Constant Region Genes Research Articles

Analysis of two IgM isotypes in Atlantic salmon and brown trout

Atlantic salmon ( Salmo salar) possesses two distinct subpopulations of polymeric IgM which are separable by anion exchange chromatography. Consistent with this finding there are two isotypic IgM heavy chain genes, CμA and CμB, in the genome of this species, presumably as a result of ancestral tetraploidy. In the present study it was shown that IgM of brown trout ( Salmo trutta) is also separated into two subpopulations by anion exchange chromatography, while IgM of rainbow trout ( Oncorhynchus mykiss) and Arctic char ( Salvelinus alpinus) are eluted in one peak. Molecular cloning of IgM heavy chain cDNAs from brown trout revealed messages of two distinct constant region genes, named CμA and CμB. As deduced from the translated cDNA sequences (and in agreement with isoelectric focusing of the corresponding proteins) the mean pI values of the heavy chains in brown trout differ with only 0.14 units, in comparison to a 0.67 unit difference in salmon. Based on the present sequence analysis we suggest that an additional cysteine near the C-terminus of CμB is critical in relation to the fractionation of IgM by anion exchange chromatography, for example by altering the overall structure of the IgM polymer and the exposure of charged residues. Most likely, the Cμ subvariant with the characteristic extra cysteine residue arose in the ancestor of Atlantic salmon and brown trout, i.e. after the three genera Salmo, Oncorhynchus and Salvelinus radiated.

The activation-induced deaminase functions in a postcleavage step of the somatic hypermutation process.

Activation of B cells by antigen fuels two distinct molecular modifications of immunoglobulin (Ig) genes. Class-switch recombination (CSR) replaces the Igμ heavy chain constant region with a downstream constant region gene, thereby altering the effector function of the resulting antibodies. Somatic hypermutation (SHM) introduces point mutations into the variable regions of Ig genes, thereby changing the affinity of antibody for antigen. Mechanistic overlap between the two reactions has been suggested by the finding that both require the activation-induced cytidine deaminase (AID). It has been proposed that AID initiates both CSR and SHM by activating a common nuclease. Here we provide evidence that cells lacking AID, or expressing a dominant negative form of the protein, are still able to incur DNA lesions in SHM target sequences. The results indicate that an intact cytidine deaminase motif is required for AID function, and that AID acts downstream of the initial DNA lesions in SHM.

The AID enzyme induces class switch recombination in fibroblasts.

The switch of the immunoglobulin isotype from IgM to IgG, IgE or IgA is mediated by class switch recombination (CSR). CSR changes the immunoglobulin heavy chain constant region (CH) gene from Cmu to one of the other CH genes. Somatic hypermutation introduces massive numbers of point mutations in the immunoglobulin variable (V) region gene, giving rise to immunoglobulin with higher affinity. Activation-induced cytidine deaminase (AID), a putative RNA-editing cytidine deaminase, is expressed strictly in activated B cells and is indispensable in both CSR and somatic hypermutation. But the exact function of AID is unknown. Here we show that ectopic expression of AID induces CSR in an artificial switch construct in fibroblasts at a level comparable to that in stimulated B cells. Sequences around recombination junctions in the artificial substrate have features similar to endogenous CSR junctions. Furthermore, AID-induced CSR in fibroblasts is dependent on transcription of the target S region, as shown in endogenous CSR in B cells. The results show that AID is the only B-cell-specific factor required for initiation of the CSR reaction in the activated locus.

Biological activity of a mouse-human chimeric immunoglobulin G2 antibody to Cryptococcus neoformans polysaccharide.

The variable regions of the heavy and light chains of the protective murine monoclonal antibody (MAb) 2H1 (m2H1) were expressed with the human constant region genes for immunoglobulin G2 (IgG2) and kappa, respectively, to construct a chimeric antibody (ch2H1). ch2H1 retains the specificity of the parent MAb, exhibits biological activity, and lacks the toxicity of the parent murine IgG1 in chronically infected mice.

