We study genes of the rabbit immune system by techniques of molecular biology coupled with immunological assays. Rabbits of the Alicia strain have a mutation (ali) that segregates with the immunoglobulin heavy chain (Igh ) locus and has a cis effect upon the expression of heavy chain variable region genes (VH) encoding the a2 allotype. We have been breeding these animals at the NIH. Genomic DNAs from homozygous mutant and wild type animals were indistinguishable by Southern analyses using a variety of restriction enzyme digests and Igh probes. However, in studies of DNA fragments from digests with infrequently cutting enzymes separated by transverse alternating field electrophoresis, we found a relatively small deletion of a segment containing 3' VH genes with important regulatory functions, the loss of which leads to the ali phenotype. Our studies of VH gene rearrangement in B-cells suggest that the 3' end of the VH locus probably plays a key role in regulation of VH gene expression in rabbits because VH gene(s) in this region are the targets of preferential VDJ rearrangements. This raises the possibility that alternative mechanisms like gene conversion are at work to generate the antibody repertoire in this species. We are currently studying five recombinants between immunoglobulin heavy chain variable region (VH) and constant region (CH) genes. Two of the VH-CH recombinants were discovered in our laboratory and three at the Basel Institute for Immunology. In order to localize the sites of the recombinations that led to the new haplotypes, we have been analyzing DNAs from the parental and recombinant haplotypes using a set of probes spanning the VH, DH, JH, C(mu) and C(gamma) regions of the Igh locus. All five recombination sites were downstream of the entire VH gene cluster; three appear to have occurred within the region containing DH genes and one downstream of C(mu). There are two copies of the gene for the rabbit kappa light chain constant region, (kappa)1 and (kappa)2. We have now shown that these genes are more than 1 Mb apart and each has associated V(kappa) as well as J(kappa) genes. We have obtained separated and pure populations of B cells expressing lambda or kappa 2 light chains and shown that the (kappa)1 gene has been deleted from many of the B cells expressing lambda light chains.
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