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1.
Nature ; 582(7810): 124-128, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32494071

RESUMO

In most species, homologous chromosomes must recombine in order to segregate accurately during meiosis1. Because small chromosomes would be at risk of missegregation if recombination were randomly distributed, the double-strand breaks (DSBs) that initiate recombination are not located arbitrarily2. How the nonrandomness of DSB distributions is controlled is not understood, although several pathways are known to regulate the timing, location and number of DSBs. Meiotic DSBs are generated by Spo11 and accessory DSB proteins, including Rec114 and Mer2, which assemble on chromosomes3-7 and are nearly universal in eukaryotes8-11. Here we demonstrate how Saccharomyces cerevisiae integrates multiple temporally distinct pathways to regulate the binding of Rec114 and Mer2 to chromosomes, thereby controlling the duration of a DSB-competent state. The engagement of homologous chromosomes with each other regulates the dissociation of Rec114 and Mer2 later in prophase I, whereas the timing of replication and the proximity to centromeres or telomeres influence the accumulation of Rec114 and Mer2 early in prophase I. Another early mechanism enhances the binding of Rec114 and Mer2 specifically on the shortest chromosomes, and is subject to selection pressure to maintain the hyperrecombinogenic properties of these chromosomes. Thus, the karyotype of an organism and its risk of meiotic missegregation influence the shape and evolution of its recombination landscape. Our results provide a cohesive view of a multifaceted and evolutionarily constrained system that allocates DSBs to all pairs of homologous chromosomes.


Assuntos
Cromossomos Fúngicos/genética , Recombinação Homóloga , Meiose , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Centrômero/genética , Segregação de Cromossomos , Cromossomos Fúngicos/metabolismo , Quebras de DNA de Cadeia Dupla , Período de Replicação do DNA , Meiose/genética , Prófase Meiótica I/genética , Recombinases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Telômero/genética , Fatores de Tempo
2.
Zhongguo Xue Xi Chong Bing Fang Zhi Za Zhi ; 32(2): 168-173, 2020 Mar 13.
Artigo em Chinês | MEDLINE | ID: mdl-32458606

RESUMO

OBJECTIVE: To establish a rapid nucleic acid detection technique for identification of Echinococcus multilocularis based on the recombinase aided isothermal amplification assay (RAA) and assess its diagnostic efficiency. METHODS: The mitochondrial gene sequence of E. multilocularis (GenBank accession number: AB018440) was used as a target sequence. The primers were designed according to the RAA reaction principle and synthesized, and RAA was performed using the generated primers. E. multilocularis genomic DNA at various concentrations and the pMD19-T (Simple) vector containing various copies of the target gene fragment were amplified using RAA to evaluate its sensitivity for detection of E. multilocularis, and RAA was em- ployed to detect the genomic DNA of E. granulosus G1 genotype, Taenia saginata, T. asiatica, T. multiceps, Dipylidium caninum, Toxocara canis, Trichuris trichiura, Giardia lamblia, Fasciola hepatica, Paragonimus westermani, Fasciola gigantica and Clonorchis sinensis to evaluate its specificity. In addition, the optimized RAA was employed to detect nine tissue specimens of E. granulosus-infected animals, 3 fecal samples from E. granulosus-infected dogs and 2 fecal samples from field infected dogs to examine its reliability and feasibility. RESULTS: The established RAA was able to detect the specific target gene fragment of E. multilocularis within 40 min. The lowest detect limit of RAA was 10 pg if E. multilocularis genomic DNA served as a template. If the re- combinant plasmid was used as a template, the minimally detectable copy number of RAA was 104. In addition, RAA was nega- tive for the genomic DNA of E. granulosus G1 genotype, T. saginata, T. asiatica, T. multiceps, D. caninum, T. canis, T. trichiura, G. lamblia, F. hepatica, P. westermani, F. gigantica and C. sinensis. The established RAA was positive for detection of the tissue specimens of infected animals, and simulated and field dog stool samples. CONCLUSIONS: A rapid, sensitive and specific RAA is established, which shows promising values in identification of E. multilocularis and gene diagnosis of alveolar echinococcosis.


