Genes encoding Xenopus laevis Ig L chains. Implications for the evolution of kappa and lambda chains.

Xenopus laevis Ig contain two distinct types of L chains, designated rho or L1 and sigma or L2. We have analyzed Xenopus genomic DNA by Southern blotting with cDNA probes specific for L1 V and C regions. Many fragments hybridized to the V probe, but only one or two fragments hybridized to the C probe. Corresponding C, J, and V gene segments were identified on clones isolated from a genomic library prepared from the same DNA. One clone contains a C gene segment separated from a J gene segment by an intron of 3.4 kb. The J and C gene segments are nearly identical in sequence to cDNA clones analyzed previously. The C segment is somewhat more similar and the J segment considerably more similar in sequence to the corresponding segments of mammalian kappa chains than to those of mammalian lambda chains. Upstream of the J segment is a typical recombination signal sequence with a spacer of 23 bp, as in J kappa. A second clone from the library contains four V gene segments, separated by 2.1 to 3.6 kb. Two of these, V1 and V3, have the expected structural and regulatory features of V genes, and are very similar in sequence to each other and to mammalian V kappa. A third gene segment, V2, resembles V1 and V3 in its coding region and nearby 5'-flanking region, but diverges in sequence 5' to position -95 with loss of the octamer promoter element. The fourth V-like segment is similar to the others at the 3'-end, but upstream of codon 64 bears no resemblance in sequence to any Ig V region. All four V segments have typical recombination signal sequences with 12-bp spacers at their 3'-ends, as in V kappa. Taken together, the data suggest that Xenopus L1 L chain genes are members of the kappa gene family.     

Immunoglobulin light chains in medaka (Oryzias latipes)

The gene segments encoding antibodies have been studied in many capacities and represent some of the best-characterized gene families in traditional animal disease models (mice and humans). To date, multiple immunoglobulin light chain (IgL) isotypes have been found in vertebrates and it is unclear as to which isotypes might be more primordial in nature. Sequence data emerging from an array of fish genome projects is a valuable resource for discerning complex multigene assemblages in this critical branch point of vertebrate phylogeny. Herein, we have analyzed the genomic organization of medaka (Oryzias latipes) IgL gene segments based on recently released genome data. The medaka IgL locus located on chromosome 11 contains at least three clusters of IgL gene segments comprised of multiple gene assemblages of the kappa light chain isotype. These data suggest that medaka IgL gene segments may undergo both intra- and inter-cluster rearrangements as a means to generate additional diversity. Alignments of expressed sequence tags to concordant gene segments which revealed each of the three IgL clusters are expressed. Collectively, these data provide a genomic framework for IgL genes in medaka and indicate that Ig diversity in this species is achieved from at least three distinct chromosomal regions.     

Description of a fugu CXC chemokine and two CXC receptor genes, and characterization of the effects of different stimulators on their expression

The primary structures of a CXC chemokine (CXCL8) and two CXC receptors (CXCR) have been characterized in fugu, Takifugu rubripes. Unlike mammalian and avian species, CXCL8 of teleosts including fugu lacks the ELR motif that appears to be important in ligand/receptor interactions on neutrophils. Genomic organization shows that fugu CXCL8 gene consists of four exons and three introns. As in other vertebrates, two CXCR genes isolated from fugu encode proteins CXCR1 and CXCR2 that possess characteristic seven transmembrane domains. Each receptor consists of two exons separated by an intron. Synteny analysis indicates that these two CXCRs were derived from whole genome duplication in teleosts, differing from mammalian CXCR1 and CXCR2. All of these genes are primarily expressed in the lymphoid tissues. Immune stimulation with PHA showed that the expression of both CXCL8 and CXCRs in PBL are upregulated even after only a short time period, but downregulated by LPS stimulation, implying that these genes are involved in the regulation of the immune response in fugu.     

Ongoing diversification of the rearranged immunoglobulin light-chain gene in a bursal lymphoma cell line.

The chicken immunoglobulin light-chain gene (IgL) encodes only a single variable gene segment capable of recombination. To generate an immune repertoire, chickens diversify this unique rearranged VL gene segment during B-cell development in the bursa of Fabricius. Sequence analysis of IgL cDNAs suggests that both gene conversion events derived from VL segment pseudogene templates (psi VL) and non-template-derived single-base-pair substitutions contribute to this diversity. To facilitate the study of postrecombinational mechanisms of immunoglobulin gene diversification, avian B-cell lines were examined for the ability to diversify their rearranged IgL gene during in vitro passage. One line that retains this ability, the avian leukosis virus-induced bursal lymphoma cell line DT40, has been identified. After passage for 1 year in culture, 39 of 51 randomly sequenced rearranged V-J segments from a DT40 population defined novel subclones of the parental tumor. All cloned V-J segments displayed the same V-J joint, confirming that the observed diversity arose after V-J rearrangement. Most sequence variations that we observed (203 of 220 base pairs) appeared to result from psi VL-derived gene conversion events; 16 of the 17 novel single nucleotide substitutions were transitions. Based on these data, it appears that immunoglobulin diversification during in vitro passage of DT40 cells is representative of the diversification that occurs during normal B-cell development in the bursa of Fabricius.     

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.     

