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1.
Nucleic Acids Res ; 48(14): 7623-7639, 2020 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-32644123

RESUMO

RNA therapeutics are a promising strategy to treat genetic diseases caused by the overexpression or aberrant splicing of a specific protein. The field has seen major strides in the clinical efficacy of this class of molecules, largely due to chemical modifications and delivery strategies that improve nuclease resistance and enhance cell penetration. However, a major obstacle in the development of RNA therapeutics continues to be the imprecise, difficult, and often problematic nature of most methods used to measure cell penetration. Here, we review these methods and clearly distinguish between those that measure total cellular uptake of RNA therapeutics, which includes both productive and non-productive uptake, and those that measure cytosolic/nuclear penetration, which represents only productive uptake. We critically analyze the benefits and drawbacks of each method. Finally, we use key examples to illustrate how, despite rigorous experimentation and proper controls, our understanding of the mechanism of gymnotic uptake of RNA therapeutics remains limited by the methods commonly used to analyze RNA delivery.


Assuntos
RNA/metabolismo , RNA/uso terapêutico , Aptâmeros de Nucleotídeos/uso terapêutico , Núcleo Celular/metabolismo , Citosol/metabolismo , Doenças Genéticas Inatas/tratamento farmacológico , Técnicas Genéticas , Humanos , MicroRNAs/uso terapêutico , Microscopia Eletrônica , Oligonucleotídeos Antissenso/uso terapêutico , RNA/química , RNA/farmacocinética , RNA Interferente Pequeno/uso terapêutico , Espectrometria de Fluorescência
2.
Mol Cell ; 79(5): 857-869.e3, 2020 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-32681820

RESUMO

Sister-chromatid cohesion describes the orderly association of newly replicated DNA molecules behind replication forks. It plays an essential role in the maintenance and faithful transmission of genetic information. Cohesion is created by DNA topological links and proteinaceous bridges, whose formation and deposition could be potentially affected by many processes. Current knowledge on cohesion has been mainly gained by fluorescence microscopy observation. However, the resolution limit of microscopy and the restricted number of genomic positions that can be simultaneously visualized considerably hampered progress. Here, we present a high-throughput methodology to monitor sister-chromatid contacts (Hi-SC2). Using the multi-chromosomal Vibrio cholerae bacterium as a model, we show that Hi-SC2 permits to monitor local variations in sister-chromatid cohesion at a high resolution over a whole genome.


Assuntos
Cromátides/fisiologia , Técnicas Genéticas , Vibrio cholerae/genética , Cromossomos Bacterianos/fisiologia , Replicação do DNA , DNA Bacteriano , Sequenciamento de Nucleotídeos em Larga Escala , Integrases/metabolismo , Conformação de Ácido Nucleico
4.
J Vis Exp ; (159)2020 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-32510510

RESUMO

As genome-wide association studies shed light on the heterogeneous genetic underpinnings of many neurological diseases, the need to study the contribution of specific genes to brain development and function increases. Relying on mouse models to study the role of specific genetic manipulations is not always feasible since transgenic mouse lines are quite costly and many novel disease-associated genes do not yet have commercially available genetic lines. Additionally, it can take years of development and validation to create a mouse line. In utero electroporation offers a relatively quick and easy method to manipulate gene expression in a cell-type specific manner in vivo that only requires developing a DNA plasmid to achieve a particular genetic manipulation. Bilateral in utero electroporation can be used to target large populations of frontal cortex pyramidal neurons. Combining this gene transfer method with behavioral approaches allows one to study the effects of genetic manipulations on the function of prefrontal cortex networks and the social behavior of juvenile and adult mice.


