Constant Region Genes Research Articles

Differential regulation of chromatin structure of the murine 3′ IgH enhancer and IgG2b germline promoter in response to lipopolysaccharide and CD40 signaling

Class switch recombination (CSR) of murine immunoglobulin heavy chain (IgH) is controlled by germline transcription-coupled modification of the accessibility of the highly repetitive switch regions (S) located upstream of the constant region genes. Activation of the 3′ IgH enhancer (3′E) is believed to regulate CSR during B cell terminal differentiation, although the detailed molecular mechanism remains unclear. Here, we show that BAF57 and BRG1, two essential subunits of murine SWI/SNF complex, differentially associate with the DNase I hypersensitive region HS1/2 of 3′E and the IgG2b germline promoter in response to LPS activation or CD40 engagement. Both LPS and CD40 signaling cause SWI/SNF complex to dissociate from HS1/2 and associate with their responsive IgG2b germline promoter, suggesting the potential fluidity of chromatin structure and specific regulatory mode for the ATP-dependent chromatin remodeler during CSR. More interesting, increase in histone acetylation is either inverse or parallel with the action of SWI/SNF complex at HS1/2 enhancer or IgG2b germline promoter, respectively. Chromatin immunoprecipitation experiments show that alteration of histone H3 and H4 acetylation has overall similarities in response to LPS and CD40 signaling, with H3 hyperacetylated and H4 hypoacetylated at the HS1/2 enhancer and reversed modification patterns at the IgG2b germline promoter. Finally, the specificity of LPS and CD40 signaling in control of CSR could be partially coded by the specific acetylation marking of H3 and H4. Our results further strengthen the notion that chromatin remodeling plays a critical role in CSR.

Absence of Immunoglobulin Class Switch in Primary Lymphomas of the Central Nervous System

Primary lymphomas of the central nervous system (PCNSLs) were investigated for their capacity to perform further maturation steps. We studied a series of 11 PCNSLs derived from immunocompetent patients for immunoglobulin (Ig) class switch recombination (CSR) by performing reverse transcriptase-polymerase chain reaction (RT-PCR) for transcripts of Ig constant region gene segments (IGHC). This analysis revealed exclusive transcription of IgM and IgD mRNA in the absence of IgG, IgA, or IgE transcription. This finding was corroborated at the protein level by the immunohistochemical demonstration of IgM on the surface of the tumor cells. The unexpected lack of CSR may be due to internal switch mu region deletions, which were detected in 7 of 11 cases. We also found that expression of activation-induced cytidine deaminase (AID), which is required for CSR and somatic hypermutation, was detectable by RT-PCR in 4 of 10 cases and by immunohistochemistry in one of three cases analyzed. This may indicate that ongoing somatic mutation, which is often observed in PCNSL, could be due to sustained AID expression in a fraction of cases and that intraclonal V gene diversity may occur in other cases at an earlier phase of tumor clone expansion, when AID may have been expressed.

Discovery of a unique Ig heavy-chain isotype (IgT) in rainbow trout: Implications for a distinctive B cell developmental pathway in teleost fish

During the analysis of Ig superfamily members within the available rainbow trout (Oncorhynchus mykiss) EST gene index, we identified a unique Ig heavy-chain (IgH) isotype. cDNAs encoding this isotype are composed of a typical IgH leader sequence and a VDJ rearranged segment followed by four Ig superfamily C-1 domains represented as either membrane-bound or secretory versions. Because teleost fish were previously thought to encode and express only two IgH isotypes (IgM and IgD) for their humoral immune repertoire, we isolated all three cDNA isotypes from a single homozygous trout (OSU-142) to confirm that all three are indeed independent isotypes. Bioinformatic and phylogenetic analysis indicates that this previously undescribed divergent isotype is restricted to bony fish, thus we have named this isotype "IgT" (tau) for teleost fish. Genomic sequence analysis of an OSU-142 bacterial artificial chromosome (BAC) clone positive for all three IgH isotypes revealed that IgT utilizes the standard rainbow trout V(H) families, but surprisingly, the IgT isotype possesses its own exclusive set of D(H) and J(H) elements for the generation of diversity. The IgT D and J segments and tau constant (C) region genes are located upstream of the D and J elements for IgM, representing a genomic IgH architecture that has not been observed in any other vertebrate class. All three isotypes are primarily expressed in the spleen and pronephros (bone marrow equivalent), and ontogenically, expression of IgT is present 4 d before hatching in developing embryos.

