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At least 235 records · Page 13Linked to original sources
PubMed1973

Inhibition of transcription of the histidine operon in vitro by the first enzyme of the histidine pathway.

An in vitro system was developed for transcription of the histidine operon of Esherichia coli carried in the genome of a defective varphi80 transducing phage. The messenger RNA (mRNA) of the histidine operon synthesized in the in vitro system was detected by hybridization to single strands of both varphi80 and varphi80dhis DNA, and by competition of this hybridization with unlabeled histidine mRNA that had been synthesized in vivo (RNA extracted from cells in which the histidine operon had been derepressed). Under the conditions used, RNA complementary to the histidine operon was about 15% of the total RNA that was synthesized in vitro from the varphi80dhis DNA template. The RNA complementary to the histidine operon was synthesized on the "sense" strand (the R strand) of varphi80dhis in the form of a polycistronic message with a sedimentation coefficient (about 38 S) very close to that observed for the histidine mRNA synthesized in vivo. Synthesis of the histidine operon RNA appears to be subject to control in vitro. Addition of the first enzyme of the pathway for histidine biosynthesis blocked transcription of the histidine operon specifically, strongly suggesting that this enzyme acts as a regulatory protein for the histidine operon.

Cell-Free System↗
PubMed1984

The use of RNAs complementary to specific mRNAs to regulate the expression of individual bacterial genes.

A naturally occurring small RNA molecule ( micF RNA), complementary to the region encompassing the Shine-Dalgarno sequence and initiation codon of the ompF mRNA, is known to block the expression of that mRNA in E. coli. We have constructed a plasmid that produces a complementary RNA to the E. coli lpp mRNA (mic[Ipp] RNA). Induction of the mic(Ipp) gene efficiently blocked lipoprotein production and reduced the amount of lpp mRNA. Two mic(ompC) genes were similarly engineered and their expression was found to inhibit drastically production of OmpC. Analysis of several types of mic(ompA) genes suggests that micRNAs complementary to regions of the mRNA likely to come in contact with ribosomes were most effective. The novel capabilities of this artificial mic system provide great potential for application in both procaryotic and eucaryotic cells.

Bacterial Proteins↗
PubMed2001

RNA-RNA noncovalent interactions investigated by microspray ionization mass spectrometry.

Electrospray ionization mass spectrometry is playing an increasing role in the study of noncovalent interactions involving biomolecules. RNA-RNA complexes are important in many areas of biology, including RNA catalysis, RNA splicing, ribosome function, and gene regulation. Here, microelectrospray mass spectrometry (microESI-MS) is used to study noncovalent base-pairing interactions between RNA oligonucleotides, an area not previously explored by this technique. Using a set of complementary RNA oligonucleotides, we demonstrate the formation of the expected double-helical RNA complexes composed of three distinct oligonucleotides. The ability to study specific RNA noncovalent interactions by microESI-MS has the potential to provide a unique method by which to analyze and assign precise molecular masses to RNA-RNA complexes.

Base Sequence↗
PubMed1986

Onc-gene expression in hyperplasia induced by tape stripping or by topical application of TPA.

Guinea-pig ears were treated topically with 20 nmol of 12-O-tetradecanoyl phorbol-13-acetate, or deprived of their horny layer by nine successive strippings. At different times after the treatment, the animals were sacrificed, the epidermis removed from the ears, and the RNA from the epidermis purified and analysed by dot-blot hybridization in order to assess and determine the amount of RNA able to hybridize to each one of the 18 onc-gene DNA probes. The following probes were used: v-src, v-fps, v-yes, v-ros, v-myc, c-myc, v-erb AB, v-myb, v-mos, v-Ha-ras, v-Ki-ras, v-abl, v-fos, v-fes, v-fms, c-sis, B-lym, v-raf. At 0 h, expression of B-lym and of myc and fos is seen. ErbAB mRNA is detected between 10 min and 4 h after stripping, as well as after TPA application. B-lym mRNA is detected for up to 36 h after stripping and for up to 8 h after TPA application. C-myc mRNa is detected for up to 36 h after tape stripping, but only for the first hour after TPA application. RNA complementary to the other onc probes was not detected, and synthesis of RNA complementary to an actin DNA probe was observed for 8 h after TPA application.

Administration, Topical↗
PubMed1973

Transcription of simian virus 40. 3. Mapping of "early" and "late" species of RNA.

