This proposal seeks to characterize a newly identified gene family designated Ly6 and to characterize the function of those genes specifically involved in lymphoid function. These antigens are involved in the generation of immunocompetent T cells which are very basic functions of lymphoid cells involved in immunoregulation. This project has been successful in that the initial probe has been cloned and used to reveal the complexity of this gene family. The complexity must be initially clarified by an extensive structural characterization of the genes. In addition, we wish to pursue several types of experiments which may give us some greater insight into the biological functions of these molecules. This will include searching for a potential natural ligand for Ly6 molecules, cross-linking surface molecules of different cells types express Ly6 and in situ hybridization studies. The goals of this project are as follows: 1 A characterization of the chromosomal segment encoding this gene family using molecular cloning, pulsed-field Southern blotting and DNA sequencing 2 Identification of the genes expressed in lymphoid cells by transfection into COS cells and analyzing for surface expression with defined monoclonal antibodies 3 Studies of the tissue specific regulatory elements in these genes using reporter (CAT) vectors and transfections into different cell types 4 Localization of the sites of expression of Ly6 sequences to specific cell types by in situ hybridization 5 An examination of the carboxyterminal sequences necessary for the addition of the phosphatidylinositol lipid linkage by site directed mutagenesis and transfection studies 6 Assessing the requirements for T cell activation b generating chimeric forms of Ly6 antigens and studying the possibility of activation of other cell types by cross-linking with antibodies 7 A search for a possible natural ligand using labelled Ly6 molecules or affinity columns 8 An attempt to characterize a human equivalent of the murine Ly6 genes
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