Assuntos
Doenças do Cão , Equinococose , Echinococcus multilocularis , Animais , Primers do DNA , Doenças do Cão/diagnóstico , Doenças do Cão/parasitologia , Cães , Equinococose/diagnóstico , Equinococose/parasitologia , Equinococose/veterinária , Echinococcus multilocularis/genética , Echinococcus multilocularis/isolamento & purificação , Fezes/parasitologia , Técnicas de Amplificação de Ácido Nucleico , Recombinases/metabolismo , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
3.
Nat Methods ; 17(4): 422-429, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32203389

RESUMO

Brain circuits comprise vast numbers of interconnected neurons with diverse molecular, anatomical and physiological properties. To allow targeting of individual neurons for structural and functional studies, we created light-inducible site-specific DNA recombinases based on Cre, Dre and Flp (RecVs). RecVs can induce genomic modifications by one-photon or two-photon light induction in vivo. They can produce targeted, sparse and strong labeling of individual neurons by modifying multiple loci within mouse and zebrafish genomes. In combination with other genetic strategies, they allow intersectional targeting of different neuronal classes. In the mouse cortex they enable sparse labeling and whole-brain morphological reconstructions of individual neurons. Furthermore, these enzymes allow single-cell two-photon targeted genetic modifications and can be used in combination with functional optical indicators with minimal interference. In summary, RecVs enable spatiotemporally precise optogenomic modifications that can facilitate detailed single-cell analysis of neural circuits by linking genetic identity, morphology, connectivity and function.


Assuntos
Genômica/métodos , Optogenética , Recombinases/metabolismo , Animais , Encéfalo/citologia , Regulação da Expressão Gênica , Engenharia Genética , Camundongos , Neurônios/metabolismo , Recombinases/genética , Peixe-Zebra
4.
PLoS Negl Trop Dis ; 14(2): e0008044, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-32069278

RESUMO

BACKGROUND: Animal trypanosomosis caused by Trypanosoma evansi is known as "surra" and is a widespread neglected tropical disease affecting wild and domestic animals mainly in South America, the Middle East, North Africa and Asia. An essential necessity for T. evansi infection control is the availability of reliable and sensitive diagnostic tools. While DNA-based PCR detection techniques meet these criteria, most of them require well-trained and experienced users as well as a laboratory environment allowing correct protocol execution. As an alternative, we developed a recombinase polymerase amplification (RPA) test for Type A T. evansi. The technology uses an isothermal nucleic acid amplification approach that is simple, fast, cost-effective and is suitable for use in minimally equipped laboratories and even field settings. METHODOLOGY/PRINCIPLE FINDINGS: An RPA assay targeting the T. evansi RoTat1.2 VSG gene was designed for the DNA-based detection of T. evansi. Comparing post-amplification visualization by agarose gel electrophoresis and a lateral flow (LF) format reveals that the latter displays a higher sensitivity. The RPA-LF assay is specific for RoTat1.2-expressing strains of T. evansi as it does not detect the genomic DNA of other trypanosomatids. Finally, experimental mouse infection trials demonstrate that the T. evansi specific RPA-LF can be employed as a test-of-cure tool. CONCLUSIONS/SIGNIFICANCE: Compared to other DNA-based parasite detection methods (such as PCR and LAMP), the T. evansi RPA-LF (TevRPA-LF) described in this paper is an interesting alternative because of its simple read-out (user-friendly), short execution time (15 minutes), experimental sensitivity of 100 fg purified genomic T. evansi DNA, and ability to be carried out at a moderate, constant temperature (39°C). Therefore, the TevRPA-LF is an interesting tool for the detection of active T. evansi infections.


Assuntos
Técnicas de Amplificação de Ácido Nucleico/métodos , Recombinases/metabolismo , Trypanosoma/isolamento & purificação , Tripanossomíase/diagnóstico , Animais , DNA de Protozoário/genética , Camundongos , Camundongos Endogâmicos C57BL , Reação em Cadeia da Polimerase/métodos , Sensibilidade e Especificidade , Trypanosoma/genética
5.
PLoS One ; 15(1): e0227476, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31935232

RESUMO

The pinewood nematode (PWN), Bursaphelenchus xylophilus, is a causative agent of pine wilt disease (PWD). To date, although several molecular diagnostic methods have been developed, rapid on-site diagnostic tools for detecting PWN in pinewood are limited. In this study, a point of care diagnostic (POCD) method for detecting PWN in pinewood using recombinase polymerase amplification (RPA) assay was developed. This method comprises quick gDNA extraction buffer (DAP buffer) for the direct extraction of gDNA of PWN from pinewood and a battery-mounted portable optical isothermal device (POID) for the detection of PWD in the field. The RPA assay can distinguish between the PWN and its conspecies which exist in pinewood and can complete diagnostic procedures within 25 min in the field. Moreover, the RPA assay can detect PWN in old wood samples in both natural and stored conditions. The POCD-RPA assay to detect PWN will be useful for epidemiological investigations in the field as well as for quarantine processes in the wood trade.


