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)

Infection of cultured mammalian cells with the Leporipoxvirus Shope fibroma virus (SFV) causes the induction of a novel uracil DNA glycosylase activity in the cytoplasms of the infected cells. The induction of this activity, early in infection, correlates with the early expression of the SFV BamHI D6R open reading frame which possesses significant protein sequence similarity to eukaryotic and prokaryotic uracil DNA glycosylases. The SFV BamHI D6R open reading frame and the homologous HindIII D4R open reading frame from the Orthopoxvirus vaccinia virus were cloned under the regulation of a phage T7 promoter and expressed in Escherichia coli as insoluble high-molecular-weight aggregates. During electrophoresis on sodium dodecyl sulfate-polyacrylamide gels, the E. coli-expressed proteins migrate with an apparent molecular mass of 25 kDa. The insoluble protein aggregate generated by expression in E. coli was solubilized in urea and, following a subsequent refolding step, displayed the ability to excise uracil residues from double-stranded plasmid DNA substrates, with the subsequent formation of apyrimidinic sites. The viral enzyme, like all other characterized uracil DNA glycosylases, is active in the presence of high concentrations of EDTA, is substrate inhibited by uracil, and does not display any endonuclease activity. Attempts to inactivate the HindIII D4R gene of vaccinia virus by targeted insertion of a dominant xanthine-guanine phosphoribosyltransferase selection marker or direct insertion of a frame-shifted oligonucleotide were uniformly unsuccessful demonstrating that, unlike the uracil DNA glycosylase described for herpesviruses, the poxvirus enzyme is essential for virus viability.
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PMID:A poxvirus-encoded uracil DNA glycosylase is essential for virus viability. 847 56

Infection with Helicobacter hepaticus causes chronic active hepatitis in certain strains of mice and is associated with hepatocellular carcinoma in A/JCr mice. Like the gastric helicobacters, H. pylori and H. mustelae, H. hepaticus possesses a high level of urease activity. However, the H. hepaticus urease structural gene sequences have not been previously determined, and the role of the urease enzyme in colonization and in pathogenesis is not known. PCR was used to amplify a portion of the urease structural genes from H. hepaticus genomic DNA. Amplified DNA fragments were cloned, and the nucleotide sequence was determined. The deduced amino acid sequence of the partial H. hepaticus ureA gene product was found to exhibit 60% identity and 75% similarity to the predicted H. pylori UreA. The deduced amino acid sequence of a partial H. hepaticus ureB gene product exhibited 75% identity and 87% similarity to the predicted H. pylori UreB. Diversity among H. hepaticus isolates was evaluated by means of a restriction fragment length polymorphism (RFLP) assay. The 1.6-kb fragments within the ureAB open reading frames, amplified from 11 independent isolates, were digested with the restriction endonuclease HhaI. Three distinct RFLP patterns were observed. Identical RFLP profiles were noted in sequential isolates of one strain of H. hepaticus during an 18 month in vivo colonization study, suggesting that the urease genes of H. hepaticus are stable. The urease genes among H. hepaticus strains were also well conserved, showing 98.8 to 99% nucleotide sequence identity among three isolates analyzed. These findings indicate that H. hepaticus has urease structural genes which are homologous to those of the gastric Helicobacter species and that these gene sequences can be used in a PCR and RFLP assay for diagnosis of this important murine pathogen.
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PMID:Development of a PCR-restriction fragment length polymorphism assay using the nucleotide sequence of the Helicobacter hepaticus urease structural genes ureAB. 970 72

Pulsed field gel electrophoresis of restriction endonuclease digested genomic DNA from a collection of clinical isolates of Rhodococcus equi was used to compare strain diversity on different Thoroughbred horse farms over time. Restricted diversity was found among the isolates tested, as the same strains were detected on multiple farms and in multiple years. Marked variation occurred in strain prevalence with some strains being represented by single isolates, and the most prevalent by 26 isolates. There were dominant strains on some farms and the prevalence of some strains differed between farms. Infection with multiple strains was noted in some cases where multiple isolates from a single foal were examined.
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PMID:Diversity of isolates of Rhodococcus equi from Australian thoroughbred horse farms. 1006 85

