Isolation of intact supercoiled mitochondrial DNA from the yeast Saccharomyces cerevisiae

: Cells of Saccharomyces cerevisiae strain PG-60 were mechanically disrupted in the presence of ethidium bromide, and the total covalently closed circular DNA (ccDNA) was prepared by EthBr-CsCl equilibrium centrifugation of lysed 15 g pellet of the homogenate. Recentrifugation of ccDNA in equilibrium neutral CsCl density gradient resolved a discrete light peak (LccDNA) with the buoyant density equal to 1.684 g/cm3 to about 10% of total ccDNA. Electron microscopy has shown that LccDNA contained circular molecules with the contour length around 25 micrometer circles and their multimeres. Well characterized linear mtDNA was transcribed with E. coli RNA-polymerase and cRNA obtained was hybridized with LccDNA 2 mu DNA and total DNA from yeast petit mutant, lacking mtDNA. Hybridization experiments have shown that the transcript of mtDNA hybridized almost exclusively with LccDNA. We have concluded that 25 micrometer circles of the LccDNA represent intact molecules of yeast mitochondrial DNA.