A Chimeric Antibody to Varicella-Zoster Virus Glycoprotein E

Varicella-Zoster virus (VZV) immune globulin (VZIG) derived from pooled human serum is currently used in immunotherapy of VZV-associated complications of chickenpox and shingles. We developed a mouse-human chimeric antibody against a VZV glycoprotein E (gE) epitope as a safer replacement for VZIG. Variable (V) heavy- and V kappa light-chain exons, derived from an anti-VZV gE antibody secreting mouse hybridoma cell line, were cloned into expression vectors containing an immunoglobulin promoter and enhancer, and human IgG1 or kappa constant (C) region genes. The expression vectors were cotransfected into mouse myeloma cell line (NSO), generating transformants that secreted chimeric human-mouse IgGs. The chimeric and the parent mouse antibody were indistinguishable in their antigen binding specificity. VZV gE chimeric antibody may prove to be a prophylactic antibody that could provide significant advantages over VZIG in having defined specificity, lessened possibility of contamination with viral pathogens, and consistent availability.

Horse cytokine/IgG fusion proteins – mammalian expression of biologically active cytokines and a system to verify antibody specificity to equine cytokines

Recombinant cytokines are valuable tools for functional studies and candidates for vaccine additives or therapeutic use in various diseases. They can also be used to generate specific antibodies to analyze the roles of different cytokines during immune responses. We generated a mammalian expression system for recombinant cytokines using the equine IgG1 heavy chain constant region as a tag for detection and purification of the expressed cytokine, demonstrated here using equine interferon-gamma (IFN-gamma), interleukin-2 (IL-2), interleukin-4 (IL4) and transforming growth factor-beta1 (TGF-beta1). The resulting IgG1 fusion proteins were composed of the C-terminal heavy chain constant region of the IgG1 (IgGa), and the N-terminal cytokine replacing the immunoglobulin heavy chain variable domain. The fusion proteins were expressed in CHO cells as dimers and their structures had similarity to that of IgG heavy chain antibodies. In contrast to other tags, the IgG1 heavy chain constant region allowed the selection for clones secreting high levels of the recombinant protein by a sensitive ELISA. In addition, the IgG1 heavy chain constant region facilitated identification of stable transfectants by flow cytometry and the secreted recombinant fusion protein by SDS-PAGE and Western blotting. To recover the cytokine from the IgG1 fusion partner, an enterokinase cleavage site was cloned between the cytokine gene and the immunoglobulin heavy chain constant region gene. The purification of the fusion protein by protein G affinity columns, the enterokinase digestion of the cytokine from the IgG1 heavy chain region after or during purification, and the biological activity of the cytokine within the fusion protein or after its isolation was demonstrated in detail for equine IFN-gamma/IgG1 by up-regulation of major histocompatibility complex (MHC) class II expression on horse lymphocytes. Biological activity could also be confirmed for the IL-2 and IL-4/IgG1 fusion proteins. To test the crossreactivity and specificity of anti-human TGF-beta1, and anti-bovine and anti-canine IFN-gamma antibodies to respective horse cytokines, the four cytokine/IgG1 fusion proteins were successfully used in ELISA, flow cytometry and/or Western blotting. In summary, equine IgG1 fusion proteins provide a source of recombinant proteins with high structural and functional homology to their native counterparts, including a convenient system for selection of stable, high expressing transfectants, and a means for monitoring specificity of antibodies to equine cytokines.

