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Title: Investigating the role of spindle checkpoint mutations in colorectal cancer
Author: Boyes, Laura
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2004
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It has been hypothesised that mutations in genes involved in chromosome segregation and mitotic checkpoints may contribute to aneuploid phenotypes in colon tumours. In support of this hypothesis deletions and missense mutations in spindle checkpoint genes BUB1 and BUBR1 have previously been reported in non-diploid colon cancers with defective mitotic checkpoints which also display chromosomal instability. We identified a deleted BUBR1 transcript in 25% of a panel of aneuploid colorectal tumours from young patients. The deletion, termed DE5, removes a protein domain that is conserved between BUB1 and BUBR1 in humans and yeast and in which mutations have previously been reported. We set out to determine the functional effects of this deletion in a yeast model system and in vivo with human cell lines. Constructs of the deleted forms of the genes were created in the S. cerevisiae homologues, BUB1 and MAD3. Functional effects of expressing deleted Bub1p and Mad3p proteins in strains null for the respective gene were studied with respect to microtubule poison sensitivity, chromosome loss and spindle checkpoint activity. Parallel control experiments used full-length gene constructs. We show that cells expressing deleted proteins are more sensitive to microtubule poisons than those expressing the full-length protein. Both the MAD3 and BUB1 DE5 mutants show a chromosome loss phenotype indicative of a defect in chromosome segregation. We observed a more severe phenotype in BUB1 DE5 deletion mutants, consistent with the chromosome loss phenotype seen in a bub1 null strain. This suggests that BUB1 may play a more significant role in mitosis than MAD3 in the yeast system. Using an assay of individual cell growth in the presence of microtubule poisons, we show that the MAD3 deletion mutants results in a defective mitotic checkpoint. A similar assay to specifically investigate spindle checkpoint function in the BUB1 DE5 mutant, revealed a similar abrogation of the spindle checkpoint.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available