To determine the orientation of transcription of the E and L strands of DNA from simian virus 40 (SV40), we used linear DNA prepared by cleavage of superhelical viral DNA by endonuclease R.R(1) from Escherichia coli as a primer.template for DNA polymerase. The resulting molecules, which were labeled only at the 3' end of each DNA strand, were then cleaved with Hemophilus parainfluenzae endonuclease Hpa I. The ensuing four DNA fragments, whose locations on the viral genome are known, were separated by electrophoresis, denatured, and hybridized to asymmetric SV40 complementary RNA. From the pattern of hybridization of the fragments containing the labeled 3' ends, we conclude that transcription of SV40 proceeds in a clockwise direction on the L strand and in a counterclockwise direction on the E strand as drawn on the conventional SV40 map. To map the "early" and "late" regions of the viral genome, we extracted RNA from lytically infected cells and hybridized it to the separated strands of the four fragments of (32)P-labeled SV40 DNA. Early after infection, RNA complementary to part of the E strand of the contiguous fragments A and C was detected. Late polysomal RNA was complementary to part of the L strand sequences of fragments A and C and to the total L strand sequence of fragments B and D.

Animals↗
PubMed1990

Catalysis of RNA cleavage by the Tetrahymena thermophila ribozyme. 1. Kinetic description of the reaction of an RNA substrate complementary to the active site.

A ribozyme derived from the intervening sequence (IVS) of the Tetrahymena preribosomal RNA catalyzes a site-specific endonuclease reaction: G2CCCUCUA5 + G in equilibrium with G2CCCUCU + GA5 (G = guanosine). This reaction is analogous to the first step in self-splicing of the pre-rRNA, with the product G2CCCUCU analogous to the 5'-exon. The following mechanistic conclusions have been derived from pre-steady-state and steady-state kinetic measurements at 50 degrees C and neutral pH in the presence of 10 mM Mg2+. The value of kcat/Km = 9 x 10(7) M-1 min-1 for the oligonucleotide substrate with saturating G represents rate-limiting binding. This rate constant for binding is of the order expected for formation of a RNA.RNA duplex between oligonucleotides. (Phylogenetic and mutational analyses have shown that this substrate is recognized by base pairing to a complementary sequence within the IVS). The value of kcat = 0.1 min-1 represents rate-limiting dissociation of the 5'-exon analogue, G2CCCUCU. The product GA5 dissociates first from the ribozyme because of this slow off-rate for G2CCCUCU. The similar binding of the product, G2CCCUCU, and the substrate, G2CCCUCUA5, to the 5'-exon binding site of the ribozyme, with Kd = 1-2 nM, shows that the pA5 portion of the substrate makes no net contribution to binding. Both the substrate and product bind approximately 10(4)-fold (6 kcal/mol) stronger than expected from base pairing with the 5'-exon binding site. Thus, tertiary interactions are involved in binding. Binding of G2CCCUCU and binding of G are independent. These and other data suggest that binding of the oligonucleotide substrate, G2CCCUCUA5, and binding of G are essentially random and independent. The rate constant for reaction of the ternary complex is calculated to be kc approximately equal to 350 min-1, a rate constant that is not reflected in the steady-state rate parameters with saturating G. The simplest interpretation is adopted, in which kc represents the rate of the chemical step. A site-specific endonuclease reaction catalyzed by the Tetrahymena ribozyme in the absence of G was observed; the rate of the chemical step with solvent replacing guanosine, kc(-G) = 0.7 min-1, is approximately 500-fold slower than that with saturating guanosine. The value of kcat/Km = 6 x 10(7) M-1 min-1 for this hydrolysis reaction is only slightly smaller than that with saturating guanosine, because the binding of the oligonucleotide substrate is predominantly rate-limiting in both cases.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1986

Molecular cloning and sequence analysis of the human parainfluenza 3 virus RNA encoding the nucleocapsid protein.

The sequence of 1690 nucleotides from the 5' end of the viral complementary RNA for the human parainfluenza 3 virus was determined by molecular cloning. One large open reading frame consisting of 1548 nucleotides was demonstrated. The encoded protein, the nucleocapsid protein (NP), consists of 515 amino acids, and has a predicted molecular weight of 57,819. A noncoding 5' sequence of 51 nucleotides is present at the end of the NP-mRNA. Two consensus sequences were identified which are homologous with sequences found in Sendai virus. One of these sequences, AGGATTAAAG, was located at the 5' end of the nucleocapsid mRNA and may function in transcription initiation. The other consensus sequence, GTAAGGGAA, was found in the viral genomic leader sequence. The nucleocapsid protein amino acid sequence was compared to other members of the Paramyxoviridae family. The parainfluenza 3 virus protein nucleocapsid amino acid sequence demonstrated a high degree of homology with the Sendai virus nucleocapsid protein. Seventy percent of the first 387 amino acids from the amino termini were identical. Little homology was observed in the distal carboxy termini.

