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)

Adenosine (beta,gamma-imido)triphosphate (AMP-PNP) and guanosine (beta,gamma-imido)triphosphate (GMP-PNP) are analogs of ATP and GTP with non-hydrolyzable gamma-phosphates. Although both AMP-PNP and GMP-PNP were used in place of ATP and GTP by Escherichia coli RNA polymerase to transcribe vaccinia virus DNA, only GMP-PNP was used by the transcriptase present within vaccinia virus cores. AMP-PNP specifically prevented initiation of transcription, since RNA initiated in the presence of ATP, GTP, and CTP was subsequently elongated by incubating the washed cores in the presence of AMP-PNP, GTP, CTP, and UTP. The RNA formed in this manner, however, was (i) several times longer than normal transcripts, indicating a defect in chain termination and/or cleavage of nascent RNA, (ii) was not polyadenylylated (although free polyadenylic acid formed), and (iii) was not extruded from the virus cores. Nearest neighbor analysis demonstrated that AMP-PNP was incorporated adjacent to all four nucleotides, and hybridization to restriction endonuclease fragments of vaccinia virus DNA indicated that the high-molecular-weight RNA was transcribed from representative fractions of the entire genome. The possibility of a block in processing rather than or in addition to a block in chain termination was suggested by the cleavage of the high-molecular-weight RNA within the core after replacement of AMP-PNP with ATP. Cleavage of purified high-molecular-weight RNA by a soluble endoribonuclease extracted from vaccinia virus cores, however, was not dependent upon ATP, nor was it inhibited by AMP-PNP. The latter results suggest that AMP-PNP blocks a step preceding cleavage.
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PMID:Multiple roles for ATP in the synthesis and processing of mRNA by vaccinia virus: specific inhibitory effects of adenosine (beta,gamma-imido) triphosphate. 69 Nov 15

Cytoplasmic extracts made from HeLa cells that have been harvested late after infection with vaccinia virus are capable of specifically transcribing templates containing vaccinia virus late-gene promoters. We applied such an extract to a phosphocellulose column and eluted the proteins with a series of buffers containing successively higher concentrations of NaCl. None of three column fractions alone was capable of specific transcription of a late-gene template. However, specific transcriptase activity could be reconstituted by mixing column fractions, with maximal activity seen when all three fractions were present. The activities present in all fractions were heat labile, resistant to micrococcal nuclease, and present only in extracts from vaccinia virus-infected cells. A quantitative complementation assay was used to further purify one factor, named VLTF-1, over subsequent columns of DEAE-cellulose and hydroxylapatite. VLTF-1 was separated from endogenous RNA polymerase, was a late-promoter-specific transcription factor, and had a sedimentation rate consistent with an apparent Mr of 45,000. The RNA polymerase-containing fraction was not only necessary for transcription with a late-promoter template but alone was capable of specifically transcribing a vaccinia virus early-gene promoter. A further difference between early and late gene transcription in this system was in the ability of the ATP analog beta-8-imidoadenosine-5'-triphosphate (AMP-PNP) to substitute for ATP in supporting specific transcription of only the late-promoter template. The system reconstituted from the various fractions retained the ability to produce the novel poly(A) sequence found on the 5' end of vaccinia virus late messages.
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PMID:Identification of factors specific for transcription of the late class of vaccinia virus genes. 247 68

In a continuation of previous efforts to study the modified ATP requirements for RNA synthesis by poIR mutants of vesicular stomatitis virus (VSV), we have used a novel reconstitution assay to show that it is the template moiety of the mutants, not the polymerase proteins, which governs both the increased utilization of the ATP analog, beta, gamma-imido ATP (AMP-PMP), and the loss of a positive cooperativity-like response to varying ATP concentrations. Assays utilized uv-irradiated virus as a source of polymerase proteins and purified N-RNA as templates. Homologous and heterologous transcriptase reactions were carried out with wild-type (wt) virus and each of the two independently isolated poIR mutants. We show that in the presence of wt N-RNA template, substitution of AMP-PNP for ATP resulted in only approximately 5% of control RNA synthesis regardless of which source of polymerase was used. Furthermore, all reactions containing wt N-RNA template responded to varying ATP concentrations with a concave, upward-shaped Lineweaver-Burke plot generally indicative of positive cooperativity effects. In contrast, all reactions which utilized N-RNA templates from the poIR mutants showed an increased utilization of AMP-PNP (greater than 20%) and a more characteristic Michaelis-Menten response to changing ATP concentrations. These findings strongly support the notion that the template-associated nucleocapsid protein modulates the utilization of an ATP site which is directly or indirectly involved in VSV RNA synthesis.
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PMID:Altered ATP utilization by the poIR mutants of vesicular stomatitis virus maps to the N-RNA template. 284 22

