THE MOLECULAR BASIS

OF

EVOLUTION


        Th e
MOLECULAR BASIS

NEW YORK - JOHN WILEY 6 SONS, INC.

London o Chapman 6 Hall, Limited

OF EVOLUTION

CHRISTIAN B. $+NFINSEN

  National Heart Institute
National institutes of Health
   Bethesda, Maryland


TO MY MOTHER

AND
TO THE MEMORY OF

MY FATHER

Copyright @ 1959 by John Wiley & Sons, Inc.

All rights reserved. This book or any part
thereof must not be reproduced in any form
without the written permission of the publisher.

Library of Congrorr Catalog Card Number: 59-11794

Printed in the United Statbs of America


PREFACE

T

     1 he writing of this book has been stimulated
by the excitement and promise of contemporary protein chem-
istry and genetics and by the possibilities of integration of these
fields toward a greater understanding of the fundamental forces
underlying the evolutionary process. It has become, inevitably,
a highly personal volume expressing the experimental and phil-
osophical outlook which has resulted from a process of self-education
in an unfamiliar area of science.  As with many biochemists, pure
biology, including genetics, has not been a major exposure in my
education, and the process of learning something about these subjects
has been both a revelation and a struggle. Various kind, and
frequently amused, friends, versed in the complexities of modem
genetics, have sifted through these pages, and I hope that most of
the misinterpretations and frank mistakes have been eliminated.

It has been highly interesting of late to observe how many scientists,
working either in protein chemistry or in genetics, or for that matter
in relatively unrelated fields, have arrived at long-range research plans
that are similar to. my own, down to almost the last detail of experi-
mental planning. This book, therefore, will undoubtedly represent the
point of view of numerous other biologically oriented individuals.  On
the other hand, some of the ideas to be discussed are so new and
controversial that for every well-informed reader of this book who,

vii


in general, approves them there may be another who considers them
nonsense.

The recent advances in the development of techniques for the study
of protein structure have made it possible to elucidate the complete
amino acid sequences of a number of rather large polypeptides pos-
sessing hormonal activity, and nearly complete sequences should soon
be available for several with enzymatic activity as well, Concomitantly,
there have been developed methods for the analysis of the finer,
more subtle, structural aspects of proteins, concerned with folding,
intramolecular bonds of various types, and intermolecular interactions.
These advances now begin to enable us to discern the vague outlines
of macromolecules, in the three-dimensional sense, and to ascribe
their physical behavior and biological activity to specific covalent and
noncovalent structural features.

We like to believe that Nature has been extremely wise and effi-
cient in the design of the chemical compounds, however large and
complicated, which make up the structure and machinery of living
things. Thus, although chemical differences are found among the
representatives of a given protein as isolated from a variety of species,
we tend to suppose that such variations, rather than being fortuitous
and unimportant irrelevancies, are part of a complicated and highly
integrated set of variations in all the functionally and structurally
important elements of the cell, the summation of which accounts for
the unique morphology and phenotypic character of the individual
organism.

In the last few years, a number of studies have shown that various
biologically active molecules may be subjected to considerable degra-
dation without loss of functional competency. It becomes necessary,
therefore, to consider that the "macromoleculariness" of proteins and
other large molecules may, in many cases, be concemed not only with
a specific biological property but with other, more subtle, phenomena
of cellular activity and engineering not yet apparent, such as adsorp-
tion to surfaces or substrates. We must, perhaps, expect a multiplicity
of variables in the "natural selection" of variants in molecules, all of
which may, together, determine the biological suitability of a particu-
lar molecular species.

An understanding of the underlying principles governing the
species specific variations in molecular structure and of the effect of
such variations on species characteristics must involve a clarification
of the process of translating information present in the genetic ma-
terial of the cell into the chemical language of enzymes, regulators,
and the like. Such considerations are only now becoming possible as

Viii                                                 PREFACE

the result of the dramatic strides taken in the last few years in genetic
theory and methodology, and a portion of the discussion in the fol-
lowing pages will have to do with the experimental background of
the analysis of genetic fine structure and with the possible significance
of such analysis to the question of protein biosynthesis.

It is abundantly clear that the metabolic organization of all living
cells, whether plant or animal, shows a remarkable uniformity.  Even
a cursory examination of the literature of comparative biochemistry
and physiology indicates that such biochemical functions as glycolysis,
proteolysis, and fatty acid degradation, as well as more integrated
processes such as electromotor activity and active transport through
membranes, are ubiquitous in nature.  Discounting the likelihood of
completely parallel evolution in the plant and animal kingdoms, and
in their major branches, we are led to conclude that, long before
significant specialization, there existed in the waters of the earth vari-
ous primeval forms of life which were endowed with representatives
of most of, if not all, the important biological processes characterizing
living things as we know them today. Although it is unlikely that we
shall ever have more than opinions regarding the origin of life, it
does seem possible to approach, experimentally, the nature of the
speciation which began when such primeval cells had become estab-
lished. This approach must involve a backward extrapolation of the
information we can obtain on the chemical and genetic factors in
organisms chosen from our modem environment.

Before examining for the reader the aspects of the mechanism of
evolution that have been particularly illuminated by recent advances
in biology and chemistry, it has been necessary to outline, in a broad
sort of way, some of the basic fundamentals of evolution and the
specific sciences, particularly genetics, that have contributed so essen-
tially to its understanding. In the opening chapter, therefore, I have
collected and rephrased some gleanings from the massive literature
of morphological evolution to serve as a background for what follows.
In several subsequent chapters is presented further preparatory ma-
terial dealing with classical and contemporary genetics and with the
basic facts of protein structure. Finally, after some discussion of the
rapidly expanding body of knowledge relating structure to function
in biological systems, I have considered a few aspects of natural selec-
tion in evolution which suggest themselves as a result of contemporary
research, as well as some experimental approaches at the molecular
level.

This book was written for pleasure, with the desire for self-en-
lightenment as the major stimulus.  Since I cannot help but feel that
PREFAC:                                                 IX


everyone in science must be interested in the evolutionary process as
the central theme of biology, I have listed a number of the original
articles and books which contributed to the subject matter of this
book.

I am greatly indebted to many of my friends and colleagues, includ-
ing Dr. W. F. Harrington, Dr. Daniel Steinberg, Dr. W. Il. Carroll,
Dr. E. D. Kom, and Dr. W. D. Dreyer, who have read and helped
improve various chapters of this book.  I should also like to express
my gratefulness to Dr. Bruce Ames for his patient help in connection
with some of the discussions of genetic subjects. My special thanks are
due Professor John T. Edsall of Harvard University and Dr. Michael
Sela of the Weizmann Institute of Science, Rehovoth, Israel, who have
read the entire manuscript and whose suggestions have been invalu-
able in the avoidance of error and in the improvement of style and
organization. Finally, I should like to thank my wife, Florence Anfin-
sen, for the cheerful and understanding support she gave to a fre-
quently rather morose husband.

Bethesda, Maryland
May 1959

CHRISTIAN B. ANFINSEN

GROUND RULES

FOR

THE READER

   A prospectus of this book was circulated to a num-
ber of experts by the publisher before the actual job of writing was
begun. The opinions received have been very helpful in establishing
what I hope is the proper slant.  One opinion, in particular, expressed
a point of view so similar to my own that I have asked its author, Dr.
William Stein, of the Rockefeller Institute for Medical Research, for
permission to reproduce it here.

It has always seemed to me, and I may be wrong in this, that when an
expert communicates with the relatively non-expert, he has a responsibility
to stay pretty close to the facts.
without scruple.         An expert can speculate to other experts
       They have the equipment to meet him on his own grounds,

evaluate the evidence and accept or reject the speculation as they choose.
The non-expert has no such basis for evaluation.  He has to accept rela-
tively uncritically what the expert tells him, and hypothesis and fact soon
become confused in his mind.  A plausible speculation-and the speculations
of the true expert are always plausible-can soon masquerade successfully
as gospel. In the present state of our ignorance, I would regard this as
unfortunate.  It would seem to me that a book such as this one should aim
to stimulate thought and experiment among practicing scientists, and
should not lull the uninitiated into thinking that we understand more than
we do.

xi

x                                                    PREFACE


We shall deal, in this book, with many contemporary hypotheses,
some of which are far from general acceptance. Consequently, I
would like to emphasize two points of caution to the reader.  First,
simple examples have purposely been chosen to illustrate the presen-
tation of subjects which in fact are sometimes complicated by excep-
tions and inconsistencies. Second, it will be apparent that the author,
like anyone else, has occasionally taken sides.

CONTENTS

1 . . . . The Time Scale and Some Evolutionary Principles   1

2.. . . Genes as Determinants of Heredity   15

3.. . . The Chemical Nature of Genetic Material   39

4.. . . The Substructure of Genes   67

5.. . . Protein Structure 98

6.. . . The Biological Activity of Proteins in Relation
       to Structure   126

7.. . . Species Variation in Protein Structure   142

8.. . . Genes as Determinants of Protein Structure   164

9.. . . On the Accuracy of Protein Synthesis   185

lo.... The Biosynthesis of Proteins   195

11 . . . . Genes, Proteins, and Evolution   211

xii

GROUND RULES FOR THE READER

xiii


chapter 1

THE TIME SCALE

AND SOME

EVOLUTIONARY PRINCIPLES

   T o most of us, paleontology is the name of a sort of
genteel outdoor science concerned with the collection and gross de-
scription of old bones and hardened mud blocks containing preserved
animal tracks. To the paleontologist and, for that matter, to any
novice who has had the good fortune to pass through what might be
called the "Darwin-to-Simpson reading stage," no definition could be
further from the truth. Just as history, to the historian, is alive and
a part of the continuing pageant of human experience, so is the study
of the life of the past a living science to its devotees.
The study of fossils cannot tell us a great deal about the natural
forces that shape the evolutionary process, but it does furnish us
with guidelines for the consideration of information derived from
other sciences. As G. S. Carter' has put it, "The part of paleontology
iu the study of evolutionary theory resembles that of natural selection
in the process of evolution; it serves to remove the inefficient but
cannot itself initiate." It is clear that we can, and should, present
only the most superficial survey of the fossil record and its interpre-


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P
z

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2                      THE MOLECULAR BASIS OF EVOLUTION

tation in the present volume. For our purposes here we need only
arrive at some general appreciation of the arbitrary divisions of geo-
logical time and outline the phylogenetic relationships that exist be-
tween the various living and extinct forms of life.

Measurements of the extent of decay of long-lived radioactive ele-
ments in the rock strata of the earth's crust enable us to make reason-
able estimates of the ages of various strata. Utilizing such data as
check points, but relying mostly on time estimates arrived at by
classical geological methods, the paleontologist can arrange the
fossilized remnants of life in a consecutive order with reasonable
accuracy. He can also, in many cases, make certain deductions con-
cerning the relation of specific upheavals and rearrangements of the
earth's surface to the changing patterns in the nature and distribution
of life as it was in the past.

For the purposes of those interested in the earth sciences, time
may be expressed perfectly well on a linear scale, as shown on the
left of Figure 1. Such a scale serves to emphasize the relatively
small fraction of global time during which life has existed on the
earth. The biologist is, however, more naturally preoccupied with
"protoplasmic" time and must magnify the portion of the time scale
that has to do with living things. The right half of Figure 1 is more
useful to the biologist and lists some of the landmarks in evolution,
assigned to their proper paleontological time period.

The earliest fossils that occur in any abundance may be assigned
to the Cambrian and Ordovician periods and include a large propor-
tion of the basic types of aquatic animals and the possible beginnings
of the vertebrates. The record for the Pre-Cambrian period is ex-
tremely sparse and is represented mostly by the relatively primitive
plants, the algae.  At the end of the Pre-Cambrian, most of the in-
vertebrate phyla were relatively well differentiated, although the ab-
sence in most instances of structural elements that could survive as
fossils makes the reconstruction of their phylogenetic tree somewhat
controversial. One scheme is presented in Figure 2. This arrange-
ment of the phyla, which includes the higher vertebrate forms for
comparison is, according to its author, L. H. Hyman, not to be taken
literally but is only suggestive. It is based on an arrangement of
animals in order of structural complexity, without separation of the
allied phyla. The bacteria, yeasts, etc., are not shown, for they
branched off at some early point in time when the momentous biologi-
cal accident occurred which led to the establishment of plant and
animal kingdoms.

Another way of looking at the phyla is shown in Figure 3, taken

THE TIME SCALE AND SOME EVOLUTIONARY PRINCIPLES                   3


Figure 2. Relationships of the phyla of the animal kingdom. The arrangement
here is based on the scheme given by L. H. Hyman in The Inoertehrates, volume
1, McGraw-Hill Book Company, p. 38, 1940.

Figure 3. A schematic diagram of the history of life. The various phyla of
animals are rcprescnted by paths, the widths of which are proportional to the
known variety of each phylum during the various biological periods. Redrawn
from G. G. Simpson, The Meanfng of EuoZutfon, 1950, by permission of Yale
University Press.

4                        THE MOLECULAR BASIS OF EVOLUTION        THE TIME SCALE AND SOME EVOLUTIONARY PRINCIPLES                   5

from George Gaylord Simpson's fascinating book, The Meaning of
Evolution. Here we see the major phyla, as they have existed through
most of biological time, in terms of their relative abundances. We
can observe here some of the correlations between geology and
biology which the paleontologist is able to make. For example, the
distinct contractions in the abundances of almost all the phyla in the
Permian and Triassic periods and the actual extinction of the
Graptolithina correlate well with the geological evidence for great
mountain building and climatic rigor during these times.

A final illustration for this phylogenetic orientation is given in
Figure 4, in which the vertebrates are arranged in their proper as-
tendency (to use an "anthropophilic" expression).  In our discussions
of the relations between the biochemistry and genetics of various or-
ganisms we shall refer from time to time to the contents of these
figures. We shall be interested, for example, in the structure of pro-
teins as they occur in various species and in the possibilities of mak-
ing some crude estimates from such data of the rates at which specific
genes have become modified.

The basic characteristics of the evolutionary process vary consid-
erably, depending on the level of evolution which is being considered.
Evolutionary change, measured broadly in terms of the origin of new
systems of animal organization, is an expression of avemge change.

,jj\ Arthropoda

Tllarslc -

Permian -

Pennsylvanian

Mismsippian

Devonian

lative variety in losril record

Ordovic!an

Duration and diversity of the prmcipal groups of ammals (baud mainly on counts 01 genera and higher groups)


Figure 4. A schematic diagram of the history of the vertebrates.  The widths of
the pattern for each vertebrate class is proportional to the known varieties of the
class in each of the geological periods.  Redrawn from G. G. Simpson, The Mean-
ing of Euolution, 1950, by permission of Yale University Press.

As Simpson has put it, "It is populations, not individuals, that
evolve." As we approach the level of immediate cause and effect,
however, certain aspects of evolution become more highly significant,
and when we consider a small experimental population of the fruit
fly, Drosophila, we must become more concerned with individual
mutations and their contribution to the survival or death of these
specific flies than with theoretical, infinitely large populations. This
example is obviously not evolution in the grand sense.  It emphasizes
"the survival of the fittest," a phrase which, in the light of modem
ideas, we know must be replaced with "the survival of the branch
of a population which is adapted well enough to its environment to
live to procreate." Nevertheless, all evolutionists will agree that the
basic cause of change must be gene mutation (although some authors
will hold out for the additional involvement of something more
ethereal in the way of causation, variously termed "aristogenesis,"
"dun u&al," "entelechy," among other names-we shall return briefly
to these terms later in this chapter).

As our store of information concerning species variations in bio-
chemical properties, and specifically in protein structure, increases,
we will do well to have before us, as a constant frame of reference,
a clear picture of the phylogenetic relationships between various forms
of life and, particularly, of the time, in terms of numbers of genera-
tions, required to accomplish these variations.

6                            THE MOLECULAR BASIS OF EVOLUTION

To develop some appreciation of the magnitude of time involved
in the differentiation of a species in relation to that required for a
more sweeping phylogenetic change, let us briefly examine those
divisions of the process called micro-, macro-, and megaevolution.
To quote the capsule summary given by Carter,* "There is, first, the
origin of the smallest evolutionary differences as seen in continuous
series of strata; secondly, there is the differentiation of the members
of a group in adaptive radiation; and thirdly, the evolution of a new
type of animal organization from its predecessors."

Microevolution

In certain favorable instances, when geological processes have re-
sulted in the formation of a continuous local succession of strata,
paleontologists have been able to reconstruct the morphological pro-
gression of a species as it took place over many hundreds of thousands
of years. An elegant example of such a reconstruction is the work
of Trueman and his collaborators on the evolution of the coiled
lamellibranch, Gryphaea, a mollusk derived from oysters of the genus
Ostrea which is frequently found in the strata of the Mesozoic era.
Mollusks of the genus Gryphuea arose frequently and independently
from flat-shelled predecessors, presumably in response to the neces-
sity for raising the mouth of the shell above its muddy environment.
Four stages in the progressive development of a line of Gryphueu are
shown in Figure 5. During the evolution from Ostrea irregulurb to
Gryphueu incurvu a number of morphological characters were modi-
fied, and each of these was changed at different rates. Any one of
these characters may be used as a measure of rate of change; in Fig-
ure 6 is shown a plot of the variations in one of these, the number
of whorls in the shell, as a function of the vertical location of the
sample studied within the superimposed strata. The populations ex-
amined by Trueman from any given stratum gave a unimodal distribu-
tion curve, strongly suggesting that the population was single and
was not a mixture of independent populations.

In a case such as this there is little question that microevolution
has occurred without any large and sudden changes (saltations).
The general characteristics of the evolution typified by the Gryphaeu,
with its succession of imperceptible gradations and with its uni-
formity around a mean, led Trueman to suggest that "such an evolv-
ing stock must be regarded as a `plexus' or `bundle of anastomosing
lineages.' " The example has been presented here mainly to illustrate

THE TIME SCALE AND SOME EVOLUTIONARY PRINCIPLES                    7


About
6x lo6
years

Ostrea I$ irregularis

Gryphaea dumortieri

Gryphaea aff obliquuta

Gryphaea aff incurva

Figure 5. Four stages in the evolution of Gryphaea from its oyster-like ancestor.
Redrawn from A. E. Trueman, Biol. Revs. Biol. Proc., Cambridge Phil. Sot., 5,
296 ( 1930).

THE MOLECULAR BASIS OF EVOLUTION

About
6 x lo6
years

Lower angulata zone

Number of whorls

Figure 6. Distribution curves showing the variation in the number of whorls of
the shells of successive populations of evolving Gryphaea.  Redrawn from A. E.
Trueman, Biol. Revs. Biol. Proc. Cambridge Phil. Sot., 5, 296 (1930).

that microevolution is a population phenomenon and that the sep-
arate development of radiating lines becomes almost impossible in a
restricted population since continual interbreeding prevents the SUC-
cessful rise of deviant groups.

Macro- and Megaevolution

When a mutation confers some benefit on an organism within the
framework of the environmental restrictions on the population to
which it belongs, the characteristic controlled by the mutant gene
may ultimately become firmly entrenched in the heredity of the en-
tire group. However, a limited horizon, such as that available to
the Gryphueu, permits only a limited phenotypic change.  Thus, even
though a few "advanced" Gryphnen might have appeared which were:

THE TIME SCALE AND SOME EVOLUTIONARY PRINCIPLES                    9

B


endowed with some unique and specially favorable character, they
would not be likely to be perpetuated as a unique strain because of
their random interbreeding with the standard average organism.

The major factor responsible for the larger changes in evolution
that lead to distinct new specializations, or to new systems of animal
or plant organization, is adaptive radiation. Adaptive radiation is the
term used by evolutionists to describe the separation of populations
into smaller groups having different natural histories. The more
mobile the group and the more demanding the environmental changes
to which adaptation must be made, the greater the diversity of form
(and the number of unsuccessful "experiments"! ) that results. This
diversity and mobility, together with the concomitant high rate of
evolutionary change, make the fossil record scattered and incomplete
as opposed to the situation for the sedentary Gryphaea. Neverthe-
less, paleontologists have been able to reconstruct the phylogeny of
numerous lines with great success, and certain distinct parameters of
macroevolution are well delineated.

The macroevolution of a particular population of organisms leads
to great complexity of form, most of the examples of which are false
starts and become extinct after a relatively short time (paleontologi-
tally speaking). For an evolutionary development to be successful,
all the various morphological parts must change in a correlated way
to insure survival. The evolutionists can express such correlations in
relative growth and development of parts by means of double
logarithmic plots, as shown in Figure 7. Here are represented the
relations between the heights of the paracones (a cusp of the molar
teeth) and the lengths of the ectolophs (the ridge on the outer
border of the crown of the same tooth) of the teeth of horses during
their progression from the primitive Eohippus to the modem animal.
Characters that may be related by such straight-line plots (of the
general form Y = bXk) are said to be undergoing allometric change,
and the slopes of the lines (k) g ive a measure of the relative rates
at which two specific bodily characters are changing.

A sudden modification in the slope of the plot relating two allo-
metric changes indicates a sudden shift in evolutionary direction.
For example, such an indication is given several times during the
evolution of the horse. As horses underwent adaptive radiation they
became exposed to new types of environmental opportunities in-
volving both new kinds of food and new terrain. The changes in
the position of the eye and in the structure of the foot and of
other physical characteristics have been described in a fascinating
way by Simpson in his book Horses.  The modification of the molars

10                         THE MOLECULAR BASIS OF EVOLUTION

Figure 7.  Changes in the structure of
the molars during the evolution of the
horses. After G. G. Simpson, Tempo
and Mode in Evolution, 1944, by per-
mission of Columbia University Press.

4-, , ,    I III1
8910 15 20 2530
   Length of ectoloph

during equine evolution is particularly instructive in connection with
our present consideration of sharp changes in evolutionary direction.
AS ecological conditions made browsing more favorable than grazing,
the whole plan of the molar was modified by natural selection in a
direction compatible with the abrasive action of hard grasses. Thus
the crown of the molar became thicker and, together with the de-
velopment of cement, permitted the animals to enjoy a fertile life
span in spite of the erosive nature of their natural food supply. A
schematic representation of the adaptive radiation of horses is shown
in Figure 8. This figu re shows the eating habits of the various suc-
cessive members on the main evolutionary line. The correlative plot
of two allometric structural features of the molars, the size of the
tooth and its height, shows that there occurred an abrupt increase in
the relative height of the tooth in the horses that converted to grazing,
whereas in another evolutionary offshoot, Hyohippus, which continued
to browse on soft, easily chewed plants, such a change did not occur.

Most authorities seem to agree that the evolution of a particular
line of organisms, like the horses, can be explained without compli-
cation on the basis of the selection of mutants that confer a survival
value on the individual and on the population to which he belongs.
The occurrence of a particularly advantageous mutation has fre-
quently led to an almost explosive change in structure or habit, and
Simpson has proposed the name "quantum evolution" for such major
jumps. The view is frequently expressed, however, that the process
of natural selection might still be an adequate explanation for these
rapid shifts. Their suddenness is perhaps overemphasized because

THE TIME SCALE AND SOME EVOLUTIONARY PRINCIPLES                   11


Figure 8. The evolution of the horses. The diagram shows the geographic dis-
tribution of the various forms and indicates their manner of securing food by
browsing or by grazing. Redrawn from G. G. Simpson, Horses, 1951, by per-
mission of Oxford University Press.

12

THE MOLECULAR BASIS OF EVOLUTION

of gaps in the fossil record that resulted from the rapidity of the
changes and the limited geographical region in which they occurred.

In discussions of those portions of evolution in which whole new
systems of biological organization arose, the terminology and interpre-
tations of experts becomes varied and, sometimes, delightfully mysti-
cal, at least to window-shoppers such as myself. We have already
mentioned the terms entelechy, e'lan vital, and aristogenesis. Such
terms have been coined to explain (explain away, perhaps) the fre-
quent, puzzling phenomena in which new structures and physiologies
have arisen in the absence of obvious adaptive value or selective in-
fluence. During the evolution of reptiles, for example, there occurred
a simplification of the jaw structure which made superfluous the
quadrate and articular bones of the reptilian jaw. Ultimately, mil-
lions of years later, these "liberated" units became involved in a major
change in the structure of the middle ear and made possible the
chain of small bones which is characteristic of this organ in mammals.
This "aristogenic" change, leading to an entirely new anatomical or-
ganization at a much later time, is not easy to explain on the basis
of selection and adaptation alone. The phenomenon has implied to
some that the evolutionary process has, built into it, some knowledge
of the future and that temporarily useless structures may be stored
away for later use according to some master plan.

From the standpoint of maintaining a more unified picture of the
evolutionary process, `aristogenesis" and the "preadaptation" of an
organism for some subsequent evolutionary event do not appear to
be necessary concepts. Simpson has pointed out that, in small pop-
ulations, a mutation which confers no adaptive value (or, indeed,
which may be detrimental) can become established, although "al-
most always this would lead to extinction."  In those rare cases when
the word "almost" applies, a change in the natural history of the or-
ganism might then cause an enormously rapid and major evolutionary
modification owing to the sudden usefulness of this otherwise dis-
advantageous gene, fortuitously harbored in the heredity of the strain.
From this point of view we may explain the whole of evolution, from
the localized, sedentary sort of microevolution to the dramatic ap-
pearance of new phyla, on the basis of mutation and selection alone.

As we shall discuss in a later chapter, certain structural parts of
biologically active proteins appear to be superfluous from the stand-
point of function. A tendency to assume that such parts are non-
essential might simply reflect the fact that we have not yet developed
sufficiently sensitive methods for the detection of subtle, second-order
relationships between structure and function.  On the other hand,

THE TIME SCALE AND SOME EVOLUTIONARY PRINCIPLES                   13


certain structural configurations may now actually be unessential and
may have been preserved as chemical vestiges of earlier molecules,
much as the bones of the mammalian ear were retained from the re-
arranging components of the reptilian jaw.
The information available to us on proteins and other chemical
components of protoplasm is, of course, insufficient to permit a
rational choice between these alternatives at the present time. We
can only hope, in analogy to the paleontologist and his "fossil record,"
that as the "protein record" relating the proteins of various species
to one another becomes more complete, some basic ground plan for
phylogenesis and speciation may begin to emerge at a molecular level
of understanding.

REFERENCES

1. G. S. Carter, Animal Evolution; a Study of Recent Views of Its Causes,
Sidgwick & Jackson, Ltd., London, 1951.
2. A. E. Trueman, Geol. Mag., 61,360 (1924).

SUGGESTIONS FOR FURTHER READING

Colbert, E. H., Evolution of the Vertebrates, John Wiley & Sons, New York,
1955.

Huxley, J. S., Evolution, the Modern Synthesis, Allen 81 Unwin, London, 1942.
Oparin, A. I., The Origin of Life on the Earth, translated from the Russian by
Ann Synge, Academic Press, New York, third edition, 1957.
Simpson, G. G., The Meaning of Eoolutfon, Yale University Press, New Haven,
third printing, 1950.
Simpson, G. G., Horses, Oxford University Press, New York, 1951.
Simpson, G. G., Life of the F'ust, Yale University Press, New Haven, 1953.
Smith, H. W., From Fish to Philosopher, Little, Brown & Company, Boston,
1953.

THE MOLECULAR BASIS OF EVOLUTION


chapter 7

L

GENES AS DETERMINANTS

OF HEREDITY

   D arwin thought of evolution as a process of adap-
tation to environment by means of the natural selection of favorable
"variations." Within the context of the knowledge of his day he
could not, of course, replace the word "variations,, with "mutations,,,
since the science of genetics had not yet been invented. However,
being a man with a strong urge to tie up loose ends, Darwin sug-
gested that "variations,,, * m&ding those that he felt might be ac-
quired in response to environmental pressures during the lifetime of
the organism, were inherited by a mechanism in which all the somatic
(body) cells contributed information to the germ cells. We know
now that acquired characteristics are not inherited and, with the
emergence of genetics, it became possible to speak of the inherited
characteristics of an organism (his phenotype) as the expression of
the sum of his chromosomal genes (his genotype).* We may now

* It should be stressed that environmental conditions, during development,
can exert a profound influence on the phenotypic expression of the genes. A
classical example of this is the effect of temperature on the number of eye facets
in Drosophila whose chromosomes bear the mutations "low-bar" and "ultra-bar."'
Two organisms with identical developmental potentialities may look or act quite
differently, although their respective offspring will be back to the .old standard

15


describe evolution in terms of the natural selection of favorable gene
mutations in a population and the perpetuation of these through re-
production.

Since this book is directed at biochemists, many of whom may
have had as little formal training in genetics as I have, it is necessary
to present, as a starting point for further reading, an abbreviated sur-
vey of the gene concept and of some of its experimental consequences.
We shall restrict ourselves to the Mendelian genetics of normal bi-
sexual reproduction as it occurs in the higher plants and animals.
The mechanisms involved, although by no means universal, can serve
as a qualitative basis for considering the reproduction of even such
specialized genetic systems as the bacterial viruses, if we are willing
to cut some comers.

Parent
generation

0
RR

\ /
     Rxr

Nearly a hundred years ago, Gregor Mendel made the observa-
tions that established the fundamental laws of genetics. Mendel
crossed strains of garden peas which differed in one contrasting char-
acter (e.g., purple or white flowers) and observed that the progeny
(the so-called F, generation) were all purple. This character was,
then, the "dominant" trait and white the "recessive." Similar dom-
inance or recessiveness was observed for many other alternative traits.

When two members of the F, generation were crossed, he observed
that about three-fourths of the progeny in the F, generation were
purple and one-fourth white. These experiments suggested that any
particular character-determining unit of heredity exists in two forms
and that these "aZZeZic" forms do not blend but maintain their identity
throughout the life of the F, organisms to separate later in the fol-
lowing generation. The units of heredity were subsequently named
"genes,, by Johanssen in 1911. An organism, like the F, peas of
Mendel, which contains both allelic forms is said to be a heterozy-
gote, and those possessing a double dose of one or the other allele
is a homozygote. We refer, genetically, to the former as Rr and to
the latter as RR or rr (homozygous for the dominant and recessive
forms respectively).

F,
generation

Mendel's experiment, summarized in Figure 9, illustrates the
"law of segregation." The frequency of occurrence of purple and
white flowered plants in the F, generation (3: 1) is to be expected
if the two allelic forms of this particular color-determining gene, one
dominant over the other, segregate to yield equal numbers of R and r
units during the formation of germ cells and then proceed to recom-
bine at random in the new generation.  Mendel checked this hy-
and the superficial characteristics acquired as the result of environmental pres-
sures will not be inherited.

Figure 9. Mendel's first law, the law of segregation; R stands for the gene for
purple and I for the gene for white flower color. Black rings and white rings
symbolize purple and white-flowered plants, respectively. Purple color is domi-
nant over white.
John Wiley & Sons, Redrawn from T. Dobzhansky, Eoolution, Genetics, and Man,
           1955.

16                         THE MOLECULAR BASIS OF EVOLUTION        GENES AS DETERMINANTS OF HEREDITY

17


Parent
generation

4
generation

F2
generation

AABB

mbh

\ AB x

AaBb

Figure IO. Mendel's second law, the law of independent assortment; A and a rep-
resent the genes for yellow and green colors, respectively, and B and b those for
smooth and wrinkled seed surfaces. Yellow is dominant over green and round
is dominant over wrinkled. Redrawn from T. Dobzhansky, Euolutfon, Genetics,
and Man, John Wiley & Sons, 1955.

pothesis by allowing the purple-flowered plants in the F, generation
to produce an F, generation. One-third of the F, plants (the RR
strain) produced only purple-flowered progeny, whereas two-thirds
(the Rr variety) produced either white- or purple-flowered progeny
in the ratio 1:3 as predicted by the principle of segregation.
In some of his experiments Mendel crossed peas which differed
in two or more traits. Thus, as summarized in Figure 10, be crossed
peas having yellow, smooth seeds with others having green, wrinkled
seeds; he knew in advance that the gene for yellow color was
dominant over that for green and the gene for round seeds was
dominant over that for wrinkled. The F, generation had seeds which
were yellow and smooth, since both dominant traits were present in
this hybrid and determined the phenotype. In the F, generation,
however, the phenotype was determined by a random combination of
the four segregated traits as shown in the figure. Seeds of the F,
progeny showed all the four possible combinations of phenotype but,
because of the dominance of yellow and smooth over green and
wrinkled, these appeared in a ratio of 9:3:3: 1 with only one-sixteenth
of the seeds having the double recessive characteristics.  This phe-
nomenon, independent assortment of genetic traits, is the second basic
"law" growing out of Mendel's studies.

The simplicity of Mendel's experiments and their ease of interpre-
tation were really due to his good fortune in choosing sets of traits
which segregated and recombined to give the theoretical 3: 1 ratio.
In many instances this ratio is not obtained, and instead certain
sets of genes may segregate together to yield what are termed "linked"
traits. To understand the linkage of genes we must first consider
the phenomena of mitosis and meiosis.
Cytologists have been aware for over a hundred years of chromo-
somes as visible rod or thread-like structures that appear in the
nucleus during cell division. The number of chromosomes per
nucleus is a characteristic constant for any given species. The
genetic information present in a cell is accurately perpetuated in each
of the daughter cells by the process of mitosis. The stages in mitosis
are shown in Figure 11 as they are observed in the root tips of
the common onion. The simplified drawing on the left side of the
figure depicts the behavior of a single chromosome of this plant.
The centromeres are represented, in this figure, by open circles.
These specialized structures within each chromosomal strand act
as points of attachment for the fibers which bind the chromosomes
to the pole of the spindle during subdivision of the cell.  The centro-
mere is replicated during the division cycle, as shown.  Occasional

GENES AS DETERMINANTS OF HEREDITY                             19

II)                      THE MOLECULAR BASIS OF EVOLUTION


Figure 11.  Mitotic cell division in the common onion: A, interphase; B, prophase;
C, metaphase; D, anaphase; E, telophase; F, daughter cells.  From T. Dobzhan-
sky, Eoolutfon, Genetfcs, and Man, John Wiley &I Sons, 1955.

cells containing chromosomes which lack a centromere, or which
have more than one, do not survive. The genetically significant
event is the exact duplication of each chromosomal daughter-strand
during the period between stages F and B, whereby hereditary con-
stancy is insured in all the somatic cells of an organism during its
growth and development.

The nucleus of the somatic cell (diploid) contains twice as many
chromosomal strands as the germ cells or gametes (haploid). The
complement of chromosomal strands in a gamete is the same as
that of somatic cells immediately following mitosis, before the ma-
chinery of the cell has had an opportunity to bring about duplica-
tion of each strand. That is, each gamete contains only a single
aIlelic form of each gene.  When two sex celIs unite, the resulting
diploid zygote contains the hereditary units of both parents arranged
in such a way that the corresponding chromosomal strands are paired
with each set of allelic genes in exact physical complementarity.

When the time comes for the cells of the reproductive tract to
produce gametes, there occurs a process termed meiosis, which is
summarized schematically in Figure 12. The sets of chromosomes
first enter a stage resembling prophase in mitosis.  The corresponding
maternal and paternal chromosome sets then proceed to find one
another by a miraculous procedure in which each bit of cytologically
discernible detail along the maternal strand pairs with its opposite
number in the paternal strand.  Each of the two strands then sub-
divides into two, and, in most organisms, the pairs of strands are
bound together at one or more points by "chiasmata" (Figure 120).

The further stages of meiosis lead to the formation of gametes
containing only one chromosome of each kind. As shown schemati-
cally in the figure, the centromere divides during the second meiotic
division. The details of these latter stages of meiosis are somewhat
different in different organisms, but the end result, haploid sex cells,
is the same.

Early in this century cytologists recognized that the phenomena of
independent assortment and segregation of heritable characteristics
were consistent with the behavior of chromosomes during cell divi-
sion. Direct evidence for such a correlation was soon forthcoming,
largely through the efforts and imagination of T. H. Morgan. Mor-
gan chose as his experimental object the fruit fly, Drosophila mekzno-
guster, which contains extremely large chromosomes in the cells of its
salivary gIands. This organism possessed a number of important
advantages for genetic research, including a high rate of multipli-
cation and a genetic apparatus having only four pairs of chromosomes.

GENES AS DETERMINANTS OF HEREDITY                              21


   --
@

Figure 12. Schematic design of the stages of meiosis. Only a single pair of
chromosomes is shown. The paternal chromosomes are in black and the maternal
in white. #The centromeres are shown as white circles. After T. Dobzhansky,
Euolution, Genettcs, and Man, John Wiley & Sons, 1955.

By crossing strains of flies which showed different inherited traits,
Morgan demonstrated that many of these traits behaved according
to the principles of Mendelian genetics.  He soon observed, however,
that a number of traits did not show independent assortment but were
frequently transmitted from parent to progeny as though they were
linked together in a genetic bundle. A consideration of the scheme
in Figure 12 will make clear the (correct) explanation put forward

22                      THE MOLECULAR BASIS OF EVOLUTION

by Morgan for these observations. Except for the segments of each
chromosomal strand that may be exchanged for their counterparts in
the course of the formation of chiasmata, the total genetic information
in each chromosome appears in any specific gamete as a unit.  Thus,
two closely linked genes (and we may think of this linkage, in
physical terms, as distance along the strand) are not likely to become
separated from one another during meiosis. Morgan and his scien-
tific followers in the field soon found that the traits with which they
dealt fell into four linkage groups and concluded that each cor-
responded to one of the four chromosomes. This conclusion was
completely supported when subsequent studies on the giant salivary
gland chromosomes of Drosophila made possible the direct com-
parison of gene mutations as detected by genetic analysis with
visible morphological changes in the individual chromosomes them-
selves (Figure 13).

Genes that are linked together frequently do show independent
assortment, in spite of their location on the same chromosome.
This separation is explainable in terms of the exchange of chromo-
somal segments that takes place between the two strands during
the formation of chiasma.  (S ee t ransfers indicated in Figure 120.)
Morgan suggested that the frequency of separation, or of recombina-
tion, of two linked genes is a function of the linear distance separating
them. Stated in other terms, the probability of a chiasma occurring
between two distant genes would be much greater than the proba-
bility of one occurring between two genes which are close to one
another. His hypothesis has been amply confirmed by a vast amount
of data on the recombination of linked genes in a variety of organisms
and, although there exist numerous examples of quantitative devi-
ation from the rule, frequency of recombination is in general a relia-
ble measure of the separation between genes.
At this juncture it may be wise to introduce an aside directed
toward the novice in genetics. The picture we have drawn of the
development of the fundamental concepts of genetics has been made
purposely rosy for simplicity's sake.  In this discussion, and in what
follows, we are interested in getting across only the most basic con-
ceptual framework of the subject and cannot consider the many
reservations and qualifications to be found in any adequate text-
book. (For example, in male Drosophila no chiasmata are formed
during the process of spermatogenesis, and consequently no linked
genes can undergo recombination in the progenies of hybrid males.
In the reproduction of bacteriophage, a matter we shall discuss at
greater length in later chapters, recombination of linked genes

GENES AS DETERMINANTS OF HEREDITY                              23


takes place, from a statistical point of uiew, in a manner quite analo-
gous to recombination in higher organisms. Estimates of the dis-
tances between two genes on the phage "chromosome" may be
based on the same general sort of calculation that we employ for
studies on sweet peas, in spite of the fact that classical reciprocal
crossover does not occur; that is, wild-type and double recombinant
phages do not, both, generally result from a single mating event.)

If genes may be thought of as being arranged in a linear fashion
along the chromosomal strand, and if the distances between them
may be estimated by linkage analysis, it is clear that a "map" can
be constructed expressing their physical relation to one another.
Such maps have been prepared for a number of species of higher
organisms and more recently for bacteria and viruses as well. A
map of some of the genes that have been studied in Drosophila mel-
anognster is shown in Figure 14. In general, the distances indicated
behveen genes can be shown to be qualitatively correct by internal
checks. Thus, in a series of crosses involving three genes A, B, and
C, if it is found that the distance between A and B is x units and
behveen B and C is y units, the distance between A and C will be
found to be approximately x plus y units.  The units used here are
"units of recombination" and are merely the percentage of the prog-
eny from any particular cross that is different from either parent
genotype.  For a variety of reasons, the "genetic distances" indicated
on maps such as that shown in Figure 13 bear only a rough corre-
spondence to the actual physical parameters of the chromosomal
strand. One factor responsible for such deviations is the apparent
greater potentiality of some parts of the chromosome to crossover
than others. Another factor involves the occurrence of multiple
crossovers. As the length between two genes becomes larger and
larger, the chance of multiple crossovers will increase and, in the
limit, there will be an equal chance of an even number and an odd
number of crossovers. Thus with widely separated genes and with
random crossover, the `map distance" would approach 50 recombi-
nation units rather than 100. Genetic maps appear, in general, to
be a reliable representation of the relative order of genes, confirming
the concept of a linear arrangement.  But it must be recognized that
the frequency of crossover varies from point to point along the
chromosome, and from species to species, and has great influence on
the additivity of distances and on the total apparent map length.

In the vast majority of cases, the translation of phenotype into the
language of genetics follows the simple rules we have attempted to
summarize. The difficulties experienced by nonspecialists in the

GENES AS DETERMINANTS OF HEREDITY                              25

24

THE MOLECULAR BASIS OF EVOLUTION


d
ID

lx

*

m

:

yellow(B) al

`27.7 lozenge (E)

           d
59.0 vermilion (E)

a.1 mtitun(W)

            J
43.0 uble (B)
u.4 garnet(E)
            b
          d\
     ii4
/56.7 forked(H) B/
-57.0 bar(E) En
59.5 flued

hy

13.0  dumpy 0)

16.5 clot(E)
         b

ee
h

81.0 dacha(B)

41.0 jammed(W) ;
           th
           rt
US black(B) D
161.0 reduced(H) ,$
,54.5 purple W)
2s btitlc (H)
`55.0  light(E) sb
57.5  cinnabar (I?) u
620 enpiled (B)$

61.0 vestigial ,w,L
           H.
72.0 lobe(E) '
75.5 curved(W) aJ

           m
w.a h=Py(B'

100.7 brown(E)
107.0 o pc&(B) M'

192 jwelin (H)

so e+(E)
26.5 hairy (Ii)

  ,dichMb (H)
-i;,&&(E)
&O eculet
47.5 &formed(E)
`46.0 pink(E)
50.0 curled(W)

,5&z  o tuhble (H)
-56.5 lpiaalev (H)
`58.7 bithow
62.0 ettQe(Bl
`63.0 @em(E)
661 delta(V)
a.5 haike
70.7 &my(B)

74.7 cmdhd(El

91.1 rourh (E'

100.7 duet(E)

ma minute-#(H)

Figure 14. Genetic maps of the chromosomes of Drosophila melanogaster.  After
C. Bridges, from Mary J. Guthrie and John M. Anderson, General Zoology, John
Wiley & Sons, 1957.

course of reading genetic literature arise from the terminology which
has been needed by experts to categorize the abnormal. A gene is
recognized only because it can be modified and appear in an ab-
normal allelic form which determines some unusual phenotypic char-
acter. We refer to such changes in genes as mutations, but we must
be constantly aware of the fact that the word has a multiplicity of
meanings and that true understanding of genie modification can only
be reached when the genetics becomes describable in chemical terms.
The appearance of a new phenotypic character may be due to a
change in the gene itself, chemical or configurational, to a deletion
or reduplication of the gene, or to one of a number of "position ef-
fects" involving the inversion or translocation of genes to new posi-
tions along the chromosome. As stated by T. Dobzhansky,2 `A
chromosome is not just a container for genes but a harmonious sys-
tem of interacting genes. The arrangement of genes in a chromosome
has developed gradually during the evolution of the organism to
which the chromosome belongs; the structure of a chromosome, like
the structure of any organ, is a product of adaptive evolution."  It is
to be hoped that the foregoing discussion of the simplest elements of
genetics will be sufficiently irritating in its compactness (and in-
completeness) to cause some readers of this book to look into a few
of the volumes listed at the end of this chapter.
Most of what follows in this book will be concerned with what
genes do, and we approach the subject in terms as chemical as pos-
sible within the limits of our present knowledge of nucleic acid and
protein structure. In the classical sense, the term "gene" has a purely
operational meaning. It may be applied to any unit of heredity that
can undergo a mutation and be detected by a change in phenotype.
As the determined distances between genes on chromosome maps be-
come less and less, the maximum size of the chemical unit which de-
termines a gene must be thought of as being smaller and smaller:
Our impression of the size of a gene, from genetic information alone,
depends entirely on the sensitivity of the methods available for the
detection of extremely infrequent crossovers. It is precisely within
this twilight zone of detectability that the classical definition of the
gene begins to break down; here contemporary research in genetics
and chemistry finds common ground. Estimates of the size of a gene
(as an operational unit) have been made by several methods which
together more or less define the upper and lower limits.  One sort of
estimate is possible from crossover data. Muller and Prokofyeva,3
for example, localized four genes on the giant salivary gland chromo-
somes of Drosophila within a distance of 0.5 micron and concluded

GENES AS DETERMINANTS OF HEREDITY                              17

26

THE MOLECULAR BASIS OF EVOLUTION


that the upper mean limit of length for each must therefore be 1250 A.
Other estimates, derived from studies of the effects of ionizing radia-
tion on the frequency of mutation, indicate that a single gene may
occupy a volume corresponding to a sphere with a diameter as small
as 10 to 100 A. The discrepancy between crossover and radiation
data is considered too large to be due to experimental or interpreta-
tive error and suggests that two different aspects of gene structure
are being measured by the two techniques, one having to do with
the crossover of the entire, intact gene (that is, a .functional unit of
genetics) and one with the modification of chemical fine structure
within its macromolecular architecture.

This conclusion appears to be supported by recent developments in
genetic fine-structure analysis, a few of which we shall review subse-
quently. To establish a bridge between the more classical concepts
of genetics and the rather revolutionary findings of the contemporary
microbial geneticist, it is instructive to consider an example of the
apparent subdivision of a single gene in the genetic material of
Drosophila.

