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Fig. 1. Progression of meiosis induced by the expression of byr1DD. (A) Amino acid sequences of H. sapiens MAPKK and S. pombe Byr1. MAPKKK phosphorylation sites of H. sapiens MAPKK and the corresponding sites of Byr1 are underlined. To make a constitutively active form of Byr1 (Byr1DD), these sites of Byr1 were replaced with aspartic acid (shown as `D'). (B) Mitotic growth defects in cells expressing byr1DD. h– wild-type (CRL239) cells were transformed with either pREP81 (vector), pREP81-byr1+ (byr1+) or pREP81-byr1DD (byr1DD). Each transformant was cultured to mid-log phase 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– wild-type (CRL239) cells carrying pREP81-byr1DD were induced to undergo ectopic meiosis in liquid EMM2 at 30°C, and fixed at 30 hours after induction. Nuclei were stained with DAPI. Bright-field images of the same cells are also shown (Phase). Arrows indicate cells with three or four nuclei, or spores. Scale bar: 5 µm. (D) Progression of meiosis in cells expressing byr1DD. h– wild-type (CRL239) cells carrying pREP81-byr1DD were induced to undergo ectopic 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. 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. (E) DNA content, measured by flow cytometry. The same cell samples used in D were examined.
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