scholarly journals A Synthetic Riboswitch to Regulate Haloarchaeal Gene Expression

2021 ◽  
Vol 12 ◽  
Author(s):  
Johannes Born ◽  
Kerstin Weitzel ◽  
Beatrix Suess ◽  
Felicitas Pfeifer
Keyword(s):  
Gene Expression ◽  
Reporter Gene ◽  
Culture Media ◽  
Fold Increase ◽  
Start Codon ◽  
Genetic Circuit ◽  
Genetic Circuits ◽  
Domains Of Life ◽  
Dose Dependent ◽  
Ribosomal Binding Site

In recent years, synthetic riboswitches have become increasingly important to construct genetic circuits in all three domains of life. In bacteria, synthetic translational riboswitches are often employed that modulate gene expression by masking the Shine-Dalgarno (SD) sequence in the absence or presence of a cognate ligand. For (halo-)archaeal translation, a SD sequence is not strictly required. The application of synthetic riboswitches in haloarchaea is therefore limited so far, also because of the molar intracellular salt concentrations found in these microbes. In this study, we applied synthetic theophylline-dependent translational riboswitches in the archaeon Haloferax volcanii. The riboswitch variants A through E and E∗ were chosen since they not only mask the SD sequence but also the AUG start codon by forming a secondary structure in the absence of the ligand theophylline. Upon addition of the ligand, the ribosomal binding site and start codon become accessible for translation initiation. Riboswitch E mediated a dose-dependent, up to threefold activation of the bgaH reporter gene expression. Raising the salt concentration of the culture media from 3 to 4 M NaCl resulted in a 12-fold increase in the switching capacity of riboswitch E, and switching activity increased up to 26-fold when the cultivating temperature was reduced from 45 to 30°C. To construct a genetic circuit, riboswitch E was applied to regulate the synthesis of the transcriptional activator GvpE allowing a dose-dependent activation of the mgfp6 reporter gene under PpA promoter control.

scholarly journals Altered Regulation of 15-Acetyldeoxynivalenol Production in Fusarium graminearum

2000 ◽  
Vol 66 (5) ◽  
pp. 2062-2065 ◽  
Author(s):  
Lifeng Chen ◽  
Susan P. McCormick ◽  
Thomas M. Hohn
Keyword(s):  
Gene Expression ◽  
Transcription Factor ◽  
Reporter Gene ◽  
Fusarium Graminearum ◽  
Large Scale ◽  
Fold Increase ◽  
Gene Clusters ◽  
Gus Expression ◽  
Plasmid Integration ◽  
Pathway Gene

ABSTRACT Most Fusarium graminearum isolates produce low or undetectable levels of trichothecenes in liquid shake cultures, making it difficult to perform biochemical studies of trichothecene biosynthesis. To develop strains with higher levels of trichothecene production under liquid shake conditions we transformed F. graminearum with both a reporter gene containing a homologous trichothecene pathway gene promoter (TRI5) and a gene encoding a heterologous trichothecene pathway transcription factor (TRI6). The TRI5 and TRI6 genes are part of the trichothecene pathway gene clusters of both Fusarium sporotrichioides and F. graminearum. These genes encode trichodiene synthase (encoded by TRI5), the first enzyme in the trichothecene pathway, and a transcription factor (encoded by TRI6) required for pathway gene expression. Transformation of F. graminearum with plasmids containing either an F. graminearum TRI5 promoter fragment (FGTRI5P ) or FGTRI5P coupled with the β-d-glucuronidase (GUS) reporter gene resulted in the identification of several transformants capable of producing 45 to 200 mg of 15-acetyldeoxynivalenol (15-ADON)/liter in liquid shake culture after 7 days. Increased 15-ADON production was only observed in transformants where plasmid integration occurred through the FGTRI5P sequence and was not accompanied by increased GUS expression. 15-ADON production was further increased in liquid culture up to 1,200 mg/liter following introduction of the F. sporotrichioides TRI6 gene (FSTRI16) into F. graminearum. The effects of FSTRI6 on 15-ADON production also depended on plasmid integration via homologous recombination of the FGTRI5P fragment and resulted in a 100-fold increase in GUS expression. High-level production of 15-ADON in liquid shake cultures provides a convenient method for large-scale trichothecene preparation. The results suggest that targeting transformation vector integration toFGTRI5P alters pathway gene expression and are consistent with the proposed conservation of TRI6 function betweenFusarium species.

