This proposal requests support to clone and characterize genes involved in hearing at the molecular level. The applicant has succeeded in producing a human fetal cochlear expression library which has yielded greater than 500 unique EST's. The work proposed in the present grant will focus largely on two genes Coch-5B2 and Coch-1D3 which were isolated from this library. The complete cDNA sequence and genomic structure of Coch-5B2 will be determined to facilitate mutation screening of the gene as a candidate for DFN1A9. Northern analysis and in situ hybridization studies, as well as immunohistochemical studies using a polyclonal antibody to the Coch-5B2 protein, will be used to define the tissue distribution of gene expression during fetal and adult life as well as the cellular and subcellular localization of the gene product. An existing mouse mutant, Asp-1, in the homologous genomic region will be screened as a positional candidate and sequence homology searches will be pursued to attempt to define the function of the gene. A homolog, antiquitin (ATQ1), for Coch-1D3 has already been discovered in the pea plant which is apparently involved in the proper maintenance of turgor. Northern analysis shows that the gene is extensively expressed in fetal cochlea, kidney, ovary, eye and heart. The gene maps to chromosome 5, and could therefore be a candidate for deafness genes that map to that chromosome. The proposed studies include Western blot analysis in adult as well as fetal tissues, and in-situ and immunohistochemical studies to investigate the site specific expression of the gene. The other major goal of the study will be to chromosomally localize the 5 percent of the greater than 500 cochlear specific EST's whose placement is not immediately apparent from sequence searches. These and other cochlear specific ESTs will then be pursued as positional candidates for mapped forms of syndromic and non-syndromic deafness.
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