The status of Ig loci rearrangements in single cells from different stages of B cell development

Boekel, Edwin ten; Melchers, Fritz; Rolink, Antonius

doi:10.1093/intimm/7.6.1013

Cited by 163 publications

(116 citation statements)

References 16 publications

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“…Ehlich et al (1994) found that the early pro-B cells in Hardy's fraction B contain 24% (9/38) of all analyzed IgH alleles in the V^DH/pj-rearranged configuration. Like wise, ten Boekel et al (1995) observed VHDH/H joining in 13% (4/30) of the IgH alleles analyzed from B220"c-]cit* pre-BI cells. Based on these two studies, we would have expected to identify 10-18 VH^H/H rearrangements among the 74 IgH alleles analyzed from Pax5-deficient pre-BI cells.…”

Section: B-cell Development Is Arrested At the Early Pro-b (Pre-bi) Cmentioning

confidence: 85%

“…3) were performed with individual B220"^c-kit'' cells that were sorted from bone marrow of twoweek-old PaxS mutant mice into single wells of a 96-well plate by a FACStar^^'^^ flow cytometer. The IgH and IgL^ gene loci of individual cells were amplified exactly according to the twostep PCR protocol of ten Boekel et al (1995), which is based on the method of Ehlich et al (1994). Only the first (Gu et al 1991) of the two DHFL16/DHSP2-specific 5' primers listed in Table 1 of ten Boekel et al (1995) was used for PCR amplification of Dj^J^-teairanged alleles which resulted in DNA fragments of the following sizes: 1880 bp (/"!…”

Section: Pcr Analysis Of Igh Gene Rearrangementsmentioning

confidence: 99%

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Essential functions of Pax5 (BSAP) in pro-B cell development: difference between fetal and adult B lymphopoiesis and reduced V-to-DJ recombination at the IgH locus.

Nutt¹,

Urbánek²,

Rolink³

et al. 1997

Genes Dev.

378

377

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Section: B-cell Development Is Arrested At the Early Pro-b (Pre-bi) Cmentioning

confidence: 85%

Section: Pcr Analysis Of Igh Gene Rearrangementsmentioning

confidence: 99%

Essential functions of Pax5 (BSAP) in pro-B cell development: difference between fetal and adult B lymphopoiesis and reduced V-to-DJ recombination at the IgH locus.

Nutt¹,

Urbánek²,

Rolink³

et al. 1997

Genes Dev.

378

377

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“…VJk PCR fragments were amplified by using the primers Vk and Jk2 for 26 cycles consisting of 1 min 94°C, 1 min at 60°C, and 1.75 min at 72°C. The final polymerization step was extended an additional 10 min, and the PCR products were detected by Southern analysis using the oligonucleotide Jk1 (25).…”

Section: Methodsmentioning

confidence: 99%

V(D)J recombination is not activated by demethylation of the kappa locus

Cherry

Beard

Jaenisch

et al. 2000

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

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V(D)J recombination is thought to be regulated by changes in the accessibility of target sites, such as modulation of methylation. To test whether demethylation of the kappa locus can activate recombination, we generated two recombinationally active B cell lines in which the gene for maintenance of genomic DNA methylation, Dnmt1, was flanked with loxP sites. Transduction with a retrovirus expressing both the cre recombinase and green fluorescent protein allowed us to purify recombinationally active cells devoid of methylation. Loss of methylation of the kappa locus was not sufficient to activate recombination, although transcription was activated in one line. It appears that demethylation of the kappa locus is not the rate-limiting step for altering accessibility and thus regulated demethylation does not generate specificity of recombination.T he V(D)J recombination machinery assembles antigen receptor genes during lymphoid development from gene segments flanked by recombination signal sequences (1). This nonhomologous site-specific recombination process is precisely controlled by a number of different regulatory mechanisms. For one thing, the coexpression of the lymphoid-specific Rag-1 and Rag-2 genes occurs restrictively in developing B and T cells, ensuring that only the appropriate cell types are recombinationally active (2). A second level of regulation also controls lineage specificity: complete Ig gene rearrangement occurs only in B cells, whereas T cell receptor genes rearrange exclusively in T cells (3). Moreover, within developing B and T cells there are temporal controls over specific gene segment usage (4, 5). Additionally, allelic exclusion ensures that each B or T cell generates only one functional antigen receptor even though two alleles exist at each locus (6).This remarkable ability of the recombination machinery to target only a very small subset of substrates within a given cell together with the fact that a single recombinase is required for this process has led to the hypothesis that the accessibility of DNA targets to the recombination machinery is tightly controlled (7-9). Recombination loci are thought to be inaccessible until they are modified to allow the recombination machinery access to target sites. Changes in chromatin structure, transcriptional activity, or DNA methylation at each locus have been identified that correlate with recombinational activity. Many studies have attempted to define the cis-acting sequences and trans-acting factors required to regulate this process. Distinct enhancer sequences present within individual loci must be controlled by different trans-acting proteins to allow developmentally regulated changes in the accessibility of each locus to occur.DNA methylation has been shown to regulate chromatin structure, leading to changes of gene expression (10). Additionally, methylation has been correlated with Ig gene regulation: both the heavy chain and light chain are hypermethylated in cells in which they are not recombined (11,12). Concomitant with V(D)J rearrang...

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“…To investigate the clonality of tumours, we measured H-chain VDJ rearrangements in tumour cell DNA by screening 7 V family members (Ehlich et al, 1994;ten Boekel et al, 1995;Corcoran et al, 1998). We used the known clonotypic B-cell line Bal-17 as a positive control which, as expected, showed a single rearrangement (Figure 5,upper panel).…”

Section: Analysis Of the Clonality Of Tumoursmentioning

confidence: 98%

Vav-promoter regulated oncogenic fusion protein NPM-ALK in transgenic mice causes B-cell lymphomas with hyperactive Jun kinase

et al. 2003

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The status of Ig loci rearrangements in single cells from different stages of B cell development

Cited by 163 publications

References 16 publications

Essential functions of Pax5 (BSAP) in pro-B cell development: difference between fetal and adult B lymphopoiesis and reduced V-to-DJ recombination at the IgH locus.

Essential functions of Pax5 (BSAP) in pro-B cell development: difference between fetal and adult B lymphopoiesis and reduced V-to-DJ recombination at the IgH locus.

V(D)J recombination is not activated by demethylation of the kappa locus

Vav-promoter regulated oncogenic fusion protein NPM-ALK in transgenic mice causes B-cell lymphomas with hyperactive Jun kinase

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