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PubMed1993

One-step RNA polymerase chain reaction for detection of hepatitis C virus RNA.

Although detection of hepatitis C virus RNA with polymerase chain reaction has become the standard for diagnosis, extensive application has been thwarted by polymerase chain reaction's labor intensiveness, risk of false-positive results through contamination and time required for individual assays. To minimize these limitations, we developed and validated a one-step hepatitis C virus RNA polymerase chain reaction assay. The one-step method was compared with traditional hepatitis C virus RNA polymerase chain reaction using primers from the highly conserved 5' untranslated region of the hepatitis C virus genome. Variables studied in the one-step method included the source and quantity of reverse transcriptase (RTase), the concentration of MgCl2 and the duration of reverse transcription and complementary DNA amplification cycles. Optimal conditions for the one-step method were obtained with 25 U of reverse transcriptase and 2 mmol/L MgCl2. The one-step method substantially reduced the time required for analysis. The sensitivity of the one-step method was comparable to that of traditional hepatitis C virus RNA polymerase chain reaction using serially diluted RNA extracted from the serum of a hepatitis C virus-infected patient. The specificity of the one-step method was confirmed on Southern-blot hybridization. The results exhibited 100% concordance with results of traditional hepatitis C virus RNA polymerase chain reaction in 50 serum samples, including those of positive and negative controls. In addition, 100% concordance was observed between the two methods' results when sera containing low levels of hepatitis C virus RNA were used.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed1982

Phosphorylation of deoxyribonucleic acid dependent RNA polymerase II by nuclear protein kinase NII: mechanism of enhanced ribonucleic acid synthesis.

RNA polymerase II was purified from Morris hepatoma 3924A by a series of ion-exchange and affinity column chromatographic fractionations, followed by sucrose gradient centrifugation in the presence of 0.3 M KC1. Purified RNA polymerase II had a specific activity of greater than 400 nmol of UMP incorporated (30 min)-1 (mg of protein)-1 by using double-stranded DNA as template. The purified enzyme contained five polypeptides (Mr 214 000, 140 000, 33 000, 25 000, and 21 000) that were present in molar quantities and two additional polypeptides (Mr 19 000 and 18 000) that had a combined molar ratio of 1.0. The cyclic AMP independent nuclear protein kinase NII, also purified from hepatoma 3924A, was able to phosphorylate RNA polymerase II polypeptides of Mr 214 000, 140 000, and 21 000. Phosphorylation of the polymerase was accompanied by enhanced transcription of double-stranded DNA, heat-denatured DNA, and poly[d-(A-T)]. The elevation in RNA polymerase activity was dependent upon the presence of hydrolyzable ATP and resulted from an increased number of RNA molecules synthesized in vitro. The average length of RNA chains was not affected by the kinase. Under similar conditions, protein kinase NII also stimulated homologous RNA polymerase I. In contrast to the phosphorylation of polymerase II, modification of polymerase I resulted in an increase in the average size, but not number, of RNA chains synthesized. The specificity of the NII kinase-catalyzed reaction was demonstrated by the inability of another homologous protein kinase, NI, to phosphorylate or activate RNA polymerase II.

Animals↗
PubMed1983

Isolation and sequence analysis of a barley alpha-amylase cDNA clone.

We have isolated a cDNA clone derived from poly(A+) RNA from barley aleurone cells stimulated with gibberellic acid. This cDNA clone contains one open reading frame coding for 438 amino acids. The cloned DNA hybridizes to a poly(A+) RNA species 1550 bases in size, the same size as the most abundant poly(A+) RNA molecules in stimulated cells. RNA complementary to this clone can be translated to make immunoprecipitable alpha-amylase in the wheat germ system and increases about 5-fold in quantity after gibberellic acid stimulation of aleurone cells. In contrast, hybridization experiments using a total cDNA probe demonstrate that the most abundant mRNA population, identical in size with our cloned sequence and presumably that for alpha-amylase, increases at least 17-fold after gibberellic acid stimulation. We therefore infer that there must be at least two populations of alpha-amylase mRNA molecules derived from separate structural genes differently influenced by gibberellic acid in aleurone cells.

Amino Acid Sequence↗
PubMedDate not supplied

Immunologic detection of protein antigens after phenol-chloroform denaturation.

