Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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 rat liver nuclear envelope fraction isolated essentially by the technique of Monneron et al. (J. Cell Biol. 55, 104-125 (1972) is characterized by high levels of glucose-6-phosphatase and 5'-nucleotidase. A broadly specific nucleoside triphosphatase activity is present. Cytochromes b5 and P-450 as well as NADPH- and NADH-cytochrome c reductase activities are present but at lower levels than found in microsomes. Cytochrome c oxidase activity is low. RNA polymerase activity is absent from the nuclear envelope fraction. Cytochemistry shows that glucose-6-phosphatase activity is strong and restricted to the nuclear envelope of nuclei. 5'-Nucleotidase shows weak reaction deposit in whole nuclei but in contrast gives clear reaction deposit in isolated nuclear envelopes. Cytochemical reaction deposit due to nucleoside triphosphatase activity is not restricted to the nuclear envelope but is found to a larger extent within the nucleus.
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PMID:An enzymic analysis of a nuclear envelope fraction. 18 34

From a genomic library of Chromatium vinosum strain D in lambda L47, a 16.5-kbp EcoRI-restriction fragment was identified by hybridization with a DNA fragment harboring the operon for Alcaligenes eutrophus poly(3-hydroxyalkanoate) (PHA) synthesis. This fragment and subfragments thereof restored the ability to synthesize and accumulate PHA in PHA-negative mutants of A. eutrophus. A region of 6977 bp was sequenced; seven open reading frames (ORFs) were identified which probably represent coding regions; six of these are most probably relevant for PHA biosynthesis in C. vinosum. The structural genes for biosynthetic acetyl-CoA acyltransferase (beta-ketothiolase; phbACv, 1188 bp) and NADH-dependent acetoacetyl-CoA reductase (phbBCv, 741 bp) were separated by ORF4 (462 bp) and ORF5 (369 bp). Downstream of phbBCv ORF7 (471 pb) was identified which was not completed at the 3' terminus. The functions of ORF4, ORF5, and ORF7 are not known. The amino acid sequences of beta-ketothiolase and acetoacetyl-CoA reductase deduced from phbACv and phbBCv, exhibited a similarity of 68.2% and 56.4% identical amino acids, respectively, to the corresponding enzymes of A. eutrophus. Antilinear to and upstream of the genes mentioned above, two genes were identified which were transcribed from a sigma 70-dependent promoter. This promoter overlapped with and was divergent to the phbACv promoter; the transcriptional start sites were mapped by S1 nuclease protection assays. These genes were ORF2 (1074 bp), whose function is not known but whose presence in Escherichia coli is essential for expression of PHA synthase activity, and the structural gene for a PHA synthase of low M(r) (phbCCv, 1068 bp). The gene products of ORF2 and phbCCv, with M(r) of 40,525 and 39,730, respectively, were expressed in E. coli applying the T7 RNA polymerase/promoter system. Although the amino acid sequence of PHA synthase deduced from phbCCv exhibited only 24.7% overall similarity with the PHA synthase of A. eutrophus, highly conserved regions were identified.
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PMID:Cloning and nucleotide sequences of genes relevant for biosynthesis of poly(3-hydroxybutyric acid) in Chromatium vinosum strain D. 139 92

Methylococcus capsulatus (Bath) uses a soluble methane monooxygenase (sMMO) to catalyse the oxidation of methane to methanol. sMMO is comprised of three components; A, B and C. Protein C (the reductase) transfers electrons from NADH to protein A (the hydroxylase) which contains the active site, and protein B regulates this electron flow. The five genes encoding the sMMO proteins and their subunits are clustered and have been cloned in Escherichia coli. A DNA fragment containing mmoB, the gene encoding protein B, was subcloned into pT7-5, a plasmid of the T7 RNA polymerase promoter expression system. Upon induction, E. coli expressed protein B which was fully functional after purification. The gene encoding protein C, mmoC, was amplified with unique restriction sites at each end using the polymerase chain reaction and then subcloned into pT7-7 (a plasmid similar to pT7-5 but containing its own ribosome-binding site and ATG start codon). Protein C expressed in E. coli was also found to be functional. This is the first report of the functional expression of methanotroph methane monooxygenase genes in a heterologous host and represents a significant step forward in our analysis of the assembly and catalysis of sMMO.
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PMID:Functional expression in Escherichia coli of proteins B and C from soluble methane monooxygenase of Methylococcus capsulatus (Bath). 151 60

