Somatic rearrangements forming active immunoglobulin mu genes in B and T lymphoid cell lines.

We have cloned an active gene for an immunoglobulin mu heavy (H) chain, bearing the variable (VH), joining (JH), and constant (C mu) sequences expressed in the IgM-secreting mouse plasmacytoma HPC-76. The mu gene was formed by somatic recombination between a VH gene and one of several JH genes, which are located about 7.7 kilobase pairs from the C mu gene in embryo DNA. The JH-C mu intervening sequence has suffered a deletion of about 2.7 kilobase pairs in HPC-76. Because the delection encompasses sequences required to switch an expressed VH-JH gene from C mu to another CH gene, it may represent a mechanism for "freezing" a lymphocyte clone at the stage of IgM expression. For the second (inactive) C mu allele in HPC-76, the entire joining and switch regions have been deleted; functional inactivation of one allele may thus represent one mechanism by which a lymphocyte clone restricts expression to a single allele (allelic exclusion). Probes generated from the cloned mu gene allowed examination of the JH locus in B, Abelson "pre-B," and T lymphoma cell lines and a myeloid line, all of which cotain RNA species bearing C mu sequences. The B and pre-B lines exhibited recombination within both alleles of the JH locus, suggesting that both alleles may be expressed in some cells. The absence of the JH gene 5' to the recombination sites favors a deletion mechanism for VH-JH joining. Recombination within the JH locus was also detected in two out of four T lymphoma lines, but not in the myeloid line. This indicates that the mechanism by which B cells generate immunoglobulin diversity is operational in some T cells. Lines that synthesize mu RNA without JH rearrangement may have activated the C mu gene directly or have undergone recombination at a more distant locus.     

Expression of an antigen receptor on T cells does not require recombination at the immunoglobulin JH-C mu locus.

Considerable evidence has accumulated suggesting that the antigen receptor(s) on T cells is coded for by genes for the variable (V) region of the immunoglobulin heavy (H) chains. In B cells, a complete gene for the immunoglobulin VH region is formed by somatic recombination of VH and joining region heavy chain (JH) gene segments [through an intermediate diversity(D) region gene segment]. In an attempt to determine whether a complete immunoglobulin VH region is expressed on T cells that bear an antigen receptor, we analyzed the restriction map of the JH-C mu locus in genomic DNA from two cloned murine cytotoxic T-lymphocyte (CTL) lines specific for the x-ray-induced leukemia RL male 1. We found no rearrangement of the JH C mu locus in the CTL lines, indicating that the T-cell antigen receptor(s) in these CTLs is not coded for by a complete immunoglobulin VH gene formed by joining of VH, (DH), and JH genes. In addition, we determined that C mu genes on both chromosomes were present and that there was no rearrangement of the C alpha, C kappa, or lambda chain genes in these CTL cells.     

Translocation of immunoglobulin VH genes in Burkitt lymphoma.

We have produced cell hybrids between mouse myeloma cells, which do not produce immunoglobulin chains, and Burkitt lymphoma cells (Daudi), which express surface IgM. Daudi Cells carry a reciprocal chromosome translocation between chromosomes 8 and 14, described as (8;14)(q24;q32). The hybrids were studied for the expression of human immunoglobulin chains and human isozyme markers, for the presence of human chromosomes, and for the presence of the human genes for heavy chain variable regions (VH) and mu and gamma chain constant (C) regions. The results indicate that the expressed mu chain gene is on normal chromosome 14 in Daudi cells. We have also determined that the chromosome 14 involved in the translocation (14q+) carries the gene for C mu and C gamma 1-4 and probably several genes for the variable region (V). Certain hybrids had lost both the chromosomes 14 but had retained the abnormal chromosome 8 (8q-) that carries the terminal end of the long arm of chromosome 14. These hybrids were studied for the presence of human VH, C mu,, and C gamma DNA sequences, and the results indicated that the hybrid cells with the 8q- chromosome contained VH genes that not C genes. Therefore, we conclude that, in the Daudi Burkitt lymphoma, the break in chromosome 14 occurred within the chromosome segment containing V region genes. As a result of the translocation some of these VH genes became associated with chromosome 8. It is possible that the expression of malignancy in Burkitt lymphoma is caused by immunoglobulin V region gene translocation resulting in activation of a gene on the long arm of human chromosome 8.     

Methylation patterns of immunoglobulin genes in lymphoid cells: correlation of expression and differentiation with undermethylation.

Different states of eukaryotic gene expression are often correlated with different levels of methylation of DNA sequences containing structural genes and their flanking regions. To assess the potential role of DNA methylation in the expression of immunoglobulin genes, which require complex rearrangements prior to expression, methylation patterns were examined in cell lines representing different stages of lymphocyte maturation. Methylation of the second cytosine in the sequence 5' C-C-G-G 3' was determined by using Hpa II/Msp I endonuclease digestion. Four CH genes (C mu, C delta, C gamma 2b, and C alpha), C kappa, V kappa, C lambda, and V lambda genes were analyzed. The results lead to the following conclusions: (i) transcribed immunoglobulin genes are undermethylated; (ii) the C gene allelic to an expressed C gene is always also undermethylated; and (iii) all immunoglobulin loci tend to become increasingly undermethylated as B cells mature.     

