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:3.1.3.1 (alkaline phosphatase)
47,916 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

When electrophoresed on an agarose gel, the DNA isolated from neocarzinostatin- (NCS-) treated HeLa cells migrates in a ladder of discrete bands indicative of preferential breakage in the linker region of the nucleosomes. The 5'-termini of the drug-induced DNA strand breaks were characterized by reduction of the nucleoside 5'-aldehyde ends to 5'-hydroxyls followed by incorporation of 32P from [gamma-32P]ATP by polynucleotide kinase and treatment of the DNA with hot alkali and alkaline phosphatase prior to the kinase assay to give the total 5'-termini. In DNA isolated from NCS-treated cells, nucleoside aldehyde accounts for 30-45% of the drug-generated 5' ends; the remainder have PO4 termini. By contrast, 5'-terminal nucleoside aldehyde in DNA cut with the drug in vitro exceeds 80% of the total 5' ends. Of the 32P representing nucleoside aldehyde in DNA from NCS-exposed cells, 77% is in TMP; the rest is in AMP much greater than CMP greater than GMP, a distribution in excellent agreement with that obtained for in vitro drug-treated DNA. DNA sequencing experiments, using the 340 base pair alphoid DNA fragment isolated from drug-treated cells, show that the pattern of breakage produced by NCS within a defined sequence of DNA in intact cells is similar to that in the in vitro reaction, with a preferential attack at thymidylate residues, but a much higher concentration of the drug was required to cause comparable breakage in intact cells.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Mechanism and base specificity of DNA Breakage in intact cells by neocarzinostatin. 295 Sep 23

DNA-dependent ATPase IV has been purified to near homogeneity from the Novikoff rat hepatoma. The enzyme is devoid of DNA polymerase, RNA polymerase, exonuclease, endonuclease, phosphomonoesterase, 3'- or 5'-phosphodiesterase, polynucleotide kinase, protein kinase, topoisomerase, helicase or DNA reannealing activities at a detection level of 10(-5) to 10(-7) relative to the ATPase activity. The enzyme is a monomer of Mr 110,000, has a sedimentation coefficient of 5.9 S, a Stokes radius of 40 A and a frictional coefficient of 1.32. In the presence of Mg2+ ion and a polynucleotide effector, ATPase IV hydrolyzes either ATP or dATP to the nucleoside diphosphate plus Pi. Other ribo- or deoxyribonucleoside triphosphates are not substrates. ATPase IV utilizes double-stranded DNA and single-stranded DNA as effector; however, it does not utilize poly(dT). The Km for dsDNA or ssDNA is 2.2 microM (nucleotide). A variety of ATP analogues were found to be competitive inhibitors of ATPase IV.
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PMID:Purification and enzymological characterization of DNA-dependent ATPase IV from the Novikoff hepatoma. 296 5

Procedures are described for identification of very infrequent in vivo 3'-ends of RNA. After purification by filter hybridization, the 3'-ends were labeled with [5'-32P] cytosine-3'-P in the RNA ligase reaction. Significantly fewer counts were incorporated in the ligase reaction than in the polynucleotide kinase reaction to label 5'-ends. The incorporation was increased by increasing the RNA concentration 5-10 fold by using only one round of filter hybridization. Non-specific RNA binding could be eliminated by RNase A treatment of the filter if a great excess of denatured heterologous DNA was immobilized along with the DNA probe. Significant amounts of DNA were released when eluting the hybrid RNA from such filters. DNA inhibited the ligase reaction, while its DNase products were even more inhibitory. Treatment of the DNase products with alkaline phosphatase completely eliminated the inhibition. We detected no spurious 5'- or 3'-ends generated in the hybrid RNA by RNase A activity used to reduce the non-specific RNA. Also, RNase T1 could be used in place of RNase A to eliminate non-specific RNA binding, but about 25 times more RNase T1 (microgram/microgram) was needed. We used partial alkali digestion to sequence 3'-ends. A major (one hit) and minor (two hit) set of products were produced which could be distinguished from each other by alkaline phosphatase treatment and homochromatography of the products.
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PMID:Isolating and sequencing the infrequent 3'-ends of a specific mRNA. 331 56