scholarly journals Generating effective models and parameters for RNA genetic circuits

2015 ◽  
Author(s):  
Chelsea Y Hu ◽  
Jeffrey D Varner ◽  
Julius B Lucks
Keyword(s):  
Gene Expression ◽  
Circuit Design ◽  
Core Gene ◽  
Equation Model ◽  
Genetic Circuit ◽  
Test Case ◽  
Genetic Circuits ◽  
Modeling Framework ◽  
Broad Array ◽  
Effective Models

RNA genetic circuitry is emerging as a powerful tool to control gene expression. However, little work has been done to create a theoretical foundation for RNA circuit design. A prerequisite to this is a quantitative modeling framework that accurately describes the dynamics of RNA circuits. In this work, we develop an ordinary differential equation model of transcriptional RNA genetic circuitry, using an RNA cascade as a test case. We show that parameter sensitivity analysis can be used to design a set of four simple experiments that can be performed in parallel using rapid cell-free transcription-translation (TX-TL) reactions to determine the thirteen parameters of the model. The resulting model accurately recapitulates the dynamic behavior of the cascade, and can be easily extended to predict the function of new cascade variants that utilize new elements with limited additional characterization experiments. Interestingly, we show that inconsistencies between model predictions and experiments led to the model-guided discovery of a previously unknown maturation step required for RNA regulator function. We also determine circuit parameters in two different batches of TX-TL, and show that batch-to-batch variation can be attributed to differences in parameters that are directly related to the concentrations of core gene expression machinery. We anticipate the RNA circuit models developed here will inform the creation of computer aided genetic circuit design tools that can incorporate the growing number of RNA regulators, and that the parameterization method will find use in determining functional parameters of a broad array of natural and synthetic regulatory systems.

scholarly journals Orthogonal tuning of gene expression noise using CRISPR–Cas

2020 ◽  
Author(s):  
Fan Wu ◽  
Jiyoung Shim ◽  
Ting Gong ◽  
Cheemeng Tan
Keyword(s):  
Gene Expression ◽  
Reporter Gene ◽  
Transcription Initiation ◽  
Mrna Translation ◽  
Genetic Circuit ◽  
Control Of Gene Expression ◽  
Gene Expression Noise ◽  
Guide Rnas ◽  
Expression Noise ◽  
Genetic Components

Abstract The control of gene expression noise is important for improving drug treatment and the performance of synthetic biological systems. Previous work has tuned gene expression noise by changing the rate of transcription initiation, mRNA degradation, and mRNA translation. However, these methods are invasive: they require changes to the target genetic components. Here, we create an orthogonal system based on CRISPR-dCas9 to tune gene expression noise. Specifically, we modulate the gene expression noise of a reporter gene in Escherichia coli by incorporating CRISPR activation and repression (CRISPRar) simultaneously in a single cell. The CRISPRar uses a single dCas9 that recognizes two different single guide RNAs (sgRNA). We build a library of sgRNA variants with different expression activation and repression strengths. We find that expression noise and mean of a reporter gene can be tuned independently by CRISPRar. Our results suggest that the expression noise is tuned by the competition between two sgRNAs that modulate the binding of RNA polymerase to promoters. The CRISPRar may change how we tune expression noise at the genomic level. Our work has broad impacts on the study of gene functions, phenotypical heterogeneity, and genetic circuit control.

