Download Perspectives

Copyright © 2005 by the Genetics Society of America
Perspectives
Anecdotal, Historical and Critical Commentaries on Genetics
Edited by James F. Crow and William F. Dove
The Favorable Features of Tryptophan Synthase for Proving
Beadle and Tatum’s One Gene–One Enzyme Hypothesis
Charles Yanofsky1
Department of Biological Sciences, Stanford University, Stanford, California 94305-5020
L
AST year we celebrated the 100th anniversary of the
birth of George Beadle. He, with Edward Tatum, in
1941, proposed one of the major conceptual advances in
biology in the 20th century: the existence of a one gene–
one enzyme relationship (Beadle and Tatum 1941).2
Unfortunately, like many biologists proposing exciting
hypotheses, they could not provide the experimental
evidence that would prove they were correct. At that
time, the existing knowledge about gene and enzyme
structure was inadequate to permit the relationship’s
verification. George Beadle was aware of this and undoubtedly regretted his inability to provide support for
their hypothesis. In a retrospective article written for
Phage and the Origins of Molecular Biology, Beadle (1966,
p. 30) states that at the Cold Spring Harbor Symposium
of 1951 “the number whose faith in one gene–one enzyme remained steadfast could be counted on the fingers of one hand—with a couple of fingers left over.”
Why? Why was their proposed basic relationship not
accepted? Why could they not provide proof for their
hypothesis?
Beadle and Tatum’s principal difficulty was that in the
1940s we knew relatively little about the chemical nature
of genetic material. The prevailing view then was that
genetic material might be protein. Existing information
on DNA structure suggested that it consisted of repetitive nucleotide sequences. If correct, it would be unlikely that DNA could specify the many different proteins present in each organism. Also, it was not yet firmly
established that polypeptides consist of linear sequences
of amino acids. Therefore, during the early years follow-
1
Author e-mail: [email protected]
A Perspectives has been written reviewing the information available
on the gene–enzyme relationship prior to Beadle and Tatum’s studies
(Hickman and Cairns 2003). A biography of George Beadle has recently been written by Paul Berg and Maxine Singer (Berg and Singer
2003).
2
Genetics 169: 511–516 (February 2005)
ing Beadle and Tatum’s proposal of the one gene–one
enzyme relationship, our understanding of the structural features of genes and enzymes was insufficient to
allow formulation of a finite molecular relationship.
Advances in the 1950s dealt with most of these issues
and suggested a plausible experimental strategy that could
be applied in comparing the structural features of a gene
with that of its corresponding protein. One of the earliest
relevant findings, by Avery et al. (1944), suggested that
the pneumococcal transforming principle—presumably
the genetic material of the organism—was very likely DNA
(see Lederberg 1994). Unfortunately, the significance
of this finding was not appreciated initially; questions
were raised about the purity of the transforming principle, our understanding of the transformation process was
incomplete, and doubts existed that pneumococcal genetic material would be structurally comparable to the
genetic material of higher organisms. Somewhat later, in
the early 1950s, Al Hershey and Martha Chase presented
their isotopic labeling studies with bacteriophageinfected Escherichia coli. Their results provided what was
considered convincing evidence that phage genetic material is in fact DNA (Hershey and Chase 1952; see
Stahl 1998). Most importantly, in 1953, Jim Watson
and Francis Crick described the double-helical structure
of DNA (Watson and Crick 1953). Their contribution
changed forever how genetic material would be viewed
by everyone. The realization that each gene probably
consists of a linear sequence of nucleotides prompted
subsequent thought on the existence of a genetic code,
an essential consideration with a direct bearing on the
gene-enzyme relationship. During this period Matt Meselson and Frank Stahl established that DNA is replicated
by synthesis of complementary DNA strands, revealing
the elegant features of DNA structure that provide for
its exact replication (Meselson and Stahl 1958). Also
of great importance, Fred Sanger showed that an insulin
polypeptide consists of a unique linear sequence of
512
C. Yanofsky
amino acids (Sanger 1952; Stretton 2002). The significance of the contributions of Sanger, Erwin Chargaff, and others are described in an article by Horace
Judson (Judson 1993). The discoveries cited, and observations by others supporting their conclusions, redefined the gene–enzyme relationship of Beadle and Tatum as the gene-protein colinearity hypothesis.
