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:2.7.7.48 (transcriptase)
9,479 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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 poliovirus RNA-dependent RNA polymerase required an oligouridylate primer or a HeLa cell protein (host factor) to initiate RNA synthesis on poliovirion RNA in vitro. The polymerase synthesized template-sized product RNA in the oligouridylate-primed reaction. In the host factor-dependent reaction, the largest product RNA synthesized by the polymerase was twice the size of the template RNA. About half of the product RNA recovered from this reaction was shown to exist in the form of a snapback sequence. Time-course reactions and pulse-chase experiments showed that the product RNA was only slightly larger than the template RNA at early reaction times and that with time it increased in size to form the dimer-sized product RNA. Inhibition of the elongation reaction by adding only [alpha-32P]UTP and ATP resulted in the formation of template-sized product RNA. The dimer-sized product RNA was unaffected by phenol extraction or proteinase K treatment but was converted to template-sized molecules by S1 nuclease. Dimer-sized poliovirus RNA that was sensitive to S1 nuclease was also isolated from poliovirus-infected cells. The results from this study indicate that the labeled negative-strand product RNA synthesized in vitro was covalently linked to the positive-strand template RNA. Thus, in vitro, the primer-dependent poliovirus RNA polymerase may initiate RNA synthesis in the presence of the host factor by using the 3' end of the template RNA as a primer.
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PMID:Poliovirus RNA-dependent RNA polymerase and host cell protein synthesize product RNA twice the size of poliovirion RNA in vitro. 298 94

RNA-dependent RNA polymerase activities were detected in purified particles of white clover cryptic viruses 1 and 2. The polymerases of the two viruses had different requirements for optimum activity. Enzyme activity was dependent upon the presence of virus particles, Mg2+, and the four ribonucleoside triphosphates, and was insensitive to actinomycin D, alpha-amanitin, and rifampicin. The labeled reaction products were dsRNAs as indicated by CF 11 column chromatography and by their ionic-strength-dependent sensitivity to hydrolysis by RNase A and resistance to S1 nuclease. The dsRNAs synthesized in vitro had the same electrophoretic mobilities as the corresponding viral templates.
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PMID:RNA-dependent RNA polymerase activity in two morphologically different white clover cryptic viruses. 335 1

Partially purified carnation cryptic virus (CarCV) preparations possessed RNA-dependent RNA polymerase activity which was absent in comparable preparations from virus-free carnations. Enzyme activity was dependent upon the presence of virus particles, Mg2+, and the four ribonucleoside triphosphates, and was insensitive to inhibitors of DNA-dependent RNA polymerases. The 32P-labeled enzyme reaction products were largely dsRNAs as indicated by resistance to S1 nuclease and RNase A at high but not low ionic strength. The in vitro synthesized dsRNAs hybridized specifically with CarCV genomic dsRNAs, and the radioactive products present in the polymerase reaction mixture sedimented with the virus particles in sucrose density gradients. The data suggest that the RNA-dependent RNA polymerase associated with CarCV particles is a replicase which catalyzes the synthesis of copies of the genomic dsRNAs.
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PMID:In vitro synthesis of double-stranded RNA by carnation cryptic virus-associated RNA-dependent RNA polymerase. 338 65

The two strands of the M double-stranded RNA species from a killer strain of Saccharomyces cerevisiae have been separated, and the 3'-terminal sequences of these strands have been determined. The positive strand programs the synthesis of the putative killer toxin precursor (M-p32) in a rabbit reticulocyte in vitro translation system. Only the negative strand hybridizes to the positive polarity transcript (m) synthesized in vitro by the virion-associated transcriptase activity. Secondary structural analysis of the extreme 3'-terminus of the negative strand using S1 nuclease is consistent with the presence of a large stem and loop structure previously proposed on the basis of RNA sequence data. This structure, and a similar structure at the corresponding 5'-terminus of the positive strand, may have functional significance in vivo.
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PMID:Structural and functional analysis of separated strands of killer double-stranded RNA of yeast. 675 69