In the course of linkage analysis, certain genetic units called
"pseudoalleles" have been detected which appear to be concerned with
the same, or at least with a closely related, function. One such set
of pseudoalleles makes up the "lozenge genes" of Drosophila melano-
gaster. A mutation in the "lozenge" region causes changes in the
pigmentation of the eyes and also certain other morphological changes.
The mutant forms are recessive to the normal allelic form of the gene;
that is, heterozygotes show normal pigmentation.  Green and Green'
have studied three mutational loci within this region of the genetic
map, all of which have "lozenge" characteristics. From an analysis
of crossover data, they have determined that all three loci fall within
a genetic distance of less than 0.1 units of recombination.  They were
further able to show that &n&e heterozygotes, in which the two
mutant alleles were on the same chromosomal strand, showed the
wild-type character, whereas an arrangement in which the two
mutants appeared on different strands of the same chromosome pro-
duced the mutant phenotype. The phenotypic consequences of the
various arrangements of two mutant loci are shown in Figure 15.
Two explanations for these observations have been offered. One
suggests that each of the individual loci controls a different enzymatic
activity which is in close physical association with the genetic locus
itself. These enzymes are pictured as components of a series of
consecutive reactions leading to the formation of an essential chem-
ical material. Such a situation might apply if the individual re-

28                          THE MOLECULAR BASIS OF EVOLUTION

Mutant
phenotype

(trams)

Wild
phenotype

(cd

Flgure 15. Schematic diagram of the cb and truns arrangements of the pseudo-
allelic "lozenge" genes in Drosophilu melunogaster. After M. M. Green and
K. C. Green, Proc. Natl. Acad. Sci. U.S., 35, 586 ( 1949).

actions were of such a nature that the operation of the reaction chain
depended on certain minimum concentrations of intermediates and
would be interrupted should diffusion (from one chromosomal strand
in the "mutant" heterozygotes of Figure 15 to another, for example)
lead to a suboptimal concentration level for any of the intermediates.
This explanation for pseudoallelism clearly involves a number of
rather large assumptions and seems less likely, at the moment,
than the second alternative, namely, that each pseudoallelic
mutation, although distinguishable, like any "gene," by  crossover,
is really a change in the stcbstructure of the functional parent gene.
Thus, we may postulate that a mutation at any of the three loci of
the lozenge gene might equally impair its function and that only with
the cis arrangement, in which one complete unmarred strand carries
the load, can the normal phenotype be expressed. To anticipate
some of our later discussion, this idea has been used by Benzer as
the basis for the coining of a new genetic term, the "cistron," by
which is meant a genetic unit of function subdivisible by genetic

GENES AS DETERMINANTS OF HEREDITY                              29


Both cistrons
functional

Only cistron A
functional

Both cistrons
functional

Both cistrons
functional
I

0  Cistron
     A

1;d
  Cis

Trans

arrangement

arrangement

Figure 16. The subdivision of a functional gene into "cistrons," both of which must
cooperate to produce an expression in the phenotype.  When two mutations occur
in the same cistron, normal function can only be expressed when the loci are in
the ck arrangement, but not when they are in the truns arrangement.  Based on
the suggestions of Seymour Benzer, The ChemlcaZ Bash of Heredity, Johns Hop-
kins Press, 1957.

tests into ultimate units of recombination termed the "recon." In
this system of terminology, two recons would belong to the same
cistron when the cb arrangement of two mutant loci in a double
heterozygote (Figure 16) results in functional adequacy and the
tram arrangement does not. The demonstration that genes are
made up of blocks of very closely linked subunits which may be
differentiated by crossover has been a tremendous stimulus to bio-
chemists interested in "genetic chemistry." The mutational effect
of ionizing radiation on a bundle of genetic matter having an esti-
mated diameter of 10 A. or so becomes a much more tangible
phenomenon when we can compare such a distance with equivalent
chemical distances, such as the separation of side chains on a
polypeptide or the molecular dimensions of a dinucleotide. As we
shall discuss in later chapters, the ultimate mutable units of genetics
do, indeed, appear to be about this size, and it is possible that we
may soon be able to equate them with individual nucleotide residues
along the polynucleotide strands of deoxyribonucleic acid.

30                        THE MOLECULAR BASIS OF EVOLUTION

An Introduction to the Concept of "Biochemical Genetics"

No summary of genetic principles would be complete without some
discussion of heredity in Neurospora. Neurospora occupies a special
niche in genetics because a great deal of the evidence relating genetic
constitution to biochemical behavior has been obtained through its
study.
It has long been evident that mutations are reflected as changes in
biochemical properties. This is essentially a paraphrase of the state-
ment that mutations are detected only because of the difference in
phenotype which they induce, the phenotype of an organism pre-
sumably being the sum of its biochemical potentialities.  The studies
on the genetic control of the structure of flower pigments by Law-
rence,5 Scott-Moncrieff, and their colleagues helped establish the
fact that individual genes determine the exact chemical structure of
these pigments by regulating the extent of methqxylation, hydroxyla-
tion, or conjugation with carbohydrate of certain heterocyclic com-
pounds called anthocyans. These studies already began to suggest
that the modification of a single gene leads to a change in some
specific biosynthetic process.

Wild strains of Neurosporn may be selected which will grow well
on an extremely simple culture medium consisting essentially of sugar,
salts, and a single vitamin, biotin. By exposing such cultures to some
mutagenic agent (e.g. X-rays), we obtain mutants that no longer
grow on the minimal medium but require the addition of nutritional
additives like yeast extract and hydrolyzed proteins and nucleic
acids. By systematic dissection of the additive mixture, it may be
determined which single nutritional requirement has been induced
by mutation. The isolation of mutant forms having clear-cut nutri-
tional requirements is not always simple, and many have been
isolated which undergo spontaneous reversion to the wild type
or which continue to grow on a minimal medium, although at a
much reduced rate.  However, a large number of stable, full-blown
mutants that require a single nutritional additive for growth has now
been isolated. These nutritional substances include a variety of
amino acids, purines and pyrimidines, and vitamins.  Because of the
conventional chromosomal system of inheritance, the position of these
mutant loci in Neurosporu may be established by orthodox crossing
over methods. The experimental approach to mapping is indicated
by a consideration of the natural history of Neurosporu, the main
points of which are shown in Figure 17.

In asexual reproduction, the haploid conidia germinate to produce

GENES AS DETERMINANTS OF HEREDITY                              31


Conidia

Conidia

Ascospo
germinat

Ascospore
germination

Ascus sac with ascospores
    4&d

Figure 17. The life cycle of Neurosporu crassa. The genetic events which occur
during the first and second meiotic divisions are illustrated in greater detail in
Figure 18. Redrawn, in part, from R. P. Wagner and H. K. Mitchell, Genetics
and Metabolism, John Wiley & Sons, 1955.

more haploid mycelia. Increase in mass also takes place by simple
growth of existing mycelia through mitosis and the utilization of
nutrients from the culture medium. In sexual reproduction cross-
fertilization takes place between two mating types, variously referred
to as A and a, or + and -. The conidia of these two types appear
to differ only in a single genetic locus on one of the chromosomes.
In a cross, the haploid nuclei of the two mating types become as-
sociated within a common cytoplasm. In subsequent events (Figure
17) the nuclei of both mating types undergo numerous equational
divisions (a,b) and subsequently fuse, side by side, into a diploid pair

32                       THE MOLECULAR BASIS OF EVOLUTION

(cd). This zygote (d) th en undergoes two rounds of meiosis (e,f)
to produce four haploid nuclei (f) which then divide mitotically, to
yield eight ascospores (g). When these ascospores are exposed to
heat or to certain other stimuli (furfural), germination is induced.

One of the advantages of Neurospora as an experimental tool in
genetics is the fact that the order of events during meiosis is faith-
fully mirrored in the final asci. As summarized in Figure 17, the
upper and lower sets of two nuclei at the four-nucleate stage are
derived from the upper and lower nuclei of the binucleate state,
and a similar regularity is preserved after the subsequent mitotic
division (stage g). The individual ascospores may be dissected
out by hand, in order.

With some mutations, which cause a visible difference in the
appearance of the final ascospore, we may estimate, without testing
the individual spores, the frequency of crossing over during meiosis,
and thus the map position of the locus in question in relation to
the centromere as a zero point. This procedure is illustrated in an
elegant way by an example taken from the work of D. R. Stadler"
on an unusual lysine-requiring mutant. This mutant, one of a num-
ber of lysine-requiring strains studied by N. Good in 1951, exhibits
delayed ascospore formation, and mutant spores may be detected
within the ascus by their colorless appearance. Perpetuation of this
abnormal strain is possible, in spite of the arrested maturation, because
the vegetative mycelium can be cultivated indefinitely without the
necessity for sexual reproduction and also because an occasional
mutant spore will mature upon aging. The photograph in Figure
18 shows the typical appearance of the asci that are produced when
the mutant is crossed with a wild-type strain.

The critical stages in meiosis following the cross are shown schc-
matically in Figure 19. The two haploid conidia first fuse to form a
zygote a,. (This zyg t
                  o e is known to be in a double-stranded form
(a,) at the start of the first meiotic division.) During this first
meiotic division crossover may or may not occur between the two
sets of parental strands. In Neurospora, the centromeres from each
parental chromosome do not divide during the first meiotic step, and
the crossed-over pairs of strands remain attached as shown in the
figure (b and c). The frequency of crossing over of a given allele
during the first meiosis is assumed to be a function of the distance of
this locus from the centromere.

During the second meiotic division each nucleus yields two daugh-
ter nuclei to give a total of four, arranged in a row, the upper and
lower set derived by division of the upper and lower of the two

GENES AS DETERMINANTS OF HEREDITY                              33


nuclei in the binucleate cell. If no crossover has occurred, the
order shown in d develops, whereas with crossover four different
arrangements may be obtained (e). When the four-nucleate cells
undergo subsequent mitosis, various asci are produced as shown in
the photograph.

The spores containing the mutant locus are easily distinguished
by their colorless appearance. Inspection of the photograph (Figure
18) indicates that in nine of the fourteen mature ascospores no
crossover has occurred; that is, the normal and the mutant forms
of the locus in question have segregated at the first meiotic division.

Figure 18. Appearance of asci produced upon crossing a wild-type strain of
Neurosporu with a lysine-requiring mutant which exhibits delayed maturation.
As discussed in the text, the approximate location of the mutant locus on its
chromosome may be deduced from the relative frequencies of first- and second-
division segregation. This photograph was obtained through the kindness of
Dr. David Et. Stadler of the University of Washington.

Five ascospores show a pattern consistent with second division segre-
gation, one alternating as in Figure 19, e, and e,, and four symmetri-
cal as in e2 and e,.  Therefore, five-fourteenths of the mature asco-
spores, during development from zygotes, have undergone crossover.
Assuming linearity of genes, a direct relationship between crossover
frequency and linear distance, and the absence of centromere division
in the first meiosis, the mutant locus would be calculated to be
5/14 X 109 or 36 per cent of the distance from the centromere to the
end of the chromosomal strand.  (Actually this map distance is to be
divided by a factor of two since the unit of mapping in Neurospom
is defined as one-half of this ratio.)

34                      THE MOLECULAR BASIS OF EVOLUTION          GENES AS DETERMINANTS OF HEREDITY                            35

             ,
Meiosis # 1
Centromere
does not divide

I      Centromere divides   I

or   1     +     +    1   fed
or   +     1     1     t   k3)
or   +    1     +    1   fed)

Figure 19. A schematic diagram of the genetic events which occur during the
development of an ascus from a zygote in Neurospora.  The left side of the dia-
gram shows the results of first division segregation, and the right side, those of
second division segregation of the two alleles, 1 and +.


The crossover frequency values obtained from cross to cross were
found by Stadler to vary over a considerable range, as is frequently
observed in genetic practice. Accurate mapping must always involve
a series of crosses between three separate markers or two markers and
the centromere, so that additivity may be used as a check. This
example is included here because it illustrates how an approximate
estimate may be made of the location of a mutant locus in Neuro-
spora, even without exhaustive crossing of progeny, when the muta-
tion produces a visible change in the convenient ascospore "re-
cording system."

The great value of the Neurospora mutant technique as a tool for
relating genetics to biochemistry will be evident from a consideration
of the following example. Three genetically distinct mutants, which
will grow on the minimal medium when this is supplemented with
one or more of the three amino acids, arginine, citrulline, and orni-
thine, have been isolated. Mutant 1 can grow only when supplied
arginine and cannot utilize citrulline or ornithine.  Mutant 2 can use
both citrulline and arginine, and mutant 3 can manage on any one
of the three nutritional additives. These observations suggest that
arginine may be produced through the sequence of reactions shown
in Figure 20. Assuming the correctness of this biochemical hypothe-
sis, we may propose that the mutant loci in the three mutants each
affect a specific enzymatic process in the reaction chain leading to
the synthesis of arginine. The correctness of this proposition is
indicated by the fact that nutritional mutants will, in general, utilize
and grow on intermediates that come after the `block" but not
those that precede it.  Indeed, in most instances, there is an accumu-
lation of intermediate metabolites preceding the block.

The particular reaction sequence leading to arginine formation is
a well-established one for many organisms. The study of the three
Neurospora mutants is, thus, mainly a confirmatory one, but it has
great historical interest since it was one of the earlier clean-cut
examples of the direct relation between the enzymatic potential of
an organism and its heredity. In many later investigations results
derived from the study of other mutants have frequently served as
the first wedge in the elucidation of new metabolic pathways.

Perhaps the most significant development growing out of the study
of the inheritance of nutritional requirements in Neurospora has been
the enunciation of the "one gene-one enzyme" hypothesis by G. W.
Beadle and E. L. Tatum and their collaborators. This hypothesis,
which proposes that a single gene controls the synthesis of only one
enzyme or other specific cellular protein, can be made quite flexible
by the proper choice of semantics. The breadth of interpretation is

36                       THE MOLECULAR BASIS OF EVOLUTION

Compounds Utilized
  by .Mutanta

- -    +    Omithine

- + t   Citrulline

+ -I- t   Arginine

k-C-C--c-COOH            Urea
\ -Y(
    0

    0
   II
NH,-C-NH     NH,

C-C-C-C-COOH

  f 0
NH -6,,
  2    I
     C-C-C-C-COOH

Figure 20. A series of biochemical reactions in the biosynthesis of arginine, the
order of which could b e established by the study of the nutritional requirements
of three mutants of Neurospora. From the work of A. M. Srb. and N. H. Horo-
witz, J. Biol. Chem., 154, 129 (1944).

directly dependent on the definition we choose to give to the word
"gene." Thus, as is true of the pseudoalleles of the "lozenge" gene
in Drosophila, finer and finer genetic analysis begins to discriminate
between loci which are part of the same functional unit. In a
relatively coarse analysis, such as the study of the three mutants
in the arginine pathway, we are not able to say with certainty
whether the blocked step in mutant 2, for example, is immediately
prior to citrulline or whether one of a number of intermediate steps
between omithine and citrulline is blocked instead. At the other
extreme, an exhaustive genetic analysis might permit the detection of
two genetic loci separated by so small a distance along the genetic
strand that they would be part of the same functional unit.  Mutation
of either of these might alter or abolish the biological activity of the
same protein molecule. This situation has, indeed, been observed
for a number of microorganisms and bacteriophages, and much
of what follows in this book will deal with this theme.
One excellent example of a direct relationship between a single
protein and a single gene is the case of the two types of tyrosinase
in Neurospora. Horowitz7 and his colleagues have shown that the
mutation of a single genetic locus causes the formation of a heat-
GENES AS DETERMINANTS OF HEREDITY                              37


labile tyrosinase which is indistinguishable from the usual, heat-
stable enzyme in all other physical and kinetic properties. The two
forms of the enzyme may be isolated in quite pure form, and there
can be no doubt that the genetic modification affects a single protein
molecule. The difference between the two forms of the enzyme is
inherited in a strictly Mendelian way; that is, a given pure strain of
Neurosporu produces only one form of the enzyme, and the progeny
of a cross between the two strains are identical with one or the
other parent strain in equal proportion.

The possibilities suggested by this and other similar gene-protein
relationships are among the most intriguing in the whole of biology.
Clearly, if slight modifications in protein structure can ultimately
be equated with equally slight changes in the molecular structure of
genetic material, there will be opened to us a whole new area of
research and speculation on the most basic aspects of the evolutionary
process.

REFERENCES

1. J. Krafka, J. Gen. Physiol., 2, 409 ( 1920).
2. T. Dobzhansky, Evolution, Genetics, and Man, John Wiley & Sons, New
York, 1955.

3. H. J. Muller and H. A. Prokofyeva, Compt. rend. acad. sci., U.R.S.S., 4, 74
(1934).

4. M. M. Green and K. C. Green, Proc. Natl. Acad. Sci. U.S., 35, 588 ( 1949).
5. W. J. C. Lawrence, in Blochemkal Sac. Symposia, Cambridge, Engl., No. 4
(1950).

6. D. R. Stadler, Genetics, 41, No. 4, 528 ( 1958).
7. N. H. Horowitz and M. Fling, in Enzymes: Units of Biological Structure and
Function, (0. G. Gaebler, editor), Academic Press, New York, p. 139, 1956.

SUGGESTIONS FOR FURTHER READING

Catcheside, D. G., The Genetics of Micro-Organizms, 1951, Pitman Publishing
Corporation, New York.
The Chemical Basis of Heredity (W. D. McElroy and B. Glass, editors), Johns
Hopkins Press, Baltimore, 1957.

Pontecorvo, G., in Advances in Enzymology, volume 13, Interscience Publish-
ers, New York, p. 121, 1952.
Wagner, Ft. P., and H. K. Mitchell, Genetics and MetaboZism, 1955, John Wiley
& Sons, New York.

38                            THE MOLECULAR BASIS OF EVOLUTION


chapter 3

THE CHEMICAL NATURE

OF

GENETIC MATERIAL

   T here can be little doubt that the major share of the
heredity of a strain is carried in the chromosomes of its germ cells.
This conclusion is based firmly on the observations of the cytologist
and the geneticist that specific mutations may be directly related to
localized morphological changes in chromosomes. It serves as the
starting point for one of the most thriving enterprises in modern bio-
logical research, namely, the identification and chemical description
of genetic material. Progress has been rapid and we can now state
with some assurance that the substance most directly associated with
the storage and perpetuation of hereditary information is the deoxy-
ribonucleic acid (DNA) of the chromosomal strands.

Aside from the circumstantial evidence furnished by the cyto-
chemical localization of DNA in chromosomes, there are a number
of other lines of evidence which give more explicit information bear-
ing on this idea. It was demonstrated by Boivin, Vendrely, and
Vendrelyl in 1948, for example, that the DNA content of somatic
cells (diploid) was constant from tissue to tissue in a single species
but that sperm cells, .(haploid) contained exactly half as much.
               i :                                  39


These observations were extended to a number of other species by
Mirsky2 and his collaborators. The latter group also showed that the
distribution and quantities of various other chemical components of
nuclei generally did not correlate in a way to be expected for
genetically critical material.  We know that the nucleus contains, in
addition to DNA, ribonucleoprotein, various arginine-rich protamines
and histones, a tryptophan-rich protein fraction, and a small amount
of lipid. None of these substances (with the exception of the prota-
mine-histone fraction, which may be directly associated with DNA)
appears to have the constancy of distribution from cell type to cell
type within a species of the sort exhibited by the DNA component.
Another type of experimental observation which suggests a genetic
role for DNA is concerned with the effects of mutagenic agents,
such as ultraviolet radiation and certain chemicals. It has been
shown that the efficiency spectrum of ultraviolet light in producing
mutations is closely related to the absorption spectrum of nucleic
acid, Such experiments are not completely convincing in themselves
since the absorption of ultraviolet photons by the nucleic acid mole-
cule might conceivably be only the first step in a chain of reactions
in which the final target could reside in molecules of a rather different
chemical nature. Experiments on the mutagenic effect of such agents
as mustard gas are similarly inconclusive since, although the nucleic
acids do appear to be much more chemically reactive to these sub-
stances in vitro than are the proteins, we cannot discount the
special sensitivity of a particularly important member of the latter
class of compounds. In spite of these possible objections, there is a
certain amount of direct evidence which indicates that when we
tamper with the chemical structure of DNA, be it by radiation or by
chemical techniques, the rate of mutation is increased. Zamen-
hof3 and his colleagues have shown that when the thymine analogue,
%bromouracil, is incorporated by a cell into the structure of its
DNA, the frequency of mutation is greatly increased. The reasons
for this stimulation of the mutation rate may be related to the pres-
ence of the abnormal pyrimidine base in the polynucleotide sequence
of the DNA molecule, or it may equally well be the result of some
aberration in the kinetics of the biosynthesis of DNA.  Whatever the
reason, we may at least conclude that the DNA molecule is impli-
cated in this chemically induced mutagenesis.
The metabolic stability of DNA also supports the close association
of DNA with genes. In spite of a number of early misleading reports,
the consensus now clearly indicates that the DNA content of chromo-
somes does not change during the stages of division, nor do the

40                       THE MOLECULAR BASIS OF EVOLUTION

subcomponents of its structure undergo equilibration with extra-
nuclear sources of DNA precursors. The most clear-cut support
for this conclusion comes from cytochemical and radiochemical
studies on the nuclei and chromosomes of growing tissues, particu-
larly of plants. Howard and Pelt,' for example, attacked the problem
by growing roots of the English broad bean, Vi& fuba, in the pres-
ence of radioactive orthophosphate. Autoradiographs of squash
preparations of the root tissue were then prepared by the stripping
film technique. An analysis of the way in which radioactivity was
associated with the nuclei in various stages of division and in the
resting stage indicated that incorporation of the isotope takes place
only in the resting, interphase nucleus and that prophase and meta-
phase nuclei are not actively synthesizing nucleic acid.  Autoradio-
graphs of root tips which had been incubated with isotope for more
extended periods permitted the further conclusion that the tagged
nucleic acids of the chromosomes are passed on to daughter cells
without intermediate degradation and resynthesis. These experi-
ments, and the resulting conclusions, have been greatly refined, both
as the result of improvements in the technique of chromosome auto-
radiography and by the study of purified isotopically labeled DNA
from various sources. We shall return to these more recent studies
after we have first considered the chemistry of DNA and the organ-
ization of the chromosome in more detail. However, the experiments
of Howard and Pelt, even without embellishment, are quite satisfy-
ing from the genetic point of view since they suggest conservation
of DNA and the physical continuity of the gene during cell repli-
cation.

Some of the best evidence for the central importance of DNA in
genetics comes from studies on "transformation." This phenomenon
discovered in pneumococcus by F. Griffith in 1928, has since beed
extended to a number of other microorganisms.  It involves the
change in the genotype of one strain of cells that is produced by
exposure to extracts of cells of a different strain.

Pneumococci generally exist in two forms. One forms "smooth"
colonies on agar plates, possesses a type-specific polysaccharide cap-
sular substance, and is virulent. The other forms "rough" colonies
and lacks both the virulence and the polysaccharide of the smooth
form. The smooth forms are genetically stable, and a strain char-
acterized by a Type II polysaccharide, for example, does not spon-
taneously mutate to Type III. "Smooth" organisms do, however,
mutate to rough forms, and this conversion appears to be irreversi-
ble. Griffith showed that when mice were subjected to mixed

THE CHEMICAL NATURE OF GENETIC MATERIAL                        41


injections of living non-encapsulated "rough" organisms and killed
"smooth" organisms, living encapsulated bacteria could be isolated
from the animal. The progeny of these transformed bacteria were
also encapsulated, and the specific polysaccharide was shown to
persist indefinitely through successive generations until spontaneous
mutation to a rough form occurred.

Avery and his collaborators," and later Hotchkiss, Zamenhof, and
others, have shown that the substance in Griffith's extracts which is
responsible for the transformation has the chemical characteristics of
DNA.g The evidence for this is now very convincing, and the trans-
formation of organisms for a host of genetic markers in addition to
that controlling encapsulation can now be attributed, with relative
certainty, to the DNA molecule. The transformation is definitely not
caused by a protein contaminant.

Many of the DNA-borne characters were originally selected by ,
exposure of bacteria to sink-or-swim situations (Figure 21). For
example, when pneumococci are grown in the presence of strepto-
mycin, essentially all the cells are killed by the drug. A very few
cells, however, survive and continue to multiply, producing a culture
which is resistant to the levels of streptomycin employed. These
organisms have acquired, by a chance mutation, the physiological
characteristics which permit them to occupy a new "ecological niche"
in nature (although a rather unnatural one). Not only do the cells
which survive streptomycin behave as a new and constant phenotype,
but samples of DNA prepared from them possess the ability to trans-
form other cells to a state of streptomycin resistance.

The genetic stability of transformable traits is highly suggestive
of true gene transfer. It is difficult, however, to prove unequivocally
that actual chromosomal material is being transferred by this process
from cell to cell. The chemistry and cytology of bacterial nuclei is
still quite obscure and, to make matters more difficult, genetic anal-
ysis of the sort that can be done with sweet peas or Drosophila is
not easy with bacteria because multiplication takes place most com-
monly by mother-daughter division rather than by sexual mating.
Sexual mating does take place fairly frequently in some microorgan-
isms, and it seems likely that the transmission of transformed char-
acters as unit particles of heredity could be studied by some direct
hybridization technique if the proper choice of bacterium and other
transformable traits were combined in one experimental system. To
my knowledge, however, such a study has not been carried out for
any of the traits that can be transferred by a purified DNA prep-
aration.

42                           THE MOLECULAR BASIS OF EVOLUTION

Parent culture

%a3
)f<> ((`, II'
~8 lb @     Culture + rare mutant, (e.g.

   - ze  c      resistant, etc., 1 in 10')

I

Klective
environment

Mutant culture (or stock variant,
e.g. encapsulated)

Purified mutant DNA
(transforming agent)

     +

Parent culture

$51 @ < J
08,  Its
%ii
r 1, x       Culture containing transformed
$33 3   D     cells (1 in 1O'to 103)
* '

Figure 21. Experimental steps in the transformation of a bacterial culture. Rr-
drawn from R. Hotchkiss, The Nucleic Acids, volume 2 (E. Chargaff and j. N.
Davidson, editors ) , Academic Press, 1954.

THE CHEMICAL NATURE OF GENETIC MATERIAL

43


There are, however, in addition to genetic stability, several other
features of transformation that support the notion that the transfer
of unit characters by DNA is closely analogous to the normal genetic
process occurring during cell division.  It has been shown, for ex-
ample, that the DNA prepared from strains of bacterial cells which
have been transformed for two transformable markers can occasion-
ally produce simultaneous transformation for both traits, as though
these were linked on the same "chromosomal" fragment,  Thus, Mar-
mur and Hotchkiss' have selected, from a strain of streptomycin-
resistant pneumococci, mutants that have also undergone a mutation
which permits them to ferment mannitol. When DNA preparations
from such doubly labeled cells were added to a culture of "wild-
type" pneumococci, three distinct types of transformed bacteria could
be isolated. The majority were either transformed for streptomycin
resistance or for mannitol utilization, but some individual cells had
clearly been transformed for both characters. The frequency of
double transformation was considerably greater than the product of
the frequencies of the two single transformations, an observation
which is genetically interpretable only in terms of the linked transfer
of two unit characters by a single event. This observation, so remi-
niscent of linkage in the chromosomes of higher organisms, certainly
suggests that a single fragment of DNA can contain the information
for the elaboration of two distinct physiological systems and that
this fragment represents a piece of the normal genetic material of
the bacterial cell from which it was derived.

These studies have since been extended by Hotchkiss and his col-
leagues to cells that contain three transformable markers. They have
shown that, during transformation with DNA, these markers may re-
main linked, be separated, or be reassembled by recombination in a
manner completely analogous to that observed with doubly marked
cells. In spite of our ignorance of the details of the process, we may
conclude with reasonable certainty that the mechanical transfer of
genetic information from cell to cell by purified DNA represents a
good model for some of the events that occur during conventional
gene transfer in dividing cells.

Chemical Structure of DNA

One of the most fascinating recent developments in biochemistry
has been the study of the structure of deoxyribonucleic acid, both
from the standpoint of its molecular composition and in terms of the

44                        THE MOLECULAR BASIS OF EVOLUTION

arrangements of its component parts in three dimensions. If DNA is
to be established as the substance responsible for the transmission of
heredity at the molecular level, we should be able to demonstrate
that the morphological behavior of chromosomes can be explained as
a function of the structure and metabolism of DNA. Amazingly
enough, such a picture has begun to emerge as the result of a series
of very skillful experiments and deductions.  There are the usual
inconsistencies and hopeful extrapolations, but the present outlook
is certainly an optimistic one.

In 1948 the conception of DNA as a regular array of repeating
tetranucleotide units was seriously shaken by the fundamental studies8
of Vischer and Chargaff and of Hotchkiss, who showed by chromato-
graphic techniques that the four heterocyclic bases in DNA are not
present in equal amounts and that there are actually more than four
such bases in some samples of DNA.  Since these studies, the list of
naturally occurring purines and pyrimidines in the DNA molecule
has grown to seven, and a complete reappraisal of the structural
details of DNA has taken place.

By an examination of the products produced from DNA by acid
or enzymatic hydrolysis we may deduce that the successive stages
of degradation are as follows: *

DNA + nucleotides -+ nucleosides + phosphoric acid +
         purine and pyrimidine bases + deoxyribose
The chemical structure of these substances is indicated in Figure
22. In most samples of DNA, four heterocyclic bases predominate-
the purines adenine and guanine and the pyrimidines thymine and
cytosine. However, some samples of DNA (e.g. from wheat germ
and the grasses in general) contain 5-methylcytosine in large amounts
and, indeed, this derivative of cytosine is found in small quantity in
preparations from mammalian tissue as well. The DNA prepared
from the `<even" members of the T bacteriophages (T2, T4, and T6)
contains 5-hydroxymethylcytosine instead of cytosine. An unusual
purine, 6-methylaminopurine has been found in the DNA of certain
bacteria. For general purposes of discussion, however, we shall take
as prototypes only the four common bases.
o The hydrolysis of ribonucleic acid (RNA), approximately nine-tenths of
which is found in the cytoplasm of cells and one-tenth in the nucleus, yields
the same set of products except for ribose in place of deoryribose, and uracil in
place of thymine ( see Figure 22). The bonds that join the nucleotide resi-
dues in RNA are basically similar to those in DNA, but much less is known
about its molecular configuration. The presumed role of RNA in the biosyn-
thesis of proteins is discussed in Chapter 10.
7HE CHEMICAL NATURE OF GENETIC MATERIAL                         45


              Y
&oxyribonucleosides
(e.g. deoxyadenmine)

Jkoxyritanucleotides
(e.g. 3'-deoxyadenylic acid)
NH2

H c:

I
o-

Ribonucleic acid
  (RNA)

The sugar, deoxyribose, together with the phosphate residues, forms
the chemical backbone of the DNA molecule. Since deoxyribose has
only three hydroxyl groups, and since one of these, at carbon 1,
is bound in an N-ribosidic linkage to one of the four heterocyclic
bases, the alternating phosphate residues must be doubly esterified
with the hydroxyl groups on positions 3 and 5. On the basis of
this information, we may already depict the general structure of DNA
as shown in Figure 23. To round out the purely chemical part of
the DNA structure we need only assign to the various 1 positions
of the deoxyribose units the proper choice of purine or pyrimidine
base.

This is a very large order. The composition of DNA varies a
great deal from source to source and, even more serious, there is
now good evidence that most samples of DNA are probably hetero-
geneous and may represent a mixture of molecules with differing
base contents and sequences. In spite of these difficulties, the ana-
lytical study of many DNA samples from a host of biological sources
has established the following relationships, which hold more or
less quantitatively throughout Nature.

6
and so on

Figure 23. The nature of the linkage between in-
dividual deoxyribonucleotides in deoxyribonucleic
acid. Individual nucleotide residues in the DNA
chain are linked through Y-5 phosphate diester
bridges. The carbon atoms of the deoxyribose
moiety are numbered l', 2', etc.  The carbon and
nitrogen atoms of the purine and pyrimidine rings
are numbered 1, 2, 3, etc., without the primes (see
Figure 22).

Figure 22. The products of hydrolysis of deoxyribonucleic acid and ribonucleic        WE CHEWCAL NATURE OF GENETIC MATERIAL

acid.

-o--P=0

47


1. There is a stoichiometric equality between the sum of the
purines and the sum of the pyrimidines.

2. There is equivalence in the contents of adenine and thymine,
and of guanine and cytosine (or the sum of cytosine and its less
common derivatives when these are present). Two types of DNA
may be recognized, the first in which the sum of adenine plus thy-
mine is larger than the sum of guanine and cytosine (the AT type),
and the reverse (the GC type) which occurs mainly in the micro-

organisms.

3. The content of &amino groups equals the content of 6-keto

groups.

We have, then, a set of requirements which data on the sequence
of bases, now meager but rapidly accumulating, must fit. These

TABLE 1

The distributions of Deoxycytidylic Acid (C) and 5Methyldeoxycytidylic
Acid (M) among the Dinucleotides from Calf Thymus DNA Produced by
Digestion with Pancreatic Deoxyribonucleases

C-Dinu-
cleotide

Mole
Fraction
of
Digest

M-Dinu-
cleotide

Mole
Fraction
of
Digest

   c-p-c-p-      1.11      M-p-M-p-      0
   C-p-T-p-      0.78      M-p-T-p-      0
   T-p-C-p-      a.34      T-p-M-p-      0
   C-p-A-p-      0        M-PA-p-      0
   A-p-C-p-      3.43      A-p-M-p-      0
   C-@-P--      0.75      M-P-G-P-      O
   G-T-C-P-      0.19      G-P-M-P-      1.03

The abbreviated formulas for dinucleotides (T-p-C-p- for example) repre-
sent the phosphate diesters formed by two mononucleotides, joined in the
manner illustrated in Figure 23.
restrictions, in themselves, tell us that DNA molecules must be con-
structed according to a meaningful plan and that the arrangement
of heterocyclic bases must be of great importance in the functional
properties of DNA. Other available evidence strongly suggests that
the sequence of bases is anything but random. For example, Sins-
heimer* has isolated from partial hydrolysates of DNA a large series
of dinucleotide fragments, some of which are shown on the left
side of Table 1. Some of the sequences listed are much more com-

48                      THE MOLECULAR BASIS OF EVOLUTION

mon than others. Recently, Shapiro and ChargafflO have reported
on their similar fractionation studies and concluded that at least
i0 per cent of the pyrimidines in various DNA samples occur as
oligonucleotide "tracts" containing three or more pyrimidines in a
row and that this sort of lumping, because of the three equality re-
lationships listed, must, therefore, also be true of the purines.
This is about as far as we can go at the moment with the critical
problem of the sequence of bases.  There has been, however, a dra-
matic advance in connection with the three-dimensional aspects of
DNA structure. A large part of this advance stems from the elegant
X-ray diffraction studies of M. H. F. Wilkins and his colleagues on
DNA from several sources. The X-ray patterns they obtained were
all remarkably similar and suggested that there existed some uniform
molecular pattern for all deoxyribonucleic acids. Their data were
consistent with the presence in DNA of two or more polynucleotide
chains arranged in a helical structure and permitted the calculation
of the rough dimensions of the helix.

Employing the data of Wilkins and his collaborators," and taking
into consideration the various restrictions on base pairing and stoichi-
ometry, J. D. Watson and F. H. CrickI were able to construct a
model which accommodates most of the experimental facts.  This
model, in spite of slight stereochemical shortcomings which have since
been adjusted by Wilkins and his colleagues (Figure 24), has been
a tremendously valuable stimulus to the field of nucleic acid chem-
istry as well as to genetics. The Watson-Crick model was based,
in addition to the analytical data on base content and the X-ray
results, on the fact that titration studies had indicated that the
polynucleotide chains in the DNA molecule were joined together
through hydrogen bonding between the base residues. The critical
assumptions made by Watson and Crick were that the number of
chains in the molecule was two, and that there was a specific sort
of base pairing which made each pair symmetrical and equivalent in
relation to the cross-linking of the two sugar-phosphate backbones.
The base pairs required by the model, adenine-thymine and guanine-
cytosine, are shown in Figure 25, joined together through hydrogen
bonds. The formulas shown in the figure are drawn from the work
of Linus Pauling and Robert Corey,13 who have considered the DNA
double-helix model in detail in relation to the crystal structure data
for purines and pyrimidines.  Their results, which show that three
hydrogen bonds can be formed between cytosine and guanine, indi-
cate that an even greater specificity of base pairing is inherent in
the DNA model than was suggested by the considerations of Watson
WE CHEMICAL NATURE OF GENETIC MATERIAL                         49


Figure 24. Photograph of a molecular model of deoxyribonucleic acid (through
the courtesy of Dr. M. H. F. Wilkins). A schematic drawing of this two-stranded
structure is shown on the right, together with certain of its dimensions.

and Crick, who assumed that two hydrogen bonds are formed be-
tween each base pair.

The model has the interesting feature that the two strands of
polynucleotide are complementary to one another and that the
arrangement of bases on one strand fixes the arrangement on the

50                          THE MOLECULAR BASIS OF EVOLUTION

Figure 25. The pairing of adenine and thymine (top) and cytosine and guanine
(bottom) by means of hydrogen bonding. Redrawn after L. Pauling and Ft. B.
Corey, Arch. B&x&em. Bbphys., 65, 164 ( 1958).

lHE CHEMICAL NATURE OF GENETIC MATERIAL

51


other. Thus, if the bases along one strand are arranged in the
order A-GGT-C-A, the opposite bases on the complementary
strand will be T-C-C-A-GT. This fact has interesting implications
in connection with the process of genetic replication, which we shall
shortly discuss.

The Watson-Crick model has received considerable support and
is certainly a splendid working hypothesis. We must remember,
however, that it is only a model and therefore examine both the pros
and the cons.

In support of the double helix can be offered the studies of
ThomasI and of Schumaker, Richards, and Schachman.15  Both have
concluded, from investigations of the changes in light scattering and
viscosity during partial enzymatic digestion, that the bulk of the
DNA molecule must be in the form of double chains which are linked
together. They have calculated that, if the molecule were a single
chain, the mean molecular weight of the fragments of DNA pro-
duced should have been considerably smaller than observed and that
the facts correspond well with a system of rather stable cross-linkages
between two polynucleotide strands.

Some of the most impressive support for a double-stranded, com-
plementary structure comes from the very recent work of Meselson
and StahP using an ultracentrifugal method developed by Vino-
gradl' and his group at the California Institute of Technology.  Vino-
grad has capitalized on the fact that strong salt solutions (e.g. of
cesium chloride) may be forced into a density gradient when exposed
to high centrifugal fields.

A macromolecular substance like DNA dissolved in the salt solu-
tion will seek its own density in the gradient and will form a rela-
tively sharp line in the centrifugal cell; the line may be detected by
its strong absorption of ultraviolet light. If two species of DNA
that differ in density are present, these will separate and form two
distinguishable zones (Figure 26). In this way it is possible to
estimate, for example, the relative proportions of normal DNA and
of DNA containing the rather dense pyrimidine, bromouracil, in a
mixture of the two.

Meselson and Stahl grew Escherichia coli in a nutrient medium
containing nitrogen of mass 15 exclusively and thus obtained a pop-
ulation of organisms which were fully labeled with this isotope.
They then transferred cells, taken during the logarithmic phase of
growth, to a medium containing Nl4 precursors.  Samples were taken
when the population had doubled and again after a second doubling.
Deoxyribonucleic acid was prepared from the original fully labeled

h 26. The density gradient centrifugation of a mixture of Nl5- and N14-
&led deoxyribonucleic acids from E. coli. Exposures were taken every 128
mim&s. Equilibrium has been attained by the time of the eighth or ninth photo-
pph. The molecular weight of the DNA is approximately 10,000,000. From
L\L bleselson and F. W. Stahl, Proc. Natl. Acad. Sci. U.S., 44, 671 (1958).

41s and from the two batches of cells that had undergone multi-
plication. Each of these preparations was then dissolved in strong
c&urn chloride solution, having a density in the neighborhood of
the apparent density of DNA, and centrifuged until density equi-
librium had been attained in the ultracentrifuge cell.  Reproductions
d the distribution patterns obtained are shown in Figure 27. The
DNA prepared from the fully labeled bacteria gives a single band
carresponding to the density to be expected for N15 material. After
one generation this band has disappeared and has been replaced with
a band having a density to be expected for equal amounts of Nl*
and N's. Finally, after two generations, two bands are observed,
one corresponding to 100 per cent N `J-DNA and one to the 50:50

52                           THE MOLECULAR BASIS OF EVOLUTION      1M CHEMICAL NATURE OF GENETIC MATERIAL                         53


1.1

0 and 1.9
mixed

0 and 4.1
mixed

Flgure 27. Ultraviolet absorption photographs showing the concentration of DNA
by density gradient centrifugation of lysates of bacteria sampled at various times
after the addition of Nr4 substrates to a growing Nrs-labeled culture.  The length
of exposure to Nl4 substrates is indicated in terms of generations.  After one
generation (fourth frame from the top) the density of the bacterial DNA lies
halfway between that of the original N lb-labeled material and that of material
containing essentially only N" (the left component in the bottom frame).  Taken
from the work of M. Meselson and F. W. Stahl, Proc. Sot. Nntl. Ad. Sci. U.S.,
44,671 (1958).

THE MOLECULAR BASIS OF EVOLUTION

Nr4-N's mixture. In experiments in which cells were permitted to
multiply through further generations, the mixed band decreased in
amount and was replaced by a greater and greater proportion of the
band corresponding to N Id-DNA. If replication of DNA had oc-
curred by a process which involved intermediate degradation into
smaller pieces with subsequent reutilization of these pieces for the
synthesis of new DNA molecules, density of the first-generation mole-
cules could not have corresponded so sharply to a specific equimolar
mixture of "light" and "heavy" chains. We would expect, rather,
a broad distribution of densities, under such circumstances, quite
unlike the results that were actually obtained.
These results are in good agreement with the predictions of the
Watson-Crick model. As shown schematically in Figure 28, each
strand of the DNA double helix might be visualized as a template on
which a new strand is fashioned during replication. If the pre-
cursor nucleotides are unlabeled, the two "daughter" helices produced
during a single replication would be expected to be half-labeled.
After a second replication two strands would be labeled and two
unlabeled. Base pairing would assure accurate assembly of comple-
mentary strands, and genetic constancy would thus be maintained.

The scheme of base pairing suggested by Watson and Crick has
recently received strong support from the enzymatic studies of Arthur
Kornberg and his colleagues'* on the in vitro synthesis of DNA-like
molecules. They reported, in 1957, that an enzyme had been puri-
fied from extracts of E. coli which could catalyze the condensation of
deoxyribonucleoside triphosphates to form large polydeoxynucleotide
chains with the liberation of pyrophosphate in the presence of a
DNA "primer." In subsequent studies the enzyme has been purified
4000-fold over the crude extract, and a number of important proper-
ties of the system have been delineated. The most dramatic finding
has been that the composition of the newly synthesized polymer
(having a molecular weight of the order of 5,000,000) exhibits the
same purine and pyrimidine base composition as that of the primer,
in spite of a constant concentration of each triphosphate added.  For
example, the addition of DNA from Aerobacter aerogenes, having a
ratio of adenine plus thymine to guanine plus cytosine of 0.82, in-
duced the formation of "DNA" with a ratio of 0.99.  With DNA from
T2 coliphage, having a ratio of 1.91, the new material showed a
value of 1.98. In keeping with the base pairing of the Watson-Crick
model, deoxyuridine triphosphate was incorporated into DNA only
in place of thymidine triphosphate and deoxyinosine triphosphate
only in place of deoxyguanosine t&phosphate.  This major break-

THE CHEMICAL NATURE OF GENETIC MATERIAL                         55


56

THE MOLECULAR BASIS OF EVOLUTION

through, in addition to supplying evidence bearing on DNA struc-
ture, may also be a key to the ultimate synthesis of polydeoxynucleo-
tides having specific biological activity.

The studies we have discussed deal with genetic material at the
molecular level. To make the results meaningful to biology, they
must be shown to apply to, or at least to be consistent with, the be-
havior of DNA at the level of the organized chromosome. The ex-
trapolation is enormous and consequently entails a good deal of faith.
We can determine, for example, that the DNA in a single haploid
cell (of Lilium) is approximately 53 X lo-l2 grams. If we assume
that this DNA is present as a single, long double helix, it can be
calculated that the length of this coil would be 1.5 X 107 microns,
or 15 meters, and that the coiled structure would make 4.4 x 10"
turns around the screw axis. The mechanical problems of unwinding
such a coil during the replication process are of a magnitude to make
a picture as simple as this one of no serious value.

In spite of the enormous gaps in knowledge between DNA struc-
ture and chromosome organization, recent experiments by Taylor,
Woods, and Hughes19 on chromosome replication make it clear that
some hypothesis must be arrived at which will accommodate both
chemical and cytological information. Taylor et al. have performed
the equivalent of the Meselson-Stahl experiment at the cellular level.
Using a technique similar to that employed by Howard and Pelt
(page 41), they h ave studied the distribution of label between
daughter cells in successive generations. Their radioautographic
technique was made considerably sharper by the use of tritium-
labeled thymidine rather than radioactive phosphate as a labeled
precursor.  Since tritium emits beta particles of rather low energy
in comparison with phosphorus, film blackening is more highly lo-
calized and the resulting autoradiographs have a high degree of cor-
respondence with cellular structure as observed by microscopic ex-
amination.

