Physical map of the 3'' region of the human immunoglobulin heavy chain locus: clustering of autoantibody-related variable segments in one haplotype.

We have constructed the physical map of the 3' region of the human immunoglobulin heavy chain variable region (VH) genes. DNA segments extending to 200 kb upstream of the JH segment were isolated in two YAC clones. Five VH segments were identified in this region in the 5' to 3' order, V(II-5), V(IV-4), V(I-3), V(I-2), and V(VI-1) segments which were all structurally normal and orientated in the same direction as the JH segments. From DNA of a different cell line we have isolated a cosmid contig containing the same DNA region which has extraordinary polymorphism. The YAC and cosmid DNAs were called haplotypes A and B, respectively. Haplotype B contained an additional VH-I segment (V(I-4.1b)) between the V(II-5) and V(IV-4) segments. V(I-4.1b) segment is almost identical to a previously published VH sequence encoding a rheumatoid factor. Another VH segment in the B haplotype (V(I-3b)) corresponding to the V(I-3) segment also showed 99.7% nucleotide sequence homology with an anti-DNA autoantibody VH sequence. However, none of the VH sequences in haplotype A showed such strong homology with autoantibody VH sequences. The results suggest that VH haplotypes may have linkage with autoantibody production.     

Ig H chain variable and C region genes in common variable immunodeficiency. Characterization of two new deletion haplotypes.

Common variable immunodeficiency, a disorder characterized by diminished antibody production, manifests clinically as an increased susceptibility to bacterial infections. We have investigated the Ig H chain V and C region gene segments in 33 patients with common variable immunodeficiency, to identify the possible role these genes may have in the molecular basis of the defect. No major deletions were recognized for the VH gene segments of the VH2, VH5, and VH6 families, nor were there any differences in the RFLP patterns of mu- or alpha- switch regions or of C gamma genes. Two new deletion haplotypes were identified for the C region genes, the first encompassing C gamma 1 on a different haplotype from the C gamma 1 deletion described previously, and the second a novel deletion encompassing both C gamma 2 and C gamma 4. Based on these and previously described deletions in the IGHC region, we postulate that homologous regions are involved in the deletion process and that other new deletions likely exist in the population.     

Deletion mapping of Ig VH gene segments expressed in human CD5 B cell lines. JH proximity is not the sole determinant of the restricted fetal VH gene repertoire.

VH gene segments expressed in a panel of monoclonal human CD5 B cell lines have been positioned on the IgH locus by deletion mapping. The analysis yielded a relative order of VH fragments of the VH2, VH4, VH5, and VH6 gene families that was consistent with, and provided a further refinement of existing maps of the human IgH locus. We demonstrate that four of six VH gene segments expressed in the CD5 B cell lines map > 500 kb from the cluster of JH segments. Two of the gene segments, positioned at approximately 850 kb (58p2) and approximately 500 kb (1-9III) from the JH segments, respectively, belong to the previously identified small cohort of second trimester fetal VH gene segments. The data show that JH proximity is not the sole determinant of restricted VH gene utilization in early human ontogeny.     

An immunoglobulin heavy chain variable region gene is generated from three segments of DNA: VH, D and JH

We have determined the sequences of separate germline genetic elements which encode two parts of a mouse immunglobulin heavy chain variable region. These elements, termed gene segments, are heavy chain counterparts of the variable (V) and joining (J) gene segments of immunoglobulin light chains. The VH gene segment encodes amino acids 1-101 and the JH gene segment encodes amino acids 107-123 of the S107 phosphorylcholine-binding VH region. This JH gene segment and two other JH gene segments are located 5' to the mu constant region gene (Cmu) in germline DNA. We have also determined the sequence of a rearranged VH gene encoding a complete VH region, M603, which is closely related to S107. In addition, we have partially determined the VH coding sequences of the S107 and M167 heavy chain mRNAs. By comparing these sequences to the germline gene segments, we conclude that the germline VH and JH gene segments do not contain at least 13 nucleotides which are present in the rearranged VH genes. In S107, these nucleotides encode amino acids 102-106, which form part of the third hypervariable region and consequently influence the antigen-binding specificity of the immunoglobulin molecule. This portion of the variable region may be encoded by a separate germline gene segment which can be joined to the VH and JH gene segments. We term this postulated genetic element the D gene segment, referring to its role in the generation of heavy chain diversity. Essentially the same noncoding sequences are found 3' to the VH gene segment and as inverse complements 5' to two JH gene segments. These are the same conserved nucleotides previously found adjacent to light chain V and J gene segments. Each conserved sequence consists of blocks of seven and ten conserved nucleotides which are separated by a spacer of either 11 or 22 nonconserved nucleotides. The highly conserved spacing, corresponding to one or two turns of the DNA helix, maintains precise spatial orientations between blocks of conserved nucleotides. Gene segments which can join to one another (VK and JK, for example) always have spacers of different lengths. Based on these observations, we propose a model for variable region gene rearrangement mediated by proteins which recognize the same conserved sequences adjacent to both light and heavy chain immunoglobulin gene segments.     

