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PubMedDate not supplied

[Rapid detection of Enterovirus by a direct method of polymerase chain reaction].

For the detection of Enterovirus, we devised a direct economical method of polymerase chain reaction which does not require a previous extraction of ribonucleic acid and uses infected cell culture supernatants. The system was developed on the basis of universal primers of Enterovirus and specific primers of vaccinal strain Sabin 1. The achieved results proved that the non-existence of methods of RNA extraction and purification prior to the reaction does not affect the susceptibility and specificity of the system, in the rapid detection of Enterovirus genomes and identification of vaccinal strains of poliovirus.

Base Sequence↗
PubMed1985

Transfection of mouse fibroblast cells with a promoterless herpes simplex virus thymidine kinase gene: number of integrated gene copies and structure of single and amplified gene sequences.

Plasmids carrying the herpes simplex virus thymidine kinase (tk) gene were used to transfect thymidine kinase-deficient cells of the mouse fibroblast cell line LM(tk-). Individual cell clones were cultivated in selective hypoxanthine-aminopterin-thymidine medium to determine the number of integrated plasmid copies which was almost always in the range of one to three copies per genome. In contrast, cells transfected with plasmids carrying a promoterless "truncated" tk gene typically contained between 10 and 25 copies per genome. Surprisingly, when the truncated tk gene was transfected together with a simian virus 40 DNA segment, including its transcriptional enhancer, the number of integrated tk gene copies was always low, between one and three copies per genome. We have analyzed the genomic organization of integrated truncated tk genes by blot hybridization of restricted cellular DNA and concluded that integrated units of plasmid DNA molecules are arranged in tandem arrays which remain stable in most cases for many cell generations. In only 1 of ca. 20 cell clones did we observe a retraction and expansion of the number of integrated promoterless tk genes as a response to the removal or readdition of selective pressure. Surprisingly, the thymidine kinase activity determined in extracts from cells growing in selective hypoxanthine-aminopterin-thymidine medium (high numbers of integrated tk gene copies) was nearly the same as the enzymatic activity in cells growing in nonselective medium (low copy numbers). Moreover, Northern blots of polyadenylated RNA, extracted from cells growing under selective and nonselective conditions, showed that, in both cases, the major species of tk-specific transcripts was ca. 1.5 kilobases in size, as expected for a tk-specific mRNA containing the entire coding region of the gene. Thus, disproportionate DNA replication appeared not to be essential for an active tk gene expression in these cells. We discuss possible pathways leading to the formation of tandem arrays of integrated truncated tk genes and the conditions required for disproportionate DNA replication in the unique case in which we found a retraction and expansion of tk gene copy numbers as a response to selective growth conditions.

Animals↗
PubMed1988

Synthesis of insulin-like growth factor II (IGF-II) in fetal rat tissues: translation of IGF-II ribonucleic acid and processing of pre-pro-IGF-II.

The single insulin-like growth factor II (IGF-II) gene is transcribed into multiple RNA species in most fetal and neonatal rat tissues. For IGF-II to serve as a local growth factor in fetal tissues, IGF-II RNA must be translated into pre-pro-rat (r) IGF-II, and the biosynthetic precursor processed to smaller biologically active forms. IGF-II RNA extracted from fetal rat liver, muscle, intestine, lung, and stomach, from rat placenta, and from fetal or neonatal mouse liver and lung directed the synthesis of 22,000 mol wt pre-pro-IGF-II in a reticulocyte lysate cell-free translation system. A biosynthetic precursor of this size had been observed previously in translation of RNA from BRL-3A rat liver cells and is predicted by the nucleotide sequence of cDNA clones encoding rIGF-II. Consistent with the developmental pattern of expression of IGF-II RNA observed in hybridization studies, RNA from adult rat liver, muscle, and intestine did not direct the synthesis of pre-pro-rIGF-II. To determine whether the IGF-II biosynthetic precursor was processed to smaller biologically active IGF-II, term fetal rat tissues were extracted with acid-ethanol, the extracts were fractionated by acid gel filtration, and the IGF pools were examined in a RIA specific for IGF-II. Levels of 1-2 micrograms/g were observed in liver, limb, lung, intestine, and brain; lower levels were observed in heart and kidney. In general, the levels of immunoreactive IGF-II corresponded to the levels of IGF-II mRNA. These results suggest that IGF-II mRNA is translated, and pre-pro-IGF-II processed to mature IGF-II in different fetal rat tissues. In contrast to IGF-I, in which alternative RNA splicing generates possible precursor molecules containing different COOH-terminal propeptide segments, we find no evidence for an IGF-II precursor in rat tissues other than 22,000 mol wt pre-pro-rIGF-II.

