Genetic transformation of the pathogenic fungus Wangiella dermatitidis.
Genetic transformation of Wangiella dermatitidis was studied using three plasmid vectors (pAN7-1, pWU44, and pKK5) and both electroporation and polyethyleneglycol-mediated methods. pAN7-1 contains the E. coli hygromycin 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 colonies/micrograms pAN7-1 on medium containing 100 micrograms HmB/ml. Strains of W. dermatitidis used in this study have innate sensitivity to HmB at a critical inhibitory concentration of 20-40 micrograms/ml. Vectors pWU44 and pKK5 contain a URA5 gene from Podospora anserina. A ura5 auxotroph of W. dermatitidis was transformed to prototrophy with pWU44 or pKK5 by complementation. Transformation frequencies for these two plasmids were between 17-50 transformants/micrograms vector DNA. Southern blotting analysis and polymerase chain reaction detection of DNA from putative transformants confirmed transformation.
Peng
M
M
Department of Microbiology, University of Texas at Austin 78712-1095, USA.
Cooper
C R
CR
Szaniszlo
P J
PJ
D001483:Base Sequence; D004271:DNA, Fungal / Q000235:genetics*; D004352:Drug Resistance, Microbial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005093:Exophiala / Q000235:genetics*; D017930:Genes, Reporter; D005822:Genetic Vectors; D006921:Hygromycin B / Q000494:pharmacology; D008969:Molecular Sequence Data; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D013104:Spheroplasts; D014162:Transfection
D004271:DNA, Fungal; D011994:Recombinant Proteins; D006921:Hygromycin B; D017853:Phosphotransferases (Alcohol Group Acceptor); C063242:hygromycin-B kinase