A mutant auxotroph for methionine was isolated in Podospora anserina during a transformation experiment. The transforming plasmid (pPAaURA5) consisted of a 1.55 kb nuclear DNA fragment of Podospora containing the URA5 gene (Begueret et al. 1984, Gene 32:487-492; Turq and Begueret 1987, Gene 53:201-209), and most (2.06 kb) of the Podospora intronic alpha mitochondrial sequence (Osiewacz and Esser 1984, Curr. Genet. 8:299-305) cloned in the pUC18 vector. The recipient strain carried the mutant ura5-6 allele (Razanamparany and Begueret 1986, Curr. Genet. 10:811-817) and the mating plus locus (mat+). [In Podospora, transformation with plasmids occurs by integration of the vector mainly outside the resident locus (Brygoo and Debuchy 1985, Mol. Gen. Genet. 200:128-131; Razanamparany and Begueret 1986, Curr. Genet. 10:811-817).]
After transformation of the ura5-6 mat+ strain with plasmid pPAaURA5 and selection of primary (ura+) transformants, the transformants were crossed to a ura5-6 mat- strain in order to purify the transformant nuclei through meiosis. In most cases, 50% ura+ and 50% ura- spores were obtained in the progeny. However, one primary transformant gave different results: of 46 monocaryotic spores tested, 20 were auxotrophic for uracil, the 26 others were methionine auxotrophs.
Three purified ura+ met- transformants were crossed with wild-type. The met- phenotype segregated as a single recessive gene. The percentage of second division segregation was about 60%. ura- spores were obtained in the progeny indicating that the parental transformant strain contained the ura5-6 allele. Furthermore, the presence of tetrads containing 2 dicaryotic spores (ura- met+) and 2 dicaryotic spores (ura+ met-) indicated the integration event of the URA5 gene occurred in a chromosome different from that carrying the URA5 locus. Three purified transformants (ura+ met-) were crossed with the ura5-6 strain. In the progeny, the two phenotypes (ura+ and met-) were associated and segregated together.
These results indicate that the met- phenotype of the primary and purified transformants resulted from the integration of the URA5 gene of plasmid pPAaURA5 in a gene involved in methionine biosynthesis. Analysis of the genomic DNA of this transformant has not been completed; we do not know if the entire plasmid or only the URA5 gene has been integrated. The strain has been called met1. It is quite fertile and stable through vegetative growth as well as through meiosis. It grows on minimal medium supplemented with methionine, cysteine or homocysteine, but is not complemented by O-acetyl-homoserine. It constitutes the first example in Podospora of the isolation of mutants by transformation-mediated gene disruption.