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PubMedDate not supplied

Reverse transcriptase and thymidine kinase as markers for tumorigenicity and viral contamination of cells.

Continuous monitoring of enzymes, particularly those involved in nucleic acid synthesis could be a useful means of detecting infections and abnormalities in cells in culture. Model systems using mouse (3T3), human (MRC-5) and chick embryo cells infected with RNA tumour viruses were studied. Reverse transcriptase activities were determined by the incorporation of (3H) nucleotides into synthetic primer-templates or into complementary DNA of endogenous RNA and characterised by their specificity for primer-templates dT12-18.rAn, dG12-18.rCn, dT12-18.DAn and dG10.rCmn, their requirements for metal ions and inhibition by antisera. Measurement of reverse transcriptase is a more sensitive method than the COFAL test for the detection of RAV infection of chick cells. Iododeoxyuridine, bromodeoxyuridine and dexamethasone, which can induce latent C viruses, have no effect on MRC-5 cells; no increases in reverse transcriptase were detected and no C particles were seen by electron microscopy. Solid tumours developed in immunosuppressed mice injected s/c with 3T3 and MRC-5 cells chronically infected with MLV but none formed after injection of cells or virus suspension alone. Thymidine kinase activities of WI-38 and MRC-5 cells are greatly increased by infection with CMV or transformation with SV40. Mammalian tumours and tumour cell lines also show a high specific activity of cytoplasmic thymidine kinase.

Cells, Cultured↗
PubMed1996

Translocation of RNA granules in living neurons.

Sorting of RNAs to specific subcellular loci occurs in diverse settings from fly oocytes to mammalian neurons. Using the membrane-permeable nucleic acid stain SYTO 14, we directly visualized the translocation of endogenous RNA in living cells. Labeled RNA was distributed nonrandomly as discrete granules in neuronal processes. The labeled granules colocalized with poly(A+) mRNA, with the 60S ribosomal subunit, and with elongation factor 1alpha, suggesting that granules represent a translational unit. A subset of labeled granules colocalized with beta-actin mRNA. Correlative light and electron microscopy indicated that the fluorescent granules corresponded to clusters of ribosomes at the ultrastructural level. Poststaining of sections with heavy metals confirmed the presence of ribosomes within these granules. In living neurons, a subpopulation of RNA granules was motile during the observation period. They moved at an average rate of 0.1 microm/sec. In young cultures their movements were exclusively anterograde, but after 7 d in culture, one-half of the motile granules moved in the retrograde direction. Granules in neurites were delocalized after treatment with microtubule-disrupting drugs. These results raise the possibility of a cellular trafficking system for the targeting of RNA in neurons.

Animals↗
PubMed1979

RNA synthesis in isolated nuclei of the dinoflagellate Crypthecodinium cohnii.

Atypical eukaryotic RNA polymerase activity was demonstrated in nuclei of Crypthecodinium cohnii, a eukaryote devoid of histones. Nuclei were isolated from growing cultures of this dinoflagellate and assayed for endogenous RNA polymerase (EC 2.7.7.6) activity. There was a biphasic response to Mg2+ with optima at approximately 0.01 and 0.02 M MgCl2, but in contrast to other eukaryotic RNA polymerases, this enzyme activity was inhibited by low MnCl2 concentrations. In the presence of 0.01 M MgCL2 the optimum (NH4)2SO4 concentration was 0.025 M, a concentration at which the nuclei were lysed. Incorporation of [3H]UMP into RNA was inhibited by actinomycin D and dependent on the presence of undergraded DNA, and the reaction product was sensitive to ribonuclease and KOH digestion. Omission of one or more ribonucleoside triphosphates greatly reduced the incorporation. Only a slight enhancement of RNA polymerase activity resulted from the addition of various amounts of native and denatured calf thymus DNA. Spermine caused a marked inhibition while spermidine had little effect on RNA synthesis in the nuclei. Under the optimum conditions described in the present paper the nuclei incorporated approximately 3 pmoles of [3H]UMP/microgram DNA at 25 C for 15 min, and approximately 80% of this activity was inhibited by the eukaryotic RNA polymerase II inhibitor, alpha-amanitin (20 micrograms/ml). A unique situation therefore exists in C. cohnii nuclei, in which absence of histones (a prokaryotic trait) is combined with alpha-amanitin-sensitive RNA polymerase activity (a eukaryotic trait).