Assuntos
Técnicas de Amplificação de Ácido Nucleico/métodos , Pinus/parasitologia , Doenças das Plantas/parasitologia , Tylenchida/genética , Animais , Sequência de Bases , DNA de Helmintos/metabolismo , Genoma Helmíntico , Limite de Detecção , Técnicas de Amplificação de Ácido Nucleico/instrumentação , Sistemas Automatizados de Assistência Junto ao Leito , Recombinases/metabolismo , Alinhamento de Sequência , Tylenchida/isolamento & purificação
6.
Genes (Basel) ; 11(1)2020 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-31936378

RESUMO

The execution of recombinational pathways during the repair of certain DNA lesions or in the meiotic program is associated to the formation of joint molecules that physically hold chromosomes together. These structures must be disengaged prior to the onset of chromosome segregation. Failure in the resolution of these linkages can lead to chromosome breakage and nondisjunction events that can alter the normal distribution of the genomic material to the progeny. To avoid this situation, cells have developed an arsenal of molecular complexes involving helicases, resolvases, and dissolvases that recognize and eliminate chromosome links. The correct orchestration of these enzymes promotes the timely removal of chromosomal connections ensuring the efficient segregation of the genome during cell division. In this review, we focus on the role of different DNA processing enzymes that collaborate in removing the linkages generated during the activation of the homologous recombination machinery as a consequence of the appearance of DNA breaks during the mitotic and meiotic programs. We will also discuss about the temporal regulation of these factors along the cell cycle, the consequences of their loss of function, and their specific role in the removal of chromosomal links to ensure the accurate segregation of the genomic material during cell division.


Assuntos
Segregação de Cromossomos/genética , Segregação de Cromossomos/fisiologia , Animais , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/fisiologia , Cromossomos/genética , Quebras de DNA de Cadeia Dupla , DNA Helicases/genética , DNA Helicases/metabolismo , Reparo do DNA/genética , Reparo do DNA/fisiologia , Recombinação Homóloga/genética , Humanos , Meiose/fisiologia , Mitose/fisiologia , Recombinases/genética , Recombinases/metabolismo
7.
Nucleic Acids Res ; 48(1): e1, 2020 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-31612958

RESUMO

Multiplex genetic assays can simultaneously test thousands of genetic variants for a property of interest. However, limitations of existing multiplex assay methods in cultured mammalian cells hinder the breadth, speed and scale of these experiments. Here, we describe a series of improvements that greatly enhance the capabilities of a Bxb1 recombinase-based landing pad system for conducting different types of multiplex genetic assays in various mammalian cell lines. We incorporate the landing pad into a lentiviral vector, easing the process of generating new landing pad cell lines. We also develop several new landing pad versions, including one where the Bxb1 recombinase is expressed from the landing pad itself, improving recombination efficiency more than 2-fold and permitting rapid prototyping of transgenic constructs. Other versions incorporate positive and negative selection markers that enable drug-based enrichment of recombinant cells, enabling the use of larger libraries and reducing costs. A version with dual convergent promoters allows enrichment of recombinant cells independent of transgene expression, permitting the assessment of libraries of transgenes that perturb cell growth and survival. Lastly, we demonstrate these improvements by assessing the effects of a combinatorial library of oncogenes and tumor suppressors on cell growth. Collectively, these advancements make multiplex genetic assays in diverse cultured cell lines easier, cheaper and more effective, facilitating future studies probing how proteins impact cell function, using transgenic variant libraries tested individually or in combination.


Assuntos
Bioensaio , Biblioteca Gênica , Plasmídeos/química , Transgenes , Animais , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Células HT29 , Humanos , Lentivirus/genética , Lentivirus/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Camundongos , Células NIH 3T3 , Oncogenes , Plasmídeos/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Recombinases/genética , Recombinases/metabolismo , Recombinação Genética , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo
8.
PLoS Genet ; 15(12): e1008217, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31790385

