Journal of Molecular Biology
Volume 255, Issue 4, 2 February 1996, Pages 564-588
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Regular article
Structural Analysis of Mitochondrial DNA Molecules from Fungi and Plants Using Moving Pictures and Pulsed-field Gel Electrophoresis

https://doi.org/10.1006/jmbi.1996.0048Get rights and content

Abstract

The size and structure of mitochondrial DNA (mtDNA) molecules was investigated by conventional and pulsed-field gel electrophoresis (PFGE) and by analyzing moving pictures during electrophoresis of individual fluorescently labelled mtDNA molecules. Little or no mtDNA that migratedd into the gel was found in circular form for fungi(Schizosaccharomyces pombe, Saccharomyces cerevisiaeandNeurospora crassa) or plants (Brassica 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 mitochondrial genome over a range of 0.06 to 570 kb.S. cerevisiae, B. hirtaand tobacco also yielded a linear mtDNA fraction containing molecules >1000 kb in size. About half the mtDNA remained in the well of the gel after PFGE. Moving pictures revealed that this well-bound (wb) mtDNA contained molecules larger than the genome size in linear form for all species (exceptN. crassa) and in multi-fibered, comet-like forms for most of the wb mtDNA ofN. crassaandSc. pombe. A minor amount of the wb mtDNA with visually interpretable structure was circular: circle sizes were both larger and smaller than the 80-kb genome ofS. cerevisiae, larger than the 19-kb genome ofSc. pombeand smaller than the 208-kb and 570-kb genomes ofB. hirtaand maize, respectively. About 25 to 75% of the wb mtDNA from cultured tobacco cells was found in circles smaller than its genome size.

Partial digestion ofSc. pombemtDNA with 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 concatemer as the most prominent form in extracted mtDNA. A pattern of bands with smears was also found for complete digests (with multiply cleaving enzymes) of mtDNA fromSc. pombe, S. cerevisiaeandN. crassa, but bands without smears were found for digests of DNA from phage lambda and several plasmids.

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