Vi&z fuba seedlings were grown in a medium containing tritiated
thymidine. During this incubation most of the twelve chromosomes
became labeled as evidenced by the correspondence between the
location of silver grains in the photographic emulsion and the micro-
scopically visible chromosomes.  Roots at this stage were then trans-
ferred to nonradioactive thymidine solutions containing colchicine
and incubated for varying lengths of time. In the presence of col-
chicine, Vi& chromosomes contract to the metaphase condition and
sister chromatids (the two separate strands of the chromosome du-
plex) are spread apart and are easily observable. Since colchicine

THE CHEMICAL NATURE OF GENETIC MATERIAL                         57


(2a)                  (2b)

Figure 29. Vicia fuba chromosomes.  Upper half, chromosomes in metaphase,
at the first division after labeling with radioactive thymidine: ( la) chromatids
spread apart but attached at the centromere; ( lb) silver grains in the photo-
graphic emulsion above the chromosomes.  Lower half, chromosomes after label-
ing, showing replication in the absence of labeled precursor: (2~) chromosomes
from a cell containing 24 chromosomes, with chromatids spread but attached at
the centromere; (2b) silver grains in the emulsion above the chromosomes in 2~.
As described in the text, these results suggest that the formation of new chromatids
takes place at the expense of low molecular weight precursors and does not involve
the degradation and reutilization of pre-existing deoxyribonucleic acid.

58                            THE MOLECULAR BASIS OF EVOLUTION

has the effect of preventing anaphase movements and the subsequent
separation of daughter cells, but does not stop chromosomes from
duplicating, a count of the number of chromosomal pairs in each cell
gives a measure of the number of rounds of replication.  Thus, cells
with 24 and 48 chromosomes have replicated once and twice, respec-
tively.
Taylor and his colleagues observed that in cells with 24 chromo-
somes only one of the sister chromatids in each metaphase pair was
labeled. It was evident that the pool of labeled precursor had been
rapidly depleted in the cells after removal from the initial solution
and that replication of chromosomes to yield 24 had involved the
use of the nonradioactive thymidine.

In cells with 48 chromosomes, analysis of all 48 was not possible.
However, of those that were sufficiently separated for clear observa-
tion, approximately half contained one labeled and one unlabeled
sister chromatid and half were completely unlabeled. In an oc-
casional "second generation" pair of chromosomes, individual sister
chromatids were labeled for only part of their length (see arrow in
Figure 29), and in such cases the corresponding portion of the other
chromatid showed radioactivity. This bebavior is to be expected for
crossing over and resembles the cytological observations that have
been made for the chromosomes of other species (e.g. the giant
salivary gland chromosomes of Drosophila).

The results are interpretable in terms of a scheme such as shown
in Figure 30. This diagram proposes the presence in each chromo-
some of two morphological halves held together at the centromere

Duplication with
labeled thymidine

First m&phase
after labeling

Duplication without
lakled thymidine

Second metaphase
after labeling

Figure 30. A schematic representation of the photographs in Figure 29. Solid
lines indicate unlabeled units in the chromosome and dashed lines represent radio-
active units formed during replication.  The dots represent grains in the photo-
graphic emulsion caused by radioactive decay of the tritiated thymidine.

THE CHEMICAL NATURE OF GENETIC MATERIAL                         59


Figure 31. A schematic diagram showing how the chromosome might be organ-
ized to account for the results summarized in Figure 29. ,The chromosome is
depmted as consisting of a central core divided into two halves.  Each half is
attached to one strand of a large number of DNA double helices.  Redrawn from
J. H. Taylor, Am. Naturalkit, 91, 209 ( 1957).

and containing two equivalent strands. During the initial labeling
period each chromatid becomes radioactive, and metaphase figures
in cells with twelve chromosomes are uniformly associated with black-
ening of the emulsion. After a second replication each chromosome
pair is still radioactive, but all the radioactivity is associated with
the original parent strand and the newly formed strand is unlabeled.'

Realizing the unlikelihood of a double helix running the full length
of the chromosome, Taylor, Woods, and Hughes have proposed an
interesting model which accommodates both their own radioactive data
at the cellular level and the theoretical implications of the double
helix. This model (see Figure 31) visualizes the chromosome as
composed of two halves, each half consisting of a central core to
which is attached one strand of each of a multitude of double helices.
When a cell enters the division cycle, the halves separate from one

* L. F. La Cour and C. R. Pele have recently repeated these experiments in
the absence of colchicine and report that radioactivity was distributed between
both chromatids.  No free labeled precursors were present during the period of
replication. These results, which are in direct contrast to those of Taylor,
Woods, and Hughes, reopen the question of whether replication is, or is not, a
completely  "conservative" mechanism in which a chromosomal strand, once
formed, is retained in the nucleus as an essentially permanent chemical struc-
ture.

60                          THE MOLECULAR BASIS OF EVOLUTION

another, each half carrying with it one of the complementary poly-
nucleotide chains. Taylor and his colleagues point out that the
model introduces a new dimension for the interpretation of crossing
over. Thus, conventional crossover might occur by exchange along
the core, whereas various minor recombinations and gene conversions
could occur by interaction or exchange among the side chains of
DNA.

Summing up, we may say that a good case can be made for the
presence of a set of intertwined helical coils of polynucleotide in
DNA. The model proposed by Watson and Crick is, at present, the
most satisfactory. Although J, DonohueZO has shown that certain
other arrangements of base pairs can be made which would permit
the construction of double-helix models, these do not agree with the
X-ray patterns that have been observed for DNA, and the pairing
of adenine with thymine and of guanine with cytosine seems most
probable at the moment. The complementarity of the two strands
forms the basis of an intriguing hypothesis for replication of genetic
material, supported by experimental evidence at both the molecular
and the cellular level.

Let us now consider briefly some of the bits of evidence that do
not fit entirely with the Watson-Crick hypothesis. These are not as
numerous as the supporting evidence, possibly because scientists, like
other citizens, are also attracted to bandwagons.
It has been suggested that the replication of DNA based solely on
complementariness of polynucleotide chains might be difficult to
reconcile with some of the analytical values for nucleotide abun-
dvance. Sinsheimer' has isolated a series of dinucleotides from calf
thymus DNA (see Table 1) which includes homologous dinucleotides
of cytosine and of 5-methylcytosine.  The results indicate a consider-
able difference in the distribution of cytosine and 5-methylcytosine;
that is, substitutions do not appear to be random. Although still
somewhat preliminary, these findings suggest that simple comple-
mentarity might not be an adequate basis for the distribution.
There is no obvious stereochemical reason for discrimination against
5methylcytosine in positions usually occupied by cytosine, and
Sinsheimer justifiably recommends that we keep our eyes open for
ancillary pathways of nucleotide metabolism or assembly that might
explain such deviations from simplicity.

Sinsheimer has also pointed out another more serious objection
to simple complementary replication. In T2 bacteriophage, in which
cytosine is completely replaced by 5-hydroxymethylcytosine, about 77
per cent of the latter compound occurs conjugated with glucose

THE CHEMICAL NATURE OF GENETIC MATERIAL                         61


(see Figure 22).  Now various studies (which we shall discuss in
the next chapter) have indicated that the DNA of bacteriophage T2
is composed of one large piece comprising about 40 per cent of the
total and a number of smaller pieces. All the unconjugated S-hy-
droxymethylcytosine is found in the large piece; that is, only about
60 per cent of the large piece is substituted, whereas all the smaller
pieces contain glucose. As stated by Sinsheimer, "Since these com-
ponents of T2 DNA are all replicated in the same cell at the same
time, the limited glucose substitution of the large piece cannot
reasonably be ascribed to some metabolic limitation.  Hence it would
appear (to put the matter mechanistically) that when the replication
of the large piece takes place, the machinery knows when to put in
glucose-substituted 5-hydroxymethylcytosine or when to insert un-
substituted 5-hydroxymethylcytosine, for the glucose content remains
unchanged through many generations." These observations could be
explained on the basis that 5-hydroxymethylcytosine and its glucose-
substituted derivative present, to the assembly line, different stereo-
chemical features which would preclude error in the determination
of sequence. The cautions advanced by Sinsheimer concerning sim-
ple complimentariness must obviously be kept well in mind, both as
brakes on overenthusiasm and for their heuristic value.

Some of the most puzzling evidence contrary to the Watson-Crick-
replication hypothesis comes from the work of G. Stent and his co-
workers on "suicide" in P@-labeled phage as the result of radioactive
decay. These experiments are not easily described, however, without
some preliminary consideration of the natural history of bacterio-
phage; consequently they will be discussed in the following chapter.

Polynucleotide "Codes"

Since there are only four (common) bases, a code founded on cor-
respondence between base pairs and amino acid residues would not
suffice, only 4 X 4 = 16 such pairs being possible.  Base triplets, on
the other hand, are too numerous (4 X 4 X 4 = 64) without the in-
troduction of restrictions. As one approach to restricting the possi-
bilities, Crick, Griffith, and Orgel have chosen to consider the case
of "nonoverlapping codes."  An illustration from their discussion
states the problem clearly. Letting A, B, C, and D represent the
four bases, we may divide a polynucleotide chain of such bases into
nonoverlapping triplets, each triplet corresponding to an amino acid
residue ( Figure 32).  "If the ends of the chain are not available, this
can be done in more than one way as shown.  The problem is how
to read the code if the commas are rubbed out, that is, a comma-less
code."

No discussion of DNA in relation to genetics would be complete
without some mention of the efforts that have been made to discover
a "code" which might permit the translation of base sequence into
protein structure. To participate in this game we need have no
special knowledge of protein chemistry beyond the following two
basic facts: first, proteins consist, for the most part, of polypeptide
chains made up of approximately twenty amino acids (exudly
twenty in codes to date) arranged in a specific sequence for each
protein species; and second, the known protein and polypeptide se-
quences have been assembled in several review articles which are

The ground rules for this particular coding puzzle were as follows:
(1) Some of the 64 triplets make sense, and some "make nonsense";
(2) all the po ssi bl e se q uences of amino acids may occur, and at every
point in the string of bases we can only read sense in the correct
way. To put ground rule 2 in other words, two triplets that make
sense can be put side by side, but overlapping triplets so formed
must always be nonsense. Thus, in the sequence ABCDAA, if ABC
and DAA make sense, BCD and CDA must always be nonsense.

To prove that 20 is the maximum number of amino acids that can
be coded in this manner is quite straightforward. Thus we may

62                            THE MOLECULAR BASIS OF EVOLUTION          THE CHEMICAL NATURE OF GENETIC MATERIAL                         63

or

. . . BCA, CDD, ABA, BDC,. . .

. . . B, CAC, DDA, BAB, DC.. ,

Figure 32. Linear arrangements of four purine or pyrimidine bases, A,B,C and
D, in which triplets, each corresponding to a specific amino acid residue, are sepa-
rated by commas. The problem is how to read the code if the commas are
rubbed out.

easily available in most libraries.  Some mathematical facility and
considerable imagination are prerequisites.

We shall consider only one such code here, the so-called "comma-
less code" of Crick, Griffith, and Orgel. It makes a good example
because experimental data have not yet appeared which can rule
out this code, and the basic cryptographic approach to "how a
sequence of four things [nucleotides] determines a sequence of
twenty things [amino acids]" is elegantly illustrated in their paper.


AB;      A
   :CB    A IA
        BD:

D   D c

Figure 33. One solution to the problem of how twenty amino acids can be coded
by four heterocyclic bases in a "comma-less" code.

eliminate AAA, BBB, etc., since, if in a sequence AAAAAA the tri-
plet AAA corresponds to amino acid a, the sequence of six may be mis-
interpreted by associating acid a with the second to fourth, or third
to fifth letters. This reduces the 64 possibilities to 60. These 60
may be grouped into twenty sets of three, each set being cyclic
permutations of one another.  If BCA stands for amino acid b, for
example, then BCABCA stands for bb, and CAB and ABC must be
ruled out as nonsense. Since only one triplet can therefore be chosen
from each cyclic set, we arrive at the "magic number" of 20.

Having shown that no more than twenty amino acids can be coded
by four bases in this system, the authors list a number of solutions,
including that shown in Figure 33. The reader may satisfy himself
that any two triplets of this set may be placed next to each other
without producing overlapping triplets which belong to the set. In
all, 288 different solutions were found which conformed to the rules.

If the structure of DNA does, indeed, have some direct relation-
ship to genetic information, we must undoubtedly take into account
many structural factors in addition to base sequence, particularly
those having to do with the geometrical arrangement of the poly-
nucleotide chain. The business of constructing codes that might
have some bearing on the problem of information transfer from DNA
to protein is, therefore, mainly of theoretical value at the moment.
In time, however, the study of DNA-protein cryptography will un-
doubtedly become a much more active field. Protein chemists are
now beginning to accumulate data relating to the chemical conse-
quences of mutation, and there should soon be a large amount of
information for consideration.  These data will be, for the most
part, very indirect. For example, the single-gene change which
leads to the formation of hemoglobin C instead of the normal hemo-
globin A is reflected in the replacement of a particular glutamic acid
residue by a residue of lysine (Chapter 8). The same replacement
has occurred, during evolution, in the ribonuclease molecule where
a lysine residue in bovine ribonuclease is replaced by glutamic acid

64                       THE MOLECULAR BASIS OF EVOLUTION

in the sheep protein (Chapter 7).  In the first case we can definitely
invoke mutation as a causative factor.  In the second, mutation is
implied but, in view of the impossibility of crossing cows with sheep,
a cause and effect relationship cannot be proved. As further ex-
amples of this sort accumulate, we shall, perhaps, be able to decide
whether there exists some uniform pattern of correspondence between
genetic composition and protein structure.

REFERENCES

1. A. Boivin, R. Vendrely, and C. Vendrely, Compt. rend., 226, 1061 (1948).
2. A. E. Mirsky, in Genetics in the 20th Century (L. C. Dunn, editor), Mac-
millan, New York ( 1951).

3. S. Zamenhof, R. DeGiovanni, and K. J. Rich, Bacteriology, 71, 60 (1956).
4. A. Howard and S. R. Pelt, Erptl. Cell Research, 2, 178 ( 1951).
5. 0. T. Avery, C. M. MacLeod, and M. McCarty, J. Erptl. Med., 79, 137
(1944).

6. These studies are reviewed by R. D. Hotchkiss in the book, The Nucleic
Acids, volume 2 (E. Chargaff and J. N. Davidson, editors), Academic
Press, New York, 1955.

7. J. Marmur and R. D. Hotchkiss, J. Biol. Chem., 214, 383 ( 1955).
8. Discussed by E. Chargaff in The Nucleic Acids, volume 1 (E. Chargaff and
J. N. Davidson, editors), Academic Press, New York, 1955.
9. R. L. Sinsheimer, Science, 125, 1123 (1957).
10. H. S. Shapiro and E. Chargaff, Biochim. et Biophys. Acta, 20, 608 ( 1957).
11. M. H. F. Wilkins, A. R. Stokes, and H. R. Wilson, Nature, 171, 738 (1953).
12. J. D. Watson and F. H. C. Crick, in Viruses, Cold Spring Harbor Symposiu
Quunt. Btology, 18, (1953).

13. L. Pauling and R. B. Corey, Arch. Biochem. Biophys., 65, 164 (1956).
14. C. A. Thomas, J. Am. Chem. Sot., 78, 1861 (1956).
15. V. N. Schumaker, E. G. Richards, and H. K. Schachman, J. Am. Chem.
Sot., 78, 4230 ( 1956).

16. M. Meselson and F. W. Stahl, Proc. Nutl. Acad. Sci. U.S., 44, 671 (1958).
17. M. S. Meselson, F. W. Stahl, and J. Vinograd, Proc. Nutl. Acud. Sci. U.S.,
43, 581 (1957).

18. J. Adler, M. J. Bessman, I. R. Lehman, H. K. Schachman, E. S. Simms, and
A. Kornberg, Federation Proc., 16, 153 ( 1957). See also Federation Proc.,
17, 178 (1958).

19. J. H. Taylor, P. S. Woods, and W. L. Hughes, Proc. Nutl. Acud. Sci. U.S.,
43, 122 (1957).

20. J. Donohue, in Molecular Structure and BioIogicuZ Specificity, (L. Pauling
and H. Itano, editors), American Institute of Biological Sciences, Publication
2, Washington, D. C., 1957.

21. F. H. Crick, J. S. Griffith, and L. E. Orgel, Proc. Nutl. Acud. Sci. U.S.,
43, 416 (1957).

THE CHEMICAL NATURE OF GENETIC MATERIAL                         65


SUGGESTIONS FOR FURTHER READING

Biochemical Society, The Structure of Nucleic Acids and Their Role in Protetn
Synthesis, Cambridge University Press, Cambridge, England, 1957.
The Chemical Bask of Heredity (W. D. McElroy and B. Glass, editors), Johns
Hopkins Press, Baltimore, 1957.

Crick, F. H. C., in Sci. American, 197, No. 3, 188 ( 1957).
Overend, W. G., and A. R. Peacocke, Endeaoour, 16, No. 62, 90 ( 1957).
Taylor, J. H., "The Time and Mode of Duplication of Chromosomes," Am.
Naturalkt, 91, 209 (1957).

66

THE MOLECULAR BASIS OF EVOLUTION


chapter 4

THE SUBSTRUCTURE

OF GENES

   A s we pointed out in Chapter 1, most evolutionists
feel that all levels of evolution can be interpreted in terms of a single
basic process, the natural selection of organisms whose phenotypes
have been modified in some favorable way by gene mutation.  Dur-
ing the earliest stages of evolutionary development, the sudden ap-
pearance of new genes must have been a frequent and important
occurrence. However, it is, perhaps, not too heretic to suggest that
the major proportion of evolution has been the result of a continual
process of modification, and integration into new systems of organi-
zation, of genetic potentialities already present in our extremely dis-
tant ancestors.  (We shall consider the evidence for the antiquity of
some of our own genes in a later chapter.)  Although evolution of the
sort undergone by the Gryphaea (page 7) is generally referred to as
microevolution, such a process is many orders of magnitude more
complex than the process that can be demonstrated in favorable test
objects like bacteriophages. With bacteriophage we may detect
"ultramicroevolutionary" changes caused by even extremely infre-
quent single-gene mutations. The morphology and general physiol-
ogy of these most elementary "organisms" are discussed in the fol-
lowing pages, together with a consideration of the progress of "fine-

67


66

TilE MOLECULAR BASIS OF EVOLUTION

Structure genetics" which has been so stimulated by work on bac-
teriophages and on their bacterial cell hosts.
Bacteriophages were discovered by D'Herelle in 1917.  He demon-
strated that these were extremely small particles, invisible by ordi-
nary microscopy, which multiply parasitically within their host and
escape in a burst when the bacterial cell wall ruptures. Later
studies by a number of investigators, among whom Delbriick,
Demerec, Luria, and Burnet deserve particular mention, showed that
the bacteriophage bacterial cell microcosm exhibited many elegant ex-
amples of evolutionary adaptation.
A particularly cogent example is presented by the case of the r
mutants of bacteriophage T4. Each wild-type T4 particle produces
a small, fuzzy plaque (Figure 34), when grown on agar containing
E. coli of strain B. This plaque type is one of the phenotypic char-
acters of this particular phage.  From time to time these wild T4
undergo a mutation which introduces a new phenotypic character.
Thus we may isolate, from individual plaques of wild T4, mutants
that produce quite a different sort of plaque, nine of which are shown
in Figure 34. In spite of the identical appearance of plaques pro-
duced by all these r mutants on E. co& B, we may easily distinguish
several different sorts of r mutants by employing a new bacterial
host. Thus, as shown in Figure 35, when the nine r mutants are
plated on E. co2i K rather than B, three subgroups become evident;
two of the mutants still form r-type plaques, one forms wild-type
plaques, and six do not multiply at all.  Whereas on strain B all the
mutants produce a visible effect, on K some are visible (the r
plaques), some are "innocuous" (wild type), and some are lethal.
Escherichia coli B, therefore, supplies something which is not sup-
plied by K, at least for the latter r mutants. In a sense, K may be
thought of as an ecological niche which is not attainable by these
"species" of T4 bacteriophages until a mutation has occurred which
makes the "something" in B no longer of importance.

4                          -

Figure 34. Plaques formed on E. toll B by wild-type bacteriophage T4 (left)
and by nine independently arising r mutants.  As described in the text, wild-
Type T4 particles infect and multiply within E. coli cells of strain B to produce
the small plaques shown. The progeny infect and lyse neighboring bacterial
cells to produce plaques, which are actually areas in the E. coZi B-agar mixture
that have become translucent owing to lysis of the bacterial suspension; r mu-
tants produce the large plaques shown in the nine photographs at the right of the
figure. The plaques may easily be distinguished from those produced by the
wild-type bacteriophage particles by their characteristic plaque morphology.
From S. Benzer, The Chetnical Basis of Heredity, Johns Hopkins Press, 1957.

THE SUBSTRUCTURE OF GENES                                   69


70

THE MOLECULAR BASIS OF EVOLUTION

Another point of evolutionary interest can be illustrated by these
observations. The r mutants which behave like wild-type phage on
E. coli K are not wild type as is shown by their behavior on B.  In
terms of evolutionary adaptation, these particles are very much analo-
gous to the reptiles which we discussed in Chapter 1. The reader
will recall that changes occurred in the structure of the jaw of
reptiles which were not of apparent adaptive value until millions
of years later when certain of the bones of the reptilian jaw became
important units in the assembly of the characteristic mammalian ear.
These changes (call them "aristogenic" if you like) could not bc
taken advantage of until the proper ecological situations made their
presence of great value to the budding mammal. The "false" wild-
type phage mutants, in a similar manner, are the possessors of muta-
tions which are, at least superficially, neither "detrimental" nor "ad-
vantageous" in a world of E. coli K, but which facilitate a marked
change in phenotype in a world of E. coli B. These r mutations
might be preserved for a long time, to become of critical importance
*hen environmental (host cell) conditions accidentally changed from
K to B. More likely, the abnormal gene would eventually back-
mutate to the normal allelic form and be forgotten as an evolutionary
ripple.

The Chemistry and Enzymology of Bacteriophages

Although a number of different sorts of bacteriophages have been
studied with respect to their chemical composition, morphology, and
genetic constitution, we shall restrict our discussion to the so-called
T-even (and particularly T2) phages since these have been most
commonly chosen for study by virologists and are by far the best
understood. These viruses, which infect and kill cells of E. coli
B, may be divided into major groups on the basis of their ability
or inability to invade E. coli B cells of certain types. Thus E. co/i
B6 will support growth of T2 and T4 viruses, B4 will support T2 and
T6 but not T4, and so on. They may be further distinguished on the
basis of their plaque morphology, the rate at which such plaques

Figure 35. When the nine r mutants, shown in Figure 34, are plated on E. coli
K rather than on strain B, three subgroups become dis*inguishable.  ,Two mutallt\
continue to form plaques similar to those formed on E. co2l B, one forms wiltl-
type plaques, and six do not multiply at all. From S. Benzer, The Chemicd
Busis of Heredity, Johns Hopkins Press, 1957.

THE SUBSTRUCTURE OF GENES                                     71


(a)

(b)

Figure 36. Electron photomicrographs of (a) bacteriophage T2 and of (b)
"ghosts" prepared by osmotic shock. These photographs were obtained through
the kindness of Dr. Roger Herriott. From R. M. Herriott and J. L. Barlow.
J. Gen. Physiol., 40, 809 ( 1957).

become visible under standard conditions of agar plate culture, and
their specific serological characteristics.

The virus particles can be visualized in the electron microscope,
and the morphology of T2 coliphage, which is more or less typical
for all the T phages, is shown in Figure 36. The particles are made
up of a rounded hexagonal head and a tail. The tip of the tail is
slightly expanded into a knob-like structure. This can be deduced
from the outlines of some of the shadows in the electron micrograph.

Intact phage particles consist of approximately 60 per cent protein
and 40 per cent DNA, together with traces of lipid material. The
nucleic acid component is entirely of the DNA variety and, as dis-
cussed earlier, contains the unusual pyrimidine 5hydroxymethylcyto-
sine partly in glucosidic combination with glucose. It was believed
for some time that the DNA and protein were tightly conjugated in
nucleoprotein form.  It can be shown, however, that the DNA is
actually physically enclosed within a protein shell, the so-called
bacteriophage "ghost," and can be released from the interior of
the particle by suitable treatment, whereupon it becomes digestible

72                            THE MOLECULAR BASIS OF EVOLUTION

by deoxyribonuclease. In Figure 36 is shown a collection of such
ghosts which have been prepared by submitting virus particles to
"osmotic shock," that is, rapid dispersion into distilled water after
prior equilibration with strong salt solution. The ghosts may be
prepared in a form essentially devoid of polynucleotide by propel
washing. The separability of protein and DNA is elegantly demon-
strated by the fact that, when phages are doubly labeled with PnZ
and S36, tie PS2 is found entirely in the DNA released by "shock"
and the S3" in the material of the ghosts.
Doubly labeled phage have also been used by Hershey and Chase
to study the fate of the various components of phage during infection
and rupture of bacterial cells. These investigators allowed the
labeled particles to infect E. coli cells and, after a brief period, ex-
posed the mixture to rapid agitation in a Waring Blendor. The
P32-labeled DNA was fiound entirely within tihe infected bacterial
cells, whereas a large part of the protein was sheared off.  The small
amount of S35-labeled protein which adhered to the bacterial cells
after the agitation procedure did not contribute to the structure of
the progeny phage produced when the infected cells were allowed to
incubate and rupture spontaneously.  Since the infected cells which
were exposed to the blender produced a normal yield of progeny,
it may be concluded that DNA alone is capable of directing the
synthesis of new particles, genotypically identical with the parent
phage, and that the protein shell around the DNA makes no contri-
bution to the process of genetic information transfer.
ing any genetic role,              Although lack-
        the protein ghost is responsible for many
features of bacteriophage infection in addition to its function as an
enclosure for DNA. Ghosts can attach to compatible host cells or
cell walls, that is, they bear the host specificity of the intact virus (see
Chapter 8). Cells which have been "infected" with ghosts are
killed and subsequently lyse, although no progeny phage are pro-
duced. Ghosts also have the mysterious ability to cause inhibition
of RNA synthesis and protein synthesis within the host cell.
It has been known for some time that small molecular fragments
of the bacterial cell wall are released shortly after infection by an
enzyme present in both intact viruses and ghosts.  This material has
been identified as a mucopeptide derivative having the interesting
structure shown in Figure 37. Now, although the nature of the
natural substrate for the enzyme, lysozyme, is not known with pre-
cision, a number of observations suggest that the function of lysozymc:
is to attack mucopolysaccharide or mucoprotein compounds. It will
degrade chitin, for example, which is a long-chained poly-N-ace@
glucosamine, although it will not attack this substance after de-

THE SUBSTRUCTURE OF GENES                                     73


l-----------.---1
N-acetylglucosamine portion  I
                    I

i?     I
        I
IjH-C-CH, f

(To preceding
of N-acetylmuramic
    acid)

/c------ ------ --,
  N-acetylmuramic acid portion   1
                     I

0-(To next residue of

,

      Ho
   CHa-CH-C-(Peptide containing
             alanine, glutamic acid,
             and diaminopimellic
L _____ -----aff??!!Y!---J

Figure 37. A proposed structure for the fragment which is produced from the
cell walls of certain Cram-positive bacteria by digestion with lysozyme.  The
arrows `indicate the probable site of attack by the enzyme. Redrawn from
W. Brumfitt, A. C. Wardlaw, and J. T. Park, Nature, 181, 1783 ( 1958 ).

acetylation. The chemical nature of the fragment split off from the
cell wall is sufficiently reminiscent of chitin to suggest that the viral
enzyme responsible for bacterial penetration is, indeed, of the lyso-
zyme variety. Dreyer and Koch' have recently compared the action
of crystalline egg white lysozyme and virus ghosts on bacterial cell
walls and have found that a very similar cleavage product is released
by both catalytic agents. Further, the enzymatic activity in the
ghosts distributes in the same way as the egg white enzyme during
purification with the specific adsorbant, Bentonite, and can be
chromatographed on the ion exchange resin, XE-64, to give a similar
elution pattern. Chemical comparison of the two lysozymes has not
yet been made, so we cannot state 6ategorically that the virus enzyme
has structural features in common tiith the egg white enzyme. The
chromatographic properties and similar molecular size of the phage
enzyme (slowly dialyzable through cellophane membranes) makes
this similarity rather likely, however, and sequential analysis should
give very interesting results. It will be of obvious importance, in
connection with the "age" of genes and with the evolutionary
process in general, to determine whether or not an enzyme designed

74                          THE MOLECULAR BASIS OF EVOLUTION

to digest cell wall material is an essential component even in "sem-
living" biological systems like the coliphages.

Morphology of 1 Phages

Some idea of the number of morphological components making up
the bacteriophage particle may be obtained by electron microscopic

Figure 38. An electron photomicrograph of a crude lysate of bacteriophage T2
prepared by treatment of infected E. coli cells with chlorbform. The photo-
graph shows intact bacteriophage particles and a collection of "heads," rods, nntl
filaments. This photograph was obtained through the kindness of Dr. E. Keller-
berger of the University of Geneva.

THE SUBSTRUCTURE OF GENES                                     75


Figure 39. An electron photomicrograph of partially purified rods from the lysate
shown in Figure 38. ~Many of these rods may be seen to terminate in a brush-
like arrangement. This photograph was obtained through the kindness of Dr.
E. Kellenberger of the University of Geneva.

examination of a phage which has been subjected to various degrada-
tive treatments (peroxide, cadmium cyanide, N-ethylmaleimide)
which split off from the body of the phage certain parts of the tail
structure. We may also examine the contents of artificially lysed
bacterial cells which are still in the process of synthesizing phage.
The electron micrograph shown in Figure 38, for example, was made
by Kellenberger and Sechaud on the crude lysate of T2 prepared by
treatment of infected E. coli cells with chloroform. The photograph
shows, in addition to intact phages, a collection of empty heads,
rods, and filaments, the latter very likely being strands of DNA
which have not yet been wrapped in their protein envelope. These
phage components may be concentrated as shown in Figure 39.  The
rods, which must constitute a large part of the tail structure, are
seen in many cases to terminate in a wire-brush-like arrangement.
In some electron micrographs we can distinguish rod-like structures
which are somewhat smaller in diameter than the intact viral tail

76                          THE MOLECULAR BASIS OF EVOLUTION

and which have been interpreted as a sort of plug in the tail which
confines the DNA to the head of the phage. Two other phage com-
ponents, not visualized by the electron microscope, are the pene-
tration enzyme (lysozyme?) and the protein material responsible
for host range specificity. The latter may be associated with one
of the morphologically distinguishable tail components.
The drawing in Figure 40 summarizes, in a schematic way, the
subunits of structure which have been detected microscopically.
A slightly different reconstruction has been proposed by Kozloff,
Lute, and Henderson.* This diagram helps to point out one of the
major puzzles of bacteriophage biosynthesis, namely, the problem of
how the DNA strand or strands are gotten into the preformed heads,
if these are indeed synthesized before DNA is. We might visualize
that either `an enzyme system, rich in genetic information, is able to

700 A.

Figure 40.
particle.  A schematic reconstruction of the morphology of- a bacteriophage
   The dimensions shown are approximate and are averaged from several
sources (see, for example, the excellent work of R. C. Williams and D. Fraser,
Virology, 2, 289 ( 1956).

THE SUBSTRUCTURE OF GENES                                     77


carry out DNA synthesis within the head (perhaps in cooperation
with the protein of the head itself) or that DNA can thread itself
into the head after synthesis on the outside. The possibility must not
be overlooked, however, that phage fragments really represent abor-
tive attempts at synthesis and that the protein shell and tail of com-
pleted phage particles are assembled around a preformed mass of
DNA. The latter possibility is supported by a certain amount of
experimental evidence and certainly makes the best sense, meta-
bolically and mechanically. For example, Hershey has shown with
isotopic tracer methods that the DNA of phage is synthesized before
the protein precursors and that the small amount of protein synthesis
which occurs before DNA synthesis may form some essential enzymes
(perhaps for the synthesis of the unique pyrimidine, or for the for-
mation of a ribonucleic acid "template") which are not otherwise
present in the bacterial host.

The Molecular Heterogeneity of Coliphage DNA

The total phosphorus content of a single T2-phage particle is
about 2 X 1047 grams. If all this phosphorus resides in a single
DNA molecule, we may calculate the total molecular weight of this
DNA to be approximately 110 million. Should the Watson-Crick
type of structure apply in phage, we are faced with the problem of
how each enormously long polynucleotide strand becomes replicated
during phage multiplication and how the two intertwined strands in
the double helix unwind during, or in preparation for, this event.

This problem has been somewhat simplified by the observations of
Levinthal and Thomas.3 Their experiments indicate that the DNA of
phage may not be in a single large unit but may be made up of
a number of smaller parts, the largest one of which makes up about
40 per cent of the DNA. The technique employed by them is a
particularly elegant example of simplicity combined with sensitivity
and precision. Levinthal and Thomas embedded bacteriophage par-
tioles, previously labeled with Ps2, in an electron-sensitive emulsion
in which the. passage of fast electrons, such as are emitted by Pan
during radioactive decay, causes the formation of a track of silver
grains. A single phage particle, acting as a "point source," can thus
cause the formation of a "star" in the emulsion, each arm of which
is produced by the decay of a single atom of phosphorus within
the DNA of the particle (Figure 41). The sensitivity of the detect-
ing method is such that the radioactivity of a phage particle, emitting

70                         THE MOLECULAR BASIS OF EVOLUTION

Figure 4 1. Photomicrograph of a "star" produced by radioactive decay of labeled
phosphorus within the DNA of a phage particle embedded in a photographic
emulsion. This photograph was obtained through the kindness of Professor Cyrus
Levinthal, Massachusetts Institute of Technology.

only about fifteen disintegrations per month, can be estimated. In
uniformly labeled electron sources (e.g. the DNA of a single phage
particle or fragments thereof) the number of tracks per star gives a
measure of the molecular size of the emitting source, relative to the
intact phage particle.

The number of tracks per star was determined for intact phage and
for first- and second-generation progeny before and after osmotic
shock. The results for the various fractions are shown in Table 2,
expressed in terms of the number of P32 atoms in the star-forming
particle, normalized to 100 for the original uniformly labeled phage.
(Put in another way, if each star formed by intact parent phage
particles has 100 tracks, each star produced by particles in suspen-
sions of "shocked" phage, having half the content of- DNA, will show
50 tracks.) The results indicate that (a) there is a single large
piece of DNA comprising about 40 per cent of the total DNA (i.e.,

THE SUBSTRUCTURE OF GENES                                     79


about 45 million in molecular weight), (b) after one generation in
nonradioactive bacteria there are, among the progeny, a few particles
in which this large piece of DNA has approximately half the P3*
atoms of the large piece in the parent phage, and (c) after a second
generation the size of this large piece does not change (the same
number of tracks per star is observed). Since some 100 to 200
progeny are formed from each parental phage, the number of labeled
particles in second generation shockates was too few to permit accu-
rate detection and track counting.

TABLE 2

The Number of Arms per "Star" for Various Suspensions of Intact or
Ruptured Phage Particles, Normalized to 100 for the Original, Intact
Par$cles3

Intact         After
Phage      Osmotic Shock

this line looked quite encouraging but, of late, the picture has become
rather murky and, after consultation with some of the investigators in
the field, it appears that the subject is best avoided at the present
time. There are, for example, several lines of evidence which
suggest rather strongly that the "40 per cent piece" is not a single
molecule but that various mild treatments can cause this aggregate to
dissociate into still smaller units having molecular weights in the
neighborhood of 12 to 15 million. The results of the original "star"
experiments seem to be unequivocal and, indeed, chromatographic
separation on special columns of ion exchange resin have also shown
the presence in T2 DNA of an entity containing about 40 per cent
of the total phosphorus. Nevertheless, any attempt to relate the
chemical structure of phage DNA to genetic behavior must be de-
ferred until much more data have been obtained.

The life Cycle and Biosynthesis of T Phages

Uniformly labeled parental phage         100          40 f 4
First-generation progeny            924 It 3         23 IL- 3
Second-generation progeny           26 f 3   Too dilute for study

The simplest and most appealing interpretation of these data is
that the large piece represents the "chromosome" of the phage and
that the two halves of its double helical structure serve as templates
for the synthesis of new DNA in the progeny. This wishful picture
is completely analogous to the scheme growing out of the Meselson
and Stahl experiments on E. coli DNA. There is, however, no com-
pelling evidence to indicate that the DNA of the T phages is in
the form of a double helix, although this is generally assumed in
most discussions of the subject for the obvious reason that it makes
discussion possible.

The Levinthal-Thomas experiments suggest a "conservative" mecha-
nism of replication during which the structure of the large piece of
DNA is not degraded or involved in exchange with external sources
of nucleotides.

It would have been pleasant, in connection with this discussion of
these experiments, to have been able to present some evidence for
the chromosome-like nature of the "big piece." Attempts have been
made to show that, during the production of progeny phage, genetic
markers, chosen at random along the genetic strand, always accom-
pany the large star-forming fragment. The early experiments along

a0

THE MOLECULAR BASIS OF EVOLUTION

It may be useful at this point to consider the events that occur
following infection of E. coli with T phages.  Most of the metabolic
consequences of infection are summarized in Figure 42, which is a
slightly modified form of that given by R. Hotchkiss in his excellent
review of 1954, and in Figure 43, taken from an article by L. Kozloff
in the Cold Spring Harbor Symposia for 1953. Figure 42 gives a
condensed view of the fates of various morphological components of
the infecting phage and the host cell and also indicates, in a rough
way, the manner in which the metabolism of the combined DNA
protein, and ribonucleic acids of the infected cell is altered. It iS
assumed that the RNA of the host is, in some way, involved in the
biosynthesis of phage protein. This assumption is based on certain
general observations on protein synthesis which we shall discuss in
more detail in Chapter 10.

Kozloffs pictorial summary of the origins of phage DNA and pro-
tein (Figure 43) emphasizes that the major share of phage protein is
derived from the nutrient medium. Of the newly synthesized phagc
DNA, it would appear that most of the phosphorus is also exogenous
in origin, but that a considerable amount of host DNA is reutilized
as a source of purines and pyrimidines.

The kinetics of the various metabolic processes that commence after
phage infection are of particular importance in connection with the
elucidation of the way in which the genetic information in phage
DNA is translated into the chemical structure of progeny phage.
THE SUBSTRUCTURE OF GENES                                    81


Phage

,DNA

-Protein

Not digested by deoxyribonuclease
until phage is damaged: 40-50s
of weight of phage.

Contains all phage sulfur, host
range specificity, penetration en-
zyme, killing power.

Host
cell

DNA

RNA

Nuclear DNA of host.

About three times as much as
DNA.

Protein

Enzymes of normal cell,  Synthe-
sis of new enzymes may be induced
by proper substrates.

-Protein

80% or more removed by Waring
Blendor.

Cell
DNA

Cytologically disorganized. Syn-
thesis stops.

Cell
protein

Normal synthesis stops.  New sys-
tems develop.

,Cell    Only slight turnover after infec-
RNA   tion.

Phage DNA now accessible to
DNase if bacteria are ruptured:
40-50% goes to progeny.

I

DNA   !ZO-35% of P derived from host

DNA

Protei,,

Mostly from endogenous sources
of amino acids.  Same host rnnge

RS parent phage.

Phage ghost still attached.

02                     THE MOLECULAR BASIS OF EVOLUTION

When the host cell becomes infected, its metabolic machinery ap-
pears to become completely devoted to phage synthesis. Protein
synthesis goes on but produces a new set of proteins.  After a short
delay, DNA synthesis proceeds actively. However, if the initial pro-
tein synthesis is prevented by the addition of a suitable inhibitor
such as chloramphenicol, the synthesis of DNA does not take place.
On the other hand, if the inhibitor is added after protein synthesis
has gotten under way, DNA synthesis can proceed. These observa-
tions suggest that an essential set of enzymes must first be formed,
perhaps for the synthesis of the unique 5hydroxymethylcytosine or
its glucose conjugate, before DNA assembly is possible.
Stent and his colleagues have carried out interesting experiments4
which have a direct bearing on the problem of the order of events
during information transfer. They infected Paz-labeled cells with
P324abeled phage, both having a sufficiently high level of isotope
to cause chemical modification of a significant fraction of the DNA
when the phosphorus atoms decayed to form sulfur atoms.  The in-
fected cells were incubated in a Pa2 medium so that tihe progeny
phage were also heavily labeled. Samples of infected cells were
frozen at -196OC. at various times after infection and were stored
in this state of suspended animation.
took place.             During this time Paa decay
    The surprising result was obtained that, in those samples
frozen approximately 10 minutes after infection, there was no de-
tectable change in the ability of the infected cells to produce infective
phage progeny in spite of the destruction of a considerable portion
of the DNA by radioactive "suicide." The results have been in-
terpreted in several ways, the most provocative being the hypothesis
that, early in the synthesis of progeny, information in the DNA of
the infecting phage particle is transferred to some substance, perhaps
protein in nature, which is not susceptible to radioactive decay.  An-
other speculation which could explain the results would require that
the structure of DNA be such that, in spite of occasional breaks in the
sugar-phosphate backbone of the molecule caused by Ps2 decay, hy-
drogen bonds hold the macromolecule intact and in a configuration
adequate for purposes of replication ( Figure 44 ).
The possible conflicts in logic that might arise from a comparison
of the I?-deoay data with the experiments of Levinthal and Thomas
or with the mass of evidence supporting the central role of a specific
4

Figure 42. The course of infection of E. co2i with bacteriophages of the T series,
Redrawn after R. D. Hotchkiss, The Nucleic Acids, volume 2, Academic Press,
1954.

THE SUBSTRUCTURE OF GENES                                    83


Liquid medium

p&O.fl 70-80%

C&-, 75-85%

P-J&,+, 75-8596

Figure 43. Schematic representation of the origin of the phosphorus, nitrogen,
and carbon of bacteriophages T2, T4, and T6. Percentages show origin of
material found in viral offspring. Redrawn from L. N. Kozloff, Cold Spring
Harbor Symposiu Qwzn. Biol., I 8,209 ( 1953).

DNA structure in the determination of heredity are not, to my
knowledge near resolution. Fortunately for the following discussion
of genetics in phage, we do not need an answer to the fascinating
problem of phage replication. Although the behavior of phage dur-
ing "mating" may only superficially resemble the process as it occurs
in more respectable organisms, the phenomena of recombination and
segregation, and of linkage, are exhibited in qualitatively the same
way.

Genetic Mapping in Bacteriophage

In most organisms it is not possible to construct a single gene
map, but instead we find that the various genes fall into several link-
age groups (see Chapter 2). However, in bacteriophage T4 and T2,
for which quite a few "genes" have been mapped, only a single

04                           THE MOLECULAR BASIS OF EVOLUTION

linkage group seems to be present, and these phages behave geneti-
cally as an organism with a single haploid chromosome. The sort
of gene map obtained depends, naturally, on the strain of bacteria
chosen as host. As we have already shown, the plaque characteristics
of mutants that are r type on E. coli B may be quite different when
the mutants are plated in E. coli K. The map obtained with a par-
ticular host cell, therefore, includes only the loci that cause a detect-
able change in phenotype in this chosen environment.
The construction of a genetic map for bacteriophage involves more
or less the same manipulations that have been used for such or-
ganisms as Drosophila or peas. A wild-type strain is chosen arbi-

Figure 44.  A schematic representation
of how breaks in the chains of the DNA
dougle helix, caused by radioactive de-
cay of P3a, might occur and still permit
the maintenance of a relatively intact
structure through the forces of hydrogen
bonding between complementary pairs
of purine and pyrimidine bases.

THE SUBSTRUCTURE OF GENES

"Nonlethal dec

ay"


trarily. Mutants are then chosen on the basis of some easily recog-
nized phenotypic character such as plaque morphology on a particular
strain of E. coli. The relative position of two mutant loci on the
"chromosome" of the phage may then be established by "mating"
the mutants and observing the proportion of the progeny that differ
from the two parental mutants. True mating does not, of course,
occur with phage. Instead, bacterial cells are mixedly infected with
the two phages in question, and recombination is allowed to take
place within the cell by some obscure process that has the superficial
characteristics of crossing over in higher organisms.  As we discussed
earlier, conventional crossing over results in the production of the
two possible recombinants, the wild type and the double recombinant
containing both mutant loci. It has been shown that, in phage, such
reciprocal crossover does not take place, but that in any single re-
combination event only one or the other recombinant is formed
[perhaps because of a "copy-choice" replication (Figure 45)].  How-
ever, since a single phage particle gives rise to several hundred
progeny, and since there appears to be about the same probability
for the formation of the two kinds of recombinants, the usual statisti-
cal treatment may be applied.

Seymour Benzef has, over the past few years, been in the process
of mapping a very large number of r mutants of bacteriophage T4.
These studies represent the first real attempt to determine the ulti-
mate limits of recombination and involve "running the map into the
ground," to use an expression attributed by Benzer to Max Delbriick.
The detection and mapping of mutations in bacteriophages are facili-
tated by several factors, including the sensitivity of the technique for
detecting mutants and the occurrence of certain mutations which
involve the "deletion" of a considerable portion of the map. The
latter factor, as we shall see, has permitted Benzer to group many
point mutations into "families," thus greatly decreasing the number
of crosses required.

In any population of T4 there occurs from time to time a spon-
taneous mutation which leads to the production of an r-type plaque
on E. coli B agar plates. Since an astronomical number of phage
particles may be plated on a single Petri dish surface, the presence
of a single mutant in as many as 109 particles is easily observed by
its r phenotype.  When a suitable number of such mutants has been
accumulated, they may be further subdivided into three subgroups
as mentioned earlier, by plating on E. coli K.  By this trick (see page
69) we may distinguish those that still show r morphology on K,
those that show the wild-t);;pe phenotype, and those that fail to grow

06                            THE MOLECULAR BASIS OF EVOLUTION

   A                (2)                    B
+                                                   +
T

+                                                   +

a                                      b

Figure 45. Three possible mechanisms of genetic recombination: A and B repre-
sent two genetic loci and o and b their alleles.  ( 1) Fragmenting crossing over;
the two parental duplexes synapse and break between the loci in question, and
heterologous pieces rejoin. (2) Nonfragmenting copy choice. (3) Fragmenting
copy choice; parent duplexes unwind and semiconservative replication occurs
along the single strands. A switchover occurs between loci A and B as the
parent chains break, and the daughter chains continue to grow complementary to
a single chain of the second duplex.  Redrawn from M. Delbriick and G. Stat
The Chemical Basis of Heredity, Johns Hopkins Press, 1957.

