The favorable features of tryptophan synthase for proving Beadle and Tatum's one gene-one enzyme hypothesis.

LAST 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 following 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 bacteriophage-infected 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 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.