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At least 343 records · Page 19Linked to original sources
PubMed1982

5-Mercaptopolyuridylic acid (MPU), a potent inhibitor of the reverse transcriptase from avian myeloblastosis virus.

The effects of partially thiolated polyuridylic acid (MPU), partially thiolated polycytidylic acid (MPC), and their unmodified counterparts, poly(U) and poly(C), respectively, on the reverse transcriptase of avian myeloblastosis is virus (AMV) have been determined, using different template-primers. Poly(C) stimulated and MPC inhibited the polymerization reaction catalyzed by the AMV reverse transcriptase in the presence of the natural viral template (endogenous RNA or purified 70S RNA), or with poly(C) . oligo(dG)12-18 but not with poly(A) . oligo(dT)12-18 as the only template-primer present in the purified enzyme system. Both poly(U) and MPU inhibited the reaction in the presence of either of the synthetic or natural templates. Using the purified enzyme with the 70S RNA template, MPU was by far the most potent inhibitor, with I50 = 0.8 microM based on the epsilon (P) value, or I50 less than 3 x 10(-9) M based on the actual, macromolecular weight of the polynucleotide.

Avian Leukosis Virus↗
PubMed1980

[Purification and properties of a releasing factor of polynucleotide phosphorylase from sarcoma M-1 of rat].

A releasing factor (RF) of polynucleotide phosphorylase (PNPase) isolated and purified from sarcoma M-1 of the rat is a protein with molecular weight of about 8000 and the isoelectric point at 4,0--4,2. The process of PNPase release from the polyribosomes of normal rat liver does not depend on the pH of the medium within the pH range of 7,2--9,0, on temperature within the interval of 0--400 degrees and on incubation time. Inhibition of phosphorolysis by endogenous RNA from normal rat liver polyribosomes requires considerable amounts of RF--approximately 170--800 molecules of RF per molecule of poly(A)+-mRNA. The data obtained suggest that PNPase release from the polyribosomes under the action of RF is not a catalytic process and is caused by competition between RF and PNPase for the binding sites on the mRNA molecule.

Animals↗
PubMed1982

[Comparison of endogenous nuclear RNA-polymerase II at different stages of mycoplasmodial growth].

The nuclei of Physarum polycephalum isolated from the 48- and 96-hour-old growing microplasmodium differ 7-8 fold by the rates of [3H]UTP incorporation in vitro accompanied by a slight (approximately 1.6 fold) change in concentration of endogenous RNA-polymerase II. Using mild fragmentation of chromatin nuclei by micrococcal nuclease and DEAE-Sephadex chromatography the bound enzyme was shown to consist of two forms differing in the degree of their binding to the template and in functional significance. Transcription in the Physarum polycephalum nuclei can be regulated by changes in the correlation of these forms.

Cell Nucleus↗
PubMed1977

Genetic variation in the RNA transcripts of endogenous virus genes in uninfected chicken cells.

Uninfected cells from two different phenotypes of chicken embryos express significant amounts of endogenous viral information, though they do not produce virus particles. Cells of the phenotype gs(+)chf(+) are positive for both group-specific (gs) antigens and chicken helper factor (chf) activity, whereas cells of a second phenotype, gs(L)chf(+)(h(E)), demonstrate noncoordinate expression of these two viral activities (very low amounts of gs antigens, but extremely high helper activity). RNA from these cells was analyzed to determine the size, genetic content, and relative abundance of virus-specific RNAs in cells of each phenotype. Two major size classes of polyadenylic acid-containing RNA, homologous to the avian leukosis virus genome, were detectable in cells of both types. The larger RNA, which contained most of the sequences of the leukosis virus genome, was of different sizes in the two phenotypes, 31S in gs(+)chf(+) cells but 35S in the noncoordinate cell type. Analysis of the viral RNA with gene-specific complementary DNA probes revealed the following characteristics. (i) The 31S RNA appeared to lack portions of the gag and pol genes. (ii) A smaller RNA species, which sedimented at 21S in both cell types, was a transcript of the 3'-proximal portion of the viral genome, consisting of the env gene and the "common" sequences. (iii) The amount of env-specific RNA in the 21S region was more than six times higher in the noncoordinate cell type than in the gs(+)chf(+) cells; this difference was concordant with the 5- to 10-fold higher chf activity in the noncoordinate cells. (iv) The endogenous viral RNA in uninfected cells and the RNA from Rous-associated virus-0 virions hybridized only partially with DNA complementary to the common region of the Rous-associated virus-2 genome, whereas the RNA of all exogenous viruses tested hybridized almost completely to this complementary DNA. Small amounts of src-specific polyadenylated RNA were also present in uninfected chicken cells. This RNA sedimented as a single peak at 26S and was not covalently linked to any other identifiable virus-specific RNA sequences. The amount of src RNA was the same in the above two types of expression-positive cells and also in cells that were gs(-)chf(-), indicating that the transcription of the cellular sequences homologous to the src gene is independent of the transcription of the other endogenous viral genes.

