Immunoglobulin Heavy Chain Constant Region Research Articles

Rate of replication of the murine immunoglobulin heavy-chain locus: evidence that the region is part of a single replicon.

We measured the temporal order of replication of EcoRI segments from the murine immunoglobulin heavy-chain constant region (IgCH) gene cluster, including the joining (J) and diversity (D) loci and encompassing approximately 300 kilobases. The relative concentrations of EcoRI segments in bromouracil-labeled DNA that replicated during selected intervals of the S phase in Friend virus-transformed murine erythroleukemia (MEL) cells were measured. From these results, we calculated the nuclear DNA content (C value; the haploid DNA content of a cell in the G1 phase of the cell cycle) at the time each segment replicated during the S phase. We observed that IgCH genes replicate in the following order: alpha, epsilon, gamma 2a, gamma 2b, gamma 1, gamma 3, delta, and mu, followed by the J and D segments. The C value at which each segment replicates increased as a linear function of its distance from C alpha. The average rate of DNA replication in the IgCH gene cluster was determined from these data to be 1.7 to 1.9 kilobases/min, similar to the rate measured for mammalian replicons by autoradiography and electron microscopy (for a review, see H. J. Edenberg and J. A. Huberman, Annu. Rev. Genet. 9:245-284, 1975, and R. G. Martin, Adv. Cancer Res. 34:1-55, 1981). Similar results were obtained with other murine non-B cell lines, including a fibroblast cell line (L60T) and a hepatoma cell line (Hepa 1.6). In contrast, we observed that IgCh segments in a B-cell plasmacytoma (MPC11) and two Abelson murine leukemia virus-transformed pre-B cell lines (22D6 and 300-19O) replicated as early as (300-19P) or earlier than (MPC11 and 22D6) C alpha in MEL cells. Unlike MEL cells, however, all of the IgCH segments in a given B cell line replicated at very similar times during the S phase, so that a temporal directionality in the replication of the IgCH gene cluster was not apparent from these data. These results provide evidence that in murine non-B cells the IgCH, J, and D loci are part of a single replicon.

Rate of Replication of the Murine Immunoglobulin Heavy-Chain Locus: Evidence that the Region is Part of a Single Replicon

We measured the temporal order of replication of EcoRI segments from the murine immunoglobulin heavy-chain constant region (IgCH) gene cluster, including the joining (J) and diversity (D) loci and encompassing approximately 300 kilobases. The relative concentrations of EcoRI segments in bromouracil-labeled DNA that replicated during selected intervals of the S phase in Friend virus-transformed murine erythroleukemia (MEL) cells were measured. From these results, we calculated the nuclear DNA content (C value; the haploid DNA content of a cell in the G1 phase of the cell cycle) at the time each segment replicated during the S phase. We observed that IgCH genes replicate in the following order: alpha, epsilon, gamma 2a, gamma 2b, gamma 1, gamma 3, delta, and mu, followed by the J and D segments. The C value at which each segment replicates increased as a linear function of its distance from C alpha. The average rate of DNA replication in the IgCH gene cluster was determined from these data to be 1.7 to 1.9 kilobases/min, similar to the rate measured for mammalian replicons by autoradiography and electron microscopy (for a review, see H. J. Edenberg and J. A. Huberman, Annu. Rev. Genet. 9:245-284, 1975, and R. G. Martin, Adv. Cancer Res. 34:1-55, 1981). Similar results were obtained with other murine non-B cell lines, including a fibroblast cell line (L60T) and a hepatoma cell line (Hepa 1.6). In contrast, we observed that IgCh segments in a B-cell plasmacytoma (MPC11) and two Abelson murine leukemia virus-transformed pre-B cell lines (22D6 and 300-19O) replicated as early as (300-19P) or earlier than (MPC11 and 22D6) C alpha in MEL cells. Unlike MEL cells, however, all of the IgCH segments in a given B cell line replicated at very similar times during the S phase, so that a temporal directionality in the replication of the IgCH gene cluster was not apparent from these data. These results provide evidence that in murine non-B cells the IgCH, J, and D loci are part of a single replicon.

Coeliac disease: genetic, immunological and environmental factors in disease pathogenesis.

