Extensive homologous chloroplast DNA recombination in the pt14 Nicotiana somatic hybrid

Summary In a previous study, six recombination sites have been confirmed in the chloroplast DNA (cpDNA) of pt14, a somatic hybrid of Nicotiana tabacum and Nicotiana plumbaginifolia. In the present study, physical mapping revealed six recombination sites in the 11.4-kb SalI fragment alone, only one of which has been previously identified. This fragment is located in the large unique region. We assume, therefore, that the pt14 cpDNA is a fine mosaic of the parental genomes with a recombination site about every 2 kb. A 748-bp region that comprised the intergenic region between ORF73 and ORF74B, and 460 bp of the petD intron have been sequenced. Parent-specific sequences in the pt14 DNA defined the regions within which recombination took place. The exact site of recombination events could not be determined because the parental sequences were identical between the polymorphic markers, and these sequences have been preserved in the pt14 line.     

Extensive homologous recombination between introduced and native regulatory plastid DNA elements in transplastomic plants

Homologous recombination within plastids directs plastid genome transformation for foreign gene expression and study of plastid gene function. Though transgenes are generally efficiently targeted to their desired insertion site, unintended homologous recombination events have been observed during plastid transformation. To understand the nature and abundance of these recombination events, we analyzed transplastomic tobacco lines derived from three different plastid transformation vectors utilizing two different loci for foreign gene insertion. Two unintended recombinant plastid DNA species were formed from each regulatory plastid DNA element included in the transformation vector. Some of these recombinant DNA species accumulated to as much as 10–60% of the amount of the desired integrated transgenic sequence in T0 plants. Some of the recombinant DNA species undergo further, “secondary” recombination events, resulting in an even greater number of recombinant plastid DNA species. The abundance of novel recombinant DNA species was higher in T0 plants than in T1 progeny, indicating that the ancillary recombination events described here may have the greatest impact during selection and regeneration of transformants. A line of transplastomic tobacco was identified containing an antibiotic resistance gene unlinked from the intended transgene insertion as a result of an unintended recombination event, indicating that the homologous recombination events described here may hinder efficient recovery of plastid transformants containing the desired transgene. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

中国口蹄疫病毒同源重组研究

从GenBank数据库中获得在我国分离的16株口蹄疫病毒全基因组序列,进而运用常规的系统发生方法分析了这16株病毒的同源重组情况,发现5株重组毒株．这些重组病毒主要来源于亚洲Ⅰ型(Asia1)和O型病毒间的重组．这些重组事件的鉴定也表明口蹄疫病毒间的交叉感染在我国比较常见．另外,在我国还出现了由于Asia1型和O型病毒重组后导致病毒血清型发生转化的现象．这些结果解释了我国口蹄疫病毒(FMDV)遗传多样性和抗原多变性的成因,提示了我国在口蹄疫预防、治疗方面所面临的复杂局面．     

Induction of homologous recombination in Saccharomyces cerevisiae

Summary We have investigated the effects of UV irradiation of Saccharomyces cerevisiae in order to distinguish whether UV-induced recombination results from the induction of enzymes required for homologous recombination, of the production of substrate sites for recombination containing regions of DNA damage. We utilized split-dose experiments to investigate the induction of proteins required for survival, gene conversion, and mutation in a diploid strain of S. cerevisiae. We demonstrate that inducing doses of UV irradiation followed by a 6 h period of incubation render the cells resistant to challenge doses of UV irradiation. The effects of inducing and challenge doses of UV irradiation upon interchromosomal gene conversion and mutation are strictly additive. Using the yeast URA3 gene cloned in non-replicating single- and double-stranded plasmid vectors that integrate into chromosomal genes upon transformation, we show that UV irradiation of haploid yeast cells and homologous plasmid DNA sequences each stimulate homologous recombination approximately two-fold, and that these effects are additive. Non-specific DNA damage has little effect on the stimulation of, homologous recombination, as shown by studies in which UV-irradiated heterologous DNA was included in transformation/recombination experiments. We further demonstrate that the effect of competing single- and double-stranded heterologous DNA sequences differs in UV-irradiated and unirradiated cells, suggesting an induction of recombinational machinery in UV-irradiated S. cerevisiae cells.     