OBJECTIVES: To compare genotypic methods for epidemiologic typing of Legionella pneumophila serogroup (sg) 1, in order to determine the best available method within Europe for implementation and standardization by members of the European Working Group on Legionella Infections. METHODS: Coded isolates (114) of L. pneumophila sg 1 comprising one epidemiologically 'unrelated' (79) and one 'related' panel of isolates (35) were sent to 12 laboratories in 11 European countries. Analysis was undertaken in each laboratory using one or more of the following methods: ribotyping, restriction fragment length polymorphism analysis, restriction endonuclease analysis, pulsed-field gel electrophoresis (PFGE), PCR using arbitrary/repeat sequence primers (AP-, AP/rep-PCR), and amplified fragment length polymorphism (AFLP) analysis. Results were analyzed visually or using gel analysis software. Each method was assessed for its: index of discrimination (D), epidemiologic concordance (E), speed of application and ease of use. In addition, phenotypic analysis was performed in two laboratories using monoclonal antibodies (mAbs). RESULTS: The D of each of the genotypic methods ranged from 0.840 for ribotyping to 0.990 for PFGE using Sfil: E ranged from 0.06 for AP- and AP/rep-PCR to 1.00 for ribotyping using Pstl/EcoRI and AFLP: in general, E was inversely related to D. Although offering only limited discrimination (D=0.838), mAb typing was both rapid and highly epidemiologically concordant (E=1.00). CONCLUSIONS: Two methods, PFGE using Sfil and AFLP, were selected for further study. AFLP is rapid and highly epidemiologically concordant (E=1.00), but is not highly discriminatory. This method will be developed as a rapid screening tool. PFGE using Sfil is highly discriminatory but, in the present study, yielded low values of E (0.12-0.71). Attempts will be made to rigorously standardize this method for use as the reference method. Primary screening of isolates by mAb subgrouping is recommended.
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PMID:A multicenter evaluation of genotypic methods for the epidemiologic typing of Legionella pneumophila serogroup 1: results of a pan-European study. 1185 90

Infections caused by fungi (mycoses) are increasingly reported in many countries owing to greater life expectancy associated with an increase in quality of medical and surgical procedures, as well as the emergence of diseases or infections that affect the immune system such as AIDS. Nosocomial outbreaks of fungal infections are sometimes reported, and typing is then necessary to find the reservoirs, analyze the modes of transmission, study the antifungal susceptibility patterns, and investigate the susceptibility of the host. In addition, the food industry is increasingly demanding typing methods that could help in selection of the best fungal strains, in order to incorporate them in the productive chains and augment the quality and security of food. This is the case for Saccharomyces cerevisiae in the wine industry: the selection and characterization of indigenous or autochthonous strains is an important objective for the production of high-quality certified wines.Several genotyping methods are now widely used for strain delineation of medically or economically important microorganisms belonging to the kingdom Fungi. Most molecular typing methods are comparable to those already described for bacteria, although the peculiarities of their nucleic acids increase the number of available methods. Although typing procedures based on the analysis of nucleic acid sequences have been developed, most genotyping methods currently in use are electrophoretically based, and the procedures include the visual comparison of nucleic acid band profiles or their reading with the help of computerized software. Here we describe some of the most frequently used genotyping methods for fungi, based on polymerase chain reactions (PCR), the isolation of chromosomal or mitochondrial DNA, and their restriction using endonuclease enzymes. The latter methods are exclusive for typing eukaryotic organisms and are based on the expected polymorphism obtained from the separation of large chromosomes using pulsed-field gel electrophoresis (PFGE) and the restriction of mitochondrial or chromosomal DNA. More sophisticated methods, such as those that combine endonuclease restriction with hybridization, are also available, although their use is less extensive and is limited mostly to research laboratories.
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PMID:Typing fungal isolates: molecular methods and computerized analysis. 1515 23

Adeno-associated virus (AAV) vectors transduce cells by multiple pathways, including integration at nonhomologous chromosomal locations by an unknown mechanism. We reasoned that spontaneous chromosome breaks may facilitate vector integration and investigated this in cells containing a specific chromosomal double-strand break created by the endonuclease I-SceI or multiple breaks created by treatment with etoposide or gamma-irradiation. Vector proviruses were found at I-SceI cleavage sites, and sequencing of vector-chromosome junctions detected microhomologies, deletions and insertions that were similar when integration occurred spontaneously at random locations or at induced double-strand breaks. Infection with AAV vectors did not increase mutation rates in normal human cells. Our results establish a mechanism for integration and suggest that AAV vectors can integrate at existing chromosome breaks rather than causing breaks themselves, which has implications for their clinical use.
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PMID:Adeno-associated virus vectors integrate at chromosome breakage sites. 1520 27

Bacteriophage PM2 has a closed circular form of double stranded DNA as a genome. This DNA from the phage is a useful source for nick-circle endonuclease assay in the fmol range. Due to difficulties in the maintenance of viral infectivity, storage conditions of the phage should be considered for the purification of PM2 DNA. The proper condition for a short-term storage of less than 2 months is to keep the PM2 phage at 4 degrees C; whereas the proper condition for a long-term storage of the PM2 phage for over 2 months is to keep it under liquid nitrogen in 7.5% glycerol. The optimal conditions for a high yield of phage progeny were also considered with the goal to achieve a successful PM2 DNA preparation. A MOI(Multiplicity Of Infection) of 0.03, in which the OD600 of the host bacteria was between 0.3 and 0.5, turned out to be optimal for the mass production of PM2 phage with a burst size of about 214. Considerations of PM2 genome size, and the concentrations and radiospecific activities of purified PM2 DNA, are required to measure the endonuclease activity in the fmol range. This study reports the proper quantitation of radioactivity and the yield of purified DNA based on these conditions.
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PMID:Biochemical quantitation of PM2 phage DNA as a substrate for endonuclease assay. 1535 2