RESUMO

During meiosis, homologous recombination repairs programmed DNA double-stranded breaks. Meiotic recombination physically links the homologous chromosomes ("homologs"), creating the tension between them that is required for their segregation. The central recombinase in this process is Dmc1. Dmc1's activity is regulated by its accessory factors including the heterodimeric protein Mei5-Sae3 and Rad51. We use a gain-of-function dmc1 mutant, dmc1-E157D, that bypasses Mei5-Sae3 to gain insight into the role of this accessory factor and its relationship to mitotic recombinase Rad51, which also functions as a Dmc1 accessory protein during meiosis. We find that Mei5-Sae3 has a role in filament formation and stability, but not in the bias of recombination partner choice that favors homolog over sister chromatids. Analysis of meiotic recombination intermediates suggests that Mei5-Sae3 and Rad51 function independently in promoting filament stability. In spite of its ability to load onto single-stranded DNA and carry out recombination in the absence of Mei5-Sae3, recombination promoted by the Dmc1 mutant is abnormal in that it forms foci in the absence of DNA breaks, displays unusually high levels of multi-chromatid and intersister joint molecule intermediates, as well as high levels of ectopic recombination products. We use super-resolution microscopy to show that the mutant protein forms longer foci than those formed by wild-type Dmc1. Our data support a model in which longer filaments are more prone to engage in aberrant recombination events, suggesting that filament lengths are normally limited by a regulatory mechanism that functions to prevent recombination-mediated genome rearrangements.


Assuntos
Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Rad51 Recombinase/metabolismo , Recombinases/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Substituição de Aminoácidos , Proteínas de Ciclo Celular/química , Troca Genética , Quebras de DNA de Cadeia Dupla , DNA Fúngico/genética , DNA Fúngico/metabolismo , Proteínas de Ligação a DNA/química , Mutação com Ganho de Função , Recombinação Homóloga , Meiose , Modelos Biológicos , Estabilidade Proteica , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química
9.
Zhongguo Xue Xi Chong Bing Fang Zhi Za Zhi ; 31(5): 468-473, 2019 Oct 16.
Artigo em Chinês | MEDLINE | ID: mdl-31713373

RESUMO

OBJECTIVE: To establish a recombinase aided isothermal amplification (RAA) assay for detection of Clonorchis sinensis. METHODS: The 18S ribosomal RNA (18S rRNA) sequence of C. sinensis was used as the target sequence, and specific primers and probes were designed, synthesized and screened to establish a rapid fluorescent RAA assay for the detection of C. sinensis. Then, the sensitivity of the fluorescent RAA assay was evaluated using the recombinant plasmids containing various copy numbers of DNA fragments and C. sinensis genomic DNA at various concentrations, and the specificity of the fluorescent RAA as say was evaluated using the genomic DNA of Ascaris lumbricoides, Echinococcus granulosus, Schistosoma japonicum, Ancylostoma duodenale and S. mansoni as templates. DNA samples were extracted from the feces containing C. sinensis eggs and freshwater fish containing metacercaria for the fluorescent RAA assay, and the performance for detection of C. sinensis-infected samples was preliminarily assessed in the field. RESULTS: A fluorescent RAA assay for detection of C. sinensis was successfully established, which was feasible for specific amplification of C. sinensis genomic DNA at 39 °C within 20 min. The lowest detection limit was 10 copies/µL if the recombinant plasmid containing various copy numbers of DNA fragments was used as a template, and the lowest detection limit was 3 pg/µL if the C. sinensis genomic DNA at various concentrations served as a template. All detections were negative if the genomic DNA of A. lumbricoides, E. granulosus, S. japonicum, A. duodenale, and S. mansoni was used as templates. In addition, the fluorescent RAA assay showed a high performance for the detection of C. sinensis-infected samples in the field, which successfully detected C. sinensis-infected human and rat fecal samples and Pseudorasbora parva samples. CONCLUSIONS: A fluorescent RAA assay is successfully established, which is simple, rapid, sensitivity and specific for detection of C. sinensis.


Assuntos
Clonorquíase , Clonorchis sinensis , Técnicas de Amplificação de Ácido Nucleico , Ácidos Nucleicos , Animais , Clonorquíase/diagnóstico , Clonorchis sinensis/genética , Primers do DNA , Fezes/parasitologia , Humanos , Limite de Detecção , Parasitologia/métodos , Ratos , Recombinases/metabolismo , Sensibilidade e Especificidade
10.
Nat Commun ; 10(1): 4845, 2019 10 24.
Artigo em Inglês | MEDLINE | ID: mdl-31649244

