Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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)

RNA is not cleaved as a consequence of the binding of RNase H to the duplex between RNA and a complementary alpha-oligodeoxyribonucleotide (oligo). In consequence targets have been selected which do not a priori require the action of RNase H to inhibit genetic expression. Two models have been used: The Friend Murine Leukemia Virus (F-MuLV) and the synthesis of rabbit beta globin.alpha-oligos trigger specific inhibitions in both systems. The functionalisation in 5' with the intercalating agent 9-NH2-ellipticine renders the oligos resistant to degradation and allows a direct action on cells.
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PMID:Comparison of anti-RNA properties of normal and ellipticine functionalized alpha and beta-oligonucleotides. 166 83

The functional domains of the avian retrovirus polymerase gene are at least tripartite in nature. Three enzymatic domains exist; the RNase H and DNA polymerase activities are located on the alpha subunit while the DNA endonuclease is located on the pp32 moiety. Virus mutants possessing deletions in the pp32 region demonstrated that this region encodes function(s) essential for replication of the virus while separate point mutations generated near the NH2 terminus of pp32 resulted in decreased replication and cell transformation. Molecular analysis of various steps in the virus replication cycle demonstrated that the synthesis of linear viral DNA, transport of viral DNA to the nucleus, and its subsequent circularization and integration into cellular DNA are apparently not affected in these point mutants. However, the synthesis of viral RNA from the integrated provirus of these point mutants appears less than that observed in wild type virus-infected cells. What role the mutated pp32 protein might have on viral transcription is discussed.
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PMID:Mutants of the Rous sarcoma virus reverse transcriptase gene are nondefective in early replication events. 240 84

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.
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PMID:Recombinant HIV-1 reverse transcriptase: purification, primary structure, and polymerase/ribonuclease H activities. 247 69

A full-length cDNA clone of the mRNA encoding the phosphoprotein (NS) of the Indiana serotype of vesicular stomatitis virus was inserted into the SP6 transcription vector. By in vitro transcription of the inserted gene followed by translation of the mRNA in a rabbit reticulocyte lysate, NS protein was synthesized. The biological activity of the protein was demonstrated by RNA synthesis in vitro by reconstitution with L protein and N-RNA template purified from virions. Using oligonucleotide-directed RNase H cleavage of the full-length NS mRNA, a series of deleted RNAs were made which gave rise to corresponding size classes of truncated NS protein after translation in vitro. The N-RNA template binding site was located at the C-terminal domain (21 amino acids) of the NS protein and the L-protein binding site was present within 14 amino acids spanning the NH2-terminal side of the N-RNA binding site. These results are similar to that obtained with the NS protein of the New Jersey serotype of VSV, indicating conservation of the functional domains within the VSV serotypes.
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PMID:The functional domains of the phosphoprotein (NS) of vesicular stomatitis virus (Indiana serotype). 284 48

The POMC gene is predominantly expressed in the pituitary gland; it is also expressed in various extrapituitary tissues. While POMC mRNAs of similar size (approximately equal to 1000 nucleotides) are present in the anterior and neurointermediate lobes of the pituitary, other POMC-expressing tissues contain POMC mRNAs of different sizes. Longer POMC mRNAs are observed in the hypothalamus. Using S1 nuclease mapping and mRNA deadenylation by RNase H, we have shown that these large hypothalamic POMC mRNAs have longer poly(A) tails than pituitary POMC transcripts but contain the same transcripted sequences. In contrast, the testes contain POMC transcripts which are smaller than pituitary POMC mRNA. RNase and S1 nuclease mapping analyses suggest that these short transcripts do not contain sequences transcribed from pituitary exons 1 and 2. Indeed, as revealed by primer-extension experiments, these transcripts appear to initiate within exon 3 sequences of the POMC gene. The heterogeneous 5'-ends of these short testicular transcripts map into the NH2-terminal portion of the precursor in the region encoding gamma MSH; if ever translated, these transcripts would produce a form of POMC that would be truncated at the NH2-terminus and therefore would be devoid of any signal peptide sequence. Interestingly, the sequence of the short testicular transcripts corresponds to that of the mouse POMC pseudogene, suggesting that this POMC pseudogene may have derived from genomic integration of testicular transcripts via a cDNA intermediate.
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PMID:Unusual proopiomelanocortin ribonucleic acids in extrapituitary tissues: intronless transcripts in testes and long poly(A) tails in hypothalamus. 285 1

Using polysomal immunoselected rat liver glutathione S-transferase mRNAs, we have constructed cDNA clones using DNA polymerase I, RNase H, and Escherichia coli ligase (NAD+)-mediated second strand cDNA synthesis as described by Gubler and Hoffman (Gubler, U., and Hoffman, B. S. (1983) Gene 25, 263-269). Recombinant clone, pGTB42, contained a cDNA insert of 900 base pairs whose 3' end showed specificity for the Yc mRNA in hybrid-select translation experiments. The nucleotide sequence of pGTB42 has been determined, and the complete amino acid sequence of a Yc subunit has been deduced. The cDNA clone contains an open reading frame of 663 nucleotides encoding a polypeptide comprising 221 amino acids with a molecular weight of 25,322. The NH2-terminal sequence deduced from pGTB42 is in agreement with the first 39 amino acids determined for a Ya-Yc heterodimer by conventional protein-sequencing techniques. A comparison of the nucleotide sequence of pGTB42 with the sequence of a Ya clone, pGTB38, described previously by our laboratory (Pickett, C. B., Telakowski-Hopkins, C. A., Ding, G. J.-F., Argenbright, L., and Lu, A.Y.H. (1984) J. Biol. Chem. 259, 5182-5188) reveals a sequence homology of 66% over the same regions of both clones; however, the 5'- and 3'-untranslated regions of the Ya and Yc mRNAs are totally divergent in their sequences. The overall amino acid sequence homology between the Ya and Yc subunits is 68%, however, the NH2-terminal domain is more highly conserved than the middle or carboxyl-terminal domains. Our data suggest that the Ya and Yc subunits of the rat liver glutathione S-transferases are products of two different mRNAs which are derived from two related yet different genes.
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PMID:Rat liver glutathione S-transferases. Construction of a cDNA clone complementary to a Yc mRNA and prediction of the complete amino acid sequence of a Yc subunit. 298 14

