Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “RNA quantity”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 757 records · Page 42Linked to original sources
PubMed1976

Stimulation of RNA polymerases I, II and III from rat liver by spermine, and specific inhibition of RNA polymerase I by higher spermine concentrations.

Spermine stimulates activities of higherly purified rat liver nuclear RNA olymerases I, II and III 3 to 4 fold. Inclusion of (NH4)2SO4 at concentrations required for maximal enzyme activities does not significantly enhance the degree of stimulation of polymerase activities by spermine, but maintains the stimulatory levels of enzymes over a broader range of spermine concentrations. The stimulatory effect of spermine at a concentration of 1 mM is a useful method for the elevation of activities of all RNA polymerases and thus provides a means to measure these enzymes when extracted from small quantities of tissues or cells. Based on the differential stimulation of the polymerases by spermine, a higher concentration of spermine (5 mM) can be selected to inhibit RNA polymerase I specifically.

Animals↗
PubMed2000

Alteration in expression profiles of a series of diabetes-related genes in db/db mice following treatment with thiazolidinediones.

We studied the effect of pioglitazone on the transcription of 42 genes associated with diabetes to examine the relationship between the antidiabetic action of thiazolidinediones (TZDs) and their ability to modulate transcription through their peroxisome proliferater-activated receptor (PPAR)-agonistic activity. Diabetic (db/db) mice were orally administered with pioglitazone for two weeks. Total RNA was prepared from liver, muscle and adipocytes and the quantity of mRNA was determined by comparative RT-PCR. The expression of diabetes-related genes was compared between lean and untreated db/db mice and between untreated and drug-treated db/db mice. The onset of diabetes was associated with a considerable alteration in the expression of a large number of diabetes-related genes. Treatment of db/db mice with pioglitazone modulated the expression of genes involved in the metabolism of glucose, lipids and lipoproteins. This included genes for phosphoenolpyruvate carboxykinase, beta-oxidation enzymes, lipoprotein lipase, apolipoprotein AI and uncoupling proteins. Most of the genes responsible for insulin signaling were unaffected. Administration of pioglitazone was also shown to induce PPARgamma expression in liver and muscle. It is therefore possible to hypothesize that TZDs may ameliorate diabetes through a mechanism of action involving a direct decrease in plasma glucose and triglyceride levels and improvements in free fatty acid-induced insulin resistance.

Adipocytes↗
PubMed1975

The regulation of transcription in bacteriophage T5-infected Escherichia coli.

The expression of bacteriophage T5-specific RNA and protein in infected cells is temporally separated into three classes: class I (preearly), class II (early), and class III (late). By immunoprecipitation techniques we have shown that T5 infection of cells leads to the synthesis of one class I polypeptide (11,000 daltons) and two class II polypeptides (90,000 and 15,000 daltons) capable of binding to the RNA polymerase of the host Escherichia coli cell. One of the class II polypeptides (90,000 daltons) is the product of gene C2, which is an essential gene product required for the initiation of class III RNA synthesis. The colicinogenic factor, ColIb, is a plasmid which prevents the normal synthesis of class II and the III bacteriophage T5-specific RNA in infected colicinogenic (ColIb+) cells. In T5-infected colicinogenic cells, only the T5 class I polypeptide is found associated with the RNA polymerase. Mutants of T5, designated T5h minus, are capable of growth on both noncolicinogenic and ColIb+ hosts. Extracts of T5h minus infected ColIb+ cells were shown to lack a small class I polypeptide (12,000 daltons) as compared to T5-infected cells. The h minus mutation, however, has no effect on the levels of the class I T5 polypeptide of similar molecular weight which is bound to the RNA polymerase. One effect of the h minus mutation is to enhance the quantities of the two class II polypeptides bound to the enzyme.

Bacterial Proteins↗
PubMed1993

Regional codon randomization: defining a TATA-binding protein surface required for RNA polymerase III transcription.

