Sequence of the Spacer in the Recombination Signal Sequence Affects V(D)J Rearrangement Frequency and Correlates with Nonrandom Vκ Usage In Vivo

Nadel, Bertrand; Tang, Alan; Escuro, Guia; Lugo-Villarino, Geanncarlo; Feeney, Ann J.

doi:10.1084/jem.187.9.1495

Cited by 77 publications

(81 citation statements)

References 48 publications

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“…2 shows that a control competition substrate containing V1 in both internal and external positions demonstrates the minimal effect of position (internal vs external) of the two competing RSS fragments in the substrates. This is consistent with the controls we have previously published using this competition recombination system (18,21). When we cloned a V H J558 RSS fragment in the competition substrate, it was vastly disfavored, with 91% of the rearrangements occurring to V1 instead of J558 (Fig.…”

Section: Analysis Of the Role Of The Rss In The Nonrandom V H Gene Usesupporting

confidence: 82%

“…It is possible that its variant spacer is the reason for its high frequency of recombination. Although the heptamer and nonamer are traditionally believed to be the most important regions of the RSS, we have previously shown that the spacer can also play a significant role in recombination frequency of V genes (18). In that study we also showed the surprising result that a RSS with a nonconsensus nonamer but an optimal spacer rearranges more frequently than a consensus RSS with a poorer spacer, thus underscoring the necessity to actually test the recombination potential of each RSS experimentally.…”

Section: Analysis Of the Role Of The Rss In The Nonrandom V H Gene Usesupporting

confidence: 49%

“…Most of the rest had a small number of changes from one of these two prevalent RSS. 81X, which rearranges the most of all genes in this family, had a unique spacer, and variations in the spacer sequence can affect recombination frequency (18,33). Thus, the hypothesis that the unique RSS of 81X was responsible for its high frequency of rearrangement was a reasonable hypothesis until we performed recombination substrate experiments (Fig.…”

Section: Discussionmentioning

confidence: 99%

“…Furthermore, Navajos, who have the allele with the nonconsensus heptamer, have a greatly increased susceptibility to H. influenzae type b disease. Thus, variations in RSS leading to relatively modest differences in rearrangement frequencies can alter the composition of the Ab repertoire and can have severe biological ramifications (18).…”

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confidence: 99%

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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

Williams

Martinez

Montalbano

et al. 2001

The Journal of Immunology

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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.

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Section: Analysis Of the Role Of The Rss In The Nonrandom V H Gene Usesupporting

confidence: 82%

Section: Analysis Of the Role Of The Rss In The Nonrandom V H Gene Usesupporting

confidence: 49%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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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

Williams

Martinez

Montalbano

et al. 2001

The Journal of Immunology

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“…In these cases, the location of the cRSS has generally been based on the position of a CAC motif (the first three residues of the consensus heptamer) near-est the recombination breakpoint that contains the highest number of additional residues that match the consensus heptamer and nonamer. However, since even authentic RSSs exhibit some degree of sequence variability in the heptamer and nonamer, and since mutations in the heptamer, nonamer, and spacer motifs have position-dependent and possibly synergistic effects on recombination efficiency (11), determining whether a given cRSS can support V(D)J recombination is problematic in the absence of functional testing. Therefore, several laboratories have applied a well established extrachromosomal V(D)J recombination assay to assess the functionality of various cRSSs in cell culture (12)(13)(14), including those suspected of mediating chromosomal translocations involving LMO2 (t(11;14)(p13; q11)) (15,16), TAL1 (t(1;14)(p34;q11)) (17), Ttg-1 (t(11; 14)(p15;q11) (18,19), and Hox11 (t(10;14)(q24;q11) (20,21) as well as interstitial deletions involving SIL/SCL (1p32) (22,23) and MTAP/p14 -16 (9p21) (13,24).…”

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confidence: 99%

V(D)J Recombinase Binding and Cleavage of Cryptic Recombination Signal Sequences Identified from Lymphoid Malignancies

