scholarly journals Redesigned TetR-Aptamer System To Control Gene Expression in Plasmodium falciparum

mSphere ◽  
2020 ◽  
Vol 5 (4) ◽  
Author(s):  
Krithika Rajaram ◽  
Hans B. Liu ◽  
Sean T. Prigge
Keyword(s):  
Gene Expression ◽  
Plasmodium Falciparum ◽  
Target Gene ◽  
Essential Genes ◽  
Untranslated Regions ◽  
Dependent Manner ◽  
Control Of Gene Expression ◽  
Content Type ◽  
Target Gene Expression ◽  
The Impact

ABSTRACT One of the most powerful approaches to understanding gene function involves turning genes on and off at will and measuring the impact at the cellular or organismal level. This particularly applies to the cohort of essential genes where traditional gene knockouts are inviable. In Plasmodium falciparum, conditional control of gene expression has been achieved by using multicomponent systems in which individual modules interact with each other to regulate DNA recombination, transcription, or posttranscriptional processes. The recently devised TetR-DOZI aptamer system relies on the ligand-regulatable interaction of a protein module with synthetic RNA aptamers to control the translation of a target gene. This technique has been successfully employed to study essential genes in P. falciparum and involves the insertion of several aptamer copies into the 3′ untranslated regions (UTRs), which provide control over mRNA fate. However, aptamer repeats are prone to recombination and one or more copies can be lost from the system, resulting in a loss of control over target gene expression. We rectified this issue by redesigning the aptamer array to minimize recombination while preserving the control elements. As proof of concept, we compared the original and modified arrays for their ability to knock down the levels of a putative essential apicoplast protein (PF3D7_0815700) and demonstrated that the modified array is highly stable and efficient. This redesign will enhance the utility of a tool that is quickly becoming a favored strategy for genetic studies in P. falciparum. IMPORTANCE Malaria elimination efforts have been repeatedly hindered by the evolution and spread of multidrug-resistant strains of Plasmodium falciparum. The absence of a commercially available vaccine emphasizes the need for a better understanding of Plasmodium biology in order to further translational research. This has been partly facilitated by targeted gene deletion strategies for the functional analysis of parasite genes. However, genes that are essential for parasite replication in erythrocytes are refractory to such methods, and require conditional knockdown or knockout approaches to dissect their function. One such approach is the TetR-DOZI system that employs multiple synthetic aptamers in the untranslated regions of target genes to control their expression in a tetracycline-dependent manner. Maintaining modified parasites with intact aptamer copies has been challenging since these repeats can be lost by recombination. By interspacing the aptamers with unique sequences, we created a stable genetic system that remains effective at controlling target gene expression.

scholarly journals A redesigned TetR-aptamer system to control gene expression in Plasmodium falciparum

2020 ◽  
Author(s):  
Krithika Rajaram ◽  
Hans B. Liu ◽  
Sean T. Prigge
Keyword(s):  
Gene Expression ◽  
Plasmodium Falciparum ◽  
Target Gene ◽  
Dna Recombination ◽  
Essential Genes ◽  
Untranslated Regions ◽  
Dependent Manner ◽  
Control Of Gene Expression ◽  
Target Gene Expression ◽  
The Impact

AbstractOne of the most powerful approaches to understanding gene function involves turning genes on and off at will and measuring the impact at the cellular or organismal level. This particularly applies to the cohort of essential genes where traditional gene knockouts are inviable. In Plasmodium falciparum, conditional control of gene expression has been achieved by using multi-component systems in which individual modules interact with each other to regulate DNA recombination, transcription or posttranscriptional processes. The recently devised TetR-DOZI aptamer system relies on the ligand-regulatable interaction of a protein module with synthetic RNA aptamers to control the translation of a target gene. This technique has been successfully employed to study essential genes in P. falciparum and involves the insertion of several aptamer copies into their 3’ untranslated regions (UTRs) which provide control over mRNA fate. However, aptamer repeats are prone to recombination and one or more copies can be lost from the system, resulting in a loss of control over target gene expression. We rectified this issue by redesigning the aptamer array to minimize recombination while preserving the control elements. As proof of concept, we compared the original and modified arrays for their ability to knock down the levels of a putative essential apicoplast protein (PF3D7_0815700) and demonstrated that the modified array is highly stable and efficient. This redesign will enhance the utility of a tool that is quickly becoming a favored strategy for genetic studies in P. falciparum.ImportanceMalaria elimination efforts have been repeatedly hindered by the evolution and spread of multidrug-resistant strains of Plasmodium falciparum. The absence of a commercially available vaccine emphasizes the need for a better understanding of Plasmodium biology in order to further translational research. This has been partly facilitated by targeted gene deletion strategies for the functional analysis of parasite genes. However, genes that are essential for parasite replication in erythrocytes are refractory to such methods, and require conditional knockdown or knockout approaches to dissect their function. One such approach is the TetR-DOZI system that employs multiple synthetic aptamers in the untranslated regions of target genes to control their expression in a tetracycline-dependent manner. Maintaining modified parasites with intact aptamer copies has been challenging since these repeats are frequently lost by recombination. By interspacing the aptamers with unique sequences, we created a stable genetic system that remains effective at controlling target gene expression.

