Determination of Gene Usage by Differential Polymerase Chain Reaction Product Hybridization

Sun, Jing; Shey, Michael R.; Butler, John E.

doi:10.1006/abio.1998.2693

Cited by 7 publications

(11 citation statements)

References 36 publications

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“…1). The V H gene specificity of these probes has been published previously 23,24 . The blots were eventually hybridized with a pan‐specific FR2 domain probe to ensure that each clone contained a VDJ insert (Fig.…”

Section: Analysis Of Vh Usagementioning

confidence: 99%

Antibody repertoire development in fetal and neonatal piglets. XI. The thymic B‐cell repertoire develops independently from that in blood and mesenteric lymph nodes

et al. 2005

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Summary The origin and function of thymic B cells is currently unresolved. In the present study we compared VH gene repertoire diversification in >3500 cloned VDJs (from 11 animals at three time‐points, using three to five animals per time‐point) that were expressed with immunoglobulin (Ig)M, IgD, IgG, IgA and IgE in thymus, mesenteric lymph nodes (MLN) and peripheral blood B cells (PBB) of newborn piglets and 5‐week‐old isolator piglets maintained germfree (GF) or colonized with Escherichia coli. The results showed that the repertoire expressed with IgM, IgD, IgG and IgA in PBB and MLN remained polyclonal, undiversified and unselected in piglets maintained GF for 5 weeks, that age and colonization resulted in significant repertoire diversification of IgG and IgA in the MLN and of IgG in blood, that the thymic B‐cell repertoire was polyclonal, unaffected by colonization and showed no clonal selection in any isotype, and that the thymic IgA and IgE repertoires were more diverse at birth than the repertoire of any isotype in MLN or PBB. IgD was seldom recovered from the PBB of newborn piglets or at any time‐point in thymus, but was recovered in the MLN of all 11 animals examined. The IgD and IgM repertoires in all tissues remained polyclonal and unselected, although VH usage by IgD transcripts did not always parallel that of IgM in the same tissue. Therefore, isotype‐switched B cells in the thymic medulla cannot be accounted for by immigration of B cells diversified by colonization of the gut, and thymic B cells undergo switch recombination and repertoire diversification before birth without clonal selection.

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Section: Analysis Of Vh Usagementioning

confidence: 99%

Antibody repertoire development in fetal and neonatal piglets. XI. The thymic B‐cell repertoire develops independently from that in blood and mesenteric lymph nodes

et al. 2005

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“…All reactions were performed in a 25-l volume containing 2.5 l of PCR buffer, 5 U of KlenTaq (AM Peptides, St. Louis, MO), 7.5 pmol of primers, 0.5 l of cDNA, 2.5 mM dNTP, and ϳ20 l of dH 2 O. Amplification was performed in a MJ-200 DNA engine under the following conditions: Initial denaturation was for 1 min at 94°C, followed by 30 cycles of denaturing at 94°C for 40 s, and then annealing at 58°C for 30 s followed by an extension at 72°C for 20 s. All products were then examined by agarose electrophoresis and Southern blotting to verify their size and identity. Five microliters of each PCR product (C, C␣, C␥, and ␤-actin) from the same cDNA preparation was applied to wells in a 96-well membrane transfer apparatus (Pierce, Rockford, IL) in 200 l of 20ϫ SSC and then blotted onto a nylon membrane as previously described (41,42). The transferred DNA was then cross-linked to the nylon membrane by exposure to UV radiation.…”

Section: Relative C C␥ and C␣ Transcript Expressionmentioning

confidence: 99%

Antibody Repertoire Development in Fetal And Neonatal Piglets. IV. Switch Recombination, Primarily in Fetal Thymus, Occurs Independent of Environmental Antigen and Is Only Weakly Associated with Repertoire Diversification

