The goal of this project is to determine the molecular basis for two unusual features of the rabbit humoral immune system: 1) that most B cells rearrange the same VH gene, VH1, in their VDJ gene rearrangements, even though the germline contains many functional VH genes that could rearrange, and 2) that the VDJ heavy chain genes undergo somatic diversification by gene conversion. These studies are important both for understanding basis mechanisms of the immune system and for identifying regulatory elements in the germline that control expression and development of the antibody repertoire.
The specific aims are to 1) determine the molecular basis for preferential rearrangement of VH1 by searching for cis acting regulatory elements such as a rearrangement enhancer and matrix associated regions (MAR), 2) develop an in vitro model for somatic gene conversion by transfecting a gene conversion substrate into cell lines and identifying one that supports gene conversion, 3) develop an in vivo model for somatic gene conversion using transgenic rabbits with a VDJ transgene linked to several upstream VH genes that can be used as donor genes, and 4) identify molecules involved in somatic gene conversion by cloning and characterizing the rabbit homologues of yeast RAD52 epistasis genes, genes known to be involved in gene conversion. These studies should help elucidate the mechanism by which cells choose VH genes to rearrange during B cell development and should begin to identify the cis-acting elements in the IgH region that regulate somatic diversification of the primary antibody repertoire.
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