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Results for “RNA, Complementary”

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PubMed1982

Polyadenylylated RNA complementary to a mouse retrovirus-like multigene family is rapidly and specifically induced by epidermal growth factor stimulation of quiescent cells.

Complementary DNA probes prepared from total polysomal poly(A)+RNA populations were used to identify clones of mouse DNA containing sequences whose expression is specifically enhanced after epidermal growth factor (EGF) stimulation of quiescent mouse embryo cells in culture. Three such clones were isolated and used to study changes in the levels of clone-specific poly(A)+RNA in the polysomes of cells after mitogenic stimulation by EGF. RNA complementary to sequences present in these clones increased approximately equal to 10-fold as a fraction of the total poly(A)+RNA by 6 hr after stimulation. All three clones were found by hybridization criteria to contain sequences related to the class of mouse retrovirus or transposon-like elements termed VL30. These VL30-related sequences were further found to be complementary to EGF-inducible poly(A)+RNAs and enhanced expression was detectable as early as 1 hr after EGF stimulation. In contrast, nine additional clones, including an AKR-type murine leukemia provirus DNA clone, contained no detectable VL30 sequence elements and were complementary to poly(A)+RNA species whose relative concentration was essentially constant in quiescent and EGF-stimulated cells. Therefore, VL30 sequence elements appear distinct in that they encompass members whose expression is specifically regulated in response to a defined peptide growth factor.

Animals↗

PubMed1977

Synthesis of complementary RNA on RNA templates using the DNA-dependent RNA polymerase of Escherichia coli.

It is shown that the DNA-dependent RNA polymerase of Escherichia coli can synthesize complementary RNA (cRNA) directly on rRNA and mRNA templates. Synthesis occurred preferentially in the presence of Mn2+ and at relatively high substrate and enzyme concentrations. No primer was required, and addition of oligo-U to a mRNA-dependent reaction gave no marked stimulation. Sedimentation analysis of cRNA made on different templates indicated that the products were mainly 2-4 S, but a fraction of the product was larger. Fingerprints of 32P-labelled cRNA made on 5 S rRNA and 18 S rRNA indicated that the complexity of the cRNAs was related to the size of the template, suggesting that a substantial portion of the templates were copied. This reaction provides a simple method for preparing cRNA of high specific activity for use in hybridisation studies, and possibly in sequence analysis. 32P-labelled cRNA made on 18 S and 28 S rRNA was a sensitive hybridisation probe for detection of the specific fragments of mouse DNA containing the rRNA genes.

DNA-Directed RNA Polymerases↗

PubMed1977

Influenza virion transcriptase: synthesis in vitro of large, polyadenylic acid-containing complementary RNA.

The influenza virion transcriptase is capable of synthesizing in vitro complementary RNA (cRNA) that is similar in several characteristics to the cRNA synthesized in the infected cell, which is the viral mRNA. Most of the in vitro cRNA is large (approximately 2.5 X 10(5) to 10(6) daltons), similar in size to in vivo cRNA. The in vitro transcripts initiate in adenosine (A) or guanosine (G) at the 5' end, as also appears to be the case with in vivo cRNA (R.M. Krug et al., 1976). The in vitro transcripts contain covalently linked polyadenylate [poly(A)] sequences, which are longer and more heterogeneous than the poly(A) sequences found on in vivo cRNA. The synthesis in vitro of cRNA with these characteristics requires both the proper divalent cation, Mg2+, and a specific dinulceside monophosphage (DNMP), ApG or GpG. These DNMPs stimulate cRNA synthesis about 100-fold in the presence of Mg2+ and act as primers to initiate RNA chains, as demonstrated by the fact that the 5'-phosphorylated derivatives of these DNMP's, 32pApG or 32pGpG, are incroporated at the 5' end of the product RNA. The RNA synthesized in vitro differs from in vivo cRNA in that neither capping nor methylation of the in vitro transcripts has been detected. The virion does contain a methylase activity, as shown by its ability to methylate exogenous methyl-deficient Escherichia coli tRNA.

Cell Line↗

PubMed1976

Synthesis of complementary RNA containing polyadenylic acid by Sendai virions in vitro.

Sendai virus synthesized, in vitro, [32P]AMP- and [3H]AMP-labeled RNA that ranged in size from 3 to 25S with major peaks at 7S and 13S. Both labeled products were predominantly single-stranded RNA and were complementary in base sequence to 50S virion RNA. Passage of the 3 to 25S in vitro RNA transcripts through a polyuridylic acid-cellulose column revealed that only the larger (predominantly 18S) RNA transcripts contained polyadenylic acid[poly(A)] segments capable of binding to the column. After treatment with a combination of RNase A and T1, the majority of the in vitro poly(A) sedimented at 6S although the product ranged in size from 3 to 9S. Proof that the RNase-resistant material was indeed poly(A) was obtained by nearest-neighbor analysis when 95% of the radioactivity was recovered in AMP.

Base Sequence↗

PubMed1975

[Conjugation of polytene chromosomes in intraspecies hybrids of the virilis group of Drosophila. II. Hybridization of complementary RNA with polytene chromosomes in specimens].

A cytological hybridization of H-3-complementary RNA synthetized from DNA a template of D. virilis with the polytene chromosomes of D. virilis and the hybrids between D. virilis and D. texana, was carried out in situ. The uridine label of RNA was shown to be located mainly over the disc of the polytene chromosomes, the silver grains in interspecies hybrids being located over both homogous chromosomes including the unpaired gions.

Animals↗

PubMed1975

Purification of influenza viral complementary RNA: its genetic content and activity in wheat germ cell-free extracts.

Influenza viral complementary RNA (cRNA) was purified free from any detectable virion-type RNA (vRNA), and its genetic content and activity in wheat germ cell-free extracts were examined. After phenol-chloroform extraction of cytoplasmic fractions from infected cells, poly(A)-containing viral cRNA is found in two forms: in single-stranded RNA and associated with vRNA in partially and fully double-stranded RNA. To purify single-stranded cRNA free of these double-stranded forms, it was necessary to employ, as starting material, RNA fractions in which cRNA was predominantly single stranded. Two RNA fractions were successfully employed as starting material: polyribosomal RNA and the total cytoplasmic RNA from infected cells treated with 100 mug of cycloheximide (CM) per ml at 3 h after infection. In WSN virus-infected canine kidney (MDCK) cells, the addition of CM at 3 h after infection stimulates the production of cRNA threefold and causes a very large increase in the proportion of the cytoplasmic cRNA which is single stranded; double-stranded RNA forms are greatly reduced in amount. Total cRNA was obtained by oligo(dT)-cellulose chromatography, and single-stranded cRNA was separated from double-stranded forms by Sepharose 4B chromatography. The cRNA preparation purified from polyribosomes consists of 95% single-stranded cRNA, with the remaining 5% apparently being double-stranded RNA forms. The cRNA preparation purified from CM-treated cells (CM cRNA) is even more pure: 100% of the radiolabeled RNA is single-stranded cRNA. Annealing experiments, in which a limited amount of 32P-labeled genome RNA was annealed to the cRNA, indicate that the purified cRNA contains at least 84 to 90% of the genetic information in the vRNA genome. Purified viral cRNA (CM cRNA) is very active in directing the synthesis of virus-specific proteins in wheat germ cell-free extracts.

Cell Line↗

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↗

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↗

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↗

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↗