Figure 1.

Spindle checkpoint mutants exhibit different rates of chromosome loss. (A) Null mutant sectoring phenotypes. The strains shown are wild type (YPH278), bub1Δ (YFP2), bub3Δ (YFS1100), mad1Δ (YFS1120), mad2Δ (YCD173), and mad3Δ (YFS1205). (B) Chromosome loss rates in null mutants determined by half-sector analysis. Wild type: 49 half-sectored colonies/61,276 total colonies (YPH278); 8/9,305 (YKH231). bub1Δ: 192/4,784 (YFP2); 89/2,121 (YRJ112). bub2Δ: 17/22,101 (YCD165); 3/3,440 (YRJ113). bub3Δ: 137/3,362 (YFS1100); 63/2,237 (YRJ114). mad1Δ: 139/12,394 (YFS1120); 32/8,552 (YMB111). mad2Δ: 87/10,153 (YCD173); 9/3,106 (YMB113). mad3Δ: 57/29,364 (YFS1205); 29/13,186 (YRJ111). (C) Immunoblots showing overexpression from a MET25 promoter. Extracts were taken after 2 h of inductionin media lacking methionine and were analyzed by Western blot using antibody specific for each protein. The left lane (vector, p423MET) shows the endogenous Bub1p expression level where detected; the right lane (–MET) shows protein expressed from the MET25 promoter. All strains were generated from YKH231 by introduction of p423MET-derived plasmids containing full-length open reading frames cloned adjacent to the MET25 promoter. (D) Chromosome loss associated with overexpression of checkpoint genes. Half-sector analysis was performed after plating the strains in C on plates lacking methionine. Vector: 14/19,030. METpBUB1: 195/17,640. METpBUB3: 17/17,875. METpMAD1: 28/11,920. METpMAD2: 92/17,065. METpMAD3: 137/12,115. Two or more additional independent transformants tested for each construct showed the same chromosome instability phenotype by colony sectoring assay. (E) Benomyl sensitivity of checkpoint null mutants. Log phase cultures were spotted in a 10-fold dilution series on rich medium (YPD) or rich medium plus Benomyl. Strains were mad1Δ (YMB111), mad2Δ (YMB113), mad3Δ (YRJ111), bub1Δ (YRJ112), and bub3Δ (YRJ114).