The repertoire of human germline VH sequences reveals about fifty groups of VH segments with different hypervariable loops.

We have used the polymerase chain reaction and VH family-based primers to clone and sequence 74 human germline VH segments from a single individual and built a directory to include all known germline sequences. The directory contains 122 VH segments with different nucleotide sequences, 83 of which have open reading frames. The directory indicates that the structural diversity of the germline repertoire for antigen binding is fixed by about 50 groups of VH segments: each group encodes identical hypervariable loops. The directory should help in mapping the VH locus, in estimating somatic mutation and VH segment usage and in designing and constructing synthetic antibody libraries.     

Organization of rabbit immunoglobulin genes. I. Structure and multiplicity of germ-line VH genes

Two rabbit germ-line VH gene segments have been isolated from a recombinant phage DNA library. Nucleotide sequence analysis indicates that both of the genes share structural and regulatory features common to mouse and human VH genes, although one appears to be a pseudogene. Comparison of the protein sequences encoded by these genes to the protein sequences of rabbit immunoglobulin V regions indicates that both genes encode VH a-negative-like molecules. Quantitative genomic blot analysis with a VH probe capable of recognizing most, if not all, germ-line VH genes indicates that there are approximately 100 VH genes in the haploid genome of rabbits. The average spacing between the germ-line VH genes was determined to be approximately 6 kb. The molecular basis for the allelic inheritance of rabbit VH allotypes is discussed in view of the structural organization of germ-line VH genes.     

Immunoglobulin heavy chain gene organization and complexity in the skate, Raja erinacea

Immunoglobulin heavy chain genes from Raja erinacea have been isolated by cross hybridization with probes derived from the immunoglobulin genes of Heterodontus francisci (horned shark), a representative of a different elasmobranch order. Heavy chain variable (VH), diversity (DH) and joining (JH) segments are linked closely to constant region (CH) exons, as has been described in another elasmobranch. The nucleotide sequence homology of VH gene segments within Raja and between different elasmobranch species is high, suggesting that members of this phylogenetic subclass may share one VH family. The organization of immunoglobulin genes segments is diverse; both VD-J and VD-DJ joined genes have been detected in the genome of non-lymphoid cells. JH segment sequence diversity is high, in contrast to that seen in a related elasmobranch. These data suggest that the clustered V-D-J-C form of immunoglobulin heavy chain organization, including germline joined components, may occur in all subclasses of elasmobranchs. While variation in VH gene structure is limited, gene organization appears to be diverse.     

Organization of variable region segments of the human immunoglobulin heavy chain: duplication of the D5 cluster within the locus and interchromosomal translocation of variable region segments.

We have studied the organization of variable region (V) genes of the human immunoglobulin heavy chain (H) by cosmid cloning. We isolated two independent immunoglobulin D5 clusters (D5-a and D5-b) from cosmid libraries of the human genome. Restriction maps of these two regions showed that downstream 15 kb portions of the 55 kb overlap were different although upstream 40 kb portions were almost identical. Four more D segments, (DM, DXP, DA and DK) were found around the D5 segment in the conserved region of each cluster. Nucleotide sequences of the corresponding D segments from each cluster were almost identical and they encoded potentially functional D regions. Analysis using human-rodent somatic cell hybrids demonstrated that both clusters were located in the immunoglobulin heavy chain (H) locus on chromosome 14, suggesting that the D5-a and D5-b regions evolved by internal duplication within this locus. We also isolated a 60 kb DNA region carrying four VH segments, designated as VH-F region, which was located on chromosome 16. Nucleotide sequences of the four VH segments were determined. Two of them encoded potentially functional VH segments, and the other two were pseudogenes. Some more VH segments were found to be located outside chromosome 14, by Southern blot hybridization of human-rodent hybrid cell DNAs. These results provide further evidence that the human VH locus has undergone recent reorganization.     

