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:3.1.27.1 (RNase)
16,360 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The nucleotide sequences were determined for the 5'-oligonucleotides obtained by complete pancreatic RNase digestion (P25) and complete T1 RNase digestion (T27) of U-2 RNA. Complete digestion of oligonucleotide P25 with snake venom phosphodiesterase produced pm3 2,2,7G, pAm, pUm, and pCp in approximately equimolar ratios. Partial digestion of these oligonucleotides with snake venom phosphodiesterase produced -Um-C-Gp and pAm-Um, indicating the sequence of the 3'-terminal portion of the 5'-oligonucleotide is pAm-Um-C-Gp. The 5'-terminal oligonucleotide did not contain a 5'-phosphate and no free nucleoside was released from the 5' end by venom phosphodiesterase digestion. Since free pm3 2,2,7G was released by digestion with nucleotide pyrophosphatase and limited digestion with snake venom phosphodiesterase, this nucleotide is apparently linked to pAm in a pyrophosphate linkage. Mass spectrometry and thin layer chromatography in borate systems showed the ribose of m3 2, 2, 7G contains no 2'O-methyl residue. Moreover, the finding that the ribose of m3 2, 2, 7G was oxidized by NaIO4 and reduced by KB3H4 in intact U-2 RNA rules out other linkages involving the 2' and 3' positions. Accordingly, it is concluded that the structure of the 5'-terminal pentanucleotide of U-2 RNA is(see article).
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PMID:Nucleotide sequence of U-2 ribonucleic acid. The sequence of the 5'-terminal oligonucleotide. 16 89

Methyl groups derived from 3H-methyl methionine were incorporated into vesicular stomatitis virus (VSV) MRNAs isolated from infected cells. Sequential degradation of the 12-18S viral mRNA species with ribonuclease T2, penicillium nuclease, and alkaline phosphatase yielded a single 3H-labeled dinucleotide. A similar resistant 32P-labeled fragment was obtained by digesting VSV mRNA uniformly labeled with 32P. This methylated and blocked oligomer was further cleaved with nucleotide pyrophosphatase, yielding two methylated 5' nucleotides. We postulate that the 5' terminal structure of the vivo 12-18S VSV mRNA contains 7-methylguanosine linked by a 5'-5' pyrophosphate bond to a methylated derivative of adenosine. In contrast to the mRNAs (+ strand), the VSV genome RNA ( MINUS STRAND) IS NOT BLOCKED.
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PMID:Methylated and blocked 5' termini in vesicular stomatitis virus in vivo mRNAs. 16 1

32P-labeled, late simian virus 40-specific RNA was isoalted from infected CV1 cells and completely degraded with RNase T2 and bacterial alkaline phosphatase. The RNase-resistant material was fractionated two dimensionally and further characterized with Penicillium nuclease and nucleotide pyrophosphatase. Two major 5' termini were identified in late simian virus 40 RNA, namely, 7-methyl Gppp 2',6-dimethyl ApUp and 7-methyl Gppp 2',6-dimethyl Ap 2'-methyl, UpUp. Both 5' termini are present in unfractionated viral RNA as well as in the separated 16S and 19S species. As both caps differ only in secondary modification, it is possible that they are derived from the same site on the DNA. The relatively higher cap II content of the 16S mRNA may be related to its slower rate of turnover.
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PMID:Characterization of the 5'-terminal capped structures of late simian virus 40-specific mRNA. 20 72

Isolated plasma membranes from mouse fibroblast lines 3T3 and its tranformant SV-3T3 contain a phosphodiesterase (oligonucleotidase, E.C. 3.1.4.19; nucleotide pyrophosphatase, E.C. 3.6.1.9) that splits capped and methylated messenger RNA obtained from both reovirus and vesicular stomatitis virus. The isolated membranes are free of demonstrable ribonuclease activity and split the mRNA to produce 7-methyl guanosine diphosphate as a product. With ATP as substrate for the phosphodiesterase enzyme, the product is AMP. Synthetic caps, AMP, ADP and ATP, but not cyclic AMP, can compete with the substrate p-nitrophenyl thymidilic acid. A possible regulatory role on messenger translation is proposed.
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PMID:Uncapping of viral messenger RNA by phosphodiesterase of fibroblast plasma membranes. 22 44

