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At least 73 records · Page 4Linked to original sources
PubMed1983

In vitro synthesis of genome length complementary RNA of vesicular stomatitis virus in the presence of inosine 5'-triphosphate.

The virion-associated RNA polymerase of vesicular stomatitis virus (VSV) synthesizes RNA in vitro when GTP is replaced by inosine 5'-triphosphate (ITP). The synthesis is optimal at an ITP concentration of 200 microM and the extent of synthesis is between 15 to 20% compared to normal transcription in the presence of GTP. Analyses of the RNA products revealed that approximately 10% of the product RNA represented plus-strand complement of the genome RNA. Defective interfering particles of VSV were also capable of synthesizing complementary RNA in the presence of ITP, in addition to 46-base RNA, although in lesser amount (2%). Since I substitution facilitates read-through of the genome RNA, these results suggest that interaction of the product RNA with the genome template or ITP-mediated modification of a viral protein may be involved in the read-through process in vitro.

Defective Viruses↗
PubMed2004

LNA (locked nucleic acid): high-affinity targeting of complementary RNA and DNA.

Locked nucleic acid (LNA) is a nucleic acid analogue containing one or more LNA nucleotide monomers with a bicyclic furanose unit locked in an RNA mimicking sugar conformation. LNA oligonucleotides display unprecedented hybridization affinity toward complementary single-stranded RNA and complementary single- or double-stranded DNA. Structural studies have shown that LNA oligonucleotides induce A-type (RNA-like) duplex conformations. The wide applicability of LNA oligonucleotides for gene silencing and their use for research and diagnostic purposes are documented in a number of recent reports, some of which are described herein.

DNA, Complementary↗
PubMed1985

Detection of preprocholecystokinin and preproenkephalin A mRNAs in rat brain by hybridization histochemistry using complementary RNA probes.

The distributions of mRNAs encoding preprocholecystokinin and preproenkephalin A in the rat brain were examined by in situ hybridization histochemistry. Complementary RNA probes 0.5-1.0 kilobase in length were synthesized in vitro with 35S-ribonucleotides using SP6 polymerase and a transcription vector containing the SP6 promotor. Following hybridization to fixed tissue sections, signals were detected by autoradiography after relatively short exposure times. These studies demonstrate that neuropeptide mRNAs can be detected within specific cells in the brain and that the patterns of hybridization match the localization of the peptides visualized by immunohistochemistry. Minimum numbers of mRNA copies encoding the peptides were determined by quantitative autoradiography.

Animals↗
PubMed2006

Locked nucleic acid: high-affinity targeting of complementary RNA for RNomics.

Locked nucleic acid (LNA) is a nucleic acid analog containing one or more LNA nucleotide monomers with a bicyclic furanose unit locked in an RNA-mimicking sugar conformation. This conformational restriction is translated into unprecedented hybridization affinity towards complementary single-stranded RNA molecules. That makes fully modified LNAs, LNA/DNA mixmers, or LNA/RNA mixmers uniquely suited for mimicking RNA structures and for RNA targeting in vitro or in vivo. The focus of this chapter is on LNA antisense, LNA-modified DNAzymes (LNAzymes), LNA-modified small interfering (si)RNA (siLNA), LNA-enhanced expression profiling by real-time RT-PCR and detection and analysis of microRNAs by LNA-modified probes.

Animals↗
PubMed1978

Segments of influenza virus complementary RNA synthesized in vitro.

