SEARCH · PubMed Health

Results for “RNA, Complementary”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 217 records · Page 12Linked to original sources

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↗

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↗

PubMed1978

[Genetics of influenza viruses].

An account is given of important fundamentals, working methods, and recent results in the context of influenza virus genetics. Reference is made to the potentials implied in gene mapping and to the possibility of elucidating the correlations between the single gene and all biological properties of the influenza virus. Discussed against that background are the polyacrylamide electrophoresis of virus RNA, hybridisation with complementary RNA, immunological and chemical differentiation of gene products, as well as the use of defect mutants and of recombinant formation. It is shown that successful elucidation of the genesis of new virus strains is possible by means of those methods which also enable the preparation of virus strains with desired properties. This is likely to open up new horizons for more progress in the context of immunoprophylaxis against virus influenza.

Genes, Viral↗

PubMed1982

Organization and expression of the poxvirus genome.

Poxviruses comprise a large group of very complex animal DNA viruses which replicate in the cytoplasm of infected cells. Vaccinia virus, the most studied poxvirus, has a linear, double stranded DNA genome with an approximate molecular weight of 120 x 10(6) (180 kilobase pairs). The two strands of the DNA molecule are naturally cross-linked at both termini. In addition, the vaccinia virus genome contains very long inverted terminal repetitions of approximately 10 kilobase pairs which are further characterized by the presence of direct tandem repeats of a 70-base-pair sequence arranged in two blocks of 13 and 17 copies, respectively. A central region of the genome is highly conserved between different orthopoxviruses. In contrast, the ends are hypervariable and may contain extensive deletions and complex, symmetrical sequences rearrangements. Vaccinia virus gene expression is divided into two stages. Early in infection, RNA complementary to one half of one strand-equivalent of the genome is transcribed within subviral particles by the virion-associated RNA polymerase. Later in infection, after DNA replication, RNA complementary to one entire strand-equivalent is transcribed. RNA made late in infection is very heterogeneous in length and a large fraction of it contains self-complementary sequences. Late genes are clustered near the central region of the genome. Vaccinia virus mRNAs do not appear to be synthesized by a splicing mechanism.

Base Sequence↗

PubMed1981

Titration of integrated simian virus 40 DNA sequences, using highly radioactive, single-stranded DNA probes.

Nick-translated simian virus 40 (SV40) [32P]DNA fragments (greater than 2 X 10(8) cpm/micrograms) were resolved into early- and late-strand nucleic acid sequences by hybridization with asymmetric SV40 complementary RNA. Both single-stranded DNA fractions contained less than 0.5% self-complementary sequences; both included [32P]-DNA sequences that derived from all regions of the SV40 genome. In contrast to asymmetric SV40 complementary RNA, both single-stranded [32P]DNAs annealed to viral [3H]DNA at a rate characteristic of SV40 DNA reassociation. Kinetics of reassociation between the single-stranded [32P]DNAs indicated that the two fractions contain greater than 90% of the total nucleotide sequences comprising the SV40 genome. These preparations were used as hybridization probes to detect small amounts of viral DNA integrated into the chromosomes of Chinese hamster cells transformed by SV40. Under the conditions used for hybridization titrations in solution (i.e., 10- to 50-fold excess of radioactive probe), as little as 1 pg of integrated SV40 DNA sequence was assayed quantitatively. Among the transformed cells analyzed, three clones contained approximately one viral genome equivalent of SV40 DNA per diploid cell DNA complement; three other clones contained between 1.2 and 1.6 viral genome equivalents of SV40 DNA; and one clone contained somewhat more than two viral genome equivalents of SV40 DNA. Preliminary restriction endonuclease maps of the integrated SV40 DNAs indicated that four clones contained viral DNA sequences located at a single, clone-specific chromosomal site. In three clones, the SV40 DNA sequences were located at two distinct chromosomal sites.

Animals↗

PubMed1999

Corticotropin-releasing factor receptor expression in the pituitary of fetal sheep after lesion of the hypothalamic paraventricular nucleus.

Both the capacity of CRF to release ACTH and the number of binding sites for CRF in the anterior pituitary decline during the final weeks of gestation in fetal sheep. The present study examined regulation of pituitary CRF receptor expression by the hypothalamic paraventricular nucleus (PVN) during late gestation in fetal sheep. Bilateral radiofrequency lesions of the PVN (PVN-Lx; n = 4) or sham lesions (SHAM; n = 5) were performed in fetal sheep at 118-122 days of gestational age (dGA). Pituitary glands from PVN-Lx and SHAM fetuses were collected at 139-142 dGA (term, approximately 148 dGA). Dual-label in situ hybridization was performed using a digoxigenin-labeled ovine POMC complementary RNA, together with a 35S-labeled ovine CRF type I (CRF1) receptor complementary RNA, to localize and quantify CRF1 receptor mRNA in POMC-hybridizing cells. Binding of [125I]-ovine CRF was also examined in the fetal pituitary of both PVN-Lx and SHAM fetuses using in situ autoradiography. The hybridization signal for the CRF1 receptor mRNA was primarily restricted to POMC-expressing cells in the anterior pituitary of both PVN-Lx and SHAM fetuses; no hybridization signal for the CRF1 receptor was observed in the neurointermediate lobe (NIL) in either group. The hybridization signal for CRF1 receptor mRNA in anterior pituitary corticotropes of PVN-Lx fetuses was significantly lower in both the inferior and superior regions of the anterior pituitary, compared with SHAM fetuses (P < 0.05). In the inferior region of the anterior pituitary, the percentage of POMC-hybridizing cells containing CRF1 receptor hybridization signal was significantly greater in PVN-Lx (90+/-7%; mean +/- SEM), compared with SHAM (67+/-6%; P < 0.05) fetuses. No differences in the percentage of POMC cells containing CRF1 receptor hybridization signal were observed in the superior region of the anterior pituitary between PVN-Lx (89+/-8%) and SHAM (87+/-9%). Binding of [125I]-ovine CRF (oCRF) was significantly greater in anterior pituitaries of PVN-Lx (140+/-19 mean arbitrary densitometry U +/- SEM), compared with SHAM (73+/-23; P < 0.05) fetuses. For both PVN-Lx and SHAM fetuses, there were no differences within group in [125I]-oCRF binding between the inferior and superior regions of the anterior pituitary. A weak, but significant (P < 0.05), autoradiographic signal for [125I]-oCRF binding was observed in the NIL of both SHAM and PVN-Lx fetal sheep. The level of [125I]-oCRF binding was significantly lower in the NIL, compared with anterior pituitary, for both SHAM (P < 0.01) and PVN-Lx fetuses. There were no differences in [125I]-oCRF binding in the NIL between SHAM and PVN-Lx fetal sheep. Our findings support a role for the PVN in regulating anterior pituitary CRF1 receptor expression in the late-gestation sheep fetus.

Animals↗