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)

High affinity sodium- and potassium-coupled L-glutamate transport into presynaptic nerve terminals and fine glial processes removes the neurotransmitter from the synaptic cleft, thereby terminating glutamergic transmission. This report describes that the purified L-glutamate transporter from pig brain is phosphorylated by protein kinase C, predominantly at serine residues. Upon exposure of C6 cells, a cell line of glial origin, to 12-O-tetradecanoylphorbol-13-acetate, about a 2-fold stimulation of L-glutamate transport is observed within 30 min. Concomitantly, the level of phosphorylation increases with similar kinetics. The phorbol ester also stimulates L-glutamate transport in HeLa cells infected with a recombinant vaccinia virus expressing T7 RNA polymerase and transfected with pT7-GLT-1. The latter is a recently cloned rat brain glutamate transporter of glial origin. Mutation of serine 113 to asparagine does not affect the levels of expressed transport but abolishes its stimulation by the phorbol ester. To our knowledge, this is the first direct demonstration of the regulation of a neurotransmitter transporter by phosphorylation.
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PMID:Phosphorylation and modulation of brain glutamate transporters by protein kinase C. 790 7

Syncytia formation in either CV-1 or HeLa T4+ cells required recombinant expression of both fusion (F) and hemagglutinin-neuraminidase (HN) glycoproteins from the human parainfluenza virus type 3 (HPIV3), human parainfluenza virus type 2 (HPIV2), and simian virus 5 (SV5). In this system, recombinant T7 transcription vectors (pT7-5 or pGEM) containing F or HN, were transfected individually or in combination into cells previously infected with a recombinant vaccinia virus expressing T7 RNA polymerase (vTF7-3). While both proteins were processed and expressed at the cell surface, syncytia formation occurred only when both glycoproteins were co-expressed. The function of HN in the fusion process could not be replaced using lectins or by co-expression of heterologous F and HN proteins. Further, cell fusion was not observed when experiments were performed using individually expressed F and HN proteins in adjacent cells. The data presented in this report support the notion that a specific interaction between both paramyxoviral glycoproteins is required for the formation of syncytia in tissue culture monolayers.
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PMID:Paramyxovirus mediated cell fusion requires co-expression of both the fusion and hemagglutinin-neuraminidase glycoproteins. 816 62

A new temperature-regulated T7 RNA polymerase-driven transcription system has been developed. This system is based on a hybrid regulatory region: the phage T7 late promoter (PT7) linked to the Escherichia coli lac operator (Olac) [Giordano et al., Gene 84 (1989) 209-219], which was located in an earlier obtained [Mashko et al., Gene 97 (1991) 259-266] temperature-controlled amplifiable plasmid, carrying cat under the control of PT7-Olac and, in addition, lambda major early promoter-operator regions and gene cIts857. Plasmids of the pT7-Olac-cat-tsr series were stably maintained at a low-copy-number when grown at low temperature (28 degrees C). In E. coli BL21(DE3), carrying the Plac-controllable T7 RNA polymerase-encoding gene, efficient repression of cat transcription was observed, that was provided by the LacI repressor and, probably, the thermolabile repressor CIts857. At low and moderate temperatures (28/37 degrees C), this 'cooperative' repression was so tight that cat expression was not observed in the cells carrying PT7-Olac on the plasmids, even after IPTG-inducible T7 RNA polymerase biosynthesis. As a result of the thermo-amplification of the recombinant plasmids and temperature-inactivation of CIts857, expression of the T7 RNA polymerase-encoding gene was derepressed due to the titration of LacI by the increasing copies of Olac which in turn, led to the highly efficient T7 RNA polymerase-driven accumulation of CAT in the cells.
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PMID:A new T7 RNA polymerase-driven expression system induced via thermoamplification of a recombinant plasmid carrying a T7 promoter-Escherichia coli lac operator. 818 58

To increase the levels of exogenous or foreign gene expression in mammalian cells, this study sought to develop an 'autogene' that will self-amplify. An autogene plasmid, pT7-G1, containing the T7 phage RNA polymerase-encoding modified gene (G1) under control of its cognate T7 promoter, was only obtained when the plasmid contained the encephalomyocarditis (EMC) untranslated sequence. In vitro transcription and translation studies confirmed that both the T7 promoter and the G1 gene were completely functional in the pT7-G1 plasmid. Expression from pT7-G1 was initiated in vivo either by co-transfection with its in vitro transcript or by transfection into NIH3T3 cell lines that stably expressed T7 RNA polymerase enzyme. Use of the pT7-G1 autogene enabled an approx. 50-fold increase in foreign protein production. Northern analysis suggested that this increased expression resulted from the self-amplification of the autogene. By allowing greater expression in cell lines with low T7 RNA polymerase expression, the pT7-G1 plasmid increases the usefulness of the T7 gene system for expression within mammalian cells.
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PMID:Self-amplifying expression from the T7 promoter in 3T3 mouse fibroblasts. 820 81

