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:3.1.27.5 (RNase)
17,967 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

Plasma membranes from 6 spontaneously metastasizing and 4 non-metastasizing rat mammary carcinomata were isolated by discontinuous sucrose density gradient centrifugation of microsomal pellets. The starting microsomal fraction contained 40-50% plasma membranes as determined by the levels of 5'-nucleotidase activity, with a negligible amount of nuclear (1%), mitochondrial (5%) and lysomal (7%) contamination. Five distinct fractions (F1-F5) were banded at densities 1 X 09, 1 X 13, 1 X 15, 1 X 17 and 1 X 21 at 25 degrees C, in addition to a pellet (F6) obtained by centrifuging at 76,000 g for 17 h. The fractions F1 through F5, all contained various concentrations of membranous structures, while the pellet (F6) contained only amorphous materials as evidenced by electron microscopy. The F3 fraction at the gradient 1 X 15 had the highest specific as well as total activity of the plasma membrane marker enzyme, with aggregates of the least contaminated plasma membranes in vesicular forms. This fraction also had the lowest specific activity for glucose-6-phosphatase (smooth ER marker) and for beta-D-glucuronidase (lysomal marker), and therefore was considered to be the "cleanest" plasma membrane fraction. When the activity of 4 additional plasma membrane marker enzymes, i.e., alkaline phosphatase, phosphodiesterase I, nucleotide pyrophosphatase and alkaline ribonuclease was determined in the same F3 fraction, their levels were significantly lower in every metastasizing tumour than in the non-metastasizing ones, with the enzyme activity decreasing in direct proportion to the metastasizing capacity. On the other hand, the marker enzymes were high in all non-metastasizing tumours, with the activity seemingly increasing with the immunogenicity of tumour cells. There was no significant difference between the 2 groups of mammary tumours in the levels of sialic acid, hexosamine, phospholipid or cholesterol in the plasma membranes. Thus, the level of plasma membrane marker enzymes is considered an accurate indicator for metastasizing capacity in the rat mammary tumour system.
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PMID:Plasma membrane associated enzymes of mammary tumours as the biochemical indicators of metastasizing capacity. Analyses of enriched plasma membrane preparations. 17 19

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 variety of 5'-3H-methyl-labeled, oxidized viral mRNAs were used as probes for detecting in wheat germ initiation complexes proteins that interact with, and can be cross-linked to, the 5'-cap structure. A limited and reproducible set of specific proteins was obtained with the different mRNAs. The binding of these proteins to the 5'-end of mRNA apparently results in protection against nucleotide pyrophosphatase digestion of the cap even in initiation complexes in which the 5'-end is susceptible to pancreatic RNase digestion. Cross-linked proteins from mammalian initiation complexes comigrated with several of the subunits of similarly treated eIF-3. A model for cap binding protein interaction with mRNA cap during initiation of translation is suggested.
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PMID:Interaction of a limited set of proteins with different mRNAs and protection of 5'-caps against pyrophosphatase digestion in initiation complexes. 49 38

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

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

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

2',5'-branched RNA was recently proposed as a key Ty1 retrotransposition intermediate, for which cleavage by lariat debranching enzyme (Dbr1p) enables reverse transcription to continue synthesizing the complete Ty1 cDNA. Because dbr1 cells can produce substantial Ty1 cDNA despite lacking Dbr1p, the obligatory intermediacy of branched RNA would require that Ty1 reverse transcriptase (RT) can read through the proposed branch site with considerable efficiency. Here we have used deoxyribozyme-synthesized 2',5'-branched RNA corresponding exactly to the proposed Ty1 branch site for a direct test of this read-through ability. Using an in vitro assay that incorporates all components known to be required for Ty1 cDNA synthesis (including the TyA chaperone protein), Ty1 RT can elongate up to the branch site. Strand transfer from the 2'-arm to the 3'-arm of the branch is observed when the Ty1 RT is RNase H+ (i.e., wild-type) but not when the Ty1 RT is RNase H-. When elongating from either the 2'-arm or the 3'-arm, Ty1 RT reads through the branch site with <or=0.3% efficiency. This is at least 60-fold lower than would be necessary to explain in vivo Ty1 cDNA synthesis in dbr1 cells, because others have reported 18% cDNA synthesis relative to wild-type cells. Our finding that Ty1 RT cannot efficiently read through the proposed Ty1 branch site is inconsistent with the hypothesis that branched RNA is an obligatory Ty1 retrotransposition intermediate. This suggests that Dbr1p acts as other than a 2',5'-phosphodiesterase during Ty1 retrotransposition.
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PMID:Ty1 reverse transcriptase does not read through the proposed 2',5'-branched retrotransposition intermediate in vitro. 1765 36

In bacteria, ribosomes stalled at the 3'-end of nonstop or defective mRNAs are rescued by the action of a specialized ribonucleoprotein complex composed of tmRNA and SmpB protein in a process known as trans-translation; for recent reviews see Dulebohn et al. [2007], Keiler [2007], and Moore and Sauer [2007]. tmRNA is a bifunctional RNA that acts as both a tRNA and an mRNA. SmpB-bound tmRNA is charged with alanine by alanyl-tRNA synthetase and recognized by EF-Tu (GTP). The quaternary complex of tmRNA-SmpB-EF-Tu and GTP recognizes stalled ribosomes and transfers the nascent polypeptide to the tRNA-like domain of tmRNA. A specialized reading frame within tmRNA is then engaged as a surrogate mRNA to append a 10 amino acid (ANDENYALAA) tag to the C-terminus of the nascent polypeptide. A stop codon at the end of the tmRNA reading frame then facilitates normal termination and recycling of the translation machinery. Through this surveillance mechanism, stalled ribosomes are rescued, and nascent polypeptides bearing the C-terminal tmRNA-tag are directed for proteolysis. Several proteases (ClpXP, ClpAP, Lon, FtsH, and Tsp) are known to be involved in the degradation of tmRNA-tagged proteins (Choy et al., 2007; Farrell et al., 2005; Gottesman et al., 1998; Herman et al., 1998, 2003; Keiler et al., 1996). In addition to its ribosome rescue and peptide tagging activities, trans-translation also facilitates the selective decay of nonstop mRNAs in a process that is dependent on the activities of SmpB protein, tmRNA, and the 3' to 5'-exonuclease, RNase R (Mehta et al., 2006; Richards et al., 2006; Yamamoto et al., 2003). Here, we describe methods and strategies for the purification of tmRNA, SmpB, Lon, and RNase R from Escherichia coli that are likely to be applicable to other bacterial species. Protocols for the purification of the Clp proteases, Tsp, and FtsH, as well as EF-Tu and other essential E. coli translation factors may be found elsewhere (Joshi et al., 2003; Kihara et al., 1996; Makino et al., 1999; Maurizi et al., 1990; Shotland et al., 2000). In addition, we present biochemical and genetic assays to study the various aspects of the trans-translation mechanism.
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PMID:Studying tmRNA-mediated surveillance and nonstop mRNA decay. 1916 51


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