The goals of this project have not changed considerably but have become broader in scope. In addition, with the advent of molecular cloning techniques, we have decided to develop this expertise and use it as an additional experimental approach to study the structure and function of Ia alloantigens. We prepared a polyclonal antibody to DS, RbO3, by immunizing rabbits with DS molecules purified from the marmoset cell line. This antibody was: (1) specific for human DS molecules; (2) did not crossreact with DR; and (3) reacted with DS molecules from all DR cell types. We have used this antibody to perform several studies which have allowed us to draw a number of conclusions about the biochemical properties of DS molecules: (1) DR and DS molecules have very similar two-dimensional gel profiles and can be distinguished in general only when analyzed as mixtures; (2) DS molecules isolated from cell lines of the same DR type are identical, suggesting strong linkage disequilibrium between DR and DS. This is analogous to the murine system, where I-A and I-E genes are tightly linked; (3) at least four DS allotypes can be distinguished by two-dimensional gel analysis. Combined with the analyses of supertypic specificities, our studies suggest that DS is as polymorphic as DR; and (4) both subunits, alpha and beta, of DS molecules are polymorphic; this is similar to observations in the murine system where both A alpha and A beta subunits have been shown to be polymorphic. We have isolated a complete cDNA clone corresponding to the DR alpha subunit. This clone has been characterized and completely sequenced. We have now applied the same method for screening cDNA clones (hybridization to a synthetic oligonucleotide) successfully to isolate a cDNA clone corresponding to the rat Thy-1 antigen. During the coming year, we hope to apply our newly learned expertise to some exciting problems. We intend to isolate the genes encoding the Ia antigens from H-2?q? mice, a strain of mouse that is highly susceptible to certain autoimmune diseases. We propose to screen a cosmid library using probes which have been provided by several other investigators. Once isolated, we will analyze and compare their structure to that of other Ia molecules. Ultimately, we plan to insert these genes into nonsusceptible mice to examine their effects on the immune system. (CS)
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