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[PMID]:28746784
[Au] Autor:Fuentes-Pardo AP; Ruzzante DE
[Ad] Endereço:Department of Biology, Dalhousie University, Halifax, NS, Canada.
[Ti] Título:Whole-genome sequencing approaches for conservation biology: Advantages, limitations and practical recommendations.
[So] Source:Mol Ecol;26(20):5369-5406, 2017 Oct.
[Is] ISSN:1365-294X
[Cp] País de publicação:England
[La] Idioma:eng
[Ab] Resumo:Whole-genome resequencing (WGR) is a powerful method for addressing fundamental evolutionary biology questions that have not been fully resolved using traditional methods. WGR includes four approaches: the sequencing of individuals to a high depth of coverage with either unresolved or resolved haplotypes, the sequencing of population genomes to a high depth by mixing equimolar amounts of unlabelled-individual DNA (Pool-seq) and the sequencing of multiple individuals from a population to a low depth (lcWGR). These techniques require the availability of a reference genome. This, along with the still high cost of shotgun sequencing and the large demand for computing resources and storage, has limited their implementation in nonmodel species with scarce genomic resources and in fields such as conservation biology. Our goal here is to describe the various WGR methods, their pros and cons and potential applications in conservation biology. WGR offers an unprecedented marker density and surveys a wide diversity of genetic variations not limited to single nucleotide polymorphisms (e.g., structural variants and mutations in regulatory elements), increasing their power for the detection of signatures of selection and local adaptation as well as for the identification of the genetic basis of phenotypic traits and diseases. Currently, though, no single WGR approach fulfils all requirements of conservation genetics, and each method has its own limitations and sources of potential bias. We discuss proposed ways to minimize such biases. We envision a not distant future where the analysis of whole genomes becomes a routine task in many nonmodel species and fields including conservation biology.
[Mh] Termos MeSH primário: Conservação dos Recursos Naturais/métodos
Genética Populacional
Genômica/métodos
[Mh] Termos MeSH secundário: Evolução Biológica
Frequência do Gene
Biblioteca Genômica
Genótipo
Haplótipos
Sequenciamento de Nucleotídeos em Larga Escala
Fenótipo
Polimorfismo de Nucleotídeo Único
Densidade Demográfica
Análise de Sequência de DNA/métodos
[Pt] Tipo de publicação:JOURNAL ARTICLE; REVIEW
[Em] Mês de entrada:1802
[Cu] Atualização por classe:180212
[Lr] Data última revisão:
180212
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:170727
[St] Status:MEDLINE
[do] DOI:10.1111/mec.14264


  2 / 5314 MEDLINE  
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[PMID]:28985510
[Au] Autor:Montalbano A; Canver MC; Sanjana NE
[Ad] Endereço:New York Genome Center, New York, NY, USA; Department of Biology, New York University, New York, NY, USA.
[Ti] Título:High-Throughput Approaches to Pinpoint Function within the Noncoding Genome.
[So] Source:Mol Cell;68(1):44-59, 2017 Oct 05.
[Is] ISSN:1097-4164
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas nuclease system is a powerful tool for genome editing, and its simple programmability has enabled high-throughput genetic and epigenetic studies. These high-throughput approaches offer investigators a toolkit for functional interrogation of not only protein-coding genes but also noncoding DNA. Historically, noncoding DNA has lacked the detailed characterization that has been applied to protein-coding genes in large part because there has not been a robust set of methodologies for perturbing these regions. Although the majority of high-throughput CRISPR screens have focused on the coding genome to date, an increasing number of CRISPR screens targeting noncoding genomic regions continue to emerge. Here, we review high-throughput CRISPR-based approaches to uncover and understand functional elements within the noncoding genome and discuss practical aspects of noncoding library design and screen analysis.
