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)

Tyrosine aminotransferase mRNA was quantitated by translation in a cell-free system derived from wheat germ followed by specific immunoprecipitation of the newly synthesized enzyme subunit. Hepatic poly(A)-containg RNA prepared from rats treated for 4 h with N6, O2'-dibutyryl cyclic AMP and theophylline was approximately 5.6 times more active in directing the synthesis of the tyrosine aminotransferase subunit relative to untreated controls. The overall template activity of the RNA prepared from control and cyclic AMP-treated animals was virtually identical, demonstrating that the cyclic nucleotide effect was specific for the tyrosine aminotransferase mRNA. At all times, after a single injection of dibutyryl cyclic AMP and theophylline, the increase in hepatic enzyme activity was accompanied by corresponding induction in the level of functional tyrosine aminotransferase mRNA. Other inducers of tyrosine aminotransferase, such as glucagon and hydrocortisone, also increased the level of tyrosine aminotransferase mRNA in proportion to their effect on enzyme activity. The RNA polymerase II inhibitor, alpha-amanitin, completely blocked the dibutyryl cyclic AMP-mediated increase in tyrosine aminotransferase mRNA activity. These studies demonstrate that, in intact animals, the induction of tyrosine aminotransferase activity by dibutyryl cyclic AMP can be completely accounted for by a corresponding increase in the level of functional mRNA coding for the enzyme.
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PMID:Increase in hepatic tyrosine aminotransferase mRNA during enzyme induction by N6,O2'-dibutyryl cyclic AMP. 2 49

Rat liver tyrosine aminotransferase has been expressed in Saccharomyces cerevisiae and Escherichia coli. In yeast, the extent of production is 20-fold higher than that in rat liver after induction by dexamethasone, and reaches 250-fold higher in an E. coli strain carrying the T7 RNA polymerase transcription system. About 250 mg pure and homogeneous enzyme was obtained from 50 g transformed E. coli cells. Determination of Mr and pI, as well as analysis of N- and C-terminal amino acids, suggest that the isolated protein is native. The catalytic properties, similar to those of the enzyme from rat liver, confirm that it is fully active and that post-translational modifications in the mammalian cells are not essential for activity. Pyridoxal 5'-phosphate strongly protects the enzyme against thermal inactivation. After denaturation, 10 thiol groups, out of 16 in the polypeptide chain, react with 5,5'-dithiobis(2-nitrobenzoic acid) whereas only five or six are accessible under native conditions. Two thiols are rapidly modified with concomitant inactivation of the apoenzyme, but pyridoxal 5'-phosphate partially protects them in the holoenzyme. The results are interpreted in the light of the structure/function relationship in this enzyme.
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PMID:Expression of mammalian tyrosine aminotransferase in Saccharomyces cerevisiae and Escherichia coli. Purification to homogeneity and characterization of the enzyme overproduced in the bacteria. 168 48

Interaction of purified eukaryotic RNA polymerase II with various synthetic palindromic DNA sequences is associated with the formation of transcriptional complexes of different stabilities, i.e. having different propensities for releasing the nascent transcript. This phenomenon was observed by using wheat-germ RNA polymerase II and a series of double-stranded template polymers containing palindromic repeating motifs of 6-16 bp, with regulatory alternating purine and pyrimidine bases such as d[ATA(CG)nC].d[TAT(GC)nG], with n = 1, 3 or 6 referred to as d(GC), d(GC)3 or d(GC)6, respectively. We also synthesized two double-stranded methylated polymers, containing the repeating units d(ATAm5CGm5C).d(TATGm5CG) and d[ATA(m5CG)6m5C].d[TAT(Gm5C)6G] [designated d(GmC) and d(GmC)6, respectively]. All of these polymers served as templates for the reaction of single-step addition of CTP to a CpG primer catalysed by wheat-germ RNA polymerase II, to an extent that seems well correlated with the number of potential initiation sites within the DNA molecules. Furthermore, in these reactions, the enzyme appears to form relatively stable transcriptional complexes, as trinucleotide product was released only very slowly. In marked contrast to the results with the CpG primer, the single-step addition reaction primed by UpA, i.e. the synthesis of UpApU proceeded at a much higher velocity and was strongly enhanced by increasing the d(G-C) content of the repeating units of the DNA polymers. Thus, taking into account the number of potential sites at which UpApU synthesis could occur, the extent of UpApU synthesis was increased about 12-fold with d(GC)6 compared to that with the d(GC) template. The catalytic nature of the reaction necessarily implies that the stability of the transcription complexes with the plant RNA polymerase II decreased as the d(G-C) content of the repeating motif increased. Furthermore, although the synthesis of CpGpC could be demonstrated with d(GmC)6 as template, the UpA-primed synthesis of UpApU could not be detected with this polymer. The results obtained in transcription of these polymers are discussed in relation to the potential involvement of palindromic DNA in transcription termination and attenuation in the presence of RNA polymerase II.
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PMID:Transcription of synthetic DNA containing sequences with dyad symmetry by wheat-germ RNA polymerase II. Increased rates of product release in single-step addition reactions. 199 1