The TATA-binding protein (TBP) is required for transcription by all three nuclear RNA polymerases. TBP was subjected to regional codon randomization, a codon-based mutagenesis method that generates complex yet compact protein libraries. Analysis of 186 temperature-sensitive TBP mutants yielded 65 specifically defective in transcription by RNA polymerase III (Pol III). These mutants map to a limited TBP surface that may interact with Tds4, a component of the Pol III transcription factor TFIIIB. Strains that contain the Pol III-defective derivatives have increased amounts of messenger RNA, which suggests that competition among TBP-interacting factors for limiting quantities of TBP determines the ratio of Pol II and Pol III transcription in vivo.

Amino Acid Sequence↗
PubMed2000

Estimation of total ribonucleic acid quantity from dilute samples by nondenaturing electrophoresis and silver staining.

Often, the amount of RNA that can be isolated from a defined tissue is very small. A method consisting of nondenaturing polyacrylamide gel electrophoresis and silver staining is described that can be used to evaluate the concentration of very dilute RNA samples. The method is a good starting point for assays dealing with small amounts of RNA, such as semiquantitative reverse transcription polymerase chain reaction (RT-PCR), making it possible to perform parallel assays from similar amounts of total RNA when quantitation by other methods is too insensitive. The method has been used successfully to monitor the amount of total RNA isolated from rat sciatic nerve and the rat C6 glioma cell line.

Electrophoresis, Polyacrylamide Gel↗
PubMed1992

Developmental switching of messenger RNA expression from the human alpha-globin cluster: fetal/adult pattern of theta-globin gene expression.

The alpha-globin gene cluster contains four functional globin genes, zeta, alpha 2, alpha 1, and theta. The developmental regulation of the embryonic zeta and fetal/adult alpha 2- and alpha 1-globin genes is well characterized at the level of protein synthesis. The developmental pattern of the theta-globin gene is not well characterized due to the inability to detect its encoded protein. Direct analysis of the globin switching at the steady-state messenger RNA (mRNA) level has been hampered by the difficulty in obtaining quantities of embryonic and early fetal mRNA sufficient for analysis. We analyzed the relative levels of the steady-state zeta-, alpha-, and theta-globin mRNAs in yolk sac in 5-, 6-, 7-, and 8-week postconception embryonic liver, and in cord and adult blood reticulocytes. We show that the switch in the alpha-globin gene cluster from the embryonic to fetal/adult pattern of expression begins at 5 to 6 weeks of gestation. Both the theta- and alpha-globin genes show similar patterns of developmental control that are reciprocal to zeta. alpha-globin RNA is barely detectable or undetectable at 5 weeks, and increases in the 6- to 8-week period, while theta-globin mRNA shows a parallel increase at 5 to 8 weeks postconception and is expressed in cord blood and adult reticulocytes. These data show that the theta-globin gene represents a fetal/adult gene, albeit expressed at a low level.

Adult↗
PubMed1999

High-throughput real-time reverse transcription-PCR quantitation of hepatitis C virus RNA.

We describe a rapid and reproducible method for assessment of the hepatitis C virus (HCV) load in serum samples. The method combines Taqman technology (Roche) and the ABI Prism 7700 (Perkin Elmer) real-time sequence detection system. We have optimized a single-tube reverse transcription-PCR (RT-PCR) that contains a dual-labeled fluorogenic probe to quantify the 5' noncoding region (5' NCR) of HCV. The probe contains a fluorescent reporter at the 5' end and a fluorescent quencher at the 3' end. The use of such a probe combined with the 5'-3' nuclease activity of Taq polymerase allows direct quantitation of the PCR product by the detection of a fluorescent reporter released in the course of the exponential phase of the PCR. For accurate quantitation of the number of copies of HCV in samples containing unknown quantities, we have used serial dilutions of a synthetic 5' NCR RNA standard of HCV that was previously quantified with an isotopic tracer. The method has a 5-log dynamic range (10(3) to 10(7)). The coefficient of regression of the standard curve was, on average, 0.98. The intra-assay and the interassay coefficients of variation of the threshold cycle were 1% and 6.2%, respectively. Seventy-nine RNA samples from the sera of infected patients were quantified by this method. Comparison of the results with those obtained by other quantitation methods (the Quantiplex 2.0 branched-DNA assay and the Superquant assay from the National Genetics Institute) revealed a significant correlation with all of the results. The mean values were also statistically comparable. In conclusion, the high sensitivity, simplicity, and reproducibility of the real-time HCV RNA quantitation which allows the screening of large numbers of samples, combined with its wide dynamic range, make this method especially suitable for monitoring of the viral load during therapy and tailoring of treatment schedules.

