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)

The phosphoenolpyruvate mutase gene from Tetrahymena pyriformis has been cloned and overexpressed in Escherichia coli. To our knowledge, this is the first Tetrahymena gene to be expressed in E. coli, a task made more complicated by the idiosyncratic codon usage by Tetrahymena. The N-terminal amino acid sequence of phosphoenolpyruvate mutase purified from T. pyriformis has been used to generate a precise oligonucleotide probe for the gene, using in vitro amplification from total genomic DNA by the polymerase chain reaction. Use of this precise probe and oligo(T) as primers for in vitro amplification from a T. pyriformis cDNA library has allowed the cloning of the mutase gene. A similar amplification strategy from genomic DNA yielded the genomic sequence, which contains three introns. The sequence of the DNA that encodes 10 amino acids upstream of the N-terminal sequence of the isolated protein was found by oligonucleotide hybridization to a subgenomic library. These 10 N-terminal amino acids are cleanly removed in Tetrahymena in vivo. The full mutase gene sequence codes for a protein of 300 amino acids, and it includes two amber (TAG) codons in the open reading frame. In Tetrahymena, TAG codes for glutamine. When the two amber codons are each changed to a glutamine codon (CAG) that is recognized by E. coli and the gene is placed behind a promoter driven by the T7 RNA polymerase, expression in E. coli is observed. The mutase gene also contains a large number of arginine AGA codons, a codon that is very rarely used by E. coli. Cotransformation with a plasmid carrying the dnaY gene [which encodes tRNA(Arg)(AGA)] results in more than 4-fold higher expression. The mutase then comprises about 25% of the total soluble cell protein in E. coli transformants. The mutase gene bears significant similarity to one other gene in the available data bases, that of carboxyphosphonoenolpyruvate mutase from Streptomyces hygroscopicus, an enzyme that catalyzes a closely related transformation. Due to the large evolutionary distance between Tetrahymena and Streptomyces, this similarity can be interpreted as the first persuasive evidence that the biosynthesis of phosphonates is an ancient metabolic process.
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PMID:Phosphonate biosynthesis: molecular cloning of the gene for phosphoenolpyruvate mutase from Tetrahymena pyriformis and overexpression of the gene product in Escherichia coli. 154 41

Human intercellular adhesion molecule-1 (ICAM-1), a specific ligand for the lymphocyte function-associated Ag-1 (LFA-1), plays an important role in leukocyte-endothelial cell interactions. It is induced by proinflammatory cytokines such as IL-1, TNF-alpha, or IFN-gamma. However, little is known concerning the intracellular regulatory mechanisms which trigger ICAM-1 up-regulation. In order to study potential regulatory elements involved in ICAM-1 induction we have cloned the human ICAM-1 gene and 5 kb of its 5'-regulatory region. The sequence of the cDNA was found to be distributed over seven exons separated by six introns, whereby each of the five extracellular Ig-like domains of ICAM-1 is encoded by its own exon. The upstream sequence harbors a number of sequence motifs implicated in the regulation and expression of eukaryotic genes, including binding sites for the transcription factors SP-1, AP-1, and NF-kB. Primer extension and S1 nuclease analysis revealed two transcription initiation sites 319 bp and 41 bp upstream of the translation start site. Consensus TATA boxes were found at the expected positions about 25 bp upstream of both start sites. Reverse transcriptase polymerase chain reaction showed differential use of the two TATA boxes in A549 and HS913T cells. Both RNA seem to code for the same for of ICAM-1 protein. For regulation studies a 1.3-kb EcoRI/SalI fragment of the 5'-flanking region was used to promote transcription of a linked luciferase reporter gene in transient-transfection assays in A549 and HS913T cells. Treatment of A549 cells with IL-1 or TNF-alpha resulted in a two- or fourfold increase in luciferase activity. Furthermore, a sixfold induction could be achieved after treatment with the phorbol ester PMA. In contrast, agents that increase intracellular cAMP levels did not induce luciferase activity. Northern blot analysis was used to investigate the kinetics of ICAM-1 mRNA synthesis upon induction with TNF-alpha and PMA. These data suggest that the up-regulation of ICAM-1 by cytokines occurs at least partly at the transcriptional level. Deletion analysis of the 1.3-kb fragment of the 5'-flanking region revealed sequences responsible for promotion and inhibition of transcription. In particular, two functionally distinct regions have been characterized: a short fragment containing an NF-kB binding site has been shown to function as an activator, followed immediately downstream by a sequence acting as a silencer element. Therefore, ICAM-1 gene expression seems to be modulated by multiple cis-acting elements.
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PMID:Cloning of the human gene for intercellular adhesion molecule 1 and analysis of its 5'-regulatory region. Induction by cytokines and phorbol ester. 168 Sep 19

