FIG. 7.
Bir1 inactivation causes a severe biorientation defect following nocodazole (NOC) treatment. (A) Equivalent 10-fold dilutions of wild-type (AY925), bir1-17 (VMY1), and dam1-S257A,S265A,S292A (BP635; nocodazole-hypersensitive control) cells were plated on YPD medium containing 8 μg/ml benomyl. (B) BIR1 (VMY33) and bir1-17 (VMY34) cells containing CEN3-(tetO)336, tetR-GFP, YFP-TUB1, and pMET3-CDC20 were arrested in G1 with α-factor at 26°C and then released for 2.5 h to a metaphase block in rich medium containing 2 mM methionine (to deplete Cdc20) and nocodazole (30 μg/ml; to depolymerize microtubules). Subsequently, nocodazole was removed and the temperature concurrently shifted to 37°C (to inactivate Bir1). Cells showing a clear bipolar spindle were scored for mono-oriented or bioriented sister CEN3 genes based on 2-min time-lapse movies taken during a 30- to 120-min time course (n is the number of cells scored). (C) Examples of cells from panel B at the point of nocodazole release (0 min) showing the absence of spindles and at 90 min showing bipolar spindles. Red, YFP-Tub1; green, sister CEN3 centromeres (tetR-GFP).