TRANSCRIPTIONAL CONTROL OF COLLAGEN A1 (IX) IN THE EYE

睫状体上皮很可能负责产生 玻璃体的大部分胶原蛋白。抑制 睫状体上皮细胞分化导致玻璃体缺失 尸体。结果，整个眼睛的发育被打乱了。 该提案将确定组织中特定的增强子区域 胶原蛋白αI(IX)基因长异构体启动子的研究 鸡眼胚胎的纤毛上皮。活体足迹 然后用高分辨率技术进行检测，结扎- 介导式聚合酶链式反应，将提供有关蛋白质结合位点的信息 在纤毛分化过程中被激活的启动子 上皮组织。以这种方式识别的序列将用于 一株用于“一杂”的报告酵母菌株的研制 用于识别转录因子的系统。GAL4-AD融合 文库将由从发育中的细胞中提纯的mRNA制成 上皮组织。负责激活该基因的转录因子(S) 睫状体上皮细胞中长型胶原αI(IX)基因 通过它们与启动子结合的能力在这个文库中进行鉴定 将序列导入报告菌株。这些cDNA将是 序列测定及其在发育眼中的表达模式 通过整装原位杂交定位。

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.