Figure 9.
Effects of Cdc5, condensin, and topoisomerase II inactivation on anaphase onset in the absence of Cdc55. (A) Strains with the indicated genotypes (W303, ySP305, ySP6130, ySP6146, ySP6121, and ySP6105) were grown in YEPD at 26°C, arrested in G1 with α-factor, and released at 37°C (time 0). At different times, cells were collected to determine the kinetics of budding and anaphase after nuclear staining with propidium iodide. (B) Serial dilutions of strains with the indicated genotypes (W303, ySP5737, ySP5929, and ySP6066) were spotted on YEPD plates and incubated at 25°C for 2 d and at 16°C for 6 d. (C) Wild-type (wt), top2-4, and cdc24 cells carrying a minichromosome (YCp50) were arrested in G1 with α-factor and released for 3 h at 37°C in the presence of nocodazole. Topological forms of plasmids isolated from these cells were examined by Southern blot after electrophoresis of total DNA in the presence of ethidium bromide using the 1.7-kb AmpR PvuI–BglII fragment of YCp50 as a probe. Purified YCp50 DNA linearized with HindIII and uncut were run in parallel to define the mobility of the various conformational forms.