FIG. 6.

Clb5 overexpression induces DNA replication in met30 mutants. (A) Wild-type cells and met30-6 mutants were synchronized in G₁ phase and released from the cell cycle block as described in the legend to Fig. 2A. Samples were taken at the time points indicated and analyzed by quantitative reverse transcriptase PCR using SYBR green. CLB5 expression levels were normalized to ACT1 levels. (B) Wild-type cells and met30-6 mutants harboring a GAL1-CLB5-9myc allele integrated at the ARS1 locus were grown in yeast extract-peptone-raffinose at 25°C to an optical density at 600 nm of 0.2, arrested with α-factor until the fraction of budded cells was less than 10%. The culture was shifted to 37°C, and incubation in the presence of α-factor was continued for 90 min. For the release, the cells were washed in prewarmed (37°C) yeast extract-peptone-raffinose and incubated in fresh prewarmed yeast extract-peptone-galactose (2% galactose, final concentration) at 37°C. Samples were taken at the time intervals indicated and analyzed by flow cytometry. Expression of Clb5-9myc was confirmed by immunoblotting using anti-Myc antibodies (data not shown). (C) Total DNA was isolated from wild-type cells and met30-6 cells expressing GAL1-CLB5-9myc for 6 h after release from the α-factor block, and the nuclear locus ACT1 as well as two mitochondrial loci ([COX1] and [COX3]) were quantified by real-time PCR using SYBR green. (D) Serial dilutions of cells with the genotypes indicated were spotted onto yeast extract-peptone-galactose plates and incubated at the temperatures specified.