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)

Several enzymes with ribonuclease H specificity have been identified in bovine lymphoid tissue. Their nomenclature and some of their properties have been reported previously. In this study the time course of the induction of the different ribonuclease H activities during the stimulation of resting bovine lymph-node cells with concanavalin A was investigated. The activity of one of these enzymes (ribonuclease H IIb) increases in parallel with the induction of uridine incorporation and is well separated from the induction of ribonuclease H I, which increases together with DNA synthesis. These data indicate that the different ribonuclease H activities serve different physiological functions. They suggest that ribonuclease H I belongs to the set of enzymes which are involved in DNA synthesis.
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PMID:Ribonuclease H levels during the response of bovine lymphocytes to concanavalin A. 85 58

Messenger RNA molecules 30-35 bases long, with sequences related to the 5'-region of cro-mRNA from lambda-phage, were prepared by T7 transcription from synthetic DNA templates. Each mRNA contained five or six internal uridine residues, which were transcribed using a mixture of UTP and thio-UTP. Initiation complexes were formed with Escherichia coli 30S ribosomes in the presence or absence of tRNA(fMet), and cross-linking of the thio-U residues was induced by UV irradiation at wavelengths greater than 300 nm. The cross-linked ribosomal proteins were identified immunologically, and cross-linked regions of the 16S RNA were isolated by excision with ribonuclease H and suitable deoxyoligonucleotides. In both cases, the particular thio-U residue involved in the cross-link was identified by ribonuclease T1 fingerprinting of the (radioactive) mRNA in the isolated cross-linked complex. The principal results were that, at thio-U positions upstream of the AUG codon, specific cross-linking occurred to protein S7 and to the 3'-terminus of the 16S RNA, in agreement with similar experiments using 70S ribosomes. Less specific cross-linking was observed to proteins S1, S18 and S21 at various positions within the mRNA. Six bases downstream from the AUG codon, a tRNA-dependent cross-link was found to position approximately 1050 of the 16S RNA, but--in contrast to similar experiments with 70S ribosomes--no cross-linking was found to the 1390-1400 region.
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PMID:The location of mRNA in the ribosomal 30S initiation complex; site-directed cross-linking of mRNA analogues carrying several photo-reactive labels simultaneously on either side of the AUG start codon. 165 Dec 32

We have studied the distribution of DNA X RNA hybrids on polytene chromosomes with the aid of a goat antibody against DNA X RNA hybrids using the immunofluorescence technique. Fixed polytene chromosomes of the sciarid Trichosia pubescens (Diptera) show distinct, stage-specific labelling patterns throughout larval development. Controls for the staining procedure - including preincubation with hybrid-specific endoribonuclease H - prove that DNA X RNA hybrids are present on fixed chromosomes. They are revealed only under mild fixation conditions which do not efficiently immobilize all chromosomal proteins, indicating that some proteins have to be removed to make the antigens accessible to antibody. Certain fixation conditions may also cause local denaturation of chromosomal DNA, and some hybrids may possibly form during specimen preparation. After incorporation of radioactive uridine, a combination of phase contrast, fluorescent, and autoradiographic images of one and the same chromosomal preparation demonstrates that hybrid fluorescence is confined to transcriptionally active regions. Two puff classes can be distinguished. The first binds antibody and includes most RNA puffs and all DNA puffs so far studied; the second, comprising some RNA puffs, does not show bright fluorescence in spite of the fact that RNA synthesis is high as revealed by 3H-uridine incorporation. DNA X RNA hybrids are not found at DNA puff sites during the DNA amplification period; these sites contain detectable hybrids only when transcription is taking place. - Combination of the fluorescent technique with its excellent resolution and autoradiography should be helpful in studying detailed topological aspects of transcriptionally active chromosomal regions.
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PMID:Immunofluorescent characterization of DNA . RNA hybrids on polytene chromosomes of Trichosia pubescens (Diptera, sciaridae). 618 42

"Uniformly" modified phosphodiester or phosphorothioate oligonucleotides incorporating 2'-deoxy-2'-fluoroadenosine, -guanosine, -uridine, and -cytidine, reported herein for the first time, when hybridized with RNA afforded consistent additive enhancement of duplex stability without compromising base-pair specificity. CD spectra of the 2'-deoxy-2'-fluoro-modified oligonucleotides hybridized with RNA indicated that the duplex adopts a fully A-form conformation. The 2'-deoxy-2'-fluoro-modified oligonucleotides in phosphodiester form were not resistant to nucleases; however, the modified phosphorothioate oligonucleotides were highly nuclease resistant and retained exceptional binding affinity to the RNA targets. The stabilizing effects of the 2'-deoxy-2'-fluoro modifications on RNA-DNA duplexes were shown to be superior to those of the 2'-O-methylribo substitutions. RNA hybrid duplexes with uniformly 2'-deoxy-2'-fluoro-modified oligonucleotides did not support HeLa RNase H activity; however, incorporation of the modifications into "chimeric" oligonucleotides has been shown to activate mammalian RNase H. "Uniformly" modified 2'-deoxy-2'-fluoro phosphorothioate oligonucleotides afforded antisense molecules with (1) high binding affinity and selectivity for the RNA target and (2) stability toward nucleases.
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PMID:Uniformly modified 2'-deoxy-2'-fluoro phosphorothioate oligonucleotides as nuclease-resistant antisense compounds with high affinity and specificity for RNA targets. 846 37

