We would like to understand how gene expression is controlled in cells of the nervous system and how that control leads to the formation of neural cell networks which can process information. A simple model of gene expression in nervous tissue will be established using the cloned gene for the mouse thy-1 glycoprotein, which we have recently isolated and characterized. This glycoprotein is the major cell-surface protein in adult brain and is evolutionarily related to the immunoglobulins, transplantation antigens and lymphoid differentiation antigens, molecules which play a crucial role in cellular recognition. We propose to characterize both alleles of the thy-1 gene from the mouse (thy-1.1 and thy-1.2) and construct a series of plasmid expression vectors containing this gene and dominant selectable markers. These genes will then be introduced into cell lines derived from mouse nerve and glial cells by DNA-mediated gene transfer. After transfection and stable integration, the expression of the cloned gene procuct on the cell surface will be assessed using allele-specific monoclonal antibodies. The levels of cell-surface expression will be compared to levels of expression of the endogenous gene in an attempt to identify critical regulators of gene expression. The DNA sequences required for thy-1 gene expression will be localized in modifying the cloned gene in vitro. These critical sequences can be compared with sequences necessary for the expression of this gene in lymphoid cells or in fibroblasts. The relative glycosylation pattern of the cloned gene produce can be compared to that of the endogenous thy-1 gene in lymphoid and neural cells. Finally, to assess possible functions for this gene product in cellular interactions in the nervous system, specific modifications will be made in the thy-1 protein by manipulating the gene in vitro. The reconstitution of gene expression at physiological levels using a cloned gene in an expression vector may allow (1) identification of regulators of other neural-specific genes, (2) identification of gene families which are expressed during neuron differentiation, or (3) development of techniques to allow nerve cell function to be specifically altered. The long-term goals of this project are to understand how gene expression during development controls the interaction of neural cells.
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