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At least 379 records · Page 21Linked to original sources
PubMed1995

A simple and efficient method for the isolation of total RNA from the fission yeast Schizosaccharomyces pombe.

A simple and efficient procedure was described for the isolation of total RNA from the fission yeast Schizosaccharomyces pombe. The present study demonstrated that the quality and the quantity of S. pombe RNA were increased by substituting phenol/chloroform mixture for phenol as a deproteinizing agent in the first vortexing step and using an ice bath instead of a dry ice-ethanol bath in the freezing step. Additionally, this protocol had the advantage of extracting total RNA without any degradation of S. pombe cells. Furthermore, the high amounts and quality of RNA extracted by this modified procedure enabled us to perform some experiments such as Northern blot, S1 mapping, primer extension, and reverse transcriptase reaction-polymerase chain reaction (RT-PCR) without further RNA purification. We suggest that this procedure is very useful to analyse primary structures and steady-state levels of RNA from S. pombe.

Base Sequence↗
PubMed2002

Comparative analysis of commercial assays for the detection and quantification of Human Immunodeficiency Virus Type 1 (HIV-1) RNA in plasma from patients infected with HIV-1 subtype C.

The commercial assays commonly used to quantify plasma human immunodeficiency virus type 1 (HIV-1) RNA in clinical settings were designed to assess HIV-1 subtype B. We compared the performance of 4 commercial assays (Amplicor versions 1.0 and 1.5 [Roche]; Quantiplex [Chiron]; and NASBA HIV-1 RNA QT [Organon Teknika]) in detecting and quantifying HIV-1 RNA in plasma from HIV-infected persons from Zambia, an area where HIV-1 subtype C is predominant. Each assay detected plasma HIV-1 RNA, but they do not all measure statistically similar quantities of plasma HIV-1 RNA.

Female↗
PubMed1984

Dexamethasone-stimulated expression of a proviral copy of mouse mammary tumor virus env mRNA.

The kinetics of dexamethasone induction of mouse mammary tumor virus RNAs in a cell line (designated line 8) which contains a deleted proviral DNA has been examined. This DNA is an apparent reverse transcription product of the env mRNA. A detectable increase in the 3.8-kilobase RNA (mouse mammary tumor virus env mRNA) was present within 15 min after hormone addition. The 2.5-kilobase RNA detected previously during hormone induction studies was also observed in these cells. Both RNAs increased in concentration up to about 4 h after hormone addition and thereafter attained the steady-state level. Pr73env was detected by immunoprecipitation and appeared to be identical to bona fide Pr73env isolated from wild-type mouse mammary tumor virus-infected cells. An additional 5.7 kilobases of RNA was detected in augmented quantities after hormone addition, but the structure of this RNA is not known.

Animals↗
PubMed1976

Maturation of ribosomes in yeast. I Kinetic analysis by labelling of high molecular weight rRNA species.

To study the maturation of ribosomes in Saccharomyces carlsbergensis, protoplasts were pulse labeled with [5-3H]uridine at 15 degrees C. Investigation of the cellular location of pulse-labelled ribosomal RNA precursor and mature ribosomal RNA shows that both the 37-S precursor RNA, common to both 17-S and 26-S rRNA, as well as the 29-S RNA, the direct precursor of 26-S rRNA, are located in the nucleus. Most of the 18-S RNA, the direct precursor of 17-S rRNA, is found in the cytoplasmic fraction. Apart from 37-S and 29-S RNA the nucleus also contains an appreciable amount of 26-S rRNA as well as a small quantity of 18-S RNA. These data indicate that processing of 29-S to 26-S RNA occurs in the nucleus, whereas the conversion of 18-S RNA to 17-S rRNA takes place in the cytoplasm. The kinetics of appearance of pulse-labelled 26-S and 17-S rRNA in the various cytoplasmic ribosomal particles indicate, that newly formed 40-S ribosomal particles are almost immediately incorporated into 80-S ribosomes and polysomes. On the other hand, there appears to exist a fairly large cytoplasmic pool of newly synthesized ribosomal particles containing 26-S rRNA and sedimenting at about 60 S. The kinetics of appearance of newly formed 26-S and 17-S rRNA in mature ribosomes show that the maturation of the large ribosomal subunit takes about twice as much time as that of the small subunit.

