scholarly journals Translation inhibition and resource balance in the TX-TL cell-free gene expression system

2017 ◽  
Vol 2 (1) ◽  
Author(s):  
Vijayalakshmi H Nagaraj ◽  
James M Greene ◽  
Anirvan M Sengupta ◽  
Eduardo D Sontag
Keyword(s):  
Gene Expression ◽  
Messenger Rna ◽  
Resource Dependence ◽  
Alternative Hypothesis ◽  
Expression System ◽  
Resource Limitation ◽  
Independent Effect ◽  
Translation Rate ◽  
Suppression Of Gene Expression ◽  
The Impact

Abstract Quantifying the effect of vital resources on transcription (TX) and translation (TL) helps to understand the degree to which the concentration of each resource must be regulated for achieving homeostasis. Utilizing the synthetic TX-TL system, we study the impact of nucleotide triphosphates (NTPs) and magnesium (Mg2+) on gene expression. Recent observations of the counter-intuitive phenomenon of suppression of gene expression at high NTP concentrations have led to the speculation that such suppression is due to the consumption of resources by TX, hence leaving fewer resources for TL. In this work, we investigate an alternative hypothesis: direct suppression of the TL rate via stoichiometric mismatch in necessary reagents. We observe NTP-dependent suppression even in the early phase of gene expression, contradicting the resource-limitation argument. To further decouple the contributions of TX and TL, we performed gene expression experiments with purified messenger RNA (mRNA). Simultaneously monitoring mRNA and protein abundances allowed us to extract a time-dependent translation rate. Measuring TL rates for different Mg2+ and NTP concentrations, we observe a complex resource dependence. We demonstrate that TL is the rate-limiting process that is directly inhibited by high NTP concentrations. Additional Mg2+ can partially reverse this inhibition. In several experiments, we observe two maxima of the TL rate viewed as a function of both Mg2+ and NTP concentration, which can be explained in terms of an NTP-independent effect on the ribosome complex and an NTP-Mg2+ titration effect. The non-trivial compensatory effects of abundance of different vital resources signal the presence of complex regulatory mechanisms to achieve optimal gene expression.

scholarly journals Translation Inhibition and Resource Balance in the Cell-Free Gene Expression System

2017 ◽  
Author(s):  
Vijayalakshmi H. Nagaraj ◽  
James M. Greene ◽  
Anirvan M. Sengupta ◽  
Eduardo D. Sontag
Keyword(s):  
Gene Expression ◽  
Resource Dependence ◽  
Alternative Hypothesis ◽  
Expression System ◽  
Resource Limitation ◽  
Independent Effect ◽  
Translation Rate ◽  
Free Gene ◽  
Suppression Of Gene Expression ◽  
The Impact

AbstractQuantifying the effect of vital resources on transcription and translation helps to understand the degree to which the concentration of each resource must be regulated for achieving homeostasis. Utilizing the synthetic transcription-translation (TX-TL) system, we study the impact of nucleotide triphosphates (NTPs) and magnesium (Mg2+), on gene expression. Recent observations of the counterintuitive phenomenon of suppression of gene expression at high NTP concentrations have led to the speculation that such suppression is due to the consumption of resources by transcription, hence leaving fewer resources for translation. In this work, we investigate an alternative hypothesis: direct suppression of the translation rate via stoichiometric mismatch in necessary reagents. We observe NTP-dependent suppression even in the early phase of gene expression, contradicting the resource limitation argument. To further decouple the contributions of transcription and translation, we performed gene expression experiments with purified mRNA. Simultaneously monitoring mRNA and protein abundances allowed us to extract a time-dependent translation rate. Measuring translation rates for different Mg2+ and NTP concentrations, we observe a complex resource dependence. We demonstrate that translation is the rate-limiting process that is directly inhibited by high NTP concentrations. Additional Mg2+ can partially reverse this inhibition. In several experiments, we observe two maxima of the translation rate viewed as a function of both Mg2+ and NTP concentration, which can be explained in terms of an NTP-independent effect on the ribosome complex and an NTP-Mg2+ titration effect. The non-trivial compensatory effects of abundance of different vital resources signals the presence of complex regulatory mechanisms to achieve optimal gene expression.