Butler

Sun

Weber

et al. 2001

The Journal of Immunology

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The epitheliochorial placenta of swine is considered a barrier to Ag and selective transport of IgG, so this species should be an excellent model with which to determine whether switch recombination is Ag dependent. Analysis of Ig levels and Ig isotype profiles in >150 normal and virus-infected fetuses from 38–110 days of gestation (DG) suggested that IgG, IgA, and IgM were most likely the result of de novo fetal synthesis. Although transcripts for IgM could be recovered at DG 50 (114 DG is full gestation) in all major fetal lymphoid tissues, those for IgG and IgA first became prominent at 60 DG in thymus, and transcription and spontaneous secretion became especially pronounced in this organ in older fetuses. Data on transcription, secretion, and serum isotype profiles suggest that although all fetal IgA and IgM may result from de novo synthesis, some IgG may result from low-level selective transport. The complementarity-determining region 3 spectratypes of thymic IgA and IgG transcripts at 70 and 90 days, respectively, were as polyclonal as that of IgM, indicating a broad repertoire of switched B cells although the VDJs transcribed with these switched isotypes in normal fetuses were not diversified in comparison to those from animals exposed to environmental Ags such as age-matched, virus-infected fetuses, colonized isolator piglets, and conventional adults. However, VDJs expressed with switched isotypes were more diversified than those expressed with IgM. Thus, switch recombination in fetal life does not appear to be driven by environmental Ag and is only weakly coupled to VDJ diversification. These findings, and the fact that the oligoclonal IgA and IgM repertoires in a noninductive site of the mucosal immune system (parotid gland) become polyclonal in piglets reared germfree, suggest that initial expansion of the switched cells in the B cell compartment of fetal and neonatal piglets is not driven by environmental Ag.

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“…In fetal piglets, 4 of these V H genes (V H A, V H B, V H C, and V H E) account for ϳ80% of the total usage of rearranged V H genes in DNA (2)(3)(4) and this preferential pattern of V H gene usage is also seen in transcripts from various tissues of young pigs (5). These observations are inconsistent with the multigenic paradigm established from studies in mice and humans.…”

mentioning

confidence: 93%

“…[The major porcine V H are the four genes that form Ͼ80% of the preimmune repertoire (2) plus V H F, V H Y, and V H Z that when combined, comprise Ͼ95% of the preimmune repertoire (see Table I). Other V H genes shown in Table I that have been recovered in full-length from DNA and/or bacterial artificial chromosomes (BACs) 3 or repeatedly in transcripts, are called minor V H genes. The term hybrid and "chimera" are used interchangeably.]…”

mentioning

confidence: 99%

“…In most of our previous studies on V H usage in swine using clonal hybridization (2,3,5) and those in humans using the same method (17), only single V H gene-specific probes were used. However, in limited studies using dual probes for CDR1 and CDR2, we identified hybrid clones, but because of the low frequency of their occurrence, they were included into a category labeled "other" (5).…”

mentioning

confidence: 99%

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Antibody Repertoire Development in Fetal and Neonatal Piglets. XIII. Hybrid VH Genes and the Preimmune Repertoire Revisited

Butler

Weber

Wertz

2006

The Journal of Immunology

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The expressed porcine VH genes belong to the VH3 family (clan), four of which, VHA, VHB, VHC, and VHE, alone comprise ∼80% of the preimmune repertoire. However, so-called “hybrid” VH genes that use CDR1 of one VH gene and the CDR2 of another are frequently encountered. We studied >3000 cloned VDJs and found that such hybrids can contribute up to 10% of the preimmune repertoire. Based on the 1) recovery of hybrid VH genes from bacterial artificial chromosome clones, 2) frequency of occurrence of certain hybrids in the preimmune repertoire, and 3) failure to recover equal numbers of reciprocal hybrids, we concluded that some chimeric genes are present in the genome and are not PCR artifacts. Two chimeric germline genes (VHZ and VHY), together with VHF and the four genes mentioned above, constitute the major VH genes and these account for >95% of the preimmune repertoire. Diversification of the preimmune IgG and IgM repertoires after environmental exposure was mainly due to somatic hypermutation of major VH genes with no evidence of gene conversion. Somatic hypermutation was 3- to 10-fold higher in CDRs than in framework regions, most were R mutations and transversions and transitions equally contributed. Data were used to 1) develop an index to quantify the degree of VH repertoire diversification and 2) establish a library of 29 putative porcine VH genes. One-third of these genes are chimeric genes and their sequences suggest that the porcine VH genome developed by duplication and splicing from a small number of prototypic genes.

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Determination of Gene Usage by Differential Polymerase Chain Reaction Product Hybridization

Cited by 7 publications

References 36 publications

Antibody repertoire development in fetal and neonatal piglets. XI. The thymic B‐cell repertoire develops independently from that in blood and mesenteric lymph nodes

Antibody repertoire development in fetal and neonatal piglets. XI. The thymic B‐cell repertoire develops independently from that in blood and mesenteric lymph nodes

Antibody Repertoire Development in Fetal And Neonatal Piglets. IV. Switch Recombination, Primarily in Fetal Thymus, Occurs Independent of Environmental Antigen and Is Only Weakly Associated with Repertoire Diversification

Antibody Repertoire Development in Fetal and Neonatal Piglets. XIII. Hybrid VH Genes and the Preimmune Repertoire Revisited

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