RESUMO

Site-specific DNA recombinases are important genome engineering tools. Chemical- and light-inducible recombinases, in particular, enable spatiotemporal control of gene expression. However, inducible recombinases are scarce due to the challenge of engineering high performance systems, thus constraining the sophistication of genetic circuits and animal models that can be created. Here we present a library of >20 orthogonal inducible split recombinases that can be activated by small molecules, light and temperature in mammalian cells and mice. Furthermore, we engineer inducible split Cre systems with better performance than existing systems. Using our orthogonal inducible recombinases, we create a genetic switchboard that can independently regulate the expression of 3 different cytokines in the same cell, a tripartite inducible Flp, and a 4-input AND gate. We quantitatively characterize the inducible recombinases for benchmarking their performances, including computation of distinguishability of outputs. This library expands capabilities for multiplexed mammalian gene expression control.


Assuntos
Temperatura Baixa , DNA/metabolismo , Engenharia Genética/métodos , Luz , Recombinases/genética , Animais , DNA Nucleotidiltransferases , Redes Reguladoras de Genes , Células HEK293 , Humanos , Integrases , Camundongos , Recombinases/metabolismo
11.
Zhongguo Xue Xi Chong Bing Fang Zhi Za Zhi ; 31(4): 388-392, 2019 Sep 19.
Artigo em Chinês | MEDLINE | ID: mdl-31612673

RESUMO

OBJECTIVE: To establish a recombinase-aided isothermal amplification (RAA) assay for detection of Cryptosporidium. METHODS: Based on Cryptosporidium-specific 18S rRNA selected as the target gene to be detected, and the primer sequences and fluorescent probes designed using the software Amplfix, and a fluorescent RAA assay was established and optimized. The fluorescent RAA assay was performed to detect 18S RNA target sequence-contained recombinant plasmids at various copies, genomic DNA of Cryptosporidium oocysts at various concentrations, and genomic DNA extracted from various numbers of Cryptosporidium oocysts to assess the sensitivity of the assay, and to detect genomic DNA extracted from Cryptosporidium oocysts, Giardia lamblia cysts, Schistosoma japonicum eggs, Ascaris lumbricoides eggs, Clonorchis sinensis eggs, Salmonella and Shigella to determine the specificity of the assay. RESULTS: A fluorescent RAA assay was successfully established, which was effective to amplify the specific 18S RNA gene fragments of Cryptosporidium within 20 min at 39 ℃. The lowest limits of the fluorescent RAA assay were 102 copies/µL for detection of 18S RNA target sequence-contained recombinant plasmids at various copies, 1 pg/µL for detection of genomic DNA of Cryptosporidium oocysts at various concentrations, and one Cryptosporidium oocyst/µL for detection of genomic DNA extracted from various numbers of Cryptosporidium oocysts, and the fluorescent RAA assay was all negative for detection of genomic DNA from G. lamblia cysts, S. japonicum eggs, A. lumbricoides eggs, C. sinensis eggs, Salmonella and Shigella. CONCLUSIONS: A novel fluorescent RAA assay is successfully established, which is simple, rapid, sensitive and specific to detect genomic DNA of Cryptosporidium oocysts.


Assuntos
Cryptosporidium , Técnicas de Amplificação de Ácido Nucleico/métodos , Técnicas de Amplificação de Ácido Nucleico/normas , Cryptosporidium/genética , DNA de Protozoário/genética , Limite de Detecção , Oocistos , RNA Ribossômico 18S/genética , Recombinases/metabolismo , Sensibilidade e Especificidade
12.
Mol Biol Rep ; 46(6): 6391-6397, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31549369

RESUMO

Newcastle disease virus (NDV) and infectious bronchitis virus (IBV) are two poultry pathogens affecting the respiratory tract of chickens, and cause major economic losses in the industry. Rapid detection of these viruses is crucial to inform implementation of appropriate control measures. The objective of our study is developing a simple, rapid and field applicable recombinase polymerase amplification (RPA)-nucleic acid lateral flow (NALF) immunoassay for detection of NDV and IBV. Isothermal amplification of the matrix protein (M) gene of NDV and the nucleoprotein (N) gene of IBV was implemented via recombinase polymerase amplification at 38 °C for 40 min and 20 min, respectively using modified labeled primers. NALF device used in this study utilizes antibodies for detection of labeled RPA amplicons. The results revealed that RPA-NALF immunoassays can detect both viruses after 40 min at 38 °C and only NDV after 20 min. The limit of detection (LOD) was 10 genomic copies/RPA reaction. The assays results on clinical samples collected from diseased chicken farms demonstrated a good performance in comparison with quantitative real time reverse transcription polymerase chain reaction (qRT-PCR). The assays established in this study can facilitate rapid, on-site molecular diagnosis of suspected cases of ND and IB viral infections as the results can be detected by the naked eye without the need for measuring fluorescence. Furthermore, the NALF device could be adapted to detect other infectious agents.


