Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.1.26.4 (RNase H)
 2,751 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have used photoaffinity labelling to examine the chloroplast RNA polymerase components which come into contact with nascent transcripts during the in vitro transcription of plastid DNA. The transcripts were synthesized in the presence of a photoactive analogue (4-thio UTP) and alpha-32P-ATP, using enriched pea chloroplast RNA polymerase preparation and a recombinant plasmid containing the plastid 16S rRNA promoter. Brief irradiation of the transcriptional complex crosslinked the photoactive nascent RNA to proximal proteins. Labelling of the transcriptional complex was dependent on 4-thio UTP and template DNA. Two polypeptides of 51 and 54 kDa were consistently crosslinked to the nascent transcripts; about 60% of the total radioactivity of the crosslinked RNA was associated with these polypeptides. In some experiments, two additional polypeptides of 38 and 75 kDa were also found to be associated with about 13% and 17% of the total crosslinked RNA radioactivity, respectively. The UV-crosslinked transcriptional complexes were stable to either DNase or S1 nuclease hydrolysis but partially sensitive to RNase T1. Insensitivity of the complex to hydrolysis with RNase H suggested that the nascent transcripts were not crosslinked to the template. The complexes could also be hydrolysed by proteinase K and thermolysin. No crosslinkage was observed when labelled RNA molecules containing 4-thio UMP residues were added after synthesis to the polymerase preparation. This suggested that the method identified only those polypeptides which came into close contact with the transcript during its synthesis. Antibodies raised against the RNA-protein complex confirmed the presence of the polypeptides in the chloroplast RNA polymerase preparation on Western blots. Preincubation of these antibodies with the chloroplast RNA polymerase inhibited plastid DNA transcription. These data showed that the transcript-binding polypeptides were functional components of the chloroplast transcriptional complex.
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PMID:Photoaffinity labelling of the pea chloroplast transcriptional complex by nascent RNA in vitro. 171 36

After thermal denaturation, an in vivo-labeled RNA was found in a temperature-sensitive initiation mutant of Bacillus subtilis (dna-37) associated with high-molecular-weight DNA. This RNA could be clearly distinguished from other RNA species by different techniques of separation, such as Sepharose 2B filtration, chromatography on nitrocellulose, and equilibrium centrifugation in density gradient. It was obtained even when HCHO was present during denaturation and chilling of nucleic acids and was still detected after a second denaturation as well as after incubation with proteinase K. Properties of the complex were not altered by prior treatment with RNase H. A control experiment using two samples of the complex treated either with pancreatic DNase or with pancreatic RNase, denatured together and centrifuged in the same density gradient, showed that no artifactual associations occur between the DNA and the RNA components of the complex. These results demonstrate that the DNA and RNA in the complex are associated by neither hydrogen bonds nor proteins, but are indicative of a DNA-RNA covalent linkage. In addition, during synchronous replication after a previous period at a nonpermissive temperature, DNA-linked RNA synthesis took place at specific times which coincided with the appearance of rifampin resistance of the first and the second replication cycles. A possible involvement of this RNA in the initiation of chromosome replication is discussed.
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PMID:Possible involvement of DNA-linked RNA in the initiation of Bacillus subtilis chromosome replication. 617 20

A single-step reverse transcription polymerase chain reaction (SRT-PCR) method was optimized for hepatitis C virus (HCV) RNA detection. Extraction procedures by proteinase K and guanidinium isothiocyanate gave similar results. The optimal MgCl2 concentration for the SRT-PCR method was 2 mM with 10 units of superscript M-MLV RNase H-reverse transcriptase and 1 unit of Taq polymerase. Shorter PCR cycling steps gave a 10-fold-increased PCR product compared with longer cycling steps. Twenty-five anti-HCV-positive sera from chronic hepatitis C patients were positive with SRT-PCR, whereas only 17 out of 25 were positive by dissociated RT and PCR (dRT/PCR). Specificity was assessed by twenty negative controls. SRT-PCR was 5-fold more sensitive (5 HCV RNA copies per assay) than dRT/PCR with an HCV RNA transcript. Our SRT-PCR method for HCV RNA detection appears fully adapted for routine use in a medical virology laboratory.
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PMID:Detection of hepatitis C virus RNA by a reliable, optimized single-step reverse transcription polymerase chain reaction. 857 10