Abstract

The long-range objective of this research program is to understand in molecular terms how the centromere/kinetochore functions to bring about proper chromosome segregation during eukaryotic mitotic and meiotic cell divisions. Research in this area is highly relevant to human health problems, such as cancer and genetic defects due to chromosome imbalances. Cancer therapy often involves treatment with drugs that interfere with normal chromosome segregation. The research supported by this grant employs combined molecular and genetic strategies to elucidate the mechanics and regulation of centromere/kinetochore function in the budding yeast Saccharomyces cerevisiae. The functional kinetochore in budding yeast is comparatively simple (125 bp of CEN DNA plus five known associated proteins), and each kinetochore binds to only one microtubule on the mitotic spindle. Thus, the yeast centromere is an excellent experimental model to analyze the molecular mechanisms involved in eukaryotic kinetochore function. In previous research supported by this grant, we isolated a multisubunit protein complex (CBF3) that binds specifically to the CEN DNA. Affinity-purified CBF3 contains a minus-end-directed, microtubule-based motor activity (Kar3p), and this complex can link and move CEN DNA on microtubules.
The specific aims of this project include 1) identification in vivo and in vitro of proteins interacting with the core kinetochore protein complex (CBF3); 2) further characterization of yeast kinetochore-microtubule interactions in vitro, including cell cycle-dependent regulation of the CBF3-Kar3p-microtubule interaction, and a study of the effects of CBF3-Kar3p on microtubule assembly-disassembly dynamics; 3) mechanistic studies on centromere/kinetochore proteins, including definition of a putative kinetochore-bound protein kinase (Cbf2p/Ndc10p) that is likely involved in regulation of kinetochore function; and 4) isolation and characterization of homologs of the essential yeast kinetochore proteins from higher eukaryotes, as a route to extend these studies to human biology.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA011034-30
Application #
2633692
Study Section
Molecular Cytology Study Section (CTY)
Program Officer
Cole, John S
Project Start
1977-01-01
Project End
2001-12-31
Budget Start
1998-01-01
Budget End
1998-12-31
Support Year
30
Fiscal Year
1998
Total Cost
Indirect Cost

  Institution

Name
University of California Santa Barbara
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
City
Santa Barbara
State
CA
Country
United States
Zip Code
93106

  Publications

Mishra, Prashant K; Baum, Mary; Carbon, John (2011) DNA methylation regulates phenotype-dependent transcriptional activity in Candida albicans. Proc Natl Acad Sci U S A 108:11965-70
Mishra, Prashant K; Baum, Mary; Carbon, John (2007) Centromere size and position in Candida albicans are evolutionarily conserved independent of DNA sequence heterogeneity. Mol Genet Genomics 278:455-65
Baum, Mary; Sanyal, Kaustuv; Mishra, Prashant K et al. (2006) Formation of functional centromeric chromatin is specified epigenetically in Candida albicans. Proc Natl Acad Sci U S A 103:14877-82
Sanyal, Kaustuv; Baum, Mary; Carbon, John (2004) Centromeric DNA sequences in the pathogenic yeast Candida albicans are all different and unique. Proc Natl Acad Sci U S A 101:11374-9
Stoyan, Tanja; Carbon, John (2004) Inner kinetochore of the pathogenic yeast Candida glabrata. Eukaryot Cell 3:1154-63
Yoon, H-J (2004) A novel small-molecule inhibitor of the chromosome segregation process in yeast. Mol Genet Genomics 271:490-8
Sanyal, Kaustuv; Carbon, John (2002) The CENP-A homolog CaCse4p in the pathogenic yeast Candida albicans is a centromere protein essential for chromosome transmission. Proc Natl Acad Sci U S A 99:12969-74
Stoyan, T; Gloeckner, G; Diekmann, S et al. (2001) Multifunctional centromere binding factor 1 is essential for chromosome segregation in the human pathogenic yeast Candida glabrata. Mol Cell Biol 21:4875-88
Yoon, H J; Carbon, J (1999) Participation of Bir1p, a member of the inhibitor of apoptosis family, in yeast chromosome segregation events. Proc Natl Acad Sci U S A 96:13208-13
Pietrasanta, L I; Thrower, D; Hsieh, W et al. (1999) Probing the Saccharomyces cerevisiae centromeric DNA (CEN DNA)-binding factor 3 (CBF3) kinetochore complex by using atomic force microscopy. Proc Natl Acad Sci U S A 96:3757-62

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