Assuntos
Comportamento Animal , Eletroporação/métodos , Técnicas Genéticas , Animais , Estudos de Viabilidade , Camundongos , Camundongos Transgênicos , Plasmídeos/genética
5.
Science ; 368(6498)2020 06 26.
Artigo em Inglês | MEDLINE | ID: mdl-32586993

RESUMO

Whole-genome duplication has played a central role in the genome evolution of many organisms, including the human genome. Most duplicated genes are eliminated, and factors that influence the retention of persisting duplicates remain poorly understood. We describe a systematic complex genetic interaction analysis with yeast paralogs derived from the whole-genome duplication event. Mapping of digenic interactions for a deletion mutant of each paralog, and of trigenic interactions for the double mutant, provides insight into their roles and a quantitative measure of their functional redundancy. Trigenic interaction analysis distinguishes two classes of paralogs: a more functionally divergent subset and another that retained more functional overlap. Gene feature analysis and modeling suggest that evolutionary trajectories of duplicated genes are dictated by combined functional and structural entanglement factors.


Assuntos
Duplicação Gênica , Genes Duplicados , Genoma Fúngico , Mapas de Interação de Proteínas/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Deleção de Genes , Redes Reguladoras de Genes , Técnicas Genéticas , Proteínas de Membrana/genética , Peroxinas/genética
6.
Nucleic Acids Res ; 48(W1): W177-W184, 2020 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-32301980

RESUMO

The Galaxy HiCExplorer provides a web service at https://hicexplorer.usegalaxy.eu. It enables the integrative analysis of chromosome conformation by providing tools and computational resources to pre-process, analyse and visualize Hi-C, Capture Hi-C (cHi-C) and single-cell Hi-C (scHi-C) data. Since the last publication, Galaxy HiCExplorer has been expanded considerably with new tools to facilitate the analysis of cHi-C and to provide an in-depth analysis of Hi-C data. Moreover, it supports the analysis of scHi-C data by offering a broad range of tools. With the help of the standard graphical user interface of Galaxy, presented workflows, extensive documentation and tutorials, novices as well as Hi-C experts are supported in their Hi-C data analysis with Galaxy HiCExplorer.


Assuntos
Cromatina/química , Software , Gráficos por Computador , Técnicas Genéticas/normas , Internet , Conformação Molecular , Reprodutibilidade dos Testes , Análise de Célula Única/normas
7.
Nucleic Acids Res ; 48(8): 4507-4520, 2020 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-32170306

RESUMO

The FinO-domain-protein ProQ is an RNA-binding protein that has been known to play a role in osmoregulation in proteobacteria. Recently, ProQ has been shown to act as a global RNA-binding protein in Salmonella and Escherichia coli, binding to dozens of small RNAs (sRNAs) and messenger RNAs (mRNAs) to regulate mRNA-expression levels through interactions with both 5' and 3' untranslated regions (UTRs). Despite excitement around ProQ as a novel global RNA-binding protein, and its potential to serve as a matchmaking RNA chaperone, significant gaps remain in our understanding of the molecular mechanisms ProQ uses to interact with RNA. In order to apply the tools of molecular genetics to this question, we have adapted a bacterial three-hybrid (B3H) assay to detect ProQ's interactions with target RNAs. Using domain truncations, site-directed mutagenesis and an unbiased forward genetic screen, we have identified a group of highly conserved residues on ProQ's NTD as the primary face for in vivo recognition of two RNAs, and propose that the NTD structure serves as an electrostatic scaffold to recognize the shape of an RNA duplex.


Assuntos
Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , RNA Bacteriano/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Proteínas de Escherichia coli/genética , Técnicas Genéticas , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Modelos Moleculares , Ligação Proteica , Domínios Proteicos
8.
Proc Biol Sci ; 287(1922): 20192995, 2020 03 11.
Artigo em Inglês | MEDLINE | ID: mdl-32126953

