Mutation and recombination in Helicobacter pylori: mechanisms and role in generating strain diversity

The pathogenic bacterium Helicobacter pylori infects half of the human population and it is one of the most genetically diverse bacterial species known. The unusual combination of high frequencies of mutation and recombination events together with a very small size of DNA fragments imported into the genome after recombination, contribute to its genetic variability. In this review article, we discuss the genetic variability of H. pylori, the mechanisms that generate diversity, and the potential relevance of genetic variability to colonization and pathogenicity.     

Genetic basis of inherited peripheral neuropathies

Progress in the elucidation of the genetic basis for inherited peripheral neuropathies has been remarkable over the last years. In particular, the molecular mechanisms underlying the autosomal dominantly inherited disorders Charcot–Marie–Tooth disease type 1A (CMT1 A), Charcot–Marie–Tooth disease type 1B (CMT1B), and hereditary neuropathy with liability to pressure palsies (HNPP) have been determined. While mutation in the gene encoding the major myelin protein, P_o has been associated with CMT1B, CMT1A and HNPP have been shown to be associated with reciprocal recombination events leading either to a large submicroscopic duplication in CMT1 A, or the corresponding DNA deletion in HNPP. Available evidence is consistent with the hypothesis that one or more genes within the relevant rearranged segment of 1.5 Mb on chromosome 17 is sensitive to gene dosage providing a novel mechanism for inherited human disorders. It is likely that the gene encoding the peripheral myelin protein PMP22 is at least one of the genes involved since the PMP22 gene maps within the CMT1A duplication (or HNPP deletion), and point mutations within it have been shown to cause a CMT phenotype in humans and comparable neuropathies in rodents (trembler and trembler^J). The mechanism(s) by which gene dosage and point mutations affecting the same gene might lead to a similar phenotype are currently unknown but recent transgenic mouse experiments suggest that similar mechanisms may also underlie other genetic diseases. © 1994 Wiley-Liss, Inc.     

Phenotypic and epistatic grouping of hypo- and hyper-rec mus mutants in Aspergillus

The mutants musK to musS of Aspergillus nidulans are sensitive to methyl-methanesulfonate (MMS) and several of them are meiotic-defective and alter mitotic recombination frequencies. All were found to be cross-sensitive to 4-nitro-quinoline-N-oxide (4-NQO) but unexpectedly none of them was hypersensitive to -rays and few to UV light. Double mus;uvs mutants were constructed to test for interactions with uvs mutations of the four epistatic groups of Aspergillus, UvsF, UvsC, UvsI, and UvsB. All meiotic-defective mus mutations caused some lethal interactions, usually with uvsF. None of them showed epistasis with UvsF or UvsB group mutants and one, musO, may represent a new group. Three mus mutations that affect recombination were assigned to the UvsC group, namely musN and K, and also musL which is recombination-defective and closely resembles uvsC. While uvsC mutants are mutators and lack UV-mutagenesis, most mus mutants had no effects on mutation. Only musR, which appeared epistatic with uvsI, showed reduced UV-reversion frequencies similar to uvsI. The recombination-proficient mus mutants appeared to be epistatic with more than one group, but in several cases sensitivities were slight and overlaps insufficient to obtain corroborating results with MMS and 4-NQO.     

B precursor acute lymphoblastic leukemia third complementarity-determining regions predominantly represent an unbiased recombination repertoire: Leukemic transformation frequently occurs in fetal life

