RecQ helicase acts before RuvABC,RecG and XerC proteins during recombination in recBCD sbcBC mutants of Escherichia coli

The RecQ helicase is required by the RecF recombination pathway that is operative in recBC(D) sbcB sbcC(D) mutants of Escherichia coli. Genetic data suggest that RecQ participates in resection of DNA ends during initiation of recombination. In vitro, RecQ can unwind a variety of DNA substrates, including recombination intermediates such as D-loops and Holliday junctions. However, its potential role in processing of recombination intermediates during the late stage of the RecF pathway has not been genetically tested. Here we studied the effect of a recQ mutation on transductional recombination and DNA repair after γ-irradiation in ΔrecBCD ΔsbcB sbcC strains deficient for RuvABC, RecG and XerC proteins. RuvABC and RecG proteins process recombination intermediates in the late stage of recombination, whereas XerC is required to resolve chromosome dimers formed upon recombination. Our results do not reveal any substantial synergistic effect between the recQ mutation, on one hand, and ruvABC, recG and xerC mutations on the other. In addition, the recQ mutation suppresses chromosome segregation defects in γ-irradiated ruvABC recG and xerC mutants. These results suggest that RecQ acts upstream of RuvABC, RecG and XerC proteins, a finding that is compatible with its primary role in initiation of the RecF recombination pathway.     

Streamlined human antibody generation and optimization by exploiting designed immunoglobulin loci in a B cell line

Monoclonal antibodies (mAbs) are widely utilized as therapeutic drugs for various diseases, such as cancer, autoimmune diseases, and infectious diseases. Using the avian-derived B cell line DT40, we previously developed an antibody display technology, namely, the ADLib system, which rapidly generates antigen-specific mAbs. Here, we report the development of a human version of the ADLib system and showcase the streamlined generation and optimization of functional human mAbs. Tailored libraries were first constructed by replacing endogenous immunoglobulin genes with designed human counterparts. From these libraries, clones producing full-length human IgGs against distinct antigens can be isolated, as exemplified by the selection of antagonistic mAbs. Taking advantage of avian biology, effective affinity maturation was achieved in a straightforward manner by seamless diversification of the parental clones into secondary libraries followed by single-cell sorting, quickly affording mAbs with improved affinities and functionalities. Collectively, we demonstrate that the human ADLib system could serve as an integrative platform with unique diversity for rapid de novo generation and optimization of therapeutic or diagnostic antibody leads. Furthermore, our results suggest that libraries can be constructed by introducing exogenous genes into DT40 cells, indicating that the ADLib system has the potential to be applied for the rapid and effective directed evolution and optimization of proteins in various fields beyond biomedicine.     

Homozygous deletion of DIS3L2 exon 9 due to non-allelic homologous recombination between LINE-1s in a Japanese patient with Perlman syndrome

Perlman syndrome is a rare, autosomal recessive overgrowth disorder. Recently, the deletion of exon 9 and other mutations of the DIS3L2 gene have been reported in patients; however, the mechanism behind this deletion is still unknown. We report the homozygous deletion of exon 9 of DIS3L2 in a Japanese patient with Perlman syndrome. We identified the deletion junction, and implicate a non-allelic homologous recombination (NAHR) between two LINE-1 (L1) elements as the causative mechanism. Furthermore, the deletion junctions were different between the paternal and maternal mutant alleles, suggesting the occurrence of two independent NAHR events in the ancestors of each parent. The data suggest that the region around exon 9 might be a hot spot of L1-mediated NAHR.     

促血管生成素-1基因重组腺病毒载体的构建及鉴定

目的构建携带小鼠促血管生成素-1(Ang-1)基因的重组腺病毒载体并鉴定。方法化学合成小鼠Ang-1基因经PCR扩增后亚克隆至穿梭质粒,重组质粒转化感受态细胞,所得转化子经PCR电泳分析并测序鉴定,携带外源基因的腺病毒穿梭质粒与携带腺病毒大部分基因组的辅助包装质粒共转染人胚肾细胞系HEK 293细胞,反复冻融获取重组腺病毒,终点稀释法测定重组腺病毒滴度,所得腺病毒转染293T细胞48 h收集培养上清,通过Western Blot分析Ang-1蛋白的表达。结果 PCR产物电泳分析可见大小约1.5 kbp的特征性条带,测序结果显示基因序列与Gen Bank提供的Ang-1序列一致,经HEK293细胞包装后腺病毒滴度为1×109pfu/ml,转染293T细胞后培养上清Western Blot分析可见大小58 k D特征性条带,即转染后能分泌正确大小的重组Ang-1蛋白。结论 Ang-1基因重组腺病毒载体可成功构建,为进一步研究Ang-1蛋白对造血干细胞动员及血管新生的作用提供实验依据。     

