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1.
Sheng Wu Gong Cheng Xue Bao ; 35(8): 1557-1565, 2019 Aug 25.
Artigo em Chinês | MEDLINE | ID: mdl-31441627

RESUMO

To construct a eukaryotic expression plasmid containing the luciferase reporter gene (Fluc) to quickly detect apoptosis. Four amino acids, Asp-Glu-Val-Asp (DEVD), the recognize motif of Caspase-3, were introduced into the middle of the Fluc-C and N fragment. Meanwhile, four amino acids, Asp-Glu-Val-Gly (DEVG), were selected as a negative control. Subsequently, the recombinant gene was cloned into the N and C terminal end of the split intein, and named as pFluc-DEVD and pFluc-DEVG. Then the plasmids were transfected into cells and renilla luciferase was co-transfected in each sample as an internal control for transfection efficiency. Then the apoptosis level was detected by the double luciferase reporter gene and the Western blotting analysis. The results showed that when apoptosis occurred, the content of firefly luciferase expressed in the pFluc-DEVD plasmid transfected group was about 3 times higher than pFluc-DEVG plasmid transfected group. Furthermore, Western blotting detection indicated that the Fluc level was significantly increased in pFluc-DEVD transfected group when pre-treated by apoptosis stimulants. The activation degree of Caspase-3 was closely related to the expression of Fluc, and had a significant statistical difference. These results confirmed that firefly luciferase protein expressed by pFluc-DEVD plasmid can be cleaved by the intracellular Caspase-3 enzyme, and this plasmid can accurately reflect the cell apoptosis level, which provides a useful method for quantitative detection of apoptosis.


Assuntos
Apoptose , Genes Reporter , Luciferases de Vaga-Lume , Transfecção
2.
Arch Virol ; 164(10): 2505-2513, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31377888

RESUMO

Infectious hematopoietic necrosis virus (IHNV) was developed as a vector to aid the construction of vaccines against viral diseases such as viral hemorrhagic septicemia virus, spring viremia of carp virus, and influenza virus H1N1. However, the optimal site for foreign gene expression in the IHNV vector has not been determined. In the present study, five recombinant viruses with the green fluorescence protein (GFP) gene inserted into different genomic junction regions of the IHNV genomic sequence were generated using reverse genetics technology. Viral growth was severely delayed when the GFP gene was inserted into the intergenic region between the N and P genes. Real-time fluorescence quantitative PCR assays showed that the closer the GFP gene was inserted towards the 3' end, the higher the GFP mRNA levels. Measurement of the GFP fluorescence intensity, which is the most direct method to determine the GFP protein expression level, showed that the highest GFP protein level was obtained when the gene was inserted into the intergenic region between the P and M genes. The results of this study suggest that the P and M gene junction region is the optimal site within the IHNV vector to express foreign genes, providing valuable information for the future development of live vector vaccines.


Assuntos
Expressão Gênica , Vetores Genéticos , Vírus da Necrose Hematopoética Infecciosa/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Fluorometria , Genes Reporter , Proteínas de Fluorescência Verde/biossíntese , Proteínas de Fluorescência Verde/genética , Reação em Cadeia da Polimerase em Tempo Real , Genética Reversa
3.
J Agric Food Chem ; 67(33): 9286-9294, 2019 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-31339733

RESUMO

Natural aryl hydrocarbon (AHR) ligands have been identified in food and herbal medicines, and they may exhibit beneficial activity in humans. In this study, white button (WB) feeding significantly decreased AHR target gene expression in the small intestine of both conventional and germ-free mice. High-performance liquid chromatography (HPLC) fractionation and ultra-high-performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) combined with an AHR-responsive cell-based luciferase gene reporter assay were used to isolate and characterize benzothiazole (BT) derivatives and 6-methylisoquinoline (6-MIQ) as AHR modulators from WB mushrooms. The study showed dose-dependent changes of AHR transformation determined by the cell-based luciferase gene reporter assay and transcription of CYP1A1 in human Caco-2 cells by BT derivatives and 6-MIQ. These findings suggested that WB mushroom contains new classes of natural AHR modulators and demonstrated HPLC fractionation and UHPLC-MS/MS combined with a cell-based luciferase gene reporter assay as a useful approach for isolation and characterization of the previously unidentifed AHR modulators from natural products.


