Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.1.4.1 (phosphodiesterase)
 18,767 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Viable mutants of simian virus 40 (SV40), with deletions ranging in size from 15 to 200 base pairs, have been obtained by infecting CV-1P cells with circularly permuted linear SV40 DNA. The linear DNA was produced by cleavage of closed circular DNA with DNase I in the presence of Mn2+, followed, in some cases, by mild digestion with lambda 5'-exonuclease. The SV40 map location and the size of each deletion were determined by using the S1 nuclease mapping procedure (Shenk et al., 1975) and the change in size of fragments produced by Hind II + III endonuclease cleavage. Deletions in at least three regions of the SV40 chromosome have slight or no effect on the rate or yield of viral multiplication and on vira-induced cellular transformation. These regions are located at the following coordinates on the SV40 physical map: 0.17 to 0.18; 0.54 to 0.59; and 0.68 to 0.74.
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PMID:Construction and analysis of viable deletion mutants of simian virus 40. 17 2

Aqueous solutions of DNA were gamma-irradiated in the presence and absence of oxygen and enzymatically hydrolysed by the combined action of pancreatic deoxyribonuclease (DNase I), snake-venom phosphodiesterase (PDE I), spleen phosphodiesterase (PDE II) and alkaline phosphatase. In contrast to unirradiated DNA, which is fully hydrolysed to nucleosides by these enzymes, gamma-irradiated DNA yields a series of oligonucleotides. Their isolation might enalbe the future identification of the chemical nature of DNA lesions.
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PMID:Enzymatic digestion of DNA gamma-irradiated in aqueous solution separation of the digests by ion-exchange chromatography. 21 Jan 33

The bacteriophage PRD1 DNA polymerase gene (gene I) has been cloned into the expression vector pPLH101 under the control of the lambda pL promoter. Tailoring of an efficient ribosome binding site in front of the gene by polymerase chain reaction led to a high level heat-inducible expression of the corresponding gene product (P1) in Escherichia coli cells. Expression was confirmed in vivo by complementation of phage PRD1 DNA polymerase gene mutants and in vitro by formation of the genome terminal protein P8-dGMP replication initiation complex. Expressed PRD1 DNA polymerase was purified to apparent homogeneity in an active form. DNA polymerase, 3'-5'-exonuclease, and P8-dGMP replication initiation complex formation activities cosedimented in glycerol gradient with a protein of 65 kDa, the size expected for PRD1 DNA polymerase. The DNA polymerase was active on DNase I-activated calf thymus DNA, poly(dA).oligo(dT) and poly(dA-dT) primer/templates as well as on native phage PRD1 genome. The 3'-5'-exonuclease activity was specific for single-stranded DNA and released mononucleotides. No 5'-3'-exonuclease activity was detected. The inhibitor/activator spectrum of the PRD1 DNA polymerase was also studied. An in vitro replication system with purified components for bacteriophage PRD1 was established. Formation of the P8-dGMP replication initiation complex was a prerequisite for phage DNA replication, which proceeded from the initiation complex and yielded genome length replication products.
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PMID:Overexpression, purification, and characterization of Escherichia coli bacteriophage PRD1 DNA polymerase. In vitro synthesis of full-length PRD1 DNA with purified proteins. 165 59

We have conducted studies to obtain practical knowledge regarding the stability, digestion, and analytical determination of the content of 8-hydroxy-2-deoxy-guanosine (8-OHdG) in oxidatively damaged DNA. Utilizing H2O2 plus uv light to form oxidatively damaged DNA, we found that storage of the DNA at -20 degrees C at alkaline pH caused a significant loss of 8-OHdG, whereas storage at -20 degrees C at neutral or acidic pH prevented loss of 8-OHdG. The 8-OHdG within DNA is stable at 100 degrees C for at least 15 min. Formation of 8-OHdG within DNA using uv light and H2O2 as a hydroxyl free radical-generating system yields the highest amounts when low levels of phosphate buffer are used; but the use of Tris or citrate buffers causes a lower yield of 8-OHdG because these buffers act as scavengers for the hydroxyl free radicals. Independent assessment of hydroxyl free radical flux by the use of salicylate trapping allows assessment of competitive radical reactions. Ethanol washing of plastic microfuge tubes prior to DNA enzymatic digestion improved the yield of 8-OHdG and reduced the variability between samples. Digestion of the oxidatively damaged DNA by the use of a method involving DNase I, endonuclease, phosphodiesterase, and alkaline phosphatase produced the highest yield of 8-OHdG.
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PMID:Conditions influencing yield and analysis of 8-hydroxy-2'-deoxyguanosine in oxidatively damaged DNA. 222 56

