The central part of the mouse immunoglobulin κ locus

Kirschbaum, Thomas; Röschenthaler, Franz; Bensch, Alexander; Hölscher, Barbara; Lautner‐Rieske, Andrea; Ohnrich, Marion; Pourrajabi, Soheil; Schwendinger, Jürgen; Zocher, Ines; Zachau, Hans G.

doi:10.1002/(sici)1521-4141(199907)29:07<2057::aid-immu2057>3.0.co;2-p

Cited by 26 publications

(39 citation statements)

References 19 publications

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“…gov). Comparisons with germ-line genes followed the nomenclature of H. G. Zachau (27)(28)(29). Analysis of V-J junctions provided the ratio of nonfunctional͞functional sequences (NF͞F ratio).…”

Section: Rna Preparation and Rt-pcrmentioning

confidence: 99%

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RNA surveillance down-regulates expression of nonfunctional κ alleles and detects premature termination within the last κ exon

Delpy

Sirac

Magnoux

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

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Random V(D)J junctions would generate nonfunctional and͞or out-of-frame sequences in about two-thirds of cases and result in abundant transcripts encoding truncated proteins. Although allelic exclusion at the DNA recombination level ensures that a single allele is functional, the frequent biallelic rearrangements need additional mechanisms to down-regulate aberrant transcripts in those cells with both a functionally and a nonfunctionally rearranged allele. The process of nonsense-mediated decay targets aberrantly rearranged Ig heavy-chain transcripts, but the situation of light-chain mRNAs is more complex, because they do not meet the usual requirements for nonsense-mediated decay and most often lack a spliceable intron downstream of the premature termination. We studied immunoglobulin heavy-chain ؊͞؊ pro-B cells in which light chain genes get rearranged and expressed in the absence of any selection for the assembly of a functional B cell receptor. Using this model, we show that the whole locus is accessible in pro-B cells and allows the assembly of a broad spectrum of V J segments, most of which are out-of-frame. This model provides an evaluation of the in vivo efficiency of RNA surveillance toward aberrant mRNAs produced in pro-B cells. Our data show that nonfunctional transcripts are excluded from the mature mRNA pool not only by detecting termination in an upstream exon but also by detecting changes in the position of termination within the last exon. Similar mechanisms efficiently down-regulate nonfunctional transcripts arising in normal mature B cells due to the biallelic transcription of rearranged genes. R andom deletions or additions at the Ig V(D)J junctions generate two-thirds of nonfunctional out-of-frame rearranged genes in B cell progenitors (1-3). Cell selection further allows the outgrowth of clones, each producing a single Ig selected for functionality and affinity for a non-self antigen determinant (4). Although some of those clones carry both functional and nonfunctional alleles of the immunoglobulin heavy-chain (IgH) and͞or light IgL loci, expression of truncated proteins is not observed in normal B cells, where aberrant transcripts are only present in very low amounts (5). For light chain (LC) genes, although the observed frequency of B cells carrying both a functional and a nonfunctional allele is Ͼ30% (1, 3), out-of-frame mature mRNA sequences were rarely obtained (6). Such an exclusion of nonfunctional Ig mRNA in B cells could involve transcriptional down-regulation and͞or mRNA quality control processes. Allelic asymmetry at the transcriptional level has been demonstrated for several cytokine genes (7,8). Concerning Ig genes, differences in the methylation status of alleles have been reported and correlated with an asymmetry in the replication pattern and the nuclear localization (9-10). However, transcriptional silencing of an excluded Ig gene allele has not been demonstrated, and, on the contrary, detection of rare out-of-frame transcripts in normal B cells indicates that this silencing...