Animals↗
PubMed2004

Detection of a novel sense-antisense RNA-hybrid structure by RACE experiments on endogenous troponin I antisense RNA.

Conformational changes in the troponin/tropomyosin complex significantly alter the mechanical properties of cardiac muscle. Phosphorylation of cardiac troponin I, part of the troponin/tropomyosin complex, reduces calcium affinity, which leads to increased relaxation of cardiac muscle. Because cardiac troponin I plays a central role in tuning the heart to different work demands, detailed knowledge of troponin I protein regulation is required. Our group previously detected naturally occurring antisense RNA for troponin I in human and rat hearts, and here, attempt to unravel the structure of rat cardiac troponin I antisense RNA. We performed rapid amplification of cDNA ends (RACE) experiments and discovered antisense sequences identical to a copy of the sense mRNA, which led us to conclude that the antisense RNA must be transcribed from troponin I mRNA in the cytoplasm. Moreover, we isolated RNA structures comprising sense and antisense sequences in one continuous molecule. As we found no homolog structures described in the literature, we called this "hybrid RNA." Because a duplex formation was demonstrated previously we concluded that hybrid RNA is a consequence of a tight interaction between sense and antisense troponin I RNA in vivo, which we discuss in the article.

Animals↗
PubMed2006

Effect of adenovirus-mediated RNA interference on endogenous microRNAs in a mouse model of multidrug resistance protein 2 gene silencing.

RNA interference with viral vectors that express short hairpin RNAs (shRNAs) has emerged as a powerful tool for functional genomics and therapeutic purposes. However, little is known about shRNA in vivo processing, accumulation, functional kinetics, and side effects related to shRNA saturation of the cellular gene silencing machinery. Therefore, we constructed first-generation recombinant adenoviruses encoding different shRNAs against murine ATP-binding cassette multidrug resistance protein 2 (Abcc2), which is involved in liver transport of bilirubin to bile, and analyzed Abcc2 silencing kinetics. C57/BL6 mice injected with these viruses showed significant impairment of Abcc2 function for up to 3 weeks, as reflected by increased serum bilirubin levels. The lack of Abcc2 function correlated with a specific reduction of Abcc2 mRNA and with high levels of processed shRNAs targeting Abcc2. Inhibition was lost at longer times postinfection, correlating with a decrease in the accumulation of processed shRNAs. This finding suggests that a minimal amount of processed shRNAs is required for efficient silencing in vivo. This system was also used to evaluate the effect of shRNA expression on the saturation of silencing factors. Saturation of the cellular silencing processing machinery alters the accumulation and functionality of endogenous microRNAs (miRNAs) and pre-miRNAs. However, expression of functional exogenous shRNAs did not change the levels of endogenous miRNAs or their precursors. In summary, this work shows that adenoviral vectors can deliver sufficient shRNAs to mediate inhibition of gene expression without saturating the silencing machinery.

ATP Binding Cassette Transporter, Subfamily B↗
PubMed1976

The use of mercurated nucleoside triphosphate as a probe in transcription studies in vitro.