The study of gene expression in cells and tissues often begins with phenol-chloroform extraction of the biologic material of interest for the isolation of intact mRNA. In most cases, the proteins denatured by phenol-chloroform are discarded. However, we found that the proteins recovered from phenol-chloroform extractions maintain their antigenicity. Therefore a method was developed for recovering the proteins from phenol-chloroform-denatured extracts that could be saved in lyophilized form until immunologic analysis. In this way, the RNA and the protein analysis can utilize exactly the same sample, and the biologic material can be saved. This is important because often these materials are available only in limited quantities. The method has been used to examine the sea urchin ets-related antigen and sea urchin ets-2 mRNA.

Animals↗
PubMed1989

Localization of Drosophila neurons that contain choline acetyltransferase messenger RNA: an in situ hybridization study.

In situ hybridization with radiolabeled complementary RNA (cRNA) probes was used to determine the location of the messenger RNA (mRNA) encoding choline acetyltransferase (ChAT) in Drosophila nervous system. Areas in the cell-rich cortical regions of the cerebrum and optic lobes hybridized with substantial concentrations of the probe. This contrasted with the cell-sparse neuropil areas where no significant concentrations of probe were observed. Although most of the cortical regions were substantially labeled, there were regions within all of the areas where labeling was sparse or nonexistent. For example in the lamina, even though the monopolar cell layer appeared to be heavily labeled, there were some neuronal profiles that were not associated with the probe. Moreover, the epithelial glia that form an arch of cell profiles subjacent to the monopolar cells were not labeled, nor were amacrine neurons in the apex of the lamina near the external optic chiasma. The highest concentration of probe (approximately 140 grains/400 microns2) was observed in the laminar monopolar cell region and the cerebral cortical rind. The next most heavily labeled region (approximately 90 grains/400 microns2) occurred over cortical cells of the medulla-lobula. In the peripheral nervous system, label over the antennal sensory neurons amounted to about 75 grains/400 microns2, and the retinular cell layer of the compound eye exhibited about 60 grains/400 microns2. The control probe did not hybridize in significant quantities in either cellular or noncellular regions. This study presents evidence that large numbers of Drosophila cortical and primary sensory neurons contain the messenger RNA necessary for the production of ChAT, the acetylcholine-synthesizing enzyme. Further, our findings provide baseline information for use in ontogenetic studies of cholinergic neurons in Drosophila, and they also provide normative data for studying the effects of mutant alleles at the Cha or Ace loci upon the transcription of ChAT messenger RNA.

Animals↗
PubMed1988

Purification of transfer RNA species by single-step ion-exchange high-performance liquid chromatography.

Anion-exchange high-performance liquid chromatography (HPLC) methods have been developed for the purification and concentration of milligram quantities of tRNA. A Waters Protein Pak DEAE 5PW 150 x 21.5 mm I.D. column was utilized for the separation of tRNA species. The chromatographic conditions chosen created non-denaturing conditions for separating the different species: 0.1 M Tris buffer (pH 7.6) at 25 degrees C, with a 0.25 M to 0.4 M sodium chloride gradient, using a 170-min gradient. The gradient form could be adjusted for optimizing purification (to over 85%) of the tRNA species of interest. The same DEAE packing in a smaller column was found to be effective for concentrating solutions of the purified tRNA. Fifty-fold concentration and recoveries above 90% have been obtained by this method. These methods were successfully applied to the purification of individual tRNA species from both Escherichia coli and yeast.

Amino Acids↗
PubMed1999

MicroSAGE: a modified procedure for serial analysis of gene expression in limited amounts of tissue.

Serial Analysis of Gene Expression (SAGE) is a powerful expression profiling method, allowing the analysis of the expression of thousands of transcripts simultaneously. A disadvantage of the method, however, is the relatively high amount of input RNA required. Consequently, SAGE cannot be used for the generation of expression profiles when RNA is limited, i.e. in small biological samples such as tissue biopsies or microdissected material. Here we describe a modification of SAGE, named microSAGE, which requires 500- to 5000-fold less starting material. Compared with SAGE, microSAGE is simplified due to incorporation of a 'single-tube' procedure for all steps from RNA isolation to tag release. Furthermore, a limited number of additional PCR cycles are performed. Using microSAGE gene expression profiles can be obtained from minute quantities of tissue such as a single hippocampal punch from a rat brain slice of 325 micrometers thickness, estimated to contain, at most, 10(5) cells. This method opens up a multitude of new possibilities for the application of SAGE, for example the characterization of expression profiles in tissue biopsies, tumor metastases or in other cases where tissue is scarce and the generation of region-specific expression profiles of complex heterogeneous tissues.

