scholarly journals Sequence-mediated regulation of adenovirus gene expression by repression of mRNA accumulation.

1997 ◽  
Vol 17 (4) ◽  
pp. 2207-2216 ◽  
Author(s):  
J C Prescott ◽  
L Liu ◽  
E Falck-Pedersen
Keyword(s):  
Gene Expression ◽  
Transcription Initiation ◽  
Early Stage ◽  
Transcription Unit ◽  
Adenovirus Infection ◽  
Mrna Accumulation ◽  
Proximal Site ◽  
A Site

Gene expression in complex transcription units can be regulated at virtually every step in the production of mature cytoplasmic mRNA, including transcription initiation, elongation, termination, pre-mRNA processing, nucleus-to-cytoplasm mRNA transport, and alterations in mRNA stability. We have been characterizing alternative poly(A) site usage in the adenovirus major late transcription unit (MLTU) as a model for regulation at the level of pre-mRNA 3'-end processing. The MLTU contains five polyadenylation sites (L1 through L5). The promoter proximal site (L1) functions as the dominant poly(A) site during the early stage of adenovirus infection and in plasmid transfections when multiple poly(A) sites are present at the 3' end of a reporter plasmid. In contrast, stable mRNA processed at all five poly(A) sites is found during the late stage of adenovirus infection, after viral DNA replication has begun. Despite its dominance during early infection, L1 is a comparatively poor substrate for 3'-end RNA processing both in vivo and in vitro. In this study we have investigated the basis for the early L1 dominance. We have found that mRNA containing an unprocessed L1 poly(A) site is compromised in its ability to enter the steady-state pool of stable mRNA. This inhibition, which affects either the nuclear stability or nucleus-to-cytoplasm transport of the pre-mRNA, requires a cis-acting sequence located upstream of the L1 poly(A) site.

scholarly journals Analysis of the estrogen regulation of the zebrafish estrogen receptor (ER) reveals distinct effects of ERalpha, ERbeta1 and ERbeta2

2004 ◽  
Vol 32 (3) ◽  
pp. 975-986 ◽  
Author(s):  
A Menuet ◽  
Y Le Page ◽  
O Torres ◽  
L Kern ◽  
O Kah ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcription Initiation ◽  
Transcription Unit ◽  
Expression Vectors ◽  
Luciferase Reporter ◽  
Estrogen Regulation ◽  
Potential Implication ◽  
Hepatic Expression

We have previously cloned and characterized three estrogen receptors (ER) in the zebrafish (zfERalpha, zfERbeta1 and zfERbeta2). We have also shown that they are functional in vitro and exhibit a distinct expression pattern, although partially overlapping, in the brain of zebrafish. In this paper, we have shown that the hepatic expression of these zfER genes responds differently to estradiol (E2). In fact, a 48-h direct exposure of zebrafish to E2 resulted in a strong stimulation of zfERalpha gene expression while zfERbeta1 gene expression was markedly reduced and zfERbeta2 remained virtually unchanged. To establish the potential implication of each zfER in the E2 upregulation of the zfERalpha gene, the promoter region of this gene was isolated and characterized. Transfection experiments with promoter-luciferase reporter constructs together with different zfER expression vectors were carried out in different cell contexts. The data showed that in vivo E2 upregulation of the zfERalpha gene requires ERalpha itself and a conserved transcription unit sequence including at least an imperfect estrogen-responsive element (ERE) and an AP-1/ERE half site at the proximal transcription initiation site. Interestingly, although in the presence of E2 zfERalpha was the most potent at inducing the expression of its own gene, the effect of E2 mediated by zfERbeta2 represented 50% of the zfERalpha activity. In contrast, zfERbeta1 was unable to upregulate the zfERalpha gene whereas this receptor form was able to tightly bind E2 and activate a reporter plasmid containing a consensus ERE. Altogether, these results indicated that the two ERbeta forms recently characterized in teleost fish could have partially distinct and not redundant functions.

scholarly journals Requirement of glucose metabolism for regulation of glucose transporter type 2 (GLUT2) gene expression in liver

1996 ◽  
Vol 314 (3) ◽  
pp. 903-909 ◽  
Author(s):  
Franck RENCUREL ◽  
Gérard WAEBER ◽  
Bénédicte ANTOINE ◽  
Francis ROCCHICCIOLI ◽  
Paulette MAULARD ◽  
...  
Keyword(s):  
Gene Expression ◽  
Glucose Metabolism ◽  
Reporter Gene ◽  
Glucose Transporter ◽  
Regulatory Region ◽  
Proximal Fragment ◽  
Mrna Accumulation ◽  
Glut2 Gene