RESUMO

Intestinal microbiota perform many functions for their host, but among the most important is their role in metabolism, especially the conversion of recalcitrant biomass that the host is unable to digest into bioavailable compounds. Most studies have focused on the assistance gut microbiota provide in the metabolism of carbohydrates, however, their role in host amino acid metabolism is poorly understood. We conducted an experiment on Mus musculus using 16S rRNA gene sequencing and carbon isotope analysis of essential amino acids (AAESS) to quantify the community composition of gut microbiota and the contribution of carbohydrate carbon used by the gut microbiome to synthesize AAESS that are assimilated by mice to build skeletal muscle tissue. The relative abundances of Firmicutes and Bacteroidetes inversely varied as a function of dietary macromolecular content, with Firmicutes dominating when mice were fed low-protein diets that contained the highest proportions of simple carbohydrates (sucrose). Mixing models estimated that the microbial contribution of AAESS to mouse muscle varied from less than 5% (threonine, lysine, and phenylalanine) to approximately 60% (valine) across diet treatments, with the Firmicute-dominated microbiome associated with the greatest contribution. Our results show that intestinal microbes can provide a significant source of the AAESS their host uses to synthesize structural tissues. The role that gut microbiota play in the amino acid metabolism of animals that consume protein-deficient diets is likely a significant but under-recognized aspect of foraging ecology and physiology.


Assuntos
Aminoácidos/metabolismo , Microbioma Gastrointestinal/fisiologia , Mamíferos/fisiologia , Animais , Isótopos de Carbono , Técnicas Genéticas , Mamíferos/genética
9.
Nucleic Acids Res ; 48(9): e50, 2020 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-32133534

RESUMO

We report a tool, Calling Cards Reporter Arrays (CCRA), that measures transcription factor (TF) binding and the consequences on gene expression for hundreds of synthetic promoters in yeast. Using Cbf1p and MAX, we demonstrate that the CCRA method is able to detect small changes in binding free energy with a sensitivity comparable to in vitro methods, enabling the measurement of energy landscapes in vivo. We then demonstrate the quantitative analysis of cooperative interactions by measuring Cbf1p binding at synthetic promoters with multiple sites. We find that the cooperativity between Cbf1p dimers varies sinusoidally with a period of 10.65 bp and energetic cost of 1.37 KBT for sites that are positioned 'out of phase'. Finally, we characterize the binding and expression of a group of TFs, Tye7p, Gcr1p and Gcr2p, that act together as a 'TF collective', an important but poorly characterized model of TF cooperativity. We demonstrate that Tye7p often binds promoters without its recognition site because it is recruited by other collective members, whereas these other members require their recognition sites, suggesting a hierarchy where these factors recruit Tye7p but not vice versa. Our experiments establish CCRA as a useful tool for quantitative investigations into TF binding and function.


Assuntos
Fatores de Transcrição/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , DNA/química , DNA/metabolismo , Expressão Gênica , Genes Reporter , Técnicas Genéticas , Sequenciamento de Nucleotídeos em Larga Escala , Regiões Promotoras Genéticas , Ligação Proteica , Saccharomyces cerevisiae/genética , Análise de Sequência de DNA
10.
Nat Commun ; 11(1): 1392, 2020 03 13.
Artigo em Inglês | MEDLINE | ID: mdl-32170178

RESUMO

Although noncanonical amino acids (ncAAs) were first incorporated into phage libraries through amber suppression nearly two decades ago, their application for use in drug discovery has been limited due to inherent library bias towards sense-containing phages. Here, we report a technique based on superinfection immunity of phages to enrich amber-containing clones, thus avoiding the observed bias that has hindered incorporation of ncAAs into phage libraries. We then take advantage of this technique for development of active site-directed ligand evolution of peptides, where the ncAA serves as an anchor to direct the binding of its peptides to the target's active site. To demonstrate this, phage-displayed peptide libraries are developed that contain a genetically encoded butyryl lysine and are subsequently used to select for ligands that bind SIRT2. These ligands are then modified to develop low nanomolar inhibitors of SIRT2.


