Reciprocal homologous recombination in or near antibody VDJ genes

To determine if rearranged heavy chain variable (VDJ) genes can recombine with each other by crossing over of DNA strands, we constructed a transgene that contained a promoter, VDJ gene, reporter gene to detect crossover events, intron enhancer, matrix attachment region, and constant gene for IgM (Cμ). Following immunization of transgenic mice, hybrid molecules were isolated from B cell DNA which contained the transgene recombined with the endogenous IgH locus. Reciprocal products of crossovers were detected by plasmid rescue and PCR amplification, and they were sequenced. Recombination occurred somewhere within 147 bp of homology that contained the J_H4 gene segment and 3′ flanking DNA. The recombined transgenes had a 20-fold increase in mutation in the VDJ region compared to nonrecombined transgenes, which indicates that DNA sequences 3′ of the Cμ gene in the endogenous IgH locus are necessary for full activity of the mutator mechanism. The recovery of recombinants between VDJ transgenes and endogenous VDJ genes raises the possibility that reciprocal recombination may somatically diversify rearranged genes between maternal and paternal alleles.     

Immunoglobulin gene rearrangement analysis in composite hodgkin disease and large B-cell lymphoma: evidence for receptor revision of immunoglobulin heavy chain variable region genes in Hodgkin-Reed-Sternberg cells?

Immunoglobulin heavy chain gene (IgH) rearrangement was studied in a patient showing the occurrence of classical Hodgkin disease and large B-cell lymphoma (LBCL) in the same lymph node. The VHDHJH region was amplified by polymerase chain reaction, the template being the DNA extracted from single Hodgkin and Reed-Sternberg and LBCL cells, microdissected on hematoxylin-eosin-stained sections by laser capture. A repeated VH4DH3JH4 segment was found in Reed-Sternberg cells, whereas a repeated VH3DH3JH4 segment was observed in LBCL cells. Rearranged VH genes carried somatic mutations in both populations, indicating a common germinal center cell origin. The IgH rearrangement found in clonally related Reed-Sternberg cells differed from the one of LBCL cells in the VH region but showed the same JH and DH segments with no variation from the respective germline sequence. The DH-JH junction is the first immunoglobulin gene segment rearranged in precursor B cells. Because the possibility of secondary Ig gene rearrangement in peripheral lymphoid organs has recently been reported, in the patient described here Reed-Sternberg and LBCL cells might originate from a common precursor in which secondary VH replacement took place during the germinal center reaction, giving rise to two different clonally related lymphomas.     

Rapid method for distinguishing clonal from polyclonal B cell populations in surgical biopsy specimens.

The polymerase chain reaction (PCR) was used to detect clonal rearrangements of the immunological heavy chain gene in frozen samples of human lymphoid tissue. DNA sequences in rearranged genes were amplified using oligomeric primers predicted from conserved sequences in the variable (VH) and joining (JH) regions. On polyacrylamide gel electrophoresis, polyclonal B cell proliferations showed a "smear", probably due to the variable lengths of the diversity (DH) region genes and the N regions separating the VH and DH and JH regions. In contrast, DNA from B cell lymphomas showed a clear single band in eight out of 10 cases. PCR undertaken on germ line DNA from non-lymphoid tumours showed no detectable bands or smears. The method can be completed within one day of biopsy, compared with several days in the case of conventional DNA blot analysis. Furthermore, it is cheaper, simpler, avoids the need for radioactive materials and requires very small amounts of DNA (about 1 micrograms).     

Third complementarity-determining region of mutated VH immunoglobulin genes contains shorter V, D, J, P, and N components than non-mutated genes

The third complementarity-determining region (CDR3) of immunoglobulin variable genes for the heavy chain (VH) has been shown to be shorter in length in hypermutated antibodies than in non-hypermutated antibodies. To determine which components of CDR3 contribute to the shorter length, and if there is an effect of age on the length, we analysed 235 cDNA clones from human peripheral blood of VH6 genes rearranged to immunoglobulin M (IgM) constant genes. There was similar use of diversity (D) and joining (JH) gene segments between clones from young and old donors, and there was similar use of D segments among the mutated and non-mutated heavy chains. However, in the mutated heavy chains, there was increased use of shorter JH4 segments and decreased use of longer JH6 segments compared to the non-mutated proteins. The overall length of CDR3 did not change with age within the mutated and non-mutated categories, but was significantly shorter by three amino acids in the mutated clones compared to the non-mutated clones. Analyses of the individual components that comprise CDR3 indicated that they were all shorter in the mutated clones. Thus, there were more nucleotides deleted from the ends of VH, D, and JH gene segments, and fewer P and N nucleotides added. The results suggest that B cells bearing immunoglobulin receptors with shorter CDR3s have been selected for binding to antigen. A smaller CDR3 may allow room in the antibody binding pocket for antigen to interact with CDRs 1 and 2 as well, so that as the VDJ gene undergoes hypermutation, substitutions in all three CDRs can further contribute to the binding energy.     

