The goals of this research are to clone and characterize the genes encoding rabbit IgA heavy chains and to determine the molecular genetic basis for recombinant IgA molecules whose VH regions are encoded by one chromosome and the CH regions by the homologous chromosome. The rabbit genome appears to contain 10 CAlpha genes and thus far, six have been cloned. The remaining four CAlpha genes will be cloned from DNA of rabbits of defined heavy chain haplotypes and the organization of these genes relative to each other and to the genes encoding CMu, CGamma and CEpsilon will be determined. Each of the 10 CAlpha genes will be subjected to nucleotide sequence analysis and each will be subcloned into expression vectors and transfected into murine plasma cells. The chimeric IgA molecules synthesized by the transfected cells will be examined for their molecular size and their reactivity with anti-allotype antisera. Antibodies specific for each heavy chain isotype will be prepared and the distribution of each of the isotypes in vivo will be determined by immunofluorescence or by RNA blots. In addition to the examination of the 10 CAlpha genes and their products, studies on the recombinant molecules will be continued. Restriction fragment length polymorphisms between two haplotypes have been identified for the JH regions and the CAlpha genes and will be used to determine the chromosomal origin of the V and C genes in the recombinant IgA cells. The recombinant IgA cells will be isolated from gut and sorted by flow cytometry after reaction with fluorescent anti-VH and anti-C antibodies. Cosmid libraries will be prepared from recombinant enriched gut plasma cells and the recombinant VH and CAlpha genes will be isolated and characterized. These studies should provide insight into the mechanism of the homologous recombination.