Adult↗
PubMed2003

Sequence and structural requirements for optimal guide RNA-directed insertional editing within Leishmania tarentolae.

The coding sequence of several mitochondrial mRNAs of the trypanosomatid family of protozoa is created by the guide RNA-directed insertion and deletion of uridylates (Us). Selection-amplification was used to explore the sequence and structure of the guide RNA and mRNA required for efficient insertional editing within a mitochondrial extract prepared from Leishmania tarentolae. This study identifies several novel features of the editing reaction in addition to several that are consistent with the previous mutagenesis and phylogenetic analysis of the reaction in Trypanosoma brucei, a distantly related trypanosomatid. Specifically, there is a strong bias against cytidines 5' of the editing sites and guanosines immediately 3' of guiding nucleotides. U insertions are directed both 5' and 3' of a genomically encoded U, which was previously assumed not to occur. Base pairing immediately flanking an editing site can significantly stimulate the editing reaction and affect the reaction fidelity but is not essential. Likewise, single-stranded RNA in the region upstream of the editing site, not necessarily immediately adjacent, can facilitate editing but is also not essential. The editing of an RNA containing many of the optimal features is linear with increasing quantities of extract permitting specific activity measurements to be made that are not possible with previously described T. brucei and L. tarentolae assays. The reaction catalyzed by the L. tarentolae extract can be highly accurate, which does not support a proposed model for editing that was based largely on the inaccuracy of an earlier in vitro reaction.

Animals↗
PubMedDate not supplied

[Analysis of hepatitis C virus proteins in hepatic cells of patients with chronic hepatitis C].

A correlation between the detection of proteins and an activity of the pathological process was analyzed in a study of the content of the C virus hepatitis (CVH) proteins in hepatic cells of patients with chronic C hepatitis (CCH). The expression of CVH proteins in frozen sections of biopsy samples of 69 CCH patients was evaluated by using the immune-histological method involving original monoclonal antibodies (MCA) to 5 CVH proteins. The results of the detection of proteins in patients were compared with an activity and stage of CCH (by using histological tests and a level of alanine aminotransferase--AAT). A set of the CVH proteins were found in the liver of 74% of patients, i.e. core proteins, NS3, NS4A, NS4B and NS5A--in 28, 43, 43, 55 and 58%, respectively. All studied proteins were detected in the cytoplasm of hepatocytes. Proteins were found in the liver more often as compared with the detection rate of CVH RNA in the blood serum (61%). This demonstrates a high sensitivity of the discussed test at detecting the CVH infection. The accumulation of the core protein was shown to correlate with the presence of the replicative form of CVH RNA in the liver and with a higher level of AAT. The quantity of NS5 A-expressing cells correlated directly with a CCH stage. The quantity of NSB- and NS3-positive hepatocytes correlated negatively with an activity of the inflammatory-and-necrotic processes in the liver. Hyper-fermentation was found more often among the antigen-positive patients. The CCH histological activity was proven to be reliably higher at a simultaneous detection of CCH proteins in the liver and of CVH RNA--in the serum.

Adolescent↗
PubMed1995

Rescue of the fission yeast snRNA synthesis mutant snm1 by overexpression of the double-strand-specific Pac1 ribonuclease.