Assuntos
Agaricus/química , Extratos Vegetais/química , Extratos Vegetais/isolamento & purificação , Receptores de Hidrocarboneto Arílico/genética , Animais , Benzotiazóis/química , Benzotiazóis/isolamento & purificação , Benzotiazóis/farmacologia , Linhagem Celular , Cromatografia Líquida de Alta Pressão , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1A1/metabolismo , Genes Reporter , Humanos , Isoquinolinas/química , Isoquinolinas/isolamento & purificação , Isoquinolinas/farmacologia , Ligantes , Camundongos , Extratos Vegetais/farmacologia , Receptores de Hidrocarboneto Arílico/metabolismo , Espectrometria de Massas em Tandem , Ativação Transcricional/efeitos dos fármacos , Verduras/química
4.
Plant Mol Biol ; 101(1-2): 149-162, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31267255

RESUMO

KEY MESSAGE: Here we describe that the regulation of MdWRKY31 on MdHIR4 in transcription and translation levels associated with disease in apple. The phytohormone salicylic acid (SA) is a main factor in apple (Malus domestica) production due to its function in disease resistance. WRKY transcription factors play a vital role in response to stress. An RNA-seq analysis was conducted with 'Royal Gala' seedlings treated with SA to identify the WRKY regulatory mechanism of disease resistance in apple. The analysis indicated that MdWRKY31 was induced. A quantitative real-time polymerase chain reaction (qPCR) analysis demonstrated that the expression of MdWRKY31 was induced by SA and flg22. Ectopic expression of MdWRKY31 in Arabidopsis and Nicotiana benthamiana increased the resistance to flg22 and Pseudomonas syringae tomato (Pst DC3000). A yeast two-hybrid screen was conducted to further analyze the function of MdWRKY31. As a result, hypersensitive-induced reaction (HIR) protein MdHIR4 interacted with MdWRKY31. Biomolecular fluorescence complementation, yeast two-hybrid, and pull-down assays demonstrated the interaction. In our previous study, MdHIR4 conferred decreased resistance to Botryosphaeria dothidea (B. dothidea). A viral vector-based transformation assay indicated that MdWRKY31 evaluated the transcription of SA-related genes, including MdPR1, MdPR5, and MdNPR1 in an MdHIR4-dependent way. A GUS analysis demonstrated that the w-box, particularly w-box2, of the MdHIR4 promoter played a major role in the responses to SA and B. dothidea. Electrophoretic mobility shift assays, yeast one-hybrid assay, and chromatin immunoprecipitation-qPCR demonstrated that MdWRKY31 directly bound to the w-box2 motif in the MdHIR4 promoter. GUS staining activity and a protein intensity analysis further showed that MdWRKY31 repressed MdHIR4 expression. Taken together, our findings reveal that MdWRKY31 regulated plant resistance to B. dothidea through the SA signaling pathway by interacting with MdHIR4.


Assuntos
Resistência à Doença , Malus/genética , Doenças das Plantas/imunologia , Reguladores de Crescimento de Planta/farmacologia , Proteínas de Plantas/metabolismo , Ácido Salicílico/farmacologia , Arabidopsis/genética , Arabidopsis/imunologia , Arabidopsis/microbiologia , Ascomicetos/fisiologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Frutas/genética , Frutas/imunologia , Frutas/microbiologia , Regulação da Expressão Gênica de Plantas , Genes Reporter , Malus/imunologia , Malus/microbiologia , Doenças das Plantas/microbiologia , Proteínas de Plantas/genética , Regiões Promotoras Genéticas/genética , Pseudomonas syringae/fisiologia , Plântula/genética , Plântula/imunologia , Plântula/microbiologia , Transdução de Sinais , Tabaco/genética , Tabaco/imunologia , Tabaco/microbiologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Técnicas do Sistema de Duplo-Híbrido
5.
World J Microbiol Biotechnol ; 35(8): 119, 2019 Jul 22.
Artigo em Inglês | MEDLINE | ID: mdl-31332541

RESUMO

The microalgal genus of Nannochloropsis is considered one of the most promising organisms for the production of biofuels due to their high lipid content. Transformation systems for marine Nannochloropsis species have been established in the recent decade, however, genetic manipulation of Nannochloropsis limnetica, the only known freshwater species in this genus, is not yet available. Based on established marine Nannochloropsis species electrotransformation protocol, nuclear genetic transformation was established in N. limnetica, meanwhile the appropriate antibiotic selection concentration and electric field strength of electroporation were determined. For the selection of transformants in N. limnetica on plates, 0.07 µg mL-1 of zeocin or 5 µg mL-1 of hygromycin B was proved sufficient, and the transformation efficiency was < 2 × 10-8 with a single pulse ranging from 2200 to 2600 V using 2-mm electroporation cuvettes. Pretreatment of N. limnetica with 10 mM lithium acetate and 3 mM dithiothreitol before electroporation increased transformation efficiency hundreds of times, and the highest transformation efficiency of 10-11 × 10-6 was obtained with an electric field strength of 12,000 V/cm. Our results help to expand the biotechnological applications of this freshwater species and provide means for successful electrotransformation of other microalgae as well. High-efficiency transformation of freshwater Nannochloropsis pretreatment of N. limnetica with 10 mM lithium acetate and 3 mM dithiothreitol before electroporation increased transformation efficiency hundreds of times.