Amanitins↗
PubMed2006

Impact of nonsense-mediated mRNA decay on the global expression profile of budding yeast.

Nonsense-mediated mRNA decay (NMD) is a eukaryotic mechanism of RNA surveillance that selectively eliminates aberrant transcripts coding for potentially deleterious proteins. NMD also functions in the normal repertoire of gene expression. In Saccharomyces cerevisiae, hundreds of endogenous RNA Polymerase II transcripts achieve steady-state levels that depend on NMD. For some, the decay rate is directly influenced by NMD (direct targets). For others, abundance is NMD-sensitive but without any effect on the decay rate (indirect targets). To distinguish between direct and indirect targets, total RNA from wild-type (Nmd(+)) and mutant (Nmd(-)) strains was probed with high-density arrays across a 1-h time window following transcription inhibition. Statistical models were developed to describe the kinetics of RNA decay. 45% +/- 5% of RNAs targeted by NMD were predicted to be direct targets with altered decay rates in Nmd(-) strains. Parallel experiments using conventional methods were conducted to empirically test predictions from the global experiment. The results show that the global assay reliably distinguished direct versus indirect targets. Different types of targets were investigated, including transcripts containing adjacent, disabled open reading frames, upstream open reading frames, and those prone to out-of-frame initiation of translation. Known targeting mechanisms fail to account for all of the direct targets of NMD, suggesting that additional targeting mechanisms remain to be elucidated. 30% of the protein-coding targets of NMD fell into two broadly defined functional themes: those affecting chromosome structure and behavior and those affecting cell surface dynamics. Overall, the results provide a preview for how expression profiles in multi-cellular eukaryotes might be impacted by NMD. Furthermore, the methods for analyzing decay rates on a global scale offer a blueprint for new ways to study mRNA decay pathways in any organism where cultured cell lines are available.

Codon, Initiator↗
PubMed1985

Posttranslational phosphorylation of specific chromosomal proteins and transcription of hnRNA genes in isolated nuclei: retention of in vivo sensitivity to 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB).

The rapidly turning over phosphorylation of specific nuclear nonhistone proteins, especially 42-, 33-, and 30-kDa polypeptides, and its relation to the transcriptional activity of hnRNA genes was investigated in isolated nuclei from salivary gland cells of Chironomus tentans. Incubation conditions promoting the phosphorylation of nonhistone proteins as well as the transcriptional activity of RNA polymerase II were established. The pattern of 32P incorporation into the nonhistone proteins found in isolated nuclei resembled that obtained in experiments with intact cells, and the endogenous RNA polymerase II retained its ability to reinitiate the transcription under in vitro assay conditions. In addition, the in vivo sensitivity of the phosphorylation of 42-, 33-, and 30-kDa polypeptides, like the sensitivity of the initiation of hnRNA transcription to 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB), were preserved in the nuclear preparation. The experimental data taken together provide further support for the idea that the activation of hnRNA genes is causally related to the phosphorylation of specific nonhistone proteins.

Animals↗
PubMed1990

An archaebacterial cell-free transcription system. The expression of tRNA genes from Methanococcus vannielii is mediated by a transcription factor.

