scholarly journals In Vivo Behavior of the Tandem Glycine Riboswitch in Bacillus subtilis

mBio ◽  
2017 ◽  
Vol 8 (5) ◽  
Author(s):  
Arianne M. Babina ◽  
Nicholas E. Lea ◽  
Michelle M. Meyer
Keyword(s):  
Gene Expression ◽  
Bacillus Subtilis ◽  
Regulate Gene Expression ◽  
Binding Domains ◽  
Expression Platform ◽  
The Impact ◽  
Organismal Fitness ◽  
Regulate Gene

ABSTRACT In many bacterial species, the glycine riboswitch is composed of two homologous ligand-binding domains (aptamers) that each bind glycine and act together to regulate the expression of glycine metabolic and transport genes. While the structure and molecular dynamics of the tandem glycine riboswitch have been the subject of numerous in vitro studies, the in vivo behavior of the riboswitch remains largely uncharacterized. To examine the proposed models of tandem glycine riboswitch function in a biologically relevant context, we characterized the regulatory activity of mutations to the riboswitch structure in Bacillus subtilis using β-galactosidase assays. To assess the impact disruptions to riboswitch function have on cell fitness, we introduced these mutations into the native locus of the tandem glycine riboswitch within the B. subtilis genome. Our results indicate that glycine does not need to bind both aptamers for regulation in vivo and mutations perturbing riboswitch tertiary structure have the most severe effect on riboswitch function and gene expression. We also find that in B. subtilis, the glycine riboswitch-regulated gcvT operon is important for glycine detoxification. IMPORTANCE The glycine riboswitch is a unique cis-acting mRNA element that contains two tandem homologous glycine-binding domains that act on a single expression platform to regulate gene expression in response to glycine. While many in vitro experiments have characterized the tandem architecture of the glycine riboswitch, little work has investigated the behavior of this riboswitch in vivo. In this study, we analyzed the proposed models of tandem glycine riboswitch regulation in the context of its native locus within the Bacillus subtilis genome and examined how disruptions to glycine riboswitch function impact organismal fitness. Our work offers new insights into riboswitch function in vivo and reinforces the potential of riboswitches as novel antimicrobial targets. IMPORTANCE The glycine riboswitch is a unique cis-acting mRNA element that contains two tandem homologous glycine-binding domains that act on a single expression platform to regulate gene expression in response to glycine. While many in vitro experiments have characterized the tandem architecture of the glycine riboswitch, little work has investigated the behavior of this riboswitch in vivo. In this study, we analyzed the proposed models of tandem glycine riboswitch regulation in the context of its native locus within the Bacillus subtilis genome and examined how disruptions to glycine riboswitch function impact organismal fitness. Our work offers new insights into riboswitch function in vivo and reinforces the potential of riboswitches as novel antimicrobial targets.

Epigenetherapy, a new concept

2011 ◽  
Vol 2 (3) ◽  
pp. 127-134
Author(s):  
Tiia Husso ◽  
Mikko P. Turunen ◽  
Nigel Parker ◽  
Seppo Ylä-Herttuala
Keyword(s):  
Gene Expression ◽  
Small Rnas ◽  
Basic Research ◽  
Clinical Applications ◽  
Regulate Gene Expression ◽  
Endogenous Gene ◽  
Rna Molecules ◽  
Regulate Gene

AbstractSmall RNAs have been shown to regulate gene transcription by interacting with the promoter region and modifying the histone code. The exact mechanism of function is still unclear but the feasibility to activate or repress endogenous gene expression with small RNA molecules has already been demonstrated in vitro and in vivo. In traditional gene therapy non-mutated or otherwise useful genes are inserted into patient's cells to treat a disease. In epigenetherapy the action of small RNAs is utilized by delivering only the small RNAs to patient's cells where they then regulate gene expression by epigenetic mechanisms. This method could be widely useful not only for basic research but also for clinical applications of small RNAs.

scholarly journals Optimization of a bacterial three-hybrid assay through in vivo titration of an RNA-DNA adapter-protein

