. It is striking that the virus thus produced
in non-primate cells shows the typical host range and serological specificity
of poliomyelitis virus. Since the structure of the protein coat is not in-
fluenced by the cell in which it is produced, it must have been strictly con-
trolled by information contained in the virus RNA.

There is a complete analogy between virus infection by RNA and
bacteriophage infection, which normally occurs by injection of DNA into
the recipient cell. Virus RNA is the carrier of information for the pro-
duction of at least the virus protein, just as DNA is the carrier of the
information which controls the synthesis of an array of phage proteins.

3. Genetic Material of Higher Organisms

Sperm heads and chromosomes are two structures known to contain
mendelian genes. Comparison of their constitution provides a first indica-
tion about the nature of the genetic material in animals. Chromosomes are
made of DNA, histones, 'residual' acidic protein and RNA (Brachet, 1942).
Residual protein may be absent from metaphase chromosomes (Casperson,
1941 ; Bloch and Godman, 1955). Sperm head in fishes contains no RNA
and no histones; it is made of DNA associated with protamines. The only
constituent that fish sperm head and chromosomes have in common there-
fore seems to be DNA which accordingly must be the carrier of genetic
determinants.

Other indications in the same direction are found in the constancy of the
DNA content per cell in any given species. Moreover, many mutagenic
agents act upon animals and plants in the same way as on bacteria and most
of them are known to react easily with DNA. The ultraviolet action
spectrum for the production of mutations in higher organism as well as in
micro-organisms resembles very much a nucleic acid absorption spectrum.
DNA from different species haVe different com.positions : the adenine/
guanine ratio seems to be characteristic of the species (Vischer and Char-
gaff, 1948; Chargaff et al, 1949; Chargaff, 1956). DNA is also one of the
most stable compounds of the animal cell and this quality is well suited to a
guardian of hereditary characters.

DNA therefore appears as the most probable carrier of genetic informa-
tion in higher organisms and although as direct an evidence as in bacterial
transformation has not been clearly obtained so far, it is the common belief
that DNA is at least the main genetic material in higher organisms.

Extensive discussions about the nature of the genetic material will be
found in reviews by Hotchkiss (1955) and by Brachet (1957).

28 THE BIOSYNTHESIS OF PROTEINS

4. The Structure of DNA

DNA as isolated from animal tissues or from micro-organisms is a macro-
molecule with a molecular weight in the range of 6-10^ (Sadron, 1959).
Chemical studies (Brown and Todd, 1952; Michelson and Todd, 1953;
Dekker et al, 1953; Carter, 1951) showed that DNA is made of a linear
backbone in which phosphoric groups and deoxyribose residues follow
each other in a quite regular manner. Each phosphate group is bound to
the C5 position of a deoxyribose and to the C3 of the next one. These long
chains of two alternating elements are identical in all the DNA samples
studied, they are perfectly monotonous and probably void of information.
But each deoxyribose residue of the chain carries on the Ci a purine or a
pyrimidine bound in N-glycosidic linkage. Adenine, guanine, thymine and
cytosine account for all but a small percentage of the bases. 5-Methylcyto-
sine (Laland et al., 1952; Wyatt, 1951), 6-methylaminopurine (Dunn and
Smith, 1955, 1960) have been found in smaller amounts in DNA.

In a few exceptional but very interesting cases, large amounts of an
unusual pyrimidine enter in DNA composition, thus T-even bacterio-
phages contain 5-hydroxymethylcytosine and a large part of it carries one
or two glucose residues on the CH2OH (Wyatt and Cohen, 1952, 1953;
Sinsheimer, 1956; Loeb and Cohen, 1959).

The base composition of DNA proved quite constant within each
species, and no significant dift'erences were found between DNA samples
obtained from different organs of a given species (Chargaff and Lipshitz,
1953). On the contrary, DNA specimens from various organisms were
found to differ considerably in base composition (Chargaff, 1950, 1958;
Wyatt, 1952) and in their chromatographic behaviour (Brown and Watson,
1953 ; Bendich et al. , 1 956) which indicates that DNA preparations are hetero-
geneous populations of molecules of different composition. It has not been
possible so far to unravel the arrangement of the purines and the pyrimi-
dines in the chains, but some indications were obtained by the study of
breakdown products formed by controlled degradation of DNA. Purines
and pyrimidines do not follow each other at random: for instance clusters
of three or more pyrimidines are more frequent than expected from a com-
pletely random distribution (Shapiro and Chargaff", 1957; Burton and
Peterson, 1960). Differential distribution analysis of the split products,
moreover, showed marked differences between DNA specimens of different
origins which had almost indistinguishable gross composition (Shapiro and
Chargaff, 1957, 1960).

DNA of different species thus differ in the proportions and in the
arrangement of the bases along the common phosphate— carbohydrate
backbone. If genetic information is a modulation of the DNA constitution,
the arrangement of the bases along the backbone must be the language in
which this information is recorded.

GENETIC CONTROL

29

Adenine

Thymine

H

Fig. 14. Chemical structure of a section of a DNA chain.

30 THE BIOSYNTHESIS OF PROTEINS

Comparison of the analytical data obtained on a great many specimens
of DNA brought to light a striking regularity or common principle of DNA
composition: whatever the origin of the DNA samples, the number of
adenine molecules was found to be equal to the number of thymine
residues, and guanine and cytosine on the other hand were always present
in equimolecular amounts (Chargaff, 1950). The fundamental significance
of this rule was fully realized only \vhen a satisfactory model of macro-
molecular structure was worked out for DNA.

Gulland et al. (1947) had observed that DNA solutions undergo irrevers-
ible changes in viscosity and in ionization characteristics outside a certain
pH range. This made it probable that DNA as isolated contains labile
bonds, e.g. hydrogen bonds, between ionizable groups. Since titration
hysteresis persists at very low DNA concentrations, the labile bonds are
probably located within each molecule, not between molecules (Jordan
et al., 1956). A considerable irreversible increase in ultraviolet absorption
is brought about by lowering the pH for a short time or by heating DNA
especially in solutions of low ionic strength (Thomas, 1951, 1953, 1954),
indicating irreversible changes in electronic state of the heterocycles.

It is clear that DNA, like proteins, can be denatured by rather mild
treatments and that its macromolecular structure is held together by labile
bonds in which ionizable groups of the ultraviolet absorbing heterocycles
are involved. DNA denaturation has been extensively studied, it manifests
itself by changes in viscosity (Doty and Rice, 1955), streaming birefringence
(Mathieson and Matty, 1955), rotatory dispersion (James and Levendahl,
1955), infrared absorption (Frick and Rosenberg, 1954; Blow and Lenor-
mant, 1955), UV absorption (Thomas, 1951, 1953, 1954), affinity for
certain dyes (Thomas, 1953), and sedimentation characteristics (Alexander
and Stacey, 1955, Oth, 1959).

X-ray diffraction studies indicated a helical structure of DNA molecules
(Wilkins et al., 1953 ; Franklin and Gosling, 1953). The size of the structural
unit and the density of DNA suggested that there must be two chains in the
unit (Crick, 1954). Model building showed that the chains are probably
held together by bonds between pairs of bases, and the best fit is obtained
by pairing adenine with thymine and guanine with cytosine (Pauling and
Corey, 1956). This was in agreement with the experimental fact that
adenine and thymine on one hand and guanine and cytosine on the other
are always found in equimolecular proportions in DNA.

Watson and Crick (1953) thus proposed the now famous double helix
structure for DNA in which the two chains run in opposite directions; each
adenine in one chain is linked by hydrogen bonding to a thymine in the
other, and each guanine to a cytosine (or a cytosine derivative). The para-
meters of this structure or of a slightly modified version of it (Feughelman
et al., 1955; Langridge et al., 1960) are in good agreement with X-ray

GENETIC CONTROL 31

diffraction data (Wilkins et al, 1953; Franklin and Gosling, 1953). The
rigidity and the shape of such a macromolecule fit with the hydrodynamic
properties of DNA solutions (Sadron, 1959). Denaturation is explained by
breakage of the purine-pyrimidine hydrogen bonds.

Adenine H u -- — 'Q\ u~^J^ Thymine Guanine _.-H— N^ Cytosine

N " W " C^H 0" \- M

'Sugar\ N-'H

/

H
Fig. 15. Hydrogen bonding of bases in DNA (Watson and Crick, 1953).

An extremely interesting feature of the Watson-Crick model is that it
provides a ready made intuitive answer to the problem of replication of the
detailed arrangement of the nucleotides during duplication. The genetic
information is contained in each chain in two complementary sets of
symbols ; it can be visualized that during duplication the chains somehow
separate and that each serves as a template for building a copy of the other.
This remarkable hypothesis cannot be considered as established; it is at
least compatible with the experimental facts at the present time.

The DNA macromolecule seems indeed admirably adapted to carrying
coded information. If the information resides in the sequence of the four
nucleotides, a linear chain of some ten thousand nucleotides can store an
enormous amount of information. The double strand system keeps the
information in a state which prepares it for duplication. DNA is chemically
stable at temperatures compatible with life and indeed more stable than
many essential cell constituents and it is metabolically inert.

Part of the DNA only exists as double helices and part in a less orderly
structure. DNA fractionation indicates that a small fraction of DNA
might exist as single polynucleotide strands (Lucy and Butler, 1954;
Bendich et al, 1956). In bacteriophage X 174, DNA is not a double helix
but a single polynucleotide chain (Sinsheimer, 1959). Studies on this
particular system will probably help to clarify the duplication process, and
the mechanism of information transfer.

5 . Structure of Virus RNA

The chemical constitution of RNA is very similar to that of DNA. The
phosphate-carbohydrate backbone is identical to that of DNA, except for
the presence of a hydroxyl group instead of a hydrogen on the C2 of the
carbohydrate which is thus ribose instead of deoxyribose. The presence of
this hydroxyl group next to a phosphodiester makes the ribonucleic acid
backbone much more susceptible to acid and alkali hydrolysis than DNA.

32

THE BIOSYNTHESIS OF PROTEINS

The sequence of the bases, adenine, guanine, cytosine and uracil along
the phosphate-carbohydrate backbone is not known for any RNA. There is
evidence, nevertheless, that it is diff-^rent in the RNA of different viruses
(Reddi, 1959). In contrast with the data on DNA composition, no rule or
regularities have been found for virus RNA, although for the mixture

Fig. 16. Molecular model of a two-strand helix of DNA (Feughelman

et al, 1955).

of RNAs extracted from animal or plant cells and from bacteria, the
number of 6-keto groups was found to be close to the number of 6-amino
groups.

X-ray studies on tobacco mosaic virus indicate that the virus RNA is
made of a single polynucleotide chain of about 6000 nucleotides which rests
in a spiral grove formed in the regularly packed protein units. This RNA

GENETIC CONTROL

33

CH2 Q Guanine

Cytosine

OH

OH

Guanine

OH

Fig. 17. Chemical structure of a section of an RNA chain.

34 THE BIOSYNTHESIS OF PROTEINS

spiral has nothing in common with the double helix structure of DNA,
the spires of the RNA strands are far apart and separated by protein
material.

For virus RNA as for DNA, it is reasonable to assume that whatever
genetic information the linear chain of RNA can carry must be recorded in
the molecule as a particular arrangement of the purines and pyrimidines.
This idea is supported by experimental facts. For instance, a very mild
treatment of the virus by nitrous acid causes the appearance of mutants
(Gierer and Mundry, 1958; Boeye, 1959; Schafer, 1959; Mundry, 1959)
and it would seem that the appearance of a given mutant results from
deamination of a single base (Gierer and Mundry, 1958; Boeye, 1959).

Tsugita and Fraenkel-Conrat (1960) have isolated a mutant of tobacco
mosaic virus which had been obtained after treatment of the isolated virus
RNA by nitrous acid. The amino acid composition of the protein coat of
this mutant seems to differ from that of the original wild strain by the re-
placement of three amino acid residues — one proline, one aspartic acid
and one threonine — by one residue each of leucine, alanine and serine.
Moreover, the amino acid sequence of the last 17 amino acids of the N-
terminal end of the protein has been established for both the original virus
and the mutant. They are shown hereafter:

parent original strain:
-Ser-Ser-Phe-Glu-Ser-Ser-Ser-Gly-Leu-Val-Try-Thr-Ser-Gly-Pro-
Ala-Thr-OH

mutant :
-Ser-Ser-Phe-Glu-Ser-Ser-Ser-Gly-Leu-Val-Try-Thr-Ser-Gly-Lez<-
Ala-Thr-OH

Fig. 18.

In the mutant, one proline has been replaced by one leucine, the rest of the
sequence remaining unchanged. It would seem that deamination of a base
in the virus RNA has caused the substitution of one amino acid for another
at a specified point within the polypeptide sequence. This is strikingly
similar to the modifications caused in human haemoglobin by spontaneous
mutations. Just as human genetic material contains information relevant
to the amino acid sequence of haemoglobin, virus RNA contains informa-
tion which controls the amino acid sequence in viral protein. This informa-
tion is modified when an amino group of certain bases within the RNA
is replaced by a keto group.

Both DNA and RNA can be visualized as long strands of undifferentiated
material, the phosphate-carbohydrate backbone, upon which purines and
pyrimidines of a few different types (usually 4 different types) are fixed
at regular intervals. The sequence of these constitutes genetic information,

GENETIC CONTROL 35

just as proper linear sequences of letters on a white sheet constitute written
language.

C. THE COLINEARITY HYPOTHESIS
1. Principle

Results reviewed in the preceding pages show that the genetic material
controls the nature and the position of individual amino acids in the
protein polypeptide chains. Since nucleic acids as well as proteins are
linear polymers and since nucleic acids differ from one another by the
arrangement of the bases just as polypeptides differ by the order of their
amino acids, it seems logical to assume a point to point correspondence
between the two linear sequences.

To test this hypothesis directly, one might think of isolating a specified
protein and the corresponding piece of DNA. Determination of the com-
plete amino acids sequence in the protein and of the nucleotides sequence
in the DNA molecule would tell to what extent and in which way they
are correlated.

One-half of such an experiment is feasible presently. The complete
sequence of amino acids has been worked out for insulin, for several
pituitary hormones, and for ribonuclease (Spackman et al., 1960). Partial
sequences are known for several other proteins. With skill and patience, the
structure of a polypeptide can now be resolved by the methods developed
by Sanger and his followers. But the other half of the experiment seems
hopeless. No ways are known to recognize by physicochemical means the
piece of DNA corresponding to a given protein and to separate it from the
rest of the macromolecules. No methods are available either for determining
the sequence of nucleotides in a polymer containing more than a few
elements.

However, although the fine chemical structure of a nucleic acid is still
beyond our reach, genetic analysis can locate with extreme accuracy muta-
tion points within a genetic locus. It should be possible then to isolate
several mutants of the locus corresponding to a given enzyme and to locate
the mutation points within the locus by genetic analysis. The abnormal
proteins corresponding to each mutant could be isolated and their struc-
tures compared. If the hypothesis is correct, the locations of the changes in
the amino acid sequence should be correlated with the positions of the
mutations within the gene.

Such an experiment will involve a considerable amount of skilled work.
It looks feasible with a suitable material : a small protein molecule easy to
isolate, produced by a micro-organism in which mapping can be performed
down to very narrow regions of the genome.

A few laboratories are at present endeavouring to do such experiments,
the results of which will be watched with great curiosity (Levinthal, 1959;
Garen, 1960).

36 THE BIOSYNTHESIS OF PROTEINS

2. Coding Problems

Although the colinearity principle is nothing more than pure hypothesis,
it looks so logical that several studies have already been devoted to the type
of correlations which might exist between the arrangement of the nucleo-
tides in DNA and the amino acid sequence in a polypeptide.

The problem is to discover and to decipher the language in which
genetic information is written. These studies are based on abstract argu-
ment and often resemble mathematical recreations. But some of these are
very ingenious and they may be fruitful if their conclusions are amenable
to experimental test or if they suggest clear experiments.

Since there are only (essentially) 4 different nucleotides for controlling
the arrangement of 20 amino acids, it is obvious that individual amino
acids cannot correspond to individual nucleotides ; they might correspond
to groups of nucleotides. There are 16 possible oriented pairs of nucleotides,
which is not enough either. The number of different sequences of three
nucleotides is 64, which is much more than what is needed.

Gamow (1954) proposed a coding system in which triplets of nucleotides
in a double helix of DNA would correspond to individual amino acids.
These triplets are overlapping, i.e. each nucleotide is part of 3 triplets and
concerns therefore 3 amino acids.

Crick et al. (1957) considered another coding principle also involving
partly overlapping triplets. Such systems would make it possible to have
about the same number («+2) of nucleotides in the DNA chain as there
are amino acids in the corresponding polypeptide. But the overlapping
triplets would impose restrictions on the possible sequences of amino acids.

Systems have also been proposed in which only a few amino acids, e.g.
the aromatic amino acids, are controlled by the gene (Schwartz, 1955),
whereas the sequences in between are not.

The most ingenious system presented so far is the 'code without commas'
(Crick et al., 1957; Crick, 1957). It is assumed that a sequence of three
nucleotides in the DNA chain corresponds to a single amino acid and that
the trios of nucleotides are contiguous in the chain, but non-overlapping.
Since in DNA thousands of nucleotides follov/ each other in a regular
linear sequence, each occupies equivalent positions in the chain. A diffi-
culty therefore arises for reading the information. One does not know how
to cut the series of nucleotides into trios. A way out of this difficulty is to
further assume that certain trios 'make sense', i.e. correspond to an amino
acid, whereas others do not, just as certain groups of letters make a word
and others are meaningless. The information will be readable 'without
commas' if each triplet which makes sense can only produce with its neigh-
bours overlapping triplets which are meaningless. A fascinating feature of
this system is that the maximum number of trios which fulfil these con-
ditions is exactly 20, i.e. exactly the number of the amino acids species to

GENETIC CONTROL 37

be arranged. There are many different languages operating on this principle,
and several types of comma-less codes have been examined (Freudenthal,
1958; Golomb et al., 1958). Such systems could code any sequence and
would impose no constraints or restrictions upon the sequence of amino
acids. But strong restrictions would exist in the arrangement of the
nucleotides in DNA.

It should not be forgotten that this is pure speculation. If taken too
seriously these entertaining schemes might obscure the situation rather
than to clarify it: the more clever they look, the greater is the danger that
they might blind us to reality. On the other hand schemes of this sort will
help to discuss and analyse the experimental data which will soon be
obtained.

Data relevant to the coding problem already emerge from the compara-
tive study of the fine structure of proteins. Thus Brenner (1957) showed
that among the sequences of amino acids known to exist in proteins, there
are more possibilities than an all overlapping code could allow (Gamow
et al., 1956). Morowitz (1959), studying the amino acids which are found
in the protein of E. coli next to a given amino acid, concluded that there are
no constraints as to the nature of the amino acid which is contiguous to
glutamic or aspartic acid or which precedes arginine or lysine. But leucine
or isoleucine are found next to arginine and lysine less frequently than
would be predicted from equally probable distribution (Morowitz and
Barra, 1959). These limited constraints may or may not be due to the
coding system.

Comparison of abnormal haemoglobins in man shows that in mutations
which result in the production of Hb S or Hb C instead of Hb A, one
glutamic acid is replaced by valine or lysine respectively, and the contigu-
ous amino acids are not affected. In the mutation of Hb A to Hb G, the
amino acid which is replaced is the one next to the glutamic acid residue
which was replaced in the mutation of Hb A to Hb S or Hb C. These facts
are not compatible with an overlapping code; they show that the actual
system allows for a large degree of independence of contiguous amino
acids.

In haemoglobin again, single mutations can cause the replacement of
glutamic acid by either valine, lysine or glycine (see Fig. 11). This suggests
that the ciphers for these four amino acids differ only slightly from one
another, perhaps by one nucleotide in each case. The number of different
substitutions of one given amino acid, e.g. glutamic acid, by another as a
result of single mutations might also be a test for certain coding systems ;
for instance, in the 'code without commas' the number of possible substitu-
tions which still 'make sense' is very limited (Levinthal, 1959). The dis-
covery of Tsugita and Fraenkel-Conrat (1960) that a mutation brought
about by deamination in virus RNA can cause the substitution of leucine

38 THE BIOSYNTHESIS OF PROTEINS

for proline in the polypeptide also suggests that the coding sequences on
RNA for leucine and proline are closely related.

Data which may be of crucial significance for the coding problem have
been obtained from comparative studies on DNA. Two DNA species of
slightly different densities can be separated by sedimentation equilibrium
in a gradient of caesium chloride (Meselson et al., 1957). Sueoka et al.
(1959) found it possible to separate by this method DNA preparations
difi^ering in their A-T/G-C ratio. It was observed that DNAs from
various bacteria differ very much from one another in their base composi-
tion but that within one species the characteristic composition is main-
tained even within rather small fragments of DNA (Rolfe and Meselson,
1959). If, as it would seem, the gross composition of the proteins is not
very different between the various species, the very marked differences in
DNA composition would indicate that the code is not the same for all the
bacterial species. Another possibility is that the code does not use 4 letters
but only 2. It is conceivable for instance that as far as coding is concerned
the important matter is the presence of either NH2 or OH in position 6 of
the purines or the corresponding position of the pyrimidines; adenine and
cytosine would then have the same meaning in terms of amino acid
(Sinsheimer, 1959). Vielmetter and Schuster (1960) were able to deaminate
preferentially the individual amino bases of the DNA of T2 phage. They
came to the conclusion that deamination of adenine and hydroxymethyl-
cytosine can cause mutations, whereas deamination of guanine can be
lethal but does not cause mutations. It is striking that the NH2 of guanine
is in position 2 whereas that of adenine and of cytosine is in position 6. This
indicates that substitution of a keto group for an animo group in position 6
changes the information, and since deamination of adenine gives hypoxan-
thine which is not found in DNA, this strongly suggests indeed that the
information might be written in a two-digit system : 6-keto or 6-amino. In
such a case, each coding unit should contain 5 nucleotides for coding one
amino acid.

Another question which can be raised in connexion with coding, is
whether there are enough nucleotides in a genetic locus to code for all the
individual amino acids of a protein molecule. Benzer tried to translate the
distances between mutation sites, as derived from genetic recombination
experiments, into number of nucleotides in DNA. He found that the ele-
ments separable by recombination in bacteriophage T4 might be as small
as two nucleotide pairs, and that a 'cistron' might contain of the order of
400 nucleotide pairs. Pontecorvo and Roper (1956) made similar estima-
tions for several loci of Aspergillus and of Drosophila, and they found, in
each case, values ranging from 1000 to 8000 nucleotide pairs. The cal-
culation involves assumptions which cannot be completely checked at
present, and the results must of course be considered a rough estimate.

GENETIC CONTROL 39

If 3 or 5 nucleotide pairs* are required for coding each amino acid, a
locus seems to be large enough for controlling the arrangement of 200-2000
amino acids. This corresponds to polypeptides weighing 20,000 to 200,000,
which is in the range of molecular weight of proteins. In the light of these
estimations, it does not appear unreasonable to assume that each amino
acid in a polypeptide might be controlled genetically. The gene might thus
contain a complete blue print of the amino acid sequence, and control all
the details of the primary structure of the corresponding protein.

* One nucleotide pair in the double helix does not carry more information than
one single nucleotide in a single strand.

CHAPTER II

The Sites of Protein Formation within
the Living Cell

A. EARLY CYTOCHEMICAL DATA

The first data on the sites of protein synthesis in the cell emerged from
cytochemical studies by Brachet and by Caspersson. Brachet (1933) had
observed that the variations of DNA content during the development of
the sea urchin egg do not parallel the changes of 'nucleic phosphorus'. This
called his attention upon the possible existence of other nucleic acids beside
DNA in this material. But at the time, there was no method available for
detecting or for determining ribosenucleic acids. Plant nucleic acid, as
RNA was then usually named, was regarded as a biochemical curiosity,
so rich in phosphate that it was probably a phosphate storage form, which
occurs in wheat germ, in yeast, and curiously enough also in pancreas.
A few years later, the purification and isolation of pancreatic ribonuclease
(Kunitz, 1940) provided a means of destroying RNA specifically. Making
use of this new tool, Brachet tried to see which structures or regions of
animal cells would be affected by ribonuclease. Brachet (1941) established
that the basophilic substance of the cytoplasm of animal tissues is speci-
fically removed by pancreatic ribonuclease. The substance responsible for
cytoplasmic basophilia was thus clearly identified as RNA, and at the same
time a very simple method for the detection and localization of RNA in
tissue sections was introduced. A screening of animal tissues by this
technique demonstrated the presence of RNA in all types of cells and
showed that RNA is responsible for the basophilia of ergastoplasm and
nucleoli. During cell division, RNA is also found in chromosomes and in
the spindle. But by far the largest amount of RNA is in the cytoplasm.

Caspersson, on the other hand, had developed a microspectrophoto-
meter with which he was able to measure light transmission at selected wave
lengths in the ultraviolet on small regions of a cell. With this apparatus,
Caspersson (1941) observed that the cytoplasm of animal cells contains
substances which strongly absorb ultraviolet light, with an absorption
spectrum similar to that of nucleic acid. Since well-known cytochemical
tests indicated that DNA was not in the cytoplasm, it was inferred that the
cytoplasm contains RNA. Later, the combined use of basic dyes, ultra-

40

SITES WITHIN THE CELL 41

violet absorption and ribonuclease test definitely confirmed that RNA is
responsible for both the basophilia and the ultraviolet absorbancy of the
cytoplasm (Gersh and Bodian, 1943; Davidson and Waymouth, 1946).

It had been known for a long time that the intensity of basophilia varies
considerably from one type of cell to another. Chemical determination of
RNA on various tissues confirmed that basophilia and gross content in
RNA go together. Animal tissues can be arranged in the following way
according to decreasing basophilia or RNA content : pancreas > intestinal
and gastric mucosa > liver > spleen > lymph nodes, testis > kidney,
muscle, heart, lung (Brachet, 1941b; Davidson and Waymouth, 1946).
Brachet (1941) and Caspersson (1941) both called the attention upon the
fact that the cells which contain large amounts of RNA are cells which pro-
duce large amounts of proteins. Systematic studies on specialized cells which
are making protein, like silk gland or frog oocytes at the time of synthesis
of yolk platelets, confirmed this rule which suggested that somehow RNA
must be involved in protein synthesis. This hypothesis exerted a very
stimulating effect on research in cytochemistry and biochemistry, and much
of the development of RNA studies in the past twenty years is due to the
recognition of a link between RNA and protein synthesis. An immediate
consequence of this idea was to suspect that the ergastoplasm is the main
site of protein synthesis.

B. FRACTIONATION OF CELL ORGANELLES

1 . Basic Observations

The work of Claude (1939) on the purification of the agent of Rous
papilloma led him to isolate from chick embryos ultramicroscopic particles
which contained RNA, lipid and protein (Claude, 1940). A survey of many
different organs of vertebrates and invertebrates, as well as plants and yeast
showed that particles comparable to those isolated by Claude are present in
all kinds of cells (Claude, 1943, 1944, 1946; Brachet and Jeener, 1944). It
was further established in these researches that the bulk of cellular RNA is
associated with these small particles. When animal tissues are homogenized
in dilute salt solution, as was commonly done in these earlier works, a great
variety of particles of different sizes are obtained; all of them seem to
contain RNA and the smaller the particles, the greater is their RNA con-
tent (Chantrenne, 1947). The fractionation procedures used at the time
were not very satisfactory. None of the well-known cell constituents, except
damaged nuclei, could be recognized in the homogenates and no clear
connexions were established between the fractions obtained by centrifuga-
tion and cell structures. A great progress was made when it was observed

42 THE BIOSYNTHESIS OF PROTEINS

that high sucrose concentrations in the extracting medium protect osmotic-
ally sensitive structures which would swell and be disrupted if transferred
into dilute salt solutions (Claude, 1946; Hogeboom et al, 1948). Under
appropriate conditions it was possible to isolate from liver homogenates
beside the nuclei, large particles which were easily identified as the
mitochondria by their shape and their staining properties. From the
supernatant, a clearly difi"erent particulate fraction was obtained; it was
made of smaller particles or vesicles (Porter et ah, 1945) of about 200 m/x
in diameter which were called microsomes. From then on, it became a
common usage to fractionate tissue homogenates by centrifugation into
four fractions, the nuclear, mitochondrial and microsomal fractions, and a
supernatant containing the substances which are not sedimentable in one
hour in the Spinco centrifuge.*

The microsomal fraction contained most of the RNA of the homogenate.
As histological studies had shown that the bulk of cellular RNA is in the
ergastoplasm or cytoplasmic ground substance, it was clear that the
microsomal fraction was derived, for a large part at least, from the cyto-
plasm. High speed centrifugation of intact liver tissue indeed confirmed
that RNA was bound to a sedimentable structure in the cell (Chantrenne,
1943; Claude, 1943 ; Brachet and Jeener, 1944; Brenner, 1947).

Fractionation of cell particulates and the availability of labelled amino
acids opened a new approach to the study of the sites of protein formation
within the living cell.

Several laboratories undertook kinetic studies on the incorporation of
labelled amino acids into the proteins of fractions isolated by centrifuga-
tion. Labelled amino acids were injected into rats ; the animals were killed
15-60 min after the injection, the liver was rapidly removed, chilled,
homogenized and the suspension was separated into nuclear, mitochondrial,
microsomal and supernatant fractions. Labelled amino acids were looked
for and determined in the proteins of these fractions. In such experiments,
Borsook et al (1950), Hultin (1950), Lee et al. (1951, 1953), Tyner et al.
(1953), Khesin (1954) established that all the fractions incorporate amino
acids, but that liver microsomes are labelled more intensively than any
of the other fractions. Allfrey et al. (1953) obtained similar results with
mouse pancreas.

More striking were later experiments in which the liver was chilled and
fractionated a few minutes after the injection of labelled amino acids
(Keller et al, 1954; Hultin, 1955 ; Loftfield, 1957). For the first ten minutes

* It should not be overlooked that these are extremely crude fractions which are
not made of pure nuclei, pure mitochondria or pure microsomes. Moreover, con-
ditions of fractionation which have been devised for rat liver, for instance, do not
necessarily apply to other tissues. (For a discussion of fractionation procedures, see
de Duve and Berthet, 1954.)

SITES WITHIN THE CELL 43

after injection of labelled alanine, leucine or valine, the specific activity of
these amino acids in microsomal proteins was four times as high as in the
supernatant proteins and eight times as high as in mitochondrial proteins.
Quite similar results were obtained with plant tissues (Stephenson et al.,
1956). Obviously, the microsomal fraction derives from cellular structures
which are more actively engaged in protein synthesis than the rest of the cell.

Several workers later succeeded in subfractionating the microsomal
fraction into components which have different metabolic activities. By
treating microsome preparations with deoxycholate, Littlefield et al.
(1955) separated a nucleoprotein containing most of the RNA and a frac-
tion made of protein and lipid. AXXirey'et al. (1953), Daly et al. (1955) used
ribonuclease and salt extraction to obtain microsomal subfractions, Hultin
used extraction and reprecipitation with salt solutions. By a similar but
somewhat more elaborate procedure, Simkin and Work (1957) separated
several protein and nucleoprotein fractions. In every case, the most rapid
incorporation of amino acids is observed in a protein material which re-
mains closely associated with RNA. Moreover, during incorporation of
labelled amino acids, the specific activity of the ribonucleoproteins very
rapidly reaches a plateau whereas that of the other protein fractions keeps
increasing slowly (Fig. 19).

If a tracer amount of radioactive amino acid is injected so as to present
the tissue with a highly radioactive precursor for a very short period only,
followed by dilution of the tracer, the specific activity of the ribonucleo-
protein particle rises abruptly during the first few minutes and then
decreases. To the contrary, the specific activity of the other microsomal
subfractions (e.g. the lipoproteins) and of the soluble fraction keeps increas-
ing for a rather long time. This is, qualitatively at least, what one would
expect if a microsomal ribonucleoprotein was an obliged intermediary
stage through which other protein fractions must pass (Littlefield et al.,
1955; Littlefield and Keller, 1957). Very clear evidence for a microsomal
ribonucleoprotein intermediate in the synthesis of globin by rabbit
reticulocyte was also presented by Rabinowitz and Olson (1956, 1959) and
by Kruh et al. (1960). Similarly, serumalbumin appears first in liver
ribonucleoproteins (Peters, 1957, 1959; Takanami, 1960) and it is later
released as soluble protein. Antibodies are first detected in the particles of
lymph nodes (Kern et ah, 1959).

Soluble proteins have often been found in a hidden form in the ribo-
nucleoprotein particles of the tissue which produces them. Earlier observa-
tions of this type were made by Jeener and Brachet (1944) for haemo-
globin in the 'small particles' of bone marrow cells and for the meianophore
expanding hormone in particles from hypophysis. More recently, Peters
(1957) and Elson (1958, 1959) found that newly made proteins can be set
free by destruction of the particles and Feldman et al. (1960) separated

44

THE BIOSYNTHESIS OF PROTEINS

antibody from ribonucleoprotein particles of lymph nodes or spleen of
immunized animals, by destroying the RNA with ribonuclease.

All the experiments reviewed above point to the microsomes and more
especially to the ribonucleoprotein they contain as important centres of
synthesis of soluble cell proteins within the living cell. It must be em-
phasized that 'microsome' is not the name of any intact cell structure. This
term simply refers to a certain type of small particles which can be separated
from cell homogenates by high speed centrifugation in an appropriate
medium. Microsome pellets are jelly-like, transparent and slightly coloured.
When redispersed in aqueous medium they form an opalescent suspension
in which regular light microscopy or even phase contrast does not reveal

700

P

iipoprotein/'

/

-o

400
300
200

r

y

/ ribo

osome-|
nucleopr

otein

/

/

/Soluble

protein

of cell

-0

^

^^

(a)

20

5 10 15 20 25 5 10 15

Time, min Time, min

Fig. 19. In vivo incorporation of I'^C leucine into microsomes of rat liver
fractionated with sodium deoxycholate.

(a) Saturating dose of ^'^C leucine.

(b) Tracer dose of ^'^C leucine.
(Littlefield et al., 1955).

any structure. Dark field examination shows a great number of particles
animated with intense brownian motion.

No such particles can be observed in living cells and it has long been
suspected that microsomes form after disruption of the cell, during the
dispersion of the cell contents in the extracting medium (Brachet and
Jeener, 1944; Claude, 1947; Porter, 1953). The fact that they contain most
of the cellular RNA indicated that microsomes originate from the ergasto-
plasm, also called cytoplasmic ground substance.

Thanks to the development of electronmicroscopy of ultrathin tissue
sections (Porter and Blum, 1953; Sjostrand, 1953), knowledge of the
structure of cytoplasm made great progress during the last few years. A
network of lamellar formations was observed in the ground substance of

SITES WITHIN THE CELL 45

many various cells ; it is especially developed in secretory cells like liver or
pancreas. This structure was described as a system of double lamellae
(Sjostrand and Hanzon, 1954; Bernhard^f a/., 1954)orof elongated vacuoles
forming a reticulum (Porter, 1953). Palade and Porter (1954) observed that
the membranes of the reticulum are lined with small granules of a rather
uniform size (100-150 A in diameter). The number of these granules
seems to be correlated with the basophilia, i.e. the RNA content of the
tissue.

By electronmicroscopy, Slautterback (1953) observed that isolated mouse
liver microsomes contain essentially two types of structures : rather large
formations of about 0-1 /i, and smaller dense particles associated with the
larger ones. Better pictures of microsomal fractions isolated from rat liver
showed that they were made of more or less swollen vesicles of various
sizes (Kuff et al, 1956) corresponding probably to the larger of Slautter-
back's particles. Littlefield et al. (1955) examined microsomal preparations
with the electron microscope before and after deoxycholate treatment.
Although the pictures they obtained were rather poor, it was clear that
deoxycholate treatment destroys or dissolves the larger components and
leaves the smaller particles. Parallel centrifugation experiments indicated
that the smaller particles were the ribonucleoproteins which had been
shown to be the most active in protein synthesis. Excellent systematic
studies by Palade and Siekevitz (1956), in which centrifugation, electron
microscopy and biochemical methods were combined, definitely estab-
lished that the so-called microsomal fraction of liver results from the dis-
ruption and dispersion of the ergastoplasm or endoplasmic reticulum into
the homogenization medium. The trabeculae and cisternae observed on
sections of osmic acid fixed tissue are found in the homogenates as vesicles
in the microsomal fraction. These vesicles react like osmometers to changes
of sucrose or salt concentration, and they dissolve in deoxycholate. The
Palade granules which line the lamellae of the reticulum do not dissolve ;
these are the ribonucleoprotein particles, most of which are normally
attached to the lamellae in vivo. It is possible, however, that the mem-
branes of the microsomal vesicles also contain some RNA (Moule et al.,
1960).

In guinea pig pancreas, the picture is similar, except that many free
ribonucleoprotein particles are observed between the lamellae, outside the
cisternae. Very large dense granules are seen within these cavities; these
are secretory granules at different stages of formation, i.e. packages of
zymogens which are the main proteins produced and excreted by the
pancreas (Siekevitz and Palade, 1958). In kinetic experiments in which
labelled amino acids are injected to the animal, the ribonucleoprotein
particles are the first to contain labelled polypeptides and also labelled
zymogens. Siekevitz and Palade (1958) have very good evidence that in

46 THE BIOSYNTHESIS OF PROTEINS

the pancreas the finished proteins arise in the ribonucleoprotein particles
attached on the outer surface of the cisternae, and that these proteins pile
up as zymogen granules inside the cisternae (Siekevitz, 1959; Hirsch,
1960). The changes in intracellular distribution of pancreatic amylase
during the secretion cycle observed by Laird and Barton (1958) are in
agreement with this picture of the secretory process.

The ergastoplasm of secretory cells can be visualized as a highly organ-
ized system for the manufacture, storage and delivery of the protein
secretion.

It is important to realize that ribonucleoprotein particles are only one of
the constituents of microsomia! fractions, and of the ergatoplasm. In order
to avoid a very frequent confusion between 'microsomes' and 'ribonucleo-
protein particles', it has been proposed to reserve the name 'ribosomes'
for the discrete ribonucleoprotein particles (Roberts, 1958). This is the
more justified in that ribosomes seem to be a fundamental constituent of all
cells, whereas the structure of the ergastoplasm and the properties of the
'microsomal fraction' in vitro differ considerably from tissue to tissue, and
are not found in bacteria.

2. Gefieral Occurrence of Ribosomes

Electronmicroscopical studies on some forty types of mammalian tissues
(Palade, 1955) revealed the presence of these dense granules in all the cells
examined except mature red blood cells of the rat which, incidentally,
make no protein. Part of the ribosomes are associated with the ergasto-
plasmic lamellae, but part are free. The distribution of ribosomes is largely
similar to that of cytoplasmic basophilia, i.e. to the distribution of RNA.
As the size and composition of the ribosomes is very uniform, the general
relationship between the amount of RNA and the intensity of protein
synthesis, which was pointed out by Brachet and by Caspersson, can now
be expressed in newer terms as follows: In animal tissues, the intensity of
protein synthesis is related to the number of ribosomes. These are sites of
protein formation.

Nucleoprotein particles similar to the Palade granules have been
isolated from the cytoplasm of plant tissues (Ts'O et ai, 1956; Ts'O, 1958)
and from micro-organisms.

The association of part of the RNA of yeast with small particles has been
known for a long time (H. Chantrenne, 1943, 1944; Schachman et al.,
1952). Yeast ribosomes were purified and studied more recently by physico-
chemical methods (Chao and Schachman, 1956). They make up a popula-
tion of nearly spherical particles with a sedimentation constant of about
80 S and a molecular weight in the range of 4-10^. About 40 per cent is
accounted for by RNA, the rest is protein. The stability of the particles
depends very much on salt concentration, and especially of the presence of

ir-:

Fig. 20. A. Section through a guinea pig pancreatic cell showing a few
mitochondria with their inner cristae, and the endoplasmic reticulum
lined with ribosomes. Free ribosomes can be seen between the cisternae.
The large intracisternal granules are the secretory products. Magnifica-
tion: X 22,000 (courtesy Dr G. E. Palade).

VHP

VHp ^^

Fig. 20. B. Microsomes from the guinea pig pancreas, showing vesicles
and ribosomes. Magnification: x 56,000 (courtesy Dr G. E. Palade).

