Protocol

Checkpoint Controls and Cancer

Volume 280 of the series Methods in Molecular Biology™ pp 275-290

Assaying the Spindle Checkpoint in the Budding Yeast Saccharomyces cerevisiae

  • Christopher M. YellmanAffiliated withDepartment of Biochemistry and Molecular Genetics, University of Virginia Medical Center
  • , Daniel J. BurkeAffiliated withDepartment of Biochemistry and Molecular Genetics, University of Virginia Medical Center

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Abstract

The spindle checkpoint is assayed in Saccharomyces cerevisiae using several criteria. Sensitivity to benzimidazole drugs is assayed in cells grown in liquid medium and cells grown on solid medium on petri plates. Cell cycle delays are measured using cells synchronized by treatment with mating pheromone α-factor, and the population is monitored by flow cytometry measuring DNA content in cells. There are two different transitions that are monitored, and cytological assays for individual cells and biochemical assays for populations of cells are presented. The metaphase to anaphase transition is assayed by monitoring sister chromatid separation using GFP-tagged chromosomes, Pds1 stability using immunofluorescence, and Mcd1/Scc1 association with chromatin using chromosome spreads. Pds1 and Mcd1/Scc1 stability is measured in populations by Western blots. The exit from mitosis is monitored by Cdc14 immunofluorescence and Clb2 Western blots.

Key Words

Mitosis spindle checkpoint regulation genetics cytology biochemistry flow cytometry