CLONING GENES INVOLVED IN HEARING

描述：该提案要求支持克隆和表征基因 在分子水平上与听觉有关。 申请人成功地 产生人胎儿耳蜗表达文库， 超过500个独特的EST。 本补助金中提出的工作将 主要集中在两个基因Coch-5 B2和Coch-1D 3，分离自 这个图书馆。 Coch-5 B2的cDNA全序列和基因组结构 将被确定为促进基因的突变筛查， DFN 1A 9的候选人。 北方分析和原位杂交研究， 以及使用多克隆抗体进行免疫组织化学研究， Coch-5 B2蛋白，将用于确定基因的组织分布 在胎儿和成人的生命以及细胞和 基因产物的亚细胞定位。 一种现有的老鼠突变体， 将同源基因组区域中的Asp-1筛选为定位突变。 将进行候选和序列同源性搜索， 定义基因的功能。 Coch-1D 3的同系物antiquitin（ATQ 1） 已经在豌豆植物中发现了， 适当地维持膨压。 北方分析表明，该基因是 在胎儿耳蜗、肾脏、卵巢、眼和心脏中广泛表达。 的 基因定位在5号染色体上，因此可能是耳聋的候选人。 与该染色体对应的基因。 建议的研究包括西 在成人以及胎儿组织中的印迹分析，以及原位和 免疫组织化学研究，以研究位点特异性表达 基因 这项研究的另一个主要目标是染色体 定位500多个耳蜗特异性EST中的5%， 从序列搜索中不能立即看出位置。 这些和 然后，将其他耳蜗特异性EST作为位置候选者进行追踪 综合征性和非综合征性耳聋的地图形式。

DESCRIPTION: 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.