Fluctuation assays

Strains to be assayed were grown overnight in 3-ml complete supplement mixture (CSM)–uracil medium, diluted 10⁻⁴, and then inoculated into 100-μl cultures so that there were approximately 1000 cells per culture. At least 24 independent cultures were used per assay, and each assay was repeated at least three times. Cultures were left overnight at 30°C until the cultures were assessed to have reached a suitable density, and then the entire culture, except for 5 μl, was plated onto predried 5-FOA plates to detect ura3 mutants that were 5-FOA–resistant. The remaining culture was pooled and diluted, and then the cell count was assayed using a Scepter cell counter. Mutation rates were calculated using the maximum likelihood method (61). To measure the background mutation rate at a site distal from the URA3 locus, the wild-type CAN1 locus was restored in G₀-URA3 and G₁₄-ORF strains. Mutation rate assays were carried out the same as above except that mutations in the CAN1 gene were detected by plating on CSM-arginine plates supplemented with canavanine (60 μg/ml).