Ordered rearrangement of immunoglobulin heavy chain variable region segments

The immunoglobulin heavy chain variable region is encoded as three separate libraries of elements in germ-line DNA: VH, D and JH. To examine the order and regulation of their joining, we have developed assays that distinguish their various combinations and have used the assays to study tumor cell analogs of B-lymphoid cells as well as normal B-lymphoid cells. Abelson murine leukemia virus (A-MuLV) transformed fetal liver cells - the most primitive B-lymphoid cell analog available for analysis - generally had DJH rearrangements at both JH loci. These lines continued DNA rearrangement in culture, in most cases by joining a VH gene segment to an existing DJH complex with the concomitant deletion of intervening DNA sequences. None of these lines or their progeny showed evidence of VHD or DD rearrangements. Heavy chain-producing tumor lines, representing more mature stages of the B-cell pathway, and normal B-lymphocytes had either two VHDJH rearrangements or a VHDJH plus a DJH rearrangement at their two heavy chain loci; they also showed no evidence of VHD or DD rearrangements. These results support an ordered mechanism of variable gene assembly during B-cell differentiation in which D-to-JH rearrangements generally occur first and on both chromosomes followed by VH-to-DJH rearrangements, with both types of joining processes occurring by intrachromosomal deletion. The high percentage of JH alleles remaining in the DJH configuration in heavy chain-producing lines and, especially, in normal B-lymphocytes supports a regulated mechanism of heavy chain allelic exclusion in which a VHDJH rearrangement, if productive, prevents an additional VH-to-DJH rearrangement.     

Allelic forms of the immunoglobulin heavy chain variable region

The complete variable region sequence of the heavy chain from a phosphorylcholine-binding myeloma protein of C57/BL allotype has been determined. When this sequence was compared with the germ line-coded heavy chain variable region sequence of BALB/c phosphorylcholine-binding proteins, five differences were observed. Four of the substitutions were located in the framework portion of the variable region and the fifth in the "J" or joining segment. Two of the framework substitutions were found at positions 14 and 16. Previous studies have shown that heavy chains from all anti-phosphorylcholine antibodies induced in C57/BL mice have the same amino acids at positions 14 and 16 as the C57/BL myeloma protein described in this communication. It has therefore been concluded that these residues are encoded in the C57/BL germ line in contrast to two alternatives in the BALB/c genome. This finding, in addition to the 96% homology found between the C57/BL and BALB/c sequences, suggests that these structures represent allelic forms of an entire variable region.     

Immunoglobulin heavy chain constant and heavy chain variable region genes in phylogenetically diverse species of bony fish

Summary Genomic DNA from 18 phylogenetically diverse species of bony fish was hybridized with probes specific for the channel catfish immunoglobulin heavy chain constant (CH) gene, as well as with immunoglobulin heavy chain variable (VH) probes specific for five channel catfish VH gene families. The results showed that CH probes strongly hybridized only to genomic fragments from other catfish species. In contrast, restricted DNA from most other species hybridized with at least two channel catfish VH probes. In those species whose DNA hybridized with multiple VH probes, the restriction pattern of hybridizing fragments was probe-dependent. These studies suggest that (1) the CH gene defined in channel catfish appears to share similarity only with CH genes in other catfish species, (2) families of VH genes appear to have diverged in early phylogenetic lineages of teleosts, and (3) VH genes similar to those defined in catfish appear to be widely represented in phylogenetically diverse species of teleosts.     

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.     

Physical mapping of the bovine immunoglobulin heavy chain constant region gene locus

Bovine antibodies have recently attracted increasing attention, as they have been shown to exhibit prophylactic and therapeutic properties in selected infectious diseases in humans. In the present study, we have isolated bacterial artificial chromosomes and cosmid clones containing the bovine JH, mu, delta, gamma 1, gamma 2, gamma 3, epsilon, and alpha genes, which allowed us to make a contig of the genes within the bovine IGHC locus. The genes are arranged in a 5'-JH-7 kb-mu-5 kb-delta-33 kb-gamma 3-20 kb-gamma 1-34 kb-gamma 2-20 kb-epsilon- 13 kb-alpha-3' order, spanning approximately 150 kb DNA. Examination of the bovine germline JH locus revealed six JH segments, two of which, JH1 and JH2, were shown to be functional although there was a strong preference for expression of the former. Sequence alignment of the bovine 5' E mu enhancer core region with those of other mammals, demonstrated an absence of the mu E3 motif and a shortened spacer between the mu A and mu B sites within the bovine E mu enhancer core region. Furthermore, the essential sequence element for class switching, switch mu, spanning approximately 3-kb repetitive sequence and abundant in the switch region motifs CTGGG (187 repeats) and CTGAG (127 repeats), was identified immediately upstream of the mu gene. A further sequence comparison revealed that the bovine IGHC genes display an extensive polymorphism leading to expression of multiple antibody allotypes.     

