Figure 4.

The ste9 mutant initiates mitosis from the G₁-phase in the absence of S-phase. In all experiments, KJ32–2A (cdc10–129) and KJ32–1A (ste9-B36 cdc10–129) were used. (A) The double mutant of ste9 cdc10 cannot grow even at the semipermissive temperature for cdc10^ts (32°C). Cells of cdc10 (left) or ste9 cdc10 (right) were cultured for 3 d at 32°C and 37°C. (B) Flow cytometric analysis of cdc10 (left) or ste9 cdc10 (right) at the semipermissive temperature. (C) Changes of the septation index. Asynchronous culture of cdc10 (open square) or ste9 cdc10 (filled circle) at the permissive temperature was shifted to 36°C. At the time indicated, cells were stained with calcofluor, and septated and nonseptated cells were counted. (D) DNA content of cdc10 (left) and ste9 cdc10 (right) mutants at 36°C. HU was added at the same time as the cells were shifted to 36°C. Addition of HU at 2 h at the restrictive temperature resulted in the same DNA profile (our unpublished observations). (E) “Cut” phenotype in the ste9 cdc10 double mutant at the restrictive temperature. Cells were incubated at 36°C for 6 h and stained with DAPI and calcofluor. (F) Lethal mitosis in the ste9 cdc10 double mutant occurred in the absence of S-phase. HU (final concentration 12 mM) was added to the cells of cdc10 (open bar) and ste9 cdc10 (black bar), which were then incubated at 28°C or 36°C for 4 h. Aliquots were plated onto YE and incubated at 25°C, and the number of colonies was counted after 3 d. Viability is expressed as the percent ratio of colony numbers of cells after 4 h of incubation relative to that of untreated cells at the beginning of the experiments (0 h). Sensitivity to HU of the cdc10 single mutant at 28°C was not examined, but the cdc10 cells were viable at 36°C in the presence of HU.