Part of the vast diversity of the repertoire of immunoglobulin molecules from the combinatorial association of the hundreds of V gene segments, and smaller numbers of D and J gene segments. However, all V,D and J genes are not used equally. Here we will determine how much non-random V, D and J gene usage in the peripheral repertoire is due to differences in the initial frequencies of rearrangement in vivo, and will determine what cis-acting sequences control the non-randomness. The recombination signal sequence, RSS, which flanks each gene segment can significantly affect the frequency of rearrangement. We will use extrachromosomal competition recombination substrates in which two V,D or J segments are in competition for recombination to a single D or J segment to determine if the RSS or adjacent flanking regions are sufficient to alter rearrangement frequencies in vitro. If so, we will determine the exact sequence which is responsible for the unequal rearrangement. A main goal is to determine the relative contribution of the classical (heptamer and nonamer sequence, spacer length variation), semi-classical (nucleotides adjacent to the heptamer) and novel (spacer sequence, more distal flanking regions parameters of actual RSS to the recombination frequencies observed in vivo. We will determine whether the differences in recombination are caused by decreased affinity of binding of RAG-1 and RAG-2 to the RSSs. We will also determine if there is any effect of chromosomal location in non-random gene usage by moving the VH81X gene into a more internal position in the VH locus by gene targeting. We will then determine if the transplanted VH81X gene still rearranges at the same high rate as the endogenous VH81X. To further analyze the control of V(D)J rearrangement, we will determine if germline transcription is required for rearrangement. Sequence-specific polyamides will be used to inhibit germline transcription of VK1 genes in an inducible cell line, and we will determine if the induced VK1 rearrangement is decreased under conditions where the polyamides block induction of transcription of the unrearranged VK1 genes. The general aim is to analyze several parameters that control V(D)J rearrangement frequencies and that could cause unequal gene usage, and to determine the extent to which these contribute to non-random gene segment utilization in vivo. Furthermore, analysis of such factors will tell us more about the V(D)J recombination mechanism itself, since we will determine in each ASE the sequence which is responsible for the preferential rearrangement.
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