Applen, Shelly2009-06-012009-06-012009-04-08https://hdl.handle.net/11299/50548Additional contributors: Laura Burrack; Judith Berman (faculty mentor).Centromere DNA and the numerous associated kinetochore proteins, are found in our cells attaching sister chromatids together and are essential for normal chromosomal segregation. The objective of this project is to characterize kinetochore mutants in Candida albicans and determine which kinetochore proteins are essential and which are necessary for chromosome stability. With GRACE (gene replacement and conditional expression) strain analysis using a tet-off promoter to regulate gene expression, we have concluded that the gene products of Dad2, Spc19 and Cse4 are absolutely essential for the growth of C. albicans, while depletion of Nuf2 and Skp1 gene products resulted in a growth defect. The growth phenotypes following depletion of other kinetochore proteins are yet to be determined. We quantified cell viability with FUN-1 vital stain and found that after prolonged depletion of Nuf2, Dad2, Skp1, and Cse4 gene products, most, but not all, cells have undergone cell death rather than remaining in a dormant state. As a second method of obtaining a knock-out strain to determine essentiality, we are transforming a Uridine-Arginine-Uridine (UAU) construct, selecting for the integration of this construct into both genome copies, and determining if all surviving cells still contain a wild-type allele resulting from triosomy or a gene duplication event. This phase is currently in progress. We also plan to determine cell size as a measure of polyploidy which could give insight as to how some cells are adapting and escaping cell death. This work will provide a better understanding of how cells react to stresses, such as kinetochore malfunction.en-USCollege of Biological SciencesDepartment of Genetics, Cell Biology and DevelopmentPresentation