DNA microarray analysis of gene expression markers of endometriosis

Objective: To use DNA microarray technology to examine differential gene expression in uterine endometrium versus endometriosis implants. Design: Pilot study. Setting: Volunteers in an academic research environment. Patient(s): Premenopausal women scheduled for surgery for suspected endometriosis. Intervention(s): Surgical excision of endometriosis tissue and uterine endometrial biopsy. Main Outcome Measure(s): Gene expression. Result(s): The expression of eight genes from a total of 4,133 genes on the DNA microarray was increased in endometriosis implants compared with uterine endometrium. The eight genes were β-actin, α-2 actin, vimentin, 40S ribosomal protein S23, Ig-λ light chain, Ig germline H chain G-E-A region γ-2 constant region gene, major histocompatibility complex class 1,C, and complement component 1 S subcomponent. Conclusion(s): The data demonstrate that the DNA microarray is an effective tool for the identification of differentially expressed genes between uterine and ectopic endometrium; further study of the genes identified herein will expand our understanding of the nature of endometriosis and assist in the eventual development of new treatments for endometriosis.

AID is required to initiate Nbs1/gamma-H2AX focus formation and mutations at sites of class switching.

Class switch recombination (CSR) is a region-specific DNA recombination reaction that replaces one immunoglobulin heavy-chain constant region (Ch) gene with another. This enables a single variable (V) region gene to be used in conjunction with different downstream Ch genes, each having a unique biological activity. The molecular mechanisms that mediate CSR have not been defined, but activation-induced cytidine deaminase (AID), a putative RNA-editing enzyme, is required for this reaction. Here we report that the Nijmegen breakage syndrome protein (Nbs1) and phosphorylated H2A histone family member X (gamma-H2AX, also known as gamma-H2afx), which facilitate DNA double-strand break (DSB) repair, form nuclear foci at the Ch region in the G1 phase of the cell cycle in cells undergoing CSR, and that switching is impaired in H2AX-/- mice. Localization of Nbs1 and gamma-H2AX to the Igh locus during CSR is dependent on AID. In addition, AID is required for induction of switch region (S mu)-specific DNA lesions that precede CSR. These results place AID function upstream of the DNA modifications that initiate CSR.

In situ class switching and differentiation to IgA-producing cells in the gut lamina propria.

One of the front lines of the immune defence is the gut mucosa, where immunoglobulin- (IgA) is continuously produced to react with commensal bacteria and dietary antigens. It is generally accepted that, after antigenic stimulation in the Peyer's patches, IgA+ lymphoblasts (B220+IgA+) migrate through the lymph and blood circulation, and eventually home to the lamina propria of the intestine. Mice that lack activation-induced cytidine deaminase (AID) are defective in class switch recombination (CSR) and somatic hypermutation. CSR changes the immunoglobulin heavy chain constant region (CH) gene being expressed from Cmu to other CH genes, resulting in a switch of the immunoglobulin isotype from IgM to IgG, IgE or IgA. AID-/- mice also secrete large amounts of immunoglobulin-mu (IgM) into faeces, and accumulate B220-IgM+ plasma cells as well as B220+IgM+ cells in the gut. Here we show that lamina propria B220+IgA+ cells have just completed CSR, as they still express both AID and transcripts from circular DNA that has been 'looped-out' during CSR. Lamina propria IgM+ B cells seem to be pre-committed to switching to IgA+ in vitro as well as in vivo. Culturing lamina propria IgM+ B cells together with lamina propria stromal cells enhances preferential switching and differentiation of B cells to IgA+ plasma cells. We conclude that IgA+ cells in the gut lamina propria are generated in situ from B220+IgM+ lymphocytes.

