HOMOLOGOUS RECOMBINATION AS THE MAIN MECHANISM FOR DNA INTEGRATION AND CAUSE OF REARRANGEMENTS IN THE FILAMENTOUS ASCOMYCETE ASHBYA-GOSSYPII

A slow and a fast growth phenotype were observed after transformation of the phytopathogenic fungus Ashbya gossypii using a plasmid carrying homologous DNA and as selectable marker the Tn903 aminoglycoside resi stance gene expressed from a strong A. gossypii promoter. Transformati ons with circular plasmids yielded slowly and irregularly growing gene ticin-resistant mycelia in which 1% of nuclei contained plasmid sequen ces. Occasionally, fast growing sectors appeared which were shown to b e initiated by homologous integration of the transforming DNA. Transfo rmants obtained with plasmids linearized within the homology region im mediately exhibited fast radial growth. In all 28 transformants analyz ed plasmid DNA was integrated homologously. Such apparent lack of nonh omologous recombination has so far not been observed in filamentous as comycetes. In 14 transformants two to four tandemly integrated plasmid copies were found. They underwent several types of genetic changes, m ainly in the older mycelium: excision of whole plasmid copies and rear rangements within the integrated DNA (inversions and deletions). These internal rearrangements involved 360-bp inverted repeats, remnants of IS-elements flanking the resistance gene, and 156-bp direct repeats, originating from the strong A. gossypii promoter. Improved vectors lac king sequence repetitions were constructed and used for stable one-ste p gene replacement in A. gossypii.