A fragment of T4 DNA (XbaI-HindIII) comprising the genes 51, 27, 28, which encodes the central plug proteins was cloned into plasmid pT7-5 and p7-6 (T7 RNA polymerase expressing system). The examined genes were only overexpressed when the orientation of cloned DNA to promoter phi 10 was as follows: promoter phi 10 and genes 51, 27, 28. This was achieved when the fragment (XbaI-HindIII) was cloned into plasmid pT7-5. Gene 27 and 28 were overexpressed when the intact fragment (XbaI-HindIII) was used. The high rate of the synthesis of proteins 27 and/or 28 had a strong inhibitory effect on the level of synthesis of the product of gene 51. For the overexpression of gene 51 in this system a deletion derivate which was devoid of gene 28 and a larger fragment of gene 27 was prepared.
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PMID:Expression of genes 51, 27, 28 coding for proteins of the central part of bacteriophage T4 baseplate in the bacteriophage T7 promoter/RNA polymerase expression system. 821 66

Expression of bacteriophage T7 RNA polymerase in mammalian cells can efficiently drive the transcription of a foreign gene controlled by the T7 promoter (Elroy-Stein et al., Proc. Natl. Acad. Sci. USA. 86, 6126-6130, 1989). We have tested the hypothesis that purified T7 RNA polymerase can be co-delivered into mammalian cells together with a reporter gene (chloramphenicol acetyltransferase, CAT) controlled by the T7 promoter (pT7-EMC-CAT) using DC-chol cationic liposomes. Indeed, significant level of CAT activity was observed in human lung adenocarcinoma (A549-1) cells which had been incubated with a complex of T7 RNA polymerase, pT7-EMC-CAT DNA and DC-chol cationic liposomes. The expression was specific in that T3 RNA polymerase could not replace the T7 RNA polymerase, and that co-delivered T7 RNA polymerase did not enhance the expression of a CAT gene controlled by the SV40 early promoter. The system was optimized in terms of enzyme, DNA and liposome concentrations. Time course experiment indicated that the expression of the T7 system was about 8-10 hours sooner than the SV40 system, consistent with the notion that T7 RNA polymerase does not enter into the nucleus and the transcription takes place in the cytoplasm of the transfected cells. The expression of the T7 system was transient; it declined after 30 hours post transfection, probably due to turnover of the phage enzyme in the mammalian cells. The expression system described here should be useful for gene transfer experiments which require a fast but transient expression of a foreign gene. We have also compared our delivery system with a commercial reagent, Lipofectin, which has been used to deliver T3 or T7 RNA polymerase with a reporter plasmid encoding the T3 or T7 promoter.
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PMID:Cytoplasmic expression of a reporter gene by co-delivery of T7 RNA polymerase and T7 promoter sequence with cationic liposomes. 833 95

Lipocortin or annexin 1 is a calcium-dependent phospholipid-binding protein which probably acts as a glucocorticoid- regulated anti-inflammatory factor. cDNA for human lipocortin 1 was cloned in the pT7.7 expression plasmid under the control of the inducible bacteriophage T7 RNA polymerase promoter. Upon induction with isopropyl thio-beta-D-galactoside, large amounts of the protein were produced and accumulated in Escherichia coli in a soluble form. The recombinant protein was purified to homogeneity by means of two subsequent ion-exchange chromatographic steps. The final yield was about 30 mg/l bacterial culture. Electrospray mass spectrometric analysis of the purified protein demonstrated that the recombinant product corresponds to the native human lipocortin 1, without the initial methionine and with a free N-terminal alanine; tryptic peptide mapping by fast-atom-bombardment mass spectrometry showed that the recombinant protein contains cysteine residues at positions 263 and 324 with free thiol groups, whereas Cys270 and Cys343 are probably involved in an intrachain disulfide bridge. Recombinant human lipocortin 1 reduces the carrageenin-induced paw oedema in rat in vivo and inhibits porcine pancreatic phospholipase A2 activity in vitro; in both cases, a dose-related response is observed.
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PMID:Structural characterization of a biologically active human lipocortin 1 expressed in Escherichia coli. 842 44

