The proposed research is part of a comprehensive research effort to molecularly define genes of the mouse, which cause oculocutaneous albinism (OCA), and in particular, the multigenic subform of OCA termed Hermansky-Pudlak Syndrome (HPS). HPS is a genetically heterogeneous, recessively inherited disease, which causes visual defects, hemorrhaging and significantly shortened lifespan due to fibrotic lung disease. A long-term goal is to devise diagnostic and therapeutic strategies for HPS, which presently has no efficacious treatment. The second long-term goal is to understand the mechanism of action of genes responsible for the normal biosynthesis of specialized mammalian subcellular organelles such as melanosomes, platelet dense granules and lysosomes. Two mouse HPS mutants, ruby eye and ruby eye-2, are of special interest, not only because they accurately model HPS, but also because they are phenotypic mimic mutants. Their molecular characterizations are therefore expected to lead to identification of a common pathway and perhaps a common protein complex involved in the disease.
The specific aims of this proposal are to: 1) complete the identification and partial characterization of the mouse ruby eye (ru) HPS gene; 2) identify and partially characterize the closely related ruby eye-2 (ru2) HPS gene; 3) isolate human homologues of the cloned ru and ru2 HPS genes and test for alterations of these genes in human kindreds; and 4) identify interactions between the products of the ru and ru2 genes. A multidisciplinary positional/candidate cloning approach will be used to accomplish these aims including construction of high-resolution genetic maps through the use of large interspecific mouse backcrosses and construction of high-resolution physical maps through identification of overlapping contigs of RPCI-23 bacterial artificial chromosomes (BACs). Critical BACs containing the gene of interest will be identified through insertion into recipient transgenic mutant mice. Transcripts within critical BACs will be identified by a combination of exon trapping, cDNA selection and complete BAC sequencing. Transcripts containing the ruby eye and ruby eye-2 mutations will be identified by qualitative and quantitative approaches including complete cDNA sequencing and ribonuclease protection assays, respectively. To identify patients with mutations in the corresponding human genes, the cDNA sequence of the human homologue of each gene and its expression level will be determined in normal individuals and in HPS patients with no mutations in known HPS genes. Each gene will be partially characterized by assays of transcript and protein tissue distribution together with subcellular localization measurements by immunofluorescence techniques. Interaction of the ruby eye and ruby eye-2 genes will be measured by a variety of phenotypic assays in mice bred to be doubly mutant for each gene together with direct tests for interaction of their protein products by co-immunoprecipitation techniques and yeast two-hybrid approaches.

National Institute of Health (NIH)
National Eye Institute (NEI)
Research Project (R01)
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Visual Sciences C Study Section (VISC)
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Chin, Hemin R
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Roswell Park Cancer Institute Corp
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