Allelic exclusion model questioned.
Explore the source record for details and available documents.
Alleles↗
Biomedical subjects
M Wabl
Publications and source records attributed to M Wabl.
Tumorigenesis mediated by an antigen receptor.
The heavy chain variable region of the immunoglobulin receptors on cells of the B lymphoma NYC are almost identical to that of other independent B-cell tumors of B/W mice. NYC IgM binds a viral antigen produced by the tumor cells; and despite extensive screening, immunoglobulin-negative variants were never found in the NYC cells, suggesting that NYC loses the capacity to grow in culture when it does not synthesize surface immunoglobulin. These findings indicate that the interaction of endogenous antigen with surface IgM continuously stimulates growth and, thus, that the tumorigenesis of B lymphomas in B/W mice is mediated by antigen receptors.
Animals↗
The murine IgG1/IgE class switch program.
Immunoglobulin class switching is controlled by cytokines. Thus, interleukin-4 (IL-4) directs class switching to both IgG1 and IgE. Consistent with this are the results reported here on restriction endonuclease analysis of active and inactive alleles of the IgH locus in IgE-producing cells. In cells that were stimulated in vitro by lipopolysaccharide and IL-4 the silent alleles preferentially switched to gamma 1, whereas in cells that were stimulated by antigen in vivo both active and inactive alleles switched to epsilon. Thirty percent of the recombined switch regions (S mu/S epsilon) contain S gamma 1 sequences, which we interpret as footprints of a previous switch to gamma 1. Since this percentage is a minimum estimate, between 30% and 100% of switching to epsilon must occur sequentially via gamma 1.
Animals↗
Bispecific antibodies that mediate killing of cells infected with human immunodeficiency virus of any strain.
Although AIDS patients lose human immunodeficiency virus (HIV)-specific cytotoxic T cells, their remaining CD8-positive T lymphocytes maintain cytotoxic function. To exploit this fact we have constructed bispecific antibodies that direct cytotoxic T lymphocytes of any specificity to cells that express gp120 of HIV. These bispecific antibodies comprise one heavy/light chain pair from an antibody to CD3, linked to a heavy chain whose variable region has been replaced with sequences from CD4 plus a second light chain. CD3 is part of the antigen receptor on T cells and is responsible for signal transduction. In the presence of these bispecific antibodies, T cells of irrelevant specificity effectively lyse HIV-infected cells in vitro.
Acquired Immunodeficiency Syndrome↗
Circular DNA is a product of the immunoglobulin class switch rearrangement.
The class of immunoglobulin is defined by the constant region of its heavy chain. When a B lymphocyte switches the class of heavy chain it produces, the constant region of mu-type heavy chain is replaced; this occurs through a DNA rearrangement that brings the gene segment encoding the new constant region close to the VDJ segment encoding the variable region. The pre-B-cell line 18-81, which switches from heavy chain mu to gamma 2b production in culture, occasionally abnormally rearranges the heavy chain locus so that DNA sequences between the switch regions of mu and gamma 2b are inverted. Because looping-out is an intermediate step in generating an inversion, the switch rearrangement could occur by looping-out and deletion. Provided that recombination is reciprocal, this would produce a circle of DNA. Indeed, circular DNA molecules have been isolated as products of rearrangement among gene segments encoding the variable regions of the T-cell receptor and of the immunoglobulin heavy chain and light chain. But whereas the breakpoints for the variable region rearrangement are precisely defined, the breakpoints for any given heavy chain class switch are scattered over a length of greater than 6 kilobases, including both switch regions. We have now isolated circular DNA containing the sequences deleted by class-switching, thereby showing that the immunoglobulin heavy chain class switch occurs through looping-out and deletion.
Animals↗
Immunoglobulin lambda gene rearrangement can precede kappa gene rearrangement.
Immunoglobulin genes are generated during differentiation of B lymphocytes by joining gene segments. A mouse pre-B cell contains a functional immunoglobulin heavy-chain gene, but no light-chain gene. Although there is only one heavy-chain locus, there are two light-chain loci: kappa and lambda. It has been reported that kappa loci in the germ-line configuration are never (in man) or very rarely (in the mouse) present in cells with functionally rearranged lambda-chain genes. Two explanations have been proposed to explain this: (a) the ordered rearrangement theory, which postulates that light-chain gene rearrangement in the pre-B cell is first attempted at the kappa locus, and that only upon failure to produce a functional kappa chain is there an attempt to rearrange the lambda locus; and (b) the stochastic theory, which postulates that rearrangement at the lambda locus proceeds at a rate that is intrinsically much slower than that at the kappa locus. We show here that lambda-chain genes are generated whether or not the kappa locus has lost its germ-line arrangement, a result that is compatible only with the stochastic theory.
Alleles↗
Beswitched. The looping out model for immunoglobulin class switching.