Animals↗
PubMed1999

Comparative study of a modified competitive RT-PCR and Amplicor HCV monitor assays for quantitation of hepatitis C virus RNA in serum.

A modified competitive RT-PCR (mcRT-PCR) to measure HCV RNA in serum and the Amplicor HCV Monitor assay were compared. For mcRT-PCR, the RNA extracted was retrotranscribed and coamplified in one step with a known amount of a DNA internal control (IC). Digoxigenin-labeled amplified products were hybridized to specific HCV DNA and IC-DNA probes and quantified by colorimetry. HCV RNA concentration was calculated by plotting the ratio of HCV/IC ODs against a calibration curve. Multiple samples were analyzed in the same round and tedious titration of each sample with a competitor was unnecessary. The mcRT-PCR assay was linear from 6 x 10(3) to 6 x 10(7) copies/ml, whereas Amplicor was linear up to 1-2 x 10(6) copies/ml. HCV RNA was measured in samples from 75 carriers. There was agreement between both methods in type 1 infections but not in type 2 or type 3 infections, in which the values measured by Amplicor were, on average, 15 times lower than those measured by the mcRT-PCR. HCV RNA measured by Amplicor was higher in type 1 infections than in type 2 or 3 infections, but no differences were found when viral load was assessed by mcRT-PCR. The binding efficiency of the Amplicor-probe was greater for type 1 than for types 2 or 3, suggesting Amplicor underestimates the viral load in the latter types. In contrast, the mcRT-PCR is not affected by genotype-related variation of HCV. This study suggests that mcRT-PCR assay is reliable for sensitive and accurate measurement of HCV RNA over a broad range of values independently of the HCV genotype.

Genotype↗
PubMed1994

Application of capillary electrophoresis to the post-polymerase chain reaction analysis of rat mRNA for gastric H+,K(+)-ATPase.

We have developed a competitive quantitative RNA/polymerase chain reaction (PCR) method using capillary electrophoresis in the post-PCR analysis for the quantitation of rat gastric H+,K(+)-ATPase mRNA. Analysis with CE allows quick and direct separation, evaluation, and characterization of DNA fragments similar in size, and it offers a convenient way of automatizing the post-PCR analysis. To estimate the magnitudes of different error contributions affecting the accuracy and reproducibility of the results, an analysis of variance was performed using the data obtained from quantitating mRNA levels in 10 different total RNA extracts from the Corpus region of Sprague-Dawley rats. The result showed that the reproducibility of the method of analysis was sufficient for the determination of H+,K(+)-ATPase mRNA levels among animals even when small fluctuations in RNA synthesis are to be measured. A comparison was also made of the mRNA levels of the beta- and alpha- subunits of H+,K(+)-ATPase, and the results were found to be in the same level.

Animals↗
PubMed1993

Glucose-dependent insulinotropic peptide: structure of the precursor and tissue-specific expression in rat.

