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Regine Kahmann - One of the best experts on this subject based on the ideXlab platform.
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REMI (Restriction Enzyme Mediated Integration) and its impact on the isolation of pathogenicity genes in fungi attacking plants
European Journal of Plant Pathology, 1999Co-Authors: Regine Kahmann, Christoph BasseAbstract:Development of molecular techniques for phytopathogenic fungi aims at the identification of fungal genes whose products are essential for successful infection of the host plant. Initial approaches have relied on isolating candidate genes and generating null-mutations by homologous recombination. Unfortunately, the results of this strategy have not been overly successful. This has led to a search for alternatives which allow an unbiased identification of pathogenicity genes. One method, which has proved successful in several systems, is a tagging mutagenesis procedure termed Restriction Enzyme mediated integration (REMI). In this mini-review we describe this procedure and review its features and results of its use when applied to the identification of fungal genes required for disease development in planta.
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tagging pathogenicity genes in ustilago maydis by Restriction Enzyme mediated integration remi
Molecular Genetics and Genomics, 1995Co-Authors: Michael Bolker, Heidi U Bohnert, Karl Heinz Braun, Johannes Gorl, Regine KahmannAbstract:In the maize pathogenic fungusUstilago maydis integration of transforming DNA at homologous or heterologous sites is often accompanied by duplications of the DNA. We show that it is possible to generate single-copy integration events with high efficiency by Restriction Enzyme-mediated integration (REMI). In about 50% of cases, a plasmid that contains a singleBamHI site is integrated at chromosomalBamHI sites, ifBamHI is added to the transformation mixtures. In the other cases it appears that integration events have also occurred preferentially atBamHI sites, but without restoration of the recognition sites. Using REMI we have generated approximately 1000 insertion mutants. Pathogenicity tests demonstrated that about 1–2% of these mutants were unable to induce symptoms when testedin planta. For two of the mutants we have shown that the phenotype is linked to the insertion event.
Ursula Kues - One of the best experts on this subject based on the ideXlab platform.
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Restriction Enzyme mediated dna integration in coprinus cinereus
Molecular Genetics and Genomics, 1997Co-Authors: Jose D Granado, Katerina Kerteszchaloupkova, Markus Aebi, Ursula KuesAbstract:Restriction Enzyme-mediated DNA integration (REMI) has recently received attention as a new technique for the generation of mutants by transformation in fungi. Here we analyse this method in the basidiomycete Coprinus cinereus using the homologous pab1 gene as a selectable marker and the Restriction Enzymes BamHI, EcoRI and PstI. Addition of Restriction Enzymes to transformation mixtures results in an earlier appearance of transformants and influences transformation rates in an Enzyme- and concentration-dependent manner. Low concentrations of Restriction Enzyme result in increased numbers of transformants compared to no addition of Enzymes. Transformation rates decrease with higher Enzyme concentrations. If protoplasts are made from cells stored in the cold, the transformation rates drop drastically even in the presence of low amounts of Enzyme. In several transformants, plasmid integration directly correlated with the action of Restriction Enzyme at random chromosomal Restriction sites. In some cases, Restriction Enzymes appear to reduce the number of integration events per transformant. Simultaneously, mutation rates can be enhanced due to the presence of Restriction Enzymes. Although Restriction Enzymes clearly promote plasmid integration into the host genome they also have cytotoxic and possibly mutagenic effects that result from processes other than plasmid integration. In consequence, for any given Enzyme used in REMI mutagenesis, the Enzyme concentration that gives the highest number of transformants must be defined experimentally. Such optimal transformation conditions should give the highest probability of obtaining mutations caused by a single Restriction Enzyme-mediated integration of the selection marker.
Jorge Chirife - One of the best experts on this subject based on the ideXlab platform.