Our understanding of the mechanism of RNA biosynthesis in archaebacteria is limited, due in part to the inability of purified RNA polymerases to transcribe purified genes accurately in vitro. In the present study, we show that cell extracts of Methanococcus vannielii and Methanococcus thermolithotrophicus purified by gradient centrifugation synthesize a distinct transcript from templates harboring a cloned homologous tRNA(Val) and tRNA(Arg) gene. The in vitro transcripts initiate with GTP at the same sites as in Methanococcus cells. About 60% of the sequence of the in vitro RNA products was analyzed by dideoxyterminated primer extension and found to be identical with that of the precursors of tRNA(Val) and tRNA(Arg). This finding indicates that this RNA polymerase fraction both initiates and terminates transcription faithfully in vitro. After purification of a cell-free extract (S-100) of M. thermolithotrophicus by phosphocellulose chromatography, the endogenous RNA polymerase has lost its ability to transcribe the tRNA(Val) gene accurately. The activity directing specific expression of this template was reconstituted by the addition of a protein-fraction devoid of RNA polymerase activity. Thus, a transcription factor appears to be required for accurate cell-free expression of tRNA genes from M. vannielii.

Base Sequence↗
PubMed1994

Gene expression mediated by bacteriophage T3 and T7 RNA polymerases in transgenic trypanosomes.

Messenger RNAs of higher eukaryotes share a functionally essential 5' monomethyl CAP structure generated during a reaction that is linked exclusively to RNA polymerase II transcription. In unicellular parasites belonging to the Kinetoplastida, however, mRNAs acquire their 5' CAP through a trans-splicing reaction which effectively uncouples pol II transcription and capping. Consequently functional mRNAs can be produced by endogenous RNA polymerase I. Here we demonstrate the extension of this flexibility to heterologous bacteriophage polymerases. Transgenic Trypanosoma brucei cell lines stably expressing functional, nuclearly localized T3 or T7 RNA polymerase were established and assayed using reporter plasmids bearing the corresponding phage promoters. In these cell lines the levels of phage promoter-driven gene expression ranges from one half to greater than 5 times that mediated by endogenous pol I. Analysis of 5' ends of transcripts synthesized by the T7 polymerase revealed that they are trans-spliced. Thus the usual eukaryotic link between mRNA production and pol II transcription can be by-passed by the introduced phage polymerases, thereby significantly expanding the critically small panel of promoters currently available for exploitation in reverse genetic approaches in T. brucei.

Animals↗
PubMed2005

Spliceosome-mediated RNA trans-splicing.

RNA repair or reprogramming is a new avenue for human gene therapy. Unlike conventional gene therapy, in which exogenous cDNAs are introduced into cells, RNA repair approaches, which are based on spliceosome-mediated pre-mRNA trans-splicing, trans-splicing ribozymes, and tRNA-splicing endonuclease, allow the correction of endogenous RNA species. Recently published accounts that in vivo phenotypic correction of a variety of inherited diseases can be achieved by RNA repair are encouraging. Nevertheless, the science of RNA repair for treatment of human diseases is just beginning and faces several scientific and technical challenges that must be addressed and surmounted. In this review, we summarize recent advances in spliceosome-mediated pre-mRNA trans-splicing. We also provide an update on the progress of this emerging technology toward the development of molecular therapy and diagnosis for human diseases and discuss the outstanding issues and challenges confronting RNA therapeutics.

Animals↗
PubMed1984

Base-pair formation between noncapped influenza virus in vitro transcripts and 18S rRNA in rabbit reticulocyte 80S ribosome-mRNA complexes detected by psoralen photoreaction.

RNA-RNA interactions between 18S ribosomal RNA and noncapped influenza cRNA were detected by psoralen photochemical cross-linking in reticulocyte 80S ribosome-cRNA complexes. In vitro transcripts of type A influenza virus synthesized by endogenous RNA polymerase with adenylyl-(3'----5')-guanosine primer formed 80S complexes with rabbit reticulocyte ribosomes. The extent of the complex formation by these noncapped cRNAs was less than that by the m7G-capped reovirus in vitro transcripts, but the former RNAs in the 80S complexes were cross-linked to 18S rRNA as efficiently as the latter RNAs by photoreaction with an RNA cross-linking agent, 4'-aminomethyl-4,5',8-trimethylpsoralen. These results suggested that mRNA with or without the cap structure on the 5'-terminal can form complexes with the ribosomes in a eukaryotic cell-free translation system by base-pair formation with 18S rRNA. Correspondingly, sequences capable of forming extensive base-pairs including four- to five-base complementarities were found between the 3'-terminal of rabbit reticulocyte 18S ribosomal RNA and the 5'-noncoding regions of either influenza virus transcripts or reovirus mRNA.

