Integrative transformation of the ascomycete Podospora anserina: identification of the mating-type locus on chromosome VII of electrophoretically separated chromosomes

Abstract

Protoplasts of wild-type strain s and a long-lived extrachromosomal mutant (AL2) of the ascomycete Podospora anserina were transformed using a plasmid (pAN7-1) which contains the hygromycin B phosphotransferase gene (hph) of Escherichia coli under the control of Aspergillus nidulans regulatory sequences. After optimizing the transformation procedure, transformation efficiencies of 15-21 transformants/micrograms plasmid DNA were obtained. Using a second selectable vector (pBT3), which contains the beta-tubuline gene of a benomyl-resistant Neurospora crassa mutant, the co-transformation rate was determined. Southern blot hybridization experiments revealed that the transforming plasmid became integrated into the genome of the recipient either as a single copy or as multiple copies. In addition, the data from molecular as well as from classical genetic analyses indicated that in independent transformants vector integration occurred at different positions. The mitotic and meiotic stability of transformants proved to be dependent on the number of integrated plasmid copies. Genetic analyses revealed a transformant in which the integrated vector is closely linked to the mating-type locus. Fractionation of whole chromosomes by pulsed field gel electrophoresis and subsequent hybridization of the immobilized DNAs against radiolabelled vector sequences indicated the largest of seven chromosomes as the chromosome containing the integrated vector and thus the mating-type locus.

Impact of a Disruption of a Pathway Delivering Copper to Mitochondria onPodospora anserinaMetabolism and Life Span

“…After 7 days of growth, mycelia of developing cultures were transferred to BMM. DNA transformation of spheroplasts of P. anserina strain s (mat Ϫ ) was performed as previously described (47). Northern analysis.…”

Over‐expression of an S‐adenosylmethionine‐dependent methyltransferase leads to an extended lifespan of Podospora anserina without impairments in vital functions

“…Production, regeneration, and integrative transformation of P. anserina spheroplasts was performed as described (Osiewacz et al, 1991;Stumpferl et al, 2004).…”

Asexual Cephalosporin C Producer Acremonium chrysogenum Carries a Functional Mating Type Locus

“…For transformation procedures, the Pa⌬MAT strain was cultivated in static liquid complete medium (45) at 27°C under constant light. The preparation and transformation of P. anserina protoplasts were performed as described previously (47). Fungal protoplasts were transformed with 15 g of cosmid DNA, and the selection of transformants occurred on solid BMM medium supplemented with 50 U of hygromycin B/ml.…”