Cloning and use of the WdURA5 gene as a hisG cassette selection marker for potentially disrupting multiple genes in Wangiella dermatitidis.
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.
Zheng
L
L
Department of Microbiology, University of Texas at Austin, Austin, TX 78712, USA.
Szaniszlo
P J
PJ
D000595:Amino Acid Sequence; D001483:Base Sequence; D002687:Chitin Synthase / Q000378:metabolism; D002874:Chromosome Mapping; D003001:Cloning, Molecular; D018274:Electroporation; D005093:Exophiala / Q000235:genetics*; D017353:Gene Deletion; D005800:Genes, Fungal; D005819:Genetic Markers; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D009962:Orotate Phosphoribosyltransferase / Q000235:genetics*; D009964:Orotidine-5'-Phosphate Decarboxylase / Q000235:genetics; D017422:Sequence Analysis, DNA; D014170:Transformation, Genetic
D005819:Genetic Markers; D002687:Chitin Synthase; D009962:Orotate Phosphoribosyltransferase; D009964:Orotidine-5'-Phosphate Decarboxylase