Cloning of immunoglobulin kappa light chain genes from mouse liver and myeloma MOPC 173

The organization of the kappa chain constant region gene was compared in DNA from an immunoglobulin-producing mouse myeloma (MOPC 173) and from liver. In situ hybridization using the Southern blotting technique revealed constant region gene-containing EcoRI-DNA fragments of 14 and 20 kb in the myeloma tissue whereas one EcoRI-DNA fragment with a length of 15 kb was found in liver DNA. After enrichment by RPC-5 chromatography and preparative electrophoresis the 14 kb fragment from MOPC 173 DNA and the 15 kb fragment from liver DNA were cloned in the bacteriophage lambda vector Charon 4A using in vitro packaging. Extensive characterization of the two fragments by restriction endonuclease mapping, in situ hybridization, and electron microscopy (R-loop and heteroduplex) showed that both fragments contain the constant region but no MOPC 173 variable region gene. Both fragments are homologous over a length of 12.5 kb including the constant region but differ from one another starting about 2.7 kb from the 5' end of the constant region gene. This indicates that the 14 kb EcoRI-DNA fragment from the myeloma tissue clearly resulted from somatic DNA rearrangement although it does not seem to carry the MOPC 173 variable region gene. These observations suggest that somatic DNA rearrangement of immunoglobulin light chain genes can involve both homologous chromosomes.Images     

A rearranged DNA sequence possibly related to the translocation of immunoglobulin gene segments.

A 5.3 kb EcoRI fragment (T3, abbreviations in ref. 2) has been cloned from DNA of a kappa light chain producing mouse myeloma. The fragment hybridizes to the k' flanking sequences of the J1 gene segment but not to C gene sequences of kappa light chain DNA. Restriction nuclease mapping and partial nucleotide sequencing showed that the fragment consists of sequences from the 5' side of the J1 and form the 3' side of a V gene segment, which apparently had been linked in a genomic rearrangement process. These rearranged flanking sequences are not the flanking sequences of the V and J gene segments which had been joined to form the two kappa light chain genes of the myeloma. Fragments with the hybridization properties of T3 have been found also in two other kappa and one lambda chain producing myelomas. The linking of flanking sequences in the myeloma genome is discussed with respect to the mechanism of recombination between V and J gene segments.     

Accessibility of expressed and non-expressed genes to a restriction nuclease.

Nuclei of mouse liver and an immunoglobulin producing myeloma were digested with HaeIII or its isoschizomer BspRI. The DNA fragment patterns after electrophoresis, blotting, and hybridization were very similar for the two types of nuclei when a probe for the non-expressed beta-globin gene was used. The constant (C) region of the kappa light chain gene, on the other hand, was more accessible to the nuclease in myeloma than on liver nuclei. In myeloma nuclei the BspRI sites were about equally sensitive, in fact the pattern resembled a partial digestion pattern of free DNA. In contrast, in liver nuclei some sites were much more protected than others. This is interpreted by assuming that this single copy non-expressed gene region is covered by nucleosomes which are preferentially located on certain DNA sequences. Restriction nuclease digestion of nuclei seems to be a promising method for the analysis of genes in their expressed and non-expressed states.     

Isolation of messenger RNA for an immunoglobulin kappa chain and enumeration of the genes for the constatn region of kappa chain in the mouse

The messenger RNA for an immunoglobulin light (kappa) chain was isolated from the mouse myeloma MOPC41 and shown to be almost twofold longer than necessary to code for its protein product. DNA complementary to the mRNA was synthesized with the RNA-directed DNA polymerase of Rous sarcoma virus. Although the individual chains of the cDNA² contained an average of only 270 nucleotides, the cDNA was heterogeneous in molecular weight, allowing the isolation of a fraction of the cDNA 620 nucleotides long. This large cDNA would be long enough to code for nearly all (95%) of the constant region if all the untranslated region of the mRNA were between the 3′ terminal poly(A) and the constant region. On the other hand, if all the untranslated region of the mRNA were at the 5′ terminus, this cDNA would code for 93% of the entire kappa chain.The specificity of nucleotide sequences in the cDNA was documented by molecular hybridization with both template RNA and RNA from various myelomas. The amount of hybridization obtained with myeloma RNA was approximately proportional to the amount of kappa chain protein produced by the various myeloma cells. In addition, there was no hybridization with RNA isolated from either BALB/c mouse liver or Escherichia coli.The genes for the constant region of the kappa chain were enumerated in the mouse genome by annealing cDNA to DNA from mouse liver and MOPC41 myeloma. The haploid genome of both tissues contained three to four genes for the constant region of kappa chain even when tested under conditions that would detect distantly related nucleotide sequences. The fact that there are only a few genes coding for the constant region of kappa chains implies that specialized genetic mechanisms are required for the generation of antibody diversity.     

