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At least 217 records · Page 12Linked to original sources
PubMed2001

Discovery of functional genes in the post-genome era by novel RNA-protein hybrid ribozymes.

Novel ribozymes were constructed that couple the site specific cleavage activity of the hammerhead ribozyme with the unwinding activity of the endogenous RNA helicase. This leads to extremely efficient cleavage of any target mRNA regardless of the secondary structure of the RNA. Since the novel hybrid ribozymes can attack any site within mRNA, libraries were made of the hybrid ribozymes with randomized binding arms and introduced into cells. This procedure made it possible to readily identify the relevant genes associated with phenotype in the apoptosis pathway. This application of a randomized library of hybrid ribozymes represents a simple powerful method for identification of genes associated with specific phenotypes.

Genes↗
PubMed1985

Purification and characterization of a transcription factor that confers promoter specificity to human RNA polymerase I.

A whole-cell HeLa extract was fractionated into two components required for accurate in vitro transcription of human rRNA. One fraction contained endogenous RNA polymerase I, and the second component contained a factor (SL1) that confers promoter selectivity to RNA polymerase I. Analysis of mutant templates suggests that the core control element of the rRNA promoter is required for activation of transcription by SL1. We purified SL1 approximately 100,000-fold by column chromatography and have shown that the addition of SL1 can reprogram the otherwise nonpermissive mouse transcription system to recognize and initiate accurate RNA synthesis from human rDNA. Antibodies raised against SL1 bind preferentially to a protein localized in the nucleolus of primate cells and specifically inhibit in vitro transcription initiating from the human rRNA promoter. By contrast, anti-SL1 does not react with the nucleolus of rodent cells and has no effect on the in vitro synthesis of mouse rRNA by a transcription system derived from mouse cells. These findings suggest that SL1 is a selectivity factor present in the nucleolus that imparts promoter recognition to RNA polymerase I and that can discriminate between rRNA promoters from different species.

Animals↗
PubMed2005

The complex interplay between plant viruses and host RNA-silencing pathways.

RNA silencing was originally identified as an immune system targeted against transposons and viruses, but is now also recognized as a major regulatory process that affects all layers of host gene expression through the activities of various small RNA species. Recent work in plants and animals indicates that viruses not only suppress, but can also exploit, endogenous RNA silencing pathways to redirect host gene expression. There are also indications that cellular, as opposed to virus-derived small RNAs, might well constitute an unsuspected defense layer against foreign nucleic acids. This complex interplay has implications in the context of disease resistance and evolution of viral genomes.

Biological Evolution↗
PubMed1976

Transcription in vitro of immunoglobulin kappa light chain genes in isolated mouse myeloma nuclei and chromatin.

Messenger RNA sequences for immunoglobulin kappa light chain were synthesized in vitro in isolated mouse myeloma nuclei using bound endogenous RNA polymerase (RNA nucleotidyltransferase; nucleoside triphosphate:RNA nucleotidyltransferase; EC 2.7.7.6) and from isolated myeloma chromatin using exogenous Escherichia coli RNA polymerase. The in vitro RNA was transcribed using 5-mercuriuridine triphosphate and separated from in vivo RNA by chromatography on an agarose sulfhydryl affinity column. Template restriction is retained in vitro since synthesis of kappa chain messenger RNA, As determined by hybridization with complementary DNA, was much more efficient in nuclei and chromatin isolated from myeloma 66.2 tissue culture cells, a kappa-chain-producing cell line, than from MOPC 315 tissue culture cells, a lambda-chain-producing cell line. Transcription of kappa chain messenger RNA was 25 times more efficient in myeloma 66.2 nuclei than in myeloma 66.2 chromatin.

Cell Nucleus↗
PubMed1978

Isolation and Characterization of a Chromatin-associated Protein Kinase from Soybean.

