Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 2.071
Filtrar
1.
Microb Cell Fact ; 18(1): 96, 2019 May 29.
Artigo em Inglês | MEDLINE | ID: mdl-31142347

RESUMO

BACKGROUND: Promoter evolution by synthetic promoter library (SPL) is a powerful approach to development of functional synthetic promoters to synthetic biology. However, it requires much tedious and time-consuming screenings because of the plethora of different variants in SPL. Actually, a large proportion of mutants in the SPL are significantly lower in strength, which contributes only to fabrication of a promoter library with a continuum of strength. Thus, to effectively obtain the evolved synthetic promoter exhibiting higher strength, it is essential to develop novel strategies to construct mutant library targeting the pivotal region rather than the arbitrary region of the template promoter. In this study, a strategy termed stepwise evolution targeting the spacer of core promoter (SETarSCoP) was established in Bacillus subtilis to effectively evolve the strength of bacterial promoter. RESULTS: The native promoter, PsrfA, from B. subtilis, which exhibits higher strength than the strong promoter P43, was set as the parental template. According to the comparison of conservation of the spacer sequences between - 35 box and - 10 box among a set of strong and weak native promoter, it revealed that 7-bp sequence immediately upstream of the - 10 box featured in the regulation of promoter strength. Based on the conservative feature, two rounds of consecutive evolution were performed targeting the hot region of PsrfA. In the first round, a primary promoter mutation library (pPML) was constructed by mutagenesis targeting the 3-bp sequence immediately upstream of the - 10 box of the PsrfA. Subsequently, four evolved mutants from pPML were selected to construction of four secondary promoter mutation libraries (sPMLs) based on mutagenesis of the 4-bp sequence upstream of the first-round target. After the consecutive two-step evolution, the mutant PBH4 was identified and verified to be a highly evolved synthetic promoter. The strength of PBH4 was higher than PsrfA by approximately 3 times. Moreover, PBH4 also exhibited broad suitability for different cargo proteins, such as ß-glucuronidase and nattokinase. The proof-of-principle test showed that SETarSCoP successfully evolved both constitutive and inducible promoters. CONCLUSION: Comparing with the commonly used SPL strategy, SETarSCoP facilitates the evolution process to obtain strength-evolved synthetic bacterial promoter through fabrication and screening of small-scale mutation libraries. This strategy will be a promising method to evolve diverse bacterial promoters to expand the toolbox for synthetic biology.


Assuntos
Bacillus subtilis/genética , Evolução Molecular Direcionada/métodos , Regiões Promotoras Genéticas , Biblioteca Gênica , Mutagênese/genética , Mutação , Biologia Sintética/métodos
2.
Molecules ; 24(9)2019 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-31067626

RESUMO

The occurrence of damage on bacterial DNA (mediated by antibiotics, for example) is intimately associated with the activation of the SOS system. This pathway is related to the development of mutations that might result in the acquisition and spread of resistance and virulence factors. The inhibition of the SOS response has been highlighted as an emerging resource, in order to reduce the emergence of drug resistance and tolerance. Herein, we evaluated the ability of betulinic acid (BA), a plant-derived triterpenoid, to reduce the activation of the SOS response and its associated phenotypic alterations, induced by ciprofloxacin in Staphylococcus aureus. BA did not show antimicrobial activity against S. aureus (MIC > 5000 µg/mL), however, it (at 100 and 200 µg/mL) was able to reduce the expression of recA induced by ciprofloxacin. This effect was accompanied by an enhancement of the ciprofloxacin antimicrobial action and reduction of S. aureus cell volume (as seen by flow cytometry and fluorescence microscopy). BA could also increase the hyperpolarization of the S. aureus membrane, related to the ciprofloxacin action. Furthermore, BA inhibited the progress of tolerance and the mutagenesis induced by this drug. Taken together, these findings indicate that the betulinic acid is a promising lead molecule in the development helper drugs. These compounds may be able to reduce the S. aureus mutagenicity associated with antibiotic therapies.


