Figure 4. bub1ΔK Cells Display High Levels of Chromosome Mis-segregation upon Nocodazole Release.
(A) Wild-type (JF004) and bub1ΔK (JF125) cells were released from G1 into media containing 30 μg/ml nocodazole and incubated at 23 °C for 3 h. Cells were subsequently released into anaphase by washing out the nocodazole. Samples were fixed in 3.7% formaldehyde for 1 h, 30 min after release, and stained with α-GFP (GFP-marked chromosome) and α-tubulin (red spindle) antibodies. DNA was stained with DAPI (blue). Percentage of nondisjunction of the GFP-marked chromatid at the first anaphase following nocodazole arrest was 2% in wild-type cells and 33% in bub1Δ K cells (n ≥ 50 anaphase cells). Scale bar represents 3 μm.
(B) Wild-type (JF004) and bub1ΔK (JF125) strains were synchronised in G1 as previously described and cells with one GFP dot (empty triangle) versus two dots (filled triangle) were counted (n = 400). Scale bar represents 2 μm.
(C) Cells from (B) were then released and incubated in media containing 30 μg/ml nocodazole at 23 °C for 3 h and released into media containing α-factor to score cells in the following G1. The number of cells with one GFP dot versus two dots were scored (n = 400).
(D) Wild-type (KH186), bub1Δ (KH127), bub1ΔK (JF098), sgo1Δ (JF188), and bub1Δ, sgo1Δ (JF185) strains were plated out in 10-fold serial dilutions on rich media and on rich media containing 8 μg/ml benomyl.
(E) Strains carrying the SUP11 artificial chromosome were grown overnight in CSM-URA media, then diluted back to OD600 0.2 and grown in YPDA media at 30 °C for 3 h. Cells were then plated out on YPD at a density of ∼500 cells per plate. Only colonies that were at least half red were scored for losing the test chromosome at the first division.