Despite these advances in the 1950s, the technology
that would have allowed either isolation of a gene or
determination of its nucleotide sequence was lacking.
Furthermore, since the concept of the genetic code was
yet to be developed, knowledge of Watson and Crick’s
double-helical structure of DNA was initially of little
help to those wishing to experimentally address the
structural relationship between a gene and the enzyme
it was proposed to specify. In the late 1950s Seymour
Benzer demonstrated that one could construct a linear
fine-structure genetic map by crossing mutants with genetically separable alterations in the same genetic region (Benzer 1957). His findings were consistent with
the interpretation that a gene consists of a specific linear
sequence of nucleotides. At approximately the same
time, Vernon Ingram identified the amino acid change
in the hemoglobin of humans with sickle cell anemia,
and he developed the protein “fingerprinting” technique, a method that could be applied in detecting
the single amino acid change in any mutant protein
(Ingram 1958; see Ingram 2004). These two advances
provided an excellent pair of experimental approaches
that could be applied immediately in testing the gene–
protein colinearity concept.
MY COMMITMENT TO PROVING THE
GENE-ENZYME RELATIONSHIP
From 1948 to 1951 I did my graduate studies at Yale
University, working with David Bonner, an alumnus of
the Beadle/Tatum group at Stanford University. My
principal research objective, like that of most members
of our group, was to attempt to provide evidence for—or
proof of—Beadle and Tatum’s one gene–one enzyme
hypothesis. This hypothesis was presented to us doctoral
students as one of the most exciting recent predictions
in the biological sciences—but one still requiring experimental proof. Beadle and Tatum’s major experimental
contribution had been the isolation of numerous biochemical mutants of Neurospora, each having a specific
nutritional requirement. Genetic analyses with these
mutants provided the exciting finding that a unique
gene was associated with each biochemical reaction.
Thus, independent mutations preventing completion
of any one specific biochemical reaction were almost
invariably located in one particular gene. Despite these
results, which certainly suggested a one gene–one enzyme relationship, very few Neurospora mutants were
defective in reactions for which the enzyme molecule
was known. Therefore it was not obvious which gene–
enzyme system could be used to show that the mutational loss of ability to perform a biochemical reaction
was due to loss of a specific enzyme. The major enzymological studies underway in the field of biochemistry
prior to 1950 unfortunately were focused on carbon
metabolism, not on amino acid, vitamin, purine, or pyrimidine biosynthesis. Thus the enzymes presumably
altered in Beadle and Tatum’s biochemical mutants
were unknown.
In my investigations on the gene-enzyme relationship,
begun in 1950 using Neurospora as the experimental
organism, I chose one of the initial enzymes of this
organism shown to catalyze a biochemical reaction for
which a specific mutant was believed to exist. This enzyme,
tryptophan desmolase, catalyzed the final reaction in tryptophan formation: the conversion of indole ⫹ l-serine to
l-tryptophan (Umbreit et al. 1946). Mitchell and Lein
(1948) had analyzed one Neurospora mutant blocked
in the indole-to-tryptophan reaction, mutant td1, and
had shown that it did in fact lack tryptophan desmolase
activity. I initially examined extracts of a second td mutant, td2, also defective in the conversion of indole to
tryptophan, and found that it, too, lacked tryptophan
desmolase activity (Yanofsky 1952). In further studies
with these td mutants, performed with Sig Suskind and
Dave Bonner (Suskind et al. 1955), we used an antiserum prepared against wild-type Neurospora tryptophan
desmolase—an antiserum that inhibited the enzyme’s
activity. Using this antiserum, we found that extracts
of mutants td2 contained a cross-reacting material that
blocked antibodies to tryptophan desmolase; this material reversed antibody inhibition of the partially purified
wild-type enzyme (Suskind et al. 1955). We named this
cross-reacting material “CRM” (Yanofsky 1956). Interestingly, extracts of the td1 mutant examined by Mitchell
and Lein lacked a td-CRM. Many additional td mutants
were subsequently isolated and analyzed in the same
manner, and they fell into the same two groups, one
producing a td-CRM and a second that did not (Suskind
et al. 1955; Yanofsky and Bonner 1955; Yanofsky 1956;
Suskind and Yanofsky 1961). From these findings it
was evident that it should be possible to identify mutants
producing many different td-CRMs—presumably inactive tryptophan desmolase proteins—proteins that
could then be isolated and analyzed to determine the
inactivating amino acid change in each.