Endogenous oligonucleotides were found in trichloroacetic acid extracts of hamster lung fibroblasts and Tetrahymena cells. Peaks of radioactivity that eluted with retention times similar to oligonucleotide markers (5- to 50-mer) were found by HPLC in cells labeled briefly with 32Pi. Only minute amounts of UV-absorbing material were detected, consistent with a rapid turnover of phosphate groups. The 32P-labeled material also migrated as oligonucleotides on 20% polyacrylamide gels; it was not hydrolyzed by alkaline phosphatase but was digested by snake venom phosphodiesterase, S1 nuclease, and pancreatic RNase and was phosphorylated by T4 polynucleotide kinase. The 32P-labeled material isolated by HPLC was alkali labile and the hydrolyzate ran as nucleotides on paper chromatography. It is concluded that the oligonucleotides are mainly oligoribonucleotides, but it is possible that oligodeoxynucleotides are also present.
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PMID:Oligonucleotides with rapid turnover of the phosphate groups occur endogenously in eukaryotic cells. 347 Jul 67

DNA synthesis was followed in vivo and in permeable Escherichia coli after ultraviolet light irradiation, irradiation and incubation in a growth medium containing chloramphenicol and in unirradiated cells. In vitro, replicative type DNA synthesis was partially restored after incubation of cells in medium containing chloramphenicol, but not in vivo. The DNA was pulse-labeled in permeable cells in the presence of deoxyribonucleoside triphosphates and ribonucleoside triphosphates. dCTP was replaced by 5-Hg-dCTP as a substrate for DNA synthesis. Hg-DNA was separated from cellular nucleic acids on thiol-agarose affinity columns. The 5' termini of newly synthesized DNA were analyzed after treatment with alkaline phosphatase and rephosphorylation with polynucleotide kinase and [gamma-32P]ATP. DNA synthesis in unirradiated permeable E. coli represents a replicative process dependent on ATP and inhibited by novobiocin. About 70% of the nascent DNA carried terminally labeled RNA moiety at its 5' end. In vitro DNA synthesis in irradiated cells was suppressed and hardly influenced by the presence of ATP or novobiocin. The 5'-RNA content of this cell population was less than 5%.
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PMID:DNA synthesis in vivo and in vitro in Escherichia coli irradiated with ultraviolet light. 354 33

The 5'-terminal nucleotide sequences of human reovirus double-stranded RNA were determined after labeling the RNA with [(32)P]phosphate by polynucleotide kinase. The 5' terminal were labeled to only a limited extent prior to sequential oxidation, beta-elimination, and phosphomonoesterase treatment, indicating that the terminal phosphates were in a modified, blocked configuration. Each genome segment, after removing the blocking group, contained the same two 5'-terminal sequences: GpApUp in one chain and G(*)pCp in the other. G(*)p is a derivative of guanylic acid, probably 2'-O-methyl-Gp, which renders the 5'-terminal sequence resistant to hydrolysis by alkali. The results indicate that the transcription of reovirus double-stranded RNA strats from the 3' end complementary to the G(*)pCp-terminal, resulting in the synthesis of single-stranded mRNA carrying the same 5' sequence as the G(*)pCp-chain. The presence of a modified nucleotide at the 5' terminus of the strand complementary to the mRNA template is a feature common to another double-stranded RNA virus, cytoplasmic polyhedrosis virus.
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PMID:The 5'-terminal nucleotide sequences of the double-stranded RNA of human reovirus. 453 Feb 78

The sites of cleavage of DNA by bleomycin A2, bleomycin B2, phleomycin, tallysomycin A, and Blenoxane (Bristol-Meyers) in reactions containing equimolar Fe2+ and atmospheric oxygen were analyzed by gel electrophoresis of 32P end labeled DNA fragments. Bleomycin A2 and bleomycin B2 reactions cleaved DNA at all sites with a frequency equal to that of Blenoxane. At high concentrations of bleomycin the site specificity of cleavage was unchanged. Bleomycin cleavage sites and phleomycin cleavage sites are a subset of sites cleaved in reactions containing tallysomycin A. The nature of 5' and 3' termini induced by bleomycin cleavage was investigated. Electrophoresis of bleomycin-induced fragments after alkaline phosphatase or polynucleotide kinase treatment indicated that 5' termini are phosphoryl groups but 3' termini are not simple phosphoryl groups. Analysis of bleomycin cleavage of single-stranded DNA substrate showed that cleavage occurs only in regions of potentially double-stranded looped-back sequences. Possible mechanisms for determination of bleomycin cleavage sequence specificity are discussed.
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PMID:Specificity of deoxyribonucleic acid cleavage by bleomycin, phleomycin, and tallysomycin. 618 7

