Restriction enzyme-mediated DNA integration in Coprinus cinereus |
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Authors: | J D Granado K Kertesz-Chaloupková M Aebi and U Kües |
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Institution: | (1) Mikrobiologisches Institut, ETH Zürich, Schmelzbergstr. 7, CH-8092 Zürich, Switzerland Fax: +41-1-632-1148; e-mail: kues@micro.biol.ethz.ch, CH |
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Abstract: | 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.
Received: 2 September 1996 / Accepted: 7 April 1997 |
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Keywords: | Coprinus cinereus Gene tagging Mutagenesis Restriction enzyme-mediated integration (REMI) Transformation |
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