Sequence of C region of L chains from Xenopus laevis Ig

A cDNA expression library, prepared from Xenopus laevis splenocytes, was screened with antibodies to Xenopus Ig. One clone, lambda XIg23, reacted with antibodies to IgY and to IgM; the insert hybridized to approximately 1.3-kb RNA from spleen, the approximate size expected for L chain mRNA. An additional clone, lambda XIg31, was identified by cross-hybridization. The inserts of lambda XIg23 and lambda XIg31 begin in the third framework region of the V region and extend through the C region to the poly(A) tail. Except for a single nucleotide difference, the two C region sequences are identical. The amino acid sequence of the C region was compared with the sequences of a variety of C kappa and C lambda, as well as to C region sequences of L chains from Rana catesbeiana and from two species of shark. The Xenopus C region resembles mouse and human C kappa slightly more than C lambda. The similarity of the Xenopus and Rana C regions to each other is approximately the same as that of either amphibian sequence to mammalian CL. The data are discussed in terms of the evolution of kappa and lambda C regions.     

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.     

Aberrant recombination events in B cell lines derived from a kappa-deficient human.

We have analyzed the structure of Ig kappa chain genes in B cell lines derived from a human individual who cannot synthesize any kappa chains, and whose Igs all contain lambda chains (1). We have characterized secondary DNA recombination events at two kappa alleles which have undergone misaligned V-J recombinations. One such secondary recombination has joined the flanking sequences of a V kappa and a J kappa 2 gene segment as if it were the reciprocal product of a V-J kappa 2 recombination, and resulted in the displacement of the recombined VJ kappa 1 gene segments from the C kappa locus. The non-rearranged form of the V kappa fragment which had recombined with the J kappa 2 flank was cloned. Nucleotide sequencing of this fragment identified a V kappa gene that differed by at least 38% from all previously sequenced human V kappa genes. The other V-J kappa segment analyzed has undergone a secondary recombination at a different site from that described above, at a site within the intervening sequence between the J kappa and C kappa gene segments, similar to the location of secondary recombinations which have occurred in lambda + B cell lines from mice and humans (2,3). These results prove that multiple recombinations can occur at one J kappa-C kappa locus.     

Sequence similarities and cross-idiotypic specificity of L chains among human monoclonal IgM kappa with anti-gamma-globulin activity

The complete amino acid sequence of five light chain variable (V) regions of human monoclonal IgM kappa rheumatoid factors (RF) was determined, and their cross-reactive idiotypes (CRI) were characterized with antibodies induced by immunization with synthetic peptides PSL2 and PSL3, corresponding to the second and third complementarity-determining regions (CDR) of the SIE light chain. Together with two additional RF studied previously, all seven RF belong to the V kappa IIIb sub-subgroup. The region encoded by the V kappa gene segment (positions 1 to 95) in all seven proteins was virtually identical in primary structure, whereas the sequence from positions 96 to 108 defined the usage of the J kappa 1 gene in three proteins and the J kappa 2 gene in four of them. Position 96 contributed by the recombination of the V kappa and J kappa gene segments showed the presence of four different amino acid residues. Both anti-PSL2 and anti-PSL3 bind efficiently to all separated L chains when analyzed by the Western blot technique, and the binding was inhibited specifically by the corresponding peptides. The results reveal that the majority of human IgM-RF light chains are derived from a single germ line V kappa gene or a family of closely related V kappa III germ line genes, and express two "primary structure-dependent" CRI, which are largely dependent on the amino acid sequence of the second and third light chain CDR.     

Gene mutations and alternate RNA splicing result in truncated Ig L chains in human gamma H chain disease

The lack of covalently associated L chains features H chain disease proteins produced in some human B cell lymphoproliferative disorders. We cloned and characterized the single rearranged kappa L chain gene from the leukemic lymphocytes of a patient (RIV) affected with gamma 1 H chain disease, to determine the molecular basis for absent L chain. This kappa allele had undergone an effective V-J rearrangement. Extensive somatic mutation focused about the V-J region created a sequence that was only 75% homologous to its germ-line counterpart. Altered acceptor (V kappa) and donor (J kappa) splice sites resulted in an aberrant splice between the leader and C kappa exons and a truncated 850-bp kappa mRNA. RIV leukemic cells as well as myeloma cells transfected with the RIV kappa gene synthesized a truncated protein. Simultaneous defects in H and L chains genes may reflect a hypermutational mechanism for Ig genes in B cells.     