Nibrin functions in Ig class-switch recombination

Nijmegen breakage syndrome (NBS) is a rare autosomal recessive disorder characterized by predisposition to hematopoietic malignancy, cell-cycle checkpoint defects, and ionizing radiation sensitivity. NBS is caused by a hypomorphic mutation of the NBS1 gene, encoding nibrin, which forms a protein complex with Mre11 and Rad50, both involved in DNA repair. Nibrin localizes to chromosomal sites of class switching, and B cells from NBS patients show an enhanced presence of microhomologies at the sites of switch recombination. Because nibrin is crucial for embryonic survival, direct demonstration by targeted deletion that nibrin functions in class switch recombination has been lacking. Here, we show by cell-type-specific conditional inactivation of Nbn, the murine homologue of NBS1, that nibrin plays a role in the repair of gamma-irradiation damage, maintenance of chromosomal stability, and the recombination of Ig constant region genes in B lymphocytes.

Vaccination against encapsulated bacteria in humans: paradoxes

Infection with encapsulated bacteria can be prevented by vaccination with capsular polysaccharides, either plain or conjugated to a protein carrier. However, results concerning these vaccinations raise several paradoxes. Polysaccharides from encapsulated bacteria are generally considered to be T cell-independent antigens which are unable to trigger a T cell-dependent germinal center reaction, but, strikingly, anti-polysaccharide antibodies are often mutated in humans. Polysaccharide–protein conjugate vaccines are able to induce a true T cell-dependent memory response with a rise in antibody titers and a switch to high-affinity IgG antibodies in children below two years of age, but neither the plain nor the conjugated vaccine can induce memory in older infants and adults. We propose some explanations for these paradoxes based on our recent observation that humans display a circulating splenic marginal zone B-cell population with a pre-diversified immunoglobulin repertoire in charge of the immune response to T cell-independent antigens. Infection with encapsulated bacteria can be prevented by vaccination with capsular polysaccharides, either plain or conjugated to a protein carrier. However, results concerning these vaccinations raise several paradoxes. Polysaccharides from encapsulated bacteria are generally considered to be T cell-independent antigens which are unable to trigger a T cell-dependent germinal center reaction, but, strikingly, anti-polysaccharide antibodies are often mutated in humans. Polysaccharide–protein conjugate vaccines are able to induce a true T cell-dependent memory response with a rise in antibody titers and a switch to high-affinity IgG antibodies in children below two years of age, but neither the plain nor the conjugated vaccine can induce memory in older infants and adults. We propose some explanations for these paradoxes based on our recent observation that humans display a circulating splenic marginal zone B-cell population with a pre-diversified immunoglobulin repertoire in charge of the immune response to T cell-independent antigens. An antigen that can induce an antibody response without the help of Th cells. Bacterial capsular polysaccharides are TI antigens. An antigen that requires interaction between Th cells and B cells to induce an antibody response. Most protein antigens are T-dependent. T-dependent responses take place in germinal centers. Mutations occurring with high frequency in the variable (V) regions of rearranged immunoglobulin (Ig) genes. In most mammalian species, hypermutation is used for the generation of high-affinity antibodies in response to td antigens. It can also be used as a diversification process (e.g. in sheep) to generate the pre-immune antibody repertoire. The process whereby IgM+ B cells switch, after antigen encounter, to the expression of a different isotype (or class, e.g. IgG, IgA or IgE) by replacing its Ig heavy chain constant region gene. Specialized structures that form transiently in secondary lymphoid organs (e.g. spleen, lymph nodes) during immune responses to TD antigens. Hypermutation, isotype switch, affinity maturation and memory B cell formation take place within these structures. Refers to the increase in the affinity of antibodies produced during the course of T cell-dependent responses. This process results from the selection within germinal centers of B cells harboring mutated receptors with higher affinity for the antigen. A B lymphocyte that has been stimulated by a TD antigen, and has differentiated into a class-switched, long-lived, non-secreting B cell-carrying mutated Ig. Memory B cells, when re-exposed to the immunizing antigen, are responsible for generating faster responses with higher affinity. A primary immunodeficiency characterized by normal or increased serum IgM levels and low or absent IgG, IgA and IgE serum levels, owing to a defect in isotype switch.

