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.

SourceResearch connectionFrom yearThrough year

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

At least 793 records · Page 44Linked to original sources

PubMed1975

Immune precipitation of immunoglobulin producing polysomes from mouse myeloma cells.

MPC-11 myeloma cells synthesizing IgG were labeled in vitro with 3H-uridine and the cell lysates, or purified polysomes treated with rabbit antiserum against the purified MPC-11 protein. The specificity of the immune precipitation was tested by rabbit anti-egg albumin and by precipitation of polysomes from "non-producer" myeloma and HeLa cells. The specific precipitation was 10 to 15% of the total cytoplasmic RNA for the IgG-producing clone of MPC-11 and about 2% for the non-producer clone or HeLa cells. Up to 20% of the total polysomal and ribosomal RNA were precipitated from the IgG producing clone. This value correlated with the IgG synthesis of these cells which is also about 20% of the total protein produced. The results suggest that myeloma cells produce a relatively large quantity of IgG due to the presence of a large amount of specific m-RNA molecules.

Animals↗

PubMed1980

Changes in Wheat Leaf Polysomal Messenger RNA Populations during the Early Stages of Rust Infection: EFFECTS OF CHLORAMPHENICOL AND LINCOMYCIN ON CELL-FREE TRANSLATION BY POLYSOMES FROM HEALTHY AND INFECTED LEAVES.

Polysomes isolated from a susceptible variety of wheat leaves (cultivar W2691) and those inoculated with the wheat stem rust fungus (f. sp. tritici, race 126-ANZ-6, 7) were incubated in a cell-free protein-synthesizing system. Under these conditions, different size classes of polypeptides, ranging in molecular weight from 10,000 to 80,000, are radiolabeled. Using double-isotope labeling technique, we show that some discrete size classes of polypeptides are synthesized in significantly greater quantitites by polysomes from inoculated leaves compared to the corresponding size classes synthesized by polysomes from healthy leaves. These results confirm our previous observation that there are significant changes in the wheat leaf polysomal messenger RNA populations at 3 days after inoculation with the rust fungus.The effects of the organelle-specific inhibitors of protein synthesis, chloramphenicol and lincomycin, on in vitro polysomal messenger RNA translation were investigated. The polypeptides synthesized by polysomes from healthy and inoculated leaves in the presence of chloramphenicol were compared. The results show that, even in the presence of this antibiotic, the polysomes from inoculated leaves synthesize greater quantities of some size classes of polypeptides. These data indicate that changes in polysomal messenger RNA populations involve, at least in part, cytoplasmic messenger RNA.

Journal Article↗

PubMed1988

Host cell-dependent homologous interference in lymphocytic choriomeningitis virus infection.

The generation of virus progeny as well as transcription, translation, and replication of the viral small RNA (S-RNA), which codes for the nucleoprotein (NP) and the glycoprotein precursor (GPC), was followed in L and MDCK cells after infection with multiplicities (m.o.i.) ranging from 0.01 to 100. In L cells, the yields of both plaque-forming units and interfering particles varied inversely with the m.o.i. Northern blot analysis revealed that early after infection with high multiplicity NP-mRNA was present, but later few or no signals of any specificity were registered. After low m.o.i. the results were negative at 8 hr, but large quantities of mRNAs for NP and GPC as well as viral genomic S-RNA and genomic-sized complementary S-RNA had been synthesized at 48 hr. In MDCK cells, throughout the range of m.o.i. both entities attained lower levels and most were generated at m.o.i. one. The degree of hybridization correlated roughly with the quantity of infectious virus to which the cells had been exposed. In the cells of both lines the NP-mRNA corresponded to the synthesis of its translation product, but once produced, most of it appeared to be retained in the phosphorylated form. We assume that the homologous interference seen in L cells after infection with high m.o.i. results from a host-dependent inhibition of viral transcription and replication mediated by NP.

Animals↗

PubMed2002

Analysis of odontoblast gene expression using a novel approach, laser capture microdissection.

Studying the mechanisms of molecular interactions in developing tissues demands sensitive molecular biological in vivo and in vitro techniques. Laser capture microdissection (LCM) allows for the isolation of mRNA in histological sections even from single cells, thus enabling the identification of in vivo gene expression products in closely circumscribed tissue areas. The aims of this study were to assess the optimal fixation, processing, and staining conditions to retrieve RNA from microdissected odontoblasts. Fluorometric assays and RT-PCR analysis of alpha 1(I) collagen, dentin sialophosphoprotein (Dspp), and osteocalcin (OC) confirmed that the total RNA isolated from day 0 and day 3 captured odontoblasts was sufficient in quantity and quality. Our results indicate that individual odontoblasts obtained by LCM are morphologically intact and chemically unaltered, allowing accurate molecular and biochemical analyses.

