Progress in Molecular and Subcellular Biology - Volume 1

Progress in Molecular and Subcellular Biology .
Volume 1
Progress in Molecular
and Subcellular Biology
Volume 1
By
B. W. Agranoff . J. Davies . F. E. Hahn· H. G. Mandel
N. S. Scott· R. M. Smillie' C. R. Woese
Editorial Board
F. E. Hahn' T. T. Puck' G. F. Springer' K. Wallenfels
Managing Editor
F. E. Hahn
Springer-Verlag New York' Heidelberg' Berlin 1969

ISBN-13: 978-3-642-46202-3 e-ISBN-13: 978-3-642-46200-9
001: 10.1007/978-3-642-46200-9
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mined by agreement with the publisher. © by Springer-Verlag Berlin Heidelberg 1969.
Softcover reprint of the hardcover 1st edition 1969
Library of ('ongress Catalog Card Number 75-79748
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Title-No. 3461
Contents
FRED E. HAHN: On Molecular Biology 1
References . . . . . . . . . . . 4
CARL R. WOESE: The Biological Significance of the Genetic Code. . . 5

I. Introduction. . . . . . . . . . 5
II. Characteristics of the Genetic Code 7
III. Conclusion 38
References 39
JULIAN DAVIES: Errors in Translation. 47

I. Introduction. . . . . 47
II. The Translation System 47
III. Errors in Translation . 50
IV. Errors Produced by Aminoglycosides 56
V. Ribosome Mutations Leading to Ambiquity 66
VI. Other Factors Causing Ambiquity in vitro . 67
VII. Does Misreading Occur in Mammalian Systems? 72
VIII. Does Misreading Occur in Living Cells? 73
IX. Finale 74
References 77
H. GEORGE MANDEL: The Incorporation of Fluorouracil into RNA and its

Molecular Consequences . . . . . . . . . . . . . 82
I. Introduction. . . . . . . . . . . . . . . . . 82
II. Incorporation of FU into Whole Cells and Tissues 83
III. FU and Ribosomes 89
VI. FU and sRNA . . . . . 99
V. FU and mRNA 105
VI. FU and Protein Synthesis 114
VII. Synthetic Polynucleotides Containing FU 125
VIII. Discussion 128
XI. Summary . 129
References 130
VI Contents
ROBERT M. SMILLIE and N. STEELE SCOTT: Organelle Biosynthesis: The

Chloroplast 136
I. Introduction . 136
II. Composition . 136
III. Chloroplast DNA 137
IV. Ribosomes and RNA 149
V. Synthesis of Chloroplast Proteins 157
VI. Photo regulation of Chloroplast Development 167
VII. General Conclusions 184
References 187
BERNARD W. AGRANOFF: Macromolecules and Brain Function-A 1969

Baedeker. . . . . 203
I. Introduction . 203
II. Growth. . . 204
III. Sensory Modalities 205
IV. Structural Localization: Brain Regions and Fractions 206
V. Human Disorders 208
VI. Learning and Memory 209
References . . . . . 210
Author Index 213

Subject Index 235
List of Contributors
BERNARD W. AGRANOFF, Chief, Section on Biochemistry, Mental Health Research
Institute and Professor, Department of Biological Chemistry, University of
Michigan, Ann Arbor, Michigan
JULIAN DAVIES, Department of Biochemistry, University of Wisconsin, Madison,
Wisconsin
FRED E. HAHN, Chief, Department of Molecular Biology, Walter Reed Army Insti-
tute of Research, Washington, D. C.
H. GEORGE MANDEL, Department of Pharmacology, The George Washington Uni-
versity School, of Medicine, Washington, D. C.
N. STEELE SCOTT, Plant Physiology Unit, C.S.I.R.O. Division of Food Preservation,
Ryde and School of Biological Sciences, The University of Sydney, Sydney,
N.S.W. 2006, Australia
ROBERT M. SMILLIE, Plant Physiology Unit, C.S.I.R.O. Division of Food Preserva-
tion, Ryde and School of Biological Sciences, The University of Sydney, Sydney,
N.S.W. 2006, Australia
CARL R. WOESE, Department of Microbiology, University of Illinois, Urbana,
Illinois
On Molecular Biology
FRED E. HAHN
The name, Molecular Biology, appeared in the scientific literature in the early
1950ies (AsTBURY, 1950; WEISS, 1953). It has been noted by F. O. SCHMITT (as cited
by WEISS, 1963) that]. E. PURKINJE used the term more than 100 years ago and it
might be interesting to the historian to trace the origin and meaning of this early
usage.
Like any science which aspires to resolving a body of observations into its deter-
minant entities and laws at the molecular level of organization, molecular biology is
a form of applied chemistry. The identical replication of DNA in vitro and the crypt-
analysis of the amino acid code have been accomplished by biochemists. Noted
biochemists regard, seriously (EDSALL, 1964) or sometimes acrimoniously (CHAR-
GAFF, 1963), the field of molecular biology as synonymous with biochemistry.
On the other hand, a not-so-"invisible college", composed mainly of microbial
geneticists and physicists with a biological bent has registered claim to have origi-
nated molecular biology (CAIRNS, STENT, and WATSON, edts., 1966) and defers to
SCHRODINGER'S treatise, What is Life? (1945) as its book of prophesy. This group
consolidated itself prominently around the genetics of bacterial viruses, looks upon
biochemistry with reservations and has shown a propensity for inductive reasoning.
Finally, KENDREW (1967) has called attention to a third scientific discipline, X-ray
study of structures of biological substances, as being one of the pivotal components
of molecular biology.
Such differences in definitions or historical outlook are spurious. It is apparent
that molecular biology owes its current prominence to biochemists, geneticists,
biophysicists, crystallographers, microbiologists and theorists of diversified back-
grounds, severally, as well as jointly.
An elegant account of the history of molecular biology by STENT (1968) is note-
worthy for at least two reasons. 1. In referring back to SCHRODINGER (1945), STENT re-
calls a speculation that the stability and continuity of the genetic endowment of orga-
nisms may require the action of hitherto unknown laws of physics. In STENT'S presenta-
tion (1968), the history of molecular biology unfolds as an attempt to recognize such
laws with the somewhat disappointing outcome that nothing of this nature has been
discovered. I rather assume that many physical scientists who have turned to biology
did so with the a priori assumption that the fundamentals of the life process consti-
tuted an unresolved form of chemistry and physics and that the primary concern
first of "quantitative" and later of molecular biology was to bring about a resolution
within the framework of the recognized and known laws of the physical universe. In
other words, molecular biology was to them not so much a vehicle for deeper pene-
tration into physics, but, conversely, the physical sciences were considered an instru-
ment of deeper penetration into the nature of life. 2. The second noteworthy feature
1 Molecular and Subcellular Biology, Vol. 1
2 FRED E.HAHN
of STENT'S discourse (1968) is the retrospective sentiment inherent in the title, That
Was The Molecular Biology That Was. The assumption is made that the postulation
and eventual proof of the Central Dogma as a fait accompli relegates the conceptual
endeavors of molecular biology to an issue of the past and that solutions to the still
remaining group of problems of developmental biology of higher organisms will
essentially be accomplished through the application and extrapolation of the know-
ledge of biochemical genetics thus far developed, but without requiring further
conceptual innovation. One senses an element of romanticism disappointed with the
failure of uncovering "other" laws of physics. A similar fin du sieele attitude was
attributed to DELBRUCK when he turned, around 1950, from genetics to the study of
sensory perception as a point of investigative departure into neurobiology. It is a
typical human fallacy to consider one's contemporary point in time the culmination
of historical development.
STENT (1968) finally does refer to mechanisms of higher nervous activities as a
"last frontier" in molecular biology where some "other" laws of physics may yet
turn up, but he also holds out the dim prospect that the human brain "may not be
capable in the last analysis of providing an explanation for itself."
This Editor would not have undertaken the task of bringing this Progress series
into existence had he been of the opinion that all that was left to the field of molecular
biology was extrapolation and application of a central dogma (in STENT'S language,
"the need to iron out the details"). Neither does he share the philosophical resigna-
tion to the thought that human cognition may fail when applied to itself as the object
of study. The history of the Theory of Relativity is one example in which mathe-
matical and scientific logic has brought to light the limitations of naive human
perception in the categories of space and time. Least of all does this Editor subscribe
to the neo-vitalistic proposition that the pursuit of molecular biology might have
brought out or may yet uncover laws of nature which are peculiarly operative in
living systems.
Some of the current scientific scene is, indeed, characterized by a carry-over of
concepts and techniques, developed in the study of genetic information of viruses and
bacteria, to problems of growth, development, morphogenesis, ontogenesis, homeo-
static maintenance and aging of large complicated organisms, hopefully of man
himself. It is intrinsic to such an approach that the framework of ideas from which the
questions are derived predetermines to some extent the answers which are obtained
in the manner of self-fulfilling prophecies. Above the phylogenetic levels of anatomi-
cal and physiological complexity at which hormonal and neural regulation mecha-
nisms come into play, one will need to ask just how "central" the Central Dogma of
molecular genetics remains according to which DNA not only specifies itself but
solely determines the totality of all other life processes.
It is possible to discern at least three areas of the possible molecular biology of the
future which can not easily be derived from the Central Dogma.
1. In living organisms the flow of matter at the molecular level is controlled and
regulated by membranes which compartmentalize organs, tissues, cells and sub-
cellular organelles. These membranes are endowed with the capability of recognizing
molecules which seems to be no less selective than that of the specificity of enzymes
for their substrates (PARDEE, 1968). But, while the "molecular biology that was" has
elucidated transcription and translation of gene structure into amino acid sequences
On Molecular Biology 3
in enzyme proteins (although the relationships between primary structure and func-
tion of enzymes remain largely unknown), we neither understand the determinants
of membrane structure nor these structures as such, nor the mechanisms underlying
the molecular specificity of membrane function. To express it differently: we do not
know the information which specifies membrane formation nor the nature of the in-
formation which membranes do contain and implement. Any attempt at conceptual
or physical reassemblage of biological structures from their functional components
must remain illusory as long as the origin, structure, and function of membranes are
unexplained.
2. Higher organisms produce regulatory chemicals, called hormones, which are
responsible for the timing and regulation of growth and development and for the
homeostatic maintenance of the fully developed organism. The production of
hormones by specialized organs is, by itself, regulated by other hormones. The
recognition of numerous hormones and the elucidation of their chemical structures
is one of the historical accomplishments of biochemistry during the past half century,
but neither do we understand the mechanism of action of even one single hormone at
the molecular level nor do we understand what predetermines, balances or changes
the state of endocrine equilibrium as a whole. To the extent to which endocrine
chemicals such as steroids have been considered inducers of messenger RNA tran-
scription at the gene, such ideas involve a seeming paradox in the Central Dogma in
that substances which are products (hormones) of gene products (enzymes) would
channel regulatory information back to DNA. Clearly, we are dealing with biological
determinism of timing (program), quality (control) and quantity (regulation) in the
chemism of living organisms on a supragenic level.
3. Mechanisms of neural control, including as its most highly developed mani-
festations processes of learning, recall and reasoning, remain an almost total mystery.
To approach, for example, the problem of learning, i.e. the acquisition, storage and
recall of "information" as a problem in molecular biology analogous to the codifica-
tion, transcription and translation of "genetic information" may be based merely on
a verbal coincidence (AGRANOFF, this volume) in using the term "information". To
approach the same problem in analogy to computer technology involves the potential
fallacy of mistaking the working principles of machine models producing resemblances
of natural occurrences, for the working of laws underlying such natural phenomena.
Medieval astronomical clocks were mechanical computers imitating and prog-
nosticating planetary movements, but failing to explain celestial mechanics in terms of
Newton's Laws.
OCHOA (1964) asked "Will it be too optimistic to hope that the next decades may
also see a breakthrough in our understanding of the molecular basis of the higher
functions of the brain?" Even in this restrained formulation, his question implies
that these functions do have a molecular basis and can, therefore, be considered a
theme in the molecular biology of the future. This Editor is mildly optimistic con-
cerning such investigative prospects, but rather doubtful that the results will lie
within the triadic relationship between DNA, RNA and protein, and the Central
Dogma operative within this relationship.
In conclusion: the Publisher and the Editors of this series, Progress in Molecular
and Subcellular Biology, are undertaking the task of selecting and serially publishing
progress reports at a time at which the scientific literature is flooded with source and
1*
4 FRED E. HAHN: On Molecular Biology
review articles and at which some molecular geneticists believe that the field has entered
into its academic phase in which further results will be merely consequences of concept-
ualizations which are by now well-established.
I have attempted to exemplify some areas of future investigations which appear
to transcend simplistic extrapolation from biochemical genetics. Molecular genetics
may be considered a field "that was," but molecular biology in a wider sense has just
begun. We intend to communicate both the academic accomplishments of the estab-
lished field and progress in the coming molecular biology.
References
ASTBURY, W. T.: Adventures in molecular biology. Harvey Lect. 46, 3 (1950-51).
CAIRNS, J., G. S. STENT, and J. D. WATSON: Phage and the origins of molecular biology.
Cold Spr. Harb. Lab. Quant. BioI. 1966.
CHARGAFF, E.: Amphisbaena. In: Essays on nucleic acids. Amsterdam: Elsevier 1963.
EDSALL, J. T.: OpeningRemarks,Proc. Plen. Sess. Sixth Int. Congr. Biochem., 3 (1964).
KENDREW, J. c.: How molecular biology started. Sci. Amer. 216 (3), 141 (1967).
OCHOA, S.: Opening Remarks, Proc. Plen. Sess. Sixth Int. Congr. Biochem., 9 (1964).
PARDEE, A. B.: Membrane transport proteins. Science 162, 632 (1968).
SCHRODINGER, E.: What is life? New York: Cambridge Univ. Press 1945.
STENT, G. S.: That was the molecular biology that was. Science 160, 390 (1968).
WEISS, P.: Medicine and society: The biological foundations. J. Mt Sinai Hosp. 19, 716
(1953).
- Personal communication 1963.
The Biological Significance of the Genetic Code
CARL R. WOESE
1. Introduction
Over the past 5 years or so we have witnessed what is often referred to as one of
the great accomplishments of science, the "solving" of the genetic code. The classical
biologist, however, may find himself hard put to grasp the biological significance of
this achievement. There can be no doubt that this is indeed a singularly great feat,
yet one can legitimately ask how the specific knowledge of just what "code" the cell
employs increases our understanding of living systems over and above the mere
knowledge that the cell does employ a "code" (of a very general kind). Would the cell
we see today have been any different had not UUU been assigned to phenylalanine?
The value of this line of questioning does not, I feel, lie so much in any answers
as in the heuristic value of the questions themselves. By so questioning we are lead to
inquire what this "genetic code" really is, what "solving" it constitutes, and so what
the problems remaining in this area are. What do we study when we study the genetic
code, i.e., what biological phenomena should be grouped under this heading? Are
we, in fact, correct in calling this a "code?" And, does our present knowledge then
constitute a "solving" of the problem? It might be argued that one has latitude to
define matters according to his own choosing, and so the "code" is "solved" if we
define things in that way. However, I do feel that definition should accord with
the nature of the biological phenomenon, not the reverse. And, in this case it is clear
that we are dealing with the immediate molecular aspects of gene expression, the
processes by which the cell effects and controls the transfer of information from one
class of macromolecules (nucleic acids) to another (proteins). When viewed in this
way the elucidation of the particular dictionary (code) the cell employs becomes only
a step in the elucidation of the overall problem.
Let us try to gain a feeling for the present status of the "coding problem" through
an analogy. Suppose we were given a particular extract from cells and we determined
it to have the following property: When nucleoside triphosphates are added to the
extract along with poly A, then poly T is synthesized, but when the poly A is re-
placed by poly T, poly C, or poly G, successively, one observes production of poly A,
poly G, or poly C, respectively. Given these and certain other experiments, one would
soon arrive at the notion of an input-output "code" for this system of the simple
composition
Input base Base specified
in polymer in output polymer
A T
C G
G C
T A
6 CARL R. WOESE
Where to proceed from here is immediately obvious in this simple example. Why are
these particular input units associated with these particular output unitS? Following
this line of questioning we would sooner or later discover that base pairing (postulat-
ed in another universe by Drs. WATSON and CRICK) lies behind all. Viewing our
knowledge of the genetic code in the light of this analogy, we see that what is now
possible is the construction of an "input-output" table, the catalog of codon assign-
ments, for the system, but what remains unknown is why this particular set of
relationships exists. The essence of the genetic code lies in those "forces" or processes
that cause UUU to be assigned to phenylalanine, or cause translation to occur in the
way that it does. And we have yet to gain the slightest appreciation for what these
are. As will be seen, if it is not already obvious, this aspect of the problem is inse-
parable from the problem of how this biological information processing system we
find in the cell could ever have arisen in the first place.
In its historical development the biological coding field is divided rather sharpyl
into eras. Initially, with the development of genetics - the concept of genotype and
phenotype - there came the realization that information somehow flowed from a
"gene" to what turned out to be a protein, the gene controlling the properties of the
protein. Subsequently with the elucidation of the structures of the polymers involved,
it became evident that this information transfer involved a simple colinear mapping
of the primary structure of a (linear) DNA molecule into that of a polypeptide. The
whole process was rather analogous to the reading of a book. The second era began
about the time of the discovery of DNA structure by WATSON and CRICK, from which
structure the coding field received a great impetus. Now, for the first time serious
attention was given to the molecular details of the gene-protein relationship. Due
mainly to the incisive thinking of men such as GAMOW, DOUNCE, and CRICK, the key
concepts of the coding field were recognized, defined, and answers to various central
questions speculated upon. In this background the third era in the genetic code began
abruptly in 1961 with the report by NIRENBERG and MATTHAEI of a feasible in vitro
system approach to elucidating the set of codon assignments. At the present time the
field seems to be entering still another era, this one characterized by the elucidation of
the molecular mechanics of the translation process -as we can see already in reports
of ribosome reassembly from subunits and studies on the mechanism of punctu-
ation.
In keeping with the above, the present review will not be confined simply to
recounting the spectacular experiments leading to the final elucidation of the codon
catalog. Rather, all facets of the problem will be juxtaposed in a way designed to
place the whole problem in a broader biological perspective. To this end it is useful
to present for each facet of the problem the historical thinking that went into defining
it, its present experimental status, and its possible relevance to the central problem of
the origin of a biological information processing system.
In considering the genetic code it is often useful to view it by analogy to the linear
information handling systems with which we are already acquainted - communica-
tion by written or spoken language, or better by analogy to general tape-tape reading
processes. In this way many of the features of the process become self-evident.
The Biological Significance of the Genetic Code 7
II. Characteristics of the Genetic Code

A. Colineari!J
Given that genetic information is stored in a linear form, the simplest and so most
reasonable assumption regarding information transfer in protein synthesis is that the
sequence of units (words) in the gene maps in the simplest way, a colinear mapping,
into the corresponding polypeptide. This is the assumption made by all of the early
thinkers on the problem, either explicitly or implicitly. Later studies showing that
protein synthesis occurred by what seemed to be a sequential assembly in time of
amino acid units beginning at the amino terminal end of the polypeptide and ending
at the carboxyl terminal (BISHOP et aI., 1960; DINTZIS, 1961), greatly strengthened
this assumption, as did a number of genetic studies on intragenic structure (see
BENZER, 1957) and the development of the message RNA concept (BRENNER et al.,
1961). Yet the thesis of colinearity did not receive a direct proof until relatively late
in the history of the genetic code. It was shown by the group working in YANOFSKY'S
laboratory (YANOFSKY et aI., 1964) that for a series of mutations in the E. coli trypto-
phan synthetase A protein gene (each of which caused a single amino acid replacement
in the corresponding protein) the linear ordering of mutations by genetic mapping
was exactly the same as the ordering of corresponding amino acid replacements in the
polypeptide (determined by amino acid sequence analyses). Another demonstration
of the validity of the colinearity thesis was performed somewhat differently using an
experimental system derived from E. coli infected with phage T 4 . A certain class of
phage mutants that mimic the properties of the peptide chain termination punctua-
tion sequences normally employed in translation, can be readily characterized in this
system by virtue of the facts: (1) that translation of any mRNA proceeds up to the
point of occurrence of one of these termination sequences (called "amber" codons)
but proceeds no further, (2) that in the T4 phage-E. coli system, the head protein or
parts thereof, can be detected in a tryptic (or chymotryptic) digest of labeled unfrac-
tionated infected cells by virtue of the fact that at the appropriate stage in the infec-
tious cycle the head protein is the major protein synthesized, and (3) amber mutant
phages can be maintained by growth on strains of the host that suppress the amber
mutation. Workers in BRENNER'S laboratory (SARABHAI et aI., 1964) were able to
utilize these facts to show that given a set of these amber mutants located at various
positions in the T 4 phage head protein gene, their positions in the genetic map
(determined by conventional genetic techniques) corresponded exactly (were colinear)
with their ranking according to the fraction of the T 4 head protein chain that they
permitted to be synthesized.
B. The Code Words- Sentence Structure and Reading; Synotryms

Given linear information transfer, one immediately thinks in terms of "letters"
and "words," the basic units in this sort of scheme, and of sentence structure. The
alphabet in which nucleic acid "sentences" (messages) are written comprises the four
different kinds of nucleotides, while the alphabet used in construction of polypeptide
"sentences" comprises 20 different units (the amino acids). That the juxtaposition of
the 4 and the 20 gave rise to a special problem in information transfer seems to have
been recognized first by CALDWELL and HINSHELWOOD (1950) and by DOUNCE
8 CARL R. WOESE
(1952). They saw that the cell would have to employ groups of units (words) in
nucleic acid language to specify the letters in amino acid language. Taken singly,
nucleotides could specify no more than four amino acids; taken in pairs 42 = 16
amino acids at most; but taken in groups of three, up to 43 = 64 amino acids can be
specified.
While these original speculations were merely to show how a limited number of
kinds of nucleotides could be used to code for some larger number of kinds of amino
acids, GAMOW saw in this nucleotide combinatorial problem the glimmerings of a far
more significant relationship: For GAMOW (1954) it was no happenstance that exactly
20 kinds of amino acids were used in the manufacture of proteins; the exact number
20 must somehow fall out of the structure of, the molecular interactions involved in,
the genetic code. Thus, the 20 assigned co dons (or codon classes) - and even the
assignments themselves - might be derivable a priori from simple considerations
such as the fact of the 4 and 20, the DNA structure, etc. - knowing no more biology
than that! Now this idea, grandiose in conception, magnetic in the challenge it
provided, and overwhelming in its potential significance, caught the imaginations of
biologist, physicist, and mathematician alike, and in one short moment set the tenor
of the coding field for nearly the next decade. This is in retrospect somewhat unfortu-
nate, for it was ultimately responsible for the term "code" being applied to the present
subject, and for the heavy overemphasis that has lead occasionally to the impression
that elucidation of the codon assignments is all that there is to the problem of the
genetic code. We shall temporarily postpone a review of specific schemes proposed to
account for the mystical number 20, and proceed with the general features of some of
these early schemes.
GAMOW'S central idea of an ordered, knowable reason for the "20" did lead him to
postulate the later-proven notion that the codon was of a uniform size of three
nucleotides (GAMOW, 1954). At the time GAMOW (1954) also called attention to
another problem involved in decoding, that of "overlapping" (and so "non-over-
lapping") translation. Since we are trained to use a non-overlapping form of verbal
or written communication, it is seldom that we are even aware of this point. In an
overlapping structure a letter is simultaneously in two or more adjacent words. For
example, given a message sequence ABCDEFGHI composed of three letter words,
a non-overlapping sentence structure would be read ABC, DEF, GHI; a completely
overlapping structure would read ABC, BCD, CDE, DEF, etc. Gamow's own
coding schemes in all cases involved an overlapping translation, this feature being
dictated by certain structural considerations, as we shall see.
A non-overlapping translation of a message presents a particular problem, again
so self-evident, so much an integral part of our language structure, that it tends to be
overlooked. How does one tell which groupings of contiguous letters in a message
constitute words, and which do not? The problem can be solved in any of three ways. In
(written) language it is solved in a !Jmbolic way, by reserving a special symbol, the
space, to designate beginnings and endings of words. The alternatives to this are a
sequential solution or a contextual solution. Let us assume that we are dealing with a
language that contains only three letter words. Any message written in this language
can then be considered to be really three messages, depending upon whether a given
letter in the message is taken to be the first, the second, or the third letter of a word.
Customarily we say that the message can be read in any of three "reading frames."
The problem in these terms is to devise a method for reading in the correct "frame."
The sequential solution is simple - one begins at a defined starting point and reads by
counting off groups of three letters at a time. Thus the starting point determines the
reading frame. Although this method makes certain exacting requirements on the
translation process (as we shall see), it is the method the cell employs to translate
message RNA (BISHOP et aI., 1960; DINTZIS, 1961; NISHIMURA et aI., 1965; WAHBA
et al., 1966).
The contextual solution was devised by CRICK and his associates (1957), and is by
far the most ingenious of the three. Suppose we were to divide all possible n letter
words into two categories, one containing words to which we assign meaning, the
other containing meaningless, undefined, or "nonsense" words. What CRICK and his
associates showed was that there is a particular way of doing this so that when a
message is constructed to contain only meaningful words (in the proper reading
frame), then reading this message in any of the improper reading frames yields only
meaningless, "nonsense" words.
Another general feature of the genetic code is the matter of degeneracy, or as it is
sometimes put, the matter of synonyms. Is a single amino acid ever represented by
more than one codon? GAMOW (1954) pictured the code as being completely de-
generate - all possible co dons assigned to amino acids - which turns out to be
nearly the true situation. In GAMOW'S case the notion again came from his basic tenet
that the number 20 is no coincidence; given the proper degeneracy rule it should be
possible to place the 64 possible triplet co dons into exactly 20 categories. CRICK, on
the other hand, pictured the code as nondegenerate, again from more fundamental
considerations (CRICK et aI., 1957). As we have just seen, one can solve the problem
of choosing the correct reading frame by creating two categories of code words,
sense and nonsense. It turns out that in such a scheme, if one employs a triplet codon
and a four letter alphabet, the maximum number of sense words is precisely 20!
The definitive experimental judgment on all the above speculation regarding the
code's general nature came during the period 1961 to 1967. The matter of code word
size was approached in a number of ways.
The first of these determinations of codon size was that of CRICK and his colla-
borators (1961). They reasoned to a very elegant experiment: If the code were highly
degenerate, if a uniform word size were employed, and if translation were non-
overlapping (or partially overlapping), then one could determine the codon size in
the following way: Suppose a message were being read in the proper reading frame,
but at a certain point one of the letters had been deleted. Upon reaching that point the
reading would thenceforth be in an improper reading frame, and so the remainder of
the message would make no sense. (Mutations producing this effect are variously
called "reading frame," "addition-deletion," "sign," or "frame-shift mutations.")
Generally such an alteration in a message RNA will lead to production of a non-
functional protein. However, suppose then that the original deletion of a base had
occurred at the nth position in an mRNA, but that at the n + ith position in this
message an addition of a nucleotide has also occurred. Now the reading of this message
is in the correct reading frame except for the stretch of i nucleotides beginning at
position n. In certain instances the region of incorrect reading could be small enough
a perturbation that sufficient sense is made of the message to produce a somewhat
functional protein. In brief then, the reading frame displacement produced by deletion
10 CARL R. WOESE
of a base could (in some instances) be corrected by a (nearby) addition of a base, or

vice versa.
If this be so, then reading frame displacements can be corrected in another
way - by deletion (or addition) of a total of n bases, where n is some exact multiple
of the codon size. This method is thus a means for determining the codon size.
In the coliphage T4 rII B cistron CRICK and his collaborators (1961) found a
genetic segment that made the above experiment feasible: While cistron function was
destroyed by extensive reading frame displacements, it was not destroyed when the
reading frame displacement was localized (and at the proximal end of this cistron).
Although we will not discuss experimental detail here, these workers isolated two
sets of mutants which had the following properties. Any member of either set would
destroy function of the rlIB cistron. Any (actually almost any) combination of one
mutation from one set with one from the other set in the same genetic segment would
restore function of the rlIB cistron. Any such combination where both mutations
came from the same set would not, however, restore function. It is clear that these two
sets of mutants had the properties predicted on theoretical grounds for mutants that
displace the reading frame. One set could be considered "deletions," the other the
compensating "additions" of nucleotides. The critical experiment was then to com-
bine in the same fashion three mutations from the same set. If the code word size were
indeed three, then only combinations of three functionally equivalent "addition" or
"deletion" mutations could restore the correct reading frame. And, of course, this is
exactly what was observed.
This determination of a codon size of three was confirmed in several ways using
the in vitro system approach. Probably the most convincing experiment was that
employing repeating sequence mRNA, done by the group working in KHORANA'S
laboratory [NISHIMURA et aI., 1965 (1,2); KHORANA et aI., 1966]. Working with
RNAs of the general structure ABABABABAB ...... , ABCABCABCABC ...... ,
etc. these workers showed that any repeating dinucleotide sequence is translated into
a repeating dipeptide sequence, repeating tetranucleotide sequences generally yield
repeating tetrapeptide sequences, but repeating trinucleotide sequences each translate
into three distinct homopeptide sequences. This is possible only if the codon size is
three nucleotides (given non-overlapping translation).
Two obvious consequences from these two studies are (1) that most if not all
co dons are assigned to amino acids - i.e., the code is highly degenerate, and (2)
translation cannot be fully overlapping.
Knowing the codon to be a nucleotide triplet, we must ask why this number is
three instead of some other number. In the past it has often been suggested that the
number three derives from the fact that three is the minimum number necessary to
supply sufficient information to encode the 20 amino acids. We shall point out the
weakness of this sort of reasoning. If this were the explanation for size of the codon,
one could question how the cell ever came to use 20 amino acids. 16 or less, say,
would do nearly as well (particularly during the early phases of cellular evolution),
and given an option as to codon size, it would seem easier to evolve a doublet code
(which could handle up to 16 amino acids) than a triplet one. Further, were a doublet
code to become established by evolution, it would hardly seem likely that it would be
replaced by a triplet code (that in any case offered so little in the way of selective ad-
vantage) when the change-over process would be so disruptive, so lethal, to the cell.
C. The Structure of the Codon Catalog

Although a number of the early theoretical speculations as to general properties
of the genetic code have turned out to be correct, none of these, with one exception,
came close to devining the general form of the set of codon assignments. Nevertheless,
there is some value in reviewing some of the early attempts as a prelude to under-
standing the experimentally determined set of assignments.
As stated, GAMOW'S thinking about this matter rested on the tenet that the number
of amino acids, 20, is not arbitrary, but is a consequence of some basic feature
(mechanism) in the genetic code, which would then make the structure of the codon
catalog knowable a priori. The first of Gamow's two coding schemes (GAMow, 1954)
envisioned amino acids as fitting into pockets on the double-stranded DNA molecule.
Each "pocket," which was roughly diamond shaped, was formed and bounded by a
base in one of the DNA chains, the adjacent base pair, and the base beyond that in the
other DNA chain. (Since the bases in a base pair mutually specify one another, the
codon in this case is in essence a triplet - i.e., two bases plus a base pair.) GAMOW
found that simply by postulating that the symmetry operations of rotation of a
"diamond" through 180 or reflection of a diamond did not alter its amino acid
0
"recognition" properties, the 64 diamonds could be placed into the requisite 20

categories - 12 fourfold degenerate categories and 8 twofold degenerate ones. (The
interested reader can readily work out the details of such a scheme.)
GAMOW subsequently produced a second scheme for generating the codon
assignments (GAMOW, 1955; GAMOW and YCAS, 1955). By that time it was fairly
certain that single-stranded RNA, not double-stranded DNA, was the actual template
for protein synthesis. Constraints tantamount to those used for the "diamond" code
could be applied to trinucleotides in RNA, giving another way of producing 20 from
64. By requiring that the order of bases in a trinucleotide not affect its coding func-
tion the 64 triplets fall into 4 sixfold degenerate categories, 12 threefold degenerate
ones, and 4 nondegenerate ones, or a total 20 categories, as Table 1 shows.
The third major coding scheme of this period was that put forth by CRICK and
his associates (1957), the socalled "comma-free code." This code had its genesis in
two considerations. First, it was a reaction to the Gamow codes, in particular a reac-
tion to GAMOW'S assumption that the nucleic acid directly "templates" the amino
acid in translation. CRICK (1958) argued that a group of nucleotides could not "re-
cognize" an amino acid. To provide for a specific pairing of amino acid to its codon,
CRICK then drew upon an idea suggested originally by DOUNCE (1952), that a set of
"molecular intermediaries" somehow recognize both amino acid and codon, and so
link them together, in an indirect linkage. In DOUNcE's case these all important inter-
mediaries were thought to be a set of "Pl enzymes," each of which recognized both
a particular amino acid and its corresponding codon(s). In CRICK'S case the inter-
mediaries were the set of "adaptors" plus the matching set of (activating) enzymes-
the enzyme recognized both amino acid and adaptor and joined them together, and
the adaptor then recognized the codon. This scheme seemed needlessly complex
at the time, but turned out to be a closer approximation to the truth then DOUNcE's
simpler one.
The second consideration in the genesis of the comma-free codes was that the
"adaptor" idea meant that translation had to be of the non-overlapping sort, which
12 CARL R. WOESE
as we have just seen raises the problem of how to determine the correct reading
frame - a problem that was solved in this case in a "contextual" way, with the
surprising result that (given a 4 letter alphabet and 3 letter words) the maximum
number of words that can make sense (be given meaning) is exactly 201 Table 2
shows such a code.
Table 1. Gamow's triangle code
Class I codons: nondegenerate

1. AAA
2. CCC
3. GGG
4. UUU
Class II codons: triply degenerate
1. AAC ACA CAA 7. GGA GAG AGG
2. AAG AGA GAA 8. GGC GCG CGG
3. AAU AUA UAA 9. GGU GUG UGG
4. CCA CAC ACC 10. UUA UAU AUU
5. CCG CGC GCC 11. UUC UCU CUU
6. CCU CUC UCC 12. UUG UGU GUU
Class III codons: sextuply degenerate
l.ACG AGC CAG CGA GAC GCA
l.ACU AUC CAU CUA UAC UCA
3.AGU AUG GAU GUA UAG UGA
4. CGU CUG GCU GUC UCG UGC
GAMOW, 1955; GAMOW and YCAS, 1955.
Table 2. A comma-free triplet code of maximum

size
ACA UCA GCA

ACC UCC GCC
ACG UCG GCG
ACU UCU GCU
AGA UGA
AGG UGG
AGU UGU
AUA
AUU
CRICK et aI., 1957.
Neither the Gamow nor the Crick schemes had any real experimental support.
The Crick scheme rested solely on the fact that the number 20 could be generated by
a very simple theoretical argument regarding an unambiguous reading of a message.
The Gamow schemes generated the number 20 from equally simple principles.
Gamow's second scheme at the time did appear to derive support from the additional
fact that it generated a frequency distribution for co dons (given the RNA composi-
tion of tobacco mosaic virus) that was remarkably like the corresponding amino acid
frequency distribution (for the virus' coat protein) (GAMOW and YeAS, 1955).
Actually Gamow's diamond code, which had the mandatory feature that translation
be of the fully overlapping sort, was known to be incorrect. In that overlapping codes
placed severe constraints upon amino acid sequences in a polypeptide, this code
could be shown to be incompatible with the then known amino acid sequence of
insulin (GAMOW et al., 1956).
Gamow's diamond code differed from the others in that it was monolithic. All
(or nearly all) features of the code derived from the "diamond" structure in the DNA
double helix - the codon size, the number of amino acids, the actual assignments, the
method of reading (overlapping), as well as certain aspects of the translation mecha-
nism and implications with regard to how this sort of a code could have evolved. In
contrast Gamow's second code, for example, did not make mandatory any mode of
translation. And the comma-free code, for example, predicted nothing about which
amino acids were assigned to which codons, or anything about the evolution of it all.
It appears in retrospect that the failing of all the coding schemes so far considered
was their overriding concern with the number 20 - all schemes had somehow to
produce exactly 20 codon categories. As we shall see, the actual codon catalog has
more than 20 codon categories (lor 2 are given over to punctuation, and a few amino
acids appear to occupy more than one category). Thus it may not be surprising that
the only theoretical code to come close to the actual situation did not take as a basic
tenet the exact generation of the number 20. This is a code known as the "axbxc
code," proposed at a later date than those just considered, actually at the time that
approximately 10 codon assignments were partially known (by composition but not
order of nucleotides within the codon) (WOESE, 1962, 1963).
Historically the axbxc code arose as follows: By the late 1950's it seemed rather
certain that the code was highly degenerate. This could be argued from the fact that
DNA compositions of various organisms varied over an extremely wide range of
G + C contents, while the amino acid compositions of the corresponding proteins
varied slightly if at all (CRICK, 1958; SUEOKA, 1961). (However, all of the first codon
assignments determined experimentally contained one or more U residues, which
lead some to mistakenly conclude that all codons must contain U and, therefore, the
code would be at best only somewhat degenerate.) In developing the axbxc code it
was argued that if this presumed degeneracy in the codon catalog were systematic,
then the very simplest way to generate it was to assume that certain bases in codons
would somehow have to be equivalent to one another in their coding function. *
More specifically it seemed reasonable to assume that bases equivalent to one another
in one position of the codon need not be so in any other position in the codon (since
there was no reason to assume the "coding function" of a base was the same in any
two positions of a codon), and that to a first approximation at least, the equivalence
of bases in any given position in co dons is independent of what bases occupy the
remaining positions in these codons. These general rules then lead to the family of
codes called the axbxc codes, a, b, and c being the number of non-equivalent bases in
each of the positions in a triplet codon, while their product, axbxc, is the number of
possible different codon categories.
The attempt to predict the specific axbxc code the cell might use was unsuccessful.
(This was a 4 X 2 X 3 code with U equivalent to G and A equivalent to C in the II
* GAMOW'S degeneracy rules more or less involved the codon as a whole, rather than
individual bases.
14 CARL R. WOESE
codon position and U equivalent to C in the III codon position- WOESE, 1962, 1963.)
Nevertheless, the codon catalog that has been experimentally determined is very like
an axbxc code - with U equivalent to C and A equivalent to G in the III codon
position. (The U-C rule is obeyed in all cases, the A-G one in most cases.)
The event that made the determination of codon assignments an experimental
reality was the development of a workable in vitro protein synthesizing system.
Sooner or later, given such a system, synthetic RNAs of simple composition would
have been tested for their capacity to stimulate amino acid incorporation into poly-
peptides - regardless of any theoretical predispositions concerning the nature of the
code. Clearly NIRENBERG and MATTHAEI (1961) were the first to realize the importance
of using RNAs of simple compositions in such a system to elucidate codon assign-
ments.
In principle, the in vitro system approach is a variation of the general approach of
elucidating codon assignments by a correlation analysis of the input RNA and the
output polypeptide compositions. However, if the input RNA composition is suf-
ficiently simple (which insures that the same will be true of the resulting polypeptide),
then no sophisticated sequence determinations are required for either polymer. It is
merely necessary to know the gross compositional analysis in both cases in order to
determine the composition (but not order) of the codon assignments. For example,
by using a (random sequence) RNA having the gross composition U: C = 2: 1, one
can determine which amino acid has the codon assignment UUU, which has CCC,
which ones have co dons containing 2 U's and 1 C, and which have co dons con-
taining 1 U and 2 C's.
All the initial work on codon assignments then involved the use of random
sequence RNA copolymers contain 1, 2, and sometimes 3 kinds of bases (polymers
that could be made using the enzyme polynucleotide phosphorylase). This approach
yielded the composition (but not order) for approximately 40 codon assignments.
[GARDNER et al., 1962; JONES and NIRENBERG, 1962; NIRENBERG et al., 1963;
WAHBA et al., 1962, 1963 (1); SPEYER et al., 1963.]
Initially it appeared that the in vitro system method might not be a workable
approach to determining the order of bases within codons, for simple RNA's whose
primary structure was (to some extent) knowable were not then available. It soon
became apparent though, that it should be possible to synthesize RNA's containing
a known short nontrivial sequence at one of the termini; (WAHBA, 1962) and such
RNAs eventually saw limited use in fixing codon assignments (STANLEY et al., 1966;
W AHBA et al., 1966). However, two new developments provided the real break-
through. One was the development by NIRENBERG'S group of the socalled "triplet
binding" technique (LEDER and NIRENBERG, 1964). The other was the devising, by
KHORANA'S group, of methods for the synthesis of RNA's containing simple known
primary structures (NISHIMURA et al., 1964).
NIRENBERG saw that it might be possible to circumvent many of the difficulties
in determining ordered codon assignments if one focused not on the completed act
of translation, the polypeptide, but instead upon the binding of the charged tRNA
to the ribosome-mRNA complex. In this case it might then be possible to employ
"mRNAs" small enough that their sequence could be readily determined - in the
limit, employing an mRNA merely the size of an individual codon. Although certain
technical difficulties and artifacts were encountered (such as a requirement for
rather high levels of Mg ion and the apparent assignment of more than one amino
acid to certain codons), a system based upon this approach proved to be workable,
and was used to ascertain all the unknown codon assignments (NIRENBERG et al.,
1965, 1966; SOLL et al., 1965).
Although the triplet binding method did yield "codon assignments," binding of
tRNA is not polypeptide synthesis, and so strictly, assignments determined by this
approach are not bona fide codon assignments. Thus, the purist was reassured on
finding that all the codon assignments taken to be reliably determined by the triplet
binding method, were confirmed through polypeptide synthesis using the repeating
Table 3. The codon catalog
UUU phe UCU ser UAU tyr UGU cys

UUC phe UCC ser UAC tyr UGC cys
UUA leu UCA ser UAA CT-l UGA CT-3

UUG leu UCG ser UAG CT-2 UGG trp
CUU leu CCU pro CAU his CGU arg

CUC leu CCC pro CAC his CGC arg
CUA leu CCA pro CAA gln CGA arg

CUG leu CCG pro CAG gln CGG arg
AUU ilu ACU thr AAU asn AGU ser

AUC ilu ACC thr AAC asn AGC ser
AUA ilu ACA thr AAA lys AGA arg

AUG met ACG thr AAG lys AGG arg
GUU val GCU ala GAU asp GGU gly

GUC val GCC ala GAC asp GGC gly
GUA val GCA ala GAA glu GGA gly

GUG val GCG ala GAG glu GGG gly
NIRENBERG et aI., 1965; BRIMACOMBE, et aI., 1965; SOLL et aI., 1965; MATIHAEI et aI.,
1966.
sequence mRNAs synthesized in the laboratory of H. G. KHORANA (summarized in

KHORANA et al., 1966). Starting from chemically synthesized DNA oligomers of
known sequence this latter group of workers were able as stated above to synthesize
RNAs of known repeating sequence - i.e. of the form ABABABAB .... , ABCABC-
ABC. . " etc. Not only were such RNAs useful in determining codon assignments
unequivocally, but as we have seen above they substantiated or proved several
general features of the genetic code.
Table 3 gives the codon catalog as determined by the various in vitro approaches.
At this point I should like briefly to note some of the obvious characteristics of
this set of assignments, reserving until later any discussion of what these may mean.
In the first place, note that this array is highly ordered. Almost all of the degeneracy
is confined to the III codon position. And, as noted previously, the degeneracy rules
16 CARL R. WOESE
(with very few exceptions) are VIII = em and Am = G m , with all four bases being
equivalent in the III codon position in a number of cases. Thus most amino acids
posses either 2 or 4 codons. A less obvious constraint (and less well defined) is that
"related" amino acids tend to be grouped with similar codons. As we shall see this
fact may have particular evolutionary significance.
D. In Vivo Confirmation of Codon Assignments

While there can be no doubt that the codon assignments determined by in vitro
approaches are the codon assignments used by the cell, it is useful to dwell briefly on
some of the in vivo approaches that have further certified these assignments. For the
most part approaches based upon the "amino acid replacement" method have been
employed. When a point mutation occurs in a gene, a change in a single amino acid
in the corresponding protein results (with certain exceptions). Thus by cataloging
what amino acids "replace" one another in a series of otherwise identical protein
sequences, one can detect what amino acids have codons differing in only 1 of the 3
codon positions. While this is purely a relative ordering of codon assignments
(independent of the actual compositions of the codons), the approach can be placed
on a partial absolute basis through the use of mutagens producing (partially) defined
base substitution mutations.
The most extensive investigations of this kind have been performed in the A
protein of tryptophan synthetase in E. coli, largely involving a single gly (GGA)
locus (for review see YANOFSKY, 1966), and also in connection with the chain termi-
nation punctuation in E. coli, as we shall see below. In all cases where it can be
ascertained that only one base in DNA has been altered, the amino acid replacement
data are in complete accord with the codon assignments determined by the in vitro
methods.
A second approach that not only confirms codon assignments but offers con-
siderable potential with regard to nucleic acid sequencing is that of "reading frame
displacement." As we have seen above, deletion or addition of a base pair in DNA
produces a translation in the incorrect reading frame distal to the point of its occur-
rence, and hence an inactive gene product. However, if the reading frame is returned
to normal by a compensating addition or deletion within a short distance of the first,
then in many instances the resulting polypeptide is not sufficiently altered to prevent
its detection and/or isolation. It should be apparent that if a genetic segment is
translated in 2 (or 3) reading frames, an unequivocal nucleotide sequence can be
deduced from the codon assignments of the amino acids. In all cases where this
approach has been applied to date, the protein data are again completely compatible
with the set of "in vitro" codon assignments (TERZAGHI et aI., 1966; BRAMMAR et aI.,
1967).
E. Punctuation
While the codon assignments can be considered as strictly formal relationships
between amino acids and codons, in considering punctuation we are forced to pass
from formality into the realm of molecular mechanisms, and so to bridge the gap
between the formal dictionary and the actual decoding act.
The need for punctuation of certain sorts is obvious from the analogy to written
language. The cell must have some mechanism for beginning and terminating the
output polypeptide tape, the "sentences," at the proper points. Likewise the synthesis
of mRNA requires similar punctuation. At the present time, however, I shall confine
attention to translation punctuation only. We shall also not discuss the extensive
subject of "modulation" sequences, the sequences responsible (directly or indirectly)
for controlling the frequency with which cistrons are transcribed and perhaps the
frequency with which messages are translated.
To begin with, punctuation could conceivably reflect any of several underlying
mechanisms. In the simplest case, punctuation may be due to nothing more than the
physical termini of the message RNA. Alternatively, sequences of bases may be
involved in specifying the termination of the polypeptide, either directly (in that they
are handled in translation much as normal codons are) or indirectly (in that the se-
quence causes a folding, etc. of the mRNA, or it is a signal for enzymatic modifica-
tion of mRNA, etc.).
For several reasons punctuation probably does not result merely from the physical
termini of messages. First the existence of polycistronic mRNAs (mRNAs coding
for more than one polypeptide) is now well established (MARTIN, 1963; OTAKA and
SPIEGELMAN, 1963). Thus all punctuation with the possible exception of the first
beginning and the last termination must somehow be encoded in terms of base
sequences. Further, the demonstration of the existence of sequences which function
specifically as punctuation marks (as we shall see) very nearly settles the matter.
1. Initiation Punctuation
The punctuation concerned with peptide chain initiation is possibly of two sorts,
that concerned with the initiation of the first peptide encoded in a polycistronic mRNA
and that concerned with the initiation of the syntheses of the "internal" polypeptides.
There exists a good deal of evidence regarding the first kind but rather less regarding
the second.
For E. coli it is known that at least one half of all proteins have a methionine
(met) residue at their N-terminal ends (WALLER, 1963). That even more translations
in E. coli than indicated by this frequency begin with met is suggested by the finding
that certain proteins which do not normally contain N-terminal met, do so when
synthesized in vitro (ADAMS and CAPPECHI, 1966; WEBSTER et al., 1966). (Their
sequence is otherwise normal.) Thus it seems that the cell may contain enzymes that
remove N-terminal met from certain proteins.
In actuality it is not met per se that is placed at the N-terminallocus in E. coli
but N-formyl met. And more importantly, it has been discovered that a special tRNA
is employed for this purpose [MARCKER and SANGER, 1964; CLARK and MARCKER,
1966 (1,2)]. In E. coli there exist two met tRNAs, one handling the placement of met
into interior positions in polypeptides. This tRNA responds to the sole met codon,
AUG, and appears in all ways to be a typical tRNA. The second met tRNA, on the
other hand, is atypical. It allows the enzymatic N-formylation of met attached to it
[MARCKER and CLARK, 1966 (1)]. It does not appear to translate interior AUG
codons (GOSH et al., 1967). It does read an initial AUG codon, however, and in
addition an initial GUG codon and possibly one or more other initial co dons [CLARK
and MARCKER, 1966 (2); GOSH et aI., 1967]. In addition this tRNA appears capable
2 Molccular and Subcellular Biology, Vol. 1
18 CARL R. WOESE
of establishing the reading frame for mRNA (SUNDARARA]AN and THACH, 1966).
When AUG appears out of the normal reading frame but very near to the 5' end of
the message, the peptide formed will begin with met. Further this tRNA appears to
fit into the "peptide" slot and playa role in 70S ribosome formation (see below)
(BRETCHER and MARCKER, 1966; LEDER and NAU, 1967; NOMURA and LOWRY, 1967;
OHTA et aI., 1967).
Initiation punctuation and N-formyl met tRNA do not, however, appear to be a
necessary condition for peptide chain initiation, at least in vitro. (If this had not been
the case NIRENBERG and MATTHAEI could not have made their epochal discovery.)
In the typical in vitro system translation of aID' RNA sequence can be obtained. How-
ever, it also appears that under these sorts of conditions the very first codon is not (al-
ways) translated - the first amino acid in a polypeptide generally corresponds to the
second codon (bases 4 to 6) in the message (and perhaps even later co dons) (SMITH
et al., 1966). The reconciliation of these ostensibly contradictory sets of fact lies in the
level of Mg ion in the environment. At Mg ion concentration higher than 10-2 M
all mRNAs can be translated in vitro about equally, but at lower Mg levels, the proba-
bility of translating a message is a function of whether it contains a terminal met
initiation codon and whether the corresponding tRNA is present [SALAS et aI.,
1967 (2); OHTA et al., 1967]. Other N-substituted amino acids may also function in
this capacity (NAKAMOTO and KOLAKOFSKY, 1966; LUCAS-LENARD and LIPMANN,
1967; ECONOMOU and NAKAMOTO, 1967). Several other factors that playa role in the
chain initiation process have been isolated from cells [SALAS et aI., 1967 (1)].
With regard to the chain initiation punctuation for polypeptides encoded by
interior cistrons of a polycistronic mRNA - (called here "secondary initiation,"
to be distinguished from the "primary initiation" occurring for the initial cistron in
that mRNA) - instances are known where the peptides involved do have N-formyl
met as their initial amino acid (VINUELA et aI., 1967). Presumably, therefore, the same
co dons are involved in secondary initiation punctuation which control primary
initiation punctuation. We then are left with the question of how this can be. How
can the sole met codon, AUG, cause both normal secondary initiation and not also
cause abnormal secondary chain initiation every time an AUG codon is encountered,
either in the proper or in the other two reading frames? The answer probably lies in
a suggestion made several times: AUG functions as secondary initiation punctuation
only when it is part of a larger unit - for example, only if preceded by some sort of
punctuation that frees the traveling ribosome of its peptidyl-tRNA.
The initiation punctuation(s) encountered may serve multiple functions, i.e., in
controlling attachment of the ribosome to the message, in setting the reading frame,
and in initiating peptide synthesis. While the latter two functions are nearly certain,
the existence of the former (especially in secondary initiation) is unclear. A role for
tRNApmet> and so AUG, in the initial association of the 30S ribosomal subunit with
mRNA has been discovered (NOMURA and LOWRY, 1967). It seems unlikely at present
that secondary initiation punctuation can effect ribosome attachment. [If this were
possible at interior cistron heads in a polycistronic mRNA, then the effect of a polar
mutation would be greater on the cistron immediately adjacent to the cistron in
which that mutation occurs than it would upon the more distal cistrons (e.g., see
MARTIN, 1967).] Nevertheless, there are suggestions to the contrary (BERBERICH et aI.,
1967; LODISH, 1968).
It is presumptuous and probably incorrect, however, to assert dogmatically at

this point that all RNAs used as messages in an E. coli cell begin simply with a 5'
initiation codon (and that all ribosomes necessarily attach at this point). Analysis of
certain RNA coliphage RNAs shows that the 5' terminal sequence does not contain
known initiation codons (as the 3' terminal sequence contains no recognized termina-
tion co dons) (SpIEGELMAN et al. 1968; DEWACHTER and FIERS, 1968).
We do not yet fully understand with regard to chain initiation punctuation the
reason, if any, for the involvement of the amino acid met and the codons AUG, GUG,
etc., as well as the need for methionine's being N-formylated. On another level one
wonders why chain initiation punctuation has evolved at all, since the in vitro system
approach reveals that punctuation is not essential to initiation of translation. With
regard to this last point I would suggest that such punctuation is perhaps a relatively
late arrival on the evolutionary scene - i.e., later than the advent of translation
itself - and is in a class with many other evolutionary "accomplishments" designed to
increase the precision of the translation process.
Although the met mechanism seems now to be the sole initiation mechanism in
the E. coli cell and perhaps in all bacterial systems, there are strong indications that
alternative initiation amino acids and sequences can be employed in the higher forms.
While the finding of N-acetyl serine and pyrrolidone carboxylic acid at the N-
terminus of proteins (WITTMAN, 1959; DOOLITTLE and BLOMBACK, 1964) in a number
of cases does not in itself prove anything, the tentative identification of a serine codon
at the 5' terminus of the tobacco necrosis satellite viral RNA (a single cistron mRNA)
strengthens the argument considerably (REICHMANN and WIMMER, unpublished).
In searching for a reason for involvement of met and perhaps ser and pyrrolidone
carboxylic acid in initiation, it is interesting to note that all of these amino acids
are capable of being cyclized under the proper conditions (GROSS and WITKOP,
1962).
The reason behind the formylation of met could lie in the fact that the amino
acid-tRNA bond is considerably weaker than the peptidyl-tRNA bond (GILBERT,
1963). If a tRNA were carrying a single amino acid to situate in the "peptide" posi-
tion on the ribosome (which it is necessary for the first amino acid in a chain to do),
this inherently weaker bond could be split before an incoming tRNA could arrive
and position another amino acid for peptide formation - unless the first amino
acyl-tRNA bond were strengthened by N-substitution. This surmise is supported
(though not proven) by the observation that unless the first codon in mRNA is an
initiation codon, it is not represented by a corresponding amino acid in the poly-
peptide output (SMITH et al., 1966) and by the above cited results with N-substituted
amino acids.
2. Punctuation Concerned with Polypeptide Chain Termination

It is interesting and noteworthy that the early theories of the code did not concern
themselves at all with the matter of punctuation. Perhaps this is related to focusing
on the mystical number 20, but there is no necessity, as we have just seen, to reserve
a separate codon just for punctuation.
Experimentalists first encountered translation punctuation in the "nonsense"
mutations found in the T4 rn gene - although their full significance was not then
2*
20 CARL R. WOESE
recognized (BENZER and CHAMPE, 1961; CHAMPE and BENZER, 1962). These muta-
tions were initially characterized by the facts that they seemed to affect translation
distal to the point of their occurrence, and could be suppressed either phenotypically
(with 5-F-uridine) or genetically (GAREN and SIDDIQI, 1962). Their full significance
emerged with the studies in S. BRENNER'S laboratory proving that these sequences
did indeed effect polypeptide chain termination, that they were triplets being read
normally in translation, and that no amino acid was assigned to these particular
triplets (SARABHAI et al., 1964; BARNETT et aI., 1967).
In all, three codons are known to produce this phenomenon of termination of
polypeptide synthesis distal from the point of their occurrence, the "amber" or
VAG, the "ochre" or VAA, and the "opal" or VGA codons. Their compositions
have been determined by the amino acid replacement approach - i.e., by deter-
mining what amino acid is placed in the polypeptide as a result of a base substitution
mutation in each of these co dons [WEIGERT and GAREN, 1965; WEIGERT et aI.,
1967 (1)]. Studies employing selective mutagens have given the same answer (BREN-
NER et aI., 1965, 1967).
Although these three co dons do cause peptide chain termination, there remains
the question of whether any of these are used normally by the cell for purposes of
punctuation. The fact that at least two of these codons (VAG and VGA) can be
suppressed in the cell at high levels (GAREN et al., 1965; SAMBROOK et al., 1967) -
of the order of 50% - without causing the cell to grow slowly, is the main argument
against their use as a major terminator in the cell. VAA suppression in most, if not
all, instances does affect cell growth rate however (KAPLAN, personal communication).
I think there is little doubt that one or all of these codons are used in the cell's chain
termination mechanism, but whether the termination sequence(s) normally em-
ployed comprise merely one of these triplets, or alternatively a larger sequence
containing one or more of these triplets is still open to argument.
The mechanism of peptide chain termination remains obscure. A factor releasing
the peptide chain from tRNA in response to the VAG codon has been detected in
cell extracts. This is not a tRNA, and attempts to detect a "terminator tRNA" have
failed to date (CAPECCHI, 1967).
F. The Translation Apparatus
Although a good deal is now known about the translation apparatus, most of this
knowledge cannot yet be related to the molecular mechanisms of translation - and
so to what we here call the genetic code. We know, for example, that the ribosome is
composed of two functionally distinguishable subunits, and each subunit contains
characteristic RNA and protein species, but we are merely at the beginning at trying
to allocate functions to all these components. Therefore, we will discuss only the
few aspects of the translation apparatus here which bear some known relationship to
the problem under discussion.
The translation process occurs in two separable stages, called here Trans I and
Trans II (BERG, 1961; WATSON, 1963). Trans I in turn can be divided into two steps
in vitro, although there is now considerable doubt that it is really divided in vivo into
separable steps (LOFTFIELD and EIGNER, 1965; YARUS and BERG, 1967). The first step
is usually termed that of amino acid activation
an + ATP + En ~ an ·AMP·En + PP.
In a sense this step is badly named, for its essence is not the activation of amino acids,
but the precise discrimination among amino acids. Discrimination is accomplished
by each amino acid's having its own "activating enzyme," capable of distinguishing
it from any other amino acid, the ratio of association constants being at least 50: 1
(BERGMANN et aI., 1961). The second step is the actual charging of tRNA
an·AMp·En +tRNAn~ an·tRNAn+En +AMP.
This step too is involved in the correct recognition of amino acids.
For amino acid polymerization (in aqueous solution) some sort of activation of
amino acids is required, and until recently it was generally thought that this activation
was inherent in the link between amino acid carboxyl group and the terminal ribose
in tRNA. However, the demonstration of the involvement GTP of in translation,
and in particular the possibility that the tRNA-bound amino acid may interact with
GTP (RAVEL, 1967; OHTA et aI., 1967; LEDER and NAU, 1967) forces a reconsidera-
tion of this matter.
Trans II comprises the actual "decoding" step, the mRNA being fed through the
ribosome tape reader in the proper reading frame, the co dons being correlated with
corresponding charged tRNAs, and the polypeptide output tape being produced.
Although we cannot relate much of what is known about ribosomes to their
function, we do know a few things, such as that the 50S and 30S ribosomal subunits
produce a functional 70S unit only when mRNA is actually present (SCHLESSINGER
et aI., 1967); the 30S subunit seems more closely associated with the mRNA and the
codon-anticodon matching, while the 50S subunit seems more involved with peptidyl
tRNA (GILBERT, 1963; PESTKA and NIRENBERG, 1966; NOMURA and LOWRY, 1967).
The 30S subunit contains about 20 protein molecules, the 50S twice that number
(MOORE et aI., 1968; WALLER and HARRIS, 1961). The peptides on the 30S particle
have an average molecular weight of about 15,000 daltons, each being a unique
species and present in a unit molar ratio (MOORE et aI., 1968).
A number of recent studies elucidating the primary structures of tRNAs now
permit us to begin relating these to tRNA coding functions (HOLLEY et aI., 1965;
ZACHAU et aI., 1966; MADISON et aI., 1966; RAJBHANDARY et aI., 1967; BAYEV et aI.,
1967; GOODMAN et al., 1968; DUBE et aI., 1968). A typical tRNA primary structure is
shown in Fig. 1. All tRNA structures so far elucidated can be made to conform to
this same type of five-armed hydrogen-bonded configuration, suggesting that such a
configuration may have some reality to it. (In most cases the lower left hand arm, as
shown, is very rudimentary); tRNA must contain at least three functional "sites"
and could have as many as five: The molecule must (1) attach to the amino acid,
(2) recognize its corresponding codon(s), (3) interact specifically with its correspond-
ing activating enzyme, and perhaps (4) interact specifically with a site(s) on the ribo-
some or (5) with a site(s) on another tRNA during peptide assembly. Site No.1,
the amino acid attachment point has long been known to be the terminal A residue,
present in all functional tRNAs (BERG, 1961). Site No.2, the anticodon, has been
definitely established as the middle three bases in the dependent loop of the molecule
as shown in Fig. 1. In all cases this triplet of bases is the exact base-pairing comple-
ment of the co dons to which a tRNA has been shown to respond [if one includes in
the base pairing rules the alternate pairings in the III position suggested by CRICK
(1966)]. The third site has not yet been definitely identified. Attempts to identify it
22 CARL R. WOESE
by competing tRNA charging with fragments obtained by digesting tRNA with

venom phosphodiesterase, suggest the site to be located in the anticodon or the lower
right hand arm of the molecule (STULBERG and ISHAM, 1967). It is quite certain that
this site can not be merely the anticodon itself (BALDWIN and BERG, 1966), but the
critical question remains whether the anticodon is included in this site. On the one
hand the existence of tRNA suppressors argues against such a suggestion; while a
number of investigations of the charging function of tRNA using agents that in-
activate specific bases suggest, on the other hand, that site No.3 and the anticodon
have features in common (BURTON et aI., 1966; MIURA, 1967; KUWANO et aI., 1968).
Studies purporting to settle the matter definitely by competing the tRNA charging
reaction with oligonucleotides of defined composition, have not been confirmed
(HAYASHI and MIURA, 1966; LETENDRE et aI., 1966).
A"'-C-U
/ 'C-G-C-C-C-C-~-G-G-G-A-G-A-C-C-AoH
G ! ! ! I ! ! I! !!!
'\. G-C-G-G-G G-G-C-U-C-U-C p
"/ \
C-y-T
/
lm U,
8 A A-A-G-8-8
II 'G'!iC-C/ "Gam
I ; ; i I
G C-G-G G
'A GFi( "A-A-I:l-I:l-I:l/
'G---C/
ItRNAtyr I y--~
Yo_A,.
C---G
p---A
I! 'c
»
Am U
A" Y'
Fig. 1
The hypothetical fourth and fifth types of sites would be common to all tRNAs,
so should then constitute sequences present in all. The GT'lj'CG contained in the
upper left arm of the tRNA shown in the figure is not only present in all tRNA
sequences so far examined, but seems to be present in all tRNAs, since the oligo-
nucleotide is present in a unit mole ratio in any total tRNA preparation (ZAMIR et al.,
1965). *
Recently a tertiary structure has been proposed for the so-called anticodon loop of
tRNA (FULLER and HODGSON, 1967). By the use of accurate molecular scale models it
has been demonstrated that 5 of the 7 bases in the nonbase-paired portion of this
arm can form a (single-stranded) helical extension of the double helical segment to
which these bases are connected. The remaining 2 of these 7 bases (the pyrimidines)
can then be strung out to complete the connection back to the other arm of the
double helical segment. This configuration leaves the anticodon bases as the topmost
three in a helix and in a position to form a base-paired structure with the codon in
mRNA. In fact, for the terminal base in the anticodon there exists a sufficient degree
* Actually, one of the variants of yeast tRNA ser and the E. coli tRNAmet-F are excep-
tions in that the final G is replaced by A - but at least one of these, and possibly both are
initiation tRNAs.
of freedom that the alternate base pairing possibilities suggested by CRICK (1966) in
his codon-anticodon interaction model can also be accommodated. Thus there is
good reason to accept tentatively, this structure as the true structure of the anticodon
loop, at least during the decoding act.
The degeneracy in codon-anticodon recognitions is itself a fascinating phenom-
enon. Although the molecular mechanisms involved can now be rationalized
through the "wobble" model of CRICK (1966) and the above-mentioned structural
model for the anticodon arm of tRNA, we are still at a loss to explain why such a
degeneracy exists (a point discussed further below). A codon-anticodon degeneracy
was suggested originally on theoretically grounds [as a possible mechanism for the
degeneracy observed in the codon catalog (WOESE, 1963)]. Initially, however, all
experimental evidence bearing on this point suggested that no such degeneracy
existed (WEISBLUM et aI., 1962; WEISBLUM et aI., 1965). (An unfortunate choice of
leu - an amino acid with six co dons and many tRNAs - as the object of study was
largely responsible for this misconception.) More refined studies on tRNA binding,
Table 4. Alternate codon-anticodon pairings -

the "wobble" model
Base in III' position Base in III codon position
of anticodon to which it pairs
A U
C G
G CorU
U AorG
I Cor U or A
CRICK, 1966.
particularly involving tRNAs whose primary structure was known to be unique,

began to build the case for a degenerate codon-anticodon interaction (BERNFIELD
and NIRENBERG, 1965; KELLOG et aI., 1966; SOLL et aI., 1967). CRICK then suggested
his well-known model for alternate base pairings that explained existing degeneracies
and predicted the general pattern to be found. In brief, the argument involved is the
following: If in making base pairs, one permits the bases to move slightly out of
their usual orientation, then one finds that base pairs other than A· U and G· Care
possible. In particular a G· U pair and several new pairs involving inosine are possible.
This then leads to the predictions for codon-anticodon pairing shown in Table 4
(CRICK, 1966).
Accuracy in the Translation Process
The most important single determinant of the evolutionary potential of a cell is
undoubtedly the accuracy with which that cell can transfer information. To date
almost all consideration of this matter has centered upon only one of the cell's in-
formation transferring processes, the replication of DNA. Mutations are the most
blatant manifestation of errors in information transfer. Although the errors en-
countered in transcription or translation are not heritable, their role in shaping the
course of evolution is no less important - though their impact on evolution is a far
24 CARL R. WOESE
more complex, subtle one. Thus I feel this matter of translation/transcription errors
to be worthy of extensive consideration.
To begin, consider a typical bacterial cell, E. coli. This organism has a mutation
rate of about 10-8 per base pair per DNA replication (Cox and YANOFSKY, 1967).
Since the haploid genome contains 5 X 106 base pairs, 0.05 mutations occur on
the average at every replication of the genome - i.e., about 5% of daughter cells are
mutants. It is possible that for this mutation rate, a genome of this size is the maxi-
mum attainable if the cell is to compete successfully and survive. In a similar way
translation or transcription error rates may control maximum length of cistrons or
polypeptides, the number of genes, the lifetime of a cell, and other properties. Let us
then consider the kinds of errors that might be encountered in the translation and
transcription processes and attempt to estimate upper limits for the various error
frequencies.
If an error occurs in the transcription of a typical structural gene, all polypeptides
translated from such a message will be incorrect. This particular error need not be a
serious one for structural genes whose mRNA output is high and/or under feed-back
control - provided that the error rate is commensurate with the production of a
large fraction of correct mRNAs. However, for genes whose mRNA output is
fixed and of the order of one copy per cell cycle, the problem becomes one of making
sure that in every cell cycle all such genes (if critical to cell function) give rise to one
perfect transcription. This in turn might limit the number of such genes the cell can
contain. [E. coli contains many genes that are apparently transcribed at the rate of
about one copy per cell cycle (MCCARTHY and BOLTON, 1964). Thus it may be that
the transcription error frequency in this organism is not more than a few orders of
magnitude greater than the mutation rate.]
A different effect of transcription error would be expected were the error to occur
in transcription of a gene coding for one of the components of the translation appara-
tus. A certain proportion of such errors should cause the component to malfunction,
to increase its own error frequency. Were this type of error to occur, for example,
during the synthesis of the anticodon region of a tRNA molecule, that tRNA would
effect an incorrect translation every time it were used. It is doubtful that the cell
could work with more than 0.01 % of its tRNAs faulty in this way, and a transcription
error rate of the order of 10-5 per base pair per transcription should assure that
condition.
Finally the transient errors in the act of translation itself (those due to codon-
anticodon mispairings, etc.) must be considered. There is good reason to assume that
over 90% of all translations are perfect. If e is then taken as the translation error
frequency (per codon per translation), then (1 - e)n is the probability of a perfect
translation, n being the number of codons in the corresponding mRNA. Taking this
probability to be 0.9 and n to be about 400 yields a value for e of 2 X 10-4 •
Thus low transcription error frequencies in the range 10-6 to 10-4 errors per base
pair per transcription, and translation error frequencies of about 10-4 per codon per
translation, seem to be about what the bacterial cell would require to function
normally.
The immediate transient translation errors are of two kinds: (1) those that are
misreadings of a codon - e.g., the substitution of one amino acid for another, and
(2) those that involve the maintenance of the correct reading frame. The class of
misreading errors includes several types. In Trans II the usual error involves re-
placing the intended amino acid by another, but punctuation errors are also con-
ceivable. In Trans I, error could occur at either of its two steps. Since the amino acid
is handled at three consecutive steps in polypeptide synthesis, errors committed at
one step in the chain will be propagated through the subsequent ones unless the cell
has evolved some way for detecting and correcting errors occurring at one step
before the system gets through the next step. And, the cell does indeed appear to
possess just such error correcting devices.
We have seen that the overall error frequency for translation should be about
10-4 per individual event. Measurements of this both in vivo and in vitro agree for the
most part with this figure (LOFTFIELD, 1963; SZER and OCHOA, 1964). (An exception
here is the frequency at which leu is incorporated into polypeptides in response to the
UUU codon, which is characterized by an order of magnitude or more higher error
rate in vitro [NIRENBERG et aI., 1963 (1); SZER and OCHOA, 1964].) However, the error
frequency for the initial amino acid activation step, in some instances appears to be
far too high for this level of fidelity. In particular, it is known that the ilu enzyme will
activate val about 1/50th as well as it will ilu (ratios of Michaelis constants taken as
the measure) (BERGMANN et al., 1961). However, this high error rate is not pro-
pagated into actual translation because the error is detected and corrected at the next,
tRNA charging, step. The val· AMp· Bilu complex interacts specifically with tRNAilu
to release the free amino acid to solution (BALDWIN and BERG, 1966). Thus, val
cannot be detected on tRNAilu.
Nevertheless, if a tRNA charging error were to get beyond this point, it would
manifest itself in the final polypeptide product. CHAPEVILLE et ai. (1962) succeeded
in reducing cys ·tRNAcys to ala·tRNAcys, and demonstrated that in protein synthesis
this tRNA chimera behaved very like, if not identically to, the normal charged tRNA
from which it was derived. Similarly, by use of heterologous charging systems it has
been possible to place ala or val on a tRNA phe (BARNETT and JACOBSON, 1964). This
in turn seems to behave as a typical charged tRNAphe (JACOBSON, 1966).
An accuracy to a few parts in 10-4also puts rather stringent requirements on the
codon-anticodon recognition. It has been argued from thermodynamic considera-
tions that the binding of an incorrect but similar anticodon to a given codon would
still involve a sufficiently strong interaction that an error rate higher than observed
would be expected (LOFTFIELD, 1963) - raising the possibility of error detecting-
correcting features in Trans II. It is known that some tRNAs will bind to mRNAs
not containing any of their proper codons; for example tRNAasp (GAg co dons)
will bind to the mRNA poly AG (repeating sequence). Yet no asp appears in the
polypeptide product made under the same conditions [NISHIMURA et aI., 1965 (1)].
(It is not impossible that this result is merely an in vitro artifact, however.) That the
ribosome itself plays a fairly sensitive role in all this can be inferred from the drastic
effect which certain agents that bind to the ribosome have on the translation error
frequency (DAVIES et aI., 1965; FRIEDMAN and WEINSTEIN, 1964). *
It has also been argued that triplet-triplet binding is not strong enough to account
for tRNA-mRNA interaction, particularly among the thermophilic bacteria. Such
an argument does not seem applicable, in that there is no need to invoke just the
* Streptomycin seems to have a particularly striking effect, but recendy it has been shown
that DNA somehow is involved in this process also (LIKOVER and KURLAND, 1967).
26 CARL R. WOESE
codon-anticodon interaction to account for this binding; other nonspecific inter-

actions are possible and would increase the overall tRNA-message binding. In any
case the structure of the anticodon "loop" (FULLER and HODGSON, 1967) itself may
have something to do both with strength of codon-anticodon interaction and with
the error frequency. If the proposed structure for the anticodon loop is correct and if
this helical structure forms (only) during and as a part of the process of decoding,
then the strength of this interaction will not be due merely to triplet-triplet inter-
action, but also to the energy of formation of the entire stacked configuration. If
stacking does increase the strength of codon binding, it might then increase its
specificity as well- i.e., if the correct codon only interacts strongly enough to force
formation of the stacked configuration.
One possibility rarely taken into account when discussing the accuracy of tRNA
reading of mRNA is that the activating enzyme may also be involved in the reaction.
Admittedly this seems remote. Nevertheless, measurements in vitro of the binding
between tRNA and its enzyme show this to be sufficiently strong (YARUS and BERG,
1967; WILLIAMS, 1968) that negligible amounts of whichever of the two molecular
types is in the minority will be left unbound. The actual situation in vivo is uncertain.
Another factor perhaps related to the accuracy of translation is the presence of
modified or atypical nucleotides in the anticodon arm of tRNA. Certainly the presence
of inosine in (HOLLEY et aI., 1965; ZACHAU et al., 1966; BAYEV et aI., 1967), and the
base pairing rules governing (CRICK, 1966), the l' anticodon position promote
ambiguity not accuracy, but a good case can be made for the fact that pseudouridine
in the II' anticodon position in yeast tRNA increases the accuracy of recognition
(WOESE et al., 1966).* Several studies suggest that "unmethylated" tRNA translates
less accurately than its normally methylated counterpart (REVEL and LITTAUER, 1966;
CAPRA and PETERKOFSKY, 1966). It is possible such modification of tRNA affects
translation accuracy by serving to "fine tune" the stacking energy of the anticodon
loop in the FULLER-HoDGSON (1967) configuration, or influence tRNA binding.
Our interest here in translation errors, as we shall shortly see, is not solely in
their magnitude. With any complex machine the errors it makes can be as diagnostic
of its nature as its normal modes of functioning, and in some cases more so. In the
case of the translation apparatus it seems that the error pattern gives insight into its
nature and into its evolution as well.
In that translation errors are normally so rare, it should be experimentally difficult
to characterize them, unless there were some way to increase their frequency without
changing their essential pattern. There appear to be a number of ways to do just this.
High Mg ion levels, and the addition of streptomycin or other related antibiotics are
convenient ways to do this in vitro (SZER and OCHOA, 1964; FRIEDMAN, WEINSTEIN,
1964; DAVIES et aI., 1964), and the latter appears to bring about errors in vivo also
[GORINI and KATAJA, 1964 (1,2)]. Applying these techniques to an in vitro system,
where mRNA of defined composition and isolated tRNA fractions can be employed,
it has been possible to elucidate the error pattern involving U and to a lesser extent C
in co dons (SZER and OCHOA, 1964; DAVIES et al., 1964, 1965, 1966; GONANO and
* In the case of the III' anticodon ambiguities, it is almost as though the evolutionary
process could not bring out a desirable degree of accuracy in the position and still maintain
an unambiguous codon response, so it went to the opposite extreme and created a totally
ambiguous response there.
EHRENSTEIN, unpublished). The general error level involving the bases A and G in
codons is far lower and may not follow any characteristic pattern, but perhaps
significantly the presence of I in co dons leads to a rather high error level (DAvms
et al., 1965). Interestingly the halogenated pyrimidines (except Huoro-substituted)
when in mRNA cause an ambiguous translation (GRUNBERG-MANAGO and MICHEL-
SON, 1964), whose pattern mimics the translation error pattern characterizing the
normal counterparts of these bases.
Translation errors (at least those involving the "high pyrimidine" codons) are to
a first approximation a function of the individual bases and not the overall composi-
tion of the codon being translated. These errors manifest two characteristics: their
frequency is a function of the position in the codon where the base occurs, while the
error pattern (what bases are mistaken for what others) is approximately independent
of position in the codon. The order of decreasing error-proneness as a function of
position in the codon is III> I > II (SZER and OCHOA, 1964; FRmDMAN and
WEINSTEIN, 1964). In each position the error pattern is Ume (read U mistaken for e),
> UmA, while UmG is least frequent or even undetectable (GONANO and EHREN-
STEIN, unpublished; SZER and OCHOA, 1964). A similar pattern is seen for e in that
emA:> emG, but emU may be a rare mistake, although the various methods used
to increase errors to an appreciable level do not all yield the same answer here
[DAvms et al., 1964, 1966; see also WOESE, 1967 (lor 2)]. These error patterns do
not bear much relationship to mistakes expected on the basis of alternate base pairing
schemes, but they seem to correlate somewhat with the stacking strengths of the
bases.
G. The Fundamental Nature and Evolution of the Genetic Code
As we have seen in the introduction, to know of base-base pairing is to under-
stand in principle the processes of nucleic acid replication and transcription. Transla-
tion presents a rather more complex situation. In the first place, from all we can now
tell there is no simple strong interaction like base pairing to serve as a foundation for
the process. Secondly, the translation apparatus is far more complex a machine than
any involved in nucleic acid replication, and as such must have had a far more
complicated evolution. Imagine the extent of evolution that was necessary to develop
50 kinds of tRNA with the enzymes necessary to create methylated bases, etc., 20
kinds of activating enzymes, with their dual functions, and a ribosome with two
highly specific RNA chains and about 40 kinds of proteins. It is quite clear that
understanding the genetic code even to the extent that we already understand nucleic
acid replication will require an extensive knowledge not only of the translation
apparatus as we now find it, but of its evolution as well.
The greatest gap in our present knowledge of living systems is that between what
we seem to know about the prebiotic environment in which living forms first arose,
and what we know about the modern (fully evolved) cell. The distance between these
two is so great that it is practically impossible even to imagine what the intermediate
stages in this evolution were like - not to speak of experimentally determining them.
Thus until serendipity provides some experimental sign posts, we have to be content
with a few naive conjectures to fill in the great gap in the code's evolution.
In a formal sense at least the evolution of the genetic code can be divided into
two main areas - the genesis of the order in the set of codon assignments, and the
28 CARL R. WOESE
genesis of the translation apparatus itself. Whether this division is justifiable - i.e.,
whether these two geneses really were in essence separable events - is open to
question. Nevertheless, most hypotheses concerned with the origin of the code have
tended to ignore the evolution of the translation machine, treating this as though it
were independent of the evolution of the codon assignments. [This splitting probably
reflects two main factors: (1) our complete ignorance in conception as well as fact,
of the evolution of the translation apparatus, and (2) the above-discussed fact that the
genetic code is often defined in a narrow sense - being restricted to the codon
assignments alone.] Actually it is hard to imagine conditions under which these two
evolutions would be separate. If it is possible to "optimize" codon assignments, this
will occur at all stages of the translation apparatus where codon assignments exist,
and so it is most unlikely that the two evolutions are not interconnected (this should
become clearer as we proceed).
The modern codon catalog is so highly ordered an array that it is necessary to
invoke interactions or constraints of some sort operating during evolution to bring
it to this state. These "forces" could be of a number of sorts. Although we are nearly
certain that strong "specificities" do not exist between amino acids of certain com-
positions and oligonucleotides of "corresponding" compositions (ZUBAY and DOTY,
1958; BRITTEN and WOESE, unpublished), there could exist weaker preferences that
would serve to guide co dons to the present relationships very gradually over the
course of evolution (WOESE et aI., 1966). Even taking such "specific" interactions
(or "codon-amino acid pairings") not to exist, we must still distinguish between a
codon catalog that is shaped by some property, some mechanism, inherent in the
translation apparatus, and a catalog whose structure is determined by constraints
not related to translation per se.
The early theorists who treated the code, generally saw fit not to deal explicitly
with the code's evolution, DOUNCE being the only exception (DOUNCE et aI., 1955).
In GAMOW'S case (GAMOW, 1954, 1955) this was no serious oversight, in that the
templating mechanisms he proposed inherently demanded a particular straight-
forward evolution. Evolution of a comma-free code (CRICK et aI., 1957), however, is
a different matter, and possibly one that would have militated against such a code.
DOUNCE (DOUNCE et aI., 1955) did argue that any system based upon "adaptors"
(see above) could not have arisen as such - he favored some loose direct association
between amino acids and oligonucleotides in the beginning. At that early stage in the
development of the coding field, as we have seen, practically all attention was directed
to rationalizing the number 20, and there tacitly existed more or less of a proscription
on considering the less rigorous (and less spectacular) problem of the code's evolu-
tion.
Once numerology was no longer the order of the day, however, proper attention
could again be paid to evolutionary considerations. SONNEBORN (1965) ushered in the
new era by suggesting a way in which a codon catalog having an order resembling
the true ordering might arise merely from selection to reduce the deleterious effect
of mutations. This scheme is representative of the class of models where the con-
straints shaping the codon catalog are external to the translation apparatus itself.
In brief, the Sonneborn model is the following: essentially all mutations are
either deleterious or neutral in their effect upon the cell. (Beneficial mutations are rare
to the point of being negligible in the present context.) A cell line capable of reducing
its burden of mutations gains then a (slight but definite) selective advantage. One
way in which this can be done is to create a set of codon assignments with the pro-
perties (1) that a codon derived by a (base substitution) mutation from another codon
has a maximal probability of being assigned to the same amino acid as is the
original codon, and (2) in those cases where the mutant codon is not assigned to the
same amino acid, it tends then to be assigned to an amino acid "related" to the
original one. A codon catalog designed to be "maximally connected" in these two
ways will present a structure very like, if not identical to, the one the cell employs.
A variation of this basic model has more recently been suggested by CRICK (1967).
The Sonneborn model postulates as its driving constraint the reduction of deleterious
effect of mutations. In the Crick model the code passes through an early phase where
a few amino acids (for reasons not mentioned here) take over the codon catalog.
Then other amino acids are introduced (Le., take over codon assignments) in ac-
cordance with their value to the evolving cell. The codon(s) a newly entering amino
acid takes is postulated to be one belonging to a "related" amino acid already encoded.
The driving constraint here, the "benefit" the cell derives from introduction of new
amino acids into the code is in a sense a "positive" rather than a "negative" one, but
like the previous case it is still a constraint external to the workings of the code.
All models based on evolving a codon catalog in a cell having a translation
apparatus in essence like that in the present day cell are open to the same criticisms -
in the main three: (1) How can such a model account for a genetic code universal
(or nearly so) in all major respects? (2) How does such a model permit codon assign-
ments to be altered? And, (3) can a codon catalog be evolved by such a route in a
finite amount of time? [WOESE, 1967 (1)]. It must be fully realized that evolution on
any of these sorts of schemes requires many steps, many trials of codon assignments,
combinations of codon assignments, etc., each (right) step in the process conferring
upon the cell a very slight selective advantage. Thus we can argue in keeping with
the first objection that since the selective advantage gained at each step is so slight,
this evolution of the code can in no way preclude the evolution in cells of other,
unrelated structures and functions. We can picture many cell lines arising, evolving
into various ecological niches, and at the same time developing different codon
catalogs. Now, many of these catalogs may be suboptimal, but since there is no one
optimal catalog according to these sorts of schemes, there is nothing to prevent some
groups of organisms in different ecological niches from evolving vastly different, but
equally optimal codon catalogs. In fact it is a rather unlikely assumption that a codon
catalog optimal for one organism would be optimal for all other organisms. (The
reader should recall the wide range of pH, temperature, pressure, oxidation-reduction
potential, and nucleic acid compositions over which organism exist and grow.) Thus,
how could just one set of codon assignments arise given the above sort of schemes
[WOESE, 1967 (1)]?
The second flaw has to do with the changing of codon assignments, an essential
element in all such models. To change a codon assignment means that the amino
acid whose assignment is removed from the rolls must then be replaced in all its
corresponding occurrences in the proteins by a different amino acid (the one now
assuming that codon assignment). Even if this is done so that an intermediary step in
the process is the creation of an ambiguous codon assignment, it is obvious that the
effect on the cell is drastic. Itis most unlikely that such a cell line would remain in existen-
30 CARL R. WOESE
ces long enough to permit establishment of a changed codon assignment. The situation
is analogous to that in physics where a system is in an energy state higher than the
minimum energy state. But to get to the minimum energy state means passing over a
high energy barrier. And the system has a vanishingly small probability of finding
itself with sufficient energy to pass the barrier [WOESE, 1967 (1)].
The final objection is a probabilistic one, a sort of entropy argument. The absolute
number of codon catalogs as highly ordered as the true one is large, but even so this
number constitutes a miniscule fraction of the total number of possible different sets
of codon assignments. This latter number is practically infinite, which means that
only an extremely small fraction of these will ever be encountered during the course
of evolution. The process of "improving" the codon catalog is, further, one that will
lead to "blind alleys" - e.g., a catalog that has been somewhat improved by a series
of, say, ten steps (codon reassignments) but now has reached a state where any further
improvement is impossible without going back a number of steps and starting over
in a different direction. There is no selective advantage in this retracing, so the system
is most unlikely to do so. And the number of these blind alleys is probably so great
that the system will never reach anything approaching an optimal codon catalog -
since there is no way of distinguishing between the blind alley and the path to an
optimal catalog [WOESE, 1967 (1)]. (This objection is based upon intuition and should
not be considered totally valid until tested by computer or some other rigorous
method.)
In view of these three objections I feel it highly unlikely that the code evolved in
any such way as just described. Let us then consider the alternative class of formula-
tions where the constraints driving the codon catalog to its form are themselves a
feature of the translation appatatus. In this case we cannot consider the evolution
of the set of assignments as being totally separate from that of the translation appara-
tus.
During its evolution, the translation apparatus must have passed through stages
where it was far simpler than it is at present* - this being a necessaty consequence
of its present complexity and the fact that in the beginning there was no such appata-
tus. This in turn essentially demands that the primitive apparatus be less accurate
than its modern counterpart. Thus one can no longer speak in terms of unique
polypeptide primary structures, of unique protein species (if these be the same size
as present ones). Instead there will be "statistical" proteins. * * A consequence of this
would be the existence of only the simplest, rudimentaty enzyme functions - the so-
called "urenzymes." All sophisticated enzymes of today would be absent, particularly
the activating enzymes (in that they have to discriminate accurately among amino
acids). Since the accuracy of translation depends for the most part upon the activating
enzymes, the evolving cell ostensibly faces a paradox: In order to have accurate
translation it must first translate accurately (WOESE, 1965).
Picture then this primitive cell, whose translation apparatus operates with a great
deal of error (or ambiguity). Let us then take the pattern of these error-ambiguities
* Data on primary structures of tRNAs are not yet extensive, but those extant suggest
that certain tRNAs shared a common ancestor.
* * A "statistical" protein comprises a group of proteins all of whose primary structures
differ from one another, but all of which are related in being a very approximate translation
of some given genetic sequence (WOESE, 1965).
to be like that seen in the cell today (see above), but take the codon catalog to be an
unordered array (using the term "assignment" a bit loosely). How can such a cell
solve the above paradox and come to translate accurately? Perhaps in this way:
Although it may be impossible to improve the accuracy of the translation mechanism
- due to the failure to make proteins of unique primary structure - it is still possible
to lessen the phenotypic consequences of these errors. This is done by reassigning
codons in accordance with the translation "error" pattern. Since errors are most
frequent in the III codon position, then a reshuffling resulting in the assignment of
a group of codons that differ only in the III position to the same amino acid will
meliorate these "errors." Similarly this constraint, which would apply in a somewhat
less compelling way to the I codon position (in that the I position is taken as some-
what less error prone), would then group I-position-related co dons to either a single
amino acid, or to a group of "related" amino acids. Another consideration would be
to place those amino acids whose positioning was the most essential to protein
function - i.e., the amino acids with functional R groups - with those co dons that
were the least error prone - i.e., the codons high in purines. This continual reshuffling
of codon assignments will then produce a continually improving capacity to translate
accurately. Eventually a point will be reached where translation becomes good enough
that the modern protein components of the translation apparatus begin to emerge,
and so the translation machine itself can actually become a more accurate mechanism
(WoEsE,1965).
The features of a codon catalog evolved on such a scheme as this are very like
those seen in the actual catalog, since the error pattern seen in cells today bears a
relationship to the order in the catalog. Thus in the actual catalog we do find (1)
almost all the degeneracy in the III codon position, (2) co dons differing just in the I
codon position being assigned to "related" amino acids (see below), and (3) the
"functional" amino acids being assigned to purine-rich codons (or II position purine
codons) - exactly as predicted by the model.
This sort of model (called the "TE" model, for translation error) copes somewhat
better with the three objections brought to the models of which that of SONNEBORN
(called the "LM" model, for lethal mutation) is the archetype. To make the TE
scheme compatible with a universal code, one argues as follows: Only after the
codon catalog has nearly or completely assumed its present form is translation
accurate enough that a better translation apparatus can begin to evolve. Therefore,
all cells that evolve a modern translation apparatus should have approximately or
exactly the same codon catalog. Possession of a "modern" translation apparatus
clearly carries with it a considerable selective advantage, so that it is not surprising
that organisms carrying more primitive translation systems, and so "primitive"
codon catalogs, have yet to be found [WOESE, 1967 (1)]. (One could make a similar
argument for universality of the LM scheme - i.e., that some special event occurred
in one cell line to make it so far superior to all others that its descendents alone came
to populate the earth. But since in the LM case the constraints driving the codon
catalog's evolution are external to the code, there is no reason to assume either that
this special event had to occur only in, or to wait upon, the evolution of a cell
line carrying the modern codon catalog. This reduces the LM scheme to postu-
lating a "miracle" in order to connect universality and a highly ordered codon
catalog.)
32 CARL R. WOESE
Given a primitive cell such as that envisioned on the TE scheme, it is relatively

easy to alter codon assignments. The level of "errors" in such a cell is so high that
altering a codon assignment does little to worsen an already chaotic situation. Of
course, as the translation capacity of the cell improves, especially after the translation
apparatus itself becomes more precise, changes in codon assignments become in-
creasingly disruptive events for the cell, until eventually a point is reached where
such an event is lethal, and from then on the set of codon assignments is essentially
"locked in" [WOESE, 1967 (1)].
The third objection, that it is highly improbable that the code could have reached
a perfected stage in a finite period of time - due to the "blind alleys" in its evolution
on the LM scheme - is also less of a compelling objection when applied to the TE
scheme. The primitive cell on the TE scheme is not really dealing with as many as
20 amino acids. In fact this type of cell should not be able to recognize individual
amino acids at all. It would do well to distinguish between groups of amino acids,
each group comprising a collection of "related" amino acids. * Since the number of
distinguishable units here is small, the number of possible codon catalogs, and so
evolutionary routes to an optimal catalog, is drastically reduced over that encountered
on the LM scheme. And so evolution tests a far larger fraction of the total possible.
Further a considerable fraction of the improved-but-not-optimal catalogs are on a
direct line to the optimal one(s) in the TE case - i.e., they do not end in blind alleys
[WOESE, 1967 (1)].
Let us finally consider the case for an evolution of the genetic code based upon
some preferential, some selective interaction between amino acids and oligonucleo-
tides. In principle such an evolution seems straightforward, in that it is a simple extra-
polation from the basic interaction - just as is the case for nucleic acid replication
and base pairing. As we have seen above, there exists no evidence for a strong
"codon-amino acid pairing" interaction, so that one has to view evolution as starting
with a weak interaction of this sort and then amplifying it, bringing it out, into an
all-or-none process. Provided these weak interactions did exist the major question is
then how on such a model one gets from a more-or-Iess direct templating mechanism
to the present mechanism, in which the amino acid associates with the codon only
through the allimportant intermediary, the tRNA "adaptor" system.
Nevertheless, were the code to evolve on the basis of such codon-amino acid
pairing interaction, one might expect to see some manifestation of the aboriginal
interaction in the translation process today. Is there some point during the translation
process where RNA "recognizes" the amino acid, even in a rudimentary fashion?
The experiments of CHAPEVILLE et al. (1962) - (see above) - and others show that
at least to a first approximation the codon-tRNA interaction is free of any codon-
amino acid pairing interactions. Trans I is a different matter, though perhaps only
through our ignorance of the process. The most intriguing feature of Trans I from
the present viewpoint is that not only does discrimination among amino acids occur
at the activation step (formation of a' AMp· E complex), but the subsequent step,
involving placement of the amino acid on the proper tRNA, also involves some
"awareness" of the amino acid. As stated above, an incorrect complex between
* Thus the strange condition could exist where the primitive proteins contain many
more than 20 kinds of amino acids, while the cell actually distinguishes among a very small
number of amino acid groups only.
val· AMP and the ilu enzyme will not place val on any tRNA (NORRIS and BERG,
1964). Although a p-F-phe is an amino acid analog that does become incorporated
into protein, a similar situation exists here too (FANGMAN and NEIDHARDT, 1964).
p-F-phe is not, as might be expected, bound as strongly by the phe enzyme as is its
normal counterpart. Even though some of the analog ends up on tRNAphe, a further
distinction between the two amino acids does occur at the tRNA charging step, the
normal amino acid again being favored. This sort of phenomenon cannot now be
given a unique interpretation, unfortunately, although one possibility is that the
activated amino acid carried by the enzyme is to some extent "recognized" by the
tRNA. However, an attempt to detect such an interaction more directly, by competing
the charging of the tRNA (by the enzyme complex) with large amounts of free
amino acid or amino acid esters has failed to demonstrate any "specific recognition"
at concentrations of competitor as high as 10-2 M (KONDO, 1967). Nevertheless, it is
of interest to note another correlation consistent with such a recognition. When
yeast tRNAs are fractionated on a column made from heavily benzoylated cellulose,
the. (uncharged) tRNAphe is bound by far the most strongly to the column (WIMMER
et al., 1968). The resemblance between the group bound to the cellulose and the phe
R group is obvious. Thus, the matter of recognition of the amino acid by tRNA
in Trans I remains unsettled, although Trans II manifests no such interaction.
1. The Universality of the Genetic Code