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dsc confirmation that vitrification is not necessary for stabilization of the Restriction Enzyme ecori dried with saccharides
Biotechnology Progress, 1999Co-Authors: Maria Del Pilar Buera, Silvia Rossi, Silvia N J Moreno, Jorge ChirifeAbstract:The glass transition temperature (Tg) of preparations of the Restriction Enzyme EcoRI, vacuum-dried in the presence of sucrose, trehalose, or raffinose, was determined using differential scanning calorimetry. Tg values were well below those expected for low-moisture sucrose, trehalose, or raffinose, and this was attributed to the presence of glycerol (a plasticizer), which was a main component of the Restriction Enzyme preparation. This was verified by determining the glass transition temperature of glycerol, which was found to be (onset value) -77 degrees C. Present results confirmed that vitrification (i.e., glass formation) was not necessary for Enzyme protection in present low-moisture saccharide systems. As shown in previous work, Enzyme EcoRI was very stable stored at 37/45 degrees C in spite of the fact that sugar matrices were completely rubbery, as unequivocally demonstrated in the present work.
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stabilization of the Restriction Enzyme ecori dried with trehalose and other selected glass forming solutes
Biotechnology Progress, 1997Co-Authors: Silvia Rossi, Maria Del Pilar Buera, Silvia N J Moreno, Jorge ChirifeAbstract:The stabilization of the Restriction Enzyme EcoRI by its incorporation into aqueous glass-forming carbohydrate or polymer solutions, followed by vacuum-drying to low moisture, has been studied. Glass-forming solutes included trehalose, sucrose, lactose, maltose, raffinose, maltodextrin DE 10, and poly(vinylpyrrolidone) (molecular weight 40,000, PVP). Among the solutes examined, trehalose and sucrose protected the Enzyme most effectively during storage at 37 and 45 degrees C. The Restriction Enzyme dried with trehalose or sucrose maintained its activity without detectable loss for at least 20 days at 37 degrees C and 12 days at 45 degrees C. In contrast, the activity of the Enzyme dried with maltodextrin or PVP was reduced during vacuum desiccation and also it decreased remarkably during storage at the same temperatures. Stored (37/45 degrees C) vacuum-dried trehalose and sucrose systems were either a dense paste or a very viscous syrup, and this indicated that they were not glassy. Moreover, no relationship was found between the glass transition temperatures (Tg) of the pure added solute and Enzyme protection during storage, since, e.g., sucrose which has significantly lower Tg values protected the Enzyme much better than either maltose, lactose, maltodextrin, or PVP. The trisaccharide raffinose offered good protection of Enzyme activity, and its role as a novel excipient matrix for labile Enzyme stabilization deserves further investigation. The stability of Enzyme EcoRI was rapidly lost when the vacuum-dried trehalose and sucrose systems were humidified to 58% relative humidity and stored at 45 degrees C, and this was attributed to disaccharide crystallization.
Myra Schink - One of the best experts on this subject based on the ideXlab platform.
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bst7i an isoschizomer of the type iis Restriction Enzyme bbvi recognizing the site
Gene, 1992Co-Authors: Thomas W. Schoenfeld, Mike Fiandt, Myra SchinkAbstract:Abstract A new type-IIS Restriction Enzyme, Bst71I, with the specificity was isolated from Bacillus stearothermophilus (Promega No. 71). This Enzyme is an isoschizomer of Bbv I with somewhat improved characteristics for use by molecular biologists.
Ting Zhou - One of the best experts on this subject based on the ideXlab platform.
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biodegradation of neonicotinoid insecticide imidacloprid by Restriction Enzyme mediated integration remi generated trichoderma mutants
Chemosphere, 2014Co-Authors: Abebe Jenberie Wubie, Qingyun Diao, Fei Xue, Zhanbo Guo, Ting ZhouAbstract:REMI (Restriction Enzyme-mediated integration) technique was employed to construct Trichoderma atroviride strain T23 mutants with degrading capability of neonicotinoid insecticide, imidacloprid. The plasmid pBluescript II KS-hph used for integration in REMI mutants was confirmed by PCR and Southern hybridization. Among 153 transformants, 57% of them have showed higher neonicotinoid insecticide, imidacloprid, degradation ability than the wild strain T23 (p<0.01). More specifically, seven single-copied T. atroviride T23 transformants have confirmed a 30% higher degradation rate than their parent isolate. Among all transformed mutants, a 95% imidacloprid degradation rate was identified as the highest. This study, thus, provided an effective approach for improving neonicotinoid insecticide-degrading capability using REMI transformed T. atroviride mutants.