Assuntos
Âmbar/metabolismo , Bacteriófagos/metabolismo , Domínio Catalítico , Peptídeos/metabolismo , Descoberta de Drogas , Técnicas Genéticas , Humanos , Ligantes , Lisina/metabolismo , Simulação de Acoplamento Molecular , Biblioteca de Peptídeos , Sirtuína 2/metabolismo
11.
J Vis Exp ; (156)2020 02 24.
Artigo em Inglês | MEDLINE | ID: mdl-32092048

RESUMO

Articles Discussed: Stahl, B. A. et al. Manipulation of Gene Function in Mexican Cavefish. Journal of Visualized Experiments. (146) (2019). Peuß, R. et al. Gamete Collection and In Vitro Fertilization of Astyanax mexicanus. Journal of Visualized Experiments. (147) (2019). Worsham, M. et al. Behavioral Tracking and Neuromast Imaging of Mexican Cavefish.Journal of Visualized Experiments. (147) (2019). Jaggard, J.B., Lloyd, E., Lopatto, A., Duboue, E.R., Keene, A.C. Automated Measurements of Sleep and Locomotor Activity in Mexican Cavefish. Journal of Visualized Experiments. (145) (2019). Luc, H., Sears, C., Raczka, A., Gross, J.B. Wholemount In Situ Hybridization for Astyanax Embryos. Journal of Visualized Experiments. (145) (2019). Riddle, M., Martineau, B., Peavey, M., Tabin, C. Raising the Mexican Tetra Astyanax mexicanus for Analysis of Post-larval Phenotypes and Whole-mount Immunohistochemistry. Journal of Visualized Experiments. (142) (2018).


Assuntos
Criação de Animais Domésticos , Characidae/fisiologia , Técnicas Genéticas , Animais
12.
Mol Cell ; 77(4): 688-708, 2020 02 20.
Artigo em Inglês | MEDLINE | ID: mdl-32001106

RESUMO

Rapidly developing technologies have recently fueled an exciting era of discovery in the field of chromosome structure and nuclear organization. In addition to chromosome conformation capture (3C) methods, new alternative techniques have emerged to study genome architecture and biological processes in the nucleus, often in single or living cells. This sets an unprecedented stage for exploring the mechanisms that link chromosome structure and biological function. Here we review popular as well as emerging approaches to study chromosome organization, focusing on the contribution of complementary methodologies to our understanding of structures revealed by 3C methods and their biological implications, and discuss the next technical and conceptual frontiers.


Assuntos
Cromossomos de Mamíferos/química , Animais , Núcleo Celular/genética , Reparo do DNA , Período de Replicação do DNA , Técnicas Genéticas , Modelos Genéticos , Análise de Célula Única , Transcrição Genética
13.
BMC Infect Dis ; 20(1): 38, 2020 Jan 14.
Artigo em Inglês | MEDLINE | ID: mdl-31937247

RESUMO

BACKGROUND: Group B Streptococcal (GBS) infections in the United States are a leading cause of meningitis and sepsis in newborns. The CDC therefore recommends GBS screening for all pregnant women at 35-37 weeks of gestation and administration of intrapartum prophylaxis (in those that tested positive) as an effective means of controlling disease transmission. Several FDA approved molecular diagnostic tests are available for rapid and accurate detection of GBS in antepartum women. METHOD: In this study, we report a clinical comparison of the Xpert GBS LB assay and a novel FDA-cleared test, Revogene GBS LB assay. A total of 250 vaginal-rectal swabs from women undergoing prenatal screening were submitted to the University of Wisconsin's clinical microbiology laboratory for GBS testing. RESULTS: We found 96.8% of samples were concordant between the two tests, while 3.2% were discordant with a positive percent agreement of 98.0% and a negative percent agreement of 96.5% between the Revogene GBS LB assay and the GeneXpert GBS LB assay. CONCLUSION: Overall, we report that both assays perform well for the detection of GBS colonization in pregnant women.