Animals↗
PubMed1979

Characterization of a retrovirus isolated from normal mink cells co-cultivated with a dog mammary tumour.

A retrovirus antigenically distinct from known type C, B and D viruses was isolated from normal mink (Mustela vison) lung cells that had been co-cultivated with 5-iododeoxyuridine- and dexamethasone-treated dog mammary tumour cells. Cytogenetic studies of the virus-releasing co-culture showed mitotic figures identical to the normal mink cell line (MvlLu) with the exception of a low frequency of cells with extensive chromosomal breakage and uncoiling. The new virus bands at a buoyant density of 1.16 g/ml, contains 60S RNA and a reverse transcriptase which prefers Mn2+ over Mg2+ for the synthesis of DNA. This enzyme utilizes poly(rA).oligo(dT) more efficiently than poly(dA).oligo(dT) and is also able to synthesize DNA copies from the endogenous RNA. Morphologically, it is a typical type C virus. Filtered virus readily infects mink, dog and other mammalian cells indicating the amphotropic nature of its cell growth requirement. Hybridization studies showed that normal mink DNA contains multiple copies of proviral sequences of this newly isolated virus. Serological analyses indicate that the mink endogenous virus contains in its core protein, in addition to the interspecies type-C determinant, an antigenic component related to one of the determinants found in the feline leukaemia virus p30 protein. This determinant is not present in the Rauscher leukaemia virus, RD114 virus or simian sarcoma virus.

Animals↗
PubMed1997

Mitochondrial glutamate dehydrogenase from Leishmania tarentolae is a guide RNA-binding protein.

To identify specific proteins interacting with guide RNAs (gRNAs) in mitochondrial ribonucleoprotein complexes from Leishmania tarentolae, fractionated and unfractionated mitochondrial extracts were subjected to UV cross-linking with added labeled gRNA and also with [alpha-32P]UTP-labeled endogenous RNA. An abundant 110-kDa protein (p110) localized in the T-V complex, which sediments in glycerol gradients at the leading edge of the 10S terminal uridylyltransferase peak, was found to interact with both types of labeled RNAs. The p110 protein was gel isolated and subjected to microsequence analysis, and the gene was cloned. The sequence revealed significant similarity with mitochondrial glutamate dehydrogenases. A polyclonal antiserum was raised against a recombinant fragment of the p110 gene and was used to demonstrate a stable and specific gRNA-binding activity by coimmunoprecipitation and competitive gel shift analyses. Complex formation was strongly inhibited by competition with poly(U) or by deletion or substitution of the gRNA 3' oligo(U) tail. Also, addition of a 3' oligo(U) tail to an unrelated transcript was sufficient for p110 binding. Both the gRNA-binding activity of the p110 protein and in vitro gRNA-independent and gRNA-dependent uridine insertion activities in the mitochondrial extract were inhibited by high concentrations of dinucleotides.

Amino Acid Sequence↗
PubMed1997

Characterization and mapping of the double-stranded regions involved in activation of PKR within a cellular RNA from 3T3-F442A cells.

PKR is a doubled-stranded RNA-dependent protein kinase which is implicated in the regulation of several cellular processes, including cell proliferation. PKR undergoes phosphorylation and activation in mouse embryonic 3T3-F442A cells in response to endogenous RNA(s). Activation of PKR is related to growth and differentiation of these cells. A cellular regulatory RNA (R-RNA) which activates PKR has been isolated from these cells and its cDNA partially sequenced. Here we have characterized the R-RNA transcript with respect to nuclease sensitivity and the extent of double-stranded structure involved in activation of PKR. The location of the activating sequence was mapped to a contiguous 226/252 nt region of the R-RNA transcript by hybridization to its cDNA fragments. Hybridization with a panel of short oligodeoxynucleotides complementary to the R-RNA, coupled with protein kinase analysis, was used to probe the 252 nt region for critical sequences. Three short non-contiguous sequences which appear most important for activation of PKR were identified within the 252 nt region. Thus, these studies have identified specific sequences most important for activation of PKR. Furthermore, since the above antisense oligodeoxynucleotides inhibit enzyme activation, our results exemplify an unusual mode of action of antisense sequences on the activation of PKR by disruption of RNA secondary structure.