[Mh] Termos MeSH primário: Sistemas CRISPR-Cas
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas
DNA Intergênico/genética
Endonucleases/genética
Edição de Genes/métodos
Genoma
[Mh] Termos MeSH secundário: Animais
DNA Intergênico/metabolismo
Endonucleases/metabolismo
Células Eucarióticas/citologia
Células Eucarióticas/metabolismo
Engenharia Genética
Biblioteca Genômica
Ensaios de Triagem em Larga Escala
Seres Humanos
RNA Guia/genética
RNA Guia/metabolismo
[Pt] Tipo de publicação:JOURNAL ARTICLE; REVIEW
[Nm] Nome de substância:
0 (DNA, Intergenic); 0 (RNA, Guide); EC 3.1.- (Endonucleases)
[Em] Mês de entrada:1710
[Cu] Atualização por classe:171126
[Lr] Data última revisão:
171126
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:171007
[St] Status:MEDLINE


  3 / 5314 MEDLINE  
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[PMID]:28906493
[Au] Autor:Anzai IA; Shaket L; Adesina O; Baym M; Barstow B
[Ad] Endereço:Department of Chemistry, Princeton University, Princeton, New Jersey, USA.
[Ti] Título:Rapid curation of gene disruption collections using Knockout Sudoku.
[So] Source:Nat Protoc;12(10):2110-2137, 2017 Oct.
[Is] ISSN:1750-2799
[Cp] País de publicação:England
[La] Idioma:eng
[Ab] Resumo:Knockout Sudoku is a method for the construction of whole-genome knockout collections for a wide range of microorganisms with as little as 3 weeks of dedicated labor and at a cost of ∼$10,000 for a collection for a single organism. The method uses manual 4D combinatorial pooling, next-generation sequencing, and a Bayesian inference algorithm to rapidly process and then accurately annotate the extremely large progenitor transposon insertion mutant collections needed to achieve saturating coverage of complex microbial genomes. This method is ∼100× faster and 30× lower in cost than the next comparable method (In-seq) for annotating transposon mutant collections by combinatorial pooling and next-generation sequencing. This method facilitates the rapid, algorithmically guided condensation and curation of the progenitor collection into a high-quality, nonredundant collection that is suitable for rapid genetic screening and gene discovery.
[Mh] Termos MeSH primário: Técnicas de Inativação de Genes/métodos
Biblioteca Genômica
Genômica/métodos
Sequenciamento de Nucleotídeos em Larga Escala/métodos
[Mh] Termos MeSH secundário: Algoritmos
Bactérias/genética
Teorema de Bayes
Mutagênese Insercional
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Em] Mês de entrada:1709
[Cu] Atualização por classe:170925
[Lr] Data última revisão:
170925
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:170915
[St] Status:MEDLINE
[do] DOI:10.1038/nprot.2017.073


  4 / 5314 MEDLINE  
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[PMID]:28749982
[Au] Autor:Yoshida Y; Koutsovoulos G; Laetsch DR; Stevens L; Kumar S; Horikawa DD; Ishino K; Komine S; Kunieda T; Tomita M; Blaxter M; Arakawa K
[Ad] Endereço:Institute for Advanced Biosciences, Keio University, Tsuruoka, Yamagata, Japan.
[Ti] Título:Comparative genomics of the tardigrades Hypsibius dujardini and Ramazzottius varieornatus.
[So] Source:PLoS Biol;15(7):e2002266, 2017 Jul.
[Is] ISSN:1545-7885
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:Tardigrada, a phylum of meiofaunal organisms, have been at the center of discussions of the evolution of Metazoa, the biology of survival in extreme environments, and the role of horizontal gene transfer in animal evolution. Tardigrada are placed as sisters to Arthropoda and Onychophora (velvet worms) in the superphylum Panarthropoda by morphological analyses, but many molecular phylogenies fail to recover this relationship. This tension between molecular and morphological understanding may be very revealing of the mode and patterns of evolution of major groups. Limnoterrestrial tardigrades display extreme cryptobiotic abilities, including anhydrobiosis and cryobiosis, as do bdelloid rotifers, nematodes, and other animals of the water film. These extremophile behaviors challenge understanding of normal, aqueous physiology: how does a multicellular organism avoid lethal cellular collapse in the absence of liquid water? Meiofaunal species have been reported to have elevated levels of horizontal gene transfer (HGT) events, but how important this is in evolution, and particularly in the evolution of extremophile physiology, is unclear. To address these questions, we resequenced and reassembled the genome of H. dujardini, a limnoterrestrial tardigrade that can undergo anhydrobiosis only after extensive pre-exposure to drying conditions, and compared it to the genome of R. varieornatus, a related species with tolerance to rapid desiccation. The 2 species had contrasting gene expression responses to anhydrobiosis, with major transcriptional change in H. dujardini but limited regulation in R. varieornatus. We identified few horizontally transferred genes, but some of these were shown to be involved in entry into anhydrobiosis. Whole-genome molecular phylogenies supported a Tardigrada+Nematoda relationship over Tardigrada+Arthropoda, but rare genomic changes tended to support Tardigrada+Arthropoda.
