scholarly journals Analysis of Promoter Function in Aspergillus fumigatus

2012 ◽  
Vol 11 (9) ◽  
pp. 1167-1177 ◽  
Author(s):  
Sanjoy Paul ◽  
J. Stacey Klutts ◽  
W. Scott Moye-Rowley
Keyword(s):  
Aspergillus Fumigatus ◽  
Reporter Gene ◽  
Reporter Genes ◽  
Antifungal Drugs ◽  
Luciferase Reporter ◽  
Pcr Analysis ◽  
Luciferase Reporter Gene ◽  
Content Type ◽  
Gene System ◽  
Reporter Gene System

ABSTRACTThe filamentous fungusAspergillus fumigatusis an important opportunistic pathogen that can cause high mortality levels in susceptible patient populations. The increasing dependence on antifungal drugs to controlA. fumigatushas led to the inevitable acquisition of drug-resistant forms of this pathogen. In other fungal pathogens, drug resistance is often associated with an increase in transcription of genes such as ATP-binding cassette (ABC) transporters that directly lead to tolerance to commonly employed antifungal drugs. InA. fumigatus, tolerance to azole drugs (the major class of antifungal) is often associated with changes in the sequence of the azole target enzyme as well as changes in the transcription level of this gene. The target gene for azole drugs inA. fumigatusis referred to ascyp51A. In order to dissect transcription ofcyp51Atranscription and other genes of interest, we constructed a set of firefly luciferase reporter genes designed for use inA. fumigatus. These reporter genes can either replicate autonomously or be targeted to thepyrGlocus, generating an easily assayable uracil auxotrophy. We fused eight differentA. fumigatuspromoters to luciferase. Faithful behaviors of these reporter gene fusions compared to their chromosomal equivalents were evaluated by 5′ rapid amplification of cDNA ends (RACE) and quantitative reverse transcription-PCR (qRT-PCR) analysis. We used this reporter gene system to study stress-regulated transcription of a Hsp70-encoding gene, map an important promoter element in thecyp51Agene, and correct an annotation error in the actin gene. We anticipate that this luciferase reporter gene system will be broadly applicable in analyses of gene expression inA. fumigatus.

Evaluation of a CRE-Directed Luciferase Reporter Gene Assay as an Alternative to Measuring cAMP Accumulation

1997 ◽  
Vol 2 (4) ◽  
pp. 235-240 ◽  
Author(s):  
Samantha E. George ◽  
Peter J. Bungay ◽  
Louise H. Naylor
Keyword(s):  
Reporter Gene ◽  
Luciferase Reporter ◽  
Luciferase Expression ◽  
Functional Assay ◽  
Ionophore A23187 ◽  
Camp Accumulation ◽  
Luciferase Reporter Gene ◽  
Gene System ◽  
Gene Assay ◽  
Reporter Gene System

A CHO reporter cell line expressing the firefly luciferase gene under the control of six cAMP response elements (CREs) was used to investigate the relationship between cAMP accumulation and cAMP dependent reporter gene expression and therefore, to assess the reporter gene system as an alternative functional assay. Timecourse experiments showed that cAMP accumulation preceded luciferase expression and that longer incubations (>2 h) were required to gain results with the reporter gene system. However, forskolin concentration dose-response studies revealed a 100-fold amplification of the signal measured by luciferase expression compared with direct cAMP accumulation, indicating that the reporter gene system afforded greater sensitivity. EC50 values determined for agonist activation of an inhibitory (5-HTlB-like) G-protein-coupled receptor (GPCR) were the same, and for a stimulatory GPCR (calcitonin Cla-like) were 10-fold lower, using the reporter gene system compared to cAMP accumulation assays, indicating the suitability of the reporter system for measuring the activity of receptors differentially coupled to the cAMP pathway. The phorbol ester, PMA, and the Ca2+ ionophore, A23187, were able to potentiate forskolin-stimulated luciferase expression but not cAMP accumulation, suggesting that the former could also be used to monitor cross-talk between different signal transduction pathways at the level of gene transcription.

Differential Effects of Components in Artemisia annua Extract on the Induction of Drug-Metabolizing Enzyme Expression Mediated by Nuclear Receptors

Planta Medica ◽  
2020 ◽  
Vol 86 (12) ◽  
pp. 867-875
Author(s):  
Xueli Zhang ◽  
Ran Meng ◽  
Haina Wang ◽  
Jie Xing
Keyword(s):  
Reporter Gene ◽  
Artemisia Annua ◽  
Constitutive Androstane Receptor ◽  
Pregnane X Receptor ◽  
Luciferase Reporter ◽  
Induction Effect ◽  
Luciferase Reporter Gene ◽  
Gene System ◽  
Drug Metabolizing Enzyme ◽  
Reporter Gene System

