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 Δtrp1 yeast strain to prototropy. 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.