Abstract
Much of the nonrandom usage of V, D, and J genes in the Ab repertoire is due to different frequencies with which gene segments undergo V(D)J rearrangement. The recombination signal sequences flanking each segment are seldom identical with consensus sequences, and this natural variation in recombination signal sequence (RSS) accounts for some differences in rearrangement frequencies in vivo. Here, we have sequenced the RSS of 19 individual V(H)7183 genes, revealing that the majority have one of two closely related RSS. One group has a consensus heptamer, and the other has a nonconsensus heptamer. In vitro recombination substrate studies show that the RSS with the nonconsensus heptamer, which include the frequently rearranging 81X, rearrange less well than the RSS with the consensus heptamer. Although 81X differs from the other 7183-I genes at three positions in the spacer, this does not significantly increase its recombination potency in vitro. The rearrangement frequency of all members of the family was determined in microMT mice, and there was no correlation between the in vitro recombination potential and V(H) gene rearrangement frequency in vivo. Furthermore, genes with identical RSS rearrange at different frequencies in vivo. This demonstrates that other factors can override differences in RSS potency in vivo. We have also determined the gene order of all V(H)7183 genes in a bacterial artificial chromosome contig and show that most of the frequently rearranging genes are in the 3' half of the region. This suggests that chromosomal location plays an important role in nonrandom rearrangement of the V(H)7183 genes.
Highlights
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We wanted to determine the sequence of the recombination signal sequence (RSS) of each VH7183 gene to determine the extent of diversity of RSS within this VH gene family and to determine whether the relative frequency of recombination of the individual family members correlated with the sequence of their RSS
Because there were Ͼ13 EcoRI bands on Southern blots that hybridized with the VH7183, and because some known genes were missing initially, we amplified some of the bacterial artificial chromosome (BAC) DNAs with framework 1 (FR1) primers and either AF269 or AM5 in an attempt to choose regions more likely to be conserved
Summary
Institut National de la Santeet de la Recherche Medical, Unite 399, Immunology and Genetics of Parasitic Diseases, Faculty of Medicine, 27 boulevard J. Additional, explanation is that each individual gene controls the frequency of its rearrangement to a large extent by virtue of its relatively unique recombination signal sequence (RSS).. Most natural TCR and Ig RSS vary from the consensus RSS, and this variation could result in differences in rearrangement frequency [17]. In these cases, the chromosomal location would be irrelevant, and could explain the inconsistent correlation of high frequency of rearrangement with proximal chromosomal location for only some, but not other, V and J loci.
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