THE SUBSTRUCTURE OF GENES

87


Bacterial
host
yn

Figure 46.  The order of the three subunits of the r region in the genetic material
of bacteriophage T4. Since rI1 and rII1 are farther apart than 711 and r1, the
frequency of recombination between rI1 and rII1 mutants should be greater than
that between rI1 and rI mutants.

at all. These three groups, termed the r1, rI1, and rII1 categories,
respectively, may be arranged in the proper linear order by perform-
ing mixed-infection crosses. As may be deduced from the scheme
in Figure 46, the frequency of recombination between members of
the rI1 and rII1 groups would be greater than between mutants of
the rI1 and r1 varieties, making the usual assumption of a linear ar-
rangement of "genes."

Benzer has chosen to concentrate mainly on the rI1 region for his
genetic analysis. The rI1 mutants have one clear advantage from
the point of view of detection, in that they do not grow on K at all
and therefore give an unequivocal phenotypic test. Some thousand
mutants of the rI1 type could thus easily be selected for the mapping
project.

The rI1 mutants were further subdivided on the basis of the cktrans
test. It will be recalled that this genetic trick enables us to de-
termine whether or not two mutants which show a similar phenotypic
abnormality are concerned with the same functional unit of heredity
or with two separate functions which interact, cooperatively, in pro-
ducing the change.  As we discussed in connection with the "lozenge"
genes of Drosophila (Chapter 2), three closely linked genetic loci all
appeared to be part of the same functional unit since crosses between
double heterozygotes bearing both mutant loci on the same strand
of the chromosome duplex (i.e., in the ci.s arrangement) produced
wild-type recombinants, whereas crosses between double heterozy-
gotes in the truns arrangement did not. The adequacy of the cis
arrangement was explained on the assumption that the one, un-
marred, strand of the chromosome supplied the required unit of
physiological function in sufficient quantity to satisfy the needs of the
organism even in the presence of one "nonfunctional" chromatid.

In phage, alth ough the genetic material behaves as though the
organism were haploid, the cis-truns test can still be applied since
mixed infection with two mutants appears to simulate the more con-
ventional diploid situation in higher organisms.  Thus, when a mutant

88                            THE MOLECULAR BASIS OF EVOLUTION

phage containing an r mutation is introduced into an E. coli K host
cell along with a wild-type phage particle, the cells are lysed and
both kinds of parent phage are liberated. The presence of the wild-
type presumably supplies the function missing in the r mutant (the
rI1 mutation is "recessive"). Although the test has not been made,
it is also assumed that mixed infection with a wild type, and with
a double r mutant bearing both mutations on the same strand, would
permit the formation of both sorts of progeny, and for the same
reason. When two r mutants are used, however, lysis of host cells
and progeny production will take place only if the two mutants are
deficient in different functiona units. The various situations are
schematized in Figure 47.  When two r mutants fail to lyse E. coli K
following mixed infection, the mutations are said to belong to the
same "cistron" (the name derived from the test employed). When
they supplement one another and cause lysis, they are said to belong
to different cistrons. By this sort of technique, Benzer divided the
rI1 mutants into two functionally different groups which he names
the A and B cistrons (Figure 48). (We might visualize that each
of the various phage-synthesizing enzymes, whose formation is in-
duced in bacteria by phage infection, is represented by a different
cistron. )

Now it is clear that to map a large number of mutant loci, even
after such further subdivision, would require an impossible number
of crosses, and Benzer has taken advantage of the fact that some of
the mutations appear to involve a change in much more than a point
locus. These unusual mutations are detected by the fact that, when

/".f pq [;:I]
   Active           Active       Presumed active

   //I: II
           Inactive         Inactive          Active

Figure 47.  Schematic diagram showing the results of ci.s and truns configurations
of double "heterozygotes" bearing two rI1 mutations. Actiue means extensive
lysis of the doubly infected cells.  lnactiue means very little lysis.  The presence
of one "unmarred" strand is pictured as being sufficient to supply the required
unit of physiological function.

THE SUBSTRUCTURE OF GENES                                     a9


A segment

----._ --._ --._
B segment    .-

    /.                                            -\
  /'                 164                         `\
//'  173      240      279  271    274  201           106
    I        I         I I        II              I
                                  \
                      IN (0'        \

                  .' cj              \ \
                /'                    \

            .M .-                  `\
          /I                        `\
      .I /c                             `\
    .r                                 `\

m = minute plaque mutant
r = raoid lvsis mutant

tu = tuibidblaque mutant

Figure 48. A partial linkage map of T4 bacteriophage. The successive drawings
indicate increasing orders of magnification of the t-11 region as revealed by the
fine-structure analysis of this region. This figure is highly schematic, and for de-
tails of its construction the reader should consult the article by S. Benzer in
"Mutation," Brookhaven Symfrosiu fn Biol., No. 8 (1956). Redrawn by permis-
sion of the author.

they are crossed with several other mutants which give wild-type
recombinants when crossed with one another, no detectable wild-
type recombinants are observed. The situation is explainable on the
basis of tbe chart shown in Figure 49. If mutant 6 represents a
"deletion" type of mutation, and 1 through 5 represent various other
ordinary point mutations, crosses between 6 and either 2, 3, or 4
will not yield wild-type recombinants since 6 is deficient in the same
properties that are missing in the other three. Wild-type recom-
binants could, however, be obtained from crosses between 6 and
either of 1 and 5 since there is no overlap. Using "deletion" mutants
for further screening within each of the two cistrons, Benzer was
able to simplify the task of mapping since it was then necessary to
perform crosses in all combinations only with the mutants that fell
within any given deletion region, rather than with all the mutants
in each cistron. We shall not describe, in detail, this part of the
mapping project. In spite of the shortcuts devised, the job was ob-
viously extremely laborious. The schematic map shown in Figure
50 was constructed from crossover data obtained on some of the
923 I mutants and shows, in addition to the location of "point" muta-
tions, the location of various of the deletion mutants (the horizontal
bars or lines) which were of such value in mapping. The map also

Figure 49. Schematic diagram showing the behavior of an "anomalous" mutant.
Mutant 6 fails to give wild-type recombinants with mutants 2, 3 and 4 located
within the same segment of the genetic strand. Wild-type recombinants are
given, however, with 1 and 5.

THE MOLECULAR BASIS OF EVOLUTION         THE SUBSTRUCTURE OF GENES                                    91

90


92

incorporates the observation, made by Benzer, that a great many
separate mutations turn out to be in the same location. In other
words, some regions of the genetic material of the rI1 zone appeared
to possess a much higher mutability than others. The word "ap-
peared" is used advisedly since it must be remembered that the
detectability of a mutant depends on the phenotypic test. We have
seen, for example, that some T mutants behave on E. coli K as
though the mutation were lethal to the phage in this environment.
Others behave like wild-type phage. These classes are, of course,
not associated with the rII region as was just discussed. However,
we may postulate (and the postulation is supported in part by certain
observations which we shall discuss in Chapter 6) that the substances
whose synthesis is controlled by one or the other of the two cistrons
in the rII region contain some structural features of greater im-
portance than others. A mutation leading to modifications in the
functionally less critical parts of structure in a phenotypic protein
might be much less frequently detected since its influence. on the
"r-ness" of an r mutant growing on K might be slight in comparison
with the effects of the mutations that cause a complete inability to
grow on K; that is, those that have caused modifications in the chem-
istry of a phenotypic protein of a magnitude which completely
abolishes function.

The cktrrm, conceived of by Benzer as the genetic unit of func-
tion, is clearIy a very sophisticated structure. It contains many dis-
tinguishable genetic subunits as is indicated by the number of indi-
vidual loci, within its length, that can be detected by recombination.
Benzer, who like Levinthal is a converted physicist, has coined names
for the operational subunits within the cistron that have a delight-
fully physical ring.  The recon is defined as the smallest element in
the one-dimensional array that is interchangeable, but not divisible,
by recombination.  The muton is defined as the smallest element of
the cistron that, when altered, gives rise to a mutant form of the
organism.

The size of the recon can be estimated by isolating and mapping
so large a number of mutants within any given region that the dis-
tance between individual points on the map begin to approach the
indivisible unit. The recon is thus an empirical unit smaller than or
is equal to the smallest nonzero interval between pairs of mutants.
The length of the muton is determined by estimating the discrep-
ancy in map distances between closely linked sets of mutants. As
shown in Figure 51, the "length" of mutation 2 is equal to the dis-
crepancy between the long distance and the sum of the two short dis-

THE SUBSTRUCTURE OF GENES                                     93

THE MOLECULAR BASIS OF EVOLUTION


I               I         1x3          I     I
                               2x3

Figure 51.  Detemlining the "length" of a mutation.  The discrepancy between the
long distance and the sum of the two short distances measures the "length" of the
central mutation.

tances. The muton would also be measurable by determining the
maximum number of mutations, separable by recombination, that can
be packed into a portion of the map.

It must be emphasized that the genetic units proposed by Benzer
are completely operational in meaning and origin. They serve the
important function of pointing up the complexity of the word "gene"
by making a clear distinction between the several operational com-
ponents of this word, namely, recombination, mutation, and function.
Although it is far too early to suggest that the observations made on
bacteriophages may be directly applicable to higher organisms, the
results at least give a rational basis for the consideration of such
phenomena as pseudoallelism. More important, the high density of
mapping attained and the indications therefrom of the size of the
recon now make it possible to discuss chemical structure and genetics
in the same breath. By making assumptions about the chemical
length of the genetic strand of DNA in phage (perhaps on the order
of 80,000 nucleotide pairs, in a double helix) and the total "genetic"
length (perhaps on the order of 800 recombination units), Benzer
is able to suggest that a single nucleotide pair might correspond to
about 0.01 recombination units. Since this distance is of the same
magnitude as that observed between some of the most closely linked
loci on the map (about 0.02 units), it is possible that the techniques

Before leaving the subject of genetic fine structure, brief mention
must be made of another technique for mapping the substructure of
genes. This technique, discovered by Zinder and Lederberg5 in
1952, relies on a phenomenon known as transduction, in which ge-
netic information in bacterial chromosomes is transmitted from cell
to cell by bacteriophage particles, Certain bacteria may be in-
habited by so-called temperate bacteriophages which exist in a sym-
biotic arrangement with their host, wherein they only rarely lyse the
bacterial cell. When such lysis does occur, the liberated phage par-
ticles can infect new host cells and, in the process, carry with them
some of the genetic peculiarities of the original host.  Only a single
phenotypic character is generally transduced at a time, as though
the phage were carrying with it a single gene. However, there is
occasional transfer of more than one genetic marker in a bundle, and
such events enable the investigator to determine the closeness of link-
age between two or more functional units of heredity.

The explanation might be offered for transduction in general that
parts of the "chromosome" of the phage have exchanged with parts
of the bacterial chromosome and that, following the establishment of
a new symbiosis, this newly acquired information is unloaded on the
new host. Many characteristics of trclnsduction are highly reminis-
cent of transformution.  In both, transfer of genetic information ap-
pears to depend on the physical transfer of DNA, either packaged as
in phage or free. Both phenomena, although generally involving
only a single genetic marker, may occasionally involve several.
Transduction has been employed by a number of investigators for
the mapping of genetic material in several bacterial species and per-
mits a degree of discrimination comparable to that achieved by
Benzer through bacteriophage crossing. However, since the con-
cept of "fine-structure genetics" is quite elegantly illustrated by the
bacteriophalge approach, we shall defer to the excellent reviews listed
below and proceed to the next item of business, the protein molecule.

94                           THE MOLECULAR BASIS OF EVOLUTION         THE SUBSTRUCTURE OF GENES                                    95

employed have, indeed, been able to distinguish the genetic effect of
a modification in as few as two pairs of nucleotides in a DNA mole-
cule. If, as we may hope, the details of protein structure in an or-
ganism ,are the reflections of the chemical structure of its genetic
material, these calculations would suggest that a mutation may be
detectable in the phenotype when as little as a single amino acid re-
placement has occurred. The reader must view these speculations
with absolute candor. The hypothesis is under active experimental
test, and we must now rely on the protein chemist to help close the
gap between genetic speculation and chemical fact.


REFERENCES

1. W. J. Dreyer and G. Koch, Virology, 6,291 ( 1958).
2. L. M. Kozloff, M. Lute, and K. Henderson, J. Biol. Chem., 228, 511 (1957).
3. Discussed by C. Levinthal and C. A. Thomas in The Chemicul Basis of Heredity.
See Suggestions for Further Reading.
4. Summarized in The Chemical Basis of Heredity.  See Suggestions for Further
Reading.

5. N. D. Zinder and J. Lederberg, 1. Bacteriology, 64, 879 (1952).

SUGGESTIONS FOR FURTHER READING

The Chemical Beds of Heredity (W. D. McElroy and B. Glass, editors), Johns
Hopkins Press, Baltimore, 1957.

Cold Spring Harbor Symposia on Quantitative Biology, 21, "Genetic Mecha-
nisms: Structure and Function" ( 1958).

Cold Spring Harbor Symposia on Quantitative Biology, 17, "Viruses" ( 1953).

96

THE MOLRCULAR BASIS OF EVOLUTION


Figure 52. A model of the myoglobin molecule based on X-ray crystallographic
data. We can deduce from the dimensions of this model, and from the known
number of amino acid residues in myoglobin, that certain portions of the poly-
peptide chain of myoglobin must be coiled to form an internal secondary struc-
ture. The gray disk-like structure shown in the photograph represents the
porphyrin prosthetic group, and the light- and dark-colored spheres represent
heavy-metal derivatives which were attached to the protein to facilitate analysis
of the diffraction data.  Dr. J. C. Kendrew of the University of Cambridge
kindly furnished this photograph.

98                             THE MOLECULAR BASIS OF EVOLUTION

chapter 5

PROTEIN STRUCTURE

   T he study of the internal structure of proteins in the
crystalline form by means of X-ray crystallography indicates a well-
defined arrangement of atoms, more or less "frozen" into a definite:
pattern which is the same for each molecule in the crystal. The
crystallographer thus obtains a picture of the structure of a protein
which is rigid and essentially invariant (Figure 52). From the
purely chemical point of view such a picture is completely satisfac-
tory.  In living cells, however, proteins are not in the solid state but
are dissolved in the intracellular fluid and can be shown to be in 3
constant state of minor rearrangement in response to shifting hydro-
gen ion concentrations and salt levels and to reversible adsorption to
intracellular surfaces.

In considering such fluctuations it is convenient to think of the
chemical fabric of proteins in terms of three distinct categories, chris-
tened by K. Linderstrem-Lang-primary, secondary, and tertiary
structure.  The term primary structure refers to the fixed amino acid
sequence of the polypeptide chain (or chains) making up the b:lck-
bone of the molecule.  This category also includes those covalent
bonds that form fixed sites of cross-linkage such 3s the disulfidc
bonds between half-cystine residues and <the phosphate diester link-
ages of certain phosphoproteins.

The concept of secondary structure is based, to 3 large extent, on

                                                       99


R" H C
@--f

Figure 53.  The dimensions of a
fully extended polypeptide chain
as derived from X-ray crystallo-
graphic data. From R. B. Corey
and J, Donahue, J. Am. Chem.
Sot., 72, 2899 (1950).

Rise per
residue I

the relatively recent discovery of helical coiling in proteins, stabilized
by hydrogen bonds between the amide (CONH) linkages of the
chain. A number of helical arrangements of the polypeptide chain
may be constructed with atomic models which fit the geometrical
restrictions imposed by the bond angles and interatomic distances
that have been determined by the X-ray crystallographer on low mo-
lecular weight substances such as di- and tripeptides (Figure 53).
These various models differ in respect to the number of amino acid
residues per turn of helix, in the pitch of the helical screw, and in the
amount of unfilled space left within their centers.  Of all the helices
that have been seriously considered as components of protein struc-
ture, the so-called a-helix appears to be the most probable. In this
structure the maximum number of intrahelical hydrogen bonds are
formed between the CONH linkages, and the structure is conse-
quently a fairly stable one. Perhaps most important, the atoms com-
prising the peptide bond fall in one plane without strain.  The like-
lihood of such a plan,ar configuration is Ihigh as judged from crystal-

Formation of
a-helix from
linear chain

Figure 54. A schematic representation of the process of coiling an extended poly-
peptide chain into the a-helical configuration. Redrawn from L. Pauling and
R. B. Corey, Proc. Intern. Wool Textile Research Conf., 9, 249 (1955).

100                          THE MOLECULAR BASIS OF EVOLUTION           PROTEIN STRUCTURE                                         101

Dimensions of an whelk

5th
turn

4th
turn

3rd
turn

18 residues
21 A.

2nd
turn

1st
turn


lographic studies of model peptides like that shown in Figure 53.
Figure 54 illustrates the way in which the extended, "p-keratin" form
of the polypeptide chain is coiled into the a-helical configuration.
Each amide group is linked to the third amide group beyond by a
hydrogen bond. A complete turn of the helix contains 3.7 amino
acid residues, each residue contributing 1.47 A. of linear translation
along the central axis. The pitch of the "screw" is thus 5.44 A.
Recent physicoohemical studies on proteins and polypeptide

model substances have shown that, in spite of the stabilization given
by the internal hydrogen bonds, the helical structure is not suffi-
ciently stable to exist in solution but unfolds into a random, disori-
ented strand. Stabilization of the helical coiling requires the pres-
ence of disulfide bridges and/or tertiary structure, and it is probable
that only those parts of proteins which are properly anchored by such
bonds can maintain the helical configuration. Tertiary bonds in-
clude suuh examples as the van der Weals interactions, the agglom-
eration of lyophobic side chains by mutual repulsion of solvent, and
such special hydrogen bonds as might exist between the hydroxyl
groups of tyrosine or the r-amino groups of lysine and electronegative
groups elsewhere along the chain (Figure 55). The variations, re-
versible and irreversible, that are possible in the secondary and ter-
tiary structure of proteins are, basically, functions of the primary,

Figure 55.  Some types of noncovalent bonds which stabilize protein structure:
(a) Electrostatic interaction; (b) hydrogen bonding between tyrosine residues
and carboxylate groups on side chains; (c) interaction of nonpolar side chains
caused by the mutual repulsion of solvent; (d) van der Waals interactions.

102                           THE MOLECULAR BASIS OF EVOLUTION

covalent structure of the protein, which is fixed and invulnerable to
modification by ordinary environmenti fluctuations. Thus, a real
understanding of the physicochemical behavior of a protein in solution
must depend on a detailed knowledge of its structure in the organic
chemical sense,

To appreciate our present state of knowledge we must attempt to
view the problem from the position occupied by protein chemistry
some fifteen years ago. In spite of a large body of rather precise
data dealing with the behavior of proteins in solution, there existed a
sort of mystical aura around macromolecular structures arising, for
the most part, from the almost complete absence of accurate informa-
tion on amino acid content and arrangement. The necessary back-
ground for the modern approach was provided when Fred Sanger
and his colleagues* undertook the study of the insulin molecule and
demonstrated that there existed, in the structural analysis of at least
one protein, no insuperable chemical difficulties.  The confidence en-
gendered by Sanger's accomplishments and the techniques he em-
ployed have led to similar structural attacks on a number of other
proteins during the past five years. This accumulated experience
has now brought us to a point where we may, with good expecta-
tions of success, undertake the elucidation of the structure of nearly
any protein whose molecular weight is within reasonable limits. At
the present time, for example, complete sequential formulas are avail-
able for the insulins and adrenocorticotropic hormones of several
species, for bovine glucagon, and for the melanocyte-stimulating hor-
mones of pig and beef pituitary glands. The sequences of several
enzymes,
completed including ribonuclease, papain, and lysozyme, should be
   within a relatively short time, and many other biologically
active proteins are under study in a number of laboratories.
Although it is outside the scope of this book to discuss, with any
degree of completeness, the methodology involved in the determina-
tion of the sequence of amino acids in polypeptide chains, some con-
sideration of this subject will be of interest. In the following sum-
mary we shall assume the availability of protein samples of proven
homogeneity. A specfic consideration of the heterogeneity of indi-
vidual proteins, and of the implications of heterogeneity with regard
to protein synthesis and genetics,. will be made in subsequent
chapters.
For reasons of personal familiarity with the protein, I have chosen
as a model substance for the discussion of methodology the enzyme
ribonuclease. This enzyme, which has been found in all cells tested
so far, catalyzes a transphosphorylation reaction which leads to the

PROTEIN STRUCTURE                                         103


I I                 I I

6, ,c,
//p'OH
0

2'9 a'mhydride of
cytidylic acid

I   bH
om3H2

Cytidind-phosphate

Figure 56. A synthetic material which is hydrolyzed by ribonuclease.

hydrolysis of certain phosphate diester linkages in ribonucleic acid
involving pyrimidine nucleotide residues. The cleavage of a variety
of synthetic substrates, of the sort shown in Figure 56, is also cata-
lyzed by the enzyme and forms the basis for a convenient enzymatic
assay. Although its specific role in cell metabolism is, as yet, un-
known, it seems likely that ribonuclease plays an important part in
some aspect of the process of protein biosynthesis (see Chapter 10).
Large quantities of ribonuclease are formed and secreted by the pan-
creas, and the pancreatic enzyme must catalyze extensive hydrolysis
of ribonucleic acid in the intestine.

The structure of bovine pancreatic ribonuclease has been under
study for several years in two laboratories in which, fortunately for
the present illustrative purposes, rather different methods have been
employed toward the same end. As the first step in the study of the
structure of any protein, it is necessary to obtain an accurate set of
analytical data on the amino acid composition of the molecule.  Such
data were furnished for ribonuclease by Hirs, Moore, and Stein,z who
established, by careful chromatographic techniques, that the protein
contained 124 amino acid residues. Their results may be expressed
in terms of the formula:

Asprs, Glur2, Gly,, Ala,,, Vale, Leu,,
Ileu,, Ser,,, ThrIO, Cy!s,, Met,, Pro,,
Phe,, Tyr,, His,, Lys,,, Arg,, (NH,),,

where each amino acid is indicated by an abbreviation derived from
the first three letters of its name.  Most readers of this book will be
familiar with these abbreviated formulas, but for the benefit of those
who are not a complete list is given in Table 3.

To determine whether or not the 124 amino acids are arranged in
a single, or in multiple, cross-linked polypeptide chains, various
chemical and physical studies were carried out as summarized in
Figure 57.  Th e presence of as many as five cross-linked chains was
possible in ribonuclease since the molecule contained four cystine
residues. The protein was, therefore, oxidized with performic acid,
which converts the sulfur atoms in disulfide bonds into sulfonic acid
groups. Ultracentrifugation and viscosity measurements permitted
calculations which indicated that the oxidized protein had essentially
the same molecular weight as the native protein. The presence of
more than one chain would have been reflected in a marked lower-
ing of the average molecular weight as estimated by these physical
measurements.

104                           THE MOLECULAR BASIS OF EVOLUTION         PROTEIN STRUCTURE                                           105

TABLE 3
Amino Acid Composition of Bovine, Pancreatic Ribonuclease2

Amino
Acid

      Number
      Residues

         per
      Molecule
Abbrevi- (mol. wt.
ations"   13,683)

Amino
Acid

      Number
      Residues

         per
      Molecule
Abbrevi- (mol. wt.
ations   13,683)

Aspartic acid
Glutamic acid
Glycine
Alanine
Valine
Leucine
Isoleucine
Serine
Threonine

Half-cystine

(Asp)
(GM
WY)
(Ala)
(VaU
We4
(Ileu)
(Ser)
(Thr)

ds,

15
1%
3
12
9
a
3
15
10

8    Total number of residues

Methionine
Proline
Phenylalanine
Tyrosine
Histidine
Lysine
Arginine
Amide NHs

W-t)
(Pro)
@`he)
Cb)
(His)
(LYS)
(Art3

4
4
3
6
4
10
(li

134

Not present in ribonuclease are tryptophan (Try) and cysteine (CySH).
Cysteic acid (CySOsH) is formed by oxidation of cysteine and cystine, and
Met02 by oxidation of methionine.  Amide nitrogens are derived from the
side-chain carboxyl groups of glutamic acid and aspartic acid and are generally
indicated as Glu(NH2) and Asp(NHs), although abbreviated forms, such
as Gn and An, seem more convenient.

8 As suggested by E. Brand and J. T. Edsall, Ann. Rev. B&hem., 16, 224
(1947).


NHz

%%3

performic acid
oxidation

NHz                            I

NHzm--------

dinitrobenzene

Mol. wt.,

Valine

,
         ("DNF'-oxidized rihoeuclenee")
       \
                               pepsin
                               digestion
  DNP,                   DNP
    TH        "I"

DNP -NH

.,-NH-

   bie-DNP-Lysine                 Dinitrophenyleted N-terminal
(the "N-terminal" amino acid)                 amino acid sequence

Figure 57. Demonstration of the presence of a single polypeptide chain in bovine
pancreatic ribonuclease. The native molecule is oxidized with performic acid
to yield an extended polypeptide chain. This derivative contains only a single
N-terminal and a single C-terminal end group.

106                      THE MOLECULAR BASIS OF EVOLUTION

The single-chain character of the protein was further indicated by
"end group analysis." The only free a-amino groups in proteins are
those present on the amino acids at the termini of polypeptide
chains. These amino groups may be reacted with reagents such as
dinitrofluorobenzene, which forms a dinitrophenyl (DNP) derivative
of the protein in which (Figure 57) the N-terminal (i.e., NH,-termi-
nal) amino acids have been converted to their DNP derivatives.
Such a modified protein may be cleaved with acid or with prote-
olytic enzymes, and the DNP-N-terminal amino acid residue, or pep-
tide, may be isolated and characterized chemically.  By this method,
evidence was obtained for only a single free a-amino group in the
protein, present in the sequence Lys.Glu.Thr.Ala. For partial char-
acterization of the C-terminal (i.e., COOH-terminal) end of the pro-
tein, the enzyme carboxypeptidase was used as one of several tools
for the specific cleavage of the peptide bond at this end of the chain.
This enzyme removed valine from ribonuclease, with only traces of
other amino acids. The presence of single N-terminal and C-ter-
minal residues in the protein confirmed the physical evidence for a
single chain.

Hydrolysis of the oxidized chain has been carried out by a variety
of proteolytic enzyme "`reagents," since these tend to catalyze a much
more limited number of peptide bond cleavages than do such agents
as mineral acids. Indeed, it now appears that nonspecific hydrolysis
by acids can, in general, be avoided at this stage and is of particular
use only in the subsequent study of the sequence of the smaller pep-
tide subunits produced by preliminary enzymatic attack.

Figure 58 is a schematic outline of two general methods of ap-
proach to the detailed structural analysis of polypeptide chains that
have been applied to the ribonuclease molecule and are of general
applicability. In both techniques the native structure of the protein
is unfolded by cleavage of the disulfide bridges, either by oxidative
methods to yield cysteic acid residues in place of half-cystine resi-
dues, or by reductive cleavage followed by chemical masking of the
resulting sulfhydryl group by some suitable agent such as iodoacetic
acid.  (Th e a 1 tt er method for cleavage has an intrinsic advantage
over the oxidative method since it can be applied to proteins con-
taining tryptophan, .an amino acid residue which is extremely labile
to the conditions of disulfide oxidation by performic acid.)

In method A one sample is hydrolyzed by trypsin which cleaves
the chain at points following lysine or arginine residues, and another
by chymotrypsin (or some other protease with different specificities
from trypsin). As indicated, a completely different, but overlapping,

PROTEIN STRUCTURE                                           107


Method A

NH2

Cross-linked
polypeptide
  chain

-OH

l'rypsin

performic acid
oxidation of disulfide bridges
(-ss- - 2so,- )
Trypsin

pain

Trypsin

Method B

CBZ         CBZ
I           I
    --------Lye ----Arg    - - -(c~4xm~;"l

trypein
1

7"            CBZ                 CBZ
                      I
(N~`$~al-----Lis---Ag + NH2R---Lys---(C~i~al
                      I

HBr t
HCOOH

I                          1 I                         I

(iV-4'-4-1;nal-----Lya---Arg + NHsR---Lya---(C~i~enal

                   separate and digest    Trypein
                   again with trypsin

/ r-l r-l r--T-l
(N-+rnnnal---Lys + NHsR--Arg + NHsR--Lye + NH2R--(Cmz;znal

set of peptide fragments is obtained; this set may then be subjected
to analysis and arranged in linear order on the basis of overlaps in
composition.

In method B trypsin cleavage is restricted to positions following
arginine only, by masking the c-amino groups of the lysine residues
with either dinitrophenyl groups or carbobenzoxy groups. In this
method, therefore, we obtain a number of fragments one greater than
the number of arginine residues in the protein. All the fragments
contain C-terminal arginine except the one derived from the C-termi-
nus of the chain (which fixes its position). Method B depends on
the subsequent determination of the sequence of small peptides iso-
lated from random hydrolysates prepared with acid or with some
very nonspecific protease such as subtilisin (a bacterial protease)
which yields short, easily manageable, peptides. These arginine-
containing sequences are then used as connecting links between the
larger fragments originally separated from the digest of the carbo-
benzoxylated protein.

The Rockefeller Institute group, following method A, have reacted
separate samples of oxidized ribonuclease with trypsin, pepsin, and
chymotrypsin. These hydrolysates have then been separated into
component peptide fragments by column chromatography. A con-
sideration of overlaps has then enabled these investigators to recon-
struct large portions of the peptide chain. As an example of such a
reconstruction, let us examine the experimental data which permitted
Hirs, Moore, and Stein3 to arrive at a partial reconstruction of the
ribonuclease chain.

A typical analytical pattern is shown in Figure 59u, obtained by
means of a Dowex-50 ion exchange column through which was passed
a tryptic digest of oxidized ribonuclease under proper conditions of
elution. The peptide peaks emerging from the column have then
been hydrolyzed and rerun separately on subsequent Dowex-50 col-

4

Figure 58. Two approaches to the determination of the sequential arrangement ol
amino acids in a polypeptide chain. In method A the chain is digested with two
or more proteolytic enzymes. The resulting fragments are then separated and
analyzed for amino &id composition.  A partial reconstruction of the molecule
can then be made on the basis of overlapping amino acid compositions.  The
symbols T, L, and A represent tyrosine, lysine, and arginine respectively. In
method B the number of cleavages caused by the proteolytic enzyme, trypsin, is
limited to those bonds in which the carbonyi of the peptide bond is donated by
an arginine residue. To arrange the fragments in their proper order we mrrst
first determine the nature of the amino acids in the immediate vicinity of each
of the arginine residues.

PROTEIN STRUCTURE                                         109


                         (Glu,Gly,8er2.Thr)L~~90,100%]
2 g                                        6
e+z 1.0 -
2.P g              (Cyn,AspNHz.Ale.Val)13.s[95.95~]
ego                                   Asp.Arg[70.90%]
                                    5
.e" ;E                     NH&wn 7
p c 3 0.5 -
2"`g
2 2!
z-         Ii/k          IA
  Effluent liters 0.5          1.0
   jl0.2N pH 3.1, 357+----- Gradually increasing pH and [Na+] -
       Ly~i~~.Thr.Ala.Ala.Ala.Lys[95,100X1
(AspNH2.GluNH2.Ala.Thr2) r
aI LYmOJOO%l       (AspNH2,Leu,Thr)Lys[100,95~]  (GluNHz,Phe)Argi75,85%]

-(0.2N pH 3.1- 2.ONpH 5.1), 35' throughout
               (a)

1.0 c                                 (AspNH,,GluNH~.

kO.ZiV pH 3.1, 35"+--Gradually increasing pH and INa+]-----

\ (Asp2(xN.,z,,-.
   Alaz,Met.Th

   ---(0.2N pH 3.1~ 2.ON pH 5.1), 35' throughout p+
                  (b)

110                         THE MOLECULAR BASIS OF EVOLUTION

umns for amino acid analysis (the results are included in the figure).
Similar analysis of a chymotryptic digest of oxidized ribonuclease
yielded the results shown in Figure 59b.

The data in Figures 59a and 59b, when considered together with
the known N-terminal sequence and with the knowledge that certain
residues occur in ribonuclease in only limited numbers (e.g. phenyl-
alanine, 3; glycine, 3; methionine and histidine, 4; etc.) permit the
partial reconstruction of the sequence of the enzyme as shown in
Figure 60. The parentheses surround the residues that may be
grouped together and thus be given approximate linear assignments
along the `chain. Amide nitrogen atoms, unless specificaMy attribut-
able to individual aspartic or glutamic acid residues, are distributed
in an arbitrary fashion to the dicarboxylic amino acids within any
given parenthesis.

The general .approach described in Figure 58, method B, yields
results that are in accord with the results obtained by method A.
Since there exist four arginine residues in ribonuclease, we may ex-
pect five peptides of varying size by the application of this method.
These peptides were, indeed, shown to be formed by trypsin diges-
tion and, following isolation, were analyzed and partially character-
ized in terms of terminal sequences. Their composition is given in
Table 4. Subsequent characterization of smaller arginine-containing
sequences isolated from digests prepared with pepsin ,or partial acid
hydrolysis permitted the alignment of these fragments in the order,
A-B-C-D-E. Comparison of the partial reconstructions possible
through the use of the two methods indicate how the different sets of
information may be fitted together to give a final picture with greater
detail than on the basis of either alone.

To construct `a final picture of the covalent structure of the pro-
tein requires, in addition to the elucidation of the complete sequence
of each peptide fragment, a study of the disulfide bridges.  The loca-
tion of these can be determined by application of the methods intro-
duced by Sanger and his colleagues in the case of insulin.  The na-

1

Figure 59. Separation of the peptides in proteolytic hydrolysates of oxidized
ribonuclease. (a) A 20-hour tryptic hydrolysate of a 200-mg. sample of protein,
chromatographed on a 150 x 1.8-cm. column of Dowex 50-X2. The points
represent the ninhydrin color value given by aliquots of each of the fractions,
expressed in leucine equivalents. Figures in brackets represent the yield of each
peptide after 3 and 20 hours of trypsin hydrolysis, respectively.  From C. H. W.
Hirs, S. Moore, and W. H. Stein, J. Biol. Chem., 219, 623 (1956).
in a chymotryptic hydrolysate of oxidized ribonuclease.     (b) Peptides
                            From C. H. W. Hirs,
W. H. Stein, and S. Moore, I. Biol. Chetn., 221, 151 (1950).

PROTEIN STRUCTURE                                          111


THE MOLECULAR !ASlS OF EVOLUTION

TABLE 4

Partial Structure of the Five Peptides Produced by Application of
"Method B" to Oxidized Ribonuclease

Peptide                   Structure

A    Lys.Glu.Thr.Ala.Ala.Ala.Lys.Phe.Glu.Arg.
       t
          A  5:  I: t T

B    Glu.(Thr,Sere,Asp3,His.Met3,Alaz,Tyr,Cys,Glu).Lys.Ser.Arg.
                        t t
                                       T     T

C    Asp.Leu.Thr.Lys.Asp.Arg.
   d  i t T

D    CySOs.(Lys3,Pro,Vals,Asps,Thr3,Phe,His,Glus,Ser~,Leu,Al~~,
     Cyse,Gly,Tyrz,Met).Ser.Ileu.Thr.(Asp,Cys).Arg.
       A t        t
                          A           T

E    Glu.(Ser~,Thr~,Glyz,Lys~,Tyr~,Proa,Asp~,Ala~,Cys2,Glue,
      Hisq,Ileuz,Vals).Phe.Asp.Ala.Ser.Val
              t t
                      P     A
-

After digestion of carbobenzoxylated, oxidized ribonuclease with trypsin,
the digest was decarbobenzoxylated by treatment with anhydrous HBr in
glacial acetic acid. The fi ve peptides were then separated by combined
paper electrophoresis and paper chromatography and each one hydrolyzed
further with acid (A), pepsin (P), trypsin (T), or chymotrypsin (C).  A study
of the composition of the fragments produced by these latter digestions per-
mitted a partial reconstruction of the secpience as shown above.

tive protein, in which these bridges are intact, is digested with
various proteases (e.g. trypsin plus chymotrypsin or subtilisin) .  From
such digests can be isolated peptides of cystine, carved out from the
structure of the cross-linked native molecule. The general approach
to the determination of disulfide bridges which has been employed
by both the group at the Rockefeller Institute and at the National
Heart Institute is given in a disarmingly simplified form in Figure 61.

Following these general procedures, Spackman, Moore, and Stein3
were able to isolate, for example, a cystine-containing peptide which,

PROTEIN STRUCTURE                                          113


              so,-        so3-

Figure 61. Steps in the determination of how half-cystine residues are paired
in disulfide linkage. The cysteic acid-containing peptides derived from each
cystine peptide are separated by chromatography and/or electrophoresis.  A con-
sideration of their amino acid compositions in terms of the amino acid sequence
of the protein will generally permit the location of each half-cystine residue
along the polypeptide chain.

upon oxidation with per-formic acid, yielded the two cysteic acid-con-
taining peptides

(CySO,-, An, Gn, Met,, Lys) and
(CySO,-, An, Ileu, Thr, Ser, Arg)r

The amino acid compositions of these peptides are such that the
cysteic acid residues could only have been derived from half-cystines
1 and 6 respectively, establishing the presence of a disulfide bridge
between these half-cystines in the native protein.  (The half-cystines
are numbered sequentially from the N-terminal end of the chain).
In a similar fashion, evidence was obtained for a disulfide bond be-
tween half-cystines 4 and 5, and, with some reservations because of
the possibility of chemical rearrangements during isolation, between
3 and 7, and 2 and 8. The results of the combined degradative stud-
ies of ribonuclease, together with the isolation and tentative charac-
terization of the cystine bridges, permit us to construct a provisional
formula for the enzyme as shown in Figure 62.  The formula includes
much of the total sequence within the parentheses in the reconstruc-
tions summarized in Figure 66 and Table 4.

114                     THE MOLECULAR BASIS OF EVOLUTION

One additional example of the determination of amino acid se-
quence is summarized in Figure 63. This figure shows, in a nutshell,
the general approach used by Li and his colleagues in the study of
the structure of the anterior pituitary hormone, adrenocorticotropin
( ACTH ) .4 The various fragments (see also Chapter S), some of
which were subjected to complete or partial sequence analysis by
methods described in some of the references at the end of this chnp-
ter, are all consistent with only one unique sequence as shown.

Figure 62. The structure of bovine pancreatic ribonuclease.  This figure has been
constructed from the combined studies of a number of individuals at the Rode-
feller Institute (including Drs. C. H. W. Hirs, S. Moore, S. H. Stein, D. Spack-
man, and L. Bailey) and at the National Heart Institute (including R. Redfield,
J. Cooke, A. Ryle, and C. B. Anfmsen). The positions of the four disuffide
bridges are probably as shown, although there is still some question about the
bridge between half-cystine residues 2 and 8 and that between half-cystine
residues 3 and 7, because of the poor yields obtained during the isolation of
cystine peptides and because of the possibility of chemical rearrangements which
might have occurred during certain steps in the sequence analysis of these pep-
tides. At the present time some 95 residues may be assigned with reasonable
certainty to definite positions in the polypeptide chain, which contains 124
residues in all. The residues that are shaded may be assigned to the general
region of the molecule indicated, but their exact position in the sequence must
await further double-checking by stepwise degradation.

PROTEIN STRUCTURE

115


THE MOLECULAR BASIS OF EVOLUTION

As we shall discuss subsequently, the knowledge of the complete
covalent structure of a protein molecule is, unfortunately, not a magic
key to the understanding of physicoehemical and catalytic behavior
in itself. A real appreciation of Nature's intent with respect to pro-
tein molecules must be sought through additional considerations of
structure in three-dimensional terms.

Secondary and Tertiary Structure

Having established the details of the covalent structure of a pro-
tein, we may proceed to a comparison of these organochemical de-
tails with physicoohemical behavior in solution.  The total number of
charges, positive and negative, along the polypeptide chains should
agree with the values determined by titrating the protein with acid
or base. A knowledge of the number of tyrosine, phenylalanine, and
tryptophan residues in the molecule should make it possible to esti-
mate, in advance, the shape and height of ultraviolet light absorption
curves in the wavelengths from 250 to 290 mp. Determinations of
the content of sulfhydryl groups in the protein should confirm the
analytical values for cysteine.
It was not surprising to the physical biochemists at work on such
comparisons to find that these predictions seldom coincide with
actuality. In general, many charges are masked or sequestered, ab-
sorption spectra are shifted from the theoretical, and sulfhydryl
groups often appear in force only after serious denaturation of the
protein molecule.  The deviations are attributable to a number of
known, and probably many unknown, parameters of secondary and
tertiary structure which we shall attempt briefly to summarize.

We have already referred to the likelihood of helical coiling in
proteins as the central theme of secondary structure. According to
the dimensions of the a-helix and to the pitch of its screw, each
residue in the polypeptide chain should contribute 1.5 A. to the
length of the helical coil.  Thus, in a helix with n residues, the total
length of the coil should be n X 1.5 A. (see Figure 54).  By an ex-
tensive series of studies of the viscosity, light scattering, and sedimen-
tation properties of the synthetic polyamino acid poly-y-benzyl-l-
glutamate in favorable solvents, Doty and his colleagues obtained
data in full agreement with this theoretical calculation; that is, ex-
perimentally determined molecular lengths, in angstroms, were equal
to the number of residues in the chain multiplied by 1.5.5  It should
be emphasized in the following that, although the word "helix" is

PROTEIN STRUCTURE                                           117


used freely for convenience, the presence of a-helical coiling in globu-
lar proteins is supported only by very circumstantial evidence. We
may safely assume that there exists some sort of ordered folding
within such proteins, but we must be wary of getting into the habit
of thinking in terms of the a-helix exclusively until more data be-
come available. It has not been established that all proteins contain
such a structure, and the examination of this point, with special ref-
erence to globular proteins, is a particularly active area of research
at the present time.

The study of the' properties of helical systems in solution depends
to a large degree on the use of optical rotatory methods. The
mathematical theory of optical rotation by proteins is extremely com-
plex and speculative.  In spite of theoretical difficulties, however,
there appears to be a high degree of consistency and predictability
in connection with optical rotatory measurements, and we can gain
an adequate appreciation of the usefulness of the method from a
purely empirical point of view.

The optical rotation of a protein is made up of several components,
one of which is the average rotation of the individual residues in the
polypeptide chains. Superimposed on this rotation is the contribution
of the ordered, repeating character of the helical configuration when
this is present in the structure. It has been found experimentally,
by studying the rotation of synthetic polyamino acids which can be
made to assume varying degrees of helical coiling by modification of
the solvent, that fully coiled chains possess an optical rotation ap-
proximately 90o (for the D line of the sodium lamp) greater than
that to be expected as an average for the residues themselves.  This
completely empirical number may be used as a basis for calculating
the content of helical coiling in a protein, according to the following
equation:

70 helical coiling = Rfolded - Runfolded
               9o"     x 100

The mean residue rotation of the folded protein, Rr,,d,a, is obtained
by measurements on the native protein in water, and Runfolded is esti-
mated by measuring in the presence of such materials as urea or
guanidine, or at elevated temperatures, under which conditions coil-
ing, stabilized by hydrogen bonds, may be assumed to be destroyed.
Some examples of optical rotatory measurements are given in Table 5
together with estimates of the amounts of helical coiling in these
proteins. A measure of the rotation of the unfolded state may also
be obtained on samples in which disulfide cross-linkages have been

118                         THE MOLECULAR BASIS OF EVOLUTION

TABLE 5

Rotatory Properties of Native Proteins in the Folded and Unfolded States8

                                  Calculated
                                  Per Cent
                         8 M urea    of Protein
    Protein       Rf olded     & nfolded     in Helix
_-

   Ribonuclease        -90.7       -126.5        40
   Insulin            -36.5       - 98.3        68
   Ovalbumin         -33.9       -106.8        81
  Lysozyme          -53.3       - 96.0        47
  Chymotrypsin       -71.9       -115.9        49
   Silk fibroinb        -40.        - 51.3        12

a C. Schellman and J. A. Schellman, Compt. rend. trav. lab. Carlsbesg, Ser.
chim., 30, 463-500 (1958).
b Silk fibroin is an example of a protein which is almost free of stabilizing
cross-linkages and consequently exists in a form nearly free of secondary
structure. Its mean residue optical rotation in urea (&+,&d) is low owing
to the high cont.ent of the optically inactive amino acid glycine.

broken by reductive or oxidative cleavage. In the absence of such
stabilizing linkages, the helix is apparently unstable, and we ob-
serve rotations such as might be expected for the average amino acid
residue, that is, about -100 to -12OO. Disulfide bridges are not
enough in themselves, however, to insure helical configuration in a
protein molecule. We must take into account the fact that agents
like urea can cause disorientation of secondary structure, even in the
presence of intact disulfide linkages.  It seems certain that a number
of tertiary s%ructural features also contribute to the determination of
protein structure in solution. The presence of such structural inter-
actions are indicated by the lack of agreement, mentioned earlier,
between the experimentally determined values for ionizable groups,
sulfhydryl groups, and the like and the values predicted from se-
quence data alone.

