REGULATION OF FIBRIL ASSOCIATED COLLAGENS IN CORNEA

角膜的透明度取决于较小的均匀直径和 胶原纤维间隔规则。“纤维状”胶原蛋白是 参与测定纤维直径的；“纤维伴生的” 胶原蛋白，一种新的类型，可能是导致纤维间质形成的原因 关系，例如间隔。我们已经分离出了两个假定的 角膜中表达“原纤维伴生的”胶原蛋白。一种是针对XIV类型的 胶原蛋白，纤维相关家族的已知成员；另一种 潜在地编码一种角膜特异性分子，具有多个、小的 胶原域。这些胶原蛋白的基因将被描述为 并定义了它们的外显子/内含子结构。顺式作用元件(DNA 序列)对基因的转录调控很重要 将会被确认。转录起始点的5‘端区域 (包含推定的促进剂/增强剂/消音剂)将被检查，如 以及第一内含子，它也可能参与转录 监管。这些区域将连接到一个报告基因和 将构建体导入皮肤和角膜成纤维细胞。准确地说 确定这些区域内的调控序列并确定 无论序列的功能是启动子、增强子还是抑制子， 部分监管区域的系统性嵌套删除 这些构建物将通过转染法进行制作和评估。这个 角膜特有的潜在顺式元件的存在将通过 活体足迹，一种允许识别和测序的方法 DNA结合蛋白(反式作用因子)所在的基因区域 都被捆绑在一起。这项技术使用聚合酶链式反应来 放大，因此可以对大量的组织进行 可以从胚胎中获得。一旦获得，这些病毒的DNA序列 顺式作用区域将被用于筛选角膜cDNAs的表达 用于分离表达DNA结合蛋白(反式作用蛋白)的cDNA文库 因素)。如果发现了角膜特有的反式作用因子，他们就会 在共转染实验中进行功能性测试。为了这个， 非角膜细胞，如皮肤成纤维细胞会同时 与编码由以下驱动的交易因子的构建体共转染 构成启动子和启动子/增强子区的构造 角膜特异的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，将表明 角膜特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。

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.