The activities in this proposal for a Mentored Clinical Scientist Development Award (MCSDA) will provide the candidate with a broad knowledge in the basic sciences of connective tissue diseases, and detailed knowledge of and experience in research writing. The basic science portion of the training leading to a PhD degree in oral biology emphasizes biochemistry, molecular biology, cellular biology and genetics. The specific research deals with collagen splicing mutations and the mechanisms of normal and aberrant RNA processing within the nucleus. This training will provide the candidate with a well rounded background in the basic sciences and the health related issues of inherited connective tissue diseases. Fibroblastic cells from individuals with osteogenesis imperfecta (OI) exhibit different mutations in collagen genes that alter the synthesis or structure of type I collagen and result in different OI phenotypes. The work detailed in this proposal is designed to identify COL1A1 mutations that result in OI phenotype because they lead to unstable mRNA products, and to determine the mechanisms by which RNA processing within the nucleus of the cell is altered. The hypothesis to be tested in the proposed studies is that specific splicing mutations affect steps in mRNA maturation, release from splicing complexes, and targeting for degradation. The first step in this project is to use cells from individuals with OI type I and to identify candidates for splicing mutations. Large overlapping segments of COL1A1 cDNA (1-1.5 kb in length) will be amplified and screened by electrophoresis in polyacrylamide gels to detect heteroduplexes, indicative of sister differences between the products of two alleles. The second step is to identify the specific mutation at the splice site, the product that results from the single mutation. The third step is to determine the fate of the abnormal mRNA species by pulse-chase and cell fractionation studies. Then the role of these protein complexes in mRNA fate will be determined by correlating mRNA nuclear protein location and amount in the nucleus and in the cytoplasm using cDNA labeled markers and immunofluorescence staining. mRNA processing will be identified and characterized. The applicant's career development plan includes a very structured and comprehensive curriculum covering oral biology and molecular biology. Classroom learning will be complemented and supplemented by the skills gained in the laboratory. The candidate will also be an active participant in an interdisciplinary seminar program.