Assuntos
Testes Diagnósticos de Rotina/métodos , Programas de Rastreamento/métodos , Técnicas de Diagnóstico Molecular/métodos , Complicações Infecciosas na Gravidez/diagnóstico , Diagnóstico Pré-Natal/métodos , Infecções Estreptocócicas/diagnóstico , Streptococcus agalactiae/genética , DNA Viral/análise , Feminino , Técnicas Genéticas , Humanos , Transmissão Vertical de Doença Infecciosa/prevenção & controle , Programas de Rastreamento/economia , Técnicas de Diagnóstico Molecular/economia , Gravidez , Complicações Infecciosas na Gravidez/virologia , Gestantes , Infecções Estreptocócicas/virologia , Fatores de Tempo , Vagina/virologia
14.
BMC Biol ; 18(1): 1, 2020 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-31898513

RESUMO

BACKGROUND: New sequencing technologies have lowered financial barriers to whole genome sequencing, but resulting assemblies are often fragmented and far from 'finished'. Updating multi-scaffold drafts to chromosome-level status can be achieved through experimental mapping or re-sequencing efforts. Avoiding the costs associated with such approaches, comparative genomic analysis of gene order conservation (synteny) to predict scaffold neighbours (adjacencies) offers a potentially useful complementary method for improving draft assemblies. RESULTS: We evaluated and employed 3 gene synteny-based methods applied to 21 Anopheles mosquito assemblies to produce consensus sets of scaffold adjacencies. For subsets of the assemblies, we integrated these with additional supporting data to confirm and complement the synteny-based adjacencies: 6 with physical mapping data that anchor scaffolds to chromosome locations, 13 with paired-end RNA sequencing (RNAseq) data, and 3 with new assemblies based on re-scaffolding or long-read data. Our combined analyses produced 20 new superscaffolded assemblies with improved contiguities: 7 for which assignments of non-anchored scaffolds to chromosome arms span more than 75% of the assemblies, and a further 7 with chromosome anchoring including an 88% anchored Anopheles arabiensis assembly and, respectively, 73% and 84% anchored assemblies with comprehensively updated cytogenetic photomaps for Anopheles funestus and Anopheles stephensi. CONCLUSIONS: Experimental data from probe mapping, RNAseq, or long-read technologies, where available, all contribute to successful upgrading of draft assemblies. Our evaluations show that gene synteny-based computational methods represent a valuable alternative or complementary approach. Our improved Anopheles reference assemblies highlight the utility of applying comparative genomics approaches to improve community genomic resources.


Assuntos
Anopheles/genética , Evolução Biológica , Cromossomos , Técnicas Genéticas/instrumentação , Genômica/métodos , Sintenia , Animais , Mapeamento Cromossômico
15.
Genome Biol ; 21(1): 10, 2020 01 14.
Artigo em Inglês | MEDLINE | ID: mdl-31937348

RESUMO

Although scRNA-seq is now ubiquitously adopted in studies of intratumor heterogeneity, detection of somatic mutations and inference of clonal membership from scRNA-seq is currently unreliable. We propose DENDRO, an analysis method for scRNA-seq data that clusters single cells into genetically distinct subclones and reconstructs the phylogenetic tree relating the subclones. DENDRO utilizes transcribed point mutations and accounts for technical noise and expression stochasticity. We benchmark DENDRO and demonstrate its application on simulation data and real data from three cancer types. In particular, on a mouse melanoma model in response to immunotherapy, DENDRO delineates the role of neoantigens in treatment response.


Assuntos
Heterogeneidade Genética , Técnicas Genéticas , Neoplasias/genética , Filogenia , Software , Animais , Humanos , Camundongos , Análise de Célula Única
16.
Development ; 147(4)2020 02 17.
Artigo em Inglês | MEDLINE | ID: mdl-31988189

