Unequal VH Gene Rearrangement Frequency Within the Large VH7183 Gene Family Is Not Due to Recombination Signal Sequence Variation, and Mapping of the Genes Shows a Bias of Rearrangement Based on Chromosomal Location

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 VH7183 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 μMT mice, and there was no correlation between the in vitro recombination potential and VH 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 VH7183 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 VH7183 genes.

Development of the Expressed Ig CDR-H3 Repertoire Is Marked by Focusing of Constraints in Length, Amino Acid Use, and Charge That Are First Established in Early B Cell Progenitors

“…In accordance with previous studies by other investigators (23,25,26,29), the prevalence of V H 7183.1 (V H 81X) declined with development (B3 F; p Ͻ 0.001). V H 81X represented 38% of the FIGURE 1.…”

“…PCR conditions were as follows: hot start at 95°C for 3 min; 35 cycles of 1 min at 94°C, 1 min at 60°C, and 1 min at 72°C; and a final extension at 72°C for 10 min. The primers were a FR1 V H 7183-specific upstream primer, AF303 (5Ј-GGGGCTCGAG GAGTCTGGGGGA-3Ј) (23), and the C exon 1 primer C 2 (5Ј-CAG GATCCGAGGGGGAAGACATTTGG-3Ј). PCR products were subcloned (TOPO-TA Cloning kit; Invitrogen Life Technologies), primed with the AF303 primer, and sequenced at the University of Alabama at Birmingham Heflin Center Sequencing Core.…”

“…We chose to look at RNA message, as this is most representative of the expressed, and thus functional, Ig repertoire. We focused on the V H 7183 family because its germline complement in IgH a alleles has been well defined (23); it represents a manageable 10% of the active repertoire (24); patterns of V H 7183 use during ontogeny and development have been well established (23,25,26); and it contributes to both self and nonself reactivities (reviewed in Ref. 27).…”

Unifying model for molecular determinants of the preselection Vβ repertoire

“…In contrast to its dominant role as a determinant for recombinational competence, RIC scores for the 23 active Vβ gene segments correlate poorly with their relative levels of rearrangement. A similar discordance between recombination frequencies and RSS qualities for a limited set of mouse VH and Vк gene segments has been described previously (54,55). These findings suggest that chromatin environment, rather than predicted RSS quality, is the dominant feature for fine-tuning Vβ use in long-range recombination.…”

Chromatin accessibility and epigenetic modifications differ between frequently and infrequently rearranging VH genes

“…For instance, the three functional members of the V H S107 family have very different rearrangement frequencies in vivo, with V1 rearranging five times more often than V11, and forty times more often than V13 (Love et al, 2000). Furthermore, all twenty V H 7183 family members rearrange at very different frequencies in vivo (Williams et al, 2001), with V H 81X rearranging considerably more often. By using freshly isolated pro-B cells, we provided the first in vivo demonstration that the extent of acetylation of histone proteins is one of the mechanisms by which rearrangement frequency of individual V H genes may be regulated (Espinoza and Feeney, 2005).…”