The long term goal of this laboratory is to define the molecular sites ln class I major histocompatibility complex (MHC) antigens which interact with the immune system by investigating the structure of class I molecules possessing standard and altered phenotypes. Information concerning class I variants, their evolution and origin will contribute to understanding the generation of the complex genetic polymorphisms which occur at the classical MHC class I loci. The goal of this proposal is to develop methods for rapid sequencing of murine class I MHC variants or alleles and to use these methods to define the structure of several MHC variants. First, locus-specific regions of nucleotide sequence homology have been identified by comparing sequences of known H-2K and H-20(L) loci. regions of homology have also been identified in other known murine class I genes. These locus and class I specific sequences will be used to make a series of synthetic deoxyoligonucleotides for use as direct sequencing primers in mRNA sequencing studies. These common sequences will also be used to design deoxyoligonucleotides for amplification, cloning and sequencing of short segments of class I genes obtained from RNA or DNA templates using the polymerase chain reaction (PCR) methodology. Next, the direct sequencing methods and the PCR methods will be used to deduce the nucleotide sequence of several variants. These Include H-2f spontaneous histogenic mutations, Kfml and Dfm2, and the corresponding standard alleles, Kf and Df. Investigation of these mutations will require sequence analysis of four class I MHC genes. The pH-2III gene of the H-2k haplotype, a potential donor gene for the Kkm1 mutation will also be investigated. Sequence analysis and genetic mapping experiments will identify its structure and location ln the MHC. Finally, genes from a class I deficient cell line, ABM-3, will be sequenced to define the structural basis for altered gene expression in these molecules. The genes of interest are Kkml (translated but not expressed on ABM-3) and Ld, (expressed on ABM-3 but not recognized by anti-Ld CTL's). Structural alterations responsible for variations in expression and function in each gene will be defined. Investigation of class I molecules by methods based on the use of standard synthetic deoxyoligonucleotides will demonstrate the feasibility of rapid methods for analysis of a variety of transplantation antigens using minimal amounts of uncloned genetic material.
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