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Pesquisa : G06.099.225.875 [Categoria DeCS]
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[PMID]:28249723
[Au] Autor:Nagashima Y; Koiwa H
[Ad] Endereço:Vegetable and Fruit Improvement Center, Department of Horticultural Sciences, Texas A&M University, College Station 77843-2133, TX, USA. Electronic address: nyukihiro@tamu.edu.
[Ti] Título:High throughput selection of antibiotic-resistant transgenic Arabidopsis plants.
[So] Source:Anal Biochem;525:44-45, 2017 May 15.
[Is] ISSN:1096-0309
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:Kanamycin resistance is the most frequently used antibiotic-resistance marker for Arabidopsis transformations, however, this method frequently causes escape of untransformed plants, particularly at the high seedling density during the selection. Here we developed a robust high-density selection method using top agar for Arabidopsis thaliana. Top agar effectively suppressed growth of untransformed wild-type plants on selection media at high density. Survival of the transformed plants during the selection were confirmed by production of green true leaves and expression of a firefly luciferase reporter gene. Top agar method allowed selection using a large amount of seeds in Arabidopsis transformation.
[Mh] Termos MeSH primário: Ágar/química
Antibacterianos/farmacologia
Arabidopsis/metabolismo
Engenharia Genética/métodos
Resistência a Canamicina/genética
Folhas de Planta/metabolismo
Plantas Geneticamente Modificadas/metabolismo
[Mh] Termos MeSH secundário: Ágar/metabolismo
Arabidopsis/efeitos dos fármacos
Arabidopsis/genética
Ensaios de Triagem em Larga Escala
Luciferases/metabolismo
Folhas de Planta/efeitos dos fármacos
Folhas de Planta/genética
Plantas Geneticamente Modificadas/efeitos dos fármacos
Plantas Geneticamente Modificadas/genética
Sementes/efeitos dos fármacos
Sementes/genética
Sementes/metabolismo
Transformação Genética
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (Anti-Bacterial Agents); 9002-18-0 (Agar); EC 1.13.12.- (Luciferases)
[Em] Mês de entrada:1705
[Cu] Atualização por classe:170508
[Lr] Data última revisão:
170508
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:170303
[St] Status:MEDLINE


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[PMID]:27572657
[Au] Autor:Johansson B; Fagerholm P; Petranyi G; Claesson Armitage M; Lagali N
[Ad] Endereço:Department of Ophthalmology and Department of Clinical and Experimental Medicine, Linköping University, Linköping, Sweden.
[Ti] Título:Diagnostic and therapeutic challenges in a case of amikacin-resistant Nocardia keratitis.
[So] Source:Acta Ophthalmol;95(1):103-105, 2017 Feb.
[Is] ISSN:1755-3768
[Cp] País de publicação:England
[La] Idioma:eng
[Mh] Termos MeSH primário: Amicacina/uso terapêutico
Úlcera da Córnea/diagnóstico
Infecções Oculares Bacterianas/diagnóstico
Resistência a Canamicina
Nocardiose/diagnóstico
[Mh] Termos MeSH secundário: Antibacterianos/uso terapêutico
Úlcera da Córnea/tratamento farmacológico
Úlcera da Córnea/microbiologia
Infecções Oculares Bacterianas/tratamento farmacológico
Infecções Oculares Bacterianas/microbiologia
Seres Humanos
Masculino
Testes de Sensibilidade Microbiana
Microscopia Confocal
Nocardia/isolamento & purificação
Nocardiose/tratamento farmacológico
Nocardiose/microbiologia
Soluções Oftálmicas
Sulfacetamida/uso terapêutico
Combinação Trimetoprima e Sulfametoxazol/uso terapêutico
Adulto Jovem
[Pt] Tipo de publicação:CASE REPORTS; JOURNAL ARTICLE
[Nm] Nome de substância:
0 (Anti-Bacterial Agents); 0 (Ophthalmic Solutions); 4965G3J0F5 (Sulfacetamide); 8064-90-2 (Trimethoprim, Sulfamethoxazole Drug Combination); 84319SGC3C (Amikacin)
[Em] Mês de entrada:1702
[Cu] Atualização por classe:170207
[Lr] Data última revisão:
170207
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:160831
[St] Status:MEDLINE
[do] DOI:10.1111/aos.13182


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[PMID]:27736762
[Au] Autor:Pholwat S; Stroup S; Heysell S; Ogarkov O; Zhdanova S; Ramakrishnan G; Houpt E
[Ad] Endereço:Division of Infectious Diseases and International Health, Department of Medicine, University of Virginia, Charlottesville, Virginia, USA.
