Download Readings Problems Background Week 7

MCB 142
MAJOR ADVANCES IN UNDERSTANDING EVOLUTION AND HEREDITY
FALL 2015
WEEK 7: OCTOBER 20 AND 22
OCTOBER 20: GENES SPECIFY ENZYMES
Inborn errors of metabolism.”One gene, one enzyme”. Conditional mutations.
OCTOBER 22: GENES AND AMINO ACID SEQUENCE. PROTEIN SYNTHESIS
Enzymes as discrete molecules. Mutations and amino acid change. The template hypothesis and the
Central Dogma. Adapters. Protein synthesis.
Readings to be Discussed Thursday October 20

Archibald Edward Garrod (1902) The incidence of Alkaptonuria: A study in chemical individuality.
Lancet, vol ii, 1616-1620.
Read the article.

George Wells Beadle and Edward Lawrie Tatum (1941) Genetic control of biochemical reactions in
Neurospora. Proceedings of the National Academy of Sciences (USA) 27: 499-506.
Read the article.

George Wells Beadle (1958) Genes and Chemical Reactions in Neurospora. Nobel Lecture,
December 11, 1958.
Read the article.

James Batcheller Sumner (1946) The Chemical Nature of Enzymes. Nobel Lecture, December 12,
1946.
Read the article.
Readings to be Discussed Thursday October 22

Linus Pauling, Harvey A. Itano, S.J. Singer and Ibert C. Wells 1949 Sickle Cell Anemia, a
Molecular Disease. Science 110: 543-548.
Read the article.

Vernon M. Ingram 1957 Gene Mutations in Human Haemoglobin: the Chemical Difference Between
Normal and Sickle Cell Haemoglobin. Nature 180: 326-328.
Read the article.

Francis Crick 1958. On protein synthesis. Symposia of the society for experimental biology 12:138163.
Read the article.
1 Study Questions
Please hand in Tuesday October 20
1. What evidence does Garrod present that alkaptonuria is inherited as a recessive Mendelian character?
2. Garrod found a higher proportion of alkaptonurics among siblings of alkaptonurics than among
children of alkaptonurics. Explain why this is expected on his hypothesis for the inheritance of
alkaptonuria?
3. If the frequency of genes for alkaptonuria is p, what is the expected frequency of alkaptonuria in the
general population? In children from first cousin marriages? Assume that the population in question
mates at random with respect to the alkaptonuria gene and that selection with respect to the gene is
negligible.
4. What result of Beadle and Tatum suggested to them that a mutation that prevents growth in minimal
medium but not in "complete" medium affects only a single enzyme? What result suggested to them that
only a single gene was mutated in the pyridoxineless mutant?
5. What led Pauling et al. to suspect, before undertaking their electrophoretic studies, that sickle cell
anemia might be caused by an aberrant hemoglobin molecule?
6. What reasons do Pauling et al. present for rejecting the possibility that the electrophoretic difference
between the hemoglobin in erythrocytes of patients with sickle cell anemia and that in normal patients
is caused by a difference only in the folding of the molecule instead of a difference in its composition?
7. How do the findings of Ingram (1957) account for the estimate of Pauling et al. regarding the amount
of charge difference between HbA and HbS at neutral pH? In what fundamental ways regarding the
nature of the difference between HbA and HbS do Ingram’s findings go beyond those of Pauling et al.?
Looking ahead to the problem of the genetic code, what kinds of code would not be compatible with
Ingram's conclusion?
8. Although the amino acid sequence Ingram deduced in his 1957 paper for the first six amino acids at
the carboxy-terminal portion of peptide number 4 in HbA and HbS is correct, the rest of the sequence he
presents is wrong and peptide number number 4 has eight amino acids, not nine. The correct sequences
of peptide number 4 from hemoglobin A and hemoglobin S were reported in 1959 by Hunt and Ingram
(Nature 184: 640-641). They are:
HbA: val-his-leu-thr-pro-glu-glu-lys
HbB: val-his-leu-thr-pro-val-glu-lys
Which acid hydrolysis products are misidentified on the chromatogram of Figure 1 in Ingram's 1957
paper?
9. What is Crick's 1958 model of the individual steps by which information passes from the sequence
of nucleotides in DNA to the sequence of amino acid residues in proteins, what molecular species are
involved and where is each molecular species synthesized?