Assuntos
Eletroporação , Água Doce/microbiologia , Microalgas/metabolismo , Estramenópilas/metabolismo , Acetatos , Regulação da Expressão Gênica , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Microalgas/genética , Plasmídeos/genética , Plasmídeos/metabolismo , Estramenópilas/genética , Transformação Genética
6.
Genome Biol ; 20(1): 140, 2019 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-31307522

RESUMO

BACKGROUND: Despite continual progress in the identification and characterization of trait- and disease-associated variants that disrupt transcription factor (TF)-DNA binding, little is known about the distribution of TF binding deactivating mutations (deMs) in enhancer sequences. Here, we focus on elucidating the mechanism underlying the different densities of deMs in human enhancers. RESULTS: We identify two classes of enhancers based on the density of nucleotides prone to deMs. Firstly, fragile enhancers with abundant deM nucleotides are associated with the immune system and regular cellular maintenance. Secondly, stable enhancers with only a few deM nucleotides are associated with the development and regulation of TFs and are evolutionarily conserved. These two classes of enhancers feature different regulatory programs: the binding sites of pioneer TFs of FOX family are specifically enriched in stable enhancers, while tissue-specific TFs are enriched in fragile enhancers. Moreover, stable enhancers are more tolerant of deMs due to their dominant employment of homotypic TF binding site (TFBS) clusters, as opposed to the larger-extent usage of heterotypic TFBS clusters in fragile enhancers. Notably, the sequence environment and chromatin context of the cognate motif, other than the motif itself, contribute more to the susceptibility to deMs of TF binding. CONCLUSIONS: This dichotomy of enhancer activity is conserved across different tissues, has a specific footprint in epigenetic profiles, and argues for a bimodal evolution of gene regulatory programs in vertebrates. Specifically encoded stable enhancers are evolutionarily conserved and associated with development, while differently encoded fragile enhancers are associated with the adaptation of species.


Assuntos
Adaptação Biológica , Elementos Facilitadores Genéticos , Evolução Molecular , Mutação , Animais , Genes Reporter , Células Hep G2 , Humanos , Camundongos Transgênicos
7.
J Agric Food Chem ; 67(31): 8649-8659, 2019 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-31283213

RESUMO

Spent coffee grounds (SCG) are the most abundant coffee byproduct and are generally discarded as waste. The horticultural use of SCG and SCG compost (SCGC) has become popular due to a growing interest in environmentally friendly measures for waste disposal. Estrogen-like endocrine disrupting chemicals in the soil can be absorbed by plants and subsequently by humans who consume these plants. The objectives of this study are to determine the phytochemical profiles of extracts of SCG and SCGC and to evaluate the estrogen-like activities of SCG, SCGC, and the major coffee phenolic acids, specifically, 5-O-caffeoylquinic acid (CQA), caffeic acid, and ferulic acid. Their inductive effects on estrogen receptor (ER)-mediated gene transcription have been examined in cultured cell lines. CQA was the most abundant phenolic acid in SCG and SCGC and was further examined for its ER-mediated estrogen-like activity using various assays. This is the first study to report the estrogen-like signaling activities of coffee byproducts and their major constituents.


Assuntos
Coffea/química , Hidroxibenzoatos/metabolismo , Fitoestrógenos/metabolismo , Extratos Vegetais/metabolismo , Receptores Estrogênicos/genética , Ativação Transcricional , Resíduos/análise , Animais , Ácidos Cafeicos/análise , Ácidos Cafeicos/metabolismo , Linhagem Celular , Compostagem , Feminino , Genes Reporter , Humanos , Hidroxibenzoatos/química , Fitoestrógenos/química , Extratos Vegetais/química , Ratos , Ratos Sprague-Dawley , Receptores Estrogênicos/metabolismo , Sementes/química
8.
Plant Cell Rep ; 38(9): 1139-1150, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31197450

