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)

A sensitive colorimetric bacterial system was developed for the detection of Hg(II) and organomercury compounds. The bioactive species, a recombinant Escherichia coli, produces proportionally elevated levels of the enzyme beta-galactosidase with increasing amounts of Hg. This is due to a reporter plasmid which carriers a Hg(II)-inducible promoter (mer promoter) from the Hg resistance transposon Tn501 regulating the transcription of a promoterless lacZ gene. Additionally, a pMB1 origin of replication without the natural RNA polymerase start site is fused downstream of the mer promoter leading to a Hg(II)-inducible plasmid replication, which results in an improved signal-to-noise ratio. To enhance the sensitivity of this cellular biosensor, the transport proteins for Hg(II) uptake are constitutively produced by a helper plasmid. To enable the detection of organically bound Hg, the Streptomyces lividans organomercurical lyase, an enzyme which catalyses the cleavage of C-Hg-bonds of organomercurial compounds, is also provided by the helper plasmid. Hg(II) and phenylmercuric acetate (PMA) concentrations as low as 5 x 10(-10) M (0.1 ppb) may be detected within a few minutes.
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PMID:Genetically modified Escherichia coli for colorimetric detection of inorganic and organic Hg compounds. 900 11

The specific interaction of the upstream element-containing promoter of the Escherichia coli acetate operon with either the RNA polymerase holoenzyme or its alpha subunit has been analyzed by the base removal method. Our results indicate that: (i) direct and specific base contacts can be detected in the acetate promoter-alpha subunit complex; (ii) base elimination in the upstream element of the acetate promoter enhances the binding of RNA polymerase. A similar effect is observed when studying the interactions between RNA polymerase and the rrnB ribosomal operon P1 promoter.
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PMID:DNA flexibility of the UP element is a major determinant for transcriptional activation at the Escherichia coli acetate promoter. 901 19

The alpha subunits of the laminin-binding integrins alpha 3 beta 1, alpha 6 beta 1, and alpha 7 beta 1 have homologous sequences and are similar in structure. Two cytoplasmic variants, A and B, have been identified for each of these alpha subunits, although the alpha 3B splice variant has been detected only at the mRNA level. We prepared a panel of mouse monoclonal antibodies specific for the A and B variants of the alpha 3 subunit to study their tissue distribution. Four monoclonal antibodies react with alpha 3A, one of which recognizes only the nonphosphorylated form; of the three anti-alpha 3B antibodies, one cross-reacts with alpha 6B. Reverse transcriptase-PCR analysis of various human tissues revealed the presence of alpha 3B mRNA in brain, heart, and skeletal muscle. Moreover, the alpha 3B protein was detected by immunoblotting in brain and heart tissue but not in skeletal muscle. In contrast, alpha 3A mRNA and protein were present in all tissues studied. Thus, the expression of alpha 3B in adult tissues is more restricted than that of alpha 3A. Immunohistochemical studies showed that in brain tissue, both variants are exclusively expressed on small blood-vessel endothelium, whereas in heart tissue their distribution patterns differ markedly. Although alpha 3A is strongly expressed on vascular smooth muscle cells, alpha 3B is detected only on endothelial cells of veins. Expression of the two variant forms of alpha 3 in K562 cells revealed that the ligand-binding specificities of alpha 3A beta 1 and alpha 3B beta 1 are identical: both bind human laminin-2 and -4, laminin-5, and laminins isolated from bovine kidney, but not bovine laminin-2 and -4, mouse laminin-1, or human fibronectin. In addition, adhesion mediated by both integrins is induced to the same extent by phorbol 12-myristate 13-acetate. The alpha 3A, but not the alpha 3B subunit, is phosphorylated; and phosphorylation of alpha 3A increases after phorbol 12-myristate 13-acetate stimulation. Thus, we found no differences between the adhesion functions of the A and B variants of alpha 3.
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PMID:The A and B variants of the alpha 3 integrin subunit: tissue distribution and functional characterization. 911 16