Abstract Artemisia annua tea is a popular dosage form used to treat and prevent malaria in some developing countries. However, repeated drinking leads to an obviously decreased efficacy, which may be related to the induction of metabolizing enzymes by artemisinin. In the present study, the ability of different components in A. annua to activate the pregnane X receptor and constitutive androstane receptor was evaluated by the dual luciferase reporter gene system. The changes in mRNA and protein expression of CYP3A4 and CYP2B6 were determined by quantitative real-time PCR and Western blotting. Results showed that in the pregnane X receptor-mediated CYP3A4 reporter gene system, chrysosplenetin and arteannuin B exhibited a weak induction effect on pregnane X receptor wt, while arteannuin A had a strong induction effect on pregnane X receptor wt and pregnane X receptor 370 and a weak induction effect on pregnane X receptor 163. In the pregnane X receptor-mediated CYP2B6 reporter gene system, arteannuin A had a moderate induction effect on pregnane X receptor wt and pregnane X receptor 379, and a weak induction effect on pregnane X receptor 403, while arteannuin B had a weak induction effect on pregnane X receptor wt and pregnane X receptor 379. Arteannuin A had a strong induction effect on constitutive androstane receptor 3 in constitutive androstane receptor-mediated CYP3A4/2B6 reporter gene systems, while arteannuin B showed a weak induction effect on constitutive androstane receptor 3 in the constitutive androstane receptor-mediated CYP2B6 reporter gene system. The mRNA and protein expressions of CYP3A4 and CYP2B6 were increased when the pregnane X receptor or constitutive androstane receptor was activated. Various components present in A. annua differentially affect the activities of pregnane X receptor isoforms and the constitutive androstane receptor, which indicates the possibility of a drug-drug interaction. This partly explains the decline in efficacy after repeated drinking of A. annua tea.

scholarly journals Evaluation of the use of the luciferase-reporter-gene system for gene-regulation studies involving cyclic AMP-elevating agents

1995 ◽  
Vol 309 (2) ◽  
pp. 385-387 ◽  
Author(s):  
O Benzakour ◽  
C Kanthou ◽  
U Dennehy ◽  
A al Haq ◽  
L P Berg ◽  
...  
Keyword(s):  
Cyclic Amp ◽  
Reporter Gene ◽  
Transcriptional Activation ◽  
Luciferase Activity ◽  
Primary Cultures ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Promoter Elements ◽  
Gene System ◽  
Reporter Gene System

The effects of cyclic AMP (cAMP)-elevating agents on the activity of cis-acting gene promoter sequences are frequently studied using the luciferase-reporter-gene system. The aim of the present study was to assess whether cAMP-elevating agents induce any changes in the level of luciferase activity independently of a transcriptional activation of promoter elements. Chloramphenicol acteyltransferase (CAT) and luciferase reporter genes under the control of the same promoter elements were transiently expressed in primary cultures of human vascular smooth-muscle cells. Transfected cells were treated with a cell-permeable and non-hydrolysable cAMP analogue, 2′-O-monobutyryl-8-bromo cAMP, or with the cAMP-elevating agents forskolin and prostaglandin E1 (PGE1). Forskolin and PGE1 induced a significant increase in the level of luciferase activity, but had no effect on CAT activity. Conclusions based solely on the use of the luciferase-reporter-gene system in studies involving promoter activation by cAMP-elevating agents could therefore be misleading.

scholarly journals Safe and Sensitive Antiviral Screening Platform Based on Recombinant Human Coronavirus OC43 Expressing the Luciferase Reporter Gene

2016 ◽  
Vol 60 (9) ◽  
pp. 5492-5503 ◽  
Author(s):  
Liang Shen ◽  
Yang Yang ◽  
Fei Ye ◽  
Gaoshan Liu ◽  
Marc Desforges ◽  
...  
Keyword(s):  
Reporter Gene ◽  
Luciferase Reporter ◽  
Wild Type Virus ◽  
Upper Respiratory Tract ◽  
Rna Helicases ◽  
Luciferase Reporter Gene ◽  
Screening Assay ◽  
Content Type ◽  
Tract Infections

ABSTRACTHuman coronaviruses (HCoVs) cause 15 to 30% of mild upper respiratory tract infections. However, no specific antiviral drugs are available to prevent or treat HCoV infections to date. Here, we developed four infectious recombinant HCoVs-OC43 (rHCoVs-OC43) which express theRenillaluciferase (Rluc) reporter gene. Among these four rHCoVs-OC43, rOC43-ns2DelRluc (generated by replacing ns2 with the Rluc gene) showed robust luciferase activity with only a slight impact on its growth characteristics. Additionally, this recombinant virus remained stable for at least 10 passages in BHK-21 cells. rOC43-ns2DelRluc was comparable to its parental wild-type virus (HCoV-OC43-WT) with respect to the quantity of the antiviral activity of chloroquine and ribavirin. We showed that chloroquine strongly inhibited HCoV-OC43 replicationin vitro, with a 50% inhibitory concentration (IC50) of 0.33 μM. However, ribavirin showed inhibition of HCoV-OC43 replication only at high concentrations which may not be applicable to humans in clinical treatment, with an IC50of 10 μM. Furthermore, using a luciferase-based small interfering RNA (siRNA) screening assay, we identified double-stranded-RNA-activated protein kinase (PKR) and DEAD box RNA helicases (DDX3X) that exhibited antiviral activities, which were further verified by the use of HCoV-OC43-WT. Therefore, rOC43-ns2DelRluc represents a promising safe and sensitive platform for high-throughput antiviral screening and quantitative analysis of viral replication.