A chromatin-associated casein-type protein kinase has been purified 500-fold from soybean (Glycine max, var. Wayne) tissue. The enzyme can be completely dissociated from isolated chromatin in 250 millimolar (NH(4))(2)SO(4). After purification, the kinase preparation is stable for at least 6 months at 0 C. The enzyme will phosphorylate casein, phosvitin, and denatured chromatin proteins, but not histones. Only ATP will serve as a phosphate donor with an apparent K(m) of 8 micromolar. Five millimolar Mg(2+) is required for maximal activity, but Mn(2+) will support phosphorylation at a lower level. The average molecular weight as determined by sucrose gradient sedimentation and gel filtration is approximately 55,000. Under conditions of low ionic strength [less than 250 millimolar (NH(4))(2)SO(4)] soybean casein kinase forms higher molecular weight aggregates with other chromosomal proteins in the preparation. The enzyme activity is not affected by cyclic AMP. Casein kinase shows a broad optimum between 7 and 8 and the isoelectric point is approximately 9. Preliminary data indicate that soybean casein kinase will not phosphorylate soybean RNA polymerases I or II, nor does it have any obvious effect on in vitro chromatin transcription by endogenous RNA polymerases.

Journal Article↗
PubMed2001

Evidence that the RNAseH activity of the duck hepatitis B virus is unable to act on exogenous substrates.

BACKGROUND: The hepadnaviral reverse transcriptase can synthesize DNA on its native RNA template within viral cores but it is usually unable to synthesize DNA employing exogenous nucleic acids as a template. The mechanism of this template commitment is unknown. Here we provide evidence that the RNAseH activity of duck hepatitis B virus reverse transcriptase may also be unable to act on exogenous substrates. RESULTS: RNAseH assays were performed under a wide variety of conditions employing substrate RNAs of Duck Hepatitis B Virus sequence annealed to complementary DNA oligonucleotides and permeabilized intracellular viral core particles. Temperature, pH, cation type, salt concentration, substrate concentration, and the sequences of the cleavage sites were varied, and the effects of ATP and dNTPs on RNAseH activity were examined. duck hepatitis B virus RNAseH activity was not detected under any of these conditions, although E. coli or Avian Myeloblastosis Virus RNAseH activity could be detected under all conditions. Access of the RNA substrate to the enzyme within the viral cores was confirmed. CONCLUSIONS: These results imply that the RNAseH activity of the DHBV reverse transcriptase may not be able to degrade exogenous RNA:DNA heteroduplexes, although it can degrade heteroduplexes of the same sequence generated during reverse transcription of the endogenous RNA template. Therefore, the RNAseH activity appears to be "substrate committed" in a manner similar to the template commitment observed for the DNA polymerase activity.

Hepatitis B Virus, Duck↗
PubMed1977

Effect of SV40 infection on [3H]uridine incorporation.

More [3H]uridine was incorporated into RNA of SV40-infected than into uninfected cells 31 h after infection. When the specific activity of the uridine triphosphate pools in infected and uninfected cells was equated by the addition of appropriate amounts of exogenous unlabelled uridine, no difference in the rate of [3H]uridine incorporation into RNA was observed. Although no difference in [3H]uridine entry or phosphorylation was demonstrable, the apparently smaller pools of endogenous RNA precursors in infected cells resulted in less isotope dilution and thus to synthesis of uridine triphosphate and RNA of higher specific activity.

Cell Line↗
PubMed1978

Expression of RNA of an endogenous replication-defective retrovirus in rat mammary adenocarcinomas induced by 7,12-dimethylbenz[a]anthracene.

An investigation into the possible relationship between chemical carcinogen induction of rat mammary tumors and the expression of an endogenous retroviral genome was initiated. Mammary tumors were induced in female SD rats with 7,12-dimethylbenz[a]anthracene (DMBA). Tumors, identified histologically as mammary adenocarcinomas, were analyzed for RNA of a replication-defective endogenous retrovirus or RNA of a helper-independent endogenous type C virus. Expression of RNA of the replication-defective virus was detected in mammary tumors weighing 0.2--2.0 g. Larger tumors, for which histologic examination revealed proportionally more fibroblastic tissue than epithelial cells, did not contain comparable concentrations of this viral RNA. RNA homologous to a helper-independent rat type C retrovirus was not detected in tumors of any size. A cell line was established from a primary DMBA-induced mammary adenocarcinoma and appeared similar to the small mammary tumors with respect to endogenous type C viral RNA expression. We discuss possible implications of the expression of endogenous replication-defective viruses for use as markers for the effects of chemical carcinogens.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1984

The interaction of adriamycin with cardiolipin in model and rat liver mitochondrial membranes.

