The goal of this project is to examine V(H) gene usage in rabbit VDJ gene rearrangements. These studies are important for understanding basic mechanisms of the immune system of mammals. Previous studies showed that rabbit B cells preferentially utilize the 3'-most germline VH gene, VH1 in their VDJ gene rearrangements.
The specific aims of this proposal are to 1) determine if the preferential usage of VH1 reflects preferential rearrangement of this germline gene or antigen selection of B cells that have utilized VH1, 2) search for a """"""""recombination enhancer"""""""" flanking VH1, 3) determine V(H) gene usage in various B cell populations including B cells that produce antibodies of defined specificity, V(H)a- B cells of mutant Alicia rabbits, V(H)a- cells of suppressed rabbits, and CD5+ B cells, 4) develop rabbit B cell lines, 5) determine the order of V(H), D(H), and J(H) gene rearrange- ments, and 6) construct a linear map of the V(H) chromosomal region. The methods used to achieve these goals include construction of recombinant phage libraries from various B cell populations, and the cloning and characterization of VDJ genes from these libraries. Vectors will be developed for use in determining if particular germline V(H) genes are preferentially rearranged and in identifying cis acting elements responsible for the rearrangement. Experiments using rabbit X mouse hybridomas and rabbit B cell lines are described, the goal being to test the hypothesis that the order of VDJ gene rearrangement in rabbit is V to D followed by VD to J, unlike other mammals that rearrange D to J, followed by V to DJ.
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