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At least 19 recordsLinked to original sources
PubMed1981

Repetitive sequences in class-switch recombination regions of immunoglobulin heavy chain genes.

Immunoglobulin class switch involves a unique recombination event that takes place at the region 5' to each heavy chain constant region gene during B lymphocyte differentiation. Such regions that are responsible for the class-switch recombination are defined as S regions (Kataoka et al., Proc. Natl. Acad. Sci. USA 77, 919, 1980). We have cloned a rearranged gamma 2b gene from a mouse myeloma (MPC11) and compared its structure with the germ line counterparts. The rearranged gamma 2b gene contained the 5' flanking region of the gamma 3 gene (S gamma 3 region) which are linked to the 5' flanking region of the gamma 2b gene (S gamma 2b region). We have determined nucleotide sequences surrounding the recombination site of the rearranged and germ line gamma 2b genes, which include the S gamma 2b and S gamma 3 regions. Both gamma 2b and S gamma 3 regions comprise tandem repetition of conserved units of 49 bp. Similar 49 bp repeating units are also found in the previously determined sequence of the S gamma 1 region in which class-switch recombination took place in MC101 myeloma. The nucleotide sequences of the S gamma 1, S gamma 2b and S gamma 3 repeating units share significant homology with each other. The S mu region, partial nucleotide sequence of which was previously determined, contains abundant short sequences such as AGCT, TGGG and AGCTGGGG which are shared in common by repeating sequences in S gamma regions. These results suggest that the recombination responsible for class switch from mu to gamma or from a gamma to another gamma, may be facilitated directly or indirectly by homology of repeating sequences in S regions.

Animals↗
PubMed1985

Probing murine male meiosis using unique DNA flanking the immunoglobulin heavy chain genes.

Murine male meiotic preparations were probed by in situ hybridization to localize 10.5 kb of unique DNA which flanks the C gamma 1 heavy chain immunoglobulin gene on the 5' side. The probe, inserted into the plasmid vector pBR 322, was labelled with I125 dCTP using nick translation. After exposing autoradiographs, made by standard techniques of in situ hybridization, for 40 days, a strong signal was obtained on one bivalent of size appropriate to pair 12. The signal was in the distal position on chromosome 12 and it could be traced from spermatogonial divisions through to mature sperm. The signal was strongest in some well-spread stages of prophase of first spermatocytes, weakest in early spermatids, then surprisingly strong in sperm heads. This variation is probably due to differences in the packaging of the target sequence at the various stages of meiosis.

Animals↗
PubMed2006

Evaluation of B-cell clonality in archival skin biopsy samples of cutaneous B-cell lymphoma by immunoglobulin heavy chain gene polymerase chain reaction.

Polymerase chain reaction (PCR)-based detection of clonal T- and B-cells is widely used in the diagnosis of various lymphomas, including those of the skin. A large number of corresponding methods have been published. Recently, for the first time, standardized PCR protocols were developed in common by 14 European centers of lymphoma diagnosis and research (Biomed-2 protocols). Here, we have applied Biomed-2 immunoglobulin heavy chain (IgH)-PCR for clonality detection in primary cutaneous B-cell lymphoma (CBCL) and compared it with previously established methods. The DNA of 43 paraffin-embedded lesional skin biopsies of confirmed CBCL cases [27 follicle center cell lymphoma (FCCL), 11 marginal zone B-cell lymphoma/immunocytoma (MZL/IC) and five large CBCL of the lower leg (CBCL-LL)] were amplified by the Biomed-2 IgH-PCR protocols as well as using four other assays, priming also the three IgH framework regions (FR) 1-3. All PCR products were analysed by fluorescence fragment analysis. Twenty-nine of 43 (67%) CBCL samples (5/5, 100% of CBCL-LL; six of 11, 54.5% of IC/MZL; 18 of 27, 66.7% of FCCL) showed monoclonal B-cell presence complementary in all of the IgH-PCR. The three Biomed-2 PCR indicated together clonality in 24 of 43 samples (56%). Considering each method separately, the Biomed-2 FR3-PCR showed the highest rate of clonality detection (20 of 43, 47%). In conclusion, the Biomed-2 FR3-PCR is recommended for detecting B-cell clonality in archival skin samples of CBCL but should be completed by FR1- and/or FR2-PCR.

