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)

An in vitro transcription system in which vesicular stomatitis virus (VSV) mRNA species have been synthesized is described. In addition to purified VSV virions, which contain an RNA-dependent RNA polymerase, this system contained a cytoplasmic cell extract that enhanced correct transcription. Gel electrophoretic analysis of the methylated polyadenylic acid [poly(A)]-containing VSV mRNA produced in this system in the presenct of S-adenosylmethionine showed the discrete VSV mRNA species. However, when unmethylated mRNA was synthesized in the presence of S-adenosylhomocysteine, the poly(A)-containing transcripts were large and heterogeneous in molecular weight and did not contain discrete VSV mRNA species. Two-dimensional fingerprint analysis of the methylated and unmethylated products suggested that identical nucleotide sequences were present in the RNAs. Further analysis showed the presence of very large heterogeneous poly(A), 200 to 2,000 nucleotides in lenght, in the unmethylated transcript. Proof that this large poly(A) was covalently linked to the correct VSV mRNA transcripts was obtained by removal of the poly(A) by hybirdization with oligodeoxythymidylic acid and digestion with RNase H. This digestion produced unmethylated VSV mRNA transcripts with the same discrete sizes as the deadenylated RNAs produced from VSV mRNA initially isolated from VSV-infected cells. The results suggest that there is a relationship between methylation at the 5'-end and polyadenylation at the 3'-end of VSV mRNA's. Furthermore, addition of the very large poly(A) does not affect the normal process of sequential transcription of the VSV genome, suggesting that this poly(A) addition is occurring independently of further transcription.
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PMID:Giant heterogeneous polyadenylic acid on vesicular stomatitis virus mRNA synthesized in vitro in the presence of S-adenosylhomocysteine. 18 93

The 5'-terminal nucleotide sequences of the avian sarcoma virus (ASV) genome are transcribed by the reverse transcriptase in vitro into a DNA transcript that represents the entire distance ( approximately 100 nucleotides) between the tRNA(Trp) primer molecule and the 5' terminus. We have used these DNA(100) transcripts in hybridization reactions with ASV-specific RNA from infected avian cells and find nucleotide sequences complementary to these transcripts on all of the various size classes of viral mRNA identified. Similar hybridization results were obtained with a specific DNA transcript complementary to viral genomic nucleotide sequences between the tRNA(Trp) primer molecule and up to, but not including, the terminal redundant sequences (DNA(70)), indicating that the observed hybridization of DNA(100) to all size classes of viral RNA in infected cells did not reflect hybridization of DNA(100) to the terminal redundant sequences at the 3' end of the viral genome. Escherichia coli RNase H hydrolysis of RNA.DNA hybrids consisting of genomic 35S RNA obtained from virus and DNA(100) transcripts indicated that viral genomic sequences complementary to these DNA transcripts were not present at sites distal to the ends of the RNA genome and therefore not adjacent to the corresponding gene sequences representing the various species of viral mRNA from infected cells. These studies suggest that the 5'-terminal genomic nucleotide sequences, or a portion thereof, are somehow added or "spliced" onto each ASV-specific mRNA species in infected cells either during or after transcription of proviral DNA for some as yet undetermined purpose.
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PMID:Evidence for splicing of avian sarcoma virus 5'-terminal genomic sequences into viral-specific RNA in infected cells. 20 93

The polycytidylic acid [poly(C)] tract in foot and mouth disease virus RNA has been located about 400 nucleotides from the 5' end of the RNA by analysis of the products from the digestion of the RNA with RNase H in the presence of oligodeoxyguanylic acid [oligo(dG)]. This treatment produces a small fragment (S) containing the small protein covalently linked to the RNA and a large fragment (L) that migrates faster than untreated RNA on low-percentage polyacrylamide gels, lacks the poly(C) tract as shown by RNase T1 digestion and oligo(dG)-cellulose binding, and is no longer infective. Polyacrylamide gel electrophoresis of fragment S suggests that it is about 400 nucleotides long, in agreement with the size estimated from the proportion of radioactivity in the fragment. Analysis of the RNase T1 digestion products of S shows that it contains only those oligonucleotides mapping close to the poly(C) tract that is situated near the 5' end of the virus RNA.
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PMID:More precise location of the polycytidylic acid tract in foot and mouth disease virus RNA. 20 92