Chicken IgL gene rearrangement involves deletion of a circular episome and addition of single nonrandom nucleotides to both coding segments

Chicken immunoglobulin light chain (IgL) gene rearrangement has been characterized. Rearrangement of the single variable (VL) segment with the single joining (JL) segment within the chicken IgL locus results in the deletion of the DNA between VL and JL from the genome. This deletion is accomplished by a molecular mechanism in which a precise joining of the IgL recombination signal sequences leads to the formation of a circular episomal element. The circular episome is an unstable genetic element that fails to be propagated during B cell development. Evidence was obtained that the formation of the circular episome is accompanied by the addition of a single nonrandom base to both the VL and JL coding segments. The subsequent joining of the VL and JL segments appears to occur at random, as we observed at least 25 unique V-J junction sequences, 11 of which are out-of-frame. A novel recombination mechanism that accounts for the observed features of chicken IgL gene rearrangement is discussed.     

Similarities and differences between the light and heavy chain Ig variable region gene repertoires in chronic lymphocytic leukemia

Analyses of Ig V(H)DJ(H) rearrangements expressed by B-CLL cells have provided insights into the antigen receptor repertoire of B-CLL cells and the maturation stages of B-lymphocytes that give rise to this disease. However, less information is available about the L chain V gene segments utilized by B-CLL cells and to what extent their characteristics resemble those of the H chain. We analyzed the V(L) and J(L) gene segments of 206 B-CLL patients, paying particular attention to frequency of use and association, mutation status, and LCDR3 characteristics. Approximately 40% of B-CLL cases express V(L) genes that differ significantly from their germline counterparts. Certain genes were virtually always mutated and others virtually never. In addition, preferential pairing of specific V(L) and J(L) segments was found. These findings are reminiscent of the expressed VH repertoire in B-CLL. However unlike the V(H) repertoire, V(L) gene use was not significantly different than that of normal B-lymphocytes. In addition, Vkappa genes that lie more upstream on the germline locus were less frequently mutated than those at the 3' end of the locus; this was not the case for Vlambda genes and is not for V(H) genes. These similarities and differences between the IgH and IgL V gene repertoires expressed in B-CLL suggest some novel features while also reinforcing concepts derived from studies of the IgH repertoire.     

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.     

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 human T cell antigen receptor is encoded by variable, diversity, and joining gene segments that rearrange to generate a complete V gene

A cDNA clone YT35 , synthesized from poly(A)+ RNA of the human T cell tumor Molt 3, exhibits homology to the variable (V), joining (J), and constant (C) regions of immunoglobulin genes. We have isolated and sequenced the germ-line V and J gene segment counterparts to YT35 from a human cosmid library, and these failed to encode 14 nucleotides of the cDNA clone between the V and J regions. We postulate that these 14 nucleotides are encoded by a third gene segment analogous to the diversity (D) gene segments of immunoglobulin heavy chain genes. This T cell antigen receptor V gene appears to be assembled from three gene segments, V, D, and J, and accordingly most closely resembles immunoglobulin heavy chain V genes.     

Diverse immunoglobulin light chain organizations in fish retain potential to revise B cell receptor specificities

We have characterized the genomic organization of the three zebrafish L chain isotypes and found they all differed from those reported in other teleost fishes. Two of the zebrafish L chain isotypes are encoded by two loci, each carrying multiple V gene segments. To understand the derivation of these L chain genes and their organizations, we performed phylogenetic analyses and show that IgL organization can diverge considerably among closely related species. Except in zebrafish, the teleost fish IgL each contain only two to four recombinogenic components (one to three V, one J) and exist in multiple copies. BCR heterogeneity can be generated, but this arrangement apparently provides neither combinatorial diversification nor an opportunity for the secondary rearrangements that, in mammals, take place during receptor editing, a process crucial to the promotion of tolerance in developing lymphocytes. Examination of the zebrafish IgL recombination possibilities gave insight into how the suppression of self-reactivity by receptor editing might be managed, including in miniloci. We suggest that, despite the diverse IgL organizations in early and higher vertebrates, two elements essential to generating the Ab repertoire are retained: the numerous genes/loci for ligand-binding diversification and the potential for correcting unwanted specificities that arise.     

Isolation and characterization of cDNA clones for mouse Ly-9.

We describe the production and characterization of a mouse mAb, S-450-33.2, recognizing the Ly-9.2 specificity. This mAb was used to purify Ly-9 molecules from lymphoid cell lines, and the amino-terminal amino acids were determined. The mAb was also used in a eukaryotic expression system, to isolate cDNA clones encoding Ly-9. Analysis of RNA showed that Ly-9 expression is lymphocyte specific, as determined by the presence of a single hybridizing 2.4-kb species found only in lymphoid cells. Genomic DNA analysis indicated that Ly-9 is encoded by a single-copy gene of 10 to 15 kb. The predicted polypeptide belongs to the Ig superfamily of cell surface molecules with four extracellular Ig-like domains, i.e., a non-disulfide-bonded V domain, a truncated C2 domain with two disulfide bonds, a second non-disulfide-bonded V domain, and a truncated C2 domain with two disulfide bonds (V-C2-V-C2). The sequence data also support the view that Ly-9 belongs to the subgroup of the Ig superfamily that includes Bcm-1, CD2, and LFA-3.