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 μg 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 μg 5′-dTMP/ml when tum1 and 24 μg 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 rho ⁰ status. Optimal stimulation of the 5′-dTMP uptaking principle in haploid TMP strains is found at 4 μg 5′-dTMP/ml when tum1 and tum2 are present.