Fig. 20. C. Rat liver ribosomes detached from microsomes by deoxy-
cholate treatment. Magnification: ■ 84,000 (Kirsch et al. 1960) (courtesy
Dr G. E. Palade).

SITES WITHIN THE CELL 47

magnesium ions. At low magnesium concentrations, the particles dissociate
into nucleoprotein fragments (Chao, 1957),

Yeast and moulds contain nuclei, mitochondria and ribosomes which
resemble those of higher organisms (Yotsuyanagi, 1955, 1956; Shatkine
and Tatum, 1959; Blondel and Turian, 1960). In bacteria, no mitochon-
dria have been recognized, the nuclear apparatus is not very sharply
separated from the surrounding material, no nuclear membrane and
no structure resembling the endoplasmic reticulum has been found.
However, electron microscopy on thin sections of bacteria shows a very
dense population of particles which resemble the ribosomes of animal cells
(Bradfield, 1956). In bacterial extracts, up to 85 per cent of the RNA is
associated with sedimentable particles (Schachman et ah, 1952). Much
attention is now being paid to these long neglected bacterial particles
(see Roberts, 1958).

The ribosomes isolated from Azotohacter vinelandii (Gillchriest and
Bock, 1958) or from E. colt (Bolton et al, 1958; Wagman and Trawick,
1958; Tissieres et al., 1959; Spahr and Tissieres, 1959) are very similar to
the yeast particles. Ribosomes from liver and from micro-organisms are
quite comparable in size range, composition, response to magnesium ion
concentration and behaviour in the ultracentrifuge (Hall and Doty, 1958;
Peterman et ah, 1958). Cheng (1957) remarked that the RNA content of a
ribosome of rat liver, pea seedlings and yeast as well, can be estimated as
equal to l-7-lO^ as expressed in molecular weight units. The same figure
was later found for certain ribosomes of E. coli (Tissieres and Watson,
1958), tobacco plant (Gierer, 1958) and mouse brain (Cheng, 1960). It
would seem therefore that a class of ribosomes might all contain about the
same absolute amount of RNA. Another striking coincidence is that the
ribonucleoprotein viruses so far studied in this respect also contain one
RNA molecule v/ith a molecular weight of about 2-10^ per virus particle.
One should probably not give too much weight to a coincidence of numbers
which may still be fortuitous. The more so, in that ribosomes can dissociate
into smaller particles (Elson and Tal, 1959; Hall and Slayter, 1959;
McCarthy, 1960; Huxley and Zubay, 1960) and sometimes constitute a
physiologically heterogeneous population (Siekevitz and Palade, 1959).
Nevertheless, Chen's observations raise the question of the existence of a
RNA 'quantum' and make one suspect similarities of structure and func-
tion between ribosome RNA and virus RNA.

In animal cells, the ribosomes of the cytoplasmic ground substance are
certainly an important part of the protein-making system, they are most
probably the sites where polypeptides arise. Their function in bacteria was
more difficult to ascertain. The total amount of ribosomes is greatest when
the bacteria are in the logarithmic phase of growth (Mendelsohn and
Tissieres, 1959). But no correlation was found between the concentration

48 THE BIOSYNTHESIS OF PROTEINS

of the 40 S compound and enzyme synthesis (Wade and Morgan, 1957;
Bowen et al., 1959). Protein synthesis is so fast in growing bacteria
(Roberts, 1957) that detailed kinetic studies on the incorporation of amino
acids into bacterial fractions comparable to the studies which were so
successful with rat liver, at first failed to show any difference in rate of
protein synthesis between ribosomal and soluble proteins, and many
workers thought that ribosomes in bacteria are of no great interest and
even that their RNA might be a waste product. Finally, McQuillen et al.
(1959) clearly estabhshed that when radioactive methionine is added to
exponentially growing E. coli, the radioactivity of the 80 S ribosomes is
buih up to saturation within 5 sec, perhaps less, and that it disappears
equally rapidly when the tracer amino acid is diluted out by addition of
non-labelled methionine. The lost radioactivity appears in soluble proteins.
The substance which is rapidly labelled in the microsome has the chemical
properties of a polypeptide and it behaves like a precursor of soluble
protein. Beside this transient protein which is continuously chased by
nascent molecules, another ribosome protein forms more slowly; it is a
permanent constituent of the ribosome structure the synthesis of which
reflects ribosome formation in the growing bacterial population.

Ribosomes can now be regarded as an essential piece of the protein
forming system in bacteria and in the cytoplasm of mould, plant and
animal cells. It is quite possible that no protein synthesis takes place except
on ribosomes, for particles which behave like ribosomes have been isolated
from nuclei (Frenster et al., 1960), and there are indications of their being
responsible for protein synthesis in these organelles as well. The existence
of ribosomes within mitochondria, however, is not quite clearly established,
although mitochondria do make proteins (see p. 53).

C. PROTEIN SYNTHESIS IN ISOLATED CELL
FRAGMENTS AND ORGANELLES

1. Protein Synthesis in Enucleate Cytoplasm

It is clear from the experiments which have been reported above that
the cytoplasmic ground substance is the main site where proteins arise in
most animal cells. On the other hand, it is well established that the primary
structure of proteins is controlled by the nuclear genes. One then wonders
how immediate is the nuclear control upon protein synthesis.

In all the in vivo experiments that have been considered so far, protein
synthesis took place in the intact cell ; and even though the cytoplasm was
the first site of protein appearance, the nucleus was always present, and one
may wonder to what extent it was involved in the process. This problem

SITES WITHIN THE CELL 49

could be partly solved by studying protein synthesis in certain types of cells
which can easily be cut into two parts, one of which is devoid of nucleus.
The enucleate fragments survive for some time and it is possible to
establish directly whether protein synthesis can take place in the absence of
a nucleus.

Amoeba proteus was used by Brachet (1955) and by Mazia and Prescott
(1955) for such experiments. The nucleus of this Amoeba is usually located
in the rear part of the cell when the animal is moving about on a glass
surface. When the cell is cut into two approximately equal parts by means
of a thin glass rod, the fragment which contains the nucleus retains all
the morphological characters and the habits of a normal Amoeba. Nucleate
fragments thrust out pseudopodia, crawl about and catch ciliates or other
prey upon which they feed. To the contrary, enucleate moieties are rather
inert; they assume a nearly spherical shape shortly after sectioning, lose
the ability to form pseudopodia and to secure food. Actually, enucleate
amoeba fragments are always starved; they survive nevertheless for 10-14
days, before collapsing. In all the experiments with Amoeba fragments, the
nucleate part to which the enucleate halves are compared must naturally
also be deprived of food.

Starvation is definitely not favourable to protein synthesis. In spite
of this, it was possible to obtain very interesting information on the
presently discussed question with this material. As can be expected, the
total amount of protein decreases in starved Amoeba. Brachet (1955)
observed that total protein diminishes more rapidly in enucleate fragments
than in starved whole Amoeba or in starved nucleate fragments. By follow-
ing, in the course of time, the changes in several individual enzymes in
both types of fragments, Brachet showed that the removal of the nucleus
results in widely difi'erent effects in the case in the individual enzymes
studied.

Thus an acid phosphatase, a dipeptidase and an esterase decreased much
faster in enucleate fragments than in nucleate parts, but other enzymes,
like a protease, enolase, adenosine triphosphatase and the respiratory
system were not more labile in the absence of the nucleus than in its
presence during starvation. It would seem therefore that the maintenance
of the individual cytoplasmic proteins is not equally dependent on the
presence of the nucleus (Brachet, 1955, 1956, 1957).

Although a net decrease of protein material is always observed in starved
Amoebae, some anabolic processes can nevertheless be observed. Mazia and
Prescott (1955), Mazia (1956), Ficq (1955) and Brachet and Ficq (1956)
showed that labelled methionine or phenylalanine are incorporated into
cytoplasmic proteins. The incorporation is 50 per cent lower in enucleate
fragments than in nucleate moieties or in intact Amoebae. However re-
duced, the incorporating capacity is maintained for up to 10 days after

50 THE BIOSYNTHESIS OF PROTEINS

enucleation. This clearly indicates that the cytoplasm contains a complete
system for incorporating amino acids into protein material and that the
pathway for this residual incorporation does not go through the
nucleus.

The drop in incorporating activity after enucleation might be due to
indirect effects, irrelevant to a nuclear control of cytoplasmic protein
synthesis, for many changes occur in the general metabolism of the Amoeba
as a result of enucleation. The enucleate part looses, for instance, the
capacity to use its lipid and glycogen stores, and it is heavily handicapped
as against nucleate parts for so basic a function as glycolysis (Brachet, 1955,
1956, 1957; Brachet and Chantrenne, 1956; Chantrenne, 1958). On the
other hand, the fact that the maintenance of various proteins is not equally
dependent on the presence of the nucleus makes one suspect that the
cytoplasm can make proteins but that a considerable fraction of the
proteins normally present in the cytoplasm of the amoeba are made under
very close nuclear control and possibly within the nucleus itself.

Another material which is very well suited for merotomy experiments is
the green marine alga Acetahularia mediterranea, which belongs to the class
of Dasycladaceae. During most of its life, the alga consists of a rather thick
filament or stalk attached to the rocks at one extremity by rhizoids. The
stalk can be 1-2 in. long ; it is made of one single cell, the nucleus of which is
always located in one of the rhizoids. At the end of the life cycle, the tip of
the stalk develops into an umbrella. When this is fully grown, the big
nucleus divides into many small nuclei which invade the cytoplasm and
finally reach the umbrella where they become enclosed in the cysts. These
are the resistant form of the organism ; each contains a few nuclei. After
a maturation period, the cysts can germinate and liberate flagellate gametes.
Conjugation of two gametes gives a uninucleate zygote. This grows into
rhizoids and a stalk, which will later develop an umbrella (see e.g. Kiihn,
1955). The complete cycle takes one year in nature and about six months
in the laboratory.

During the part of the life cycle which extends from the formation of the
zygote up to the complete development of the umbrella, Acetahularia is a
single cell ; the nucleus is located in a rhizoid and the cytoplasm is uniformly
filled with chloroplasts. The cell is surrounded by a tough polysaccharide
casing. If the alga is cut across the stem, both fragments recover very well
and can be kept alive for more than two months. The fragment with the
rhizoids contains the nucleus, the other one is purely cytoplasmic. Both
fragments are amply provided with chloroplasts, and photosynthesis is not
impaired by enucleation (Brachet et al., 1955). Extremely interesting ex-
periments were performed on Acetahularia by Hammerling (1934, 1946,
1953) and by his collaborator Beth (1943). If the nucleate part of one
strain is grafted on an enucleate part of another strain of Acetahularia, the

SITES WITHIN THE CELL

51

morphology of the umbrella which develops corresponds to the strain of
the nucleate part. Since the structure of the umbrella is a hereditary char-
acter, it is quite clear that genetic determinants are provided by the
nucleate moiety. This is important for the present discussion.

In his early studies, Hammerling (1934) had already observed that
enucleated fragments can elongate for some time and that the number of
chloroplasts increase. This made it probable that proteins were formed in

adult
cap

Zygote
^;^l^ 2 doys

8 days
Fig. 21. Life cycle of Acetabularia mediterranea (courtesy of J. Brachet).

cytoplasmic fragments. Direct biochemical studies by Brachet and his
collaborators established that the rate of incorporation of i*C02 or of
labelled glycine into the proteins of Acetabularia is not changed in the
enucleate fragment. The rate of incorporation is the same in both halves at
least during the first two weeks after cutting (Brachet and Chantrenne,
1951, 1952; Chantrenne et ah, 1953). The total protein content increases at
the same rate in nucleate as in non-nucleate fragments (Vanderhaeghe,

52 THE BIOSYNTHESIS OF PROTEINS

1954; Brachet etal., 1955; Clauss, 1958). This means that in Acetahularia a.
complete system for making protein material is contained in the cytoplasm
and can operate in the absence of the nucleus.

Studies on non-nucleate fragments of sea urchin eggs (Malkin, 1954)
or of newt eggs (Tiedemann and Tiedemann, 1954) also showed that
various labelled protein precursors can be incorporated into proteins in a
cytoplasm without a nucleus. Rabbit reticulocytes is another example of an
enucleate cell which can incorporate labelled amino acids into its proteins
(London et al., 1950; Borsook et al., 1952; Koritz and Chantrenne, 1954;
Nizet and Lambert, 1953).

However, in all the experiments mentioned above there was no evidence
that the protein material synthesized in the absence of the nucleus was
made of normal protein. Some data on reticulocytes made it probable that
regular haemoglobin was made in these enucleate cells (Hammarsten et al.,
1953). But this important question has been answered only recently in
studies on the net synthesis of specific enzymes in fragments of Aceta-
hularia. Thus enolase is made in the absence of the nucleus at the same rate
as total protein material for at least eleven days after enucleation (Baltus,
1959) and net synthesis of phosphorylase and of invertase goes on at an
almost normal rate for two or three weeks in enucleate cells (Clauss, 1959).
This means that cytoplasmic fragments of Acetahularia contain a perfect afid
complete system for making certain proteins, including the genetic information
required for the synthesis of active enzymes.

This conclusion must, however, be qualified. Protein synthesis is inde-
pendent of the nucleus /or some time only: a change occurs in the non-
nucleate fragments about two weeks after the algae have been cut. Between
the twelfth and the fifteenth days after section, the rate of incorporation of
^'*C02 into the non-nucleate fragments drops to about 30 per cent below
that of the nucleate halves, and net synthesis of proteins stops altogether,
although the enucleate fragments can survive for at least two months there-
after (Brachet and Chantrenne, 1951 ; Vanderhaeghe, 1954; Brachet et al.,
1955; Richter, 1959). Protein synthesis in older algae is more independent
of the nucleus than in younger ones (Hammerling, 1956). It would appear
that some substance produced by the nucleus is required for maintaining
protein synthesis and that this substance is slowly exhausted when the
cytoplasm is deprived of the nucleus. There is, however, no evidence that
this hypothetic substance is a specific gene product: it might be quite a
common biochemical originating in the nucleus (Brachet, 1952, 1954;
Brachet and Chantrenne, 1956).

Moreover, in Acetahularia as well as in Amoeha, the formation of certain
proteins, e.g. acid phosphatase (Keck and Clauss, 1958) appears to depend
more directly on the presence of the nucleus; for it stops shortly after
enucleation, i.e. at a time when the bulk of cytoplasmic proteins and

SITES WITHIN THE CELL 53

enzymes are still being synthesized at a normal pace. Some other species of
algae, e.g. Spirogyra (Van Wysselingh, 1908) or Elodea (Yoshida, 1959)
behave probably very much like Acetabularia; others, like Micrasterias
(Waris, 1951), do not stand enucleation as well.

It remains that enucleation experiments establish that in several of the
cell types examined a large part of the cytoplasmic proteins can be made in
the absence of the nucleus. The cytoplasm must therefore contain centres
of synthesis which can produce perfect protein molecules and are able to
operate in the absence of the nucleus for a considerable time period. This
conclusion has received new support from recent experiments on isolated
mitochondria, which will be examined presently.

2. Protein Synthesis in Isolated Mitochondria

Kinetic studies on amino acid incorporation into cellular components
in vivo pointed to the microsomes as the most active centres of protein
synthesis. In liver, kidney or pancreas, amino acid incorporation into
proteins of the mitochondria represents but a minor fraction of total incor-
poration and for a long time mitochondria did not retain the attention as
sites of protein synthesis. In muscle, however, the picture is different.
Mitochondria come close to microsomes in rate of amino acid incorporation
in vivo. Indeed, for the first five minutes, incorporation into the mito-
chondrial fraction is somewhat higher than in the microsomes (Simpson
and McLean, 1955).

Thanks to studies on oxidative phosphorylation in isolated mitochondria,
conditions have been worked out for isolating and preserving mitochondria
in vitro in relatively good conditions (e.g. Slater and Holton, 1954). In
such preparations from muscle and even from liver, a fairly rapid incorpora-
tion of amino acids into protein material could be observed (McLean et al.,
1958; Campbell and Greengard, 1959; Reiss et al., 1959). Incorporation
was later shown to take place in well defined proteins, like cytochrome-c
(Bates et al., 1958, 1960) and this line of research culminated in the observa-
tion of the net synthesis of cytochrome-c in isolated heart muscle mito-
chondria (Bates and Simpson, 1959). A weighable amount of cytochrome-c
was actually synthesized in vitro in these experiments, and good evidence
was obtained that a synthesis of the protein moiety was taking place in the
process.

This is a very important achievement for several reasons. It shows that
the ergastoplasm is not the only centre of cytoplasmic protein synthesis.
On the other hand, these experiments, like those on enucleate cytoplasm,
show that complete systems for making specific proteins exist in cyto-
plasmic organelles and can accomplish their function without the direct
participation of the nuclear genetic material. Liver or muscle mitochondria
are relatively large elaborate structures and attempts have been made at

54 THE BIOSYNTHESIS OF PROTEINS

locating, among the mitochondrial components, the first centres of protein
synthesis. The results at the time of writing are not quite clearcut. Rendi
(1959, 1960) has isolated from disrupted mitochondria a sedimentable
fraction which contains most of the RNA of the mitochondria and may
have some similarity with ribosomes. On the other hand, Kalf and Simpson
(1959) showed that when mitochondria are exposed to sonic vibrations
followed by differential centrifugation, components which are not sedi-
mented in 8 hrs at 105,000g possess a pronounced capacity to incorporate
amino acids into protein material. The protein synthesizing system in this
supernatant must comprise RNA as an essential part, for ribonuclease
suppresses the amino acid incorporation (see also Kalf et ah, 1959). The
discrepancy between these two results probably reflects differences in
degree of disorganization of the preparations used. Both groups of work,
however, point to some RNA containing element as being again at the
heart of the process of protein synthesis.

3. Protein Synthesis in Chloroplasts

In green plant tissues, much less information has been obtained so far
on the kinetics of amino acid incorporation. Stephenson et al. (1956) and
Sissakian (1957) studied the incorporation of labelled amino acids in vivo
into various cell constituents of tobacco leaves and found that microsomes
are preferentially labelled and contain the bulk of radioactivity for the first
few minutes, but that chloroplasts thereafter incorporate amino acids at a
fairly high rate, as if they were also important centres of protein synthesis.
Plashkov and Ivanko (1956) found the highest in vivo incorporation of
methionine into chloroplasts and mitochondria. Racusen and Hobson
(1959) using ^^002 as the precursor found no significant difference between
the synthesis of chloroplast protein and the other proteins, in swiss chard
leaves.

That chloroplasts and microsomes contain independent centres of
protein synthesis was actually established by earlier data which passed
unnoticed (Chantrenne et al., 1953 ; Brachet et al., 1955). In parallel experi-
ments in which 1^002 and ^^lycine were used as precursors, it was found
that glycine incorporation is more rapid in microsomes than in chloroplasts,
whereas i'*C02 to the contrary is more rapidly incorporated into the
proteins of the chloroplasts than into microsomal protein. This clearly
indicates that amino acids which arise within the chloroplasts as a result
of photosynthetic carbon dioxide fixation can be used for protein synthesis
directly within the chloroplast without going through the microsomes.
Since, on the other hand, an exogenous amino acid is incorporated more
readily in the microsomes than into the chloroplasts, it must be concluded
that microsomes and chloroplasts are two independent sites of protein
synthesis.

SITES WITHIN THE CELL 55

4. Synthesis of Protein in the Cell Nucleus

Cytochemical data, especially observations of amino acid incorporation
into cell protein by autoradiographic techniques, indicated that the nucleus
is not as active a centre of protein synthesis as the ergastoplasm. Neverthe-
less, labelled amino acids were found within a relatively short time in the
nuclear protein ; and in liver and lymph nodes, the in vivo incorporation of
labelled phenylalanine into the nucleus, as judged from radioautography
experiments, represents a considerable fraction of total incorporation
(Ficq and Brachet, 1956; Ficq, 1959; Ficq and Errera, 1959; Gavosto and
Ficq, 1954; Zalokar, 1960b). More recent observations (Zalokar, 1960a)
on Drosophila show that protein synthesis begins a little later in the
nucleus than in the cytoplasm.

The drop in amino acid incorporation following enucleation of Amoeba
(Mazia and Prescott, 1955), the decrease of certain enzymes under the same
conditions (Brachet, 1955-1957) or the passage of ^53 labelled proteins
from a grafted labelled nucleus to the unlabelled nucleus (Goldstein, 1959)
could be interpreted as indications that certain proteins were made in the
nucleus. Daly et al. (1952) showed that certain nuclear proteins of various
mouse tissues incorporate labelled glycine in vivo at a rate comparable with
the average cytoplasmic proteins. Similar results were later obtained by
Smellie et al. (1953) for rat liver. It is not quite clear, however, whether the
labelled proteins found in the nuclei were made within the nuclei, or
whether they originated in the cytoplasm.

AUfrey (1954) succeeded in isolating nuclei of thymus which are able to
incorporate amino acids in vitro into their proteins. Adequate preparations
can be obtained by fractional centrifugation of a calf thymus homogenate
in sucrose solutions. Allfrey et al. (1955, 1956, 1957) thoroughly studied
the requirements for the incorporation of labelled amino acids and estab-
lished convincingly that labelled alanine, glycine and lysine are incorpor-
ated to various extents into several typical nuclear proteins in isolated
thymus nuclei. Radioautography experiments (Ficq and Errera, 1958, 1959)
confirmed that the incorporation (of phenylalanine) really occurs in clean
nuclei and is not due to contamination with remnants of cytoplasm or with
thymocytes which are usually present in small numbers in the prepara-
tions. Since only the L-amino acid isomers are taken up, and since an
amino acid once incorporated into protein is not exchanged with free
amino acids in the medium, it can be taken as most probable that
the incorporation represents true synthesis of nuclear proteins (Allfrey
et al, 1957).

Thymus nuclei were for several years the only type of nuclei with which
incorporation could be observed in vitro. Logan et al. (1959) recently
obtained similar results with isolated nuclei from rat liver. All these
observations make it clear that nuclei contain all that is necessary for

E

56 THE BIOSYNTHESIS OF PROTEINS

incorporating amino acids into proteins and most probably possess a com-
plete system for the synthesis of proteins.

Summing Up

From the results reviewed in the foregoing pages, it is clear that there are
many sites of protein synthesis in the living cell. Nuclei, mitochondria,
chloroplasts make their own protein constituents. In bacteria, the cell mem-
brane appears as a site of intense protein formation, but the ribosomes are
the most actively engaged in protein synthesis. The cytoplasmic ground
substance of higher organisms is the site of synthesis of the bulk of the
proteins of many cells. In the ground substance, protein appears first in the
ribonucleoprotein particles (ribosomes) which are responsible for the pro-
duction of the soluble cytoplasmic proteins. In secretory cells, the ergasto-
plasm has a remarkably elaborate structure which probably plays a part in
the process of secretion and with which many ribosomes are associated.

Ribosomes are in all cases an essential part of the protein-making
machinery.

CHAPTER III

Nucleic Acids and Protein Synthesis

A. THE POSITION OF DNA IN PROTEIN
SYNTHESIS

For reasons which have been considered in Chapter I of the present
book, it is almost certain that DNA ultimately controls the primary struc-
ture of the individual proteins. A question then arises — are the amino
acids arranged in the correct sequence under the immediate action of the
gene, is specific DNA the protein-forming system?

At the present time it is convenient to examine separately the data
obtained with higher organisms and with bacteria.

1. Higher Organisms

In animal or plant tissue, asking whether DNA is directly and 'person-
ally' involved in assembling the proteins amounts to asking whether all
proteins are made in the chromatin. From what we have seen in the pre-
ceding chapters, the answer to this question is definitely negative. Complete
proteins endowed with their normal biochemical properties or enzymic
activity can be made in the absence of the nucleus, e.g. in isolated pieces of
cytoplasm or in isolated mitochondria, which contain no DNA. Most
cytoplasmic proteins are made in the cytoplasm, and DNA is not a consti-
tuent of the system which is immediately involved in their making.

The synthesis of nuclear proteins must be examined more closely.
Fractionation of isolated nuclei after incorporation of amino acids indi-
cated that the rate of labelling differs considerably among the various
nuclear proteins. Histone is almost completely inert, whereas two protein
fractions which are found in close association with DNA and with RNA
respectively incorporate amino acids very rapidly.

In vitro, the incorporation is inhibited and finally suppressed in the
presence of deoxyribonuclease, which destroys DNA. The degree of
impairment becomes greater as more and more of the DNA is depolymer-
ized and removed from the nucleus. However, the amino acid incorporating
ability is not irreversibly destroyed by treatment with deoxyribonuclease,
for it can be largely restored by the addition of a DNA supplement (AUfrey
et al, 1955 ; Mirsky et. al, 1956).

These very striking results at first were taken as direct evidence that

57

58 THE BIOSYNTHESIS OF PROTEINS

DNA is required for protein synthesis in isolated nuclei, and that DNA is
involved personally in the process. However, further data from the same
laboratory (AUfrey and Mirsky, 1957) showed that after inhibition by
deoxyribonuclease, amino acid incorporation into nuclear proteins can be
restored by partially degraded DNA just as well as by native DNA. Even
RNA would restore the amino acid uptake. More strangely still, polyadeny-
lic acid, heparin, chondroitinsulphate and as unnatural a polyanionic
compound as polyethylene sulphonate could restore much of the incorpor-
ating capacity of deoxyribonuclease inactivated nuclei. If polyethylene
sulphonate is added immediately after the depolymerization of DNA has
occurred, it is possible to remove 75 per cent of the DNA without change
in nuclear ability to incorporate amino acids into protein (Allfrey and
Mirsky, 1959).

Moreover, protein synthesis is not the only function of isolated nuclei to
be impaired by deoxyribonuclease; the enzyme deeply damages other
metabolic processes in nuclei and especially oxidative phosphorylation
(Allfrey and Mirsky, 1959). Again, exogeneous DNA, RNA and poly-
anionic substances restore ATP production. The effects on amino acid
incorporation might thus be a mere consequence of the inhibition of
phosphorylation.

The experiments with isolated nuclei cannot be taken any more as
evidence of DNA being directly involved in the synthesis of nuclear
proteins. To the contrary they underline that the presence of intact DNA
is unimportant for the incorporation process, since DNA can be sub-
stituted for by any one of several polyanionic substances including synthe-
tic resins.

It is not known how deoxyribonuclease inhibits phosphorylation. A
possibility is that deoxyribonuclease would bind reversibly some negatively
charged compounds like phospholipids or perhaps some enzyme involved
in the process, and could be displaced by an excess of exogeneous poly-
anionic compounds (cf. Sekiguchi and Sibatani, 1958).

Whatever the explanation of deoxyribonuclease inhibition proves to be,
one must admit that there is at present no evidence that DNA is directly
involved in the synthesis of even nuclear proteins.

2. Bacteria

The most recent studies on DNA formation indicate that at the level
of individual bacteria in exponentially growing cultures the synthesis of
DNA is essentially continuous (Schaechter et al., 1959; Young and Fitz-
James, 1959; McFall and Stent, 1959). Under the usual laboratory con-
ditions, protein synthesis and DNA synthesis thus occur simultaneously
in bacteria. It is possible, however, to dissociate protein synthesis from
DNA formation in a number of different ways. Thymidine starvation of

ROLE OF NUCLEIC ACIDS 59

Lactobacillus acidophilus suppresses DNA synthesis, but the bacteria con-
tinue to make RNA and protein. No division occurs, and the bacteria grow
into long filaments. Under conditions which prevent any measurable
synthesis of DNA, the amount of protein can increase by a factor ten
(Jeener and Jeener, 1952). Similar observations were made later by Cohen
and Earner (1955) with thymidine requiring strains of E. coli. In these
experiments the synthesis of enzymes was studied, and the results clearly
showed that perfect enzymatically active proteins can be made in the
absence of DNA synthesis. Very small doses of ultraviolet light (257 mja)
inhibit DNA synthesis without preventing protein formation (Kelner,
1953; Kanazir and Errera, 1954). High doses of X-rays can abolish DNA
synthesis completely without inhibiting the formation of enzymes, in
bacteria and yeast (Baron et ah, 1953; Chantrenne and Devreux, 1959).
Mustard gas also inhibits DNA synthesis in E. coli, without impairing
j8-galactosidase formation (Pardee, 1954), and the slight inhibition of
protein synthesis by N-mustard, which is observed occasionally, is due to
general toxicity (Clark et al., 1957).

All these data constitute good evidence that the synthesis of DNA is not
necessary for protein synthesis in bacteria. This conclusion is an interesting
one, but more important still would be to establish whether the structural
integrity of the DNA gene is necessary for continued protein production,
since information relative to protein primary structure is contained in
DNA.

When micro-organisms are irradiated with heavy doses of X-rays, their
DNA may be damaged. DNA extracted from yeast which had received
200,000 r was degraded in such a way that it could not be precipitated any
more by acid (Chantrenne, 1958; Chantrenne and Devreux, 1959). This
depolymerization of DNA may not be a direct effect of ionizing radiations;
it may be due to the action of some nucleases released in the cell by mem-
brane breakdown (F. Hutchinson, personal communication). Whatever the
mode of action of the X-rays may be, yeast cells containing largely degraded
DNA were able to produce proteins at a normal rate ; actually the synthesis
was somewhat stimulated.

The first studies on the effect of DNA removal or DNA destruction on
protein synthesis in bacterial systems are due to Gale. Staphylococcus
aureus cells disrupted by supersonic vibrations are able to make enzymes
(Gale and Folkes, 1955). It is possible to remove nucleic acids (both
RNA and DNA) from such preparation by M NaCl extraction at 37°
and the degree of nucleic acids depletion can be controlled to a certain
extent. It was found that removal of the nucleic acids progressively reduces
enzyme production. The synthesis of various enzymes can be restored by
adding RNA if the depletion has been moderate, but DNA becomes an
additional requirement when nucleic acid depletion has been severe. This

60 THE BIOSYNTHESIS OF PROTEINS

would indicate that DNA is required for the synthesis of certain enzymes.
The results, however, would be very interesting if the system required a
quite specific type of DNA. In some experiments it looked as if DNA from
the same bacterial species only was able to reactivate protein synthesis.
However, substances were later found among the split products of yeast
RNA which are able to reactivate the system just as well as nucleic acids.
The substances involved have already been purified to a considerable
extent by Gale but they are not as yet identified. They are low-molecular-
weight substances of very high activity. The meaning of these very
intriguing results is not clear at present (Gale, 1956, 1957, 1958); the
situation in this system reminds one to some extent of the case of isolated
thymus nuclei mentioned before; an agent which destroys DNA sup-
presses protein synthesis, but this process can be restored by other
substances beside DNA. Other experiments which are in some way com-
parable to Gale's were made by Spiegelman on protoplasts of B. mega-
teriiim (Spiegelman, 1957; Landman and Spiegelman, 1955). Lysozyme
catalyses the hydrolysis of cell walls of various bacteria. When living B.
megaterium or B. siibtilis are treated by lysozyme, their cell wall dissolves
more or less completely; if the osmotic pressure of the medium is suffi-
ciently high, the bacterial content is not dispersed in the medium and the
bacterial body changes into spherical droplets, the protoplasts (Weibull,
1953), which preserve most of the biochemical activities of intact bacteria,
including the synthesis of proteins (Lester, 1953; Beljanski, 1954; Bridoux
and Hanotier, 1954; McQuillen, 1955), including active enzymes (Wiame
et al., 1955), and bacteriophage production (Brenner and Stent, 1955;
Salton and MacQuillen, 1955). Treatment of the protoplasts by deoxy-
ribonuclease under suitable conditions (Spiegelman, 1957) results in the
removal of up to 99 per cent of the DNA; this has no adverse effect on
enzyme synthesis, which is often stimulated. Since deoxyribonuclease
splits the DNA molecule into very small pieces, it is obvious that the
physical integrity of DNA is not required for enzyme production in these
protoplasts. Spiegelman went a step further in the dissociation of the
system in using protoplasts which had been broken by osmotic shock.
From the disintegrated protoplasts, sedimentable systems were obtained
which retained the ability to make enzymes. Like Gale's disrupted Staphy-
lococci, these preparations consisted of membranous bodies containing only
a small fraction of the original DNA of the protoplasts. Treatment by
deoxyribonuclease removed most of the residual DNA and did not impair
enzyme synthesis. It must be mentioned, however, that much DNA was
reformed in these disrupted preparation at the same time as enzymes were
produced.

It would seem that DNA is not a rate limiting factor of protein synthesis
in disrupted bacterial preparation since DNA can be damaged and elimin-

ROLE OF NUCLEIC ACIDS 61

ated almost completely without effects on enzyme synthesis. These works
indicate that in bacteria as well as in higher organisms many proteins can
be made in the absence of DNA.

A completely different approach to the same problem was that of Fuerst
and Stent (1956), who studied the consequences of breakages in DNA
chains caused by the decay of ^^P previously incorporated in the macro-
molecule. E. colt was grown in a medium containing phosphate of fairly
high specific radioactivity. The bacteria were then frozen and kept at a very
low temperature in order to interrupt all metabolic activities and let the
32p decay. After periods of time sufficient for a notable fraction of the ^^p
to have disintegrated, the bacteria were plated and tested for viability
(ability to form a colony). The experiments showed that the bacteria lose
their viability as a result of 32p decay. With thymidine-less mutants in
which the synthesis of DNA can be controlled, it could be shown that the
loss of viability is largely due to the decay of ^'^P atoms in DNA.

A very striking fact is that the capacity of making /3-galactosidase is lost
at the same pace as viability, although this enzyme is not necessary to cell
maintenance or multiplication under the conditions used. Moreover, a few
hundred disintegrations of ^sp per bacterium in the DNA are enough to
reduce the fraction of viable bacteria, and the enzyme production to 1 per
cent. Since E. coli contains several thousand ^ap atoms in DNA, this indi-
cates that a relatively small damage to DNA chains can block enzyme
production entirely (McFall et ah, 1958).

These results no doubt indicate a very close dependence of protein
synthesis on the integrity of the genetic material and they are in sharp con-
trast with the data quoted previously. However, a very puzzling impli-
cation of these observations is that the synthesis of a specific protein,
j8-galactosidase for instance, is abolished as a result of the decay of ^^p
atoms occurring outside the genetic locus of the enzyme. For ^^p decay is
certainly random and there are at least a thousand genes; the chances that
one 32p decay occurs within the enzyme locus are much too small to explain
the observed effects by local action within the locus of j8-galactosidase
(Pardee, 1959). Moreover, experiments by Jacob and Wollman (1958) on
bacterial conjugation show that ^ap decay can break the linear structure
which contains the genes without inactivating the individual loci.

Since in Stent's experiments destruction in other regions of the genetic
material of the bacteria can suppress the synthesis of the enzyme, the effects
of 32p decay are not directly relevant to the problem that we are considering
at present, namely the personal involvement of the gene in putting together
the protein. The effects of ^'^P decay obviously do not concern the control
of the structure of an individual protein by a specific DNA region, they are
probably more relevant to the control of the concerted operation of bacter-
ial biosynthesis by the bacterial genome as a whole. One might be observing

62 THE BIOSYNTHESIS OF PROTEINS

here a very interesting aspect of the regulation of bacterial protein
synthesis.

A direct attempt was made by Jacob and Pardee (1959) to find out
whether genetic DNA participates in making the corresponding protein.
They studied the kinetics of ^-galactosidase appearance in bacteria which
originally lack the corresponding gene, when the gene is introduced by
bacterial conjugation. It was shown that the enzyme is produced at the
maximal rate within a few minutes, and possibly immediately after the
introduction of the piece of genome containing the competent gene. More-
over, the amount of j8-galactosidase synthesized in a population of such
zygotes is proportional to the square of time. This can be interpreted in the
following way: as the number of zygotes formed is known to be propor-
tional to time, the production of protein molecules must again be directly
proportional to the time elapsed after zygote formation for the experi-
mental quadratic function to be satisfied. This is exactly what one would
expect if the gene would act directly as a catalyst in the production of the
protein. If the gene were producing a stable intermediary catalyst at a
constant rate, which in turn would make the enzyme at a constant rate, the
enzyme production would be a function of the third power of time. The
experimental data are therefore incompatible with the continuous pro-
duction at a constant rate under the action of the genetic locus of a stable
catalyst which in turn produces enzymes linearly (Jacob, 1959). They could
not, however, be taken as evidence for the direct participation of DNA in
the making of j8-galactosidase. A few minutes is a long time for E. colt;
which is able to make a protein within 5 sec (McQuillen et al., 1959) and
very complex processes may take place within the 5 min or so which follow
conjugation. It is conceivable that some stable catalyst is being made
rapidly in a limited number of samples during this period. Another pos-
sibility is that an unstable agent is produced continuously in the presence
of DNA only.

3. Conclusion

There is no positive evidence at present for a direct participation of DNA
in protein synthesis in any of the systems studied so far. On the other hand,
there is ample and clear evidence that many proteins can be made without
the direct participation of DNA.

This raises a fundamental question. If DNA carries coded information
concerning the primary structure of proteins, and if its presence and inte-
grity are not a prerequisite of protein synthesis, it must be concluded that
the genetic information is transferred from DNA to some other cell con-
stituent in which it can be preserved for some time and eventually used in
protein synthesis. This is a logical necessity. But no one knows to what
substance the information is transferred, neither in what form, nor when

ROLE OF NUCLEIC ACIDS 63

the transfer occurs. Lack of experimental evidence has encouraged specu-
lation. It seems obvious that a substance able to carry the information
required for controlling the primary structure of a protein must be a rather
large molecule, most probably with a linear structure. There are good
reasons to suspect that the information carrier might be made of ribose-
nucleic acid.

B. RIBOSENUCLEIC ACIDS AND PROTEIN SYNTHESIS

The first pieces of evidence for a participation of RNA in protein syn-
thesis are found in the works of Brachet and of Caspersson on the dis-
tribution of RNA in various animal tissues. A striking correlation was
shown to exist between the amount of RNA and the intensity of protein
synthesis (cf. p. 41). The fact that newly-formed proteins are first detected
in ribosomes (cf. p. 43) is further evidence for the participation of RNA in
protein synthesis. Before considering what is known or suspected at present
about the function of ribosenucleic acids in protein synthesis, it is essential
to be aware of their extreme diversity.

1. Plurality and Metabolic Heterogeneity of Cellular RNAs

It is known from cytological studies that RNA is present in several cell
constituents : ergastoplasm, nucleoli, chromatin, mitochondria and certain
membranes (Brachet, 1940, 1942, 1957; Caspersson and Schulz, 1939;
Caspersson, 1941, 1950). During mitosis, RNA is also found in chromo-
somes and in the spindle (Brachet, 1942, 1957).

Autoradiographic studies show in the most striking manner that differ-
ences in metabolism exist between these differently located RNAs. Thus in
the salivary glands of Drosophila (Herbert, 1954; McMaster-Kaye and
Taylor, 1958) radioactive phosphate is incorporated more rapidly into
the RNA of the nuclei and of the chromosomes than into cytoplasmic
RNA. Tritiated cytidine is found in nucleolus RNA earlier and in greater
amount than in any other cell constituent (Ficq, 1959; Perry, 1960). The
loops of the lampbrush chromosomes of amphibian oocytes are the site of
a rapid incorporation of adenine into RNA (Ficq et al, 1959; Sirlin, 1960 a,
b). in the giant chromosomes of diptera, labelled cytidine is incorporated
into RNA at well-defined positions along the chromosomes, at certain times
of the life cycle of the larvae (Rudkin and Woods, 1959). Differences in
RNA metabolism are thus found between different cell regions, even
within one subcellular structure.

Biochemical studies on isolated cell constituents had also revealed the
metabolic heterogeneity of cellular RNA. Radioactive phosphate was
injected into an animal, and the liver was isolated and homogenized. The

64 THE BIOSYNTHESIS OF PROTEINS

homogenate was fractionated by centrifugation into nuclear, mitochon-
drial and microsomal fractions, and a supernatant. All these fractions con-
tain RNA, and the specific radioactivity of their RNA phosphorus showed
marked differences: the nuclear RNA had always a higher radioactivity
than the other fractions. Difi^erences in rate of incorporation were also
observed between the cytoplasmic fractions (Marshak and Calvet, 1949;
Jeener and Szafarz, 1950; Barnum andHuseby, 1950; Jeener, 1952; Tyner
et ah, 1953; Moldave and Heidelberger, 1954; Sacks and Samarth, 1956).
The use of other precursors of RNA, like glycine, formate (Smellie and
Davidson, 1956) and orotic acid (Hurlbert and Potter, 1952) led to similar
observations. Changes in physiological conditions differently affect the
metabolism of these various fractions (Jeener and Szafarz, 1950; Reid and
Stevens, 1956; Moldave, 1954; De Lamirande et al., 1958).