Nucleotide sequence and restriction fragment length polymorphisms of the equine Cε gene

IgE is the dominant immunoglobulin isotype involved in type I hypersensitivities in mammals. The heavy chain constant region domains of equine IgE are encoded by a single gene, the Cε gene. By restriction analysis of cDNA from 15 unrelated horses, we have now identified two Cε alleles, characterised by a Sma I restriction fragment length polymorphism, which we designated Cε a and Cε b. Sequence analysis of both, Cε a and Cε b cDNA, showed in addition two single base exchanges resulting in two amino acid substitutions. Both sequences have only 95.9% homology of the coding region sequence with the published equine Cε sequence, which could represent a third haplotype. Polymorphism of the IgE heavy chain constant region gene, as described here, might well impose genetic variability on the effector functions of equine IgE predisposition to allergic diseases in horses.

Intraspecies heterogeneity of immunoglobulin alpha-chain constant region genes in rhesus macaques.

Immunoglobulin A (IgA) is the major antibody class present in external secretions and is also an important component of serum immunoglobulins. On mucosal surfaces, IgA represents a first line of defence by neutralizing invading pathogens. The number of IgA constant-region genes (C alpha) present in different mammalian species is variable. Immunoglobulin C alpha genes differ mainly in the sequences located in the hinge region. IgA molecules, whose hinge regions are remarkably similar to those of the respective human molecules, are present in hominoid primates. In this report, we show that two alleles of a single immunoglobulin C alpha are present in rhesus macaques (Macaca mulatta). In addition, we show that intraspecies immunoglobulin C alpha allelic polymorphism is very high in this non-human primate species. Specifically, five different hinge regions, some of which are proline-rich, were identified from a total of eight rhesus macaque immunoglobulin C alpha-chains. The five hinge regions were different from those present in hominoid primates, both in length and in sequence. These results represent the first example of high levels of intraspecies immunoglobulin constant-region variability and suggest that IgAs of variable structure and function may be present in rhesus macaques. As rhesus macaques are widely used as animal models for the development of vaccines for acquired immune deficiency syndrome (AIDS), the possible presence of structurally and functionally variable IgA molecules in different animals should be taken into account when designing experimental strategies to induce mucosal antibody responses to human immunodeficiency virus (HIV).

Class switch recombination was specifically targeted to immunoglobulin (Ig)G4 or IgA in Hodgkin's disease-derived cell lines.

In T cell-dependent immune responses, class switch recombination occurs in germinal centres. There is now evidence that Hodgkin/Reed-Sternberg cells are derived from germinal centre B cells. Cytokines specifically determine the direction of class switching, i.e the isotype of the new antibodies. We performed restriction analyses and polymerase chain reaction on the immunoglobulin heavy chain loci for five Hodgkin's disease-derived B-cell lines and one Hodgkin's disease-derived T-cell line in order to analyse class switch recombination. In all the B-cell lines, class switch recombination had been targeted to Calpha4 or Calpha1/2. This showed that cell-line precursors had undergone class switching, probably under the influence of TH2 or TH3 cell-derived cytokines. Deletions comprising several constant region genes were observed in cell lines L428, L1236, L591 and KMH2. Karyotype analyses of two of these revealed translocational breakpoints within the immunoglobulin heavy chain gene locus. Our data support the view that a chromosomal instability may occur during class switch recombination in Hodgkin/Reed-Sternberg cells causing chromosomal breaks. Thus, as in other germinal centre B cell-derived lymphomas, the immunoglobulin gene locus may be frequently involved in structural chromosomal aberrations in Hodgkin's disease.