3T3 Cells↗
PubMed1996

Complete replication in vitro of tobacco mosaic virus RNA by a template-dependent, membrane-bound RNA polymerase.

A crude membrane-bound RNA polymerase, obtained by differential centrifugation of extracts of tomato leaves infected with tobacco mosaic tobamovirus (tomato strain L) TMV-L), was purified by sucrose density gradient centrifugation. Removal of the endogenous RNA template with micrococcal nuclease rendered the polymerase template dependent and template specific. The polymerase was primer independent and able to initiate RNA synthesis on templates containing the 3'-terminal sequences of the TMV-L positive or negative strands. TMV-vulgare RNA was a less efficient template, while RNAs of cucumber mosaic cucumovirus and red clover necrotic mosaic dianthovirus, or 5'-terminal sequences of TMV-L positive or negative strands, did not act as templates for the polymerase. A main product of the reaction with TMV-L genomic RNA as a template, carried out in the presence of [alpha-32P]UTP, was genomic-length single-stranded RNA. This was shown to be the positive strand and uniformly labelled along its length, demonstrating complete replication of TMV-L RNA. Genomic-length double-stranded RNA, labelled in both strands, and small amounts of RNAs corresponding to the single- and double-stranded forms of the coat protein subgenomic mRNA were also formed. Antibodies to N-terminal and C-terminal portions of the 126-kDa protein detected the 126-kDa protein and the 183-kDa readthrough protein in purified RNA polymerase preparations, whereas antibodies to the readthrough portion of the 183-kDa protein detected only the 183-kDa protein. All three antibodies inhibited the template-dependent RNA polymerase, but none of them had any effect on the template-bound enzyme.

Antibodies↗
PubMed1977

Multiple forms of DNA-dependent RNA polymerase I from immature chick liver. Selective effect of 17beta-estradiol.

1. We found that a single injection of 17beta-estradiol into immature chicks resulted within 24 h in a 2-fold increase of the transcripitional capacity of liver nuclei which involved both endogenous RNA polymerase I and II actvities. Similarly, RNA polymerase activity I of purified nucleoli was also doubled. 2. During purification of RNA polymerase I from controls and treated chicks the difference in activity was almost completely lost. 3. RNA polymerase I could be resolved into two forms, IA and IB, on CM-Spehadex. Form IA increased and form IB decreased after estrogen treatment while the sum of the two remained constant. Form IA sedimented more slowly than form IB in glycerol gradients and was predominant in purified nucleoli. 4. These observations suggest that there may be an equilibrium between the two forms of RNA polymerase I which may change in order to produce the translational shift-up which occurs after estrogen treatment.

Animals↗
PubMed1983

Inhibitory effect of N-phenyl-N'-3-hydroxyphenylthiourea (PTU-23) on the reproduction of encephalomyocarditis virus in Krebs-II cells.