Assuntos
Farmacorresistência Bacteriana/efeitos dos fármacos , Recombinases Rec A/genética , Staphylococcus aureus/genética , Triterpenos/farmacologia , Ciprofloxacino/efeitos adversos , Ciprofloxacino/farmacologia , DNA Bacteriano/efeitos dos fármacos , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Humanos , Mutagênese/efeitos dos fármacos , Mutagênese/genética , Resposta SOS (Genética)/efeitos dos fármacos , Resposta SOS (Genética)/genética , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/patogenicidade , Fatores de Virulência/genética
3.
Int J Mol Sci ; 20(9)2019 Apr 26.
Artigo em Inglês | MEDLINE | ID: mdl-31027327

RESUMO

The MAPPIT (mammalian protein protein interaction trap) method allows high-throughput detection of protein interactions by very simple co-transfection of three plasmids in HEK293T cells, followed by a luciferase readout. MAPPIT detects a large percentage of all protein interactions, including those requiring posttranslational modifications and endogenous or exogenous ligands. Here, we present a straightforward method that allows detailed mapping of interaction interfaces via MAPPIT. The method provides insight into the interaction mechanism and reveals how this is affected by disease-associated mutations. By combining error-prone polymerase chain reaction (PCR) for random mutagenesis, 96-well DNA prepping, Sanger sequencing, and MAPPIT via 384-well transfections, we test the effects of a large number of mutations of a selected protein on its protein interactions. The entire screen takes less than three months and interactions with multiple partners can be studied in parallel. The effect of mutations on the MAPPIT readout is mapped on the protein structure, allowing unbiased identification of all putative interaction sites. We have thus far analysed 6 proteins and mapped their interfaces for 16 different interaction partners. Our method is broadly applicable as the required tools are simple and widely available.


Assuntos
Mutagênese/genética , Mapeamento de Interação de Proteínas/métodos , Animais , Humanos , Ligação Proteica
5.
Molecules ; 24(6)2019 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-30889828

RESUMO

The members of the Old Yellow Enzyme (OYE) family are capable of catalyzing the asymmetric reduction of (E/Z)-citral to (R)-citronellal-a key intermediate in the synthesis of L-menthol. The applications of OYE-mediated biotransformation are usually hampered by its insufficient enantioselectivity and low activity. Here, the (R)-enantioselectivity of Old Yellow Enzyme from Saccharomyces cerevisiae CICC1060 (OYE2y) was enhanced through protein engineering. The single mutations of OYE2y revealed that the sites R330 and P76 could act as the enantioselectivity switch of OYE2y. Site-saturation mutagenesis was conducted to generate all possible replacements for the sites R330 and P76, yielding 17 and five variants with improved (R)-enantioselectivity in the (E/Z)-citral reduction, respectively. Among them, the variants R330H and P76C partly reversed the neral derived enantioselectivity from 32.66% e.e. (S) to 71.92% e.e. (R) and 37.50% e.e. (R), respectively. The docking analysis of OYE2y and its variants revealed that the substitutions R330H and P76C enabled neral to bind with a flipped orientation in the active site and thus reverse the enantioselectivity. Remarkably, the double substitutions of R330H/P76M, P76G/R330H, or P76S/R330H further improved (R)-enantioselectivity to >99% e.e. in the reduction of (E)-citral or (E/Z)-citral. The results demonstrated that it was feasible to alter the enantioselectivity of OYEs through engineering key residue distant from active sites, e.g., R330 in OYE2y.


Assuntos
Aldeídos/metabolismo , Engenharia Metabólica/métodos , Monoterpenos/metabolismo , NADPH Desidrogenase/química , Saccharomyces cerevisiae/enzimologia , Sequência de Aminoácidos , Substituição de Aminoácidos , Biocatálise , Modelos Moleculares , Mutagênese/genética , Proteínas Mutantes/metabolismo , NADPH Desidrogenase/metabolismo , Oxirredução , Estereoisomerismo
6.
Methods Mol Biol ; 1957: 251-269, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30919359

RESUMO

ß-Arrestins (ß-arrs) were originally appreciated for the roles they play in the desensitization and internalization of G protein-coupled receptors (GPCRs). They are also now known to act as molecular scaffolds, providing control in multiple signalling pathways. Through their scaffolding properties, ß-arrs dynamically regulate the activity and/or subcellular distribution of protein partners giving rise to an appropriate cellular response. There are two ß-arr isoforms, namely, ß-arr1 and ß-arr2, which share high sequence homology and structural conservation. While the ß-arrs often display conserved overlapping roles, decisive differences between the isoforms also exist. A striking example of this is the subcellular distribution of the ß-arr isoforms. While ß-arr1 is distributed both in cytoplasmic and nuclear compartments, ß-arr2 displays an apparent cytoplasmic distribution. Both ß-arrs are actively imported into the nucleus, but ß-arr2 is constitutively exported by a leptomycin B-sensitive pathway due to a nuclear export signal in its C-terminus that is absent in ß-arr1. ß-arr2 therefore undergoes constitutive nucleocytoplasmic shuttling enabling the displacement of nuclear binding cargoes, such as Mdm2. Here, we describe methods to explore the differential nucleocytoplasmic shuttling capacities of the ß-arrs.