Even the more elaborate among the presently used fractionation pro-
cedures are still very crude, and each time it has been possible to further
separate one of the cellular fractions into several subfractions, e.g. by com-
bining centrifugation with extraction in salt, in phenol or in detergents, it
was found that the RNAs contained in the subfractions again differ in their
physiological or metabolic activity (Vincent, 1952, 1957, 1958; Bhargava
etai, 1958; Schneider and Potter, 1958; Logan, 1957; OsRwaetaL, 1958;
Sihataniet al, 1959, 1960; Osawa, 1959; Goldthwait, 1959; Georgky et al.,
1960; Antoni et al, 1960; Scholtissek, 1960).

Among the various RNAs so far detected, special mention must be
made of the so-called 'soluble RNA'. In their studies on incorporation of
amino acid into proteins of liver homogenates, Hoagland et al. (1957, 1958)
observed that labelled amino acids are bound to a special RNA fraction
which is not sedimented with the particle bound RNA (see Chapter IV).

This 'soluble RNA' is not a single substance, but a mixture of several
molecular species (Bloemendal and Bosch, 1959). It could be separated by
chromatography on cationic starch exchanger or by counter-current distri-
bution into fractions selectively binding certain amino acids (Smith et al.,
1959;Uo\\eyetal., 1959).

It is quite possible that soluble RNA preparations contain, beside
amino acid binding RNAs, other RNAs which play a different role or have
a different type of metabolism (Canellakis and Herbert, 1960).

In bacteria also, RNA fractions differing in their metabolism have been
detected and separated (Countryman and Volkin, 1959). Immediately
after infection by a bacteriophage, a limited amount of a special high turn-
over RNA is formed (Volkin and Astrachan, 1956; Astrachan and Volkin,
1958; Watanabe and Kiho, 1958). In ultraviolet irradiated (Suzuki and Ono,
1959) or in chloramphenicol treated bacteria (Neidhart and Gros, 1957),
labile RNA fractions have been detected. Column fractionation of extracts
of normal E. coli after short time incorporation of radioactive phosphate

ROLE OF NUCLEIC ACIDS 65

shows unequal distribution of 32p in the RNA of the collected fractions
(Roberts et al, 1958).

Differences in nucleotide composition have been observed between
various RNA fractions isolated from a single tissue. For instance, the com-
position of RNA is not the same for all the fractions isolated from rat liver
homogenate (De Lamirande et al.., 1955 ; Elson et al., 1955). Vincent (1952)
showed that RNA of isolated nucleoli of starfish oocytes contains more
guanine and less uracil than the average cytoplasmic RNA (see also Harris,
1959; Vincent and Baltus, 1960).

In yeast and in pancreas, soluble RNA contains a 5-ribosyluridine
nucleotide which is not found in particle RNA (Davis and Allen, 1957;
Kemp and Allen, 1958; Osawa and Otaka, 1959; Yu and Allen, 1959;
Cohn, 1959; Scannell et al, 1959; Otaka et al, 1959). Methylated purine
are also more abundant in this RNA (Bergquist and Matthews, 1959).

Differential salt extraction, enzyme or acid hydrolysis (Jeener, 1948;
Sacks et al, 1955; Sacks and Samarth, 1956; Martin and Morton, 1956;
Osawa et al, 1958) also reveal the presence of RNAs which may differ in
the way they are associated with other cell constituents (Martin and
Morton, 1956; Brachet, 1959; Bell, 1959; Shigeura and Chargaff, 1960).

All these results leave no doubt that a living cell contains a population of
many different molecular species of RNA which have independent
physiological activities, just as a cell contains many protein species. Good
methods of fractionation are urgently needed and there is little doubt
that progress in the study of the function of RNA will be crippled until
suitable methods are found. Attempts at fractionating ribosenucleic acids
by physicochemical methods have been made repeatedly (Chantrenne,
1945; Bacher and Allen, 1950; Ghuysen, 1950; Delcambe, 1950; Del-
cambe and Desreux, 1950; Desreux and Ghuysen, 1951; Ghuysen and
Desreux, 1952; Mallette and Lamanna, 1953; Roberts et al, 1958). The
fractions thus obtained from yeast RNA differ in molecular weight but not
much in composition (Ghuysen and Desreux, 1952; Miura and Suzuki,
1956; Miura et al, 1958; Smith, 1960). By countercurrent distribution,
RNA of different compositions can be partly separated (Kirby, 1960).
Electrophoresis also permits some fractionation (Harris and Davies, 1960).

The development of methods for isolation of proteins has been facilitated
by the fact that many proteins have individual features which make it easy to
recognize and to determine them : typical solubility, characteristic colours,
specific enzymic activity. Nothing quite comparable is known for RNA. Two
recent developments indicate, however, that one might witness important
progress in this field in the coming years : the so-called soluble RNAs con-
tain abnormal nucleotides and bind amino acids by ester bond, whereas
the bulk of RNA does not. It is to be expected that several RNAs will be
characterized by their capacity to bind individual amino acids in this

66 THE BIOSYNTHESIS OF PROTEINS

fashion. On the other hand the isolation of infectious RNA from several
viruses is a fundamental progress not only for the understanding of the
process of virus multiplication, but also for the development of our know-
ledge of RNA in general. With these RNAs which are endowed with
specific biological activities, biochemists will learn how to handle RNA
without destroying the structural features which are essential to their
biological activities.

It is very important to keep in mind that so far all the studies on RNA
(except a few recent works on virus RNA) have been performed on mix-
tures of various RNA fractions, which were most of the time partly
degraded. This has been an inexhaustible source of confusion. Innumerable
works on nucleic acid metabolism are extremely difficult to interpret for
the results always concern a mixture of nucleic acids. When tracers are
used, new sources of hidden difficulties are involved. Beside the familiar
complications due to dilution of labelled precursors, existence of several
pools of precursors, interconnexions between metabolic pathways, a very
serious type of complication has been appreciated only recently (Vincent
and Baltus, 1959). It was found that certain RNA fractions readily bind
two molecules of cytidylic acid and one of adenylic acid at the end of their
molecule (Heidelberger et al., 1956; Canellakis, 1957; Edmonds and
Abrams, 1957; Paterson and Lepage, 1957; Hecht et al., 1958; Herbert,
1958; Allfrey and Mirsky, 1959; Harbers and Heidelberger, 1959; Okazaki
and Okazaki, 1959). This process was often taken as reflecting nucleic acid
synthesis ; it now appears that it corresponds to a metabolic activity which
might be connected with protein synthesis.

All this serves to emphasize the physiological heterogeneity of nucleic
acids, the complexities of their metabolism, and the primitive stage in
which we are still in our attempts to analyse the function of these substances
in biochemical terms. It is important to keep this in mind and to avoid
being too dogmatic in all interpretations of RNA metabolism in terms of
RNA formation.

2. Metabolism of RNA and Protein Synthesis

One of the first working hypotheses retained by workers interested in the
function of RNA in protein synthesis, was that RNA and protein must be
made simultaneously or that the synthesis of protein depends on RNA
formation. A very great amount of research has been devoted to testing
these ideas, and it would be hopeless to try and analyse the results of all
these contributions. It has repeatedly been reported, for instance, that in
growing tissues or in growing bacteria, protein and RNA synthesis go
together, or that the rate of incorporation of precursors into the two types
of macromolecules depends on the same basic conditions, like the provision
of energy, a carbon source, a nitrogen source and active phosphorylations.

ROLE OF NUCLEIC ACIDS 67

Obviously, these facts are irrelevant to the existence of a connexion be-
tween RNA synthesis and protein formation. They are a mere reflexion of
the general requirements of cells for growth or for any type of synthesis.
In the same way, the fact that a population of growing bacteria usually
make protein and nucleic acid simultaneously does not imply that the
mechanism of protein synthesis is such that one type of macromolecule
cannot be made without the other being made at the same time.

More significant are the attempts made at dissociating the two processes,
e.g. by varying growth-rate, by using inhibitors or by causing specifically
the synthesis of a protein. Let us consider briefly a few typical data.

Spiegelman and Kamen (1947) showed that the phosphorus of yeast
RNA is renewed very slowly in actively respiring yeast which is deprived of
nitrogen source. If ammonium salts are added, proteins are synthesized,
the uptake of radioactive phosphate is increased in all phosphorus com-
pounds of the cell, but much more so in RNA than in any other cellular
constituent, as if the synthesis of RNA was indeed connected with protein
synthesis. Abrams et al. (1949) made similar observations for the incorpora-
tion of precursors into RNA purines; however, it was later found that
X-rays markedly reduce this RNA metabolism without inhibiting protein
synthesis (Abrams, 1951). Grenson (1952) studied incorporation of phos-
phate into various types of animal tissues which have in common a high
rate of protein synthesis, but which differ physiologically. She found no
simple or uniform correlation between phosphorus incorporation into RNA
and the production of protein. Similar observations were made for RNA
synthesis during synthesis and secretion of amylase by pigeon liver pan-
creas (Hokin and Hokin, 1953, 1954, 1956), or during the period of recovery
after starvation in muscle or liver (Laird et al., 1955 ; Zak and Gutmann,
1960). In continuous cultures of a flagellate, a simple correlation was
clearly observed between RNA synthesis and protein formation when the
organisms were growing at a constant rate. However, strong disharmonies
appeared as soon as the rate of growth was abruptly changed by a sudden
modification of the medium composition. A net loss of RNA for instance
could even be observed at times when protein synthesis was stimulated
(Jeener, 1952). Price (1952) and more recently Magasanik et al. (1959),
Peabody and Hurwitz (1960) observed comparable phenomena with
bacteria: if the substance serving as carbon source is replaced by another
one to which the organism is not adapted, growth slows down very much
while the bacteria make enzymes which will metabolize the new carbon
source. RNA synthesis is very much depressed during this period.

Direct comparison of RNA fonnation and protein synthesis thus led to
conflicting results for many years: a rather convincing correlation was
found in certain systems but it failed to appear in crucial cases. The syn-
thesis of protein and of RNA may very often occur simultaneously, but

68 THE BIOSYNTHESIS OF PROTEINS

they are not necessarily connected. Moreover, the positive correlations
which were occasionally observed between incorporation of precursors
into protein and net RNA synthesis for instance have lost much of their
interest since the plurality and the metabolic heterogeneity of cellular
ribosenucleic acids have been recognized. If a cell contains quite an assort-
ment of various ribosenucleic acids, data on total net synthesis of RNA, or
incorporation into total cellular RNA give but average values; important
changes involving certain RNA fractions might pass unnoticed because
they can be masked by other processes occurring at the same time in other
RNA fractions. It is clear that a direct comparison of the metabolism of
total RNA and total protein synthesis cannot be very informative, and one
should look for data on the possible connexions between RNA metabolism
and protein synthesis at the level of different ribonucleoprotein fractions.
A good start in this direction can be found in a metabolic investigation of
the ribonucleoprotein particles of guinea pig pancreas by Siekewitz and
Palade (1959). Five ribonucleoprotein fractions were isolated by fractional
centrifugation and compared as for the rate of in vivo incorporation of
adenine into RNA and of amino acids into proteins. The rates and the
kinetics of incorporation differ very much between the five fractions. It is
striking that fractions especially active in protein synthesis showed
practically no incorporation of adenine into their RNA, whereas another
fraction incorporated adenine very actively but was very poor in protein
synthesis. This indicates that the synthesis of protein and that of nucleic
acids are not associated at the level of nucleoproteins.

A completely different approach to the problem discussed in the present
chapter consists in interfering specifically in various ways with the meta-
bolism of RNA and in watching the effects on protein formation. The first
attempt along this line was probably made by Jeener and Jeener (1952)
with Thermohacterium acidophilus. This is an exacting bacterium which
requires, among a score of various substances, uracil and thymidine for
growth. Deprivation of thymidine stops DNA synthesis and bacterial
division, but it has no immediate effect on protein synthesis. On the con-
trary, uracil starvation almost immediately stops protein formation. These
early observations have been confirmed by Okazaki and Okazaki (1958).
Similarly, in pyrimidine-less mutants of E. coli the synthesis of enzymes
depends on the exogeneous supply of pyrimidines, except under special
conditions when these are provided by the breakdown of certain RNA
fractions (Pardee, 1954, 1955; Earner and Cohen, 1958). The ability of
resting yeast to make the enzyme a-glucosidase depends on the level of the
free nucleotide pool, and the best way of depleting this pool is to force
yeast to produce proteins (Spiegelman et ai, 1955).

Deprivation of nucleic acid precursors, in all these cases, was thus found
to be damageable to protein production. Conversely, the provision of

ROLE OF NUCLEIC ACIDS 69

purines, pyrimidines and their derivatives favours it in some systems
(Hancock, 1957; Reiner, 1960). In disrupted Staphylococcus aureus, a
supplement of purines or pyrimidines stimulates protein and enzyme
formation (Gale and Folkes, 1953; Creaser, 1955; Gale, 1956). A ribonu-
clease hydrolysate of RNA also contains substances which stimulate the
incorporation of amino acids into the proteins of such systems; but this
effect which was first attributed to oligonucleotides (Gale and Folkes,
1955) is in fact due to other substances which have not been identified yet
(Gale et ah, 1958). Webster observed that the incorporation of amino acid
into cytoplasmic particles from pea roots is markedly promoted by a
mixture of the four ribonucleosides, whereas desoxyribosides are inactive.
A mixture of the four ribonucleoside triphosphates ATP, GTP, CTP and
UTP also stimulated amino acid incorporation (Webster and Johnson,
1955; Webster, 1957).

Thus protein synthesis can, in certain systems, be limited by lack of
purine, pyrimidine and derivatives. As the latter substances can serve as
precursors of RNA, these observations have often been taken as evidence
that protein synthesis requires the synthesis of new RNA molecules.
Apparent confirmation of this conclusion was found in the inhibition of
protein synthesis by purine and pyrimidine analogues, and by the direct
observation of an increased incorporation of labelled adenine or uracil into
RNA during induced enzyme formation (Gale and Folkes, 1955; Chan-
trenne, 1956). It seemed highly probable therefore that the continuous
synthesis of some RNA fraction was necessary for protein synthesis (see
review by Spiegelman, 1956). Although this possibility is in no way
excluded, it is realized at present that other interpretations of the experi-
mental facts are equally probable. The fact that RNA precursors are
required for some process does not prove that the synthesis of new RNA
molecules is required. Many 'RNA precursors' also participate in the
metabolism of low molecular weight material. As will be shown in the next
chapter, purine and pyrimidine analogues do interfere with protein syn-
thesis, but in most cases it is not by preventing RNA synthesis that they do
so. The increased incorporation of adenine which accompanies enzyme
induction in yeast is a rather involved process, in some way connected with
the breakdown of certain RNA fractions (Chantrenne, 1958). The evidence
for a synthesis of new RNA molecules linked to protein synthesis therefore
is certainly not compelling. But it remains that metabolic processes'hwolving
some RNA fractions are most probably involved in protein synthesis. The
nature and significance of these processes is at present anyone's guess. The
results of recent investigations have indicated that soluble RNA can bind in
succession at the end of its polynucleotidic chains two pyrimidine nucleo-
tides followed by a purine nucleotide. The attachment of this terminal
sequence of three nucleotides is a prerequisite for the fixation of activated

70 THE BIOSYNTHESIS OF PROTEINS

amino acids on to soluble RNA (Heidelberger et ah, 1956; Canellakis,
1957; Hecht et al, 1958; Edmonds and Abrams, 1957; Paterson and Le
Page, 1957; Okazaki and Okazaki, 1959; Harbers and Heidelberger, 1959;
Vincent and Baltus, 1959). The requirement for purines and pyrimidines
for protein synthesis might very well find its explanation in this end chain
metabolism of soluble RNA, or in other reactions that are still to be dis-
covered.

3. Importance of the Structural Integrity of Ribonucleic Acids

(a) Effects of rihonuclease. Highly purified ribonuclease from beef pan-
creas was found to inhibit completely the incorporation of labelled amino
acids into proteins of homogenized pancreas or liver (Siekevitz, 1952;
AWir &y et al., 1953; Zamecnik and Keller, 1954). In similar preparations,
ribonuclease was without any effect on other complex biosynthetic pro-
cesses such as lipid formation. The selective action of ribonuclease upon
amino acid incorporation thus provided a very direct evidence to the
participation of some RNase sensitive substance, most probably RNA, in
protein formation. Observations of the same kind were made on homo-
genates of animal or plant cells. Disrupted bacteria are able to incorporate
amino acids into their proteins under appropriate conditions; here again
the incorporating system is inactivated by ribonuclease, which destroys a
large part of the RNA in the preparation (Gale, 1956, 1957; Gale and
Folkes, 1955). Protoplasts (i.e. bacteria which have been stripped of their
cell wall and protected from osmotic effects by an adequate concentration of
sugar or salts) keep the capacity of making proteins, including inducible
enzymes (Wiame et ah, 1955 ; Landman and Spiegelman, 1955 ; Spiegelman,
1957). In the presence of ribonuclease, incorporation of amino acids into
protoplasts protein is completely inhibited (Lester, 1953; Beljanski, 1954).
Protoplasts are very fragile structures, and ribonuclease easily causes their
disruption, followed by dispersal of their content (Brenner, 1955). It can
be shown, however, that the inhibition of protein synthesis occurs before
the disruption of the protoplasts (Bridoux and Hanotier, 1956). Spiegel-
man and Landman (1955) established conditions which ensure stability and
preserve the metabolic activity of the protoplasts in the presence of ribonu-
clease for a longer time. The enzyme invariably suppresses protein forma-
tion under these conditions, in a rather selective manner.

More striking still are the effects of ribonuclease on otherwise intact
living cells. Crude ribonuclease preparations had been shown to inhibit
cell division when injected into an amphibian egg (Thomas et ah, 1946).
This experiment was later repeated with highly purified enzyme on divid-
ing eggs at the two and four cells stages; it was observed that micro-
injection of ribonuclease into one blastomere inhibits the division not only
of the injected cell but of the other blastomeres as well, as if ribonuclease

ROLE OF NUCLEIC ACIDS 71

was able to pass from one cell into the next one (Ledoux et ah, 1954;
Brachet and Ledoux, 1955). Indeed, injection of the enzyme is not neces-
sary; ribonuclease readily enters the cells of amphibian embryos at the
morula stage when these are merely immersed in a dilute ribonuclease
solution in tap water. This offered an easy way of affecting RNA in vivo
and of exploring the function of RNA in protein synthesis. Simple experi-
ments that biochemists had never attempted because of their school years
prejudices about permeability of cell membranes, now looked sensible and
were soon performed ; many types of cells were found to pick up ribonu-
clease. Amoeba proteus for instance rapidly takes up ribonuclease from the
medium (Brachet, 1954—55; Schumaker, 1958), most probably by a process
known as pinocytosis (Lewis, 1931; Holter and Marshall, 1954; Holter,
1959), which consists in the engulfment of droplets of medium; the basic
proteins contained in the medium penetrate the cell for they are later found
in the cytoplasm and even in the nucleus. In a ribonuclease solution,
Amoebae lose part of their basophilia (i.e. RNA is partly destroyed) and at
the same time their capacity for incorporating labelled amino acids is
almost completely abolished ; respiration is not affected and ATP produc-
tion continues, an accumulation of labile phosphate esters is even observed
(Skreb-Guilcher, 1955). Ribonuclease thus can inhibit protein synthesis
under such conditions that the general metabolic processes are main-
tained.

Another type of ribonuclease sensitive system is the onion root. As
observed by Kaufmann et al. (1954, 1957), the pancreatic enzyme causes
mitotic abnormalities. The absorption of RNase and other proteins by
onion root cells has been confirmed by direct experiments with ^H labelled
protein (Jensen and McLaren, 1960); labelled RNase was observed in
association with the nucleolus. Brachet (1954) showed that incorporation
of amino acids and growth of the root are stopped by ribonuclease. An
important observation (Brachet, 1954, 1955, 1956) is that protein synthesis
is almost completely suppressed by a rather short ribonuclease treatment
(e.g. 30 min). Under these conditions, the extent of RNA degradation is
very limited; even after 18 hrs action only 20 per cent of the RNA is re-
moved. A limited damage to RNA or the destruction of a certain type of
RNA only is enough to suppress protein synthesis. A quite similar con-
clusion was reached by Yakeyama et al. (1958) who observed a complete
inhibition of fibroin synthesis by ribonuclease in isolated silk glands of
Bombyx mori under such conditions that only a limited RNA destruction
had occurred. Roots which had been treated by RNase for 30 min, and in
which protein synthesis was thus almost completely suppressed, were
homogenized and fractionated by high speed centrifugation. No significant
difference in RNA content was found in any of the sedimentable fractions
between RNase treated and control roots. But a 50 per cent decrease in the

F

72 THE BIOSYNTHESIS OF PROTEINS

RNA to protein ratio in the 'soluble' fraction was consistently observed.
This indicates that the inhibition of protein synthesis in living onion roots
caused by ribonuclease is probably due to partial degradation of a soluble
RNA fraction by the enzyme (Brachet and Six, 1959).

The possibility, however, remains that RNase might also interfere with
protein synthesis in other ways, for instance, by slowly damaging ribosome
RNA or by forming complexes with it. Ribosome RNA is less sensitive to
the lytic action of the enzyme than soluble RNA (Shigeura and Chargaff,
1960); other basic proteins like histone, salmine and to a lesser extent
cytochrome-c indeed partly inhibit protein synthesis in roots (Brachet,
1956) and in ascites cells (Becker and Green, 1960) by combining with
RNA.

Finally, in certain types of animal cells, ribonuclease causes deep changes
in RNA metabolism. For instance, in ascites cells in a medium supple-
mented with free nucleotides, the enzyme first stimulates RNA synthesis
(Ledoux, 1956; Pileri et al, 1957), causing a rapid uptake of pyrimidine
nucleotides, thus leading to the formation of abnormal RNA, which is later
degraded (Ledoux and Vanderhaeghe, 1957). The effect on protein synthe-
sis is not striking in this case, however, and respiration is depressed (Ledoux
and Baltus, 1954).

Ribonuclease was also found to inhibit growth of Bacillus megaterium
(Groth, 1956) and of £". coli (Jerne and Maaloe, 1957). In many cases, lysis
of the bacteria occurs, but it is possible to isolate strains which are not
lysed by the enzyme, and which will continue to grow in its presence
(Jeener, 1959a). Using a lysogenic strain of Bacillus megaterium resistant
to ribonuclease lysis, Jeener showed that the synthesis of phage protein is
partly inhibited whereas bacterial growth is not. The phage proteins made
in the presence of the enzyme have abnormal immunological and chromato-
graphic properties (Jeener et al., 1960). They can be integrated into phage-
like structures, but these are poorly adsorbed on receptive bacteria, and they
are also very fragile. Incomplete phage particles have indeed been isolated
by serological precipitation (Jeener, 1959b). In these experiments, also, a
small fraction only of the total RNA could have been degraded, for the
RNA/DNA ratio was practically unaffected. But the composition of RNA
was changed: a higher content in uridylic acid was consistently found in
the bacteria which had been treated with ribonuclease. This suggests that
the abnormalities observed in the proteins produced might result from a
structural modification of some RNA fraction (Jeener, 1959b).

It should be mentioned again here that ribonuclease does not affect pro-
tein synthesis in intact mitochondria, but it suppresses completely amino
acid incorporation into proteins in extracts obtained from mitochondria
(Kalf and Simpson, 1959; Kalf et al, 1959). It is probable therefore that
protein synthesis in mitochrondria can go on in a medium containing

ROLE OF NUCLEIC ACIDS 73

ribonuclease simply because the enzyme cannot enter intact liver mito-
chondria.

The results reviewed above show that ribonuclease acting on living cells
brings about various types of damages. In favourable cases, it can selec-
tively inhibit the synthesis of protein or of certain proteins without acting
upon general metabolism and energy production, or without affecting the
synthesis of many various cell constituents. The mode of action of the
enzyme is not completely analysed, but inhibition of protein synthesis
coincides with the breakdown of certain RNA fractions especially soluble
RNA.

It would seem that a limited degradation or modification of certain RNA
fractions is enough to stop protein synthesis, or to cause modifications in
the properties of the proteins produced.

(b) Modification of RNA by analogues of purines and pyrimidines. Many
analogues of the natural purines and pyrimidines have been prepared by
chemical synthesis. Some of them proved to be potent inhibitors of growth
for certain animal or plant cells and for certain strains of bacteria (Elion
et al., 1954). It is likely that substances of this kind interfere with the meta-
bolism, the synthesis or the function of nucleic acids or nucleotidic com-
pounds. Present knowledge of the mode of action of these analogues is
actually rather poor, restricted as it is to the case of a few compounds and a
few organisms or biochemical systems. It is, however, sufiicient already to
show that purine and pyrimidine analogues can interfere in many ways
with the metabolism of the natural parent products, and more indirectly
with other metabolic processes as well. This is illustrated by the following
examples: 5-fluoro-uracil inhibits methylation in the synthesis of thymine
(Bosch et al, 1958; Cohen et al, 1958; Heidelberger ^^ a/., 1957; Eidinoff
et al., 1957; Harbers et al., 1959) and interferes with cell wall formation
(Tomasz and Borek, 1959); 6-mercaptopurine affects acetate and formate
utilization, probably by interfering with the formation of purine containing
cofactors (Bolton and Mandel, 1957); 5-hydroxyuridine exerts several
different effects including inhibition of purine utilization (Slotnick et al.,
1953); 8-azaguanine inhibits adenosine deaminase (Feigelson and David-
son, 1956); 6-azauracil causes the inhibition of polynucleotide phosphory-
lase (Skoda et al., 1959) and it blocks the conversion of orotic acid into
uracil (Skoda and Sorm, 1958, 1959; Sells, 1959-1960). Several analogues
can substitute for the natural parent constituent in the feed-back control
exerted by this compound upon its own synthesis (Gots and Gollub, 1959;
Trudinger and Cohen, 1956; Smith and Sullivan, 1960).

Different types of cells also react differently or to various degrees to a
purine or pyrimidine analogue, and a type or effect which is shown in one
organism does not necessarily occur in others. Therefore the effect of an
analogue on a given organism cannot be predicted, and careful analysis is

74 THE BIOSYNTHESIS OF PROTEINS

necessary in each case. This situation may look depressing; on the other
hand, it makes it possible to find organisms and analogues which will be par-
ticularly suitable for the study of a given biochemical problem. Cases which
are especially interesting for the problem considered in the present chapter,
namely the importance of RNA integrity for protein synthesis, are those of
organisms which incorporate purine or pyrimidine analogues into their
nucleic acids. Thus 8-azaguanine is incorporated into RNA of various
organisms (Mitchel et al, 1950; Heinrich et al, 1952; Bennett et ah, 1953;
Mandel et al, 1954; Matthews, 1953, 1957; Lasnitski et al, 1954; Mat-
thews and Smith, 1955, 1956; Mandel and Markham, 1958). 2-Thiouracil
incorporation into RNA of tobacco mosiac virus (Jeener and Rosseels,
1953; Jeener, 1957; Matthews, 1956; Mandel et al, 1957) and of Bacillus
megaterium (R. Hamers, 1956; Amos et al, 1958) has been established.
5-Fluorouracil can also be incorporated into RNA in several organs of rat
and mouse (Heidelberger et al, 1957; Harbers et al, 1959), in Escherichia
coli (Horowitz et al, 1958; Horowitz and Chargaff, 1958; Gros, 1959;
Brockman et al, 1960) and in tobacco mosaic virus (Gordon and Staehelin,
1959).

As 2-thiouracil is being incorporated into RNA by Bacillus megaterium,
the bacteria continue to grow and to make proteins, but growth becomes
linear (Hamers, 1956). In E. coli, a similar change in the shape of the
growth curve is observed, but the effect is not as rapidly established as in
B. megaterium. Protein synthesis is but slightly inhibited by thiouracil in
E. coli, but the production of certain enzymes is drastically reduced. The
differential rate of increase in j8-galactosidase activity, for instance, is
reduced by about 90 per cent. The residual increase is due to the produc-
tion of a protein material which is precipitated by an antiserum prepared
against regular j8-galactosidase. But titration by the immunological method
of Cohn and Torriani (1952) shows that this material has less enzyme
activity per unit of serologically precipitable protein than regular j3-
galactosidase. This indicates that a slightly abnormal enzyme is produced
in the presence of thiouracil (Hamers and Hamers-Casterman, 1959).

Bacteriophage production by a lysogenic strain of Bacillus megaterium
can also be inhibited by 2-thiouracil. The major action of the analogue in
the present case is a drastic inhibition of phage DNA synthesis; the
formation of phage proteins is also decreased, but to a lesser extent, and the
protein produced in the presence of thiouracil retain normal immunological
properties, as well as the capacity of fixation upon bacteria (Jeener et al,
1959).

5-Fluorouracil inhibits growth of £". coli by 50 per cent. Certain enzymes,
like catalase, succinic dehydrogenase or phosphatase continue to be pro-
duced whereas j3-galactosidase synthesis is almost completely stopped
(Horowitz et al, 1958; Horowitz and Chargaff", 1959; Gros, 1960). While

ROLE OF NUCLEIC ACIDS 75

^-galactosidase production is thus drastically reduced, the synthesis of a
protein closely related to the enzyme but catalytically inactive can be
demonstrated by serological reactions (Bussard et ah, 1960).

The analogue inhibits growth of E. coli only when it can be incorporated
into nucleotidic compounds, since mutants lacking the enzymes for making
the nucleotide of fluorouracil are resistant to the analogue (Brockman et al.,
1960). As the effects of the analogue are observed without delay, the damage
caused by fluorouracil incorporation must concern one of the RNA
fractions which are rapidly renewed (Naono and Gros, 1960). The damaged
RNA seems to be involved in the process of selection or of arrangement of
the amino acids, for the proline and tyrosine content of the total bacterial
protein material made in the presence of fluorouracil is lower than normal.

The reason why certain enzymes are deeply affected by such errors in
amino acid selection, whereas other enzymes are rather insensitive, can be
visualized easily. It is clear that the active centre of a given enzyme, or the
folding which creates this centre, may eventually be affected by amino
acid replacements. If, however, the number of changes in the amino acid
sequence is not too large, it is conceivable that the enzyme retains a prac-
tically normal tertiary structure, together with its catalytic and serological
properties. Ribonuclease, for instance, can stand a considerable amount of
damage indeed, without losing its catalytic properties: certain peptide
bonds can be broken (Richards, 1958), a whole section of polypeptide can
even be removed (Anfinsen et al., 1955; Rogers and Kalnitzky, 1957)
without much change of activity. The resulting molecules are simply more
fragile than the intact enzyme.

In contrast with thiouracil and fluorouracil, another analogue, 6-azau-
racil, which also inhibits bacterial growth, does not interfere with protein
synthesis more than with the formation of other cell constituents (Sells,
1959). It is important to realize that 6-azauracil is not incorporated into
RNA in any detectable amount (Handschumaker, 1957); it inhibits nucleic
acid synthesis by blocking the pathways of uridylic acid synthesis (Skoda
and Sorm, 1958, 1959).

The rate of incorporation of 8-azaguanine into the nucleic acids varies
very much according to the organism (Matthews, 1957). Bacillus cereus has
received special attention because it takes up quite a large amount of the
analogue. Detailed studies by Smith and Matthews (1957), Mandel and
Markham (1958) showed that 8-azaguanine replaces up to 40 per cent of the
guanine in the RNA made in its presence, and that the composition of the
nucleic acid is not otherwise changed. Synthesis of RNA continues at a
somewhat increased rate in the presence of the analogue, DNA is made
at a closely normal pace, but protein synthesis is drastically inhibited
(Chantrenne, 1958, 1959; Chantrenne and Devreux, 1958, 1959, 1960;
Mandei and Altman, 1960).

76 THE BIOSYNTHESIS OF PROTEINS

It must be emphasized that many cell constituents are still synthesized
normally, which indicates that basic metabolism and energy production are
not touched. The process of RNA synthesis also continues but confusion
of azaguanine for guanine leads to abnormal nucleic acids. Under these
conditions protein synthesis is obliterated, although the utilization of
amino acids by the bacteria is not prevented, neither their condensation
into peptides. Cell wall material for instance, including the peptides they
contain, continue to be made at a normal rate (Chantrenne and Devreux,
1958; Richmond, 1959; Roodyn and Mandel, 1960).

In these cases, the analogue partly replaces the normal purine or
pyrimidine compound, and this results in the formation of modified RNA.
It is striking that in all the cases where such an incorporation of abnormal
bases into RNA occurs, protein synthesis is interfered with. Let us con-
sider these facts a little more closely.

There are good reasons to believe that the inhibition of protein synthesis
is linked to the incorporation of azaguanine into nucleic acids. For instance
mutant strains of Streptococcus foecalis resistant to azaguanine have been
shown to differ from the wild strain in that the resistant mutant has lost
the ability to incorporate azaguanine (and guanine) into nucleotidic com-
pounds. The same difference was observed for sensitive and resistant lines
of a leukaemia (Brockman et al., 1957-58). In Tetrahymena geleii, inhibition
of growth by azaguanine is observed only in the presence of uracil which is
required for RNA synthesis in this organism (Kidder et ai, 1951 ; Heinrich
et al.y 1952). Recent research in the author's laboratory has shown that this
also applies to protein synthesis in the same organism. It is only when
azaguanine can be incorporated into RNA that protein synthesis is inhibited
by the analogue. On the other hand, kinetic studies on the incorporation of
azaguanine into RNA and of amino acids into protein indicate that in
Bacillus cereus the inhibition of protein synthesis is already fully expressed
at a time when the amount of RNA made in the presence of azaguanine
represents only about 10 per cent of the total RNA (Chantrenne, 1958).
Fractionation of bacterial content by centrifugation shows that at that time
the 'non-sedimentable' RNA is already heavily loaded with azaguanine,
whereas ribosome RNA contains relatively little of the analogue. This
observation, together with the facts reported above, suggests that the incor-
poration of azaguanine into a soluble RNA might be responsible for the
general inhibition of protein synthesis caused by the analogue.

This is one more piece of evidence that the integrity of some soluble
RNA is a requirement for protein synthesis.

The action of azaguanine on B. cereus can be completely prevented by
several purines or purine derivatives (Mandel, 1957). Thus in the presence
of an adequate concentration of guanosine, no azaguanine is incorporated
into RNA and no inhibition of protein synthesis is observed. Guanosine

ROLE OF NUCLEIC ACIDS

77

can even release the inhibition produced by azaguanine and completely
restore protein synthesis and bacterial growth if added some time after the
inhibition by azaguanine is established. Under those conditions, a large
part of the azaguanine that had been incorporated into RNA is rejected and
found in the medium mostly as azaguanosine (Matthews and Smith, 1956;
Smith and Matthews, 1957; Mandel, 1957; Mandel and Markham, 1958).
Restoration of protein synthesis by guanosine, however, becomes more and

Penicillinase

750-

120 240
min

120 240
min

Fig. 22. Restoration by guanosine of the synthesis of protein material (a)
and of constitutive penicillinase (b), in B. cereiis after azaguanine
inhibition. The numbers at the end of the curves indicate the time in
minutes elapsed between the additions of azaguanine and of guanosine
(Chantrenne and Devreux, 1960).

more sluggish when the period of azaguanine inhibition increases. An
unexpected feature of the recovery of protein synthesis under the action of
guanosine is that the synthesis of an enzyme like constitutive penicillinase
is not restored at the same time as net synthesis of the average protein
material. This is illustrated in Fig. 22. For instance, when guanosine is
added 45 min after the analogue, the synthesis of protein is rapidly
restored, whereas penicillinase formation is not yet restored 2 hrs. later (Chan-
trenne, 1959; Chantrenne and Devreux, 1960). There is evidence that

78 THE BIOSYNTHESIS OF PROTEINS

proteins made during this restoration period are abnormal; catalase for
instance has an abnormal temperature coefficient (Chantrenne, in press).
Beside the lesion which blocks all protein synthesis, and which is easily
repaired by an early addition of guanosine, continued action of azaguanine
causes another type of lesions which do not affect the synthesis of all the
proteins equally. These secondary lesions develop rather slowly, but they
are also cured very slowly by guanosine. A closer study of RNA during
azaguanine inhibition and of its reversal by guanosine showed that an
increasing amount of azaguanine is incorporated into RNA 'irreversibly',
in this sense that it fails to be displaced by guanosine. Identification of this
fraction of RNA will eventually make it possible to locate the site of the
secondary lesions which affect differently the synthesis of the individual
proteins. The specificity of synthesis of the individual proteins depends
probably on the integrity of this fraction of RNA.

However, this conclusion cannot be taken for certain at present, since
DNA also has been shown to take up some azaguanine (Smith and Mat-
thews, 1957; Mandel et ah, 1957). Although the amount incorporated into
DNA is very small as compared to RNA, it might be enough to explain the
specific effects on protein synthesis.

In Bacillus cereus, the uptake of azaguanine into soluble RNA is rapid
and the degree of guanine replacement by azaguanine very high. This
rapidly leads to an almost complete inhibition of all protein synthesis. It is
only during the recovery caused by guanosine that differential effects on the
synthesis of individual proteins can be observed. In other systems which
are somewhat less sensitive to the guanine analogue, differential effects are
observed during the action of the drug. Thus Creaser (1955-56) noticed
that in a Staphylococcus azaguanine can inhibit the synthesis of ^-galacto-
sidase much more than that of catalase. Jeener et al. (1959) also observed
that within a well-chosen range of concentrations, azaguanine depresses
the synthesis of bacteriophage protein much more than that of bacterial
proteins in B. megaterinm. More recently Heyes (1959) reported that
azaguanine inhibits protein synthesis in roots of pea seedlings only
partially, and that the degree of inhibition is not the same for the four
individual enzymes which were studied. Disturbances of the development
of embryos (Waddington et al., 1955; Nishimara and Nimura, 1958; De
Vincentis, 1960) or of fern gametocyte (Hotta and Oswa, 1958; Hotta et al.,
1959), or of tumour growth (Kidder et al, 1951 ; Mandel et al, 1954) might
be due to a more or less selective action of azaguanine upon the synthesis of
individual proteins at certain stages of development (O'Brien, 1959). In
other systems, general inhibition of protein synthesis prevails (Dutton et al,
1958).

(c) Summing Up. It can be concluded from the foregoing that slight
modifications of RNA brought about in vivo by ribonuclease or by the

ROLE OF NUCLEIC ACIDS 79

fraudulent incorporation of abnormal purines or pyrimidines, result in deep
changes in protein synthesis. The effects can be more or less drastic:
protein synthesis can be stopped or only reduced to a certain extent. Under
these latter conditions, different proteins can be affected to various degrees
and abnormal proteins can be produced. This clearly indicates that the
integrity of certain RNA fractions is a necessary requirement for the syn-
thesis of normal protein. That changes in protein structure result from
changes in RNA composition is probably the most convincing evidence for
a control of protein structure by RNA. Of all cellular RNAs, soluble
RNAs are the first to be affected by the destructive action of RNase and the
first to be deeply changed by analogues. It would seem that modification of
some soluble RNA fraction is enough to block or to upset the specificity
of the process. Evidence from the restoration of protein synthesis after
inhibition by ribonuclease or by azaguanine indicates, however, that
soluble RNA is rather easily replaced by the cell when favourable condi-
tions are returned. To the contrary, damage caused by the same agents,
most probably to another type of RNA, which also affect the specificity of
protein synthesis, becomes easily irreversible.

A thorough study of changes brought about in protein structure by
modifications of RNA may afford a means of studying the most puzzling
aspect of protein synthesis, namely the control of specificity.

Beside the agents mentioned above — purine and pyrimidine analogues
and ribonuclease — the use of nitrous acid for the limited deamination of
nucleic acid adenine, guanine and cytosine might prove very informative
in this respect. Hexetidine has also been shown to alter the specificity of
protein synthesis in a bacterium (Halvorson and Gorman, 1959).