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.     

Diverse organization of immunoglobulin VH gene loci in a primitive vertebrate.

The immunoglobulin (Ig) heavy chain variable (VH) gene family of Heterodontus francisci (horned shark), a phylogenetically distant vertebrate, is unique in that VH, diversity (DH), joining (JH) and constant region (CH) gene segments are linked closely, in multiple individual clusters. The V regions of 12 genomic (liver and gonad) DNA clones have been sequenced completely and three organization patterns are evident: (i) VH-D1-D2-JH-CH with unique 12/22 and 12/12 spacers in the respective D recombination signal sequences (RSSs); VH and JH segments have 23 nucleotide (nt) spacers, (ii) VHDH-JH-CH, an unusual germline configuration with joined VH and DH segments and (iii) VHDHJH-CH, with all segmental elements being joined. The latter two configurations do not appear to be pseudogenes. Another VH-D1-D2-JH-CH gene possesses a D1 segment that is flanked by RSSs with 12 nt spacers and a D2 segment with 22/12 spacers. Based on the comparison of spleen, VH+ cDNA sequences to a germline consensus, it is evident that both DH segments as well as junctional and N-type diversity account for Ig variability. In this early vertebrate, the Ig genes share unique properties with higher vertebrate T-cell receptor as well as with Ig and may reflect the structure of a common ancestral antigen binding receptor gene.     

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.     

Structural repertoire of the human VH segments.

The VH gene segments produce the part of the VH domains of antibodies that contains the first two hypervariable regions. The sequences of 83 human VH segments with open reading frames, from several individuals, are currently known. It has been shown that these sequences are likely to form a high proportion of the total human repertoire and that an individual's gene repertoire produces about 50 VH segments with different protein sequences. In this paper we present a structural analysis of the amino acid sequences produced by the 83 segments. Particular residue patterns in the sequences of V domains imply particular main-chain conformations, canonical structures, for the hypervariable regions. We show that, in almost all cases, the residue patterns in the VH segments imply that the first hypervariable regions have one of three different canonical structures and that the second hypervariable regions have one of five different canonical structures. The different observed combinations of the canonical structures in the first and second regions means that almost all sequences have one of seven main-chain folds. We describe, in outline, structures of the antigen binding site loops produced by nearly all the VH segments. The exact specificity of the loops is produced by (1) sequence differences in their surface residues, particularly at sites near the centre of the combining site, and (2) sequence differences in the hypervariable and framework regions that modulate the relative positions of the loops.     

Biased reading frames of pre-existing DH--JH coding joints and preferential nucleotide insertions at VH--DJH signal joints of excision products of immunoglobulin heavy chain gene rearrangements.

During B cell differentiation immunoglobulin (Ig) DH segments join to JH segments, followed by joining of VH to DJH. Although circular excision products of DH--JH rearrangements have been characterized, excision products of VH to DJH joining have never been isolated. We selectively denatured chromosomal DNA of mouse splenocytes and enriched circular DNA spanning the long distance between VH and DH. Subsequent PCR amplifications allowed the identification of signal joints of VH to DJH. Sequence analysis indicated that preexisting DH--JH coding joints of excision products showed a strong bias for reading frame 1, and the absence of reading frame 2, which would allow the expression of a truncated mu chain called D mu protein. When comparing the joints of the VH--DJH and DH--JH rearrangements we observed N-nucleotide insertions to be abundant at the VH--DH signal joint, but very sparse at the DH--JH signal joint, while the coding joints of both contained abundant N-insertions. These differences in N region insertions at the signal joints suggest a differential control of the D--J and V--DJ rearrangements.     

VH gene segments in the mouse and human genomes

We have examined the mouse genome sequence to determine its VH gene segment repertoire. In all, 141 segments are mapped to a 3 Mb region of chromosome 12. There is evidence that 92 of these are functional in the mouse strain used for the genome sequence, C57BL/6J; 12 are functional in other mouse strains, and 37 are pseudogenes. The mouse VH gene segment repertoire is therefore twice the size of that in humans. The mouse and human loci bear no large-scale similarity to each other. The 104 functional segments belong to one of the 15 known sequence subgroups, which have been further clustered into eight sets here. Seven of these sets, comprising 101 sequences, are related to five of the human VH families and have the same canonical structures in their hypervariable regions. Duplication of members of one set in the distal half of the locus is mainly responsible for the larger size of the mouse repertoire. Phylogenetic analysis of the VH segments indicates that most of the sequences in the human and mouse VH loci have arisen subsequent to the divergence of the two organisms from their common ancestor.     