At this point let us take a more detailed look at the "universality" of the genetic
code, for this feature is one of the main clues we possess with regard to the code's
nature. The facts of the matter are these: (1) With the exceptions to be noted all
evidence suggests the same set of codon assignments to be used in all organisms
(SPEYER et al., 1963; SAGER et al., 1963; MARSHALL et al., 1967; LEVINTHAL et at,
1962). Exceptions are various microbial strains where certain codons, particularly
the peptide chain termination punctuation ones, have ambiguous assignments - i.e.,
strains in which these codons are suppressed [WEIGERT and GAREN, 1965; WEIGERT
et al., 1967 (1); BRENNER et al., 1966]. There appear to be similar phenomena in some
of the higher forms (MAGNI and PUGLISI, 1966). (2) The Trans II process seems to be
in essence universal, in that charged tRNAs from E. coli and other microbial sources
can bring about the correct placement of amino acids in an in vitro system (derived
from reticylocytes) that is synthesizing hemoglobin (WEISBLUM et al., 1965; GONANO,
1967). (3) The Trans I process, however, is not universal in two ways: For one, a
given amino acid activating enzyme from one source often does not charge the
corresponding tRNAs when they are derived from an "unrelated" organism (SUEOKA,
1965). (But often this heterologous system does yield the expected chargings.) Then,
in a small fraction of cases, this heterologous pairing of enzymes and tRNA can result
in charging of the incorrect tRNA, the best studied case being the charging E. coli
tRNA vai and tRNAala by a Neurospora phe enzyme (BARNETT and JACOBSON, 1964).
The problem is now to explain how a universality with exceptions that remain almost
trivially few, can exist.
The fact of universality must mean either (1) that all existing organisms derive
from a common ancestor cell line that already had, before divergent evolution began,
those features of the code found to be universal, and that subsequent evolution has
34 CARL R. WOESE
been unable to alter these features, or (2) constraints on the evolving system must
exist which force all evolving forms of the code to assume and maintain those features
found to be universal. The alternatives can be referred to as "divergent" as opposed
to "convergent" evolutionary models.
We are not justified at the present time in assuming that the fact of universality
has no significance, unless we are disposed to allow the existence of "miracles," i.e.,
universality is a happenstance. The problems encountered in trying to rationalize
models such as the LM scheme with universality have just been discussed, as have
those when the constraints shaping the code are of a particular type internal to the
translation apparatus (the TE scheme). Although the latter model can be rationalized
with a universal code, it still requires a certain amount of special pleading. In this
case, as with the LM scheme, more than one codon catalog could be optimal, and to
arrange universality it is necessary to assume that only one code has the opportunity
to achieve this optimal condition.
The only scheme that is unequivocally compatible with universality is one where
there can be only one optimal codon catalog, which in turn suggests a codon-amino
acid pairing scheme for the code's evolution. This cannot be considered strong
support for such a scheme, however.
What is to be made of the exceptions to universality of the codon catalog? The
most telling point here seems to be that although high level suppression constitutes
a mechanism for altering codon assignments and has undoubtedly existed throughout
the whole of evolutionary history, the extent of nonuniversality caused by its existence
is nearly negligible. The usual explanation for the failure of suppression to randomize
the codon catalog is that the changing of a codon assignment would be tantamount to
creating many hundreds of mutations simultaneously in the genome, and this would
obviously be lethal for any cell. However, we know that the UAG termination codon
can be translated as an amino acid two of every three times without seeming to cause
the cell any trouble (KAPLAN et aI., 1965). Also a 50% gly (AGA) suppressor strain
of E. coli is known to exist (CARBON et aI., 1966). If such strains can become established
in nature, which is not totally unlikely, then eventually the suppression might
become "locked in" (Le., it becomes essential to the cell to translate a given codon
in two ways), which in turn could lead to a gradual reduction of the frequency of
occurrence of that particular codon throughout the genome - a condition conducive
to changing codon assignments. * In one strain of mice an instance of 100% sup-
pression - i.e., a change of codon assignment - has actually been reported, at least
in some tissues (RIFKIN et aI., 1966).
The degree of nonuniversality in the translating mechanism is slight enough that
it can be viewed as perhaps having no functional significance. Certainly the cistrons
controlling the translation apparatus are among the slowest to drift in evolution. The
importance of the slight degree of nonuniversality that appears in Trans I, lies in
whether or not it means that the absolute configurations of the matching sites on tRNA
and its corresponding activating enzyme are unimportant - provided only that they
maintain certain proper relative configurational relationships. The alternative is that
the absolute configurations are important in that they relate to the amino acid being
* Something resembling this may have happened to the CGX-arg codons in some
higher forms, and the AGPur-arg co dons in E. coli [SUBAK-SHARPE et aI., 1966; CARBON
et aI., 1966; WOESE, 1967 (1)]. In any case, these codons are seldom used in these organisms.
carried by the tRNA * - i.e., a codon-amino acid pairing mechanism exists. This
latter mechanism ostensibly requires Trans I to be universal. However, one can
imagine trivial reasons for the failure of heterologous Trans I systems to charge
correctly, reasons that do not vitiate the essence of this sort of hypothesis. In con-
clusion then, the fact of universality strongly suggests that the constraints driving
the codon catalog to its final form were somehow inherent within the translation
apparatus, although there is no strong suggestion that a codon-amino acid pairing
mechanism had to be operative.
Table 5. Amino acid polar requirement,
UUU phe 5.0 UCU UAU tyr 5.4 UGU cys 4.8
UUC UCC UAC UGC
ser 7.5
UUA (leu) UCA UAA UGA trp 5.2
UUG UCG UAG UGG
CUU CCU CAU his 8.4 CGU

CUC CCC CAC CGC
leu 4.9 pro 6.6 arg 9.1
CUA CCA CAA gln 8.6 CGA
CUG CCG CAG CGG
AUU Heu 4.9 ACU AAU asn 10.0 AGU (ser)

AUC ACC AAC AGC
thr 6.6
AUA Heu ACA AAA lys 10.1 AGA (arg)
AUG met 5.3 ACG AAG AGG
GUU GCU GAU asp 13.0 GGU

GUC GCC GAC GGC
val 5.6 ala 7.0 gly 7.9
GUA GCA GAA glu 12.5 GGA
GUG GCG GAG GGG
WOESE et ai., 1966.
What evidence or argument do we have for the elusive codon-amino acid pairing
types of interactions playing a crucial role in the evolution of the genetic code? As
stated above, such a mechanism should leave its trace in the order shown in the codon
catalog. And, indeed the ordering of amino acids by codon assignments does bear a
striking parallel to their ordering according to certain criteria of "relatedness"
(WOESE et aI., 1966). An ordering of the amino acids according to their chromato-
graphic characteristics in pyridine-like solvents appears in Table 5. The ways in
which this ordering relates to amino acid ordering by codon assignment are: (1) amino
acids, all of whose co dons differ only in the III codon position, are seen in all cases to
be classed together by chromatographic criteria. (2) For the II position pyrimidine
co dons, the amino acids whose co dons differ only in the I codon position are also
grouped by chromatographic properties. The other less obvious correlations will not
be discussed here. The correlations shown are sufficiently striking that there is no
* Alternatively they could relate to the anticodons.
3*
36 CARL R. WOESE
doubt that they are real. The only question is whether such an ordering means that
the code stems basically from specific interactions between amino acids and the
heterocyclic bases. Alternatively the orderings seen could stem merely from some
general properties of amino acids and so, for example, reflect merely the fact that
"related" amino acids perform "related" functions in a protein molecule (see the LM
and TE models above). Since any interaction between two molecules should reflect
the properties of both, it should be possible to distinguish between these two alter-
natives. However, the possibilities cannot now be distinguished because of the im-
possibility of defining within reasonable limits what "related amino acids" means in
the context of protein functions. I would venture to guess, however, that amino acid
pairs such as gIn-his, asn-Iys, trp-cys, ala-pro, met-val, which are classed as "related"
both by codon assignments and by the pyridine chromatographic criterion, would not
generally be functionally equivalent in their roles in protein.
Attempts to show a more direct relationship between amino acids and nucleo-
tides, etc. have been less successful- due in a large part to the interactions between
the two being weak, and to the limited solubility of the nucleic acid derivatives.
Chromatographic experiments do show that compounds that are analogs of the amino
acid R groups distinguish among the nucleic acid bases (WELTON and WOESE,
unpublished), but data are yet too few to detect whether correlations between such
separations and the codon assignments exist.
2. The Evolution of the Translation Apparatus

Finally we turn to what is the most inscrutable aspect of the genetic code, the
evolution of the actual translation apparatus. The prebiotic beginnings of this
process had to be in simple interactions and structures. Our difficulty then lies in
charting the course from these simple beginnings to the present state of complexity.
Having decided to discount "miracles" as playing a role in the code's evolution, we
take this process to be a gradual, connected progression - eventually yielding to a
reasoned understanding. In attempting to circumscribe the problem let us first review
the essential features of the translation process, the features that will require an
evolutionary explanation. First there is the basic colinear mapping relationship,
involving the 3: 1 ratio for mapping bases into amino acids. Second, the amino acid-
codon correspondence is unique, i.e., unambiguous and accurate. Third, the mapping
is a time sequential process, i.e., it is a "tape reading". Fourth, the reading utilizes
the "adaptor" mechanism, the interposition of one or more molecular species be-
tween the amino acid and the codon, with the burden for "recognition" of both
parties falling upon the "adaptor" system. Lastly, one has the particular structures of
the various components of the mechanism.
Since the primitive versions of the translation apparatus had necessarily to be far
simpler than the present one, one should be able to make an educated guess as to
what components of the present translation system might not have been present in
the more primitive versions, and so simplify considerations. As stated above it is
very likely that primitive translation was an inaccurate process, and so the (precise-
function) protein components of the translation apparatus could not have been pres-
ent initially. Thus the essence of primitive translation had to lie in a nucleic acid
mediated process. What then would the evolutionary precursors of the ribosome and
tRNA be like?
Evidence points strongly to the existence of a smaller primitive ribosome. The

23S rRNA (derived from the 50S ribosomal subunit) from E. coli will hybridize with
essentially all the DNA to which the 16S rRNA (from 30S ribosomes) will hybridize.
More interestingly, the reverse of this also holds (MANGIAROTTI et al., 1968). In the
light of what is known about rRNA hybridization the most reasonable interpretation
of these data is that the 16S rRNA and both halves of the 23S rRNA all derive from
one common ancestor rRNA, and so at some stage a primitive ribosome no bigger
than today's 30S particle must have existed. RNA homologies to this extent then
suggest a proto-ribosome that may have comprised a collection of still smaller identical
subunits. Since the modern 30S particle contains 20 polypeptide subunits (MOORE
et ai., 1968), it is possible that its primitive ancestor at some stage did likewise, and
that this proto-ribosome, therefore, might have been a regular icosahedron, and so
the proto-16S rRNA a 20-fold redundant piece of RNA. * Taking the 16S rRNA to
be 0.55 X 106 daltons in weight, the weight of the basic unit would then be 28 X 1()3
daltons, or approximately the weight of a tRNA or 5S RNA molecule (BROWNLEE
et ai., 1967). Preliminary studies on the oligonucleotide patterns derived from rRNA
digested by nucleases, do give suggestions of a redundancy within the 16S subunit
(WOESE, unpublished).
Going one step further it is imaginable that at some very early stage in evolution
there existed a still smaller primitive oligonucleotide subunit that played a major role
in the origin and evolution of both rRNA and tRNA. And, I wish to suggest that
such a primitive subunit was the prototype for the "arms" found in tRNA today -
see Fig. 1. Each "arm" is a single nucleic acid chain of roughly 20 nucleotides length.
The central section contains a "loop" of anywhere from three to nearly ten nucleo-
tides which are unpaired, while the two terminal segments of the strand, five or so
bases long, complement one another and so form a double helical segment. 5S RNA
structure also appears to contain at least two of these structures, and it is conceivable
the 16S and 23S rRNAs will also contain them as well, which would make such
structures common to all functional RNAs. These structures will henceforth be
called PRINADS (for Primitive Nucleic Acid Duplex Structures).
The prebiotic existence of PRINADS can be rationalized on the grounds that
self-complementarity confers upon these structures a high degree of stability, which
would cause them to accumulate on the primitive earth. The number of base pairs
(less than ten) is sufficiently small that the structure also would be (under readily
attainable environmental conditions) in a rapid equilibrium between a random coil and
duplex, thereby facilitating their replication. Further, the unpaired bases in the
"loop" would be exposed to the environment, and so could function in a catalytic or
other reactive manner - making PRINADS the basis for primitive "enzymatic"
activity. The giving of such reactivity to PRINADS could then rationalize their
association with amino acids, and begin to rationalize groups of these evolving into
proto-ribosomes and proto-tRNAs.
The core of the translation apparatus today is the socalled "adaptor" system. The
reason generally accepted for its existence is the same one given for postulating
adaptors in the first place- they exist because nucleic acids cannot recognize amino acids
directly (CRICK, 1958). Actually there are a number of feasible alternatives to this
* It is tempting to try to equate this 20 to the number of amino acids in the codon
catalog, but atavistic Gamow-ian thinking is no longer appreciated.
38 CARL R. WOESE
raison d'etre, including the exact opposite explanation; adaptors might have arisen
because nucleic acids (e.g., PRINADS) could recognize amino acids. What is im-
portant at this juncture is to realize alternatives exist and so maintain an open attitude.
Perhaps the two most telling features of the adaptor system are the polypeptide-
tRNA relationship and the codon-anticodon relationship. The amino acid is linked
covalently to tRNA to begin with, and the growing polypeptide chain remains so
throughout translation. Does this intimate linkage bespeak some very ancient asso-
ciation between a proto-tRNA and some kind of polypeptide synthesis? The codon-
anticodon relationship in translation in turn resembles somewhat nucleic acid re-
plication. Is this purely fortuitous or could the tape reading feature have arisen out of
nucleic acid replication? The mRNA· tRNA· ribosome interaction in translation is
complex enough that one questions whether it was preceded by a simpler, two body
interaction. Could rRNA itself have been the original message (the original genome)?
Or is this three body interaction related to, say, a three stranded complex between a
nucleic acid duplex and a basic protein, etc. ?
All the above considerations point to and are a part of a general question: Did
translation originate as a translation - a colinear mapping of nucleic acid primary
structure into polypeptide primary structure? If so, was the original relationship a
direct "templating" or did it encompass some adaptor feature (indirect amino acid-
codon relationship) to begin with? Various nucleoprotein complexes provide prece-
dent for a colinear direct "templating" amino acid-nucleotide relationship. And a
reasonable case can be made for the suggestion that one or the other kind of polymer
may have played a primitive catalytic role in the synthesis of polymers of the opposite
type (WOESE, 1968). But, it is difficult to envision the tRNA molecule arising as an
intermediary if its prototype were not associated with the amino acid to begin with.
The aboriginal tRNA could even have been a catalyst producing polypeptides in a
nontranslating manner (WOESE, 1963).
Nucleic acid-polypeptide interactions provide no precedent for the observed
coding ratio of 3: 1. As pointed out above, it is the weakest of arguments to maintain
that this features arose merely because the cell needed a triplet to provide sufficient
information to encode its 20 amino acids. What seems far more likely is that the
triplet arose as an unavoidable consequence of the way the evolving translation
mechanism worked - a triplet may be, for example, the minimal (or in some way
"optimal" or available) number of bases in one of the PRINAD loops. Or it may
relate to the fact that AAA is more distinguishable from GGG than A is from G.
III. Conclusion
In conclusion the evolution of the genetic code is the major remaining problem
in the coding field. This problem is also the central one in the evolution of the first
"modern" cell. At present we have very little concept of what the stages and events
in this most intricate process were. Understanding in this area is probably more
impeded by this lack of a concept than it is by a lack of facts. Barring miracles, the
code's evolution should be a gradual step-wise process, utilizing and conforming to
simple interactions between nucleic acids and polypeptides and/or their derivatives,
and so readily understandable. What seems needed most at present is a broadening of
our view of the code, so that it is not completely tied to and shaped by our conception
of the translation apparatus used by cells today. Only in this way can we hope to
envision its more primitive stages.
References
ADAMS, J. M., and M. R. CAPECCHI: N-formylmethionyl-sRNA as the initiator of protein
synthesis. Proc. nat. Acad. Sci. (Wash.) 55, 147-154 (1966).
BALDWIN, A. N., and P. BERG: Purification and properties of isoleucyl ribonucleic acid
synthetase from Escherichia coli. J. bioI. Chem. 241, 831-838 (1966).
BARNE1T, L., S. BRENNER, F. H. C. CRICK, R. G. SHULMAN, and R. J. WA1TS-ToBIN: Phase-
shift and other mutants in the first part of the cistron of bacteriophage T4. Phil. Trans. B.
252,487-560 (1967).
BARNE1T, W. E.: Interspecies aminoacyl-sRNA formation: Fractionation of neurospore
enzymes involved in anamolous aminoacylation. Proc. nat. Acad. Sci. (Wash.) 53,
1462-1467 (1965).
- , and K. B. JACOBSON: Evidence for degeneracy and ambiguity in interspecies aminoacyl-
sRNA formation. Proc. nat. Acad. Sci. (Wash.) 51, 642-647 (1964).
BAYEV, A. A., T. V. VENKSTERN, A. D. MIRZABEKOV, A. I. KRUTELINA, L. LI, and V. D.
AXELROD: Mol. BioI. (USSR) 1, 754 (1967).
BENZER, S.: Fine structure of a genetic region in bacteriophage, Proc. nat. Acad. Sci. (Wash.)
41, 344-354 (1955).
- , and S. P. CHAMPE: Ambivalent rII mutants of phage T4. Proc. nat. Acad. Sci. (Wash.)
47, 1025-1032 (1961).
BERBERICH, M. A., J. S. KOVACH, and R. F. GOLDVERGER: Chain initiation in a poly-
cistronic message: Sequential versus simultaneous derepression of the enzymes for
histidine biosynthesis in Salmonella typhimurium. Proc. nat. Acad. Sci. (Wash.) 57, 1857 to
1864 (1967).
BERG, P.: Ann. Rev. Biochem. 30, 293 (1961).
BERGMANN, F. H., P. BERG, and M. DIECKMANN: The enzymic synthesis of amino acyl
derivatives of ribonucleic acid. J. bioI. Chem. 236, 1735-1740 (1961).
BERNFIELD, M., and M. W. NIRENBERG: Science 147, 479 (1965).
BISHOP, J., J. LEAHY, and R. SCHWEET: Formation of the peptide chain of hemoglobin.
Proc. nat. Acad Sci. (Wash.) 46, 1030-1038 (1960).
BRAMMER, W. J., H. BERGER, and C. YANOFSKY: Altered amino acid sequences produced by
reversion of frameshift mutants of tryptophan synthetase A gene of E. coli. Proc. nat.
Acad. Sci. (Wash.) 58, 1499-1506 (1967).
BRENNER, S., and J. R. BECKWITH: Ochre mutants, a new class of suppressible nonsense
mutants. J. molec. BioI. 13, 629-637 (1965).
- , F. JACOB, and M. MESELSON: An unstable intermediate carrying information from genes
to ribosomes for protein synthesis. Nature (Lond.) 190, 576-581 (1961).
- , A. O. W. STRE1TON, and S. KAPLAN: Genetic code: The "nonsense" triplets for chain
termination and their suppression. Nature (Lond.) 206, 994-998 (1965).
- - - Identify ofN2 and ochre nonsense mutants. J. molec. BioI. 19, 574-575 (1966).
- , L. BARNE1T, E. R. KATZ, and F. H. C. CRICK: UGA: A third nonsense triplet in the
genetic code. Nature (Lond.) 213, 449--450 (1967).
BRETCHER, M. S., and K. A. MARCKER: Polypeptidyl-sRNA and aminoacyl-sRNA binding
sites on ribosomes. Nature (Lond.) 211, 380-384 (1966).
BRIMACOMBE, R., J. TRUPIN, M. NIRENBERG, P. LEDER, M. BERNFIELD, and T. JAUONI:
RNA code words and protein synthesis VIII. Nucleotide sequences of synonym codons
for arginine, valine, cysteine, and alanine. Proc. nat. Acad. Sci. (Wash.) 54, 954-960
(1965).
BRODY, S., and C. YANOFSKY: Suppressor gene alteration of protein primary structure.
Proc. nat. Acad. Sci. (Wash.) 50,9-16 (1963).
BROWNLEE, G. G., F. SANGER, and B. G. BARRELL: Nucleotide sequence of 5S ribosomal
RNA from Escherichia coli. Nature (Lond.) 215, 735-736 (1967).
40 CARL R. WOESE
BURTON, K., N. VARNEY, and P. ZAMECNIK: Action of osmium tetroxide on amino acid
transfer ribonucleic acid: Correlations between the genetic code and the sensitivity of
acceptor activity. Biochem. J. 99, 29C (1966).
CALDWELL, P., and C. HINSHELWOOO: J. chern. Soc. 1950,3156.
CAPECCHI, M.: Polypeptide chain termination in vitro: Isolation of a release factor. Proc. nat.
Acad. Sci. (Wash.) 58, 1145-1151 (1967).
CAPECCHI, M. R., and G. N. GUSSIN: Suppression in vitro: Identification of a serine sRNA as
a "nonsense" suppressor. Science 149, 417-422 (1965).
CAPRA, J. D., and A. PETERKOFSKY: The coding properties of methyldeficient leucyl-
transfer RNA. ]. molec. BioI. 21, 455-465 (1966).
CARBON,]., P. BERG, and C. YANOFSKY: Studies ofmissence suppression of the tryptophan
synthetase A-protein mutant A36. Proc. nat. Acad. Sci. (Wash.) 56, 764-771 (1966).
CHAMPE, S. P., and S. BENZER: Reversal of mutant phenotypes by 5-fluorouracil: An ap-
proach to nucleotide sequences in messenger-RNA. Proc. nat. Acad. Sci. (Wash.)
48, 532-546 (1962).
CHAPEVILLE, F., F. LIPMANN, G. VON EHRENSTEIN, B. WEISBLUM, W. RAY, and S. BENZER:
On the role of soluble ribonucleic acid in coding for amino acids. Proc. nat. Acad. Sci.
(Wash.) 48, 1086-1092 (1962).
CLARK, B. F. c., and K. A. MARCKER: (1) The role ofN-formly-methionyl-sRNA in protein
biosynthesis. J. molec. BioI. 17,394-406 (1966).
- - (2) N-formyl-methionyl-sRNA and chain initiation in protein biosynthesis. Nature
(Lond.) 211, 378-380 (1966).
Cox, E. c., and C. YANOFSKY: Altered base ratios in the DNA of an Escherichia coli mutator
strain. Proc. nat. Acad. Sci. (Wash.) 58, 1895-1902 (1967).
CRICK, F. H. c.: On protein synthesis. Symp. Soc. expo BioI. 12, 138-163 (1958).
- The present position of the coding problem. Brookhaven Symp. in BioI. 12, 35-38
(1959).
- Codon-anticodon pairing: The wobble hypothesis. J. molec. BioI. 19, 548-555 (1966).
- Origin of the genetic code. Nature (Lond.) 213, 119 (1967).
- , J. GRIFFITH, and L. E. ORGEL: Codes without commas. Proc. nat. Acad. Sci. (Wash.)
43,416-421 (1957).
- , L. BARNETT, S. BRENNER, and R. J. WATTS-ToBIN: General nature of the genetic code for
proteins. Nature (Lond.) 192, 1227-1232 (1961).
DAVIES, J., W. GILBERT, and L. GORINI: Streptomycin suppression and the code. Proc. nat.
Acad. Sci. (Wash.) 51, 883-890 (1964).
-, L. GORINI, and B. DAVIS: Misreading of RNA codewords induced by aminoglycoside
antibiotics. J. Mol. Pharm. 1, 93-110 (1965).
- , D. S. JONES, and H. G. KHORANA: A further study of misreading of co dons induced by
streptomycin and neomycin: Ribopolynucleotides containing two nucleotides in alter-
nating sequence as templates. J. molec. BioI. 18, 48-57 (1966).
DEWACHTER, R., and W. FIERS: Studies on the bacteriophage MS2. IV The 3'-OH terminal
undecanucleotide sequence of the viral RNA chain. J. molec. BioI. 30, 507-528 (1968).
DINTZIS, H.: Assembly of the peptide chains of hemoglobin. Proc. nat. Acad. Sci. (Wash.) 47,
247-261 (1961).
DOOLITTLE, R. F., and B. BLOMBACK: Amino acid sequence investigations of fibrinopeptides
from various mammals: Evolutionary implications. Nature (Lond.) 202, 147-152
(1964).
DOUNCE, A.: Enzymologia 15, 251 (1952).
- , M. MORRISON, and K. J. MONTY: Role of nucleic acid and enzymes in peptide chain
synthesis. Nature (Lond.) 176, 597-598 (1955).
DUBE, S. K., K. A. MARCKER, B. F. C. CLARK, and S. CORY: Nucleotide sequence of N-
formyl-methionyl-transfer RNA. Nature (Lond.) 218, 232-233 (1968).
ECONOMOU, A., and J. NAKAMOTO: Further studies on the initiation of protein synthesis with
N-formylmethionine in E. coli extracts. Proc. nat. Acad. Sci. (Wash.) 58, 1033-1039
(1967).
EHRENSTEIN, G. VON, and D. DIAS: A leucine acceptor sRNA with ambiguous coding
properties in a polynucleotide stimulated polypeptide synthesis. Proc. nat. Acad. Sci.
(Wash.) 50, 81-86 (1963).
FANGMAN, W. L., and F. C. NEIDHARDT: Demonstration of an altered aminoacyl ribonucleic

acid synthetase in a mutant of Escherichia coli. J. bioI. Chern. 239, 1839-1843 (1964).
FLEISSNER, E.: Polypeptide synthesis with methyl deficient soluble RNA. Biochemistry 6,
621-632 (1967).
FRIEDMAN, M., and I. B. WEINSTEIN: Lack of fidelity in the translation of synthetic poly-
ribonucleotides. Proc. nat. Acad. Sci. (Wash.) 52, 988-996 (1964).
FULLER, W., and A. HODGSON: Conformation of the anticodon loop in tRNA. Nature
(Lond.) 215, 817-821 (1967).
GALLUCCI, E., and A. G AREN: Suppressor genes for nonsense co dons II. The Su-4 and Su-5
suppressor genes of E. coli. J. molec. BioI. 15, 193-200 (1966).
GAMOW, G.: Possible relation between deoxyribonucleic acid and protein structures. Nature
(Lond.) 173, 318 (1954).
- BioI. Medd. danske Vidensk. Selsk. 22, (8) (1955).
- , A. RICH, and M. YCAS: Advanc. bioI. med. Phys. 4, 23 (1956).
- , and M. Y CAS: Statistical correlation of protein and ribonucleic acid composition. Proc.
nat. Acad. Sci (Wash.) 1955, 1011-1019.
GARDNER, R. S., A. J. WAHBA, C. BASILIO, R. S. MILLER, P. LENGYEL, and J. SPEYER:
Synthetic polynucleotides and the amino acid code VII. Proc. nat. Acad. Sci. (Wash.)
48,2087-2095 (1962).
GAREN, A., and O. SIDDIQI: Suppression of mutations in the alkaline phosphatase structural
cistron of E. coli. Proc. nat. Acad. Sci. (Wash.) 48, 1121-1127 (1962).
- , S. GAREN, and R. C. WILHELM: Suppressor genes for nonsense mutations I. Su-1, Su-2,
and Su-3 genes of Escherichia coli. J. molec. BioI. 14, 167-178 (1965).
GILBERT, W.: Polypeptide synthesis in E. coli II. The polypeptide chain and sRNA. J.
molec. BioI. 6, 380---403 (1963).
GOLDSTEIN, A., J. B. KIRSHBAUM, and A. ROMAN: Direction of synthesis of messenger RNA
in cells of E. coli. Proc. nat. Acad. Sci. (Wash.) 54, 1669-1675 (1965).
GONANO, F.: Specificity of serine transfer ribonucleic acids in the synthesis of hemoglobin.
Biochemistry 6,977-983 (1967).
GOODMAN, H. M., J. ABELSON, A. LANDY, S. BRENNER, and J. D. SMITH: Nature (Lond.)
217, 1019 (1968).
GORINI, L., and E. KATAJA: Phenotypic repair of streptomycin of defective genotypes in
E. coli. Proc. nat. Acad. Sci. (Wash.) 51, 487-493 (1964).
- - Streptomycin-induced oversuppression in E. coli. Proc. nat. Acad. Sci. (Wash.) 51,
995-1001 (1964).
GHOSH, H. P., and H. G. KHORANA: Studies on polynucleotides LXXXIV. On the role of
ribosomal subunits in protein synthesis. Proc. nat. Acad. Sci. (Wash.) 58, 2455-2461 (1967).
- , D. SOLL, and H. G. KHORANA: Studies on polynucleotides LXVII. Initiation of protein
synthesis in vitro as studied by using ribopolynucleotides with repeating nucleotide se-
quences as messengers. J. molec. BioI. 25, 275-298 (1967).
GROSS, E., and B. WITKOP: Nonenzymatic cleavage of peptide bonds: The methionine
residues in bovine pancreatic ribonuclease. J. bioI. Chern. 237, 1856-1860 (1962).
GRUNBERG-MANAGO, M., and A. MICHELSON: Polynucleotide analogues. II. Stimulation of
amino acid incorporation by polynucleotide analogues. Biochim. biophys. Acta (Arnst.)
80,431---442 (1964).
HAYASHI, H., and K. MIURA: Functional sites in transfer ribonucleic acid. Nature (Lond.)
209,376-378 (1966).
HOLLEY, R. W., J. APGAR, G. A. EVERETT, J. T. MADISON, M. MARQUISEE, J. R. PENWICK,
and A. ZIMIR: Structure of a ribonucleic acid. Science 147,1462-1467 (1965).
JACOBSON, K. B.: A test of tRNA as amino acid adaptor in hemoglobin synthesis. Cold Spr.
Harb. Symp. quant. BioI. 31, 719-722 (1966).
JONES, 0., and M. W. NIRENBERG: Qualitative survey oftRNA code words. Proc. nat. Acad.
Sci. (Wash.) 48, 2115-2123 (1962).
KAPLAN, S., A. O. W. STRETTON, and S. BRENNER: Amber suppressors: Efficiency of chain
propagation and suppressor specific amino acids. J. molec. BioI. 14, 528-533 (1965).
KELLOGG, D. A., B. P. DOCTOR, J. E. LOEBEL, and M. W. NIRENBERG: RNA co dons and
protein synthesis. IX. Synonym codon recognition by multiple species of valine-, alanine-,
and methionine-sRNA. Proc. nat. Acad. Sci. (Wash.) 55,912-919 (1966).
42 CARL R. WOESE
KHORANA, H. G., H. BUCHI, H. GOOSH, N. GUPTA, T. M. JACOB, H. KOSSEL, R. MORGAN,

S. A. NARANG, E. OHTSUKA, and R. D. WELLS: Polynucleotide synthesis and the genetic
code. Cold Spr. Harb. Symp. quant. BioI. 31, 39-50 (1966).
KOLAKOFSKY, D., and T. NAKAMOTO: The initiation of viral protein synthesis in E. coli
extracts. Proc. nat. Acad. Sci. (Wash.) 56, 1786-1793 (1966).
KONDO, M.: Studies on transfer RNA. Ph. D. thesis, University of Illinois 1967.
KOSSEL, H., A. R. MORGAN, and H. G. KHORANA: Studies on polynucleotides LXXIII.
Synthesis in vitro of polypeptides containing tetrapeptide sequences dependent upon
DNA-like polymers containing repeating tetranucleotide sequences: Direction of
reading of message RNA. Proc. nat. Acad. Sci. (Wash.) 56, 1786-1793 (1966).
KUWANO, M., Y. HAYASHI, H. HA YASHI, and K. MIURA: Photochemical modification of trans-
fer RNA and its effect on amino acyl RNA synthesis. J. molec. BioI. 32, 659-671 (1968).
LEDER, P., and M. NAU: Initiation of protein synthesis III. Factor-gtP-codon dependent
binding ofF-mat-tRNA to ribosomes. Proc. nat. Acad. Sci. (Wash.) 58, 774-781 (1967).
- , and M. W. NIRENBERG: RNA code words and protein synthesis. II. Nucleotide sequence
of a valine RNA code word. Proc. nat. Acad. Sci. (Wash.) 52, 420-427 (1964).
LETENDRE, c., A. M. MICHELSON, and M. GRUNBERG-MANAGO: Oligonucleotide inhibition
of amino acid attachment. Cold Spr. Harb. Symp. quant. BioI. 31, 171-176 (1966).
LEVINTHAL, c., E. SIGNER, and K. FETHEROLF: Reactivation and hybridization of reduced
alkaline phosphatase. Proc. nat. Acad. Sci. (Wash.) 48, 1230-1237 (1962).
LIKOVER, T. E., and C. G. KURLAND: The contribution of DNA to translation errors
induced by streptomycin in vitro. Proc. nat. Acad. Sci. (Wash.) 58,2385-2392 (1967).
LODISH, H. : Independent translation of the genes of bacteriophage f2 RNA. J. molec. BioI.
32, 681-685 (1968).
LOFTFIELD, R. B.: Errors in protein synthesis. Biochem. J. 89, 82-87 (1963).
- , and E. A. EIGNER: A soluble ribonucleic acidindiced increase in the specificity of E. coli
isoleucine activating enzyme. J. bioI. Chern. 240, PC 1482 (1965).
LUCAS-LENARD, J., and F. LIPMANN: Initiation of polyphenylalanine synthesis by N-acetyl
phenylalanine. Proc. nat. Acad. Sci. (Wash.) 57, 1050-1057 (1967).
MCCARTHY, B. J., and E. T. BOLTON: Interaction of complementary RNA and DNA. J.
molec. BioI. 8, 184-200 (1964).
MADISON, J. T., G. A. EVERETT, and H. KUNG: Oligonucleotides from yeast tyrosine trans-
fer ribonucleic acid. J. bioI. Chern. 242, 1318-1323 (1966).
MAGNI, G. E., and P. O. PUGLISI: Mutagenesis of super-suppressors in yeast. Cold Spr.
Harb. Symp. quant. BioI. 31, 699-704 (1966).
MANGIAROTTI, G., D. APIRION, D. SCHLESSINGER, and L. SILENGP: Biosynthetic precursors
of 30S and 50S ribosomal particles in Escherichia coli. Biochemistry 7, 456-472 (1968).
MARCKER, K., and F. SANGER: N-formyl-methionyl-sRNA. J. molec. BioI. 8, 835-840 (1964).
MARSHALL, R. E., C. T. CASKEY, and M. W. NIRENBERG: Fine structure of RNA codewords
recognized by bacterial, amphibian, and mammalian transfer RNA. Science 155, 820 to
826 (1967).
MARTIN, R. G.: The one operon-one messenger theory of transcription. Cold Spr. Harb.
Symp. quant. BioI. 28, 357-360 (1963).
- Frameshift mutants in the histidine operon of Salmonella typhimurium. J. molec. BioI. 26,
311-328 (1967).
MATTHAEI, J. H., and M. W. NIRENBERG: Characteristics and stabilization of DNAasesensi-
tive protein synthesis inE. coliextracts. Proc. nat. Acad. Sci. (Wash.) 47, 1580-1588 (1961).
- , H. P. VOIGH, G. HELLER, R. NETH, G. SCHOCH, H. KIBLER, F. AMELUNXEN, G. SANDER,
and A. P ARMIGGIANI: Specific interactions of ribosomes in decoding. Cold Spr. Harb. Symp.
quant. BioI. 31, 25-38 (1966).
MIURA, K.: Specificity in Structure of Transfer RNA. Prog. Nucleic Acid Res. and Mol.
BioI. 6, 39-83 (1967).
MOORE, P. B., R. R. TRAUT, H. NOLLER, P. PEARSON, and H. DELIUS: Ribosomal proteins of
Escherichia coli. II. Proteins from the 30S subunit. J. molec. BioI. 31, 441-462 (1968).
MORGAN, A. R., R. D. WELLS, and H. G. KHORANA: Studies on polynucleotides LIX.
Further codon assignments from amino acid incorporation directed by ribopolynucleo-
tides containing repeating trinucleotide sequences. Proc. nat. Acad. Sci. (Wash). 56,
1899-1906 (1966).
- - - Studies on polynucleotides LXXIV. Direct translation in vitro of single stranded

DNA-like polymers with repeating sequence in the presence of Neomycin B. J. molec.
Biol. 26, 477-498 (1967).
MUNDRY, K. W., u. A. GIERER: Z. Vererbungsl. 89, 614 (1958).
NAKAMOTO, T., and D. KOLAKOFSKY: A possible mechanism for initiation of protein syn-
thesis. Proc. Acad. Sci. (Wash.) 55, 606-613 (1966).
NIRENBERG, M. W., O. W. JONES, P. LEDER, B. F. C. CLARK, W. S. SLY, and S. PESTKA: (2)
On the coding of genetic information. Cold Spr. Harb. Symp. quant. BioI. 28,
549-557 (1963).
- , and H. J. MATTHAEI: The dependence of cell-free protein synthesis in E. coli upon
naturally occurring and synthetic polyribonucleotides. Proc. nat. Acad. Sci. (Wash.)
47, 1588-1602 (1961).
- , and P. LEDER: RNA codewords and protein synthesis. Science 145, 1399-1407 (1964).
- , J. H. MATTHAEI, O. JONES, R. MARTIN, and S. BARONDES: (1) Fed. Proc. 22, 55
(1963).
- , P. LEDER, M. BERNFIELD, R. BRIMACOMBE, J. TRUPIN, F. ROTTMAN, and C. O'NEAL:
RNA codewords and protein synthesis VII. On the general nature of the RNA code.
Proc. nat. Acad. Sci. (Wash.) 53, 1161-1168 (1965).
NISHIMURA, S., T. M. JACOB, and H. G. KHORANA: Synthetic deoxyribonucleotides as
templates for ribonucleic acid polymerase; the formation and characterization of a ribo-
polynucleotide with a repeating trinucleotide sequence. Proc. nat. Acad. Sci. (Wash.)
52, 1494-1501 (1964).
- , D. S. JONES, E. OHTSUKA, H. HAYATSU, T. M. JACOB, and H. G. KHORANA: (1) Studies
on polynucleotides XLVII. The in vitro synthesis of homopeptides as directed by a ribo-
polynucleotide containing a repeating trinucleotide sequence. New codon sequences for
lysine, glutamic acid and arginine. J. molec. Biol. 13,283-301 (1965).
- - , and H. G. KHORANA: (2) Studies on polynucleotides XLVIII. The in vitro synthesis
of a copeptide containing two amino acids in alternating sequence dependent upon a
DNA-like polymer containing two nucleotides in alternating sequence. J. molec. BioI.
13, 302-324 (1965).
NOMURA, M., and C. B. LOWRY: Phage f2 RNA-directed binding of formyl-methionyl tRNA
to ribosomes and the role of 30S ribosomal subunits in initiation of protein synthesis.
- - , and C. GUTHRIE. The initiation of protein synthesis; joining of the 50S ribosomal
subunit to the initiation complex. Proc. nat. Acad. Sci. (Wash.) 58, 1487-1493 (1967).
NORRIS, A. T., and P. BERG: Mechanism of aminoacyl RNA synthesis: studies with isolated
amino-acyl adenylate complexes of isoleucyl RNA synthetase. Proc. nat. Acad. Sci.
(Wash.) 1964, 330-337.
OHTA, T., I. SHIMADA, and K. IMAHORI: Conformational change of tyrosyl-RNA synthetase
induced by its specific transfer RNA. J. molec. BioI. 26, 519-524 (1967).
-, S. SARKAR, and R. E. THACH: The role of guanosine-5'-triphosphate in the initiation of
peptide synthesis III. Binding of formyl methionyl-tRNA to ribosomes. Proc. nat. Acad.
Sci. (Wash.) 58, 1638-1644 (1967).
OHTAKA, Y., and S. SPIEGELMAN: Translational Control of protein synthesis in a cell free
system directed by a polycistronic viral RNA. Science 142, 493-497 (1963).
PAULING, L., and M. DELBRUCK: The nature of the intermolecular forces operative in
biological processes. Science 92, 77-82 (1940).
PELC, S. R., and M. G. E. WELTON: Stereochemical relationships between coding triplets
and amino acids. Nature (Lond.) 209, 868-869 (1966).
PESTKA, S., and M. W. NIRENBERG: Regulatory mechanisms and protein synthesis X. Codon
recognition on 30S ribosomes. J. molec. BioI. 21, 145-172 (1966).
RAJBHANDARY, U. L., S. H. CHANG, A. STUART, R. D. FAULKNER, R. M. HOSKINSON, and
H. G. KHORANA: Studies on polynucleotides LXVIII. The primary structure of yeast
phenylalanine transfer RNA. Proc. nat. Acad. Sci. (Wash.) 57, 751-758 (1967).
RAVEL, J. M.: Demonstration of a guanosine triphosphate-dependent enzymatic binding of
amino acyl-ribonucleic acid to Ejcherichia coli ribosomes. Proc. nat. Acad. Sci. (Wash.)
57, 1811-1816 (1967).
44 CARL R. WOESE
REVEL, M., and U. Z. LI1TAUER: The coding properties of methyldeficient phenylalanine

transfer RNA from E. coli. J. molec. BioI. 15,389-394 (1966).
RIFKIN, D. B., D. 1. HIRSH, M. R. RIFKIN, and W. KONIGSBERG: A possible ambiguity in
the coding of mouse hemoglobin. Cold Spr. Harb. Symp. quant. BioI. 31, 715-718
(1966).
SAGER, R., I. B. WEINSTEIN, and Y. ASHKENAZI: Coding ambiguity in cell-free extracts of
chlamydomonas. Science 140, 304-306 (1963).
SALAS, M., M. B. HILLE, J. A. LAST, A. J. WAHBA, and S. OCHOA: (1) Translation of the
genetic message II. Effect of initiation factors on the binding of formyl-methionyl-
tRNA to ribosomes. Proc. nat. Acad. Sci. (Wash.) 57, 387-394 (1967).
- , M. J. MILLER, A. J. WAHBA, and S. OCHOA: (2) Translation of the genetic message V.
Effect of Mg+ + and formylation of methionine in protein synthesis. Proc. nat. Acad. Sci.
(Wash.) 57, 1865-1869 (1967).
SAMBROOK, J. F., D. P. FARR, and S. BRENNER: A strong suppressor specific for UGA.
Nature (Lond.) 214, 452---453 (1967).
SARABHAI, A. S., A. O. W. STRETTON, S. BRENNER, and A. BOLLE: Colinearity of the gene
with the polypeptide chain. Nature (Lond.) 201, 13-17 (1964).
SCHLESSINGER, D., G. MANGIAROTTI, and D. APIRION: The formation and stabilization of
30S and 50S ribosome couples in Escherichia coli. Proc. nat. Acad. Sci. (Wash.) 58,
1782-1789 (1967).
SMITH, M. A., M. SALAS, W. M. STANLEY JR., A. J. WAHBA, and S. OCHOA: Direction of
reading of the genetic message II. Proc. nat. Acad. Sci. (Wash.) 55, 141-147 (1966).
SOLL, D., E. OHTSUKA, D. S. JONES, R. LOHRMANN, H. HAYATSU, S. NISHIMURA, and H. G.
KHORANA: Studies on polynucleotides XLIX. Stimulation of the binding of amino acyl
tRNA to ribosomes by ribotrinucleotides and a survey of codon assignments for 20
amino acids. Proc. nat. Acad. Sci. (Wash.) 54, 1378-1385 (1965).
- , J. D. CHERAYIL, and R. M. BOCK: Studies on polynucleotides LXXV. Specificity of
tRNA for codon recognition as studied by the ribosomal binding technique. J. molec.
BioI. 29,97-122 (1967).
- , and U. L. RAJBHANDARY: Studies on polynucleotides LXXVI. Specificity of transfer
RNA for codon recognition as studied by amino acid incorporation. J. molec. BioI. 29,
113-124 (1967).
SONNEBORN, T. M.: Degeneracy of the genetic code: Extent, nature, and genetic implica-
tions. In: Evolving genes and proteins, p. 377. BRYSON, V., and H. VOGEL, Eds. New
York: Academic Press 1965.
SPEYER, J., P. LENGYEL, C. BASILIO, and S. OCHOA: Synthetic polynucleotides and the
amino acid code IV. Proc. nat. Acad. Sci. (Wash.) 48, 441---448 (1962).
- , A. WAHBA, R. GARDNER, and S. OCHOA: Synthetic polynucleotides and the amino
acid code. Cold Spr. Harb. Symp. quant. BioI. 28, 559-567 (1963).
SPIEGELMAN, S., D. BISHOP, D. MILLS, and N. PACE: Mss in preparation.
STANLEY, W. M., JR., M. A. SMITH, M. B. HILLE, and J. A. LAST: Studies on the translation
of the genetic message 1. Preparation of oligonucleotide messengers of specified base
sequence. Cold Spr. Harb. Symp. quant. BioI. 31, 99-102 (1966).
STULBERG, M. P., and K. R. ISHAM: Studies on the locus of the enzyme recognition site in
phenylalanine transfer RNA. Proc. nat. Acad. Sci. (Wash.) 57, 1311-1316 (1967).
SUBAK-SHARPE, H., W. M. SHEPARD, and J. HAY: Studies on sRNA coded by Herpes Virus.
Cold Spr. Harb. Symp. quant. BioI. 31,583-594 (1966).
SUEOKA, N.: Correlation between base composition of desocyribonucleic acid and amino
acid composition of protein. Proc. nat. Acad. Sci. (Wash.) 47, 1141-1148 (1961).
- Variation and heterogeneity of base composition of desoxyribonucleic acids: A compila-
tion of old and new data. J. molec. BioI. 3,31---40 (1961).
- In: Evolving genes and proteins, p. 479. BRYSON, V., and H. VOGEL, Eds. New York:
Academic Press 1965.
- , J. MARMUR, and P. DOTY: Dependence of density of desocyribonucleic acids on guanine-
cytosine content. Nature (Lond.) 183, 1429-1431 (1959).
- , and T. YAMANE: Fractionation of amino acyl-acceptor RNA on a methylated albumin
column. Proc. nat. Acad. Sci. (Wash.) 48, 1454-1461 (1962).
SUNDARARA]AN, T. A., and R. E. THACH: Role of the formylmethionine codon AUG in

phasing translation of synthetic messenger RNA. J. molec. BioI. 19,74-90 (1966).
SWARTZ, J. H., J. M. EISENSTADT, G. BRAWERMAN, and N. D. BINDER: Synthesis ofphagef2
coat protein in Euglena in vitro system. Proc. nat. Acad. Sci. (Wash.) 53, 195-200 (1965).
SZER, W., and S. OCHOA: Complexing ability and coding properties of synthetic poly-
nucleotides. J. molec. BioI. 8, 823-834 (1964).
TERZAGHI, E., Y. OKADA, G. STREISINGER, J. EMRICH, M. INOUYE, and A. TSUGITA: Change
of a sequence of amino acids in phage T4lysozyme by acridine induced mutations. Proc.
nat. Acad. Sci. (Wash.) 56, 500-507 (1966).
THACH, R. E., M. A. CECERE, T. A. SUNDARARA]AN, and P. DOTY: The polarity of messen-
ger RNA translation in protein synthesis. Proc. nat. Acad. Sci. (Wash.) 54,1167-1173
(1965).
TRAUB, P., and M. NOMURA: Structure and function of E. coli ribosomes V. Reconstitution
of functionally active 30S ribosomal particles from RNA and proteins. Proc. nat. Acad.
Sci. (Wash.) 58, 777-784 (1968).
TRAUT, R. R., P. B. MOORE, H. DELIVS, H. NOLLER, and A. TISSIERS: Ribosomal proteins of
Escherichia coli I. Demonstration of different primary structures. Proc. nat. Acad. Sci.
(Wash.) 57, 1294-1301 (1967).
VINUELA, E., M. SALAS, and S. OCHOA: Translation of the genetic message III. Pormylation
as initiator of proteins programmed by polycistronic messenger RNA. Proc. nat. Acad.
Sci. (Wash.) 57, 729-734 (1967).
WAHBA, A. J., C. BASILIO, J. SPEYER, P. LENGYEL, R. S. MILLER, and S. OCHOA. Synthetic
polynucleotides and the amino acid code, VI. Proc. nat. Acad. Sci. (Wash.) 48, 1683 to
1686 (1962).
- , R. S. GARDNER, C. BASILIO, R. S. MILLER, J. F. SPEYER, and P. LENGYEL: (1) Synthetic
polynucleotides and the amino acid code VII. Proc. nat. Acad. Sci. (Wash.) 49,116-122
(1963).
-, R. S. MILLER, C. BASILIO, R. S. GARDNER, P. LENGYEL, and J. SPEYER: (2) Synthetic
polynucleotides and the amino acid code IX. Proc. nat. Acad. Sci. (Wash.) 49, 880-885
(1963).
- , M. SALAS, and W. M. STANLEY, JR.: Studies on the translation of the genetic message II.
Translation of oligonucleotide messengers of specified base sequence. Cold Spr. Harb.
Symp. quant. BioI. 31, 103-112 (1966).
WALLER, J. P.: The NH 2-terminal residues of the proteins from cell free extracts of E. coli.
J. molec. BioI. 7, 483-496 (1963).
- , and J. I. HARRIS: Studies on the composition of the protein from Escherichia coli ribo-
somes. Proc. nat. Acad. Sci. (Wash.) 47, 18-23 (1961).
WATSON, J. D.: The involvement of RNA in the synthesis of protein. Science 140, 17-26
(1963).
- , and F. H. C. CRICK: Cold Spr. Harb. Symp. quant. BioI. 18, 123 (1953).
WEBSTER, R. E., D. L. ENGELHARDT, and N. D. ZINDER: In vitro protein synthesis: chain
initiation. Proc. nat. Acad. Sci. (Wash.) 55, 155-161 (1966).
WEIGERT, M., and A. GAREN: Base composition of nonsense codons in Escherichia coli 1.
The NI codon, UAG. Nature (Lond.) 206, 992-995 (1965).
- , E. LANKA, and A. GAREN: (1) Base composition of nonsense co dons in Escherichia coli II.
The N2 codon, UAA. J. molec. BioI. 23, 391-400 (1967).
- - - (2) Amino acid substitutions resultins from suppression of nonsense mutations III.
Tyrosine insertion by the Su-4 gene. J. molec. BioI. 23, 401-404 (1967).
WEISBLUM, B., S. BENZER, and R. W. HOLLEY: A physical basis for degeneracy in the amino
acid code. Proc. nat. Acad. Sci. (Wash.) 48, 1449-1454 (1962).
- , F. GONANA, G. VON EHRENSTEIN, and S. BENZER: A demonstration of coding degeneracy
for leucine in the synthesis of protein. Proc. nat. Acad. Sci. (Wash.) 53, 329-334 (1965).
WILLIAMS, J. A.: Ph. D. Thesis, University of Illinois 1968.
WIMMER, E., I. MAXWELL, and G. M. TENER: Biochemistry 7 (in press).
WITTMAN, H. G.: Comparison of the tryptic peptides of chemically induced and spontaneous
mutants of tobacco mosaic virus. Virology 12, 609-612 (1959).
WOESE, C. R.: Nature of the biological code. Nature (Lond.) 194, 1114-1115 (1962).
46 CARL R. W OESE: The Biological Significance of the Genetic Code
WOESE, C. R.: The genetic code - 1963. ICSU Rev. 5, 210-240 (1963).
- On the evolution of the genetic code. Proc. nat. Acad. Sci. (Wash.) 54, 1546-1552 (1965).
- (1) The genetic code: The molecular basis of genetic expression. New York: Harper and
Row 1967.
- (2) The present status of the genetic code. Prog. Nucleic Acid Res. and Mol. BioI. 7,
107-172 (1967).
- The fundamental nature of the genetic code: pre biotic interactions between polynucleo-
tides and polyamino acids or their derivatives. Proc. nat. Acad. Sci. (Wash.) 59, 110-117
(1968).
- , D. H. DUGRE, W. C. SAXINGER, and D. A. DUGRE: The molecular basis to the genetic
code. Proc. nat. Acad. Sci. (Wash.) 55, 966-974 (1966).
YAMANE, T., and N. SUEOKA: Enzymic exchange of leucine between different components
ofleucine acceptor RNA in E. coli. Proc. nat. Acad. Sci. (Wash.) 51, 1178-1184 (1964).
YANOFSKY, c., B. C. CARLTON, J. R. GUEST, D. R. HELINSKI, and U. HENNING: On the co-
linearity of gene structure and protein structure. Proc. nat. Acad. Sci. (Wash.) 51,
266-272 (1964).
-, J. ITo, and V. HORN: Amino acid replacements and the genetic code: Cold Spr. Harb.
Symp. quant. BioI. 31, 151-162 (1966).
YARUS, M., and P. BERG: Recognition of tRNA by aminoacyl tRNA synthetase. J. molec.
BioI. 28, 479 (1967).
ZACHAU, VON H. G., D. DUTTING und H. FELDMAN: Angew. Chern. 78, 392 (1966).
ZAMIR, A., R. W. HOLLEY, and M. MARQUISEE: Evidence for the occurrence of a common
pentanucleotide sequence in the structures of transfer ribonucleic acids. J. bioI. Chern.
240, 1267-1273 (1965).
ZUBAY, G., and P. DOTY: Nucleic acid interactions with metal ions and amino acids. Bio-
chim. biophys. Acta (Arnst.) 29, 47-58 (1958).
Errors in Translation
JULIAN DAVIES
I. Introduction
During the process of information transfer from gene to protein there are a
number of steps which require that this information be transmitted from one type of
biological molecule to another. This problem of information transfer and the ac-
companying problem of recognition is at its most complex in the translation process
in which the information encoded in messenger RNA (mRNA) molecules is used to
direct the synthesis of proteins. It is known that the translation process consists of
three, and perhaps four steps which involve a specific rapport between two different
biological molecules (ATTARD!, 1967). The laws of chance alone would dictate a
higher probability of error in the translation process than in the (formally) simpler
transcription or replication processes; accurate estimates of the error frequency in
transcription or replication cannot be made, but this is certainly beyond the limits of
accuracy of experimental methods such as nearest neighbor analysis.
II. The Translation System

The steps involved in translation are shown in Fig. 1 and intramolecular recogni-
tion of high specificity has to be maintained in these stages to preserve the fidelity of
protein synthesis. These reactions, in more detail are:
A. Reaction oj an Amino-Acid with its Specific Aminoaryl-tRNA Synthetase

These enzymes, of which a number have been isolated and purified, are known to
have two functions: 1) to form a specific complex with an amino acid, and 2) to
transfer this amino acid to its corresponding tRNA (NOVELLI, 1967). There being
20 amino acids in protein, at least 20 aminoacyl-tRNA synthetases are necessary and
in a number of cases there are thought to be multiple synthetases for the same amino
acid although the evidence is not convincing in Escherichia coli. These enzymes, in
addition to their role in the charging of different tRNA molecules, are intimately
involved in the control of the synthesis of macromolecules (NEIDHART, 1966). It is
obvious that errors of recognition in either of the two reactions mentioned above,
such that 1) an amino acid forms a complex with the wrong synthetase or, 2) an amino
acid when complexed with the correct synthetase reacts with the "wrong" tRNA
would lead to the same end result, viz. incorrect "charging" - an amino acid attached
to the wrong tRNA.
Fortunately, as we shall discuss later, there would appear to be a built - in pro-
tection against some of these mistakes.
48 JULIAN DAVIES
Charging
A+ATP aminoacy' .. A-AMP-mAS
sy~nthetase
(AS)
AS+AMP
l tRNApOOJ
+
I
I
I
I
I
I
I
I
----------1,
I
I
,
----------,
I
I
I
I
I
_________ 1
,
I
I
J
----------- I
,,
As _____________ )
A4 A3A2A1F
termination signal finished polypeptide

AS A4 A3 A2Aj F-AS A4 A3 A2 Aj
a
ATP + AA2 + AS ~ AA2-AMP-AS + PPi

AA2-AMP-AS + tRNA2 ~ AA2-tRNA2 + AS + AMP
AA -tRNA2 + polysome-tRNAt-AA t
l....
2
factors, etc. /tRNA2-AA2

---------:>~ polysome .............
J tRNAt-AA t
polysome-tRNA2-AA2-AAt-oE<~---------'
,
I polymerizing
I repeat enzyme(s)
I
'. - - - - - - - - - - - ~ polysome-tRNAn -AAn -AAn-t - -AA2-AA t
mRNA + ribosomes + tRNAn J
release .
+ AAn-AAn-t--AA2-AAt ....<:;:,----------
(tRNAt-AA t represents initiating aminoacyl-tRNA)
b
Fig. 1. The translation process. (A, amino acid; A-, aminoacyl-tRNA; F-, N-formyl
methionyl-tRNA; e, 30S ribosomal subunit; 0, 50S ribosomal subunit)
Errors in Translation 49
B. Recog1Jition Involving tRNA

Molecules of tRNA arc polyribonucleotides of some 80 bases which carry amino
acids attached to their - CCA terminal sequence; the structures of several tRNA's
have now been determined and the primary sequence, in those cases known, is
consistent with a "clover-leaf" structure with the three bases complementary to
mRNA co dons - the anticodon - being found in one of the loops of this structure;
there are multiple tRNA's for a large number of the amino acids (MIURA, 1967). These
molecules are the adapters required for the reading of the RNA code in terms of
amino acids, and as such they require two recognition sites. One of these sites must
identify the correct aminoacyl-tRNA synthetase complex and so ensure transfer of
an amino acid to its specific tRNA; an error in this step would result in an amino acid
being charged to the wrong tRNA (the same outcome as in A2 above). The second
property built into the tRNA structure is the ability to recognize coding triplets in
mRNA; as we have mentioned previously, the anticodon, which is complementary to
the codon in antiparallel fashion, assumes this particular role. Any change in tRNA
structure altering the primary, secondary or tertiary structure of these two recognition
sites could destroy or modify tRNA function and accordingly, cause incorrect placing
of an amino acid in a protein chain. A third site on tRNA is required for attachment to
ribosomes, this site is not directly concerned with the fidelity of the translation
process.
C. Messenger RNA
These molecules are large single stranded polynucleotides which are comple-
mentary to the "sense" strand of DNA and contain information necessary to dictate
the amino acid sequence of a protein in the form of a linear sequence of trinucleotides
(codons) (SINGER and LEDER, 1966). These molecules bind to the 30S subunits of
ribosomes and direct the attachment of aminoacyl-tRNA molecules to the peptide-
bond-forming site on the ribosome (OKAMOTO and TAKANAMI, 1963; MATTHAEI et aI.,
1964; KAJl, SUZUKI and KAJI, 1966; PESTKA and NIRENBERG, 1966). Errors would
occur if a codon could be made to recognize more than one anticodon, and this
situation would most likely arise from the presence of bases in the mRNA molecule
which would be capable of pairing with bases other than those prescribed by the
normal pairing rules (AID = G/c). Tautomerization of bases could well lead to such
ambiguity; substitution of 5-fluorouracil for uracil is believed to induce mistakes in
translation by such a mechanism (CHAMPE and BENZER, 1962). For mistakes to occur
at the mRNA level, we require that normal pairing of one or a few bases be modified
without completely destroying the ability of the co dons containing these changed
bases to recognize an anticodon.
D. Ribosomes
Ribosomes are large polynucleotide-protein complexes which are made up of two
discrete subunits, each playing a separate role in the mechanism of peptide bond
formation. Messenger RNA and one tRNA molecule bind to the smaller (30S)
subunit, which is also the subunit to which N-formylmethionyl-tRNA binds when
protein synthesis is initiated (NOMURA and LOWRY, 1967). It seems that the larger
(50S) subunit is more crucially involved with actual peptide bond formation - there
would appear to be separate aminoacyl-tRNA binding sites on each subunit (IGARASHI
and KAJI, 1967). The chemical nature of the role of the ribosome in the recognition
50 JULIAN DAVIES
between mRNA and tRNA molecules is not clear but there is suggestive evidence
that ribosomes do exert a positive role in this process and to some extent define the
accuracy of this recognition. This leads to the expectation that certain mutational
changes or chemical modifications in ribosome structure would lead to ambiguity in
translation.
It should be realized that so very little is known of the actual chemistry of the
specific interactions or enzymatic reactions involved in translation that it is not
possible to consider mechanisms by which various agents induce ambiguity in
translation process. Conversely, while they define the overall reactions in greater
detail, agents affecting translation have so far contributed little to our knowledge of
the chemistry of translation. It is highly probable that this situation will change within
the next few years. We will now consider, in more detail, some examples of errors in
recognition occurring at various stages in translation.
III. Errors in Translation

A. Aminoacyl-tRNA Synthetases
These enzymes are required for the enzymatic esterification of a tRNA with its
correct amino acid; the enzyme must, by definition, recognize an amino acid and a
tRNA molecule. It has been shown recently (YARUS and BERG, 1967) that the syn-
thetase will recognize its specific tRNA even if it is not complexed to the amino acid;
in other words, this recognition is the property of the enzyme alone. R. B. LOFTFIELD
and coworkers (LOFTFIELD, 1963) have carried out a series of experiments which give
some idea of the error frequency in charging, by studying the competition, if any,
between two closely related amino acids e.g. valine and isoleucine in these steps. It
was found that with these two natural amino acids, the error in charging tRNAval
with isoleucine, or tRNAisoleu with valine was less than 1 in 10,000. With an amino
acid analogue (not natural) the error frequency was considerably higher: alloisoleu-
cine could be attached to either tRNA val or tRNAileu in competition with the natural
amino acids to an extent of about 5%. The same high error frequency is found with
other close analogues e.g. p-fluorophenylalanine and phenylalanine.
The charging of tRNA has more recently been studied in greater detail and it
would appear that the first reaction in this process, complexing of an amino acid with
an aminoacyl-tRNA synthetase is often indiscriminate (NORRIS and BERG, 1964).
Thus far, it has been shown that isoleucyl-tRNA synthetase can accept both isoleucine
or valine, valyl-tRNA synthetase can accept valine and threonine (BERGMANN, BERG
and DIECKMANN, 1961), and lysyl-tRNA synthetase can accept lysine and hydroxy-
lysine (STERN and MEHLER, 1965). The high specificity demonstrated for charging
by LOFTFIELD et al. must then occur in the process of transfer of the amino acid to
tRNA, and this expectation has been convincingly demonstrated by NORRIS and
BERG who showed that although isoleucyl-tRNA synthetase can accept both iso-
leucine or valine, only isoleucine is transferred to tRNA ileu ' In a detailed study of this
reaction it was found that there was very little difference in stability between the two
enzyme-aminoacyl adenylate complexes in the presence of pyrophosphate, but in the
presence of tRNA lleu the isoleucyl complex transferred the amino acid to the tRNA,
while in the presence of tRNAval the valyl complex was broken down without
transferring valine (BALDWIN and BERG, 1966). Therefore this transfer has a much
higher specificity with respect to the amino acid than does the formation of the
enzyme-aminoacyl adenylate complex.
Other aminoacyl-tRNA synthetases have been extensively purified but tests of
their error frequency in complexing and transfer have not yet been fully reported. It
would seem, on the basis of the above experiments, that errors at the level of tRNA
charging would be extremely low; recent corroboration comes from experiments of
Y ARUS and BERG (1967) who have demonstrated the high specificity of the enzyme-
tRNA interaction by a membrane filter technique.
However, studies on the use of heterologous systems for charging have indicated
that errors can occur if a tRNA preparation from one source is charged with an
aminoacyl-tRNA synthetase obtained from another species. BARNETT and JACOBSON
(1964) have shown that if E. coli tRNA is charged using an enzyme prepared from
Neurospora, phenylalanine can be attach-
1000,--------------------,
ed to several E. coli tRNA molecules in
addition to tRNAphe ' Such a system does « r:f'-----Q.. .... ,
then introduce errors in translation and ~ 800

'"
continued work (BARNETT, 1965; IMA- ',<
01
=<- ,
MOTa, YAMANE and SUEOKA, 1965) on this 8 600-
\
\
heterologous system has shown that the ?

\
\
phenylalanyl-tRNA synthetase from Neuro- c 400--
\
\
spora can be separated into two distinct E \
\
\
synthetases, El and E 2 , by DEAE column ----
C 200-
(J)
\
\
chromatography. These two enzymes have :J \
8 \
the following properties: 1) enzyme El

charges phenylalanine to E. coli tRNA phe , OL-----~--~I~--~--~
50 60 70 80
whileE2 charges phenylalanine to two other Temperature
E. coli tRNA's (val and ala) which do not Fig. 2. The effect of temperature on the
normally accept phenylalanine; 2) Neuro- specificity of isoleucyl-tRNA synthetase.
spora tRN~he is charged in the presence of (ARCA, FRONTAL! and TEccE, 1965)
El but not E 2 • An interesting further deve-
lopmenthas shown that in Neurospora one of the phenylalanyl-tRNA synthetases (E1)
and a phenylalanine tRNA are of mitochondrial origin; it is the cytoplasmic pheny-
lalanyl-tRNA synthetase from Neurospora (E2) which can attach phenylalanine to E. coli
tRNAval and tRNAala (BARNETT and EPLER, 1966; BARNETT and BROWN, 1967;
BARNETT, BROWN and EPLER, 1967).
Errors may also be induced in the tRNA charging process when the experiments
are carried out under certain extreme or nonpermissive conditions; this indicates that
particular structural conformations of RNA or protein molecules are essential for
fidelity. The isoleucyl-tRNA synthetase from the thermophile B. stearothermophilus
(growth at 65°, no growth at 37°) shows the same lack of discrimination (when
tested at 50°C) in formation of aminoacyl adenylate enzyme complex as the corres-
ponding E. coli synthetase (ARCA et al., 1963, 1964). That is, at this temperature both
isoleucine and valine can complex with the enzyme, but only isoleucine can be trans-
ferred to tRNAileu ' If, however, this reaction is carried out at 75° specificity in the
enzyme-tRNA recognition is lost and valine is transferred to tRNAiJeu with concomi-
tant decrease in isoleucine transfer (Fig. 2). Valine is found esterified to the terminal
adenosine of the tRNA molecule in the usual manner; recent results indicate that
4*
52 JULIAN DAVIES
ambiguous transfer of serine and threonine to tRNAileu can occur under similar con-
ditions of high temperature (ARCA, FRONTAL! and TEccE, 1965).
Although these abnormal conditions provide a clear demonstration ot lack of
fidelity in tRNA charging, the underlying mechanism is far from clear; is the misre-
cognition due to thermally induced changes in the tRNA or in the enzyme recogni-
tion sites? A close relationship between the melting curve of tRNAileu and the kinetics
of formation of valyl-sRNAileu has been taken to indicate that anomalous charging
was the result of modification of the tRNA molecule (TEccE, pers. communication).
B. Do Translation Errors Due to Anomalous Charging Occur in Whole Cells?

A considerable number of E. coli mutants with thermolabile aminoacyl-tRN A
synthetases are known (FANGMAN and NEIDHART, 1964; YANIV, JACOB and GROS,
1965) and it is conceivable that some of these altered enzymes would show anomalous
charging properties at the nonpermissive temperature. No reports of translational
mistakes have yet been reported for any of these enzymes and a systematic survey
for such mistakes does not seem to have been made.
PRINTZ and GROSS (1967) have described a conditional lethal mutant of Neuro-
spora crassa which apparently synthesizes defective proteins at the nonpermissive
temperature. These authors propose that the lethal mutation occurs in an aminoacyl-
tRNA synthetase which functions normally at low temperature but which introduces
a level of translational mistakes sufficient to prevent growth at the higher temperature.
The nature of the errors in this case, and the way in which they are induced, has not
been rigorously established, but there is a significant difference in the properties of
the wild type and mutant leucyl-tRNA synthetase as judged by a different K. for
leucine in the formation of leucyl-tRNA.
C. Transfer RNA
A tRNA molecule must posses two recognition sites built into its nucleotide
sequence, one for its cognate aminoacyladenylate-tRNA synthetase and the other
(the anticodon) for the mRNA codon. The first recognition site involves protein and
RNA, while the second (in its simplest form) involves an RNA-RNA interaction.
There is some controversy at the present time as to whether these two sites are
related or not, and competition experiments or selective inactivation experiments
have failed to resolve this point (LETENDRE, MICHELSON and GRUNBERG-MANAGO,
1966; HAYASHI and MIURA, 1966). It is evident that a single base change in the
anticodon which occurs in Su+ strains does not alter the specificity of charging
(LANDY et al., 1967) and the weight of the evidence favors the notion that the two
sites are not related. This particular problem does not concern us here, however, as it
should be quite clear that any chemical modification or substitution which alters a
recognition sequence in tRNA would be expected to produce misrecognition errors
of one kind or the other, whereas major modification of these sites would presumably
result in complete inactivation.
Many attempts have been made to modify selectively either the anticodon or the
enzyme-amino acid recognition properties of tRNA by chemical means; these
methods all suffer from lack of specificity. Although a particular chemical reagent
may selectively attack only one kind of base, it is unlikely that anyone particular base
in the primary sequence could be modified. The results, at present, of this chemical
groping are somewhat disappointing, although with more and more tRNA's being
sequenced, together with the finding of a number of rare bases which may only occur
once in a particular tRNA sequence, it is to be hoped that more selective attack will
be possible. In most of the published work on the chemical modification of tRNA,
the main concern has been with complete inactivation of recognition rather than
modification. However, EBEL and his coworkers (BAKES et aI., 1965; WElL, 1965)
have considered this consequence of reaction between tRNA and "specific" chemical
reagents; bromination of tRNA has been most studied in their hands. E. coli tRNA
was brominated in dimethylformamide, charged with amino acids, and used in an
in vitro polypeptide synthesizing system with poly U as messenger. Under these
conditions, in addition to incorporation of phenylalanine (UUU) into TCA-precipi-
table material, substantial amounts of glutamic acid (GAA), isoleucine (AUU) ,
lysine (AAA), and valine (GUU) were also incorporated. Examination of the bro-
minated tRNA indicated marked changes in secondary structure as determined by
melting curves. These results could be explained in terms of alterations in the ability
of the tRNA to accept an amino acid, or in the ability to transfer; evidence in favor
of the latter effect was provided by experiments in which the tRNA was charged first
and then brominated, when essentially the same incorporation patterns were ob-
tained. As already pointed out, an accurate interpretation of these experiments in
specific chemical terms is not possible; whether gross alterations in secondary
structure of tRNA, or modification of bases in recognition sequences are responsible
for this ambiguity is also open to question.
Other experiments have been carried out in which tRNA is modified by chemical
means such as chemical alkylation, UV-irradiation, reaction with nitrous acid, or
formaldehyde, etc. but examination of the products for induced coding errors has not
been reported (WElL, 1965).
The possible effects of enzymatic methylation of tRNA on transfer have also been
studied extensively. It is well known that tRNA deficient in methyl groups can be
isolated from certain bacterial mutants; this tRNA has been isolated and partially
purified and compared in properties with normal tRNA (MANDEL and BOREK, 1963).
One can also examine the changes which occur when this submethylated tRNA is
enzymatically methylated in extracts. Since only a small number of methyl groups are
attached to the polynucleotide sequence of tRNA in an enzymatic reaction subsequent
to the synthesis of polynucleotide backbone, it has been considered likely that these
methyl groups must playa crucial role in either charging or transfer. Several groups
of workers (LITTAUER, REVEL and STERN, 1966; PETERKOVSKY, ]ESENKY and CAPRA,
1966) have come to the conclusion that small differences in coding properties (binding
response to a given RNA triplet) do occur when submethylated tRNA species are
tested in the NIRENBERG-LEDER assay. It would seem unlikely that such small differ-
ences in codon recognition represent a major class of translational errors, and in
common with the other tRNA modifications it cannot be said whether the ambiguity
is due to modification of overall tRNA secondary structure or of a specific site. It is
probable that more specific methods of chemical attack will become available now
that the complete structure (including the anticodon) is known for several tRNA's.
Such chemical modification must certainly help to define the aminoacyl-tRNA
synthetase recognition site of tRNA's.
54 JULlAN D AVlES
D. Messenger RNA
Any change in a base or bases in mRNA which would result in ambigous pairing
by this base, would presumably lead to translation errors. It is probable that only
modifications in mRNA primary structure would have this effect, as any change in
secondary structure would have more drastic effects on the process of protein syn-
thesis; it is thought that double-strandedness decreases template activity (SINGER,
JONES and NIRENBERG, 1963).
Probably the best example of an agent producing translation errors due to
substitution in mRNA is 5-fluorouracil (FU). This base analogue is incorporated into
the RNA of growing cells or viruses and can cause phenotypic suppression of non-
sense mutations together with the production of enzymatically inactive immunolo-
gically cross reacting proteins (BuSSARD et aI., 1960; NAKADA and MAGASANIK, 1964).
The ability of this compound to allow suppression has been most fruitful in genetic
and biochemical studies of gene expression. Although there have been attempts to
demonstrate the action ofFU in vitro (BU]ARD and HEIDELBERGER, 1966; GRUNBERG-
MANAGO and MICHELSON, 1964), by showing that its incorporation into ribopoly-
nucleotide chains and subsequent translation leads to ambiguous products, none of
these attempts have been successful. However, the evidence from suppression of
nonsense codons is fully consistent with the notion that FU can replace U in a mes-
senger molecule and then occasionally be read as C in translation.
Other halogenated bases have been tested for their ability to influence fidelity in
translation. The work of GRUNBERG-MANAGO and MICHELSON (1964) has shown that
the substitution of a halogen atom in uracil or cytidine residues in mRNA can give
rise to errors in translation in vitro (Table 1).
In addition, polybromo C incorporates proline at higher efficiency than poly C
and the halogenated polymer also directs the incorporation of threonine but not of
histidine. The findings that polybromo U gives no tyrosine incorporation and poly-
bromo C no histidine incorporation are interesting, in that they suggest that this
type of misreading may be more specific than that induced by streptomycin (Table 2);
thus 5-BrU or 5-BrC can be read as A when in the 5' position, but not in the internal
position in the coding triplet.
Methylation of mRNA did not cause misreading, although this might be consider-
ed a much more "physiological" process; methylation of mRNA is believed not to
take place in vivo.
Errors can also be induced by UV irradiation of mRNA; if poly U is irradiated
with UV light in aqueous solution, the resulting polymer, when used in an in vitro
protein synthesizing system, shows a reduced capacity to direct the incorporation of
phenylalanine and a greatly increased capacity for serine incorporation (GROSSMANN,
1962, 1963). Isolation of a phenylalanylserine dipeptide indicated that both amino
acids were incorporated into the same peptide chain. There are two photoproducts
known to be produced by UV irradiation of poly U, one a uracil-uracil dimer and the
other the 4,5-water adduct of uracil. The former was shown to be responsible for the
reduction in phenylalanine incorporation and the latter for serine incorporation.
GRUNBERG-MANAGO and MICHELSON (1964) apparently did not test for serine in-
corporation in their experiments using poly 5-hydroxyuracil as messenger, as this
would seem to be a direct analogy.
Thus far studies on these particular modifications, leading to ambiguities in

translation, have been confined to homopolymers, so it cannot be said whether or not
some of these changes might cause ambiguity by neighboring base effects.
Table 1. Stimulation of amino acid in~orporation by poly U and poly C derivatives. [(GRUNBERG-
MANAGO and MICHELSON, 1964 (1)]
Controls (without polymers): isoleucine, 36; leucine, 128; phenylalanine, 69; serine, 58;
tyrosine, 56; cysteine, 44; glycine, valine, 11; tryptophan, lysine, 28. The incubation mixture
is the same as for Table 1, except for isoleucine where the final pH for incorporation in
presence of poly BrU was 7.4. Specific activity of (14qamino acids: isoleucine, 24.2; leucine,
24.7; phenylalanine, 18.2; serine, 21.9; tyrosine, 9.6; glycine, 21.9; valine, 28.6; tryptophan,
9.3; lysine, 11; [35S]cysteine, 30. Poly-T, 320 fLg/ml; Ho-U, 336 fLg/ml (for phenylalanine
only 240 and 208 fLg/ml of polymer were used respectively); U",U, 246 fLg/ml; BrU2 , 400 fLgl
ml; copolymers 200 fLg/ml. For leucine and phenylalanine, 448 fLg/ml of s-RNA were added.
S30 (5 mg/ml) was used instead of ribosomes for the experiment with U",U
Incorporation in Illlmoles/mgribosnmes above control
U",U, BrU poly THoU Copolymers
Isoleucine 316 383 o o 433 (U/A = 1.4)

Leucine 817 1440 156 100 672 (U)
Phenylalanine 2130 1100 632 289 1780 (U)
Serine 68 99 578 (CfU = 1.27)
Tyrosine 28 22 334 (U/A = 1.4)
Cysteine 23 o 175 (U/G = 3.2)
Valine 27 o 414 (U/G = 0.94)
Glycine, lysine, tryptophan o
& Ratio: U:",U = 2.5.
Controls: proline, 50; threonine, 117; histidine, 70; glutamine, 84; alanine, arginine, serine,
aspartic and glutamic acids, 23. Same experimental conditions as inTable 1. Specific activity
of amino acids: proline, 10.85; threonine, 20.8; histidine, 23.3; glutamine, 4.5; lysine, 11;
alanine, 44; arginine, 19.56; aspartic acid, 58.5. sRNA, 200 fLg/ml; BrC, 160 fLg/ml; copoly-
mers, 200 fLg/ml. Incorporation was measured by the radioactive material precipitated with
tungstate 0.25% in trichloroacetic acid 5% pH 2 (the trichloroacetic acid being brought to
pH 1.6 to 1.7 with 4N NaOH before Na tungstate was added). The incubation mixture was
heated 10 min with N NaOH at 37° so as to hydrolyse all the amino acids on the sRNA's
before acidic precipitation
Amino acids Incorporation in fLfLmoles/mg ribosomes
above control
BrC Copolymers
Proline 626 71(q

Threonine 510 208 (A/C = 1.4)
Histidine o 68 (A/C = 0.33)
Glutamine 25 165 (A/C = 1.4)
Lysine o 929 (A)
Serine, alanine, arginine, aspartic acid,
glutamic acid, and asparagine o
We next go on to a consideration of the effects of aminoglycoside antibiotics; it

has not yet been established whether these drugs induce errors in mRNA reading by
a direct effect, or indirectly by some effect on the conformation of the ribosome which
56 JULIAN DAVIES
influences codon-anticodon pairing. However, we shall consider these agents in the

section on ribosomes.
E. Ribosomes
The precise role of the ribosome in polypeptide synthesis is not known; super-
ficially they can be considered as structural supports which allow juxtaposition of
mRNA and anticodons such that the correct base pairing can take place. There are a
number of sites on the ribosome specific for the binding of aminoacyl-tRNA or
polypeptidyl-tRNA presumably arranged in such a way to best facilitate the enzymatic
formation of peptide bonds. It is assumed that the enzymatic processes concerned
with peptide bond formation and tRNA binding have no role in the actual recogni-
tion process. If, as it thought, there is a particular conformation of the ribosome
(probably only on the 30S subunit) which favors faithful interactions between codon
and anticodon, then any factor which deranges this conformation would induce
misreading of the code. Such alterations could occur as the result of mutational or
chemical changes which alter the structure of a crucial component (RNA or protein)
of the ribosome, or as a consequence of the attachment of some agent which inter-
feres with the structural integrity of the unit. It should be made clear at this point
that knowledge of the chemistry of ribosomes is incomplete and we know nothing of
the nature of the various sites which exist on them; whether ribosomal RNA or
ribosomal protein, or both, are unvolved in the functioning of ribosomes in protein
synthesis also remains a mystery.
IV. Errors Produced by Aminoglycosides

Only in the case of the aminog0,coside antibiotics can it be said definitely that the site
of action is on the ribosome. Although there are a number of agents which can
introduce errors in translation, only aminoglycoside antibiotics are known to induce
Properties of reconstituted ribosomes
30s 30s
("Ix:~n')
~('T") (~r')~
resistant sensitive resistant sensitive
Fig. 3. Properties of reconstituted ribosomes
misreading as a result of attachment to ribosomes. The evidence for this is quite clear
and comes from the finding that if ribosomal subunits from streptomycin-sensitive
and streptomycin-resistant strains are interchanged and tested in vitro, then only those
70S units which carry a 30S subunit from a sensitive parent are sensitive, no matter
what the source of the 50S subunit (Cox, WHITE and FLAKS, 1964; DAVIES, 1964);
see Fig. 3.
A. Streptomycin
The present status of work on the mechanism of action of streptomycin has been
the subject of an excellent review by JACOBY and GORINI (1967) and it suffices only to
outline the major features of the drugs' action before discussing translation errors in
more detail. In view of the fact that the bulk of the genetic and biochemical evidence
refers to the action of Sm, we shall consider other aminoglycosides separately.
The proposal that Sm can cause errors in protein synthesis derived from the
findings by GORINI and KATAJI (1964) that Sm would allow phenotypic suppression
of mutational defects in E. coli; there is now a wealth of evidence corroborating
phenotypic suppression by Sm in a number of bacterial and bacteriophage systems.
These effects of the drug on whole cells lead to a reexamination of the effect of Sm on
polypeptide synthesis in vitro, with the subsequent detection of induced errors in
translation (DAVIES, GILBERT and GORINI, 1964). Although no direct causal relation-
ship between the two has been demonstrated, there is a good correlation between the
effects of the drug on polypeptide synthesis in vitro and the effects of whole cells.
Thus, 1) Sm in low concentrations causes misreading of a large number of poly-
nucleotide-directed polypeptide synthesis in vitro, when extracts or ribosomes from
Sm" cells are used, 2) the drug concentration required for these effects in vitro is of the
same order of magnitude as that required for its bactericidal action (FLAKS, Cox and
WHITE, 1962), 3) extracts or ribosomes from Smr , and Smd cells are in general resistant
to the misreading effects of Sm *, but their sensitivity to other drugs which are not
cross-resistant with Sm is unaffected (DAVIES, GILBERT and GORINI, 1964).
To obtain more information on the way in which Sm affects reading of the code,
experiments have been carried out with different polynucleotides and the amino acid
substitutions produced by Sm have been interpreted in terms of effects on the reading
of bases in codons. The first of such experiments indicated the general nature of the
changes induced in the translation of poly U and also a non-U polymer, poly CA
(DAVIES, GILBERT and GORINI, 1964). Further, more definitive, experiments with
homopolymers allowed a more accurate description of the effects of Sm on RNA
co dons (DAVIES, GORINI and DAVIES, 1965):
1) Sm causes misreading of only one base at a time in UUU and CCC triplets, but
in AAA and III all three may be misread at the same time; misreading of A is, how-
ever, extremely low and probably not significant. If one considers I as a rather special
case, then all misreadings induced by Sm are "connected."
2) Sm causes misreading of bases in the 5' and internal positions in pyrimidine
codons; in the case of CCC the drug apparendy favors the 5' position.
3) The highly susceptible pyrimidine bases can be read as pyrimidine or purine
in the presence of Sm. A summary of these results is shown in Table 2.
We have mentioned that poly I is a special case; this base, which is highly sensitive
to Sm-induced misreading, does not normally act as a messenger, does not occur in
mRNA, and is probably not truly representative of the behavior of G. It has been
discovered recently (ANDERSON, DAVIES and DAVIS, 1967) that poly I can be made to
function as a messenger at high Mg++ concentrations, when it directs the incorpora-
* The exceptions to this statement are the extracts obtained from "competent" Sm r
strains. A low level of misreading is detected in such extracts, consistent with the finding
that competent strains are capable of phenotypic suppression by Sm (ANDERSON, GORINI
and BRECKENRIDGE, 1967).
58 JULIAN DAVIES
tion of substantial amounts of valine (GUC) and glycine (GGG); it can be said that
this polymer appears to have an elevated spontaneous level of ambiguity.
With the goal of characterising the Sm-induced changes in translation to a higher
degree of accuracy, a study of the effects of Sm on the translation of chemically
defined polydiribopolynucleotides was performed (DAVIES, JONES and KHORANA,
Table 2. Stimulation of amino acid incorporation by aminogfycoside antibiotics. (DAVIES, GORINI

and DAVIS, 1965)-
Homopolymer Amino acid Level of Codons

codon incorporated incorporationb
UUU Phenylalanine H UUU, UUC

Isoleucine I AUU,AUC
Serine I-H UCU,UCC,UCA,UCG,AGU,AGC
Tyrosine I-H UAU, UAC
Leucine L CUU, CUC, CUA, CUG, UUA, UUG
CCC Proline H CCU,CCC,CCA,CCG
Histidine H CAU,CAC
Serine H UCU, UCC, UCA, UCG, AGU, AGC
Threonine I ACU, ACC, ACA, ACG
Leucine I-H CUU, CUC, CUA, CUG, UUA, UUG
Alanine L CCU, GCC, GCA, GCG
AAA Lysine H AAA,AAG
Valine L GUU, GUC, GUA, GUG
Arginine L CGU, CGC, CGA, CGG, AGA
Aspartic acid,
glutamic acid L GAU, GAC, GAA, GAG
Glycine L GGU, GGC, GGA, GGG
III Glycine H GGU, GGC, GGA, GGG
Valine H GUU, GUC, GUA, GUG
Tyrosine H UAU, UAC
Arginine I CGU, CGC, CGA, CGG
Lysine L AAA,AAG
Serine L UCU, UCC, UCA, UCG, AGU, AGC
- These data were taken from TRUPIN et aI., with the exception of the AGA triplet for
arginine, which has been experimentally determined by H. G. KHORANA (pers. communica-
tion). Sequence in codons is 5'-terminal, internal, and 3 /-terminal. Co dons in boldface type
are connected to input codons; those in italics are co=ected with U and C interchanged in
the 3 /-terminal position.
b H, large effect of drug on incorporation; I, intermediate effect; and L, small effect.
1966). These experiments, the results of which are found in Table 3 for poly UC and
poly UG, amplify the results already obtained with homopolymers in showing some
degree of specificity in Sm-misreading. In summary, it was concluded from these more
refined experiments that:
1) Only pyrimidine bases can be misread.
2) Bases in the 5 ' -position of a coding triplet are very much more susceptible to
Sm than internal bases.
Table 3. Effect of streptomycin and neomycin on amino acid

incorporation directed by repeating dinucleotides. The top line
represents normal incorporation. (DAVIES, JONES and
KHORANA, 1966)
...... UCUCUCUCUCUC ..... .
.j, .j,
ser leu
Amino acids incorporated in the presence of:
Streptomycin Neomycin
phe (UU~) phe, pro, his

pro (CC~) arg (CG~)
[his (CA~)] ileu (AU~)
ser and leu inhibited thr (AC~)
tyr (UA~)
[gin (CA~)]
[cys (UG~)]
ser and leu inhibited
...... UGUGUGUGUGUG ..... .

