Fig. 3. Mitotic growth defects in mutant cells expressing byr1^DD. (A) Amino acid sequences of H. sapiens MAPK and S. pombe Spk1. MAPKK phosphorylation sites of H. sapiens MAPK and the corresponding sites of Spk1 are underlined. To make unphosphorylatable forms of Spk1, these sites in Spk1 were replaced with either or both alanine (A) and phenylalanine (F). These mutated Spk1 constructs are designated Spk1^AY, Spk1^TF, and Spk1^AF as indicated. (B) h^– spk1⁺ (TG7) cells, h^– spk1 (TP106-3C) cells, h^– spk1^AF (TG43) cells, h^– spk1^AY (TG44) cells, h^– spk1^TF (TG45) cells, h^– mei2 (CRL544) cells, and h^– mei3 (TG30) cells were transformed with pREP81-byr1^DD. Each transformant was cultured in liquid EMM2 with thiamine. After removing thiamine, fivefold serial dilutions of each transformant were spotted onto EMM2 plates with (right panel, `Repressed') or without thiamine (left panel, `Induced'). Plates were photographed after 4 days incubation at 30°C. (C) h^– spk1 (TP106-3C) cells, h^– mei2 (CRL544) cells, and h^– mei3 (TG30) cells carrying pREP81-byr1^DD were cultured in liquid EMM2 at 30°C to induce ectopic meiosis, and fixed at 30 hours after induction. The nuclei were stained with DAPI. Bright-field images of the same cells are also shown (Phase). Scale bar: 5 µm. (D) Progression of meiosis in mei3 cells expressing byr1^DD. h^– mei3 (TG30) cells carrying pREP81-byr1^DD were induced to undergo meiosis in liquid EMM2 at 30°C. The number of nuclei per cell was counted every 2 hours from 13 to 29 hours after induction (open symbols). As a control, h^– wild-type (CRL239) cells carrying pREP81-byr1^DD were examined (filled symbols). At least 300 cells were scored for each time point. During analysis, the cell concentration was kept between 1x10⁶ and 2x10⁷ cells/ml by diluting with EMM2.