Chicken IgA H chains. Implications concerning the evolution of H chain genes.

A cDNA clone (A1) encoding for a novel chicken Ig H chain isotype was isolated. In sequence comparison to mammalian H chains, A1 C region was most closely related to the alpha isotype. For example, identities of 35%, 32%, and 31% at the amino acid level to rabbit C alpha, C mu, and C gamma were observed, respectively. Distribution of the glucosamine acceptor sites (Asn-X-Ser/Thr) in A1 C region was typical of alpha H chains. Moreover, A1 C region probe hybridized to a 2.2-kb RNA species expressed in the epithelial lymphoid tissues. Thus, A1 was identified as the avian homologue for mammalian alpha H chains. Interestingly, the chicken C alpha was structurally consistent with four complete CH domains, whereas only three domains are present in the mammalian C alpha genes. In addition, interdomain sequence alignments suggested that the homologue for the chicken C alpha 2 domain is missing from the mammalian alpha H chains. Thus, the present data suggest evolution of the IgA isotype before the segregation of avian and mammalian species. Also, the first C alpha gene may have consisted of four CH domains, whereas reorganization of the C alpha 2 region led to the generation of hinge region in the mammalian alpha H chains.     

Sequences of mouse immunoglobulin light chain genes before and after somatic changes.

We have determined the nucleotide sequences of the germ line gene as well as a corresponding somatically mutated and rearranged gene coding for a mouse immunoglobulin lambdaI type light chain. These sequencing studies were carried out on three Eco RI-DNA fragments which had been cloned from BALB/c mouse embryos or a lambdaI chainsecreting myeloma, H2020. The embryonic DNA clone Ig 99lambda contains two protein-encoding segments, one for the majority of the hydrophobic leader (L) and the other for the rest of the leader and the variable (V) region of the lambda0 chain (Cohn et al., 1974); these segments are separated by a 93 base pair (bp) intervening sequence (I-small). The coding of the V region ends with His at residue 97. The second embryonic DNA clone Ig 25lambda includes a 39 bp DNA segment (J) coding for the rest of the conventionally defined V region (that is, up to residue 110), and also contains the sequence coding for the constant (C) region approximately 1250 untranslated bp (I-large) away from the J sequence. The J sequence is directly linked with the V-coding sequence in the myeloma DNA clone, Ig 303lambda, which has the various DNA segments arranged in the following order: 5' untranslated region, L, l-small, V linked with J, l-large, C, 3' untranslated sequence. The lg 303lambda V DNA sequence codes for the V region synthesized by the H2020 myeloma and is different from the lg 99lambda V DNA sequence by only two bases. No silent base change was observed between the two DNA clones for the entire sequence spanning the 5' untranslated regions and the V-coding segments. These results confirm the previously drawn conclusion that an active complete lambdaI gene arises by somatic recombination that takes place at the ends of the V-coding DNA segment and the J sequence. No sequence homology was observed at or near the sites of the recombination.     

The kinetics of V-J joining throughout 3.5 megabases of the mouse Ig kappa locus fit a constrained diffusion model of nuclear organization

To gain insight into the nuclear organization of the mouse Ig kappa locus and how it may relate to the formation of synapses during recombination, we have studied the kinetics of rearrangement of different V kappa gene families to J kappa gene segments in the pre-B cell line, 103bcl2. Remarkably, V kappa gene families separated by more than 3.5 Mb from J kappa gene segments rearranged with nearly identical kinetics to those as close as 18 kb to J kappa gene segments. These results fit a model of nuclear organization in which the entire V kappa J kappa region resides within a single nuclear subcompartment and is capable of exhibiting multiple reversible contacts through diffusion and Brownian motion.     

Somatic hypermutation of immunoglobulin kappa may depend on sequences 3'' of C kappa and occurs on passenger transgenes.