Four 25-nt oligonucleotides consisting of sequences of dA and dT (D1-4) have been synthesized. As shown in a companion paper (Rippe et al., 1989), the two combinations D1.D3 and D2.D4 form normal antiparallel duplexes, whereas the pairs D1.D2 and D3.D4 constitute duplexes with the same sequences, but with the two strands parallel to each other. The activities of the following DNA processing enzymes and chemical reagents on the parallel stranded (ps) and antiparallel stranded (aps) duplexes were tested. (i) The restriction endonucleases DraI, SspI, and MseI do not cut the ps duplexes. (ii) DNase I and exonuclease III exhibit a much lower activity with the ps duplexes. (iii) The nuclease activities of S 1 nuclease, micrococcal nuclease (S 7), phage lambda 5'-exonuclease, and the 3'-5' nuclease activity of Escherichia coli DNA polymerase I and its large fragment are higher with the ps than with the aps substrates. (iv) Bal 31 nuclease and the chemical nuclease 1,10-phenanthroline-copper ion [(OP)2Cu+] degrade ps-DNA and aps-DNA at approximately the same rate but show preferred cutting sites only with the aps molecules. (v) The iron(II)-EDTA complex has equivalent nuclease activities with the ps and the aps molecules. (vi) The ps duplex is not a substrate for blunt-end ligation with phage T4 DNA ligase.
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PMID:Substrate properties of 25-nt parallel-stranded linear DNA duplexes. 255 23

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 macromolecule binding 3H-methylcholanthrene (3H-MCA) and 3H-benzo(a)pyrene (3H-BaP) and sedimenting in the 4-5 S region of sucrose gradient (4.5 S) was identified in rat liver cytosol. The binding was displaced by 100-fold molar excess unlabeled ligands whereas 2,3,7,8-tetrachloro-dibenzo-p-dioxin (TCDD) was ineffective. The dissociation constant for both polycyclic aromatic hydrocarbons (PAHs) was of the order of 10(-8) M or lower. Both 3H-MCA and 3H-BaP bound to 4.5 S in a non covalent manner, since 92% of the bound radioactivity was extractable with ethyl ether. Furthermore the binding was strongly reduced by urea 8 M and by guanidine. HCl 4 M (99 and 70% respectively). Thin layer chromatography of the ethyl ether-solubilized radioactivity showed a peak comigrating with PAHs used as standards. When chromatographed on Sephadex G-200, 4.5 S was eluted as a sharp peak with an apparent molecular weight of 50-60,000 daltons. Enzyme treatment of liver cytosol showed that the 4.5 S binding sites were destroyed by micrococcal nuclease (92% of inhibition). Papain and phosphodiesterase I and II reduced the binding to 50%, whereas DNase I, DNase II, RNase, phospholipase A2 and C and trypsin were ineffective. These data suggest that the PAHs binding macromolecule of rat liver cytosol is a protein associated with a polynucleotide. The binding of both PAHs was enhanced by increasing the incubation temperature, the maximum being reached after 20-30 min at 37 degrees C. After 2.5 min at 65 degrees C, binding sites were completely destroyed. The same temperature-induced "activation" was obtained also by prewarming the cytosol at 37 degrees C in the absence of ligands.
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PMID:Polycyclic aromatic hydrocarbon binding macromolecules. Identification, characterization and temperature activation of a 4.5 S binding nucleoprotein. 406 Feb 44