The Schizosaccharomyces pombe temperature-sensitive mutant snm1 maintains reduced steady-state quantities of the spliceosomal small nuclear RNAs (snRNAs) and the RNA subunit of the tRNA processing enzyme RNase P. We report here the isolation of the pac1+ gene as a multi-copy suppressor of snm1. The pac1+ gene was previously identified as a suppressor of the ran1 mutant and by its ability to cause sterility when overexpressed. The pac1+ gene encodes a double-strand-specific ribonuclease that is similar to RNase III, an RNA processing and turnover enzyme in Escherichia coli. To investigate the essential structural features of the Pac1 RNase, we altered the pac1+ gene by deletion and point mutation and tested the mutant constructs for their ability to complement the snm1 and ran1 mutants and to cause sterility. These experiments identified four essential amino acids in the Pac1 sequence: glycine 178, glutamic acid 251, and valines 346 and 347. These amino acids are conserved in all RNase III-like proteins. The glycine and glutamic acid residues were previously identified as essential for E. coli RNase III activity. The valines are conserved in an element found in a family of double-stranded RNA binding proteins. Our results support the hypothesis that the Pac1 RNase is an RNase III homolog and suggest a role for the Pac1 RNase in snRNA metabolism.

Amino Acid Sequence↗
PubMed1993

Localization of intestinal interleukin 1 activity and protein and gene expression to lamina propria cells.

BACKGROUND: Interleukin 1 (IL-1) is a key mediator of bowel inflammation, but there is limited knowledge about the amount and site of production of this cytokine in the gastrointestinal tract under physiological or pathological conditions. METHODS: Epithelial and lamina propria mononuclear cells were isolated from control, and Crohn's disease- and ulcerative colitis-involved mucosa to investigate the capacity of these cells to generate IL-1 bioactivity, IL-1 alpha and IL-1 beta immunoreactivity, and gene expression. RESULTS: Control lamina propria mononuclear cells produced substantial amounts of IL-1 alpha and IL-1 beta, which increased dramatically when inflammatory bowel disease cells were used. Epithelial cells from control, Crohn's disease, and ulcerative colitis intestine displayed no IL-1 bioactivity or immunoreactivity. Lamina propria mononuclear cells contained moderate to large quantities of IL-1 alpha and IL-1 beta messenger RNA (mRNA), respectively, whereas epithelial cells had none. The absence of IL-1 transcripts in epithelial cells was selective, because mRNA for HLA-DR antigens was present in control and inflammatory bowel disease cells. CONCLUSIONS: In normal and inflamed human intestine there is a distinct compartmentalization of IL-1, as mononuclear but not epithelial cells generate this cytokine. The high levels of IL-1 in inflammatory bowel disease may explain several of its local and systemic manifestations, and blockade by specific antagonists could have important therapeutic effects.

Adolescent↗
PubMed1997

HCF-1, a hydrophobin from the tomato pathogen Cladosporium fulvum.

A hydrophobin, named HCF-1, was isolated from the culture medium of Cladosporium fulvum, the causal agent of tomato leaf mould. The protein forms insoluble aggregates when the medium is vigorously aerated. These aggregates can be dissociated by trifluoroacetic acid into monomers which migrate as 10-kDa molecules on SDS-PAGE. HCf-1 is encoded by a single gene, HCf-1. The gene contains two small introns and is translated into a 105-amino acid protein which is then processed to give a mature 83-amino acid protein. The position of the eight cysteine residues and the predicted hydrophobicity profile are typical of fungal hydrophobins. HCf-1 RNA is expressed in growing mycelium and conidia but its quantity diminishes transiently after germination; its abundance does not change when the fungus is grown on nitrogen- or carbon-deficient medium. This is the first step in evaluating the role of hydrophobins in establishment of basic compatibility between C. fulvum and tomato.

Amino Acid Sequence↗
PubMed1993

Dependence of nucleic acid degradation on in situ free-radical production by adriamycin.

