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)

We have demonstrated that oligoribonucleotides that lack a 3'-OH group and cannot be extended by RNA polymerase can hybridize to the single-stranded DNA formed inside the transcription initiation bubble (or open complex) and inhibit transcription. Using the lacUV5/Escherichia coli RNA polymerase or trpEDCBA/E. coli RNA polymerase transcription system as a model, we have found that effective inhibitors are five nucleotides in length and must be complementary to the DNA template strand in the region from -5 to +2 about the transcription start site (designated +1). We have used the DNA cleavage activity of 1,10-phenanthroline-copper to confirm that the mechanism of inhibition is via oligoribonucleotide hybridization to the open complex and have used this cleavage chemistry to demonstrate that these oligonucleotide inhibitors hybridize in an antiparallel orientation to their DNA target. Systematic modification of the parent phosphodiester oligoribonucleotide pentamer revealed that the phosphorothioate backbone-containing analogs have increased open complex binding affinity and are more effective transcription inhibitors than their phosphodiester counterparts.
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PMID:Oligoribonucleotide-based gene-specific transcription inhibitors that target the open complex. 1118 Oct 35

The momP1 promoter of the bacteriophage Mu mom operon is an example of a weak promoter. It contains a 19-base pair suboptimal spacer between the -35 (ACCACA) and -10 (TAGAAT) hexamers. Escherichia coli RNA polymerase is unable to bind to momP1 on its own. DNA distortion caused by the presence of a run of six T nucleotides overlapping the 5' end of the -10 element might prevent RNA polymerase from binding to momP1. To investigate the influence of the T(6) run on momP1 expression, defined substitution mutations were introduced by site-directed mutagenesis. In vitro probing experiments with copper phenanthroline ((OP)(2)Cu) and DNase I revealed distinct differences in cleavage patterns among the various mutants; in addition, compared with the wild type, the mutants showed an increase (variable) in momP1 promoter activity in vivo. Promoter strength analyses were in agreement with the ability of these mutants to form open complexes as well as to produce momP1-specific transcripts. No significant role is attributed to the overlapping and divergently organized promoter, momP2, in the expression of momP1 activity, as determined by promoter disruption analysis. These data support the view that an intrinsic DNA distortion in the spacer region of momP1 acts in cis as a negative element in mom operon transcription. This is a novel mechanism of regulation of toxic gene expression.
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PMID:Intrinsic DNA distortion of the bacteriophage Mu momP1 promoter is a negative regulator of its transcription. A novel mode of regulation of toxic gene expression. 1127 87

A number of reagents have been used to define the sequence-specific protein-DNA contacts by footprinting analysis. We report a new in vivo technique using the complex of 1,10-phenanthroline and copper [(OP(2))Cu] as a probe to study various intracellular DNA-protein interactions in whole cells. The versatility of the protocol is demonstrated by applying the technique to address various processes. The protocol is applied to (i) detect structural alterations in DNA as a result of single base substitution, (ii) footprint site-specific DNA-binding proteins, (iii) analyze promoter occupancy by RNA polymerase and (iv) analyze molecular interactions during transcription initiation. The results demonstrate that in vivo (OP)(2)Cu probing is a useful tool in studying important cellular processes involving DNA-protein interactions and has potential applications in post-genomic research.
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PMID:A versatile in vivo footprinting technique using 1,10-phenanthroline-copper complex to study important cellular processes. 1169 42

The genomes of Archaea harbor homologs of the global bacterial regulator leucine-responsive regulatory protein (Lrp). Archaeal Lrp homologs are helix-turn-helix DNA-binding proteins that specifically repress the transcription of their own genes in vitro. Here, we analyze the interaction of Pyrococcus LrpA with components of the archaeal transcriptional machinery at the lrpA promoter. DNA-protein complexes can be isolated by electrophoretic mobility shift assays that contain both LrpA and the two archaeal transcription factors TBP and TFB. Phenanthroline-copper footprinting experiments showed that the DNA-binding sites of LrpA and TBP/TFB do not overlap. These results and the finding that association of RNA polymerase with the TBP-TFB promoter complex was inhibited in the presence of LrpA indicate that Pyrococcus LrpA interferes with RNA polymerase recruitment. A DNA motif required for the LrpA-DNA interaction was inferred from dimethylsulfate methylation interference experiments.
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PMID:A Pyrococcus homolog of the leucine-responsive regulatory protein, LrpA, inhibits transcription by abrogating RNA polymerase recruitment. 1180 82

