ELEMENTS REQUIRED FOR MYOSIN VIIA--USH1B TRANSCRIPTION

肌球蛋白VIIA--USH1B转录所需的元素

• 批准号: 2014914
• 负责人: DANA Jo ORTEN
• 金额: $ 4.9万
• 依托单位: FATHER FLANAGAN'S BOYS' HOME
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-05-01 至 1999-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2014914
• 关键词: congenital deafness; ear hair cell; enzyme activity; gene mutation; genetic promoter element; genetic transcription; human genetic material tag; myosins; neurogenesis; nucleic acid sequence; oligonucleotides; retinal pigment epithelium; retinitis pigmentosa; tissue /cell culture; transcription factor; visual photoreceptor

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.

肌球蛋白VIIa是基因USH 1B的产物，基因USH 1B是遗传位点 Usher综合征1B型突变与 综合症包括先天性耳聋和逐渐失明。的 正常蛋白质在前庭系统的毛细胞中表达， 耳蜗，在光感受器、视网膜色素上皮（RPE）细胞中，以及 在肾脏和睾丸中。 在光感受器和毛细胞中， 参与构建和维持它们不同的细胞骨架 专业化。 肌球蛋白VIIa是第一个克隆的基因， 证明了毛细胞的功能需求;了解 它的表达是如何被控制的可以揭示转录控制 其更一般地在毛细胞和/或视网膜细胞中起作用 分化 研究者的目的是定义监管要素， 肌球蛋白VIIa的组织特异性表达。 使用删除分析 在假定的启动子区域中，主要焦点是定义 转录所需的启动子元件。 地区 克隆的基因组DNA包含约4. 5 kb上游和 包括转录起始位点的基因与报道基因连接 编码荧光素酶的基因。 这个结构，以及其中的衍生物， 逐渐增加的部分被删除，将被 瞬时转染到视网膜色素上皮细胞系中， 也表达肌球蛋白VIIa。 荧光素酶活性（光输出） 细胞提取物将被定量。 初步分析表明， 证实了报告基因表达的激活由全长 构建体和衍生物，但不是细胞类型特异性表达; 在肌球蛋白表达的非 表达细胞。 初步研究表明， 睾丸中的转录。 RT-PCR和RNA酶保护的组合 提出了测定来解决这个问题。 所需的DNA序列 进行Usher综合征突变筛查 I型家庭