Genomic instability arises from quiescent cells during chronological aging. (A) Density-gradient separation of nonquiescent (upper) and quiescent (lower) cells from chronologically aging cultures. (B) Calcofluor staining of upper and lower fractions from wild-type and sch9Δ mutant cultures. The arrows indicate budding cells. Bar, 10 µm. (C) Percentage of upper and lower fractions in the culture. (D) Budding indices of upper and lower fractions (n = 6–7). (E) Mutation frequency in CAN1 gene (Can^r mutants/10⁶ cells) in the upper and lower fractions (n = 6–7). (F) Mutation frequency in the CAN1 gene before and after allowing the cells to undergo one population doubling during a standard chronological life span study (n = 4). Error bars indicate ±SEM. (C–E) Data represent the mean ± SEM (error bars); *, P < 0.05; **, P < 0.01; two-tailed t test, sch9Δ versus wild type (WT). ^, P < 0.05; ^^, P < 0.01; two-tailed t test, day 5 or day 7 versus day 3 of the same strain.