We also observed presumed revertants in cultures of
several of our CRM⫹ mutants, mutants that could grow
without an indole or tryptophan supplement. Genetic
analyses revealed that growth of some of these was due
to a change in a gene unlinked to the td locus—a suppressor mutation (Yanofsky 1952). When we examined
our CRM⫺ mutants, particularly td1, we were unable
to obtain a suppressed strain (Yanofsky and Bonner
1955). (In retrospect, this result is surprising. We would
expect that some of the mutations in these mutants
Perspectives
would be amber or ochre suppressible nonsense mutations. This observation was not examined further, and
there are several possible explanations.) One interesting
observation made in these studies was that each suppressed mutant appeared to produce both the wild-type
enzyme and the mutant td-CRM. This pattern allowed
me to conclude that suppression does not alter the
mutant td gene; rather, it permits some form of mistranslation of the altered genetic information in this gene,
restoring some wild-type enzyme activity (Yanofsky
1956). This conclusion was reached prior to the discovery of messenger RNA, transfer RNA, or the mechanism
of translation. Since our suppressible td mutants fell
into several distinct classes—i.e., each class responded
to a different suppressor gene—I assumed that these
mutants must have nonidentical changes in the td gene
and protein (Yanofsky 1956). Unfortunately, my attempts to purify wild-type Neurospora tryptophan desmolase were unsuccessful, leading me to conclude that
the Neurospora td gene-tryptophan desmolase system
would not be suitable for establishing the gene-enzyme
relationship.
During this period George Beadle and Ed Tatum
were very supportive of my studies; each communicated
several of my papers to the Proceedings of the National
Academy of Sciences. I am sure that each must have
been responsible for my being invited to speak at various
symposia. Beadle occasionally contacted me by mail,
requesting updates on our accomplishments on colinearity.
MY MOVE TO WESTERN RESERVE
UNIVERSITY MEDICAL SCHOOL
In 1954, while the above advances were being made,
I accepted a faculty position as assistant professor in
the Department of Microbiology at Western Reserve
University Medical School. Since I was unaware of any
gene–enzyme system that would be ideal for testing
gene–protein colinearity, and since, as a new recruit, I
felt it would be wise to choose a project that would allow
me to be productive, I decided to focus my attention
on identifying all the remaining intermediates, reactions, and enzymes of the tryptophan biosynthetic pathway. I adopted E. coli as my principal experimental organism, primarily because I was aware of the outstanding
contributions of Joshua Lederberg in establishing this
organism’s attractiveness for mutational and genetic
analyses (see Lederberg 1987). However, throughout
this period, as I recall, the colinearity question continued to be foremost in my mind. In fact I spent the
summer of 1956 at the Cold Spring Harbor Laboratory
learning about phage-P22-mediated transduction in Salmonella and its use in genetic analysis. My hope was to
use transduction to prepare a fine-structure genetic map
of a trp gene of E. coli. While at Cold Spring Harbor,
Ellis Englesberg and I became very good friends, and
513
we exchanged thoughts, results, and advice, repeatedly,
for many years thereafter. Upon returning to Western
Reserve I contacted Ed Lennox at the University of
Illinois who had been characterizing the E. coli transducing phage P1kc. He and I collaborated in establishing
that this phage could be used to prepare a fine-structure
genetic map of any of the trp genes of E. coli (Yanofsky
and Lennox 1958).
I MOVE AGAIN—TO STANFORD UNIVERSITY—
AND ADDRESS COLINEARITY
In 1958, with some regrets, I decided to leave Western
Reserve Medical School and move to the Department of
Biological Sciences at Stanford University. For someone
dedicated to proving colinearity, as I was, I could not
have relocated into more exciting quarters. I was assigned the lab space previously occupied by Ed Tatum
and his group, just down the hall from George Beadle’s
former lab in the basement of Jordan Hall. I had decided then that the time had come for me to mount an
all-out effort to prove or disprove colinearity. On the
basis of my studies at Western Reserve, I decided that
the tryptophan desmolase of E. coli might be an ideal
enzyme for this task. I felt that all the necessary experimental approaches that would allow me to prepare a
fine-structure map and to determine the amino acid
changes in inactive mutant proteins existed.