Loss of miR-83 extends lifespan and affects target gene expression in an age-dependent manner in Caenorhabditis elegans

2018 ◽  
Vol 45 (12) ◽  
pp. 651-662 ◽  
Author(s):  
Emmanuel Enoch Dzakah ◽  
Ahmed Waqas ◽  
Shuai Wei ◽  
Bin Yu ◽  
Xiaolin Wang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Caenorhabditis Elegans ◽  
Target Gene ◽  
Dependent Manner ◽  
Target Gene Expression ◽  
Age Dependent

scholarly journals The Impact of Leadered and Leaderless Gene Structures on Translation Efficiency, Transcript Stability, and Predicted Transcription Rates in Mycobacterium smegmatis

2020 ◽  
Vol 202 (9) ◽  
Author(s):  
Tien G. Nguyen ◽  
Diego A. Vargas-Blanco ◽  
Louis A. Roberts ◽  
Scarlet S. Shell
Keyword(s):  
Gene Expression ◽  
Mycobacterium Tuberculosis ◽  
Mycobacterium Smegmatis ◽  
Half Life ◽  
Untranslated Regions ◽  
Translation Efficiency ◽  
Production Rates ◽  
Content Type ◽  
The Impact ◽  
Mrna Half Life

ABSTRACT Regulation of gene expression is critical for Mycobacterium tuberculosis to tolerate stressors encountered during infection and for nonpathogenic mycobacteria such as Mycobacterium smegmatis to survive environmental stressors. Unlike better-studied models, mycobacteria express ∼14% of their genes as leaderless transcripts. However, the impacts of leaderless transcript structures on mRNA half-life and translation efficiency in mycobacteria have not been directly tested. For leadered transcripts, the contributions of 5′ untranslated regions (UTRs) to mRNA half-life and translation efficiency are similarly unknown. In M. tuberculosis and M. smegmatis, the essential sigma factor, SigA, is encoded by a transcript with a relatively short half-life. We hypothesized that the long 5′ UTR of sigA causes this instability. To test this, we constructed fluorescence reporters and measured protein abundance, mRNA abundance, and mRNA half-life and calculated relative transcript production rates. The sigA 5′ UTR conferred an increased transcript production rate, shorter mRNA half-life, and decreased apparent translation rate compared to a synthetic 5′ UTR commonly used in mycobacterial expression plasmids. Leaderless transcripts appeared to be translated with similar efficiency as those with the sigA 5′ UTR but had lower predicted transcript production rates. A global comparison of M. tuberculosis mRNA and protein abundances failed to reveal systematic differences in protein/mRNA ratios for leadered and leaderless transcripts, suggesting that variability in translation efficiency is largely driven by factors other than leader status. Our data are also discussed in light of an alternative model that leads to different conclusions and suggests leaderless transcripts may indeed be translated less efficiently. IMPORTANCE Tuberculosis, caused by Mycobacterium tuberculosis, is a major public health problem killing 1.5 million people globally each year. During infection, M. tuberculosis must alter its gene expression patterns to adapt to the stress conditions it encounters. Understanding how M. tuberculosis regulates gene expression may provide clues for ways to interfere with the bacterium’s survival. Gene expression encompasses transcription, mRNA degradation, and translation. Here, we used Mycobacterium smegmatis as a model organism to study how 5′ untranslated regions affect these three facets of gene expression in multiple ways. We furthermore provide insight into the expression of leaderless mRNAs, which lack 5′ untranslated regions and are unusually prevalent in mycobacteria.

