The spindle checkpoint monitors the bipolar attachment of sister chromatids to the mitotic spindle. A screen for Saccharomyces cerevisiae mutants sensitive to the microtubule depolymerising drug, benomyl, isolated 6 components of the spindle checkpoint; <i>MAD1</i>, <i>MAD2, MAD3, BUB1, BUB2, BUB3. </i>None of these components are essential for growth under normal conditions. <i>MPS1</i>, which encodes an essential kinase involved in spindle pole duplication, was also found to have a dual role in the spindle checkpoint. This work identifies the Saccharomyces cerevisiae open reading frame, YJL013c, as encoding for the <i>MAD3</i> gene. Mutations in this ORF are benomyl sensitive and divide faster on benomyl plates in a manner similar to that of previously characterised spindle checkpoint components. Mad3p localises to the nucleus when overexpressed and its expression levels are not cell cycle regulated. Mad3p shares two regions of homology with Bub1p; region I and region II. This work shows that region I of Mad3p is required for association with the target of the spindle checkpoint, Cdc20p. Region II is required for binding to Bub3p. A two hybrid assay using Mad3p as bait identified 8 possible interactors one of which is a regulatory subunit of the yeast protein phosphatase, Glc7p, a protein recently implicated in chromosome segregation. The loss or gain of all or part of a chromosome could lead to or propagate a cancerous cell. The mitotic checkpoint prevents the unequal segregation of sister chromatids and so protects against the loss or gain of chromosomes. Using an assay developed by Hieter <i>et al </i>the chromosome loss rates of spindle checkpoint mutants were calculated. Surprisingly, mutants in the different components had significantly different loss rats suggesting additional roles for Bub1p and Bub3p in the efficient segregation of sister chromatids. Point mutants in <i>mad3 </i>and <i>bub1 </i>were analysed in further detail and the effects of overexpression of checkpoint components was also analysed.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:653042 |
| Date | January 2004 |
| Creators | Johnston, Raymond Carron |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/12214 |
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