Nonhomologous recombination at sites within the mouse JH-C delta locus accompanies C mu deletion and switch to immunoglobulin D secretion.

Plasma cells secrete immunoglobulins other than immunoglobulin M (IgM) after a deletion and recombination in which a portion of the immunoglobulin heavy-chain locus (IgH), from the 5'-flanking region of the mu constant-region gene (C mu) to the 5'-flanking region of the secreted heavy-chain constant-region gene (CH), is deleted. The recombination step is believed to be targeted via switch regions, stretches of repetitive DNA which lie in the 5' flank of all CH genes except delta. Although serum levels of IgD are very low, particularly in the mouse, IgD-secreting plasmacytomas of BALB/c and C57BL/6 mice are known. In an earlier study of two BALB/c IgD-secreting hybridomas, we reported that both had deleted the C mu gene, and we concluded that this deletion was common in the normal generation of IgD-secreting cells. To learn how such switch recombinations occur in the absence of a switch region upstream of the C delta 1 exon, we isolated seven more BALB/c and two C57BL/6 IgD-secreting hybridomas. We determined the DNA sequences of the switch recombination junctions in eight of these hybridomas as well as that of the C57BL/6 hybridoma B1-8. delta 1 and of the BALB/c, IgD-secreting plasmacytoma TEPC 1033. All of the lines had deleted the C mu gene, and three had deleted the C delta 1 exon in the switch recombination event. The delta switch recombination junction sequences were similar to those of published productive switch recombinations occurring 5' to other heavy-chain genes, suggesting that nonhomologous, illegitimate recombination is utilized whenever the heavy-chain switch region is involved in recombination.     

High-frequency gene conversion between repeated C mu sequences integrated at the chromosomal immunoglobulin mu locus in mouse hybridoma cells.

The occurrence of mitotic recombination between repeated immunoglobulin mu gene constant (C mu) region sequences stably integrated at the haploid chromosomal immunoglobulin mu locus in murine hybridoma cells was investigated. Recombination events are detected as changes in hapten-specific immunoglobulin M production. Recombination occurs with high frequency (0.5 to 0.8%) by a mechanism consistent with gene conversion. A double-strand break repair-like mechanism is suggested by the finding that repair of a 2-bp deletion mutation and a 2-bp insertion mutation occurs with parity in a donor-directed manner. The results also suggest that the gene conversion process is directional in that the 5' C mu region sequence is preferentially converted.     

Polyclonal activation of the murine immune system by an antibody to IgD. VIII. Stimulation of IgD secretion

The low levels of serum IgD found in mice and the lack of a typical DNA switch sequence between C delta and C mu raise the possibility that the generation of murine IgD-secreting cells results from a chance "mistake" rather than a controlled process. The recent observation that injection of mice with purified IgD upregulates IgD receptor expression on helper T cells and enhances the ability of these T cells to induce B cells to differentiate into antibody secreting cells led us to look for evidence of controlled differentiation of B cells into IgD-secreting cells. To do this, we injected mice with a goat antibody to IgD (GaM delta), because this antibody stimulates large increases in IgM, IgG1, IgG2a, and IgE secretion. Mice injected with GaM delta demonstrated a large increase in splenic content of mRNA specific for the secreted form of delta-chain, as well as a greater than 100-fold increase in the percentage of splenic IgD-containing plasmablasts. The secretory IgD response was totally T-dependent. Production of the secretory form of IgD was not seen until 7 days after GaM delta injection, and peaked sharply on day 8, whereas by day 6 IgM secretion had already peaked and IgG1 and IgG2 secretion had attained substantial levels. This observation suggests that: 1) either cells that synthesize large quantities of the secretory form of delta-chain, unlike cells that synthesize large quantities of the secretory forms of gamma-, epsilon-, or alpha-chains, do this without deleting C mu, or, despite the absence of a typical DNA switch sequence between C mu and C delta, controls must exist to effect the C mu deletion and VDJ-C delta joining; and 2) if secreted IgD has a role in the regulation of a humoral immune response it most likely is involved in later processes, such as memory cell generation or response termination, rather than in relatively early processes, such as helper T cell activation.     