scholarly journals E2f1, E2f2, and E2f3 Control E2F Target Expression and Cellular Proliferation via a p53-Dependent Negative Feedback Loop

2007 ◽  
Vol 27 (1) ◽  
pp. 65-78 ◽  
Author(s):  
Cynthia Timmers ◽  
Nidhi Sharma ◽  
Rene Opavsky ◽  
Baidehi Maiti ◽  
Lizhao Wu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Target Gene ◽  
Target Genes ◽  
Cellular Proliferation ◽  
Kinase Activity ◽  
Cyclin Dependent Kinase ◽  
Control Of Gene Expression ◽  
Target Gene Expression ◽  
Rb Phosphorylation ◽  
P53 Target Genes

ABSTRACT E2F-mediated control of gene expression is believed to have an essential role in the control of cellular proliferation. Using a conditional gene-targeting approach, we show that the targeted disruption of the entire E2F activator subclass composed of E2f1, E2f2, and E2f3 in mouse embryonic fibroblasts leads to the activation of p53 and the induction of p53 target genes, including p21 CIP1 . Consequently, cyclin-dependent kinase activity and retinoblastoma (Rb) phosphorylation are dramatically inhibited, leading to Rb/E2F-mediated repression of E2F target gene expression and a severe block in cellular proliferation. Inactivation of p53 in E2f1-, E2f2-, and E2f3-deficient cells, either by spontaneous mutation or by conditional gene ablation, prevented the induction of p21 CIP1 and many other p53 target genes. As a result, cyclin-dependent kinase activity, Rb phosphorylation, and E2F target gene expression were restored to nearly normal levels, rendering cells responsive to normal growth signals. These findings suggest that a critical function of the E2F1, E2F2, and E2F3 activators is in the control of a p53-dependent axis that indirectly regulates E2F-mediated transcriptional repression and cellular proliferation.

scholarly journals Functional Genetic Elements for Controlling Gene Expression inCupriavidus necatorH16

2018 ◽  
Vol 84 (19) ◽  
Author(s):  
Swathi Alagesan ◽  
Erik K. R. Hanko ◽  
Naglis Malys ◽  
Muhammad Ehsaan ◽  
Klaus Winzer ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dynamic Range ◽  
Loop Structure ◽  
Stem Loop ◽  
Control Of Gene Expression ◽  
Content Type ◽  
Genetic Elements ◽  
Stem Loop Structure ◽  
Inducible Systems ◽  
The Impact

ABSTRACTA robust and predictable control of gene expression plays an important role in synthetic biology and biotechnology applications. Development and quantitative evaluation of functional genetic elements, such as constitutive and inducible promoters as well as ribosome binding sites (RBSs), are required. In this study, we designed, built, and tested promoters and RBSs for controlling gene expression in the model lithoautotrophCupriavidus necatorH16. A series of variable-strength, insulated, constitutive promoters exhibiting predictable activity within a >700-fold dynamic range was compared to the native PphaC, with the majority of promoters displaying up to a 9-fold higher activity. Positively (AraC/ParaBAD-l-arabinose and RhaRS/PrhaBAD-l-rhamnose) and negatively (AcuR/PacuRI-acrylate and CymR/Pcmt-cumate) regulated inducible systems were evaluated. By supplying different concentrations of inducers, a >1,000-fold range of gene expression levels was achieved. Application of inducible systems for controlling expression of the isoprene synthase geneispSled to isoprene yields that exhibited a significant correlation to the reporter protein synthesis levels. The impact of designed RBSs and other genetic elements, such as mRNA stem-loop structure and A/U-rich sequence, on gene expression was also evaluated. A second-order polynomial relationship was observed between the RBS activities and isoprene yields. This report presents quantitative data on regulatory genetic elements and expands the genetic toolbox ofC. necator.IMPORTANCEThis report provides tools for robust and predictable control of gene expression in the model lithoautotrophC. necatorH16. To address a current need, we designed, built, and tested promoters and RBSs for controlling gene expression inC. necatorH16. To answer a question on how existing and newly developed inducible systems compare, two positively (AraC/ParaBAD-l-arabinose and RhaRS/PrhaBAD-l-rhamnose) and two negatively (AcuR/PacuRI-acrylate and CymR/Pcmt-cumate) regulated inducible systems were quantitatively evaluated and their induction kinetics analyzed. To establish if gene expression can be further improved, the effect of genetic elements, such as mRNA stem-loop structure and A/U-rich sequence, on gene expression was evaluated. Using isoprene production as an example, the study investigated if and to what extent chemical compound yield correlates to the level of gene expression of product-synthesizing enzyme.

