Genetic suppressors of bacteriophage t5 amber mutants.

Nonsense mutations in structural genes appear to abort polypeptide chain synthesis at the amino acid site governed by the mutant codon. The two presently recognized types of nonsense mutant, amber and ochre, have been assigned the codons UAG and UAA, respectively. Certain bacteria harbor one or more genetic suppressors that counteract the effect of appropriate nonsense mutations in their own structural genes as well as in those of infecting viruses. The suppressors, which may become active (su+) or inactive (su-) by mutation, permit completion of the affected polypeptide chains by inserting an amino acid at the nonsense site. The type of amino acid inserted depends on the suppressor. At the phenotypic level, a given amber suppressor works with most amber mutants but no ochre mutants. A given ochre suppressor works with most ochre mutants and also with some amber mutants. When a mutant fails to respond to a suppressor that works with other mutants of the same class, the current theory suggests that the polypeptide chain is completed, but is defective owing to the nature of the inserted amino acid. Recent papers, which give additional references, are those by R. Hill and G. S. Stent (Biochem. Biophys. Res. Commun. 18:757, 1965), S. Kaplan, A. 0. W. Stretton, and S. Brenner (J. Mol. Biol. 14:528, 1965), E. Gallucci and A. Garen (J. Mol. Biol. 15:193, 1966), T. K. Gartner and E. Orias (J. Bacteriol. 91:1021, 1966), and R. E. Webster, D. L. Engelhardt, and N. D. Zinder (Proc. Natl. Acad. Sci. U.S. 55:155, 1966). To extend our genetic analysis of phage T5 (W. D. Fattig and F. Lanni, Genetics 51:157, 1965; Y. T. Lanni, F. Lanni, and M. J. Tevethia, Science 152:208, 1966), we needed an Escherichia coli strain that would both allow rapid phage adsorption and suppress amber (am) mutants. The mutants, obtained by mutagenesis with 5bromodeoxyuridine, were identified as amber mutants by their ability to grow in strain CR63, which has a classical amber suppressor, but not in an su-derivative of CR63 or, in the many cases tested, strain B, a classical sustrain. Strain F, the only available strain allowing fast adsorption of T5, is sufor all our am mutants. We report here the isolation and some properties of su+ derivatives. The source was a histidine auxotroph, which we derived from strain F by mutagenesis with 2aminopurine. If the auxotroph happened to have arisen by nonsense mutation, phenotypic reversion could occur by mutation to su+. Such suppressed mutants might permit growth of our T5 am mutants. Five cultures of the auxotroph, started from separate colonies, were grown overnight in nutrient broth at 37 C, washed thoroughly with non-nutrient buffer, and plated on minimal agar. All of the 154 revertant colonies, from a total of about 2 x 109 plated cells, were picked to broth and spot-tested with one T5 am mutant. The 21 su+ isolates were easily sorted into four well-defined groups (Table 1) with a set of 13 representative am mutants. Tests with one isolate from each su+ group were extended to a total of 48 am mutants, which represent 21 cistrons according to current enumeration. To increase the discrimination, we used 50 to 100 plaque formers per spot instead of the much higher number often employed. Within the class of positive responses, we were therefore able to note differences in plaque size, which generally may be expected to correlate with the more tediously estimated average burst size. In spot tests at 30 C, 42 am mutants responded to all four su+ testers, and the remainder responded at least to two. Thus, among 192 combinations of 48 mutants and four suppressors, 184 gave a clearly positive result. Judged from plaque sizes, the responses were, however, variable. With rare exception, and then only when plaques were very tiny, the plaque number was similar on all testers; and plaques on a given tester were generally fairly uniform, regardless of their average size. The heterogeneity of positive responses can be seen in Table 2, which was assembled from a larger body of concurrent tests. The following comments, often referring to Table 2 for examples, are based on our whole ex-