The interaction of adriamycin with cardiolipin in model membranes and in various membrane preparations derived from rat liver mitochondria was studied and the results are analyzed in the light of a possible specific interaction between adriamycin and cardiolipin. It was found that adriamycin binds to cardiolipin-containing model membranes with a fixed stoichiometry of two drug molecules per cardiolipin. Furthermore, the extent of drug complexation by mitochondria and mitoplasts (inner membrane plus matrix) is in reasonable agreement with their cardiolipin content. In contrast, adriamycin-binding curves of inner membrane ghosts and submitochondrial particles reveal considerable association to an additional site, presumably RNA. The evidence for the potential importance of RNA as a target comes from experiments on outer membranes and microsomes which both appear to bind substantial amounts of adriamycin. Removal of the major part of the RNA associated with these fractions by EDTA treatment is accompanied by a dramatic reduction of binding capacity. We propose that endogenous RNA present in mitochondria and mitoplasts is not accessible for adriamycin at low concentrations of the drug due to the presence of an intact lipid barrier. This potential site comes to expression in ghosts and submitochondrial particles, due to the absence of an intact lipid bilayer and due to the inside-out orientation of the limiting membrane, respectively. Electron microscopical studies show that adriamycin induces dramatic changes in mitochondrial morphology, similar to the uncoupler-induced effects described by Knoll and Brdiczka (Biochim. Biophys. Acta 733, 102-110 (1983). Adriamycin has an uncoupling effect on mitochondrial respiration and oxidative phosphorylation. The concentration dependence of this effect correlates with the adriamycin-binding curve for mitochondria which implies that only bound adriamycin actively inhibits respiration.

Animals↗
PubMed1998

Specific telomerase RNA residues distant from the template are essential for telomerase function.

The reverse transcriptase telomerase is a ribonucleoprotein complex that adds telomeric repeats to chromosome ends, using a sequence within its endogenous RNA component as a template. Although templating domains of telomerase RNA have been studied in detail, little is known about the roles of the remaining residues, particularly in yeast. We examined the functions of nontemplate telomerase residues in the telomerase RNA of budding yeast Kluyveromyces lactis. Although approximately half of the RNA residues were dispensable for function, four specific regions were essential for telomerase action in vivo. We analyzed the effects of mutating these regions on in vivo function, in vitro telomerase activity, and telomerase RNP assembly. Deletion of two regions resulted in synthesis of stable RNAs that appeared unable to assemble into a stable RNP. Mutating a region near the 5' end of the RNA allowed RNP assembly but abolished enzymatic activity. Mutations in another specific small region of the RNA led to an inactive telomerase RNP with an altered RNA conformation.

Deoxyribonucleases, Type II Site-Specific↗
PubMed1980

Regulation of gene transcription by estrogen and progesterone. Lack of hormonal effects on transcription by Escherichia coli RNA polymerase.

Estrogen and progesterone markedly stimulate transcription of ovalbumin and conalbumin (transferrin) genes in chick oviduct as measured by hybridization of labeled RNA synthesized in isolated nuclei to immobilized plasmid DNA containing these gene sequences. Using this direct assay for specific gene transcription, we explored the basis of previous reports indicating that steroid hormones also cause changes in oviduct chromatin structure that can be detected by Escherichia coli RNA polymerase. We observed no effect of these hormones on the ability of E. coli RNA polymerase to transcribe specifically the conalbumin and ovalbumin genes 8 1/2 h after hormone administration when transcription of these genes by endogenous RNA polymerase was elevated 5- and 30-fold, respectively. Furthermore, we were unable to detect any significant effect of either of these hormones on the total number of E. coli RNA polymerase binding sites in oviduct nuclei or chromatin. In contrast, after several days of hormone administration, we detected an apparent preferential ovalbumin RNA synthesis by E. coli RNA polymerase and this effect could be transferred to unstimulated nuclei by a 0.35 M salt extract of active nuclei. However, further experiments revealed that this preferential ovalbumin RNA synthesis is an artifact produced by transcription from contaminating ovalbumin mRNA. We conclude that E. coli RNA polymerase does not recognize steroid hormone-induced changes in oviduct chromatin.