VDJ-Switch Region Analysis in Multiple Myeloma Patients Reveals Homogeneity and Long-Term Stability of Switch Junctions, and Ongoing Mutation Upstream of Switch Mu.

During class switch recombination (CSR) B cells change their immunoglobulin heavy chain (IgH) constant region genes by a deletional recombination mechanism involving well characterized switch (S) regions several Kb downstream of IgH variable region (VDJ) sequences. Unlike VDJ recombination, the junction formed by CSR between pre-switch S-mu (IgM) and post-switch S-gamma (IgG) is imprecise, and the key factors mediating this process are not fully understood. In multiple myeloma, a bone marrow cancer characterized by post-switch plasma cells bearing a unique clonotypic VDJ, switch regions have been shown to mediate translocations in a subset of MM patients, suggesting a role in transformation. In this study we cloned and sequenced the VDJ-hybrid immunoglobulin switch (VDJ-S) regions of post-switch multiple myeloma (MM) cells at diagnosis and a second time point to determine if the switch junction was normal, homogeneous within a time point, and stable over the course of malignancy.The VDJ-S of five stage III IgG MM patients were examined in this study. Each patient has a specific clonotypic VDJ region and downstream hybrid switch junction (S-mu/S-gamma) arising after class switch recombination (CSR) from IgM to IgG. For each patient two samples were tested: a diagnosis sample and a second sample taken between 1.6 and 4.2 years later. The 5–7 Kb VDJ-S was amplified by long distance PCR using bulk DNA samples from bone marrow and blood as template, CDR2 specific sense primers and S-gamma specific antisense primers. In each case a single DNA product was amplified, cloned, and sequenced with primers covering the VDJ-S region. Patient specific CDR2 primers were used to confirm that the fragments were clonotypic. Unique switch junctions for patients 1–5 were detected at positions 125, 251, 435, 590, and 750 of S-mu, respectively. This agrees with previous studies mapping MM switch junctions to the 5′ portion of S-mu. The sequence and mutation profile of the switch junctions, with more frequent mutations in the S-mu region upstream of the hybrid junction, suggests that these junctions arise from normal CSR. A single hybrid junction was detected in all patients, corresponding to the single VDJ-Switch fragment amplified by long-distance PCR. The switch junction sequences also remained constant between time point samples, suggesting switch junction stability. Interestingly, new mutations were observed in the 3.5 Kb region upstream of the switch region, in the second time point samples of 2/5 patients. Most notably, 19 new mutations were detected in one patient, including 3 bp and 64 bp deletions 135 and 223 bp downstream of the intronic enhancer, respectively. The significance of these mutations is unclear, considering previous studies showing mutations, insertions, and deletions in this region in normal CSR. Nevertheless, it seems this process continues in clonotypic cells of some patients throughout malignancy. This ongoing mutation may lead to ‘remodeling' of the VDJ-S region, which is significant in the context of immunoglobulin translocations where proximity with immunoglobulin enhancers may confer a selective advantage.

Combination Therapy of Humanized Chimeric-Anti-Human VLA4 Monoclonal Antibodies and Anti-Cancer Drugs for AML.

