In accordance with the Physician Scientist Award (PSA), the candidate, the sponsor, and the Phase I advisory committee, in conjunction with the Department of Pediatrics, have constructed a detailed 2-phase 5-year training program intended to provide the candidate with an intensive research experience. Phase I consists of participation in seven graduate division courses in 4 departments, regular attendance at various seminars, and three laboratory rotations. This experience, modeled after the graduate Ph.D. program in Endocrinology, will fulfill the Phase I requirements of the PSA. Phase Ill is 100% lab research under the direct supervision of the primary sponsor. During Phase II the candidate's research effort will focus on the investigation of Gene X (a tandemly duplicated gene recently discovered in the laboratory. of the sponsor). We propose the following specific aims: (1) Complete the structural characterization of the X gene locus (which lies in the P45Oc2l locus). Thus far only XA has been cloned and sequenced completely. Only a portion of XB has been identified (over 20kb of genomic DNA has been sequenced). The remainder of XB will be cloned through use of a randomly primed DNA library and polymerase chain reaction technology. The full-length cDNAs can then be used to construct bacterial expression vectors and produce large amounts of X proteins for raising antibodies which in turn can be used to establish the location of these proteins in tissues (#2). (2) Characterize the gene X transcriptional regulation through transient transfection of various cell lines with expression vectors containing various portions of the XA and XB regulatory regions fused to a reporter gene and assaying reporter gene activity under specific conditions. (3) Determine the tissue distribution of XA, XB, and tenascin by in situ hybridization and by immunohistochemistry. We postulate that XA and XB encode extracellular matrix proteins, based on their deduced amino acid similarity to the extracellular matrix protein tenascin. We will study the co-localization of the XA, XB, and tenascin mRNAs and proteins in order to; first, determine if they are present in the extracellular matrix and second, study their specific patterns of gene expression. (4) Determine if the X proteins affect cell adhesion. As noted in #3, the structural similarity of XB and tenascin suggests that they share functional similarities. We will utilize an in vitro cell attachment assay to determine if the X proteins affect cell adhesion. Successful completion of this training program will equip the candidate to initiate a successful, independent research career investigating the molecular basis of endocrine function.
