Improved Method for the Isolation of Thymine-Requiring Mutants of Escherichia coli

Abstract

Thymine-requiring mutants of Escherichia coli were rarely found before the observation by Okada, Yanagisawa, and Ryan (Z. Vererbungslehre 92:403, 1961) that cultures of bacteria grown in the presence of high concentrations of aminopterin, thymine, purines, and serine contain a surprising number of cells unable to synthesize their own supply of thymine. They showed that these thymine-dependent mutants could grow much faster in the presence of aminopterin than the parent strains, even though both were supplied with adequate amounts of those metabolites whose syntheses are blocked by aminopterin. These observations provided the basis for a widely used method of isolation of this special class of mutants. The greater efficiency of a simplified medium was reported by Okada, Homma, and Sonohara (J. Bacteriol. 84:602, 1962). We also found that the yield of thymutants was greatly increased if a completely minimal medium, usually M9, containing only thymine (50 ,g/ml) in addition to aminopterin (-300 ,ug/ml) was used as the isolation medium.

Effect of the DNA replication velocity on the response of Salmonella typhimurium to mild heating

“…This was based on the methods of Stacey & Simpson (1965) and Ibe (1973). A 5 ml bottle of M9TT medium (containing 50 mg/l of thymine and 10 mg/l of trimethoprim) was inoculated with a loopful of wild type Salm.…”

Phosphoribosylpyrophosphate (PRPP)‐less mutants of Escherichia coli

“…Thymine {thyA} and low-thymine {thyA deoS)-requiring strains were selected as described previously (Breitman and Bradford. 1964;Stacey and Simpson, 1965). Udp*, DeoB* or DeoD*" transductants were selected on medium with cytidine, thymidine or inosine, respectively, as the carbon sources (Hammer-Jespersen, 1983).…”

Role of glucose-6-phosphate in the regulation of aspartate semialdehyde dehydrogen ase in Escherichia coli

“…Fraenkel [11]; PP1 is a Thy* Pgm-conjugant of a Thy-derivative (obtained by trimethoprim resistance [12]) of POC 20 (1ys1120, lysC1008, ilvA, pgi-2, thrA 1101, metLMlO00; manuscript in preparation) with H fr PGM 3 (pgm-3; [6]);Pgm phenotype was scored by the blue test described in [6]. b Culturing was performed in minimal medium [ 13] supplemented with 1 tzg/ml of thiamine HC1, and 2 mg/ml of sugars; "repression conditions" were 4 mM L-lysine, 0.05 mM DL-meso-diaminopimelate (DAP), 5 mM DL-threonine, 2 mM L-isoleucine and 2 mM DL-methionine.…”