Bone marrow (BM) minimal residual disease causes relapse after chemotherapy in AML. We have previously reported that VLA4-positive leukemic cells acquired resistance to drug-induced apoptosis through the PI-3K/AKT/Bcl-2 signaling pathway, which is activated by the interaction of VLA4 and fibronectin on BM stromal cells. This resistance was negated by mouse-anti-human VLA4Ab (mouse VLA4Ab). In human leukemia SCID mouse model, we demonstrated a 100% survival rate with combination of mouse VLA4 Ab and AraC, while with AraC alone, only slight prolongation of survival was attained. In clinical study, overall survival at 5 years was 90% for 10 VLA4− patients and 25% for 15 VLA4+ patients (Matsunaga T et al, Nature Med 2003, 9, 1158–1165). In the present study, to perform the translational research, we first examined the myelosuppressive effect of the combination of rat-anti-mouse VLA4 Ab and AraC in C57/BL6 mice, and found that CBC data were almost the same as those of the mice treated with AraC alone. We next produced humanized chimeric-anti-human VLA4 Ab (chimeric-VLA4Ab), and examined its efficacy in combination with anti-cancer drugs in vitro and in vivo (human leukemia SCID mouse model). Chimeric-VLA4Abs were produced as follows: (i) total RNA of mouse VLA4 Abs were extracted from two hybridomas (SG/17 and SG/73), (ii) cDNA were synthesized by reverse transcriptase, (iii) variable region gene of mouse VLA4 Abs were amplified by 5′RACE method, (iv) TA cloning of amplified gene was performed, (v) sequence of mouse VLA4Abs gene was determined, (v) cloned variable region gene of mouse VLA4 Abs and constant region gene of human IgG1 were inserted into expression vector, and the expression vector was transfected into 293T cells, (vi) supernatant of the 293T cells was collected and purified to obtain chimeric-VLA4 Abs. The effects of chimeric-VLA4 Abs thus obtained in in vitro and in vivo (human leukemia SCID mouse model) were comparable to those of mouse VLA4 Abs. To perform the clinical study, we are presently producing the GMP-graded chimeric-VLA4 Abs.

Silencing of the immunoglobulin heavy chain locus by removal of all eight constant-region genes in a 200-kb region

Silencing or removal of individual C (constant)-region genes and/or adjacent control sequences did not generate fully deficient Ig (immunoglobulin) − mice. A reason is that different C genes share many functional tasks and most importantly are individually capable of ensuring lymphocyte differentiation. Nevertheless, incomplete arrests in B-cell development were found, most pronounced at the onset of H-chain expression. Here we show that removal of 200 kb accommodating all C genes, Cμ–Cδ–Cγ3–Cγ1–Cγ2b–Cγ2a–Cε–Cα, stops antibody production. For this two loxP targeting constructs were introduced into the most 5′ C gene and the distal α 3′ enhancer. Cre– loxP-mediated in vivo deletion was accompanied by extensive germ-line mosaicism, which could be separated by breeding. Homozygous C-gene deletion mice did not express Ig H or L chains and flow cytometry revealed a complete block in B-cell development. However, C-gene removal did not affect DNA rearrangement processes following locus activation, as recombination efficacy appears to be similar to what is found in normal mice.

Structure and organization of the T cell receptor alpha chain genes in Atlantic salmon

A cDNA fragment of the T cell receptor (TCR) α chain mRNA in Atlantic salmon ( Salmo salar) was amplified by PCR and used as a probe to isolate a full-length clone from a leukocyte cDNA library. Additionally, a genomic lambda clone comprising the TCR α chain constant region (Cα) gene and flanking regions was isolated and partially sequenced. The Cα gene consists of three exons corresponding to the immunoglobulin (Ig) domain, the hinge region and the transmembrane peptide/cytoplasmatic tail, and two exons corresponding to the untranslated tail of the mRNA. Remnants of a transposase gene and a partial duplication of the Cα gene were found nearby the intact gene. One J segment was found 1.5 kb upstream of the Cα gene. Twenty-six other J elements were identified among cDNA fragments covering the V/J/Cα junction. Representatives of five Vα gene families were identified by PCR amplification of genomic DNA fragments. PCR amplification of Cα fragments from another individual revealed a slightly different Cα gene which most likely represents an allelic variant.

Immunoglobulin class-switch recombination in mice devoid of any Sμ tandem repeat

AbstractImmunoglobulin heavy-chain class-switch recombination (CSR) occurs between highly repetitive switch sequences located upstream of the constant region genes. However, the role of these sequences remains unclear. Mutant mice were generated in which most of the Iμ-Cμ intron was deleted, including all the repeats. Late B-cell development was characterized by a severe impairment, but not a complete block, in class switching to all isotypes despite normal germ line transcription. Sequence analysis of the Iμ-Cμ intron in in vitro activated–mutant splenocytes did not reveal any significant increase in activation-induced cytidine deaminase (AID)–induced somatic mutations. Analysis of switch junctions showed that, in the absence of any Sμ repeat, the Iμ exon was readily used as a substrate for CSR. In contrast to the sequence alterations downstream of the switch junctions, very few, if any, mutations were found upstream of the junction sites. Our data suggest that the core Eμ enhancer could be the boundary for CSR-associated somatic mutations. We propose that the core Eμ enhancer plays a central role in the temporal dissociation of somatic hypermutation from class switching.