To facilitate further studies of flavivirus transcription, cell extraction methods and in vitro reaction conditions which increased West Nile virus (WNV) RNA-dependent RNA polymerase activity were determined. Subcellular fractions from WNV-infected BHK-21/W12 cells were characterized with regard to their protein and RNA content and in vitro polymerase activity. In both a cytoplasmic fraction, designated S1, and a fraction enriched for outer nuclear membranes, designated S2, seven virus-specific proteins, NS5 (96 kilodaltons [kDa]), NS3 (67 kDa), E (48 kDa), NS1 (47 kDa), ns4a (26 kDa), ns2a (17 kDa), and ns2b (14.5 kDa), were detected. The fractions also contained virus-specific RNA and cellular rRNA and mRNA. Polymerase activity in S1 and S2 fractions from WNV-infected cells was concentrated by pelleting and consisted of two types of enzyme activities: the WNV RNA-dependent RNA polymerase and terminal transferases of cellular origin. Enhanced levels of WNV polymerase activity were obtained from these cell fractions by altering several of the in vitro reaction conditions. Although Mg2+ was the divalent cation preferred by WNV polymerase, virus-specific in vitro transcription was detected at reduced levels when Mn2+ (0.05 or 0.5 mM) was present as the sole divalent cation. Product analysis revealed that the viral polymerase incorporated radiolabeled ribonucleotides into three distinct RNA species. Free single-stranded genome-sized RNA which was LiCl insoluble and RNase sensitive was found by fingerprint analysis to have an oligonucleotide pattern similar to that of WNV genomic RNA. RNA molecules which comigrated as a broad band near the top of the gel were separable into LiCl-insoluble, partially RNase-sensitive replicative-intermediate RNA and LiCl-soluble, RNase-resistant replicative-form RNA. The cellular transferases added UMP or AMP residues to the 3'-termini of cellular mRNA, tRNA, and 18S and 28S rRNA. Although a cellular terminal transferase has been reported to function in initiation of poliovirus transcription, no labeling of the WNV RNA by either of these cellular enzymes was detected. Therefore, they appear to play no specific role in flavivirus RNA synthesis.
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PMID:Characterization of West Nile virus RNA-dependent RNA polymerase and cellular terminal adenylyl and uridylyl transferases in cell-free extracts. 302 63

The presence of terminal nucleotidyl transferase activities catalyzing the addition of AMP, CMP, GMP, and UMP residues to the 3' ends of oligonucleotide primers was detected in healthy tomato plants. These enzyme activities copurify with RNA-dependent RNA polymerase during the initial stages of purification. Their separation from RNA-dependent RNA polymerase is finally achieved by DEAE chromatography: terminal transferase activities are retained on DEAE while RNA-dependent RNA polymerase does not bind in the presence of 20 mM MgCl2. Elution by a linear gradient of 0 to 400 mM NH4Cl releases all four terminal transferase activities from the DEAE column at a concentration of 270 mM NH4Cl, thus suggesting that they may belong to one enzyme molecule; this question, however, needs further clarification. The enzyme activities are completely dependent on the presence of an RNA primer and are strongly influenced by its base composition as well as its chain length. Characterization of the respective reaction products by electrophoresis on 15% polyacrylamide sequencing gels reveals striking differences as to the number of nucleotides added to a given primer. In the case of UMP transfer to U8 or A8 and in the case of GMP transfer to A8 only 1 to 6 nucleoside monophosphates are added to the 3' terminus of the oligonucleotide primer, whereas in the case of AMP transfer to A8 or U8, the CMP transfer to A8, and the GMP transfer to U8, longer chains of minimally 30 nucleotides are added to the respective primer. After gradient elution from DEAE the transferase preparation displays no nucleolytic activity when incubated in the presence of 3H-labelled ribosomal RNA or [3H]poly(A) X poly(U). Only in the case of [3H]poly(A) and [3H]poly(U) or [3H]poly(C) 10 to 15% of the radioactivity is transferred to acid-soluble counts.
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PMID:Simultaneous presence of terminal adenylyl, cytidylyl, guanylyl, and uridylyl transferase in healthy tomato leaf tissue: separation from RNA-dependent RNA polymerase and characterization of the terminal transferases. 628 7