The sugar beet mitochondrial genome: A complex organisation generated by homologous recombination

Summary The complete physical map of the mitochondrial genome of the Owen cytoplasm of sugar beet has been determined from overlapping cosmid clones. The genome is 386 kb in size and has a multicircular organisation generated by homologous recombination across repeated DNA elements. The location of the rRNA genes and several polypeptide genes has been determined. In addition the mitochondrial genome was found to contain a sequence of chloroplast DNA including part of the 16 S rRNA gene.     

Multilocus sequence analysis of homologous recombination and diversity in Arthrobacter sensu lato named species and glacier-inhabiting strains

Members of the bacterial genus Arthrobacter sensu lato are Gram-positive actinomycetes distributed worldwide and found in numerous environments including soil, water, glacier ice, and sewage. Homologous recombination is an important driving force in bacterial evolution, but its impact on Arthrobacter sensu lato evolution is poorly understood. We evaluated homologous recombination among 41 Arthrobacter sensu lato named species, using multilocus sequence analysis (MLSA). A high level of recombination was found, associated with strong diversification and a reticulate evolutionary pattern of Arthrobacter sensu lato. We also collected a total of 31 cold-adapted Arthrobacter sensu lato strains from two cold glaciers located in northwest China and two temperate glaciers in southwest China, and evaluated their diversity and population structure by MLSA. The glacier strains displayed high diversity, but rates of recombination among the four glacier groups were quite low, indicating that barriers to homologous recombination formed in the past among the populations on different glaciers. Our findings indicate that historical glaciation events shaped the contemporary distributions, taxonomic relationships, and phylogeographic patterns of Arthrobacter sensu lato species on glaciers.     

RadB acts in homologous recombination in the archaeon Haloferax volcanii,consistent with a role as recombination mediator

Homologous recombination plays a central role in the repair of double-strand DNA breaks, the restart of stalled replication forks and the generation of genetic diversity. Regulation of recombination is essential since defects can lead to genome instability and chromosomal rearrangements. Strand exchange is a key step of recombination – it is catalysed by RecA in bacteria, Rad51/Dmc1 in eukaryotes and RadA in archaea. RadB, a paralogue of RadA, is present in many archaeal species. RadB has previously been proposed to function as a recombination mediator, assisting in RadA-mediated strand exchange. In this study, we use the archaeon Haloferax volcanii to provide evidence to support this hypothesis. We show that RadB is required for efficient recombination and survival following treatment with DNA-damaging agents, and we identify two point mutations in radA that suppress the ΔradB phenotype. Analysis of these point mutations leads us to propose that the role of RadB is to act as a recombination mediator, which it does by inducing a conformational change in RadA and thereby promoting its polymerisation on DNA.     

反向遗传学技术在猪瘟病毒研究中的应用

猪瘟目前在许多国家流行并对养猪业造成巨大损失。虽然常规疫苗(如中国猪瘟兔化弱毒疫苗,即C株)在猪瘟防控中发挥巨大作用,但近年来在猪瘟防控中出现的新情况,如非典型感染、持续性感染及免疫失败等;同时目前世界上许多国家正开展的猪瘟扑灭计划使得弱毒疫苗的应用受到很大限制。因此,加强猪瘟病毒在致病机理、传播机制等方面的研究以及加快新型猪瘟疫苗的开发是当务之急。近年来,反向遗传学技术的发展为猪瘟病毒基因功能研究和疫苗制备方面开辟了新思路。以下回顾了反向遗传操作技术在猪瘟病毒基因功能研究与标记疫苗株构建方面的研究进展,同时提出了该领域目前面临的问题,并对其未来发展方向进行了展望。     

Involvement of the inverted repeat of the yeast 2-micron plasmid in Flp site-specific and RAD52-dependent homologous recombination