A subcellular system is described which is capable of in vitro synthesis of large nuclear RNA and the formation of both cap I [m7G(5')pppXmpYp] and capII [m7G(5')-pppXmpYmpZp] structures. This system, which consists of partially purified intact nuclei and residual cytoplasmic tags, carries out both guanosine addition, utilizing GTP, and the appropriate methylation reactions, utilizing S-adenosylmethionine as the methyl donor. The general structure of the caps was verified by analyses of methylated derivatives recovered after RNase T2 hydrolysis and after digestion with P1 nuclease, bacterial alkaline phosphatase,and nucleotide pyrophosphatase. Cap formation in large nuclear RNA species was found to be closely associated with transcription, as indicated by alpha-manitin sensitivity and a requirement for the presence of all four nucleoside triphosphates. Recovery of a class of cap II structures, in which only the methyl group at position Y is labeled, as well as capII structures in which all methylated constituents are labeled, indicates the presence of at least two independent methylation events in the in vitro system.
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PMID:Synthesis methylation, and capping of nuclear RNA by a subcellular system. 99 Feb 62

Phosphodiesterase I [EC 3.1.4.1] was purified from normal human urine in a highly purified state free from phosphodiesterase II, RNase, DNase I, DNase II, and phosphatase by column chromatographies of DEAE-Toyopearl, butyl-Toyopearl, Affi-Gel blue, and Sephadex G-150. The molecular weight of the enzyme was 1.9 x 10(5) and the pH optimum around 9.0 with p-nitrophenyl deoxythymidine 5'-phosphate as the substrate. The enzyme hydrolyzed the 3'-5' linkage of various dinucleoside monophosphates at approximately the same rate and the phosphodiester bonds of cyclic 3',5'-mononucleotides to produce mononucleoside 5'-phosphate. The enzyme also hydrolyzed ADP to 5'-AMP and Pi, ATP to 5'-AMP and PPi, and NAD+ to 5'-AMP and NMN. The enzyme activity was abolished by removal of metal ions with EDTA, and the metal-free enzyme was reactivated on the addition of Zn2+. The enzyme activity was also abolished by some reducing agents and the inhibition was reversed by Zn2+. The metal-free enzyme was less stable than the native enzyme, and Zn2+ and Co2+ restored the stability of the metal-free enzyme to the level of the native enzyme. The enzyme degraded oligonucleotides and high molecular nucleotides stepwise from the 3'-termini to give 5'-mononucleotides. The enzyme hydrolyzed single-stranded DNA more preferentially than double-stranded DNA. The enzyme also nicked superhelical covalently closed circular phi X174 DNA to yield first open circular DNA and then linear DNA.
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PMID:Phosphodiesterase I in human urine: purification and characterization of the enzyme. 282 85

A single capped oligonucleotide is released from Trypanosoma brucei poly(A)+ RNA upon digestion with RNase T2. This observation supports the hypothesis that all T. brucei mRNAs share a common leader sequence. Digestion of the T2-resistant species with nucleotide pyrophosphatase shows that the capping nucleotide is 7-methylguanosine 5'-monophosphate (pm7G). Additional characterization of the T2-resistant fragment indicates that modifications are present on the first four transcribed nucleotides; the 5' termini of T. brucei mRNAs can, therefore, be described as "cap 4" structures. Identical 5'-cap structures are found on the T. brucei spliced leader (SL) RNA; an observation compatible with the hypothesis that the small SL RNA acts as a donor of the SL for the mRNA. However, we find that within a population of purified SL RNAs are species that are capped but incompletely modified. The presence of these unmodified and partially modified species allowed us to analyze the 5' sequence of the SL RNA transcript. The results indicate that transcription begins four nucleotides upstream of the reported 5' end. Therefore, the T. brucei SL transcript is actually 39 rather than 35 nucleotides long. We have also analyzed the capped oligonucleotide of a distantly related Trypanosomatid, Leptomonas collosoma and find it to be identical to that of T. brucei. The potential significance of these results is discussed in light of observations of trypanosome gene expression.
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PMID:Trypanosome mRNAs have unusual "cap 4" structures acquired by addition of a spliced leader. 312 Jan 86

