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)

Gene expression in Legionella pneumophila, the etiological agent of Legionnaires' disease, can be controlled by alternative forms of RNA polymerase programmed by distinct sigma factors. To understand the regulation of L. pneumophila flagellin expression, we cloned the sigma factor (FliA) of RNA polymerase responsible for the transcription of the flagellin gene, flaA. FliA is a member of the sigma28 class of alternative sigma factors identified in several bacterial genera. The gene fliA has been isolated from an expression library of L. pneumophila isolate Corby in Escherichia coli K-12. This library was transformed into a fliA mutant of E. coli K-12 containing a plasmid carrying the L. pneumophila-specific flaA promoter fused to the reporter gene luxAB. Screening the obtained transformants for luciferase activity, we isolated the major part of the fliA gene on a 1.64-kb fragment. This fragment was sequenced and used for reverse PCR in order to recover the complete fliA gene. The resulting 1.03-kb fragment was shown to contain the entire fliA gene. L. pneumophila FliA has 55 and 43% amino acid identity with the homologous sequences of Pseudomonas aeruginosa and E. coli. Furthermore, the L. pneumophila fliA gene was able to restore the flagellation and the motility defect of an E. coli fliA mutant. This result suggests that the L. pneumophila sigma28 protein can bind to the E. coli core RNA polymerase to direct transcription initiation from the flaA-specific promoter.
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PMID:The alternative sigma factor sigma28 of Legionella pneumophila restores flagellation and motility to an Escherichia coli fliA mutant. 898 75

An oxidant stress has been shown to prevail in experimental and clinical nephrotic syndrome. Such oxidant stress may be induced by a reduced activity of antioxidant systems. We examined the altered expression of manganese-superoxide dismutase (Mn-SOD), an antioxidant enzyme, in patients with idiopathic nephrotic syndrome, in whom an increased oxidant stress had been demonstrated. The Mn-SOD activities in peripheral blood mononuclear cells obtained from 12 patients with active nephrotic syndrome (6.0 +/- 1.1 years of age, mean +/- SE) and hypoproteinemia were 42% lower (p < 0.05) than in 12 control subjects (5.5 +/- 0.5 years of age) with normal serum total protein concentrations. Reverse-transcriptase polymerase chain reaction also demonstrated that Mn-SOD messenger RNA expression in the patients with nephrotic syndrome was, on average, 59% lower than in control subjects. Because expressions of some genes are sensitive to serum, the serum dependency of Mn-SOD gene transcription was studied in glomerular endothelial cells transfected with a luciferase reporter gene fused with a rat Mn-SOD DNA fragment of -806 to +22 bp of the transcription initiation site (-806:+22). When these cells were exposed to different concentrations of fetal bovine serum (0.5% to 15%), the transcriptional activities determined by luciferase activities were proportional to serum concentrations. This serum-dependent transcriptional activation was also demonstrated by the fragment (-220:+22) but not by the fragment (-220:-20). When glomerular endothelial cells transfected with the fragment (-220:+22) were treated with 5% serum from patients with active nephrotic syndrome, transcriptional activation was more than 80% less than that by 5% serum from control subjects without nephrosis. These results indicate that Mn-SOD gene transcription is regulated at least in part by serum, and that the serum-dependent transcription of the gene is diminished in patients with idiopathic nephrotic syndrome. The regulatory region of serum-dependent gene transcription resides within its early promoter region. Our findings suggest that down-regulation of antioxidant enzyme transcription may contribute increased oxidant stress in idiopathic nephrotic syndrome.
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PMID:Down-regulation of manganese-superoxide dismutase gene expression in idiopathic nephrotic syndrome. 915 91