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Section: Rna Preparation and Rt-pcrmentioning

confidence: 99%

“…1). This pro-B repertoire involved V segments dispersed throughout the locus (including the most upstream hf24) and belonging to the four V contigs (Table 1) (27)(28)(29). No transcript included the J 3 segment known to lack a functional recombination signal (33).…”

Section: Features Of Sequences Expressed In Pro-b Cellsmentioning

confidence: 99%

RNA surveillance down-regulates expression of nonfunctional κ alleles and detects premature termination within the last κ exon

Delpy

Sirac

Magnoux

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

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“…As shown in Fig. 1, based on previous physical mapping studies (1,18,19,(23)(24)(25)(26), ML is predicted to contain all three functional members of the V2 gene family and one functional member of the V21 gene family, along with uninterrupted sequences spanning from V21G to the 3Ј RS. This predicted structure was verified by numerous assays (see below).…”

Section: Structure Of Ig Minilocimentioning

confidence: 97%

“…(1,18,19,(23)(24)(25)(26)61). Not shown are the positions of nonfunctional V2, V9, and V20 pseudogenes (23)(24)(25)(26). Shown below are the three parent DNA constructs that were fused together to create 5ЈEML.…”

Section: Isolation Of Ig-expressing B Cellsmentioning

confidence: 99%

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High-Level Rearrangement and Transcription of Yeast Artificial Chromosome-Based Mouse Igκ Transgenes Containing Distal Regions of the Contig

Hammer

George-Raizen

et al. 2000

The Journal of Immunology

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The mouse Igκ L chain gene locus has been extensively studied, but to date high-level expression of germline transgenes has not been achieved. Reasoning that each end of the locus may contain regulatory elements because these regions are not deleted upon Vκ-Jκ joining, we used yeast artificial chromosome-based techniques to fuse distal regions of the contig to create transgene miniloci. The largest minilocus (290 kb) possessed all members of the upstream Vκ2 gene family including their entire 5′ and 3′ flanking sequences, along with one member of a downstream Vκ21 gene family. In addition, again using yeast artificial chromosome-based technology, we created Igκ miniloci that contained differing lengths of sequences 5′ of the most distal Vκ2 gene family member. In transgenic mice, Igκ miniloci exhibited position-independent and copy number-dependent germline transcription. Igκ miniloci were rearranged in tissue and developmental stage-specific manners. The levels of rearrangement and transcription of the distal and proximal Vκ gene families were similar to their endogenous counterparts and appeared to be responsive to allelic exclusion, but were differentially sensitive to numerous position effects. The minilocus that contained the longest 5′ region exhibited significantly greater recombination of the upstream Vκ2 genes but not the downstream Vκ21 gene, providing evidence for a local recombination stimulating element. These results provide evidence that our miniloci contain nearly all regulatory elements required for bona fide Igκ gene expression, making them useful substrates for functional analyses of cis-acting sequences in the future.

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The 5′ part of the mouse immunoglobulin κ locus

et al. 1999

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The 5' region of the mouse kappa locus comprises 63 Vkappa genes in six contigs of together 1.5 Mb, including one which links the region to the central part of the locus. The structures of the contigs were established by detailed restriction mapping of cosmid clones prepared from libraries of mouse C57BL/6 DNA and of yeast and bacterial artificial chromosomes (YACs, BACs with mouse DNA inserts). Pulsed-field gel electrophoresis of yeast artificial chromosome digests indicated that the gaps between the contigs were 10 to 60 kb, comprising together about 160 kb. The region of the kappa locus described here contains Vkappa1, Vkappa2, Vkappa9/10, Vkappa11, Vkappa12/13, Vkappa20, Vkappa24, Vkappa32, Vkappa33/34 and Vkappa38C genes as well as the VkappaRF gene and, towards the center of the locus, a number of Vkappa4/5 genes. Near the 5' end of the locus interspersed alpha-tubulin gene-like sequences were found. At its 3' side the region borders on the Vkappa4/5 contigs of the central region of the locus which is described in the accompanying report (Eur. J. Immunol. 1999. 29: 2057-2064). Structural details are to be found in the Internet at http://www.med.uni-muenchen.de/biochemie/zach au/kappa.htm. In a concluding section the main features of the structure of the mouse kappa locus are summarized.

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The central part of the mouse immunoglobulin κ locus

Cited by 26 publications

References 19 publications

RNA surveillance down-regulates expression of nonfunctional κ alleles and detects premature termination within the last κ exon

RNA surveillance down-regulates expression of nonfunctional κ alleles and detects premature termination within the last κ exon

High-Level Rearrangement and Transcription of Yeast Artificial Chromosome-Based Mouse Igκ Transgenes Containing Distal Regions of the Contig

The 5′ part of the mouse immunoglobulin κ locus

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