Coeliac disease in humans is activated by the dietary ingestion of wheat gliadins and similar proteins in other grains. We have studied genetic, immunological and environmental factors that may play a role in the pathogenesis of disease. In mice, two genetic regions, the major histocompatibility complex (H-2) and the immunoglobulin heavy chain constant region, were shown to regulate the production of anti-gliadin antibody. In coeliac disease patients on a gluten-free diet, elevated levels of antigliadin antibody were associated with the immunoglobulin heavy chain allotype marker G2m(n). Studies of additional environmental factors involved in coeliac disease revealed a region of amino acid sequence homology and immunological crossreactivity between A-gliadin, a wheat gliadin component known to activate coeliac disease and the Elb early region protein of human adenovirus 12, an adenovirus serotype usually isolated from the human intestinal tract. Specific HLA markers may be associated with coeliac disease because they reflect the host's response to virus.

Mechanisms of divergence and convergence of the human immunoglobulin α1 and α2 constant region gene sequences

Nucleotide sequences of the human α1 and two allelic α2 immunoglobulin heavy chain constant region genes are presented. The genes contain three exons, each encoding a single constant region protein domain. The protein hinge region is encoded at the 5′ end of the second exon, and the rapid evolutionary changes in length of the hinge correspond to duplications or deletions within the hinge-coding region, probably facilitated by repeats in the DNA sequence. Alignment of the α1 and α2 gene sequences reveals an unusual coupled deletion-duplication in the 5′-flanking region, which can be explained in terms of a slipped-strand mispairing model. Comparison of nucleotide sequences of the α1 gene and two alleles of the α2 gene indicates a localized transfer of genetic information from the 3′ end of the α1 gene to one of the α2 alleles, probably by a gene conversion. At one end of the region within which conversion apparently occurred, there is a 40 bp sequence of the type that can form Z-DNA.

Activation of the c-myc oncogene in B and T lymphoid tumors.

The cellular myc protio-oncogene, which is homologous to the transforming gene of the acute avian retrovirus MC29, is strongly implicated in the development of B lymphomas. Most murine plasmacytomas and human Burkitt lymphomas display translocations now known to represent recombination of the cellular mc gene with the immunoglobulin heavy chain constant region (CH) locus. Since the findings by several groups that led to this important conclusion have been reviewed recently by Klein (1983), Perry (1983), and Leder et al. (1983), we will limit discussion here to our own recent data concerning the nature of the recombination event and its consequences for c-myc transcription. We also present evidence that c-myc can participate in oncogenesis within the T as well as the B lymphoid lineage.

Genes for the mouse T cell alloantigens Tpre, Tthy, Tind, and Tsu are closely linked near Igh on chromosome 12.

Genes encoding the T cell alloantigens Tpre, Tthy, Tind, and Tsu have been mapped to a short segment of chromosome 12 using Igh recombinant strains of mice. These loci are located in a tightly linked cluster between the immunoglobulin heavy chain constant region gene cluster, Igh-C, and the serum prealbumin locus Pre-1, in this manner: centromere--Igh-C--(Tthy, Tind)-Tsu-Tpre--Pre-1.

Arrangement of human immunoglobulin heavy chain constant region genes implies evolutionary duplication of a segment containing gamma, epsilon and alpha genes.

Cosmid clones containing the human gamma, epsilon and alpha heavy chain constant region genes and an epsilon pseudogene have been isolated. All these genes have a switch sequence detectable by hybridization. We have studied overlapping cosmids covering two separate regions of the genome, and the gene order in each of these regions was found to be gamma-gamma-epsilon-alpha. This implies an evolutionary duplication in this multigene family involving gamma, epsilon and alpha genes.

DNA sequence associated with chromosome translocations in mouse plasmacytomas.