Monodehydroascorbate radical (MDA) reductase, an FAD-enzyme, is the first enzyme to be identified whose substrate is an organic radical and catalyzes the reduction of MDA to ascorbate by NAD(P)H. Its cDNA has been cloned from cucumber seedlings (Sano, S., and Asada, K. (1994) Plant Cell Physiol. 35, 425-437), and a plasmid was constructed in the present study that allowed a high level expression in Escherichia coli of the cDNA-encoding MDA reductase using the T7 RNA polymerase expression system. The recombinant MDA reductase was purified to a crystalline state, with a yield of over 20 mg/liter of culture, and it exhibited spectroscopic properties of the FAD similar to those of the enzyme purified from cucumber fruits during redox reactions with NADH and MDA. The red semiquinone of the FAD of MDA reductase was generated by photoreduction. p-Chloromercuribenzoate inhibited the reduction of the enzyme-FAD by NADH, and dicumarol suppressed electron transfer from the reduced enzyme to MDA. The specificity of electron acceptors of the recombinant enzyme appeared to be similar to that of MDA reductase, even though the amino acid sequence encoded by the cDNA was somewhat different from that of the enzyme purified from cucumber fruits. The Km values for NADH and NADPH of the recombinant enzyme indicated a high affinity of the enzyme for NADH. The reaction catalyzed by the enzyme did not exhibit saturation kinetics with MDA up to 3 microM. A second order rate constant for the reduction of the enzyme-FAD with NADH was 1.25 x 10(8) M-1 s-1, as determined by a stopped-flow method, and its value decreased with increases in ionic strength, an indication of the enhanced electrostatic guidance of NADH to the enzyme-FAD.
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PMID:Molecular characterization of monodehydroascorbate radical reductase from cucumber highly expressed in Escherichia coli. 754 69

The activity of the proline catabolic enzyme pyrroline-5-carboxylate dehydrogenase (EC 1.5.1.12) was induced up to three-hundred-fold by the addition of three hundred proline to the growth medium of the Gram-positive bacterium Streptomyces coelicolor A3(2). Rifampicin, an inhibitor of RNA polymerase activity, abolished induction, implying that regulation was at the level of activation of gene transcription. The enzyme was purified and SDS-PAGE of the highly purified enzyme preparation revealed a single subunit with M(r) 68,000. A single band of protein, which also stained for enzyme activity, was observed after native gel electrophoresis. The M(r) of the enzyme was estimated to be approximately 265,000 by native gel electrophoresis and approximately 305,000 by gel filtration, which indicated that the enzyme had a tetrameric quaternary structure. The apparent Km for pyrroline-5-carboxylate was 109 +/- 7.3 microM, whilst that for NAD+ was 43.3 +/- 2.5 microM. Product inhibition by NADH (apparent Ki 0.6mM) was observed. The observed Vmax was 22.0 +/- 1 mol min-1 (mg protein)-1. Neither 1 nor 5 mM proline had any effect on enzyme activity, whilst glutamate was a very weak inhibitor.
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PMID:Interaction between primary and secondary metabolism in Streptomyces coelicolor A3(2): role of pyrroline-5-carboxylate dehydrogenase. 755 Oct 40

1,3-Propanediol dehydrogenase (EC 1.1.1.202) was purified to homogeneity from Citrobacter freundii grown anaerobically on glycerol in continuous culture. The enzyme is an octamer of a polypeptide of 43,400 Da. When tested as a dehydrogenase, the enzyme was most active with substrates containing two primary alcohol groups separated by one or two carbon atoms. In the physiological direction, 3-hydroxypropionaldehyde was the preferred substrate. The apparent Km values of the enzyme for 3-hydroxypropionaldehyde and NADH were 140 and 33 microM, respectively. The enzyme was inhibited by chelators of divalent cations but could be reactivated by the addition of Fe2+. The dhaT gene, encoding the 1,3-propanediol dehydrogenase, was cloned, and its nucleotide sequence (1,164 bp) was determined. The deduced dhaT gene product (387 amino acids, 41,324 Da) showed a high level of similarity to a novel family (type III) of alcohol dehydrogenases. The dhaT gene was overexpressed in Escherichia coli 274-fold by using the T7 RNA polymerase/promoter system.
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PMID:Purification of 1,3-propanediol dehydrogenase from Citrobacter freundii and cloning, sequencing, and overexpression of the corresponding gene in Escherichia coli. 772 5

FNR is a transcriptional regulator that controls gene expression in response to oxygen limitation in Escherichia coli. The NADH dehydrogenase II gene (ndh) is repressed by FNR under anaerobic conditions. Repression is not simply due to occlusion of the promoter (-35 and -10) region by FNR because adjacent pairs of FNR monomers were found to bind at two sites centred at -50.5 and -94.5 in the ndh promoter region without preventing RNA polymerase binding. However, contact between RNA polymerase and the -132 to -62 region of the non-coding strand of ndh DNA, and RNA polymerase-mediated open complex formation, were prevented by bound FNR. The upstream FNR-binding site (-94.5) was needed for efficient FNR-dependent repression of ndh transcription in vitro, and also for repression of an ndh-lacZ fusion in vivo. Anaerobic ndh repression may thus involve the binding of two pairs of FNR monomers upstream of the -35 region, which prevents essential RNA polymerase-DNA contacts in the upstream region as well as inhibiting RNA polymerase function by direct FNR interaction. Expression of the ndh-lacZ fusion in an fnr deletion strain was enhanced by anaerobic growth in rich medium or minimal medium supplemented with amino acids. Furthermore, two proteins (M(r) 12,000 and 35,000) which interact with and may activate transcription from the ndh promoter under these conditions were detected by gel retardation analysis. These putative amino acid-responsive activators may thus offset FNR-mediated repression and maintain a low level of anaerobic ndh expression for regulating the NAD+/NADH ratio during growth in rich media.
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PMID:Regulation of transcription at the ndh promoter of Escherichia coli by FNR and novel factors. 806 61