Zhang

Swanson

2008

Journal of Biological Chemistry

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To understand the molecular basis for these large differences, we investigated the binding and cleavage of these cRSSs by the RAG1/2 proteins that initiate V(D)J recombination. We find that the RAG proteins comparably bind all cRSSs tested, albeit more poorly than a consensus RSS. We show that four cRSSs that support levels of V(D)J recombination above background levels in cell culture (LMO2, TAL1, Ttg-1, and SIL) are also cleaved by the RAG proteins in vitro with efficiencies ranging from 18 to 70% of a consensus RSS. Cleavage of LMO2 and Ttg-1 by the RAG proteins can also be detected in cell culture using ligation-mediated PCR. In contrast, Hox11 and SCL are nicked but not cleaved efficiently in vitro, and cleavage at other adventitious sites in plasmid substrates may also limit the ability to detect recombination activity at these cRSSs in cell culture.The antigen binding domains of immunoglobulins and T cell receptors are encoded in germ line arrays of V, D, and J gene segments that are assembled into functional variable exons by V(D)J recombination during lymphocyte development (1). The site of recombination is directed by a recombination signal sequence (RSS) 2 that flanks each receptor gene segment and consists of a conserved heptamer (consensus 5Ј-CACAGTG-3Ј) and nonamer (consensus 5Ј-ACAAAAACC-3Ј) separated by either 12 or 23 Ϯ 1 nucleotides of more highly varied sequence. V(D)J recombination can be conceptually divided into two phases, a cleavage phase and a joining phase (2). In the cleavage phase, the lymphoid cell-specific RAG1 and RAG2 (recombination activating gene-1 and -2, respectively) proteins assemble a multiprotein synaptic complex with two RSSs (generally one 12-RSS and one 23-RSS) and introduce a DNA double strand break at each RSS between the heptamer and adjacent coding segment via a nick-hairpin mechanism to yield a blunt 5Ј-phosphorylated signal end and a coding end terminating in a covalently sealed DNA hairpin structure. In the joining phase, the signal ends are generally joined precisely to form signal joints, and the coding ends are processed and joined to form coding joints that typically contain nucleotide additions or deletions at the junction. These processes are normally mediated by components of the nonhomologous end-joining DNA repair pathway.

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The variable genes and gene families of the mouse immunoglobulin κ locus

et al. 1999

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In this report 118 mouse V O genes are described which, together with the 22 V O genes reported previously (T. Kirschbaum et al., Eur. J. Immunol. 1998. 28: 1458-1466 amount to 140 genes that had been cloned and sequenced in our laboratory. For 73 of them cDNAs are known, i. e. they have to be considered functional genes, although 10 genes of this group have 1-bp deviations from the canonical promoter, splice site or heptanucleotide recombination signal sequences. Twenty V O genes have been defined as only potentially functional since they do not contain any defect, but no cDNAs have been found (yet) for them. Of the 140 V O genes 47 are pseudogenes. There are indications that two to five V O genes or pseudogenes exist in the O locus which we have not yet been able to clone. The 140 V O genes and pseudogenes were assigned to 18 gene families, 4 of them being one-member families. This differs from previous enumerations of the families only by the combination of the V O 9 and V O 10 families and by the addition of the V O dv gene as a new separate family. Sequence identity usually was 80 % or above within the gene families and 55-80 % between genes of different families. Many of the mouse V O gene families show significant homologies to the human ones, indicating that in evolution V O gene diversification predated the divergence of the primate and rodent clades.

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Sequence of the Spacer in the Recombination Signal Sequence Affects V(D)J Rearrangement Frequency and Correlates with Nonrandom Vκ Usage In Vivo

Cited by 77 publications

References 48 publications

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

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

V(D)J Recombinase Binding and Cleavage of Cryptic Recombination Signal Sequences Identified from Lymphoid Malignancies

The variable genes and gene families of the mouse immunoglobulin κ locus

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