Ectopic recombination within homologous immunoglobulin mu gene constant regions in a mouse hybridoma cell line.

We have transferred a pSV2neo vector containing the wild-type constant region of the immunoglobulin mu gene (C mu) into the mutant hybridoma igm482, which bears a 2-bp deletion in the third constant-region exon of its haploid chromosomal mu gene (C mu 3). Independent igm482 transformants contain the wild-type immunoglobulin C mu region stably integrated in ectopic chromosomal positions. We report here that the wild-type immunoglobulin C mu region can function as the donor sequence in a gene conversion event which corrects the 2-bp deletion in the mutant igm482 chromosomal C mu 3 exon. The homologous recombination event restores normal immunoglobulin M production in the mutant cell.     

A switch region inversion contributes to the aberrant rearrangement of a mu immunoglobulin heavy chain gene in MPC-11 cells.

We describe the unique features of an aberrantly rearranged mu immunoglobulin heavy chain gene isolated from MPC-11 cells (a gamma 2b producing Balb/c plasmacytoma). A novel rearrangement has occurred 1.5 Kb 5' of the MPC-11 mu gene (denoted 18b mu) resulting in the deletion of the majority of the repetitive switch region (S mu) and 5' flanking DNA including the Joining (JH) sequences. The remainder (275 bp) of the S mu repeat has undergone a complete sequence inversion. DNA sequences 5' of the inverted S mu sequence do not resemble Variable (VH), Diversity (D), JH or their conserved flanking sequences. A DNA sequence localized 5' of the inverted S mu sequence, (p18b mu-1.4) detects a small family of homologous sequences in Balb/c DNA. The 18b mu-1.4 like sequences lack homology to S mu, exhibit flanking sequence polymorphisms in 5 out of 6 inbred mouse strains and undergo partial or complete deletion in 5 out of 10 plasmacytomas tested. Two 18b mu-1.4 homologous sequences display a higher copy number in C57Bl/6, AL/N and CAL9 mouse strains.     

Isotype switching in human B lymphocyte malignancies occurs by DNA deletion: evidence for nonspecific switch recombination

The mechanism and specificity of isotype switching operative in human B lymphocytes was investigated by a determination of immunophenotype and immunoglobulin heavy and light chain gene status in a panel of human Ig-, IgM, IgG, and IgA B cell malignancies. Regardless of specific tumor type or switched immunophenotype, isotype switching was accompanied by the rearrangement of the expressed CH gene downstream of VDJH, with concomitant deletion of upstream CH genes in all cases. On the allelically excluded chromosome, 25% of the IgG or IgA tumors have retained C mu, and 75% have deleted C mu. The 5' recombination breakpoints for both productive and excluded alleles lie within or near S mu, 3' of the enhancer. No correlation between the extent of allelically excluded CH deletions and the isotype produced by the tumor was observed. Excluded chromosome deletion endpoints were found 5', equal to, or 3' of productive chromosome deletion endpoints. Furthermore, we have identified at least one IgM+ tumor that has undergone abortive CH gene deletions and have observed several unanticipated switch region deletions and potential translocations. The data suggest that isotype switching in human B cells occurs by a nonsubclass- and nonclass-specific switch recombinase.     

The complete amino-acid sequence of the heavy chain of the human myeloma protein WIE, an immunoglobulin D.

The human myeloma protein WIE is a lambda-type immunoglobulin D; the amino-acid sequence of its Fc part and aminoethylated heavy chain was completely determined. The VH-part (subgroup III) begins N-terminally with 5-oxoproline, and it contains a long, unique CDR3 region. Since the constant part differs from known delta chains by one amino-acid substitution in the hinge region, IgD WIE probably represents an allotypic variant. As in other protein delta chains, O-glycosylations are confined to the hinge region. Furthermore, the ratios of N-glycosylations at the three positions are identical in IgD WAH [Takahashi, N. et al. (1984) J. Chromatogr. 317, 11-26.] and IgD WIE (100%, 50%, 100%). From the most conserved constant domain, C delta 3, a three-dimensional model was constructed to clarify the role of its delta-specific substitutions and glycosylation.     