Animals↗

PubMed1988

An algorithm for comparing multiple RNA secondary structures.

A new distributed computational procedure is presented for rapidly determining the similarity of multiple conformations of RNA secondary structures. A data abstraction scheme is utilized to reduce the quantity of data that must be handled to determine the degree of similarity among multiple structures. The method has been used to compare 200 structures with easy visualization of both those structures and substructures that are similar and those that are vastly different. It has the capability of processing many more conformations as a function of research requirements. The algorithm is described as well as some suggestions for future uses and extensions.

Algorithms↗

PubMed1978

Regulation of the synthesis of Sindbis virus-specified RNA: role of the virion core protein.

Cells infected with seven different RNA+ mutants of Sindbis virus were found to accumulate a virus-specified polypeptide of mol. wt. 144000 (p144) during incubation at the non-permissive temperature, while at the same time synthesis of the virus structural proteins was drastically reduced. Mapping of the tryptic peptides of p144 showed that it contained the amino acid sequences of all the virus structural proteins. At the non-permissive temperature cells infected with the same seven mutants (out of 28 examined) also showed increased synthesis of 26S RNA, the mRNA for the virus structural proteins, relative to 42S RNA, and the virus genome, compared with infections by wild-type virus. We propose that both these phenotypic effects are the results of a single mutational step and that the primary defect in the processing of the virus structural protein precursor induces the relatively increased rate of synthesis of structural protein mRNA. Temperature-shift experiments with mutant-infected cells showed that p144 itself is not the agent of this effect. The failure of exposure to zinc ions to alter the RNA ratio in wild-type virus-infected cells suggested that the virus envelope proteins are not involved either, since their synthesis is preferentially inhibited under these circumstances. It is possible that it is the failure to synthesize the proper quantity of core protein in the mutant-infected cells which causes the shift of RNA synthesis in favour of structural protein mRNA.

Culture Techniques↗

PubMed2006

The application of laser microdissection to in planta gene expression profiling of the maize anthracnose stalk rot fungus Colletotrichum graminicola.

Laser microdissection (LM) offers a potential means for deep sampling of a fungal plant-pathogen transcriptome during the infection process using whole-genome DNA microarrays. The use of a fluorescent protein-expressing fungus can greatly facilitate the identification of fungal structures for LM sampling. However, fixation methods that preserve both tissue histology and protein fluorescence, and that also yield RNA of suitable quality for microarray applications, have not been reported. We developed a microwave-accelerated acetone fixation, paraffin-embedding method that fulfills these requirements and used it to prepare mature maize stalk tissues infected with an Anemonia majano cyan fluorescent protein-expressing isolate of the anthracnose stalk rot fungus Colletotrichum graminicola. We successfully used LM to isolate individual maize cells associated with C. graminicola hyphae at an early stage of infection. The LM-derived RNA, after two-round linear amplification, was of sufficient quality and quantity for global expression profiling using a fungal microarray. Comparing replicated LM samples representing an early stage of stalk cell infection with samples from in vitro-germinated conidia, we identified 437 and 370 C. graminicola genes showing significant up- or downregulation, respectively. We confirmed the differential expression of several representative transcripts by quantitative reverse-transcriptase polymerase chain reaction (RT-PCR) and documented extensive overlap of this dataset with a PCR-subtraction library enriched for C. graminicola transcripts in planta. Our results demonstrate that LM is feasible for in planta pathogen expression profiling and can reveal clues about fungal genes involved in pathogenesis. The method in this report may be advantageous for visualizing a variety of cellular features that depend on a high degree of histochemical preservation and RNA integrity prior to LM.

Acetone↗

PubMed1997

Laminin chain-specific gene expression during mouse oocyte maturation.

