FIG. 3.

Cell cycle arrest phenotype of rpn3 mutants. (A) Wild-type YE46 (RPN3) cells and YE100 mutant (rpn3-4) cells were grown at 25°C to the early log phase and shifted to 37°C. Cell samples were withdrawn before (0 h) and every hour after the temperature upshift and analyzed for nuclear DNA content by flow cytometry. 1N and 2N indicate cells with unreplicated and fully replicated nuclear DNA, respectively. (B) Nuclear DNA profile of wild-type and ts rpn3 cells presynchronized in G₁ with α-factor (αF) and released to 38.5°C. YE46 (RPN3), YE100 (rpn3-4), and YE101 (rpn3-7) strains were grown in YEPD medium to the early log phase, incubated with α-factor for 3.5 h at 25°C [αF 210 (25°C)], shifted to 38.5°C for an additional hour [αF 60 (38.5°C)], and finally resuspended in medium preequilibrated to 38.5°C and without the pheromone (0 min). Upon release from the G₁ arrest, cell aliquots were taken at 20-min intervals and processed for flow cytometric analysis of nuclear DNA content. 1N and 2N indicate cells with unreplicated and fully replicated nuclear DNA, respectively.