The immortalized human chondrocyte cell line C-20/A4 has the ability to produce superoxide constitutively at low levels of 5.4 x 10(-2) nmol/min/10(6) cells (S.E.M. = +/-0.5, n = 30) and at raised levels upon stimulation with ionomycin and phorbol 12-myristate 13-acetate. Priming and anti-priming effects of interleukin (IL)-1 beta and IL-4, respectively, are also demonstrated. Reverse transcriptase polymerase chain reaction (RT-PCR) amplification using oligonucleotide primers to components of the NADPH oxidase enzyme complex showed mRNA expression of p22-phox, p40-phox and p47-phox. Western blot analysis using polyclonal antisera indicated the presence of the p47-phox p67-phox polypeptide components. These results show that the C-20/A4 cells contain an NADPH oxidase-like complex, similar to that found in other cell types, which produces superoxide anions.
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PMID:Detection of protein and mRNA of various components of the NADPH oxidase complex in an immortalized human chondrocyte line. 918 52

The rpoF gene of Escherichia coli codes for the RNA polymerase sigmaF (or sigma28) subunit, which is involved in transcription of the flagellar and chemotaxis genes. Both sigmaF and sigma70 (the major sigma subunit in growing cells) were overexpressed, purified to homogeneity, and compared with respect to activity and specificity. The affinity of sigmaF to core RNA polymerase (E) is higher than that of sigma70, as measured by gel filtration high-pressure liquid chromatography. In an in vitro transcription system, the holoenzyme (E sigmaF) containing sigmaF selectively transcribed the flagellar and chemotaxis genes, all of which could not be transcribed by E sigma70. This strict promoter recognition property of sigmaF is similar to those of other stress response minor sigma subunits but different from those of the principal sigma subunits, sigma70 and sigma38. sigma70-dependent transcription in vitro is inhibited at high concentrations of all salts tested, showing maximum activity at 50 mM. In contrast, sigmaF-dependent transcription was maximum at 50 mM KCI and then decreased to negligible level at 300 mM; in the cases of potassium acetate and potassium glutamate, maximum transcription was between 200 and 300 mM. DNase I foot printing of the fliC and fliD promoters indicated that sigmaF alone is unable to bind DNA, but E sigmaF specifically recognizes -10 and -35 regions of the sigmaF-dependent promoters with rather long upstream protection. Alteration of the promoter structure after binding of E sigmaF was suggested.
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PMID:Promoter selectivity of Escherichia coli RNA polymerase sigmaF holoenzyme involved in transcription of flagellar and chemotaxis genes. 920 42

The effect of potassium glutamate was examined on the DNA-directed in vitro protein synthesizing system of Salmonella typhimurium which conventionally contained acetate as a sole counter anion. The glutamate replacement increased the potassium optimum by about 70% and improved the expression of different DNA templates, but selectively. The biggest improvements in expression (about 8-fold) were seen with a lacUV5 (from Escherichia coli) template and with a mutant promoter his operon (from S. typhimurium) template. In contrast, the expression of a leuV promoter (from Escherichia coli) template was relatively unaffected by the glutamate replacement. The chain-growth-rate of mRNA and polypeptide syntheses in the DNA-directed in vitro protein synthesizing system were unaffected by the glutamate replacement. It was concluded that at least a part of the effect of glutamate replacement is on RNA polymerase-promoter interaction, and most likely the association step. Glutamate replacement did not alter the ppGpp-mediated positive and negative regulation of the his and leuV promoter, respectively, in the in vitro system. Taken together, the results suggest that the use of potassium glutamate in place of potassium acetate in DNA-directed in vitro synthesis provides a physiologically more relevant approximation of the ionic environment in vivo.
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PMID:The DNA-directed in vitro protein synthesizing system of Salmonella typhimurium: the effect of glutamate substitution. 925 65