Labeled, purified 30S RNA from Moloney murine sarcoma virus was annealed to an excess of Moloney murine leukemia virus complementary DNA. Upon treatment of the resulting DNA.RNA hybrids with RNase H followed by sucrose gradient sedimentation, and undigested 18S RNA molecule was recovered. This RNA molecule was shown to represent the "sarcoma-specific" region of the virus. The unintegrated linear DNA provirus of murine sarcoma virus 124 was isolated from newly infected cells and a physical map of the sarcoma-specific region was obtained. First, unintegrated full-length linear proviral DNA molecules were cleaved by several restriction endonucleases. The reciprocal position and orientation with respect to the viral RNA of the resulting fragments were established. The location of the sarcoma-specific region was determined by competition-hybridization with 125I-labeled viral genomic RNAs and proviral DNA fragments. A 1500-base-pair fragment was obtained by cleavage with HindIII + Bgl II. This fragment mapped between 750 and 2250 base pairs from the right end of the proviral DNA (corresponding th the 3' terminus of the viral RNA) and contained the whole set of the sarcoma-specific information. This murine sarcoma virus proviral restriction fragment is approximately of the same size and map position as the isolated 18S sarcoma-specific RNA.
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PMID:The "sarcoma-specific" region of Moloney murine sarcoma virus 124. 20 71

A DNA endonuclease, Endo-I, which cleaves superhelical DNAs, has been isolated from avian myeloblastosis virions stripped of their coats by mild detergent treatment. The enzyme has a broad pH optimum around 7.5-8.0 and requires Mg2+ for activity. A second endonuclease, Endo-II, with a requirement for Mn2+, also present in viral cores, copurified with avian myeloblastosis virus alpha beta DNA polymerase (reverse transcriptase, RNA-dependent DNA nucleotidyltransferase) and similarly cleaved superhelical DNAs. Heat denaturation and sodium fluoride and N-ethylmaleimide inhibition studies were carried out to demonstrate a possible relationship between the two endonucleases and the viral DNA polymerase and RNase H activities. It appears that Endo-II may be an intrinsic activity of the polymerase.
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PMID:DNA endonucleases associated with the avian myeloblastosis virus DNA polymerase. 22 53

We investigated the influence of viral RNase H on the transcription of the avian sarcoma virus RNA in a virion-associated reaction. The ability of RNase H to degrade the RNA moiety of the initially formed RNA-DNA hybrid at the 5' end of the viral genome was found to be greatly dependent on the exact concentration of nonionic detergent used to activate the reaction. At a detergent concentration optimal for extensive and faithful in vitro transcription of avian sarcoma virus RNA by the virion-associated RNA-dependent DNA polymerase, most of the 5' terminus of the RNA was digested in 30 min at 41 degrees C. At higher than optimal detergent concentrations, however, little of that RNA was digested. We conclude that removal of the 5'-terminal redundancy in the RNA after its transcription into DNA is a prerequisite for base pairing of the DNA to the 3'-terminal redundant sequence. Lack of removal of this sequence leads to incorrect elongation and substantial reduction of DNA synthesis. When tested with a synthetic RNA-DNA hybrid, virion-associated RNase H did not reveal a detergent dependence.
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PMID:Effect of viral RNase H on the avian sarcoma viral genome during early transcription in vitro. 22 44

Foot and mouth disease virus RNA has been treated with RNase H in the presence of oligo (dG) specifically to digest the poly(C) tract which lies near the 5' end of the molecule (10). The short (S) fragment containing the 5' end of the RNA was separated from the remainder of the RNA (L fragment) by gel electrophoresis. RNA ligase mediated labelling of the 3' end of S fragment showed that the RNase H digestion gave rise to molecules that differed only in the number of cytidylic acid residues remaining at their 3' ends and did not leave the unique 3' end necessary for fast sequence analysis. As the 5' end of S fragment prepared form virus RNA is blocked by VPg, S fragment was prepared from virus specific messenger RNA which does not contain this protein. This RNA was labelled at the 5' end using polynucleotide kinase and the sequence of 70 nucleotides at the 5' end determined by partial enzyme digestion sequencing on polyacrylamide gels. Some of this sequence was confirmed from an analysis of the oligonucleotides derived by RNase T1 digestion of S fragment. The sequence obtained indicates that there is a stable hairpin loop at the 5' terminus of the RNA before an initiation codon 33 nucleotides from the 5' end. In addition, the RNase T1 analysis suggests that there are short repeated sequences in S fragment and that an eleven nucleotide inverted complementary repeat of a sequence near the 3' end of the RNA is present at the junction of S fragment and the poly(C) tract.
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PMID:The nucleotide sequence at the 5' end of foot and mouth disease virus RNA. 23 62