In order to elucidate the components of the oxygen sensory complex in HepG2 cells which regulates the production of erythropoietin, we have microinjected recombinant variants of the human small GTP-binding protein hRac1 and measured their effects on the production of reactive oxygen species (ROS) by the dihydrorhodamine-123 technique. The dominant-negative mutant hRac1(T17N) inhibits the NADH-stimulated production of ROS in HepG2 cells, whereas the constitutively activated hRac1(G12V) leads to an increase in intracellular ROS concentration. Reverse transcriptase PCR analysis showed that the hRac1, but not the hRac2, gene is expressed in HepG2 cells. These results demonstrate that hRac1, and not hRac2, is involved in the regulation of ROS production in HepG2 cells and suggest that hRac1 specifically functions in the non-phagocytic NAD(P)H oxidase complex.
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PMID:Rac1, and not Rac2, is involved in the regulation of the intracellular hydrogen peroxide level in HepG2 cells. 957 45

Mitochondrial adenosine triphosphate (ATP) generation plays a major role in insulin secretion in pancreatic islet beta cells. The relationship between age and nutritional status of the islet and mitochondrial gene messenger RNA (mRNA) expression was investigated. Three animal groups were studied: infant (12-day-old) rats fed either mother's milk or a high carbohydrate (HC) diet; young (2 to 4-month-old) rats; and old (12 to 14-month-old) rats. The expression of mitochondrial cytochrome oxidase (CYO) (subunits I, II, and III), beta-nicotinamide adenine dinucleotide, reduced form dehydrogenase subunit 4 (NADH-DH4), and ATP synthase (subunit 6) (ATP-SYN6) mRNAs was characterized by semiquantitative reverse-transcriptase polymerase chain reaction (RT-PCR). The mitochondrial gene mRNAs were identified in each of the groups of rat islets and in RINm5F cells. CYO-II mRNA expression in young and old rat pancreatic islets was 12.7- and 8.2-fold higher, respectively, compared with the level in infant rat islets. The expression of NADH-DH4 and ATP-SYN6 mRNAs was 47% and 40% lower, respectively, in young rat islets compared with the level in infant rat islets. CYO-I, CYO-III, and cytoplasmic glyceraldehyde-3-phosphate dehydrogenase (GPDH) mRNA expression did not differ between experimental groups. Artificial rearing of infant rat pups on a HC diet for 8 days lead to a 3.3-fold increase in islet CYO-II mRNA expression compared with mother-fed pups. However, glucose (11 mmol/L) stimulation of cultured isolated islets from young and old rats for 4 days failed to affect the expression level of mitochondrial gene mRNAs. Thus, aging affected the differential expression of CYO-II, NADH-DH4, and ATP-SYN6 mRNAs in rat islets. CYO-II mRNA expression was modulated only in infant rat islets after in vivo administration of carbohydrate.
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PMID:Mitochondrial-encoded gene regulation in rat pancreatic islets. 1122 30

Whether red algae are related to green plants has been debated for over a century. Features present due to their shared photosynthetic habit have been interpreted as support for an evolutionary sisterhood of the two groups but, until very recently, characters endogenous to the host cell have provided no reliable indication of such a relationship. In this investigation, we examine three molecular data sets that have provided key evidence of a possible relationship between green plants and red algae. Analyses of an expanded alignment of DNA-dependent RNA polymerase II largest subunit sequences indicate that their support for independent origins of rhodophytes and chlorophytes is not the result of long-branch attraction, as has been proposed elsewhere. Differences in the pol II C-terminal domain, an essential component of plant mRNA transcription, also suggest different host cell ancestors for the two groups. In contrast, concatenated sequences of two groups of mitochondrial genes, those encoding subunits of NADH-dehydrogenase as well as cytochrome c oxidase subunits plus apocytochrome B, appear to cluster red algal and green plant sequences together because both groups have evolved relatively slowly and share a super-abundance of ancestral positions. Finally, analyses of elongation factor 2 sequences demonstrate a strong phylogenetic signal favoring a rhodophyte/chlorophyte sister relationship, but that signal is restricted to a contiguous segment comprising approximately half of the EF2 gene. These results argue for great caution in the interpretation of phylogenetic analyses of ancient evolutionary events but, in combination, indicate that there is no emerging consensus from molecular data supporting a sister relationship between red algae and green plants.
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PMID:Are red algae plants? A critical evaluation of three key molecular data sets. 1144 56


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