Molecular characterization of the A/J J558 family of heavy chain variable region gene segments

The nucleotide sequences of the coding as well as the flanking regions of 11 A/J J558 heavy chain variable region (VH) gene segments are presented. Among these J558 VH segments was the unrearranged germline VH gene segment recruited in the predominant A strain-specific anti-arsonate response. Three other VH gene segments that are greater than 92% related to the p-azophenylarsenate (Ars) A VH gene segment were also isolated. Detailed analysis of the nucleotide sequences of these as well as the remaining seven J558 VH gene segments reveal that the J558 VH gene family is composed of distinct, but related, J558 VH subfamilies. Deletion mapping analyses were used to position the Ars A VH gene segment proximally with respect to the DH-JH clusters within the J558 VH gene family and distally with respect to its own J558 subfamily. The documentation of J558 VH subfamilies is discussed in the context of J558 VH family evolution and diversification.     

Strain-dependent expression of VH gene families

The tremendous diversity of the antibody specificity repertoire stems from the ability of each developing B cell to select one out of many possible variable, diversity, and joining gene segments by specific rearrangement of the DNA. The mechanism by which V region gene segments is selected is not known. Moreover, evidence for both random and nonrandom expression of VH genes in mature B cells has been presented previously. In this report, the technique of in situ hybridization is used to accurately measure at the single cell level VH gene family expression in LPS-induced cells from several strains. In this way, at least one-third of the B cells are stimulated and a large sampling of activated splenocytes from each strain analyzed. The use of in situ hybridization eliminates any potential biases resulting from transformation protocols. In addition, because all populations of cells are analyzed by both in situ hybridization and immunocytochemical staining with anti-IgM, the proportion of cells detected by in situ hybridization could be compared with the proportion of B cells, blasts, and plasma cells in the population. It was concluded from these comparisons that the cells being detected by in situ hybridization under the conditions described are plasmablasts and plasma cells. Therefore, an accurate measure of the functional and expressed VH gene repertoire could be made. The results clearly demonstrate strain-dependent variation in VH gene family expression, particularly VH 7183 and VH J558 with up to three-fold differences observed. Thus, either there is considerable strain variation in the number of functional VH gene family segments or the expression of VH genes is not entirely random.     

VH gene expression by nontransformed pre-B cells upon differentiation in vitro

Mice have more than 1000 VH gene segments, and each pre-B cell must choose a single one for rearrangement to encode the V portion of the antibody H chain. Presumably, all or most of the functional VH gene segments must be chosen by the population of B lymphocytes if the organism is to express the diversity that is observed in the immune system. Control of the selection of a VH gene segment for expression is not understood. We have found that the members of the VH gene family closest to the constant genes, the 7183 family, are transcribed in a manner that is specific for the stage of B cell development after pre-B cells derived from spleens of 6- to 8-wk-old nude mice are induced to differentiate in vitro by a mixture of dendritic cells and mitogen-activated T lymphocytes (DC-T). DC-T from spleens and lymph nodes induce transient high levels of synthesis of RNA from the 7183 VH family, whereas DC-T from Peyer's patches of mice of the same age as those from which spleen and lymph node DC-T were prepared did not induce the expression of RNA from that gene family. Spleen and Peyer's patch DC-T induce secretion of similar total amounts of antibody. Therefore, the RNA synthesis from members of at least one VH gene family is specific both for the lymphoid tissue in which B cell differentiation occurs and for the developmental stage of the B lymphoid cells.     

Dispersed localization of D segments in the human immunoglobulin heavy-chain locus.

We have studied the organization of the human immunoglobulin heavy-chain genes by pulse field gel electrophoresis as well as by isolation of cosmid clones. The total length of the heavy-chain variable region locus was estimated to be approximately 3000 kb. We found that D segments including a recently isolated D5 segment were dispersed among VH segments. We identified a pseudo V segment 18 kb 3' to the D5 segment in isolated cosmid clones. A 300 kb fragment produced by MluI digestion contained VH, D, JH segments and the distance between VH and D was estimated to be approximately 240 kb. Overlapping cosmid clones containing the human D1, D2, D3, D4, JH, Cmu and C delta genes were isolated. Restriction maps of these regions indicated that the distance between D and JH is about 22 kb. A partial restriction map of the VH locus was constructed using the pulse field gel electrophoresis technique and deletion of VH segments in B cells.