Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.7.6 (RNA polymerase)
 34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In subcellular extracts of Kunjin virus-infected cells prepared by lysis and differential centrifugation, the viral RNA polymerase, RNA and proteins were associated mainly with cytoplasm. When the cytoplasmic extract (500 g supernate) of infected cells labelled for 3 h from 24 h post-infection was further fractionated by rapid centrifugation through a sucrose density gradient, all viral products were located only in dense or "heavy membrane" fractions, which contained three types of virus-induced morphologically distinct membrane structures. These dense fractions were treated with 0.5% NP40 and the soluble material was again centrifuged through a sucrose gradient for analyses as before. Viral RNA polymerase activity was retained and was associated with replicative intermediate RNA and some replicative form RNA in the peak enzyme fractions sedimenting at 20S to 40S. Enrichment of NS3 and of the small nonstructural proteins NS2A and NS2B/NS4A was apparent in these fractions which were well separated from the slow sedimenting structural proteins. No detergent-resistant structures in the "heavy membrane" fractions other than ribosome-like particles were visible. The data show that the RNA polymerase complex cosedimented with virus-induced membrane structures and remained associated with specific nonstructural proteins and replicative intermediate RNA after detergent treatment.
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PMID:Molecular and ultrastructural analysis of heavy membrane fractions associated with the replication of Kunjin virus RNA. 132 51

We have examined the role of ras-related rab proteins in transport from the ER to the Golgi complex in vivo using a vaccinia recombinant T7 RNA polymerase virus to express site-directed rab mutants. These mutations are within highly conserved domains involved in guanine nucleotide binding and hydrolysis found in ras and all members of the ras superfamily. Substitutions in the GTP-binding domains of rab1a and rab1b (equivalent to the ras 17N and 116I mutants) resulted in proteins which were potent trans dominant inhibitors of vesicular stomatitis virus glycoprotein (VSV-G protein) transport between the ER and cis Golgi complex. Immunofluorescence analysis indicated that expression of rab1b121I prevented delivery of VSV-G protein to the Golgi stack, which resulted in VSV-G protein accumulation in pre-Golgi punctate structures. Mutants in guanine nucleotide exchange or hydrolysis of the rab2 protein were also strong trans dominant transport inhibitors. Analogous mutations in rab3a, rab5, rab6, and H-ras did not inhibit processing of VSV-G to the complex, sialic acid containing form diagnostic of transport to the trans Golgi compartment. We suggest that at least three members of the rab family (rab1a, rab1b, and rab2) use GTP hydrolysis to regulate components of the transport machinery involved in vesicle traffic between early compartments of the secretory pathway.
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PMID:GTP-binding mutants of rab1 and rab2 are potent inhibitors of vesicular transport from the endoplasmic reticulum to the Golgi complex. 142 35

We have tested the hypothesis that the flavivirus nonstructural protein NS3 is a viral proteinase that generates the termini of several nonstructural proteins by using an efficient in vitro expression system and monospecific antisera directed against the nonstructural proteins NS2B and NS3. A series of cDNA constructs was transcribed by using T7 RNA polymerase, and the RNA was translated in reticulocyte lysates. The resulting protein patterns indicated that proteolytic processing occurred in vitro to generate NS2B and NS3. The amino termini of NS2B and NS3 produced in vitro were found to be the same as the termini of NS2B and NS3 isolated from infected cells. Deletion analysis of cDNA constructs localized the protease domain within NS3 to the first 184 amino acids but did not eliminate the possibility that sequences within NS2B were also required for proper cleavage. Kinetic analysis of processing events in vitro and experiments to examine the sensitivity of processing to dilution suggested that an intramolecular cleavage between NS2A and NS2B preceded an intramolecular cleavage between NS2B and NS3. The data from these expression experiments confirm that NS3 is the viral proteinase responsible for cleavage events generating the amino termini of NS2B and NS3 and presumably for cleavages generating the termini of NS4A and NS5 as well.
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PMID:In vitro processing of dengue virus type 2 nonstructural proteins NS2A, NS2B, and NS3. 214 43

