Restriction enzyme-mediated DNA integration in Coprinus cinereus

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 pabI 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 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.

Fruiting body development in Coprinus cinereus: regulated expression of two galectins secreted by a non-classical pathway The GenBank accession number for the sequence reported in this paper is AF130360

“…Generation of the trp1 + and trp1 + A on transformants of the monokaryons PG78 (A6 B42 trp-1.1,1.6), FA2222 (A5 B6 trp-1.1,1.6) and 218 (A3 B1 trp-1.1,1.6) was as described previously . Strains were grown on solid YMG or YMG\T medium (Rao & Niederpruem, 1969 ;Granado et al, (1998) 1997) in dark and light conditions as described by KerteszChaloupkova! et al (1998).…”

“…Fruiting body development was followed by growing strains at 37 mC on YMG\T in the dark for 5 d and then incubating the cultures at 25 mC in an alternating 12 h light\dark cycle (Granado et al, 1997). For the nutritional studies, homokaryon AmutBmut was grown at 37 mC in continuous dark on Coprinus minimal medium supplemented with 5 mg para-aminobenzoic acid ml − " (Granado et al, 1997), containing various concentrations of glucose or asparagine. For all of the experiments the cultures were grown from single explants inoculated in the centre of 90 mm Petri dishes.…”

Oligonucleotide sequences forming short self-complimentary hairpins can expedite the down-regulation of Coprinopsis cinerea genes

“…The Coprinopsis cinerea auxotroph strain PG78 (A6 B42 pab1 trp1.1, 1.6; (Granado et al, 1997)) was used for GFP transformations and was the negative non-transformed control.…”

Biomass measurement by flow cytometry during solid‐state fermentation of basidiomycetes

“…Extensive sample disruption is required in order to obtain strong and reproducible signals. In preliminary experiments, a gentle biological method of disruption involving enzymatic treatment of the SSF samples based on the work of Granado et al (52) was investigated. Unfortunately, this method was ineffective.…”