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

---

Query: EC:2.7.7.6 (RNA polymerase)
34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have constructed a full-length cDNA clone of the virulent T48 strain of Ross River virus, a member of the alphavirus genus. Infectious RNA can be transcribed from this clone using SP6 or T7 RNA polymerase. The rescued virus has properties indistinguishable from those of the T48 strain of Ross River virus. We have used this clone, together with a full-length cDNA clone of Sindbis virus, to construct chimeric plasmids in which the 5' and the 3' nontranslated regions of the Sindbis and Ross River genomes were exchanged. The nontranslated regions of the two viral genomes differ in both size and sequence although they maintain specific conserved sequence elements. Virus was recovered from all four chimeras. Chimeras containing heterologous 3' nontranslated regions had replicative efficiencies equal to those of the parents. In contrast, the chimeras containing heterologous 5' nontranslated regions were defective in RNA synthesis and virus production, and the severity of the defect was dependent upon the host. Replication of a virus containing a heterologous 5' nontranslated region may be inefficient due to the formation of defective protein-RNA complexes, whereas, the presumptive complexes formed between host or virus proteins and the 3' nontranslated region to promote RNA synthesis appear to function normally in the chimeras.
...
PMID:Infectious RNA transcripts from Ross River virus cDNA clones and the construction and characterization of defined chimeras with Sindbis virus. 167 12

Tagetitoxin, a chlorosis-inducing phytotoxin produced by Pseudomonas syringae pv. tagetis, inhibits RNA synthesis directed by chloroplast RNA polymerase. In isolated chloroplasts, tagetitoxin quickly and specifically reduced the incorporation of [3H]uridine into RNA. When it was added to transcriptionally active chloroplast protein extracts, the toxin directly inhibited incorporation of [32P]UTP into RNA. In addition, tagetitoxin inhibited in vitro RNA synthesis directed by the RNA polymerase from Escherichia coli. In vitro transcription reactions directed by chloroplast RNA polymerase or E. coli RNA polymerase are inhibited at tagetitoxin concentrations less than 1 microM. Nuclear RNA polymerase II purified from wheat germ was only affected at tagetitoxin concentrations greater than 100 microM during in vitro transcription. Tagetitoxin concentrations as high as 1 mM did not affect in vitro transcription reactions directed by RNA polymerase from bacteriophage T7 or SP6.
...
PMID:Tagetitoxin inhibits RNA synthesis directed by RNA polymerases from chloroplasts and Escherichia coli. 168 34

Previous studies revealed that antisense oligodeoxynucleotides to specific regions of the human immunodeficiency virus-1 (HIV-1) are potent inhibitors of replication of HIV-1 in vitro (Zamecnik, P. C., Goodchild, J., Taguchi, Y., and Sarin, P. S. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 4143-4146). We now report that antisense RNA, synthesized in vitro using T7 and SP6 RNA polymerase, displayed an anti-HIV-1 effect in the HTLV-IIIB/H9 system in vitro. Treatment of HIV-1-infected H9 cells with viral env region antisense RNA encapsulated in liposomes targeted by antibodies specific for the T cell receptor molecule CD3 almost completely inhibited HIV-1 production. The viral env segment covered a part of exon II of HIV-1 tat gene. No anti-HIV activity could be detected with similarly targeted liposome-encapsulated sense env RNA or with pol RNA synthesized in either the sense or antisense orientations, or with env region antisense RNA free in solution, or encapsulated in liposomes in the absence of the targeting antibody. A semiquantitative evaluation revealed that 4000-7000 RNA molecules became cell-bound in targeted liposomes; the half-life of the intracellularly present hybridizable antisense env RNA was approximately 12 h. Western blots showed that antisense env RNA suppressed tat gene expression by approximately 90% and gp160 production by 100%. These data were confirmed by immunoprecipitation studies. Northern blots (using an env probe) demonstrated the existence of all major HIV RNA species (9.3-, 4.3-, and 2.0-kb mRNA) in HIV-infected cells treated with antisense env RNA although at a reduced level. We conclude that the antisense env RNA inhibited viral protein production at the translational level.
...
PMID:Inhibition of expression of human immunodeficiency virus-1 in vitro by antibody-targeted liposomes containing antisense RNA to the env region. 169 56

