Expression of a VHC kappa chimaeric protein in mouse myeloma cells

The heavy (H) and light (L) chains of antibodies consist of variable (V) and constant (C) regions. The V regions of the heavy and light chains form the antibody combining site. To determine whether a V region could be functional when joined to a polypeptide other than its own C region, we constructed a chimaeric gene encoding the V region of a mouse heavy chain and the C region of a mouse kappa light chain ( VHC kappa). The heavy-chain gene is derived from an A/J mouse hybridoma cell line 36-65 whose antibody product (gamma 1, kappa) is specific for the hapten azophenylarsonate. We report here that, when introduced into a mouse myeloma cell line, the chimaeric gene is expressed and a protein of the expected molecular weight is secreted into the medium. As light chains tend to dimerize we expected that the VHC kappa protein might associate with light chain from the cell line 36-65 to form an antibody-binding molecule. Affinity binding experiments and Ka determination indicate that this is the case. Dimers of this type offer a novel and interesting alternative to existing antibody-binding molecules.     

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 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.     

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.     

RNA splicing generates a variant light chain from an aberrantly rearranged kappa gene

Both C kappa regions in MPC 11 cells are rearranged into active transcripion units, one producing a normal kappa chain and the other an internally deleted kappa fragment lacking a V region. The gene coding for the kappa fragment mRNA is aberrantly rearranged and lacks a site for V leads to C kappa splicing. An alternative splicing event which deletes the V region from the nuclear RNA precursor generates the kappa fragment mRNA.     

A kappa-immunoglobulin gene is formed by site-specific recombination without further somatic mutation.

The active gene for a kappa light chain is formed by a somatic recombination event that joins one of several hundred variable region genes to one of a series of recombination sites (J-segments) encoded close to the kappa constant region gene. The nucleotide sequences of cloned germ line and somatically recombined genes define the precise organisation of these genetic segments and the site and nature of the recombination event that joined them. Apart from somatic recombination, no further alteration of ther germ line sequence has occurred. The J-segment is of special interest as it encodes signals for both DNA and RNA splicing and provides a means of generating further immunoglobulin gene diversity.     

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.     

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.     

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.     

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.     

A mutant immunoglobulin light chain is formed by aberrant DNA- and RNA-splicing events

A mutant immunoglobulin gene has been formed by an abnormal (non V/J) recombination event such that abnormal RNA splicing is required to form a mutant light chain. The structure of the gene suggests that the small palindrome thought to be involved in V/J joining also provides the basis for this abnormal DNA recombination and that the absence of a J segment and RNA splice signal allows an abnormal RNA splicing reaction to occur.     

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.     

Dispersed human immunoglobulin kappa light-chain genes

The gene segments encoding the constant and variable regions of human immunoglobulin light chains of the kappa type (C kappa, V kappa) have been localized to chromosome 2. The distance between the C kappa and V kappa genes and the number of germline V kappa genes are unknown. As part of our work on the human V kappa locus, we have now mapped two solitary V kappa gene and a cluster of three V kappa genes to chromosomes 1, 15 and 22, respectively. The three genes that have been sequenced are nonprocessed pseudogenes, and the same may be true for the other two genes. This is the first time that V-gene segments have been found outside the C-gene-containing chromosomes. Our finding is relevant to current estimates of the size of the V kappa-gene repertoire. Furthermore, the dispersed gene regions have some unusual characteristics which may help to clarify the mechanism of dispersion.     

Conserved organization of the human and murine T-cell receptor beta-gene families

Generation of an immune response depends on the interaction of haematopoietic cell types, among which T cells and their receptors are of central importance. The T-cell receptor is a heterodimer consisting of disulphide-linked alpha and beta-chains, each chain divided into variable (V) and constant (C) regions. The beta-chain is encoded by the rearrangement of separate variable (V beta), diversity (D beta) and joining (J beta) gene segments during T-cell differentiation. To examine the mechanisms of somatic DNA rearrangement and evolution of the beta-gene segments, we have constructed a physical map of the human T-cell receptor beta-chain family containing 40 V beta gene segments as well as both C beta gene clusters. A comparison of the published nucleotide sequences of human and murine V beta gene segments reveals 12 examples of gene segments sharing 65% or more interspecies homology. The relative order of these human and murine V beta gene segment homologues is also conserved along the chromosome, apart from more extensive human gene duplication, presumably as a consequence of constraints imposed on evolutionary mechanisms operating to diversify these gene families or of selective pressures operating to maintain order.