CLONING OF THE CDNA ENCODING A CENTROSOME AUTOANTIGEN

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DESCRIPTION (adapted from applicant's abstract): The centrosome, which is composed of a centriole pair and an amphorous cloud of pericentriolar material, is the principal microtubule organizing center in mammalian cells. It has been demonstrated that it is the pericentriolar material, and not the centrioles that contains the microtubule nucleating capacity within the centrosome complex. How the pericentriolar material is able to interact with tubulin and microtubules is unknown, due in part to the fact that the biochemical composition of the pericentriolar region remains largely unknown. In preliminary experiments, a human fetal liver cDNA expression library has been screened using a human autoimmune antiserum that recognized proteins within the pericentriolar region, and two clones have been identified that contained cDNAs encoding a portion of a 185/299 kd centrosome protein. Experiments are detailed that will allow for the structural and functional characterization of this high molecular weight centrosome protein. This will be achieved by cloning and sequencing the full-length cDNA encoding the centrosome protein and then using the cloned cDNA to produce a bacterial expression system. The expressed centrosome protein fragments then will be used to investigate centrosome-microtubule interactions using in vitro microtubule assays, to identify the major autoantigenic epitopes, and to generate monospecific anticentrosome antibodies. The monospecific antibodies, as well as the autoimmune anticentrosome antiserum, then will be microinjected into cultured cells to identify the in vivo function of the centrosome protein. Specific techniques to be used include molecular assembly assays, cell culture, microinjection, SDS-PAGE immunoblotting, and various forms of microscopy. Cell lines to be used include HeLa, CHO, and PtK2. Rabbits will be used for antibody production.