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)

The HBV P gene encodes a multifunctional polyprotein which contains the major enzymatic activities required for hepadnaviral reverse transcription (protein primer, DNA polymerase, and RNase H). Mutant HBV genomes unable to synthesize fully functional P gene products were analysed for their potential to be rescued by a second mutant HBV genome that either contained a wild type P gene (intergenic complementation) or a mutation in a functionally different P gene domain (intragenic complementation). This analysis was carried out by cotransfecting two mutants at a time into HepG2 cells and assaying for the production of core particles containing DNA polymerase activity. The results obtained demonstrate the existence of intergenic, but not of intragenic complementation. This indicates that the primary P gene product is not processed before core assembly, and furthermore that there is a rather free mixing of all HBV gene products in the HBV infected cell which can lead to the efficient propagation of defective viral genomes.
Gastroenterol Jpn 1990 Sep
PMID:Complementation analysis of mutants defective in different steps of HBV reverse transcription. 222 68

The reverse transcriptase polymerase of the human T-cell lymphotropic virus/lymphadenopathy-associated virus has been cloned into an expression vector and expressed in Escherichia coli. Two polypeptides of 66 and 51 kDa molecular mass are detectable in polymerase-expressing bacterial lysates with human patient sera. They are processed from a short-lived 120-kDa polyprotein precursor equivalent to a region consisting of polymerase, protease, and endonuclease. The 51 kDa protein appears to originate from the 66-kDa molecule; additional processing products are 32- and 15-kDa proteins. The bacterially expressed polymerase is enzymatically active and exhibits the template specificities, ion requirements, and response to inhibitors of the authentic enzyme. It was purified by DEAE-cellulose-, phosphocellulose-, and poly(rC)-agarose column chromatography followed by glycerol density gradient centrifugation. It copurifies with an RNase H activity, suggesting the existence of a virus-coded DNA polymerase-RNase H complex. The purified bacterial enzyme allows a safe large-scale screening for inhibitors of both activities.
J Biol Chem 1987 Sep 15
PMID:RNase H activity associated with bacterially expressed reverse transcriptase of human T-cell lymphotropic virus III/lymphadenopathy-associated virus. 244 62

Mechanisms that could operate to initiate pBR322 DNA replication in the absence of RNase H and DNA polymerase I are described. Two different pathways leading to extensive unwinding of pBR322 DNA have been observed under DNA replication reaction conditions in vitro. In the presence of RNA polymerase and DNA gyrase, specifically initiated RNA II (the leading-strand primer precursor) can form an RNA-DNA hybrid with the template that starts just upstream of the origin of DNA replication and continues for about 3 kilobases. Subsequent digestion of the RNA in this RNA-pBR322 DNA hybrid results in the formation of a highly unwound DNA termed form I. If DNA gyrase is absent during the RNA polymerase-catalyzed elongation of RNA II, a stable RNA-pBR322 DNA hybrid can still form that is localized to the origin region of the genome. Formation of this hybrid activates the primosome assembly site present on the lagging-strand DNA template, by displacing it to a single-stranded conformation, thereby allowing preprimosome assembly. Once assembled, the DNA helicase activity of the preprimosome, in the presence of the single-stranded DNA binding protein and DNA gyrase but in the absence of any further transcription, can also result in extensive unwinding of pBR322 DNA. The product of this reaction, form I DNA, is more unwound than form I DNA. The formation of both form I and form I DNA is inhibited by the presence of excess RNA I, as well as by RNase H at concentrations sufficient to catalyze the normal processing of RNA II required for initiation of leading-strand DNA synthesis. These results suggest that RNA II-pBR322 DNA hybrid formation is essential to permit preprimosome assembly during pBR322 DNA replication under conditions where both RNase H and DNA polymerase I are absent.
J Biol Chem 1989 Sep 05
PMID:Transcriptional activation of pBR322 DNA can lead to duplex DNA unwinding catalyzed by the Escherichia coli preprimosome. 247 95

