scholarly journals A precisely adjustable, variation-suppressed eukaryotic transcriptional controller to enable genetic discovery

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Asli Azizoglu ◽  
Roger Brent ◽  
Fabian Rudolf
Keyword(s):  
Cellular Protein ◽  
Cell Cycle Synchronization ◽  
Basal Expression ◽  
Expression Of Genes ◽  
Precise Adjustment ◽  
Conditional Expression ◽  
Biological Discovery ◽  
Dependent Growth ◽  
Cell Variation ◽  
Genetic Discovery

Conditional expression of genes and observation of phenotype remain central to biological discovery. Current methods enable either on/off or imprecisely controlled graded gene expression. We developed a 'well-tempered' controller, WTC846, for precisely adjustable, graded, growth condition independent expression of genes in Saccharomyces cerevisiae. Controlled genes are expressed from a strong semisynthetic promoter repressed by the prokaryotic TetR, which also represses its own synthesis; with basal expression abolished by a second, 'zeroing' repressor. The autorepression loop lowers cell-to-cell variation while enabling precise adjustment of protein expression by a chemical inducer. WTC846 allelic strains in which the controller replaced the native promoters recapitulated known null phenotypes (CDC42, TPI1), exhibited novel overexpression phenotypes (IPL1), showed protein dosage-dependent growth rates and morphological phenotypes (CDC28, TOR2, PMA1 and the hitherto uncharacterized PBR1), and enabled cell cycle synchronization (CDC20). WTC846 defines an 'expression clamp' allowing protein dosage to be adjusted by the experimenter across the range of cellular protein abundances, with limited variation around the setpoint.

scholarly journals A precisely adjustable, variation-suppressed eukaryotic transcriptional controller to enable genetic discovery

2019 ◽  
Author(s):  
Asli Azizoğlu ◽  
Roger Brent ◽  
Fabian Rudolf
Keyword(s):  
Gene Expression ◽  
Gene Dosage ◽  
Cellular Protein ◽  
Native Promoter ◽  
Basal Expression ◽  
Expression Of Genes ◽  
Precise Adjustment ◽  
Conditional Expression ◽  
Dependent Growth ◽  
Cell Variation

AbstractMethods to express genes conditionally into phenotype remain central to biological experimentation and biotechnology. Current methods enable either on/off or imprecisely controlled graded gene expression. We developed a “well-tempered” controller, WTC846, for precisely adjustable, graded and growth condition independent conditional expression of genes in Saccharomyces cerevisiae. In WTC846 strains, the controlled genes are expressed from a strong, native promoter engineered to be repressed by the prokaryotic TetR protein and induced by tetracycline and analogues. A second instance of this promoter drives TetR itself. This autorepression loop exhibits low cell-to-cell variation in gene expression and allows precise adjustment of the steady state abundance of any protein with inducer. A second, constitutively expressed zeroing repressor abolishes basal expression in the absence of inducer. WTC846-controlled, stable (Cdc42, Tpi1) and unstable (Ipl1) proteins recapitulated known knockout and overexpression phenotypes. WTC846::CDC20 strains enabled inducer regulated cell cycle synchronization. WTC846 alleles of CDC28, TOR1, PBR1 and PMA1 exhibited expected gene dosage-dependent growth rates and morphological phenotypes, and WTC846::WHI5 strains exhibited inducer controlled differences in cell volume. WTC846 controlled genes comprise a new kind of “expression clamped” allele, for which variation in expression is minimized and gene dosage can be set by the experimenter across the range of cellular protein abundances. In yeast, we expect WTC846 alleles to find use in assessment of phenotypes now incompletely penetrant due to variable dosage of the causative protein, and in genome-wide epistasis screens. Implementation in higher cells should enable experiments now impossible due to cell-to-cell variation and imprecise control.