scholarly journals Marfan Syndrome Variability: Investigation of the Roles of Sarcolipin and Calcium as Potential Transregulator of FBN1 Expression

Genes ◽  
2018 ◽  
Vol 9 (9) ◽  
pp. 421 ◽  
Author(s):  
Louise Benarroch ◽  
Mélodie Aubart ◽  
Marie-Sylvie Gross ◽  
Marie-Paule Jacob ◽  
Pauline Arnaud ◽  
...  
Keyword(s):  
Gene Expression ◽  
Marfan Syndrome ◽  
Messenger Rna ◽  
Calcium Influx ◽  
Surrogate Endpoint ◽  
Connective Tissue Disorder ◽  
Eqtl Analysis ◽  
Chromosome 11 ◽  
Fbn1 Gene ◽  
The Impact

Marfan syndrome (MFS) is an autosomal dominant connective tissue disorder that displays a great clinical variability. Previous work in our laboratory showed that fibrillin-1 (FBN1) messenger RNA (mRNA) expression is a surrogate endpoint for MFS severity. Therefore, an expression quantitative trait loci (eQTL) analysis was performed to identify trans-acting regulators of FBN1 expression, and a significant signal reached genome-wide significant threshold on chromosome 11. This signal delineated a region comprising one expressed gene, SLN (encoding sarcolipin), and a single pseudogene, SNX7-ps1 (CTD-2651C21.3). We first investigated the region and then looked for association between the genes in the region and FBN1 expression. For the first time, we showed that the SLN gene is weakly expressed in skin fibroblasts. There is no direct correlation between SLN and FBN1 gene expression. We showed that calcium influx modulates FBN1 gene expression. Finally, SLN gene expression is highly correlated to that of the neighboring SNX7-ps1. We were able to confirm the impact of calcium influx on FBN1 gene expression but we could not conclude regarding the role of sarcolipin and/or the eQTL locus in this regulation.

scholarly journals MicroRNA-377 Alleviates Myocardial Injury Induced by Hypoxia/Reoxygenation via Downregulating LILRB2 Expression

Dose-Response ◽  
2020 ◽  
Vol 18 (2) ◽  
pp. 155932582093612 ◽  
Author(s):  
Mengwei Xie ◽  
Chunlan Hu ◽  
Delin Li ◽  
Shifeng Li
Keyword(s):  
Gene Expression ◽  
Myocardial Infarction ◽  
Messenger Rna ◽  
Myocardial Injury ◽  
Cell Activity ◽  
Gene Expression Omnibus ◽  
Luciferase Reporter ◽  
Data Set ◽  
The Impact ◽  
Hypoxia Reoxygenation

Background: miR-377 is closely related to myocardial regeneration. miR-377-adjusted mesenchymal stem cells abducted ischemic cardiac angiogenesis. Nevertheless, there were rarely reports about the impact of miR-377 on myocardial ischemia injury. The purpose of this work is that whether miR-377 can protect against myocardial injury caused by hypoxia/reoxygenation (H/R). Methods: Gene expression omnibus database ( http://www.ncbi.nlm.nih.gov/geo/ ; no. GSE53211) was utilized to study the differential expression of miR-377 in patients with an acute ST-segment elevation myocardial infarction and healthy controls. The luciferase activity was determined utilizing the dual-luciferase reporter system. Quantitative real-time polymerase chain reaction and Western blotting were used to measure the messenger RNA and protein level. Results: Low expression of miR-377 and high expression of leukocyte immunoglobulin-like receptor B2 (LILRB2) were identified in patients with myocardial infarction from analyzing the Gene Expression Omnibus data set. Besides, miR-377 expression was downregulated in cardiomyocyte exposed to H/R. Additionally, overexpression of miR-377 could visibly improve cardiomyocyte injury by regulating cell activity and apoptosis. Conclusions: In short, our findings suggested that miR-377/LILRB2 might regard as a hopeful therapeutic target for myocardial ischemic.

