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)

Enzymes involved in (methyl)phenol degradation of Pseudomonas putida H are encoded by the catabolic operon (phlA-L) on plasmid pPGH1. Transcription of this operon by the sigma54 (RpoN)-containing RNA polymerase is positively controlled by the gene product of the divergently transcribed phlR in response to the availability of the respective substrate. Additionally, phenol degradation is subject to carbon catabolite repression induced by organic acids (e.g., succinate, lactate, and acetate) or carbohydrates (e.g., glucose and gluconate). Analysis of lacZ fusion to the catabolic promoter and quantified primer extension experiments indicate that carbon catabolite repression also occurs at the transcriptional level of the catabolic operon. In this study, it is furthermore shown that carbon catabolite repression is a negative control. Titration of the postulated negative controlling factor was exclusively observed when extra copies of functional phlR gene were present in the cell. We therefore conclude that PhlR is the target and that carbon catabolite repression of phenol degradation occurs by interfering with the activating function of PhlR.
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PMID:Carbon catabolite repression of phenol degradation in Pseudomonas putida is mediated by the inhibition of the activator protein PhlR. 860 80

The 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate (HOPD) hydrolase-encoding gene (bpdF) in the biphenyl (BP)/polychlorinated biphenyl (PCB)-degrading bacterium, Rhodococcus sp. M5 (M5), was found to be located within a 4.5-kb HindIII-BamHI genomic DNA that was 5.4 kb downstream from the bpdC1C2BADE gene cluster. The deduced amino acid (aa) sequence of bpdF revealed that the hydrolase contains 297 aa (32679 Da) that was verified by expression in the Escherichia coli T7 RNA polymerase/promoter system. Unlike previously known HOPD hydrolases, the aa sequence of BpdF appears unique. Interestingly, all HOPD hydrolases and related proteins from the phenol and toluene/xylene degradation pathways, were found to have a bias in the codon usage in the catalytic Ser within the conserved VGNS(M/F)GG motif.
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PMID:Location and sequence analysis of a 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate hydrolase-encoding gene (bpdF) of the biphenyl/polychlorinated biphenyl degradation pathway in Rhodococcus sp. M5. 867 30

To better understand the mechanisms that regulate stable RNA synthesis, we have analyzed the RNA polymerase I and III transcriptional activities of extracts isolated from cells propagated under a variety of conditions. Under balanced growth conditions the levels of both RNA polymerase I- and III-specific transcription increased proportionally with growth rate. Upon nutritional starvation, RNA polymerase I transcription rapidly declined, followed by 5 S rDNA and eventually tDNA transcription. Transcriptional activities in extracts were restored when the nongrowing cultures were resuspended in fresh medium, although growth did not resume. The differential expression of 5 S rDNA and tDNA genes in extracts prepared from cells subjected to partial starvation was traced to a 5 S rDNA-specific inhibitor and not to a defect in any RNA polymerase III transcription factor. Characterization of this inhibitor indicated that it was not 5 S rRNA. It was sensitive to phenol extraction and resistant to RNase, and its target did not appear to be transcription factor IIIA. Not all treatments that slowed or stopped growth down-regulated the stable RNA transcription apparatus. Cells that have been subjected to either energy starvation or cycloheximide treatment still retain the ability to synthesize stable RNA in vitro, suggesting the presence of alternative regulatory mechanisms.
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PMID:Regulation of the RNA polymerase I and III transcription systems in response to growth conditions. 870 32

