Figure 2

glc7-E101Q mutant exhibits slow growth but no appreciable delay in the cell cycle or chromosome segregation defect. (A) The growth of wild type (GLC7) vs glc7-E101Q strains in rich medium (YPAD) was determined from early log-phase cultures. (B) FACs analysis for DNA content (propidium iodide staining) of log-phase wild type and glc7-E101Q cells. Cell counts are shown plotted against fluorescence intensity (PI). (C) Budding index of wild type and glc7-E101Q mutant strains. (D) Fluorescence microscopy of anaphase cells (mitotic spindle >2.5 μm) wild type and glc7-E101Q cells expressing a chromosome [GFP-LacI (green) and centromeric (CEN15) repeats of the lactose operon (lacO)] and spindle pole body (SPB) [Spc29-CFP (red)] markers. The percentage of cells with chromosomes localized to both SPBs is indicated at the bottom (n = 3, 300 cells/experiment). (E) Test for chromosome segregation using a sectoring assay. Wild type GLC7, glc7-E101Q and ame1-6 strains lacking a functional ADE2 gene (ade2Δ-101) and transformed with a Circle III/SUP11 were plated on to medium lacking adenine (YPD) and scored for appearance of red sectors after 4-day incubation. (F) Spotting assay of glc7-E101Q vs. wild type on DMSO control or benomyl plates (5-fold serial dilutions of 1.0 × 10⁶ cells/ml; 5 μl/spot). Benomyl-sensitive (tub1-1) and resistant (tub2-104) strains were plated as controls.