scholarly journals Up to 100-Fold Increase of Apparent Gene Expression in the Presence of Epstein-Barr Virus oriP Sequences and EBNA1: Implications of the Nuclear Import of Plasmids

1998 ◽  
Vol 72 (7) ◽  
pp. 6181-6185 ◽  
Author(s):  
Françoise Längle-Rouault ◽  
Volker Patzel ◽  
Annie Benavente ◽  
Martine Taillez ◽  
Nathalie Silvestre ◽  
...  
Keyword(s):  
Gene Expression ◽  
Reporter Gene ◽  
Epstein Barr Virus ◽  
Luciferase Activity ◽  
Fold Increase ◽  
Luciferase Gene ◽  
Reporter Gene Activity ◽  
Barr Virus ◽  
Epstein Barr ◽  
Cytoplasmic Injection

ABSTRACT A 100-fold increase in luciferase activity was observed in 293 cells, stably expressing Epstein-Barr nuclear antigen 1 (EBNA1; 293-EBNA1 cells), that had been transiently transfected with plasmids carrying Epstein-Barr virus (EBV) oriP sequences. This increase was observed in comparison to reporter gene activity obtained after transfection with a plasmid carrying no oriPsequences. The luciferase gene on these plasmids was under the control of either the cytomegalovirus immediate-early 1 gene enhancer-promoter (CMV IE1) or the Rous sarcoma virus promoter. The increase of reporter gene activity was not due to plasmid replication, since a similar enhancement was observed in the presence of aphidicolin, an inhibitor of replicative DNA synthesis, or after deletion of the dyad symmetry (DS) element within oriP. Luciferase production was not increased in the presence of only the DS element. Microinjection of plasmids carrying the CMV IE1 promoter-driven luciferase gene with or without oriP sequences into the nuclei of 293-EBNA1 cells resulted in a 17-fold increase in luciferase activity. Cytoplasmic injection of these plasmids led to an enhancement of luciferase activity of up to 100-fold. This difference in the factor of activation after nuclear or cytoplasmic injection could be ascribed to increased transport of plasmids carrying oriP from the cytoplasm to the nucleus in the presence of EBNA1. These data suggest the possibility of substantially increasing the apparent expression of a gene under the control of a strong constitutive promoter in the presence of oriP sequences and EBNA1. This improvement in expression is due to intranuclear enhancement of gene expression.oriP-specific transport of plasmid DNA from the cytoplasm of 293-EBNA1 cells to the nucleus seems to contribute to the observed effect.

scholarly journals PPARα Gene Expression in the Developing Rat Kidney: Role of Glucocorticoids

2001 ◽  
Vol 12 (6) ◽  
pp. 1197-1203
Author(s):  
FATIMA DJOUADI ◽  
JEAN BASTIN
Keyword(s):  
Gene Expression ◽  
Fatty Acids ◽  
Fatty Acid ◽  
Culture Media ◽  
Rat Kidney ◽  
Fold Increase ◽  
Kidney Cortex ◽  
Peroxisome Proliferator ◽  
Rat Kidney Cortex