We have compared the pattern of somatic mutation in different immunoglobulin kappa transgenes and suggest that an element(s) located between 1 kb and 9 kb 3' of C kappa is necessary for somatic hypermutation of the antibody V gene. The sequences of transgenic and endogenous Ig V regions were determined in antigen-specific B cell hybridomas specific for 2-phenyloxazolone from independent lines of hyperimmunized transgenic mice. We analysed somatic mutation of the transgene both in hybridomas in which the transgenic kappa chain contributes to the antigen combining site as well as in hybridomas in which the transgene is a passenger with the expressed antibody being composed of endogenously-encoded heavy and light chains. In both cases, nucleotide changes in the transgene are correctly targeted to the V region and are absent from the C region. They accumulate at a similar rate to that in the endogenous Ig genes within the same cell and we find that, irrespective of whether or not the transgene kappa is directly selected by antigen, somatic mutation occurs at a similar rate and involves only single base substitutions. Furthermore, the pattern of mutations in passenger transgenes gives information about the intrinsic sequence specificities of the somatic hypermutation mechanism.     

In vitro and in vivo expression of a nephritogenic Ig heavy chain determinant: pathogenic autoreactivity requires permissive light chains

Lymphocyte antigen receptors are promising targets for immune intervention strategies in disorders marked by repertoire skewing or expansion of lymphocyte subsets. Appropriate application of immune receptor modulation is predicated on understanding the role of a particular receptor in pathogenesis and disease regulation. The VHB/W16 gene, restricted to mice carrying the j haplotype for the J558 family, is overexpressed by murine lupus anti-DNA Ig. This gene is also expressed recurrently among nephritogenic anti-DNA Ig recovered from several autoimmune strains, suggesting that cells expressing this pathogenic receptor are positively selected during disease progression. To explore the extent and mechanisms by which Ig H chains expressing this gene contribute to autoimmunity, an Ig H chain gene was engineered for in vitro and in vivo recombination studies. Site-directed mutagenesis generated unique restriction sites to link PCR-amplified V region (VDJ) cDNA to previously isolated genomic fragments containing Ig regulatory and signal sequences. The new 3 kb VDJ gene was then ligated to a 9 kb fragment encoding the IgM constant region. Transfection of H chain loss variant myeloma with the complete 12 kb construct, termed 238H-Cmicro, resulted in secretion of intact Ig pairing 238H-Cmicro, with a lambda L chain; however, transfectant Ig lacked autoreactivity and pathogenicity. Introduction of the 238H-Cmicro H chain as a transgene onto the non-autoimmune C57BL/6 background resulted in abundant B cell surface expression of 238H-Cmicro, however, four transgenic Ig recovered by fusion of LPS-stimulated splenocytes and formed by combination of 238H-Cmicro, with endogenous kappa chains do not bind DNA or laminin. These results indicate that the antigen binding sites encoded by this disease-associated gene and/or H chain must associate with permissive L chains to specify autoimmunity. The 238H-Cmicro, transgenic model should prove useful in dissecting the in vivo fate of 238H-Cmicro, L combinations that produce pathogenic autoreactive receptors and in evaluating receptor-targeted interventions.     

Somatic point mutations in unrearranged immunoglobulin gene segments encoding the variable region of lambda light chains.

Somatic point mutations are usually found in the coding and flanking regions of functionally and aberrantly rearranged immunoglobulin variable region gene segments. Mutations in the unrearranged V gene segments of myelomas or hybridomas have not been described so far. We have cloned and sequenced unrearranged V lambda gene segments from several cell lines. There were no nucleotide changes in four unrearranged V lambda segments: one V lambda 1 from a lambda 3-producing hybridoma and one V lambda 2 from a lambda 1-producing myeloma (J558) and two V lambda 2 from a kappa-producing myeloma (P3X63). However, we found somatic mutations in the unrearranged V lambda segments from the lambda 2-producing myeloma MOPC315. The unrearranged V lambda 1 gene segment had two mutations in the coding region and the unrearranged V lambda 2 had one mutation in the 3' flanking region. We also cloned and sequenced the unrearranged J lambda and C lambda gene segments of MOPC315 and found no sequence alterations. This is consistent with the notion that the overall mutation rate is not higher in this cell line. Therefore, we suggest that the somatic hypermutation system can use unrearranged V gene segments as substrates. The extensive sequencing required for this work revealed a number of errors in the reported nucleotide sequences of the Ig lambda locus in BALB/c mice.     

