GENETIC STRUCTURE AND FUNCTION IN HUMAN CELLS

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The objectives of this proposal are: (1) To map sites on HLA class II molecules which carry out the component functions involved in antigen presentation; (2) to determine the genes and molecular sites on HLA class II molecules involved in presentation of the target autoantigens in HLA class II associated autoimmune diseases; (3) to determine whether different HLA class II alleles are expressed at different levels, and, if so, whether this variation is correlated with susceptibility to autoimmune diseases; (4) to generate new chromosome 6p deletion breakpoints for purposes of gene mapping. These aims will be pursued by (1) isolation of HLA class II mutants affected for presentation of soluble antigens to T cell lines and clones; (2) DNA sequencing of class II mutant genes which code for functionally defective class II molecules; (3) molecular characterization and functional analysis of the mutant class II molecules; and (4) evaluation of the levels of class II mRNAs and glycoproteins in cells with different class II alleles. Mutations will also be introduced into HLA class II genes by site specific mutagenesis, and the mutant genes will be introduced into cells for purposes of mapping molecular sites involved in antigen presentation. Mutational analysis as outlined above will be applied to presentation of the target autoantigens to T cells derived from patients with myasthenia gravis, and, if possible, type I diabetes and multiple sclerosis. It is hoped that these studies will lead to a better understanding of the molecular features of HLA class II molecules involved in presentation of soluble antigens to T cells, and of the differences in class II structure or expression which result in predisposition to autoimmune diseases.