Identification and Characterization of Components of the Mitotic Spindle Checkpoint Pathway in Fission Yeast

摘要

During anaphase of mitosis, sister chromatids are separated by the mitotic spindle. The spindle assembly checkpoint protects the integrity of the genome by initiating a cell cycle delay if chromosomes are not property attached to the spindle. Cells lacking a functional spindle checkpoint may gain or lose genetic information, which can cause cell death or predispose cells to cancer. For example, mutations in human Bub1 p have been identified in cancer cell lines and altered expression of the bub1 gene has been shown to correlate with tumor cell proliferation and chromosomal instability. Most human spindle checkpoint components were identified by their similarity to yeast checkpoint proteins that were discovered through genetic screens. Many aspects of spindle checkpoint function are not yet understood, and genetic evidence indicates there are additional checkpoint proteins that have not been identified. By using a genetic screen in fission yeast, this project aims to identity and characterize novel spindle checkpoint components and novel mutant alleles of known spindle checkpoint genes. To date, mutations in three known yeast spindle checkpoint genes have been identified by this screen. A novel bub1 mutant, called bub1- A78V, was selected for further study because it expresses stable Bub1p but is spindle checkpoint defective, and the bub1-A78V mutation occurs inside the Bub1p Mad3-like region, a domain which is conserved from yeast Bub1p to human Bub1p, but the function of this region remains unknown. Our studies indicate that the Mad3-like region of Bub1p is required for correct localization of Bub1p, Bub3p, and Mad3p inside the nucleus In cycling cells and to unattached kinetochores when the spindle checkpoint is activated.