Fig. 2. Ectopic expression of cru1. (A) Growth ability of cells expressing an ectopic copy of cru1 gene was examined by spotting serial dilutions of exponential cultures of UMP28 and TAU54 strains in solid Complete Medium with either 2% glucose (CMD, noninduction conditions) or 2% arabinose (CMA, induction conditions). Plates were incubated for 3 days at 28°C. (B) Micrographs showing the cell morphology of TAU54 cells after 9 hours of growth in CMA liquid cultures (inducing conditions). Notice the elongated shape, and the presence of a single nucleus (DAPI staining) and a microtubule network compatible with a G2-like state (GFP-Tub1 epifluorescence). Bars, 10 µm. (C) FACS analysis of DNA content of UMP28 and TAU54 cells in noninducing conditions (CMD) and inducing conditions (CMA). Samples were removed after 0, 3, 6 and 9 hours after transfer to conditional medium. The shift to higher than 2C DNA content observed in TAU54 cells incubated in CMA after 9 hours is due to mitochondrial DNA staining. (D) Western analysis of cyclin levels after cru1 overexpression. Protein extracts from the indicated strains, carrying epitope tagged versions of U. maydis B-type cyclins, were obtained after incubation in noninducing (CMD) and inducing (CMA) conditions at the indicated times (in hours). Clb1 was detected by using an anti-VSV antibody, whereas Clb2 was detected with an ani-MYC antibody. As loading control we used Cdk1 levels, detected with an anti-PSTAIRE antibody.