A DNA sequence that generates aberrantly rearranged immunoglobulin heavy chain constant region genes in murine plasmacytomas is shown to participate in a chromosome translocation. We have previously termed this DNA sequence NIARD for non-immunoglobulin-associated rearranging DNA. NIARD rearrangements were found frequently in murine plasmacytomas but were not detected in normal lymphocytes. These rearrangements occasionally involve the switch region of the C alpha gene. In this study, DNA samples obtained from mouse-Chinese hamster somatic cell hybrid lines were digested with various restriction endonucleases and analyzed by the Southern transfer technique with a NIARD hybridization probe. These experiments show that NIARD resides on chromosome 15 in the mouse germ line. Since NIARD is found adjacent to the C alpha gene (located on chromosome 12) in some plasmacytomas, it is apparent that a translocation involving these two chromosomes has occurred. We have proposed a rcpT(12;15) model to explain our data. The implications of NIARD rearrangements for malignant transformation are discussed.

Constant region determinants on the antigen-binding chain of the suppressor T-cell factor.

Antigen recognition units on T cells, including isolated antigen receptors and antigen-specific T-cell factors, have been found to have antigen-binding moieties like conventional immunoglobulins and to carry the determinants analogous to the variable (V) region of the immunoglobulin1–5. However, the immunoglobulin heavy chain constant region (Igh-C) determinants have not been demonstrated on T-cell receptor molecules6–9. If they possess unique constant region determinants, they must therefore be entirely distinct from the Igh-C. By production of an antiserum between Igh allotype congeneic mice Owen et al. suggest that a T-cell constant region determinant (Tsud) is encoded by a gene located near the Igh-C genes on the 12th chromosome10,11. In addition, we have also demonstrated that a BALB/c anti-CB-20 antiserum detects at least two distinct allotypic determinants, either of which is expressed on the antigen-specific suppressor or augmenting T-cell factor12. We now demonstrate that the new allotypic determinants detected by the BALB/c anti-CB-20 antiserum are expressed on the constant region of the antigen-binding molecule of an antigen-specific suppressor T-cell factor (TsF), and that this molecule is encoded by genes located on the right side of the Igh- V gene locus on the 12th chromosome.

Rapid isolation of cloned isotype switch variants using fluorescence activated cell sorting.

We have used highly specific, directly fluorescein-conjugated heterologous (conventional) and monoclonal antibodies directed against mouse immunoglobulin isotypes in conjunction with the fluorescence activated cell sorter (FACS) to enrich and clone hybridoma cells producing new immunoglobulin heavy chain constant regions. Each variant retains the parental heavy chain variable region and the parental immunoglobulin light chain; thereby each variant binds the same dansyl (DNS) hapten. These isotype switch variants occur at frequencies of approximately 10-5 to 10-6. We were able to isolate the variants by first sorting for an approximate 1000-fold enrichment of the desired immunoglobulin-producing cells, growing these cells for five to nine days, followed by a second 1000-fold enrichment and direct cell cloning into 96 well culture trays. Clones were screened only 3-5 weeks after the original selection for secretion of dansyl-binding immunoglobulin of the selected isotype. Judicious combination of existing methods permits improved analytical techniques using the cell sorter. These include: first, "red" fluorescence staining of dead cells with ethidium bromide or propidium iodide and using the red fluorescence measurement to exclude dead cells from the green fluorescence selection; and second, the use logarithmic amplification of fluorescence signals, allowing for more succinct selection of fluorescence parameters for sorting.

Immunoglobulin heavy chain gene rearrangement and transcription in murine T cell hybrids and T lymphomas.

We have examined the arrangement of immunoglobulin heavy chain constant (CH) and joining (JH) region genes in murine T cell hybrid lines and in T lymphomas. CH genes derived from both parental cell types were present in all hybrids for which polymorphism in sequences flanking CH genes permitted us to distinguish parental CH genes. All T lymphomas and T cell hybrids retained the C alpha gene in germ-line configuration and all but one cell line had germ-line C mu genes. Novel DNA fragments reactive with JH probes were observed in six of nine T cell hybrids, as well as in two T lymphomas, WEHI7.1 and YAC-1, but not in the fusion parent, BW5147. No RNA homologous to C gamma 2b, C alpha, or lambda genes was detected in any of the T cell lines. T cell lines contained poly(A)+ RNA homologous to a C mu cDNA probe. More importantly, in several cell lines the C mu RNAs were associated with membrane-bound polyribosomes. These results suggest that both JH rearrangements and C mu RNA production occur in at least some mature, antigen-specific T cells. They may therefore reflect events in normal T cell development and function related to those involved in the generation of the T receptor for antigen.