Functional groups which exhibit modified properties can be studied
in a quantitative way. An excellent example comes from the spec-
trophotometric examination of proteins. It was first observed bv
Crammer and Neuberger6 in 1943 that the spectrum of ovalbumin in
the region attributable to tyrosine residue absorption was shifted to
shorter wavelengths as compared with the spectrum of tyrosine itself.
These results indicated that the absorption of light energy by the
tyrosine chromophore bad been modified in some way by the environ-

PROTEIN STRUCTURE                                          119


I     I      I     I      I

Wave length A

b)

Wave length A

Figure 64.  ( a) Spectra of inactive ribonuclease ( top curve ), of inactive deriva-
tive of ribonuclease isolated from limited pepsin digests (middle curve), and of
an exhaustive pepsin digest of ribonuclease (bottom curve).  (b) Differences
in extinction at various wavelengths between native ribonuclease and a complete
pepsin digest of ribonuclease (upper curve), of the inactive pepsin derivative
(second curve), of ribonuclease in 8 M urea (third curve), and of ribonuclease
in 8 M urea in the presence of 0.003 M phosphate ions (bottom curve).  From
C. B. Anfinsen, Federation hoc., 16, 783 ( 1957).

ment of the tyrosine residues within the structure of the protein
molecule. Similar observations were subsequently made for a num-
ber of other proteins including ribonuclease, lysozyme, and serum
albumin. It was ultimately shown that the shift, characteristic of the
abnormal spectrum (shown for ribonuclease in Figure 64), could be
abolished by agents like urea which cause the rupture of hydrogen
bonds.  Simultaneously there were released, in a titratable form, car-
boxy1 groups which had hitherto been masked and unavailable to
titration by alkali. The number of such unmasked acid groups ap-
proximated the number of tyrosine residues which, it could be cal-
culated, were responsible for the shift in extinction maximum.  These
findings furnished evidence for the presence of tyrosine hydroxyl-

120                           THE MOLECULAR BASIS OF EVOLUTION

carboqlate interactions of the type we have already shown schemati-
cally in Figure 55." The stage was, of course, set for the detection
of these particular hydrogen-bonded interactions since there is avail-
able here a simple method ,of observation, namely the spectrophotom-
eter. Other hydrogen-bonded combinations, for example, between
amino groups and carboxyl groups or between two carboxyl groups,
may also be expected to exist in proteins, but they have so far escaped
detection because of the lack of an adequate assay procedure for them.

Our consideration of the protein structure up to this point has
been concerned with static or equilibrium properties. The covalent
bonds in proteins can be relied on to resist most of the environmental
fluctuations to which proteins are normally exposed.  In a sense, they
determine the three-dimensional potentialities by establishing a fixed
distribution of charges and lyophobic groupings along the polypeptide
chain and by acting as the basis for rigid and permanent cross-link-
ages. The covalent bonds do, however, have a certain degree of
flexibility, and the slight deformations in bond angles between atoms,
together with the rotation of atoms around single bonds, make it
possible for a protein to assume a vast number of slightly different
configurations in solution. When we measure the viscosity, optical
rotation, or ultracentrifugal behavior of a protein preparation, the re-
sults obtaiced relate only to the average molecule in the solution.  In
general, the deviation from this *average is probably fairly small be-
cause some arrangements have a much higher stability (i.e., a lower
total free energy of configuration). The small fluctuations in con-
figuration which do occur are, however, undoubtedly of considerable
importance in connection with the biological activity of proteins and
fortunately can be studied by means of a few specialized techniques
which measure the dynamic aspects of protein structure.

As we have discussed earlier, it is possible to estimate the extent
of helical coiling in proteins by measuring optical rotatory properties
if we are willing to equate "repeating units of asymmetry" with
"helix." The theoretical and experimental support for this assump-
* It will be noted that the spectral characteristics of tyrosine in native ribo-

nuclease (and in other proteins in which similar effects have been observed)
resemble to some extent those exhibited on ionization of the phenolic hydroxyl
group of free tyrosine. In both instances a shift of the spectrum toward longer
wavelengths as well as an increase in absorption occurs. It seems likely that
formation of a hydrogen bond could bring about spectral changes analogous to
those of ionization by increasing the phenolic O-H bond distance.  Indeed :I
number of spectrophotometric studies on substituted phenols have demonstrated
a shift of the spectrum toward the red under conditions favoring hydrogen bond
formation.

PROTEIN STRUCTURE                                           121


-m

tion is good enough to satisfy most experts as a working hypothesis.
By assuming, then, the presence of a certain amount of helical coiling
in a protein like insulin, various models may be constructed based
on the covalent structure derived by Sanger and his colleagues. In
constructing such models it is necessary to guess where the helical
portion belongs.  Guessing is made somewhat easier by the ob-
servation that random polypeptide chains without internal cross-
linkages do not spontaneously coil up into organized structures, as
judged from viscosity studies and from the effect on optical rotatory
properties when disulfide cross-linkages are ruptured by oxidation or
reduction. Thus, in the case of insulin, whose structure is shown
in Figure 65u, we may assume that the 60 per cent or so of the
polypeptide chain which appears, polarimetrically, to be in the
a-helix form is most likely to be located within the sterically stabilized
region between the disulfide bridges joining the A chain to the R
chain. The schematic drawing (Figure 65b) is intended to convey
the idea of steric hindrance within the coiled portions of the chains.
Within such a region of a protein molecule, we might expect to find
that the CONH hydrogens involved in the hydrogen bonding of the
helix are much less likely to exchange with the hydrogens of the
water in which the protein is dissolved than are the hydrogens of
side-chain groups or of CONH bonds outside the helical region. It
is precisely this relative stability which the technique of deuterium
exchange can measure, and this teohnique constitutes one of the
most sensitive methods for the measurement of the dynamic state of
protein structure.'  Its particular value lies in the fact that, even
when other physical measurements indicate that the average, equi-
librium form of a protein contains some helical structure, it can
measure the degree of deviation from the average resulting from the
reversible opening and closing of hydrogen bonds with accordion-like
flapping of the helix.

In practice, a protein is "loaded" with deuterium by solution in
deuterium oxide for a time sufficient to allow all exchangeable hydro-
gens to come into equilibrium with the deuterium of the solvent.
The protein is then carefully dried. Upon resolution in ordinary
water, deuterium atoms equilibrate with the water, and the rate of
appearance of deuterium, as measured by density change in the
solvent, permits the division of total exchangeable atoms into those
that are instantaneously, rapidly, and slowly exchangeable.

In insulin there are 91 theoretically exchangeable hydrogen atoms.
These include the hydrogen atoms on the side chains of various amino
acid residues and the CONH hydrogens forming the polypeptide

PROTEIN STRUCTURE                                          123

122

THE MOLECULAR BASIS OF EVOLUTION


I%?
Val
lispNH,
GiuNH 2
H'ia
IL"
Cl6
G!Y
Ser
tiis
L-3-J
V'al
G)"
A$
Iau
T;r
Leu
v+
QS
G!Y
G!u
-4.v
GUY
Pile
P'he
Tg*
Tir
P,kO
LYS
Ala

Figure 66. The relative degrees
of "excbangeabihty" of various
hydrogen atoms in insulin with
deuterium atoms in the sol-
vent: solid circles, instantaneous
exchange; crosshatched circles,
exchange at intermediate rates;
open  circles,  restricted ex-
change. From K.   Linder-
strflm-Lang,  Symposium on
Pepttde  Chemistry,  Special
Publications 2, The Chemical
Society, London, 1955.

chain. When deuterium-loaded insulin is dissolved in water at OOC.,
60 of these 91 exohange instantaneously. With further incubation
additional exchange of other hydrogen atoms occurs, but at this tem-
perature the rate of equilibration is rehitively slow. In the presence
of denaturing agents like urea, or at higher temperatures, the rate
of equilibr'ation is greatly increased, and all 91 of the deuterium
atoms are ultimately exchanged.

These results have been interpreted by Linderstdm-Lang' as
shown in Figure 66. The slowly exchangeable hydrogen atoms (in-
dicated by open circles) have been assigned to the region of insulin
where the helical configuration is assumed to reside. The assign-
ment of "rapidly" exchangeable atoms is somewhat more arbitrary,
but can be made, in part, to that region of the A chain within the
disulfide "loop" which, when forced into a helical arrangement in
models, permits the formation of the intrachain disulfide bridge only
with considerable bond deformation.

This interesting method furnishes perhaps the most clear-cut evi-
dence for the existence of a continual flux of local minor rearrange-
ment within the structure of proteins in solution. It brings out, in a
particularly dramatic way, the intrinsic instability of the three-dimen-

124                       THE MOLECULAR BASIS OF EVOLUTION

sional fabric of proteins which can lead to denaturation and loss of
biological activity, and it emphasizes some of those structural com-
ponents whose presence was not even suspected only a few years ago.

REFERENCES

1. F. Sanger in Currents in Biochetntcal Research (D. E. Green, editor), Inter-
science Publishers, New York, p. 434, 1956.

2. C. H. W. Him, W. G. Stein, and S. Moore, J. Biol. Chem., 211, 941 ( 1954).
3. S. Moore and W. H. Stein in The Huroey Lectures 1956-1957, Academic

4. C. H. Li. I. Geschwind. It. D. Cole, I. Raacke, J. Harris, and J. Dixon, NR-

Press, New York, p. 119, 1958.
ture, 176, 687 (1955). '

5. B. W. Low in The Proteins, volume 1, Part A (H. Neurath and K. Bailey,
editors), Academic Press, New York, Chapter 4, 1957.
6. J. L. Crammer, and A. Neuberger, Biochem. J., 37, 302 (1943).
7. K. V. Linderstrem-Lang in Symposium on Peptide Chemistry, Special Pub-
lication 2, The Chemical Society, London, 1955.

SUGGESTIONS FOR FURTHER READING

Anfinsen, C. B., and R. R. Redfield, in Aduances in Protein Chemistry, volume
11, Academic Press, New York, 1956.
Crick, F. H. C., and J. C. Kendrew, in Aduances in Protein Chemistry, volume
12, Academic Press, New York, 1957.
Edsall, J. T., and J. Wyman, Riophysical Chemistry, volume 1, Academic Press,
New York, 1958.

Linderstrem-Lang, K. V., Lane Medical Lectures, volume 6, Stanford University
Press, Stanford, 1952.

Low, B. W. and J. 1'. Edsall in Currents in Biochemical Research, (D. E. Green,
editor), Interscience Publishers, New York, p. 379, 19'6.
Pauling, L., and R. B. Corey, Proc. Natl. Acad. Sci. U.S., 37, 235 ( 1951).
Pauling, L., Il. B. Corey, and H. R. Branson, Proc. Natl. Acad. Sci. U.S., 37,
205 ( 1951).

PROTEIN STRUCTURE

125


chapter 6

   THE BIOLOGICAL ACTIVITY

OF PROTEINS

IN RELATION TO STRUCTURE

   W e may almost define the life sciences as those
concerned with the elucidation of the mechanisms by which molecules
exert their specific actions on living cells. In the case of many simple
inorganic ions and organic molecules it has been possible to arrive
at an approximate understanding of their mechanism of action. We
have some understanding, for example, of the physiological sequelae
of raising or lowering the tonicity of body fluids by the administration
or withdrawal of sodium chloride.  Similarly, the aberrations in the
synaptic transmission of nerve impulses following the administration
of physostigmine may be partially explained by the action of this
drug on the enzyme acetylcholinesterase. It is a tribute to cellular
complexity that even such well-studied systems as these continue to
be frontiers of research and speculation among those individuals who
are fully conversant with them.

Protein chemists naturally feel that the most likely approach to the

126

understanding of cellular behavior lies in the study of structure and
function of protein molecules. This is, perhaps, not an entirely un-
reasonable point of view. Except for those rare phenomena in biol-
ogy which are purely physical, the "aliveness" of cells is basically the
summation of enzymic catalysis and its regulation.

The field of protein chemistry has now reached a stage of relative
sophistication `allowing us to think of proteins as organic chemicals
rather than as conglomerates of amino acids. In spite of their
enormous complexity we now can measure the extent of such phc-
nomena as "denaturation" in terms of rather well-defined changes in
specific types of chemical bonds, some of which have been discussed
in Chapter 5. Because of this happy situation, we may approach,
in a rational way, the relation of specific aspects of covalent and non-
covalent protein chemistry to biological activity. Proteins, as mol-
ecu&, must be thought of as consisting of one or more polypeptide
chains, cross-linked and coiled through a variety of chemical bonds
of varying strengths.  Modifications of any of these lead to an entity
which is not identical to the original native molecule and which,
in a purist sense, may be considered "denatured." From the stand-
point of function, however, we may take a somewhat narrower view.
The nativeness of an enzyme, as estimated by its ability to catalyze a
reaction, need not involve the whole of its structure.

Studies of the effect of specific degradation on biologically activr
proteins are fairly recent.  It was observed, however, more than
twenty years ago, that various reactive groupings on proteins could
be modified by substitution, or conversion to other forms, without loss
of function, Perhaps the best-known example of such work is the
series of investigations by Herriott and Northrop of the activity of
pepsin during progressive acetylation.'  Pepsin was treated with
ketene which converted free amino groups and tyrosine hydroxyl
groups to their acetyl derivatives. By this method Herriott was able
to prepare a crystalline derivative which contained seven acetyl
groups per mole of pepsin and had approximately 60 per cent of the
enzyme activity of the original pepsin. He demonstrated that the
ultraviolet absorption spectrum of this 60 per cent active material
was modified to the extent to be expected for the masking of three
tyrosine hydroxyl groups. Further, by cautious hydrolysis at pII
0.0 or at pH 10.0, three acetyl groups could be removed with a simul-
taneous increase in enzyme activity.  These, and other, studies indi-
cated that tyrosine residues were somehow involved in activity and
also that acetylation of a number of the free amino groups in the pro-
tein had no eflect on function.

THE BlOLOGlCAL ACTIVITY OF PROTEINS IN RELATION TO STRUCTURE         127


Experiments of this sort have now become relatively commonplace,
and there is no doubt that the structures of a large number of en-
zymes and hormones may be tampered with without inactivation.
In spite of such information, the concept persisted, until very re-
cently, that the structure of biologically active proteins was more or
less "inviolable" and that the complete and integrated three-dimen-
sional architecture of these proteins was required for function.  This
concept was nurtured by several theoretical considerations in which
the protein molecule was pictured as a network of resonating path-
ways through which electrons might surge and redistribute during
the process of catalysis. The observations of immunology added fur-
ther support to this concept, since it was well known that relatively
minor changes in, for example, the structure of a hapten could
lead to a profound modification in the effectiveness of reaction with
a specific antibody.

The idea of the "inviolability" of protein structure is now slowly
being replaced with the idea of the "functional adequacy of less than
the whole." Shortly after Sanger and his colleagues had completed
their monumental studies on beef insulin, it was shown by Lens2 that
a known, degradative change in the hormone, namely, the removal
of the C-terminal alanine residue of the B chain, did not lead to loss
of biological activity.  (The evolutionary implications of this observa-
tion were not particularly obvious at the time since the experiment
was the first of its kind and had to be thought of as an isolated ex-
ample. Now, however, as the result of many similar observations,
we must concern ourselves (see Chapter 11) with the question of
why the C-terminal alanine residue is preserved in insulin as a con-
stant structural feature if it serves no function in the biological activity
of the hormone.)

Insulin has been subjected to other, more extensive, studies of this
sort. However, for the purpose of illustration of the extent to which
proteins may be degraded without inactivation, we shall consider
instead three other examples about which we have somewhat more
information: the pituitary hormone, ACTH, the pancreatic enzyme,
ribonuclease, and the plant enzyme, papain. In the following dis-
cussion of these examples, two quite different approaches to the struc-
tural basis of biological activity will be considered more or less simul-
taneously. One aim is to show that active polypeptides can be de-
graded without loss of function, and is concerned mainly with indi-
cating the magnitude of unessential parts of structure.  The other is
directed deliberately toward the delineation of the essential parts,
that is, the active centers.

128                         THE MOLECULAR BASIS OF EVOLUTION

Adrenocorticotropic Hormone

Complete sequences have been elucidated for adrenocorticotropic
hormone (ACTH) isolated from three species, hog, beef, and sheep.
The problem of species variation will be discussed elsewhere. For
our present purposes we shall consider only the material isolated
from hog pituitaries since the relation between structure and function
appears to be the same for the hormones of all three species.
Adrenocorticotropic ,hormone is one of a large number of polypep-
tides which can be demonstrated in extracts of the anterior pituitary
gland. The hormone has been prepared in highly purified form and
has been shown to cause the selective stimulation of the adrenal cor-
tex with the release of adrenal steroid hormones.  It also causes de-
pletion of the ascorbic acid and cholesterol stores of the adrenal gland
and is capable of stimulating the repair of histological changes owing
to hypophysectomy.

The sequence of pig ACTH is shown in Figure 67. The polypep-
tide is composed of 39 amino acid residues and contains several
intriguing groups of amino acids, notably the sequence of three hy-
droxyamino acids, Ser.Tyr.Ser, at the N-terminus and the highly
basic grouping, Lys.Lys.Arg.Arg, in positions 15 to 18. The action
of carboxypeptidase removes three residues from the C-terminal end
of the chain withovt loss in hormonal activity. A more severe deg-
radation results during limited pepsin digestion with the removal of
the eleven C-terminal amino acids, still without change in activity.
These experiments establish that, as a maximum, only the first 28
residues of ACTH are required for hormonal function.  Paul Bell
and his co-workers have recently reduced this estimate, reporting that
by mild `acid hydrolysis of the chain the last 15 residues may be re-
moved without inactivation.3

It has also been possible to characterize the activity of ACTH in
terms of specific aspects of structure which are essential.  Thus it has
been found that degradation of the N-terminal end of the chain with
leucine aminopeptidase results in complete loss of activity after only
one or two residues have been removed.  Recent studies on the
action of fibrinolysin by White and Gross4 have further underlined
the importance of the first half of the hormone. As is shown in
Figure 67, this proteolytic enzyme cleaves the chain at two points,
following residues 8 and 15, with complete inactivation.

These various degradative studies establish that at least 16, and
perhaps as many as 24, of the amino acids of ACTH are essential
ones. The ultimate elucidation of the exact structural requirements

THE RIOlOGICAL ACTIVITY OF PROTEINS IN RELATION TO STRUCTURE         129


w-
ASP

Figure 67. The structure of porcine
chymotrypsh            a-corticotropin. Points of attack by
by the arrows. (C), trypsin (T), pepsin (P), and fibrinolysin (F) are indicated

for ACTH activity must probably await the step-by-step synthesis
of the sequence, and such studies are in progress in Klaus Hofmann's
laboratory,5 where the unequivocal synthesis of a peptide correspond-
ing to at least the first 14 amino acids in the polypeptide has been
achieved. At last report, the synthetic material had not yet reaohed
sufficient length to exhibit hormonal activity. In the meantime,
Boissonnas and his colleagues6 have reported the synthesis of a biologi-
cally active peptide consisting of the first 20 N-terminal amino acids
of ACTH. Although the synthesis is not entirely unequivocal, the
results give strong support to the conclusions drawn from the
degradative work.

The studies on ACTH are of particular interest since they have to
do with a substance which is assayed by in uiuo methods. Investiga-
tions of the changes in activity during the degradation of enzymes
must generally be controlled by in vitro tests. The possibility that
functional adequacy in the test tube does not give a true picture of
activity in an organized cellular environment adds an aspect of un-
certainty to the interpretations. Discounting the rather slim possi-
bility that degraded ACTH is reconverted to native ACTH during
activity assays, we have, for this hormone, clear-cut evidence for
the "functional adequacy of less than the whole."

Papain

Papain is a powerful proteolytic enzyme present in papaya latex
which has been crystallized, both as the native protein and as the

130                      THE MOLECULAR BASIS OF EVOLUTION

mercuri-derivative, by Emil Smith and his colleagues.' The enzyme
is of `considerable interest as a model for the study of the relation-
ships between structure and function because it appears to contain
a great deal of structure which is "superfluous" for activity. The
molecule contains six sulfhydryl groups, five of which are not reactive
with the usual sulfhydryl reagents in the native protein but appear
from within the inner regions of the structure only after suitable
denaturation. The single reactive sulfhydryl is essential for activity,
and complete inactivation occurs when two molecules of the protein
react with a single molecule of mercuric ion to form the dimer mer-
captide. No other sulfur is present beyond the six cysteine residues,
and cross-linkage of the 180 amino acid residue chain through cystine
bridges is therefore excluded.

The most exciting development in the studies on papain has been
the observation by Hill and Smith8 that approximately 80 residues
may be removed from the N-terminal end of the chain by leucine
aminopeptidase without loss in catalytic activity. The remaining
C-terminal stretch of amino acids is relatively poor in basic amino
acids but rich in tyrosine and valine. The noncritical nature of
lysine side chains is indicated by the fact that the r-amino groups
may be converted to the guanido derivatives, by treatment with
0-methylisourea, without activation.

Papain is reversibly inactivated by strong urea solutions and, to
some extent, irreversibly by guanidinium ions.  Under such condi-
tions of denaturation the shape of the molecule is considerably altered
as evidenced by changes in the optical rotatory, viscosity, and ultra-
centrifugal properties. Therefore, although only a portion of the
papain chain is essential for catalysis and is apparently not dependent
on covalent cross-linkages, there are obviously important secondary
and tertiary structural features involved in the determination of its
catalytically active center.

Ribonucleare

The present status of our knowledge of the sequence of ribonuclease
has been reviewed in an earlier chapter together with a considera-
tion of some of the physical properties of this enzyme, both in its
native state and under denaturing conditions. With this background
we can now examine some of the covalent and noncovalent aspects
of its structure in connection with the problem of catalytic activity.
As we have seen, the protein contains two rather longish "tails," one
at the N-terminal end consisting of 25 residues and one at the

THE BlOLOGlCAL ACTWIlY OF PROTEINS IN RELATION TO STRUCTURE        131


C-terminal end consisting of 14 (Figure 62, Chapter 5). Both of
these parts of the chain have been subjected to controlled hydrolysis
with proteolytic enzymes. It was first shown by F. M. Richard@
that native ribonuclease, when digested with the bacterial enzyme
subtilisin, was rapidly and specifically hydrolyzed at the Ala.Ser bond
(residues 20-21) in the N-terminal "tail." The resulting derivative,
in which the 20 residues of the "tail" portion were still attached to the
body of the enzyme through some particularly strong noncovalent
interaction, could be isolated on ion exchange columns and was
shown to retain the full activity of the original protein.  This active,
degraded ribonuclease molecule now also contains, in addition to the
iv-terminal lysine residue characteristic of the native enzyme, a new
N-terminal serine residue.

The C-terminal end of the chain can also be degraded without
inactivation. It has been possible to remove the C-terminal valine
residue by carboxypeptidase treatment, together with a fraction of
the serine and alanine residues which immediately precede the valine.
In such samples of degraded protein no evidence for inactivation has
been observed.

The site of these noninasctivating cleavages with proteolytic enzymes
may be assigned to specific portions of the linear polypeptide chain,
Some active products of more extensive digestion have also been de-
scribed for which, unfortunately, the exact sites of cleavage cannot
be pinpointed at present. Uziel, Stein, and Moore,`" for example,
have reported the isolation of active derivatives of ribonuclease which
has been subjected to trypsin digestion under conditions of partial
unfolding of the protein in dilute urea solutions.
sky and Rogers               Similarly, Kalnit-
             I1 describe experiments using carboxypeptidase diges-
tion in which the digesting enzyme apparently contained other pro-
teases of unknown types which caused extensive degmdation of the
molecule without loss of much activity. As the analysis of these
latter studies becomes more complete and it becomes possible to as-
sign the changes that have occurred to definite parts of the polypep-
tide chain, we shall be able to make much more useful statements
about the location and nature of the "active center" of this enzyme.

We may also derive useful information from experiments designed
to determine the minimum changes necessary to cause inactivation of
the enzyme. Two particular experiments are of special interest here.
The first has to do with the inactivation of ribonuclease activity dur-
ing photooxidation. Weil and Seibles'* have shown that the enzyme,
when exposed to light in the presence of methylene blue, undergoes
slow loss of activity and that this loss parallels, at least during the

132                        THE MOLECULAR BASIS OF EVOLUTION

early stages of photooxidation, the disappearance of one (possibly
two) of the four histidine residues that occur in this protein. Al-
though the exact location of the histidine residue (or residues) in-
volved has not been determined, it should be noted that two of the
four are found in the C-terminal twenty amino acids of the chain.

A second sort of inactivating treatment which may ultimately help
to elucidate the chemical basis of ribonuclease activity involves pep-
sin digestion under carefully controlled conditions. When the en-
zyme is exposed to pepsin at pH values in the neighborhood of 1.8,
there occurs an extremely rapid hydrolysis of what appears to be a
single, particularly labile, peptide bond.13 The macromolecular por-
tion of the reaction mixture may be isolated on ion exchange columns
and is completely devoid of activity. The only other detectable
fragment is a tetrapeptide, Asp.Ala.Ser.Val, the sequence of which
indicates that it was derived from the C-terminal end of the chain
by the cleavage of the peptide bond following the phenylalanine
residue at position 120 in the polypeptide chain (see Figure 62).
It has not been possible to detect the presence of cleavages elsewhere
in the inactive macromolecular derivative, and both physical studies
and C-terminal amino acid analysis support the conclusion that
nothing else has happened beyond this unique hydrolysis.

Since the earlier studies on the carboxypeptidase treatment indicate
the nonessentiality of the three C-terminally located amino acid resi-
dues, these results with pepsin suggest that the aspartic acid residue
located at position 121 has some special function in the maintenance,
or mechanics, of the catalytically active center.  We shall discuss this
point further in connection with observations on the spectral prop-
erties of ribonuclease and its various active and inactive derivatives.

Although the combined consideration of the degradative experi-
ments indicates that some portions of the sequence of ribonuclease
have greater importance in activity than others, we cannot conclude
that catalysis by this enzyme requires only a simple linear sequence
of amino acids. Both oxidized ribonuclease and ribonuclease that
has been converted to a random, open chain by reduction of the
disulfide bridges are completely inactive. It seems very likely that
one or more portions of the sequence, perhaps quite widely separated
in the sense of linear distance along the chain, are essential, and that
their relation to one another in space is fixed by restrictions intro-
duced by disulfide bridges and by other bridges of a noncovalent
nature.

Some recent experiments on the relation between controlled, step-
wise reduction of the disulfide bridges in relation to activity are of

THE MOLOGICAL ACTIVITY OF PROTEINS IN RELATION TO STRUCTURE         133


Figure 68. Activity of ribonuclease at various stages of reduction (expressed as
percentage of the specific activity of native ribonuclease) as a function of the
number of moles of sulfhydryl per mole of enzyme. Solid triangle, reduction in
absence of urea; solid circle, reduction in 8 M urea; open square, reoxidation of
fully reduced, inactive ribonuclease; open circle, reoxidation of samples contain-
ing more than six sulfhydryl groups per average molecule; open triangle, reoxida-
tion of samples containing about four sulfhydryl groups per average molecule.
From M. Sela, F. H. White, and C. B. Anl?nsen, Science, 125, 691 ( 1957).

interest in this regard.ls The disulfide bonds may be reduced by
thioglycollic acid and by various other reducing agents as discussed
in Chapter 5. It has been found that the reduction of one of the
four bridges in ribonuclease goes fairly rapidly in the absence of urea,
but that in 8 M urea, in which the three-dimensional structure of the
protein has been disoriented and "loosened," all four are easily
cleaved. During reduction the activity falls off in a manner suggest-
ing that one, and perhaps two, of the bridges may be dispensed with
from the standpoint of enzymatic activity (Figure 68). Thus when
an average of one SS link has been reduced, we can still demonstrate
the presence of 80 to 90 per cent of the initial activity in spite of the
absence of all but traces of native enzyme. Even at an average level
of four SH groups per mole of protein (equivalent to the rupture of
two SS `bridges), considerably more than half the activity remains.
It has recently been possible to separate some of the intermediates
of early stages of reduction on ion exchange columns, and the iden-
tification of the bridges that are most susceptible to reductive cleavage
is now under investigation. Preliminary results indicate that nearly

134                      THE MOLECULAR BASIS OF EVOLUTION

full activity is retained after the disulfide bridge between the first
and sixth half-cystine residue is cleaved.

The reduction experiments, and the proteolytic studies discussed
earlier, permit us to perform a certain amount of paper surgery on
the enzyme and to construct a two-dimensional picture of the max-
imum structural requirement for activity (Figure 69). It seems likely
that amputations of other parts of this structure may be possible
without inactivation. We can already safely conclude, however, that
considerable portions of the covalent structure of ribonuclease must
either be unimportant evolutionary vestige or involve aspects of
ribonuclease function and intracellular behavior about which we are
still in complete ignorance.

SH groups

Active after reduction /
of l-6 disulfide
   bdge

  L-----f------
Inactivated by cleavage here    t -:---- -i
with pepsin at pH 1.8                 Actwe after removal of
                          C-terminal residue with
                       carboxypeptidaae

Figure 69. A schematic drawing of the ribonuclease molecule in two dimensions
(see also Figure 62 ). Various experimental modifications of the native molecule
are indicated, together with the consequences of such modifications in terms of
enzyme activity. Also included are some results of studies on the comparative
structures of bovine and ovine pancreatic ribonucleases (see Chapter 7). The
data summarized in this figure make it tempting to suggest that much of or all
the N-terminal "tail" of the molecule, together with only a portion of the "core,"
might eventually be shown to be sufficient for catalytic activity. The "active
center" must obviously be a complicated three-dimensional structure but may not
necessarily involve more than a fraction of the entire protein. This drawing is
extremely speculative and is included here only to indicate some of the directions
that research on the biological activity of this enzyme may take in the future.
THE BIOLOGICAL ACTIVITY OF PROTEINS IN RELATION TO STRUCTURE        135


Before leaving the subject of enzymatic structure in relation to
function (and the reader must excuse my preoccupation with a spe-
cific enzyme about whose properties I happen to be able to write
with less labor than certain others of equal illustrative value), let us
consider a few aspects of noncovalent structure in terms of catalytic
activity.13 We have referred, in Chapter 5, to some of the physical
characteristics of ribonuclease under conditions of reversible dena-
turation by urea. The rather large changes that occurred in the
structure of the enzyme under such conditions (e.g., the intrinsic vis-
cosity of ribonuclease in 8 M urea shifts to about 0.035 as compared
with a value of 0.036 in dilute salt solution) made it of interest to ex-
amine whether or not activity was retained in 8 M urea solutions.
It was quite surprising to find that hydrolysis of RNA was unimpaired
and, indeed, even somewhat stimulated under these conditions of hy-
drogen bond disorientation. The conclusion was drawn that activity
was independent of organized, three-dimensional structure.  That
this conclusion might be premature, however, was suggested when
it was subsequently observed that low concentrations of phosphate
ions or other polyvalent anions (e.g. arsenate) could reverse the un-
folding effect of urea.  The natural substrate for ribonuclease, RNA,
is itself a polyanion and might also cause refolding of the enzyme
under the conditions of assay. Various technical difhculties having
to do with the spectral and optical properties of ribonucleic acid
made the direct test of this hypothesis difficult. However, model
substances such as polymetaphosphate and uridine3'-phosphate
(which is the product of ribonuclease attack on the synthetic sub-
strate, uridylic-2',3'+yclic-phosphate) could also cause a refolding of
the molecule as based on measurements of its spectrum, optical ro-
tation, and intrinsic viscosity (Table 6). The refolding was not com-
plete, as evidenced by the somewhat lower optical rotation of the
enzyme in 8 M urea in the presence of these agents as compared with
that of the native enzyme in water, and by an incomplete regenera-
tion of viscosity behavior. However, spectral differences were com-
pletely abolished.

Although these experiments suggested that complete "nativeness"
might not be essential, as indicated by the partial but significant ir-
reversibility of the optical rotatory and viscosity properties of the en-
zyme to anions, it appeared that the portion of the three-dimensional
structure of the enzyme which is responsible for the peculiar spectral
properties of several of the six tyrosine residues (page 120, Chap-
ter 5) must be in the proper configuration.

In an effort to support this hypothesis, the spectra of various ac-

136                        THE MOLECULAR BASIS OF EVOLUTION

TABLE 6
Some Physical Properties of Ribonuclease and Modified Ribonucleases

       Unfolding
Ribonuclease   Agent     Anion    b 6. lo-`.   bl;     %p/c b

Native             -          -

Native        8 M urea       -

Native        8Murea 0.4M
                   chloride
Native        8Murea O.lBM
               phosphate
Native        8 M urea  0.15 M
                      arsenate
Native        8 M urea  0.15 M
                uridylate
Oxidized            -          -

Reduced and car-
boxymethylated -          -
Pepsin inactivated -          -
Subtilisin digested -          -

-

1120    -71.7O    0.036
  0    -103.7"    0.085
110    -98.8O

1120    -81 .O"    0.050

11eo

0    -91.6O    0.116

  0
  0
11520

--80.7O

0.052

        0.149
-83.fP    0.039

The concentration of the proteins was %6-2.7 grams/liter for spectro-
photometric readings, S-30 grams/liter for polarimetric, and 14-15 grams/
liter for viscosimetric measurements.  All measurements were carried out
at pH 6-7.  Anions were added as their sodium salts.
The active derivative of ribonuclease produced by limited subtilisin diges-

tion was purified according to Richards (see page 13%).

n Change in extinction relative to the absorption of ribonuclease in 8 M
urea in the absence of salts, at 985 rnp.

b Reduced viscosity, in (grams/100 ml)-`. All viscosity measurements
were carried out in 0.1 M KCI.

tive and inactive derivatives of ribonuclease were examined.  It was
found that all the derivatives of ribonuclease that possessed activity
showed a normal "shifted' `spectrum, whereas all (those that were inac-
tive possessed the spectral characteristics of tyrosine residues with
unmodified resonance properties. We might tentatively conclude
from these studies that the presence of a shifted, "native," spectrum
leads to a "diagnosis" of catalytic activity and that factors which
cause changes in the spectrum may be expected to inactivate.

To summarize our impressions at this point, it appears that at least
one bond in the N-terminal and part of the C-terminal "tail" are dis-
pensable, and that one, ,and perhaps two, of the SS bridges may be
opened with impunity. Various findings suggest that a part of the

THE BIOLOGICAL ACTIVITY OF PROTEINS IN RELATION TO STRUCTURE         137


structure in the vicinity of the C-terminal "tail" has special impor-
tance, specifically the aspartic acid residue at position 121. Finally,
there appears to be good correlation between the presence of a
"shifted" spectrum, as occurs in the native enzyme, and activity.
Since the evidence is now rather convincing that such shifted spectra
may involve hydrogen bonding between carboxylate groups in the
protein and fhydroxyl groups on tyrosine residues, it may be sug-
gested, as one alternative, that the free carboxylate group of the
aspartic acid residue at position 121 is involved in such a linkage
and functions as one of the determinants of the three-dimensional
form of the active center of the enzyme.

The importance of considering the enzymatic activity of a protein
in terms of three-dimensional structure is strongly emphasized by an
especially intriguing aspect of ribonuclease chemistry, recently re-
ported by F. M. Richards8  In a continuation of his studies on the
enzymatically active derivative prepared by limited subtilisin diges-
tion, he has investigated the nature of the noncovalent bond or
bonds (see page 132) which hold the N-terminal "tail" peptide to the
macromolecular portion of the protein. The attachment may be
broken by treatment of the derivative with trichloroacetic acid, and
the N-terminal peptide fragment may be separated by dialysis and
purified by electrophoretic methods. Richards has shown that both
the peptide and the macromolecular component are inactive, but that
upon mixing the two activity is completely regenerated as shown in
Figure 70.

Richards has also examined the spectral properties of the separate
fragments and of the reconstituted, active mixture.  The 20-amino
acid peptide, which contains no tyrosine, of course shows no absorp-
tion at 280 rnp. The large piece, however, has the expected absorp-
tion in this region of the ultraviolet, but shows none of the spectral
shift observed with the native protein or with the subtilisin-digested
enzyme before separation of the two fragments.  Its spectrum is ex-
tremely similar to that observed for native ribonuclease in urea or
after treatment with inactivating reagents such as pepsin.  Upon
mixing the two components, the shifted tyrosine spectrum reappears
almost completely, indicating that the peptide "tail" plays an impor-
tant part in the determination of that part of the three-dimensional
structure of the protein which is responsible for the anomalous tyro-
sine absorption.

We have, in Richards' experiments, an instance of biological func-
tion in a relatively small peptide which is strongly reminiscent of the
action of some of the peptide hormones of the anterior and posterior

138                         THE MOLECULAR BASIS OF EVOLUTION

-

          I        A I        I
   5        10         " 25       50
Volume of N-terminal peptide solution added, ~1.

Figure 70.  The regeneration of ribonuclease activity by the addition of the N-
terminal peptide (which is split off from the native molecule by subtilisin) to the
inactive macromolecular "core."  As described in the text, the "core" was pre-
cipitated from the subtilisin digest with trichloroacetic acid. The N-terminal
polypeptide, containing twenty amino acid residues, causes complete regenera-
tion of activity when it has been added in amounts approximately equimolar with
the concentration of the macromolecular component.   From F. M. Richards,
Proc. Sot. Natl. Acad. Scl. U.S., 44, 162 ( 1958).

parts of the pituitary gland. The complete regeneration of ribonu-
clease activity occurs at concentrations of the order of 10-o M, a level
within quite reasonable limits for hormonal action.  On the basis of
these studies alone, the N-terminal peptide fragment does not neces-
sarily have to be an intrinsic part of the active center.  It may con-
ceivably function only as a determinant of proper folding in the rest
of the molecule. Whatever its role, the forces that bind this small
polypeptide to the body of ribonuclease must be multiple and highly
specific.

In addition to being an essential partner to the catalytic "center" of
ribonuclease, Richards' peptide fragment also appears to possess the
ability to stabilize the rest of the molecule. The active subtilisin
derivative is stable in aqueous solution, reflecting the considerable
strength of the association between the two components of the com-
plex. In urea, however, where these components dissociate, as they
do in trichloroacetic acid, the macromolecular fraction is rapidly de-
natured and cannot be reactivated by the addition of the peptide,
even following removal of the dissociating agent. The specific, cata-

THE BIOLOGICAL ACTIVITY OF PROTEINS IN RELATION TO STRUCTURE         139


lytically active configuration in the intact subtilisin derivative is thus
dependent on the participation of certain bonds and amino acid resi-
dues contributed by the macromolecular body of the derivative, sta-
bilized by the peptide "tail" fragment.

It is much too early to attempt to synthesize the various observa-
tions on the chemical basis of ribonuclease into a coherent whole.
It is a tribute to the progress of modern protein chemistry, however,
that we can even hope to do so in the near future. The pattern al-
ready emerging for ribonuclease, in which certain widely separated
areas of the protein are strongly implicated as components of an
active center, and other portions (at least one disulfide bond and an
assortment of amino acid residues Nat various points in the chain) are
not, is an object lesson in the necessity for reorienting our thinking
about proteins from planar to solid geometry.

REFERENCES

1. Described in Crystalline Enzymes, John H. Northrop, Moses Kunitz, and
Roger M. Herriott, second edition, Columbia University Press, New York,
1948.

2. J, Lens, Biochim. et Biophys. Acta, 3,367 ( 1949).
3. P. H. Bell, K. S. Howard, Ft. G. Shepherd, B. M. Finn, and J. H. Meisen-
helder, J. Am. Chem. Sot. 785059 ( 1956).
4. W. F. White and A. M. Gross, J. Am. Chem. Sot., 79, 1141 (1957).
5. K. Hofmann, T. A. Thompson, and E. T. Schwartz, 1. Am. Chem. Sot., 79,
6087 (1957).

8. R. A. Boissonnas, S. Guttmann, J, P. Waller, and P. A. Jaquenoud, Er-
perientiu, 12, 446 ( 1956).

7. E. L. Smith, Federation Proc., 16, 801 ( 1957).
8. Ft. L. Hill and E. L. Smith, Biochim. et Biophys. Acta, 19, 376 ( 1956).
9. F. M. Richards, Proc. Natl. Acad. Sci. US., 44, 162 ( 1958).
10. M. Uziel, W. H. Stein, and S. Moore, Federation Proc., 16, 263 (1957).
11. G. Kalnitsky and W. I. Rogers, B&him. et Biophys. Acta, 20, 378 ( 1956).
12. L. Weil and T. S. Seibles, Arch. Biochem. Biophys., 54, 368 (1955).
13. C. B. Anfinsen, Federation Proc., 16, 783 (1957); M. Sela and C. B. Anfin-
sen, Biochim. et Biophys. Acto, 24, 229 (1957); M. Sela, C. B. Anfinsen,
and W. F. Harrington, Biochim. et Biophys. Actu. 26, 502 (1957); M. Sela,
F. H. White, Jr., and C. B. Anfinsen, Science, 125, 691 (1957).

SUGGESTIONS FOR FURTHER READING

Anfinsen, C. B., and R. R. Redfield in Advances in Protein Chemistry, volume
11, Academic Press, New York, 1956.

140                           THE MOLECULAR BASIS OF EVOLUTION

Enzymes: Units of Biological Strucfure and Function (0. H. Gaebler, editor),
Academic Press, New York, 1956.
Molecular Structure anal Biological Specificity, edited by L. Pauling and H. A.
Itano, Publication 2, American Institute of Biological Sciences, Washington,
D. C., 1957.

Symposium on Protein Structure (A. Neuberger, editor), John Wiley & Sons,
New York, 1958.

THE BIOLOGICAL ACTIVITY OF PROTEINS IN RELATION TO STRUCTURE         141


chapter 7

SPECIES VARIATION

IN

PROTEIN STRUCTURE

   0 ne of the major questions to be answered in ar-
riving at a clear understanding of the phylogenetic relationships be-
tween different forms of life is whether there exist identical, or
closely homologous, genes in widely separated species, or whether
similarities in phenotype are due to analogous genes which determine
equivalent appearance or function by different pathways. The tech-
niques of experimental genetics permit us to compare the genetic
makeup of only those organisms that can be successfully crossed.
We know, for example, that the eye pigments of a wide variety of
species contain the same light-sensitive compound. However, we
have no genetic way of testing whether the synthesis of this com-
pound in different organisms is under the control of the same set of
genes, structurally modified perhaps in some slight manner but still
essentially identical, or whether completely different genes are in-
volved which act in concert to achieve the same end result.

All genetic analyses depend on the availability of some recogniz-
able phenotypic character, be it morphological, functional, or meta-

142

bolic. When the character being used as the criterion for the pres-
ence or absence of a functional gene or set of genes is a gross mor-
phological one, we cannot attempt to distinguish homology of genes
from analogy of genes.  This is true even in those instances in which
we can demonstrate the presence, in widely separated species, of
identical chemical structures such as the creatine phosphate in the
tissues of all vertebrates.  The production of such a substance could
be carried out by analogous, rather than homologous, enzyme sys-
tems in the different species, and the genes that exert the basic con-
trol on the synthetic process might conceivably be quite different in
terms of chemical organization. There does appear to be one ap-
proach, however, which might give definitive information about the
persistence of particular genes throughout the phyla. The tech-
niques of isolation and structural analysis now available enable the
protein chemist to make exact comparisons of proteins isolated from
a wide variety of biological sources. If we accept the hypothesis
that the proteins represent a primary, if perhaps a fuzzy, "print" of
genetic information, we may then conclude that two organisms have
the same gene, or gene set, when they both contain the same protein
molecule.  (Many readers will not be willing to swallow, whole, the
thesis that proteins represent the direct translation of genetic infor-
mation. We shall consider some of the arguments, pro and con, in
Chapter 8. )

It is clear that we must expect to find differences between the
"same" protein in various species.  This is particularly true since, as
we have discussed in the past chapter, certain parts of biologically
active protein molecules are relatively more "dispensable" than oth-
ers from the standpoint of function.  Mutations that lead to changes
in the sequence of amino acids in the last three C-terminal amino
acids of ribonuclease, for example, might cause little change in the
life expectancy or fertility of the affected animal.  On the other hand,
a mutation that led to a critical modification in the sequence of the
"active center" of the enzyme might well be lethal, and the gene, so
mutated, would not be perpetuated.
A comparison of the structures of homologous proteins (i.e., pro-
teins with the same kinds of biological activity or function) from
different species is important, therefore, for two reasons. First, the
similarities found give a measure of the minimum structure which
is essential for biological function.  Second, the clifferetlces found
may give us important clues to the rate at which successful mutations
have occurred throughout evolutionary time and may also serve as an
additional basis for establishing phylogenetic relationships.

SPECIES VARIATION IN PROTEIN STRUCTURE                           143


The proteins and polypeptides for which complete comparisons of
covalent structure can be made at the present time are relatively few
in number. We ,can list, in this category, insulin, adrenocorticotropin,
melanotropin, vasopressin, oxytocin, and hypertensin. The complete
structure of glucagon (hypoglycemic factor) has been elucidated,
but no comparisons of this hormone from different species have as yet
appeared. Among the enzymes, only ribonuclease has been suffi-
ciently studied to permit essentially complete comparison of species
differences. The structure of a tetradecapeptide portion of cyto-
chrome c, in the vicinity of the heme prosthetic group, has been ex-
amined for a fairly large number of organisms.  Finally, there exists
a large literature on the composition, end group, and end sequence
analysis of various sets of homologous proteins, and on their physical,
enzymatic, and immunologic properties, which allows us to make at
least some educated guesses about similarities and differences.

Ribonuclease and the "Fingerprinting" Technique

The detection and study of chemical differences between homolo-
gous proteins is generally carried out by one or another variety of
"fingerprinting" technique.  In essence, this involves the use of re-
producible physical methods for the separation of peptide fragments
produced by digestion of the proteins with proteolytic enzymes.
After establishing the distribution pattern of these fragments for the
original "reference standard" protein, differences obtained on digests
of the protein from other biological sources may then be easily de-
tected, and the nature of the chemical modification may be deter-
mined by classical methods of amino acid and sequence analysis.
An example of a "fingerprint" comparison is presented in Figures 71
and 72. This figure shows the patterns for beef and sheep ribonu-
cleases. The differences are quite clear and are completely repro-
ducible from digest cto digest. In this instance, digestion was carried
out first with trypsin and subsequently with chymotrypsin. The
protein was oxidized with performic acid prior to digestion to avoid
steric complications which might be introduced by the disulfide
bridges.

