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Fig. 2. Analysis of the steady-state level of Byr4p in SIN mutants. (A) The indicated mutants were grown in YE medium to exponential phase and incubated for 4 hours at 36°C. Protein extracts were analysed by western blotting with the indicated antibodies. (B) leu1::nmt1-spg1(ura4+) ura4-D18 cells were grown in EMM2 plus leucine in the presence of 2 µM thiamine (off) or without thiamine for 18 hours at 29°C (on) to induce spg1 expression. Cells overexpressing spg1 showed a multiseptated phenotype. Protein extracts were prepared, and western blots were probed with the indicated antibodies. The three arrows indicate the most rapidly migrating form, which predominates in cdc16-116, and two others that represent different states of phosphorylation. How these relate to the forms observed in Fig. 1 will be the subject of future experiments. (C) The mutant spg1-Q41A-HA was expressed from pREP3 in a spg1::ura4+ background, in the presence of 2 µM thiamine at 25°C, and cells were then shifted to 19°C for 9 hours or to 36°C for 5 hours. Cells were fixed and stained with DAPI and Calcofluor. Bar, 10 µm. (D) Protein extracts from the experiment shown in C, as well as from wild-type and spg1::ura4+ cells rescued by expression of either pREP41-cdc7 (without thiamine) or pREP3-spg1 (grown in the presence of thiamine) were analysed by western blotting with the indicated antibodies. The bottom panel shows the steady-state level of Spg1p-Q41A–HA at the indicated temperatures.
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