Animals↗
PubMed1973

Transcription of simian virus 40. II. Hybridization of RNA extracted from different lines of transformed cells to the separated strands of simian virus 40 DNA.

The amount of simian virus 40 (SV40) DNA present in various SV40-transformed mouse cell lines and "revertants" isolated from them was determined. The number of viral DNA copies in the different cell lines ranged from 1.35 to 8.75 copies per diploid quantity of mouse cell DNA and from 2.2 to 14 copies per cell. The revertants had the same number of viral DNA copies per diploid quantity of mouse cell DNA as their parental cell lines. (However, they showed an increased number of viral DNA copies per cell due to their increased amount of DNA.) By using separated strands of SV40 DNA, the extent of each DNA strand transcribed into stable RNA species was determined for the transformed and "revertant" cell lines. From 30 to 80% of the "early" strand and from 0 to 20% of the "late" strand was present as stable RNA species in the cell lines tested. There was no alteration in the pattern of the stable viral RNA species present in three concanavalin A-selected revertants, whereas in a fluorodeoxyuridine-selected revertant there appeared to be less viral-specific RNA present in the cells.

Animals↗
PubMed1999

Prenatal diagnosis of Lowe syndrome by OCRL1 messenger RNA analysis.

Prenatal screening of oculo-cerebro-renal syndrome of Lowe (OCRL; McKusick 309000) was performed using cultured amniocytes. Following identification of defective mRNA expression in the OCRL; gene of the proband's fibroblasts, the mRNA size and quantity of the cultured amniocytes were compared. Based on this analysis, the fetus was diagnosed as being normal and was subsequently delivered as a healthy boy. This is the first reported successful prenatal screening of OCRL using a comparison with defective mRNA of OCRL1 from affected subjects.

Adolescent↗
PubMed1998

Comparisons of Nitric Oxide Synthases in Normal Human Placenta from 37 to 41 Weeks Gestation: Qualitative and Quantitative Analyses.

> Objective: The purpose of this study was to determine the type of nitric oxide synthase isoform and to measure the quantities of nitric oxide synthase mRNA in the human placenta. Methods: The isoforms of nitric oxide synthase were determined in ten placentas of normal pregnant women using information regarding calcium dependence and inhibition by arginine analogs and findings from Western blot analyses and reverse transcriptase-polymerase chain reaction. Results: The activity of nitric oxide synthase was largely calcium dependent, although a small element of calcium-independent activity was observed. A stronger inhibition was shown with Nomega-nitro-l-arginine than with Nomega-monomethyl-l-arginine. On Western blots endothelial nitric oxide synthase was detected as a band of 140 kilodaltons, without evidence of inducible nitric oxide synthase. Messenger RNA of endothelial nitric oxide synthase was readily detected by reverse transcriptase-polymerase chain reaction, but that of inducible nitric oxide synthase was barely detected. The quantity of mRNA of inducible nitric oxide synthase was about 100-fold less than that of endothelial nitric oxide synthase. Conclusions: These results point to the constitutive endothelial type as the predominant isoform of nitric oxide synthase in human placenta from 37 to 41 weeks gestation, although protein and mRNA of inducible nitric oxide synthase may exist.

Journal Article↗
PubMed2005

Quantification of the differences between quenched and annealed averaging for RNA secondary structures.

The analytical study of a disordered system is usually difficult due to the necessity to perform a quenched average over the disorder. Thus, one may resort to the easier annealed ensemble as an approximation to the quenched system. In the study of RNA secondary structures, we explicitly quantify the deviation of this approximation from the quenched ensemble by looking at the correlations between neighboring bases. This quantified deviation then allows us to propose a constrained annealed ensemble which predicts some physical quantities much closer to the results of the quenched ensemble without becoming technically intractable.

Binding Sites↗
PubMed2005

Ultrasound-accelerated formalin fixation of tissue improves morphology, antigen and mRNA preservation.