The impact of a 48-h fast on SIRT1 and GCN5 in human skeletal muscle

2016 ◽  
Vol 41 (9) ◽  
pp. 953-962 ◽  
Author(s):  
Brittany A. Edgett ◽  
Trisha D. Scribbans ◽  
James P. Raleigh ◽  
Jennifer B.L. Matusiak ◽  
Kristen Boonstra ◽  
...  
Keyword(s):  
Gene Expression ◽  
Skeletal Muscle ◽  
Messenger Rna ◽  
Animal Studies ◽  
Human Skeletal Muscle ◽  
Muscle Protein ◽  
Peak Oxygen Uptake ◽  
The Impact ◽  
Whole Muscle ◽  
Activation Status

The present study examined the impact of a 48 h fast on the expression and activation status of SIRT1 and GCN5, the relationship between SIRT1/GCN5 and the gene expression of PGC-1α, and the PGC-1α target PDK4 in the skeletal muscle of 10 lean healthy men (age, 22.0 ± 1.5 years; peak oxygen uptake, 47.2 ± 6.7 mL/(min·kg)). Muscle biopsies and blood samples were collected 1 h postprandial (Fed) and following 48 h of fasting (Fasted). Plasma insulin (Fed, 80.8 ± 47.9 pmol/L; Fasted, not detected) and glucose (Fed, 4.36 ± 0.86; Fasted, 3.74 ± 0.25 mmol/L, p = 0.08) decreased, confirming participant adherence to fasting. Gene expression of PGC-1α decreased (p < 0.05, –24%), while SIRT1 and PDK4 increased (p < 0.05, +11% and +1023%, respectively), and GCN5 remained unchanged. No changes were observed for whole-muscle protein expression of SIRT1, GCN5, PGC-1α, or COX IV. Phosphorylation of SIRT1, AMPKα, ACC, p38 MAPK, and PKA substrates as well as nuclear acetylation status was also unaltered. Additionally, nuclear SIRT1 activity, GCN5, and PGC-1α content remained unchanged. Preliminary findings derived from regression analysis demonstrate that changes in nuclear GCN5 and SIRT1 activity/phosphorylation may contribute to the control of PGC-1α, but not PDK4, messenger RNA expression following fasting. Collectively, and in contrast with previous animal studies, our data are inconsistent with the altered activation status of SIRT1 and GCN5 in response to 48 h of fasting in human skeletal muscle.

scholarly journals The transcriptomic blueprint of molt in rooster using various tissues from Ginkkoridak (Korean long-tailed chicken)

BMC Genomics ◽  
2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Clémentine Charton ◽  
Dong-Jae Youm ◽  
Byung June Ko ◽  
Donghyeok Seol ◽  
Bongsang Kim ◽  
...  
Keyword(s):  
Gene Expression ◽  
Nervous System ◽  
Messenger Rna ◽  
Ribosomal Proteins ◽  
Reproductive Tract ◽  
Gene Transcript ◽  
Cell Dynamics ◽  
Tissue Samples ◽  
Physiological Functions ◽  
The Impact