In the presence of Mg(2+) and a specific primer, ApG or GpG, the influenza WSN virion transcriptase synthesizes large, polyadenylic acid-containing complementary RNA (cRNA) (Plotch and Krug, J. Virol., 21:24-34, 1977). After removal of its polyadenylic acid with RNase H in the presence of polydeoxythymidylic acid, the in vitro cRNA distributed into seven discrete bands during electrophoresis in acrylamide gels containing 6 M urea. The eight known segments of virion RNA (vRNA) also distributed into seven bands under these conditions as two, rather than the expected three, large-sized segments were resolved. Each of the in vitro cRNA segments migrated slightly faster than the corresponding vRNA segment. To determine whether this difference in mobility reflects a difference in size between cRNA and vRNA, the double-stranded RNA formed by annealing labeled in vitro cRNA to unlabeled vRNA was subjected to various nuclease treatments and was analyzed by gel electrophoresis. Hybrids treated with RNase T2 or a combination of RNase T2 and RNase H migrated slightly faster than those treated only with RNase H, indicating that RNase T2 removed an RNA sequence other than polyadenylic acid, most probably a short sequence of vRNA not hydrogen bonded to cRNA. These results suggest that the in vitro cRNA segments are shorter than, and thus incomplete transcripts of the corresponding vRNA segments. All eight hybrids were resolved by gel electrophoresis, indicating that all eight vRNA segments are transcribed into cRNA in vitro. We also present evidence suggesting that the ApG primer initiates in vitro transcription exactly at the 3' end of vRNA.

Cell-Free System↗
PubMed1976

A genetic approach to studies on the synthesis of the complementary RNA's of fowl plague virus.

The rate of incorporation of 5-fluorouracil into complementary strands of replicating RNA of fowl plague virus (FPV) has been studied. The efficiency of incorporation was estimated by determination of the reversion frequency in s-mutants with known types of base transitions in the RNA of the virus particle. It was established that maximum incorporation of 5-fluorouracil into progeny virus particle RNA took place between 2 and 4 h after infection. The maximum incorporation of the mutagen into complementary RNA (plus strands) occurred when the cells were exposed to 5-fluorouracil from 1 to 2 h after infection.

Base Sequence↗
PubMed1995

Transient expression assay for antisense RNAs using episomal replication of plasmids: effective reduction of retinoblastoma gene (Rb-1) product by its antisense RNA complementary to 3'-untranslated region.

We have developed a transient expression assay for selection of effective antisense RNAs using episomal replication of plasmids in COS-7 cells, an African green monkey kidney-derived cell line expressing SV40 large T antigen. The transient expression assay was enabled by a liposome-mediated DNA transfection method, by which about 70% of the cells were reproducibly transfected with exogenous DNAs. Plasmids expressing antisense RNAs for the retinoblastoma gene (Rb-1) mRNA and harboring SV40 ori were constructed and introduced into COS-7 cells to examine their inhibitory effect on the accumulation of endogenous Rb protein (pRb). Only the antisense RNA complementary to the 3'-untranslated region (UTR) in Rb-1 mRNA was expressed stably at high levels for 3 days after the transfection. This antisense RNA reduced by 73% the content of endogenous pRb 70 h after transfection. A similar inhibition was detected in mouse mammary carcinoma cells (FM3A) that were stably transfected with the antisense RNA expressing vector directed to 3'UTR. In contrast, no obvious change in pRb was observed with antisense RNAs complementary to the coding region of Rb-1 mRNA. The cellular content of these antisense RNAs was lowered by degradation; thus these RNAs did not affect the levels of pRb in COS-7 and FM3A cells. These results, taken together, suggest that the expression levels and the stability of antisense RNAs are involved in their repressive activity, and our transient expression assay provides a rapid and easy system for evaluation of ectopic antisense RNA activity in COS-7 cells.

Animals↗
PubMed1979

Transfer of 5'-terminal cap of globin mRNA to influenza viral complementary RNA during transcription in vitro.