2020 ◽  
Author(s):  
Clara D. Wang ◽  
Rachel Mansky ◽  
Hannah LeBlanc ◽  
Chandra M. Gravel ◽  
Katherine E. Berry
Keyword(s):  
Gene Expression ◽  
Protein Interactions ◽  
Regulate Gene Expression ◽  
E Coli ◽  
Binding Energetics ◽  
Response To Stress ◽  
First Time ◽  
Regulate Gene

ABSTRACTNon-coding RNAs regulate gene expression in every domain of life. In bacteria, small RNAs (sRNAs) regulate gene expression in response to stress and are often assisted by RNA-chaperone proteins, such as Hfq. We have recently developed a bacterial three-hybrid (B3H) assay that detects the strong binding interactions of certain E. coli sRNAs with proteins Hfq and ProQ. Despite the promise of this system, the signal-to-noise has made it challenging to detect weaker interactions. In this work, we use Hfq-sRNA interactions as a model system to optimize the B3H assay, so that weaker RNA-protein interactions can be more reliably detected. We find that the concentration of the RNA-DNA adapter is an important parameter in determining the signal in the system, and have modified the plasmid expressing this component to tune its concentration to optimal levels. In addition, we have systematically perturbed the binding affinity of Hfq-RNA interactions to define, for the first time, the relationship between B3H signal and in vitro binding energetics. The new pAdapter construct presented here substantially expands the range of detectable interactions in the B3H assay, broadening its utility. This improved assay will increase the likelihood of identifying novel protein-RNA interactions with the B3H system, and will facilitate exploration of the binding mechanisms of these interactions.

scholarly journals Effects of Vitrification on the Blastocyst Gene Expression Profile in a Porcine Model

2021 ◽  
Vol 22 (3) ◽  
pp. 1222
Author(s):  
Cristina Cuello ◽  
Cristina A. Martinez ◽  
Josep M. Cambra ◽  
Inmaculada Parrilla ◽  
Heriberto Rodriguez-Martinez ◽  
...  
Keyword(s):  
Gene Expression ◽  
Gene Expression Analysis ◽  
Survival Rates ◽  
Control Group ◽  
P Value ◽  
Transcriptome Profile ◽  
Embryo Viability ◽  
The Impact

This study was designed to investigate the impact of vitrification on the transcriptome profile of blastocysts using a porcine (Sus scrofa) model and a microarray approach. Blastocysts were collected from weaned sows (n = 13). A total of 60 blastocysts were vitrified (treatment group). After warming, vitrified embryos were cultured in vitro for 24 h. Non-vitrified blastocysts (n = 40) were used as controls. After the in vitro culture period, the embryo viability was morphologically assessed. A total of 30 viable embryos per group (three pools of 10 from 4 different donors each) were subjected to gene expression analysis. A fold change cut-off of ±1.5 and a restrictive threshold at p-value < 0.05 were used to distinguish differentially expressed genes (DEGs). The survival rates of vitrified/warmed blastocysts were similar to those of the control (nearly 100%, n.s.). A total of 205 (112 upregulated and 93 downregulated) were identified in the vitrified blastocysts compared to the control group. The vitrification/warming impact was moderate, and it was mainly related to the pathways of cell cycle, cellular senescence, gap junction, and signaling for TFGβ, p53, Fox, and MAPK. In conclusion, vitrification modified the transcriptome of in vivo-derived porcine blastocysts, resulting in minor gene expression changes.

scholarly journals Impact of the acidic environment on gene expression and functional parameters of tumors in vitro and in vivo

2021 ◽  
Vol 40 (1) ◽  
Author(s):  
Mandy Rauschner ◽  
Luisa Lange ◽  
Thea Hüsing ◽  
Sarah Reime ◽  
Alexander Nolze ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Death ◽  
Western Blot ◽  
Tumor Cell ◽  
Low Ph ◽  
Strong Increase ◽  
Acidic Ph ◽  
The Impact