scholarly journals Nutritional Control of Antibiotic Resistance via an Interface between the Phosphotransferase System and a Two-Component Signaling System

2013 ◽  
Vol 58 (2) ◽  
pp. 957-965 ◽  
Author(s):  
Holly Snyder ◽  
Stephanie L. Kellogg ◽  
Laura M. Skarda ◽  
Jaime L. Little ◽  
Christopher J. Kristich
Keyword(s):  
Gene Expression ◽  
Antibiotic Resistance ◽  
Dependent Manner ◽  
Signaling System ◽  
Gi Tract ◽  
Phosphotransferase System ◽  
Control Of Gene Expression ◽  
Content Type ◽  
Two Component ◽  
Cephalosporin Resistance

ABSTRACTEnterococci are ubiquitous inhabitants of the gastrointestinal (GI) tract. However, antibiotic-resistant enterococci are also major causes of hospital-acquired infections. Enterococci are intrinsically resistant to cephalosporins, enabling growth to abnormally high densities in the GI tract in patients during cephalosporin therapy, thereby promoting dissemination to other sites where they cause infection. Despite its importance, many questions about the underlying basis for cephalosporin resistance remain. A specific two-component signaling system, composed of the CroS sensor kinase and its cognate response regulator (CroR), is required for cephalosporin resistance inEnterococcus faecalis, but little is known about the factors that control this signaling system to modulate resistance. To explore the signaling network in which CroR participates to influence cephalosporin resistance, we employed a protein fragment complementation assay to detect protein-protein interactions inE. faecaliscells, revealing a previously unknown association of CroR with the HPr protein of the phosphotransferase system (PTS) responsible for carbohydrate uptake and catabolite control of gene expression. Genetic and physiological analyses indicate that association with HPr restricts the ability of CroR to promote cephalosporin resistance and gene expression in a nutrient-dependent manner. Mutational analysis suggests that the interface used by HPr to associate with CroR is distinct from the interface used to associate with other cellular partners. Our results define a physical and functional connection between a critical nutrient-responsive signaling system (the PTS) and a two-component signaling system that drives antibiotic resistance inE. faecalis, and they suggest a general strategy by which bacteria can integrate their nutritional status with diverse environmental stimuli.

scholarly journals MiR-124 synergism with ELAVL3 enhances target gene expression to promote neuronal maturity

2020 ◽  
Author(s):  
Ya-Lin Lu ◽  
Yangjian Liu ◽  
Matthew J. McCoy ◽  
Andrew S. Yoo
Keyword(s):  
Gene Expression ◽  
Mirna Target ◽  
Target Gene ◽  
Target Genes ◽  
Dependent Manner ◽  
Neuronal Function ◽  
Mirna Target Gene ◽  
Target Gene Expression ◽  
Human Neurons

SummaryNeuron-enriched microRNAs (miRNAs), miR-9/9* and miR-124 (miR-9/9*-124), direct cell fate switching of human fibroblasts to neurons when ectopically expressed by repressing anti-neurogenic genes. How these miRNAs function after the onset of the transcriptome switch to a neuronal fate remains unclear. Here, we identified direct targets of miRNAs by Argonaute (AGO) HITS-CLIP as reprogramming cells activate the neuronal program and reveal the role of miR-124 that directly promotes the expression of its target genes associated with neuronal development and function. The mode of miR-124 as a positive regulator is determined by a neuron-enriched RNA-binding protein, ELAVL3, that interacts with AGO and binds target transcripts, whereas the non-neuronal ELAVL1 counterpart fails to elevate the miRNA-target gene expression. Although existing literature indicate that miRNA-ELAVL1 interaction can result in either target gene upregulation or downregulation in a context-dependent manner, we specifically identified neuronal ELAVL3 as the driver for miRNA target gene upregulation in neurons. In primary human neurons, repressing miR-124 and ELAVL3 led to the downregulation of genes involved in neuronal function and process outgrowth, and cellular phenotypes of reduced inward currents and neurite outgrowth. Results from our study support the role of miR-124 promoting neuronal function through positive regulation of its target genes.

