We studied genes of the rabbit immune system by techniques of molecular biology and immunology. PCR allowed us to specifically identify excision circles resulting from DH to JH and Dbeta to Jbeta DNA rearrangements. Extrachromosomal circular DNA purified from rabbit bone marrow cells was assayed by PCR to determine the relative in vivo rearrangement frequencies of Ig DH to JH genes. DH genes rearranged to individual JH genes with different frequencies. This bias did not correlate with potential sequence overlaps in the DH or JH coding sequences. The JH2 and JH4 genes were the preferred targets of recombination in primary rearrangements. Analyses of genomic VDJH indicated that B cells expressing VDJH4 heavy chains survived and dominated in the bone marrow due to post-rearrangement selection. We obtained new data that support the proposal made many years ago that the rabbit appendix could be a bursal equivalent. The DNA sequences of rearranged heavy chain variable region genes from B cells in the light and dark zones of appendix germinal centers from 6-week-old rabbits were highly diversified in CDR2, probably through both gene conversion and somatic hypermutation; some LZ sequences were closer to germline. We derived simple evolutionary trees that showed the relationships of cells with different sequences to each other and to single progenitors. Potential donors that could have participated in gene conversion-like alterations of rearranged genes were identified. We prepared a cosmid library containing 35-45 kb fragments of rabbit genomic DNA in order to further characterize VH gene expression and diversification in the rabbit. We found and characterized an upstream VH gene that may act as a donor for gene conversion-like alterations of rearranged VH-gene sequences that lead to production of VH structures resembling those encoded by a deleted VH1 gene in mutant ali/ali rabbits.We also found and are characterizing germline DH and JH genes. We produced anti-RAG-2 antibodies and used them in studies of developing rabbit thymus and appendix tissues.
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