Stable allele replacement and unstable non-homologous integration events during transformation of Ascobolus immersus.

An homologous transformation system for the filamentous fungus Ascobolus immersus has been developed, based on the complementation of a met2 mutation by the wild-type (wt) allele gene encoding homoserine O-transacetylase. Transformation of A. immersus met2 mutants occurs with moderate frequencies (about 50 transformants per microgram input DNA). Analysis of the DNA of the met2+ transformants showed that transformation resulted either in a single integration of the donor DNA into the genome by many different nonhomologous recombination events or in the substitution of the endogenous met2 mutation by the wt transforming allele. The relative frequencies of both events depended on the vector sequences carrying the cloned met2 gene. Whereas the substitution event led, as expected, to genetically stable transformants, the non-homologous integration was always associated with a strong instability when transformants were crossed and underwent meiosis.