Abstract Background Annual molt is a critical stage in the life cycle of birds. Although the most extensively documented aspects of molt are the renewing of plumage and the remodeling of the reproductive tract in laying hens, in chicken, molt deeply affects various tissues and physiological functions. However, with exception of the reproductive tract, the effect of molt on gene expression across the tissues known to be affected by molt has to date never been investigated. The present study aimed to decipher the transcriptomic effects of molt in Ginkkoridak, a Korean long-tailed chicken. Messenger RNA data available across 24 types of tissue samples (9 males) and a combination of mRNA and miRNA data on 10 males and 10 females blood were used. Results The impact of molt on gene expression and gene transcript usage appeared to vary substantially across tissues types in terms of histological entities or physiological functions particularly related to nervous system. Blood was the tissue most affected by molt in terms of differentially expressed genes in both sexes, closely followed by meninges, bone marrow and heart. The effect of molt in blood appeared to differ between males and females, with a more than fivefold difference in the number of down-regulated genes between both sexes. The blueprint of molt in roosters appeared to be specific to tissues or group of tissues, with relatively few genes replicating extensively across tissues, excepted for the spliceosome genes (U1, U4) and the ribosomal proteins (RPL21, RPL23). By integrating miRNA and mRNA data, when chickens molt, potential roles of miRNA were discovered such as regulation of neurogenesis, regulation of immunity and development of various organs. Furthermore, reliable candidate biomarkers of molt were found, which are related to cell dynamics, nervous system or immunity, processes or functions that have been shown to be extensively modulated in response to molt. Conclusions Our results provide a comprehensive description at the scale of the whole organism deciphering the effects of molt on the transcriptome in chicken. Also, the conclusion of this study can be used as a valuable resource in transcriptome analyses of chicken in the future and provide new insights related to molt.

Application of RNAi technology in forest trees.

2021 ◽  
pp. 54-71
Author(s):  
Matthias Fladung ◽  
Hely Häggman ◽  
Suvi Sutela
Keyword(s):  
Gene Expression ◽  
Tree Species ◽  
Messenger Rna ◽  
Forest Tree ◽  
Rna Molecules ◽  
Forest Tree Species ◽  
Advantages And Disadvantages ◽  
Rnai Technology ◽  
Suppression Of Gene Expression ◽  
The Stability

Abstract A diverse set of small RNAs is involved in the regulation of genome organization and gene expression in plants. These regulatory sRNAs play a central role for RNA in evolution and ontogeny in complex organisms, including forest tree species, providers of indispensable ecosystem services. RNA interference is a process that inhibits gene expression by double-stranded RNA and thus causes the degradation of target messenger RNA molecules. Targeted gene silencing by RNAi has been utilized in various crop plants in order to enhance their characteristics. For forest tree species, most of the successful RNAi modification has been conducted in poplar. Over the past 20 years, successful RNAi-mediated suppression of gene expression has been achieved with a variety of economically important traits. Moreover, the stability of RNAi-mediated transgene suppression has been confirmed in field-grown poplars. In this chapter, we describe examples of successful RNAi applications mainly in poplar but also provide some information about application of RNAi in pest control in forest tree species. Advantages and disadvantages of this technology with respect to the particular features of forest tree species will be discussed.

Application of RNAi technology in forest trees.

2021 ◽  
pp. 54-71
Author(s):  
Matthias Fladung ◽  
Hely Häggman ◽  
Suvi Sutela
Keyword(s):  
Gene Expression ◽  
Tree Species ◽  
Messenger Rna ◽  
Forest Tree ◽  
Rna Molecules ◽  
Forest Tree Species ◽  
Advantages And Disadvantages ◽  
Rnai Technology ◽  
Suppression Of Gene Expression ◽  
The Stability

Abstract A diverse set of small RNAs is involved in the regulation of genome organization and gene expression in plants. These regulatory sRNAs play a central role for RNA in evolution and ontogeny in complex organisms, including forest tree species, providers of indispensable ecosystem services. RNA interference is a process that inhibits gene expression by double-stranded RNA and thus causes the degradation of target messenger RNA molecules. Targeted gene silencing by RNAi has been utilized in various crop plants in order to enhance their characteristics. For forest tree species, most of the successful RNAi modification has been conducted in poplar. Over the past 20 years, successful RNAi-mediated suppression of gene expression has been achieved with a variety of economically important traits. Moreover, the stability of RNAi-mediated transgene suppression has been confirmed in field-grown poplars. In this chapter, we describe examples of successful RNAi applications mainly in poplar but also provide some information about application of RNAi in pest control in forest tree species. Advantages and disadvantages of this technology with respect to the particular features of forest tree species will be discussed.