10. What are the most important aspects in which Crick's model differs from present knowledge?
2 11. What argument does Crick give for invoking the existence of adapter molecules?
12. What argument does Crick give for adapters not being proteins and instead being or at least
containing nucleic acids?
13. What argument does Crick give for thinking that the adapters themselves cannot recognize the amino
acid they carry and that a separate enzyme exists to join each adapter to its specific amino acid?
14. What argument does Crick give for rejecting the possibility that the adapters are molecules in the
soluble RNA fraction and for speculating that adapters might instead be produced by the breakdown of
the soluble RNA? (The argument here is incorrect. The adapters, transfer-RNA molecules, are in the
soluble fraction.).
15. How would you describe Crick's way of thinking in this reading? Does it remind you of any of the
other readings in this course? Explain.
Genes and Enzymes-Some Background
Evidence that a Mendelian “factor” might somehow determine the presence or absence
of an enzymatic activity was first put forward in 1902 by the French biologist Lucien
Cuenot and by the English physician Archibald Garrod. Their work also provided the
first demonstration of Mendelian inheritance in animals.
Based on the results of crosses of mice with different fur
colors and pre-existing evidence for two different fur color pigments
– black and yellow – Cuenot proposed that a Mendelian gene (his
term was mnemon, the word “gene” was not coined until 1909) was
responsible for the production of a precursor which is converted into
Archibald Garrod
black or yellow pigment by the action of two different “diastases” (enzymes), each of
1857-1936
which is produced by a separate mnemon. “Diastase” is an old term for enzyme,
originally the isolable activity that depolymerizes starch, the first enzyme to be
discovered. (It was not known until many years later that enzymes are proteins and that proteins are
specific molecular entities.) Cuenot was also the first person to report the existence of multiple alleles,
different forms of the same gene. Cuenot’s work was long unrecognized by most geneticists (important
exceptions being William Castle at Harvard and Sewall Wright at the University of Chicago, both of
whom were conducting crosses with mice) until after George Beadle and Edward Tatum, then at
Stanford University, published evidence from the bread mold Neurospora crassa for the “one-gene oneenzyme” hypothesis in 1946 (see below).
Lucien Cuenot
1866-1951
More remarkable than the failure of geneticists to note the work of Cuenot, was the failure to
note the much more widely published work on hereditary defects in metabolism by the prominent
English physician Archibald Garrod, who, in 1902, proposed that alkaptonuria results from
homozygosity for a recessive Mendelian gene. Alkaptonuria is a condition in which the urine turns a
dark color upon exposure to oxygen. It was already known at the time that the condition results from the
accumulation of homogentisic acid in the urine and that homogentisic acid reacts with oxygen to give a
dark substance, subsequently shown to be a complex polymer. It was also known that homogentisic acid
fed experimentally to alkaptonurics is excreted unchanged but is not excreted if fed to normal patients,
indicating an inability of alkaptonurics to decompose it – an “error of metabolism”, as Garrod called it.
3 In his 1902 paper, Garrod summarized data showing that (i) alkaptonurics tend to occur in
clusters within sibships; (ii) patients with alkaptonuria are far more likely to be the children of first
cousins than are non-alkaptonuric individuals; and (iii) many more alkaptonurics have first cousins as
parents than have alkaptonurics as parents. Suspecting a genetic basis for the condition, Garrod credited
the English geneticist and early Mendelian, William Bateson, with telling him that these observations
were consistent with inheritance of alkaptonuria as a recessive Mendelian character. In subsequent
publications, Garrod proposed similar explanations for two additional recessive metabolic defects,
albinism and cystinuria. By 1909, evidence had appeared that the aromatic ring of homogentisic acid
could be broken by a “ferment” present in liver extracts. Garrod, in his book of that year, “Inborn Errors
of Metabolism”, wrote “We may further conceive that the splitting of the benzene ring of homogentisic
acid in normal metabolism is the work of a special enzyme, that in congenital alkaptonuria this enzyme
is wanting…”.
At the time and for almost two decades more, it was widely thought that
enzymes might be complex colloidal entities rather than molecules with specific
structure. This misconception persisted even several years after the enzyme urease,
which catalyses the hydrolysis of urea to carbon dioxide and ammonia, was prepared
from jackbeans in pure crystalline form by the American biochemist James Sumner
at Cornell in 1926, after nine years of effort. (Crystallization is evidence of purity -do you see why?) Only after several other proteins had been crystallized and shown
by sedimentation analysis and electrophoretic analysis to be monodisperse was it
generally agreed by biochemists that enzymes are specific molecular entities.
