Analysis of centromere function in Saccharomyces cerevisiae using synthetic centromere mutants |
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| Authors: | Michael R Murphy Dana M Fowlkes Molly Fitzgerald-Hayes |
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| Institution: | (1) Department of Biochemistry, Program in Molecular and Cellular Biology, 901 Lederle Graduate Research Center, University of Massachusetts at Amherst, 01003 Amherst, MA, USA;(2) Department of Pathology, Curriculum in Genetics, University of North Carolina at Chapel Hill, 601 Brinkhous-Bullit 228 H, 27514 Chapel Hill, NC, USA;(3) Present address: Laboratory of Cellular and Developmental Biology, NIDDK, National Institutes of Health, Building 6, Room B1-26, 20892 Bethesda, MD, USA |
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| Abstract: | We constructed Saccharomyces cerevisiae centromere DNA mutants by annealing and ligating synthetic oligonucleotides, a novel approach to centromere DNA mutagenesis that allowed us to change only one structural parameter at a time. Using this method, we confirmed that CDE I, II, and III alone are sufficient for centromere function and that A+T-rich sequences in CDE II play important roles in mitosis and meiosis. Analysis of mutants also showed that a bend in the centromere DNA could be important for proper mitotic and meiotic chromosome segregation. In addition we demonstrated that the wild-type orientation of the CDE III sequence, but not the CDE I sequence, is critical for wild-type mitotic segregation. Surprisingly, we found that one mutant centromere affected the segregation of plasmids and chromosomes differently. The implications of these results for centromere function and chromosome structure are discussed.by M. Yanagida |
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