Biochemistry - The Chemical Reactions of Living Cells, Volume 3 - D. Metzler 1980

Biochemical Genetics and the Synthesis of Nucleic Acids and Proteins
Genetic Methods
Establishing Correlation Between Genetic Maps and Amino Acid Sequences

Although studies of the rII region of the bacteriophage T4 chromosome demonstrated that genetic mapping could be carried out at the level of individual DNA NUCLEOTIDES, it was still necessary to prove the existence of a linear correspondence between nucleotide sequences in DNA and Amino acid sequences in Proteins. This was successfully accomplished by Yanofsky et al. [134] in experiments with E. coli Tryptophan synthetase, as well as by Sarabhai et al. [134a] in their Study of the T4 phage coat protein.

Tryptophan synthetase (p. 141) consists of two subunits, A and B (or $\alpha$ and $\beta$), the first of which contains a mere 268 Amino Acids. The Fine Structure of the A Gene was mapped in the following manner. A large number of mutant Bacteria unable to grow on a medium lacking tryptophan (tryptophan auxotrophs) were isolated. Genetic crosses were performed using a specialized transducing bacteriophage designated as phage Pike [134]. During Replication in susceptible bacteria, transducing Bacteriophages occasionally incorporate a segment of the bacterial chromosome into their own DNA. Subsequently, when such a phage infects other bacteria, a portion of its Genetic information can be transferred via recombination into the Chromosomes of the surviving host bacteria. By utilizing a series of deletion mutants, much like the mapping of the rII gene, it was possible to divide the A gene into A number of segments, while recombination frequency analysis enabled precise mapping.

The second part of the proof for collinearity between The nucleotide sequence in DNA and the Amino Acid Sequence in proteins involved determining the complete amino acid sequence of tryptophan synthetase and mapping the peptide fragments of the mutant Enzymes (Chap. 2, Sec. 3.2). Peptide maps made it possible to identify defective Peptides and precisely establish The Nature of Amino Acid Substitutions in A wide variety of tryptophan auxotrophs. Once this was achieved, it turned out that Mutations located very close to one another corresponded to amino acid substitutions in directly (or very closely) adjacent Regions of the polypeptide chain.

The same problem was investigated by Sarabhai et al. [134a], who studied nonsense mutations (Sec. 5) leading to premature termination of Polypeptide chain synthesis. During the late stages of E. coli infection by phage T4, Protein Synthesis is directed predominantly toward The production of a single protein, namely the viral HEAD protein. Following Protein synthesis in infected Cells in the presence of specifically ¹⁴C-labeled amino acids, The Cell extracts were treated with Trypsin or Chymotrypsin; the phage head protein peptides were separated by Electrophoresis, and autoradiographs were prepared from the resulting electrophoretograms. It was shown that a series of T4 phage nonsense mutants, with mutations localized within the coat protein gene, synthesized incomplete chains of the phage coat protein, with the resulting peptide fragments varying in length. By examining the autoradiographs of the enzymatically fragmented peptides, the mutants could be ordered According to the decreasing length of the peptides produced. Furthermore, it was demonstrated that the sequence obtained in this manner corresponded to the sequence predicted from genetic mapping data.

It has already been mentioned that the collinearity of codon and amino acid sequences was proved by direct determination of nucleotide sequences in RNA and DNA molecules and the corresponding amino acid sequences in proteins (Sec. B, 2, c).

Even before the triplet nature of codons was definitively established, Crick and his coworkers made clever use of frameshift mutations to prove that METABOLISM/28.html">The Genetic Code is indeed composed of nucleotide triplets. Consider what happens when two strains of bacteria are mated, each carrying a frameshift mutation (for example, a -1 deletion). Genetic recombination may generate mutants containing both frameshift mutations. However, such recombinants are difficult to identify because (according to virtually any coding theory) they continue to produce completely defective proteins. Nevertheless, Crick and his colleagues succeeded in introducing a third frameshift mutation of the same type into the same gene and observed that recombinants carrying all three deletions (or insertions) were capable of synthesizing at least partially active proteins. The explanation for this is straightforward. Deletions of one or two nucleotides completely inactivate the gene, whereas the deletion of three nucleotides located within the same gene and close to one another shortens the gene by a mere three nucleotides. In this case, the gene will contain only a small region with altered codons. The encoded protein will be normal, save for a short segment in which Some amino acids are replaced and one is entirely absent. We already know that in most proteins, only a relatively small fraction of amino acids is strictly invariant. Consequently, a gene with a modified local region very often retains The ability to synthesize functionally active products, provided that no reading frame shift has occurred.