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Vol. 16, Issue 12, 5649-5660, December 2005
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* Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, WA 98109-1042;
Molecular and Cellular Biology Program, University of Washington and Fred Hutchinson Cancer Research Center, Seattle, WA 98195
Submitted August 17, 2005;
Revised September 16, 2005;
Accepted September 22, 2005
Monitoring Editor: Kerry Bloom
Kinetochores mediate chromosome attachment to the mitotic spindle to ensure accurate chromosome segregation. Budding yeast is an excellent organism for kinetochore assembly studies because it has a simple defined centromere sequence responsible for the localization of >65 proteins. In addition, yeast is the only organism where a conditional centromere is available to allow studies of de novo kinetochore assembly. Using a conditional centromere, we found that yeast kinetochore assembly is not temporally restricted and can occur in both G1 phase and prometaphase. We performed the first investigation of kinetochore assembly in the absence of the centromeric histone H3 variant Cse4 and found that all proteins tested depend on Cse4 to localize. Consistent with this observation, Cse4-depleted cells had severe chromosome segregation defects. We therefore propose that yeast kinetochore assembly requires both centromeric DNA specificity and centromeric chromatin.
Abbreviations used: 
F, factor; CenH3, centromeric histone H3 variant; ChIP, chromatin immunoprecipitation; cCEN, conditional centromere; dox, doxycycline; eCEN, endogenous centromere; FACS, fluorescence-activated cell sorter; IP, immunoprecipitate; SPB, spindle pole body.
The online version of this article contains supplemental material at MBC Online (http://www.molbiolcell.org).
Present address: University of California, Santa Cruz, ETOX, 1156 High St., Santa Cruz, CA 95064.
Address correspondence to: Sue Biggins (sbiggins{at}fhcrc.org).
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