Figure 3.

Deletion of the GIN4 gene causes a prolonged mitotic arrest in cells that are dependent upon Clb2 for survival. Cells were grown overnight at 30°C until they reached an OD of 0.5 (control cells) or 0.8 (Δgin4, Δclb^1,3,4, Δbar1 cells), and α-factor was then added to 2 μg/ml at t = 0. At each time point, 1.5 ml of culture was removed and analyzed for Clb2 levels or for the presence of mitotic spindles, as previously described (Kellogg and Murray, 1995; Pringle et al., 1991). The strains used in this experiment carry deletions of the BAR1 gene to prevent them from breaking through the α-factor arrest. (A) A plot of the percentage of cells with a mitotic spindle as a function of time after the addition of α-factor to log phase cultures of a Δgin4, Δclb^1,3,4, Δbar1 strain and a Δclb^1,3,4, Δbar1 control strain. The percentage of cells with mitotic spindles was determined by counting spindles in random fields of cells. Over 200 cells were counted for each data point. After 3 h, the spindles began to appear unusually thick and bent, and it became difficult to accurately count the number of cells with spindles. (B) A Western blot showing the amount of Clb2 present as a function of time after the addition of α-factor to log phase cultures of the same strains shown in A. (C) Examples of the short spindles observed in the Δgin4, Δclb^1,3,4, Δbar1 strain 2 h after addition of α-factor to a log phase culture.