scholarly journals MicroRNAs as Potential Biomarkers in Atherosclerosis

2019 ◽  
Vol 20 (22) ◽  
pp. 5547 ◽  
Author(s):  
Alexey Churov ◽  
Volha Summerhill ◽  
Andrey Grechko ◽  
Varvara Orekhova ◽  
Alexander Orekhov
Keyword(s):  
Molecular Mechanisms ◽  
Expression Patterns ◽  
Inflammatory Cells ◽  
Cellular Protein ◽  
Cholesterol Homeostasis ◽  
Transcriptional Level ◽  
High Morbidity ◽  
Industrialized Countries ◽  
Rna Molecules ◽  
Expression Of Genes

Atherosclerosis is a complex multifactorial disease that, despite advances in lifestyle management and drug therapy, remains to be the major cause of high morbidity and mortality rates from cardiovascular diseases (CVDs) in industrialized countries. Therefore, there is a great need in reliable diagnostic/prognostic biomarkers and effective treatment alternatives to reduce its burden. It was established that microRNAs (miRNAs/miRs), a class of non-coding single-stranded RNA molecules, can regulate the expression of genes at the post-transcriptional level and, accordingly, coordinate the cellular protein expression. Thus, they are involved not only in cell-specific physiological functions but also in the cellular and molecular mechanisms of human pathologies, including atherosclerosis. MiRNAs may be significant in the dysregulation that affects endothelial integrity, the function of vascular smooth muscle and inflammatory cells, and cellular cholesterol homeostasis that drives the initiation and growth of an atherosclerotic plaque. Besides, distinct expression patterns of several miRNAs are attributed to atherosclerotic and cardiovascular patients. In this article, the evidence indicating the multiple critical roles of miRNAs and their relevant molecular mechanisms related to atherosclerosis development and progression was reviewed. Moreover, the effects of miRNAs on atherosclerosis enabled to exploit them as novel diagnostic biomarkers and therapeutic targets that may lead to better management of atherosclerosis and CVDs.

scholarly journals FpvA Receptor Involvement in Pyoverdine Biosynthesis in Pseudomonas aeruginosa

2002 ◽  
Vol 184 (12) ◽  
pp. 3268-3275 ◽  
Author(s):  
Jiangsheng Shen ◽  
Allison Meldrum ◽  
Keith Poole
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Iron Uptake ◽  
Mature Protein ◽  
Wild Type ◽  
Deletion Derivative ◽  
Siderophore Receptors ◽  
Expression Of Genes ◽  
Peptide Moiety ◽  
N Terminus ◽  
Dependent Growth

ABSTRACT Alignment of the Pseudomonas aeruginosa ferric pyoverdine receptor, FpvA, with similar ferric-siderophore receptors revealed that the mature protein carries an extension of ca. 70 amino acids at its N terminus, an extension shared by the ferric pseudobactin receptors of P. putida. Deletion of fpvA from the chromosome of P. aeruginosa reduced pyoverdine production in this organism, as a result of a decline in expression of genes (e.g., pvdD) associated with the biosynthesis of the pyoverdine peptide moiety. Wild-type fpvA restored pvd expression in the mutant, thereby complementing its pyoverdine deficiency, although a deletion derivative of fpvA encoding a receptor lacking the N terminus of the mature protein did not. The truncated receptor was, however, functional in pyoverdine-mediated iron uptake, as evidenced by its ability to promote pyoverdine-dependent growth in an iron-restricted medium. These data are consistent with the idea that the N-terminal extension plays a role in FpvA-mediated pyoverdine biosynthesis in P. aeruginosa.

scholarly journals A Vitamin B12-Based System for Conditional Expression Reveals dksA To Be an Essential Gene in Myxococcus xanthus