Most Sinorhizobium meliloti strains lack several key genes involved in microbial biotin biosynthesis, and it is assumed that this may be a special adaptation which allows the microbe to down-regulate metabolic activities in the absence of a host plant. To further explore this hypothesis, we employed two different strategies. (i) Searches of the S. meliloti genome database in combination with the construction of nine different gusA reporter fusions identified three genes involved in a biotin starvation response in this microbe. A gene coding for a protein-methyl carboxyl transferase (pcm) exhibited 13.6-fold-higher transcription under biotin-limiting conditions than cells grown in the presence of 40 nM biotin. Consistent with this observation, biotin-limiting conditions resulted in a significantly decreased survival of pcm mutant cells compared to parental cells or cells grown in the presence of 40 nM biotin. Further studies indicated that the autoinducer synthase gene, sinI, was transcribed at a 4.5-fold-higher level in early stationary phase in biotin-starved cells than in biotin-supplemented cells. Lastly, we observed that open reading frame smc02283, which codes for a putative copper resistance protein (CopC), was 21-fold down-regulated in response to biotin starvation. (ii) In a second approach, proteome analysis identified 10 proteins which were significantly down-regulated under the biotin-limiting conditions. Among the proteins identified by using matrix-assisted laser desorption ionization-time of flight mass spectrometry were the pi subunit of the RNA polymerase and the 50S ribosomal protein L7/L12 (L8) subunit, indicating that biotin-limiting conditions generally affect transcription and translation in S. meliloti.
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PMID:Biotin limitation in Sinorhizobium meliloti strain 1021 alters transcription and translation. 1257 Oct 48

Metalaxyl is a systemic fungicide used to control plant diseases caused by Oomycete fungi. Its formulations include granules, wettable powders, dusts, and emulsifiable concentrates. Application may be by foliar or soil incorporation, surface spraying (broadcast or band), drenching, and seed treatment. Metalaxyl registered products either contain metalaxyl as the sole active ingredient or are combined with other active ingredients (e.g., captan, mancozeb, copper compounds, carboxin). Due to its broad-spectrum activity, metalaxyl is used world-wide on a variety of fruit and vegetable crops. Its effectiveness results from inhibition of uridine incorporation into RNA and specific inhibition of RNA polymerase-1. Metalaxyl has both curative and systemic properties. Its mammalian toxicity is classified as EPA toxicity class III and it is also relatively non-toxic to most nontarget arthropod and vertebrate species. Adequate analytical methods of TLC, GLC, HPLC, MS, and other techniques are available for identification and determination of metalaxyl residues and its metabolites. Available laboratory and field studies indicate that metalaxyl is stable to hydrolysis under normal environmental pH values, It is also photolytically stable in water and soil when exposed to natural sunlight. Its tolerance to a wide range of pH, light, and temperature leads to its continued use in agriculture. Metalaxyl is photodecomposed in UV light, and photoproducts are formed by rearrangement of the N-acyl group to the aromatic ring, demethoxylation, N-deacylation, and elimination of the methoxycarbonyl group from the molecule. Photosensitizers such as humic acid, TiO2, H2O2, acetone, and riboflavin accelerate its photodecomposition. Information is provided on the fate of metalaxyl in plant, soil, water, and animals. Major metabolic routes include hydrolysis of the methyl ester and methyl ether oxidation of the ring-methyl groups. The latter are precursors of conjugates in plants and animals. In soils the most relevant metabolite is the metalaxyl acid, which is formed predominantly by soil microorganisms. Plant uptake, microbial degradation, photodecomposition, and leaching are the major route of metalaxyl dissipation. It has a tendency to migrate to deeper soil horizons with a potential to contaminate groundwater, particularly in soils with low organic matter and clay content. Therefore, precautions should be taken for the continuous application of metalaxyl to crops. If use of metalaxyl is greately increased, the risk of occurrence in groundwater must be reassessed, as by monitoring studies in the most vulnerable areas in main use regions. The R-isomer of metalaxyl (mefenoxam) has recently been registered as the only active compound. Therefore, quantitative studies on the fate of this specific isomer are needed, including appropriate analytical methods. As the use rates of mefenoxam are approximately one-half those recommended for metalaxyl and mefenoxam dissipates more rapidly, concerns for mefenoxam reaching groundwater are even less justified.
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PMID:Metalaxyl: persistence, degradation, metabolism, and analytical methods. 1258 32

Ceramidases deacylate ceramides, important intermediates in the metabolic pathway of sphingolipids. In this study, we report the cloning and characterization of a novel mouse alkaline ceramidase (maCER1) with a highly restricted substrate specificity. maCER1 consists of 287 amino acids, and it has a 28 and 32% identity to the Saccharomyces alkaline ceramidases (YPC1p and YDC1p) and the human alkaline phytoceramidase, respectively. Reverse transcriptase-PCR analysis demonstrated that maCER1 was predominantly expressed in skin. maCER1 was localized to the endoplasmic reticulum as revealed by immunocytochemistry. In vitro biochemical characterization determined that maCER1 hydrolyzed D-erythro-ceramide exclusively but not D-erythro-dihydroceramide or D-ribo-phytoceramide. Similar to other alkaline ceramidases, maCER1 had an alkaline pH optimum of 8.0, and it was activated by Ca2+ but inhibited by Zn2+,Cu2+, and Mn2+. maCER1 was also inhibited by sphingosine, one of its products. Metabolic labeling studies showed that overexpression of maCER1 caused a decrease in the incorporation of radiolabeled dihydrosphingosine into ceramide and complex sphingolipids but led to a concomitant increase in sphingosine-1-P (S1P) in HeLa cells. Mass measurement showed that overexpression of maCER1 selectively lowered the cellular levels of D-erythro-C24:1-ceramide, but not other ceramide species and caused an increase in the levels of S1P. Taken together, these data suggest that maCER1 is a novel alkaline ceramidase with a stringent substrate specificity and that maCER1 is selectively expressed in skin and may have a role in regulating the levels of bioactive lipids ceramide and S1P, as well as complex sphingolipids.
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PMID:Cloning and characterization of a mouse endoplasmic reticulum alkaline ceramidase: an enzyme that preferentially regulates metabolism of very long chain ceramides. 1278 75

