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:3.1.30.2 (endonuclease)
18,621 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Estrogen-mediated accumulation of the avian apolipoprotein (apo) II mRNA is in part due to its stabilization. To identify the biochemical activity responsible for this effect, radiolabeled, capped, and polyadenylated apoII mRNA was incubated in vitro in liver cytosolic extracts from roosters who received either estrogen (estrogen-treated extract) or the vehicle (control extract) parenterally. The mRNA was very stable in estrogen-treated extract but was rapidly degraded in control extract. The RNA was degraded predominantly by endonuclease rather than exonuclease activity. The addition of the estrogen-treated extract to the control extract prevented the degradation of the mRNA in trans. This biochemical activity was heat labile and was also destroyed by proteinase K but not by micrococcal nuclease, indicating that estrogen treatment resulted in the expression of a protein in the liver that stabilized the apoII mRNA by inhibiting its nucleolytic degradation. This mRNA stabilization factor was labile around 60 degrees C, whereas the RNase remained stable up to 80 degrees C. Studies on mRNA protein interaction showed that both control and estrogen-treated extracts contain mRNA-binding (mRNP) proteins that bind apoII mRNA. An increased binding to apoII mRNA by a subset of these proteins was observed with estrogen-treated extract as compared with the control extract. This activity, although it afforded complete protection from nucleolytic degradation to apoII and apo A1 mRNAs, appeared to provide less protection to mRNAs encoding chicken serum albumin and vitellogenin, suggesting differential stabilization of mRNAs. These studies indicate that a cytosolic mRNA-stabilization factor, providing apoII mRNA complete protection from nucleolytic degradation, is expressed in the avian liver upon estrogen treatment. This appears to be the first time that a biochemical activity responsible for hormone-mediated stabilization of mRNAs and estrogen induction of mRNA binding by specific mRNPs have been identified and partially characterized in vitro.
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PMID:In vitro characterization of an estrogen-regulated mRNA stabilizing activity in the avian liver. 877 38

We have investigated the endonuclease activity of the influenza A virus RNA polymerase in an in vitro assay with an artificial influenza-like mRNA containing a cap structure at its 5' terminus, followed by a 10 nt beta-globin mRNA sequence, and the 5' and 3' conserved termini of a truncated nucleoprotein (NP) cRNA influenza sequence. Results showed that partially purified virion ribonucleoprotein complexes (RNPs) and micrococcal nuclease treated RNPs cleaved the artificial influenza-like mRNA substrate specifically at positions near the 5' terminus to generate capped 14 and 15 nucleotide long RNA fragments which subsequently served as primers to initiate transcription. The endonuclease activity was completely blocked by addition of cap analog and competitively inhibited by added globin mRNA. Furthermore, an in vitro reconstituted influenza RNA transcription reaction containing a truncated NP vRNA as template, micrococcal nuclease treated RNPs and globin mRNA as primer, synthesized capped and uncapped full length (+) sense products. Enzyme kinetics showed that capped RNA was made earlier in the reaction; it reached a peak at 120 min and then declined. However, uncapped cRNA synthesis appeared later and remained as the dominant product later in the reaction. The nature of these products was confirmed by ribonuclease protection assays and by primer extension.
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PMID:Influenza A virus RNA-dependent RNA polymerase cleaves influenza mRNA in vitro. 880 82

In Escherichia coli, ribonuclease E (RNase E) is a key endonuclease in mRNA decay. We have analysed the role of E coli RNase E on the degradation of a heterologous cytochrome c3 (cyc) mRNA from Desulfovibrio vulgaris Hildenborough. The decay of the cyc transcript in wild-type and mutant E coli cells was followed and the degradation intermediates analysed by Northern blotting and S1 protection analysis. The half-life of total cyc mRNA intermediates was increased in the RNase E mutant. A number of degradation intermediates were stabilised, and new species arose. However, some species decayed faster in the met5 mutant at the non-permissive temperature, suggesting that RNase E might inhibit their degradation. The results indicate that RNase E is involved in cyc mRNA degradation, and, interestingly, decay of certain intermediates could be reduced by this enzyme activity. This may suggest a functional interaction between RNase E and exonucleases, like polynucleotide phosphorylase.
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PMID:RNase E can inhibit the decay of some degradation intermediates: degradation of Desulfovibrio vulgaris cytochrome c3 mRNA in E coli. 887 97