Amino Acid Sequence↗
PubMed1989

Production of anti-idiotypic antibodies by immunization with a pair of complementary peptides.

Previous investigations have suggested that pairs of peptides specified by complementary RNA sequences (termed complementary peptides) can interact with one another. In the light of this finding, we hypothesized that an antibody directed against a peptide might interact with an antibody against its complementary peptide at the antigen combining site. To address this possibility, polyclonal antibodies against a peptide, Leu-Glu-Arg-Ile-Leu-Leu (LERILL), and its complementary peptide, Glu-Leu-Cys-Asp-Asp-Asp (ELCDDD), were made monospecific by affinity chromatography. Using radioimmunoassays, anti-ELCDDD antibodies were shown to interact with 125I-anti-LERILL antibodies but not with 125I-control antibodies. More importantly, the interaction of the two antibodies could be blocked using either peptide antigen, but not with control peptides. Furthermore, 125I-anti-LERILL binding to LERILL could be blocked with anti-ELCDDD antibody and vice versa. We concluded therefore that antibody/antibody binding occurred at or near the antigen combining site, demonstrating that this interaction was an idiotypic/anti-idiotypic one.

Amino Acid Sequence↗
PubMed1991

The 3' promoter region involved in RNA synthesis directed by the turnip yellow mosaic virus genome in vitro.

We have previously shown that the last 100 nucleotides from the 3' end of turnip yellow mosaic virus (TYMV) RNA compete in vitro with genomic RNA for the TYMV-specific RNA-dependent RNA polymerase (RdRp). To further characterize the promoter on genomic RNA that produces complementary RNA strands, shorter fragments corresponding to the 3' region of the viral RNA were generated and used in in vitro assays. Fragments as short as 38 nucleotides corresponding to the 3' end of TYMV RNA compete with the viral RNA for the RdRp suggesting that the 3' promoter on plus strand RNA is probably less than or equal to 38 nucleotides long. These transcripts are themselves used as templates in vitro.

Base Sequence↗
PubMed1986

Use of [32P]RNA probes for the dot-hybridization detection of potato spindle tuber viroid.

A dot-hybridization assay using 32P-labelled RNA probes (+RNA and cRNA) transcribed from potato spindle tuber viroid (PSTV) cDNA was described. A complete cDNA copy of PSTV, originally cloned in pBR 322 (pAV 401) was subcloned in the BamHI site of a 'Riboprobe' cloning vectors pSP 64 and pSP 65 in opposite orientations. The reconstructed plasmids were designated pDX 1 and pDX 4, respectively. Transcription of pDX 1 and pDX 4 plasmids by SP6 RNA polymerase resulted in the generation of PSTV-specific RNA (+RNA) and PSTV complementary RNA (cRNA), respectively. The cRNA probe was much more sensitive than the +RNA probe and the nick-translated cDNA probe from the plasmid pAV 401 for the detection of PSTV in clarified plant sap. As little as 1.4 pg of purified PSTV mixed in clarified sap from uninoculated tomato leaves has been detected using cRNA probe. A relatively simple procedure using cetyltrimethyl ammonium bromide (CTAB) as nucleic acid precipitant and an enrichment step for the purification of PSTV was described.

DNA Restriction Enzymes↗
PubMed2005

A cellular screening assay to test the ability of PKR to induce cell death in mammalian cells.

Long double-stranded RNA (>30 bp), usually expressed in cells infected with RNA viruses, triggers antiviral responses that induce apoptosis of the infected cells. PKR can be selectively activated in glioblastoma cells by in situ generation of dsRNA following introduction of antisense RNA complementary to an RNA expressed specifically in these cells. Harnessing PKR for the selective killing of cancer cells is potentially a powerful strategy for treating cancer, but we were unable to induce apoptosis by this approach in a T cell lymphoma. We therefore established a cellular screening assay to test the ability of PKR to induce death in cell lines, especially those originating from human cancers. This "PKR killing screen" is based on the infection of cells with an adenoviral vector encoding GyrB-PKR, followed by coumermycin treatment. Cancers represented by cell lines in which PKR activation leads to cell death are good candidates for the dsRNA killing approach, using antisense to RNA molecules specifically expressed in these cells. The PKR killing screen may also serve as a tool for exploring PKR signaling and other related pathways, by identifying new cases in which PKR signaling is inhibited or impaired.