Site-specific recombination within the Saccharomyces cerevisiae 2-micron DNA plasmid is catalyzed by the Flp recombinase at specific Flp Recognition Target (FRT) sites, which lie near the center of two precise 599-bp Inverted Repeats (IRs). However, the role of IR DNA sequences other than the FRT itself for the function of the Flp reaction in vivo is not known. In the present work we report that recombination efficiency differs depending on whether the FRT or the entire IR serves as the substrate for Flp. We also provide evidence for the involvement of the IR in RAD52-dependent homologous recombination. In contrast, the catalysis of site-specific recombination between two FRTs does not require the function of RAD52. The efficiency of Flp site-specific recombination between two IRs cloned in the same orientation is about one hundred times higher than that obtained when only the two FRTs are present. Moreover, we demonstrate that a single IR can activate RAD52-dependent homologous recombination between two flanking DNA regions, providing new insights into the role of the IR as a substrate for recombination and a new experimental tool with which to study the molecular mechanism of homologous recombination. Received: 14 June 1999 / Accepted: 3 November 1999     

A role for alternative end-joining factors in homologous recombination and genome editing in Chinese hamster ovary cells

CRISPR technologies greatly foster genome editing in mammalian cells through site-directed DNA double strand breaks (DSBs). However, precise editing outcomes, as mediated by homologous recombination (HR) repair, are typically infrequent and outnumbered by undesired genome alterations. By using knockdown and overexpression studies in Chinese hamster ovary (CHO) cells as well as characterizing repaired DNA junctions, we found that efficient HR-mediated genome editing depends on alternative end-joining (alt-EJ) DNA repair activities, a family of incompletely characterized DNA repair pathways traditionally considered to oppose HR. This dependency was influenced by the CRISPR nuclease type and the DSB-to-mutation distance, but not by the DNA sequence surrounding the DSBs or reporter cell line. We also identified elevated Mre11 and Pari, and low Rad51 expression levels as the most rate-limiting factors for HR in CHO cells. Counteracting these three bottlenecks improved precise genome editing by up to 75%. Altogether, our study provides novel insights into the complex interplay of alt-EJ and HR repair pathways, highlighting their relevance for developing improved genome editing strategies.     

Construction of the first shuttle vectors for gene cloning and homologous recombination in Mycoplasma agalactiae

Mycoplasma agalactiae is a worldwide ruminant pathogen that causes significant economic losses by inflicting contagious agalactia in sheep and goats. The development of efficient control strategies requires a better understanding of the mycoplasma factors that promote successful infection. However, lack of genetic tools has been a major impediment in studying the pathogenic mechanisms of M. agalactiae. This study describes the identification and cloning of the M. agalactiae origin of replication (oriC) in order to construct the first shuttle vectors for targeted gene disruption, gene complementation and expression studies. Additionally, this report provides the first evidence of the occurrence of homologous recombination and the functionality of heterologous tetM determinant in this pathogen.     

Isogenic auxotrophic mutant strains in the <Emphasis Type="Italic">Aspergillus fumigatus</Emphasis> genome reference strain AF293

Aspergillus fumigatus is a ubiquitous fungus that is a frequent opportunistic pathogen in immunosuppressed patients. Because of its role as a pathogen, it is of considerable experimental interest. A set of auxotrophic isogenic strains in the A. fumigatus genome reference strain AF293 has been developed. Using molecular genetic methods, arginine and lysine auxotrophs were made by deletion of argB and lysB, respectively. Transformation of these auxotrophic strains with plasmids carrying argB or lysB, respectively, results in efficient integration at these loci. Finally, these strains are able to form stable diploids, which should further facilitate analysis of gene functions in this fungus. Furthermore, the development of this isogenic set of auxotrophic strains in the AF293 background will enable investigators to study this important opportunistic human pathogen with greater facility.     

Ecological genomics in Xanthomonas: the nature of genetic adaptation with homologous recombination and host shifts

Background

Comparative genomics provides insights into the diversification of bacterial species. Bacterial speciation usually takes place with lasting homologous recombination, which not only acts as a cohering force between diverging lineages but brings advantageous alleles favored by natural selection, and results in ecologically distinct species, e.g., frequent host shift in Xanthomonas pathogenic to various plants.