1. Purified form A RNA polymerase and the endogenous, nuclear form A RNA polymerase are shown to incorporate 5-mercuri-uridine 5'-triphosphate (Hg-UTP) into RNA in vitro. The Km for both Hg-UTP and UTP are in the region of 10 muM for the purified enzyme. 2. The RNA products formed in nucleoli by endogenous RNA polymerase A have similar base compositions (G + C-rich) whether UTP or Hg-UTP is provided as the substrate in vitro. 3. Sulphydryl-Sepharose chromatography of RNA synthesised in vitro by nucleoli allows separation of this material from the endogenous RNA, when the former is synthesised in the presence of Hg-UTP. 4. In-vitro-synthesised nucleolar RNA hybridises with cot profiles similar to 28-S ribosomal RNA, when made with either Hg-UTP or UTP. 5. Hybridisation studies using DNA excess suggest that little competition occurs between the in vitro transcripts and the endogenous nucleolar RNA. 6. Size analysis of in vitro transcripts show that although some degradation occurs during isolation, purification and hybridisation of the RNA species, most of the RNA remains larger than 5 S throughout.

Animals↗
PubMed1976

Natural immunity in mice to structural polypeptides of endogenous type C RNA viruses.

The immunological responses of inbred mice to structural components of one class of endogenous virus were investigated by means of radioimmunoassays utilizing highly purified viral proteins. Naturally occurring antiviral antibodies were demonstrated only in those strains possessing information for induction of a mouse cell-tropic endogenous virus. Moreover, these antibodies invariably appeared subsequent to the detection of spontaneous replication of this virus in the same animal. The immune responses elicited were much stronger against endogenous viral gp70 than p30, consistent with previous findings of tolerance in the mouse to the major structural antigen of its endogenous virus. However, the demonstration of an immune response to p30 under conditions of both natural and experimental immunization establishes that tolerance to this viral antigen can be overcome.

AKR murine leukemia virus↗
PubMed1978

Estrogen withdrawal in chick oviduct. Characterization of RNA synthesized in isolated nuclei using a mercurated precursor.

1. As a prerequisite for analyzing the effect of estrogen on transcription in chick oviduct, we describe suitable methods for the synthesis (under conditions restricting reinitiation), and isolation of RNA transcripts from oviduct nuclei in vitro, utilizing mercurated UTP (Hg-UTP) as an RNA precursor and chromatography on sulphydryl-Sepharose (SH-Sepharose) to recover mercurated RNA (Hg-RNA). The techniques described include treatment of Hg-RNA with p-hydroxymercuribenzoate, to improve the efficiency of binding to SH-Sepharose, and elution of Hg-RNA from SH-Sepharose after treatment with 60% formamide at 90 degrees C, to eliminate contamination by aggregated nucleic acid. 2. RNA synthesized by endogenous form B RNA polymerase (using either UTP or Hg-UTP as precursor) was recovered in nuclear lysates in the form of 30--85-S heterogeneous RNA . protein complexes, and after removal of protein, was 10--12 S in size. 3. The nature of RNA transcripts synthesized in vitro was examined by hybridization. More than 90% of the RNA was complementary to "unique" DNA sequences, and 50--60% of the hybridized RNA could be competed with homologous, steady-state nuclear RNA, indicating a significant degree with homologous, steady-state nuclear RNA, indicating a significant degree of homology between in vitro transcripts and in vivo RNA. The level of homology was similar whether RNA synthesis was performed in low salt, or in high salt in the presence of heparin. Possible reasons for only partial competition in these experiments are discussed. 4. Withdrawal of estrogen from chicks leads to a 50% reduction in endogenous RNA polymerase activities in nuclei within 48 h. Similar levels of competition with Hg-RNA transcripts for "unique" DNA were obtained using oviduct nuclear RNAs isolated before or after estrogen withdrawal, and even with liver nuclear RNA. Thus, in oviduct, those sequences present in primary transcripts, and analyzed under our experimental conditions, are present in different hormonal states and also in other chick tissues.