Chloromycetine strongly inhibits protein synthesis in bacteria. The
action of this antibiotic closely resembles the effects of azaguanine in many
respects; it probably acts at about the same point upon the mechanism
of protein synthesis. Closer analysis of the various substances which dis-
sociate protein and RNA synthesis in different ways (Gale and Folkes,
1953 ; Gale, 1958) remains certainly one of the promising approaches to the
understanding of the exact function of RNA.

4. RNA as the Genetic Messenger

At the end of Chapter II, it was concluded that DNA is not directly
involved in the synthesis of most proteins, and that the genetic informa-
tion it contains must be transferred to some other substance before serving
in the synthesis of cytoplasmic proteins.

The common belief is at present that the substance which carries the
genetic information from DNA to cytoplasmic centres of protein synthesis
must be a RNA. This is one side of what Crick (1958) called the 'central
dogma', thus making it clear that this opinion rests largely on a priori

80 THE BIOSYNTHESIS OF PROTEINS

considerations. The intermediate depositary of genetic information must
be able to receive the information from DNA, to record it in some way and
to convey it to the sites of protein synthesis. As we shall see, RNA seems
to fulfil these a priori requirements.

(a) Do RNA molecules carry genetic information? Virus RNA can indeed
transfer to a living cell the specific information required for making at least
the protein of the virus (see Chapter I). It is therefore quite reasonable to
suspect that some fraction of cellular RNA might play a similar role, with-
out prejudice as to what fraction this may be.

There is every reason to believe that the genetic information contained
in DNA or in virus RNA consists in a specific arrangement of the purines
and pyrimidines on the phosphate-carbohydrate backbone. The two
families of nucleic acids are so closely related, that they must speak closely
similar languages. The translation of DNA information into RNA language
must be a rather simple matter. The easiest way to visualize how RNA
could receive information from DNA and store it in its own structure, is to
assume that specific RNA is made under direct control of DNA. Interesting
attempts have been made at formulating in terms of structural chemistry
possible template processes by which a unique sequence of bases might be
imposed upon a nascent RNA chain which would take shape in close
contact with DNA. Thus Lockingen and De Busk (1956) developed this
idea in rather general terms, and suggested that the strands of the DNA
double helix could separate and a RNA chain would organize under the
influence of the bases of single DNA strands. Although the DNA structure
which was then assumed by the authors is probably outdated by now, the
same idea might soon be considered again in a revised form since single
strand DNA have been shown to exist (Sinsheimer, 1959).

Stent (1958) and Zubay (1958) developed models in which an RNA chain
would be organized in the deep grove of a DNA double helix. The se-
quence in RNA is supposed to be imposed by that of the pairs of bases in
DNA (Fig. 23).

Experimental results obtained by Rich and associates with synthetic
polynucleotides make processes of this type rather plausible. When equiva-
lent amounts of polyadenylic and polyuridylic acids are mixed together in
dilute salt solution, a double helix forms in which each adenine in the
polyadenylic is hydrogen bonded to a uracil in the polyuridylic chain (Rich,
1957; Felsenfeld, 1958; Steiner and Beers, 1959). The structure of this
double helix is very similar to that of DNA. If enough magnesium ions
or polyamines are added, the double helix is able specifically to bind
another polyuridylic acid chain. A three-stranded molecule is thus formed,
one component of which is loosely bound to a very stable double helix
(Felsenfeld and Rich, 1957; Zubay, 1958; Rich, 1959a, 1959b).

ROLE OF NUCLEIC ACIDS

ADENINE ADENINE THYMINE

81

ADENINE
CYTOSiNE / GUANINE

CYTOSiNE

, Guanine

(a) According to Stent (1958).

^(23"),

'0--' \(Rl

/Guoniney--^' „*.- N>

(DNA)r ^elix ^

>H \IV2i0A

. :.A^ r/(22'')

(NHj) 3-OA. ^ / ^

/Cytosine
IN A)/

(b) According to Zubay (1958b).

Fig. 23. Purine-pyrimidine triplets which may be involved in directing
the synthesis of an RNA chain upon a DNA double helLx.

82 THE BIOSYNTHESIS OF PROTEINS

Hybrid helices can also form in which one strand with a DNA backbone
wraps around another strand with a RNA backbone in such a manner that
the strands are held together by complementary hydrogen bonds formed
between purine and pyrimidine residues (Rich, 1960). Should RNA be
copied from DNA by such a process, a complete correlation between
RNA and DNA composition would be expected only if both DNA chains
were copied and if each DNA unit would produce about the same number
of RNA replicas.

Belozersky and Spirin (1958) determined the base composition of DNA
and total RNA of nineteen bacterial species belonging to various groups.
As shown in the tables, the composition of DNA varies widely, from
species in which the Adenine Thymine pair accounts for 70 per cent of the
bases, to types in which it accounts for about 25 per cent only. In the same
organisms, the composition of total RNA differs much less from species to
species. However, a certain correlation is observed between the composi-

/Q_i_p\ /

tion of RNA and DNA: the ratio ,—-, — — -' seems to vary as a linear func-

/(A+U) ^

tion ofrj^—r^ in DNA (Belozersky, 1959).

Although the composition of global RNA does not correspond to that
of global DNA of the same organism, the observed correlation together
with the fact that DNA composition in a given species is rather homogen-
ous down to the level of smaller molecules (Rolfe and Meselson, 1959)
suggests that a fraction of the RNA has indeed a composition which corre-
sponds to that of DNA. Belozersky's results are therefore compatible with
the idea that a fraction of bacterial RNA is organized under the control of
DNA, and might possibly carry specific genetic information.

Volkin and Astrachan (1957) made very interesting observations on RNA
synthesis at the beginning of phage infection. The distribution of ^sp
incorporated into RNA indicates that the small RNA fraction which rapidly
forms after infection has the same base composition as phage DNA (with
uracil substituted for thymine). Using the same method. Yeas and Vincent
(1960) reached the conclusion that yeast contains a relatively small fraction
of RNA with a high turnover rate, whose composition is very similar to
that of yeast DNA, with uridylic acid replacing thymidylic acid. The
authors, for this reason, suspect that this fraction may be a primary gene
product, a specific RNA copied from DNA and which carries information
to centres of protein synthesis. A new RNA fraction which may deserve
special attention has recently been isolated by Sibatani et al. (1960) from
animal tissues. It seems to be made at the same time as DNA and to be
metabolically as inert as DNA.

None of these observations can be taken as definite evidence that specific

ROLE OF NUCLEIC ACIDS

83

ribosenucleic acids carrying genetic information for the synthesis of
proteins do exist in cells. However, they point towards directions where
such evidence should be sought.

DNA COMPOSITION IN DIFFERENT BACTERIA

Content of bases

Species

in molar

per cent

Pur.

G + T

G+C

G

A

C

T

Pyr.

A + C

A + T

Clostridium perfringens

15-8

34-1

15-1

35-0

1-00

1-03

0-45

Staph, poyogenes aureus

17-3

32-3

17-4

33-0

0-98

1-01

0-53

Pasteurella tularensis

17-6

32-4

17-1

32-9

1-00

1-02

0-53

Proteus vulgaris

19-8

30-1

20-7

29-4

1-00

0-97

0-68

Escherichia coli

26-0

23-9

26-2

23-9

1-00

1-00

1-09

Proteus morganii

26-3

23-7

26-7

23-3

1-00

0-98

M3

Shigella dysenteriae

26-7

23-5

26-7

23-1

1-01

0-99

M5

Salmonella typhosa

26-7

23-5

26-4

23-4

1-01

1-00

M4

Salmonella typhi-muriuni

27-1

22-9

27-0

23-0

1-00

1-00

M8

Ervinia carotovora

27-1

23-3

26-9

22-7

1-02

0-99

117

Corynebact. diphtheriae

27-2

22-5

27-3

23-0

0-99

101

1-20

Azotobacter agile

28-3

21-4

26-5

23-8

0-99

1-08

1-21

Azotobacter vinelandii

27-4

22-1

28-9

21-7

0-98

0-97

1-28

Azotobacter chroococcum

28-7

20-5

28-5

22-2

0-97

1-04

1-34

Aerobacter aerogenes

28-8

21-3

28-0

21-9

1-00

1-03

1-31

Mycobacterium vadosum

29-2

20-7

28-5

21-6

1-00

1-03

1-37

Brucella abortus

29-0

21-0

28-9

211

1-00

1-00

1-37

Alcaligenes faecalis

33-9

16-5

32-8

16-8

0-98

103

2-00

Pseudomonas aeruginosa

33-0

16-8

34-0

16-2

0-99

0-97

2-03

Mycobacter. tuberculosis BCG

34-2

16-5

33-3

160

1-03

101

2-08

Sarcina lutea

36-4

13-6

35-6

14-4

1-00

1-03

2-57

Actinomyces globisporus strep-

tomycini

36-1

13-4

37-1

13-4

0-98

0-98

2-73

Actinomyces globisporus flave-

olus

36-3

13-8

37-2

12-7

1-04

0-96

2-77

Actinomyces griseus

35-8

13-8

37-4

13-0

0-97

0-96

2-73

Actinomyces viridochromoge-

nes

36-6

13-3

37-2

12-9

0-99

0-98

2-80

Proactinomyces citreus

35-3

14-2

36-7

13-8

0-98

0-96

2-57

Micromonospora coerulea

36-2

14-3

35-6

13-9

1-02

1-04

2-53

Abbreviations: G = guanine; A = adenine; C = cytosine; T = thymine;
Pur. = purine bases; Pyr. = pyrimidine bases.

Fig. 24. (Belozersky, 1959).

(b) Is cytoplasmic RNA made in close contact with DNA? The pathways
of RNA synthesis remain very obscure. It is true that Grunberg-Manago
and Ochoa (1955) discovered an enzyme which catalyses the condensation

84

THE BIOSYNTHESIS OF PROTEINS

of ribonucleoside diphosphates into polynucleotides the basic chemical
structure of which is identical to that of RNA (Grunberg-Manago et al.,
1956; Ochoa, 1959). DNA does not seem to be required for this reaction,
but the enzyme also lacks specificity, and the nature of the polynucleotides
which are made under its action depends essentially on the composition of
the mixture of nucleoside diphosphates. It is only little influenced by the
nature of the primer.

RNA COMPOSITION IN DIFFERENT BACTERIA

Nucleotide content

Species

in molar

per cent

Pur.

G+U

G+C

G

A

C

U

Pyr.

A + C

A + U

Clostridium perfringens

29-5

28-1

22-0

20-4

1-36

1-00

1-06

Staphyl. pyogenes aureus

28-7

26-9

22-4

22-0

1-25

1-03

1-05

Pasteurella tularensis

29-8

27-3

21-0

21-9

1-33

1-07

103

Proteus vulgaris

31-0

26-3

24-0

18-7

1-34

0-99

1-22

Escherichia coli

30-7

26-0

24-1

19-2

1-31

1-00

1-21

Proteus morganii

3M

26-0

23-7

19-2

1-31

1-01

1-21

Shigella dysenteriae

30-4

25-9

24-4

19-9

1-29

0-99

1-21

Salmonella typhosa

30-8

26-1

24-0

19-1

1-32

1-00

1-21

Salmonella typhi-murimn

31-0

26-1

23-8

191

1-33

1-00

1-21

Ervinia carotovora

29-5

26-5

23-7

20-3

1-27

0-99

114

Corynebact. diphtheriae

31-6

23-1

23-8

21-5

1-21

M3

1-24

Azotobacter agile

31-0

24-2

26-0

18-7

1-23

0-99

1-33

Azotobacter vinelandii

30-3

23-9

25-5

20-2

1-19

102

1-27

Azotobacter chroococcum

30-4

24-7

24-7

20-1

M8

1-02

1-23

Aerobacter aerogenes

30-3

26-0

24-1

19-6

1-29

1-00

119

Mycobacterium vadosmn

31-7

23-8

23-5

21-0

1-25

M2

1-23

Brucella abortus

30-2

25-4

24-9

19-5

1-26

0-99

1-23

Alcaligenes faecalis

30-9

25-7

24-1

19-3

1-31

1-01

1-22

Pseudomonas aeruginosa

31-6

25-1

23-8

19-5

1-31

1-05

1-24

Mycobacter. tuberculosis BCG

33-0

22-6

26-1

18-3

1-25

1-05

1-45

Sarcina lutea

32-7

23-2

24-2

19-9

1-27

Ml

1-32

Actinomyces globisporus strep-

tomycini

311

23-8

25-2

19-9

1-22

1-04

1-29

Abbreviations:

G = guanylic acid; A
U = uridylic acid; Pur. =
nucleotides.

= adenylic acid; C = cytidylic acid;
purine nucleotides; Pyr. = pyrimidine

Fig. 25. (Belozersky, 1959).

No one knows whether the function of polynucleotidephosphorylase in
vivo is to make RNA or to destroy it. There are reasons to believe that the
synthesis of RNA follows another pathway, in which amino acids are some-
how involved. In Staphylococcus aureus and in Escherichia coli, amino acid

^.,

'*^'

?r«S

■*- uk-v' ,.*y*"

Fig. 26. Portion of a lampbrush chromosome showing many loops of
various lengths. Phase contrast. Magnification 700 (Gall, 1956).

ROLE OF NUCLEIC ACIDS 85

deficiency suppresses not only protein synthesis but RNA synthesis as well
(Sands and Roberts, 1952; Gale and Folkes, 1953; Borek et al, 1955). In
yeast, sulphur starvation prevents the synthesis of both protein and nucleic
acid, although the formation of soluble nucleotides is not impaired
(Schmidt et al., 1956). Such observations at first suggested that RNA
synthesis may depend on protein synthesis. However, chloramphenicol
inhibits protein synthesis without preventing RNA formation (Gale, 1953 ;
Wisseman et al., 1954) and amino acids stimulate RNA synthesis even
when protein synthesis is suppressed by the antibiotic (Gale and Folkes,
1953).

RNA synthesis in Escherichia coli requires the simultaneous presence of
all the amino acids even when protein synthesis is 98 per cent inhibited by
chloramphenicol (Gros and Gros, 1956; Pardee and Prestidge, 1956; Yeas
and Brawerman, 1957). With amino acids requiring strains, under such
conditions, the addition of a very small amount of the limiting amino acid
will cause the synthesis of a large amount of RNA. One amino acid mole-
cule makes possible the polycondensation of at least eight nucleotide
residues (Gros and Gros, 1957). These observations can be accounted
for by assuming, e.g. that the RNA precursors are amino acid-
nucleotide compounds. Further developments will show whether this
assumption is correct. Finally, an enzymic synthesis of RNA from
ribonucleoside triphosphates has been reported (Chung et al., 1960;
Weiss, 1960).

The possible involvement of DNA in RNA synthesis has not been clearly
observed so far in metabolic studies (see, however, Chung et al., 1960). But
our present knowledge on the pathways of RNA synthesis must be regarded
as quite rudimentary.

Histochemical research indicates that the nucleus is an active centre
of RNA synthesis, and that some RNA probably forms in the vicinity of
DNA. The lampbrush chromosomes of amphibian oocytes (Fig. 26) and
the giant chromosomes of insect salivary glands display protusions into
the nuclear sap, which are called loops in the first case, puffs in the second.
These contain RNA. Autoradiography studies, completed by ribonuclease
treatment of the preparations, clearly established that the loops of lamp-
brush chromosomes and the puffs of giant chromosomes are the sites of a
very active incorporation of radioactive precursors into this RNA (Taylor
et al., 1955; Ficq and Pavan, 1957; Ficq et al., 1959). This probably
reflects a net synthesis of RNA, since corresponding increases in basophilia
were observed at similar locations (Pavan and Breuer, 1955).

In roots of Vicia faba, the RNA which is present in small amounts in
chromatin, the DNA containing structure in resting nuclei, incorporates
cytidine very rapidly (Woods, 1959; Woods and Taylor, 1959). Human
amnion cells in tissue culture incorporate cytidine-^H in the extranucleolar

86 THE BIOSYNTHESIS OF PROTEINS

region faster than in nucleoli and much faster than in cytoplasm (Gold-
stein and Micou, 1959).

A nuclear element in which RNA accumulates in considerable amount
is the nucleolus. Nucleoli are often observed to stay in close contact with
certain regions of the chromosomes and there are many indications that the
nucleoli are in some way produced by regions of the chromosome, often
called nucleolar organizers (for a discussion see Brachet, 1957). During
oogenesis in amphibians, the lampbrush chromosomes to which we have
referred before are especially developed at the time when synthesis of yolk
protein is most active, and nucleoli are also very numerous at this stage.
The nucleoli might be regarded as a transitory storage place for RNA
made in the chromosomes. It must be noted, however, that the nucleolar
organizers are heterochromatic regions of the chromosomes, i.e. regions
characterized by special staining properties, and which are believed to
contain genetic controlling element involved in the quantitative expression
of characters, whereas the genes of the sort we have considered so far in
relation with protein structure are located in the euchromatin.

The histochemical observations on the whole support the idea that some
RNA may be built up within the chromosome or in close contact with
the genetic material. They are compatible with the hypothesis that RNA
receives genetic information from DNA, although they cannot be taken as
evidence for it.

The next task that the genetic messenger should accomplish is getting
out of the nucleus and reaching the centres of protein synthesis in the cyto-
plasm. What is the evidence for the transfer of RNA from nucleus to
cytoplasm?

Precursors of RNA are usually incorporated more rapidly into nuclear
RNA than into the average cytoplasmic RNA (Jeener and Szafarz, 1950;
Smellie et ah, 1953, 1955; Barnum et al., 1953). From the kinetics of the
incorporation, it was concluded that the bulk of RNA in liver cytoplasm
does not derive from the nucleus, but that the possibility exists that some
cytoplasmic RNA might be produced in the nucleus (Smellie et al, 1953;
Barnum et «/., 1953).

This conclusion rests on several assumptions which are very difficult to
check and which now appear as oversimplifications, since it is known how
heterogeneous both nuclear and cytoplasmic RNA are.

Enucleation experiments were designed to establish whether RNA can
be formed at all in the absence of the nucleus. In Acetahularia, or in sea
urchin eggs, enucleation has no effect on incorporation of labelled orotic
acid or glycine into RNA (Malkin, 1954; Brachet and Szafarz, 1956;
Brachet et ah, 1955). Precursors are also incorporated into the RNA of
rabbit reticulocytes (Kruh and Borsook, 1955). Incorporation of precursors
into total cytoplasmic RNA cannot, however, be equated to RNA synthesis,

ROLE OF NUCLEIC ACIDS 87

it might possibly be due, e.g. to chain end metaboHsm (see p. 66). Direct
determination of the RNA content of batches of nucleate and enucleate
parts of Acetahidaria indicated a net RNA synthesis of short duration in
enucleate fragments (Brachet et al., 1955). Other groups of workers
observed an immediate cessation of RNA synthesis after enucleation
(Richter, 1957; Noara et al, 1959), or even a loss of RNA. It is probable
therefore that the RNA found in the cytoplasm comes largely from the
nucleus. On the other hand, cytoplasmic RNA in Acetahularia is not inert,
it seems to undergo a turnover process (degradation and resynthesis), which
is independent of the nucleus. When this cytoplasmic RNA metabolism is
interfered with by ultraviolet irradiation, net loss is observed. This is, how-
ever, followed by recovery and a net synthesis of RNA is then observed in
enucleate fragments (Richter, 1959). Conflicting results obtained by differ-
ent groups are probably due to differences in physiological conditions of
the algae under study in the individual cases. Different adjustment of the
cytoplasmic steady state of RNA may result in net synthesis (Brachet et ah,
1955), absence of variation (Richter, 1957) or net decrease of RNA content
of the cytoplasm (Naora et al., 1959). Chloroplastic RNA forms in enucleate
cytoplasm, apparently at the expense of other cytoplasmic RNAs (Naora
et al., 1960). The cytoplasmic RNA turnover observed by Richter (1959)
also explains that incorporation of various precursors into cytoplasmic
RNA is largely independent of the nucleus. All the data on net synthesis of
RNA in Acetahularia nevertheless make it clear that this process is closely
dependent on the presence of the nucleus, and much more so than protein
synthesis which continues undisturbed after enucleation. Recent results by
Richter (1959) are especially convincing: if a nucleate fragment oi Acetahul-
aria is grafted on an enucleate part in which net RNA synthesis had
stopped, the amount of RNA again starts to increase in the old cytoplasmic
fragment.

This is all very suggestive of a nuclear production of RNA which is
secreted into the cytoplasm. The important point would be to know whether
this special RNA fraction is bringing with it information for protein syn-
thesis. Although this question cannot be answered yet, interesting results
relevant to this question were reported by Stich and Plant (1958). Ribo-
nuclease inhibits protein synthesis both in nucleate and enucleate frag-
ments of Acetahularia. When the fragments are transferred to normal
medium after a few days of ribonuclease action, protein synthesis is
restored in nucleate but not in enucleate moieties. This shows that a sub-
stance which is inactivated by ribonuclease and which is involved in protein
synthesis requires the presence of the nucleus for its formation. It is exactly
what would be expected if the 'central dogma' was true.

In Amoeha, the importance of the nucleus for the synthesis of cyto-
plasmic RNA is even more striking than in the case of Acetahularia. Thus

G

88 THE BIOSYNTHESIS OF PROTEINS

the RNA content of enucleate fragments of Amoeba proteus decreases
quickly and strongly during starvation whereas it is maintained in nucleate
halves (Brachet, 1955). If living Amoebae are transferred into a medium
containing ribonuclease, the enzyme enters the cells and destroys a large
part of the RNA both in the cytoplasm and in the nucleus. When the
Amoebae are later returned to normal medium or better to a medium con-
taining yeast RNA, nucleic acid is reformed in many Amoebae; under such
conditions, RNA appears first in the nucleoli and later in the cytoplasm,
and enucleate fragments are unable to restore their nucleic acids (Brachet,
1955 b, c).

More direct evidence for the passage of nuclear RNA into the cyto-
plasm in Amoeba proteus was obtained by Goldstein and Plant (1955).
Amoebae were fed with micro-organism which had been grown in a medium
containing 22PO4. The nuclei of labelled Amoebae were grafted into non-
labelled Amoebae. Autoradiography studies and ribonuclease test indicated
that radioactive phosphorus contained in the nuclear RNA at the beginning
of the experiment was lost by the nucleus and later found in cytoplasmic
RNA. Although in these experiments it is actually the fate of phosphorus
atoms which was studied, not that of RNA molecules, these data strongly
suggested that part of the cytoplasmic RNA originates in the nucleus.

Incorporation experiments using radioactive adenine and uracil in
enucleate cytoplasm of Amoeba invariably showed that enucleation drastic-
ally reduces the incorporation into cytoplasmic RNA (Plaut and Rustad,
1956; Prescott, 1957). Conflicting results have been reported as to whether
cytoplasm can make some RNA in the absence of the nucleus or whether
all cytoplasmic RNA comes from the nucleus. The latter opinion was
advocated by Prescott (1957, 1959) who believes that the low residual
incorporation observed in enucleate Amoeba proteus is due to the activity
of micro-organisms recently phagocyted by the Amoeba. Indeed with an
Acanthamoeba, which can be grown in sterile medium, no detectable incor-
poration of adenine, orotic acid or uracil takes place in enucleate fragments
(Prescott, 1960). An uncertainty which remains is whether the precursors
enter the enucleate parts at all, for absorption of various substances can be
afl^ected by enucleation (see Mazia and Prescott, 1955).

Recent kinetic studies on RNA synthesis in populations of animal cells
also provide evidence that some RNA of nuclear origin can be secreted
into the cytoplasm. Thus cells of the human amnion grown in tissue culture
were exposed for a short time to cytidine-^H, and then replaced in a
medium containing non-labelled cytidine. Autoradiography showed a
progressive movement of the label from nucleus to cytoplasm. All the
label that was in the RNA of the nuclei at the time of transfer into 'cold'
medium was found in cytoplasmic RNA 24 hr later (Goldstein and Micou,
1959).

ROLE OF NUCLEIC ACIDS 89

Perry and Errera (1960) showed that in Hela cells irradiation of the
nucleolus with a microbeam of ultraviolet light (257 mfx) inhibits the
incorporation of tritiated cytidine into both cytoplasmic and nuclear
(extranucleolar) RNA. Kinetic studies are interpreted as indicating that
RNA synthesis takes place independently both in the nucleoli and in the
nucleus (outside the nucleoli), and that both fractions pass into the cyto-
plasm. According to the same studies, less than 10 per cent of the cyto-
plasmic RNA can be formed in the cytoplasm of Hela cells (Perry, 1960).
Working with a similar material Feinendegen et al. (1960) came to the con-
clusion that RNA synthesis begins in the chromatin portion of the nucleus,
and that RNA later passes into the cytoplasm. Amano and Leblond (1960)
also concluded that, in liver, nucleolar and other nuclear RNAs are made
independently and that they behave as precursors of cytoplasmic RNA.

Further support to the hypothesis of a nuclear origin of cytoplasmic
RNA will be found in studies on the labelling of RNA of Drosophila larvae
by radioactive phosphate (Herbert, 1954) or tritiated uridine (Zalokar,
1960).

Clear evidence in the same direction was obtained by Zalokar (1959,
1960b) in experiments with hyphae of Neurospora crassa the content of
which had been stratified by high speed centrifugation. In short exposures
to tritiated uridine followed by dilution of the isotopic precursors with
non-labelled uridine, the nuclear RNA is labelled first, tritium-marked
compounds later appear in the cytoplasm and the labelling of ergastoplasm
increases with time at the expense of nuclear label. Again there remains
the possibility that the labelled molecules which pass from the nucleus to
the cytoplasm are not intact RNA molecules, but products from which
cytoplasmic RNA is built up. But the possibility that RNA does pass is very
good.

The works reviewed above dealt with several types of animal and plant
cells and made use of various experimental approaches. Their results are
not always in entire agreement with one another; however, a general con-
clusion can be safely drawn, namely that the nucleus is a very important
centre of ribosenucleic acid synthesis ; moreover, it is likely that a large part
of the cytoplasmic RNA is made within the nucleus in the vicinity of
chromatin. Clearly, these are very good reasons to retain the hypothesis that
the genetic messenger which carries genetic information from DNA to the
cytoplasmic centres of protein formation is probably made of RNA.

(c) Where is the genetic messenger? It should not be forgotten, however,
that the very existence of a messenger carrying the genetic information has
not been established by direct experiments, it is postulated only because
most proteins arise at a distance from DNA or can be made in the absence
of DNA. The experiments which come closest to establish directly the exist-
ence of genetic information outside DNA, and to provide some indications

90 THE BIOSYNTHESIS OF PROTEINS

about the nature or the state of the compound which carries it, are un-
fortunately not in perfect agreement with each other. Schweet et al. (1958)
isolated microsomes from reticulocytes and showed that the non-sediment-
able fraction was required together with the microsomes for amino acids to
be incorporated into Hb. Liver supernatant can be substituted for reticulo-
cytes supernatant, but liver microsomes even in the presence of reticulocytes
supernatant do not catalyse incorporation into Hb. This indicates that part
at least of the specific information must lay in the microsomes (see also
Allen and Schweet, 1960; Allen et al, 1960). On the other hand, Wain-
wright (1960) working with extracts of Neurospora reached a different
conclusion. The synthesis of tryptophane synthetase could be observed
in cell-free extracts of conidia. Mixed system containing soluble and
sedimentable fractions prepared from wild strain or from a mutant lacking
tryptophane synthetase were used. When a mutant 'particle' fraction was
supplemented with wild type soluble fraction, considerable tryptophane
synthetase was produced. Conversely, a mixture of wild type 'particles'
with mutant 'soluble' fraction failed to develop any detectable activity. The
defective component of the mutant extract has thus been located in the
soluble, non-sedimentable fraction.

It is probable that in such systems, and with the progress of techniques
for isolation of biologically active RNA, it will soon be possible to check
whether the carrier of genetic information is an RNA, or else to discover its
nature.

Reports from several laboratories have indicated that RNA extracted
from micro-organisms might cause the production of certain specific
proteins or might confer specific characters to homologous cells (Mina-
gawa et ah, 1951; Minagawa, 1955; Reiner and Goodman, 1955; Kramer
and Straub, 1956; Hunter and Butler, 1956; Hrubesova et al, 1959;
Kessler, 1957). It must be admitted that the results obtained in these
interesting works are not always sufficiently clear and reproducible. At
present, it is very difficult to evaluate them. If these observations prove to
be correct, they will acquire a fundamental significance comparable only
to bacterial transformation by DNA and to transmission of virus diseases
by isolated RNA.

5. Concluding Remarks

The participation of RNA in protein synthesis has been known for
twenty years. So far, a function has been clearly attributed to only one class
of RNA — the 'transfer RNAs' which are specific carriers of activated
amino acids (see p. 102). But these represent only a small fraction of total
RNA. The exact function of ribosomal RNA is not yet clarified. The
evidence for the participation of this RNA in protein synthesis remains the
correlation between their amount and the intensity of protein synthesis.

ROLE OF NUCLEIC ACIDS 91

and the fact that proteins arise in contact with the ribosomes. The nature
of the evidence has not changed for twenty years; it has simply become
more and more convincing as more numerous and more refined experi-
ments were made. The exact function of ribosomal RNA in protein syn-
thesis is still an enigma.

CHAPTER IV

Chemical Pathways of Protein Biosynthesis

A. ENERGY REQUIREMENT

For many years, it was assumed that the synthesis of protein is brought
about by proteolytic enzymes. An enzyme, acting as a true catalyst, should
be able to promote a reaction in both directions. Since a mixture of pro-
teolytic enzymes split protein into smaller peptides and finally into amino
acids in vitro, it seemed reasonable to assume that the same agents are
responsible for amino acid condensation into proteins in vivo. The con-
ditions within the living cell were supposed to be such that the hydrolytic
process was reversed. Plastein formation in concentrated protein hydro-
lysate and the formation of relatively insoluble peptides under the action of
proteolytic enzymes, provided some support for this theory. However, it
was untenable on thermodynamical grounds. The standard free energy of
hydrolysis of a dipeptide is about —3 kcal. The figure becomes less
negative when the peptide chain becomes longer but it is still about —1
kcal for a soluble polypeptide of 'infinite length' (Borsook and Huffman,
1945; Linderstrom-Lang, 1949, 1952). The concentration of the amino
acids in the cell is low; the amount of a dipeptide in equilibrium with the
corresponding pair of amino acids must be very small indeed (Linder-
strom-Lang, 1949; Borsook, 1954), and for each further condensation the
equilibrium concentration will rapidly decrease and become vanishingly
small. These considerations led to the prediction that the condensation of
amino acids into elementary peptides and into polypeptides will not pro-
ceed to any appreciable extent unless it is driven by some process making
energy available for the reaction (Lipmann, 1941 ; Linderstrom-Lang,
1949, 1952).

Borsook and Dunoff (1940) were the first to check this prediction ex-
perimentally in the case of hippuric acid formation ; they determined the
equilibrium constant of hippuric acid hydrolysis, and were able to show
that the amount of hippuric acid actually synthesized in vitro by kidney or
liver slices from various animals is 60-75 times greater than the amount
corresponding to the thermodynamic equilibrium. Coupling with an
energy-yielding process was also indicated by the fact that inhibition of
respiration either by cyanide or by oxygen deprivation completely in-
hibited the synthesis. Lipmann (1945) later showed that the acetylation of

92

CHEMICAL PATHWAYS 93

amines which also results in the formation of a peptide-like linkage, is
blocked by dinitrophenol, indicating that the energy required for peptide
bond formation is provided by oxidative phosphorylation. With a system
of soluble enzymes from pigeon liver, the requirement for ATP was
established, and it was found that one mole of ATP is split for each mole of
amine acetylated. Similar conclusions were rapidly extended to the synthesis
of other small peptides: hippuric acid (Borsook and Dubnoff, 1947),
/)-aminohippuric acid (Cohen and McGilvery, 1946, 1947) and glutathione
(Johnston and Bloch, 1951 ; Webster, 1953). Evidence that the synthesis of
enzymes in bacteria and in yeast depends on phosphorylation had mean-
while been obtained (Monod, 1944; Spiegelman, 1946).

Peptidic bond formation in small peptides was thus regarded as a model
of possible mechanisms of polypeptide biosynthesis and these studies had
indeed a fundamental influence on the discovery of what is known at
present about the pathways of protein formation. The advent of i^C-
labelled amino acids was the other element which made possible the rapid
progresses of the last decade in this field.

As soon as the requirement for energy coupling was discovered for small
peptidic compounds, it was readily established that the incorporation of
labelled amino acids into protein of tissue slices or of homogenates also
depends on respiration (Frantz et al, 1947, 1948; Winnick et al., 1947;
Melchior et al, 1948; Peterson and Greenberg, 1952). Amino acid in-
corporation, like the synthesis of small peptides, is driven by oxidative
phosphorylation since the inhibition of the incorporation process by dinitro-
phenol, runs parallel to the inhibition of phosphorylation (Frantz et al.,
1948). Finally, it was clearly established that ATP or systems able to
regenerate it are required for protein synthesis in homogenates (Peterson
and Greenberg, 1952; Siekevitz, 1952; Zamecnik and Keller, 1954). It
was probable therefore that the condensation of amino acids into polypep-
tides involved — at one stage at least — a coupling with ATP splitting. A
great many works have now confirmed these data and extended them to
many various tissues and organisms. Coupling with ATP utilization is a
quite general requirement for amino acid incorporation into protein.

Most workers in the field were long reluctant to equate amino acid in-
corporation to de novo protein synthesis from amino acids. The possibility
that incorporation corresponds to replacement of an old amino acid unit
by a new one within polypeptides has been a constant source of worry.
This question had already been raised by Schoenheimer et al. (1939) who
were the first to observe amino acid incorporation into protein in living
animals, and it became more acute in works with tissue slices or homo-
genates where incorporation is always very low and where no measurable
net increase in protein was observed. Exchange incorporation found
apparent support in certain facts. For instance the amino acid 'analogue'

94 THE BIOSYNTHESIS OF PROTEINS

/)-fluorophenylalanine suppresses the synthesis of enzymes and depresses
the incorporation of phenylalanine ; but it does not affect the incorporation
of the other amino acids (Halvorson and Spiegelman, 1952; Rabinovitzeia/.,
1954). The first interpretation of these facts was that the amino acid ana-
logue blocks phenylalanine utilization and net protein synthesis, and that
the unaffected incorporation of the other amino acids must therefore be due
to an exchange process. This interpretation later proved incorrect ; actually,
^-fluorophenylalanine does not prevent protein synthesis, it competes with
phenylalanine and it is incorporated into proteins instead of the normal
amino acid (Baker et ah, 1954; Munier and Cohen, 1956, 1959; Kerridge,
1959; Vaughan and Steinberg, 1960; Richmond, 1960). The incorporation
of all the other amino acids in the presence of the analogue is due to net
synthesis of abnormal proteins in which a large part of the phenylalanine is
replaced by the analogue. Many of the abnormal enzymes made in the
presence of fluorophenylalanine are inactive, hence the observation that the
synthesis of enzymes (as measured by the increase of enzymic activity) is
inhibited. Other amino acid analogues act in the same way (Sharon and
Lipmann, 1957; Pardee etal, 1957; Brawerman and Yeas, 1957; Gross and
Tarver, 1955; Vaughan and Steinberg, 1959; Munier and Cohen, 1959;
Cowie and Cohen, 1957 ; Cohen et ah, 1958 ; Rabinovitz ef «/., 1955). Clearly,
there is no reason to call upon exchange processes for explaining the experi-
mental results.

Another group of observations which were regarded as direct evidence
for exchange processes was made by Gale (1957) on disrupted Staphylo-
cocci. The time curve of the incorporation of glutamic acid into acid
insoluble compounds is not the same when glutamic acid is added alone or
together with all the other amino acids. When a mixture of amino acids is
provided, the incorporation is linear and largely irreversible, for the
addition of non-labelled glutamic acid causes no loss of incorporated I'^C.
To the contrary when the system is presented with glutamic acid alone,
incorporation is fast but it rapidly stops ; the time curve resembles a satura-
tion or adsorption curve. Moreover, non-labelled glutamic acid causes a
release of part of the previously incorporated labelled compound, indicat-
ing a replacement by mere exchange (Gale, 1957). These facts, no doubt,
were well observed but their interpretation at present must be re-examined ;
at the time they were made, the existence of soluble RNA and its capacity
of binding amino acids reversibly was not known ; it is possible that part
of the exchangeable glutamic acid was bound in this way or at the end of
polypeptide (Webster, 1959). It was not known either that in bacterial
preparations amino acids can be incorporated into peptide constituents of
cell wall material (Mandelstam and Roger, 1958; Chantrenne and Devreux,
1958, 1960; Hancock and Park, 1958; Richmond, 1959; Roodyn and
Mandel, 1960) and it would seem that part of the glycine or glutamic acid

CHEMICAL PATHWAYS 95

incorporated into acid-insoluble material in the absence of other amino
acids, is actually incorporated into cell wall material rather than into
protein (Gale and Folkes, 1958; Mandelstam and Rogers, 1959). Thus, the
apparent evidence provided by the quoted experiments in favour of direct
exchange of free amino acids with amino acid residue in a protein cannot
any more be regarded as valid.

It has been most clearly shown that in exponentially growing bacteria
amino acid incorporation into protein is essentially irreversible (Rotman
and Spiegelman, 1954; Hogness et al., 1955). In animal or plant tissue,
in yeast and in resting bacteria, a protein turnover does occur (Halvorson,
1958; Mandelstam, 1957, 1958), but there is no reason to assume that it is
due to an amino acid exchange; protein turnover probably reflects the
continued formation of new molecules from amino acids released by des-
truction of other protein molecules. Exchange process can still be called
upon for explaining unequal labelling of proteins in various positions, but
it is only one of several possible interpretations of this phenomenon which
is still completely obscure (see p. 115).

In conclusion, it may be assumed that amino acid incorporation within
polypeptide chains is due to net synthesis of protein material; the only
restriction being that it may not always correspond to the formation of
complete or perfect protein molecules.

ATP requirement for amino acid incorporation confirmed that protein
synthesis is driven by energy yielding reactions, and indicated ATP as the
essential energy distributor in this process as well as in many other synthe-
ses. The mechanism of the energy coupling has been the object of many
studies. An early working hypothesis assumed that ATP was used in the
making of small peptides, and that polypeptides resulted from rearrange-
ments of amino acids between these peptides by a series of transpeptidation
reactions (Fruton, 1950, 1952, 1957; Hanes et al, 1950; Waelsch, 1952;
Linderstrom-Lang, 1949, 1952; Virtanen, 1950). The justification of this
hypothesis was that it accounted for the energy coupling and ATP require-
ment on one hand, and that it implicated on the other hand many well
observed transpeptidation processes catalysed by proteolytic enzymes.
Transpeptidations involving esters or amides of amino acids or peptides can
indeed result in the formation of rather long peptides in vitro (Fruton et al.,
1951, 1953 ; Jones ^^«/., 1952; Durell and Fruton, 1954; Waley and Watson,
1954; Blau and Waley, 1954; Schweet et al, 1948; Brenner et al, 1950;
Tauber, 1952; Kaganova and Orekhovich, 1954; Watson, 1956; Neumann
et al , 1 959). Glutamine and glutathione received special attention as possible
initiators of protein formation, because they can transfer their glutamyl
residue to a variety of amino acids and peptides (Hanes et al, 1950, 1952,
1953; Waelsch, 1952; Hird and Springell, 1954) and it was conceivable
that a series of transfers of carboxyl or amino moieties of small peptides could

96 THE BIOSYNTHESIS OF PROTEINS

result in polypeptide formation. However attractive this hypothesis may
have looked for some time, no convincing evidence for the operation of such
a mechanism has been provided, and direct attempts to test the idea of the
participation of glutathione for instance as an initiator of amino acid
incorporation have led to a negative conclusion (Hendler and Greenberg,
1954; Flavin and Anfinsen, 1954; Askonas et ah, 1955; Barry, 1956).
Besides, glutamyl transpeptidases are found only in a very few tissues
(Revel and Ball, 1959). If polypeptides were formed by a series of trans-
peptidations at the energy level of the peptide bond, the concentration of
free intermediary peptides would be rather high, for the equilibrium
constant of such transpeptidations should be close to unity. And no free
peptides have been found to accumulate in living cell to any considerable
extent. There is at present no positive reason to think that proteolytic
enzymes play an important part in protein synthesis, neither that they play
any part in the process. It is not excluded, however, that they might be
involved in the finishing steps of protein formation (Horowitz and Hauro-
witz, 1959); limited proteolysis is clearly involved in zymogen activation
(Davie and Neurath, 1955; Desnuelle and Fabre, 1955).