Results

Using whole-genome sequences, we examined the genetic divergence in Xanthomonas campestris that infected Brassicaceae, and X. citri, pathogenic to a wider host range. Genetic differentiation between two incipient races of X. citri pv. mangiferaeindicae was attributable to a DNA fragment introduced by phages. In contrast to most portions of the genome that had nearly equivalent levels of genetic divergence between subspecies as a result of the accumulation of point mutations, 10% of the core genome involving with homologous recombination contributed to the diversification in Xanthomonas, as revealed by the correlation between homologous recombination and genomic divergence. Interestingly, 179 genes were under positive selection; 98 (54.7%) of these genes were involved in homologous recombination, indicating that foreign genetic fragments may have caused the adaptive diversification, especially in lineages with nutritional transitions. Homologous recombination may have provided genetic materials for the natural selection, and host shifts likely triggered ecological adaptation in Xanthomonas. To a certain extent, we observed positive selection nevertheless contributed to ecological divergence beyond host shifting.

Conclusion

Altogether, mediated with lasting gene flow, species formation in Xanthomonas was likely governed by natural selection that played a key role in helping the deviating populations to explore novel niches (hosts) or respond to environmental cues, subsequently triggering species diversification.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1369-8) contains supplementary material, which is available to authorized users.     

Role of PCNA and TLS polymerases in D-loop extension during homologous recombination in humans

Homologous recombination (HR) is essential for maintaining genomic integrity, which is challenged by a wide variety of potentially lethal DNA lesions. Regardless of the damage type, recombination is known to proceed by RAD51-mediated D-loop formation, followed by DNA repair synthesis. Nevertheless, the participating polymerases and extension mechanism are not well characterized. Here, we present a reconstitution of this step using purified human proteins. In addition to Pol δ, TLS polymerases, including Pol η and Pol κ, also can extend D-loops. In vivo characterization reveals that Pol η and Pol κ are involved in redundant pathways for HR. In addition, the presence of PCNA on the D-loop regulates the length of the extension tracks by recruiting various polymerases and might present a regulatory point for the various recombination outcomes.     

High-throughput cloning of human liver complete open reading frames using homologous recombination in Escherichia coli

In this article, we describe a high-throughput cloning method, seamless enzyme-free cloning (SEFC), which allows one-step assembly of DNA fragments in vivo via homologous recombination in Escherichia coli. In the method, the desired open reading frame (ORF) is amplified by use of ORF-specific primers with flanking sequences identical to the two ends of a linearized vector. The polymerase chain reaction (PCR) product and the linearized vector are then cotransformed into E. coli cells, where the ORF is incorporated into the vector in vivo. SEFC is a simple, reliable, and inexpensive method of cloning in which PCR fragments are fused into expression vectors without unwanted amino acids or extra in vitro manipulations apart from the single PCR amplification step. Using this method, we successfully cloned human liver complete ORFs into the yeast AD and DB vectors and generated a clone resource of 4964 AD-ORFs and 4676 DB-ORFs in 3 months. This approach will be useful for daily DNA cloning and for creating proteome-scale clone resources.     

N-linked glycosylation status of classical swine fever virus strain Brescia E2 glycoprotein influences virulence in swine

E2 is one of the three envelope glycoproteins of classical swine fever virus (CSFV). Previous studies indicate that E2 is involved in several functions, including virus attachment and entry to target cells, production of antibodies, induction of protective immune response in swine, and virulence. Here, we have investigated the role of E2 glycosylation of the highly virulent CSFV strain Brescia in infection of the natural host. Seven putative glycosylation sites in E2 were modified by site-directed mutagenesis of a CSFV Brescia infectious clone (BICv). A panel of virus mutants was obtained and used to investigate whether the removal of putative glycosylation sites in the E2 glycoprotein would affect viral virulence/pathogenesis in swine. We observed that rescue of viable virus was completely impaired by removal of all putative glycosylation sites in E2 but restored when mutation N185A reverted to wild-type asparagine produced viable virus that was attenuated in swine. Single mutations of each of the E2 glycosylation sites showed that amino acid N116 (N1v virus) was responsible for BICv attenuation. N1v efficiently protected swine from challenge with virulent BICv at 3 and 28 days postinfection, suggesting that glycosylation of E2 could be modified for development of classical swine fever live attenuated vaccines.     