[Mh] Termos MeSH primário: Extremófilos/genética
Regulação da Expressão Gênica
Proteoma/metabolismo
Tardígrados/genética
[Mh] Termos MeSH secundário: Animais
Sequência de Bases
Mapeamento Cromossômico/veterinária
DNA/química
DNA/metabolismo
Dessecação
Extremófilos/crescimento & desenvolvimento
Extremófilos/fisiologia
Perfilação da Expressão Gênica/veterinária
Transferência Genética Horizontal
Ligação Genética
Tamanho do Genoma
Estudo de Associação Genômica Ampla/veterinária
Biblioteca Genômica
Sequenciamento de Nucleotídeos em Larga Escala/veterinária
Família Multigênica
Filogenia
Proteoma/genética
Reprodutibilidade dos Testes
Especificidade da Espécie
Tardígrados/crescimento & desenvolvimento
Tardígrados/fisiologia
[Pt] Tipo de publicação:COMPARATIVE STUDY; JOURNAL ARTICLE; VALIDATION STUDIES
[Nm] Nome de substância:
0 (Proteome); 9007-49-2 (DNA)
[Em] Mês de entrada:1708
[Cu] Atualização por classe:170829
[Lr] Data última revisão:
170829
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:170728
[St] Status:MEDLINE
[do] DOI:10.1371/journal.pbio.2002266


  5 / 5314 MEDLINE  
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[PMID]:28674067
[Au] Autor:Chan CH; Levar CE; Jiménez-Otero F; Bond DR
[Ad] Endereço:BioTechnology Institute, University of Minnesota-Twin Cities, St. Paul, Minnesota, USA.
[Ti] Título:Genome Scale Mutational Analysis of Geobacter sulfurreducens Reveals Distinct Molecular Mechanisms for Respiration and Sensing of Poised Electrodes versus Fe(III) Oxides.
[So] Source:J Bacteriol;199(19), 2017 Oct 01.
[Is] ISSN:1098-5530
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:generates electrical current by coupling intracellular oxidation of organic acids to the reduction of proteins on the cell surface that are able to interface with electrodes. This ability is attributed to the bacterium's capacity to respire other extracellular electron acceptors that require contact, such as insoluble metal oxides. To directly investigate the genetic basis of electrode-based respiration, we constructed transposon-insertion sequencing (Tn-Seq) libraries for growth, with soluble fumarate or an electrode as the electron acceptor. Libraries with >33,000 unique insertions and an average of 9 insertions/kb allowed an assessment of each gene's fitness in a single experiment. Mutations in 1,214 different genomic features impaired growth with fumarate, and the significance of 270 genes unresolved by annotation due to the presence of one or more functional homologs was determined. Tn-Seq analysis of -0.1 V versus standard hydrogen electrode (SHE) electrode-grown cells identified mutations in a subset of genes encoding cytochromes, processing systems for proline-rich proteins, sensory networks, extracellular structures, polysaccharides, and metabolic enzymes that caused at least a 50% reduction in apparent growth rate. Scarless deletion mutants of select genes identified via Tn-Seq revealed a new putative porin-cytochrome conduit complex ( ) crucial for growth with electrodes, which was not required for Fe(III) oxide reduction. In addition, four mutants lacking components of a putative methyl-accepting chemotaxis-cyclic dinucleotide sensing network ( ) were defective in electrode colonization but grew normally with Fe(III) oxides. These results suggest that possesses distinct mechanisms for recognition, colonization, and reduction of electrodes compared to Fe(III) oxides. Since metal oxide electron acceptors are insoluble, one hypothesis is that cells sense and reduce metals using the same molecular mechanisms used to form biofilms on electrodes and produce electricity. However, by simultaneously comparing thousands of transposon mutants undergoing electrode-dependent respiration, we discovered new cytochromes and chemosensory proteins supporting growth with electrodes that are not required for metal respiration. This supports an emerging model where recognizes surfaces and forms conductive biofilms using mechanisms distinct from those used for growth with metal oxides. These findings provide a possible explanation for studies that correlate electricity generation with syntrophic interspecies electron transfer by and reveal many previously unrecognized targets for engineering this useful capability in other organisms.