The sort of fingerprinting used here is rapid and technically sim-
ple and serves, adequately, for the preliminary detection of differ-
ences. Indeed, if we are happy with micro techniques of the sort
that served Sanger and his colleagues so well in their studies of in-
sulin structure, a complete comparison of the corresponding peptides

144                         THE MOLECULAR BASIS OF EVOLUTION

Chromatography. using
n-butanol-acetic acid-water; 4~15 ------+

Figure 71. A "fingerprint"
pancreatic ribonuclease.  of a proteolytic enzyme digest of oxidized bovine
the left of the figure.  An aliquot of the digest was applied in a small spot at
        This material was then subjected to descending paper

chromatography, as indicated by the arrow, and, after allowing the solvent to
evaporate, the pnper was moistened with buffer solution and subjected to high-
voltage electrophoresis. The sheet of paper was then sprayed with ninhydrin
solution to stain those areas containing peptides.  These areas were cut out
(from a lightly stained paper), and the peptides were eluted.  The amino acid
composition of each was determined, after hydrolysis with acid, by paper chroma-
tography. The composition of the various peptide components is given in Table
8. For further details consult an article by C. B. Anfinsen, S. E. G. Aqvist,
Juanita P. Cooke, and Bijrje Jonsson, J. BioE. Chem., 234, No. 5 (1959).


/,
cl
I

30 3b

Chromatography, using
n-butanol-acetic acid-water; 4:1:5 --

Figure 72. A "fingerprint"
nuclease.        of performic acid-oxidized ovine pancreatic ribo-
   The techniques employed were the same as those described in Figure
71, and the composition of the various peptide components is given in Table 8.

shown in such a fingerprint can be made with relative assurance.
The use of such methods always involves an uncertainty in regard
to the minor ninhydrin-positive components that routinely plague the
paper chromatographer, and a certain amount of personal judgment
is frequently involved in deciding whether a trace peptide compo-

Asp
Thr
Ser
Ghl
Pro
GlY
Ala
CYS
Val
Met
Ileu
Leu
`Jh
Phe
T .YS
His
A rg
__-

15         15.65          15.20     -1 (?)
10         9.77           9.06       -1
15         13.95          16 15      +e
12         11.75           13.3      +1to+e
4          3.!)5           3.06
3         3. 03          3. I4
1%         11.87          11.1        -1
4          3.36           3.60
9          8.48          8.87
4         3.62           3.73
3         1.83           1.76
2         2.05           1.99
6          5.7           5.71
3         3.27          3.41
10         11.25           9.88       -1
4         3.51           3.83
4         3.76          3.78

9 -~

a From unpublished experiments of S. Aqvist, C. B. Anfinsen, J. Cooke, and
B. Jiinsson.

146                       THE MOLECULAR BASIS OF EVOLUTION       SPECIES VARIATION IN PROTEIN STRUCTURE                          147

nent is due to a fleck of dirt on the paper or to a bona fide structural
fragment. For this reason the purist will often prefer the use of ion
exchange columns over paper chromatography and electrophoresis,
since he can then make more quantitative estimates of the recovery
of fragments in relation to theoretical expectation.  The latter course
is obviously to be recommended in principle. However, when a
large series of proteins are to be compared, and when experience has
indicated the limits of error involved, the more rapid and flexible
paper method will probably be employed to establish the major gen-
eralities of structure.

The study of species differences in the structure of enzymes is of
special interest because with these proteins we can, in many in-
stances, consider such variations in terms of the ability of the enzyme
to catalyze a specific, chemical reaction. When, for example, a suc-

TABLE 7

Amino Acid Analyses of Beef and Sheep Pancreatic Ribonuclease"

Amino
Acid

Beef Enzyme

Sheep Enzyme

      Average Observed  Average Observed Estimated
       Number of Resi-   Number of Resi-  Changes in
      dues per Mole   dues per Mole    Sheep
"Theory"  (mol. wt. 13,683)  (mol. wt. 13,683)   Enzyme
                                            ~.__


TABLE 8
Analyses of Peptides from Fingerprints of Trypsin and Chymotrypsin
Digestions of Oxidized Pancreatic Beef and Sheep Ribonuclease'

   Beef No.             Composition           Sheep No.

     3b      Lysz,Glu,Thr,Ala3   (beef)
          Lyr,Glu,Ser, Ala   (sheep)              3a and 3b
     6b      Phe+Glu,Arg.                       6b
      -f      Asp,Glu,Thr,Ser3,Met,His                     -0

      -c      Asp,Alan,Sera,Tyr                          -E

     16       Cys,Asp,Glu,M&,Lys                      16

      1       Ser,Arg                                 1

      2      Asp,Leu,Thr,Lys                           -

     13       AspArg
          Asp,Leu,Thr,Glu,Arg   (sheep)               10

      9      Cys,Asp,Val.Pro,Thr,T.ys,Ph-                  9

     18       Glu3,Vala,~,,-u,Serz,His, Asp,Alaz,Cys            18

     14       Asp,Cys,AIa,Val,Lys                      14

     PO       Cys,Aspz,Glu,Gly,Thr,Tyr                  20

      7       Glu,Ser,Tyr                              7

     13        Thr,Ser,fiIet                             12

     21       Cys,Asp,Ileu,Thr,Ser,Arg                    21

     15       Glu,Gly,Ser2,Thr,T,ya                       1.5
     Ah      Cys,Asp,Ala, Z'yrt,Pro,Lys                8b

      SC      Asp,Glu,Ala, Thr2.Lys
      -       Lys,Glu,Ala, Thr                         23

     28      His,VaI,Ileu~,Cys,Asp,Glu,Gly,Ala,Pro,Tyr        as

      4       VaZz,Pro,His,Phe                         4

     a2       Asp,Ala,Ser,Val                        22

From carbohenzoxy- Lya,Ser,Glu,Ala,Phe,Arg                  s3
oxidized sheep
ribonuclease    Cys,Lys,His,Asp,Glu,Ser,Met,Thr,Ala,Tyr,Arg     Sl
a Amide nitrogens cannot be assigned to specific glutamic or aspartic acid residues
since they are split off by the acid hydrolysis prior to chromatography.
b According to earlier observations (see Figure 60, Chapter 5). cleavages should have
occurred between phenylalanine and glutamic acid in peptide 6 and in such a way as
to remove the lysine residue from peptide 8.  However, only traces of free phenylalanine
and lysine were detected on the fingerprint patterns.
c These two peptides were not detected hy the ninhydrin-staining reaction but have
been accounted for in peptide Sl. which was prepared by trypsin digestion of the
carbobenzoxylated polypeptide chain (see Chapter 5), rather than by combined trypsin-
chymotrypsin digestion of oxidized ribonuclease.
The spots labeled 5 and 17 in the fingerprint of digests of beef ribonuclease were
present in such small quantities that their amino acid compositions could not be deter-
mined with any assurance.  Th e Same was true for the component labeled "X" in the
                      .
beef ribonurlease fingerprint.
The amino acids on the paper rhromatograms (Figure 73) which gave an unusually
strong ninhydrin reaction are italicized.  The subscripts, when present, indicate
the number of moles of each amino acid in the peptide under consideration, as

cessful mutation has occurred which leads to the substitution of a
charged amino acid residue for an uncharged one in a sequence, this
information permits us to make some helpful conclusions regarding
the nature and location of the binding site for substrate molecules on
the enzyme surface.

The amino acid analyses for bovine and sheep pancreatic ribonu-
clease are shown in Table 7. These data show that sheep ribonucle-
ase contains less lysine, threonine (and perhaps less aspartic acid 1,
and more serine and glutamic acid than does the beef enzyme.  End
group analysis indicates that both proteins contain N-terminal lysine,
and sedimentation constants determined in the ultracentrifuge are es-
sentially identical. When the peptides separated by the fingerprint-
ing procedure illustrated in Figures 71 and 72 were eluted and
analyzed, qualitatively, for amino acid composition, the results sum-
marized in Table 8 were obtained. Examples of the chromato-
graphic comparison of hydrolysates of a few sheep and beef peptides
are shown in Figure 73. The peptides obtained from hydrolysatcs
of the beef protein are those to be expected from the combined nc-
tion of trypsin and chymotrypsin on oxidized ribonuclease, as may be
deduced from the partial formula for this polypeptide shown in Fig-
ure 60. Corresponding peptides from the sheep material are also
easily assignable to particular areas of the formula.  Those sequences
in sheep ribonuclease which differ from the beef structure may be al-
located to specific areas of the chain on the basis of their composi-
tions. At one point in the chain of the sheep enzyme, the absence
of a trypsin-sensitive sequence involving lysine had precluded cleav-
age where the beef enzyme wus split, and a single, longer peptide
(sheep peptide 10 Figure 73e), embodying two of the beef frag-
ments, resulted.

The studies relating various aspects of structure to function which
were reviewed in the last chapter have suggested that the disulfide
bridge joining half-cystines 1 and 6 may be reduced without
complete destruction of catalytic activity. The species variation oc-
curring at residue 37, where a positively charged amino acid, lysine,
has been replaced by a negatively charged amino acid, glutamic acid,
also suggests the unessentiality for substrate adsorption and hydroly-
sis of this part of the polypeptide chain.
-___ ___  ~___
determined for hcrf ribonu&=asr (Figure 60, Chapter 5). Thus for Ijcef peptitlr :$I,
the earlier quantitative an:IIysrs. xhirh drmonstrated the presence of two rcsitlllrs
of lysinc alId three of alanine for each residue of glutamic acid and threoninr, \vere
confirmed by thr staining reaction which was correspondingly stronger for the t 11-o
former amino acids.

148

THE MOLECULAR BASIS OF EvoLUTlON        SPECIES VARIATION IN PROTEIN STRUCTURE                           149


(f)

Figure 73. Two dimensional chromatography of the amino acids
produced by acid hydrolysis of some of the peptide components
shown in Figures 71 and 72.  (a) Peptide 22 from beef ribonuclease.
This is the C-terminal tetrapeptide sequence of the enzyme and has the
structure Asp.Ala.Ser.Val.  (b) The C-terminal tetrapeptide of sheep
ribonuclease, having the same composition as that obtained from the
beef enzyme. (c) Peptide component 18 from bovine pancreatic
ribonuclease. This peptide is derived from residues 47 through 61
in the polypeptide chain. (d) The amino acid composition of
peptide 18 from the ovine enzyme.
tical from both species.  (e) P pt'd Peptide 18 appears to be iden-
                      e I e component 10 from sheep ribo-
nuclease.  This peptide is derived from the amino acids in positions
34 through 39. The lysine residue, present in the structure of bo-
vine ribonuclease within this portion of the sequence, has been re-
placed by glutamic acid in the sheep enzyme. The cleavage with
trypsin which occurs at residue 37 of the beef enzyme can thus not

occur for the sheep protein, and a single hexapeptide sequence is obtained instead of a tetrapeptide and dipeptide.
ponent 3b from sheep ribonuclease.                                  (f) Peptide com-
             The peptide represents the N-terminal heptapeptide sequence of the enzyme.
3b from the beef fingerprint, the N-terminal heptapeptide sequence.                     (g) Component

by the replacement of serine by threonine.           The beef enzyme differs, in this region, from the sheep enzyme
                  See Figure 62 for details of structure.


Adrenocorticotropin IACTH)

The complete amino acid sequences are known for corticotropins
isolated from the anterior pituitary glands of three different species,
pig, beef, and sheep. The structure of sheep ACTH was discussed
in the last chapter, and the sequences shown in Table 9 include only
those areas of the three molecules where differences are to be found.
Although some difference between the content of amide nitrogen
groups has been reported for the three species, these are not included
in the figure since it has not been possible to rule out, with certainty,
the possibility that these variations are due, in part, to the rigors of
the isolation and purification techniques employed.

TABLE 9

Variations in Amino Acid Sequences Among Different Preparations of
ACTH
                           Residue No.

Preparation

Species    525 26 27 528 `29 30    31 33 33

@-Corticotropin  sheep
           beef* 1
Corticotropin A pig

Ala.Gly.Glu.Asp.Asp.Glu   Ala.Ser.Glu.NHr
Asp.Gly.Ala.Glu.Asp.Glu   Leu.Ala.Glu
                              -__

' Identity with sheep hormone not absolutely certain but very probable as
judged from the nearly complete sequence analysis by J. S. Dixon and C. H. Li
(personal communication to the author).

Two points are of particular interest in regard to the sequences
shown. First, the corticotropins of sheep and beef are identical and
differ from that of the pig. This finding is consonant with the
closer phylogenetic relationship of sheep and cows to each other than
of either to pigs. Second, chemical differences are found only in that
portion of the ACTH molecule which has been shown to be unessen-
tial for hormonal activity. Genetic mutations leading to such differ-
ences might, therefore, not be expected to impose significant disad-
vantages in terms of survival, and these genes could become estab-
lished in the gene pools of the species.

Melanotropin (MSH 1

Melanotropin, like the other hormones considered in this chapter,
is a typically chordate polypeptide. Indeed, the demonstration of
melanocyte-stimulating activity in extracts of tunicates constitutes an

152                          THE MOLECULAR BASIS OF EVOLUTION   SP~~,~~ VARIATION IN PROTEIN STRUCTURE


important bit of evidence supporting the assignment of these organ-
isms to the main thoroughfare of evolution between invertebrates and
chordates.

Melanotropins have been isolated in pure form from both pig and
beef pituitaries (posterior-intermediate lobes). Only a single poly-
peptide having MSH activity has been isolated from beef tissues
whereas two different chemical entities termed (Y and p-MSH have
been isolated and characterized from hog pituitaries.  The struc-
tures of these substances is given in Figure 74 together with that for
porcine ACTH. We shall consider further the provocative similar-
ity between the structures of MSH and ACTH in Chapter 10 in re-
lation to protein biosynthesis. This similarity is undoubtedly re-
sponsible for the fact that adrenocorticotropic hormone exhibits
marked melanocyte-stimulating activity.
Beef P-MSH differs from porcine P-MSH only in the replacement
of the glutamic acid residue in position 2 by serine.  Porcine (Y-MSH,
however, is considerably different from both the other hormones and
is actually identical with the sequence of the first thirteen amino
acids in pig ACTH, except for the presence of a masking, acyl, group
on the N-terminal amino group and an amide nitrogen group at the
C-terminus. Lee and Lerner,* who isolated a-MSH, have suggested
that this form of the hormone is the major one in pituitary extracts
since it accounts, in their experiments, for the largest share of activ-
ity, although other investigators have not so far confirmed the pres-
ence of a-MSH.*
Bovine P-MSH possesses considerably less biological activity than
does porcine /3-MSH, and, until comparisons can be made of synthetic
samples of these two materials, this difference in potency must be
ascribed to the amino acid substitution at position 2.

Insulin

Sanger and his colleagues have determined the amino acid se-
quences for insulins derived from five different species.  Differences
* C. H. Li and his colleagues have also recently isolated a-MSH from both
porcine and bovine pituitary glands.
in both species.         The structures were found to be the same
       ( Personal communication. )

The sequence of a-MSH has been confirmed by total synthesis in the lab-
oratories of Klalls Hofmann and of R. Boissonnas. The activity of the synthetic
material is critically dependent on the presence of the acetyl group on the
N-terminal serine residue. Thus, in the experiments of Roissonnas and his co-
workers, the activity of the acetylated polypeptide was approximately 70 times
greater than before acetylation.
Boissonnas. )         (Persona1 communications from Hofmann and

154

THE MOLECULAR BASIS OF EVOLUTION

. . . CySOsH.Ala.Ser.Val . . . (beef)
. . . CyS03H.Thr.Ser.Ileu . . . (pig)
. . . CySOaH.Ala.Gly.Val . . . (sheep)
. . . CyS03H.Thr.Gly.Ileu . . . (horse)
. . . CySOsH.Thr.Ser.Ileu . . . (sperm whale)
. . . CySOaH.Ala.Ser.Thr . . . (sei whale)

Figure 75. Species differences in the amino acid sequences of insulins from
various biological sources. These differences all occur within the disulfide "loop"
of the A chain.

were limited to the amino acids within the disulfide "loop" of the A
chain (Figure 75) and the R chain was identical in all instances (see
Figure 65). Of the five insulins examined, only those from the pig
and the sperm whale exhibited the same structure.  The fact that all
the observed differences were restricted to the sequence within the
"loop" suggests that the amino acids in this region of the insulin
molecule are not particularly critical ones from the standpoint of
hormonal activity. On the other hand, several investigators have ob-
tained evidence indicating that insulin loses its biological activity
when disulfide bridges are reductively cleaved. The species differ-
ence results with insulin suggest that only the steric configuration of
the loop is essential and that the "spacers" between the half-cystine
residues may be varied through mutation of the corresponding gene
or genes. It is of interest that sequence variations have not been ob-
served in the C-terminal region of the B chain, an area which does
appear to be essential for activity as shown by the inactivation of
insulin following the removal of the last seven residues in the chain.

Hypertensins

The hypertensins are peptides present in serum which possess
pressor activity.  Two forms have been isolated from horse serum,'
the first being convertible to the second by the action of an enzyme
in plasma according to the equation:

Asp.Arg.Val.Tyr.Ileu.His.Pro.Phe.His.Leu -+

Asp.Arg.Val.Tyr.Ileu.His.Pro.Phe. + His.Leu

The precursor compound is not active in an in vitro test system, but
after cleavage of the critical peptide bond it becomes active both
in viva and in vitro. The precursor form has also been isolated from

SPECIES VARIATION IN PROTEIN STRUCTURE                           155


p . Ala . C.lu . Cy . His .
?        s

CH3 dH-CH, CH3 AH-CH,
/y;,y/
Hc'u;yH
     . .

CH3 FHz   dH2 CH,
  CHz     CHz

   COOH     COOH

`t'hr . Vat . Glu .

. Ala . Glu . Cy . His . Thr . Val . Glu . Lys

. .         . .

CH3 CH2
   I    CH, tiH3
         I
   CH2     CH2

   COOH     COOH

bovine serum3 and is identical with that from horse serum except
for the substitution of isoleucine by dine. These two amino acids
are extremely similar in structure and the substitution in this case
represents one of the more minimal changes possible given the avail-
able selection of naturally occurring amino acids.

Cytochrome c

or

The electron-transporting enzyme, cytochrome c, furnishes one of
the most interesting examples of species variations in protein struc-
ture, since it has been isolated in pure form from a particularly wide
assortment of species. Unfortunately, studies on variations in se-
quence have been carried out for only a relatively small portion of
the total chain, and preliminary amino acid analyses indicate that
there may be modifications elsewhere in the molecule as well.  Never-

Figure 76. Structure of the peptide-porphyrin compound isolated from trypsin
digests of cytochrome c.
1024 (1954).     After H. Tuppy and G. Bodo, Monatsch. Chem., 85,

Beef
Horse
Pig I

Salmon

Chicken

Silkworm

Yeast

Rhodospirit-

    NH2       NH2
    I       I
. . . Vat.Glu.Lys.Cys.Als.Glu.Cys.His.Thr.V~t.~lu.l,ys . .

    NH*       NH2
   I
. . . V;~I.Gtu.I,ys.Cy.s.Al:~.Gtu.Cys.~lis.Tt~r.V~~I.Gtu . . .

    NH2       Nlt2
    I       I
. . . V~~t.Gtu.Lys.Cys.Ser.Gl~~.~~ys.lIis.Tt~r.Vat.~~tu . . .

    NH2       NIl2
    I       I
. . . Val.Gtu.Arg.Cys.Ata.Glu.Cys.His.Tt~r.Val.Glu . _ .

Phe.Lys.Tt~r.Arg.Cys.Glu.IRu.Cys.Hirc.Tt~r.Vat.Glu . . .
                              NH2
. . . Lys 1

or .Cys.Leu.At:~.Cys.lti.s.`~t~r.Phe.Asp.Gtu.Gty.Ala.Asp.I,ys . . .
Arg

lum ruhrum

         1,y.s
Common sequcwe: or 1 .Cys.;Y.Y.Cys.lIis.Ttlr.
          Aw

Figure 77. Variations in the sequence of the polypeptide chain of cytochrome c
from species to species. From H. T~~ppy, Symposic~~ on Protein Structure (A.
Neuberger, editor), John Wiley & Sons, 1958.

THE MOLECULAR BASIS OF EVOLUTION        SPECIES VARIATION IN PROTEIN STRUCTURE                           157

156


theless, the investigations of H. Tuppy, S. PaIt&, and G. Rodo,4 on
this ubiquitously distributed enzyme, lend the most convincing sup-
port to the argument that certain units of the universal gene pool
may be extremely ancient.

In the degradative studies of the enzyme, advantage was taken
of the finding of H. Theorel15 that the heme prosthetic group of cyto-
chrome c is attached through stable thioether linkages to the protein
moiety. After proteolytic degradation with trypsin (and in later
studies with pepsin), that portion of the polypeptide chain which
is attached to the heme nucleus was isolated. The structure of the
heme-peptide compound as determined for cytochrome c from horse
heart tissue is shown (in two alternatively possible forms) in
Figure 76.

In subsequent investigations corresponding sequences from cyto-
chrome obtained from a variety of other species have been elucidated
as shown in Figure 77.

Somatotropinr I Growth Hormones) and Prolactin

Pure growth hormone has been isolated from the pituitaries of the
species listed in Table 10 by C. H. Li and his colleagues. These
proteins have been subjected to both physical and chemical study,
and, although even partially complete sequences are not yet available,
a great deal can already be said about species variability.  Molecular
weights vary over nearly a twofold range, and the differences in the
number of chains and the cystine content are striking.  The beef and
sheep hormones are, as in the case of several other proteins we have
discussed earlier, quite similar, reflecting once again the close phylo-
genetic relationship between these two species.

The prolactins of sheep and beef are also extremely similar (Table
11 ), the only difference between them so far observed being a slightly
greater tyrosine content in the beef hormone.  The absence of a
chemically detectable C-terminal amino acid residue is another ex-
ample of "masked" end groups.
known.           The nature of the masking is un-

Hemoglobin

The hemoglobins have been studied, from the phylogenetic point
of view, perhaps more than any other class of proteins.  Most of

158                       THE MOLECULAR BASIS OF EVOLUTION

TABLE 10
N- and C-Terminal Sequences of Somatotropins from Various Species'

                   Terminal Sequences
                                              -

Somatotropins         Amino End        Carboxyl End

  Bovine

  Ovine

  Whale
  Monkey
  Human
-___

Phe.Ala . . .
Ala.Phe.Ala . . .
Phe . . .
Ala . . .
Phe.AspNHt.Lys . . .
Phe.Ala.Thr . . .
Phe.Ser.Thr . . .

. . . Ala.Phe.Phe

. . . Try.Ala.Phe

. . . Leu.Ala.Phe

. . . Ala.Gly.Phe
. . . Tyr.Leu.Phe

Some Physicochemical Properties of Various Somatotropins

Physicochemical
Characteristicsb

Bovine   Ovine

Whale

Monkey   Human

s20,w             3.19     2.76     2.84     1.88     9.47
D20,w x 10'         7.23     5.25     6.56     7.20     8.88
v20              0.76     0.733    0.737    0.796    0.739
Molecular weight    45,000   47,800   39,900   45,400   27,100
f/f0              1.31     1.68     1.45     1.57     1.23
PI               0.85     6.8      (5.9      5.5      4.9
Cystine            4        5        3        4        4
N-Terminal
Residue(s)       Phe,Ala  Phe,Ala    Phe     Phe     Phe
C-Terminal
Residue           Phe     I'he     I'he     Phe     I'he

              ___-
8 From C. H. Li, Symposium 012 Protein Structure (A. Neuberger, editor),
John Wiley & Sons, 1958.
b S~O,~ in Svedhergs; D~o,~ in cm.2/sec.; p in ct./gram; f/f@, dissymmetry
constant; PI, isoelectric point; cystine in residues per mole.  For details on
the determination and significance of the physical constants consult the
volumes entitled The Proteins (H. Neurath and K. Bailey, editors), Academic
Press, 1953, 1954.

the available information on the hemoglobins has to do with the
chemical and spectrophotometric characteristics of the various pros-
thetic groups and with oxygen and carbon dioxide-combining proper-
ties. Consequently, this information is not directly pertinent to our
present discussion of species differences in protein structure. Re-

SPECIES VARIATION IN PROTEIN STRUCTURE                         159


TABLE 11
Some Physical and Chemical Properties of Prolactin from Ovine
and Bovine Pituitary Glands'
Physical and Chemical Properties         Ovine        Bovine

Molecular weight
Sedimentation-diffusion
Osmotic pressure
Analytical data
Diffusion coefficient (Dzs)
Sedimentation constant (Szs,,)
Partial specific volume (V20)
Isoelectric point, pH
Specific rotation
Partition coefficient (%butanol/O.%? %
  aqueous trichloroacetic acid)
Tyrosine, y0
Tryptophan, ye
Cystine, residue/mole
N-terminal amino acid
C-terminal amino acid

24,200
26,500        46,000
24,100
8.44 x 10-7
2.19
0.739
5.73           5.73
-40.5O       -4O.P

1.58          2.07
5.26          6.62
1.69           1.75
3             3
Threonine      Threonine

none            none

s From C. H. Li, Symposium on Protein Structure (A. Neuberger, editor),
John Wiley & Sons, 1958.

cently, however, following the important initial studies of R. Porter
and F. Sanger,6 a number of investigators have begun to examine
amino acid sequences in the hemoglobins.  Such studies are becoming
increasingly more meaningful as the result of physicochemical inves-
tigations on the number of peptide chains per molecule and on the
size of the monomer subunit, It now appears that, with the possible
exception of foetal hemoglobin and the hemoglobin of the chicken
(perhaps birds in general), the vertebrate hemoglobins contain two
types of chains. These are present, under physiological conditions,
in the form of a molecule with a molecular weight of about 65,000,
composed of four chains, two of each type, held together through
noncovalent linkages.  (The earlier results, which indicated the pres-
ence of six chains in the hemoglobin of the horse, are probably in-
correct on the basis of recent electrophoretic and ultracentrifugal in-
vestigations. )  Valine is the N-terminal amino acid residue on both
polypeptide chains of the vertebrate hemoglobins so far examined,
except for the goat, sheep, and cow, in which one of the chains be-
gins with methionine. A summary of the available end group data
is given in Table 12. It is far too early to attempt to make any

160                          THE MOLECULAR BASIS OF EVOLUTION

TABLE 12

End Group Data on Vertebrate Hemoglobins

Species

N-Terminal Amino Acids or Sequences8

Human adult'*3          Val.T,eu
Human foetaIl*          Val.-
Dog2                 Val.Leu
Horse,**2*4 pig*           Val.Leu
COW,`*? goat,"2 sheep',2      Val.Leu
Guinea pig2            Val.Leu
Rabbit,* snake*           Val.Leu
Chicken2              Val.Leu

Val.-
Val.-
Val.GIy
Val.Glu.Leu
Met.Gly
Val.Ser
Val.Gly

(Val.Asp)t
(Val.Gly)2

(Val.Asp)2

8 The presence of a third iv-terminal sequence has been reported only by
Ozawa and Satake.2

1. K. Porter and F. Sanger, Biochem. J., 42, 887 (1948).
2. H. Ozawa and K. Satake, J. Biochem. (Japan), 42, 641 (1955).
3. M. S. Masri and K. Singer, Arch. B&hem. BiopAys., 68, 414 (1955).
4. D. B. Smith, A. Haug, and S. Wilson, Federation Proceedings, 16,766 (1957).

evolutionary sense out of this information.  However, it might be
pointed out that the N-terminal sequence, Val.Leu, seems to be pres-
ent throughout the species examined, including the representatives
of the reptiles and the birds. We may speculate on the possibility
that the "valyl-1eucyl" chain represents a relatively early invention
of evolution, and that the addition (and modification) of a second
type of chain accompanied later differentiations in the vertebrate
phylum.

The elegant studies of Pauling, Itano, and their colleagues, and
of Ingram on the normal and abnormal human hemoglobins,  are
discussed in the next chapter. The information gained from these
studies should be of great value as a baseline for the investigation
of hemoglobin structure in other species.

Species Comparisons of Serum Proteins

The chemist, interested in comparative protein chemistry, generally
studies proteins that have unique and interesting biological activities.
This is the reason why most of our knowledge of protein structure
concerns enzymes, hormones, and pigment-associated proteins.  The
odds in favor of being chosen for study are, in a sense, fixed in favor
of exactly those proteins that Nature might find it necessary to pre-
serve in reasonably unmodified form during the evolutionary process.

SPECIE.5 VARIATION IN PROTEIN STRUCTURE                          161


TABLE 13
Precipitin Tests with Antihuman Serum'

(Antihuman serum was prepared in rabbits by periodic injection
         with human serum)

        Origin of Serum        Amount of Precipitate
                         Relative to Human
Primates
Man                              100
Chimpanzee
Gorilla                   130 (loose precipitum)
                                  64
Orang                              49
Mandrill                            42
Guinea baboon                         29
Spider monkey                         a9
Carnivores

Dog
Jackal
Himalayan bear
Genet
Cat
Persian lynx
Tiger

3
10 (loose precipitum)
8
3
3
3
a

IJngulates
ox                                10
Sheep                             10
Water buck                             7
Hog deer                              7
Reindeer                               7
Goat                                  0
Horse                                 a
Swine                                   0
Rodents
Guinea pig                                0
Rabbit                                   0

Insectivore.9
Tenrec                                  0
Marsupials
Six species: rock and nail-tailed wallabies,
  kangaroo, Tasmanian wolf                 0
8 After G. H. F. Nuttall, from Biochemical Evolution; G. Wald in Trends in
Phgsiology and Biochemistry (E. S. G. B arron, editor), Academic Press, 195%.

162                    THE MOLECULAR BASIS OF EVOWTION

On the other hand, the "permissible" degree of change in proteins
that require less rigid engineering, such as certain of the serum pro-
teins, the less dynamic elements of tissues such as the collagens and
elastins, and various proteins of the hair and skin, is likely to be quite
large.

One of the earliest studies of the comparative biochemistry of pro-
teins was carried out by Nuttall and his collaborators.  These inves-
tigators used immunological techniques to study the phylogenetic re-
lationships between the serum proteins of a wide variety of species.
They employed the extent of the precipitin reaction between anti-
human serum and the serums of other species as a measure of sim-
ilarity (Table 13). We know that the precipitin reaction is not an
absolutely specific one and, therefore, that cross reactions which do
occur do not require the presence of molecules identical to human
serum protein molecules. The results suggest that the serum pro-
teins of the species examined form a graded series of macromolecules
in which only serums from phylogenetic "neighbors" can cross react
significantly. Nuttall strengthened this conclusion by cross-reacting
serums from more closely related animals. A dramatic example is
his observation that antifrog serum reacts strongly with serums of
other tail-less amphibia, but not at all with those of tailed amphibia.
In spite of the complete lack of immunochemical similarity between
the serum albumins of distant species, the more obvious functional
aspects of the protein may nevertheless be retained. For example,
the serum albumins of rat and man carry out such physiological
functions as fatty acid binding and transport and osmotic pressure
regulation, in essentially the same manner and with equal facility.
We may guess, until experimentation has a chance to prove us wrong,
that the modifications which led to immunological differences spared,
or at least only slightly remodeled, the functionally critical parts of
serum albumin structure.

REFERENCES

1. T. G. Lee and A. B. Lerner, I. Biol. Chem., 221, 943 (1956).
2. L. T. Skeggs, Jr., K. E. Lentz, J. R. Kahn, N. P. Shumway, and K. R. Woods,

J. Exptl. Med., 104, 193 (1958).

3. D. F. Elliott and W. S. Peart, Nature, 177, 527 (1956).
4. These studies are summarized by H. Tuppy in Symposium on Protein Struc-

ture (A. Neuberger, editor), Methuen, London, 1958.
5. H. Theorell, Biochem. Z., 298, 242 ( 1938); EnzymoZogiu, 6, 88 ( 1939).
6. R. R. Porter and F. Sanger, Btochem. J., 42,287 ( 1948).

SPECIES VARIATION IN PROTEIN STRUCTURE                         163


chapter 8

GENES AS DETERMINANTS

OF

PROTEIN STRUCTURE

    3 tudies on the biochemical effects of mutations have
given strong support to the notion that individual genes are con-
cerned with the biosynthesis of individual proteins. In a large num-
ber of instances it has been possible to attribute the absence, or
modification of an enzyme to a single gene mutation. The study of
such biochemical lesions, not only in microorganisms like Neurospora
and E. coli but in man and other higher organisms as well, has led
to the concept of a "one gene-one
have already introduced in Chapter 2. enzyme" relationship, which we

The term "gene" as used in this context has, until quite recently,
been employed to convey the purely abstract concept of a unit of
heredity. It represented a quantum of genetic information that in
some way controlled the biosynthesis of a single protein or, in more
cautious terms, of some "functional unit." The recent advances in
the biochemistry of the chromosome and of DNA, and in the mapping
of genetic "fine structure" of the sort we have discussed in relation
to bacteriophage, now make it possible to speculate about gene action

164

in chemical terms instead of formal abstraction.
of S. Benzer, G. Streisinger,         The investigations
             M. Demerec and his collaborators,
G. Pontecorvo, and many others have indicated that the idea of
a one-dimensional array of "genes," divisible by genetic recombina-
tion, may very likely be extended down to molecular dimensions.
Their results suggest that the word "pseudoallelism" needed to be
invented only because of the difficulties of demonstrating extremely
rare recombinations in unfavorable biological material.
If we accept the generalities of the "one gene-one enzyme" con-
cept, and if we are willing to go along with the present trend of
opinion on the role of DNA as the basic determinant of heredity, we
must seriously consider the conclusion that the information which
governs details of protein structure is present in the chemical struc-
ture of the DNA molecule. It is an undeniable temptation to sug-
gest further that a point mutation is really just a very localized change
in the sequence or the three-dimensional relationships within a poly-
nucleotide chain and that such a localized change might reflect itself
in the sequence and folding of the protein concerned. In spite of
the fact that many investigators properly accept the generality as a
working hypothesis, such speculations are, at present, mostly fancy
with little fact. The pathway from gene structure to phenotypic
protein may be a long and tortuous one, and we cannot rule out such
possible complications as the combined action of several genes in the
synthesis of a single protein or the involvement of cytoplasmic heredi-
tary factors which might modify, or even initiate, steps in a biosyn-
thetic pathway.

If this hypothesis is an approximately correct one, however, we
should, as N. Horowitz has pointed out, be able to demonstrate
mutations that lead to qualitative as well as quantitative changes in
enzymes and other proteins. It should be possible, for example, to
show that various mutations within a given protein-determining re-
gion of the genetic material of an organism can lead to "mutant"
forms of a biologically active protein which exhibit varying degrees
of functional adequacy. Mutations affecting portions of protein struc-
ture that are essential for function should be lethal ones, whereas
those affecting less essential regions might either be undetected or
"leaky," to use the genetic patois.

In spite of the fact that hundreds of examples have been found of
gene-protein relationships, it has been possible to demonstrate a cor-
relation between the mutation of a single gene and the chemical and
physical properties of a homogeneous protein molecule in only a few
instances. Many of these positive correlations have emerged from
GENES AS DETERMINANTS OF PROTEIN STRUCTURE                      165


studies on proteins of higher organisms for the simple reason that
protein samples of sufficient purity are easier to come by with red
cells, milk, and plasma than with microorganisms.  However, the ad-
vantages offered by microorganisms in respect to genetic mapping
has been a tremendous stimulus to gene-minded protein chemists, and
it is likely that many of the major advances in this area will be made
on material from such sources. If, for example, the protein whose
biosynthesis is under the control of that region of genetic material in
T4 bacteriophage so elegantly mapped by Benzer (see Chapter 4)
could be identified and isolated in pure form, it is clear that a direct

TABLE 14

Alterations in Proteins Attributable to Mutations

Protein

Species

Demonstrated or Possible
Effects of Mutation

Hemoglobin1*2           Man
               Sheep
                   Mouse
&Lactoglobulin** 2         Cattle
Haptoglobin'            Man
Pantothenate-synthesizing
enzyme'              E. coli
Tyrosinase'          Neurmpora cr.
Glutamic acid dehydrogenase'  Neurospora cr.

For more detailed reference see:

Composition and charge
Charge
Charge
Charge
Charge

Thermostability
Thermostability
Reversible heat activation

1. N. Horowitz, Federation hoc., 16, 818 (1956).

4. D. Steinberg and E. Mihalyi, Ann. Reu. &o&em., 26, 373 (1957).
test, in enormous detail, could be made for the existence of a corre-
spondence between "cistron" and protein.  Such detail could never
be achieved with human proteins because extensive gene mapping
in man is limited by his lengthy generation time and his eugenic
mores.

A partial list of those proteins for which gene-linked modification
has been demonstrated is presented in Table 14. With one excep-
tion, human hemoglobin, the difference between the normal protein
and that obtained from the mutant has been in electrophoretic mo-
bility, heat stability, and serological behavior. The net charge, stabil-
ity, and serology of a protein are, of course, quite distinctive charac-
teristics, and the proteins in Table 14 which have been studied in
respect to these parameters can almost certainly be assumed to exist

166                      THE MOLECULAR BASIS OF EVOLUTION

in forms whose differences are related to allelomorphic genes.  Never-
theless, small organic molecules, tightly bound to proteins, can modify
charge, and polysaccharides or other haptenic substances may in-
fluence antigenicity. For such reasons, the case of human hemoglobin
is a particularly favorable one, since for this protein the electro-
phoretic and solubility differences between mutant forms are at-
tributable to actual modifications in amino acid sequence.

In 1949, L. Pauling, H. A. Itano, S. J. Singer, and I. C. Wells' made
the important observation that the hemoglobin of sickle-cell anemics
is electrophoretically abnormal and that in individuals with sickle-cell
trait (an asymptomatic condition) a mixture of the abnormal sickle-
cell and the normal forms could be demonstrated. Extensive study
of the familial relationships of sickle-cell anemia has indicated that
this frequently fatal disease is inherited in a Mendelian fashion.
By an analysis of the genetic relationships between sickle-cell anemia
and sickle-cell trait, J. V. Neel established that the production of the
abnormal hemoglobin was due to the presence of a single mutant
gene. Genetically, the anemic may be characterized as homozygous
for the sickling gene and the individual with the trait as heterozygous.

The studies of Pauling and Itano and their collaborators, together
with the discovery by H. Hijrlein and G. Weber2 of a congenital
methemoglobinemia involving an abnormal globin component, stim-
ulated the search for other genetically linked aberrations in hemo-
globin synthesis. At present writing a dozen or more types of ab-
normal hemoglobins which may be detected by their unusual physi-
cal properties are known.  In addition, there are a number of clinical
situations in which detection depends on hematologic examination
but no changes in the physical properties of hemoglobin have been
observed. Such abnormal individuals have microcytic red cells or
cells showing some other deviation from the normal morphology of
erythrocytes. These instances of inhibition of synthesis of normal
hemoglobin are collectively named thalassemia, and Allison has pro-
posed, on the basis of the observation that the locus controlling the
thalassemia effect does not appear to be allelomorphic with the nor-
mal hemoglobin gene, Hb *, that the locus for thalassemia be desig-
nated Th. The normal gene at this locus would then be termed
ThN and the thalassemia allele, ThT.
Examples of the clinical nomenclature and genotypic designations
for a number of abnormalities involving the hemoglobin molecule are
given in Table 15. This compilation is taken from the excellent re-
view by Itano to which the reader is referred for more detailed in-
formation. For our present purposes it is sufficient to recognize,

GENES AS DETEkMlNANTS OF PROTEIN STRUCTURE                      167


TABLE 15
The Human Hemoglobins'

     Method of Detection

Method of Detection

A   Normal adult
F   Foetal
x   Electrophoresis
S   Electrophoresis
  Solubility
   Tactoid formation
C   Electrophoresis

E   Electrophoresis
G   Electrophoresis
H   Electrophoresis
I  ' Electrophoresis
J   Electrophoresis
M  Spectrophotometry

D  Electrophoresis and solubility

Nomenclature of Syndromes Associated with
Abnormalities in Hemoglobin Metabolism

Genotype

       Condition

                        -
Homozygous
Normal
Sickle-cell anemia
Hemoglobin C disease
Thalassemia major
Thalassemia major
Heterozygous
Sickle-cell trait
Hemoglobin C trait
Sickle-cell hemoglobin C disease
Thalassemia minor
Thalassemia minor
Sickle-cell thalassemia disease
Hemoglobin C thalassemia disease
Doubly heterozygous
Sickle-cell thalassemia disease
Hemoglobin C thalassemia disease

Hb Locus

HbAHbA
HbsHbs
HbcHbc
HbthHbth

HbAHbS
HbAHbC
HbSHbC
HbAHbLh

HbSHbth
HbCHbth

HbAHbS
HbAHbC

Th Locus

ThN ThN

ThTThT

ThNThT

ThN ThT
ThN ThT

* From a review by H. Itano, ~duances in Protein Chemiatfg, volume 12
(C. B. Anfinsen, M. L. Anson, K. Bailey, and J. T. Edsall, editors), Academid
Press, p. 215, 1957.

168

THE MOLECULAR BASIS OF EVOLUTION

first, that some of the various abnormal hemoglobins (Hb', Hb',
etc.) are under the control of a series of genes which seem to be
allelic (they are, perhaps, pseudoallelic) and, second, that certain
other abnormalities, inclusively termed thallassemias ( ThT 1, Th'r, etc. )
involve genetic abnormalities for which no physical or chemical
reflection in the structure of the hemoglobin molecule has been ob-
served and which appear to be associated with genetic loci different
from the HbA locus.

Let us now examine what chemical data we have.  The differences
observed by Pauling, Itano, and their colleagues in the electrophoretic
mobility of normal and sickle-cell hemoglobin might be ascribed to
modifications in the amino acid sequence leading to the introduction
or deletion of charged side-chain groups. On the other hand, such
charge differences might be apparent only and could reflect the man-
ner of folding of the polypeptide chains of the protein to expose or
to mask charged groups in response to configurational change. A
direct test of these hypotheses has been made by V. Ingram,3 who
has examined the details of sequence in the molecule (Figure 78)
by means of the sensitive "fingerprinting" technique described in the
previous chapter. His investigations have made it extremely likely
that both sickle-cell hemoglobin and hemoglobin C differ from normal
hemoglobin in only a single amino acid residue.  The affected portion
of the protein is shown in Figure 79. A glutamic acid residue in
HbA has been replaced with valine and lysine, respectively, in Hbs
and HbC. The corresponding changes in net charge per mole (plus
2 for Hbs and plus 4 for Hba, with respect to HbA) agree with that
to be expected from the electrophoretic measurements, and no evi-
dence has been obtained for other changes in sequence in the rest of
the molecular structure of the protein.  We have here, then, a direct
test of the proposition that a mutation in a specific genetic locus
causes a specific change in the covalent structure of the phenotypic
protein related to this locus. Indeed, Ingram's experiments are a test
with a vengeance. Not only do the allelic Mendelian genes HbA,
Hb", and HbC have to do with a very restricted aspect of structure,
but they all appear to be related to the same aspect, namely the se-
quence at one unique point. If the sequence of nucleotides in the
polynucleotide chain of DNA determines polypeptide sequence, how
can we explain the fact that these three genetically segregatable loci
all influence the same position in the polypeptide?

A particularly intriguing possibility for explaining Ingram's results
comes from a consideration of the theoretical model of Watson and
Crick for DNA structure. The obligatory pairing of heterocyclic

GENES AS DETERMINANTS OF PROTEIN STRUCTURE                     169


          (0)               (h)

Figure 78. "Fingerprints" of the peptides produced by digestion of normal hemo-
globin (a) and sickle-cell hemoglobin (b) with trypsin.  The "fingerprints" were
obtained by a combination of electrophoresis and chromatography, more or less
as described in Figures 71 and 72.
the fingerprints differ significautly.  The encircled areas in the figure show where
                From V. M. Ingram, Nature, 100, 326 (1957).

bases in this structure has, as we have discussed earlier, been sug-
gested as a basis for the accurate self-duplication of DNA strands.
The specific sequences of the bases in the complementary strands of
the double helix have also been viewed as a set of coded genetic
information which might serve as the fundamental template for pro-
tein synthesis. The most popular code form has been one based on
"triplets," in which various sets of three nucleotides correspond to a
specific amino acid. Employing this idea, we may arbitrarily trans-
late the sequence of amino acids in hemoglobin that differs in the
three mutant forms into a corresponding nucleotide code as shown
in Figure 80. The replacement of a single nucleotide with another
within the critical trinucleotide sequence would give us the required

170                          THE MOLECULAR BASIS OF EVOLUTION

change in code.  (The reader will obviously not take all this too
seriously. The most improbable hypotheses in science have turned
out to be true, however, and this one certainly deserves some serious
consideration for its novelty and coherence.)

One very interesting question is raised by the existence of three
mutant forms of hemoglobin differing from one another in respect to
a single "locus." Why, with some 300 ,amino acid residues in a
hemoglobin monomer to choose from, has the accident of mutation
occurred, and been perpetuated, in the same place three times?  The
phenomenon is qualitatively reminiscent of the results obtained by
Benzer in bis analysis of mutants in the rII region of bacteriophage
T4 where he ,observed that, out of many hundreds of mutant colonies
selected, a disproportionately great number involved mutation in the
Same genetic ~locus, whereas others were modified only rarely. The
nonrandom distribution of affected loci, both in the bacteriophage
case for which we have a good deal of genetic information, and for
human hemoglobin for which we unfortunately have very little, might
mean that only certain mutations are "permissible" and that the de-
gree of permissibility is slight in most of the genetic material. We
might equally well suggest, however, that some unsuspected peculiari-
ties of DNA structure favor the modification of some lengths of
nucleotide sequence more than others. Most probably, the mutant
hemoglobin genes have been preserved because of the selective ad-
vantage Ithey have conferred on the affected individuals. , (Sickle-cell
anemia, for example, is correlated with decreased susceptibility to
clinical malaria.)

