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)

Examinations were made on substances that enhance or inhibit the induction of hepatoma in rats previously fed 3'-methyl-4-(dimethylamino)azobenzene (3'-Me-DAB) for a brief period. The substances tested were stilbene, 4-nitrostilbene, 4,4'-dihydroxystilbene, diethylstilbestrol, 17beta-estradiol, and methyltestosterone. Male Donryu rats were fed 0.5 g of 3'-Me-DAB by being maintained on a diet containing 0.06% 3'-Me-DAB, and then they were fed 0.25 or 0.5 g of a test substance with the basal diet. Comparison of the development and yield of hepatomas indicated that 4-nitrostilbene and methyltestosterone had an activity of enhancing 3'-Me-DAB carcinogenesis, whereas diethylstilbestrol and 17beta-estradiol had an activity to retard it. Other substances showed no such activities. The enhancement by 4-nitrostilbene and inhibition by diethylstilbestrol of 3'-Me-DAB carcinogenesis was correlated with their effect on liver nucleic acid metabolism. Feeding of 4-nitrostilbene caused a selective inhibition of Mn2+-(NH4)2SO4-activated RNA polymerase activity of liver nuclei and reduced liver RNA content. The deleterious alteration of liver RNA metabolism was followed by the enhancement in the incorporation of ip-injected 3H-thymidine into DNA of liver nuclei. On the other hand, feeding of diethylstilbestrol increased tissue RNA content without effect on RNA polymerase activity of liver nuclei, and had an activity of increasing the incorporation of 3H-thymidine into DNA. The possible implication of these results with regard to the enhancement and inhibition of hepatocarcinogenesis is discussed.
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PMID:Enhancing and inhibitory effects of some stilbene and steroid compounds on induction of hepatoma in rats fed 3'-methyl-4-(dimethylamino)azobenzene. 20 6

An acidic nucleolar phosphoprotein with a subunit M(r) of 70,000 was purified as an apparent dimer of 139,000 from isolated nuclei of the slime mold Physarum polycephalum. The protein was purified without the aid of strong dissociating agents after its selective phosphorylation in isolated nuclei by a polyamine-mediated reaction. Its amino acid composition resembled that of a nucleolar phosphoprotein from Novikoff hepatoma ascites cells. The phosphoprotein stimulated rRNA synthesis 5-fold by RNA polymerase I within a nucleolar, ribosomal deoxyribonucleoprotein complex isolated from nucleoli of P. polycephalum. It was also identified as a component of the complex. It bound with high affinity and specificity to the palindromic ribosomal DNA of 38 x 10(6)M(r) from P. polycephalum, which contained two coding sequences for 5.8S, 19S, and 26S rRNA. It also bound to three fragments of ribosomal DNA of M(r) 21.2 x 10(6), 17.1 x 10(6), and 8.1 x 10(6), prepared by cleavage with restriction endonucleases HindIII, PstI, and BamHI, respectively. All of these fragments included the symmetry axis of the palindromic ribosomal DNA. The phosphoprotein that had been treated with alkaline phosphataseagarose to hydrolyze the phosphate groups did not stimulate transcription and did not bind to ribosomal DNA or to the restriction fragments indicated. We have thus isolated a specific phosphoprotein with the capacity to stimulate transcription of a specific set of genes in a eukaryote. These findings suggest that this phosphoprotein may specifically regulate functions of ribosomal DNA in a manner dependent on its degree of phosphorylation.
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PMID:Polyamine-mediated phosphorylation of a nucleolar protein from Physarum polycephalum that stimulates rRNA synthesis. 28 43

