Figure 5. Overproduction of Erf2 and Erf4 in proliferating cells disrupts vegetative growth and induces aberrant meiosis and sporulation inpat1-114 cells.

erf2 OE erf4 OE refers to strain 7 from Figure 4B that co-overexpresses erf2 and erf4 at high levels. These cells were grown in the presence of nutrients at permissive temperature, and co-overexpression of erf2 and erf4 was induced by switching cells to thiamine-free medium (see Materials and Methods). Indicated times or time intervals refer to time after induction of erf2 and erf4 co-overexpression. (A) Fold change in OD₅₉₅ of cultures during indicated 12 h intervals. OD₅₉₅ was maintained <0.6. (B) DNA content analysis. (C) Blankophor staining of cells (top panels). Dimensions of septated cells (cell length and width, n = 20) and percentage of septated cells (septation index, n≥200) were determined by measuring and counting blankophor-stained cells (bottom panels, left to right). Error bars, SD. (D) Percentage of cells with 1, 2, or >2 nuclei (n≥200) was determined by DAPI staining. (E) DAPI (top) and DIC (bottom) images of cells under conditions where erf2 and erf4 overexpression were repressed (+Thiamine) as well as 96 h postinduction of erf2 and erf4 co-overexpression. Scale bars, 10 µm. (F) Left panel, percentage of cells with spores (n≥200) at indicated times postinduction. Right panel, DIC images of cells 96 h postinduction before (top) and after (bottom) β-glucuronidase digestion, which specifically kills vegetative cells but not spores. Nonoverexpressing pat1-114 cells continue vegetative growth under the same conditions (see Figure S5).