Neutral microepidemic evolution of bacterial pathogens

Understanding bacterial population genetics is vital for interpreting the response of bacterial populations to selection pressures such as antibiotic treatment or vaccines targeted at only a subset of strains. The evolution of transmissible bacteria occurs by mutation and localized recombination and is influenced by epidemiological as well as molecular processes. We demonstrate that the observed population genetic structure of three important human pathogens, Streptococcus pneumoniae, Neisseria meningitidis, and Staphylococcus aureus, can be explained by using a simple evolutionary model that is based on neutral mutational drift, modulated by recombination, and which incorporates the impact of epidemic transmission in local populations. The predictions of this neutral "microepidemic" model are found to closely fit observed genetic relatedness distributions of bacteria sampled from their natural population, and it provides estimates of the relative rate of recombination that agree well with empirical estimates. The analysis suggests the emergence of neutral bacterial population structure from overlapping microepidemics within clustered host populations and provides insight into the nature and size distribution of these clusters. These findings challenge the assumption that strains of bacterial pathogens differ markedly in relative fitness.     

Alu‐mediated large deletion of the CDSN gene as a cause of peeling skin disease

Peeling skin disease (PSD) is an autosomal recessive skin disorder caused by mutations in CDSN and is characterized by superficial peeling of the upper epidermis. Corneodesmosin (CDSN) is a major component of corneodesmosomes that plays an important role in maintaining epidermis integrity. Herein, we report a patient with PSD caused by a novel homozygous large deletion in the 6p21.3 region encompassing the CDSN gene, which abrogates CDSN expression. Several genes including C6orf15, PSORS1C1, PSORS1C2, CCHCR1, and TCF19 were also deleted, however, the patient showed only clinical features typical of PSD. The deletion size was 59.1 kb. Analysis of the sequence surrounding the breakpoint showed that both telomeric and centromeric breakpoints existed within Alu‐S sequences that were oriented in opposite directions. These results suggest an Alu‐mediated recombination event as the mechanism underlying the deletion in our patient.     

