scholarly journals Transcriptional Regulation of the ant Operon, Encoding Two-Component Anthranilate 1,2-Dioxygenase, on the Carbazole-Degradative Plasmid pCAR1 of Pseudomonas resinovorans Strain CA10

2004 ◽  
Vol 186 (20) ◽  
pp. 6815-6823 ◽  
Author(s):  
Masaaki Urata ◽  
Masatoshi Miyakoshi ◽  
Satoshi Kai ◽  
Kana Maeda ◽  
Hiroshi Habe ◽  
...  
Keyword(s):  
Transcriptional Unit ◽  
Northern Hybridization ◽  
Luciferase Expression ◽  
Transcription Start Point ◽  
Rt Pcr ◽  
Transcription Start ◽  
Pseudomonas Resinovorans ◽  
Two Component ◽  
Degradative Plasmid ◽  
Strain Ca10

ABSTRACT The carbazole-degradative plasmid pCAR1 of Pseudomonas resinovorans strain CA10 has two gene clusters, carAaAaBaBbCAcAdDFE and antABC, which are involved in the conversions of carbazole to anthranilate and anthranilate to catechol, respectively. We proved that the antABC gene cluster, encoding two-component anthranilate 1,2-dioxygenase, constitutes a single transcriptional unit through Northern hybridization and reverse transcription-PCR (RT-PCR) analyses. The transcription start point of antA was mapped at 53 bp upstream point of its translation start point, and the −10 and −35 boxes were homologous to conserved σ70 recognition sequence. Hence the promoter of the ant operon was designated P ant . 5′ Deletion analyses using luciferase as a reporter showed that the region up to at least 70 bp from the transcription start point of antA was necessary for the activation of P ant . Luciferase expression from P ant was induced by anthranilate itself, but not by catechol. Two probable AraC/XylS-type regulatory genes found on pCAR1, open reading frame 22 (ORF22) and ORF23, are tandemly located 3.2 kb upstream of the antA gene. We revealed that the product of ORF23, designated AntR, is indispensable for the stimulation of P ant in Pseudomonas putida cells. Northern hybridization and RT-PCR analyses revealed that another copy of P ant , which is thought to be translocated about 2.1 kb upstream of the carAa gene as a consequence of the transposition of ISPre1, actually drives transcription of the carAa gene in the presence of anthranilate, indicating that both ant and car operons are simultaneously regulated by AntR.

scholarly journals Transcription Analysis of a Lantibiotic Gene Cluster from Bifidobacterium longum DJO10A

2011 ◽  
Vol 77 (17) ◽  
pp. 5879-5887 ◽  
Author(s):  
Ju-Hoon Lee ◽  
Xiulan Li ◽  
Daniel J. O'Sullivan
Keyword(s):  
Gene Expression ◽  
Response Regulator ◽  
Bifidobacterium Longum ◽  
Regulatory System ◽  
Broth Culture ◽  
Northern Hybridization ◽  
Transcription Start ◽  
Transcription Analysis ◽  
Content Type ◽  
Two Component

ABSTRACTBifidobacterium longumDJO10A was previously demonstrated to produce a lantibiotic, but only during growth on agar media. To evaluate the feasibility of production of this lantibiotic in broth media, a transcription analysis of thelanAgene was undertaken. Comparative microarray analysis of broth and agar cultures ofB. longumDJO10A revealed that the lantibiotic production, modification, transport/peptidase, and immunity genes were significantly upregulated in agar cultures, while the two-component regulatory genes were expressed equally under both conditions. This suggested that the signal transduction regulatory system should function in broth cultures. Real-time PCR and Northern hybridization confirmed thatlanAgene expression was significantly repressed in broth cultures. A crude lantibiotic preparation from an agar-grown culture was obtained, and its antimicrobial spectrum analysis revealed a broad inhibition range. Addition of this extract to broth cultures ofB. longumDJO10A inducedlanAgene expression in a dose-dependent fashion. Subinoculation using >10% of an induced broth culture maintainedlanAexpression. The expression oflanAwas log-phase specific, being significantly downregulated in stationary phase. Transcription start analysis oflanArevealed a 284-bp 5′ untranslated region, which was proposed to be involved in repression of transcription, while an inverted repeat structure located at bp −75 relative to the transcription start was strategically located to likely function as a binding site for the two-component response regulator. Understanding the transcription regulation of thislanAgene is the first step toward enabling production of this novel and potentially interesting lantibiotic in broth cultures.

