Aberrant rearrangement of the kappa light-chain locus involving the heavy-chain locus and chromosome 15 in a mouse plasmacytoma

The creation of a functional antibody gene requires the precise recombination of gene segments initially separated on the chromosome. Frequently errors occur in the process, resulting in the formation of a non-functional gene. The non-functional genes can be generated by incomplete rearrangements, frameshifts, or the use of pseudo V or J joining segments. It is likely that these aberrant rearrangements arise by the same mechanism as is used in generating functional genes, a process which we have suggested may involve unequal sister chromatid exchange. Aberrant rearrangements of immunoglobulin genes occur in normal lymphocytes and play a major part in allelic exclusion. However, it has recently been suggested that aberrant rearrangements involving immunoglobulin and non-immunoglobulin genes may be involved in tumorigenesis. This suggestion has been stimulated by the frequent occurrence of translocations involving chromosomes known to carry immunoglobulin genes in B-cell malignancies. The rearrangement of non-immunoglobulin DNA to the heavy-chain locus has recently been reported. Some aberrant rearrangements of the kappa locus appear to be due to rearrangements to sites that do not include the conventional sequence for V gene segment joining. Here we describe an aberrant kappa rearrangement that has led to the joining of DNA from chromosomes 15, 6 and 12, and so appears to be the result of chromosomal translocations or transpositions. As 15/6 or 15/12 translocations have frequently been found in mouse plasmacytomas (as have analogous translocations in human lymphocyte tumours) this aberrant kappa rearrangement may be unique to the plasmacytoma from which it was isolated.     

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.     

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.     

An unusual translocation of immunoglobulin gene segments in variants of the mouse myeloma MPC11

Immunoglobulin light chain genes of the mouse are composed in germ-line DNA of four separate segments, the leader, V (variable), J (joining) and C (constant) segments. In immunocompetent cells a V and J gene segment are joined by a site-specific recombination event. In variants of the mouse myeloma MPC11 a so-called kappa (k) light chain fragment is expressed which consists of the MOPC321 leader peptide, joined to the kappa constant region peptide. Using the Southern blotting technique we found that the gene coding for the light chain fragment has apkparently been generated by an aberrant translocation of a V gene segment identical or very similar to the MOPC321 V gene segment into the large intervening sequence between the J and the C gene segments. The resulting deletion of the splice signals of the J segments could be the reason for the observed splicing between leader and C region sequences, a phenomenon which may be of general interest for the understanding of the splicing mechanism.     

A conserved sequence in the immunoglobulin J kappa-C kappa intron: possible enhancer element

Several functionally important genetic elements (such as the TATA box, mRNA splice sequences, poly(A) addition signal) were first detected as short segments of unexplained sequence homology within non-coding regions of different genes. A short region of unknown sequence in the intron between the human J kappa and C kappa immunoglobulin coding regions was found to be sufficiently homologous to the corresponding segment of the mouse gene to form stable heteroduplexes. Although no specific function has yet been definitely ascribed to this region (which we call the kappa intron conserved region, or KICR), some functional significance has been inferred from the findings that (1) activation of B lymphocytes induces a DNase hypersensitivity site in this region and (2) deletions including this region reduce expression of kappa genes introduced into lymphoid cells. To delineate the KICR more precisely and to test the generality of the sequence conservation in a third species, we have sequenced this region of the human and mouse genes and have examined the corresponding region of a recently cloned rabbit kappa gene. We find a segment of about 130 base pairs (bp) that shows striking conservation in all three species, demonstrating homology significantly higher than within the C kappa coding region itself. Two short sequences from the J kappa-C kappa intron that were noted by other investigators to be homologous to proposed 'enhancer' sequences both lie within the conserved region.     

The gene encoding the T-cell receptor alpha-chain maps close to the Np-2 locus on mouse chromosome 14

Serological and molecular genetic analyses of T-cell clones have shown that the T-cell antigen receptor apparently comprises two glycosylated, disulphide-linked polypeptide chains (alpha and beta), both of which span the cell membrane. Cloning of the genes encoding the two chains from mouse and human DNA has shown that the alpha- and beta-chains are composed of variable (V) and conserved (C) regions in agreement with peptide mapping data. Gene segments encoding variable and conserved domains of the beta-chain have been identified and undergo rearrangements during T-cell differentiation. The genes encoding the alpha-chain, so far described at the level of complementary DNA clones, also identify DNA rearrangements. Thus, the genes encoding the T-cell receptor show the same structure and dynamic behaviour as immunoglobulin genes, indicating that the two gene families belong to the same supergene family; this evolutionary relationship is supported by the fact that the genes encoding the beta-chain of the T-cell receptor are closely linked to immunoglobulin kappa light-chain genes on chromosome 6 in mouse. In man, however, the beta genes map to chromosome 7 (ref. 14) whereas the kappa-chain genes are located on chromosome 2, indicating that linkage between the two gene families is not needed for proper expression. Here we describe genomic clones encoding the constant portion of the T-cell receptor alpha-chain and map the gene to chromosome 14 in mouse, close to the gene for purine nucleoside phosphorylase (Np-2) which, in man, has been associated with T-cell immunodeficiencies.     

