Figure 6.

Overexpression of Cse4p increases CEN, but not 2 μm, plasmid loss and does not affect plasmid DNA content under selective growth conditions. (A) Normalized DNA levels of CEN FZO1HA plasmid were detected by qPCR using two plasmid-specific primer pairs (FZO1HA and vector/LEU2 junction) on DNA harvested from WT cells, psh1Δ cells, or cells overexpressing Cse4p from the chromosome (pGAL-FLAG-CSE4) for 24 hr in selective media. LEU2 primers could not be used, as the mutated leu2-3,112 allele is present in the genome of these strains. DNA content was normalized to ACT1 levels and graphed as in Figure 4C. See Figure S3A in File S1 legend for further detail. (B) The fraction of CEN FZO1HA plasmid-bearing was analyzed as in Figure 4D after 0 and 48 hr growth in media without selection for the CEN plasmid. Cells were grown in medium containing galactose for 24 hr prior to the shift to nonselective medium, and galactose induction was continued during growth without selection. Error bars represent the SD of at least three biological replicates. (C) The fraction of 2 μm FZO1HA plasmid-bearing WT cells, psh1Δ cells, or cells overexpressing Cse4p from the chromosome (pGAL-FLAG-CSE4) was analyzed as in (B) For (A–C) P-values are calculated from a two-tailed t-test; * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, and ns = P > 0.05.