Search results for "Reporter"
showing 10 items of 166 documents
The 3'-UTR of the mRNA coding for the major protein kinase C substrate MARCKS contains a novel CU-rich element interacting with the mRNA stabilizing …
2003
The expression of the major protein kinase C substrate MARCKS (myristoylated alanine-rich C kinase substrate) is controlled by the stability of its mRNA. While the MARCKS mRNA is long living in quiescent fibroblasts (t1/2 = 14 h), its half-life time is drastically reduced (t1/2 = 2 h) in cells treated with phorbol esters to activate protein kinase C (PKC) or treated with growth factors. In a first step to study the underlying mechanism we identified both a cis-element on the MARCKS mRNA and the corresponding trans-acting factors. Fusing the complete 3'-UTR or specific regions of the 3'-UTR of the MARCKS gene to a luciferase reporter gene caused a drastic decrease in luciferase expression to…
3'-Untranslated regions of oxidative phosphorylation mRNAs function in vivo, as enhancers of translation
2000
Recent findings have indicated that the 3´-untranslated region (3´-UTR) of the mRNA encoding the β-catalytic subunit of the mitochondrial H+-ATP synthase has an in vitro translation-enhancing activity (TEA) [Izquierdo and Cuezva, Mol. Cell. Biol. (1997) 17, 5255–5268; Izquierdo and Cuezva, Biochem. J. (2000) 346, 849–855]. In the present work, we have expressed chimaeric plasmids that encode mRNA variants of green fluorescent protein in normal rat kidney and liver clone 9 cells to determine whether the 3´-UTRs of nuclear-encoded mRNAs involved in the biogenesis of mitochondria have an intrinsic TEA. TEA is found in the 3´-UTR of the mRNAs encoding the α- and β-subunits of the rat H+-ATP syn…
Cutting Edge: An IL-17F-CreEYFP Reporter Mouse Allows Fate Mapping of Th17 Cells
2009
Abstract The need for reporter lines able to faithfully track Th17 cells in vivo has become an issue of exceptional importance. To address this, we generated a mouse strain in which Cre recombinase is expressed from the IL-17F promoter. Crossing the IL-17F-Cre allele to a conditional enhanced yellow fluorescent protein (EYFP) reporter mouse yielded the IL-17F-CreEYFP strain, in which IL-17F expression is twinned with EYFP in live IL-17F-expressing cells. Although we demonstrate that IL-17F expression is restricted to CD4+ T cells during experimental autoimmune encephalomyelitis, IL-17F-CreEYFP CD8 T cells robustly expressed IL-17F in response to TGF-β, IL-6, and IL-23. Fate mapping of IL-17…
Inactivation of PadR, the repressor of the phenolic acid stress response, by molecular interaction with Usp1, a universal stress protein from Lactoba…
2009
ABSTRACT The phenolic acid decarboxylase gene padA is involved in the phenolic acid stress response (PASR) in gram-positive bacteria. In Lactobacillus plantarum , the padR gene encodes the negative transcriptional regulator of padA and is cotranscribed with a downstream gene, usp1 , which encodes a putative universal stress protein (USP), Usp1, of unknown function. The usp1 gene is overexpressed during the PASR. However, the role and the mechanism of action of the USPs are unknown in gram-positive bacteria. Therefore, to gain insights into the role of USPs in the PASR; (i) a usp1 deletion mutant was constructed; (ii) the two genes padR and usp1 were coexpressed with padA under its own promo…
Distinct 5' SCL enhancers direct transcription to developing brain, spinal cord, and endothelium: neural expression is mediated by GATA factor bindin…
1999
The SCL gene encodes a basic helix-loop-helix transcription factor with a pivotal role in the development of endothelium and of all hematopoietic lineages. SCL is also expressed in the central nervous system, although its expression pattern has not been examined in detail and its function in neural development is unknown. In this article we present the first analysis of SCL transcriptional regulation in vivo. We have identified three spatially distinct regulatory modules, each of which was both necessary and sufficient to direct reporter gene expression in vivo to three different regions within the normal SCL expression domain, namely, developing endothelium, midbrain, and hindbrain/spinal …