[Mh] Termos MeSH primário: Compostos Férricos/metabolismo
Genoma Bacteriano
Geobacter/genética
Geobacter/metabolismo
Mutação
[Mh] Termos MeSH secundário: Proteínas de Bactérias/metabolismo
Biofilmes/crescimento & desenvolvimento
Elementos de DNA Transponíveis
Eletrodos
Transporte de Elétrons
Fumaratos/metabolismo
Fumaratos/farmacologia
Biblioteca Genômica
Geobacter/efeitos dos fármacos
Geobacter/crescimento & desenvolvimento
Oxirredução
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (Bacterial Proteins); 0 (DNA Transposable Elements); 0 (Ferric Compounds); 0 (Fumarates); 1K09F3G675 (ferric oxide)
[Em] Mês de entrada:1710
[Cu] Atualização por classe:171003
[Lr] Data última revisão:
171003
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:170705
[St] Status:MEDLINE


  6 / 5314 MEDLINE  
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[PMID]:28599654
[Au] Autor:Mo X; Shi C; Gui C; Zhang Y; Ju J; Wang Q
[Ad] Endereço:Shandong Key Laboratory of Applied Mycology, School of Life Sciences, Qingdao Agricultural University, Qingdao, 266109, China. xhmo2013@163.com.
[Ti] Título:Identification of nocamycin biosynthetic gene cluster from Saccharothrix syringae NRRL B-16468 and generation of new nocamycin derivatives by manipulating gene cluster.
[So] Source:Microb Cell Fact;16(1):100, 2017 Jun 09.
[Is] ISSN:1475-2859
[Cp] País de publicação:England
[La] Idioma:eng
[Ab] Resumo:BACKGROUND: Nocamycins I and II, produced by the rare actinomycete Saccharothrix syringae, belong to the tetramic acid family natural products. Nocamycins show potent antimicrobial activity and they hold great potential for antibacterial agent design. However, up to now, little is known about the exact biosynthetic mechanism of nocamycin. RESULTS: In this report, we identified the gene cluster responsible for nocamycin biosynthesis from S. syringae and generated new nocamycin derivatives by manipulating its gene cluster. The biosynthetic gene cluster for nocamycin contains a 61 kb DNA locus, consisting of 21 open reading frames (ORFs). Five type I polyketide synthases (NcmAI, NcmAII, NcmAIII, NcmAIV, NcmAV) and a non-ribosomal peptide synthetase (NcmB) are proposed to be involved in synthesis of the backbone structure, a Dieckmann cyclase NcmC catalyze the releasing of linear chain and the formation of tetramic acid moiety, five enzymes (NcmEDGOP) are related to post-tailoring steps, and five enzymes (NcmNJKIM) function as regulators. Targeted inactivation of ncmB led to nocamycin production being completely abolished, which demonstrates that this gene cluster is involved in nocamycin biosynthesis. To generate new nocamycin derivatives, the gene ncmG, encoding for a cytochrome P450 oxidase, was inactivated. Two new nocamycin derivatives nocamycin III and nocamycin IV were isolated from the ncmG deletion mutant strain and their structures were elucidated by spectroscopic data analyses. Based on bioinformatics analysis and new derivatives isolated from gene inactivation mutant strains, a biosynthetic pathway of nocamycins was proposed. CONCLUSION: These findings provide the basis for further understanding of nocamycin biosynthetic mechanism, and set the stage to rationally engineer new nocamycin derivatives via combinatorial biosynthesis strategy.
