MHC Class I antigens are polymorphic membrane glycoproteins involved in tissue rejection. This project aims to analyze structure-function relationships of these antigens. Employing site-directed mutagenesis, we have studied the polymorphic sites involved in the immune functions of these antigens. Four step mutagenesis has been undertaken to introduce five amino acid substitutions in position 63-73 of the mouse H-2Ld antigen. These positions are replaced by the H-2Dd type amino acid in the mutant antigens. Our rational for this mutagenesis is based on our initial efforts to predict immunologically functional sites of the MHC Class I antigens. The mutagenesis to replace four amino acids is complete, and the mutated genes have been introduced into L-cells. We plan to characterize functions of the mutated H-2Ld gene products by testing their reactivities with more than 50 monoclonal antibodies. Further, we plan to test reactivities of T-cells specific for using H-2 restricted cytotoxic T-cells (CTL), and alloreactive CTLs. In a collaborative study, we have sequenced the H-2DP gene located from B10 mice. The H-2P haplotype is remote in its origin from other laboratory strains. We found the H-2DP gene is very homologous to known MHC Class I genes, the highest homology being found with the H-2Ld and H-2Db genes, rather than other D regin genes. Unlike most other structural genes, greater numbers of nucleotide substitutions are found in exons than in introns suggesting evolutionary force to increase the polymorphism. Chimeric Class I genes were constructed which have the first and the second domain sequences derived from two different Dp or Dd. Characterization of transformants is underway to study contributions of conformational structures generated by both domains to antigenicity and to functions of Class I antigens.