.j, .j,
cys val
Amino acids incorporated in the presence of:
Streptomycin Neomycin
arg (CG~, AGa) arg, ser

[ser (AG~, UC~)] [ala (GC~)]
cys and val inhibited [gIn (CA~)]
[gly (GG~, GG~)]
[leu (UU~, CU~, CU~)]
[pro (CC~, CC~)]
[tyr (UA~)]
[his (CA~)]
cys and val inhibited
3) Pyrimidine bases in the 5' -position are misread almost exclusively as the cor-
responding pyrimidine; by contrast, internal pyrimidines can be misread as pyrimi-
dine or purine.
4) Misreading of a base is subject to a "neighbouring base effect." This is especially
true of G which can strongly restrict the misreading of an adjacent base in the same
codon; a summary of the probable misreading pattern for Sm is seen in Table 4.
Although there are some minor inconsistencies in the results obtained in these
different analyses it seems safe to conclude that, with two bases probably inert to
misreading and the existence of "neighbouring base effects," not all of 64 co dons
would be susceptible to misreading. At least half would be unaffected by Sm and the
"neighbouring base effects" would impose further restrictions (DAVIES, 1966).
60 JULIAN DAVIES
Table 4. Summary of misreading patterns induced by strepto-

mycin
5'-base Internal base 3'-base
U -+C,A U -+ C wobble
C -+ U C -+ U
A} no misreading, A) no misreading,
neighbouring neighbouring
G base effects G base effects
less marked
How does Sm cause these coding changes? Evidence has been presented to show
that the effects of Sm are expressed during the codon-anticodon interaction on the
ribosome. This was convincingly demonstrated by PESTKA, MARSHALL and NIREN-
BERG (1965) who showed that the specificity of trinucleotide-stimulated binding of
aminoacyl-tRNA to ribosomes could be upset by the presence of Sm. A poly U
template, which under normal conditions directs the strong binding of phenylalanyl-
tRNA, could stimulate the binding of isoleucyl-, leucyl- and seryl-tRNAs to Sm'
ribosomes in the presence of Sm; ribosomes from an Sm r strain showed no such
effects.
Somewhat surprising was the effect of chain length of the template on misrecogni-
tion; an oligo U chain of at least six nucleotides was required to show binding of
isoleucyl-tRNA in the presence of Sm, perhaps indicating that two tRNA binding
sites are involved in Sm action. For inhibition, as distinct from misreading, a tri-
nucleotide was sufficient and the UUU-directed binding of phenylalanyl-tRNA to
ribosomes was inhibited by Sm. This was one of the first indications that inhibition
might not be a consequence of amino acid substitution (misreading). Similar binding
experiments were later presented by KAJI and KAJI (1965).
The effect of Sm on the actual process of recognition was further dissected in
studies of polynucleotide-directed aminoacyl-tRNA binding to the 30S ribosomal
subunit, which can take place under the same conditions required for binding to
complete 70S particles. KAJI, SUZUKA and KAJI (1966) found that Sm inhibited the
binding of phenylalanyl-tRNA to 30S particles in the presence of poly U; however,
in contrast to the effects of Sm on 70S ribosomes, a marked concentration effect was
found and substantially higher Sm concentrations were required to inhibit binding
to the 30S compared to the 70S. Subunits isolated from an Smr strain were inert to
this inhibition, so there is definite relationship to genotype.
When binding of isoleucyl-tRNA to 30S subunits was studied, it was found that
Sm did not promote this binding and no misrecognition could be demonstrated on
30S subunits with Sm or high magnesium concentrations; codon recognition on 30S
subunits would thus appear to have a much higher degree of fidelity than that on 70S
ribosomes (PESTKA, 1967). When 50S subunits were added to the 30S subunits
completing the 70S ribosomal unit, both Sm and Mg++-induced misreading were
again evident.
The present notion of the ribosomal cycle during protein synthesis requires that
the 70S ribosome dissociates into 30S and 50S particles on completion with release of
polypeptide chains and reinitiation of protein synthesis involves only the 30S sub-
unit. When these 30S particles, with attached formylmethionyl-tRNA, have re-
attached to the mRNA, the 50S subunit adds to complete the 70S ribosome and pro-
tein synthesis continues (NOMURA and LOWRY, 1967; MANGTAROITI and SCHLESSINGER,
1966).
We have seen that in vitro experiments indicate different effects of Sm on the 30S
and 70S particles in protein synthesis, and although these experiments were done
with the artificial poly U system it is not unreasonable to propose that Sm might also
affect the natural ribosomal cycle in a similar way.
A likely proposal is that Sm causes one effect on protein synthesis as a result of an
interaction with free 30S subunits during the initiation process, and this may lead to
inhibition of protein synthesis. A second effect of Sm, misreading, may be the result
of an interaction of Sm with the complete 70S particle during polyribosomal poly-
peptide bond formation. It would be emphasized that these are merely suggestions,
but they should be experimentally testable. Yet another action of Sm may be re-
sponsible for the phenotypic suppression of certain chain-terminating mutations
(GORINI and DAVIES, 1968).
Several workers have found that the method of preparation of ribosomes can
affect the response of these ribosomes to Sm in polypeptide synthesis. There is a
significant difference in properties between ribosomes which have been dissociated
or sheared during purification as measured by aminoacyl-tRNA binding studies; non-
dissociated ribosomes are more susceptible to the action of Sm than are dissociated-
reassociated ribosomes (PESTKA, 1966). The differences between these two prepara-
tions are less marked when they are tested in polypeptide synthesis; the binding
assay is substantially less sensitive than peptide bond synthesis for Sm effects, so this
may only reflect on the method of assay rather than the preparation.
A more striking result of extensive ribosome and supernatant factor purification
has been revealed by LIKOVER and KURLAND (1968), these workers found that there
are two separable effects of Sm on poly U-directed polypeptide synthesis. One is
stimulation of leucine incorporation which seems to be independent of the origin of
the ribosomes, and the second is stimulation of isoleucine and serine incorporation
which requires that Sm' ribosomes be used. This latter stimulation requires the
addition of denatured DNA to the incorporation system, the DNA acting as some sort
of cofactor; such experiments cast doubt on the interpretation of the experiments
demonstrating misreading by Sm but the various Sm phenotypes are conserved in
this case. As with any of these in vitro experiments, it is rather difficult to assess these
results in terms of effects of Sm on whole cells or on polypeptide synthesis in vitro,
as the components used in such experiments are carried through various processing
procedures and generally the published experiments relate only to poly U-directed
synthesis.
Thus far, we have confined the discussion only to the effects of Sm on amino acid
incorporation directed by synthetic templates; a different situation obtains with the
use of natural mRNAs, such as bacteriophage or viral RNA. The results obtained with
such RNA species are rather more difficult to interpret in terms of a straightforward
misreading - inhibition hypothesis.
The experiments of VAN KNIPPENBERG et al. [1965 (1,2)] have shown that the
effect of Sm on the translation of viral RNAs (TMV, TYMV) in vitro is highly
dependent on the magnesium concentration. It is seen in Fig. 4 that at low Mg++
62 JULIAN DAVIES
concentration (5 mM) peptide bond synthesis is inhibited by Sm, whereas at high

Mg++ (15 mM) the drug enhances polypeptide synthesis. These authors tested for
misreading by measuring ratios of incorporation of individual amino acids over this
concentration range, and found no evidence of distortion of these ratios by Sm.
Stimulation of polypeptide synthesis by Sm at high Mg++ concentrations cannot be
explained at the moment, it could have something to do with spurious chain initiation
or release; at the lower and (presumably) more physiological Mg++ concentrations Sm
has aninhibitory effect, as is observed in whole cells. The existence of misreading in
these particular experiments cannot be decided until fingerprints of the synthesized
protein products are compared.
What might be the mechanism of inhibition produced by Sm? We have already
suggested that this may be the result of interference with initiation on 30S subunits,
Sensit ive Res istant

16000r-------------------.-------------------,
b
f-
r1\ \
r
.0'-"\
-1 /\
,
I, f
f 0 '\..~
~\\
o
""",-0
\
o
I , , I
20 28 0 12 20 28
Mg2+ concn. (pmoles/ml)
Fig. 4. Effect of Sm and Mg++ on the translation of TYMV-RNA [VAN KNIPPENBERG et ai.,
1965 (1)]
and this would explain why inhibition is not found at high Mg++ concentrations where
the normal initiation mechanism is by-passed. Alternatively, spurious release or inter-
ference with normal release would satisfactorily account for this; if peptides are not
released from polyribosomes when synthesized in the presence of Sm one would
expect to find a high proportion of "stuck 70S" particles (70S ribosomes protected
from dissociation in low Mg++ by the bound polypeptide chain) in the treated
extract and indeed this seems to be the case, as was shown by HERZOG (1964).
Experiments using RNA from RNA-containing bacteriophages lend support to
the conclusions of experiments with viral RNA; the drug inhibits peptide bond
synthesis at low Mg++ and stimulates at high Mg++ concentration (BISSELL, un-
published).
There is more suggestive evidence that Sm does cause misreading in phage RNA-
directed synthesis. SCHWARTZ (1965) has used a system in which polypeptide syn-
thesis directed by f2 RNA is limited because of the enzymatic destruction of asparagine
during the course of reaction. If Sm is added to such an incubation system it stimulates
polypeptide synthesis considerably, presumably substituting (by misreading) another
amino acid for the missing asparagine. Further work suggests that it is substitution of
lysine for asparagine in the presence of Sm which allows continued translation in this
case (SCHWARTZ, 1967).
In view of the possible artifacts arising from experiments under in vitro conditions,
it has been considered necessary to confirm that the amino acid incorporations in-
duced by Sm in vitro are in fact substitutions in the synthesized polypeptide chain.
In two independent sets of experiments it has been demonstrated by enzymatic and
chemical digestion of the peptide product that with poly V as messenger, Sm induces
the formation of a random polypeptide containing phenylalanine, leucine and iso-
leucine residues (OLD and GORINI, 1965; BODLEY and DAVIE, 1966).
B. Other Aminoglycoside Antibiotics

There are several other aminoglycoside antibiotics which have been shown to
affect translation in vitro (DAVIES, GORINI and DAVIS, 1965). These other drugs are
shown in Table 5, they differ both qualitatively and quantitatively from Sm in that
they do not induce misincorporation of the same amino
acids to the same levels. These other drugs have been Table 5. Aminoglycoside
less well studied than Sm, particularly from a genetic antibiotics which cause
point of view, mainly because of the difficulty of ob- translation errors
taining resistant bacterial mutants. Some progress has
Streptomycin
been made in studies with paromomycin which appears to Dihydrostreptomycin
be interchangable with Sm in many suppression expe- Bluensomycin
riments (GORINI, ROSSET and ZIMMERMAN, 1967); how- Kanamycin
ever, there is no reason to think that these drugs act in Neomycin (B, C)
Neamine
any way differently from Sm in causing translation errors Paromomycin
over a substantial concentration range. Table 5 shows Gentamicin
amino acid incorporations induced by neomycin com· Nebramycin
pared to Sm; the other drugs fall roughly between Hygromycin B
these two extremes. More conspicuous comparisons
can be made between these drugs by examining the effects of changes in drug
concentration on total misreading (DAVIES and DAVIS, 1968). Figs. 5 and 6 show
the effects of these drugs on poly V-directed synthesis in Sm' and Sm' extracts,
the other polymers behave in almost exactly the same way, so that the concentration
dependence curves are characteristic for each drug. Neomycin is the most potent drug
(highest level of misreading at lowest drug concentration), but is effective over a
narrow range only; gentamicin exhibits a high level of misreading throughout the
range of concentrations tested, being capable of inducing the highest levels of mis-
reading of any drug. Whether or not these effects at high concentration, inhibition in
the case of neomycin, or stimulation in the case of gentamicin and kanamycin, are due
solely to effects on codon-anticodon recognition has not yet been established. Since
the concentration dependence curves are typical for each drug it should be a simple
matter to use them to detect changes in the activity of the drugs following chemical
modification, and so provide a facile method for determining structure-activity
relationships; this avoids many of the problems of working with whole cells (DAVIES,
1968). As an example, it can be seen that replacement of an amino group by a hy-
droxyl group in a neosamine residue of neomycin, to give paramomycin, results in a
very marked change in concentration dependence curve (Fig. 6).
64 JULIAN DAVIES
Some structure-activity studies have been carried out with these drugs and by
testing the biological activity of the three sugar moieties of kanamycin in vitro,
TANAKA, MASUKA WA and UMEZAWA (1967) have concluded that the deoxystreptamine
L
0...
U
10 100 1000
Fig. 5. Misreading of poly V-directed synthesis by various amino glycoside antibiotics,
using Sm s extract. (DAVIES and DAVIS, 1968)
poly U misreading-Smr
12000
10000 -
8000
L
0...
U 6000
4000
2000 --"- _ _- - - 7 - Hen
0
-
..
_e
~
10- 6
~-------;,.-
~
10-5
Sm
10-1.
Concentration of antibiotic (M)
I I I
10 100 1000
Approximate drug: ribosome ratio
Fig. 6. Misreading of poly V-directed synthesis by various aminoglycoside antibiotics,
using Sm R extract. (DAVIES, 1967)
residue (Fig. 7) is responsible for misreading activity. This finding leads to the
proposal that streptamine or deoxystreptamine residues are required for misreading;
aminoglycoside antibiotics which do not contain these sugar residues, such as
spectinomycin and kasugamycin, do not cause misreading. However, it is clear from
Kanamycin
6-glucosamine 3-glucosamine
(Kanosamine)
OH
~------~yr------~
Deoxystreptamine
Fig. 7. The structure of kanamycin
a number of experiments that the presence of streptamine and deoxystreptamine

residues alone cannot be responsible for the action of these drugs, and other func-
tional groups in the molecule playa role in their activity (DAVIES, 1968).
C. Structllre-Activity Relationships
It should be clear from the preceding discussion that very little is known of the
chemistry of Sm action, and there would seem to be two major barriers to further
understanding of this problem. First, not enough is known of the structure of Sm in
terms of what substituents are responsible for what particular effect, binding, mis-
reading, and inhibition. Second, the site of action of the drug is the ribosome, a very
complex ribonucleoprotein with numerous specific functions, but with poorly under-
stood structure. Although some progress has been made in breaking down ribosomes
into functional units smaller than the 30S and 50S particles e.g. sensitivity and re-
sistance of Sm are properties of the 23S core of the 30S subunit (STAEHELIN and
MESELSON, 1966; TRAUB, HOSOKAw A and NOMURA, 1966) there is little or no informa-
tion which relates to the role of ribosome in codon-anticodon recognition.
To restate the facts apparent at this time, one Sm molecule when bound to the 30S
ribosomal subunit (LEON and BROCK, 1967; KAJI and TANAKA, 1068) can interfere
with codon recognition in such a way as to provoke mispairing mistakes in the
5'-terminal position (preferentially) of the codon triplet. It is not all clear why the
5'-base of each codon along a messenger RNA should be more accessible to external
perturbations; perhaps it is more exposed. Since pyrimidine to pyrimidine changes
characterize Sm misreading, the changes induced are such that U in the codon can
66 JULIAN DAVIES
now pair with G in the anticodon and C in the codon pairs with A in the anticodon;
these pairings are strongly excluded in the normal sense.
It has been pointed out by BROCK (1966) that it is not necessary to assume that Sm
exerts its effects through the mediation of the ribosome. It is equally possible that the
drug attaches to the ribosome in close proximity to the mRNA binding site and func-
tions as a result of a direct interaction on the messenger molecule. In other words,
does Sm work in a direct or allosteric fashion?
To conclude this section on the effects of aminoglycoside antibiotics, mention
should be made of their most extreme effect; promotion of the template activity of
DNA. It was discovered by MCCARTHY and HOLLAND (1965) that denatured DNA,
in the presence of aminoglycoside antibiotics can perform as a most effective messen-
ger for polypeptide synthesis in extracts. Various RNA fractions (ribosomal RNA,
tRNA) were also converted into efficient messengers in the presence of the drugs.
In many respects the template activity of DNA induced by aminoglycosides is
similar to misreading of polyribonucleotides. The drugs vary in potency, with Nm
being the most active and Sm the least active, and the intact drug molecule is required
for maximum effect. There were also found to be some differences, notably the con-
centration dependence curves, in which a simple drug: ribosome relationship for
activity was not found for Sm (MCCARTHY, HOLLAND and BUCK, 1966). Neamine, a
constituent of neomycin was found to be much more active on DNA templates than
it is on polyribonucleotide messenger molecules.
It is not clear whether or not Sm' extracts are resistant to the DNA effect; the
work of MCCARTHY, HOLLAND and BUCK (1966) includes experiments on an Sm'
strain which is sensitive to the DNA template effect, but according to the published
data extracts from this particular Sm' strain are not truly resistant when tested with
poly U in the presence and absence of Sm. MORGAN, WELLS and KHORANA (1967)
have examined the effect of aminoglycosides on the translation of chemically defined
deoxypolynucleotides and have found that in the presence of neomycin, these poly-
mers act as good templates and in most cases direct the synthesis of the copolypeptide
expected if the corresponding ribopolymer were used, without misreading. Streptomy-
cin was inactive in these experiments.
No satisfactory explanation has been offered for this phenomenon, but as it is
known that the deoxypolynucleotides will direct the binding of aminoacyl-tRNA to
ribosomes, the aminoglycosides would seem to be acting at some stage other than
recognition. It is quite possible that the drugs modify the secondary structure of the
DNA strands, thus allowing them to be bound to the ribosome in the correct con-
formation for polypeptide synthesis. A similar situation probably exists in the case of
poly I, which is a poor template for polypeptide synthesis presumably because of its
high degree of secondary structure; in the presence of aminoglycosides, however,
poly I is a very efficient template. Poly I too, can direct the binding of aminoacyl-
tRNA's.
v. Ribosome Mutations Leading to Ambiquity

There are only a few cases in which it is thought that a bacterial strain carries a
mutation in a ribosomal gene which leads to a certain degree of ambiguous transla-
tion; in none of these cases has it been proven that such a situation actually exists. The
simplest case of such ribosomal induced ambiguity would be the existence of ri-
bosomal mutations which act as nonspecific suppressor mutations; here one would
hope for fortuitous induced ambiguity to afford a reasonable level of an acceptable
amino acid substitution at a particular mutated codon in the required mRNA, without
affecting all other mRNA codons to any appreciable extent; in any proposal of efficient
suppression (.-.5%) by ambiguity it is necessary to assume a certain degree of speci-
ficity.
APIRION (1966) has described the isolation of a temperature sensitive revertant of
a tryptophan auxotroph in which reversion is presumably the result of a suppressor
mutation which acts normally at 30° but not at 43°. The temperature sensitive site
was thought to be a protein of the 50S ribosomal subunit; thus at 43° the capacity of
the ribosomes for protein synthesis would be disturbed and possibly cause translation
errors. In support of this proposal, it was found that when the suppressed temperature-
sensitive mutant was grown at 43°, the specific activity of certain enzymes was
substantially reduced. However, as APIRION points out, more convincing proof that
ribosomal suppression is taking place in this mutant is required.
Other possible candidates for ribosomal-based suppression come from the
"over-suppressed" mutants of GORINI and KATAJA (1964), and revertants of Sm-
dependence (HASHIMOTO, 1960; BROWNSTEIN and LEWANDOWSKI, 1967). In the
former case, the lesion has not definitely been mapped as a ribosome locus, and in the
latter case there is an equal possibility that the mutant is a second-site revertant and
not a true suppressor of the Smd mutation.
VI. Other Factors Causing Ambiguity in Vitro

The first hint that polynucleotide-directed polypeptide synthesis might be
ambiguous came from early work with poly V-directed synthesis; in addition to
promoting the incorporation of phenylalanine into peptide, substantial incorporation
of leucine was also detected (BRETSCHER and GRUNBERG-MANAGO, 1962; MATTHAEI
et al., 1962). The amounts of leucine were some 5 to 25% of the phenylalanine in-
corporation, so that this represents a high frequency error which seems to take place
under conditions when the in vitro incorporation system is operating at maximum
efficiency. It has now been established that one of the leucine tRNA's can respond to
both poly VA and poly V (BENNETT, GOLDSTEIN and LIPMANN, 1965), this would be
a large error if such substitution occurred in whole cells.
A. Divalent Cations
In a lucid discussion of the genetic code and its expression, WOESE (this volume)
has suggested that there is a "natural" error frequency in translation which can be
magnified in a number of ways, the leucine error mentioned above appears to be of
somewhat different character. There are a number of environmental changes which
produce errors, of which the best studied are changes in Mg++ or cation concentra-
tion, pH, temperature and the addition of organic solvents and of course, amino-
glycoside antibiotics. There are, in addition, some reagents which tend to reduce the
error frequency, changes in tRNA concentration and the addition of urea have this
property.
5*
68 JULIAN DAVIES
The effects of magnesium and cation concentration changes have been observed
by several groups of workers (DAVIES, GILBERT and GORINI, 1964; SZER and OCHOA,
1964; FRIEDMAN and WEINSTEIN, 1964; So and DAVIE, 1964). Fig. 8shows the effects
of changes in magnesium concentration on poly U directed synthesis. It can be seen
from this figure that Smr ribosomes, which are essentially resistant to the misreading
effects of Sm are susceptible to Mg++-induced ambiguity, therefore the effects of the
two agents are different. Only in the case of aminoglycoside antibiotics is the site of
action - the ribosome - definitely established. Not surprisingly, certain other
cations can also induce misreading; this is especially true of spermidine which can
8 1\\
I
I \
I \
I \
)<,
/
I \
'"
\
CD \\ /
~ \
\
ii: I
I '/
\/
\
6 I x \
<t
<t I
I \\
I \
c I \
0 l' \
\
'5 I
I
x_ SmS \
0a. I
x- - -x Sm' < Sm
x
4 I
0u I 0 - - - 0 Sm'
I
.S I 0-- - -0 Sm'. Sm
I
<I> I
.S I
u
:::J
<I>
0 2 , I
I
p-----O' ....
~ I
I
"" " ' ....
I
I
.-d
,," "
~x
~ ...
-,-,";' .,..0- ......
0
10 15 20
mM Mg<+
Fig. 8. Sm and Mg++ -induced misreading of poly U-directed synthesis in Sms and Sm R
extracts. (DAVIES, GILBERT and GORIN I, 1964)
most effectively replace magnesium as the essential divalent cation in polypeptide

synthesis. Spermine, on the other hand, tends to be quite inhibitory (FRIEDMAN and
WEINSTEIN, 1964; DAVIES, unpublished).
Misreading induced by Mg++ concentration changes has not been as extensively
studied as that produced by amino glycoside antibiotics, but in those cases which
allow comparison of the two effects, e.g. poly U-directed synthesis, the amino acids
incorporated (isoleucine, serine, tyrosine) are the same. The levels are, however,
considerably different since Sm at 10-6 M induces a 10-fold higher degree of mis-
reading than that seen when the Mg++ concentration is raised from 1.3 X 10- 2 to
2.5 X 10-2 M. Different aminoglycoside antibiotics, as mentioned before, show
different misreading spectra, which is another difference between cation and amino-
glycoside-induced misreading. Certain ambiguities in triplet recognition may be a
consequence of the elevated Mg++ concentration used (2 X 10- 2 M) in such experi-
ments (NIRENBERG et aI., 1966); and it has been suggested that a chain terminating
triplet in R17 RNA can be read through in vitro in a high magnesium ion concentra-
tion (CAPECCHI, 1967).
B. Temperature Changes
Temperature changes produce marked effects on polymer-directed polypeptide
synthesis; this is clearly demonstrated by the results of Fig. 9. As the temperature of
reaction is changed from 20° to 37° to 45° the optimum magnesium concentration
increases to 0.012, 0.016, and 0.022 M, respectively (SZER and OCHOA, 1964).
-;;
e
Cl.
6
.cI
.;:
/
-.. /"
C)
E 5
1/1
"0
E 4 Ie
/
.sc
:l..
3
.2
a(5
Cl. 2
(5
..
(.)
.S
.S
1/1
>-
...J
0 5
Fig. 9. Effect of temperature and Mg++ on poly A-directed polylysine synthesis. (SZER and
OCHOA, 1964)
An interesting effect of temperature is found in experiments with extracts of

thermophilic bacteria. When the protein synthetic activity of extracts of Bacillus
stearothermophilus is examined (FRmDMAN and WEINSTEIN, 1964) at the normal
growth temperature (65°) and at a lower temperature (37°) it is found that although
the overall activity is not greatly altered, changes in magnesium concentration at
these two temperatures markedly affect the leucine-phenylalanine ambiguity (Table 6).
As has been pointed out by SZER and OCHOA (1964), temperature effects may be most
conveniently interpreted in terms of secondary structure changes, but there is not yet
convincing evidence that this is the explanation of temperature induced ambiguity,
although reversible conformational changes in tRNA molecules are known to occur,
and to affect aminoacylation. In a similar way magnesium concentration changes can,
as a result of subtle changes in tRNA structure, completely inactivate these molecules
in their normal function (FRESCO et aI., 1966). It is not at all unlikely that magnesium
concentration or temperature changes could cause relaxation or tightening of a
ribosomal conformation which is crucial for accurate translation.
C. Organic Solvents
DAvm and his coworkers (So and DAVIE, 1964, 1965; So, BODLEY and DAvm,
1964) have examined the effects of a number of parameters on translation fidelity
70 JULIAN DAVIES
Table 6. Effects of temperature and Mg++ on leucine-phenylalanine ambi-

guiry in B. stearothermophilus extracts. (FRIEDMAN and WEINSTEIN,1964)
Temp. COc) Mg++ (M) Net poly U stimulation(f1.f1.moles)

Phe Leu Leu/phe (%)
65 0.010 42 4 10
0.018 61 10 16
37 0.006 116 3 3
0.010 170 56 32
0.018 74 87 118
Reaction mixtures contained in a 0.4 ml volume the following
components (in f1.moles unless otherwise specified): Tris IICl buffer,
pH 7.8, 4.0; potassium chloride, 24.0; ,B-mercaptoethanol, 2.4;
ATP, 0.25; GTP, 0.01; CTP, 0.01; UTP, 0.01; phosphoenolpyru-
vate, 1.25; phosphoenolpyruvate kinase, 12 f1.g; a mixture of 02-L-
amino acids,4 excluding the radioactive amino acid, 0.0125 of each;
the indicated 04-L-amino acid, 0.5 to 1.2 mf1.moles; and the S-30
fraction of B. stearothermophilus (approximately 1.4 mg protein and
0.8 mg RNA). Magnesium acetate was added at the final concentra-
tions indicated in the table. The mixtures were incubated at the speci-
fied temperature for 20 min in the absence and presence of 100 f1.g
of polyuridylic acid. Reactions were stopped with TCA, heated at
85 DC for 30 min, washed by membrane filtration, and assayed for
radioactivity as previously described. 5 The specific activities of the
04-L-amino acids, obtained from Schwarz BioResearch, Inc., were
in the range of 850 to 1000 (Joc/mg. Values represent net polymer
stimulations (see Methods). In the absence of poly U, the incorpora-
tion of phenylalanine and leucine was in the range of 2 to 6 and
4 to 10 (Jof1.moles, respectively.
in vitro; the most dramatic changes were induced by the addition of organic solvents
to the incorporating system. It was found that the addition of methanol, ethanol,
2-propanol, acetone, dioxane, or ethylene glycol in concentrations between 0.2 to
1.0 M markedly stimulated poly U-directed phenylalanine incorporation; poly C-
directed proline incorporation was enhanced to an even greater degree. Initial studies
on the phenylalanine-leucine ambiguity indicated that this ratio was altered with
increase in alcohol concentration; over the higher range of alcohol concentrations,
incorporation of isoleucine was also noticeable (Fig. 10). In like manner, leucine and
threonine incorporation were incorporated when poly C was used as messenger.
It is not possible to compare these effects directly with those of Sm, as there is no
report of other ambiguities induced by alcohol addition; Sm, with poly C, induces the
incorporation of serine, histidine, threonine, and leucine in addition to stimulating
proline. The effects of Sm and alcohol were found to be more than additive and
massive ambiguity levels were introduced when both agents were used simultaneously.
In addition to studies of organic solvent-induced misreading, these workers con-
firmed the effects of Mg++ concentration changes and tRNA concentration changes
on translation. The latter tends to nullify induced translation errors; the effect of urea
(more polar than water and alcohol) on the translation of homopolymers was also
studied and this compound also reduces the poly U-induced leucine and isoleucine
ambiguity.
So and DAVIE (1965) concluded their studies on the rather complex interrelation-
ships between polar and nonpolar agents and magnesium ion concentration by
suggesting that induced ambiguity may be caused by tautomerism of uracil residues
in the RNA moieties involved in polypeptide synthesis; at this time there is no con-
vincing experimental evidence in support of this notion. SARIN and ZAMECNIK (1965)
have found that organic solvents do affect the amino acid esterification process and in
addition, ethylene glycol for example, has a marked effect on the conformation of RNA
and ribosomes as measured by optical rotatory dispersion. It seems safe to assume that
organic solvents may influence several components of the in vitro incorporation
system to induce ambiguity.
4200r-----------------,
c
c 'Qj
a
e D.. 3000
.2
8..-0
6 E
u 0
c VI
;~
U
o 01
E
co __
.- VI
Ji"*E
::l..
2-
o 2.56
Ethanol conc. (M)
Fig. 10. Effect of ethanol on poly V-directed synthesis. (So and DAVIE, 1964)
Yet another environmental change which affects translation fidelity is a change in

pH (GRUNBERG-MANAGO and DONDoN, 1965); in general an increase in pH from
6.5 to 8 decreases poly V-directed phenylalanine formation and increases ambiguous
incorporation (leucine, isoleucine, and serine). As with other examples of agents
inducing ambiguity there is no experimental evidence which might suggest a mecha-
nism for this type of change. GRUNBERG-MANAGO and DONDoN think that both pH
and Mg++ concentration changes might decrease the stability of the ribosome-tRNA-
messenger complex such that faulty tRNA insertions can be made.
In contrast to the agents that we have discussed so far, when excessive amounts
of tRNA are put into an in vitro incorporating system the level of ambiguity is re-
duced rather than enhanced. A number of reports of this effect (DAVIES, GORINI and
DAVIS, 1965; PESTKA, MARSHALL and NIRENBERG, 1965; So and DAVIE, 1965;
GRUNBERG-MANAGO and DONDoN, 1965) have been made and excess tRNA reduces
the ambiguity induced by base analogue-containing polymers, Sm, alcohols and
magnesium. It is perhaps interesting that excess tRNA reduces these errors whereas
it does not seem to reduce the "natural" leucine error; this is consistent with the
proposal by LIKOVER and KURLAND (1968), that the leucine ambiguity is a pheno-
menon separate and distinct from the poly V induced-isoleucine and serine errors.
72 JULIAN DAVIES
Several proposals have been aired to explain the corrective effect of tRNA, it seems
most likely that it is a mass action effect. If one considers the phenylalanine-isoleucine
ambiguity, it can be assumed that under certain conditions (those which lead to
ambiguity) ileu-tRNA can compete effectively with the phe-tRNA for the ribosomal
binding site; the higher affinity of the phe-tRNA for the site would tend to reduce
this competition when high concentrations of phe-tRNA are present.
VII. Does Misreading Occur in Mammalian Systems?

We have concerned ourselves, up to this point, solely with translation errors in
bacterial incorporation systems and it is of interest to consider whether such errors
can occur in mammalian systems, which have somewhat different features (80S
rather than 70S ribosomes, etc.). This problem has been addressed by WEINSTEIN
and coworkers (WEINSTEIN, FRIEDMAN and OCHOA, 1966), who have concluded that
mammalian systems are less susceptible to the fluctuations which can be induced in
bacterial systems; however, earlier experiments with incorporating systems from
several mammalian sources indicated that the phenylalanine-leucine ambiguity was as
pronounced as in bacterial extracts (WEINSTEIN, OCHOA and FRIEDMAN, 1966).
Further experiments with these systems have led WEINSTEIN, FRIEDMAN and OCHOA
to conclude that Mg++ concentration changes, or the addition of Sm, polyamines, or
alcohol have little or no effect on the translation of poly U in rat liver, or rabbit
reticulocyte extracts. Experiments using heterologous systems of aminoacyl-tRNAs
from mammalian sources and ribosomes from bacteria and vice versa indicated that the
ribosomes from mammalian sources were responsible for the increased fidelity in such
systems. Aminoacyl-tRNA's from mammalian systems, when used in a protein
synthetic system with E. coli ribosomes lead to a high phenylalanine/leucine ambi-
guity, whereas when homologous mammalian ribosomes were used, little or no
ambiguity was detected. Unfortunately, experiments on induced ambiguity in these
mixed systems were somewhat unclear as Sm did not introduce the normal high level
of ambiguity when bacterial ribosomes were used.
In contrast to these findings are the experiments of LAMFROM and GRUNBERG-
MANAGO (1967) with a more purified incorporation system derived from rabbit
reticulocytes in which Mg++ concentration and pH changes did produce substantial
ambiguities. Leucine and isoleucine misreading increased as the Mg++ concentration
was raised, and at 0.015 M Mg++ leucine was incorporated to 40% of the phenyl-
alanine level. The leucine ambiguity of poly U was enhanced below pH 7.8 in this
purified cell-free system from reticulocytes. On the other hand, in agreement with
WEINSTEIN, FRIEDMAN and OCHOA (1966), streptomycin had very little effect on
translation fidelity.
WEINSTEIN, FRIEDMAN and OCHOA (1966) have extended their experiments to
examine the fidelity of protein synthesis in extracts of mammalian cells when directed
by native messenger RNA and under these circumstances the fidelity of the system
appears to be lower. Thus, lowering the temperature from 37° to 20° and raising the
Mg++ concentration from 0.005 to 0.008 M produced a lowering of the ratio of
leucine to phenylalanine incorporated into protein from 136% to 65%. Spermine,
spermidine, ethanol, raised Mg++ concentration, and Sm produced similar changes on
this endogenous mRNA-directed reaction. As these agents all caused general inhibi-
tion of protein synthesis it is perhaps more correct to say that, in general, leucine
incorporation was more strongly inhibited than phenylalanine incorporation.
Whether or not this genuine misreading as a result of amino acid substitution cannot
be said at present; it is clear however that misreading in mammalian systems has
characteristics different from that in bacteria.
VIII. Does Misreading Occur in Living Cells ?

In bacteria, only circumstantial evidence supports the notion that translation
errors can be induced in whole cells:
1) Phenotypic suppression by aminoglycoside antibiotics (GORINI and KATAJA,
1964) and alcohol (GADO and HORVATH, 1963) is presumed to occur by a misreading
mechanism. There is a strong correlation between the effects observed in vitro and in
whole cells, in terms of effective concentrations, resistance and permissiveness, e.g.
different spectra of misreading in extracts can be taken to suggest that the agents will
differ in their ability to suppress certain mutations in cells (GORINI and KATAJA,
1965).
2) The production of immunologically cross reacting material (CRM). If the
amino glycoside antibiotics produce misreading in cells, one would expect that in a
drug-treated cell that loss of a particular enzyme activity be coincident with the pro-
duction of an enzymatically inactive CRM - this has been demonstrated in Sm and
Nm treated E. coli (BISSELL, 1965).
3) Other evidence of misreading, faulty amino acid substitution, comes from the
finding that in many cases the enzyme activity restored to a mutant by phenotypic
suppression with aminoglycosides is temperature sensitive (GORINI, unpublished;
DAVIES, unpublished). This suggests that the amino acid replacement, although
sufficient for part restoration of enzyme activity has given rise to an enzyme with
altered tertiary structure.
It is obvious that this "evidence" is far from convincing and demonstration of
altered primary protein sequence by biochemical methods is awaited. Experiments in
which bacterial amino acid auxotrophs are killed by "forced" feeding of amino acids
have been quoted as examples of translation errors (KINDLER and BEN-GURION,
1965). In one such experiment a phe- his- pro- strain of E. coli K12 was starved of
phenylalanine in the presence of large amounts of leucine, which resulted in a signi-
ficant loss of viability; the addition of phenylalanine reversed this effect. No further
details have been made available on this experiment and in view of the complicated
regulatory controls existing in bacteria it would not be surprising if this effect was
the result of impaired regulation rather than errors in translation.
In mammalian cells the situation seems to be reversed since there is evidence for
(possible) translation errors in the primary sequence of a number of proteins, but
there is no suggestion as to how these replacements took place. One of the most
fascinating problems of recent years has been the finding by VON EHRENSTEIN (1966)
and by RIFKIN et al. (1966) of amino acid multiplicities in certain positions in the
sequence of hemoglobins. Thus in the (X-chain of rabbit hemoglobin there are at least
six positions for which more than one amino acid has been found, so far only neutral
amino acids are involved (VON EHRENSTEIN, 1966). Table 7 shows the substitutions
and the corresponding co dons, and it can be seen that several of the substitutions can
74 JULIAN DAVIES
be explained by a single base change, which may occur most readily by errors in
translation. This notion is strengthened by the finding of all four possible sequence
permutations involving positions 48 to 49 in a single individual; this is however not
completely random as there are certain restrictions on this sequence.
These results, and those of RIFKIN et al. (1966) have not been fully explained to
date. The ambiguities may be the result of changes at the chromosome level but a
more likely explanation would be that there are certain minor, but specific adapters
(tRNA or amino acyl synthetases) which serve only certain positions in the poly-
peptide chain. Further results on this problem are eagerly awaited.
Another problem involving ambiguity or heterogeneity in a polypeptide chain
concerns the immunoglobulins. This problem, in its present state, appears to be an
order of magnitude more difficult than any others that we have so far discussed. It is
now well established that there is enor-
Table 7. Amino acid multipli- mous heterogeneity in the sequence of the
citie.r in the a-chain of rabbit 107 amino acids in the NH2-terminal
hemoglobin. (VON EHRENSTEIN,
1966) half of the light chain (Bence-Jones pro-
tein) of immunoglobulins; variations have
Position Amino acids
been found in at least 40 of these posi-
17 tions. In some of these cases 4 or 5 dif-
{ val (GUX)
leu (CUX) ferent amino acids have been found to
48 { leu (CUX) occupy the variable site. The heavy chains
phe (UUX) of immunoglobulins seem to have a sim-
49 { ser (UCX) ilar variable section; these sections of
thr (ACX)
70 variability are probably involved in the
{ val (GUX)
thr (ACX) many different antibody combining sites
76 { leu (CUX) and a consideration of their derivation
val (GUX) and function is of great importance in
80 { ser (UCX) understanding the antibody-antigen re-
leu (UUX)
action. There are a number of theories
which have been put forward to explain this extraordinary variability in protein
structure, none of these theories has yet been put to the test (LENNOX and COHN,
1965). One of these ideas, proposed by POTTER, ApPELLA and GEISSER (1965),
suggests that the variation arises from errors in the translation process. Thus it is
assumed that the positions of variation are characterized by unusual coding triplets
which can recognize a number of special aminoacyl-tRNA molecules. The tRNA
molecules involved have to be capable of accepting several different amino acids
(Le. recognizing several aminoacyl adenylate-enzyme complexes), but can recognize
only the special codons in the variable positions.
IX. Finale
The agents which are known to induce translation errors are listed in Table 8.
We conclude this chapter by asking a number of questions:
1) What is the chemical mechanism for the bringing about of translation errors
in vitro?
2) Do translation errors occur in living cells?
3) If translation errors occur in cells, do they have any biochemical significance,

or are they just "noise?"
None of these questions have been satisfactorily answered.
Table 8. Factors influencing translation fidelity

in vitro
Cations (K+, NH+4 , Mg++)

Polyamines (spermidine)
Aminoglycosides (streptomycin)
Urea
pH changes
Temperature changes
Organic solvents (ethanol)
Changes in sRNA or amino acid concentration
Abbreviations
Sm, streptomycin; Nm, neomycin; Km, kanamycin; Hm, hygromycin; Gm,
gentamicin; Pm, paromomycin; poly D, polyuridylate; poly C, polycytidylate;
mRNA, messenger ribonucleic acid; tRNA, transfer ribonucleic acid; A, adenine;
G, guanine; C, cytidine; D, uracil; I, inosine; bromo D, bromouracil; bromo C,
bromocytidine; poly DC, repeating copolymer of D and C; poly UG, repeating
copolymer of U and G; su, suppressor; TYMV; tobacco yellow mosaic virus.
Addendum in Proof
Transfer RNAs with altered charging or coding characteristics have been obtained
by both physiological and chemical means. Thus bacteriophage infection alters the
coding properties of some tRNA classes and it is thought that these modifications
are responsible for changes in protein synthesis following phage infection (SUEOKA
et al., 1966; NIRENBERG et al., 1966). In addition, modified phenylalanine tRNA's
have been isolated from E. coli following iron starvation, these modified species seem
to have altered acceptor activity (WETTSTEIN and STENT, 1968).
It has been possible to isolate tRNA with 100% replacement of uracil by 5 fluor-
ouracil, following growth of cells in the antimetabolite. Such tRNA has an altered
secondary structure as determined by thermal denaturation experiments, and appears
to accept amino acids normally. When used in in vitro polypeptide synthesis, FU-
containing lysyl-tRNA responded normally to poly A but was not functional in
polypeptide synthesis when directed by the phage messenger R17 RNA. It is thought
that this may be due to ambiguous pairing (LOWRIE and BERGQUIST, 1968; GIEGE
et al., 1969). Experiments with a missense suppressor tRNA (CARBON and CURRY,
1968) have shown that this modified tRNA, when obtained from su + strains or when
derived chemically does have an altered capacity for accepting its cognate amino acid.
If this missense tRNA is produced by a single base change in the anticodon, it would
appear that there is some relationship in tRNA between the recognition site for
aminoacylation and the anticodon.
Three forms of SUitI tyrosine tRNA have been isolated and found to differ in
modification of a adjacent to the anticodon (GEFTER and RUSSELL, 1969). These
forms do not differ with respect to charging properties but do differ markedly in
76 JULIAN DAVIES
their abilities to function in polypeptide synthesis. The latter differences have been
shown to be due to differences in the abilities of these species to bind to ribosomes
in the presence of trinucleotides. Thus, alterations in bases outside of the anticodon
can presumably affect codon-anticodon pairing.
The effects of ultraviolet light on translational characteristics of messenger RNA
have been studied by OTTENSMEYER and WHITMORE [1968 (1,2)] who have shown
that uracil dimer can be read either as guanine-uracil or uracil-uracil, and that uracil
hydrate (as previously shown by GROSSMAN, 1963) can be read as cytosine. By
examining the products obtained in polypetide synthesis directed by irradiated U-
containing polynucleotides, it was concluded that uracil hydrate is more likely to be
read as cytosine when in the 5'-, than when in the internal position of a codon.
Based on these findings, OTTENSMEYER and WHITMORE predicted that the amber
nonsense codon might be read as CAG following conversion of the 5'-U to the
hydrate by UV irradiation, and they have presented evidence for UV-induced pheno-
typic reversion of an amber mutation in vivo.
Two addition papers pertain to translational errors due to altered ribosomes.
GAUSE and GRUNBERGER (1968) have compared the properties of the ribosomes
obtained from B. paracoli (48% GC) and a mutant strain of this organism with a
70% GC content. As judged by the translation of poly U, the two ribosome pre-
parations were identical except for small differences in response to various antibiotics.
In addition, the mutant ribosomes showed a reduced capacity to recognize valyl-tRNA
in response to the GUA codon.
A genetic determinant for ribosomal ambiguity has been identified by ROSSET
and GORINI (1969). The genetic locus for this character (ram locus) maps near
streptomycin and spectinomycin resistance and has been shown to determine a com-
ponent of the 30S subunit of the ribosome. Ribosomes from a ram mutant show an
elevated level of translation errors in response to polynucleotide messengers in vitro,
and a strain carrying a ram mutation is capable of suppressing nonsense mutants of
phage T4. These mutants would appear to be the first true representatives of a class
of non-specific ribosomal suppressors.
A ribosomal preparation from rabbit spleen has been tested for translational
ambiguity in poly U- and poly UC-directed synthesis (STAVY, 1968), and varied
magnesium ion concentrations were found to give different ratios of leucine and
phenylalanine incorporated into polypeptide. Although magnesium ion induced
misreading, the system was inert to the effects of streptomycin or organic solvents.
Although this result was considered to support the possibility that immunoglobulin
variations could be due to translational errors, additional experiments on hemoglobin
variations tend to suggest that translational ambiguity is not responsible for the
amino acid changes in hemoglobin lX-chains. BILSE and Popp (1968) have separated
(by solubility differences) different forms of the lX-chain from the same source, and
propose that these discrete chains would arise by gene duplication.
A recent review summarizes the present state of knowledge on antibiotics (includ-
ing aminoglycosides) which affect ribosomes (WEISBLUM and DAVIES, 1969) and the
various stages of protein synthesis.
References
ANDERSON, P., J. DAVIES, and B. D. DAVIS: Effect of spectinomycin on polypeptide syn-
thesis in extracts of Escherichia coli. J. molec. BioI. 29, 203 (1967).
ANDERSON, W. F., L. GORINI, and L. BRECKENRIDGE: Role of ribosomes in streptomycin-
activated suppression. Proc. nat. Acad. Sci. (Wash.) 54, 1076 (1965).
ASPIRION, D.: Altered ribosomes in a suppressor strain of Escherichia coli. J. molec. BioI. 16,
285 (1966).
ARCA, M., L. FRONTALI, and G. TECCE: Lack of specificity in the formation of aminoacyl-
sRNA as a possible source of coding errors. Biochim. biophys. Acta (Amst.) 108, 326
(1965).
- , C. CALVORI, L. FRONTALI, and G. TECCE: Thermal denaturation and amino acid binding
ability of soluble RNA. Biochem. biophys. Res. Commun. 10, 117 (1963).
- - - - The enzymic synthesis of aminoacyl derivatives of soluble ribonucleic acid from
Bacillus stearothermophilus. Biochim. biophys. Acta (Amst.) 87, 440 (1964).
ATl'ARDI, G.: The mechanism of protein synthesis. Ann. Rev. Microbiol. 21, 383 (1967).
BALDWIN, A. N., and P. BERG: Transfer ribonucleic acid-induced hydrolysis of valyladenylate
bound to isoleucyl ribonucleic acid synthetase. J. bioI. Chem. 241, 839 (1966).
BAKES, J., N. BEFORT, J. H. WElL, and J. P. EBEL: Modification of the aminoacid code after
bromination of transfer RNA. Biochem. biophys. Res. Commun. 19, 84 (1965).
BARNETl', W. E.: Interspecies aminoacyl-sRNA formation: fractionation of Neurospora
enzymes involved in anomalous aminocaylation. Proc. nat. Acad. Sci. (Wash.) 53, 1462
(1965).
- , and D. H. BROWN: Mitochondrial transfer ribonucleic acids. Proc. nat. Acad. Sci. (Wash.)
57,452 (1967).
- , and J. L. EPLER: Fractionation and specificities of two aspartyl-ribonucleic acid and two
phenylalanyl-ribonucleic acid synthetases. Proc. nat. Acad. Sci. (Wash.) 55, 184 (1966).
-, and K. B. JACOBSON: Evidence for degeneracy and ambiguity in interspecies aminoacyl-
sRNA formation. Proc. nat. Acad. Sci. (Wash.) 51, 642 (1964).
- , D. H. BROWN, and J. L. EPLER: Mitochondrial specific aminoacyl-RNA synthetases.
Proc. nat. Acad. Sci. (Wash.) 57, 1775 (1967).
BENNETl', T. P., J. GOLDSTEIN, and F. LIPMANN: Coding and charging specificities ofsRNA's
isolated by countercurrent distribution. Proc. nat. Acad. Sci. (Wash.) 53, 385 (1965).
BERGMANN, F. H., P. BERG, and M. DIECKMANN: The enzymic synthesis of amino acyl
derivatives of ribonucleic acid. J. bioI. Chem. 236, 1735 (1961).
BISSELL, D. M.: Formation of an altered enzyme by Escherichia coli in the presence of neomy-
cin. J. molec. BioI. 14,619 (1965).
BODLEY, J. W., and E. W. DAVIE: A study of the mechanism of ambiguous amino acid
coding by poly U: the nature of the products. J. molec. BioI. 18, 344 (1966).
BRETSCHER, M. S., and M. GRUNBERG-MANAGO: Polyribonucleotide-directed protein
synthesis using an E. coli cell-free system. Nature (Lond.) 195,283 (1962).
BROCK, T. D.: Streptomycin, in Biochemical studies of antimicrobial drugs. Sixteenth
symposium Soc. Gen. Microbiol, p. 131. New York: Cambridge Univ. Press 1966.
BROWNSTEIN, B., and L. J. LEWANDOWSKI: A mutation suppressing streptomycin depend-
ence. I. An effect on ribosome function. J. molec. BioI. 25, 99 (1967).
BUJARD, H., and C. HEIDELBERGER: Flourinated pyrimidines XXVII. Attempts to determine
transcription errors during formation of fluorouracil containing messenger ribonucleic
acid. Biochemistry 5, 3339 (1966).
BUSSARD, A., S. NAONO, F. GROS et J. MONOD: Effets d'un analogue de l'uracil sur les
proprietes d'une proteine enzymatique synthetisee en sa presence. C. R. Acad. Sci.
(Paris) 250, 4049 (1960).
CAPECCHI, M. R.: Polarity in vitro. J. molec. BioI. 30, 213 (1967).
CARBON, J., and J. B. CURRY. Genetically and chemically derived missense suppressor transfer
RNA's with altered enzymic aminoacylation rates. J. Mol. BioI. 38, 201 (1968).
CHAMPE, S. P., and S. BENZER: Reversal of mutant phenotypes by 5-fluorouracil: an ap-
proach to nucleotide sequences in messenger-RNA. Proc. nat. Acad. Sci. (Wash.) 48,
532 (1962).
78 JULIAN DAVIES
Cox, E. c., J. R. WHITE, and J. G. FLAKS: Streptomycin action and the ribosome. Proc. nat.
Acad. Sci. (Wash.) 51, 703 (1964).
DAVIES, J.: Studies on the ribosomes of streptomycin-sensitive and resistant strains of
Escherichia coli. Proc. nat. Acad. Sci. (Wash.) 51, 659 (1964).
- Streptomycin and the genetic code. Cold Spr. Harb. Symp. quant. BioI. 31, 665
(1966).
- Structure-activity relationships among aminoglycoside antibiotics. Antimicrobial agents
and chemotherapy, 1967,297.
- , and B. D. DAVIS: Misreading of RNA codewords induced by aminoglycoside anti-
biotics: the effect of drug concentration. J. bioI. Chern. 243, 3312 (1968).
- , W. GILBERT, and L. GORINI: Streptomycin, suppression, and the code. Proc. nat. Acad.
Sci. (Wash.) 51, 883 (1964).
-, L. GORINI, and B. D. DAVIS: Misreading of RNA codewords induced by aminoglycoside
antibiotics. Mol. Pharmacol. 1, 93 (1965).
- , D. S. JONES, and H. G. KHORANA: A further study of misreading of codons induced by
streptomycin and neomycin using ribopolynucleotides containing two nucleotides in
alternating sequence as templates. J. molec. BioI. 18,48 (1966).
EHRENSTEIN, G. VON: Translational variations in the amino acid sequence of the ",-chain
of rabbit hemoglobin. Cold Spr. Harb. Symp. quant. BioI. 31, 705 (1966).
FANGMAN, W. L., and F. C. NEIDHARDT: Demonstration of an altered aminoacyl ribo-
nucleic acid synthetase in a mutant of Escherichia coli. J. bioI. Chern. 239, 1839 (1964).
FLAKS, J. G., E. C. Cox, and J. R. WHITE: Inhibition of polypeptide synthesis by strepto-
mycin. Biochem. biophys. Res. Commun. 7, 385 (1962).
FRESCO, J. R., A. ADAMS, R. ASCIONE, D. HENLEY, and T. LINDAHL: Tertiary structure in
transfer RNAs. Cold Spr. Harb. Symp. quant. BioI. 31, 527 (1966).
FRIEDMAN, S. M., and 1. B. WEINSTEIN: Lack of fidelity in the translation of synthetic
polyribonucleotides. Proc. nat. Acad. Sci. (Wash.) 52, 988 (1964).
- - Protein synthesis in a subcellular system for Bacillus stearothermophilus. Biochim.
biophys. Acta (Arnst.) 114, 593 (1966).
GADO, 1., and 1. HORVATH: The effect of methanol on the growth of a streptomycin-depend-
ent strain of Escherichia coli in streptomycin-free media. Life Sci. 10, 741 (1963).
GAUSE, G. G., and D. GRUNBERGER: Translational characteristics of ribosomes and coding
properties of transfer RNA in the mutant of bacterium paracoli with the increased GC
content of DNA. Biochim. biophys. Acta (Arnst.) 166, 538 (1968).
GEFTER, M. L., and R. RUSSELL: Role of modifications in tyrosine transfer RNA: a modified
base affecting ribosome binding. J. Mol. BioI. 39, 145 (1969).
GIEGE, R., J. HEINRICH, J.-H. WElL, and J.-P. EBEL: Etude de proprietes biologiques des
acides ribonucIeiques de transfert de levure ayant incorpore du 5-fluorouracile. Biochim.
GORINI, L., and E. KATAJA: Phenotypic repair by streptomycin of defective genotypes in
E. coli. Proc. nat. Acad. Sci. (Wash.) 51, 487 (1964).
- - Streptomycin-induced oversuppression in E. coli. Proc. nat. Acad. Sci. (Wash.) 51,
995 (1964).
- - Suppression activated by streptomycin and related antibiotics in drug sensitive
strains. Biochem. biophys. Res. Commun. 18, 656 (1965).
- , and J. DAVIES: The effect of streptomycin on ribosomal function. In: Current Topics in
Microbiology and Immunology, Vol. 44, p. 100. Berlin-Heidelberg-New York: Springer
1968.
- , R. ROSSET, and R. A. ZIMMERMANN: Phenotypic masking and streptomycin dependence.
Science 157, 1314 (1967).
GROSSMANN, L.: The effects of ultraviolet-irradiated polyuridylic acid in cell-free protein
synthesis in E. coli. Proc. nat. Acad. Sci. (Wash.) 48, 1609 (1962).
- The effects of ultraviolet-irradiated polyuridylic acid in cell-free protein synthesis in
Escherichia coli. II. The influence of specific photoproducts. Proc. nat. Acad. Sci. (Wash.)
50, 657 (1963).
GRUNBERG-MANAGO, M., and J. DONDON: Influence of pH and sRNA concentration on
coding ambiguities. Biochem. biophys. Res. Commun. 18,517 (1965).
- , and A. M. MICHELSON: (1) Polynucleotide analogues. II. Stimulation of amino acid

incorporation by polynucleotide analogues. Biochim. biophys. Acta (Amst.) 80, 431
(1964).
- - (2) Polynucleotide analogues. IV. Polyfluorouridylic acid and co-polymers containing
fluorouridylic acid. Biochim. biophys. Acta (Amst.) 87, 593 (1964).
HASHIMOTO, K.: Streptomycin resistance in Escherichia coli analyzed by transduction.
Genetics 45, 49 (1960).
HAYASHI, H., and K. MIURA: Anticodon sequence as a possible site for the activity of
transfer RNA. Cold Spr. Harb. Symp. quant. BioI. 31, 63 (1966).
HERZOG, A.: An effect of streptomycin on the dissociation of Escherichia coli 70S ribosomes.
Biochem. biophys. Res. Commun. 15, 172 (1964).
HILSE, K., and R. A. Popp: Gene duplication as the basis for amino acid ambiguity in the
alpha-chain polypeptides of mouse hemoglobins. Proc. NatI. Acad. Sci. (Wash.) 61,
930 (1968).
IGARASHI, K., and A. KAJI: On the nature of two ribosomal sites for specific sRNA binding.
IMAMOTO, F., T. YAMANE, and N. SUEOKA: Existence of two phenylalanyl-sRNA synthe-
tases in Neurospora crassa. Proc. nat. Acad. Sci. (Wash.) 53, 1456 (1965).
JACOBY, G. A., and L. GORINI: The effect of streptomycin and other aminoglycoside
antibiotics on protein synthesis. Antibiotics. Vol. 1, p. 726. Berlin-Heidelberg-New
York: Springer 1967.
KAJI, H., and A. KAJI: Specific binding of sRNA to ribosomes: effect of streptomycin.
- , 1. SUZUKA, and A KAJI: Binding of specific soluble ribonucleic acid to ribosome~.
Binding of soluble ribonucleic acid to the template-30S subunit complex. J. bioI. Chem.
241, 1251 (1966).
- , and Y. TANAKA: Binding of dihydrostreptomycin to ribosomal subunits. J. molec. BioI.
32,221 (1968).
KINDLER, S. H., and R. BEN-GURIaN: Lethal effect ofL-leucine on E. coli, in vivo corrobora-
tion of coding ambiguity. Biochem. biophys. Res. Commun. 18, 337 (1965).
KNIPPENBERG, P. H. VAN, J. C. VAN RAVENSWAAY CLAASEN, M. GRIJM-VOS, H. VELDSTRA,
and L. BOSCH: (1) Stimulation and inhibition of polypeptide synthesis by streptomycin
in ribosomal systems of Escherichia coli, programmed with various messengers. Biochim.
biophys. Acta (Amst.) 95, 461 (1965).
- , M. GRIJM-VOS, H. VELDSTRA, and L. BOSCH: (2) Effects of streptomycin on the transla-
tion of turnip yellow mosaic virus RNA in vitro. Biochem. biophys. Res. Commun. 20,
4 (1965).
LAM FROM, H., and M. GRUNBERG-MANAGO: Ambiguities of translation of poly U in the
rabbit reticulocyte system. Biochem. biophys. Res. Commun. 27, 1 (1967).
LANDY, A., J. ABELSON, H. M. GOODMAN, and J. D. SMITH: Specific hybridization of tyrosine
transfer nucleic acids with DNA from a transducing bacteriophage 1> 80 carrying the
amber suppressor gene SUm. J. molec. BioI. 29, 457 (1967).
LENNOX, E. S., and M. COHN: Immunoglobulins. Ann. Rev. Biochem. 36, 647 (1967).
LEON, S. A., and T. D. BROCK: Effect of streptomycin and neomycin on physical properties
of the ribosome. J. molec. BioI. 24, 391 (1967).
LETENDRE, c., A. M. MICHELSON, and M. GRUNBERG-MANAGO: Oligonucleotide inhibition
of amino acid attachment. Cold Spr. Harb. Symp. quant. BioI. 31, 71 (1966).
LIKOVER, T. E., and C. G. KURLAND: The contribution of DNA to translation errors
induced by streptomycin in vitro. Proc. nat. Acad. Sci. (Wash.) 58, 2385 (1967).
LITTAUER, U. Z., M. REVEL, and R. STERN: Coding properties of methyl-deficient phenyl-
alanine transfer RNA. Cold Spr. Harb. Symp. quant. BioI. 31, 501 (1966).
LOFTFIELD, R. B.: The frequency of errors in protein biosynthesis. Biochem. J. 89, 82
(1963).
LOWRIE, R. ]., and P. L. BERGQUIST: Transfer ribonucleic acids from Escherichia coli treated
with 5-fluorouraciI. Biochemistry 7,1761 (1968).
MANDEL, L. R., and E. BOREK: The nature of the RNA synthesized during conditions of
unbalanced growth in E. coli K 12W6. Biochemistry 2,560 (1963).
80 JULIAN DAVIES
MANGIAROTTI, G., and D. SCHLESSINGER: Polyribosome metabolism in Escherichia coli.

I. Extraction of polyribosomes and ribosomal subunits from fragile, growing Escherichia
coli. J. molec. BioI. 20, 123 (1966).
MATIHAEI, J. H., O. W. JONES, R. G. MARTIN, and M. W. NIRENBERG: Characteristics and
composition of RNA coding units. Proc. nat. Acad. Sci. (Wash.) 48, 666 (1962).
-, F. AMELUNXEN, K. ECKERT und G. HELLER: Zum Mechanismus der Proteinbiosynthese.
I. Die Bindung von matrizen-RNA und aminoacyl-RNS an Ribosomen. Ber. Bunsenges.
Physikal. Chemie. 68, 735 (1964).
MCCARTHY, B. J., and J. J. HOLLAND: Denatured DNA as a direct template for in vilro
protein synthesis. Proc. nat. Acad. Sci. (Wash.) 54, 880 (1965).
- - , and C. A. BUCK: Single-stranded DNA as a template for in vilro protein synthesis.
Cold Spr. Harb. Symp. quant. BioI. 31, 683 (1966).
MIURA, K.-I.: Specificity in the structure of transfer RNA. Progr. in Nucl. Acid Res. and
Molec. BioI. 6, 39 (1967).
MORGAN, A. R., R. D. WELLS, and H. G. KHORANA: Studies on polynucleotides LXXIV.
Direct translation in vivo of single-stranded DNA-like polymers with repeating nucleotide
sequences in the presence of neomycin B. J. molec. BioI. 26, 477 (1967).
NAKADA, D., and B. MAGASANIK: The roles of inducer and catabolite repressor in the syn-
thesis of ,B-galactosidase by E. coli. J. molec. BioI. 8, 105 (1964).
NEIDHARDT, F. c.: Roles of amino acid activating enzymes in cellular physiology. Bact. Rev.
30, 701 (1966).
NIRENBERG, M. W., T. CASKEY, R. MARSHALL, R. BRIMACOMBE, D. KELLOG, B. DOCTOR,
D. HATFIELD, J. LEVIN, F. RorrMAN, S. PESTKA, M. WILCOX, and F. ANDERSON: The
RNA code and protein synthesis. Cold Spr. Harb. Symp. quant. BioI. 31, 11 (1966).
NOMURA, M., and C. V. LOWRY: Phage f2 RNA-directed binding offormylmethionyl-tRNA
to ribosomes and the role of 30S ribosome subunits in initiation of protein synthesis.
- - , and C. GUTHRIE: The initiation of protein synthesis: joining of the 50S ribosomal
subunit to the initiation complex. Proc. nat. Acad. Sci. (Wash.) 58, 1487 (1967).
NORRIS, A. T., and P. BERG: Mechanism of aminoacyl RNA synthesis: studies with isolated
aminoacyl adenylate complexes of isoleucyl RNA synthetase. Proc. nat. Acad. Sci.
(Wash.) 52, 330 (1964).
NOVELLI, G. D.: Amino acid activation for protein synthesis. Ann. Rev. Biochem. 36, 449
(1967).
OKAMOTO, T., and M. TAKANAMI: Interaction of ribosomes and some synthetic polyribo-
nucleotides. Biochim. biophys. Acta (Amst.) 68, 325 (1963).
OLD, D., and L. GORINI: Amino acid changes provoked by streptomycin in a polypeptide
synthesized in vilro. Science 150, 1290 (1965).
OTTENSMEYER, F. P., and G. F. WHITMORE: Coding properties of ultraviolet photoproducts
of uracil. I. Binding studies and polypeptide synthesis. J. Mol. BioI. 38, 1 (1968).
- , - Coding properties of ultraviolet photoproducts of uracil. II. Phenotypic reversion of
the amber mutation: implication of the uracil hydrate. J. Mol. BioI. 38, 17 (1968).
PESTKA, S.: Studies on the formation of transfer ribonucleic acidribosome complexes. I.
The effect of streptomycin and ribosomal dissociation on 14C-aminoacyl transfer ribo-
nucleic acid binding to ribosomes. J. bioI. Chem. 341, 367 (1966).
- The action of streptomycin on protein synthesis in vitro. Bull N.Y. Acad. Med. 43, 126
(1967).
-, and M. W. NIRENBERG: Code-word recognition on 30S ribosomes. Cold Spr. Harb.
Symp. quant. BioI. 31, 641 (1966).
- , R. MARSHALL, and M. W. NIRENBERG: RNA codewords and protein synthesis. V.
Effects of streptomycin on the formation of ribosome-sRNA complexes. Proc. nat.
Acad. Sci. (Wash.) 53, 639 (1965).
PETERKOVSKY, A., C. JESENSKY, and J. D. CAPRA: The role of methylated bases in the biolo-
gical activity of E. coli leucine tRNA. Cold Spr. Harb. Symp. quant. BioI. 31, 515 (1966).
POTIER, M., E. ApPELLA, and S. GEISSER: Variations in the heavy polypeptide chain structure
of gamma myeloma immunoglobulins from an inbred strain of mice and a hypothesis as
to their origin. J. molec. BioI. 14, 361 (1965).
PRINTZ, D. B., and S. R. GROSS: An apparent relationship between mistranslation and an

altered leucyl-tRNA synthetase in a conditional lethal mutant of Neurospora crassa. Ge-
netics 55,451 (1967).
RIFKIN, D. B., D. 1. HIRSCH, M. R. RIFKIN, and W. KONIGSBERG: A possible ambiguity in
the coding of mouse hemoglobin. Cold Spr. Harb. Symp. quant. BioI. 31, 715 (1966).
RossET, R., and L. GORINI: A ribosomal ambiguity mutation. J. Mol. BioI. 39, 95 (1969).
SARIN, P. S., and P. C. ZAMECNIK: Modification of amino acid acceptance and transfer
capacity of s-RNA in the presence of organic solvents. Biochem. biophys. Res. Commun.
19, 198 (1965).
SCHWARTZ, J. H.: An effect of streptomycin on the biosynthesis of the coat protein of
coliphage f2 by extracts of E. coli. Proc. nat. Acad. Sci. (Wash.) 53, 1133 (1965).
- Modification of the coat protein of coliphage f2 synthesized in cell-free extracts of
E. coli in the presence of streptomycin. Fed. Proc. 26, 865 (1967).
SINGER, M. F., and P. LEDER: Messenger RNA: an evaluation. Ann. Rev. Biochem. 35,
195 (1966).
- , O. W. JONES, and M. W. NIRENBERG: The effect of secondary structure on the template
activity of polyribonucleotides. Proc. nat. Acad. Sci. (Wash.) 49, 392 (1963).
So, A. G., and E. W. DAVIE: The effects of organic solvents on protein biosynthesis and
their influence on the amino acid code. Biochemistry 3, 1165 (1964).
- - Effects of amino acids, sRNA, and ethanol on coding ambiguity with polyuridylic acid.
Biochemistry 4, 1973 (1965).
- , J. W. BODLEY, and E. W. DAVIE: The influence of environment on the specificity of
polynucleotide dependent amino acid incorporation into polypeptide. Biochemistry 3,
1977 (1964).
STAEHELIN, T., and M. MESELSON: Determination of streptomycin sensitivity by a subunit
of the 30S ribosome of Escherichia coli. J. molec. BioI. 19, 207 (1966).
STAVY, L.: Miscoding in a cell-free system from spleen. Proc. nat. Acad. Sci. (Wash.) 61,
347 (1968).
STERN, R., U. A. H. MEHLER: Lysyl soluble RNA synthetase from Escherichia coli. Biochem.
Z. 342, 400 (1965).
SUEOKA, N., T. KANO-SUEOKA, and W. J. GARTLAND: Modification of sRNA and regulation
of protein synthesis. Cold Spr. Harb. Symp. Quant. BioI. 31, 571 (1966).
SZER, W., and S. OCHOA: Complexing ability and coding properties of synthetic poly-
nucleotides. J. molec. BioI. 8, 823 (1964).
TANAKA, N., H. MASUKAWA, and H. UMEZAWA: Structural basis of kanamycin for miscoding
activity. Biochem. biophys. Res. Commun. 26, 544 (1967).
TRAUB, P., K. HOSOKAWA, and M. NOMURA: Streptomycin sensitivity and the structural
components of the 30S ribosomes of Escherichia coli. J. molec. BioI. 19,211 (1966).
WElL, J. H.: Effet des modifications chimiques sur l'activite biologique du RNA de transfert.
Bull. Soc. Chim. bioI. (Paris) 47, 1303 (1965).
WEINSTEIN, 1. B., M. OCHOA JR., and S. M. FRIEDMAN: Fidelity in the translation of mess en-
ger ribonucleic acids in mammalian subcellular systems. Biochemistry 5, 3322 (1966).
- , S. M. FRIEDMAN, and M. OCHOA JR.: Fidelity during translation of the genetic code.
Cold Spr. Harb. Symp. quant. BioI. 31, 671 (1966).
WEISBLUM, B., and J. DAVIES: Antibiotic inhibitors of the bacterial ribosome. Bacteriol.
Revs. 32, 493 (1968).
WETTSTEIN, F. 0., and G. S. STENT: Physiological induced changes in the property of
phenylalanine tRNA in Escherichia coli. J. Mol. BioI. 38, 25 (1968).
WOESE, C. R.: The genetic code. The molecular basis for genetic expression. New York:
Harper and Row 1967.
YANIV, M., F. JACOB et F. GROS: Mutations thermosensibles des systemes activant la valine
chez E. coli. Bull. Soc. Chim. bioI. (Paris) 48, 1609 (1965).
Y ARUS, M., and P. BERG: Recognition of tRNA by amino acyl tRNA synthetases. J. molec.
BioI. 28,479 (1967).
6 Molecular and Subcellular BIology, Vol. 1

The Incorporation of 5-Fluorouracil Into RNA
and its Molecular Consequences*
H. GEORGE MANDEL
r. Introduction
It was recognized about two decades ago that analogs of nucleic acid bases could
be utilized as substrates for incorporation into polynucleotides, resulting in the forma-
tion of "fraudulent" nucleic acids containing an aberrant base. Among the compounds
most extensively studied which are able to replace a base in nucleic acids are 8-
azaguanine, 2-thiouracil, 5-bromouracil, 5-fluorouracil, 6-azathymine and 6-thiogua-
nine. The total number of analogs which are incorporated into polynucleotides is
quite limited. A general review on metabolite analog incorporation appeared in 1958
(MATTHEWS, 1958) which described the functions associated with the presence of such
an abnormal base in nucleic acids. Because most of the analogs had carcinostatic
activity, the anabolism and catabolism of such compounds in relation to their bio-
chemical and chemotherapeutic actions was the subject of another review (MANDEL,
1959).
In view of the great progress that has been made in the intervening years in under-
standing the formation and functions of subcellular components, it was felt appro-
priate to reexamine the literature with respect to our present knowledge of the in-
corporation of a characteristic nucleic acid base analog. 5-Fluorouracil (FU) was
chosen because this drug, developed in 1957, has become of great interest in clinical
and experimental cancer chemotherapy, and has been most useful as a biochemical
tool. Since probably it is the base analog incorporated into RNA which has received
the greatest attention by investigators, the present review concerns itself exclusively
with the formation and functions of FU-RNA. The incorporation of FU into the
various subcellular RNA components and the consequent biochemical effects are
described in detail in the present review. This survey covers the literature published
up to the middle of 1968, and is intended to be comprehensive of all the more pertinent
reports available.
The interest of CHARLES HEIDELBERGER and colleagues to synthesize 5-fluorouracil
was prompted by the report of RUTMAN et al. (1954) that uracil was preferentially used
for nucleic acid biosynthesis of tumors. It was believed that this fluoro analog might
have tumor-inhibitory properties and might interfere at one or more sites in the syn-
thesis or functions of nucleic acids. One potential site of sensitivity concerned the me-
thylation of the C-5 position of uracil to form the corresponding thymine compound,
since the C-5 position would be substituted by the inert fluorine atom. It was also
* This review was supported in part by USPHS Grant CA 02978 from the National
Cancer Institute, N.I.H., Bethesda, Maryland.
The Incorporation of 5-Fluorouracil into RNA and its Molecular Consequences 83
considered likely that FU, because of its close structural similarity to U, could be
incorporated into polynucleotides and thus might provide a new tool for the ex-
ploration of intermediary metabolism and nucleic acid synthesis as well as function.
In 1957 HEIDELBERGER et ai. (1957) first reported their results with FU and other
synthesized derivatives (DUSCHINSKY et aI., 1957). Evidence was provided for the
incorporation of this analog into the total nucleic acids of Ehrlich ascites tumors,
livers and spleens of mice. They established that the recovery of radiocarbon in the
nucleic acids from 14C-FU treated tumor· bearing mice was due to the presence of FU
as an integral moiety of the polynucleotides. The possibility of adsorption of conta-
minating free base during the isolation process had been excluded. The analog was
recovered from RNA hydrolysates and characterized chemically as fluorouracil.
FU bears close structural resemblance to U; the F atom has an atomic radius of
1.35 A compared to 1.2 A for hydrogen. Because of the greater electronegativity of
F than H, FU has a lower pKa than does U (8.15 and 9.45, respectively) (HEIDEL-
BERGER, 1965). As a result, at physiological pH, FU is more dissociated than is U.
This difference is felt to be of significance in explaining some of the properties of the
analog, and will be discussed later.
A large number of biochemical and pharmacological alterations produced by FU
have been recognized and have been the subject of thorough literature reviews
(HEIDELBERGER, 1965; also BROCKMAN and ANDERSON, 1963; ELION and HITCHINGS,
1965). Among the drug's major biochemical effects, in addition to its incorporation
into polyribonucleotides, are the inhibition of thymidylate synthetase and therefore
DNA synthesis, inhibition of the conversion of pyrimidine precursors into RNA, and
interference with normal bacterial cell wall biosynthesis.
The purpose of the present review is to focus on only those published reports
which relate specifically to the incorporation of FU into nucleic acids. Unfortunately,
the mechanism of many effects of FU can not yet be explained fully, and may be asso-
ciated with the inhibition of DNA synthesis and its resulting consequences. Such
reports have been excluded whenever possible.
Section II of the present review deals with the incorporation of FU into whole
cells, organs or other tissues which have not been fractionated into subcellular
components. The following Sections will describe the incorporation of FU into the
ribosomal, soluble and messenger RNA fractions. The molecular consequences of
the formation of such analog-containing RNA, principally in relation to the biosyn-
thesis of proteins, will be discussed. Information on the synthesis and properties of
FU incorporated into polynucleotides in vitro is also provided.
II. Incorporation of FU into Whole Cells and Tissues

FU can be recovered from the RNA of most species to which the analog has been
administered. Table 1 provides a comprehensive list of the various mammalian tissues,
plants and microorganisms which have been examined and in which FU-RNA has
been demonstrated. In all cases reported so far, FU replaced only U in RNA. The
relative composition of the other major bases of RNA was unchanged, and the sum of
FU plus U equaled the U component of normal tissues. Fluorocytosine has not been
recovered from RNA, and significant incorporation of FU into DNA has not been
established (DE KLOET, 1968; HOROWITZ and CHARGAFF, 1959; BOSCH et al., 1958;
6*
84 H. GEORGE MANDEL
CHAUDHURI et aI., 1958). It is important to stress that in many investigations no

effort had been made to achieve maximal values for incorporation of FU or replace-
ment of U. Variations in route of administration, dose, duration of exposure to the
drug, etc., could undoubtedly have provided higher values than those reported here,
but cell death due to inhibition of DNA synthesis would limit FU-RNA formation.
For Table 1, the highest value for each species reported has been quoted.
A. Formation of FU-RNAfrom FU
Mammalian tissue. The earliest detailed report of incorporation of FU into RNA
was by CHAUDHURI et al. (1958) who described the isolation of FU-RNA (Fig. 1 a)
and established the purity and identity of the analog, recovered as the monoribo-
nucleotide FUMP. The authors could find no evidence of FC in RNA after the ad-
ministration of FU, in contrast to the conversion into RNA uracil and cytosine
following exogenous uracil. These authors also were the first to conclude that FU
a
I
:::l..
E AMP GMP UMP FUMP
g ~
N
-0 J!'
:~
<II
v v
C CI
.2
.8 "U
<:; CI
Q::
III
.0
<{
0
Tube no.
b CMP AMP GMP

3
:::l..
E
0
<D
N
c; 2
<II
V
C
CI
.0
(; UMP
III
.0
<{
Tube no.
Fig. 1. Elution diagrams of hydrolysate of RNA synthesized in the presence of FU, indi-
cating recovery of FUMP and signifying FU in internucleotide form in RNA. Top (a):
Analysis of RNA from tissues of tumor-bearing mice, using Dowex-1-formate ion exchange
columns. Open peaks refer to absorbance at 260 mfL ; shaded peak, radioactivity from 14C_FU
experiments. From CHAUDHURI et ai., 1958. Bottom (b): Analysis of RNA from E. coli,
indicating large quantity of FUMP recovered from microorganisms, using Dowex-2-
formate columns. Only absorbance measured. (From HOROWITZ and CHARGAFF, 1959)
Table 1. Incorporation of PU into RNA of variour rpecier
Species %ofU Comment Reference ~
replaced n
......
ij
n
70 Partially reversed by U ANDOH and CHARGAFF, 1965; 0
Escherichia coli B
HILLS and HOROWITZ, 1966
.a
Escherichia coli, uracil auxotroph 25 Essentially unchanged by addition of U, HOROWITZ and CHARGAFF, 1959
...i»0
FU partially replaces U requirement
g.
ij
Bacillus subtilis 40-60 SAUNDERS et aI., 1968 0.....,
Bacillus cereus 15 Reversed by U, enhanced by TdR REICH and MANDEL, 1966; U1
I
REICH and MANDEL, 1964
Staphylococcus aureus 5 Tested only in presence of U HIGNEIT, 1964 ~
Pediococcus cerevisiae * Reversed by U WHITE and NICHOL, 1963 ...00
Reversed by U C
Saccharomyces carlsbergensis 70 DE KLOET, 1968; DE KLOET and STRI1KERT, 1966 ~
Candida utilis 10 Reversed by U; incorporation of FU, KEMPNER, 1961 g.
equal to that of U ::r....
Trichoderma viride GRESSEL and GALUN, 1966 0
Tobacco mosaic virus 56 Reversed by UR STAEHELIN, 1960; KRAMER et aI., 1964 :;d
Polio virus 36 Reversed by UR MUNYON and SALZMANN, 1962 Z
Phage MS2 80 SHIMURA, et aI., 1965 >-
i»
ij
Phage R 17 28 Reversed by U GRAHAM and KIRK, 1965 0.-
Phage f2 LODISH et al., 1965 ....
CI>
Soybean hypocotyl Incorporation FUR; UR = 0.5 KEY, 1966
~
Xanthium pensylvanicum bud + leaf Incorporation of FU: orotate = 0.1, BONNER and ZEEVAART, 1962
reversed by oro tate ~
n
Tobacco leaf chloroplasts < 4 Incorporation not firmly established STAEHELIN and GORDON, 1960 ~
Housefly eggs 0.05 Eggs less viable KILGORE and PAINTER, 1966 ...
(j
Mouse liver 0.6 4 h after administration to mouse CHAUDHURI et aI., 1958 0
ij
CI>
Mouse embryos Mother had received FU DAGG et al., 1966 n
Ehrlich ascites tumor 6 4 h after administration to mouse CHAUDHURI et aI., 1958 og
n
Sarcoma 180 3.5 4 h after administration to mouse CHAUDHURI et aI., 1958 ij
n
n
Human tissues Variable MUKHER1EE et al., 1963 CI>
* 6.9 !Lg FU/mg dry weight. 00

U1
86 H. GEORGE MANDEL
was not incorporated into DNA. Numerous acid-soluble products containing FU

have been identified, but these will not be discussed here.
More FU was incorporated into the RNA of ascites cells of Sarcoma 180 than into
that of spleen or liver after FU administration to tumor-bearing mice, and the maxi-
mum quantity of analog incorporated into the tissues was reached at 4 h after ad-
ministration. As shown in Table 1, the replacement by FU of 6% of the U content of
RNA was remarkably high considering the slower rate of cellular biosynthesis of
tumor cells than microorganisms, for instance. The analog was also incorporated into
human tumors and normal tissues, but the quantity of FU recovered in the different
fractions varied considerably (MUKHERJEE et aI., 1963).
The incorporation of FU into RNA of ascites cells in vitro has also been reported
(BOSCH et aI., 1958). In general, RNA of rapidly metabolizing tissues, such as intestinal
mucosa and more cellular tumors, incorporated increased quantities of FD. It might
be expected that such tissues also had increased capacity to anabolize FU to inter-
mediary nucleotides, so that the greater incorporation into RNA was largely a re-
flection of enhanced anabolism and an increased availability of analog-containing
precursors (MUKHERJEE et aI., 1963). Four times more FU was incorporated into
RNA of regenerating rat liver than into RNA of normal liver (NEMETH, 1962).
When 14C-FU at teratogenic doses was injected into pregnant mice, radioactivity
was recovered in the RNA fraction of the embryos, the extra-embryonic membranes
and deciduas (DAGG et aI., 1966).
Insects. KILGORE and PAINTER (1962) observed that when flies were fed a diet of
labeled FU the eggs contained FU-RNA which was maximal in eggs deposited during
the first day of oviposition. Egg viability was inversely related to the FU content
(KILGORE and PAINTER, 1966). FU was recovered in the eggs only after it had been
administered to the female mosquito, since, as might be expected, insufficient 14C-FU
for detection was transferred to the fertilized egg from the male fly. Larvae emerging
from these eggs were still radioactive (KILGORE and PAINTER, 1962).
Plants. FU was incorporated into bud RNA of Xanthium pensylvanicum (BONNER
and ZEEVAART, 1962) when applied to the plant leaves or the bud directly; some in-
corporation into plant leaves following direct application was also detected. The
soybean hypocotyl also incorporated FU into RNA (KEY, 1966).
Microorganisms. The incorporation of FU into RNA of microorganisms takes
place most readily and leads to extensive replacement of U. An elution diagram of
hydrolysate of E. coli RNA formed in the presence of FU is shown in Fig. 1 b, and
indicates the massive amounts of analog that have been recovered from FU-RNA
following hydrolysis to FUMP (HOROWITZ and CHARGAFF, 1959). Because of the
extensive incorporation of FU into RNA and the accumulated experience on sub-
cellular fractionation in microorganisms, these systems have been widely investigated
and have provided considerable information on the formation and functions of
different RNA species containing the analog. These will be the subject of the sub-
sequent Sections of this review.
B. Conversion of Related Drugs to FU-RNA

FO (STAEHELIN and GORDON, 1960; HARBERS et aI., 1959; CHAUDHURI et aI.,
1958), FUR (STAEHELIN and GORDON, 1960; HARBERS et aI., 1959) and FUdR
(LAMPKIN-HIBBARD et aI., 1963; WHITE and NICHOL, 1963; HARBERS et al., 1959)
were converted to FU ribonucleotides and incorporated as FU in RNA of mammalian
or microbial tissue. Under comparable conditions when FU, FO, FUR and FUdR
were incubated with Ehrlich ascites cells, 2.3, 1.3, 11.1 and 1.1 % of the added analogs
was recovered as FU-RNA. The percentages of the incorporation into RNA of the
corresponding nonfluorinated derivatives were almost identical (HARBERS et al.,
1959). The ribonucleoside, therefore, can be a very efficient source of FU-RNA in
tumors, and its relatively greater toxicity has been linked to its facilitated uptake
and incorporation into RNA (HEIDELBERGER, 1965). Since FUdR can be cleaved to
the free base, the use of FUdR as a tool to relate observed effects ofFU to interference
with DNA synthesis exclusively may be hazardous. 14C-FC apparently was incorpo-
rated into Candida albicans and was recovered as FU-RNA. When given to rodents this
derivative was deaminated in the GI tract and then was reutilized as FU (KOECHLIN
et aI., 1966).
C. Factors Mod!fyingQuantity oj FU-RNA Formation
Various reports have indicated diminished incorporation of FU into RNA with
decreasing sensitivity to the drug. The observations were usually based on the decreas-
ed ability to convert FU to anabolite nucleotides, and the diminished formation of
these nucleic acid precursors then limited analog incorporation into RNA as a conse-
quence.
1. Resistance
The most apparent example was reported by BROCKMAN et al. (1960) who found
that FU-RNA formation by Escherichia coli cells resistant to FU was only 0.3% of that
of the sensitive strain. Results from other microbial systems were less well defined.
No difference in FU incorporation was reported in two strains of Bacillus subtilis, one
sensitive and another resistant to the drug (BODMER and GRETHER, 1965) although
sufficient data were not provided. Four resistant substrains of Pediococcus cerevisiae
incorporated significantly less FU, whereas one showed only a slight reduction
(WHITE and NICHOL, 1963) in comparison with the wild strain.
As a rule, incorporation of FU into sensitive and resistant tumors differed only by
a factor of 2 or 3. The L-1210 ascites tumor sensitive to FU contained only slightly
more FU in RNA than the resistant line (GOLDBERG et al., 1966); the content of FU-
RNA in P-1798 lymphomas increased as these tumors developed increasing sensitivity
to FU (LAMPKIN-HIBBARD et aI., 1963); and an FU-sensitive Ehrlich ascites carci-
noma incorporated more analog than did the related resistant line both in vivo [HEI-
DELBERGER et aI., 1960 (1)] and in vitro [HEIDELBERGER et al., 1960 (2)]. These small
differences in FU incorporation undoubtedly did not account for the greatly altered
response of the tumor subline to the drug. In the last case, for example, the mechanism
of resistance to FU was due to the decreased inhibition of DNA formation, rather
than the diminished incorporation of analog into RNA.
2. Presence of other Pyrimidines

The simultaneous administration of pyrimidines with FU altered the extent of
incorporation of the analog, and it frequently changed the system's response to the
drug (Table 1). In general, in microorganisms, the administration of U with FU
prevented any alterations produced by the analog on RNA synthesis and reduced the
88 H. GEORGE MANDEL
incorporation of FU into RNA without appreciably reversing inhibition of DNA

formation. It should be emphasized that the extent of reversal depended on the relative
utilization of the pyrimidines by the tissue system, the doses employed, and the time
of addition of the pyrimidines in relation to the damage already produced by FU.
Occasionally UR was found to be more effective than U for recovery, apparently due
to the greater ability of many tissues to utilize pyrimidine nucleosides (even though
the flu oro base analog was inhibitory). Thymine or TdR usually had little effect on FU
incorporation into RNA (WHITE and NICHOL, 1963) while these supplementations
were effective in restoring DNA synthesis. In order to study FU-RNA formation or
the effects on RNA biosynthesis, therefore, FU and TdR have occasionally been
administered together to mask the drug's effect on DNA synthesis. The effectiveness
of this procedure is subject to some question, however. In B. cereus, for example, TdR
actually increased the incorporation of FU into RNA (REICH and MANDEL, 1966).
There is as yet no explanation for this observation. Usually the combination of U and
TdR or T overcame all of the inhibitory actions of FU in microbial systems (REICH
and MANDEL, 1966; WACHSMAN et ai., 1964; HOROWITZ et ai., 1960; COHEN et ai.,
1958).
No information has been reported on the reversal of FU-RNA formation in tumor
cells, but neither TdR nor UR prevented the inhibitory effect of FU in tissue culture.
On the other hand, growth inhibition by FUdR was overcome by the co-administra-
tion of TdR (RICH et al., 1958).
D. Distribution of FU within RNA Chains

Various methods of analysis were used to locate the relative position of FU within
the polyribonucleotide chain. The absence of appreciable quantities of FUR as a
product of alkaline hydrolysis of FU-RNA isolated from Sarcoma 180 implied that
most of the analog was not terminal (CHAUDHURI et al., 1958). Correspondingly,
hydrolysis of tobacco mosaic virus containing FU with pancreatic ribonuclease did
not produce fluorouridine-3 /, 5' -diphosphate. Unlike similar experiments with
thiouracil-RNA (MANDEL et ai., 1957), FU, therefore, does not appear to be concen-
trated at the ends of polynucleotide chains (GORDON and STAEHELIN, 1959). Such a
conclusion is not surprising in view of the relatively large quantity of FU that can be
incorporated, all at the expense of U.
E. Incorporation into Nuclear and Cytoplasmic RNA

As has been shown with other precursors, nuclear RNA became labeled more
rapidly than cytoplasmic RNA after the administration of 14C-FD. This difference
diminished with time (CHAUDHURI et al., 1958). Experiments with 14C-FUR provided
identical results (HARBERS et al., 1959).
F. Effect of FU-RNA on Growth: Whole Cells

Since FU-RNA formation in cells was usually accompanied by the presence of
FU-containing ribonucleotides, it is difficult to assign specific inhibitory effects due
solely to the incorporation ofFU into RNA. A positive correlation between sensitivity
ofleukemia cells and total FU nucleotides including FU-RNA has been described, for
example (KESSEL et al., 1966). The situation is further complicated in that FU,
following conversion to FUdRP, inhibits thymidylate synthetase (COHEN et al., 1958).
Although the effect on total RNA synthesis was relatively minor quantitatively,
there usually was redistribution of RNA among the subcellular fractions because of
inhibition of ribosome formation (see Section III A). In addition, some changes in
composition of tRNA have been reported, as discussed in Section IV C. In micro-
organisms, alterations in cell wall formation are known to take place (THOMAS and
BOREK, 1962; ROGERS and PERKINS, 1960). Consequently it is difficult to relate specific
biological responses to FU-RNA formation in whole cells.
In tumor cells, the inhibition of DNA synthesis rather than the alteration of RNA
synthesis was believed to be more closely associated with the drug's carcinostatic
properties, whereas toxicity resulted from the latter drug effects (BOSCH et al., 1958).
This conclusion is based in part on the chemotherapeutic efficacy of FUdR which has
little effect on RNA synthesis, and the toxicity of FUR, since this ribonucleoside is so
readily anabolized to FU-RNA (HEIDELBERGER, 1965). However, the inability to
prevent the inhibitory action of FU with TdR indicates that the drug acts by several
mechanisms in tumor cells (RICH et al., 1958).
In microbial systems also it appears that the inhibition of DNA synthesis is not the
only cause of growth inhibition. The addition ofTdR to cells inhibited by FU usually
led to at least partial restoration of DNA synthesis (REICH and MANDEL, 1966;
WACHSMAN et al., 1964; HOROWITZ et al., 1960). Whether the reversal of the blockage
of DNA synthesis was synonymous with recovery of DNA function is not clear,
however; in B. megaterium grown in the presence of FU, for example, TdR was
effective in restoring DNA content but did not prevent the loss of viability of the
cells (WACHSMAN et al., 1964) presumably related to "thymine-less" death (COHEN
et al., 1958). The supplementation of cultures with TdR usually did not overcome
growth inhibition produced by FU, indicating that other actions of the drug, un-
doubtedly including FU-RNA, were producing growth inhibition.
III. FU and Ribosomes

FU produces major alterations in ribosome formation, and is itself incorporated
into these subcellular components which normally account for the majority of the
cell's RNA.
A. FU Incorporation and Ribosomal Alterations
The first studies revealing an effect of FU on ribosomes were those of ARONSON
[1961 (2)], GROS et al. (1961) and KEMPNER (1961). These authors observed that FU
inhibition of E. coli led to the formation of abnormal ribosomes which could not be
readily converted to larger particles, even upon removal of the analog [ARONSON, 1961
(2)]. Extracts from cells grown in the presence of14C-FU when examined at 10- 4M Mg++
contained radioactivity in what appeared to be 30S particles, whereas little label was
transferred to the 50S ribosomal subunit (GROS et al., 1961). In Candida utilis, FU
reduced the incorporation of uracil into rRNA and permitted the accumulation of
sRNA. Labeling with 14C-FU followed a pattern similar to that of 14C-U in the pres-
ence of unlabeled FU (KEMPNER, 1961). It was concluded that FU decreased ribo-
somal RNA synthesis by 40%, and that small ribosomal particles of about 30S
90 H. GEORGE MANDEL
accumulated which were not converted to the larger ribosomes (KEMPNER and
MILLER, 1963).
E. coli. In general, following density gradient centrifugation the elution profiles of
ribosomes or ribosomal subunits differed appreciably from normal when cells had
been grown in the presence of FU. Qualitatively identical conclusions were drawn
regardless of whether a labeled purine, pyrimidine, amino acid, phosphate or FU was
used to mark the newly formed components. When the fractionation experiments had
been carried out at 10- 2 M Mg++, extracts from FU-treated cells contained fewer
70S to 1OOS particles than did extracts from normal cells, and these particles differed from
1. 5r - -- - - - -- -------,30000
ft
50S
30S
1\ ff\ . -
-;
tl.O I- 20000
~
N 0-
Ci ::2:
w a..
u u
c::
0
.0
j ff \\ I \l
(;
III
«
.0 0.5I- 10000
J .Vj • \~ /Ii \
A
V. t!;V ~d\
l><>
t7
'l"""<f"<)(K><1 I
I I 0
0 10 20 30
Fraction no.
Fig. 2. Sucrose gradient sedimentation analysis of extract from E. Goli grown with 14C_
adenine in presence of FU.e, Absorbance at 260 mfJ., indicating characteristic normal 50S
and 30S ribosomal subunits and sRNA peaks; 0, radioactivity associated with RNA labeled
during FU treatment, distributing only near 30S peak and sRNA. Experiments with 14C_FU
provided a very similar pattern of radioactivity. (From KONO and OSAWA, 1964)
normal ribosomes in having less sedimentation homogeneity and in exhibiting greater

sensitivity to RNase. In contrast to normal cells, a large proportion of the material
had sedimentation values of 50S and less (HILLS and HOROWITZ, 1966). Some reaggre-
gation of the FU ribosomal particles to 70S units at 10-2 M Mg++ concentrations has
been claimed (ANDOH and CHARGAFF, 1965).
Investigations carried out in the presence of 10-4 M Mg++ confirmed that "50S"
particles were no longer synthesized after FU treatment [HILLS and HOROWITZ, 1966;
IWABUCHI et aI., 1966; SELLS and CRUDUP, 1966; NAKADA, 1965; KONO and OSAWA,
1964; ARONSON, 1961 (2)]. Ribosomal particles formed in the presence ofFU differed
from the normal 30S ribosomal subunit in having sedimentation values between
27 and 33S (Fig. 2) with considerable heterogeneity, and in being appreciably more
sensitive to RNase (HILLS and HOROWITZ, 1966; ANDOH and CHARGAFF, 1965;
KONO and OSAWA, 1964). Small amounts of other components (40 to 47S) were also
uncovered, especially after prolonged inhibition by FU (HILLS and HOROWITZ, 1966;
ANDoH and CHARGAFF, 1965). Proteins synthesized during FU treatment also sedi-
mented in the 30S region with the ribosomal particles (IWABUCHI et aI., 1966).
The ribosomal particles made in the presence ofFU differed from the 1SS and 25S
particles which accumulate during chloramphenicol treatment. When chloramphenicol
and FU were present simultaneously, particles characteristic of chloramphenicol
500
50S 1\
Vt
I
I
I
I
1
)
~ 400 I I
200
~
\
If
o
<D I I
N
01 I
oCI> 300 ;1' \ 305
I
.1
ff
I
u
.v
C
d
-eo
,.J!
VI
~ 200 100
/
/
100
0~----------~10~----------~2Z0~----~ 0
Fraction no.
Fig. 3. Sucrose gradient sedimentation analysis of extract of E. coli grown with I4C-FU but
in the presence of U. Note normal distribution of radioactivity, in contrast to Fig. 2 . • , Ab-
sorbance at 260 mfL; 0, radioactivity. (From HILLS and HOROWITZ, 1966)
inhibition predominated, indicating that the two drugs inhibited different steps of
ribosomal synthesis. When the antibiotic was removed but FU was still present, the
chloramphenicol particles were converted to FU particles (KONO and OSAWA, 1964).
No abnormal ribosomal subunits were observed when uracil was present together
with FU, even though FU was still incorporated into such components (Fig. 3).
These ribosomes were stable in contrast to the abnormal FU-ribosomal particles, and
FU was not lost during subsequent growth, even in the presence of uracil. Less FU
was incorporated per mg RNA formed when FU and U were present simultaneously,
compared to results when only FU was administered (HILLS and HOROWITZ, 1966).
The ribosomal RNA from FU-treated cells was approximately 17S and 22 to 23S
(Fig.4a) (HILLS and HOROWITZ, 1966; ANDOH and CHARGAFF, 1965; KONO and
OSAWA, 1964), which suggested that it resembled RNA from nascent rather than
92 H . GEORGE MANDEL
--==~~
0
N ~
ci
c
c
ci a
c
~~
.,
u
=
It
52
..,
d d d ~ d
(-I nw 09Z \\) a:)U\)qJosq~
~~----~----~~-----r~----~o
( 0 ) VoId:>
~ ~ ~ N 0 ~
I ~ ___ --~----<T I~
~P ____
e::;:_________________________ _g
'('l:> \!2
CX)
0 d
c c
~ 0
-.,
C C
0 0
0 a
---..------- u
It
8
~
0
0
.,
~
It
~ ~.r::::.:--J7------- _52
-~~~) -,
CX)
n!
.L I I .L 1 I I
o o
~ d
I-I rlLU09Z lD a::>uDqJOsq~
Fig. 4
from mature ribosomes (16S and 23S) (KONO et aI., 1964). The possibility that the
increased weight of the FU-RNA was responsible for the slightly heavier sedimenta-
tion coefficient should also be considered, however, since the two rRNA fractions
contained considerable quantities of FU. The base composition of the rRNA fraction
was unchanged by FU, except that 70% of U had been replaced by FU (Table 2).
FU-rRNA demonstrated slightly less hyperchromicity than normal rRNA. The
resolution of normal rRNA and FU-rRNA was greatly enhanced using chromato-
graphy on methylated albumin columns (ANDOH and CHARGAFF, 1965). The pres-
ence of 22 to 23S rRNA in the absence of 50S ribosomal subunits into which they are
normally converted has been observed in several reports (HILLsandHoRowITZ, 1966;
ANDOH and CHARGAFF, 1965; NAKADA, 1965; KONO and OSAWA, 1964); in other
experiments much less of this RNA was synthesized (SELLS and CRUDUP, 1966;
GROS et al., 1962).
Other microbial systems. In Stap~lococcllS aureus growing in the presence of U, FU
replaced only 4% of the U in ribosomal RNA, whereas 14% of U of sRNA was
replaced by the analog (HIGNETI, 1964). Examination of the ribosomal fraction
from an FU culture at 10-3 M Mg++ revealed a greater proportion of slowly sedi-
menting particles compared to a control. The heavier ribosomal subunit (41S) was
not found, and instead of a 30S subunit, particles with sedimentation values of 28S,
26S and 19S were formed. At 10-2 M Mg++ the patterns were more similar but
showed an extra heavy component (71S) in the FU-treated culture and a greater re-
lative proportion of particles of 49S. The abnormal particles were more sensitive to
RNase than controls (HIGNETI, 1966). Sufficient information was not supplied to
further characterize these components or to explain the observations. Although the
sedimentation coefficients differed from those reported for E. coli, the overall effects
of FU on the pattern of distribution of ribosomes were similar in the two systems.
In Bacillus cereus FU produced an imbalance in RNA synthesis in that more sRNA
was formed at the expense of rRNA. Whereas normal cells contained approximately
equal quantities of these components, almost four times the amount of sRNA com-
pared to rRNA was synthesized in the presence of FU (HAHN and MANDEL, 1967)
Fig. 4 a-d. Profiles of RNA from cells of various species, with alterations produced by FU
treatment. a, RNA from E. coli separated by density gradient centrifugation: e, Normal
cells labeled with 14C-adenine, elution of characteristic 23S and 16S rRNA, 4S sRNA;
0, cells labeled with 3H-FU; note slightly shifted rRNA peaks, relatively greater proportion
of label in sRNA. (From KONO et al., 1964). b, RNA from B. cereus, separated by density
gradient centrifugation. Experimental conditions similar to those in a. e, Normal cells
labeled with 14C-guanine; 0, cells labeled with 14C_FU. Note virtual absence of labeling of
rRNA peaks, with sRNA synthesized as major component. (From HAHN and MANDEL).
c, RNA from S. carlsbergensis, separated by density gradient centrifugation. Left, control
yeast cells labeled with 14C-adenine; right, cells following exposure to FU and 14C-adenine ;
---,absorbance at 260 m(L, 0, radioactivity. Peaks identified in a but with FU, synthesis
of rapidly sedimenting high molecular weight D-RNA. (From DE KLOET, 1968). d, RNA
from soybean hypocotyl separated on methylated albumin columns: top, normal cells
labeled with 32P-phosphate for 4h; bottom, cells grown in presence ofFU and 32P. ---Ab-
sorbance at 260 m(L, - - - radioactivity. Peaks I through V represent sRNA, DNA, rRNA..,
rRNAb and D-RNA, respectively. Note reduction of label in rRNA peaks while formation
of D-RNA continued. Labeling with 14C-FUR apparently similar to results in bottom profile.
(From KEY, 1966)
\0
.j:>.
Table 2. Relative composition of RNA fractions from E. coli following treatment with PU for 30 min and 6 h, and for 2 h following recovery from 30 min exposure
to PU. Radiophosphate was used to locate and quantitate major bases as ribonucleotide!
(From ANDOH and CHARGAFF, 1965)
Conditions RNA Nucleotide constituents, % of total radioactivity
preparation FUxlOO Purines:
~~---
A G C U FU U+FU pyrimidines
U+FU
Fe
Normal sRNA 22.1 33,2 29.2 15.5 1.24 c;J
16S-RNA 26.7 30.3 22.0 21.0 1.33 tn
0
23S-RNA 26.3 30.7 21.7 21.2 1.33 :>0
Cl
tn
FU, 30 min sRNA 19.6 30.0 29.8 6.0 14.7 20.7 71.0 0.98 ~
>
16S-RNA 26.3 28.3 22.7 7.8 14.8 22.6 65.4 1.21 z
tl
23S-RNA 25.6 30.2 22.3 7.0 14.9 21.9 68.0 1.26 tn
t"'
FU, 6 h sRNA 19.7 27.3 28.6 6.9 17.6 24.5 71.8 0.89
16S-RNA 25.4 29.0 23.6 6.9 15.2 22.1 68.8 1.19
23S-RNA 26.7 28.1 22.0 7.3 15.9 23.2 68.5 1.21
Recovered from 30 min FU sRNA 20.3 30.6 28.5 13.8 6.8 20.6 33.0 1.04
rRNA 25.7 31.3 22.4 18.7 1.9 20.6 9.2 1.33
total RNA 23.1 31.7 25.4 17.4 2.4 19.8 12.1 1.21
(Fig. 4 b). The extent of this effect was dependent on the concentration of FU used,
and also was directly related to the increase in generation time of the culture.
The quantity of 70S ribosomes formed was drastically reduced during growth in the
presence ofFU. Instead, at 10-2 M Mg++, the existence of new and abnormal particles
sedimenting in the 30S and 50S regions was evident. The distribution profile of
ribosomal RNA differed from that of the control in that the quantity was considerably
reduced, and much less 23S rRNA was synthesized (HAHN and MANDEL). FU-rRNA
had a slightly lower Tm than did the control product. Less ribosomal and more
soluble protein was formed during inhibition by FU, and the ratio of RNA to protein
was reduced in ribosomes but was increased in the soluble fraction (HAHN and
MANDEL).
FU altered the normal pattern of rRNA in the fungus, Trichoderma viride, and
particles of 17S and 23S, plus an unidentified component of higher sedimentation
coefficient, were produced (GRESSEL and GALUN, 1966).
In a recent study on Saccharomyces carlsbergensis (DE KLOET, 1968; DE KLOET and
STRI]KERT, 1966) FU also inhibited the formation of ribosomal subunits. Again the
more rapidly sedimenting subunit was essentially unlabeled by 14C-adenine during
growth in the presence of FU or by 14C-FU, whereas sRNA and the region of the
gradient corresponding approximately to the lighter subunit contained most of the
radioactivity. The distribution of ribosomal RNA was also altered appreciably and
contained relatively large amounts of a high molecular weight RNA. Base analyses of
this high molecular weight RNA, formed by the yeast in the presence of FU, sug-
gested that this RNA resembled DNA ("D-RNA") (Fig.4c).
Plants. In the soybean hypocotyl, FU apparently selectively inhibited rRNA and
sRNA synthesis without affecting the formation of a D-RNA (Fig. 4d). The analog
administered as the nucleoside was incorporated into the soybean hypocotyl RNA,
principally the D-RNA fraction, but relatively little was present in the ribosomal
fraction (KEY, 1966). This D-RNA fraction was sensitive to the action of actinomy-
cin, and growth of the plant tissue appeared to require the continued formation of the
component. Its mean life was about 2 h, and the fraction exhibited messenger-like
properties. Corn mesocotyl and radish cotyledon tissue behaved similarly to the
soybean hypocotyl in this regard (KEY and INGLE, 1964). FU also selectively inhibited
ribosomal synthesis in the excised Xanthium bud, whereas the D-RNA fraction
continued to be produced (CHERRY and VAN HUYSTEE, 1965).
Animals. In the only related and still preliminary experiment on mammalian
tissues, ribosomal synthesis in the liver of rats treated with FU was reduced, and
nuclear and perhaps nucleolar precursors were not converted to cytoplasmic ribo-
somes. With low concentrations of FU, considerable quantities of an unidentified
product which may have messenger properties were labeled after 3H-cytidine ad-
ministration. High concentrations of FU inhibited all RNA synthesis (WILLEN and
STENRAM, 1967).
B. PU Incorporation into AlreatlY Abnormal Ribosomal Particles