The acute monocytic leukemia cell line THP-1 secretes predominantly IL-1 beta after treatment with bacterial lipopolysaccharide and tumour promoting phorbol ester (PMA). IL-1 alpha is also secreted, but represents less than 10% of the total IL-1 activity. This differential is reflected at the level of mRNA as IL-1 beta mRNA is more abundant than IL-1 alpha mRNA. Studies of transcription in isolated nuclei however indicate that each gene is transcribed at a similar rate, suggesting that post-transcriptional mechanisms regulate the relative abundance of IL-1 alpha and IL-1 beta mRNA. Measurement of RNA half life after addition of alpha-amanitin (an inhibitor of RNA polymerase II) indicate that IL-1 alpha mRNA is not as stable as IL-1 beta mRNA suggesting one mechanism for the different relative levels of RNA.
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PMID:Post-transcriptional control of IL-1 gene expression in the acute monocytic leukemia line THP-1. 326 53

Mutans streptococci have been shown to give rise to variants in terms of expression of surface protein antigens by repeated subculturing of the organisms, which in turn induces changes in colonial morphologies. A 2,850-bp upstream region of the gene (pag) for a surface protein antigen, PAg, of Streptococcus sobrinus MT3791 was determined. Analysis of the nucleotide sequence revealed the existence of three open reading frames (ORFs) located upstream of the pag gene. ORF1 extended from an undetermined further upstream sequence to the termination codon TAG lying 1,943 bp upstream of the pag gene. ORF2, consisting of 609 bp lying 1,689 bp upstream of the pag gene, encoded a protein of 23,347 Da and a protein of 22,792 Da. The synthesis of these proteins (protein antigen regulators) was demonstrated by using the in vitro T7 RNA polymerase/promoter system. ORF3, extending from 314 bp upstream of the pag gene to 712 bp upstream of the pag gene, encoded a protein of 14,802 Da. Disruption of chromosomal ORF2 of parent strain MT3791 by allelic exchange resulted in isogenic mutants, termed PAREm-1 and PAREm-2, that synthesized larger amounts of cell-free and cell-associated PAg than did the parent strain. RNA dot blot analysis demonstrated that expression of PAg-specific mRNA transcripts by mutants PAREm-1 and PAREm-2 was about 32-fold higher than that by strain 3791. Mutants PAREm-1 and PAREm-2 were found to be more hydrophobic than strain MT3791. Resting cells of these mutants attached in larger numbers to saliva-coated hydroxyapatite than did those of the parent strain. These results suggest that protein antigen regulator regulates the expression of PAg gene in a negative fashion, affecting the colonization of tooth surfaces by the organism. Thus, ORF2 is concluded to be a negative regulator gene of PAg synthesis and was designated par.
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PMID:Molecular characterization of a negative regulator of Streptococcus sobrinus surface protein antigen gene. 833 Oct 66