[Ti] Título:eis Promoter C14G and C15G Mutations Do Not Confer Kanamycin Resistance in Mycobacterium tuberculosis.
[So] Source:Antimicrob Agents Chemother;60(12):7522-7523, 2016 Dec.
[Is] ISSN:1098-6596
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:We studied the significance of particular eis mutations on Mycobacterium tuberculosis drug resistance using a specialized transduction strategy. Recombinant strains harboring eis promoter mutations C14T, C12T, and G10A exhibited kanamycin resistance with MICs of 40, 10, and 20 µg/ml, respectively, while recombinant strains harboring C14G and C15G mutations were kanamycin susceptible (MIC, 2.5 to 5 µg/ml). Each of the eis mutants tested remained amikacin susceptible (MIC, 0.5 to 4 µg/ml). The identification of specific eis mutations is needed for accurate genotypic susceptibility testing for kanamycin.
[Mh] Termos MeSH primário: Antígenos de Bactérias/genética
Antituberculosos/farmacologia
Proteínas de Bactérias/genética
Canamicina/farmacologia
Mutação
Mycobacterium tuberculosis/efeitos dos fármacos
[Mh] Termos MeSH secundário: Acetiltransferases
Amicacina/farmacologia
Antígenos de Bactérias/metabolismo
Proteínas de Bactérias/metabolismo
Expressão Gênica
Genótipo
Resistência a Canamicina/genética
Mycobacterium tuberculosis/genética
Mycobacterium tuberculosis/crescimento & desenvolvimento
Regiões Promotoras Genéticas
Transdução Genética
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (Antigens, Bacterial); 0 (Antitubercular Agents); 0 (Bacterial Proteins); 59-01-8 (Kanamycin); 84319SGC3C (Amikacin); EC 2.3.1.- (Acetyltransferases); EC 2.3.1.- (Eis protein, Mycobacterium tuberculosis)
[Em] Mês de entrada:1708
[Cu] Atualização por classe:171116
[Lr] Data última revisão:
171116
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:161014
[St] Status:MEDLINE


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[PMID]:27655632
[Au] Autor:Béguin P; Charpin N; Koonin EV; Forterre P; Krupovic M
[Ad] Endereço:Unité Biologie Moléculaire du Gène chez les Extrêmophiles, Institut Pasteur, 25 rue du Docteur Roux, 75015 Paris pierre.beguin@pasteur.fr.
[Ti] Título:Casposon integration shows strong target site preference and recapitulates protospacer integration by CRISPR-Cas systems.
[So] Source:Nucleic Acids Res;44(21):10367-10376, 2016 Dec 01.
[Is] ISSN:1362-4962
[Cp] País de publicação:England
[La] Idioma:eng
[Ab] Resumo:Casposons are a recently discovered group of large DNA transposons present in diverse bacterial and archaeal genomes. For integration into the host chromosome, casposons employ an endonuclease that is homologous to the Cas1 protein involved in protospacer integration by the CRISPR-Cas adaptive immune system. Here we describe the site-preference of integration by the Cas1 integrase (casposase) encoded by the casposon of the archaeon Aciduliprofundum boonei Oligonucleotide duplexes derived from the terminal inverted repeats (TIR) of the A. boonei casposon as well as mini-casposons flanked by the TIR inserted preferentially at a site reconstituting the original A. boonei target site. As in the A. boonei genome, the insertion was accompanied by a 15-bp direct target site duplication (TSD). The minimal functional target consisted of the 15-bp TSD segment and the adjacent 18-bp sequence which comprises the 3' end of the tRNA-Pro gene corresponding to the TΨC loop. The functional casposase target site bears clear resemblance to the leader sequence-repeat junction which is the target for protospacer integration catalyzed by the Cas1-Cas2 adaptation module of CRISPR-Cas. These findings reinforce the mechanistic similarities and evolutionary connection between the casposons and the adaptation module of the prokaryotic adaptive immunity systems.