The enzymatic domains of the avian retrovirus polymerase (pol) gene have been mapped by the use of peptide antibodies and COOH-terminal amino acid analysis. The processed pol beta polypeptide is cleaved in vivo to yield alpha and pp32. Rabbit antibodies were directed against synthetic peptides whose sequence was deduced from the known pol sequence of Rous sarcoma virus, Prague C (Schwartz, D.E., Tizard, R., and Gilbert, W. (1983) Cell 32, 853-869). The RNase H active site of pol was located in the NH2-terminal region of the alpha DNA polymerase subunit. The COOH terminus of the alpha subunit was found to be immediately adjacent to the NH2 terminus of the pp32 pol protein. COOH-terminal amino acid analysis of pp32 revealed that this protein is also processed. From the deduced amino acid sequence of pol, it appears likely that pol encodes an additional 4100-dalton polypeptide located at its extreme COOH terminus. The enzymatic domains on beta appear to map in the following order: RNase H-DNA polymerase-DNA endonuclease. Hydrophilicity analysis and secondary structure predictions of wild type Rous sarcoma virus pol products and mutated pp32 possessing single amino acid changes permit further structural evaluation of the multifunctional pol protein.
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PMID:Structural characterization of the avian retrovirus reverse transcriptase and endonuclease domains. 298 84

The NH2-terminal amino acid sequence of the pp32 DNA binding protein has been determined, thus establishing its precise coding region in the polymerase gene of Rous sarcoma virus. Specific mutations were constructed in molecularly cloned Prague A DNA near the NH2- and COOH-termini of pp32 and the effects were assayed by transfection on chick embryo fibroblasts. Out-of-frame deletions at both sites and an in-frame deletion near the NH2 terminus rendered the DNA noninfectious and transformation negative. Single point mutations near the NH2 terminus reduced the transfection efficiency and the rate of virus replication. Biochemical studies indicated that the RNA-directed DNA polymerase and RNase H activities of the mutant viruses were not affected but the processing of the viral beta polypeptide was altered.
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PMID:Requirement of the avian retrovirus pp32 DNA binding protein domain for replication. 609 34

The gene for Escherichia coli ribonuclease H has been studied by use of a plasmid which contains a segment of the E. coli chromosome. The genomic DNA was subcloned from pLC28-22 to pBR322 by use of various restriction enzymes. Such subcloning limited the RNase H gene to a piece of DNA no longer than 760 base pairs. Cells bearing plasmids containing the RNase H gene produce as much as 10-15 times the normal amount of RNase H without any drastic effect on maintenance of the plasmid or cell growth. DNA sequence analysis has permitted the prediction of a protein whose molecular weight is 17,559 (155 amino acid residues). The predicted sequence was confirmed by amino acid analysis, NH2-terminal amino acid sequence, and size determination of highly purified RNase H.
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PMID:DNA sequence of the gene coding for Escherichia coli ribonuclease H. 629 74

Syntheses of non ionic oligodeoxynucleoside phosphoramidates (P-NH2) and mixed phosphoramidate- phosphodiester oligomers were accomplished on automated solid supported DNA synthesizer using both H-phosphonate and phosphoramidite chemistries, in combination with t-butylphenoxyacetyl for N-protection of nucleoside bases, an oxalyl anchored solid support and a final treatment with methanolic ammonia. Thermal stabilities of the hybrids formed between these new analogues and their DNA and RNA complementary strands were determined and compared with those of the corresponding unmodified oligonucleotides, as well as of the phosphorothioate and methylphosphonate derivatives. Dodecathymidines containing P-NH2 links form less stable duplexes with DNA targets, d(C2A12C2) (deltaTm/modification -1.4 degrees C) and poly dA (deltaTm/modification -1.1 degrees C) than the corresponding phosphodiester and methylphosphonate analogues, but the hybrids are slightly more stable than the one obtained with phosphorothioate derivative. The destabilization is more pronounced with poly rA as the target (deltaTm/modification -3 degrees C) and could be compared with that found with the dodecathymidine methylphosphonate. The modification is less destabilizing in an heteropolymer-RNA duplex (deltaTm/modification -2 degrees C). As expected, the P-NH2 modifications are highly resistant towards the action of various nucleases. It is also demonstrated that an all P-NH2 oligothymidine does not elicit Escherichia coli RNase H hydrolysis of the poly rA target but that the modification may be exploited in chimeric oligonucleotides combining P-NH2 sections with a central phosphodiester section.
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PMID:Oligodeoxynucleoside phosphoramidates (P-NH2): synthesis and thermal stability of duplexes with DNA and RNA targets. 865 64


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