Sumner, John Northrop at Rockefeller and Wendell Stanley at UC Berkeley
James Sumner
shared
the 1946 Nobel Prize in chemistry for showing that enzymes are pure chemical
1887-1955
entities (Sumner and Northrup) and that Tobacco Mosaic Virus could be crystallized
(Stanley). Sumner’s engaging 1946 Nobel Prize speech is included as light reading for this week.
Following Garrod’s work, cases were found in which Mendelian factors could be traced to
specific known biochemical reactions in flower color of plants and, later, in Drosophila eye color. But
starting with a phenotype resulting from a single biochemical step and showing it to display Mendelian
inheritance left open the possibility that the responsible gene is atypical. Perhaps most genes govern
many biochemical steps or other biological processes but go unstudied because alteration in such a
multi-function gene is likely to be lethal to the organism and therefore not studied. The notion that most
genes might govern many processes reflected the then common view that the individual gene is a
complex organelle-like entity, capable of directly governing numerous biosynthetic reactions and other
processes and might itself be an enzyme or group of enzymes.
An important step forward was the demonstration in 1941
by George Beadle and Edward Tatum, that the effect of X-ray
induced mutations rendering the bread mold Neurospora unable to
grow in minimal culture medium (a solution of salts, sucrose and
biotin) could be overcome by adding a single specific vitamin or
metabolite to the minimal medium. By starting with mutations
unable to grow in minimal medium, for which the number of
affected biochemical reactions or other processes was initially
unknown, they reversed the earlier procedure by starting with a
mutation that could, in principal, disable many vital functions and
Edward Tatum
George Beadle
then showing that, in fact, only a single vitamin or metabolite
1909-1975
1903-1989 could restore the ability to grow. Then, to complete the argument,
they performed crosses to wild-type to show that such mutations
segregate in meiosis as single Mendelian factors. The protocol was to irradiate Neurospora prior to
meiosis with X-rays to induce mutations, prepare single-spore cultures in rich medium (yeast extract,
4 malt extract, glucose), and test Neurospora from each such culture for the ability to grow on minimal
medium. (The mutagenic action of X-rays had been shown in 1927 by Herman Muller working with
Drosophila.) Lines that grow on rich medium but are unable to grow on minimal medium were assumed
to lack one or more substance that is present in rich medium. The question was “one or more?”
The next step was to test many known vitamins and nutrients one-by-one for the ability to restore
Neurospora growth in minimal medium. It was found that a single vitamin or other single nutrient would
remedy the growth defect in most mutants and also that such mutations segregate like single recessive
alleles in crosses. Initially the full procedure was done with only a single mutant, one requiring
pyroxidine for growth in minimal medium, but soon many more mutants were tested, with the same
result. So, at least the mutations identified in this manner each segregated like a single Mendelian factor
and appeared to specify only a single biochemical step, not multiple steps, as though affecting only a
single enzymatic activity. Hence “one gene-one enzyme”.
But the objection was made by physicist-turned-geneticist Max Delbruck, at CalTech, that the
only mutants Beadle and Tatum could recover had at least to be able to grow on complex medium in
order to be cloned out of an X-rayed mass culture of Neurospora. The recovered mutants might therefore
represent only a small unrepresentative sub-set of all genes. The argument was that the more substances
a single gene specified, the more likely it would be that at least one of them could not be provided even
by complex media and therefore would have been lethal and not even seen by Beadle and Tatum. Such
substances would include those that are simply not present in complex medium, or are present but
cannot enter Neurospora cells. An attempt to deal with this possibility was made by Norman Horowitz
and Urs Leupold working at CalTech, where Beadle had by then become chairman of the Biology
Division. Their idea was to employ temperature-sensitive conditional lethals. Such mutants can grow in
minimal medium at one temperature but not at a higher temperature. Working with Neurospora and also
with E. coli, Horowitz and Leupold then tested each temperature-sensitive mutant (induced by X-rays or
ultraviolet light) to see if it would grow at the higher temperature in complex medium. Their assumption
was that most or many essential cell components that might be absent in complex medium or unable to
enter cells from the medium might nevertheless be susceptible to intracellular heat inactivation. In that
case, the kind of mutations that Delbruck thought would be missed in previous experiments could be
obtained as temperature sensitives.