[Mh] Termos MeSH primário: Sítios de Ligação
Sistemas CRISPR-Cas
Elementos de DNA Transponíveis/genética
Endodesoxirribonucleases/metabolismo
[Mh] Termos MeSH secundário: Ordem dos Genes
Resistência a Canamicina/genética
Mutagênese Insercional
Conformação de Ácido Nucleico
Plasmídeos/genética
Sequências Repetidas Terminais
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (DNA Transposable Elements); EC 3.1.- (Endodeoxyribonucleases)
[Em] Mês de entrada:1705
[Cu] Atualização por classe:170922
[Lr] Data última revisão:
170922
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:160923
[St] Status:MEDLINE


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[PMID]:27595984
[Au] Autor:Tikhe CV; Martin TM; Howells A; Delatte J; Husseneder C
[Ad] Endereço:Department of Entomology, Louisiana State University Agricultural Center, Baton Rouge, LA, USA. ctikhe1@lsu.edu.
[Ti] Título:Assessment of genetically engineered Trabulsiella odontotermitis as a 'Trojan Horse' for paratransgenesis in termites.
[So] Source:BMC Microbiol;16(1):202, 2016 Sep 05.
[Is] ISSN:1471-2180
[Cp] País de publicação:England
[La] Idioma:eng
[Ab] Resumo:BACKGROUND: The Formosan subterranean termite, Coptotermes formosanus is an invasive urban pest in the Southeastern USA. Paratransgenesis using a microbe expressed lytic peptide that targets the termite gut protozoa is currently being developed for the control of Formosan subterranean termites. In this study, we evaluated Trabulsiella odontotermitis, a termite-specific bacterium, for its potential to serve as a 'Trojan Horse' for expression of gene products in termite colonies. RESULTS: We engineered two strains of T. odontotermitis, one transformed with a constitutively expressed GFP plasmid and the other engineered at the chromosome with a Kanamycin resistant gene using a non- disruptive Tn7 transposon. Both strains were fed to termites from three different colonies. Fluorescent microscopy confirmed that T. odontotermitis expressed GFP in the gut and formed a biofilm in the termite hindgut. However, GFP producing bacteria could not be isolated from the termite gut after 2 weeks. The feeding experiment with the chromosomally engineered strain demonstrated that T. odontotermitis was maintained in the termite gut for at least 21 days, irrespective of the termite colony. The bacteria persisted in two termite colonies for at least 36 days post feeding. The experiment also confirmed the horizontal transfer of T. odontotermitis amongst nest mates. CONCLUSION: Overall, we conclude that T. odontotermitis can serve as a 'Trojan Horse' for spreading gene products in termite colonies. This study provided proof of concept and laid the foundation for the future development of genetically engineered termite gut bacteria for paratransgenesis based termite control.
[Mh] Termos MeSH primário: Enterobacteriaceae/genética
Técnicas de Transferência de Genes
Engenharia Genética/métodos
Isópteros/microbiologia
[Mh] Termos MeSH secundário: Animais
Biofilmes/crescimento & desenvolvimento
Elementos de DNA Transponíveis
Sistema Digestório/microbiologia
Sistema Digestório/patologia
Enterobacteriaceae/metabolismo
Enterobacteriaceae/fisiologia
Microbioma Gastrointestinal
Genes Bacterianos
Canamicina/farmacologia
Resistência a Canamicina/genética
Controle Biológico de Vetores/métodos
Recombinação Genética
Transformação Bacteriana
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (DNA Transposable Elements); 59-01-8 (Kanamycin)
[Em] Mês de entrada:1706
[Cu] Atualização por classe:170601
[Lr] Data última revisão:
170601
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:160907
[St] Status:MEDLINE
[do] DOI:10.1186/s12866-016-0822-4


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[PMID]:27576542
[Au] Autor:Georghiou SB; Seifert M; Lin SY; Catanzaro D; Garfein RS; Jackson RL; Crudu V; Rodrigues C; Victor TC; Catanzaro A; Rodwell TC
[Ad] Endereço:Department of Medicine, University of California San Diego, La Jolla, CA, USA. sgeorghi@ucsd.edu.