Context-dependent Pax-5 repression of a PU.1/NF-κB regulated reporter gene in B lineage cells
2001
Enhancers located in the 3' end of the locus in part regulate immunoglobulin heavy chain (IgH) gene expression. One of these enhancers, HS 1,2, is developmentally regulated by DNA binding proteins like NF-kappaB, Pax-5 and the protein complex NF-alphaP in B lineage cells. Here we report that NF-alphaP is the ets protein PU.1. A glutathione-S-transferase (GST)-pulldown assay demonstrated that PU.1 can physically interact with NF-kappaB in solution. Experiments in COS cells showed that PU.1 and NF-kappaB (p50/c-Rel) can activate transcription of an enhancer linked reporter gene. The paired domain protein Pax-5 has previously been shown to repress enhancer-dependent transcription. Additional c…
Functional characterization of the enhancer blocking element of the sea urchin early histone gene cluster reveals insulator properties and three esse…
2000
Insulator elements can be functionally identified by their ability to shield promoters from regulators in a position-dependent manner or their ability to protect adjacent transgenes from position effects. We have previously reported the identification of a 265 bp sns DNA fragment at the 3' end of the sea urchin H2A early histone gene that blocked expression of a reporter gene in transgenic embryos when placed between the enhancer and the promoter. Here we show that sns interferes with enhancer-promoter interaction in a directional manner. When sns is placed between the H2A modulator and the inducible tet operator, the modulator is barred from interaction with the basal promoter. However, th…
Molecular Basis for Endocrine Disruption by Pesticides Targeting Aromatase and Estrogen Receptor
2020
The intensive use of pesticides has led to their increasing presence in water, soil, and agricultural products. Mounting evidence indicates that some pesticides may be endocrine disrupting chemicals (EDCs), being therefore harmful for the human health and the environment. In this study, three pesticides, glyphosate, thiacloprid, and imidacloprid, were tested for their ability to interfere with estrogen biosynthesis and/or signaling, to evaluate their potential action as EDCs. Among the tested compounds, only glyphosate inhibited aromatase activity (up to 30%) via a non-competitive inhibition or a mixed inhibition mechanism depending on the concentration applied. Then, the ability of the thr…
Genetic Systems for Monitoring Interactions of Transmembrane Domains in Bacterial Membranes
2013
In recent years several systems have been developed to study interactions of TM domains within the inner membrane of the Gram-negative bacterium Escherichia coli. Mostly, a transmembrane domain of interest is fused to a soluble DNA-binding domain, which dimerizes in E. coli cytoplasm after interactions of the transmembrane domains. The dimeric DNA-binding domain subsequently binds to a promoter/operator region and thereby activates or represses a reporter gene. In 1996 the first bacterial system has been introduced to measure interactions of TM helices within a bacterial membrane, which is based on fusion of a transmembrane helix of interest to the DNA-binding domain of the Vibrio cholerae …
Study of the aryl hydrocarbon receptor mediated effects through in silico modeling and in vitro bioassays
2020
The aryl hydrocarbon receptor (AhR) is a cytoplasmatic sensor of diverse endogenous and exogenous substances. In a toxicological context, the former known as “dioxin receptor” has been investigated as a xenobiotic chemoreceptor and due to its roles in mediating carcinogenesis, endocrine disruption, among other immunological, hepatic, cardiovascular, and dermal toxicity mechanisms. The deep physiological implications of AhR in cellular proliferation, adhesion, division, differentiation, as well as in the reproductive, immunological and cardiovascular homeostasis have opened a new field of research in order to harness AhR’s pharmacological potential. Hence, AhR has become a therapeutic target…