chapter 10

THE BIOSYNTHESIS
OF PROTEINS

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

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

195
and anuclear halves, the latter fragment temporarily synthesizes pro-
tein at a rate even more rapid than that of the intact cell, although
this synthetic activity soon disappears. If the biosynthesis of a spe-
cific, chemically definable protein like hemoglobin can continue in
the absence of an intact nucleus, it becomes essential to focus our
attention on the mechanism by which the requisite information might
be transferred to, and perhaps temporarily stored in, the cytoplasm
of cells.

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

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

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

196 THE MOLECULAR BASIS OF EVOLUTION

 

Figure 90. Electron photomicrograph of a section through a cell from the pancreas
of the guinea pig. Magnification, 56,000x. The layered structures at the center
of the photograph are the “endoplasmic reticulum” and consist of lipoidal mem-
branes studded with particles rich in RNA. The oval, cross-channeled structures
at the top are mitochondria. A portion of the cell nucleus is visible at the bottom
of the photograph. This photograph was obtained through the kindness of
Dr. George Palade of the Rockefeller Institute for Medical Research. From “The
Endoplasmic Reticulum,” J. Biophys. Biochem. Cytology, 2, (suppl.) 85 (1956).

THE BIOSYNTHESIS OF PROTEINS 197
  
 
 

  

Cytoplasmic
compartment

   

 

 

 
 
 

Mitochondrion

  

Granule

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

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

198 THE MOLECULAR BASIS OF EVOLUTION

arrangement would furnish the cell with a large surface as a site for
metabolic activity and would also serve as a natural divider of the
“genetic” and “assembly line” portions of the cell.

It must be emphasized that the scheme presented in Figure 91 is
only the distillation of several possibilities considered reasonable
by expert cytologists. It is included here only to indicate to the
reader the degree of sophistication that has been reached in the study
of cellular substructure. Uniformity of interpretation by experts is
more than can be expected in any rapidly advancing field, and it is
gratifying that most of the differences of opinion among cytologists
revolve around relatively minor points.

When a tissue is homogenized, the endoplasmic reticulum is dis-
integrated. From recent analyses it would appear that the so-called
microsome fraction is composed mainly of granules to which pieces
of reticulum still adhere. Thus, treatment of microsomal preparations
with lipoprotein-disrupting agents such as deoxycholate yields par-
ticles containing most of the RNA of the original preparation but
only a small proportion (about one-sixth) of the original protein.
With ribonuclease, on the other hand, which digests and depoly-
merizes the RNA, only membranous material is observed upon elec-
tron microscopic examination. In some tissues, like the hen’s oviduct,
the ergastoplasm is not as friable, and, even after fairly vigorous
homogenization, a relatively intact membrane particle complex may
be isolated by centrifugation at relatively low speeds.

The origin of the ergastoplasm is not established. It has recently
been shown that, in the hepatic cells of animals fed after a prolonged
fasting period, regeneration of membranes begins near the periphery
of the cell. These membranes are free of granules and only later
assume the studded appearance which characterizes them in an ac-
tively secreting cell. It has been suggested that the reticulum might
be the product of the continuing process of pinocytosis (water im-
bibition) and phagocytosis ( particle imbibition) at the cell surface.
Electron microscope studies have indicated that engulfed fluids and
solids are surrounded with a sheath of the external plasma membrane
of the cell, pinched off during the passage of nutrients across the cell
surface. This membrane ultimately becomes continuous with the
endoplasmic reticulum. The observations, if substantiated, would re-
quire some rather vigorous metabolism. For example, as Swerdlow,
Dalton, and Birks? have recently pointed out, if the incorporation of
the plasma membrane into actively imbibing cells like macrophages
were continuous, the cells would soon consist of nothing else. Such
cells would obviously require active processes both for the regenera-

THE BIOSYNTHESIS OF PROTEINS 199
tion of new plasma membrane and for the breakdown of reticulum
as it accumulated and pressed in on the nucleus.