Immunoglobulins of the non-galliform birds: antibody expression and repertoire in the duck

Galliform and non-galliform birds express three immunoglobulin isotypes, IgM, IgA and IgY. Beyond this we should not generalize because differences in gene organization may have functional consequences reflected in the immune response. At present, studies on non-galliform birds are largely restricted to ducks. Ducks express an alternatively spliced form of their IgY heavy chain (upsilon) gene, the IgY(DeltaFc), that lacks the Fc region and Fc-associated secondary effector functions. It is not known how common the expression of the IgY(DeltaFc) is among birds, nor the functional consequences. It is also not known whether the unusual organization of the duck IgH locus, also shared with the chicken, having the gene order of mu, alpha and upsilon, with alpha inverted in the locus, is unique to the galloanseriform lineage. Ducks, like chickens, have a single immunoglobulin light chain of the lambda (lambda) type. Evidence suggests that ducks, like chickens, generate their immunoglobulin repertoire through a single functional rearrangement of the variable (V) region, and generate diversity through gene conversion from a pool of pseudogenes. In Southern blots of germline and rearranged bursal DNA, both the heavy and light chain loci of ducks appear to each undergo one major rearrangement event. For both heavy and light chains, the functional V region element and the pseudogenes appear to consist of a single gene family. Further analysis of 26 heavy chain joining (JH) and 27 light chain JL segments shows there is use of a single J segment in ducks, which is diversified presumably through somatic mutations and gene conversion events. Despite this limitation on the rearrangement of immunoglobulin genes, analysis of 26 DH and 122 VL sequences suggests that extensive sequence diversity is generated.     

Organization of the murine immunoglobulin VH complex: placement of two new VH families (VH10 and VH11) and analysis of VH family clustering and interdigitation

During B cell development, there is an ordered expression of heavy chain variable region (VH) genes during ontogeny such that JH proximal VH genes are rearranged and expressed before the more JH distal VH genes. Thus, the relative chromosomal position of VH genes is biologically significant. We have previously employed deletion mapping to order the nine described murine VH gene families as follows: 3609-J558-(J606/VGAM3-8/S107)-3660-(X24/Q52/7183 ). (Families within parentheses were not mapped relative to each other.) In this report we continue this analysis by mapping two recently described heavy chain variable region gene families (VH10 and VH11). VH10 is located at the JH proximal end of the major cluster of J558 VH gene segments. VH11 (a very small family) is intermingled with the 3660 family. Although in general VH genes are thought to be clustered, we and others have reported some interspersion between families. To further address this issue, we have analyzed 80 recombinant phage clones containing J558 VH gene segments for the presence of other VH family genes. Our data indicate that the J558 and 3609 VH families are extensively intermingled as has recently been described for the most JH proximal Q52 and 7183 families.     

中国人群慢性淋巴细胞白血病免疫球蛋白重链可变区基因组成及突变分析

目的 研究中国慢性淋巴细胞白血病(chronic lymphocytic leukemia,CLL)患者免疫球蛋白重链可变区(immunoglobulin variable heavy chain region,IGVH)基因各个片段的组成以及突变情况.方法 应用多重PCR技术扩增64例CLL患者的IGVH基因片段,纯化PCR扩增产物后直接测序,应用IMGT/V-QUEST及IGBlast数据库分析,明确VH基因有无突变和突变位置,以及VH、DH,JH家族各个成员组成情况.结果 64例CLL患者中,VH3家族31例(48%)、VH4家族26例(41%)、VH1家族4例(6%)、VH2家族2例(3%)、VH7家族1例(2%).44例患者发生体细胞突变,占CLL患者的69%;20例无突变,占CLL患者的31%,其中6例(9%)VH基因的同源性为100%.VH4家族中有9例无突变(9/26,35%);VH3家族中8例无突变(8/31,26%);VH1家族中1例无突变(1/4,25%).在所检测的CLL标本中,VH4-34是最常见的VH基因片段,检测出8例(13%),其中无突变3例;其次为VH4-59,检测出7例(11%),无突变者3例.DH基因中DH3家族最常见,有25例(39%);其中11例(11/20,55%)出现在无突变组中.无突变组中最常见的DH基因片段为DH3-10和DH3-22,各为4例(4/20,20%).JH基因中JH6家族最常见,检测出23例(36%),其中9例出现在无突变组中(9/20,45%).结论 中国CLL患者IGVH基因家族表达比例和突变情况与西方国家存在显著差异,推测可能与不同的种族和环境中的抗原选择有关,其预后意义有待进一步探讨.     

