This research project proposal is directed at the molecular and cellular mechanisms which control the qualitative and quantitative utilization of variable region genes during B cell development. The studies proposed are based on the premise that constraints in V gene use reflect the evolution of the rearrangement process to ensure the generation of a functional and protective pre-immune repertoire. Ongoing studies on V gene utilization focus on two well-characterized VK genes which are utilized at markedly different frequencies in BALB/c mice and for which there is evidence of rearrangement frequency differences, cellular selection, and intrinsic junctional bias. New studies are directed at identifying alterations of higher order chromatin structure within the Igh-V locus associated with VH gene rearrangement. These studies are based on the hypothesis that V gene rearrangement is tightly regulated by chromatin structure. Nuclease hypersensitivity studies will be used to determine the degree of VH gene exposure (accessibility) in different subregions throughout the Igh-V locus in non-rearranging and actively rearranging pre-B cell lines. Attempts will be made to identify a postulated cis-regulatory region responsible for regulating Igh-V locus rearrangement via VH exon accessibility. Other studies will identify regulatory elements associated with individual VH genes which function during V gene rearrangement. These studies on the molecular mechanisms and cellular mechanisms of antibody repertoire formation will provide insight into a process fundamental to the understanding of immune responsiveness. The available cell lines, molecular probes, and detailed mapping studies of the Igh locus provide a unique opportunity to examine the regulation of a large chromosomal region. These studies also impact directly on questions relating to V gene utilization biases of certain B cell neoplasms and the ability to reconstitute the antibody repertoire in bone marrow transplant patients.
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