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. 2002 Sep;13(9):3029–3041. doi: 10.1091/mbc.E02-04-0203

Figure 4.

Figure 4

Overexpression of Bub1p or Bub1p fragments both disrupts the spindle checkpoint and causes damage. (A) Disruption of a ctf18Δ-induced checkpoint delay. ctf18Δ strains containing p423MET plasmids expressing no (vector), full-length (BUB1), or partial (1–210, 1–367, 211–367, 1–608) alleles of BUB1 were created by transformation of YFS377. Cells were grown to early log phase in the absence of methionine for 18–24 h, and were prepared for flow cytometry (as in Hanna et al., 2001). A representative histogram is given for each genotype; four independent transformants were analyzed. The fractions of budded and unbudded cells were determined and are shown as mean ± SD (3 d.f.). (B) Disruption of the ctf18Δ delay results in decreased viability. Two independent isolates from each of the ctf18Δ strains described in A were grown to early log phase in media containing methionine. Cells were spotted onto solid medium without methionine in a 10-fold serial dilution series. Overexpression of bub1-[211-367]p in wild-type or bub3Δ cells did not result in a significant reduction in mortality (bottom). (C) Competence to arrest in response to MPS1 overexpression. Log phase cells grown in raffinose media lacking methionine (to induce expression of the BUB1 alleles) were treated with 3% galactose to induce MPS1 expression. Samples were taken at t = 0 and t = 4 h, formaldehyde fixed, DAPI stained, and scored for bud and nuclear morphology. The graph shows the percentage arrested (large-budded uninucleate) cells at each time point. Two independent transformants were analyzed (average ± range indicated). All strains were derived from YML101 (GAL-MPS1). All plasmids overexpressing BUB1 alleles in this strain were derived from p423MET (vector). YCD362 (bub1Δ) was included as a control for the assay. (D) Chromosome missegregation in a mad3Δ host. Chromosome loss rates were determined by half-sector analysis on plates lacking methionine. Vector: 5/3,327. pBUB1: 18/2,295. p[1–210]: 4/2,408. p[1–367]: 51/2,500. p[1–608]: 42/2,766. p[211–367]: 37/2,349. Data for a representative transformant are shown; at least two independent transformants were analyzed for each construct. The strains were YFS1205 derivatives created by introduction of p423MET (vector) and related BUB1 allele overexpression plasmids.