Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.1.30.1 (S1 nuclease)
 3,660 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The presence of a nuclear DNA polymerase in mouse sperm from adult testes has been confirmed and the properties of this enzyme further investigated. This activity was shown to be greatly enhanced by treating the spermatozoa with methanol or ethanol before incubation in the reaction medium or by their addition in small amounts to this medium. It was protected against degradation by nuclear proteases by adding soybean trypsin inhibitor and was stimulated by ATP. It was found to be Mg2+ dependent (optimum concentration: 7.5 mM), DNA dependent, and all four deoxynucleoside triphosphates were needed for optimal reaction. The radioactive acid-precipitable product of polymerization was not eliminated by organic solvents, nor by pronase, ribonuclease or by nuclease S1; however, it was converted to a large extent to acid-soluble products by pancreatic deoxyribonuclease. Since it was only partially solubilized by Triton X-100, it therefore did not appear to be preferentially associated with the nuclear membranes. The activity recovered after incubation depended also on the pH (optimum at pH 8.3) and did not work well in a medium for DNA polymerase alpha. The temperature for maximum incorporation of nucleotides was found to be 32 degrees C and, under our conditions, the reaction was linear for 30 min. The DNA polymerase activity was inhibited by low and high concentrations of KCl. It was not lowered by N-ethylmaleimide or p-hydroxymercuribenzoate; urea slightly stimulated the reaction and this stimulation was reversed by subsequent treatment with N-ethylmaleimide. Actinomycin D (40 mug/ml), ethidium bromide (25--50 muM), netropsin (5--50 mug/ml), and spermidine (0.5--2.5 mM) lowered the polymerization of DNA precursors. The nuclear enzyme could shift from the endogenous template to activated exogenous calf thymus DNA, the resulting nuclear radioactivity being reduced. The endogenous DNP template ability was not increased by deoxyribonuclease activation according to the method of Aposhian and Kornberg (J. Biol. Chem. (1962) 237, 519--525) suggesting that the amount of DNA polymerase associated with chromatin was probably limiting the reaction. The DNA polymerase activity detected in mouse sperm nuclei has numerous properties of low molecular weight DNA polymerases (DNA polymerase beta) reported in several eukaryotic organisms.
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PMID:Further characterization of a DNA polymerase activity in mouse sperm nuclei. 1 3

Single-strand-specific nuclease S1 from Aspergillus oryzae is shown to degrade DNA and RNA in lysates of HeLa cells in the presence of 9 M urea and sodium dodecylsulfate. Free dodecylsulfate inhibits S1 nuclease. However, if the detergent is complexed with proteins prior to the addition of the enzyme, S1 nuclease can degrade nucleic acids at dodecylsulfate concentrations which would inhibit the enzyme completely if no other proteins were present. In lysates prepared from HeLa cells by treatment with dodecylsulfate and urea, the detergent is complexed by cellular proteins and therefore S1 nuclease can be used to digest DNA and RNA. DNA can be completely degraded but, even after heat-denaturation, only 60% of the cellular RNA is converted into acid-soluble material. Analysis of the acid-insoluble RNA fragments by gel filtration reveals that the majority of the degradation products is approximately of tRNA size.
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PMID:Degradation of nucleic acids in cell lysates by S1 nuclease in the presence of 9 M urea and sodium dodecylsulfate. 90 32

S1 nuclease isolated from Aspergillus oryzae has been used to investigate the secondary structure of rabbit globin messenger RNA (mRNA). The enzyme, which is specific for single stranded nucleotides, digests globin mRNA to a limited extent, with 65-75% of the mRNA nucleotides resistant to digestion under mild conditions. This limited digestion is not due to enzyme inactivation, but rather to the normal activity of the single-strand nuclease. The reaction was studied as a function of temperature, salt and enzyme concentration. Analysis of the products of digestion on 20% acrylamide- 7M urea slab gels reveals a stable pattern of unique fragments ranging in size from 9 to 71 nucleotides. Separated alpha and beta globin mRNAs show similar, but not identical gel patterns, indicating strong structural similarities between the two species. The high degree of nuclease resistance, along with the fragment patterns seen on polyacrylamide gels, gives evidence to support a model of rabbit globin mRNA which contain specific, rather than random, helical structure.
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PMID:Specific hydrolysis of rabbit globin messenger RNA by S1 nuclease. 90 77