Intervening sequences divide the gene for the constant region of mouse immunoglobulin mu chains into segments, each encoding a domain.

To elucidate the structure of the gene for the constant region of immunoglobulin mu chains, we have cloned a 9.9-kilobase-pair fragment of mouse DNA bearing a gene for the constant region of the mu chain (C mu gene) from an IgM-secreting mouse plasmacytoma. The sequence around this gene has apparently undergone somatic rearrangement; the gene occurs in an EcoRI restriction endonuclease fragment of a different size from that in embryo or liver DNA and no C mu-bearing fragment of embryo size remains in the plasmacytoma. The cloned sequence lacks a variable region gene; hence, if this C mu gene is active, its position within the clone indicates that the gene for the variable region of a heavy chain (VH gene) must be more than 3.7 kilobase pairs away. The C mu gene is divided by three intervening sequences into four coding segments, each of which encodes one of the domains (homology units) of the polypeptide. The nucleotide sequence coding for amino acids near the V-C junction is not present within the C mu clone or clones bearing homologous embryonic VH genes. This suggests that an immunoglobulin heavy chain, in common with light chains, is encoded not only by a V and C gene, but also by an independent joining region (JH) gene.     

Polymorphisms of a human variable heavy chain gene show linkage with constant heavy chain genes.

In this study, restriction fragment length polymorphisms (RFLPs) were identified with an immunoglobulin variable heavy chain region (Ig VH) probe and the inheritance of the polymorphisms was analyzed in families. Linkage within the VHII gene cluster and between the VHII and Ig CH genes was investigated by lod (logarithm of odds) score analysis. In addition, the position of the VHII genes was determined in relation to another polymorphic locus--D14S1, which is tightly linked and centromeric to the CH genes. Genetic associations between genes in the CH and VH clusters were analyzed. These RFLPs represent genetically characterized VH region polymorphisms and it is hoped that they will facilitate the study of disease correlations as well as further the understanding of the genetics of the immunoglobulin heavy chain genes in humans.     

Immunoglobulin JH, C mu, and C gamma gene rearrangements in human B lymphocytes clonally transformed by Epstein-Barr virus.

Somatic rearrangements and deletions of immunoglobulin gene segments have been demonstrated in several types of murine B cells. In addition, rearrangements of the JH, C mu, and light chain immunoglobulin gene segments have been reported in human pre-B-cell leukemias and B-cell lymphomas. We have used recombinant DNA probes for the human JH, C mu, and C gamma immunoglobulin gene loci to analyze the genetic events associated with heavy chain gene expression in human B cells clonally transformed by Epstein-Barr virus. Southern hybridization analysis of BamHI-digested cell clone DNAs shows that these human B-cell clones often have bi-allelic JH rearrangements and that heavy chain isotype switching is associated with bichromosomal C mu and C gamma gene rearrangements. Deletions of germ line C mu and C gamma segments were observed that were sometimes bi-allelic. Overall, the observed rearrangements and deletions of heavy chain constant region genes suggest that human heavy chain class switching proceeds in a general order consistent with the proposed order of the heavy chain gene classes along chromosome 14.     

Complete nucleotide sequence of an immunoglobulin heavy-chain gene and analysis of immunoglobulin gene organization in a primitive teleost species.

The immunoglobulin heavy-chain variable region (VH) locus in a phylogenetically primitive teleost (Elops saurus) has been characterized by a strategy that relied initially on cross-hybridization between genomic VH segments and a murine VH probe. Using a homologous (Elops) VH probe and DNA sequencing, this gene family has been shown to be complex and to contain overt pseudogenes. A homologous probe also has been used to isolate a full copy length cDNA containing constant (CH) as well as joining (JH) and VH regions. Genomic analyses using CH-, JH-, and VH-specific probes have demonstrated the presence of only a single hybridizing CH and several JH elements. JH-CH linkage is less than or equal to 3.6 kilobases (kb) and VH-CH linkage is less than or equal to 100 kb, as estimated by field-inversion gel electrophoresis. An additional VH family sharing less than 50% nucleotide identity with the prototype Elops VH sequence is described. Taken together, these results suggest that the immunoglobulin VH locus in a comparatively primitive teleost resembles the VH locus in mammals, but not that found in the more phylogenetically distant elasmobranchs. The evolutionary radiations of cartilaginous and bony fishes are associated with a dramatic change in the organization and, presumably, regulation of immunoglobulin genes. The origins of the modern VH gene locus can be traced to the primitive teleost fishes.     

The chromosome 14 breakpoint in neoplastic B cells with the t(11;14) translocation involves the immunoglobulin heavy chain locus.

