Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.7.6 (RNA polymerase)
 34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The increasing incidence of tuberculosis and other mycobacterial infections due to AIDS epidemic resulted in the need of rapid and accurate identification of isolated mycobacteria. The correct identification result leads to the selection of an appropriate therapeutic regimen. Polymerase chain reaction and restriction enzyme analysis (PCR-REA) has been developed since 1992 and used as the rapid method for identifying mycobacteria. Several genes or sequences have been used as an amplified target for PCR-REA. The present study aims to evaluate the potential use of PCR-REA of gene-encoding heat shock protein 65 kDa (hsp65) and beta-subunit RNA polymerase (rpoB) for the identification of mycobacteria compared with conventional biochemical identification. Two hundreds clinical isolates, consisting of 50 isolates of Mycobacterium tuberculosis and 150 isolates of nontuberculous mycobacteria (NTM), were submitted for identification using PCR-REA and biochemical method. The results demonstrated that PCR-REA identified 188 isolates of both M. tuberculosis and NTM concordantly with biochemical identification. Discordant identification results obtained from 12 isolates, comprised of 8 M. scrofulaceum, 1 M. avium complex, 1 M. malmoense, 1 M. terrae complex, and 1 M. chelonae/abscessus. Overall, the concordant percentage of results obtained from PCR-REA compared with biochemical method was 100%, 98.8%, and 83.3% for M. tuberculosis complex, rapidly growing, and slowly growing mycobacteria, respectively, and the results of hsp65 PCR-REA was in agreement with those obtained from rpoB PCR-REA. From this study, PCR-REA appears to be a simple, rapid, and reliable method for identifying mycobacteria in a routine microbiology laboratory.
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PMID:Comparative evaluation of polymerase chain reaction and restriction enzyme analysis: two amplified targets, hsp65 and rpoB, for identification of cultured mycobacteria. 1576 1

Mycobacterium tuberculosis RNA polymerase is 1,000-fold more sensitive to rifampin than Escherichia coli RNA polymerase. Chimeric E. coli RNA polymerase in which the beta-subunit segment encompassing rifampin regions I and II (amino acids [aa] 463 through 590) was replaced with the corresponding region from M. tuberculosis (aa 382 through 509) did not show an increased sensitivity to the antibiotic. Thus, the difference in amino acid sequence between the rifampin regions I and II of the two species does not account for the difference in rifampin sensitivity of the two polymerases.
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PMID:Different rifampin sensitivities of Escherichia coli and Mycobacterium tuberculosis RNA polymerases are not explained by the difference in the beta-subunit rifampin regions I and II. 1579 46

The previously established two-plasmid system in Escherichia coli for the identification of Mycobacterium tuberculosis promoters that are recognized by RNA polymerase containing the stress response sigma factor sigmaF was optimized. Expression of the M. tuberculosis sigmaF encoded by sigF gene was under the control of the isopropyl beta-D-thiogalactopyranoside (IPTG)-dependent Ptrc promoter. A low level of IPTG induced a nontoxic but sufficient level of sigmaF to interact with the core enzyme of RNA polymerase. Such an RNA polymerase holoenzyme recognized the known sigmaF-dependent promoter, usfXp1, which was cloned in the compatible promoter probe plasmid, upstream of a promoterless lacZalpha reporter gene. Primer extension analysis of the usfXp1 promoter in the E. coli two-plasmid system after IPTG-induced expression of M. tuberculosis sigF revealed a transcription start point that was identical as in M. tuberculosis. This new system has been shown to be useful for identification of M. tuberculosis sigmaF-dependent promoters.
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PMID:Optimization of a two-plasmid system for the identification of promoters recognized by RNA polymerase containing Mycobacterium tuberculosis stress response sigma factor, sigmaF. 1588 4