Biopsy↗
PubMed1988

DNA polymorphism 5' to the JH region of the human immunoglobulin heavy chain gene and immunoglobulin gene rearrangements in leukemia.

DNA samples of two patients with acute myeloblastic leukemia were noted to have a variant band on digestion with HindIII and hybridization to a probe for the joining region of the immunoglobulin (Ig) heavy chain gene (JH). Further analysis showed that the variant band represented a constitutional DNA polymorphism rather than an Ig gene rearrangement. The HindIII fragment detected by the JH probe includes a highly polymorphic region 5' to the Ig heavy chain locus, and hence variant germline bands may be seen on autoradiography that can be mistaken for Ig gene rearrangements. The use of several different restriction enzymes and the analysis of the constitutional DNA allows a clear distinction to be made between somatic gene rearrangements and DNA polymorphisms.

Acute Disease↗
PubMed1998

Analysis of clonal diversity in mouse immunoglobulin heavy chain genes selected for size of the antigen combining site.

Size-diversity of Ig and T cell receptor antigen binding (CDR3) regions can be visualized by "CDR3 fingerprinting", and provides an estimate of B- or T-cell repertoire complexity. The method does not identify clonal diversity, however, which can only be determined by random sequencing of the CDR3s. In this study we demonstrate that a combination of fingerprinting and single strand conformation polymorphism (SSCP) analysis can be used for a rapid estimation of clonal diversity within mouse Ig antigen binding regions selected for size. This application may be useful in the analysis of clonal expansion within B- and T-cell repertoires.

Amino Acid Sequence↗
PubMed1988

neu protooncogene fused to an immunoglobulin heavy chain gene requires immunoglobulin light chain for cell surface expression and oncogenic transformation.

The protein encoded by the neu protooncogene (human gene symbol NGL for neuro/glioblastoma-derived) is a member of the surface receptor/tyrosine kinase family. Though its structure suggests that it can transduce a transmembrane signal, neither its extracellular ligand nor its critical intracellular substrates are known. To explore the functional properties of the protein encoded by neu, we created a fusion gene that joins the cytoplasmic domain of neu to the extracellular portion of an immunoglobulin heavy chain. The localization of the fusion polypeptide can then be controlled by coexpression with immunoglobulin light chain. In the absence of light chain, the heavy chain-neu polypeptide is expressed intracellularly and has no transforming activity. By contrast, in the presence of light chain the fusion polypeptide is expressed at the cell surface and produces tumorigenic foci. Thus, transformation apparently requires expression at the cell surface, where the neu intracellular domain can interact with components that are localized to the plasma membrane. The fusion protein is active in cellular transformation when the transmembrane domain is derived either from neu or from immunoglobulin, indicating that the neu transmembrane domain is not specifically required for transformation, although neu activation in tumors is known to result from a point mutation in this region. The extracellular immunoglobulin heavy and light chain domains of the fusion protein form a functional binding site that allows antigen to modulate its activity, reversing the transforming effect.

Animals↗
PubMed1989

The t(5;14) chromosomal translocation in a case of acute lymphocytic leukemia joins the interleukin-3 gene to the immunoglobulin heavy chain gene.

Chromosomal translocations have proven to be important markers of the genetic abnormalities central to the pathogenesis of cancer. By cloning chromosomal breakpoints one can identify activated proto-oncogenes. We have studied a case of B-lineage acute lymphocytic leukemia (ALL) that was associated with peripheral blood eosinophilia. The chromosomal translocation t(5;14) (q31;q32) from this sample was cloned and studied at the molecular level. This translocation joined the immunoglobulin heavy chain joining (Jh) region to the promotor region of the interleukin-3 (IL-3) gene in opposite transcriptional orientations. The data suggest that activation of the IL-3 gene by the enhancer of the immunoglobulin heavy chain gene may play a central role in the pathogenesis of this leukemia and the associated eosinophilia.

Base Sequence↗
PubMed1986

Biallelic heavy chain immunoglobulin gene rearrangement in acute nonlymphocytic leukemia.

