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S U M M A R YTwo methionine auxotrophs (metG) of Salmonella typhimurium with the structural genes of the regulon intact have been studied. They possessed abnormal growth kinetics and the effect of the metG mutation on protein, ribonucleic acid and DNA synthesis suggested that these strains were impaired in their ability to synthesize protein; since they were able to synthesize methionine, but still required it for growth, they might have been defective in methionine activation for protein synthesis. To test this, the activity of methionyl-sRNA synthetase (L-methionine : sRNA ligase (AMP) EC 6 . I . I . 10) was determined in enzymic extracts of wild-type and metG strains. By using an acylation reaction the activation of methionine for protein synthesis was shown to be very decreased in metG extracts and this was reflected in vivo by a decreased level of charged methionyl-sRNA in mutant bacteria; in the pyrophosphate exchange assay mutants showed greatly increased K,,, (methionine) values. The release of [3H]methionine from [3H]methionylsRNA was catalysed by wild-type extract, provided that pyrophosphate was present in the assay mixture, but not by the mutant extract. These results are discussed in relation to the two-part reaction catalysed by methionylsRNA synthetase. Mutant and wild-type enzyme behaviour differed at different pH values but not when subjected to chromatography on DEAEcellulose or gel-filtration on Sephadex-Gzoo. MetG mutants grown with limiting methionine had decreased values of all the biosynthetic enzymes except cystathionase, which was apparently de-repressed, suggesting that methionyl-sRNA was not the co-repressor for the methionine biosynthetic pathway.

Six mutants, allelic to ade3, were isolated after mutagenic treatment of a prototrophic strain of yeast. All six grow on medium supplemented with adenine alone and four respond to histidine. Supplementation with adenine plus histidine or methionine inhibits growth, but a mixture of these three is stimulatory. Heteroallelic diploids formed by the new mutants with the standard ade3 can resemble either parent or show an intermediate phenotype. The new mutants, unlike standard ade3, are not fully epistatic to ade2. The activities of three enzymes concerned in tetrahydrofolate metabolism have been assayed in the new and standard ade3 mutants and wild type. The only difference detected between the new and standard ade3 was in the levels of 10-formyltetrahydrofolate synthetase. Activity in the new mutants ranged from 36 to 109% of wild type compared with 10 to 12% in the standard ade3. Possible mechanisms to account for the varied phenotypes at the ade3 locus are discussed.

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