Figure 2.

Stability of Cdc6p N-terminal deletion mutants. Strain BJ5459 was transformed with integrating plasmids bearing wild-type or mutant GAL-CDC6.-MYC9 (GAL plasmids described in Table 3), and transformants carrying the integrated gene were isolated. Cells were grown overnight in YPR to a cell density of 2 × 10⁶ cells/ml. Solid galactose was added to 2%, and cells were incubated for 2 h. Nocodazole was added to a final concentration of 15 μg/ml, and cells were incubated for an additional 3 h, at which point the population was arrested, as determined by morphology and flow cytometry (our unpublished observations). Transcription was repressed by removing cells from galactose–raffinose medium and resuspending in YPD, and samples were taken at the times indicated. To determine levels of Cdc6p, extracts were prepared as described in MATERIALS AND METHODS and 10 μg of protein were electrophoresed on a 10% gel, blotted to a polyvinylidene difluoride membrane (Millipore, Bedford, MA), and probed with anti-myc monoclonal antibody 9E10. The numbers on the left refer to the amino acids deleted. NLS refers to the KRKK to AAAA mutant described in the text.