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At least 1,135 records · Page 63Linked to original sources
PubMed1978

The isolation of recombinants between related orbiviruses.

Temperature-sensitive mutants of the related orbiviruses, Wallal and Mudjinbarry, recombine with high frequency when grown in pairs in cell culture. The genome of each virus consists of discrete segments of double-stranded RNA and high frequency recombination suggests that reassortment of genome segments occurs rather than classical recombination. Electrophoresis in acrylamide gels of RNA extracted from the progeny of a cross between Wallal ts 101 and Mudjinbarry ts 3 mutants, revealed that three plaque isolates of 60 tested differed in RNA pattern from each of the parent viruses and from each other. Further analysis of the electrophoretic profiles suggested that the isolates were recombinants with RNA segments derived from each of the parent viruses.

Arboviruses↗
PubMed1996

Direct 5S rRNA Assay for Monitoring Mixed-Culture Bioprocesses.

This study demonstrates the efficacy of a direct 5S rRNA assay for the characterization of mixed microbial populations by using as an example the bacteria associated with acidic mining environments. The direct 5S rRNA assay described herein represents a nonselective, direct molecular method for monitoring and characterizing the predominant, metabolically active members of a microbial population. The foundation of the assay is high-resolution denaturing gradient gel electrophoresis (DGGE), which is used to separate 5S rRNA species extracted from collected biomass. Separation is based on the unique migration behavior of each 5S rRNA species during electrophoresis in denaturing gradient gels. With mixtures of RNA extracted from laboratory cultures, the upper practical limit for detection in the current experimental system has been estimated to be greater than 15 different species. With this method, the resolution was demonstrated to be effective at least to the species level. The strength of this approach was demonstrated by the ability to discriminate between Thiobacillus ferrooxidans ATCC 19859 and Thiobacillus thiooxidans ATCC 8085, two very closely related species. Migration patterns for the 5S rRNA from members of the genus Thiobacillus were readily distinguishable from those of the genera Acidiphilium and Leptospirillum. In conclusion, the 5S rRNA assay represents a powerful method by which the structure of a microbial population within acidic environments can be assessed.

Journal Article↗
PubMed2005

Involvement of a short interspersed element in epigenetic transcriptional silencing of the amoebapore gene in Entamoeba histolytica.

Transcriptional silencing of an amoebapore (ap-a) gene occurred in Entamoeba histolytica following the transfection of plasmids containing a DNA segment (473 bp) homologous to the 5' upstream region of the gene (R. Bracha, Y. Nuchamowitz, and D. Mirelman, Eukaryot. Cell 2:295-305, 2003). This segment contains the promoter region of the ap-a gene, a T-rich stretch, followed by a truncated SINE1 (short interspersed element 1) that is transcribed from the antisense strand. Transfection of plasmids containing truncated SINE1 sequences which lack their 3' regulatory elements upstream of the ap-a gene was essential for the downstream silencing of the ap-a gene while transfection with plasmids containing the entire SINE1 sequence or without the T-rich stretch promoted the overexpression of the ap-a gene. Both the T-rich stretch and sequences of the 5' SINE1 were essential for the transcription of SINE1. RNA extracts from gene-silenced cultures showed small amounts of short (approximately 140-nucleotide), single-stranded molecules with homology to SINE1 but no short interfering RNA. Chromatin immunoprecipitation analysis with an antibody against methylated K4 of histone H3 showed a demethylation of K4 at the domain of the ap-a gene, indicating transcriptional inactivation. These results suggest the involvement of SINE1 in triggering the gene silencing and the role of histone modification in its epigenetic maintenance.

Animals↗
PubMed1978

Isolation and characterization of a virus-specific ribonucleoprotein complex from reticuloendotheliosis virus-transformed chicken bone marrow cells.

Chicken bone marrow cells transformed by reticuloendotheliosis virus (REV) produce in the cytoplasm a ribonucleoprotein (RNP) complex which has a sedimentation value of approximately 80 to 100S and a density of 1.23 g/cm3. This RNP complex is not derived from the mature virion. An endogenous RNA-directed DNA polymerase activity is associated with the RNP complex. The enzyme activity was completely neutralized by anti-REV DNA polymerase antibody but not by anti-avian myeloblastosis virus DNA polymerase antibody. The DNA product from the endogenous RNA-directed DNA polymerase reaction of the RNP complex hybridized to REV RNA but not to avian leukosis virus RNA. The RNA extracted from the RNP hybridized only to REV-specific complementary DNA synthesized from an endogenous DNA polymerase reaction of purified REV. The size of the RNA in the RNP is 30 to 35S, which represents the subunit size of the genomic RNA. No 60S mature genomic RNA was found within the RNP complex. The significance of finding the endogenous DNA polymerase activity in the viral RNP in infected cells and the maturation process of 60S virion RNA of REV are discussed.