sae is essential for expression of the staphylococcal adhesins Eap and Emp

Microbiology ◽  
2005 ◽  
Vol 151 (6) ◽  
pp. 1789-1800 ◽  
Author(s):  
Niamh Harraghy ◽  
Jan Kormanec ◽  
Christiane Wolz ◽  
Dagmar Homerova ◽  
Christiane Goerke ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Extracellular Matrix ◽  
Virulence Factor ◽  
Eukaryotic Cells ◽  
Transcription Start Point ◽  
Chronic Infections ◽  
Transcription Start ◽  
Regulatory Mutants ◽  
Global Regulators ◽  
Glucose Analysis

Eap and Emp are two Staphylococcus aureus adhesins initially described as extracellular matrix binding proteins. Eap has since emerged as being important in adherence to and invasion of eukaryotic cells, as well as being described as an immunomodulator and virulence factor in chronic infections. This paper describes the mapping of the transcription start point of the eap and emp promoters. Moreover, using reporter-gene assays and real-time PCR in defined regulatory mutants, environmental conditions and global regulators affecting expression of eap and emp were investigated. Marked differences were found in expression of eap and emp between strain Newman and the 8325 derivatives SH1000 and 8325-4. Moreover, both genes were repressed in the presence of glucose. Analysis of expression of both genes in various regulatory mutants revealed that sarA and agr were involved in their regulation, but the data suggested that there were additional regulators of both genes. In a sae mutant, expression of both genes was severely repressed. sae expression was also reduced in the presence of glucose, suggesting that repression of eap and emp in glucose-containing medium may, in part, be a consequence of a decrease in expression of sae.

scholarly journals RT-PCR and Northern blot analysis in search for a putative Paramecium beta-adrenergic receptor.

1999 ◽  
Vol 46 (3) ◽  
pp. 813-821
Author(s):  
A Płatek ◽  
J Wiejak ◽  
E Wyroba
Keyword(s):  
Northern Blot ◽  
Blot Analysis ◽  
Adrenergic Receptor ◽  
Northern Blot Analysis ◽  
Molecular Probes ◽  
Northern Hybridization ◽  
Rt Pcr ◽  
Beta Adrenergic Receptor ◽  
Beta Adrenergic ◽  
Sequence Tagged Sites

RT-PCR and Northern blot analysis were performed in order to search for a putative beta-adrenergic receptor (beta-AR) in Paramecium using several beta2-adrenergic-specific molecular probes. Under strictly defined RT-PCR conditions DNA species of expected molecular size about 360 bp were generated with the primers corresponding to the universal mammalian beta2-AR sequence tagged sites (located within the 4th and the 6th transmembrane regions of the receptor). This RT-PCR product hybridized in Southern blot analysis with the oligonucleotide probe designed to the highly conservative beta2-AR region involved in G-proteins interaction and located within the amplified region. Northern hybridization was performed on Paramecium total RNA and mRNA with human beta2-AR cDNA and two oligonucleotide probes: the first included Phe 290 involved in agonist binding (Strader et al., 1995) and the second was the backward RT-PCR primer. All these probes revealed the presence of about 2 kb mRNA which is consistent with the size of beta2-AR transcripts found in higher eukaryotes.

Constitutive expression of cat-86 associated with a change in the transcription start point

Gene ◽  
1991 ◽  
Vol 105 (1) ◽  
pp. 113-117 ◽  
Author(s):  
Nicholas P. Ambulos ◽  
Un Jin Kim ◽  
Elizabeth J. Rogers ◽  
Paul S. Lovett
Keyword(s):  
Constitutive Expression ◽  
Transcription Start Point ◽  
Transcription Start

scholarly journals Analysis of a Growth-Phase-Regulated Two-Component Regulatory System in the Periodontal Pathogen Treponema denticola

2008 ◽  
Vol 190 (18) ◽  
pp. 6162-6169 ◽  
Author(s):  
Jesse R. Frederick ◽  
Elizabeth A. Rogers ◽  
Richard T. Marconi
Keyword(s):  
Growth Phase ◽  
Regulatory Networks ◽  
Response Regulator ◽  
Regulatory System ◽  
Open Reading Frames ◽  
Periodontal Pathogen ◽  
Treponema Denticola ◽  
Rt Pcr ◽  
Two Component ◽  
Transcriptional Regulatory Mechanisms

