The centromere regulates the movements of the chromosomes in mitosis. Work from this lab, among others, has used antibody probes to show that the centromere is composed of several subdomains in addition to the one prominent subdomain recognized by electron microscopy: the kinetochore. The kinetochore has recently become the focus of wide interest since it may contain the mechanochemical motor responsible for anaphase movement of the sister chromatids to the spindle poles. The ultimate goal of our studies is to construct a structural and functional map of the outer domains of the human centromere. Using human autoantibodies, we identified a family of centromere proteins, CENP-A (17 kDa), CENP-B (80 kDa), and CENP-C (140 kDa) in previous work supported by this grant. We cloned, sequenced, and began an in depth characterization of the molecular structure and biological role of CENP-B. We also obtained preliminary clones of CENP-C. In the experiments proposed for the upcoming grant period we will continue our analysis of CENP-B function, we will carry out a detailed molecular and functional analysis of CENP-C, and we will continue to screen for new components of the centromere and kinetochore. CENP Antigens, General. We will continue to characterize the disruption of chromosome movements in mitosis caused by injection of purified autoantibodies into cultured cells. CENP-B. We will examine the association between CENP-B with alpha-satellite DNA by in vitro binding experiments using cloned proteins and crude chromosomal extracts and by immunocytological investigation of abnormal chromosomes with translocated alpha-satellite domains. We will attempt to determine the role of CENP-B in vivo by creating dominant disruptions of CENP-B function, using transfection and/or injection of cultured cells with plasmid constructs expressing different CENP-B subdomains. Such constructs will also be used to identify those portions of CENP-B required for correct targeting and assembly into the centromere. We will examine the interaction of CENP-B with other chromosomal proteins by chemical crosslinking and by affinity chromatography using various CENP-B subdomains expressed in bacteria. We will characterize the patterns of transcription and posttranslational modification of CENP-B across the cell cycle. CENP-C. We will isolate full-length cDNA clones for CENP-C, obtain the DNA sequence, and use an immunological approach to demonstrate that these clones encode bona fide CENP-C. We will map the distribution of CENP-C in the centromere by immunoelectron microscopy.We will perform analyses similar to those listed above in order to determine the biological role of CENP-C in centromere structure and function. New Centromere Components. We will use a newly designed shotgun cloning method to identify new components of the centromere and kinetochore. Either mitotic chromosome scaffolds or an isolated human minichromosome will be used as antigen. Newly identified antigens will be used to elucidate the network of protein-protein interactions within the centromere. Antigens of suitable interest will be characterized as above for CENPs B and C.
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