Fig. 5. Ectopic overexpression of mek1⁺ in vegetative cells causes Cdc25-dependent G2/M cell cycle arrest. (A) Wild-type cells (PN22) transformed with the pREP3X vector or with plasmid pSS123 (nmt-mek1⁺) were streaked out on plates containing thiamine (nmt1 promoter OFF) or lacking thiamine (nmt1 promoter ON) and incubated for 3 days at 30°C. (B) Cells from strain S1297, which contain an integrated nmt1-mek1-GFP construct, were incubated in nmt1-repressing conditions (OFF) or nmt1-inducing conditions (ON) for 24 hours. Nuclei were stained with DAPI. Backlight reveals cell bodies. (C) Wild-type (PN22), wee1-50 (S145), cdc2-3w (S176) and cdc2-3w cdc25 (S898) cells transformed with pSS124 were grown on plates containing or lacking thiamine (nmt-mek1⁺ OFF and nmt-mek1⁺ ON, respectively). For wildtype, cdc2-3w and cdc2-3w cdc25, plates were incubated at 30°C, whereas wee1-50 cells were incubated at 36°C to inactivate Wee1 function. Microcolonies were photographed after 30 hours. Note that cdc25 cells do not elongate in response to mek1⁺ overexpression. (D) Strains S1297 (wildtype) and S1302 (cdc25-9A), which contain nmt1-mek1-GFP integrated in the genome were incubated in the absence (OFF) or in the presence (ON) of thiamine for 22 hours. Cells were visualized at the fluorescence microscope using a GFP filter. Backlight reveals cell bodies. Note that in the absence of thiamine Mek1-GFP accumulates in the nucleus of both wildtype and cdc25-9A, but cdc25-9A cells elongate considerably less than wildtype; in addition, cdc25-9A binucleate dividing cells are observed frequently.