A VH gene is located within 95 Kb of the human immunoglobulin heavy chain constant region genes

Using cosmids covering about 117 Kb upstream of the human immunoglobulin chain C mu gene, we have identified a potentially functional VH gene, belonging to the VHVI subgroup. This VHVI gene is only about 95 Kb from the C mu gene and is probably the first functional VH segment of the Igh locus. These results illustrate the proximity of the human VH, DH and JH segments involved in creation of the complete heavy chain genes.     

Molecular characterization of the VH region of murine autoantibodies from neonatal and adult BALB/c mice

We report on the molecular characterization of the heavy (H) chain variable (V) region of two murine autoantibodies reacting with a conventional self antigen, thyroglobulin (Tg), originated from unimmunized/unstimulated neonatal mice (clone B10H2) and from an adult hyperimmunized mouse (clone 62), respectively. Serologically, both hybridoma antibodies express the same idiotype (Id). By cloning and sequencing we demonstrated that their VH regions are encoded by identical VDJ gene elements including the VD and DJ joints. This VH belongs to the VH 7183 gene family, the most 3'-end proximal family in the murine genome. The D segment is unique and only 50% similar to any murine D segment. The J segment utilized is germ-line JH4. The cloned DNA rearrangement was transfected into the J558L myeloma cells and the secreted antibody was found to express the reference Id on the H chain, hence proving that the productive VDJ rearrangement had been identified. These results show that a spontaneously arising and an antigen-induced autoantibody use an identical VDJ gene rearrangement and that after hyperimmunization somatic mutation did not occur. The significance of this finding with respect to ontogeny of the B cell repertoire is discussed.     

The genetic basis of antibody production: a single heavy chain variable region gene encodes all molecules bearing the dominant anti-arsonate idiotype in the strain A mouse

A nucleic acid probe specific for heavy chains bearing the cross-reactive idiotype (Id) associated with the anti-p-azophenylarsonate response of strain A mice has been prepared. Analysis of arsonate-binding Id+ hybridoma cell lines has revealed that all of them contain the same germ-line VH gene rearranged to the JH2 segment. An Id+ hybridoma which is unable to bind arsonate utilized the same VH gene, but it has apparently rearranged to the JH4 segment. Id- cell lines contain other rearranged VH genes. Analysis of DNa of strain A mice revealed that there is apparently only one germ line gene that can give rise to Id+ heavy chains. Since the Id is expressed as a large collection (greater than 50) of related but nonidentical heavy chain sequences, we conclude that their diversity is the result of a somatic mutation process. Analysis of a single hybridoma cell line (45-59) reveals that somatic mutation can operate on an Id-encoding gene and result in an antigen-binding molecule that has lost all of its Id determinants. Further analysis of the genome of strain A mice has revealed the presence of germ-line genes differing from the Id-encoding gene by at least 8 base pairs. These genes, however, apparently do not contribute to the anti-arsonate Id response.     

T cell receptor DJ but not VDJ rearrangement within a recombination substrate introduced into a pre-B cell line

To elucidate mechanisms that regulate ordered and tissue-specific assembly of Ig and TCR variable region gene segments, we have introduced a recombination substrate comprised of germline TCR beta V, D, and J gene segments into an Abelson murine leukemia virus-transformed pre-B cell line that actively rearranges endogenous Ig H chain variable region gene segments but does not rearrange endogenous light chain or TCR variable region gene segments. We find that these cells efficiently join D beta segments to J beta segments within the mini-locus, but that they do not make any detectable site-specific rearrangements of the introduced V beta segment even though it is closely linked in the same construct to the D beta. These findings suggest that factors necessary for V beta to (D beta)J beta joining may be absent in these pre-B cells and also imply that the order in which TCR V beta, D beta, and J beta segments are rearranged can be influenced by factors other than the 12/23 recombination rule. Furthermore, in agreement with the an accessibility model of VDJ recombinase control, the D beta region of the construct was found to be relatively more sensitive to DNAase I digestion in isolated nuclei when compared to the unrearranged V beta region.     

Molecular mechanisms involved in receptor editing at the Ig heavy chain locus

In receptor editing, a phenomenon that has recently come to light and into favor, a rearranged VDJ or VJ gene segment encoding a variable region of an Ig chain is replaced by another. In this commentary, the molecular mechanisms involved in the editing process are examined in some detail. Editing is most likely mediated by the same V(D)J recombinase activity responsible for the formation of the original VDJ or VJ segment. An embedded heptamer, which is present near the 3' end of many VH elements, is used as the recombination signal sequence at the Ig heavy chain locus. It has been postulated that the mediation of receptor editing is the evolutionary force maintaining the embedded heptamer. Some of the evidence for and against this hypothesis is discussed.