Recombinant HIV-1 reverse transcriptase (RT) was stably overproduced as a soluble protein in Escherichia coli using a double-plasmid expression system in which an RT precursor protein was expressed and processed in vivo by HIV-1 protease produced in trans. The RT thus produced consisted of an equimolar mixture of two polypeptides, p66 and p51, which were copurified to greater than 90% homogeneity and were found to share a common NH2 terminus as judged by sequence analysis of the polypeptide mixture. The observed sequence confirmed correct in vivo cleavage by protease at the protease-RT polyprotein junction to yield an NH2 terminus identical to that of genuine viral RT (M. M. Lightfoote et al. (1986) J. Virol. 60, 771-775; F. diMarzo Veronese et al. (1986) Science 231, 1289-1291). The bacterially expressed RT had a specific activity similar to that of viral RT and inhibition studies with phosphonoformate confirmed that it was indistinguishable from the viral enzyme with respect to sensitivity to this inhibitor. Polymerase activated gel analysis of the mixture indicated that p66 was associated with a higher level of RT activity than p51. RNase H activated gel analysis suggested that the purified preparation of recombinant RT was free of endogenous E. coli RNase H, and that the RNase H activity of RT was exclusively associated with the p66 polypeptide, supporting the hypothesis that the RNase H domain is located in the COOH-terminal region of the molecule.
Arch Biochem Biophys 1989 Sep
PMID:Recombinant HIV-1 reverse transcriptase: purification, primary structure, and polymerase/ribonuclease H activities. 247 69

We report the inhibition of encephalomyocarditis virus (EMCV) RNA translation in cell-free rabbit reticulocyte lysates by antisense oligonucleotides (13-17-base oligomers) complementary to (a) the viral 5' non-translated region, (b) the AUG start codon and (c) the coding sequence. Our results demonstrate that the extent of translation inhibition is dependent on the region where the complementary oligonucleotides bind. Non-complementary and 3'-non-translated-region-specific oligonucleotides had no effect on translation. A significant degree of translation inhibition was obtained with oligonucleotides complementary to the viral 5' non-translated region and AUG initiation codon. Digestion of the oligonucleotide:RNA hybrid by RNase H did not significantly increase translation inhibition in the case of 5'-non-translated-region-specific and initiator-AUG-specific oligonucleotides; in contrast, RNase H digestion was necessary for inhibition by the coding-region-specific oligonucleotide. We propose that (a) 5'-non-translated-region-specific oligonucleotides inhibit translation by affecting the 40S ribosome binding and/or passage to the AUG start codon, (b) AUG-specific oligonucleotides inhibit translation initiation by inhibiting the formation of an active 80S ribosome and (c) the coding-region-specific oligonucleotide does not prevent protein synthesis because the translating 80S ribosome can dislodge the oligonucleotide from the EMCV RNA template.
Eur J Biochem 1989 Sep 01
PMID:Antisense oligonucleotide inhibition of encephalomyocarditis virus RNA translation. 247 10

Oligodeoxynucleotides with different arrangements of methylphosphonate linkages were examined for nuclease sensitivity in vitro, stability in tissue culture, and ability to form RNase H-sensitive substrates with complementary RNA. After nuclease treatment, resistance was demonstrated by the ability to alter the electrophoretic mobility of a labeled complementary phosphodiester oligodeoxynucleotide. Both 5'- and 3'-exonuclease activities were retarded by methylphosphonate linkages. Methylphosphonate-containing oligodeoxynucleotides with 1-5 adjacent phosphodiester linkages were tested as substrates for the endonucleases DNase I and DNase II. The results indicated that a span of three or fewer contiguous internal phosphodiester linkages led to the greatest resistance to endonuclease. However, in serum-supplemented culture medium half-lives of these oligodeoxynucleotides were independent of the number of contiguous phosphodiester linkages. Methylphosphonate-containing oligodeoxynucleotides were hybridized to RNA runoff transcripts and tested as substrates for RNase H. The results indicated that a span of three internal phosphodiester linkages in the oligodeoxynucleotide was necessary and sufficient to direct cleavage of the RNA in the duplex.
Nucleic Acids Res 1989 Sep 25
PMID:Number and distribution of methylphosphonate linkages in oligodeoxynucleotides affect exo- and endonuclease sensitivity and ability to form RNase H substrates. 247 96

Ribonuclease H IIb, which seems to play a physiological role during transcription, was purified from calf thymus tissue. A polyclonal antibody, raised against the most purified ribonuclease H IIb fraction, recognizes in crude extracts almost exclusively a 52-kDa protein band. By immunoaffinity chromatography and immunoprecipitation experiments, we are able to deplete enzyme extracts from the crossreacting 52-kDa protein band and from ribonuclease H IIb activity. Enzyme activity is eluted from the immunoaffinity matrix in association with a 52-kDa protein under denaturing conditions. Immunoaffinity chromatography enables us also to calculate a purification factor of around 20,000 from the crude extract. The native molecular mass for the enzyme of around 45 kDa, as determined by gel filtration, suggests that calf thymus ribonuclease H IIb is most probably monomeric. The enzyme possesses an isoelectric point of 7.0. It requires Mg2+ ions for activity, is inhibited by N-ethylmaleimide, and exhibits a pH optimum of 9.0-9.5. The enzyme releases oligoribonucleotides with 3'-OH and 5'-phosphate ends, probably in an exonucleolytical manner. The third largest subunit of yeast RNA polymerase A (I) displays ribonuclease H activity [Huet et al. (1976) Nature 261, 431-433]. We discuss our findings in the light of a possible association of ribonuclease H IIb and RNA polymerase A (I) in higher eukaryotes.
Eur J Biochem 1989 Sep 15
PMID:Serological analysis and characterization of calf thymus ribonuclease H IIb. 255 85