The single-strand specific nuclease S1 from Aspergillus oryzae (EC 3.1.4.21) was purified 600-fold in 16% yield from dried mycelia. Determination of the isoelectric point of S1 nuclease as 4.3-4.4 allowed adjustment of chromatographic conditions such that the enzyme was isolated free of contaminating ribonucleases T1 and T2. S1 nuclease so purified was used for removal of single-stranded portions from the RNA of the Escherichia coli phage MS2, which has a helical content of about 65% in vitro. At 23 degrees, increasing amounts of enzyme converted the RNA to mononucleotides in about equimolar base ratios. No small intermediates of chain length 2-8 were found. At 0 degrees, MS2 RNA hydrolysis was slower and reached, in exhaustive digests, a plateau where 70% of the substrate RNA remained insoluble in 66% EtOH. With [32P]MS2 RNA, strip chart counting of 6% acrylamide-6 M urea electrophoresis patterns of such digests gave recoveries of 80-91% in the form of defined oligomer bands. On 2.5% acrylamide-0.5% agarose gels, the molecular weights of the major oligomers were found to range from 25,000 to 41,000. Similar to purified tRNAArg used as a control, these oligomers were not resistant to pancreatic RNase-RNase T1 hydrolysis at 37 degrees, and were not bound on hydroxylapatite at 50 degrees in 0.14 M sodium phosphate (pH 6.8). Melting of the oligomers gave complex profiles without a clear Tm and showed an increase in A260 of 35% at 93 degrees over that at 28 degrees. Upon formaldehyde denaturation of MS2 RNA prior to S1 nuclease hydrolysis, no resistant oligomers were found.
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PMID:S1 nuclease hydrolysis of single-stranded nucleic acids with partial double-stranded configuration. 118 98

The relative levels of mitochondrial specific gene transcripts were compared in two murine large cell lymphoma cell lines that differ in their propensities to form liver metastases and in their sensitivity to macrophage mediated antitumor cytostasis and cytolysis. Full-length transcripts of the mitochondrial genes were hybridized on electroblots from citrate/urea gels with specific gene prodes. The mitochondrially encoded gene NADH dehydrogenase subunit 5 (ND5), that encodes a component of NADH dehydrogenase (complex I) of the electron transport chain, was significantly overexpressed in the highly metastatic RAW117-H10 compared to low metastatic RAW117-P cells. Results from analysis of RNA blots were confirmed in an S1 nuclease protection assay. Since RAW117-H10 cells are significantly more resistant to cytostasis by activated macrophages in coculture and such macrophage activity can inhibit RAW117 tumor cell respiration and growth, a mechanism was suggested that allows RAW117 cell escape from certain host effector mechanisms that block cellular respiration by an increase in the in vivo concentrations of translatable messenger RNA (mRNA) that codes for key components of the electron transport chain.
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PMID:Transcripts of the mitochondrial gene ND5 are overexpressed in highly metastatic murine large cell lymphoma cells. 138 22

We describe here a novel approach to the dissection of chromatin structure by extracting DNA fragments from digested nuclei irreversibly immobilized (via proteins) on Celite columns. Three successive gradients (NaCl, LiCl-urea, temperature) are used to release three families of DNA fragments: namely, the 'DNA adherence' classes DNA-0, DNA-I and DNA-II, respectively. This 'protein image' DNA chromatography separates DNA fragments in accordance with the tightness of their bonds with proteins in situ. There are at least two DNA-skeleton attachment sites differing from each other by their resistance to the dissociating agents used as well as their susceptibility to DNAase I and S1 nuclease treatments, DNA cross-linking and single-stranded breaks. Several lines of evidence show a specific, topological rather than chemical, DNA-protein linkage at the tight attachment site. A hierarchy of chromatin loops demarcated by these attachment sites was determined. The technique described is generally applicable and can be used both to probe DNA-protein interactions and to map specific DNA sequences within the chromatin domain.
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PMID:Differential dissociation of chromatin digests: a novel approach revealing a hierarchy of DNA-protein interactions within chromatin domains. 193 69

