Somatic hypermutation and junctional diversification at Ig heavy chain loci in the nurse shark

We estimate there are approximately 15 IgM H chain loci in the nurse shark genome and have characterized one locus. It consists of one V, two D, and one J germline gene segments, and the constant (C) region can be distinguished from all of the others by a unique combination of restriction endonuclease sites in Cmu2. On the basis of these Cmu2 markers, 22 cDNA clones were selected from an epigonal organ cDNA library from the same individual; their C region sequences proved to be the same up to the polyadenylation site. With the identification of the corresponding germline gene segments, CDR3 from shark H chain rearrangements could be analyzed precisely, for the first time. Considerable diversity was generated by trimming and N addition at the three junctions and by varied recombination patterns of the two D gene segments. The cDNA sequences originated from independent rearrangements events, and most carried both single and contiguous substitutions. The 53 point mutations occurred with a bias for transition changes (53%), whereas the 78 tandem substitutions, mostly 2-4 bp long, do not (36%). The nature of the substitution patterns is the same as for mutants from six loci of two nurse shark L chain isotypes, showing that somatic hypermutation events are very similar at both H and L chain genes in this early vertebrate. The cis-regulatory elements targeting somatic hypermutation must have already existed in the ancestral Ig gene, before H and L chain divergence.     

The evolution of multiple isotypic IgM heavy chain genes in the shark

The IgM H chain gene organization of cartilaginous fishes consists of 15-200 miniloci, each with a few gene segments (V(H)-D1-D2-J(H)) and one C gene. This is a gene arrangement ancestral to the complex IgH locus that exists in all other vertebrate classes. To understand the molecular evolution of this system, we studied the nurse shark, which has relatively fewer loci, and characterized the IgH isotypes for organization, functionality, and the somatic diversification mechanisms that act upon them. Gene numbers differ slightly between individuals ( approximately 15), but five active IgM subclasses are always present. Each gene undergoes rearrangement that is strictly confined within the minilocus; in B cells there is no interaction between adjacent loci located > or =120 kb apart. Without combinatorial events, the shark IgM H chain repertoire is based on junctional diversity and, subsequently, somatic hypermutation. We suggest that the significant contribution by junctional diversification reflects the selected novelty introduced by RAG in the early vertebrate ancestor, whereas combinatorial diversity coevolved with the complex translocon organization. Moreover, unlike other cartilaginous fishes, there are no germline-joined VDJ at any nurse shark mu locus, and we suggest that such genes, when functional, are species-specific and may have specialized roles. With an entire complement of IgM genes available for the first time, phylogenetic analyses were performed to examine how the multiple Ig loci evolved. We found that all domains changed at comparable rates, but V(H) appears to be under strong positive selection for increased amino acid sequence diversity, and surprisingly, so does Cmicro2.     

Partitioning of rearranged Ig genes by mutation analysis demonstrates D-D fusion and V gene replacement in the expressed human repertoire

The accurate partitioning of Ig H chain V(H)DJ(H) junctions and L chain V(L)J(L) junctions is problematic. We have developed a statistical approach for the partitioning of such sequences, by analyzing the distribution of point mutations between a determined V gene segment and putative Ig regions. The establishment of objective criteria for the partitioning of sequences between V(H), D, and J(H) gene segments has allowed us to more carefully analyze intervening putative nontemplated (N) nucleotides. An analysis of 225 IgM H chain sequences, with five or fewer V mutations, led to the alignment of 199 sequences. Only 5.0% of sequences lacked N nucleotides at the V(H)D junction (N1), and 10.6% at the DJ(H) junction (N2). Long N regions (>9 nt) were seen in 20.6% of N1 regions and 17.1% of N2 regions. Using a statistical analysis based upon known features of N addition, and mutation analysis, two of these N regions aligned with D gene segments, and a third aligned with an inverted D gene segment. Nine additional sequences included possible alignments with a second D segment. Four of the remaining 40 long N1 regions included 5' sequences having six or more matches to V gene end motifs, which may be the result of V gene replacement. Such sequences were not seen in long N2 regions. The long N regions frequently seen in the expressed repertoire of human Ig gene rearrangements can therefore only partly be explained by V gene replacement and D-D fusion.     

Chromatin remodeling at the Ig loci prior to V(D)J recombination.

