The joining of V and J gene segments creates antibody diversity

The variable regions of mouse kappa (kappa) chains are coded for by multiple variable (V) gene segments and multiple joining (J) gene segments. The V kappa gene segments code for residues 1 to 95; the J kappa gene segments code for residues 96 to 108 (refs 1-3). This gene organisation is similar to that encoding the V lambda regions. Diversity in V kappa regions arises from several sources: (1) there are multiple germ-line V kappa gene segments and J kappa gene segments; (2) combinatorial joining of V kappa gene segments with different germline J kappa gene segments; and possibly, (3) somatic point mutation, as postulated for V lambda gene segments. Also, from a comparison of the number of germ-line J kappa gene segments and amino acid sequences, it has been suggested that J kappa region sequences may be determined by the way V kappa and J kappa gene segments are joined. This report supports this model by directly associating various J kappa sequences with given J kappa gene segments.     

Preferential expression of a defined T-cell receptor beta-chain gene in hapten-specific cytotoxic T-cell clones

A multitude of different antigens can be recognized by T cells through specific receptors. Both the alpha- and beta-chains of the T-cell receptor contribute to the antigen recognition portion. The repertoire of beta-chain variable region (V beta) gene segments is limited to some 20 elements which seem to be used randomly in different T cells. Diversity at the beta-chain level can be created in several ways: a multiplicity of germline gene segments; combinatorial diversity by rearranging different V, diversity (D), joining (J) and constant (C) region elements; junctional diversity by joining gene segments at different sites; N-region diversity, that is, insertion of random nucleotides at junctional sites; and somatic mutation. However, the major sources and the extent of diversity of the T-cell receptor are unclear. To address this issue, 42 H-2Kb-restricted, 2,4,6-trinitrophenyl (TNP)-specific cytotoxic T-cell (Tc) clones from C57BL/6 mice were characterized with respect to expression of different beta-chain gene segments in messenger RNA using specific oligonucleotide probes. We report here that nearly half of the Tc clones use identical elements for productive beta-chain gene rearrangement. Thus, there is a restriction in the use of beta-chain gene segments in this panel of Tc clones which favours a particular V beta--D beta--J beta--C beta combination with a defined D beta element.     

A novel type of aberrant recombination in immunoglobulin genes and its implications for V-J joining mechanism

Functional kappa light chain genes are formed during B-lymphocyte differentiation by the joining of initially separate V and J gene segments. It has been suggested that the intervening DNA is deleted, however the recent reports of what appear to be the reciprocal products of V and J recombination (back-to-back conserved V and J flanking sequences, called f-fragments) in DNA from mature lymphocytes make a simple deletion model unlikely. An alternative scheme involving unequal sister chromatid exchange has been proposed, supported by the evidence that the f-fragments seem to have segregated from the chromosome carrying the reciprocal complete kappa light chain gene (this and other schemes are briefly reviewed in ref. 8). We report here the analysis of a mouse myeloma (MOPC 41), in which a productive (kappa+) and a non-productive (kappa-) rearrangement has occured, which may help to clarify the mechanism of V-J joining. The aberrant rearrangement has led to the joining of a J1 gene segment to a sequence unrelated to any V gene (L10), and which in the germ line is flanked by a sequence resembling a V region recombination signal sequence. In this case no segregation of the reciprocal recombination products (kappa-41 and f41), which is a required step in sister chromatid exchange models, has taken place. An inversion model provides the simplest explanation of this J rearrangement.     

Recombination between an expressed immunoglobulin heavy-chain gene and a germline variable gene segment in a Ly 1+ B-cell lymphoma

The early stages of murine B-cell differentiation are characterized by a series of immunoglobulin gene rearrangements which are required for the assembly of heavy(H) and light(L)-chain variable regions from germline gene segments. Rearrangement at the heavy-chain locus is initiated first and consists of the joining of a diversity (DH) gene segment to a joining (JH) gene segment. This forms a DJH intermediate to which a variable (VH) gene segment is subsequently added. Light-chain gene rearrangement follows and consists of the joining of a VL gene segment to a JL gene segment: once a productive light-chain gene has been formed the cell initiates synthesis of surface immunoglobulin M (sIgM) receptors (reviewed in ref. 1). These receptors are clonally distributed and may undergo further diversification either by somatic mutation or possibly by continued recombinational events. Such recombinational events have been detected in the Ly 1+ B-cell lymphoma NFS-5, which has been shown to rearrange both lambda and H-chain genes subsequent to the formation of sIgM (mu kappa) molecules. Here we have analysed a rearrangement of the productive allele of NFS-5 and found that it is due to a novel recombination event between VH genes which results in the replacement of most or all of the coding sequence of the initial VHQ52 rearrangement by a germline VH7183 gene. Embedded in the VH coding sequence close to the site of the cross-over is the sequence 5' TACTGTG 3', which is identical to the signal heptamer found 5' of many DH gene segments. This embedded heptamer is conserved in over 70% of known VH genes. We suggest that this heptamer mediates VH gene replacement and may play an important part in the development of the antibody repertoire.     