RESUMO

Cellular proliferation is a basic process during organ development, tissue homeostasis and disease progression. Likewise, after injury typically multiple cell lineages respond to various cues and proliferate to initiate repair and/or remodeling of the injured tissue. Unravelling the specific role of proliferation of one cell type and its lineage in the context of the whole organism during tissue regeneration and/or disease progression would provide valuable information on these processes. Here, we report a new genetic system that allows cell proliferation to be inhibited in a tissue-specific manner. We generated Cre- or Dre-inducible p21-GFP (ip21-GFP) transgenic mice that enable experimentally induced permanent cell cycle arrest of specific cell lineages of interest, while genetically marking these cells. This system allows for the inhibition of pathogenic cell proliferation. We found that cardiac fibroblast proliferation inhibition significantly reduced scar formation, and promoted neovascularization and cardiomyocyte survival. Additionally, we found that inhibition of one type of cell proliferation (namely, hepatocytes) induces the lineage conversion of another type cells (i.e. ductal cells) during tissue regeneration. These results validate the use of ip21-GFP mice as a new genetic tool for cell lineage-specific inhibition of cell proliferation in vivo.


Assuntos
Proliferação de Células , Regulação da Expressão Gênica , Técnicas Genéticas , Alelos , Animais , Linhagem da Célula , Inibidor de Quinase Dependente de Ciclina p21/fisiologia , Feminino , Fibroblastos/fisiologia , Proteínas de Fluorescência Verde , Coração/crescimento & desenvolvimento , Coração/fisiologia , Hepatócitos/citologia , Hepatócitos/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Infarto do Miocárdio/patologia , Miócitos Cardíacos/citologia
17.
Cell Rep ; 30(4): 1235-1245.e4, 2020 01 28.
Artigo em Inglês | MEDLINE | ID: mdl-31995761

RESUMO

DNA-protein crosslinks (DPCs) are a frequent form of DNA lesion and are strongly inhibitive in diverse DNA transactions. Despite recent developments, the biochemical detection of DPCs remains a limiting factor for the in-depth mechanistic understanding of DPC repair. Here, we develop a sensitive and versatile assay, designated ARK, for the quantitative analysis of DPCs in cells. ARK uses sequential chaotropic and detergent-based isolation of DPCs and substantially enhances sample purity, resulting in a 5-fold increase in detection sensitivity and a 10-fold reduction in background reading. We validate the ARK assay with genetic mutants with established deficiencies in DPC repair and demonstrate its robustness by using common DPC-inducing reagents, including formaldehyde, camptothecin, and etoposide. In addition, we show that the Fanconi anemia pathway contributes to the repair of DPCs. Thus, ARK is expected to facilitate various studies aimed at understanding both fundamental biology and translational applications of DNA-protein crosslink repair.


Assuntos
Reagentes para Ligações Cruzadas/farmacologia , Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Camptotecina/farmacologia , Reparo do DNA/genética , Etoposídeo/farmacologia , Anemia de Fanconi/genética , Anemia de Fanconi/metabolismo , Técnicas de Inativação de Genes , Técnicas Genéticas , Células HeLa , Humanos , Inibidores da Topoisomerase I/farmacologia
18.
Plant Cell Rep ; 39(4): 501-510, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31915913

RESUMO

KEY MESSAGE: An efficient and improved transformation method for functional genetics studies in S. italica, being a boon for the Setaria scientific community and for crop improvement. Foxtail millet (Setaria italica) is a short life cycle C4 plant, with sequenced genome, and a potential model plant for C4 species. S. italica is also important on a global food security and healthiness context due to its importance in arid and semi-arid areas. However, despite its importance, there are just few transformation protocols directed to this species. The current protocols reached about 5.5-9% of efficiency, which do not make it a valuable model organism. Different types of explants were used in the above mentioned methods, such as immature and mature inflorescence and shoot apex. However, these techniques have many limitations, such as unavailability of explants throughout the year and a crucial, laborious and considerable time-consuming selection. Aiming a simplified and efficient methodology, we adopted dry mature seeds as explants, which are available in abundance, are constant along the year and well responsive to tissue culture, in addition to a differentiated approach that reaches on an average 19.2% transformation efficiency of S. italica. Thus, we propose a protocol that optimizes the transformation efficiency of this cereal crop allowing a high increase on transformation and regeneration rates. Our transformation protocol provides an interesting tool for Setaria community research as well as enables new strategies for breeding enhanced productivity in the species.


