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)

The ribonuclease protection assay procedure described enables the relative quantitation of either single mRNAs or multiple mRNA species simultaneously in a sample of total RNA and demonstrates its applicability to two systems of relevance to the study of apolipoproteins, namely, liver tissue and liver-derived cell lines in culture. The main requirements of the method are the availability of cDNA cloned into a vector that directs the transcription of antisense RNA for the preparation of radioactive probes, and choice of suitable restriction endonuclease sites for linearizing the cDNA so that the final protected products of the various mRNA species are sufficiently different in size to allow their separation. For moderately abundant apolipoprotein mRNAs in rat liver, the method is sensitive down to 1 microgram total RNA. Other experimental sources of RNA or the assay of less abundant mRNA species may require a larger amount of starting material. These aspects of the assay need to be determined for each probe/ tissue system to be studied. The need for adjustments to the specific radioactivity of the probes will depend on the relative abundance of the target mRNA molecules and can be readily determined empirically. The rationale for varying the specific radioactivities to facilitate multiple assays as suggested here is both simple and effective. A preliminary assay provides information on the relative levels of the mRNA species of interest, and the step of preparing, in parallel, a sample of unlabeled antisense RNA provides the means for quantitative dilution of specific radioactivity of the 32P-labeled RNA probe where required.
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PMID:Determination of apolipoprotein mRNA levels by ribonuclease protection assay. 874 22

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

Androgen receptor (AR) plays a key role in cell growth both in the normal prostate and in prostate cancer. Androgen ablation and prolonged antiandrogen therapy can give rise to AR-dependent prostate tumors, which nonetheless can grow in the androgen-deprived milieu. Here we describe the ribozyme approach to selectively degrading the AR mRNA and thereby inhibiting AR function. A trans-acting hammerhead ribozyme was designed to cleave the rat AR mRNA at the position +1827/ 1828, a region predicted to be minimally involved in generating stable secondary structures. Using AR mRNA fragments as substrates, it was established that this ribozyme can specifically cleave the RNA target in a sequence-specific manner. Kinetic experiments determined a Km for the substrate of 77 nM and a kcat/Km value of 1.8 x 10(7) M(-1) x min(-1), suggesting a catalytic efficiency similar to that of protein enzymes such as the relatively nonspecific ribonuclease A and a sequence-specific endonuclease EcoRI. Transient cotransfections of prostate-derived PC3 cells with three plasmids, an AR-inducible chloramphenicol acetyltransferase (CAT) reporter, an AR expression vector, and a ribozyme expression vector, showed that the ribozyme was capable of reducing the functional activity of AR. At an equimolar ratio of the AR expression plasmid to ribozyme expression plasmid, androgen-inducible CAT activity was inhibited 70%. Similar extents of inhibition were also observed at the cellular mRNA level using ribonuclease protection assays, indicating that the ribozyme functioned as an AR mRNA cleaving enzyme in cellulo. Immunocytochemical examination revealed a decline of AR immunoreactivity in ribozyme-transfected cells. In addition, no morphologically detectable cellular abnormalities were associated with ribozyme expression, indicating the absence of deleterious side effects. These results offer a new avenue for the control of AR function and cell growth, especially in the case of androgen-resistant, but AR-dependent, prostate cancer cells.
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PMID:Catalytic cleavage of the androgen receptor messenger RNA and functional inhibition of androgen receptor activity by a hammerhead ribozyme. 977 79