Assuntos
Núcleo Celular/metabolismo , Biologia Molecular/métodos , beta-Arrestinas/metabolismo , Transporte Ativo do Núcleo Celular , Sequência de Aminoácidos , Bioensaio , Imunofluorescência , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Células HeLa , Humanos , Processamento de Imagem Assistida por Computador , Cinética , Modelos Biológicos , Mutagênese/genética , Saccharomyces cerevisiae/metabolismo , Transdução de Sinais , Transformação Genética , beta-Arrestinas/química
7.
Nature ; 566(7743): 275-278, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30700905

RESUMO

Genetic instability, a heritable increase in the rate of genetic mutation, accelerates evolutionary adaptation1 and is widespread in cancer2,3. In mammals, instability can arise from damage to both copies of genes involved in DNA metabolism and cell cycle regulation4 or from inactivation of one copy of a gene whose product is present in limiting amounts (haploinsufficiency5); however, it has proved difficult to determine the relative importance of these two mechanisms. In Escherichia coli6, the application of repeated, strong selection enriches for genetic instability. Here we have used this approach to evolve genetic instability in diploid cells of the budding yeast Saccharomyces cerevisiae, and have isolated clones with increased rates of point mutation, mitotic recombination, and chromosome loss. We identified candidate, heterozygous, instability-causing mutations; engineering these mutations, as heterozygotes, into the ancestral diploid strain caused genetic instability. Mutations that inactivated one copy of haploinsufficient genes were more common than those that dominantly altered the function of the mutated gene copy. The mutated genes were enriched for genes functioning in transport, protein quality control, and DNA metabolism, and have revealed new targets for genetic instability7-11, including essential genes. Although only a minority (10 out of 57 genes with orthologues or close homologues) of the targets we identified have homologous human genes that have been implicated in cancer2, the remainder are candidates to contribute to human genetic instability. To test this hypothesis, we inactivated six examples in a near-haploid human cell line; five of these mutations increased instability. We conclude that single genetic events cause genetic instability in diploid yeast cells, and propose that similar, heterozygous mutations in mammalian homologues initiate genetic instability in cancer.


Assuntos
Evolução Molecular , Instabilidade Genômica/genética , Heterozigoto , Modelos Genéticos , Mutação , Neoplasias/genética , Saccharomyces cerevisiae/genética , Linhagem Celular , Diploide , Haploinsuficiência/genética , Humanos , Mutagênese/genética , Taxa de Mutação , Mutação Puntual
8.
Int J Mol Sci ; 20(3)2019 Feb 12.
Artigo em Inglês | MEDLINE | ID: mdl-30759774

RESUMO

Lipases with unique substrate specificity are highly desired in biotechnological applications. In this study, a putative marine Geobacillus sp. monoacylglycerol lipase (GMGL) encoded gene was identified by a genomic mining strategy. The gene was expressed in Escherichia coli as a His-tag fusion protein and purified by affinity chromatography with a yield of 264 mg per liter fermentation broth. The recombinant GMGL shows the highest hydrolysis activity at 60 °C and pH 8.0, and the half-life was 60 min at 70 °C. The GMGL is active on monoacylglycerol (MAG) substrate but not diacylglycerol (DAG) or triacylglycerol (TAG), and produces MAG as the single product in the esterification reaction. Modeling structure analysis showed that the catalytic triad is formed by Ser97, Asp196 and His226, and the flexible cap region is constituted by residues from Ala120 to Thr160. A mutagenesis study on Leu142, Ile145 and Ile170 located in the substrate binding tunnel revealed that these residues were related with its substrate specificity. The kcat/Km value toward the pNP-C6 substrate in mutants Leu142Ala, Ile145Ala and Ile170Phe increased to 2.3-, 1.4- and 2.2-fold as compared to that of the wild type, respectively.