Glucose-dependent insulinotropic peptide (GIP) is a 42-amino acid gastrointestinal regulatory peptide that stimulates insulin secretion from pancreatic beta cells in the presence of glucose. Approximately 7.8 x 10(5) recombinant clones of a neonatal rat intestinal cDNA library were screened by using plaque hybridization, and three clones were identified and sequenced with the dideoxynucleotide chain-termination method. The translated amino acid sequence deduced from the nucleotide sequence of the cDNA indicated that rat GIP was derived by proteolytic processing of a 144-amino acid precursor polypeptide. The mature peptide is flanked by a 43-amino acid NH2-terminal peptide that contains a 21-amino acid signal peptide and by a 59-amino acid COOH-terminal peptide. Analysis of the nucleotide and amino acid sequence of rat GIP revealed only two substitutions from the known human GIP peptide. The use of high-stringency RNA blot-hybridization analysis of total RNA extracted from various organs demonstrated expression of the GIP gene in the duodenum and jejunum and, to a lesser extent, in the ileum. In addition, expression of the GIP gene was observed in the submandibular salivary gland both by RNA analysis and RIA. In response to duodenal perfusion of a 20% Lipomul meal for 60 min, duodenal mucosal GIP mRNA concentrations increased by 42.8% and 48.2% at 30 and 60 min, respectively.

Amino Acid Sequence↗
PubMed1990

Diabetes-induced changes in guanine-nucleotide-regulatory-protein mRNA detected using synthetic oligonucleotide probes.

Synthetic oligonucleotide probes were designed to detect the alpha-subunits of the guanine-nucleotide-regulatory proteins (G-proteins) Gi-1, Gi-2, Gi-3 and Gs (Gi is inhibitory and Gs is stimulatory). Each probe detected a single major mRNA species in Northern blots of RNA extracted from a variety of tissues. A probe was designed to identify the two forms of G-protein beta-subunits, beta 1 and beta 2. This probe hybridised with a single 1.8-kb transcript (beta 2) in RNA from all tissues studied except for brain, where a less-abundant 3.4-kb transcript (beta 1) was also detected. These probes were used to assess whether the induction of diabetes, using streptozotocin, altered the levels of mRNA coding for specific G-protein components. In hepatocytes, diabetes caused a significant reduction in the number of transcripts coding for alpha-Gs, alpha-Gi-2 and alpha-Gi-3; mRNA for alpha-Gi-1 was undectable. In adipocytes, diabetes increased dramatically the mRNA coding for alpha-Gi-1 and alpha-Gi-3, whilst no significant changes occurred in the fractions coding for alpha-Gi-2 and alpha-Gs. No significant changes in the mRNA coding for G-protein alpha-subunits were observed in either brain, heart, skeletal muscle or kidney. Diabetes did not cause any significant changes in the mRNA coding for beta 2 in any tissue or cell population studied. Such results on the relative levels of mRNA encoding G-protein components was obtained by comparing equal amounts of total RNA from tissues of control and diabetic animals. G-protein mRNA levels were expressed relative to ribosomal 28S RNA levels and, in some instances, relative to transcripts for a structural protein called CHO-B. The total cellular levels of both RNA and DNA were assessed in the various tissues and cells studied. Major falls in RNA levels/cell appeared to occur in hepatocytes and to a lesser extent in adipocytes and skeletal muscle. Thus major reductions in G-protein transcripts occurred in hepatocytes. The detected changes in G-protein mRNA are discussed in relation to the available evidence on G-protein expression. We suggest that diabetes causes tissue-specific changes in the levels of mRNA for particular G-protein species; this may have consequences for the functioning of cellular signal-transduction mechanisms in the affected tissues.

Animals↗
PubMedDate not supplied

[Identification of the hop latent virus on the varieties of Ukrainian selection].

Hop latent virus diagnostic of the hop gardens of the modern varieties in Ukraine has been made. Biomolecular diagnosticum has been developed using own primers and conditions for PCR method in combination with classical techniques of biological testing, electron microscopy and double strain RNA extraction from the infected plants.

Base Sequence↗
PubMed1990

Subgroup classification of porcine group-A rotaviruses, using monoclonal antibodies in an enzyme-linked immunosorbent assay.