RESUMO

KEY MESSAGE: Transcription factors from mammals and plants, which play a role in innate immunity, interact with the same microbe-associated molecular pattern (MAMP)-responsive sequences from Arabidopsis thaliana. The interaction of mouse NF-κB p65 with MAMP-responsive sequences containing the core motif GACTTT of the WT-box was investigated. This revealed one sequence, derived from the promoter of the A. thaliana gene At1g76960, a gene with unknown function, to activate NF-κB p65 dependent reporter gene expression in plant cells very strongly. A bioinformatic analysis predicts three putative NF-κB p65 binding sites in this sequence and all three sites are required for reporter gene activation and binding. The sequence is one of the weakest MAMP-responsive sequences previously isolated, but the introduction of a GCC-box increases its MAMP responsivity in combination with upstream WT-box sequences. Although a bioinformatic analysis of the unmutated cis-sequence only predicts NF-κB p65 binding, A. thaliana WRKY40 was selected in a yeast one-hybrid screen. WRKY40, which is a transcriptional repressor, requires the sequence TTTTCTA for direct binding. This sequence is similar to the WK-box TTTTCCAC, previously shown to interact with tobacco NtWRKY12. In summary, this work supports the similarity in binding site recognition between NF-κB and WRKY factors.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Fator de Transcrição RelA/genética , Fatores de Transcrição/genética , Animais , Sítios de Ligação , Genes Reporter , Camundongos , Regiões Promotoras Genéticas/genética
9.
Plant Mol Biol ; 101(1-2): 41-61, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31183604

RESUMO

KEY MESSAGE: Several classes of transcription factors are involved in the activation of defensins. A new type of the transcription factor responsible for the regulation of wheat grain specific defensins was characterised in this work. HD-Zip class IV transcription factors constitute a family of multidomain proteins. A full-length cDNA of HD-Zip IV, designated TaGL7 was isolated from the developing grain of bread wheat, using a specific DNA sequence as bait in the Y1H screen. 3D models of TaGL7 HD complexed with DNA cis-elements rationalised differences that underlined accommodations of binding and non-binding DNA, while the START-like domain model predicted binding of lipidic molecules inside a concave hydrophobic cavity. The 3'-untranslated region of TaGL7 was used as a probe to isolate the genomic clone of TdGL7 from a BAC library prepared from durum wheat. The spatial and temporal activity of the TdGL7 promoter was tested in transgenic wheat, barley and rice. TdGL7 was expressed mostly in ovary at fertilisation and its promoter was active in a liquid endosperm during cellularisation and later in the endosperm transfer cells, aleurone, and starchy endosperm. The pattern of TdGL7 expression resembled that of genes that encode grain-specific lipid transfer proteins, particularly defensins. In addition, GL7 expression was upregulated by mechanical wounding, similarly to defensin genes. Co-bombardment of cultured wheat cells with TdGL7 driven by constitutive promoter and seven grain or root specific defensin promoters fused to GUS gene, revealed activation of four promoters. The data confirmed the previously proposed role of HD-Zip IV transcription factors in the regulation of genes that encode lipid transfer proteins involved in lipid transport and defence. The TdGL7 promoter could be used to engineer cereal grains with enhanced resistance to insects and fungal infections.


Assuntos
Defensinas/genética , Regulação da Expressão Gênica de Plantas , Fatores de Transcrição/genética , Triticum/genética , DNA Complementar/genética , Grão Comestível/genética , Grão Comestível/metabolismo , Genes Reporter , Hordeum/genética , Hordeum/metabolismo , Especificidade de Órgãos , Oryza/genética , Oryza/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Regiões Promotoras Genéticas/genética , Fatores de Transcrição/metabolismo , Triticum/metabolismo , Técnicas do Sistema de Duplo-Híbrido
10.
Nat Commun ; 10(1): 2822, 2019 06 27.
Artigo em Inglês | MEDLINE | ID: mdl-31249300

RESUMO

Interactions between proteins play an essential role in metabolic and signaling pathways, cellular processes and organismal systems. We report the development of splitFAST, a fluorescence complementation system for the visualization of transient protein-protein interactions in living cells. Engineered from the fluorogenic reporter FAST (Fluorescence-Activating and absorption-Shifting Tag), which specifically and reversibly binds fluorogenic hydroxybenzylidene rhodanine (HBR) analogs, splitFAST displays rapid and reversible complementation, allowing the real-time visualization of both the formation and the dissociation of a protein assembly.