Superoxide dismutase (SOD) cDNA, mSOD2, encoding cytosolic copper/zinc SOD (CuZnSOD) cDNA was isolated from suspension-cultured cells of cassava (Manihot esculenta Crantz) by cDNA library screening, and its expression was investigated in relation to environmental stress. mSOD2 is 774 bp in length with an open reading frame (ORF) of 152 amino acids, corresponding to a protein of predicted molecular mass 15 kDa and a pI of 5.22. One copy of the mSOD2 gene was found to be present in the cassava genome by Southern analysis using an mSOD2 cDNA-specific probe. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis revealed diverse expression patterns for the mSOD2 gene in various tissues of intact cassava plants, at various stages of the growth in suspension cultures, and in the leaf tissues exposed to different stresses. The mSOD2 gene was highly expressed in suspension-cultured cells and in the stems of intact plants. However, it was expressed at low levels in leaves and roots. During suspension cell growth, the mSOD2 transcript progressively increased during culture. Moreover, the mSOD2 gene in excised cassava leaves responded to various stresses in different ways. In particular, it was highly induced in leaf tissue by several abiotic stresses, including high temperature (37 degrees C), chilling (4 degrees C), methyl viologen (MV) exposure, and wounding treatment. These results indicate that the mSOD2 gene is involved in the antioxidative process triggered by oxidative stress induced by environmental change.
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PMID:Molecular characterization of a cDNA encoding copper/zinc superoxide dismutase from cultured cells of Manihot esculenta. 1576 66

This paper describes the bioconjugation of histidine-tagged enzymes and other proteins to the surface of composite "magnetomicelles" consisting of magnetic gamma-Fe2O3 nanoparticles encapsulated within cross-linked polystyrene-block-polyacrylate copolymer micelle shells. Free carboxylic acid groups on the magnetomicelle surface were converted to Cu2+-iminodiacetic acid (IDA) for protein capture. The conjugation of T4 DNA ligase and enhanced green fluorescent protein to magnetomicelles revealed that proteins were captured with a high surface density and could be magnetically separated from reaction mixtures and subsequently released from the nanoparticle surface. Additionally, bioconjugation of T7 RNA polymerase yielded a functional enzyme that maintained its biological activity and could be recycled for up to three subsequent transcription reactions. We propose that protein-magnetomicelle bioconjugates are effective for protein bioseparation and enzymatic recycling and further strengthen the idea that nanoparticle surfaces have utility in protein immobilization.
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PMID:Encapsulated magnetic nanoparticles as supports for proteins and recyclable biocatalysts. 1722 72

Dietary fat accelerates the ageing process and causes a greater mortality by accumulating lipid hydroperoxide (LPO) in Drosophila melanogaster. The present study found that the life span of D. melanogaster was shortened from 54 to 6 days in a dose-dependent manner when fat in diet increased from 0% to 25%. The results showed that supplementation of both green tea catechins (GTC) and broccoli extract (BE) reversed partially the fat-induced mortality. The maximum life span was 44 days for the control group fed with a 5% fat, whereas it increased to 50 and 59 days in the GTC- and BE-supplemented groups, respectively. The 50% survival time for the control flies fed with a 5% fat diet was 30 days. In contrast, it increased to 32 and 48 days when GTC and BE were supplemented in the diet. This was consistent with a significant reduction in total body LPO level in D. melanogaster maintained on the GTC- and BE-supplemented diet. Accordingly, catalase and superoxide dismutase (SOD) activities increased significantly in the flies fed with a GTC or a BE diet compared with those fed with a control 5% fat diet. Reverse transcriptase-polymerase chain reaction analysis indicated that the increase in enzymatic activities of catalase and SOD was accompanied by up-regulation of genes for catalase, copper-zinc containing SOD and manganese-containing SOD. It was concluded that GTC and BE reversed the fat-induced mortality in D. melanogaster, most likely but necessarily solely, by up-regulation of endogenous antioxidant enzymes.
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PMID:Green tea catechins and broccoli reduce fat-induced mortality in Drosophila melanogaster. 1770 29


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