The IL-1R antagonist protein (IRAP) is a competitive inhibitor of IL-1, which is predominantly synthesized by monocytes. We show that this molecule is also expressed in human synovial fibroblasts and dermal fibroblasts (CRL 1445). IRAP mRNA was regulated in a time- and dose-dependent manner by IL-1 alpha, TNF-alpha, LPS, and PMA. Maximal induction of IRAP mRNA was observed between 8 and 16 h after stimulation with IL-1 alpha (1 U/ml), TNF-alpha (10 U/ml), LPS (50 ng/ml), and PMA (10 ng/ml). Their relative efficacy was as follows: PMA > LPS > IL-1 alpha > TNF-alpha. Potentiation was observed when fibroblasts were treated with IL-1 alpha plus basic fibroblast growth factor and IL-1 alpha plus platelet-derived growth factor-BB homodimer. Although LPS and PMA were the best inducers of IRAP mRNA, quantitation of the IRAP protein revealed that its synthesis and release were differentially regulated. Immunoprecipitation and SDS-PAGE of culture supernatant from LPS-treated cells and cell lysates of fibroblasts treated with LPS or PMA showed a single IRAP band with a molecular mass of approximately 22 kDa. Very little IRAP was detected in culture supernatants of cells treated with PMA. Quantitation of IRAP revealed that LPS induced the synthesis of secreted IRAP that was released, whereas the majority of the protein induced by PMA remained cell-associated. Reverse transcriptase-polymerase chain reaction amplification demonstrated that although LPS and PMA induced both transcripts, LPS preferentially induced secreted IRAP, whereas PMA differentially induced intracellular IRAP mRNA. Fibroblasts synthesize at least two different forms of IRAP depending on the inducing signal, and may regulate the inflammatory response by dampening the proinflammatory effects of IL-1 via a negative feedback mechanism with IRAP. The relative importance of fibroblast sIRAP vs intracellular IRAP in regulating the inflammatory response by the connective tissue remains to be determined.
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PMID:Regulation of expression of IL-1 receptor antagonist protein in human synovial and dermal fibroblasts. 847 46

The N protein coded by bacteriophage lambda plays an essential role in the completion of lambda transcription by recognizing the boxB sequence in nascent transcripts and then aggregating with Escherichia coli RNA polymerase and four other E. coli proteins into an unstoppable transcription complex. In order to explore the functionality of N protein and the specific recognition between N and boxB, 14 amino acid positions near the amino-terminal end of N lambda were mutated extensively. The mutant proteins were scored for N function in vivo by a two-plasmid construct that visualizes readthrough transcription as lacZ expression in colonies of E. coli. Mutation was achieved by single TAG replacements, translated through suppression into 13 different amino acids, or by scrambling at assorted three-codon sets. Of the 14 amino acid positions tested (Tables 5 and 6), six remained functional with a wide variety of substitutions, while substitution was sometimes deleterious at one Ala and two Gln positions. At each of the five Arg positions, however, maintenance of Arg occupancy proved important for N function. Despite effective screening for increased N function at boxBs with defective loops, no N mutant, simple or complex, was found to change the order of preference of wild-type N lambda for boxBs with defective loops. Thus, although multiple amino-terminal Arg positions are found to be important for N function, mutations in the region spanning the five Arg residues were not found to compensate for defects in boxB loop.
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PMID:Clustered arginine residues of bacteriophage lambda N protein are essential to antitermination of transcription, but their locale cannot compensate for boxB loop defects. 851 Jan 51

The absorption of water and electrolytes by the gallbladder seems to be largely dependent upon a Na+/H+ exchange at the apical membrane of the gallbladder epithelium. To find out if the exchanger involved is the NHE3 isoform, as in other absorbing epithelia, two studies were performed using the rabbit gallbladder. First, we studied 22Na absorption in Ussing chambers with Krebs buffer as a control solution, and in the presence of amiloride (100, 200 or 1000 microM), ethyl-isopropyl-amiloride (EIPA, 1 or 5 microM), or the phorbol ester, phorbol 12-myristate 13-acetate (PMA, 1 microM). A net mucosal-to-serosal Na+ flux was observed with control buffer. No inhibition of this net flux was observed with 5 microM EIPA, and the IC50 for amiloride was found to be 200 microM. PMA induced a reduction of absorption by 30% that was prevented by incubation with calphostin C. Resistance to amiloride and EIPA, and inhibition by PMA are consistent with the involvement of the NHE3 isoform. The second study involved reverse-transcriptase polymerase chain reaction (RT-PCR) of total gallbladder RNA, with two primers designed to amplify a 645-base-pair fragment from NHE3 mRNA. A cDNA fragment of the expected size was actually obtained from gallbladder RNA, while RT-PCR of RNA from the liver, which does not contain NHE3, gave negative results. A sequence of 492 nucleotides of the amplified product was determined, which was almost superimposable onto the known sequence of the corresponding fragment of rabbit NHE3. It is concluded that, in rabbit gallbladder, neutral NaCl absorption is, at least in part, dependent on the NHE3 isoform of the Na+/H+ exchanger.
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PMID:Role of the NHE3 isoform of the Na+/H+ exchanger in sodium absorption by the rabbit gallbladder. 877 28