Cell Fractionation↗
PubMed1980

Influence of nucleotides, cations and nucleoside triphosphatase inhibitors on the release of ribonucleic acid from isolated rat liver nuclei.

The reasons underlying reported discrepancies in the effects of ATP, ADP, adenosine 5'-[beta gamma-methylene]triphosphate, AMP + PPi, P-chloromercuribenzoate and F- on RNA efflux from isolated rat liver nuclei and on nuclear envelope nucleoside triphosphatase activity were investigated. The stimulatory effect of ADP was attributed to myokinase activity associated with the nuclei; this activity was eluted on repeated washing with nuclear incubation medium. In the absence of Ca2+ and Mn2+, ATP, adenosine 5'[beta gamma-methylene]triphosphate and AMP +PPi were found to promote release of both DNA and RNA. In the presence of 0.5 mM-Ca2+ and 9.3 mM-Mn2+, only ATP promoted RNA efflux to a significant extent. In the absence of spermidine, Ca2+ and Mn2+, nuclei released large quantities of DNA and RNA into the medium; this effect was promoted by p-chloromereuribenzoate. In the presence of the three cations, however, p-chloromercuribenzoate inhibited RNA efflux. F- caused a slight leakage of DNA from nuclei. The results are discussed in terms of models for the effects of ATP and analogues on RNA efflux and nuclear stability.

Adenosine Diphosphate↗
PubMed1988

An excised SV40 intron accumulates and is stable in Xenopus laevis oocytes.

Xenopus laevis oocytes injected with simian virus 40 (SV40) DNA synthesize abundant quantities of viral late region RNA. In a previous analysis of the 5' ends of oocyte SV40 late RNAs, it was observed that, in contrast to the majority of the late RNA species, an abundant class of viral late RNAs, whose 5' ends mapped at or near nucleotide 294, was not polyadenylated. The structure of this RNA class has now been characterized further. We have shown that this species consists of a class of small uncapped RNA molecules with heterogeneous 3' ends mapping between nucleotides 417 and 433. This corresponds well with the position of a 139-nucleotide intron within the leader region of late 16S RNA (nucleotides 294-433). The identification of this RNA class as an excised intron was strongly supported by the fact that it displayed anomalous mobilities on different percentage polyacrylamide gels, a property of lariat introns. Furthermore, incubation of oocyte RNA with a HeLa cell extract with lariat debranching activity converted the small RNA to a class that now migrated as less than or equal to 140 nucleotides in length in 8% gels, consistent with the size of the linear intraleader intron. Additional analysis of this RNA showed that it is primarily nuclear in localization and is probably the most stable viral RNA species in the oocyte. These data suggest that oocytes accumulate large quantities of the 16S intraleader intron because of their failure to debranch this RNA efficiently.

Animals↗
PubMed1979

Effect of capping upon the mRNA properties of satellite tobacco necrosis virus ribonucleic acid.

The mRNA guanyltransferase-mRNA methyltransferases of vaccinia virions can be used to introduce a 5'-terminal m7g(5')pp(5')Apm... capping group onto the RNA of satellite tobacco necrosis virus (STNV RNA) to yield intact capped STNV RNA. Studies with an in vitro system from wheat germ and limiting quantities of capped and uncapped STNV RNA show that the rates and extents of formation of initiation complexes of protein synthesis by intact capped and uncapped STNV RNA are identical, suggesting that 5'-terminal cap groups cannot function in the translation of STNV RNA. Also, the cap analogue pm7G equally inhibits the initiation and the translation of limiting quantities of both capped and uncapped STNV RNA. These contrasting observations suggest that the wheat germ system contains a pm7G sensitive protein and that STNV RNA has a tertiary structure that restricts the function of an added 5'-terminal capping group. This theory is supported by observations that fragmented capped STNV RNA is better at forming initiation complexes than is equally fragmented uncapped STNV RNA.

Adenine Nucleotides↗
PubMed1997

The effect of transfection of antisense cDNA for procollagen alpha 1 (IV) on stimulated proliferation in rat glomerular endothelial cells.