Animals↗
PubMed2002

Gene expression profile of mouse myocardium with transgenic overexpression of A1 adenosine receptors.

Transgenic mice with cardiac-specific overexpression of adenosine A(1) receptors (A(1)AR) have demonstrated metabolic and functional tolerance to myocardial ischemia. We utilized cDNA microarrays to test the hypothesis that the cardioprotective mechanism(s) of A(1) overexpression involves altered gene expression. Total RNA extracted from the left ventricles from A(1) transgenic (n = 4) and wild-type (n = 6) mice was hybridized to Affymetrix mgU74A chips. Comparison of RNA expression levels in transgenic to wild-type myocardium revealed approximately 636 known genes with expression significantly altered by greater than 25%. We observed increased expressions of genes including NADH dehydrogenase, the GLUT4 glucose transporter, Na-K-ATPase, sarcolemmal K(ATP) channels, and Bcl-xl in A(1)AR-overexpressing hearts. We also observed decreased expression of pro-apoptotic genes including a 50% reduction in message level of caspase-8. Protein expression of GLUT4 and caspase-8 was also altered comparable to the differences in gene expression. These data illustrate genes with chronically altered patterns of expression in A(1) transgenic mouse myocardium that may be related to adenosine receptor overexpression-mediated cardioprotection.

Adenosine Triphosphate↗
PubMed2006

Isolation of RNA from tumor samples: single-step guanidinium acid-phenol method.

The guanidinium acid-phenol method of RNA extraction is relatively fast (4 h) and is useful for the processing of large numbers of samples, without the need for ultracentrifugation. This protocol produces total RNA that includes ribosomal, transfer, and messenger RNA. This high-quality RNA is suitable for Northern blot analysis, dot-blot hybridization, poly (A) RNA selection, in vitro translation, cDNA library construction, reverse transcriptase-polymerase chain reaction, ribonuclease protection assay, and primer extension experiments.

Guanidine↗
PubMed1982

Participation of poly(ADP-ribosyl)ation in the depression of RNA synthesis caused by treatment of mouse lymphoma cells with methylnitrosourea.

When mouse lymphoma cells (L-1210) are treated with methylnitrosourea, a DNA-damaging agent, polyadenosine diphosphoribose (poly(ADP-ribose)) synthetase activity increases 5-8-fold in 2-3 h, while RNA polymerase activity remains constant for an initial 2 h and then gradually decreases to 25-30% of the control level in 5 h. Both alpha-amanitin-sensitive and -resistant RNA polymerase activities are depressed to the same degree by the treatment with methylnitrosourea. The depression in RNA synthesis is virtually prevented when the treated cells are cultured in the presence of 3-aminobenzamide, a specific inhibitor of poly(ADP-ribose) synthetase. Analyses of the RNA extracted from the cells labeled with [3H]uridine by agarose gel electrophoresis and by poly(U)-Sepharose column chromatography show that the contents of ribosomal precursor RNA and poly(A)-containing RNA are both low in the methylnitrosourea-treated cells as compared with those in the untreated cells and that the reduction in the contents of these kinds of RNA is almost completely prevented by the addition of 3-aminobenzamide to the culture medium. These results suggest that the enhancement of poly(ADP-ribosyl)ation causes the decrease in both synthesis of ribosomal RNA and messenger RNA.

Animals↗
PubMed1998

Detection of picobirnavirus in HIV-infected patients with diarrhea in Argentina.