The vast majority of nuclease activity in yeast mitochondria is due to a single polypeptide with an apparent molecular weight of 38,000. The enzyme is located in the mitochondrial inner membrane and requires non-ionic detergents for solubilization and activity. A combination of heparin-agarose and Cibacron blue-agarose chromatography was employed to purify the nuclease to approximately 90% homogeneity. The purified enzyme shows multiple activities: 1) RNase activity on single-stranded, but not double-stranded RNA, 2) endonuclease activity on single- and double-stranded DNA, and 3) a 5'-exonuclease activity on double-stranded DNA. Digestion products with DNA contain 5'-phosphorylated termini. Antibody raised against an analogous enzyme purified from Neurospora crassa (Chow, T. Y. K., and Fraser, M. (1983) J. Biol. Chem. 258, 12010-12018) inhibits and immunoprecipitates the yeast enzyme. This antibody inhibits 90-95% of all nuclease activity present in solubilized mitochondria, indicating that the purified nuclease accounts for the bulk of mitochondrial nucleolytic activity. Analysis of a mutant strain in which the gene for the nuclease has been disrupted supports this conclusion and shows that all detectable DNase activity and most nonspecific RNase activity in the mitochondria is due to this single enzyme.
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PMID:Purification and properties of the major nuclease from mitochondria of Saccharomyces cerevisiae. 328 39

Ribonuclease II is a processive 3'- to 5'-exoribonuclease in Escherichia coli with two binding sites: a catalytic site associated with the first few 3'-nucleotides and an anchor site binding nucleotides approximately 15 to 25 from the 3'-end. When RNase II degrades single-stranded helical poly(C), the enzyme-substrate complex dissociates at discrete intervals of 12 nucleotides. RNase II stalled at the last rC of single-stranded 3'-(rC)(n)(dC)(m) oligonucleotides. The more residues released, the faster the stalled complex dissociated and the less it inhibited RNase II activity, i.e. the enzyme-substrate association weakened progressively. Using phosphodiesterase I (PDE I) as a probe, a method was developed to identify cytidine residues in (32)P-oligonucleotides interacting with a protein. PAGE bands corresponding to nucleotides 1-6 from the 3'-end were consistent with interaction at the catalytic site, and following a gap, bands approximately 15 to 25 from the 3'-end, with anchor site association. Both 3' and 5' binding were necessary to maintain the complex. Of most significance, the original anchor site nucleotides remained fixed at the anchor site while the 3'-end was pulled, or threaded, through the catalytic site, i.e. the substrate did not 'slide' through the enzyme. DNA oligonucleotides with double-stranded stem-loops were good competitive inhibitors of RNase II. A 3'-single-stranded arm was essential, while optimal binding required both 5'- and 3'-arms. PDE I probing indicated that the nucleotides at the anchor site were specified by the spatial distance from the catalytic site, and on only one of the duplex strands. When degradation of a structured RNA paused or stopped, the RNase II-product commenced cycles of dissociation-reassociation. Duplex strand binding by RNase II made complex DNA or RNA structures accessible to degradation by other nucleases and further verified the PDE I footprinting method.
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PMID:The reaction mechanism of ribonuclease II and its interaction with nucleic acid secondary structures. 1044 70

In the murine model for respiratory syncytial virus (RSV) infection, cytokine patterns induced by vaccinations with either killed (i.e. formalin-inactivated, alum-precipitated) virus (KV) or live virus (LV) have been shown to influence disease expression. To determine the mRNA expression of the cytokines IL-4 and IFN-gamma in BALB/c mice challenged with RSV, a real-time quantitative reverse-transcriptase PCR assay was developed. This assay uses 5'-exonuclease fluorogenic probes and is performed on the ABI PRISM 7700 Sequence Detector System (TaqMan). The relative quantitative levels of mRNA for IL-4 and IFN-gamma were compared with those measured by an RNase protection assay (RPA) and an enzyme immunoassay (EIA), which are methods used to measure the levels of mRNA and protein, respectively. Results obtained by the TaqMan assay showed that mice primed with KV induces increased IL-4 mRNA production while LV induces increased IFN-gamma mRNA, which is in agreement with conventional methods. IL-4 and IFN-gamma relative quantities obtained from TaqMan were highly correlated to those determined by RPA (r=0.96 for IFN-gamma, P<0.01) and EIA (r=0.90 for IL-4 and r=0.75 for IFN-gamma, P<0.01). Assay reproducibility was examined by testing a same sample in triplicate at three experiments. Minimal deviation values were observed in both intra- and inter-assays. TaqMan, which is rapid, sensitive and reproducible, provides an alternative tool for the quantitative analysis of cytokine mRNA expression in the murine model of RSV immunopathogenesis.
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PMID:Cytokine expression in respiratory syncytial virus-infected mice as measured by quantitative reverse-transcriptase PCR. 1250 27


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