With the use of a high yield prokaryotic expression system, large amounts of human eosinophil cationic protein (ECP) have been obtained. This has allowed a thorough kinetic study of the ribonuclease activity of this protein. The catalytic efficiencies for oligouridylic acids of the type (Up)nU>p, mononucleotides U>p and C>p, and dinucleoside monophosphates CpA, UpA, and UpG have been interpreted by the specific subsites distribution in ECP. The distribution of products derived from digestion of high molecular mass substrates, such as poly(U) and poly(C), by ECP was compared with that of RNase A. The characteristic cleavage pattern of polynucleotides by ECP suggests that an exonuclease-like mechanism is predominantly favored in comparison to the endonuclease catalytic mechanism of RNase A. Comparative molecular modeling with bovine pancreatic RNase A-substrate analog crystal complexes revealed important differences in the subsite structure, whereas the secondary phosphate-binding site (p2) is lacking, the secondary base subsite (B2) is severely impaired, and there are new interactions at the po, Bo, and p-1 sites, located upstream of the P-O-5' cleavable phosphodiester bond, that are not found in RNase A. The differences in the multisubsites structure could explain the reduced catalytic efficiency of ECP and the shift from an endonuclease to an exonuclease-type mechanism.
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PMID:Kinetic and product distribution analysis of human eosinophil cationic protein indicates a subsite arrangement that favors exonuclease-type activity. 1033 57

In this report, we describe the molecular cloning and characterization of DLAD, a novel mammalian deoxy-ribonuclease homologous to DNase II. The full length cDNA for mouse DLAD has been cloned by polymerase chain reaction. The cDNA contains a 1065 bp open reading frame (ORF) encoding a 354 amino acid protein with a calculated molecular mass of 40 767. The predicted protein for DLAD shares 34.4% identity with DNase II. DLAD is also homologous to three predicted proteins, C07B5.5, F09G8.2 and K04H4.6, from the nematode Caenorhabditis elegans. Furthermore, the third ORF of the fowlpox virus genome is found to encode a DLAD homologue showing 37. 1% identity at the amino acid level. Northern blot analysis reveals that expression of the DLAD mRNA is highly restricted to the liver. DLAD mainly exists as a cytoplasmic protein with divalent cation-independent endonuclease activity and cleaves DNA to produce 3'-phosphoryl/5'-hydroxyl ends. It is active under a wide range of pH with maximum activity at pH 5.2. Among known DNase inhibitors tested, aurintricarboxylic acid and Zn(2+)are found to be effective inhibitors of the DLAD activity.
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PMID:DLAD, a novel mammalian divalent cation-independent endonuclease with homology to DNase II. 1049 74

The 62 residue peptide, SSR(1-62), whose sequence corresponds to that of ribonuclease (RNase) from Sulfolobus solfataricus, and its related peptides, SSR(1-22) and SSR(10-62), were chemically synthesized and their RNase activity and DNA-binding activity were examined. The RNase activity assay using yeast RNA or tRNA(fMet) as substrate showed that the synthetic peptide SSR(1-62) did not hydrolyze yeast RNA or tRNA(fMet). These data were not consistent with previous reports that both the native peptide isolated from S. solfataricus [Fusi et al. (1993) Eur. J. Biochem. 211, 305-311] and the recombinant peptide expressed in Escherichia coli [Fusi et al. (1995) Gene 154, 99-103] were able to hydrolyze tRNA(fMet). However, the synthetic SSR(1-62) exhibited DNA-binding activity. In the presence of synthetic SSR(1-62), the cleavage of DNA (plasmid pUCRh2-4) by restriction endonuclease (EcoRI) was not observed, suggesting that synthetic SSR(1-62) bound to DNA protected DNA from its enzymatic digestion. Neither SSR(1-22) nor SSR(10-62) prevented DNA from being cleaved by a restriction enzyme. These findings strongly suggest the importance of not only the N-terminal region of SSR(1-62) but also the C-terminal region for DNA-binding. Circular dichroism spectroscopy of synthetic SSR(1-62) indicated a beta-sheet conformation, in contrast with synthetic SSR(1-22), which exhibited an unordered conformation.
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PMID:Amino acids and peptides. LVII. Synthetic peptide with a sequence of ribonuclease from Sulfolobus solfataricus, SSR(1-62), does not function as an RNase. 1068 31

