Figure 4.

Pds1p does not inhibit cyclin degradation in G₁. The strains used were as follows: wild-type (OCF1517.0, WT₁), GAL1–pds1-db (OCF1517.2), wild-type (K1534, WT₂), and cdc16-123 (K4438). All strains carried a plasmid encoding for GAL–CLB2–HA (pWS944 in WT₁ and pds1-db; pOC16 in WT₂ and cdc16-123). All strains were grown at 23°C in nonselective medium (YPR), and arrested in G₁ with α factor (10⁻⁶ m final concentration), or in mitosis with nocodazole (15 μg/ml final concentration). When all cultures were arrested, they were shifted to 37°C, to inactivate the cdc16-123 gene product. Thirty minutes later, while maintaining the temperature at 37°C, dextrose (D) or galactose (G) were added (2% final concentration). The samples shown here were taken 1 hr later and processed for Western blot analysis as described in Materials and Methods. (A) The levels of Clb2p–HA with and without the galactose induction, in mitosis (top) and in G₁ (bottom), detected by using anti-HA antibodies. (B) The levels of β-tubulin in each of the samples from the G₁ arrested cultures, to show equal protein loading in all lanes. (C) Pds1-db in G₁ and mitosis, in the presence of dextrose (D) or galactose (G), demonstrating that the lack of Clb2p stabilization in G₁ in the GAL–pds1-db strain is not due to the lack of Pds1p-db induction.