Abstract. The α isoform of peroxisome proliferator-activated receptor (PPARα), which is highly expressed in the kidney, can stimulate the expression of genes that are involved in fatty acid catabolism and therefore might be involved in the control of renal fatty acid β-oxidation. PPARα expression and its regulation in the immature kidney are not well documented. This study delineated the developmental pattern of PPARα expression in the rat kidney cortex and the medulla between postnatal days 10 and 30 and investigated the role of glucocorticoids in regulating PPARα expression. In the cortex, PPARα mRNA and protein increased 2- and 1.8-fold, respectively, from 10 to 21 d and then decreased 1.5- and 2.4-fold from 21 to 30 d. In the medulla, PPARα mRNA and protein increased continuously 3.3- and 2.4-fold, respectively. It is shown here that acute treatment by dexamethasone of 10-d-old rats precociously induced a 4- to 6-fold increase in PPARα mRNA and a 1.8-fold increase in protein within 6 h in each part of the kidney. Chronic injection of dexamethasone for 3 d also increased PPARα mRNA 3.8- and 2.2-fold in the cortex and the medulla, respectively, with a 1.5- and 2-fold increase in protein. Furthermore, adrenalectomy prevented the increases in PPARα mRNA and protein in both the cortex and the medulla between postnatal days 16 and 21, and these could be restored by dexamethasone treatment. Finally, with the use of an established renal cell line, it was shown that glucocorticoids stimulate gene expression of PPARα and of medium chain acyl-CoA dehydrogenase (MCAD, a PPARα target gene) 2- to 4-fold and 1.5-fold, respectively, and that addition of fatty acids in the culture media led to a 2.2-fold increase in MCAD mRNA. Altogether, these results demonstrated that glucocorticoids are potent regulators of PPARα development in the immature kidney and that these hormones act in concert with fatty acids to regulate MCAD gene expression in renal cells.

scholarly journals Golden Gate Assembly of Aerobic and Anaerobic Microbial Bioreporters

2021 ◽  
Author(s):  
Aaron J. Hinz ◽  
Benjamin Stenzler ◽  
Alexandre J. Poulain
Keyword(s):  
Gene Expression ◽  
Escherichia Coli ◽  
Synthetic Biology ◽  
Reporter Gene ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Fluorescent Microscopy ◽  
Genetic Circuit ◽  
Regulatory Sequences ◽  
Golden Gate

Microbial bioreporters provide direct insight into cellular processes by producing a quantifiable signal dictated by reporter gene expression. The core of a bioreporter is a genetic circuit in which a reporter gene (or operon) is fused to promoter and regulatory sequences that govern its expression. In this study, we develop a system for constructing novel Escherichia coli bioreporters based on Golden Gate assembly, a synthetic biology approach for the rapid and seamless fusion of DNA fragments. Gene circuits are generated by fusing promoter and reporter sequences encoding yellow fluorescent protein, mCherry, bacterial luciferase, and an anaerobically active flavin-based fluorescent protein. We address a barrier to the implementation of Golden Gate assembly by designing a series of compatible destination vectors that can accommodate the assemblies. We validate the approach by measuring the activity of constitutive bioreporters and mercury and arsenic biosensors in quantitative exposure assays. We also demonstrate anaerobic quantification of mercury and arsenic in biosensors that produce flavin-based fluorescent protein, highlighting the expanding range of redox conditions that can be examined by microbial bioreporters. IMPORTANCE Microbial bioreporters are versatile genetic tools with wide-ranging applications, particularly in the field of environmental toxicology. For example, biosensors that produce a signal output in the presence of a specific analyte offer less costly alternatives to analytical methods for the detection of environmental toxins such as mercury and arsenic. Biosensors of specific toxins can also be used to test hypotheses regarding mechanisms of uptake, toxicity, and biotransformation. In this study, we develop an assembly platform that uses a synthetic biology technique to streamline construction of novel Escherichia coli bioreporters that produce fluorescent or luminescent signals either constitutively or in response to mercury and arsenic exposure. Beyond the synthesis of novel biosensors, our assembly platform can be adapted for numerous applications, including labelling bacteria for fluorescent microscopy, developing gene expression systems, and modifying bacterial genomes.

scholarly journals Inotropic and lusitropic, but not arrhythmogenic, effects of adipocytokine resistin on human atrial myocardium

2020 ◽  
Vol 319 (3) ◽  
pp. E540-E547
Author(s):  
Hamish M. Aitken-Buck ◽  
Aram A. Babakr ◽  
Ingrid C. Fomison-Nurse ◽  
Isabelle van Hout ◽  
Philip J. Davis ◽  
...  
Keyword(s):  
Epicardial Adipose Tissue ◽  
Culture Media ◽  
Fold Increase ◽  
Human Myocardium ◽  
Adrenergic Agonists ◽  
Maximal Level ◽  
Atrial Myocardium ◽  
Human Atrial Trabeculae ◽  
Dose Dependent ◽  
Arrhythmogenic Effects