Rearrangement of Ig H and L chain genes is highly regulated and takes place sequentially during B cell development. Several lines of evidence indicate that chromatin may modulate accessibility of the Ig loci for V(D)J recombination. In this study, we show that remodeling of V and J segment chromatin occurs before V(D)J recombination at the endogenous H and kappa L chain loci. In recombination-activating gene-deficient pro-B cells, there is a reorganization of nucleosomal structure over the H chain J(H) cluster and increased DNase I sensitivity of V(H) and J(H) segments. The pro-B/pre-B cell transition is marked by a decrease in the DNase I sensitivity of V(H) segments and a reciprocal increase in the nuclease sensitivity of Vkappa and Jkappa segments. In contrast, J(H) segments remain DNase I sensitive, and their nucleosomal organization is maintained in mu(+) recombination-activating gene-deficient pre-B cells. These results indicate that initiation of rearrangement is associated with changes in the chromatin structure of both V and J segments, whereas stopping recombination involves changes in only V segment chromatin. We further find an increase in histone H4 acetylation at both the H and kappa L chain loci at the pro-B cell stage. Although histone H4 acetylation appears to be an early change associated with B cell commitment, acetylation alone is not sufficient to promote subsequent modifications in Ig chromatin.     

Polymorphisms in human H chain V region genes from the VHIII gene family

Polymorphisms of the Ig H chain V region (VH) genes were examined with probes from the coding and flanking regions of a gene from the largest VH gene family, VHIII. The 5'-flanking probe gave the simplest pattern and revealed the largest number of polymorphic fragments. Analysis of unrelated individuals and of families identified five polymorphic loci. Two alleles were detected for each of two of the loci, whereas a polymorphic band was scored as present or absent for the other three loci. The polymorphic fragments segregated in the expected Mendelian fashion and parental haplotypes could be assigned in all cases. Comparison of the patterns obtained with the flanking and coding region probes suggests that the human VHIII gene family is highly polymorphic and may contain several hundred V genes. This method, as well as the polymorphism detected, can be used to investigate the organization and germ-line variation of H chain V genes and their inheritance in normal individuals and in individuals with immunologic disorders.     

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.     

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 natural autoantibody repertoire of nonobese diabetic mice is highly active

Analysis of spontaneous hybridomas generated from nonobese diabetic (NOD) mice indicates that the natural autoantibody repertoire of NOD mice is highly active compared with C57BL/6 and BALB/c mice. This property of increased B cell activity is present early in life (4 wk) and persists in older mice of both sexes. Even when selected for binding to a prototypic beta cell Ag, such as insulin, NOD mAb have characteristics of natural autoantibodies that include low avidity and broad specificity for multiple Ags. Analyses of the variable region of Ig H chain (V(H)) and variable region kappa L chain genes expressed by six insulin binding mAb show that V gene segments are often germline encoded and are identical with those used by autoantibodies, especially anti-dsDNA, from systemic autoimmune disease in MRL, NZB/W, and motheaten mice. V(H) genes used by four mAb are derived from the large J558 family and two mAb use V(H)7183 and V(H)Q52 genes. The third complementarity-determining region of Ig H chain of these mAb have limited N segment diversity, and some mAb contain DNA segments indicative of gene replacement. Genetic abnormalities in the regulation of self-reactive B cells may be a feature that is shared between NOD and conventional systemic autoimmune disorders. In NOD, the large pool of self-reactive B cells may fuel autoimmune beta cell destruction by facilitating T-B cell interactions, as evidenced by the identification of one mAb that has undergone Ag-driven somatic hypermutation.     

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.     

On the genomics of immunoglobulins in the gray,short-tailed opossum <Emphasis Type="Italic">Monodelphis domestica</Emphasis>

Annotated maps of the IGH, IGK, and IGL loci in the gray, short-tailed opossum Monodelphis domestica were generated from analyses of the available whole genome sequence for this species. Analyses of their content and organization confirmed a number of previous conclusions based on characterization of complementary DNAs encoding opossum immunoglobulin heavy and light chains and limited genomic analysis, including (a) the predominance of a single immunoglobulin heavy chain variable region (IGHV) subgroup and clan, (b) the presence of a single immunoglobulin (Ig)G subclass, (c) the apparent absence of an IgD, and (d) the general organization and V gene complexity of the IGK and IGL light chain loci. In addition, several unexpected discoveries were made including the presence of a partial V to D, germline-joined IGHV segment, the first germline-joined Ig V gene to be found in a mammal. In addition was the presence of a larger number of IGKV subgroups than had been previously identified. With this report, annotated maps of the major histocompatibility complex, T-cell receptor, and immunoglobulin loci have been completed for M. domestica, the only non-eutherian mammalian species for which this has been accomplished, strengthening the utility of this species as a model organism. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Ig V region gene expression in small lymphocytic lymphoma with little or no somatic hypermutation