[Ti] Título:Shedding light on the performance of a pyrosequencing assay for drug-resistant tuberculosis diagnosis.
[So] Source:BMC Infect Dis;16:458, 2016 Aug 31.
[Is] ISSN:1471-2334
[Cp] País de publicação:England
[La] Idioma:eng
[Ab] Resumo:BACKGROUND: Rapid molecular diagnostics, with their ability to quickly identify genetic mutations associated with drug resistance in Mycobacterium tuberculosis clinical specimens, have great potential as tools to control multi- and extensively drug-resistant tuberculosis (M/XDR-TB). The Qiagen PyroMark Q96 ID system is a commercially available pyrosequencing (PSQ) platform that has been validated for rapid M/XDR-TB diagnosis. However, the details of the assay's diagnostic and technical performance have yet to be thoroughly investigated in diverse clinical environments. METHODS: This study evaluates the diagnostic performance of the PSQ assay for 1128 clinical specimens from patients from three areas of high TB burden. We report on the diagnostic performance of the PSQ assay between the three sites and identify variables associated with poor PSQ technical performance. RESULTS: In India, the sensitivity of the PSQ assay ranged from 89 to 98 % for the detection of phenotypic resistance to isoniazid, rifampicin, fluoroquinolones, and the injectables. In Moldova, assay sensitivity ranged from 7 to 94 %, and in South Africa, assay sensitivity ranged from 71 to 92 %. Specificity was high (94-100 %) across all sites. The addition of eis promoter sequencing information greatly improved the sensitivity of kanamycin resistance detection in Moldova (7 % to 79 %). Nearly all (89.4 %) sequencing reactions conducted on smear-positive, culture-positive specimens and most (70.8 %) reactions conducted on smear-negative, culture-positive specimens yielded valid PSQ reads. An investigation into the variables influencing sequencing failures indicated smear negativity, culture negativity, site (Moldova), and sequencing of the rpoB, gyrA, and rrs genes were highly associated with poor PSQ technical performance (adj. OR > 2.0). CONCLUSIONS: This study has important implications for the global implementation of PSQ as a molecular TB diagnostic, as it demonstrates how regional factors may impact PSQ diagnostic performance, while underscoring potential gene targets for optimization to improve overall PSQ assay technical performance. TRIAL REGISTRATION: ClinicalTrials.gov ( #NCT02170441 ). Registered 12 June 2014.
[Mh] Termos MeSH primário: Antituberculosos/farmacologia
Tuberculose Extensivamente Resistente a Medicamentos/diagnóstico
Mycobacterium tuberculosis/genética
[Mh] Termos MeSH secundário: Antituberculosos/uso terapêutico
Tuberculose Extensivamente Resistente a Medicamentos/tratamento farmacológico
Tuberculose Extensivamente Resistente a Medicamentos/microbiologia
Fluoroquinolonas
Genes Bacterianos
Seres Humanos
Isoniazida/farmacologia
Canamicina/farmacologia
Resistência a Canamicina/genética
Testes de Sensibilidade Microbiana
Técnicas de Diagnóstico Molecular
Tipagem Molecular
Mutação
Mycobacterium tuberculosis/efeitos dos fármacos
Regiões Promotoras Genéticas
Rifampina/farmacologia
Sensibilidade e Especificidade
Análise de Sequência de DNA
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (Antitubercular Agents); 0 (Fluoroquinolones); 59-01-8 (Kanamycin); V83O1VOZ8L (Isoniazid); VJT6J7R4TR (Rifampin)
[Em] Mês de entrada:1706
[Cu] Atualização por classe:170613
[Lr] Data última revisão:
170613
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:160901
[Cl] Clinical Trial:ClinicalTrial
[St] Status:MEDLINE
[do] DOI:10.1186/s12879-016-1781-y


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[PMID]:27208106
[Au] Autor:Lipscomb GL; Conway JM; Blumer-Schuette SE; Kelly RM; Adams MW
[Ad] Endereço:Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia USA.
