A degenerate DNA transposon, Pat, was identified in the genomes of various wild-type strains of the filamentous fungus Podospora anserina. In these strains, the number (approximately 20-25 copies per genome) and location of Pat sequences appear to be conserved. Two copies of Pat, one complete and one partial, were cloned and characterized. The sequence of the complete element is 1856 bp long and contains imperfect inverted terminal repeats (ITRs) of 53 bp. The target site duplication comprises the sequence TA. The amino acid sequence derived from one reading frame of Pat shows significant homology to members of the Fot1 family of transposons. However, this reading frame is interrupted by numerous stop codons. Since no transcripts of Pat were identified in different P. anserina strains grown under standard conditions and under increased stress, we conclude that none of the copies of Pat is active in the strains analyzed, under the environmental conditions investigated. Comparison of the sequences of the two cloned Pat sequences revealed 89% (589/747 nucleotides) identity. Most of the differences (82%, 129/158) can be attributed to transitions preferentially at CpA:TpG and CpT:ApG dinucleotides. The dinucleotide ratios in Pat are similar to those in a Neurospora crassa transposon which was subject to repeat-induced mutation (RIP), but differ significantly from those found in single-copy genes of P. anserina and in fungal DNA transposons not modified by this mechanism. Molecular analysis of the progeny of a cross between the wild-type strain and a transgenic strain in which a nuclear gene was duplicated by transformation yielded the first clear evidence that a RIP-like process is active in P. anserina.

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.

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