Assuntos
Geobacillus/genética , Monoacilglicerol Lipases/genética , Mutagênese/genética , Sequência de Aminoácidos , Catálise , Clonagem Molecular/métodos , Diglicerídeos/genética , Escherichia coli/genética , Esterificação/genética , Concentração de Íons de Hidrogênio , Hidrólise , Proteínas Recombinantes/genética , Alinhamento de Sequência , Especificidade por Substrato/genética , Triglicerídeos/genética
9.
Nucleic Acids Res ; 47(4): 1964-1976, 2019 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-30605516

RESUMO

Random mutagenesis for the hyperthermophilic archaeon Thermococcus kodakarensis was established by the insertion of an artificial transposon designed to allow easy identification of the transposon-inserted locus. The phenotypic screening was applied for the isolation of thermosensitive mutants of T. kodakarensis, which resulted in the isolation of 16 mutants showing defective growth at the supraoptimal temperature 93°C. The high occurrence of the mutants suggested that the high thermotolerance of hyperthermophiles was achieved by a combination of diverse gene functions. The transposon insertion sites in two-thirds of the mutants were identified in a group of genes responsible for tRNA modifications including 7-formamidino-7-deaza-guanosine (archaeosine), N1-methyladenosine/N1-methylinosine, N4-acetylcytidine, and N2-dimethylguanosine/N2,N2-dimethylguanosine. LC-MS/MS analyses of tRNA nucleosides and fragments exhibited disappearance of the corresponding modifications in the mutants. The melting temperature of total tRNA fraction isolated from the mutants lacking archaeosine or N1-methyladenosine/N1-methylinosine decreased significantly, suggesting that the thermosensitive phenotype of these mutants was attributed to low stability of the hypomodified tRNAs. Genes for metabolism, transporters, and hypothetical proteins were also identified in the thermosensitive mutants. The present results demonstrated the usefulness of random mutagenesis for the studies on the hyperthermophile, as well as crucial roles of tRNA modifications in cellular thermotolerance.


Assuntos
Guanosina/análogos & derivados , Mutagênese/genética , RNA de Transferência/genética , Thermococcus/genética , Sequência de Bases , Cromatografia Líquida , Regulação da Expressão Gênica em Archaea , Guanosina/química , Guanosina/genética , Nucleosídeos/química , Nucleosídeos/genética , Espectrometria de Massas em Tandem , Temperatura Ambiente
10.
Nucleic Acids Res ; 47(4): 1871-1879, 2019 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-30605521

RESUMO

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone is a potent nicotine carcinogen that leads to many DNA lesions, the most persistent being the O2-[4-oxo-4-(3-pyridyl)butyl]thymine adduct (POB-T). Although the experimental mutagenic profile for the minor groove POB-T lesion has been previously reported, the findings are puzzling in terms of the human polymerases involved. Specifically, while pol κ typically replicates minor groove adducts, in vivo studies indicate pol η replicates POB-T despite being known for processing major groove adducts. Our multiscale modeling approach reveals that the canonical (anti) glycosidic orientation of POB-T can fit in the pol κ active site, but only a unique (syn) POB-T conformation is accommodated by pol η. These distinct binding orientations rationalize the differential in vitro mutagenic spectra based on the preferential stabilization of dGTP and dTTP opposite the lesion for pol κ and η, respectively. Overall, by uncovering the first evidence for the replication of a damaged pyrimidine in the syn glycosidic orientation, the current work provides the insight necessary to clarify a discrepancy in the DNA replication literature, expand the biological role of the critical human pol η, and understand the mutational signature in human cancers associated with tobacco exposure.


Assuntos
Carcinógenos/química , Adutos de DNA/genética , Dano ao DNA/efeitos dos fármacos , Replicação do DNA/efeitos dos fármacos , Biologia Computacional , Adutos de DNA/química , Humanos , Mutagênese/genética , Mutagênicos/química , Mutação , Nitrosaminas , Timina/química , Tabaco/efeitos adversos , Tabaco/química
11.
Dis Model Mech ; 12(1)2019 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-30626575

RESUMO

A wide range of genetic mouse models is available to help researchers dissect human disease mechanisms. Each type of model has its own distinctive characteristics arising from the nature of the introduced mutation, as well as from the specific changes to the gene of interest. Here, we review the current range of mouse models with mutations in genes causative for the human neurodegenerative disease amyotrophic lateral sclerosis. We focus on the two main types of available mutants: transgenic mice and those that express mutant genes at physiological levels from gene targeting or from chemical mutagenesis. We compare the phenotypes for genes in which the two classes of model exist, to illustrate what they can teach us about different aspects of the disease, noting that informative models may not necessarily mimic the full trajectory of the human condition. Transgenic models can greatly overexpress mutant or wild-type proteins, giving us insight into protein deposition mechanisms, whereas models expressing mutant genes at physiological levels may develop slowly progressing phenotypes but illustrate early-stage disease processes. Although no mouse models fully recapitulate the human condition, almost all help researchers to understand normal and abnormal biological processes, providing that the individual characteristics of each model type, and how these may affect the interpretation of the data generated from each model, are considered and appreciated.