[Mh] Termos MeSH primário: Actinomycetales/genética
DNA Bacteriano/genética
Família Multigênica
Compostos Orgânicos/metabolismo
[Mh] Termos MeSH secundário: Actinomycetales/metabolismo
Biblioteca Genômica
Conformação Molecular
Compostos Orgânicos/química
Análise de Sequência de DNA
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (DNA, Bacterial); 0 (Organic Chemicals); 63748-09-4 (nocamycin)
[Em] Mês de entrada:1711
[Cu] Atualização por classe:171108
[Lr] Data última revisão:
171108
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:170611
[St] Status:MEDLINE
[do] DOI:10.1186/s12934-017-0718-5


  7 / 5314 MEDLINE  
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[PMID]:28586923
[Au] Autor:Kandathil AJ; Breitwieser FP; Sachithanandham J; Robinson M; Mehta SH; Timp W; Salzberg SL; Thomas DL; Balagopal A
[Ad] Endereço:From Johns Hopkins University and Johns Hopkins Bloomberg School of Public Health, Baltimore, Maryland.
[Ti] Título:Presence of Human Hepegivirus-1 in a Cohort of People Who Inject Drugs.
[So] Source:Ann Intern Med;167(1):1-7, 2017 Jul 04.
[Is] ISSN:1539-3704
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:Background: Next-generation metagenomic sequencing (NGMS) has opened new frontiers in microbial discovery but has been clinically characterized in only a few settings. Objective: To explore the plasma virome of persons who inject drugs and to characterize the sensitivity and accuracy of NGMS compared with quantitative clinical standards. Design: Longitudinal and cross-sectional studies. Setting: A clinical trial (ClinicalTrials.gov: NCT01285050) and a well-characterized cohort study of persons who have injected drugs. Participants: Persons co-infected with hepatitis C virus (HCV) and HIV. Measurements: Viral nucleic acid in plasma by NGMS and quantitative polymerase chain reaction (PCR). Results: Next-generation metagenomic sequencing generated a total of 600 million reads, which included the expected HIV and HCV RNA sequences. HIV and HCV reads were consistently identified only when samples contained more than 10 000 copies/mL or IU/mL, respectively, as determined by quantitative PCR. A novel RNA virus, human hepegivirus-1 (HHpgV-1), was also detected by NGMS in 4 samples from 2 persons in the clinical trial. Through use of a quantitative PCR assay for HHpgV-1, infection was also detected in 17 (10.9%) of 156 members of a cohort of persons who injected drugs. In these persons, HHpgV-1 viremia persisted for a median of at least 4538 days and was associated with detection of other bloodborne viruses, such as HCV RNA and SEN virus D. Limitation: The medical importance of HHpgV-1 infection is unknown. Conclusion: Although NGMS is insensitive for detection of viruses with relatively low plasma nucleic acid concentrations, it may have broad potential for discovery of new viral infections of possible medical importance, such as HHpgV-1. Primary Funding Source: National Institutes of Health.
[Mh] Termos MeSH primário: Infecções por HIV/virologia
Hepatite C/virologia
Hepevirus/isolamento & purificação
Abuso de Substâncias por Via Intravenosa/virologia
Viremia/diagnóstico
[Mh] Termos MeSH secundário: Coinfecção
Estudos Transversais
Feminino
Biblioteca Genômica
Infecções por HIV/complicações
Hepatite C/complicações
Hepevirus/genética
Seres Humanos
Estudos Longitudinais
Masculino
Reação em Cadeia da Polimerase
Análise de Sequência de DNA
Análise de Sequência de RNA
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Em] Mês de entrada:1707
[Cu] Atualização por classe:170828
[Lr] Data última revisão:
170828
[Sb] Subgrupo de revista:AIM; IM
[Da] Data de entrada para processamento:170607
[St] Status:MEDLINE
[do] DOI:10.7326/M17-0085


  8 / 5314 MEDLINE  
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[PMID]:28542173
[Au] Autor:Gao B; Vorwerk H; Huber C; Lara-Tejero M; Mohr J; Goodman AL; Eisenreich W; Galán JE; Hofreuter D
[Ad] Endereço:Department of Microbial Pathogenesis, Yale University School of Medicine, New Haven, Connecticut, United States of America.
