Selection by genetic transformation of a Saccharomyces cerevisiae mutant defective for the nuclear uracil-DNA-glycosylase.
A coliphage M13 chimer containing the Saccharomyces cerevisiae TRP1 gene and ARS1 replication origin (mPY2) was grown on an ung- dut- strain of Escherichia coli. The resulting single-stranded phage DNA had 13% of thymine residues substituted by uracil. This DNA failed to transform a delta trp1 yeast strain to prototrophy. However, when a mutagenized yeast stock was transformed with uracil-containing single-stranded mPY2 DNA, unstable transformants were obtained. After plasmid segregation, about half of these were retransformed at a high frequency by uracil-containing single-stranded mPY2 DNA. In vitro, these mutants were defective for uracil-DNA-glycosylase activity. They were designated ung1. Strains containing the ung1 mutation have an increased sensitivity to sodium bisulfite and sodium nitrite but a wild-type sensitivity to methyl methanesulfonate, UV light, and drugs that cause depletion of the thymidylate pool. They have a moderate mutator phenotype for nuclear but not for mitochondrial genes. A low mitochondrial uracil-DNA-glycosylase activity was demonstrated in the mutant strains.
D002467:Cell Nucleus / Q000201:enzymology; D002678:Chimera; D045647:DNA Glycosylases; D004277:DNA, Single-Stranded / Q000378:metabolism; D005800:Genes, Fungal; D008741:Methyl Methanesulfonate / Q000494:pharmacology; D008928:Mitochondria / Q000201:enzymology; D009154:Mutation; D009699:N-Glycosyl Hydrolases / Q000235:genetics*; D010641:Phenotype; D010957:Plasmids; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D014466:Ultraviolet Rays; D014498:Uracil / Q000378:metabolism*; D051981:Uracil-DNA Glycosidase
D004277:DNA, Single-Stranded; D014498:Uracil; D008741:Methyl Methanesulfonate; D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; D051981:Uracil-DNA Glycosidase