Abstract Background The low extracellular pH (pHe) of tumors resulting from glycolytic metabolism is a stress factor for the cells independent from concomitant hypoxia. The aim of the study was to analyze the impact of acidic pHe on gene expression on mRNA and protein level in two experimental tumor lines in vitro and in vivo and were compared to hypoxic conditions as well as combined acidosis+hypoxia. Methods Gene expression was analyzed in AT1 prostate and Walker-256 mammary carcinoma of the rat by Next Generation Sequencing (NGS), qPCR and Western blot. In addition, the impact of acidosis on tumor cell migration, adhesion, proliferation, cell death and mitochondrial activity was analyzed. Results NGS analyses revealed that 147 genes were uniformly regulated in both cell lines (in vitro) and 79 genes in both experimental tumors after 24 h at low pH. A subset of 25 genes was re-evaluated by qPCR and Western blot. Low pH consistently upregulated Aox1, Gls2, Gstp1, Ikbke, Per3, Pink1, Tlr5, Txnip, Ypel3 or downregulated Acat2, Brip1, Clspn, Dnajc25, Ercc6l, Mmd, Rif1, Zmpste24 whereas hypoxia alone led to a downregulation of most of the genes. Direct incubation at low pH reduced tumor cell adhesion whereas acidic pre-incubation increased the adhesive potential. In both tumor lines acidosis induced a G1-arrest (in vivo) of the cell cycle and a strong increase in necrotic cell death (but not in apoptosis). The mitochondrial O2 consumption increased gradually with decreasing pH. Conclusions These data show that acidic pHe in tumors plays an important role for gene expression independently from hypoxia. In parallel, acidosis modulates functional properties of tumors relevant for their malignant potential and which might be the result of pH-dependent gene expression.

scholarly journals Enhancers Predominantly Regulate Gene Expression in vivo Via Transcription Initiation

2019 ◽  
Author(s):  
Martin Stephen Charles Larke ◽  
Takayuki Nojima ◽  
Jelena Telenius ◽  
Jacqueline A. Sharpe ◽  
Jacqueline A. Sloane-Stanley ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcription Initiation ◽  
Regulate Gene Expression ◽  
Regulate Gene

scholarly journals KH domain containing RNA-binding proteins coordinate with microRNAs to regulate Caenorhabditis elegans development

2021 ◽  
Vol 11 (2) ◽  
Author(s):  
Dustin Haskell ◽  
Anna Zinovyeva
Keyword(s):  
Gene Expression ◽  
Caenorhabditis Elegans ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Regulate Gene Expression ◽  
C Elegans ◽  
Binding Domains ◽  
Kh Domain ◽  
Regulate Gene

Abstract MicroRNAs (miRNAs) and RNA-binding proteins (RBPs) regulate gene expression at the post-transcriptional level, but the extent to which these key regulators of gene expression coordinate their activities and the precise mechanisms of this coordination are not well understood. RBPs often have recognizable RNA binding domains that correlate with specific protein function. Recently, several RBPs containing K homology (KH) RNA binding domains were shown to work with miRNAs to regulate gene expression, raising the possibility that KH domains may be important for coordinating with miRNA pathways in gene expression regulation. To ascertain whether additional KH domain proteins functionally interact with miRNAs during Caenorhabditis elegans development, we knocked down twenty-four genes encoding KH-domain proteins in several miRNA sensitized genetic backgrounds. Here, we report that a majority of the KH domain-containing genes genetically interact with multiple miRNAs and Argonaute alg-1. Interestingly, two KH domain genes, predicted splicing factors sfa-1 and asd-2, genetically interacted with all of the miRNA mutants tested, whereas other KH domain genes showed genetic interactions only with specific miRNAs. Our domain architecture and phylogenetic relationship analyses of the C. elegans KH domain-containing proteins revealed potential groups that may share both structure and function. Collectively, we show that many C. elegans KH domain RBPs functionally interact with miRNAs, suggesting direct or indirect coordination between these two classes of post-transcriptional gene expression regulators.

