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)

In 1990, 17 adult Rhipicephalus turanicus ticks were collected in the south of France. Two spotted fever group rickettsiae, Mtu1 and Mtu5, were isolated from the hemolymphs of two of these ticks by the centrifugation shell-vial technique by using HEL cells. These isolates were compared with reference spotted fever group rickettsial serotypes by using three identification methods: microimmunofluorescence serologic typing, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and polymerase chain reaction followed by restriction endonuclease fragment length polymorphism analysis. The results obtained by all these techniques showed that Mtu1 and Mtu5 are each previously undescribed rickettsial serotypes. A comparison of the three methods used to identify the isolates led us to the conclusion that, in large-scale epidemiological studies, the simplest way to identify isolates in ticks is to first use the polymerase chain reaction-restriction fragment length polymorphism analysis directly on triturated ticks as a screening method to detect interesting rickettsiae, and then attempt to isolate rickettsiae from ticks for identification by microimmunofluorescence and SDS-PAGE, both of which are time-consuming and expensive to carry out.
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PMID:Comparison of serologic typing, sodium dodecyl sulfate-polyacrylamide gel electrophoresis protein analysis, and genetic restriction fragment length polymorphism analysis for identification of rickettsiae: characterization of two new rickettsial strains. 135 21

This study was undertaken to assess the discriminatory value of restriction endonuclease (RE) digestion patterns of Streptococcus suis chromosomal DNA using polyacrylamide gel electrophoresis (SDS-PAGE) and DNA-rDNA hybridization. For the RE digestion patterns, DNAs were digested separately with the enzymes BamHI and BglII and the resultant fragments were separated by SDS-PAGE. An Escherichia coli rDNA probe derived from pKK3535 was used for the hybridization. Twenty-three S. suis capsular type 2 isolates recovered from diseased and clinically healthy pigs, from a human case, and from a cow were compared in this study. The majority of isolates associated with septicaemia belonged to one restriction endonuclease analysis (REA) profile group. Isolates associated with pneumonia belonged either to the REA profile group of isolates associated with septicaemia or to a second REA profile group. The REA profiles of isolates from clinically healthy animals were more heterogeneous. The REA profile of the type 2 reference strain, S735, which was originally isolated from a pig, was very different from those of the porcine and bovine isolates but similar to the profile of the human isolate. The profiles obtained after rDNA hybridization were more homogeneous. Although different patterns were detected in the 23 isolates, there was no correlation between the source of the isolate and the patterns observed with this technique.
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PMID:Molecular analysis of isolates of Streptococcus suis capsular type 2 by restriction-endonuclease-digested DNA separated on SDS-PAGE and by hybridization with an rDNA probe. 136 84

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

In Escherichia coli the mutY (or micA)-dependent DNA mismatch repair pathway can convert A degrees G and A degrees C mismatches to C.G and G.C base pairs, respectively, through a short repair-tract mechanism. The MutY protein has been purified to near homogeneity from an E. coli overproducer strain. Purified MutY has been shown to contain both N-glycosylase and 3' apurinic/apyrimidinic (AP) endonuclease activities. The N-glycosylase removes the mispaired adenines of A degrees G and A degrees C mismatches, and the AP endonuclease acts on the first phosphodiester bond 3' to the AP sites. The N-glycosylase and the nicking (combined N-glycosylase and AP endonuclease) activities copurified through multiple chromatographic steps without a change in relative specific activities. Furthermore, both N-glycosylase and AP endonuclease activities can be recovered by renaturation of a single polypeptide band from an SDS/polyacrylamide gel. Renaturation required the presence of iron and sulfide. These findings suggest that the MutY protein, like endonuclease III, is an iron-sulfur protein. DNA fragments with A degrees C mismatches were 20-fold less active than DNA with A degrees G mispairs as a substrate for purified MutY.
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PMID:Escherichia coli MutY protein has both N-glycosylase and apurinic/apyrimidinic endonuclease activities on A.C and A.G mispairs. 138 98

Sodium dodecyl sulphate-polyacrylamide gel electrophoresis of whole cell protein, immunoblotting with normal human serum and restriction endonuclease analysis using Taq I enzyme were applied to 38 clinically significant isolates of Moraxella (Branhamella) catarrhalis obtained during a suspected outbreak of nosocomial infection. Each of 18 strains had individual profiles by at least two of the three methods (unique strains). The remaining 20 strains were assigned to five groups (A-E) on the basis of similarity by at least two of the three methods. Isolates within groups A, D and E were homologous by all three methods. Immunoblot groups B and C had two distinct whole cell protein profiles (B1 and B2) but indistinguishable restriction endonuclease profiles (group B/C). This emphasizes the need to use more than one technique in characterizing strains from suspected outbreaks of nosocomial infection. Grouped strains were more likely to originate from the same hospital ward than unique strains and were associated with a significantly longer median time from patient admission to strain isolation (14 versus 3.5 days, p less than 0.005). Furthermore, the beta-lactamase activity was homologous within the groups. The results suggest that nosocomial infection involving several distinct Moraxella catarrhalis strains persisted over a period of months, involving at least 20 patients on three different wards. Such infection is probably common in wards harbouring suitably predisposed patients. The mode of transmission remains to be elucidated, but the above three techniques possess sufficient reproducibility and discriminatory ability to constitute suitable investigative tools.
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PMID:Use of molecular methods to characterize Moraxella catarrhalis strains in a suspected outbreak of nosocomial infection. 139 49

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

In order to alter the primary metabolism of C. acetobutylicum, we have constructed E. coli- or B. subtilis-C. acetobutylicum shuttle vectors that could be used to deliver homologous fermentative genes into C. acetobutylicum ATCC 824. The plasmid copy number and plasmid stability in C. acetobutylicum for several of these plasmids were determined. We have also developed a protocol for the electrotransformation of C. acetobutylicum ATCC 824. Difficulty in the transformation of C. acetobutylicum ATCC 824 with vectors containing DNA from E. coli plasmids was found to be due to the existence of a restriction system in this strain. This type II restriction endonuclease, named Cac824I, recognizes the sequence 5'-GCNGC-3' and cuts ColE1 plasmids frequently. One of the vectors, pFNK1, possessing a variety of unique cloning sites was used in the amplification of one acid (PTB) and one solvent (AADC) formation gene. The corresponding enzyme activities were amplified in C. acetobutylicum as shown by enzyme assays and SDS-PAGE gels of cell extracts.
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PMID:Vector construction, transformation, and gene amplification in Clostridium acetobutylicum ATCC 824. 141 17

NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH) is a key enzyme involved in the catabolism of the prostaglandins. The cDNA for human placental 15-PGDH has been expressed in Escherichia coli as a catalytically active protein. The polymerase chain reaction was used to introduce restriction endonuclease sites at each end of the 15-PGDH coding sequence. The 15-PGDH DNA was then inserted into the bacterial expression plasmids pUC-18 and pUC-19 which contain the isopropyl-l-thio-beta-D-galactopyranoside (IPTG) inducible lacZ promoter. Extracts from E. coli containing these expression plasmids exhibited 15-PGDH activity which was inducible with (IPTG). Crude extracts from E. coli expressing 15-PGDH activity were found to contain proteins of the predicted sizes in stained SDS-polyacrylamide gels and in Western blots using human placental 15-PGDH antiserum. The specific activity in E. coli extracts was several hundred-fold higher than that seen in extracts from human placenta.
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PMID:Expression of the cDNA for NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase as a catalytically active enzyme in Escherichia coli. 150 55


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