The lack of simple and efficient methods for extraction of DNA from Nocardia spp. has hampered molecular manipulation of the DNA for diagnostic purposes. In the present study, a method for the rapid extraction of undegraded genomic nocardial DNA was established. Briefly, 14 pathogenic Nocardia strains were grown at 37 degrees C for 3 to 5 days in Sauton broth containing 0.05% Tween 80. Subsequently, the cultures were treated for 48 h with 1.2 mg of cycloserine per ml (final concentration). Cells were then harvested by centrifugation and treated with a lysis solution containing 3 mg of lysozyme per ml. This was followed by the addition of proteinase K and sodium dodecyl sulfate to final concentrations of 0.2 mg/ml and 0.5%, respectively, and incubation for 1 h at 50 degrees C. DNA was precipitated with isopropanol after phenol-chloroform-isoamyl alcohol extractions and RNase treated before being quantitated and analyzed by agarose gel electrophoresis. The average undegraded DNA yields obtained were 101 micrograms for Nocardia brasiliensis and 121 micrograms for N. asteroides. This DNA was suitable for restriction endonuclease digestion and PCR amplification, which are methods being applied to the characterization and diagnosis of slowly growing organisms such as Nocardia spp.
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PMID:A rapid and gentle method for isolation of genomic DNA from pathogenic Nocardia spp. 887 44

Previous work from this laboratory [Dompenciel,R.E., Garnepudi,V.R. and Schoenberg,D.R. (1995)J. Biol. Chem.270, 6108-6118] described the purification and properties of an estrogen-regulated endonuclease isolated from Xenopus liver polysomes that is involved in the destabilization of albumin mRNA. The present study mapped cleavages made by this enzyme onto the secondary structure of the portion of albumin mRNA bearing the major cleavage sites. The predominant cleavages occur in the overlapping APyrUGA sequence AUUGACUGA present in a single-stranded loop region, and in AUUGA located within a bulged AU-rich stem. A structural mutation which converted the major loop cleavage site to a hairpin bearing one APyrUGA element eliminated cleavage at the intact site. This confirms that the polysomal RNase is specific for single-stranded RNA. Additional point mutations in the major loop characterized the nucleoside sequence requirements for cleavage. Finally, snake venom exonuclease was used to demonstrate the polysomal RNase generates products with a 3' hydroxyl. Binding of an estrogen-induced protein to a portion of the 3'UTR of vitellogenin mRNA may be involved in its stabilization by estrogen [Dodson,R.E. and Shapiro,D.J. (1994)Mol. Cell. Biol.14, 3130-3138]. The core binding site for this protein bears the sequence APyrUGA, suggesting that stabilization may be accomplished by occlusion of a cleavage site for the polysomal RNase.
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PMID:Cleavage properties of an estrogen-regulated polysomal ribonuclease involved in the destabilization of albumin mRNA. 901 22

The Escherichia coli rnc-era-recO operon encodes ribonuclease III (RNase III; a dsRNA endonuclease involved in rRNA and mRNA processing and decay), Era (an essential G-protein of unknown functions and RecO (involved in the RecF homologous recombination pathway). Expression of the rnc and era genes is negatively autoregulated: RNase III cleaves the rncO 'operator' in the untranslated leader, destabilizing the operon mRNA. As part of a larger effort to understand RNase III and Era structure and function, we characterized rnc operon structure, function and regulation in the closely related bacterium Salmonella typhimurium. Construction of a S typhimurium strain conditionally defective for RNase III and Era expression showed that Era is essential for cell growth. This mutant strain also enabled selection of recombinant clones containing the intact S typhimurium rnc-era-recO operon, whose nucleotide sequence, predicted protein sequence, and predicted rncO RNA secondary structure were all highly conserved with those of E coli. Furthermore, genetic and biochemical analysis revealed that S typhimurium rnc gene expression is negatively autoregulated by a mechanism very similar or identical to that in E coli, and that the cleavage specificities of RNase IIIs.t. and RNase IIIE.c. are indistinguishable with regard to rncO cleavage and S typhimurium 23S rRNA fragmentation in vivo.
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PMID:Structure and regulation of the Salmonella typhimurium rnc-era-recO operon. 915 Aug 81