Fifty-six samples of feces and intestinal contents from nonvaccinated diarrheal pigs with rotavirus infections were tested, using a subgroup (SGP)-specific ELISA, to determine rotavirus SGP classification. Forty-one percent (23/56) were SGP 1, 25% (14/56) were SGP 2, and 34% (19/56) were not classifiable. For classifiable samples, the geographic distribution for SGP 1 and SGP 2, respectively was: 60%/40% from Ohio (n = 15), 63%/37% from other midwestern states (Iowa, Minnesota, Nebraska, South Dakota; n = 16), and 67%/33% from Canada (n = 6). Thirty-seven SGP-classifiable samples were categorized according to age of pigs. Of pigs less than or equal to 1 week old, 22% of samples were SGP 1 (n = 8), and 14% (n = 5) were SGP 2. Of samples from 1- to 2-week-old pigs, 8% were SGP 1 (n = 3), and 5% were SGP 2 (n = 2). Of samples from 2- to 3-week-old pigs, 5% were SGP 1 (n = 2), and 8% were SGP 2 (n = 3). Of samples from 3- to 4-week-old pigs, 5% were SGP 1 (n = 2), and 3% were SGP 2 (n = 1). Of samples from pigs greater than 4 weeks old, 22% were SGP 1 (n = 8) and 8% were SGP 2 (n = 3). Double-stranded RNA extracted from positive controls and from 10 selected field samples (5 from SGP 1 and 5 from SGP 2) was electrophoresed in polyacrylamide gels to detect correlation between subgroup classification by ELISA and long or short double-stranded RNA electrophoretic-migration patterns. All SGP-1 and -2 rotavirus samples tested had typical long double-stranded RNA electrophoretic-migration patterns.

Age Factors↗
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↗
PubMed1977

[Effect of chronic administration of carbon tetrachloride on turnover of individual RNA fractions in rat liver tissue].

During the carbon tetrachloride treatment of rats (for 24 weeks) the 5-H3-uridine content and incorporation into different rat liver RNA fractions after a short-term exposure of labeled precursor were studied. These fractions were obtained by preparative 2.5% polyacrylamide gel electrophoresis from the nuclear and cytoplasmic RNA extracted by thermal phenol fractionation. CCl4 administration resulted in a decrease in the level of transfer and ribosomal RNAs in the rat liver. Chronic CCl4 treatment led to disturbances of the turnover simultaneousness of different components of rapidly labled RNA.

Animals↗
PubMed2006

Antimicrobial peptides from the skin of the Japanese mountain brown frog Rana ornativentris: evidence for polymorphism among preprotemporin mRNAs.

A previous study led to the isolation of antimicrobial peptides belonging to the temporin and brevinin-2 families from a pooled extract of the skin of adult specimens of the Japanese mountain brown frog Rana ornativentris Werner 1903. In order to ascertain whether individual frogs expressed the full complement of temporin genes, we individually cloned cDNAs encoding the temporin precursors from total RNA extracted from the skins of 12 frogs by RT-PCR using a set of preprotemporin-specific primers. All the specimens examined contained mRNAs directing the synthesis of the novel, but inactive, temporin-1Oe (ILPLLGNLLNGLL x NH2). Nucleotide sequence analysis revealed marked polymorphism among individual frogs. Twenty-seven distinct preprotemporin-1Oe mRNAs were identified that contained synonymous substitutions in the antimicrobial peptide region and both synonymous and non-synonymous substitutions in the signal peptide and intervening sequence regions. Up to eight preprotemporin-1Oe mRNA variants were found within a single frog. In addition, several cDNAs encoding preprotemporin-1Oa and -1Ob and a single cDNA encoding preprotemporin-1Oc were characterized. Peptidomic analysis of norepinephrine-stimulated skin secretions revealed the presence of temporin-1Oe, temporin-1Of (SLILKGLASIAKLF x NH2), temporin-1Og (FLSSLLSKVVSLFT x NH2), four members of the ranatuerin-2 family and one member of the palustrin-2 family in addition to previously characterized temporin and brevinin-2 peptides.

