TRANSCRIPTIONAL CONTROL OF COLLAGEN A1 (IX) IN THE EYE

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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.