Centromeres are chromosomal elements that are essential to the attachment of chromosomes in the mitotic spindle, and to the coordinated separation of chromatids in anaphase. Several centromere specific proteins have been identified using antibodies derived from patients with the CREST variant of scleroderma. Most research effort has centered on three proteins believed to be centromere specific in higher eucaryotes, designated CENP-A, CENP-B and CENP-C. We have focused our attention on the 17 kD antigen designated CENP-A. We have identified CENP-A as a centromere specific histone, and have extensively characterized this protein. Recently, we have determined that CENP-A is selectively retained in bovine sperm nuclei, while the bulk histones are nearly quantitatively replaced by protamine. This discovery has been of substantial practical significance, as it has allowed us to purify CENP-A to homogeneity. We have subjected the protein to sequence analysis, and with approximately 50% of the sequence determined, we find It has regions of substantial homology with histone H3, and also contains several cleavage peptides that are entirely unique. We Intend to use sequence Information to generate specific antibodies to CENP-A to determine CENP-A localization on chromosomes, and for microinjection assay of CENP-A function in mitosis. Sequence data will also be used to generate oligonucleotide probes for molecular cloning and sequencing of CENP-A cDNA, and to form expression vectors for the purpose of molecular analysis of CENP-A function in mitosis. We will use anti-CENP-A antibodies to immunoaffinity purify chromatin fragments containing CENP-A. We will then clone the CENPA associated DNA for the purpose of identifying specific CENP-A associated DNA sequences. This project will entail the use of immunocytochemistry and electron microscopy, and procedures for protein and DNA purification and characterization, column chromatography, and electrophoresis. The project will also entail isolation and sequence analysis of CENP-A cDNA, and of centromere specific DNA sequences. Proper function of centromeres is important to euploid distribution of chromosomes in mitosis. The aneuploidy which accompanies transformation suggests abnormal centromere function in these cells. Basic information about centromere components is essential to understanding their proper function during mitosis.
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