The sigma factor sigma 73 of the obligate intracytoplasmic bacterium Rickettsia prowazekii was overexpressed and purified from Escherichia coli. The rickettsial rpoD gene encoding sigma 73 was cloned into a Ndel-BamHI-cleaved pET-15b vector under control of T7 transcription and translation signals. The recombinant plasmid encoded a 75 kDa fusion protein that was overproduced in E. coli BL21(DE3) and purified from inclusion bodies after solubilization with guanidine hydrochloride and using His. Bind metal chelation resin. The N-terminal His. Tag sequence of the 75 kDa fusion protein was removed by thrombin treatment to obtain R. prowazekii sigma 73T. The R. prowazekii sigma 73T as well as the 75 kDa fusion protein had the ability to bind to core DNA-dependent RNA polymerase of both R. prowazekii and E. coli and to stimulate their interaction with a rickettsial promoter.
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PMID:Rickettsia prowazekii sigma factor sigma 73 can be overexpressed in Escherichia coli and promotes RNA polymerase binding and transcription. 893 16

By database search analysis, we identified three Arabidopsis EST (Expression Sequence Tag) entries having similarity to eubacterial RNA polymerase sigma factors. cDNA clones corresponding to these partial sequences were isolated, and the complete nucleotide sequences were determined. All three sequences encode proteins highly homologous to cyanobacterial and plastid sigma factors, and the gene products have N-terminal extensions which are assumed to function as plastid-targeting transit peptides. Thus we have concluded that the gene products are RNA polymerase sigma factors of plastids, and named sigA, sigB and sigC, respectively. Expression of these genes was analyzed by RNA gel-blot analysis and shown to be induced by illumination after a short-term dark adaptation. This strongly suggests that light regulation of the nuclear encoded sigma factor genes is involved in light-dependent activation of plastid promoters.
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PMID:Characterization of three cDNA species encoding plastid RNA polymerase sigma factors in Arabidopsis thaliana: evidence for the sigma factor heterogeneity in higher plant plastids. 928 Mar 3

A homolog of the multiple-stress-responsive transcription factor sigmaB of Bacillus subtilis was predicted from the DNA sequence analysis of a region of the Staphylococcus aureus chromosome. A hybrid between the coding sequence of the first 11 amino acids of the gene 10 leader peptide of phage T7 (T7.Tag) and the putative sigB gene of S. aureus was constructed and cloned into Escherichia coli BL21(DE3)pLysS for overexpression from a T7 promoter. A homogeneous preparation of the overproduced protein was obtained by affinity chromatography with a T7.Tag monoclonal antibody coupled to agarose. The amino-terminal amino acid sequence of the first 22 residues of the purified protein matched that deduced from the nucleotide sequence. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified protein, designated sigmaSB, indicated that it migrated as an approximately 39-kDa polypeptide. Promoter-specific transcription from the B. subtilis sigmaB-dependent PB promoter of the sigB operon was stimulated by sigmaSB in a concentration-dependent fashion when reconstituted with the S. aureus core RNA polymerase (RNAP). Specific transcript from the predicted sigmaB-dependent PB promoter of the sigB operon of S. aureus was obtained by the reconstituted RNAP in a runoff transcription reaction. The sar operon of S. aureus contains three promoter elements (P1, P2, and P3) and is known to partly control the synthesis of a number of extracellular toxins and several cell wall proteins. Our in vitro studies revealed that transcription from the P1 promoter is dependent on the primary sigma factor sigmaSA, while that of the P3 promoter is dependent on sigmaSB. As determined by primer extension studies, the 5' end of the sigmaSB-initiated mRNA synthesized in vitro from the sar P3 promoter is in agreement with the 5' end of the cellular RNA.
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PMID:Alternative transcription factor sigmaSB of Staphylococcus aureus: characterization and role in transcription of the global regulatory locus sar. 933 83


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