We have recently demonstrated that globin mRNAs are effective primers for influenza viral RNA transcription in vitro catalyzed by the virion transcriptase [Bouloy, M., Plotch, S. J. & Krug, R. M. (1978) Proc. Natl. Acad. Sci. USA 75, 4886-4890]. Here, we present direct evidence that the 5'-terminal methylated cap of the globin mRNAs is transferred to viral complementary RNA (cRNA) during transcription. Chemical (beta-elimination) or enzymatic removal of the cap of globin mRNAs eliminated essentially all their priming activity. Much of this activity could be restored by recapping the beta-eliminated globin mRNAs with the vaccinia virus guanylyl and methyl transferases. Globin mRNAs containing (32)P label only in the cap (m(7)G(32)pppm(6)A(m)-) were prepared by recapping beta-eliminated globin mRNAs with the vaccinia virus enzymes, [alpha-(32)P]GTP, and unlabeled S-adenosylmethionine. By using this labeled globin mRNA as primer and unlabeled nucleoside triphosphates as precursors, the viral cRNA segments that were synthesized were shown to contain a (32)P-labeled 5'-terminal cap structure. Gel electrophoretic analysis indicated that the globin mRNA-primed cRNA segments were 10-15 nucleotides longer at their 5' end than ApG-primed cRNA segments, which initiate exactly at the 3' end of the virion RNA templates. This suggests that, in addition to the cap, about 10-15 other nucleotides are also transferred from the globin mRNA to viral cRNA. A mechanism for the priming of influenza viral cRNA synthesis by globin mRNA is proposed.

Base Sequence↗
PubMed1979

Cap and internal nucleotides of reovirus mRNA primers are incorporated into influenza viral complementary RNA during transcription in vitro.

Reovirus mRNA's containing a 5'-terminal methylated cap structure (m(7)GpppG(m)) were shown to be effective primers for influenza viral RNA transcription in vitro catalyzed by the influenza virion transcriptase. Priming activity required the presence of methyl groups in the cap since reovirus mRNA's with 5'-terminal GpppG were inactive as primers. Both the cap and internal nucleotides were physically transferred from radiolabeled reovirus mRNA to influenza viral complementary RNA (cRNA) during transcription in vitro. By using reovirus mRNA's with methyl-(3)H-labeled caps as primers, we showed that the influenza viral cRNA synthesized in the presence of unlabeled nucleoside triphosphates contained [methyl-(3)H]m(7)GpppG(m), identical to that found in the reovirus mRNA primer. To demonstrate transfer of internal residues, reovirus mRNA's synthesized in the presence of all four alpha-(32)P-labeled ribonucleoside triphosphates were used as primers. The resulting influenza viral cRNA was (32)P-labeled. Diethyl-aminoethyl-Sephadex chromatography of the RNase T2 digest of this cRNA demonstrated (32)P radiolabel in both internal residues (charge -2) and the cap (charge -4.6). Approximately 25 internal nucleotides along with the cap of reovirus mRNA were transferred to each chain of influenza viral cRNA. Gel electrophoretic analysis indicated that the segments of influenza viral cRNA primed by reovirus mRNA were approximately the same size as those primed by a different mRNA, globin mRNA, strongly suggesting that the influenza virion transcriptase complex transfers approximately the same number of nucleotides plus the cap from different mRNA primers to the 5' end of influenza viral RNA transcripts.

Base Sequence↗
PubMed1981

Detection of complementary RNA intermediates of viroid replication by Northern blot hybridization.

Molecular hybridization by the Northern blot technique in combination with 125I-labeled PSTV (+) RNA and 32P-labeled PSTV cDNA as probes has been applied to detect viroid-specific sequences in healthy and viroid(PSTV)-infected tomato plants. Conditions are described which allow differentiation of (+) and (-) viroid sequences on the basis of the different thermostabilities of the corresponding hybrid molecules. By this experimental approach, it is documented that no viroid-specific DNA sequences can be detected and that viroid replication proceeds via complementary RNA intermediates. Out of the seven (-) RNA species found, six are apparently larger than the circular viroid (+) RNA and one is about the same size as the linear (+) RNA molecule.

Base Sequence↗
PubMed1997

Importance of structural differences between complementary RNA molecules to control of replication of an IncB plasmid.