Structure and organization of the T cell receptor alpha chain genes in Atlantic salmon

A cDNA fragment of the T cell receptor (TCR) α chain mRNA in Atlantic salmon (Salmo salar) was amplified by PCR and used as a probe to isolate a full-length clone from a leukocyte cDNA library. Additionally, a genomic lambda clone comprising the TCR α chain constant region (Cα) gene and flanking regions was isolated and partially sequenced. The Cα gene consists of three exons corresponding to the immunoglobulin (Ig) domain, the hinge region and the transmembrane peptide/cytoplasmatic tail, and two exons corresponding to the untranslated tail of the mRNA. Remnants of a transposase gene and a partial duplication of the Cα gene were found nearby the intact gene. One J segment was found 1.5 kb upstream of the Cα gene. Twenty-six other J elements were identified among cDNA fragments covering the V/J/Cα junction. Representatives of five Vα gene families were identified by PCR amplification of genomic DNA fragments. PCR amplification of Cα fragments from another individual revealed a slightly different Cα gene which most likely represents an allelic variant.

53BP1 links DNA damage-response pathways to immunoglobulin heavy chain class-switch recombination

The mammalian protein 53BP1 is activated in many cell types in response to genotoxic stress, including DNA double-strand breaks (DSBs). We now examine potential functions for 53BP1 in the specific genomic alterations that occur in B lymphocytes. Although 53BP1 was dispensable for V(D)J recombination and somatic hypermutation (SHM), the processes by which immunoglobulin (Ig) variable region exons are assembled and mutated, it was required for Igh class-switch recombination (CSR), the recombination and deletion process by which Igh constant region genes are exchanged. When stimulated to undergo CSR, 53BP1-deficient cells exhibited no defect in C(H) germline transcription or AID expression, however these cells had a profound decrease in switch junctions. The current findings, in combination with the known 53BP1 functions and how it is activated, implicate the DNA damage response to DSBs in the joining phase of class-switch recombination.

The generation of antibody diversity through somatic hypermutation and class switch recombination.

Antibody-mediated immunity is critical to the resistance of vertebrate species to pathogenic organisms. Although low-affinity immunoglobin (Ig) M antibodies circulate in the blood prior to encountering pathogens, high-affinity IgG and IgA antibodies are required to inactivate toxins, neutralize viruses, and promote the clearance of microorganisms. Individuals, such as those with hyper-IgM syndrome (HIGM), who lack the ability to make such high-affinity IgG and IgA antibodies, are unable to combat bacterial and viral infections and usually die at a young age (Revy et al. 2000; Imai et al. 2003). Prior to exposure to antigen, the initial generation of a diverse antibody repertoire is achieved early in B-lymphocyte development by the successful rearrangement of the V, D, and J gene segments to produce B cells, each of which makes a unique Ig heavyand light-chain variable (V) region (Fig. 1A; Tonegawa 1983). These V regions encode the antigen binding sites of antibodies that are then expressed on the surface of a B lymphocyte and its clonal progeny. Following specific antigen recognition by its cognate B lymphocyte and costimulation by helper T lymphocytes, the B lymphocyte enters the germinal center of peripheral lymphoid organs to become a centroblast B cell. There, a second wave of antibody diversification occurs through somatic hypermutation (SHM) and/or gene conversion (GC) of the V region to generate high-affinity antigen binding sites (Fig. 1B; MacLennan 1994). SHM is the predominant mechanism in mice and humans, whereas GC occurs in chickens and some other species (Weill and Reynaud 1996). In the same centroblast B cell, the heavy-chain V regions encoding the antigen binding sites are rearranged down the chromosome through class switch recombination (CSR) so that they can be expressed with one of the constant (C) region genes to carry out many different effector functions and be distributed throughout the body (Fig. 1B; Manis et al. 2002b). SHM and CSR are largely targeted to the Ig genes, but their targeting to other genes causes many of the B-cell lymphomas in mice and humans (Pasqualucci et al. 2001). The biochemical mechanism and regulation of SHM and CSR are the topic of this review.