Replacement recombinant events targeted at immunoglobulin heavy chain DNA sequences in mouse myeloma cells

The rate of gene conversions and double crossovers between transfected and integrated mu heavy chain immunoglobulin genes was measured in myeloma cells. The assay relies on correction of an integrated and defective mu heavy chain expression unit, present in a repeated head to tail array in the genome of the myeloma cell line J558L. Following electroporation of these cells with restriction fragments containing normal immunoglobulin sequences, targeted recombination events are identified by a complement-mediated haemolytic plaque assay measuring production of functional IgM. Recombination results in replacement of a segment of the target sequence with the exogenous sequence. Different crossover positions are possible, giving rise to alternative rearrangements of the target site. In the case of one of the recombinants we analysed, more than one of the repeated targets had undergone a conversion event. The efficiency of homologous recombination was shown to depend on the extent of homology between transfected and target DNA. A targeting efficiency of 1 x 10(-5) to 2 x 10(-5) was achieved when the exogenous DNA contained 10,000 bases of sequence homologous with the target.     

Nucleotide sequences of switch regions of immunoglobulin C epsilon and C gamma genes and their comparison

Immunoglobulin class switch involves a unique recombination event that takes place at the switch (S) region which is located 5' to each constant region (C) gene of the heavy (H) chain. For example, differentiation of the B lymphocyte from a mu-chain producer to an epsilon-chain producer is mediated by the switch recombination between the S mu and S epsilon regions. In order to elucidate the molecular mechanism for the switch recombination, we have determined nucleotide sequences surrounding the class switch recombination sites of the C epsilon and C gamma 3 genes and those in the 5' flanking regions of the C gamma 2a and C delta genes. The results indicate that the 5' flanking regions of all the CH genes except for the C delta gene contain the S regions which comprise tandem repetition of short unit sequences in agreement with the previous analyses of the S gamma 1, S gamma 2b, S mu, and S alpha regions. Comparison of the nucleotide sequences of all the S regions revealed that length as well as nucleotide sequences of the S regions vary among different classes of the CH gene, but they share short common sequences, (G)AGCT and TGGG(G). The nucleotide sequence of the S mu region is homologous to those of the other S regions in the decreasing order of the S epsilon, S alpha, S gamma 3, and (S gamma 1, S gamma 2b, s gamma 2a) regions. We have compared the nucleotide sequences immediately adjacent to the recombination sites of seven rearranged genes and have always fund tetranucleotides TGAG and/or TGGG, except for one case. Such tetranucleotides may constitute a part of the recognition sequence of a putative recombinase. These results provide further support for our previous proposal that the switch recombination may be facilitated by short common sequences dispersed in all the S regions.     

Nucleotide sequence and properties of the murine gamma 3 immunoglobulin heavy chain gene switch region: implications for successive C gamma gene switching

During B lymphocyte differentiation, immunoglobulin heavy chain constant region (CH) genes undergo a unique series of DNA recombination events culminating in the CH class switch. CH switch (S) regions are located 2 kb 5' of each CH gene except delta (i.e. mu, gamma 3, gamma 1, gamma 2b, gamma 2a, epsilon and alpha). We describe the structural features of the gamma 3 switch region. Hybridization experiments show that S gamma 3 has remarkable homology to both S mu and other S gamma regions while S mu possesses limited homology to the other S gamma sequences. However, S mu possesses extensive sequence homology with S epsilon and S alpha. The nucleotide sequence of S gamma 3 reveals higher densities of S mu repetitive sequences (GAGCT and GGGGT) and another S region common sequence (YAGGTTG) than observed for S gamma 1, S gamma 2b or S gamma 2a. In addition, the conservation of S mu like repetitive sequences in S gamma regions is correlated with the 5' leads to 3' gamma gene order (i.e. S gamma 3 greater than S gamma 1 greater than S gamma 2b greater than S gamma 2a). A model is presented which suggests that the unique features of S gamma 3 may allow for successive switches from C mu to any C gamma gene.     

