The lingering enigma of the allelic exclusion mechanism

B lymphocytes produce diverse antibody specificities by "randomly" assembling antibody genes from germline segments. Yet, though each B lymphocyte has multiple allelic loci for the different antibody chains, each clonally derived mature B lymphocyte expresses a single species of antibody with a unique specificity via a process termed allelic exclusion. Despite some progress, the precise mechanism of allelic exclusion remains an enigma.     

Bacteriophage lambda repressor allelic modulation of the Rex exclusion phenotype

The sensitivity of delta red-gam delta ren mutants of bacteriophage lambda to Rex exclusion by lambda rexA+ rexB+ lysogens is modulated by the prophage cI repressor allele. We show the following: (i) lambda spi156 delta nin5 forms plaques on a cI+-rexA+-rexB+ lysogen with 10(5)-fold higher efficiency than on cI[Ts]-rexA+-rexB+ derivatives. (ii) The cI[Ts]857 allele augmentation of Rex exclusion is recessive to cI+. (iii) The cI857-mediated increase in Rex exclusion activity involves the participation of a genetic element mapping outside of cI-rexA-rexB.     

Evidence for allelic exclusion in Chinese hamster ovary cells.

Earlier results suggested that the functional hemizygosity of genes in pseudodiploid Chinese hamster ovary (CHO) cells is due to the silencing of one allele by DNA methylation. From this one could make a strong prediction that we have now been able to confirm by genetic experiments, using thymidine kinase (TK) alleles. TK- mutants induced by ethylmethane sulphonate (EMS) were all revertible to TK+ at high frequency by the demethylating agent 5-azacytidine (5-aza-CR). This revertibility was due to reactivation of a silent nonmutant TK allele. Further mutagenesis by EMS yielded TK- derivatives that were no longer revertible by 5-aza-CR; these are assumed to have mutations in both alleles. TK- cells were also transfected with equine herpes virus TK+ DNA, and the TK+ derivatives were shown to be markedly less stable than cells with the normal TK+ gene. CHO cells lack metallothionein activity (sensitive to cadmium), and also require proline for growth, because genes have become silenced during the establishment of the cell line. In both cases 5-aza-CR reactivates these genes to give the cadmium resistant and proline independent phenotypes. Long-term experiments with reactivants in the absence of selection showed that the genes become silent, presumably as a result of de novo methylation. A strain resistant to cytosine arabinoside (araCR) was also resistant to 5-azadeoxycytidine (5-aza-CdR), but not to 5-aza-CR, which would be expected if the araCR strain lacked deoxycytidine kinase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Prominent roles for odorant receptor coding sequences in allelic exclusion

Mammalian odorant receptors (ORs) are crucial for establishing the functional organization of the olfactory system, but the mechanisms controlling their expression remain largely unexplained. Here, we utilized a transgenic approach to explore OR gene regulation. We determined that although olfactory sensory neurons (OSNs) are capable of supporting expression of multiple functional ORs, several levels of control ensure that each neuron normally expresses only a single odorant receptor. Surprisingly, this regulation extends beyond endogenous ORs even preventing expression of transgenes consisting of OR-coding sequences driven by synthetic promoters. Thus, part of the intrinsic feedback system must rely on elements present in the OR-coding sequence. Notably, by expressing the same transgenic ORs precociously in immature neurons, we have overcome this suppression and established a generic method to express any OR in approximately 90% of OSNs. These results provide important insights into the hierarchy of OR gene expression and the vital role of the OR-coding sequence in this regulation.     

Evidence that Smith-McCort dysplasia and Dyggve-Melchior-Clausen dysplasia are allelic disorders that result from mutations in a gene on chromosome 18q12

Smith-McCort dysplasia is a rare autosomal recessive osteochondrodysplasia characterized by short limbs and a short trunk with a barrel-shaped chest. The radiographic phenotype includes platyspondyly, generalized abnormalities of the epiphyses and metaphyses, and a distinctive lacy appearance of the iliac crest. We performed a genomewide scan in a consanguineous family from Guam and found evidence of linkage to loci on chromosome 18q12. Analysis of a second, smaller family was also consistent with linkage to this region, producing a maximum combined two-point LOD score of 3.04 at a recombination fraction of 0 for the marker at locus D18S450. A 10.7-cM region containing the disease gene was defined by recombination events in two affected individuals in the larger family. Furthermore, all affected children in the larger family were homozygous for a subset of marker loci within this region, defining a 1.5-cM interval likely to contain the defective gene. Analysis of three small, unrelated families with Dyggve-Melchior-Clausen syndrome, a radiographically identical disorder with the additional clinical finding of mental retardation, provided evidence of linkage to the same region, a result consistent with the hypothesis that the two disorders are allelic.     

