Identification and nucleotide sequence of a diversity DNA segment (D) of immunoglobulin heavy-chain genes

A putative diversity segment of immunoglobulin heavy-chain genes (D segments) has been identified 700 base pairs 5' to JH1 DNA on the germ-line genome of the mouse. This 10-base pair D segment is flanked by two sets of sequences related to (SEE FORMULAR IN TEXT) which are possible recognition sites for a recombinase. The spacer separating the heptamer and the nonamer is 12 base pairs long on both sides of the D segment. As the space separating the two signal sequences in VH DNAs and JH DNAs is 23 +/- 1 base pairs long, the two recombinations required for creation of a complete immunoglobulin VH gene, a VH--D joining and a D--JH joining, follow a 12/23-base pair spacer rule. Allelic exclusion is discussed with respect to D segments.     

A novel VH to VHDJH joining mechanism in heavy-chain-negative (null) pre-B cells results in heavy-chain production

During B-cell development, the VH genes of immunoglobulin heavy (H) chains are assembled from three different germline components: the variable (VH) segment, the diversity (D) segment and the joining (JH) segment. The joining between two segments involves the recognition of conserved nonamer-heptamer sequences bordering each segment, double-stranded cuts at the heptamer-segment border, and the re-ligation of the two segment ends which have frequently been modified by the deletion and addition of nucleotides. The flexibility of the joint increases VHDJH variability. However, it also results in many pre-B cells which do not produce immunoglobulin H chains and have non-functional VHDJH complexes carrying the VH and JH coding sequences in different reading frames. We show here that such 'null cells' are not dead-end products of the B-cell developmental pathway but can perform a novel VH to VHDJH joining using a 5' VH segment to replace the VH sequence of the VHDJ-H complex. This process can result in the generation of a VHDJ+H complex and the subsequent expression of an immunoglobulin heavy chain.     

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.     

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.     

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.     

Recombination signal sequences restrict chromosomal V(D)J recombination beyond the 12/23 rule

The genes encoding the variable regions of lymphocyte antigen receptors are assembled from variable (V), diversity (D) and joining (J) gene segments. V(D)J recombination is initiated by the recombinase activating gene (RAG)-1 and -2 proteins, which introduce DNA double-strand breaks between the V, D and J segments and their flanking recombination signal sequences (RSSs). Generally expressed DNA repair proteins then carry out the joining reaction. The conserved heptamer and nonamer sequences of the RSSs are separated by non-conserved spacers of 12 or 23 base pairs (forming 12-RSSs and 23-RSSs). The 12/23 rule, which is mediated at the level of RAG-1/2 recognition and cutting, specifies that V(D)J recombination occurs only between a gene segment flanked by a 12-RSS and one flanked by a 23-RSS. Vbeta segments are appended to DJbeta rearrangements, with little or no direct Vbeta to Jbeta joining, despite 12/23 compatibility of Vbeta 23-RSSs and Jbeta12-RSSs. Here we use embryonic stem cells and mice with a modified T-cell receptor (TCR)beta locus containing only one Dbeta (Dbeta1) gene segment and one Jbeta (Jbeta1) gene cluster to show that the 5' Dbeta1 12-RSS, but not the Jbeta1 12-RSSs, targets rearrangement of a diverse Vbeta repertoire. This targeting is precise and position-independent. This additional restriction on V(D)J recombination has important implications for the regulation of variable region gene assembly and repertoire development.     

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.     

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.     

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.     

Unusual sequences in the murine immunoglobulin mu-delta heavy-chain region

The delta heavy (H) chain of mouse immunoglobulin D (IgD) is unusual both in its structure and in its differential expression relative to immunoglobulin M (IgM; reviewed in ref. 1). The region of DNA between IgM and IgD H-chain constant-region genes is probably implicated in this control. So far only fragments of the area have been sequenced. Now, however, we present the complete sequence as well as the sequence of the introns of the C delta gene. We have found several interesting features (Fig. 1), including an open reading frame (ORF) between Cmu and C delta which encodes 146 amino acids that might represent a previously unsuspected domain-like protein; three blocks of simple repetitive sequences; a 162-base pair (bp) unique-sequence inverted repeat; and a domain-like pseudogene in the large intron of C delta. We have not found, however, any sequence 5' of C delta resembling the switch (S) recombination sequences associated with class switching in other heavy chains. Moreover, we have determined the 3' deletion end point of an IgD-producing myeloma and find no sequences reminiscent of switch sites nearby.     

