Figure 2.

The progression of mitotic phase in arp4S23A/D159A cells is impaired. (A) Wild-type (YHO800) and arp4S23A/D159A (YHO820) cells were grown in YPAD at 23°C for 3 h with 15 μg/ml nocodazole. The culture was shifted to 37°C and incubated in the presence of nocodazole for 1 h. Cells were released from the block by washing in prewarmed (37°C) YPAD and incubated in fresh prewarmed YPAD medium at 37°C. Samples were taken at the time points indicated and analyzed by flow cytometry. (B) Wild-type (YHO800) and arp4S23A/D159A (YHO820) cells were synchronized in G2/M phase with nocodazole for 3 h at 23°C and cultured for 1 h at non-permissive temperature (37°C). Equal quantities of yeast spheroplasts were digested with 2 U/μl micrococcal nuclease, which preferentially digests DNA in linker regions between nuclesomes, for 0, 2, 5, 15 and 30 min. The purified DNA was resolved on a 1.5% agarose 0.5 × TBE gel and visualized by ethidium bromide staining.