Replication of the IncB miniplasmid pMU720 is dependent on the expression of repA, the gene encoding replication initiator protein RepA. Binding of a small antisense RNA (RNAI) to its complementary target (stem-loop I [SLI]) in the RepA mRNA prevents the participation of SLI in the formation of a pseudoknot that is an enhancer of translation of this mRNA. Thus, RNAI regulates the frequency of replication of pMU720 by controlling the efficiency of translation of the RepA mRNA. Mutational analysis of the two seven-base complementary sequences involved in formation of the pseudoknot showed that only the five central bases of each were critical for the formation of the pseudoknot. Physical analysis of SLI showed that despite the complete complementarity of its sequence to that of RNAI, the structures of the two molecules are different. The most prominent difference between the two structures is the presence of a 4-base internal loop immediately below the hairpin loop of SLI but not that of RNAI. Closure of this internal loop in SLI resulted in a 40-fold reduction in repA expression and loss of sensitivity of the residual expression to inhibition by RNAI. By contrast, repA expression was largely unaffected by the closure of a lower internal loop whose presence in SLI and RNAI is essential for effective interaction between these two molecules. These results suggest that the interaction of SLI with the distal pseudoknot bases is fundamentally different from the RNAI-SLI binding interaction and that the differences in structure between RNAI and SLI are necessary to allow SLI to be able to efficiently bind RNAI and to participate in pseudoknot formation.

Bacterial Proteins↗
PubMed1999

Suppression of nonsense mutations induced by expression of an RNA complementary to a conserved segment of 23S rRNA.

We identified a short RNA fragment, complementary to the Escherichia coli 23S rRNA segment comprising nucleotides 735 to 766 (in domain II), which when expressed in vivo results in the suppression of UGA nonsense mutations in two reporter genes. Neither UAA nor UAG mutations, examined at the same codon positions, were suppressed by the expression of this antisense rRNA fragment. Our results suggest that a stable phylogenetically conserved hairpin at nucleotides 736 to 760 in 23S rRNA, which is situated close to the peptidyl transferase center, may participate in one or more specific interactions during peptide chain termination.

Base Sequence↗
PubMed1987

RNA complementary to a herpesvirus alpha gene mRNA is prominent in latently infected neurons.

In initial attempts to define the molecular events responsible for the latent state of herpes simplex virus, in situ hybridization was utilized to search for virally encoded RNA transcripts in latently infected sensory neurons. The use of cloned probes representing the entire viral genome indicated that transcripts encoded within terminal repeats were present. When the alpha genes encoding ICP-0, ICP-4, and ICP-27 and the gamma 1 gene encoding VP-5 were employed, only RNA transcripts hybridizing to the ICP-0 probe were detected. In latently infected cells, the ICP-0--related transcripts were localized principally in the nucleus; this was not the case in acutely (productively) infected neurons or in neurons probed for RNA transcripts coding for actin. In Northern blotting experiments, an RNA of 2.6 kilobases was detected with the ICP-0 probe. When single-stranded DNAs from the ICP-0 region were used as probes, RNA from the strand complementary to that encoding ICP-0 messenger RNA (mRNA) was the major species detected. This RNA species may play a significant role in maintaining the latent infection.

Animals↗
PubMed1989

Antisense RNA complementary to 3' coding and noncoding sequences of creatine kinase is a potent inhibitor of translation in vivo.

Antisense RNA is a potentially powerful tool for creating dominant negative mutations, but one of the limitations of this strategy has been the relative inefficiency of antisense transcripts in blocking target gene expression. To identify more effective target sequences, helper-free retrovirus-mediated gene transfer was used to introduce antisense RNAs complementary to multiple functional regions of the human creatine kinase B (CK-B) mRNA into U937 cells. Antisense RNA complementary to the last third of the coding and all of the noncoding regio of this mRNA is highly effective; one or two antisense transcripts is sufficient to block the expression of one CK-B mRNA. In contrast, antisense RNA from which sequences complementary to the last 17 codons and all the 3' noncoding region have been deleted has no effect on CK-B expression. Neither antisense RNA alters the abundance of the target message, processing of the primary transcript, egress of the CK-B message from the nucleus, or the polysome profile of CK-B mRNA in sucrose gradients. These results point to a direct effect of the antisense transcript on translation and suggest that this effect may be explained at least in part by an inhibition of elongation or termination as a consequence of the duplex formed in the distal coding and/or 3' noncoding region.