Assuntos
Esclerose Amiotrófica Lateral/genética , Esclerose Amiotrófica Lateral/fisiopatologia , Modelos Animais de Doenças , Animais , Marcação de Genes , Camundongos Transgênicos , Mutagênese/genética , Mutação/genética
12.
Dis Model Mech ; 12(1)2019 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-30626588

RESUMO

Over the past decade, new methods and procedures have been developed to generate genetically engineered mouse models of human disease. This At a Glance article highlights several recent technical advances in mouse genome manipulation that have transformed our ability to manipulate and study gene expression in the mouse. We discuss how conventional gene targeting by homologous recombination in embryonic stem cells has given way to more refined methods that enable allele-specific manipulation in zygotes. We also highlight advances in the use of programmable endonucleases that have greatly increased the feasibility and ease of editing the mouse genome. Together, these and other technologies provide researchers with the molecular tools to functionally annotate the mouse genome with greater fidelity and specificity, as well as to generate new mouse models using faster, simpler and less costly techniques.


Assuntos
Pesquisa Biomédica , Modelos Animais de Doenças , Animais , Células-Tronco Embrionárias/metabolismo , Edição de Genes , Camundongos , Mutagênese/genética , Interferência de RNA
13.
Plant Cell Rep ; 38(4): 487-501, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30684023

RESUMO

KEY MESSAGE: The analysis of 93 mutant alleles in 18 genes demonstrated that CRISPR-Cas9 is a robust tool for targeted mutagenesis in maize, permitting efficient generation of single and multiple knockouts. CRISPR-Cas9 technology is a simple and efficient tool for targeted mutagenesis of the genome. It has been implemented in many plant species, including crops such as maize. Here we report single- and multiple-gene mutagenesis via stably transformed maize plants. Two different CRISPR-Cas9 vectors were used allowing the expression of multiple guide RNAs and different strategies to knockout either independent or paralogous genes. A total of 12 plasmids, representing 28 different single guide RNAs (sgRNAs), were generated to target 20 genes. For 18 of these genes, at least one mutant allele was obtained, while two genes were recalcitrant to sequence editing. 19% (16/83) of mutant plants showed biallelic mutations. Small insertions or deletions of less than ten nucleotides were most frequently observed, regardless of whether the gene was targeted by one or more sgRNAs. Deletions of defined regions located between the target sites of two guide RNAs were also reported although the exact deletion size was variable. Double and triple mutants were created in a single step, which is especially valuable for functional analysis of genes with strong genetic linkage. Off-target effects were theoretically limited due to rigorous sgRNA design and random experimental checks at three potential off-target sites did not reveal any editing. Sanger chromatograms allowed to unambiguously class the primary transformants; the majority (85%) were fully edited plants transmitting systematically all detected mutations to the next generation, generally following Mendelian segregation.


Assuntos
Sistemas CRISPR-Cas/genética , Técnicas de Inativação de Genes/métodos , Zea mays/genética , Edição de Genes , Genoma de Planta/genética , Mutagênese/genética
14.
Artigo em Inglês | MEDLINE | ID: mdl-30678824

RESUMO

Titanium dioxide nanoparticles (TiO2-NPs) are widely used in the cosmetics, health, and food industries, but their safety and genotoxicity remain a matter of debate. We investigated whether TiO2-NPs could induce gene mutations in mouse lymphoma L5178Y cells and Salmonella typhimurium strains TA97a, TA98, TA100, TA102, and TA1535. Following preliminary tests, 2 mg/mL for the mouse lymphoma gene mutation assay and 1.25 mg/plate for the in vitro bacterial reverse mutation assay (Ames test) were selected as the highest concentrations. Exposure to TiO2-NPs for 4 or 24 h with or without S9 metabolic activation did not increase mutation frequency for any of the concentrations tested in L5178Y cells. In the Ames test, TiO2-NPs did not induce reverse mutation in the bacterial strains. No positive mutagenic responses were observed in either test system, and therefore we cannot classify TiO2-NPs as mutagenic; further testing will be required to determine conclusively whether TiO2-NPs are genotoxic.