Amino Acid Sequence↗
PubMed2005

Molecular misreading: the frequency of dinucleotide deletions in neuronal mRNAs for beta-amyloid precursor protein and ubiquitin B.

Human neuronal cells contain mutant beta-amyloid precursor protein (APP) and ubiquitin B (UBB) mRNAs, in which dinucleotide deletions ('Delta') are generated in/around GAGAG-motifs by an unknown mechanism referred to as 'Molecular Misreading.' The encoded frameshifted (+1) proteins accumulate in the neuropathological hallmarks of Alzheimer's disease (AD) and in other neurodegenerative and age-related diseases. To measure the concentration of Delta mRNAs, we developed a highly sensitive and specific assay, utilizing peptide nucleic acid-mediated PCR clamping, followed by cloning and colony hybridization with sequence-specific oligonucleotide probes. We found only a few molecules of Delta mRNA/microg of cellular RNA, at levels <10(-5) to 10(-6) x the concentration of WT mRNA, in RNA extracted from: (i) cultured human neuroblastoma cells grown under a variety of conditions, (ii) the frontal half of brains from wild type and XPA(-/-) DNA repair-deficient mice, and (iii) post-mortem temporal cortices from humans. Importantly, in RNA from the temporal cortices of AD and Down Syndrome patients that contain betaAPP+1 and UBB+1 immunoreactive cells, we found the same low levels of Delta mRNA. We infer that the accumulation of +1 proteins in neurons of these patients is not caused by an increase in the concentration of Delta mRNAs.

Adult↗
PubMed1999

Comparison of specific and random priming in the reverse transcriptase polymerase chain reaction for genotyping group A rotaviruses.

This study describes an approach to the molecular typing of rotaviruses which requires only a single RNA extraction and reverse transcription (RT) reaction using random primers. Random-primed RT provides complementary DNA (cDNA) which can be used not only for G- and P-typing polymerase chain reactions (PCR), but also for the detection of other RNA viruses which may act as enteric pathogens. It is a sensitive and specific method that can detect 10 virus particles/ml of 10% faecal suspension provided the cDNA is amplified in a nested typing-PCR. Of 121 specimens positive for rotavirus by EM and analysed using this method, only 8% could not be G- or P-genotyped. The untyped samples were tested again performing the RT reaction with G- and P-specific primers, achieving a 5% increase in sensitivity. Comparing G-genotyping against G-serotyping, 92% were genotyped through random priming RT-PCR whereas only 64% were serotyped using G-serotype specific monoclonal antibodies (MAbs).

Aged↗
PubMed1995

Identification of a novel receptor tyrosine kinase expressed in acute myeloid leukemic blasts.

Using the polymerase chain reaction with degenerate oligonucleotides derived from conserved motifs within the catalytic kinase domain of protein tyrosine kinases, and RNA extracted from embryonic stem cells, sequences that encode a segment of the kinase domain of several potentially novel receptor tyrosine kinases (RTKs) have been identified. One of these was selected for further study because in Northern analysis it hybridized to RNA from multipotential hematopoietic cell lines, but not from lines representative of lineage-committed cells. A cDNA for this receptor, designated developmental tyrosine kinase (DTK), was isolated and encodes a protein with structural similarities to AXL. Together these receptors form a new class of RTK. DTK is expressed in a number of human leukemic cell lines, and in the blasts of 6 of 11 patients with acute myeloid leukemia (AML) analyzed. The structure of DTK suggests that it may function as a cell adhesion molecule, and mediate cell-to-cell or cell-matrix interactions between hematopoietic cells and their respective microenvironments.

Amino Acid Sequence↗
PubMedDate not supplied

Complete regression of a guinea pig hepatocarcinoma by immunotherapy with "tumor-immune" RNA or antibody to fibrin fragment E.

