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MBC in Press, published online ahead of print January 26, 2005
Mol. Biol. Cell 10.1091/mbc.E04-09-0788

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Submitted on September 8, 2004
Revised on January 10, 2005
Accepted on January 13, 2005

Identification of Xenopus CENP-A and an Associated Centromeric DNA Repeat

Nathaniel S. Edwards and Andrew W. Murray

Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138

Monitoring Editor: Kerry Bloom

Kinetochores are the proteinaceous complexes that assemble on centromeric DNA and direct eukaryotic chromosome segregation. The mechanisms by which higher eukaryotic cells define centromeres are poorly understood. Possible molecular contributors to centromere specification include the underlying DNA sequences and epigenetic factors like binding of the centromeric histone, CENP-A. Frog egg extracts are an attractive system for studying centromere definition and kinetochore assembly. To facilitate such studies, we cloned a Xenopus laevis homolog of CENP-A (XCENP-A). We identified centromere-associated DNA sequences by cloning fragments of DNA that copurified with XCENP-A by chromatin immunoprecipitation. XCENP-A associates with Fcr1 (frog centromeric repeat 1), a 174 base pair repeat containing a possible CENP-B box. Southern blots of partially digested genomic DNA revealed large ordered arrays of Fcr1 in the genome. Fluorescent in situ hybridization with Fcr1 probes stained most centromeres in cultured cells. By staining lampbrush chromosomes, we specifically identified the 11 (of 18) chromosomes that stain consistently with Fcr1 probes.

Address correspondence to: E-mail: amurray{at}mcb.harvard.edu




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