Assuntos
Vírus da Bronquite Infecciosa/isolamento & purificação , Vírus da Doença de Newcastle/isolamento & purificação , Doenças das Aves Domésticas/virologia , Recombinases/metabolismo , Animais , Galinhas , Imunoensaio , Vírus da Bronquite Infecciosa/genética , Limite de Detecção , Vírus da Doença de Newcastle/genética , Técnicas de Amplificação de Ácido Nucleico/métodos , Proteínas Virais/genética
13.
Proc Natl Acad Sci U S A ; 116(37): 18391-18396, 2019 09 10.
Artigo em Inglês | MEDLINE | ID: mdl-31420511

RESUMO

The circular chromosomes of bacteria can be concatenated into dimers by homologous recombination. Dimers are solved by the addition of a cross-over at a specific chromosomal site, dif, by 2 related tyrosine recombinases, XerC and XerD. Each enzyme catalyzes the exchange of a specific pair of strands. Some plasmids exploit the Xer machinery for concatemer resolution. Other mobile elements exploit it to integrate into the genome of their host. Chromosome dimer resolution is initiated by XerD. The reaction is under the control of a cell-division protein, FtsK, which activates XerD by a direct contact. Most mobile elements exploit FtsK-independent Xer recombination reactions initiated by XerC. The only notable exception is the toxin-linked cryptic satellite phage of Vibrio cholerae, TLCΦ, which integrates into and excises from the dif site of the primary chromosome of its host by a reaction initiated by XerD. However, the reaction remains independent of FtsK. Here, we show that TLCΦ carries a Xer recombination activation factor, XafT. We demonstrate in vitro that XafT activates XerD catalysis. Correspondingly, we found that XafT specifically interacts with XerD. We further show that integrative mobile elements exploiting Xer (IMEXs) encoding a XafT-like protein are widespread in gamma- and beta-proteobacteria, including human, animal, and plant pathogens.


Assuntos
Bacteriófagos/genética , Integrases/metabolismo , Recombinases/metabolismo , Recombinação Genética , Vibrio cholerae/metabolismo , Vibrio cholerae/virologia , Proteínas de Bactérias/metabolismo , Sequência de Bases , Toxina da Cólera , Cromossomos Bacterianos/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli , Integrases/genética , Proteínas de Membrana/genética , Plasmídeos , Vibrio cholerae/genética
14.
Genes Dev ; 33(17-18): 1191-1207, 2019 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-31371435

RESUMO

The vast majority of eukaryotes possess two DNA recombinases: Rad51, which is ubiquitously expressed, and Dmc1, which is meiosis-specific. The evolutionary origins of this two-recombinase system remain poorly understood. Interestingly, Dmc1 can stabilize mismatch-containing base triplets, whereas Rad51 cannot. Here, we demonstrate that this difference can be attributed to three amino acids conserved only within the Dmc1 lineage of the Rad51/RecA family. Chimeric Rad51 mutants harboring Dmc1-specific amino acids gain the ability to stabilize heteroduplex DNA joints with mismatch-containing base triplets, whereas Dmc1 mutants with Rad51-specific amino acids lose this ability. Remarkably, RAD-51 from Caenorhabditis elegans, an organism without Dmc1, has acquired "Dmc1-like" amino acids. Chimeric C. elegans RAD-51 harboring "canonical" Rad51 amino acids gives rise to toxic recombination intermediates, which must be actively dismantled to permit normal meiotic progression. We propose that Dmc1 lineage-specific amino acids involved in the stabilization of heteroduplex DNA joints with mismatch-containing base triplets may contribute to normal meiotic recombination.