Adriamycin (Adr) is one of the most powerful antitumor drugs. Its therapeutic effect may be due to its cyclic reduction-oxidation and, thus, generation of oxygen radicals. Using the spin-trap 5,5'-dimethyl-1-pyrroline N-oxide (DMPO) and EPR we have demonstrated that in an enzymatic system consisting of NADPH, NADPH-cytochrome P-450 reductase, and Fe(EDTA)2 Adr stimulates formation of .OH radicals in the presence of DNA or RNA with equal efficiency. Incubation of nucleic acids in the Adr-dependent reaction generating .OH radicals resulted in extensive degradation of double- and single-stranded DNA, but did not effect RNA. In contrast, both DNA and RNA were effectively destroyed in a footprinting system, ascorbate-Fe(EDTA)2-H2O2, which generates .OH radicals in massive quantities. Fluorescence assays indicated that Adr forms stable complexes with ds- and ss-DNA but reacts only slightly with RNA. We conclude that the formation of Adr-nucleic acid complex is necessary for .OH radical-mediated cleavage of the latter, and thus, Adr may be regarded as a chemical nuclease acting in situ.

DNA↗
PubMed2001

Safety, vaccine virus shedding and immunogenicity of trivalent, cold-adapted, live attenuated influenza vaccine administered to human immunodeficiency virus-infected and noninfected children.

OBJECTIVE: To assess the safety of live, attenuated influenza vaccine (LAIV) administered to relatively asymptomatic or mildly symptomatic HIV-infected children and non-HIV-infected children. METHODS: Twenty-five non-HIV and 24 HIV-infected children (CDC Class N or A1,2) were enrolled into this double blind, placebo-controlled study. Children were randomized within each HIV status group to one of two dosing regimens: Regimen 1, Dose 1 = LAIV, Dose 2 = placebo, Dose 3 = LAIV; or Regimen 2, Dose 1 = placebo, Dose 2 = LAIV, Dose 3 = LAIV. Study doses were separated by 28 to 35 days. Reactogenicity events within 10 days and adverse events within 28 to 35 days after each study dose were recorded. Blood HIV RNA concentrations, CD4 counts and CD4% were measured throughout the study on HIV-infected children. Quantitative influenza cultures were performed on nasal aspirates collected periodically from all children up to 28 to 35 days after each study dose. Influenza isolates were assessed for retention of the temperature-sensitive phenotype. Serum influenza HAI antibodies were measured before and after each LAIV vaccination. RESULTS: No significant differences were found in rates of reactogenicity events and vaccine-related adverse events after placebo or the first dose of LAIV within each HIV status group, nor were differences found between HIV-infected and HIV-uninfected children after each dose of LAIV. Overall none of the HIV-infected children experienced a significant LAIV-related serious adverse event or influenza-like illness, making the one sided 95% CI of such a serious event occurring after LAIV 0 to 12%. No significant changes in geometric mean HIV RNA concentrations, CD4 counts or CD4% or prolonged or increased quantity of LAIV virus shedding occurred in HIV-infected children after receiving either dose of LAIV. All recovered influenza isolates retained the temperature-sensitive phenotype. After two doses of LAIV, 83% of the non-HIV-infected and 77% of the HIV-infected children had a > or = 4-fold rise in influenza antibody to at least one of the three LAIV strains. CONCLUSION: If relatively healthy HIV-infected children become exposed to LAIV inadvertently, then serious adverse outcomes would not be expected to occur frequently.

Child↗
PubMed1994

Rapid RT-PCR amplification from limited cell numbers.

We describe a rapid and efficient RT-PCR method particularly suited to procedures involving limited cell and target gene copy numbers. Purified leukocytes and myeloid colonies derived from patients with chronic myelogenous leukemia (CML) in chronic phase were used for direct RT-PCR. Purified cells and colonies were lysed using a small quantity of DEPC-treated water containing RNasin as an RNA inhibitor. The untreated lysate was either used immediately for RT-PCR or frozen at -70 degrees C for later use. By this method we were able to consistently amplify bcr-abl transcripts from as few as 10 cells. No noticeable difference was observed between products amplified from fresh and frozen samples.

Base Sequence↗
PubMed1994

Hepatitis C virus NS3 serine proteinase: trans-cleavage requirements and processing kinetics.