Amanitins↗
PubMed1976

Type C particle-positive and type C particle-negative rat cell lines: characterization of the coding capacity of endogenous sarcoma virus-specific RNA.

Various rat cell lines have been analyzed for expression of endogenous RNA homologous either to RT21C, a typical rat type C virus, or to Kirsten sarcoma virus. Cells have been found that express either (i) high levels of RNA homologous to RT21C rat type C virus and low levels of RNA homologous to Kirsten sarcoma virus (RT21Chigh,sarclow) or (ii) high levels of RNA homologous to Kirsten sarcoma virus and low levels of RNA homologous to typical rat type C virus (sarchigh, RT21Clow). The properties of these two classes of cell lines have been compared. Each type of cell contains an equal amount of the expressed RNA on polysomes. Cell lines that are RT21Chigh produce abundant rat p30 nad p12 structural proteins and release rat type C particles containing viral RNA and reverse transcriptase into supernatant fluids from these cultures. Cell lines that are sarchigh,RTC21Clow have no detectable rat viral p12 protein and no p30 protein immunoreactive in even broad interspecies radioimmunoassays, and do not release type C particles into the supernatant from the cultures. When the particle-negative cell lines are superinfected with heterologous mouse or wooly type C viruses or are producing typical rat type C virus particles, the endogenous sarcoma virus-specific RNA is secreted from these cells. The sarcoma virus-specific RNA can be transcribed in complementary DNA in the endogenous reverse transcriptase reactions carried out in vitro with such virus preparations. However, exposure of cells that are permissive to the helper virus with the particles containing sarcoma virus-specific RNA has not yet resulted in cell transformation or in the synthesis of these RNA sequences. The results suggest: (i) that the first step in the genesis of sarcoma viruses involves the packaging of this expressed sarcoma virus-specific RNA in helper viral particles; (ii) that efficient transmission of the sarcoma virus-specific RNA requires additional events; and (iii) that the formation of a stable sarcoma virus by recombination between the helper viral genome and part of the rescued sarcoma virus-specific RNA is much less common event than the rescue process itself.

Base Sequence↗
PubMed1993

Application of nonisotopic RNA in situ hybridization to detect endogenous gene expression in pathologic specimens.

Detection of endogenous messenger RNA in surgical pathology tissue samples is a useful technique for identifying or characterizing populations of normal or neoplastic cells. Potential problems with this technique include degradation of target RNA in routinely processed specimens, and low concentration of target RNA sequences in the cells, necessitating the use of radiolabeled probes for detection. In this report, we demonstrate detection of nonmuscle actin messenger RNA in fixed, paraffin-embedded lymphoid tissues and bone marrow aspirate smears, using routinely obtained surgical and hematologic specimens. In addition, increased sensitivity was achieved by hybridization with full-length RNA probes, rather than oligonucleotide DNA probes; these probes were labeled with digoxigenin-11-UTP, allowing colorimetric detection of hybridization and avoiding the use of isotopes. The advantages and potential applications of this technique as an aid in pathologic diagnosis are discussed.

Actins↗
PubMed2004

Inhibition of porcine endogenous retroviruses by RNA interference: increasing the safety of xenotransplantation.

Transplantation of porcine xenografts into human recipients is a realistic option to overcome the growing worldwide shortage of suitable allogeneic organs. However, there remains the risk of infection by porcine endogenous retroviruses (PERVs) that cannot be eliminated like that by other microorganisms by breeding pigs under specified pathogen-free conditions. To reduce the release of PERVs by porcine transplants, a new approach, RNA interference (RNAi), was applied. Here, we show significant reduction of PERV expression by synthetic short interfering RNAs (siRNAs) corresponding to different parts of the viral genes gag, pol, and env. The most inhibitory sequences were selected and expressed as short hairpin RNAs (shRNAs) by a polymerase III vector system leading to persistent suppression of PERV replication. Cells or organs from transgenic pigs producing such shRNAs should increase the safety of xenotransplantation.

Animals↗
PubMed1976

Endogenous type C RNA virus of Odocoileus hemionus, a mammalian species of New World origin.