The Vent-Like Homeobox Gene VENTX Promotes Human Myeloid Development and Is Highly Expressed In Acute Myeloid Leukemia

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 739-739
Author(s):  
Vijay P. S. Rawat ◽  
Natalia Arseni ◽  
Farid Ahmed ◽  
Medhanie A. Mulaw ◽  
Silvia Thoene ◽  
...  
Keyword(s):  
Gene Expression ◽  
Progenitor Cells ◽  
Cell Lines ◽  
Myeloid Cells ◽  
Lymphoid Cells ◽  
Hematopoietic Development ◽  
Cd34 Cells ◽  
The Impact

Abstract Abstract 739 Recent studies suggest that a variety of regulatory molecules active in embryonic development such as clustered and non-clustered homeobox genes play an important role in normal and malignant hematopoiesis. Since it was shown that the Xvent-2 homeobox gene is part of the BMP-4 signalling pathway in Xenopus, it is of particular interest to examine the expression profile and function of its only recently discovered human homologue VENTX in hematopoietic development. Expression of the VENTX gene was analyzed in normal human hematopoiesis and AML patients samples by microarray and qPCR. To test the impact of the constitutive expression of VENTX on human progenitor cells, CD34+ cord blood (CB) cells were retrovirally transduced with VENTX or the empty control vector and analyzed using in vitro and in vivo assays. So far we and others have not been able to identify a murine Xenopus xvent gene homologue. However, we were able to document the expression of this gene by qPCR in human lineage positive hematopoietic subpopulations. Amongst committed progenitors VENTX was significantly 13-fold higher expressed in CD33+ BM myeloid cells (4/4 positive) compared to CD19+ BM lymphoid cells (5/7 positive, p=0.01). Of note, expression of VENTX was negligible in normal CD34+/CD38− but detectable in CD34+ BM human progenitor cells. In contrast to this, leukemic CD34+/CD38− from AML patients (n=3) with translocation t(8,21) showed significantly elevated expression levels compared to normal CD34+ BM cells (n=5) (50-fold higher; p≤0.0001). Furthermore, patients with normal karyotype NPM1c+/FLT3-LM− (n=9), NPM1c−/FLT3-LM+ (n=8) or patients with t(8;21) (n=9) had an >100-fold higher expression of VENTX compared to normal CD34+ BM cells and a 5- to 7.8-fold higher expression compared to BM MNCs. Importantly, lentivirus-mediated long-term silencing of VENTX in human AML cell lines (mRNA knockdown between 58% and 75%) led to a significant, reduction in cell number compared to the non-silencing control construct (>79% after 120h). Suggesting that growth of human leukemic cell lines depends on VENTX expression in vitro. As we observed that VENTX is aberrantly expressed in leukemic CD34+ cells with negligible expression in normal counterparts, we assessed the impact of forced VENTX gene expression in normal CD34+ human progenitor cells on the transcription program. Gene expression and pathway analysis demonstrated that in normal CD34+ cells enforced expression of VENTX initiates genes associated with myeloid development (CD11b, CD125, CD9,CD14 and M-CSF), and downregulates genes involved in early lymphoid development (IL-7, IL-9R, LEF1/TCF and C-JUN) and erythroid development such as EPOR, CD35 and CD36. We then tested whether enforced expression of VENTX in CD34+ cells is able to alter the hematopoietic development of early human progenitors as indicated by gene expression and pathway analyses. Functional analyses confirmed that aberrant expression of VENTX in normal CD34+ human progenitor cells induced a significant increase in the number of myeloid colonies compared to the GFP control with 48 ± 6.5 compared to 28.9 ± 4.8 CFU-G per 1000 initially plated CD34+ cells (n=11; p=0.03) and complete block in erythroid colony formation with an 81% reduction of the number of BFU-E compared to the control (n=11; p<0.003). In a feeder dependent co-culture system, VENTX impaired the development of B-lymphoid cells. In the NOD/SCID xenograft model, VENTX expression in CD34+ CB cells promoted generation of myeloid cells with an over 5-fold and 2.5-fold increase in the proportion of human CD15+ and CD33+ primitive myeloid cells compared to the GFP control (n=5, p=0.01). Summary: Overexpression of VENTX perturbs normal hematopoietic development, promotes generation of myeloid cells and impairs generation of lymphoid cells in vitro and in vivo. Whereas VENTX depletion in human AML cell lines impaired their growth.Taken together, these data extend our insights into the function of human embryonic mesodermal factors in human hematopoiesis and indicate a role of VENTX in normal and malignant myelopoiesis. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Identification of a Mutation in the Bacillus subtilis S-Adenosylmethionine Synthetase Gene That Results in Derepression of S-Box Gene Expression

