Figure 6.

Overproduction of Plo1 resulted in the association of Cdc7 with the SPB in G2-arrested cdc25.22 cells. (A) Two independent synchronous cultures were generated by elutrient centrifugation 13 and 15 h after the removal of thiamine from IH1548 (h⁻ cdc25.22 leu1.32 pHN 204) cells cultured in EMM2 medium at 25°C to induce overexpression of the plo1⁺ gene. The temperature of each synchronous culture was immediately shifted to the restrictive temperature of 36°C after its generation. Although inappropriate septation was induced in each culture, many more septa were seen in the 15-h culture (filled squares) than the 13-h culture (open diamonds) as each was followed into the cdc25.22 arrest. As a control the temperature of an uninduced asynchronous culture was shifted to 36°C (filled circles). Septation was inhibited in this culture for 4 h, after which some cells began to leak past the cdc25.22 arrest point. (This is a common feature of an asynchronous culture. In contrast to the synchronized cells, some members of this population had been at the cdc25.22 arrest point for the full 4-h time course of the experiment.) Staining of the 15-h and control culture showed that Spg1 was activated to recruit Cdc7 to the interphase SPBs as a consequence of Plo1 overproduction. (C) Cdc7 and DAPI/phase-contrast images of the same field of cells taken from the 210-min time point of the 15-h culture (indicated by a star in A). Cells 1–3 have Cdc7 staining of their SPBs in nuclei, which have been bisected by the inappropriately initiated septum.