Acquisition of mitochondrial DNA by a transformation vector for Ustilago violacea

Plasmid pUCH1 is a 5.2-kb pUC18 construct bearing the hygB gene fused to a promoter from Cochliobolus heterostrophus. Haploid cells of the basidiomycete, Ustilago violacea, were transformed with this plasmid. In addition to multiple integrations of plasmid sequences into U. violacea nuclear DNA, vector sequences independent of the nuclear genome were indicated by Southern-blot analysis using all or part of pUCH1 as a probe. Hybridization also revealed intact pUCH1 and several larger derivatives in satellite bands from CsCl-bis-benzamide gradients of whole cellular DNA and in DNA from purified mitochondria [mitochondrial (mt) DNA preparations] of transformed U. violacea; circular DNAs consistent with the sizes of DNAs in these satellite bands were seen in electron microscope analyses of the same mt DNA preparations as well. The plasmids could be detected in mt DNA preparations even after 30 generations of transformant growth under selective pressure. Transformation of Escherichia coli by these mt DNA preparations produced bacterial transformants bearing intact pUCH1, as well as several pUCH1 derivatives, including pUCH2, an approx. 8.0-kb plasmid. A 2.5-kb EcoRI fragment from pUCH2 showed only weak hybridization with pUCH1. This unique fragment did hybridize strongly with mt DNA from untransformed U. violacea. This derivative thus appears to have acquired mt sequences from U. violacea.