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Results for “RNA, Complementary”

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PubMed1979

The synthesis and processing of a nuclear RNA precursor to rat pregrowth hormone messenger RNA.

A recombinant DNA plasmid, pBR322-GH1, which contains about 80% of the sequences of rat pregrowth hormone (pGH) mRNA, allowed an analysis of nuclear RNA from GH3 cells for possible precursors of cytoplasmic pGH mRNA. A single 20-22S RNA SPECIES ABOUT 2-3 TIMes larger than pGH mRNA was detected in nuclear RNA from GH3 cells labeled for 5 min. with 3H-uridine. After longer label times a 12S RNA indistinguishable in size from cytoplasmic 12S pGH mRNA became the predominant labeled RNA complementary to the plasmid pBR322-GH1. Both of these nuclear RNA species contained poly (A). Kinetic analysis of the labeling of nuclear and cytoplasmic pGH mRNA sequences showed that the 20S and 12S nuclear RNA molecules were labeled before significant labeling of cytoplasmic pGH mRNA was detected, and also indicated that there is complete conservation of nuclear pGH mRNA sequences in the production of cytoplasmic pGH mRNA. These results indicate that cytoplasmic pGH mRNA is generated by nuclear processing of a larger nuclear RNA molecule.

Animals↗

PubMed1977

Amount and distribution of virus-specific sequences in giant RNA molecules isolated from polyoma-infected mouse kidney cells.

A two-step hybridization with polyoma DNA was used to study the composition of giant RNA molecules synthesized in mouse kidney cells late in productive infection by polyoma virus. Giant molecules longer than a complete transcript of the polyoma genome were purified from cells that had been pulse-labeled for 30 min with [3H]uridine and annealed, under mild conditions (50% formamide, 37 degrees C), with polyoma DNA loaded on nitrocellulose filters. Hybridized RNA (6 to 7% of the entire population of 3H-labeled molecules and up to 15% of the molecules containing polyadenylic acid [poly(A)]] was eluted and annealed a second time with polyoma DNA under more stringent conditions. In this second step, 75% of the 3H-labeled RNA formed an RNase-resistant hybrid. Under the same conditions, complementary RNA hybridized with polyoma DNA to a maximal extent of 80%. Since the difference between 75 and 80% is within the experimental error of the hybridization assay, it is inferred that the giant molecules selected by the first hybridization may consist entirely of virus-specific sequences or contain, at the most, a minor fraction of nonviral sequences. To examine the possibility that such nonviral sequences are clustered at the 3'-terminus of these molecules, poly(A)+ giant RNA, which had not been preselected by hybridization with polyoma DNA, was fragmented by a limited alkaline hydrolysis. Fragments linked to the poly(A) segment were separated from the rest of the cleavage products. A one-step hybridization with polyoma DNA revealed that both fractions contain 8 to 10% of virus-specific sequences. These results indicate that the 3'-termini of the poly(A)+ polyoma-specific giant RNA molecules consist of viral rather than nonviral sequences.

Animals↗

PubMed1986

Ambisense RNA viruses: positive and negative polarities combined in RNA virus genomes.

The coding strategies of arenaviruses (family Arenaviridae) and members of the Phlebovirus genus of the Bunyaviridae differ from those of other negative-sense RNA viruses in that some proteins are coded in viral-complementary RNA sequences and others are coded in the viral RNA sequence. The term ambisense RNA has been proposed to denote these unique coding arrangements. The implications of the ambisense RNA coding strategy for the evolution and infection processes of these viruses are discussed.

Arenaviridae↗

PubMed2006

Oocyte expression with injection of purified T7 RNA polymerase.

The Xenopus oocyte is a widely used system for protein expression. Investigators have had the choice between two different techniques: injection into the cytoplasm of in vitro transcribed complementary RNA (cRNA) or injection into the nucleus of complementary DNA (cDNA). We report on a third expression technique that is based on the combined injection of cDNA and purified T7 RNA polymerase directly into the cytoplasm of oocytes.

Animals↗

PubMed1988

Single RNA species injected in Xenopus oocyte directs the synthesis of active choline acetyltransferase.

