Integrative transformation of the ascomycete Podospora anserina: identification of the mating-type locus on chromosome VII of electrophoretically separated chromosomes |
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Authors: | Hienz D. Osiewacz Adriane Skaletz Karl Esser |
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Affiliation: | (1) Lehrstuhl für Allgemeine Botanik, Ruhr-Universität, Postfach 102148, W-4630 Bochum, Federal Republic of Germany;(2) Deutsches Krebsforschungszentrum, Institut für Virusforschung, Projekt: Molekularbiologie der Alterungsprozesse, Im Neuenheimer Feld 506, W-6900 Heidelberg, Federal Republic of Germany |
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Abstract: | Summary 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/ plasmid DNA were obtained. Using a second selectable vector (pBT3), which contains the -tubuline gene of a benomyl-resistant Neurospora crassa mutant, the cotransformation 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. Fractination 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.Offprint requests to: K. Esser |
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