To determine whether the ALL (acute lymphoblastic leukemia) CDR3 (third complementarity-determining region) repertoire represents the recombination repertoire, or shows evidence of selectional processes inherent to normal B cell differentiation or malignant transformation, we analyzed 68 ALL CDR3 regions and included 127 previously published sequences in the analyses. We found no evidence of selection prior to malignant transformation as recombination was random with 1/3 “in frame” and 2/3 “out of frame” joinings and usage of all three D reading frames was observed. D and J_H gene segments were predominantly umutated which allowed a detailed analysis of gene usage and rearrangement characteristics. J_H⁴ and J_H⁶ usage (both 32.2%) was significantly different (p = 0.005) from that observed in peripheral B lymphocytes. D gene family usage roughly represented D gene family size with the exception of the D_XP and D_A/K family which were over- and underrepresented (p = 0.05), respectively. D-D fusions were found in 26.2% of CDR3 regions. If less stringent criteria were applied DIR homology was found in 40/65 sequences, suggesting the frequent involvement of DIR gene segments in human CDR3 formation. The rearranged D genes were evenly distributed over the D locus, suggesting that D recombination is a predominantly random process, independent of physical location at the locus. Also, there was no correlation between J_H gene usge and physical location of the rearranged D gene segment, which excludes a major contribution of the DJ_H replacement recombination mechanism. In 36.1% of CDR3 regions N-nucleotides at the DJ_H junction were absent. This frequency is higher than observed for peripheral B lymphocytes. It is suggested that for a number of ALL the initial transformational event took place in early fetal life. We conclude that ALL CDR3 sequences show no evidence of selection prior to malignant transformation, nor of extensive changes subsequent to malignant transformation.     

The smt-0 mutation which abolishes mating-type switching in fission yeast is a deletion

Mating-type switching in the fission yeast, S. pombe, is initiated by a DNA double-strand break (DSB) between the mat1 cassette and the H1 homology box. The mat1-cis-acting mutant, smt-0, abolishes mating-type switching and is shown here to be a 263-bp deletion. This deletion starts in the middle of the H1 homology box, 31 bp from the site of the DSB, and extends into the flanking region distal to mat1. The sequence of the region distal to H1 in the wild-type is also presented. In this region we observe a bias in the distribution of purine residues between the two DNA strands.     

Small interspersed sequences that serve as recombination sites at the cox2 and atp6 loci in the mitochondrial genome of soybean are widely distributed in higher plants

Mitochondrial DNA (mtDNA) fragments that contain cox2 and atp6 were cloned from a wild soybean (Glycine soja, accession `B09002') and from a cultivated soybean (G. max, `Harosoy'). Comparison of these DNAs revealed that two sets of repeated sequences, namely, 299 bp and 23 bp, were present in the 5′ regions of cox2 and atp6. The 299-bp and 23-bp repeats were present close to each other on the 5′ flanking region and the 5′ part of the coding region of cox2 in both `Harosoy' and `B09002', as well as on the 5′ flanking region of atp6 in `Harosoy', while these two repeats were separated by a 706-bp nucleotide sequence that contained a truncated sequence of nad3 at the 5′ flanking region of atp6 in `B09002'. The mtDNA configurations upstream from atp6 and cox2 found in `Harosoy' appeared to have been generated from configurations of cox2 and atp6 found in `B09002' via recombination across the 299-bp or 23-bp repeated sequences, or vice versa, in the mitochondrial genome of the hypothetical progenitor of these plants. The 299-bp sequence was found to be interspersed in the mitochondrial genome. Eight loci were identified that include mtDNA configurations that are inter-convertible with each other via recombination across this sequence in `B09002'. Various loci on the mitochondrial genomes of higher plants that harbor segments of the 299-bp repeats in Glycine were identified. Received: 16 August / 15 December 1997     