Porcine articular chondrocytes have the capacity to release superoxide in response to the addition of the calcium ionophore ionomycin in a concentration-dependent manner. This activity was not stimulated by the addition of fMetLeuPhe or the kinase activator phorbol myristate acetate (PMA). However, this release of superoxide was inhibited by iodonium diphenyl (IDP), suggesting the involvement of NADPH oxidase. Reverse transcriptase polymerase chain reaction (RT-PCR) using oligonucleotides designed against the known sequences for the human phagocyte NADPH oxidase showed the expression of p22-phox, p40-phox, and p47-phox mRNA, while Western blot analysis of chondrocyte extracts using polyclonal antisera raised against the human phagocyte NADPH oxidase suggested the presence of the p67-phox polypeptide. These results suggest that porcine articular chondrocytes can release reactive oxygen species using a NADPH oxidase-like complex.
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PMID:Detection of superoxide and NADPH oxidase in porcine articular chondrocytes. 929 50

Brain-derived neurotrophic factor (BDNF) has potent trophic and protective actions on CNS neurons, including mesencephalic dopaminergic neurons, ventral forebrain cholinergic neurons and spinal motor neurons. To evaluate the effects of calcium and other second messengers on BDNF gene transcription, C6 glioma cells were treated for 4 h with the calcium ionophore A23187, forskolin + isobutyl-methyl-xanthine (IBMX), or the phorbol ester, 12-O-tetradecanoyl-phorbol-13-acetate. Semi-quantitative RT-PCR analysis revealed that A23187 treatment increased BDNF transcripts containing the protein coding exon by 4.4-6.4-fold. Alternate BDNF transcripts were elevated to varying degrees after treatment with this ionophore and a subset of these transcripts was elevated following forskolin + IBMX treatment. When co-incubated with the RNA polymerase inhibitor, actinomycin D, A23187-induced increases were reduced or abolished, suggesting that calcium-mediated regulation of BDNF mRNA expression occurs at transcriptional as well as post-transcriptional levels. Transient transfection experiments employing reporter constructs containing serial 5' deletions of alternate BDNF promoters suggested that A23187-induced elevations in BDNF exon 1b, 1d and 1e containing transcripts are mediated by putative calcium-responsive regions flanking all three of these exons.
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PMID:Localization of putative calcium-responsive regions in the rat BDNF gene. 940 30

A monomercury derivative of fluoresceine acetate (FMMA) was previously suggested as a specific reagent reacting with only one of four cysteine (Cys) residues in the alpha. subunit of Escherichia coli RNA polymerase. Here, we analyzed the reactivity against FMMA of both isolated alpha subunit and alpha subunit assembled in the holoenzyme. In both cases, the highest reactivity was identified for Cys-269 positioned in the regulatory helix of C-terminal domain (CTD) which includes the contact sites for both class-I transcription factors and DNA UP elements. Substitution of Ala for both Cys-269 and Cys-176 completely eliminates the reactivity of alpha subunit against the fluorescent dye, supporting the prediction that another reactive amino acid under native conformation is Cys-176, which is positioned within or near the region important for alpha dimerization and its binding of beta' subunit. In the isolated alpha subunit, the reactivity against FMMA is different between these two Cys residues and the order is from Cys-269 to Cys-176. Mutant alpha-subunits, bearing only one Cys residue at either 269 or 176, could be reconstituted into locally modified and active enzymes. This FMMA modification system may provide a tool suitable for studies of intra- and intermolecular interactions of this subunit.
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PMID:Specific fluorescent labeling of two functional domains in RNA polymerase alpha subunit. 948 26

In response to dibutyryl cyclic AMP (dbcAMP) and all-trans retinoic acid (ATRA), HL60 cells differentiate into granulocyte-like cells. Membrane-associated phospholipase D (PLD) activity in response to guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) or phorbol myristate acetate (PMA) was upregulated by these treatments. Reverse transcriptase-polymerase chain reaction (RT-PCR) analyses revealed that both hPLD1a and hPLD1b mRNAs were expressed in HL60 cells and that their expression levels increased during differentiation. hPLD2 mRNA levels rose dramatically during differentiation. These results suggest that the PLD genes undergo changes in transcriptional regulation during granulocytic differentiation of HL60 cells.
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PMID:Increased mRNA expression of phospholipase D (PLD) isozymes during granulocytic differentiation of HL60 cells. 951 45


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