Highly efficient promoters of coliphage T5 were identified by selecting for functional properties. Eleven such promoters belonging to all three expression classes of the phage were analyzed. Their average AT content was 75% and reached 83% in subregions of the sequences. Besides the well-known conserved sequences around -10 and -33, they exhibited homologies outside the region commonly considered to be essential for promoter function. Interestingly, the consensus hexamers around -10 (TAT AAT) and -35 (TTG ACA) were never found simultaneously within the sequence of highly efficient promoters. Several of these promoters compete extremely well for Escherichia coli RNA polymerase and can be used for the efficient in vitro synthesis of defined RNA species. In addition, some of these promoters accept 7-mGpppA as the starting dinucleotide, thus producing capped mRNA in vitro which can be utilized in various eucaryotic translation systems.
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PMID:Promoters recognized by Escherichia coli RNA polymerase selected by function: highly efficient promoters from bacteriophage T5. 390 50

Time- and dose-dependence of the formation of the different cytoplasmic hormone-protein complexes were studied in the rat liver after administration in vivo of [3H]cortisol or [3H]dexamethasone and compared with the stimulation of RNA polymerase B and induction of tyrosine aminotransferase and tryptophan oxygenase. No correlation could be found between formation in vivo of any of the five cytoplasmic hormone-protein complexes found and stimulation of RNA polymerase B activity or enzyme induction. After administration of [3H]cortisol, different metabolites of cortisol could be demonstrated in the isolated hormone-protein complexes. No time- or dose-dependence of the metabolite patterns could be observed after application of hormone doses that were in the range of the biologically active doses. After administration of [3H]dexamethasone, the same hormone-protein complexes were observed, which contained, however, the injected steroid instead of metabolites. These results seem to indicate that the cytoplasmic binding components present in the rat liver are enzymes involved in the metabolism of the glucocorticosteroids and that dexamethasone binds to these enzymes as a substrate analogue.
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PMID:No correlation between binding of glucocorticosteroids to specific cytoplasmic proteins in vivo and enzyme induction in the rat liver. 613 71

Analysis of primary transcripts made by Escherichia coli RNA polymerase on T4 DNA containing an intact or partially deleted tRNA gene cluster demonstrates that the T4 tRNA genes are transcribed from two promoters differing in their strength. The stronger (P1) and the weaker (P2) promoters are located at distances of 1 kb and 1.5 kb from the tRNA genes, respectively. Selective initiation of individual transcripts with dinucleotides shows that P1 and P2 promoters contain the sequences TAT and CAC respectively. The two-promoter organisation of the tRNA cluster may reflect two superimposed mechanisms of gene expression in T4-infected bacteria.
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PMID:IN vitro transcription of bacteriophage T4 tRNA gene cluster from two different promoters. 722 Mar 46

Mounting experimental evidence suggests that the TAT protein, released from human immunodeficiency virus-1 (HIV-1)-infected inflammatory cells, may genetically reprogram targeted cells within a localized environment to develop highly vascularized tumors of mesenchymal origin. The fibroblast growth factor (FGF) family of polypeptides has gained general acceptance as initiators of angiogenesis and functions as potent mitogens for mesoderm-derived cells. To evaluate a potential biological relationship between TAT and acidic FGF (FGF-1), primary murine embryonic fibroblasts either were transfected with the viral transactivator or were transduced (retrovirally mediated) with a secreted, chimeric form of the human polypeptide growth factor, human stomach tumor/Kaposi's sarcoma (hst/KS)FGF-1. Reverse transcriptase-polymerase chain reaction, Western blotting, in situ immunohistochemical, heparin affinity, DNA synthesis, and transient transfection techniques were used to confirm expression, localization, and functionality of the transgenes. Both transfected and transduced cells constitutively expressing either TAT or (hst/KS)FGF-1 adopted a transformed phenotype, maintained aggressive growth behavior, and demonstrated both induction of FGF-specific phosphotyrosyl proteins and nuclear association of FGF-1 and FGF-1 receptor. Increased levels of endogenous, murine FGF-1 mRNA (reverse transcriptase-polymerase chain reaction) and protein (immunoblot analysis) were apparent in both (hst/KS)FGF-1- and TAT-transformed cells. Medium conditioned by (hst/KS)FGF-1-transduced cells contained steady-state levels of biologically active FGF-1 which exhibited a representative molecular weight. Limited sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of the conditioned medium from TAT-transformed cells demonstrated the appearance of FGF-1 as latent, high molecular weight complexes requiring reducing agents to activate full biological activity. Collectively, these results suggest that TAT induces the expression and secretion of FGF-1, which may be potentially relevant to the pathophysiological development of AIDS-Kaposi's sarcoma.
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PMID:The HIV-1 TAT protein induces the expression and extracellular appearance of acidic fibroblast growth factor. 754 39