Patients with hepatocellular carcinoma (HCC), gastrointestinal, lung, and ovarian cancers were shown to have autoantibodies to nuclear and nucleolar antigens as detected by immunofluorescence on cell substrates. The frequency of antinuclear antibodies (ANAs) was significantly higher (P less than 0.001) in patients with HCC (57/184 = 31%) than in patients with chronic hepatitis or liver cirrhosis (25/187 = 13%). Although a range of fluorescence patterns was observed, a higher percentage of nucleolar fluorescence was detected in HCC, and three of these nucleolar antigens were identified. They were NOR-90, nucleolus organizer region doublet polypeptides of 93 and 89 kDa involved in RNA polymerase I transcription; fibrillarin, a 34 kDa protein of the nucleolar U3 ribonucleoprotein particle which is engaged in preribosomal RNA processing; and nucleophosmin/protein B23, a 37 kDa polypeptide which is associated with ribosome maturation and cellular proliferation. All these antigens are nucleolar components that are engaged in some aspect of ribosome biosynthesis. Since autoantibodies to these nucleolar antigens have also been found in systemic autoimmune diseases, they do not represent autoimmune reactions unique to cancer but might reflect reaction pathways related to immune responses that are antigen-driven. The ANA response in HCC appears to be dynamic reactions to this antigen-drive since some patients with chronic liver disease showed seroconversion to ANA positivity, marked increase in titer and/or change in antibody specificity preceding or coincident with clinical detection of HCC. These changes in ANA showed a close temporal relationship with transformation from long-established chronic liver disease to HCC.
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PMID:Nucleolar antigens and autoantibodies in hepatocellular carcinoma and other malignancies. 131 27

We have determined the sequence of cDNA for the human histidyl-tRNA synthetase (HRS) in a hepatoma cell line and confirmed it in fetal myoblast and fibroblast cell lines. The newly determined sequence differs in 48 places, including insertions and deletions, from a previously published sequence. By sequence specific probing and by direct sequencing, we have established that only the newly determined sequence is present in genomic DNA and we have sequenced 500 hundred bases upstream of the translation start site. The predicted amino acid sequence now clearly demonstrates all three motifs recognized in class 2 aminoacyl-tRNA synthetases. Alignment of E. coli, yeast, and when available, mammalian predicted amino acid sequences for three of the four members of the class 2a subgroup (his, pro, ser, and thr) shows strong preservation of amino acid specific signature regions proximal to motif 2 and proximal to motif 3. These probably represent the active site binding regions for the proximal acceptor stem and for the amino acid. The first two exons of human HRS contain a 32 amino acid helical motif, first described in human QRS, a class 1 synthetase, which is found also in a yeast RNA polymerase, a rabbit termination factor, and both bovine and human WRS, suggesting that it may be an RNA binding motif.
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PMID:Human histidyl-tRNA synthetase: recognition of amino acid signature regions in class 2a aminoacyl-tRNA synthetases. 154 69

In this study we describe the activation of a protein kinase which phosphorylates a peptide, T669, comprising amino acids 663-681 of the epidermal growth factor receptor and containing the phosphate acceptor site Pro-Leu-Thr669-Pro. In the human epidermoid carcinoma cell line KB, T669 kinase activity in cytosolic extracts peaked (up to 15-fold compared with basal levels) 15-30 min after addition of interleukin-1 (IL-1) and closely paralleled receptor occupancy with a half-maximally effective concentration of approximately 100 pM IL-1 alpha. IL-1 treatment elevated T669 kinase activity to a variable extent in selected fibroblast lines, the hepatoma cell line HepG2, and the murine thymoma EL4 6.1. An IL-1 receptor-negative EL4 variant and the B cell lines 70Z/3, CB23, and RPMI 1788 did not respond in this way. All of the cell lines except 70Z/3 showed increased levels of T669 kinase when treated with the protein kinase C activator phorbol myristate acetate and/or with epidermal growth factor. This finding is in agreement with a previous study (Countaway, J. L., Northwood, I. C., and Davis, R. J. (1989) J. Biol. Chem. 264, 10828-10835). Activators of protein kinase A did not mimic the ability of IL-1 to stimulate T669 kinase activity, nor did the protein kinase C inhibitor staurosporine abrogate the effect of IL-1. T669 kinase activity from IL-1-stimulated KB cells was partially purified by ion exchange, hydrophobic interaction, and size exclusion chromatography. The partially purified enzyme phosphorylated myelin basic protein, a characteristic substrate of microtubule-associated protein-2 kinase (MAP-2 kinase) and the peptide Arg-Arg-Arg-(Tyr-Ser-Pro-Thr-Ser-Pro-Ser)4 from RNA polymerase II. Western blotting of chromatographic fractions revealed that T669 kinase activity corresponded with two proteins of 43 and 45 kilodaltons which cross-reacted with antibodies raised against peptide sequences of rat extracellular signal-regulated kinase-1/microtubule-associated protein-2 kinase. T669 kinase activity was critically dependent on the presence of phosphatase inhibitors. Since both the 43- and 45-kDa proteins, immunoprecipitated from [32P]phosphate-labeled cells, demonstrated a dramatic increase in their levels of serine, threonine, and tyrosine phosphorylation after brief treatment with IL-1, we conclude that IL-1 modulates the activity of these extracellular signal-regulated kinase/microtubule-associated protein-2 kinases by altering the level of their phosphorylation.
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PMID:Interleukin-1 represents a new modality for the activation of extracellular signal-regulated kinases/microtubule-associated protein-2 kinases. 165 5