Genomic organization of the genes encoding mouse T-cell receptor alpha-chain

The vertebrate immune system uses two kinds of antigen-specific receptors, the immunoglobulin molecules of B cells and the antigen receptors of T cells. T-cell receptors are formed by a combination of two different polypeptide chains, alpha and beta (refs 1-3). Three related gene families are expressed in T cells, those encoding the T-cell receptor, alpha and beta, and a third, gamma (refs 4-6), whose function is unknown. Each of these polypeptide chains can be divided into variable (V) and constant (C) regions. The V beta regions are encoded by V beta, diversity (D beta) and joining (J beta) gene segments that rearrange in the differentiating T cell to generate V beta genes. The V gamma regions are encoded by V gamma, J gamma and, possibly, D gamma gene segments. Studies of alpha complementary DNA clones suggest that alpha-polypeptides have V alpha and C alpha regions and are encoded by V alpha and J alpha gene segments and a C alpha gene. Elsewhere in this issue we demonstrate that 18 of 19 J alpha sequences examined are distinct, indicating that the J alpha gene segment repertoire is much larger than those of the immunoglobulin (4-5) or beta (14) gene families. Here we report the germline structures of one V alpha and six J alpha mouse gene segments and demonstrate that the structures of the V alpha and J alpha gene segments and the alpha-recognition sequences for DNA rearrangement are similar to those of their immunoglobulin and beta-chain counterparts. We also show that the J alpha gene-segment organization is strikingly different from that of the other immunoglobulin and rearranging T-cell gene families. Eighteen J alpha gene segments map over 60 kilobases (kb) of DNA 5' to the C alpha gene.     

Double-stranded cleavage by cell extracts near recombinational signal sequences of immunoglobulin genes

The genes encoding the variable regions of murine immunoglobulin light chains are present in the germ line in two separate segments, V and J. During B lymphocyte differentiation these segments are brought together to form a single unit (for review see ref. 1). Although much is known about the structures of V and J segments, both in germ-line configuration and after rearrangement, essentially nothing is known about the biochemical mechanism of V-J recombination. One possible step in proposed mechanisms of immunoglobulin gene rearrangement is endonucleolytic cleavage of the participating DNA segments before joining. In an attempt to detect such an activity, we have developed an assay for the detection of site-specific double- or single-strand endonucleolytic activity in crude soluble extracts. Using this assay we have detected an activity in extracts of nuclei from mouse B-lymphoid lines and from mouse L cells that is capable of introducing duplex breaks near the recombinational signal sequences of immunoglobulin JK segments. We report the activity here because of its intrinsic interest although we lack any direct evidence that it has a role in V-J recombination.     

Regulated progression of a cultured pre-B-cell line to the B-cell stage

The variable (V) regions of heavy and light immunoglobulin chains are encoded by multiple germline DNA elements which are assembled into complete variable-region genes in precursor(pre-) B lymphocytes. The heavy-chain V region (VH) is assembled from three separate germline DNA elements, the variable (VH), diversity (D) and joining (JH) segments; whereas light-chain variable regions of either the kappa or lambda type are assembled from two elements, the VL and JL. Analysis of tumour cell lines or sorted cell populations which represent early and late pre-B cells has suggested that heavy-chain assembly and expression generally precedes that of light chains; but, primarily because of the lack of appropriate model systems to study the phenomenon, the mechanism and significance of this apparently orderly differentiation process are much debated. Here we describe for the first time a transformed cell line, 300-19, which sequentially undergoes all of the immunoglobulin gene rearrangement and expression events associated with the differentiation of pre-B cells to surface immunoglobulin-positive B lymphocytes. Analysis of the in vitro differentiation of 300-19 cells provides direct evidence for distinct differentiation phases of first VH and subsequently VL assembly during B-cell differentiation. Furthermore, these analyses suggest that the mu heavy chain, resulting from a productive VHDJH rearrangement, has both a positive and a negative regulatory role in mediating this ordered differentiation process, that is, signalling the cessation of VH gene assembly and simultaneously signalling the onset of VL assembly.     