                               - -
HbA... ~is.Vsl.Leu.Leu.Thr.Pro.Uu.Glu.~ys . .
     T                  t

                                   -
Hb S . . . ;Iis.Val.Leu.Leu.Thr.Pro.VaZ.Glu.&s . . .
    t                  t

HbC.. . &is.Val.Leu.Leu.Thr.Pro.ip Glu.L+ys . . .
     t              r t

Figure 79.  The differences in amino acid sequence between normal hemoglobin,
sickle-cell hemoglobin and hemoglobin C.  The arrows indicate the points of
attack by trypsin which have lead to the production of the peptide fragments
shown in the figure.

GENES AS DETERMINANTS OF PROTEIN STRUCTURE                      171


DNA         Protein         DNA        Protein         DNA        Protein
    3.                         and subsequent study of their chemical structure, may well lead to
                           I       ;:.,.        xs      B

another situation like that of the hemoglobins for which the direct
chemical consequences of mutation can be shown.  Others of the
protein systems under investigation, listed in Table 14, also promise
to be extremely informative, particularly those involving easily iso-
lated proteins like the ,&lactoglobulins of milk. Because of their
flexibility as regards genetic analysis, however, the bacteria and bac-
teriophages are, at present, receiving the most concerted attention.
For example, no less than three laboratories are in the midst of the
particular problem of determining the effects of mutation in the h
region of bacteriophage T2 on the chemical nature of the phage
particle.
The host range (h) region of the genetic material of bacteriophage
T2 determines whether or not a phage particle will adsorb to a
specific bacterial <cell host. Thus the wild-type phage, T2h+, will
adsorb to and infect E. coli of the B strain but not of the B/2
strain, whereas h mutants will attack both B and B/2. Thus wild-.
type particles ( h+ ), when grown on a Petri dish containing agar in
which is uniformly distributed a mixture of B and B/2 E. coli, will
lyse only the B cells and a turbid plaque will be formed. On the
other hand, h mutants will attack both B and B/2 and a completely
clear plaque will result.  (It is convenient, in what follows, to think
of an h+ mutation as a "defect" in the "normal" h region of the genetic

Figure 80. A hypothetical scheme showing how the structure of deoxyribonucleic
acid might be related to the structures of hemoglobin A (normal hemoglobin),
hemoglobin S (sickle-cell hemoglobin), and hemoglobin C. The diagram sug-
gests a correspondence between triplets of purine and pyrimidine bases and in-
dividual amino acid residues. A change in the base sequence corresponding to
the third amino acid from the top of the drawing could conceivably lead to the
changes in code required for the modifications in sequence shown in Figure 79.
The reader should be very much aware of the completely speculative nature of
this diagram.

In the absence of further chemical data of the sort available for the
human hemoglobins, it may be of value to examine in more detail
some of the research now in progress whioh can be expected to settle
some of the problems that we have posed. Many groups of investi-
gators are busily engaged in attempts to isolate and characterize par-
ticular proteins from organisms that differ in genotype by a single
mutation. We have already referred to the studies of Horowitz and
Fling (Chapter 2) on the tyrosinases of Neurospora mutants in which
differences in heat stability and activation energies of thermal inacti-
vation were demonstrated. Rigid purification of these tyrosinases;

strand. ) This difference in phenotypic behavior can be made the
basis for a quantitative estimate of the proportion of h ,and hf par-
ticles and has been applied by Streisinger and Franklin4 for the lo-
cation of various h mutants along the linear genetic map in a manner
much like that employed by Benzer for the mapping of r mutants
in bacteriophage T4.

Since use of the technique of fine-structure mapping in bacterio-
phage will become more and more common, it will be instructive to
examine briefly the general approach to the mapping of the h region
as an additional example.

An h-type phage arbitrarily named h,O, was plated on mixed B
and B/2 cells as above described, and the turbid plaques were chosen
as examples of ,reversions to the hf genotype.
obtained `a series of mutants of the h+ variety. .ln this way there was
                        Fourteen h+ mutants
having low reversion indices" were then crossed wi.th each other as
* All the h+ mutants were examined for their propensity to revert spon-
taneously to the h phenotype, and those having a high "reversion index" were
discarded since such mutants would have introduced technical difficulties in sub-
sequent studies of the ability of pairs of h' mutants to yield h phenotypes by
genetic recombination.

172                     THE MOLECULAR BASIS OF EVOLlJTldN         GENES AS DETERMINANTS OF PROTEIN. STRUCTURE                     173


The "defective," h+ loci are encircled. During the formation of
progeny a process analogous to crossing over takes place.  The proba-
bility that recombination of hl and IQ will occur in the doubly infected
cell, A, is four times greater than the probability that hl and hl will
recombine in B.

The relative positions of hl+, IQ+, and hs+ might then be

3x      *
  0
     hz+

Of

The correct order, hl+-h3+--&+, may be established by crossing
mutant h2+ with h3+.  Recombination here will correspond to 3~
rather than 5x.

Figure 81. Establishing the relative separation and order of h+ loci by two fac-
tor crosses.

174                      THE MOLECULAR BASIS OF EVOLUTION

Possibility 1                 Possibility 2

   `p           hz
B.---i.---, '
         l       /-------
             I /`-----
          I
          I   I ,I
A w-----&z=z:_:

`22

hz

0
).+     hl  0
           hz+

               Ii
A ----w-w-,' .-_-_----
               I\--------

              hl

2 h genotypes

with many more r+ since only one
"crossover" is involved

2 h genotypes

  8 r+h2h
  @9 ~22Ml
with many more r2:!

Thus, order is 722-h&? if rf are in excess,
or       rtz-hz-hl if 1-22 are in excess.

Figure 82. Establishing the absolute order of h+ loci by three-factor crosses
(f&+ X *+h,+).

well as with the original h + strain by mixed infection of E. coli B.
The progeny were examined (by the "turbid-or-clear" plaque test)
for the relative proportion of h phenotypes that had formed through
recombination ( Figure 81) . Each pair of h+ mutants was found to
yield h recombinants with a characteristic and reproducible fre-
quency. All these frequencies were low, however (less than 1 per
cent), indicating that the h+ mutants examined all occurred within
a region along the genetic map of less .than two recombination units.
(As discussed in Chapter 4, the total "length" of the genetic material
in T2 phage may correspond to as much as 800 such units.)

Having established that all the various h+ mutations occurred
within a small region of the map, it was necessary to determine the
order in which they were arranged with respect to one another.  This
was done through the use of three-factor crosses, a procedure with
a forbidding name but one that is perfectly straightforward when
thought of in terms of a simple model (Figure 82).

Crosses were made by mixed infection of strain B bacteria with
bacteriophages containing, in addition to one of the hf loci, either
the wild type, r+, or the mutant, rz2 (which belong to the so-called
GENES AS DETERMINANTS OF PROTEIN STRUCTURE                      175


4, +

hw'

`22 3

o.co4 !: 0.0015
,026 + 0.035

0.29 2 0.03

Figure 83. Genetic map of h+ mutants. Taken from the studies of G. Streisinger
and N. Franklin on the genetic determination of host range in bacteriophage T2,
Cold Spring Harbor Sympodu Quant. Biology, 21, 103 ( 1956).

plaque-type mutants that were mapped by Benzer). The prepara-
tion of these doubly marked mutants requires a considerable amount
of technical manipulation involving repeated back-crossing and selec-
tion and we shall not attempt to describe the details of the chore.
Suffice it to say that strains of bacteriophage were obtained which
permitted crosses of the type r,,h, +h, x r+h,h,+ to be made, where
the rz2 locus is situated 24 recombination units from the h region as
determined by two-factor crosses.

The relative location of two h+ loci, h,+ and h2+, with respect to
the r2* locus may be determined by the estimation of the proportion
of the h mutants formed by recombination which is r+ in character.
The applicability of this test becomes clear upon inspection of the
schematic representation shown in the figure. If the order of loci
is r22-h,-h, rather than r2., -h,-h,, a far greater proportion of h-type
recombinants will be r+ smce the incorporation of the segregated h
alleles into one functional unit requires only one "crossover" in the
first instance and two in the second. All of this deduction involves,
of course, the assumptions we have mentioned earlier, including the
reality of a linear arrangement of genetic loci in phage and the avail-
ability of a mechanism of crossover at least analogous to that gen-
erally invoked for recombination in the chromosomes of higher or-

176                       THE MOLECULAR BASIS OF EVOLUTION

ganisms. These assumptions are, operationally speaking, applicable
in the present case. The order of h+ loci shown on the map in
Figure 83, which were determined from the three-factor cross data,
are compatible with the distances between the loci which was indi-
cated by the preliminary two-factor cross experiments.
Before considering these genetic observations in terms of the heredi-
tary control of phage chemistry, one further observation needs to be
reviewed. This concerns the demonstration of the functional unity
of the h region. Do all the h+ mutations in the "map" shown in
Figure 83 belong to a single unit of function (a "cistron"), or is it
possible that they are divided into more than one group and act co-
operatively in the determination of host range specificity? A de-
cision may be made by use of the &-tram test which we have previ-
ously described in relation to the r mutants. Streisinger' demon-
strated that all the h mutants belong to a single functional unit by
comparing the effectiveness of crosses of the cis type (h x h+ ) and
of the tram type (h, + X h,+ ) in producing h phenotypes (i.e., phage
which adsorbs to both B and B/2 bacteria). If, in Figure 84, each

I

hl

  Progeny of which only
+  about 3% are h-
     phenotypes

Ck?

Progeny of which more
than 60% are
A-phenotypes

Figure 84. Application of the cis-truns test to determine the functional unity of
the h region in the genetic material of bacteriophage T2. We may conclude
that portions I and II of the h region of the hereditary material of bacteriophage
T2 cannot act cooperatively to yield an "unblemished" h phenotype.
h region must be free of h* loci.                     The entire

GENES AS DETERMINANTS OF PROTEIN STRUCTURE                      177


of the indicated portions of the genetic strand acts separately, and
they cooperatively produce a normal h phenotype, all the progeny in
such a mixed infection should have the h character. It was found,
however, that only a very small proportion of the progeny were h
in phenotype (about 3 per cent, of the order of that to be expected
from crossover and other sequelae of recombination). In the case
of the ci.s arrangement (Figure 84), a high percentage of h pheno-

Figure 85. Electron photomicrograph of bacteriophage T2 adsorbed on cell walls
of E. coli B. Some of the adsorbed virus particles have lost their DNA, pre-
sumably by injection into the bacterial cell (see arrows). This photograph was
obtained through the kindness of Dr. Thomas F. Anderson of the Institute for
Cancer Research, Philadelphia, Pa.

178                      THE MOLECULAR BASIS OF EVOLUTION

types was observed (about 80 per cent).  (Enough phenotypically h
material is presumably made by the all-h strand to confer this charac-
ter on some of the h+ genomes as well as the h. That is, genetically
hf phage may have, associated with their protein coats, some h-type
host-range protein). It may be concluded, therefore, that only a func-
tionally complete h region will suffice for the expression of the h
character.

We are now in a position to consider the genetic map of the h
region in terms of what it does for the bacteriophage particle.  Our
attention must, of course, be directed at that part of the chemistry
(and morphology) of T2 which has to do with its adsorption to host
cells. Phage particles attach to bacterial cells by the tips of their
tails (Figure 85). The same sort of attachment occurs with phage
"ghosts" prepared by suddenly exposing intact phage to an osmotic
shock. Since the phage ghost is essentially all protein, except for
traces of carbohydrate present in such small amounts that its func-
tional importance is fairly unlikely, it may be concluded that the busi-
ness of attachment involves a specific protein component. Further
support for the protein nature of the adsorbing substance comes from
the fact that the kinetics of inactivation of the adsorptive capacity by
agents like urea are very similar to those of protein denaturation.  It
has also been observed that the blocking of amino groups in phage
prevents attachment to bacteria.

We may approach the problem of isolating and characterizing the
protein component responsible for host range specificity in two ways.
First, we may proceed to isolate various fragments from disrupted
phage particles. Such studies have been carried out by S. Brenner
and his colleagues in the Cavern&h Laboratory at the University of
Cambridge. These investigators have concluded that the host-range
function is carried in the slender fibers that are attached to, and
wrapped around, the tail of the virus particle (Figure 86) and have
prepared highly purified concentrates of free fibers for chemical
study.

A second approach to the problem involves the fractionation of
the total protein mixture making up phage ghosts in the same way
that we would approach the isolation of an enzyme from a crude
tissue extract. Phage ghosts may be solubilized in a number of ways
that should not cause modification in the covalent structure of the
component proteins, and solutions prepared with such agents as urea
and guanidine appear to be amenable to study by chromatographic,
electrophoretic, and ultracentrifugal techniques.  (See Figures 87
and 88, for example. )

GENES AS DETERMINANTS OF PROTEIN STRUCTURE                     179


Figure 06. Electron photomicrograph
of T2 bacteriophage, disrupted by
treatment with N-ethyl maleimide.
This photograph was obtained through
the kindness of Mrs. E. R. Kauf-
man and Dr. A. M. Katz of the Na-
tional Institutes of Health, Bethesda,
Maryland.

Both the morphological and "chemical" attacks on the fractionation
problem require a test for functional activity. Although not direct,
a test has been devised based on the fact that the antigenicity of the
T-even bacteriophages against rabbit antiphage antibody is con-
trolled by the same genetic locus as that which determines the host
range. Thus, Streisinger6 has shown that no measurable recombina-
tion occurs between the determinants of host range and the determi-
nants of serotype. (The reader must be asked to assume the validity
of this conclusion; he may, however, wish to read the elegant paper
of Streisinger,5 in which the details and arguments are presented.)
We may, then, hopefully assay any given protein fraction or morpho-
logical fraction for activity by estimating its ability to block the
phage-neutralizing action of an inactivating antibody preparation,

The studies on the chemical consequences of mutation in the h
region of bacteriophage have only just begun, and the problems of
isolation must first be solved. It seems likely, however, that these
investigations, as well as others concerned with other regions of the

180                        THE MOLECULAR BASIS OF EVOLUTION

GENES A5 DETERMINANTS OF PROTEIN STRUCTURE


0.6 ,     I     I     I     I     I     I     I     I   I

0 IL                    Serum blocking power
0.1 -

           l J-L-L    -

0     10    20   30 40 50 60 70 80    90

0.01 M ,
      t -
phosphate

Gradient to
0.1 M phosphate

Gradtent to -__- +I
1.0 M phosphate

Fraction number

Figure 87. Partial purification of the protein component in ghosts of bacterio-
phage T2 which is responsible for serum blocking power (see text) and which
presumably determines the host range of the phage. A preparation of "ghosts"
was dissolved in ice-cold 5.2 M urea at pH 7.4 and chromatographed on a column
of the cation exchanger XE-64. From W. J. Dreyer, A. Katz, and C. B. Anfinsen,
Federation Proc., I 7, 214 ( 1958).

genetic map of phage, should ultimately enable us to make direct
point-by-point comparisons of genetic changes and structural modi-
fications in protein molecules. The particular power of the bacterio-
phage approach, and of similar studies on other microbial systems,*
is the extreme discrimination of the genetic mapping for these "OI-

' For example, C. Levinthal and A. Garen, of the Massachusetts Institute of
Technology, have recently begun the mapping of the "cistron" which controls
the synthesis of an alkaline phosphatase in E. coli. This enzyme is synthesized
in large quantities when phosphate is limiting in the culture medium.  Organ-
isms are grown on plates, containing medium or low phosphate concentration,
which are then sprayed with nitrophenylphosphate. Alkaline phosphatase-con-
taining cells cleave the phosphate ester to yield the yellow-colored nitrophenol.
Bacterial colonies containing the enzyme thus become yellow, some mutants re-
main white, and certain mutants, having enzyme of intermediate activity, are
weakly colored, The enzymes, active or inactive, may be isolated from the
various strains and are at present being subjected to structural analysis.

182                      THE MOLECULAR BASIS OF EVOLUTION

ganisms." If Benzer's calculation of the length of the "recon" in
terms of nucleotide units in the DNA chain proves to be reasonably
correct, we may expect to be able to distinguish, genetically, between
loci as closely packed as those determining the three hemoglobins in-
vestigated by Ingram. In the h region, for example, the locus hal+
appears to be only 0.004 recombination units from ho+. Translated
into Benzer's nucleotide language, this distance would correspond to
about the distance between one nucleotide pair. In spite of the
wishful chemical thinking and genetic uncertainty involved in all
these speculations, it must be very clear why the biochemist is will-
ing to risk the gamble of time and effort required to test the gen-

E' 0.6 -
t2
2 0.5 -
ii
g  0.4 -
iG
.$
0" 0.3 -

0.2 5

0.9     ,
  86.0 '
3     I    I    I    I   I      I       I
         4.8
0.8 -

0.7 - "

  - 0.1 M phbsphate, pH 6.5        0.2 M phosphate.
                              , -         0.2M phosphate,
   \              ,   pH 6.5    I- PH 6.5 + 0.2 M
                                             NaCl

Fraction number, 22 ml./fraction

Figure 88. The purification of lysozyme from lysates of E. calf on a column of the
cation exchanger, XE-64. The small chromatographic peak at the far right of
the chromatogram contains the lysozyme activity (the dotted curve). Lysozyme
may also be isolated from bacteriophage ghosts.
however, is an E. coli lysate.          The starting material of choice,
          Enough lysozyme is presumably synthesized follow-
ing infection of E. co2i cells with phage to more than satisfy the needs for the
formation of progeny.  The excess enzyme within the infected bacterial cells is
then released into the surrounding culture medium upon lysis. The enzyme
emerging from the ion exchange column is purified several thousandfold over its
concentration in the crude lysate. From unpublished experiments of Dr. W. J.
Dreyer, National Heart Institute, Bethesda, Maryland.
GENES AS DETERMINANTS OF PROTEIN STRUCTURE                      183


era1 hypothesis. With luck, the answers might begin to clarify some
of the most central problems of biology.

REFERENCES

1. L. Pauling, H. A. Itano, S. J. Sanger, and I. C. Wells, Science, 110, 543
(1949).

2. H. Harlein and G. Weber, Deut. med. Wochschr., 73, 878 (1948).
3. V. M. Ingram, Nature, 180,326 (1957).
4. G. Streisinger and N. C. Franklin, "Genetic Mechanisms: Structure and Func-
tion," Cold Spring Harbor Symposia on Quant. Biol., 21, 103 (1956).

5. G. Streisinger, Virology, 2, 388 ( 1956).

184

THE MOLECULAR BASIS 0~ EVOLUTION


chapter 9

ON THE ACCURACY

OF

PROTEIN SYNTHESIS

   E xcept for the relatively minor influence of environ-
mental factors, the phenotypic potentialities of an individual are de-
termined by the genetic information in his chromosomes. In this
book we have taken as a working hypothesis an even more specific
proposition, namely, that the phenotypic picture presented by an or-
ganism is the summation of the effects, physical and catalytic, pro-
duced by the complement of protein molecules characterizing the
species in question. That is to say, we assume that the genetic in-
formation available to an organism is first translated into protein
structure, each gene determining specific details of a corresponding
protein molecule, either alone or in collaboration with other genes.
For example, we may think of the structure and physiological prop-
erties of hemoglobin as being determined by a single hemoglobin
gene or "cistron" (see Chapter 4) or perhaps by several cooperating
genes having to do with each of the peptide chains of hemoglobin
and with the folding and cross attachments between them. The
biosynthesis and arrangement of nonprotein substances such as car-

185


bohydrates and fats, and the distribution and integration of these sec-
ondary products into characteristic cellular systems, might then be
considered to be the business of the proteins as agents of the geno-
type, acting as enzymes, hormones, and structural subunits of mor-
phology.

Since we know that even a single gene mutation, causing a very
limited change in the structure of a single protein species (e.g. the
sickle-cell hemoglobin case discussed in the previous chapter), can
induce a marked variation in phenotypic behavior, it becomes of
prime importance in these considerations to examine the accuracy of
the mechanisms by which proteins are synthesized and to try to ap-
praise the degree to which errors in these mechanisms may occur.
If a change in the structure of a protein involving a single amino
acid residue can cause a marked change in function, we may justifi-
ably be rather concerned by the influence of random heterogeneities
in structure caused by biosynthetic errors. When we speak of the
"control" of protein synthesis by genes, we would like to have some
idea of the limits within which this control is exercised.

The highly developed techniques now available for the physico-
chemical study of proteins have made it possible to detect inhomo-
geneities in protein preparations caused by differences as small as the
presence or absence of a single amide nitrogen group in a fraction of
a population of molecules in solution. The apparent inhomogeneity
of "pure" proteins may become even more marked as time goes on.
In spite of such elegant tools as ion exchange chromatography, coun-
tercurrent distribution, and refined electrophoresis machines, we must
recognize that the criteria of homogeneity are only relative and that
heterogeneities not observable by presently available methods may,
tomorrow, be detected on the basis of new, more discriminating
procedures.

Since proteins are very complicated organic chemicals, they may
naturally exist in a number of isomeric forms. We can conveniently
classify such variations under two major headings, using terms sug-
gested by D. Steinberg and E. Mihalyi in their recent review on pro-
tein chemistry.l Variations in the structure of a protein may be at-
tributed to sequential or to configurational isomerism. The former
classification refers to differences in amino acid sequence between in-
dividual molecules and, for convenience, is defined to include other
aspects of structure which involve covalent bonds such as disulfide
bridges, phosphate ester linkages, and amide nitrogen groups.  These
are the stable, black-or-white parameters of structure, not modified
by the ordinary methods of handling proteins during purification and

186                        THE MOLECULAR BASIS OF EVOLUTION

storage. Configurational isomerism, on the other hand, refers to dif-
ferences in the mode of coiling of peptide chains or to the location,
frequency, and stability of noncovalent bonds.  Such isomerism is to
be expected in aqueous solutions since the bonds responsible for the
secondary and tertiary structure of proteins are strongly affected by
the acidity, polarity, and temperature of the environment.
Colvin, Smith and Cook, in their review2 written in 1954, have
presented a list of examples from the literature in which inhomogene-
ity has been demonstrated in protein preparations, presumably of a
high degree of purity. Thus, to quote only a few examples, lyso-
zyme, ribonuclease, and ovalbumin showed reversible boundary
spreading upon electrophoretic analysis, human gamma globulin ap-
peared heterogeneous both by electrophoretic and by ultracentrifu-
gal criteria, and insulin could be resolved into two components by
countercurrent distribution techniques. These authors have chosen
to interpret the experimental results in terms of a "microheterogene-
ity" in structure and suggest that `it seems more correct to describe a
native protein, not in terms of a finite number of definite chemical
entities, but as a population of closely related individuals which may
differ either discretely or continuously in a number of properties."
They have suggested that, should "microheterogeneity" be observed
for all native proteins, the cellular mechanisms for the synthesis of
proteins need not be specific and rigid, and that there might exist a
broad spectrum of individual protein "subspecies" within any single
"species," differing in enzymic, hormonal, or physicochemical prop-
erties. This conclusion was certainly an understandable one on the
basis of the information available in 1954. It is now apparent, how-
ever, that many of the examples which supported the concept of
"microheterogeneity" could be included in the list quoted only be-
cause of the inadequacy of the available knowledge about the chem-
istry of these proteins. In the case of beef pancreatic ribonuclease,
for example, we can separate on proper chromatographic columns
two major and two very minor components. A similar family of
ribonucleases may be shown to be present in sheep pancreas.3  These
four components appear to be present normally in pancreas tissue
since they may be separated both from purified, crystalline starting
materials and from crude extracts of pancreas glands.  Electrometric
titrations on the two major components isolated from beef pancreas
have suggested that they differ by a single carboxyl group (or con-
versely by a single amide nitrogen group). Rather than assume a
broad microheterogeneity in the sense of a spectrum of related ma-
terials, we might equally well conclude that "ribonuclease" is not a

ON THE ACCURACY OF PROTEIN SYNTHESIS                          187


statistical population of related proteins but rather a limited group of
well-defined chemical entities.

The complete absence of sequential isomerism in any sample of
protein can only be established by the quuntitatiue recovery of nZZ the
fragments on which the sequence reconstruction is based. In prac-
tice the optimal situation has never been reached, and, for the pro-
teins and polypeptides that have been studied in detail hitherto, se-
quential purity can only be inferred from the fact that aberrant se-
quences of amino acid residues, not fitting into the final reconstruc-
tion, have not been observed. In the studies of Sanger and his col-
leagues on the structure of insulin, for example, an enormous array
of peptide fragments was examined, and none of these was found to
be incompatible with the sequences of the two chains as they finally
emerged. Such studies give strong presumptive evidence for the se-
quential purity of the starting material.  In other work, such as that
of Shepherd and his collaborators on the structure of ACTH,4 and in
the careful chromatographic separation of the enzymatically pro-
duced fragments of ribonuclease by C. H. W. Hirs, S. Moore, W. H.
Stein and L. Bailey (Chapter 5), careful balance sheets of recoveries
were kept, and for many portions of the over-all sequences recov-
eries were quantitative within the experimental error of determination.
In the case of ACTH none of the fragments was isolated in less than
70 per cent yield, and total recovery, on the average, was 93 per cent
of the total starting material. In those fragments recovered in yields
less than quantitative it was shown, by paper chromatography, by
terminal amino acid analysis, and by the presence of integral molar
ratios of constituent amino acids, that sequential purity was extremely
likely.

These examples illustrate two of the ways in which we may ap-
praise the homogeneity of a protein; first, by an examination of the
internal consistency between the sequences of a large number of
small peptide fragments in terms of the final reconstructed sequence
of the polypeptide chain representing the common denominator and,
second, by a consideration of the completeness of recovery of these
fragments. Both methods are as good as the accuracy of methods of
detection or analysis available for peptides and give a lower limit for
the degree of purity. Except for special sorts of inhomogeneity
which we shall discuss more fully later, most proteins or polypeptides
that have been examined will probably appear to be at least 90 per
cent or more pure by these criteria. The last 5 to 10 per cent (or 3
to 5 per cent when analysis is done by careful ion exchange chroma-
tography) remains an unknown quantity and might conceivably ob-

188                           THE MOLECULAR BASIS OF EVOLUTION

scure the presence of small amounts of physically similar but struc-
turally different protein material.
Another more worrisome factor in this regard has to do with the
extent to which closely related proteins might be removed from the
major fraction during isolation procedures. The purification of a
protein such as hemoglobin presents no problem of this sort, since
the starting material, washed red cells, contains only insignificant
amounts of other proteins and the yield of hemoglobin can be made
nearly quantitative by careful experimental manipulation.  Most en-
zymes and hormones, however, must be concentrated many hundreds
or even thousands of times from their initial in uiuo state of purity.
Modem purification procedures are tremendously discriminating.
Under circumstances in which two forms of the same protein, differ-
ing only by a carboxylic acid group, may be separated, it is not un-
likely that very minor variations in charge or polarity within a fam-
ily may result in the complete separation of related molecules.

For these reasons we cannot be categorical about the absence of
microheterogeneity, even in the sense in which this term was used by
Colvin, Smith and Cook. There is, however, no evidence which re-
quires that we take the "broad spectrum" point of view, and it would
seem unnecessary at the present time to think of protein biosynthesis
as an inaccurate or arbitrary process.

A more optimistic alternative may be given as an explanation for
the bald fact that a well-defined sequential isomerism does occur in
certain proteins. Isomerism, as observed for the p-lactoglobulins,
hemoglobins, serum haptoglobin, etc., may be thought of as the re-
flection of heterozygosity in the corresponding genetic material. In
those instances in which adequate genetic analyses have been carried
out, the occurrence of more than one form of a single protein has
been attributable to the presence of sets of allelic genes. In most
cases only two forms are observed. We do not find, for example,
more than one abnormal hemoglobin in any one individual (except
for varying amounts of the fetal form which many believe to be ex-
tremely similar or identical with a portion of adult homoglobin).  On
the other hand, multiple forms of certain other proteins have been
observed in some instances. Proteins in the P-globulin fraction of
the serum of cattle, for example, may be divided, by the sensitive
starch gel electrophoresis method of 0. Smithies,5 into four or five
well-separated subcomponents. It is important to emphasize, how-
ever, that these are sepuruble and that what is obtained is not a
smear of overlapping substances but rather a well-defined and repro-
ducible pattern. By some techniques such as gradual salting-out

ON THE ACCURACY OF PROTEIN SYNTHESIS                          189


precipitation, even highly purified human hemoglobin from normal
individuals appears to be subdivisible into several components as ob-
served by Roche and his colleagues.6 Although most workers in the
field consider this phenomenon to be due to artifacts introduced by
interactions with salts and buffer molecules, it is still quite possible
that the effect is a real one, one that is simply not demonstrable by
the usual electrophoretic analysis employed for the study of the
hemoglobin series.

We have discussed, in a previous chapter, the concept of genetic
fine structure which suggests that each "gene" may be composed of a
large number of subgenic chemical units, each contributing a small
piece of information to the protein biosynthetic process.  The occur-
rence of many abnormal forms of a protein in addition to the normal
one cannot be excluded if this concept is generally applicable.  Thus,
the portion of the genetic material that has to do with a particular
protein molecule might involve more than one "cistron." The syn-
thesis of separate chains, or even of parts of the same chain of a pro-
tein, might be controlled by different genetic functional units. If
this were the case, and if the mutations were not lethal ones and still
permitted the synthesis of a functionally adequate protein, we can
easily see that a multiplicity of closely related proteins could result
through the cooperation of the several cistrons involved. The
hemoglobins that have so far been separated and studied have dif-
fered in electric charge. Since electrophoresis would not distin-
guish between two hemoglobins differing, let us say, by the substitu-
tion of valine for isoleucine or of serine for threonine, other tech-
niques for fractionation will be required to test this possibility.

The Significance of Amino Acid Analogue Incorporation

A large number of structural analogues of amino acids have been
synthesized (Figure 89) and tested for their utilizability in protein
biosynthesis. These include the methionine analogues, selenomethi-
onine and ethionine, the phenylalanine analogues, o- and p-fluoro
phenylalanine and /3-B-thienylalanine, and the tryptophane analogue,
`I-azatryptophane. They may be synthesized in radioactive form
and thus furnish a powerful and sensitive tool for testing whether
the mechanisms of protein biosynthesis are absolutely precise and
specific or whether alternative structures may be formed by replace-
ment of natural amino acid residues with man-made substitutes.

The results of such tests are clear-cut. Abnormal amino acids

190                         THE MOLECULAR BASIS OF EVOLUTION

can be used. Cowie and Cohen,r for example, have grown a methi-
onine-requiring mutant of E. coli in a medium completely free of
methionine but containing selenomethionine instead. The cells were
able to synthesize certain enzymes in a relatively normal manner in
spite of the unusual nutritional circumstance, and the proteins of the
daughter cells were of the selenomethionine variety. In another
similar study, M. Gross and H. Tarver have shown that the proteins
of Tetrahymenu pyriformis can incorporate Cr4-labeled ethionine.
The incorporation represents true peptide bond formation since it
was found that ethionine-containing peptides could be isolated from
partial acid hydrolysates. An interesting experiment on analogue
incorporation has been carried out by D. Steinberg and M. Vaughan,
who studied the in vitro uptake of tritium-labeled o-fluorophenyl-
alanine (see Figure 89) into the proteins of the minced hen's ovi-
duct.8 Pure lysozyme was then isolated from the tissue and digested
with trypsin and chymotrypsin. The digest was subjected to finger-
printing as described in Chapter 7, and the peptides thus separated
were analyzed for the presence of aromatic amino acids. A small
proportion of the peptides that normally contained phenylalanine
were found to contain the radioactive analogue in place of the nat-
ural amino acid.

Although analogues may be incorporated into proteins, the com-
promise with normalcy does not seem to be a happy one, for most of
them also cause marked inhibition of growth. We can say nothing
at the moment about the mechanism of this inhibition. Studies are
now in progress in several laboratories to determine the efficiency'of
incorporation of analogues as a function of their degree of dissimilar-
ity from the natural amino acid they mimic.

Nature appears to have been extremely clever in her choice of
standard amino acids and has managed to choose some twenty which
differ sufficiently to preclude mistakes in recognition. Valine and
isoleucine residues are extremely similar from the point of view of
three-dimensional structure, and it would not be too surprising to
find an occasional lapse in the precision of protein assembly at points
of protein structure involving one or the other of these amino acids.
To my knowledge, however (within the limits of analytical accuracy
mentioned earlier in this chapter ) , valine-isoleucine interchange has
not been observed, except in samples of the same protein or polypep-
tide isolated from two different species or from an individual who is
"heterozyg,ous" for the material in question. On the other hand,
D. Cowie and his colleagues have recently shown that a considerable
amount of the methionine in E. coli proteins may be replaced by

ON THE ACCURACY OF PROTEIN SYNTHESIS                         191


(4

(b)

Figur* 89. The ~~~le~ular structure Of some amino acids ant1 amino acid an;,.
lopes. (a) Phenyl~ 1, ~1 anine and p-fluoroplicnylala~~i~le, ( 1) ) tyrosine and o-fluoro-
phenykthnine, (c) norleucine and methionine, (d) isoleucine and leucine. AI-

192

THE MOLECULAR BASIS OF EVOLUTION

(4

though norlcwine is a chemical isomer of lwcinc and isoleucinc, its molecrdar
shape is much more similar to tllat of the sulfur-containing amino acid methioninc
than to that of isolcucine or lcucine.

ON THE ACCURACY OF PROTEIN SYNTHESIS

193


norleucine,D an amino acid which is not normally found in proteins
but which exhibits a remarkable similarity in molecular appearance
(Figure 89) to methionine.

Protein synthesis is not an absolutely precise process.  Some amino
acid analogues can substitute for natural amino acids.  Nevertheless,
the weight of evidence indicates that no mistakes are detectable
under normal circumstances and that the protein assemble mecha-
nism must have built into it an extraordinary capacity for structural
discrimination.

REFERENCES

1. D. Steinberg and E. Mihalyi, Ann. Reo. Biochem., 28, 373 (1957).
2. J. R. Colvin, D. B. Smith, and W. H. Cook, Chern. Rem., 54, 687 ( 1954).
3. S. Aqvist and C. B. Anfinsen, J. Biol. Chem., 234, No. 5 (1959); C. B. Anfin-
sen, S. Aqvist, J. Cooke, and B. Jonsson, 1. Biol. Chem., 234, No. 5 ( 1959).
4. ll. G. Shepherd, S. D. Wilson, K. S. Howard, P. H. Bell, D. S. Davies, S. B.
Davis, E. A. Eigner, and N. E. Shakespeare, J. Am, Chem. Sot., 78, 5067
(1958).

5. 0. Smithies, Biochem. J., 81,629 ( 1955).
6. J. Roche, Y. Derrien, and M. Roques, Bull. sot. chfm. btol., 35, 933 (1953).
7. D. B. Cowie and G. N. Cohen, Biochfm. et Biophys. Actu, 26, 252 (1957).
8. I am grateful to Dr. Daniel Steinberg and Dr. Martha Vaughan of the Na-
tional Heart Institute, Bethesda, Md., for information on these studies prior to
publication.

9. Personal communication from Dr. D. B. Cowie of the Carnegie Institution,
Department of Terrestrial Magnetism, Washington, D. C.

194

THE MOLECULAR BASIS OF EVOLUTION


chapter 10

THE BIOSYNTHESIS

OF PROTEINS

    I here can be little doubt that the specific informa-
tion necessary for the biosynthesis of proteins is, in some way, woven
into the structure of the deoxyribonucleic acids of the chromosome.
Ample support for this conclusion is given by the numerous observa-
tions that have related Mendelian genes to individual protein mole-
cules.  As we have seen, the most direct evidence has come from in-
stances in which the genetic results could be compared with the
chemical and physical properties of isolated, homogeneous proteins,
such as hemoglobin, tyrosinase, and p-lactoglobulin. Equally con-
vincing are the results obtained by bacteriologists and virologists who
have demonstrated that highly purified samples of DNA are capable
of modifying both the genotype and phenotype of recipient cells, or
of inducing the formation of the relatively complicated protein com-
plex which characterizes the bacteriophage particle.

It is clear, however, that protein synthesis can take place outside
the nucleus itself. In the reticulocyte, for example, hemoglobin syn-
thesis proceeds at a rapid rate, and not until the cell has become a
mature erythrocyte does such synthesis cease.  Similarly, in the alga
Acetabuluria mediterranea, whose cell may be separated into nuclear

195


and anuclear halves, the latter fragment temporarily synthesizes pro-
tein at a rate even more rapid than that of the intact cell, although
this synthetic activity soon disappears. If the biosynthesis of a spe-
cific, chemically definable protein like hemoglobin can continue in
the absence of an intact nucleus, it becomes essential to focus our
attention on the mechanism by which the requisite information might
be transferred to, and perhaps temporarily stored in, the cytoplasm
of cells.

The biosynthesis of protein is among the biological phenomena
that are highly dependent on structural organization. Even when
synthesis can proceed in the absence of a nucleus, the process is only
temporarily maintained (although admittedly this degeneration might
be due to deficiencies in any one of a number of metabolic factors
only indirectly related to protein synthesis per se). Because of this
dependence on structural integrity, the recent investigations of the
nature of cellular substructure have contributed perhaps the most im-
portant advance toward an ultimate understanding of the nature of
the biosynthetic mechanism. These studies, in spite of their empha-
sis on static morphology, are beginning to give us a picture of the
cell as a highly organized system of interconnected metabolic units
into which all the dramatic observations of the enzyme chemist and
the geneticist must ultimately be fitted.

Two relatively new techniques-electron microscopy of ultrathin
sections and differential sedimentation of cellular components in su-
crose solutions-have been of particular importance in this process of
the description of cellular architecture. The latter of these methods
permits the isolation of more or less homogeneous samples of mito-
chondria, microsomes, nuclei, and other cell inclusions, allowing the
study of the relative abilities of these particulate fractions to incorpo-
rate labeled precursors into nucleic acids and proteins.  We shall dis-
cuss these observations later, but first let us look at some of the results,
obtained through electron microscopy, which indicate how these
functional components are arranged within the intact cell.

The electron micrograph in Figure 90, taken by Dr. George Palade
of the Rockefeller Institute, is of guinea pig pancreas.  Careful ex-
amination and measurement of many such photographs have estab-
lished the presence in the cytoplasm of concentrically arranged mem-
branes, having a thickness of about 40 A. These membranes, vari-
ously called the "endoplasmic reticuliim," the "ergastoplasm," or
simply the "intracellular cytoplasmic membrane," are studded
throughout with small electron-dense granules. These are the gran-

196                           THE MOLECULAR BASIS OF EVOLUTION

Figure 90. Electron photolnicrograph of a section through a cell from the pancreas
of the guinea pig. hlagnification, 56,000~.
of the photograph are the         The layered structures at the center
         "endoplasmic reticulum" and consist of lipoidal mem-

branes studded with particles rich in RNA.  Th e oval, cross-channeled structures
at the top are mitochondrin.  A portion of the ccl1 nucleus is visible at the bottom
of the photograph. This photograph was obtained through the kindness of
Dr. Gcorgc l'alatlc of the Rockcfcllcr Institute for Medical Research.  From "The
Entlopl:lsmic Rrticrtlum," J. Biophp. Biochen~ Cytology, 2. (snppl. ) 85 ( 19.56 ).

THE BIOSYNTHESIS OF PROTEINS                                  197


Nuclear compaltmcnt

Figure 91. A schematic diagram of how some of the structural elements within
a cell might be organized. The cytoplasm of the cell is visualized as being
divided into two portions by the folded and invaginated endoplasmic reticulum,
the "internal" portion being continuous with the outer surface of the nucleus and
possibly directly connected with the nucleolus through a small channel. #The
"external" portion of the cytoplasm occupies a space between the endoplasmic
reticulum and the plasma membrane of the cell and contains the mitochondria.

ules which, following homogenization of a tissue, may be isolated as
a discrete fraction by differential centrifugation, attached to pieces
of fragmented membrane. Sjiistrand and Hanzonl have reported
that, in their experience (and this is generally confirmed by many
investigators), the granule-studded membranes are always arranged
so that they face the mitochondria, the cell membrane, or each other
with their rough side, but that around the nucleus the smooth side of
the membrane is presented. Such an arrangement would be com-
patible with the situation outlined schematically in Figure 91.  Here
the reticulum is visualized not as a number of independent mem-
branes but as a large balloon-like structure surrounding the nucleus
and crumpled upon itself.  This would lead to the sort of orientation
of granules suggested by Sjostrand and Hanzon and would serve to
divide the cell up into two major compartments, one containing the
nucleus and the other the mitochondria, together with the cytoplas-
mic fluid in which they are bathed. Thus, the "crumpled balloon"

198                          THE MOLECULAR BASIS OF EVOLUTION

arrangement would furnish the cell with a large surface as a site for
metabolic activity and would also serve as a natural divider of the
"genetic" and "assembly line" portions of the cell.
It must be emphasized that the scheme presented in Figure 91 is
only the distillation of several possibilities considered reasonable
by expert cytologists. It is included here only to indicate to the
reader the degree of sophistication that has been reached in the study
of cellular substructure. Uniformity of interpretation by experts is
more than can be expected in any rapidly advancing field, and it is
gratifying that most of the differences of opinion among cytologists
revolve around relatively minor points.

When a tissue is homogenized, the endoplasmic reticulum is dis-
integrated. From recent analyses it would appear that the so-called
microsome fraction is composed mainly of granules to which pieces
of reticulum still adhere. Thus, treatment of microsomal preparations
with lipoprotein-disrupting agents such as deoxycholate yields par-
ticles containing most of the RNA of the original preparation but
only a small proportion (about one-sixth) of the original protein.
With ribonuclease, on the other hand, which digests and depoly-
merizes the RNA, only membranous material is observed upon elec-
tron microscopic examination.  In some tissues, like the hen's oviduct,
the ergastoplasm is not as friable, and, even after fairly vigorous
homogenization, a relatively intact membrane particle complex may
be isolated by centrifugation at relatively low speeds.
The origin of the ergastoplasm is not established. It has recently
been shown that, in the hepatic cells of animals fed after a prolonged
fasting period, regeneration of membranes begins near the periphery
of the cell. These membranes are free of granules and only later
assume the studded appearance which characterizes them in an ac-
tively secreting cell. It has been suggested that the reticulum might
be the product of the continuing process of pinocytosis (water im-
bibition) and phagocytosis (particle imbibition) at the cell surface.
Electron microscope studies have indicated that engulfed fluids and
solids are surrounded with a sheath of the external plasma membrane
of the cell, pinched off during the passage of nutrients across the cell
surface. This membrane ultimately becomes continuous with the
endoplasmic reticulum. The observations, if substantiated, would re-
quire some rather vigorous metabolism. For example, as Swerdlow,
Dalton, and Birks* have recently pointed out, if the incorporation of
the plasma membrane into actively imbibing cells like macrophages
were continuous, the cells would soon consist of nothing else.  Such
cells would obviously require active processes both for the regenera-

THE BIOSYNTHESIS OF PROTEINS                                  199


tion of new plasma membrane and for the breakdown of reticulum
as it accumulated and pressed in on the nucleus.

Cytological Aspects of Protein Synthesis

In 1940, T. Caspersson3 suggested a general theory of protein bio-
synthesis which is, in essence, still in accordance with observation.
In his theory the genetic information within the DNA of the nucleus
was pictured as being transmitted to the nucleolus through the
medium of basic nucleohistones, rich in arginine and lysine. The
nucleolus then converted this information into ribonucleoprotein
which, in turn, induced and directed the biosynthesis of proteins by
enzyme systems in the cytoplasm.

One aspect of this theory can probably be safely discarded. The
role postulated for the nucleohistones was one of precursor for the
purines and pyrimidines of the ribonucleoprotein. We now know,
from isotope studies, that the building blocks of RNA are simple
substances such as glycine, formate, and carbon dioxide and are not
basic amino acids. We cannot, however, rule out the possibility that
nucleohistones might serve as agents of information transfer, acting,
in a sense, as negative prints of the specifically arranged nucleotide
sequences of DNA.

In this connection, Wilkins and his colleagues have suggested the
interesting hypothesis that protamines and nucleohistones might be
wrapped around the double helix of the DNA strand (shown in
profile in Figure 92). This suggestion, admittedly based on very
preliminary X-ray studies, is consistent with certain amino acid se-
quence data by Felix, Fischer, and Krekels.'  About one-third of the
residues in protamines are nonbasic, and these residues would have
to occur at folds in the polypeptide chain to permit all basic side
chains to associate with phosphate groups. The sequence analyses
do in fact show that nonbasic residues occur in pairs and that folds
such as those indicated in the figure would thus be possible.

  What evidence can we marshal for the second step in Caspersson's
theory, namely, that the nucleolus is concerned with the synthesis
of cytoplasmic ribonucleoprotein? Here we find ourselves immersed
in a mass of data, much of it conflicting. The fact, for example, that
some enucleated cells can continue to form protein suggests that
cytoplasm is autonomous in this respect. On the other hand, we
may postulate that ribonucleoprotein, produced in the nucleus and
containing genetic information, has a certain "life expectancy" in the

   200                        THE MOLECULAR BASIS OF EVOLUTION
-

Figtwo 92. A diagram showing
how protamine might be wrapped
in a spiral fashion around the
DNA double helix. The poly-
peptide chain is wound around
the small groove of the helix.
Phosphate groups of the DNA
coincide with the basic ends of the
arginine chains (black circles) of
the protamine  molecule. Non-
basic residues are shown in pairs
at the folds in the polypeptide
chain. From M. H. F. Wilkins,
Cold Spring Harbor Symposia
Quad. Bfol., 21, 75 (1956).

cell which varies from species to species, and that protein synthesis
may proceed as long as some of this material remains functional.
Two twes of experimental observation have a direct bearing on
                                                      - .

this quei6on. The- first, involving the measurement of rates of in-
corporation of various nucleic acid and protein precursors into the
ribonucleoproteins of nucleus and cytoplasm, indicates that such
substances are subject to a much greater metabolic flux in the former
cellular compartment. As shown in Figure 93, nuclear RNA attains
a much higher specific radioactivity than does cytoplasmic RNA
when an animal is administered radioactive phosphate.  Further, the
peak of radioactivity is attained much more rapidly in the nuclear
material. Thus, although the amount of RNA in the nucleolus is
generally relatively small in comparison with that in the cytoplasm,
the rate of turnover of the former is such that it might serve as a
precursor of at least part of the cytoplasmic RNA.
A second type of approach has been made by Ficq and her col-

THE BIOSYNTHESIS OF PROTEINS


IMicrosomal RNA

I   I   I   I   I   I

0
0   2   4   6   8   10  12  14  16  18  20  22  24

             Time, hours

Figure 93. Relative specific activities of inorganic phosphate, nuclear RNA,
anal microsomal RNA of mouse liver tissue at various times after the administration
of Pn. Redrawn from R. M. S. Smellie, The Nucleic Acids, volume 2 (E. Chargaff
and J. N. Davidson, editors), Academic Press, 1955.

leagues,6 using autoradiographic methods. After administration of
radioactive glycine to rats and to starfish ogcytes, she observed that
the rate of labeling of nuclear proteins was considerably higher than
that for cytoplasmic proteins. In the starfish oijcyte the well-defined
nucleolus showed particularly marked activity. Much of this radio-
activity is rapidly washed out of nuclei when they are isolated from
homogenized tissues.