2009 ◽  
Vol 191 (9) ◽  
pp. 3108-3119 ◽  
Author(s):  
Diana García-Moreno ◽  
María Carmen Polanco ◽  
Gloria Navarro-Avilés ◽  
Francisco J. Murillo ◽  
S. Padmanabhan ◽  
...  
Keyword(s):  
Transcriptional Control ◽  
Essential Gene ◽  
Myxococcus Xanthus ◽  
Stringent Response ◽  
Multicellular Development ◽  
New Genes ◽  
Expression Of Genes ◽  
Conditional Expression ◽  
Dividing Cells

ABSTRACT Myxococcus xanthus is a prokaryotic model system for the study of multicellular development and the response to blue light. The previous analyses of these processes and the characterization of new genes would benefit from a robust system for controlled gene expression, which has been elusive so far for this bacterium. Here, we describe a system for conditional expression of genes in M. xanthus based on our recent finding that vitamin B12 and CarH, a MerR-type transcriptional repressor, together downregulate a photoinducible promoter. Using this system, we confirmed that M. xanthus rpoN, encoding σ54, is an essential gene, as reported earlier. We then tested it with ftsZ and dksA. In most bacteria, ftsZ is vital due to its role in cell division, whereas null mutants of dksA, whose product regulates the stringent response via transcriptional control of rRNA and amino acid biosynthesis promoters, are viable but cause pleiotropic effects. As with rpoN, it was impossible to delete endogenous ftsZ or dksA in M. xanthus except in a merodiploid background carrying another functional copy, which indicates that these are essential genes. B12-based conditional expression of ftsZ was insufficient to provide the high intracellular FtsZ levels required. With dksA, as with rpoN, cells were viable under permissive but not restrictive conditions, and depletion of DksA or σ54 produced filamentous, aberrantly dividing cells. dksA thus joins rpoN in a growing list of genes dispensable in many bacteria but essential in M. xanthus.

scholarly journals A Propionate-Inducible Expression System for Enteric Bacteria

2005 ◽  
Vol 71 (11) ◽  
pp. 6856-6862 ◽  
Author(s):  
Sung Kuk Lee ◽  
Jay D. Keasling
Keyword(s):  
Fluorescent Protein ◽  
Expression System ◽  
Enteric Bacteria ◽  
Expression Vectors ◽  
Gene Encoding ◽  
Basal Expression ◽  
Expression Of Genes ◽  
Catabolic Genes ◽  
Wide Range ◽  
Green Fluorescent

ABSTRACT A series of new expression vectors (pPro) have been constructed for the regulated expression of genes in Escherichia coli. The pPro vectors contain the prpBCDE promoter (P prpB ) responsible for expression of the propionate catabolic genes (prpBCDE) and prpR encoding the positive regulator of this promoter. The efficiency and regulatory properties of the prpR-P prpB system were measured by placing the gene encoding the green fluorescent protein (gfp) under the control of the inducible P prpB of E. coli. This system provides homogenous expression in individual cells, highly regulatable expression over a wide range of propionate concentrations, and strong expression (maximal 1,500-fold induction) at high propionate concentrations. Since the prpBCDE promoter has CAP-dependent activation, the prpR-P prpB system exhibited negligible basal expression by addition of glucose to the medium.

scholarly journals Role of NF-kB RelB in Aryl Hydrocarbon Receptor-Mediated Ligand Specific Effects

2019 ◽  
Vol 20 (11) ◽  
pp. 2652 ◽  
Author(s):  
Yasuhiro Ishihara ◽  
Sarah Y. Kado ◽  
Christiane Hoeper ◽  
Shelly Harel ◽  
Christoph F. A. Vogel
Keyword(s):  
Aryl Hydrocarbon Receptor ◽  
Functional Expression ◽  
Dependent Manner ◽  
Induced Expression ◽  
Aryl Hydrocarbon ◽  
Regulatory Enzymes ◽  
Basal Expression ◽  
Expression Of Genes ◽  
Ahr Gene

