The frequency of multiple recombination events occurring at the human Ig kappa L chain locus

Products of Ig kappa L chain gene rearrangement in a variety of human B cell samples were investigated by sequential Southern blot hybridization analysis. By application of four region-specific probes (C kappa, J kappa, U' kappa and kappa de) a complete spectrum of kappa rearrangements, including both predicted and novel products, were detected. Nearly 30% of the products detected reflect multiple recombination of the kappa locus. The kappa-deleting element was responsible for 70% of the multiple rearrangements that were detected. Interestingly, eight kappa-expressing samples exhibited rearrangement of the kappa-deleting element. The remaining multiple recombination products were characteristic of double V kappa-J kappa rearrangement. This frequency reveals that secondary V-J rearrangement may significantly contribute to the expression of kappa L chains in humans.     

Linkage between a gene for a serum protein and the gene for the Ig kappa light chain in rabbits.

A rabbit serum protein (Prt) that migrates ahead of transferrin in polyacrylamide gel electrophoresis is inherited in a simple Mendelian fashion. The gene for this protein is linked to the gene for the Ig kappa light chain and the degree of linkage is calculated as 23.5 +/- 4.7 centimorgans.     

Identification of enhancer sequences 3' of the rabbit Ig kappa L chain loci

The rabbit is useful for studies of Ig L chain gene expression because of a great disparity in expression of two isotypic forms of the kappa L chain. Normally, K1 is expressed at high levels and K2 is almost silent; expression of K2 increases in mutant or experimentally allotype-suppressed animals. The reasons for the preferential utilization of the K1 isotype have not been fully elucidated. We were interested in looking for second enhancers 3' of the C kappa genes because the absence of a 3' enhancer in the K2 locus could explain the preferential utilization of the K1 isotype. However, we found a strong region of enhancer activity about 7 kb downstream of the C kappa 2 gene. Sequences in this region are highly conserved between rabbit, man, and mouse. There also appears to be a homologous 3' enhancer region in the rabbit K1 locus. We also confirmed earlier reports that the rabbit K1 intron enhancer is inactive in transient transfections into mouse B cells but find that the same construct has low but significant activity in a human B cell line. In a comparable construct the K2 intron enhancer is without activity suggesting possible differential activity of the intronic enhancers.     

Differential accessibility at the kappa chain locus plays a role in allelic exclusion

Gene rearrangement in the immune system is always preceded by DNA demethylation and increased chromatin accessibility. Using a model system in which rearrangement of the endogenous immunoglobulin kappa locus is prevented, we demonstrate that these epigenetic and chromatin changes actually occur on one allele with a higher probability than the other. It may be this process that, together with feedback inhibition, serves as the basis for allelic exclusion.     

DNA conformational variations in the in vitro torsionally strained Ig kappa light chain gene localize on consensus sequences

We have analyzed the localization and the dependence upon superhelical density of the DNA sites which modify their conformation under torsional strain in a mouse Ig L kappa gene. The conformational variations occur on DNA sites which have been defined as protein interaction sites and consensus sequence motifs: the 5'-upstream regulatory decanucleotides, the TATA sequence, the consensus heptanucleotides of the J recombinational sequences.     

Beta-globin gene cluster haplotypes in the Corsican and Sardinian populations

The distribution of beta-globin cluster haplotypes has been studied in the populations of Corsica (France) and Sardinia (Italy). The analysis was carried out using five restriction fragment length polymorphism markers on chromosome 11 inside the beta-globin cluster using the restriction enzymes HincII and HindIII. The results show a remarkable heterogeneity within the two islands. However, the presence of rare haplotypes common to the most conservative areas (Nuoro and Corte) of the two islands is particularly interesting. These data support the hypothesis of a common origin of the populations of Sardinia and Corsica during the middle and upper Paleolithic periods and could be interpreted as a founder effect.     