scholarly journals Studying the Function of Phytoplasma Effector Proteins Using a Chemical-Inducible Expression System in Transgenic Plants

2021 ◽  
Vol 22 (24) ◽  
pp. 13582
Author(s):  
Keziah M. Omenge ◽  
Florian Rümpler ◽  
Subha Suvetha Kathalingam ◽  
Alexandra C. U. Furch ◽  
Günter Theißen
Keyword(s):  
Gene Expression ◽  
Laser Scanning ◽  
Expression System ◽  
Inducible Expression ◽  
Effector Proteins ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Protein Accumulation ◽  
Disease Phenotypes ◽  
The Impact

Phytoplasmas are bacterial pathogens that live mainly in the phloem of their plant hosts. They dramatically manipulate plant development by secreting effector proteins that target developmental proteins of their hosts. Traditionally, the effects of individual effector proteins have been studied by ectopic overexpression using strong, ubiquitously active promoters in transgenic model plants. However, the impact of phytoplasma infection on the host plants depends on the intensity and timing of infection with respect to the developmental stage of the host. To facilitate investigations addressing the timing of effector protein activity, we have established chemical-inducible expression systems for the three most well-characterized phytoplasma effector proteins, SECRETED ASTER YELLOWS WITCHES’ BROOM PROTEIN 11 (SAP11), SAP54 and TENGU in transgenic Arabidopsis thaliana. We induced gene expression either continuously, or at germination stage, seedling stage, or flowering stage. mRNA expression was determined by quantitative reverse transcription PCR, protein accumulation by confocal laser scanning microscopy of GFP fusion proteins. Our data reveal tight regulation of effector gene expression and strong upregulation after induction. Phenotypic analyses showed differences in disease phenotypes depending on the timing of induction. Comparative phenotype analysis revealed so far unreported similarities in disease phenotypes, with all three effector proteins interfering with flower development and shoot branching, indicating a surprising functional redundancy of SAP54, SAP11 and TENGU. However, subtle but mechanistically important differences were also observed, especially affecting the branching pattern of the plants.

scholarly journals Heavy Water Reduces GFP Expression in Prokaryotic Cell-Free Assays at the Translation Level While Stimulating Its Transcription

2013 ◽  
Vol 2013 ◽  
pp. 1-9 ◽  
Author(s):  
Luisa S. Hohlefelder ◽  
Tobias Stögbauer ◽  
Madeleine Opitz ◽  
Thomas M. Bayerl ◽  
Joachim O. Rädler
Keyword(s):  
Gene Expression ◽  
Heavy Water ◽  
Expression System ◽  
Inhibitory Effect ◽  
Dependent Manner ◽  
Gfp Expression ◽  
Antiproliferative Agent ◽  
Underlying Mechanisms ◽  
The Impact

Thein vitroproliferation of prokaryotic and eukaryotic cells is remarkably hampered in the presence of heavy water (D2O). Impairment of gene expression at the transcription or translation level can be the base for this effect. However, insights into the underlying mechanisms are lacking. Here, we employ a cell-free expression system for the quantitative analysis of the effect of increasing percentages of D2O on the kinetics ofin-vitroGFP expression. Experiments are designed to discriminate the rates of transcription, translation, and protein folding using pDNA and mRNA vectors, respectively. We find that D2O significantly stimulates GFP expression at the transcription level but acts as a suppressor at translation and maturation (folding) in a linear dose-dependent manner. At a D2O concentration of 60%, the GFP expression rate was reduced to 40% of an undisturbed sample. We observed a similar inhibition of GFP expression by D2O in a recombinantEscherichia colistrain, although the inhibitory effect is less pronounced. These results demonstrate the suitability of cell-free systems for quantifying the impact of heavy water on gene expression and establish a platform to further assess the potential therapeutic use of heavy water as antiproliferative agent.

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