Infection with equine herpesvirus-4 (EHV-4) is a major cause of respiratory tract disease, equine rhinopneumonitis, in horses. Although the full sequence of EHV-4 has been reported, genomic differences among EHV-4 field isolates have not yet been characterized. In this study, the genomic diversity between 23 Japanese EHV-4 isolates was analyzed by digestion with restriction endonucleases (BamHI, BgIII, EcoRI, SacI, and SalI) and polymerase chain reaction (PCR). The restriction endonuclease digestion patterns of the EHV-4 field isolates showed distinct differences which included mobility shifts of some fragments as well as loss and/or gain of fragments. Two EHV-4 genes containing repeat sequences, ORFs 24 and 71, were amplified by PCR and the amplified fragments were compared among the field isolates. The sizes of the amplified fragments varied among epizootiologically unrelated isolates, while the fragments of related isolates had the same size. The observed genomic diversity among EHV-4 field isolates may be a useful tool for epidemiological study of equine rhinopneumonitis by EHV-4 infection.
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PMID:Genomic diversity among equine herpesvirus-4 field isolates. 1599 81

In this study, we demonstrated that the methyltransferase activity associated with Dam was essential for attenuation of Aeromonas hydrophila virulence. We mutated aspartic acid and tyrosine residues to alanine within the conserved DPPY catalytic motif of Dam and transformed the pBAD/damD/A, pBAD/damY/A, and pBAD/damAhSSU (with the native dam gene) recombinant plasmids into the Escherichia coli GM33 (dam-deficient) strain. Genomic DNA (gDNA) isolated from either of the E. coli GM33 strains harboring the pBAD vector with the mutated dam gene was resistant to DpnI digestion and sensitive to DpnII restriction endonuclease cutting. These findings were contrary to those with the gDNA of E. coli GM33 strain containing the pBAD/damAhSSU plasmid, indicating nonmethylation of E. coli gDNA with mutated Dam. Overproduction of mutated Dam in A. hydrophila resulted in bacterial motility, hemolytic and cytotoxic activities associated with the cytotoxic enterotoxin (Act), and protease activity similar to that of the wild-type (WT) bacterium, which harbored the pBAD vector and served as a control strain. On the contrary, overproduction of native Dam resulted in decreased bacterial motility, increased Act-associated biological effects, and increased protease activity. Lactone production, an indicator of quorum sensing, was increased when the native dam gene was overexpressed, with its levels returning to that of the control strain when the dam gene was mutated. These effects of Dam appeared to be mediated through a regulatory glucose-inhibited division A protein. Infection of mice with the mutated Dam-overproducing strains resulted in mortality rates similar to those for the control strain, with 100% of the animals dying within 2 to 3 days with two 50% lethal doses (LD50s) of the WT bacterium. Importantly, immunization of mice with a native-Dam-overproducing strain at the same LD50 did not result in any lethality and provided protection to animals after subsequent challenge with a lethal dose of the control strain.
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PMID:Mutations within the catalytic motif of DNA adenine methyltransferase (Dam) of Aeromonas hydrophila cause the virulence of the Dam-overproducing strain to revert to that of the wild-type phenotype. 1698 54

Klebsiella pneumoniae is a gram-negative bacterium of significant clinical importance. This study examines the differential pulmonary host anti-bacterial responses towards two clinical isolates of K. pneumoniae. Intratracheal inoculation with 7 x 10(4)CFU of strain 43816 induced 100% mortality in C57BL/6J mice within 5 days post infection, whereas infection with 5 x 10(5)CFU of strain IA565 resulted in 100% survival. Infection with strain 43816 resulted in significant pulmonary and peripheral blood bacterial burden and induction of the chemokines MIP-2, KC and MCP-1 by 24h post infection. In contrast, IA565-infected mice displayed basal chemokine levels and no detectable bacteria by 24h post inoculation were isolated from lungs or peripheral blood. These data indicate an apparent lack of pathogenicity of strain IA565. Since little is known about Klebsiella-specific virulence genes, we have utilized PCR-based genomic DNA and cDNA suppressive subtractive hybridization and identified nine DNA sequences unique to the pathogenic strain of K. pneumoniae 43816. These sequences were highly homologous to enteric bacterial genes regulating iron uptake, fimbrial-mediated adhesion, energy production and conversion, transcriptional regulation, signal transduction, restriction endonuclease activity, and membrane transport.
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PMID:Identification of Klebsiella pneumoniae genes uniquely expressed in a strain virulent using a murine model of bacterial pneumonia. 1736 11


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