Abnormal ribosomal particles accumulating in the presence of chloramphenicol
are converted to mature ribosomes upon removal of the antibiotic. When these
abnormal particles had been formed in the absence of FU, this maturation of ribo-
somes, including synthesis of ribosomal protein, took place even in the presence of
96 H . GEORGE MANDEL
FU (IWABUCHI et aI., 1966). When these particles were formed in the presence of
chloramphenicol and FU simultaneously, however, maturation was prevented by FU
after chloramphenicol was removed. Similarly, abnormal ribosomal particles due to
puromycin inhibition could be converted to mature ribosomes only if the FU had
been added after the puromycin was removed (SELLS and CRUDUP, 1966).
Another comparable situation is that of a "relaxed" methionine-requiring mutant
of E. coli in which RNA synthesis continues even though protein synthesis has been
blocked by methionine starvation. Under these conditions, abnormal ribosomal
particles are formed. The addition of methionine (corresponding to recovery from
the antibiotics in the above examples) then leads to the combination of the accu-
mulated "relaxed" particles with ribosomal protein to form mature ribosomes
a5r---------------.----------------r--------------~50
a b c
40 -;-
r n
} 1\
f : \I
, I I
/ \I 10
'--../
I /
/ v
/
Fraction no.
Fig. 5a-c. Sucrose gradient sedimentation analyses of extracts of E. coli "relaxed" mutants
recovering from methionine starvation. a, cells following starvation received 14C-Ieucine
and methionine, and incorporated radioactivity into normal ribosomal subunits; b, as in a
except that FU was administered with the 14C-leucine and methionine, and again radio-
activity incorporated into normal ribosomal subunits even though FU was present during
this incorporation; c, cells previously starved in presence of FU now received 14C-leucine,
methionine as well as U. These cells did not incorporate 14C-Ieucine into normal ribosomal
subunits but made soluble protein. Total incorporation in all cases was equal even though
this is not apparent in frame c, for unexplained reasons; - - - absorbance at 254 mfL. e. radio-
activity in hot trichloroacetic acid-precipitated residue. (From NAKADA, 1965)
(NAKADA, 1965). FU present during the methionine starvation was incorporated into
the ribosomal particles, and prevented the formation of mature ribosomes even after
methionine had been restored. Thus, the normally occurring preferential synthesis of
ribosomal protein had been inhibited under these conditions, and soluble protein
was made instead. However, FU added after restoration of methionine to the starved
cells permitted the preferential synthesis of ribosomal protein and led to the comple-
tion of normal ribosomes from the preformed "relaxed" particles. During this period
of maturation of ribosomes from preformed "relaxed" particles, the analog was
being incorporated into new ribosomal particles and inhibited the formation of new
ribosomes (Fig. 5).
From these experiments it was concluded that the ribosomal RNA contained in
the "relaxed" particles acted as messenger for the synthesis of ribosomal protein. The
incorporation of FU into these particles apparently had interfered with their template
activity. These experiments also implied that the messenger half life was longer than
that of the usual mRNA. It was observed that whereas "relaxed" particles stimulated
the incorporation of lysine into 30S and 50S ribosomes in a cell-free amino acid
incorporating system, particles containing FU, while incorporating an equivalent
quantity of amino acid, formed only small amounts of ribosomal protein (NAKADA,
1965). Thus, the residual protein made did not combine with the "relaxed" particles
(Fig. 6).
Precursor particles accumulated during puromycin treatment also had template
activity (SELLS and CRUDUP, 1966). As in the case of the relaxed mutants, FU inhibited
0.6 300
a 50S
I'
b c
I' I'
('\
..!.. 0.5
I I'
II I' I \I
::to , I ,
E
,
I I I I
04 , I I 200
I I t
~
1/', II
III 305
N
I
I
f\ ~
c; 03
,
fJ
I
I
I I
\
,
I
f
I
I
I ,
I :::E
a..
'"c: ,
"v I I
, I
<.>
I t I V U
)r/4
0 I I \
.0 02 100
5Ul I \
I
.0 I
« I
I
I \
I I
I
0
0 0 30
Fraction no.
Fig. 6 a-c. Sucrose density gradient analysis of cell-free preparations from E. coli "relaxed"
mutants that had incorporated 14C-Iysine in vitro. The ribosomal fraction of the incubation
system was prepared from cells, a, during normal growth; b, 30 min after initiation of methio-
nine starvation in the absence ofFU ;and c, 30 min after ini dation of methionine starvation in the
presence of FU. The mixture after 14C-Iysine incorporation was then centrifuged and ribo-
somal components fractionated. Total 14C-Iysine incorporation into polypeptides of incu-
bation mixture in all three cases was almost identical. Thus, "relaxed" particles clearly had
messenger activity, in contrast to ribosomes. FU-containing relaxed particles did not permit
ribosomal protein to be synthesized although amino acids were incorporated into poly-
peptides. - - - Ultraviolet absorbance, e, radioactivity. (From NAKADA, 1965)
the programming for ribosomal protein biosynthesis normally associated with the
accumulated precursor particles. The association of messenger activity with ribo-
somal particles accumulating during inhibition of protein synthesis by chlorampheni-
col (OTAKA et aI., 1964) and 8-azaguanine (GRUNBERGER and MANDEL, 1965) has also
been reported.
C. Fate of FU Ribosomal Particles During Recovery from the Analog

When FU was removed from a suspension of E. coli inhibited by the drug, 30S
and 50S ribosomal subunits began to appear very gradually (SELLS and CRUDUP, 1966;
GROS et aI., 1962). The recovery process was accelerated when uracil was added. The
newly formed "normal" ribosomes now contained FU (HILLS and HOROWITZ, 1966;
IWABUCHI' et aI., 1966; KONO and OSAWA, 1964). The question therefore arose as to
98 H. GEORGE MANDEL
whether the abnormal FU-containing ribosomal particles had served as precursors for
mature ribosomes after the removal of FU. Alternatively, it was possible that the
particles had been degraded and then had reformed into mature ribosomes. The latter
turned out to be the case (HILLS and HOROWITZ, 1966; IWABUCHI et al., 1966; ANDOH
and CHARGAFF; 1965).
During recovery, FU was released from ribosomal particles only when uracil had
been added. However, even in the absence of exogenous U the FU particles were
unstable, as concluded by two lines of evidence: (1) 14C-FU in ribosomal particles
could be chased out by 12C-FU (HILLS and HOROWITZ, 1966; SELLS and CRUDUP,
1966); and (2) with actinomycin D, which inhibits new RNA synthesis, most of the
FU in the particles was lost during recovery even in the absence of exogenous U, and
the remaining FU particles were not converted to mature ribosomes (HILLS and HORO-
WITZ, 1966; ANDOH and CHARGAFF, 1965). Under similar conditions, ribosomal
particles formed in the presence of chloramphenicol were fully utilized once the
antibiotic was withdrawn. It was concluded that FU, during recovery, was ejected
from the ribosomal particles but then was reincorporated to form normal ribosomes.
Normal components of the abnormal particles were reutilized for ribosome forma-
tion. When U had been added to accelerate recovery from FU inhibition, it competed
with FU for reutilization, and some FU therefore was lost into the medium. It was
stated that the protein synthesized during FU inhibition was not used for normal
ribosome formation once the FU had been removed from the medium (I w ABUCHI
et al., 1966), although this point was not fully established; soluble protein apparently
was not preferentially used for this purpose, however.
The abnormal ribosomal pattern resulting from the effect ofFU on S. carlsbergensis
very slowly converted towards a more normal ribosomal distribution during recovery
in the presence of U (DE KLOET, 1968). Reincubation of 14C-FU-treated cells in a
medium containing the unlabeled analog led to loss of the label from the RNA,
indicating instability of the FU-RNA. These results resembled those reported for
E. coli (HILLS and HOROWITZ, 1966; SELLS and CRUDUP, 1966).
In B. cereus, where FU treatment exerted an even greater inhibition of ribosomal
synthesis than in E. coli, the addition of U 30 min after FU led to reversal of growth
inhibition for at least one generation. During this time FU was lost from the RNA and
ribosomal synthesis again resumed. On the other hand, if TdR was added to a culture
inhibited by FU, ribosomal synthesis did not recover, and growth inhibition con-
tinued (HAHN and MANDEL).
D. Conclusions: FU and Ribosomes

In considering all of the above information, a number of conclusions may be
reached which are similar for all the microorganisms studied.
1. FU strongly inhibited the formation of normal ribosomes. The new ribosomal
particles synthesized in the presence of the analog differed from normal in that:
a) they contained FU;
b) they were more sensitive to enzymatic degradation;
c) they had different sedimentation characteristics which became most pronounced
upon examination of the subribosomal particles. The 50S particle usually was missing
altogether, and the 30S particle was abnormal and heterogeneous by sedimentation
analysis. The ribosomal RNA, on the other hand, appeared to be more nearly normal,
even though FU replaced a considerable proportion of the normal U complement. In
various systems the 23S rRNA component was less readily formed.
2. FU, probably in the form of FU-rRNA, interfered with the combination of
proteins with ribosomal particles and prevented normal ribosomal maturation (HILLS
and HOROWITZ, 1966; IWABUCHI et al., 1966; NAKADA, 1965; KONO and OSAWA, 1964)
It did not diminish the total quantity of amino acids incorporated into polypeptides.
Whether this effect was due to an alteration in the physical or chemical properties of
the protein formed, or to the problems associated with binding to FU-containing
rRNA rather than to normal rRNA, is still unresolved.
3. A small amount of FU could apparently be tolerated in ribosomes since such
ribosomes appeared to behave normally.
4. During recovery from FU inhibition new ribosomes were formed from the
abnormal ribosomal particles following the selective elimination of much of the FU,
and reutilization of the remaining components.
Too little information was available on the effects of FU on ribosomes of plants
or mammalian tissues. At the present time, however, there is little reason to suspect
effects on ribosomes different from those described for microorganisms. The overall
effects of FU on labeling of subcellular particles of these tissues allowed for the
preferential synthesis of a large D-RNA fraction containing FU.
IV. FU and sRNA

The effect of FU on soluble RNA has not been investigated as extensively as that
on ribosomes. Frequently this fraction has not been resolved, in which case it is
designated sRNA. Where an effort of purification has been made, transfer RNA
(tRNA) fractions are isolated.
A. PU Incorporation and sRNA Alterations

In E. coli, radioactivity from labeled phosphate in the presence of FU, or 14C-FU,
was distributed about equally between ribosomes and sRNA (ANDOH and CHARGAFF,
1965) or favored ribosomes (HILLS and HOROWITZ, 1966), although comparisons
among different laboratories may be complicated by differences in extraction tech-
niques and growth conditions. In B. cereus, most of the RNA made after the addition
of FU was sRNA (HAHN and MANDEL, 1967).
In S. aureus, replacement of U by FU was greater in sRNA than rRNA (14%
versus 4%, respectively) (HIGNETT, 1964). In E. coli, this difference was small (Table
2) (ANDoH and CHARGAFF, 1965). The highest substitution of U by FU in tRNA
reported is 82%, which was obtained following special fractionation (LOWRIE and
BERGQUIST, 1968) (see below). It was calculated, however, that tRNA of E. coli
synthesized after the addition of FU had 100% of the U replaced by FU. In B. cereus
there was much more FU replacement in sRNA than in rRNA. TdR, which increased
total incorporation of the analog when added to the inhibited cells (REICH and
MANDEL, 1966), further increased the proportion of FU in sRNA compared to that in
rRNA (HAHN and MANDEL).
7*
100 H. GEORGE MANDEL
The sRNA peak labeled with FU eluted somewhat more slowly from methylated
albumin columns or sedimented more rapidly in sucrose gradients than a correspond-
ing normal sRNA isolated from E. coli (LOWRIE and BERGQUIST, 1968; HILLS and
HOROWITZ, 1966; ANDOH and CHARGAFF, 1965; SUEOKA and YAMANE, 1963) or
Trichoderma (GRESSEL and GALUN, 1966). This difference in behavior was due to
changes in physical properties of sRNA produced by the replacement of U by FU
(SUEOKA and YAMANE, 1963).
A new sRNA peak was demonstrated after labeling spores of the fungus, Tri-
choderma viride, in the presence of FU with either 3H-adenosine or 3H-FU (Fig. 7).
9000 30
8000
40
7000
50
6000
0- I
U 5000
;:!
';I!. 60 1000
Q;
.
u
L c: 800 _
4000
(L
u ~ 70
°E r.-
3000 III
c: 600'"'_
E 80 L
I-
2000 400 ~
1CXXl 200
Tube noo
Fig. 7. Methylated albumin column chromatography of extracts of RNA from Trichoderma
labeled with either 3H-FU or 14C-U, followed by chase with excess unlabeled U. Pattern
reveals three FU-Iabeled areas at left, corresponding to 4.4S (tube 5), 5S (tube 16) and an
intermediary peak (tube 8), whereas U-Iabeled peaks are only those corresponding to 4.4S
and 5S. This new peak was characteristic of inhibition by FU and was also demonstrable
when adenosine was used as label in presence of FU. Note lack of 3H but not 14C in DNA
peak (tube 25) and altered rRNA. - - - % transmittance; 0, 14C-radioactivity; .,3H_
radioactivity. (From GRESSEL and GALUN, 1966)
This new component had a sedimentation coefficient between the usual 4.4S and 5S
peaks and was stable during a subsequent chase with U. Its significance is as yet
unknown (GRESSEL and GALUN, 1966).
B. Fate of FU-sRNA During Recovery from the Analog

During recovery from FU inhibition, only a small percentage of the FU in sRNA
was ejected, in contrast to the much greater loss of the analog from rRNA. For
instance, while about 70% of U was replaced by FU in sRNA or the two rRNA
components after 30 min of inhibition by FU, after recovery only 9% of the U com-
ponent of the rRNA fractions consisted of the analog, while sRNA still had 33% of its
U replaced by FU (ANDOH and CHARGAFF, 1965) (Table 2). These results were con-
firmed by HILLS and HOROWITZ (1966) and IWABUCHI et al. (1966). However, lack of
information on biosynthesis during the recovery period makes it difficult to evaluate
how much of the analog was actually lost from this fraction, and how much was
merely diluted by the formation of normal sRNA.
C. Effect of FU on sRNA Composition

It was anticipated that FU might interfere with the formation of pseudouridylic
acid in sRNA (ANDOH and CHARGAFF, 1965), since the addition of a ribose moiety
to the C-5 position of polynucleotide uracil is required for the formation of this minor
base component of RNA. This, of course, is the position substituted by the fluorine
Table 3. Relative composition of tRNA from E. coli following treatment with FU. Radiophosphate
was used to locate and quantitate bases as ribonucleotides following alkaline hydrolYsis of tRNA
(From LOWRIE and BERGQUIST, 1968)
Component of tRNA Time after FU addition, in generation times
0 0.25 0.5 0.75 1.0 1.5 2.0
Cytidylic acid 24.8 23.8 24.1 24.2 24.3 24.4 23.8

Adenylic acid 19.0 19.1 19.0 19.1 19.0 18.9 18.9
Guanylic acid 30.4 30.1 30.0 30.0 31.5 30.5 30.7
Uridylic and 5-fluoro-
uridylic acids 17.6 19.1 20.2 20.2 20.3 20.3 20.4
5-FluorouridyIic acid 3.1 9.7 10.2 11.4 14.0 14.2
Pseudouridylic acid 1.65 0.95 0.27 0.18 0.14 0.13 0.11
Ribothymidylic acid 1.35 0.57 0.12 0.07 0.06 0.03 0.03
Inosinic acid 0.52 0.50 0.63 0.57 0.59 0.54 0.56
2-Methylinosinic acid 0.18 0.12 0.21 0.23 0.22 0.21 0.18
N2-Methylguanylic acid and
1-methylguanylic acid 0.06 0.08 0.05 0.06 0.06 0.06 0.05
N2-Dimethylguanylic acid 0.03 0.02 0.03 0.03 0.04 0.02 0.02
2-Methyladenylic acid 0.02 0.02 0.02 0.03 0.02 0.02 0.03
N6-Methyladenylic acid 0.01 0.02 0.02 0.02 0.01 0.01 0.02
5-Methylcytidylic acid 1.61 1.63 1.71 1.76 1.68 1.72 1.69
Guanosine 3 /, 5'-diphosphate 3.29 3.45 2.53 3.38 3.34 2.93 2.90
atom. The inhibition of the C-5 methylation of deoxyuridylate by FUdR had been
demonstrated earlier (COHEN et aI., 1958; BoscH et aI., 1958). Indeed it was demon-
strated that the administration of FO (but not FU) to the rat reduced the liver 1pMP
content from a normal of 20% of the uridine nucleotides of sRNA to about 12%
(WAGNER and HEIDELBERGER, 1962). Furthermore, the incorporation of 14C-orotate
into1pMP in the presence ofFOwas reduced to 14% of control, while the concomitant
conversion to UMP was about 45% of normal. FU administration had produced no
such effect (see also SKODA and HANDSCHUMACHER, 1963), but it was observed that FU
was not incorporated into liver RNA. FO, on the other hand, had been extensively
converted to FU derivatives, and then was incorporated into the liver RNA in the form
of FU and inhibited 1pMP formation specifically. SKODA and HANDSCHUMACHER (1963)
found no specific effect of FU on the urinary excretion of 1pR in mice or on the specific
activity of urinary 1pR following 14C-orotate administration.
LOWRIE and BERGQUIST (1968) have demonstrated clearly that incorporation of

FU into tRNA of E. coli produced no changes in major nucleotide composition other
than replacement of FU for U. On the other hand, the relative contents of the minor
base constituents, ribothymidylic acid and tpMP were exclusively and dramatically
reduced to values even below those anticipated if merely all new synthesis had stopped
(Table 3).
D. Effect of FU on Function of sRNA
Preliminary reports (GRas et aI., 1962; GRaS and NAONO, 1961) suggested that
sRNA from E. coli treated with FU possessed the same number of binding sites for
arginine, proline or tyrosine in vitro as did normal sRNA, and that FU-sRNA for
tyrosine functioned normally (NAKADA, 1965). On the other hand, GRAY and RACH-
MELER (1967) concluded that FU treatment had altered amino acid acceptor activity
for a number of amino acids tested. However, they compared tRNA which they had
prepared from FU-treated E. coli K12, with a commercial sample of E. coli B tRNA,
and in addition their incorporation values were abnormally low (LOWRIE and BERG-
QUIST, 1968). As a consequence, it is difficult to evaluate these findings. Using yeast
sRNA, EBEL et al. (1965) have also reported that FU altered the amino acid acceptance
differently depending on the particular compound studied. The mechanism was
uncertain and insufficient experimental detail was provided.
In the investigations of LOWRIE and BERGQUIST (1968) the presence of FU in the
tRNA, with activating enzymes from either control or FU-treated E. coli, did not
alter the capacity of acceptance of at least five amino acids, and the rates of loading
for the two amino acids tested, phenylalanine and lysine, were identical. Similarly,
the rate and extent of incorporation of phenylalanine or lysine into polypeptides in an
E. coli system, with poly U or poly A as messengers, respectively, were unaltered
when the FU-tRNA replaced the normal tRNA. It was concluded, therefore, that
FU-tRNA's for lysine and phenylalanine could function normally in the incorporation
of these amino acids (Table 4). When bacteriophage R17 RNA served as messenger
instead of the synthetic polymers, however, FU-tRNA did not transfer lysine into
polypeptides, and phenylalanine incorporation was reduced. Even in the presence
of normal tRNA, FU-tRNA inhibited the transfer of amino acids. This inhi-
bition of lysine incorporation was not due to miscoding, as tested by the relative
binding to ribosomes of FU- or control lysine-tRNA with copolymer messengers
consisting of varying proportions of A and G. It was reasoned that if the FU
in the anticodon sequence of lysine, normally UUU, was to behave like C rather
than U (see Section V Don miscoding), some FU-tRNA would then pair preferentially
with G rather than A of the copolymer and would bind increasingly as the relative
content of G in the copolymer was enhanced. This effect would not be true for
control tRNA, since U does not normally base pair with G. However, as the relative
content of G to A of the copolymers was varied, the ratio of binding to ribosomes by
FU-tRNA and tRNA remained constant, and therefore miscoding by lysine was ex-
cluded. It is significant, however, that the binding of FU-tRNA was consistently only
70% of that of control tRNA. When calculated for the completely FU-substituted
tRNA, this reduction in ribosome binding would be about 50%.
It is intriguing to speculate on the mechanism of inhibition of lysine transfer by
FU-tRNA as programmed by R17 RNA. Since tpMP forms part of the anticodon
sequence of yeast tyrosine-tRNA (MADISON et al., 1966), replacement of this minor

base component by FU could have important consequences. Unfortunately, there is
as yet no definitive evidence for such an effect. LOWRIE and BERGQUIST (1968) believe
that the translation of the R17 message, which functioned largely for phage coat
protein production, was inhibited after the third of the four phenylalanine residues in
the coat, and therefore the subsequent translation of the cistron leading to incorpora-
tion of lysine and the fourth phenylalanine was halted. This mechanism, which would
Table 4. Stimulation of polypeptide vnthesis in vitro by control and

FU-tRNA. R1l refers to bacteriophage RNA which served as
source of mRNA. S26 was another strain of E. coli from which
tRNA had been prepared. Reaction mixture consisted of S30 frac-
tion, tRNA, messenger source, 14C-amino acid and 19 unlabeled
amino acids, and usual other components. Amino acid incorporation
expressed as fLfLmoles/100 fLg ribosomes/20 min
(From LOWRIE and BERGQUIST, 1968)
mRNA tRNA Amino acid % stimulation
incorporated
14C-phenylalanine
Poly U 172 0
Poly U Control 540 214
Poly U FU 527 206
Poly U 526 550 220
R17 37 0
R17 Control 59 60
R17 FU 49 32
R17 526 71 92
14C-Iysine
Poly A 50 0
Poly A Control 105 110
Poly A FU 96 92
Poly A 526 112 124
R17 32 0
R17 Control 57 81
R17 FU 29 0
R17 526 65 104
agree with the results of Table 4, could well be due to the lack of functional activity
of an FU-tRNA specific for some amino acid other than lysine or phenylalanine. It
was suggested that this amino acid might be tyrosine.
E. HighlY Purified FU-tRNA

Studies on the function of tRNA containing FU or, for that matter, any other
analog-containing components with long half-lives are complicated by the unavoidable
presence of the normal components synthesized by the cells before the addition of the
analog. Thus, experiments on FU containing tRNA really deal with mixtures of FU-
tRNA and normal tRNA, and it is necessary, therefore, to ascertain the functions of
undiluted FU-tRNA. A preliminary report by SUEOKA and YAMANE (1963) revealed
an extra component when arginine or phenylalanine· charged tRNA from FU-treated

cells was eluted on methylated albumin columns, in comparison to the corresponding
elution pattern for control cells. The tRNA fractions from FU-treated cells could thus
be resolved into one component corresponding essentially to tRNA containing little
FU (16% U replaced by FU) and synthesized mainly before the introduction of the
analog, and another component consisting of the tRNA containing the bulk of the
FU (82% U replaced by FU) and synthesized after the addition of FU (LOWRIE and
BERGQUIST, 1968) (Fig. 8). If the resolution were absolute, one would expect a com-
plete separation of the normal tRNA from the FU-tRNA.
Preliminary experiments with the FU-tRNA again indicated ability to accept
amino acids. Lack of quantitative loading was believed to have been due to partial
0..5 to. .--------------------------------------------,20.
I 0..8
--
0.4 15 -;
:::t.
a
E
CD
N
0.3
?:
.~
<5
0..6 _~~~- -- ---- __ - - 0
'"S
E -- 10. x
d ::E
.., a..
'c"
u
0.2 Ci 04
III
u
"(;
.0
U1
.0 5
« 0..1 0..2
0.
Fraction no.
Fig. 8. Methylated albumin column chromatography of 3H-phenylalanine-tRNA and 14C_
phenylalanine-FU-tRNA. By this procedure it was possible to resolve amino acid-charged
FU-tRNA into two components, one of which behaved like the normal tRNA component
formed before the addition of FU to E. coli, and the other was the highly FU-substituted
amino acid-charged FU-tRNA. - - - Absorbance at 260 mIL; 0, 3H-radioactivity; •• 14C_
radioactivity; - - - salt gradient. (From LOWRIE and BERGQUIST. 1968)
degradation. This FU-tRNA possessed less sharply defined thermal transition and a
diminished hyperchromicity than normal tRNA, indicating a less ordered secondary
structure.
F. Conclusions: FU and sRNA
FU was shown to be extensively incorporated into sRNA, and complete replace-
ment of U in this fraction appears to take place. Some evidence was described for the
formation of a new macromolecular component in this fraction which contained the
analog. The physical properties of tRNA differed slightly from those of control
tRNA, and it appears that certain functions of tRNA may be selectively altered by
analog incorporation. Since 1j!MP content is greatly diminished by FU treatment,
possibly incorporation into proteins of amino acids such as tyrosine, whose anti-
codon can contain '1pMP, may be specifically affected. Alternatively, it is possible that
continued to be synthesized in the presence of FU which contained appreciable

quantities of the analog (KEY, 1966). The "D-RNA" which was essential for growth
of the plant tissues, also continued to be present in corn mesocotyl and radish coty-
ledon (KEY and INGLE, 1964) and the Xanthium bud (CHERRY and VAN HUYSTEE,
1965). It is apparent that in the yeast and the plants a messenger-like fraction exists
which continues to be formed in the presence of FU and into which the analog was
incorporated.
A previous section (III B) described the formation of abnormal ribosomal
particles with messenger activity. These particles also contained the analog when FU
was present during their formation (IwABucHI et aI., 1966; SELLS and CRUDUP, 1966;
NAKADA, 1965).
B. FU Incorporation into RNA Viruses
Since viruses and phage upon infection greatly reduce normal nucleic acid syn-
thesis of the host while permitting extensive replication of viral polynucleotides, the
effects of a pyrimidine analog on viral nucleic acid synthesis could be studied speci-
fically. The RNA of these viruses possesses genetic information and thus acts as
mRNA.
1. Tobacco Mosaic Virus
Among the first reports of FU on virus synthesis was that of GORDON and STAEHE-
LIN (1958) who inoculated tobacco leaves with TMV in the presence of FU and
collected virus at a yield of 50% of normal. Analysis of this virus indicated 30%
replacement of U by FU, without additional alterations in RNA composition.
DAVERN and BONNER (1958) independently reported similar findings. The effect of
FU on virus yield was greatest when the analog was added immediately after infection
and could be completely reversed if UR (STAEHELIN and GORDON, 1960) but not U
(DAVERN and BONNER, 1958) was present concomitantly with FU. The virus content
of FU or its yield were independent of the plant host in which the virus was produced
(HOLOUBEK, 1963; STAEHELIN and GORDON, 1960). Preincubation of the plant leaves
with FU reduced virus yield upon infection.
FU-containing virus (47% of U replaced by FU) possessed normal sedimentation
characteristics and size distribution (GORDON and STAEHELIN, 1959) as determined by
electron microscopy, even though it contained up to 800 molecules of FU in the
nucleic acids of a virus particle made up of about 6,000 nucleotides (STAEHELIN,
1960). The progeny of FU virus grown without FU again had normal composition
(GORDON and STAEHELIN, 1959). Although the FU-virus, in the absence of FU, had
normal infectivity when tested by counting the number of lesions produced after the
local application of virus to host leaves (in comparison with an equal concentration
of control virus), the rate of replication of the FU-virus was reduced when virus
production was measured (GORDON and STAEHELIN, 1959). The latter procedure was
considered a more sensitive criterion.
Although some differences have been reported in the literature regarding in-
fectivity of FU-TMV, it is clear that the analog did not make the resulting RNA non-
functional. Most authors concluded normal infectivity (LOZERON and GORDON, 1964;
HOLOUBEK, 1963; GORDON and STAEHELIN, 1959). DAVERN and BONNER (1958)
reported a reduction in infectivity only for TMV with high FU content. Even in
virus in which 36% of U was replaced by FU, infectivity was reduced by only 50%
FU may replace a 'lj'MP nucleotide in the anticodon sequence. It is known, however,

(see Section VI on proteins) that effects of FU on amino acid incorporation are diffi-
cult to document and are quite subtle, and thus large-scale alterations in amino acid
recognition as a consequence of FU incorporation are unlikely. Further investigation
may reveal to what extent FU-sRNA contributes to the overall responses associated
with FU. For future experiments additional information is required regarding the
functions of the minor base components of the tRNA species.
v. FU and mRNA
A . FU Incorporation into mRNA
Pulse labeling of bacterial cells with labeled FU readily revealed that the analog
was incorporated into a subcellular RNA species distinct from rRNA or sRNA
(GROS et aI., 1961) (Fig. 9). This fraction had been associated with messenger activity
~' I\
50 ~
i I ~
I II ,
I
I I • I '
-;;-
0.400
I
I
I
,6 I
I
""A /'
:, I1"'\\ I>: \ 1:' I
20
~ j
I
~ I. {
.
0
I b
~ I
I
\ I ' \ I -
.~
0 /0
ill I
N
0
0/
I V \
I
(j)
I
u I
0.200
c
f 10
-e" I
I
\.
0
U1 sl
.D I
« d
I
I
•
P
qU.,i7
."sf" • •
~ ....<r • •
.~ . o
0 10
Tube no.
Fig. 9. Sucrose gradient sedimentation analysis of extract of E. coli after a 30-sec pulse of
14C-FU. Radioactivity appeared in region corresponding to mRNA. 0, absorbance at
260 mIL, e, radioactivity. (From GROS et al., 1961)
(SINGER and LEDER, 1966). Longer periods of exposure to 14C-FU resulted in the
more extensive incorporation of the analog into ribosomal particles and sRNA. Var-
ious other investigators have confirmed this observation (HOROWITZ and KOHLMEIER,
1967; HILLS and HOROWITZ, 1966; SELLS and CRUDUP, 1966; NAKADA, 1965; NAKADA
and MAGASANIK, 1964).
Saccharomyces carlsbergensis incubating in the presence of FU synthesized large
quantities of a high molecular weight RNA which had a base composition inter-
mediate between that of RNA and DNA, in which about 75% of the U had been
replaced by FU (DE KLOET, 1968). In the soybean hypocotyl a DNA-like RNA
(KRAMER et aI., 1964). Since it is unreasonable to assume that two populations of

TMV were synthesized in which one type was entirely normal and the other com-
pletely substituted by the analog (GORDON and STAEHELIN, 1959), it must be con-
cluded that FU functioned like U during infection and replication.
Although the earlier studies detected no virus mutants (GORDON and STAEHELIN,
1959), KRAMER et al. (1964) have reported an increase in the number of mutants in the
FU-TMV population. The relative ability of TMV to produce necrotic lesions on
two species of tobacco plants, Java and Xanthi, was used as a measure of mutation
rate. These mutants appear to have an alteration in the amino acid content of the
virus protein [WITTMANN-LIEBOLD and WITTMANN, 1965 (2)], and are described in
Section VI D.
FUdR had only a slight inhibitory action on virus production, in contrast to FO
and FUR, which were active (STAEHELIN and GORDON, 1960). Both FO and FUR
were incorporated into TMV as FU.
2. Polio Virus
Similar to the results with TMV, FU reduced the yield of polio virus produced
in cultures of infected HeLa cells in the presence of analog, and was incorporated into
virus RNA replacing 36% of the U (MUNYON and SALZMAN, 1962). The FU-virus,
in the absence of FU, possessed unaltered infectivity, and the size and rate of forma-
tion of plaques resembled that of control virus (confirmed by TERSHAK, 1966, 1964).
The virus specificity for different host cell lines was unaffected by the presence of the
analog.
Preincubation of host cells with FU had no effect on subsequent yield or rate of
virus formation, indicating that the FU effect on virus synthesis was associated with
the presence of the drug during or after infection (TERSHAK, 1964). UR partially
overcame the reduction of virus yield in the presence of FU (COOPER, 1964).
Polio virus growing on embryo rabbit kidney cells in the presence of higher
concentrations of FU showed the results of mutation (COOPER, 1964). In addition,
polio virus polymerase from cells infected with FU virus had altered properties. These
observations are discussed in Section VI D.
3. Phage MS 2
The yield of MS 2, an RNA phage growing on E. coli, was inhibited by FU
(SHIMURA et aI., 1965), depending on the time of addition of the analog. When FU
was added immediately after infection there was practically no phage produced; with
longer intervals between infection and the addition of FU, progressively greater
amounts of infectious particles were formed, with a maximum yield of about 50%
of control. These phage particles had up to 80% of U replaced by FU.
It was possible, using cesium chloride density gradient centrifugation (Fig. 10),
to separate the FU-substituted phage from control phage synthesized before the
addition of the analog (SHIMURA and NATHANS, 1964). The slightly higher density of
FU phage (1.436) than control phage (1.424) was associated with the RNA compo-
nent, and was directly related to the percent of U replaced by FU. Sedimentation
values for FU- or control RNA were almost identical, and S values for their respective
RNA components were also comparable to each other, indicating little change in
gross structure. Melting profiles differed only slightly from normal and showed
slightly less hyperchromicity.
The specific infectivity of the progeny phage varied between 5% and 25 % of

control. Thus, even with 80% ofU replaced by FU, the FU-phage possessed biological
activity. The reduction in infectivity measured as the ratio of plaque forming units to
ultraviolet absorbance is demonstrated in Fig. 11. The major fraction of the infective
phage banded in a sharp peak corresponding to the density of normal phage, whereas
a smaller peak of infectivity coincided with the absorbance of the FU-phage. By this
means of separation SHIMURA et al. (1965) have purified the FU-phage from conta-
minating normal MS 2.
""
tj,\. ·
, II
I \ 1\ 300
1.50
I.:r~--txI
!) \
1000
N
~
a.: '\ \ 200 _
'".!...
•
...,r.-
,
~ 1.40
a.. , I x
() \ ~
500 ,I
,
,
II a..
()
--'. ---' il ~
100
~
iJ·
Fraction no.
Fig. 10. Cesium chloride gradient sedimentation analysis of E. coli phage. Sequential labeling
in same flask: 3H-adenosine, followed by chase, to label normal phage; thereafter FU and
32P-phosphate to label more dense FU-phage. e, 3H radioactivity; 0, 32p radioactivity;
x, buoyant density. (From SHIMURA et ai., 1965)
A subsequent report by the same authors (SHIMURA et aI., 1967) revealed that
phage produced in the presence of FU consisted of two components. In addition to
the somewhat more dense than normal infectious FU-phage just described, another
particle was produced which had a lighter buoyant density than control phage. These
new particles were defective in that they were noninfectious. Their RNA content was
only 65% of that of normal phage, and again had 80% of the U replaced by FU. The
particles did not adsorb to E. coli receptor sites and showed diminished reactivity
with antiserum. The RNA from the particles was heterogeneous, had a diminished
sedimentation coefficient, but had the same T m and hyperchromicity as the RNA of
infectious FU-phage. The RNA from the defective particles exhibited increased
messenger activity in directing the synthesis of phage-specific protein compared to
control phage RNA. (See also Section VI D.)
4. fCAN-1
FU inhibited the growth of fCAN-I phage if added early after infection, but still
reduced yield when added later (DAVERN, 1964). Uridine completely restored growth
when added together with FU. The ability of phage-infected cells to yield infective
progeny declined with time of incubation in the presence of FU. Some of the particles
produced were noninfectious even though they contained phage-specific antigens.
About 25% of the progeny were mutants with increased temperature sensitivity
compared to the wild type.
0.30.0. 6
0.200 "~4
0..10.0. - 2
.-
~ 0
~ ""
~
0 )(
<D 0. 0.
N
So. 55 E
0 0..600 ......
3 :j
'"u
C
Cl
~
.0
(5
Vl
.0
~ 0..40.0. 2
Fraction no.
Fig. 11. Relative infectivity of MS2 phage fractionated by cesium chloride gradient sedi-
mentation. Top, absorbance (e) measured for control and FU phage (see also Fig. 10).
Infectivity (0) was greater for normal phage (peak near tube 44) than FU phage (peak near
tube 40), although FU phage was still infective. FU phage (fraction 39) was then purified by
recentrifugation and slightly different elution procedure. Infectivity of FU phage is de-
monstrated at bottom. x, buoyant density. (From SHIMURA et al., 1965)
5. f2
Bacteriophage f2 synthesized in the presence of FU was defective, and its proper-
ties resembled those described for MS 2 (LODISH et ai., 1965). The particles which
contained FU reacted to anti f2 serum but were nonviable, lacked a normal comple-
ment of RNA, contained heterogeneous RNA, and did not adsorb appreciably to
cellulose nitrate filters (LODISH et ai., 1964). A variable amount of the FU-phage was
more dense than the control phage. The FU-defective phage resembled closely in its
characteristics the "su-1 dead" phage mutant of f2 which had immunological proper-
ties corresponding to those of the wild phage, lacked a normal complement of RNA,
and did not adsorb to bacteria or to cellulose nitrate filters.
6. R17
GRAHAM and KIRK (1965) have elaborated the time course of phage R17 produc-
tion. Added at time of infection, FU blocked phage synthesis and that of infectious
RNA. 5 min after infection, FU still blocked infectious RNA formation, but active
phage were formed from the RNA made before the addition of the analog. Uracil
reversed this effect if added within 20 min of infection. No loss of infectivity was
reported for phage in which 28% of U had been replaced by FU, although the yield
of this phage had been reduced to 15% of normal.
C. FU- Viruses and Altered Sensitization to Ultraviolet Light

This area of investigation is still insufficiently advanced to associate a specific
RNA with the enhanced sensitivity to ultraviolet radiation. The presence of FU in the
RNA of bacterial cells did not make these cells more sensitive to radiation by ultra-
violet light, in contrast to microorganisms in which bromouracil had been incor-
porated into the DNA (KAPLAN et al., 1962). Since the only reports of increased radio-
sensitization following FU incorporation into RNA deal with viruses, this informa-
tion is presented here.
The enhanced radio sensitization of cells following analog incorporation into DNA
prompted BECAREVIC et al. (1963) to test the effect of ultraviolet light on FU-RNA
in a system where this RNA was the sole carrier of genetic information. Tobacco
mosaic virus containing FU showed a fivefold increase in susceptibility to ultraviolet
radiation compared to the normal virus, as measured by reduction in virus infectivity.
Since nucleic acids isolated from FU-TMV were more susceptible to ultraviolet light
also, the photosensitivity was apparently linked to the RNA portion of the virus
(LOZERON and GORDON, 1964). Sensitization was enhanced as the FU content was
increased up to 50% replacement of U, and was greater at a wavelength of radiation
of 296.7 mil than of 253.7 mil. Although the mechanism of ultraviolet sensitization
is not clear, absorbancy differences between FU and U were believed to contribute to
the relative susceptibility at different wavelengths.
The relationship of the formation of 5,6-dihydro-6-hydroxy-FU derivatives from
the corresponding FU compounds upon radiation by ultraviolet light, and the pos-
sible toxicity of these compounds, are not known (FIKUS et aI., 1964; LOZERON et al.,
1964).
TERSHAK (1964) established increased sensitivity of FU-substituted polio virus
to ultraviolet light, although progeny of this virus produced in the absence of FU had
not inherited this property. The sensitivity to ultraviolet light was associated with the
infectious center derived from the viruses. Kinetic studies revealed that at least part
of the original RNA that had invaded the cell remained functional for 3 h or more.
The sensitization was not associated with the virus-producing ability of the host cells
or the incorporation of the analog into the host. Cells infected with normal virus
showed a higher degree of resistance to ultraviolet light 3 h after infection than those
infected with FU-virus, again indicating the increased sensitivity of the FU-virus to
ultraviolet light.
D. Miscoding by FU-mRNA
Because of the intermediary role of mRNA in transcription and translation, it is
possible to visualize two major alterations resulting from the incorporation of FU
H H F
\c H ---------1;tc
C'N-H--------1;tC c
c H
~ N _______ H _______ H
C C
~c RP RP
o o
A U FU (keto form)
H
I H o F
cO-:H~~H
H
~P ~:*H
O N_H--------
_______________ 0
/
H
G c FU (enol form)
Fig. 12. Potential base pairing of FU in keto form with adenine like uracil, or in enol form
with guanine like cytosine. Uracil, with higher pKa than FU, is entirely in keto form
Table 5. Schemes for miscoding due to FU-mRNA

Condition Base pairs Transcribed into Recog- Alteration from
DNA mRNA as nized by normal in code
sRNA as recognition
I 1 normal T-A U U
2 normal A-T A A
3 normal G-C G G
4 normal C-G C C
5 transcription error C-G b FU U C ..... U
6 transcription +
translation error C-G b FU b C (error corrected)
II 1 normal T-A U U
2 translation error T-A a FU b C U ..... C
3 replacement T-A FU a U (none)
Virus Replicating
RNA form
III 1 normal A U U
2 normal G C C
3 normal C G G
4 normal U A A
5 most likely replication FU a A A (none)
6 replication error FU b G G A ..... G
7 replication error G b FU U C-.-U
8 replication error A a FU b C U -.- C
a FU pairs like U, b FU pairs like C.

into mRNA. Both are related to the lower pKa of FU compared to U, and the re-
sultant greater enolization of FU at physiological pH values. Thus, FU may have a
greater tendency than U to pair with G (Fig. 12). The major schemes for the conse-
quent miscoding are shown in Table 5.
Mechanism I. Some of the G in the transcribed strand of DNA might pair with FU
instead of C, and RNA polymerase would then incorporate the analog into mRNA
instead of C (Table 5, 1-5). Once in the mRNA, FU would then behave as U during
the translation process. Thus, sRNA would recognize a U instead of a C. Clearly, two
sequential mispairings would be equivalent to a correction to normal (Table 5, 1-6).
80
No FU FU
701-
f! 60 -
C
0
U
+...
50 -
'0
.,
C
.,~
a.
40-
.!::
."
30 -
~
>.
~ 20 -
c o HB 118 & N 12
.c o NT 332
a.. • AP 100
& M 12 • 131
lO- • AP 100
• 131
o 10 20 30 40 50 60 0 10 20 30 40 50 60
Minutes of incubation after infection (26°C)
Fig. 13. Yield of various rII phage mutants in E. coli in the absence (left) and presence (right)
of FU. Certain mutants proliferated only after the addition of FU which had "corrected"
original mutation. (From CHAMPE and BENZER, 1962)
Mechanism II. FU could be incorporated into mRNA in place of U but then occa-
sionally base pair as C instead of U during translation (Table 5, Il-2).
Some evidence exists for both possibilities. CHAMPE and BENZER (1962) made the
remarkable observation that certain rIl mutants of phage T4, which normally were
unable to grow on E. coli K because of a base alteration in their DNA, proliferated
after the addition of FU (Fig. 13). This analog thus had reversed the defective pheno-
type and had allowed almost normal proliferation. This stimulation of growth by FU
was prevented by U. FU addition was most effective immediately following in-
fection during the period of active synthesis of mRNA while rRNA and sRNA
were not formed. The mutants were defective because of an alteration in DNA (AT
instead of GC) which thus transcribed U instead of C into mRNA. The incorporation
of FU into mRNA instead of this specific U then corrected the mutation by again
coding as C (Mechanism II). Such a result was observed in almost half of the mutants,
suggesting that only one strand of DNA was transcribed. There was also some indica-
tion that FU could correct mutants possessing a GC instead of an AT pair in DNA,
and thus could be incorporated instead of C into mRNA (Mechanism I). However,
this correction was uncommon.
It is quite likely that these explanations are oversimplifications. Because the phage
particles replicated in the presence of FU, additional mechanisms to explain the ob-
servation become possible, since an extra step is introduced into the process [see also
(HEIDELBERGER, 1965) and Section VI D below]. An example of this replication
process is indicated in Table 5, III, where a replicating form is produced from mRNA
which then acts as template. The incorporation of FU into either strand of the repli-
cating form permits additional substitution (Table 5, III 6 to 8).
Effects similar to the phage mutants were noted with phosphatase-negative E. coli
mutants whose defect could be suppressed by FU (CHAMPE and BENZER, 1962). Thus,
it has been possible to convert a nonsense mutation to a sense configuration in 13 out
of 15 mutants (GAREN and SIDDIQI, 1962). In the presence of FU the amount of
enzyme synthesized in some cases was the same as that of normal cells, and appeared
normal by electrophoretic mobility, heat stability and specific activity. ROSEN (1965)
reported that FU produced a 200-fold increase in alkaline phosphatase synthesis
("synthesis" based on sensitivity to 5-methyltryptophan inhibition) in E. coli mutants.
Again the enzyme appeared to be identical with that of the wild type as judged by
behavior during chromatography, electrophoretic mobility, stability to a proteolytic
enzyme and temperature dependence. Synthesis of the wild type enzyme was also
doubled by FU. This unexplained observation unfortunately complicates the pheno-
menon.
FU also permitted growth of 10 of 24 mutants of Neurospora induced by nitrous
acid and differing from the wild type by a single DNA base pair substitution. This
growth stimulation by FU was believed to be due to reversion in phenotype but not
genotype, and was reversed by uridine (BARNETT and BROCKMAN, 1962). The in-
crease in growth in the presence of FU of Neurospora mutants lacking glutamate
dehydrogenase may be another example of phenotypic reversal. It, too, was compli-
cated by the fact that the wild strain grew better in the presence ofFU, suggesting that
the FU effect may have been based at least in part on a different mechanism (SUN-
DARAM, 1967).
BUJARD and HEIDELBERGER (1966) have attempted to provide evidence for
Mechanism I but were not able to establish significant transcription errors during the
formation of FU-mRNA. Using a synthetic DNA consisting of one strand of alter-
nating T-G sequences and the other of alternating A-C sequences, the RNA poly-
merase was made to copy only the former strand and to prepare A-C sequences of
RNA. The incorporation of added 32P-FUTP, or 32P-UTP as control, was followed
by hydrolysis to measure incorporation of FU or U into positions adjacent to A in
the RNA synthesized. No more incorporation ofFU than U could be shown, and the
frequency of incorporation of these bases (errors) was less than one in 3,000 cytosines
in RNA. This technique probably ",as limited by an unavoidable trace of DNA in the
added RNA polymerase which then acted as template for the observed incorporation
ofU or FU.
8 Moleculat and Subcellular Biology, Vol. 1
E. Conclusions: FU and mRNA

The incorporation of FU into mRNA of growing cells has been established. Be-
cause of the short-lived nature of this molecular species in bacteria, no analytical data
on replacement of U by FU are available. In plants, where this fraction is more stable,
incorporation of FU was extensive. In viruses, where RNA exists solely for the pur-
pose of viral replication, FU was readily used for RNA formation, and FU-RNA
functioned sufficiently to allow growth (though slowed) and a high degree of in-
fectivity. Careful analysis of the FU-viruses, however, provided some evidence of
mutations involving differences in viability, immunological properties and physico-
chemical characteristics. These will be described in greater detail in the next section
(VI D).
VI. FU and Protein Synthesis

Because of the early report on incorporation of FU into mRNA (GROS et al.,
1961), a search was begun for evidence of miscoding leading to incorrect translation
of messenger RNA. The inhibition of the formation of inducible enzymes (HORO-
WITZ et al., 1958), and claims for major changes in amino acid content resulting from
FU-treatment [NAONO and GROS, 1960 (1)] were rationalized as a result of FU-mRNA
formation. Subsequent studies, however, have suggested that these conclusions may
represent oversimplifications, as discussed below.
A. Effect of FU on Composition of Total Protein

NAONO and GROS [1960 (1)] were first to conclude that FU had altered the incor-
poration patterns of specific amino acids into protein, and amino acid binding to RNA
of E. coli or B. megaterium. The incorporation of labeled proline and tyrosine, for
example, was depressed in the presence of FU, that of arginine was stimulated, where-
as that of nine other amino acids was almost unchanged. Alkaline phosphatase
isolated and purified from E. coli grown with 14C-proline in the presence of FU, had
a specific radioactivity 25% lower than normal [NAONO and GROS, 1960 (2)]. On the
other hand, a DEAE chromatographic elution profile of an extract of sulfate-labeled
cells grown in the presence of FU indicated that amounts and classes of the various
proteins were unchanged from those of extracts for control cells (GROS and NAONO,
1961). It was concluded that the composition of total protein and enzymes was
altered during growth in the presence of FU, even though amino acid content had not
been measured.
CERNA et al. (1963) also observed differences produced by FU on the incorpora-
tion of amino acids into E. coli. The inhibition by FU of tyrosine incorporation was
greater than that of proline and arginine, differing from the report ofNAoNo and GROS
[1960 (1)]. The relative degree of inhibition of amino acid incorporation increased
with the time of exposure to FU, implying that the effect may have been unrelated to
FU-mRNA formation, which should have produced an immediate effect.
It is difficult to reconcile these observations with the bulk of the evidence which
contradicts an effect on composition of total protein formed in the presence of FU. It
is known now that pool sizes of amino acids may be altered by FU (KEMPNER and
MILLER, 1962), and cellular permeability may have been affected by FU, leading to
the utilization of compensatory pathways for amino acid synthesis. Since cell wall
formation in microorganisms, including E. coli (TOMASZ and BOREK, 1962; ROGERS

and PERKINS, 1960), is known to be impaired by FU, further effects on amino acid
incorporation may be anticipated. The selective effect on amino acid incorporation
described by CERNA et al. (1963) could not be reversed by U, suggesting that it might
even be related to some secondary effect resulting from inhibition of DNA synthesis.
These reports have been discussed here at length because they have been widely
quoted, even though most other investigators could not confirm their conclusions.
Among the other reports, ARONSON [1961 (2)] found no effect of FU on uptake
of labeled sulfate, proline or leucine into E. coli protein, and demonstrated by amino
acid analysis that protein composition was unaffected by FU. HARBERS et al. (1959)
could observe no alteration in the incorporation of lysine into various protein frac-
tions of Ehrlich ascites cells, and the incorporation of valine into total liver protein
of the guinea pig was unchanged by FU (NEMETH, 1962). KEMPNER and MILLER
(1962) reported normal composition of protein of Candida utilis, even though FU had
altered the pool sizes of individual amino acids. In B. cereus treated with FU, the in-
corporation of labeled tyrosine, proline and arginine was inhibited to precisely the
same extent as was total protein synthesis (REICH and MANDEL, 1966). Samples of
RNA from control and FU-treated E. coli were equally effective in stimulating the
in vitro incorporation of leucine, serine, arginine or proline (SOFFER, 1964).
It is apparent, of course, that minor changes in amino acid composition produced
by growth in the presence of FU would be undetected since analysis of total protein is
relatively insensitive. Evidence for subtle changes in composition has now been
produced in two laboratories during amino acid sequence analysis of viral or phage
mutants [TOOZE and WEBER, 1967; WITTMANN-LIEBOLD and WITTMANN, 1965 (1)].
See Section VI D.
B. Effect of FU on Enzyme Synthesis
HOROWITZ et al. (1958) first reported that FU prevented the induction of fJ-
galactosidase in an E. coli U-requiring mutant while total cellular protein biosynthesis
continued. It was then concluded that in E. coli inducible enzymes were no longer
synthesized in the presence of FU, whereas among the constitutive enzymes some
continued to be formed and others were no longer produced. Thus, it was established
quite early that the effect of FU was not directed exclusively at inducible enzymes.
HOROWITZ et al. (1960) also observed that the addition of FU before the inducer
lactose completely blocked induction of fJ-galactosidase (see also NAKADA and
MAGASANIK, 1964), whereas the simultaneous addition merely reduced the rate of
enzyme formation. The addition of the inhibitor 2 min after the inducer allowed for
additional enzyme synthesis, but still less than that of a control culture (PARDEE and
PRESTIDGE, 1961). After 4 min following induction and removal of the inducer, FU
no longer was active in inhibiting enzyme synthesis (NAKADA and MAGASANIK, 1964).
The simultaneous presence of U partially restored the induction.
A large body of information has accumulated on various enzymes whose syn-
thesis was inhibited by FU while protein production continued. These results have
been tabulated (Table 6). In these examples the inhibitory effect of FU was probably
related to an effect on RNA formation, and frequently could be reversed by U. It
is important to emphasize that other enzymes continued to be formed in the presence
of FU. For example, two inducible enzymes were synthesized in Saccharomyces
8*
carlsbergensis even though 70% of U had been replaced by FU in the new RNA
(DE KLOET, 1968). See Table 6.
The mechanism of the specific inhibition by FU of certain enzymes, generally
acknowledged to be due to the incorporation of FU into mRNA, resulting in failure
to code for specific proteins, has now been challenged. New evidence suggests that
repression of enzyme synthesis was due to the accumulation of catabolites in the
medium of cells grown in the presence of FU (HOROWITZ and KOHLMEIER, 1967). It
is recognized that catabolite repression can inhibit ,a-galactosidase synthesis (NAKADA
and MAGASANIK, 1964). It has now been observed that ,a-galactosidase could be form-
ed in E. coli in the presence of FU depending on the supply of nutrients. For example,
in the presence of glycerol, a good carbon source, practically no enzyme was synthe-
sized by the FU-treated cells (Fig. 14 a). However, when succinate, a poor carbon
source, was substituted for the glycerol (Fig. 14 b), or when the glycerol was re-
moved (even 60 min after the FU addition) (Fig. 14 c), or when oxygen was ex-
cluded, almost normal quantities of ,a-galactosidase, in relation to the rate of total
protein synthesis, were produced (HOROWITZ and KOHLMEIER, 1967). During this
time FU was incorporated into the mRNA fraction (HOROWITZ and KOHLMEIER,
1967; NAKADA and MAGASANIK, 1964), thus implying that FU-mRNA could be
functional with respect to ,a-galactosidase formation. On the other hand, catabolite
repression inhibited the formation of ,a-galactosidase mRNA (NAKADA and MAGASA-
NIK, 1964). The continued incorporation of FU into the messenger fraction, therefore,
apparently was not specific for this enzyme. Normal production of ,a-galactosidase in
the simultaneous presence of U and FU was cited as further support for the independ-
ence of ,a-galactosidase synthesis and the concurrent FU-RNA formation (HOROWITZ
and KOHLMEIER, 1967). However, the necessary details regarding incorporation of
FU into mRNA or the relative replacement of U by FU under these conditions were
not available. Nevertheless, the dependence on the environmental conditions of the
quantity of ,a-galactosidase formed in the presence of FU strongly suggests that
catabolite repression was at least partially responsible for the inhibition of p-galactosi-
dase synthesis by FU. It is not known at this time whether the FU-related inhibition
of the formation of other enzymes was also associated with catabolite repression.
Partial support for the belief that FU-mRNA formation was not responsible for
the decrease in ,a-galactosidase formation comes from the report of BEN-HAMIDA and
SCHLESSINGER (1965). These authors observed that enzyme synthesis in the presence
of the inducer continued for a short time whether FU-RNA was being synthesized or
mRNA synthesis was being inhibited (completely, presumably) by actinomycin or
proflavine.
C. Ejject oj FU on Function and Pf?ysical Properties oj Protein

NAoNo and GROS [1960 (2)] reported that purified alkaline phosphatase from FU-
treated E. coli, though having normal enzymatic activity, Ks and Kr values, was more
rapidly inactivated by heat (Fig. 15). Unfortunately no additional information on
this system has become available. BUSSARD et al. (1960) have proposed that ,a-galac-
tosidase from constitutive strains of E. coli exposed to FU consisted of a protein
giving a specific serological cross-reaction to normal ,B-galactosidase but having
diminished enzymatic properties. No qualitative changes in the inducible enzyme
were produced by FU. NAKADA and MAGASANIK (1964) have confirmed that ,a-
Table 6. Effects of PU on enzymatic activity
Enzyme Source Effect Comments Reference ~
S-
o
0
p-galactosidase E. coli inhibition inducible and constitutive; HOROWITZ et aI., 1960; .a
0
occasionally reversed by U; GROS and NAONO, 1961 ;;J
decreased enzyme specific activity ::t.
0
Succinate dehydrogenase E. coli stimulation constitutive HOROWITZ et aI., 1960 t:l
Catalase E. coli none constitutive HOROWITZ et al., 1960 a.
inducible l.I\
D-serine dehydrase E. coli inhibition HOROWITZ et aI., 1960
,8-glucuronidase E. coli inhibition constitutive (?) GROS and NAONO, 1961 :!1s::
Serine deaminase E. coli none GROS and NAONO, 1961 0t;
Glucose-6-phosphate dehydrogenase E. coli none 0
GROS and NAONO, 1961 s::
Alkaline phosphatase E. coli none decreased thermostability NAONO and GROS, 1960 ;;J
Ribonuclease S. aureus inhibition inconclusive HIGNETT,1965 a:
Deoxyribonuclease S. aureus none inconclusive HIGNETT,1965 5·
...
Ribonuclease B. subtilis inhibition exoenzyme KADOWAKI et al., 1965 0
~
Alkaline phosphatase B. subtilis inhibition inducible KADOWAKI et aI., 1965 Z
~-amylase B. subtilis none exoenzyme; thermostability KADOWAKI et aI., 1965 >
unchanged ~p..
Aconitase B. cereus inhibition constitutive KLU1!ES and HARTMANN, 1969 ::0.
Malate dehydrogenase B. cereus inhibition constitutive KLUBES and HARTMANN, 1969 m
Fumarase B. cereus inhibition constitutive KLUBES and HARTMANN, 1969
Succinate dehydrogenase B. cereus none constitutive KLUBES and HARTMANN, 1969 ~
0
NADH oxidase B. cereus none constitutive KLU1!ES and HARTMANN, 1969 E..
Penicillinase B. cereus none inducible REICH and MANDEL, 1966 t;
d-aminolevulinate synthetase Rhodopseudo- inhibition U reversed effect BULL and LASCELLES, 1963
'"
monas
~
t:l
m
spheroides n
Succinate dehydrogenase R. spheroides none BULL and LASCELLES, 1963
.g
n
~-glucosidase Saccharomyces none inducible; no qual. change t:l
DE KLOET, 1968 0
n
carlsbergensis en
Galactosidase S. carlsbergensis none inducible; no qual. change DE KLOET, 1968
....
....
-...J
......
......
(Xl
Table 6 (continued)
Enzyme Source Effect Comments Reference
Nitrate reductase I radish none inducible KEY and INGLE, 1964

cotyledon
corn mesacotyl
Ribonuclease I soybean none inducible KEY and INGLE, 1964
hypocotyl
Threonine dehydrase rat liver inhibition FO converted to FU-RNA PITOT and PERAINO, 1964
Ornithine transaminase rat liver inhibition FO converted to FU-RNA PITOT and PERAINO, 1964
d-aminolevulinate synthetase rat liver inhibition MARVER et aI., 1966
;:r:
Tyrosine transaminase rat liver none FU admin. 7 h after hydrocortisone GARREN et aI., 1964 0t<1
increase FU admin. 9 h after hydrocortisone 0
~
Tryptophan pyrrolase adult rat liver none inducible NEMETH, 1962; Gl
t<1
KROGER and GREVER, 1965 ~
inhibition inducible; related to diet GAETANI and SPADONI, 1961
Tryptophan pyrrolase
z>
adult rat liver none FU admin. 7 h after GARREN et aI., 1964 tl
t<1
prevents FU admin. 9 h after GARREN et aI., 1964 t"
decrease
Tryptophan pyrrolase rat, regen. liver inhibition tryptophan-induced; reversed by UR NEMETH, 1962;
KROGER and GREVER, 1965
Tryptophan pyrrolase adrenalectomized inhibition cortisone-induced KROGER and GREVER, 1965
rat liver
Tryptophan pyrrolase newborn guinea inhibition tryptophan-induced; developmental; NEMETH, 1962
pig liver reversed by UR
Tryptophan pyrrolase young guinea slight tryptophan-induced; adaptive; NEMETH, 1962
pig liver inhibition reversed by UR
~
~
o
a b rC /.
0.10 ./
.ao
~.
-E / g
E 0.5 2.0 Jl a,
Vt
~
- ~
~
·c ~o
~ 0.4 ~
ru ~
8s::
:g ~ 0.05 ;3
~ 0.3 g 2.:
1.0 Ci. s·
~
£o
8"
~I 0.2 ~
~
~
b
p..
r:{ I I I ~
~ OJ~u ....
fA
o
LI Protein oq/ml
t
Fig. 14a-c. Increase in p-galactosidase content in relation to total protein synthesis to indicate differential rate of enzyme syn-
"~
thesis in E. coli B. a, culture grew on glycerol-salts medium in presence (D) or absence (0) of FU; induction with lactose.
...
b, as in a but culture grew on succinate medium; induction with methyl P-D-thiogalactoside. Cultures grew more slowly in bl
I:l
fA
this medium, with less catabolite repression under these conditions. c, glycerol carbon source was removed after growth of cul- (1)
ture in this source and FU was then added (e). Glycerol restored in one culture (0) only. Catabolite accumulated only in presence .g
of glycerol and reduced enzyme synthesis resulted. (From HOROWITZ and KOHLMEIER, 1967) ~(1)
fA
....
....
\0
galactosidase induced during the presence of FU exhibited 30% less enzymatic

activity relative to its serological cross reactivity (Fig. 16). The enzyme formed in the
presence of FU but induced in the absence of the analog appeared to have a normal
ratio of cross-reactivity to enzymatic activity.
In the only report of its kind, ribosome-free soluble protein from S. aureus treated
with FU behaved differently during electrophoresis compared with the fraction from
normal cells (HIGNETT, 1964). No further information has become available.
100~-----------,
.;:;-
":;:
u
<{
10
gc
-
0
u 5
0
o
Min at 90°C
Fig. 15. Thermal inactivation of alkaline phosphatase from normal (e) and FU-treated (0)
E. coli. The enzymes had been purified by ammonium sulfate fractionation and chromato-
graphy on DEAE columns. (From NAONO and GROS, 1960)
/1
~ 100 ./.
Q)
.0
.;:
/ ,I
80 I-
---.~
C
:J
~ 60 I-
'C
Q)
Q;
> 40 I-
0
u I //
~ I II
Q) 20 l- I II
I "
E
>-
• / ••/~I
N
c o60• 1'1. 1.1'''·1 i,' 1 ~
w 80 100 120 140 160 180 200 240
Enzyme added (units/ tube)
Fig. 16. Enzyme recovered in supernatant fraction uncombined with anti-p-galactosidase
serum, as a function of total amount of enzyme added. Different quantities of p-galactosidase-
containing extracts were mixed with a constant quantity of antiserum. e, Extracts of control
cells induced and grown in the absence of FU; ... extracts of cells induced in the absence of
FU, but then grown in the presence ofFU; • extracts of cells induced in the presence ofFU,
but then incubated in the absence of FU. For identical extent of protein cross reaction,
extract. in which FU was present during induction contained less enzyme activity. (From
NAKADA and MAGASANIK, 1964)
D. Effect of FU on Virus Proteins

The relative simplicity of viral proteins coded by viral RNA containing FU was
considered a useful opportunity for determining specific effects of the incorporation
of the analog on protein formation and functions.
TMV. The composition and function of protein from TMV containing FU has
been subject to considerable examination. Amino acid analysis of TMV in which
19% of U had been replaced by FU revealed no alterations (HOLOUBEK, 1963), and
reconstitution using normal virus RNA with protein from either FU- or normal
virus provided the same yield of virus. From these experiments it was concluded that
the incorporation of FU into the virus had not led to errors in the reading of the
message for the synthesis of virus protein. No change in antigenic properties of the
coat protein could be determined (SUTIC and D]ORD]EVIC, 1964).
On the other hand, more sophisticated analyses of amino acids of tryptic peptides
from the protein of mutant viruses produced by infection in the presence of FU have
indicated amino acid replacements corresponding to miscoding by C instead ofU, or
by G instead of A [WrrTMANN-LIEBOLD and WITTMANN, 1965 (1);WITTMANN, 1964]
(Table 7). The first of these exchanges has been described as a translation error
(Table 5, I1-2). It is possible to explain the miscoding of G for A because of aberrant
replication of the template prior to translation (Table 5, I11-6) (KRAMER et aI., 1964).
The presence of FU during this process and the incorporation of FU into either chain
of the replicating form, however, permits sufficient opportunities for replacement to
rationalize almost any codon substitutions (Table 5, III 6 to 8).
Polio virus. The first report (MUNYON and SALZMAN, 1962) indicated that the pro-
tein of polio virus was unaltered by the presence of FU. The kinetics of neutralization
with rabbit antiserum of the FU-containing and control viruses were identical, and
thermal inactivation probably was unchanged. The amino acid composition of the
two viruses could not be shown to be different. Subsequently it was discovered that
FU produced a high incidence of heat-defective mutants (COOPER,1964). Although
infectivity at 30° for FU and control virus were not appreciably different, at increasing
temperatures (up to 40°), FU-substituted virus progeny formed fewer plaques than
did normal progeny of the same strains. The studies of TERSHAK (1966) confirmed
this alteration which was found to occur to a greater extent when virus was grown
with increasing concentrations of FU. Subcultures of the FU virus in the absence of
FU showed retention of the heat-defective character. The mutation rate was considered
to be up to tenfold that of normal virus (COOPER, 1964).
Extracts of polio virus polymerase from cell cultures infected with FU-virus (in
the absence of FU) were more sensitive to inhibition by Mn++ and more heat resistant
than were corresponding extracts from control virus. These alterations in enzyme
activity were heritable, strongly suggesting that the source of the polymerase was of
viral rather than cellular origin. The parental virus RNA apparently had served as the
messenger, since enzyme extracts from cells infected with normal virus in the presence
or absence of FU behaved identically (TERSHAK, 1966).
Phages. The formation of defective FU particles by MS 2 coliphage with di-
minished RNA content was considered a mutation produced by the analog (SHIMURA
et al., 1967). These noninfective particles adsorbed poorly to the host cells and showed
serological differences from normal phage, although no composition differences of
peptic digests could be demonstrated. The inability to detect differences in the finger-
.......
~
Table 7. Alterations in amino acid composition of virus or phage mutants

Species Mutant Position Amino acid and code Exchange Reference
altered normal mutant
~
()
TMV FU-27 58 valine, GUU alanine, GCU U -+C WI1TMANN-LIEBOLD and WITTMANN, 1965 (2) n1
0
~
TMV FU-41 28 threonine, ACU alanine, GCU A-+G WITTMANN-LIEBOLD and WITTMANN, 1965 (2) C'l
n1
TMV FU-243 65 serine, AGU glycine, GGU A -+G WI1TMANN-LIEBOLD and WITTMANN, 1965 (2) ~
>
R17 amber B24, B29 6 glutamine, CAG serine, UAG C-+U TOOZE and WEBER, 1967 Z
0
n1
t"'
R17 amber B22 50 glutamine, CAG serine, UAG C-+U TOOZE and WEBER, 1967
R17 amber B17, B18, B19 54 glutamine, CAG serine, UAG C-+U TOOZE and WEBER, 1967
print analysis of phage-specific proteins synthesized in an in vitro incorporating system

stimulated by FU-RNA (80% FU replacing U) may have rested on the lack of
sufficiently sensitive techniques to pinpoint a minute but important chemical altera-
tion. It was considered possible that abnormal coat protein subunits were synthesized
which, because of a less compact fitting, allowed for the partial degradation of the
RNA. Alternatively, a viral maturation protein was assumed to be missing, and non-
functional protein was synthesized. A very similar conclusion was drawn for the
related f2 phage produced in the presence of FU which closely resembled the "su-1
dead" phage mutant (LODISH et al., 1965).
Results with fCAN-1 phage revealed that phage production in the presence of FU
was greatly diminished, although phage-specific antigen was decreased to a lesser
extent. Thus, ineffective mutant phage particles had been produced which still pos-
sessed phage-specific antigenic properties. A considerable percentage of the FU phage
were heat-sensitive, in contrast to normal phage.
Various amber mutants of phage R17, which had mutated because of exposure to
FU, were subjected to amino acid sequence analysis (TOOZE and WEBER, 1967). The
sites of the mutations in the coat protein cistron were found to be due to replacement
of glutamine by serine (Table 7). This miscoding may be of type 1-5 in Table 5, but
again it could be of Type III.
E. Implied Alterations Resultingfrom FU-mRNA on Protein and Cell Functions

JACOB and MONOD (1961) have provided the general hypothesis for the functions
of messenger RNA altered by the incorporation of a base analog. In commenting on
the effect of FU in inhibiting the induction of enzymes or producing possible altera-
tions in their biophysical properties, they stated that such drug responses cannot
result from the mere presence of FU in the cells but must reflect incorporation of FU
into a constituent involved in the information transfer system. The kinetics of this
response in turn must reflect the kinetics of FU incorporation into that constituent.
It has been observed that the onset of the drug's actions is immediate; the effect
appears to be homogeneous and does not increase with time. If the constituent
responsible for the drug action were stable, one would expect the populations of
molecules made in the presence of FU to be heterogeneous, and the fraction of
abnormal molecules to increase progressively with time. Thus, the responsible
constituent must be formed and must decay very rapidly. They concluded, therefore,
that messenger RNA was the most likely constituent to be responsible for the ob-
served drug effects, although other cell constituents could also be involved conco-
mitantly.
In addition to the described effects based on the observed incorporation of FU
into mRNA, there are numerous other biochemical actions of FU which probably
rely on altered functions due to incorporation of FU into mRNA. In many cases the
evidence is still incomplete, and cannot yet be separated from some other drug action.
Among the actions probably involving FU-mRNA which deserve emphasis is the
effect of the drug on induction of tryptophan pyrrolase during different stages of
development of host liver (NEMETH, 1962). For example, FU blocked completely the
development of this enzyme in the newborn guinea pig, inhibited adaptation ap-
preciably in the young guinea pig, but had no effect on enzyme formation in adult rat
liver unless liver was regenerating.
The inhibition of sporulation by FU, both in bacteria (ARONSON and DEL VALLE,
1964; DEL VALLE and ARONSON, 1962) and in a fungus (GALUN and GRESSEL, 1966),
has been described and is similar to that of induction. FU prevented sporulation of
B. cereus if added rapidly after initial inoculation with the spores, whereas later treat-
ment was ineffective. A similar effect was produced by actinomycin. It was concluded
that spore formation relied on the formation of mRNA whose function was altered
by these drugs. Chloramphenicol was inhibitory even when added at the later time,
suggesting that protein synthesis followed the commitment to sporulation which
involved messenger. In Trichoderma, FU applied immediately prior to photoinduction
suppressed sporulation without greatly affecting growth. Sporulation did occur when
FU exposure for the same duration took place several hours before or after photo-
induction (KEY and INGLE, 1964). FU inhibited photoinduction in the Xanthium
bud only if applied at the beginning of an inductive dark period. This effect could be
reversed by orotate only if the latter compound was applied within 8 h of FU treat-
ment. RNA synthesis was required for the photoperiodic induction which was
inhibited by FU (BONNER and ZEEVAART, 1962).
The continued growth of RNA viruses and phage in the presence of FU, because
DNA synthesis was not required (COOPER and ZINDER, 1962), has been described
above in detail (Section V B). On the other hand, synthesis of DNA viruses was
usually inhibited by FU (reviewed in HEIDELBERGER, 1965). The relative effectiveness
of U rather than TdR in overcoming the inhibition of the DNA-bacteriophage T3 or
T7 was postulated as being due to the formation of FU-mRNA which then led to
alterations in the required structural proteins of the phage [GOODMAN, 1963; see also
ARONSON, 1961 (1)].
The replacement by FU of the U requirement for growth of bacteriophage T2H in
the presence of TdR implied that FU-mRNA had some biological activity (GOOD-
MAN, 1965). The growth of a uracil auxotroph of E. coli in the presence of FU, which
synthesized some protein and RNA while incorporating FU to replace 20% of U,
undoubtedly relied on the same basis (HOROWITZ and CHARGAFF, 1959; HOROWITZ
et aI., 1958).
Attempts to link a small degree of resistance to ultraviolet radiation with FU-
mRNA formation must be considered premature. The effect became maximal 2 min
after the addition ofFU to E. coli suspensions, was reversed equally rapidly following
removal of FU, and apparently was prevented altogether by UR (BEN-ISHAI et aI.,
1965).
F. Conclusions: FU and Proteins

It is apparent from the data that FU produced alterations in protein synthesis. The
evidence is strongest in the experiments with viruses and phage, where mutant forms
have been isolated. The exchange of amino acids both in tobacco mosaic virus and the
amber phage mutants represented FU-related effects resulting from miscoding.
Although the misreading was undoubtedly produced by FU-mRNA, the exact
mechanism of this miscoding is still not clear.
The alterations in protein synthesis were relatively minute, and it is doubtful if
they can be discerned during amino acid analysis of total protein. On the other hand,
alterations in enzyme specific activity, heat sensitivity, serological cross reactions,
adsorbability, host specificity and sensitivity to cations have been observed following
FU treatment when particular protein molecules were examined. These observations
have not yet been associated with definitive alterations in chemical composition.
It is quite likely that the inhibitory action of the analog on enzyme induction is
related, at least in part, to the formation of FU-mRNA, although catabolite repression
seems to account for some of these observations as well. Nevertheless, an important
effect of FU can be associated with subtle alterations in protein formation.
VII. Synthetic Polynucleotides Containing FU

The availability of enzymes capable of catalyzing the formation of FU-containing
nucleic acid polymers has provided information on in vitro mechanisms of synthesis
and various properties of analog-substituted molecules.
A. PolYnucleotide Phosphorylase CatalYsis
LENGYEL et al. (1961) first prepared poly FU by the condensation of FUDP using
polynucleotide phosphorylase from Azotobacter vinelandii. The copolymers poly U-FU
(WAHBA et al., 1963) as well as poly A-FU, polyI-FU and poly C-FU (GRUNBERG-
MANAGO and MICHELSON, 1964) were synthesized by condensation of the appropriate
nucleoside diphosphates at suitable ratios of concentrations.
The rate of polymerization of FUDP was slower than that of UDP (GRuNBERG-
MANAGO and MICHELSON, 1964). Yield could be enhanced by increasing the concen-
tration of Mg++ beyond that normally used for poly U synthesis (SZER and SHUGAR,
1963). Phosphorolysis of poly FU was less rapid than that of poly U (GRUNBERG-
MANAGO and MICHELSON, 1964). Poly FU was slightly less stable to hydrolysis at
pH 11 than was poly U, and also reacted with poly A to form twin or triple-stranded
complexes (SZER and SHUGAR, 1963). A copolymer with adenine was less stable and
had a lower Tm than the corresponding poly A-U (MASSOULIE et al., 1966; SZER and
SHUGAR, 1963). Above 0°, poly FU had little if any organized secondary structure
under the usual range of salt conditions. SZER and SHUGAR (1963) reported hyper-
chromicity and a Tm well below that for poly V, and concluded that poly FU at room
temperature was in the form of a random coil (SZER and SHUGAR, 1963). The pI(,. of
poly FU was 8.1, compared to that of 9.81 for poly U (MASSOULIE et al., 1966). By
contrast, the corresponding values for the monomers were 7.6 and 9.5, respectively.
In polymeric form, ionization of FU was somewhat impeded (MASSOULIE et al., 1966;
SZER and SHUGAR, 1963).
Although earlier studies (WAHBA et al., 1963; LENGYEL et al., 1961) did not de-
monstrate poly FU-produced stimulation of amino acid incorporation in an E. coli
system, poly V-FU was found to be almost as active as poly U. In the experiments of
GRUNBERG-MANAGO and MICHELSON (1964) poly FU coded for phenylalanine ex-
clusively, and did not exhibit the ambiguity of poly U which can permit leucine in-
corporation up to 25% of that of phenylalanine (Table 8). Poly FV was less efficient
than poly V, and at its optimal conditions it was only half as effective as the corre-
sponding amount of poly U. Whereas slight increases in temperature from 25° to 37°
raised the stimulatory functions of poly V, similar alterations diminished the func-
tions of poly FU. Lowering the pH of the incubation mixture from 8 to 7 increased
the activity of poly FU, probably because ionization was depressed. Most important,
however, FU resembled U in its coding properties, with the above limitations, and
in vitro did not demonstrate the additional coding behavior of C, G or A. Thus, for
example, poly C-FU or poly A-FU did not code for threonine (ACU, ACC, ACA,
ACG) or histidine (CAU, CAC), and poly FU did not code for valine (GUU, GUC,
GUA, GUG). These results are listed in Table 8.
B. RNA PolYmerase CatalYsis
Polymers containing FU were also prepared using DNA-directed RNA poly-
merase, with FUTP replacing UTP as one of the substrates. Under these conditions
the presence of the analog slowed the rate of polymerization, depending on the source
of DNA used as primer (KAHAN and HURWITZ, 1962; also SLAPIKOFF and BERG,
1967). Because of the lower pKa of FUTP compared to that of UTP (7.9 versus 9.3,
respectively), the analog was more dissociated at physiological pH and showed
diminished ability to form H bonds with the opposing 6-amino group of the DNA
primer, thereby delaying condensation.
It was observed that even though FU was distributed next to all the component
nucleotides, the nearest neighbor frequency differed from that characteristic of the
position of U in the newly formed RNA. When UTP and FUTP competed for con-
densation, U was preferentially incorporated next to A and C residues, whereas no
such preference existed for the incorporation of U or FU next to G (SLAPIKOFF and
BERG, 1967). It was postulated that the altered neighbor frequency rested on the
selective copy of the enzyme of certain regions of DNA where the analog was
present (KAHAN and HURWITZ, 1962) or else was related to differences in base stacking
ability of the substrates with the nearest neighbors (SLAPIKOFF and BERG, 1967).
Altogether, however, FU was a specific replacement for U only, and base ratios were
unchanged except for the replacement of U.
C. DNA PolYmerase CatalYsis
FdUTP was incorporated into T2 bacteriophage DNA where it replaced 9% of
the incorporation of dTTP (ApOSHIAN and KORNBERG, 1962). However, FdUTP has
not been detected in any cells following FU treatment (HEIDELBERGER, 1965).
D. Conclusions: FU-RNA in vitro and in vivo
The formation of FU-RNA in vitro provided results quite analogous to those
reported in vivo. No evidence for coding of FU different from that of U has so far
been demonstrated in vitro. FUhasalso been shown to code like U in vivo except for a few
isolated instances where miscoding like C occurred as a rare event. It is likely that the in
vitro experiments were not sufficiently sensitive to allow a similar observation. The de-
creased ambiguity in the messenger activity of poly FU compared to poly U probably
needs further substantiation, and its biological significance is still unclear. It is reason-
able to assume that the greater dissociation ofFU and its compounds at physiological
pH, compared to that of the corresponding U derivatives, and the resulting shift to
increased enolization of FU explain many of the biochemical alterations.
Decreases in hyperchromicity and melting temperatures have been reported for
nucleic acid polymers prepared in vitro. Several authors have reported decreased
hyperchromicity and a lowered melting temperature of FU-RNA isolated from in vivo
preparations as well, such as in E. coli rRNA (ANDOH and CHARGAFF, 1965), B.
cereus rRNA (HAHN and MANDEL), E. coli tRNA (LOWRIE and BERGQUIST, 1968),
and MS2 phage RNA (SHIMURA et aI., 1965).
~......
::I
8
Table 8. Incorporation of amino acids in presence of different polymers. Incubation mixture included 14C-amino acid plus 19 other amino acids, sRNA, S-30 frac- .ao
tion in usual in vitro {)Istem. Composite of different experiments. Poly U 240 (Jog/ml, poly PU about 800 (Jog/mi. Incorporation expressed as (Jo(Jomoles amino acid
per mg ribosomal protein ~.
(From GRUNBERG-MANAGO and MICHELSON, 1964) ::I
S,
Incorporation of amino acids in presence of different polymers <Jl
Amino acid Code Normal base Incorporation FU polymers Incorporation Effect of FU :n

~
polymers o
Phenylalanine VUpyc poly U 7700 poly FU 700- replaces V ~

Leucine UUpu, CUpy, CUpu poly U 900- poly FU 0 less ambiguity ~
Valine GUpy, GVpu poly UG 470 poly FU 7b no coding like G ~.
Serine UCpu, VCpy, AGpy poly CU 501 poly V-FU 0 no coding like C
:;d
Serine VCpu, VCpy, AGpy poly CU 329 poly C-FU 575 replaces V Z
Threonine ACpy, ACpu poly CA 353 poly A-FU 0 no coding like C >
Histidine CApy poly CA 128 poly A-FU 0 no coding like C
Histidine CApy poly CA 128 poly C-FU 0 no coding like A
'&."
::;.
Isoleucine AUpy, AUA poly VA 618 poly U-FU 3 no coding like A
Isoleucine AUpy, AUA poly UA 441 poly A-FU 121 replaces U
'"
~
Leucine VUpu, CUpy, CUpu poly UA 331 poly A-FU 70 replaces U o
0'
n
Leucine VUpu, CUpy, CUpu poly U 461 poly U-FU 311 replaces V ~
Tyrosine VApy poly UA 350 poly A-FU 91 replaces V ~
Phenylalanine VUpy poly VA 1359 poly A-FU 260 replaces U n
o
::I
• Estimated. b FVDP equally effective as poly FU. • py = pyrimidines U or C, pu = purines G or A '"
..c
'"
~
::I
'"
~
'"
.....
N
-..J
The significance of a decrease in secondary structures of the FU-polymers is not

obviously related to major alterations in biochemical function, although it un-
doubtedly plays a role. It has been observed, however, that in almost all instances the
FU-RNA had less potency than the corresponding normal RNA in vivo and in vitro.
VIII. Discussion
A. Consequences of FU-RNA
It is apparent from all these reports that, except in viruses, an evaluation of the
biological functions of FU-RNA usually cannot be dissociated from several con-
comitant events. Among these may be listed:
1. Obviously, the presence of FU instead of U in RNA. This steric alteration is
associated with increased dissociation, greater enolization, and decreased secondary
structure. A number of other physicochemical changes have been described (Section
VII). In many instances the analog replaced U without major qualitative deviations,
even though quantitatively the FU-RNA usually was less active than the correspond-
ing normal RNA (Section IV).
2. The lack ofU in the newly formed RNA. There is as yet no information on any
major functions of U that FU or its compounds do not possess, except perhaps the
inability of poly FU to code for leucine, which contrasts with the ambiguous behavior
of poly U (Table 8). Nevertheless, such functions may still exist.
3. The failure to make normal ribosomes. Although existing ribosomes allowed
continuation of protein synthesis in the presence of FU, cells cannot grow indefinitely
in the absence of new mature ribosomes (Section III).
4. The lack of certain constituents in tRNA, such as pseudouridylate and ribo-
thymidylate. At the present time there is insufficient information about the alterations
resulting from the absence of these minor base nuc1eotides (Section IV).
5. The effect of growth inhibition by FU on the accumulation of catabolites
repressing the synthesis of certain enzymes, and other indirect results (Section VI).
6. The presence of intermediary FU ribonuc1eotides which have pharmacological
properties (HEIDELBERGER, 1965).
7. The occasional failure to produce normal cell wall in several bacterial species
(ToMAsz and BOREK, 1962; ROGERS and PERKINS, 1960). This drug effect, not con-
sidered here, can produce cell lysis.
8. The failure to form DNA. This drug response is extremely fundamental, and
though not specifically considered in this review has been discussed as a potential site
of drug action producing a large variety of secondary responses (HEIDELBERGER,
1965).
B. Unexplored areas
It is not surprising that while research on the incorporation of FU into RNA has
opened up new areas of investigations major uncertainties in our present state of
knowledge have also been revealed. For example, although considerable information
has been obtained on the drug's interaction with subcellular fractions, almost all
efforts have been directed at microbial systems. Research on plants has been quite
rewarding, but with one exception (WILLEN and STENRAM, 1967) there was no infor-
mation on the effect of FU on subcellular components in animal systems. Since FU is
extensively used clinically in cancer chemotherapy, the effect of this drug on sub-
cellular fractions of tumor cells should be investigated.
Another approach which is now feasible deals with the amino acid sequence of
enzymes synthesized in the presence of FU and having altered biophysical or func-
tional properties. Several reports have indicated drug-produced alterations in alkaline
phosphatase [NAONO and GROS, 1960 (2)] and ,B-galactosidase (NAKADA and MAGA-
SANIK, 1964; BUSSARD et aI., 1960), and virus proteins appear to have been affected
also (SHIMURA et aI., 1967; TOOZE and WEBER, 1967; TERSHAK, 1966; LODISH et al.,
1965; COOPER, 1964; KRAMER et al., 1964). It would seem appropriate to examine
several other enzymes for such mutational changes, and then to carry out amino acid
sequence analyses.
Additional information is necessary to explain why FU inhibits the maturation of
ribosomes. Relatively few investigators have examined ribosomes at 10-2 M Mg++
(HAHN and MANDEL, HIGNETT, 1966; HILLS and HOROWITZ, 1966; ANDOH and
CHARGAFF, 1965). In several systems inhibited by FU the amount of 23S rRNA was
diminished (HAHN and MANDEL, SELLS and CRUDUP, 1966; GROS et aI., 1962), and the
presence of newly formed 50S ribosomal subunits could not be demonstrated at
10- 4 M Mg++ (see Section III A). Although ribosomes do not mature because of the
failure to combine FU-rRNA with protein, it is uncertain whether this drug-produced
inhibition is related to the lack of formation of a proper protein for complexing with
rRNA, or to the inability of the proteins to use FU-rRNA for ribosome formation.
Various results with cells recovering from FU inhibition suggest the destruction of
the faulty rRNA, but the protein complement has not been examined sufficiently. It
should also be recalled that cells can tolerate some FU in their ribosomes without
demonstrating any deleterious effects (HILLS and HOROWITZ, 1966).
The role of FU-mRNA needs to be further elucidated. The fact that certain
inducible enzymes are no longer formed while others are synthesized in the presence
of FU requires a more careful analysis to ascertain the relation of FU incorporation
into mRNA and messenger function. The apparent effect of catabolite repression
(HOROWITZ and KOHLMEIER, 1967) should be further evaluated, since such an ex-
planation may account for many of the other observations reported.
IX. Summary
A comprehensive literature review on the incorporation of the carcinostatic
pyrimidine analog, 5-fluorouracil (FU), into ribonucleic acid has revealed a wide
series of drug effects apparently resulting from the formation of FU-RNA in animals,
plants and microorganisms. The consequences of the replacement of uracil by FU
included inhibition of growth, of developmental changes, and reduction of infectivity.
At the subcellular level, alterations in ribosomal composition and properties, changes
in the functions, properties and base composition of transfer RNA, and modifications
in the functions of messenger RNA have been described in detail. FU was shown to
be incorporated into all of these RNA species instead of uracil, and replacement by
FU approached 100% in several instances where only the newly synthesized fraction
was examined. Some evidence of miscoding was provided, as indicated by a few
instances of exchange of amino acids in viral protein. Numerous examples of altera-
tions in biological or physical properties of specific proteins have been reported. In-
hibition of enzyme induction probably was related in part to the formation of faulty
messenger RNA which contained FU. Altogether, however, careful analytic examina-
tion was required to locate biochemical pathways in which FU could not function
as U.
Abbreviations
FU, 5-Buorouracil; FC, 5-fluorocytosine; FO, 5-fluoroorotate; FUR, Buorouri-
dine; FUdR, Buorodeoxyuridine; FUMP, fluorouridylate; FUDP, fluorouridine
diphosphate; FUTP, fluorouridine triphosphate; FdUTP, fluorodeoxyuridine
triphosphate; FU-RNA, FU incorporated into RNA; FU-TMV, FU incorporated
into tobacco mosaic virus.
U, uracil; UR, uridine; UMP, uridylate; 'l/'R, pseudouridine; 'l/'MP, pseudouridy-
late; A, adenine; G, guanine; C, cytosine; I, inosine; T, thymine; TdR, thymidine;
and dTTP, thymidine triphosphate.
References
ANDOH, T., and E. CHARGAFF: Formation and fate of abnormal ribosomes of E. coli cells
treated with 5-fluorouracii. Proc. nat. Acad. Sci. (Wash.) 54, 1181-1189 (1965).
ApOSHIAN, H. V., and A. KORNBERG: Enzymatic synthesis of deoxyribonucleic acid. IX.
The polymerase formed after T2 bacteriophage infection of Escherichia coli: A new en-
zyme. ]. bioI. Chern. 237, 519-525 (1962).
ARONSON, A. I.: (1) The inhibition of bacteriophage protein synthesis by 5-fluorouracii.
Biochim. biophys. Acta (Arnst.) 49, 89-97 (1961).
- (2) The effect of 5-fluorouracil on bacterial protein and ribonucleic acid synthesis.
- , and M. R. DEL VALLE: RNA and protein synthesis required for bacterial spore formation.
BARNEIT, W. E., and H. E. BROCKMAN: Induced phenotypic reversion by 8-azaguanine and
5-fluorouracii. Biochem. biophys. Res. Commun. 7, 199-203 (1962).
BECAREVIC, A., B. DJORDJEVIC, and D. SUTIc: Effect of ultra-violet light on tobacco mosaic
virus containing 5-fluorouracii. Nature (Lond.) 198, 612-613 (1963).
BEN-HAMIDA, F., and D. SCHLESSINGER: Stability of p-galactosidase messenger ribonucleic
acid in Escherichia coli. ]. Bact. 90, 1611-1616 (1965).
BEN-IsHAI, R., B. Z. CAVARI, H. GOLDIN, and S. KERPEL: On the mechanism of 5-fluoro-
uracil-induced resistance to ultraviolet irradiation in Escherichia coli. Biochim. biophys.
Acta (Arnst.) 95, 291-301 (1965).
BODMER, W. F., and S. GRETHER: Uptake and incorporation of thymine, thymidine, uracil,
uridine, and 5-fluorouracil into the nucleic acids of Bacillus subtilis. ]. Bact. 89, 1011-1014
(1965).
BONNER, ]., and]. A. D. ZEEVAART: Ribonucleic acid synthesis in the bud, an essential
component of floral induction in Xanthium. Plant Physioi. 37, 43-49 (1962).
BOSCH, L., E. HARBERS, and C. HEIDELBERGER: Studies on fluorinated pyrimidines. V.
Effects on nucleic acid metabolism in vitro. Cancer Res. 18, 335-343 (1958).
BROCKMAN, R. W., and E. P. ANDERSON: Pyrimidine analogues. In: Metabolic inhibitors, I.
pp. 239-285. HOCHSTER, R. M., and]. H. QUASTEL, Eds. New York: Academic Press
1963.
- , ]. M. DAVIS, and P. STUITS: Metabolism of uracil and 5-fluorouracil by drug-sensitive
and by drug-resistant bacteria. Biochim. biophys. Acta (Arnst.) 40, 22-32 (1960).
BUJARD, H., and C. HEIDELBERGER: Fluorinated pyrimidines. XXVII. Attempts to deter-
mine transcription errors during the formation of fluorouracil-containing messenger
ribonucleic acid. Biochemistry 5,3339-3345 (1966).
BULL, M. ]., and ]. LASCELLES: The association of protein synthesis with the formation of
pigments in some photosynthetic bacteria. Biochem. ]. 87, 15-28 (1963).
BUSSARD, A., S. NAoNo, F. GROS et ]. MONOD: Effets d'un analogue de l'uracile sur les
proprietes d'une proteine enzymatique synthetisee en sa presence. C. R. Acad. Sci.
(Paris) 250, 4049-4051 (1960).
CERNA, ]., ]. RYCHLiK, D. GRUNBERGER, and F. SORM: The effect of 5-fluorouracil-con-

taining ribonucleic acid on protein synthesis by Escherichia coli in vivo. Coll. Czech.
Chern. Commun. 28, 1215-1223 (1963).
CHAMPE, S. P. and S. BENZER: Reversal of mutant phenotypes by 5-fluorouracil: An approach
to nucleotide sequences in messenger-RNA. Proc. nat. Acad. Sci. (Wash.) 48, 532-546
(1962).
CHAUDHURI, N. K., B. ]. MONTAG, and C. HEIDELBERGER: Studies on fluorinated pyrimi-
dines. III. The metabolism of 5-fluorouracil-2-C14 and 5-fluoroorotic-2-C'4 acid in vivo.
Cancer Res. 18, 318-328 (1958).
CHERRY, J. H., and R. VAN HUYSTEE: Effects of 5-fluorouracil on photoperiodic induction
and nucleic acid metabolism of Xanthium. Plant Physiol. 40, 987-993 (1965).
COHEN, S. S.,]. G. FLAKS, H. D. BARNER, M. R LOEB, and J. LICHTENSTEIN: The mode of
action of 5-fluorouracil and its derivatives. Proc. nat. Acad. Sci. (Wash.) 44, 1004-1012
(1958).
COOPER, P. D.: The mutation of poliovirus by 5-fluorouracil. Virology 22,186-192 (1964).
COOPER, S., and N. D. ZINDER: The growth of an RNA bacteriophage: The role of DNA
synthesis. Virology 18, 405-411 (1962).
DAGG, C. P., A. DOERR, and C. OFFUTT: Incorporation of 5-fluorouracil-2-C'4 by mouse
embryos. BioI. Neonat. (Basel) 10,32-46 (1966).
DAVERN, C. I.: The inhibition and mutagenesis of an RNA bacteriophage by 5-fluorouracil.
Austral. J. bioI. Sci 17, 726-737 (1964)
- , and J. BONNER: The influence of 5-fluorouracil on tobacco-mosaic virus production in
tobacco-leaf discs. Biochim. biophys. Acta (Arnst.) 29, 205-206 (1958).
DE KLOET, S. R.: Effects of 5-fluorouracil and 6-azauracil on the synthesis of ribonucleic acid
and protein in Saccharomyces carlsbergensis. Biochem. ]. 106, 167-178 (1968).
- , and P. ]. STRIJKERT: Selective inhibition of ribosomal RNA synthesis in Saccharomyces
carlsbergensis by 5-fluorouracil. Biochem. biophys. Res. Commun. 23, 49-55 (1966).
DEL VALLE, M. R., and A. I. ARONSON: Evidence for the synthesis of stable informational
RNA required for bacterial spore formation. Biochem. biophys. Res. Commun. 9,
421-425 (1962).
DUSCHINSKY, R., E. PLEVEN, and C. HEIDELBERGER: The synthesis of 5-fluoropyrimidines.
]. Amer. chern. Soc. 79, 4559-4560 (1957).
EBEL, J. P., ]. H. WElL, B. RETHER et J. HEINRICH: Nature des sites responsables de l'acti-
vite acceptrice du s-RNA. Bull. Soc. Chim. bioI. (Paris) 47, 1599-1608 (1965).
ELION, G. B., and G. H. HITCHINGS: Metabolic basis for the actions of analogs of purines
and pyrimidines. In: Advanc. Chemotherapy 2,91-177 (1965).
FIKUS, M., K. 1.. WIERZCHOWSKI, and D. SHUGAR: Reversible photochemical transformation
of 5-fluorouracil analogues and poly-5-fluorouridylic acid. Biochem. biophys. Res.
Commun. 16, 478-483 (1964).
GAETANI, S., and M. A. SPADONI: Effect of 5-fluorouracil on hepatic induced increase of
tryptophan-pyrrolase. Nature (Lond.) 191, 1296-1297 (1961).
GALUN, E., and]. GRESSEL: Morphogenesis in trichoderma: Suppression of photoinduction
by 5-fluorouracil. Science 151, 696-698 (1966).
GAREN, A., and O. SIDDIQI: Suppression of mutations in the alkaline phosphatase structural
cistron of E. coli. Proc. nat. Acad. Sci. (Wash.) 48, 1121-1127 (1962).
GARREN, L. D., R. R. HOWELL, G. M. TOMKINS, and R. M. CROCCO: A paradoxical effect
of actinomycin D: The mechanism of regulation of enzyme synthesis by hydrocortisone.
GOLDBERG, A. R., ]. H. MACHLEDT, JR., and A. B. PARDEE: On the action of fluorouracil
on leukemia cells. Cancer Res. 26, 1611-1615 (1966).
GOODMAN, F.: Nucleic acid metabolism and bacteriophage multiplication: Effects of 5-flu-
orouracil on coliphage synthesis. Virology 21,249-257 (1963).
- Partial satisfaction of a uracil requirement by 5-fluorouracil.]. Bact. 89, 1156-1157 (1965).
GORDON, M. P., and M. STAEHELIN: The incorporation of 5-fluorouracil into the nucleic
acid of tobacco mosaic virus. ].A mer. chern. Soc. 80,2340-2341 (1958).
- - Studies on the incorporation of 5-fluorouracil into a virus nucleic acid. Biochim.
biophys. Acta (Arnst.) 36, 351-361 (1959).
9*
GRAHAM, A. F., and C. KIRK: Effect of 5-fluorouracil on the growth of bacteriophage R17.
]. Bact. 90, 928-935 (1965).
GRAY, P. N., and M. RACHMELER: The effects of 5-fluorouracil and 6-thioguanine incorpo-
ration on the amino acid acceptor activity of Escherichia coli tRNA. Biochim. biophys.
Acta (Arnst.) 138,432-435 (1967).
GRESSEL, ]., and E. GALUN: Effect of 5-fluorouracil on the soluble RNA of Trichoderma.
Biochem. biophys. Res. Commun. 24, 162-168 (1966).
GROS, F., W. GILBERT, H. H. HIATT, G. ATTARDI, P. F. SPAHR, and]. D. WATSON: Mole-
cular and biological characterization of messenger RNA. Cold Spr. Harb. Symp. quant.
BioI. 26, 111-126 (1961).
- , and S. NAONO: Bacterial synthesis of "modified" enzymes in the presence of a pyrimidine
analogue. In: Protein biosynthesis, pp. 195-205. Harris, R.].C.,Ed. London: Academic
Press 1961.
-- - , D. HAYES, F. HAYES et ]. D. Watson: Etude du role de l'ARN dans Ie transfert de
l'information genetique. Colloq. Intern. Centre Nat. Rech. Sci (Paris) 106, 437-459
(1962).
GRUNBERG-MANAGO, M., and A. M. MICHELSON: Polynucleotide analogues IV. Polyfluorou-
ridylic acid and copolymers containing fluorouridylic acid. Biochim. biophys. Acta
(Arnst.) 87, 593-600 (1964).
GRUNBERGER, D., and H. G. MANDEL: Enhanced messenger activity of RNA from 8-aza-
guanine-treated Bacillus cereus. Molec. Pharmacoi. 1, 157-162 (1965).
HAHN, G. A., and H. G. MANDEL: To be submitted.
- - Effectof5-fluorouracil on ribosomal synthesis inBacilluscereus. Fed. Proc. 26, 729 (1967).
HARBERS, E., N. K. CHAUDHURI, and C. HEIDELBERGER: Studies on fluorinated pyrimidines.
VIII. Further biochemical and metabolic investigations. ]. bioI. Chern. 234, 1255-1262
(1959).
HEIDELBERGER, c.: Fluorinated pyrimidines. In: Progr. Nuci. Acid Res. and Molec. BioI. 4,
1-50 (1965).
- , N. K. CHAUDHURI, P. DANNEBERG, D. MOOREN, L. GRIESBACH, R. DUSCHINSKY, R. ].
SCHNITZER, E. PLEVEN, and]. SCHEINER: Fluorinated pyrimidines, a new class of tumor-
inhibitory compounds. Nature (Lond.) 179, 663-666 (1957).
- , A. GHOBAR, R. K. BAKER, and K. L. MUKHERJEE: (1) Studies on fluorinated pyrimidines.
X. In vivo studies on tumor resistance. Cancer Res. 20, 897-902 (1960).
- , G. KALDOR, K. L. MUKHERJEE, and P. B. DANNEBERG: (2) Studies on fluorinated
pyrimidines. XI. In vitro studies on tumor resistance. Cancer Res. 20, 903-909 (1960).
HIGNETT, R. c.: The incorporation of 5-fluorouracil by Staphylococcus aureus (strain Duncan).
II. Biochim. biophys. Acta (Arnst.) 91, 584-591 (1964).
- The incorporation of 5-fluorouracil by Staphylococcus aureus (strain Duncan). III. Biochim.
biophys. Acta (Arnst.) 95, 538-543 (1965).
- Interference of 5-fluorouracil in the biosynthesis of ribosomes in Staphylococcus aureus
(strain Duncan). Biochim. biophys. Acta (Arnst.) 114, 559-564 (1966).
HILLS, D. c., and]. HOROWITZ: Ribosome synthesis in Escherichia coli treated with 5-fluoro-
uracil. Biochemistry 5, 1625-1632 (1966).
HOLOUBEK, V.: The composition of tobacco mosaic virus protein after the incorporation of
5-fluorouracil into the virus. ]. molec. BioI. 6, 164-166 (1963).
HOROWITZ, ]., and E. CHARGAFF: Massive incorporation of 5-fluorouracil into a bacterial
ribonucleic acid. Nature (Lond.) 184, 1213-1215 (1959).
- , and V. KOHLMEIER: Formation of active p-galactosidase by Escherichia coli treated with
5-fluorouracii. Biochim. biophys. Acta (Arnst.) 142, 208-218 (1967).
- , ]. ]. SAUKKONEN, and E. CHARGAFF: Effect of 5-fluorouracil on a uracil-requiring mu-
tant of Escherichia coli. Biochim. biophys. Acta (Arnst.) 29, 222-223 (1958).
- - - Effects of fluoropyrimidines on the synthesis of bacterial proteins and nucleic
acids. ]. bioI. Chern. 235, 3266-3272 (1960).
IWABUCHI, M., E. OTAKA, M. KONO, and S. OSAWA: The effect of 5-fluorouracil on the
ribosome formation in Escherichia coli. Biochim. biophys. Acta (Arnst.) 114, 83-94 (1966).
]ACOB, F., and ]. MONOD: Genetic regulatory mechanism in the synthesis of proteins.
]. molec. BioI. 3, 318-356 (1961).
KADOWAKI, K., J. HOSODA, and B. MARUO: Effects of actinomycin D and 5-fluorouracil on