Previous studies have shown that glucose increases the glucose transporter (GLUT2) mRNA expression in the liver in vivo and in vitro. Here we report an analysis of the effects of glucose metabolism on GLUT2 gene expression. GLUT2 mRNA accumulation by glucose was not due to stabilization of its transcript but rather was a direct effect on gene transcription. A proximal fragment of the 5´ regulatory region of the mouse GLUT2 gene linked to a reporter gene was transiently transfected into liver GLUT2-expressing cells. Glucose stimulated reporter gene expression in these cells, suggesting that glucose-responsive elements were included within the proximal region of the promoter. A dose-dependent effect of glucose on GLUT2 expression was observed over 10 mM glucose irrespective of the hexokinase isozyme (glucokinase Km 16 mM; hexokinase I Km 0.01 mM) present in the cell type used. This suggests that the correlation between extracellular glucose and GLUT2 mRNA concentrations is simply a reflection of an activation of glucose metabolism. The mediators and the mechanism responsible for this response remain to be determined. In conclusion, glucose metabolism is required for the proper induction of the GLUT2 gene in the liver and this effect is transcriptionally regulated.

scholarly journals Control of Adenovirus Early Gene Expression during the Late Phase of Infection

1998 ◽  
Vol 72 (5) ◽  
pp. 4049-4056 ◽  
Author(s):  
Shawn P. Fessler ◽  
C. S. H. Young
Keyword(s):  
Gene Expression ◽  
Dna Replication ◽  
Transcription Initiation ◽  
Late Phase ◽  
Transcription Unit ◽  
Early Gene ◽  
Wild Type ◽  
Mrna Accumulation ◽  
Early Gene Expression ◽  
Late Protein

ABSTRACT The adenovirus gene regulatory program occurs in two distinct phases, as defined by the onset of DNA replication. During the early phase, the E1A, E1B, E2, E3, and E4 genes are maximally expressed, while the major late promoter (MLP) is minimally expressed and transcription is attenuated. After the onset of DNA replication, the IVa2 and pIX genes are expressed at high levels, transcription from the MLP is unattenuated and fully activated, and early gene expression is repressed. Although the cis elements andtrans-acting factors responsible for the late-phase activation of the MLP have been identified and characterized and the role of DNA replication in activation has been established, the mechanism(s) underlying the commensurate decrease in early gene expression has yet to be elucidated. The results of this study demonstrate that this decrease depends on a fully functional MLP. Specifically, virus mutants with severely deficient transcription from the MLP exhibit a marked increase in expression of the E1A, E1B, and E2 early genes. These increases were observed at the level of transcription initiation, mRNA accumulation, and protein production. In addition, expression from the late gene pIX, which is not contained within the major late transcription unit (MLTU), is also markedly increased. To begin the analysis of the mechanisms underlying these late-phase effects, mixed-infection experiments with mutant and wild-type viruses were performed. The results show that the effects on early gene expression, as measured both at the protein and RNA levels, are mediated in trans and not in cis. These observations are consistent either with a model in which one or more late protein products encoded by the MLTU acts as a repressor of early gene expression or with one in which the wild-type MLP competes with early promoters for limiting transcription factors.

scholarly journals SigB-Dependent In Vitro Transcription of prfA and Some Newly Identified Genes of Listeria monocytogenes Whose Expression Is Affected by PrfA In Vivo

2005 ◽  
Vol 187 (2) ◽  
pp. 800-804 ◽  
Author(s):  
Marcus Rauch ◽  
Qin Luo ◽  
Stefanie Müller-Altrock ◽  
Werner Goebel
Keyword(s):  
Gene Expression ◽  
Listeria Monocytogenes ◽  
Transcription Initiation ◽  
In Vitro Transcription ◽  
Direct Link ◽  
Class Iii ◽  
New Genes ◽  
Class Iii Genes

ABSTRACT Recent studies have identified several new genes in Listeria monocytogenes which are positively or negatively affected by PrfA and grouped into three classes (E. Milohanic et al., Mol. Microbiol. 47:1613-1625, 2003). In vitro transcription performed with promoters of some class III genes showed strict SigB-dependent but PrfA-independent transcription initiation. Transcription starting at the prfA promoter PprfA2 was also optimal with SigB-loaded RNA polymerase, suggesting a direct link between SigB- and PrfA-dependent gene expression.