Amanitins↗
PubMed1975

The RNA polymerase activity of the preimplantation mouse embryo.

The activation of the mouse embryo genome has been studied during early cleavage, in vivo. Individual embryos, prepared as whole mounts, were assayed for endogenous RNA polymerase activity. RNA synthesis was detected by autoradiography as the incorporation of [3H]UMP into an acid-insoluble product. No RNA polymerase activity could be detected in the pronuclei of one-cell embryos. Radioactive incorporation was first evident in the nuclei of two-cell embryos. This appeared to be confined to the nucleoplasm and could be abolished by alpha-amanitin but not by low concentrations of actinomycin D. Polymerase activity which was not affected by alpha-amanitin was first detected in the four-cell embryo, predominantly at the peripheries of the nucleoli. Nucleolar labelling increased markedly between subsequent cleavages, reaching a peak in early morulae. In one- and two-cell embryos, label incorporation could be found in the nucleus of the persisting polar body.

Amanitins↗
PubMed2006

RNA interference is not involved in natural antisense mediated regulation of gene expression in mammals.

BACKGROUND: Antisense transcription, yielding both coding and non-coding RNA, is a widespread phenomenon in mammals. The mechanism by which natural antisense transcripts (NAT) may regulate gene expression are largely unknown. The aim of the present study was to explore the mechanism of reciprocal sense-antisense (S-AS) regulation by studying the effects of a coding and non-coding NAT on corresponding gene expression, and to investigate the possible involvement of endogenous RNA interference (RNAi) in S-AS interactions. RESULTS: We have examined the mechanism of S-AS RNA base pairing, using thymidylate synthase and hypoxia inducible factor-1alpha as primary examples of endogenous genes with coding and non-coding NAT partners, respectively. Here we provide direct evidence against S-AS RNA duplex formation in the cytoplasm of human cells and subsequent activation of RNAi. CONCLUSION: Collectively, our data demonstrate that NAT regulation of gene expression occurs through a pathway independent of Dicer associated RNAi. Moreover, we introduce an experimental strategy with utility for the functional examination of other S-AS pair interactions.

Cell Line↗
PubMed1994

Faithful degradation of soybean rbcS mRNA in vitro.

The mRNA encoding the soybean rbcS gene, SRS4, is degraded into a set of discrete lower-molecular-weight products in light-grown soybean seedlings and in transgenic petunia leaves. The 5'-proximal products have intact 5' ends, lack poly(A) tails, lack various amounts of 3'-end sequences, and are found at higher concentrations in the polysomal fraction. To study the mechanisms of SRS4 mRNA decay more closely, we developed a cell-free RNA degradation system based on a polysomal fraction isolated from soybean seedlings or mature petunia leaves. In the soybean in vitro degradation system, endogenous SRS4 mRNA and proximal product levels decreased over a 6-h time course. When full-length in vitro-synthesized SRS4 RNAs were added to either in vitro degradation system, the RNAs were degraded into the expected set of proximal products, such as those observed for total endogenous RNA samples. When exogenously added SRS4 RNAs already truncated at their 3' ends were added to either system, they too were degraded into the expected subset of proximal products. A set of distal fragments containing intact 3' ends and lacking various portions of 5'-end sequences were identified in vivo when the heterogeneous 3' ends of the SRS4 RNAs were removed by oligonucleotide-directed RNase H cleavage. Significant amounts of distal fragments which comigrated with the in vivo products were also observed when exogenous SRS4 RNAs were degraded in either in vitro system. These proximal and distal products lacking various portions of their 3' and 5' sequences, respectively, were generated in essentially a random order, a result supporting a nonprocessive mechanism. Tagging of the in vitro-synthesized RNAs on their 5' and 3' ends with plasmid vector sequences or truncation of the 3' end had no apparent effect on the degradation pattern. Therefore, RNA sequences and/or structures in the immediate vicinity of each 3' end point may be important in the degradation machinery. Together, these data suggest that SRS4 mRNA is degraded by a stochastic mechanism and that endonucleolytic cleavage may be the initial event. These plant in vitro systems should be useful in identifying the cis- and trans-acting factors involved in the degradation of mRNAs.