[Ti] Título:Metabolic and fitness determinants for in vitro growth and intestinal colonization of the bacterial pathogen Campylobacter jejuni.
[So] Source:PLoS Biol;15(5):e2001390, 2017 May.
[Is] ISSN:1545-7885
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:Campylobacter jejuni is one of the leading infectious causes of food-borne illness around the world. Its ability to persistently colonize the intestinal tract of a broad range of hosts, including food-producing animals, is central to its epidemiology since most infections are due to the consumption of contaminated food products. Using a highly saturated transposon insertion library combined with next-generation sequencing and a mouse model of infection, we have carried out a comprehensive genome-wide analysis of the fitness determinants for growth in vitro and in vivo of a highly pathogenic strain of C. jejuni. A comparison of the C. jejuni requirements to colonize the mouse intestine with those necessary to grow in different culture media in vitro, combined with isotopologue profiling and metabolic flow analysis, allowed us to identify its metabolic requirements to establish infection, including the ability to acquire certain nutrients, metabolize specific substrates, or maintain intracellular ion homeostasis. This comprehensive analysis has identified metabolic pathways that could provide the basis for the development of novel strategies to prevent C. jejuni colonization of food-producing animals or to treat human infections.
[Mh] Termos MeSH primário: Proteínas de Bactérias/metabolismo
Infecções por Campylobacter/microbiologia
Campylobacter jejuni/fisiologia
Proteínas de Transporte de Cátions/metabolismo
Gastroenterite/microbiologia
Modelos Biológicos
[Mh] Termos MeSH secundário: Absorção Fisiológica
Aminoácidos/metabolismo
Animais
Antibacterianos/efeitos adversos
Proteínas de Bactérias/genética
Campylobacter jejuni/crescimento & desenvolvimento
Campylobacter jejuni/isolamento & purificação
Proteínas de Transporte de Cátions/genética
Elementos de DNA Transponíveis
Disbiose/induzido quimicamente
Disbiose/microbiologia
Deleção de Genes
Estudos de Associação Genética
Genoma Bacteriano
Biblioteca Genômica
Camundongos Endogâmicos C57BL
Viabilidade Microbiana
Mutagênese Insercional
Mutação
[Pt] Tipo de publicação:COMPARATIVE STUDY; JOURNAL ARTICLE
[Nm] Nome de substância:
0 (Amino Acids); 0 (Anti-Bacterial Agents); 0 (Bacterial Proteins); 0 (Cation Transport Proteins); 0 (DNA Transposable Elements); 0 (KtrB protein, Bacteria)
[Em] Mês de entrada:1709
[Cu] Atualização por classe:170906
[Lr] Data última revisão:
170906
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:170526
[St] Status:MEDLINE
[do] DOI:10.1371/journal.pbio.2001390


  9 / 5314 MEDLINE  
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[PMID]:28446608
[Au] Autor:Pan S; Nikolakakis K; Adamczyk PA; Pan M; Ruby EG; Reed JL
[Ad] Endereço:From the Department of Chemical and Biological Engineering, University of Wisconsin-Madison, Madison, Wisconsin 53706.
[Ti] Título:Model-enabled gene search (MEGS) allows fast and direct discovery of enzymatic and transport gene functions in the marine bacterium .
[So] Source:J Biol Chem;292(24):10250-10261, 2017 Jun 16.
[Is] ISSN:1083-351X
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:Whereas genomes can be rapidly sequenced, the functions of many genes are incompletely or erroneously annotated because of a lack of experimental evidence or prior functional knowledge in sequence databases. To address this weakness, we describe here a odel- nabled ene earch (MEGS) approach that (i) identifies metabolic functions either missing from an organism's genome annotation or incorrectly assigned to an ORF by using discrepancies between metabolic model predictions and experimental culturing data; (ii) designs functional selection experiments for these specific metabolic functions; and (iii) selects a candidate gene(s) responsible for these functions from a genomic library and directly interrogates this gene's function experimentally. To discover gene functions, MEGS uses genomic functional selections instead of relying on correlations across large experimental datasets or sequence similarity as do other approaches. When applied to the bioluminescent marine bacterium , MEGS successfully identified five genes that are responsible for four metabolic and transport reactions whose absence from a draft metabolic model of caused inaccurate modeling of high-throughput experimental data. This work demonstrates that MEGS provides a rapid and efficient integrated computational and experimental approach for annotating metabolic genes, including those that have previously been uncharacterized or misannotated.