After androgen ablation by castration, the epithelial cells of the rat ventral prostate are eliminated by apoptosis. The number of cells showing apoptotic chromatin degradation increases with time up to day 3 after castration as verified by in situ end labeling of fragmented DNA. Apoptotic chromatin degradation is catalyzed by a Ca2+, Mg2+-dependent endonuclease. Recently, evidence has been presented that suggests deoxyribonuclease I (DNase I) is identical or very closely related to the apoptotic endonuclease (Peitsch, M.C., B. Polzar, H. Stephan, T. Crompton, H.R. MacDonald, H.G. Mannherz, and J. Tschopp. 1993. EMBO [Eur. Mol. Biol. Organ.] J. 12:371-377). Therefore, the expression of DNase I in the ventral prostate of the rat was analyzed before and after androgen ablation at the level of protein, enzymatic activity, and gene transcripts using immunohistochemical and biochemical techniques. DNase I immunoreactivity was detected only in a few single epithelial cells before androgen ablation. After castration, a time-dependent increase in DNase I immunoreactivity was observed within the epithelial cells. It first appeared after about 12 h in the apical region of a large number of epithelial cells. Up to day 3 after castration, the intracellular DNase I antigenicity continuously increased, and the cell nuclei gradually became DNase I positive. At day 5, almost all nuclei of the epithelium were stained by anti-DNase I. DNase I immunoreactivity was particularly concentrated in cells showing morphological signs of apoptosis, like nuclear fragmentation, and in many cases was found to persist in apoptotic bodies. DNase I gene transcripts were detected in control animals using dot and Northern blotting as well as RNase protection assay. After androgen ablation, the amount of DNase I gene transcripts in total extractable RNA was found unchanged or only slightly decreased up to day 5. Their exclusive localization within the epithelial cells was verified by in situ hybridization. Before castration, the DNase I gene transcripts were homogeneously distributed in all epithelial cells. At day 3, DNase I-specific mRNA was found to be highly concentrated in cells of apoptotic morphology. Using the zymogram technique, a single endonucleolytic activity of about 32 kD was detected in tissue homogenates before castration. After androgen ablation, the endonucleolytic activity increased about four- to sevenfold up to day 3. At day 5, however, it had dropped to its original level. At day 1, three new endonucleolytic variants of higher molecular mass were expressed. At day 3, the predominant endonucleolytic activity exhibited an apparent molecular mass of 32 kD. Enzymatic analysis of the endonucleases present in prostate homogenates before and after castration demonstrated properties identical to DNase I. They were inhibited by chelators of divalent cations, Zn2+ ions and monomeric actin. Immunodepletion was achieved by immobilized antibodies specific for rat parotid DNase I. A polyclonal antibody raised against denatured DNase I was shown by Western blotting to stain a 32-kD band after enrichment of the endonuclease from day 0 and 3 homogenates by preparative gel electrophoresis. The data thus indicate that androgen ablation leads to translational upregulation of an endonucleolytic activity with properties identical to DNase I in rat ventral prostate, followed by its intracellular retention and final nuclear translocation in those epithelial cells that are destined to apoptotic elimination.
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PMID:Androgen ablation leads to an upregulation and intranuclear accumulation of deoxyribonuclease I in rat prostate epithelial cells paralleling their apoptotic elimination. 915 93

RNase E is encoded by the rne (also known as ams or hmp) gene and is the principal enzyme that controls the chemical decay of bulk mRNA in Escherichia coli. Earlier work has shown that RNase E degrades its own mRNA, autoregulating production of the RNase E protein. Here we show that in cells lacking RNase E activity, the 3.6-kilobase rne gene transcript is cleaved site specifically at two locations near its center by a novel endonuclease whose activity is modulated by the presence or absence of amino acids in the culture medium. These cleavages produce a 2-kilobase RNase E-sensitive RNA fragment corresponding to the 3' half of the transcript. Using primer extension and RNase protection analysis, we mapped RNase E-independent cleavages to sites 1558 and 1576 nucleotides from the 5' end of the rne transcript (coordinates 1738 and 1747 of the rne gene). Our results indicate the existence of a previously unknown RNase E-independent mechanism for degradation of rne transcripts and further demonstrate that this mechanism responds to changes in cell growth conditions.
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PMID:Processing of the rne transcript by an RNase E-independent amino acid-dependent mechanism. 918 86

We have previously shown that a molecule consisting of a fusion of a Ca(2+)-dependent nuclease (from Staphylococcus aureus) to a retroviral coat protein specifies a potent antiviral specific for that retrovirus. Genes specifying such fusion proteins can be delivered to virus-susceptible cells, providing an antiviral gene therapy aimed at limiting virus spread. We report here the results of experiments to vary the nuclease moiety of such fusion proteins. We found that one nuclease. Serratia marcescens nuclease, was extremely toxic to host cells and hence not likely to be useful for therapeutic purposes. A second nuclease, Escherichia coli RNase Hl was found to be nontoxic and highly effective against a murine leukemia virus when it was fused to the leukemia virus coat protein. The fusion protein was enzymatically active and stably expressed, without apparent toxicity to host cells. Reduction in infectious virus output was as high as 97-99%. These studies provide a model system for the development of gene therapeutic agents aimed at combating retroviral infections in vivo.
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PMID:Antiretroviral effect of a gag-RNase HI fusion gene. 923 Oct 76

A new method for isolation of prawn baculovirus and subsequent extraction of viral DNA was developed. No density gradient centrifugation, ultracentrifugation or phenol-chloroform extraction steps were involved. Phenylmethylsulfonyl fluoride (PMSF) was used to prevent proteinase degradation, DNase and RNase were used to degrade prawn DNA and RNA respectively. The nucleocapsid was a bacilliform virion, about 58 62 nm in width and 300-350 nm in length as observed by transmission electron microscopy. Intact viral DNA was obtained by lysing nucleocapsids with guanidine hydrochloride and degrading protein with proteinase K. As the viral DNA was digested with restriction endonuclease and separated by electrophoresis, restriction fragments were clearly shown on the agarose gel. The size of the DNA was estimated approximately to be 290 kb. The virus which appeared to be a prawn baculovirus was named prawn white spot baculovirus (PWSBV) due to the white spots which appeared on the inside surface of the crust of infected prawns.
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PMID:A simple and efficient method for purification of prawn baculovirus DNA. 927 11


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