Introduction and maintenance of prokaryotic DNA in Ustilago violacea

A strain of the basidiomycete, Ustilago violacea, was transformed with a prokaryotic plasmid, pMP4-1, which confers resistance to neomycin. U. violacea transformants were selected at a frequency of 5 per microgram pMP4-1 DNA. Such transformants were at least 8-fold more resistant to neomycin than was the untransformed recipient U. violacea. Enzyme activity associated with the neomycin resistance gene was also found in the transformants. Southern DNA-DNA hybridization detected pMP4-1-derived sequences in both nuclear and mitochondrially-associated DNAs from transformants. The patterns of hybridization suggested integration of pMP4-1 sequences into the respective genomes. DNA from the nuclear fraction of U. violacea transformants failed to produce E. coli transformants resistant to neomycin or to carbenicillin. In contrast, DNA from the mitochondrially-associated fraction in U. violacea transformants produced E. coli transformants resistant to neomycin. The E. coli transformants contained a pMP4-1-derivative, pWP8, which was subsequently shown by Southern blot analysis to harbor U. violacea mitochondrial DNA. Thus, a prokaryotic plasmid can be used to transform the eukaryote U. violacea and acquire endogenous sequences from this organism.