We have investigated a number of genes for possible overexpression upon induction of transformation, using mouse cells transformed with a temperature-sensitive mutant of simian virus 40 [SV40Ts(A)-transformed BALB-3T3, line A255], which express the transformed phenotype at 33 degrees C and suppress it as 39 degrees C. We examined both RNA polymerase II transcripts belonging to selected cellular oncogenes and RNA polymerase III transcripts of the B2 class. The selection was predicated on the basis of previous reports of an overexpression of these genes in transformed cell lines or tumors. Among the oncogenes, only cellular myc mRNA was found in increased amounts after transfer to the permissive temperature. However, nuclear run-on transcription assays indicated accelerated transcription rates for both c-myc and c-ras, with 2-3-fold and 4-5-fold increases at 2 and 6 hr post-induction, respectively. This acceleration was preceded by a 16-fold increase in the transcription rate of mouse B2 genes occurring within 30 min of the transfer to 33 degrees C. The sizeable and immediate burst in the rate of B2 transcription following the induction shift and the temporal order of activation of c-myc and c-ras are suggestive of a causal progression leading to full expression of the transformed phenotype.
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PMID:Increased rates of specific RNA polymerase III transcription precede overexpression of cellular oncogenes upon induction of transformation. 247 9

The E1a gene of adenovirus encodes two proteins, 289 and 243 amino acids long, which have positive (transactivator) and negative (enhancer repressor) RNA polymerase II transcriptional regulatory properties and cell transformation activities including cooperation with an activated ras gene. The E1a transforming functions more closely correlate with the repressor property than with transactivation in that both E1a proteins express the repressor and transformation functions while only the 289-amino-acid protein is an efficient transactivator. To understand whether the transcriptional regulatory activities of E1a are related to its ras cooperation activity, we generated a series of mutant E1a expression vectors by linker insertion mutagenesis of the 289-amino-acid protein. Here we describe a new class of mutants which although defective for enhancer repression still can cooperate with the ras oncogene in cell transformation. The mutants are also defective in transcription transactivation. Our data suggest that enhancer repression and transformation via ras cooperation are separate E1a functions and that cooperation with ras does not rely on either of the RNA polymerase II transcription regulatory functions of E1a. We also show that mutations which inactivate enhancer repression are not confirmed to a single critical domain necessary for repression. We therefore propose that the integrity of the overall configuration of the E1a proteins is important for the repression activity.
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PMID:Adenovirus E1a ras cooperation activity is separate from its positive and negative transcription regulatory functions. 296

Transcription factor CTF, which is responsible for selective recognition of eukaryotic promoters that contain the sequence CCAAT, was purified to apparent homogeneity by sequence-specific DNA affinity chromatography. Binding sites for CTF in the human Ha-ras and alpha-globin promoters were highly homologous to sequences recognized by nuclear factor I (NF-I), a cellular DNA-binding protein that is required for the initiation of adenovirus DNA replication in vitro. To determine the relationship between CTF and NF-I, we compared the biochemical properties of these two proteins. CTF and NF-I were found to be indistinguishable in polypeptide composition, DNA-binding properties, immunological cross-reactivity, and in vitro stimulation of DNA replication and transcription initiation. We conclude that CTF/NF-I can serve both as a transcription selectivity factor for RNA polymerase II and as an initiation factor for adenovirus DNA replication.
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PMID:A cellular DNA-binding protein that activates eukaryotic transcription and DNA replication. 302 47

The modulation of RNA polymerase II processivity through the untranslated N-myc first exon represents an important mechanism governing N-myc mRNA levels during normal development. In this study, we employed the rat embryo fibroblast (REF) cooperation assay as a functional means to (i) map N-myc first exon sequences involved in the regulation of N-myc gene expression and specifically their contribution to controlling a block to transcriptional elongation and (ii) determine whether this transcriptional control mechanism plays a role in governing the oncogenic activity of N-myc. Transfection of N-myc expression constructs harboring various deletions within the untranslated first exon revealed that a region encoding a potential stem-loop structure followed by a thymine stretch (stem-loop/T region) was required for efficient transcriptional attenuation. The increase in transcriptional read-through associated with all deletions involving the stem-loop/T region also resulted in a more aggressive malignant phenotype as evidenced by a significant enhancement in the subcloning efficiency of N-myc/ras transformed foci. Most significantly, when cell lines generated from overexpression of the intact N-myc expression construct were selected for anchorage-independent growth, a strong block to transcriptional elongation was completely eliminated in all cases examined. Since the subcloning efficiency of transformed foci and the capacity of permanently established cell lines for anchorage-independent growth are direct correlates of more advanced stages of malignant transformation, our findings suggest that loss of transcriptional attenuation represents an important genetic event in the progression of N-myc-induced cellular transformation.
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PMID:Loss of transcriptional attenuation in N-myc is associated with progression towards a more malignant phenotype. 747 16