Nascent replicative form type II (RFII) DNA of coliphage M13 synthesized in an Escherichia coli mutant deficient in the 5' leads to 3' exonuclease associated uith DNA polymerase I contains ribonucleotides that are retained in the covalently closed RFI DNA sealed in vitro by the joint action of T5 phage DNA polymerase and T4 phage DNA ligase. These RFI molecules are labile to alkali and RNase H, unlike the RFI produced either in vivo or from RFII with E. coli DNA polymerase I and E. coli DNA ligase. The ribonucleotides are located at one site and predominantly in one strand of the nascent RF DNA. Furthermore, these molecules contain multiple small gaps, randomly located, and one large gap in the intracistronic region.
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PMID:Structure of nascent replicative form DNA of coliphage M13. 27 30

In the presence of RNA polymerase, RNase H, discriminatory factors alpha and beta, Escherichia coli binding protein, DNA elongation factor I, DNA elongation factor II preparation, DNA polymerase III, and ATP, UTP, GTP, CTP, dATP, dTTP, dGTP, and dCTP, fd viral DNA can be quantitatively converted to RFII containing a unique gap in the linear minus strand. This gap, mapped with the aid of restriction endonucleases HinII and HpaII, is located within Fragment Hpa-H of the fd genome. The discrimination reaction has been resolved into two steps: Step A, fd viral DNA, E. coli binding protein, and discriminatory factors alpha and beta form a protein DNA complex; Step B, the complex isolated by agarose gel filtration selectively forms fd RFII when supplemented with RNase H, RNA polymerase, and the DNA elongation proteins. The omission of any of the proteins described above during the first reaction resulted in either no discrimination or a decrease in discrimination when the missing protein was added during the second step. Results are presented which indicate that E. coli binding protein, discriminatory factors alpha and beta, and RNase H must be present during the time RNA synthesis occurs in order to selectively form RFII from fd DNA and not phiX RFII. The amount of fd and phiX174 RNA-DNA hybrid formed in vitro is directly related to the DNA synthesis observed. Thus, under discriminatory conditions, only fd viral DNA leads to fd RNA-DNA complexes and no phiX RNA-DNA hybrid is formed. Under nondiscriminatory conditions, both DNAs yield RNA-DNA hybrids and DNA synthesis. In the absence of discriminatory factor alpha, no RNA-DNA hybrid is formed with either DNA, and in turn, no DNA synthesis is detected with either DNA template.
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PMID:Selective inhibition of phiX RFII compared with fd RFII DNA synthesis in vitro. II. Resolution of discrimination reaction into multiple steps. 32 48

T7 gene 6 exonuclease has been shown to have an RNase H activity as well as a double-strand specific DNase activity by the following experiments: The RNase H activity coelutes with the DNase activity from DEAE-cellulose, phosphocellulose, hydroxyapatite, and Sephadex G-200 columns. Gene 6 exonuclease specified by a T7 strain with a temperature sensitive mutation in gene 6 has an extremely heat-labile RNase H activity as well as a heat-labile DNase activity. T7 gene 6 exonuclease degrades the RNA region of a poly(A) . poly(dT) hybrid polymer exonucleolytically from the 5' terminus, releasing a ribonucleoside 5'-monophosphate product. When the RNA strand of a 0X174 RNA . DNA hybrid molecule synthesized with E. coli RNA polymerase is degraded, a ribonucleoside triphosphate is produced from the 5'-triphosphate terminus. Participation of T7 gene 6 exonuclease in the removal of primer RNA in discontinuous replication of T7 DNA is discussed.
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PMID:T7 gene 6 exonuclease has an RNase H activity. 36 24


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