ABSTRACT Nothing is currently known regarding the global regulatory networks of Treponema denticola and other oral spirochetes. In this report, we assess the properties and potential phosphotransfer capability of a putative two-component regulatory system (TCS) of T. denticola that is formed by the products of open reading frames tde0032 (a sensor kinase) and tde0033 (a response regulator), henceforth designated AtcS and AtcR, respectively. Using PCR and DNA sequence analyses, atcS and atcR were demonstrated to be widely distributed and conserved among T. denticola isolates. Reverse transcription-PCR (RT-PCR) analyses revealed that these genes are cotranscribed and may also be expressed as part of a larger operon that includes several flanking genes. Analyses using 5′ rapid amplification of cDNA ends identified the transcriptional start sites for these operons and provided evidence that some of these genes may be independently transcribed from internal promoters. Real-time RT-PCR and Western blot analysis revealed significant upregulation of atcRS during late-stage growth, indicating growth-phase-dependent expression. Lastly, the phosphorelay capability of the AtcRS system was assessed and demonstrated using recombinant proteins. AtcS was found to undergo autophosphorylation and to transfer phosphate to AtcR. These analyses represent the first description of a functional TCS in an oral spirochetes and provide insight into the transcriptional regulatory mechanisms of these important bacteria.

scholarly journals AtypicalCTSKTranscripts andARNTTranscription Read-Through intoCTSK

2005 ◽  
Vol 6 (5-6) ◽  
pp. 268-276
Author(s):  
Fabienne S. Giraudeau ◽  
Jean-Philippe Walhin ◽  
Paul R. Murdock ◽  
Nigel K. Spurr ◽  
Ian C. Gray
Keyword(s):  
Negative Impact ◽  
Cathepsin K ◽  
Chromosome 1 ◽  
Rt Pcr ◽  
Transcription Start ◽  
Aryl Hydrocarbon ◽  
Pcr Product ◽  
Close Proximity ◽  
Pcr Products ◽  
Human Chromosome 1

The aryl hydrocarbon receptor nuclear translocator (ARNT) and cathepsin K (CTSK) genes lie in a tandem head-to-tail arrangement on human chromosome 1. The two genes are in extremely close proximity; the usualCTSKtranscription start site is less than 1.4 kb downstream of the end of the longest reportedARNTtranscript. By generating an RT-PCR product that overlaps both the 3′ end ofARNTand the 5′ end ofCTSK, we show thatARNTtranscripts may extend through theARNT–CTSKintergenic region and progress into theCTSKgene. Furthermore, by using quantitative RT-PCR from several tissues to detect theARNTexpression signature inCTSKintrons, we show thatARNTtranscripts can read through intoCTSKas far asCTSKintron 3, extending approximately 3.7 kb downstream of the end of the longest previously describedARNTmRNA. Given thatARNTandCTSKare expressed in an overlapping range of tissues,ARNTread-through may have a negative impact onCTSKtranscript levels by interfering withCTSKexpression. We also present evidence for novelCTSKtranscripts following sequence analysis ofCTSK-derived ESTs and RT-PCR products. These transcripts show alternate 5′ splicing and or 5′ extension and are sometimes initiated from a cryptic alternative promoter which is upstream of the knownCTSKpromoter and possibly in the 3′ UTR ofARNT.

A Second Promoter for the Human KCl Cotransporter 1 (KCC1) Gene Drives Differential Expression of a Variant Isoform in Sickle Versus Normal Reticulocytes.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3592-3592
Author(s):  
Kathleen Anderson ◽  
Scott C. Crable ◽  
Suzan M. Hammond ◽  
Clinton H. Joiner ◽  
Patrick G. Gallagher
Keyword(s):  
Promoter Activity ◽  
Regulatory Elements ◽  
Dominant Negative ◽  
Jurkat Cells ◽  
Pcr Analysis ◽  
Luciferase Expression ◽  
Alternative Transcript ◽  
Red Cell ◽  
Rt Pcr ◽  
Tissue Samples