An alternative to the hypothesis considered above for explaining how
energy is funnelled into protein synthesis, is to assume that the amino
acids, or at least some of them, are activated directly in some process
involving ATP, and that the activated forms of the amino acids then con-
dense into polypeptides.

B. THE ACTIVATION OF AMINO ACIDS

1 . Amino Acid Activation Enzymes

Analysis of the enzymic acetylation of aromatic amines by Lipmann's
group led to the basic discovery that ATP, which is used up stoichio-
metrically in the process, is not split in the 'usual' way into ADP and
phosphate; instead, it is broken into AMP and inorganic pyrophosphate
(Lipmann, 1952; Jones et al., 1953). This was the first observation of the
direct utilization of the second energy rich bond of ATP. Another example
of this new type of ATP cleavage was soon found by Maas and Novell!
(1953) in pantothenic acid synthesis from pantoic acid and ^-alanine. Ex-
periments with purified preparations of the enzyme provided evidence that
the formation of pantothenic acid involves the production of an enzyme-
bound pantoyl adenylate as an intermediate. The evidence was as follows :
the purified preparation catalyses an exchange of the two terminal phos-
phates of ATP with inorganic pyrophosphate, but this exchange requires
the presence of pantoate specifically. In the presence of concentrated

CHEMICAL PATHWAYS 97

hydroxylamine, pantoylhydroxamic acid and inorganic pyrophosphate are
formed in stoichiometric amounts, but this reaction is much slower than
the exchange. These experimental facts can be explained by the following
scheme (Maas, 1955) —

E + ATP ^-"-"- "^ E-AMP ~pantoate + PP
E-AMP ^pantoate + NH^OH ^ E + AMP + pantoyl-NHOH

The formation of a bond between AMP and pantoate was confirmed by the
observation of a transfer of i^O between the carboxyl group of pantoic acid
and AMP.

It was soon found that a relatively slow pyrophosphate-ATP exchange,
which takes place in soluble extracts of rat liver, is strongly stimulated by a
mixture of the natural amino acids, and that hydroxamates are formed in
the presence of amino acids and high concentration of hydroxylamine;
leucylhydroxamate was identified among them (Hoagland, 1955). Com-
pletely similar observations were made by De Moss and Novelli (1955)
with extracts of many bacterial strains as well as yeast and moulds. That
the reactions involved are quite similar to those of pantoate activation was
soon confirmed. Leucyladenylate prepared by chemical synthesis when
incubated with these bacterial extracts and inorganic pyrophosphate forms
ATP, and free leucine appears (De Moss et al, 1956). Heavy oxygen is
exchanged between the carboxyl of an amino acid and the AMP moiety of
ATP (Hoagland et al., 1957; Bernlohr and Webster, 1958) showing that
at some time during the process the carboxyl group of the amino acid
shares one oxygen atom with AMP, not with the pyrophosphate residue.

These experiments clearly established the existence of a direct activation
of amino acids. Amino acid activation enzymes were then sought and found
in all kinds of animal or plant tissues and in micro-organisms (see reviews
by Novelli and De Moss, 1957, and by Chantrenne, 1960).

The discoverers of the amino acid activation enzymes noticed that if the
total concentration of amino acids is kept constant, the ATP-pyrophos-
phate exchange is promoted as the number of individual amino acids is
increased. This strongly suggested that the different amino acids did not
compete for a common activation enzyme, and that there were probably
several enzymes with different specificities. When activation was studied
separately for each individual amino acid, great differences in efficiency in
promoting the pyrophosphate exchange were observed between them (De
Moss and NovelH, 1955, 1956; Hoagland et al, 1956; Berg, 1956). Some of
the natural amino acids had so little effect on the exchange that for a time it
was believed that a few of the natural amino acids only can be activated in
such reactions. Later on, activation of all amino acids was observed in
bacteria (Nisman et al., 1957, 1958; Nisman, 1959) in animal and plant
tissues (Lipmann, 1958; Webster, 1959). But it remains that the reaction

98 THE BIOSYNTHESIS OF PROTEINS

of some amino acids, like proline, histidine, threonine, tryptophan, leucine
is usually strong, whereas that of arginine, glutamic acid or asparagine is
often so weak as to be difficult to show (Lipmann, 1958). This may be due,
in part at least, to differences in stability of the individual activation
enzymes in tissue extracts, as well as to exacting requirements of some of
them (Schweet et ah, 1957; Nisman, 1959; Webster, 1959). For instance
a purified alanine-activation enzyme from rat liver is very labile, it loses its
activity by freezing and thawing, and it is rapidly inactivated by oxygen
(Holley and Goldstein, 1959).

Davie et al. (1956) were able to isolate from beef pancreas an almost pure
enzyme which is highly specific for L-tryptophan ; the only other amino
acids which react with this enzyme are certain tryptophan analogues
(Sharon and Lipmann, 1957). Enzymes which activate other amino acids
specifically have now been isolated or purified to various degrees: the
enzyme for methionine activation was purified from yeast (Berg, 1956),
tryptophan activation enzyme was separated from the enzymes for threo-
nine and serine in an extract of pig pancreas (Cole et al., 1957). Tyrosine
activation enzymes were also partly purified from the same tissue (Schweet
et al., 1957) and from yeast (Van de Ven et al., 1958). Serine activation
enzyme was purified from beef pancreas (Webster and Davie, 1959) and a
threonine specific enzyme from calf liver (Lipmann et al., 1959). Recently,
activation of the dipeptides L-leucyl-L-tyrosine and glycyl-L-leucine has
been reported (Tuboi and Huzino, 1960; Brown, 1960).

Little is known at present about the structure of these activation enzymes
and their mode of action. Their active centre probably contains a — SH
group, for ^-chloromercuribenzoate or oxygen inhibit the tryptophan
enzyme from pancreas (Davie et al., 1956) and the alanine enzyme from
rat liver (Holley and Goldstein, 1959). The activity of several activation
enzymes is protected by reduced glutathione (Allen et al, 1960). The
participation of — SH groups in activation enzymes reminds one of the
case of triosephosphate dehydrogenase. This enzyme also contains — SH
groups (Rapkine, 1938) to which the activated carboxyl of phosphoglyceric
acid is transitorily bound as a thioester (Krimsky and Racker, 1952).
Activated carboxyls are also carried by coenzyme A as thioesters. One may
therefore suspect the participation of a thioester of amino acids at some
stage of the activation process.

Some activation enzymes cause a rapid formation of hydroxamates
although they catalyse the pyrophosphate exchange very poorly (Novelli
and De Moss, 1957). Further analysis of this apparent discrepancy
might throw light on the reaction mechanism.

An interesting fact is that the aminoacyl adenylates are very strongly
bound to the enzymes. During the pyrophosphate-ATP exchange catalysed
by the enzyme in the presence of the corresponding amino acid, no net

CHEMICAL PATHWAYS

99

change of ATP concentration is observed (De Moss et al., 1956), and no
free aminoacyl adenylate could ever be detected (Hoagland et al., 1956).
Using large amounts of pure tryptophan activation enzyme, Kingdon et al.
(1958) and Karasek et al. (1958) were able to liberate tryptophanyl adenylate
by denaturing the enzyme. The amount of anhydride obtained is com-
patible with the existence of one molecule of tryptophanyl adenylate per
molecule of enzyme. The aminoacyl adenylate might therefore be described
as a prosthetic group of the enzyme, rather than as a substrate or reaction
product. Actually, free aminoacyl adenylates would rapidly disappear in the
cell and be wasted in all kinds of reactions; it is quite certain that their
being bound to the activation enzyme protects them from reacting at
random (Askonas et ah, 1957; Moldave et al., 1959). They are anhydrides
of a carboxylic acid with adenylic acid, and belong therefore to the class of
mixed anhydrides of a carboxylic acid with a phosphoric group which
carries a substituent. Substances of this type have been shown to be rather

OH OH
Fig. 27. Amino acyl adenylate.

Stable in water, and much more so than the corresponding compound with
unsubstituted phosphoric group like acetylphosphate (Chantrenne, 1948).
On the other hand, they react very rapidly with amino acids to form a pep-
tide bond, even in 10"^ M solution at pH 74 and this is a purely non-enzymic
reaction (Chantrenne, 1947, 1948, 1949, 1950; Katchalski and Paecht,
1954; Avison, 1955; Moldave et al, 1959). Free aminoacyl adenylates in
neutral or slightly alkaline medium would rapidly form polypeptides at
random or produce a large variety of peptides by reacting with any amino
acid present. The formation of well-defined proteins from such substances
is possible only if they are in some way prevented from reacting at random
(Chantrenne, 1950). That their binding to the enzyme protects the amino-
acyl adenylates from reacting with amines is clearly shown by their behav-
iour in the presence of hydroxylamine. Whereas in the free state they would
react extremely readily with neutral 10~^ m hydroxylamine, it is necessary
to use at least 0-3 m, sometimes 3 m hydroxylamine to trap these 'activated'
carboxyl groups (Hoagland, 1955 ; De Moss and Novelli, 1955 ; Davie et al..

100 THE BIOSYNTHESIS OF PROTEINS

1956; Berg, 1956). But at such high concentrations, hydroxylamine reacts
even with common esters (Chantrenne, 1948; Raacke, 1958). The reactivity
towards amines of enzyme bound aminoacyl adenylates is thus much lower
than that of the free form. Incidentally, the high reactivity of aminoacyl
adenylates raised doubts as to the meaning of amino acid incorporation
into proteins in the presence of activation enzymes which produce amino
acid adenylates: could not the observed incorporation be explained by a
non-enzymic reaction of the mixed anhydride with — NH2 groups of pre-
existent proteins (Zioudrou et ah, 1958; Zioudrou and Fruton, 1959)? The
low reactivity of the enzyme bound anhydride with common amines
permits us to discard this objection ; but it would be a fundamental mistake
to provide an experimental system with ready made free aminoacyl
adenylates, for these — at least the fraction in excess to what can be bound
by the corresponding enzyme — would immediately react at random with
most available — NH2 groups (Wong et ah, 1959).

Aminoacyl adenylates are anhydrides of the amino acids, but they cannot
leave the enzyme, and they are not very reactive in this bound form even
with as eager an acceptor as hydroxylamine. One may wonder then whether
such an activated form can be an intermediate in protein synthesis, and
what its normal acceptor is.

These two questions are actually linked. Let us consider first the fate of
the activated amino acid.

In amine acetylation (Lipmann, 1950), in the synthesis of hippuric acid
(Chantrenne, 1951; Schachter and Taggart, 1954), the active acyl residue
of the enzyme-bound acyladenylate is picked by coenzyme A, a typical
acyl carrier which conveys it to the condensing enzyme where the acyl
radical will meet the acceptor amine and condense with it in a practically
irreversible reaction. But aminoacyl residues are not transferred to
coenzyme A (Jencks and Lipmann, 1957).

The discovery of the amino acid acceptor was a major achievement of
work on tissue homogenates and disrupted cells, which will now be con-
sidered briefly. The first experiments designed with the hope of observing
incorporation of labelled amino acids into homogenates gave depressingly
low incorporation indeed. It was established, however, that the incorpora-
tion was real and not due to contamination, and that it depended on
respiration (Winnick et al., 1948) or more precisely on oxidative phos-
phorylation (Frantz et ah, 1948 ; Borsook et al, 1949). The amino acids were
shown to be incorporated within polypeptide chains (Winnick, 1949, 1950)
and the incorporation of one labelled amino acid was stimulated by a mix-
ture of the others. The recognition of a few pitfalls (Borsook et al, 1949;
Winnick, 1950; Peterson and Greenberg, 1952) and their elimination, and
progress in the methods of preparation of homogenates (Schneider and
Hogeboom, 1951; Bucher, 1953) provided extracts in which amino acid

CHEMICAL PATHWAYS 101

incorporation was better and continued at a linear rate for about 15 min
(Siekevitz, 1952; Zamecnik and Keller, 1954); it was possible then to
obtain reproducible and reliable results. The factors required for incor-
poration and the interactions between several components of the system
could be studied more easily and with more confidence than before.

It is in the microsomal fraction that the highest rate of incorporation was
observed, in homogenates of liver as well as in the liver of the living animal.
The microsomes alone were almost inactive, but they would incorporate
readily in the presence of both mitochondria and the soluble fraction
(Siekevitz, 1952). The function of the mitochondria in such a system is
actually limited to the regeneration of ATP, for they can be dispensed with,
provided substances able to furnish phosphate-bond-energy by anaerobic
processes are available (e.g. hexosediphosphate, phosphocreatin, phos-
phoenolpyruvate). Dialysis of the soluble fraction suppressed the incor-
poration, but this could be restored to a large extent by a supplement of
ATP and adequate metabolizable substrate to regenerate it (Zamecnik
and Keller, 1954).

In later studies, Keller and Zamecnik (1956) observed that the fraction
of this supernatant which plays a part in amino acid incorporation is pre-
cipitated when the pH of the dialysed supernatant is adjusted to 5. This
fraction, often called 'pH 5 enzymes', is of course a very crude mixture of
many components which precipitate together at that pH. The incorpora-
tion system could then be reconstituted from the microsomal fraction, the
redissolved pH 5 precipitate, ATP and an ATP regenerating system.
Reprecipitation or treatment of the pH 5 fraction by the anion exchanger
Dowex-1 inhibited the system almost completely. Reactivation was
achieved by ATP plus a small amount of guanosinetriphosphate. This is
one more compound required for amino acid incorporation (Keller and
Zamecnik, 1956; Littlefield and Zamecnik, 1957). The pH 5 fraction then
received special attention. Hoagland (1955) had found that the supernatant
fraction of a liver homogenate catalyses the exchange of pyrophosphate
with ATP in the presence of several amino acids, and the formation of
hydroxamates of the amino acids in the presence of hydroxylamine, thus
indicating the presence of amino acid activation enzymes in this super-
natant. Association of these activities with the pH 5 precipitate obtained
from the crude supernatant (Hoagland et al., 1956), strongly suggested
that these activation enzymes are the constituents of the pH 5 precipitate
which are required for amino acid incorporation.

The system at this stage seemed to consist of an energy source capable of
regenerating ATP, the activation enzymes which will cause the condensa-
tion of the amino acids with ATP, the microsomal particles which are the
place where newly-formed polypeptides are first observed, and guanosine-
triphosphate. In between the activation enzymes and the microsomal

102 THE BIOSYNTHESIS OF PROTEINS

particles there was a big gap. Since the amino acid adenylates are strongly-
bound to the activation enzymes, the problem was to find out how activated
amino acids were transferred to the microsomal particles.

Hultin (1956) obtained evidence for the accumulation in homogenates
of some form of activated amino acids which can serve as precursors of
proteins in the absence of ATP. The addition of a one thousand-fold excess
of non-labelled leucine to a homogenate in which radioactive leucine is
being incorporated causes a sudden and radical drop of specific radio-
activity of the free leucine, but incorporation is not stopped immediately:
it goes on undisturbed for a few minutes, before being completely inhibited.
This indicates that some intermediate between free amino acids and
precipitable protein piles up in the homogenate. The intermediate is in the
soluble fraction, and it is in a form which does not equilibrate rapidly with
free amino acids as the amino acid adenylates would do (Hultin and
Beskow, 1956).

Holley (1957) obtained indications on the probable polyribonucleotidic
nature of the acceptor by observing that ribonuclease suppresses a AMP-
ATP exchange which is catalysed by the pH 5 fraction and which depends
on alanine. Hoagland et ah (1957) found that the 'pH 5 enzymes' prepara-
tion from rat liver contains about 5 per cent ribonucleic acid, and that
when this preparation is incubated with ATP and labelled leucine, the
RNA subsequently isolated from this fraction is labelled. Leucine is thus
bound to the RNA of the pH 5 fraction. The bond is relatively stable in
acid medium, and alkali labile. Bound leucine does not exchange with non-
labelled free leucine. When the leucine-labelled RNA is incubated with
concentrated hydroxylamine, leucylhydroxamic acid is formed, indicating
that leucine is bound to RNA through the carboxyl in such a way that this
group is moderately reactive. When leucine-labelled RNA, isolated by the
phenol method (Kirby, 1956) is added to a microsomal suspension, leucine
leaves the RNA and it is transferred to microsomal protein material, pro-
vided guanosinetriphosphate is present (Hoagland et al., 1957, 1958;
Zamecnik et al., 1958).

Thus some ribonucleic acid which is contained in the soluble fraction,
and in the pH 5 precipitate obtained therefrom, can act as a transitory
carrier of activated amino acids in between the activation step catalysed by
the activation enzymes, and the microsomal particles where polypeptides
first appear.

2. Transfer RNA

The discovery of amino acid binding by soluble RNA from rat liver
was soon confirmed, and RNAs with similar acceptor properties were
found in other animal tissues, e.g. pancreas (Weiss et al, 1958), mammary
gland (Fraser and Gutfreund, 1958), in a protozoan (Mager and Lipmann,

CHEMICAL PATHWAYS 103

1958), in bacteria (Berg, 1958) and in yeast (Osawa, 1960; Otaka and
Osawa, 1960; Monier et al, 1960). Although no systematic survey has been
reported, it seems probable that RNAs able to bind activated amino acids
will be found everywhere. It is important to realize that activated amino
acids cannot be transferred to all kinds of RNA. Ribosomal nucleic acids,
which make up around 85 per cent of total cellular RNA, do not accept
activated amino acids. It is only from the soluble fraction that adequate
acceptor RNAs have been isolated.

The very first studies on soluble RNA (Hoagland et al, 1957, 1958)
already indicated that crude soluble RNA from rat liver can bind several
individual amino acids, and that there is no competition between these.
When the RNA had been saturated first with a given amino acid, it could
still bind all the other amino acids. This suggested that crude soluble RNA
contained independent specific acceptor sites for each kind of amino acid.
Similar observations were made for soluble RNA from E. coli (Berg and
Ofengand, 1958; Nisman, 1959) and from reticulocytes (Schweet et al.,
1958). A similar system was found in plant cells (Webster, 1959). An im-
portant question was to find out whether there was one molecular species
of RNA able to bind the individual amino acids at specific sites distributed
along its molecule, or whether there were several molecular species of RNA,
each specific for one single type of amino acids. Fractionation of nucleic
acids is not an easy matter. However, Goldthwait (1958) using an Ecteola
column. Smith et al. (1959) with a cation starch exchanger, Holley et al.
(1959) by means of counter-current distribution, Lipmann, et al. (1959)
with column electrophoresis, all achieved some degree of resolution of
soluble RNA. When the fractionation was applied to a crude soluble RNA,
which had been loaded with various labelled amino acids, a definite al-
though only partial separation of fractions binding difl:'erent amino acids
was obtained. Thus Holley et al. (1959) quite clearly separated a RNA
fraction labelled with i^C alanine from another which was loaded with I'^C
leucine. The acceptor RNA for leucine was also partly separated from the
acceptor for threonine (Lipmann et ah, 1959) and from that for tyrosine
(Smith et al., 1959). Brown (1960) has succeeded in separating almost com-
pletely the acceptor RNAs for histidine and tyrosine from all the others, by
coupling chemically to polydiazostyrene the histidine and tyrosine bound
to RNA. The other amino acids do not react with the resin under the condi-
tions used; consequently, only histidine and tyrosine with their attached
specific RNA are retained with the polymer. Mild alkali treatment splits
the amino acid-RNA bond and releases the specific acceptor RNAs.

Zamecnik et al. (1960) have recently developed a chemical method which
is, in principle, applicable to the isolation of all the individual acceptor
RNAs.

The specific acceptors of the individual amino acids thus belong to

H

104 THE BIOSYNTHESIS OF PROTEINS

different molecular species of RNA, which can be separated by several
methods.

The average molecular weight of soluble RNA has been estimated by-
different methods to be in the range of 10,000-50,000 (Hoagland, 1958;
Zamecnilc et al., 1958; Goldthwait, 1958; Hoagland et al, 1958). The
more recent figures being 16,000 (Brown, 1960), 25,000 for E. coli (Tissieres
et al., 1959) and 27,000 for yeast (Otaka and Osawa, 1960). These figures
can only be rough approximations since the molecular weight determina-
tions were made on preparations known to be mixtures of many different
molecular species of RNA. They indicate that soluble RNAs are molecules
containing of the order of 50-80 nucleotide residues. An end group deter-
mination gave an estimate of about 100 residues (Singer and Cantoni,
1960; Allen ^^ a/., 1960).

It was shown, moreover, that the RNA acceptors of proline, leucine and
valine all have that same molecular size (Klee and Cantoni, 1960). When
crude soluble RNA is loaded with leucine, the maximum amount it can
bind corresponds to one leucine residue for about two thousand nucleotides
(Goldthwait, 1958; Hecht et al., 1959). Since there are twenty different
amino acids, and since the acceptor RNAs are all of the same size, assuming
equivalent proportions of each, one can estimate that there is one acceptor
site per one hundred or so nucleotides. Other estimates gave one amino
acid residue per 70-90 nucleotides (Allen et ah, 1960). This being roughly
the size of one soluble RNA molecule, it would seem that soluble RNAs
most probably carry one single acceptor site per molecule.

Soluble nucleic acids differ from ribosomal nucleic acids on several
accounts: they are soluble in molar NaCl, whereas the bulk of cellular
RNA is precipitated (Smith, 1960), they contain considerable amounts of
5-ribosyl uracil nucleotide, which differs from normal uridylic acid by the
position in which uracil is bound to the Ci of ribose (Dunn, 1959, 1960;
Scannell, et al, 1959; Osawa and Otaka, 1959). Soluble RNA is also
adsorbed by charcoal (Brown, 1960) and by Ecteola (Otaka and Osawa, 1960)
much more readily than the bulk of RNA. Several precursors of RNA are
incorporated much more rapidly into soluble RNA than into microsomal
RNAs, but the results of recent investigations indicate that in homogenates
adenylic or cytidylic nucleotides can be incorporated at the end of the
chains of soluble RNA in a process which does not correspond to the
synthesis of new RNA molecules (Heidelberger et al., 1956; Paterson and
Lepage, 1957; Edmonds and Abrams, 1957; Canellakis, 1957; Canellakis
and Herbert, 1960; Herbert and Canellakis, 1960). It would seem that
soluble RNAs are able to bind in succession two cytidylic nucleotides,
followed by an adenylic residue which thus comes to occupy a terminal
position in the polynucleotide chain. This last adenylic residue is bound
through its 5' phosphate group to the 3' position of the last cytidylic

CHEMICAL PATHWAYS

105

nucleotide, and accordingly both 2' and 3' positions of the ribose in the
terminal adenylic nucleotide are free. This is established by periodate
oxidizability of the terminal adenylic residue, and by the fact that when i^C-
labelled ATP is used as a source of terminal adenylic acid, most of the label
in the alkali hydrolysate is recovered as adenosine.

CHg

Cytosine

Cytosine

Adenine

Cytosine

Cytosine

Adenine

OH OH

Fig. 28. Terminal sequence of soluble transfer RNA with and without
attached amino acid.

Prolonged incubation of soluble RNA removes the terminal nucleotides,
and fixation of amino acids is possible only after the reformation of the
terminal cytidyl-cytidyl adenosine (C-C-A) sequence (Hecht et aL, 1958,
1959). Singer and Cantoni (1960), on the other hand, provided evidence that

106 THE BIOSYNTHESIS OF PROTEINS

the Other end of these RNA molecules terminate by guanosine 5' phosphate.
Harbers and Heidelberger (1959) have separated RNAs having the usual
terminal C-C-A sequence from yet another group in which the final
sequence is uridyl-uridyl guanosine. It is not known whether the latter can
bind amino acids.

Evidence discussed before indicates that the carboxyl group of the
amino acids is bound to soluble RNA in such a way that it is only moder-
ately reactive with hydroxylamine. The reactivity of this bond resembles
that of an aminoacyl ester more than that of a mixed phosphoric anhydride
(Raacke, 1958; Hoagland et ah, 1958; Lipmann et ah, 1959) and binding
of the carboxyl of the amino acid somewhere else than on the phosphate
residue is therefore indicated. A soluble RNA loaded with amino acids was
hydrolysed by ribonuclease and the split products separated by paper
electrophoresis. The amino acids were not released, they were found to be
associated with adenosine, from which they could be separated by mild
alkali. Since the adenosine amino acid compound does not reduce periodate,
it must be concluded that the amino acids are bound to an —OH in position
2' or 3' of adenosine (Zachau et ah, 1958), for a compound with hydroxyl
groups on two consecutive carbons (like 2' and 3' of adenosine) would be
oxidized by periodate.

The amino acid composition of the mixed adenosine-amino acid com-
pounds thus isolated from RNA indicated the presence in various amounts
of almost all the amino acids (Lipmann et ah, 1959). Preiss et ah (1959)
studying soluble RNA from E. coli obtained similar results. They showed
that periodate treatment destroys the acceptor capacity of RNA. More-
over, if an amino acid residue is linked to the RNA prior to treatment with
periodate, the acceptor site specific for the bound amino acid is protected
against inactivation while the others are destroyed. This provides an ele-
gant demonstration of the high degree of specificity of the acceptor RNA,
and confirms that a free glycol structure is necessary for the fixation of all
the individual amino acids.

In the present stage of our knowledge, it seems that there might exist
twenty soluble RNAs, each able to accept specifically one single amino
acid. This is bound by ester linkage to the —OH in position 2' or 3' of the
adenosine residue of the terminal cytidyl-cytidyl adenosine sequence.

Two important questions must now be asked; how do the specific RNAs
pick up the amino acids, and how do they transfer them further? The puri-
fication of several activation enzymes, and the possibility of isolating soluble
RNAs by the phenol method which most probably destroys all the enzymes
present, made it possible to study the requirement for the transfer of
activated amino acid from the enzyme to soluble RNA. Apparently nothing
else is required beside the pure activation enzyme and the RNA (Schweet
et ah, 1958; Preiss et ah, 1959; Wong et ah, 1959). Capacity of a threonine

CHEMICAL PATHWAYS 107

activation enzyme to activate the amino acid and to transfer it to RNA
remained strictly proportional in the course of purification (Lipmann et al.,
1959). Studies by HoUey (1957), HoIIey and Goldstein (1959) are also
relevant to the mechanism of the transfer. The purified alanine activation
enzyme which catalyses the pyrophosphate ATP exchange in the presence
of alanine, catalyses also the exchange of i^C-AMP with ATP in the
presence of both alanine and soluble RNA. This last exchange is com-
pletely abolished by ribonuclease. This fact strongly suggests that the
reaction between enzyme bound alanine adenylate and soluble RNA is
direct. Mager and Lipmann (1958) provided evidence for the reversibility
of the amino acid transfer to s-RNA in Tetrahymena preparations, by
showing that reversion is obtained by the addition of pyrophosphate and
AMP. Inhibition of the pyrophosphate exchange by s-RNA also supports
a direct interaction between s-RNA and the activation enzyme (Goldstein
and Holley, 1960). The aminoacyl group alone is transferred to soluble
RNA, not the adenylic acid residue, which is liberated (Wong and Mol-
dave, 1960).

One must conclude that activation of the amino acid with formation of
the enzyme-bound aminoacyl adenylate, and the transfer of the aminoacyl
residue to the RNA are both brought about by the activation enzyme. The
donor specificity of the activation enzymes must be such that they can
distinguish between the twenty diflferent acceptor RNAs and deliver the
activated acyl group to the right one only. It has been briefly reported that
two activation enzymes both specific for alanine were isolated from pig
liver: the one from the cytoplasm, the other from nuclei (Weber, 1960).
The cytoplasmic enzyme is said to transfer alanine to cytoplasmic RNA
from muscle and liver, but not to nuclear RNA or yeast RNA. There are
indications that a certain degree of species specificity might also exist at this
level. The activation enzymes of guinea pig will transfer to animal s-RNA,
but E. colt RNA is a bad acceptor (Allen et al, 1960).

What happens to the activated amino acids which are bound to their
specific soluble RNA? They can be transferred to the ribosomes where they
first appear as polypeptides. This was shown first by Hoagland et al. (1957,
1958) with rat liver homogenates. More recently Lacks and Gros (1959)
established that the amino acids bound to soluble RNA in E. coli are very
rapidly renewed and that any loss of labelled amino acid from the soluble
RNA is accompanied by a gain of i^C in the proteins.

Extremely little is known about this transfer at present, except that
guanosine triphosphate (GTP) is required at this stage, possibly also ATP
(Hoagland et al, 1957, 1958; Webster, 1959; Ogata et al, 1960). Some
other factor, which is especially abundant in regenerating liver, also
favours the passage of amino acids from soluble RNA to microsomal
protein (Rendi, 1959: Grossi and Moldave, 1959, 1960; Nathans and

108 THE BIOSYNTHESIS OF PROTEINS

Lipmann, 1960). Evidence for the participation of the so-called S-protein
(Sachs, 1957) or of some — SH containing protein at this stage was ob-
tained by vonderDecken and Hultin (1960) and by Hulsmann and Lipmann,
1960. In certain bacteria, the transfer is inhibited by chloramphenicol (Lacks
and Gros, 1959), streptomycin (Erdos and Ullmann, 1959) or puromycin
(Yarmolinski and de la Haba, 1959).

No doubt, the most obscure steps in protein synthesis are here, and
unfortunately this is the heart of the matter. The amino acids on soluble
RNA are bound in an activated state to specific carriers, but they are still
isolated from one another. The next step must be their condensation into
the genetically controlled sequence. Before venturing in considerations
upon this still completely mysterious process, let us return to the present
scheme of amino acid activation and examine how much confidence we can
place in it.

There is no doubt that amino acids can follow this pathway and be
incorporated into polypeptides. Most amino acids can be activated by
specific 'activation enzymes' and transferred to specific ribonucleic
acceptors; these in turn will loose the bound amino acids, which will
eventually be recovered as polypeptides in the ribosome fraction. If an
aminoacyl adenylate on the activation enzyme is labelled in the carboxyl
by 1^0, heavy oxygen is later found in the proteins (Boyer and Stulberg,
1958). Molecules of the amino acid which had been activated by this
process were therefore used in making proteins. The ubiquity of the
activation enzymes indicates that they fulfil a quite general function com-
mon to all kinds of cells of animals, plants and bacteria. A more convincing
indication in favour of the importance of activation enzymes for protein
synthesis was obtained by Sharon and Lipmann (1957): several tryptophan
analogues are known to be incorporated instead of tryptophan into the
proteins of micro-organisms and animal tissues, whereas other tryptophan
analogues are not incorporated. It is a striking fact that the analogues which
are incorporated are those which are activated by the pure enzyme. This
identical specificity of the activation enzyme and of protein synthesis as a
whole, is good circumstantial evidence for the participation of this activa-
tion enzyme in protein synthesis. The mistakes made by this enzyme are
reflected in the protein formed.

The exact function of soluble RNA in the process is not completely
clarified. It can act as a depositary of activated amino acids. But one may
wonder whether its function resembles more that of a store-house in a
side street or that of a carrier of amino acids on the highway. In support of a
function of carrier or obligatory intermediate, one may quote the rapid
inhibition of protein synthesis by ribonuclease, which is known to degrade
soluble RNA rapidly and to act more slowly on microsomal RNA (Brachet
and Six, 1959). The experiments upon which the classical scheme of pro-

CHEMICAL PATHWAYS 109

tein synthesis is founded were tracer experiments in which incorporation
of amino acids into crude protein material was studied. But Campbell et al.
(1960) and Ogata et al. (1960) obtained evidence for the validity of this
scheme for labelling of a specific protein, serumalbumin, in a liver homo-
genate. The requirements are a source of ATP, the pH 5 fraction and GTP;
ribonuclease treatment of the pH 5 fraction which contains the soluble
RNA inhibits the incorporation into serumalbumin. Finally, in living
E. coll, chasing experiments in which non-labelled amino acids where
added after a period of incorporation of labelled ones showed that the
amino acids bound to soluble RNA behave as if they were intermediates on
the way of protein synthesis (Lacks and Gros, 1960). These results which
were obtained with intact exponentially growing bacteria indicate that the
pathway under consideration is indeed operative in bacteria as well as in
animal tissues.

However, other observations indicate that we do not know the whole
story yet, even for the activation steps perhaps. In the experiments just
mentioned with living E. colt for instance, it would seem that the rate of
renewal of the amino acids bound to soluble RNA can account only for
part of the total incorporation into proteins in exponentially growing bacteria,
as if an alternative pathway contributed to the incorporation. Silkworm
fibroin contains 42 per cent glycine and 28 per cent alanine. However, the
activation enzymes for tryptophan and for tyrosine are much more active
than the one for glycine (Heller et al., 1959). Although the lability of the
activation enzymes makes a comparison of absolute activities difficult, and
its significance questionable, these observations nevertheless again raise
the question of the existence of alternative pathways.

C. OTHER FACTORS INVOLVED IN PROTEIN
SYNTHESIS

(a) The incorporation enzymes. Beljanski and Ochoa (1958) isolated
from bacteria a protein fraction which is required for the incorporation in a
cell free system from Alkaligenes foecalis, in the absence of amino acid
activation enzymes (Beljanski, 1960). This fraction 'replaces' the pH 5
fraction in the incorporation of leucine into proteins of rat liver microsomes
(in this sense that it causes a very strong stimulation of incorporation, as
the activation enzymes would do). This 'incorporation enzyme', however,
does not catalyse the amino acid promoted exchange of pyrophosphate
with ATP. Instead, this purified fraction causes the exchange of each
i4C-labelled nucleoside diphosphate (ADP, GDP, UDP, CDP) with the
corresponding triphosphate. Differential thermal inactivation of the ex-
change of the four pairs of nucleotides points to the existence of four

no THE BIOSYNTHESIS OF PROTEINS

different enzymes in the preparation, one for each type of nucleotide. It is
conceivable that these enzymes catalyse the activation of amino acids by
a process of the type illustrated in the synthesis of glutamine or of gluta-
thione. The enzymes involved in the formation of these peptides have been
partly purified (Elliot, 1951, 1953; Snoke and Bloch, 1952, 1955; Webster
and Varner, 1954). The carboxyl group of glutamic acid must be activated
in the process, for in the presence of ATP and hydroxylamine, glutamyl
hydroxamic acid is formed. No free glutamic acid derivative with a reactive
carboxyl could ever be isolated however, and it is assumed that the activated
compound is bound to the enzyme. A bound glutamyl-phosphate inter-
mediate is postulated (Webster and Varner, 1954; Varner and Webster,
1955 ; Kowalsky et ah, 1956), because heavy oxygen is transferred stoichio-
metrically to inorganic phosphate, and not at all to the ADP moiety of
ATP; besides, the exchange of inorganic phosphate with the terminal
phosphate of ATP requires the presence of both glutamic acid and the
acceptor (e.g. ammonia in the synthesis of glutamine). The following
scheme accounts for these experimental facts, although the detailed work-
ing of the enzyme cannot be considered as completely clarified.

Enzyme + ATP + Glu ^====i ( y- Glu~P) Enzyme + ADP

( Y- Glu~P) Enzyme +NH3 ^ = ===^ y- GIU-NH2 + Pi + Enzyme

Fig. 29.

A similar reaction scheme may be assumed for the 'incorporation
enzyme'. It would account for the exchange data and for the fact that
amino acids cause a net liberation of inorganic phosphate from the nucleo-
side triphosphates in the presence of the enzyme preparation. It would also
explain the transfer of heavy oxygen from the amino acid to inorganic
phosphate in bacterial preparations (Bernlohr and Webster, 1958). An
interesting observation is that the individual amino acids do not cause the
liberation of phosphate from all four nucleoside triphosphates equally well.
Thus glycine appears to react readily with ATP, less with GTP, leucine
reacts with UTP and CTP, phenylalanine with CTP only (Beljanski,
I960).

It is indeed impossible at present to decide whether these reactions play
a part in protein synthesis or not. One favourable indication is that the
amino acid promoted liberation of inorganic phosphate is inhibited by
chloramphenicol, a typical inhibitor of protein synthesis in bacteria. The
possibility that the 'incorporation enzyme' might be involved together
with the activation enzymes must be kept in mind.

(b) The S-protein. An additional factor in amino acid incorporation in
certain systems, is a protein material isolated by Sacks (1957) from the
soluble fraction of liver homogenate, but v/hich does not contain any
activation enzymes. This S-protein enhances the incorporation of amino

CHEMICAL PATHWAYS 111

acids into microsomal particles. Its effect is especially marked with micro-
somes that have been lyophilized or treated with acetone (Sachs, 1957).
Microsomes treated under certain conditions by the detergent lubrol or
by perfluoro-octanoate can incorporate amino acids in the absence of the
amino acid activation enzymes (Cohn, 1959); in this system the S-protein
is necessary. A crucial question is the dispensability of the amino acid
activation enzymes. If this is well established and if the observed incor-
poration reflects protein synthesis, then one must conclude that there are
two pathways of amino acid activation leading eventually to protein via
s-RNA. This raises questions that the presently available data cannot
answer. Are there two ahernative pathways for the synthesis of any protein,
or are certain proteins made by one process and others by the other one?
Such a duality of proteins certainly does not appeal to anyone, but one
should be prepared to the eventuality that e.g. the constitutive proteins of
the ribosomes might be made in another way than the enzymes and the
other common proteins or that certain cell organelles might not make
proteins in exactly the same way as the cytoplasmic ground substance. This
is supported by a few observations. Hexetidine disturbs protein synthesis
in bacteria in a very strange way : in the presence of this agent the synthesis
of all proteins is not uniformly affected. Incorporation of labelled pre-
cursors seems to occur in a few protein fractions only, as if the synthesis of
most proteins was inhibited, leaving the formation of a few others unaffected
(Halvorson and Gorman, 1959). Chloramphenicol at rather high concen-
tration inhibits protein synthesis in isolated nuclei and mitochondria, but
not into ribonucleoprotein particles (Rendi, 1959). Chloramphenicol and
chlortetracyclin inhibit amino acid incorporation into large particles con-
tained in a homogenate of Tetrahymena but not into the microsomal
fraction (Mager, 1960).

(c) Lipids. Hendler (1958, 1959) presented evidence for another carrier
of amino acids besides soluble RNA. A lipid fraction from hen oviduct was
found to incorporate amino acids very rapidly; the amino acids seem to be
held by a highly labile bond in this lipid fraction and to be rapidly renewed.
Hunter et al (1959) also observed that a lipoprotein fraction isolated from
cytoplasmic membranes of B. megaterium takes up labelled amino acids
very rapidly. Moreover, they obtained indications that if both soluble RNA
and lipids are concerned in protein synthesis, the lipids are involved at a
stage subsequent to the action of soluble RNA. A lipoidic substance con-
taining carbohydrates was shown to stimulate amino acid incorporation
in homogenates (Hradec and Stroufova, 1960). These systems are still very
crude, and it is not quite clear at present whether the lipids are involved in
protein synthesis or in some other process, e.g. amino acid transport.
Further research is certainly needed along this line.

(d) Incorporation factors. One further point which still awaits clarifica-

112 THE BIOSYNTHESIS OF PROTEINS

tion is the nature and functions of the amino acid 'incorporation factors'
discovered by Gale, Disrupted Staphylococcus cells incorporate amino
acids readily under a variety of conditions (Gale and Folkes, 1955; Gale,
1955, 1957). Removal of the nucleic acids by salt extraction almost com-
pletely suppresses the incorporation. The process can be restored by the
addition of staphylococcal RNA or better of a ribonuclease digest of RNA.
The activity is not due to any nucleotide or oligonucleotide fragment of
the RNA as one would have anticipated. The activity is associated with
several substances present in minute amounts in such digests. A purified
preparation of the so-called 'glycine incorporation factor' promotes the
incorporation of glycine, phenylalanine, aspartic acid, leucine, glutamic
acid, arginine and lysine, and to a lesser extent that of valine, isoleucine,
tyrosine and proline (Gale, 1958). The same preparation also stimulates
the incorporation of adenine into nucleic acids (Gale and Folkes, 1958).
The chemical nature of these factors is completely unknown. The glycine
incorporation factor is reasonably stable to acid or alkali, it is devoid of
charge, except at high pH values, when it becomes slightly positively
charged.

