GENETIC-TRANSFORMATION OF THE PATHOGENIC FUNGUS WANGIELLA-DERMATITIDIS

Genetic transformation of Wangiella dermatitidis was studied using thr ee plasmid vectors (pAN7-1, pWU44, and pKK5) and both electroporation and polyethyleneglycol-mediated methods, pAN7-1 contains the E. coli h ygromycin B (HmB) phosphotransferase (hph) gene. Expression of the hph gene confers resistance to antibiotic HmB. Selection for resistance, indicative of transformation, resulted in 10-203 HmB-resistant colonie s/mu g pAN7-1 on medium containing 100 mu g HmB/ml. Strains of W. derm atitidis used in this study have innate sensitivity to HmB at a critic al inhibitory concentration of 20-40 mu g/ml. Vectors pWU44 and pKK5 c ontain a URA5 gene from Podospora anserina. A ura5 auxotroph of W. der matitidis was transformed to prototrophy with pWU44 or pKK5 by complem entation. Transformation frequencies for these two plasmids were betwe en 17-50 transformants/mu g vector DNA. Southern blotting analysis and polymerase chain reaction detection of DNA from putative transformant s confirmed transformation.