The naturally occurring nucleotide 3-(3-amino-3-carboxy-propyl)uridine (acp3U) at position 47 of tRNA(Phe) from Escherichia coli was modified with a diazirine derivative and bound to ribosomes in the presence of suitable mRNA analogues under conditions specific for the ribosomal A, P or E sites. After photo-activation at 350 nm the cross-links to ribosomal proteins and RNA were identified by our standard procedures. In the 30S subunit protein S19 (and weakly S9 and S13) was the target of cross-linking from tRNA at the A site, S7, S9 and S13 from the P site and S7 from the E site. Similarly, in the 50S subunit L16 and L27 were cross-linked from the A site, L1, L5, L16, L27 and L33 from the P site and L1 and L33 from the E site. Corresponding cross-links to rRNA were localized by RNase H digestion to the following areas: in 16S rRNA between positions 687 and 727 from the P and E sites, positions 1318 and 1350 (P site) and 1350 and 1387 (E site); in the 23S rRNA between positions 865 and 910 from the A site, 1845 and 1892 (P site), 1892 and 1945 (A site), 2282 and 2358 (P site), 2242 and 2461 (P and E sites), 2461 and 2488 (A site), 2488 and 2539 (all three sites) and 2572 and 2603 (A and P sites). In most (but not all) cases, more precise localizations of the cross-link sites could be made by primer extension analysis.
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PMID:The ribosomal neighbourhood of the central fold of tRNA: cross-links from position 47 of tRNA located at the A, P or E site. 852 54

The naturally occurring nucleotide 3-(3-amino-3-carboxy-propyl) uridine ("acp3U") at position 20:1 of lupin tRNAMet was coupled to a photoreactive diazirine derivative. Similarly, the 4-thiouridine at position 8 of Escherichia coli tRNAPhe was modified with an aromatic azide. Each of the derivatized tRNAs was bound to E. coli ribosomes in the presence of suitable mRNA analogues, under conditions specific for the A, P, or E sites. After photoactivation of the diazirine or azide groups, the sites of crosslinking from the tRNAs to 16S or 23S rRNA were analyzed by our standard procedures, involving a combination of ribonuclease H digestion and primer extension analysis. The crosslinked ribosomal proteins were also identified. The results for the rRNA showed a well-defined series of crosslinks to both the 16S and 23S molecules, the most pronounced being (1) an entirely A-site-specific crosslink from tRNA position 20:1 to the loop-end region (nt 877-913) of helix 38 of the 23S RNA (a region that has not so far been associated at all with tRNA binding), and (2) a largely P-site-specific crosslink from tRNA position 8 to nt 2111-2112 of the 23S RNA (nt 2112 being a position that has previously been identified in footprinting studies as belonging to the ribosomal E site). The data are compared with results from a parallel study of crosslinks from position 47 (also in the central fold of the tRNA), as well as with previously published crosslinks from the anticodon loop (positions 32, 34, and 37) and the CCA-end region (position 76, and the aminoacyl residue).
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PMID:The ribosomal environment of tRNA: crosslinks to rRNA from positions 8 and 20:1 in the central fold of tRNA located at the A, P, or E site. 859 57

Reduced tension of O2 slows the degradation rate of mRNA for tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine synthesis, in the pheochromocytoma (PC12) clonal cell line. The observed increase in half-life (30 h versus 10 h) correlates with enhanced binding of a 66-kDa protein (hypoxia inducible protein) to the pyrimidine-rich tract located between bases 1552 1578 in the 3 -untranslated region of TH mRNA (hypoxia-inducible protein binding site (HIPBS)). The present study investigates the protein binding site within the 27-base HIPBS, first by using specific cleavages of HIPBS and its flanking sequences with antisense oligodeoxynucleotides and RNase H and then by using mutational analysis of the binding properties. We found that the 27-base HIPBS oligoribonucleotide was sufficient to bind the protein in vitro in a hypoxia-stimulated manner. We further identified the optimal hypoxia-inducible protein binding site that is represented by the motif (U/C)(C/U)CCCU, where the core binding site is indicated by the underlined cytidines. Substitutions of either one of the cytidines with purine or uridine abolished the protein binding. The mutations within HIPBS, which partially reduced binding, did not prevent stimulation of protein binding for extracts from hypoxic cells. The hypoxia-induced increase in complex formation was proportional to the strength of binding using proteins from normoxic cells. The HIPBS element is conserved in TH mRNAs derived from different species.
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PMID:Characterization of the hypoxia-inducible protein binding site within the pyrimidine-rich tract in the 3'-untranslated region of the tyrosine hydroxylase mRNA. 862 33