Critical test of a sister chromatid exchange model for the immunoglobulin heavy-chain class switch

B lymphocytes may switch from producing an immunoglobulin heavy chain of the mu class to that of the gamma, epsilon or alpha class. To maintain the specificity, the new heavy chain must keep the original variable (V) region; this is achieved by deleting DNA sequences so that the V (consisting of joined VH, diversity (DH) and joining (JH) gene segments) and C (constant) gene segments coding for the new heavy chain are brought into close proximity (reviewed in ref. 5; we do not consider here the mu-delta situation). There are, in principle, three types of chromosomal rearrangements that yield a deletion: rearrangement within a chromatid; unequal sister chromatid exchange (as suggested by Obata et al.); and unequal recombination between chromosomal homologues. We have analysed the arrangement of C mu DNA in clones of the pre-B-cell line 18-81 that switches in vitro from mu to gamma 2b. The clones examined produce either mu, gamma 2b or no immunoglobulin chain. We report here that all the gamma 2b clones had lost at least one copy of C mu and no clones contained three copies of C mu. These findings formally exclude both unequal sister chromatid exchange and recombination between homologues as mechanisms for creating a gene encoding the gamma 2b chain.     

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.     

High-affinity binding site for a specific nuclear protein in the human IgM gene

Proteins binding to specific regions of DNA with high affinity frequently govern or regulate reactions at the gene level. We have identified a high-affinity binding site in the immunoglobulin mu gene that binds a specific nuclear protein, and have now characterized it fully using nuclear factor 1 (NF-1), a protein purified from the nuclei of HeLa cells and required for the in vitro replication of adenovirus (Ad) DNA. NF-1 protects a 25-base pair (bp) double-stranded segment of DNA which shares a consensus sequence, 5' TGGA/CNNNNNGCCAA 3', with similar binding sites in the Ad-5 terminal repeat and the human c-myc gene. Although this site differs from the enhancer region, a biological function is suggested by the fact that it is DNase I hypersensitive in immunoglobulin-producing lymphoblastoid cells. The binding site for the NF-1 protein in the mu gene, by analogy with the site in the Ad-5 terminal repeat, may represent one component of a cellular origin of replication; alternatively, it may be responsible for the activation of the chromatin in this region.     

Functional V region formation during in vitro culture of a murine immature B precursor cell line

B lymphocytes originate from pluripotential haematopoietic stem cells and differentiate into immunoglobulin (Ig)-producing cells. B-cell lineage differentiation is accompanied by two types of immunoglobulin gene rearrangements--rearrangement of V, D and J gene segments to create a functional V gene and rearrangement of CH genes for heavy-chain switching. These results, however, have been obtained mainly by analysis of immunoglobulin gene organization of myeloma cells. Baltimore and his colleagues have established Abelson murine leukaemia virus (A-MuLV)-transformed cell lines and found a few lines capable of carrying out kappa-gene rearrangement or undergoing isotype switching during in vitro culture. To study early B-cell lineage differentiation events, we have now also established A-MuLV-transformed cell lines which are capable of differentiating from mu- to mu+ and of undergoing continuing rearrangement of heavy-chain genes in culture. Analysis of immunoglobulin gene organization of these transformed cells revealed that mu- cells have already undergone DNA rearrangements involving JH segments but an additional rearrangement of JH segments is required for initiation of mu-chain synthesis. Southern blot analysis of the DNA and two-dimensional gel electrophoresis of intracytoplasmic mu-chain show that mu-chain diversity with respect to antigen specificity may be generated during this second rearrangement process. As no rearrangement of light-chain genes takes place in these cells, this implies that light-chain gene rearrangement requires some further change, or a different enzyme.     

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.     

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.