Formalin fixation and paraffin embedding are conventional tissue preservation and processing methods used for histologic diagnosis in over 90% of cases. However, formalin fixation has three disadvantages: (1) slow fixation (16-24 h) hinders intraoperative decision making, (2) slow quenching of enzymatic activity causes RNA degradation, and (3) extensive molecule modification affects protein antigenicity. Applying high-frequency, high-intensity ultrasound to the formalin fixative cuts fixation time to 5-15 min. Fixation of various tissues such as lymph node, brain, breast, and prostate suggests that, compared to the conventional method, implementation of ultrasound retains superior and more uniform tissue morphology preservation. Less protein antigenicity is altered so that rapid immunohistochemical reactions occur with higher sensitivity and intensity, reducing the need for antigen retrieval pretreatment. Better RNA preservation results in stronger signals in in situ hybridization and longer RNA fragments extracted from fixed tissues, probably due to rapid inhibition of endogenous RNase activity. Molecules extracted from ultrasound-fixed tissues are of greater integrity and quantity compared to conventionally fixed tissues, and thus better support downstream molecular analyses. Overall, ultrasound-facilitated tissue preservation can provide rapid and improved morphological and molecular preservation to better accommodate both traditional and molecular diagnoses.

Antigens↗
PubMed1984

Differential expression of mRNAs for alpha- and beta-tubulin during differentiation of the parasitic protozoan Leishmania mexicana.

The parasitic protozoan Leishmania mexicana amazonensis has two developmental stages: a motile flagellated promastigote stage and a sessile intracellular amastigote stage. In our previous work, cells of the promastigote stage were found to synthesize more tubulin protein than those of the amastigote stage. Here, tubulin mRNAs in these leishmanias were analyzed. Based on dot blot hybridization between total leishmanial RNA and tubulin-specific cDNA probes derived from chicken brain, amastigotes and promastigotes were found to have approximately equal amounts of alpha- and beta-tubulin mRNAs. RNA blotting of leishmanial RNA, using chicken tubulin cDNA probes, showed that amastigotes and promastigotes both gave a single mRNA species of 2100 nucleotides for alpha-tubulin in roughly similar quantities. However, such analysis for beta-tubulin revealed mainly a single mRNA species of 3600 nucleotides for amastigotes and three species of 2800, 3600, and 4400 nucleotides for promastigotes, the smallest mRNA being the most predominant. Thus, regulation of gene expression appears to be different only for beta-tubulin between the two developmental stages of this protozoan.

Animals↗
PubMed1983

Effects of formaldehyde exposure on the extractability of DNA from proteins in the rat nasal mucosa.

Reaction of an homogenate of the rat nasal mucosa with formaldehyde (CH2O) followed by solubilization and extraction in a strongly denaturing aqueous-immiscible organic solvent mixture decreased the quantity of nucleic acids that remained in aqueous solution in comparison with a control homogenate untreated with CH2O. The absent DNA and RNA were located in the interface between the aqueous and organic phases, from which they could be recovered only after enzymatic proteolysis. It is concluded that interfacial nucleic acids were cross-linked to proteins by CH2O. The concentration of cross-links was estimated with 14CH2O; under conditions that rendered 48% of the DNA nonextractable from proteins, there was less than one cross-link per 28,000 nucleotide residues. Exposure of rats to airborne CH2O at concentrations of 0, 2, 6, 15, and 30 ppm (6 hr/day for 2 days) resulted in a statistically significant increase in the percentage of the total DNA from the respiratory mucosa that was located in the interface at concentrations of 6 ppm and higher. However, the percentage of DNA from the olfactory mucosa located in the interface was not increased by CH2O exposure. Analysis of nasal mucosal DNA by ultracentrifugation in CsCl density gradients provided no evidence of a change in the buoyant density of the DNA caused by reaction with CH2O. The results indicate that CH2O may induce DNA-protein cross-links in the respiratory but not in the olfactory mucosa at concentrations equal to or greater than 6 ppm.

Animals↗
PubMed1998

Cyclic AMP regulation of G(i alpha2) and G(i alpha3) mRNAs and proteins in astroglial cells.