Previous studies with U937 cells, a human monocyte cell line, have shown that the activity of cyclic nucleotide phosphodiesterase 4 (PDE4) is increased by agents that elevate cyclic AMP content. The present experiments were conducted to determine 1) whether an increase in PDE4 steady-state message and/or protein accompanies the up-regulation of PDE4 activity and 2) whether the up-regulation changes the functional responses of U937 cells to activators of adenylyl cyclase. To up-regulate PDE4 activity, U937 cells were treated for 4 h with a combination of 1 microM salbutamol, a beta-adrenoceptor agonist, and 30 microM rolipram, a PDE4 inhibitor. Cells were washed extensively to remove drugs and used immediately in various experimental protocols. Reverse transcriptase-polymerase chain reactions conducted with primers specific for the four PDE4 subtypes suggested that pretreatment with salbutamol and rolipram increased steady-state mRNA levels of PDE4A and PDE4B, but not PDE4C or PDE4D. Immunoblot analyses using two rabbit polyclonal antibodies, one directed against human recombinant PDE4A and PDE4D and a second directed against human recombinant PDE4B, revealed bands of immunoreactivity corresponding to approximately 125 kDa (PDE4A) and approximately 70 kDa (PDE4B), respectively, that increased in intensity after cells were treated with salbutamol and rolipram. As demonstrated in both time course and concentration-response studies with prostaglandin E2 (PGE2), an agent that activates adenylyl cyclase by a non-beta-adrenoceptor-mediated mechanism, cAMP accumulation was substantially decreased in cells in which PDE4 activity had been up-regulated. The difference in PGE2-stimulated cAMP accumulation between control and PDE4 up-regulated cells was greatly reduced in the presence of rolipram, consistent with the notion that an increase in PDE4 activity was responsible for the heterologous desensitization. Functionally, up-regulation of PDE4 markedly decreased the ability of PGE2 to inhibit LTD4-induced Ca2+ mobilization in intact cells. A hypothetical implication of these results is that increasing PDE4 activity in vivo by administering beta-adrenoceptor agonists could exacerbate inflammatory processes by decreasing the activity of endogenous anti-inflammatory agents such as PGE2.
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PMID:Salbutamol up-regulates PDE4 activity and induces a heterologous desensitization of U937 cells to prostaglandin E2. Implications for the therapeutic use of beta-adrenoceptor agonists. 755 25

The catalytic properties of electrophoretically homogeneous RNA-directed RNA polymerase (RdRP, EC 2.7.7.48) from tomato leaf tissue were studied with the aid of oligonucleotides of defined sequence. It was found that RdRP catalyzes in vitro the transcription of short single-stranded RNA and DNA molecules into precisely complementary RNA copies up to the full length of these templates. The transcription of RNA- and DNA-oligonucleotide templates was equally effective. Differences in transcription efficiency were found to depend on nucleotide sequence rather than on the RNA or DNA nature of the single-stranded nucleic acid. Double-stranded nucleic acids such as poly(A).poly(U) and a double-stranded DNA 14-mer were not transcribed. The RdRP-directed transcription could be primed because RNA and DNA dinucleotides and trinucleotides complementary to the 3'-terminal nucleotides of the template were extended by the enzyme. The unprimed transcription was shown to start preferentially at the 3'-terminal nucleotides of the template. RdRP is capable of adding a single noncomplementary nucleotide to the 3' terminus of about 50% of the runoff transcripts. AMP was preferred over GMP, whereas CMP and UMP were terminally added at very low frequency.
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PMID:RNA-directed RNA polymerase from tomato leaves. II. Catalytic in vitro properties. 768 23

There is increasing evidence that the membrane-bound thyrotropin receptor (TSHR) may be mediating clinically important direct effects of thyrotropin (TSH) and of TSHR antibodies (TSHRab) in extra-thyroidal tissues. TSHR mRNA has formerly been detected in thyroid, retroorbital muscle and fibroblasts, peripheral lymphocytes and rodent fat. It is well known that thyroid disease may aggravate or induce heart disease, but the pathophysiological role of TSH and TSHRab is not clear. The aim of this study was to investigate if TSHR is present in cardiac muscle. Reverse transcriptase polymerase chain reactions revealed TSHR in human heart and Northern blot on extracted RNA showed a RNA species of 4.4 kb. TSH stimulation of cultured mouse AT-1 cardiomyocytes elevated the levels of intracellular second messenger 3',5'-cyclic AMP. This effect of TSH could be inhibited by TSHR antibodies. In solution hybridization levels of TSHR mRNA in AT-1 cells were 50% of mRNA in crude mouse heart. In conclusion functional TSHR is present in cardiomyocytes.
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PMID:Evidence for the presence of functional thyrotropin receptor in cardiac muscle. 779 53

