ELEMENTS REQUIRED FOR MYOSIN VIIA--USH1B TRANSCRIPTION

肌球蛋白VIIa是USH1B基因的产物，USH1B是 亚瑟综合征1B型基因突变。与关联的功能 综合征包括先天性耳聋和渐进性失明。这个 正常蛋白在前庭系统的毛细胞中表达， 在光感受器、视网膜色素上皮(RPE)细胞中，以及 在肾脏和睾丸。在感光器和毛细胞中，它可能是 参与构建和维护它们不同的细胞骨架 专业化认证。肌球蛋白VIIa是第一个克隆的具有 证明毛细胞的功能要求；理解 它的表达如何被控制可能揭示转录控制 更普遍地在毛细胞和/或视网膜细胞中工作 差异化。 调查员的目标是定义规定以下内容的监管要素 肌球蛋白VIIa的组织特异性表达。使用删除分析 在推测的启动子区域中，主要的焦点是定义 转录所需的启动子元件。地区 克隆的基因组DNA包含约4.5kb的上游和 包括转录起始点在内的一名记者将加入 编码荧光素酶的基因。这种结构，以及其中的衍生品 越来越大的片段已被删除，将被删除 瞬时转染人视网膜色素上皮细胞系 也表达肌球蛋白VIIa。荧光素酶活性(光输出) 细胞提取物是要定量的。初步化验结果显示 通过全长激活报告基因的表达 构造和派生，但不是特定于细胞类型的表达； 在表达肌球蛋白的非肌球蛋白中观察到同样的相对激活 表达细胞。初步研究表明，有多个起点 睾丸中的转录。RT-PCR与核糖核酸酶保护的结合 为了解决这一问题，人们提出了检测方法。需要DNA序列 将对Usher综合征的突变进行筛查 I类族。

Myosin VIIa is the product of the gene USH1B, the locus of genetic mutations in Usher syndrome type 1B. Features associated with the syndrome include congenital deafness and gradual blindness. The normal protein is expressed in hair cells of the vestibular system and cochlea, in photoreceptors, retinal pigment epithelium (RPE) cells, and in kidney and testis. In photoreceptors and hair cells, it may be involved in constructing and maintaining their different cytoskeletal specializations. Myosin VIIa is the first cloned gene with a demonstrated functional requirement in hair cells; an understanding of how its expression is controlled could reveal transcriptional controls that operate more generally in hair cell and/or retinal ell differentiation. The investigator aims to define the regulatory elements that specify the tissue-specific expression of myosin VIIa. Using deletion analysis of the putative promoter region, the primary focus is to define promoter elements that are required for transcription. Regions of cloned genomic DNA encompassing some 4. 5kb upstream of and including the transcriptional start site are to be joined to a reporter gene encoding luciferase. This construct, and derivatives in which progressively larger segments have been deleted, are to be transiently transfected into a retinal pigment epithelium cell line that also expresses myosin VIIa. The luciferase activity (light output) in cell extracts is to be quantitated. Preliminary assays have demonstrated activation of reporter gene expression by the full-length construct and derivatives, but not cell type-specific expression; the same relative activation was observed in myosin-expressing an non- expressing cells. Initial studies suggest multiple start sites for transcription in testis. A combination of RT-PCR and RNAse protection assays are proposed to resolve this issue. DNA sequences required for transcription are to be screened for mutations in Usher syndrome type I families.