The adipocytokine resistin is released from epicardial adipose tissue (EAT). Plasma resistin and EAT deposition are independently associated with atrial fibrillation. The EAT secretome enhances arrhythmia susceptibility and inotropy of human myocardium. Therefore, we aimed to determine the effect of resistin on the function of human myocardium and how resistin contributes to the proarrhythmic effect of EAT. EAT biopsies were obtained from 25 cardiac surgery patients. Resistin levels were measured by ELISA in 24-h EAT culture media ( n = 8). The secretome resistin concentrations increased over the culture period to a maximal level of 5.9 ± 1.2 ng/mL. Coculture with β-adrenergic agonists isoproterenol ( n = 4) and BRL37344 ( n = 13) had no effect on EAT resistin release. Addition of resistin (7, 12, 20 ng/mL) did not significantly increase the spontaneous contraction propensity of human atrial trabeculae ( n = 10) when given alone or in combination with isoproterenol. Resistin dose-dependently increased trabecula-developed force (maximal 2.9-fold increase, P < 0.0001), as well as the maximal rates of contraction (2.6-fold increase, P = 0.002) and relaxation (1.8-fold increase, P = 0.007). Additionally, the postrest potentiation capacity of human trabeculae was reduced at all resistin doses, suggesting that the inotropic effect induced by resistin might be due to altered sarcoplasmic reticulum Ca2+ handling. EAT resistin release is not modulated by common arrhythmia triggers. Furthermore, exogenous resistin does not promote arrhythmic behavior in human atrial trabeculae. Resistin does, however, induce an acute dose-dependent positive inotropic and lusitropic effect.

scholarly journals Comparison of 7α-methyl-19-nortestosterone effectiveness alone or combined with progestins on androgen receptor mediated-transactivation

Reproduction ◽  
2012 ◽  
Vol 143 (2) ◽  
pp. 211-219 ◽  
Author(s):  
Rocío García-Becerra ◽  
David Ordaz-Rosado ◽  
Gabriela Noé ◽  
Bertha Chávez ◽  
Austin J Cooney ◽  
...  
Keyword(s):  
Gene Expression ◽  
Androgen Receptor ◽  
Reporter Gene ◽  
Gene Transcription ◽  
Detrimental Effect ◽  
Synergistic Effects ◽  
Estrogen Receptor Α ◽  
Male Contraceptive ◽  
Estrogen Activity ◽  
Dose Dependent

7α-methyl-19-nortestosterone (MENT) is an androgen with potent gonadotropin inhibitory activity and prostate-sparing effects. These attributes give MENT advantages over testosterone as a male contraceptive, but, as in the case of testosterone, a partial dose-dependent suppression of spermatogenesis has been observed. Combination of testosterone or MENT with synthetic progestins improves the rate of azoospermia; however, it is unknown whether these combinations affect hormone androgenicity or exert synergistic effects via progestational or androgenic interaction. Herein, using transactivation assays, we examined the ability of MENT alone or combined with several 19-nor-derived synthetic progestins to activate androgen receptor (AR)-dependent gene transcription. In addition, the capability of 7α-methyl-estradiol (7α-methyl-E2), an aromatized metabolite of MENT, to transactivate gene transcription via estrogen receptor α (ERα; ESR1) or ERβ (ESR2) was also investigated. As expected, MENT induced gene transactivation through either the progesterone receptor (PGR) or the AR. MENT was as efficient as progesterone in activating PGR-mediated reporter gene expression, but it was ten times more potent than testosterone and dihydrotestoterone in activating of AR-driven gene expression. The addition of increasing concentrations of other 19-nortestosterone derivatives (norethisterone or levonorgestrel) did not affect, in a significant manner, the ability of MENT to activate AR-dependent reporter gene transcription. The same results were obtained with different cell lines. 7α-Methyl-E2 resulted in potent estrogen activity via both ER subtypes with efficiency similar to natural E2. These results suggest that the addition of 19-nortestosterone-derived progestins, as a hormonal adjuvant in male fertility strategies for effective spermatogenic suppression, does not display any detrimental effect that would interfere with MENT androgenic transcriptional activity.