Deletion of the human T-cell receptor delta-gene by a site-specific recombination

The newly described T-cell receptor (TCR) delta locus is located inside the TCR alpha locus, between variable region (V)alpha and joining region (J)alpha. Although the delta and alpha TCR genes are physically linked on the same chromosome, they are sequentially expressed during T-cell development. This implies the existence of a highly efficient regulatory mechanism by which these two genes are independently rearranged. We have recently described a genetic element 'T early alpha' (TEA) in humans transcribed in foetal thymocytes, spliced alternatively to constant region (C)alpha, and located between the TCR-delta locus (5') and the group of J alpha segments (3'). Importantly, TEA flanks a common site of rearrangement in the thymus, and distinguishes cells using TCR-gamma/delta (TEA in germline configuration) from cells using TCR-alpha/beta (TEA deleted on both chromosomes). In order to understand this TEA-associated recombination we analysed genomic clones representing these thymic rearrangements. We show that the TEA-associated recombination deletes the delta locus before productive (V delta D delta J delta) rearrangement. The diversity (D)delta and J delta regions, which provide the major source of delta gene diversity, are eliminated as a consequence of delta gene deletion and cannot then be used in conjunction with an alpha-TCR. We propose that the TEA-associated deletion of TCR-delta precedes the formation of an alpha-TCR and could down-regulate TCR-delta formation in maturing thymus.     

Organization and sequences of the variable, joining and constant region genes of the human T-cell receptor alpha-chain

An essential property of the immune system is its ability to generate great diversity in antibody and T-cell immune responses. The genetic and molecular mechanisms responsible for the generation of antibody diversity have been investigated during the past several years. The gene for the variable (V) region, which determines antigen specificity, is assembled when one member of each of the dispersed clusters of V gene segments, diversity (D) elements (for heavy chains only) and joining (J) segments are fused by DNA rearrangement. The cloning of the beta-chain of the T-cell antigen receptor revealed that the organization of the beta-chain locus, which is similar to that of immunoglobulin genes, is also composed of noncontiguous segments of V, D, J and constant (C) region genes. The structure of the alpha-chain seems to consist of a V and a C domain connected by a J segment. We report here that the human T-cell receptor alpha-chain gene consists of a number of noncontiguous V and J gene segments and a C region gene. The V region gene segment is interrupted by a single intron, whereas the C region contains four exons. The J segments, situated 5' of the C region gene, are dispersed over a distance of at least 35 kilobases (kb). Signal sequences, which are presumably involved in DNA recombination, are found next to the V and J gene segments.     

Rearrangement of two distinct T-cell gamma-chain variable-region genes in human DNA

Selective cloning procedures for T-cell-specific complementary DNAs have revealed the existence of a gene designated gamma as well as the main antigen receptor alpha- and beta-chain genes. The gamma-chain genes undergo rearrangement during T-cell differentiation but the patterns and complexity of such rearrangements differ markedly in mouse and human. In mouse, a panel of cytotoxic T-lymphocyte clones exhibit the same rearrangement pattern with a gamma-chain gene probe and a set of three gamma-chain variable (V) genes have been identified in the DNA. Clonal diversity in mouse seems to be confined to V-J (joining) regions. In contrast, human T-cell lines exhibit diverse rearrangements suggestive of a family of differing V gamma genes variously rearranging to the two gamma-chain constant (C) region genes. Here we report the cloning of two very different V gamma genes rearranged to J segments upstream of the two human C gamma genes. Both V gamma genes are rearranged productively but nucleotide sequence comparison shows that they possess very little homology with each other. This shows that human T-cell V gamma genes exist which differ significantly from each other at the nucleotide level and that such diverse genes can be usefully rearranged in different T cells.     

Isotype exclusion and transgene down-regulation in immunoglobulin-lambda transgenic mice

A given B lymphocyte makes an antibody containing either kappa- or lambda-light chains, but not both. This isotype exclusion is effected at the level of the rearrangement of the immunoglobulin gene segments, although by an unknown mechanism. An attractive possibility is that, following productive rearrangement of one of the light-chain loci, the newly synthesized light-chain polypeptide inhibits DNA rearrangement for the other isotype. To test such feedback regulation, we have created transgenic mice carrying a rearranged lambda 1-gene. By contrast with the B cells in normal newborn mice which are mainly kappa+lambda-, the B cells in the newborn transgenic mice express lambda- but not kappa-chains. We propose that the synthesis of any light chain, be it kappa or lambda, that allows expression of IgM on the cell surface results in a cessation of all V-J joining. Interestingly, the limited light-chain repertoire of the transgenic mice does not persist and most adult B cells express endogenous kappa-rearrangements and down-regulate the transgene.     