Reverse transcriptase PCR (RT-PCR) is an important tool for Mycobacterium tuberculosis research and diagnostics. A standard procedure using N-acetyl-L-cysteine (NALC) and NaOH has been widely adopted for digestion and decontamination of sputum specimens for mycobacterial culture. The objective of this study was to determine the compatibility of this method with the recovery of RNA for RT-PCR assays. Nineteen sputum specimens were collected from smear-positive, pretreatment tuberculosis patients. After homogenization with NALC and glass beads, specimens were further processed by the addition of either NaOH, as per the standard decontamination protocol, or phosphate buffer. RNA was prepared by using a modified guanidine-phenol extraction method developed specifically for sputum sediments. DNA was isolated from the same specimens. Reverse transcriptions of alpha antigen (85B protein) mRNA and 16S rRNA were performed together, and aliquots were removed for separate PCRs. In all specimens, the 85B mRNA target was greatly diminished by treatment with NaOH; however, the 16S rRNA target remained unaffected. Storing sputum specimens for 48 h at 4 degrees C before processing did not seem to affect the integrity or yield of RNA; however, some degradation occurred by 72 h. Data suggest that the NaOH-NALC method for processing sputum samples is not suitable for detecting mRNA targets in RT-PCR assays.
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PMID:Alkaline decontamination of sputum specimens adversely affects stability of mycobacterial mRNA. 888 Apr 95

The soil bacterium Pseudomonas putida is capable of degrading many aromatic compounds, including benzoate, through catechol as an intermediate. The catabolism of catechol is mediated by the catBCA operon, whose induction requires the pathway intermediate cis,cis-muconate as an inducer and the regulatory protein, CatR. CatR also regulates the plasmid-borne pheBA operon of P. putida PaW85, which is involved in phenol catabolism. We have used an in vitro transcription system to study the roles of CatR, cis,cis-muconate, Escherichia coli RNA polymerase, and promoter sequences in expression of the cat and phe operons. The assay confirmed the requirement of both CatR and cis,cis-muconate for transcript formation. We also examined the in vitro transcription of three site-directed mutants of the catBCA promoter; the results obtained compared favorably with previous in vivo data. The requirement of the alpha subunit of RNA polymerase for expression of the catBCA and the pheBA transcripts was also examined. The C-terminal region of the alpha subunit of RNA polymerase has been implicated in direct protein-protein contact with transcriptional regulatory proteins and/or direct contact with the DNA. We show that the carboxyl terminus of the alpha subunit is required for the expression of the catBCA and the pheBA operons because RNA polymerases with truncated alpha subunits were deficient in activation. Further experiments demonstrated the arginine at position 265 and the asparagine at position 268 of the alpha subunit as possible amino acids involved in activation. On the basis of these and previous results, we propose a model to explain the interaction of the different regulatory components leading to CatR-dependent activation of the catBCA operon.
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PMID:Activation of the catBCA promoter: probing the interaction of CatR and RNA polymerase through in vitro transcription. 907 7

A reversible target capture viral RNA extraction procedure was combined with a reverse-transcriptase nested polymerase chain reaction (PCR) to develop a capture PCR assay providing a rapid and safe prediction method for circulating bluetongue virus in infected ruminants. This new assay was compared with virus isolation and a recently developed antigen-capture enzyme-linked immunosorbent assay (ELISA) for the detection of bluetongue virus. Eight Warhill crossbred sheep were inoculated subcutaneously with bluetongue virus serotype 10, and blood samples were taken sequentially over a period of 28 days. The capture PCR detected the peak of viremia, as determined by virus isolation and antigen-capture ELISA, from day 5 to day 14 after challenge. The results indicate that the rapid-capture bluetongue virus PCR provides a rapid indicator of samples in which virus can be isolated. In addition, this capture bluetongue virus PCR procedure does not require a lengthy phenol extraction or the use of the highly toxic methyl mercury hydroxide denaturant.
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PMID:Bluetongue virus detection: a safer reverse-transcriptase polymerase chain reaction for prediction of viremia in sheep. 921 Dec 28

