Met30 is required for G₁/S transition and M-phase, but not for S-phase: (A) Wild-type cells and met30-6 mutants were grown in yeast extract-peptone-dextrose at 25°C to an optical density at 600 nm of 0.2, arrested with α-factor (20 ng/ml final concentration) 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-dextrose and incubated in fresh prewarmed yeast extract-peptone-dextrose medium at 37°C. Samples were taken at the time intervals indicated and analyzed by flow cytometry. (B) Wild-type cells and met30-6 mutants were grown as for panel A, but instead of α-factor, hydroxyurea (400 mM final concentration) was added. When >90% of cells showed a “large-budded” morphology, cells were shifted to 37°C and incubation in the presence of hydroxyurea was continued for 90 min. Cells were released and analyzed from the hydroxyurea block as described for panel A. (C and D) Same as for panel B, but nocodazole (15-μg/ml final concentration) was used to arrest cells instead of hydroxyurea. Nuclear division was scored by counting the number of nuclei/cell.

Cln2 overexpression does not overcome the G₁ arrest of met30 mutants. (A) Wild-type cells and met30-6 mutants harboring a GAL1-CLN2-HA3 allele integrated at the LEU2 locus were grown in yeast extract-peptose-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 below 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-peptose-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 immunoblotting with anti-HA (12CA5) antibodies to detect Cln2-HA3 and anti-PSTAIRE antibodies for detection of Cdc28. Cln2-associated kinase activity was assayed using anti-HA-associated immunocomplexes to phosphorylate histone H1. (B) Samples from the experiments described for panel A were analyzed by flow cytometry. (C) Serial dilutions of the strains used panel A were spotted on yeast extract-peptone-galactose plates and incubated at the temperatures indicated.

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.

Cell cycle arrest of temperature sensitive met30 mutants: (A) Wild-type cells and temperature sensitive met30-6 mutants were grown in yeast extract-peptone-dextrose at 25°C to an optical density at 600 nm of 0.2, the temperature was shifted to 37°C and samples were taken at the time intervals indicated. For each sample cell number was counted. (B) DNA content of the samples as in panel A was analyzed by flow cytometry. (C) Wild-type cells and met30-6 mutants were grown in yeast extract-peptone-dextrose at 25°C to an optical density at 600 nm of 0.2 and shifted to a semipermissive temperature (30°C) for 2 h, and filamentous actin stained with rhodamine-phalloidin was visualized by fluorescence microscopy.

Efficient expression of G₁ cyclins requires Met30. (A) α-Factor arrest and release were as described in the legend to Fig. 2A. Samples were analyzed by immunoblotting: wild-type cells and met30-6 mutants expressing both Cln2-HA3 and Cln3-18myc were used to detect Cln2 and Cln3; strains expressing both Swi4-6myc and Swi6-HA3 for detection of Swi4 and Swi6. Cdc28 was detected with anti-PSTAIRE antibodies as a loading control. (B) Total RNA was analyzed by Northern blotting with radiolabeled probes to detect CLN1 and CLN2, respectively. (C) Strains as indicated were replica plated to plates lacking uracil and plates containing 5-fluoro-orotic acid and incubated at 25°C.

Deletion of SIC1 does not suppress the G₁ arrest of met30 mutants. (A) Serial dilutions of cells with the indicated genotypes were spotted onto yeast extract-peptone-dextrose plates and incubated at the temperatures specified. (B) sic1Δ and sic1Δ met30-6 mutants were synchronized with α-factor and released from the cell cycle block as described in the legend to Fig. 2A. Samples were analyzed by flow cytometry.

Met30 is required for efficient origin binding of replication factors. (A) Wild-type cells (PY1202) and met30-6 mutants (PY1203) expressing Cdc45-HA3 from its chromosomal locus were grown at 25°C to an optical density at 600 nm of 0.5, 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. Cells were released from the G₁ arrest at 37°C, and samples were taken at the time intervals indicated and analyzed by chromatin immunoprecipitation followed by detection of ARS305 by real-time PCR using SYBR green. Wild-type (wt) cells and met30-6 mutants (PY283) expressing untagged Cdc45 were processed in parallel, and values obtained in the PCR detection were subtracted as background in the graphs shown. (B) Wild-type cells (PY1204) and met30-6 mutants (PY1207) expressing Mcm7-HA3 from its chromosomal locus were grown and analyzed as for panel A. (C) Wild-type cells and met30-6 mutants expressing Mcm4-HA3 (PY1058 and PY1059) or Mcm7-HA3 from their chromosomal loci were grown and arrested with α-factor as described for panel A. Cells were shifted to 37°C for 90 min in the presence of α-factor, and samples were taken and analyzed as for panel A. (D) Total cell lysates prepared from α-factor-arrested samples after 90 min at 37°C (time point 0′ in panels A, B, and C) were analyzed by immunoblotting with anti-HA (12CA5) antibodies to detect Cdc45-HA3, Mcm4-HA3, and Mcm7-HA3. anti-PSTAIRE antibodies were used for detection of Cdc28 as a loading control.