The cleavage specificities of the RNase P holoenzymes from Escherichia coli and the yeast Schizosaccharomyces pombe and of the catalytic M1 RNA from E. coli were analyzed in 5'-processing experiments using a yeast serine pre-tRNA with mutations in both flanking sequences. The template DNAs were obtained by enzymatic reactions in vitro and transcribed with phage SP6 or T7 RNA polymerase. The various mutations did not alter the cleavage specificity of the yeast RNase P holoenzyme; cleavage always occurred predominantly at position G + 1, generating the typical seven base-pair acceptor stem. In contrast, the specificity of the prokaryotic RNase P activities, i.e. the catalytic M1 RNA and the RNase P holoenzyme from E. coli, was influenced by some of the mutated pre-tRNA substrates, which resulted in an unusual cleavage pattern, generating extended acceptor stems. The bases G - 1 and C + 73, forming the eighth base pair in these extended acceptor stems, were an important motif in promoting the unusual cleavage pattern. It was found only in some natural pre-tRNAs, including tRNA(SeCys) from E. coli, and tRNAs(His) from bacteria and chloroplasts. Also, the corresponding mature tRNAs in vivo contain an eight base pair acceptor stem. The presence of the CCA sequence at the 3' end of the tRNA moiety is known to enhance the cleavage efficiency with the catalytic M1 RNA. Surprisingly, the presence or absence of this sequence in two of our substrate mutants drastically altered the cleavage specificity of M1 RNA and of the E. coli holoenzyme, respectively. Possible reasons for the different cleavage specificities of the enzymes, the influence of sequence alterations and the importance of stacking forces in the acceptor stems are discussed.
...
PMID:Sequence changes in both flanking sequences of a pre-tRNA influence the cleavage specificity of RNase P. 170 37

The synthesis of cRNA probes for in situ hybridization is usually accomplished with transcription systems using cloning vectors that contain bacteriophage RNA polymerase promoters. In this report we describe an alternative technique for generating cRNA probes that obviates the need for cDNA cloning by using the polymerase chain reaction. The segment of DNA corresponding to the desired RNA sequence was amplified from genomic DNA by the polymerase chain reaction. The bacteriophage SP6 and T7 RNA polymerase promoters were incorporated into the amplified DNA by including the promoter sequences in the 5' termini of the oligonucleotides used to prime the polymerase chain reaction. The promoters permitted the subsequent transcription of sense and antisense cRNA from the amplified DNA. The antisense cRNA was radiolabeled to high specific activity for use as a probe in Northern and mRNA in situ hybridization analyses. This technique offers the advantages of speed and simplicity over conventional transcription systems, which require cDNA cloning. Bypassing the need for cloning in the synthesis of cRNA probes may facilitate the use of these probes in research and diagnostic applications of mRNA in situ hybridization.
...
PMID:Transcription of cRNA for in situ hybridization from polymerase chain reaction-amplified DNA. 170 28

Ricin B chain (RTB) is an N-glycosylated galactose-specific lectin which folds into two globular domains. Each domain binds one galactoside. The x-ray crystallographic structure has shown that the two binding sites are structurally similar and contain key binding residues which hydrogen bond to the sugar, and a conserved tripeptide, Asp-Val-Arg. We have used oligonucleotide site-directed mutagenesis to change either the binding residues or the homologous tripeptide in one or other or in both of the sites. The 5' signal sequence and RTB coding region were excised from preproricin cDNA and fused in frame to generate preRTB cDNA. Transcripts synthesized in vitro from wild-type or mutant preRTB cloned into the Xenopus transcription vector pSP64T using SP6 RNA polymerase, were microinjected into Xenopus oocytes. The recombinant products were segregated into the oocyte rough endoplasmic reticulum and core-glycosylated, and the N-terminal signal peptide was removed. Mutating sugar binding sites individually did not abrogate the lectin activity of RTB. When both sites were changed simultaneously, RTB was produced which was soluble and stable but no longer able to bind galactose. Changing the Asn residues of the two RTB N-glycosylation sites to Gln showed that oligosaccharide side chains were essential for both the stability and biological activity of recombinant RTB.
...
PMID:Mutational analysis of the galactose binding ability of recombinant ricin B chain. 171 62

We have examined the effects of nucleosome cores on the initiation and elongation of RNA transcripts by phage T7 RNA polymerase in vitro. A transcription template, pT207-18, was constructed containing tandemly repeated 207 base-pair (bp) nucleosome positioning sequences from a sea urchin (Lytechinus variegatus) 5 S RNA gene inserted between the T7 and SP6 transcription promoters of pGEM-3Z. Nucleosome cores were reconstituted onto supercoiled, closed circular pT207-18 DNA and double label transcription experiments were performed to determine the effects of nucleosome cores on the initiation and elongation of transcripts by T7 RNA polymerase. Both transcript initiation and elongation were inhibited, the extent of the inhibition being directly proportional to the number of nucleosome cores reconstituted onto the pT207-18 DNA templates. Time course transcription experiments indicated that nucleosome cores caused a reduction in the equilibrium length of transcripts and not mere retardation of elongation rates. Continuous regularly spaced linear arrays of nucleosomes were obtained by digesting reconstituted nucleosomel pT207-18 templates with DraI, for which a unique restriction site lies within the nucleosome positioning region of the 207 bp 5 S rDNA repeat sequence. After in vitro transcription with T7 RNA polymerase an RNA ladder with 207 nucleotide spacing was obtained, indicating that transcription can occur through continuous arrays of positioned nucleosome cores. It is demonstrated that nucleosome cores partially inhibit the elongation of transcripts by T7 RNA polymerase, while allowing passage of the transcribing polymerase through each nucleosome core at an upper limit efficiency of 85%. Hence, complete transcripts are produced with high efficiency from short nucleosomal templates, while the production of full-length transcripts from long nucleosomal arrays is relatively inefficient. The results indicate that nucleosome cores have significant inhibitory effects in vitro not only on transcription initiation but on transcription elongation as well, and that special mechanisms may exist to overcome these inhibitory effects in vivo.
...
PMID:Nucleosome arrays inhibit both initiation and elongation of transcripts by bacteriophage T7 RNA polymerase. 173 Oct 87