Assuntos
Nanopartículas Metálicas/toxicidade , Mutagênese/efeitos dos fármacos , Testes de Mutagenicidade , Titânio/farmacologia , Animais , Linhagem Celular Tumoral , Dano ao DNA/genética , Camundongos , Mutagênese/genética , Taxa de Mutação , Salmonella typhimurium/genética
15.
Methods Mol Biol ; 1917: 121-143, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30610633

RESUMO

Precise genome engineering can be efficiently made using the revolutionary tool named CRISPR/Cas (clustered regularly interspaced short palindromic repeat/CRISPR-associated protein) systems. Adapted from the bacterial immune system, CRISPR/Cas systems can generate highly specific double-strand breaks (DSBs) at the target site, and desired sequence modifications can be introduced during the DSB repair process, such as nonhomologous end-joining (NHEJ) or homology-directed repair (HDR) pathways. CRISPR/Cas9 is the most widely used genome editing tool for targeted mutagenesis, precise sequence modification, transcriptional reprogramming, epigenome editing, disease treatment, and many more. The ease of use and high specificity make CRISPR/Cas9 a great tool not only for basic researches but also for crop trait improvements, such as higher grain yield, better tolerance to abiotic stresses, enhanced disease resistance, and better nutritional contents. In this protocol, we present a step-by-step guide to the CRISPR/Cas9-mediated targeted mutagenesis in maize Hi II genotype. Detailed procedures will guide through the essential steps including gRNA design, CRISPR/Cas9 vector construction, Agrobacterium-mediated maize immature embryo transformation, and molecular analysis of the transgenic plants to identify desired mutant lines.


Assuntos
Agrobacterium/genética , Sistemas CRISPR-Cas/genética , Edição de Genes/métodos , Zea mays/genética , Mutagênese/genética , Transformação Genética/genética
16.
Methods Mol Biol ; 1917: 269-281, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30610643

RESUMO

Various CRISPR/Cas9 systems have been extensively applied for targeted mutagenesis to generate mutants that impaired in genes of interest. Clustered regularly interspersed short palindromic repeats (CRISPR) from Prevotella and Francisella 1 (Cpf1) is new RNA-directed endonuclease possessing some differences as compared to Cas9. Several papers have shown that Cpf1 could be a versatile tool in plant genome engineering. Cfp1 from Francisella novicida (FnCpf1) recognizes TTN as its protospacer adjacent motif (PAM). TTN is a shortest PAM among other known Cpf1s such as AsCpf1 or LbCpf1, which use TTTN as PAM. The length of PAM can be the restriction of the number of target sequences. Cpf1 generates cohesive DNA end after the digestion of target sequences. Sticky DNA end is thought to appropriate for in vivo ligation rather than blunt DNA end created by Cas9. Therefore, FnCpf1 is practical for targeted mutagenesis experiments. The application of FnCpf1-mediated targeted mutagenesis to the plant genome engineering could accelerate molecular breeding of crops. Here, we describe procedures for targeted mutagenesis in tobacco using FnCpf1.


Assuntos
Genoma de Planta/genética , Mutagênese/fisiologia , Proteínas de Plantas/metabolismo , Tabaco/genética , Mutagênese/genética , Proteínas de Plantas/genética
17.
Methods Mol Biol ; 1931: 61-73, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30652283

RESUMO

Induced mutagenesis is a powerful approach to generate variations for elucidation of gene function and to create new traits for breeding. Here, we described a procedure to develop a pedigreed mutant library through chemical mutagenesis with ethylmethane sulfonate (EMS) treated seeds in sorghum and discussed its potential to generate new traits for sorghum improvement. Unlike random mutagenesis, a pedigreed mutant library, once properly established, can serve as a powerful resource to isolate and recover mutations of both agronomical and biological importance. With the development of affordable and high-throughput next-generation sequencing technologies, identification of causal mutations from a mutant library with a uniform genetic background becomes increasingly efficient and cost-effective. Fast causal gene discovery from mutant libraries combined with precise genome editing techniques will accelerate incorporation of new traits and revolutionize crop breeding.


