title: Insertional mutagenesis of Aspergillus fumigatus.
abstract: We have investigated transformation with heterologous DNA as a method for insertional mutagenesis of Aspergillus fumigatus. Two methods, polyethylene glycol-mediated transformation of protoplasts and electroporation of germinating spores, were used to establish conditions leading to single-copy integration of transforming DNA at different genomic sites. We have assessed the effect of restriction enzyme-mediated integration (REMI) for both methods. Non-REMI protoplast transformation led to integration of multiple copies of transforming DNA in the majority of transformants. Results of REMI with protoplast transformation varied depending on the enzyme used. Low concentrations of several restriction enzymes stimulated transformation, but of ten enzymes investigated only REMI with XhoI and KpnI resulted in single-copy integration of transforming DNA for the majority of transformants. For protoplast transformation with XhoI- or KpnI-based REMI, 50% and 76% of insertions, respectively, were due to integrations at a genomic enzyme site corresponding to the enzyme used for REMI. Electroporation of spores without addition of restriction enzyme resulted in a high transformation efficiency, with up to 67% of transformants containing a single copy of transforming DNA. In contrast to protoplast transformation, electroporation of spores in the presence of a restriction enzyme did not improve transformation efficiency or lead to insertion at genomic restriction sites. Southern analysis indicated that for both protoplast transformation with REMI using KpnI or XhoI and for electroporation of spores without addition of restriction enzymes, transforming DNA inserted at different genomic sites in a high proportion of transformants.
authors.affiliation: Department of Infectious Diseases, Imperial College School of Medicine, Hammersmith Hospital, London, UK.
authors.forename: J S
authors.initials: JS
authors.lastname: Brown
authors.forename: A
authors.initials: A
authors.lastname: Aufauvre-Brown
authors.forename: D W
authors.initials: DW
authors.lastname: Holden
mesh_terms: D001232:Aspergillus fumigatus / Q000235:genetics* / Q000472:pathogenicity; D004262:DNA Restriction Enzymes; D004271:DNA, Fungal / Q000235:genetics; D004274:DNA, Recombinant / Q000008:administration & dosage / Q000235:genetics; D018274:Electroporation; D016681:Genome, Fungal; D016254:Mutagenesis, Insertional / Q000379:methods*; D009693:Nucleic Acid Hybridization; D010957:Plasmids / Q000235:genetics; D011523:Protoplasts; D013172:Spores, Fungal / Q000235:genetics; D014170:Transformation, Genetic; D014774:Virulence / Q000235:genetics
chemical_list: D004271:DNA, Fungal; D004274:DNA, Recombinant; D004262:DNA Restriction Enzymes