Figure 6.
Cds1 kinase response in the spp1 mutants. (A) Cds1 kinase activity in spp1 mutants. Extracts were prepared from wild-type (w-t) and the spp1 mutants at either the permissive temperature (26°C) or after incubation at the nonpermissive temperature (36.5°C) for 3.5 h. Cds1 kinase was immunoprecipitated from cell extract and assayed for kinase activity using MBP as substrate as described in MATERIALS AND METHODS. Top: a control blot of 1/30th of the extract used for immunoprecipitation; bottom: autoradiograph of the Cds1 kinase activity of the immunoprecipitates. Fold induction was calculated using the activity of the wild-type control at the respective temperature as 1.0 and was quantified using Phosphorimager software. (B) Cds1 kinase activity induced by hydroxyurea (HU) arrested wild-type and spp1 mutant cells. Cell cultures were synchronized with 11 mM HU for 3.5 h at 26oC after which fresh HU (11 mM) was added to each culture, and cultures immediately were transferred to a water bath for further incubation at the nonpermissive temperature of 36.5oC. Cell samples were removed at the indicated times and lysed. Cds1 protein was immunoprecipitated and assayed for Cds1 kinase activity. Top: a control blot with anti-Cds1 antibodies of 1/30th of the cell extract used for immunoprecipitation; bottom: autoradiograph of Cds1 kinase activity using MBP as substrate. (C) Left: quantification of the Cds1 kinase activity shown in B; right: percentage of cells displayed aberrant mitosis determined by microscopic examination of DAPI-stained cells at the indicated times. (D) The percentage of cells displaying aberrant mitosis in wild-type and spp1-arrested cells at 36.5°C, either in the absence (left) or presence of 11 mM HU (right) added 2.5 h after shift to the nonpermissive temperature. Cells were scored by DAPI staining of ethanol-fixed samples taken at the indicated times.