Acute nonlymphocytic leukemia (ANLL) was diagnosed in 18 patients based on morphological, cytochemical and immunological criteria. Leukemic cells of these cases were subjected to Southern blot analysis and subsequent hybridization to heavy chain immunoglobulin and T-cell-receptor gene probes. A rearrangement within the immunoglobulin joining region was detected in 2 cases, while the T-cell-receptor beta-chain gene was in germline configuration in all samples investigated. These data confirm recent reports indicating that immunoglobulin heavy chain gene rearrangements are not restricted to B-lineage neoplasms.

Adult↗
PubMed1990

The physical organization of the human immunoglobulin heavy chain gene complex.

Two dimensional DNA electrophoresis (2D-DE) was used to map the variable (VH) region of the human heavy chain immunoglobulin gene cluster. Seventy-six VH gene segments were mapped to specific SfiI, BssHI and NotI fragments by 2D-DE. We have determined that a common insertion/deletion polymorphism of 80 kb, involving three VH gene segments, occurs in the VH region. The physical map suggests that the evolution of the human IGH gene complex involved duplication of blocks containing different VH families. This physical map will allow comparison of the usage of VH loci in human ontogeny with their proximity to the CH region. Knowledge of the germline repertoire of a particular DNA source studied in essential as the number of the dispersed VH gene segments of VH families, especially of the VH5 family, is variable. 2D-DE, as illustrated here for the IGH gene cluster, has general application in the development of large scale physical maps of gene and repeat families.

Chromosome Deletion↗
PubMed2001

The application of a PCR technique for the detection of immunoglobulin heavy chain gene rearrangements in fresh or paraffin-embedded skin tissue.

Although detection of a clonal sequence of the heavy chain gene of immunoglobulin by the polymerase chain reaction (PCR) is frequently used to assess lymphoid infiltrates in skin biopsy specimens, there are no data on the sensitivity and specificity of this test in detecting clonal B cell populations. Having refined a PCR technique for the detection of immunoglobulin heavy chain (IgH) gene rearrangement in both fresh and formalin-fixed, paraffin-embedded skin samples, we undertook to define the role of this assay in the diagnostic setting. Thirty-one cases of cutaneous B cell lymphoma (CBCL), 19 cases of B cell pseudolymphoma (lymphocytoma cutis), 34 cases of benign lymphocytic infiltrates of the skin and one case of cutaneous T cell lymphoma (CTCL) were studied using the polymerase chain reaction assay. All biopsies were formalin-fixed, paraffin-embedded skin sections apart from 13 of the 31 CBCL specimens which were fresh skin specimens. DNA from the framework region 3 (FR3) sequence of the IgH genes was amplified to ascertain the presence of a clonal IgH gene rearrangement. The findings were correlated with histological and immunophenotyping results on all samples. The assay performed with 73% sensitivity and 100% specificity, comparable to results obtained examining fresh lymphoid tissue specimens from patients with B cell tumours. The results indicate that this technique is a useful tool in the work up of suspected CBCL and in differentiating between CBCL and mixed lymphocytic infiltrates, a clearly important distinction with regards to prognosis and treatment.

Clone Cells↗
PubMed1987

The cis-acting regulatory elements of immunoglobulin heavy chain gene involved in enhanced immunoglobulin production after lipopolysaccharide (LPS) stimulation.

In the present study, the transient expression of the human immunoglobulin heavy chain gene (HIG1) was analyzed in a mouse pre-B-cell line, 70Z/3, after LPS stimulation. HIG1 gene and its recombinant plasmids were transfected by the calcium phosphate method into 70Z/3 cells and the cells were stimulated with lipopolysaccharide (LPS) 48 hr after DNA transfection. The amounts of human heavy chain gene products greatly increased in 70Z/3 cells after LPS stimulation, but the increment was diminished by deletion of the heavy chain gene enhancer element (MluI-HpaI fragment) in the J-C intron from the HIG1 gene. The gene, delta 3, which contained the 5' promoter region and the rearranged VDJ region of HIG1 but lacked the enhancer element, was weakly transcribed in 70Z/3 cells after LPS stimulation. Insertion of the enhancer element into the delta 3 gene greatly enhanced the transcription of the VDJ gene. The highest enhancement of the VH gene transcription rate was obtained when the 3' half of the enhancer element was ligated to the delta 3 gene. The present data suggest that the 3' half of the enhancer element of the heavy chain gene may play an important role in the enhanced production of immunoglobulin which is induced with LPS stimulation.