In vitro synthesized complementary RNA (cRNA) transcribed from a non-full length Drosophila choline acetyltransferase (ChAT) cDNA clone will direct the synthesis of enzymatically active and immunologically recognizable protein when injected into Xenopus oocytes. The levels of ChAT activity expressed in injected oocytes are proportional, over 4 orders of magnitude difference, to the concentration of injected 'sense' orientation cRNA. GpppG capping of the in vitro synthesized cRNA is not necessary for expression of active ChAT but inclusion of the capping compound during in vitro transcription results in higher levels of enzyme expression at lower levels of cRNA injection. In addition the capped cRNA results in increasing ChAT expression by the oocytes up to 7 days after injection while uncapped cRNA results in maximum enzyme activity after a single day and decreasing levels of activity during subsequent days. A single immunologically detectable protein is produced by oocytes injected with 'sense' cRNA which has a molecular size of 75 kDa and is indistinguishable from the major form of ChAT present in Drosophila. Oocytes making enzymatically active ChAT also accumulate significant levels of acetylcholine. We conclude from these results that our our non-full length Drosophila ChAT cDNA clone contains all the necessary coding information to make a functional protein which appears to have the same size and activity as native Drosophila ChAT.

Acetylcholine↗

PubMed1975

Temperature-sensitive mutants of influenza WSN virus defective in virus-specific RNA synthesis.

Influenza WSN virus temperature-sensitive (ts) mutants were examined for defects in viral complementary RNA (cRNA) synthesis. The synthesis of viral cRNA was determined by hybridizing RNA from infected cells to radiolabeled virion RNA of known specific activity. Mutants in complementation groups I and III synthesized little, or no, cRNA at the nonpermissive temperature (39.5 C). When cells infected by these mutants were incubated for 5 h at the permissive temperature (33 C) and were then shifted to 39.5 C, net synthesis of cRNA ceased. This strongly suggests that mutants in these two complementation groups possess a ts defect in the transciptase complex. Mutants in group II and group V synthesize reduced amounts of cRNA at 39.5 C. In contrast to the group I and group III mutants, cRNA synthesis in cells infected by a group II or a group V mutant continues after a shift-up. This indicated that these mutants do not possess a ts transcriptase complex and that these mutants are most probably defective in some step in the amplification of cRNA synthesis. As will be discussed, the most likely defect in these mutants is in the synthesis of virion-type RNA. These results suggest that there are two influenza viral gene functions required for transcription and most likely two additional gene functions required for RNA replication.

Cell Line↗

PubMed2006

Solid-phase detection of RNA using bispyrene-modified RNA probe.

Bispyrene modified 2'-O-methyl oligoRNA (OMUpy2), which is a useful fluorescent probe for specific RNA detection, was immobilized on a glass substrate via various linker molecules to develop a more convenient RNA detection chip for gene expression study. As the fluorescence intensity of OMUpy2 enhanced greatly when it hybridized with its complementary RNA, it was expected that RNA detection by OMUpy2-immobilized chip (RNA-chip) required neither fluorescent labels to target nucleic acid nor washing protocol after the hybridization. As the linker molecules, oligo(dT), agarose, and poly(ethylene glycol) (PEG) were chosen, and the fluorescence from the chip was measured by a fluorescence microscope. Results indicated that the RNA-chip was able to detect the complementary oligoribonucleotide (cORN) without target labeling and washing protocols. Furthermore, the background emission was reduced as the length of the linker increased. In the case of PEG linker, 25-fold enhancement of the fluorescence intensity of the OMUpy2 was observed upon the addition of cORN.

Fluorescent Dyes↗

PubMed2002

Implications of high-affinity hybridization by locked nucleic acid oligomers for inhibition of human telomerase.

Oligonucleotides that contain locked nucleic acid (LNA) bases have remarkably high affinity for complementary RNA and DNA sequences. This increased affinity may facilitate the recognition of nucleic acid targets inside cells and thus improve our ability to use synthetic oligonucleotides for controlling cellular processes. Here we test the hypothesis that LNAs offer advantages for inhibiting human telomerase, a ribonucleoprotein that is critical for tumor cell proliferation. We observe that LNAs complementary to the telomerase RNA template are potent and selective inhibitors of human telomerase. LNAs can be introduced into cultured tumor cells using cationic lipid, with diffuse uptake throughout the cell. Transfected LNAs effectively inhibited intracellular telomerase activity up to 40 h post-transfection. Shorter LNAs of eight bases in length are also effective inhibitors of human telomerase. The melting temperatures of these LNAs for complementary sequences are superior to those of analogous peptide nucleic acid oligomers, emphasizing the value of LNA bases for high-affinity recognition. These results demonstrate that high-affinity binding by LNAs can be exploited for superior recognition of an intracellular target.