Physical Mapping of the Bovine Immunoglobulin Heavy Chain Constant Region Gene Locus

Bovine antibodies have recently attracted increasing attention, as they have been shown to exhibit prophylactic and therapeutic properties in selected infectious diseases in humans. In the present study, we have isolated bacterial artificial chromosomes and cosmid clones containing the bovine JH, mu, delta, gamma 1, gamma 2, gamma 3, epsilon, and alpha genes, which allowed us to make a contig of the genes within the bovine IGHC locus. The genes are arranged in a 5'-JH-7 kb-mu-5 kb-delta-33 kb-gamma 3-20 kb-gamma 1-34 kb-gamma 2-20 kb-epsilon- 13 kb-alpha-3' order, spanning approximately 150 kb DNA. Examination of the bovine germline JH locus revealed six JH segments, two of which, JH1 and JH2, were shown to be functional although there was a strong preference for expression of the former. Sequence alignment of the bovine 5' E mu enhancer core region with those of other mammals, demonstrated an absence of the mu E3 motif and a shortened spacer between the mu A and mu B sites within the bovine E mu enhancer core region. Furthermore, the essential sequence element for class switching, switch mu, spanning approximately 3-kb repetitive sequence and abundant in the switch region motifs CTGGG (187 repeats) and CTGAG (127 repeats), was identified immediately upstream of the mu gene. A further sequence comparison revealed that the bovine IGHC genes display an extensive polymorphism leading to expression of multiple antibody allotypes.

Elucidation of a downstream boundary of the 3′ IgH regulatory region

Class switch recombination (CSR) changes the immunoglobulin heavy chain (IgH) constant region gene (C H) in B cells from IgM to IgG, IgA, or IgE, without modifying the variable region gene segment. This process requires transcription through switch (S) regions located upstream of the C H genes targeted for CSR, a process that relies on the activity of an uncharacterized regulatory region at the 3′ end of the C H locus (3′ IgH RR) that has been implicated via the effects of pgk- neo cassettes inserted into the locus. The 30 kb region just downstream of the most 3′ C H gene (Ca) contains four known enhancer elements including HS3a, HS1,2, HS3b, and HS4. Replacement of either of the proximal two enhancer elements (HS3a or HS1,2) with a pgk- neo gene cassette disrupted germline transcription of and CSR to most C H genes. However, replacement of either of the enhancers with a loxP sequence had no effect on CSR indicating that these elements are not critical for CSR. Insertion of a pgk- neo cassette at various sites within the C H locus inhibited CSR to upstream, but not downstream C H genes, supporting the notion that the pgk- neo cassette insertion into the locus short-circuits the ability of the 3′ RR to facilitate CSR of dependent C H genes upstream of the insertion. These analyses also indicated that the key elements of the 3′ IgH RR were downstream from HS1,2. In this study, we have sought to localize the 3′ IgH RR by defining its 3′ boundary. For this purpose, a pgk- neo gene cassette was targeted 2 kb downstream of the HS4 element in ES cells that had normal ability to undergo CSR. We then employed Rag-2 deficient blastocyst complementation to generate chimeric mice that harbored B cells homozygous for this mutation. Such chimeras exhibited normal reconstitution of the splenic compartment and had normal serum immunoglobulin levels. Upon in vitro activation, transcription from the pgk- neo cassette was induced in B cells, however, CSR to all measured IgH isotypes occurred at normal levels. These findings, coupled with previous pgk- neo insertion studies, suggest that key elements of the 3′ IgH RR lie within a 17 kb region between HS1,2 and 2 kb downstream of HS4.