Organization and evolution of variable region genes of the human immunoglobulin heavy chain

We have isolated 23 different cosmid clones of the heavy-chain variable region genes (VH) of human immunoglobulin. These clones encompass about 1000 X 10(3) base-pairs of DNA containing 61 VH genes. Characterization of the 23 clones by Southern blot hybridization showed that VH genes belonging to different families were physically linked in many regions. Cluster 71, which was analyzed in detail, comprised seven VH segments arranged in the same orientation with different intervals. This clone contained internal homology regions, each carrying two VH segments of different families. Comparison of the nucleotide sequences of VH segments within each family showed that profiles of accumulation of mutations in framework (FR) and complementarity-determining (CDR) regions were different. CDR had more mutations at amino-acid-substituting positions than at silent positions, whereas FR had the reverse distribution of mutations. Five out of seven VH segments of this cluster were pseudogenes containing various mutations. VH pseudogenes were classified into two distinct groups; one with a few replacement mutations (conserved pseudogenes), and the other with rather extensive mutations (diverged pseudogenes). The possibility that conserved pseudogenes serve as a reservoir of VH segments is discussed.     

Evolutionary dynamics of the immunoglobulin heavy chain variable region genes in vertebrates

Immunoglobulin heavy chains are polypeptides encoded by four genes: variable (IGHV), joining (IGHJ), diversity (IGHD), and constant (IGHC) region genes. The number of IGHV genes varies from species to species. To understand the evolution of the IGHV multigene family, we identified and analyzed the IGHV sequences from 16 vertebrate species. The results show that the numbers of functional and nonfunctional IGHV genes among different species are positively correlated. The number of IGHV genes is relatively stable in teleosts, but the intragenomic sequence variation is generally higher in teleosts than in tetrapods. The IGHV genes in tetrapods can be classified into three phylogenetic clans (I, II, and III). The clan III and/or II genes are relatively abundant, whereas clan I genes exist in small numbers or are absent in most species. The genomic organization of clan I, II, and III IGHV genes varies considerably among species, but the entire IGHV locus seems to be conserved in the subtelomeric or near-centromeric region of chromosome. The presence or absence of specific IGHV clan members and the lineage-specific expansion and contraction of IGHV genes indicate that the IGHV locus continues to evolve in a species-specific manner. Our results suggest that the evolution of IGHV multigene family is more complex than previously thought and that several factors may act synergistically for the development of antibody repertoire. Electronic supplementary materials The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Variability of the immunoglobulin heavy chain constant region locus: a population study

The Immunoglobulin Heavy chain Constant region (IGHC) locus is a multigene family composed of highly homologous segments often involved in unequal crossings over that lead to deleted and duplicated haplotypes. The frequencies of these haplotypes in 558 individuals from Lombardy, Veneto, Puglia and Sardinia were determined by Pulsed Field Gel Electrophoresis (PFGE), followed by Southern blotting with four IGHC probes, and compared with those observed in 110 subjects from Piedmont. Twenty deletions and 60 duplications were characterized, all in heterozygous individuals except for 2 homozygous deletions. The differences in frequency between the five populations were not significant. The deletions/duplications involved one or more genes: GP-A2, A1-E and G4 duplications, and A1-E and GP-A2 deletions were the most common. Four new duplications are described: three, involving the genes from GP to A2, from G2 to G4, and G4, are counterparts of known deletions. The fourth duplication spans from GP to G2. A G1 deleted heterozygous individual never previously described in Italy is reported. All the rearranged haplotypes seem to be the result of unequal crossing over. The difference between the number of duplications and deletions was significant in Sardinia, Lombardy, Puglia and in the total of 668 subjects (P < 0.001). This may be due to selection or genetic drift.     

A novel family of variable region genes of the human immunoglobulin heavy chain

We isolated and sequenced six variable-region (V) gene segments of the human immunoglobulin heavy-chain (H) using the V71-2 segment as probe. These VH segments were more than 90% homologous to each other and less than 65% homologous to members of the three known VH families. The VH fragments hybridized to an identical set of restriction fragments on Southern blots of human placenta DNA. The new family was designated as the VH-IV family. The complexity of the VH-IV family was estimated to be at least nine genes, of which the sequenced seven were functional genes. The VH-IV family is homologous (76%) to the mouse Vh36-60 family.     

Phage antibody fragments library combining a single human light chain variable region with immune mouse heavy chain variable regions

We describe the construction of a phage antibody fragments library which combines, in a single cloning step, a synthetic human light chain variable region (V(L)) with a diverse set of heavy chain variable regions, from a mouse immunized with the prostate specific antigen (PSA). Despite V(L) restriction, selection from this library rendered two different single chain Fv antibody fragments, specifically recognizing PSA. The human V(L), used as a general partner for mouse heavy chains, was constructed by linking the germline A27 gene and the J(K)1 minigene segment, both of which are prominently involved in human antibody responses. Our approach offers a fast and simple way to produce half-human molecules, while keeping the advantage of immunizing animals for high affinity antibodies.     