Immunologic compensation in a patient with a large IgH constant region deletion

Background: Deficiencies of serum Ig of the IgG isotype typically predispose individuals to recurrent infections in some but not all cases. Patients with large deletions of the Ig heavy chain genes are free of recurrent and severe infections. Objective: We sought to determine a mechanism of immunologic compensation that would possibly explain the reason for this patient’s paucity of infection despite lacking several classes of serum Ig. Methods: The patient is a 50-year-old white man. Serum Ig levels and specific antibody titers were measured by using various methods, including nephelometry, enzyme immunoassay, and radial immunodiffusion. The status of the Ig heavy chain genes was examined by means of Southern blotting of genomic DNA isolated from EBV-transformed B cells. Results: The patient’s serum lacked detectable IgG1, IgG2, IgG4, and IgA1 levels. Southern blot analysis demonstrated a large heavy chain constant (C) region gene deletion that included Cγ1, Cα1, ψCγ, Cγ2, and Cγ4. Antibody responses to capsular pneumococcal and hemophilus polysaccharide antigens were essentially absent. However, IgG3 antibodies against the protein antigen tetanus toxoid were present. Relatively high antibody titers were found against pneumococcal surface proteins as well. Conclusion: We conclude that our patient’s relative freedom from serious infection may be as a result of production of IgG3 antibodies to pneumococcal capsular proteins. (J Allergy Clin Immunol 2001;107:1051-5.)

A new coding region polymorphism of human IgLC2.

A new allelic form of the human IgLC2 gene is described. The marker involves a T to C substitution in the C lambda 2 constant region gene, a silent substitution at amino acid coding position 178 (YAASSYLSL) and two substitutions in the 3'-flanking region. Analysis of IgLC2 alleles in a total of 60 individuals has indicated a frequency of 0.32 for the new allele, which has been designated IgLC2*B2. The *B1 and *B2 alleles encode T and C, respectively, at nucleotide position 212 in the IgLC2 coding region. Both the *B1 and *B2 alleles are found in individuals homozygous for the single-copy RFLP allele of IgLC2/IgLC3 (8 kb EcoRI). Knowledge of alleles of this marker will be important for studies on the expression of the IgLC2 and IgLC3 isotypes in normal and autoimmune lymphocyte populations, as the coding regions of the two isotypes differ only at this position. The marker will also be useful in further studies of linkage with other IgLV and IgLC markers and to establish possible correlations with susceptibility to autoimmune disorders.

A new coding region polymorphism of human IgLC2

AbstractA new allelic form of the human IgLC2 gene is described. The marker involves a T to C substitution in the Cλ2 constant region gene, a silent substitution at amino acid coding position 178 (YAASSYLSL) and two substitutions in the 3′‐flanking region. Analysis of IgLC2 alleles in a total of 60 individuals has indicated a frequency of 0.32 for the new allele, which has been designated IgLC2*B2. The *B1 and *B2 alleles encode T and C, respectively, at nucleotide position 212 in the IgLC2 coding region. Both the *B1 and *B2 alleles are found in individuals homozygous for the single‐copy RFLP allele of IgLC2/IgLC3 (8 kb EcoRI). Knowledge of alleles of this marker will be important for studies on the expression of the IgLC2 and IgLC3 isotypes in normal and autoimmune lymphocyte populations, as the coding regions of the two isotypes differ only at this position. The marker will also be useful in further studies of linkage with other IgLV and IgLC markers and to establish possible correlations with susceptibility to autoimmune disorders.

IgG2a and IgA Co-Expression by the Natural Autoantibody-producing Murine B Lymphoma T560

