scholarly journals Cap-Independent Translational Enhancement of Turnip Crinkle Virus Genomic and Subgenomic RNAs

2000 ◽  
Vol 74 (3) ◽  
pp. 1085-1093 ◽  
Author(s):  
Feng Qu ◽  
T. Jack Morris
Keyword(s):  
Coat Protein ◽  
Translational Control ◽  
Firefly Luciferase ◽  
Plant Viruses ◽  
Luciferase Reporter ◽  
Translational Efficiency ◽  
Genomic Rna ◽  
Luciferase Reporter Gene ◽  
Independent Manner ◽  
Subgenomic Rna

ABSTRACT The presence of translational control elements and cap structures has not been carefully investigated for members of theCarmovirus genus, a group of small icosahedral plant viruses with positive-sense RNA genomes. In this study, we examined both the 5′ and 3′ untranslated regions (UTRs) of the turnip crinkle carmovirus (TCV) genomic RNA (4 kb) as well as the 5′ UTR of the coat protein subgenomic RNA (1.45 kb) for their roles in translational regulation. All three UTRs enhanced translation of the firefly luciferase reporter gene to different extents. Optimal translational efficiency was achieved when mRNAs contained both 5′ and 3′ UTRs. The synergistic effect due to the 5′-3′ cooperation was at least fourfold greater than the sum of the contributions of the individual UTRs. The observed translational enhancement of TCV mRNAs occurred in a cap-independent manner, a result consistent with the demonstration, using a cap-specific antibody, that the 5′ end of the TCV genomic RNA was uncapped. Finally, the translational enhancement activity within the 5′ UTR of 1.45-kb subgenomic RNA was shown to be important for the translation of coat protein in protoplasts and for virulent infection in Arabidopsis plants.

scholarly journals Measurement of Effects of Antibiotics in Bioluminescent Staphylococcus aureus RN4220

2001 ◽  
Vol 45 (12) ◽  
pp. 3456-3461 ◽  
Author(s):  
Mervi Tenhami ◽  
Kaisa Hakkila ◽  
Matti Karp
Keyword(s):  
Staphylococcus Aureus ◽  
High Throughput Screening ◽  
Pathogenic Bacteria ◽  
Antimicrobial Agents ◽  
Light Emission ◽  
Detection System ◽  
Firefly Luciferase ◽  
Luciferase Reporter ◽  
Luciferase Expression ◽  
Luciferase Reporter Gene

ABSTRACT The spread of antibiotic resistance among pathogenic bacteria is a serious threat to humans and animals. Therefore, unnecessary use should be minimized, and new antimicrobial agents with novel mechanisms of action are needed. We have developed an efficient method for measuring the action of antibiotics which is applied to a gram-positive strain,Staphylococcus aureus RN4220. The method utilizes the firefly luciferase reporter gene coupled to the metal-induciblecadA promoter in a plasmid, pTOO24. Correctly timed induction by micromolar concentrations of antimonite rapidly triggers the luciferase gene transcription and translation. This sensitizes the detection system to the action of antibiotics, and especially for transcriptional and translational inhibitors. We show the results for 11 model antibiotics with the present approach and compare them to an analytical setup with a strain where luciferase expression is under the regulation of a constitutive promoter giving only a report of metabolic inhibition. The measurement of light emission from intact living cells is shown to correlate extremely well (r = 0.99) with the conventional overnight growth inhibition measurement. Four of the antibiotics were within a 20% concentration range and four were within a 60% concentration range of the drugs tested. This approach shortens the assay time needed, and it can be performed in 1 to 4 h, depending on the sensitivity needed. Furthermore, the assay can be automatized for high-throughput screening by the pharmaceutical industry.

scholarly journals DDX3 Participates in Translational Control of Inflammation Induced by Infections and Injuries