We established a hybrid baculovirus-T7 RNA polymerase system for transient expression in mammalian cells. Two recombinant baculoviruses carrying cDNA of bacteriophage T7 RNA polymerase, with or without a nuclear localization signal, under the control of a mammalian promoter were constructed. High level expression of T7 RNA polymerase was observed in various mammalian cell lines after infection with the recombinant baculoviruses. After transfection of plasmids containing the luciferase gene under the control of the T7 promoter, high luciferase activity was detected in cells infected with the recombinant baculoviruses. We also constructed a plasmid containing an entire cDNA clone of type 1 poliovirus under the T7 promoter. Two days after transfection of the plasmid into the cells infected with the recombinant baculoviruses, a high titer of poliovirus was recovered. The use of the recombinant baculoviruses did not cause any cytopathic effects even at a high multiplicity of infection. The lack of replication ability and low toxicity are the advantageous features of the hybrid baculovirus-T7 polymerase system in comparison with the widely used vaccinia-T7 polymerase system for gene expression and recovery of infectious viruses from its cDNA.
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PMID:A hybrid baculovirus-T7 RNA polymerase system for recovery of an infectious virus from cDNA. 916 81

Development of methodologies for gene transfer into the central nervous system (CNS) is important for fundamental research as well as clinical studies for gene therapy. Cationic liposomes (CL) are attractive vectors because of their safety and ease of use. However, to date only low rates of success have been reported. We succeeded in obtaining high transfection efficiencies into the newborn mouse brain in vivo by CL and a cytoplasmic gene expression system based on T7 RNA polymerase and T7 RNA polymerase- and the luciferase-gene with the T7 promoter sequence. This system showed an efficiency rate 2 orders of magnitude higher than the standard system, which used CL and luciferase genes with a Rous sarcoma virus promoter, pRSVL. In addition, in vitro experiments using LLCMK2 cells showed that cytoplasmic gene expression occurred rapidly (within 6 h) after transfection. In contrast, pRSVL required 24-48 h for induction of luciferase expression. Our results suggest that the cytoplasmic gene expression system is useful for gene delivery into the CNS.
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PMID:Cytoplasmic gene expression system enhances the efficiency of cationic liposome-mediated in vivo gene transfer into mouse brain. 916 51

The growth-promoting activity of GH, the principal hormonal determinant of body size, is mediated by insulin-like growth factor I (IGF-I). Most of the IGF-I in plasma circulates in a 150-kDa complex that contains IGF-binding protein-3 (IGFBP-3) and an acid-labile subunit (ALS). The 150-kDa complex serves as a reservoir of IGF-I and determines its bioavailability to the tissues. Formation of the 150-kDa complex depends upon the synthesis of ALS, which is synthesized primarily in liver and is regulated by GH. The present study demonstrates that GH stimulates ALS gene transcription in rat liver and ALS promoter activity in a rat hepatoma cell line. ALS messenger RNA (mRNA) and ALS nuclear transcripts were decreased to similar extents in the livers of GH-deficient hypophysectomized rats. GH increased hepatic ALS mRNA within 3-4 h to about 65% of the levels seen in sham-operated control rats. To confirm that GH stimulated ALS gene transcription, we transiently transfected an ALS promoter-luciferase reporter gene construct into H4-II-E rat hepatoma cells and primary rat hepatocytes. Recombinant human GH (hGH) stimulated promoter activity about 3-fold. In contrast, basal promoter activity was lower, and GH stimulation was absent when the ALS reporter construct was transfected into GH-responsive 3T3-F442A mouse preadipocyte fibroblasts. GH stimulation of ALS promoter activity in H4-II-E cells was mediated by functional GH receptors; nonprimate (rat and bovine) GH gave identical stimulation to hGH, and stimulation by hGH occurred at physiological concentrations. Reverse transcriptase-PCR analysis indicated that GH receptor mRNA was present in H4-II-E cells at approximately 40% of the level seen in rat liver. GH also induced the expression of the endogenous c-fos gene, indicating that the signaling pathway necessary for the activation of gene expression by GH was intact in H4-II-E cells. Thus, H4-II-E cells are a GH-responsive liver cell line that should provide a useful system in which to study the molecular mechanism of transcriptional regulation by GH of ALS and other hepatic genes.
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PMID:Growth hormone stimulates transcription of the gene encoding the acid-labile subunit (ALS) of the circulating insulin-like growth factor-binding protein complex and ALS promoter activity in rat liver. 917 59