PTU-23, an effective in vivo wide-range enterovirus inhibitor, suppresses the reproduction of encephalomyocarditis (EMC) virus in Krebs-II ascites carcinoma cells at concentrations of 20-50 micrograms/ml, not affecting the cellular synthetic processes. The virus-specific RNA synthesis is distinctly inhibited. The studies on the kinetics of this effect point to its leading role in the inhibitory action the compound exerts on the production of infectious virions. The sedimentation profile in sucrose density gradient of the viral RNA isolated from PTU-23-treated cells by phenol extraction shows a clear inhibition of the synthesis of the single-stranded (ss) 37S RNA and to a lesser extent of the double-stranded (ds, RF) 20S RNA. The effect of the inhibitor on the RNA-dependent RNA polymerase in a cell-free RNA-synthesizing system has been studied, using an an enzyme preparation the 40000 g fraction of a nuclear-free extract from infected Krebs-II cells, containing the enzyme bound to the endogenous RNA template. It is established that the compound does, not affect the synthesis of the enzyme which could be probably explained by the incomplete (45-70%) inhibition of the viral RNA synthesis, its translatory function remaining unperturbed. The observed insignificant inhibition (20-26%) of the enzyme activity during the application of the inhibitor to the RNA-synthesizing reaction mixture cannot explain its effect on the viral RNA synthesis. An interaction of PTU-23 with another virus-specific protein component of the replication complex is suggested.

Animals↗
PubMed1992

Localization of an RNA-binding domain in the nucleocapsid protein of the coronavirus mouse hepatitis virus.

The interaction between the nucleocapsid (N) protein of mouse hepatitis virus (MHV) and RNA was studied in an effort to define portions of the N molecule that participate in binding to RNA. N mRNAs transcribed from SP6 and T7 vectors were translated in a rabbit reticulocyte lysate. Analysis of synthesized N protein in a nondenaturing gel system showed that it bound in vitro to an endogenous RNA in the reticulocyte lysate but not to its own mRNA. A set of deletion mutants was constructed in order to localize the RNA-binding activity of the N protein. It was found that removal of as much as 135 amino-terminal or 57 carboxy-terminal amino acids from the molecule had little or no effect on RNA binding. Moreover, deletion mutants lacking both termini still retained RNA-binding ability. By contrast, internal deletions or truncations extending beyond these two limits effectively abolished RNA binding by N protein. Thus, the RNA-binding region of N has been mapped to the second (central) of the three structural domains of the molecule.

Amino Acid Sequence↗
PubMed1997

Regulation of gene expression by natural antisense RNA transcripts.

The use of synthetic antisense oligonucleotides as specific inhibitors of gene expression exploits the susceptibility of mRNA to functional blockade at several levels, including mRNA processing, transport, translation and degradation. It is becoming increasingly apparent that the actions of these synthetic oligomers are analogous to those of endogenous RNA molecules involved in the regulation of gene expression in both prokaryotes and eukaryotes. A growing number of eukaryotic genes are now thought to be regulated at least in part by natural antisense RNA transcribed from the presumptive non-coding DNA strand. This possibility is supported by the presence of a complex system of double-stranded (ds) RNA-specific proteins and dsRNA-induced signal transduction pathways in eukaryotic cells. The presence of functional open reading frames in a number of recognized natural antisense RNA transcripts indicates that, in addition to regulating gene function at the RNA level, the antisense strand of many genes may code for as yet unidentified proteins. In the present study we review the current literature on the role(s) played by natural antisense RNA in eukaryotic cells, with an emphasis on genes for which clear evidence of regulation, or potential regulation by natural antisense RNA is available.

Animals↗
PubMed2004

High affinity binding of the Translin/Trax complex to RNA does not require the presence of Y or H elements.

Translin and its partner protein, Trax, are components of an RNA binding complex that has been implicated in suppressing translation of several mRNAs by binding to Y and H cis elements contained in these transcripts. However, it is unclear which features of these elements are critical for conferring high affinity binding to the Translin/Trax complex, information that might be useful in identifying other candidate transcripts targeted by this complex. To help clarify this issue, we have assessed the effect of truncating or mutating a segment of the 3'UTR of the protamine-2 transcript which contains both Y and H elements and binds to this complex with high affinity. Our results indicate that high affinity binding to this segment is preserved following extensive mutation of the Y and H elements as long as clusters of G residues are retained. Thus, our findings indicate that the Translin/Trax complex recognizes clusters of G residues rather than RNA sequences that closely match the primary sequence of the Y and H elements. This revised view of the cis elements recognized by the Translin/Trax complex may be useful in future studies aimed at identifying endogenous RNA species targeted by this complex.

3' Untranslated Regions↗