Assuntos
Mapeamento de Interação de Proteínas/métodos , Proteínas/química , Linhagem Celular , Fluorescência , Genes Reporter , Humanos , Proteínas Luminescentes/química , Proteínas Luminescentes/metabolismo , Ligação Proteica , Proteínas/metabolismo , Rodanina/química , Proteínas de Ligação a Tacrolimo/química , Proteínas de Ligação a Tacrolimo/metabolismo
11.
Ecotoxicol Environ Saf ; 181: 214-223, 2019 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-31195230

RESUMO

In the aftermath of the Great East Japan Earthquake of March 11, 2011, marine fish in Kesennuma Bay, Japan, have been contaminated with heavy oil containing polycyclic aromatic hydrocarbons (PAHs). To estimate the risk of six PAHs (benzo[α]pyrene, dibenzothiophene, phenanthrene, 2,3,5-trimethylnaphthalene, acenaphthene, and 1-methylphenanthrene), which have been detected at high levels in the tissues of fish from Kesennuma Bay, we attempted to evaluate the effects of these PAHs on the fish aryl hydrocarbon receptor (AHR) signaling pathway. We initially measured PAH concentrations and cytochrome P4501A catalytic activities (EROD: ethoxyresorufin-O-deethylase and MROD: methoxyresorufin-O-demethylase) as markers of AHR activation in greenlings (Hexagrammos otakii) collected from Kesennuma Bay in 2014. The results showed that alkylated PAH concentrations and EROD/MROD activities were higher in sites close to the oil-spilled sites than in the control site, suggesting AHR activation by spilled alkylated PAHs. We then investigated AHR-mediated responses to these PAHs in the in vitro reporter gene assay system where red seabream (Pagrus major) AHR1 (rsAHR1) or rsAHR2 expression plasmids were transiently transfected into COS-7 cells. The in vitro assay showed rsAHR isoform-, PAH-, and dose-dependent transactivation potencies. The relative effective concentrations of benzo[α]pyrene, dibenzothiophene, phenanthrene, 2,3,5-trimethylnaphthalene, acenaphthene, and 1-methylphenanthrene that induce 20% of the maximum benzo[α]pyrene response (REC20-BaP) for rsAHR1 activation were 0.052, 38, 79, 88, 270 nM, and no response, respectively, and those for rsAHR2 activation were 0.0049, 32, 53, 88, 60 nM, and no response, respectively. The results showed that the REC20-BaP values of benzo[α]pyrene for both the rsAHR1 and rsAHR2 isoforms were lower than the concentrations (0.041-0.20 nM) detected in the muscle tissue of fish from Kesennuma Bay, while the REC20-BaP values of other PAHs were higher than their tissue concentrations. In silico rsAHR homology modeling and subsequent ligand docking simulation analyses indicated that the rsAHR activation potencies of PAHs could be predicted from a rsAHR2 model. This study shows that in vitro and in silico rsAHR analyses may be a useful tool for assessing the risks to fish contaminated with PAHs.


Assuntos
Peixes/metabolismo , Poluição por Petróleo , Hidrocarbonetos Policíclicos Aromáticos/análise , Receptores de Hidrocarboneto Arílico/metabolismo , Animais , Células COS , Cercopithecus aethiops , Simulação por Computador , Citocromo P-450 CYP1A1/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Genes Reporter , Japão , Perciformes/metabolismo , Petróleo , Hidrocarbonetos Policíclicos Aromáticos/química , Hidrocarbonetos Policíclicos Aromáticos/metabolismo , Hidrocarbonetos Policíclicos Aromáticos/toxicidade , Receptores de Hidrocarboneto Arílico/química , Receptores de Hidrocarboneto Arílico/genética , Medição de Risco , Dourada/genética
12.
BMC Bioinformatics ; 20(1): 309, 2019 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-31185910

RESUMO

BACKGROUND: Fluorescent reporter genes have become widely used for monitoring gene expression in living cells. When a microbial strain carrying a reporter gene is grown in a microplate reader, the fluorescence and the absorbance (optical density) of the culture can be automatically measured every few minutes in a highly parallelized way. The extraction of useful information from the resulting large amounts of data is not easy to achieve, because the fluorescence and absorbance measurements are only indirectly related to promoter activities and protein concentrations, requiring mathematical models of the expression of reporter genes for their interpretation. Although the principles of the analysis of reporter gene data are well-established today, there is a lack of general-purpose bioinformatics tools based on generic measurement models and sound inference procedures. This has motivated the development of WellInverter, a web application based on well-known methods for regularized linear inversion. RESULTS: We present a new version of WellInverter that considerably improves the performance and usability of the original application. In particular, we have put in place a parallel computing architecture with a load balancer to distribute analysis queries over several back-end servers, we have completely redesigned the graphical user interface to better support the different analysis steps, and we have developed a plug-in system for the parsing of data files produced by microplate readers from different manufacturers. We illustrate the functioning of WellInverter by analyzing data of the expression of a fluorescent reporter gene controlled by a phage promoter in growing Escherichia coli populations. We show that the expression pattern in different growth media, supporting different growth rates, corresponds to the pattern expected for a constitutive gene. CONCLUSIONS: The new version of WellInverter is a robust, easy-to-use and scalable web application, which has been deployed on two publicly accessible web servers and which can also be installed locally. A demo version of the application with two sample datasets is available on-line.