Abstract The K+Cl- cotransporter plays a significant role in the maintenance of red cell volume. During cellular maturation, this cotransporter actively moves K+ and Cl- out of the cell. The accompanying movement of water results in dehydration and shrinking of the red cell. Because KCl cotransporter activity is higher in sickle compared to normal reticulocytes, it has been considered a potential modifier gene for sickle cell disease. We have evidence for expression of three KCC genes in human reticulocytes and have investigated the promoter for KCC1. While the expression of the principal KCC1 transcript did not differ in SS compared to normal reticulocytes, we now describe an alternative transcript of the KCC1 gene emanating from a second promoter and exhibiting a restricted tissue distribution. Investigation of the EST databases revealed spliced ESTs corresponding to the use of four distinct N-terminal exons in the KCC1 gene, each reported multiple times in the dataset. Primers were developed for these 5′ regions (exon1, 1a, 1b, and 1c) and used in an RT-PCR reaction with human reticulocyte RNA. The exon1 form and the exon1b variant were expressed. When the relative levels of these forms were compared, expression of the exon1 transcript was unchanged, while significantly higher levels of the exon1b variant was evident in the AA reticulocyte RNA compared to numerous SS samples. In an analysis of seven other human tissue samples, the exon1b isoform was highly expressed in kidney, lung, and heart, while the KCC1ex1 transcript was expressed at a constant level in all tissues. Although the transcript for this variant could arise from the KCC1 promoter we have previously characterized, the pattern of expression suggested control from a second promoter. A 915bp region corresponding to −787 to +128 was isolated and cloned into a reporter construct to test for promoter activity. This clone was compared with KCC1 promoter constructs in transient transfection assays. The exon1b construct not only exhibited promoter activity by directing high levels of luciferase expression in K562 cells, it also demonstrated tissue-specificity with a low level of activity in Jurkat cells. This recapitulates the endogenous levels detected by RT-PCR analysis of these cell lines. The −787/+128 exon1b construct is also 3-fold more active than the ubiquitously expressed exon1 promoter. To identify the control elements for this promoter, we produced a series of deletion constructs; the smallest construct contained 181bp. No reduction in reporter gene activity was evident, indicating the major regulatory elements lie very close to this promoter. Since exon1 encodes 39aa and exon1b only 7aa, the use of this smaller first exon effectively produces an N-terminal truncation in the protein. Studies with the mouse KCC1 cDNA have demonstrated that proteins produced by an N-terminal truncation are not only inactive for K+Cl- cotransport, but also function as dominant negative regulators of a full-length KCC1 protein. High level expression of this variant in AA cells compared to SS cells would therefore be consistent with the low reported activity in the AA reticulocytes. Induction or modulation of the expression of the KCC1ex1b variant may be an key factor in the control of red cell hydration.

Localization of a transcription start point within the human Hinf element

Gene ◽  
1989 ◽  
Vol 83 (1) ◽  
pp. 173-179 ◽  
Author(s):  
T.Herbert Manoharan ◽  
T. Somasundaram ◽  
G. Chinnadurai
Keyword(s):  
Transcription Start Point ◽  
Transcription Start

scholarly journals The Two-Component System GrvRS (EtaRS) RegulatesaceExpression in Enterococcus faecalis OG1RF

2014 ◽  
Vol 83 (1) ◽  
pp. 389-395 ◽  
Author(s):  
Jung Hyeob Roh ◽  
Kavindra V. Singh ◽  
Sabina Leanti La Rosa ◽  
Ana Luisa V. Cohen ◽  
Barbara E. Murray
Keyword(s):  
Urinary Tract Infection ◽  
Urinary Tract ◽  
Tract Infection ◽  
Regulatory System ◽  
Northern Hybridization ◽  
Infection Model ◽  
Content Type ◽  
Infection Models ◽  
Two Component ◽  
Main Regulator

Expression oface(adhesin tocollagen ofEnterococcus faecalis), encoding a virulence factor in endocarditis and urinary tract infection models, has been shown to increase under certain conditions, such as in the presence of serum, bile salts, urine, and collagen and at 46°C. However, the mechanism oface/Ace regulation under different conditions is still unknown. In this study, we identified a two-component regulatory system GrvRS as the main regulator ofaceexpression under these stress conditions. Using Northern hybridization and β-galactosidase assays of anacepromoter-lacZfusion, we found transcription ofaceto be virtually absent in agrvRdeletion mutant under the conditions that increaseaceexpression in wild-type OG1RF and in the complemented strain. Moreover, agrvRmutant revealed decreased collagen binding and biofilm formation as well as attenuation in a murine urinary tract infection model. Here we show that GrvR plays a major role in control ofaceexpression andE. faecalisvirulence.

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