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.     

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.     

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.     

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.     

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.     

An unusual type of V-J joining diversifies the primary repertoire of mouse lambda 1 light chains

It is well established that B lymphocytes can achieve an almost unlimited antibody repertoire by using combinations of at least three different basic mechanisms. First, the mammalian genome has multiple, distinct germline variable (V), joining (J) and diversity (D) gene segments which can presumably combine randomly to give V-D-J joins in the heavy (H)-chain loci and V-J joins in the light (L)-chain loci during the formation of functional antibody genes. Second, the actual joining points between any two combining gene segments can vary considerably, increasing the germline diversity. Further variability is generated in the heavy-chain locus by de novo addition of extra nucleotides between the combining gene segments. Finally, somatic mutations independent from joining events can accumulate in V regions during the lifetime of B lymphocytes. Here we report that when V and J regions join in the formation of functional lambda 1 light-chain genes, 'lethal' out-of-frame joins can be compensated for by the deletion of nucleotides several bases upstream of the actual joining points; this generates small stretches of nucleotides in a new frame between the deletion and the V-J joining point, thus creating additional diversity in the third hypervariable regions of the mouse lambda 1 light chains.     

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.     

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.     

Mapping T-cell receptor-peptide contacts by variant peptide immunization of single-chain transgenics.

To test models of T-cell recognition, mice transgenic for T-cell receptor alpha or beta chain have been immunized with variant peptides that force changes in the resulting T-cell response. In particular, charge substitutions on the peptide often elicit reciprocal charges in the junctional (CDR3) sequences of T-cell receptor V alpha or V beta chains, indicating direct T-cell receptor-peptide contact, and allowing derivation of a topology for the T-cell receptor-MHC interaction. At one position on the peptide, variants transformed a homogeneous V beta response into a very heterogeneous one.     

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.     

Two monoclonal antibodies against different antigens using the same VH germ-line gene

Immunoglobulin diversity seems to arise largely by three mechanisms: (1) the existence of several germ-line genes, which must be rearranged before expression--that is, V and J for the light (L) chains, V, D and J for the heavy (H) chains; (2) somatic events, including mutations and gene conversion; and (3) combinatorial association of heavy and light chains, leading to the proposal that random pairing of p X H and q X L chains might generate p X q antibody molecules expressing discrete specificities. As heavy and light chains derived from the same immunoglobulin molecule would frequently reassociate preferentially, it is likely that only a fraction of potential heavy--light pairs actually provides "valid' antibodies. As a consequence of combinatorial heavy--light chain pairing, antibodies of discrete specificities sharing the same VH region, associated with distinct light chains (or vice versa) should be encountered. We report here that two heavy chains, derived from the same VH germ-line gene, may be present in anti-NP or anti--GAT antibodies, depending on their association with a specific lambda or kappa light chain, respectively.     

Complete nucleotide sequence of an immunoglobulin VH gene homologue from Caiman, a phylogenetically ancient reptile

Immunoglobulin variable (V) gene regions typify extensive multigenic families in terms of overall size, chromosomal arrangement and presence of large numbers of apparent pseudogenes. A unique mechanism of somatic reorganization involving recombination of VH, D and JH or VL and JL segments accompanies the differentiation of lymphoid cells and together with somatic mutation and other types of recombination accounts for V-region diversity. Although these processes have been well characterized in higher mammals, little is known concerning their origin and diversification during phylogenetic time. Previously, we described the blot-hybridization characteristics of murine VHIII probes with restriction enzyme-digested genomic DNA isolated from several phylogenetically critical species, including Caiman crocodylus, a modern representative of an ancient reptilian subclass. Here we have used a murine probe, S107V, to select homologous clones from a library of Caiman genomic DNA constructed in a lambda bacteriophage. The complete nucleotide sequence of a Caiman gene homologous to the murine VH gene and its adjacent 5' and 3' region is described. Comparison of the sequence with mammalian prototypes shows evidence of considerable organizational and structural homology extending outside the presumed VH-coding region and including elements believed to be involved in somatic recombination. Inferences about the evolution of this multigenic family can now be extended to the level of phylogenetic class.     

Substitution of murine for human CD4 residues identifies amino acids critical for HIV-gp120 binding

Human CD4 is the receptor for the gp120 envelope glycoprotein of human immunodeficiency virus and is essential for virus entry into the host cell. Sequence analysis of CD4 has suggested an evolutionary origin from a structure with four immunoglobulin-related domains. Only the two NH2-terminal domains are required to mediate gp120 binding. The extracellular segment of murine CD4 has an overall 50% identity with its human counterpart at the amino-acid level, but fails to bind gp120. To define those residues of human CD4 critical for gp120 binding, we have taken advantage of this species difference and substituted all non-conserved murine for human CD4 residues between amino-acid positions 27-167. We used oligonucleotide-directed mutagenesis to create each of 16 individual mutant human CD4 molecules containing from 1-4 amino-acid substitutions. Introduction of as few as three amino acids into corresponding positions of human CD4 abrogates gp120 binding. Furthermore, these critical residues are located in domain I with a contribution from domain II. Modelling studies using the three-dimensional coordinates of the V kappa Bence-Jones REI homodimer localize the site in domain I to the C" beta strand within CDR2 but projecting away from the homologues of principle antigen-binding regions CDR 1 and 3.     

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.     

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.