Defective interfering (DI) particles of equine herpesvirus type 1 (EHV-1) are capable of mediating persistent infection (S. A. Dauenhauer, R. A. Robinson, and D. J. O'Callaghan, J. Gen. Virol. 60:1-14, 1982; R. A. Robinson, R. B. Vance, and D. J. O'Callaghan, J. Virol. 36:204-219, 1980). Sequence analysis of cloned DI particle DNA revealed that portions of two regulatory genes, ICP22 (IR4) and ICP27 (UL3), are linked in frame to form a unique hybrid open reading frame (ORF). This hybrid ORF, designated as the IR4/UL3 gene, encodes the amino-terminal 196 amino acids of the IR4 protein (ICP22 homolog) and the carboxy-terminal 68 amino acids of the UL3 protein (ICP27 homolog). Portions of DNA sequences encoding these two regulatory proteins, separated by more than 115 kbp in the standard virus genome, were linked presumably by a homologous recombination event between two identical 8-bp sequences. Reverse transcriptase-PCR and S1 nuclease analyses revealed that this unique ORF is transcribed by utilizing the transcription initiation site of ICP22 and the polyadenylation signal of ICP27 in DI particle-enriched infection. Immunoprecipitation and Western blot (immunoblot) analyses with antisera to the ICP22 and ICP27 proteins demonstrated that a 31-kDa hybrid protein was synthesized in the DI particle-enriched infection but not in standard virus infection. This 31-kDa hybrid protein was expressed at the same time as the ICP22 protein in DI particle-enriched infection and migrated at the same location on polyacrylamide gel electrophoresis as the protein expressed from a cloned IR4/UL3 expression vector. These observations suggested that the unique IR4/UL3 hybrid gene is expressed from the DI particle genome and may play a role in DI particle-mediated persistent infection.
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PMID:Expression of an equine herpesvirus 1 ICP22/ICP27 hybrid protein encoded by defective interfering particles associated with persistent infection. 852 42

Cancers from patients with tumor-induced hypercalcemia usually produce a circulating factor that mimics the parathyroid hormone activity, termed parathyroid hormone-related protein. Incidence of tumor-induced hypercalcemia appears to be high in patients with squamous cell carcinoma of the esophagus, and the presence of parathyroid hormone-related protein have been shown in some primary esophageal cancers. In the present study, we have investigated the presence of parathyroid hormone-related protein in a patient with metastasized squamous cell carcinoma of the esophagus complicated with tumor-induced hypercalcemia. Protein was searched by immunohistochemistry, and messenger RNA was investigated by reverse transcriptase-polymerase chain reaction and S1 nuclease assay. Both messenger RNA and protein were detected in hepatic metastases, whereas normal esophageal mucosa and primary cancer did not express detectable protein or messenger RNA using the S1 nuclease assay. Reverse transcriptase-polymerase chain reaction was positive in all these tissues, including normal esophageal mucosa. In conclusion, the present case suggests that tumor-induced hypercalcemia due to esophageal squamous cell carcinoma may be caused by parathyroid hormone-related protein mostly released by liver metastases.
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PMID:Parathyroid hormone-related protein in an esophageal squamous cell carcinoma with tumor-induced hypercalcemia. 904 Feb 21

Complementary DNA of marmoset CYP1A2 was isolated by means of screening the cDNA library and reverse-transcriptase polymerase chain reaction. The deduced amino acid sequence of marmoset CYP1A2 consisted of 516 residues and showed 88.2 and 90.0% identities to corresponding forms in human and cynomolgus monkey, respectively. S1 nuclease protection assay demonstrated that CYP1A2 mRNA was expressed constitutively in the liver, but not in the lung, kidney and small intestine. The level of CYP1A2 mRNA in the liver was increased by treatment with 3-methylcholanthrene and polychlorinated biphenyls. Marmoset CYP1A2 expressed in recombinant yeast activated 2-amino-3-methylimidazo [4,5-f]quinoline (IQ) and 2-amino-3,8-dimethylimidazo [4,5-f]quinoxaline (MeIQx) efficiently, and also activated 2-amino-1-methyl-6-phenylimidazo [4,5-b]pyridine (PhIP), but at a relatively lower rate in the umu mutagenicity test. Marmoset CYP1A2 also showed ethoxyresorufin O-de-ethylase activity. Based on these results, we demonstrate that marmosets constitutively express CYP1A2 in the liver as in humans.
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PMID:Marmoset CYP1A2: primary structure and constitutive expression in livers. 936 10