Culture of rat astrocytes in the presence of 10 microM forskolin, isoproterenol, a nonselective beta-adrenergic agonist, or 8-bromo-cyclic AMP increased transiently in a time-dependent manner the levels of G(i alpha2) and G(i alpha3) mRNAs as measured by northern blot analysis. G(i alpha1) mRNA was not expressed in these cells. After 6-9 h of culture with forskolin or isoproterenol the amounts of G(i alpha2) and G(i alpha3) mRNAs were maximal (150-300% over control). In cells challenged with 8-bromo-cyclic AMP, the level of G(i alpha2) mRNA level was maximal after 1 h of culture, while the maximal for G(i alpha3) mRNA was reached after 6 h of culture. For prolonged exposure times (24 h) to these agents the levels of G(i alpha2) and G(i alpha3) mRNAs decreased to the values observed in control cells. The forskolin-induced increase in G(i alpha2) protein expression measured by western blot analysis was similar to the increase in G(i alpha2) mRNA amount. In contrast, forskolin induced only a modest increase in the quantity of G(i alpha3) protein (150% over control) despite a large increase of G(i alpha3) mRNA content. Transcription rates and RNA stability were measured simultaneously after isolation of newly synthesized mRNA was performed. The half-lives of G(i alpha) mRNAs were approximately 0.7 and 3 h for G(i alpha2) and G(i alpha3), respectively. Culturing astrocytes in the presence of forskolin resulted in an increase in the half-lives of G(i alpha2) mRNA and G(i alpha3) mRNA by five- and twofold, respectively. The relative transcription rate of the G(i alpha3) gene was slightly increased by approximately 1.3-1.5-fold in forskolin-treated cells but not that of G(i alpha2) gene. These results suggest that cyclic AMP enhanced G(i alpha2) and G(i alpha3) mRNA expression by both transcriptional and posttranscriptional mechanisms, with an increase of G(i alpha) mRNA stability as one of the major checkpoints.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1977

State of the viral DNA in rat cells transformed by polyma virus. II. Identification of the cells containing nonintegrated viral DNA and the effect of viral mutations.

F2408 rat cells transformed by polyoma virus contained integrated and nonintegrated viral DNA. The presence of nonintegrated viral DNA is under control of the A early viral function. Polyoma ts-a-transformed rat cells lose the free viral DNA when growth at the nonpermissive temperature (40 degrees C), but they reexpress it 1 to 3 days after they are shifted back to the permissive temperature. In contrast, rat cells transformed by a late viral mutant, ts-8, contain free viral DNA at both permissive and nonpermissive temperatures. Treatment of the transformed rat cells with mitomycin C produces a large increase in the quantity of free viral DNA and some production of infectious virus. Experiments of in situ hybridization, with 3H-labeled polyoma complementary RNA as a probe, show that only a minority (approximately 0.1%) of the transformed cells contain nonintegrated viral DNA at any given time. These results suggest that the presence of free viral DNA in polyoma-transformed rat cells is caused by a spontaneous induction of viral DNA replication, occurring with low but constant probability in the transformed cell population, and that the free viral DNA molecules originate from the integrated ones, probably through a phenomenon of excision and limited replication.

Animals↗
PubMed2002

X chromosomes of American marsupials contain minimal amounts of euchromatin.

The karyotypes of four South American didelphid marsupials, representing diploid numbers of 2n = 14 and 18, have been analyzed by a variety of banding techniques. The 2n = 14 karyotypes display a high degree of homoeology, but there also exist distinct similarities between the 2n = 14 and 2n = 18 karyotypes. The interspecific differences found are due to centric fissions, pericentric inversions, and variations in the amount and composition of the constitutive heterochromatin. Contrary to the evolutionary conservation of the banding patterns in all autosomal arms, there are multiple differences in the number and chromosomal location of the nucleolus organizer regions. In species with X-linked nucleolus organizers, the 18S + 28S ribosomal RNA genes escape inactivation in female cells. Measurements on the X chromosomes of Marmosa fuscata and Micoureus demerarae unexpectedly reveal the lowest quantities of euchromatin so far determined in the X chromosomes of mammals: 1.5% and 1.8%, respectively, of their haploid female genomes. This is significantly less than the amount of euchromatin in the basic X chromosomes of other marsupials (3%) or eutherians (5%).

Animals↗