Abstract

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.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM032022-11
Application #
2176415
Study Section
Cellular Biology and Physiology Subcommittee 1 (CBY)
Project Start
1991-09-30
Project End
1996-07-31
Budget Start
1994-08-01
Budget End
1996-07-31
Support Year
11
Fiscal Year
1994
Total Cost
Indirect Cost

  Institution

Name
University of Washington
Department
Biochemistry
Type
Schools of Medicine
DUNS #
135646524
City
Seattle
State
WA
Country
United States
Zip Code
98195

  Publications

Palmer, D K; Angello, J C; Margolis, R L (1997) 2-Aminopurine induces spindle cell morphology in MM14 myoblasts in the absence of differentiation signals. Exp Cell Res 230:262-74
Bosc, C; Cronk, J D; Pirollet, F et al. (1996) Cloning, expression, and properties of the microtubule-stabilizing protein STOP. Proc Natl Acad Sci U S A 93:2125-30
Martineau, S N; Andreassen, P R; Margolis, R L (1995) Delay of HeLa cell cleavage into interphase using dihydrocytochalasin B: retention of a postmitotic spindle and telophase disc correlates with synchronous cleavage recovery. J Cell Biol 131:191-205
Andreassen, P R; Margolis, R L (1994) Microtubule dependency of p34cdc2 inactivation and mitotic exit in mammalian cells. J Cell Biol 127:789-802
Cool, D E; Andreassen, P R; Tonks, N K et al. (1992) Cytokinetic failure and asynchronous nuclear division in BHK cells overexpressing a truncated protein-tyrosine-phosphatase. Proc Natl Acad Sci U S A 89:5422-6
Andreassen, P R; Margolis, R L (1992) 2-Aminopurine overrides multiple cell cycle checkpoints in BHK cells. Proc Natl Acad Sci U S A 89:2272-6
Andreassen, P R; Margolis, R L (1991) Induction of partial mitosis in BHK cells by 2-aminopurine. J Cell Sci 100 ( Pt 2):299-310
Palmer, D K; O'Day, K; Trong, H L et al. (1991) Purification of the centromere-specific protein CENP-A and demonstration that it is a distinctive histone. Proc Natl Acad Sci U S A 88:3734-8
Andreassen, P R; Palmer, D K; Wener, M H et al. (1991) Telophase disc: a new mammalian mitotic organelle that bisects telophase cells with a possible function in cytokinesis. J Cell Sci 99 ( Pt 3):523-34
Garel, J R; Job, D; Margolis, R L (1987) Model of anaphase chromosome movement based on polymer-guided diffusion. Proc Natl Acad Sci U S A 84:3599-603

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