2018 ◽  
Vol 39 (1) ◽  
Author(s):  
Yu-Chang Ku ◽  
Min-Hua Lai ◽  
Chen-Chia Lo ◽  
Yi-Chuan Cheng ◽  
Jian-Tai Qiu ◽  
...  
Keyword(s):  
Translational Control ◽  
Fluorescent Protein ◽  
Mitogen Activated Protein Kinase ◽  
Luciferase Reporter ◽  
Antibody Array ◽  
Transgenic Zebrafish ◽  
Pathway Enrichment Analysis ◽  
Translational Efficiency ◽  
Macrophage Migration ◽  
Target Mrnas

ABSTRACT Recent studies have suggested that DDX3 functions in antiviral innate immunity, but the underlying mechanism remains elusive. We previously identified target mRNAs whose translation is controlled by DDX3. Pathway enrichment analysis of these targets indicated that DDX3 is involved in various infections and inflammation. Using immunoblotting, we confirmed that PACT, STAT1, GNB2, Rac1, TAK1, and p38 mitogen-activated protein kinase (MAPK) proteins are downregulated by DDX3 knockdown in human monocytic THP-1 cells and epithelial HeLa cells. Polysome profiling revealed that DDX3 knockdown reduces the translational efficiency of target mRNAs. We further demonstrated DDX3-mediated translational control of target mRNAs by luciferase reporter assays. To examine the effects of DDX3 knockdown on macrophage migration and phagocytosis, we performed in vitro cell migration assay and flow cytometry analysis of the uptake of green fluorescent protein-expressing Escherichia coli in THP-1 cells. The DDX3 knockdown cells exhibited impaired macrophage migration and phagocytosis. Moreover, we used a human cytokine antibody array to identify the cytokines affected by DDX3 knockdown. Several chemokines were decreased considerably in DDX3 knockdown THP-1 cells after lipopolysaccharide or poly(I·C) stimulation. Lastly, we demonstrated that DDX3 is crucial for the recruitment of phagocytes to the site of inflammation in transgenic zebrafish.

scholarly journals Novel Whole-Cell Antibiotic Biosensors for Compound Discovery

2007 ◽  
Vol 73 (20) ◽  
pp. 6436-6443 ◽  
Author(s):  
Andreas Urban ◽  
Stefan Eckermann ◽  
Beate Fast ◽  
Susanne Metzger ◽  
Matthias Gehling ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
High Throughput ◽  
High Throughput Screening ◽  
Protein Biosynthesis ◽  
Firefly Luciferase ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Environmental Biology ◽  
Drug Candidates ◽  
Novel Drug

ABSTRACT Cells containing reporters which are specifically induced via selected promoters are used in pharmaceutical drug discovery and in environmental biology. They are used in screening for novel drug candidates and in the detection of bioactive compounds in environmental samples. In this study, we generated and validated a set of five Bacillus subtilis promoters fused to the firefly luciferase reporter gene suitable for cell-based screening, enabling the as yet most-comprehensive high-throughput diagnosis of antibiotic interference in the major biosynthetic pathways of bacteria: the biosynthesis of DNA by the yorB promoter, of RNA by the yvgS promoter, of proteins by the yheI promoter, of the cell wall by the ypuA promoter, and of fatty acids by the fabHB promoter. The reporter cells mainly represent novel antibiotic biosensors compatible with high-throughput screening. We validated the strains by developing screens with a set of 14,000 pure natural products, representing a source of highly diverse chemical entities, many of them with antibiotic activity (6% with anti-Bacillus subtilis activity of ≤25 μg/ml]). Our screening approach is exemplified by the discovery of classical and novel DNA synthesis and translation inhibitors. For instance, we show that the mechanistically underexplored antibiotic ferrimycin A1 selectively inhibits protein biosynthesis.

scholarly journals Use of Leishmania donovani Field Isolates Expressing the Luciferase Reporter Gene in In Vitro Drug Screening

