Figure 2.

bub1 ⁺ is required for spindle checkpoint function. (A) bub1 mutants are hypersensitive to benomyl. Strains were grown in YES medium to 0.5–2 × 10⁷ cells/ml. About 200 cells were spotted onto YES plates containing the indicated concentrations of the anti-microtubule drug benomyl and incubated at 32°C for 3 d. (B–D) bub1 mutants are unable to maintain a mitotic arrest. Small G2 cells of nda3KM311 Δswi6 and nda3KM311 Δbub1 were shifted to 18°C, which disrupts spindle microtubules due to the nda3 defect, and samples were taken every hour. (B) Unlike the Δswi6 cells which arrest with high levels of histone H1 kinase activity (left), the Δbub1 cells fail to maintain such an arrest and the kinase activity drops after 3 h (right). DAPI and calcofluor staining shows that this is followed by a wave of septation (C) from 4–6 h as the Δbub1 cells (solid squares) progress through the cell cycle, and ultimately leads to a cut phenotype (D) as septation occurs without chromosome segregation. The Δswi6 cells maintain their arrest with hypercondensed chromosomes (data not shown) and do not septate (C, open circles). (E) Δbub1 cells are unable to prevent sister chromatid separation in the absence of a spindle. Cells from strain 379 (nda3KM311) and 401 (nda3KM311 Δbub1) were grown in YES medium at 32°C to 3 × 10⁶ cells/ml, shifted to 18°C for 8 h, and then fixed and processed for FISH using a probe detecting all three centromeres (cenFISH). Nuclear DNA was stained with DAPI. Bar, 5 μm.