Finding that at least half of the temperature-sensitive mutants they isolated could indeed grow on
complex medium at the higher temperature, Horowitz and Leupold concluded that no more than half of
the temperature-sensitive mutations affected substances that were not present in complex medium or that
were present but could not enter cells. On their assumption that most substances specified by genes
could be rendered temperature sensitive by mutation, this was taken to mean that, contrary to Delbruck's
reservation, a substantial proportion of genes governed individual enzymes required for the production
of a substance that was present in rich medium and could enter cells. What genes are and how they
specify proteins would become known only later, after knowledge of the structure of DNA and of the
processes of transcription and translation could be brought to bear on the problem. And in doing so it
became clear that the term “gene” may be used in several different ways, to be discussed later.
Genes, Amino Acid Sequence and Protein Synthesis-Some Background
We have seen how the experiments of Cuenot (1902), Garrod (1902-1909) and Beadle and
Tatum (1941) provided early evidence that genes specify enzymes, generally in one-to-one
correspondence, and how the work of Sumner (1926), Northrup (1929) and others established that
enzymes and other proteins are reasonably discrete chemical entities. But how do genes specify the
amino acid sequence of proteins? Or perhaps only the folding of proteins? Indeed, do proteins have
unique amino acid sequences or is the sequence of a given enzyme even if specified by a given gene
5 somewhat variable? And can individual mutations substitute only one amino acid for another or must an
individual mutation change more than one amino acid?
The first solid evidence that a particular Mendelian allele specifies the amino acid composition
of a particular protein came from the comparison of sickle cell hemoglobin with normal hemoglobin by
Linus Pauling and his colleagues at CalTech in 1949. Sickle cell disease is manifested by the presence
of sickle-shaped red blood cells in venous blood at low oxygen tension, whereas normal red cells are
biconcave in shape.
Having shown in 1936 by measurements of diamagnetism (Pauling and Coryell, PNAS 22: 210216) that oxygenation of hemoglobin causes a change in the binding of iron within the hemoglobin
molecule and upon learning several years later (in 1945) that sickling is much more pronounced in
venous (deoxygenated) blood than in arterial (oxygenated) blood, Pauling surmised that the disease must
be caused by a structural abnormality in the hemoglobin molecule itself, not by some more complex
abnormality of the red blood cell. In Pauling’s recollection of a conversation with Dr. William B. Castle
(son of the Harvard zoologist William E. Castle, an early mouse geneticist) during a train trip from
Denver to Chicago written many years later:
“...Bill Castle, a physician from Peter Bent Brigham Hospital and Children's Hospital in Boston, talked
about sickle cell anemia. I was not especially interested in what he was saying, because at that time I
felt that diseases were just too complicated for me to attempt to understand. He mentioned that the red
blood cells were twisted out of shape in the blood of patients with this disease. I thought to myself, there
are probably thousands of different chemical substances in the red blood cell, and I don't think I can
understand why a cell would be twisted out of shape. Then, he went on to explain that they twisted out of
shape in venous blood, but resume their normal flattened spherical shape in arterial blood. Within a few
seconds the idea flashed through my mind that red cells contain a great amount of hemoglobin - about
one-seventh of the content of a cell is hemoglobin molecules - and that the difference between venous
blood and arterial blood is that in venous blood the hemoglobin is present as hemoglobin itself, whereas
in arterial blood it becomes oxyhemoglobin because there are oxygen molecules attached to the iron
atoms. .... It immediately occurred to me that sickle-cell anemia must be a disease of the hemoglobin
molecule, with sickle-cell hemoglobin having an abnormal structure...”
Linus Pauling in
Sickle-cell Disease: Basic Principles and Clinical Practice.
1994 Raven Press, NY.
This insight led to the discovery by Pauling, Itano, Singer and Wells (1949), that hemoglobin
from sickle-cell patients (HbS) and hemoglobin from normal individuals (HbA) have opposite net
charges at neutral pH. From their titration and electrophoretic data they calculated that at neutral pH the
HbS molecule has 2-4 more positive charges than the HbA molecule. While it is conceivable that a
change in folding could shield or unshield ionizable amino acid side chains, thereby altering the
isoelectric pH, this was made unlikely by measurements of diffusion and sedimentation constants
showing no difference in molecular conformation between HbA and HbS. Finding no change in the
molecular structure of the porphyrin moiety, it was concluded that there must be a difference in protein
composition, rather than merely in folding, between HbS and HbA.