2006 ◽  
Vol 188 (10) ◽  
pp. 3674-3681 ◽  
Author(s):  
Brooke A. McDaniel ◽  
Frank J. Grundy ◽  
Vineeta P. Kurlekar ◽  
Jerneja Tomsic ◽  
Tina M. Henkin
Keyword(s):  
Gene Expression ◽  
Bacillus Subtilis ◽  
Rna Structure ◽  
Synthetase Activity ◽  
Gene Encoding ◽  
Sam Synthetase ◽  
Transcription In Vitro ◽  
Adenosylmethionine Synthetase

ABSTRACT Genes in the S-box family are regulated by binding of S-adenosylmethionine (SAM) to the 5′ region of the mRNA of the regulated gene. SAM binding was previously shown to promote a rearrangement of the RNA structure that results in premature termination of transcription in vitro and repression of expression of the downstream coding sequence. The S-box RNA element therefore acts as a SAM-binding riboswitch in vitro. In an effort to identify factors other than SAM that could be involved in the S-box regulatory mechanism in vivo, we searched for trans-acting mutations in Bacillus subtilis that act to disrupt repression of S-box gene expression during growth under conditions where SAM pools are elevated. We identified a single mutant that proved to have one nucleotide substitution in the metK gene, encoding SAM synthetase. This mutation, designated metK10, resulted in a 15-fold decrease in SAM synthetase activity and a 4-fold decrease in SAM concentration in vivo. The metK10 mutation specifically affected S-box gene expression, and the increase in expression under repressing conditions was dependent on the presence of a functional transcriptional antiterminator element. The observation that the mutation identified in this search affects SAM production supports the model that the S-box RNAs directly monitor SAM in vivo, without a requirement for additional factors.

scholarly journals A multiplexed, automated evolution pipeline enables scalable discovery and characterization of biosensors

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Brent Townshend ◽  
Joy S. Xiang ◽  
Gabriel Manzanarez ◽  
Eric J. Hayden ◽  
Christina D. Smolke
Keyword(s):  
Small Molecules ◽  
Biosynthetic Pathway ◽  
De Novo ◽  
Living Cells ◽  
Regulate Gene Expression ◽  
Liquid Handling ◽  
Regulate Gene

AbstractBiosensors are key components in engineered biological systems, providing a means of measuring and acting upon the large biochemical space in living cells. However, generating small molecule sensing elements and integrating them into in vivo biosensors have been challenging. Here, using aptamer-coupled ribozyme libraries and a ribozyme regeneration method, de novo rapid in vitro evolution of RNA biosensors (DRIVER) enables multiplexed discovery of biosensors. With DRIVER and high-throughput characterization (CleaveSeq) fully automated on liquid-handling systems, we identify and validate biosensors against six small molecules, including five for which no aptamers were previously found. DRIVER-evolved biosensors are applied directly to regulate gene expression in yeast, displaying activation ratios up to 33-fold. DRIVER biosensors are also applied in detecting metabolite production from a multi-enzyme biosynthetic pathway. This work demonstrates DRIVER as a scalable pipeline for engineering de novo biosensors with wide-ranging applications in biomanufacturing, diagnostics, therapeutics, and synthetic biology.

Sign in / Sign up

Export Citation Format

Share Document