Assuntos
Mutação/genética , Sorghum/genética , Grão Comestível/efeitos dos fármacos , Grão Comestível/genética , Metanossulfonato de Etila/farmacologia , Biblioteca Gênica , Genoma de Planta/efeitos dos fármacos , Genoma de Planta/genética , Genótipo , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Mutagênese/efeitos dos fármacos , Mutagênese/genética , Mutação/efeitos dos fármacos , Linhagem , Melhoramento Vegetal/métodos , Sorghum/efeitos dos fármacos
18.
Methods Mol Biol ; 1898: 69-80, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30570724

RESUMO

We describe a recombineering-based method for the genetic manipulation of lytically replicating bacteriophages, focusing on mycobacteriophages. The approach utilizes recombineering-proficient strains of Mycobacterium smegmatis and employs a cotransformation strategy with purified phage genomic DNA and a mutagenic substrate, which selects for only those cells that are competent to take up DNA. The cotransformation method, combined with the high rates of recombination obtained in M. smegmatis recombineering strains, allows for the efficient and rapid generation of bacteriophage mutants.


Assuntos
Bacteriófagos/genética , DNA/genética , Mycobacterium smegmatis/genética , Recombinação Genética , Bacteriófagos/química , DNA/química , Eletroquimioterapia , Eletroporação , Engenharia Genética , Mutagênese/genética , Mycobacterium smegmatis/virologia
19.
Mutagenesis ; 34(1): 33-40, 2019 03 06.
Artigo em Inglês | MEDLINE | ID: mdl-30541036

RESUMO

Valid and predictive models for classifying Ames mutagenicity have been developed using conformal prediction. The models are Random Forest models using signature molecular descriptors. The investigation indicates, on excluding not-strongly mutagenic compounds (class B), that the validity for mutagenic compounds is increased for the predictions based on both public and the Division of Genetics and Mutagenesis, National Institute of Health Sciences of Japan (DGM/NIHS) data while less so when using only the latter data source. The former models only result in valid predictions for the majority, non-mutagenic, class whereas the latter models are valid for both classes, i.e. mutagenic and non-mutagenic compounds. These results demonstrate the importance of data consistency manifested through the superior predictive quality and validity of the models based only on DGM/NIHS generated data compared to a combination of this data with public data sources.


Assuntos
Testes de Mutagenicidade/tendências , Mutagênicos/toxicidade , Relação Quantitativa Estrutura-Atividade , Simulação por Computador , Japão , Mutagênese/genética
20.
Environ Mol Mutagen ; 60(2): 100-121, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30536466

RESUMO

The interpretation and significance of DNA adduct data, their causal relationship to mutations, and their role in risk assessment have been debated for many years. An extended effort to identify key questions and collect relevant data to address them was focused on the ubiquitous low MW N7-alkyl/hydroxyalkylguanine adducts. Several academic, governmental, and industrial laboratories collaborated to gather new data aimed at better understanding the role and potential impact of these adducts in quantifiable genotoxic events (gene mutations/micronucleus). This review summarizes and evaluates the status of dose-response data for DNA adducts and mutations from recent experimental work with standard mutagenic agents and ethylene oxide and propylene oxide, and the importance for risk assessment. This body of evidence demonstrates that small N7-alkyl/hydroxyalkylguanine adducts are not pro-mutagenic and, therefore, adduct formation alone is not adequate evidence to support a mutagenic mode of action. Quantitative methods for dose-response analysis and derivation of thresholds, benchmark dose (BMD), or other points-of-departure (POD) for genotoxic events are now available. Integration of such analyses of genetox data is necessary to properly assess any role for DNA adducts in risk assessment. Regulatory acceptance and application of these insights remain key challenges that only the regulatory community can address by applying the many learnings from recent research. The necessary tools, such as BMDs and PODs, and the example datasets, are now available and sufficiently mature for use by the regulatory community. Environ. Mol. Mutagen. 60: 100-121, 2019. © 2018 The Authors. Environmental and Molecular Mutagenesis published by Wiley Periodicals, Inc. on behalf of Environmental Mutagen Society.


Assuntos
Adutos de DNA/genética , Mutagênese/efeitos dos fármacos , Mutação/efeitos dos fármacos , Adutos de DNA/química , Adutos de DNA/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , Compostos de Epóxi/toxicidade , Óxido de Etileno/toxicidade , Humanos , Peso Molecular , Mutagênese/genética , Mutagênicos/toxicidade , Mutação/genética , Medição de Risco
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA