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)

A factor stimulating RNA polymerase II from Ehrlich ascites tumor cells was purified. The final preparation appeared almost homogeneous on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and had a molecular weight of 38 000. The endonuclease activity of about 10 mug of purified factor, if any was well below the 10(-5) mug equivalent of pancreatic deoxyribonuclease, indicating that the stimulation of RNA synthesis by this factor was not due to contaminating endonuclease. This factor specifically stimulated RNA polymerase II on native DNA as template and did not affect RNA polymerase I at all. The molecular size of RNA synthesized in the presence of this factor increased markedly compared with that synthetized by RNA polymerase II alone.
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PMID:Purification of a factor from Ehrlich ascites tumor cells specifically stimulating RNA polymerase II. 99 Feb 65

A soluble extract prepared from T7-infected E. coli is able to initiate DNA synthesis on an exogenous T7 DNA template. We have developed a fractionation procedure to resolve and identify the proteins required for T7 DNA synthesis. By this method we have purified the following T7 replication-related proteins (each greater than 50% pure as judged by sodium dodecyl sulfate gel electrophoresis): T7 DNA-binding protein (27,000 daltons), T7 RNA polymerase (105,000 daltons), T7 DNA polymerase (gene 5-protein, 85,000 daltons, plus host-factor), T7 DNA ligase (40,000 daltons), and T7 DNA-priming protein (65,000 daltons). The T7 DNA-priming protein, synthesized between 7.5 and 15 min following infection, was not detectable if the infecting phage carried an amber mutation in gene 4. Using an in vitro complementation assay which specifically measures the stimulation of DNA synthesis in an extract prepared from T7 gene 4-mutant infected cells, we have purified the DNA-priming protein about 2,000-fold. The purified priming protein preparations are essentially free of endonuclease, exonuclease, DNA ligase and DNA polymerase activity, but they do contain measurable DNA-dependent RNA synthetic acitvity. The enzyme is rapidly inactivated by heating to 46 degrees C and by treatment with N-ethylmalemide. In the presence of T7 DNA-binding protein and all four ribonucleoside triphosphates, the DNA-priming protein enables T7 DNA polymerase to initiate DNA synthesis on intact duplex T7 DNA. Closer studies of its enzymatic function as well as of the possible roles of the other proteins in the T7 replication system will be presented in the accompanying paper.
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PMID:Studies on bacteriophage T7 DNA synthesis in vitro. I. Resolution of the T7 replication system into its components. 110 17

The culture medium of Pseudomonas BAL 31 contains endonuclease activities which are highly specific for single-stranged DNA and for the single-stranded or weakly hydrogen-bonded regions in supercoiled closed circular DNA. Exposure of nicked DNA to the culture medium results in cleavage of the strang opposite the sites of preexisting single-strand scissions. At least some of the linear duplex molecules derived by cleavage of supercoiled closed circular molecules contain short single-stranded ends. Single-strand scissions are not introduced into intact, linear duplex DNA or unsupercoiled covalently closed circular DNA. Under these same reaction conditions, 0X174 phage DNA is extensively degraded and PM2 form I DNA is quantitatively converted to PM2 form III linear duplexes. Prolonged exposure of this linear duplex DNA to the concentrated culture medium reveals the presence of a double-strand exonuclease activity that progressively reduces the average length of the linear duplex. These nuclease activities persist at ionic strengths up to 4 M and are not eliminated in the presence of 5% sodium dodecyl sulfate. Calcium and magnesium ion are both required for optimal activity. Although the absence of magnesium ion reduces the activities, the absence of calcium ion irreversibly eliminates all the activities.
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PMID:Extracellular nucleases of Pseudomonas BAL 31. I. Characterization of single strand-specific deoxyriboendonuclease and double-strand deoxyriboexonuclease activities. 117 26