Cytological Aspects of Protein Synthesis

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

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

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

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

200 THE MOLECULAR BASIS OF EVOLUTION

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

 

Arginine side
chains

Polypeptide
CREPD cain

cell which varies from species to species, and that protein synthesis
may proceed as long as some of this material remains function® on
Two types of experimental observation have a direct caring on
this question. The first, involving the measurement of rates ° a -
corporation of various nucleic acid and protein precursors, in 0 we
ribonucleoproteins of nucleus and cytoplasm, indicates at s a
substances are subject to a much greater metabolic flux in t ie om ‘
cellular compartment. As shown in Figure 93, nuclear k a tains
a much higher specific radioactivity than does cytop asmic ‘
when an animal is administered radioactive phosphate. Furt i e
peak of radioactivity is attained much more rapidly in the nuc ear
material. Thus, although the amount of RNA in the nucleo rs is
generally relatively small in comparison with that in the cytop asm,
the rate of turnover of the former is such tt it might serve as
at least part of the cytoplasmic .
Pecan type of approach has been made by Ficq and her col-

201
THE BIOSYNTHESIS OF PROTEINS
40

Inorganic P

w
o

Nuclear RNA

Relative specific activity
nN
°

_
o

 

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

Time, hours

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

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

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

202 THE MOLECULAR BASIS OF EVOLUTION

processes in enucleated and otherwise disturbed cells that represent
the dying gasps of a partially functional but abnormal mechanism,
is a source of some of the more exciting speculations in the field.
Some very recent work does seem to offer strong support for the
latter alternative. Stich and Plaut have examined the potentials for
growth, protein synthesis, and differentiation of nuclear and anu-
clear halves of Acetabularia after exposure to ribonuclease action.
Their results, summarized in F igure 94, show that, in the absence of
a nucleus, normal growth and synthesis cannot be resumed after
RNA degradation. Stich and Plaut suggest that the nuclear product
which effects the recovery of nucleated halves is RNA, but they em-
phasize that this suggestion should not be extended to include all the
cytoplasmic RNA but may apply only to a small and highly critical
fraction produced by the nucleus. With this reservation, these ob-
servations are in good agreement with the results of Moldave and

 

 

 

 

 

 

 

 

  

 

 

 

 

 

 

 

 

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

THE BIOSYNTHESIS OF PROTEINS 203
Heidelberger already mentioned and also with studies recently re-
ported by McMaster-Kaye and Taylor,’ who show that what has
hitherto been referred to as “nuclear” RNA may be divided into nu-
cleolar and chromosomal fractions, only the former of which has an
especially dynamic metabolism. It would begin to seem that the
nucleolus may, as Caspersson suggested, occupy a very central posi-
tion in the over-all process of protein biosynthesis.

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

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

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

204 THE MOLECULAR BASIS OF EVOLUTION

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

Protein Synthesis in Ruptured-Cell Preparations®

Tissue homogenates, although they incorporate amino acids only
weakly and lack the integrated information system of the cell, have
the advantage that the need for certain essential cell components be-
comes emphasized. Various cofactors or substrates may then be
screened for their capacity to stimulate the synthesis of proteins.

It was observed, in a number of preliminary studies, that homog-
enates could support the incorporation of labeled amino acids into
proteins when ATP was added as an energy source. Mitochondria,
which serve as the site of ATP production through oxidative phos-
phorylation, could substitute for added ATP in large part. The in-
corporated amino acids in such systems are found mainly in the mi-
crosome fraction of the homogenate (although mitochondria would
also appear to support the synthesis of some proteins, e.g. cyto-
chrome c).

A major contribution was made by P. C. Zamecnik and E. B.
Keller? when they observed that, if more gentle homogenizing tech-
niques were employed, a reconstructed system consisting of micro-
somes and a particle-free supernatant would incorporate labeled pre-
cursors when supplied with an energy source such as phosphocre-
atine or phosphoenolypyruvate. This incorporation was specific for
the natural L-amino acids and was abolished by the addition of ribo-
nuclease. The true synthesis of peptide bonds was demonstrated by
the isolation of peptides from partial hydrolysates of the protein
which contained C14.