.I;=1 GE]
   0  I  2  3  4  5  6  7  8  9  10 11 12 13 14       0 2
                               1 3 4 5 6 7 8 9 IO II 12 13 11

These observations are consistent with the idea that cytoplasmic
ribonucleoprotein stems from the nucleus.  As usual, however, there
is another side to the coin. Brachet and Szafarz have shown, for
example, that cytoplasmic RNA of Acetabulnria continues to incorpo-
rate the purine precursor, erotic acid, in spite of previous enucleation.5
Further, Moldave and HeidelbergerC have found that the ribonucleic
acids of the nuclei, microsomes, and mitochondria show different
chemical properties, such as variations in the ratios of heterocyclic
bases and in nucleotide sequences. We are obviously far from an
answer to the question of the essentiality of direct nuclear control of
protein synthesis. Whether the present confusion is attributable to
the presence of two distinct types of protein synthesis, one nuclear
and one cytoplasmic, as has been suggested by Brachet and others,
or whether experimental inconsistencies are due to the occurrence of

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I     RNaS.2    I          ' RNase 1

Figure 94. The effect of ribonuclease action on the growth and protein synthesis
of nucleated and enucleated cell fragments of Acetabuhiu medfterranea.  The
top curves (left, nucleated; right, enucleated) show the effects of ribonuclease on
growth and the bottom curves those on protein synthesis.  Fragments were trans-
ferred to normal medium at the times indicated by the bars at the bottom of each
graph. In the absence of a nucleus, normal growth and synthesis could not be
resumed after RNA degradation.
Btochem. Cytology, 4, 119 ( 1958). From H. Stich and W. Plaut, I. Biophys.

202                          THE MOLECULAR BASIS OF EVOLUTION           THE BIOSYNTHESIS OF PROTEINS                                  203

processes in enucleated and otherwise disturbed cells that represent
the dying gasps of a partially functional but abnormal mechanism,
is a source of some of the more exciting speculations in the field.

Some very recent work does seem to offer strong support for the
latter alternative. Stich and Plaut have examined the potentials for
growth, protein synthesis, and differentiation of nuclear and anu-
clear halves of Acetabulariu after exposure to ribonuclease action.
Their results, summarized in Figure 94, show that, in the absence of
a nucleus, normal growth and synthesis cannot be resumed after
RNA degradation. Stich and Plaut suggest that the nuclear product
which effects the recovery of nucleated halves is RNA, but they em-
phasize that this suggestion should not be extended to include ntl the
cytoplasmic RNA but may apply only to a small and highly critical
fraction produced by the nucleus. With this reservation, these ob-
servations are in good agreement with the results of Moldave and


Heidelberger already mentioned and also with studies recently re-
ported by McMaster-Kaye and Taylor,' who show that what has
hitherto been referred to as "nuclear" RNA may be divided into nu-
cleolar and chromosomal fractions, only the former of which has an
especially dynamic metabolism. It would begin to seem that the
nucleolus may, as Caspersson suggested, occupy a very central posi-
tion in the over-all process of protein biosynthesis.

The third step in Caspersson's scheme involves the production of
cytoplasmic proteins by a process dependent on the ribonucleopro-
teins, whose possible origin we have just discussed.  Here we seem to
be on reasonably firm ground, at least from the biochemical point of
view. It has long been known that the rate at which a tissue can
synthesize protein is correlated with its RNA content. Studies with
the ultraviolet microscope and with conventional staining methods
demonstrated, rather early, that such actively proliferating tissues as
root tips and yeast cells and such secretory cells as the exocrine cells
of the pancreas and liver were all rich in RNA.  It was also observed
that enucleation of amoebae caused a rapid fall in the level of RNA,
paralleled by a fall in the rate of labeled amino acid incorporation.

The most direct evidence for the essential role of RNA in protein
synthesis comes from studies on isolated cellular particles. When
the tissues of an animal which has been administered a radioactive
amino acid are homogenized and subjected to differential sedimenta-
tion, it is found that the proteins of the microsomes have become
most rapidly labeled.  (We must keep in mind, however, the obser-
vations of Ficq on the extractability of protein from the isolated nu-
cleus when appraising data obtained on isolated cell particles in gen-
eral.) Subdivision of microsomal matter into arbitrary fractions by
differential extraction showed that those fractions containing the
highest levels of RNA were also most rapidly labeled, although, in
later samples, radioactivity had been rapidly transferred to the lipo-
protein matrix to which the ribonucleoprotein is attached or in which
it is dissolved.

The endoplasmic reticulum disappears during mitosis, as well as
during starvation, as we have discussed earlier, and it is of interest
in connection with Caspersson's general hypothesis that this behavior
is shared by the nucleolus, which we have indicated might be impli-
cated as a central site of ribonucleoprotein synthesis. The parallel
disappearance and reappearance of ribonucleoprotein-rich reticulum
on the one hand, and of the ribonucleoprotein-producing nucleolus
on the other, furnish inferential evidence for the idea that these two

204                       THE MOLECULAR BASIS OF EVOLUTION

cellular components are somehow linked in the process of establish-
ing information-rich biosynthetic machinery in the cytoplasm.

Protein Synthesis in Ruptured-Cell Preparations8

Tissue homogenates, although they incorporate amino acids only
weakly and lack the integrated information system of the cell, have
the advantage that the need for certain essential cell components be-
comes emphasized. Various cofactors or substrates may then be
screened for their capacity to stimulate the synthesis of proteins.
It was observed, in a number of preliminary studies, that homog-
enates could support the incorporation of labeled amino acids into
proteins when ATP was added as an energy source. Mitochondria,
which serve as the site of ATP production through oxidative phos-
phorylation, could substitute for added ATP in large part. The in-
corporated amino acids in such systems are found mainly in the mi-
crosome fraction of the homogenate (although mitochondria would
also appear to support the synthesis of some proteins, e.g. cyto-
chrome c ) .

A major contribution was made by P. C. Zamecnik and E. B.
Keller9 when they observed that, if more gentle homogenizing tech-
niques were employed, a reconstructed system consisting of micro-
somes and a particle-free supernatant would incorporate labeled pre-
cursors when supplied with an energy source such as phosphocre-
atine or phosphoenolypyruvate. This incorporation was specific for
the natural L-amino acids and was abolished by the addition of ribo-
nuclease. The true synthesis of peptide bonds was demonstrated by
the isolation of peptides from partial hydrolysates of the protein
which contained 04.

Pursuing the dissection of this system, Zamecniks and his col-
leagues found that a protein fraction could be prepared from the su-
pernatant by precipitation at pH 5. This fraction no longer con-
tained bound nucleotides, which were present in the original crude
extract, and could support amino acid incorporation only when ATP
and either guanosine triphosphate or guanosine diphosphate were
added.

It was suggested by F. Lipmann in 1941 that amino acids might be
raised to an energy level, sufficient to permit them to undergo peptide
bond condensations, by conjugation with high-energy phosphate groups.
Support for this prediction was obtained by M. Hoagland and others,
THE BIOSYNTHESIS OF PROTEINS                                205


The Question of Intermediates in Protein Biosynthesis

            11
        I
1. Enzyme + NHz-CH-C    + ATP v~
             \
                  OH

Enz-(AMP - CO-CH-NH?) + Pyrophosphate

R

2. Enz-(AMP - CO-CH--NH*) + NH@H -+

R

NHzCH-CONHOH + AMP + Enzyme

Figure 95. A postulated mechanism for the activation of amino acids for protein
synthesis. In step 1 the amino acid is converted to an enzyme-bound acyl phos-
phate derivative of adenylic acid (AMP). This complex may be utilized for
intracellular protein synthesis or, as shown in the figure, may be reacted with
hydroxylamine to yield the amino acid hyclroxamate.

who showed that during the incubation of amino acids with ATP and
an enzyme fraction prepared from the "pH 5 enzyme" derivatives
were formed which had the characteristics' of an active acyl com-
pound; that is, they formed hydroxamates upon treatment with hy-
droxylamine.8 Although the active intermediate compounds have
not been isolated in more than trace amounts, the fact that hydrox-
amates are formed and that pyrophosphate is liberated during the re-
action strongly suggests that the chemical events may be described
by the formulas in Figure 95. Several investigators have recently
synthesized the postulated amino acid adenylates and report that
they can undergo nonspecific condensations with pre-existing free
amino groups in proteins.  The decision whether AMP-amino acid
conjugates are real, or only apparent, participants in the pathway be-
tween free amino acid and protein must therefore depend on future
research. The postulated `reactions are appealing because of their
analogy to already proven activation reactions for other substrate
molecules, such as fatty acids. They are also an attractive possibil-
ity for the simple reason that enzymes which form amino acid
adenylates do exist in the cytoplasm of cells.

206                       THE MOLECULAR BASIS OF EVOLUTION

Perhaps the greatest puzzle in protein biosynthesis is the question
of how sequences are determined and how cross-linking and folding
of peptide chains is brought about in a way that yields biologically
specific proteins. It seems likely that much of this process must in-
volve RNA, since this is the only obvious substance in the cytoplasm
which bears some structural (and probably metabolic) relationship
to the DNA of the nucleus, from which most of or all the informa-
tion that specifies the patterns of the protein molecules must orig-
inally stem. Indeed, a number of investigators have recently oh-
tained suggestive evidence for the involvement of a soluble, non-
microsomal, RNA fraction of the cytoplasm which is extremely active
in incorporating labeled amino acids and which may act as an inter-
mediate transport system between activated amino acids and the ribo-
nucleoproteins of the endoplasmic reticulum.*

If we assume that RNA serves as some sort of "template" for the
assembly of proteins, must there be a separate ribonucleoprotein
"template" for each cellular protein, or is it possible that there exists
some facet in protein synthesis which permits the utilization of com-
mon information for the synthesis of many different kinds of protein
molecules? The evidence we have for the presence of repeating pat-
terns in protein biosynthesis is, at present, very meager but sugges-
tive enough to make the idea worthy of serious consideration.  Sev-
eral groups of investigators have gone to considerable trouble in as-
sembling the known amino acid sequences in proteins (making up a
total of some 499 or 590 residues) and subjecting their sequential ar-
rangement to a rough statistical analysis for the occurrence of "re-
peats." As yet, no definite pattern has emerged from such efforts, al-
though certain special sequences are found with surprising frequency.
The dipeptide sequence, Ser.Arg, for example, has been demon-
strated in ribonuclease, chymotrypsin, lysozyme, salmine ( twice),
and phosphorylase (and this statement is based on only a cursory
search of the literature on protein structure). The phosphoproteins
so far examined involve phosphoserine-containing sequences with
remarkable similarity in structure.  Table 16 contains a list of some
of these, derived from proteins with no obvious connection except,
perhaps, the fact that they are all secretory proteins. In every

* Several articles on the chemical nature of intermediates in protein synthesis
and on the kinetics of the assembly of proteins are listed at the end of this chap-
ter. `lo I** ". " These aspects of protein biosynthesis are under active study in
many laboratories, but the field is still too confused for constructive discussion.

THE BIOSYNTHESIS OF PROTEINS                                  207


case the phosphoserine residue is either preceded or followed by a
dicarhoxyic amino acid.

Even more striking is the similarity in the sequence of amino acids
associated with the "active centers" of a number of enzymes. A large
number of enzymes have been found to be sensitive to reagents of
the sort typified by the compound, di-isopropylfluorophosphate
(DFP). Such reagents presumably inactivate by reacting with spe-

TABLE 16

Amino Acid Sequences Containing 0-Phosphorylserine (SerP)'

Pepsin
Egg albumin

a-Casein

Thr.SerP.Glu
Glu.SerP.Ala
Asp.SerP.Glu.Ileu.Ala
  SerP.Glu
  SerP.Ala
Glu.SerP

a A general discussion of phosphorus-containing proteins is given by G. Perl-
mann, Advances k Protein Chemiafry, volume 10, (M. L. Anson, K. Bailey,
and J. T. Edsall, editors), Academic Press, 1955.

cific serine hydroxyl groups to yield the 0-di-isopropyl phosphate
derivative (DIP protein). After partial hydrolysis of DIP proteins,
the acidic phosphopeptides may be isolated and characterized.  Four
enzymes, trypsin, chymotrypsin, phosphoglucomutase, and thrombin,
have been studied in particular detail in relation to the DFP reac-
tion, and it has been found that the amino acid sequence in the
neighborhood of the tagged serine residue is probably identical in
all four cases.`O Although the complete sequence has been estab-
lished unequivocally for only two of the proteins, it is almost certain
from the preliminary data that all four involve the sequence

The determination of ammo acid sequences in proteins and poly-
peptides is still at an early stage, and unfortunately we lack sufficient
data for a proper statistical analysis of the results.  Nevertheless, the
data we do have make it tempting to postulate that the "templates"
responsible for the biosynthesis of peptide chains involve common
sets of "instructions" which correspond to recurring chemical fine
structure in the genetic material of the nucleus. Alternatively, vari-
ous common peptide intermediates that have structures suited to
specific functional requirements might be involved in the assembly
of different classes of proteins. These hypotheses are attractive from
the standpoint of evolutionary theory, which suggests to us a com-
mon origin for living things, both present and past and a gradual
process of change in variety and kind through the modification of the
genotypes available at any moment. This thesis, which is discussed
in greater detail in the following chapter, would propose that "prim-
itive" chemical structures having generalized enzymatic or hormonal
functions were modified during evolution to yield families of more
specialized molecules.

Gly.Asp.SerP.Gly.Glu.Ala

REFERENCES

in which the phosphate group is derived from the phosphorylating
reagent except in the case of phosphoglucomutase, which contains
phosphate as an integral part of the enzyme. The presence of an
identical sequence of amino acids in these four proteins, particularly
in association with areas of the molecules that are strongly implicated
in catalytic function, is almost too much to attribute to chance alone.

1. F. S. Sjiistrand and V. Harmon, Exptl. Cell Research, 7, 393 (1954); ibid.,
p. 415.

2. M. Swerdlow, A. J. Dalton, and L. S. Birks, Anal. Gem., 28, 597 (1956).
3. T. Caspersson, Cell Growth nnd Cell Function, Norton, New York, 1950.
4. K. Felix, H. Fischer, and A. Krekels, Progress in Biophysics, volume 6,
pergamon Press, London, p. 1, 1956.

Another example of similarity in sequence is found in the hormones
of the pituitary gland whose structures were discussed in Chapters 6
and 7. The formulas for adrenocorticotropic hormone (ACTH) and

5. For a discussion of these, and related studies, see the review by J. Brachet
in The Nucleic Acids, volume 2 (E. Chargaff and J. N. Davidson, editors),
Academic Press, New York, 1955.

6. K. Moldave and C. Heidelberger, I. Am. Chem. Sot., 76, 679 (1954).
7. R. McMaster-Kaye and H. J. Taylor, J. Biophys. Biochem. Cytology, 4, 5

208                            THE MOLECULAR BASIS OF EVOLUTION           THE BIOSYNTHESIS OF PROTEINS                               209

for two forms of the melanocyte stimulating hormone (MSH>: of
porcine pituitary tissue are shown in Figure 74 (page 153). The
material known as /3-MSH contains a heptapeptide sequence which is
identical with residues 4 to 10 of ACTH. An even more striking ex-
ample of recurring sequence is a-MSH, which has a structure identi-
cal with the first thirteen residues of ACTH except for the addition
of a C-terminal amide group and an N-terminal acyl radical. The
similarities in these structures are particularly interesting because of
the fact that ACTH is formed in the posterior lobe of the pituitary
gland, whereas MSH appears to be synthesized in the pars intermedia.


( 1958). (The reader should be cautioned that a direct relationship be-
tween nuclear and cytoplasmic RNA is far from established-see for ex-
ample, J. W. Woodward, J. Biophys. B&hem. Cytology, 4, 383 ( 1958 ).
8. Reviewed in a series of papers in Proc. Nat. Aced. Sci. U.S., 44, No. 2
(1958).

9. P. C. Zamecnik and E. B. Keller, J. Btol. Chem., 209, 337 (1954).
10. J. A. Gladner and K. Laki, J. Am. Chem. Sot., 80, 1263 (1958).
11. R. Loftfield, in Progress in Biophysics and Bfochemktry, Pergamon Press,
London, 1958.

12. S. Spiegelman in The Chemical Basis of Heredity (W. D. McElroy and
B. Glass, editors), Johns Hopkins Press, Baltimore, 1957.
13. J. L. Simkin and T. S. Work, Nature, 179, 1214 (1957).
14. D. Steinberg, M. Vaughan, and C. B. Anfinsen, Science, 124, 389 (1956).

210

THE MOLECULAR BASIS OF EVOLUTION


chapter 11

GENES, PROTEINS,

AND

EVOLUTION

4,

  . . . we must remember that heredity, development,
and evolution are essentially epigenetic and not preformistic.
We do not inherit from our ancestors, close or remote,
separate characters, functional or vestigial.  What we do
inherit is, instead, genes which determine the pattern of
developmental processes. . . . "

T. DOBZHANSKY, Evolution, Genetics, and Man,

   A s the genes of a species are modified and reshuf-
fled, occasional organisms will appear within a restricted population
having phenotypic characteristics that enable them to explore desir-
able ecological niches which were unattainable by their predecessors.
The individual changes are generally quite small.  Many generations
must come and go, during which forays into formerly forbidden ter-
ritory by this developing branch of the population become more fre-

GENES, PROTEINS, AND EVOLUTION                                211


quent and are of longer duration as the result of further reorganiza-
tion of the gene pool by random mutation and natural selection.  In
time the summation of these changes results in a new species, fully at
home in its new environment and su5ciently different in physiology
from its distant ancestors that cross-fertilization is no longer possible.

We have discussed, in several earlier chapters, the techniques used
by the geneticist for the analysis and description of limited portions
of such a chain of events. As long as crosses can be made between
different family lines, phenotypic changes can generally be related to
specific genes and the spread of these genes through a population can
be fairly accurately mapped. Thus, the basic assumptions of evolu-
tionary theory may be directly tested, and with some precision, when
the segment of time under consideration is small, and we are able to
describe the process in terms of changes in genotype.  When we deal
with evolution on a larger scale, however, the tools of the geneticist
are no longer applicable. The evolutionist must now rely on the
study of relative morphology and ecology as deduced from the fossil
record, or on the comparative anatomy and physiology of living rep-
resentatives of surviving species.

The principal aim of this book has been to examine the basic prin-
ciples underlying another possible method for the study of evolution.
This method is based on the hypothesis that the individual proteins
which characterize a particular species are unique reflections of the
genes which control their synthesis. The examination of the chem-
istry of a series of homologous proteins is, of course, a purely pheno-
typic approach to the problem.  Nevertheless, the evidence available
to us, even at this early date, suggests that the. structure of proteins
may be a relatively direct expression of gene structure and that com-
parative protein chemistry may furnish a qualitative view of geno-
typic differences and similarities. If we accept the general hypothe-
sis, we are led to infer, for example, that the "insulin-determining"
genes of the pig and the sperm whale are identical, like the insulins
whose structures they determine. Indeed, should several genes be
concerned with the synthesis of insulin, the same would also be true
for these.

Another interesting potentiality of comparative protein chemistry
is that it might permit us to determine whether the same phenotypic
characteristic, shown by two completely unrelated organisms, is at-
tributable to analogous or to homologous genes. For example, both
bacteriophage T2 and chicken's eggs contain proteins that have lyso-
xyme activity. The genetic material of both coliphage and chickens
must be said to contain information that can direct the formation of

212                          THE MOLECULAR BASIS OF EVOLUTION

proteins with this function. Is it possible that these two organisms
contain nearly identical (that is, homologous) stretches of genetic
material, or are the genes for lysozyme synthesis, and the lysozymes
themselves, entirely different? This would appear to be the sort of
question that might be attacked directly by the comparative study of
protein structure. As we have already seen, in connection with cyto-
chrome c, ribonuclease, hemoglobin, and other proteins, there is ex-
cellent evidence which indicates that many homologous genes do ap-
pear to have survived happily through long periods of time, some
well exceeding the span of the fossil record.

A Biochemical `Approach to the Species Problem

Th e paleontologist, in estimating the rates and directions of evolu-
tion, must depend almost entirely on morphological evidence.  Even
with this relatively crude sort of yardstick, he can begin to distin-
guish patterns of change such as we discussed in Chapter 1, in con-
nection with the characteristics of tooth structure in the evolving
horses. He is limited, however, to the results of evolution and can
never hope to elucidate the underlying physiological changes that
participate to produce new phyla.

of Although most of the ancient species disappeared, representatives
almost all the phyla escaped extinction by adapting to their new

environments, thus perpetuating large parts of heredity. We have
available to us, then, a contemporary sample of the life of the past
from which we should be able to deduce a great deal about the fac-
tors that were decisive in phylogenesis long ago. The study of
"biochemical evolution" has already been of considerable value in the
establishment of biological interrelationships. For example, the oc-
currence of melanocyte-stimulating, oxytocic, and vasopressor hor-
monal activity in extracts of the neural gland if tunicates furnishes
strong evidence in support of the assignment of this subphyllum, the
Urochordata, to the direct pathway between the invertebrates (which
lack MSH activity) and vertebrates. The presence of both arginine
phosphate (an invertebrate phosphagen) and creatine phosphate (the
typically vertebrate phosphagen) in tunicates adds additional support
to this assignment.

We shall not attempt to discuss here the numerous contributions
of this sort that biochemistry has made to evolutionary theory.  The
reader will find this material summarized in a number of comprehen-
sive essays and books." 2, 3 Our present concern is primarily with
GENES, PROTEINS, AND EVOLUTION                               213


Figure 96. An electron photomicrograph of collagen fibrils from bovine skin.
Magnification x 42,000. Obtained through the kindness of Dr. Jerome Gross,
Massachusetts General Hospital, Harvard University Medical School.

214

THE MOLECULAR BASIS OF EVOLUTION

the biochemical changes in protein molecules that are much nearer,
in metabolic terms, to the genes themselves than are such products
of enzymatic action as the phosphagens, or the eye pigments of
Drosophila. As \Vald has put it "It is a truism in biochemistry that
each species of animal and plant possesses specifically different pro-
teins." The full understanding of speciation must, almost certainly,
be sought in the structllre of proteins.  To expand this point a bit,
let us consider two elegant examples of speciation that are demon-
strably related to changes in protein strrlcture.

Th e protein collagen is largely responsible for the physical prop-
erties of such structural tissues as skin and cartilage.  LVhen collagen
fibrils (Figure 96) are exposed to heat they change markedly in in-
ternal structure and yield the molecular form known iis gelatin.
Now recent studies by X-ray crystallography have shown that the
collagen molecule is very likely composed of three strands of poly-
peptide, cross-linked through a system of hydrogen bonds of con-
siderable strength.'  The amino acid sequence, Gly.Pro.IIypro, ap-
pears fairly frequently along the chains, and the hydroxyl groups on
the hydroxyproline residues are presumably major contributors to the
hydrogen bond network.  \Vhen collagen is heated in solution, the
hydrogen bonds become ruptured at a critical temperature, known
as the "shrinkage temperature," and the organized structure is quickly
disoriented to form the mow globular and amorphous gelatin strnc-
ture. Although the exact mechanism of this rearrangement is not
known, it is possible, on the basis of the results of current work on
the properties of synthetic polyproline and of mixed polymers of
proline and glycine, that the shrinkage may be associated with a
cis-trclns isomcrization at l)rolil7c-l"olinc Or proline-hydroxyproline
bonds," in conjunction with ordinary "entropic" denatrwation.
On the basis of these chemical and physical observations we might

srqqmsc that collagcw molewlcs,  suited to either cold or warm
habitats, co~~ld bc devised hy natlu'ca through the introduction or de-
letion of hydroxyproline residues.  Animals living in climates tending
to be very warm would do well to lltilizc collagens with I$.$ shrink-
agcl trnll~c,r;lturc,.~, ant1 thaw liviug in cold clirnatcs cor~ltl do with
considerably fcwcbr sitw for cross-linkage and with lowc~r shrinkage
tcmperatwcs.

The studies of K. If. Gustavson and of T. Takahashi on the col-
lngens of fishes suggest that this is precisely the mechanism which
has been employed." The shrinkage tempcraturcs of cold-\vater fishes
arc always lower than those of warm-water fishes, and an amazingly
linear relationship exists between shrinkage temperatnre and the con-

GENES, PROTEINS, AND EVOLUTION                                215


13 ,         I          I           I          I            TABLE 17

40         50         60         70
Shrinkage temperature, z, "C

Figure 97. The relationship between the hydroxyproline contents of the collagens
of various fishes and their "shrinkage temperature."'

tent of hydroxyproline (Figure 97), although the vertebrate collagens
are otherwise extremely similar in composition.  It is a provocative
fact that collagen shrinkage temperatures seem to fall about 15 or
20o above the highest temperatures likely to be encountered by a
species, as though this margin of safety were adequate in the ordi-
nary course of climatic events.

The relationship between the visual pigments of marine fishes and
the depths of their habitats is another dramatic example of adapta-
tion through modification of protein structure. Denton and War-
ren,' Munz,s Wald and his colleagues,D and many other investigators
have studied the chemical structure and the spectral properties of a
variety of fish rhodopsins. Rhodopsin, composed of a vitamin A
derivative complexed with a protein, opsin, constitutes the light-
sensitive element of the retinal rods. The vitamin A-like prosthetic
group, retinene, responsible for light absorption, has been found to
be identical in all the species listed in Table 17. Since opsins do
not themselves absorb light in the spectral interval between 480 and
503 rnp., the shifts in the position of the absorption maxima shown
in Figure 98 must be attributed to the effects of the opsins on the

216                            THE MOLECULAR BASIS OF EVOLUTION

Spectral Properties of Rhodopsins from Various Fishes

        Species
Summer flounder (Paralichthys den#atus
Linnaeus)
Scup (Stenotomus uer~ico20~ Mitchell)
Butterfish (Poronotus triacanthus Peck)
Barracuda (Sphyraena bore&r DeKay)
Cod (Gadus callarias Linnaeus)
Cusk (Rrosme brosme Miiller)
Lancet-fish (Alepiaarusferoz Lowe)

Summer
Range of
Depth,
fathoms

a-10     503      0.695
l-20      498      0.686
l-30      499      0.610
l-10      498      0.575
5-76      496      0.530
lo-100     494      0.455
>a00     480      0.%50

E54o/&,ax

spectral properties of retinene. The mechanism by which conjuga-
tion with opsin can induce a change in the spectral properties of
retinene is quite obscure. We have previously discussed a related in-
stance of a spectral shift, where the absorption characteristics of the

0.8

up, butterfish, barracuda

500            550            600
Wave length, rnfi

Figure 98. Absorption spectra of rhodopsins of marine fishes in 2 per cent aqueous
digitonin solution. The maximum absorption (A,,,.=) shifts toward shorter wave-
lengths in rough correlation with the depth of habitat.  See Table 17.

GENES, PROTEINS, AND EVOLUTION                                217


tyrosine residue are modified by hydrogen bonding of the hydroxyl
group.  The shift in this case was small, of the order of a few milli-
microns. In the rhodopsin absorption system maxima differ by as
much as 23 mp as we move from the summer flounder to the lancet
fish. This large shift implies a major change in the nature of the
interaction between protein and prosthetic group.

The biological observation that makes all this of special interest is
the fact that a correlation is observed between the mean depth of
habitat of the various species of fishes and the spectral properties of
their visual pigments.  The correlation is not at all exact and, as the
data in Table 17 show, a wide spread of A,,,~, exists at all depths.
Nevertheless, the information available is sufficient to form the basis
of a strong hypothesis. Over twenty years ago G. L. Clarke observed
that the increasing blueness of light with depth in the ocean raises
"the question of the possibility of a shift in the sensitivity of the eye
of a deep-water fish toward the blue end of the spectrum." This
possibility is realized in the spectroscopic observations just listed, and
it is now possible to apply the techniques of protein chemistry to the
elucidation of the details of this fascinating chapter in biochemical
ecology. The study of the structural modifications in opsin which
have taken place during the evolution of the fishes will be especially
interesting since, as we have seen for the insulins and cytochromes c,
large spans of evolutionary time may pass without too extensive a
change in a particular protein molecule. The changes in the opsin
molecule may be so cleverly contrived and so incisive that extensive
alterations in sequence and folding have been unnecessary. On the
other hand, if alterations hnve been extensive, we shall be required
to rationalize a very complex set of interactions between protein and
prosthetic group. Both alternatives are intriguing, to say the least,
and the study of the chemistry of the opsins should make a most
valuable contribution to the understanding of evolution at the molecu-
lar level.

The Rate of Evolution

As G. G. Simpson has pointed out, the question "How fast has evo-
lution occurred?" is meaningless without the addition of the qualifica-
tions, "the evolution of what organisms, of which of their structures,
and at what time in their history." The opossum, for example, has
changed relatively little in the past 80,OOO~OOO years, whereas the

218                          THE MOLECULAR BASIS OF EVOLUTION

evolution of the horses during the past 60,000,OOO years has involved
at least eight distinct genera.

Just as the anatomical organization of some organisms has changed
much more rapidly than that of others, it seems likely that we shall
find a large spread in the rates at which specific protein molecules
have been modified during evolution. Although our basis for discus-
sion of this point is still very thin, it is already evident that some pro-
teins have undergone far greater structural change than others over
an equivalent period. Compare, for example, the somatotropins of
the sperm whale and the sheep with the insulins of these same species.
Although the changes in insulin structure have been restricted to very
minor modifications in a limited part of one polypeptide chain, the
somatotropins are quite markedly modified in molecular weight, in
cystine content, and in the number of polypeptide chains.  Equally
striking differences in degrees of modification exist between numerous
others of the examples discussed in Chapter 7.

How are we to plan our experimental approach in attempting to
establish some chemical coherence in the tremendous puzzle of specia-
tion? We must, it would seem to me, begin with the basic assump-
tion that the phenotypic character of a species is primarily determined
by its unique spectrum of proteins. We may then proceed to a
study of the extent to which each of the individual proteins within
any spectrum may be modified without loss of biological function.
As we already know, the degree of "violability" of different proteins
may vary enormously as judged from the results of in vitro studies on
denaturation and chemical modification in relation to function.  Even
here, however, many of the observed differences in sensitivity may
be overemphasized and may depend on the choice of methods used
for modification. Even though two proteins may be very similar in
regard to the proportion of their total structure that is essential for
function, one set of reagents may attack critical parts of one and not
seriously alter the other. Amino groups, for example, may be acety-
lated with essentially no effect in pepsin, but at least some of these
same groups appear to be critical for the activity of lysozyme. A
proper comparison of two biologically active proteins thus must de-
pend on the use of a wide variety of inactivating reagents, and ulti-
mately on the deliberate degradative sort of study that aims to reduce
proteins to their minimum, functionally adequate size.

Since, however, proteins can be modified without loss of function,
it seems certain that the permissible degree of modification, in terms
of fractions of their total structure, will vary somewhat from molecu-

GENES, PROTEINS, AND EVOLUTION                                219


lar species to species.  It does not seem too farfetched to think of
the proteins of a given organism as being subdivisible into those that
have structures quite closely tailored to an essential functional re-
quirement, those that are designed with only moderate "efficiency"
or whose function is relatively dispensable, and those that are inter-
mediate.  Once again, illustrations come to mind.  Several individuals
exhibiting only slight clinical abnormality have been shown to be
completely devoid of serum albumin.  These individuals, able to lead
a normal existence, are living evidence for the dispensability of this
protein under the ecological circumstances peculiar to humans. On
the other hand, no one will question the inability of most species to
survive in the absence of cytochrome c or of the enzymes necessary
for oxidative phosphorylation.

If we accept these subdivisions of the protein spectrum, we may
`express" a species in terms of a hierarchy of protein structures rang-
ing in violability from none to very much. The further evolution
of this species, involving the usual mutation and natural selection,
would then be reflected in a change in its proteins, one end of the
spectrum remaining relatively fixed while the other may change con-
siderably. Thus the cytochrome c molecule, which we might think
of as a relatively "primitive" protein, and indispensable for most life,
would be stubbornly perpetuated in the evolving phyla with minimal
change, whereas the structure of the serum albumins might fluctuate
with the shifting parameters of natural selection.  From time to time,
entirely new protein structures might arise, as in the dramatic ap-
pearance of insulin and of other hormones at the point in evolution
when the protovertebrates and vertebrates appeared. The molecu-
lar basis for such "explosive" appearance of new protein entities is,
of course, completely obscure. Only a thorough understanding of
the processes of protein biosynthesis and of genetic information trans-
fer will enable us to choose between such alternatives as the de now
creation of a whole new gene as opposed to the fortuitous reshuffling
of already available genetic units.

We may safely predict that the patterns of change observed in the
protein spectrum by future biochemists will not always be smooth
and tidy. The criteria of natural selection will differ greatly from
species to species, from environment to environment, and from period
to period, and the survival value of gene mutations in a population,
and of their images in the phenotype, will be quite varied.

A large number of important aspects of evolution have been omitted
from this book. Some of these omissions may be attributed to type-

220                        THE MOLECULAR BASIS OF EVOLUTION

GENES, PROTEINS, AND EVOLUTION

221


writer fatigue. Most of them, however, have been purposely omitted
because of the lack of adequate factual material for discussion, and
this book is already well supplied with speculation. We might, for
example, have taken up the question of the spatial organization of
genes in relation to function. The recent studies on the mapping of
genes related to histidine biosynthesis in Salmonella (Figure 99) by
Hartmann, Demerec, and others indicate that the various cistrons
associated with the series of intermediate enzymes occur in the same
region of the genetic strand and that these genetic determinants are
arranged on the gene map in the same order as the reaction sequence
itself. A schematic representation of this linkage map is given in
Figure 99. The evolutionary implication that linked biochemical steps
have been added, successively, in sequence along the chromosome is
a very exciting one but is clearly not general.  In Neurospora, for ex-
ample, genetic loci for closely related enzymatic steps are scattered
at random throughout the chromosomal apparatus.
We might, also, have spent some time on the question of cytoplasmic
heredity, which we know to be of importance in many biological sys-
tems. Here again the scarcity of published information is a limiting
factor. The study of inheritance of traits in a non-Mendelian fashion
is likely to be difficult and confusing, and the genetic or biochemical
study of such traits might receive disproportionately little attention.
As Nanney has recently suggested, "It is perhaps only natural that in-
vestigations of `messy' characteristics are discontinued before publica-
tion and that investigators move on to traits more readily analyzed."
The omnivorous reader will find Nanney's reviewl" of the subject of
cytoplasmic heredity in The Chemical Basis of Heredity excellent
reading.
The list of omissions can be extended. The chemistry of RNA and
its genetic properties, the rearrangements of genes within the chromo-
some and the phenotypic consequences of such rearrangements, the
problem of polyploidy, the interactions of nonallelic genes-many of
these might, even now, be discussed with some intelligence in bio-
chemical terms.

The relationships between genotype and phenotype will, predict-
ably, become a major preoccupation of more and more "pure" and
medical scientists during the coming years. This book has grown out
of my own attempts to arrive at some sort of appreciation of the
potentialities of chemical genetics and the evolutionary approach.
It will have been well worth the effort if it can help to stimulate
the growing interest in evolution as the central theme in the life
sciences.

222                         THE MOLECULAR BASlS OF EVOLUTION

REFERENCES

1. J. B. S. Haldane, The Biochemistry of Genetics, Allen & Unwin, London,
second impression 1956.

2. M. Florkin, Biochemical Eoolution, Academic Press, New York, 1949.
3. G. Wald, in Modern Trends in Physiology and Biochemistry, Academic
Press, New York, 1952.

4. A. Rich and F. H. C. Crick, Nature, 176,915 (1955).
5. W. F. Harrington, Natrcre, 181, 997 ( 1958).
6. K. H. Gustavson, The Chemistry and Reactioity of Collagen, Academic
Press, New York, 1956. The analytical studies of Takahashi are discussed
on pages 224-227.

7. E. J. Denton and F. J. Warren, Nature, 178, 1059 (1956).
8. F. W. Munz, Science, 125, 1142 ( 1957).
9. G. Wald, P. K. Brown and P. S. Brown, Nature. 180,969 ( 1957).
10. D. L. Nanney, The Chemical Basis of Heredity (W. D. McElroy and
B. Glass, editors), Johns Hopkins Press, Baltimore, 1957.

GENES, PROTEINS, AND EVOLUTION

223


INDEX

.4cetabularta mediterranea, protein syn-
  thesis in, 195, 203
ACTH, 115, 116
Activation of amino acids for protein
  synthesis, 205, 206
"Active centers," chemical nature of,
   268
Adrenocorticotropic hormone, active
  degradation products of, 129
Adrenocorticotropin, structure of, 115,
   116
Adrenocorticotropins, species differ-
  ences in, 152
Alkaline phosphatase, genetic de-
  termination of, 182
Allelic genes, 16
Allometric change, 10
Amino acid activation, 205, 207
Amino acid analogues, incorporation
  into proteins, 190
Amino acid sequences, repetition of in
  proteins, 207-269
Amino acids, abbreviations for, 105
Aristogenesis, 6, 13

Bacteriophage, plaque morphology of,
  67-71

r-mutants of, 68-71
Bacteriophage proteins, fractionation of,
  179, 182, 183

Bacteriophages, chemistry and enzy-
mology of, 71

life cycle and biosynthesis of, 81
morphology of T group, 7.5-77, 180,
  181

Biochemical evolution, 213
Biosynthesis of proteins, 195

Carbobenzoxylated polypeptide chains,
trypsin digestion of, 108, 109
Carboxypeptidase, use of in protein
  structure studies, 107
Centromeres, 19,21, 22, 33, 35
Chiasmata, 21, 23
Chromatography, ion exchange, 169,
  110

Chromosome, organization of, 59-61
Chromosomes, 19, 21, 24, 2628
Cis-trans test, 29, 30, 88, 89
applied to host range function, 177
Cistron, 29, 36, 89, 93
Code, "commaless," 63, 64
"Codes," genetic, 62-65
Collagen, characteristics of in relation
  to hydroxyproline content, 215
species differences in, 215, 216
structure of, 214, 215
Configurational isomerism in proteins,
  186

a-Corticotropin, porcine, structure of,
  130

Cytochrome c, species differences in,
  157

Cytoplasmic heredity, 222

Denaturation, 127
Deoxyribonucleic acid, chemical struc-
  ture of, 4456
conservation of during cell replica-
   tion, 41, 57-60
in chromosomes, 394.1, 44
molecular dimensions of, 4951,
    78-81
synthesis of, 55, 56
X-ray diffraction analysis of, 49
Deuterium exchange, 123-125

225


Differentiation, of teeth, 10
reptilian jaw, 13
Di-isopropylfluorophosphate, 208
Dinitrophenylation, 106, 107
Diploid cells, 21
Disulfide bridges, location of in pro-
  teins, 113, 114

2%~~ oitd, 6, 13
Endoplasmic reticulum, 196, 197, 199
Entelechy, 6, 13
Enzymatic digestion of polypeptide
  chains, 106-111, 113
Ergastoplasm, 196
Evolution, rate of, 218

"Fingerprinting" technique for pep-
  tides, 144

Gamete, 21
Genes, 15, 16
analogous, 142
determinants of protein structure,
    164
dominant, 16
homologous, 142
linked, 22, 23
molecuhir size of, 27, 28
recessive, 16
substructure of, 67
Genetic codes, 62-85
Genetic fine structure, 173
Genetic maps, 24, 25, 26
fine structure, 86, 88-95, I73
in bacteriophage, 84, 173-176
Genetic recombination, 16
Genetics of host range in bacterio-
  phage, 173
Genotype, 15
Growth hormones, species differences
  in, 158
Gryphaea, evolution of, 7-10

Haploid cells, 21
a-Helical coiling in globular proteins,
  118, 119
Helical coiling in proteins, 100, 101
a-Helix, 100-102
dimensions of, 101

226

llcmoglobins, 167-171
abnormal, structural differences in,
   16%171

species differences in, 158, 160, 161
Heredity, cytoplasmie, 222
Heterozygote, 16
Homozygote, 16
IIorses, evolution of, 10-12
IIost range genetics in bacteriophage,
  173

Hypertensins, species differences in,
  155, 157

Immunochemical comparisons of serum
  proteins, 162, 163
Independent assortment, law of, 18, 19
Insulin, active degradation product of,
  128

structure of, 122
Iusulins, species difference in, 154, 155

Law of independent assortment, 18, 19
Law of segregation, 16, 17
Linkage, 22, 23
Linkage groups, 23, 26
Linkage map, for histidine biosynthesis
  in SaZmotaella, 221, 222
of T4 bacteriophage, 90, 92
Lozenge genes, 28, 29
Lysozyme, of bacteriophage, 73-75,
  183. 212

specificity of, 74

Macroevolution, 7, 9, 10
Maps, genetic, in bacteriophage, 84,
  173-176
Megaevolution, 7, 9,
Meiosis, 19, 21, 22
stages in, 22
Mclanotropins, species differences in,
   154
structures of, 153
Membrane, cytoplasmic, 196-199
Mendelian genetics, 16-19
Microevolution, 7
"M icroheterogeneity," 187
Mitosis, 19, 20
stages in, 20
Mlltagenesis, chemical, 40
Mutants, deletion, 86, 89, 91
Mutation, 27

THE MOLECULAR BASIS OF EVOLUTION

Mutations, effects on protein structure,
  166
Muton, 93

Myoglobin, structure of, 98

Natural selection, 11, 15
Neurospcm, 3138
nutritional mutants of, 31, 32, 34,
   36, 37

Neurospora crassa, life cycle of, 32
Nucleus, composition of, 40

"One gene-one enzyme" hypothesis, 36
Opsins, species variations in, 216-218
Optical rotation of proteins, 118, 119
Ostrea, evolution of, 7
Oxidation of disulfide groups in pro-

teius, 107, 108

Papain, active degradation products of,
  130, 131

Paper chromatography, of amino acids,
  150, 151

of peptides, 145, 146
Paper electrophoresis of peptides, 14.5,
  146

Peptide "fingerprints," 144
Pcptide patterns as "fingerprints" of
  proteins, 144

Peptide separation on paper, 144-146
Phenotype, 15
Phosphoserine, in proteins, 208
Phylogenetic relationships, -3-6
l'olypeptide chain, dimensions of, 100
Preadaptation, 13
Prolactins, species differences in, 158,
   160
Protamines, conjugation of with DNA,
  200, 201
Protein biosynthesis, 195
in Acetdmlaria mediterruneu, 195,
    203
in ruptured-cell preparations, 205
Protein structure, configurational iso-
  merism in, 186
sequential isomerism in, 18G
species variation in, 142
Proteins, bacteriophage, fractionation
  of, 179, 182, 183
biological activity of, in relation to
   structure, 126

INDEX

Proteins, heterogeneity of, 185
Pseudoalleles, 28, 29

"Quantum evolution," 11

Radiation, adaptive, 10
mutational effect of, 28, 29
Ilcct)lllbination frccluency, 23
Recombination units, 25
Recon, 30, 93
Replication, and structure of deoxy-
  ribonucleic acid, 55
"copy-choice," 86, 87
Reversible denaturation, of papain, 131
of ribonuclease, 136, 137
Hibonuclease, action of, 103, 104
composition of, 104, 105
partial reductidn with retention of
activity, 134, 135
pepsin digestion of, 133, 135, 137,
    138

peptidc "fingerprint" of, 144, 14G,
  146-151

photooxidation of, 132, 133
reduction of, 133-135
reversible denaturation of, 136, 137
structure-function relationships, 131
structure of, 112, 115
subtilisin digestion of, 132, 135,
  137-140

synthetic substrate for, 104
Ribonucleases, beef and sheep, compo-
  sitions of, 147
species differences in structures of,
    149

Ribonucleic acid, chemical structure of,
  4Fj, 46

nuclear, turnover of, 201, 202
role in protein synthesis, 203, 204,
  207

Suln~onelka, chemical genetics of, 221,
  222
Saltation, 7
Secondary structure, 117
"Scgrcgation, law of," 16, 17
Sq~cntial isomerism in proteins, 186
Serum albumin, absence from sera of
  certain illdividuals, 220
Scrllm proteins, species differences in,
  161-163

227


Species variation in protein structure,
  142

Spectroscopic properties of proteins, 119
Structure, of proteins, 99
  in relation to function, 126
primary, 99

secondary, 99-102
tertiary, 99, 102

Teeth, differentiation of, 10
Tertiary structure, 117
Three-factor cross, 175
Transduction, 95

   22%
-

Transformation, by DNA, 41-44
of linked genes, 44
Tyrosine, anomolous light absorption
of, in proteins, 119, 120
-carboxylate interactions, 120, 121

Urea, effect of on protein structure,
  120, a4

Visual pigments, species differences in,
  216218

Zygote, 21, 32, 33, 35

THE MOLECULAR BASIS OF EVOLUTION