Differential accessibility at the kappa chain locus plays a role in allelic exclusion

Gene rearrangement in the immune system is always preceded by DNA demethylation and increased chromatin accessibility. Using a model system in which rearrangement of the endogenous immunoglobulin kappa locus is prevented, we demonstrate that these epigenetic and chromatin changes actually occur on one allele with a higher probability than the other. It may be this process that, together with feedback inhibition, serves as the basis for allelic exclusion.     

A change in the structure of Vbeta chromatin associated with TCR beta allelic exclusion

To investigate chromatin control of TCR beta rearrangement and allelic exclusion, we analyzed TCR beta chromatin structure in double negative (DN) thymocytes, which are permissive for TCR beta recombination, and in double positive (DP) thymocytes, which are postallelic exclusion and nonpermissive for Vbeta to DbetaJbeta recombination. Histone acetylation mapping and DNase I sensitivity studies indicate Vbeta and DbetaJbeta segments to be hyperacetylated and accessible in DN thymocytes. However, they are separated from each other by hypoacetylated and inaccessible trypsinogen chromatin. The transition from DN to DP is accompanied by selective down-regulation of Vbeta acetylation and accessibility. The level of DP acetylation and accessibility is minimal for five of six Vbeta segments studied but remains substantial for one. Hence, the observed changes in Vbeta chromatin structure appear sufficient to account for allelic exclusion of many Vbeta segments. They may contribute to, but not by themselves fully account for, allelic exclusion of others.     

Structural plasmid evolution as a result of coupled recombinations at bom and cer sites

We have studied the recombination of plasmids bearing bom and cer sites. The bom ( basis of mobilization) site is required for conjugative transfer, while the cer ( Col E1 resolution) site is involved in the resolution of plasmid multimers, which increases plasmid stability. We constructed a pair of parent plasmids in such a way as to allow us select clones containing recombinant plasmids directly. Clone selection was based on the McrA sensitivity of recipient host DNA modified by M. Ecl18kI, which is encoded by one of the parent plasmids. The recombinant plasmid contains segments originating from both parental DNAs, which are bounded by bom and cer sites. Its structure is in accordance with our previously proposed model for recombination mediated by bom and cer sequences. The frequency of recombinant plasmid formation coincided with the frequency of recombination at the bom site. We also show that bom-mediated recombination in trans, unlike in cis, is independent of other genetic determinants on the conjugative plasmids.     

An important role of phospholipase Cgamma1 in pre-B-cell development and allelic exclusion

Phospholipase Cgamma1 (PLCgamma1) has been reported to be expressed predominantly in T cells and to play an important role in T-cell receptor signaling. Here we show that PLCgamma1 is expressed throughout B-cell development, with high expression in B-cell progenitors, and is involved in pre-B-cell receptor (pre-BCR) signaling. Reduced expression of PLCgamma1, in the absence of PLCgamma2 (PLCgamma1+/-PLCgamma2-/-), impedes early B-cell development at the pro-B- to pre-B-cell transition and impairs immunoglobulin heavy chain allelic exclusion, hallmarks of defective pre-BCR signaling. In contrast, early B-cell development is largely normal, whereas late B-cell maturation is impaired in the absence of PLCgamma2 alone (PLCgamma2-/-) and overexpression of PLCgamma1 in PLCgamma2-/- mice fails to restore BCR-mediated B-cell proliferation and maturation. These studies reveal an essential role of PLCgamma1, distinct from that of PLCgamma2, in B-cell development.     

Attenuation of IL-7 receptor signaling is not required for allelic exclusion

Allelic exclusion prevents pre-B cells from generating more than one functional H chain, thereby ensuring the formation of a unique pre-BCR. The signaling processes underlying allelic exclusion are not clearly understood. IL-7R-dependent signals have been clearly shown to regulate the accessibility of the Ig H chain locus. More recent work has suggested that pre-BCR-dependent attenuation of IL-7R signaling returns the H chain loci to an inaccessible state; this process has been proposed to underlie allelic exclusion. Importantly, this model predicts that preventing pre-BCR-dependent down-regulation of IL-7R signaling should interfere with allelic exclusion. To test this hypothesis, we made use of transgenic mice that express a constitutively active form of STAT5b (STAT5b-CA). STAT5b-CA expression restores V(D)J recombination in IL-7R(-/-) B cells, demonstrating that IL-7 regulates H chain locus accessibility and V(D)J recombination via STAT5 activation. To examine the effects of constitutively active STAT5b on allelic exclusion, we crossed STAT5b-CA mice (which express the IgM(b) allotype) to IgM(a) allotype congenic mice. We found no difference in the percentage of IgM(a)/IgM(b)-coexpressing B cells in STAT5b-CA vs littermate control mice; identical results were observed when crossing STAT5b-CA mice with hen egg lysozyme (HEL) H chain transgenic mice. The HEL transgene enforces allelic exclusion, preventing rearrangement of endogenous H chain genes; importantly, rearrangement of endogenous H chain genes was suppressed to a similar degree in STAT5b-CA vs HEL mice. Thus, attenuation of IL-7R/STAT5 signaling is not required for allelic exclusion.     