Major reorganization of immunoglobulin VH segmental elements during vertebrate evolution

In mammals, the immunoglobulin heavy-chain variable region (VH) locus is organized in a linear fashion; individual VH, diversity (DH), joining (JH) and constant (CH) region segments are linked in separate regions. During somatic development, coding segments flanked by characteristic short recombination signal sequences, separated by intervening sequence regions that may exceed 2,000 kilobases (kb), are recombined. Combinatorial joining of different segments as well as imprecision in this process contribute to the diversity of the primary antibody response; subsequent mutation further alters functionally rearranged genes. This basic somatic reorganization mechanism is shared by six major families of genes encoding antigen receptors. Previously, we have shown that multiple germline genes and mammalian-like recombination signal sequences are associated with the VH gene family of Heterodontus francisci (horned shark), a primitive elasmobranch. Studies presented here demonstrate that segmental reorganization involving mammalian-like DH and JH segments occurs in the lymphoid tissues of this species. In marked contrast to the mammalian system, we find multiple instances of close linkage (approximately 10 kb) between individual VH, DH, JH, and CH segments. This unique organization may limit combinatorial joining and be a factor in the restricted antibody response of this lower vertebrate.     

The structure, rearrangement and expression of D beta gene segments of the murine T-cell antigen receptor

It has been postulated that the variable region of the beta-polypeptide of the murine T-cell antigen receptor is encoded by three distinct germ-line gene segments--variable (V beta), diversity (D beta) and joining (J beta)--that are rearranged to generate a V beta gene. Germ-line V beta and J beta gene segments have been isolated previously. Here we report the isolation and characterization of two germ-line D beta gene segments that have recognition signals for DNA rearrangement strikingly similar to those found in the three immunoglobulin gene families and in V beta and J beta gene segments. The D beta and J beta segments can join in the absence of V beta gene segment rearrangement and these rearranged sequences are transcribed in some T cells.     

Sequences at the somatic recombination sites of immunoglobulin light-chain genes.

The entire nucleotide sequence of a 1.7-kilobase embryonic DNA fragment containing five joining (J) DNA segments for mouse immunoglobulin kappa chain gene has been determined. Each J DNA segment can encode amino acid residues 96--108. Comparison of one of the five J DNA sequences with those of an embryonic variable (V) gene and a complete kappa chain gene permitted localisation of a precise recombination site. The 5'-flanking regions of J DNA segments could form an inverted stem structure with the 3'-non-coding region of embryonic V genes. This hypothetical structure and gel-blotting analysis of total embryo and myeloma DNA suggest that the somatic recombination may be accompanied by excision of an entire DNA segment between a V gene and a J DNA segment. Antibody diversity may in part be generated by modulation of the precise recombination sites.     

Human T-cell gamma genes contain N segments and have marked junctional variability

The gamma-chain genes are encoded by immunoglobulin-like gene segments in germline DNA which rearrange during the somatic development of T cells to form an active gene. The protein produced by these genes has not been identified and the diversity of the proteins that the genes can express has not been determined. We expect that the diversity of expressed gamma-chains is produced by the same three mechanisms that produce diversity of other immunoglobulin-like genes: (1) germline variable (V) and joining (J) region repertoires; (2) somatic mutation; and (3) junctional diversity. To define the contribution of each of these mechanisms to the generation of gamma-chain diversity, several gamma-chain complementary clones and rearranged gamma-chain genes have been characterized. Most of these clones seem to encode a defective gamma-chain, the variable- and constant-region portions being joined such that they would not be translated in the same reading frame. Here we report that the germline J-region diversity of the human T-cell gamma-chain is very limited and that somatic mutation does not contribute to the diversity of the gamma-chains encoded by the cloned segments. However, the junctional diversity of these gamma-chain genes is extensive. We suggest that N sequences (template-independent sequences) have been inserted enzymatically into all of the gamma-chain genes characterized.     

Amplification of immunoglobulin lambda constant genes in populations of wild mice

The lambda immunoglobulin light chain (Ig lambda) locus of BALB/c inbred mice consists of two variable region gene segments (V lambda)1-3, and four constant region gene segments (C lambda)1,2,4,5. Each C lambda gene segment is associated with a unique joining segment (J lambda)2,4-7, and they are organized in two paired units, J3C3-J1C1 and J2C2-J4C4 (refs 4, 8). Using cDNA probes specific for C lambda 1 and C lambda 2 (ref. 9) we have analysed the genomic organization of the C lambda gene segments in wild-derived and inbred strains of mice. Although Southern blots of the genomic DNA of inbred mice show a constant pattern of hybridization, wild-derived mice show a high degree of variation in the number, size and intensity of hybridizing fragments. We have now found that, per haploid genome, mice of a Mus musculus musculus stock isolated from Sladeckovce, Czechoslovakia (CzII) have at least 12 C lambda segments, and mice of a Mus musculus domesticus stock 'Centreville Lights' from Centreville, Maryland (CL) have at least 8 C lambda segments. There appears to have been relatively recent amplifications of the C lambda gene segments in wild mice.