The second postdoc to join my group, Irving Crawford, arrived at Stanford at the same time I did. He set
out to determine the characteristics of the tryptophan
synthase of E. coli. (The name of the enzyme was changed
from desmolase to synthetase and, finally, to synthase.)
Building on our prior clues, Crawford showed that tryptophan synthase of E. coli is actually an enzyme complex,
consisting of two separable polypeptides, designated
TrpA and TrpB. This enzyme complex catalyzes the
last two reactions in tryptophan formation, whereas the
separate TrpA and TrpB proteins each catalyzes only
one of these reactions (Crawford and Yanofsky 1958).3
Most importantly, he observed that the activity of each
protein in the reaction that it performs alone is increased appreciably, from 30- to 100-fold, when it is
3
The 3D structure of the tryptophan synthase enzyme complex
of Salmonella typhimurium has been determined (Hyde et al. 1988).
Structural explanations have also been provided for enzyme inactivation resulting from the amino acid changes introduced by our trpA
missense mutations (Miles 1995). Tryptophan synthase of E. coli and
S. typhimurium is an ␣␤␤␣ tetramer, with TrpA as ␣ and TrpB as ␤.
Each ␣-chain is bound to the surface of one of the ␤-chains of a ␤2
dimer. Each ␣-chain active site is connected to a ␤-chain active site
by an intraenzyme tunnel, and indole, enzymatically generated from
indoleglycerol phosphate in the ␣-active site, traverses through this
tunnel to the ␤-active site, where it is combined with l-serine to form
l-tryptophan. In the tryptophan synthase (desmolase) of Neurospora
crassa the ␣-domain is fused to the ␤-domain in the ␣␤ order, with a
connecting amino acid sequence linker between the two domains.
The active enzyme from Neurospora is a dimer of this ␣-␤ fusion
polypeptide.
514
C. Yanofsky
mixed with the other protein (Crawford and Yanofsky
1958; Yanofsky 1959). This monumental observation
suggested that each inactive missense TrpA and TrpB
protein probably could be assayed enzymatically by measuring ability to activate the unaltered other partner
polypeptide, using the reaction performed by that second polypeptide. Thus each missense TrpA protein,
although inactive in the indoleglycerol phosphate →
indole reaction, would activate the wild-type TrpB protein 30-fold in the indole ⫹ serine → tryptophan reaction. We went ahead and used the indole-to-tryptophan
assay to follow the purification of each CRM⫹ mutant
TrpA protein.
Barbara Maling, Don Helinski, and Ulf Henning
performed our initial TrpA protein studies, purifying
and characterizing selected missense mutant proteins
(Maling and Yanofsky 1961; Helinski and Yanofsky
1962; Henning and Yanofsky 1962a). To accomplish
this, they capitalized on an earlier observation, which
had regulatory significance, namely, whenever any trpA
tryptophan auxotroph was grown in a medium containing a growth-limiting level of indole or tryptophan,
it overproduced the mutant TrpA protein, to ⵑ1–2%
of the soluble protein. Under these conditions, mutant
TrpA proteins were overproduced and easily purified,
exploiting our additional finding that TrpA is relatively
stable to acidic conditions, whereas TrpB and most E. coli
proteins are not. Column chromatography provided the
final purification. Each mutant protein was then digested with trypsin, chymotrypsin, or both, and 2D fingerprints of the peptide digests were prepared by electrophoresis and paper chromatography, as described by
Ingram (Yanofsky et al. 1961). The fingerprint of each
mutant protein was then searched for a peptide with
altered mobility, resulting from a specific amino acid
change. The fingerprints of about half of the mutant
proteins that we examined initially displayed a single
peptide with altered mobility. The amino acid change
in each was then determined by sequencing the altered
peptide. By 1962, we had completed our initial examination of selected TrpA mutant proteins using these approaches. We observed that trpA mutants with genetic
alterations near one another on the trpA genetic map
had amino acid changes in the same tryptic peptide
(Helinski and Yanofsky 1962; Henning and Yanofsky
1962a). This initial finding buoyed our confidence that
our approaches would be successful and that we would
be able to test fully the gene-protein colinearity hypothesis.