Block in development at the pre-B-II to immature B cell stage in mice without Ig kappa and Ig lambda light chain

Silencing individual C (constant region) lambda genes in a kappa(-/-) background reduces mature B cell levels, and L chain-deficient (lambda(-/-)kappa(-/-)) mice attain a complete block in B cell development at the stage when L chain rearrangement, resulting in surface IgM expression, should be completed. L chain deficiency prevents B cell receptor association, and L chain function cannot be substituted (e.g., by surrogate L chain). Nevertheless, precursor cell levels, controlled by developmental progression and checkpoint apoptosis, are maintained, and B cell development in the bone marrow is fully retained up to the immature stage. L chain deficiency allows H chain retention in the cytoplasm, but prevents H chain release from the cell, and as a result secondary lymphoid organs are B cell depleted while T cell levels remain normal.     

Contributions of amino acid side chains to the kinetics and thermodynamics of the bivalent binding of protein L to Ig kappa light chain

Protein L is a bacterial surface protein with 4-5 immunoglobulin (Ig)-binding domains (B1-B5), each of which appears to have two binding sites for Ig, corresponding to the two edges of its beta-sheet. To verify these sites biochemically and to probe their relative contributions to the protein L-Ig kappa light chain (kappa) interaction, we compared the binding of PLW (the Y47W mutant of the B1 domain) to that of mutants designed to disrupt binding to sites 1 and 2, using gel filtration, BIAcore surface plasmon resonance, fluorescence titration, and solid-phase radioimmunoassays. Gel filtration experiments show that PLW binds kappa both in 1:1 complexes and multivalently, consistent with two binding sites. Covalent dimers of the A20C and V51C mutants of PLW were prepared to eliminate site 1 and site 2 binding, respectively; both the A20C and V51C dimers bind kappa in 1:1 complexes and multivalently, indicating that neither site 1 nor site 2 is solely responsible for kappa binding. The A20R mutant was designed computationally to eliminate site 1 binding while preserving site 2 binding; consistent with this design, the A20R mutant binds kappa in 1:1 complexes but not multivalently. To probe the contributions of amino acid side chains to binding, we prepared 75 point mutants spanning nearly every residue of PLW; BIAcore studies of these mutants revealed two binding-energy "hot spots" consistent with sites 1 and 2. These data indicate that PLW binds kappa at both sites with similar affinities (high nanomolar), with the strongest contributions to the binding energy from Tyr34 (site 2) and Tyr36 (site 1). Compared to other protein-protein complexes, the binding is insensitive to amino acid substitutions at these sites, consistent with the large number of main chain interactions relative to side chain interactions. The strong binding of protein L to Ig kappa light chains of various species may result from the ambidextrous binding of the B1-B5 domains and the unimportance of specific side chain interactions.     

Variation in the crossover point of kappa immunoglobulin gene V-J recombination: evidence from a cryptic gene

Analysis of amino acid and nucleotide sequences of kappa immunoglobulin chains and their genes has led to the hypothesis that the exact site of V-J joining in these genes varies and that this variation is partially responsible for generating amino acid diversity at the recombination site. To assess this hypothesis we have cloned and determined the sequence of one of the two V-J recombinant genes carried by the plasmacytoma MOPC173 and the V region germline precursor of this gene. We find that this V region has been joined to a J segment at a crossover point that indeed differs from the one previously described. This recombination has occurred in such a way as to produce an out of phase, missense reading frame and, hence, a cryptic light chain gene. This result directly supports the cross-over-point variation hypothesis but also indicates that the flexibility of this reaction is accompanied by a cost to the organism in terms of the generation of missense genes.     

The rheumatoid factor reactivity of a human IgG monoclonal autoantibody is encoded by a variant V kappa II L chain gene

To determine the genetic and molecular basis for rheumatoid factor (RF) autoantibody reactivity in patients with destructive, erosive arthritis, we established a human lymphoblastoid cell line (hRF-1) from a patient with polyarthritis that produced an IgG RF mAb, mAb hRF-1. Studies of isolated H and L chains showed that the specificity of RF reactivity is conferred by mAb hRF-1 L chains. The L chain gene was cloned from a cDNA library prepared from hRF-1 cells. The nucleotide sequence was similar to known V kappa II L chains except for a two nucleotide change corresponding to a change of two amino acids in an invariable region of FR3. A germ-line gene with one of the nucleotide changes was identified by polymerase chain reaction in multiple cell lines, including K562 that does not rearrange Ig genes, but the other nucleotide change appeared to be due to mutation. Either or both of these amino acid changes may contribute to the RF reactivity, because an antibody with the same V kappa II L chain except for these two amino acid changes in FR3 did not have RF reactivity. The RF reactivity of isolated L chains from mAb hRF-1 was confirmed by transfecting COS cells with an expression vector encoding the hRF-1 kappa-chain and showing that the secreted k-chains had RF reactivity. Expression of this variant V kappa II L chain gene may form the basis for RF autoantibody reactivity in some patients.     