A terminal adenylyl transferase (TATase) activity has been identified in preparations of purified poliovirus RNA-dependent RNA polymerase (3Dpol). Highly purified 3Dpol is capable of adding [32P]AMP to the 3' ends of chemically synthesized 12-nucleotide (nt)-long RNAs. The purified 52-kDa polypeptide, isolated after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and renatured, retained the TATase activity. Two 3Dpol mutants, purified from Escherichia coli expression systems, displayed no detectable polymerase activity and were unable to catalyze TATase activity. Likewise, extracts from the parental E. coli strain that harbored no expression plasmid were unable to catalyze formation of the TATase products. With the RNA oligonucleotide 5'-CCUGCUUUUGCA-3' used as an acceptor, the products formed by wild-type 3Dpol were 9 and 18 nt longer than the 12-nt oligomer. GTP, CTP, and UTP did not serve as substrates for transfer to this RNA, either by themselves or when all deoxynucleoside triphosphates were present in the reaction. Results from kinetic and stoichiometric analyses suggest that the reaction is catalytic and shows substrate and enzyme dependence. The 3'-terminal 13 nt of poliovirus minus-strand RNA also served as an acceptor for TATase activity, raising the possibility that this activity functions in poliovirus RNA replication. The efficiency of utilization and the nature of the products formed during the reaction were dependent on the acceptor RNA.
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PMID:Identification of terminal adenylyl transferase activity of the poliovirus polymerase 3Dpol. 805 62

1. 5-Hydroxytryptamine (5-HT) elicited a dose-dependent stimulation of intracellular adenosine 3': 5'-cyclic monophosphate (cyclic AMP) accumulation in cultured astrocytes derived from neonatal rat (Sprague Dawley) thalamic/hypothalamic area with a potency (pEC50) of 6.68 +/- 0.08 (mean +/- s.e. mean). 2. In order to characterize the 5-HT receptor responsible for the cyclic AMP accumulation the effects of a variety of compounds were investigated on basal cyclic AMP levels (agonists) and 5-carboxamidotryptamine (5-CT) stimulated cyclic AMP levels (antagonists). The rank order of potency for the agonists investigated was 5-CT (pEC50 = 7.81 +/- 0.09) > 5-methoxytryptamine (5-MeOT) (pEC50 = 6.86 +/- 0.36) > 5-HT (pEC50 = 6.68 +/- 0.08). The following compounds, at concentrations up to 10 microM, did not affect basal cyclic AMP levels 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT), cisapride, sumatriptan, DOI and RU 24969. The rank order of potency of antagonists was methiothepin (pKi = 7.98 +/- 0.25) > mesulergine (pKi = 7.58 +/- 0.18) > ritanserin (pKi = 7.20 +/- 0.24) > clozapine (pKi = 7.03 +/- 0.19) > mianserin (pKi = 6.41 +/- 0.19). The following compounds, at concentrations up to 10 microM, were inactive: ketanserin, WAY100635, GR127935. This pharmacological profile is consistent with that of 5-HT7 receptor subtype-mediated effects. 3. The cultured astrocytes exhibited regional heterogeneity in the magnitude of cyclic AMP accumulation (Emax). Cells cultured from the thalamic/hypothalamic area had significantly higher Emax values (588 +/- 75% and 572 +/- 63% of basal levels for 5-CT and 5-HT, respectively) compared to brainstem (274 +/- 51% and 318 +/- 46%, respectively) and colliculus astrocytes (244 +/- 15% and 301 +/- 24%, respectively). No significant differences in pEC50 (for either 5-HT or 5-CT) values were observed. 4. Reverse transcriptase-polymerase chain reaction (RT-PCR) with primers specific for the 5-HT7 receptor confirmed expression of messenger RNA for this receptor subtype by the cultured astrocytes derived from all regions investigated. Primers specific for the 5-HT6 receptor also amplified a cDNA fragment from the same samples. 5. From these findings, we conclude that astrocytes cultured from a number of brain regions express functional 5-HT receptors positively coupled to adenylyl cyclase and that the level of receptor expression or the efficiency of receptor coupling is regionally-dependent. The pharmacological profile of the receptor on thalamic/hypothalamic astrocytes suggests that the 5-HT7 receptor is the dominant receptor that is functionally expressed even though astrocyte cultures have the capacity to express both 5-HT6 and 5-HT7 receptor messenger RNA.
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PMID:Identification of 5-hydroxytryptamine receptors positively coupled to adenylyl cyclase in rat cultured astrocytes. 903 57


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