During the switch in expression of an immunoglobulin class, the gene segment encoding the constant region of the heavy chain is replaced in a way that leads to a deletion. Three different models of how this deletion is generated have been proposed: recombination between homologs, unequal sister chromatid exchange, and looping out and deletion. While none of the predicted recombination products of the first two models have been found, the products of the looping out--inversions and circular DNA--have been isolated. Thus looping out and deletion appears to be the appropriate model to explain the genetic events leading to the immunoglobulin heavy chain class switch.
Animals↗
Translation affects immunoglobulin mRNA stability.
When termination codons were introduced into exons of the gene for Ig mu chain, steady-state levels of mu mRNA were reduced, both at the pre-B cell stage and at the plasma cell stage. A termination codon in the variable region gene segment and a termination codon in the second exon of the constant region gene segment had effects of similar magnitude. When the termination codon was deleted, the original level of mRNA was restored. The rate of mu gene transcription was the same whether or not a termination codon was present. Therefore, the termination codons must reduce the amount of the mRNA by reducing its stability. Since the introduced termination codons prematurely terminate translation and, in so doing, change the ribosome load on the mRNA, we conclude that mu mRNA stability is conferred in part by ribosomal protection from enzymatic degradation. We propose that the differences in mu mRNA stability during B lymphocyte differentiation are due to different amounts of ribosomes available for translation.
B-Lymphocytes↗
Immunoglobulin mRNA stability varies during B lymphocyte differentiation.
During differentiation of B lymphocytes, the change in the amount of immunoglobulin heavy chain produced is reflected by a change in the steady state level of heavy chain mRNA. At the pre-B cell stage, the earliest stage at which immunoglobulin chain is produced, and later at the small resting B cell stage, there is a low steady state level of heavy chain mRNA. After the small B cell has differentiated to become a plasma cell, the steady state level of heavy chain mRNA is much higher. We confirm that the transcription rate at the immunoglobulin mu heavy chain gene does not change during differentiation from the pre-B cell to the plasma cell stage. In contrast, we show here that differences in the stability of mu mRNA are sufficient to account for the differences in the steady state level at the various differentiation stages.
B-Lymphocytes↗
Looping out and deletion mechanism for the immunoglobulin heavy-chain class switch.
In the mouse pre-B-cell line 18-81, cells can switch production in vitro from immunoglobulin mu chain to gamma 2b chain. The gene encoding the gamma 2b chain is created by a rearrangement of the mu gene. This rearrangement always takes place within a homolog. In cells with a gamma 2b gene, most of the time the gene segment encoding the constant region of the mu chain is deleted, but often the rearrangement leads to cells that produce no immunoglobulin, and all DNA sequences are retained. The latter result is due to an inversion. Inversions exclude the unequal sister chromatid exchange model of the heavy-chain class switch. Looping out is an intermediate step in the process of generating an inversion. Our findings demonstrate that the switch rearrangement occurs by looping out and deletion.
Animals↗
High rates of deletions in the constant region segment of the immunoglobulin mu gene.
Spontaneous deletions at the immunoglobulin heavy-chain locus are frequently found in myelomas, hybridomas and pre-B-cell lines. We have measured the rates for large and small deletions within the constant-region gene segment for mu chain in a pre-B-cell lines. The large deletions, which include the entire first and second exons, occurred at a rate of 1.7 X 10(-5) per cell generation. The small deletions, which span a few base pairs, occurred at a rate of 1.4 X 10(-7) per cell generation. The rate for the reversion of a termination codon in the second exon is even less than that for the small deletions and is 1000 times lower than the reversion rate that had been determined for the variable-region gene segment. Therefore, the variable-region gene segment is likely to be the preferred target for hypermutation.
B-Lymphocytes↗
Allelic inclusion in a pre-B-cell line that generates immunoglobulin heavy chain genes in vitro.
In a pre-B-cell line that rearranges its heavy chain gene segments in vitro, we found that the rate of productive rearrangement on one allele was not influenced by the presence of heavy chain protein encoded by the other allele. This shows that allelic exclusion of heavy chain genes is not regulated at the genetic level.
Alleles↗
Measurements of mutation rates in B lymphocytes.
It is established that somatic mutation is an important source of antibody diversity in vivo. It is also established that Igh-V gene segments are hypermutable in vitro. This is not a completely satisfactory situation. While there is no reason to believe that Igh-V genes are not hypermutable in vivo as well, direct experimental evidence is lacking. Perhaps experiments with transgenic mice will soon fill this gap. It is not so clear how much higher than normal the rate of hypermutation is. As far as we are aware, there are no direct measurements of mutation rates per base pair per cell generation in mammals, certainly not for lymphocyte cell lines. For a variety of reasons, it is difficult to measure very low mutation rates. The general consensus is that the normal rate should be somewhere between 10(-10) and 10(-12) mutations per base pair per cell generation. Therefore, an experiment designed to directly determine a rate using the compartmentalization test would involve hundreds of cultures, each containing at least 10(9) cells. It is not a trivial problem to find one or a few mutants among so many cells. It is simple to study mutation to resistance to a drug, for example, ouabain or azaguanine, but, as we discussed, there are technical and conceptual pitfalls. The vast excess of dead cells influences the growth of a few mutant cells, particularly in lymphocyte cell lines. Even if this problem could be solved, the mutation rate so obtained would be "per gene(s)" and not "per base pair". The problems associated with cytotoxic agents can be avoided by immunofluorescence methods in conjunction with selective cloning or cell sorting. Using these techniques, we have carried out extensive experiments to determine whether the immunoglobulin mutator system acts, at least partially, on genetic elements other than those in or near the heavy chain variable region gene segment. For an opal termination codon in a heavy chain constant region gene segment, the rate of reversion was less than 10(-7) per base pair per cell generation. This upper limit was fixed by the high rate of small deletions at the heavy chain locus. For an allotype mutation at B2m, the gene encoding beta 2 microglobulin, the rate of mutation was less than 10(-8). This upper limit could be lowered by at least two orders of magnitude by using a high-speed cell sorter.
