DIFFERENTIAL GENE EXPRESSION IN CILIARY EPITHELIUM

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The ocular ciliary epithelium is the site of aqueous humor formation in the mammalian eye. Compiled evidence indicates that along the ocular ciliary epithelium there are anatomical regional differences that underlie biochemical, physiological and molecular differences. The long term objective of this proposal is to characterize at the molecular level the differentiated properties of the nonpigmented (NPE) and pigmented (PE) ciliary epithelial cells in normal and diseased ocular tissue. Information on ciliary epithelial cell-specific genes may elucidate key functions that may be impaired in human diseased ciliary epithelial cells. The specific aims are as follows: A) Preparation of subtractive NPE and PE cDNA libraries from two directional cDNA libraries constructed from poly A+ mRNA of NPE and PE cells respectively in Uni-ZAP XR vectors. using complementary hybridization we can obtain an enrichment of NPE and PE-specific clones. We will immunoscreen bovine expression and/or human expression cDNA libraries constructed in lambda-gtll and Uni-ZAP XR vectors with a group of monoclonal and polyclonal antibodies distinguishing NPE and PE cells antigenically. B) Characterization of NPE and PE-specific DNA sequences by restriction endonuclease mapping and DNA sequence analysis. We have already isolated and partially characterized several bovine cDNA clones from bovine ciliary epithelial cDNA libraries. These bovine cDNA clones, which display cellular and regional specificity, will be used to isolate and characterize the corresponding human full length clones. C) Assessing the level of gene expression for the NPE and PE-specific clones by in situ hybridization in the ciliary epithelium. Identification of cell-specific clones encoding known proteins should allow antibodies to be designed to function as technical tools for the analysis of protein biogenesis, assembly and structure. We will assay the role of retinoids and transforming growth factor-beta (TGF-beta) in maintaining differentiated gene expression using NPE and PE cells.. In particular the expression of the cellular retinaldehyde-binding protein (CRALBP) in these cells could provide information on how retinoids are transported into the aqueous humor. D) Expression of ciliary epithelial genes in pathological eye conditions. We will identify and examine the level of expression of cDNA clones which have altered gene expression in the ocular ciliary epithelium of pathological cases (i.e. glaucoma, cataracts or ocular inflammation).