The T560 B lymphoma produces polyreactive IgG2a with the features of natural autoantibody. All T560 cells bear and secrete IgG2a but a small fraction spontaneously co-express IgA. Cells secreting IgA alone cannot be detected. IgA secretion is enhanced by interaction of T560 cells either with activated T cells and cognate antigen, or with LPS, but not with cytokines, including 1L-5 and TGF-fl IgA and IgG2a mRNAs have identical VI86.2, DFL 16 and JH1 sequences from framework 2 through JH1. PCR analysis reveals that previous recombination events have led to deletion of the μ., γ3, γl, γ2b constant region genes from both the productive and the unproductive chromosome but the former has retained γ2a, and α, the latter only α. Digestion-circularization (DC)-PCR experiments provide formal proof of DNA recombination between Ca and the intron upstream of Cα. Evidently, the productive chromosome has switched only as far as γ2a, the unproductive all the way to the a constant region gene. The unproductive allele is transcriptionally active as evidenced by the presence of mRNA encoding Cal inappropriately spliced to a cryptic splice site in the downstream intron of DQ52 (eliminated from the productive chromosome). A specific RT-PCR using oligonucleotide primers derived from the upstream initiation site of the la exon and from Cal discloses that T560 cells contain a-germ line mRNA, presumably transcribed from the la-region of the productive chromosome, spliced to Cα. Treatment with LPS stops production of these spliced transcripts suggesting that it may promote either DNA recombination in cells spontaneously transcribing la or a change in splicing such that la sequence is no longer joined to Ca. Verification of the DC-PCR product by sequencing reveals that the T560 and B10.A IgA (Ig2b allotype) hinge is different from the BALB/c IgA (Ig2a allotype) hinge: it has two extra Cys and has eliminated the first Thr, a potential glycosylation site in BALB/c IgA.

Baculovirus expression cassette vectors for rapid production of complete human IgG from phage display selected antibody fragments

For the expression of human intact IgG antibodies, we have constructed a set of baculovirus expression vectors designed to facilitate rapid insertion of heavy and light chain genes of Fab or scFv antibodies derived from phage display antibody libraries. By linking them to human constant or Fc regions, expression of complete human immunoglobulin molecules was achieved in insect cells by infection with recombinant baculovirus. The IgG expression cassette vectors are based on the backbone vector which contains two back to back polyhedron and p10 promoters. The IgG expression cassette elements, including the authentic IgG lambda or kappa and heavy chain signal sequences, as well as light chain (lambda or kappa) and heavy chain constant region genes are combined in a single vector and are controlled by the p10 and polyhedron promoter respectively. Either of VL or Fab-L and VH or Fab-Fd genes from common phage display systems can be directly inserted into one of the cassette vectors through in-frame cloning sites. This design of a single cassette vector combining heavy and light chain expression elements allowed rapid production and secretion of correctly processed and assembled intact immunoglobulins from recombinant baculovirus infected insect cells. The recombinant antibodies showed the expected molecular size of the H2L2 heterodimer in non reducing SDS–PAGE. No apparent differences were found between the expression level of heavy and light chains, and antigen binding function was preserved. For various antibodies, yields between 6 and 18 mg/l IgG were obtained.

A DNase I hypersensitive site near the murine gamma1 switch region contributes to insertion site independence of transgenes and modulates the amount of transcripts induced by CD40 ligation.

Several cis-acting elements regulate the expression of germline transcripts of heavy chain constant region genes and their subsequent switch recombination. To study such elements in the murine gamma1 gene, we have utilized a transgenic approach. In this study we focused on a DNase I hypersensitive site (termed 'Site II') that lies about 2 kb 3' of the gamma1 promoter region and I exon, just 5' to the gamma1 switch region. We have reported that gamma1 transgenes with Site II display the characteristics of a locus control region (LCR) in that they are insertion site independent and copy number dependent. For the present study we prepared six lines of transgenic mice that have the promoter region and I exon, but lack Site II. Expression of RNA from gamma1 transgenes that lack Site II is not correlated with transgene copy number; expression is insertion site dependent. This result indicates that DNase hypersensitive Site II is an important part of the LCR-like elements in the murine gamma1 gene. RNA expression from the gamma1 transgenes that lack Site II is inducible by IL-4 and by CD40 ligation. However, the induction of transgenic RNA expression by CD40 ligation is greater than expected, suggesting that elements within Site II participate in negative regulation of the amount of germline transcripts after CD40 ligation.