In vitro transcription of hepatitis B virus DNA (HBV DNA) was studied using nuclear extracts of human hepatoma cell lines. RNA polymerase II-dependent run-off transcription of pre-S mRNA under the control of pre-S1 promoter was observed in nuclear extracts obtained from HepG2 and PLC/PRF/5 cells, and the efficiencies in these extracts were significantly higher than those in nuclear extracts of non-liver cells such as HeLa, Molt-4, and Ehrlich. Analysis of run-off transcripts by the pre-S1 promoter, using deletion mutants of HBV DNA as templates and synthetic oligonucleotides as competitors, showed that hepatocyte nuclear factor 1 was necessary for initiation of in vitro transcription of pre-S mRNA. The run-off transcript of pregenome RNA was also detected and its initiation site was determined. Nuclear extracts of not only hepatoma cells but non-liver cells were active in transcription of pregenome RNA in vitro. However, run-off transcripts of S mRNA and X mRNA were not observed in this system. These results suggest that there were some differences between the mechanisms of HBV DNA transcription in vitro and in vivo. This in vitro transcription system will be useful for clarifying the mechanism regulating transcription of HBV DNA since the biochemical and functional characteristics of the nuclear factors can readily be analyzed.
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PMID:In vitro transcription of the hepatitis B virus gene by nuclear extracts of human hepatoma cells. 185 Sep 18

Human interleukin-6 or B-cell stimulatory factor-2 is a cytokine involved in acute phase and immune response. Cloning of cDNA for human interleukin-6 in the pT7.7 expression plasmid under the control of a bacteriophage T7 RNA polymerase promoter system allows rapid production of the cytokine in Escherichia coli. Upon cell induction with isopropyl thiogalactopyranoside, recombinant human interleukin-6 is overexpressed and forms insoluble inclusion bodies. Solubilization of the protein with 6 M guanidine hydrochloride and refolding in the presence of a reduction/oxidation system results in a quantitative recovery of recombinant human interleukin-6. This material is already 70% pure and can be further purified to homogeneity with a single passage over a weak anionic-exchange column. Extended structural characterization of the purified protein by electrospray mass spectrometry, automated Edman degradation and peptide mapping by high-pressure liquid chromatography/fast-atom-bombardment mass spectrometry demonstrates that recombinant human interleukin-6 is identical to the natural protein both in amino acid sequence and S-S bridge content. However, it contains a minor component accounting for about 20% of the entire translated protein which exhibits a Met-Ala dipeptide extension at the N-terminus. Purified recombinant human interleukin-6 is biologically active because it is able to induce at least 70-fold the human C-reactive promoter transfected in human hepatoma Hep 3B cells and is stable for several months in 10% glycerol at 4 degrees C. The expression system described in the present work has the main advantage of producing a high yield of recombinant human interleukin-6 (about 25 mg/l) combined with a very simple purification scheme.
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PMID:Single-step purification and structural characterization of human interleukin-6 produced in Escherichia coli from a T7 RNA polymerase expression vector. 205 Jan 35