Assuntos
Biologia Computacional/métodos , Genes Reporter , Internet , Software , Algoritmos , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Fluorescência , Genes Bacterianos , Regiões Promotoras Genéticas , Interface Usuário-Computador
13.
Planta ; 250(2): 657-665, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31147828

RESUMO

MAIN CONCLUSION: The grapevine VvßVPE promoter is specifically expressed in the seed. The - 1306~- 1045 bp core region restricts expression in other tissues and organs. Vacuolar processing enzyme (VPE) is a cysteine proteinase regulating vacuolar protein maturation and executing programmed cell death (PCD) in plants. Vitis vinifera (Vv)ßVPE is a ß-type VPE showing seed-specific expression that processes seed proteins during ovule development. However, the regulation of the seed-specific gene expression is far from understood. In this study, we characterize VvßVPE promoter (pVvßVPE) from 12 seeded and seedless grape genotypes. 94.56% of the pVvßVPE coding sequence is consistent. Two ßVPE promoters were constructed and transformed into Arabidopsis thaliana via ß-glucuronidase (GUS) fused expression vectors, using cv. Pinot Noir and cv. Thompson as seed and seedless candidates. GUS staining in different tissues and organs revealed that VvßVPE expresses specifically in the embryo, including the cotyledon, hypocotyl and suspensor, but not in the leaf, stem, root or flowers of the seedling. Using promoter deletion analysis, we created four incomplete VvßVPE promoters and found each pVvßVPE deletion could drive GUS gene to express in seeds. Interestingly, seed specificity disappeared when the promoter missed the core - 1306~- 1045 bp region. All deletion promoters presenting various quantified GUS activities indicate that the region - 1704~- 1306 bp inhibits, and the region - 705~- 861 bp promotes gene expression of VvßVPE. Our results demonstrate that pVvßVPE is a seed-specific promoter in both seeded and seedless grapes. Moreover, the core region of pVvßVPE (- 1306~- 1045 bp) is the key one responsible for seed-specific expression.


Assuntos
Cisteína Endopeptidases/genética , Regiões Promotoras Genéticas/genética , Sementes/genética , Vitis/genética , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Clonagem Molecular , Regulação da Expressão Gênica de Plantas , Genes Reporter , Especificidade de Órgãos , Óvulo Vegetal/genética , Óvulo Vegetal/crescimento & desenvolvimento , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas , Plântula/genética , Plântula/crescimento & desenvolvimento , Sementes/crescimento & desenvolvimento , Vitis/crescimento & desenvolvimento
14.
Zhongguo Shi Yan Xue Ye Xue Za Zhi ; 27(3): 641-645, 2019 Jun.
Artigo em Chinês | MEDLINE | ID: mdl-31204911

RESUMO

OBJECTIVE: To clone the promoter sequence of acute monocytic leukemia new antigen gene.MLAA-34 and identify its promoter core region. METHODS: The full-length fragment of MLAA-34 gene promoter region was amplified by PCR, then was ligated into pGL3-Basic vector, and the recombinant plasmid was cloned. Constructed a series of MLAA-34 gene promoter 5' flanking region truncated plasmid. These recombinant plasmids were transfected into U937 and HEK293 cells, and the dual luciferase reporter gene was used to detect the promoter activity of each fragment to determine the minimum active region. Transcription factor binding sites were analyzed by bioinformatics methods. RESULTS: The recombinant plasmid containing MLAA-34 promoter sequence and its truncated plasmid were successfully constructed, and the promoter activity was significantly increased as compared with the empty vector (P<0.001). The minimal active region of MLAA-34 located between 402 bp and 200 bp. It contained multiple transcription factor binding sites such as E2F1, MZF-1, SP1, USF2 and STAT3. CONCLUSION: The promoter of luciferase reporter gene has been successfully constructed with different deletion fragments of MLAA-34, and its core promoter region may contain multiple transcription factor sequence.