The nascent DNA synthesized by permeable cells of Bacillus subtilis in the presence of 5'-mercurideoxycytidine triphosphate and 2',3'-dideoxyATP has been isolated and characterized. The newly synthesized DNA was isolated free from other cellular nucleic acids by affinity chromatography on thiol-substituted agarose. The number average chain length of the nascent DNA synthesized in one minute at 25 degrees C was 33 nucleotide residues, due to the chain-terminating action of 2',3'-dideoxyATP. Several lines of evidence indicated that at least 90% of the DNA thus isolated carried a terminally phosphorylated RNA moiety at its 5'-end: (1) the nascent DNA was resistant to exonucleolytic degradation by spleen phosphodiesterase unless first hydrolyzed by strong alkali or ribonuclease; (2) the 5'-termini of nascent DNA could not be phosphorylated by polynucleotide kinase unless first treated with alkaline phosphatase or subjected to hydrolysis by strong alkali or ribonuclease; (3) alkaline hydrolysis of nascent DNA labeled with 32P at the 5'-end released unlabeled DNA with a free 5'-terminus and 32P-labeled ribonucleoside 3',5'-bisphosphates; (4) ribonuclease degradation of similarly labeled material produced an unlabeled DNA-containing polynucleotide fraction and 32P-labeled ribo-oligonucleotides; (5) chromatography on dihydroxyboryl cellulose showed that the RNA moiety lacked a 3'-terminal cis-diol grouping (even after treatment with alkaline phosphatase) unless first subjected to the 3'-exonucleolytic action of bacteriophage T4 DNA polymerase. The sequence of the ribonucleotide chains was elucidated by end-group labeling with polynucleotide kinase and digestion with various ribonucleases. The ribonucleotide moiety was primarily three and four residues in length with the predominant sequence (pp)pApG(pC)1-2pDNA. The possibility that it represents a primer for discontinuous DNA synthesis is discussed.
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PMID:Analysis of the 5'-termini of nascent DNA chains synthesized in permeable cells of Bacillus subtilis. 618 36

Commercial preparations of the enzymes used in the analysis of RNA primary structure (bacterial alkaline phosphatase, polynucleotide kinase, and RNA ligase) are virtually always more or less contaminated with RNases. This leads to degradation of initial RNAs in the course of labeling and formation of a set of spurious labeled fragments. We have shown that bentonite present in the incubation medium in a concentration of 0.04% selectively inhibits the contaminating RNases, not affecting the activities of bacterial alkaline phosphatase, polynucleotide kinase, and RNA ligase.
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PMID:Inhibition of ribonuclease contamination in preparations of T4 RNA ligase, polynucleotide kinase, and bacterial alkaline phosphatase with bentonite. 620 51

Covalent joining of the two half molecules of tRNAAla by T4 RNA ligase to form a reconstituted whole molecule was investigated. The two half molecules consisting, respectively, of residues 1-35 and 36-75 were prepared by partial degradation of tRNAAla with RNAase T1. The 5'-half molecule was treated with alkaline phosphatase to remove the 3'-terminal phosphate group, and the 5'-OH group of the 3'-half molecule was phosphorylated with [gamma-32P]ATP by polynucleotide kinase. The two terminal nucletides to be joined were identified as Guo and Cyd. Prior to the covalent joining reaction, the two modified half molecules in an equimolar mixture were annealed, and the rejoined half molecules, separated by gel electrophoresis, served as the substrate for T4 RNA ligase. Optimum conditions for this ligation, such as RNA ligase concentration, pH, Mg2+ concentration, reaction temperature and time of reaction, were investigated. Under the optimum conditions a yield of about 70% joining of the reconstituted whole molecule was obtained as shown by gel electrophoresis, resistance to hydrolysis by alkaline phosphatase, nearest neighbour analysis and alanine acdeptor activity.
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PMID:Joining of yeast alanine transfer ribonucleic acid half molecules to form a whole molecule by T4 RNA ligase. 626 Jan 88


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