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 genes of the AccI restriction-modification system specific for GT(A/C) (G/T)AC were cloned from the chromosomal DNA of Acinetobacter calcoaceticus, and their nucleotides sequenced. The restriction and modification genes coded for polypeptides with calculated molecular weights of 42,494 and 63,078, respectively. Both the enzymes were coded by the same DNA strand and the restriction gene was upstream of the methylase gene, separated by 2 bp. The restriction gene was significantly expressed in E. coli cells, so that the AccI restriction endonuclease could be purified to homogeneity. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration indicated that the catalytically active form of the endonuclease was tetrameric. Sequence comparison with related enzymes indicated that AccI methylase contained a segment of tetra-amino acids, NPPY, characteristic of N6-adenine methylases. In addition, some homologous regions were found in the sequence of HincII methylase specific for GT(C/T) (A/G)AC.
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PMID:Cloning and nucleotide sequences of the AccI restriction-modification genes in Acinetobacter calcoaceticus. 136 3

A new restriction endonuclease, designated as ApaLI, was purified from cell-free extracts of Acetobacter pasteurianus IFO 13753 by streptomycin treatment, ammonium sulfate fractionation, combined column chromatographies on heparin-Sepharose CL-6B and DEAE-Sepharose CL-6B and Fast Protein Liquid Chromatography on Mono Q HR 5/5. The purified enzyme was homogeneous on polyacrylamide gel disc electrophoresis. The molecular weight of the purified enzyme was calculated as 26,000 daltons by gel filtration using Sephadex G-200, and the isoelectric point of the purified enzyme was 4.8 by ampholine sucrose-density gradient isoelectric focusing. The purified enzyme cleaved lambda, Ad2, SV40, M13mp18 RF 1, psi X174 RF I and pBR322 DNAs at 4, 7, 0, 0, 1 and 3 sites, respectively. The purified enzyme worked best at 37 degrees C and pH 8.0 in a reaction mixture (50 microliters) containing 1.0 micrograms lambda DNA, 10mM Tris-HCl, 7 mM 2-mercaptoethanol, 7 mM MgCl2 and 25mM NaCl. However, the purified enzyme did not require NaCl necessarily for the enzyme reaction. The purified enzyme recognized the palindromic hexanucleotide DNA sequence, 5'-GTGCAC-3' and cut between G and T, producing a 5'-cohesive tetranucleotide extension.
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PMID:Purification, properties and recognition sequence and cleavage site determinations of restriction endonuclease from Acetobacter pasteurianus IFO 13753 (ApaLI). 136 91

A new restriction endonuclease, designated as AgeI, was purified from cell-free extracts of a marine bacterium, "Agrobacterium gelatinovorum" IAM 12617 by streptomycin treatment, ammonium sulfate fractionation, combined column chromatographies on heparin-Sepharose CL-6B and DEAE-Sepharose CL-6B and FPLC on Mono Q (HR 5/5) and Superose 12 (HR 10/30). The purified enzyme was homogeneous on SDS-polyacrylamide gel disc electrophoresis and free from other phosphatase and exonuclease activities on ligation-recutting test. The relative molecular mass of the enzyme was 24,000 daltons by SDS-polyacrylamide gel disc electrophoresis. The gel filtration using Superose 12 (HR 10/30) gave the same calculation (23,000 daltons). These data indicated that the enzyme is a monomer. The isoelectric point of the enzyme was 6.5. The purified enzyme cleaved lambda and Ad2 DNAs at 10 or more and 5 sites, respectively. However, the purified enzyme did not cleave SV40, phi X174 RF I, M13mp 18 RF I or pBR322 DNAs. pBR328 DNA was cleaved at 1 site by the purified enzyme. The purified enzyme worked best at 37 degrees C and pH 7.5 in a reaction mixture (50 microliters) containing 1.0 micrograms lambda DNA, 10 mM Tris-HCl, 7 mM 2-mercaptoethanol, 7 mM MgCl2 and 50 mM NaCl. The purified enzyme did not require monovalent cations necessarily for the enzyme reaction. The enzyme recognized the palindromic hexanucleotide DNA sequence 5'-ACCGGT-3' and cut between A and C, producing a 5'-cohesive tetranucleotide extension.
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PMID:Purification, properties and determinations of recognition sequence and cleavage site of restriction endonuclease from "Agrobacterium gelatinovorum" IAM 12617, a marine bacterium (AgeI). 136 92