Animals↗
PubMed1983

A tissue-specific transcription enhancer element is located in the major intron of a rearranged immunoglobulin heavy chain gene.

We have studied the DNA sequences required for high level expression of a cloned heavy chain immunoglobulin gene stably introduced into mouse myeloma cells by DNA transfection. We found that DNA sequences derived from the germ line JH-C mu region are required for accurate and efficient transcription from a functionally rearranged VH promoter. Similar to viral transcriptional enhancer elements, these cellular sequences stimulate transcription from either the homologous VH gene segment promoter or a heterologous SV40 promoter. They are active when placed on the 5' or 3' side of the rearranged VH gene segment and they function when their orientation is reversed. However, unlike viral enhancers, the Ig gene enhancer appears to act in a tissue-specific manner, since it is active in mouse B cells but not in mouse fibroblasts. The nucleotide sequence of the Ig enhancer region contains repeating elements that closely resemble sequence the possible role of tissue-specific transcription in cell differentiation and malignant transformation.

Animals↗
PubMedDate not supplied

Transcription enhancer identified near the human C mu immunoglobulin heavy chain gene is unavailable to the translocated c-myc gene in a Burkitt lymphoma.

In Burkitt lymphoma the c-myc gene, the cellular homologue of the viral oncogene v-myc, has been implicated in the aetiology of this human B-cell malignancy. Burkitt lymphoma cells possess specific chromosomal rearrangements involving the region proximal to the c-myc gene and one of the three human immunoglobulin loci. The nature of the effect exerted by the immunoglobulin loci on the translocated c-myc gene is controversial: whereas some reports have suggested c-myc transcription is elevated in Burkitt lymphoma cells, others have suggested the level of transcription is unaffected by the translation. Recently, transcription enhancer elements have been identified in the intron between the JH and C mu segments of the heavy-chain immunoglobulin gene in mice. If similar enhancers exist in humans they may lead to increased transcription of the translocated c-myc gene and thus contribute to oncogenesis in Burkitt lymphoma. We report here the identification of an enhancer element adjacent to the human C mu gene on normal chromosome 14, but this enhancer does not remain on the abnormal chromosome 14 to which the c-myc gene has been translocated in the Burkitt lymphoma cell line Raji. This element is, therefore, not available for control of the translocated c-myc gene in this case.

Base Sequence↗
PubMed2003

Receptor revision of immunoglobulin heavy chain genes in human MALT lymphomas.

BACKGROUND/AIMS: Rearrangement of immunoglobulin gene segments, leading to B cells with functional receptors, is thought to be largely restricted to developing immature B cells in bone marrow. However, accumulating evidence suggests that mature B cells occasionally modify their antigen specificity by VH segment replacements during the germinal centre reaction to enhance antigen affinity, or to overcome self reactive antigen receptors. Although malignant B cells maintain the features of their normal counterparts in most instances, to date, such replacements have not been described for human B cell lymphomas. METHODS: Rearranged immunoglobulin heavy chain genes from two extranodal marginal zone B cell lymphomas were amplified, cloned, and sequenced. Sequences with identical CDR3 regions were selected and aligned to each other and public databases. RESULTS: VH replacements were seen in two extranodal marginal zone B cell lymphomas. In line with the hypothesis that in mature B cells these replacements are associated with active somatic hypermutation, in addition to VH replacement, different mutation patterns were seen in the revised VH portions. In the remaining common 3'-VH regions, these mutations could be used to establish a phylogenetic relation between the sequences, rendering the possibility of artefactual chimaeric polymerase chain reaction products very unlikely. CONCLUSIONS: These results support the view that VH replacements are a further mechanism for reshaping antigen affinity and specificity, and indicate that these receptor modifications are not restricted to normal and reactive germinal centre B cells, but may also occur in close association with the development of malignant B cell lymphomas.