We hybridized neoplastic cells from a patient with chromic lymphocytic leukemia of the B-cell type, which carried a reciprocal chromosomal translocation between chromosomes 11 (q13) and 14 (q32) with mouse plasmacytoma cells. The hybrid cells were studied for the presence, rearrangement, and expression of the human immunoglobulin mu chain locus. The results indicate that the expressed mu chain gene is located on the normal chromosome 14, whereas the 14q+ translocation chromosome carries the excluded immunoglobulin constant (C) region mu chain allele (C mu) but does not contain variable (V) region heavy chain genes (VH). Since we found that the heavy chain joining region DNA (JH) of the excluded mu chain gene is on the 14q+ chromosome, we can conclude that the chromosomal break observed in the leukemic cells occurred in a chromosomal region within or 5' of the JH region. With these results, it is logical to postulate that a gene, for which we suggest the name bcl-1, is located on band q13 of chromosome 11 and is activated by its translocation into close proximity with the rearranged heavy chain locus on chromosome 14q+, contributing to the neoplastic transformation of the B cells with the t(11;14) chromosomal translocation.     

Cloned pairs of variable region genes for immunoglobulin heavy chains isolated from a clone library of the entire mouse genome.

To investigate the organization of immunoglobulin genes, we have constructed a clone library containing 10(6) randomly generated fragments of mouse embryo DNA, corresponding to eight equivalents of the genome. The cloning method involved methylation of embryo DNA at EcoRI recognition sites, partial digestion by EcoRI* endonclease activity, and direct ligation of the resulting large fragments to the lambda phage vector Charon 4A. The library was searched for sequences homologous to a cloned complementary DNA copy of a mu heavy chain mRNA. Nine clones bearing variable heavy chain (VH) sequences were isolated, representing at least eight distinct VH genes. Thus, multiple related VH genes are available in the genome to contribute to immunoglobulin diversity. Each of the two clones carries a pair of VH genes, one pair separated by 15 +/- 1 kilobase pairs of mouse DNA and the other by 14 +/- 2 kilobase pairs. This indicates that related VH genes are clustered and may occur in a tandem array having a repeating unit of 14--16 kilobase pairs. The large spacer sequences between VH genes cannot, however, be highly conserved.     

Homologous recombination can restore normal immunoglobulin production in a mutant hybridoma cell line.

We report here the occurrence of homologous recombination between transferred and chromosomal immunoglobulin genes. Specifically, we have corrected a chromosomal immunoglobulin gene mutation by transferring pSV2neo vectors encoding the constant region of the immunoglobulin mu heavy chain to mutant hybridoma cells that bear a 2-base-pair deletion in the third constant region exon of their chromosomal mu gene. After DNA transfer, we detected G418-resistant transformants that produce normal IgM. Analysis of the DNA structure of the mu gene in these transformants indicates that in four of five cases the mu gene has been restored as a result of the integration of a single copy of the transfer vector by a reciprocal homologous recombination event; the fifth case seems to have resulted from gene conversion or double crossover. These results suggest that this technology might be adapted for mapping immunoglobulin gene mutations by marker rescue and for more convenient engineering of specifically altered immunoglobulin.     

Nucleotide sequence of immunoglobulin heavy chain joining segments between translocated VH and mu constant regions genes.

To investigate the mechanism of recombination of immunoglobulin heavy chain variable and constant region genes, we have determined the nucleotide sequence of a large portion of the recombination region between an active C mu gene and its associated VH gene, isolated from an IgM-secreting mouse plasmacytoma, HPC76. By comparison with the sequence of the mu mRNA, we determined the exact boundaries of the intervening sequence between the VH76 and C mu genes. The rearranged VH76 gene encodes up to amino acid 116 without interruption, the 3' 39 nucleotides (the JH76 region) being derived from an embryonic JH segment (JH315) whose sequence was recently determined [Early, P., Huang, H., Davis, M., Calame, K. & Hood, L. (1980) Cell 195, 981-992]. The active JH76 does not use the first two codons of the embryonic JH315 from which it is derived. This indicates that V-J recombination is important in generating diversity within the third hypervariable region of heavy chains. We have identified another JH segment (JHA4), located 336 nucleotides 3' to the rearranged JH76 segment. This JH segment is expressed in the heavy chains of anti-levan myeloma proteins, which have truncated third hypervariable regions. We propose that the nucleotide sequence 5' to JHA4 is important for generating V region genes with shortened third hypervariable regions.     

A 16/6 idiotype-positive anti-DNA antibody is encoded by a conserved VH gene with no somatic mutation

Recently, the heavy chain of 16/6 idiotype-positive human anti-DNA antibodies was found to be similar, but not identical, to the VH26 gene. We resequenced the VH26 gene and found that its coding sequence is actually identical to the complementary DNA sequence of the anti-DNA antibodies previously described. Together with the previous data, our findings demonstrate that some human autoantibodies are encoded directly by immunoglobulin V region genes, and that these V region genes are remarkably conserved in populations.     

A 16/6 idiotype—positive anti-DNA antibody is encoded by a conserved VH gene with no somatic mutation

Recently, the heavy chain of 16/6 idiotype—positive human anti-DNA antibodies was found to be similar, but not identical, to the VH26 gene. We resequenced the VH26 gene and found that its coding sequence is actually identical to the complementary DNA sequence of the anti-DNA antibodies previously described. Together with the previous data, our findings demonstrate that some human autoantibodies are encoded directly by immunoglobulin V region genes, and that these V region genes are remarkably conserved in populations.