The tyrosinase promoter has been used to target expression of the mutated human T24 Ha-ras oncogene in pigment-producing cells of transgenic mice. Two independent founder mice carrying the transgene survived and showed the same distinct phenotype of mutated coat color, deeply pigmented skin with multiple nevi, and twirling behavior. The offspring of one of these founders were developed into a line that stably expressed the same phenotype. Histopathological analysis of the tissues revealed hyperpigmentation and/or melanocytic hyperplasia in the skin, eyes, inner ear, and meningeal membranes in the brain. Reverse transcriptase-polymerase chain reaction analysis revealed expression of the transgene in skin, brain, and spleen. We propose that these transgenic mice will be a model for studying the process of multistage melanoma carcinogenesis and a system for evaluating potential chemopreventive agents.
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PMID:Hyperpigmentation and melanocytic hyperplasia in transgenic mice expressing the human T24 Ha-ras gene regulated by a mouse tyrosinase promoter. 766 20

IL-8 is a chemotactic cytokine with proinflammatory and growth-promoting activities. Recently it has been shown to influence several functions of keratinocytes, including HLA-DR expression, chemotaxis, and proliferation by binding to a specific receptor. Because psoriasis vulgaris is characterized by epidermal hyperproliferation and infiltration of inflammatory cells, we investigated the expression of IL-8 and its receptor in normal and psoriatic epidermis using semiquantitative reverse-transcriptase-polymerase chain reaction. In addition the mRNA levels of the proto-oncogenes c-ras, c-raf, c-myc, and HER-2 were also investigated as potential growth-promoting stimuli in psoriatic epidermis. IL-8 mRNA was only detected in lesional psoriatic epidermis, and IL-8R-specific mRNA was found to be 10 times increased in lesional psoriatic epidermis. There was no significant difference in the protooncogene mRNA levels. In order to test the relevance of the massively increased IL-8R levels in psoriatic epidermis, we investigated the effect of the antipsoriatic drug FK-506 on specific IL-8 and IL-8R mRNA expression. FK-506 dose dependently inhibited IL-8R expression and function. Our data suggest that in psoriatic skin, elevated IL-8 levels and markedly increased IL-8R expression may act in concert to induce the cardinal signs of psoriasis--epidermal hyperproliferation and leukocyte infiltration. IL-8R may prove a molecular target for antipsoriatic drugs such as FK-506.
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PMID:Increased expression of epidermal IL-8 receptor in psoriasis. Down-regulation by FK-506 in vitro. 769 48

The hepatitis C virus H strain (HCV-H) polyprotein is cleaved to produce at least 10 distinct products, in the order of NH2-C-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B -COOH. An HCV-encoded serine proteinase activity in NS3 is required for cleavage at four sites in the nonstructural region (3/4A, 4A/4B, 4B/5A, and 5A/5B). In this report, the HCV-H serine proteinase domain (the N-terminal 181 residues of NS3) was tested for its ability to mediate trans-processing at these four sites. By using an NS3-5B substrate with an inactivated serine proteinase domain, trans-cleavage was observed at all sites except for the 3/4A site. Deletion of the inactive proteinase domain led to efficient trans-processing at the 3/4A site. Smaller NS4A-4B and NS5A-5B substrates were processed efficiently in trans; however, cleavage of an NS4B-5A substrate occurred only when the serine proteinase domain was coexpressed with NS4A. Only the N-terminal 35 amino acids of NS4A were required for this activity. Thus, while NS4A appears to be absolutely required for trans-cleavage at the 4B/5A site, it is not an essential cofactor for serine proteinase activity. To begin to examine the conservation (or divergence) of serine proteinase-substrate interactions during HCV evolution, we demonstrated that similar trans-processing occurred when the proteinase domains and substrates were derived from two different HCV subtypes. These results are encouraging for the development of broadly effective HCV serine proteinase inhibitors as antiviral agents. Finally, the kinetics of processing in the nonstructural region was examined by pulse-chase analysis. NS3-containing precursors were absent, indicating that the 2/3 and 3/4A cleavages occur rapidly. In contrast, processing of the NS4A-5B region appeared to involve multiple pathways, and significant quantities of various polyprotein intermediates were observed. NS5B, the putative RNA polymerase, was found to be significantly less stable than the other mature cleavage products. This instability appeared to be an inherent property of NS5B and did not depend on expression of other viral polypeptides, including the HCV-encoded proteinases.
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PMID:Hepatitis C virus NS3 serine proteinase: trans-cleavage requirements and processing kinetics. 796 6


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