Diarrhea due to enteric pathogens is an important complication of advanced HIV infection. Picobirnaviruses are agents recently linked with human enteritis. In total, 197 fecal samples collected from HIV-infected and noninfected patients with and without diarrhea were investigated for the presence of rotavirus and picobirnavirus by polyacrylamide gel electrophoresis. Picobirnavirus was detected in 8.8% of 57 HIV-infected patients with diarrhea, but it was detected in neither those without diarrhea (p<.018) nor in the group of subjects uninfected with HIV (p<.022). All genomic electropherotypes of picobirnavirus strains had a wide pattern. Picobirnavirus genome segments varied in size between 2.4 and 2.7 and 1.6 and 1.9 kbp for the slow and fast migrating bands, respectively. Rotaviruses were not detected in any of the clinical groups studied. Two methods for the extraction of nucleic acid-phenol/chloroform and guanidinium thiocynate (GTC)/silica-were compared. Detection of picobirnavirus by polyacrylamide gel electrophoresis was 2.5 times more sensitive following guanidinium thiocynate RNA extraction. This investigation offers preliminary results about the circulation of picobirnavirus in HIV-infected patients in Córdoba, Argentina.

Adult↗
PubMed2005

Combining laser capture microdissection with quantitative real-time PCR: effects of tissue manipulation on RNA quality and gene expression.

Laser capture microdissection (LCM) is increasingly being used in quantitative gene expression studies of the nervous system. The current study aimed at determining the impact of various tissue manipulations on the integrity of extracted RNA in LCM studies. Our data indicate that various tissue preparation strategies prior to microdissection may decrease RNA quality by as much as 25%, thus affecting expression profiles of some genes. To circumvent this problem, we developed a strategy for reverse transcriptase real-time PCR that has considerable sensitivity and can be used to calculate relative changes in gene expression. This approach was validated in subregions of the rat cerebellum. Accordingly, expression of glial gene markers - myelin-associated glycoprotein and proteolipid protein 1 - was found 70-160-fold higher in the white matter layer of the cerebellar cortex as compared to the neuron-enriched granular layer. In contrast, expression of a specific neuronal maker, neuron-specific enolase, was found seven-fold higher in the granular layer, as compared to the white matter layer. Furthermore, this approach had high sensitivity and specificity as we were able to detect a 38% decrease in the expression of neuron-specific enolase without a change in the expression of glial markers following administration of the neurotoxin, ibotenic acid. These results demonstrate feasibility of performing accurate semi-quantitative gene expression analyses in LCM samples.

Animals↗
PubMed1991

Latency-associated transcripts in corneas and ganglia of HSV-1 infected rabbits.

Herpes simplex virus (HSV) establishes latent infection in the sensory neuron and possibly in non-neuronal tissue, particularly the cornea. During latency only one region of the HSV genome is transcribed, producing RNAs known as latency associated transcripts (LAT). The gene for LAT overlaps with the HSV gene for the protein ICPO in the downstream regions of both genes. Latency can occur in the absence of LAT. This study reports the detection of ICPO/LAT and thymidine kinase (TK) gene fragments by the polymerase chain reaction in DNA extracted from the corneas and trigeminal ganglia of latently infected rabbits. Both genes were detected in four of four trigeminal ganglia tested and in three of five corneas tested. More importantly, this study reports the first detection of LAT in RNA extracted from 9% of corneas from latently infected rabbits (n = 22) by the polymerase chain reaction. LAT was detected in RNA from 100% of the corresponding trigeminal ganglia (n = 22). Although LAT is not essential for latency, it remains the only known molecular marker for latent HSV infections. Detection of LAT in these rabbit corneas suggests that HSV latency may occur in this non-neuronal tissue and that reactivation from non-neuronal tissue may occur at a low frequency in animals in which HSV latency has been established.

Animals↗
PubMed1985

Alpha 1-antitrypsin and serum albumin mRNA accumulation in normal, acute phase and ZZ human liver.

Alpha 1-Antitrypsin and albumin mRNA levels of 4 human livers were assessed using a newly sequenced cDNA clone of the carboxyterminal third of alpha 1-antitrypsin and a previously cloned albumin cDNA sequence. The relative concentration of alpha 1-antitrypsin mRNA was the same in poly(A)-containing RNA isolated from acute phase (MM) and alpha1-antitrypsin deficient (ZZ) individuals. In the acute phase liver relative to the normal (MM) liver, total RNA extracts showed a marked decrease in albumin mRNA concentration but no increase in alpha 1-antitrypsin mRNA. The ZZ liver showed decreased total and poly(A)-containing RNA content but the same proportion of alpha 1-antitrypsin to albumin mRNA as in the normal (MM) liver. This supports other evidence that ZZ alpha 1-antitrypsin deficiency is due to a defect in polypeptide processing (secretion) rather than a deficiency in mRNA accumulation.

Adolescent↗
PubMed1988

Taurine and beta-alanine act on both GABA and glycine receptors in Xenopus oocyte injected with mouse brain messenger RNA.