Mapping of the chicken immunoglobulin heavy‐chain constant region gene locus reveals an inverted α gene upstream of a condensed υ gene

Chicken antibodies are increasingly being used as diagnostic and therapeutic tools. As only the genomic organization of the micro encoding gene was previously known, we analysed the chicken immunoglobulin Y (IgY) and IgA (upsilon and alpha chain) immunoglobulin heavy-chain constant region (IGHC) genes and the organization of the chicken IGHC locus. The alpha gene is encoded by four separate exons, whereas, surprisingly, there is no intervening DNA sequence between the CH1 and CH2 domains of the IgY heavy chain, which is thus encoded by three exons separated by two introns. DNA sequence analysis shows that the exon boundaries of the chicken IGHC genes are not consistent with published domain borders. Furthermore, differences in DNA sequence confirm the existence of IgY, IgA and IgM allotypes in chickens. Finally, our results show that the IGHC genes of chicken (IgY, IgA and IgM) are all colocated on chromosome E18C15W15, where the alpha gene is located upstream of the upsilon gene in an inverted transcriptional orientation. The distances between the mu and alpha genes and the alpha and upsilon genes are about 18 and 15 kilobases, respectively, and thus, the size of the whole chicken IGHC locus is approximately 67 kilobases.

Site-specific and directional gene replacement mediated by Cre recombinase

A novel method for the site-specific introduction of genes into eukaryotic cells using the prokaryotic Cre- Lox P recombination system is presented. Cre recombinase catalyzes recombination between two Lox P sites or between two mutant Lox P 511 sites. However, recombination is not catalyzed between a Lox P and a Lox P 511 site. We now demonstrate that it is possible to catalyze accurate exchange between two DNA segments each flanked by a Lox P and a Lox P 511 site. In the example presented, expression of the Cre recombinase resulted in the replacement of a murine IgA constant region gene with a LoxP site at the 5′ end and a LoxP 511 site at the 3′ end by a human IgA constant region gene flanked by the same wild type and mutant Lox P sites. This method provides a novel approach for the site-specific substitution of specific genes.

Transcription-induced Cleavage of Immunoglobulin Switch Regions by Nucleotide Excision Repair Nucleases in Vitro

Immunoglobulin (Ig) heavy chain class switch recombination (CSR) mediates isotype switching during B cell development. CSR occurs between switch (S) regions that precede each Ig heavy chain constant region gene. Various studies have demonstrated that transcription plays an essential role in CSR in vivo. In this study, we show that in vitro transcription of S regions in their physiological orientation induces the formation of stable R loops. Furthermore, we show that the nucleotide excision repair nucleases XPF-ERCC1 and XPG can cleave the R loops formed in the S regions. Based on these findings, we propose that CSR is initiated via a mechanism that involves transcription-dependent S region cleavage by DNA structure-specific endonucleases that function in general DNA repair processes. Such a mechanism also may underlie transcription-dependent mutagenic processes such as somatic hypermutation, and contribute to genomic instability in general.

Sequence and diversity of the rat delta T-cell receptor.

The cDNA sequence of the delta T-cell receptor (TCRD) in the adult Lewis rat thymus was determined using the technique of rapid amplification of cDNA ends. Sixteen variable region genes (TCRDV), two diversity regions (TCRDD), two joining regions (TCRDJ), and a single constant region gene (TCRDC) were identified. The sixteen unique TCRDV genes identified represented eight different subfamilies in the rat and were highly conserved (>80% nucleotide identity) to corresponding mouse sequences. Extensive junctional diversity was observed in the rat, with both TCRDD regions (TCRDD1 and TCRDD2) utilized in the majority of cDNA clones identified. The two TCRDJ genes were highly conserved and corresponded to TCRDJ1 and TCRDJ2 in the mouse; the majority of clones utilized TCRDJ1. The TCRDC region in the rat was 91.1% identical to the mouse TCRDC gene and was highly conserved to other species. Although extensive sequence information about mouse gamma-delta T-cell receptor genes is available, current knowledge of rat gamma-delta T-cells is limited. The sequence analysis presented in this study adds to our understanding of gamma-delta T-cells in general, and it may be utilized to study the role of gamma-delta T-cells in immune-mediated disease and transplantation models previously established in the rat.