Polarized gene conversion at the bz locus of maize

Nucleotide diversity is greater in maize than in most organisms studied to date, so allelic pairs in a hybrid tend to be highly polymorphic. Most recombination events between such pairs of maize polymorphic alleles are crossovers. However, intragenic recombination events not associated with flanking marker exchange, corresponding to noncrossover gene conversions, predominate between alleles derived from the same progenitor. In these dimorphic heterozygotes, the two alleles differ only at the two mutant sites between which recombination is being measured. To investigate whether gene conversion at the bz locus is polarized, two large diallel crossing matrices involving mutant sites spread across the bz gene were performed and more than 2,500 intragenic recombinants were scored. In both diallels, around 90% of recombinants could be accounted for by gene conversion. Furthermore, conversion exhibited a striking polarity, with sites located within 150 bp of the start and stop codons converting more frequently than sites located in the middle of the gene. The implications of these findings are discussed with reference to recent data from genome-wide studies in other plants.Gene conversion in organisms where all four products of a meiotic tetrad can be recovered refers to a departure from the normal 2:2 segregation of alleles and arises from the repair of meiotic double-strand breaks (DSBs) by a homologous recombination mechanism (1). Gene conversion represents the nonreciprocal, but faithful, transfer of information between two homologous DNA sequences, usually located in homologous chromosomes. The stretch of DNA that is transferred during a gene conversion event, called the conversion tract, can vary in yeast from a few hundred bases to more than 12 kb and is composed of sequences found in only one of the parental chromosomes, i.e., it is continuous, not patchy. Conversion polarity within a gene, which is the tendency of markers located near one end of the gene to convert more frequently than those located at the opposite end, has been reported in several Ascomycete fungi: Ascobolus (2), Neurospora (3), yeast (4), and Aspergillus (5). The high conversion end is usually the 5′ end (6), but can also be the 3′ end (7). Conversion frequency gradients are generally accepted to reflect a preferential initiation site for recombination that is located at the high conversion end of the gene (8, 9).In most organisms that undergo meiosis, only one of the four meiotic products is ordinarily recovered, so it is not possible to identify gene conversion on the basis of aberrant segregation ratios. A notable exception is the Arabidospsis quartet1 (qrt1) mutant that allows pollen tetrad analysis and has been used to demonstrate gene conversion events unambiguously by their classic 3:1 segregation (10) to estimate genome-wide conversion frequencies (11, 12) and to measure the tract lengths of such conversion events (12, 13). Usually, however, gene conversions have been identified by the flanking marker arrangement of intragenic recombinants (IGRs). This convention derives from the observation that, in yeast asci displaying gene conversion of a central marker flanked by two closely linked outside markers, the convertant spore could carry a parental or a recombinant arrangement of flanking markers with about equal frequency, on average (14, 15). Therefore, geneticists studying recombination in organisms where tetrad analysis is not possible have tended to refer to IGRs bearing a parental or noncrossover (NCO) arrangement of flanking markers as convertants, a convention that we also follow in this paper. In these cases, a convenient way of detecting conversion polarity is to compare the relative frequencies with which the two parentally marked IGRs are recovered from heteroallelic combinations (5, 16).In contrast to observations in fungi, it was noted repeatedly in maize recombination experiments that the vast majority of IGRs were crossovers (COs), i.e., associated with an exchange of flanking markers (17). Most of those studies dealt with polymorphic heterozygotes in which the recombining heteroalleles were derived from progenitor alleles that differed by single nucleotide and indel polymorphisms in as much as 1.6% of their sequences (18) and often included a transposon insertion allele. This experimental setup helped to place recombination junctions, but affected the outcome of the experiment. A different picture emerged from recombination studies with dimorphic heterozygotes at the bz locus, in which the recombining heteroalleles were derived from the same progenitor and differed only at the two sites between which recombination was being measured (19). In dimorphic heterozygotes, one CO class still predominated (that expected from the relative location of the mutations in the gene), but the NCO classes occurred in much higher numbers, so that the CO/NCO ratio was often less than 1. Similar results have been obtained at the r locus (20). Thus, the CO/NCO ratio in maize can be allele dependent. The variability in the CO/NCO ratio for different recombination hotspots observed in a highly polymorphic yeast hybrid (21) may reflect the extent of allelic polymorphisms among different loci.The sharp difference in the outcome of experiments involving polymorphic and dimorphic heterozygotes was explained in terms of the dual recombination pathway proposed by Allers and Lichten (22) and supported by other work (23–25), whereby repair of the initiating DSB produces COs via a double Holliday junction (DHJ) intermediate and NCOs via a synthesis-dependent strand annealing (SDSA) pathway. In this model, the decision to repair a DSB as a CO (via a DHJ) or a NCO (via SDSA) would happen at or soon after the initial step of strand invasion. The bz and r data suggest that that decision is affected by the extent of mismatches in the heteroduplex DNA formed by the invading strand. In the absence of extensive heterologies, mismatch repair proteins would not bind to the heteroduplex and the newly synthesized strand would be displaced readily leading to the recovery of NCO products.In the studies with bz dimorphic heterozygotes, the two NCO classes occurred in roughly similar numbers, i.e., the NCO class that carried the flanking markers of the proximal (5′) allele was recovered approximately as frequently as the NCO class that carried the flanking markers of the distal (3′) allele. Thus, there was no indication of preferential conversion of the proximal or distal allele (26, 27). However, the sample of mutant sites used in those studies did not include any at either end of the gene. Because most conversion gradients in yeast show strong 5′ or 3′ polarity, we could have missed a conversion gradient at bz that was steep at one or both ends but hardly detectable thereafter. To examine at greater depth the issue of conversion polarity within a higher plant gene, we now isolated a series of new bz mutations from the Bz-McC progenitor allele and extended our analysis to include sites at both the 5′ and 3′ ends of the bz gene. Mutations covering the length of the gene were tested in all possible pairwise combinations in two large diallels, with remarkably similar results in the two experiments. We find that bz mutants derived from the Bz-McC allele, which is flanked by single-copy DNA sequences on either side (28), show a U-shaped conversion gradient, with higher conversion frequencies at both the 5′ and 3′ ends than at the center. The implications of these findings are discussed with reference to recent data from genome-wide studies in several organisms.