SIMPLE METHOD FOR ISOLATION AND CHARACTERIZATION OF THYMIDYLATE UPTAKING MUTANTS IN SACCHAROMYCES-CEREVISIAE

The mutant tmp1-10ts 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 3-4 generations in the presence of 10-2 M 5''-dTMP. Thymidylate utilizing mutants tum mutants) were isolated which can grow in the presence of 12-24 .mu.g 5''-dTMP/ml. Genetical analysis revealed one of these mutants strains to be a double mutant, tum1 tum2. For normal growth haploid thymidylate auxotrophic strains require approximately 360 .mu.g 5''-dTMP/ml when tum1 and 24 .mu.g 5''-dTMP when tum2 is present, respectively. Cells prototrophic for thymidylate (TMP) harboring 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 labeled 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 .mu.g 5''-dTMP/ml when tum1 and tum2 are present.