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Eukaryotic Cell, July 2005, p. 1298-1307, Vol. 4, No. 7
1535-9778/05/$08.00+0     doi:10.1128/EC.4.7.1298-1307.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Deletion and Allelic Exchange of the Aspergillus fumigatus veA Locus via a Novel Recyclable Marker Module

Sven Krappmann,* Özgür Bayram, and Gerhard H. Braus

Institute of Microbiology & Genetics, Department of Molecular Microbiology and Genetics, Georg-August-University Göttingen, Göttingen, Germany

Received 22 February 2005/ Accepted 26 April 2005

Detailed evaluation of gene functions in an asexual fungus requires advanced methods of molecular biology. For the generation of targeted gene deletions in the opportunistic pathogen Aspergillus fumigatus we designed a novel blaster module allowing dominant selection of transformants due to resistance to phleomycin as well as dominant (counter)selection of a Cre recombinase-mediated marker excision event. For validation purposes we have deleted the A. fumigatus pabaA gene in a wild-type isolate by making use of this cassette. The resulting pabaA::loxP strain served as the recipient for subsequent targeting of the velvet locus. Homologous reconstitution of the deleted gene was performed by an allele whose expression is driven in a nitrogen source-dependent manner, as validated by Northern analyses. Overexpression of the veA locus in A. fumigatus does not result in any obvious phenotype, whereas the sporulation capacities of the veA null mutant are reduced on nitrate-containing medium, a phenotype that is completely restored in the reconstituted strain.

* Corresponding author. Mailing address: Institute of Microbiology & Genetics, Department of Molecular Microbiology and Genetics, Grisebachstr. 8, D-37077 Göttingen, Germany. Phone: 49 551 39-3772. Fax: 49 551 39-3820. E-mail: skrappm{at}gwdg.de.

Eukaryotic Cell, July 2005, p. 1298-1307, Vol. 4, No. 7
1535-9778/05/$08.00+0     doi:10.1128/EC.4.7.1298-1307.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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