Adenocarcinoma↗
PubMed1993

Computer-aided search for effective antisense RNA target sequences of the human immunodeficiency virus type 1.

For the biological and therapeutic application of antisense nucleic acids, there is a need to identify effective local target regions of given cellular target mRNAs or viral single-stranded nucleic acids. One critical parameter for the effectiveness of antisense nucleic acids could be the potential of intramolecular folding of a given sequence element of the target strand and the antisense strand, respectively. The folding potential of such subsequences was calculated by using an established secondary structure prediction algorithm. For the genomic RNA and the complementary RNA strand of the human immunodeficiency virus type 1 (HIV-1), an energy profile was calculated that monitors the local folding potential of each sequence position surrounded by a window of given length ranging from 50 to 400 nucleotides. The resulting energy profile was compared to the effectiveness of HIV-1-directed antisense RNAs. It was found that significant minima of the local folding potential (high delta G values) correlated with antisense RNA target regions involved in strong inhibition of HIV-1 replication that had been measured independently in two earlier studies by using different experimental approaches. Conversely, antisense RNAs directed against local subregions with a high folding potential (low delta G values) showed weak or no antiviral effect in human cells. The results indicate that analyses of the local folding potential of a given target RNA can support the selection of effective target sequences for antisense RNA.

Antiviral Agents↗
PubMed1982

Use of specific single stranded DNA probes cloned in M13 to study the RNA synthesis of four temperature-sensitive mutants of HK/68 influenza virus.

Specific single stranded DNA probes have been obtained for both influenza virion RNA (vRNA) and complementary RNA (cRNA) by cloning a hemagglutinin gene fragment in the single stranded DNA phase M13. These probes were used for hybridization with the total labeled RNA from cytoplasmic extracts of infected cells. MDCK cells were infected with temperature-sensitive mutants of influenza HK/68 and the production of the virus specific RNA species was analysed at both permissive and restrictive temperatures. Results show that two NP mutants which undergo intracistronic complementation exhibit two different phenotypes at the non permissive temperature: ts2C is poly A cRNA and vRNA negative whereas ts463 is RNA positive. Two mutants of P genes were also analysed and we discuss the relationship existing between the synthesis of the three RNA species especially between poly A and non poly A cRNA.

Animals↗
PubMed1992

Comparison of two reconstituted systems for in vitro transcription and replication of influenza virus.

The transcription and replication of influenza RNA can be studied in vitro by the reconstitution of functional ribonucleoprotein (RNP) complex from viral core proteins including the RNA polymerase (complex of three P protein subunits) and nucleoprotein (NP), and model templates. Here, two different core protein preparations, one based on CsCl centrifugation (CS enzyme) and the other on micrococcal nuclease treatment of viral cores (MN enzyme), were compared side-by-side. Short model RNA templates and their 3'-half molecules of both viral RNA (vRNA) and complementary RNA (cRNA) senses were reconstituted with the core protein preparations in parallel, and RNA polymerase activity was tested either in the presence or absence of ApG or globin mRNA as primers. Both enzyme preparations were active in the syntheses of short vRNA and cRNA transcripts using ApG as a primer, although the synthesis of cRNA was 2-10-fold higher (depending on the template used) than the synthesis of vRNA. The MN enzyme, however, was more active per weight of total protein than the CS enzyme, probably because of its higher content of RNA polymerase. Both enzymes failed to show primer-independent synthesis of vRNA. The differences observed in the synthesis of short transcripts using globin mRNA as a primer are discussed.

Base Sequence↗
PubMed1973

RNA-dependent RNA polymerase in nuclei of cells infected with influenza virus.

Nuclei purified from chicken embryo fibroblast cells infected with influenza (fowl plague) virus contain an RNA-dependent RNA polymerase. The in vitro activity of this enzyme is insensitive to actinomycin D, and is completely destroyed by preincubation with ribonuclease. Enzyme induction is prevented if cells are treated with actinomycin D or cycloheximide at the time of infection. RNA-dependent RNA polymerase activity increases rapidly in cell nuclei from 1 h postinfection, reaches a maximum at 3 to 4 h, then declines; a similar RNA polymerase activity in the microsomal cell fraction increases from 2 h postinfection and reaches a maximum at 5 to 6 h. The characteristics of the nuclear and microsomal enzymes in vitro are similar with respect to pH and divalent cation requirements. The in vitro products of enzyme activity present in the nuclear and microsomal fractions of cells infected for 3 and 5 h were characterized by sucrose density gradient analysis, and annealing to virion RNA. The microsomal RNA polymerase product contained 67 and 93% RNA complementary to virion RNA at 3 and 5 h, respectively; for the nuclear RNA polymerase product these values were 40% in each case.