Glomerular endothelial cells were stably transfected with a pMAMneo-Blue vector recombinant for procollagen alpha 1 (IV) cDNA in the sense (S) or antisense (AS) orientation utilizing a calcium phosphate precipitation technique. Cellular clones resistant to G418 antibiotic were selected and expanded for further analysis. Immunofluorescence microscopy demonstrated less Type IV collagen in the AS clones (1.0 +/- 0.3) than in control parent (P) and S clones (2.0 +/- 0.4) (P < 0.05). Western analysis showed that the AS clones synthesized 20 +/- 10% of the 205-kd alpha 1 (IV) chain of Type IV collagen compared with P cells (P < 0.05). As transfected clones demonstrated similar basal proliferation rates as control cells when cultured in 0.5% fetal calf serum (FCS), but failed to undergo fetal calf serum (FCS)-stimulated hyperplasia when grown on standard fibronectin-coated surfaces in 40% FCS (P < 0.05, compared with P- and S-transfected control cells). There were significant linear relationships between the presence of Type IV collagen as detected by either immunofluorescence microscopy or alpha 1 (IV) peptide chain quantitation by Western analysis and the ability of cells to undergo FCS-stimulated hyperplasia when grown on fibronectin (P < 0.05). Growth on a surface comprised of fibronectin plus Type IV collagen restored the capacity of AS transfected cells to respond to FCS stimulation (P < 0.001), but had no significant effect on the proliferative behavior of P or S cells. Measurements of AS RNA levels in these cells suggest that the inhibition of stimulated proliferation is determined by the presence of a threshold quantity of cellular AS RNA. These data demonstrate that Type IV collagen plays a critical role in conditioning glomerular endothelial cells to respond to proliferative stimuli.

Animals↗
PubMed2003

Nuclear factor-kappaB translocation mediates double-stranded ribonucleic acid-induced NIT-1 beta-cell apoptosis and up-regulates caspase-12 and tumor necrosis factor receptor-associated ligand (TRAIL).

The mechanism of induction of apoptosis by double-stranded RNA (dsRNA) is not fully characterized. The dsRNA is normally present in extremely low quantities in cells, but following infection with RNA viruses, large quantities of the dsRNA viral replicative intermediate may be produced triggering the antiviral response as well as cell death. In this report, transfection of polyinosinic-polycytidylic acid [poly(I:C)] into NIT 1 cells has been used as a model of intracellular dsRNA-induced beta-cell apoptosis. At 18 h post transfection, 45% of the cells were apoptotic as indicated by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling (TUNEL) staining, and this was accompanied by an increase in nuclear factor kappaB (NF-kappaB) p50/p65 nuclear translocation and cleavage of caspases 3 and 8. The NF-kappaB inhibitor peptide, SN50, significantly reduced caspase-3 activity and the percentage of TUNEL-positive cells, substantiating a role for NF-kappaB in inducing intracellular dsRNA-mediated apoptosis. Concomitantly, RNA-dependent protein kinase activity was observed at 3 h post transfection along with phosphorylation and degradation of inhibitory kappaB-alpha. Expression of TRAIL (TNF-related apoptosis-inducing ligand), Fas, IL-15, and caspase-12 mRNAs was up-regulated in the presence of poly(I:C) but not when SN50 was also added. In contrast, there was no change detected in Fas, Fas-associated death domain, Bcl-2, Bcl-xl, Bax, p53, or XIAP(X-linked inhibitor of apoptosis protein) expression up to 12 h after poly(I:C) transfection. In addition, caspase-12 was cleaved, and phosphorylation of eukaryotic initiation factor 2alpha occurred, suggesting that an endoplasmic reticulum stress pathway was involved in addition to NF-kappaB induction of an extrinsic pathway, possibly mediated by TNF-related apoptosis-inducing ligand.

Animals↗
PubMed1970

Some effects of triton x-100 on pea etioplasts.

When pea etioplast preparations were treated with Triton X-100, the membranes disappeared, the pigments were solubilized, and the organelles appeared to disintegrate. Low speed centrifugation (2000g) of the preparations following treatment with Triton X-100 resulted in a pellet which contained considerable quantities of plastid material. This included RNA polymerase and DNA polymerase activity, much of the DNA, about 30% of the RNA, and 50% of the protein of the washed plastid. The amount of RNA polymerase and DNA polymerase activity associated with the low speed pellet was dependent on the pH during Triton treatment. Significant quantities of the RNA polymerase activity of chloroplasts from spinach, peas, and tobacco were also recovered in the pellet after Triton treatment.

Journal Article↗
PubMed1974

Intracellular viral RNA species in mouse cells nonproductively transformed by the murine sarcoma virus.