scholarly journals Development of a New Reporter Gene System-dsRed/Xanthine Phosphoribosyltransferase-Xanthine for Molecular Imaging of Processes Behind the Intact Blood-Brain Barrier

2003 ◽  
Vol 2 (2) ◽  
pp. 153535002003031
Author(s):  
Mikhail Doubrovin ◽  
Vladimir Ponomarev ◽  
Inna Serganova ◽  
Suren Soghomonian ◽  
Tadashi Myagawa ◽  
...  
Keyword(s):  
Blood Brain Barrier ◽  
Reporter Gene ◽  
Fusion Gene ◽  
Reporter Genes ◽  
Fluorescent Imaging ◽  
Brain Barrier ◽  
Gene System ◽  
Blood Brain ◽  
Reporter Gene System

We report the development of a novel dual-modality fusion reporter gene system consisting of Escherichia coli xanthine phosphoribosyltransferase (XPRT) for nuclear imaging with radiolabeled xanthine and Discosoma red fluorescent protein for optical fluorescent imaging applications. The dsRed/XPRT fusion gene was successfully created and stably transduced into RG2 glioma cells, and both reporters were shown to be functional. The level of dsRed fluorescence directly correlated with XPRT enzymatic activity as measured by ribophosphorylation of [14C]-xanthine was in vitro ( Ki = 0.124 ± 0.008 vs. 0.00031 ± 0.00005 mL/min/g in parental cell line), and [*]-xanthine octanol/water partition coefficient was 0.20 at pH = 7.4 (log P = 0.69), meeting requirements for the blood-brain barrier (BBB) penetrating tracer. In the in vivo experiment, the concentration of [* C]-xanthine in the normal brain varied from 0.20 to 0.16 + 0.05% dose/g under 0.87 + 0.24% dose/g plasma radiotracer concentration. The accumulation in vivo in the transfected flank tumor was to 2.4 ± 0.3% dose/g, compared to 0.78 ± 0.02% dose/g and 0.64 ± 0.05% dose/g in the control flank tumors and intact muscle, respectively. [14C]-Xanthine appeared to be capable of specific accumulation in the transfected infiltrative brain tumor (RG2-dsRed/XPRT), which corresponded to the 585 nm fluorescent signal obtained from the adjacent cryosections. The images of endogenous gene expression with the “sensory system” have to be normalized for the transfection efficiency based on the “beacon system” image data. Such an approach requires two different “reporter genes” and two different “reporter substrates.” Therefore, the novel dsRed/XPRT fusion gene can be used as a multimodality reporter system in the biological applications requiring two independent reporter genes, including the cells located behind the BBB.

scholarly journals Study on the Mechanism of Mirna-21 Affecting the Differentiation of Bone Marrow Mesenchymal Stem Cells into Cardiomyocyte-Like Cells By Targeting Ajuba / Isl1 Axis

2021 ◽  
Author(s):  
Wengang Yang ◽  
Song Xue ◽  
Hui Zheng ◽  
Jianggui Dan ◽  
Lei Zhao ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Reporter Gene ◽  
Luciferase Reporter ◽  
Myoid Cells ◽  
Luciferase Reporter Gene ◽  
Slow Virus ◽  
Marrow Mesenchymal Stem Cells ◽  
Reporter Gene System

Abstract Purpose: To study the mechanism of miRNA-21 targeting ajuba/ Isl1 axis to affect BMSC differentiation to cardiac myoid cells. Methods: BMSC was cultured and miRNA-21 was constructed to infect BMSC. The miRNA-21 was directly regulated by luciferase reporter gene system. The expression of cTnI, ajuba and Isl1 was detected by RT-qPCR and WB. The expression of cTnI, ajuba and Isl1 was detected by RT-qPCR and WB, and miR-21 was detected by RT-qPCR; The differentiation ability of BMSC in all groups was detected by RT-qPCR and WB; To evaluate the effect of ajuba and Isl1 on the differentiation ability of BMSC. Results: BMSC was cultured successfully, BMSC was successfully constructed by mir-21-OE, mir-21-KD, ajuba-OE and ajuba KD slow virus; RT-qPCR and WB were used to detect the high expression of cTnI in mir-21-OE and ajuba KD groups, and the expression of miR-21 was increased, the expression of ajuba was inhibited and Isl1 expression was enhanced. Conclusion: The expression of ajuba was inhibited by up regulation of miRNA-21 target, and the expression of Isl1 enhanced to promote BMSC differentiation, that is, miRNA-21 regulated the axis of ajuba/ Isl1 to influence the differentiation of BMSC to cardiomyocyte-like cells.

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