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Fig. 5. Conditional removal of Wee1. (A) Levels of wee1 mRNA in the conditional strain. The wild-type FB1 and conditional UMC23 (wee1^nar) strain were grown for 8 hours in permissive (minimal medium with nitrate, MMNO₃) or restrictive conditions (rich medium, YPD). The RNA was extracted and analyzed by northern blotting, loading 10 µg total RNA per lane. 18s rRNA was used to control for loading. (B) Growth of conditional strain in solid medium. Serial tenfold dilutions of FB1 (WT) and UMC23 (wee1^nar) cultures were spotted in solid rich medium (YPD) and minimal medium with nitrate (MMNO₃). YPD plates were incubated for 2 days and the nitrate plates for 4 days at 28°C. (C) Western blotting of inhibitory phosphorylation following wee1 depletion. Protein extracts from the strains indicated were obtained after incubation in repressive conditions (YPD) at the times indicated (in hours). The Cdk1 levels were determined with anti-phospho-Cdc2 (Tyr15) and anti-PSTAIRE antibodies. (D) Flow cytometry of wild-type and UMC23 cells grown in permissive and restrictive conditions. Cells grown in MMNO₃ were centrifuged, washed twice in minimal medium without nitrogen, and resuspended in the appropriate medium. Samples were taken for FACS analysis at the times indicated. (E) Length of wild-type and UMC23 cells growing in permissive conditions (MMNO₃). In the upper plot, the length of the major axis of FB1 and UMC23 mother cells was measured and plotted as function of the number of cells. Below, the length of the buds was measured from the same population. A sample of 110 cells was used for each measurement.

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