细菌内同源重组高效制备含绿色荧光蛋白和抗凋亡基因bcl-XL的重组腺病毒载体

目的:制备含绿色荧光蛋白和抗凋亡基因bcl-X_L的重组腺病毒载体。方法:采用PCR方法从质粒pEGFP-C3-bcl-X_L中扩增出bcl-X_L基因,再亚克隆至腺病毒穿梭质粒中,形成转移质粒pAdTrack-CMV-bcl-X_L;然后与pAdEasy-1共转化BJ5183菌,采用细菌内同源重组法构建重组腺病毒质粒pAdEasy-1-gfp-bcl-X_L,筛选同源重组的阳性克隆;抽提的pAdEasy-1-gfp-bcl-X_L经PacI线性化后,再用LIPOFECTAMINE^TM2000介导转染至人胚肾293细胞内包装扩增出重组腺病毒颗粒;采用PCR方法对重组腺病毒进行鉴定;用氯化铯超高速梯度离心纯化获取高滴度的重组腺病毒rAd-gfp-bcl-X_L。结果:由pAdTrack-CMV-bcl-X_L和pAdEasy-1共转化BJ5183菌16-20h后,可获得35%的阳性重组体细菌克隆。由重组质粒DNA经293细胞包装后产生的重组腺病毒,经PCR检测表明含有目的基因bcl-X_L。氯化铯纯化所得重组腺病毒滴度约为65×10¹²PFU/L。结论:用细菌内同源重组法可以高效、简便、快捷地制备出重组腺病毒质粒载体,重组体病毒质粒经293细胞包装、扩增和氯化铯纯化后可制备出高滴度的重组腺病毒颗粒rAd-gfp-bcl-X_L,为人类相关疾病的基因治疗提供良好的基因转染载体。     

Rearrangement of upstream DH and VH genes to a rearranged immunoglobulin variable region gene inserted into the DQ52-JH region of the immunoglobulin heavy chain locus

We investigated gene rearrangements in the mutant IgH locus of a mouse strain generated by insertion of a rearranged heavy chain variable region gene (V_T15) into the DQ52-J_H region through gene targeting. In more than half of the B cells of heterozygous mutant mice, the mutant IgH locus was silenced by the rearrangement of an endogenous D_H or D_H and V_H gene to the inserted V_T15 gene. In these cases, a functional V_HD_HJ_H gene was present on the wild-type allele. The silencing rearrangement appeared to be mediated by recombination signal sequence (RSS)-like elements present in the “recipient” V_T15 gene. Among the many such elements on the inserted V_T15 gene, which apparently met the requirement for an RSS with respect to nucleotide sequence, only two were observed in the actual rearrangements. This indicates that targeting of the recombination machinery involves sequences in addition to the RSS motifs as they have been characterized so far. In homozygous mutant mice, most B cells appeared to carry the intact V_T15 gene on both mutant IgH alleles, although single-cell polymerase chain reaction revealed that silencing rearrangements occured frequently in B cell progenitors in the bone marrow. This observation indicates that once silencing rearrangements are initiated in a cell, they involve both V_T15 genes in most cases, reminiscent of normal D_H-J_H rearrangement. B cells which did not initiate such rearrangements develop to populate the peripheral B cell compartment.     

Mutations in the RNA-binding domains of tombusvirus replicase proteins affect RNA recombination in vivo

RNA recombination, which is thought to occur due to replicase errors during viral replication, is one of the major driving forces of virus evolution. In this article, we show evidence that the replicase proteins of Cucumber necrosis virus, a tombusvirus, are directly involved in RNA recombination in vivo. Mutations within the RNA-binding domains of the replicase proteins affected the frequency of recombination observed with a prototypical defective-interfering (DI) RNA, a model template for recombination studies. Five of the 17 replicase mutants tested showed delay in the formation of recombinants when compared to the wild-type helper virus. Interestingly, two replicase mutants accelerated recombinant formation and, in addition, these mutants also increased the level of subgenomic RNA synthesis (Virology 308 (2003), 191-205). A trans-complementation system was used to demonstrate that mutation in the p33 replicase protein resulted in altered recombination rate. Isolated recombinants were mostly imprecise (nonhomologous), with the recombination sites clustered around a replication enhancer region and a putative cis-acting element, respectively. These RNA elements might facilitate the proposed template switching events by the tombusvirus replicase. Together with data in the article cited above, results presented here firmly establish that the conserved RNA-binding motif of the replicase proteins is involved in RNA replication, subgenomic RNA synthesis, and RNA recombination.