Here, we investigate the role of RelB in the regulation of genes which were identified to be induced in an aryl hydrocarbon receptor (AhR)-dependent manner and critically involved in regulation of immune responses. We analyzed the expression of genes of the AhR gene battery, cytokines, and immune regulatory enzymes in bone marrow-derived macrophages (BMM) and thymus of B6 wildtype (wt) mice and RelB knockout (RelB−/−) mice after treatment with various AhR ligands. The 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced expression of indoleamine 2,3-dioxygenase 1 (IDO1) and IDO2 was significantly repressed in thymus of RelB−/− mice but not in BMM derived from RelB−/− mice. Interestingly, the induced and basal expression of the cytokines interleukin (IL)-17A, IL-22, and CCL20 required the functional expression of RelB. The RelB-dependent expression of CCL20 was induced by the AhR ligands TCDD and 6-formylindolo[3,2-b]carbazole (FICZ), whereas indole-3-carbinol (I3C) suppressed CCL20 in lipopolysaccharide (LPS)-activated wt BMM. The LPS-induced expression of IL-6 and IL-10 was enhanced by TCDD and FICZ, whereas I3C significantly suppressed these cytokines in BMM. The exposure to FICZ led to higher increases of IL-17A and IL-22 mRNA compared to the effect of TCDD or I3C in thymus of wt mice. On the other hand, TCDD was the strongest inducer of CYP1A1, AhR Repressor (AhRR), and IDO2. In summary, these findings provide evidence for the important role of RelB in the transcriptional regulation of cytokines and enzymes induced by AhR ligands.

scholarly journals Zinc limitation in Klebsiella pneumoniae profiled by quantitative proteomics influences transcriptional regulation and cation transporter-associated capsule production

2019 ◽  
Author(s):  
Arjun Sukumaran ◽  
Jennifer Geddes-McAlister
Keyword(s):  
Klebsiella Pneumoniae ◽  
Environmental Conditions ◽  
Metal Ion ◽  
Cellular Protein ◽  
Pcr Analysis ◽  
Phenotypic Analysis ◽  
Growth And Survival ◽  
Expression Of Genes ◽  
Capsule Production ◽  
The Impact

Abstract Background: Microbial organisms encounter a variety of environmental conditions, including changes to metal ion availability. Metals ions play an important role in many biological processes for growth and survival. As such, microbes alter their cellular protein regulation and secretion patterns in adaptation to changing environmental conditions. This study focuses on Klebsiella pneumoniae, an opportunistic bacterium responsible for nosocomial infections and by using K. pneumoniae, we aim to determine how a nutrient-limited environment (e.g., zinc) modulates the cellular proteome and secretome of the bacteria. This information will inform on protein-level regulation of bacterial responses to nutritional immunity within the host and improve our understanding of the dynamic and complex relationship between host and pathogen during infection. Results: Analysis of intra- and extracellular changes identified 2,380 proteins from the total cellular proteome (cell pellet) and 246 secreted proteins (supernatant). Specifically, hutC, a repressor of the histidine utilization operon, showed significantl increases abundance under replete conditions, which coincided with an expected reduction in expression of genes within the hut operon from our validation qRT-PCR analysis. Additionally, we characterized a putative cation transport regulator, chaB that was significantly abundant under zinc-replete conditions. Phenotypic analysis of a chaBdeletion strain observe a reduction in capsule production, greater tolerance to high extracellular zinc concentrations, and unimpaired virulence when compared to the WT strain. Conclusions: This is first study to comprehensively profile the impact of zinc availability on the proteome and secretome of K. pneumoniae and uncover a novel connection between zinc transport and capsule production in the bacterial system.

scholarly journals Constricted Flux through the Branched-Chain Amino Acid Biosynthetic Enzyme Acetolactate Synthase Triggers Elevated Expression of Genes Regulated by rpoS and Internal Acidification