That same year, James Neel at the University of Michigan produced evidence that sickle cell
anemia (also called sickle cell disease) resulted from homozygosity of a gene which when heterozygous
caused little if any clinically evident illness. Until then, it had mistakenly been suspected that the disease
was due to a gene with dominant clinical expression. Neel examined blood samples from the clinically
normal parents of sickle cell anemia patients under conditions of very low oxygen tension. The test was
6 done with blood from both parents of 13 sickle cell anemia patients and from one parent of another 16
sickle cell anemia patients. Red blood cells from every one of the 42 samples sickled when the oxygen
tension was greatly lowered, proving that all the parents were heterozygous carriers of the sickle cell
gene and that clinical expression as sickle cell anemia required homozygosity.
Apparently independent of Neel’s genetic evidence that the parents of sickle cell anemia patients
are heterozygous, Pauling, et al. (1949), finding that HbA and HbS are both present in the blood of such
parents in roughly equal amounts, reached the same conclusion. The electrophoretic and genetic
evidence, taken together, constitute the first demonstration that a Mendelian gene directs the amino acid
composition of a protein.
[Digression on Heterosis. Soon after the report of Pauling et al. (1949), Anthony Allison investigating
the distribution of human blood group types among different populations in East Africa discovered an
unexpectedly high (20-30 percent) frequency of people with sickle cell trait (A/S heterozygotes), as
determined by the characteristic sickling of red blood cells when oxygen is removed. Finding very few
adults with clinically identifiable sickle cell anemia (S/S homozygotes), he reasoned that the lethality of
homozygosity must be balanced in such populations by a selective advantage of heterozygotes relative to
A/A homozygotes acting before reproductive age. The postulated advantage was then traced to a lower
frequency of fatal malaria among young people with sickle cell trait (A/S) as compared with A/A
homozygotes, thereby maintaining the S allele in the population. (Although well-established in this case,
and likely in a few other known human conditions, such heterozygote advantage or "heterosis" in
humans has been difficult to establish with certainty except for A/S.]
Even though the electrophoretic studies of Pauling et al. showed almost certainly
that the sickle cell mutation changes the amino acid composition of hemoglobin, it was
not then established that specific proteins had unique amino acid sequences. The first
demonstration that this is so was accomplished in 1951 by the sequencing of the 30amino acid B chain of bovine insulin in 1951, by Frederick Sanger and Hans Tuppy
for which Sanger was awarded the 1956 Nobel Prize in Chemistry, not only for the
sequence determination itself but also for developing methods for
such sequencing when no suitable methods had previously existed.
(Sanger went on to develop methods for the sequencing of RNA and
DNA, for which he was awarded a second Nobel Prize, in 1980, the
only person besides Marie Curie to receive two Nobel Prizes in science.)
Although it was accepted from the work of Sanger and others that proteins
had definite amino acid sequences, it remained to determine the nature of the change
Vernon Ingram
in amino acid sequence corresponding to specific mutation. This was first
1924-2006
accomplished in 1956 by Vernon Ingram, then at the University of Cambridge.
Seeking to determine the nature of the amino acid difference between sickle cell
hemoglobin and wild type hemoglobin, Ingram examined tryptic digests of HbA and HbS by a
combination of paper electrophoresis followed by paper chromatography, giving rise to a twodimensional separation of the various tryptic fragments. This revealed that the only difference between
HbA and HbS is in one particular tryptic fragment of the B chain, which Ingram called peptide number
4. The following year, in the paper we read this week, Ingram reported that the difference between
peptide number 4 from HbA and the corresponding peptide from HbS was the replacement of a glutamic
acid residue by a valine residue. Thus, the mutation causing sickle cell anemia changes only a single
amino acid at a particular site and the replacement of an acidic amino acid residue with a neutral one
explains the charge difference at neutral pH found by Pauling et al. in 1949. Ingram's finding
demonstrated that a mutation can change the amino acid at a single site in a protein without any other
Frederick Sanger
1918-2013 7 change in amino acid sequence. (Incidentally, it also constituted the first experimental evidence that the
genetic code is not "overlapping". We will discuss the structure of the genetic code in Week 9.)