Evolutionary stability of the immunoglobulin heavy chain variable region gene families in teleost

 A comparison between related species would allow us to study the evolutionary changes in complex gene families. To investigate the evolution of immunoglobulin VH gene families in lower vertebrates, we compared cDNA VH clones from two related teleost fish species, Arctic charr (Salvelinus alpinus) and rainbow trout (Oncorhynchus mykiss), which are separated from their common ancestor by 12–20 million years (MY). The results showed that randomly isolated charr VH genes could be closely grouped to known VH genes of rainbow trout, suggesting that the VH family structure is stable during 12–20 MY and that the total number of VH families changes only gradually over a longer period. This finding also led us to define eight VH gene families of Arctic charr, designated Salalp VH I, VH II, and so on. The presence of species-specific amino acids suggests that non-reciprocal genetic exchanges (e.g., gene duplication) play an important role in shaping the evolution of the V gene family. Received: 23 July 1997 / Revised: 6 October 1997     

A genetic marker in the variable region of rabbit immunoglobulin heavy chain.

A previous study [Mole et al. (1971) Biochem. J. 124, 301-318] showed several differences in sequence between the variable (V) regions of rabbit immunoglobulin Aa1 and Aa3 heavy chains. The inheritance of one such difference has been followed in a family of 38 rabbits by a radioautographic peptide-'map' technique and is shown to segregate in a Mendelian fashion. This clearly demonstrates the presence of a genetic marker in the rabbit heavy-chain V region, although the finding that Aa2 and Aa3 heavy chains have identity of sequence in the region studied obscures the relationship of this genetic marker to the a locus.     

Genetic and antigenic characterization of fragments of the pheasant immunoglobulin Y heavy chain constant region

Few studies have attempted to characterize the pheasant (Phasianus colchicus) immunoglobulin Y (IgY) heavy chain constant region. In the present study, fragments of the pheasant IgY heavy chain constant region were cloned, analyzed, and expressed. The cross-reactivity of IgY or immunoglobulin G (IgG)s with antigens from other vertebrate species was determined using dot-enzyme-linked immunosorbent assay and western blot analysis. Five peptides of the pheasant IgY heavy chain constant region were synthesized to determine its immunoregulatory activity in vitro. The IgY heavy chain constant region from pheasant showed the highest homology with that from chicken (71.2 %) and duck (49.1 %). Phylogenetic analysis for IgY showed that pheasant was closely related to chicken and duck than to any other analyzed vertebrate species. The rabbit anti-chicken IgG showed immunologic cross-reactivity with recombinant proteins of the pheasant IgY heavy chain constant region. Four peptides were able to induce significant up-regulation of interleukin (IL)-1β, IL-4, and interferon-γ in chicken peripheral blood lymphocytes, suggesting a new role of avian IgY in immune regulation.     

Identification of a matrix-associated region 5'' of an immunoglobulin heavy chain variable region gene.

In the accompanying report (C. F. Webb, C. Das, S. Eaton, K. Calame, and P. Tucker, Mol. Cell. Biol. 11:5197-5205, 1991), we characterize B-cell-specific protein-DNA interactions at -500 and -200 bp upstream of the mu immunoglobulin heavy chain promoter whose abundances were increased by interleukin-5 plus antigen. Because of the high A + T/G + C ratio of these sequences and the consistent findings by others that enhancer- and promoterlike regions are often located near matrix-associated regions, we asked whether these sequences might also be involved in binding to the nuclear matrix. Indeed, DNA fragments containing the -500 binding site were bound by nuclear matrix proteins. Furthermore, UV cross-linking studies showed that the DNA binding site for interleukin-5-plus-antigen-inducible proteins could also bind to proteins solubilized from the nuclear matrix. Nuclear matrix-associated sequences have also been demonstrated on either side of the intronic immunoglobulin heavy chain enhancer. Our data suggest a topological model by which interactions among proteins bound to the promoter and distal enhancer sequences might occur.     

人源抗EPO基因工程抗体重链可变区的克隆和鉴定

利用噬菌体抗体显示技术筛选 EPO的人源抗体 ,得到了抗 EPO的人源抗体的重链基因。此抗体基因在噬菌体表面呈现的抗体分子具有良好的抗体活性和特异性。为制备完整的、具有更高亲和力的抗体打下了基础。     

The G4 gene is duplicated in 44% of human immunoglobulin heavy chain constant region haplotypes

The structure of the human immunoglobulin heavy chain constant region (IGHC), on chromosome 14q32, comprises nine CH genes and two pseudogenes, all originating from multiple duplication events. Continuing evolution of the region is demonstrated by the finding of various types of duplicated and deleted haplotypes, which together add up to 6%. Here we provide molecular and genetic evidence that the G4 gene is duplicated in 44% of IGHC haplotypes in the Italian population. The duplication spans about 20 kb of genomic DNA and probably originated through unequal crossing over. Refined characterisation of the genomic region downstream from the G4 gene improves our knowledge of the evolutionary history of CH genes. Received: 4 December 1996 / Accepted: 10 February 1997