The temporal order of replication of murine immunoglobulin heavy chain constant region sequences corresponds to their linear order in the genome.

The time of replication during the S phase in a murine erythroleukemia (MEL) cell line was determined for immunoglobulin heavy chain constant region C alpha, C gamma 2b and C mu sequences whose boundaries are defined by EcoR1 restriction endonuclease sites (EcoR1 segments). Logarithmically growing cultures of MEL cells with an S phase of about 7.5 hours were pulse labelled with 20 micrograms/ml of 5-bromodeoxyuridine (BUdR). The cells were then fractionated by centrifugal elutriation into 10-12 distinct populations containing cells in different stages of the cell cycle. Flow microfluorimetric (FMF) analysis of DNA content, measurements of cell volume and autoradiography after 3H-thymidine pulse labelling were used to determine position in the cell cycle. Fractions were pooled to represent four selected intervals of S in which BU-DNA was synthesized for 2.5 hrs or less. Newly replicated DNA which had incorporated BUdR into one strand was isolated, cleaved with EcoR1, and separated on neutral Cs2S04 gradients. Equal amounts of BU-DNA replicated during these four intervals of S were electrophoresed in 0.8% agarose gels, transferred to diazotized aminobenzyloxymethyl paper and hybridized with 32p probes containing the C alpha, C gamma 2b and C mu genes and flanking sequences. The relative amounts of segments replicated were assessed by quantitation of the appropriate bands on the autoradiograms by microdensitometry. The results indicate that the 2.8 kb C alpha, 6.6 kb C gamma 2b and 12 kb C mu EcoR1 segments in these MEL cells replicated during defined intervals of the first half of the S phase. The order of replication of these EcoR1 segments as the cells proceeded through S was C alpha, C gamma 2b, C mu, corresponding to the linear order of the genes determined by restriction endonuclease mapping.

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.

Mapping of the Fv-4 mouse gene controlling resistance to murine leukemia viruses.

Fv-4, a gene controlling susceptibility to ecotropic murine leukemia viruses, was linked to both Pre-I (prealbumin) and Igh-I (immunoglobulin heavy-chain constant region) loci on chromosome 12 in mice. The gene order was Fv-4 - Pre-I - Igh-I, and the estimated frequency of recombination between Fv-4 and Pre-I was about 20%.

(AGCTG) (AGCTG) (AGCTG) (GGGTG) as the Primordial Sequence of Intergenic Spacers: The Role in Immunoglobulin Class Switch

The means employed for immunoglobulin heavy chain class switch appears to be no different from that by which meiotic intergenic crossing-overs at accomplished. As with other intergenic spacers, the 5' noncoding sequence of each Ig CH (immunoglobulin heavy chain constant region) gene apparently undergoes unconstrained sequence changes due to randomly sustained base substitutions, deletions, and duplications. Yet, there remains sufficient regional sequence homology between the Ig Cmu 5' noncoding sequence and those of its somatic recombination partners, e.g., Ig C gamma 1, Ig C gamma 2b, Ig C alpha, because each of these 5' noncoding sequences is made of multiple copies in various stages of degeneracy of one primordial 20 base pair-long sequence: (AGCTG) (AGCTG) (AGCTG) (GGGTG).

Human immunoglobulin heavy chain genes: evolutionary comparisons of C mu, C delta and C gamma genes and associated switch sequences.

Human immunoglobulin heavy chain constant region genes have been characterised in isolated clones. The human c mu gene comprises discrete domains for C mu 1, C mu 2, C mu 3 and C mu 4 + tp separated by short intervening sequences. The C delta gene has been located about 5 kb downstream of C mu 4. Furthermore, the coding segments for the membrane form of mu have been located 1.9 kb downstream of C mu 4. Tandemly repeated sequences implicated in the heavy chain class switch occur upstream of the C mu and the C gamma genes, but none were detected near the C delta gene. These tandem repeats are very homologous to those of mouse. Particularly common is the sequence G-A-G-C-T. These data suggest that the mu to gamma switch in humans involves DNA rearrangements of the CH-genes and subsequent deletion of DNA, but that the coexpression of C mu and C delta genes results from different mechanisms.

