FIG. 5.

Degradation of Dbf4p is dependent on Cdc23p function. Wild-type (YCH192) and cdc23-1 (YCH208) cells were transformed with a 2μm-based plasmid carrying GAL1-HA-DBF4 (pCH920). For both strains an overnight culture was grown selectively at 23°C in SC lacking histidine. This culture was inoculated into YEP plus raffinose, grown to early log phase at 23°C, and arrested in G₁ with α-factor. When >90% of the cells, as determined by microscopy, exhibited the arrest state, the temperature of the culture was shifted to 37°C, the restrictive temperature for cdc23-1 cells. FACS analysis of these blocked samples indicated that >90% of the cells had a 1N content of DNA and were in G₁ (data not shown). After 30 min at the restrictive temperature, galactose was added at time −30 to induce expression of HA-DBF4 from the GAL promoter. Thirty minutes later, at time zero, glucose was added to turn off expression of HA-DBF4 from the GAL promoter while maintaining the G₁ arrest at the restrictive temperature. FACS analysis of these blocked samples also indicated that >90% of the cells had a 1N content of DNA and were in G₁ (data not shown). Samples of equal density were taken at the indicated time points and assayed by immunoblotting for HA-Dbf4p and actin. Actin detection was used as an internal loading control.