The restriction endonuclease AatII was purified from cell-free extracts of Acetobacter aceti IFO 3281 by streptomycin treatment, ammonium sulfate fractionation, combined column chromatographies on DEAE-Toyopearl 650S, heparin-Sepharose CL-6B and DEAE-Sepharose CL-6B and FPLC on Mono Q and on Superose 12 (gel filtration). The purified enzyme was homogeneous on SDS-polyacrylamide gel disk electrophoresis. The relative molecular mass of the purified enzyme was 190,000 daltons by gel filtration. The SDS-polyacrylamide gel disk electrophoresis gave the relative molecular mass of 47,500 daltons. These data indicated that the purified, native enzyme is a tetramer (190,000 daltons) composed of four 47,500-dalton subunits. The isoelectric point of the enzyme was 6.0. The purified enzyme was intensely activated by manganese ion (50-fold increase or more when compared with magnesium ion). The enzyme worked best at 37 degrees C and pH 8.5 in a reaction mixture (50 microliters) containing 1.0 micrograms lambda DNA, 10 mM Tris-HCl, 7 mM 2-mercaptoethanol, 7 mM MnCl2 and 50 mM NaCl. The enzyme recognizes the same palindromic hexanucleotide sequence 5'-GACGTC-3', cuts between T and C and produces a 3'-tetranucleotide extension in the presence of MnCl2, as it does in the presence of MgCl2.
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PMID:Purification of restriction endonuclease from Acetobacter aceti IFO 3281 (AatII) and its properties. 136 9

A restriction endonuclease, designated as DmaI, was purified from cell-free extracts of Deleya marina IAM 14114 by streptomycin treatment, ammonium sulfate fractionation and two steps of chromatographies on heparin-Sepharose CL-6B and Mono Q (HR 5/5, FPLC). The purified enzyme was homogeneous on SDS-polyacrylamide gel disk electrophoresis and a ligation-recutting test. The relative molecular mass measurements of the purified enzyme gave 28,000 daltons by SDS-polyacrylamide gel disk electrophoresis and 56,000 daltons by gel filtration. These data indicated that the purified enzyme (56,000 daltons) has a dimeric structure composed of two 28,000-dalton subunits. The isoelectric point was 5.5. The purified enzyme worked best at 37 degrees C in a reaction mixture (50 microliters) containing 1.0 micrograms lambda DNA, 10 mM Tris-HCl, 7 mM 2-mercaptoethanol, 7 mM MgCl2 and 100 mM NaCl (pH 7.5). The enzyme was stable up to 55 degrees C and between pH 7.0 and 9.0. The purified enzyme recognizes the palindromic hexanucleotide DNA sequence 5'-CAGCTG-3', cuts between G and C and produces a flush end (isoschizomer of PvuII).
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PMID:Purification and properties of restriction endonuclease from Deleya marina IAM 14114, a marine bacterium (DmaI). 136 11

Thirty-three clinical isolates from male nongonococcal urethritis and 28 isolates from soft tissue infections and ulcers were identified as Bacteroides ureolyticus by conventional bacteriological tests and were compared with five reference strains of the species. Whole-cell proteins from these clinical isolates and the reference strains were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The majority of the strains from the two sources could be divided into five different groups, named phenons I to V; phenons I to IV have been described previously by others, while phenon V has been described recently by us. Digestion of chromosomal DNA from 16 of the clinical isolates (including strains representative of each of the five SDS-PAGE phenons) and the five reference strains was attempted with restriction endonucleases EcoRI, PstI, SmaI, and HindIII. After electrophoresis in agarose gels, good digestion was observed with HindIII only, and 12 different banding patterns (restriction endonuclease analysis [REA] profiles) were obtained for the 19 strains digested; one nongonococcal urethritis isolate and one reference strain did not show any digestion. From the agarose gels, HindIII-digested fragments of DNA were transferred to nylon membranes by use of vacuum blotting and subjected to hybridization with 32P-labelled 16S-23S rRNA from Escherichia coli. The resultant pattern of bands (ribotypes), which depends on the restriction fragment length polymorphisms in the rRNA genes, was used as a measure of genomic variation within the species. In total, 13 different ribotypes were obtained for the 19 strains. For some strains, good correlation was achieved among the SDS-PAGE phenons, REA profiles, and ribotypes. However, for others, REA analysis and ribotyping were able to discriminate between strains which shared the same SDS-PAGE phenon. Interestingly, these two techniques of DNA characterization were able to differentiate between isolates from the genital tract and those associated with soft tissue infections and ulcers.
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PMID:Restriction endonuclease analysis and ribotyping differentiate genital and nongenital strains of Bacteroides ureolyticus. 140 Oct 7

