Print Email Facebook Twitter A novel strategy for the isolation of defined pyrG mutants and the development of a site-specific integration system for Aspergillus awamori Title A novel strategy for the isolation of defined pyrG mutants and the development of a site-specific integration system for Aspergillus awamori Author Gouka, R.J. Hessing, J.G.M. Stam, H. Musters, W. van den Hondel, C.A.M.J.J. Centraal Instituut voor Voedingsonderzoek TNO Publication year 1995 Abstract A homologous gene transfer system for Aspergillus awamori for site-specific integration is described, based on two components. First, a defined A. awarnori pyrG mutant strain constructed by a selection strategy for gene-replacement in fungi. Second, a vector with a homologous pyrG selection marker containing a defined mutation at a site different from that of the mutations in the pyrG gene of the defined mutant strain. Defined mutation in the A. awarnori pyrG gene, isolated from a genomic library by heterologous hybridisation with the A. niger pyrG gene as a probe, were introduced by specifically altering sequences at restriction sites in the coding region of the gene. After transformation of the A. awamori wild-type strain with vectors containing these mutated pyrG genes, and selection for 5-fluoro-orotic acid resistance (5-FOAR), on the average 60% of the 5-FOAR colonies originated from replacement of the wild-type pyrG gene by the mutated pyrG allele. After transformation of a mutant strain, carrying a mutation near the 5' end of the pyrG gene with vectors containing a mutation near the 3' end of the pyrG gene, 35% of the resulting transformants contained one copy of the vector at the pyrC locus. Subject BiologyAmino Acid SequenceAspergillusBase SequenceDNA, FungalGene Transfer TechniquesGenes, FungalGenetic VectorsMolecular Sequence DataMutagenesis, Site-DirectedRestriction MappingTransformation, GeneticAspergillus awamoriAspergillus nigerFungi To reference this document use: http://resolver.tudelft.nl/uuid:432d5533-290f-43bf-8f24-2937dd9c293e DOI https://doi.org/10.1007/bf00314444 TNO identifier 67195 ISSN 0172-8083 Source Current Genetics, 27 (6), 536-540 Document type article Files To receive the publication files, please send an e-mail request to TNO Library.