The histidyl-tRNA synthetase gene (hisS) from Streptococcus equisimilis was cloned and sequenced. The gene for this aminoacyl-tRNA synthetase has an open reading frame of 1278 nucleotides. The deduced amino acid sequence encodes a protein of 426 amino acids with MW = 47,932. The protein is predicted to be soluble with a pl = 5.27. The protein sequence has extensive overall identity/similarity with the Escherichia coli and the yeast histidyl-tRNA synthetases (approximately 58% and approximately 20%, respectively). A putative promoter for gene transcription lies within two hundred nucleotides of the polypeptide start codon. The enzyme was overexpressed, to a level of about 18% of total cellular protein, as a fusion protein (containing an additional 15 amino acids) in E. coli using the pT7 expression system containing the T7 RNA polymerase/promoter (Tabor and Richardson, Proc. Natl. Acad. Sci. U.S.A. 82:1074-1078, 1985). The predicted MW for the hisS gene product is in good agreement with the size of the fusion protein determined by SDS-PAGE (M(r) = 53,700). Amino acid sequencing of the intact fusion protein and proteolytic fragments confirmed the deduced sequence of the synthetase at many positions throughout the protein. The expressed protein catalyzed the specific aminoacylation of tRNA(His) in vitro.
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PMID:Molecular cloning, sequence, structural analysis and expression of the histidyl-tRNA synthetase gene from Streptococcus equisimilis. 844 73

Here, we describe a modification of a plasmid, pT7-7 [Tabor and Richardson, Proc. Natl. Acad. Sci. USA 262 (1985) 1074-1078], that allows expression of inserted genes from the phage T7 RNA polymerase promoter. The modification is designed to suppress readthrough transcription from cryptic promoters and start points on the plasmid, in order to reduce expression in the absence of T7 RNA polymerase and thus improve the vector for use in the expression of highly toxic gene products. This vector (pT7SC) was used to stably clone the POL3 gene (encoding DNA polymerase delta) of Saccharomyces cerevisiae, which destabilizes all other cloning and expression vectors tested. Previously described expression strategies proved ineffective in overexpressing the POL3 gene. A new strategy was developed which relies on induction by infection with mutant T7 phage. This system efficiently overproduced the POL3 gene product.
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PMID:A new cloning vector and expression strategy for genes encoding proteins toxic to Escherichia coli. 848 92

The Escherichia coli gene murB, encoding the enzyme uridine-5'-diphospho-N-acetyl-2-amino-2-deoxy-3-O-lactylglucosenicoti namide adenine dinucleotide phosphate oxidoreductase (EC 1.1.1.158) (EP-reductase), the second enzyme in the peptidoglycan biosynthetic pathway, has been amplified using PCR technology with the Kohara recombinant lambda phage E11C11 (534) as template. The synthetic gene was subcloned into the NdeI and BamHI restriction sites of the expression vector pT7-7, designed to utilize T7 RNA polymerase to direct transcription of the target gene, in a two-step procedure. The first step involved the directional insertion of the 590-bp NdeI to BamHI restriction fragment of murB into the pT7-7 vector to give the plasmid pT7-7-murB-590. The construction of the desired overproducing plasmid was completed by the bidirectional insertion of the 442-bp BamHI to BamHI restriction fragment of murB into a similarly restricted pT7-7-murB-590 plasmid followed by restriction digestion to select the properly oriented insert, pT7-7-murB. Overexpression of EP-reductase from the E. coli strain BL 21 (DE 3) containing the pT7-7-murB gene, after induction, allowed the production of 36 mg of target protein per 3 wet grams of E. coli cells. The EP-reductase was purified in a single step utilizing dye-ligand chromatography to yield 30 mg of pure protein. The availability of these levels of reductase will allow the mechanism of this pivotal enzyme to be thoroughly studied as a potential target for the design of a new generation of antibiotics. In addition, the EP-reductase generated in this study has been utilized as a coupling enzyme to assay the first enzyme in the peptidoglycan biosynthetic pathway, UDP-N-acetylglucosamine enolpyruvyl transferase, and these results are also presented.
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PMID:Overproduction and one-step purification of Escherichia coli UDP-N-acetylglucosamine enolpyruvyl reductase. 874 27


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