The human ADH1, ADH2, and ADH3 genes are closely related members of a gene family which are differentially expressed during liver development. To begin examining the mechanism of this tissue-specific and stage-specific expression, the 5'-flanking nucleotide (nt) sequences of the three genes were determined and the transcription start point (tsp) were identified. Sequences of all three genes indicated a high degree of homology (greater than 80% nt sequence identity) from the AUG translation start codon to about nt -780 relative to the tsp. Transient transfection assays of a set of plasmids containing various lengths of ADH 5'-flanking DNA fused to cat were performed in the HepG2 and Hep3B human hepatoma cell lines. The results indicated that the ADH2 promoter-proximal region was transcriptionally active in the absence of upstream sequences. To identify potential cis-acting elements in the ADH2 promoter-proximal region, a DNase I footprinting assay using a rat liver nuclear extract was used. Protection occurred in several locations including one, between nt -51 and -10, which shares homology with known binding sites for a previously identified rat-liver transcription factor called CCAAT/enhancer binding protein (C/EBP). Purified C/EBP was shown by footprint analysis to bind at two distinct sites in the ADH2 promoter located at nt -51 to -31 and -21 to -10. The TATA-box promoter element at nt -30 to -22 was not protected by C/EBP, but was partially protected by a factor in the rat liver nuclear extract. Thus, it is possible that the flanking C/EBP molecules may create a novel binding pocket for TFIID, the TATA-binding general transcription factor for RNA polymerase II. Alternatively, the C/EBP molecules may block access to the TATA box, and stimulate transcription of ADH2 by interacting with some component(s) other than TFIID.
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PMID:Promoters for the human alcohol dehydrogenase genes ADH1, ADH2, and ADH3: interaction of CCAAT/enhancer-binding protein with elements flanking the ADH2 TATA box. 216 44

The sequences preceding the albumin mRNA start site are able to direct efficient transcription only upon introduction into cells expressing the endogenous albumin gene. In transient expression assays, the activity of a reporter gene (CAT) linked to this promoter is 100-fold higher in H4II differentiated hepatoma cells than in H5 dedifferentiated cells which no longer express their albumin gene. This tissue specificity depends on the very proximal promoter region, composed of a CCAAT box, the proximal element and a TATA box. Deletion of the CCAAT box leads to a two- to threefold decrease in activity, deletion of the proximal element (PE) results in loss of activity. The PE is a high-affinity binding site for HNF1/APF, a strictly liver specific trans-acting factor. When the affinity of this factor for PE is decreased by bacterial methylation (PE includes a dam methylase site), by mutation, or by its replacement with the homologous element from the alpha-fetoprotein gene (AFP), the activity of the short promoter (PE plus the TATA box) is abolished. This activity can be rescued in the presence of the more upstream elements: DEII, DEI and the CCAAT box (recognized, respectively, by the NF1/CTF, C/EBP and NFY/ACF factors) which are then absolutely required. Our results suggest that the upstream elements contribute to promoter activity by stabilizing the HNF1-PE complex and not by direct interaction with TFIID or the RNA polymerase. It is probable that these elements, essentially dispensable in already differentiated hepatoma cells, play a crucial role during development or differentiation to activate the promoter in cells that contain a low concentration of HNF1 and/or an HNF1 unable to open inactive chromatin alone.
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PMID:Anatomy of the rat albumin promoter. 218 62

The protein components that direct and activate accurate transcription by rat RNA polymerase I were studied in extracts of Novikoff hepatoma ascites cells. A minimum of at least two components, besides RNA polymerase I, that are necessary for efficient utilization of templates were identified. The first factor, rat SL-1, is required for species-specific recognition of the rat RNA polymerase I promoter and may be sufficient to direct transcription by pure RNA polymerase I. Rat SL-1 directed the transcription of templates deleted to -31, the 5' boundary of the core promoter element (+1 being the transcription initiation site). The second factor, rUBF, increased the efficiency of template utilization. Transcription of deletion mutants indicated that the 5' boundary of the domain required for rUBF lay between -137 and -127. Experiments using block substitution mutants confirmed and extended these observations. Transcription experiments using those mutants demonstrated that two regions within the upstream promoter element were required for optimal levels of transcription in vitro. The first region was centered on nucleotides -129 and -124. The 5' boundary of the second domain mapped to between nucleotides -106 and -101. DNase footprint experiments using highly purified rUBF indicated that rUBF bound between -130 and -50. However, mutation of nucleotides -129 and -124 did not affect the rUBF footprint. These results indicate that basal levels of transcription by RNA polymerase I may require only SL-1 and the core promoter element. However, higher transcription levels are mediated by additional interactions of rUBF, and possibly SL-1, bound to distal promoter elements.
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PMID:Characterization of factors that direct transcription of rat ribosomal DNA. 234 70


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