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Results for “RNA, Complementary”

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PubMed1987

Localization of neuropeptide Y mRNA in neurons of human cerebral cortex by means of in situ hybridization with a complementary RNA probe.

The distribution of mRNA encoding neuropeptide Y (NPY) in neurons of the normal human cerebral cortex in surgical biopsy specimens and postmortem brain was studied by in situ hybridization techniques. A 32P-labeled complementary RNA (cRNA) probe was used on cryostat sections of 13 formaldehyde-fixed cortical biopsy specimens. Hybridization to NPY mRNA was found in all samples: after autoradiography, discrete deposits of silver granules were observed on neuronal cell bodies abundantly distributed in the deep layers of the cortex, particularly laminae IV and VI, and on smaller cell bodies in the white matter. The localization of the neurons hybridized for NPY mRNA was comparable to that of NPY-immunoreactive cells as shown in sections from the same tissue blocks immunostained by using NPY antibodies. The specificity of the in situ hybridization technique was confirmed by blot hybridization analysis of electrophoretically fractionated RNA. This study clearly demonstrated the consistent localization of NPY gene transcription and expression in normal mature human cortical neurons.

Antibodies↗

PubMed1978

Polyoma virus complementary RNA directs the in vitro synthesis of capsid proteins VP1 and VP2.

Polyoma virus complementary RNA, synthesized in vitro by using highly purified Escherichia coli RNA polymerase and nondefective form I polyoma DNA, was translated in a wheat germ cell-free system. Polypeptides were synthesized that comigrated on sodium dodecyl sulfate-polyacrylamide gels with the polyoma capsid proteins VP1 and VP2, although most of the cell-free products were of smaller molecular weights. The VP1-size protein specifically immunoprecipitated with anti-polyoma virus serum, and upon digestion by trypsin yielded [35S]methionine-labeled tryptic peptides that co-chromatographed with the [3H]methionine-labeled tryptic peptides of virion-derived VP1 on both cation-exchange and anion-exchange resins. The VP2-size in vitro product contained all the virion VP2 methionine-labeled tryptic peptides, as shown by cation- and anion-exchange chromatography and two-dimensional fingerprinting on cellulose. We conclude that full-length polyoma VP1 and VP2 are synthesized in response to complementary RNA and consequently that the viral capsid proteins VP1, VP2, and VP3 are entirely virus coded.

Capsid↗

PubMed1989

NMR study of the possible interaction in solution of angiotensin II with a peptide encoded by angiotensin II complementary RNA.

The potential binding of angiotensin II (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe) (AII) to a peptide encoded by its complementary RNA (Lys-Gly-Val-Asp-Val-Tyr-Ala-Val) (IIA) has been studied by monitoring the 1H NMR spectrum of IIA in aqueous phosphate or Tris.HCl buffer (2H2O) as it is titrated with AII. For molar ratios of AII/IIA ranging from 0.2 to 1.8, the NMR spectra are unchanged as compared to the spectra of the isolated peptides. Based on these findings, the Kd for the putative biomolecular complex of the two peptides under these conditions is calculated to be greater than 10(-4) M. This result does not support the suggestion of Elton et al. [Elton, T. S., Dion, L.D., Bost, K. L., Oparil, S. & Blalock, J. E. (1988) Proc. Natl. Acad. Sci. USA 85, 2518-2522] that AII and IIA engage in high-affinity binding (Kd approximately 5 x 10(-8) M) with each other.

Amino Acid Sequence↗

PubMed1979

Localization of viral DNA-replication in sections of human warts by nucleic acid hybridization with complementary RNA of human papilloma virus Type 1.

Using complementary RNA of human papilloma virus type 1 (HPV1) and in situ hybridization techniques the localization of viral DNA replication was studied in sections of 38 human virus acanthomata from 31 different patients. In five cases significant labeling was detected by autoradiography. Labeling started always in the first or the second suprabasal cell layer and was strongly limited to the nuclei. A remarkable early beginning of the vacuolated process seemed to be correlated with the visible DNA replication. No labeling could be detected in the basal cell layer. This suggests that these cells contain at the most only a small number of viral genomes. Our findings represent only the situation in human warts definitely caused by HPV 1. We are not able to say, whether our negative hybridization results in the remaining warts indicate either the scarcity of viral genomes within these sections or their infection by a different agent.

DNA Replication↗

PubMed1977

Human beta-globin messenger RNA. I. Nucleotide sequences derived from complementary RNA.

Sequence analysis studies were carried out on human beta-globin mRNA (beta-mRNA) prepared from alpha-thalassemic, sickle cell, and Hb A reticulocytes. Highly purified beta-mRNA served as substrate for the preparation of cDNA by RNA-dependent DNA polymerase. The cDNA was transcribed by Escherichia coli RNA polymerase and the resulting cRNA was analyzed. Over 300 nucleotides were assigned to the beta-mRNA coding region and 37 nucleotides were assigned to the 3'-terminal noncoding region. The normal termination codon is UAA which is separated by 28 nucleotides from an out of phase UAA triplet. The origin of each of the abnormally long beta-globin variants Tak and Cranston is consistent with reduplication of dinucleotides prior to the normal termination codon, and both globin variants can terminate at the out of phase UAA.

Anemia, Sickle Cell↗

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↗

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↗

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↗

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↗