Expression patterns of laminin chain mRNAs (A, B1, and B2) during mouse oocyte maturation were examined using the competitive reverse transcription-polymerase chain reaction (RT-PCR) method. Total and poly (A)-rich mRNAs isolated from various stages of maturing oocytes in vitro were subjected to RT-PCR and the precise amount of laminin chain-specific mRNA transcripts was estimated by adding externally known amounts of in vitro transcribed mutant cRNA transcripts as an internal control. The estimated copy numbers for A, B1, and B2 chain mRNAs in a single germinal vesicle-stage oocyte were 1.34 +/- 0.19 x 10(5), 6.95 +/- 0.32 x 10(6), and 2.0 +/- 0.56 x 10(5), respectively. Although notable changes of all laminin chain mRNA levels were not observed at any stage of meiotic maturation in total mRNA preparation, chain- and meiotic stage-dependent alterations of poly (A)-tailed mRNA quantities were observed in poly (A)-rich mRNA preparation. A potent RNA synthesis blocker, alpha-amanitin did not influence the changes of mRNA levels, implying the presence of posttranscriptional regulation mechanism in the expression of laminin chains during mouse oocyte maturation. Discrete and time-dependent deadenylation of A and B1 chain, but not B2 chain mRNA, was observed during oocyte maturation by a rapid amplification of cDNA ends (RACE)-PCR. In germinal vesicle (GV)-stage oocytes, only B1 chain was found to be present in a highly polyadenylated state and subsequent deadenylation was observed as meiosis progressed. The poly (A) tail modification was dependent on the initiation of meiotic resumption. Although all laminin chain mRNAs were found in fully grown and meiotically competent mouse oocytes, Western blot analysis detected the B1 chain polypeptide only in GV- and polar body (PB)-stage eggs. These results suggest that the expression of laminin B1 chain in mouse oocytes may be due to its large amount of mRNA transcripts and/or high level of polyadenylation state that is efficient for translational activation.

Animals↗

PubMed2002

Maculavirus, a new genus of plant viruses.

Maculavirus is a new genus of plant viruses typified by Grapevine fleck virus (GFkV). A possible second member is Grapevine redglobe virus (GRGV). Maculaviruses are phloem-limited non-mechanically transmissible viruses with isometric particles c. 30 nm in diameter that have a rounded contour and prominent surface structure. Vectors, if any, are unknown. GFkV preparations contain two centrifugal components, T made up of empty protein shells and B, which contains 35% RNA. The coat protein (CP) has a molecular mass of 24 kDa. The genome is a single-stranded RNA that has c. 50% cytosine residues. It is 7564 nt in size, excluding the poly(A) tail and contains four putative open reading frames (ORF) that encode a 215.4 kDa polypeptide with the conserved motifs of replication-associated proteins of positive-strand RNA viruses (ORF1), the CP (ORF2), and one (GRGV) or two (GFkV) proline-rich polyproteins of 31.4 kDa (ORF3) and 15.9 kDa (ORF4), respectively, with unknown function. Replication-associated proteins and CP are phylogenetically related to those of members of the genera Tymovirus and Marafivirus. GFkV-infected grapevine cells contain vesiculated mitochondria, the possible site of RNA replication. In the natural host, GFkV particles accumulate in great quantity, sometimes in crystalline arrays in phloem cells.

Open Reading Frames↗

PubMed1992

Regulation of aromatase cytochrome P-450 (estrogen synthetase) transcripts in the quail brain by testosterone.

The aromatase cytochrome P-450 (P-450AROM) cDNA, which was identified by homologies in the DNA and in the deduced amino acid sequences with human P-450AROM cDNA, was isolated from a brain cDNA library of Japanese quail, demonstrating the presence of RNA transcripts of P-450AROM in the quail brain. To determine trace amounts of P-450AROM mRNA in the brain and to examine the effects of testosterone on its expression, a quantitative PCR method of RNA transcripts was developed. Brain total RNA was subjected to reverse transcription reaction and then quantitated by PCR from cDNA with a fluorescent dye-labeled primer. The quantity of P-450AROM mRNA was calculated by using an internal standard of modified P-450AROM (m-P-450AROM) RNA. The brain P-450AROM was primarily transcribed in the hypothalamus area (1.15 +/- 0.14 amol/micrograms of RNA) and traces of transcripts only were detected in the cerebellum (0.038 +/- 0.005 amol/micrograms of RNA). The P-450AROM mRNA in the hypothalamus of castrated quail was low (0.270 +/- 0.078 amol/micrograms of RNA) and increased 4- to 5-fold following treatment with testosterone. These results demonstrate, for the first time, that the increase in P-450AROM activity that is observed in the brain following treatment with testosterone results from a pretranslational regulation of the P-450AROM by androgens.

Androgens↗

PubMed1985

13C-NMR of ribosyl A-A-A, A-A-G, and A-U-G. Synthesis and assignment.

The three RNA trinucleotides; ApApA, ApApG, and ApUpG, have been synthesized in sufficient quantity to obtain natural abundance 13C(1H)-NMR spectra at strand concentrations between 4 and 100 mM. Comparisons between 70 degrees C spectra of the three trimers and their consistuent dimers ApA, ApG, ApU, and UpG allow secure assignments to be made for most of the resonances. This paper describes the syntheses and 13C assignments of the oligomers.