My research assistant, Ginny Horn, focused on isolating numerous trpA mutants; with these, she performed
fine-structure genetic analyses, using phage P1kc, and
constructed a fine-structure genetic map. As I recall,
UV treatment was initially our preferred method of mutagenesis. The subset of tryptophan auxotrophs that
grew on indole, and accumulated indoleglycerol phosphate, was identified; these auxotrophs were presumed,
correctly, to be trpA mutants. Good growth vs. poor
growth on indole distinguished between missense and
CRM⫺ (nonsense) trpA mutants. In other genetic studies, we exploited the fact that the tonB locus, conferring
resistance to phage T1, was located adjacent to trpA
on the map. This knowledge allowed us to screen for
spontaneous T1 resistant (ton B) mutants in which a
deletion extended into the trp operon—into trpA or into
other genes of the operon. The overlapping deletion
endpoints in trpA were located by mapping them against
our trpA point mutations. In turn, point mutations were
crudely located by crossing them with the overlapping
deletion mutants, essentially as had been done by Benzer
for T4rII (Benzer 1959). A linked outside marker, cysB,
was used in three point crosses to order trpA point mutations located near one another. Many trpA point mutants
were crossed pairwise to determine recombination frequencies, relative to recombination with an independent,
unlinked marker; these crosses provided normalized map
distances separating each pair of mutationally altered sites
(Yanofsky et al. 1964).
In the initial verification of these approaches, Don
Helinski and Ulf Henning established that two sets of
mutants altered near the same site in trpA changed the
same wild-type amino acid, glycine-to-arginine and glycineto-glutamic acid, respectively (Helinski and Yanofsky
1962; Henning and Yanofsky 1962a). (They also observed that several independently isolated mutants altered at the same site in the trpA gene had exactly the
same amino acid changes, glycine-to-arginine or the
same glycine-to-glutamic acid.) When two mutants with
distinct amino acid changes at this glycine site were
crossed with one another, wild-type-like recombinants
with glycine were produced, suggesting that the wildtype coding sequence (codon) specifying glycine could
be changed to two different codons, one specifying arginine and the second specifying glutamic acid (Henning
and Yanofsky 1962b). Clearly, this was an example of
intracodon recombination, the result expected if several
nucleotides compose the codon for each amino acid
and genetic exchange is possible between adjacent nucleotides. In subsequent studies John Guest examined
the products of crosses of several mutants, each containing
a different amino acid at the same position in the protein,
and demonstrated intracodon recombination consistent
with what was then known about the genetic code (Guest
and Yanofsky 1965). His findings extended the evidence
that each codon consists of several nucleotides. These and
subsequent in vivo studies (Yanofsky et al. 1969) provided
support for some of the codon assignments deduced
from the in vitro studies by Nirenberg, Khorana, and
others (Khorana et al. 1966; Nirenberg et al. 1966).
By 1963 we had obtained considerable genetic and
protein data with many of our trpA missense mutants,
all of which were consistent with the colinearity concept.
Our missense mutants had alterations throughout much
of the trpA gene. The relationship between genetic map
distance and distance between corresponding altered
amino acids was observed to vary somewhat. Since none
Perspectives
of our trpA missense mutants were altered near either
end of the trpA gene, we selected specific nonsense
mutants with alterations near these ends and isolated
pseudo-wild-type (partial) revertants of each. The locations of the amino acid changes in these partial revertants allowed us to confirm colinearity for the entire
gene and protein.
I initially presented our exciting findings establishing
colinearity for the trpA gene and TrpA protein of E. coli
at the 1963 Cold Spring Harbor Symposium (Yanofsky
1963). Our first detailed article was published the following year (Yanofsky et al. 1964) and was coauthored by
the postdocs who were responsible for the bulk of our
protein findings, Bruce Carlton, John Guest, Donald
Helinski, and Ulf Henning. Not content with anything
less than having the complete amino acid sequence of
the TrpA protein, Gabriel Drapeau, John Guest, and
Bruce Carlton of my lab persisted on this most tedious
aspect of the project. They completed their determination of the complete amino acid sequence of the TrpA
protein in 1967 (Yanofsky et al. 1967). At the time, this
was the longest polypeptide to have been sequenced.