The glucocorticoid-responsive units (GRUs) of the rat tyrosine aminotransferase were associated with the regulatory sequences of a cellular gene expressed ubiquitously--that coding for the largest subunit of RNA polymerase II. In transient expression assays, glucocorticoid responsiveness of the hybrid regulatory regions depends on the spatial relationship and number of regulatory elements. Two parameters affect the ratio of induction by glucocorticoids: the basal level of the hybrid promoter that is affected by the RNA polymerase II regulatory sequences and the glucocorticoid-induced level that depends on the distance between the GRUs and the TATA box. A fully active glucocorticoid-responsive hybrid gene was used to generate transgenic mice. Results show that a composite regulatory pattern is obtained: ubiquitous basal expression characteristic of the RNA polymerase II gene and liver-specific glucocorticoid activation characteristic of the tyrosine aminotransferase GRUs. This result demonstrates that the activity of the tyrosine aminotransferase GRUs is cell-type-specific not only in cultured cells but also in the whole animal.
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PMID:Tissue specificity of a glucocorticoid-dependent enhancer in transgenic mice. 763 67

A 32-kDa polypeptide corresponding to NAC, the product of the Klebsiella aerogenes nac gene, was overexpressed from a plasmid carrying a tac'-'nac operon fusion and purified to near homogeneity by taking advantage of its unusual solubility properties. NAC was able to shift the electrophoretic migration of DNA fragments carrying the NAC-sensitive promoters hutUp, putPp1, and ureDp. The interaction between NAC and hutUp was localized to a 26-bp region centered approximately 64 bp upstream of the hutUp transcription initiation site. Moreover, NAC protected this region from DNase I digestion. Mobility shift and DNase I protection studies utilizing the putP and ureD promoter regions identified NAC-binding regions of sizes and locations similar to those found in hutUp. Comparison of the DNA sequences which were protected from DNase I digestion by NAC suggests a minimal NAC-binding consensus sequence: 5'-ATA-N9-TAT-3'. In vitro transcription assays demonstrated that NAC was capable of activating the transcription of hutUp by sigma 70-RNA polymerase holoenzyme when this promoter was presented as either a linear or supercoiled DNA molecule. Thus, NAC displays the in vitro DNA-binding and transcription activation properties which have been predicted for the product of the nac gene.
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PMID:The nitrogen assimilation control protein, NAC, is a DNA binding transcription activator in Klebsiella aerogenes. 776 65

In order to clone candidate tumor suppressor genes whose loss contributes to the pathogenesis of neuroblastoma (NB), we performed polymerase chain reaction (PCR) screening using a high-density sequence tagged site-content map within a commonly deleted region (chromosome band 1p36) in 24 NB cell lines. We found a approximately 480 kb homozygously deleted region at chromosome band 1p36.2 in one of the 24 NB cell lines, NB-1, and cloned the human homologue (KIF1B-beta) of the mouseKif1B-beta gene in this region. The KIF1B-beta gene had at least 47 exons, all of which had a classic exon-intron boundary structure. Mouse Kif1B is a microtubule-based putative anterograde motor protein for the transport of mitochondria in neural cells. We performed mutational analysis of the KIF1B-beta gene in 23 cell lines using 46 sets of primers and also an allelic imbalance (AI) analysis of KIF1B-beta in 50 fresh NB samples. A missense mutation at codon 1554, GTG (Gly) to ATG (Met), silent mutations at codon 409 (ACG to ACA) and codon 1721 (ACC to ACT), and polymorphisms at codon 170, GAT (Asp) to GAA (Glu), and at codon 1087, TAT (Tyr), to TGT (Cys), were all identified, although their functional significances remain to be determined. The AI for KIF1B-beta was slightly higher (38%) than those for the other two markers (D1S244, D1S1350) (35 and 32%) within the commonly deleted region (1p36). Reverse transcriptase-PCR analysis of the KIF1B-beta gene revealed obvious expression in all NB cell lines except NB-1, although decreased expression of the KIF1B-beta gene was found in a subset of early- and advanced-stage NBs. These results suggest that the KIF1B-beta gene may not be a candidate for tumor suppressor gene of NB.
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PMID:Genomic structure and mutational analysis of the human KIF1B gene which is homozygously deleted in neuroblastoma at chromosome 1p36.2. 1152 94


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