Base Sequence↗
PubMed1982

Localization of human variable and constant region immunoglobulin heavy chain genes on subtelomeric band q32 of chromosome 14.

Analysis of a group of human/rodent somatic cell hybrids with nucleic acid probes prepared from cloned human variable region (VH), junctional (JH), and constant region (C epsilon) heavy chain immunoglobulin genes indicates that all of these IgH genes are localized on the subtelomeric (q32) band of chromosome 14. Somatic cell hybrids were isolated in selective medium after fusing human fibroblasts with hprt- Chinese hamster cells. The human parental cells contained two translocation chromosomes representing a reciprocal translocation between chromosomes X and 14. Only those hybrid cell lines retaining a complete human autosome 14 or the X/14 translocation chromosome (i.e. containing band 14q32) retained the human IgH genes. Retention of these genes did not correlate with the presence of the other translocation chromosome, 14/X. These results indicate that all human IgH genes (VH, JH, and CH) map to the same chromosomal band (14q32) which is commonly involved in reciprocal translocations with human chromosome 8 (8q24) in B-cell neoplasms.

Animals↗
PubMed2002

Immunoglobulin heavy chain gene analysis in lymphomas: a multi-center study demonstrating the heterogeneity of performance of polymerase chain reaction assays.

Determination of monoclonality through an evaluation of immunoglobulin heavy chain (IgH) gene rearrangements is a commonly performed and useful diagnostic assay. Many laboratories that perform this assay do so by the polymerase chain reaction (PCR). To evaluate current methods for performing IgH gene testing, 19 different Association of Molecular Pathology (AMP) member laboratories analyzed 29 blinded B cell and T cell lymphoid neoplasm samples of extracted DNA and formalin-fixed, paraffin-embedded (FFPE) tissue and were asked to complete a technical questionnaire. From this study, it is clear that Southern blot analysis remains the diagnostic gold standard, with a 100% diagnostic sensitivity and specificity. There was, however, remarkable heterogeneity in the performance of, and results obtained from, IgH PCR assays with diagnostic sensitivity ranging from over 90% to as low as 20%, when evaluating the same specimens. Many laboratories overestimate the diagnostic sensitivity of their IgH PCR assay, and there was a significant, and under appreciated, drop-off (from 61.3% to 41.8%) in detection in paired FFPE as compared with fresh/frozen tissues. Fixation has a dramatic impact on the inability to perform the test on FFPE (43.1%) versus DNA already extracted from fresh or frozen tissue (2.8%). A number of variables that affected the outcome of IgH PCR were identified. Strategies that improved the detection of monoclonal IgH rearrangements include: the addition of FRII to the FRIII upstream primer (increasing detection from 57.3% to 73.6%) and the use of the FR3A rather than the FR3 FRIII primer (increasing detection from 54.7% to 69.7%). Although numerous variables (from DNA extraction to PCR product detection) were evaluated, making it difficult to mandate alterations in laboratory practice, these findings ought to prompt diagnostic molecular pathology laboratories to reevaluate their claims of sensitivity, as well as their methodologies. Both pathologists and surgeons need to ensure that not all submitted material is fixed, if there is adequate sample. Importantly, there is a need for greater standardization to reduce the unacceptably high false negative rate of this crucial diagnostic assay.

Blotting, Southern↗
PubMed1983

c-myc Gene rearrangements involving gamma immunoglobulin heavy chain gene switch regions in murine plasmacytomas.

In murine plasmacytomas, the c-myc gene has frequently been found to undergo rearrangement by virtue of a T(12;15) chromosome translocation. The immunoglobulin heavy chain gene switch region (S alpha) constitutes the target for most of these recombinations particularly in IgA producing plasmacytomas. We sought to identify non-S alpha myc target sites in several IgG producing tumors. The c-myc target in MPC-11 (a BALB/c IgG2b producing plasmacytoma) has been cloned, localized to the Igh-C locus and identified as the gamma 2a heavy chain gene switch region (S gamma 2a). Furthermore, by Southern blot hybridization, we have determined that the S gamma 2b region is the c-myc target in two NZB IgG2b producing plasmacytomas. The potential relation between Ig class expressed and c-myc translocation target is discussed.

Animals↗