Selection by genetic transformation of a Saccharomyces cerevisiae mutant defective for the nuclear uracil-DNA-glycosylase

Area of Science:

• Molecular Biology
• Genetics
• Microbiology

Background:

• Coliphage M13 and Saccharomyces cerevisiae genetic elements were combined to create a chimeric phage.
• Phage DNA incorporating uracil instead of thymine was generated by growing the chimeric phage in a specific Escherichia coli strain.

Purpose of the Study:

• To investigate the biological consequences of uracil incorporation into phage DNA.
• To identify and characterize yeast mutants defective in uracil-DNA-glycosylase activity.

Main Methods:

• Transformation of yeast strains with uracil-containing single-stranded phage DNA.
• In vitro enzymatic assays to assess uracil-DNA-glycosylase activity.
• Sensitivity testing of mutant strains to various mutagens and DNA-damaging agents.

Main Results:

• Uracil-containing phage DNA failed to transform wild-type yeast but yielded unstable transformants in a mutagenized stock.
• Mutant yeast strains (ung1) were isolated, exhibiting defective uracil-DNA-glycosylase activity.
• The ung1 mutation conferred sensitivity to sodium bisulfite and nitrite, a moderate nuclear mutator phenotype, and reduced mitochondrial uracil-DNA-glycosylase activity.

Conclusions:

• Uracil incorporation into DNA poses a challenge for yeast transformation and genome stability.
• The ung1 mutation highlights the critical role of uracil-DNA-glycosylase in DNA repair and preventing mutagenesis.
• Deficiencies in uracil-DNA-glycosylase can lead to specific mutational sensitivities and altered gene expression patterns.