Animals↗

PubMed2005

3'-modified oligo (2'-O-methylribonucleotides) as improved probes for hybridization with RNA.

A series of octa (2-O-methylribonucleotides) with an additional 3'-terminal deoxynucleoside (T, dC, dA or dG) linked by the 3'-3' (inverted) bond was synthesized. The exceptional stability of these oligomers to a 3'-exonuclease (SVP) and nucleases in culture medium containing 10% heat-inactivated fetal calf serum was demonstrated. It was shown that the addition of the 3'-dangling inverted deoxynucleoside increases substantially the thermal stability of the duplexes of oligo(2'-O-methylribonucleotides) with complementary RNA and DNA in the case of a relatively weak terminal AmU(T) pair and enhances the mismatch sensitivity.

Base Pair Mismatch↗

PubMed1992

A new method for reconstituting influenza polymerase and RNA in vitro: a study of the promoter elements for cRNA and vRNA synthesis in vitro and viral rescue in vivo.

The influenza RNA polymerase is known to catalyse three distinct copying activities: (i) transcription of minus-sense virion RNA (vRNA) into mRNA, (ii) transcription of vRNA into full-length complementary RNA (cRNA), and (iii) transcription of cRNA to vRNA. Ever since the discovery of the conserved 13 and 12 long sequences at each end of all the influenza RNA segments, these have been good candidates for promoters of transcription. By devising a new, simple method for preparing influenza polymerase complex capable of transcribing in vitro added short model RNA templates without interference from endogenous viral RNA, we have now tested the promoter hypothesis. We conclude that the 13 long and the 12 long 3' conserved sequences of cRNA and vRNA of influenza A virus are by themselves sufficient to promote vRNA and cRNA synthesis in vitro. Using our new method, we also show that chloramphenicol acetyl transferase (CAT) activity can be detected in MDBK (bovine kidney) cells, after transfection of influenza polymerase assembled with a negatively stranded CAT RNA, even in the absence of helper virus. As in a previously described method (Luytjes et al., 1989), CAT activity is amplified by helper virus and can be rescued in infectious recombinant virus.

Base Sequence↗

PubMed2000

Straightening of bulged RNA by the double-stranded RNA-binding domain from the protein kinase PKR.

The human interferon-induced protein kinase, PKR, is an antiviral agent that is activated by long stretches of double-stranded (ds)RNA. PKR has an N-terminal dsRNA-binding domain that contains two tandem copies of the dsRNA-binding motif and interacts with dsRNA in a nonsequence-specific fashion. Surprisingly, PKR can be regulated by certain viral and cellular RNAs containing non-Watson-Crick features. We found that RNAs containing bulges in the middle of a helix can bind to p20, a C-terminal truncated PKR containing the dsRNA-binding domain. Bulges are known to change the global geometry of RNA by bending the helical axis; therefore, we investigated the conformational changes of bulged RNA caused by PKR binding. A 66-mer DNA-RNA(+/- A(3) bulge)-DNA chimera was constructed and annealed to a complementary RNA strand. This duplex forces the protein to bind in the middle. A 66-mer duplex with a top strand composed of DNA-DNA(+/-A(3) bulge)-RNA was used as a control. Gel mobility-shift changes among the RNA-protein complexes are consistent with straightening of bulged RNA on protein binding. In addition, a van't Hoff analysis of p20 binding to bulged RNA reveals a favorable DeltaDeltaH degrees and an unfavorable DeltaDeltaS degrees relative to binding to straight dsRNA. These thermodynamic parameters are in good agreement with predictions from a nearest-neighbor analysis for RNA straightening and support a model in which the helical junction flanking the bulge stacks on protein binding. The ability of dsRNA-binding motif proteins to recognize and straighten bent RNA has implications for modulating the topology of RNAs in vivo.

Base Sequence↗

PubMed1982

Expression of early viral gene products in adenovirus type 12-infected and -transformed cells.