[Ti] Título:A Highly Thermostable Kanamycin Resistance Marker Expands the Tool Kit for Genetic Manipulation of Caldicellulosiruptor bescii.
[So] Source:Appl Environ Microbiol;82(14):4421-8, 2016 Jul 15.
[Is] ISSN:1098-5336
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:UNLABELLED: Caldicellulosiruptor bescii, an anaerobic Gram-positive bacterium with an optimal growth temperature of 78°C, is the most thermophilic cellulose degrader known. It is of great biotechnological interest, as it efficiently deconstructs nonpretreated lignocellulosic plant biomass. Currently, its genetic manipulation relies on a mutant uracil auxotrophic background strain that contains a random deletion in the pyrF genome region. The pyrF gene serves as a genetic marker to select for uracil prototrophy, and it can also be counterselected for loss via resistance to the compound 5-fluoroorotic acid (5-FOA). To expand the C. bescii genetic tool kit, kanamycin resistance was developed as a selection for genetic manipulation. A codon-optimized version of the highly thermostable kanamycin resistance gene (named Cbhtk) allowed the use of kanamycin selection to obtain transformants of either replicating or integrating vector constructs in C. bescii These strains showed resistance to kanamycin at concentrations >50 µg · ml(-1), whereas wild-type C. bescii was sensitive to kanamycin at 10 µg · ml(-1) In addition, placement of the Cbhtk marker between homologous recombination regions in an integrating vector allowed direct selection of a chromosomal mutation using both kanamycin and 5-FOA. Furthermore, the use of kanamycin selection enabled the targeted deletion of the pyrE gene in wild-type C. bescii, generating a uracil auxotrophic genetic background strain resistant to 5-FOA. The pyrE gene functioned as a counterselectable marker, like pyrF, and was used together with Cbhtk in the ΔpyrE background strain to delete genes encoding lactate dehydrogenase and the CbeI restriction enzyme. IMPORTANCE: Caldicellulosiruptor bescii is a thermophilic anaerobic bacterium with an optimal growth temperature of 78°C, and it has the ability to efficiently deconstruct nonpretreated lignocellulosic plant biomass. It is, therefore, of biotechnological interest for genetic engineering applications geared toward biofuel production. The current genetic system used with C. bescii is based upon only a single selection strategy, and this uses the gene involved in a primary biosynthetic pathway. There are many advantages with an additional genetic selection using an antibiotic. This presents a challenge for thermophilic microorganisms, as only a limited number of antibiotics are stable above 50°C, and a thermostable version of the enzyme conferring antibiotic resistance must be obtained. In this work, we have developed a selection system for C. bescii using the antibiotic kanamycin and have shown that, in combination with the biosynthetic gene marker, it can be used to efficiently delete genes in this organism.
[Mh] Termos MeSH primário: Firmicutes/genética
Firmicutes/efeitos da radiação
Instabilidade Genômica/efeitos da radiação
Temperatura Alta
Resistência a Canamicina
Biologia Molecular/métodos
Seleção Genética
[Mh] Termos MeSH secundário: Genética Microbiana/métodos
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Em] Mês de entrada:1710
[Cu] Atualização por classe:171002
[Lr] Data última revisão:
171002
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:160522
[St] Status:MEDLINE
[do] DOI:10.1128/AEM.00570-16


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[PMID]:27010218
[Au] Autor:Willby MJ; Green KD; Gajadeera CS; Hou C; Tsodikov OV; Posey JE; Garneau-Tsodikova S
[Ad] Endereço:Division of Tuberculosis Elimination, National Center for HIV/AIDS, Viral Hepatitis, STD, and TB Prevention, Centers for Disease Control and Prevention , Atlanta, Georgia 30329, United States.
[Ti] Título:Potent Inhibitors of Acetyltransferase Eis Overcome Kanamycin Resistance in Mycobacterium tuberculosis.
[So] Source:ACS Chem Biol;11(6):1639-46, 2016 06 17.