Genes for urea cycle enzymes including liver-type arginase are expressed mainly in the liver and are regulated developmentally, nutritionally, and hormonally in a coordinated manner. The promoter region of the rat arginase gene was investigated with an in vitro transcription system using nuclear extracts prepared from rat tissues. Accurate initiation of the transcription in liver nuclear extracts was confirmed by runoff analysis and S1 nuclease mapping. The arginase promoter was transcribed more efficiently in liver nuclear extracts than in brain extracts, reproducing the in vivo tissue specificity qualitatively. Analysis of deletion mutants of the 5'-flanking region in liver nuclear extracts revealed a positive regulatory region spanning nucleotides -90 to -51 relative to the transcription start site. Overlapping this region, two protected areas were detected by DNase I footprinting. Competition analysis with synthetic oligonucleotides showed that the more downstream protected area was occupied, in a mutually exclusive manner, by two factors each related to CTF/NF-1 and Sp1. The other more upstream protected area was recognized by a factor related to the liver-enriched transcription factor C/EBP, which was recently shown to interact with regulatory regions of two other urea cycle enzyme genes.
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PMID:In vitro analysis of the rat liver-type arginase promoter. 202 18

Heat shock-induced small cytoplasmic RNA (HSI scRNA) from Drosophila culture cell was larger in size after dimethylchloroacetal treatment than in 7 M urea. Judging from its thermal denaturation profile and the nuclease S1 digestion experiment it was supposed that HSI scRNA is double-stranded. Nucleotide sequence analysis of the scRNA also suggested that it is double-stranded RNA with the blunt-ends.
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PMID:Low molecular weight RNA of Drosophila cells which is induced by heat shock--II. Structural properties. 246 34

Partially purified S1 nuclease was bound through its carbohydrate moiety to Con A-Sepharose containing increasing amounts of lectin. The retention of activity was high, varying essentially from 75% on the "low lectin" matrix (1 mg Con A/mL of Sepharose), to no detectable activity on the "high lectin" matrix (8 mg Con A/mL of Sepharose). However, approximately 50% activity could be restored in "high lectin" matrix when the coupling was carried out in the presence of glucose, suggesting that the loss of activity on the "high lectin" matrix is caused by conformational changes brought about by the multiple attachment of the enzyme to the matrix. Interaction of Con A with S1 nuclease was used to predict the nature of carbohydrate moiety and its location with respect to the active site of the enzyme. Immobilization resulted in an increase in the optimum temperature, pH, and temperature stabilities, but it did not affect the pH optimum. A marginal increase in the apparent Km was observed. The bound enzyme also showed enhanced stability toward 8 M urea. On repeated use, the bound enzyme retained more than 80% of its initial activity after 6 cycles. These results are discussed taking into consideration the factors affecting immobilized enzymes. In addition, the potential use of immobilized S1 nuclease as an analytical tool is discussed.
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PMID:Influence of lectin concentration on the catalytic properties of S1 nuclease bound to Concanavalin A-sepharose. 250 51

This report describes methods for quantifying specific sequences in preparations of single-stranded or double-stranded nucleic acids. We use saturating amounts of hybrid-selected, strand-specific probes and perform hybridizations in urea solutions. Hybrids (RNA-DNA or DNA-DNA) are then analyzed by either of two methods. The first employs standard S1 nuclease digestion, precipitation of resistant material on glass fiber filters, and assay by liquid scintillation counting. This method is generally chosen in the assay of rare transcripts in total nucleic acid extracts as well as preparations of polyadenylated RNA. The second employs the separation of excess probe from probe-target hybrids by gel electrophoresis, recovery of hybrids on ion-exchange paper, and determination of cpm bound by liquid scintillation counting. This method is of particular value in the assay of double-stranded sequences and the quantitation of restriction fragment length polymorphisms. Whereas both methods are accurate over ranges of target abundance representing at least several orders of magnitude, the latter ("NA45 assay") is most sensitive, and the selection of M13-bound or unbound DNA fragments by this method can be exploited in a variety of other applications.
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PMID:Quantitation of genes and transcripts by saturation hybridization in urea solutions using strand-selected probes. 289 Mar 16


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