At the time of Ingram's discovery, evidence had just been produced by Pauling and his
colleagues that there are two kinds of peptide chain in hemoglobin, now called alpha and beta, both with
valine at their N-terminal ends, and that the hemoglobin molecule consists of two alpha chains and two
beta chains. The difference between hemoglobin A and hemoglobin S is in the beta chain. X-ray
diffraction studies of hemoglobin crystals by Max Perutz and his colleagues at the University of
Cambridge showed hemoglobin to be made up of two identical half-molecules. Thus, the hemoglobin
molecule is made up of two identical subunits, each of which comprises one alpha chain and one beta
chain, both starting with valine. The alpha chain of human hemoglobin has 141 amino acids and the beta
chain has 146.
Although the amino acid sequence Ingram deduced for the first six amino acids at the carboxyterminal portion of peptide number 4 in HbA and HbS is correct, the amino acid sequence he reported
for the rest of the peptide is wrong. As shown in later work by Ingram and Tim Hunt, the correct
sequences, starting at the N-terminal end, are val-his-leu-thr-pro-glu/val-glu-lys. The valine of peptide
number 4 is the first amino acid in beta hemoglobin and therefore the glu/val substitution is at position 6
counting from the N-terminal end of the beta chain. Still, this error does not affect Ingram's evidence for
there being only a single amino acid difference between HbA and HbS .
Once it was shown that DNA is the nuclear carrier of heredity and that its structure is that of a
linear polymer in which its four nucleotides can occur in any order, and that proteins are definite
molecular entities with specific amino acid sequences, the stage was set to ask the grand question of just
how the sequence of nucleotides specifies the sequence of amino acids. The question had two parts:
What is the molecular mechanism of protein synthesis? And what is the code, the set of rules by which
one could read the amino acid sequence of a gene-specified protein from the DNA sequence of the
corresponding gene? Although it was not proven until several years after these questions came to the
forefront of molecular biological research, it was generally assumed that the sequence of nucleotides in
the DNA of a gene, the genetic map of its mutations, and the sequence of amino acids in the specified
protein are all co-linear.)
Seldom during the attack on a fundamental problem in biology has there been written as lucid an
account of the state of knowledge and theory when the problem was about half-solved as is the analysis
of the problem of protein synthesis circa 1957 by Francis Crick. Much of the picture described in his
1958 essay "On Protein Synthesis" proved to be correct, notably:
(i) The "Sequence Hypothesis", the idea that the linear sequence of nucleotides in DNA specifies
the linear sequence of amino acids in protein and the implication that nucleotide and amino acid
sequences are co-linear, an idea that immediately raises the "coding problem".
(ii) The "Central Dogma", the idea that information may pass from nucleic acid to nucleic acid
and from nucleic acid to protein but not from protein to protein or from protein to nucleic acid. (Note
that reverse transcription does not violate the Central Dogma.)
(iii) The hypothesis that the amino acid sequence of protein is specified on an RNA template
which itself is copied from DNA (except in the case of RNA viruses). Although Crick and others at the
time mistakenly thought the RNA template for protein synthesis was ribosomal RNA, this hypothesis
nevertheless raised the problem of how the amino acids are assembled in the correct order, a problem
that led to:
8 (iv) The "Adapter Hypothesis", the idea that in addition to the RNA template for protein
synthesis there must be small RNA molecules, "adapters" at least one kind for each kind of amino acid,
and that each is loaded with its specific amino acid by a specific enzyme and then diffuses to the RNA
template where it recognizes via base-pairing the correct site at which the amino acid should be placed
in the peptide chain that is being synthesized.
(v) The coding problem. Here Crick outlines some of the major questions regarding the genetic
code: What is the coding ratio? Is the code overlapping? Is it degenerate? How are code-words
delineated? Is the code "comma-free"? (The correct idea, that the code might be read in words of fixed
size, such as nucleotide triplets, starting from a fixed starting point, was not considered at the time.)
Except for the adapter hypothesis, which was original to Crick, the above ideas were, in one
form or another, part of the general thinking by the rather small number of investigators focused on the
general problem of the genetic specification of proteins. Two of the most fundamental features of
protein synthesis are missing from Crick's 1958 conception. First, as implied by kinetic studies of the
expression of the E. coli lac operon conducted as early as 1952 but apparently not realized until several
years later, the template for protein synthesis is a short-lived intermediate shown, in 1961, to be mRNA,
not ribosomal RNA. Second, as also discovered in 1961, the DNA code for a particular amino acid
sequence is read in constant size "words" (nucleotide triplets) from a fixed starting point.
9