An understanding of the mechanisms involved in the generation of antibody diversity to the tremendous variety of antigens and potential pathogens has long been a major goal of immunologists. It is now known that each of the l00 or so VH genes are theoretically able to associate with each of several D and JH genes to generate H-chain diversity. This combinatorial diversity is augmented by junctional diversity, somatic mutation, the insertion of additional nucleotides at junctions (N segments) and possibly gene conversion. The relative contribution of the various diversity generating mechanisms to an immune response is poorly understood, as is the extent of the reserve diversity potential. To address these questions, a unique model system will be used in which a set of VH genes which ordinarily makes only a very minor contribution to the total array of Ig molecules will be forced to generate the entire VH contribution to the immune defense of the organism, thus revealing the full diversity-generating potential of these genes. Lymphoid tissue from a rabbit permitted (through allotype suppression) to express only a few VH genes will be processed to yield both Mu and Gamma VH cDNA libraries form H-chain mRNA. A germline DNA library will also be constructed using nonlymphoid tissues from the same rabbit. Selected cloned cDNAs will be sequenced for comparison to each other and to their progenitor germline genes to assess the relative contributions and possible interrelationships of somatic mutation, gene conversion, D and JH associations with VH, or other diversity-generating mechanisms to diversity. The contribution of germline genes and the diversity-generating mechanisms to the expressin of latent allotypes will also be investigated. Latent allotypes appear to be genetically unexpected VH regions which occur spontaneously at low levels in some rabbits. Latent allotypes can also be reliably induced in all rabbits thus far tested through the injection of anti-allotype antibody. It has been postulated that latent allotypes may play a role in a pre-existing allotype/idiotype regulatory network. The VH region from latent al IgG will be first sequenced at the protein level. This information will be used to construct oligonucleotide probes to be used to identify the corresponding cDNA and germline genes.
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