Base Sequence↗
PubMed1987

Modification of cell surface glycoproteins, macrophage cytostasis, and blood-borne metastatic properties of the murine RAW117 large cell lymphoma by virus superinfection.

Highly metastatic sublines of the murine RAW117-P large cell lymphoma parental line have been selected sequentially in vivo for enhanced blood-borne organ colonization. We found that the subline RAW117-H10, selected 10 times for liver colonization, formed more than 200 times as many liver tumor nodules than the RAW117-P line and showed loss of cell surface RNA tumor virus Mr 70,000 envelope glycoprotein and sensitivity to activated macrophage-mediated cytostasis. Superinfection of RAW117-H10 cells with endogenous RNA tumor virus isolated from RAW117-P cells caused increases in Mr 70,000 envelope glycoprotein expression and sensitivity to activated macrophage-mediated cytostasis concomitant with loss of liver metastatic properties. The results suggest that RAW117 cell surface Mr 70,000 envelope glycoprotein is involved in host macrophage-mediated surveillance mechanisms and metastatic properties.

Animals↗
PubMed1970

Nucleic acid synthesis in Bacillus subtilis infected with bacteriophage beta-22.

Changes in the synthesis of host and phage nucleic acid after infection of Bacillus subtilis with virulent bacteriophage beta22 were analyzed by deoxyribonucleic acid (DNA)-DNA and ribonucleic acid (RNA)-DNA hybridization. Host DNA replication continued during the first third of the 55-min latent period and then ceased at approximately the time replication of the phage genome was initiated. Host-specific RNA was synthesized concurrently with phage RNA during the first half of the latent period but was repressed late in the infection. For much of the latent period, the population of phage-specific RNA changed continually as new species were transcribed and earlier species were repressed; detectable changes ceased coincidentally with the appearance of intracellular phage. Control over transcription of phage DNA was to some degree an intrinsic property of the interaction of B. subtilis DNA-dependent RNA polymerase and the phage genome, since only the early species of phage RNA were synthesized in vitro by B. subtilis polymerase and pure beta22 DNA. In vitro transcription of late functions was demonstrated by using the endogenous RNA polymerase activity of the nucleoprotein complex (nuclear fraction) from infected cells.

Bacillus subtilis↗
PubMed1995

In vitro RNA editing-like activity in a mitochondrial extract from Leishmania tarentolae.

A mitochondrial extract from Leishmania tarentolae directs the incorporation of uridylate (U) residues within the pre-edited domain of synthetic cytochrome b (CYb) and NADH dehydrogenase subunit 7 mRNA. This has several characteristics of an in vitro RNA editing activity, but no direct evidence for involvement of guide RNAs was obtained. Inhibition by micrococcal nuclease suggests a requirement for some type of endogenous RNA. The limitation of internal U-incorporation to the pre-edited region in the CYb mRNA and the inhibition by deletion or substitution of both mRNA anchor sequences for CYb gRNA-I and -II could be consistent either with a gRNA-mediated process or a secondary structure-mediated process. A low level of incorporation of [alpha-32P]CTP occurs at the same sites as UTP. Internal U-incorporation activity is selectively inhibited by heterologous RNAs, suggesting an involvement of low affinity RNA-binding proteins which can be competed by the added RNA.

Animals↗
PubMed1990

An RNA-dependent UMP-incorporating activity is associated with the small subunit of cytoplasmic ribosomes in bloodstream forms of Trypanosoma brucei.

Trypanosoma brucei cytoplasm contained a UMP-incorporating activity which was dependent upon the presence of endogenous RNA, stimulated by exogenous RNA and precipitable by ammonium sulphate. The activity cosedimented with ribosomes, and after ribosome dissociation using EDTA was mainly associated with the small (31S) subunit. Ribosome-associated activity was selective for UTP as a substrate and greatly stimulated by the presence of at least one other nucleoside triphosphate.

Animals↗