Since the technology used in our colinearity studies
was current and well known, other investigators adopted
similar procedures and applied them with their preferred gene and enzyme, determined to provide proof
for colinearity. Alan Garen, Frank Rothman, and Cy
Levinthal selected the alkaline phosphatase of E. coli.
Rothman has written an essay (Rothman 1987) describing their studies, its background, approaches, and the
solution of the colinearity problem. George Streisinger
selected T4 phage lysozyme and presented beautiful
analyses of the consequences of frameshift mutations
(Streisinger et al. 1966). Seymour Benzer continued
his studies with the phage T4 rII locus and devoted
considerable attention to deducing the specificity of
different mutagens. Each group contributed new findings of significance supporting the concept of colinearity as well as providing data that improved our understanding of how genes and proteins are altered by
mutation. At that time, we were all using our respective
systems to address the nature of the genetic code.
Although most scientists attempting to prove or disprove colinearity were using strategies similar to the one
we adopted, Sydney Brenner, Tony Stretton, and Anand
Sarabhai developed a more imaginative, radically different approach (Sarabhai et al. 1964). They selected for
study the gene for the head protein of phage T4D,
a gene encoding a major protein product of phage
replication. They chose to characterize the head proteins of a set of suppressible amber nonsense mutants,
each altered in the head-protein gene. They prepared
a fine-structure genetic map ordering these mutational
alterations. To locate the positions of the corresponding
changes in the head proteins of these mutants, they
simply determined the length of each head-protein fragment produced as a consequence of the amber chain
termination mutation. They infected cells with phage
515
bearing the different head-protein gene amber mutations and labeled newly synthesized proteins with different radioactive amino acids. The cells were then lysed,
the nucleic acids removed, and the total protein hydrolyzed with trypsin or chymotrypsin. The resulting
peptides were then separated by high-voltage paper electrophoresis, and the labeled peptides were detected by
autoradiography. Sarabhai et al. (1964) compared the
set of labeled peptides obtained from each mutant headprotein fragment with the peptides produced from the
wild-type head protein. They observed that each mutant
head protein generated a subset of labeled peptides
with an endpoint corresponding to the location of the
respective amber mutation on the genetic map of the
T4 head-protein gene. Their results were definitive, establishing gene-polypeptide colinearity.
We could not have used this approach in our colinearity studies with the tryptophan synthase TrpA protein.
Our prematurely terminated TrpA polypeptides produced by nonsense mutants undoubtedly are degraded,
since we were unable to detect TrpA-CRM in extracts
of any of our nonsense mutants. The excessive overproduction of the T4 phage coat protein, and possibly the
characteristics of this protein, appeared to permit headprotein fragments lacking carboxy segments of the protein to survive proteolysis, thereby allowing polypeptide
fragment analysis.
Despite these accomplishments by several groups,
each strongly supporting gene–protein colinearity, it
became routine some years later simply to isolate and
sequence any specific gene, and, with knowledge of the
genetic code, predict its specified amino acid sequence
from its nucleotide sequence. However, it took many
additional years—until 1979—to determine the complete nucleotide sequence of trpA of E. coli (Nichols
and Yanofsky 1979). The conclusions reached previously on the basis of genetic characterization of genes
encoding proteins were validated, confirming the basic
relationship proposed by George Beadle and Edward
Tatum in 1941 on the basis of work in Neurospora.
Ironically, our studies establishing gene–protein colinearity were performed with an enzyme consisting of two
polypeptides encoded by adjacent genes in E. coli.
I am deeply indebted to David Perkins and Don Helinski for their
valuable comments on this manuscript. I would also like to thank the
granting agencies that provided support for the research conducted in
my laboratory: the National Institutes of Health, the National Science
Foundation, the American Heart Association, and the American Cancer Society. It is a particular pleasure to acknowledge the important
contributions by the members of my group during the years we analyzed gene-protein colinearity.
LITERATURE CITED
Avery, O. T., C. M. MacLeod and M. McCarty, 1944 Studies on
the chemical nature of the substance inducing transformation
of Pneumococcal types. J. Exp. Med. 79: 137–157.