2005 ◽  
Vol 49 (9) ◽  
pp. 3776-3783 ◽  
Author(s):  
Ashutosh ◽  
Suman Gupta ◽  
Ramesh ◽  
Shyam Sundar ◽  
Neena Goyal
Keyword(s):  
Reporter Gene ◽  
High Throughput ◽  
High Throughput Screening ◽  
Leishmania Donovani ◽  
Luciferase Activity ◽  
Firefly Luciferase ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Field Isolates

ABSTRACT Currently available primary screens for the selection of candidate antileishmanial compounds are not ideal. These techniques are time-consuming, laborious, and difficult to scale and require macrophages, which limit their use for high-throughput screening. We have developed Leishmania donovani field isolates that constitutively express the firefly luciferase reporter gene (luc) as a part of an episomal vector. An excellent correlation between parasite number and luciferase activity was observed. luc expression was stable, even in the absence of drug selection, for 4 weeks. The transfectants were infective to macrophages, and intracellular amastigotes exhibited luciferase activity. The suitability of these recombinant field isolates for in vitro screening of antileishmanial drugs was established. The luciferase-expressing sodium stibogluconate-resistant cell lines offer a model for the screening of compounds for resistance. The system is in routine use at the Central Drug Research Institute, Lucknow, India, for high-throughput screening of newly synthesized compounds.

scholarly journals The Coding sequence of firefly luciferase reporter gene affects specific hyperexpressionArabidopsis thaliana cpl1mutant

2017 ◽  
Author(s):  
Hisashi Koiwa ◽  
Akihito Fukudome
Keyword(s):  
Reporter Gene ◽  
Firefly Luciferase ◽  
Luciferase Reporter ◽  
Wild Type ◽  
Coding Region ◽  
Luciferase Reporter Gene ◽  
Coding Sequence ◽  
Firefly Luciferase Reporter ◽  
Endogenous Genes ◽  
Firefly Luciferase Reporter Gene

AbstractForward genetic screening of mutants using firefly luciferase (LUC) reporter gene became a standard practice in plant research. Such screenings frequently identified alleles ofCPL1(Carboxyl-terminal Phosphatase-Like 1) regardless of promoters or pathways studied. Expression of the corresponding endogenous genes often shows the minimal difference between wild type andcpl1.Here we show that theLUCcoding sequence is responsible for the high expression incpl1,using a classicalRD29a-LUC. Deletion of theLUC3’-UTR did not change hyperactivation ofLUCincpl1.However, a codon-modifiedLUC(LUC2) produced similar expression levels both in wild type and incpl1. These results indicate that the coding region ofLUCis responsible for thecpl1-specificLUCoverexpression uncoupled with the expression of the endogenous counterpart.

scholarly journals Transient Transfection of the Zoonotic Parasite Babesia microti

Pathogens ◽  
2020 ◽  
Vol 9 (2) ◽  
pp. 108 ◽  
Author(s):  
Mingming Liu ◽  
Shengwei Ji ◽  
Mohamed Abdo Rizk ◽  
Paul Franck Adjou Moumouni ◽  
Eloiza May Galon ◽  
...  
Keyword(s):  
Genetic Manipulation ◽  
Firefly Luciferase ◽  
Transient Transfection ◽  
Babesia Microti ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Transfection Method ◽  
Firefly Luciferase Reporter ◽  
Zoonotic Parasite ◽  
Firefly Luciferase Reporter Gene

The development of genetic manipulation techniques has been reported in many protozoan parasites over the past few years. However, these techniques have not been established for Babesia microti. Here, we report the first successful transient transfection of B. microti. The plasmids containing the firefly luciferase reporter gene were transfected into B. microti by an AMAXA 4D Nucleofection system. Twenty-four-hour synchronization, the 5′-actin promoter, program FA100, and 50 μg of plasmid DNA constituted the best conditions for the transient transfection of B. microti. This finding is the first step towards a stable transfection method for B. microti, which may contribute to a better understanding of the biology of the parasite.

scholarly journals Glycyrrhizin Acid and Glycyrrhetinic Acid Modified Polyethyleneimine for Targeted DNA Delivery to Hepatocellular Carcinoma