Hypothesis of the initiation of DNA methylation de novo and allelic exclusion by small RNA

We have discovered that 5'-CG-3' dinucleotide and 5'-CNG-3' trinucleotide are found in published sequences of small interfering RNA and microRNA more often than they should be found in a random sequence. This circumstance is evidence of an important biological purpose of 5'-CG-3' dinucleotides and 5'-CNG-3' trinucleotides in small RNA sequences. We suppose that small RNAs containing mentioned di- and trinucleotides participate in creation of chromatin marks of epigenetic information through high-specific search of DNA sequences liable to repression and through initiation of the methylation de novo of 5'-CG-3' and 5'-CNG-3' sites in DNA fragments, which appeared to be bound complementary with small RNA. Several genes can be inactivated simultaneously when they contain the motif which is recognized by small RNA. Allelic exclusion appears, to our opinion, as a result of initiation by small RNA of de novo DNA methylation of all alleles but one that exist in the cell. The predecessor of this small RNA is transcribed from the antiparallel allele chain. Those alleles are inactivated which antiparallel chain is less actively read by RNA-polymerase, which, as we suppose, releases DNA from attached to it small RNA in the process of transcribing. But the quantity of small RNA which is transcribed from just one allele is insufficient to overcome the level when the repression process of this allele de novo starts.     

Quantitative analysis of idiotypic mimicry and allelic exclusion in mice with a mu Ig transgene

Analysis of C57BL/6 mice (IgM allotype, Igh-6b or mu b) that carry an Ig H chain transgene of a different allotype (mu a) shows that IgM molecules of mixed allotype (mu a mu b) are present among serum antibodies. The finding was extended to hybridomas prepared from nonimmune transgenic mice, many of which also failed to exhibit allelic exclusion. The proportions of mu a and mu b secreted by individual hybridomas varied markedly, and the product of an individual hybridoma was found to be heterogeneous with respect to the allotype content of individual molecules. The ratio of mu a:mu b chains secreted by individual hybridomas was found to correlate with the number of transgene copies remaining in each hybridoma, and several hybridomas that secrete only mu b-positive molecules had apparently lost all but one copy of the transgene. An idiotype characteristic of the transgene was found to be present only in association with the transgenic (mu a) allotype, and indirect evidence strongly suggests that the idiotype was present only on mu a polypeptide chains. Thus, there is no evidence in this system for the induction of idiotypically cross-reactive endogenous molecules.     

Hypothesis of initiation of DNA methylation de novo and allelic exclusion by small RNAs

We have established that 5′-CG-3′ dinucleotide and 5′-CNG-3′ trinucleotide are found in published sequences of small interfering RNA and microRNA more often than they should be in random DNA sequences. This circumstance indicates the important biological role played by 5′-CG-3′ dinucleotides and 5′-CNG-3′ trinucleotides in small RNA sequences. We suggest that small RNAs containing these di- and trinucleotides participate in the creation of chromatin marks of epigenetic information through a highly specific search for repressible DNA sequences and through the initiation of the methylation de novo of 5′-CG-3′ and 5′-CNG-3′ sites in DNA fragments appearing to be bound complementary to small RNAs. Several genes can be inactivated simultaneously if they contain the motif recognized by small RNA. Allelic exclusion appears, in our opinion, as a result of initiation by small RNAs of DNA methylation de novo of all but one of the alleles that exist in the cell. The predecessor of this small RNA is transcribed from the antiparallel allele chain. Alleles whose antiparallel chains are less actively read by RNA polymerase, which, as we suggest, in the process of transcribing, releases DNA from small RNA bound to it, are inactivated. However, the quantity of small RNA transcribed from only one allele is insufficient to overcome the level above which the repression process of this allele is initiated de novo.     

Two different genomes that produce the same result

Despite considerable differences in genomic sequence, the developmental program of gene expression between two similar Dictyostelium species is remarkably similar.     

Analysis of the frequency of allelic exclusion of immunoglobulin light chain genes and molecular characteristics of immunoglobulins secreted by hybridomas with expression of both allelic genes

The investigation of 750 B-lymphocyte hybridoma clones obtained by fusion of mouse myeloma and newborn heterozygous Igk-la/Igk-1b rat splenocytes has revealed that 9,8% of Ig kappa-chain genes are rearranged productively. Seventeen hybridomas secrete kappa-chains of both allelic variants. The analysis of IgM molecules of nine such clones demonstrated that in six cases only one L-chain allotype is present in IgM. Thus for the first time the high frequency of selective association of H and L chains was shown. Evidently this selectively may function as one of the allelic exclusion mechanisms at the Ig assembly stage.