Beadle, G. W., 1966 Biochemical genetics: some recollections, pp.
23–32 in Phage and the Origins of Molecular Biology, edited by J.
516
C. Yanofsky
Cairns, G. S. Stent and J. D. Watson. Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, NY.
Beadle, G. W., and E. L. Tatum, 1941 Genetic control of biochemical reactions in Neurospora. Proc. Natl. Acad. Sci. USA 27: 499–
506.
Benzer, S., 1957 The Chemical Basis of Heredity, pp. 70–93. Johns
Hopkins University Press, Baltimore.
Benzer, S., 1959 On the topology of the genetic fine structure. Proc.
Natl. Acad. Sci. USA 45: 1607–1620.
Berg, P., and M. Singer, 2003 George Beadle, an Uncommon Farmer.
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
Crawford, I. P., and C. Yanofsky, 1958 On the separation of the
tryptophan synthetase of Escherichia coli into two protein components. Proc. Natl. Acad. Sci. USA 44: 1161–1170.
Guest, J. R., and C. Yanofsky, 1965 Amino acid replacements associated with reversion and recombination within a coding unit. J.
Mol. Biol. 12: 793–804.
Helinski, D. R., and C. Yanofsky, 1962 Correspondence between
genetic data and the position of amino acid alteration in a protein.
Proc. Natl. Acad. Sci. USA 48: 173–183.
Henning, U., and C. Yanofsky, 1962a An alteration in the structure
of a protein predicted on the basis of genetic recombination
data. Proc. Natl. Acad. Sci. USA 48: 183–190.
Henning, U., and C. Yanofsky, 1962b Amino acid replacements
associated with reversion and recombination within the A gene.
Proc. Natl. Acad. Sci. USA 48: 1497–1504.
Hershey, A. D., and M. Chase, 1952 Independent functions of viral
protein and nucleic acids in growth of bacteriophage. J. Gen.
Physiol. 36: 39–56.
Hickman, M., and J. Cairns, 2003 The centenary of the one-gene
one-enzyme hypothesis. Genetics 163: 839–841.
Hyde, C. C., S. A. Ahmed, E. A. Padlan, E. W. Miles and D. R.
Davies, 1988 Three-dimensional structure of the tryptophan
synthase ␣2␤2 multienzyme complex from Salmonella typhimurium. J. Biol. Chem. 263: 17857–17871.
Ingram, V. M., 1958 Abnormal human hemoglobins. 1. The comparison of normal human and sickle-cell hemoglobins by fingerprinting. Biochem. Biophys. Acta 28: 539–545.
Ingram, V. M., 2004 Sickle-cell anemia hemoglobin: the molecular
biology of the first “molecular disease”—the crucial importance
of serendipity. Genetics 167: 1–7.
Judson, H. F., 1993 Frederick Sanger, Erwin Chargaff, and the metamorphosis of specificity. Gene 135: 19–23.
Khorana, H. G., H. Buchi, H. Ghosh, N. Gupta, T. M. Jacob et
al., 1966 Polynucleotide synthesis and the genetic code. Cold
Spring Harbor Symp. Quant. Biol. 31: 26–38.
Lederberg, J., 1987 Gene recombination and linked segregation
in Escherichia coli. Genetics 117: 1–4.
Lederberg, J., 1994 The transformation of genetics by DNA: an
anniversary celebration of Avery, MacLeod and McCarty. Genetics
136: 423–426.
Maling, B. D., and C. Yanofsky, 1961 The properties of altered
proteins from mutants bearing one or two lesions in the same
gene. Proc. Natl. Acad. Sci. USA 47: 551–566.
Meselson, M., and F. W. Stahl, 1958 The replication of DNA in
Escherichia coli. Proc. Natl. Acad. Sci. USA 44: 671–682.
Miles, E. W., 1995 Tryptophan synthase: structure, function, and
protein engineering. Subcell. Biochem. 24: 207–254.
Mitchell, H. K., and J. Lein, 1948 A Neurospora mutant deficient
in the enzymatic synthesis of tryptophan. J. Biol. Chem. 175:
481–482.
Nichols, B. P., and C. Yanofsky, 1979 Nucleotide sequences of
trpA of Salmonella typhimurium and Escherichia coli : an evolutionary
comparison. Proc. Natl. Acad. Sci. USA 76: 5244–5248.