Expression of messenger RNA (mRNA) in both embryonic and adult cells may be profoundly influenced by untranslated sequences in the 3'-end. Elements in the 3'-untranslated regions (UTRs) of messengers are known to influence messenger stability, polyadenylation, and translation. We have examined the effects of the 3'-UTR of Xenopus laevis c-mycI (either alone or in combination with the 5'-first exon) on the expression of a chloramphenicol acetyltransferase (CAT) reporter in Xenopus embryos. The Xenopus c-mycI 3'-UTR enhanced messenger translation independent of the 5'-UTR. RNase H analysis indicated that the Xenopus c-mycI 3'-UTR can promote the cytoplasmic polyadenylation of CAT mRNA in embryos. The result suggests that the post-fertilization enhancement of translation caused by the c-mycI 3'-UTR may be a consequence of cytoplasmic polyadenylation. A uridine (U)-rich sequence in the Xenopus c-mycI 3'-UTR that may be responsible for polyadenylation is similar to an element that destabilizes mammalian c-myc transcripts. We discuss the possibility that U-rich sequences may play a dual role by destabilizing growth-related transcripts in adult cells and stimulating their polyadenylation during development, and we propose that a switch in the role of such sequences in adult cells could lead to stabilization of these messengers, increased translation, and abnormal growth control.
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PMID:Stimulation of translation and cytoplasmic polyadenylation by the Xenopus c-mycI 3'-untranslated region. 940

Two analog uridine triphosphates tethering additional functionality, one a primary amino group and the second a mercapto group, were prepared and tested for their compatibility with in vitro RNA selection procedures. 5-(3-Aminopropyl)uridine triphosphate (UNH(2)) as a uridine substitute was a more effective substrate for T7 RNA polymerase than 5-(2-mercaptoethyl)uridine triphosphate (USH). However, both functioned in transcription assays of 100 nt templates to generate RNA transcripts in quantities sufficient to initiate RNA selection procedures. Transcription of RNA pools with T7 RNA polymerase and UNH(2) or USH occurred with efficiencies of 43 and 29%, respectively, of the values obtained for native UTP transcription. In addition, the transcribed RNA containing roughly 25% UNH(2) residues exhibited better substrate properties for SuperScript(TM) II RNase H reverse transcriptase than did RNA transcripts containing approximately 25% of the USH analog. With either analog, both transcription and reverse transcription proceeded with high fidelity for insertion of the analog residue.
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PMID:Expanding the structural and functional diversity of RNA: analog uridine triphosphates as candidates for in vitro selection of nucleic acids. 1095

The high rate of mutation which is inherent in reverse transcription of the HIV genome is a result of the lack of proof-reading function of the reverse transcriptase enzyme. This has allowed the HIV virus to develop resistance to multiple antiviral agents. It may be possible to use this viral property to advantage by treatment with an antiviral nucleoside analogue which is a close structural isostere of uridine and deoxyuridine. The drug is unable to form hydrogen bonds with adenine and will be excluded from host cell DNA by its 3' to 5' proof-reading exonuclease activity. However, reverse transcriptase, which has no such mechanism, will allow incorporation of the drug into proviral DNA. The drug will have an inhibitory effect on RNase H function. It will also be expected to cause delay in elongation at those sites in the template strand that contain two or more adjacent adenine bases, because two drug molecules will, for practical purposes, never be inserted in the same strand next to each other. The length of the delay in strand elongation will therefore be a function of the availability of the natural NTP or dNTP. Both the rate and fidelity of protein synthesis will be affected by the drug. There will be decreased stability of the proviral double stranded DNA and if the proviral DNA is able to integrate into the host cell chromosome, double stranded breaks may be produced by the host cells' DNA repair mechanisms. Finally there will be a specific 'strand trade' mutation that the drug will induce specifically into viral but not into cellular genetic material.
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PMID:A nucleoside analogue of 2, 4-difluoropyridine has potential as an antiretroviral agent with multiple and unique mechanisms of action, and may be effective against the HIV organism. 1105 20


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