On the basis of earlier studies with both detergent-disrupted virions (the endogenous reaction) and an in vitro reconstructed reaction, the RNase H activity associated with Moloney murine leukemia virus reverse transcriptase has been implicated in the generation of plus-strand RNA primers during reverse transcription. Here we used an oligonucleotide extension assay to show that the RNA primers remaining bound to the plus DNA strands initiated at the normal origin in the in vitro reaction are heterogeneous in length. This result indicates that, although a precise cleavage generates the 3' end of the priming RNA, RNase H exhibits less specificity at other break sites. During the endogenous reaction, a kinetic analysis of the synthesis of plus strands corresponding to different regions of the genome suggested that additional sites for the initiation of plus-strand DNA existed upstream of the normal origin. Direct analysis of fragments produced in the endogenous reaction, as well as in the in vitro reaction, confirmed the existence of upstream plus-strand initiation sites. Several of these sites were mapped to the nucleotide level by the oligonucleotide extension method. A comparison of the nucleotide sequences surrounding the upstream initiation sites with the sequence at the normal plus-strand origin revealed common features, which suggests a mechanism for plus-strand priming based on sequence recognition by the RNase H/reverse transcriptase protein. Although primer removal by RNase H is highly efficient for DNA fragments initiated at the normal origin, the RNA primers were inefficiently removed from the fragments initiated at the upstream sites. This result suggests that primer removal, like primer generation, involves sequence recognition by the enzyme.
J Virol 1987 Sep
PMID:The role of Moloney murine leukemia virus RNase H activity in the formation of plus-strand primers. 303 72

Pre-mRNA splicing in vitro is preceded by complex formation (spliceosome assembly). U2 small nuclear RNA (snRNA) is found in the earliest form of the spliceosome detected by native gel electrophoresis, both in Saccharomyces cerevisiae and in metazoan extracts. To examine the requirements for the formation of this early complex (band III) in yeast extracts, we cleaved the U2 snRNA by oligonucleotide-directed RNase H digestion. U2 snRNA depletion by this means inhibits both splicing and band III formation. Using this depleted extract, we were able to design a chase experiment which shows that a pre-mRNA substrate is committed to the spliceosome assembly pathway in the absence of functional U2 snRNP. Interactions occurring during the commitment step are highly resistant to the addition of an excess of unlabeled substrate and require little or no ATP. Sequence requirements for this commitment step have been analyzed by competition experiments with deletion mutants: both the 5' splice site consensus sequence and the branch point TACTAAC box sequence are necessary. These experiments strongly suggest that the initial assembly process requires a trans-acting factor(s) (RNA and/or proteins) that recognizes and stably binds to the two consensus sequences of the pre-mRNA prior to U2 snRNP binding.
Mol Cell Biol 1988 Sep
PMID:Early commitment of yeast pre-mRNA to the spliceosome pathway. 306 22

We have used RNA from human hypothalamus as template for the production of cDNAs encoding insulin-like growth factor II (IGF-II). The prohormone coding sequence of brain IGF-II RNA is identical to that found in liver; however, the 5' untranslated sequence of the brain cDNA has no homology to the 5' untranslated sequence of the previously reported liver cDNAs. By using hybridization to specific probes as well as a method based on the properties of RNase H, we found that the human IGF-II gene has at least three exons that encode alternative 5' untranslated regions and that are expressed in a tissue-specific manner. A probe specific to the brain cDNA 5' untranslated region hybridizes to a 6.0-kilobase transcript present in placenta, hypothalamus, adrenal gland, kidney, Wilms tumor, and a pheochromocytoma. The 5' untranslated sequence of the brain cDNA does not hybridize to a 5.3-kilobase transcript found in liver or to a 5.0-kb transcript found in pheochromocytoma. By using RNase H to specifically fragment the IGF-II transcripts into 3' and 5' fragments, we found that the RNAs vary in size due to differences in the 5' end but not the 3' end.
Proc Natl Acad Sci U S A 1987 Sep
PMID:Tissue-specific expression of insulin-like growth factor II mRNAs with distinct 5' untranslated regions. 347 48


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