Nirenberg, M., T. Caskey, R. Marshall, R. Brimacombe, D. Kellogg et al., 1966 The RNA code and protein synthesis. Cold
Spring Harbor Symp. Quant. Biol. 31: 11–24.
Rothman, F. G., 1987 Gene-protein relationships in Escherichia coli
alkaline phosphatase: competition and luck in scientific research,
pp. 307–311 in Phosphate Metabolism and Cellular Regulation in
Microorganisms, edited by A. Torriani-Gorini, F. G. Rothman,
S. Silver, A. Wright and E. Yagil. ASM Press, Washington DC.
Sanger, F., 1952 The arrangement of amino acids in proteins. Adv.
Protein Chem. 7: 1–28.
Sarabhai, A. S., A. O. W. Stretton, S. Brenner and A. Bolle, 1964
Co-linearity of the gene with the polypeptide chain. Nature 201:
13–17.
Stahl, F. W., 1998 Hershey. Genetics 149: 1–6.
Streisinger, G., Y. Okada, J. Emrich, J. Newton, A. Tsugita et al.,
1966 Frameshift mutations and the genetic code. Cold Spring
Harbor Symp. Quant. Biol. 31: 77–84.
Stretton, A. O. W., 2002 The first sequence: Fred Sanger and
insulin. Genetics 162: 527–532.
Suskind, S. R., and C. Yanofsky, 1961 Control Mechanisms in Cellular
Processes, pp. 3–21. Ronald Press, New York.
Suskind, S. R., C. Yanofsky and D. M. Bonner, 1955 Allelic strains
of Neurospora lacking tryptophan synthetase: a preliminary immunochemical characterization. Proc. Natl. Acad. Sci. USA 41: 577–
582.
Umbreit, W. W., W. A. Wood and I. C. Gunsalus, 1946 The activity
of pyridoxal phosphate in tryptophan formation by cell-free enzyme preparations. J. Biol. Chem. 165: 731–732.
Watson, J. D., and F. H. C. Crick, 1953 Molecular structure of
nucleic acids. Nature 171: 737–738.
Yanofsky, C., 1952 The effects of gene change on tryptophan desmolase formation. Proc. Natl. Acad. Sci. USA 38: 215–226.
Yanofsky, C., 1956 Gene interactions in enzyme synthesis, pp. 147–
160 in Henry Ford Hospital International Symposium: Enzymes, Units
of Biological Structure and Function. Academic Press, New York.
Yanofsky, C., 1959 A second reaction catalyzed by the tryptophan
synthetase of Escherichia coli. Biochim. Biophys. Acta 31: 408–416.
Yanofsky, C., 1963 Discussion following article by W. Gilbert, protein synthesis in Escherichia coli. Cold Spring Harbor Symp. Quant.
Biol. 28: 296–297.
Yanofsky, C., and D. M. Bonner, 1955 Gene interactions in tryptophan synthetase formation. Proc. Natl. Acad. Sci. USA 40: 761–
769.
Yanofsky, C., and E. S. Lennox, 1958 Transduction and recombination study of linkage relationships among the genes controlling
tryptophan synthesis in Escherichia coli. Virology 8: 425–447.
Yanofsky, C., D. R. Helinski and B. D. Maling, 1961 The effects
of mutations on the composition and properties of the A protein
of Escherichia coli tryptophan synthetase. Cold Spring Harbor
Symp. Quant. Biol. 26: 11–24.
Yanofsky, C., B. C. Carlton, J. R. Guest, D. R. Helinski and U.
Henning, 1964 On the colinearity of gene structure and protein
structure. Proc. Natl. Acad. Sci. USA 51: 266–272.
Yanofsky, C., G. R. Drapeau, J. R. Guest and B. C. Carlton, 1967
The complete amino acid sequence of the tryptophan synthetase
A protein (␣ subunit) and its colinear relationship with the genetic map of the A gene. Proc. Natl. Acad. Sci. USA 57: 296–298.
Yanofsky, C., H. Berger and W. J. Brammar, 1969 In vivo studies
on the genetic code. Proc. XII Intern. Congr. Genet. 3: 155–165.