REGULATION OF FIBRIL-ASSOCIATED COLLAGENS IN CORNEA

角膜透明度取决于小的均匀直径和 胶原纤维的规则间隔。 “纤维状”胶原蛋白是 参与决定原纤维直径;“原纤维相关” 胶原蛋白，一种新的类别，可能是负责纤维间 关系，如间距。 我们分离了两个假定的 角膜中表达的“原纤维相关”胶原蛋白。 一种是XIV型 胶原蛋白，纤维相关家族的已知成员;其他 可能编码角膜特异性分子， 胶原结构域。 这些胶原蛋白的基因将被表征 并定义了它们的外显子/内含子结构。 顺式作用元件（DNA 序列），这些序列对于基因的转录调节是重要的 将被识别。 转录起始位点的5'区 （含有推定的启动子/增强子/沉默子）将被检查， 以及第一个内含子，其也可能参与转录调控。 调控 这些区域将连接到报告基因，并且将这些区域连接到报告基因。 转染到皮肤和角膜成纤维细胞中的构建体。 精确 鉴定这些区域内的调控序列，并确定 无论序列起启动子、增强子或沉默子的作用， 系统性巢式缺失的部分调控区， 制备这些构建体并通过转染进行评价。 的 角膜特有的潜在顺式元件的存在将通过 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） 都被束缚住了 该技术采用聚合酶链反应， 扩增，并且因此可以对组织的量进行扩增。 可从胚胎中获得。 一旦获得，这些DNA序列 将利用顺式作用区筛选角膜cDNA表达 文库以分离表达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， 在共转染实验中测试功能。 为此， 非角膜细胞，如皮肤成纤维细胞，将同时 与编码由以下驱动的反式作用因子的构建体共转染 组成型启动子和启动子/增强子区的构建体 角膜特异性胶原基因启动子连接到一个报告基因。 如果观察到报告基因的转录，则表明 角膜特有的反式作用因子能够直接影响 在非角膜细胞中的角膜特异性基因的调节。

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.