Assuntos
Aminoácidos/metabolismo , Rad51 Recombinase/química , Rad51 Recombinase/metabolismo , Recombinases/química , Recombinases/metabolismo , Recombinação Genética/genética , Aminoácidos/genética , Animais , Pareamento Incorreto de Bases , Caenorhabditis elegans/enzimologia , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Sequência Conservada , Mutação , Rad51 Recombinase/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Recombinases/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
15.
Mol Cell ; 76(1): 27-43.e11, 2019 10 03.
Artigo em Inglês | MEDLINE | ID: mdl-31447390

RESUMO

Cancer cells acquire unlimited proliferative capacity by either re-expressing telomerase or inducing alternative lengthening of telomeres (ALT), which relies on telomere recombination. Here, we show that ALT recombination requires coordinate regulation of the SMX and BTR complexes to ensure the appropriate balance of resolution and dissolution activities at recombining telomeres. Critical to this control is SLX4IP, which accumulates at ALT telomeres and interacts with SLX4, XPF, and BLM. Loss of SLX4IP increases ALT-related phenotypes, which is incompatible with cell growth following concomitant loss of SLX4. Inactivation of BLM is sufficient to rescue telomere aggregation and the synthetic growth defect in this context, suggesting that SLX4IP favors SMX-dependent resolution by antagonizing promiscuous BLM activity during ALT recombination. Finally, we show that SLX4IP is inactivated in a subset of ALT-positive osteosarcomas. Collectively, our findings uncover an SLX4IP-dependent regulatory mechanism critical for telomere maintenance in ALT cancer cells.


Assuntos
Neoplasias Ósseas/enzimologia , Proteínas de Transporte/metabolismo , Osteossarcoma/enzimologia , RecQ Helicases/metabolismo , Homeostase do Telômero , Telômero/metabolismo , Animais , Neoplasias Ósseas/genética , Neoplasias Ósseas/patologia , Proteínas de Transporte/genética , Proliferação de Células , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Feminino , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Células HeLa , Humanos , Camundongos Knockout , Camundongos SCID , Osteossarcoma/genética , Osteossarcoma/patologia , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , RecQ Helicases/genética , Recombinases/genética , Recombinases/metabolismo , Transdução de Sinais , Telômero/genética , Telômero/patologia
16.
Talanta ; 205: 120155, 2019 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-31450450

RESUMO

Although isothermal nucleic acid amplification is advantageous in pathogen detection in resource-limited settings, an electricity-dependent heating module is often required. Here, we developed a wearable microfluidic device combined with recombinase polymerase amplification (RPA) for simple and rapid amplification of HIV-1 DNA using human body heat. The human body temperature at the human wrist varied from 33 to 34 °C in the ambient environment, which is sufficient to perform RPA reactions. With the aid of a cellphone-based fluorescence detection system, this device detected HIV-1 DNA quantitatively ranging from 102 to 105 copies/mL with a log linearity of 0.98 in 24 min. These results demonstrate that this wearable point-of-care (POC) nucleic acid testing method is advantageous over traditional PCR and other isothermal nucleic acid amplification methods in terms of time, portability and independence on electricity. This wearable microfluidic device in conjunction with a cellphone-based fluorescence detection system can be potentially used for the detection of HIV-1 and adapted for POC detection of a broad range of infectious pathogens in resource-limited settings.


Assuntos
DNA Viral/análise , DNA Viral/genética , HIV-1/genética , Dispositivos Lab-On-A-Chip , Técnicas de Amplificação de Ácido Nucleico/instrumentação , Recombinases/metabolismo , Dispositivos Eletrônicos Vestíveis , Temperatura Corporal , Humanos , Limite de Detecção , Fatores de Tempo
17.
BMC Vet Res ; 15(1): 294, 2019 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-31412870

RESUMO

BACKGROUND: Glaesserella parasuis (G. parasuis) is an influential pathogen of the pig, which induces high morbidity and mortality in naive pig populations in the pig industry. Accurate and rapid detection of the agent is important for disease control. In this study, a simple recombinase polymerase amplification (RPA) with a Lateral flow (LF) strip (RPA-LF-GPS) was developed to detect G. parasuis. RESULTS: The RPA-LF-GPS can specifically detect G. parasuis a limit of 100 CFU from other common related pathogens causing arthritis in the pig. The RPA-LF-GPS assay can use boiled synovial fluid samples as a template with the same sensitivity as other DNA extraction methods. In the detection of clinic positive synovial fluid sample, RPA-LF-GPS is equally sensitive (98.1%) compared with that of PCR (90.4%) (P > 0.05). The whole procedure of the RPA-LF-GPS assay could be finished in 1 hour without professional equipment. CONCLUSIONS: RPA-LF-GPS assay is a rapid and simple method for point-of-care diagnostic testing for G. parasuis infection.