Structural organization of the proopiomelanocortin gene in Xenopus laevis. 5'-end homologies within the toad and mammalian genes

This study reports the isolation and characterization of the entire proopiomelanocortin (POMC) gene of the amphibian Xenopus laevis. The Xenopus POMC gene consists of three exons of which the main exon 3 codes for all of the bioactive domains of the precursor protein. Intron A (2.6 kb) separates the segments encoding the 5'-untranslated mRNA region and intron B (2.5 kb) interrupts the protein-coding sequence near the signal peptide coding region. In that this structural organization of the Xenopus POMC gene is similar to those of the mammalian genes, apparently the POMC gene has been remarkably stable during 350 million years of vertebrate evolution. A comparative analysis of the 5'-flanking sequences of the Xenopus and mammalian POMC genes reveals the presence of several conserved regions. One of these regions is homologous with sequences located upstream of the capping sites of other glucocorticoid-regulated genes and another region contains a segment reminiscent of a viral enhancer consensus sequence.     

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.     

Two induced anti-Z-DNA monoclonal antibodies use VH gene segments related to those of anti-DNA autoantibodies

The cDNA for H and L chain V regions of two anti-Z-DNA mAb, Z22 and Z44, were cloned and sequenced. These are the first experimentally induced anti-nucleic acid antibody sequences available for comparison with autoantibody sequences. Z22 and Z44 are IgG2b and IgG2a antibodies from C57BL/6 mice. They recognize different facets of the Z-DNA structure. They both use VH10 family genes and share 95% sequence base sequence identity in the VH and leader sequences; however, they differ in the 5'-untranslated region of the VH mRNA, indicating they arise from different germline genes. Both use JH4 segments. They differ from each other very extensively in the CDR3 of both H and L chains. The most closely related H chains in the current GenBank/EMBL data base are two mouse IgG anti-DNA autoantibodies, one from an MRL-lpr/lpr mouse (MRL-DNA4) and one from an NZB/NZW mouse (BV04-01). Z22 and Z44 share 95% sequence identity with these antibodies in the VH segment. In addition, Z22 is identical to MRL-DNA4 at 91% of the positions in the 5'-untranslated region of the H chain mRNA. The two antibodies share 95% base sequence identity in the V kappa segment. The most closely related L chains, with 97 to 98% sequence identity, are the V kappa 10b germline gene for Z22 and the V kappa 10a germ line gene, which is associated with A/J anti-arsonate antibodies and BALB/c anti-ABO blood group substance antibodies, for Z44. Z22 and Z44 share several structural features (similarities in VH, JH, and V kappa) but differ very markedly in the L chain CDR1 and both H and L chain CDR3 sequences; these regions may determine the differences in their specific interactions with Z-DNA.     

H chain V region sequences of three human monoclonal IgM with anti-myelin-associated glycoprotein activity.

The amino acid sequence corresponding to the V region H chain gene used by three monoclonal IgM directed to the myelin-associated glycoprotein (MAG) is presented. They all belonged to the VHIII variability subgroup, but each may well represent a new member of this family inasmuch as their homology with previously sequenced VHIII genes was less than 80%. Strikingly, there was no greater homology between the H chain V regions of the anti-MAG IgM. Partial amino acid sequence data indicated that these V regions were joined to as yet unidentified DH segments; however, two H chains used very similar DH, possibly indicating that this sequence was involved in the fine specificity of the IgM for MAG. All H chains included a JHIV region. These data, together with results obtained from the sequence of the three kappa L chains of the same IgM molecules (Mihaesco, E., H. Ayadi, N. Congy, M. C. Gendron, J. P. Roy, H. Heyermann, B. Frangione, and J. C. Brouet. 1989. J. Biol. Chem. 264:21481), indicate that the repertoire of VL and VH gene segments used by anti-MAG IgM is quite diverse, in contrast to previous structural data obtained for other human monoclonal IgM autoantibodies. Possibly, these differences reflect distinct pathogenesis.     