An RNAse protection assay is described that increases substantially the degree of precision with which one can measure the mRNA levels in cells and tissues through the use of the internal standard. The assay can be used to measure any mRNA for which the corresponding cDNA is available. We describe here the use of the assay to measure the apolipoprotein (apo)-A-I, apo-B, and apo-E mRNA levels in tissues from the cynomolgus monkey. cDNA fragments derived from each mRNA were subcloned into pGEM-9Zf(-), a vector containing a polylinker that is flanked by the SP6 and T7 RNA polymerase promoters. That series of plasmids, called RNA quantitation vectors (pRQV-AI, B, or E), permitted the synthesis of a sense RNA strand and an antisense RNA strand for the gene of interest. The sense stand was used as the internal standard and added to the RNA to be analyzed just prior to initiation of the assay. The radiolabeled antisense strand served as the probe. By including some nucleotides derived from the vector, we were able to design both the internal standard and the probe such that, after solution hybridization and RNAse digestion, the size of the protected internal standard-probe fragments was different from that of the authentic mRNA-probe fragments. Those fragments were then separated by gel electrophoresis, and the radioactivity in the authentic mRNA band was compared to that in the internal standard band. The mass of the authentic mRNA could then be calculated from the ratio of the radioactivity in each band and the mass of the internal standard.
...
PMID:mRNA quantitation by a simple and sensitive RNAse protection assay. 177 97

Recombinant plasmids containing sequences derived from the genome of a tomato ringspot virus (TomRSV) isolate associated with both stem pitting disease of stone fruits and apple union necrosis and decline were constructed. Selected inserts were subcloned into the polylinker region of the SP6 transcription vector pSP64. Using the SP6 promoter flanking this region, high specific activity 32P-labelled cRNA probes were generated by SP6 RNA polymerase. cRNA probes were specific for TomRSV RNA 2 present in purified virions or in extracts from woody and herbacous hosts. No sequence relatedness was detected between TomRSV RNA 2 and genomic RNA from tobacco ringspot, arabis mosaic, strawberry latent ringspot, or cucumber mosaic virus in Northern blot analysis using TomRSV cRNA probes. These probes detected TomRSV infection in woody and herbaceous hosts in dot-blot hybridization assays.
...
PMID:cDNA cloning and analysis of RNA 2 of a Prunus stem pitting isolate of tomato ringspot virus. 179 54

A cell-free system containing rotavirus subviral particles (SVPs), rabbit reticulocyte lysate, and [35S]methionine was programmed to synthesize viral protein by the addition of messenger RNA (mRNA). Electrophoretic analysis of single-shelled particles recovered from the system by CsCl centrifugation showed that newly made VP6 assembled into the particles in vitro. Electrophoretic analysis also showed that the newly made VP6 which bound to single-shelled particles in vitro was arranged in trimeric units. To identify the domain within VP6 essential for assembly into single-shelled particles, amino- and carboxyl-truncated species of VP6 were assayed for the ability to associate with single-shelled particles in the cell-free system. The truncated proteins were introduced into the system by adding VP6 mRNAs containing 5'- and 3'-terminal deletions. The terminally deleted mRNAs were prepared using SP6 RNA polymerase to transcribe portions of cDNAs of the rotavirus SA11 gene for VP6 (gene 6). Analysis of the ability of truncated VP6 to associate with single-shelled particles showed that a domain essential for assembly resides at the carboxyl-end of VP6 located between amino acid residues 251 and 397. To contrast the domain for assembly with that for trimerization, amino- and carboxyl-truncated species of VP6 were also examined by electrophoretic assay for the ability to trimerize in vitro. The results showed that the domain for trimerization resides near the center of VP6 located between amino acid residues 105 and 328. Comparison of the domains for assembly and trimerization showed that they are unique but may overlap. The fact that some truncated species of VP6, although able to bind to single-shelled particles were unable to form trimers in vitro, suggests that trimerization of VP6 is not prerequisite for the assembly of single-shelled particles.
...
PMID:Rotavirus morphogenesis: domains in the major inner capsid protein essential for binding to single-shelled particles and for trimerization. 184 94


<< Previous 1 2 3 4 5 6 7 8 9 10 Next >>

---