[Mh] Termos MeSH primário: Aliivibrio fischeri/genética
Organismos Aquáticos/genética
Proteínas de Bactérias/genética
Sistemas Especialistas
Genômica/métodos
Modelos Genéticos
[Mh] Termos MeSH secundário: Aliivibrio fischeri/crescimento & desenvolvimento
Aliivibrio fischeri/metabolismo
Animais
Aquicultura
Organismos Aquáticos/metabolismo
Proteínas de Bactérias/metabolismo
Simulação por Computador
Decapodiformes/crescimento & desenvolvimento
Decapodiformes/microbiologia
Escherichia coli/genética
Escherichia coli/crescimento & desenvolvimento
Escherichia coli/metabolismo
Deleção de Genes
Teste de Complementação Genética
Biblioteca Genômica
Hawaii
Sequenciamento de Nucleotídeos em Larga Escala
Anotação de Sequência Molecular
Fases de Leitura Aberta
Oceano Pacífico
Proteínas Recombinantes/metabolismo
Reprodutibilidade dos Testes
Especificidade da Espécie
[Pt] Tipo de publicação:COMPARATIVE STUDY; JOURNAL ARTICLE; VALIDATION STUDIES
[Nm] Nome de substância:
0 (Bacterial Proteins); 0 (Recombinant Proteins)
[Em] Mês de entrada:1707
[Cu] Atualização por classe:170706
[Lr] Data última revisão:
170706
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:170428
[St] Status:MEDLINE
[do] DOI:10.1074/jbc.M116.763193


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[PMID]:28319150
[Au] Autor:Liu G; Lanham C; Buchan JR; Kaplan ME
[Ad] Endereço:Department of Molecular and Cellular Biology; University of Arizona, Tucson, Arizona, United States of America.
[Ti] Título:High-throughput transformation of Saccharomyces cerevisiae using liquid handling robots.
[So] Source:PLoS One;12(3):e0174128, 2017.
[Is] ISSN:1932-6203
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:Saccharomyces cerevisiae (budding yeast) is a powerful eukaryotic model organism ideally suited to high-throughput genetic analyses, which time and again has yielded insights that further our understanding of cell biology processes conserved in humans. Lithium Acetate (LiAc) transformation of yeast with DNA for the purposes of exogenous protein expression (e.g., plasmids) or genome mutation (e.g., gene mutation, deletion, epitope tagging) is a useful and long established method. However, a reliable and optimized high throughput transformation protocol that runs almost no risk of human error has not been described in the literature. Here, we describe such a method that is broadly transferable to most liquid handling high-throughput robotic platforms, which are now commonplace in academic and industry settings. Using our optimized method, we are able to comfortably transform approximately 1200 individual strains per day, allowing complete transformation of typical genomic yeast libraries within 6 days. In addition, use of our protocol for gene knockout purposes also provides a potentially quicker, easier and more cost-effective approach to generating collections of double mutants than the popular and elegant synthetic genetic array methodology. In summary, our methodology will be of significant use to anyone interested in high throughput molecular and/or genetic analysis of yeast.
[Mh] Termos MeSH primário: Automação Laboratorial/instrumentação
Robótica
Saccharomyces cerevisiae/genética
Transformação Genética
[Mh] Termos MeSH secundário: Automação Laboratorial/métodos
Canavanina/toxicidade
Meios de Cultura
Técnicas de Inativação de Genes
Biblioteca Genômica
Temperatura Alta
Saccharomyces cerevisiae/efeitos dos fármacos
Fatores de Tempo
[Pt] Tipo de publicação:JOURNAL ARTICLE; VIDEO-AUDIO MEDIA
[Nm] Nome de substância:
0 (Culture Media); 3HZV514J4B (Canavanine)
[Em] Mês de entrada:1708
[Cu] Atualização por classe:170829
[Lr] Data última revisão:
170829
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:170321
[St] Status:MEDLINE
[do] DOI:10.1371/journal.pone.0174128



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