Two novel immunotherapeutic regimens were developed for a uniformly lethal, intradermally growing transplantable ascites variant (line 10) of a diethylnitrosamine-induced hepatoma in strain 2 guinea pigs. In an apparently tumor-specific immunotherapy model, 32 guinea pigs were cured by the injection into the tumor area, five or seven days after tumor challenge, of syngeneic or xenogeneic RNA extracts obtained from lymphoid tissues of line 10-immune strain 2 guinea pigs or rhesus monkeys, as part of a total regimen which included syngeneic nonsensitive peritoneal exudate cells injected prior to, and tumor-specific antigen injected after, the RNA. In another immunotherapy model, not tumor-specific, 18 strain 2 guinea pigs were cured by the injection into the tumor area, 6 and 16 days after tumor challenge, of antibody specific for fibrin fragment E (FFE), an essential component in the formation of a fibrin matrix considered to be important in tumor development. When therapy was delayed to 12 days in the RNA test system, or to 16 days in the anti-FFE test system, complete abrogation of the tumors did not occur. The long-term survival of the 50 successfully treated animals and their immunity to further tumor challenge indicated that both immunotherapeutic procedures had systemic effects. To test this further, line 10 cells were injected intradermally simultaneously at two sites and only one site was treated. When the one tumor location was treated with anti-FFE, complete regression of the treated tumor and a 30% retardation in the development of the untreated tumor were observed. When this tumor location was treated with the RNA regimen, complete regression of the tumors occurred at both the treated and the untreated sites. Optimal conditions for both immunotherapeutic models and their combination have yet to be establshed. Nonetheless, both immunotherapeutic regimens were more effective than any other immunotherapy thus far reported for this tumor, including the use of BCG or its derivatives.

Animals↗
PubMed1980

Regulation of endogenous murine leukemia virus-related nuclear and cytoplasmic RNA complexity in C57BL/6J mice of increasing age.

The nucleotide sequence complexity of murine leukemia virus *MuLV)-related RNA has been measured by RNA-complementary DNA hybridization analysis in nuclear and cytoplasmic RNA isolated from liver and brain of low-leukemia-strain C57BL/6J mice of different ages. In these two tissues, an approximate 1.5- to 2-fold increase in the complexity of steadystate nuclear MuLV-related RNA sequences was observed as a function of age. Maximum complexity was observed with nuclear RNA extracts from old mice and corresponded to roughly 70 to 75% of the total MuLV genome. In contrast to the age-related increase in complexity of nuclear MuLV genome was detected in liver and brain steady-state cytoplasmic RNA, irrespective of animal age. These data suggest that control mechanisms regulating the transcription and/or stabilization of nuclear RNA transcripts of endogenous mouse MuLV-related genomes become less stringent with animal age even in low-tumor mouse strains. The data also support the existence of independent posttranscriptional mechanisms which prevent accumulation of these MuLV-related transcripts in steady-state cytoplasmic RNA and which do not seem to be as subject to the relaxation of stringency as a function of age.

Aging↗
PubMed1999

Sequence data suggests big liver and spleen disease virus (BLSV) is genetically related to hepatitis E virus.

A monoclonal antibody (mAb) that reacted specifically with a 16 kDa big liver and spleen disease virus (BLSV) protein was used to identify the protein in western immunoblots of infected liver extracts and enable partial amino acid sequence analysis of the protein. Based on this sequence, a degenerate primer was designed that was used in conjunction with random hexamers in a reverse transcriptase-POR (RT PCR), to amplify a 523 bp product from RNA extracted from homogenates of BLSV-infected livers. There was 62% nucleotide sequence identity between this sequence and the sequence of the helicase gene of human hepatitis E virus (HEV). POR primers designed from this 523 bp fragment were able to amplify a 490 bp product from livers of virus-infected chickens but not chickens from virus-free flocks.

Animals↗