Type C RNA viruses have been isolated from several Old World vertebrates, and an even larger number of Old World species have been shown to contain endogenous viral genetic sequences. The present report describes the first isolation of type C virus endogenous to a species originating in the New World. This virus, isolated from cells of the Columbian black-tailed deer, Odocoileus hemionus, is shown to possess genetic sequences in common with DNA of its species of origin. While it shares biochemical and immunologic characteristics with other mammalian type C viruses, its immunologic properties readily distinguish it from known endogenous viruses.

Animals↗
PubMed2001

Quantitative analyses of messenger RNA of human endogenous retrovirus in patients with systemic lupus erythematosus.

OBJECTIVE: Human endogenous retrovirus (HERV) has emerged as a possible causative agent of systemic lupus erythematosus (SLE). To investigate the role of HERV in the etiology of SLE, we performed quantitative analyses of messenger RNA (mRNA) of the HERV clone 4-1 in patients with SLE. METHODS: Reverse transcriptase (RT)-polymerase chain reaction (PCR) and real-time quantitative PCR (RQ-PCR; TaqMan methodology) were used in this experiment. RESULTS: The quantities of mRNA of the HERV clone 4-1 gag region in patients with SLE were significantly higher than in healthy controls, and the amounts of such mRNA in the patients were decreased by steroid treatment. CONCLUSION: These phenomena may be related to the production of viral components derived from HERV clone 4-1 and contribute to the pathogenesis of SLE; studies using a larger number of patients are required to confirm these points.

Adult↗
PubMed1987

Endogenous cytokinins in the ribosomal RNA of higher plants.

Endogenous cytokinin-active ribonucleosides were isolated from the rRNA and tRNA of pea epicotyls (Pisum sativum L., var Alaska) and of wheat germ (Triticum aestivum). The RNA preparations were analyzed for cytokinins by enzymic hydrolysis, ethyl acetate extraction, and Sephadex LH-20 fractionation in several solvents. Tentative identification of the cytokinins was based on cochromatography with synthetic cytokinin standards in several systems and on activity in the tobacco bioassay. Both the rRNA and tRNA from 10 day old pea epicotyls contained ribosylzeatin, isopentenyladenosine, and 2-methylthioribosylzeatin. The latter compound was the most active fraction in the pea rRNA, but was the least active fraction in the tRNA, where isopentenyladenosine activity was predominant. The 2-methylthioribosylzeatin from pea rRNA was identified by gas chromatography-mass spectrometry. Wheat germ rRNA contained cis and trans ribosylzeatin and 2-methylthioribosylzeatin. The tRNA contained isopentenyladenosine in addition. The specific cytokinin activity (activity per A(260) unit) of the tRNA was over forty times that of the rRNA. Significant contamination of the rRNA preparations by cytokinin-containing tRNA is considered unlikely on the basis of quantitative differences in the cytokinin content of the rRNA and tRNA preparations, electrophoretic analysis of rRNA purity and cytokinin analysis of fractionated oligonucleotide digests.

Journal Article↗
PubMed2006

Human endogenous retroviral pol RNA and protein detected and identified in the blood of individuals with schizophrenia.

Retrovirus has been speculated as one of the potential infectious agents involved in the development of schizophrenia. Here we used nested RT-PCR to detect the RNA of HERV pol gene in blood from schizophrenic patients and normal human. We found retroviral pol genes expressed in blood from 20 of 58 (34.5%) individuals with recent-onset schizophrenia, but not from 38 normal persons (p<0.01). Sequence analysis revealed that the expressed gene was homologous to those of the human endogenous retroviral (HERV) family. The ERV9 family was the closest, with 90% homology in the gene sequence. In addition, Western blots showed that antibody against ERV9 pol protein in serum from the HERV+ schizophrenia patients, but not from control (p<0.01). Our data suggested that the transcriptional activation of certain retroviral elements might be associated with the development of schizophrenia in some patients. Further characterization of retroviral elements in subjects with schizophrenia may aid in better diagnosis and treatment of this disorder.

Adolescent↗