A Mycoplasma gallisepticum strain designated 6/85 (MGI) exhibiting reduced virulence for both chickens and turkeys was sequentially passaged 10 times in each species. DNA extracted from organisms before passage and those isolated after the third, sixth, and 10th passages was studied by restriction endonuclease DNA analysis using BamHI, BglII, EcoRI, HindIII, and PstI endonucleases. The virulent-type strain designated S6 was used as a comparison. Comparison of DNA fragment patterns of MGI and S6 strains showed distinct differences, although some similarities were evident. Passage of the strain in vivo did not affect DNA fragment patterns of the MGI strain. Electrophoretic protein patterns produced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed very similar band patterns in both the MGI and S6 strains. The most notable differences were seen in bands located in the molecular-mass regions of approximately 46.5, 50-54, 58-64, and 105-140 kilodaltons. Alteration of band pattern profiles following in vivo passage of the MGI strain was apparent in a single band at approximately 86 kilodaltons that appeared to stain more intensely following passage.
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PMID:Demonstration of the genetic stability of a Mycoplasma gallisepticum strain following in vivo passage. 132 7

Mycoplasma synoviae (MS) isolates made in 1988-89 from turkey flocks in North Carolina, Missouri, and Ontario, Canada, were compared with each other and MS reference strains (WVU-1853, F10-2AS, Neb-3S, and K1968) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of cell proteins and restriction endonuclease analysis (REA) of DNA. SDS-PAGE and REA indicated considerable homology among MS reference strains and recent field isolates. However, sufficient differences were resolved to identify the MS reference strains as different from each other and the field isolates, and to classify seven of nine recent field isolates as a cluster of nearly identical strains. The results suggest that flocks infected with members of the cluster were epizootiologically associated, possibly by a common or point source of infection.
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PMID:An outbreak of Mycoplasma synoviae infection in North Carolina turkeys: comparison of isolates by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and restriction endonuclease analysis. 132 12

Exopolysaccharides interfere with the isolation and characterization of plasmid DNA from gram-negative bacteria. To repress capsular polysaccharide production, bacteria were cultured in medium containing bismuth nitrate and sodium salicylate. Rapid removal of other contaminating bacterial surface components was achieved by mild acidic zwitterionic detergent extraction. After treatment, bacterial cells were more readily lysed in alkaline detergents. The resulting plasmid preparations contained virtually no capsular polysaccharide and relatively small quantities of lipopolysaccharide and protein, yet they produced yields of nucleic acids similar to those of conventional plasmid preparations. Conventional preparations from encapsulated organisms were largely insoluble and appeared as smears following agarose gel electrophoresis, with indefinite plasmid banding. Plasmids prepared by the new method were highly soluble in conventional buffers and exhibited high-resolution plasmid banding patterns in agarose gels. Plasmids as large as 180 kbp could be isolated and visualized, without apparent nicking, and were readily digested by restriction endonuclease enzymes. The method proved effective with encapsulated or mucoid strains of Klebsiella pneumoniae, Escherichia coli, Acinetobacter anitratus, Salmonella typhimurium, and Enterobacter species. The complete method for plasmid isolation was not suitable for Pseudomonas aeruginosa because of the inhibitory effects of bismuth. Thus, removal of contaminating bacterial surface structures enabled the rapid isolation and characterization of plasmids from mucoid clinical isolates, without the use of organic solvents, CsCl gradients, or expensive, disposable columns.
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PMID:Rapid plasmid DNA isolation from mucoid gram-negative bacteria. 133 82

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

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

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

Heparin-agarose chromatography was used to isolate a restriction endonuclease (ENase) from the cellulolytic Gram+ anaerobe, Ruminococcus albus 8. The enzyme, Ral8I, was eluted from the column using 230-310 mM Na+. However, the preparation was active only with DNA substrates that were not Dam-methylated. Moreover, the restriction fragment pattern generated from simian virus 40 (SV40) DNA was not consistent with the expected number of Dam-methylation sites. Alignment of the Dam-methylation sites in SV40 DNA indicated that Ral8I may actually recognize the asymmetric sequence, GGATC. This was confirmed by nucleotide (nt) sequence analysis and, further, Ral8I was found to cause cleavage of the DNA approx. 5 nt downstream from the recognition sequence. Ral8I can therefore be classified as a type-IIS restriction endonuclease and is an isoschizomer of AlwI, BinI and BthII.
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PMID:Partial purification and characterization of Ral8I, a class-IIS restriction endonuclease from Ruminococcus albus 8 which recognizes 5'-GGATC. 154 46


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