We have analysed early viral gene products expressed in adenovirus type 12 (Ad12)-infected cells as well as in two Ad12-transformed hamster cell lines, and Ad12-induced rat tumour cell lines by cell-free translation of virus-specific RNA which was selected by hybridization to cloned restriction endonuclease fragments of virus DNA. Proteins synthesized in vitro were analysed by one- and two-dimensional gel electrophoresis. It was found that RNA encoded by early region E1A directs the synthesis of at least eight polypeptides with apparent mol. wt. 38K, 36K, 30K, 28K, 26K, 25K, 24K and 22K. All these proteins are related to each other. E1B-specific RNA directs the synthesis of three proteins: 59K, 19K and 17K. Early region E2a codes for a 61K polypeptide which probably represents the single-strand DNA-binding protein of Ad12. RNA complementary to region E3 directs the synthesis of a 16K protein, and RNA transcribed from region E4 the synthesis of polypeptides with mol. wt. 20K, 18K and 11.5K. We have mapped a 67K polypeptide into the region within 11 to 28 map units (E2b). The analysis of proteins directed by virus-specific RNAs prepared from two Ad12-transformed hamster cell lines (T637, HA12/7) and one Ad12-induced rat tumour line (RBT12/3) showed that early regions E1 and E4 are expressed in all three Ad12-transformed cell lines. RNA transcribed from early regions E2 and E3 have been detected in lines T637 and RBT12/3. The virus RNA prepared from the Ad12-transformed cell lines directed synthesis of polypeptides with mol. wt. very similar to those of early virus proteins from infected cells. However, in all three Ad12-transformed cell lines mentioned above we have found RNAs which directed the synthesis of additional polypeptides of early regions E1 (34K) and E4 (25K, 24K) not detected in infected cells. The DNA sequence between 11 and 28 map units (coding for the 67K protein) is not expressed in the Ad12-transformed cells.

Adenoviruses, Human↗

PubMed1982

Herpesvirus infection alters the steady-state levels of cellular polyadenylated RNA in polyoma virus-transformed BHK cells.

Polyoma-transformed BHK cells are permissive for the replication of herpes simplex virus type 1. The effect of herpes infection on the steady-state levels of bulk mRNA sequences in these cells was studied by using cDNA to polyadenylated cytoplasmic RNA from uninfected cells. The principal findings were: (i) herpes simplex virus type 1 infection caused a pronounced reduction in the cytoplasmic levels of moderately abundant mRNAs' (ii) after infection, increased amounts of RNA complementary to the cDNA were isolated as part of the nonadenylated cytoplasmic RNA fraction.

Animals↗

PubMed2005

Production and characterization of amplified tumor-derived cRNA libraries to be used as vaccines against metastatic melanomas.

BACKGROUND: Anti-tumor vaccines targeting the entire tumor antigen repertoire represent an attractive immunotherapeutic approach. In the context of a phase I/II clinical trial, we vaccinated metastatic melanoma patients with autologous amplified tumor mRNA. In order to provide the large quantities of mRNA needed for each patient, the Stratagene Creator SMART cDNA library construction method was modified and applied to produce libraries derived from the tumors of 15 patients. The quality of those mRNA library vaccines was evaluated through sequencing and microarray analysis. RESULTS: Random analysis of bacterial clones of the library showed a rate of 95% of recombinant plasmids among which a minimum of 51% of the clones contained a full-Open Reading Frame. In addition, despite a biased amplification toward small abundant transcripts compared to large rare fragments, we could document a relatively conserved gene expression profile between the total RNA of the tumor of origin and the corresponding in vitro transcribed complementary RNA (cRNA). Finally, listing the 30 most abundant transcripts of patient MEL02's library, a large number of tumor associated antigens (TAAs) either patient specific or shared by several melanomas were found. CONCLUSION: Our results show that unlimited amounts of cRNA representing tumor's transcriptome could be obtained and that this cRNA was a reliable source of a large variety of tumor antigens.

Journal Article↗

PubMed1994

Persistent expression of a truncated form of the luteinizing hormone receptor messenger ribonucleic acid in the rat testis after selective Leydig cell destruction by ethylene dimethane sulfonate.