Assuntos
Antígenos de Neoplasias/genética , Proteínas Reguladoras de Apoptose/genética , Leucemia Monocítica Aguda , Adulto , Clonagem Molecular , Genes Reporter , Células HEK293 , Humanos , Leucemia Monocítica Aguda/genética , Luciferases , Regiões Promotoras Genéticas
15.
Nat Commun ; 10(1): 2673, 2019 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-31209208

RESUMO

Alternative splicing performs a central role in expanding genomic coding capacity and proteomic diversity. However, programming of splicing patterns in engineered biological systems remains underused. Synthetic approaches thus far have predominantly focused on controlling expression of a single protein through alternative splicing. Here, we describe a modular and extensible platform for regulating four programmable exons that undergo a mutually exclusive alternative splicing event to generate multiple functionally-distinct proteins. We present an intron framework that enforces the mutual exclusivity of two internal exons and demonstrate a graded series of consensus sequence elements of varying strengths that set the ratio of two mutually exclusive isoforms. We apply this framework to program the DNA-binding domains of modular transcription factors to differentially control downstream gene activation. This splicing platform advances an approach for generating diverse isoforms and can ultimately be applied to program modular proteins and increase coding capacity of synthetic biological systems.


Assuntos
Processamento Alternativo/genética , Regulação da Expressão Gênica/genética , Engenharia Genética/métodos , RNA/genética , Fatores de Transcrição/genética , Motivos de Aminoácidos/genética , Animais , Linhagem Celular , Biologia Computacional , Sequência Consenso/genética , Éxons/genética , Biblioteca Gênica , Genes Reporter/genética , Humanos , Íntrons/genética , Mutagênese Sítio-Dirigida/métodos , Domínios Proteicos/genética , Isoformas de Proteínas/genética , RNA/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Genética
16.
Nat Commun ; 10(1): 2787, 2019 06 26.
Artigo em Inglês | MEDLINE | ID: mdl-31243281

RESUMO

Continuity, robustness, and regeneration of cell lineages relies on stem cell pools that are established during development. For the mammalian spermatogenic lineage, a foundational spermatogonial stem cell (SSC) pool arises from prospermatogonial precursors during neonatal life via mechanisms that remain undefined. Here, we mapped the kinetics of this process in vivo using a multi-transgenic reporter mouse model, in silico with single-cell RNA sequencing, and functionally with transplantation analyses to define the SSC trajectory from prospermatogonia. Outcomes revealed that a heterogeneous prospermatogonial population undergoes dynamic changes during late fetal and neonatal development. Differential transcriptome profiles predicted divergent developmental trajectories from fetal prospermatogonia to descendant postnatal spermatogonia. Furthermore, transplantation analyses demonstrated that a defined subset of fetal prospermatogonia is fated to function as SSCs. Collectively, these findings suggest that SSC fate is preprogrammed within a subset of fetal prospermatogonia prior to building of the foundational pool during early neonatal development.


Assuntos
Células-Tronco Germinativas Adultas/fisiologia , Linhagem da Célula , Testículo/embriologia , Animais , Diferenciação Celular , Desenvolvimento Embrionário , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Genes Reporter , Masculino , Camundongos , Camundongos Transgênicos , RNA/genética , Espermatogênese/fisiologia , Espermatogônias/fisiologia
17.
Dokl Biochem Biophys ; 485(1): 138-140, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31201635

RESUMO

To study the mechanisms of transcriptional regulation, a convenient experimental approach is to use the artificial chimeric constructs carrying the regulatory elements of interest. In the present work, we describe the creation and characterization of a novel genetic construct that makes it possible to study the transcriptional regulation of the early-late gene of the ecdysone cascade. Using the data of genome-wide experiments, we have isolated the main regulatory region of the hr4 gene, which was successfully used to create a chimeric reporter construct expressing a fluorescent protein upon the treatment with the ecdysone hormone. This reporter system can be used to study the mechanisms of the ecdysone response, both in cell culture and in tissues, at various stages of the Drosophila development.


Assuntos
Proteínas de Drosophila , Ecdisona/metabolismo , Genes Reporter , Receptores Citoplasmáticos e Nucleares , Transcrição Genética , Animais , Linhagem Celular , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Estudo de Associação Genômica Ampla , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo
18.
Nat Neurosci ; 22(7): 1182-1195, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31209381

RESUMO

Understanding the diversity of cell types in the brain has been an enduring challenge and requires detailed characterization of individual neurons in multiple dimensions. To systematically profile morpho-electric properties of mammalian neurons, we established a single-cell characterization pipeline using standardized patch-clamp recordings in brain slices and biocytin-based neuronal reconstructions. We built a publicly accessible online database, the Allen Cell Types Database, to display these datasets. Intrinsic physiological properties were measured from 1,938 neurons from the adult laboratory mouse visual cortex, morphological properties were measured from 461 reconstructed neurons, and 452 neurons had both measurements available. Quantitative features were used to classify neurons into distinct types using unsupervised methods. We established a taxonomy of morphologically and electrophysiologically defined cell types for this region of the cortex, with 17 electrophysiological types, 38 morphological types and 46 morpho-electric types. There was good correspondence with previously defined transcriptomic cell types and subclasses using the same transgenic mouse lines.


