REGULATION OF FIBRIL ASSOCIATED COLLAGENS IN CORNEA

角膜中纤维相关胶原的调节

• 批准号: 2162664
• 负责人: MARION K GORDON
• 金额: $ 18.1万
• 依托单位: TUFTS UNIVERSITY BOSTON
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-05-01 至 1996-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2162664
• 关键词: DNA binding protein; DNA footprinting; RNA splicing; chick embryo; collagen; computer assisted sequence analysis; cornea; fibroblasts; gene expression; genetic library; genetic manipulation; genetic mapping; genetic regulatory element; genetic transcription; in situ hybridization; nucleic acid probes; polymerase chain reaction; reporter genes; tissue /cell culture; transcription factor; transfection

Corneal transparency depends on the small uniform diameter and the regular spacing of collagen fibrils. The "fibrillar" collagens are involved in determining fibril diameter; the "fibril-associated" collagens, a new class, may be responsible for interfibrillar relationships, such as spacings. We have isolated cDNAs for two putative "fibril associated" collagens expressed in cornea. One is for type XIV collagen, a known member of a fibril-associated family; the other potentially encodes a cornea-specific molecule with multiple, small collagenous domains. The genes for these collagens will be characterized and their exon/intron structures defined. Cis-acting elements (DNA sequences) that are important for transcriptional regulation of the genes will be identified. The region 5' to the transcriptional start site (containing putative promoters/enhancers/silencers) will be examined, as well as the first intron, which may .also be involved in transcriptional regulation. These regions will be ligated to a reporter gene and the constructs transfected into skin and corneal fibroblasts. To precisely identify the regulatory sequences within these regions and to determine whether the sequences function as promoters, enhancers, or silencers, systematic nested deletions of portions of the regulatory regions of these constructs will be made and evaluated by transfection. The presence of potential cis-elements unique to cornea will be examined by in vivo footprinting, a method which allows identification and sequencing of regions of genes to which DNA-binding proteins (trans-acting factors) are bound. The technique employs the polymerase chain reaction for amplification, and thus can be performed on the amounts of tissues available from embryos. Once obtained, the DNA sequences of these cis-acting regions will be utilized to screen a corneal cDNA expression library to isolate cDNAs expressing DNA binding proteins (trans-acting factors). If trans-acting factors unique to cornea are found they will be tested for functionality in co-transfection experiments. For this, non-corneal cells, such as skin fibroblasts will be simultaneously co-transfected with a construct encoding the transacting factor driven by a constitutive promoter, and a construct of the promoter/enhancer regions of the cornea-specific collagen gene promoter linked to a reporter gene. If transcription of the reporter gene is observed, it will indicate that a trans=acting factor unique to cornea is able to effect directly the regulation of a cornea-specific gene in a non-corneal cell.

角膜透明度取决于较小的均匀直径和 胶原原纤维的规则间距。 “纤维状”胶原蛋白是 参与确定原纤维直径； “原纤维相关” 胶原蛋白是一类新的胶原蛋白，可能是导致纤维间纤维化的原因 关系，例如间距。 我们已经分离出两个假定的 cDNA 在角膜中表达的“原纤维相关”胶原蛋白。 一种适用于 XIV 型 胶原蛋白，原纤维相关家族的已知成员；另一个 可能编码具有多个小分子的角膜特异性分子 胶原域。 这些胶原蛋白的基因将被表征 及其外显子/内含子结构的定义。 顺式作用元件（DNA 对基因转录调控很重要的序列） 将被识别。 转录起始位点 5' 区域 （包含假定的启动子/增强子/沉默子）将被检查，因为 以及第一个内含子，也可能参与转录 规定。 这些区域将连接到报告基因和 转染到皮肤和角膜成纤维细胞中的构建体。 准确地说 鉴定这些区域内的调控序列并确定 序列是否充当启动子、增强子或沉默子， 部分调控区域的系统性嵌套删除 这些构建体将通过转染来制备和评估。 这 角膜特有的潜在顺式元素的存在将通过以下方式进行检查 体内足迹法，一种允许识别和测序的方法 DNA 结合蛋白（反式作用因子）所结合的基因区域 被绑定。 该技术采用聚合酶链式反应 放大，因此可以对组织的数量进行 可以从胚胎中获得。 一旦获得这些DNA序列 顺式作用区将用于筛选角膜 cDNA 表达 用于分离表达 DNA 结合蛋白（反式作用 因素）。 如果发现角膜特有的反式作用因子，他们将 在共转染实验中测试其功能。 为了这， 非角膜细胞，例如皮肤成纤维细胞将同时 与编码驱动因子的构建体共转染 组成型启动子和启动子/增强子区域的构建体 与报告基因连接的角膜特异性胶原蛋白基因启动子。 如果观察到报告基因的转录，则表明 角膜独有的反式作用因子能够直接影响 非角膜细胞中角膜特异性基因的调节。