GnRH receptors belong to the family of G protein-coupled receptor proteins and have been localized to the anterior pituitary, brain and reproductive organs as well as many steroid-dependent tumor tissues. Recently, cDNAs for the GnRH receptors of several species including the human have been cloned. To determine the structure of the gene encoding the human GnRH receptor, we isolated the receptor gene clones from the human genomic libraries. Comparison of the genomic and cDNA sequences revealed that the human GnRH receptor gene is composed of three exons and two introns and spans over 20 kb in size. Exon 1 encodes the 5' untranslated sequence and nucleotide +1 to +522 in the open reading frame, exon 2 encodes nucleotide +523 to +742 and exon 3 encodes nucleotide +743 to +987 in the open reading frame as well as the 3' untranslated sequence. Southern blot analysis of genomic DNA and localization of the GnRH receptor gene to a single site on human chromosome 4 (4q13) indicate the presence of a single copy of the gene in the human genome. Several regulatory sequences for various hormones and other regulatory factors were identified, including PEA-3, AP-1, AP-2, and Pit-1 sites. In addition, glucocorticoid/progesterone response element thyroid hormone response element, and cAMP response element sequences were identified. Reverse transcriptase-primer extension and 5' RACE analysis of the human pituitary RNA demonstrated the presence of multiple transcriptional start sites upstream of the translational start site. Analysis of the 5' flanking region of the gene also revealed the presence of multiple TATA and CAAT sequences. The finding of multiple transcriptional start sites raises the possibility of tissue-specific regulation and the existence of variable size transcripts. Chimeras containing 1.26 kb (-534 to 728) of the 5' flanking region of the receptor gene and the luciferase (Luc) gene expressed a significant luciferase activity when transfected into a human endometrial tumor cell line (HEC-1A) and a breast tumor cell line (MCF-7) but not in a mouse pituitary gonadotrope cell line (alpha T3-1), suggesting the existence of multiple promoter elements in the gene. These findings indicate a multiplicity of regulation of expression of the GnRH receptor and provide the substrate for detailed investigation in the reproductive system.
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PMID:Molecular structure of the human gonadotropin-releasing hormone receptor gene. 1102 3

The MHC is an essential contributor to autoimmunity. Lmp2 and Tap1 are genes located in the MHC class II region, and they encode proteins participating in the generation and transport of endogenous peptides for T cell education. A mutation (T-->A) has now been detected in the shared bidirectional promoter of the Lmp2 and Tap1 genes in the nonobese diabetic (NOD) mouse. The nucleotide substitution (TCATTC-->TCAATC) in NOD mice eliminates an initiator (Inr) element (TCATTC) thought to be important for RNA polymerase II positioning in the Lmp2 orientation. It also created a CAAT-like box and an inverted CAAT-like box in the Lmp2 and Tap1 orientations, respectively. Northern blot revealed reduced amounts of Tap1 and Lmp2 mRNA in NOD mice, and 5'-rapid amplification of cDNA ends revealed the loss of a transcription start site of Lmp2 in these animals. The Tap1-Lmp2 promoter from NOD mice showed reduced transcriptional activity in transient transfection assays with luciferase reporter constructs for both Tap1 and Lmp2 genes. Observed altered substrate specificity of Lmp2 containing proteasomes isolated from NOD mice was consistent with reduced Lmp2 activity. The beneficial influence of non-MHC genes (NOR mice) and gender factors (male NOD mice) influencing the penetrance of the promoter polymorphism further confirmed the essential gender and hormonal context of the mutation. This study identifies the first specific mutation in the MHC of the NOD mouse that specifically impacts the activity of genes involved in peptide presentation, a process essential for T cell education.
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PMID:Reduced expression of Tap1 and Lmp2 antigen-processing genes in the nonobese diabetic (NOD) mouse due to a mutation in their shared bidirectional promoter. 930 Jul 32

