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

The mutant tmpl--10ts which 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 mug 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 mug 5'-dTMP/ml when tuml and 24 mug 5'-dTMP when tum2 is present, respectively. Cells prototrophic for thymidylate (TMP) harbouring tuml 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 mug5'-dTMP/ml when tuml and tum2 are present.