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

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

THE BIOSYNTHESIS OF PROTEINS 205
R Oo
| 74
1. Enzyme + NH,»—-CH—C— - ATP >

\
OH

R

Enz—(AMP ~ CO—-CH—NH)) + Pyrophosphate
R

2. Enz—(AMP ~ CO—CH—NH,) + NH.OH —
R

NH2,CH—CONHOH + AMP + Enzyme

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

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

206 THE MOLECULAR BASIS OF EVOLUTION

The Question of Intermediates in Protein Biosynthesis

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

If we assume that RNA serves as some sort of “template” for the
assembly of proteins, must there be a separate ribonucleoprotein
“template” for each cellular protein, or is it possible that there exists
some facet in protein synthesis which permits the utilization of com-
mon information for the synthesis of many different kinds of protein
molecules? The evidence we have for the presence of repeating pat-
terns in protein biosynthesis is, at present, very meager but sugges-
tive enough to make the idea worthy of serious consideration. Sev-
eral groups of investigators have gone to considerable trouble in as-
sembling the known amino acid sequences in proteins (making up a
total of some 400 or 500 residues) and subjecting their sequential ar-
rangement to a rough statistical analysis for the occurrence of “re-
peats.” As yet, no definite pattern has emerged from such efforts, al-
though certain special sequences are found with surprising frequency.
The dipeptide sequence, Ser.Arg, for example, has been demon-
strated in ribonuclease, chymotrypsin, lysozyme, salmine (twice),
and phosphorylase (and this statement is based on only a cursory
search of the literature on protein structure), The phosphoproteins
so far examined involve phosphoserine-containing sequences with
remarkable similarity in structure. Table 16 contains a list of some
of these, derived from proteins with no obvious connection except,
perhaps, the fact that they are all secretory proteins. In every

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

THE BIOSYNTHESIS OF PROTEINS 207
case the phosphoserine residue is either preceded or followed by a
dicarboxyic amino acid.

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

TABLE 16
Amino Acid Sequences Containing O-Phosphorylserine (SerP)*
Pepsin Thr.SerP.Glu
Egg albumin Glu.SerP.Ala
Asp.SerP.Glu.Tleu. Ala
o-Casein SerP.Glu
SerP.Ala
Glu.SerP

« A general discussion of phosphorus-containing proteins is given by G. Perl-
mann, Advances in Protein Chemistry, volume 10, (M. L. Anson, K. Bailey,
and J. T. Edsall, editors), Academic Press, 1935.

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

Gly.Asp.SerP.Gly.Glu.Ala

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

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

208 THE MOLECULAR BASIS OF EVOLUTION

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

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

REFERENCES

1. F, S. Sjéstrand and V. Hanzon, Exptl. Cell Research, 7, 393 (1954); ibid,
. 415.

2. M. Swerdlow, A. J. Dalton, and L. S. Birks, Anal. Chem., 28, 597 (1956).

3. T. Caspersson, Cell Growth and Cell Function, Norton, New York, 1950.

4, K. Felix, H. Fischer, and A. Krekels, Progress in Biophysics, volume 6,
Pergamon Press, London, p. 1, 1956.

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

6. K. Moldave and C. Heidelberger, J. Am. Chem. Soc., 76, 679 (1954).

7. R. McMaster-Kaye and H. J. Taylor, J. Biophys. Biochem. Cytology, 4, 5

THE BIOSYNTHESIS OF PROTEINS ‘ 209
10.
ll.

12

13
14

2

(1958). (The reader should be cautioned that a direct relationship be-
tween nuclear and cytoplasmic RNA is far from established—see for ex-
ample, J. W. Woodward, J. Biophys. Biochem. Cytology, 4, 383 (1958).

. Reviewed in a series of papers in Proc. Nat. Acad. Sci. U.S., 44, No. 2
(1958).

. P. C. Zameenik and E. B. Keller, J. Biol. Chem., 209, 337 (1954).

J. A. Gladner and K, Laki, J. Am. Chem. Soc., 80, 1263 (1958).

R. Loftfield, in Progress in Biophysics and Biochemistry, Pergamon Press,

London, 1958.

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

. J. L. Simkin and T. S. Work, Nature, 179, 1214 (1957).

. D. Steinberg, M. Vaughan, and C. B. Anfinsen, Science, 124, 389 (1956).

10 THE MOLECULAR BASIS OF EVOLUTION