Heavy chain variable region contribution to the NPb family of antibodies: somatic mutation evident in a γ2a variable region

To examine germ line genes of the heavy chain variable region (V_H) that might contribute to formation of antibodies of the NP^b family, we have derived cDNA clones from two hybridomas making NP^b antibodies. One, B1–8, made an IgM protein and was derived during a primary response; the other, S43, made an IgG_2a protein and was derived during a hyperimmune response. Sequence comparison of the two clones showed that they differed by only 10 bp in the V_H region, had very different D segments and had identical J segments (J₂). A set of closely related germ line V_H genes was then cloned from a partial Eco RI library of C57BI/6 DNA. By comparing the germ line V_H regions to the cDNA V_H regions, we identified seven potential candidates for encoding the V_H regions of NP^b antibodies. The seven V_H regions were sequenced, and one V(186-2) contained exactly the DNA sequence found in the clone derived from B1–8. None of the DNA sequence differences that distinguished the S43-derived clone from the B1–8 clone was found in any of the other six germ line genes. Because the S43 sequence was more closely related to the V(186-2) germ line sequence than to any of the other V_H genes, we conclude that the differences between the genes resulted from somatic mutation and that the two hybridomas derived their V_H regions from the same germ line gene. Certain of the sequenced V_H genes contain crippling mutations; the repertoire of germ line V_H genes that can contribute to the diversity of antibodies may therefore be less than the total number of genes detectable by hybridization.     

Somatic mutation of immunoglobulin light-chain variable-region genes

A single germline immunoglobulin kappa-variable-region gene, V_κ167, is rearranged and expressed in two myelomas, MOPC167 and MOPC511. Only this single germline gene displays close homology to the expressed genes. Neither of the rearranged, functional genes, however, has a nucleotide sequence that is identical to the germline V_κ167 gene. Both active genes display several single-base-pair mutations with respect to the germline sequence. The nucleotide sequence data predict the alteration of a restriction-enzyme-recognition site within the V_κ167 gene between germline cells and cells producing the MOPC167 light-chain protein. Based on this restriction-site alteration, Southern blot analysis proves unambiguously that no gene present in the germline BALB/c mouse genome contains the exact V_κ167 nucleotide sequence found in cells committed to MOPC167 antibody production. Instead, the alterations found in the expressed MOPC167 and MOPC511 V-region genes have apparently arisen by a process of somatic mutation during cellular differentiation. Since nucleotide alterations are found in framework and hypervariable portions of the variable region, the mechanism of somatic mutation is not limited to hypervariable sequences. In addition, Southern blot hybridization indicates that the observed mutations did not arise by recombinational events, but are single-base-pair substitutions. Based on the distribution of mutations that have been found in expressed immunoglobulin variable-region genes, a model that links the introduction of somatic mutations to DNA replication during the V-J joining event is proposed.     

Arrangement of lambda light chain genes in mutant clones of the MOPC 315 mouse myeloma cells

The synthesis of lambda light chains and the arrangement of the lambda-chain genes was examined in cells of the mouse myeloma MOPC 315, which is an alpha lambda 2 producer, and in several mutants derived from it. The mutants produce lambda 2 chains only (MOPC 315.26, MOPC 315.34, and MOPC 315.37) or fail to produce alpha and lambda 2 chains (MOPC 315.25 and MOPC 315.36). Messenger RNA from the lambda 2 chain-producing cells directed the synthesis of a lambda 2 chain precursor and a fragment of the lambda 1 chain (lambda 1 F) in a wheat embryo cellfree system, whereas mRNA from the cells that do not produce lambda 2 chains directed the synthesis of lambda 1 F only. DNA from the parental MOPC 315 cells and from the lambda 2 chain-producing cells contained discrete EcoRI restriction fragments coding for rearranged lambda 1 and lambda 23 chain genes and their respective germ-line V and J-C regions. DNA from the no-Ig-producing cells contained fragments coding for the rearranged lambda 1 chain gene and the germ-line V lambda 2 region, but it lacked the sequences coding for the rearranged lambda 2 chain gene and the germ-line V lambda 1 and J-C lambda 1 regions. These results suggest that rearrangements of the lambda 1 and lambda 2 chain genes occur on different chromosomes in MOPC 315 cells and imply that rearrangements of the lambda 1 and lambda 2 chain genes on the same chromosome may be mutually exclusive.     