According to Wagle et al. (1960), the glycine incorporation factor
increases the (very low) incorporation which is observed in liver micro-
somes in the absence of the pH 5 fraction. The same laboratory has also
shown that a derivative of vitamin B12 stimulates very much amino acid
incorporation into the proteins of homogenates of liver from vitamin B12
deficient animals (Barker, 1958, 1959). The site of action of this substance
has not been located exactly, but it does not seem to concern the activation
steps (Mehta et «/., 1959 ; Wagle et «/., 1958 ; Szafranski et al, 1960) and the
observed effects might actually be indirect (Eraser and Holdsworth, 1959;
Arnstein and Simkin, 1959). Becarevic (1957) had also noted a restoration
by vitamin B12 of protein synthesis in ultraviolet-irradiated yeast.

Yoshikawa and Maruo (1960) have isolated an amine which is strongly
bound to RNA preparations and which stimulates amylase formation by
B. subtilis.

Many experimental facts remain to be explained and eventually inte-
grated into a scheme of amino acid incorporation. It is possible that the
presently accepted pathway involving activation enzymes, soluble RNA
and microsomes is only a first approximation to a more complex reality.

D. THE SEQUENTIAL CONDENSATION OF AMINO
ACIDS INTO POLYPEPTIDES

In the evolution of ideas on protein formation, two main working
hypotheses which were regarded as alternative and mutually exclusive

CHEMICAL PATHWAYS 113

retained the attention. One hypothesis assumed that proteins are made
stepwise as the other substances, the other one that they are assembled in
one stroke by a template process. The template hypothesis remained sterile
for a long time because it was beyond the reach of experimentation ; the
current developments of genetics and molecular biology have now given so
much consistency to this old daydream that it appears at present an almost
certain reality.

The hypothesis of a stepwise formation of polypeptides has not been very
successful; it never tried to account for the genetic control of protein
synthesis. But it suggested many experiments, and gathered a number of
data which will have to be integrated in the final scheme of protein synthesis.

As will be seen, the two hypotheses are incompatible only when they are
both taken in their extreme form, and it is quite possible that the actual
mechanism of protein synthesis presents facets in which both hypotheses
can find some justification.

1. Are there Free Peptide Intermediates?

The question as to the existence of peptide intermediates between amino
acids and proteins was the subject of much discussion a few years ago ; it
was thereafter disregarded, but it cannot yet be answered unequivocally at
the present time. Intermediate stages must of course exist between
separate amino acids and a polypeptide containing for instance thirty amino
acid residues. The problem is to know whether polypeptides are made
stepwise from oligopeptides or peptide derivatives having an independent
existence and being handled by a series of separate catalysts, or whether
all the linkages between the amino acids which make a polypeptide are
formed, in more or less rapid succession, under the control of one single
catalyst, the organizer molecule or template.

The direct search for oligopeptides in living cells has not been very
rewarding. If common small peptides, like glutathione or carnitine, are
discarded — for they do not seem to be intermediates in protein synthesis
— no notable amount of free peptides have been detected in cells. Excep-
tions may be some plant material (Fogg, 1952; Dagley and Johnson, 1956)
and resting bacteria maintained under adverse conditions (Gale and Van
Halteren, 1952; Weinbaum and Malette, 1959; Sorm and Cerna, 1960).
Tracer experiments designed to find out whether growing bacteria could
form enzymes from inactive precursors (Monod et ah, 1952; Halvorson
and Spiegelman, 1952; Rotman and Spiegelman, 1954; Hogness et al,
1955) did not detect any accumulation of complex intermediates between
amino acids and proteins. These experiments of course do not prove that
intermediate peptides do not exist, but they show that if they exist their
concentration is so low as to escape detection by the sensitive tracer
methods used, and that they are renewed very rapidly.

114 THE BIOSYNTHESIS OF PROTEINS

On the other hand, indirect and circumstantial evidence for the existence
of peptide intermediates has been presented on several occasions. Peptides
obtained by the action of proteolytic enzymes on proteins stimulate protein
synthesis more than a mixture of amino acids in certain systems (Kihara
and Snell, 1955; Rychlik and Sorm, 1956; Fox and Krampitz, 1956). The
rather frequent occurrence of individual tripeptides in certain classes of
proteins (Sorm, 1957, 1959, 1960) makes one wonder whether these tri-
peptides might not be made separately and serve as precursors for several
related proteins. So far, however, all the studies on the utilization of
labelled di- or tripeptides have shown that the amino acids are used more
readily than the peptides and that these must actually be split into amino
acids to be utilized. The amino acids are the real precursors. This was
found for instance in the case of the synthesis of haemoglobin in reticulo-
cytes (Nizet and Lambert, 1954), and of casein in the goat (Godin and
Work, 1956), for amino acid incorporation in tissue slices (Hendler and
Greenberg, 1954) and for bacterial growth (Meinhart and Simmonds,
1955). The stimulatory effect of peptides obtained by enzymic hydrolysis
of proteins is not completely explained. It is due in certain cases to glutam-
ine and asparagine (Rychlik and Sorm, 1957), which are contained in
enzymic hydrolysates of proteins, but not in acid hydrolysates since these
amides are split by acid into ammonia and glutamic or aspartic acid re-
spectively. It is indeed established at present that glutamine and asparagine
are incorporated as such into proteins (Barry, 1954, 1956; Rabinovitz et ah,
1956; Sansom and Barry, 1958). They should be regarded as individual
amino acids which are just as different from glutamic and aspartic acids
as e.g. leucine or glycine, as far as incorporation into protein is concerned.
The 'complete' amino acid mixtures used in the overwhelming majority of
the experiments did not contain any asparagine or glutamine, and were
therefore incomplete mixtures of protein precursors.

Another possibility is that peptides might in certain cases be degraded
by living cells in such a way that they would provide activated amino acids
directly (Walter et ah, 1956). This also deserves further investigation.

A more striking phenomenon which might reflect the existence of
peptide intermediates of some sort is the non-uniform labelling of proteins
as observed most clearly with in vitro systems. Pieces of hen oviduct in-
corporate the carbon of ^'^002 in vitro into ovalbumin and other proteins.
Ovalbumin which had been labelled in this manner was split by a bacterial
protease into two fragments, according to Ottesen and Wollenberger (1953).
The larger fragment, named plakalbumin, can be isolated in a crystalline
form, the smaller one is a hexapeptide. The specific radioactivity of the
aspartic acid residues of these two fragments differed by a factor greater
than four (Steinberg and Anfinsen, 1952). A similar result was later ob-
tained for the incorporation of labelled phenylalanine and glycine into

CHEMICAL PATHWAYS 115

insulin and into ribonuclease in slices of beef pancreas. This time, each
protein was split into many peptides ; the specific radioactivity of the same
amino acid was notably different in the different peptides originating from
the same protein (Vaughan and Anfinsen, 1954). More recently, non-
uniform labelling was reported for collagen (Gehrman et al., 1956) and
procollagens (Orekhovich et al., 1959), for silk fibroin (Shimura et ah, 1956),
for amylase in B. siibtilis (Yoshida and Tobita, 1960), for haemoglobin in
the rabbit (Kruh et al., 1960) and for cytochrome-c in isolated muscle
mitochondria (Simpson, personal communication). In other cases, how-
ever, no inequalities of labelling were detected (Muir et al., 1952; Heim-
berg and Velick, 1954; Askonas et al., 1955; Simpson, 1955) but the
experiments were made with intact animals and the incorporation was of
relatively long duration. Non-uniform labelling is best observed for short
periods of amino acid uptake and with poorly active or damaged systems.

The meaning of unequal labelling is not clear. Good discussions on the
subject have been presented by Steinberg et al. (1956) and by Borsook
(1956). The first interpretation proposed was that amino acids are first
incorporated into various oligopeptides or peptide derivatives which are
later combined to make a protein. According to the size of each peptide
pool and to their turnover rate, a given labelled amino acid would be
diluted to difi^erent degrees in each oligopeptide, and the protein formed by
association of the peptides would not be uniformly labelled. The incorpora-
tion is so low in most of the in vitro systems considered that a pool of
peptides amounting to some 2 per cent of the free amino acid pool could
account for the experimental data.

Another way of explaining non-uniform labelling is to assume that in the
presence of protein forming system, the peptide linkages of proteins can
be labilized in such a way that an amino acid comprised within the poly-
peptide chain can exchange with free amino acids (Chantrenne, 1952; Gale,
1953; Borsook, 1956). Differences in exchange rate at different positions
in the protein chains would explain differences in specific radioactivity. An
alternative interpretation, which avoids the assumption of exchange
processes for which there is no compelling evidence, is that the time
required for making a protein is long in the poorly active system considered ;
in this sense that each individual polypeptide molecule stays on the weaving
machine long enough for the specific radioactivity of an amino acid, e.g.
glycine, to change considerably between two additions of this amino acid
at different places along the polypeptide (Dalgliesh, 1953). If protein
synthesis takes places by such a 'stepwise template process' (Steinberg et ah,
1956) one should expect to find peptide intermediates bound to some
structures in the experimental systems in which unequal labelling is
observed.

In the last few years, the occurrence of nucleotide-amino acid and

116 THE BIOSYNTHESIS OF PROTEINS

nucleotide-peptide compounds has been reported from several laboratories.
Ascites tumour cells contain aspartic acid associated with a uridylic nucleo-
tide (Reith, 1956), and chicken liver a compound tentatively described as
adenosinediphosphoglutamic acid (Hansen and Hageman, 1956). Several
compounds containing a few amino acids were isolated from micro-organ-
isms (Hase et al, 1959; Brown, 1959). The peptide linked nucleotides
which were detected in yeast (Koningsberger et al., 1957; Harris and
Davies, 1958; Koningsberger, 1960) and in liver (Szafranksi et al., 1959,
1960; Steinberg et al., 1960) might be of special interest, for the terminal
carboxyl of the peptide moiety reacts with hydroxylamine. The com-
pounds might thus be activated peptides acting as intermediates in some
anabolic process. The chemical structure of some of the compounds from
yeast has now been established (Davies and Harris, 1960). They are mixed

HN:C-NH2 HN-.C-NHj

NH NH

CH2 ^^2

CH, CHp CH3 CH

°' CHa'O'P-OCO-CHNHCOCHNHCOCH NHC0CH-NH2
OH

Fig. 30. Structure of a nucleopeptide from yeast (Davies and Harris,

1960).

anhydrides in which the terminal carboxyl of the peptide is condensed
with the 5' phosphate group of the uridylic moiety of a dinucleotide (Fig.
30). Some of these compounds are extracted only by cold acid treatment,
as if they were weakly bound to other cell constituents in neutral extracts.
One may wonder whether the peptides found in yeast by Turba and Esser
(1955) have the same origin.

The uridylic acid peptide compounds discovered by Parks (1952), and
other clearly related substances (Strominger and Threnn, 1959; O'Brien
and Zilliken, 1959) have not been mentioned here, because they are known
to be precursors of bacterial cell wall. This serves to remind one that
amino acids and peptides are found in other substances beside proteins,
and that nucleotide-peptide compounds might have nothing to do with
protein synthesis. However, until their function is discovered, they must

CHEMICAL PATHWAYS 117

be regarded as likely intermediates, the study of which will be pursued with
great interest.

2. Are Polypeptides Made on a Template?

In its present state of development, biochemistry offers essentially one
type of explanation for each of the metabolic steps it describes, namely the
presence in the cell of an adequate enzyme which promotes the reaction
considered. When several possibilities exist from a purely chemical point of
view, the specificity of the enzyme or of the enzyme assortment is again
invoked. For instance, a great variety of fatty acids, terpenes, carotenoids,
steroids can be made from acetate ; the nature and assortment of the pro-
ducts elaborated by a cell is explained by its particular complement of
enzymes.

If this type of stepwise assembly process is assumed to operate in the
specific synthesis of the individual proteins, a fundamental difiiculty is
raised by the number of enzymes which would be specifically involved in
the production of each single specific protein. This time, simply referring
to the specificity of the enzymes would amount merely to displacing the
problem, for the enzymes are specific proteins and they are also made in the
same cell. The difficulty has been appreciated by biochemists for more than
a quarter of a century, and it was suggested several times, on purely logical
grounds, that the structure of the enzymes or at least of their specific part
must be controlled by a model, a mould or a template, i.e. by a substance
having a structure complementary to that of the enzyme synthesized.

Current developments of molecular biology (see Chapter I) give a great
vitality to this old idea. Indeed it is now established that the primary
structure of the proteins, i.e. the sequence of the amino acids in the
polypeptide chains, is controlled by mendelian genes. The information
relative to the arrangement of the amino acids is recorded on the genetic
material which is made of nucleic acids. The purine and pyrimidine bases
compose the ciphers in which this information is coded, since replacement
of one base by another or substitution of one keto for an amino group in one
base can change the information and result in the specific replacement of
one amino acid by another at one specified place in the polypeptide chain.
All the specific information relative to the primary structure of a protein
is comprised in a unique segment of the genetic material, the locus. The
size of the locus is such that it might contain enough information for con-
trolling every single amino acid in the polypeptide. These facts almost
inescapably lead to the conviction that the arrangement of the amino acids
in the polypeptide correspond point to point to that of the purines and
pyrimidines (or of their 6-keto and 6-amino groups) in DNA. Moreover,
the information concerning a protein molecule must be transferred to the
protein making system in one single package or in a very small number of

118 THE BIOSYNTHESIS OF PROTEINS

packages : the information required for the synthesis of a piece of protein
of the size of a complete polypeptide chain cannot be used unless it is
delivered en bloc. This almost certainly means that all the building blocks
which make up a polypeptide are assembled under the influence of one
single organizer macromolecule.

The remarkable results of the studies on the mechanism of DNA
replication, in providing evidence for the operation of a rigid template
process in this case (Kornberg, 1958; Lehman et aL, 1958), indirectly sup-
port the conviction that a mechanism of this nature operates in protein
synthesis. Unveiling of the template mechanism is the task for the coming
years ; it is felt that our curiosity will not have to wait for a very long time
to be satisfied.

Current ideas about this most important step of protein synthesis have
been deeply influenced by the brilliant theory presented by Crick (1957,
1958) as a development of the coding principle called 'code without com-
mas'. For reasons that have been discussed in Chapter I of the present
book, Crick suggested that each amino acid of a protein is coded in the
corresponding DNA by a sequence of three bases ; there are twenty such
sequences, each coding for an amino acid. The coding sequences for each
amino acid are such that if they were located in a row next to one another
no reading mistake could be made: the triplets corresponding to each
amino acid are such that any triplet formed from contiguous parts of any
two of them is nonsense, i.e. does not correspond to any amino acid. This
is the coding principle.

The gene DNA which carries this coded information transmits it to some
RNA by controlling the arrangement of the nucleotides in RNA by a tem-
plate process of the kind discussed by Stent and by Zubay (1958). This
specific RNA is the template for protein synthesis. The difiiculty here is
that amino acids must find their right place along a polynucleotide in order
to obey the coded information. Crick suggests that specific enzymes, able to
recognize amino acids and short nucleotide sequences, bind the individual
amino acids to specific trinucleotides which are complementary to the cod-
ing sequences in the RNA. These serve as adaptors which will carry the
amino acids and will find their right place by forming hydrogen bonds with
the complementary bases on the RNA template. When the template is
covered with adaptors carrying amino acids, the amino acids unite into
a polypeptide chain, which has thus the primary structure specified by
the gene.

If there are only two coding digits (6-keto and 6-amino) instead of four
as first assumed by Crick (see p. 36), triplets should be replaced by
groups of five nucleotides, but this does not change the principle of this
extremely attractive template mechanism.

The experimental data reviewed before establish that amino acids are

CHEMICAL PATHWAYS

119

bound individually to specific ribonucleic acids (transfer RNA) and it is
very tempting to consider these as the adaptors which will carry the
activated amino acids to the template and locate them at their right position
according to Crick's scheme. This hypothetical mechanism (Hoagland
et al., 1959) is depicted on Fig. 31. The template carrying specific informa-
tion is supposed to be microsomal RNA. So far, experimentation has been
unable to attribute a clear function to the RNA of the ribosome, which
constitutes some 80 per cent of cellular or bacterial RNA. But it has been
recognized a long time ago that the intensity of protein synthesis is related

E3(AMP-aa3) +

Fig. 31. A hypothetical scheme for the interaction of microsomal RNA
and transfer RNAs with amino acids attached (Hoagland etal., 1959).

to the amount of RNA, and essentially of ribosome RNA (Brachet, 1941).
Nascent polypeptides, in liver and bacteria, are found in association with
the ribosomes. The assumption that ribosome RNA is the template or is
part of it, is therefore reasonable. The fact that modifications of soluble
RNA can stop protein synthesis is quite compatible with the above
scheme.

There is one difficulty, in this hypothetical template process, it is the
size of soluble RNA. A sequence of three to five nucleotides should be
sufficient to code for an amino acid. If we add to it the terminal cytidyl-
cytidyl-adenosine sequence to which the amino acid is bound, this makes

I

120 THE BIOSYNTHESIS OF PROTEINS

eight nucleotides at most; but soluble RNA contain probably 10 times as
many. Such molecules seem too bulky to operate as adaptators in the sense
of Crick and it would seem that if soluble RNA is really an obligatory
intermediate on the pathway of protein synthesis, the template mechanism
must operate in a somewhat different way.

It may be not necessary to assume that the template is at one time
covered with nucleotidic 'adaptors' each carrying its own amino acid; it is
conceivable that each specific soluble RNA adds in turn its amino acid to
the growing polypeptide. Such a process would of course be slower than the
other one. Protein synthesis takes several minutes in animal cells (Peters,
1957; Craddock and Dalgliesh, 1957; Loftfield and Eigner, 1958; Dintzis
et ah, 1958; Morris and Dickman, 1960), and only a few seconds in micro-
organisms (Boeye, 1957; McQuillen et ah, 1959; Cowie and McClure,
1958; Zalokar, in press). These are short times as compared to the usual
duration of biochemical experiments, but exceedingly long times in terms
of absolute rate of reaction. If it is assumed that soluble RNA and ribo-
somes must collide for the activated amino acids to be linked to the poly-
peptides, the collision frequency which can be estimated is sufficient to
support the observed rate of peptide bond synthesis, if more than one
collision out of ten thousand results in amino acid transfer (Ts'O and
Lubell, 1960). Sequential addition therefore does not seem excluded. It
would be compatible with many experimental data (Webster, 1959; Bishop
et al., 1960) including the apparent absence of intermediates in rapidly
synthesizing systems like living bacteria, and the non-uniform labelling in
extremely slow systems like homogenates of animal tissues.

In a template process outlined by Dalgliesh (1953), several peptide
chains in different stages of development were attached simultaneously to
the templates, each by a small region as shown in Fig. 32. The polypeptides
all grow in the same direction by addition of activated amino acids, and peel
off behind the assembly zone. Future developments in molecular biology
will tell whether such a scheme is possible topologically.

The experimental data available at present are not sufficient for a useful
discussion of a template process in terms of chemical reactions. But several
of the present lines of research might soon provide information relevant to
the template mechanism : further data on the relations between soluble and
ribosomal RNA, a better understanding of the function of amino acids in
RNA synthesis, further study on the function of peptide nucleotide com-
pounds will be very valuable.

A metabolic relationship seems to exist between soluble and ribosomal
RNA, for energy-dependent transfers of oligonucleotides in both directions
have been demonstrated (Bosch et al., 1959). Hultin and von der Decken
(1959) and Moldave (1960) also observed a transfer of soluble RNA to the
microsomes and GTP seemed to promote the transfer. Clarification of these

CHEMICAL PATHWAYS

121

processes will certainly throw light upon the real function of soluble RNA
and upon the mechanism of amino acid transfer to the ribosome.

It will be important also to know a little better the conditions for coapta-
tion between polynucleotides, a promising field of studies opened by the
work of Rich (1959).

A completely unexplained fact is the necessity of a complete assortment
of amino acids for RNA synthesis. The fact that analogues of amino acids
inhibit RNA formation is another reason to believe that the individual
amino acids have a specific function in RNA synthesis. There is indeed an
intriguing reciprocity between the requirement for amino acids in RNA
synthesis and the requirement for precursors of RNA in protein synthesis.
This led several workers to the suggestion that RNA and protein might be
assembled from common precursors, visualized as some kind of nucleotide
amino acid compounds (Gros and Gros, 1957; Yeas and Brawerman, 1957;

v. ... v.... v.- V..-

Growing

peptide

chains

Tcmplot*
surface

Direction o1 chain growth

Fig. 32.

Pardee et ah, 1957; Spiegelman, 1957). The recently discovered inter-
actions between amino acids and nucleoside triphosphates (Beljanski, 1960)
might prove of great interest in this respect. The relation between amino
acids and the synthesis of RNA might be the key to the template mechan-
ism for the synthesis of proteins and nucleic acids. Interesting attempts at
formulating in chemical terms template processes taking this idea into
account have been made by Simkin and Work (1957), Kiepal-Kochanska
(1958), Michelson (1958). Raacke (1958) has proposed a more elaborate
process which features attachment of the carboxyl activated amino acids to
the template by their — NH2 groups, and which has the merit of making
predictions and suggesting experiments. Future research will tell the value
of these various hypotheses.

122 THE BIOSYNTHESIS OF PROTEINS

E. FORMATION OF THE PROTEIN MOLECULE

(a) Folding

When the amino acids have united into the genetically controlled se-
quence, a polypeptide is formed, not a protein molecule. The polypeptide
must fold in a specific way, hydrogen bonds be established between neigh-
bouring parts of the polypeptide, certain regions of the chain will form a
helix for instance, whereas others will not. The helix is not straight, it folds
upon itself, sometimes in a very intricate way, as illustrated by the structure
of myoglobin (Kendrew et al., 1960) (see Figs. 4 and 33). It is only when the
tertiary structure is established that the protein molecule possesses its all
important physiological properties : enzymic activity, serological character-
istics, capacity of associating with other substances in a specific way.

It is assumed at present that once the primary structure is there, folding
does not raise any further problems (Perutz et al., 1960) ; folding is regarded
as a spontaneous process which is determined by the amino acid sequence
and by conditions prevailing in the cell, like pH, ionic strength, nature of
the ions. Reversibility of limited denaturation indicates that the right
folding of polypeptide segments can indeed re-form spontaneously. Com-
plete uncoiling is usually irreversible, for there are too many possibilities
of formation of bonds between different parts of the chain or between
chains. If the polypeptides were released from the assembly line as ran-
domly coiled molecules, their remoulding into the right configuration would
be difficult. On the contrary, if the end of the polypeptide is allowed to fold
spontaneously as it comes oft' the template, then folding might go on in a
regular and unique manner as the chain grows. The tertiary structure
would then be strictly determined, at each step of its formation, by the
nature of the amino acid residues and by the shape and position of the
already formed parts of the molecule. Folding might conceivably be
influenced by other substances present in the vicinity of the template. But
it must be realized that the data on the secondary and tertiary structures of
individual proteins are really too scanty for deciding whether folding can
always occur spontaneously or whether it requires in certain cases the
active participation of special cellular mechanisms. A case like trypsinogen
for instance might raise difficulties. In this molecule, part of the polypep-
tide is not folded in a helix ; it is maintained in a somewhat extended state ;
the splitting of one bond in this particular region allows the polypeptide to
assume a helical structure, as shown by a change in rotatory power. There
is therefore a structural feature in the trypsinogen molecule which prevents
it from assuming the more coiled configuration of active trypsin (Neurath,
1957). Entirely too little is known at present to realize whether the forma-
tion of a protein molecule with an inner tension, like trypsinogen, raises
new problems or not.

-/ ^ J

(^|MK

^^\

h

P^^j^", ^^^W f ^ ^P

«^^CX1

^

^^mmjBSi^^

v:j -"'

(a)

(6)
Fig. 33. (a) Tertiary structure of myoglobin.

(b) The course of the polypeptide chain.
(Kendrew et al., 1958).

CHEMICAL PATHWAYS 123

(b) Bisulphide Bridges

Most of the proteins the primary structure of which has been unravelled
contain cystine. The disulphide bridges maintain a certain folding of the
polypeptide upon itseh' as in ribonuclease or they connect together different
polypeptide chains as in insulin. Disulphide bridges are essential to the
stability of the protein and to its physiological properties. It is not known
whether the formation of the SS bridges requires a specific enzymic
oxidation, or not; the oxidoreduction state of the — SH groups of proteins
and its relations, in the different regions of the cell, with free — SH con-
taining substances like glutathione are very obscure, a few steps in the
analysis of these interactions have only been made recently.

(c) Association of Polypeptides

Certain proteins are made of several polypeptide chains, either different
or identical, and the physiological properties of these proteins depend on
the association of the parts. Globin is a good example : each molecule is
made of four polypeptides: two a chains, two /S chains, assembled in an
intricate structure (Perutz et al., 1960). The association of the a and ^
chains into the finished protein occurs spontaneously in vitro under
adequate conditions. The formation of an active enzyme by mixing extracts
of two different mutants strains of Neurospora in the cold (see p. 18) is best
explained by the spontaneous association of polypeptides into active
enzyme. A related phenomenon is the association of two proteins into an
active enzyme (Yanofsky and Crawford, 1959). Again the correct associa-
tion arises spontaneously.

(d) Further Tra?isformations

Further changes occur in certain protein molecules, which transform
them into other proteins with quite different properties, as for instance the
transformation of a zymogen into active enzyme. It is a matter of taste to
consider these transformations as finishing stages of the synthesis of
certain proteins or as modifications undergone by finished proteins. We
will here adopt the latter attitude, because these further changes do not
concern all protein molecules. The transformations result from the action
of specific enzymes; they are of various types and each would deserve
special study; a few examples will simply be quoted here. Thus trans-
formations of pepsinogen, trypsinogen or chymotrypsinogen into the cor-
responding enzymes are interesting cases of limited proteolysis ; so is the
cascade of protein transformations involved in blood clotting. Phosphory-
lase-fl results from the phosphorylation of four serine residues of phos-
phorylase-6 (Fischer et al, 1959), followed by dimerization (Keller and
Cori, 1953; Krebs and Fischer, 1956). Collagen and certain plant proteins
contain hydroxyproline. At variance with the twenty common amino acids,

124 THE BIOSYNTHESIS OF PROTEINS

ready made hydroxyproline is not used for the synthesis of these proteins
(Steward and Pollack, 1958; Green and Lowther, 1959) or it is a very
inefficient precursor if it is used at all (Mitoma et ah, 1959). Hydroxy-
proHne exists only in bound form, it arises by a modification of proline
after this has been incorporated into polypeptides (Stetten, 1949; Jackson
and Smith, 1957; Wolf and Berger, 1958). A special oxidase is involved in
the transformation (Van Robertson et ah, 1959).

Unusual observations have been reported on amylase formation in
pancreas extracts. The active enzyme is released from an inactive precursor,
it is not made de novo from amino acids in the homogenate. The appearance
of amylase activity requires the presence of ATP, threonine and arginine,
although these amino acids are not incorporated into the active enzyme
(Straub et al., 1953; Straub and Ullmann, 1957; Straub, 1958; Grabowski
and Munro, 1960). An activation process of a new type might be operating
here.

Processes of maturation of protein systems may also be mentioned at
this point. Reserve proteins of pea seeds undergo a slow evolution after
they have been made: the electrophoretic pattern changes, indicating
association of many protein components into a few larger units (Danielsson,
1952; Snellman and Danielsson, 1953; Raacke, 1957). These changes recall
the transformations of gluten which involve oxidation of — SH groups
(De Deken et ah, 1953). But here we may have crossed the limit of processes
which are not relevant to protein biosynthesis.

The attachment of a prosthetic group may be spontaneous. This is the
case of certain flavoproteins, as shown a long time ago by Theorell (1935).
The porphyrin nucleus of catalase can probably find its proper place in the
protein moiety since the apo-catalase made by a porphyrin-less mutant of
E. coli can combine in vitro with haemin, resulting in a complex endowed
with catalase activity (Beljanski, 1955). Certain Staphylococci also make
apo-catalase when deprived of haemin, and complete the synthesis of the
enzyme if haemin is added ; this later step apparently involves the partici-
pation of coenzyme A (Jensen, 1957). Incidentally, these results indicate
that the proper folding of the polypeptide chain did not depend on the
prosthetic group since apo-catalase was made in its absence.

These processes give the finishing touch which confers to the individual
proteins their typical features and physiological properties, but the bio-
chemical problems raised are minor as compared to the making of the
chains with their genetically controlled sequence of amino acids.

(e) Integration of Proteins into Structures

Association of proteins into structures of a higher level can also occur
spontaneously. The best example of this phenomenon is the association of
the protein of tobacco mosiac virus into rods resembling the virus, or better

CHEMICAL PATHWAYS 125

the formation in vitro of the complete virulent virus by spontaneous
association of some 3000 molecules of protein and one RNA chain into the
well-known structure.

The association of proteins with lipids and other substances into micro-
scopical structures like membranes, fibres, lamellae, mitochondria, etc. . . . will
raise other problems which have practically not been touched at the mole-
cular level so far. These organelles do not seem to arise de novo, they
develop from similar or related pre-existing structures, as if they could only
grow by accretion of new material on to structures which insure the cor-
rect arrangement of the building blocks.

The thiol-disulphide equilibrium seems to be determining in the forma-
tion of structures. Embryologists have reported on many occasions striking
inhibitions of morphogenesis by sulphydril reagents like iodoacetamide
(see Brachet, 1946, 1960). Certain of these effects are due to the inhibition
of some common SH enzymes, or to the inhibition of mitosis. But more
subtle changes in morphogenesis caused by various thiols or disulphide
containing compounds certainly reflect processes of a completely diff"erent
nature. As a rule, free thiols disturb morphogenesis; on the contrary
disulphide substances improve it, if anything. This was observed in widely
different morphogenetic processes: development of amphibian embryos,
regeneration of the tail in amphibian tadpoles, regeneration of the head of
planarians, and cap formation in Acetabularia mediterranea (Brachet, 1959).
This last-mentioned case is especially interesting, for the alga consists of a
single cell (see p. 50) and the morphogenetic processes here do not depend
on cell division or cell migration. Growth continues in the presence of
thiols, synthesis of new material is not prevented, but the newly made
material does not form the normal macroscopic structure. Instead of a well-
shaped hat, the alga develops a swollen club-like structure. It would seem
that the thiol-disulphide equilibrium is of great importance for the relative
positions in space that the substances synthesized during growth come to
occupy. This strongly suggests that when the formation of disulphide
bridges is interfered with, proteins cannot associate correctly into structures,
either because the folding of the structure proteins themselves is not right,
or because disulphide bridges play a fundamental part in the association of
constituents — proteins or otherwise — into microscopic structures.

A new field of study will soon develop here, for the progress of electron-
microscopy, X-ray diffraction analysis and light microscopy will certainly
make it possible to bridge the gap between biochemistry and morphology,
and eventually give the first molecular interpretation of form.

CHAPTER V

Regulation of Protein Synthesis

A. ENZYMIC ADAPTATION

The rate of protein synthesis, like that of all the anabolic processes,
depends on temperature, pH, energy production, availability of building
blocks, etc. . . . More interesting is the specific control of the rate of syn-
thesis of the individual proteins, exerted by external or internal agents.

The assortment of proteins actually made by a cell is conditioned by the
genetic material ; the presence of the gene, however, confers but a potential-
ity, the expression of which depends on specific controlling systems. Let us
ignore for a moment the problem of differentiation and consider only
micro-organisms ; even for these relatively simple cells, the assortment of the
proteins actually produced does not depend on the genetic material alone :
it can be influenced by changes of the medium composition. Such mould
will start producing a protease when transferred on to a medium which
contains milk. A bacterium which makes a phosphatase in given surround-
ings may stop to manufacture the enzyme if inorganic phosphate is added to
the medium. Certain substances can thus specifically induce a cell to make
certain proteins, others can specifically repress the production of an enzyme
without affecting the synthesis of the other proteins. Induction and re-
pression of enzyme synthesis have been studied mostly in bacteria and
yeasts; comparable effects have been observed in plants and animals, but
they are much more complex especially in animals where homeostatic
mechanisms oppose changes of conditions and where superposed hormonal
actions make the results extremely intricate (see p. 170).

It is not the purpose of the present chapter to discuss thoroughly the
problem of enzymic adaptation and repression; many excellent reviews
and discussion have been published on the subject. Reading them in the
chronological order gives an extremely vivid picture of the detours of
discovery in the field, with a succession of bright hypotheses, blind alleys
and break through, resulting in the slow elaboration of a more and more
accurate picture of the phenomenon (Wortman, 1882; Duclaux, 1900;
Dienert, 1900; Karstrom, 1936; Stephenson and Yudkin, 1936; Yudkin,
1938; Monod, 1947, 1956, 1958, 1960; Spiegelman, 1950; Stanier, 1951;
Mandelstam, 1956; Spiegelman and Campbell, 1956; Pollock and Mandel-
stam, 1958; Vogel, 1958; Pollock, 1956, 1959; Pardee et al, 1959). Only a

126

REGULATION 127

few facts which throw hght upon the mechanism of protein synthesis and
upon its control will be considered here.

1 . Induced Sy?ithesis of Enzymes in Micro-organisms

Enzymic adaptation refers to the adjustment of a specific enzyme
activity caused by a change of the medium under such conditions that
selection of mutants can be excluded. In several cases, it has been clearly
established that the adaptive increase of certain enzyme activities in micro-
organisms corresponds to the complete de novo synthesis of the enzyme
proteins (e.g. Cohn and Toriani, 1953). For instance, E. coli was grown in
a medium containing radioactive protein precursors so that every protein
present in the bacteria was strongly labelled. The bacteria were then trans-
ferred into a non-labelled medium and induced by a galactoside to make
^-galactosidase. The enzyme was isolated in a state of great purity, and its
radioactivity was measured. Practically no label was found in the enzyme ;
^-galactosidase had been synthesized completely from non-radioactive pre-
cursors, it did not pre-exist in the cell in any form before the addition of the
inducer galactoside. The inducer therefore caused the complete synthesis
of the enzyme from amino acids (Rotman and Spiegelman, 1954; Hogness
et al., 1955). The usual genetic control of enzyme synthesis remains, how-
ever, for certain mutant strains of E. coli are unable to make jS-galactosidase
even in the presence of galactosides (Lederberg, 1951); these strains result
from mutations which are all located within a narrow region of the genome,
called the z locus (Pardee et al., 1959; Monod, 1960). Transfer of a small
piece of genome containing the z locus to a Shigella confers to this bacter-
ium the capacity of making a galactosidase which is identical to the coli
enzyme (Cohn et al., 1960). The z region of the genome must be the locus
which specifically controls the primary structure of the enzyme.

What then is the function of the inducer? Does it add a little piece of
structural information to the main information provided by the gene? Does
it select among several possibilities the folding corresponding to the active
enzyme? Does it pull the enzyme off^ the template? Does it control the
operation of the genetic locus or that of the assembly template in some
specific way?

Sometimes, a variety of substances are able to induce the formation of the
same enzyme; these various inducers are structurally related to the sub-
strate of the enzyme. Thus ^-galactosidase synthesis in E. coli (Monod etal.,
1951) or in B. megateriuni (Landman, 1957) can be induced by various
^-galactosides or thiogalactosides, some of which are not split by the
enzyme. A comparable situation exists in the induction of a glucosidase in
yeast (Duerksen and Halvorson, 1959) and of penicillinase in B. cereus
(Pollock, 1956). In spite of differences of structure of the inducers, the
properties of the enzyme produced are always the same, although

128 THE BIOSYNTHESIS OF PROTEINS

the amount of enzyme may depend on the inducer. One can induce the
synthesis of another enzyme, galactokinase, in E. coli in the absence of any
exogeneous inducer by causing the development of a temperate phage which
had introduced the corresponding structural gene by transduction (Buttin
eifl/., 1960).

Mutants which normally produce the enzyme in the absence of any
added inducers have been isolated in a few cases; these are 'constitutive'
mutants. Penicillinase produced by a constitutive mutant of B, cereus was
compared to the induced enzyme made by the adaptive parent strain. None
of the physical, chemical, enzymological and serological tests applied could
reveal any difference between the constitutive and the induced enzymes,
which had both been highly purified and crystallized (Kogut et ah, 1956;
Pollock, 1956). Similar, although less complete evidence for identity of
constitutive and inducible enzyme were obtained for the penicillinase of
B. suhtilis (Manson et ah, 1954) and for the j8-galactosidaseof £". co//(Monod
and Cohn, 1951) and oi Neurospora crassa (Landman, 1954).

The capacity of constitutive mutants to produce perfect enzyme in the
absence of added inducer raises an interesting dilemma: are these strains
constitutive because they can really make the enzyme without any parti-
cipation of an inducer, or is it because they themselves manufacture the
inducer, whereas the adaptive strains cannot do so? The second alternative
at first looked very promising: it provided a unitary view (Cohn and Monod,
1956) in which all the enzymes were regarded as inducible, the constitutive
enzymes being induced by an endogeneous inducer. Moreover, the
occurrence of endogeneous inducers was indicated by numerous cases of
sequential induction (Stanier, 1947, 1950, 1955; Suda et ah, 1949).

When the synthesis of an enzyme is induced by the substrate, the inducer
substrate is acted upon by the induced enzyme and it often happens that
the product of this enzymic transformation induces the formation of a
second enzyme. Addition of the first inducer in such a case results in the
sequential synthesis of two enzymes. The second one is actually induced
by an endogeneous inducer. If this process repeats itself several times, a
series of enzymes can be induced in succession. The classical example is
the sequential induction in P. fluorescens of seven enzymes which catalyse
the oxidation of the indol ring of tryptophan into ^-keto-adipic acid in seven
successive steps. It is assumed that each intermediary metabolite acts as an
inducer for the synthesis of the next enzyme. It is remarkable that in many
cases the different enzymes are actually made in succession. Kinetic studies
on the utilization of intermediates or on the formation of enzymes made it
possible to unravel several metabolic pathways by making use of sequential
induction (Stanier, 1947; Karlson and Barker, 1948; Ajl, 1950; Wiame,
1951).

If all the enzymes were induced either by an endogeneous or by an

REGULATION 129

exogeneous inducer, it could be visualized that the inducer brings a comple-
ment of structural information, and that it is involved for instance in
organizing the active centre of the enzyme. Should the inducer cause the
polypeptide chain of the nascent enzyme to fold in a specific way, the
remarkable coaptation of enzyme with substrate would receive a rational
interpretation ; sequential induction would even partly explain the establish-
ment of metabolic chains of successive reactions (Pollock, 1953).

The unitarj'^ hypothesis of general induction in enzyme synthesis looked
indeed very promising. It was difficult to test experimentally, however, for
the failure to isolate from a constitutive mutant a substance which can
induce an inducible strain does not prove much ; success has actually been
claimed (Kramer and Straub, 1956) in penicillinase induction, but the
results proved difficult to reproduce and the nature of the active agent could
not be quite clearly established. The problem has recently been approached
by the methods of genetics in the exceptionally favourable case of j3-
galactosidase of E. coli K 12. As mentioned in Chapter I, the genome of a
Hfr bacterium, or part of it, can be transferred during conjugation to an
F~ recipient bacterium. Crosses of this type were made between bacteria
which differed by the presence of normal or mutated genes either in the z
locus controlling the structure of ^-galactosidase, or in the i region which
controls the inducible or constitutive character of the synthesis of the
enzyme (Pardee et al., 1959). Let us call z+ the normal form of the z locus
and z" any mutated form of this locus which prevents the formation of
active enzyme protein. In the same way, i+ will refer to inducibility, i~ to
constitutivity. Let us consider the two genetic formulae z+i+ and z~i~; none
of these strains will produce /S-galactosidase in the absence of added
inducer: the former because it must be induced to do so, the second because
it does not possess the correct structural information for making the
enzyme. Zygotes resulting from the conjugation of a donor z^i" and of a
recipient z+i+ do not produce the enzyme in the absence of exogeneous
inducer, although they possess both the structural information z+ and the
constitutivity character i~. On the other hand, if a z~i~ strain which is
potentially constitutive but does not possess the structural information
receives the information z+ together with i+, i.e. inducibility, from a donor
strain, it immediately starts making the enzyme, but the synthesis stops
after some time; it will then be obtained again if an inducer is added. The
zygote at first behaved like a constitutive, but it became adaptive after some
time. When both forms of the i gene are present in the same bacterium, the
phenotype corresponding to inducibility prevails ; the 'inducible' character
is dominant, 'constitutive' is recessive. The constitutive bacterium, how-
ever, contains all that is necessary for making the enzyme, this is an obvious
certainty. Therefore, it must be concluded that the i+ gene which causes
inducibility prevents the spontaneous production of the enzyme by

130 THE BIOSYNTHESIS OF PROTEINS

establishing some kind of specific inhibition. Since in the second type of
crossing inducibiHty (as opposed to constitutivity) takes some time to be
established, it would seem that the development or the operation of the
inhibitory mechanism is rather slow. On the other hand, an exogeneous
inducer is able to release the inhibition rapidly.

