bub1 + is required for spindle checkpoint function. ( A ) bub1 mutants are hypersensitive to benomyl. Strains were grown in YES medium to 0.5–2 × 10 7 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 × 10 6 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.
{}