Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.7.49 (reverse transcriptase)
 31,746 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Single-strand conformation polymorphism (SSCP) analysis was used to characterize genetic polymorphisms among 12 isolates of dengue-2 virus, which were previously genetically characterized by RNase T1 oligonucleotide mapping and by sequencing the viral envelope (E) gene. Specific cDNA fragments from the dengue-2 isolates were amplified by the reverse transcriptase-polymerase chain reaction. The viral E, premembrane (prM), and nonstructural 5 (NS5) gene cDNAs of 291 basepairs (bp), 291 bp, and 201 bp, respectively, were denatured, rapidly chilled to promote intrastrand reassociation, electrophoretically separated on nondenaturing polyacrylamide gels, and SSCP patterns were observed by silver staining. The SSCP analysis revealed polymorphisms among a number of dengue-2 isolates from the same topotype, and these were markedly different between isolates of different topotype (distinct genetic group). Comparison of nucleotide sequence and SSCP analyses of the 291-bp E cDNA demonstrated that virus isolates that produced identical SSCP patterns contained 0-7 nucleotide substitutions, whereas isolates that showed different SSCP patterns contained 4-25 nucleotide substitutions. Positive predictive value and negative predictive value as measures of certainty for predicting identical and different sequences were 26% and 100%, respectively. The SSCP patterns of the 12 dengue-2 isolates suggested greater genetic variation in the prM gene region than in either the E or NS5 gene regions. The SSCP analyses should allow easy, sensitive, and rapid screening of dengue viruses isolates and the assessment of variation at a number of sites in the virus genome. Additionally, SSCP screening of dengue-2 virus for genetic variability may reveal the introduction of new viral genotypes in a given geographic area. These genetic variants of the virus could serve as markers of the epidemic potential of the virus strain.
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PMID:Rapid characterization of genetic diversity among twelve dengue-2 virus isolates by single-strand conformation polymorphism analysis. 934 56

RNAs made in the mitochondrion of Physarum polycephalum are edited relative to their template by the precise addition of nonencoded nucleotides, while they are being synthesized. This insertional editing has been reproduced in vitro during run-on extension of RNAs initiated in vivo, within partially purified mitochondrial transcription elongation complexes (mtTECs), but it does not occur when the mitochondrial polymerase initiates transcription on exogenous cloned DNA. This chapter describes in vitro transcription systems in which mtTEC RNAs are elongated on repositioned parts of the genome or exogenous DNA, in order to investigate how the nontemplated insertions are directed. Restriction enzyme digestion and DNA ligation are used to generate the chimeric templates, and the RNA products are analyzed directly by nuclease dissection (S1 protection followed by RNase T1 digestion) or by reverse transcriptase-polymerase chain reaction (RT-PCR) followed by restriction enzyme analysis or cloning and sequencing.
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PMID:Chimeric templates and assays used to study physarum cotranscriptional insertional editing in vitro. 1510 80

Rapid detection of viral pathogens is crucial for antiviral therapy. High-density 60-70-mer oligonucleotide microarrays have been explored for broad detection of many viruses. However, relatively low specificity and the complex analytical processes are the major limitations when pan-viral oligonucleotide microarrays are used to detect viral pathogens. In this study, genus-specific oligonucleotides were used as probes and modified sample preparations were carried out to improve the specificity and accuracy of the pan-viral oligonucleotide microarray. Genus-specific 63-mer oligonucleotide probes were used for screening human pathogenic RNA viruses. A total of 628 oligonucleotide probes covering 32 RNA viral genera from 14 viral families were used. The number of oligonucleotide probes was decreased to simplify the analytical process of hybridization and to minimize cross-hybridization. Host genomes were removed by DNase I/RNase T1 digestion before viral nucleic acid extraction, and non-ribosomal hexanucleotides were used for reverse transcription to minimize interference of host genomes. Cultured viruses were used for microarray validation. The microarray was validated by cultured isolates that belonged to five viral genera. By using DNase I/RNase T1 digestion before viral nucleic acid extraction and non-ribosomal hexanucleotides for reverse transcription, the specificity of the microarray was improved. Furthermore, the analytical process of hybridization results was simplified. The specificity of pan-viral microarray could be improved by using genus-specific oligonucleotides as probes and by using non-ribosomal hexanucleotides for reverse transcription. Combined with subsequent degenerate reverse transcriptase-polymerase chain reaction and sequencing processes, this improved genus-specific oligonucleotides microarray provides a relatively flexible strategy for diagnosis of RNA virus diseases.
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PMID:Improvement of the specificity of a pan-viral microarray by using genus-specific oligonucleotides and reduction of interference by host genomes. 2173 54


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