An acid deoxyribonuclease has been purified from rat small intestinal mucosa by a procedure including ammonium sulfate fractionation, chromatographies on DEAE-cellulose, CM-cellulose and SE-Sephadex and finally isoelectric focusing. Polyacrylamide gel electrophoresis of the purified enzyme preparation showed one major and two minor bands, and the enzyme activity corresponded to one of the minor bands. The enzyme preparation was free of contaminating DNase I, DNase III, alkaline RNase, acid and alkaline phosphatases and nonspecific phosphodiesterase, but slight activities of DNase IV and acid RNase were detected. The enzyme did not require divalent cations for activity, had a pH optimum of 4.5 in 0.33 M sodium acetate buffer, and had an optimum temperature of 50 to 60 degrees C when assayed for 30 min. The rate of hydrolysis of native DNA was about 2.5-fold faster than that observed with denatured DNA. Its molecular weight was found to be 9.0 +/- 0.1. The enzyme catalyzes the endonucleolytic cleavage of native and denatured DNA, yielding oligonucleotides which have an average chain length of about 7, and which contain 3'-phosphoryl termini. The mode of action of the enzyme is double-strand scission.
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PMID:Purification and properties of an acid deoxyribonuclease from rat small intestinal mucosa. 663 Jan 62

The adjacent, divergently transcribed glpACB and glpTQ operons of Escherichia coli encode the anaerobic glycerol 3-phosphate dehydrogenase and glycerol 3-phosphate transporter/phosphodiesterase, respectively. These operons are negatively controlled by glp repressor binding to operators that overlap the glpA promoter elements. Using DNase I footprinting, three additional operators (OT1-3) were identified at positions +307 to +359 within the glpT coding region. To assess a potential regulatory role for these remote operators in vivo, a glpT-lacZ transcriptional fusion containing all of the glpA and glpT operators was constructed. The response of this fusion to the glp repressor was compared to fusion constructs in which OT1 and OT3 were inactivated, either by deletion or by site-directed mutagenesis. It was found that repression of glpT conferred by binding of glp repressor to glpA operators was increased about three- to fourfold upon introduction of the remote glpT operators. In addition, two integration host factor (IHF) binding sites were identified downstream of the glpT transcriptional start site at positions +15 to +51 and +193 to +227. A regulatory role for IHF was demonstrated by showing that repression of glpT mediated by GlpR was decreased about twofold in strains deficient in IHF and that mutations in IHF1 and/or IHF2 decreased repression about two- to threefold. The effect of IHF was apparent only when the remote operators were present. All of the results are consistent with a model of repression involving GlpR binding simultaneously to the glpA and remote glpT operators, with intervening DNA forming a loop.
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PMID:Action at a distance for glp repressor control of glpTQ transcription in Escherichia coli K-12. 917 45

DNA formed an insoluble complex on mixing with chitosan (poly-D-glucosamine) in solution. The DNA content of the complex was about 50% and the DNA remained insoluble in aqueous media of pH 2-7%; e.g., on treatment of the DNA-chitosan complex with phosphate-buffered saline at pH 7 and 37 degrees C for 26 h, the DNA released into the aqueous phase was less than 0.05%. Obviously, DNA and chitosan formed a tight complex due to ionic interactions. The DNA can be solubilized by treatment with 0.1 N NaOH. RNA and other polynucleotides formed similar insoluble complexes with chitosan. The DNA attached to chitosan can be digested with a mixture of DNase I and phosphodiesterase. Cytosine residues in the DNA (denatured DNA) can be deaminated by treatment with sodium bisulfite, forming uracil DNA-chitosan. The uracil DNA-chitosan served as a substrate for uracil DNA glycosylase. Using polynucleotide-chitosan as an adsorbent, the affinities of reagents for polynucleotides can be determined directly. With this technique it was found that carcinogenic heterocyclic amines have an affinity for RNA as well as DNA. The results with homopolyribonucleotide-chitosans as adsorbents for 4 heterocyclic amines indicated that the binding occurs in a purine nucleotide-specific manner. These results suggest that the polynucleotides in the chitosan complex are accessible to enzymes and reagents. This new derivative may be useful in chemical and biological studies of polynucleotides and substances interacting with polynucleotides.
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PMID:Polynucleotide-chitosan complex, an insoluble but reactive form of polynucleotide. 930 Oct 36


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