the formation of enzymes in Bacillus subtilis. Biochim. biophys. Acta (Arnst.) 103, 311 -
318 (1965).
KAHAN, F. M., and J. HURWITZ: The role of deoxyribonucleic acid in ribonucleic acid
synthesis. IV. The incorporation of pyrimidine and purine analogues into ribonucleic
acid. J. bioI. Chern. 237, 3778-3785 (1962).
KAPLAN, H. S., K. C. SMITH, and P. A. TOMLIN: Effect of halogenated pyrimidines on radio-
sensitivity of E. coli. Radiat. Res. 16,98-113 (1962).
KEMPNER, E. S. : The selection and utilization of metabolic analogs for nucleic acid synthesis.
- , and J. H. MILLER: Alteration of carbon metabolism by a base analog. Biophys. J. 2,
327-337 (1962).
- - The mechanism of action of purine and pyrimidine analogs in microorganisms. Bio-
chim. biophys. Acta (Arnst.) 76,341-346 (1963).
KESSEL, D., T. C. HALL, and I. WODINSKY: Nucleotide formation as a determinant of
5-fluorouracil response in mouse leukemias. Science 154, 911-913 (1966).
KEY, J. L.: Effect of purine and pyridimine analogues on growth and RNA metabolism in
the soybean hypocotyl: The selective action of 5-fluorouracil. Plant Physiol. 41, 1257-
1264 (1966).
- , and J. INGLE: Requirement for the synthesis of DNA-like RNA for growth of excised
plant tissue. Prec. nat. Acad. Sci. (Wash.) 52, 1382 -1388(1964).
KILGORE, W. W., and R. R. PAINTER: The effect of 5-fluorouracil on the viability of house
fly eggs. J. Econ. Entomology 55, 710-712 (1962).
- - Insect chemosterilants: Incorporation of 5-fluorouracil into house fly eggs. J. Econ.
Entomology 59, 746-747 (1966).
KLUBES, P., and K. L. HARTMANN: The differential effects of 8-azaguanine, 5-fluorouracil,
6-mercaptopurine, and 6-thioguanine on the activities of some Krebs cycle enzymes in
cell-free extracts from Bacillus cereus. Biochem. Pharmacol., in press (1969).
KOECHLIN, B. A., F. RUBIO, S. PALMER, T. GABRIEL, and R. DUSCHINSKY: The metabolism
of 5-fluorocytosine-2-14 C and of cytosine- 14 C in the rat and the disposition of 5-fluore-
cytosine-2-14 C in man. Biochem. Pharmacol. 15, 435----446 (1966).
KONO, M., and S. OSAWA: Intermediary steps of ribosome formation in Escherichia coli.
-, E. OT AKA, and S. OSA WA: Changes in sedimentation properties of ribosomal ribonuc-
leic acids during the course of ribosome formation in Escherichia coli. Biochim. biophys.
Acta (Arnst.) 91, 612-618 (1964).
KRAMER, G., H. G. WITTMANN und H. SCHUSTER: Die Erzeugung von Mutanten des
Tabakmosaikvirus durch den Einbau von Fluoruracil in die Virusnucleinsaure. Z. Natur-
forsch. 19b, 46-51 (1964).
KROGER, H., und B. GREUER: Einflufi von Antagonisten des Nucleinsaurestoffwechsels und
von Rontgenstrahlen auf die Induktion von Enzymen. Biochem. Z. 341,190-198 (1965).
LAMPKIN-HIBBARD, J. M., K. L. MUKHERJEE, and C. HEIDELBERGER: Effects of steroids and
fluoropyrimidines on lymphomas. II. In vivo studies on tumor resistance and collateral
sensitivity. Cancer Res. 23, 468-476 (1963).
LENGYEL, P., J. F. SPEYER, and S. OCHOA: Synthetic polynucleotides and the amino acid
code. Proc. nat Acad. Sci. (Wash.) 47, 1936-1942 (1961).
LODISH, H. F., S. COOPER, and N. D. ZINDER: Host-dependent mutants of the bacteriophage
f2. IV. On the biosynthesis of a viral RNA polymerase. Virology 24, 60-70 (1964).
- , K. HORIUCHI, and N. D. ZINDER: Mutants of the bacteriophage f2 V. On the production
of noninfectious phage particles. Virology 27, 139-155 (1965).
LOWRIE, R. J., and P. L. BERGQUIST: Transfer ribonucleic acids from Escherichia coli treated
with 5-fluorouracil. Biochemistry 7,1761-1770 (1968).
LOZERON, H. A., and M. P. GORDON: Ultraviolet sensitization and photoreactivation of tobacco
mosaic virus ribonucleic acid containing 5-fluorouracil. Biochemistry 3,507-510 (1964).
- , T. GABRIEL, W. TAUTZ, and R. DUSCHINSKY: The photochemistry of 5-fluorouracil.
Biochemistry 3, 1844-1850 (1964).
MADISON, J. T., G. A. EVERETr, and H. KUNG: Nucleotide sequence of a yeast tyrosine

transfer RNA. Science 153, 531-534 (1966).
MANDEL, H. G.: The physiological disposition of some anticancer agents. PharmacoI. Rev.
11, 743-838 (1959).
- , R. MARKHAM, and R. E. F. MATrHEWS: The distribution of thiouracil in nucleic acid of
tobacco mosaic virus. Biochim. biophys. Acta (Amst.) 24,205-206 (1957).
MARVER, H. S., A. COLLINS, D. P. TSCHUDY, and M. RECHCIGL, JR.: t5-Aminolevulinic acid
synthetase. II. Induction in rat liver. J. bioI. Chem. 241,4323-4329 (1966).
MASSOULIE, J., A. M. MICHELSON, and F. POCHON: Polynucleotide analogues VI. Physical
studies on 5-substituted pyrimidine polynucleotides. Biochim. biophys. Acta (Amst.) 114,
16-26 (1966).
MATrHEWS, R. E. F.: Biosynthetic incorporation of metabolic analogues. PharmacoI. Rev.
10,359-406 (1958).
MUKHERJEE, K. L., A. R. CURRERI, M. JAVID, and C. HEIDELBERGER: Studies on fluorinated
pyrimidines. XVII. Tissue distribution of 5-fluorouracil-2-C\4 and 5-fluoro-2'-deoxyuri-
dine in cancer patients. Cancer Res. 23, 67-77 (1963).
MUNYON, W., and N. P. SALZMAN: The incorporation of 5-fluorouracil into poliovirus.
Virology 18. 95-101 (1962).
NAKADA, D.: Formation of ribosomes by a "relaxed" mutant of Escherichia coli. J. molec.
BioI. 12, 695-725 (1965).
-, and B. MAGASANIK: The roles of inducer and catabolite repressor in the synthesis of
p-galactosidase by Escherichia coli. J. molec. BioI. 8, 105-127 (1964).
NAONO, S., et F. GROS: (1) Effets d'un analogue de base nucleique sur la biosynthese de
proteines bacteriennes. Changements de la composition glob ale des proteines. C. R. Acad.
Sci. (Paris) 250, 3527-3529 (1960).
- - (2) Synthese par E. coli d'une phosphatase modifiee en presence d'un analogue
pyrimidique. C. R. Acad. Sci. (Paris) 250, 3889-3891 (1960).
NEMETH, A. M.: The effect of 5-fluorouracil on the developmental and adaptive formation
of tryptophan pyrrolase. J. bioI. Chem. 237, 3703-3706 (1962).
OrAKA, E., S. OSAWA, and A. SIBATANI: Stimulation of 14C-Ieucine incorporation into
protein in vitro by ribosomal RNA of Escherichia coli. Biochem. biophys. Res. Commun. 15,
568-574 (1964).
PARDEE, A. B., and L. S. PRESTIDGE: The initial kinetics of enzyme induction. Biochim
biophys. Acta (Amst.) 49, 77-78 (1961).
PITOT, H. c., and C. PERAINO: Studies on the induction and repression of enzymes in rat liver.
I. Induction of threonine dehydrase and omithine-t5-transaminase by oral intubation of
casein hydrolysate. J. bioI. Chem. 239, 1783-1788 (1964).
REICH, M., and H. G. MANDEL: Uracil: Failure to restore DNA synthesis while relieving
5-fluorouracil-induced inhibition. Science 145, 276-277 (1964).
- - Dissociation of cellular functions in Bacillus cereus by 5-fluorouraciI. J. Bact. 91,
517-523 (1966).
RICH, M. A., J. L. BOLAFFI, J. E. KNOLL, L. CHEONG, and M. L. EIDINOFF: Growth inhibi-
tion of a human tumor cell strain by 5-fluorouracil, 5-fluorouridine, and 5-fluoro-2'-
deoxyuridine - Reversal studies. Cancer Res. 18, 730-735 (1958).
ROGERS, H. J., and H. R. PERKINS: 5-Fluorouracil and mucopeptide biosynthesis by Staphylo-
coccus aureus. Biochem. J. 77, 448-459 (1960).
ROSEN, B.: Characteristics of 5-fluorouracil-induced synthesis of alkaline phosphatase. J.
molec. BioI. 11, 845-850 (1965).
RUTMAN, R. ]., A. CANTAROW, and K. E. PASCHKIS: Studies in 2-acetylaminofluorene
carcinogenesis. III. The utilization of uracil-2-C14 by preneoplastic rat liver and rat
hepatoma. Cancer Res. 14, 119-123 (1954).
SAUNDERS, P. P., G. A. SCHULTZ, R. E. BASS, and G. F. SAUNDERS: Effects of 5-fluorouracil
on a uracil and thymine requiring strain of Bacillus sublilis. Bact. Proc. 1968, 131.
SELLS, B. H., and K. CRUDUP: Ribosome production during recovery from puromycin
treatment: Influence of 5-fluorouraciI. Biochim. biophys. Acta (Amst.) 123, 253-264
(1966).
SHIMURA, Y., and D. NATHANs: The preparation of coliphage MS2 containing 5-fluoro-
uracil. Biochem. biophys. Res. Commun. 16, 116-120 (1964).
- , R. E. MOSES, and D. NATHANs: Coliphage MS2 containing 5-fluorouracil. I. Preparation
and physical properties. J. molec. BioI. 12, 266-279 (1965).
- - - Coliphage MS2 containing 5-fluorouracil. II. RNA-deficient particles formed in the
presence of 5-fluorouraciI. J. molec. BioI. 28, 95-102 (1967).
SINGER, M. F., and P. LEDER: Messenger RNA: An evaluation. Ann. Rev. Biochem. 35,
195-230 (1966).
SKODA, J., and R. E. HANDSCHUMACHER: The influence of certain antimetabolites on the
incorporation of orotic acid into pseudouridine in animals. Biochim. biophys. Acta
(Arnst.) 68, 481-483 (1963).
SLAPIKOFF, S., and P. BERG: Mechanism of ribonucleic acid polymerase action. Effect of
nearest neighbors on competition between uridine triphosphate and uridine triphos-
phate analogs for incorporation into ribonucleic acid. Biochemistry 6,3654-3658 (1967).
SOFFER, R. L.: Studies on the biological activity of ribonucleic acid isolated from Escherichia
coli after exposure to 5-fluorouracil. Biochim. biophys. Acta (Arnst.) 87,416-422 (1964).
STAEHELIN, M.: Chemical modifications of virus infectivity: Reactions of tobacco mosaic
virus and its nucleic acid. Experientia (Basel) 16, 473-483 (1960).
-, and M. P. GORDON: Effects of halogenated pyrimidines on the growth of tobacco mosaic
virus. Biochim. biophys. Acta (Arnst.) 38, 307-315 (1960).
SUEOKA, N., and T. YAMANE: Fractionation of aminoacyl-acceptor RNA and the coding
problem. In: Informational Macromolecules, pp. 205-227. VOGEL, H. J., V. BRYSON,
and J. O. LAMPEN, Eds. New York: Academic Press 1963.
SUNDARAM, T. K.: Phenotypic reversal by 5-fluorouracil of the auxotrophy of am mutants of
Neurospora crassa. Biochim. biophys. Acta (Arnst.) 138,611-613 (1967).
SUTIC, D., and B. D JORDJEVIC: Effect of 5-fluorouracil on antigenic properties of tobacco
mosaic virus. Nature (Lond.) 203, 434-435 (1964).
SZER, W., and D. SHUGAR: Preparation of poly-5-fluorouridylic acid and the properties of
halogenated poly-uridylic acids and their complexes with poly-adenylic acid. Acta bio-
chim pol. 10,219-231 (1963).
TERSHAK, D. R.: Effect of 5-fluorouracil on poliovirus growth. Virology 24, 262-269 (1964).
- Effect of5-fluorouracil on poliovirus-induced RNA polymerase. J. molec. Biol. 21, 43-50
(1966).
TOMASZ, A., and E. BOREK: The mechanism of an osmotic instability induced in E. coli
K-12 by 5-fluorouracil. Biochemistry 1, 543-552 (1962).
TOOZE, J., and K. WEBER: Isolation and characterization of amber mutants of bacteriophage
R17. J. molec. BioI. 28, 311-330 (1967).
WACHSMAN, J. T., S. KEMP, and L. HOGG: Comparative effects of 5-fluorouracil on strains
of Bacillus megaterium. J. Bact. 87, 1011-1018 (1964).
WAGNER, N. J., and C. HEIDELBERGER: Some effects of 5-fluoroorotic acid and 5-fluoroura-
cil on the soluble ribonucleic acid of rat liver. Biochim. biophys. Acta (Arnst.) 61, 373-
379 (1962).
WAHBA, A. J., R. S. GARDNER, C. BASILIO, R. S. MILLER, J. F. SPEYER, and P. LENGYEL:
Synthetic polynucleotides and the amino acid code. VIII. Proc. nat. Acad. Sci. (Wash.)
49, 116-122 (1963).
WHITE, P. J., and C. A. NICHOL: Effects of uracil and thymidine on the development of
resistance to 5-fluorouracil in Pediococcus cerevisiae. J. Bact. 85, 97-105 (1963).
WILLEN, R., and U. STENRAM: RNA synthesis in the liver of rats treated with 5-fluorouracil.
Arch. Biochem. Biophys. 119, 501-503 (1967).
WITTMANN, H. G.: Proteinanalysen von chemisch induzierten Mutanten des Tabakmosaik-
virus. Z. VererbungsI. 95, 333-344 (1964).
WITTMANN-LIEBOLD, B., and H. G. \'V'ITTMANN: (1) Lokalisierung von Aminosaureaus-
tauschen bei Spontanmutanten und nach Fluoruracileinbau isolierten Mutanten des Tabak-
mosaikvirus. Z. Vererbungsl. 97,218-225 (1965).
- - (2) Lokalisierung von Aminosaureaustauschen bei Nitritmutanten des Tabakmosaik-
virus. Z. Vererbungsl. 97, 305-326 (1965).
Organelle Biosynthesis: The Chloroplast
ROBERT M. SMILLIE and N. STEELE SCOTT
1. Introduction
The cells of higher organisms contain highly differentiated bodies or organelles,
each of which is delimited from the surrounding cytoplasm by an outer membrane.
Organelles carry out specialized cellular functions; they possess complex and unique
internal structures and contain a characteristic complement of enzymes and other
constitutents. Clearly, knowledge of the genetic and metabolic regulatory systems
involved in the differentiation of these cellular structures is crucial to our comprehen-
sion of the biochemistry of growth and differentiation of the cells of higher organisms.
In this review we have discussed the organization of cellular mechanisms for the
synthesis of a specialized organelle of plant cells, the chloroplast, and comparisons
have also been made with the mechanisms involved in the synthesis of the mitochon-
drion. Emphasis has been placed on the nucleic acids of the chloroplasts, their pro-
perties and synthesis, and their respective roles in the synthesis of chloroplast proteins.
In the last section of the review we have examined the photo regulation of these
processes.
Several works reviewing various aspects of the biogenesis of chloroplasts have
been published in recent years including "The Plastids" by KIRK and TILNEy-BASSETT
(1967), the "Biochemistry of Chloroplasts," Volumes I and II (edited by T. W.
GOODWIN) and reviews by GRANICK (1955, 1961) and SCHIFF and EpSTEIN (1965).
The biosynthesis of the lipid components of chloroplasts as well as the structure,
function and inheritance of chloroplasts were not specifically considered in this
review; for reviews of these aspects the reader is referred to the references given above
and to recent publications edited by SHIBATA et al. (1968), OLSON et al. (1967) and
VERNON and SEELY (1966).
II. Composition
A. Chloroplasts
KIRK (see KIRK and TILNEy-BASSETT, 1967) has made an estimate of the chemical
composition of spinach chloroplasts based on a number of published analyses. About
69% of the dry weight consists of protein and of this about 55% is water-soluble
protein and 45% is membrane protein (see also Section V.B.3). Lipids make up 21 %
of the chloroplast (chlorophylls 5%, carotenoids 0.7%) and nucleic acids 1 to 8%.
In carbohydrate-storing chloroplasts, polysaccharides can account for a substantial
part of the dry weight, but the amount varies widely depending on the type of plastid,
its age, and the environmental conditions. Estimates of the lipid composition of the
Organelle Biosynthesis: The Chloroplast 137
internal lipoprotein membranes of chloroplasts have been given by ALLEN et al.

(1966), PARK (1966), and KIRK and TILNEy-BASSEIT (1967).
From one-quarter to one-half of the water-soluble protein of chloroplasts can be
separated as a single protein fraction (Fraction I protein) with a molecular weight of
about 5 X 105• Only one enzymic activity has been associated with this protein, that
of the first enzyme of the Calvin cycle, ribulose-l,5-diphosphate carboxylase. This
and other enzymes found in chloroplasts are listed in Table 1. Several other enzymic
activities which have been associated with chloroplasts in earlier studies (see reviews
by WEIER and STOCKING, 1952 and SISSAKIAN, 1958) are omitted from this list, since
their presence or absence has not been confirmed using the improved methods for
isolating chloroplasts now available. In this connection, LEECH (1966) has discussed
the possible occurrence of NAD-malate dehydrogenase in chloroplasts.
Besides chloroplasts, several other types of plastids are found in plant cells in-
cluding those containing predominantly carotenoids (chromoplasts), starch (amylo-
plasts), oil (elaioplasts) and storage protein (proteoplasts). The formation of these
plastids has been investigated in only a few instances and it will be interesting to
determine if they also contain DNA and a protein-synthesizing system. Protein-
containing bodies, which are possibly a type of proteoplast, have been isolated from
wheat endosperm by MORTON and RAISON (1963) and shown to incorporate amino
acids into their protein.
B. Proplastids
The word proplastid is usually used to describe partially differentiated plastids
found in the meristematic cells of multicellular algae or higher plants and in etiolated
cells (cells in which chlorophyll synthesis has been suppressed by growth in the dark).
KIRK (see KIRK and TILNEy-BASSEIT, 1967) has proposed the name etioplast to
describe the proplastid of etiolated cells. The composition of proplastids varies widely
depending on the organism and its stage of growth (see Section VI). They lack chloro-
phyll but contain small amounts of protochlorophyllide [0.1 to 0.3% of the concentra-
tion of chlorophyll in chloroplasts, BOGORAD, 1967 (1)]. Proplastids from etiolated
bean leaves contain comparable amounts of RNA [BOARDMAN, 1966 (1)] and ap-
proximately one-third as much protein and lipid per plastid (MEGO and JAGENDORF,
1961) as mature chloroplasts. The structure of proplastids has been reviewed by
GUNNING [1965 (2)].
III. Chloroplast DNA

A. Occurrence and Properties
The existence of DNA in chloroplasts and other organelles is now well established
on the basis of cytological, chemical and physical evidence (for a recent review see
KIRK, 1966). Table 2 lists those organisms in which chloroplast DNA has been shown
to occur and compares properties of chloroplast DNA with DNA from other sources.
The base compositions of chloroplast DNA from different higher plants are remark-
ably similar and all have a comparable or higher content of guanine and cytosine than
the corresponding nuclear DNA. By contrast, the chloroplast DNA of unicellular
algae have higher contents of adenine and thymine than the corresponding nuclear
DNA. 5-methyl cytosine, which is usually found in nuclear DNA, is absent from the
>-'
Table 1. Enzymes found in chloroplasts ,»
ex>
Function Enzyme References
CO 2 fixation: Calvin cycle RuDP carboxylase

3-phosphoglycerate kinase
NADP-glyceraldehyde-3-P
dehydrogenase
Triosephosphate isomerase
FDP aldolase PETERKOFSKY and RACKER (1961),
Transketolase HEBER et al. (1963), SMILLIE (1963) ?:I
2Cylulose-5-P epimerase
oc;
tti
Ribose-5-P isomerase
~
Ribulose-5-P kinase
Sedoheptulose-1, 7-diphosphatase ~
rJl
Alkaline FDPase ;;::
FDPase (ferredoxin-activated) BUCHANAN et al. (1967) P
t-<
Carbonic anhydrase EVERSON and SLACK (1968) t;J
C4 -dicarboxylic pathway Pyruvate-P;-dikinase HATCH and SLACK (1967) b
0.-
Phosphoenolpyruvate carboxylase SLACK and HATCH (1967)
Photosynthetic electron transfer and
:z:
rJl
phosphorylation Cytochrome f DAVENPORT and HILL (1952) t;l
tti
Cytochrome b 559 LUNDEGARDH (1962), t-<
tti
BOARDMAN and ANDERSON (1967) rJl
Cytochrome b 562 HILL (1954) n
o
Ferredoxin SAN PIETRO and LANG (1958) ::j
Plastocyanin KATOH et al. (1961)
Rubimedin HENNINGER et al. (1966)
Ferredoxin-NADP-reductase KEISTER et al. (1960)
(Pyridine nucleotide transhydrogenase)
Coupling factor (ATPase) MCCARTY and RACKER (1966)
Protein-binding? Structural protein CRIDDLE (1966)
Porphyrin synthesis 5-aminolaevulinate dehydratase CARELL and KAHN (1964)
Ferrochelatase JONES (1967), PORRA and LASCELLES (1968)
Chlorophyllase SUDYINA (1963)
Table 1. (Continued)
Function Enzyme References
Lipid metabolism Fatty acid synthetase STUMPF et al. (1967).

Stearoyl-acyl-carrier protein desaturase NAGAI and BLOCH (1966)
Galactolipid hydro lases SASTRY and KATES (1964)
Mevalonate kinase ROGERS et al. [1967 (1)]
Carotenoid oxidase FRIEND and MAYER (1960)
Carbohydrate metabolism Phosphorylase STOCKING (1959) o...
UDPG: F-6-P glucosyltransferase cra
UDPG: fructose glucosyltransferase o
(1)
UDPG pyrophosphorylase ~
BIRD et al. (1965) IJ:l
Phosphoglucomutase
Glucose-P isomerase
o·
(J>
Sucrose-6-phosphatase ].
(1)
Nucleic acid and nucleotide metabolism DNA polymerase SPENCER and WHITFELD (1967), ;!'l.
(J>
TEWARI and WILDMAN (1967), SCOTT et al. (1968)
RNA polymerase KIRK [1964 (1), (2)] ;1
Ribonuclease HEBER (1963), SZARKOWSKI et al. (1962) (1)
Aminoacyl-sRNA synthetase BOVE and RAACKE (1959),

HENSHALL and GOODWIN [1964 (2)] ~...
Adenosine-5' -triphosphatase BENNUN and AVRON (1964) .g
Adenylate kinase SMILLIE (1963) ~
Pyrimidine nucleotide synthesis )OUSSAUME and BOURDU (1966) ~
Nitrite reduction Nitrite reductase RITENOUR et al. (1967)

Sulphate reduction Adenylylsulphate kinase
DAVIES et al. (1966)
Sulphate adenylyl-transferase
Other enzymes Inorganic pyrophosphatase SMILLIE et al. (1963)
Glutamate-oxalacetate transaminase HEBER (1960)
Oxalate oxidase NAGAHISA and HATTORI (1964)
Acid phosphatase RAGLETTI et al. (1966)
>-'
~
140 ROBERT M. SMILLIE and N. STEELE SCOTT
chloroplast DNA of Euglena (BRAWERMAN and EISENSTADT, 1964; RAY and HANA-
WALT, 1964), snapdragon (RUPPEL and VAN WYK, 1965) and tobacco (TEWARI and
WILDMAN, 1966).
The validity of some of the measurements on chloroplast DNA, particularly in
higher plants, could be challenged on the grounds of possible bacterial contamina-
tion of chloroplast preparations; in the majority of the studies no specific attempt was
made to deal with this problem. TEWARI and WILDMAN (1966) grew their plants
aseptically to avoid the problem of bacteria. For many algae, axenic cultures are
available and we have found less than 1 bacterium/200,000 chloroplasts in prepara-
tions of chloroplasts from Euglena (SCOTT et aI., 1968).
The problem of contamination of chloroplasts with nuclear DNA is less easy to
solve unequivocally. DNA extracted from isolated chloroplasts generally contains
some nuclear DNA (see references in Table 2) and this makes the identification of a
DNA unique to chloroplasts more difficult. Provided the densities differ sufficiently,
separation of the nuclear and chloroplast DNA can be achieved by centrifugation
in a CsCI gradient. Where both have similar densities, gradient centrifugation may be
insufficient in itself to demonstrate the existence of a separate chloroplast DNA and
other means must be employed. KIRK (1963) used base ratio determinations to
distinguish small differences in the nucleotide composition of nuclear and chloroplast
DNA of broad bean chloroplasts. TEWARI and WILDMAN (1966) found that chloro-
plast DNA from tobacco renatured much more easily than nuclear DNA, and the
application of this renaturation test in future studies should prove valuable in
distinguishing between organelle and nuclear DNA. In the case of isolated mitochon-
dria or mitochondrial fragments, deoxyribonuclease can be used to remove conta-
minating nuclear DNA as the mitochondrial structure apparently protects the orga-
nelle DNA from degradation (TEWARI et al., 1965; EDELMAN et aI., 1966; NASS, 1966).
This technique has not been applied to the purification of chloroplast DNA, but the
inhibition of RNA synthesis in isolated chloroplasts by deoxyribonuclease (see
Section IV. C) suggests the DNA within the chloroplast, unlike the DNA of mitochon-
dria, is susceptible to attack by deoxyribonuclease.
While it is generally considered that the nuclear DNA contaminating preparations
of isolated chloroplasts is due to the presence of nuclear fragments or to adsorption
of nuclear DNA released from broken nuclei onto chloroplasts, the possibility has
not been entirely eliminated that some of this apparent nuclear DNA is actually
associated with the chloroplast in vivo.
Estimates of the amount of DNA per chloroplast (Table 2) are usually about the
same as values reported for the DNA content of a bacterium (0.3-1 X 10-14g).
Molecular weights of up to 4 X 107 have been reported for chloroplast DNA after
isolation (see references in Table 2), but these values could be low because of shearing,
and a recent electron micrograph shows chloroplast DNA up to 150!L long (WOOD-
COCK and FERNANDEZ-MoRAN, 1968), which is equivalent to a molecular weight of
30 X 107•
Chloroplast DNA appears to have a higher molecular weight than mitochondrial
DNA, since the maximum value reported for the latter is 107 • The DNA from animal
mitochondria is circular with a length of 5.4 !L (VAN BRUGGEN et aI., 1966; SINCLAIR
and STEVENS, 1966; NASS, 1966), which is equivalent to a molecular weight of 107 • It
is not known if DNA from plant mitochondria and chloroplasts is circular.
Satellite DNA has been found in the purple photosynthetic bacteria Chromatium
and Rhodospirillum rubrum by SUYAMA and GIBSON (1966), but there is no evidence to
link this DNA with the chromophores or photosynthetic capability of these bacteria.
B. The Localization oj DNA within the Chloroplast

Cytological evidence for the localization of DNA in chloroplasts has come from
both light and electron microscopy.
RIS and PLAUT (1962) found material which was Feulgen positive and stained
with acridine orange in the chloroplast of Chlamydomonas moewusii. The stained mate-
rial was found in areas of low electron density proximate to the pyrenoid.
Electron micrographs of the same cells revealed the presence of microfibrils,
attributed to DNA, in these areas and similar areas containing micro fibrils have been
found occurring randomly in electron micrographs of chloroplasts of higher plants
[GUNNING, 1965 (1); KISLEV et aI., 1965], a green algae [BISALPUTRA and BISALPUTRA,
1967 (1)] a chrysomonad (GIBBS, 1967) and brown algae [BISALPUTRA and BISALPUTRA,
1967 (2)]. Microfibrils were not visible in sections pretreated with deoxyribonuclease,
but such experiments should be interpreted with caution since the enzymic treatment
may cause the loss, not only of the DNA, but of other stainable material which is
structurally integrated with the DNA.
Although the microfibrils occur only in electron clear areas of the chloroplast
their presence in the more electron dense areas may be obscured. Consequently, it is
not known if DNA is localized in several discrete areas of the chloroplast or if it is
distributed throughout the chloroplast. Nor is it known if there are one or several
molecules of DNA per chloroplast.
Chloroplast DNA may also be associated with membranes of the chloroplast.
DYER and LEECH (1968) found an association between DNA and the lamellar fraction
of chloroplasts, and the DNA seen in the electron micrographs of whole chloroplasts
may be loops or broken ends of bound DNA. WOODCOCK and FERNANDEZ-MoRAN
(1968) found strands of DNA in electronmicrographs of shadow cast lysates of
spinach chloroplasts. In some areas they found the DNA characteristically associated
with the chloroplast membrane and here the conformation of the DNA led them to
suggest that- the DNA was in a single stranded form. The longest strand of DNA
found (150 (1.) would weigh about 0.5 X 10-15 g, which could mean that at least 10%
of the DNA in spinach chloroplasts (Table 2) was in one strand. The DNA in
mitochondria also appears to be closely associated with membranes (Section lILA).
There areno reports of his tones associated with satellite DNA and possibly mem-
brane associations take their place. An association of DNA, DNA polymerase and
chloroplast membranes may account for the specificity of chloroplast DNA poly-
merase toward chloroplast DNA (see Section III. C.l) found in Euglena, especially if the
DNA is single stranded in these areas.
C. Synthesis of Chloroplast DNA

Irradiation of the cytoplasm of Euglena with ultraviolet light results in a loss of the
capacity of daughter cells to form chloroplasts (bleaching) and this condition is not
rectified in subsequent generations by genetic information contained in the nucleus
(GIBOR and GRANICK, 1962). The bleaching is associated with a loss of chloroplast
Table 2. The occu"ence of DNA in chloroplasts of various plant and algal species. The density of chloroplast DNA is compared with the density of other DNA i!3
-
species. The percentages of the satellite DNA components in the total DNA of the organism is shown where it can be calculated from or is shown in the original
report. IlVhere possible, the amount of DNA per chloroplast and the % of guanine and cytosine in DNA are given. The latter value mqy be calculated from the
relationship between density and base content (SCHILD KRAUT et al., 1962). The nuclear DNA figures are uncorrected for methylation (KIRK, 1967)
Species Nuclear Chloroplast Other DNAI Chloroplast References

Density % GC Density % GC Density % GC chloroplast DNA as
gms/cc glee glee (g X 1014) % total DNA
~
Acetabularia medite"anea 0.01 GIBOR and IZAWA (1963) 0
to
0.03-0.4 BALTUS and BRACHET (1963) tTl
l"
..;
Chlorella ellipsoida 1.716 57 1.695 36 1.0-1.2 IWAMURA (1960),
CHUN et al. (1963) ~
CFl
Chlamydomonas reinhardi 1.721 62 1.694 35 1.711 42 1.2 SAGER and ISHIDA (1963) ;;::
Chlamydomonas reinhardi 1.723 64 1.695 36 LEFF et al. (1963) t::
t"'
Chlamydomonas reinhardi 1.723 64 1.695 36 CHUN et al. (1963) ;;
~
Chlamydomonas reinhardi 1.723 64 1.695 36 1.715 46 0.8 6 CHIANG and SUEOKA (1967) ::l
0..
Euglena gracilis, Z. 1.708 51& 1.684 25a 1.692 25 1.1 2-5 BRAWERMAN and EISENSTADT
~
(1964) CFl
RAY and HANAWALT (1964) >-l
Euglena gracilis var. bacillaris 1.702 43 1.685 26 1.690 31 tTl
tTl
Euglena gracilis var. bacillaris 1.707 52a 1.686 26a 1.691 b 32 EDELMAN et al. (1964) t"'
tTl
Antirrhinum major 41a 37a 0.56 RUPPEL and VAN WYK (1965) CFl
n
(snapdragon) 0
>-l
Antirrhinum major 1.697 38 1.709 50 GREEN and GORDON (1967) >-l
Vicia faba (broad bean) 40a 37a 0.3 KIRK (1963, 1966)
Beta vulgaris (beet) 1.695 36 1.705 46 1.719 60 CHUN et al. (1963)
Beta vulgaris vat. cicla 1.689 30 1.700 41 KISLEV et al. (1965)
(Swiss chard)
Spinacia oleracea (spinach) 1.695 36 1.705 46 1.719 60 CHUN et al. (1963)
3.8-6.9 CHIBA and SUGUHARA (1957)
4 BIGGINS and PARK (1964)
Table 2. (Continued)
Nieotiana tabaeum (tobacco) 1.690 31 1.703 44 SHIPP et al. (1965)

1.696 37 1.706 47 1.711 52 GREEN and GORDON (1965) 0...
aq
1.697 38 1.702 43 TEWARI and WILDMAN (1966) 10
::l
co
Nieotiana gllltinosa 1.697 38 1.702 43 0.47 9 TEWARI and WILDMAN ~
co
(1966, 1968) to
Phaseolus vulgaris (bean)c 35 35 BERIDZE et al. (1967)
o·
til
1.694 ) 1.694 ) '<
1.703 44 1.703 44 ::l
Er
co
Brassiea rapa (turnip) 1.692 33 1.695 36 SUYAMA and BONNER (1966) g!.
til
Equisetum sp. (horsetail) 1.697 38 1.713 54 GREEN and GORDON (1967)

..
>-l
Ranuneullis repens (buttercup) 1.697 38 1.703 44 GREEN and GORDON (1967) ::r
co
n
Dianthus earyophyllus 1.695 36 1.706 47 GREEN and GORDON (1967) g:
(carnation) ...00
'0
Tagetes patula (marigold) 1.692 33 1.702 43 1.707 48 GREEN and GORDON (1967) .,
~
• Determined by chemical analyses. b Attributed to mitochondrial DNA (EDELMAN et aI., 1966) .
c Two components of nuclear DNA and two of chloroplast DNA were separated. The DNA from the chloroplast was more easily renatured.
.....
~
V>
144 ROBERT M. SMILLIE and N. STEELE Scon
DNA (EDELMAN, SCHIFF and EpSTEIN, 1965). Thus it can be inferred that the chloro-
plast exerts some control over the synthesis of its own DNA independently of the
nucleus. Studies demonstrating the synthesis of chloroplast DNA in isolated chloro-
plasts have confirmed this.
1. Synthesis of DNA in the Isolated Chloroplast

In enucleated Acetabu/aria mediterranea 14C02 was incorporated into DNA under
conditions in which the chloroplasts were known to replicate (GIBOR, 1967). The
DNA (density 1.685 glee) was isolated from a chloroplast fraction.
OD6 -A260
___ 3H
1500
1000
E
c
E5j 500 c
~-4 "e
....::1:-';;
C
0 ::>
0
u
Fraction number
Fig. 1. The synthesis of chloroplast DNA in isolated chloroplasts. Isolated Euglena chloro-
plasts were incubated with 3H-thymidine 5'-triphosphate, the DNA isolated from the chloro-
plasts and the chloroplast DNA (e = 1.686) separated from the nuclear DNA (e = 1.707) by
centrifugation on a esC! gradient. In the lower graph, the DNA was treated with deoxy-
ribonuclease before centrifugation. (Data from Scon et at, 1968)
SPENCER and W HITFELD [1967 (1)] found isolated spinach chloroplasts would
rapidly incorporate 3H-thymidine 5'-triphosphate into an acid-stable fraction. The
uptake was inhibited by pretreating the chloroplasts with deoxyribonuclease or
actinomycin D. That most of the incorporated label was in chloroplast DNA was
demonstrated by the ease of denaturation and renaturation of the labelled material
(see Section III.A.). There was also a smaller incorporation of3H into nuclear DNA,
apparently catalyzed by a separate polymerase. Similar results have been reported by
TEWARI and WILDMAN (1967) for the incorporation oPH-adenosine and 3H-thymi-
dine 5'-triphosphates into DNA of tobacco chloroplasts. Centrifugation of the la-
belled products on gradients of CsCI showed they had the same density as chloroplast
DNA. Hybridization studies (see Section III.D.2) with the newly synthesized 3H-DNA
indicated that it had more base sequences in common with chloroplast DNA than
with nuclear DNA. The base ratio of the 3H-DNA was also similar to chloroplast
DNA.
Synthesis of chloroplast DNA has also been found in isolated chloroplasts from
Euglena (SCOTT et aI., 1968). The chloroplast and nuclear DNA of this organism can
be separated by centrifugation in a gradient of CsCl and this enabled us to demonstrate
that the uptake of3H-thymidine 5'-triphosphate by isolated chloroplasts was essen-
tially into the chloroplast DNA alone (Fig. 1). The reason for the low incorporation
into the nuclear DNA fraction is not known, but the result suggests a specific
mechanism for synthesizing chloroplast DNA that does not recognize nuclear DNA
as a primer. For instance chloroplast DNA may be bound to membranes (see Section
IILB.) and the chloroplast DNA polymerase may also be bound in such a way as to
produce a favourable juxtaposition between enzyme and substrate. SPENCER and
WHITFELD [1967 (1)] noted that both the chloroplast DNA and the DNA polymerase
did not leach out of chloroplasts even after extensive washing. Whether these systems
represent de novo or repair synthesis of DNA remains to be established.
The incorporation of3H-precursors of DNA into isolated chloroplasts of several
plants and algae has also been demonstrated by autoradiography (see PARTHIER and
WOLLGIEHN, 1966).
2. Synthesis of DNA in vivo
Studies with synchronously dividing cells of Euglena and Chlamydomonas have
disclosed that chloroplast and nuclear DNA replicate at different stages of cell growth.
PETROPOLOUS (1964) and COOK and HUNT (1965) grew cells of Euglena synchronously
by alternating light and dark periods, and determined the period of growth during
which the cells showed maximum sensitivity to bleaching by ultraviolet light. This
period was equated with the period in which chloroplast DNA was most likely to be
single stranded and therefore replicating. Conflicting results have come from these
studies as maximum sensitivity has been reported both at the beginning (COOK and
HUNT), and in the middle (PETROPOLOUS) of the light period. In a subsequent auto-
radiographic study, COOK (1966) showed that maximum rates of3H-adenine uptake
occurred at the beginning of the light period and again towards the end of this period
and he interpreted this as being due to chloroplast and nuclear DNA synthesis,
respectively.
Using the now classical technique of following the replication of DNA with 15N
and 14N (MESELSON and STAHL, 1958), CHIANG and SUEOKA (1967) clearly demonstrat-
ed that chloroplast DNA of Chlamydomonas reinhardi replicated during the light
period in synchronously growing cultures, while the cells and nuclear DNA replicated
in the dark phase of growth.
Tobacco seedlings incorporated 32p more rapidly into chloroplast DNA than into
nuclear DNA (GREEN and GORDON, 1966) and this again is consistent with a turnover
of chloroplast DNA independent of nuclear DNA.
D. Function of Chloroplast DNA

1. Information Content
The amount of genetic information in the DNA of Euglena chloroplasts can be
calculated by making a few simple approximations. From DNA analyses (KEMPNER
and MILLER, 1965), it can be calculated that each Euglena cell contains 3.2 X 10-12 g
DNA. As about 2 to 5% of this DNA is in the chloroplast (Table 2) and there are
about 10 chloroplasts per autotrophic cell (EpSTEIN and SCHIFF, 1961), then each
chloroplast contains about 10-14 g of DNA. For DNA containing equal amounts of
all four bases the average molecular weight of a sodium mononucleotide unit would be
332 and using A vogadros number we can calculate that each chloroplast contains ap-
proximately 2 X 107 individual base units. If only half of the DNA duplex is used for
coding there are 107 bases available, and of these 1% are polycistronic for ribosomal
RNA (Scon and SMILLIE, 1967). A small amount, probably less than 1%, could be
used for coding for the relatively small transfer RNA. Of the remaining DNA, if 3
mononucleotides code for an amino acid and an average amino acid molecular weight
is 140, then there is enough DNA to code for 14,000 different polypeptide chains of
molecular weight 30,000. Some chloroplasts contain less DNA than Euglena chloro-
plasts (Table 2) and in these the amount of information available would be less. It is
not known to what extent sections or even whole strands of DNA are duplicated with-
in a chloroplast. This could considerably reduce the amount of information available,
but it seems likely that the chloroplast would still contain sufficient information to
code for several hundred polypeptide chains.
SUYAMA and BONNER (1966) estimated that mitochondria from various plant species
contain about 3.5 X 10-16 g of DNA. Mitochondrial DNA is circular and has a
molecular weight of 107 (see Section IILA.); thus there would be about 21 strands of
DNA per mitochondrion. If all these strands were identical, each would have enough
information to code for ribosomal RNA and about 15 polypeptide chains of molecular
weight 30,000. On the other hand, if the strands are all different and there is only one
with a cistron for ribosomal RNA, then there would be enough information for about
500 chains.
These calculations suggest that there is more potential genetic information in a
chloroplast than in a mitochondrion.
2. Molecular Hybridization
One of the most powerful tools for analysing the sources of transcription of
genetic information is the technique of DNA-RNA hybridization. This technique
depends on the observation that RNA will bind specifically to the DNA which
presumably coded for its synthesis (HALL and SPIEGELMAN, 1961). This and many
similar observations have been interpreted as demonstrating a specific pairing, base
for base, along the RNA chain and its DNA cistron.
The hybridization reaction depends on denaturing the DNA to a single stranded
form and then incubating it with RNA under conditions which allow the RNA to
unfold and then anneal with the DNA, forming a stable duplex. After a suitable in-
cubation period, unpaired RNA is hydrolyzed with ribonuclease and the hybrid
material in the RNA-DNA duplex, which is resistant to ribonuclease, is measured.
The measurement can be quantitated if both the DNA and RNA are isotopically
labelled. To avoid renaturation, the DNA is immobilized in the single stranded form
in agar (MCCARTHY and BOLTON, 1963) or more simply on a nitrocellulose membrane
filter (GILLESPIE and SPIEGELMAN, 1965). Care must be taken to ensure the ribo-
nuclease is free of deoxyribonuclease and the DNA and RNA are well characterized
and free of nucleases capable of acting over the relatively long time required for the
hybridization reactions.
The minimum criterion of a successful hybridization should be the demonstration
of a monophasic curve showing eventual saturation of the DNA with RNA, as the
RNA input to the reaction is increased. If saturation is not attained and the curve
continues to rise at a constant or varying rate it may indicate the presence of more
than one species of RNA in the hybrid, nonspecific competition or hybridization and,
as the incubation proceeds, hybrid breakdown and RNA degradation.
By applying the technique of DNA-RNA hybridization to the study of organelle
biogenesis it has been possible to demonstrate that one function of chloroplast DNA
is to code for the RNA of chloroplast ribosomes (SCOTT and SMILLIE, 1967). Experi-
ment 1 in Table 3 shows the amount oflabelled chloroplast ribosomal RNA bound to
Table 3. Hybridization of 32P-RNA with Chloroplast DNA. Chloroplast DNA (3 ILg) was
annealed with 29 ILg of 32 P-ribosomal RNA (3,200 counts/min/lLg) from chloroplasts in experiments
Nos. 1 to 5, and with RNA from dark-grown Euglena cells in experiment No.6. In experiments
Nos. 2 to 5 inclusive, an excess of unlabelled ribosomal R1VA (100 lLg) from different sources was
included in the annealing mixtures. The amount of hybrid formed is expressed in the last column as a
percentage of the amount of hybridformed between chloroplast DNA and RNA in experiment No.1.
(Data from SCOtT and SMILLIE, 1967)
Ex- Source of Source of Counts/100 min in Related to
periment 32P-RNA unlabelled RNA hybrid No.1 as 100
No.
1 chloroplasts 9,594 ± 3.2% 100

2 chloroplasts autotrophic cells 7,157 ± 4.8% 75
3 chloroplasts chloroplasts 5,906 ± 6.8% 62
4 chloroplasts dark-grown cells 9,082 ± 2.4% 95
5 chloroplasts ZUV-l cells 10,566 ± 5.0% 110
6 dark-grown cells 133 ± 80% 1
chloroplast DNA at saturation. Definite hybridization between chloroplast DNA and

chloroplast ribosomal RNA was obtained, but the amount of cytoplasmic RNA
bound to the DNA was not significant. The DNA in each chloroplast contains about
20 to 40 cistrons for chloroplast ribosomal RNA and apparently none for the cyto-
plasmic ribosomal RNA (SCOTT and SMILLIE, 1967).
Similar results have been obtained for tobacco chloroplast DNA (TEW ARI and
WILDMAN, 1968). The saturation of chloroplast DNA with chloroplast ribosomal
RNA was similar in both reports (0.5 to 0.65% - TEWARI and WILDMAN, 1 % -
SCOTT and SMILLIE), but there were only four chloroplast ribosomal RNA cistrons per
tobacco chloroplast. These chloroplasts have less DNA than Euglena chloroplasts
(Table 2) and the differences may reflect a higher degree of ploidy of the Euglena
chloroplast DNA. TEWARI and WILDMAN also found cistrons for cytoplasmic
ribosomal RNA in the nucleus. Saturation and competition experiments showed that
these nuclear and chloroplast cistrons were specific for the cytoplasmic and chloro-
plast RNA, respectively. Binding of chloroplast ribosomal RNA to nuclear DNA was
also found although the specificity of this binding was not closely examined. TEW ARI
and WILDMAN calculated that the 0.3% hybridization between nuclear DNA and
cytoplasmic ribosomal RNA was equivalent to about 2000 cistrons. As they found
10*
0.1 %hybridization between nuclear DNA and chloroplast ribosomal RNA, it can be
calculated that there are about 800 cistrons for the chloroplast RNA (allowing for the
different molecular weights of chloroplast and cytoplasmic ribosomal RNA). As
tobacco contains 300 to 500 chloroplasts per nucleus (TEWARI and WILDMAN, 1968),
there would be about 1200 to 2000 non-nuclear cistrons per cell for chloroplast
ribosomal RNA on chloroplast DNA, as well as the proposed nuclear cistrons.
Another important test of specific hybridization is the competition experiment. If
unlabelled RNA identical to the labelled RNA is included in the hybridization mix-
ture, then the amount of labelled RNA in the hybrid should be reduced in proportion
to the amount of competing unlabelled RNA added. In the experiment in Table 3,
these conditions were not rigorously observed owing to the difficulties of obtaining
matching labelled and unlabelled RNA preparations. However, the amount of la-
belled chloroplast ribosomal RNA hybridizing with chloroplast DNA was signi-
ficantly reduced by addition of unlabelled chloroplast ribosomal RNA, but not by
addition of RNA from dark-grown cells or from a bleached mutant unable to form
chloroplasts.
Hybridization between DNA from different sources has been used to study base
sequence homology in DNA (WARNAAR and COHEN, 1966; DENHARD, 1966; RI-
CHARDS, 1967). Such experiments must be interpreted with caution and some of the
difficulties have been discussed by MCCARTHY (1967). The experiments are charac-
terized by an inability to achieve saturation and currently there is no enzyme available
to digest away all but DNA-DNA duplexes formed on the membrane filter.
Using this technique, RICHARDS (1967) found binding between chloroplast and
nuclear DNA from Euglena and concluded they contained common base sequences,
but because of the technical difficulties inherent in the technique other interpretations
are possible. Using an agar gel variation of the technique, SHIPP et al. (1965) found
no annealing between nuclear DNA and chloroplast DNA from tobacco. With
improvements to the technique of DNA-DNA hybridization it should be possible to
establish whether or not copies of chloroplast (and mitochondrial) DNA are retained
in the nucleus.
E. Origin of Chloroplast DNA

The evolution of chloroplasts and other organelles such as the mitochondrion
has aroused much interest and speculation but the existing evidence is far too meagre
to allow definite conclusions to be drawn. Two main theories to explain the origin of
chloroplasts and their DNA have been proposed. The first, an episomal theory of
origin, proposes that a chloroplast DNA arose from a piece of nuclear DNA establish-
ing and evolving in the cytoplasm of a cell. The second theory suggests that the DNA
originated from a procaryotic photosynthetic cell, e.g. a blue-green alga or photo-
synthetic bacterium, which established itself as an endosymbiont within a eucaryotic
cell.
Analyses of base compositions of chloroplast DNA (Table 2) do not show any
marked relations between chloroplast DNA and nuclear DNA (Section lILA). How-
ever, DNA-DNA and DNA-RNA hybridization experiments have raised the pos-
sibility that homologous base sequences do exist in nuclear and chloroplast DNA
(RICHARDS, 1967; TEWARI and WILDMAN, 1967). If substantiated by future experi-

ments, this could mean that at least parts of the nuclear and chloroplast DNA have
common origins.
There are also many similarities between the nucleic acids of chloroplasts and
bacteria and the resemblances between their 70S ribosomes will be discussed (Sec-
tion IV.A). The DNA in chloroplasts also shows several similarities with DNA of
bacteria (and blue-green algae) and some of these are listed below:
(i) The similarity in appearance of DNA images seen in electron micrographs of
chloroplasts, bacteria and blue-green algae.
(ii) The comparable amount of DNA in a chloroplast and a bacterium (Section
III. B).
(iii) The absence of 5-methyl cytosine from chloroplast (Section lILA). and
bacterial DNA
(iv) The apparent absence of histones associated with DNA in chloroplasts and
bacteria (Section III.B).
(v) Chloroplast DNA, like bacterial DNA, is easily renatured (Section 3.1).
(vi) The sensitivity of both chloroplast and bacterial DNA synthesis to inhibition
by nalidixic acid (Section VLE.).
This evidence is consistent with the evolution of the chloroplast from a procaryotic
photosynthetic cell and indeed examples of endosymbiotic associations between blue-
green algae and eucaryotic cells are not uncommon. The cryptomonad Cyanophora
paradoxa, for instance, contains a blue-green algal endosymbiont which has lost its
cell wall, divides along with the host cell and contains DNA very similar in composi-
tion to that of free-living members of its taxonomic group (EDELMAN et aI., 1967).
On the other hand, the apparent similarities of chloroplast DNA with bacterial
and blue-green algal DNA may instead be a reflection of the particular functions
fulfilled by nonchromosomal genetic systems in the cell of a higher organism e.g.,
the possible requirement for a system with inherent resistance to genetic variability
(see SAGER, 1966). If a large number of the structural genes for individual chloroplast
proteins are eventually shown to reside in the nucleus, and much of the genetic
evidence already suggests that this is the case (see KIRK, 1966), the endosymbiont
theory of chloroplast origin will be more difficult to uphold.
IV. Ribosomes and RNA

In 1962 LYTTLETON made the important discovery of the existence of organelle-
localized ribosomes distinct in size and properties from ribosomes in the neighbouring
cytoplasm. He showed that chloroplasts of spinach contained a class of ribosome
which differed in size (LYITLETON, 1962) and protein composition (LYTTLETON, 1968)
from cytoplasmic ribosomes. Ribosomes and polyribosomes have now been isolated
from the chloroplasts of many algae and plants. The mitochondrion also appears to
possess a distinct species of ribosome although these have not been as well charac-
terized.
In Table 4 ribosomes have been classified into three groups on the basis of sedi-
mentation coefficients of the ribosome itself, its subunits and its RNA. Ribosomes
from animals and higher plants have sedimentation coefficients of about 80S, but they
differ in the size of their ribosomal RNA components, while bacteria have 70S
ribosomes. Ribosomes from other organisms appear to fall into one of these three
classes. Ribosomes from blue-green algae, like bacterial ribosomes, are 70S (TAYLOR
et aI., 1967). Ribosomes within each group may show differences in their binding
of specific antibiotics and their stability in different concentrations of Mg++ ions.
Table 4. Classes of ribosomes. A possible classification of ribosomes based on

their S values and the S values of their subunits and ribosomal RNA compo-
nents
Source of ribosomes S values
Ribosomes Subunits RNA
Animal (cytoplasm) 80 60,40 29,18

Plant (cytoplasm) 80 60,40 25,16 or 18
Bacteria 70 50,30 23,16
A. The Chloroplast Ribosome

Table 5 summarizes data published on the sedimentation coefficients of chloro-
plast ribosomes, their subunits and RNA. For comparison, some of the available data
on cytoplasmic and mitochondrial ribosomes is included. Chloroplast ribosomes with
the possible exception of those from Euglena fall into the 70S class. STUTZ and NOLL
(1967) found that ribosomes from pinto bean chloroplasts were indistinguishable
from E. coli ribosomes in sucrose gradients. Lower S values for ribosomes from
Euglena chloroplasts have been reported (EISENSTADT and BRAWERMAN, 1964;
GNANAM and KAHN, 1967), but these should be reinvestigated especially since the
RNA subunits of the ribosomes are typical of 70S ribosomes (see Section IV.B).
Relatively high Mg++ concentrations (5 to 10 mM) are required to prevent
chloroplast ribosomes breaking down into subunits (BOARDMAN et al., 1965; SAGER
and HAMILTON, 1967; LYTTLETON, 1968) and this requirement is also a characteristic
of bacterial ribosomes (PETERMAN, 1964). In Chlamydomonas reinhardi the 80S cyto-
plasmic ribosomes also require 5 to 10 mM Mg++ for stability (SAGER and HAMILTON,
1967), but in higher organisms the 80S ribosomes are generally stable in low concen-
trations of Mg++ (1 to 2 mM). The sensitivity of ribosomes to Mg++ concentration is
well known and HSIAO (1964) has pointed out that lower organisms require higher
Mg++ concentrations for ribosomal integrity.
O'BRIEN and KALF (1967) have reported the isolation of undegraded 55S ribo-
somes from rat liver mitochondria. These ribosomes however could be the large
subunit of a 70S or 80S ribosome as they did not appear to break down into subunits
themselves. In Neurospora, mitochondrial 80S ribosomes have been found (RIFKIN,
WOOD and LUCK, 1967) and these are sensitive to the concentrations of Mg++ ions
breaking down at low concentrations (lmM).
The similar behaviours of chloroplast, mitochondrial and bacterial ribosomal
systems towards antibiotics is discussed in another section (Section V.B.l).
A gel electrophoresis study of ribosomal proteins in spinach has shown that the
chloroplast ribosomal proteins are qualitatively different from the cytoplasmic
ribosomal proteins (LYTTLETON, 1968). Species differences in cytoplasmic ribosomal
Table 5. "5" value! of chloroplast and mitochondriall'ibosome! and their constituent RNA
Species Chloroplast ribosomes Cytoplasmic Ribosomes References
Whole Subunits RNA Whole Subunits RNA
Euglena gracilis 43 30,23 49 36,27 BRAWERMAN (1963)

60 36,30 19, 14 70 19 EISENSTADT and BRAWERMAN (1964)
43 30,17 23, 16 65 47,38 GNANAM and KAHN (1967)
Chlamydomonas reinhardi 70 80 SAGER and HAMILTON (1967)
Acetabularia mediterranea 28, 18 BALTUS and QUERTIER (1966)
Various higher plants 70 79-81 ODINTSOVA et al. (1964) 0
...
OIl
70 85 BOARDMAN et al. (1965) II>
t:I
16 SPENCER and WHITFELD (1966)
28, 18 POLLARD et al. (1966)
"1=
"b:I
23, 16 LOENING and INGLE (1967) o·
5pinacia oleracea (spinach) 66 47,33 LYTI'LETON (1962)
70 50,30 80 SPENCER (1965), ~'"
go
SPENCER and WHITFELD (1966)
"f!!.
Nicotianum tabacum (tobacco) 70 CHEN and WILDMAN (1967) '"
Pisum sativum 70 45,32 80 56,40 SVETAILO et al. (1967) ~
Brassica pekinensis (Chinese cabbage) 68 83 55,40 24, 16 CLARK et al. (1964) "
Phaseolus vulgaris (pinto bean) 70 23, 16 80 25,16 STUTZ and NOLL (1967) ~
0
Phaseoleus vulgaris (bean) 70 80 BOARDMAN [1966 (1)]
...
0
'0
67 23, 16 79 KUNTZELL and NOLL (1967) Ii>
Triticum vulgare 70 80 BARTELS et al. (1967) '"....
Clivia miniata 78 MIKULSKA et al. (1963)
Chenopodium album (Lamb's quarters) 67 48.7,30.4 MIKULSKA et al. (1963)
Mitochondrial ribosomes
Yeast 23, 16 WINTERSBERGER (1966)
23, 18 80 ROGERS et al. [1967 (2)]
Rat liver 55 80 O'BRIEN and KALF (1967)
Neurospora crassa 81 61,47 25, 19 28,18 RIFKIN et al. (1967)
73 23, 16 77 26,17 KUNTZELL and NOLL (1967)
.....
U1
.....
proteins were also found and such differences may extend to chloroplast ribosomal
proteins.
Thus it can be concluded that chloroplast ribosomes are similar to bacterial
ribosomes in their overall size, sensitivities to Mg++ concentration and antibiotics
and size of their RNA components (Section IV .B), but subsequent more detailed studies
of chloroplast ribosomes may well provide a limit to this analogy.
B. Chloroplast RNA
Leaves of higher plants contain about 140 to 175 !Jog RNA/mg chlorophyll and
of this about 15 to 28% is in the chloroplast (WOLLGIEHN et aI., 1966). In Euglena
about 17 to 29% of the RNA is in the chloroplast (SMILLIE et aI., 1963). Most of the
chloroplast RNA is ribosomal.
Two RNA components are obtained from ribosomes, one from each subunit.
The larger component has an S value of 23 in bacteria, 25 in higher plants and 29 in
animals (Table 4). The smaller component is 16S in bacteria and usually 18S in higher
organisms although values of 16S have been reported for some plants. Thus values of
16S were found in pinto beans (STUTZ and NOLL, 1967), pea seedlings and potato
tuber (CLICK and TINT, 1967) but 18S in pea and bean seedlings (LOENING and INGLE,
1967) and wheat leaves (HADZIYEV, et al., 1968).
The 70S bacterial ribosomes yield 23S and 16S RNA in an approximate mass ratio
of 2: 1 (molar ratio 1 : 1) and current evidence suggests that a molar ratio of large to
small ribosomal RNA of 1 : 1 might be expected from all ribosomal systems irrespec-
tive of the size of the individual components (CLICK and TINT, 1967). Since chloro-
plast ribosomes are also 70S, one might expect to find chloroplast ribosomal RNA of
the order of 23S and 16S in the mass ratio 2: 1. However, RNA extracted from
chloroplasts usually shows more 16S than 23S RNA (SPENCER and WHITFELD, 1966;
STUTZ and NOLL, 1967; LOENING and INGLE, 1967) and in other instances larger
subunits (28S and 18S) have been reported (BALTUS and QUERTIER, 1966; POLLARD
et aI., 1966). Euglena also shows anomalous values with 19S and 14S ribosomal RNA
being found in the chloroplasts and 19S and 14S RNA in the cytoplasm (EISENSTADT
and BRAWERMAN, 1964; ZELDIN and SCHIFF, 1967).
We have compared RNA extracted from Euglena chloroplasts with E. coli ribo-
somal RNA and found the two to be indistinguishable on polyacrylamide gels (Fig. 2).
The 23S and 16S components were in a mass ratio of about 2: 1 on the basis of their
absorbancies at 260 nm. However, the 23S component in particular is easily degraded
and during storage of chloroplast RNA prepared by conventional methods, there is
progressive breakdown to products with S values approaching those noted above
for Euglena. Possibly, improved preparative and analytical methods will show that the
70S ribosomes of other chloroplasts contain large and small ribosomal RNA (probably
23S and 16S) in the molecular ratio 1: 1 and this would be analogous to the situations
pertaining in animal and plant cytoplasm and bacterial ribosomes (CLICK and TINT,
1967; STUTZ and NOLL, 1967).
The 23S and 16S ribosomal RNA from Bacillus megaterium originate from different
cistrons of the DNA (YANKOFSKY and SPIEGELMAN, 1963). There are differences in
the base composition of the two cytoplasmic ribosomal RNA in plants and CLICK
and HACKETT (1966) have suggested that these may also originate from individual
cistrons. We have now demonstated in hybridization experiments that the 23 S and

16 S ribosomal RNA of Euglena chloroplasts anneal with separate cistrons on the
chloroplast DNA (unpublished experiments).
Smaller ribosomal RNA components (4 to 5S) have been extracted from many
tissues and recently DYER and LEECH (1968) have found a 5S ribosomal RNA peculiar
to bean leaf chloroplasts. They also have found a 5S soluble RNA species charac-
teristic of chloroplasts and different from cytoplasmic soluble RNA.
Differences in base composition between cytoplasmic and chloroplast ribosomal
RNA have been shown in some cases (BRAWERMAN and EISENSTADT, 1964; POLLARD
23
16
la
Distance travelled (cm)

Fig.2a-d. Chloroplast RNA in Euglena. Electrophoresis through polyacrylamide gels
(LoENINGand INGLE 1967) has been used to compare Euglena chloroplast RNA with ribosomal
RNA from E. coli. (a) E. coli ribosomal RNA, (b) Euglena chloroplast RNA, (c) a mixture
of the two and (d) a blank gel (SCOTT and SMILLIE, 1969)
et aI., 1966; BALTUS and QUERTIER, 1966), but not in others (e.g. LYTTLETON, 1962).
As in the case of chloroplast DNA, such differences are not essential to prove the
existence of a distinct chloroplast species since the important difference is the base
sequence.
C. Synthesis of RNA in the Isolated Chloroplast
The occurrence of both DNA and RNA in chloroplasts led to the view that
chloroplasts may carry out DNA-dependent RNA synthesis.
KIRK [1964 (1,2)] showed that chloroplasts from the leaves of broad bean carried
out a DNA-dependent incorporation of 14C-adenosine 5'-triphosphate into RNA.
Similar results have been obtained with chloroplasts isolated from tobacco (SEMAL
et aI., 1964), Euglena (SHAH and LYMAN, 1966), Acetabularia (BERGER, 1967), spinach
[SPENCER and WHITFELD, 1967 (2)] and Chinese cabbage (BOVE et aI., 1967). The in-
corporations were dependent upon the presence of all four nucleoside triphosphates
and were inhibited by pretreatment of the chloroplasts with deoxyribonuclease,
ribonuclease or actinomycin D. The polymerase activity in the broad bean chloro-
plasts was higher than the activity in the nuclei and it showed a different sensitivity to
metal ions. This suggests the existence of different polymerases in the chloroplasts
and nuclei. Chloroplasts also appear to contain enzymes for nucleotide synthesis since
chloroplasts from Bryopf?yllum incorporated orotic acid into both free pyrimidine
nucleotides and RNA (JOUSSAUME and BOURDU, 1966).
SPENCER and WHITFELD [1967 (2)] separated the products of the chloroplast and
nuclear RNA polymerases of spinach by centrifugation in a sucrose gradient and
found the chloroplast product had a sedimentation constant of 11 S and was more
polydisperse than the 8S product of the nuclear enzyme. They suggested the 11S
product was largely messenger RNA as a similar product could be isolated from
chloroplasts after short-term uptake oPH-uridine into spinach plants. However, the
llS product could also represent precursor material of ribosomal RNA.
Thus chloroplasts contain polymerase enzymes for the synthesis of DNA (Section
III.C.2) and RNA. Differences between the chloroplast and nuclear systems have been
indicated but there is insufficient evidence to conclude if these are due to polymerases
with different properties or to the effects of the physical and chemical environment in
chloroplast and nucleus on the enzymic activities.
D. RNA Synthesis in the Developing Chloroplast
Synthesis of RNA is one of the earliest biosynthetic events associated with the
development of proplastids into mature chloroplasts. It precedes the synthesis of most
of the chloroplast protein and lipid and results in the formation of species of RNA
which are unique to chloroplasts and are required for synthesis of chloroplast protein.
Several lines of evidence can be advanced to support these statements. (1) Green cells
of Euglena and Chlorella contained more RNA than dark-grown or bleached cells
[SMILLIE and KROTKOV, 1960; BRAWERMAN et aI., 1962; AOKI and HASE, 1964 (1)].
(2) The incorporation of labelled precursors into the RNA of dark-grown Euglena *
(BRAWERMAN and CHARGAFF, 1959; SMILLIE et aI., 1963; ZELDIN and SCHIFF, 1967)
or maize leaves [BOGORAD, 1967 (1,2)] was stimulated by light. (3) When dark-
adapted, nondividing cells of Euglena were allowed to green in light, there was a net
increase in cellular RNA content, mostly ribosomal RNA, and this increase preceded
the synthesis of chlorophyll and chloroplast protein (BRAWERMAN et aI., 1962).
Similarly, if plastids of glucose-bleached cells of Chlorella* were allowed to develop,
the production of cellular protein was preceded by synthesis of RNA [AOKI and
HASE, 1964 (1)]. (4) The changes in RNA synthesis which were induced by light were
accompanied by the appearance of new RNA species in Euglena (BRAWERMAN and
CHARGAFF, 1959; POGO et aI., 1962). BRAWERMAN (1966) has interpreted these changes
in Euglena in terms of a synthesis of specific plastid ribosomes, since shifts in the
nucleotide composition of RNA during greening were consistent with the formation
of new chloroplast ribosomes (which differed in nucleotide composition from
cytoplasmic ribosomes). (5) Specific inhibitors of RNA synthesis prevented chloro-
plast development. Thus actinomycin D, which blocks RNA synthesis dependent
* see footnote on p. 186
upon DNA, inhibited chlorophyll synthesis in both Euglena (SMILLIE et aI., 1963;
POGO and POGO, 1964; MCCALLA and ALLAN, 1964; SHAH and LYMAN, 1966), Chlorella
[AOKI and BASE, 1964 (2)] and higher plants (BOGORAD and JACOBSON, 1964).
5-fluorouracil, which prevents the formation of new functional ribosomes, and
hadacidin, an inhibitor of adenine synthesis, also inhibited chloroplast development
(SMILLIE, 1963; SMILLIE et aI., 1963; AOKI et aI., 1965; MEGO, 1964). (6) Illumination
of dark-grown seedlings of maize promoted rapidaincrease in plastid RNA polymerase
[BOGORAD, 1967 (1,2)].
These studies showed that light induced synthesis of RNA in etiolated cells and
that apparently this synthesis was essential for the formation of mature chloroplasts.
What then, was the nature of the newlyformed RNA and were the changes largely
confined to plastid RNA? SMILLIE et aI. (1963) extracted RNA from Euglena at various
stages of chloroplast development and separated ribosomal and soluble RNA by
centrifugation in a sucrose gradient. Both types of RNA increased during greening,
the most pronounced changes occurring during the first 24 h of illumination. The
existence of a light-inducible synthesis of both soluble and ribosomal RNA associated
with chloroplast development has been confirmed by ZELDIN and SCHIFF (1967) and
a similar conclusion was reached by AOKI and BASE (1967) who found that the ratio
of soluble RNA to ribosomal RNA of 30: 70 in glucose-bleached Chlorella remained
constant throughout greening. Light almost certainly induces a synthesis of messenger
RNA as well and the inhibition of chlorophyll synthesis by actinomycin D in bean
leaves which, in contrast to Euglena, synthesize most of their plastid ribosomal RNA
independently of light [BOARDMAN, 1966 (1), see also Section VLD) may be due pri-
marily to interference in messenger RNA synthesis (BOGORAD and JACOBSON, 1964).
The light-induced synthesis of these major classes of RNA is not confined to the
plastids. BRAWERMAN et aI. (1962) examined the intracellular distribution of RNA
formed during chloroplast development in Euglena. The RNA of the plastids in-
creased, but so did the RNA of other subcellular fractions. SMILLIE et aI. (1963)
estimated that the RNA in the plastids was insufficient to account for all of the RNA
synthesized in light and more recent experiments by ZELDIN and SCHIFF (1967) and
AOKI and BASE (1967) have supported this conclusion. AOKI and BASE (1967) made
the interesting observation that the onset of the maximum rate of synthesis of chloro-
plast ribosomal RNA lagged behind that of cytoplasmic ribosomal RNA in Chlorella
and this was consistent with the rapid light-induced synthesis of cytoplasmic enzymes
noted by SMILLIE et aI. (1963) which preceded the synthesis of chloroplast proteins in
Euglena.
Virtually all of the ribosomes essential for completion of the synthesis of chloro-
plasts are produced during the lag period of chlorophyll synthesis. This was shown in
a series of experiments using the uracil analogue, 5-fluorouraciI. At the same time
these experiments have provided some insight into the functional role of these
ribosomes, in particular the identities of the proteins synthesized on them.
EVANS and SMILLIE (1962) showed that 5-fluorouracil inhibited chlorophyll
synthesis in Euglena. This inhibitor blocks the production of new ribosomes in bac-
teria while only slightly interfering with the turnover of existing ribosomes (ARON-
SON, 1961). The chlorophyll synthesis was inhibited only if 5-fluorouracil was added
during the early stages of chloroplast development in Euglena (SMILLIE et aI., 1963)
and similar results were obtained with Chlorella (AOKI et aI., 1965). These experiments
156 ROBERT M . SMILLIE and N. STEELE SCOTT
indicated that sufficient ribosomes (and messenger RNA?) were formed during first
10 to 15 h of illumination to allow completion of chloroplast development. On the
other hand, the most active period of chlorophyll and chloroplast protein synthesis
was between 15 and 72 h of continuous illumination.
Since 5-fluorouracil added during the early period of chloroplast development
prevents the production of new ribosomes which are essential for chloroplast develop-
ment, it should also prevent the appearance of proteins normally synthesized on these
ribosomes. One might also expect the inhibition to be fairly specific for these pro-
teins, since although 5-fluorouracil may inhibit the synthesis of new cytoplasmic
ribosomes, dark-adapted cells probably contain a sufficient reserve of cytoplasmic
U'I
:3 2.0
<X>5!
"P- __ _
;r/ ------
I "'-,
I ,
,
I ,
,
I '
I "
'"
3
Days in light
Fig. 3. Inhibition of synthesis of chlorophyll and a chloroplast cytochrome (cytochrome-552),
but not glucose-6-phosphate dehydrogenase, by 5-fluorouracil (FU) during chloroplast
development in Euglena. Cells were grown heterotrophically in the dark, transferred to an
inorganic medium, shaken in the dark for 2 days and then illuminated. FU was added just
before the cultures were placed in the light. (Data from SMILLIE et aI., 1963)
ribosomes to sustain a reasonable rate of cytoplasmic protein synthesis. Thus the

synthesis of respiratory enzymes observed when dark-adapted cells of Euglena were
illuminated, was not inhibited by 5-fluorouracil (SMILLIE et aI., 1963). Fig. 3 shows
one such example. Glucose-6-phosphate dehydrogenase increased during the first day
of illumination then declined and 5-fluorouracil did not inhibit this increase. Similarly,
the light-induced utilization of storage carbohydrate ({3-1,3-g1ucan) which accompanies
chloroplast development (SMILLIE et aI., 1963) was only marginally affected by 5-
fluorouracil (DWYER, 1968). However, the synthesis of cytochrome-552, a component
of the photosynthetic electron transfer pathway, was inhibited. Thus the ribosomes
produced presumably in the plastids during the first 10 to 15 h of illumination appear
to be essential for the synthesis of cytochrome-552 as well as chlorophyll and the
same holds for the chloroplast b-type cytochrome, ferredoxin-NADP-reductase and
ribulose-1,5-diphosphate carboxylase (SMILLIE et aI., 1968).
In contrast to results obtained with Euglena and Chlorella, the partially expanded
leaves of etiolated bean seedlings contain almost as many 70S plastid ribosomes as the
green leaves, and BOARDMAN [1966 (1)] concluded that synthesis of chloroplast
ribosomes in bean leaves is not a light-dependent process. An explanation for this
apparent discrepancy between the results obtained with algae and bean leaves is
given in Section VLH.1.
v. Synthesis of Chloroplast Proteins

A. Protein Synthesis in the Isolated Chloroplast
Labelled amino acids or CO2 supplied to leaf tissue are incorporated into chloro-
plast protein (STEPHENSON et al., 1956; HEBER, 1962; PARTHIER, 1964, 1965). HEBER
(1962) showed the label from 14C02 was rapidly incorporated into chloroplast protein,
Table 6. Protein synthesis in isolated chloroplasts
Source of chloroplasts Reference
Algae:
Acetabularia mediterranea GOFFEAU and BRACHET (1965)
Euglena gracilis EISENSTADT and BRAWERMAN (1963, 1964)
Higher plants:
Tobacco SPENCER and WILDMAN (1964), PARTHIER (1964),
FRANCKI et al. (1965), BOARDMAN et al. (1965,1966),
VAN KAMMEN (1967)
Tomato HALL and COCKING (1966) (1), (2)
Spinach PARTHIER (1965), SPENCER (1965)
Pea SISSAKIAN et al. (1965)
Bean PARENTI and MARGULIES (1967)
Wheat BAM]I and ]AGENDORF (1966)
whereas there was a slight lag before it appeared in cytoplasmic protein and he
concluded that protein is synthesized within chloroplasts. Subsequently, chloroplasts
capable of incorporating amino acids into protein have been isolated from many
plants and algae (Table 6). Difficulties in interpreting earlier reports (not listed
in Table 6) of protein synthesis by isolated chloroplasts because of the possibility of
contamination by bacteria have been discussed by App and ]AGENDORF (1964).
Amino acid activating enzymes (aminoacyl-sRNA synthetases) have also been
demonstrated in chloroplasts isolated from several plants [CLARK, 1958; BovE and
RAAKE, 1959; MARCUS, 1959; HENSHALL and GOODWIN, 1964 (2); FRANCKI et al.,
1965; SISSAKIAN et al., 1965).
The amino acid incorporating capacity of chloroplasts is dependent on their 70S
ribosomes, and these ribosomes can continue to synthesize peptides when removed
from chloroplasts. In extracts of young tobacco and spinach leaves, chloroplasts ac-
counted for over 90% of the protein-synthesizing activity (SPENCER and WILDMAN,
1964; SPENCER, 1965), but in Euglena the chloroplast and cytoplasmic activities were
comparable on an RNA basis (EISENSTADT, 1967). The activities of chloroplast and
cytoplasmic ribosomes differed in their response to added messenger RNA (EISEN-

STADT and BRAWERMAN, 1964) and to variations in the concentration of Mg++ ions.
Chloroplast ribosomes from tobacco showed optimal activity at 11 to 15 mM Mg++,
while the optimum concentration for cytoplasmic ribosomes was 5 mM (BOARDMAN et
aI., 1966). The incorporation ofphenylalanine into protein by spinach chloroplasts was
dependent on adenosine 5'-triphosphate and was stimulated by other amino acids and
guanosine 5' -triphosphate. Ribonuclease, puromycin, and chloramphenicol were
strongly inhibitory, but actinomycin D and deoxyribonuclease had only slight effects
(SPENCER, 1965). In contrast, actinomycin D inhibited amino acid incorporation by chlo-
roplasts prepared from Acetabularia 11 days after enucleation (GOFFEAu and BRACHET,
1965). Properties of the protein-synthesizing system of chloroplasts have been sum-
marized recently by PARTHIER and WOLLGIEHN (1966), BOARDMAN (1967), EISEN-
STADT (1967), KIRK and TILNEy-BASSETT (1967), and WILDMAN (1967).
An important question yet to be resolved is the identity of proteins synthesized
by isolated chloroplasts. About 23% of the activity from 14C-valine incorporated into
spinach chloroplasts was recovered in soluble protein (obtained by subjecting the
chloroplasts to osmotic shock followed by centrifugation) and of the remaining
activity about half was released by treatment with 3.5% Triton X-l00 (SPENCER,
1965). About 20% of the activity incorporated into tobacco 70S ribosomes could be
recovered in a soluble protein fraction (4S to 18S) and more than half of the activity
remaining on the ribosomes was transferred to the soluble fraction by incubating the
ribosomes with puromycin. These results may be contrasted with studies on isolated
mitochondria where amino acids were incorporated almost exclusively into particulate
proteins (ROODYN et aI., 1962; WINTERSBERGER, 1965; WHEELDON and LEHNINGER,
1966; BEATTIE et al., 1967; KADENBACH, 1967; NEUPERT et al., 1967).
The incorporation of activity into soluble chloroplast protein is consistent with
the conclusions based on studies with inhibitors that chloroplast ribosomes syn-
thesize enzymes of the Calvin cycle and Fraction I protein (Section V.B.l).
There is little information about the range of sizes of polyribosomal clusters in
chloroplasts, although it is known their formation in oat plastids is stimulated follow-
ing exposure of etiolated leaves to light (BROWN and GUNNING, 1966) and in chloro-
plasts and cytoplasm of Chinese cabbage they show diurnal fluctuations (CLARK et al.,
1964). The size of a polyribosomal cluster is usually related to the size of the poly-
peptide chain being synthesized. For instance a myosin peptide subunit of molecular
weight 170,000 to 200,000 is synthesized in the leg muscle of chick embryo on
clusters containing 50 to 60 individual ribosomes (HEYWOOD et al., 1967). Although
25 to 50% of the soluble protein of chloroplasts consists of Fraction I protein, which
has a high molecular weight (ca. 500,000), very large polyribosomal clusters may not
necessarily occur in chloroplasts as their size will be related to the number and size of
individual polypeptide chains in Fraction I protein.
B. Cellular Sites oj Synthesis oj Chloroplast Protein

The time course of the synthesis of enzymes and appearance of photosynthetic
activities during the growth and differentiation of chloroplasts has been summarized
by KIRK and TILNEy-BASSETT (1967) and will not be discussed here, except to mention
that while the increase in chlorophyll usually provides a reasonable indication of the
increase in total chloroplast protein, individual proteins may be synthesized at

different times. The elucidation of the various mechanisms responsible for the syn-
thesis of the many and diverse proteins found in chloroplasts is likely to be a long and
difficult task, and we might begin by asking if all chloroplast proteins are synthesized
within the chloroplasts on 70S ribosomes or whether some of them are synthesized
in the cytoplasm on 80S ribosomes and subsequently transferred to the chloroplast.
1. Synthesis of Fraction I Protein and Enzymes of the Calvin Cycle

The intracellular site of synthesis of Calvin cycle enzymes and Fraction I protein
has been studied using the antibiotic chloramphenicol. Both GALE (1963) and PETER-
MANN (1964) have pointed out that chloramphenicol, which inhibits protein synthesis
by combining with the 50S subunit of the bacterial ribosome (VAZQUEZ, 1966), is
ineffective in most non-bacterial systems. However, several reports have described
inhibition of the growth of photoautotrophic algae by chloramphenicol, and further,
it interferes with the synthesis of both chlorophyll and chloroplast protein in cells
containing developing chloroplasts (Table 7). The reason for the inhibition of algal
growth by chloramphenicol became more apparent from studies of its effect on
photoautotrophic verus heterotrophic growth. The growth of photoautotrophic
Euglena was inhibited by chloramphenicol, but growth on suitable carbon growth
substrates other than CO2 , either in the light or in the dark, was only marginally
affected by chloramphenicol (SMILLIE et al., 1963). More recently, experimental con-
ditions have been devised in which chlorophyll synthesis was severely repressed by
concentrations of chloramphenicol which had no inhibiting effect on the rate of cell
division (AARONSON et al., 1967; SMILLIE et aI., 1967). Thus inhibition appeared to
be selectively directed towards the formation of functional chloroplasts, presumably
by interfering with the synthesis of chloroplast protein. In higher plants, the syn-
thesis of chloroplast protein was also preferentially inhibited by chloramphenicol
when compared with its effect on the synthesis of cytoplasmic protein (PARTHIER,
1965).
The incorporation of amino acids into proteins by ribosomes isolated from
chloroplasts was sensitive to low concentrations of chloramphenicol (EISENSTADT
and BRAWERMAN, 1964; SPENCER and WILDMAN, 1964; GOFFEAU and BRACHET, 1965;
SISSAKIAN et al., 1965; SPENCER, 1965; BAM]I and jAGENDORF, 1966), but for
cytoplasmic ribosomes it was less sensitive e.g. Euglena (EISENSTADT and BRAWERMAN,
1964) or maize endosperm (GRAEBE and NOVELLI, 1966). Ribosomes isolated from
the chloroplasts of Euglena or higher plants bound chloramphenicol more strongly
than cytoplasmic ribosomes from the same cells (ANDERSON and SMILLIE, 1966). This
finding offers a possible explanation for the selective action of chloramphenicol in
photosynthetic organisms, since VAZQUEZ (1964) had previously demonstrated that
bacterial 70S ribosomes had a much greater capacity to bind chloramphenicol than
the 80S ribosomes of chloramphenicol-insensitive higher organisms. Cytoplasmic
ribosomes have a higher affinity for messenger RNA than the 70S ribosomes of either
chloroplasts or bacteria and chloramphenicol may compete more effectively for
binding sites on the 70S ribosomes (cE. EISENSTADT, 1967).
These studies indicate that chloramphenicol inhibits the synthesis of protein on
the 70S ribosomes of chloroplasts. Conversely, the identity of individual proteins
...
g;
Table 7. The inhibitory action of chloramphenicol on photoautotrophic growth, chlorophyll synthe.ris and synthesis of chloroplast proteins
Organism Inhibition of: References

Growth Chlorophyll Chloroplast
synthesis protein
synthesis
:;>;:I
Algae: 0
til
ttl
Chlorella pyrenoidosea + GALLOWAY and KRAUSS (1959) el
C. ellipsoidea + TAMIYA et aI. (1962)
C. ellipsoidea + CZYGAN (1964) ~
rJ)
C. protothecoides + AOKI et aI. (1965) ;;::
C. vulgaris ECHLIN and MORRIS (1965)
...t""
+
Polyhedriella helvetica + ECHLIN and MORRIS (1965) ...t""ttl
Chlamydomonas reinhardi ECHLIN and MORRIS (1965) I»
+ t:I
p..
Chlamydomonas reinhardi + HUDOCK and LEVINE (1964)
S cenedesmus quadricauda + TAYLOR (1965) ;z:
Ankistrodesmus braunii CZYGAN (1964) rJ)
+
Coelastrum proboscideum var. mlatatum + CZYGAN (1964) ttl
""
ttl
Euglena gracilis SMILLIE et aI. (1963), POGO and POGO (1965), t""
+ + + ttl
AARONSON et aI. (1967), LINNANE and STEWART rJ)
(1967) n
Acetabularia crenulata + SWEENEY et al. (1967) §
Leaves:
Bean + MARGULIES (1964, 1966)
Pumpkin + + MOLOTKOVSKII and SMIRNOV (1963)
Tobacco + + PARTHIER et al. (1964), PARTHIER (1965)
Pea, bean, tobacco, Horlen.ria + MOLOTKOVSKIl and SMIRNOV (1963)
Organelle Biosynthesis: The Chlotoplast 161
synthesized on the 70S tibosomes can be ascertained by the effect of chloramphenicol

on their synthesis under conditions where cytoplasmic protein synthesis is not in-
hibited. SMILLIE et al. (1967,1968) examined the effect of chloramphenicol on the
synthesis of several enzymes of the Calvin cycle during chloroplast devdopment in
both non-dividing and dividing ( + glucose) cells of Euglena. In dividing cells, the chlo-
ramphenicolfailed to inhibit, and in fact slightly stimulated, the rate of cell division, the
synthesis of respiratory enzymes and the uptake ofglucose from the medium. However,
the synthesis of chlorophyll, ribulose-l,5-diphosphate carboxylase, NADP+-glyceral-
dehyde-3-phosphate dehydrogenase and Fraction I protein was inhibited. Inhibition
of the synthesis of the carboxylase, dehydrogenase and transketolase by chlor-
amphenicol in higher plants has also been reported (MARGULIES, 1964; ZIEGLER and
ZIEGLER, 1965; FEIERABEND, 1966). It can be concluded that either the enzymes of
the Calvin cycle and Fraction I protein are synthesized within the chloroplasts on the
chloroplast ribosomes, or alternatively, they are synthesized in the cytoplasm by
Table 8. Action of chloramphenicol and cycloheximide on protein synthesif

Chloramphenicol Cycloheximide
Bacteria +
Blue-green algae +
Cytoplasm: fungi +
green algae, plants +
mammals +
Mitochondria +
Chloroplasts +
some mechanism which requires the concomitant synthesis of protein within the
chloroplasts.
By employing a second specific inhibitor of protein synthesis, cycloheximide,
it has been possible to decide between these alternatives. Both cycloheximide and
chloramphenicol inhibit peptide formation on the ribosome, but their inhibitory
effects are complementary: chloramphenicol-sensitive ribosomes are insensitive to
cycloheximide and cycloheximide-sensitive ribosomes are insensitive to chloram-
phenicol (Table 8). Thus cycloheximide had little effect on either the growth of
bacteria (HALL et al., 1951) or the incorporation of amino acids into protein by
bacterial ribosomes (ENNIS and LUBIN, 1964). Similarly, the growth of blue-green
algae was not affected by cycloheximide (L. BOGORAD, private communication) but
was inhibited by chloramphenicol (GALLOWAY and KRAUSS, 1959; ECHLIN and
MORRIS, 1965). The lack of inhibition by chloramphenicol of protein synthesis
involving 80S cytoplasmic ribosomes has already been mentioned, but cycloheximide
at 1 to 2 (Lgjml was a potent inhibitor of growth and cell-free protein synthesis in
yeast (KERRIDGE, 1958; SIEGEL and SISLER, 1965), Chlorella (MORRIS, 1967), Euglena
(KIRK and ALLEN, 1965) and mammalian cells (ENNIS and LUBIN, 1964; KAy and
KORNER, 1966).
In contrast, protein synthesis localized in chloroplasts or mitochondria behaved
more like the bacterial or blue-green algal systems in its response to these inhibitors.
Evidence for the inhibition of protein synthesis in chloroplasts and mitochondria by
chloramphenicol is cited elsewhere in this Section and Section V.B.2. Cycloheximide,

on the other hand, is without effect. The incorporation of amino acids into isolated
spinach chloroplasts was not significantly reduced by cycloheximide at 28 [Lg/ml
(D. SPENCER, private communication), while concentrations as high as 500 [Lg/ml
failed to inhibit protein synthesis by mitochondria isolated from yeast (BEATTIE et aI.,
1967). Thus if any chloroplast proteins are synthesized in the cytoplasm and then
migrate to the chloroplasts, their synthesis should be inhibited by cycloheximide,
whereas synthesis within chloroplasts might possibly continue for some time in the
presence of cycloheximide even though all other growth in the cell is virtually im-
mobilized. Table 9 shows results obtained by SMILLIE et aI. (1967, 1968) on the
synthesis of two enzymes of the Calvin cycle in Euglena when dark-grown cells were
illuminated in the presence of either cycloheximide (15 [Lg/ml) or chloramphenicol
Table 9. Action of chloramphenicol and rycloheximide on the {),nthesis in vivo of enzymes of the Calvin
rycle.
(Data from SMILLIE et aI., 1967)
Conditions Increase during illumination for 72 h
Ribulose-l,5- NADP+-glyceral-
diphosphate dehyde-3-phosphate
carboxylase dehydrogenase
([J.moles substrate! ([J.moles substrate!
min!10 9 cells) min!10 9 cells)
Control 0.62 13.0

Chloramphenicol 0.D7 -0.6
Cycloheximide 1.34 15.3
% inhibition with chloramphenicol 89 100
Cycloheximide treatment as % of control 216 118
(1 mg/ml) Chloramphenicol drastically inhibited the formation of both enzymes, but

there was no inhibition by cycloheximide. Similarly the net synthesis of Fraction I
protein was blocked by chloramphenicol, but cycloheximide, even at the relatively
high concentration used in these experiments, had only a marginal effect. Two con-
clusions can be drawn from these results. First, the synthesis of two Calvin cycle
enzymes and Fraction I protein took place within the chloroplasts on the 70S chloro-
plast ribosomes and second, there was no obligatory interdependence of protein syn-
thesis within the chloroplast with protein synthesis in the cytoplasm, since the former
proceeded at almost normal rates in the absence of the latter.
Many of the enzymes catalyzing reactions of the Calvin cycle are also found in the
cytoplasm. Possibly, separate isoenzymes exist in the chloroplast and cytoplasm, but
there is no evidence for this except in the case of fructose-1,6-diphosphate aldolase
(RUTTER, 1964; RUSSEL and GIBBS, 1966). Chloroplasts of Euglena contain ametal-inde-
pendent aldolase, while the cytoplasmic aldolase is metal-requiring (G. K. RUSSEL, perso-
nal communication). Table 10 shows the effects of chloramphenicol and cycloheximide
on the cellular concentrations of the two Euglena aldolases during chloroplast develop-
ment in dividing cells. Cells kept in the dark contained mostly the metal-dependentaldo-
lase. Upon illumination, the activity of the chloroplast aldolase increased but the
increase was less in chloramphenicol-treated cells. Cells grown in the presence of
cycloheximide showed an even greater increase in the chloroplast enzyme compared

with control cells, while the activity of the cytoplasmic enzyme declined. These
results are consistent with synthesis of the metal-independent aldolase on chloroplast
ribosomes.
It is crucial to the interpretation of these experiments that cycloheximide effec-
tively blocked cytoplasmic protein synthesis. At 2 to 5 fLg/ml, cycloheximide com-
pletely inhibited cell division, uptake of glucose from the medium and increases in
cytoplasmic enzymes (SMILLIE et at, 1968). Probably the most convincing evidence for
the effective inhibition of cycloplasmic protein synthesis by cycloheximide in greening
cells would be to determine its effect on the synthesis of some cytoplasmic enzyme
which can be induced independently of chloroplast development. We have studied
the ethanol-induced formation ofisocitrate lyase in greening cells of Euglena and found
it to be inhibited by cycloheximide (unpublished experiments). The synthesis of this
Table 10. Activities of fructose-I,6-diphosphate aldolases in Euglena. Dark-grown cells were illu-
minated for 66 h in the presence of chloramphenicol (1.5 mg/ml)or cycloheximide (3 fLg/ml). The "dark"
cells were maintained in the dark throughout this period. Glucose was added as a growth substrate to all
cultures. The metal-independent aldolase was assayed in the presence of 5 mM ethylenediamine tetra-
acetate which inhibited the metal-requiring aldolase
Treatment of culture Activity (fLmoles FDP/10 9 cells/min)
Total Metal- Metal-
independent requiring
enzyme enzyme
Dark 9.7 1.7 8.0

Light 10.7 4.8 5.9
Light + chloramphenicol (1.5 mg/ml) 7.3 2.6 4.7
Light + cycloheximide (3 fLg/ml) 9.3 7.6 1.7
enzyme in seedlings of watermelon is also inhibited by cycloheximide (HOCK and