RNase E is an important regulatory enzyme that plays a key role in RNA processing and degradation in Escherichia coli. Internal cleavage by this endonuclease is accelerated by the presence of a monophosphate at the RNA 5' end. Here we show that the preference of E. coli RNase E for 5'-monophosphorylated substrates is an intrinsic property of the catalytically active amino-terminal half of the enzyme and does not require the carboxy-terminal region. This property is shared by the related E. coli ribonuclease CafA (RNase G) and by a cyanobacterial RNase E homolog derived from Synechocystis, indicating that the 5'-end dependence of RNase E is a general characteristic of members of this ribonuclease family, including those from evolutionarily distant species. Although it is dispensable for 5'-end-dependent RNA cleavage, the carboxy-terminal half of RNase E significantly enhances the ability of this ribonuclease to autoregulate its synthesis in E. coli. Despite similarities in amino acid sequence and substrate specificity, CafA is unable to replace RNase E in sustaining E. coli cell growth or in regulating RNase E production, even when overproduced sixfold relative to wild-type RNase E levels.
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PMID:Regions of RNase E important for 5'-end-dependent RNA cleavage and autoregulated synthesis. 1076 47

Previous work from this laboratory identified a polysome-associated endonuclease whose activation by estrogen correlates with the coordinate destabilization of serum protein mRNAs. This enzyme, named polysomal ribonuclease 1, or PMR-1, is a novel member of the peroxidase gene family. A characteristic feature of PMR-1 is its ability to generate in vitro degradation intermediates by cleaving within overlapping APyrUGA elements in the 5'-coding region of albumin mRNA. The current study sought to determine whether the in vivo destabilization of albumin mRNA following estrogen administration involves the generation of decay intermediates that could be identified as products of PMR-1 cleavage. A sensitive ligation-mediated polymerase chain reaction technique was developed to identify labile decay intermediates, and its validity in identifying PMR-1-generated decay intermediates of albumin mRNA was confirmed by primer extension experiments performed with liver RNA that was isolated from estrogen-treated frogs or digested in vitro with the purified endonuclease. Ligation-mediated polymerase chain reaction was also used to identify decay intermediates from the 3'-end of albumin mRNA, and as a final proof of principle it was employed to identify in vivo decay intermediates of the c-myc coding region instability determinant corresponding to sites of in vitro cleavage by a polysome-associated endonuclease.
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PMID:Identification of in vivo mRNA decay intermediates corresponding to sites of in vitro cleavage by polysomal ribonuclease 1. 1115 74

Estrogen induces a global change in the translation profile of Xenopus hepatocytes, replacing serum protein synthesis with production of the yolk protein precursor vitellogenin. This is accomplished by the coordinate destabilization of serum protein mRNAs and the transcriptional induction and subsequent stabilization of vitellogenin mRNA. Previous work identified an endonuclease activity whose appearance on polysomes correlated with the disappearance of serum protein mRNAs. This enzyme, polysomal ribonuclease 1 (PMR1), is a novel member of the peroxidase gene family. The current study examined the association of PMR1 with its mRNA targets on polysomes and mRNPs. The highest amount of polysome-bound PMR1 was observed prior to estrogen induction of mRNA decay. Its distribution on sucrose density gradients matched the absorbance profile of polysome-bound mRNA, suggesting that PMR1 forms a latent complex with mRNA. Following dissociation with EDTA the 62 kDa PMR1 sedimented with a larger complex of >670 kDa. Estrogen induces a 22-fold increase in unit enzymatic activity of polysome-bound PMR1, and a time-dependent loss of PMR1 from polysomes in a manner that mirrors the disappearance of albumin mRNA. These data suggest that the key step in the extensive estrogen-induced change in mRNA decay in Xenopus liver is activation of a latent mRNA endonuclease associated with its target mRNA.
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PMID:Polysomal ribonuclease 1 exists in a latent form on polysomes prior to estrogen activation of mRNA decay. 1122 65


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