Skip to main content
. 2017 Sep 19;7(11):3731–3743. doi: 10.1534/g3.117.300227

Figure 4.

Figure 4

Loss of Psh1p increases mRNA levels of genes expressed from CEN plasmids due to higher plasmid DNA content achieved through plasmid missegregation. (A) Normalized mRNA expression of the endogenous, genome-expressed genes FZO1, MIA40, SEC63, or ALG1 in WT and psh1Δ cells grown in YPD medium, not expressing a plasmid, was determined using RT-qPCR analysis. Expression was normalized to ACT1 levels and is graphed relative to WT cells; error bars represent the SD of at least three biological replicates with three technical replicates each. (B) Normalized mRNA expression of FZO1HA, mia40-4HA, and LEU2 in WT and psh1Δ cells carrying the indicated CEN plasmid, and normalized mRNA expression of genomic SEC63 and ALG1 in WT and psh1Δ cells expressing the CEN FZO1HA plasmid were determined as in (A). (C) Normalized DNA levels of CEN plasmid FZO1HA, mia40-4HA, and LEU2 in DNA harvested from WT and psh1Δ cells carrying the indicated CEN plasmid as determined by qPCR on DNA samples. Plasmid DNA content was normalized to the genomic level of ACT1 and graphed as in (A). (D) DNA levels of genome-expressed ACT1 and SEC63 in the DNA used in (C) from cells expressing the FZO1HA CEN plasmid as determined by qPCR. Values are graphed relative to WT cells; error bars represent the SD of at least three biological replicates with three technical replicates each. (E–G) The fraction of FZO1HA CEN (E) mia40-4HA CEN (F) or FZO1HA 2 μm plasmid-bearing WT and psh1Δ cells after 0 and 48 hr without selection for the plasmid was assessed by comparing the number of cells able to grow on selective media to the cell number on YPD. Error bars represent the SD of at least three biological replicates. (H) Normalized DNA levels of 2 μm and CEN plasmid FZO1HA and LEU2 in WT and psh1Δ cells, determined as in (C). (I) DNA levels of genome-expressed ACT1 and SEC63 in the DNA used in (H) as determined by qPCR, graphed and analyzed as in (D). For (A–I) P-values are calculated from a two-tailed t-test and * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, and ns = P > 0.05.