Adenine Nucleotides↗

PubMed1985

Dexamethasone inhibits alpha-fetoprotein gene transcription in neonatal rat liver and isolated nuclei.

The effect of dexamethasone on rat alpha-fetoprotein (AFP) expression has been further examined. Quantitation of serum AFP levels from newborns treated with dexamethasone showed a dose-response relationship between the quantity of dexamethasone administered and the reduction in AFP serum level. RNA blots, utilizing cloned AFP cDNA as probe, showed a marked reduction in AFP mRNA in dexamethasone treated livers. The extent of AFP mRNA depletion was correlated with dexamethasone dosage. The effect of dexamethasone on AFP mRNA concentration was relatively rapid; a substantial reduction occurred 12 hours after a single injection. The effect of dexamethasone appeared to be irreversible as hormone withdrawal did not cause AFP mRNA levels to rise. One putative AFP nuclear RNA precursor was identified which rapidly disappeared following dexamethasone treatment. AFP mRNA synthesis was also diminished in nuclei transcribed in vitro. The direct inhibitory effect of glucocorticoid hormone on AFP gene transcription was demonstrated in a reconstituted cell-free nuclear system.

Animals↗

PubMed2001

Quantitative analysis of mRNA amplification by in vitro transcription.

Effective transcript profiling in animal systems requires isolation of homogenous tissue or cells followed by faithful mRNA amplification. Linear amplification based on cDNA synthesis and in vitro transcription is reported to maintain representation of mRNA levels, however, quantitative data demonstrating this as well as a description of inherent limitations is lacking. We show that published protocols produce a template-independent product in addition to amplifying real target mRNA thus reducing the specific activity of the final product. We describe a modified amplification protocol that minimizes the generation of template-independent product and can therefore generate the desired microgram quantities of message-derived material from 100 ng of total RNA. Application of a second, nested round of cDNA synthesis and in vitro transcription reduces the required starting material to 2 ng of total RNA. Quantitative analysis of these products on Caenorhabditis elegans Affymetrix GeneChips shows that this amplification does not reduce overall sensitivity and has only minor effects on fidelity.

Animals↗

PubMedDate not supplied

Virulence gene expression during conidial germination in Cochliobolus carbonum.

The fungal pathogen Cochliobolus carbonum race 1 produces a host-selective toxin (HC-toxin) that is responsible for increased virulence on susceptible genotypes of maize. The toxin is synthesized by a peptide synthetase, which is a product of the HTS1 gene. Because the toxin is not stored in dormant conidia, early expression of HTS1 is crucial for extensive colonization of susceptible leaf tissue. To detect the HTS1 transcript and determine the onset of HTS1 gene expression, we analyzed RNA preparations from ungerminated and germinated conidia by reverse transcription-polymerase chain reaction using oligonucleotide primers within the 15.7-kb open reading frame of HTS1. With primer pairs near both the 3'- and the 5'-termini, amplified products of the HTS1 transcript were detected in RNA prepared from dormant conidia. With all primer pairs used, the quantities of transcript increased substantially during germ tube emergence and elongation, indicating that expression of HTS1 is up regulated during spore germination. Digestion with restriction endonucleases confirmed the identity of the amplified products. Amplification of the constitutively expressed beta-tubulin transcript, which is processed to remove introns, as well as the absence of amplification products with primers spanning the HTS1 coding sequence established that cDNA was amplified and not contaminating genomic DNA.

Amino Acid Isomerases↗

PubMed1994

Characterization of a virus variant produced by L cells persistently infected with lymphocytic choriomeningitis virus.

Continuous cultivation of murine L cells infected with lymphocytic choriomeningitis virus strain Armstrong leads to production of L(Arm) cells, which produce a predominantly cell-associated attenuated variant, the L(Arm) virus. The relatively few infectious particles that are released have lost the ability to form plaques on L cells and to cause illness in mice even if inoculated intracerebrally. Based on equal protein M(r)s, antigenicity and protein kinase activity, essentially identical results were obtained for the purified Armstrong and L(Arm) viruses. There was also no difference in production and release of particles with the potential to cause homologous interference. Such particles consisted of two types, one of which was highly susceptible to u.v.-irradiation, the other was highly resistant. In the case of the L(Arm) virus interfering particles, it appears that the u.v.-irradiation-susceptible forms represented infectious virus. Purified L(Arm) virus particles contained considerable quantities of subgenomic forms of (small) S- and (large) L-RNA and their complementary counterparts, which all appeared to be replicated autonomously in an unenriched manner.