Molecular characterization of drug resistance of Mycobacterium tuberculosis strains of different origins can generate information useful for developing molecular methods that are widely applicable for rapid drug resistance detection. Using DNA sequencing and allele-specific polymerase chain reaction (AS-PCR), we investigated genetic mutations associated with isoniazid (INH) and rifampin (RIF) resistance among 29 drug-resistant clinical isolates of M. tuberculosis collected from Malatya, Turkey, including 19 multi-drug-resistant (MDR) isolates. Point mutations were detected at codons 531, 516, 526, and 513 of the RNA polymerase beta- subunit gene (rpoB) in 10 (47.6%), five (23.8%), three (14.3%), and three (14.3%) of the 21 RIF-resistant isolates, respectively. Of the five isolates having mutations in codon 516, three also had mutations at codon 527; one had a concurrent mutation at codon 572. Mutations at codon 315 of the catalase-peroxidase-encoding gene (katG) were found in 17 (63.0%) of the 27 INH-resistant isolates. Interestingly, the katG codon 315 mutation was observed at a much higher frequency in MDR isolates than in INH-mono-resistant isolates ( approximately 79% vs. 25%). This study provided the first molecular characterization of INH and RIF resistance of M. tuberculosis clinical isolates from Eastern Turkey, and extended our knowledge of molecular basis of M. tuberculosis drug resistance.
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PMID:Molecular characterization of isoniazid and rifampin resistance of Mycobacterium tuberculosis clinical isolates from Malatya, Turkey. 1591 Feb 21

Rifampin resistance in Mycobacterium tuberculosis is mainly due to a small number of mutations in the rpoB gene coding for the beta subunit of RNA polymerase. Heteroresistance, which is defined as a mixture of wildtype and resistant subpopulations in the same culture, can influence the sensitivity of molecular tests for determining drug resistance by leading to false negative or indeterminable results. In our laboratory, we identified one heteroresistant strain during sequencing of the rpoB gene mutations, which are responsible for Rifampin resistance in M. tuberculosis. The sequence analysis of this isolate demonstrated the mixture of different strains, and the exact nature of the mutation could not be determined. In order to solve this problem, the possible mutation site was digested with Tsp509I restriction endonuclease, which made us separate the different strains. Sequencing of the separated mutated product revealed the mutation responsible for Rifampin resistance. From this result, we propose that, when the suggested mutation site creates and/or deletes a restriction endonuclease recognition site in heteroresistant populations, restriction endonuclease analysis can be used to ease the determination of resistance mutations by sequencing.
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PMID:Restriction endonuclease analysis as a solution for determining rifampin resistance mutations by automated DNA sequencing in heteroresistant Mycobacterium tuberculosis strains. 1591 Feb 27

Lack of maturation of phagosomes containing pathogenic Mycobacterium tuberculosis within macrophages has been widely recognized as a crucial factor for the persistence of mycobacterial pathogen. Host molecule tryptophan-aspartate containing coat protein (TACO) has been shown to play a crucial role in the arrest of such a maturation process. The present study was addressed to understand whether or not polyphenols derived from green tea could down-regulate TACO gene transcription. And if yes, what impact TACO gene down-regulation has on the uptake/survival of M. tuberculosis within macrophages. The reverse-transcriptase polymerase chain reaction and reporter assay technology, employed in this study, revealed that the major component of green tea polyphenols, epigallocatechin-3-gallate had the inherent capacity to down-regulate TACO gene transcription within human macrophages through its ability to inhibit Sp1 transcription factor. We also found out that TACO gene promoter does contain Sp1 binding sequence using bioinformatics tools. The down-regulation of TACO gene expression by epigallocatechin-3-gallate was accompanied by inhibition of mycobacterium survival within macrophages as assessed through flow cytometry and colony counts. Based on these results, we propose that epigallocatechin-3-gallate may be of importance in the prevention of tuberculosis infection.
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PMID:Green tea polyphenol inhibits Mycobacterium tuberculosis survival within human macrophages. 1635 57