Within its intermediate host, Toxoplasma gondii switches between two forms: a rapidly replicating tachyzoite and an encysted bradyzoite. Bradyzoites persist within the host throughout its life, hidden from antimicrobial agents and the immune system. The signals that mediate switching are poorly understood. A gene trap was employed to isolate genes whose expression is up-regulated early in the switching of bradyzoites via the negative and positive selectable marker hypoxanthine-xanthine-guanine phosphoribosyltransferase (HXGPRT). T. gondii was transfected with promoterless HXGPRT and negatively selected with 6-thioxanthine to inhibit the growth of tachyzoites expressing HXGPRT. The surviving tachyzoites were then induced for in vitro bradyzoite formation and treated with mycophenolic acid and xanthine to positively select for parasites in which the construct had integrated downstream of a bradyzoite-specific gene. Strains were checked for their ability to differentiate by using Dolichos biflorus agglutinin (a bradyzoite-specific lectin) and a monoclonal antibody against P36 (a bradyzoite-specific surface antigen). After differentiation, all gene-trapped clones had Dolichos immunofluorescence and all but one expressed P36. The sequences flanking the insertion site of this P36-negative strain were homologous to the Toxoplasma family of surface antigens, strongly suggesting that P36 is encoded by the disruptive gene. Genetic mapping and complementation of the P36-negative strain further indicated that the disrupted gene is P36. Reverse transcriptase PCR and S1 nuclease digestion were used to compare mRNA levels during the tachyzoite and bradyzoite stages. The presumptive P36 gene does not appear to regulate its mRNA levels between the two stages, indicating a posttranscriptional mechanism of regulation for early bradyzoite-specific genes.
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PMID:Isolation of developmentally regulated genes from Toxoplasma gondii by a gene trap with the positive and negative selectable marker hypoxanthine-xanthine-guanine phosphoribosyltransferase. 944 77

Vibrio ordalii is a major cause of vibriosis in wild and cultured marine salmonids and carries pMJ101, a 30-kb cryptic plasmid that replicates in the absence of DNA polymerase I without producing single-stranded intermediates. A recombinant derivative harboring the pMJ101 replication region proved to be compatible with pJM1, a plasmid containing the iron acquisition system required for the virulence of V. anguillarum 775, another important pathogen that causes vibriosis. Sequence analysis of a 1.56-kb fragment harboring the pMJ101 replication region revealed the presence of typical features found in DNA origins including an AT-rich region, 11 dam-methylation sites of which 5 are within the putative ori region, and five copies of the 9-bp consensus sequence for DnaA binding. Gel retardation assays demonstrated that the latter replication element indeed binds DnaA purified from Escherichia coli. A potential open reading frame encoding a hydrophilic protein with a predicted pI of 10.3 and an M(r) of 33,826 was found adjacent to the ori region. Although these properties are typical of DNA-binding proteins, no significant homology was found between this predicted protein, named RepM, and other previously characterized proteins. Reverse transcriptase-polymerase chain reaction analysis of total RNA demonstrated the presence of repM mRNA in V. ordalii. The major initiation site of this mRNA was located 187 nucleotides upstream of the GTG initiation codon as determined by nuclease S1 protection assays. This transcription initiation site is preceded by putative -10 and -35 promoter sequences that control the expression of the repM replication gene. These results demonstrate that the replication region of pMJ101 shares some structural and sequence similarities with other DNA replication regions, which include DnaA binding and methylation sites and an open reading frame encoding a distinct protein required for its replication.
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PMID:Analysis of the replication elements of the pMJ101 plasmid from the fish pathogen Vibrio ordalii. 1041 62


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