A second gene, VpreB in the lambda 5 locus of the mouse, which appears to be selectively expressed in pre-B lymphocytes.

The murine gene lambda 5 is selectively expressed in pre-B lymphocytes. Of the three exons encoding lambda 5, exons II and III show strong homologies to immunoglobulin lambda light (L) chain gene segments, i.e. to J lambda intron and exon, and C lambda exon sequences respectively. We have now found, 4.6 kb upstream of lambda 5, another gene composed of two exons which is selectively expressed in pre-B cell lines as a 0.85 kb mRNA potentially coding for a protein of 142 amino acids including a 19 amino acid-long signal peptide. The 5' sequences of this gene show homologies to sequences encoding the variable regions of kappa and lambda L chains and of heavy (H) chains. The deduced amino acid sequence contains the consensus cysteine residues as well as other consensus amino acids at positions which characterize immunoglobulin (Ig) domains. We call the second gene VpreB. The 3' end of VpreB encoding the 26 carboxyl terminal amino acids shows no homology to any known nucleotide sequence. The putative protein encoded by VpreB is a potential candidate for association with the putative protein encoded by lambda 5, and thereby a candidate for association with H chains in pre-B cells. Southern blot analysis of DNA from liver (germ line) and 70Z/3 pre-B cell lines reveals two genes which hybridize to the VpreB gene. We call VpreB1 the gene which is found 5' of lambda 5. The other gene, called VpreB2, which has not yet been located within the genome, shows 97% nucleotide sequence homology to VpreB1 in an area of 1 kb which covers the coding region of the gene.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evolution of immunoglobulin light chain genes: analysis of Xenopus IgL isotypes and their contribution to antibody diversity.

The amphibian Xenopus laevis expresses several types of immunoglobulin light chain (IgL). cDNA clones for two IgL isotypes, C sigma 1 and C sigma 2, were analysed. C sigma is expressed in spleen and mitogen-stimulated B cells, like another Xenopus IgL type, termed C rho. C sigma shares less than 33% residues with C rho or with CL regions of shark, chicken and mammals. This suggests that C sigma diverged from a common ancestor of CL regions before or at the emergence of amphibians. Two families of VL elements, V sigma 1 and V sigma 2 are associated with C sigma (but not with C rho). They rearrange to their own set of JL elements, J sigma 1 and J sigma 2, which are poorly related to other J elements of the Ig gene family. The Xenopus genome contains a few V sigma 2 and multiple V sigma 1 elements (comparable with mammalian V kappa), but only two C sigma genes. Thus, the organization and expression of Xenopus IgL loci are apparently similar to mammalian IgL loci but different from shark and chicken IgL loci. Only a few VL elements are expressed, since cDNA clones show extensive sharing of CDR1 and CDR2 sequences; some clones differ only in CDR3. Rearranging VL and JL elements increases CDR3 diversity in both V sigma families, but abortive rearrangements are frequent in V sigma 1 regions. The very poor heterogeneity of expressed VL elements therefore appears to limit antibody diversity in Xenopus.     

Polymorphism of kappa variable region (V kappa-1) genes in inbred mice: relationship to the Ig kappa-Ef2 serum light chain marker

We describe here the cloning and complete sequence analysis of the rearranged kappa variable region gene from the V kappa-1A-producing myeloma (C.AL20-TEPC-105). A 5'-flanking region probe from the V kappa-1A gene has been used to study the V kappa-1 germ-line gene family in strains of mice differing at the Ig kappa-Ef2 locus. All Ef2a strains examined possess an identical pair of BamHI restriction fragments strongly hybridizing to the 5' probe. Surprisingly, only two of the six strains of mice previously designated Ef2b (NZB and C58) possessed clearly altered restriction fragment sizes for V kappa-1 genes. The remaining four Ef2b strains, namely BDP/J, CE/J, I/LnJ, and P/J, appear to carry V kappa-1 genes similar to those of Ef2a strains. It is suggested that these strains may carry a third form of the V kappa-1A gene, differing in the protein coding region but indistinguishable at the DNA level with the use of BamHI or EcoRI. Alternatively, these strains may fail to express V kappa-1A light chains due to a regulatory defect involving this subgroup of kappa genes.