To correlate the process of ethylene dimethane sulfonate (EDS)-induced disappearance and repopulation of Leydig cells with LH receptor (LHR) expression, testicular messenger RNA (mRNA) and binding of LHR were analyzed in male rats, 5, 15, 20, and 40 days after treatment with 75 mg EDS/kg BW. Five and 15 days after EDS treatment, the serum testosterone level was reduced by 90% (P < 0.01) and testicular [125I]iodo-hCG binding was nearly undetectable (P < 0.01). Multiple splice variants of the LHR mRNA, with sizes of 6.8, 4.2, 2.7, and 1.8 kilobases, were detected in control testes upon Northern hybridization. Interestingly, 5 and 15 days after EDS injection, only the 1.8-kilobase band, previously reported to correspond to a truncated form of the LHR mRNA and encoding its extracellular part, remained, whereas the other mRNA species disappeared. On days 20 and 40 after EDS treatment, the pattern of hybridization gradually returned to that resembling the control pattern. To increase the sensitivity of mRNA detection, testicular RNA was reverse transcribed and amplified by polymerase chain reaction, using primers complementary to various parts of the LHR complementary DNA. The specificity of the complementary DNAs generated was verified by Southern hybridization with nested oligonucleotide primers. Five and 15 days after EDS treatment, only truncated mRNA forms, encoding regions of the extracellular part of the LHR, could be amplified. At 20 and 40 days, the pattern of amplification was similar to that in control testes, with amplification of the whole coding sequence. In situ hybridization was performed on day 5 after EDS treatment, when the interstitial space was devoid of morphologically discernible Leydig cells. An antisense complementary RNA probe, corresponding to the extracellular domain of the receptor, hybridized in the interstitial space to apparent precursor Leydig cells. Taken together, these results strongly suggest that the precursor Leydig cells, resistant to the cytotoxic action of EDS, express truncated forms of the LHR mRNA in the early stages of their differentiation. The full-length mRNA of LHR gradually appears when the functional Leydig cells recover and attain their differentiated functions. These data are analogous with our previous findings on testicular ontogeny; the fetal Leydig cell precursors constitutively express a truncated form of the LHR gene as well, and a similar change occurs in its alternative splicing during testicular maturation. Hence, the truncated form of the LHR mRNA is an early sign of Leydig cell differentiation, whether it occurs during ontogeny or in adulthood upon recovery from cytotoxic treatment.

Animals↗

PubMed2002

Selective strand annealing and selective strand exchange promoted by the N-terminal domain of hepatitis delta antigen.

We have previously shown that the N-terminal domain of hepatitis delta virus (NdAg) has an RNA chaperone activity in vitro (Huang, Z. S., and Wu, H. N. (1998) J. Biol. Chem. 273, 26455-26461). Here we investigate further the basis of the stimulatory effect of NdAg on RNA structural rearrangement: mainly the formation and breakage of base pairs. Duplex dissociation, strand annealing, and exchange of complementary RNA oligonucleotides; the hybridization of yeast U4 and U6 small nuclear RNAs and of hammerhead ribozymes and cognate substrates; and the cis-cleavage reaction of hepatitis delta ribozymes were used to determine directly the role of NdAg in RNA-mediated processes. The results showed that NdAg could accelerate the annealing of complementary sequences in a selective fashion and promote strand exchange for the formation of a more extended duplex. These activities would prohibit NdAg from modifying the structure of a stable RNA, but allow NdAg to facilitate a trans-acting hammerhead ribozyme to find a more extensively matched target in cognate substrate. These and other results suggest that hepatitis delta antigen may have a biological role as an RNA chaperone, modulating the folding of viral RNA for replication and transcription.

Base Sequence↗

PubMed1991

Enzymatic aminoacylation of single-stranded RNA with an RNA cofactor.

A chemically synthesized single-stranded ribonucleotide tridecamer derived from the 3' end of Escherichia coli alanine tRNA can be charged with alanine in the presence of short complementary RNA oligonucleotides that form duplexes with the 3' fragment. Complementary 5' oligomers of 9, 8, 6, and 4 nucleotides all confer charging of the 3' fragment. Furthermore, in the presence of limiting 5' oligomer, greater than stoichiometric amounts of the single-stranded 3' acceptor fragment can be aminoacylated. This is due to a reiterative process of transient duplex formation followed by charging, dissociation of the 5' oligomer, and then rebinding to an uncharged single-stranded ribotridecamer so as to create another transient duplex substrate. Thus, a short RNA oligomer serves as a cofactor for a charging enzyme, and it thereby makes possible the aminoacylation of single-stranded RNA. These results expand possibilities for flexible routes to the development of early charging and coding systems.

Alanine-tRNA Ligase↗

PubMed2005

Cooperation of metal-ion fixation and target-site activation for efficient site-selective RNA scission.

Iminodiacetate-DNA conjugates and acridine-DNA conjugates were synthesized and combined for site-selective RNA hydrolysis by Lu(III). When these conjugates form a ternary complex with complementary RNA, the Lu(III)-iminodiacetate complex is placed near the target phosphodiester linkage of RNA which is in front of the acridine and is activated by noncovalent interactions. The site-selective hydrolysis by these combinations is several times as fast as that achieved by combining unmodified DNA (without iminodiacetate) and the acridine-DNA conjugate.

Base Sequence↗