The hepatitis C virus H strain (HCV-H) polyprotein is cleaved to produce at least 10 distinct products, in the order of NH2-C-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B -COOH. An HCV-encoded serine proteinase activity in NS3 is required for cleavage at four sites in the nonstructural region (3/4A, 4A/4B, 4B/5A, and 5A/5B). In this report, the HCV-H serine proteinase domain (the N-terminal 181 residues of NS3) was tested for its ability to mediate trans-processing at these four sites. By using an NS3-5B substrate with an inactivated serine proteinase domain, trans-cleavage was observed at all sites except for the 3/4A site. Deletion of the inactive proteinase domain led to efficient trans-processing at the 3/4A site. Smaller NS4A-4B and NS5A-5B substrates were processed efficiently in trans; however, cleavage of an NS4B-5A substrate occurred only when the serine proteinase domain was coexpressed with NS4A. Only the N-terminal 35 amino acids of NS4A were required for this activity. Thus, while NS4A appears to be absolutely required for trans-cleavage at the 4B/5A site, it is not an essential cofactor for serine proteinase activity. To begin to examine the conservation (or divergence) of serine proteinase-substrate interactions during HCV evolution, we demonstrated that similar trans-processing occurred when the proteinase domains and substrates were derived from two different HCV subtypes. These results are encouraging for the development of broadly effective HCV serine proteinase inhibitors as antiviral agents. Finally, the kinetics of processing in the nonstructural region was examined by pulse-chase analysis. NS3-containing precursors were absent, indicating that the 2/3 and 3/4A cleavages occur rapidly. In contrast, processing of the NS4A-5B region appeared to involve multiple pathways, and significant quantities of various polyprotein intermediates were observed. NS5B, the putative RNA polymerase, was found to be significantly less stable than the other mature cleavage products. This instability appeared to be an inherent property of NS5B and did not depend on expression of other viral polypeptides, including the HCV-encoded proteinases.

Amino Acid Sequence↗
PubMed1994

Immunodetection of the ribosomal transcription factor UBF at the nucleolus organizer regions of fish cells.

A human autoimmune serum to nucleolus organizer regions (NORs) has been used to localize these structures at the light microscopic level in carp and trout tissue culture cells. In interphase cells, the immunofluorescence pattern indicates that the NORs autoantigens are contained exclusively within the nucleolus of carp epithelial (EPC) and trout gonad (RTG) cells. This fluorescence is punctuate rather than uniform, and presumably represents transcriptional complexes of ribosomal DNA. During mitosis, the autoantigens are detected by immunofluorescence microscopy at the chromosomal nucleolus organizer regions of condensed chromosomes, indicating that a considerable quantity of the molecule(s) remains bound to the ribosomal RNA genes. The major nucleolus autoantigen, defined in mammals as the upstream ribosomal binding factor (UBF), has been identified on immunoblots as a 90 kDa protein in extracts from fish cell lines and tissues. Thus, NORs appear to function as nucleation centers for ribosomal RNA together with a complex set of well-conserved protein factors, such as UBF. Our results suggest evolutionary conservation from fish to mammals with respect to ribosomal RNA biosynthesis driven by RNA polymerase I.

Animals↗
PubMed1979

Relationship between single-stranded DNA isolated from cultured muscular cells during differentiation and the transcription of messenger RNA.

Single-stranded DNA (ssDNA), equivalent to about 2% of the total nuclear DNA, was isolated by an improved method of hydroxyapatite chromatography from native nuclear DNA of rat myoblast cells and myotubes of the L6 line. Small quantities of 125I-labelled ssDNA were annealed with a large excess of unlabelled DNA, cytoplasmic RNA and mRNA from myoblasts or myotubes. The results indicated that ssDNA belongs to the non-repetitious portion of the cell genome and is formed of two distinct molecular fractions. The major ssDNA fractions (75%) consist of non-self-reassociating DNA sequences and the minor fraction (25%) consists of self-reassociating DNA sequences. About 30--32% and 25--26% of ssDNA from myoblast represent DNA sequences complementary to total cytplasmic RNAs and polyadenylated RNAs respectively. Hybridizations of ssDNA with an excess of RNA from myoblasts and/or myotubes show differences in the abundance and the diversity of mRNA during mascular differentiation. These differences were confirmed by DNA-driven reactions between 125I-labelled polyadenylated RNA and ssDNA in great excess.

Animals↗