Assuntos
Infecções por Haemophilus/veterinária , Haemophilus parasuis , Recombinases/metabolismo , Doenças dos Suínos/microbiologia , Líquido Sinovial/microbiologia , Animais , Infecções por Haemophilus/microbiologia , Técnicas de Amplificação de Ácido Nucleico/veterinária , Reação em Cadeia da Polimerase/veterinária , Recombinases/química , Recombinases/genética , Sensibilidade e Especificidade , Suínos , Doenças dos Suínos/diagnóstico
18.
Mol Cell Probes ; 47: 101434, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31401295

RESUMO

Brucellosis is a worldwide re-emerging zoonosis. It has an economic impact due to abortion and loss of fertility in livestock. In this study, Real-time recombinase polymerase amplification (RT-RPA-BP26) targeting Brucella spp. bp26 gene and Lateral flow dipstick (LFD-RPA-IS711) combined with SYBR- Green recombinase polymerase amplification (RPA) targeting insertion sequence IS711 region of Brucella spp. bp26 gene, was developed to detect Brucella spp. from different sample types in domestic animals. The sensitivity and specificity of the two developed RPAs were compared with real-time PCR, PCR, and Rose Bengal Plate Test (RBPT). The analytical sensitivity and detection limit of Real-time RPA and LFD RPA were four and six copies per reaction respectively. The detection of six colony forming units (CFU) of the bacteria-bearing construct with the target sequence was within 20 min at 40 °C for Real-time RPA and 37 °C for LFD RPA. The LFD RPA could work at temperatures between 30 and 35 °C and could be completed within 10-30 min. No significant differences were observed when comparing the results from Real-time RPA and LFD RPA to Real-time PCR and PCR. Both methods showed no cross reactivity with Chlamydia abortus, Toxoplasma gondii, Salmonella typhimurium, and Escherichia coli. In conclusion, RPA is a useful and convenient field and point of care test for brucellosis.


Assuntos
Brucella/isolamento & purificação , Brucelose/diagnóstico , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Recombinases/metabolismo , Animais , Proteínas de Bactérias/genética , Brucella/genética , Brucelose/veterinária , Bovinos , Feminino , Corantes Fluorescentes/química , Gado/microbiologia , Sensibilidade e Especificidade , Ovinos
19.
Methods Mol Biol ; 2021: 121-127, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31309501

RESUMO

Mannose-resistant Proteus-like (MR/P) fimbriae, produced by the uropathogen Proteus mirabilis, are major contributors to urinary tract infection. Expression of mrp genes is controlled by an invertible element in the mrp operon promoter, and promoter orientation is controlled by a single recombinase, MrpI, which determines whether the element is in the on or off orientation. Detailed here is a simple assay to determine the orientation of the invertible element in a population of P. mirabilis, a rapid screen to detect element-locked mrpI mutants, and quantification of mixtures of on and off bacteria.


Assuntos
Fímbrias Bacterianas/genética , Regiões Promotoras Genéticas , Proteus mirabilis/metabolismo , Fímbrias Bacterianas/metabolismo , Ensaios de Triagem em Larga Escala , Mutação , Óperon , Reação em Cadeia da Polimerase , Proteus mirabilis/genética , Recombinases/metabolismo
20.
Bull Exp Biol Med ; 167(1): 57-61, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31177451

RESUMO

We studied the effect of estriol, chorionic gonadotropin, oncostatin M, and hormone-cytokine combinations on the expression of recombinase RAG-1 in T-regulatory (Treg) and T helper 17 (Th17) lymphocytes. It was found that estriol in a concentration corresponding to the first trimester of pregnancy increased the level of Treg (CD4+FoxP3+) cells and suppressed the formation of Th17 (CD4+RORC+) lymphocytes. This effect was nor observed after individual administration of chorionic gonadotropin and oncostatin M, but some combinations of the studied hormones with oncostatin M increased the percentage of CD4+FOXP3+ cells. In the presence of oncostatin M, the studied hormones enhanced the expression of RAG-1 in CD4+FoxP3+ cells, but not in CD4+RORC+ cells, thereby initiating of Treg T-cell receptor (TCR) revision. The mechanisms of hormone cytokine control of induction of the immune tolerance during pregnancy are discussed.


Assuntos
Gonadotropina Coriônica/farmacologia , Estriol/farmacologia , Oncostatina M/farmacologia , Recombinases/metabolismo , Linfócitos T Reguladores/efeitos dos fármacos , Linfócitos T Reguladores/enzimologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/metabolismo , Células Cultivadas , Feminino , Fatores de Transcrição Forkhead/metabolismo , Humanos
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