scholarly journals A tight cold-inducible switch built by coupling thermosensitive transcriptional and proteolytic regulatory parts

2019 ◽  
Vol 47 (21) ◽  
pp. e137-e137 ◽  
Author(s):  
Yang Zheng ◽  
Fankang Meng ◽  
Zihui Zhu ◽  
Weijia Wei ◽  
Zhi Sun ◽  
...  
Keyword(s):  
Gene Expression ◽  
High Performance ◽  
Target Gene ◽  
Dynamic Range ◽  
Culture Media ◽  
Basic Research ◽  
Control Of Gene Expression ◽  
Target Gene Expression ◽  
Engineered Systems ◽  
Bacterial Genes

Abstract Natural organisms have evolved intricate regulatory mechanisms that sense and respond to fluctuating environmental temperatures in a heat- or cold-inducible fashion. Unlike dominant heat-inducible switches, very few cold-inducible genetic switches are available in either natural or engineered systems. Moreover, the available cold-inducible switches still have many shortcomings, including high leaky gene expression, small dynamic range (<10-fold) or broad transition temperature (>10°C). To address these problems, a high-performance cold-inducible switch that can tightly control target gene expression is highly desired. Here, we introduce a tight and fast cold-inducible switch that couples two evolved thermosensitive variants, TFts and TEVts, as well as an additional Mycoplasma florum Lon protease (mf-Lon) to effectively turn-off target gene expression via transcriptional and proteolytic mechanisms. We validated the function of the switch in different culture media and various Escherichia coli strains and demonstrated its tightness by regulating two morphogenetic bacterial genes and expressing three heat-unstable recombinant proteins, respectively. Moreover, the additional protease module enabled the cold-inducible switch to actively remove the pre-existing proteins in slow-growing cells. This work establishes a high-performance cold-inducible system for tight and fast control of gene expression which has great potential for basic research, as well as industrial and biomedical applications.

scholarly journals Methodological considerations on selection of stable reference genes for RT-qPCR in the neonatal rat brain in hypoxia and hypothermia

2019 ◽  
Author(s):  
M. Bustelo ◽  
M.A. Bruno ◽  
C.F. Loidl ◽  
H.W.M. Steinbusch ◽  
A.W.D. Gavilanes ◽  
...  
Keyword(s):  
Gene Expression ◽  
Rat Brain ◽  
Statistical Methods ◽  
Target Gene ◽  
Expression Profiles ◽  
Neonatal Rat ◽  
Experimental Conditions ◽  
Target Gene Expression ◽  
The Impact ◽  
Neonatal Rat Brain

AbstractReal-time reverse transcription PCR (qPCR) normalized to an internal reference gene (RG), is a frequently used method for quantifying gene expression changes in neuroscience. Although RG expression is assumed to be constantly independent of physiological or experimental conditions, several studies have shown that commonly used RGs are not expressed stably. The use of unstable RGs has a profound effect on the conclusions drawn from studies on gene expression, and almost universally results in spurious estimation of target gene expression. Approaches aimed at selecting and validating RGs often make use of different statistical methods, which may lead to conflicting results. The present study evaluates the expression of 5 candidate RGs (Actb, Pgk1, Sdha, Gapdh, Rnu6b) as a function of hypoxia exposure and hypothermic treatment in the neonatal rat cerebral cortex –in order to identify RGs that are stably expressed under these experimental conditions– and compares several statistical approaches that have been proposed to validate RGs. In doing so, we first analyzed the RG ranking stability proposed by several widely used statistical methods and related tools, i.e. the Coefficient of Variation (CV) analysis, GeNorm, NormFinder, BestKeeper, and the ΔCt method. Subsequently, we compared RG expression patterns between the various experimental groups. We found that these statistical methods, next to producing different rankings per se, all ranked RGs displaying significant differences in expression levels between groups as the most stable RG. As a consequence, when assessing the impact of RG selection on target gene expression quantification, substantial differences in target gene expression profiles were observed. As such, by assessing mRNA expression profiles within the neonatal rat brain cortex in hypoxia and hypothermia as a showcase, this study underlines the importance of further validating RGs for each new experimental paradigm considering the limitations of each selection method.

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