Antigen-binding repertoire and Ig H chain gene usage among B cell hybridomas from normal and autoimmune mice

LPS-stimulated B cells were used to generate a panel of mAb that were a random sample of the preimmune repertoire of C57BL/6 and highly autoimmune, viable motheaten mice. These mAb were tested for reactivity to a number of "self" and foreign Ag. Binding that could be detected only at nM mAb concentrations or less was considered significant. We found that a surprisingly high number of the mAb bound one or more of the Ag tested, and many mAb bound more than a single Ag. Ag-induced mAb were likewise tested and found to have greatly reduced cross-reactivities. We found no significant differences, either in frequency of Ag binding or degree of cross-reactivity, between normal and autoimmune mice. Furthermore, the frequency with which a given Ag was bound by our panel of mAb was found to be proportional to the size of the Ag. The frequency with which individual VH gene families were expressed by our panel was consistent with a stochastic usage of VH genes in the preimmune repertoire. We interpret these data as showing that the preimmune repertoire is highly cross-reactive and that the activation of autoreactive clones in autoimmune animals is due to a defect in cellular regulation rather than a difference in repertoire.     

HLA genes and haplotypes in Ryukyuans suggest recent gene flow to the Okinawa Islands.

Polymorphism of HLA genes was investigated in a population sample of Ryukyuans living on the main island of Okinawa (n = 197), in the southwestern islands of Japan. Serological typing was applied to class I loci (HLA-A, -B, and -C) and to HLA-DRB1; nucleotide sequence-level typing was performed using PCR microtiter plate hybridization and PCR single-strand conformation polymorphism methods. Ryukyuans showed a higher frequency of DRB1*0405 and lower frequencies of DRB1*1502 and DRB1*1302 compared with Hondo Japanese living on main islands. Principal components and phylogenetic analyses of 12 East Asian populations, including Ryukyuans, were performed based on the allele frequencies of HLA-A, -B, and -DRB1. In the principal components analysis 3 Japanese populations (Ryukyuans, Hondo Japanese, and Ainu) formed a cluster and showed the highest affinity to 2 Korean populations. In the phylogenetic tree Ryukyuans and Ainu were neighbors, but the genetic distance between them was larger than the distances between Ryukyuans and Hondo Japanese and between Ryukyuans and Korean populations. The geographic cline of the predominant haplotype in Ryukyuans, A*24-B*54-DRB1*0405, suggests that an ancestral population possessing A*24-B*54-DRB1*0405 moved into the Okinawa Islands after the divergence of Ryukyuans from the Ainu. Such a recent gene flow, probably from South China to the Okinawa Islands, is considered the major cause of difference in genetic characteristics between Ryukyuans and the Ainu.     

Multiple sequences related to a constant-region kappa light chain gene in the rabbit genome

Four allelic genes control kappa light chain allotypes in the rabbit. Amino acid sequence studies have revealed an extensive divergence (22%--33%) in the alternative forms of the kappa constant region (b4, b5, b6 and b9). Furthermore, independent studies have shown that a rabbit could express a wrong allotype. To assess the hypothesis that b allotypes are encoded by duplicated genes with a polymorphic control mechanism, we have analyzed the DNAs of four different homozygous rabbits using the Southern blot hybridization technique, with a cloned b4 C kappa probe. DNAs of individual b4, b5, b6 and b9 rabbits were cleaved with Eco RI, Kpn I and Pst I restriction endonucleases. Comparative analysis of restriction patterns shows that all four rabbit DNAs contain multiple DNA fragments hybridizing with the probe under low- and high-stringency washing conditions. In addition, each restriction pattern is distinct, suggesting that the C kappa genes are organized differently in animals expressing different allotypes.     

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.