Corynebacterium glutamicum, a soil Gram-positive bacterium used for industrial amino acid production, was found to grow optimally at pH 7.0-9.0 when incubated in 5 litre fermenters under pH-controlled conditions. The highest biomass was accumulated at pH 9.0. Growth still occurred at pH 9.5 but at a reduced rate. The expression of the pH-regulated F0 F1 ATPase operon (containing the eight genes atpBEFHAGDC) was induced at alkaline pH. A 7.5 kb transcript, corresponding to the eight-gene operon, was optimally expressed at pH 9.0. The same occurred with a 1.2 kb transcript corresponding to the atpB gene. RT-PCR studies confirmed the alkaline pH induction of the F0 F1 operon and the existence of the atpI gene. The atpI gene, located upstream of the F0 F1 operon, was expressed at a lower level than the polycistronic 7.5 kb mRNA, from a separate promoter (P-atp1). Expression of the major promoter of the F0 F1 operon, designated P-atp2, and the P-atp1 promoter was quantified by coupling them to the pET2 promoter-probe vector. Both P-atp1 and P-atp2 were functional in C. glutamicum and Escherichia coli. Primer extension analysis identified one transcription start point inside each of the two promoter regions. The P-atp1 promoter fitted the consensus sequence of promoters recognized by the vegetative sigma factor of C. glutamicum, whereas the -35 and -10 boxes of P-atp2 fitted the consensus sequence for sigma(H)-recognized Mycobacterium tuberculosis promoters C(C)/(G)GG(A)/(G)AC 17-22 nt (C)/(G)GTT(C)/(G), known to be involved in expression of heat-shock and other stress-response genes. These results suggest that the F0 F1 operon is highly expressed at alkaline pH, probably using a sigma (H) RNA polymerase.
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PMID:Transcriptional analysis of the F0F1 ATPase operon of Corynebacterium glutamicum ATCC 13032 reveals strong induction by alkaline pH. 2375 42

In vitro transcription constitutes an important tool in the study of the regulation of gene expression. Here, we present a fast and easy procedure to prepare Mycobacterium tuberculosis RNA polymerase for in vitro transcription assays. RNA polymerase is assembled from recombinant proteins expressed in Escherichia coli, thus eliminating the need for biosafety containment facilities, and is mixed with any of the 13 M. tuberculosissigma factors. We show that the recombinant RNA polymerase is free from contaminating sigma factors, produces transcriptional start sites matching those characterized in vivo and allows the formal identification of sigma factors involved in the expression of genes of interest.
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PMID:A recombinant Mycobacterium tuberculosis in vitro transcription system. 1643 73

Among transcription factors that bind to bacterial RNA polymerase (RNAP) and modulate its activity, a number of small molecules irreversibly inhibit RNAP thereby causing cell death. To be of clinical significance such inhibitors must (1) inhibit a broad range of bacterial RNAPs but not affect human cells, (2) penetrate bacterial cell walls and (3) circumvent bacterial resistance mechanisms. Rifamycins, the only class of RNAP inhibitors that have found their way into clinical practice, are widely used in the treatment of tuberculosis and leprosy. However, the practical value of this class of antibiotics is limited by a rapid rise in resistant bacterial isolates. In this review we focus on recent advances in studies of prokaryotic transcription that allow a detailed structural and functional characterization of a number of RNAP/rifamycins complexes, thereby opening new opportunities for the design of superior antibacterial agents.
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PMID:Is it easy to stop RNA polymerase? 1647 53

Bovine tuberculosis, caused by Mycobacterium bovis (Mycobacterium tuberculosis complex), is a zoonotic disease that affects cattle worldwide. The bacterium infects other animal species, both domesticated and wild, and this range of hosts complicates attempts to control or eradicate the disease. Despite advances in the characterization of the mechanisms involved in host-pathogen interactions and host cell responses to M. tuberculosis complex in human, bovine and mouse cells, differentially expressed genes in tissue biopsies of naturally occurring tuberculous and nontuberculous exposed individuals have been poorly characterized. In this study, differential gene expression was analysed using suppression-subtractive hybridization in oropharyngeal tonsils and mandibular lymph nodes of field-collected tuberculous and nontuberculous European wild boars from a tuberculosis-endemic area of Spain. Real-time PCR and semiquantitative reverse-transcriptase PCR of selected genes confirmed the results of the suppression-subtractive hybridization analysis. Protein expression of selected differentially expressed genes was analysed by radial immunodiffusion or immunohistochemistry. Differential gene expression varied among tuberculous and non-tuberculous groups and between tonsils and lymph nodes. Single and multiple cellular mechanisms were affected, including signal transduction, immune response, inflammation, stress, apoptosis/antiapoptosis, cell structure, adhesion and transport, protein and DNA/RNA metabolism and enzymatic processes. These results demonstrate the modulation of gene expression by mycobacterial infection in tonsils and mandibular lymph nodes of European wild boars naturally exposed to M. bovis, and provide a basis for defining host-pathogen interactions and the mechanism of protective immunity.
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PMID:Genes differentially expressed in oropharyngeal tonsils and mandibular lymph nodes of tuberculous and nontuberculous European wild boars naturally exposed to Mycobacterium bovis. 1648 12


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