Increased Expression of u-PA and u-PAR on Monocytes by LDL and Lp(a) Lipoproteins – Consequences for Plasmin Generation and Monocyte Adhesion

1999 ◽  
Vol 81 (04) ◽  
pp. 594-560 ◽  
Author(s):  
Florence Ganné ◽  
Marc Vasse ◽  
Jean-Louis Beaudeu ◽  
Jacqueline Peynet ◽  
Arnaud François ◽  
...  
Keyword(s):  
Fold Increase ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Atheromatous Plaque ◽  
Monocyte Adhesion ◽  
Blood Monocytes ◽  
Proteolytic Cascade ◽  
Ecm Degradation ◽  
Dose Dependent Increase ◽  
Dose Dependent

SummaryMonocyte-derived foam cells figure prominently in rupture-prone regions of atherosclerotic plaque. As urokinase/urokinase-receptor (u-PA/u-PAR) is the trigger of a proteolytic cascade responsible for ECM degradation, we have examined the effect of atherogenic lipoproteins on monocyte surface expression of u-PAR and u-PA. Peripheral blood monocytes, isolated from 10 healthy volunteers, were incubated with 10 to 200 µg/ml of native or oxidised (ox-) atherogenous lipoproteins for 18 h and cell surface expression of u-PA and u-PAR was analysed by flow cytometry. Both LDL and Lp(a) induced a dose-dependent increase in u-PA (1.6-fold increase with 200 μg/ml of ox-LDL) and u-PAR [1.7-fold increase with 200 μg/ml of ox-Lp(a)]. There is a great variability of the response among the donors, some of them remaining non-responders (absence of increase of u-PA or u-PAR) even at 200 μg/ml of lipoproteins. In positive responders, enhanced u-PA/u-PAR is associated with a significant increase of plasmin generation (1.9-fold increase with 200 μg/ml of ox-LDL), as determined by an amidolytic assay. Furthermore, monocyte adhesion to vitronectin and fibrinogen was significantly enhanced by the lipoproteins [respectively 2-fold and 1.7-fold increase with 200 μg/ml of ox-Lp(a)], due to the increase of u-PAR and ICAM-1, which are receptors for vitronectin and fibrinogen. These data suggest that atherogenous lipoproteins could contribute to the development of atheromatous plaque by increasing monocyte adhesion and trigger plaque weakening by inducing ECM degradation.

Increased Protein Synthesis by Human Platelets during Phagocytosis of Latex Particles in Vitro

1976 ◽  
Vol 35 (02) ◽  
pp. 350-357 ◽  
Author(s):  
Hana Bessler ◽  
Galila Agam ◽  
Meir Djaldetti
Keyword(s):  
Protein Synthesis ◽  
Fold Increase ◽  
Human Platelets ◽  
Polystyrene Latex ◽  
Latex Particles ◽  
The Third ◽  
Platelet Protein ◽  
Dose Dependent ◽  
Phagocytotic Activity

SummaryA three-fold increase of protein synthesis by human platelets during in vitro phagocytosis of polystyrene latex particles was detected. During the first two hours of incubation, the percentage of phagocytizing platelets and the number of latex particles per platelet increased; by the end of the third hour, the first parameter remained stable, while the number of latex particles per cell had decreased.Vincristine (20 μg/ml of cell suspension) inhibited platelet protein synthesis. This effect was both time- and dose-dependent. The drug also caused a decrease in the number of phagocytizing cells, as well as in their phagocytotic activity.

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