IN VITRO SELECTION AND CHARACTERIZATION OF AN MHC VARIANT CARRYING AN ALTERED H-2K(B) GENE PRODUCT

Abstract

The H-2 major histocompatibility complex located on chromosome 17 of the mouse is a complex of genes involved in recognition and reaction in the immune system. A potentially fruitful approach to the study of biological function, genetic properties and structural basis in the H-2 system is to develop somatic cell lines which carry H-2 mutations. To this end we have used an Abelson virus induced lymphoblastoid line (R8) which is heterozygous at the H-2 locus expressing the K and D genes of the "b" and "d" haplotypes (alleles), to construct a functionally haploid (hemizygous) line, expressing exclusively the H-2K('b) haplotype. Antibody and complement selection against the K('b) gene, in mutagenized cells was used in order to isolate a series of H-2 negative variants.;The identification of a structural mutation in the H-2K('b) gene product became our main interest since the availability of the primary amino acid sequence of H-2K('b) enables us to analyze any such mutation in great detail. Variants that expressed no H-2K('b) on their cell surface were isolated and further examined by analyzing the cell lysate since possible H-2 products may be synthesized but not integrated into the membrane and hence will be internally located. One such variant was obtained and a H-2K('b) mutant product was identified and characterized. The molecule obtained is glycosylated, shows the same molecular weight as the parent H-2K('b) and binds B2-microglobulin. Comparative tryptic maps indicated that the H-2K('b) molecule in the mutant contained at least one new peptide absent in the parent, and was thus structurally different from it. These changes in the mutant may be responsible for the inability of this molecule to be recognized on the cell surface.