Animals↗
Possible target of Abelson virus phosphokinase in cell transformation.
By fusing interphase cells to cells undergoing mitosis, the interphase chromosomes can be visualized. When analyzed in this way, chromosomes of normal mouse cells show characteristic undercondensed centromeric regions. We have found that the centromeric regions of chromosomes from Abelson virus-transformed cells are fully condensed. Abelson virus transforms mouse cells by introducing into them a virally encoded phosphokinase that is expressed constitutively. Thus, we propose that the condensation of centromeric chromatin is a result of overphosphorylation by the Abelson virus phosphokinase, and that the centromeric region is the relevant target of overphosphorylation in transformed cell growth.
Abelson murine leukemia virus↗
High rate of somatic point mutation in vitro in and near the variable-region segment of an immunoglobulin heavy chain gene.
The "silent" allele at the immunoglobulin heavy-chain locus in the pre-B-lymphocyte line 18-81 contains a correctly assembled gene. However, an amber termination codon within the variable-region gene segment prematurely terminates translation into complete heavy chain. Revertants that do produce heavy chain are generated at a high rate, which is termed hypermutation. By DNA sequencing of subclones, we have confirmed that whenever mu chain is produced by the usually silent allele, a true reversion is found in the DNA. Mutations are not confined to the position of the amber termination codon but are also found at other sites in and near the variable-region gene segment.
Base Composition↗
Hypermutation at the immunoglobulin heavy chain locus in a pre-B-cell line.
Most cells in the well-known pre-B-lymphocyte line 18-81 have correctly assembled genes for both alleles at the immunoglobulin heavy chain locus. Only one allele is "active"; the other, "silent" allele contains an amber termination codon. The rate of reversion of this amber codon was determined to be 0.3-1 X 10(-4) per cell generation. This high rate is termed hypermutation.
Alleles↗
A theory of allelic and isotypic exclusion for immunoglobulin genes.
Heavy (H) chain binding protein (BiP), which binds to free immunoglobulin H chain of the mu and gamma classes, can be demonstrated in pre-B-cells. It is proposed that the displacement of BiP from H chain by light (L) chain terminates the activity of the enzyme system, L-generase, which catalyzes DNA rearrangement at the L chain loci, generating the complete gene which may or may not be functional. This ensures allelic and isotypic exclusion for the L chain loci. It is further proposed that those cells that productively rearrange both alleles at the H chain locus are eliminated by the "H chain toxicity" effect.
Alleles↗
The role of DNA rearrangement and alternative RNA processing in the expression of immunoglobulin delta genes.
We have established the exon-intron structure of the gene coding for the constant (C) region of the mouse immunoglobulin delta heavy chain, using DNA clones isolated from BALB/c embryos and the delta mRNA extracted from two delta-producing hybridomas, B1-8. delta 1 and GCL2.8. At least three types of C delta gene structures are identified. A 2.7 kb delta mRNA reveals six exons. This delta mRNA may code for a membrane-bound delta chain. A second delta mRNA of 1.8 kb shares the first (5' side relative to direction of transcription) three exons with the 2.7 kb delta mRNA and in addition contains a fourth exon unique to this mRNA species. This delta mRNA most likely codes for a secreted delta chain. A third delta mRNA, also of 1.8 kb, shares the first four exons and a part of the fifth exon with the 2.7 kb mRNA. Its function, if any, remains unclear. We investigated the question of how a lymphocyte can produce the mu and delta heavy chains simultaneously, using the hybridoma GCL 2.8, which makes both IgM and IgD. Results of Southern gel blot analysis and gene cloning experiments indicate that this cell utilizes the same rearranged VH gene for the synthesis of the mu and delta chains, and yet maintains the embryonic configuration for the C mu and C delta genes and for the intervening region. Based on these results, we conclude that the VH sequence is spliced alternatively to the C mu or C delta sequence during processing of the primary RNA transcript. An alternative mechanism for the expression of the delta gene is found in hybridoma B1-8. delta 1, which actively secretes delta chains and synthesizes no mu chain. This mechanism involves deletion of the C mu gene, which brings the complete VH gene closer to the C delta gene.
Animals↗