Allelic variants of rearranged immunoglobulin heavy and light chain genes in hybridoma PTF-02 and parent myeloma

The hybridoma PTF-02 secretes an antibody against pig transferrin. Rearranged genes for heavy and light immunoglobulin chains have been studied in the genomes of this hybridoma and in the parent myeloma P3-X63.Ag8.653. The hybridoma was shown to contain three rearranged allelic variants of the heavy chain gene's locus. The gene H2, responsible for synthesis of the heavy chain of the antibody to transferrin, was transmitted in the hybridoma cell from a lymphocyte. Two other genes (H1 and H3) were found both in the hybridoma and parent myeloma genomes. The gene H1 was identified in MOPC21 myeloma, which is a precursor of the X63.Ag8 descendent line. Rearranged k genes were also identified both in the hybridoma and parent myeloma. A functional (K2) gene and a fetal (F) gene appeared in the hybridoma genome from an antigen-stimulated normal lymphocyte. The fetal gene was lost in the course of continuous cultivation of the hybridoma PTF-02 cell line. The gene K1 was transmitted from the myeloma used for fusion. In such a way, the pedigree of rearranged heavy and light chain genes in the hybridoma PTF-02 was established. The results obtained in this work may be relevant to many hybridomas whose immortalizing fusion partner is a MOPC21 derivative, and allow one to identify and isolate functional variable genes to create recombinant constructions.     

Untranslated immunoglobulin kappa light chain mRNA in a lambda light chain-producing mouse myeloma, MOPC104E

Fourteen clones were isolated in culture from a mouse myeloma, MOPC104E. All clones had kappa and lambda types of light chain mRNAs in approximately equimolar quantity as assayed by hybridization with specific complementary DNA (cDNA). However, the myeloma produces and secretes only lambda-type light chain protein. Both kappa- and lambda-type mRNAs in these clones were indistinguishable from kappa- and lambda-type mRNAs of other myelomas with respect to (a) adsorption to oligo-(dT) cellulose, (b) molecular size (12.6 S), and (c) thermal stability of the hybrids formed with corresponding cDNA. The kappa chain mRNA of MOPC104E cells, however, was translated very inefficiently both in vivo and in vitro, whereas the lambda chain mRNA was translated efficiently. These results indicate that each cell of MOPC104E myeloma synthesizes a crippled kappa chain mRNA in addition to a normal lambda chain mRNA.     

A V region determinant (idiotope) expressed at high frequency in B lymphocytes is encoded by a large set of antibody structural genes.

Idiotope Ac38, a V region determinant of the lambda 1 chain-bearing, germ line encoded antibody B1-8, is expressed at high frequency (approximately 1/40) in lambda 1 chain-bearing B cells. Here, we describe the isolation of lambda-positive hybridomas from C57BL/6 mice which had been immunized with antibody Ac38, the antibody recognizing idiotope Ac38. In Northern blot analysis, mRNA isolated from 10 such hybridomas hybridizes with a cDNA probe from the VH gene expressed in the cell line B1-8. Amino acid sequence analysis of the VH regions of four of the hybridoma proteins reveals that they are all derived from related, though distinct, germ line VH genes. In one case the sequence data suggest that extensive somatic mutation has taken place. Only one of the four sequences derives from the same VH gene that is expressed in the cell line B1-8. Together with earlier evidence, the present data demonstrate that the Ac38 idiotope is a marker for at least five VH and three D region genes in the C57BL/6 germ line. This explains the high frequency at which this idiotope is expressed in the B cell population. In addition, our sequence determinations identify two VH genes in the C57BL/6 strain which are closely related (and possibly allelic) to two known BALB/c VH genes. One of these genes is the gene expressed in the BALB/c myeloma MOPC 104E.     