Gene expression-based in vivo and in vitro prediction of liver toxicity allows compound selection at an early stage of drug development

2010 ◽  
Vol 25 (3) ◽  
pp. 183-194 ◽  
Author(s):  
Adrian Roth ◽  
Franziska Boess ◽  
Christian Landes ◽  
Guido Steiner ◽  
Christian Freichel ◽  
...  
Keyword(s):  
Gene Expression ◽  
Drug Development ◽  
Liver Toxicity ◽  
Early Stage ◽  
Compound Selection

scholarly journals Combinatorial Regulation of thedevOperon by MrpC2 and FruA during Myxococcus xanthus Development

2014 ◽  
Vol 197 (2) ◽  
pp. 240-251 ◽  
Author(s):  
Ashleigh Campbell ◽  
Poorna Viswanathan ◽  
Terry Barrett ◽  
Bongjun Son ◽  
Shreya Saha ◽  
...  
Keyword(s):  
Myxococcus Xanthus ◽  
Developmental Expression ◽  
Cooperative Binding ◽  
Fruiting Bodies ◽  
Content Type ◽  
Combinatorial Regulation ◽  
Proximal Site ◽  
A Site

Proper expression of thedevoperon is important for normal development ofMyxococcus xanthus. When starved, these bacteria coordinate their gliding movements to build mounds that become fruiting bodies as some cells differentiate into spores. Mutations in thedevTRSgenes impair sporulation. Expression of the operon occurs within nascent fruiting bodies and depends in part on C signaling. Here, we report that expression of thedevoperon, like that of several other C-signal-dependent genes, is subject to combinatorial control by the transcription factors MrpC2 and FruA. A DNA fragment upstream of thedevpromoter was bound by a protein in an extract containing MrpC2, protecting the region spanning positions −77 to −54. Mutations in this region impaired binding of purified MrpC2 and abolished developmental expression of reporter fusions. The association of MrpC2 and/or its longer form, MrpC, with thedevpromoter region depended on FruAin vivo, based on chromatin immunoprecipitation analysis, and purified FruA appeared to bind cooperatively with MrpC2 to DNA just upstream of thedevpromoterin vitro. We conclude that cooperative binding of the two proteins to this promoter-proximal site is crucial fordevexpression. 5′ deletion analysis implied a second upstream positive regulatory site, which corresponded to a site of weak cooperative binding of MrpC2 and FruA and boosteddevexpression 24 h into development. This site is unique among the C-signal-dependent genes studied so far. Deletion of this site in theM. xanthuschromosome did not impair sporulation under laboratory conditions.

scholarly journals Detection and analysis of gene expression during infection by in vivo expression technology

2000 ◽  
Vol 355 (1397) ◽  
pp. 587-599 ◽  
Author(s):  
D. Scott Merrell ◽  
Andrew Camilli
Keyword(s):  
Gene Expression ◽  
Gene Fusion ◽  
Ex Vivo ◽  
Gene Cassette ◽  
Chromosome Loss ◽  
In Vivo Expression ◽  
Reporter Systems ◽  
A Site

Many limitations associated with the use of in vitro models for study of bacterial pathogenesis can be overcome by the use of technologies that detect pathogen gene expression during the course of infection within an intact animal. in vivo expression technology (IVET) accomplishes this with versatility: it has been developed with a variety of reporter systems which allow for either in vivo selection or ex vivo screening. Selectable gene fusion systems generally allow for the complementation of a bacterial metabolic defect that is lethal in vivo, or for antibiotic resistance during the course of in vivo antibiotic challenge. In contrast, the screenable gene fusion system uses a site–specific DNA recombinase that, when expressed in vivo, excises a selectable gene cassette from the bacterial chromosome. Loss of this cassette can then be either screened or selected for ex vivo . The recombinase–based IVET can be used to detect genes that are transcriptionally induced during infection, including those expressed transiently or at low levels and, in addition, can be used to monitor the spatial and temporal expression of specific genes during the course of infection.

scholarly journals Domains within RbpA Serve Specific Functional Roles That Regulate the Expression of Distinct Mycobacterial Gene Subsets