These genetical studies do not indicate the nature of the inhibitory
system. They are not incompatible with the general induction theory. The
i+ gene might be responsible for the production of an enzyme D which
destroys the endogeneous inducer; since this endogeneous inducer must be
present in the constitutive cytoplasm, the z+ gene would be expressed as
soon as introduced; to the contrary, the expression of the dominant i+ gene
would take the time required for the enzyme D to reduce the steady state
concentration of the endogeneous inducer below the induction threshold.
A completely different interpretation of the experimental results can also
be considered, which rejects the generalized induction theory. It may be
assumed that the i+ gene (inducibility) controls the production of a sub-
stance which blocks specifically the synthesis of galactosidase, and the
action of which is antagonized by exogeneous inducers. The delay in the
establishment of the 'inducible' character in the second type of crossing is
then explained by the time required for reaching the threshold concentra-
tion of the repressing agent.

Recent data by Pardee and Prestidge (1959) indicate that the i+ gene
is able to express itself under conditions which prevent protein synthesis
almost completely. If this conclusion is correct, the first model can be dis-
carded. Besides, these experiments indicate that the i+ gene does not con-
trol the structure of any protein, and — adopting the second of the models
discussed above — that the repressing agent is not a protein.

The generalized repression theory departs from the generalized induc-
tion theory in that it leaves all the structural information to the structural
gene, the inducer and the repressor are mere regulators of the enzyme
producing systems. The generalized repression theory unites in a single
concept the phenomena of induction and of repression of enzyme synthesis,
which will now be examined briefly.

2. Repression of Enzyme Synthesis in Micro-organisms

Galactose inhibits the synthesis of constitutive ^-galactosidase in E. colt.
Tryptophan and certain of its analogues inhibit the formation of tryptophan
synthetase in the same organism (Monod and Cohen-Bazire, 1953). The
specificity of these inhibitions indicated that the substances interfere with
the process of induction of the enzymes (Cohn and Monod, 1953). More
cases of such inhibitions have been observed and further analysed. Thus
Gorini and Maas (1957) and Vogel (1957), who coined the name repression
for this phenomenon, observed that the synthesis of each enzyme of the

REGULATION 131

anabolic chain which makes arginine starting from acetylornithine is in-
hibited by arginine. VaHne represses the synthesis of vahne transaminase
(Adelberg, 1953), uracil represses the synthesis of at least three constitutive
enzymes of the metabolic chain leading to orotic acid (Yates and Pardee,
1957); similar repression phenomena were found in the pathways of syn-
thesis of the purines (Magasanik, 1957, 1958), of methionine and cysteine
(Cohn et al., 1953, Wyesundera and Woods, 1953, 1960; Bourgeois et al,
1959, 1960) and histidine (Ames and Garry, 1959). Inorganic phosphate
represses the synthesis of alkaline phosphatase in E. coli (Horiuchi et al.,
1959; Torriani, 1960).

Inhibition of the synthesis of certain enzymes by glucose had been
known for a long time, especially in cases of induced enzyme synthesis
(Dienert, 1900; Stephenson and Yudkin, 1936; Gale, 1943). This glucose
effect is well illustrated by diauxie (Monod, 1942). When bacteria are grown
in a medium containing two carbon sources, e.g. glucose and maltose, they
use glucose first ; growth slows down very much when most of the glucose
has been used up, and it is only then that the synthesis of the enzyme which
attacks maltose is permitted. The utilization of the new carbon source
makes possible a restoration of growth, resulting in the familiar diauxie
growth curve. The glucose effect, which had not been analysed very much
because it was observed for many enzymes and therefore seemed to be
unspecific, is a case of repression (Neidhardt and Magasanik, 1956, 1957;
Magasanik, 1957; Magasanik et al, 1959).

Repression is thus as common a phenomenon as induced synthesis of
enzymes. As a rule, enzyme synthesis is induced by the substrate and
repression is caused by the product of reaction or by the end product of the
anabolic chain in which the enzyme is involved. Induction and repression
of enzyme synthesis thus afford a remarkable adjustment system which
selects within the genetic potentialities of the cell an enzyme assortment
adapted to the conditions of the medium. Induction has an immediate or
very rapid elTect on the level of the enzyme ; repression changes much less
rapidly the level of enzyme: it simply stops further production of an
enzyme which was being made, the level of the enzyme usually decreases as
it is being diluted in the increasing cell mass of the growing bacterial
population.

3. Mechanisms of Repression and Induction

The formal antagonism of inducer and repressor suggests that these
substances might both act in opposite ways on the same controlling system ;
there might exist between them a relation similar to that of the substrate
of an enzyme and a competitive inhibitor. Recent experiments by Gorini
(1960) indeed showed that ornithine can reverse the repressive eff^ect of
arginine on the formation of ornithine transcarbamylase, and induction by

132 THE BIOSYNTHESIS OF PROTEINS

ornithine occurs only under conditions of partial repression by arginine.
The effects of ornithine, the inducer, and of arginine, the repressor, seem
to be competitive.

Sulphate represses tyrosinase formation in Neurospora (Horowitz et ah,
1960) and aromatic amino acids release the repression. Tyrosinase thus
behaves as an adaptive or as a constitutive enzyme depending on the con-
centration of sulphate in the medium. Here it is difficult to visualize how
sulphate and aromatic amino acids could compete for the same site; it
should be realized that there is no reason to think that the exogeneous
inducers and repressors act as such on the protein making machinery. The
necessity for the transformation of the added inducer into the actual induc-
ing agent has been postulated in several cases of enzyme induction. Clayton
(1960) provided evidence for the elaboration of an intermediary substance
responsible for induction of catalase by the exogeneous inducers oxygen
or hydrogen peroxide. New words will be needed to distinguish between
the inducers and repressors that the experimenter adds to the cell sus-
pension and the resulting active intracellular agents which operate the
regulatory mechanism. Competition probably takes place between these
'elaborated' inducers and repressors.

Since induction and repression are highly specific, inducers and repres-
sors must exert their action on the system which controls the specificity of
synthesis of the individual proteins. Three sites of action of the regulators
can be envisaged at present: the structural gene, the genetic messenger
which carries the information to the enzyme making system, and the
template itself. Presently available data do not permit to decide. Enuclea-
tion experiments should make it possible to establish whether induced
enzyme synthesis can take place in the absence of the gene. Unfortunately,
the type of cells which can be easily enucleated, so far proved unfavourable
to enzyme induction studies. An increase of catalase activity was caused by
hydrogen peroxide in anucleate as well as in intact Acetahiilaria mediter-
ranea (Brachet and Chantrenne, 1953), but it could not be clearly established
whether catalase was synthesized in the process (Chantrenne, 1955b).
Induced enzyme formation in yeast is not inhibited by very high doses of
ionizing radiation, which cause such damage to the DNA of the cell that it
cannot be precipitated with acid after alkaline extraction (Chantrenne and
Devreux, 1959). Induced enzyme synthesis has been observed in bacterial
preparations from which most of the DNA had been removed by salt
extraction or destroyed by deoxyribonuclease (Spiegelman, 1956). These
facts suggest that the site of action of the inducer is not the DNA; unfor-
tunately, the results are not entirely convincing, because the nature of the
damage caused to DNA by radiation is not clearly understood, and the
disrupted bacterial system from which most of the DNA was extracted
synthesized new DNA while making enzymes.

REGULATION 133

Since RNA is most probably the specific constituent of the template
which organizes proteins, the question as to the requirement of specific
RNA synthesis for enzyme induction is relevant to the problem under
discussion. If the inducer or repressor operate at the level of the gene and
regulate the production of cytoplasmic replicas made of RNA, one would
expect enzymic induction to be accompanied by the synthesis of new
specific RNA molecules, the organizers of the induced enzyme. Several
years ago, some experimental data were quoted in favour of such a view.
Thus Creaser (1955) observed that the induced synthesis of a /3-galactosidase
in Staphylococcus aureus is stimulated by purines and pyrimidines. The
ability of resting yeast to form an induced a-glucosidase is strongly de-
pendent upon the level of the free nucleotide pool (Spiegelman et al., 1955).
Several purine and pyrimidine analogues were reported to inhibit the
induced synthesis of enzymes much more than the synthesis of constitutive
enzymes. However, this conclusion proved unfounded (for a discussion see
Chantrenne, 1958). The incorporation of labelled uracil or adenine is
stimulated during enzyme induction in disrupted Staphylococci (Gale and
Folkes, 1955) and in yeast (Chantrenne, 1956, 1958) ; this at first sight could
be taken as indicating the synthesis of new RNA molecules. Further
studies on the yeast system, however, showed that the stimulated incorpora-
tion was probably linked in an indirect way only to the induction process,
and it cannot be taken as convincing evidence for the synthesis of new RNA
molecules (Chantrenne, 1958; Chantrenne and Devreux, 1959). The dis-
covery of chain end renewal in soluble RNA (see Chapter III) adds one
more difficulty in evaluating the significance of increased incorporation
of precursors into total cellular RNA.

Release of repression does not seem to require any RNA synthesis. The
formation of ornithine transcarbamylase is repressed by arginine ; it starts
as soon as arginine is removed from the medium. With a uracil-less strain,
it is possible to prevent RNA synthesis by uracil deprivation. Even so,
removal of arginine causes immediate synthesis of the enzyme under con-
ditions which prevent net RNA synthesis (Rogers and Novelh, 1959).
Studies on diauxie also indicated that little RNA is made during the lag
which corresponds to the release of repression (Magasanik et al, 1959).
It must be realized, however, that the synthesis of an amount of RNA
representing 1 per cent of total RNA would escape observation; and the
amount of RNA that one would expect to be synthesized during adaptation
would not exceed that value : there are several hundred different proteins
in a bacterial species, there must be as many specific RNA varieties ; the
appearance of one new type of template RNA among several hundred pre-
existing RNAs would indeed be very difficult to detect by quantitative bio-
chemical determinations. Moreover, the absence of net synthesis does not
mean that no new molecular species of RNA can form; Earner and Cohen

134 THE BIOSYNTHESIS OF PROTEINS

(1958) indeed showed that a rapid RNA turnover takes place in uracil-less
mutants during protein synthesis.

The question as to whether enzyme induction requires the synthesis of
new specific RNA molecules is not solved. Several data which were taken
as circumstantial evidence for a necessary RNA synthesis now appear
irrelevant, and no positive evidence has been afforded so far. On the other
hand, the pieces of evidence against the synthesis of specific RNA are not
completely convincing either. The problem therefore is still open.

A more direct approach has been the search for specific RNA in adapted
cells. More or less crude preparations containing RNA were extracted from
induced bacteria and added to suspensions of non-adapted cells. In a few
cases, it was reported that these extracts induced the synthesis of the
enzyme that was looked for, although there was no regular inducer in the
preparations used (Minagawa, 1955 ; Reiner and Goodman, 1955 ; Hunter
and Butler, 1956; Nomura and Yoshikawa, 1959). Kramer and Straub
(1956, 1957) reported that an extract, obtained by treating a penicillinase
constitutive mutant of B. cereus with m NaCl at 100° C, causes the in-
ducible strain to produce penicillinase, provided the recipient bacteria
have been pre-treated with ribonuclease. Penicillinase production lasts
but for 20-30 min, and it is inhibited by chloramphenicol. Ribonuclease
abolishes the activity of the sodium chloride extract. Further analysis of
this interesting phenomenon is needed in order to establish whether the
effect is specific and whether it corresponds to an actual synthesis of enzyme
(Pollock, 1959). It is difficult to evaluate the significance of these observa-
tions; the fact that they are difficult to duplicate and that they involved
crude preparations is no reason to disregard them.

Lack of clear and positive evidence for the formation of new specific
RNA organizers during enzyme induction encouraged the idea that induc-
tion and repression of enzyme synthesis rest on the control of pre-existing
protein forming machines (Monod, 1958; Vogel, 1957; Magasanik et ah,
1959). The inducers and repressors could for instance facilitate or prevent
the separation of the nascent protein from the assembly template (Vogel,
1957, 1958; Monod, 1958), or activate it in any other way.

More recent data on the genetic control of /S-galactosidase synthesis in
E. coli indicate that the inducer and repressor (or more precisely the real
controlling agents derived from them) might actually operate a switch of
some sort which blocks or releases the operation of the protein weaving
machine. Jacob et al. (1960) showed that the regulatory genetic determinant
i which controls the induced versus constitutive nature of ^-galactosidase
synthesis controls simultaneously the expression of several structural
genes which are closely linked on the genetic map. A similar situation was
observed in the pathways of tryptophan formation (Cohen and Jacob,
1959) and of histidine synthesis: histidine represses the synthesis of the

REGULATION 135

four enzymes involved in the conversion of imidazolglycerolphosphate into
histidine (Ames and Garry, 1959). The use of a choice of mutants makes it
clear that the effect cannot be explained by the prevention of sequential
inductions. The four genes corresponding to the four enzymes are con-
tiguous (Hartman, 1956). It looks as if a group of closely linked genes could
be put into operation or blocked simultaneously. The existence of a lock or
'operator' is supported by direct genetical studies in the case of /3-galactosi-
dase (Jacob et al, 1960) where mutants have been isolated which have
the properties one would predict if the mutation had occurred within the
operator. It was also shown that in heterozygotes the operator controls the
expression of the genes when they are in cis position with the operator, and
does not act on the genes which are in a piece of genetic material separated
from the homologous one which contains the operator. These facts can be
tentatively interpreted in the following way (Jacob et al., 1960) : besides the
locus which contains the structural information for the synthesis of an
enzyme, one should distinguish a new genetical unit, the operon, made of
an operator and a group of closely linked loci, the expression of which is
controlled by the operator. The operator is acted upon by a repressor pro-
duced under the action of the controlling gene (for instance, in the case
of galactosidase). The repressor blocks the operon; the locked operon can
be released by an inducer.

This fascinating scheme contains implications which are amenable to
experimental test; future research will tell whether it is a good picture of
reality. It provides an interesting interpretation for the clustering of genes
controlling the enzymes of a single metabolic pathway (Hartman, 1956).
At first sight, the operator hypothesis might be taken to mean that the
repressor acts at the level of the genome. Actually, the available data are
just as compatible with an action of the repressor on cytoplasmic replicas of
the operator. This would, however, imply that the operon is copied as a unit
and that the cytoplasmic templates corresponding to the several genetic
loci of an operon work in a co-ordinate manner. It will be interesting to
compare the size of the operon with those of the DNA molecule and of
ribosomal RNA. The DNA molecule is large enough to accommodate
several loci, and the RNA molecule of the ribosome large enough to serve
as a template for several proteins. One may wonder whether the new
physiological unit of expression, the operon, will be superposable to the
chemical unit of structure, the molecule, and to the RNA quantum of the
ribosomes.

How can one visualize at the molecular level the process of repression
and induction in this perspective? The keys which lock or release the system
must be of such a nature that they can interact with the lock. The operator
may be assumed to be a region of a DNA molecule or of its cytoplasmic
RNA replica. The key should be able to interact in a specific way with a

K

136 THE BIOSYNTHESIS OF PROTEINS

nucleotide sequence. In view of the present knowledge of the interactions
between polynucleotides a sensible hypothesis is that the active repressing
agent contains a nucleotide sequence complementary to that of the operator
or to its cytoplasmic replica. The polynucleotidic repressing agent could be
made by the controlling gene (i+ for galactosidase) which indeed does not
seem to control the structure of any protein (Pardee and Prestidge, 1959).
A more difficult matter is to fit the exogeneous, or endogeneous, inducers
like lactose into the picture ; do they cause or prevent the production of the
repressor at the level of the gene, do they become associated with a specific
nucleotide sequence (Szilard, 1960), butthenby what enzyme action? Where
does the competition take place? Thus we are confronted with the same kind
of questions as before. But because they are asked in new terms, these
questions will certainly suggest new experim.ents which will throw new
light upon the mechanism of induction and repression of enzyme synthesis.

Torriani (1956) and Halvorson and Jackson (1956) observed that the
early phase of induction is exceptionally sensitive to ultraviolet irradiation.
During the lag period of maltase induction by maltose in resting yeast, a
fraction of RNA becomes acid soluble without being degraded to free
nucleotides, and this fraction is reincorporated into acid insoluble RNA
when the synthesis of the enzyme begins (Gobert — see Chantrenne, 1958).
This small RNA fraction is released from the ribosomes (Gobert, unpubl.).
One may wonder whether these observations have some bearing on the
transitory participation of fragile nucleotidic compounds in the process
which triggers enzyme synthesis.

Finally, it may be remarked that if induction and repression of the
synthesis of enzymes is a process in which the specific templates are put to
operation or switched off^, the rate of production of an enzyme must be
determined by the number of templates which are in operation. In a con-
stitutive strain, the rate of production of an enzyme must depend on the
total number of templates corresponding to the enzyme. Almost nothing
is known about the number of identical templates in the cell, neither is
it known when the templates are produced: are they being continuously
produced or only at one stage of cell division (Mitchinson, 1958; Plaut,
personal communication)? Do all the structural genes produce the same
number of cytoplasmic replica, or is another regulation mechanism operat-
ing on the production of templates? In haemoglobin synthesis in man, the
templates depending on two homologous genes present in a heterozygote
produce their specific protein at about the same rate, since the amounts of
normal and sickle cell haemoglobins produced are comparable.

It will be noticed that a large part of the present picture of induction and
repression of enzyme synthesis is derived from studies on a few micro-
organisms and a few enzymes. By far the most thoroughly analysed case is
^-galactosidase synthesis in E. coli; it has been the object of exceptionally

REGULATION 137

far-reaching genetical studies. The uniformity of fundamental biochemical
mechanisms in all kinds of living cells allows one to assume that the mechan-
isms involved in the control of ^S-galactosidase synthesis are probably of wide
significance. It would, however, be misleading to suppose that these solve all
the problems of regulation and that no other mechanisms can be operating.
Other types of processes may be superimposed upon those described
above. For instance, evidence that enzyme transformation occurs in certain
cases has been presented recently.

4. Induced Transformation of an Enzyme

In anaerobically grown yeast, oxygen induces the formation of the
enzymes and electron carriers of the respiratory chain (Ephrussi and
Slonimski, 1950; Slonimski, 1953, 1954, 1955) and of several peroxidases
(Chantrenne, 1954, 1955; Chantrenne and Courtois, 1954). Complete
synthesis of the cytochrome-c protein during this adaptation has been
demonstrated (Yeas and Drabkin, 1955), but more complex processes also
occur. Changes in the absorption spectrum of the yeast cells suggest the
replacement of certain haemoproteins by others (Slonimski, 1953; Yeas,
1956). This might be due to protein turnover, for at variance with bacteria,
yeast readily makes new enzymes in the absence of any external source of
nitrogen, at the expense of the intracellular pool of free amino acids and of
proteins which can be degraded to amino acids (Halvorson, 1958). How-
ever, the formation of lactic dehydrogenase during respiratory adaptation
seems to involve a direct transformation of enzymes.

In anaerobically grown yeast, there is a lactic acid dehydrogenase which
catalyses the dehydrogenation of D-lactic acid only; on the contrary, after
respiratory adaptation, an enzyme can be isolated from yeast which oxidizes
the L-isomer of lactic acid specifically. Meanwhile, the anaerobic enzyme
almost completely disappears (Slonimski and Tysarowski, 1958; Labeyrie
et al., 1959; Nygaard, 1959, 1960). This replacement of an enzyme by
another related enzyme is not due to the destruction of one protein while
the other is being made ; there is evidence that the anaerobic D-lactic acid
dehydrogenase is transformed into the L-enzyme normally present in
aerobic cells (Kattermann and Slonimski, 1960). Amino acid analogues
preclude the formation of active catalase, cytochrome oxidase, cytochrome
peroxidase, presumably by being incorporated into the proteins ; but these
analogues do not interfere with the replacement of the anaerobic lactic
dehydrogenase by the aerobic type of enzyme. Stereospecificity is not the
only diff'erence between the two enzymes : the aerobic type reduces cyto-
chrome-c, the anaerobic does not. Thus the transformation involves the
acquisition of cytochrome reductase activity beside the change in stereo-
specificity. An intermediary stage in the transformation was detected: the
anaerobic enzyme which is specific for D-lactic acid first acquires the

138 THE BIOSYNTHESIS OF PROTEINS

property of transferring electrons to cytochrome, and it is later changed
into the L-specific enzyme. The complete transformation takes more time
than the synthesis of cytochrome oxidase or catalase, which seem to behave
according to the classical scheme of induced enzyme synthesis (Nygaard,
1960).

5. Permeases and Efizyme Synthesis

Certain exogeneous inducers induce enzyme synthesis only if they can
enter the bacterium and if their concentration at their site of action is
sufficient. Small molecular weight substances sometimes can enter a cell
by diffusion. More complex processes are involved in most cases; the
'permeability' of cells is very specific and cannot in any way be compared
to the old misleading model of the semipermeable membrane which retains
certain molecular species and allows water and other substances to diffuse
freely. Some cells absorb and concentrate proteins readily but are com-
pletely impermeable to glycin or succinic acid for instance. The proteins
are taken up by a complex process of pinocytosis. Moreover, all types of
cells are able to concentrate various substances, like amino acids or sugars,
from an outer dilute solution. Thus E. colt grown on lactose is able to
absorb and concentrate several j8-galactosides (Monod, 1956; Cohen and
Monod, 1957); the concentration inside the bacterium can be a hundred
times the outer concentration, and its maintenance requires a continuous
expenditure of energy. Azide, which prevents phosphorylations, inhibits
the concentration mechanism. The accumulated galactosides seem to be
free within the bacterium and this considerably increases the inner osmotic
pressure; if the cell wall is damaged by enzyme action, its mechanical
strength is reduced, and the internal osmotic pressure resulting from the
accumulation of the galactosides can be high enough to cause the rupture of
the bacteria (Rickenberg et al., 1956; Sistrom, 1958). This permeation
system, which was called 'permease', is not present in bacteria which have
not been in contact with galactosides; it is an inducible system which
responds to the same inducers as /3-galactosidase.

When a non-adapted bacterium is confronted with an adequate galacto-
side, some galactoside enters the cell by diffusion, and there is a certain
probability that the formation of one molecule of permease is induced.
Once this molecule is formed, the internal concentration of galactoside
increases, and this results in the induction of more permease and of j8-
galactosidase at the same time (Novick and Weiner, 1957). The permease —
the chemical nature of which is unknown — thus influences the induced
formation of the enzyme j3-galactosidase in an indirect way, by concentrat-
ing the inducer of the enzyme within the cell. The kinetics of j3-galactosi-
dase formation actually reflects that of the formation of permease.

Rather complex consequences of this situation can be observed. If

REGULATION 139

non-induced cells are grown in a medium containing glucose and a little
^-galactoside, the permease and the enzyme are not produced, due to re-
pression by glucose. However, if the bacteria are first grown on lactose,
and later transferred into the medium which contains glucose and a galacto-
side, they will continue to make both the permease and ^-galactosidase,
because the permease they contain to start with is able to concentrate the
galactoside enough to release the glucose repression. Thus two populations
of identical genotype in an identical environment can be made to differ in
their assortment of enzymes. This situation, once it has been established,
is very stable ; it could be maintained for more than 150 generations (Cohn,
1958).

If unadapted bacteria are given a threshold concentration of inducer for
a short time, the probability of acquiring the first molecule of permease is
not great; therefore in a population of bacteria certain individual cells will
adapt, and others will not. By increasing then the glucose to inducer ratio
in the medium adequately, it is possible to maintain in the same population
individual cells which do make the enzyme and others which do not. The
formation or non-formation of the enzyme is passed on clonally by the
cells to their descendants, as if it were a regular hereditary character (Cohn
and Horibata, 1959). Maintenance in the adapted cells is of course condi-
tioned by the presence of the inducer in the medium.

This illustrates well the very complex consequences of the occurrence of
permeases for the regulation of enzyme synthesis. Comparable per-
meation systems have been found for amino acids and for various sugars and
derivatives.

6. Enzymic Adaptation in Animal Tissues

A few cases of enzymic adaptation in animals have been reported. The
tryptophan peroxidase activity of rat liver increases when tryptophan is
injected into the animal (Knox and Mehler, 1950, 1951); the same enzyme
can be evoked in dissociated embryonic cells (Stearns and Kostellow, 1958).
Tyrosine transaminase is increased by injections of tyrosine (Lin and Knox,
1957). A strong increase of xanthinoxidase activity in the liver of mice is
observed about a week after they have received injections of xanthine
(Dietrich, 1954). Formation of adenosine deaminase in chick embryos after
administration of adenosine was also reported (Gordon and Roder, 1953);
however, systematic studies by Solomon (1960) did not confirm this
observation. A strong decrease in kidney glutaminase is caused by
glutamate injection; this might be an example of repression (Goldstein,
1959).

Enzymic adaptation in animals does not show the schematic simplicity
of the process found in micro-organisms. A review by Knox et al. (1956)
shows very well how intricate and complex is the regulation of the level of

140 THE BIOSYNTHESIS OF PROTEINS

enzyme activity in animals. The system which has best been studied from
the biochemical point of view is the stimulation of tryptophan peroxidase
activity of rat liver. It was soon observed (Knox, 1951) that an increase of
this enzyme activity can be obtained not only by tryptophan, but also by
the injection of histidine or of adrenaline. Adrenalectomy reduces the
normal basic level of the enzyme. Due to this and other hormonal actions,
involved controlling effects are observed. Cortisone and hydroxycortisone
stimulate the production of the enzyme (Knox and Auerbach, 1955).
Adrenocorticotropic hormone (ACTH) stimulates peroxidase activity, and
hypophysectomy depresses it. This effect of ACTH is probably mediated
through the steroid hormones since ACTH causes the synthesis of corti-
costeroids (Koritz et ah, 1957). Reserpin (Canal et ah, 1959) and insulin
(Schor and Frieden, 1958) also increase the level of the enzyme. The stimu-
lating effects of tryptophan and of insuline or hydroxycortisone are
additive, as if these agents were acting in different ways (Civen and Knox,
1959; Schor and Frieden, 1958).

Increase in tryptophan peroxidase activity was obtained in isolated
perfused liver (Price and Dietrich, 1957) and in liver slices (Ephimotchkina,
1954). Such systems make it possible to avoid the hormonal effects. Even in
cell-free systems, an increase of tryptophan peroxidase was caused by
tryptophan (Clouet and Gordon, 1959), but the increment of enzyme
activity was correlated with changes in permeability of the mitochondria,
which strongly suggests that the increase in activity might reflect changes
in the accessibility of the enzyme rather than an actual de novo synthesis
(Gordon and Rydziel, 1959). There were indications that a net synthesis
of tryptophan peroxidase occurs during in vivo induction by tryptophan
(Gros et al., 1956). On the other hand, convincing evidence was recently
obtained that substrate induction of this enzyme activity may be due — at
least in part — to an intracellular translocation of the enzyme or of an
activator (Greengaard and Feigelson, 1960); this reminds one of certain
observations by Lee and Williams (1953) pointing in the same direction.
It is therefore doubtful whether tryptophan peroxidase 'induction' has
anything in common with the induced synthesis of enzymes as observed in
micro-organisms; it might be a case of adjustment of the activity of pre-
existing enzyme systems. This may explain the rapid disappearance of the
enzyme activity during deadaptation (Feigelson et al., 1959).

The activity of tyrosine a-ketoglutarate transaminase can also be
increased by injecting tyrosine or hydroxycortisone (Lin and Knox, 1958).
It would seem at present that the increase rests on a process of activation or
release and not on protein synthesis, as shown by experiments combining
the use of labelled amino acids and the serological isolation of the enzyme
(Kenney, 1960).

One should therefore be very careful in assimilating these effects with

REGULATION 141

the induced synthesis of proteins. Enzymic adaptation in animals — at
least for the presently analysed cases — may be irrelevant to the control of
enzyme synthesis.

B. NON-MENDELIAN HEREDITARY FACTORS IN
ENZYME SYNTHESIS

According to the presently accepted theory of enzyme synthesis, the
nuclear gene contains information relative to the arrangement of the amino
acids in the polypeptides. The information is transferred to some cyto-
plasmic constituent, most probably a specific RNA, and the amino acids
condense in the genetically determined sequence on the surface of the
ribosome. The resulting polypeptide is released and it folds in the correct
way, thus giving rise to the active enzyme.

At some point along this sequence of events, controlling agents intervene,
either to trigger or to inhibit the formation of individual proteins or of
restricted groups of proteins.

A few important experimental facts are not clearly integrated into this
scheme. For instance, the ability of yeast and moulds to make several
enzymes of aerobic metabolism is known to be carried over from parent to
progeny in a manner which does not obey mendelian laws of heredity. Let
us summarize first some of the observations made by Ephrussi and his
group on yeast.

When a culture of Saccharomyces cerevisiae is plated on a solid medium
containing glucose, most of the colonies which develop are of nearly
identical size; however, a few colonies of distinctly smaller diameter are
always observed. This is not due to contamination or to heterogeneity of
the yeast strain used, for the same result is obtained when the culture is
initiated with one single cell isolated from a large colony. Such cells con-
stantly produce in their progeny a majority of cells identical to themselves,
and a few cells of a difierent type, recognizable by the small size of the
colonies they form. To the contrary, cells from small colonies give rise to
small colonies exclusively; they never recover the capacity of forming large
colonies (Ephrussi, 1949, 1953). The change from large colony to small
colony type is hereditary, and it is irreversible as far as the present data
indicate; the difference in size of the colonies is due to a difference in rate
of multiplication of the cells. Actually, this difference is observed only when
the cells are grown in the presence of air; in a nitrogen atmosphere, normal
yeast does not grow faster than the mutant. It was indeed clearly established
that the small colonies mutant is deprived of a functional respiratory
system, and that it lacks several important enzymes of the respiratory
chain, for instance C)^ochrome oxidase, cytochrome-a and cytochrome-6

142 THE BIOSYNTHESIS OF PROTEINS

(Tavlitzki, 1949; Slonimski and Ephrussi, 1949; Ephrussi and Slonimski,
1950; Slonimski, 1949, 1953).

The mutation responsible for the appearance of respiration deficiency
differs from normal mutations in several respects. It occurs spontaneously
with a frequency of about 3-10""^ per cell per generation, whereas usual
genetic mutations occur one thousand times less frequently. The rate of
mutation is as high in diploid as in haploid cells. It can be increased
enormously by various treatments: effect of acridine dyes (Ephrussi, 1949,
1953; Ephrussi and Hottinguer, 1950), triphenyltetrazolium chloride
(Laskowsky, 1954), manganous ions (Nagai and Nagai, 1958), an elevated
temperature (Yeas 1956; Sherman, 1959), ultraviolet light (Raut, 1954)
and by various other agents (Lindegren, 1959). In the presence of euflavine,
under proper conditions, practically every bud formed is a respiration
deficient mutant. The mutation affects simultaneously the formation of
several enzymes. However, when the yeast cells are treated by a threshold
concentration of acriflavine, some of the freshly arisen small colony cells
can still give rise to both normal and respiration deficient cells ; it looks as
if they remained for a few generations in an unstable state from which they
can still revert to the normal type or become irreversibly respiratory
deficient (Ephrussi and Hottinguer, 1951). All these features indicate that
this mutation is indeed of an exceptional type. It is exceptional also in its
hereditary transmission, as we shall see presently.

Haploid Saccharomyces cerevisiae of opposing mating types can be
crossed; the diploid zygotes multiply by budding for unlimited periods,
remaining diploid. They can, however, be induced to sporulate by being
transferred onto adequate medium. The asci contain four haploid spores.
The spores, if isolated, give rise to haploid cultures which will remain
indefinitely haploid unless crossed with a haploid of the opposite mating

type.

When two haploid yeasts differing by one single mendelian character (A'
versus A) are crossed, and the resulting diploid is caused to sporulate, two
spores in each ascus contain the allele A, the other two contain the allele
A'. This is a regular mendelian segregation of the character. When a normal
haploid yeast is crossed with a respiration deficient mutant obtained as
indicated above, different results can be obtained, depending on the par-
ticular mutant used. Two extreme types have been encountered: the so-
called neutral and the suppressive mutants. Crosses of normal yeast with
neutral small colony mutant give normally respiring zygotes, and the four
spores in each ascus derived from such zygotes are all of the normal
respiratory type. Respiration deficiency is not transmitted to any of the
spores, no mendelian segregation of the character is observed, the character
simply disappears, although in the very same asci typical mendelian
markers were shown to segregate in the regular manner. These and other

Fig. 34. Colonies formed on normal solid medium by yeast grown in

normal medium (A), and in media containing 1/100,000 (B) or 1/10,000

(C) of acriflavine (Ephrussi, 1950).

REGULATION

143

genetic experiments established that the mutation to respiration deficiency
does not concern a chromosomal gene. It must consist in the loss or
irreversible inactivation of an extrachromosomal factor which is necessary
for the synthesis of respiratory enzymes (Chen et ah, 1950). This extra-
chromosomal factor is perpetuated during cell proliferation, since it is
transmitted indefinitely through generations in normal yeast.

Crosses involving the 'suppressive' type of small colony mutant give a
slightly different picture. Zygotes formed by fusion of a normal with a
suppressive are able to respire and they can be made to sporulate.

But in this case, the four spores in each ascus all produce respiratory

Fig. 35. Result of a cross between a normal strain (G) and a cytoplasmic

mutant (P) of yeast. The capacity to form respiratory enzymes is indicated

by stippling (Ephrussi, 1953).

deficient mutants which are of the suppressive type. Here again the
respiration capacity fails to segregate as mendelian markers do, indicating
that it depends for its transmission on an extrachromosomal factor. This
time, the small colony character prevails; it would seem that the sup-
pressive mutant not only fails to perpetuate the normal extrachromosomal
object, but that it also prevents its reproduction in the cytoplasm of the
hybrid.

Comparable observations were made on Neurospora crassa, for the
synthesis of the same group of respiratory enzymes. Several respiration
deficient mutants were isolated by Mitchell and Mitchell (1952). Some of

144 THE BIOSYNTHESIS OF PROTEINS

them are maternally inherited; this was well established for the mutant
'poky' and for a few others designated by the symbol mi (for maternally
inherited). The character again does not segregate as a mendelian marker.
In these mutants, the respiratory system shows various abnormalities in the
cytochromes system (Mitchell et aL, 1953; Tissieres et al., 1953; Tissieres
and Mitchell, 1954).

In Neurospora as well as in yeast, the formation of several enzymes of the
respiratory chain thus depends on some hereditary non-mendelian factor.
In Neurospora, just as in yeast, it looks as if an extrachromosomal element
were required, as if this object could ensure its own continuity through
generations of cells, and as if it were in certain cases able to replace the
corresponding factor of the wild type (Pittenger, 1956).

To complete the picture, it must be emphasized that although the
synthesis of the respiratory enzymes in yeast and mould depends on the
presence of an extrachromosomal factor, it also depends nevertheless on
typical mendelian genes, just like any other enzyme synthesis. This last
point is established by the existence of still another type of respiration
deficient mutants, in which the character segregates in a perfectly mende-
lian manner. The mutation, this time, concerns the synthesis of a single
protein (Chen et ah, 1950; Mitchell et al., 1953). For instance, the mende-
lian mutants C-115 and C-117 of Neurospora lack cytochrome-^ and cyto-
chrome-6 respectively. The synthesis of these proteins, like that of any
other enzyme, is therefore controlled by regular mendelian genes; the
extrachromosomal factor is a further requirement for the formation of the
respiratory enzymes, which has not been observed for the common
enzymes.

Another group of enzymes which depend on cytoplasmic hereditary
factors are those of the chloroplast in green plants. Certain green flagellates
possess one single chloroplast per cell; this chloroplast normally divides
before cell division and each daughter cell receives one chloroplast.
Occasionally, the plast division lags behind cell division, and the daughter
cells separate at a time when the division of the chloroplast is not com-
pleted; under such circumstances, one of the daughter cells is deprived of
chloroplast. The striking fact is that cells which have thus lost the chloro-
plast, as a result of a mechanical accident, never recover the capacity of
producing a new chloroplast. The progeny of such cells is for ever devoid
of photosynthesis apparatus (Lwofi^, 1949).

Because of these facts and of observations made on mosaics in higher
plants, chloroplasts are commonly described as self-duplicating organelles.
This is a mere expression of direct microscopical observation.

It is possible to influence by various means the relative rate of division of
the cell and of the chloroplasts in protozoans like Euglena, by changing the
medium or the temperature or by growing the cells in the dark. The num-

REGULATION 145

ber of chloroplasts per cell can thus be considerably reduced, and some
cells may eventually lose their last plast, thus giving rise to a clone of per-
manently white cells. Ultraviolet irradiation also causes the appearance of
many white cells. A mass transformation of green Euglena into white cells
can even be brought about by growth in the presence of streptomycin
(Provasoli et ah, 1951). Loss of the photosynthesis apparatus under these
conditions is irreversible as far as one can judge at present. However, this
permanent loss is preceded by a reversible lesion. When Euglena are kept
in streptomycin for 2 hr (1/10 of the mean generation time) and then trans-
ferred to normal medium, many of the descendants of the treated cells are
white and at each generation some green cells give rise to new white cells
(De Deken-Grenson and Messin, 1958).

Certain of the white cells are apparently changed irreversibly, for their
progeny remains white for ever. Others, however, can produce a normal
green progeny after more than ten white generations. A certain unstable
state can thus result from a short streptomycin treatment, just as yeasts
which have been kept in low concentrations of acridine remain during a
few generations in an unstable state in which respiratory deficient cells can
produce either respiratory deficient or normal buds (Ephrussi and Hot-
tinguer, 1951).

The hereditary loss of chloroplasts in Euglena has indeed much in com-
mon with the hereditary loss of the respiratory system in yeast (De Deken-
Grenson, 1960), Both appear as a mutation, the frequency of which can be
increased enormously and in a specific way by the action of chemicals, or
by unfavourable conditions.

No genetical studies comparable to those made on yeast or Neurospora
could be applied to Euglena, because of the absence of sexual reproduction.
But the importance of an extrachromosomal factor is shown by the fact
that the mechanical loss of the last chloroplast results in permanently white
cells; this indicates that the factor is the chloroplast itself or one of its
constituents. On the other hand, it is almost certain that the formation of
at least certain of the chloroplastic enzymes is undernuclear genetic control,
since several typical mutants blocked at various stages of chlorophyll
synthesis have been isolated from Chlorella (Granick, 1950; Bogorad and
Granick, 1953).

Although the lack of direct genetic analysis makes any conclusion un-
certain, it is most probable that the formation of the photosynthesis
apparatus, like that of the respiratory system of yeast, depends on both
nuclear genes and extrachromosomal factors (chloroplastic in the green
plants).

What are the respective functions of the mendelian gene and of the
extrachromosomal factor in the formation of the respiratory enzymes in
yeast and mould or of chloroplastic proteins in Euglena}

146 THE BIOSYNTHESIS OF PROTEINS

When the experimental facts led to the conclusion that an extra-
chromosomal factor was involved, that this factor was perpetuated through
generations, that it could eventually be lost irreversibly, it was perfectly
normal to visualize this factor as a kind of free lance gene. It was indeed
commonly referred to as plasmagene, self-dividing particle, self-duplicating
particle, extranuclear autoreproducing particulate factor, etc. ... In this
perspective it was assumed that acridines, high temperature, tetrazolium
salts, etc., interfered with the multiplication of this particle 'endowed with
genetic continuity'. The nuclear gene was regarded as controlling the
multiplication of the self-reproducing cytoplasmic particle or its function.
This was very satisfactory especially in view of the fact that several other
biological phenomena appeared to fit into a very similar picture (see Sym-
posium sur les Unites biologiques douees de continuite genetique, 1948).