Nonstructural proteins 2C, 3CD, 3C, and 3D, and the cellular protein actin, are present in highly purified preparations of foot-and-mouth disease virus (FMDV) and poliovirus. They remain bound in variable amounts to the RNAs when the RNAs are extracted from the viruses with phenol or phenol-sodium dodecyl sulfate (SDS) and, for FMDV, when the RNA is released from the particles by a lowering of the pH below 7. RNA prepared by these methods is rapidly degraded at 37 degrees C, particularly in the presence of NH4+ ions, but hydrolysis can be prevented by antibody against Escherichia coli-expressed 3D, indicating that it is the RNA polymerase that has nuclease activity. In contrast, virion RNA from which the nonstructural proteins and actin have been removed by extraction with guanidine thiocyanate-phenol-chloroform or proteinase K-phenol is stable at 37 degrees C, although its specific infectivity is lower than that of the RNA extracted with phenol or phenol-SDS. The possible implications of the close association of replication complex proteins with the RNA in virus particles are discussed.
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PMID:Foot-and-mouth disease virus and poliovirus particles contain proteins of the replication complex. 931 48

Infectious bursal disease virus (IBDV), a member of the birnaviridae family, contains a bisegmented double-stranded RNA (dsRNA) genome. The segments are linked covalently at 5' termini by a large (90 kDa) viral genomic protein that migrates similar to viral RNA dependent RNA polymerase of IBDV. A proteinase K digestion based approach and acid-guanidium-phenol-chloroform (AGPC) RNA extraction method were used to extract dsRNA of IBDV from infected bursae. After extraction, dsRNA of IBDV was purified by precipitation with lithium chloride. The yield and purity of dsRNA of IBDV extracted by AGPC method was lower than that of proteinase K digestion based approach. This observation correlates with the presence of a genome-linked protein in IBDV. Although dsRNA obtained by both methods are suitable for reverse transcription-polymerase chain reaction (RT-PCR) amplification of at least up to 1201 base pairs (bp) of cDNA, dsRNA extracted by the proteinase K digestion method is more suitable than that by AGPC method for the amplification of longer fragments (1958 bp) of IBDV cDNA by PCR.
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PMID:A comparison of two RNA isolation methods for double-stranded RNA of infectious bursal disease virus. 977 17

The viral factor responsible for triggering the acute phase response, or 'flu' syndrome, associated with many acute viral infections is not defined. One candidate viral factor is double-stranded RNA (dsRNA) generated during viral replication. In this report we demonstrate by reverse-transcriptase polymerase-chain reaction that nuclease-stable viral RNA was released from influenza-infected MDCK epithelial cells at the time of cell lysis. Removal of virion-associated RNA by ultracentrifugation left equal amounts of positive- and negative-strand viral RNA in the medium that resisted degradation by endogenous RNase in the medium and by exogenous RNase added prior to phenol extraction. These data are the first demonstration that viral RNA with characteristics of dsRNA is spontaneously released from dying influenza virus-infected cells, and thus is available to amplify cytokine induction and contribute to systemic disease.
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PMID:Spontaneous release of stable viral double-stranded RNA into the extracellular medium by influenza virus-infected MDCK epithelial cells: implications for the viral acute phase response. 993 Jan 93

Three Candida albicans strains were tested in the presence of 17-beta-estradiol (10-6 M and 10-9 M) for increased growth and for enhanced survival during incubation at nonpermissive temperatures. All 3 test organisms showed increased growth in the presence of estradiol compared with estrogen-free controls. Likewise, all 3 strains, when treated with estradiol, survived incubation at 48 degrees C better than did controls. Cytoplasmic extracts were probed with an anti-hsp90 antibody, and results suggested that intracellular hsp90 was up-regulated in the presence of 10-9 M 17-beta-estradiol. The results were confirmed by reverse-transcriptase polymerase chain reaction with primers specific for C. albicans hsp90. A kinetic study revealed that peak hsp90 expression occurred within 2 h of exposure to 17-beta-estradiol. In addition, estrogen increased the amount of cdr1 (Candida multidrug resistance) mRNA compared with cells not treated with estrogen. Coumarin and phenol also up-regulated hsp90 and cdr1 mRNAs, indicating that the estrogen-sensing and -response systems in C. albicans may lack specificity.
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PMID:Estrogen effects on Candida albicans: a potential virulence-regulating mechanism. 1076 74


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