title: Heterologous transformation of Agrocybe aegerita with a bacterial neomycin-resistance gene fused to a fungal promoter-like DNA sequence.
abstract: DNA sequences of the basidiomycete Agrocybe aegerita were cloned in E. coli based on their ability to drive the expression of the bacterial promoterless tetracycline (Tc)-resistance gene. A 0.48% frequency of the cloned sequences promoted antibiotic-resistance. The sequence conferring the highest Tc resistance (40 μg/ml) was selected to drive the expression in E. coli of two other promoterless genes encoding chloramphenicol and neomycin resistance. One of the derivative vectors, pN13-A2, carrying a chimeric neomycin-resistance gene, was used to transform an A. aegerita neomycin-sensitive strain by protoplast electroporation. Transformation frequencies ranged from 1 to 2.8 transformants per μg of DNA per 10(3) viable cells, in a relatively high background of spontaneous-resistant colonies (2% of the surviving protoplasts). Molecular analyses showed that transformation had occurred by the integration of pN13-A2 sequences, either ectopically or at the resident locus carrying the A. aegerita promoter-like sequence, with probable molecular rearrangements. The nucleotide sequence of the promoter-like fragment revealed the presence of a CT motif that is known to be involved in a promoter function in some highly expressed genes of filamentous fungi.
authors.affiliation: Laboratoire de Génétique Moléculaire et Amélioration des Champignons Cultivés, Université de Bordeaux II-INRA, C.R.A. de Bordeaux, F-33883, Villenave d'Ornon Cédex, France.
authors.forename: T
authors.initials: T
authors.lastname: Noël
authors.forename: P
authors.initials: P
authors.lastname: Simoneau
authors.forename: J
authors.initials: J
authors.lastname: Labarère