Using the polymerase chain reaction we examined for specific Ig kappa-L chain V region gene (V kappa gene) rearrangement in small lymphocytic non-Hodgkin's lymphomas that express Ig bearing a major kappa-L chain associated cross-reactive Id, designated 17.109. Previously, we identified the 17.109-cross-reactive Id in chronic lymphocytic leukemia as a serologic marker for expression of a highly conserved V kappa gene, designated Humkv325. Using sense-strand oligonucleotides specific for the 5'-end of this V kappa gene and antisense oligonucleotide specific for a J kappa region consensus sequence, we could amplify specifically Humkv325 when juxtaposed with J kappa through Ig gene rearrangement. This allowed us to amplify rearranged V kappa genes from DNA isolated from minute amounts of lymphoma biopsy material for molecular analyses. Our studies demonstrate that 17.109-reactive SL NHL, with or without associated CLL, rearrange, and presumably express, Humkv325 without substantial somatic diversification. Our data suggest that malignant B cells in SL NHL, in contrast to NHL of follicular center cell origin, may express immunoglobulin variable region genes with little or no somatic hypermutation.     

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.     

The multiple shark Ig H chain genes rearrange and hypermutate autonomously

Sharks and skates are representatives of the earliest vertebrates with an immune system based on V(D)J rearrangement. They possess a unique Ig gene organization consisting of 15 to >50 individual IgM loci, each with one VH, two DH, one JH, and one set of constant region exons. The present study attempts to understand how multiple Ig genes are regulated with respect to rearrangement initiation and to targeting during somatic hypermutation. The linkage of three single-copy IgH genes was determined, and single-cell genomic PCR studies in a neonatal animal were used to examine any relationship between relative gene position and likelihood of rearrangement. Our results show that one to three IgH genes are activated independently of linkage or allelic position and the data best fit with a probability model based on the hypothesis that V(D)J rearrangement occurs as a sequence of trials within the B cell. In the neonatal cell set, two closely related IgH, G2A, and G2B, rearranged at similar frequencies, and their membrane forms were expressed at similar levels, like in other young animals. However, older animals displayed a bias in favor of the G2A isotype, which suggests that although rearrangement at G2A and G2B was randomly initiated during primary repertoire generation, the two very similar IgM sequences appear to be differentially expressed with age and exposure to Ag. We performed genomic single-cell PCR on B cells from an immunized individual to study activation-induced cytidine deaminase targeting and found that hypermutation, like V(D)J rearrangement, occurred independently among the many shark IgH.     

Heavy chain V region diversity in the duck-billed platypus (Ornithorhynchus anatinus): long and highly variable complementarity-determining region 3 compensates for limited germline diversity

In this work, to study the emergence of the H chain V region repertoire during mammalian evolution, we present an analysis of 25 independent H chain V regions from a monotreme, the Australian duck-billed platypus, Ornithorhynchus anatinus. All the sequences analyzed were found to form a single branch within the clan III of mammalian V region sequences in a distance tree. However, compared with a classical V gene family this branch was more diversified in sequence. Sequence analysis indicates that the apparent lack of diversity in germline V segments is well compensated for by relatively long and highly diversified D and N nucleotides. In addition, extensive sequence variation was observed in the framework region 3. Furthermore, at least five and possibly seven different J segments seem to be actively used in recombination. Interestingly, internal cysteine bridges in the complementarity-determining region (CDR)3 loop, or between the CDR2 and CDR3 loops, are found in approximately 36% of the platypus V(H) sequences. Such cysteine bridges have also been observed in cow, camel, and shark. Internal cysteine bridges may play a role in stabilizing long and diversified CDR3 and thereby have a role in increasing the affinity of the Ab-Ag interaction.     

V gene rearrangement is required to fully activate the hypermutation mechanism in B cells

Nonproductively rearranged H and L chain loci of B cell hybridoma lines expressing heavily mutated antibodies were cloned and partially sequenced. The results confirm earlier data showing that somatic point mutations are as frequent in nonproductively rearranged loci containing a rearranged V gene as in productively rearranged loci. They establish in addition that in nonproductive H chain loci which bear a DJH rearrangement the frequency of somatic mutations is more than 10 times lower (0.2%) than in VDJH loci expressed by the same cells (2.5%). Thus, the hypermutation mechanism operating in B cell differentiation is targeted at V genes rearranged to the J locus and may require nucleotide sequences associated with both V and J elements in order to be fully activated. An inversion of the JH2 segment was detected in one DJH locus. This inversion appears to be the result of a secondary joining event occurring occasionally in the course of B cell development.