Assuntos
Conjuntos de Dados como Assunto , Neurônios/classificação , Córtex Visual/citologia , Potenciais de Ação , Animais , Forma Celular , Bases de Dados Factuais , Genes Reporter , Camundongos , Camundongos Transgênicos , Técnicas de Patch-Clamp , Transcriptoma , Córtex Visual/fisiologia
19.
J Biomed Nanotechnol ; 15(3): 518-530, 2019 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-31165697

RESUMO

This paper aimed to find an effective method to destroy cancer cells by targeting breast cancer cells with natural killer (NK) cells transfected with the human ferritin heavy chain (hFTH1) gene by polyethylene glycol (PEG)-modified dendrimerentrapped gold nanoparticles (Au DENPs). In this study, fifth-generation polyamidoamine (G5 PAMAM) dendrimers modified with PEG were used as templates to entrap gold nanoparticles to transfect hFTH1 into NK cells. Our results revealed that the prepared Au DENPs/FTH1 provided high-quality imaging performance (hypointensity on T2-weighted MR imaging) and efficient transfection efficiency (reaching 80.2%) at a N/P ratio (ratio of the number of surface primary amines on {(Au0)25-G5 · NH2-mPEG17} to the number of phosphate groups in the hFTH1 backbone) of 5:1. Interestingly, the results showed that Au DENPs/FTH1 effectively guided NK-92 cells to concentrate around tumor cells for effective gene therapy without severely impacting their activity. This work will provide a new research platform for immunotherapy based on NK cells and lead to the optimization and even individualization of breast cancer immunotherapy through nanomolecular visualization research, which has a broad scope for future clinical applications.


Assuntos
Dendrímeros , Nanopartículas Metálicas , Apoferritinas , Linhagem Celular Tumoral , Genes Reporter , Ouro , Humanos , Imagem por Ressonância Magnética , Polietilenoglicóis
20.
Ecotoxicol Environ Saf ; 181: 164-171, 2019 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-31185430

RESUMO

Short-chain chlorinated paraffins (SCCPs) are frequently detected in environmental matrices and human tissues. It was hypothesized that SCCPs might interact with the peroxisome proliferator-activated receptor α (PPARα). In the present study, an in vitro, dual-luciferase reporter gene assay and in silico molecular docking analysis were employed together to study the interactions between SCCPs congeners and PPARα. Expressions of genes downstream in pathways activated by PPARα in liver of rats exposed to 1, 10, or 100 mg/kg bm/d of C10-13-CPs (56.5% Cl) for 28 days were examined to confirm activation potencies of SCCPs toward PPARα signaling. Effects of exposure to C10-13-CPs (56.5% Cl) on fatty acid metabolism in rat liver were also explored via a pseudo-targeted metabolomics strategy. Our results showed that C10-13-CPs (56.5% Cl) caused a dose-dependent greater expression of luciferase activity of rat PPARα. Molecular docking modeling revealed that SCCPs had a strong capacity to bind with PPARα only through hydrophobic interactions and the binding affinity was dependent on the degree of chlorination in SCCPs congeners. In livers of male rats, exposure to 100 mg/kg bm/d of C10-13-CPs (56.5% Cl) resulted in up-regulated expressions of 11 genes that are downstream in the PPARα-activated pathway and regulate catabolism of fatty acid. Consistently, accelerated fatty acid oxidation was observed mainly characterized by lesser concentrations of ∑fatty acids in livers of rats. Overall, these results demonstrated, for the first time, that SCCPs could activate rat PPARα signaling and thereby disrupt metabolism of fatty acid in livers of male rats.


Assuntos
Ácidos Graxos/metabolismo , Fígado/efeitos dos fármacos , PPAR alfa/metabolismo , Parafina/toxicidade , Animais , Expressão Gênica/efeitos dos fármacos , Genes Reporter , Halogenação , Fígado/metabolismo , Luciferases/genética , Masculino , Simulação de Acoplamento Molecular , PPAR alfa/química , Parafina/química , Ratos , Transdução de Sinais , Regulação para Cima
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