Activation of the budding yeast spindle assembly checkpoint without mitotic spindle disruption

Science

Volumn 273, Issue 5277, 1996, Pages 953-956

  Hardwick, Kevin G ^a   Weiss, Eric ^b   Luca, Francis C ^b   Winey, Mark ^b   Murray, Andrew W ^a  

^a UNIVERSITY OF CALIFORNIA   (United States)

^b UNIVERSITY OF COLORADO   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

ARTICLE; CELL CYCLE; CHROMOSOME SEGREGATION; MITOSIS; MITOSIS SPINDLE; PRIORITY JOURNAL; YEAST;

SACCHAROMYCETALES;

EID: 0029791321     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.273.5277.953     Document Type: Article

References (27)

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24. Cells were grown in rich growth medium [yeast extract, peptone, and dextrose (YPD)] for 3 hours at the indicated temperatures (Fig. 1). Nocodazole was used at 20 μg/ml in combination with benomyl at 30 μg/ml. Both drugs were necessary to maintain the arrest in wild-type cells at 37°C. Cell lysates were prepared, and immunoblotting was done as described (8). Strains are wild type (KH146), mps1-1 (WX241-2b), mps2-1 (WX178-3c), and cdc31-2 (ELW65-9b). The cdc31 cells exhibited delayed mitosis even when grown at 24°C.
25. 2-terminally myc-tagged MPS1 gene in an Afl III to Swa I fragment into pNZ2 digested with Nco I and Sma I. The pELW325 was then transformed into yeast strain BJ2168, and galactose-driven overexpression of Mps1p was confirmed by immunoblotting with the 9E10 antibody (19) (Fig. 2A, inset) Cells (ELW175) grown in medium containing 2% raffinose or exposed to 3% galactose for 6 hours were prepared for EM (20) and viewed on a Philips CM10 microscope (Mahwah. NJ). Spindle lengths were measured from spindle pole body to spindle pole body directly on the negatives from the EM and corrected for magnification (Fig. 2B). Several spindles traversed serial sections and required spindle length correction for section thickness (70 to 80 nm) by triangulation. KH135 cells (which contain pAFS120, a GAL1-Nmyc-MPS1 integration construct made by polymerase chain reaction to introduce Xho I and Eco RI sites at either end of the NmycMPS1 fragment, allowing it to be subcloned into pDK20) were grown overnight in yeast-extract peptone (YEP) containing 2% raffinose, arrested oy exposure to 4% galactose for 5 hours, and prepared for immunofluorescence as described (Fig. 2C) (8). ELW200 cells (integrated GAL1-NmycMPS1) were grown in YEP containing 2% dextrose or shifted into YEP with 3% galactose for 6 hours and prepared for flow cytometry as described (Fig. 2D) (21). The DNA stained by propidium iodide in 5000 cells per sample was detected on a Becton Dickinson FACScan flow cytometer.
26. Unbudded cells containing integrated copies of GAL-MPS1 were picked individually and placed on slabs containing 4% galactose (Fig. 3A). The cells were grown at 30°C, and the number of cells that had divided and rebudded were counted at the times shown. Each point is an average from at least 50 cells. Strains are wild type (KH153), mad1Δ.1 (KH155), mad2-1 (KH157), mad3-2 (KH150), bub1Δ (KH161), bub2-1 (KH163), and bub3Δ (KH165). Cells of the strains listed above were grown in YEP with 2% raffinose to mid-log phase, collected, and incubated in fresh medium for 90 min with the addition of alpha factor to a final concentration of 10 μM. The cells were collected and resuspended in YEP containing 2% raffinose, 10 μM alpha factor, and 3% galactose and incubated for 2 hours. Finally, cells were collected, rinsed once in medium without alpha factor, and released into YEP containing 2% raffinose and 3% galactose. Timing began at the release from mating factor arrest, and samples were taken every 20 min. Cells were fixed with 70% ethanol and examined microscopically to determine the fraction of large budded cells as described (3). Cells of the previously mentioned strains were grown overnight in YEP with 4% raffinose and then galactose was added to a final concentration of 4%. Samples were taken after 3 and 6 hours of growth at 30°C. Control (0) cells were grown for 3 hours in dextrose (Fig. 3C).
27. We thank A. Straight for the integrating GAL1-MPS1 construct, T. Giddings for help with the EM analysis, A. Hoyt and B. T. Roberts for bub strains and deletion constructs, and all the members of our labs for their advice and encouragement. pNZ2 was provided by G. N. Zecherle of the University of Washington, Seattle, and pDK20 was provided by D. Kellogg of the University of California, Santa Cruz, K.G.H. and F.C.L. are Fellows of the Leukemia Society of America and E.W.W. was a trainee of NIH. This work was supported by grants to A.W.M. from NIH, the March of Dimes, and the David and Lucile Packard Foundation; and to M.W. from NIH, the American Cancer Society, and the Pew Scholars Program in the Biomedical Sciences.