Blotting, Northern↗
PubMed1986

Cytoplasmic RNA complementary to pre-albumin intron V.

We have detected an RNA species, containing sequences complementary to pre-albumin intron V in poly(A)+ RNA from rat liver cytoplasm (but not in nuclear RNA). Its relative abundance roughly parallels that of mature albumin mRNA, when comparing control with acute phase preparations.

Animals↗
PubMed1975

Polyadenylated RNA complementary to repetitive DNA in mouse L-cells.

Complementary DNA, synthesized with L-cell polyadenylated RNA as template, renatured with total L-cell DNA to about 70%. About 30% complementary to unique sequence DNA and another 10 and 30% corresponded to sequences about 20- and 500-fold repetitive. Complementary DNA was fractionated after partial hybridization with total polyadenylated RNA to obtain preparations enriched or impoverished in complements of the most frequent polyadenylated RNA. Renaturation of these complementary DNA fractions with L-cell DNA revealed that most frequent RNAs are transcribed from repetitive DNA sequences, Complementary DNA, density labeled with bromodeoxyuridine, was fractionated by renaturation with L-cell DNA to yield fractions enriched in repetitive and unique sequence DNA. The denisty labeled complementary DNA was purified by equilibrium centrifiguation in an alkaline Cs2SO4 gradient. The complementary DNA representing mainly repetitive DNA sequences hybridized preferentially to frequent polyadenylated RNA.

Adenine Nucleotides↗
PubMed1978

[Complementary RNA in the chicken sarcoma cells and Rous sarcoma virus].

The subcellular localization in chicken Rous sarcoma of nucleotide sequence, complementary to Rous sarcoma virus RNA was examined by RNA/RNA molecular hybridization. The preparations of radioiodinated virion RNA were annealed with RNAs from different fractions (nuclei, mitochondria, free and membrane-bound polyribosomes) isolated from chicken Rous sarcoma. Formation of RNA-ase resistant hybrids between the viral 125I-RNA and RNA from the mitochondria and membrane-bound polyribosomes was revealed. The latter were characterized by a higher relative redundancy of nucleotide sequences complementary to virion RNA than that in the former, by factor 446. The role of complementary ribonucleotide sequences is discussed.

Animals↗
PubMed1974

Synthesis of complementary RNA sequences during productive adenovirus infection.

Liquid RNA.DNA hybridization with separated strands of adenovirus type 2 DNA revealed that late nuclear RNA can hybridize to about 85% of the 1-strand and 10-15% of the h-strand, whereas late cytoplasmic RNA hybridizes to 65-70% and 25% of the l- and h-strand, respectively. With separated strands from the six EcoRI fragments of adenovirus type 2 DNA as probes, it was shown that late nuclear RNA hydridizes to 85-90% of the l-strand from all six EcoRI fragments. Since late cytoplasmic RNA hybridizes to 40-50% of the h-strand from both fragments EcoRI-B and EcoRI-C, complementary viral RNA sequences are synthesized during adenovirus infection. Complementarity between nuclear and cytoplasmic RNA could also be demonstrated by showing that late cytoplasmic RNA which had been preincubated with late nuclear RNA hybridized to a smaller fraction of the h-strand of fragment EcoRI-C than without preincubation. Double-stranded RNA which contains sequences that correspond to at least 60% of the viral genome was isolated from infected cells. However, less than 2% of the newly synthesized late RNA became double-stranded after incubation under annealing conditions, which suggests that RNA derived from one of the strands is present at a low concentration. Accordingly, it was shown that nearly all viral cytoplasmic RNA which is synthesized late after infection is derived from the l-strand.

Adenoviridae↗