This proposal focuses on mechanisms of gene regulation in the developing avian retina, with emphasis on genes expressed in the Muller glial cells. The regulatory systems under examination include glial filamin and the glucocorticoid hormone induction of glutamine synthetase. Filamin proteins are actin cross linking agents thought to play an important role in regulating cell shape and motility. We propose to characterize filamin gene structure, through genomic and cDNA cloning, and determine the primary structure of the filamin protein, through DNA sequencing of cDNA clones. Subsequent efforts will focus on the role alterations in chromatin structure play in restricting glial filamin expression to Muller cells during terminal differentiation using nuclease sensitivity assays with cloned hybridization probes. Our long term objectives include examination of regulatory DNA sequences on the filamin gene postulated to be required for Muller cell specific gene expression, by DNA-mediated transfection of Muller cell derived cultured """"""""flat cells"""""""". Using cloned hybridization probes, we will examine the molecular mechanism(s) responsible for eliciting the several hundred fold rise in glutamine synthetase enzyme protein during terminal differentiation of the retina, exclusively in the Muller glial cells. Emphasis will be placed on the role of the glucocorticoid hormone inducer and the mechanism by which disruption of cell-cell contacts in retina organ cultures inhibits glutamine synthetase induction. Through these gene regulatory studies, we hope to enhance our understanding of the mechanisms mediating the Muller cell phenotype during terminal differentiation of the retina.
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