Disruption of the Aspergillus fumigatus argB gene using a novel in vitro transposon-based mutagenesis approach

• Published in: Current genetics Vol. 45; no. 4; pp. 235 - 241
• Main Authors: Jadoun, J, Shadkchan, Y, Osherov, N
• Format: Journal Article
• Language: English
• Published: United States Springer Nature B.V 01-04-2004
• Subjects: argB gene; arginine; Aspergillus fumigatus; Aspergillus fumigatus - genetics; Aspergillus fumigatus - metabolism; auxotrophs; Blotting, Southern; carboxy-lyases; DNA Transposable Elements; Fungi; Gene Deletion; genes; Genetic Complementation Test; Genetic Vectors; Mutagenesis; Neurospora crassa; ornithine carbamoyltransferase; Ornithine Carbamoyltransferase - genetics; Ornithine Carbamoyltransferase - metabolism; orotidine-5'-monophosphate decarboxylase; Polymerase Chain Reaction; pyr4 gene; transposons
• ISSN: 0172-8083; 1432-0983
• DOI: 10.1007/s00294-003-0480-6

We disrupted the Aspergillus fumigatus argB gene, encoding ornithine transcarbamylase, using a novel in vitro transposon-based mutagenesis approach. This approach utilizes a modified transposon containing the Neurospora crassa pyr4 gene, which is randomly inserted in vitro into a target sequence of interest. Clones in which the gene of interest has been disrupted are identified by PCR and used to transform a pyrG-deficient strain of A. fumigatus. Using this approach, we obtained arginine auxotrophs of A. fumigatus. Full characterization of the argB insertion was performed by Southern blot analysis. These strains can be supplemented by addition of arginine into the culture medium and can be fully rescued to arginine prototrophy by transformation with the intact A. fumigatus argB gene.