Animals↗
PubMed1974

Transcribing complexes in cells infected by vesicular stomatitis virus and rabies virus.

We analyzed cell extracts from BHK(21) cells infected with vesicular stomatitis virus (VSV) and rabies virus for in vitro RNA polymerase activity. Cells infected with VSV B virions exhibited several complexes with in vitro RNA polymerase activity in sucrose gradients. These complexes synthesize VSV transcriptase product (4 to 18S) polyadenylated in RNA complementary to virion RNA. Cells infected with a high multiplicity of B virions and T particles show only one RNA polymerase complex active in vitro. This complex sediments at 110S and makes only small (2S) RNA. Carrier BHK(21) cells persistently noncytopathically infected with VSV contain several complexes active in RNA polymerase, but both exhibit very low activity. Cytoplasms of cells noncytopathically infected with rabies virus also show very low levels of a complex containing RNA polymerase activity. No transcriptase nor any other in vitro polymerase activity could be found associated with purified rabies virions, although they do carry out primary transcription in cells treated with actinomycin D and cycloheximide before infection.

Animals↗
PubMed1976

Characterization of polysome-associated RNA from influenza virus-infected cells.

Virus-specific polysome-associated RNA (psRNA) and RNA after dissociation of polysomes were analyzed by direct hybridization with unlabeled viral RNA (vRNA) and complementary RNA (cRNA). psRNA after a 30-min pulse with [3H]uridine contained 28% labeled cRNA, 70% host RNA, and no vRNA. After dissociation, psRNA sedimented heterogeneously. Heavy RNA (greater than 60S), ribosomal subunit RNA (rsuRNA, 30-60S), free mRNA (fmRNA, 10-30S), and light RNA (less than 10S) contained 16%, 54%, 70% and 28% cRNA, respectively, but no vRNA. When actinomycin D (AcD) was added at 2 h postinfection, the nature of the psRNA depended on the concentration of AcD and the condition of the labeling. At AcD concentrations of 1 mug or more per ml, no detectable vRNA or cRNA was associated with polysomes. At 0.2 mug of AcD per ml (a concentration that partially inhibited cRNA synthesis) and 2 h of labeling at 2.5 h postinfection, psRNA contained 40% viral-specific RNA, which included both vRNA and cRNA in almost equal amounts. When polysomes were dissociated, however, viral-specific fm RNA from AcD-treated cells contained exclusively cRNA and no detectable vRNA. Increasing amounts of labeled vRNA were present in the heavy region of the gradient (and in the pellet), which also contained varying amounts of cRNA. The labeled vRNA appears to be associated with polysomes in a cesium chloride density gradient (rho = 1.525 g/ml). Although we have ruled out the trivial explanation of viral ribonucleoprotein contamination,the nature of the complex containing both polysomes and vRNA is unknown.

Animals↗
PubMed2005

Activation of dsRNA dependent protein kinase PKR in Karpas299 does not lead to cell death.

Activated double-stranded RNA (dsRNA)-dependent protein kinase PKR is a potent growth inhibitory protein that is primarily activated in virally infected cells, inducing them to die. We have recently shown that PKR can be selectively activated in cancer cells, by in situ generation of dsRNA following introduction of antisense RNA complementary to an RNA expressed specifically in the cancer cell. The feasibility of this approach was demonstrated using a glioblastoma line that overexpresses a truncated form of the EGFR. PKR and its signaling pathway are not restricted to a given cell line; therefore, in principle, this dsRNA killing approach can be applied to any cancer that expresses unique RNA sequences. Nonetheless, applying this approach to Karpas299 cells, from a T-cell non-Hodgkin's lymphoma that harbors the NPM/ALK translocation, did not result in cell death, implying that PKR signaling pathway is repressed in this cell line. Indeed, the phosphorylation of eIF2alpha by PKR was impaired in Karpas299 cells. Furthermore, levels of the cellular inhibitor p67 were elevated in these cells. Long antisense, as well as RNAi for p67, delivered into Karpas299 cells by adenoviruses, reduced p67 levels. The reduction in p67 levels led to increased phosphorylation of eIF2alpha, and an additive effect was achieved by coinfection with NPM/ALK-AS encoding adenoviruses. Infection with these adenoviruses, however, did not promote growth inhibition. These findings imply that anti-apoptotic mechanisms counteract PKR signaling in this T-cell non-Hodgkin's lymphoma.

Adenoviridae↗