2019 ◽  
Vol 20 (20) ◽  
pp. 5074 ◽  
Author(s):  
Cao ◽  
Gao ◽  
Zhan ◽  
Qiu ◽  
Piao ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Hepg2 Cells ◽  
Transfection Efficiency ◽  
Gene Transfection ◽  
Glycyrrhetinic Acid ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Independent Manner ◽  
Endocytosis Pathway

In the last 2–3 decades, gene therapy represented a promising option for hepatocellular carcinoma (HCC) treatment. However, the design of safe and efficient gene delivery systems is still one of the major challenges that require solutions. In this study, we demonstrate a versatile method for covalent conjugation of glycyrrhizin acid (GL) or glycyrrhetinic acid (GA) to increase the transfection efficiency of Polyethyleneimine (PEI, Mw 1.8K) and improve their targeting abilities of hepatoma carcinoma cells. GA and GL targeting ligands were grafted to PEI via N-acylation, and we systematically investigated their biophysical properties, cytotoxicity, liver targeting and transfection efficiency, and endocytosis pathway trafficking. PEI-GA0.75, PEI-GL10.62 and PEI-GL20.65 conjugates caused significant increases in gene transfection efficiency and superior selectivity for HepG2 cells, with all three conjugates showing specific recognition of HepG2 cells by the free GA competition assay. The endocytosis inhibition and intracellular trafficking results indicated that PEI-GA0.75 and GL10.62 conjugates behaved similarly to SV40 virus, by proceeding via the caveolae- and clathrin-independent mediated endocytosis pathway and bypassing entry into lysosomes, with an energy independent manner, achieving their high transfection efficiencies. In the HepG2 intraperitoneal tumor model, PEI-GA0.75 and PEI-GL10.62 carrying the luciferase reporter gene gained high gene expression, suggesting potential use for in vivo application.

scholarly journals Cytoplasmic polyadenylation-element-binding protein (CPEB)1 and 2 bind to the HIF-1α mRNA 3′-UTR and modulate HIF-1α protein expression

2008 ◽  
Vol 417 (1) ◽  
pp. 235-246 ◽  
Author(s):  
Sonja Hägele ◽  
Uwe Kühn ◽  
Melanie Böning ◽  
Dörthe M. Katschinski
Keyword(s):  
Translational Control ◽  
Binding Protein ◽  
Hypoxia Inducible Factor ◽  
Luciferase Reporter ◽  
Insulin Stimulation ◽  
Luciferase Reporter Gene ◽  
Cytoplasmic Polyadenylation ◽  
Protein Levels ◽  
Hypoxia Inducible Factor 1 ◽  
Element Binding Protein

The heterodimeric HIF (hypoxia-inducible factor)-1 is a transcriptional master regulator of several genes involved in mammalian oxygen homoeostasis. Besides the well described regulation of the HIF-1α subunit via hydroxylation-mediated protein stability in hypoxia, there are several indications of an additional translational control of the HIF-1α mRNA, especially after growth factor stimulation. We identified an interaction of CPEB (cytoplasmic polyadenylation-element-binding protein) 1 and CPEB2 with the 3′-UTR (untranslated region) of HIF-1α mRNA. Overexpression of CPEB1 and CPEB2 affected HIF-1α protein levels mediated by the 3′-UTR of HIF-1α mRNA. Stimulation of neuroblastoma SK-N-MC cells with insulin and thus activation of endogenous CPEBs increased the expression of a luciferase reporter gene fused to the 3′-UTR of HIF-1α as well as endogenous HIF-1α protein levels. This could be abrogated by treating the cells with CPEB1 or CPEB2 siRNAs (short interfering RNAs). Injection of HIF-1α cRNA into Xenopus oocytes verified the elongation of the poly(A)+ (polyadenylated) tail by cytoplasmic polyadenylation. Thus CPEB1 and CPEB2 are involved in the regulation of HIF-1α following insulin stimulation.

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