The responding pathway (process from agonist binding to channel opening) of taurine and beta-alanine was investigated in Xenopus oocytes injected with mouse brain poly(A)+ RNA. Responses to gamma-aminobutyric acid (GABA), glycine, taurine and beta-alanine were induced in oocytes injected with poly(A)+ RNA extracted from 3 regions, cerebrum, cerebellum and brainstem of the mouse brain. From comparison, responses to these 4 inhibitory amino acids in each regional poly(A)+ RNA-injected oocytes were categorized into at least 3 groups: (1) GABA, (2) glycine, and (3) taurine and beta-alanine. No cross-desensitization was observed between GABA response and glycine response, but taurine and beta-alanine responses cross-desensitized both the GABA and glycine responses. Taurine and beta-alanine responses were partially inhibited by the GABA antagonist, bicuculline, and also by the glycine antagonist, strychnine. The results suggest that the taurine or the beta-alanine response in the brain is caused through both the GABA receptor and the glycine receptor.

Alanine↗
PubMed2005

Combined application of RT-PCR and immunohistochemistry on paraffin embedded sentinel lymph nodes of prostate cancer patients.

The detection of tumor cells in the sentinel lymph node (SLN) is of great importance for the prognosis of cancer patients. At present, immunohistochemistry and RT-PCR for tumor marker expression are the most sensitive techniques available for this analysis. However, so far, most RT-PCR-based analyses of SLNs have been performed on fresh material, excluding a direct comparison with the (immuno)histologic results. In our view, this does not entirely aid routine diagnosis. We established an efficient method for RNA extraction and RT-PCR from paraffin sections of SLNs from prostate cancer patients and compared the results with the (immuno)histologic data of adjacent sections. Amplifiable RNA was obtained from 133 SLNs of 68 prostate cancer patients. Correlation of PSA-specific RT-PCR with (immuno)histologic findings showed a positive and negative predictive value of 83% and 100%, respectively, for the prostate cancer patients investigated. Four of 12 patients with biochemical relapse, but without (immuno)histologically detectable tumor cells were RT-PCR-positive for PSA. We found that single sections of paraffin-embedded SLNs are suitable for routinely performed RT-PCR. Combined with (immuno)histology, PSA-specific RT-PCR is a revealing supplementary technique for the detection of tumor cells in SLNs of prostate cancer patients.

Adenocarcinoma↗
PubMed1975

Ribonucleic acid synthesis in Plasmodium knowlesi maintained both in vivo and in vitro.

RNA extracted from purified parasites of Plasmodium knowlesi was fractionated using agarose gel electrophoresis. Preparations from para sites grown both in vivo and in vitro contained species of RNA with sedimentation coefficients of 4-0S, 5-0S, 16-6S, 24-2S, 31-4S, 38-0S and 48-3S. There was less RNA present in parasites grown in vitro than the equivalent stage parasites grown in vivo but the proportional amounts of the various species of RNA was similar in both cases. It is suggested that the 24-2S and 16-6S species of RNA are ribosomal and that the high molecular weight 31-4S, 38-0S and 48-0S species are ribosomal precursors. Ribosomal RNA synthesis occurs throughout the cell cycle during growth from the ring to the schizont stage; maximum incorporation of [H3]-adenosine occurs at the late trophozoite stage before nuclear division.

Animals↗
PubMed1981

Effects of (hydroxymethyl)trimethylpsoralen on structure and function of bacteriophage MS2 ribonucleic acid.

Treatment of bacteriophage MS2 with 4'-(hydroxymethyl)-4,5',8-trimethylpsoralen and 360-nm light caused a dose-dependent decline in the infectivity of the virus. Covalent photobinding of a single psoralen molecule on the phage genome was a lethal event. Ribonucleic acid (RNA) extracted from psoralen and light-treated virus had a dose-dependent 385-nm fluorescence emission but was unaltered in its physical properties compared to control RNA samples. Phage adsorption and penetration in Escherichia coli host cells were unaffected, but in vivo replication of the treated virus was affected to the same extent as infectivity. The cell-free translational activity of the MS2 RNA was also severely reduced after psoralen and light treatment of the phage. Examination of the in vitro translation products revealed that the synthesis of the viral replicase protein was most substantially affected. Psoralen treatment of purified, protein-free MS2 RNA promoted an even greater reduction in cell-free synthesis of all viral proteins. This difference in translational function was consistent with the observation that virion-free RNA bound approximately 4 times as much psoralen as did RNA treated within the phage capsid. It was concluded that the replicase gene is the most sensitive region of the viral RNA molecule for psoralen binding.