Immunoglobulin kappa-genes. Cloning, hybridization and structural analysis

The principal problems in molecular and genetic immunology to be resolved are the structure of Ig-genes and the regulation of their expression. The isolation of mRNA for light and heavy Ig-chains would be a first step along this line. A combination of two approaches may be the best strategy in mRNA preparation. Affinity purification allows one to obtain pure mRNA in a one-step procedure whereas immunoprecipitation makes it possible to prepare mRNAs for both heavy and light immunoglobulin chains in considerable amounts. In the series of experiments, conditions for synthesis of long and short cDNA chains were elaborated and the clone containing the fragment of kappa-chain gene was isolated and characterized. This clone was used as a probe for hybridization with genomic DNA from myeloma and hybridoma cells. It was shown that both allelic genes on homologous chromosomes were rearranged in myeloma MOPC21 cells. The original cell line of hybridoma PTF02 contains the embryonic gene as well as the differentiated genes. However, only differentiated genes can be detected in a similar experiment conducted with the same hybridoma after passage on mice. In conclusion, the coordination of homologous and heterologous chromosome expression in B cells in discussed in terms of the feed-back control.     

Characterization of allelic V kappa-1 region genes in inbred strains of mice

Germ line genes encoding mouse Ig kappa-chains belonging to the V kappa-1 group have been isolated from BALB/c, NZB, and CE, three inbred strains of differing kappa haplotype. The V kappa-1A and V kappa-1C germ line genes isolated from BALB/c (Ig kappa c) were identical to those previously described. These are the two major V kappa-1 germ line genes in BALB/c and together account for 40 of the 53 expressed V kappa-1 sequences that have been reported to date. Allelic differences in a single germ line variable region gene (V kappa-1A) in different strains of mice explain the differences in L chain IEF patterns previously associated with the Ig kappa-Ef2 locus. The rearranged kappa-gene expressed in the BALB/c myeloma MOPC-460 has been isolated and found to represent a V kappa-1A somatic variant differing by three nucleotides from the germ line V kappa-1A gene. Germ line genes isolated from NZB (Ig kappa b) and CE (Ig kappa f) show greater than 95% identity with the BALB/c genes over the 1700 nucleotides compared. Comparison by region indicated the greatest conservation of sequence occurs in and around the leader exon followed by the V-region exon. The NZB gene encodes the amino acid sequence found in the myeloma PC-2205, previously designated V kappa-1B. The V kappa-1 gene isolated from CE is likely an allele of the BALB/c V kappa-1C gene as the two share greater than 96% identity over 1700 nucleotides. The CE gene has been designated V kappa-1Cf. Ancient remnants of LINE-1 repetitive elements were detected approximately 400 bp downstream of all of the V kappa-1 genes. These possess greater homology with repetitive elements found near other kappa genes than they do with the native L1Md sequence.     

Somatic DNA rearrangement generates functional rat immunoglobulin kappa chain genes: the J kappa gene cluster is longer in rat than in mouse

The kappa immunoglobulin (Ig) genes from rat kidney and from rat myeloma cells were cloned and analyzed. In kidney DNA one C kappa species is observed by Southern blotting and cloning in phage vectors; this gene most likely represents the embryonic configuration. In the IR52 myeloma DNA two C kappa species are observed: one in the same configuration seen in kidney and one which has undergone a rearrangement. This somatic rearrangement has brought the expressed V region to within 2.7 kb 5' of the C kappa coding region; the rearrangement site is within the J kappa cluster which we have mapped. The rat somatic Ig rearrangement, therefore, closely resembles that seen in mouse Ig genes. In the rat embryonic fragment two J kappa segments were mapped at 2 and 4.3 kb 5' from the C kappa coding region. Therefore, the rat J kappa cluster extends over about 2.3 kb, a region much longer than the 1.4 kb of the mouse and human J kappa clusters. In the region between C kappa and the expressed J kappa of IR52 myeloma DNA, and XbaI site present in the embryonic kappa gene has been lost. A somatic mutation has therefore occurred in the intervening sequence DNA approx. 0.7 kb 3' from the V/J recombination site. Southern blots of rat kidney DNA hybridized with different rat V kappa probes showed non-overlapping sets of bands which correspond to different subgroups, each composed of 8-10 closely related V kappa genes.     

Molecular analysis of immunoglobulin heavy chain genes coding for idiotypic and anti-idiotypic antibodies involved in B-B cellular interaction.