Baboon immunoglobulin constant region heavy chains: identification of four IGHG genes.

The increasing use of nonhuman primate models in biomedical research and especially in vaccine development requires the characterization of their immunoglobulin genes and corresponding products. Therefore, we sequenced, cloned and characterized the four immunoglobulin gamma chain constant region genes ( IGHG) present in baboons. These four genes were designated IGHG1, IGHG2, IGHG3 and IGHG4 on the basis of sequence similarities with the four human genes encoding the IgG1, IgG2, IgG3 and IgG4 subclasses and the three known rhesus macaque IGHG genes. Specifically, the baboon IgG1, IgG2, IgG3 and IgG4 sequences exhibit 90.3%, 88.3%, 86.7% and 89.6% amino acid identity to their human counterpart. The percent of amino acid identity of baboon IgG1, IgG2 and IgG3 to the corresponding rhesus macaque sequences is 98.5, 93.1 and 94.4, respectively. Therefore, baboon and rhesus macaque IGHG genes are highly homologous to each other. The majority of differences existing between baboon and human sequences are clustered in the hinge region, with the upper hinge being the most diverse and containing several proline residues. Similar to rhesus macaques, the hinge regions of all baboon IGHG genes consist of a single exon, whereas in humans the IgG3 molecule is encoded by multiple exons. These results confirm the evolutionary instability of the hinge region and indicate that functional properties associated with the hinge regions of baboon and human IgG molecules might differ between the two species.

Replication and subnuclear location dynamics of the immunoglobulin heavy-chain locus in B-lineage cells.

The murine immunoglobulin heavy-chain (Igh) locus provides an important model for understanding the replication of tissue-specific gene loci in mammalian cells. We have observed two DNA replication programs with dramatically different temporal replication patterns for the Igh locus in B-lineage cells. In pro- and pre-B-cell lines and in ex vivo-expanded pro-B cells, the entire locus is replicated early in S phase. In three cell lines that exhibit the early-replication pattern, we found that replication forks progress in both directions through the constant-region genes, which is consistent with the activation of multiple initiation sites. In contrast, in plasma cell lines, replication of the Igh locus occurs through a triphasic pattern similar to that previously detected in MEL cells. Sequences downstream of the Igh-C alpha gene replicate early in S, while heavy-chain variable (Vh) gene sequences replicate late in S. An approximately 500-kb transition region connecting sequences that replicate early and late is replicated progressively later in S. The formation of the transition region in different cell lines is independent of the sequences encompassed. In B-cell lines that exhibit a triphasic-replication pattern, replication forks progress in one direction through the examined constant-region genes. Timing data and the direction of replication fork movement indicate that replication of the transition region occurs by a single replication fork, as previously described for MEL cells. Associated with the contrasting replication programs are differences in the subnuclear locations of Igh loci. When the entire locus is replicated early in S, the Igh locus is located away from the nuclear periphery, but when Vh gene sequences replicate late and there is a temporal-transition region, the entire Igh locus is located near the nuclear periphery.

Identification of Potential Regulatory Elements in the Human Immunoglobulin Loci

ABSTRACT In this post-genomic era, it is necessary to formulate specific questions and develop bioinformatics tools to understand the vast amounts of information stored in DNA sequence. Using the combinatorial pattern discovery algorithm called Teiresias developed by the Bioinformatics and Pattern Discovery Group at IBM, we have identified novel conserved motifs present in the immunoglobulin loci. In the human VH promoter regions, two new putative regulatory elements have been implicated in basal transcriptional regulation of immunoglobulin. In the intergenic regions of the immunoglobulin constant region genes segments, elements were identified that are absent in similar regions of the Igβ gene. Since the expression patterns of Igβ and the Ig genes are similar such elements may have functions in activities specific to the Ig genes such as somatic hypermutation. These elements represent V gene-specific motifs identified through the use of a pattern discovery algorithm.