O6-Alkylguanine-DNA alkyltransferase (ATase) activity was determined in crude sonicates of tissues obtained from the F2 offspring of human ATase transgenic founder mice. In certain cases, samples were analyzed both before and after administration of zinc sulfate in the drinking water for 2 wk to upregulate the mouse metallothionein-1 promoter that controls the expression of the transgene. In liver samples obtained by partial hepatectomy, the ATase activities of nontransgenic mice ranged from 63 to 139 fmol/mg total protein (mean of 10 mice, 95.3 +/- 23 fmol/mg), whereas in positive transgenic mice, the range was from 503 to 2119 fmol/mg (mean of 10 mice, 963 +/- 475 fmol/mg). The difference between the mean ATase values for these two groups of mice is highly significant (P less than 0.001). All positive mice expressed ATase and in those examined using the human ATase coding sequence as a probe, isoschizomeric-restriction endonuclease digestion showed no evidence of cytosine methylation in the transgene. After zinc sulfate induction, the ATase levels in residual liver tissue were for the controls 84-191 fmol/mg (mean of 10 mice, 123 +/- 31.5 fmol/mg) and for positive mice 908-3273 fmol/mg (mean of 10 mice, 1960 +/- 724 fmol/mg). Induction thus caused a 1.4- to 3.2-fold increase in ATase activity in the tissues of individual transgenic mice (mean, 1.8-fold; P less than 0.003), with the greatest increase generally occurring in those mice that had the lowest preinduction levels. Hepatic ATase levels were thus increased up to 28 times higher in transgenic mice than in nontransgenic mice. When data from other groups of transgenic and nontransgenic mice (eight of each) was included and analyzed in an independent rather than paired fashion, the mean values for zinc-treated controls and transgenic mice, respectively, were 106 fmol/mg and 1415 fmol/mg, still a highly significant (P less than 0.001) difference. In two mice given a single intraperitoneal dose of cadmium chloride, hepatic ATase increased 2.1- and 4.9-fold, respectively. The effect of partial hepatectomy alone was also considered: for transgenic mice the mean ATase level increased from 453 to 661 fmol/mg protein after 48 h. In other offspring subjected to either unilateral nephrectomy or orchidectomy, induction of ATase activity by zinc sulfate was also seen in kidney (5.7- and 8.4-fold) and testis (1.7- and 3.1-fold), although these observations were made with small numbers of mice.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Tissue-specific expression and induction of human O6-alkylguanine-DNA alkyltransferase in transgenic mice. 150 42

Crystals have been obtained of the extracellular endonuclease from the bacterial pathogen Serratia marcescens. This magnesium-dependent enzyme is equally active against single and double-stranded DNA, as well as RNA, without any apparent base preference. The Serratia nuclease is not homologous with staphylococcal nuclease, the only other broad specificity endonuclease for which a structure exists, nor is it homologous with other nucleases that have been solved by X-ray diffraction. The structure of this enzyme should, therefore, provide new information about this class of enzyme. At present we have succeeded in obtaining large, high quality crystals using ammonium sulfate. They crystallize in the orthorhombic space group P2(1)2(1)2(1), with cell dimensions a = 106.7 A, b = 74.5 A, c = 68.9 A, and diffract to beyond 2 A. Low-resolution native data sets have been recorded and a search is under way for heavy-atom derivatives.
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PMID:Crystallization and preliminary crystallographic analysis of a novel nuclease from Serratia marcescens. 165 38

Two isoforms of an extracellular endonuclease, nucleases Sm1 and Sm2, were purified from culture fluid of Serratia marcescens strain BIO MI by ligand-exchange chromatography on phosphocellulose and DEAE-Toyopearl 650S. The pI-values for nucleases Sm1 and Sm2 were found to be 7.1 and 6.7, respectively. The amino acid analysis and N-terminal amino acid sequencing of the proteins showed a significant degree of homology between the enzymes. The nuclease Sm1 has been crystallized from ammonium sulfate solution by the vapour diffusion technique. The crystals belong to the space group P2(1)2(1)2(1) with unit cell constants a = 69.0, b = 106.7, c = 74.8 A, contain two molecules in an asymmetric unit, packing density Vm = 2.3 A/Da, and diffract to at least 1.5 A resolution. The Pt- and UO2-derivatives of the protein were obtained. Preliminary X-ray investigation of nuclease Sm2 crystals was carried out.
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PMID:Two isoforms of Serratia marcescens nuclease. Crystallization and preliminary X-ray investigation of the enzyme. 166 39

The intestinal anaerobic spirochetes Treponema hyodysenteriae B78T (T = type strain), B204, B169, and A-1, Treponema innocens B256T and 4/71, Treponema succinifaciens 6091T, and Treponema bryantii RUS-1T were compared by performing DNA-DNA reassociation experiments, sodium dodecyl sulfate-polyacrylamide gel electrophoresis of cell proteins, restriction endonuclease analysis of DNA, and 16S rRNA sequence analysis. DNA-DNA relative reassociation experiments in which the S1 nuclease method was used showed that T. hyodysenteriae B78T and B204 had 93% sequence homology with each other and approximately 40% sequence homology with T. innocens B256T and 4/71. Both T. hyodysenteriae B78T and T. innocens B256T exhibited negligible levels of DNA homology (less than or equal to 5%) with T. succinifaciens 6091T. The results of comparisons of protein electrophoretic profiles corroborated the DNA-DNA reassociation results. We found high levels of similarity (greater than or equal to 96%) in electrophoretic profiles among T. hyodysenteriae strains, moderate levels of similarity (43 to 49%) between T. hyodysenteriae and T. innocens, and no detectable similarity between the profiles of either T. hyodysenteriae or T. innocens and those of T. succinifaciens, T. bryantii, and Escherichia coli. Restriction endonuclease analysis of DNA was not useful in assessing genetic relationships since there was heterogeneity even between strains of T. hyodysenteriae. Partial 16S rRNA sequences of the intestinal spirochetes were determined by using a modified Sanger method and were compared in order to evaluate the phylogenetic relationships among these and other spirochetes.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Reclassification of Treponema hyodysenteriae and Treponema innocens in a new genus, Serpula gen. nov., as Serpula hyodysenteriae comb. nov. and Serpula innocens comb. nov. 170 92


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