We recently reported that a unique B cell clone (B19-1d), specific for a cross-reactive idiotype (CRI) on MOPC104E myeloma protein (M104E), enhances Igh-restricted CRI+ antibody production. In this paper, we report the nucleotide sequences of immunoglobulin heavy chain variable regions (VH) of both M104E and B19-1d-derived hybridoma (HB19) antibodies. The sequence data revealed that both belong to the J558 germ line VH gene subfamily. Strikingly, not only the VH region, but also the leader sequences of M104E and HB19 are very similar to each other at 88% (VH) and 91% (leader) homology, but they use different D and J segments. The VH region sequence similarity is highest among the germ line VH gene sequences of the BALB/c J558 subfamily so far screened. Southern hybridization data, using 5'-noncoding regions of either M104E or HB19 genomic VH gene clones as probes, revealed that both VH genes are conserved in the M104E CRI producer strains of mice. Moreover, these probes show the restriction length polymorphism pattern of mouse VH genes in various strains. That the HB19 VH gene locates to the 5' upper arm of the M104E VH gene on the chromosome was suggested by Southern blot hybridization. Immunoglobulin VH gene restriction of idiotypic and antiidiotypic B-B cellular interaction is discussed from a molecular point of view.     

Rearranged and germline immunoglobulin kappa genes: different states of DNase I sensitivity of constant kappa genes in immunocompetent and nonimmune cells

The rearrangement of a variable (V) and a constant (C) gene appears to be a necessary prerequisite for immunoglobulin gene expression. Multiple different rearranged kappa genes were found in several mouse myelomas, although these cells produce only one type of kappa chain [Wilson, R., Miller, J., & Storb, U. (1979) Biochemistry 18, 5013--5021]. It is therefore of interest to understand how only one allele within a lymphoid cell becomes expressed, while the other allele remains nonfunctional ("allelic exclusion"). We have studied the chromatin conformation of kappa genes by making use of the preferential digestion of potentially active genes by DNase I described, for example, for globin genes [Weintraub, H., & Groudine, M. (1976) Science (Washington, D.C.) 193, 848--856]. The DNase I sensitivity of kappa genes in myeloma tumors, in a B cell lymphoma, and in liver was determined by hybridization with DNA on Southern blots. It was found that rearranged C kappa genes are DNase I sensitive in myelomas in which several kappa genes are rearranged, regardless of whether the rearranged genes code for the kappa chains synthesized by the cell. Furthermore, the C kappa gene in germline configuration is also DNase I sensitive in a B cell lymphoma; i.e., it is in the same chromatin state as the rearranged C kappa gene which probably codes for the kappa chains produced by the cell. The altered chromatin state appears to be localized: V kappa genes in germline context are not DNase I sensitive in myeloma or B lymphoma cells while C kappa genes present in a kappa gene cluster on the same chromosomes are sensitive. When rearranged, however, the V kappa genes are as sensitive to DNase I as are rearranged C kappa genes. V lambda and C lambda genes are not DNase I sensitive in kappa myelomas. Thus, commitment to kappa gene expression is apparently correlated with a chromatin conformation which confers increased DNase I sensitivity to the DNA in the vicinity of all C kappa genes in the cell. "Allelic exclusion" does not operate on the level of chromatin conformation which can be detected by altered DNase I sensitivity.     

Analysis of immunoglobulin light chain rearrangements in the salivary gland and blood of a patient with Sjögren's syndrome

Patients with Sjögren's syndrome (SS) have characteristic lymphocytic infiltrates of the salivary glands. To determine whether the B cells accumulating in the salivary glands of SS patients represent a distinct population and to delineate their potential immunopathologic impact, individual B cells obtained from the parotid gland and from the peripheral blood were analyzed for immunglobulin light chain gene rearrangements by PCR amplification of genomic DNA. The productive immunglobulin light chain repertoire in the parotid gland of the SS patient was found to be restricted, showing a preferential usage of particular variable lambda chain genes (Vλ2E) and variable kappa chain genes (VκA27). Moreover, clonally related V_L chain rearrangements were identified; namely, VκA27–Jκ5 and VκA19–Jκ2 in the parotid gland, and Vλ1C–Jλ3 in the parotid gland and the peripheral blood. Vκ and Vλ rearrangements from the parotid gland exhibited a significantly elevated mutational frequency compared with those from the peripheral blood (P < 0.001). Mutational analysis revealed a pattern of somatic hypermutation similar to that found in normal donors, and a comparable impact of selection of mutated rearrangements in both the peripheral blood and the parotid gland. These data indicate that there is biased usage of V_L chain genes caused by selection and clonal expansion of B cells expressing particular V_L genes. In addition, the data document an accumulation of B cells bearing mutated V_L gene rearrangements within the parotid gland of the SS patient. These results suggest a role of antigen-activated and selected B cells in the local autoimmune process in SS.