Cloning and use of theWdURA5gene as ahisGcassette selection marker for potentially disrupting multiple genes inWangiella dermatitidis

Abstract

A genomic clone encoding the Wangiella dermatitidis orotidine monophosphate pyrophosphorylase gene (WdURA5) was isolated by screening a subgenomic plasmid DNA library of this phaeohyphomycotic agent using a PCR amplification product of the gene as a probe. When plasmid DNA containing the cloned WdURA5 gene was introduced by electroporation into a wdura5 auxotrophic recipient strain derived previously by selection with 5-fluoroorotic acid (5-FOA), an apparent gene repair event occurred at high frequency without any evidence of integration of the plasmid DNA. Therefore, the hygromycin B resistance gene (the hph gene) was used as a dominant selective marker for the disruption of WdURA5 to generate a new, more stable, wdura5 auxotrophic strain. Transformation of this strain was then achieved with high efficiency and high frequency by site-specific integration using WdURA5 as a selective marker. To initiate attempts to use this marker repeatedly for multiple chitin synthase (WdCHS) gene disruptions in single strains of W. dermatitidis, a hisG_WdURA5_hisG cassette was constructed and used to disrupt WdCHS2. The WdURA5 gene in the disruptant was then successfully recycled under selection for resistance to 5-FOA.