Overexpression of cyclin E does not influence homologous recombination in Chinese hamster cells

Overexpressed cyclin E in tumours is a prognosticator for poor patient outcome. Cells that overexpress cyclin E have been shown to be impaired in S-phase progression and exhibit genetic instability that may drive this subset of cancers. However, the origin for genetic instability caused by cyclin E overexpression is unknown. Homologous recombination plays an important role in S-phase progression and is also regulated by the same proteins that regulate cyclin E-associated kinase activity, i.e., p53 and p21. To test the hypothesis that overexpressed cyclin E causes genetic instability through homologous recombination, we investigated the effect of cyclin E overexpression on homologous recombination in the hprt gene in a Chinese hamster cell line. Although cyclin E overexpression shortened the G1 phase in the cell cycle as expected, we could see no change in neither spontaneous nor etoposide-induced recombination. Also, overexpression of cyclin E did not affect the repair of DNA double-strand breaks and failed to potentiate the cytotoxic effects of etoposide. Our data suggest that genetic instability caused by overexpression of cyclin E is not mediated by aberrant homologous recombination.     

细菌内同源重组法制备FMDV聚蛋白编码基因重组腺病毒

采用PCR方法从重组质粒pMD18_T/PP中扩增出FMDV的聚蛋白(PP)编码基因,再亚克隆至腺病毒穿梭质粒中,形成重组穿梭质粒rpAd_CMV/PP;将获得的重组穿梭质粒与腺病毒骨架载体通过在大肠杆菌内质粒间同源重组获得重组腺病毒质粒rpAd/PP。将腺病毒载体线性化后用脂质体介导转染293细胞从而获得含有口蹄疫病毒PP编码基因的重组腺病毒。通过倒置显微镜观测,可见明显的细胞病变,利用荧光显微镜可观测到报告基因绿色荧光蛋白的表达,并在电镜下观察到FMDV的空衣壳。结果证明已成功获得了含有口蹄疫病毒PP编码基因的重组腺病毒rAd/PP,并成功表达组装FMDV空衣壳,为FMDV腺病毒活载体疫苗的研究奠定了基础。     

Homologous recombination as an evolutionary force in the avian influenza A virus

Avian influenza A viruses (AIVs), including the H5N1, H9N2,and H7N7 subtypes, have been directly transmitted to humans,raising concerns over the possibility of a new influenza pandemic.To prevent a future avian influenza pandemic, it is very importantto fully understand the molecular basis driving the change inAIV virulence and host tropism. Although virulent variants ofother viruses have been generated by homologous recombination,the occurrence of homologous recombination within AIV segmentsis controversial and far from proven. This study reports threecirculating H9N2 AIVs with similar mosaic PA genes descendedfrom H9N2 and H5N1. Additionally, many homologous recombinantsare also found deposited in GenBank. Recombination events canoccur in PB2, PB1, PA, HA, and NP segments and between lineagesof the same/different serotype. These results collectively demonstratethat intragenic recombination plays a role in driving the evolutionof AIVs, potentially resulting in effects on AIV virulence andhost tropism changes.     

Impairment of homologous recombination control in a Fanconi anemia-like Chinese hamster cell mutant

DNA interstrand cross-links (ICL)-inducing agents such as cisplatin, mitomycin C (MMC) and nitrogen mustards are widely used as potent antitumor drugs. Although ICL repair mechanism is not yet well characterized in mammalian cells, this pathway is thought to involve a sequential action of nucleotide excision repair (NER) and homologous recombination (HR). The importance of unraveling ICL repair pathways is highlighted by the hypersensitivity to ICL-inducing agents in cells of patients with the genetic disease Fanconi anemia (FA) and in cells mutated in the Breast Cancer susceptibility genes BRCA1 and BRCA2. To better characterize the involvement of HR in the sensitivity to ICL-inducing agents, we examined spontaneous and ICL-induced HR in rodent FA-like V-H4 cells. In this report, we show that MMC-hypersensitive V-H4 cells exhibit an increased spontaneous homology-directed repair (HDR) activity compared to the resistant V79 parental cells. Elevated HDR activity results mainly in increased conservative Rad51-dependent recombination, without affecting non-conservative single-strand annealing process (SSA). We also show that HDR activity is enhanced following MMC treatment in parental cells, but not in rodent FA-like V-H4 cells. Moreover, our data indicate that Rad51 foci formation is significantly delayed in these FA-like cells in response to crosslinking agent. These findings provide evidence for an impairment of HR control in V-H4 cells and emphasize the involvement of the FA pathway in HR-mediated repair.