Multiple transcriptional start sites have been identified for the gene encoding the rat Type I isozyme of hexokinase (White, J.A., Liu, W., and Wilson, J. E., Arch. Biochem. Biophys. 335, 161-172, 1996); these are clustered at positions approximately -460, -300, and -100 relative to the translational start codon (ATG, with A being +1). PC12 cells and H9c2 cells were transfected with luciferase reporter constructs containing genomic sequence between positions -3366 and -171. Marked (85%) decrease in promoter activity was associated with deletion of sequence between -742 and -516. In DNase I footprinting experiments, two regions, called P1 (-552 to -529) and P2 (-480 to -458) boxes, were protected by proteins present in nuclear extracts from PC12 cells. Mutation or deletion of the P2 box had no effect on promoter activity; protection in this region, which includes the most upstream cluster of transcriptional start sites, is attributed to binding of RNA polymerase II or associated factors. In contrast, mutations or deletions in the P1 box had markedly detrimental effects on promoter activity and on binding of proteins in PC12 cell nuclear extracts. Maintenance of a consensus Sp1 binding site centrally located in the P1 box was critical for both promoter activity and binding. A second Sp1 site (-570), just upstream from the P1 box, was also shown to be functionally important but no protection of this region was detected in footprinting experiments, presumably reflecting lower affinity at this site under the conditions used. Supershift experiments demonstrated the involvement of Sp1, Sp3, and Sp4 in formation of complexes with the P1 box region and implicate these transcription factors in regulating promoter activity associated with this region. Another series of reporter constructs, including sequence between -171 and -1, permitted detection of an additional promoter activity downstream from -364. While not yet extensively characterized, it is already evident that the cis elements influencing the downstream promoter activity are distinct from the Sp factors determined to be important in expression from the upstream promoter region.
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PMID:Two Sp sites are important cis elements regulating the upstream promoter region of the gene for rat type I hexokinase. 932 94

Many eukaryotic RNA polymerase II promoters contain initiator elements which direct accurate transcription in a TATA-independent manner. The PEA3/Ets-binding site (PEA3/EBS) is a common enhancer element in eukaryotic genes and is also found near the transcriptional start sites of many TATA-less promoters. We demonstrate that two PEA3/EBSs driving expression of the luciferase reporter gene, function as a minimal transcriptional initiator element. Maximal levels of transcription was achieved when two PEA3/EBSs, in either orientation, were located on the same face of the DNA helix, and the sites could be separated by up to three helical turns. In vitro transcription start sites directed by PEA3/EBS elements were clustered on either side of the upstream PEA3/EBS and were abolished by immunodepletion of GA-binding protein (GABP) from FM3A cell nuclear extracts. In vivo, co-transfection of GABPalpha and GABPbeta expression vectors enhanced reporter gene expression driven from PEA3/EBS initiator elements. Like other initiator elements, the PEA3/EBS elements were activated synergistically by upstream Sp1-binding sites. Thus, our results establish GABP as both a transcriptional activator factor and as an initiator factor.
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PMID:GA-binding protein-dependent transcription initiator elements. Effect of helical spacing between polyomavirus enhancer a factor 3(PEA3)/Ets-binding sites on initiator activity. 936 Sep 80

A genetic engineering approach was made to generate a recombinant non-segmented negative-strand RNA virus, Sendai virus (SeV) of the family Paramyxoviridae, that expresses firefly luciferase. The DNA construct containing the entire open reading frame (ORF) of the luciferase gene followed by the SeV transcription stop and restart signals connected with the conserved intergenic three nucleotides was inserted immediately before the ORF of the viral 3'-proximal nucleocapsid (N) protein gene in a full-length SeV cDNA copy. After intracellular expression of full-length antigenomic transcripts from the engineered cDNA and of the viral n ucleocapsid protein and RNA polymerase from the respective plasmids, a recombinant SeV expressing luciferase activity at a high level was recovered, although the tendency of this particular reporter gene product to aggregate in cells made it difficult to estimate the maximum level of expression. The increase in genome length brought about by inserting 1728 nucleotides into the 15,384 nucleotide parental SeV was associated with reduced plaque size, slightly slower replication kinetics and a severalfold decrease in yield of the virus. The inserted luciferase gene was stably maintained after numerous rounds of replication by serial passages in chick embryos. These results indicate the potential utility of SeV as a novel expression vector.
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PMID:Creation of an infectious recombinant Sendai virus expressing the firefly luciferase gene from the 3' proximal first locus. 936 67


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