This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The immunoglobulin heavy chain (IgH) locus undergoes large-scale contraction in B cells poised to undergo IgH V(D)J recombination. We considered the possibility that looping of distinct IgH V regions plays a role in promoting long-range interactions. To address this issue we have simultaneously visualized three subregions of the IgH locus, using 3D-fluorescence in situ hybridization. We demonstrated looping of IgH V-regions in developing B cells with relative high frequency as compared to hematopoietic progenitor or CD8 T-lineage cells. Looping of a subset of IgH V-regions was also observed in RAG-deficient pro-B cells. These data indicated that in developing B cells, IgH V regions loop in proximity of the IgH D/J cluster prior to the onset of Ig gene recombination. Based on these observations, we have proposed that Ig loci are repositioned by a looping mechanism prior to rearrangement to facilitate the joining of Ig variable, joining and diversity segments. We propose to examine the structure of the immunoglobulin gene in T and B lineage cells using electron microscopy. Briefly, BAC probes will be labeled using nick-translation with biotinylated nucleotides. Fixed cells will be permeabilized, hybridized with the BAC probes and incubated with streptavidin beads conjugates with quantum particles. The structure of the various segments, Variable, diversity, joining and enhancer regions will then be analyzed in B cells undergoing gene rearrangement. Additinonally, T cell will be analyzed as control cells that do not undergo gene rearrangement.
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