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.     

A uniform deleting element mediates the loss of kappa genes in human B cells

Human immunoglobulin light-chain genes become rearranged in an ordered fashion during pre-B-cell development such that rearrangement generally occurs in kappa genes before lambda genes (refs 1,2). This ordered process includes an unanticipated deletion of the constant kappa (C kappa) gene and kappa enhancer sequence which precedes lambda rearrangement, and the site of this deletional recombination was located 3' to the joining (J kappa) segments in 75% of cases studied. We have now characterized the recombinational element responsible for this event on three separate alleles and found them to be identical. This kappa-deleting element recombined site-specifically with a palindromic signal (CACAGTG) located in the J kappa-C kappa intron. All losses of C kappa genes in other human B cells were mediated by this determinant, including the 25% of instances when this element recombined with sequences 5' to J kappa. In contrast, the kappa-deleting element remained in its germline form on all successful kappa-producing alleles. Moreover, kappa loss is an evolutionarily conserved event, as the kappa-deleting element appears to be the human homologue of the murine RS sequence. Our results suggest that this element may help ensure isotypic and allelic exclusion of light chains and may be involved in the ordered use of human light-chain genes.     

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.     

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.     

In situ nick-translation distinguishes between active and inactive X chromosomes

Template-active regions of chromatin are structurally distinct from nontranscribing segments of the genome. Recently, it was suggested that the conformation of active genes which renders them sensitive to DNase I may be maintained even in fixed mitotic chromosomes. We have developed a technique of mitotic cell fixation and DNase I-directed nick-translation which distinguishes between active and inactive X chromosomes. We report here that Gerbillus gerbillus (rodent) female cells contain easily identified composite X chromosomes each of which includes the original X chromosome flanked by two characteristic autosomal segments. After nick-translation the active X chromosome in each cell is labelled specifically in both the autosomal and X-chromosomal regions. The inactive X chromosome is labelled only in the autosomal regions and in a small early replicating band within the late replicating 'original X' chromosome. Our technique opens the possibility of following the kinetics of X-chromosome inactivation and reactivation during embryogenesis, studying active genes in the inactive X chromosome and mapping tissue-specific gene clusters.     

DNase I hypersensitive sites in the chromatin of human mu immunoglobulin heavy-chain genes

An immunoglobulin polypeptide chain is encoded by multiple gene segments that lie far apart in germ-line DNA and must be brought together to allow expression of an immunoglobulin gene active in B lymphocytes. For the immunoglobulin heavy chain genes, one of many variable (V) region genes becomes joined to one of several diversity (D) segments which are fused to one of several joining (J) segments lying 5' of the constant region (C) genes. Here we show that the rearranged mu genes of an IgM-producing human B-lymphocyte cell line exhibit pancreatic deoxyribonuclease (DNase I) hypersensitive sites in the JH-C mu intron that are absent in naked DNA or the chromatin of other differentiated cell types. DNA sequence analysis reveals that the major hypersensitive site maps to a conserved region of the JH-C mu intron recently shown to function as a tissue-specific enhancer of heavy-chain gene expression. A similar association of an enhancer-like element with a DNase I hypersensitive site has been reported for the mouse immunoglobulin light-chain J kappa-C kappa intron. These results implicate disruption of local chromatin structure in the mechanism of immunoglobulin enhancer function.     

