A novel strategy for the isolation of defined pyrG mutants and the development of a site-specific integration system for Aspergillus awamori
article
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.
Molecular Sequence Numbers: GENBANK: S79674;
Chemicals/CAS: DNA, Fungal; Genetic Vectors
Molecular Sequence Numbers: GENBANK: S79674;
Chemicals/CAS: DNA, Fungal; Genetic Vectors
Topics
TNO Identifier
67195
ISSN
01728083
Source
Current Genetics, 27(6), pp. 536-540.
Pages
536-540
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