In the present perspective, however, this interpretation looks less con-
vincing than it was only a few years ago. The function of the nuclear gene
is better understood at present: it is known that the gene controls the
sequence of amino acids in the polypeptide chains (see Chapter I). As a
result, questions about the nature and function of the extrachromosomal
factor necessary for the synthesis of respiratory enzymes can now be asked
in a different manner. One wonders especially whether the object contains
an extra piece of information about the arrangement of amino acids in cyto-
chrome-a, h and cytochrome oxidase, or whether it is involved in some
regulation process.

The irreversibility of the non-chromosomal mutation, the existence
of two types of cytoplasmic respiratory deficient mutants in yeast, the
fact that the induction of the mutation is subordinated to cell multi-
plication, no doubt suggest that it is due to a change in a particulate object
which carries with itself some features necessary to its own perpetuation.
But these facts in no way imply that the extrachromosomal factor carries
any piece of specific structural information required for its own duplication.
There is no positive evidence either that the extrachromosomal factor
carries any piece of structural information for the synthesis of the respira-
tory enzymes.

It has been mentioned that the progeny of certain white Euglenae which
form after a short streptomycin treatment can recover the capacity of
making chloroplasts, although they had no visible chloroplast left. In the
same way, some respiratory deficient yeasts in the unstable state which
follows a moderate acriflavin treatment can give rise to normal respiring
cells. When they are in this unstable state, it is quite certain that yeasts or
Euglenae have not lost any part of the structural information required for
the synthesis of the respiratory enzymes or of the chloroplastic constitu-
ents; it is only the expression of this information that was blocked for
several generations (De Deken-Grenson, 1960).

REGULATION 147

When the capacity of making respiratory enzymes or chloroplasts is lost
permanently, it is not possible so far to decide whether this is due to the
eventual loss of some structural information or to the permanent jamming
of the mechanism of its expression, but the latter possibility looks very
probable in view of the facts just mentioned.

In the case of the 'poky' mutant of Neurospora crassa, there is clear
evidence that no structural information is lost: young cultures of this
maternally inherited mutant are devoid of 'succinoxidase' and cytochrome
oxidase, but older organisms actually make these enzyme systems in
amounts which exceed 50 per cent of the titre of the normal wild strain
(Haskins et al., 1953). Therefore, the complete structural information for
these proteins is contained in 'poky' ; the abnormality is that, in spite of the
presence of the structural information, the synthesis of the enzymes failed
to occur before the fifth or sixth day of growth. This strongly suggests that
the extrachromosomal factor in 'poky' in some way regulates or conditions
the biochemical process of synthesis of the respiratory enzymes.

If this is so, the extrachromosomal hereditary factor need not be regarded
any more as a wandering gene-like particle or plasmagene or self-duplicat-
ing entity; it should rather be considered as a piece of a controlling mechan-
ism. One may even wonder whether the experimental facts could not be
explained by the existence of alternative and mutually exclusive steady
states maintained by some kind of feed back system; the non-chromosomal
mutation might be an irreversible switch from one of these states to the
other. It has indeed been long realized that alternative stable steady states
could mimic heredity (Wright, 1945; Delbruck, 1949; Pollock, 1953; De
Deken-Grenson, 1957; Beale, 1958).

In the case of Euglena, the rate of cytoplasmic mutation depends entirely
on the physiological state of the cell at the time the mutagenic agent is
applied (De Deken-Grenson, 1960). A steady state system would account
for these observations more easily than a plasmagene model.

In recent discussions on cytoplasmic inheritance (Ephrussi, 1958;
Lederberg, 1958; Nanney, 1958; Catcheside, 1958; De Deken-Grenson,
1960) the respective merits of the plasmagene versus steady states were
compared; efforts were made in most cases to advocate one of the models
or to decide into which picture a given phenomenon can best be fitted.
But it is more and more clearly apparent from these discussions that each
type of explanation has valuable features, although none accounts satis-
factorily for all the aspects of cytoplasmic heredity. In the author's opinion,
a satisfactory model should have the sharpness of the plasmagene theory
and the flexibility of the steady state principle.

A unifying theory could possibly be derived from the concept of 'auto-
catal)rtic particle' which was proposed (Campbell, 1960) as an interpreta-
tion of long term adaptation of yeast to galactose (Winge and Roberts,

148 THE BIOSYNTHESIS OF PROTEINS

1948; Spiegelman et al, 1951; Campbell and Spiegelman, 1956). A clear
distinction is made here between autoduplicating particles like genes or
viruses, which carry structural information for their own multiplication,
and aiitocatalytic objects which are required, albeit remotely, for the
formation of identical objects because they fulfil a certain biochemical or
biophysical function. In this broad sense, all the 'indispensable' enzymes
of a living cell are autocatalytic objects, and so are many other cell con-
stituents, e.g. the phospholipids of the membranes. For if the activity of
the enzyme is blocked or if the phospholipid is destroyed, the cell dies and
stops making the enzyme and the phospholipid.

If the physiological activity of an autocatalytic object X was required for
the synthesis of the respiratory enzymes, but completely unnecessary for
the life of the cell and for its multiplication, that autocatalytic object would
indeed have many properties in common with the 'extrachromosomal
hereditary factor' of yeast. Accidental loss or destruction would obviously
deprive the cell of the capacity of making the respiratory enzymes and of
perpetuating the factor.

But it must be clearly realized that the loss of the object X per se is not
the essential thing; what is all important is the disappearance of the
biochemical or h'lo-ph.y&icdX function that it normally fulfilled. This special
feature of the autocatalytic particle has two fundamental consequences.
The first is that any inhibitor of the activity or function of X is a potential
mutagen. If the biochemical activity or the biophysical futiction of the
object X is interrupted, the synthesis of more X is blocked, together with
that of the respiratory enzymes. In resting cells, the only observable fact
will be the inhibition of the synthesis of the respiratory chain. But if the
cells do multiply, while X cannot, X will be lost by dilution for part of
the progeny, and mutants will appear. This is exactly what happens in the
presence of euflavine in yeast. Euflavine first inhibits the synthesis of the
respiratory enzymes, and respiratory mutants appear a few hours later
(Slonimski, 1953; Marcovich, 1958). Among the acridines, only those
which specifically interfere with the synthesis of cytochrome oxidase are
mutagenic (Slonimski, 1953). A fairly good correlation between the condi-
tions which depress chlorophyll synthesis and those which favour mutation
in Eiiglena was also observed by De Deken-Grenson (1960) who suggested
that the hereditary transformation might be a consequence of the primary
phenotypic inhibition of chlorophyll synthesis.

A second consequence of the properties of an autocatalytic object like X
is that its loss is not irreversible-by-nature as would be that of a structural
gene. It will be reversible or irreversible depending on whether or not the
function normally accomplished by X can be temporarily fulfilled by an
action of the experimentator, or due to a change in conditions. This will
indeed restore the synthesis of X which will then take over its normal

REGULATION 149

function. A beautiful illustration of such a phenomenon is the loss and
recovery of galactosidase synthesis in E. coli. The factor X here is obviously
the permease. It is an autocatalytic object, since its function is to pump
galactosides into the bacterium, and since the achievement of a certain
concentration of galactoside within the cell is required for the induction of
permease formation and /S-galactosidase synthesis. Loss of the permease
occurs during growth under conditions which abolish the function of the
permease, e.g. when there is no galactoside available to be pumped into the
cell. Loss of the permease is reversible because when a sufficient concentra-
tion of galactoside is applied from the outside, free diffusion can com-
pensate momentarily for the function of the permease, and thus induce its
formation, together with j3-galactosidase synthesis. The adapted state can
then be transmitted clonally as long as the conditions allow the permease to
function. Long-term adaptation of yeast to galactose, for which the model
of autocatalytic particle was first proposed (Campbell, 1960) is another
example of reversible loss of the particle.

In other cases, of course, the loss will be irreversible for all practical
purpose simply because it will be impossible to compensate for the bio-
chemical or biophysical function of the autocatalytic factor long enough to
allow the synthesis of it to be restored. For instance, if the function of the
autocatalytic factor is to be an essential piece of some subcellular structure
it might be altogether impossible to restore this function from the outside.

The concept of an autocatalytic object which does not carry any genetic
information but simply accomplishes a certain physiological function
necessary for its own formation and eventually for other anabolic processes
as well, thus leads to a general theory which accommodates cytoplasmic
heredity and other phenomena which mimic heredity. Besides, it leads to
predict puzzling and somewhat conflicting features of cytoplasmic
heredity: the loss of the cytoplasmic factor as if it occurred by dilution of
particles, the mutagenic effect of inhibitors of certain enzyme synthesis.
The unstable state in which yeast or Eiiglenae can exist after a threshold
treatment by the mutagen might be a state in which the activity of X is just
sufficient to insure its own synthesis but not that of the enzymes; slight
changes of the medium can tilt the equilibrium either towards recovery or
towards irreversible loss.

Any theory can of course be stretched to accommodate any experimental
facts and it would be without interest for the present discussion to elaborate
further the concept of autocatalytic element. This model is presented in an
attempt at making a unifying synthesis. The only merit that it claims is of
placing old facts in a new perspective and, in so doing, of suggesting new
experiments.

Whatever the ultimate nature of the non-mendelian factors in enzyme
synthesis may be, it is becoming more and more probable that these factors

150 THE BIOSYNTHESIS OF PROTEINS

are 'epigenetic' or 'epinucleic', to take the same words as Nanney (1958)
and Lederberg (1958), i.e. 'metabolic' (De Deken-Grenson, 1960), rather
than of the nature of wandering genes or virus-hke elements. That is the
justification for considering non-mendehan heredity in the chapter of the
present book which deals with the regulation of protein synthesis.

C. CHANGES IN PROTEIN SYNTHESIS DURING
DIFFERENTIATION

The unique cell out of which a metazoan grows, the fertihzed egg, con-
tains all the information for making all the specific proteins of the complete
organism. The egg does not grow and multiply like a bacterium which
gives rise to an offspring of identical bacteria. The egg first divides a few
times without growing. This 'cleavage' results in a group of cells which are
not identical to each other and which are disposed in space according to a
certain pattern. Some of these cells, or blastomeres, contain more mito-
chondria, others contain more storage material ; some are larger, some are
smaller; droplets of lipids may be found mostly in one region of the
segmented egg, grains of pigment in others. Enzymes also are differently
distributed among the blastomeres : certain regions of the blastula reduce
oxidized dyes much more actively than others, the oxidation enzymes,
sulphhydril rich proteins, ribonucleoproteins, phosphatases, are distributed
in the cells according to certain gradients which in some cases coincide
with the morphogenetic 'fields' defined by the embryologists to describe
and explain development. During the segmentation period, little protein
synthesis, if any, takes place. But after the egg material has been frag-
mented and distributed into so many cells, with their special position in
space, growth begins and with it — as a part of it — protein synthesis. Cer-
tain regions of the segmented egg will grow faster than others, sheets of
cells will move under or above other groups of cells ; interactions between
these will cause visible changes in the morphology and arrangements of the
individual cells, and thus the embryo will begin to take shape. Specialized
structures, tissues and organs appear; differences in shape and properties
of the various regions of the embryo become clearer and clearer as develop-
ment progresses.

Thus one fertilized egg gives rise to a very heterogeneous offspring of cells,
the properties of which diverge more and more during development, until
they are fully 'differentiated' in the adult organism. It is barely necessary
to emphasize the differences in the assortment of enzymes and proteins
between liver, brain, muscle and pancreas. Clearly, these cells make a com-
pletely different set of proteins. When and how do such differences in the
specificity of protein synthesis arise during development?

REGULATION 151

Consider haemoglobin synthesis in the embryo. The cells of the gastrula
do not make haemoglobin ; it is certain that the cells from which the blood
islets originate contained the gene which specifies the sequence of amino
acids in haemoglobin; synthesis of this protein, however, did not take
place to any appreciable extent, it was prevented in some way. Controlling
or regulatory processes therefore certainly play a part in the differentiation
of protein synthesis.

Interesting studies were made on the appearance of a few highly
specialized or characteristic proteins during differentiation. Thus lens
proteins are not detected by a serological precipitation method before the
appearance of the lens itself (Flickinger et al., 1955; ten Cate and van
Doorenmaalen, 1950). Even in the head ectoderm wherefrom the lens is
formed, or in the eye vesicles, which induce the transformation of head
ectoderm into lens, no lens antigen is found by the precipitation method
(Woerdeman, 1955). With the Coons method for localizing antigen
on histological sections which uses a fluorescent antiserum as a specific
stain, a characterized localization was found only when the lens was formed ;
before this, some lens antiserum was bound everywhere. It may be there-
fore that typical lens proteins were made before, but in small amounts, by
all the cells of the embryo, and that their formation was later restricted to a
few territories.

Ebert (1954, 1958, 1959) did a similar study on the appearance of heart
myosin in the chick embryo. At the end of gastrulation, a protein which
reacts with anticardiac myosin serum arises everywhere in the embryo;
later this protein is restricted to the heart-forming area. Actin, on the con-
trary, is not detected except in this area, and only when myosin localization
is already restricted to it.

The appearance of skeletal muscle myosin and of collagen have also been
studied by similar and other methods ; and a wealth of data on the changes
in many enzymic activities during development can be found in the
literature of chemical embryology (see Brachet, 1944, 1960).

In a way, all these data can be taken as more elements to be added to the
morphological observations ; they are an extension of morphology down to
the chemical level. As a rule, specific proteins appear indeed at the same
time as the corresponding organ. It may be argued that the question as to
which comes first, specific proteins or morphological differentiation, is not
very consistent: in so intricate a process as development, where each step
may influence the steps to come in very indirect ways, it is certainly not
easy to distinguish causes from effects. But proteins are the most immediate
expression of the genetic characters, and the enzymes do control all the
biochemical operations by which cell material is made. Proteins therefore
must occupy a key position in differentiation. As the mechanism of protein
synthesis and its control are becoming better and better understood, the

L

152

THE BIOSYNTHESIS OF PROTEINS

Study of changes of protein synthesis during development is certainly a
promising approach to some of the basic problems of embryology.

A fundamental question is whether two differentiated cells of the same
organism produce two different sets of proteins because they have a differ-
ent complement of mendelian genes. This is, in newer words and in a more
restrictive sense, the problem of the equipotentiality of the nuclei which
retained the attention of Driesch (1894) and of Morgan (1934).

The amount of DNA in a diploid somatic cell is just double that of the
spermatozoon. The number of chromosomes and their general shape

Z7A Myosin

Actin + Myosin

Fig. 36. Distribution of actin and myosin in the early chick embryo at
successive stages of development, as shown by Coon's method (Ebert,

1954).

usually do not change during development. In insects, certain tissues con-
tain enormously enlarged chromosomes, and a correlation has been estab-
lished between the visible bands of the chromosomes and the genetic map
computed from recombination frequencies. The same pattern of bands is
found in the chromosomes of the salivary gland and of the midgut of
Drosophila (Berger, 1940) or in the rectum, the Malpighian tubes, the
midgut and the salivary glands of Sciara (Beermann, 1952); there is no
evidence of any change in structure of these chromosomes during differ-
entiation. These are indications that not many genes are lost on the way;
but subtle changes in the state of the genes might escape these crude

REGULATION 153

observations. The actual presence of a gene can be shown only by its
function. But when a character fails to show up, unless the character
reappears in the offspring of the cell, it is impossible to tell whether the
gene had disappeared or whether its expression was blocked. It is not known
whether irreversibly differentiated cells have lost certain genetic determin-
ants or whether the expression of the corresponding character was made
irreversibly impossible. What we need to answer such a question is a way
of testing the activity of the genetic material in a non-differentiated cell or
in a cell which underwent another type of differentiation.

It has not been feasible so far to exchange the DNA or the chromosomes
of two differentiated cells to see what happens. But nuclear transfer comes
closest to it. Spemann (1928) and Seidel (1932) had found that during
early cleavage, the nuclei are equipotential : the nucleus of a dorsal blasto-
mere can be replaced by a nucleus from the ventral part without affecting
embryonic development. Up to a time when the embryo contains about one
hundred cells, the nuclei remain equipotential, they are not irreversibly
differentiated. But it should be realized that growth and synthesis (except
for nuclear material) had not started yet at that time. Briggs and King
(1953, 1959) succeeded in replacing the nucleus of frog eggs by nuclei taken
from frog embryos at more advanced stages of development. When the
nucleus was taken from a blastula or even from an early gastrula, a stage
at which protein synthesis just begins, it was able to replace the nucleus of
the egg and ensure a complete development of the embryo into a tadpole.
Clearly, the nuclei at those stages had retained all their potentialities, they
possessed the same genetic information as the nucleus of a fertilized egg. If
the nucleus was taken at a slightly later stage, segmentation took place
normally but development was blocked during gastrulation, as if this
nucleus had lost some of its capacities. More striking still are the serial
transplantations in which the nuclei were taken at an early stage from
embryos which had themselves originated from nuclear transfer, and whose
development had stopped at an early stage. These never permit full develop-
ment (King and Briggs, 1956, 1957).

Similar experiments were made on another amphibian, Xenopus, by
Fischberg et al. (1959) who succeeded in obtaining complete development
after transferring nuclei taken from somites, i.e. from a region of the embryo
which is already clearly differentiated and which will soon form skeletal
muscle ; some of the nuclei were still totipotential 9 hr before the somites
became contractile. It would seem therefore that these cells which were
well advanced on the way of differentiation still possessed all the genes and
that their nuclei were still able to accomplish all the functions of an egg
nucleus, when returned to the cytoplasm of an egg. At later stages, however,
they became unable to ensure development.

These experiments indicate that some differentiation can take place

154 THE BIOSYNTHESIS OF PROTEINS

without irreversible change occurring in the nucleus. The significance of the
loss of capacities of the transplanted nuclei taken at later stages is of course
much more uncertain, for it is very difficult to establish whether the change
is due to differentiation or to damages caused to the nuclei during trans-
plantation; the operation becomes indeed more and more difficult as
development progresses. It is not certain at present whether the nuclei
undergo a differentiation process.

In giant chromosomes, metabolic processes have been observed which
suggest that some differentiation might take place within the chromosomes
themselves. Although the pattern of the bands of the giant chromosomes
is the same in the different organs in which they are found, very striking
metabolic processes occur at specific regions of the chromosomes. Certain
typical euchromatic bands, i.e. bands which are supposed to contain men-
delian genes, are changed into large bulbs (Breuer and Pavan, 1955). During
this process, a very active incorporation of thymidine takes place, indicating
localized DNA synthesis (Ficq and Pavan, 1957; Ficq et al, 1958); RNA
is also produced at the same locations. It is remarkable that these metabolic
processes occur at the level of well-defined bands, and at certain stages of
larval development only. This might possibly reflect a process of nuclear
differentiation.

The sequence of events which result in cytoplasmic and eventually
nuclear differentiation is unknown. There is clear evidence that the cyto-
plasm influences the state of the nucleus, and that both must in some way
be adjusted to one another for proper co-operation. Hybrids between two
sea urchins Paracentrotiis and Arbacia, or between two frogs, Rana pipiens
and Rana sylvatica, or Rana esculenta with R. temporaria are blocked more
or less early during development, usually at gastrulation. In these lethal
hybrids, the nuclei of many cells are abnormal, they are overloaded with
RNA(Brachet, 1944, 1954; Zeller, 1956).

If the nucleus of the fertilized egg of R. pipiens is transferred into an
enucleate unfertilized egg of R. sylvatica, development starts, but it is
blocked at the blastula stage. If the nucleus of one of the blastomeres is now
transferred back into an enucleate R. pipiens egg, development is blocked
at the beginning of gastrulation. This suggests that the nucleus had been
irreversibly changed due to its reduplication in a foreign cytoplasm
(Moore, 1958, 1959).

It is therefore quite reasonable to consider that changes in the properties
of the nuclei might be the consequence of differences in cytoplasmic pro-
perties. But changes in nuclear activity certainly influence cytoplasm. A
cumulative effect will thus progressively be observed, and it is easy to
visualize that once nucleus and cytoplasm are engaged in such a cascade
of changes which determine one another, the resulting evolution of the cell
line will rapidly become irreversible.

Si:.>^

Fig. 37. Radioautograph of the distal segment of the salivary C chromo-
some of Rhyncosciara avgelae at three different stages of larval develop-
ment. The larvae were fixed 24 hr after receiving an injection of ^H-
Thymidine. Heavy blackening shows localized strong incorporation of
thymidine (Ficq et al., 1959) (courtesy Dr A. Ficq).

REGULATION 155

By what kind of nuclear and cytoplasmic changes is the specificity of
protein synthesis changed during development? No answer can be pro-
vided at present to this question. If one adopts the common — but un-
checked — assumption that each cell of a metazoan contains the complete
collection of mendelian genes, then the changes must concern the control
of protein synthesis. The difficult point is that these changes, although they
may not be genie, are transmitted clonally. One is left with guesses in-
pired by observations on simpler phenomena which share certain features
with differentiation, or on special cases of controlling mechanisms.

The interplay of inducers and repressors of protein synthesis, for in-
stance, indicate a mechanismi by which the production of specific groups
of enzymes could be switched on or off. Not very complicated modalities
of such systems can indeed ensure the clonal maintenance of certain
metabolic states, and of specific protein synthesis (see p. 138). The known
cases of cytoplasmic mutations, in yeast, Eiiglena or Paramaecium, provide
a model for clonally transmitted restrictions of the capacity to make certain
specific proteins (see p. 141). For these reasons, the theories proposed for
explaining cytoplasmic heredity — whether they involved plasmagenes or
steady states — were often suggested as models of differentiation. The
wandering controlling elements found by McClintock (1956) to inhibit
certain genes in maize, or the episomic elements of bacteria (Jacob et al.,
1960) also suggest mechanisms by which the mathematical rigidity of
mendelian control can be tempered.

Processes of differentiation are often presented as being irreversible by
essence; or rather irreversibility is often taken as a criterion of differentia-
tion, i.e. as part of its definition. Although differentiation 'properly speak-
ing' may be irreversible, this irreversibility should not blind us, for it
may be merely a usual consequence of the type of process involved in
differentiation. Changes of cell constitution or cell structures which are not
irreversible, might be brought about by the same fundamental processes as
differentiation during development, but remain reversible because they are
less extent and less intricate. It might be very informative to know more
about the mechanisms of changes like sporulation, spore germination or
even the changes in enzyme constitution during the growth phase of
micro-organisms, changes in the mating type, i.e. in the constitution of the
cilia in Paramaecium; differentiation during growth of the slime mould
(Sussman, 1958; Wright and Anderson, 1958) might provide a transition
between micro-organisms and metazoans. It is true that in these simpler
processes differentiation is controlled or triggered by changes of the
medium, whereas in higher organisms interactions between cells (in-
ductions) play an essential part. But a few known facts indicate that the
outer agents of differentiation are not always very involved; simple
chemicals can have rather specific actions in some aspects at least of

156 THE BIOSYNTHESIS OF PROTEINS

embryonic development. Differentiation of cells of the neural crest into
pigment cells normally occur as a result of interactions with the mesoderm
which lies below. The appearance of the pigment cells can be prevented by
analogues of phenylalanine and, in explants of ventral mesoderm, different-
iation is obtained when phenylalanine is added to the medium. It seems
that the production of phenylalanine by the mesoderm and its utilization
by the ectoderm is an essential factor in the differentiation of these cells
(Wilde, 1955, 1956). Specific nutritional requirements have also been
observed in cultures of organs (Wolff, 1957). Differences in requirements
are simply a sign of chemical differentiation, but they also point to the
possibility that certain tissues might depend on their neighbours for simple
compounds. These elementary interactions may be of fundamental impor-
tance. Studies on the formation of specific proteins in such systems might
be illuminating.

Pioneer work on the transfer of specificity by cell extracts and especially
by microsomes must be mentioned here. When liver microsomes are
spread on the chorioallantoic membrane of a developing chick embryo, a
thickening of the membrane is observed and the amount of glucose-6-
phosphatase, an enzyme characteristic of liver microsomes, increases
considerably (Le Clerc, 1954) as if the microsomes had carried over the
capacity of making this enzyme. More recently, Ebert (1959) implanted
on the chorioallantoic membrane a microsomal fraction from adult cardiac
muscle together with Rous sarcoma virus. In the tumour masses which
developed, muscle tissue was observed to form in 27 per cent of the
cases. These observations open new perspectives and a wide field of new
possibilities to the experimental study of differentiation.

D. THE SYNTHESIS OF ANTIBODIES

If bacteria, foreign cells, foreign proteins or polysaccharides are injected
to an adult rabbit, new y-globulins appear in the serum of the animal a
few days after the injection. These special y-globulins are recognizable by
their ability to form complexes with the injected substance or to bind
specifically on the surface of the injected foreign cells. These new y-
globulins are the antibodies, the injected cells or substances the antigens.
The antibodies are highly specific of the antigen which caused their appear-
ance in the blood.

The antibodies do not arise by transformation of pre-existing y-globulins:
when i^C labelled normal y-globulins are injected into the blood of a rabbit
immunized against Pneiimococciis, the antibodies do not become labelled
(Gros et al., 1952; Green and Anker, 1954). On the other hand, when
labelled amino acids are injected to an animal which is producing anti-

REGULATION 157

bodies, these become strongly labelled (Green and Anker, 1954; Askonas
et ah, 1956; Taliaferro, 1957). Production of antibodies therefore rests on
the synthesis of new specific proteins all the way from amino acids.

The use of a protein antigen coupled with a fluorescent dye makes it
possible to locate on tissue sections the cells which contain the corre-
sponding antibodies: the fluorescent antigen is bound or precipitated in
situ by the antibodies (Coons and Kaplan, 1950; Coons, 1958). It was shown
by this method, completed by tracer experiments (Askonas and White,
1956) that antibodies are produced by individual cells or by groups of cells
of the reticuloendothelial system, the plasma cells, which are found
especially in the lymph nodes and in the spleen.

Antibody production thus reflects the de novo synthesis of specific
y-globulins by specialized cells under the stimulus of an antigen. Super-
ficially at least, this phenomenon is comparable to the induced synthesis of
enzymes. The antigen, like the substrate of an enzyme, induces the
synthesis of a protein which combines specifically with it.

Obviously antibody formation raises the same problems as induced
enzyme synthesis. The most fundamental question, in the present perspec-
tive of protein synthesis, is whether the antigen brings to the antibody
producing cell part of the structural information relative to the antibody,
or whether the antigen simply triggers a ready-made machine to produce a
protein the structure of which is completely specified by a gene.

The first theory of antibody production (Ehrlich, 1900) assumed that the
antigen, through chance affinity, binds certain receptors or special out-
growths of a cell surface ; the cell reacts by replacing the bound receptors
by two or more new ones, part of which are cast oflF in the blood as anti-
bodies. According to this view, the antibody structures pre-exist, the
antigen simply stimulates their production and their release into circula-
tion; it selects an antibody out of many, but it does not bring any structural
information to the antibody producing system.

Landsteiner (1933) coupled proteins with various azaaryl groups under
conditions which do not cause denaturation. He showed that such sub-
stituted proteins are good antigens, and that the antibodies that they
evoke in the rabbit are quite specific of the artificial chemical groups
bound to the protein. Using substituents in which tartaric acid residues
had been introduced, he showed that the antibodies can even distinguish
between L and D isomers. These fundamental studies established that
antibodies are specific of individual chemical groups in the antigen mole-
cule, rather than of the molecule as a whole. It was clear also that anti-
bodies can be evoked by many kinds of chemical structures, including
some which are not likely to ever come in contact with a rabbit, were it not
for the deliberate intervention of an organic chemist. It seemed incredible
that the animal could possess a ready-made specific antibody to cope with

158 THE BIOSYNTHESIS OF PROTEINS

each synthetic antigen. It was deemed much more probable that the
antigen itself participates in the molding of the corresponding specific
antibody.

Thus Breinl and Haurowitz (1930) advanced the view that the antigen
interferes with the process of globulin synthesis so that globulin molecules
complementarily adjusted to the shape of the specific groups of the antigen
are formed. Proteins can difter by the amino acid sequence in the chains
or (and) by the way these chains are folded. Pauling (1940) elaborated a
model of antibody synthesis in which it was assumed that the polypeptide
chains of the globulins or certain regions of them were likely to assume
many different types of folding. In the absence of an antigen molecule, the
newly synthesized polypeptide chain might fold up in any one of a number
of ways, representing the maximum stability under the conditions then
existent in the cell. If, however, an antigen molecule were present, the
polypeptide chain would fold up into a configuration complementary in
structure to the antigen molecule. The specific shape of the antibody mole-
cule would thus be imprinted upon the plastic polypeptide chain of the
globulin molecule by the antigen itself.

It must be noted here that the structural information brought by the
antigen adds to the unchanged genetic information, and that it is of a
completely different nature. The additional information controls the estab-
lishment of the secondary and tertiary structures of the polypeptide only.
It is a kind of structural information which is disregarded in the present
concepts of protein synthesis in which all the attention is focused on the
primary structure of the protein (see p. 122). The antigen-template would
act after the nucleic acid template has assembled the amino acids into a
polypeptide in the genetically controlled sequence.

The direct antigen template hypothesis has implications which could,
in principle, be checked experimentally. It assumes that the antigen must
be present for antibody production to continue; it predicts that all anti-
bodies, or at least many antibodies, should have the same amino acid
sequence. This prediction is amenable to experimental check. Five different
antibodies produced by the rabbit against ovalbumin and against four
strains of Pneumococcus respectively, all terminate by the same amino acid
sequence Ala-Leu-Val-Asp-Glu and four more antibodies which have not
been studied as thoroughly also have a terminal Ala (Porter, 1950a;
McFadden and Smith, 1955). Within experimental limitations, the amino
acid composition is the same for all (Smith et al., 1955). These facts lend
support to the above hypothesis. It should not be overlooked, however, that
the globulins are large molecules containing some 1500 amino acid
residues and that the replacement of a few amino acids by others would
not be detected easily. But the techniques for determining the amino acid
sequence have advanced considerably in the last few years, and it is to be

REGULATION 159

hoped that the interesting works quoted above will be extended and that
the primary structure of a large part of the antibody molecule will be un-
ravelled. This does not appear beyond the reach of experimentation, for
the large antibody molecule can be split into fragments some of which
contain the specific groups which react with the antigen (Porter 1950b,
1958). Knowledge of the primary structure of these fragments would be of
fundamental importance. It would provide not only a test for the direct
template hypothesis but also an essential foundation of any coherent theory
of immunological response.

According to the direct template hypothesis, antibody can be produced
only as long as the antigen is present. It is a fact that immunity can last
for several years after the last injection of antigen ; unfortunately it is very
difficult to find out experimentally whether the antigen does persist for
years in antibody producing cells and it is impossible as well to prove that
it does not persist, for in a template process a few molecules per active cell
might be sufficient to ensure continued antibody production.

A theory, which derives from the preceding one but avoids the require-
ment for the persistence of the antigen, was proposed by Burnet and
Fenner (1949). Here the antigen is assumed to modify the agents of globu-
lin synthesis permanently and in such a way that they will produce a new
globulin, the antibody, even when the antigen has been eliminated. This
is the indirect antigen-template theory. The permanent modification is
assumed to concern a piece of the protein making machine which is respon-
sible for the specificity of the synthesis. For instance, a modification of the
DNA of the antibody producing cell has been suggested as an obvious
possibility (Schweet and Owen, 1957).

Recently, the old natural selection theory of Ehrlich was revived in a
new form by Jerne (1955). Ehrlich's theory has been abandoned because
many workers found it incredible that antibodies specific for strange artifi-
cial organic groups could pre-exist in the organism. But this opinion was
based on the more or less implicit assumption that each antigen evokes an
antibody which is different from any other globulin and which is perfectly
adapted to the antigen. Actually, the response is not as perfect and the
specificity of the reaction not so absolute. Many proteins are bad antigens,
not all chemical groups have antigenic properties, and all the animals do
not react equally well to an individual antigen. In response to an antigen,
an animal often produces a heterogeneous group of antibodies (Talmadge,
1957; Lapresle, 1959). Cross reactions occasionally occur between 'un-
related' antigens (Dixon and Maurer, 1955). The blood of a non-immunized
animal often contains proteins which manifest a great affinity for certain
foreign substances (Jerne, 1956). It is conceivable therefore that an animal
produces a broad but finite assortment of y-globulins able to form com-
plexes with many various kinds of chemical groups. When introduced into

160 THE BIOSYNTHESIS OF PROTEINS

an organism, a foreign substance may combine by chance affinity with
those of the y-globuhns which happen to have such a configuration that
they form a rather stable complex with the injected substance. Let us
assume that the organism reacts by producing more of this diverted
y-globulin, the production of specific antibodies will be accounted for.
This is essentially Ehrlich's theory reworded in molecular instead of
morphological terms. The next question is how the organism is stimulated
to produce specifically the y-globulins which happen to combine with the
exogeneous substance. Jerne (1955) imagines that the y-globulin-antigen
complexes are picked up by competent cells and that they stimulate these
cells to copying the particular y-globulin contained in the complex. A very
interesting feature of this theory is that it predicts an increasing fitness of
the antibody produced in the course of immunization, for the globulins
which have the greatest affinity for the antigen will be produced in greater
amount and will eventually overwhelm all the others. The assumption that
the swallowed y-globulin brings into antibody producing cells the structural
information for the synthesis of identical y-globulin molecules is difficult to
integrate into the present views on protein synthesis : information is known
to flow from the nucleic acids to the proteins, but there is no evidence that
proteins can ever transmit the structural information for their own repro-
duction to a protein making system. Whatever the value of the hypothetical
mechanism proposed by Jerne, the revival of a selection theory of antibody
synthesis was very stimulating. Newer hypotheses were soon developed in
which the selection was assumed to occur between cell lines rather than
between molecules (Burnet, 1959; Lederberg, 1959). These theories also
account for several other experimental data which should now be sum-
marized briefly.

When an antigen is injected for the first time into an animal, antibody
appears in its blood after a few days ; the titre of antibody then decreases
gradually and it may eventually become very low, or undetectable. This is
the primary response. If a few weeks or a few months later the animal
receives a new injection of the same antigen, it soon produces large amounts
of antibody and keeps producing it for a long period. This secondary
response indicates that although antibody production ceased, the organism
had kept memories of its first contact with the antigen, since it was made
more responsive to a second contact. This memory ert'ect should be
accounted for. Another puzzling feature of antibody synthesis is that it
is caused by foreign substances only, as if the organism was able to distin-
guish self from foreign. Moreover, embryos or newly-born animals do not
respond to an injection of antigen by producing antibodies; nevertheless
the injected substance can change the antibody producing system in such
a way that the animal in its adult life will not produce antibodies against
the foreign antigen to which it has been exposed during its fetal or early

REGULATION 161

life; it has acquired an 'enduring immune tolerance' towards this foreign
substance (Billingham et ah, 1953). An unresponsive period in which an
aduh behaves in this respect as a newly-born animal can be observed after
irradiation with a high dose of X-rays (Dixon and Maurer, 1955). Injection
of a very large amount of antigen can have similar effects (for a review see
Chase, 1959). The recognition of self and non-self, and the adoption as
'self of an antigen injected during an unresponsive period raise the most
challenging problems of the control of antibody synthesis. Cytological
observation with fluorescent antigens shows that after the first antigen
injection, thousands of cells in the lymph nodes may pick up the antigen;
but antibody appears in a much smaller number of these cells, and after a
few days antibody is demonstrable only in a few cells. In a secondary
response, many hundreds of cells containing traces of antibody can be seen ;
they are scattered throughout the areas where the antigen was deposited
after the first injection. These cells seem to spring up independently and to
multiply, so that their descendants often coalesce to form clumps of cells
(Coons, 1958).

Immunological response is thus associated with processes of cell
multiplication and difterentiation.

If two antigens A and B are injected into the same animal, and the antibody
producing cells are located by the Coons' method using a diff"erent
fluorescent marker for each antigen, it is observed that antibodies reacting
with each individual antigen are often produced by difl'erent plasma cells,
as if certain cells had specialized in the production of antibodies against
antigen A, and others against antigen B (White, 1958). The same fact was
observed by a completely diff"erent method. Tissues from an immunized
animal can continue to produce antibodies in vitro (Fagraeus, 1948). Cells
of lymph nodes of a rabbit immunized against two different bacteria were
isolated in microdrops, and tested for the production of antibodies against
each bacterium species. About 10 per cent of the isolated cells produced
one or the other antibody, but none were found to produce both (Nossal
and Lederberg, 1958). However, in similar experiments with rabbits im-
munized against phages T2 and T5, 8 per cent of the isolated cells produced
antibody against one or the other phage exclusively, but about 2 per cent
produced both types of antibodies (Attardi et al., 1959). These results
indicate that when an animal is injected with two different antigens, in-
dividual cells often form one species of antibody only, but formation of
one antibody does not prevent the cell from making another antibody at
the same time.

Burnet (1959) has proposed a new theory which takes in consideration
the enduring immune tolerance, the fact that antibodies are not produced
by all individual cells of the competent organs, and that antibody producing
cells do multiply. Burnet assumes that the mesenchymal tissues are made

162 THE BIOSYNTHESIS OF PROTEINS

of a heterogeneous population of cells with various protein synthesis
abilities; this heterogeneity arises in the course of development and
differentiation. There are a certain number of different clones (i.e. classes
of cells all derived from one single cell by mitotic divisions) which are
each competent towards a certain antigenic determinant. If the relevant
determinant comes in contact with such a cell, it stimulates this to pro-
liferate into plasma cells which produce the corresponding antibodies. It is
further assumed that if an antigen reacts with the cell at a certain early
stage of differentiation, that cell is functionally eliminated. This would
account for the elimination of cells able to produce antibodies against
antigenic substances present in the host or introduced into it during early
life. One should eventually obtain a residual population of clones able to
react only with foreign antigens.

The fact that different cells often specialize in the production of one or
the other antibody (Nossal and Lederberg, 1958) is certainly in line with a
selection theory in which the selective process operates at the cellular level.
The observation that certain cells can produce two antibodies simultane-
ously (Attardi et ah, 1959) conflicts with Burnet's theory which in its most
extreme form indeed assumes that each antibody is produced by a different
clone of cells, but it is not incompatible with the basic idea of the theory,
which can easily be readjusted accordingly.

Lederberg's views (1959) are very similar to Burnet's; it is assumed that
the stem line of antibody forming cells is genotypically heterogeneous
owing to relatively frequent mutation in the y-globulin gene. An antigen
functions by recognizing those cells already competent to produce a globu-
lin which reacts with the antigen. These cells are selected in the sense that
they are stimulated to proliferate; on the other hand, their particular
ability to produce antibody is also stimulated, or allowed to express itself.
The event which is assumed to cause heterogeneity among the stem cells
is mutation of a highly mutable globulin gene. Obviously, a 'cytoplasmic'
mutation, or any kind of change which can be clonally transmitted would
serve the same purpose (Lederberg, 1959; Schultz, 1959).

It has been mentioned earlier (p. 139) that in bacteria an adapted state,
once it has been established, can be maintained clonally through many
generations under conditions which could not cause adaptation (Cohn,
1957; Novick and Weiner, 1957); this is due to the special properties of a
permease system. A model of antibody formation has been derived from
this phenomenon by Monod (1959). In the primary response, the antigen
is assumed to induce in certain cells both the antibody and a specific
permease or a specific cellular antibody which captures the antigen. The
cells which have acquired this specific antigen-capture system will exhibit
an increased sensitivity to a further antigen injection since they are now
able to concentrate it specifically. If they multiply, this increased sensitivity

REGULATION 163

will be transmitted to the clone. Heterogeneity among the population will
be achieved through the channel of this inducible antigen-capture system,
without change in the genetic material.

Szilard (1960) also developed a theory which rests entirely on the
presently accepted views about induction and repression of enzyme
synthesis and on feedback mechanisms. It gives an interpretation of all the
major facts of antibody production, but this is at the cost of several
assumptions.

All these theories of antibody formation are extremely attractive; they
are unfortunately highly speculative, because facts are scarcer than ideas in
the present state of biochemistry of antibody synthesis. Progress in cell
cultivation methods and the possibility of studying antibody production
in "vitro might pave the way for a new type of experimental approach. One
may hope that these brilliant working hypotheses will help to design clear
experiments and will thus eventually lead to a better understanding of this
intriguing process, which is not of academic interest only. A broad field
lays open for experimentation.

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