The human T-cell receptor alpha-chain gene maps to chromosome 14

The T-cell receptor for antigen has been identified as a disulphide-linked heterodimeric glycoprotein of relative molecular mass (Mr) 90,000 comprising an alpha- and a beta-chain. The availability of complementary DNA clones encoding mouse and human beta-chains has allowed a detailed characterization of the genomic organization of the beta-chain gene family and has revealed that functional beta-chain genes in T cells are generated from recombination events involving variable (V), diversity (D), joining (J) and constant (C) gene segments. Recently, cDNA clones encoding mouse and human alpha-chains have been described; the sequences of these clones have indicated that functional alpha-chain genes are also generated from multiple gene segments. It is possible that chromosomal translocations involving T-cell receptor alpha- and beta-chain genes have a role in T-cell neoplasms in much the same way as translocations involving immunoglobulin genes are associated with oncogenic transformation in B cells. In the latter case, the chromosomal localization of the immunoglobulin genes provided one of the first indications of the involvement of such translocations in oncogenic transformation. The chromosomal assignment of the alpha- and beta-chain genes may, therefore, provide equally important clues for T-cell neoplastic transformation. The chromosomal location of the mouse and human beta-chain gene family has been determined: the murine gene lies on chromosome 6 (refs 12, 13) whereas the human gene is located on chromosome 7 (refs 13, 14). Here we use a cDNA clone encoding the human alph-chain to map the corresponding gene to chromosome 14.     

Identical V beta T-cell receptor genes used in alloreactive cytotoxic and antigen plus I-A specific helper T cells

T lymphocytes involved in the cellular immune response carry cell-surface receptors responsible for antigen and self recognition. This T-cell receptor molecule is a heterodimeric protein consisting of disulphide-linked alpha- and beta-chains with variable (V) and constant (C) regions. Several complementary DNA and genomic DNA clones have been isolated and characterized. These analyses showed that the genomic arrangement and rearrangement of T-cell receptor genes using VT, diversity (DT), joining (JT) and CT gene segments is very similar to the structure of the known immunoglobulin genes. We have isolated two cDNA clones from an allospecific cytotoxic T cell, one of which shows a productive V beta-J beta-C beta 1 rearrangement without an intervening D beta segment. This V beta gene segment is identical to the V beta gene expressed in a helper T-cell clone specific for chicken red blood cells and H-21. The other clone carries the C beta 2 gene of the T-cell receptor, but the C beta 2 sequence is preceded by a DNA sequence that does not show any similarity to V beta or J beta sequences.     

Somatic variants of murine immunoglobulin lambda light chains

Studies of the murine lambda light chains produced by myeloma cells provided the first evidence for somatic point mutation of germ-line variable (V) region genes. An examination of the variable regions of 19 lambda 1 chains revealed seven which differed from a common sequence by one to three amino acid substitutions. Subsequently, one of these presumed somatic variants of the single lambda 1 V gene was characterized by DNA sequence analysis of the rearranged functional gene. The predicted DNA sequence alteration was observed and no silent mutation was evident. These studies of lambda chain variants suggested that the hypervariable, complementarity-determining regions (CDRs) ht be a preferred site of somatic mutation because all seven characterized variants contained substitutions only in these regions. By contrast, comparisons of closely related kappa chain variable region amino acid sequences, and more recently VK and VH genes, have suggested that somatic mutation probably occurs in codons for both framework and CDR residues. To examine this apparent discrepancy between the sites of somatic mutations in lambda and kappa genes, we have determined the nucleotide sequence of two lambda 1 gene from hybridomas and a lambda 2 gene from a myeloma. These sequences demonstrate that somatic mutation in lambda genes can occur in both the framework and CDR residues.     

Variability and repertoire size of T-cell receptor V alpha gene segments

The immune system of higher organisms is composed largely of two distinct cell types, B lymphocytes and T lymphocytes, each of which is independently capable of recognizing an enormous number of distinct entities through their antigen receptors; surface immunoglobulin in the case of the former, and the T-cell receptor (TCR) in the case of the latter. In both cell types, the genes encoding the antigen receptors consist of multiple gene segments which recombine during maturation to produce many possible peptides. One striking difference between B- and T-cell recognition that has not yet been resolved by the structural data is the fact that T cells generally require a major histocompatibility determinant together with an antigen whereas, in most cases, antibodies recognize antigen alone. Recently, we and others have found that a series of TCR V beta gene sequences show conservation of many of the same residues that are conserved between heavy- and light-chain immunoglobulin V regions, and these V beta sequences are predicted to have an immunoglobulin-like secondary structure. To extend these studies, we have isolated and sequenced eight additional alpha-chain complementary cDNA clones and compared them with published sequences. Analyses of these sequences, reported here, indicate that V alpha regions have many of the characteristics of V beta gene segments but differ in that they almost always occur as cross-hybridizing gene families. We conclude that there may be very different selective pressures operating on V alpha and V beta sequences and that the V alpha repertoire may be considerably larger than that of V beta.