1998 ◽  
Vol 180 (4) ◽  
pp. 785-792 ◽  
Author(s):  
Tina K. Van Dyk ◽  
Brenda L. Ayers ◽  
Robin W. Morgan ◽  
Robert A. Larossa
Keyword(s):  
Acetolactate Synthase ◽  
Inducible Promoter ◽  
Weak Acid ◽  
Open Reading Frames ◽  
New Members ◽  
Basal Expression ◽  
Expression Of Genes ◽  
Elevated Expression ◽  
Valine Biosynthesis ◽  
Branched Chain

ABSTRACT The first common enzyme of isoleucine and valine biosynthesis, acetolactate synthase (ALS), is specifically inhibited by the herbicide sulfometuron methyl (SM). To further understand the physiological consequences of flux alterations at this point in metabolism,Escherichia coli genes whose expression was induced by partial inhibition of ALS were sought. Plasmid-based fusions of randomE. coli DNA fragments to Photorhabdus luminescens luxCDABE were screened for bioluminescent increases in actively growing liquid cultures slowed 25% by the addition of SM. From more than 8,000 transformants, 12 unique SM-inducible promoter-lux fusions were identified. Thelux reporter genes were joined to seven uncharacterized open reading frames, f253a, f415,frvX, o513, o521, yciG, and yohF, and five known genes, inaA,ldcC, osmY, poxB, andsohA. Inactivation of the rpoS-encoded sigma factor, ςS, reduced basal expression levels of six of these fusions 10- to 200-fold. These six genes defined four new members of the ςS regulon, f253a, ldcC,yciG, and yohF, and included two known members,osmY and poxB. Furthermore, the weak acid salicylate, which causes cytoplasmic acidification, also induced increased bioluminescence from seven SM-inducible promoter-lux fusion-containing strains, namely, those with fusions of the ςS-controlled genes and inaA. The pattern of gene expression changes suggested that restricted ALS activity may result in intracellular acidification and induction of the ςS-dependent stress response.

scholarly journals Defining a rob Regulon in Escherichia coliby Using Transposon Mutagenesis

2000 ◽  
Vol 182 (13) ◽  
pp. 3794-3801 ◽  
Author(s):  
Marjon H. J. Bennik ◽  
Pablo J. Pomposiello ◽  
Derek F. Thorne ◽  
Bruce Demple
Keyword(s):  
Gene Expression ◽  
Organic Solvents ◽  
Deletion Mutant ◽  
Northern Analysis ◽  
Promoter Regions ◽  
Mobility Shift ◽  
Basal Expression ◽  
Transcription Cofactor ◽  
Expression Of Genes ◽  
Range Of Functions

ABSTRACT The Rob protein of Escherichia coli is a member of the AraC-XylS family of prokaryotic transcriptional regulators and is expressed constitutively. Deletion of the rob gene increases susceptibility to organic solvents, while overexpression of Rob increases tolerance to organic solvents and resistance to a variety of antibiotics and to the superoxide-generating compound phenazine methosulfate. To determine whether constitutive levels of Rob regulate basal gene expression, we performed a MudJ transposon screen in arob deletion mutant containing a plasmid that allows for controlled rob gene expression. We identified eight genes and confirmed that seven are transcriptionally activated by normal expression of Rob from the chromosomal rob gene (inaA, marR, aslB,ybaO, mdlA, yfhD, andybiS). One gene, galT, was repressed by Rob. We also demonstrated by Northern analysis that basal expression ofmicF is significantly higher in wild-type E. coli than in a rob deletion mutant. Rob binding to the promoter regions of most of these genes was substantiated in electrophoretic mobility shift assays. However, Mu insertions in individual Rob-regulated genes did not affect solvent sensitivity. This phenotype may depend on changes in the expression of several of these Rob-regulated genes or on other genes that were not identified. Rob clearly affects the basal expression of genes with a broad range of functions, including antibiotic resistance, acid adaptation, carbon metabolism, cell wall synthesis, central intermediary metabolism, and transport. The magnitudes of Rob's effects are modest, however, and the protein may thus play a role as a general transcription cofactor.

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