COLLAGEN GENE EXPRESSION IN THE CORNEA

鸟类角膜原始基质具有独特的结构。它由以下内容组成 由I型和I型胶原纤维组成的垂直排列的层 II由角膜上皮细胞产生的胶原蛋白。初级阶段 基质还含有IX型胶原，至少作为一种暂时性的 成分，我最近的研究表明，这种角膜的形状 独特的细胞外基质分子不同于 在软骨中表达。而软骨IX型分子则含有 位于纤维周围基质中的大球状氨基末端结构域 沿着胶原纤维，这个结构域在IX型角膜中缺失。 胶原蛋白分子。我建议，对这种差异的分析将 提供新颖和令人兴奋的线索来回答一般的问题 细胞用来构建独特的细胞外基质的机制 来自不同组织的一组共同的细胞外基质基因。 作为分析的第一步，DNA克隆和测序技术 将用于表征IX型角膜的结构 胶原蛋白。此外，旋转阴影电子显微镜，Western 印迹和免疫沉淀将被用来表征 角膜IX型胶原蛋白。二、差异化表达 在两种不同的IX型胶原中，角膜和软骨形成， 将通过不同的核糖核酸酶保护方法进行检查 角膜、巩膜、晶状体、视网膜、脊索、神经管等组织 上皮和软骨。第三，精准定位细胞 用原位杂交法表达两种类型的IX型胶原 研究将使用含有角膜和 软骨特异的IX型胶原核苷酸序列。最后， Al(IX)基因将在旨在破译 角膜型别表达的分子机制 九号胶原蛋白。

The avian corneal primary stroma has a unique structure. It consists of orthogonally arranged layers of collagen fibrils, composed of type I and II collagens that are produced by corneal epithelial cells. The primary stroma contains also type IX collagen, at least as a transitory component, and my recent studies show that the corneal form of this unique extracellular matrix molecule is different from that which is expressed in cartilage. Whereas cartilage type IX molecules contain a large globular amino-terminal domain located in the perifibrillar matrix along collagen fibrils, this domain is absent in corneal type IX collagen molecules. I propose that analysis of this difference will provide novel and exciting clues to answer the general question of what mechanisms cells utilize to build unique extracellular matrices in different tissues from a common set of extracellular matrix genes. As a first step in this analysis, DNA cloning and sequencing techniques will be applied to characterize the structure of corneal type IX collagen. In addition, rotary shadowing electron microscopy, western blotting and immunoprecipitation will be used to characterize the corneal type IX collagen protein. Second, the differential expression of the two different type IX collagens, the corneal and cartilage forms, will be examined by means of RNase protection methods in different tissues such as cornea, sclera, lens, retina, notochord, neural tube epithelium and cartilage. Third, to precisely locate the cells expressing the two forms of type IX collagen, in situ hybridization studies will be performed using DNA probes containing cornea and cartilage specific type IX collagen nucleotide sequences. Finally, the al(IX) gene will be examined in experiments aimed at deciphering the molecular mechanisms involved in expression of the corneal form of type IX collagen.