[Is] ISSN:1554-8937
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:A major cause of tuberculosis (TB) resistance to the aminoglycoside kanamycin (KAN) is the Mycobacterium tuberculosis (Mtb) acetyltransferase Eis. Upregulation of this enzyme is responsible for inactivation of KAN through acetylation of its amino groups. A 123 000-compound high-throughput screen (HTS) yielded several small-molecule Eis inhibitors that share an isothiazole S,S-dioxide heterocyclic core. These were investigated for their structure-activity relationships. Crystal structures of Eis in complex with two potent inhibitors show that these molecules are bound in the conformationally adaptable aminoglycoside binding site of the enzyme, thereby obstructing binding of KAN for acetylation. Importantly, we demonstrate that several Eis inhibitors, when used in combination with KAN against resistant Mtb, efficiently overcome KAN resistance. This approach paves the way toward development of novel combination therapies against aminoglycoside-resistant TB.
[Mh] Termos MeSH primário: Acetiltransferases/antagonistas & inibidores
Antituberculosos/farmacologia
Proteínas de Bactérias/antagonistas & inibidores
Óxidos S-Cíclicos/farmacologia
Resistência a Canamicina/efeitos dos fármacos
Mycobacterium tuberculosis/efeitos dos fármacos
Tiazóis/farmacologia
[Mh] Termos MeSH secundário: Antituberculosos/química
Cristalografia por Raios X
Óxidos S-Cíclicos/química
Desenho de Drogas
Ensaios de Triagem em Larga Escala
Canamicina/metabolismo
Canamicina/farmacologia
Mycobacterium tuberculosis/enzimologia
Staphylococcus aureus/efeitos dos fármacos
Relação Estrutura-Atividade
Tiazóis/química
[Pt] Tipo de publicação:JOURNAL ARTICLE; RESEARCH SUPPORT, N.I.H., EXTRAMURAL; RESEARCH SUPPORT, NON-U.S. GOV'T
[Nm] Nome de substância:
0 (Antitubercular Agents); 0 (Bacterial Proteins); 0 (Cyclic S-Oxides); 0 (Thiazoles); 59-01-8 (Kanamycin); EC 2.3.1.- (Acetyltransferases); EC 2.3.1.- (Eis protein, Mycobacterium tuberculosis)
[Em] Mês de entrada:1709
[Cu] Atualização por classe:170919
[Lr] Data última revisão:
170919
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:160325
[St] Status:MEDLINE
[do] DOI:10.1021/acschembio.6b00110


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[PMID]:26426390
[Au] Autor:Schneider K; Schiermeyer A; Dolls A; Koch N; Herwartz D; Kirchhoff J; Fischer R; Russell SM; Cao Z; Corbin DR; Sastry-Dent L; Ainley WM; Webb SR; Schinkel H; Schillberg S
[Ad] Endereço:Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Aachen, Germany.
[Ti] Título:Targeted gene exchange in plant cells mediated by a zinc finger nuclease double cut.
[So] Source:Plant Biotechnol J;14(4):1151-60, 2016 Apr.
[Is] ISSN:1467-7652
[Cp] País de publicação:England
[La] Idioma:eng
[Ab] Resumo:Genome modification by homology-directed repair (HDR) is an attractive tool for the controlled genetic manipulation of plants. Here, we report the HDR-mediated gene exchange of expression cassettes in tobacco BY-2 cells using a designed zinc finger nuclease (ZFN). The target contained a 7-kb fragment flanked by two ZFN cutting sites. That fragment was replaced with a 4-kb donor cassette, which integrates gene markers for selection (kanamycin resistance) and for scoring targeting (red fluorescent protein, RFP). Candidates resulting from cassette exchange were identified by molecular analysis of calli generated by transformation via direct DNA delivery. The precision of HDR-mediated donor integration was evaluated by Southern blot analysis, sequencing of the integration locus and analysis of RFP fluorescence by flow cytometry. Screening of 1326 kanamycin-resistant calli yielded 18 HDR events, 16 of which had a perfect cassette exchange at the insert junction and 13 of which produced functional RFP. Our results demonstrate that ZFN-based HDR can be used for high frequency, precise, targeted exchange of fragments of sizes that are commercially relevant in plants.