BEEVERS, 1966).
2. Proteins of the Photosynthetic Electron Transfer Pathway

Chloramphenicol inhibits the formation of photosynthetic electron transfer
proteins in Euglena. The synthesis of cytochrome-552 (c-type cytochrome), b-type
cytochrome and ferredoxin-NADP+-reductase, and the incorporation of 54Mn into
chloroplast lamellae were all inhibited at 1 mgjml (SMILLIE et at, 1963, 1967, 1968).
However, cycloheximide also inhibited chlorophyll synthesis and the formation of
electron transfer proteins (KIRK and ALLEN, 1965; EVANS et at, 1967; SMILLIE et at,
1967, 1968), but higher concentrations than those required to inhibit cytoplasmic synthe-
sis were needed. Thus at lor 2 fLg/ml, inhibition of chlorophyll synthesis was restricted
to the first few hours of chloroplast development and after this period, there was
either no inhibition or an enhancement of chlorophyll synthesis (EVANS et at, 1967).
Concentrations of about 15 fLg/ml were required to achieve severe inhibition of
chlorophyll synthesis but even this concentration was ineffective if the dark adapted
cells were cultured in the presence of chloramphenicol and then illuminated in a
medium containing cycloheximide (15 fLgjml) but not chloramphenicol (SMILLIE
11*
et al., 1968). Since conditions have been found for the continued synthesis of photo-
synthetic electron transfer proteins in the presence of relatively high concentrations
of cycloheximide, it seems likely that these proteins also are synthesized on the chloro-
plast ribosomes.
Related experiments on the synthesis of mitochondrial electron transfer proteins
have led to the opposite conclusion, namely that it is not organelle-localized ribo-
somes but instead cytoplasmic ribosomes that are responsible for the synthesis of
many mitochondrial respiratory proteins. Chloramphenicol inhibited the incorpora-
tion of amino acids into protein by isolated mitochondria (MAGER, 1960; DAS et aI.,
1964; KROON, 1963; LAMB et al., 1968), yet it did not depress the synthesis of cyto-
chrome c in the yeast Saccharo1l1yces cerevisiae (HUANG et al., 1966) thus indicating that
cytochrome c was synthesized extramitochondrially. This conclusion was supported
by the lack of incorporation of amino acids into cytochrome c or other soluble
proteins by isolated mitochondria (SIMPSON et al., 1961; ROODYN et aI., 1962; WHEEL-
DON and LEHNINGER, 1966; KADENBACH, 1967) and by the observation ofBEAITIE et
al. (1966) that the rate of in vivo incorporation of amino acids into cytochrome c was
correlated with the rate of incorporation into cytoplasmic protein rather than into
total mitochondrial protein. A direct transfer of microsomal protein to mitochondria
in vitro has been demonstrated by KADENBACH (1967). Enzymes of the tricarboxylic
acid cycle also appear to be synthesized in the cytoplasm, since the oxygen-induced
synthesis of fumarase, malate dehydrogenase and succinate dehydrogenase in yeast
was not inhibited by chloramphenicol provided the cells were supplied with certain
essential lipids (DUNCAN and STEWART, 1968).
The intracellular site of synthesis of structurally bound cytochromes of mito-
chondria has not been determined with certainty. Their synthesis in yeast was
inhibited by chloramphenicol (HUANG et aI., 1966; CLARK-WALKER and LINNANE,
1967; DUNCAN and STEWART, 1968), but on the other hand, KADENBACH (1967) was
unable to find evidence for incorporation of labelled amino acids into particulate
cytochrome by isolated mitochondria. The inhibition by chloramphenicol could point
to synthesis of certain electron transfer proteins in mitochondria; alternatively, these
proteins could be synthesized in the cytoplasm, the chloramphenicol acting by inter-
fering with the synthesis of mitochondrial membrane protein to which the electron
transfer proteins are bound.
3. Membrane Proteins
Information on the synthesis of the proteins of the lamellae (other than electron
transfer proteins) and the outer membrane of chloroplasts is meagre. The stage of
chloroplast development when protein subunits of the lamellar membranes are syn-
thesized is not known. Neither has the intracellular site of synthesis of this protein
been established. nor the extent to which its synthesis is regulated by light.
Studies on the biosynthesis of chloroplast membrane proteins have been impeded
by a lack of suitable methods for fractionating and separating the different membrane
proteins. However, considerable work in this area is now in progress and the separa-
tion of two pigment-protein complexes, the one containing photosystem I and the
other photosystem Il, has been achieved by treatment of chloroplast lamellae with
detergents followed by fractionation by differential centrifugation (ANDERSON and
BOARDMAN, 1966; BRIANTAIS, 1966; ARNON et al., 1968) or gel electrophoresis [OGAWA
et al., 1966; SIRONVAL et al., 1966; THORNBER et al., 1967 (1), (2)]. Procedures for
fractionating the outer and inner membranes or mitochondria are available (SOTTa-
CASA et al., 1967), but methods for separating the outer membrane of the chloroplast
from the lamellae have not been developed.
Other smaller membrane-bound proteins have also been extracted from lamellae
by treatment with detergents or other substances. Extraction of lamellae with 1 mM
ethylenediamine tetraacetate at pH 8.0 released a protein, approximately 100 A in
diameter, which contains latent Ca++-dependent adenosine-5'-triphosphatase activity
and is thought to couple between the electron transfer pathway and the phosphory-
lation of adenosine 5'-diphosphate (MCCARTY and RACKER, 1966; MOUDRIANAKIS
et aI., 1968). A red protein (rubimedin), approximately 75 A in diameter, has been
isolated from lamellae by HENNINGER et al. (1966).
CRIDDLE (1966) has isolated a lamellar protein fraction, which he has named
structural protein, by extraction with detergent (sodium deoxycholate or sodium
dodecyl sulphate) followed by precipitation with 12 to 16% saturated ammonium
sulphate. Structural protein has a molecular weight of 25,000 and contains aspartic
acid as the only N-terminal amino acid. Its possible physiological role was indicated
by its capacity to bind chlorophyll and certain proteins. Mitochondria similarly
contain structural protein which is capable of binding proteins and coenzyme
nucleotides. The structural protein from Neurospora mitochondria specifically inter-
acts with mitochondrial malate dehydrogenase (MUNKRES and WOODWARD, 1967).
Respiratory incompetence may arise from mutations resulting in structural alterations
to either the enzyme (MUNKRES and WOODWARD, 1966) or to the structural protein
itself (WOODWARD and MUNKRES, 1966) that prevents this interaction.
Euglena cells grown in the presence of sufficient chloramphenicol to inhibit
chlorophyll synthesis continue to produce structural proteins and it has been sug-
gested that a protein-synthesizing system either inside or outside the chloroplasts and
comparatively insensitive to chloramphenicol is involved (SMILLIE et aI., 1968).
Further work in this area is needed since one might expect the chloroplast ribosome
to be the site of synthesis of lamellar membrane protein by analogy with isolated
mitochondria which incorporate amino acids almost exclusively into membrane
protein of the inner membrane (ROODYN et al., 1962; WINTERSBERGER, 1965; WHEEL-
DON and LEHNINGER, 1966; KADENBACH, 1967). The protein of the outer membrane
of the mitochondria does not become appreciably labelled (BEATTIE et al., 1967;
NEUPERT et al., 1967) suggesting that extramitochondrial systems are involved in its
synthesis. Since the chloroplast appears to be the site of synthesis of all the chloro-
plast proteins which have been investigated so far, it would be of interest to determine
where proteins of the outer membrane of the chloroplast are synthesized. On the
basis of electron microscopic studies, FREy-WYSSLING (1967) has proposed that the
outer membranes of both chloroplasts and mitochondria are cytoplasmic in origin.
4. Other Enzymes
The majority of the non-protein components of chloroplasts, such as porphyrins,
quinones, fatty acids, etc. are related structurally to compounds found elsewhere in
the cell and often they share common precursors. For instance, cytochromes and
chlorophyll are synthesized from 5-aminolaevulinate, and there are common pre-
cursors for the synthesis of the polyisoprenoid side-chain of chlorophyll, carotenoids,
and chloroplast and mitochondrial quinones. A single pathway may exist in the cell
for the synthesis of a common precursor; alternatively, the same precursor may be
synthesized, at: more than one site in the cell. Recent investigations indicate the
existence of dual pathways. Thus chloroplasts contain an independent enzymic
system for the synthesis of fatty acids from acetate (STUMPF et aI., 1967) and for the
synthesis of terpenoids (TREHARNE et aI., 1966) and enzymes functioning in such
pathways may occur in both the cytoplasm and chloroplast e.g., mevalonate kinase
[ROGERS et aI., 1967 (1)]. The determination of whether such enzymes are cyto-
plasmic in origin or whether there are separate sites of synthesis in organelles and
cytoplasm must await future studies.
Separate pathways for porphyrin synthesis may exist both within and outside
chloroplasts (GRANICK, 1967). The first enzyme in the pathway leading to the
synthesis of cytochromes and chlorophyll, 5-aminolaevulinate synthetase, has not
been demonstrated in chloroplasts, but both it and the next enzyme in the pathway,
5-aminolaevulinate dehydrase, are induced by light in the photosynthetic bacterium
Rhodospseudomonas spheroides (LASCELLES, 1959). Soluble and membrane-bound forms
of the dehydrase have been detected in Euglena (CARELL and KAHN, 1964). Both
forms increase when dark-grown cells are illuminated and the effects of chlorampheni-
col and cycloheximide indicate part of the increase is due to protein synthesis within
the chloroplast and part to synthesis in the cytoplasm (our unpublished experiments).
Ferrochelatase, the enzyme inserting Fe2+ ions into dicarboxylic prophyrins to
yield the corresponding haem is present both in chloroplasts and mitochondria
(PORRA and LASCELLES, 1968). Because most chloroplast enzymes appear to be
synthesized within the organelle, whereas many mitochondrial enzymes are produced
extramitochondrially, it will be interesting to determine the intracellular sites of
synthesis of the two ferrochelatases.
Another enzyme apparently synthesized on chloroplast ribosomes is nitrite re-
ductase. This enzyme, along with nitrate reductase, is induced in plants receiving their
nitrogen in the form of nitrate. Although the reductases act sequentially in the reduc-
tion of nitrate to ammonium ions, they are localized in different regions of the cell.
The leaf nitrate reductases of maize and foxtail are cytoplasmic, but the corresponding
nitrite reductases are localized in the chloroplasts (RITENOUR et aI., 1967). In this
connection it is interesting that NADH is the preferred electron donor for nitrate re-
ductase (BEEVERS et aI., 1965) whereas reduced ferredoxin, a component of the photo-
synthetic electron transfer pathway, is the donor for nitrite reductase (RAMIREZ et aI.,
1966). LOSADA et al. (1965) have demonstrated a direct coupling between nitrate
reductase and photosynthetic electron transfer reactions via flavin mononucleotide,
but relatively high concentrations of flavin are required and the physiological signi-
ficance of this reaction is doubtful.
SCHRADER et al. (1967) found that chloramphenicol inhibited the induction of
nitrite reductase in maize. The induction of nitrate reductase was not inhibited under
the same experimental conditions, but its activity rapidly decreased in leaves treated
with cycloheximide at 2 [kg/ml (INGLE et aI., 1966). These results are consistent with
the synthesis of nitrite reductase in chloroplasts and the synthesis of nitrate reductase
in the cytoplasm.
A comparison of the nitrate and nitrite reductases of higher plants with those of
blue-green algae also provides an interesting example of a relationship between
enzyme specificity and intracellular compartmentation. The photosynthetic lamellar
system of blue-green algae, unlike that in other algae and higher plants, is not se-
parated from the cytoplasm by a limiting membrane and reduced ferredoxin can act
as an electron donor for both reductases (HATTORI and UESUGI, 1968).
VI. Photoregulation of Chloroplast Development

There is considerable variation throughout the plant kingdom in the way in which
light regulates chloroplast development. In some plants chloroplasts develop normally
in the dark, while in others several different photosystems combine to influence chloro-
plast development. Further, light may induce pronounced morphological changes
making it difficult to decide whether developmental changes in chloroplasts are
directly photoregulated, or are causally related to morphogenetic changes induced by
cytoplasmic photoregulators.
In general, nonflowering plants including algae do not require light to produce
mature chloroplasts, but within most groups there are exceptions. Thus Euglena does
not form chlorophyll in the dark and this is also true of some species of Chlorella.
Ochromonas danica (Chrysophyceae) shows a light requirement, although small
amounts of chlorophyll (1 to2% of normal) are produced in dark-grown cells (GIBBS,
1962). Mosses and ferns can produce chlorophyll in the dark but in some instances
chlorophyll synthesis is stimulated by light. Conifers can also accumulate chlorophyll
in the dark, but the capacity to do this varies among the species; spruce seedlings
produce nearly equal amounts of protochlorophyllide and chlorophyll in the dark,
pine mostly chlorophyll, and larch mostly protochlorophyllide (SUDYINA, 1963). In
both monocotyledons and dicotyledons, chlorophyll development is dependent upon
light and these plants become etiolated when grown in the dark.
In all plants in which chloroplast development is light-dependent, continuous light
is required for the conversion of protochlorophyllide to chlorophyllide a. But light
can influence chloroplast development through other photosystems. Phytochrome
is involved in the photoregulation of chloroplast synthesis in the angiosperms.
Although red light activates both the protochlorophyll and phytochrome systems,
blue light also is required for certain early structural changes during the light-
dependent differentiation of chloroplasts from etiolated plastids and blue light is
most effective for promoting chlorophyll formation in glucose-bleached Chlorella.
Protein synthesis in etiolated plastids is stimulated by continuous illumination with
low intensity far-red light, and qualitative and quantitative changes in photosynthetic
products caused by changes in light quality and intensity affect chloroplast develop-
ment.
A. Conversion of Protochlorophyllide to Chlorophyllide a
One of the most important photo systems controlling chloroplast development
involves the conversion of protochlorophyllide (magnesium 2-vinyl pheoporphyrin
as) to chlorophyllide a. The process is one of reduction and light absorbed by proto-
chlorophyllide itself activates the system (KOSKI et aI., 1951). The identity of the
hydrogen donor is unknown.
Although the photoreduction of protochlorophyllide is usually studied by illumi-

nating etiolated cells, it is also thought to be part of the main pathway for chlorophyll
a synthesis in normal green cells [see BOARDMAN, 1966 (2)]. The properties of proto-
chlorophyllide and the mechanism of its conversion to chlorophyllide a have been
reviewed by BOARDMAN [1966 (2)].
Etiolated tissues contain protochlorophyll (protochlorophyllide containing a
phytol side chain) as well as protochlorophyllide, but the latter predominates (WOLFF
and PRICE, 1957). For this reason we shall refer only to the photo conversion of proto-
chlorophyllide to chlorophyllide a, even though simultaneous conversion of the
protochlorophyll to chlorophyll a may take place. The addition of phytol to chloro-
phyllide a to yield chlorophyll ais believed to be independent oflight (WOLFF and PRICE,
1957; VIRGIN, 1960).
Measurements of the phototransformations occurring in etiolated cells upon
illumination are complicated by the discovery that protochlorophyllide can exist
Pchl- 635 - - - - - - - Pchl-650
jU9hl lughl
Dark
Chl - 673 ""'!~!"----_ Chl - 685
lughl
___ Physical
Chl - 683
Disruption
Fig. 4. Spectral shifts in protochlorophyll and chlorophyll in vivo
in vivo in two forms (HILL et aI., 1953; SHIBATA, 1957), one showing an absorption
maximum at 635 nm (Pchl-635), the other at 650 nm (Pchl-650). The 650 form
predominates in etiolated cells and it is converted by light to chlorophyllide showing
a maximum at 684 to 685 nm (chl-685). But after several minutes in the dark there is
a fairly abrupt spectral shift to 672 to 673 nm (chl-673) (SHIBATA, 1957; BUTLER and
BRIGGS, 1966; BOARDMAN, 1967). At a still later stage corresponding to the commen-
cement of lamellae formation there is a shift back to a higher wavelength indicative
of the appearance of yet another form of chlorophyll (chl-683) (BUTLER, 1965;
BUTLER and BRIGGS, 1966). These changes are illustrated in Fig. 4.
The cause of these spectral shifts to higher or lower wavelengths is obscure; nor
have the spectral shifts been correlated satisfactorily with known structural changes
occurring within the differentiating plastid (BOARDMAN, 1967). Structural disruption
of plastids by freezing and thawing or by grinding resulted in a shift to lower wave-
lengths and BUTLER and BRIGGS (1966) inferred that the in vivo dark conversion of
chl-685 to chl-673 following illumination was also due to a structural change, possibly
disaggregation of the pigment molecules as the three dimensional lattices of the tubules
of the prolamellar bodies broke down.
Other explanations for these changes have been proffered such as phytolation of
the porphyrin (SIRONVAL et al., 1965) and a change in the bonding of porphyrin to
protein (BOARDMAN, 1967). The pertinence of the spectral shifts in etiolated cells to
chlorophyll synthesis in the normal green cell is debatable. It could be they are
peculiar to the type of system studied. In etiolated Euglena cells, which do not contain
prolamellar bodies showing a crystalline appearance, similar spectral shifts were not
found and only the forms of protochlorophyllide and newly-formed chlorophyll
absorbing at the shorter wavdengths are present (BUTLER and BRIGGS, 1966). The
relative roles of the protochlorophyllideJchlorophyllide system and other photo-
systems controlling the differentiation of chloroplasts will be considered in the re-
mainder of this section.
B. Photoregulation of the Synthesis of Chloroplast Protein in Algae

Chloroplast proteins are synthesized in most algae independently of light although
light can of course exert an indirect effect through the provision of energy and carbon
by photosynthesis. In some algae, and in Euglena, chloroplasts fail to develop in the
absence of light and in these organisms the synthesis of chloroplast proteins appears
to be photoinducible. Continuous light is required and presumably the photoacceptor
involved is protochlorophyllide. Dark-adapted cells of Euglena contain protochloro-
phyllide which is transformed to chlorophyll a upon illuminating the cells (NISHI-
MURA and HUZISIGE, 1959). Continuous light is also necessary for the synthesis of
chloroplast protein and if greening cells are returned to darkness there is cessation
not only of chlorophyll synthesis, but also of the formation of new chloroplast pro-
teins and the utilization of storage polysaccharide (fJ-1,3-glucan) for this synthesis
(DWYER, 1968). The cessation of protein synthesis is not always immediate and a
carryover effect from the light has been reported for both Euglena (SpmR, 1964) and
Chlamydomonas (HUDOCK and LEVINE, 1964). Ribonucleic acids produced in the light
apparently can continue to function in chloroplast protein synthesis for some time
in the dark, but eventually this synthesis comes to a halt. Aside from the requirement
for continuous light, there is little other evidence which would implicate proto-
chlorophyllide as the primary photoregulator of chloroplast protein synthesis and the
possible existence of other photoregulators cannot entirdy be discounted. The action
spectrum for the light-stimulated synthesis of chlorophyll in Chlorella corresponds
neither with protochlorophyllide or chlorophyll (SOKAWA and HASE, 1967). Phyto-
chrome has been isolated from the green alga Mesotaenium(TAYLOR and BONNER, 1967)
but here its function appears to be in phototaxis rather than in chloroplast develop-
ment (HAUPT, 1959).
Thus only two mechanisms by which light can influence the synthesis of chloro-
plast proteins in Euglena and those algae which become etiolated in the dark have been
reasonably well characterized. The light-induced conversion of protochlorophyllide
to chlorophyll a can directly regulate the synthesis of chloroplast proteins and possible
derepression mechanisms are discussed by SCHIFF and EpSTEIN (1965). In addition,
the intensity and spectral qualities of light available for photosynthesis will affect the
concentrations of cellular metabolites, some of which may exert a control on the
synthesis of chloroplast proteins by feedback mechanisms. This aspect is discussed in
greater detail in Section VI.F.
C. Photoregulation of the Synthesis of Chloroplast Protein in Higher Plants

Protochlorophyllide is clearly implicated as the photoacceptor responsible for
controlling the synthesis of chlorophyll a in the leaves of higher plants [BOARDMAN,
1966 (2)]. Since the synthesis of chlorophyll is accompanied by the synthesis of
chloroplast-specific enzymes (HALL et al., 1959; SMILLIE, 1963), it might be assumed
that protochlorophyllide is also the primary photoregulator of the synthesis of chloro-
plast proteins. However, evidence is accumulating that a different photoacceptor
regulates the synthesis of many of the proteins found in the chloroplasts of angio-
sperms.
Several lines of evidence showing differences between the photoregulation of
chlorophyll synthesis and the synthesis of chloroplast proteins are considered below.
1. Light-independent Synthesis of Chloroplast Protein

Table 11 compares the relative amounts of various proteins found in proplastids
and mature chloroplasts of different plants. The activities found in etiolated or green
Table 11. Activities of plastid enzymes in etiolated leaves compared with green leaves
Enzyme % in Plant Reference
etiolated
compared
with green
leaves
RuDP carboxylase 43 BarleY" HALL et al. (1959)

38 Ryeb FEIERABEND and PIRSON (1966)
14 Pea· GRAHAM et al. (1968) (1)
3.3 Peab GRAHAM et al. (1968) (1)
Fraction I protein 104 Bean· BOARDMAN (1966) (1)
13 Beanb BOARDMAN (1966) (2)
NADP-glyceraldehyde-
3-P dehydrogenase 24, 78 Bean" MARGULIES (1965)
7 Beanb MARGULIES (1965)
25 Beano MARGULIES (1965)
16 Beano MARCUS (1960)
Transketolase 64 Ryeb FEIERABEND and PIRSON (1966)
Phosphoriboisomerase 57 Ryeb FEIERABEND and PIRSON (1966)
Transhydrogenase 14,19,48 Bean" KEISTER et al. (1962)
NADPH-diaphorase:
soluble 1.5 Peab GRAHAM et al. e
particulate 0.037 Peab GRAHAM et al. e
Plastid RNA-polymerase 15d Maize" BOGORAD (1967) (1), (2)
Pyruvate-Prdikinase 8.5 Maizeb GRAHAM et al. e
PEP carboxylase 14 Maizeb GRAHAM et al. e
" per unit fresh weight; b per leaf or stem apex; 0 per unit soluble protein;
d etiolated compared with partially-greened leaves;
e data from GRAHAM et al. (1968) (1), (2) and unpublished experiments.
tissues will vary with the pretreatment the tissues have received so that the percentage
increases in protein content upon illuminating etiolated tissues will in part reflect
experimental conditions. Nevertheless, two conclusions are evident from the data
in Table 11. Proplastids synthesize significant amounts of chloroplast-specific proteins
in the complete absence of light and the amounts synthesized can show large varia-
tions depending on the species of plant. The levels of some of the enzymes of the
Calvin cycle in etiolated cereal leaves approach those found in the corresponding green
leaves and thus increases resulting from exposure of etiolated leaves to continuous
light are only about 1- to 3-fold (per leaf basis) contrasted with increases in excess of
10-fold in pea leaves.
Thus while chloroplast protein synthesis in many plants is photoregulated, in none
of the plants so far studied is this regulation absolute; variable amounts of the chloro-
plast-specific proteins being formed in the dark depending on the organism, its stage
of development, and the environmental conditions.
2. Relationship Between Chlorophyll Synthesis and the Synthesis of Chloroplast

Protein
In most greening cells there is a good correlation between synthesis of chlorophyll
and synthesis of chloroplast protein, but there does not appear to be a close depend-
ence of one on the other.
BRIGGS (1920) showed that partially greened leaves continued to increase their
photosynthetic capacity when placed in darkness and SMITH (1954) found that etio-
lated barley leaves, if illuminated with white light for 10 min and then returned to
darkness, acquired a small capacity for light-dependent evolution of oxygen during
the dark period. This capacity was considerably enhanced following a second brief
exposure to light even though little additional chlorophyll was formed. The inference
from these studies that chloroplast proteins can be synthesized in the absence of
chlorophyll synthesis was confirmed by direct measurements of Fraction I protein.
This protein continued to increase in greening bean leaves after the leaves were
returned to darkness although chlorophyll synthesis ceased immediately (KuPKE,
1962; RACUSEN and FOOTE, 1965).
The persistence of an increased rate of protein synthesis in leaves following a
period of illumination has also been demonstrated by experiments in vitro. WILLIAMS
and NOVELLI (1964) irradiated the shoots of etiolated maize, bean or soy bean seed-
lings with white light for 60 min and returned the plants to darkness for a further
120 min. Ribosomes isolated from these leaves showed a 50 to 200% stimulation in
the rate of incorporation of leucine into acid-insoluble products when compared
with ribosomes prepared from the corresponding non-illuminated plants.
While synthesis of chloroplast protein can continue in the absence of chlorophyll
synthesis, the reverse is also true. The chlorophyll content of illuminated leaves may
continue to increase even after the maximum capacity for photosynthesis (SINGH and
LAL, 1935; SESTAK and CATSKY, 1962; SMILLIE, 1962) and the optimallevels ofphoto-
synthetic enzymes (SMILLIE, 1962) have been reached. In bean leaves chloramphenicol
inhibits the development of photosynthetic activity and the synthesis of some
chloroplast proteins while showing only a marginal effect on chlorophyll synthesis
(MARGULIES, 1962, 1964).
These studies do not preclude protochlorophyllide as a significant photoregulator
of the synthesis of chloroplast protein, but they do demonstrate that the latter is
not necessarily linked to the synthesis of chlorophyll.
3. The Effect of Pre-Illumination on Chlorophyll Synthesis

When etiolated leaves are illuminated the synthesis of chlorophyll does not
begin for some hours, except for the initial photoconversion of the existing proto-
chlorophyllide to chlorophyllide a (WOLFF and PRICE, 1957; VIRGIN, 1960). The period
between the commencement of continuous illumination and the ensuing synthesis of
chlorophyll varies with the age of the plant (SISLER and KLEIN, 1963), but it may also
be influenced by environmental factors such as pretreatment with light (WITHROW
et aI., 1956; VIRGIN, 1957, 1958, 1960; LIVERMAN, 1960; PRICE and KLEIN, 1961),
ionizing radiation (GAILEY and TOLBERT, 1958; PRICE and KLEIN, 1962), growth
substrates (WOLFF and PRICE, 1957, 1960) and other chemicals (SISLER and KLEIN,
1963). For instance, rapid synthesis of chlorophyll a in etiolated bean leaves did not
begin until after 2 h of continuous illumination. However, if the etiolated plants were
pretreated with red light (about 660 nm) for a few minutes and returned to darkness
for 5 to 15 h before illumination, the lag in the onset of chlorophyll synthesis was
eliminated (WITHROW et aI., 1956). VIRGIN (1957) found that with etiolated wheat
seedlings, an interposed dark period of at least 6 h was required to achieve the maxi-
mum stimulation of chlorophyll synthesis by pretreatment with red light. Because of
the relatively long period of darkness required, it appeared likely that the pretreatment
with red light stimulated some biosynthetic process during the subsequent dark
period, possibly the capacity to form precursors of chlorophyll a that were limiting in
dark-grown plants. VIRGIN'S experiments gave further credence to this idea. In etio-
lated barley leaves which had not been pretreated with light, the rate of chlorophyll a
formation after illumination was related to the rate of protochlorophyll formation in
darkness (VIRGIN, 1955). This capacity to synthesize protochlorophyll in the dark was
increased by pretreating seedlings with red light (VIRGIN, 1958). The ability of
excised barley shoots to accumulate protochlorophyllide when incubated in darkness
with a porphyrin precursor, 5-aminolaevulinate (GRANICK,1959, 1964), suggested
that the availability of this precursor might limit the rate of protochlorophyllide
formation and hence chlorophyll production when etiolated leaves were first illumi-
nated. This suggestion was supported by experiments in which etiolated barley
leaves infiltrated with 5-aminoleavulinate showed no lag in the onset of chlorophyll
synthesis and pretreatment with red light no longer effected an increase in chloro-
phyll synthesis (SISLER and KLEIN, 1963).
The synthesis of chlorophyll a in etiolated leaves then, may be determined by the
availability of 5-aminolaevulinate, and the synthesis of the latter may be induced by
red light. An important question however remains, namely whether red light merely
stimulates enzymic reactions leading to the production of 5-aminolaevulinate or
whether there is an actual increase in enzymes catalysing its synthesis in the plastids.
5-aminolaevulinate is synthesized from glycine and succinate by the enzymes 5-
aminolaevulinate synthetase and succinate thiokinase but direct measurements of
these activities in etiolated and greening tissues of higher plants are unavailable.
GASSMAN and BOGORAD [1967] (2) have, however, indirectly measured the activity of
the 5-aminolaevulinate-synthesizing system in maize and bean by the capacity of the
leaves to synthesize protochlorophyllide capable of being photo converted to chloro-
phyllide a. The etiolated leaves were illuminated for 1 min with light of sufficient
intensity to convert all the endogenous protochlorophyllide to chlorophyllide a. The
amount of protochlorophyllide synthesized during a subsequent 4-h period of dark-
ness was determined spectrophotometrically by the increase in the 650 nm absorption

band of protochlorophyllide. Preincubation of the leaves for 4 h with 5 mM chlor-
amphenicol or 1 mM puromycin resulted in little or no new protochlorophyllide
[BOGORAD, 1967 (1); GASSMAN and BOGORAD, 1967 (2)]. Preincubation with a
relatively high concentration of cycloheximide (50 [Lg/ml) for 16 h did not inhibit the
regeneration of protochlorophyllide. The inhibition by chloramphenicol was largely
reversed by supplying 5-aminolaevulinate to the leaves. Thus the inductive effect of
light on the synthesis of protochlorophyllide appeared to act by promoting the
synthesis of 5-aminolaevulinate, and this in turn required the synthesis of new
protein. Furthermore, the pattern of the inhibition, that is inhibition by chlorampheni-
col and puromycin but not by cycloheximide, implicated chloroplast ribosomes
(Section V.B.). It will be important to establish if one effect of chloramphenicol is to
prevent the synthesis of a plastid-ocalized 5-aminolaevulinate synthetase.
From the foregoing it can be inferred that the effectiveness of pretreatment with
red light in decreasing the lag phase of chlorophyll synthesis would be diminished by
chloramphenicol. Experimental verification of this has been obtained by MARGULIES
(1967).
4. Photoregulation by Phytochrome
The studies discussed above provide evidence of a regulatory action by red light
upon the production of enzymes catalyzing the synthesis of precursors of porphyrins.
As these changes do not require continuous red light, the primary photo regulator
may be some photoacceptor other than protochlorophyllide. The demonstration by
WITHROW et al (1956) that the effect of pretreatment with red light in reducing the
normal lag in chlorophyll synthesis was nullified by brief exposure to far-red light
(>- 700 nm) immediately following the red illumination implicated the pigmented
protein phytochrome. PRICE and KLEIN (1961) and MITRAKOS (1961) alternated
doses of light at 660 nm and 730 nm and found the type of red light given last always
determined the level of response; only plants receiving the 660 nm irradiation last
showed a stimulated rate of chlorophyll synthesis. These responses to light at 660 nm
and 730 nm are characteristic of phytochrome.
Many morphogenetic changes responsive to light are mediated by phytochrome
(e.g. see MOHR, 1966). Phytochrome exists in two forms, a physiologically active
form with a maximum absorption band at 725 nm (P725) and an inactive form showing
a maximum absorption band at 665 nm (P665). Both forms are photointerconvertible
and this explains the failure of pretreatment with red light to stimulate chlorophyll
synthesis if followed by irradiation with far-red light.
P665 Light at 665 nm P725
(inactive) Light at 725 n~ (active)
At photostationary states only 81 % of P 665 is converted to P725 by irradiation with
665 nm light (BUTLER et aI., 1964) owing to considerable overlap of the two spectra
at this wavelength. However, irradiation with light at a wavelength >- 725 nm con-
verts 99% of P725 to P665 (BUTLER et al., 1963). The exact nature of the molecular
events occurring during phototransformations are unknown, but besides a change in
the spectral properties of the chromophore, the different reactivities of P665 and P725
towards sulfhydryl-reacting compounds such as p-mercuribenzoate and N-ethyl
maleimide suggest a conformational change in the protein (SIEGELMAN and BUTLER,

1965). The identity of the chromophore has not been established, but it may be a
tetrapyrrole.
As is evident from the foregoing discussion, one physiological consequence of
the photoconversion of P665 to P 725 in etiolated leaves is very likely an increased
synthesis of an enzyme or enzymes catalyzing the synthesis of 5-aminolaevulinate. Is
this the full extent of the photo regulation of protein synthesis in the developing
plastid or does this control extend to the synthesis of other proteins? Certainly
carotenoid synthesis is stimulated by activation of phytochrome [COHEN and GOOD-
WIN, 1962; HENS HALL and GOODWIN, 1964 (1)]. Red light enhances the disappearance
of starch and sugar in excised leaves of etiolated maize seedlings (KLEIN et al., 1963;
PRICE et al., 1964, 1965) and the uptake of sucrose by excised epicotyls of etiolated pea
seedlings (GOREN and GALSTON, 1966). These instances of light-induced carbohydrate
Table 12. Changes in plastids of etiolated bean leaves induced by short exposure to light. The values from
MEGO and JAGENDORF (1961) are taken from several different experiments. Etiolated seedlings were
pretreated by illumination with red (1 h) or white light (1.5 h). The plants were kept in the dark, in
continuous light or illuminated intermittently for the next 5 days
------------------------
Light treatment Leaf wet Plastid
weight Diameter Dry Nitrogen Non-
(mg) ([L) weight ([L[Lg) chlorophyll
([L[Lg) lipid ([L[Lg)
Dark 17 3.3 3.6 0.30 0.7

Short exposure to light,
then dark 38 4.3 6.1 0.73 1.3
White light 134" 6.0b 7.9" 0.8()a 2. ()a
" 14 h light per day; b continuous light.
utilization most likely occur in response to an increased synthesis of protein, although

not necessarily to an increased synthesis of plastid protein.
MEGO and] AGENDORF (1961) were the first to make a detailed examination of the
possible wider involvement of phytochrome in regulating the synthesis of chloro-
plast protein. They examined gross changes in plastid composition and structure of
etiolated plastids of Black Valentine bean induced by brief exposure to red or white
light. Etiolated seedlings were exposed to light and then returned to darkness for
several days. After 5 days in the dark, the average diameter of the plastids had
increased from 3.3 [L to 4.3 [L and the dry weight and nitrogen and lipid contents of
the plastids had risen 2-fold (Table 12). Thus the brief exposure to light resulted in
major developmental changes in the plastids. Further, since greening plants showed
comparable increments in the nitrogen content of their plastids, continuous light did
not appear to be necessary for the synthesis of virtually all the plastid protein.
Subsequent studies have established conclusively that the synthesis of individual
plastid proteins in etiolated leaves is responsive to brief irradiation by red light. The
activity of NADP-glyceraldehyde-3-phosphate dehydrogenase in etiolated bean
leaves increased 2- to 3-fold per unit soluble protein following irradiation of the
leaves with red light for a few minutes (MARCUS, 1960; MARGULIES, 1965). Results
obtained with this enzyme are unequivocal as the enzyme is found only in chloro-
plasts in green tissues (HEBER et ai., 1963). Several other enzymes of the Calvin cycle
were investigated by FEIERABEND and PIRSON (1966) using rye seedlings. The activity
of ribulose-1,5-diphosphate carboxylase of etiolated seedlings increased 2.6-fold
(per shoot) in continuous white light and 2.2-fold after treatment with red light
(661 nm), whereas treatments with far-red light (726 nm), red followed by far-red, or
continuous blue light (435 nm) resulted in much smaller increases. Transketolase
behaved similarly except the increases in activity were smaller (lo6-fold in continuous
light). Ribose-5-phosphate isomerase also increased after red light treatment. Chloro-
phyll was synthesized in the leaves illuminated with continuous blue light, thus
demonstrating that the photo conversion of protochlorophyllide to chlorophyll was
Table 13. Changes in plastid proteins in stem apices of etiolated pea seedlings irradiated briefly with
red or far-red light. Data from GRAHAM et al. (1958) (1). Values are per stem apex and the value
for non-illuminated plants is taken as 1.0. The plants were grown for 7 days in the dark and irradiations
were carried out daily for the next 5 days with red (661 nm) light for 5 min, far-red (733 nm) light
for 20 min or red followed by far-red light. Total light energies: 661 nm, 216 Kiloergs cm-2 ; 733 nm,
240 Kiloergs cm-2
Dark 661 nm 661 + 733nm
733nm
Fresh weight 1.0 5.1 2.5 1.6

Soluble protein 1.0 5.3 2.7 1.7
Enolase 1.0 3.3 1.9 1.4
NADP-isocitrate dehydrogenase 1.0 2.9 1.5 1.3
Malate dehydrogenase 1.0 3.6 1.9 1.3
RuDP carboxylase 1.0 91 36 17
NADP-glyceraldehyde-3-phosphate
dehydrogenase 1.0 35 18 6
Alkaline fructose-l,6-bisphosphatase 1.0 15 3.1 1.9
Fraction I protein 1.0 11 5.1 3.3
not in itself sufficient to induce the synthesis of enzymes of the Calvin cycle. Un-
fortunately, in none of these studies were changes in the activities of non-plastid
enzymes ascertained, and it is impossible do decide if the phytochrome-mediated
effects on enzyme activity are specific to plastid enzymes or if they reflect a general
increase in the activities of cellular enzymes brought about by altered patterns of leaf
development.
The net increases per leaf in plastid enzymes induced by intermittent or continuous
irradiation of etiolated bean or cereal leaves are relatively small, usually about 2- or
3-fold, and this can be attributed to the high levels of enzymic activity already
present in the etiolated plastids of these plants. Larger phytochrome-mediated
responses might be expected in plants such as the pea where plastid development is not
as far advanced in dark-grown seedlings. Table 13 shows results obtained by GRAHAM
et al. [1968 (1)] for pea seedlings. The plastids of the dark-grown plants contained low
amounts of Calvin cycle enzymes compared with normal chloroplasts (Table 11), but
plants subjected to short exposures of red light showed large increases in ribulose-
1,5-diphosphate carboxylase, NADP-glyceraldehyde-3-phosphate dehydrogenase and
alkaline fructose-1,6-diphosphatase. The increases in several growth parameters were
much smaller, approximately 5-fold in fresh weight and soluble protein and 3- to
4-fold in respiratory enzymes including enolase, NADP-isocitrate dehydrogenase
and malate dehydrogenase. Chlorophyll synthesis in the dark following the red light
treatments was negligible. Amino acyl-sRNA synthetases also increase proportio-
nately to the increase in dry weight [HENSHALL and GOODWIN, 1964 (2)].
The analyses on Fraction I protein demonstrated for the first time net synthesis of
a specific chloroplast protein as the result of red irradiation. The increase was not as
large as the increase in its associated enzymic activity, ribulose-1,5-diphosphate
carboxylase, and may have been due to an initial light-induced activation of the
enzyme. This latter result was variable and it was not established with certainty
whether such an activation actually occurred although both rapid activation and
synthesis of this enzyme by light has been reported by others (BASSHAM and KIRK,
1968; CHEN et aI., 1967). After the second day of irradiation, the carboxylase acti-
vity increased concomitantly with Fraction I protein [GRAHAM et aI., 1968 (1)].
The responses induced by red light shown in (Table 13) were reduced, but not entire-
ly reversed, by subsequent exposure to light at 733 nm. Further, the magnitude of the
increases in plastid enzymes resulting from the exposure to light at 661 nm approached
values attained in green leaves. For instance, the activity of ribulose-1,5-diphosphate
carboxylase was 0.4 to 0.5 times the values found in leaves exposed to white light
either continuously or for 16 h per day.
Three conclusions can be drawn from these studies:
(1) Phytochrome rather than photochlorophyllide is the primary photoregulator
for the synthesis of Calvin cycle enzymes within the plastids.
(2) The magnitude of the phytochrome-mediated changes approach those found
in plants exposed to continuous illumination.
(3) The increases in the plastid proteins are greater than those shown by other
growth parameters.
What of proteins of the photosynthetic electron transfer pathway? These proteins
are localized in the lamellae in mature chloroplasts, yet there is little structural change
concomitant with the red light induced increase in soluble plastid protein, and there
is no formation of lamellae (MEGO and jAGENDORF, 1961; KLEIN et al., 1964). The
activity of ferredoxin NADP-reductase (assayed as NADP-specific pyridine nucleo-
tide transhydrogenase) in etiolated bean leaves increased by 50% in the dark following
a short exposure to red light, but the increase was small compared with the 2.5- to
8-fold increases found in continuous light (KEISTER et al., 1962). Our studies using
pea seedlings have stressed the need to distinguish between the membrane-bound and
soluble forms of this enzyme. Illumination of dark-grown pea seedlings each day
with light at 661 nm for 3 min increased the activity of the soluble reductase (measured
by substituting the dye 2,6-dichlorophenol indophenol for ferredoxin in the assay), but
the response of the particulate enzyme was far more significant; it increased 610-fold
per stem apex to a value which was 1/5 to 1/4 the value found in continuously illuminated
leaves (Table 14). These results provide evidence of a definite effect of red light in
promoting the synthesis of a plastid membrane-bound protein. Since photoacceptors
other than phytochrome initiate formation of lamellae and synthesis of at least one
membrane component, chlorophyll, much more information on photoregulation of
the synthesis of membraneous proteins is required.
Aside from its function in regulating the synthesis of Calvin cycle enzymes,
phytochrome may well act as a photoregulator of the synthesis of enzymes of an
alternative pathway of photosynthetic CO2 fixation described by HATCH and SLACK
(1966). The activities of two key enzymes of this pathway, pyruvate-Pt-dikinase
and phosphoenolpyruvate carboxylase (see Table 1), increased 12-fold and 7-fold,
respectively (per unit fresh weight of leaf), after briefly irradiating dark-grown
maize seedlings with light at 661 nm [GRAHAM et al., 1968 (2)]. Respiratory enzymes
Table 14. Light-induced changes in NADPH-diaphorase in etiolated

pea leaves. The experimental conditions are described by GRAHAM et al.
(1968) (1). The values in brackets are relative to a value of 1.0for non-
illuminated plants
NADPH-diaphorase
«(Lmoles substrate/min/100
leaf apices)
Soluble Membrane-
bound
Dark 0.173 (1.0) 0.0055 (1.0)

Red (661 mm, 3 min) then dark 6.8 (39) 3.35 (610)
White (continuous) 11.3 (65) 15.0 (2730)
including NADP-isocitrate dehydrogenase, glucose-6-phosphate dehydrogenase and

acid phosphatase showed slight changes only. The effect of far-red light on this
system has not yet been ascertained.
D. Photoregulation of Synthesis of Chloroplast RNA

The light-induced synthesis of RNA which precedes the synthesis of chloroplast
protein in Euglena has already been discussed in Section IV.D Here light exerts a
regulatory action through protochlorophyll or some other photoacceptor. In the
angiosperms, phytochrome appears to be the primary photoregulator of the syn-
thesis of plastid protein and it may be asked if the photo conversion of P665 to P 725
also induces a specific synthesis of RNA within the plastids. The synthesis of plastid
proteins in dark-grown plants after brief illumination with red light eliminates the
possibility of some critical requirement for continuous light in the synthesis of the
plastid RNA that is responsible for the synthesis of these proteins.
The total RNA content of barley leaves showed little response to light; changes
in the first leaf during growth were similar whether the seedlings were grown in the
light or in darkness. In each case the RNA content rose to above 20 !log RNA-NJleaf
and then decreased (RHODES and YEMM, 1966). Plastids from etiolated leaves of rye
contained three times as much RNA per mg N as chloroplasts (SZARKOWSKI and
GOLASZEWSKI, 1961) and since the protein content of plastids rose 3-fold when
etiolated rye was illuminated (FEIERABEND and PIRSON, 1966), it can be concluded
that there is little change in the RNA content of plastids during greening. DURANTON
(1966) arrived at a similar conclusion for maize. Boardman [1966 (1)] found that 70S
plastid ribosomes were synthesized in etiolated bean leaves. Exposure to light did not
induce a large secondary increase in plastid ribosome content, nor greatly affect the
proportions of plastid to cytoplasmic ribosomes (1 :2.86 in etiolated leaves and

1 :2.14 in green leaves). These results are consistent with the high activities of plastid
enzymes found in dark-grown bean plants (Table 11).
The absence of net changes in ribosome content may not necessarily reflect the
true state of events, since light could possibly induce a synthesis of new ribosomes
concomitant with the degradation of existing ribosomes and, in fact, other studies
have provided evidence for a light-stimulated synthesis of both RNA and RNA poly-
merase in etiolated leaves. Actinomycin D blocked chlorophyll production after
illumination of etiolated leaves [BOGORAD and JACOBSON, 1964; GASSMAN and BOGO-
RAD, 1967 (1)], which suggests, since the etiolated plastids already contained most
of their ribosomes, that light stimulated the production of messenger RNA. Light
also stimulated the activity of RNA polymerase. Illuminating etiolated maize for
30 min followed by a 90-min period of darkness increased the activity of RNA
polymerase in the plastids by 6.5 times [BOGORAD, 1967 (1), (2)].
There is no direct evidence for photoregulation of plastid RNA by phytochrome,
but the studies cited above show that light can affect the rate of synthesis of plastid
RNA and when these are considered together with studies on the photoregulation of
plastid protein synthesis it seems reasonable to suppose that phytochrome can
mediate in regulating the synthesis of plastid RNA. Furthermore, the response of
RNA synthesis in etiolated plastids to illumination is likely to be greater in a plant
such as the pea, which shows a large phytochrome-mediated synthesis of plastid
protein (Section VI.CA), compared with bean or cereal plants. More information on
effects of red and far-red light on the synthesis of plastid ribosomal RNA in different
plants would be especially pertinent since this RNA is coded by plastid DNA (Sec-
tion III.D.2) and is presumably synthesized within the plastids.
E. Light and the Synthesis of Chloroplast DNA

During oxygen-induced development of mitochondria in the yeast Saccharomyces
cerevisiae there is a rapid synthesis of mitochondrial DNA (SWIFT et aI., 1967). So far
there is no indication of a similar synthesis of organelle DNA associated with the
induction of chloroplast development in nondividing cells. Our own experiments
have not disclosed any marked stimulation in 32p incorporated into the chloroplast
DNA of Euglena during such development. This result is consistent with studies in
which DNA synthesis was inhibited with antibiotics or by irradiation with ultraviolet
light while chloroplast development was unaffected.
Nalidixic acid (1-ethyl-1 ,4-dihydro-7-methyl-4-oxo-1 ,8-naphthyridine-3-carboxylic
acid), a specific inhibitor of DNA synthesis in bacteria, inhibited chloroplast replica-
tion in Euglena without affecting chloroplast development (LYMAN, 1967). Another
inhibitor of DNA synthesis, mitomycin c, failed to inhibit chloroplast development
in Chlorella. AOKI and HASE (1965) found that concentrations of the antibiotic from
200 to 1000 !1-g/ml had no apparent effect whatever on chloroplast development in
"glucose-bleached" cells while cell division was suppressed throughout most of the
greening period.
SCHIFF et aI. (1961) showed that exposure of dark-grown cells of Euglena to a
dosage of ultraviolet light sufficient to induce 100% bleaching in daughter colonies
did not preclude a normal transition of proplastids into mature chloroplasts. The
bleaching phenomenon is thought to be brought about by specific interference in the

replication of plastid DNA through a dimerization of neighbouring thymine moieties
induced by the ultraviolet irradiation.
While DNA synthesis in plastids does not appear to be mandatory for chloroplast
development in non-dividing cells of dark-adapted Euglena or "glucose-bleached" cells
of Chiorella, IWAMURA (1960) noted that following synchronous division of Chlorella
cells and while the cells were still small and very active photosynthetically, a minor
component of the cellular DNA continued to turnover rapidly. He has equated this
metabolically active component with a second, minor species of chloroplast DNA
(IWAMURA and KUWASHIMA, 1968). The apparent stimulation of synthesis of this
minor chloroplast DNA by light may not be a direct effect, but rather be related to
the stage of growth of the cells, since, as has already been mentioned (Section IILC.2),
the replication of chloroplast DNA in synchronously grown Chlamydomonas occurs
during the light period while that of nuclear DNA is restricted to the dark period.
Thus light may influence the timing and rate of synthesis of chloroplast DNA in as,
much as it affects the rate of photosynthesis and growth of the cells.
F. Photosynthesis and the Regulation of Chloroplast Development

Active photosynthesis is not obligatory for the formation of mature chloroplasts,
as Euglena (SCHIFF et al., 1967; Dwyer, 1968), Chlorella (MATSUKA and HASE, 1966)
and bean leaves (KLEIN and NEUMAN, 1966) can form chloroplasts in the presence of
inhibitors of photosynthetic oxygen evolution provided an exogenous or endogenous
source of carbon other than CO2 is available. Nevertheless, in cells in which the pro-
duction of energy and carbon are partially dependent upon photosynthesis, cellular
synthetic processes including those responsible for synthesis of chloroplast compo-
nents would be affected by the nature and rate of formation of photosynthetic pro-
ducts. The latter in turn would be influenced not only by the intensity and duration
of the illumination, but also by its spectral composition. Many common metabolites
exhibit catabolite repression of the synthesis of chloroplast proteins and one way in
which photosynthesis may conceivably alter the course of chloroplast development
is through shifts in cellular concentrations and relative proportions of critical meta-
bolites. Thus Chlorella, which when grown in blue light incorporated more carbon
from CO2 into amino acids and less into glycollate (HAUSCHILD et al., 1962), also
showed an increase in the protein content of the cell (KOWALLIK, 1965). It would be
interesting to determine if this effect of blue light is primarily on synthesis of chloro-
plast protein or whether the protein metabolism of the cell as a whole is affected. A
close relationship between the types of photosynthetic assimilates produced and the
relative amounts of carbohydrate, protein and lipids synthesized in the chloroplasts is
not unexpected especially as photosynthetic products appear to be used preferentially
for the synthesis of chloroplast constituents. Thus HEBER'S results (1962) suggested
preferential utilization of 14C02 for the synthesis of chloroplast protein compared
with the synthesis of cytoplasmic proteins and WIECKOWSKI and GOODWIN (1967)
have shown that 14C02 was incorporated readily into plastid terpenoids but less so
into cytoplasmic sterols, whereas the reverse was true in feeding experiments with
a terpenoid precursor mevalonate. Amino acids as well as sugars and phosphorylated
compounds are produced during photosynthesis (HOLM-HANSEN et al., 1959) and
12*
these amino acids may be maintained in separate metabolic pools within the chloro-
plasts (SMITH et aI., 1961) where they could be used for the synthesis of chloroplast
proteins. Probable control points in the Calvin cycle that determine the relative flows of
photosynthetic products into proteins and lipids as opposed to carbohydrates have
been discussed by BASSHAM and KIRK (1968).
ZIEGLER and ZIEGLER [1965, 1966 (1), (2)] have demonstrated a relation between
photosynthesis and the synthesis of a chloroplast enzyme. Illumination of a variety of
green plants resulted in a rapid (within 20 min) and reversible (in the dark) synthesis
ofNADP+-glyceraldehyde-3-phosphate dehydrogenase. The synthesis appeared to be
linked to photosynthesis; inhibitors of photosynthesis blocked synthesis of the
enzyme. Activation of Calvin cycle enzymes by light has been demonstrated (BUCHA-
nan et aI., 1967; BASSHAM and KIRK, 1968), but the ZIEGLERS considered the increase
in NADP-glyceraldehyde-3- phosphate dehydrogenase was due to enzyme synthesis
since it was blocked by chloramphenicol and by the amino acid analogues p-fluoro-
phenylalanine and ethionine.
It is well known that light intensity determines the eventual size and anatomical
characteristics of chloroplasts. The optimum light intensity for greening in Euglena is
well below the optimum for photosynthesis, and intensities above 200 ft.-candles
result in decreased cellular levels of chlorophyll. This is not simply photobleaching
of chlorophyll, as other chloroplast lipids are similarly affected (CONSTANTOPOULOS
and BLOCH, 1967). We shall not attempt to summarize here the literature on the
relationship between light intensity and the chlorophyll content of photosynthetic
cells, but from the available evidence one can make the generalization that conditions
favouring the formation of an abundance of growth substrates result in increased
cellular growth, but decreased synthesis of chloroplast constituents. Decreasing the
availability of these substrates (e.g. by low light intensity) or preventing their utiliza-
tion for cellular growth (e.g. by addition of cycloheximide, see Section V.B.1) circum-
vents the repression of chloroplast formation. The repression of chloroplast pro-
duction in Euglena even at low light intensities by exogenously supplied growth sub-
strates (App and ]AGENDORF, 1963; HARRIS and KIRK, 1968) is consistent with this
thesis and again the repressive action is nullified by inhibiting cytoplasmic protein
synthesis with cycloheximide (Section V.B.1).
The phenomenon of repression of chloroplast formation by high light intensity is
also common in higher plants but is more complicated since it is usually accompanied
by pronounced anatomical and physiological changes in the leaves. Sun and shade
leaves show many differences in their anatomy as well as in their chlorophyll con-
tent per unit leaf area, maximum rate of photosynthesis, net assimilatory rate per day
and transpiration (see EVENARI, 1965). Various mechanisms to explain these changes
have been suggested including photo bleaching of chlorophyll at high light intensities
and control through protochlorophyll or even phytochrome but, as in the case of
unicellular organisms, the most important factor is probably photosynthesis itself,
with conditions of high light intensity favouring rapid production of photosynthates
and consequent repression of chloroplast development through feedback mechanisms.
The question of whether these photomorphogenetic changes can be explained solely
on the basis of changes in the levels of cellular metabolites brought about by light
energy utilized in photosynthesis, or whether other photo systems are involved,
remains for future studies.
G. A Requirement for Blue Light

Illumination of etiolated leaves by white or red light results not only in reduction
of photochlorophyllide but also in structural alterations to the prolamellar bodies of
the plastids (VON WETTSTEIN, 1966). Membraneous tubes in the prolamellar body
rearrange to form vesicles and these subsequently disperse to form either ordered
rows which radiate across the plastids or concentric rings. The second process, that
of vesicle dispersion, requires light of higher intensity (VIRGIN et al., 1963). Further,
only blue light is effective, the action spectrum showing a sharp maximum at 450 m[L,
and red or far-red absorbing pigments do not appear to be involved (HENNINGSEN,
1967). The identity of the blue-light acceptor is unknown. Xanthophylls and fJ-caro-
tene seem to be excluded since vesicle dispersion still occurs in carotenoid-deficient
mutants of Helianthus annuus (WALLES, 1965, 1967). This requirement for blue light
has been demonstrated only in dark-grown plants with proplastids containing pro-
lamellar bodies and its significance, if any, in the formation of chloroplasts in plants
grown in continuous or intermittent light has yet to be established.
H. Conclusions
1. Role of Phytochrome
Table 15 lists chloroplast enzymes whose synthesis is induced in dark-grown
plants by a brief exposure to red light. All of these enzymes are synthesized to some
extent in the dark-grown plants, but their further synthesis is regulated by light.
Table 15. List of chloroplast enzymes whose .rynthesis is photoregulated by pqytochrome

Enzyme Plant
Pea Bean Rye Maize
Fraction I protein +
RuDP carboxylase + +
NADP+-Ga-3-P dehydrogenase + +
Alkaline FDPase +
Transketolase +
Ribose-5-P isomerase + +
Transhydrogenase +
Aminoacyl-sRNA synthetase +
Enzymes synthesizing protochlorophyll +
Pyruvate-Pj-dikinase- +
PEP carboxylase +
-Reversibility by far-red light not yet demonstrated.
Phytochrome and not protochlorophyllide appears to be the primary regulator of the

synthesis of enzymes functioning in pathways of CO2 fixation. The results with
membrane-boundNADPH-diaphorase suggests the same may hold for proteins of the
photosynthetic electron transfer pathway, but nothing can be said of other membrane-
ous proteins since the effect of red light on their synthesis has not been studied. How
does phytochrome effect this regulation and why is part of this synthesis, especially
in the monocotyledons, independent of light?
If phytochrome exerts a direct control on the rate of protein synthesis in plastids,

one might expect to find it localized in plastids. Unfortunately, the intracellular distri-
bution of phytochrome is difficult to ascertain since it rapidly precipitates below pH
7.2, but is easily extracted above this pH (SmGELMAN and BUTLER, 1965), and no
evidence for its existence in plastids has been obtained. Compared with other parts
of the plant, the leaves of etiolated seedlings contain one of the highest concentra-
tions of phytochrome (FURUYA and HILLMAN, 1964; BRIGGS and SmGELMAN, 1965),
but this distribution is most likely to be related to the rate of growth of the different
tissues. Thus, there is no compelling evidence indicating direct participation by
phytochrome in chloroplast development and by considering data obtained on three
different types of plants, peas, beans and rye, an alternative explanation for the red
light effect can be given.
Leaf expansion, especially in dicotyledons, is influenced by light (PARKER et al.,
1949) and phytochrome has been implicated as the photoregulator (LIVERMAN et al.,
Leaf Development
..
Cel! Division---.... Cel! Expansion
Cytoplasmic Protein
.. Synthesis ..
.. Plastid Protein Synthesii
Aproximate
Development
in Dark I I1
Pea Bean Rye
Fig. 5. Diagrammatic representation of the main periods of cytoplasmic and chloroplast
protein synthesis in developing leaves. The vertical dashed lines represent the approximate
development of pea, bean and rye leaves in the dark in terms of the synthesis of their cytoplas-
mic and plastid proteins, the right-hand end of the rectangle indicating the situation in fully-
expanded, green leaves. For other details see text
1955). In the dark the leaves of cereals expand and in terms of content (Table 11)
and size their plastids show appreciable development. Photoactivation of the phyto-
chrome system results in a further increase in plastid protein and relatively small
changes in leaf area and the activities of respiratory enzymes. Thus cytoplasmic
protein synthesis and a substantial portion of the synthesis of plastid proteins is
completed in the absence of light. In contrast, activation of the phytochrome system
of etiolated pea plants results in considerable expansion of the leaves and comparable
increases in weight, protein content and activities of cytoplasmic enzymes. Still greater
increases are shown by the plastid proteins. Although the bean plant is also a dico-
tyledon, its leaves expand much more in the dark, and development of the plastids in
the dark is intermediate between that of pea and rye.
These differences can be explained by assuming that the synthesis of plastid protein
is highly dependent upon the stage of growth of the cell, and that in the differentiating
cell the synthesis of plastid protein is not linearly related to the synthesis of cyto-
plasmic protein but instead lags behind the latter, being associated primarily with the
period of cell expansion (some overlap of the main periods of cytoplasmic and plastid
protein synthesis would be expected). These ideas are diagramatically represented in
Fig. 5. It would be of interest to determine if the synthesis of plastid DNA similarly
lags behind the synthesis of nuclear DNA. This scheme would explain the apparently
greater effect of phytochrome on plastid protein synthesis in peas compared with the
synthesis of cytoplasmic proteins, as well as the variable amounts of plastid proteins
synthesized in the dark in different plants. Further, it obviates the necessity to
postulate a direct effect of phytochrome in regulating plastid differentiation. Instead,
phytochrome would act on some other cellular process which promotes cell develop-
ment, the ensuing changes in the plastids being causally related to the extent of this
development. The continued synthesis of Fraction I protein and increase in ribulose-
1,5-diphosphate carboxylase in leaves which continue to slowly expand in the dark
(KUPKE, 1962; RACUSEN and FOOTE, 1965) is consistent with this scheme. Recent
studies by AKOYUNOGLOV and SIEGELMAN (1968) are also pertinent. They showed
that the protochlorophyllide content of bean leaves was extremely low during the
first few days of growth in the dark and the maximal rate of increase did not occur
until between the 4th and 8th day of growth. If protochlorophyllide content is used
as an index of the growth of proplastids, it would appear that proplastid formation
occurs at a comparatively late stage in the differentiation of the leaf cells.
How does the photoconversion OfP665 to P725 act to promote cell development?
HENDRICKS and BORTHWICK (1967) believe that photoconversion of membrane-bound
phytochrome may possibly change membrane permeability. Interactions with plant
hormones may also be involved since light is known to modify the action of gibberellic
acid (LOCKART, 1964) and the latter in turn inhibits the phytochrome-stimulated uptake
of sucrose by excised etiolated pea buds (GOREN and GALSTON, 1967). Whatever the
mechanism of phytochrome action, growth of leaves in the dark slows down after a
time (hormone imbalance?) and the stage of growth at which this happens varies
in different plants. Activation of the phytochrome system allows growth and differ-
entiation to proceed further.
The proposed mechanism of photoregulation of plastid development emphasizes
cytoplasmic control of plastid differentiation. One consequence of this is that al-
though plastids contain a semi-autonomous biosynthetic mechanism, the differentiation
of chloroplasts in higher plants is essentially a cellular process and not one of inde-
pendent organelle development. Thus factors which regulate the differentiation of
cells also regulate the differentiation of plastids.
2. Role of Continuous Light

If the synthesis of plastid RNA and protein is related to the stage of cell differentia-
tion, the latter being subject to regulation by the phytochrome system, the question
of the role of continuous light in chloroplast differentiation still remains. Apart from
effects of light utilized for photosynthesis on chloroplast development, continuous
light is required for the synthesis of chlorophyll. How then does a block in the con-
version of protochlorophyllide to chlorophyll a repress biosynthetic processes in
developing plastids? The answer to this question is not at all clear. The synthesis of
chlorophyll is not a prerequisite for formation of lamellae. Mutants of barley (VON
WETTSTEIN, 1959) and the alga Cyanidium caldarium (BOGORAD et al., 1963) deficient
in chlorophyll a could still form lamellae although in the barley mutant, aggregation of
lamellae to form grana did not occur. Nevertheless, in the normal cell the formation
of lamellae does not proceed in the absence of light and it is possible the synthesis
of some other components essential for membrane assembly require continuous

light. The synthesis of chlorophyll b is dependent upon continuous light since it is
formed from chlorophyll a, but it too does not appear to be an essential structural
component of lamellae (VON WETTSTEIN, 1959; GOODCHILD et al., 1966). Carotenoid
synthesis is stimulated by activation of the phytochrome system, but, interestingly
enough, only carotenoids characteristic of dark-grown cells are formed [COHEN and
GOODWIN, 1962; HENSHALL and GOODWIN, 1964 (1)]. Continuous light absorbed by
some other photoreceptor is necessary to produce the carotenoid pattern characteristic
of chloroplast. CLAES (1967) has determined the action spectrum for carotene syn-
thesis in a mutant of Chlorella vulgaris and concluded that the photoreceptor is most
likely chlorophyll. The synthesis of other lamellar lipids is also influenced by light.
High light intensities resulted in a parallel decrease in chlorophyll and the galactosyl
glycerides, but the percentage of polyunsaturated fatty acids in the chloroplasts rose
sharply (CONSTANTOPOULOS and BLOCH, 1967) and the possibility that continuous
light is required for the synthesis of some essential structural lipids of the lamellae
cannot be discounted.
So far it has not been possible to point to a single protein in angiosperm plastids
requiring continuous light for its synthesis. The synthesis of all soluble proteins we
have examined, as well as at least one particulate electron transfer protein, are photo-
regulated by the phytochrome system. More information on photoregulation of the
synthesis of lamellar proteins is obviously required and the plant which has been
grown in the dark exept for brief exposures to red light should provide a good
starting point for such experiments.
VII. General Conclusions

The original discoveries made by genetic analysis that non-Mendelian genes are
involved in the formation of certain organelles have been borne out with the identi-
fication of organelle DNA. Chloroplasts and mitochondria both contain DNA as do
the mitochondria-like kinetoplasts and basal bodies found in certain protozoa (GUTT-
MAN and EISENMAN, 1965; RANDALL and DISBREY, 1965) and the function of non-
chromosomal DNA in the formation of still other cellular structures seems probable,
since chloroplasts and mitochondria do not account for all of the cytoplasmic DNA
found in plant cells (HOTTA et al., 1965; FISHER and JENSEN, 1967). Whether non-
chromosomal DNA is associated only with highly developed organelles enveloped by
double membranes or whether it is also associated with other less complex lamellar
systems such as the Golgi apparatus, peroxisomes, tonoplast and plasmalemma is not
known, but answers to questions such as this and as to the relative roles of nuclear
and organelle DNA in the biosynthesis of various organelles will be required before
basic mechanisms of cell differentiation can be fully understood.
The amount of information contained in organelle DNA and its contribution to
organelle synthesis (compared with that of nuclear DNA) might be expected to be
related to the complexity of the organelle itself. If this is the case, the chloroplast,
which is perhaps the most highly organized and complex organelle found in cells,
would afford an excellent opportunity for studying the cellular function of organelle
DNA. Chloroplasts contain about the same amount of DNA as a bacterium. The
mitochondrion contains considerably less and we would expect the lamellar organel-
les mentioned above to have even less, assuming they have any at all.
What then is the function of chloroplast DNA ? The chloroplasts contain a protein-
synthesizing system and some of the proteins synthesized by this system have been
identified. Our DNA-RNA hybridization experiments (SCOrI' and SMILLIE, 1967) and
those of TEWARI and WILDMAN (1968) suggest that the ribosomal RNA of this
system is coded by chloroplast DNA. The protein components of the chloroplast
ribosome differ from those of the 80S cytoplasmic ribosome and these perhaps are
also coded by chloroplast DNA, but there is no direct evidence for this. The postu-
lated synthesis of chloroplast ribosomal RNA directed by chloroplast DNA is
supported circumstantially by autoradiographic evidence of RNA synthesis at DNA-
containing sites in chloroplasts (GIBBS, 1967) and by evidence of DNA-dependent
RNA synthesis by isolated chloroplasts (Section IV.C.). It would not be surprising if
the RNA polymerase involved is also coded by chloroplast DNA. Mitochondrial
DNA also may have a similar function to chloroplast DNA in coding for organelle
ribosomes. Hybridization experiments indicate this (FUKUHARA, 1967; SUYAMA, 1967),
and LINNANE et al. (1968) have shown that a cytoplasmic determinant (mitochondrial
DNA?) is involved in resistance of yeast to erythromycin, which in turn is associated
with an alteration in the mitochondrial ribosome.
Chloroplasts possibly possess unique transfer ribonucleic acids (DYER and LEECH,
1968) and Neurospora mitochondria contain transfer ribonucleic acids (BARNErI' and
BROWN, 1967) and amino acyl-RNA synthetases (BARNErI' et al., 1967) which differ
from the cytoplasmic ribonucleic acids and enzymes. If transfer ribonucleic acids
unique to chloroplasts are identified and purified, it should be possible to determine
if they, like chloroplast ribosomal RNA, are coded by chloroplast DNA.
One of the most important problems for future studies on organelle biosynthesis
will be to establish the origin of the messenger RNA for individual chloroplast
proteins. The synthesis of a polypeptide chain of a chloroplast protein on 70S chloro-
plast ribosomes does not necessarily mean that the messenger RNA involved originated
from chloroplast DNA and, in fact, genetic evidence points to the nucleus containing
most of the structural genes for chloroplast proteins. Examples of nuclear mutations
resulting in mutants with blocks in a single step in the synthesis of some chloroplast
component, or even mutants deficient in a single chloroplast protein (e.g. see GORMAN
and LEVINE, 1966), are sufficiently common for KIRK (1966) to have concluded that
most of the structural genes for chloroplast proteins are located in the nucleus. He
suggested that the genetic information in the chloroplast may be mainly regulatory
genes.
Structural genes for several mitochondrial enzymes are located in the nucleus.
Convincing evidence for the coding of the primary structure of yeast iso-1-cyto-
chrome c by a chromosomal gene has been provided by SHERMAN et al. (1966) and
mitochondrial malate dehydrogenase (MUNKRES and RICHARDS, 1965; DAVIDSON
and CORTNER, 1967) and aconitate dehydratase (OGUR et al., 1964) similarly appear to
be coded by nuclear genes.
WOODWARD and MUNKRES (1966), on the other hand, have concluded that
structural protein in mitochondria is coded by mitochondrial DNA and we have
postulated an analogous role for the DNA of chloroplasts (SMILLIE et aI., 1968).
Thus chloroplast DNA appears to be directly involved in the formation of a 70S

ribosomal system for protein synthesis in chloroplasts. It probably also direcdy codes
for some chloroplast proteins, e.g. lamellar structural protein, but other proteins are
probably coded by the nuclear DNA. The assignation of the respective coding
functions of nuclear and chloroplast DNA towards individual chloroplast proteins
may prove to be complicated by the possible retention of copies of chloroplast DNA
in the nucleus (see Section III.D.2).
Inhibition of the synthesis of Calvin cycle enzymes, Fraction I protein, nitrite
reductase and electron transfer proteins in greening cells by chloramphenicol, but not
by cycloheximide, suggests that these proteins are synthesized within the chloroplasts.
However, the role of the 70S plastid ribosomes in proplastid synthesis and also in
synthesis of the outer membrane and membrane proteins of the chloroplast lamellae
is obscure and other protein-synthesizing systems, possibly cytoplasmic, may be
involved (see SMILLIE et al., 1968). While many, if not most, of the enzymes respon-
sible for synthesis of the lipid components of chloroplasts are localized in the chloro-
plasts, we again know very little about their formation. Future studies no doubt will
be directed towards ascertaining the stage of chloroplast differentiation when these
enzymes first appear-are they for instance synthesized at about the same time as
chlorophyll and the enzymes which function in CO2 fixation, or are they synthesized,
as is the case with plastid ribosomal RNA, during the lag phase of the synthesis of
chlorophyll and the other proteins? In spite of these uncertainties, the chloroplast
seems capable of synthesizing a large number of organelle proteins, and in this
respect may be contrasted with the mitochondrion which synthesizes a relatively
small number of its own proteins. While some proteins of the inner mitochondrial
membranes, including structural proteins and cytochrome oxidase, are probably
synthesized within the mitochondrion, many mitochondrial proteins including
cytochrome c, catalase, succinate dehydrogenase and soluble enzymes of the tri-
carboxylic acid cycle appear to be synthesized outside on the endoplasmic reticulum.
From these observations we have concluded (SMILLIE et al., 1967, 1968) that one
major difference between the developing chloroplast and mitochondrion is the
capacity of the chloroplast to synthesize a much greater number of its own proteins.
Acknowledgements
The assistance of Miss S. FINCH, Dr. B. D. PATTERSON and Dr. K. J. SCOTT in the
preparation of this article is gratefully acknowledged.
* (see p. 154). The majority of studies on chloroplast development in unicellular organisms

have been carried out with dark-adapted cells of Euglena gracilis or glucose-bleached cells of
Chlorella protothecoidcs. Heterotrophic growth ofEuglena in the dark completely represses chlo-
rophyll synthesis and chloroplast development can be studied in nondividing cells by trans-
ferring dark-grown cells to a medium lacking carbon growth substrates and subsequently
illuminating the culture. If, on the other hand, carbon sources other than CO 2 are added at the
time the cells are illuminated, chloroplast development and cell division can be studied simul-
taneously. In Chlorella, growth in darkness only partially represses chlorophyll synthesis,
but if glucose is added to the medium the repression is complete. Chloroplast development
can then be studied by illuminating these cells in a medium containing a nitrogen source,
but not glucose.
References
AARONSON, S., B. B. ELLENBOGEN, L. K. YELLEN, and S. H. HUTNER: In IIillo differentiation
of Euglena cytoplasmic and chloroplast protein synthesis with chloramphenicol and D!.-
ethionine. Biochem. biophys. Res. Commun. 27, 535 (1967).
AKOYUNOGLOU, G. A., and H. W. SIEGELMAN: Protochlorophyllide resynthesis in dark-
grown bean leaves. Plant Physiol. 43, 66 (1968).
ALLEN, C. F., O. HIRAYAMA, and P. GOOD: Lipid composition of photosynthetic systems.
In: Biochemistry of chloroplasts, Vol. 1, p. 195. T. W. GOODWIN, Ed. London and New
ANDERSON, J. M., and N. K. BOARDMAN: Fractionation of the photochemical systems of
photosynthesis. 1. Chlorophyll contents and photochemical activities of particles iso-
lated from spinach chloroplasts. Biochim. biophys. Acta (Arnst.) 112, 403 (1966).
ANDERSON, L. A., and R. M. SMILLIE: Binding of chloramphenicol by ribosomes from
chloroplasts. Biochem. biophys. Res. Commun. 23, 535 (1966).
AOKI, S., and E. HASE: (1) De- and re-generation of chloroplasts in the cells of Chlorella
protothecoides. 1. Synthesis of nucleic acids and protein in relation to the process of re-
generation of chloroplast. Plant and Cell Physiol. 5, 473 (1964).
- - (2) De- and re-generation of chloroplasts in the cells of Clorella protothecoides. II.
Effects of actinomycin on greening of "glucose-bleached" and "etiolated" algal cells.
Plant and Cell Physiol. 5, 485 (1964).
- - De- and re-generation of chloroplasts in the cells of Chlorella protothecoides. ill. Effects
of mitomycin C on the processes of greening and division of "glucose-bleached" algal
cells. Plant and Cell Physiol. 6, 347 (1965).
- - Ribonucleic acids appearing during the process of chloroplast regeneration in the
"glucose-bleached" cells of Chlorella protothecoides. Plant and Cell Physiol. 8, 181 (1967).
-, J. K. MATSUBARA, and E. HASE. De- and re-generation of chloroplasts in the cells of
Chlorella protolhecoides. IV. Effects of 5-fluorouracil, dihydrostreptomycin, chlorampheni-
col and acridine orange on the processes of greening and division of "glucose-bleached"
algal cells. Plant and Cell Physiol. 6,475 (1965).
APp, A. A., and A. T. JAGENDORF: Repression of chloroplast development in Euglena
gracilis by substrates. J. Protozool. 10, 340 (1963).
- - 14C-amino acid incorporation by spinach chloroplast preparations. Plant Physiol.
39, 772 (1964).
ARONSON, A. 1.: The effect of 5-fluorouracil on bacterial protein and ribonucleic acid
synthesis. Biochim. biophys. Acta (Arnst.) 49, 98 (1961).
ARNON, D. 1., H. Y. TSU]IMOTO, B. D. MCSWAIN, and R. K. CHAIN: Separation of two
photochemical systems of photosynthesis by fractionation of chloroplasts. In: Compara-
tive biochemistry and biophysics of photosynthesis, p. 113. SHIBATA, K., A. TAKAMIYA,
A. T. JAGENDORF, and R. C. FULLER Eds. Tokyo: University of Tokyo Press 1968.
BALTUS, E., and J. BRACHET: Presence of deoxyribonucleic acid in the chloroplasts of
Acelabu/aria medile"anea. Biochim. biophys. Acta (Amst.) 76, 490 (1963).
- , and J. QUERTIER. A method for the extraction and characterization of RNA from sub-
cellular fractions of Acetabularia. Biochim. biophys. Acta (Arnst.) 119, 192 (1966).
BAM]I, M. S., and A. T. JAGENDORF. Amino acid incorporation by wheat chloroplasts.
Plant Physiol. 41, 764 (1966).
BARNETr, W. E., and D. H. BROWN: Mitochondrial transfer ribonucleic acids. Proc. nat.
Acad. Sci. (Wash.) 57, 452 (1967).
- - , and J. L. EPLER: Mitochondrial-specific aminoacyl-RNA synthetases. Proc. nat.
Acad. Sci. (Wash.) 57, 1775 (1967).
BARTELS, P. G., K. MATSUDA, A. Siegel, and T. E. WEIER: Chloroplastic ribosome forma-
tion: Inhibition by 3-amino-l, 2, 4-triazole. Plant Physiol. 42, 736 (1967).
BASSHAM, J. A., and M. KIRK: Dynamic metabolic regulation of the photosynthetic carbon
reduction cycle. In: Comparative biochemistry and biophysics of photosynthesis, p. 365.
SHIBATA, K., A. TAKAMIYA, A. T. JAGENDORF, and R. C. FULLER, Eds. Tokyo: Uni-
versity of Tokyo Press 1968.
BEATTIE, D. S., R. E. BASFORD, and S. B. KORITZ: Studies on the biosynthesis of mito-

chondrial protein components. Biochemistry 5, 926 (1966).
- - - The inner membrane as the site of the in vitro incorporation of L-[14C] leucine into
mitochondrial protein. Biochemistry 6, 3099 (1967).
BEEVERS, L., L. E. SCHRADER, D. FLESHER, and R. H. HAGEMAN: The role of light and
nitrate in the induction of nitrate reductase in radish cotyledons and maize seedlings.
BENNUN, A., and M. AVRON: Light-dependent and light-triggered adenosine triphosphatase
in chloroplasts. Biochim. biophys. Acta (Arnst.) 79, 646 (1964).
BERGER, S.: RNA-synthesis in Ace/abularia. II. RNA-synthesis in isolated chloroplasts.
Protoplasma (Wien) 64, 13 (1967).
BERIDZE, T. G., M. S. ODINTSOVA, and N. M. SISAKYAN: Distribution of DNA components
of bean leaves in all fractions. Mol BioI. 1, 142 (1967); Chern. Abstr. 67, 79652 d (1967).
BIGGINS, J., and R. B. PARK: Nucleic acid content of chloroplasts of spinach isolated by a
non-aqueous techniques. Nature (Lond.) 203, 425 (1964).
BIRD, I. F., H. K. PORTER, and C. R. STOCKING: Intracellular localisation of enzymes asso-
ciated with sucrose synthesis in leaves. Biochim. biophys. Acta (Arnst.) 100, 366 (1965).
BISALPUTRA, T., and A. A. BISALPUTRA: (1) The occurrence of DNA fibrils in chloroplasts of
Laurencia spectabilis. J. Ultrastruct. Res. 17, 14 (1967).
- - (2) Chloroplast and mitochondrial DNA in a brown alga Egregia menziesii. J. Cell
BioI. 33, 511 (1967).
BOARDMAN, N. K.: (1) Ribosome composition and chloroplast development in Phaseolus
vulgaris. Exp. Cell Res. 43, 474 (1966).
- (2) Protochlorophyll. In: The Chlorophylls, p. 437. VERNON, L. P., and G. R. SEELY,
Eds. New York and London: Academic Press 1966.
- Chloroplast structure and development. In: Harvesting the sun: Photosynthesis in plant
life, p. 211. SAN PIETRO, A., F. A. GREER, and T. J. ARMY, Eds. New York and London:
- , and J. M. ANDERSON: Fractionation of the photochemical systems of photosynthesis.
II. Cytochrome and carotenoid contents of particles isolated from spinach chloroplasts.
Biochim. Acta (Arnst.) 143, 187 (1967).
- , R. I. B. FRANCKI, and S. G. WILDMAN: Protein synthesis by cell-free extracts from
tobacco leaves. II. Association of activity with chloroplast ribosomes. Biochemistry 4,
872 (1965).
- - - Protein synthesis by cell-free extracts of tobacco leaves. III. Comparison of the
physical properties and protein synthesizing activities of 70S chloroplast and 80S cyto-
plasmic ribosomes. J. molec. BioI. 17, 470 (1966).
BOGORAD, L.: (1) Aspects of chloroplast assembly. In: Harvesting the sun: Photosynthesis
in plant life, p. 191. SAN PIETRO, A., F. A. GREER, and T. J. ARMY, Eds. New York and
London: Academic Press 1967.
- (2) Biosynthesis and morphogenesis in plastids. In: Biochemistry of chloroplasts, Vol. II,
p. 615. T. W. GOODWIN, Ed. London and New York: Academic Press 1967.
- , and A. B. JACOBSON: Inhibition of greening of etiolated leaves by actinomycin D. Bio-
chern. biophys. Res. Commun. 14, 113 (1964).
-, F. V. MERCER, and R. MULLENS: Studies with Cyanidium caldarium. II. The fine structure
of pigment-deficient mutants. In: Photosynthetic mechanisms of green plants, Misc Publ.
1145, p. 560. Nat Acad. Sci.-Nat. Res. span Council ,Washington 1963.
BovE, J. M., C. BovE, M.-J. RONDOT, and G. MOREL: Chloroplasts and virus-RNA synthesis.
In: Biochemistry of chloroplasts, Vol. II, p. 329. T. W. GOODWIN, Ed. London and
New York: Academic Press 1967.
BovE, J., and I. D. RAACKE: Amino acid-activating enzymes in isolated chloroplasts from
spinach leaves. Arch. Biochem. 85, 521 (1959).
BRAWERMAN, G.: The isolation of a specific species of ribosomes associated with chloroplast
development in Euglena gracilis. Biochim. biophys. Acta (Arnst.) 72, 317 (1963).
- Nucleic acids associated with the chloroplasts of Euglena gracilis. In: Biochemistry of
chloroplasts, Vol. I, p. 301. T. W. GOODWIN, Ed. London and New York: Academic Press
1966.
- , and E. CHARGAFF: Changes in protein and ribonucleic acid during the formation of
chloroplasts in Euglena gracilis. Biochim. biophys. Acta (Amst.) 31, 164 (1959).
- , and J. M. EISENSTADT: Deoxyribonucleic acid from the chloroplasts of Euglena gracilis.
Biochim. biophys. Acta (Arnst.) 91, 477 (1964).
- , A. o. POGO, and E. CHARGAFF: Induced formation of ribonucleic acids and plastid
protein in Euglena gracilis under the influence of light. Biochim. biophys. Acta (Amst.)
55, 326 (1962).
BRIANTAIS, J. -M.: Isolement et activite de particules chloroplastiques correspondant aux
deux systemes photochimiques. Photochem. Photobiol. 5, 135 (1966).
BRIGGs, G. E.: Experimental researches on vegetable assimilation and respiration. XIII. The
development of photosynthetic activity during germination. Proc. roy. Soc. B 91,249
(1920).
BRIGGS, W. R., and H. W. SIEGELMAN: Distribution of phytochrome in etiolated seedlings.
BROWN, F. A. M., and B. E. S. GUNNING: Distribution of ribosome-like particles in Avena
plastids. In: Biochemistry of chloroplasts, Vol. I, p. 365. T. W. GOODWIN, Ed. London
and New York: Academic Press 1966.
VAN BRUGGEN, E. F. J., P. BORST, G. J. C. M. RUTTENBERG, M. GRUBER, and A. M. KROON:
Circular mitochondrial DNA. Biochim. biophys. Acta (Arnst.) 119,437 (1966).
BUCHANAN, B. B., P. P. KALBERER, and D. I. ARNON: Ferredoxin-activated fructose di-
phosphatase in isolated chloroplasts. Biochem. biophys. Res. Commun. 29, 74 (1967).
BUTLER, W. L.: Development of photosynthetic systems 1 and 2 in a greening leaf. Biochim.
biophys. Acta (Amst.) 102, 1 (1965).
- , and W. R. BRIGGS: The relation between structure and pigments during the first stages
of proplastid greening. Biochim. biophys. Acta (Amst.) 112, 45 (1966).
-, S. B. HENDRICKS, and H. W. SIEGELMAN: Action spectra of phytochrome in vitro. Photo-
chem. Photobiol. 3, 521 (1964).
-, H. C. LANE, and H. W. SIEGELMAN: Nonphotochemical transformations of phyto-
chrome in vivo. Plant Physiol. 38, 514 (1963).
CARELL, E. F., and J. S. KAHN: Synthesis of porphyrins by isolated chloroplasts of Euglena.
Arch. Biochem. 108, 1 (1964).
CHEN, J. L., and S. G. WILDMAN: Functional chloroplast polyribosomes from tobacco
leaves. Science 155, 1271 (1967).
CHEN, S., D. McMAHON, and L. BOGORAD: Early effects of illumination on the activity of
some photosynthetic enzymes. Plant Physiol. 42, 1 (1967).
CHIANG, K. S., and N. SUEOKA: Replication of chloroplast DNA in Chlam.ydomonas reinhardi
during vegetative cell cycle: Its mode and regulation. Proc. nat. Acad. Sci. (Wash.) 57,
1506 (1967).
CHIBA, Y., and K. SUGAHARA: The nucleic acid content of chloroplasts isolated from spinach
and tobacco leaves. Arch. Biochem. 71, 367 (1957).
CHUN, E. H. L., M. H. VAUGHAN JR., and A. RICH: The isolation and characterization of
DNA associated with chloroplast preparations. J. molec. BioI. 7, 130 (1963).
CLAES, H.: Action spectrum of light-dependent carotenoid synthesis in Chlorella vulgaris. In:
Biochemistry of chloroplasts, Vol. II, p. 441. T. W. GOODWIN, Ed. London and New
CLARK J. M., JR.: Amino acid activation in plant tissues. J. bioI. Chem. 233, 421 (1958).
CLARK, M. F., R. E. F. MATTHEWS, and R. K. RALPH: Ribosomes and polyribosomes in
Bra.r.rica pekinensis. Biochim. biophys. Acta (Amst.) 91, 289 (1964).
CLARK-WALKER, G. D., and A. W. LINNANE: The biogenesis of mitochondria in Saccharo-
myces cerevi.riae. A comparison between cytoplasmic respiratory-deficient mutant yeast
and chloramphenicol-inhibited wild type cells. J. Cell BioI. 34, 1 (1967).
CLICK, R. E., and D. P. HACKETT: Evidence that the two ribosomal RNA species of plant
tissue are synthesized on different genetic loci. J. molec. BioI. 17, 279 (1966).
-, and B. L. TINT: Comparative sedimentation rates of plant, bacterial and animal ribo-
somal RNA. J. molec. BioI. 25, 111 (1967).
CoHEN, R. Z., and T. W. GOODWIN: The effect of red and far-red light on carotenoid syn-
thesis by etiolated maize seedlings. Phytochem. 1, 67 (1962).
190 ROBERT M. SMILLIE and N. STEELE SCOTI
CONSTANTOPOULOS, G., and K. BLOCH: Effect of light intensity on the lipid composition of
Euglena gracilis. ]. bio!. Chern. 242, 3538 (1967).
COOK, J. R.: The synthesis of cytoplasmic DNA in synchronized Euglena. ]. Cell BioI. 29,
369 (1966).
- , and W. HUNT: Ultraviolet bleaching of synchronized Euglena. Photochem. Photobiol. 4,
877 (1965).
CRIDDLE, R. S.: Protein and lipoprotein organization in the chloroplast. In: Biochemistry of
chloroplasts, Vol. I, p. 203. T. W. GOODWIN, Ed. London and New York: Academic
Press 1966.
CZYGAN, F.-C.: Untersuchungen tiber die Wirkung von Chloramphenicol auf den Auf- und
Abbau der Pigmente einiger Grunalgen. Arch. Mikrobiol. 47, 251 (1964).
DAS, H. K., S. K. CHATIERJEE, and S. C. Roy: Protein synthesis in plant mitochondria.
II. Glutamate and glutamine incorporation and a study of initial steps and streptomycin
effect. Biochim biophys. Acta (Arnst.) 87, 478 (1964).
DAVENPORT, H. E., and R. HILL: The preparation and some properties of cytochrome f.
Proc. roy. Soc. B 139, 327 (1952).
DAVIDSON, R. G., and]. A. CORTNER: Mitochondrial malate dehydrogenase: A new genetic
polymorphism in man. Science 157, 1569 (1967).
DAVIES, W. H., E. I. MERCER, and T. W. GOODWIN: Some observations on the biosynthesis
of the plant sulpholipid by Euglena gracilis. Biochim. ]. 98, 369 (1966).
DENHARDT, D. T.: A membrane-filter technique for the detection of complementary DNA.
Biochem. biophys. Res. Commun. 23, 641 (1966).
DUNCAN, M. ]., and P. R. STEWART: Lipids, protein synthesis and the induction of respira-
tory enzymes in Saccharomyces cerevisiae. Proc. Aust. Biochem. Soc. p. 94 (1968).
DURANTON, ].: Proteogenese et formation des chloroplastes au cours du verdissement de
Zea mays L. In: Currents in photosynthesis: Proceedings of the second Western-Europe
Conference on photosynthesis, p. 321. THOMAS, ]. B., and]. C. GOODHEER, Eds. Rotter-
dam: A. Donker 1966.
DWYER, M. R.: The development of chloroplasts in Euglena. Ph. D. Thesis, University of
Sydney 1968.
DYER, T. A., and R. M. LEECH: Chloroplast and cytoplasmic low-molecular-weight ribo-
nucleic acid components of the leaf of Vicia faba L. Biochem. ]. 106, 689 (1968).
ECHLIN, P., and I. MORRIS: The relationship between blue-green algae and bacteria. BioI.
Rev. 40, 143 (1965).
EDELMAN, M., C. A. COWAN, H. T. EpSTEIN, and]. A. SCHIFF: Studies of chloroplast deve-
lopment in Euglena. VIII. Chloroplast-associated DNA. Proc. nat. Acad. Sci. (Wash.) 52,
1214 (1964).
- , ]. A. SCHIFF, and H. T. EpSTEIN: Studies of chloroplast development in Euglena. XII.
Two types of satellite DNA. ]. molec. BioI. 11, 769 (1965).
- , H. T. EpSTEIN, and]. A. SCHIFF: Isolation and characterization of DNA from the mito-
chondrial fraction of Euglena. ]. molec. Bio!. 17,463 (1966).
- , D. SWINTON,]. A. SCHIFF, H. T. EpSTEIN, and B. ZELDIN: Deoxyribonucleic acid of the
blue-green algae (Cyanophyta). Bact. Rev. 31, 315 (1967).
EISENSTADT, ]. M.: Protein synthesis in chloroplasts and chloroplast ribosomes. In: Bioche-
mistry of chloroplasts, Vol. II, p. 341. T. W. GOODWIN, Ed. London and New York:
- , and G. BRA WERMAN: The incorporation of amino acids into the protein of chloroplasts
and chloroplast ribosomes of Euglena gracilis. Biochim. biophys. Acta (Arnst.) 76, 319
(1963).
- - The protein-synthesizing systems from the cytoplasm and the chloroplasts of Euglena
gracilis. ]. molec. BioI. 10,392 (1964).
ENNIS, H. L., and M. LUBIN: Cycloheximide: Aspects of inhibition of protein synthesis in
mammalian cells. Science 146, 1474 (1964).
EpSTEIN, H. T., and]. A. SCHIFF: Studies of chloroplast development in Euglena. 4. Electron
and fluorescence microscopy of the prop las tid and its development into a mature
chloroplast. ]. Protozool. 8,427 (1961).
EVANS, W. R., and R. M. SMILLIE: The role of nucleic acids in the formation of chloroplasts.
Plant Physiol. 37, (Suppl.). XXXVIII (1962).
- , R. WALENGA, and C. JOHNSON: Effect of cycloheximide on chloroplast development in
Euglena. Fourth Annual Research Report of the Charles F. Kettering Research Labora-
tory, p. 99. Yellow Spring (Ohio): The Ampersand Press 1967.
EVENARI, M.: In: Recent progress in photobiology, p. 161. E. J. BOWEN, Ed. Oxford:
Blackwell Scientific Publ. 1965.
EVERSON, R. G., and C. R. SLACK: Distribution of carbonic anhydrase in relation to the C 4
pathway of photosynthesis. Phytochem. 7, 581 (1968).
FEIERABEND, J.: Enzymbildung in Roggenkeimlingen wahrend der Umstellung von hetero-
trophem auf autotrophes Wachstum. Planta 71, 326 (1966).
- , and A. PIRSON: Die Wirkung des Lichtes auf die Bildung von Photosynthese-Enzymen
in Roggenkeimlingen. Z. Pflanzenphysiol. 55. 235 (1966).
FISHER, D. B., and W. A. JENSEN: A radioautographic study of nuclear and cytoplasmic
DNA synthesis by cotton embryos. J. Cell BioI. 35, 40A, Abstract 78 (1967).
FRANCKI, R. 1. B., N. K. BOARDMAN, and S. G. WILDMAN: Protein synthesis by cell-free
extracts from tobacco leaves. 1. Amino acid incorporating activity of chloroplasts in
relation to their structure. Biochemistry 4, 865 (1965).
FREy-WYSSLING, A: The organelles of the cell, their comparative ultrastructure and physio-
logy. Aust. J. Sci. 29, 458 (1967).
FRIEND, J., and A. M. MAYER: The enzymic destruction of carotenoids by isolated chloro-
plasts. Biochim. biophys. Acta (Amst.) 41, 422 (1960).
FUKUHARA, H.: Informational role of mitochondrial DNA studied by hybridization with
different classes of RNA in yeast. Proc. nat. Acad. Sci. (Wash.) 58, 1065 (1967).
FURUYA, M., and W. S. HILLMAN: Observations on spectrophotometrically assayable phyto-
chrome in vivo in etiolated Pisum seedlings. Planta 63,31 (1964).
GAILEY, F. B., and N. E. TOLBERT: Effect of ionizing radiation on the development of
photosynthesis in etiolated wheat leaves. Arch. Biochem. 76, 188 (1958).
GALE, E. F.: Mechanism of antibiotic action. Pharmacol. Rev. 15, 481 (1963).
GALLOWAY, R. A., and R. W. KRAUSS: The differential action of chemical agents, especially
polymyxin-B, on certain algae, bacteria and fungi. Amer. J. Botany 46, 40 (1959).
GASSMAN, M., and L. BOGORAD: (1) Control of chlorophyll production in rapidly greening
bean leaves. Plant Physiol. 42, 774 (1967).
- - (2) Studies on the regeneration of protochlorophyllide after brief illumination of
etiolated bean leaves. Plant Physio!. 42, 781 (1967).
GIBBS, S. P.: Chloroplast development in Ochromonas danica. J. Cell BioI. 15, 343 (1962).
- Synthesis of chloroplast RNA at the site of chloroplast DNA. Biochem. biophys. Res.
Commun. 28, 653 (1967).
GIBOR, A.: DNA synthesis in chloroplasts. In: Biochemistry of chloroplasts, Vol. II, p. 321.
T. W. GOODWIN, Ed. London and New York: Academic Press 1967.
- , and S. GRANICK: Ultraviolet sensitive factors in the cytoplasm that affect the differentia-
tion of Euglena plastids. J. Cell Bio!. 15, 599 (1962).
- , and M. IZAWA: The DNA content of the chloroplasts of Acetabularia. Proc. nat. Acad.
Sci. (Wash.) 50, 1164 (1963).
GILLESPIE, D., and S. SPIEGELMAN: A quantitative assay for DNA-RNA hybrids with DNA
immobilized on a membrane. J. molec. BioI. 12,829 (1965).
GNANAM, A., and J. S. KAHN: Biochemical studies on the induction of chloroplast develop-
ment in Euglena gracilis. III. Ribosome metabolism associated with chloroplast develop-
ment. Biochim. biophys. Acta (Amst.) 142,493 (1967).
GOFFEAU, A., and J. BRACHET: Deoxyribonucleic acid-dependent incorporation of amino
acids into the proteins of chloroplasts isolated from anucleate Acetabu/aria fragments.
Biochim. biophys. Acta (Amst.) 95, 302 (1965).
GOODCHILD, D. J., H. R. HIGH KIN, and N. K. BOARDMAN: The fine structure of chloro-
plasts in a barley mutant lacking chlorophyll b. Exp. Cell Res. 43, 684 (1966).
GOREN, R., and A. W. GALSTON: Control by phytochrome ofl4C-sucrose incorporation into
buds of etiolated pea seedlings. Plant Physio!. 41, 1055 (1966).
192 ROBERT M. SMILLIE and N. STEELE SCO'IT
- - Phytochrome controlled 14C-sucrose uptake into etiolated pea buds: Effects of

gibberellic acid and other substances. Plant Physiol. 42, 1087 (1967).
GORMAN, D. S., and R. P. LEVINE: The photosynthetic electron transfer chain of Chlamy-
domonas reinhardi. VI. Electron transport in mutant strains lacking either cytochrome 553
or plastocyanin. Plant Physiol. 41, 1648 (1966).
GRAEBE, J. E., and G. D. NOVELLI: Amino acid incorporation in a cell-free system from
submerged tissue cultures of Zea mays L. Exp. Cell Res. 41, 521 (1966).
GRAHAM, D., A. M. GRIEVE, and R. M. SMILLIE: (1) Phytochrome as the primary photo-
regulator of the synthesis of Calvin cycle enzymes in etiolated pea seedlings. Nature
(Lond.) 218, 89 (1968).
- -, M. D. HATCH, C. R. SLACK, and R. M. SMILLIE: (2) Phytochrome as a primary photo-
regulator in the synthesis of enzymes of photosynthetic carbon dioxide fixation in higher
plants. Proceedings of International Congress of photobiology. Hanover, New Hamp-
shire, 1968.
GRANICK, S.: Plastid structure, development and inheritance. In: Encyclopaedia of plant
physiology, Vol. I, p. 507. W. RUHLAND, Ed. Berlin-Gottingen-Heidelberg: Springer
1955.
- Magnesium porphyrins formed by barley seedling treated with «5-amino levulinic acid.
Plant Physiol. 34, Suppl. XVIII (1959).
- The chloroplasts: Inheritance, structure and function. In: The Cell, Vol. II, p. 489.
BRACHET, J., and A. E. MIRSKY, Eds. New York and London: Academic Press 1961.
- Induction of the synthesis of «5-amino levulinic acid in liver parenchyma cells in culture
by chemicals that induce acute porphyria. Proc. 6th Int. Congo Biochem. (New York).
Oxford: Vol. VI. Pergamon Press Ltd. 1964.
- The heme and chlorophyll biosynthetic chain. In: Biochemistry of chloroplasts, Vol. II,
GREEN, B. R., and M. P. GORDON: Satellite DNA's of tobacco. Fed. Proc. 24, 539 (1965).
- - Replication of chloroplast DNA of tobacco. Science 152, 1071 (1966).
- - The satellite DNA's of some higher plants. Biochim. biophys. Acta (Amst.) 145, 378
(1967).
GUNNING, B. E. S.: (1) The fine structure of chloroplast stroma following aldehyde osmium-
tetroxide fixation. J. Cell BioI. 24, 79 (1965).
- (2) The greening process in plastids. 1. The structure of the prolamellar body. Proto-
plasma (Wien) 60, 111 (1965).
GU'ITMAN, H. N., and R. N. EISENMAN: Acriflavin-induced loss of kinetoplast deoxyribo-
nucleic acid in Crithidia fasciculata (Culex pipiens strain). Nature (Lond.) 207, 1280
(1965).
HADZIYEV, D., S. L. MEHTA, and S. ZALIK: Studies on the ribonucleic acid from wheat
leaves and chloroplasts. Plant Physiol. 43, 229 (1968).
HALL, B. D., and S. SPIEGELMAN: Sequence complementarity of T2-DNA and T 2-specific
RNA. Proc. nat. Acad. Sci. (Wash.) 47, 137 (1961).
HALL, D.O., R. C. HUFFAKER, L. M. SHANNON, and A. WALLACE: Influence of light on
dark carboxylation reactions in etiolated barley leaves. Biochim. biophys. Acta (Amst.)
35, 540 (1959).
HALL, E. A., F. KAVANAGH, and I. N. ASHESHOV: Action of 45-antibacterial substances on
bacterial viruses. Antibiot. and Chemother. 1, 369 (1951).
HALL, T. c., and E. C. COCKING: (1) Amino acid incorporation into protein by aseptic cell-
free systems from tomato cotyledons and leaves. Biochim. biophys. Acta (Amst.) 123,
163 (1966).
- - (2) Studies on protein synthesis in tomato cotyledons and leaves. II. Intermediate
stages of protein synthesis. Plant Physiol. 7, 343 (1966).
HARRIS, R. c., and J. T. O. KIRK: Control of chloroplast formation in Euglena gracilis:
Antagonism between C and N sources. Biochem. J. 106, 34P (1968).
HATCH, M. D., and C. R. SLACK: Photosynthesis by sugar cane leaves. A new carboxylation
reaction and the pathway of sugar formation. Biochem. J. 101, 103 (1966).
- - The participation of phosphoenolypyruvate synthetase in photosynthetic CO2
fixation of tropical grasses. Arch. Biochem. 120, 224 (1967).
HATIORI, A., and I. UESUGI: Ferredoxin-dependent photoreduction of nitrate and nitrite

by subcellular preparations of Anabaena cylindrica. In: Comparative biochemistry and
biophysics of photosynthesis, p. 201. SHIBATA, K., A. TAKAMIYA, A. T. JAGENDORF, and
R. C. FULLER, Eds. Tokyo (Japan): University of Tokyo Press 1968.
HAUPT, W. Die Chloroplastendrehung bei Mougeotia. Planta 53, 484 (1959).
HAUSCHILD, A. H. W., C. D. NELSON, and G. KROTKOV: The effect of light quality on the
products of photosynthesis in Chlorella vulgaris. Canad. J. Botany 40, 179 (1962).
HEBER, U.: Vergleichende Untersuchungen an Chloroplasten, die durch Isolierungsopera-
tionen in nicht-wiiBrigem und in wiiBrigem Milieu erhalten wurden. II. Kritik der Rein-
heit und Fermentlokalisationen in Chloroplasten. Z. Naturforsch. 15 b, 100 (1960).
- Protein synthesis in cloroplasts during photosynthesis. Nature (Lond.) 195, 91 (1962).
- Ribonucleinsiiuren in den Chloroplasten der Blattzelle. Planta 59, 600 (1963).
- , N. G. PON, and M. HEBER: Localization of carboxydismutase and triosephosphate
dehydrogenases in chloroplasts. Plant Physiol. 38, 355 (1963).
HENDRICKS, S. B., and H. A. BORTHWICK: The function of phytochrome in regulation of
plant growth. Proc. nat. Acad. Sci. (Wash.) 58, 2125 (1967).
HENNINGER, M. D., C. GELARDI, and F. L. CRANE: Rubimedin particles on chloroplast
lamellae. Exp. Cell Res. 44, 655 (1966).
HENNINGSEN, K. W.: An action spectrum for vesicle dispersal in bean plastids. In: Biochem-
istry of chloroplasts, Vol. II, p. 453. T. W. GOODWIN, Ed. London and New York:
HENSHALL, J. D., and T. W. GOODWIN: (1) The effect of red and far red light on carotenoid
and chlorophyll formation in pea seedlings. Photochem. Photobiol. 3, 243 (1964).
- - (2) Amino acid-activating enzymes in germinating pea seedlings. Phytochem. 3, 677
(1964).
HEYWOOD, S. M., R. M. DOWBEN, and A. RICH: The identification of polyribosomes syn-
thesizing myosin. Proc. nat. Acad. Sci. (Wash.) 57, 1002 (1967).
HILL, R.: The cytochrome b component of chloroplasts. Nature (Lond.) 174, 501 (1954).
- , J. H. C. SMITH, and C. S. FRENCH: Carnegie Inst. Wash. Year Book 52, 153 (1953).
HOCK, B., and H. BEEVERS: Development and decline of the glyoxylate-cycle enzymes in
watermelon seedlings (Citrullus vulgaris SCHRAD). Z. Pflanzenphysiol. 55, 405 (1966).
HOLM-HANSEN, 0., K. NISHIDA, V. MOSES, and M. CALVIN: Effects of mineral salts on
short-term incorporation of carbon-dioxide in Chlorella. J. expo Botany 10, 109 (1959).
HOTIA, Y., A. BASSEL, and H. STERN: Nuclear DNA and cytoplasmic DNA from tissues of
higher plants. J. Cell BioI. 27, 451 (1965).
HSIAO, T. c.: Characteristics of ribosomes isolated from roots of Zea mays. Biochim. biophys.
Acta (Arnst.) 91, 598 (1964).
HUANG, M., D. R. BIGGS, C. D. CLARK-WALKER, and A. W. LINNANE: Chloramphenicol
inhibition of the formation of particulate mitochondrial enzymes of Saccharomyces cere-
visiae. Biochim. biophys. Acta (Arnst.) 114, 434 (1966).
HUDOCK, G. A., and R. P. LEVINE: Regulation of photosynthesis in Chlamydomonas reinhardi.
INGLE, J., K. W. JOY, and R. H. HAGEMAN: The regulation of activity of the enzymes
involved in the assimilation of nitrate by higher plants. Biochem. J. 100, 577 (1966).
I WAMURA, T.: Distribution of nucleic acids among subcellular fractions of Chlorella. Biochim.
- , and S. KUWASHIMA: DNA species in chloroplasts of Chlorella. In: Comparative bio-
chemistry and biophysics of photosynthesis, p. 354. SHIBATA, K., A. TAKAMIYA, A. T.
JAGENDORF, and R. C. FULLER, Eds. Tokyo: University of Tokyo Press 1968.
JONES, O. T. G.: Haem synthesis by isolated chloroplasts. Biochem. biophys. Res. Commun.
28, 671 (1967).
JOUSSAUME, M., and R. BOURDU: Conversion of carbon-14-labelled orotic acid into pyrimidine
nucleotides by chloroplasts. Nature (Lond.) 210, 1363 (1966).
KADENBACH, B.: Synthesis of mitochondrial proteins: Demonstration of a transfer of pro-
teins from microsomes into mitochondria. Biochim. biophys. Acta (Arnst.) 134,430 (1967).
VAN KAMMEN, A.: Characteristics of in vitro amino acid incorporation by the ribosomal
fraction from tobacco leaves. Arch. Biochem. 118, 517 (1967).