The size and quantity of virus-specific RNA in five non-virus-producing mouse cells transformed by the Moloney isolate of murine sarcoma virus (MSV) was determined. Hybridization of RNA from transformed cells with the [(3)H]DNA product of the RNA-directed DNA polymerase of the murine sarcoma-leukemia virus was used to detect and quantitate virus-specific RNA. The amount of virus-specific RNA in non-virus-producing cells was less than one-sixth of that found in virus-producing cells. A striking correlation was found between the amount of intracellular virus-specific RNA and the degree of agglutination by conconavalin A previously reported for the four non-virus-producing NIH/3T3 cell lines (Salzberg and Green, 1974). A major RNA subunit sedimenting at 26 to 28S was detected in all five MSV-transformed non-virus-producing cells. This could represent the RNA genome of defective MSV.

Animals↗
PubMed1978

Immunofluorescent detection of nuclear double-stranded RNA in situ in Vero and mosquito cells.

Double-stranded RNA (dsRNA) was detected in situ by indirect immunofluorescence with antibodies to dsRNA. It was seen in nuclei of Vero and Aedes albopictus cells, but not in BHK cells, KB cells, chick embryo fibroblasts, or HeLa cells. Reactive dsRNA was present in the nucleoplasm, but not in nucleoli or cytoplasm. Extracted RNA from the whole cell contained from 0.08 percent (BHK) to 0.46 percent (HeLa) dsRNA, as estimated by serological methods. This dsRNA, found in molecules having the size distribution of heterogeneous nuclear RNA, did not renature rapidly after denaturation. The quantity of dsRNA in total extracted RNA did not correlate with the presence or absence of nuclear staining in situ.

Cell Line↗
PubMed1982

Structure and function of rat liver polysome populations. II. Characterization of polyadenylate-containing mRNA associated with subpopulations of membrane-bound particles.

Poly(A)+RNA fractions prepared from free and loosely and tightly membrane-bound polysome populations (poly(A)+RNAfree, poly(A)+RNAloose, and poly(A)+RNAtight) were used to drive cDNA in homologous and heterologous hybridization reactions. A large fraction by mass of sequences was shared among the three poly(A)+RNA populations, but shared sequences exhibited distinct frequency distributions within the different populations. 13-15 in vitro translation products of poly(A)+RNAfree and poly(A)+RNAloose detected by gel electrophoresis were shared. Most of these were produced in different relative quantities by the two RNA populations. Five or six higher mol wt polypeptides were produced by poly(A)+RNAloose that were not detected as products of either poly(A)+free or poly(A)+RNAtight. We suggest that loosely bound polysomes may not be artifactually derived as reflected in their quantitatively distinct poly(A)+RNA population. Two tightly membrane-bound RNP fractions were prepared from rat liver on the basis of their release from or retention on purified rough microsomes or a crude membrane fraction after in vitro disaggregation of polysomes with high-salt and puromycin. Homologous and heterologous hybridizations involving their poly(A)+RNA fractions revealed that a large portion by mass of sequences was shared but that these sequences exhibited distinct frequency distributions in the two fractions. The RNA fractions produced exhibited distinct frequency distributions in the two fractions. The RNA fractions produced an identical set of in vitro translation products but individual polypeptides were produced in different relative quantities. This indicates that the two RNP fractions do not arise by any random artifactual process and suggests that they may represent functionally distinct populations.

Animals↗
PubMed1974

The fractionation of t-RNA on N,N'-bis(3-aminopropyl)-piperazine substituted-Sepharose.

An anion exchange agarose has been prepared by modifying sepharose 6B with N,N'-bis (-3-aminopropyl) piperazine. This material (BAPP-Sepharose) has been used for the fractionation of t-RNA from E.coli by column chromatography. The results obtained with gram quantities of crude t-RNA at pH 4.6 and pH 8.0 as measured by the elution patterns of alanyl, arginyl, aspartyl, leucyl, lysyl, methionyl, phenylalanyl, prolyl, seryl, tyrosyl, and valyl t-RNA are described.

Chromatography, Ion Exchange↗
PubMed1997

The use of RNA probes for the analysis of gene expression.

