On the Colinearity of Gene Structure and Protein Structure.

In the late 1950s, much thought and research were focused on deciphering the genetic code, determining how proteins are synthesized, and explaining how genetic information is translated into proteins. Whether or not the linear nucleotide sequence of a gene corresponds to the linear amino acid sequence of a polypeptide, “colinearity” was of major concern. But how could this relationship be examined experimentally? Specific genes could not be isolated, much less sequenced. Similarly, detecting single amino acid changes in a set of mutant proteins seemed beyond the realm of possibility. Benzer's studies in the late 1950s established that a fine-structure genetic map could suffice as a representation of the nucleotide sequence of a gene. Further, with the development of the peptide “fingerprinting” technique by Vernon Ingram in 1958, there was the hope that single amino acid changes could be detected in some proteins (V. E. Ingram, Biochim. Biophys. Acta 28:539–545, 1958). Colinearity of gene structure and protein structure was demonstrated in the early 1960s by my group and by Sydney Brenner and his co-workers (A. S. Sarabhai, A. O. W. Stretton, S. Brenner, and A. Bolle, Nature 201:13–17, 1964). Interestingly, the types of mutants examined and the experimental approaches used by the two groups were quite different. Our studies dealt with missense mutants and determination of the single amino acid changes in the corresponding protein. Brenner's group examined amber nonsense mutants and demonstrated changes in the length of a prematurely terminated protein. The unique feature of the protein we selected for study, the tryptophan synthetase α subunit of Escherichia coli, was its role as a component of an enzyme complex. Mutant missense α subunits could be readily recognized because they activated the β subunit of the enzyme. The positions of amino acid changes in purified missense proteins were then related to a fine structure genetic map of the trpA gene. Sydney Brenner's group solved colinearity by using amber nonsense mutants altered in the head protein of bacteriophage T4. They exploited the finding that, during the late stages of T4 infection, >50% of the protein synthesized by the host is head protein. Head proteins produced by amber mutants were isotopically labeled and then digested with trypsin or chymotrypsin, and the labeled peptides were separated by electrophoresis. Scoring the presence or absence of each peptide allowed the linear ordering of the terminated proteins with respect to the genetic map. We were all delighted to find that nature had not played one of its masterful tricks. Imagine our surprise when mRNA splicing was discovered! CHARLES YANOFSKY