2018 ◽  
Vol 200 (13) ◽  
Author(s):  
Jerome Prusa ◽  
Drake Jensen ◽  
Gustavo Santiago-Collazo ◽  
Steven S. Pope ◽  
Ashley L. Garner ◽  
...  
Keyword(s):  
Gene Expression ◽  
Mycobacterium Tuberculosis ◽  
Transcription Initiation ◽  
Dependent Manner ◽  
Interaction Domain ◽  
Structural Domains ◽  
Content Type ◽  
Group I

ABSTRACT The RNA polymerase (RNAP) binding protein A (RbpA) contributes to the formation of stable RNAP-promoter open complexes (RP o ) and is essential for viability in mycobacteria. Four domains have been identified in the RbpA protein, i.e., an N-terminal tail (NTT) that interacts with RNAP β′ and σ subunits, a core domain (CD) that contacts the RNAP β′ subunit, a basic linker (BL) that binds DNA, and a σ-interaction domain (SID) that binds group I and group II σ factors. Limited in vivo studies have been performed in mycobacteria, however, and how individual structural domains of RbpA contribute to RbpA function and mycobacterial gene expression remains mostly unknown. We investigated the roles of the RbpA structural domains in mycobacteria using a panel of rbpA mutants that target individual RbpA domains. The function of each RbpA domain was required for Mycobacterium tuberculosis viability and optimal growth in Mycobacterium smegmatis . We determined that the RbpA SID is both necessary and sufficient for RbpA interaction with the RNAP, indicating that the primary functions of the NTT and CD are not solely association with the RNAP. We show that the RbpA BL and SID are required for RP o stabilization in vitro , while the NTT and CD antagonize this activity. Finally, RNA-sequencing analyses suggest that the NTT and CD broadly activate gene expression, whereas the BL and SID activate or repress gene expression in a gene-dependent manner for a subset of mycobacterial genes. Our findings highlight specific outcomes for the activities of the individual functional domains in RbpA. IMPORTANCE Mycobacterium tuberculosis is the causative agent of tuberculosis and continues to be the most lethal infectious disease worldwide. Improved molecular understanding of the essential proteins involved in M. tuberculosis transcription, such as RbpA, could provide targets for much needed future therapeutic agents aimed at combatting this pathogen. In this study, we expand our understanding of RbpA by identifying the RbpA structural domains responsible for the interaction of RbpA with the RNAP and the effects of RbpA on transcription initiation and gene expression. These experiments expand our knowledge of RbpA while also broadening our understanding of bacterial transcription in general.

scholarly journals Positive and Negative Autoregulation ofREB1 Transcription in Saccharomyces cerevisiae

1998 ◽  
Vol 18 (7) ◽  
pp. 4368-4376 ◽  
Author(s):  
Kevin L.-C. Wang ◽  
Jonathan R. Warner
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Binding Sites ◽  
Transcription Initiation ◽  
Transcriptional Control ◽  
Initiation Site ◽  
Transcription Unit ◽  
Pol I ◽  
Activation Of Transcription

ABSTRACT Reb1p is a DNA binding protein of Saccharomyces cerevisiae that has been implicated in the activation of transcription by polymerase (Pol) II, in the termination of transcription by Pol I, and in the organization of nucleosomes. Studies of the transcriptional control of the REB1 gene have led us to identify three Reb1p binding sites in the 5′ region of the its gene, termed A, B, and C, at positions −110, −80, and +30 with respect to transcription initiation. In vitro, Reb1p binds to the three sites with the relative affinity of A ≥ C > B. Kinetic parameters suggest that when both A and C sites are present on the same DNA molecule, the C site may recruit Reb1p for the A site. In vivo the A and B sites each contribute to the transcription activity ofREB1 in roughly additive fashion. Mutation of both A and B sites abolishes transcription. On the other hand, the C site is a negative element, reducing transcription by 40%. In cells overexpressing Reb1p, the C site reduces transcription by more than 80%. This effect can be transposed to another transcription unit, demonstrating that the effect of Reb1p binding at the C site does not depend on interaction with upstream Reb1p molecules. Relocation of the C site to a position 105 bp downstream of the transcription initiation site abolishes its effect, suggesting that it does not act as a conventional attenuator of transcription. We conclude that binding of Reb1p at the C site hinders formation of the initiation complex. This arrangement of Reb1p binding sites provides a positive and negative mechanism to autoregulate the expression of REB1. Such an arrangement could serve to dampen the inevitable fluctuation in Rep1p levels caused by the intermittent presence of its mRNA within an individual cell.

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