The monomeric bacteriophage RNA polymerases allow the synthesis of virtually any RNA molecule in unlimited quantity. In this protocol, we describe the preparation of plasmid and PCR-derived templates. A basic transcription protocol is provided with several optional modifications. The use of RNA probes in Northern blot hybridization and in RNase protection assays is described. The relative advantages and pitfalls of these two methods to quantitatively detect mRNA targets are discussed.

Animals↗
PubMed2006

Extraction of RNA from archival tissues and measurement of thrombospondin-1 mRNA in normal, dysplastic, and malignant oral tissues.

Thrombospondin-1 (TSP-1) is an extracellular matrix glycoprotein implicated in the regulation of angiogenesis and tumour development. Our objectives were to ascertain the quantity and quality of RNA extracted from archival, formalin-fixed, paraffin embedded, oral tissues and their application in measuring the concentrations of TSP-1 mRNA in these tissues. We compared three techniques of isolation of RNA as well as related experimental variables. TSP-1 mRNA was measured in specimens of normal, dysplastic, and malignant oral tissues by real-time reverse transcriptase polymerase chain reaction (RT-PCR). RNA suitable for analysis by real-time RT-PCR was obtained by the three techniques tested, although the yield varied depending on the protocol used (range 0.2-3.6 microg/mm(3)). The mean (S.D.) concentrations of TSP-1 mRNA relative to 18S were 21.1 (7.2) in normal oral tissues (n=9), 11.0 (8.2) in dysplastic tissue (n=8) and 7.3 (5.3) in carcinomatous tissue (n=17). The difference between normal and carcinomatous specimens was significant (p=0.01). This reduction in expression of TSP-1 mRNA from normal to dysplasia to carcinoma may favour the angiogenic drive that accompanies the development of oral tumours.

Carcinoma, Squamous Cell↗
PubMed2003

High-throughput confirmation of differential display PCR results using reverse Northern blotting.

Nylon filter arrays spotted with differential display PCR (DD-PCR) clones and hybridized with radiolabeled cRNA generated from the source RNA pool (reverse Northern blot) provide a high-throughput means to screen clones for artifacts. Reverse Northern blots also confirm differential gene expression in parallel and require modest quantities of the source RNA pool. We describe a strategy to screen multiple candidates from DD-PCR by high-throughput ligation and transformation, followed by reverse Northern blotting. Purification of re-amplified DD-PCR clones and fabrication of nylon arrays was facilitated by a batch-processing protocol using the widely available Biomek laboratory robot and Bioworks scripts (available from the authors). A strategy to screen out DD-PCR product artifacts of an inappropriate size was also employed. Using these approaches, we identified several mRNAs that are differentially expressed in response to venlafaxine, fluoxetine or desipramine antidepressant treatment in rat C6 glioma cell lines and are candidates for full length clone isolation using 5'-RACE. Such an approach provides a rapid means to eliminate the high percentage of false positive clones from DD-PCR and enables independent confirmation of differential gene expression patterns generated by various experimental conditions.

Animals↗
PubMed2004

Micronutrient supplementation increases genital tract shedding of HIV-1 in women: results of a randomized trial.

To test the hypothesis that micronutrient supplementation decreases genital HIV-1 shedding, a double-blind, randomized, placebo-controlled trial of 6 weeks of multivitamin plus selenium supplementation vs. placebo was conducted among 400 HIV-1-seropositive, nonpregnant, antiretroviral-naive women in Mombasa, Kenya. Primary outcome measures included cervical and vaginal shedding of HIV-1-infected cells and RNA. Secondary outcomes included plasma viral load and CD4 count. Surprisingly, the odds of detection of vaginal HIV-1-infected cells were 2.5-fold higher (P = 0.001) and the quantity of HIV-1 RNA in vaginal secretions was 0.37 log10 copies/swab higher (P = 0.004) among women who received micronutrients in comparison to placebo, even after adjustment for potential confounders including baseline HIV-1 shedding and CD4 count. The increase in vaginal HIV-1 shedding was greatest among women who had normal baseline selenium levels. Micronutrient supplementation resulted in higher CD4 (+23 cells/microL, P = 0.03) and CD8 (+74 cells/microL, P = 0.005) counts compared with placebo but did not alter the plasma viral load. In this randomized trial, micronutrients resulted in higher levels of genital HIV-1 shedding compared with placebo. The potential benefit of micronutrient supplementation in HIV-1-seropositive women should be considered in relation to the potential for increased infectivity.

Adolescent↗