The kinetochore is the specialized chromosomal region which forms the attachment point for the microtubules of the mitotic spindle. Little is known about either the composition or mode of action of this structure. This proposal presents a plan for the identification, structural mapping, and functional characterization of kinetochore proteins. These experiments will exploit autoimmune sera from patients with the CREST syndrome of scleroderma. These sera contain antibodies which bind specifically to the centromere region of human chromosomes. The possible significance of these antibodies in the pathogenesis of the CREST syndrome is currently unknown. In immunoblots they react with three minor chromosomal proteins CENP-A (Mr = 17kd), CENP-B (80kd) and CENP-C (140kd), as well as with several other antigens. We have used affinity-purified antibodies to show that the CENP species are found at the centromere. In an effort to obtain biochemical amounts of these extremely minor proteins, we will use the lambda GT11 expression vector system to clone cDNAs encoding the CENP antigens. This will enable us to purify sufficient amounts of CENP: B-galactosidase fusion proteins from bacterial lysogens for injection into rabbits. In this way we will obtain high titer antibodies of known specificity directed against individual CENP species. These antibodies will be used to probe isolated taxol-stabilized mitotic apparatus in experiments designed to show which, if any, of the CENP species interact closely with spindle microtubules. They will also be used in conjunction with an in vitro microtubule assembly assay in order to probe the role of individual CENP species in kinetochore function. The rabbit antibodies will also be used for immunoelectron microscopy to map the distribution of individual CENP proteins in the trilaminar kinetochore structure. In addition, the cDNA clones will be used to determine the pattern of synthesis of the CENP proteins throughout the cell cycle by Northern blotting, the organization of the CENP genes by Southern blotting, and finally, the sequence of the CENP proteins. These data will demonstrate whether the CENP species are the products of a multi-gene family, as suggested by previous immunoblotting experiments. The CENP species are not the only chromosomal proteins recognized by the CREST patient sera. The above experimental design will also be applied to the study of other antigens whose chromosomal location is currently unknown.
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