HOMOLOGOUS TRANSFORMATION OF THE EDIBLE BASIDIOMYCETE AGROCYBE-AEGERITA WITH THE URA1 GENE - CHARACTERIZATION OF INTEGRATIVE EVENTS AND OF REARRANGED FREE PLASMIDS IN TRANSFORMANTS

The URA1 gene, encoding dihydroorotate dehydrogenase of the pyrimidine pathway, cloned into pUC18 (pUral-1) was used to develop an homologous transformation system for the cultivated mushroom Agrocybe aegerita. Protoplasts of a ural auxotrophic strain were transformed by electroporation with efficiencies ranging from 1 to 26 transformants per mu g of DNA. The phenotype of the stable Ura+ transformants suggested a strong nuclear heterogeneity further confirmed by Southern-blot analysis. All transformants acquired extrachromosomal forms derived from pUral-1. Integration of pUral-1 into chromosomal DNA occurred for some transformants. Plasmids containing the integrant of pUC18 recombined to different parts of the URAl gene were rescued from A. aegerita transformants through transformation of E. coli. Their molecular analysis indicated that they represent products of the continuous excision of primary-integrated vector sequences rather than ARS-dependent autoreplicative forms.