Isolation of t cell membrane proteins (IgT) with antisera to non-isotypic determinants of immunoglobulins: Evidence thaT IgT ‘Light’ chains are not identical to B cell kappa chains

Radiolabelled cell membrane proteins obtained by detergent lysis of murine T cells labelled by lactoperoxidase-catalysed radioiodination were specifically immunoprecipitated with an antiserum to immunoglobulin Fab determinants. Absorption of the antiserum by myeloma proteins or normal immunoglobulins coupled to Sepharose and the use of antibody proteins eluted from these absorbents indicated that the antibody activity in the serum responsible for binding detergent-soluble T cell membrane proteins was directed towards variable region and not to the constant region determinants of kappa light chains. Immunoglobulin light chain sized polypeptides isolated from T cells with this antiserum were found to possess serological characteristics which differed from serum or B cell membrane immunoglobulin kappa chains. Evidence is presented which suggests that a portion of the light chain sized polypeptides isolated from T cells may be proteolytic fragments of a larger polypeptide chain. The results suggest that immunoglobulin-like detergent-soluble T cell membrane proteins isolated with antisera to immunoglobulins (IgT) possess neither immunoglobulin heavy nor light chain constant regions determinants but do carry determinants associated with immunoglobulin variable regions.

Studies on the nature and germ-line stability of DNA sequences flanking the mouse immunoglobulin heavy-chain constant-region genes.

Studies on the nature and germ-line stability of DNA sequences flanking the mouse immunoglobulin heavy-chain constant-region genes.

The immunoglobulin mu constant region gene is expressed in mouse thymocytes.

It has been a matter of controversy whether the functional capacity of T cells to discriminate between antigens is mediated via immunoglobulin, an immunoglobulin-like molecule, or by the product(s) of unrelated genes. The progenitors of immunoglobulin-secreting cells, B cells, express membrane-bound immunoglobulin as the antigen-specific receptor on their surface. For T cells, although products of immunoglobulin heavy chain variable region genes are implicated as receptor components, there has been no compelling immunochemical evidence for participation of either immunoglobulin light chains or heavy chain constant regions (see refs 2-6 for the disparate views). Recently, using cloned immunoglobulin DNA sequences as hybridization probes, we have demonstrated that the immunoglobulin Cmu gene, but not the Cmu gene, is expressed as polyadenylated RNA in some T cell tumour (T lymphoma) cell lines. Individual T lymphoma lines yielded up to three discrete mu RNA species of different sizes (1.9, 2.2 and 3.0 kilobases), each species being different in size from the major mu RNA species present in B lymphoma cells (2.4 and 2.7 kilobases). We show here that cells from the normal mouse thymus contain mu RNA species, indistinguishable in size from those in T lymphoma cells, but contain little if any kappa RNA.

Nucleotide sequence of immunoglobulin heavy chain joining segments between translocated VH and mu constant regions genes.

To investigate the mechanism of recombination of immunoglobulin heavy chain variable and constant region genes, we have determined the nucleotide sequence of a large portion of the recombination region between an active C mu gene and its associated VH gene, isolated from an IgM-secreting mouse plasmacytoma, HPC76. By comparison with the sequence of the mu mRNA, we determined the exact boundaries of the intervening sequence between the VH76 and C mu genes. The rearranged VH76 gene encodes up to amino acid 116 without interruption, the 3' 39 nucleotides (the JH76 region) being derived from an embryonic JH segment (JH315) whose sequence was recently determined [Early, P., Huang, H., Davis, M., Calame, K. & Hood, L. (1980) Cell 195, 981-992]. The active JH76 does not use the first two codons of the embryonic JH315 from which it is derived. This indicates that V-J recombination is important in generating diversity within the third hypervariable region of heavy chains. We have identified another JH segment (JHA4), located 336 nucleotides 3' to the rearranged JH76 segment. This JH segment is expressed in the heavy chains of anti-levan myeloma proteins, which have truncated third hypervariable regions. We propose that the nucleotide sequence 5' to JHA4 is important for generating V region genes with shortened third hypervariable regions.