Recombination between Two Identical Sequences within the Same Retroviral RNA Molecule

As a consequence of being diploid viruses, members of the Retroviridae have a high recombination rate. To measure recombination between two identical sequences within the same RNA molecule per round of retroviral replication cycle, a murine leukemia virus based vector (JZ442 + 3' Hyg) has been constructed. It carries a drug resistance gene, hyg, and a 290-bp repeat sequence of the 3' hyg gene inserted into the 3' untranslated region of the green fluorescent protein gene (gfp). Under fluorescence microscopy, Hygr cells containing the recombinant proviruses were clear, while a green color was observed in the drug-resistant cells carrying the parental proviruses. The rate of recombination was determined by the ratio of the number of clear colonies to the total number of Hygr colonies (green and clear colonies). The rate of recombination was found to be 62% by this method. The intermolecular recombination rate between an infectious virus bearing two copies of the 290-bp segment and a noninfectious chimeric RNA virus containing only a single copy of this sequence was also measured.     

Switch circular DNA formed in cytokine-treated mouse splenocytes: evidence for intramolecular DNA deletion in immunoglobulin class switching

We have characterized circular DNA in mouse splenocytes treated with the mitogen lipopolysaccharide (LPS) and various cytokines, including transforming growth factor beta (TGF-beta) and interleukin 4 (IL-4). Using probes of immunoglobulin heavy chain constant genes (CH), excision products of class switch recombination were identified. The majority of the clones contained the 3' portion of the switch mu (S mu) region and the 5' portion of other switch regions. Some clones contained 3'-S gamma sequences instead of 3'-S mu. This indicates that isotype switching may occur not only from C mu, but also from one of the C gamma genes to other CH genes further down-stream. In the presence of LPS, the cytokine TGF-beta enhanced the detection of 5'-S alpha-positive clones, while the lymphokine IL-4 enhanced 5'-S gamma 1 positives. The data support the notion that TGF-beta and IL-4 can direct isotype-specific class switching.     

The repair and recombination enzyme ERCC1 is not required for immunoglobulin class switching

Class switch recombination (CSR) is a programmed gene rearrangement in which a B cell which is producing IgM and IgD antibody develops into an IgG-, IgA- or IgE-expressing cell. This is achieved by recombination between switch regions located 5' of each of the immunoglobulin heavy chain constant regions, except Cdelta. The mechanism of CSR has not been resolved but it is thought to involve a double-strand break followed by end joining. It has previously been suggested that the nucleotide excision repair protein ERCC1 may be involved in CSR due to its known roles in removal of 3' single-stranded tails in various types of recombination. In this study, we examined class switching in cultured splenocytes from ERCC1-deficient mice and found no evidence of any deficiency.     

Molecular components of the B cell antigen receptor complex of class IgD differ partly from those of IgM

Two classes of immunoglobulin, IgM and IgD, are present as antigen receptors on the surface of mature B lymphocytes. We show here that IgD molecules are noncovalently associated in the B cell membrane with a heterodimer consisting of two proteins of 35 kd (IgD-alpha) and 39 kd (Ig-beta), respectively. The two novel proteins are not found in the IgD-expressing myeloma J558L delta m, which fails to bring IgD antigen receptor onto the cell surface. In a surface IgD positive variant line of this myeloma, however, membrane-bound IgD molecules are associated with the heterodimer, suggesting that the formation of an antigen receptor complex is required for surface IgD expression. We further demonstrate that the IgD-associated heterodimer differs partly from that of the IgM antigen receptor and that its binding to the heavy chain only requires the presence of the last constant domain and the transmembrane part of the delta m chain.