KATOH, S., 1. SUGA, 1. SHIRATORI, and A. TAKAMIYA: Distribution of pI as to cyanin in plants,

with special reference to its localization in chloroplasts. Arch. Biochem. 94, 136 (1961).
KA Y, J. E., and A. KORNER: Effect of cycloheximide on protein and ribonucleic acid syn-
thesis in culrured human lymphocytes. Biochem. ]. 100, 815 (1966).
KEISTER, D. L., A. T. ]AGENDORF, and A. SAN PIETRO: Development of bean-leaf trans-
hydrogenase in etiolated leaves. Biochim. biophys. Acta (Arnst.) 62, 332 (1962).
- , A. SAN PIETRO, and F. E. STOLZENBACH: Pyridine nucleotide transhydrogenase from
spinach. 1. Purification and properties. ]. bioI. Chern. 235, 2989 (1960).
KEMPNER, E. S., and]. H. MILLER: The molecular biology of Euglena gracilis. 1. Growth
conditions and cellular composition. Biochim. biophys. Acta (Arnst.) 104, 11 (1965).
KERRIDGE, D.: The effect of actidione and other antifungal agents on nucleic acid and
protein synthesis in Saccharomyces carlsbergensis. ]. gen. Microbiol. 19,497 (1958).
KIRK, ]. T. 0.: The deoxyribonucleic acid of broad bean chloroplasts. Biochim. biophys.
Acta (Arnst.) 76, 417 (1963).
(1) DNA-dependent RNA synthesis in chloroplast preparations. Biochem. biophys. Res.
Commun. 14,393 (1964).
(2) Studies on RNA synthesis in chloroplast preparations. Biochem. biophys. Res.
Commun. 16,233 (1964).
- Nature and function of chloroplast DNA. In: Biochemistry of chloroplasts, Vol. I, p. 319.
T. W. GOODWIN, Ed. London and New York: Academic Press 1966.
- Effect of methylation of cytosine residues on the buoyant density of DNA in caesium
chloride solutions. ]. molec. BioI. 28, 171 (1967).
- , and R. L. ALLEN: Dependence of chloroplast pigment synthesis on protein synthesis:
Effect of actidione. Biochem. biophys. Res. Commun. 21, 523 (1965).
- , and R. A. E. TILNEy-BASSETT: The plastids: their chemistry, structure, growth and
inheritance. London and San Francisco: W. H. Freeman and Co. 1967.
KISLEV, N., H. SWIFT, and L. BOGORAD: Nucleic acids of chloroplasts and mitochondria in
Swiss chard. ]. Cell BioI. 25, 327 (1965).
KLEIN, S., and J. NEUMAN: The greening of etiolated bean leaves and the development of
chloroplast fine structure in absence of photosynthesis. Plant and Cell Physiol. 7,115 (1966).
- , G. BRYAN, and L. BOGORAD: Early stages in the development of plastid fine structure in
red and far-red light. ]. Cell BioI. 22, 433 (1964).
KLEIN, W. H., L. PRICE, and K. MITRAKOS: Light stimulated starch degradation in plastids
and leaf morphogenesis. Photochem. Photobiol. 2, 233 (1963).
KOSKI, V. M., C. S. FRENCH, and ]. H. C. SMITH: Action spectrum for the transformation
of protochlorophyll to chlorophyll a in normal and albino com seedlings. Arch. Biochem.
31, 1 (1951).
KOWALLIK, W.: Die Proteinproduktion von Chlorella im Licht verschiedener Wellenlangen.
Planta 64, 191 (1965).
KROON, A. M.: Protein synthesis in heart mitochondria. 1. Amino acid incorporation into
the protein of isolated beef-heart mitochondria and a fraction derived from them by sonic
oscillation. Biochim. biophys. Acta (Arnst.) 72, 391 (1963).
KUNTZELL, H., and H. NOLL: Mitochondrial and cytoplasmic polysomes from Neurospora
crassa. Narure (Lond.) 215, 1340 (1967).
KUPKE, D. W.: Correlation of a soluble leaf protein with chlorophyll accumulation. ]. bioI.
Chern. 237, 3287 (1962).
LAMB, A. ]., G. D. CLARK-WALKER, and A. W. LINNANE: The biogenesis of mitochondria.
4. The in vitro differentiation of mitochondrial and cytoplasmic protein synthesizing
systems by antibiotics. Biochim. biophys. Acta (Arnst.) (1968) (in press).
LASCELLES, ].: Adaptation to form bacteriochlorophyll in Rhodopseudomonas spheroides:
Changes in activity of enzymes concerned with pyrrole synthesis. Biochem. ]. 72, 508
(1959).
LEECH, R. M.: Comparative biochemistry and comparative morphology of chloroplasts
isolated by different methods. In: Biochemistry of chloroplasts, Vol. I, p. 65. T. W.
GOODWIN, Ed. London and New York: Academic Press 1966.
LEFF, ]., M. MANDEL, H. T. EpSTEIN, and]. A. SCHIFF: DNA satellites from cells of green
and aplastidic algae. Biochem. biophys. Res. Commun. 13, 126 (1963).
LINNANE, A. W., and P. R. STEWART: The inhibition of chlorophyll formation in Euglena by

antibiotics which inhibit bacterial and mitochondrial protein synthesis. Biochem. biophys.
Res. Commun. 27, 511 (1967).
-, A. J. LAMB, C. CHRISTODOULOU, and B. B. LUKINS: Biogenesis of Mitochondria. 6. The
biochemical basis of the resistance of Saccharomyces cerevisiae toward antibiotics which
specifically inhibit mitochondrial protein synthesis. Proc. nat. Acad. Sci. (Wash.) 59,
1288 (1968).
LIVERMAN, J. L.: Control of growth and reproductive processes by red and far-red light.
Radiat. Res. Suppi. 2, 133 (1960).
- , M. P. JOHNSON, and L. STARR: Reversible photoreaction controlling expansion of
etiolated bean leaf disks. Science 121, 440 (1955).
LOCKHART, J. A.: Physiological studies on light sensitive stem growth. Planta 62, 97 (1964).
LOENING, U. E., and J. INGLE: Diversity of RNA components in green plant tissues.
Nature (Lond.) 215, 363 (1967).
LOSADA, M., J. M. RAMIREZ, A. PANEQUE, and F. F. DEL CAMPO: Light and dark reduction of
nitrate in a reconstituted chloroplast system. Biochim. biophys. Acta (Arnst.) 109, 86
(1965).
LUNDEGARDH, B.: Quantitative relations between chlorophyll and cytochromes in chloro-
plasts. Physioi. Plant. 15, 390 (1962).
LYMAN, H.: Specific inhibition of chloroplast replication in Euglena gracilis by nalidixic acid.
J. Cell BioI. 35, 726 (1967).
L YTTLETON, J. W.: Isolation of ribosomes from spinach chloroplasts. Exp. Cell Res. 26,
312 (1962).
- Protein constituents of plant ribosomes. Biochem. biophys. Acta (Arnst.) 154, 145 (1968).
MAGER, J.: Chloramphenicol and chlortetracycline inhibition of amino acid incorporation
into proteins in a cell-free system from Tetrahymena pyriformis. Biochim. biophys. Acta
(Arnst.) 38, 150 (1960).
MARCUS, A.: Amino acid dependent exchange between pyrophosphate and adenosine tri-
phosphate in spinach preparations. J. bioI. Chern. 234, 1238 (1959).
- Photocontrol of formation of red kidney bean leaf triphosphopyridine nucleotide linked
triosephosphate dehydrogenase. Plant Physioi. 35, 126 (1960).
MARGULIES, M.: Effect of choramphenicol on light-dependent development of seedlings of
Phaseolus vulgaris var. Black Valentine with particular reference to development of photo-
synthetic activity. Plant Physioi. 37, 473 (1962).
Effect of chloramphenicol on light-dependent synthesis of proteins and enzymes of
leaves and chloroplasts of Phaseolus vulgaris. Plant Physioi. 39, 579 (1964).
- Relationship between red light mediated glyceraldehyde-3-phosphate dehydrogenase
formation and light dependent development of photosynthesis. Plant Physioi. 40, 57
(1965).
- Effect of chloramphenicol on chlorophyll synthesis of bean leaves. Plant Physioi. 42, 218
(1967).
MATSUKA, M., and E. Base: The role of respiration and photosynthesis in the chloroplast
regeneration in the "glucose-bleached" cells of Chlorella protothecoides. Plant and Cell
Physioi. 7, 149 (1966).
Mc CALLA, D. R., and R. K. ALLAN: Effect of actinomycin D on Euglena chloroplast forma-
tion. Nature (Lond.) 201, 504 (1964).
MCCARTHY, B. J.: Arrangement of base sequences in deoxyribonucleic acid. Bact. Rev. 31,
215 (1967).
- , and E. T. BOLTON: An approach to the measurement of genetic relatedness among
organisms. Proc. nat. Acad. Sci. (Wash.) 50, 156 (1963).
- , and E. RACKER: Effect of a coupling factor and its antiserum on photophosphorylation
and hydrogen ion transport. In: Energy conversion by the photosynthetic apparatus.
Brookhaven Symp. BioI. 19,202 (1966).
MEGO, J. L.: The effect of hadacidin on chloroplast development in non-dividing Euglena
cells. Biochim. biophys. Acta (Arnst.) 79, 221 (1964).
- , and A. T. JAGENDORF: Effect of light on growth of Black Valentine bean plastids.
13*
MESELSON, M., and F. W. STAHL: The replication of DNA in E.fcherichia coli. Proc. nat.
Acad. Sci. (Wash.) 44, 671 (1958).
MIKULSKA, E. 1., M. S. ODINTSOVA, and N. M. SISAKYAN: Isolation and characteristics of
ribosomes from chloroplasts. Biokhimiya 27, 899 (1963).
MITRAKOS, K.: The participation of the red far-red reaction system in chlorophyll-meta-
bolism. Physio!. Plant. 14, 497 (1961).
MOHR, H. : Differential gene activation as a mode of action of phytochrome 730. Photochem.
Photobio!' 5, 469 (1966).
MOLOTKOVSKII, Y., and A. M. SMIRNOV: Action of chloramphenicol on protein synthesis in
plants. Fizio!' Rast. 10, 325 (1963).
MORRIS, 1. The effect of cycloheximide (Actidione) on protein and nucleic acid synthesis by
ChloreJla. J. expo Botany 18, 54 (1967).
MORTON, R. K., and J. K. RAISON: A complete intracellular unit for incorporation of amino-
acid into storage protein utilizing adenosine triphosphate generated from phytate.
Nature (Lond.) 200, 429 (1963).
MOUDRIANAKIS, E. N., S. H. HOWELL, and A. E. KARU: Characterization of the "quanta-
some" and its role in photosynthesis. In: Comparative biochemistry and biophysics of
photosynthesis, p. 67. SHIBATA, K., A. TAKAMIYA, A. T. JAGENDORF, and R. C. FULLER,
Eds. Tokyo: University of Tokyo Press 1968.
MUNKRES, K. D., and F. M. RICHARDS: Genetic alteration of Neuro.fpora malate dehydro-
genase. Arch. Biochem. 109, 457 (1965).
- , and D. O. WOODWARD: On the genetics of enzyme locational specificity. Proc. nat. Acad.
Sci. (Wash.) 55, 1217 (1966).
- - Interaction of Neuro.fpora mitochondrial structural protein with other proteins and
coenzyme nucleotides. Biochim. biophys. Acta (Arnst.) 133, 143 (1967).
NAGAHISA, M., and A. HATTORI: Studies on oxalic acid oxidase in green leaves. Plant
and Cell Physio!. 5, 205 (1964).
NAGAI, J., and K. BLOCH: Enzymatic de saturation of stearyl acyl carrier protein. J. bio!.
Chern. 241, 1925 (1966).
NASS, M. M. K.: The circularity of mitochondrial DNA. Proc. nat. Acad. Sci. (Wash.) 56,
1215 (1966).
NEUPERT, W., D. BRDICZKA, and TH. BUCHER: Incorporation of amino acids into the outer
and inner membrane of isolated rat liver mitochondria. Biochem. biophys. Res. Commun.
27,488 (1967).
NISHIMURA, M., and H. HUZISIGE: Studies on chlorophyll formation in Euglena gracili.f with
special reference to the action spectrum of the process. J. Biochem. (Tokyo) 46, 225
(1959).
O'BRIEN, T. W., and G. F. KALF: Ribosomes from rat liver mitochondria. 1. Isolation
procedure and contamination studies. J. bio!. Chern. 242, 2172 (1967).
ODINTSOVA, M. S., E. V. GOLUBEVA, and N. M. SISSAKIAN: Chloroplast ribosomes. Nature
(Lond.) 204, 1090 (1964).
OGAWA, T., F. OBATA, and K. SHIBATA: Two pigment proteins in spinach chloroplasts.
Biochim. biophys. Acta (Arnst.) 112,223 (1966).
OGUR, M., L. COKER, S. OGUR, and A. ROSHANMANESH: Tricarboxylic acid cycle mutants in
yeasts. Fed. Proc. 23, 378 (1964).
OLSON, J. M., G. HIND, H. LYMAN, and H. W. SIEGELMAN, Eds.: Energy conversion by the
photosynthetic apparatus. Brookhaven Symp. Bio!. 19, (1967).
PARENTI, F., and M. M. MARGULIES: In vitro protein synthesis by plastids of Pha.feolu.f
vulgari.f. 1. Localization of activity in the chloroplasts of a chloroplast containing fraction
from developing leaves. Plant Physio!. 42, 1179 (1967).
PARK, R. B.: Chloroplast structure. In: The chlorophylls, p. 283. VERNON,L. P., and G. R.
SEELY, Eds. London and New York: Academic Press 1966.
PARKER, M. W., S. B. HENDRICKS, H. A. BORTHWICK, and F. W. WENT: Spectral sensitivities
for leaf and stem growth of etiolated pea seedlings and their similarity to action spectra
for photoperiodism. Amer. J. Botany 36, 194 (1949).
PARTHIER, B.: Proteinsynthese in griinen Bliittern. Z. Naturforsch. 19b, 235 (1964).
- Chloramphenicol-Wirkung auf eine durch Licht stimulierte Proteinsynthese in Blattero

und in isolierten Chloroplasten. Z. Naturforsch. 20b, 1191 (1965).
- , B. MALAVIYA, and K. MOTHES: Effects of chloramphenicol and kinetin on uptake and
incorporation of amino acids by tobacco leaf disks. Plant and Cell Physiol. 5, 401 (1964).
- , R. WOLLGIEHN: Nucleinsauren und Proteinsynthese in Plastiden. In: Probleme der
biologischen Reduplikation. P. SITTE, Hrsg. Berlin-Heidelberg-New York: Springer
1966.
PETERKOFSKY, A., and E. RACKER: The reductive pentose phosphate cycle. m. Enzyme
activities in cell-free extracts of photosynthetic organisms. Plant Physiol. 36, 409 (1961).
PETERMANN, M. L.: The physical and chemical properties of ribosomes. Amsterdam:
Elsevier Publ. Co. 1964.
PETROPULOS, S. F.: Ultraviolet inactivation of chloroplast formation in synchronously
dividing Euglena gracili.r. Science 145, 392 (1964).
POGO, A. 0., G. BRAWERMAN, and E. CHARGAFF: New ribonucleic acid species associated
with the formation of the photosynthetic apparatus in Euglena gracili.r. Biochemistry 1,
128 (1962).
POGO, B. G. T., and A. O. POGo: DNA dependence of plastid differentiation inhibition by
actinomycin D. J. Cell BioI. 22,296 (1964).
- - Inhibition by chloramphenicol of chlorophyll and protein synthesis and growth in
Euglena gracili.r. J. Protozool. 12, 96 (1965).
POLLARD, C. ]., A. STEMLER, and D. F. BLAYDES: Ribosomal ribonucleic acids of chloro-
plastic and mitochondrial preparations. Plant Physio!. 41, 1323 (1966).
PORRA, R. ]., and]. LASCELLES: Studies on ferrochelatase. The enzymic formation of haem
in proplastids, chloroplasts and plant mitochondria. Biochem. ]. 108,343 (1968).
PRICE, L., and W. H. KLEIN: Red, far-red response and chlorophyll synthesis. Plant Physio!.
36, 733 (1961).
- - Chlorophyll synthesis in X-irradiated etiolated bean leaf tissue. Radiat. Botany 1, 269
(1962).
- , MITRAKOS, and W. H. KLEIN: Photomorphogenesis and carbohydrate changes in etio-
lated tissue. Quart. Rev. BioI. 39, 11 (1964).
- - - Some kinetical aspects of light-induced carbohydrate utilization in etiolated leaf
tissue. Plant Physiol. 18, 540 (1965).
RACUSEN, D., and M. FOOTE: Protein synthesis in dark-grown bean leaves. Canad. ]. Bot.
43,817 (1965).
RAGETLI, H. W. J., M. WEINTRAUB, and U. M. RINK: Latent acid phosphatases in chloro-
plasts. Canad. ]. Bot. 44, 1723 (1966).
RAMIREZ,]. M., F. F. DEL CAMPO, A. PANEQUE, and M. LOSADA: Ferredoxin-nitrite reductase
from spinach. Biochim. biophys. Acta (Amst.) 118, 58 (1966).
RANDALL, ]., and C. DISBREY: Evidence for the presence of DNA at basal body sites in
Tetrahymena pyriformi.r. Proc. roy. Soc. B 162,473 (1965).
RAY, D. S., and P. C. HANAWALT: Properties of the satellite DNA associated with the
chloroplasts of Euglena gracili.r. ]. molec. Bio!. 9, 812 (1964).
RHODES, M. ]. c., and E. W. YEMM: The development of chloroplasts and photosynthetic
activities in young barley leaves. New Phytol. 65, 331 (1966).
RICHARDS, O. c.: Hybridization of Euglena gracili.r chloroplast and nuclear DNA. Proc. nat.
Acad. Sci. (Wash.) 57, 156 (1967).
RIFKIN, M. R., D. D. WOOD, and D. ]. L. LUCK: Ribosomal RNA and ribosomes from
mitochondria of Neuro.rpora cra.r.ra. Proc. nat. Acad. Sci. (Wash.) 58, 1025 (1967).
RIS, H., and W. PLAUT: Ultrastructure of DNA-containing areas in the chloroplast of
Chlamydomona.r. ]. Cell BioI. 13, 383 (1962).
RITENOUR, G. L., K. W. JOY, ]. BUNNING, and R. H. HAGEMAN: Intracellular localization of
nitrate reductase, nitrite reductase, and glutamic acid dehydrogenase in green leaf tissue.
ROGERS, L. ]., S. P. ]. SHAH, and T. W. GOODWIN: (1) The intracellular localization of
mevalonate activating enzymes: Its importance in the regulation of terpenoid biosyn-
thesis. In: Biochemistry of chloroplasts, Vol. II, p. 283. T. W. GOODWIN, Ed. London
and New York: Academic Press 1967.
ROGERS, P. J., B. N. PRESTON, N. B. TrrcHENER, and A. W. LINNANE: (2) Differences be-

tween the sedimentation characteristics of the ribonucleic acids prepared from yeast
cytoplasmic ribosomes and mitochondria. Biochem. biophys. Res. Commun. 27, 405
(1967).
ROODYN, D. B., J. W. SurrIE, and T. S. WORK: Protein synthesis in mitochondria. 2. Rate
of incorporation in lIi/ro of radioactive amino acids into soluble proteins in the mito-
chondrial fraction, including catalase, malic dehydrogenase and cytochrome c. Biochem.
J. 83,29 (1962).
RUPPEL, H. G., u. D. VAN WYK: Dber die Desoxyribonucleinsaure in Chloroplasten von
Anlirrhinum majus. Z. Pflanzenphysioi. 53, 32 (1965).
RUSSELL, G. K., and M. GIBBS: Regulation of photosynthetic capacity in Chlamydomonas
mundana. Plant Physioi. 41, 885 (1966).
RUTTER, W. J.: Evolution of aldolase. Fed. Proc. 23, 1248 (1964).
SAGER, R.: Mendelian and non-Mendelian heredity: A reappraisal. Proc. roy. Soc. B. 164,
290 (1966).
- , and M. G. HAMILTON: Cytoplasmic and chloroplast ribosomes of Chlamydomonas:
Ultracentrifugal characterization. Science 157, 709 (1967).
- , and M. R. ISHIDA: Chloroplast DNA in Chlamydomonas. Proc. nat. Acad. Sci. (Wash.) 50,
725 (1963).
SAN PIETRO, A., and H. M. LANG: Photosynthetic pyridine nucleotide reductase. 1. Partial
purification and properties of the enzyme from spinach. J. bioI. Chern. 231, 211
(1958).
SASTRY, P. S., and M. KATES: Hydrolysis of monogalactosyl and digalactosyl diglycerides
by specific enzymes in runner-bean leaves. Biochemistry 3, 1280 (1964).
SCHIFF, J. A., and H. T. EpSTEIN: The continuity of the chloroplast in Euglena. In: Repro-
duction: Molecular, subcellular and cellular, p. 131. M. LOCKE, Ed. New York: Academic
Press 1965.
- , H. LYMAN, and H. T. EpSTEIN: Studies of chloroplast development in Euglena. III.
Experimental separation of chloroplast development and chloroplast replication.
- , M. H. ZELDIN, and J. RUBMAN: Chlorophyll formation and photosynthetic competence
in Euglena during light-induced chloroplast development in the presence of 3,(3, 4-di-
chlorophenyl) 1,1-dimethyl urea (DCMU). Plant Physioi. 42,1716 (1967).
SCHILDKRAUT, C. L., J. MARMUR, and P. DOTTY: Determination of the base composition of
DNA from its buoyant density in CsO. J. molec. BioI. 4, 430 (1962).
SCHRADER, L. E., L. BEEVERS, and R. G. HAGEMAN; Differential effects of chloramphenicol
on the induction of nitrate and nitrite reductase in green leaf tissue. Biochem. biophys.
Res. Commun. 26, 14 (1967).
SCOTT, N. S., and R. M. SMILLIE: Evidence for the direction of chloroplast ribosomal RNA
synthesis by chloroplast DNA. Biochem. biophys. Res. Commun. 28, 598 (1967).
- - Ribosomal RNA in chloroplasts of Euglena gracilis. Currents. Mod. BioI. 2, 339
(1969).
-, V. C. SHAH, and R. M. SMILLIE: Synthesis of chloroplast DNA in isolated chloroplasts.
J. Cell BioI. 38, 151 (1968).
SEMAL, J., D. SPENCER, Y. T. KIM, and S. G. WILDMAN: Properties of a ribonucleic acid
synthesizing system in cell-free extracts of tobacco leaves. Biochim. biophys. Acta
(Arnst.) 91, 205 (1964).
SESTAK, Z., and J. CATSKY: Intensity of photosynthesis and chlorophyll content related to
leaf age in Nicotiana sanderae hort. BioI. Plant. (Praha) 4, 131 (1962).
SHAH, V. c., and H. LYMAN: DNA-dependent RNA synthesis in chloroplasts of Euglena
gracilis. J. Cell BioI. 29, 174 (1966).
SHERMAN, F., J. W. STEWART, E. MARGOLIASH, J. PARKER, and W. CAMPBELL: The struc-
tural gene for yeast cytochrome c. Proc. nat. Acad. Sci. (Wash.) 55, 1498 (1966).
SHIBATA, K.: Spectroscopic studies on chlorophyll formation in intact leaves. J. Biochem.
44, 147 (1957).
-, A. TAKAMIYA, A. T. JAGENDORF, and R. C. FULLER, Eds.: Comparative biochemistry and
biophysics of photosynthesis. Tokyo: University of Tokyo Press 1968.
SHIPP, W. S., F. J. KIERAS, and R. HASELKORN: DNA associated with tobacco chloroplasts.
SIEGEL, M. R., and H. D. SISLER: Site of action of cycloheximide in cells of Saccharomyces
parlorianur. ill. Further studies on the mechanism of action and the mechanism of resist-
ance in Saccharomycer species. Biochim. biophys. Acta (Arnst.) 103, 558 (1965).
SIEGELMAN, H. W., and W. L. BUTLER: Properties of phytochrome. Ann. Rev. Plant PhysioI.
16, 383 (1965).
SIMPSON, M. V., D. M. SKINNER, and J. M. LUCAS: On the biosynthesis of cytochrome c.
J. bioI. Chem. 236, PC81 (1961).
SINCLAIR, J. H., and B. J. STEVENS: Circular DNA filaments from mouse mitochondria.
SINGH, B. N., and K. N. LAL: Investigations of the effect of age on assimilation of leaves.
Ann. Botany 49, 291 (1935).
SIRONVAL, C., H. CLI]STERS, J.-M. MICHEL, R. BRONCHART, and R. M. MICHEL-WOLWERTZ:
On two classes of chlorophyll-lipoprotein complexes and on the organization of the
chloroplast lamellae. In: Currents in photosynthesis: Proceedings of the second Westem-
Europe Conference on Photosynthesis, p. 111. THOMAS, J. B., and J. C. GOEDHEER, Eds.
Rotterdam: A. Donker 1966.
- , M. R. MICHEL-WOLwERTz,and A. MADSEN: On the nature and possible functions of the
673- and 684-m(L forms in IIivo of chlorophyll. Biochim. biophys. Acta (Amst.) 94, 344
(1965).
SISLER, E. C., and W. H. KLEIN: The effect of age and various chemicals on the lag phase of
chlorophyll synthesis in dark-grown bean seedlings. PhysioI. Plant. 16, 315 (1963).
SISSAKIAN, N. M.: Enzymology of the plastids. Advanc. EnzymoI. 20, 201 (1958).
-,1. 1. FILIPPOVICH, E. N. SVETAILO, and K. A. ALIYEV: On the protein-synthesizing
system of chloroplasts. Biochim biophys. Acta (Amst.) 95,474 (1965).
SLACK, C. R., and M. D. HATCH: Comparative studies on the activity of carboxylases and
other enzymes in relation to the new pathway of photosynthetic carbon dioxide fixation
in tropical grasses. Biochem. J. 103, 660 (1967).
SMILLIE, R. M.: Photosynthetic and respiratory activities of growing pea leaves. Plant
PhysioI. 37, 716 (1962).
- Formation and function of soluble proteins in chloroplasts. Canad. J. Bot. 41, 123
(1963).
- , W. R. EVANS, and H. LYMAN: Metabolic events during the formation of a photo-
synthetic from a nonphotosynthetic cell. Brookhaven Symp. BioI. 16, 89 (1963).
- , D. GRAHAM, M. R. DWYER, A. GRIEVE, and N. F. TOBIN: Evidence for the synthesis
in IIillo of proteins of the Calvin cycle and of the photosynthetic electron transfer pathway
on chloroplast ribosomes. Biochem. biophys. Res. Commun. 28, 604 (1967).
- , and G. KROTKOV: Phosphorus-containing compounds in Euglena gracilir grown under
different conditions. Arch. Biochem. 89, 83 (1960).
- , N. S. SCOTT, and D. GRAHAM: Biogenesis of chloroplasts: Roles of chloroplast DNA and
chloroplast ribosomes. In: Comparative biochemistry and biophysics of photosynthesis,
p. 332. SHmATA, K., A. TAKAMIYA, A. T. JAGENDORF, and R. C. FULLER, Eds. Tokyo:
University of Tokyo Press 1968.
SMITH, D. c., J. A. BASSHAM, and M. KIRK: Dynamics of the photosynthesis of carbon
compounds. II. Amino acid synthesis. Biochim. biophys. Acta (Amst.) 48, 299 (1961).
SMITH, J. H. C.: The development of chlorophyll and oxygen-evolving power in etiolated
barley leaves when illuminated. Plant and Cell PhysioI. 29, 143 (1954).
SOKAWA, Y., and E. HASE: Effect of light on the chlorophyll formation in the "glucose-
bleached" cells of Chlorella prolothecoider. Plant and Cell PhysioI. 8, 495 (1967).
SOTTOCASA, G. 1.., B. KUYLENSTIERNA, 1.. ERNSTER, and A. BERGSTRAND: An electron-
transport system associated with the outer membrane of liver mitochondria. A bio-
chemical and morphological study. J. Cell BioI. 32, 415 (1967).
SPENCER, D.: Protein synthesis by isolated spinach chloroplasts. Arch. Biochem. 111, 381
(1965).
- , and P. R. WHITFELD: The nature of the ribonucleic acid of isolated chloroplasts. Arch.
Biochem. 117, 337 (1966).
200 ROBERT M. SMILLm and N. STEELE SCOTT
SPENCER, D., and P. R. WHITFELD: (1) DNA synthesis in isolated chloroplasts. Biochem.
biopbys. Res. Commun. 28, 538 (1967).
- - (2) Ribonucleic acid synthesizing activity of spinach chloroplasts and nuclei. Arch.
Biochem. 121, 336 (1967).
- , and S. G. WILDMAN: The incorporation of amino acids into protein by cell-free extracts
from tobacco leaves. Biochemistry 3, 954 (1964).
SPIER, R. (1964): Quoted in: The plastids, their chemistry, structure, growth and inheri-
tance, p. 463. KIRK, J. T. 0., and R. A. E. TILNEy-BASSETT, Eds. London and San
Francisco: W. H. Freeman and Co. 1967.
STEPHENSON, M. L., K. V. THIMANN, and P. C. ZAMECNIK: Incorporation of 04-amino
acids into proteins of leaf disks and cell-free fractions of tobacco leaves. Arch. Biochem.
65, 194 (1956).
STOCKING, C. R.: Chloroplast isolation in nonaqueous media. Plant Physiol. 34, 56
(1959).
STUMPF, P. K., J. BROOKS, T. GALL lARD, J. C. HAWKE, and R. SIMONI: Biosynthesis of fatty
acids by photosynthetic tissues of higher plants. In: Biochemistry of chloroplasts, Vol. II,
STUTZ, E., and H. NOLL: Characterization of cytoplasmic and chloroplast polysomes in
plants: Evidence for three classes of ribosomal RNA in nature. Proc. nat. Acad. Sci.
(Wash.) 57, 774 (1967).
SUDYINA, E. G.: Chlorophyllase reaction in the last stage of biosynthesis of chlorophyll.
Photochem. Photobiol. 2, 181 (1963).
SUYAMA, Y.: The origins of mitochondrial ribonucleic acids in Tetrahymena pyriformi.r. Bio-
chemistry 6, 2829 (1967).
- , and W. D. BONNER: DNA from plant mitochondria. Plant Physiol. 41, 383 (1966).
- , and J. GIBSON: Satellite DNA in photosynthetic bacteria. Biochem. biophys. Res.
Commun. 24, 549 (1966).
SVETAILO, E. N., 1. 1. PHILIPPOVICH, and N. M. SISSAKIAN: Differences in sedimentation
properties of chloroplast and cytoplasmic ribosomes from pea seedlings. J. molec. BioI.
24, 405 (1967).
SWEENEY, B. M., C. F. TUFFLI, JR., and R. H. RUBIN: The circadian rhythm in photo-
synthesis in Ace/abularia in the presence of actinomycin D, puromycin, and chlor-
amphenicol. J. gen. Physiol. 50, 647 (1967).
SWIFT, H., M. RABINOWITZ, and G. GETZ: DNA and mitochondrial morphology in anaerobic
and adapting yeast. J. Cell BioI. 35, 131A (1967).
SZARKOWSKI, J. W., u. T. GOLASZEWSKI: RNA-Gehalt der Plastiden von griinen und etio-
lierten Pflanzen. Naturwissenschaften 48, 457 (1961).
- - , und M. OMBACH: Die Ribonukleaseaktivitat in Plastiden. Naturwissenschaften 49,
135 (1962).
TAMIYA, H., Y. MORIMURA, and M. YOKOTA: Effects of various antimetabolites upon the
life cycle of Chlorella. Arch. Mikrobiol. 42, 4 (1962).
TA YLOR, A. 0., and B. A. BONNER: Isolation of phytochrome from the alga Me.rotaenium and
liverwort Spaerocarpo.r. Plant Physiol. 42, 762 (1967).
TAYLOR, F. J.: Chloramphenicol inhibition of the growth of green algae. Nature (Lond.)
207, 783 (1965).
TAYLOR, M. M., J. E. GLASGOW, and R. STORCK: Sedimentation coefficients of RNA from
70S and 80S ribosomes. Proc. nat. Acad. Sci. (Wash.) 57, 164 (1967).
TEWARI, K. K., and S. G. WILDMAN: Chloroplast DNA from tobacco leaves. Science 153,
1269 (1966).
- - DNA polymerase in isolated tobacco chloroplasts and nature of the polymerized
product. Proc. nat. Acad. Sci. (Wash.) 58, 689 (1967).
- Function of chloroplast DNA. 1. Hybridization studies involving nuclear and chloro-
plast DNA with RNA from cytoplasmic (80S) and chloroplast (70S) ribosomes. Proc.
nat. Acad. Sci. (Wash.) 59, 569 (1968).
- , J. JAYARAMAN, and H. R. MAHLER: Separation and characterization of mitochondrial
DNA from yeast. Biochem. biophys. Res. Commun. 21, 141 (1965).
THORNBER, J. P., R. P. F. GREGORY, C. A. SMITH, and J. L. BAILEY: (1) Studies on the nature
of the chloroplast lamella. 1. Preparation and some properties of two chlorophyll-protein
complexes. Biochemistry 6, 391 (1967).
- , J. C. STEWART, M. W. C. HATTON, and J. L.BAILEY: (2) Studies on the nature of chloro-
plast lamellae. II. Chemical composition and further physical properties of two chloro-
phyll-protein complexes. Biochemistry 6, 2006 (1967).
TREHARNE, K. J.,E. 1. MERCER, andT. W.GOODWIN: Incorporation of (14q carbon dioxide
and [2_14q mevalonic acid into terpenoids of higher plants during chloroplast develop-
ment. Biochem. J. 99, 239 (1966).
VAZQUEZ, D.: Uptake and binding of chloramphenicol by sensitive and resistant organisms.
Nature (Lond.) 203, 257 (1964).
- Mode of action of chloramphenicol and related antibiotics. Symp. Soc. general Microbiol.
16, 169 (1966).
VERNON, L. P., and G. R. SEELY, Eds.: The chlorophylls. London and New York: Academic
Press 1966.
VIRGIN, H. 1.: Protochlorophyll formation and greening in etiolated barley leaves. Physiol.
Plant. 8, 630 (1955).
- Chlorophyll a content and transpiration of etiolated wheat leaves after pre-treatment
with a short light impulse followed by dark periods of varying lengths. Physiol. Plant. 10,
445 (1957).
- Studies on the formation of proto chlorophyll and chlorophyll a under varying light
treatments. Physiol. Plant. 11, 347 (1958).
- Pigment transformations in leaves of wheat after irradiation. Physiol. Plant. 13, 155
(1960).
- , A. KAHN, and D. VON WETTSTEIN: The physiology of chlorophyll formation in relation
to structural changes in chloroplasts. Photochem. Photobiol. 2, 83 (1963).
WALLES, B.: Plastid structures of carotenoid-deficient mutants of sun flower (HeNanthu.
annu. L.). 1. The white mutant. Hereditas (Lund) 53, 247 (1965).
Use of biochemical mutants in analyses of chloroplast morphogenesis. In: Biochemistry
of chloroplasts, Vol. II, p. 633. T. W. GOODWIN. Ed. London and New York: Academic
Press 1967.
WARNAAR, S. 0., and J. A. COHEN: A quantitative assay for DNA-DNA hybrids using
membrane filters. Biochem. biophys. Res. Commun. 24, 554 (1966).
WEIER, T. E., and C. R. STOCKING: The chloroplast: Structure, inheritance, and enzymology.
II. Botan. Rev. 18, 14 (1952).
VON WETTSTEIN, D.: The formation of plastid structures. Brookhaven Symp. BioI. 11, 138
(1959).
- On the physiology of chloroplast structures. In: Biochemistry of chloroplasts, Vol. I,
WHEELDON, L. W., and A. L. LEHNINGER: Energy-linked synthesis and decay of membrane
proteins in isolated rat liver mitochondria. Biochemistry 5, 3533 (1966).
WIECKOWSKI, S., and T. W. GOODWIN: Incorporation of DL_[2_14q mevalonic acid lactone
into f3-carotene and the phytol side chain of chlorophyll in cotyledons of four species of
pine seedlings. Biochem. J. 105, 89 (1967).
WILDMAN, S. G.: The organization of grana-containing chloroplasts in relation to location
of some enzymatic systems concerned with photosynthesis, protein synthesis, and ribo-
nucleic acid synthesis. In: Biochemistry of chloroplasts, Vol. II, p. 295. T. W. GOODWIN,
Ed. London and New York: Academic Press 1967.
WILLIAMS, G. R., and G. D. NOVELLI: Stimulation of an in vitro amino acid incorporating
system by illumination of dark-grown plants. Biochem. biophys. Res. Commun. 17,23
(1964).
WINTERSBERGER, E.: Proteinsynthese in isolierten Hese-Mitochondrien. Biochem. Z. 341,
409 (1965).
- Synthesis and function of mitochondrial ribonucleic acid. In: The regulation of metabolic
processes in mitochondria, p. 439. TAGER, W. M., S. PAPA, E. QUAGLIARIELLO, and E. C.
SLATER, Eds. Amsterdam: Elsevier 1966.
202 ROBERT M. SMILLIE and N. STEELE SCOTT: Organelle Biosynthesis: The Chloroplast
WITHROW, R. B., J. B. WOLFF, and L. Price: Elimination of the lag phase of chlorophyll
synthesis in dark-grown bean leaves by a pretreatment with low irradiances of mono-
chromatic energies. Plant Physioi. 31, Suppi. XIII (1956).
WOLFF, J. B., and L. Price: Terminal steps of chlorophyll a biosynthesis in higher plants.
Arch. Biochem. 72, 293 (1957).
- - The effect of sugars on chlorophyll biosynthesis in higher plants. J. bioI. Chern. 235,
1603 (1960).
WOLLGIEHN, R., M. RUESS, and D. MUNSCHE: Ribonucleic acids of chloroplasts. Flora 157,
92 (1966).
WOODCOCK, C. L. F., and H. FERNANDEZ-MoRAN: Electron microscopy of DNA conforma-
tions in spinach chloroplasts. J. molec. BioI. 31, 627 (1968).
WOODWARD, D.O., and K. D. MUNKRES: Alterations of a maternally inherited mito-
chondrial structural protein in respiratory-deficient strains of Neurospora. Proc. nat.
Acad. Sci. (Wash.) 55, 872 (1966).
YANKOFSKY, S. A., and S. SPIEGELMAN: Distinct cistrons for the two ribosomal RNA com-
ponents. Proc. nat. Acad. Sci. (Wash.) 49, 538 (1963).
ZELDIN, M. H., and J. A. SCHIFF: RNA metabolism during light-induced chloroplast
development in Euglena. Plant Physioi. 42, 922 (1967).
ZIEGLER, H., u. I. ZIEGLER: Der Ein£luB der Belichtung auf die NADP+-abhangige Glycerin-
aldehyd-3-phosphat-Dehydrogenase. Planta (Bed.) 65, 369 (1965).
- - (1) Die lichtinduzierte Synthese der NADP+-abhiingigen Glycerinaldehyd-3-phos-
phat-Dehydrogenase III. Die Bedeutung des Chlorophylls und der Ein£luB von Stoff-
wechselinhibitoren. Planta (Beri.) 69, 111 (1966).
- - (2) Die lichtinduzierte Aktivitatssteigerung der NADP+-abhiingigen Glycerinnal-
dehyd-3-phosphat Dehydrogenase. IV. Der EinfluB auf die Photosyntheseintensitat.
Biochim biophys. Acta (Arnst.) 126, 449 (1966).
Macromolecules and Brain Function
A 1969 Baedeker
B. W. AGRANOFF
1. Introduction
The inclusion of a neurobiological topic in a volume dedicated to molecular
biology might assume a common ground which does not exist. The equivalence of
the term "information" to describe macromolecular coding of genetic traits, as well
as the storing of behavioral patterns, is not based on experimental grounds and is
more properly a linguistic coincidence. Yet, the idea that a specific behavior results in
the formation of a novel RNA or protein, and that following injection into a recipient
animal such molecules can then elicit the original behavior, has been proposed and
reported [44]. Like extrasensory perception, this idea awaits repeated experimental
verification before receiving further note. Like extrasensory perception, negative
experiments do not dismay the faithful. Reports that memory is transferred via
injection require that the putative effective molecules can enter the brain following
intraperitoneal injection, that they can find relevant cells in the brain before they are
degraded and, further, that such molecules have interspecies activity.
While I am of the school of skeptics, I do nevertheless concede that reports of
such experiments have stimulated many scientists to ask the question, "Well then,
what does go on in the brain? Do the concepts and tools of molecular biology
provide us with new insights or techniques for studying higher brain function?"
What follows is an attempt to describe a science in its infancy. Like a travel
guidebook, it will hopefully soon be obsolete. Neurobiology is a field which, like a
newly formed, underdeveloped country, has great potentiality. As an emigre of 10
or so years, I feel sufficient allegiance to be concerned about its growth, resources
and tourism. It is my hope to convey the present excitement in both its wide open
spaces and in its more civilized, highly-populated regions.
The molecular approach to the nervous system can be properly traced back to
the beginnings of modern biochemistry and the revolutionary challenges to vitalism
personified in the form of the 19th century neurochemist, Thudichum [59]. Much of
the field called neurochemistry, which developed in the past 50 years, deals with
analytical and metabolic aspects of the brain. Many recent reviews and symposium
volumes have summarized our present state of knowledge of the specialized bio-
chemistry of this organ [26, 39, 50, 51]. This chapter deals primarily with those studies
which at present appear to hold greatest promise for the future.
204 B. W. AGRANOFF
II. Growth
A simple conceptual model for the storage of behavioral information in the brain
might require that new cells form as a result of experience. However the cells of the
brain that are implicated in brain function from anatomical and electrophysiological
considerations are the neurons, and they are completely or almost completely formed
at birth [17]. There is evidence that some interneuronal connections (synapses) continue
to form after birth. In the mouse cortex nerve endings continue to form during
early infancy and can be studied in tissue explants [5]. Brains of rats raised in a
stimulating, as opposed to a deprived environment show anatomical differences.
Such observations have been used to postulate a plasticity of the brain [9]. More
recently it has been observed that the reported changes in brain size and enzyme
content are transitory and thus more likely to be related to phenomena associated
with learning than with memory formation [54]. While neurons do not divide, the
supporting glial cells do, and they have been postulated to play an important role in
behavior [281. Recent studies in our laboratory indicate that DNA synthesis (and
thereby perhaps cell replication) does not appear to be required for the formation of
memory [14].
It becomes apparent that understanding the development of the nervous system
and its responses to the environment await a better understanding of cellular differ-
entiation and morphogenesis itself. At the present time, there are few clues in our
recently gained knowledge about cell replication which pertain directly to develop-
ment. It is possible, on the other hand, that some exciting models for developmental
biology lie in the nervous system. One such example is the nerve growth factor, a
substance which stimulates growth of sympathetic nerve chains [41]. Antibodies to
the nerve growth factor, when injected into developing animals, prevent the for-
mation of the sympathetic chains. There is a cross-species specificity for this sub-
stance and considerable evidence to involve a nuclear site of action of the factor.
Studies on the nature of the factor itself suggest a protein with complex subunit
interactions [60].
Another interesting neural phenomenon which may yield clues concerning
morphogenesis is the observed regrowth in the central nervous system of adult
lower vertebrates and of invertebrates. It is known that when the dorsal skin of a
developing frog is transplanted to the ventral surface, the skin will become reinner-
vated so as to be read in the central nervous system as being of ventral origin [45]. In
other words, nerve which regrows outward from the central nervous system re-
cognizes the original skin location. Similarily, when the optic nerve of a fish or
salamander is cut, and the eyeball rotated 1800 and reinserted into the orbit, nerves
regrow to their original connections [57]. If the rotation is performed early in life,
then the animal reconnects the eye to the brain in a way which respecifies the retinal
attachments, and the adult does not have an "upside-down eye." It has recently been
postulated that the specificity of the eye position is established at the time when the
ganglion cells are formed in the retina. Horizontal and vertical axes are established at
somewhat different times [35]. It has been suggested that each nerve fiber has a
specific chemical messenger which allows it to find its way back to its central con-
nection or, alternatively, that a matrix for specification is created by chemical gra-
dients. Two independent gradients could specify a large number of fibers. A simplified
Macromolecules and Brain Function - A 1969 Baedeker 205
model for the study of this phenomenon may be offered by recent studies in the leech,
in which specific neuronal regrowth occurs in skin segments involving a small
number ofaxons [48].
III. Sensory Modalities

Of the various biological mechanisms for transducing various exteroceptive
stimuli into electrical impulses, vision has probably been worked on most extensively.
The rhodopsin retinine cycle is now familiar [62], but much remains to be learned
about the physicochemical nature of the interactions of these two substances brought
on by light. The chemical basis of color vision is not so well understood.
Of increasing interest, particularly to chemists, are taste and smell, the study of
chemoreception. In an amusing account of 140 years ago, BRILLAT-SAVARIN com-
bined his culinary and scientific skills to write "The Physiology of Taste" [12]. We do
not know much more about chemoreception today. In man, we can easily identify
taste and smell, the former being mediated via the 7th, 9th and 10th cranial nerves and
the latter by the first. The olfactory mucosa of higher animals detects only volatile
substances while taste buds must contact a solution of sapient material. On the basis
of human perception studies, smell has been proposed to involve some 7 to 9 types
of receptors, termed floral, musky, ethereal, etc. [6]. Taste appears to involve four
receptors, namely bitter, sweet, sour and salty. Reception of these modalities is
assigned to specific regions of the human tongue. There is considerable controversy
today concerning the neural coding of taste [29, 32]. It is claimed on the one hand
that specific receptors exist for each taste modality in higher animals [8], while others
think that a common receptor with specific codes for transmission of various tastes
along the same nerve exists [49]. By plotting psychophysical measurements such as
the degree of taste or the number of impulses conducted along a fiber as a function of
the concentration of sapient substances, a concentration-effect curve can be plotted [7].
Its temperature-dependency can also be studied. On the basis of such studies, it has
been proposed that interactions of sapient molecules with taste receptors are of a
low energy variety, such as might be expected in the change of conformation of a
protein. The isolation of a protein which might be a sweet taste receptor from cow
tongue has been reported [19]. By means of difference spectra, changes in the ultra-
violet absorption and refractive index have been claimed with various sugars as well
as synthetic sweeteners. A possible model system for such interaction might be in the
footpad of the housefly which can detect glutathione. A genetic dimorphism exists in
humans for taste of various substances. That is, there is a genetic trait for the tasting
of phenylthiourea [30], quinine [22] and other substances.
Another interesting aspect of taste detection is the nature of the detector cells. A
specialized epithelium in the tastebud is attached presumably by a synapse to a
neuronal dendrite. It appears that the presynaptic region of this synapse is constantly
being regenerated, while the postsynaptic portion remains.
Several substances have been reported that will temporarily destroy the taste of
sweet substances, and also of sour ones [321. In aquatic animals, the distinction of taste
and smell is more complex than in higher animals. Most fishes have nares which may
or may not communicate with the pharynx. In salmon, olfaction appears to modulate
the homing instinct [31]. This smell may be in the form of nonvolatile dissolved
206 B. W. AGRANOFF
substances. Tastebuds are present on the tongue and on the integument around the
mouth. In some animals such as catfish, barbels with both taste and smell detectors
are present.
The phenomenon of chemotaxis may be considered to involve a form of chemo-
reception. Bacteria have been observed to travel in a chemical gradient, such as of
oxygen [1]. If one could find an acceptable model for behavior in a bacterium, it
would be tempting to study it, since microorganisms can be subjected to elegant
genetic techniques. Specificity of the stimulus and of the response in a microorganism
leaves something to be desired. Chemotaxis was studied extensively many years ago
with polymorphonuclear leukocytes from blood. New techniques for the quantitative
study of chemotaxis have recently been reported [11].
Many theories have been advanced to explain the nature of the olfactory receptor.
For example, it has been postulated that infrared absorption maxima correlate well
with subjective odor [64]. Such a theory would predict no difference between D and
L isomers. While this would seem easy to test, it is not. Olfaction and taste, like
other senses are detected logarithmically. Contamination of one part per thousand in an
odorant could seriously affect results. It has been stated that skatole, one of the most
powerful odorants for man, is itself not detected when pure. Recent purification
techniques such as gas chromatography and zone melting should provide ultrapure
substances to facilitate olfactory studies.
Another theory proposes that molecular size and shape play a role [6]. It is
claimed that any given odor can be reconstructed by "playing a chord" with various
classes of odors in the nose. An objection raised to both the infrared and the shape
receptor mechanisms has been the statement that the odor of almonds is obtained with
both benzaldehyde and hydrogen cyanide [27]. The two molecules do not have similar
shapes no do they have similar infrared patterns. Further, some individuals have a
genetic anosmia for cyanide but not for benzaldehyde. I believe that a clue lies in the
compound amygdalin. When almonds are crushed, a nitrile is cleaved to yield
carbohydrate, HCN and benzaldehyde. What we generally perceive as the almond
odor is due to both benzaldehyde and HCN. We recognize either one of them as
almond odor by a process of generalization.
Simple models for olfaction may exist in the pheromones of insects [37]. These
substances have eluded detection until recently because of their great potency. Sex
attractants are detected by the antennae of male insects in concentrations as low as a
few parts per billion. Many of these substances are low molecular weight alcohols of
simple structure. The chemical nature of the receptors is unknown.
IV. Structural Localization: Brain Regions and Fractions

As the anatomical approach to the nervous system has become chemical, so have
chemical studies taken greater cognizance of the complex histological and cytological
architecture of the brain. For the microanatomist, the silver stain has been used more
than any other type of technique to characterize neuronal types and interconnections
for nearly a century. The denseness of the neural network of the brain does not
readily permit identification of single cells by conventional staining. It is possible
with silver mc::thods selectively to stain only 1 in every 100 or so brain cells and yet
to stain such cells completely, including all their dendritic endings and the full length
of the axon. By focusing through a thick section of brain after silver staining, the
entire extent of a neuron can be visualized within the dense forest of other neurons
and glial cells. Whether the stained cells are inherently more reactive or whether the
functional state of the cell at the time of tissue fixation is a factor in stainability is
unknown. Since a neuronal process can extend several feet in a large animal, another
technique has proved valuable for tracing functional pathways. When brain or spinal
cord neuronal axons are cut, that portion of the cell on the side of the cut attached to
the cell body survives while the distal portion degenerates. By cutting a tract of
neurons, waiting some time and then staining for degenerated fibers, the direction of
the tract and its final destination can be traced. Special stains for studying early
degeneration have been devised l47]. The chemical nature of the neuronal silver
stain and its variations including those for degeneration are not understood and
constitute challenging questions for the future.
Primarily on the basis of electrophysiological and pharmacological studies, neuro-
transmitter substances in nerve have been proposed. Storage of these substances near
presynaptic terminals mediate transmission of electrical impulses between neurons.
Regulation at the level of synthesis, release, combination with receptor, presynaptic
reuptake and enzymatic degradation have been postulated [40]. Serotonin, norepine-
phrine, dopamine, gaba and acetylcholine have variously been assigned such a role.
Discovery of fluorescence following treatment of lyophilized brain slices with formal-
dehyde [18] has permitted visualization of individual cells in brain regions which
appear to have specific neurotransmitters in their cytoplasm.
Separation of neurons and glia by means of microdissection of the brain [33] or
by sedimentation in density gradients [53] has permitted studies of these two major
classes of brain cells. Homogenates of brain subjected to subcellular fractionation
show that nerve endings containing synaptic regions are pinched off and re-
sealed [21]. Such particles are also termed synaptosomes [63]. They contain mito-
chondria and vesicles presumably filled with neurotransmitter, but no easily identified
ribosomes.
The use of chemical methods to demonstrate functional relationships between
various regions in the brain has been attempted many ways. Following section of
nerve trunk connectives in the cockroach thoracic ganglion, a characteristic change
in the distribution of RNA in the bodies of various giant neurons may be detected
by a histochemical method [16]. This permits assignment of nerve processes in the
cut nerve to specific numbered cells. Using radioactive Krypton-85 as a measure of
cerebral blood flow, it has been possible to demonstrate way stations in the visual
system of the cat. Autoradiograms are made with sections of cat brain visual stimu-
lation and intravenous injection of the labeled gas [56]. An increase in protein
synthesis in the occipital cortex has been reported following visual stimulation [58].
Compared to other organs, brain has many distinguishing characteristics. Although
it uses glucose as the sole source of metabolic energy, and does not take up amino
acids readily from the blood in vivo, broken cell preparations of brain show active
protein synthesis [61]. This is a reflection of the functional separation of the brain
from the body, the blood-brain barrier. Large amounts of the acidic amino acids,
glutamate and aspartate are present in brain. In addition, glutamine and N-acetyl
aspartate are prominent. Cystathionine is found in increased amounts in the brains of
primates only. In lower animals forms, taurine and isethionic acid are characteristic
208 B. W. AGRANOFF
anions. A family of acidic proteins specific to nerve tissue have been described [46].
Antibodies to one of these substances react with brain; but not other organs, in a wide
variety of animals [42].
An enzyme, glutamic decarboxylase, is characteristic of nervous tissue. It gives
rise to gamma-aminobutyric acid, an inhibitory neurotransmitter [52]. In the lobster,
analysis of the axoplasm of the excititory and inhibitory fibers shows 10 times higher
gaba concentrations in the latter than in the former [40].
The white matter of brain contains myelin, specialized membranes containing
unusual lipids such as galactocerebrosides and the phosphatidyl inositol phosphates.
The phosphatidyl inositol phosphates are present in all animal membranes [55] but
are found in largest amounts in excitable tissues such as brain. In all tissues, the mono-
ester phosphate part of the lipid turns over rapidly.
V. Human Disorders
Various genetic defects are known to be associated with mental retardation. Even
when the genetic defect is fairly well established, correlation with the functional
lesion may be obscure. Phenylketonuria is a case in point. This autosomal recessive
disease is characterized by an absent or defective enzyme, phenylalanine hydroxylase.
Highest activities are normally found in liver. Progress in understanding the disease
would be enhanced by the discovery of a suitable experimental animal with this
lesion. Since tyrosine is an intermediate on the pathway of degradation of phenyl-
alanine, alternate metabolites of phenylalanine are present in high concentrations in
the disease state. They include phenylpyruvate, phenylacetate and o-tyrosine. One
or more of these and related substances may be toxic to brain development. The
disease is presently diagnosed early in life by the presence of phenylpyruvate in the
urine or by a high level of phenylalanine in the blood. Patients are placed on a diet
which is low in phenylalanine content. Presumably after 2 to 3 years, the brain is
sufficiently formed so that the enzyme deficiency will no longer influence the brain.
Routine screening of urines for the past few years has revealed about twice the inci-
dence of that expected from the number of institutionalized phenylketonurics. There
are undoubtedly individuals who excrete phenylpyruvate (phenylketonurics) but
do not have diagnosed mental retardation. Because of massive screening programs,
institutionalized children probably have a higher probability of detection of a meta-
bolic disorder than do children in the general population. Thus, of the 20-odd inborn
errors in metabolism presently thought to be associated with mental retardation, it
may well be that in many instances there is no causal relationship between the mental
retardation and the metabolic lesion. Another aspect of phenylketonuria has serious
implications for the future. Female phenylketonurics raised on low phenylalanine
diets during childhood may now develop normally, then switch to a more palatable,
normal diet. Should they remain on a normal diet during pregnancy, all of their
children may be retarded [43]. If unmanaged, our present treatment of phenylketon-
uria could result in more retarded children than had the disease not been diag-
nosed.
VI. Learning and Memory

"Ohne Phosphor, keine Gedank", has been attributed to BUCHNER [32] and to
COEURBE [59]. The statement arose from the discovery of large amounts of phos-
phorus in the brain which we now know reflects the large amount of phospholipid
present. Putting this finding together with the high phosphorus content in fish
prompted Agassiz to perpetrate the myth that "fish is good brain food" [36]. Studies
that attempted to relate brain chemistry and mental function came only recently.
Numerous experiments of HYDEN over the past 10 years in which hand dissected
groups of neurons and glial cells have been analyzed, suggest that there are changes
in the amount of RNA and in the base ratios of different cells [33] as well as the
migration rate of soluble proteins [34] following imposition of various physiological
factors such as stress and learning. Elegant microtechniques have been developed in
the Gothenborg laboratory in the course of these experiments.
More recently, changes in RNA labeling have been reported in the brains of mice
undergoing shock-avoidance training when compared to brains of "yoked" and
resting controls. In these experiments, 14C or 3H uridine was injected into each of two
mice and their brains combined prior to extraction of RNA and distribution on a
gradient. In general, the trained animal showed a higher rate of incorporation of the
isotope it received. The increase was not localized, but rather distributed throughout
the gradient [65]. Other experiments which may implicate macromolecular synthesis
in memory formation employed antibiotic antimetabolites. FLEXNER reported that
puromycin injected into bilateral intracerebral sites in a mouse a few days after
training in a right-left discrimination task, obliterated memory of the training [25].
Subsequent experiments with acetoxycycloheximide (AXM), another antibiotic anti-
metabolite which like puromycin, inhibits protein synthesis, did not produce memory
loss. These experiments were further complicated by experiments which suggest
that an intracerebral injection may produce electrical effects which could be amnestic
[15]. Injection of puromycin before training produced no effect on learning but
memory was lost shortly thereafter. The latter experiments are very similar to the
results obtained in the author's laboratory using goldfish as the experimental animal
but with an intracranial rather than an intracerebral injection. By not touching the
brain, the disadvantages of intracerebral injection [10,24] are avoided. Because of the
thin skull of the goldfish and the lack of well-formed membranes, 10 !Ll of a potential
disruptive agent can be injected easily into the unanesthetized fish, and it can be shown
that the agent rapidly produces its effect over the entire brain [13]. (The brain of a
10 g goldfish weighs about 80 mg.) A shock-avoidance task is used. A light signal is
initiated on the side of a shuttlebox in which the fish has been placed, and he has
20 sec to swim over a barrier in the center of the apparatus in order to avoid a pun-
ishing electrical shock administered through the water [3]. The sequence is then
initiated on the other side of the box. With increasing trials, fish show an increasing
tendency to avoid the shock. We have been able to demonstrate the phenomenon
of consolidation [2]. Drug injected immediately following training produces severe
memory loss while the same amount of drug injected a few hours later has no effect.
Both puromycin and acetoxycycloheximide block memory formation but not learning.
Actinomycin D, an inhibitor of RNA synthesis, produces a similar memory effect
with no detectable action on learning or acquisition [4]. An inhibitor of DNA syn-
210 B. W. AGRANOFF
thesis, cytosine arabinoside, does not affect memory although it blocks thymidine
incorporation into DNA in the goldfish brain [14]. The fish experiments have led to
the postulation of a short-term memory associated with acquisition which is not
effected by inhibitors of DNA, RNA or protein synthesis. The two latter steps
do appear to be required during a critical post-trial period in which long-term, or
permanent memory is formed. In other experiments, we have found evidence that
the onset of the memory fixing process appears to be triggered by conditions of the
animals' external environment [20].
Our results are in general compatible with the hypothesis that some sort of
induction or growth process is involved when permanent memory is formed. This
does not mean that a macromolecule is formed which is specific to a given behavioral
sequence. I rather prefer to consider that an animal has a repertoire of behavioral
responses which arise by natural selection and mutation similar to that postulated for
immune body formation.
During learning, the time in which the animal acquires a higher probability of
responding in a given situation, he is selecting and arranging in temporal fashion
from a large number of possible responses which have been established genetically.
The memory-forming process which we believe we can disrupt, may be anatomically
or only temporally related to the observed memory. In the first instance, it is possible
that a series of neurons establish new relationships at their synaptic connections
during the consolidation period. Alternatively, a region or system within the brain
may be responsible for generating a signal such as the secretion of a small molecule
which "fixes" all synaptic connections remaining in a heightened state following
training and inhibitory agents simply block this "fixing" process. In either instance,
it is not anticipated that a unique protein will be made. As stated by KETY, "There may
some day be a biochemistry or a biophysics of memory-but not of memories" [38].
References
1. ADLER, J.: Chemotaxis in bacteria. Science 153, 708 (1966).
2. AGRANOFF, B. W., R. E. DAVIS, and J. J. BRINK: Memory tormation in the goldfish.
3. - - - Chemical studies on memory fixation in goldfish. Brain Res. 1, 303 (1966).
4. - - , L. CASOLA, and R. LIM: Actinomycin D blocks formation of memory of shock
avoidance in the goldfish. Science 158, 1600 (1967).
5. ALTMAN, J., and G. D. DAs: Post-natal origin of microneurones in the rat brain. Nature
(Lond.) 207, 953 (1965).
6. AMOORE, J. E.: Current status of the steric theory of odor. Ann. N.Y. Acad. Sci. 116,457
(1964).
7. BEIDLER, L. M.: A theory of taste stimulation. J. gen. Physiol. 38, 133 (1954).
8. VON BEKESY, G.: Taste theories and the chemical stimulation of single papillae. J. appl.
Physiol. 21, 1 (1966).
9. BENNETT, E. L., M. C. DIAMOND, D. KRECH, and M. R. ROSENZWEIG: Chemical and
anatomical plasticity of brain. Science 146, 610 (1964).
10. BOHDANECKA, M., Z. BOHDANECKY, and M. E. JARVIK: Amnesic effects of small bila-
teral brain puncture in the mouse. Science 157, 334 (1967).
11. BOYDEN, S. V.: The chemotactic effect of mixtures of antibody and antigen on poly-
morphonuclear leukocytes. J. expo Med. 115, 453 (1962).
12. BRILLAT-SAVARIN: The physiology of taste, 1825, modern English translation by JEAN
ANTHELME. New York: Dover Publications 1960.
13. BRINK, J. J., R. E. DAVIS, and B. W. AGRANOFF: Effects of puromycin, acetoxycyclo-

heximide and actinomycin D on protein synthesis in goldfish brain. J. Neurochem.
13, 889 (1966).
14. CASOLA, L., R. LIM, R. E. DAVIS, and B. W. AGRANOFF: Behavioral and Biochemical
Effects of Intracranial Injection of Cytosine Arabinoside in Goldfish. Proc. nat. Acad.
Sci. (Wash.) 60, 1389 (1968).
15. COHEN, H. D., F. ERVIN, and S. H. BARONDES: Puromycin and cycloheximide: Different
effects on hippocampal electrical activity. Science 154, 1557 (1966).
16. COHEN, M. J., and J. W. JACKLET: Neurons of insects. RNA changes during injury and
regeneration. Science 148, 1237 (1965).
17. CRAIN, S. M.: Biolectric activities in CNS cultures. In: International Review of Neuro-
biology, Vol. 9, p. 1. PFEIFFER, C. c., and J. R. SMYTHIES, Eds. New York: Academic
Press 1966.
18. DAHLSTROM, A., and K. FUXE: Evidence for the existence of monoamine neurones in
the central nervous system. Acta physiol. scand. Supl. 147 (64), 5 (1965).
19. DASTOLI, F. R., and S. PRICE: Sweet-sensitive protein from bovine taste buds: Isolation
and assay. Science 154, 905 (1966).
20. DAVIS, R. E., and B. W. AGRANOFF: Stages of memory formation in the goldfish:
Evidence for an environmental trigger. Proc. nat. Acad. Sci. (Wash.) 55, 555 (1966).
21. DE ROBERTIS, E., A. PELLEGRINO DE IRALDI, G. RODRIGUEZ, and C. J. GOMEZ: On the
isolation of nerve endings and synaptic vesicles. J. biophys. biochem. Cytol. 9,229
(1961).
22. FISCHER, R., and F. GRIFFIN: Quinine dimorphism: A cardinal determinant of taste
sensitivity. Nature (Lond.) 200, 343 (1963).
23. FISHBEIN, M.: Shattering health superstitions, p. 32. New York: Horace Liveright, Inc.
1930.
24. FLEXNER, J. B., and L. B. FLEXNER: Restoration of expression of memory lost after
treatment with puromycin. Proc. nat. Acad. Sci. (Wash.) 57, 1651 (1967).
25. - - , and E. STELLAR: Memory in mice as affected by intracerebral puromycin. Science
141,57 (1963).
26. FOLCH-PI,J.: Chemical pathology of the nervous system. New York: Pergamon Press 1961.
27. FULLMAN, B.: Stereochemical theory of olfaction. Nature (Lond.) 199, 912 (1963).
28. GALAMBOS, R.: A glial-neural theory of brain function. Proc. nat. Acad. Sci. (Wash.) 47,
129 (1961).
29. HARPER, H. W., J. R. JAY, and R. P. ERICKSON: Chemically evoked sensations from
single human taste papillae. Physiol. Behav. 1,319 (1966).
30. HARRIS, H., and H. KALMUS: The measurement of taste sensitivity to phenylthiourea.
Ann. Eugen. (Lond.) 15,24 (1950).
31. HASLER, A. D.: Olfactory and gustatory sense of fishes. In: The physiology of fishes, II.
New York: Academic Press 1957.
32. HORNSTEIN, L, and R. TERANISHI: The chemistry of flavor. Chern. and Eng. News,
April 3, 92 (1967).
33. HYDEN, H., and E. EGYHAZI: Nuclear RNA changes of nerve cells during a learning
experiment in rats. Proc. nat. Acad. Sci. (Wash.) 48, 1366 (1962).
34. - , and P. W. LANGE: Protein synthesis in the hippocampal pyramidal cells of rats
during a behavioral test. Science 159, 1370 (1968).
35. JACOBSON, M.: Development of neural specificity in retinal ganglion cells of Xenopus.
Develop. BioI. 17,202 (1968).
36. JENSEN, L. B.: Man's foods, p. 108. Champaign (Illinois): The Garrard Press 1953.
37. KARLSON, P., and M. LUSCHER: Pheromones: A new term for a class of biologically
active substances. Nature (Lond.) 183, 55 (1959).
38. KETY, S. S.: The true nature of a book. An allegory. Nat. Inst. Hlth Record 12, 3 (1960).
39. KETY, S. S., and J. ELKES: Regional Neurochemistry. New York: Pergamon Press 1961.
40. KRAVITZ, E. A.: Acetylcholine, p-aminobutyric acid, and glutamic acid: Physiological
and chemical studies related to their roles as neurotransmitter agents. In: The neuro-
sciences, a study program, p. 433. QUARTON, G. c., T. MELNECHUK, and F. O.
SCHMITT, Eds. New York: Rockefeller Univ. Press 1967.
14*
212 B. W. AGRANOFF: Macromolecules and Brain Function - A 1969 Baedeker
41. LEVI-MoNTALCINI, R.: The nerve growth factor: Its mode of action on sensory and
sympathetic cells. Harvey Lect. 60, 217 (1966).
42. LEVINE, L.: Immunochemical approaches to the study of the nervous system. In: The
neurosciences, a study program, p. 220. QUARTON, G. C, T. MELNECHUK, and F. O.
SCHMITT, Eds. New York: Rockefeller Univ. Press 1967
43. MABRY, C, ]. C DENNISTON, and ]. G. COLDWELL: Mental retardation in children of
phenylketonuric mothers. New Engl. ]. Med. 275, 1331 (1966).
44. MCCONNELL, ]. V.: The biochemistry of memory. In: Chemistry of learning, p. 310.
CORNING, W. C., and S. C RATNER, Eds. New York: Plenum Press 1967.
45. MINER, N.: Integumental specification of sensory fibers in the development of cutaneous
local sign. ]. compo Neurol. 105, 101 (1956).
46. MOORE, B. W., and D. MCGREGOR: Chromatographic and electrophoretic fractionation
of soluble proteins of brain and liver. ]. bioI. Chern. 240, 1647 (1965).
47. NAUTA, W. H. M., and R. A. GYGAX: Silver impregnation of degenerating axons in the
central nervous system: a modified technique. Stain Technol. 29, 91 (1954).
48. NICHOLLS, ]., and D. A. BAYLOR: Receptive fields, synaptic connections, and regenera-
tion patterns of sensory neurons in the CNS of the leech. In: Society of general
physiologists symposium of Physiological and biochemical aspects of nervous
integration. F. D. CARLSON, Ed. Engleword Cliffs, N. ].: Prentice-Hall (1968).
49. PFAFFMANN, C: Gustatory nerve impulses in rat, cat and rabbit. ]. Neurophysiol. 18,
427 (1955).
50. RICHTER, D.: Metabolism of the nervous system. New York: Pergamon Press 1957.
51. ROBERTS, E., and C F. BAXTER: Neurochemistry. Ann. Rev. Biochem. 32, 513 (1963).
52. - , and S. FRANKEL: y-Aminobutyric acid in brain: Its formation from glutamic acid.
]. bioI. Chern. 187, 55 (1950).
53. ROSE, S. P. R.: Preparation of enriched fractions from cerebral cortex containing iso-
lated, metabolically active neuronal cells. Nature (Lond.) 206, 621 (1965).
54. ROSENZWEIG, M. R., and A. L. LEIMAN: Brain functions. A=. Rev. Psychol. 19, 55
(1968).
55. SEIFFERT, U. B., and B. W. AGRANOFF: Isolation and separation of inositol phosphates
from hydrolysates of rat tissues. Biochim. biophys. Acta (Arnst.) 90, 574 (1965).
56. SOKOLOFF, L.: In: New research techniques of neuroanatomy. W. F. WINDLE, Ed.
Springfield: C C Thomas 1957.
57. SPERRY, R. W.: Mechanisms of neural maturation. In: Handbook of experimental
psychology, p. 236. S. S. STEVENS, Ed. New York: Wiley 1951.
58. TALWAR, G. P., B. K. GOEL, S. P. CHOPRA, and B. D'MoNTE: Brain RNA. In: Macro-
molecules and behavior. ]. GAITO, Ed. New York: Appleton-Century-Crofts 1966.
59. TOWER, D. B.: Origins and development of neurochemistry. Neurology (Mi=eap.) 8,
Suppl. 1 (1958).
60. VARON, S., ]. NOMURA, and E. M. SHOOTER: The isolation of the mouse nerve growth
factor protein in a high molecular weight form. Biochemistry 6, 2202 (1967).
61. W AELSCH, H., and A. LAJTHA: Protein metabolism in the nervous system. Physiol. Rev.
41, 709 (1961).
62. W ALD, G.: The molecular organization of visual systems. In: Light and life, p. 724.
McELROY, W. D., and B. GLASS, Eds. Baltimore: Johns Hopkins Press 1961.
63. WHITTAKER, V. P.: Pharmacological studies with isolated cell components. Biochem.
Pharmacol. 9, 61 (1962).
64. WRIGHT, R. M.: Odor and molecular vibration. Nature (Lond.) 190, 1101 (1961).
65. ZEMP,]. W., J. E. WILSON, K. SCHLESINGER, W. O. BOGGAN, and E. GLASSMAN: Brain
function and macromolecules. 1. Incorporation of uridine into RNA of mouse brain
during short-term training experience. Proc. nat. Acad. Sci. (Wash.) 55, 1423 (1966).
Author Index
Aaronson, S., B. B. Ellenbogen, L. K. Apirion, D., see Mangiarotti, G. 37, 42
Yellen, and S. H. Hutner 159, 160, - see Schlessinger, D. 44
187 Aposhian H. V., and A. Kornberg 126,
Abelsohn,]., see Goodman,H.M. 21,41 130
- see Landy, A. 52, 79 App, A. A., and A. T. Jagendorf 157,
Adams, A., see Fresco, J. R. 69, 78 180,187
Adams,]. M., and M.R.Capecchi 17,39 Appella, E., see Potter, M. 74, 80
Adler, J. 206,210 Arca, M., C. Calvori, L. Frontall, and
Agranoff, B. W. 3 G. Tecce 51, 77
- R. E. Davis, and J. J. Brink 209,210 - L. Frontall, and G. Tecce 52, 77
- - L. Casola, and R. Lim 209,210 Arnon, D. I., H. Y. Tsujimoto, B. D.
- see Brink, J. J. 209,211 McSwain, and R. K. Chain 165, 187
- see Casola, L. 204, 209, 211 - see Buchanan, B. B. 138, 180, 189
- see Davis, R. E. 210, 211 Aronson, A. I. 89,90,115,124,130,155,
- see Seiffert, U. B. 208, 212 187
Akoyunoglou, G. A., and H. W. Siegel- - and M. R. del Valle 123, 130
man 183,187 - see del Valle, M. R. 123, 131
Aliyev, K. A., see Sissakian, N. M. 157, Ascione, R., see Fresco, J. R. 69,78
159, 199 Asheshov, I. N., see Hall, E. A. 161,170,
Allan, R. K., see McCalla, D.R. 155,195 192
Allen, C. F., O. Hirayama, and P. Good Ashkenazi, Y., see Sager, R. 33,43
137,187 Aspirion, D. 67, 77
Allen, R. L. see Kirk, J. T. O. 161, 163, Astbury, W. T. 1,4
194 Attardi, G. 47, 77
Altman, J., and G. D. Das 204,210 - see Gros, F. 89, 105, 114, 132
Amelunxen, F., see Matthaei, J. M. 15, Avron, M., see Bennun, A. 139, 188
42,49,80 Axelrod, V. D., see Bayev, A. A. 21, 26,
Amoore, J. E. 205, 206, 210 39
Anderson, E. P., see Brockman,R. W. 83,
130 Bailey,]. L., see Thornber,]. P. 165,201
Anderson, F., see Nirenberg, M. W. 69, Baker, R. K., see Heidelberger, C. 87, 132
75,80 Bakes, J., N. Befort, J. H. Weil, and J. P.
Anderson, J. M., and N. K. Boardman Ebel 53,77
164, 187 Baldwin, A. N., and P. Berg 22, 25, 50,77
- see Boardman, N. K. 138,188 Baltus, E., and J. Brachet 142, 187
Anderson, L. A., and R. M. Smillie 159, - and]. Quertier 151, 152, 153, 187
187 Bamji, M. S., and A. T. Jagendorf 157,
Anderson, P., J. Davies, and B. D. Davis 159,187
57,77 Barner, H. D., see Cohen, S. S. 88, 89,
Anderson, W. F., L. Gorini, and L. Brek- 101, 131
kenridge 77 Barnett, L., S. Brenner, F. H. C. Crick,
Andoh, T., and E. Chargaff 85, 90, 91, R. G. Shulman, and R. J. Watts-Tobin
93, 94, 98, 99, 100, 101, 126, 129, 130 20,39
Aoki, S., and E. Hase 154, 155, 160, 177, - see Brenner, S. 7, 20, 39
187 - see Crick, F. H. C. 9, 10, 40
- , J. K. Matsubara, and E. Hase 155, Barnett, W. E. 39, 51, 77
160, 187 and H. E. Brockman 113, 130
Apgar, ]., see Holley, R. W. 21, 26, 41 - and D. H. Brown 51,77,185,187
214 Author Index
Barnett, W. E., D. H. Brown, and J. L. - see Bergmann, F. H. 21, 25, 39, 50,
Epler 51, 77, 185, 187 77
- and J. L. Epler 51,77 - see Carbon, J. 34, 40
- and K. B. Jacobson 20,25,33,39,51, - see Norris, A. T. 32, 50, 80
77 - see Slapikoff, S. 126, 135
Barondes, S. H., see Cohen, H. D. 209, - see Yarus, M. 20, 26, 45, 50, 51, 81
211 Berger, H., see Brammer, W. J. 16,39
- see Nirenberg, M. W. 43 Berger, S. 153, 188
Barrell, B. G., see Brownlee, G. G. 37,39 Bergmann, F. H., P. Berg, and M. Dieck-
Bartels, P. G., K. Matsuda, A. Siegel, and mann 21, 25, 39, 50, 77
T. E. Weier 151, 187 Bergquist, P. L., see Lowrie, R. J. 75,79,
Basford, R. E., see Beattie, D. S. 158, 100, 101, 102, 103, 104, 126, 133
162, 164, 165, 188 Bergstrand, A., see Sottocasa, G. L. 165,
Basilio, c., see Gardner, R. S. 14, 41 199
- see Speyer, J. 14,33,44 Beridze, T. G., M. S. Odintsova, and N. M.
- see Wahba, A. J. 14,45,125, 135 Sissakian 143, 188
Bass, R. E., see Saunders, P. P. 85, 134 Bernfield, M., and M. W. Nirenberg 39
Bassel, A., see Hotta, Y. 184, 193 - see Brimacombe, R. 15,39
Bassham, J. A., and M. Kirk 176, 180, - see Nirenberg, M. W. 15, 43
187 Biggins, J., and R. B. Park 142, 188
- see Smith, D. C. 180, 199 Biggs, D. R., see Huang, M. 164,193
Baxter, C. F., see Roberts, E. 203,212 Binder, N. D., see Schwartz, J. H. 44
Bayev, A. A., T. V. Venkstem, A. D. Bird, 1. F., H. K. Porter, and C. R.
Mirzabekov, A. 1. Krutelina, L. Li, Stocking 139, 188
and V. D. Axelrod 21, 26, 39 Bisalputra, A. A., see Bisalputra, T. 141,
Baylor, D. A., see Nicholls, J. 212 188
Beattie, D. S., R. E. Basford, and S. B. Bisalputra, T., and A. A. Bisalputra 141,
Koritz 158,162, 164, 165, 188 188
Becarevic, A. B., Djordjevic, and D. Sutic Bishop, D. 7, 9
110,130 Bishop, D., see Spiegelmann, S. 19,44
Beckwith, J. R., see Brenner, S. 7,20,39 Bissell, D. M. 62, 73, 77
Beevers, H., see Hock, B. 163, 193 Blaydes, D. F., see Pollard, C. J. 151, 152,
Beevers, L., L. E. Schrader, D. Flesher, 153, 197
and R. H. Hageman 166,188 Bloch, K., see Constantopoulos, G. 180,
- see Schrader, L. E. 166, 198 184, 190
Befort, N., see Bakes, J. 53, 77 - see Nagai, J. 139, 196
Beidler, L. M. 205, 210 Blomback, B., see Doolittle, R. F. 19,40
von Bekesy, G. 205, 210 Boardman, N. K. 137, 151, 155, 157, 158,
Ben-Gurion, R., see Kindler, S. H. 73, 79 168, 169, 170, 188
Ben-Hamida, F., and D. Schlessinger 116, - and J. M. Anderson 138,188
130 - R. 1. B. Francki, and S. G. Wildman
Ben-Ishai, R., B. Z. Cavari, H. Goldin, and 150, 151, 157, 158, 188
S. Kerpel 124, 130 - see Anderson, J. M. 164, 187
Bennett, E. L., M. C. Diamond, D. Krech, - see Francki, R. 1. B. 157, 191
and M. R. Rosenzweig 204, 210 - see Goodchild, D. J. 184, 191
Bennett, T. P., J. Goldstein, and F. Lip- Bock, R. M., see Soll, D. 23,44
mann 67,77 Bodley, J. W., and E. W. Davie 63,77
Bennun, A., and M. Avron 166, 188 - see So, A. G. 69, 81
Benzer, S., and S. P. Champe 20,39 Bodmer, W. F., and S. Grether 87,130
- see Champe, S. P. 20,40,49,77,112, Bogard, L., and A. B. Jacobson 155, 178,
113, 131 188
- see Chapeville, F. 25, 32,40 - F. V. Mercer, and R. Mullens 183,
- see Weisblum, B. 23, 33, 45 188
Berberich, M. A., J. S. Kovach, and R. F. Boggan, W.O., see Zemp, J. W. 209,212
Goldverger 18, 39 Bogorad, L. 137,154,155,161,170,173,
Berg, P. 20, 21, 39 177, 188
- see Baldwin, A. N. 22, 25, 50, 77 - see Chen, S. 176,189
Author Index 215
Bogorad, L., see Gassman, M. 172, 173, - see Crick, F. H. C. 9, 10, 40

177, 191 - see Goodman, H. M. 21, 41
- see Kislev, N. 141, 142, 194 - see Kaplan, S. 34, 41
- see Klein, S. 176, 194 - see Sambrook, ]. F. 20, 44
Bohdanecka, M., Z. Bohdanecky, and - see Sarabhai, A. S. 7, 20, 44
M. E. ]arvik 209,210 Bretscher, M. S., and M. Grunberg-
Bohdanecky, Z., see Bohdanecka, M. Manago 67, 77
209,210 - and K. A. Marcker 18,39
Bolaffi, ]. L., see Rich, M. A. 88, 89, 134 Briantais, ].-M. 165, 189
Bolle, A., see Sarabhai, A. S. 7, 20, 44 Briggs, G. E. 171,189
Bolton, E. T., see McCarthy, B.]. 24, Briggs, W. R., and H. W. Siegelman 182,
42, 146, 195 189
Bonner, B. A., see Taylor, A. O. 169,200 - see Butler, W. L. 168, 169, 189
Bonner, ]., and]. A. D. Zeevaart 85,86, Brillat-Savarin 205,210
124, 130 Brimacombe, R., J. Trupin, M. Niren-
- see Davern, C. 1. 106,107, 131 berg, P. Leder, M. Bernfield, and
Bonner, W. D., see Suyama, Y. 143, 146, T. ]auoni 15,39
200 - see Nirenberg, M. W. 15, 43, 69, 75,
Borek, E., see Mandel, L. R. 53, 79 80
- see Tomasz, A. 89, 115, 128, 135 Brink, ]. J., R. E. Davis, and B. W.
Borst, P., see van Bruggen, E. F.]. 140, Agranoff, 209, 211
189 - see Agranoff, B. W. 209,210
Borthwick, H. A., see Hendricks, S. B. Britten, and C. P. Woese 28
183, 193 Brock, T. D. 66, 77
- see Parker, M. W. 182, 196 - see Leon, S. A. 65, 79
Bosch, L., E. Harbers, and C. Heidel- Brockman, R. W., and E. P. Anderson
berger 83, 86, 89, 101, 130 83, 130
- see van Knippenberg, P. H. 61,62, 79 - J. M. Davis, and P. Stutts 87,130
Bourdu, R., see ]oussaume, M. 139,154, Brockmann, H. E., see Barnett, W. E.
193 113,130
Bove, c., see Bove, ]. M. 154, 188 Brody, S., and C. Yanofsky 39
Bove, ]. M., C. Bove, M.-]. Rondot, and Bronchart, R., see Sironval, C. 165, 199
G. Morel 154,188 Brooks, J., see Stumpf, P. K. 139, 166,
- and]. D. Raacke 139, 157, 188 200
Boyden, S. V. 206,210 Brown, D. H., see Barnett, W. E. 51,77,
Brachet, ]., see Baltus, E. 142, 187 185,187
- see Goffeau, A. 157, 158, 159, 191 Brown, F. A. M., and B. E. S. Gunning
Brammer, W. ]., H. Berger, and C. 158, 189
Yanofsky 16,39 Brownlee, G. G., F. Sanger, and B. G.
Brawerman, G. 151,154,188 Barrell 37,39
- and E. Chargaff 154, 189 Brownstein, B., and L. J. Lewandowski
- and ]. M. Eisenstadt 140, 142, 153, 67,77
189 Bruggen, E. F. J. van, P. Borst, G.]. C. M.
- A. O. Pogo, and E. Chargaff 154,155, Ruttenberg, M. Gruber, and A. M.
189 Kroon 140,189
- see Eisenstadt, ]. M. 150, 151, 152, Bryan, G., see Klein, S. 176, 194
157,158,159, 190 Buchanan, B. B., P. P. Kalberer, and
- see Pogo, A. O. 154, 197 D. 1. Arnon 138, 180, 189
- see Schwartz, ]. H. 44 Bucher, Th., see Neupert, W. 158, 196
Brdiczka, D., see Neupert, W. 158, 196 Buck, C. A., see McCarthy, B. J. 66, 80
Breckenridge, L., see Anderson, W. F. 77 Bujard, H., and C. Heidelberger 54, 77,
Brenner, S., L. Barnett, E. R. Katz, and 113,130
F. M. C. Crick 7,20,39 Bull, M. ]., and J. Lascelles 117, 130
- and]. R. Beckwith 7,20,39 Bunning, ]., see Ritenour, G. L. 139,
- A. o. W. Stretton, and S. Kaplan 7, 166, 197
20,33,39 Burton, K., N. Varney, and P. C. Zamecnik
- see Barnett, L. 20, 39 22,39
216 Author Index
Bussard, A., S. Naono, F. Gros, and J. - see Harbers, E. 86,87,88,115, 132

