TRANSCRIPTIONAL CONTROL OF COLLAGEN A1 (IX) IN THE EYE

睫状上皮可能负责产生 玻璃体的大部分胶原蛋白。抑制 睫状上皮分化导致玻璃体缺失 身体结果，整个眼睛的发育被破坏。 该建议将确定组织特异性增强子区域， 胶原蛋白α I（IX）基因的长同种型的启动子， 鸡胚眼的睫状上皮。体内足迹 然后用高分辨率技术检测，连接- 介导的PCR，将提供有关蛋白质结合位点的信息 在纤毛细胞分化过程中被激活的启动子中， 上皮以这种方式鉴定的序列将用于 用于“单杂交”的报道酵母菌株的制备 用于鉴定转录因子的系统。GAL 4-AD融合 文库将由从发育中的细胞中纯化的mRNA制成。 上皮转录因子负责激活 睫状体上皮中的长型胶原α I（IX）基因将被 通过它们与启动子结合的能力在该文库中鉴定 引入报告菌株的序列。这些cDNA将 测序及其在发育中的眼表达模式 通过整体原位杂交定位。

It is likely that ciliary epithelium is responsible for production of the most of the collagenous proteins of the vitreous body. Inhibition of ciliary epithelium differentiation leads to absence of the vitreous body. As a result, the development of the entire eye is disrupted. This proposal will identify the tissue-specific enhancer regions in the promoter of the long isoform of the collagen alphaI (IX) gene in the ciliary epithelium of the embryonic chicken eye. In vivo footprinting followed by detection with the high resolution technique, ligation- mediated PCR, will provide information about protein binding sites in the promoter that are activated during differentiation of the ciliary epithelium. Sequences identified in this manner will be used for preparation of a reporter yeast strain to be used in the "one-hybrid" system for identifying transcription factors. A GAL4-AD fusion library will be made from mRNA purified from developing ciIiary epithelium. The transcription factor(s) responsible for activating the long-form of collagen alphaI (IX) gene in ciliary epithelium will be identified in this library by their ability to bind to the promoter sequences introduced into the reporter strain. These cDNAs will be sequenced and their expression patterns in the developing eye mapped by whole mount in situ hybridization.