Assuntos
Regeneração/genética , Setaria (Planta)/genética , Transformação Genética , Agrobacterium tumefaciens/genética , Técnicas de Cultura de Células/métodos , Células Cultivadas , Grão Comestível/genética , Grão Comestível/metabolismo , Técnicas Genéticas , Vetores Genéticos , Fenótipo , Melhoramento Vegetal , Folhas de Planta/genética , Folhas de Planta/crescimento & desenvolvimento , Folhas de Planta/metabolismo , Plantas Geneticamente Modificadas , Interferência de RNA , Regeneração/fisiologia , Sementes/efeitos dos fármacos , Sementes/genética , Setaria (Planta)/metabolismo , Setaria (Planta)/microbiologia , Setaria (Planta)/fisiologia
19.
Plant Cell Rep ; 39(4): 511-525, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31938834

RESUMO

KEY MESSAGE: A simple and robust Agrobacterium-mediated gene expression system in the C4 panicoid model crop, foxtail millet has been developed with up to 27 % transformation efficiency. Foxtail millet (Setaria italica L.) is a model crop to study C4 photosynthesis, abiotic stress tolerance, and bioenergy traits. Advances in molecular genetics and genomics had identified several potential genes in this crop that would serve as candidates for imparting climate-resilient traits in related millets, cereals, and biofuel crops. However, the lack of an efficient genetic transformation system has been impeding the functional characterization of these genes in foxtail millet per se. Given this, an easy and efficient regeneration and transformation protocol was optimized using mature seeds as a choicest explant. The suitability of secondary embryogenic calli over primary calli is underlined due to their high competence. The use of perfect combinations of plant growth regulators together with the ionic strength of organic and inorganics salts was found to influence regeneration and genetic transformation. We studied and optimized various crucial factors that affect the genetic transformation of foxtail millet calli using Agrobacterium tumefaciens-mediated approach. Secondary embryogenic calli and LBA44404 strain were found to be the best targets for transformation. The use of high sucrose and glucose, together with freshly prepared tobacco leaves extract, Silwet L-77 and acetosyringone, improved the efficiency of the genetic transformation of foxtail millet. Moreover, the use of an in vitro regeneration system with 84% callusing efficiency and 70-74% regeneration frequency led to a high recovery of transformants. Altogether, the present study reports a highly efficient (~ 27%) transformation system in foxtail millet that will expedite forward and reverse genetic studies in this important crop.


Assuntos
Agrobacterium tumefaciens/genética , Produtos Agrícolas/genética , Setaria (Planta)/genética , Transformação Genética , Técnicas de Cultura de Células/métodos , Células Cultivadas , Técnicas Genéticas , Vetores Genéticos , Fenótipo , Células Vegetais/efeitos dos fármacos , Células Vegetais/microbiologia , Células Vegetais/fisiologia , Reguladores de Crescimento de Planta/metabolismo , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Plantas Geneticamente Modificadas/fisiologia , Regeneração/genética , Regeneração/fisiologia , Sementes/efeitos dos fármacos , Sementes/genética , Sementes/metabolismo , Sementes/microbiologia , Setaria (Planta)/metabolismo , Setaria (Planta)/microbiologia
20.
Mol Biol Evol ; 37(1): 291-294, 2020 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-31432070

RESUMO

ModelTest-NG is a reimplementation from scratch of jModelTest and ProtTest, two popular tools for selecting the best-fit nucleotide and amino acid substitution models, respectively. ModelTest-NG is one to two orders of magnitude faster than jModelTest and ProtTest but equally accurate and introduces several new features, such as ascertainment bias correction, mixture, and free-rate models, or the automatic processing of single partitions. ModelTest-NG is available under a GNU GPL3 license at https://github.com/ddarriba/modeltest , last accessed September 2, 2019.


Assuntos
Substituição de Aminoácidos , Evolução Molecular , Técnicas Genéticas , Modelos Genéticos , Software
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