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Fig. 4. Ectopic expression of wee1. (A) The growth of cells expressing an ectopic copy of the wee1 gene was examined by spotting serial dilutions of exponential cultures of FB1 (wild type) and UMC33 (Pcrg1:wee1) strains in solid rich medium with either 2% glucose (YPD, non-inducing conditions) or 2% arabinose (YPA, inducing conditions). Plates were incubated for 3 days at 28°C. (B) Micrographs showing the cell morphology of FB1 and UMC33 cells after 6 hours of growth in YPA liquid cultures (inducing conditions). Note the elongated shape and the presence of a single nucleus (DAPI staining). Scale bars: 20 µm. (C) Microtubule network of UMC41 cells, carrying an 
-tubulin-GFP fusion and expressing high levels of wee1 (GFP-Tub1 epifluorescence; Scale bar: 25 µm. (D) FACS analysis of cell DNA content of FB1 and UMC33 in non-inducing (YPD) and inducing conditions (YPA). Samples were removed 0, 2, 4 and 6 hours after transfer to conditional medium. The shift to a DNA content higher than 2C observed in UMC33 cells incubated in CMA for 6 hours was due to mitochondrial DNA staining. (E) Western analysis of inhibitory phosphorylation after wee1 overexpression. Protein extracts from the FB1 and UMC33 cultures incubated in inducing conditions for the times indicated were obtained (YPA; in hours). The overexpressed wee1 allele was myc-tagged and detected with an anti-MYC antibody. Cdk1 was visualized with anti-phospho-Cdc2 (Tyr15) and anti-PSTAIRE antibodies.
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