Monod 54, 77, 116, 129, 130 - see Heidelberger, C. 83, 132
Butler, W. L. 168, 189 Chen, J. L., and S. G. Wildman 151,189
- and W. R. Briggs 168,169, 189 Chen, S., D. McMahon, and L. Bogorad
- S. B. Hendricks, and H. W. Siegelman 176, 189
173, 189 Cheong, L., see Rich, M. A. 88, 89, 134
- H. C. Lane, and H. W. Siegelman Cherayil, J. D., see SoIl, D. 23,44
173, 189 Cherry, J. H., and R. van Huystee 95,
- see Siegelman, H. W. 174,182,199 106, 131
Chiang, K. S., and N.Sueoka 142,!145,189
Cairns, J., G. S. Stent, and J. D. Watson Chiba, Y., and K. Sugahara 142, 189
1,4 Chopra, S. P., see Talwar, G. P. 212
Caldwell, P., and C. Hinshelwood 7,39 Christodoulou, c., see Linnane, A. W.
Calvin, M., see Holm-Hansen, O. 179, 185, 195
193 Chun, E. H. L., M. H. Vaughan Jr., and
Calvori, c., see Arca, M. 51, 77 A. Rich 142, 189
Campbell, W., see Sherman, F. 185, 198 Claes, H. 184, 189
Cantorow, A., see Rutman, R. J. 82,134 Clark, B. F. c., and K. A. Marcker 17,40
Capecchi, M. R. 20,39,69, 77 - see Dube, S. K. 21, 40
- and G. N. Gussin 40 - see Nirenberg, M. W. 14, 25, 42
- see Adams, J. M. 17,39 Clark J. M., Jr. 157,189
Capra, J. D., and A. Peterkofsky 26, 40 Clark, M. F., R. E. F. Matthews, and
- see Peterkofsky, A. 53, 80 R. K. Ralph 151,158, 189
Carbon, J., P. Berg, and C. Yanofsky 34, Clark-Walker, G. D., and A. W. Li=ane
40 164, 189
- and J. B. Curry 75, 77 - see Huang, M. 164, 193
Carell, E. F., and J. S. Kahn 138, 166, 189 - see Lamb, A. J. 164, 194
Carlton, B. c., see Yanofsky, C. 7,45 Click, R. E., and D. P. Hackett 152, 189
Caskey, C. T., see Marshall, R. E. 33,42 - and B. L. Tint 152, 189
- see Nirenberg, M. W. 69,75,80 Clijsters, H., see Sironval, C. 165, 199
Casola, L., R. Lim, R. E. Davis, and Cocking, E. C., see Hall, T. C. 157, 192
B. W. Agranoff 204, 210, 211 Cohen, H. D., F. Ervin, and S. H. Barondes
Casola, L., see Agranoff, B. W. 209,210 209,211
Catsky, J., see Sestak, Z. 171, 198 Cohen, J. A., see Warnaar, S. O. 148,201
Cavari, B. Z., see Ben-Ishai, R. 124, 130 Cohen, M. J., and J. W. Jacklet 207,211
Cecere, M. A., see Thach, R. E. 44 Cohen, R. Z., and T. W. Goodwin 174,
Cerna, J., J. Rychlik, D. Grtinberger, and 184, 189
F. Sorm 114, 115, 131 Cohen, S. S., J. G. Flaks, H. D. Barner,
Chain, R. K., see Amon, D. I. 165, 187 M. R. Loeb, and J. Lichtenstein 88,
Champe, S. P., and S. Benzer 20, 40, 49, 89,101,131
77,112,113,131 Cohn, M., see Lennox, E. S. 74,79
- see Benzer, S. 20, 39 Coker, L., see Ogur, M. 185, 196
Chang, S. H., see Rajbhandary, U. L. 21, Coldwell, J. G., see Mabry, C. 208, 212
43 Collins, A., see Marver, H. S. 118, 134
Chapeville, F., F. Lipman, G. von Ehren- Constantopoulos, G., and K. Bloch 180,
stein, B. Weisblum, W. Ray, and S. 184, 190
Benzer 25,32,40 Cook, J. R. 145, 190
Chargaff, E. 1,4 - and W. Hunt 145, 190
- see Andoh, T. 85, 90, 91, 93, 94, 98, Cooper, P. D. 107, 121, 129, 131
99, 100, 101, 126, 129, 130 Cooper, S., and N. D. Zinder 124, 131
- see Brawerman, G. 154, 155, 189 - see Lodish, H. F. 109, 133
- see Horowitz, J. 83, 84, 85, 86, 88, Cortner, J. A., see Davidson, R. G. 185,
89, 114, 115, 117, 124, 132 190
- see Pogo, A. O. 154, 197 Cory, S., see Dube, S. K. 21, 40
Chatterjee, S. K., see Das, H. K. 164, 190 Cowan, C. A., see Edelman, M. 142,190
Chandhuri, N. K., B. J. Montag, and Cox, E. c., J. R. White, and J. G. Flaks
C. Heidelberger 84,85,86,88, 131 56,78
Author Index 217
Cox, E. c., and C. Yanofsky 24, 40 - see Brink, J.]. 209,211

- see Flaks, J. G. 57,78 - see Casola, L. 204, 210, 211
Crain, S. M. 204, 211 de Kloet, S. R. 83, 85, 93, 95, 98, 106,
Crane, F. L., see Henninger, M. D. 138, 116,117,131
165, 193 - and P. ]. Strijkert 85, 95, 131
Crick, F. H. C. 6, 11, 13, 21, 23, 26, 40 Delbriick, M. 2
- L. Barnett, S. Brenner, and R. ]. - see Pauling, L. 43
Watts-Tobin 9, 10, 40 del Campo, F. F., see Losada, M. 166, 195
- J. Griffith, and L. E. Orgel 9, 11,28, - see Ramirez, J. M. 166, 197
40 Delius, H., see Moore, P. B. 21, 37, 42
- see Barnett, L. 20, 39 - see Traut, R. R. 45
- see Brenner, S. 7, 20, 39 del Valle, M. R., and A. I. Aronson 123,
- see Watson, J. D. 6,45 131
Criddle, R. S. 138, 165, 190 Denhardt, D. T. 148, 190
Crocco, R. M., see Garren, L. D. 118,131 Denniston, J. c., see Mabry, C. 208,212
Crudup, K., see Sells, B. H. 90, 93, 96, de Robertis, E., A. Pellegrino de Iraldi,
97, 98, 105, 106, 129, 134 G. Rodriguez, and C. J. Gomez 207,
Curreri, A. R., see Mukherjee, K. L. 85, 211
86,134 De Wachter, R., and W. Fiers 19,40
Curry, J. B., see Carbon, J. 75, 77 Diamond, M. c., see Bennett, E. L. 204,
Czygan, F.-C. 160, 190 210
Dias, D., see Ehrenstein, G. von 40
Dagg, C. P., A. Doerr, and C. Offutt 85, Dieckmann, M., see Bergmann, F. H. 21,
86, 131 35, 39, 50, 77
Dahlstrom, A., and K. Fuxe 207,211 Dintzis 7,9
Danneberg, P. B., see Heidelberger, C. Disbrey, c., see Randall, J. 184, 197
83, 132 Djordjevic, B., see Becarevic, A. 110, 130
Das, G. D., see Altman, J. 204,210 - see Sutic, D. 121, 135
Das, H. K., S. K. Chatterjee, and S. C. Roy d'Monte, B., see Talwar, G. P. 212
164, 190 Doctor, B. P., see Kellogg, D. A. 23,41
Dastoli, F. R., and S. Price 205, 211 - see Nirenberg, M. W. 69, 75, 80
Davenport, H. E., and R. Hill 138, 190 Doerr, A., see Dagg, C. P. 85,86, 131
Davern, C. I. 109, 131 Dondon, ]., see Grunberg-Manago, M.
- and]. Bonner 106, 107, 131 71,78
Davidson, R. G., and J. A. Cortner 185, Doolittle, R. F., and B. Blomback 19,40
190 Dotty, P., see Schildkraut, C. L. 198
Davie, E. W., see Bodley, J. W. 63,77 - see Sueoka, N. 44
- see So, A. G. 68, 69, 71, 81 - see Thach, R. E. 44
Davies, J. 56, 60, 63, 64, 65, 68, 71, 73, - see Zubay, G. 28,45
78 Dounce, A. 6,7,11,40
- and B. D. Davis 63, 64, 78 - M. Morrison, and K. J. Monty 28,40
- W. Gilbert, and L. Gorini 25,26,27, Dowben, R. M., see Heywood, S. M.
40,57,68, 78 158, 193
- L.Gorini,andB.D.Davis 57,63,71,78 Dube, S. K., K. A. Marcker, B. F. C.
- D. S. Jones, and H. G. Khorana 58, Clark, and S. Cory 21, 40
59,78 Dugre, D. A., see Woese, C. R. 26,28,
- see Anderson, P. 57, 77 35,45
- see Gorini, L. 61, 78 Duncan, M. J., and P. R. Stewart 164,
- see Weisblum, B. 76,81 190
Davies, W. H., E. I. Mercer, and T. W. Duranton, J. 177,190
Goodwin 139, 190 Duschinsky, R., E. Pleven, and C. Heidel-
Davis, B. D., see Anderson, P. 57, 77 berger 83, 131
- see Davies, J. 57,63, 64, 78 - , see Heidelberger, C. 83, 132
Davis, J. M., see Brockman, R. W. 87, - see Koechlin, B. A. 87,133
130 - see Lozeron, H. A. 110, 133
Davis, R. E., and B. W. Agranoff 210,211 Dutting, D., see Zachau, H. G. von 21,
- see Agranoff, B. W. 209,210 26,46
218 Author Index
Dwyer, M. R. 156,169,179, 190 Evans, W. R., and R. M. Smillie 155,191

- see Smillie, R. M. 161, 162, 163, 165, - R. Walenga, and C. Johnson 163,191
186, 199 - see Smillie, R. M. 139, 152, 154, 155,
Dyer, T. A., and R. M. Leech 141,153, 156, 159, 160, 163, 199
185,190 Evenari, M. 180, 191
Everett, G. A., see Holley, R. W. 21, 26,
Ebel, J. P., J. H. Weil, B. Rether, and 41
J. Heinrich 102,131 - see Madison, J. T. 21,42,103, 134
- see Bakes, J. 53, 77 Everson, R. G., and C. R. Slack 138, 191
- see Giege, R. 75, 78
Echlin, P., and 1. Morris 160,161, 190 Fangman, W. L., and F. C. Neidhardt 33,
Eckert, K., see Matthaei, J. H. 49,80 40,52,78
Economou, A., and J. Nakamoto 18, 40 Farr, D. P., see Sambrook, J. F. 20, 44
Edelman, M., C. A. Cowan, H. T. Ep- Faulkner, R. D., see Rajbhandary 21,43
stein, and J. A. Schiff 142, 190 Feierabend, J. 161, 191
- H. T. Epstein, and J. A. Schiff 140, - and A. Pirson 170,175,177,191
143, 190 Feldmann, H., see Zachau, H. G. von 21,
- J. A. Schiff, and H. T. Epstein 144, 26,46
190 Fernandez-Morau, H., see Woodcock,
- D. Swinton, J. A. Schiff, H. T. Ep- C.L.F. 140,141,202
stein, and B. Zeldin 149, 190 Fetherolf, K., see Levinthal, C. 42
Edsall, J. T. 1,4 Fiers, W., see DeWachter, R. 19,40
Egyhazi, E., see Hyden, H. 207,209, 211 Fikus, M., K. L. Wierzchowski, and
Ehrenstein, G. von 73, 74, 78 D. Shugar 110, 131
- and D. Dias 40 Filippovich, I. I., see Sissakian, N. M.
- see Chapeville, F. 25, 32, 340 157,159, 199
- see Gonano, F. 26,27 Fischer, R., and F. Griffin 205, 211
- see Weisblum, B. 23,33,45 Fishbein, M. 211
Eidinoff, M. L., see Rich, M. A. 88, 89, Fisher, D. B., and W. A. Jensen 184, 191
134 Flaks, J. G., E. C. Cox, and J. R. White
Eigner, E. A., see Loftfield, R. B. 20, 42 57,78
Eisenman, R. N., see Guttman, H. N. - see Cohen, S. S. 88,89,101, 131
184, 192 - see Cox, E. C. 56, 78
Eisenstadt, J. M. 157, 158, 159, 190 Fleissner, E. 40
- and G. Brawerman 150,151,152,157, Flesher, D., see Beevers, L. 166, 188
158, 159, 190 Flexner, J. B., and L. B. Flexner 209,211
- see Brawerman, G. 140, 142, 153, 189 - L. B. Flexner, and E. Stellar 209, 211
- see Schwartz, J. H. 44 Flexner, L. B., see Flexner, J. B. 209,211
Elion, G. B., and G. H. Hitchings 83, 131 Folch-Pi, J. 203, 211
Elkes, J., see Kety, S. S. 203,211 Foote, M., see Racusen, D. 171,183, 197
Ellenbogen, B. B., see Aaronson, S. 159, Frankel, S., see Roberts, E. 208, 212
160, 187 Francki, R. 1. B., N. K. Boardman, and
Emrich, J., see Terzaghi, E. 16,44 S. G. Wildman 157,191
Engelhardt, D. L., see Webster, R. E. 17, - see Boardman, N. K. 150, 151, 157,
45 158, 188
Ennis, H. L., and M. Lubin 161, 190 French, C. S., see Hill, R. 168, 193
Epler, J. L., see Barnett, W. E. 51, 77, - see Koski, V. M. 167, 194
185, 187 Fresco, J. R., A. Adams, R. Ascione,
Epstein, H. T., and J. A. Schiff 146, 190 D. Henley, and T. Lindahl 69, 78
- see Edelman, M. 140, 142, 143, 144, Frey-Wyssling, A. 165, 191
149, 190 Friedman, S. M., and 1. B. Weinstein 25,
- see Lefl', J. 142, 194 26, 27, 40, 68, 69, 70, 78
- see Schiff, J. A. 136, 169, 177, 198 - see Weinstein,!. B. 72, 81
Erickson, R. P., see Harper, H. W. 205, Friend, J., and A. M. Mayer 139, 191
211 Frontall, L., see Area, M. 51, 52, 77
Ernster, L., see Sottocasa, G. L. 165, 199 Fukuhara, H. 185, 191
Ervin, F., see Cohen, H. D. 209, 211 Fuller, R. C., see Shibata, K. 136, 198
Author Index 219
Fuller, W., and A. Hodgson 22, 26, 40 Gilbert, W. 19,21,41

Fullman, B. 206, 211 - see Davies, J. 25, 26, 27, 40, 57, 68,
Furuya, M., and W. S. Hillman 182, 191 78
Fuxe, K., see Dahlstrom, A. 207, 211 - see Gros, F. 89, 105, 114, 132
Gillespie, D., and S. Spiegelman 146, 191
Gabriel, T., see Koechlin, B. A. 87, 133 Glasgow, J. E., see Taylor, M. M. 150,
- see Lozeron, H. A. 110, 133 200
Gado, 1., and 1. Horvath 73, 78 Glassman, E., see Zemp, J. W. 209,212
Gaetani, S., and M. A. Spadoni 131 Gnanam, A., and J. S. Kahn 150, 151,
Gailey, F. B., and N. E. Tolbert 172,191 191
Galambos, R. 204, 211 Goel, B. K., see Talwar, G. P. 212
Gale, E. F. 159, 191 Goffeau, A., and J. Brachet 157, 158,
Galliard, T., see Stumpf, P. K. 139, 166, 159, 191
200 Golaszewski, T., see Szarkowski, J. W.
Galloway, R. A., and R. W. Krauss 160, 139,177,200
161, 191 Goldberg, A. R., J. H. Machledt Jr., and
Gallucci, E., and A. Garen 41 A. B. Pardee 87, 131
Galston, A. W., see Goren, R. 174, 183, Goldin, H., see Ben-Ishai, R. 124, 130
191 Goldstein, A., J. B. Kirshbaum, and A.
Galun, E., and J. Gressel 123, 131 Roman 41
- see Gressel, J. 85, 95, 100, 132 Goldstein, J., see Bennett, T. P. 67,77
Gamow, G. 6, 8, 9, 11, 28, 41 Goldverger, R. F., see Berberich, M. A.
- A. Rich, and M. Y cas 13, 14 18,39
- and M. Ycas 11,12 Golubeva, E. V., see Odintsova, M. S.
Gardner, R. S., A. J. Wahba, C. Basilio, 151, 196
R. S. Miller, P. Lengyel, and]. Speyer Gomez, C. J., see de Robertis, E. 207,
14,41 211
- see Speyer, J. 14,33,44 Gonano, F. 26,33,41
- see Wahba, A. J. 45,125, 135 - and G. von Ehrenstein 26,27
Garen, A., and O. Siddiqi 20,41,113,131 - see Weisblum, B. 23,33,45
- S. Garen, and R. C. Wilhelm 20, 41 Good, P., see Allen, C. F. 137, 187
- see Gallucci, E. 41 Goodchild, D. J., H. R. Highkin, and
- see Weigert, M. 20,33,45 N. K. Boardman 184, 191
Garen, S., see Garen, A. 20,41 Goodman, F. 124,131
Garren, L. D., R. R. Howell, G. M. Goodman, H. M., J. Abelsohn, A. Landy,
Tomkins, and R. M. Crocco 118, 131 S. Brenner, and J. D. Smith 21,41
Gartland, W. J., see Sueoka, N. 75, 81 - see Landy, A. 52, 79
Gassman, M., and L. Bogorad 172, 173, Goodwin, T. W. 136
177,191 - see Cohen, R. Z. 174, 184, 189
Gause, G. G., and D. Grunberger 76, 78 - see Davies, W. H. 139,190
Gefter, M. L., and R. Russell 75, 78 - seeHenshall,J.D. 139,157,174,176,
Geisser, S., see Potter, M. 74, 80 184, 193
Gelardi, c., see Henninger, M. D. 138, - see Rogers, L. J. 197
165, 193 - see Treharne, K. J. 166,201
Getz, G., see Swift, H. 178, 200 - see Wieckowski, S. 179, 201
Ghobar, A., see Heidelberger, C. 87,132 Gordon, M. P., and M. Staehelin 88, 106,
Ghosh, H. P., and H. G. Khorana 17,41 107,131
- D. SoIl, and H. G. Khorana 17,41 - see Green, B. R. 142, 143, 145, 192
Gibbs, M., see Russell, G. K. 162, 198 - see Lozeron, H. A. 107, 110, 133
Gibbs, S. P. 141, 167, 185, 191 - see Staehelin, M. 85,86,106,107, 135
Gibor, A. 144, 191 Goren, R., and A. W. Galston 174, 183,
- and S. Granick 141, 191 191
- and M. Izawa 142, 191 Gorini, L. 73
Gibson, J., see Suyama, Y. 141,200 - and J. Davies 61, 78
Giege, R., J. Heinrich, J.-H. Weil, and - and E. Kataja 26,41,57,67,73,78
J.-P. Ebel 75, 78 - R. Rosset, and R. A. Zimmermann
Gierer, A., see Mundry, K. W. 42 63,78
220 Author Index
Gorini, L., see Anderson, W. F. 77 Grunberg-Manago, M., and J. Dondon

- see Davies, J. 25, 26, 27, 40, 57, 63, 71,78
68,71,78 - and A. M. Michelson 27,41,54, 55,
- see Jacoby, G. A. 57,79 79,125, 127, 132
- see Old, D. 63, 80 - see Bretscher, M. S. 67, 77
- see Rosset ,R. 76,81 - see Lamfrom, H. 72, 79
Gorman, D. S., and R. P. Levine 185, - see Letendre, C. 22, 42, 52, 79
192 Guest, J. R., see Yanofsky, C. 7,45
Graebe, J. E., and G. D. Novelli 159,192 Gunning, B. E. S. 137,141, 192
Graham, A. F., and C. Kirk 85, 110, 132 - see Brown, F. A. M. 158, 189
Graham, D., A. M. Grieve, and R. M. Gussin, G. N., see Capecchi, M. R. 40
Smillie 170, 175, 176, 177, 192 Guthrie, c., see Nomura, M. 43, 80
- - M. D. Hatch, C. R. Slack, and R. M. Guttman, H. N., and R. N. Eisenman
Smillie 170,175,176,177,192 184, 192
- see Smillie, R. M. 161, 162, 185, 186, Gygax, R. A., see Nauta, W. H. M. 207,
199 212
Granick, S. 136,166,172, 192
- see Gibor, A. 141,191 Hackett, D. P., see Click, R. E. 152, 189
Gray, P. N., and M. Rachmeler 102, 132 Hadziyev, D., S. L. Metha, and S. Zalik
Green, B. R., and M. P. Gordon 142, 152, 192
143, 145, 192 Hageman, R. G., see Schrader, L. E.
Gregory, R. P. F., see Thornber, J. P. 166, 198
165,201 Hageman, R. H., see Beevers, L. 166, 188
Gressel, J., and E. Galun 85,95,100,132 - see Ingle, J. 166, 193
- see Galun, E. 123, 131 - see Ritenour, G. L. 139,166, 197
Grether, S., see Bodmer, W. F. 87, 130 Hahn, G. A., and H. G. Mandel 93,95,
Greuer, B., see Kroger, H. 118, 133 98, 99, 126, 129, 132
Griffith, J., see Crick, F. H. C. 9, 11,28, Hall, B. D., and S. Spiegelman 146, 192
40 Hall, D.O., R. C. Huffaker, L. M. Shannon,
Griesbach, L., see Heidelberger, C. 83, and A. Wallace 192
132 Hall, E. A., F. Kavanagh, and I. N.
Grieve, A. M., see Graham, D. 170,175, Asheshov 161,170, 192
176,177,192 Hall, T. c., and E. C. Cocking 157, 192
Grieve, A., see Smillie, R. M. 161, 162, - see Kessel, D. 89, 133
163,165,186, 199 Hamilton, M. G., see Sager, R. 150, 151,
Griffin, F., see Fischer, R. 205,211 198
Grijm-Vos, M., see van Knippenberg, Hanawalt, P. c., see Ray, D. S. 140, 142,
P. H. 61, 62, 79 197
Gros, F., W. Gilbert, H. Hiatt, G. Attardi, Handschumacher, R. E., see Skoda, J.
P. F. Spahr, and J. D. Watson 89, 101,135
105,114,132 Harbers, E., N. K. Chandhuri, and C.
- and S. Naono 102,114,117,132 Heidelberger 86,87,88, 115, 132
- S. Naono, D. Hayes, F. Hayes, and - see Bosch, L. 83,86,89,101, 130
J. D. Watson 93,97, 102, 129, 132 Harper, H. W., J. R. Jay, and R. P.
- see Bussard, A. 54, 77, 116, 129, 130 Erickson 205, 211
- see Naono, S. 114,116,117,120,129, Harris, H., and H. Kalmus 205, 211
134 Harris, J. I., see Waller, J. P. 21,45
- see Yaniv, M. 52,81 Harris, R. c., and J. T. O. Kirk 180,192
Gross, E., and B. Witkop 19, 41 Hartmann, K. L., see Klubes, P. 117,133
Gross, S. R., see Printz, D. B. 52,81 Hase, E., see Aoki, S. 154,155,177, 187
Grossmann, L. 54, 76, 78 - see Matsuka, M. 179, 195
Gruber, M., see van Bruggen, E. F. J. - see Sokawa, Y. 169, 199
140, 189 Haselkorn, R., see Shipp, W. S. 143, 199
Grilnberger, D., and H. G. Mandel 97, Hashimoto, K. 67, 79
132 Hasler, A. D. 205, 211
- see Cerna, J. 114,115, 131 Hatch, M. D., and C. R. Slack 138,177,
- see Gause, G. G. 76, 78 192
Author Index 221
Hatch, M. D., see Graham, D. 170, 175, Henning, U., see Yanofsky, C. 7,45
176, 177, 192 Henninger, M. D., C. Gelardi, and F. L.
- see Slack, C. R. 138, 199 Crane 138, 165, 193
Hatfield, D., see Nirenberg, M. W. 69, Henningsen, K. W. 181, 193
75,80 Henshall, J. D., and T. W. Goodwin
Hatton, M. W. c., see Thornber, J. P. 139, 157, 174, 176, 184, 193
165,201 Herzog, A. 62, 79
Hattori, A., and 1. Uesugi 167, 193 Heywood, S. M., R. M. Dowben, and
- see Nagahisa, M. 139, 196 A. Rich 158, 193
Haupt, W. 169,193 Hiatt, H., see Gross, F. 89,105,114, 132
Hauschild, A. H. W., C. D. Nelson, and Highkin, H. R., see Goodchild, D. J.
G. Krotkov 179,193 184, 191
Hawke, J. c., see Stumpf, P. K. 139,166, Hignett, R. C. 85,93,117,120,129,132
200 Hill, R. 138, 193
Hayashi, H., and K. Miura 22,41,52, 79 - J. H. C. Smith, and C. S. French 168,
- see Kuwano, M. 22, 41 193
Hayashi, Y., see Kuwano, M. 22,41 - see Davenport, H. E. 138, 190
Hayatsu, H., see Nishimura, S. 9, 10, 25, Hille, M. B., see Salas, M. 18, 43
43 - see Stanley, W. M. Jr. 14, 44
- see SolI, D. 15,44 Hillman, W. S., see Furuya, M. 182, 191
Hayes, D., see Gros, F. 93, 97, 102, 129, Hills, D. c., and J. Horowitz 85,90,91,
132 93, 97, 98, 99, 100, 101, 105, 129, 132
Hayes, F., see Gros, F. 93, 97, 102, 129, Hilse, K., and R. A. Popp 76,79
132 Hind, G., see Olson, J. M. 136, 196
Heber, M., see Heber, U. 138, 175, 193 Hinshelwood, c., see Caldwell, P. 7,39
Heber, U. 139,157,175, 193 Hirayama, 0., see Allen, C. F. 137, 187
- N. G. Pon, and M. Heber 138, 175, Hirsh, D. 1., see Rifkin, D. B. 34, 43,
193 73,74,81
Heidelberger, C. 82,83, 87, 89, 113, 124, Hitchings, G. H., see Elion, G. B. 83,131
126, 128, 132 Hock, B., and H. Beevers 163, 193
- N. K. Chandhuri, P. B. Danneberg, Hodgson, A., see Fuller, W. 22, 26, 40
D. Mooren, L. Griesbach, R. Du- Hogg, L., see Wachsman, J. T. 88, 89,
schinsky, R. J. Schnitzer, E. Pleven, 135
and J. Scheiner 83, 132 Holland, J. J., see McCarthy, B. J. 66,80
- A. Ghobar, R. K. Baker, and K. L. Holley, R. W., J. Apgar, G. A. Everett,
Mukherjee 87,132 J. T. Madison, M. Marquisee, J. R.
- G. Kaldor, K. L. Mukherjee, and Penwick, and A. Zimir 21,26,41
P. B. Danneberg 87,132 - , see Weisblum, B. 23,45
- see Bosch, L. 83, 86, 89, 101, 130 - see Zamir, A. 22,45
- see Bujard, H. 54, 77, 113, 130 Holm-Hansen, 0., K. Nishida, V. Moses,
- see Chandhuri, N. K. 84, 85, 86, 88, and M. Calvin 179,193
131 Holoubek, V. 106, 107, 121, 132
- see Duschinsky, R. 83, 131 Horiuchi, K., see Lodish, H. F. 85, 109,
- see Harbers, E. 86, 87, 88, 115, 132 123, 129, 133
- see Lampkin-Hibbard, J. M. 87, 133 Hornstein, 1., and R. Teranishi 205, 209,
- see Mukherjee, K. L. 85, 86, 134 211
- see Wagner, N. J. 101, 135 Horowitz, J., and E. Chargaff 83,84,85,
Heinrich, J., see Ebel, J. P. 102, 131 86,124,132
- see Giege, R. 75, 78 - and V. Kohlmeier 105, 116, 119, 129,
Helinski, D. R., see Yanofsky, C. 7,45 132
Heller, G., see Matthaei, J. H. 15, 42, - J. J. Saukkonen, and E. Chargaff 88,
49, 80 89,114,115,117,124,132
Hendricks, S. B., and H. A. Borthwick - see Hills, D. C. 85,90,91, 93, 97, 98,
183, 193 99, 100, 101, 105, 129, 132
- see Butler, W. L. 173, 189 Horvath, 1., see Gado, 1. 73, 78
- see Parker, M. W. 182, 196 Hoskinson, R. M., see Rajbhandary, U. L.
Henley, D., see Fresco, J. R. 69, 78 21,43
222 Author Index
Hosoda, J., see Kadowaki, K. 117, 133 - see Keister, D. L. 138, 170, 176, 194
Hosokawa, K., see Traub, P. 65, 81 - see Mego, J. L. 137,174,176, 195
Hotta, Y., A. Bassel, and H. Stem 184, - see Shibato, K. 136, 198
193 Jarvik, M. E., see Bohdanecka, M. 209,
Howell, R. R., see Garren, L. D. 118, 131 210
Howell, S. H., see Moudrianakis, E. N. Javid, M., see Mukherjee, K. L. 85, 86,
165, 196 134
Hsiao, T. C. 150,193 Javoni, T., see Brimacombe, R. 15,39
Huang, M., D. R. Biggs, C. D. Clark- Jay, J. R., see Harper, H. W. 205,211
Walker, and A. W. Linnane 164,193 Jayaraman, J., see Tewari, K. K. 140,200
Hudock, G. A., and R. P. Levine 160, Jensen, L. B. 209, 211
169, 193 Jensen, W. A., see Fisher, D. B. 184, 191
Huffaker, R. c., see Hall, D. O. 192 Jesensky, c., see Peterkofsky, A. 53,80
Hunt, W., see Cook, J. R. 145, 190 Johnson, c., see Evans, W. R. 163, 191
Hurwitz, J., see Kahan, F. M. 126, 133 Johnson, M. P., see Liverman, J. L. 182,
Hutner, S. H., see Aaronson, S. 159, 160, 195
187 Jones, D. S., see Davies, J. 58,59, 78
Huystee, R., van see Cherry, J. H. 95, - see Nishimura, S. 9, 10, 25, 43
106, 131 - see Soll, D. 15,44
Huzisige, H., see Nishimura, H. 169, 196 Jones, O. T. G. 138,193
Hyden, H., and E. Egyhazi 207,209,211 Jones,O. W., and M. W. Nirenberg 14,41
- and P. W. Lange 209, 211 - see Matthaei, J. H. 67,80
- see Nirenberg, M. W. 43
Igarashi, K., and A. Kaji 49, 79 - see Singer, M. F. 54,81
Imahori, K., see Ohta, T. 18, 21, 43 Joussaume, M., and R. Bourdu 139,154,
Imamoto, F., T. Yamane, and N. Sueoka 193
51,79 Joy, K. W., see Ingle, J. 166, 193
Ingle, J., K. W. Joy, and R. H. Hageman - see Ritenour, G. L. 139,166, 197
166,193
- , see Key, J. L. 95, 106, 118, 124, Kadenbach, B. 158, 164, 165, 193
133 Kadowaki, K., J. Hosoda, and B. Maruo
- see Loening, U. E. 151, 152, 195 117,133
Inouye, M., see Terzaghi, E. 16,44 Kahan, F. M., and J. Hurwitz 126, 133
Isham, K. R., see Stulberg, M. P. 22, 44 Kahn, A., see Virgin, H. 1. 181,210
Ishida, M. R., see Sager, R. 142, 198 Kahn, J. S., see Carell, E. F. 138, 166, 189
Iwabushi, M., E. Otaka, M. Kono, and - see Gnanam, A. 150,151, 191
S. Osawa 90, 91, 96, 97, 98, 99, 101, Kaji, A., see Igarashi, K. 49, 79
106, 132 - see Kaji, H. 49, 60, 79
Iwamura, T. 142,179, 193 Kaji, H., and A. Kaji 60, 79
- and S. Kuwashima 179, 193 - 1. Suzuka, and A. Kaji 49, 60, 79
Izawa, M., see Gibor, A. 142, 191 - and Y. Tanaka 65,79
Kalberer, P. P., see Buchanan, B. B. 138,
Jacklet, J. W., see Cohen, M. J. 207,211 180, 189
Jacob, F., and J. Monod 123, 132 Kaldor, G., see Heidelberger, C. 87, 132
- see Yaniv, M. 52,81 Kalf, G. F., see O'Brien, T. W. 150, 151,
Jacob, T. M., see Nishimura, S. 9,10,14, 196
25,43 Kalmus, H., see Harris, H. 205, 211
Jacobson, A. B., see Bogard, L. 155,178, Kammen, A. van 157, 193
188 Kano-Sueoka, T., see Sueoka, N. 75, 81
Jacobson, K. B. 25,41 Kaplan, H. S., K. C. Smith, and P. A.
- see Barnett, W. E. 20, 25, 33, 39, 51, Tomlin 110,133
77 Kaplan, S., A. O. Stretton, and S. Brenner
Jacobsen, M. 204,211 20,34,41
Jacoby, G. A., and L. Gorini 57, 79 - see Brenner, S. 7, 20, 33, 39
Jagendorf, A. T., see App, A. A. 157, Karlson, P., and M. Luscher 206, 211
180, 187 Karu, A. E., see Moudrianakis, E. N.
- see Bamji, M. S. 157,159, 187 165, 196
Author Index 223
Kataja, E., see Gorini, L. 26,41, 57, 67, Klein, S., and J. Neuman 179, 194
73,78 - G. Bryan, and L. Bogorad 176, 194
Kates, M., see Sastry, P. S. 139, 198 Klein, W. H., L. Price, and K. Mitrakos
Katoh, S., I. Suga, I. Shiratori, and A. 174, 194
Takamiya 138, 194 - see Price, L. 172,173,174, 197
Katz, E. R., see Brenner, S. 39 - see Sisler, E. C. 172, 199
Kavanagh, F., see Hall, E. A. 161, 170, Klubes,P.,andK.L.Hartmann 117,133
192 Knippenberg, P. H., van, J. C. van
Kay, J. E., and A. Komer 161,194 Ravenswaay Claasen, M. Grijm-Vos,
Keister, D. L., A. T. Jagendorf, and A. H. Veldstra, and L. Bosch 61,62, 79
San Pietro 138, 170, 176, 194 - M. Grijm-Vos, H. Veldstra, and L.
- A. San Pietro, and F. E. Stolzenbach Bosch 61,79
194 Knoll, J. E., see Rich, M. A. 88,89, 134
Kellogg, D. A., B. P. Doctor, J. E. Koechlin, B. A., F. Rubio, S. Palmer, T.
Loebel, and M. W. Nirenberg 23,41 Gabriel, and R. Duschinsky 87, 133
- see Nirenberg, M. W. 69, 75, 80 Kohlmeier, V., see Horowitz, J. 105,
Kemp, S., see Wachsman, J. T. 88, 89, 116, 119, 129, 132
135 Kolakofsky, D., and T. Nakamoto 41
Kempner, E. S. 85,89, 133 - see Nakamoto, T. 18, 42
- and J. H. Miller, 90, 114, 115, 133, Kondo, M. 33,41
145, 194 Konigsberg, W., see Rifkin, D. B. 34,43,
Kendrew, J. c. 1,4 73,74,81
Kerpel, S., see Ben-Ishai, R. 124, 130 Kono, M., and S. Osawa 90,91,93,97,
Kerridge, D. 161,194 99,133
Kessel, D., T. C. Hall, and I. Wodinsky - E. Otaka, and S. Osawa 93, 133
89, 133 - see Iwabushi, M. 90, 91, 96, 97, 98,
Kety, S. S. 210, 211 99, 101, 106, 132
- and J. Elkes 203,211 Koritz, S. B., see Beattie, D. S. 158, 162,
Key, J. L. 85,86,93,95,106,133 164, 165, 188
- and J. Ingle 95,106,118,124, 133 Kornberg, A., see Aposhian, H. V. 126,
Khorana, H. G. 10, 15, 58 130
- see Davies, J. 58, 59, 78 Komer, A., see Kay, J. E. 161,194
- see Ghosh, H. P. 17, 41 Koski, V. M., C. S. French, and J. H. C.
- see Kossel, H. 41 Smith 167,194
- see Morgan, A. R. 42, 66, 80 Kassel, H., A. R. Morgan, and H. G.
- see Nishimura, S. 9, 10, 14, 25, 43 Khorana 41
- see Rajbhandary, U. L. 21,43 Kovach, J. S., see Berberich, M. A. 18,
- see Soll, D. 15, 44 39
Kibler, H., see Matthaei, J. H. 15,42 Kowallik, W. 179, 194
Kieras, F. J., see Shipp, W. S. 143, 199 Kramer, G., H. G. Wittmann, and H.
Kilgore, W. W., and R. R. Painter 85, 86, Schuster 85, 107, 121, 129, 133
133 Krauss, R. W., see Galloway, R. A. 160,
Kim, Y. T. see Semal. J. 153, 198 161, 191
Kindler, S. H., and R. Ben-Gurion 73, 79 Kravitz, E. A. 207, 208, 211
Kirk, c., see Graham, A. F. 85, 110, 132 Krech, D., see Bennett, E. L. 204,210
Kirk, J. T. O. 137, 139, 140, 142, 149, Kroger, H., and B. Greuer 118, 133
153,185, 194 Kroon, A. M. 164,194
- and R. L. Allen 161, 163, 1194 - see van Bruggen, E. F. J. 140, 189
- and R. A. E. Tilney-Bassett 136, 137, Krotkov, G., see Hauschild, A. H. W.
158, 194 179, 193
- see Harris, R. C. 180, 192 - see Smillie, R. M. 154, 199
Kirk, M., see Bassham, J. A. 176, 180, Krutelina, A. I., see Bayev, A. A. 21, 26,
187 39
- see Smith, D. C. 180, 199 Kung, H., see Madison, J. T. 21, 42,
Kirshbaum, J. B., see Goldstein, A. 41 103, 134
Kislev, N., H. Swift, and L. Bogorad 141, Ktintzell, H., and H. Noll 151, 194
142, 194 Kupke, D. W. 183,194
224 Author Index
Kurland, C. G., see Likover, T.E. 25, - see Hudock, G. A. 160,169, 193
42, 61, 71, 79 Levinthal, c., E. Signer, and K. Fetherolf
Kuwano, M., Y. Hayashi, H. Hayashi, 42
and K. Miura 22, 41 Lewandowski, L. J., see Brownstein, B.
Kuwashima, S., see Iwamura, T. 179,193 67,77
Kuylenstierna, B., see Sottocasa, G. L. Li, L., see Bayev, A. A. 21,26,39
165, 199 Lichtenstein, J., see Cohen, S. S. 88, 89,
101,131
Lajtha, A., see Waelsch, H. 207,212 Likover, T. E., and C. G. Kurland 25,42,
Lal, K. N., see Singh, B. N. 171,199 61,71,79
Lamb, A. J., G. D. Clark-Walker, and Lim, R., see Agranoff, B. W. 209,210
A. W. Linnane 164,199 - see Casola, L. 204, 210, 211
- see Linnane, A. W. 185,195 Lindahl, T., see Fresco, J. R. 69, 78
Lamfrom, H., and M. Grunberg-Manago Linnane, A. W., and P. R. Stewart 160,
72,79 195
Lampkin-Hibbard, J. M., K. L. Mukherjee, - A. J. Lamb, C. Christodoulou, and
and C. Heidelberger 87, 133 H. B. Lukins 185, 195
Landy, A., J. Abelson, H. M. Goodman, - see Clark-Walker, G. D. 164, 189
and J. D. Smith 52,79 - see Huang, M. 164, 193
- see Goodman, H. M. 21, 41 - see Lamb, A. J. 164, 194
Lane, H. c., see Buder, W. L. 173, 189 - see Rogers, P. J. 139, 151, 166, 298
Lang, H. M., see San Pietro, A. 138, 198 Lipmann, F., see Bennett, T. P. 67, 77
Lange, P. W., see Hyden, H. 209,211 - see Chapeville, F. 25, 32, 40
Lanka, E., see Weigert, M. 20,33,45 - see Lucas-Lenard, J. 18, 42
Lascelles, J. 166, 194 Littauer, U. Z., M. Revel, and R. Stern
- see Bull, M. J. 117,130 53,75
- see Porra, R. J. 138, 166, 197 - see Revel, M. 26,43
Last, J. A., see Salas, M. 18,43 Liverman, J. L. 172, 195
- see Stanley, W. M., Jr. 14, 44 - , M. P. Johnson, and L. Starr 182,155
Leder, P., and M. Nau 18,21,42 Lockhart, J. A. 183, 195
- and M. W. Nirenberg 14, 42 Lodish, H. F. 18, 42
- see Brimacombe, R. 15, 39 - K. Horiuchi, and N. D. Zinder 85,
- see Nirenberg, M. W. 14, 15, 25, 42, 109, 123, 129, 133
43 - , S. Cooper, and N. D. Zinder 109, 133
- see Singer, M. F. 49,81,105, 135 Loeb, M. R., see Cohen, S. S. 88,89, 101,
Leech, R. M. 137,194 131
- see Dyer, T. A. 141,153,185, 190 Loebel, J. E., see Kellogg, D. A. 23, 41
Leff, J., M. Mandel, H. T. Epstein, and Loening, U. E., and J. Ingle 151, 152,
J. A. Schiff 142, 194 195
Lehninger, A. L., see Wheeldon, L. W. Loftfield, R. B. 25, 42, 50, 79
158, 164, 165,201 - , and E. A. Eigner 20, 42
Leiman, A. L., see Rosenzweig, M. R. Lohrmann, R., see Soll, D. 15, 44
204,212 Losada, M., J. M. Ramirez, A. Paneque,
Lengyel, P., J. F. Speyer, and S. Ochoa and F. F. del Campo 166,195
125, 133 - see Ramirez, J. M. 166,197
- see Gardner, R. S. 14,41 Lowrie, R. J., and P. L. Bergquist 75,79,
- see Speyer, J. 14,33,44 100, 101, 102, 103, 104, 126, 133
- see Wahba, A. J. 14,45,125, 135 Lowry, C. B., see Nomura, M. 18, 21, 43
Lennox, E. S., and M. Cohn 74, 79 Lowry, C. V., see Nomura, M. 49, 61,
Leon, S. A., and T. D. Brock 65, 79 80
Letendre, c., A. M. Michelson, and M. Lozeron, H. A., and M. P. Gordon 107,
Grunberg-Manago 22, 42, 52, 79 110,133
Levi-Montalcini, R. 204, 212 - T. Gabriel, W. Tautz, and R. Du-
Levin, J., see Nirenberg, M. W. 69, 75, schinsky 110, 133
80 Lubin, M., see Ennis, H. L. 161, 190
Levine, L 212 Lucas, J. M., see Simpson, M. V. 164,
Levine, R. P., see Gorman, D. S. 185,192 199
Author Index 225
Lucas-Lenard, J., and F. Lipmann 18,42 Marquisee,M., see Holley,R. W. 21,26,41

Luck, D. J. L., see Rifkin, M. R. 150,151, - see Zamir, A. 22,45
197 Marshall, R. E., C. T. Caskey, and M. W.
Lukins, H. B., see Linnane, A. W. 185, Nirenberg 33,42
195 - see Nirenberg, M. W. 69, 75, 80
Lundegardh, H. 138, 195 Marshall, R., see Pestka, S. 60, 71, 80
Luscher, M., see Karlson, P. 206,211 Martin, R. G. 17,18,42
Lyman, H. 178,195 - see Matthaei, ]. H. 67,80
- see Olson, J. M. 136,196 - see Nirenberg, M. W. 43
- see Schiff, J. A. 177, 198 Marno, B., see Kadowaki, K. 117, 133
- see Shah, V. C. 153, 155, 198 Marver, H. S., A. Collins, D. P. Tschudy,
- see Smillie, R. M. 139, 152, 154, 155, and M. Rechcigl, Jr. 118,134
156,159,160,163, 199 Massoulie, J., A. M. Michelson, and F.
Lyttleton, ]. W. 149, 150, 151, 153, 195 Pochon 125, 134
Masukawa, H., see Tanaka, N. 64, 81
Mabry, c., J. c. Denniston, and J. G. Matsubara, J. K., see Aoki, S. 155, 160,
Coldwell 208, 212 187
Machledt, J. M. Jr., see Goldberg, A. R. Matsuda, K., see Bartels, P. G. 151, 187
87, 131 Matsuka, M., and E. Hase 179, 195
Madison, J. T., G. A. Everett, and H. Matthaei, J. H., F. Amelunxen, K. Eckert,
Kung 21,42,103, 134 and G. Heller 49, 80
- see Holley, R. W. 21, 26, 41 - and M. W. Nirenberg 42
Madsen, A., see Sironval, C. 168, 199 - O. W. Jones, R. G. Martin, and M. W.
Magasanik, B., see Nakada, D. 54, 80, Nirenberg 67,80
105, 115, 116, 120, 129, 134 - H. P. Voigh, G. Heller, R. Neth, G.
Mager, J. 164, 195 Schoch, H. Kibler, F. Amelunxen, G.
Magni, G. E., and P. O. Puglisi 33,42 Sander, and A. Parmiggiani 15, 42
Mahler, H. R., see Tewari, K. K. 140, - see Nirenberg, M. W. 6,14,18,25,43
200 Matthews, R. E. F. 82, 134
Malaviya, B., see Parthier, B. 160, 197 - see Clark, M. F. 151,158, 189
Mandel, H. G. 82, 143 - see Mandel, H. G. 88,134
- , R. Markham, and R. E. F. Matthews Maxwell, 1., see Wimmer, E. 33,45
88, 134 Mayer, A. M., see Friend, J. 139,191
- see Grunberger, D. 97,132 McCalla, D. R., and R. K. Allan 155, 195
- see Hahn, G. A. 93, 95, 98, 99, 126, McCarthy, B. J. 148,195
129, 132 - and E. T. Bolton 24,42, 146, 195
- see Reich, M. 85,88,89,99,115,117, - and J. ]. Holland 66, 80
134 - J. J. Holland, and C. A. Buck 66,80
Mandel, L. R., and E. Borek 53, 79 - and E. Racker 138, 165, 195
Mandel, M., see Leff,]. 142, 194 McConnell, J. V. 203,212
Mangiarotti, G., and D. Schlessinger 61, McGregor, D., see Moore, B. W. 212
80 McMahon, D., see Chen, S. 176,189
- D. Apirion, D. Schlessinger, and McSwain, B. D., see Arnon, D. 1. 165,
L. Silengp 37, 42 187
- see Schlessinger, D. 44 Mego, J. L. 155, 195
Marcker, K. A., and F. Sanger 17,42 - and A. T. Jagendorf 137, 174, 176,
- see Bretcher, M. S. 18, 39 195
- see Clark, B. F. C. 17,40 Mehler, A. H., see Stern, R. 50,81
- see Dube, S. K. 21,40 Mehta, S. L., see Hadziyev, D. 152, 192
Marcus, A. 157, 170, 174, 195 Mercer, E. 1., see Davies, W. H. 139, 190
Margoliash, E., see Sherman, F. 185, 198 - see Treharne, K. ]. 166, 201
Margulies, M. M. 160,161,170,171,173, Mercer, F. V., see Bogard, L. 183,188
174, 195 Meselson, M., and F. W. Stahl 145, 196
- see Parenti, F. 157, 196 - see Staehelin, T. 65, 81
Markham, R., see Mandel, H. G. 88, 134 Michel, J.-M., see Sironval, C. 165, 199
Marmur, J., see Schild kraut, C. L. 198 Michelson, A. M., see Grumberg-Manago,
- see Sueoka, N. 44 M. 27,41,54,55, 79, 125,127, 132
226 Author Index
Michelson, A. M., see Letendre, C. 22, - see Lampkin-Hibbard, J. M. 87, 133

42,52,79 Mullens, R., see Bogard, L. 183,188
- see Massoulie, J. 125, 134 Mundry, K. W., and A. Gierer 42
Michel-Wolwertz, R. M., see Sironval, C. Munkres, K. D., and F. M. Richards 185,
165, 168, 199 196
Mikulska, E. 1., M. S. Odintosova, and - and D. O. Woodward 165,196
N. M. Sissakian 151, 196 - see Woodward, D. O. 165, 185, 202
Miller, J. H., see Kempner, E. S. 90,114, Munsche, D., see Wollgiehn, R. 152,202
115,133,145,194 Munyon, W., and N. P. Salzman 85, 107,
Miller, M. J., see Salas, M. 18,43 121, 134
Miller, R. S., see Gardner, R. S. 14, 41
- see Wahba, A. J. 14,45,125, 135 Nagahisa, M., and A. Hattori 139, 196
Mills, D., see Spiegelmann, S. 19,44 Nagai, J., and K. Bloch 139,196
Miner, N. 204,212 Nakada, D. 90, 93, 96, 97, 99, 102, 105,
Mirzabekov, A. D., see Bayev, A. A. 21, 106,134
26,39 - , and B. Magasanik 54, 80, 105, 115,
Mitrakos, K. 173, 196 116, 120, 129, 134
- see Klein, W. H. 174, 194 Nakamoto, J., see Economou, A. 18,40
- see Price, L. 174, 197 Nakamoto, T., and D. Kolakofsky 18, 42
Miura, K. 22, 42 - see Kolakofsky, D. 41
- see Hayashi, H. 22,41,52, 79 Nass, M. M. K. 140, 196
- see Kuwano, M. 22, 41 Naono, S., and F. Gros 114, 116, 117,
Miura, K.-1. 49, 80 120, 129, 134
Mohr, H. 173, 196 - see Bussard, A. 54, 77, 116, 129, 130
Molotkovskii, Y., and A. M. Smirnov - see Gros, F. 102,114,117, 132
160, 196 Nathans, D., see Shimura, Y. 85, 107,
Monod, J., see Bussard, A. 54, 77, 116, 108, 109, 121, 126, 129, 135
129, 130 Nau, M., see Leder, P. 18, 21, 42
- see Jacob, F. 123,132 Nauta, W. H. M., and R. A. Gygax 207,
Montag, B. J., see Chaudhuri, N. K. 84, 212
85, 86, 88, 131 Neidhardt, F. C. 47, 80
Monty, K. J., see Dounce, A. 28,40 - see Fangman, W. L. 33, 40, 52, 78
Moore, B. W., and D. McGregor 208, Nelson, C. D., see Hauschild, A. H. W.
212 179, 193
Moore, P. B., R. R. Traut, H. Noller, Nemeth, A. M. 86, 115, 118, 123, 134
P. Pearson, and H. Delius 21, 37, 42 Neth, R., see Matthaei, J. H. 15,42
- see Traut, R. R. 45 Neuman, ]., see Klein, S. 179, 194
Mooren, D., see Heidelberger, C. 83, 132 Neupert, W., D. Brdiczka, and Th. Bucher
Morel, G., see Bove, J. M. 154, 188 158, 196
Morgan, A. R., R. D. Wells, and H. G. Nichol, C. A., see White, P. J. 85,87,88,
Khorana 42, 66, 80 135
- see Kossel, H. 41 Nicholls, J., and D. A. Baylor 205,212
Morimura, Y., see Tamiya, H. 160, 200 Nirenberg, M. W. 14, 23, 42
Morris,1. 161, 196 - T. Caskey, R. Marshall, R. Brima-
- see Echlin, P. 160, 161, 190 combe, D. Kellog, B. Doctor, D.
Morrison, M., see Dounce, A. 28, 40 Hatfield, J. Levin, F. Rottman, S.
Morton, R. K., and J. K. Raison 137,196 Pestka, M. Wilcox, and F. Anderson
Moses, R. E., see Shimura, Y. 85, 107, 69,75,80
108, 109, 121, 126, 129, 135 - P. Leder, B. F. C. Clark, W. S. Sly, and
Moses, V., see Holm-Hansen, O. 179,193 S. Pestka 14,25,42
Mothes, K., see Parthier, B. 160, 197 - and P. Leder 43,53
Moudrianakis, E. N., S. H. Howell, and - and H. J. Matthaei 6,14,18,25,43
A. E. Karu 165,196 - J. H. Matthaei, O. Jones, R. Martin,
Mukherjee, K. L., A. R. Curreri, M. and S. Barondes 43
Javid, and C. Heidelberger 85, 86, - P. Leder, M. Bernfield, R. Brima-
134 combe, J. Trupin, F. Rottman, and
- see Heidelberger, C. 87, 132 C. O'Neal 15,43
Author Index 227
Nirenberg, M. W., see Bemfield, M. 39 - S. Sarkar, and R. E. Thach 18,21,43

- see Brimacombe, R. 15,39 Ohtaka, Y., and S. Spiegelmann 17,43
- see Jones, O. 14,41 Ohtsuka, E., see Nishimura, S. 9, 10, 25,
- see Kellogg, D. A. 23,41 43
- see Leder, P. 14,42 - see Soli, D. 15, 44
- see Marshall, R. E. 33, 42 Okada, Y., see Terzaghi, E. 16, 44
- see Matthaei, J. H. 42,67,80 Okamoto, T., and M. Takanami 49, 80
- see Pestka, S. 21, 43, 49, 60, 71, 80 Old, D., and L. Gorini 63, 80
- see Singer, M. F. 54, 81 Olson, J. M., G. Hind, H. Lyman, and
Nishida, K., see Holm-Hansen, o. 179, H. W. Siegelman 136,196
193 Ombach, M., see Szarkowski, J. W. 139,
Nishimura, M., and H. Huzisige 169, 196 200
Nishimura, S., T. M. Jacob, and H. G. O'Neal, c., see Nirenberg, M. W. 15, 43
Khorana 14,43 Orgel, L. E., see Crick, F. H. C. 9,11,28,
- D. S. Jones, and H. G. Khorana 9, 40
10,25,43 Osawa, S., see Iwabushi, M. 90, 91, 96,
- - E. Ohtsuka, H. Hayatsu, T. M. 97, 98, 99, 101, 106, 132
Jacob, and H. G. Khorana 9,10,25,43 - see Kono, M. 90, 91, 93, 97, 99, 133
- see SolI, D. 15, 44 - see Otaka, E. 97, 134
Noll, H., see Kiintzell, H. 151, 194 Otaka, E., S. Osawa, and A. Sibatani 97,
- see Stutz, E. 150, 151, 152, 200 134
Noller, H., see Moore, P. B. 21,37,42 - see Iwabushi, M. 90, 91, 96, 97, 98,
- see Traut, R. R. 45 99, 101, 106, 132
Nomura, J., see Varon, S. 204,212 - see Kono, M. 93, 133
Nomura, M., and C. B. Lowry 18, 21, Ottensmeyer, F. P., and G. F. Whitmore
43,49,61, 80 76,80
- - and C. Guthrie 43,80
- see Traub, P. 44, 65, 81 Pace, N., see Spiegelmann, S. 19,44
Norris, A. T., and P. Berg 32,50,80 Painter, R. R., see Kilgore, W. W. 85,86,
Novelli, G. D. 47, 80 133
- see Graebe, J. E. 159,192 Palmer, S., see Koechlin, B. A. 87, 133
- see Williams, G. R. 171,201 Paneque, A., see Losada, M. 166, 195
- see Ramirez, J. M. 166, 197
O'Brien, T. W., and G. F. Kalf 150, 151, Pardee, A. B. 2, 4
196 - and L. S. Prestidge 115, 134
Obata, F., see Ogawa, T. 165, 196 - see Goldberg, A. R. 87, 131
Ochoa, M., Jr., see Weinstein, 1. B. 72,81 Parenti, F., and M. M. Margulies 157,
Ochoa, S. 3, 4 196
- see Lengyel, P. 125,133 Park, R. B. 137, 196
- see Salas, M. 18,43 - see Biggins, J. 142, 188
- see Smith, M. A. 18, 19, 44 Parker, J., see Sherman, F. 185,198
- see Speyer, J. 14,33,44 Parker, M. W., S. B. Hendricks, H. A.
- see Szer, W. 24,26,27, 44, 68, 69, 81 Borthwick, and F. W. Went 182, 196
- see Vinuela, E. 18,45 Parmiggiani, A., see Matthaei, J. H. 15,
- see Wahba, A. J. 14,45 92
Odintsova, M. S., E. V. Golubeva, and Parthier, B. 157,160, 196
N. M. Sissakian 151, 196 - and R. Wollgiehn 145,158, 197
- see Beridze, T. G. 143,188 - B. Malaviya, and K. Mothes 160,197
- see Mikulska, E.1. 151, 196 Paschkis, K. E., see Rutman, R. J. 82,
Offutt, c., see Dagg, C. P. 85,86, 131 134
Ogawa, T., F. Obata, and K. Shibata 165, Pauling, L., and M. Delbriick 43
196 Pearson, P., see Moore, P. B. 21, 37, 42
Ogur, M., L. Coker, S. Ogur, and R. Pelc, S. R., and M. G. E. Welton 43
Roshanmanesh 185, 196 Pellegrino de Iraldi, A., see de Robertis, E.
Ogur, S., see Ogur, M. 185, 196 207,211
Ohta, T., 1. Shimada, and K. Imahori 18, Penwick, J. R., see Holley, R. W. 21,26,
21,43 41
15*
228 Author Index
Peraino, C, see Pitot, H. C 118, 134 Rabinowitz, M., see Swift, H. 178, 200
Perkins, H. R., see Rogers, H. J. 89,115, Rachmeler, M., see Gray, P. N. 102, 132
128, 134 Racker, E., see McCarthy, B. J. 138,
Pestka, S. 61, 80 165,195
- R. Marshall, and M. W. Nirenberg - see Peterkofsky, A. 138,197
60,71,80 Racusen, D., and M. Foote 171,183,197
- and M. W. Nirenberg 21, 43, 49, 80 Ragetli, H. W. J., M. Weintraub, and
- see Nirenberg, M. W. 14, 25, 42, 69, U. M. Rink 139, 197
75,80 Raison, J. K., see Morton, R. K. 137,196
Peterkofsky, A., and E. Racker 138, 197 Rajbhandary, U. L., S. H. Chang, A.
- C Jesensky, and J. D. Capra 53,80 Stuart, R. D. Faulkner, R. M. Hoskin-
- see Capra, J. D. 26,40 son, and H. G. Khorana 21,43
Petermann, M. L. 150,159, 197 - see Soll, D. 23,44
Petropulos, S. F. 145, 197 Ralph, R. K., see Clark, M. F. 151, 158,
Pfaffmann, C 205, 212 189
Philippovich, 1. 1., see Svetailo, E. N. Ramirez, J. M., F. F. del Campo, A.
151,200 Paneque, and M. Losada 166, 197
Pirson, A., see Feierabend, J. 170, 175, - see Losada, M. 166,195
177,191 Randall, J., and C Disbrey 184, 197
Pitot, H. C, and C Peraino 118, 134 Ravel, J. M. 21,43
Plaut, W., see Ris, H. 141,197 Ravenswaay Claasen, J. C van, see
Pleven, E. see Duschinsky, R. 83, 131 Knippenberg, P. H. van 61, 62, 79
- see Heidelberger, C 83, 132 Ray, W., see Chapeville, F. 25,32,40
Po chon, F., see Massoulie, J. 125, 134 Ray, D. S., and P. C Hanawalt 140, 142,
Pogo, A. 0., G. Brawerman, and E. 197
Chargaff 154,197 Rechcigl, M., Jr., see Marver, H. S. 118,
- see Brawerman, G. 154, 155, 189 134
- see Pogo, B. G. T. 155,160, 197 Reich, M., and H. G. Mandel 85, 88, 89,
Pogo, B. G. T., and A. O. Pogo 155,160, 99,115,117,134
197 Reichmann, and E. Wimmer 19
Pollard, C J., A. Stemler, and D. F. Rether, B., see Ebel, J. P. 102,131
Blaydes 151, 152, 153, 197 Revel, M., and U. Z. Littauer 26,43
Pon, N. G., see Heber, U. 138, 175, 193 - see Littauer, U. Z. 53, 79
Popp, R. A., see Hilse, K. 76, 79 Rhodes, M. J. C, and E. W. Yemm 177,
Porra, R. J., and J. Lascelles 138, 166, 197
197 Rich, A., see Chun, E. M. L. 142, 189
Porter, H. K., see Bird, 1. F. 139, 188 - see Gamow, G. 13,41
Potter, M., E. Appella, and S. Geisser 74, - see Heywood, S. M. 158, 193
80 Rich, M. A., J. L. Bolam, J. E. Knoll,
Prestidge, L. S., see Pardee, A. B. 115, L. Cheong, and M. L. Eidinoff 88, 89,
134 134
Preston, B. N., see Rogers, P. J. 139,151, Richards, F. M., see Munkres, K. D. 185,
166, 198 196
Price, L., and W. H. Klein 172,173, 197 Richards, O. C 148,149, 197
- K.Mitrakos,and W.M.Klein 174,197 Richter, D. 203, 208, 212
- see Klein, W. H. 174, 194 Rifkin, D. B., D. 1. Hirsch, M. R. Rifkin,
- see Withrow, R. G. 172,173,202 and W. Konigsberg 34,43, 73, 74, 81
- see Wolff, J. B. 168,172,202 Rifkin, M. R., D. D. Wood, and D. J. L.
Price, S., see Dastoli, F. R. 211 Luck 150, 151, 197
Printz, D. B., and S. R. Gross 52,81 - see Rifkin, D. B. 34, 43, 73, 74, 81
Puglisi, P.O., see Magni, G. E. 33,42 Rink, U. M., see Ragetli, H. W. J. 139,
Purkinje, J. E. 1 197
Ris, H., and W. Plaut 141, 197
Quertier, J., see Baltus, E. 151, 152, 153, Ritenour, G. L., K. W. Joy, J. Bunning,
187 and R. H. Hageman 139,166, 197
Roberts, E., and C F. Baxter 203,212
Raacke,1. D., see Bove, J. 139,157, 188 - and S. Frankel 208, 212
Author Index 229
Rodriguez, G., see de Robertis, E. 207, Sander, G., see Matthaei, J. H. 15,42
211 Sanger, F., see Brownlee, G. G. 37, 39
Rogers, H. J., and H. R. Perkins 89,115, - see Marcker, K. 17,42
128,134 San Pietro, A., and H. M. Lang 138, 198
Rogers, L. J., S. P. J. Shah, and T. W. - see Keister, D. L. 138, 170, 176, 194
Goodwin 197 Sarabhai, A. S., A. O. W. Stretton, S.
Rogers, P. J., B. N. Preston, N. B. Titche- Brenner, and A. Bolle 7, 20, 44
ner, and A. W. Linnane 139,151,166, Sarkar, S., see Ohta, T. 18,21,43
198 Sarin, P. S., and P. C. Zamecnik 71, 81
Roman, A., see Goldstein, A. 41 Sastry, P. S., and M. Kates 139, 198
Rondot, M.-J., see Bove, J. M. 154, 188 Saukkonen, J. J., see Horowitz, J. 88,
Roodyn, D. B., J. W. Suttie, and T. S. 89, 114, 115, 117, 124, 132
Work 158,164,165, 198 Saunders, G. F., see Saunders, P. P. 85,
Rose, S. P. R. 207,212 134
Rosen, B. 113,134 Saunders, P. P., G. A. Schultz, R. E. Bass,
Rosenzweig, M. R., and A. L. Leiman and G. F. Saunders 85, 134
204,212 Saxinger, W. c., see Woese, C. R. 26,28,
- see Bennett, E. L. 204,210 35,45
Roshanmanesh, R., see Ogur, M. 185,196 Scheiner, J., see Heidelberger, C. 83, 132
Rosset, R., and L. Gorini 76,81 Schiff, J. A., and H. T. Epstein 166,169,
- see Gorini, L. 63, 78 198
Rottman, F., see Nirenberg, M. W. 15, - H.Lyman, and H. T.Epstein 177,198
43,69, 75, 80 - M. H. Zeldin, and J. Rubman 179,
Roy, S. c., see Das, H. K. 164, 190 198
Rubin, R. H., see Sweeney, B. M. 160, - see Edelman, M. 140, 142, 143, 144,
200 149, 190
Rubio, F., see Koechlin, B. A. 87, 133 - see Epstein, H. T. 146, 190
Rubman, J., see Schiff, J. A. 179,198 - see Leff, J. 142, 194
Ruess, M., see Wollgiehn, R. 152, 202 - see Zeldin, M. H. 152, 154, 155,202
Ruppel, H. G., and D. van Wyk 140, 142, Schildkraut, C. L., J. Marmur, and P.
198 Dotty 198
Russel, G. K. 162 Schlesinger, K., see Zemp, J. W. 209,212
- and M. Gibbs 162,198 Schlessinger, D., G. Mangiarotti, and
Russel, R., see Gefter, M. L. 75, 78 D. Apirion 44
Rutman, R. J., A. Cantarow, and K. E. - see Ben-Hamida, F. 116, 130
Paschkis 82, 134 - see Mangiarotti, G. 37,42,61,80
Ruttenberg, G. J. C. M., see van Bruggen, Schmitt, F. O. 1
E. F. J. 140,189 Schnitzer, R. J., see Heidelberger, C. 83,
Rutter, W. J. 162,164, 198 132
Rychlik, J., see Cerna, J. 114,115,131 Schoch, G., see Matthaei, J. H. 15, 42
Schrader, L. E., L. Beevers, and R. G.
Sager, R. 149, 198 Hageman 166, 198
- and M. G. Hamilton 150, 151, 198 - see Beevers, L. 166, 188
- and M. R. Ishida 142, 198 Schrodinger, E. 1, 4
- 1. B. Weinstein, and Y. Ashkenazi 33, Schultz, G. A., see Saunders, P. P. 85,
43 134
Salas, M., M. B. Hille, J. A. Last, A. J. Schuster, H., see Kramer, G. 85, 107,
Wahba, and S. Ochoa 18, 43 121, 129, 133
- M. J. Miller, A. J. Wahba, and S. Schwartz, J. H. 62,63, 81
Ochoa 18,43 - J. M. Eisenstadt, G. Brawerman, and
- see Smith, M. A. 18, 19, 44 N. D. Binder 44
- see Vinuela, E. 18,45 Scott, N. S., and R. M. Smillie 146,147,
- see Wahba, A. J. 9, 14, 45 153,185, 198
Salzman, N. P., see Munyon, W. 85, 107, - V. C. Shah, and R. M. Smillie 140,
121,134 144, 145, 198
Sambrook, J. F., D. P. Farr, and S. - see Smillie, R. M. 161, 162, 185, 186
Brenner 20, 44 199
230 Author Index
Seely, G. R., see Vernon, L. P. 136, 201 Sissakian, N. M. 137, 199

Seiffert, U. B., and B. w. Agranoff 208, - J. J. Filippovich, E. N. Svetailo, and
212 K. A. Aliyer 157, 159 199
Sells, B. H., and K. Crudup 90, 93, 96, - see Beridze, T. G. 143, 188
97, 98, 105, 106, 129, 134 - see Mikulska, E. I. 151, 195
Semals, J., D. Spencer, Y. T. Kim, and - see Odintsova, M. S. 151, 196
S. G. Wildman 153,198 - see Svetailo, E. N. 151, 200
Sestak, Z., and J. Catsky 171, 198 Skinner, D. M., see Simpson, M. V. 164,
Shah, V. c., and H. Lyman 153,155, 198 199
- see Scott, N. S. 140,144,145, 198 Skoda, J., and R. E. Handschumacher
Shah, S. P. J., see Rogers, L. J. 197 101, 135
Shannon, L. M., see Hall, D. O. 192 Slack, C. R., and M. D. Hatch 138, 199
Sherman, F., J. W. Stewart, E. Margoliash, - see Everson, R. G. 138, 191
J. Parker, and W. Campbell 185, 198 - see Graham, D. 170, 175, 176, 177,
Shibata, K. 168, 198 192
- A. Takamiya, A. T. Jagendorf, and - see Hatch, M. D. 138,177, 192
R. C. Fuller 136, 198 Slapikoff, S., and P. Berg 124, 135
- see Ogawa, T. 165,196 Sly, W. S., see Nirenberg, M. W. 19,25,
Shimada, I., see Ohta, T. 18, 21, 43 42
Shimura, Y., and D. Nathans 107, 135 Smillie, R. M. 138, 139, 155, 170, 171,
- R. E. Moses, and D. Nathans 85,107, 199
108, 109, 121, 126, 129, 135 - W. R. Evans, and H. Lyman 139,
Shipp, W. S., F. J. Kieras, and R. Hasel- 152, 154, 155, 156, 159, 160, 163,
korn 143, 199 199
Shiratori, I., see Katoh, S. 138, 194 - and G. Krotkov 154,199
Shooter, E. M., see Varon, S. 204,212 - N. S. Scott, and D. Graham 161, 162,
Shugar, D., see Fikus, M. 110, 131 185, 186, 199
- see Szer, W. 125,135 - D. Graham, M. R. Dwyer, A. Grieve,
Shulman, R. G., see Barnett, L. 20,39 and N. F. Tobin 161, 162, 163, 165,
Sibatani, A., see Otaka, E. 97, 134 186, 199
Siddiqi, 0., see Garen,A. 20,41,113,131 - see Anderson, L. A. 159,187
Siegel, A., see Bartels, P. G. 151, 187 - see Evans, W. R. 155, 191
Siegel, M. R., and H. D. Sisler 161, 199 - see Graham, D. 170, 175, 176, 177,
Siegelman, H. W., and W. L. Butler 174, 192
182, 199 - see Scott, N. S. 140, 144, 145, 146,
- see Akoyunoglou, G. A. 183, 187 147,153, 185, 198
- see Briggs, W. R. 182, 189 Smirnov, A. M., see Molotkovskii, Y.
- see Butler, W. L. 173, 189 160, 196
- see Olson, J. M. 136, 196 Smith, C. A., see Thornber, J. P. 165,
Signer, E., see Levinthal, C. 42 201
Silengp, L., see Mangiarotti, G. 37, 42 Smith, D. c., J. A. Bassham, and M. Kirk
Simoni, R., see Stumpf, P. K. 139, 166, 180, 199
200 Smith, J. D., see Goodman, H. M. 21,41
Simpson, M. V., D. M. Skinner, and J. M. - see Landy, A. 52, 79
Lucas 164, 199 Smith, J. H. C. 171,199
Sinclair, J. H., and B. J. Stevens 140, 199 - see Hill, R. 168, 193
Singer, M. F., O. W. Jones, and M. W. - see Koski, V. M. 167, 194
Nirenberg 54, 81 Smith, K. c., see Kaplan, H. S. 110, 133
- and P. Leder 49,81,105, 135 Smith, M. A., M. Salas, W. M. Stanley Jr.,
Singh, B. N., and K. N. Lal 171, 199 A. J. Wahba, and S. Ochoa 18,19,
Sironval, c., M. R. Michel-Wolwertz, and 44
A. Madsen 168, 199 - see Stanley, W. M. Jr. 14,44
- H. Clijsters, J.-M. Michel, R. Bron- So, A. G., J. W. Bodley, and E. W. Davie
chart, and R. M. Michel-Wolwertz 69,81
165, 199 - and E. W. Davie 68, 69, 71, 81
Sisler, E. c., and W. H. Klein 172, 199 Soffer, R. L. 115,135
Sisler, H. D., see Siegel, M. R. 161, 199 Sokawa, Y., and E. Hase 169, 199
Author Index 231
Sokoloff, L. 207,212 Stem, H., see Hotta, Y. 184, 193

SolI, D., E. Ohtsuka, D. S. Jones, R. Stem, R., and A. H. Mehler 50, 81
Lohrmann, H. Hayatsu, S. Nishimura, - see Littauer, U. Z. 52, 79
and H. G. Khorana 15,44 Stevens, B. J., see Sinclair, J. H. 140,199
- J. D. Cherayil, and R. M. Bock 23,44 Stewart,J.c., see Thornber, J.P. 165,201
- and D. L. Rajbhandary 23,44 Stewart, J. W., see Sherman, F. 185, 198
- see Ghosh, H. P. 17,41 Stewart, P. R., see Duncan, M. J., 164,190
Sonneborn, T. M. 28,31,44 - see Linnane, A. W. 160, 195
Sorm, F., see Cerna, J. 114,115,131 Stocking, C. R. 139, 200
Sottocasa, G. L., B. Kuylenstierna, L. - see Bird,!. F. 139, 188
Ernster, and A. Bergstrand 165, 199 - see Weier, T. E. 137, 201
Spadoni, M. A., see Gaetani, S. 131 Stolzenbach, F. E., see Keister, D. L. 194
Spahr, P. F., see Gros, F. 89, lOS, 114, Storck, R., see Taylor, M. M. 150, 200
132 Streisinger, G., see Terzaghi, E. 16, 44
Spencer, D. 151, 157, 159, 162, 199 Stretton, A. O. W., see Brenner, S. 7,20,
- and P. R. Whitfeld 139,144,145,151, 33,39
152, 154, 199, 200 - see Kaplan, S. 34, 41
- and S. G. Wildman 157, 159, 200 - see Sarabhai, A. S. 7, 20, 44
- see Semal, J. 153, 198 Strijkert, P. J., see de Kloet, S. R. 85,95,
Sperry, R. W. 204,212 131
Speyer, J. 44 Stuart, A., see Rajbhandary, D. L. 21,43
- P. Lengyel, C. Basilio, and S. Ochoa Stulberg, M. P., and K. R. Isham 22, 44
44 Stumpf, P. K., J. Brooks, T. Galliard,
- - - A. Wahba, R. Gardner, and J. C. Hawke, and R. Simoni 139,166,
S. Ochoa 14, 33, 44 200
- see Gardner, R. S. 14,41 Stutts, P., see Brockman, R. W. 87, 130
- see Wahba, A. J. 14,45 Stutz, E., and H. Noll 150,151,152,200
Speyer, J. F., see Lengyel, P. 125, 133 Sudyina, E. G. 138, 167, 200
- see Wahba, A. J. 125, 135 Sueoka, N. 13, 33, 44
Spiegelmann, S., D. Bishop, D. Mills, and - T. Kano-Sueoka, and W. J. Gartland
N. Pace 19,44 75,81
- see Gillespie, D. 146, 191 - J. Marmur, and P. Dotty 44
- see Hall, B. D. 146,192 - and T. Yamane 44,100,103, 135
- see Ohtaka, Y. 17,43 - see Chiang, K. S. 142,145,189
- see Yanofsky 152,202 - see Imamoto, F. 51,79
Spier, R. 169,200 - see Yamane, T. 45
Staehelin, M. 85, 106, 135 Suga, 1., see Katoh, S. 138, 194
- and M. P. Gordon 85, 86, 106, 107, Sugahara, K., see Chiba, Y. 142, 189
135 Sundaram, T. K. 113, 135
- see Gordon, M. P. 88, 106, 107, 131 Sundararajan, T.A.,andR.E. Thach 18,44
Staehelin, T., and M. Meselson 65,81 - see Thach, R. E. 44
Stahl, F. W., see Meselson, M. 145, 196 Sutic, D., and B. Djordjevic 121, 135
Stanley, W. M. Jr., M. A. Smith, M. B. - see Becarevic, A. 110, 130
Hille, and J. A. Last 14,44 Suttie, J. W., see Roodyn, D. B. 159, 164,
- see Wahna, A. J. 9, 14, 45 165, 198
- see Smith, M. A. 18,19,44 Suyama, Y. 185, 200
Starr, L., see Liverman, J. L. 182, 195 - and W. D. Bonner 143, 146, 200
Stavy, L. 76, 81 - and J. Gibson 141,200
Stellar, E., see Flexner, J. B. 209, 211 Suzuka, 1., see Kaji, H. 49, 60, 79
Stemler, A., see Pollard, C. J. 151, 152, Svetailo, E. N., 1. 1. Philippovich, and
153, 197 N. M. Sissakian 151,200
Stenram, D., see Willen, R. 95, 128, 135 - see Sissakian, N. M. 157,159, 199
Stent, G. S. 1, 2, 4 Sweeney, B. M., C. F. Tuffli, Jr., and R. H.
- see Cairns, J. 1,4 Rubin 160, 200
- see Wettstein, F. O. 75, 81 Swift, H., M. Rabinowitz, and G. Getz
Stephenson, M. L., K. V. Thimann, and 178,200
P. C. Zamecnik 157, 200 - see Kislev, N. 141,142, 194
232 Author Index
Swinton, D., see Edelman, M. 149, 190 Tomasz, A., and E. Borek 89, 115, 128,
Szarkowski, J. W., and T. Golaszewski 135
177,200 Tomkins, G. M., see Garren, L. D. 118,
- T. Golaszewski, and M. Ombach 139, 131
200 Tomlin, P. A., see Kaplan, H. S. 110, 133
Szer, W., and S. Ochoa 25,26,27,44,68, Tooze, J., and K. Weber 115, 122, 123,
69,81 129, 135
- and D. Shu gar 125, 135 Tower, D. B. 203,209,212
Traub, P., K. Hosokawa, and M. Nomura
Takamiya, A., see Katoh, S. 138, 194 65,81
- see Shibata, K. 136, 198 - and M. Nomura 44
Takanami, M., see Okamoto, T. 49, 80 Traut, R. R., P. B. Moore, H. Delius,
Talwar, G. P., B. K. Goel, S. P. Chopra, H. Noller, and A. Tissiers 45
and B. d'Monte 212 - see Moore, P. B. 21, 37, 42
Tamiya, H., Y. Morimura, and M. Yokota Treharne, K. J., E. J. Mercer, and T. W.
160,200 Goodwin 166,201
Tanaka, N., H. Masukawa, and H. Trupin, J., see Brimacombe, R. 15,39
Umezawa 64, 81 - see Nirenberg, M. W. 15,43
Tanaka, Y., see Kaji, H. 65, 79 Tschudy, D. P., see Marver, H. S. 118,
Tautz, W., see Lozeron, H. A. 110, 133 134
Taylor, A. 0., and B. A. Bonner 169,200 Tsugita, A., see Terzaghi, E. 16,44
Taylor, F. J. 160,200 Tsujimoto, H. Y., see Arnon, D. 1. 165.
Taylor, M. M., J. E. Glasgow, and R. 187
Storck 150, 200 Tuffli, C. F. Jr., see Sweeney, B. M. 160,
Tecce, G 52 200
- see Area, M. 51,52,77
Teranishi, R., see Hornstein,!' 205, 209, Uesugi, T., see Hattori, A. 167, 193
211 Umezawa, H., see Tanaka, N. 64, 81
Tershak, D. R. 107, 110, 121, 129, 135
Terzaghi, E., Y. Okada, G. Streisinger, Valle, M. R. del, see Aronson. A. 1. 123,
J. Emrich, M. Inouye, and A. Tsugita 130
16,44 Varney, N., see Burton, K. 22,39
Teuer, G. M., see Wimmer, E. 33,45 Varon, S., J. Nomura, and E. M. Shooter
Tewari, K. K., and S. G. Wildman 139, 204,212
140, 143, 144, 147, 148, 149, 185, 200 Vaughan, M. H. Jr., see Chun, E. H. L.
- J. Jayaraman, and H. R. Mahler 140, 142, 189
200 Vazquez, D. 159,201
Thach, R. E., M. A. Cecere, T. A. Veldstra, H., see Knippenberg, P. H. van
Sundararajan, and P. Dotty 44 61,62,79
- see Ohta, T. 18, 21, 43 Venkstern, T. V., see Bayev, A. A. 21,
Thach, R. E., see Sundararajan, T. A. 18, 26,39
44 Vernon, L. P., and G. R. Seely 136, 201
Thimann, K. V., see Stephenson, M. L. Vinuela, E., M. Salas, and S. Ochoa 18,
157,200 45
Thornber, J. P., R. P. F. Gregory, C. A. Virgin, H. 1. 168, 172,201
Smith, and J. L. Bailey 165,201 - A. Kahn, and D. von Wettstein 181,
- J. c. Stewart, M. W. C. Hatton, and 201
J. L. Bailey 165,201 Voigh, H. P., see Matthaei, J. H. 15, 42
Tilney-Bassett, R. A. E., see Kirk, J. T. O.
136, 137, 158, 194 Wachsman, J. T., S. Kemp, and L. Hogg
Tint, B. L., see Click, R. E. 152, 189 88,89, 135
Tissiers, A., see Traut, R. R. 45 Waelsch, H., and A. Lajtha 207,212
Titchener, N. B., see Rogers, P. J. 139, Wagner, N. J., and C. Heidelberger 101,
151, 166, 198 135
Tobin, N. F., see Smillie, R. M. 161, 162, Wahba, A. J. 14
163, 165, 186, 199 C. Basilio, J. Speyer, P. Lengyel, R. S.
Tolbert, N. E., see Gailey. F. B. 172,191 Miller, and S. Ochoa 14,45
Author Index 233
Wahba, A. ]., R. S. Gardner, C. Basilio, - see Virgin, H. I. 181, 201

R.S.Miller, ].F. Speyer, and P.Lengyel Wettstein, F. 0., and G. S. Stent 75,81
45, 125, 135 Wheeldon, L. W., and A. L. Lehninger
- M. Salas, and W. M. Stanley Jr. 9,14, 158, 164, 165,201
45 White, ]. R., see Cox, E. C. 56,78
- see Gardner, R. S. 14,41 - see Flaks, ]. G. 57, 78
- see Salas, M. 18,43 White, P. ]., and C. A. Nichol 85,87,88,
- see Smith, M. A. 18, 19, 44 135
- see Speyer,]. 14,33,44 Whitfeld, P. R., see Spencer, D. 139,144,
Wald, G. 205,212 145, 151, 152, 154, 199,200
Walenga, R., see Evans, W. R. 163, 191 Whitmore, G. F., see Ottensmeyer, F. P.
Wallace, A., see Hall, D. O. 192 76,80
Waller, ]. P. 17,45 Whittaker, V. P. 207,212
- and]. 1. Harris 21,45 Wierzchowski, K. L., see Fikus, M. 110,
Walles, B. 181, 201 131
Warnaar, S. 0., and]. A. Cohen 148,201 Wieckowski, S., and T. W. Goodwin
Watson,]. D. 20,45 179,201
- and F. H. C. Crick 6, 45 Wilcox, M., see Nirenberg. M. W. 69, 75,
- see Cairns,]. 1,4 80
- see Gros, F. 89,93,97, 102, 105, 114, Wildman, S. G. 158,201
129, 132 - see Boardman, N. K. 150, 151, 157,
Watts-Tobin, R. ]., see Barnett, L. 20,39 158, 188
- see Crick, F. H. C. 40 - see Chen, ]. L. 151, 189
Weber, K., see Tooze,]. 115, 122, 123, - see Francki, R. 1. B. 157, 191
129,135 - see Semal, ]. 153, 198
Webster, R. E., D. L. Engelhardt, and - see Spencer, D. 157, 159,200
N. D. Zinder 17,45 - see Tewari, K. K. 139, 140, 143, 144,
Weier, T. E., and C. R. Stocking 137,201 147, 148, 149, 185, 200
- see Bartels, P. G. 151, 187 Wilhelm, R. c., see Garen, A. 20,41
Weigert, M., and A. Garen 20, 33, 45 Willen, R., and U. Stenram 95, 128, 135
- E. Lanka, and A. Garen 20,33,45 Williams, G. R., and G. D. Novelli 171,
Weil, ]. M. 53,81 201
- see Bakes, ]. 53, 77 Williams,]. A. 26,45
- see Ebel,]. P. 102, 131 Wilson, ]. E., see Zemp, J. W. 209,212
- see Giege, R. 75, 78 Wimmer, E., I. Maxwell, and G. M. Teuer
Weinstein, I. B., S. M. Friedman, and S. 33,45
Ochoa Jr. 72, 81 - see Reichmann 19
- ' M. Ochoa]r., and S. M. Friedman 72, Wintersberger, E. 151, 158, 165, 201
81 Withrow, R. B., J. B. Wolff, and L. Price
- see Friedman, M. 25, 26, 27, 40, 68, 172,173,202
69,70,78 Witkop, B., see Gross, E. 19,41
- see Sager, R. 33, 43 Wittmann, H. G. 19,45,121, 135
Weintraub, M., see Ragettli, H. W. ]. - see Kramer, G. 85, 107, 121, 129,
139, 197 133
Weisblum, B., S. Benzer, and R. W. Holley - see Wittmann-Liebold, B. 107, 115,
23,45 121, 122, 135
and ]. Davies 76,81 Wittmann-Liebold, B., and H. G. Witt-
- F. Gonana, G. von Ehrenstein, and mann 107, 115, 121, 122, 135
S. Benzer 23, 33, 45 Wodinsky, I., see Kessel, D. 89, 133
- see Chapeville, F. 25,32,40 Woese, C. R. 13, 14, 23, 27, 29, 30, 31,
Weiss, P. 1,4 32, 37, 38, 45, 67, 81
Wells, R. D., see Morgan, A. R. 42,66, - D. H. Dugre, W. C. Saxinger, and
80 D. A. Dugre 26,28,35,45
Welton, M. G. E., and C. R. Woese 36 - see Britten 28
- see Pelc, S. R. 43 - see Welton, M. G. E. 36
Went, F. W., see Parker, M. W. 182, 196 Wolff, ]. B., and L. Price 168, 172, 202
Wettstein, D. von 181, 183, 184, 201 - see Withrow, R. B. 172,173,202
234 Author Index
Wollgiehn, R., M. Ruess, and D. Munsche Yellen, L. K., see Aaronson, S. 159, 160,
152,202 187
- see Parthier, B. 145,158, 197 Yemm, E. W., see Rhodes, M. J. C. 177,
Wood, D. D., see Rifkin, M. R. 150,151, 197
197 Yokota, M., see Tamiya, H. 160,200
Woodcock, C. L. F., and H. Fernandez-
Moran 140,141,202 Zachau, H. G. von, D. Dutting, and
Woodward, D.O., and K. D. Munkres H. Feldman 21,26,46
165,185,202 Zalik, S., see Hadziyev, D. 152,192
- see Munkres, K. D. 165,196 Zamecnik, P. c., see Burton, K. 22,39
Work, T. S., see Roodyn, D. B. 158,164, - see Sarin, P. S. 71, 81
165, 198 - see Stephenson, M. L. 157,200
Wright, R. M. 206,212 Zamir, A., R. W. Holley, and M. Marquisee
van Wyk, D., see Ruppel, H. G. 140,142, 22,45
198 Zeevaart, J. A. D., see Bonner, J. 85,86,
124, 130
Yamane, T., and N. Sueoka 45 Zeldin, B., see Edelman, M. 149, 190
- see Imamoto, F. 51, 79 Zeldin, M. H., and J. A. Schiff 152, 154,
- see Sueoka, N. 44,100,103, 135 155,202
Yaniv, M., F. Jacob, and F. Gros 52, 81 - see Schiff, J. A. 179, 198
Yanofsky, C. 16 Zemp, J. W., J. E. Wilson, K. Schlesinger,
- B. C. Carlton, J. R. Guest, D. R. W. O. Boggan, and E. Glassman 209,
Helinski, and U. Henning 7,45 212
- see Brammer, W. J. 16,39 Ziegler, H., and I. Ziegler 161, 180, 202
- see Brody, S. 39 Ziegler, I., see Ziegler, H. 161, 180, 202
- see Carbon, J. 34, 40 Zimir, A., see Holley, R. W. 21, 26, 41
- see Cox, E. C. 24, 40 Zimmermann, R. A., see Gorini, L. 63,
Yanofsky, S. A., and S. Spiegelman 152, 78
202 Zinder, N. D., see Cooper, S. 124,131
Yarus, M., and P. Berg 20,26,46,50,51, - see Lodish, H. F. 85, 109, 123, 129, 133
81 - see Webster, R. E. 17,45
Yeas, M., see Gamow, G. 11, 12, 13,41 Zubay, G., and P. Dotty 28, 45
Subject Index
Abnormal ribosomes, resulting from in- - , photo regulation of RNA synthesis 177
corporation of 5-Fluorouracil 90 - , protein synthesis 157-167
Acetone, producing misreading 70 - , RNA 152-157
N-acetyl aspartate in brain 207 - , RNA synthesis 153-157
Acetylcholine in brain function 207 - , ribosomes 149-152
N-acetyl serine 19 Coding problem 5, 6, 7-14, 28, 29
Acidic amino acids in brain 207 Codons 10,11-20
Actinomycin, producing memory loss 209 - , amber 7
Amino acids, activation 21 - , ochre 20
Amino acyl transfer RNA, synthesis 47, - , opal 20
48,50-52 - , "relatedness" to amino acids 35
Aminoglycosides, structure-activity rela- Control, humoral 3
tionships for induced misreading 65 Control, neural 3
- , translation errors produced by 56-66 Cycloheximide, as inhibitor of chloroplast
protein synthesis 161
Blue light, requirement for chloroplast Deoxystreptamine residue, responsible for
formation 181 misreading 64
Bluensomycin, translation errors produced
by 63 Developmental biology 2
- - , nervous system 204
Calvin cycle enzymes in chloroplasts 159 Dihydrostreptomycin, translation errors
Carotenoids in chloroplasts 166 produced by, 63
Central Dogma 2 Dioxane, producing misreading 70
Chemotaxis 206 Dopamine in brain function 207
Chloramphenicol, as inhibitor of chloro-
plast protein synthesis 159, 163, 165, Ethanol, producing misreading 70
166 Ethylene glycol, producing misreading 70
Chlorophyll synthesis in chloroplasts,
effect of pre-illumination 172 Ferrochelatase in chloroplasts 166
Chlorophyllides, conversions in chloro- 5-Fluorodeoxyuridine triphosphate as sub-
plasts 167 strate of DNA polymerase 126
Chloroplasts 136-186 5-Fluorouracil, bacterial resistance to, 87
- , composition 136 - , conversion of related compounds into,
Chloroplast, DNA 137 87
- - , evolutionary origin 148 - , distribution within RNA chains 88
- - , function 184 - , effects on cell growth 88, 89
- - , Hybridization with ribosomal RNA , on enzyme synthesis 115
146 - , - on function of transfer RNA 102
- - , Localization within the chloroplast - , - on physical properties of proteins
141 116
- - , synthesis 141-145 - , - on viral proteins 120
- development, regulation and photo- - in mammalian ribosomes 95
synthesis 179-184 - in ribosomes of Bacillus cereus 93
- , enzyme synthesis 159 - - - of E. coli 90
- , membrane proteins 164 - - - offungi 95
- , photoregulation of development 167 - - - of plants
- , photoregulation of protein synthesis - - - of S. aureus 93
169-177 - - - of yeast 95
236 Subject Index
5-Fluorouracil, in synthetic polynucleotides Kasugamycin, failure to induce misreading

125 65
- , in transfer RNA 99
- in viral RNAs 106-110 Magnesium ion in translation 18, 62, 68
- , incorporation into cells and tissues ~embranes 2, 164
83-86 ~emory consolidation 209
- , - into E. coli 84, 86 - transfer 203
- , - into insect eggs 86 ~essenger RNA, polycistronic 17
- , - into messenger RNA 105 ~ethanol, producing misreading 70
- , - into neoplastic cells 86 ~iscoding by FU-messenger RNA
- , - into plants 86 110-113
- , - into RNA 82-130 ~olecular biology 1,203
- , - into ribosomes 89-99 - - , history 1
- , influence on protein composition 114
- , teratogenic doses 86 Nalidixic acid, inhibiting chloroplast DNA
- , translation errors produced by 54, synthesis 178
110-114 Neamine, translation errors produced by, 63
5-Fluorouridine diphosphate as substrate Nebramycin, translation errors produced
for polynucleotide phosphorylase 125 by 63
- triphosphate as substrate of RNA Neighbouring base effect in misreading 60
polymerase 126 Neomycin (B, C), translation errors pro-
N-formyl methionine 17,49 duced by 63
Neo-vitalism 2
Gamma aminobutyric acid in brain func- Nerve growth factor 204
tion 207 Neurochemistry 203
Genetic code 5-39 Neurons 204
- - , codon-anticodon pairing 23 Neurotransmitter substances 207
- - , codon catalog 11-16 Nitrite reductase in chloroplasts 166
- - , colinearity 7 Norepinephrine in brain function 207
- - , "comma-free" 11 Organic solvents, producing misreading
- -,degenracy 9 69-72
- - , evolution 27-33 Paromomycin, translation errors produced
- - , initiation punctuation 17-19 by 63
- - , misreading in mammalian systems 72
- - , in vivo 73 pH changes, producing misreading 71
- - , overlapping 8 Phenotypic suppression by streptomycin
- - , punctuation 16-20 57,61
- - , reading frame 9-10 Phenylketonuria 208
- - , - - , displacement 10, 16 Pheromones (insects) 206
- - , termination punctuation 19-20 Phospholipids in brain 208, 209
- - , translation 20-23, 47-50 Photosynthetic Pathway in chloroplasts
- -,-,accuracy 23-27 163
- - , - , evolution of apparatus 36-38 Phytochrom in chloroplasts, role in
- - , universality 33-36 photoregulation 173
- - , wobble hypothesis 23 Porphyrine synthesis in chloroplasts 166
Gentamicin, translation errors produced by 2-Propanol, producing misreading 70
63 Puromycin, producing memory loss 209
Glutamic decarboxylase in brain 208 Pyrimidine bases, misreading of 58
Glutamine in brain 207
Regrowth of nerves 204
Hygromycin B, translation errors pro- Ribosomes, conformational changes 71
duced by 63 - , evolutionary precursors 36, 37
- , mutations leading to ambiguity 66,76
Information transfer 6, 7 - , recovering from the incorporation of
5-Fluorouracil 97
Kanamycin, translation errors produced by - , subunits 21,49
63 RNA and brain function 209
Subject Index 237
Serotonine in brain function 207 Transcription, errors 24

Silver stain of brain cells 206 Transfer RNA, compositional changes
Smell 205, 206 resulting from incorporation of 5-
Spectinomycin, failure to induce misread- fluorouracil 101
ing 65 - - , conformational changes 71
Streptomycin, binding to 30s ribosome - - , function 49, 52-56
subunits 65 - - structure 21, 22, 185
- , inhibition of protein synthesis by 61 Translation errors 24-27,47-76
- , translation errors produced by 57-63 - - , resulting from alterations in transfer
Suppressor mutations, temperature sensi- RNA 75
tivity of 67 Temperature changes, resulting in misread-
Synaptosomes 207 ing 69
Taste of glutathione (housefly) phenyl- Uracil dimers, in messenger RNA 54,

thiourea quinine 205 76
- , receptors 205 Urea, producing misreading 75

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Progress in Molecular and Subcellular Biology .

Springer-Verlag New York' Heidelberg' Berlin 1969

CARL R. WOESE: The Biological Significance of the Genetic Code. . . 5

JULIAN DAVIES: Errors in Translation. 47

H. GEORGE MANDEL: The Incorporation of Fluorouracil into RNA and its

ROBERT M. SMILLIE and N. STEELE SCOTT: Organelle Biosynthesis: The

BERNARD W. AGRANOFF: Macromolecules and Brain Function-A 1969

Author Index 213

II. Characteristics of the Genetic Code

B. The Code Words- Sentence Structure and Reading; Synotryms

of a base could (in some instances) be corrected by a (nearby) addition of a base, or

C. The Structure of the Codon Catalog

"recognition" properties, the 64 diamonds could be placed into the requisite 20

Class I codons: nondegenerate

GAMOW, 1955; GAMOW and YCAS, 1955.

Table 2. A comma-free triplet code of maximum

ACA UCA GCA

CRICK et aI., 1957.

Table 3. The codon catalog

UUU phe UCU ser UAU tyr UGU cys

UUA leu UCA ser UAA CT-l UGA CT-3

CUU leu CCU pro CAU his CGU arg

CUA leu CCA pro CAA gln CGA arg

AUU ilu ACU thr AAU asn AGU ser

AUA ilu ACA thr AAA lys AGA arg

GUU val GCU ala GAU asp GGU gly

GUA val GCA ala GAA glu GGA gly

sequence mRNAs synthesized in the laboratory of H. G. KHORANA (summarized in

D. In Vivo Confirmation of Codon Assignments

It is presumptuous and probably incorrect, however, to assert dogmatically at

2. Punctuation Concerned with Polypeptide Chain Termination

by competing tRNA charging with fragments obtained by digesting tRNA with

Table 4. Alternate codon-anticodon pairings -

particularly involving tRNAs whose primary structure was known to be unique,

codon-anticodon interaction to account for this binding; other nonspecific inter-

Given a primitive cell such as that envisioned on the TE scheme, it is relatively

1. The Universality of the Genetic Code

Table 5. Amino acid polar requirement,

CUU CCU CAU his 8.4 CGU

AUU Heu 4.9 ACU AAU asn 10.0 AGU (ser)

GUU GCU GAU asp 13.0 GGU

WOESE et ai., 1966.

2. The Evolution of the Translation Apparatus

Evidence points strongly to the existence of a smaller primitive ribosome. The

FANGMAN, W. L., and F. C. NEIDHARDT: Demonstration of an altered aminoacyl ribonucleic

KHORANA, H. G., H. BUCHI, H. GOOSH, N. GUPTA, T. M. JACOB, H. KOSSEL, R. MORGAN,

- - - Studies on polynucleotides LXXIV. Direct translation in vitro of single stranded

REVEL, M., and U. Z. LI1TAUER: The coding properties of methyldeficient phenylalanine

SUNDARARA]AN, T. A., and R. E. THACH: Role of the formylmethionine codon AUG in

II. The Translation System

A. Reaction oj an Amino-Acid with its Specific Aminoaryl-tRNA Synthetase

termination signal finished polypeptide

ATP + AA2 + AS ~ AA2-AMP-AS + PPi

factors, etc. /tRNA2-AA2

B. Recog1Jition Involving tRNA

III. Errors in Translation

then introduce errors in translation and ~ 800

heterologous system has shown that the ?

the following properties: 1) enzyme El

B. Do Translation Errors Due to Anomalous Charging Occur in Whole Cells?

Thus far studies on these particular modifications, leading to ambiguities in

Isoleucine 316 383 o o 433 (U/A = 1.4)

Proline 626 71(q