Animals↗

PubMed1976

Production of large quantities of interferon with calf testicular cells and leucocytes.

Interferon (IF) was induced in calf testicle monolayer cultures and leucocycte suspension cultures with Semliki forest virus and Newcastle disease virus to evaluate the potency of those cells for producing IF in large quantities for clinical experiments. Interferon activity was measured by an RNA-inhibition test using Semliki forest virus as challenge. To produce 10(6) units of IF, leucocytes from 67-5 litres of blood or 7-2 X 10(9) testicle cells were needed.

Animals↗

PubMed1981

Quantitative isolation of oligo- and polyadenosine-diphosphoribosylated proteins by affinity chromatography from livers of normal and dimethylnitrosamine-treated Syrian hamsters. In vivo and in vitro metabolism of the homopolymer.

Polyadenosine- and adenosine-diphosphoribosylated proteins of hamster liver were quantitatively isolated with the aid of m-aminophenyl boronic acid glutaryl hydrazide polyacrylamide affinity resin by selective adsorption at pH 8.2 and elution at pH 4.0. Polymer-free proteins, DNA, and RNA are readily separated from adenosine-diphosphoribosylated proteins. The total quantity of proteins that is covalently modified by the homopolymer is 14.3 micrograms/mg of DNA or 37.4 micrograms/g of liver in controls and 38.7 micrograms/mg of DNA or 116 micrograms/g of liver in dimethylnitrosamine-treated hamsters. Polymer content increases from 9 to 15 nmol/mg of DNA to 42 to 118 nmol/mg of DNA following treatment with dimethylnitrosamine. Pulse labeling with [14C]ribose results in a parallel doubling in dimethylnitrosamine-treated animals of the specific activities of adenosine- diphosphoribose and NAD+ and of the [14C]ribose content of polyadenosine-diphosphoribose of chain length between 20 and 40, indicating chain elongation of pre-existing larger polymers. Two groups of proteins that are isolated as polyadenosine-diphosphoribose adducts are increased significantly after treatment with dimethylnitrosamine, one minor component of a mass between 100-112 X 10(3) daltons, and a major group exhibiting a mass of 158-162 X 10(3) daltons. Polyadenosine-diphosphoribose synthetase activity of isolated hepatic nuclei is increased by 32-37% after dimethylnitrosamine treatment, and since the change in glycohydrolase activity is negligible relative to the increase in synthetase, the augmentation of polyadenosine-diphosphoribosylated proteins can be explained by the increased synthetase of nuclei. The molecular size distribution of DNA in liver nuclei of control and dimethylnitrosamine-treated hamsters is indistinguishable.

Adenosine Diphosphate Ribose↗

PubMed1997

Contributions of increased efficiency and capacity of protein synthesis to rapid cardiac growth.

Rapid cardiac growth depends upon faster synthesis than degradation of protein. The rate of protein synthesis is determined by the efficiency with which the existing components of the ribosome cycle make protein and by the quantity of the components that are present. The tissue content of RNA is taken as an index of the capacity of synthesis and efficiency is expressed as the amount of protein formed per amount of RNA over a certain time period. The efficiency of synthesis is regulated by hormones, including insulin, agents that increase cAMP, alpha-adrenergic agonists, endothelin I and angiotensin II. In addition, provision of non-carbohydrate substrates and mechanical factors such as stretch and contraction increase efficiency. Impaired energy availability as occurs in anoxic or ischemic muscle decreases efficiency. Increased phosphorylation of ribosomal protein, S6, or of the peptide chain initiation factor, elF-4E, have been suggested as mechanisms to regulate efficiency of mRNA translation. Increased efficiency of synthesis accounts for cardiac growth in the first few days following aortic banding, pulmonary artery constriction and thyroxine administration. Decreased efficiency accounts for cardiac atrophy in heterotopic transplanted hearts during the first 3 days following transplantation. The capacity of synthesis is increased by insulin, thyroid hormone, activators of protein kinase C, agents that increase cAMP, and endothelin-1. Stretch of the ventricular wall and contraction of cultured neonatal myocytes accelerates ribosome formation. An increased rate of ribosomal DNA transcription accounts for accelerated ribosome formation and depends on increased activity of a transcription factor, upstream binding factor (UBF). The activity of UBF is increased either by increased rates of synthesis or by phosphorylation of the protein. Increased capacity of synthesis is a major contributor to rapid cardiac growth in the newborn heart and after several days of pressure overload.

Animals↗