[Mh] Termos MeSH primário: Desoxirribonucleases/metabolismo
Marcação de Genes/métodos
Tabaco/genética
[Mh] Termos MeSH secundário: Southern Blotting
Desoxirribonucleases/genética
Citometria de Fluxo/métodos
Resistência a Canamicina/genética
Proteínas Luminescentes/genética
Proteínas Luminescentes/metabolismo
Células Vegetais
Plantas Geneticamente Modificadas
Reparo de DNA por Recombinação/genética
Tabaco/citologia
Dedos de Zinco
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (Luminescent Proteins); 0 (red fluorescent protein); EC 3.1.- (Deoxyribonucleases)
[Em] Mês de entrada:1612
[Cu] Atualização por classe:161230
[Lr] Data última revisão:
161230
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:151002
[St] Status:MEDLINE
[do] DOI:10.1111/pbi.12483


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[PMID]:26646012
[Au] Autor:Harmer CJ; Hall RM
[Ad] Endereço:School of Molecular Bioscience, The University of Sydney, Sydney, Australia.
[Ti] Título:IS26-Mediated Precise Excision of the IS26-aphA1a Translocatable Unit.
[So] Source:MBio;6(6):e01866-15, 2015 Dec 08.
[Is] ISSN:2150-7511
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:UNLABELLED: We recently showed that, in the absence of RecA-dependent homologous recombination, the Tnp26 transposase catalyzes cointegrate formation via a conservative reaction between two preexisting IS26, and this is strongly preferred over replicative transposition to a new site. Here, the reverse reaction was investigated by assaying for precise excision of the central region together with a single IS26 from a compound transposon bounded by IS26. In a recA mutant strain, Tn4352, a kanamycin resistance transposon carrying the aphA1a gene, was stable. However, loss of kanamycin resistance due to precise excision of the translocatable unit (TU) from the closely related Tn4352B, leaving behind the second IS26, occurred at high frequency. Excision occurred when Tn4352B was in either a high- or low-copy-number plasmid. The excised circular segment, known as a TU, was detected by PCR. Excision required the IS26 transposase Tnp26. However, the Tnp26 of only one IS26 in Tn4352B was required, specifically the IS26 downstream of the aphA1a gene, and the excised TU included the active IS26. The frequency of Tn4352B TU loss was influenced by the context of the transposon, but the critical determinant of high-frequency excision was the presence of three G residues in Tn4352B replacing a single G in Tn4352. These G residues are located immediately adjacent to the two G residues at the left end of the IS26 that is upstream of the aphA1a gene. Transcription of tnp26 was not affected by the additional G residues, which appear to enhance Tnp26 cleavage at this end. IMPORTANCE: Resistance to antibiotics limits treatment options. In Gram-negative bacteria, IS26 plays a major role in the acquisition and dissemination of antibiotic resistance. IS257 (IS431) and IS1216, which belong to the same insertion sequence (IS) family, mobilize resistance genes in staphylococci and enterococci, respectively. Many different resistance genes are found in compound transposons bounded by IS26, and multiply and extensively antibiotic-resistant Gram-negative bacteria often include regions containing several antibiotic resistance genes and multiple copies of IS26. We recently showed that in addition to replicative transposition, IS26 can use a conservative movement mechanism in which an incoming IS26 targets a preexisting one, and this reaction can create these regions. This mechanism differs from that of all the ISs examined in detail thus far. Here, we have continued to extend understanding of the reactions carried out by IS26 by examining whether the reverse precise excision reaction is also catalyzed by the IS26 transposase.
[Mh] Termos MeSH primário: Elementos de DNA Transponíveis
Recombinação Genética
[Mh] Termos MeSH secundário: DNA Bacteriano/química
DNA Bacteriano/genética
Escherichia coli/efeitos dos fármacos
Escherichia coli/genética
Resistência a Canamicina
Dados de Sequência Molecular
Plasmídeos
Análise de Sequência de DNA
Transposases/metabolismo
[Pt] Tipo de publicação:JOURNAL ARTICLE; RESEARCH SUPPORT, NON-U.S. GOV'T
[Nm] Nome de substância:
0 (DNA Transposable Elements); 0 (DNA, Bacterial); EC 2.7.7.- (Transposases)
[Em] Mês de entrada:1608
[Cu] Atualização por classe:151209
[Lr] Data última revisão:
151209
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:151210
[St] Status:MEDLINE



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