A simple method for the isolation and characterization of thymidylate uptaking mutants in Saccharomyces cerevisiae

Summary The mutant tmp1–10^tswhich confers thermosensitive auxotrophy for thymidylate is employed for the selection of 5-dTMP uptaking mutants. At the nonpermissive temperature yeast cells phenotypically wild type for thymidylate uptake can grow for only 3 to 4 generations in the presence of 10^–2 M 5-dTMP. Thymidylate utilizing mutants (tum mutants) were isolated which can grow in the presence of 12 to 24 g 5-dTMP/ml. Genetical analysis revealed one of these mutant strains to be a double mutant, tuml tum2. For normal growth haploid thymidylate auxotrophic strains require approximately 360 g 5-dTMP/ml when tum1 and 24 g 5-dTMP when tum2 is present, respectively. Cells prototrophic for thymidylate (TMP) harbouring tum1 tum2 will also take up 5-dTMP and incorporate it specifically into their DNA. Thymidylate utilization in such strains is independent of functional mitochondria, as similar incorporation of labelled 5-dTMP is found in isogenic strains with rho⁺, rho^– and rho⁰ status. Optimal stimulation of the 5-dTMP uptaking principle in haploid TMP strains is found at 4 g 5-dTMP/ml when tum1 and tum2 are present.