A high efficiency transformation system for the basidiomycete Ustilago violacea employing hygromycin resistance and lithium-acetate treatment.
A basidiomycete phytopathogenic fungus, Ustilago violacea, was transformed with pUCH1, a bacterial plasmid containing the hygromycin (Hyg)-resistance hygB gene fused to a promoter from the ascomycete Cochliobolus heterostrophus. After lithium acetate/polyethylene glycol treatment of whole sporidial cells, U. violacea transformants appeared on Hyg-agar at a frequency of 60-80 per microgram pUCH1 DNA. The Hyg phenotype was 100% stable in these transformants for at least 30 generations of mitotic growth under non-selective conditions. Southern DNA-DNA hybridization revealed multiple integrations of the pUCH1 plasmid into the U. violacea nuclear DNA. In addition, Escherichia coli transformants appeared at a frequency of 12 per microgram nuclear fraction DNA from Hyg U. violacea transformants; these E. coli consistently contained a deleted pUCH1 plasmid. This latter result suggested the low-frequency production of circular molecules by recombination within the integrated sequences.
Bej
A K
AK
Department of Biology, University of Louisville, KY 40292.
Perlin
M H
MH
D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001487:Basidiomycota / Q000235:genetics*; D002934:Cinnamates; D004271:DNA, Fungal / Q000235:genetics; D004352:Drug Resistance, Microbial / Q000235:genetics; D005800:Genes, Fungal; D005819:Genetic Markers; D006921:Hygromycin B / Q000031:analogs & derivatives / Q000494:pharmacology*; D008094:Lithium / Q000494:pharmacology*; D010957:Plasmids; D014170:Transformation, Genetic; D014588:Ustilago / Q000235:genetics*
D000900:Anti-Bacterial Agents; D002934:Cinnamates; D004271:DNA, Fungal; D005819:Genetic Markers; D006921:Hygromycin B; C026273:hygromycin A; D008094:Lithium