STRUCTURAL-ANALYSIS OF MITOCHONDRIAL-DNA MOLECULES FROM FUNGI AND PLANTS USING MOVING-PICTURES AND PULSED-FIELD GEL-ELECTROPHORESIS

The size and structure of mitochondrial DNA (mtDNA) molecules was inve stigated by conventional and pulsed-field gel electrophoresis (PFGE) a nd by analyzing moving pictures during electrophoresis of individual f luorescently labelled mtDNA molecules. Little or no mtDNA that migrate d into the gel was found in circular form for fungi (Schizosaccharomyc es pombe, Saccharomyces cerevisiae and Neurospora crassa) or plants (B rassica hirta, tobacco, voodoo lily and maize). Most mtDNA migrated as a smear of linear DNA sizes from about 50 to 100 or 250 kilobases (kb ), depending on the species, irrespective of the size of the mitochond rial genome over a range of 0.06 to 570 kb. S. cerevisiae, B. hirta an d tobacco also yielded a linear mtDNA fraction containing molecules > 1000 kb in size. About half the mtDNA remained in the well of the,eel after PFGE. Moving pictures revealed that this well-bound (wb) mtDNA c ontained molecules larger than the genome size in linear form for all species (except N. crassa) and in multi-fibered, comet-like forms for most of the wb mtDNA of N. crassa and Sc, pombe. A minor amount of the wb mtDNA with visually interpretable structure was circular: circle s izes were both larger and smaller than the 80-kb genome of S. cerevisi ae, larger than the 19-kb genome of Sc. pombe and smaller than the 208 -kb and 570-kb genomes of B. hirta and maize, respectively. About 25 t o 75% of the wb mtDNA from cultured tobacco cells was found in circles smaller than its genome size. Partial digestion of Sc, pombe mtDNA wi th restriction endonucleases that cleave once per genome revealed gel bands at about 38 kb and 19 kb with a smear of sizes between the bands and below the 19-kb band, suggesting a head-to-tail genomic concateme r as the most prominent form in extracted mtDNA. A pattern of bands wi th smears was also found for complete digests (with multiply cleaving enzymes) of mtDNA from Sc, pombe, S. cerevisiae and N. crassa, but ban ds without smears were found. for digests of DNA from phage lambda and several plasmids. (C) 1996 Academic Press Limited