Coliphages↗
PubMed1977

Effects of estradiol on in vitro nucleoside incorporation and methylation of rat uterine ribonucleic acid.

The in vitro nucleoside incorporation and methylation of uterine RNA was examined by incubating immature rat uteri with either 3H-uridine, 3H-uridine-3H-cytidine, and/or 14C-methyl-methionine. Radioactivity was determined in extracted RNA which was fractionated by gel electrophoresis or by differential solubility in zine acetate or lithium chloride. Estradiol (E2)-treated uteri respond by showing a maximum in vitro incorporation of nucleosides into total RNA and high M.W. RNA within one hr of in vivo treatment. Nucleoside incorporation into low M.W. RNA increased gradually throughout the first 3 hrs of a 4-hr hormone treatment period. E2 in vivo also caused increased in vitro methylation of total, high, and low M.W. uterine RNA fractions. Methylation of all fractions increased through the first 3 hrs of hormone treatment, but greater methylation of low M.W. RNA was apparent. The data indicate that in vivo E2 treatment stimulates an initial transitory stage of in vitro nucleoside incorporation into uterine RNA followed by a more prolonged interval of RNA methylation.

Animals↗
PubMed1981

Processing of naked 45-S ribosomal RNA precursor in vitro by an RNA-associated endoribonuclease.

A processing endoribonuclease was isolated from the cytoplasm of chick embryos. The enzyme was easily obtained using an RNA extraction procedure based on a mild deproteinization with Sarkosyl and cold phenol/chloroform. This technique assured the recovery of several proteins and the endoribonuclease in association with the RNA. It was demonstrated that this endoribonuclease was capable of promoting, in vitro, a precise processing of naked 45-S ribosomal RNA precursor to molecules resembling the intermediates as well as the 28-S and 18-S cytoplasmic RNAs found in vivo. The presence of magnesium ions was required for the correct processing function of the enzyme. In addition, under the same conditions, the mature ribosomal RNA substrates were degraded at a slower rate by this RNA-associated RNase. It was possible to fractionate the enzymatic preparation into two different populations by means of a sucrose gradient: one associated and the other partially free of an RNA component. The effect of the intrinsic RNA associated with the endoribonuclease on the enzymatic activity was tested by analyzing both the enzymatic populations and the total enzymatic preparation treated with pronase or with immobilized pancreatic RNase. In all cases in which the RNA component was present, the enzyme showed processing activity. On the other hand, when the RNA component was absent or at least partially degraded the enzyme proved to be more active in processing precursor molecules and in promoting extensive degradation of mature RNA species. Although the presence of RNA in association with the enzyme was demonstrated, its role in the regulation of the enzymatic activity is yet not clear.

Animals↗
PubMed1968

Replication of Western equine encephalomyelitis virus. II. Cytoplasmic structure involved in the synthesis and development of the virions.

Analysis of the cytoplasmic fraction of chick embryo cells during the exponential phase of Western equine encephalomyelitis (WEE) virus growth showed that the viral ribonucleic acid (RNA) labeled by a short pulse with (3)H-uridine was associated with a structure which sedimented in sucrose density gradients with a coefficient of 65S. The RNA extracted from this structure sedimented in sucrose density gradients at 26S. After a longer period of exposure to (3)H-uridine, the radio-active viral RNA was associated with a structure which sedimented in sucrose density gradients as would materials with coefficients of about 140S. The 140S structure contained viral RNA and viral protein. It was shown that the 140S structures are not virus-induced polysomes. The 140S structure contained predominantly the 40S type of viral RNA and some 26S type. Electrophoretic analysis of the disrupted virion revealed that at least two proteins (types I and II) were present in the purified virion. Only type II protein was present in the 140S structure. Unlike the virion, the 140S structure did not contain any lipid which could be detected by the incorporation of (14)C-choline. These data suggest that the 140S structure represents the internal nucleoprotein part of the virion. The rate of appearance of labeled virus lags behind that of the formation of the 140S structure in infected cells. Pulse-chase experiments with (3)H-leucine suggest that the 140S structure may represent a precursor to the virus particle. The results are discussed in terms of the maturation of WEE virus in the infected cells.

Acrylates↗