Use of mitochondrial DNA as a sensitive and specific marker for localization of mitochondria in fractionated plant extracts

Upon subfractionation of certain plant seed homogenates on sucrose density gradients, we encountered problems in defining the location and amount of mitochondria using marker enzymes. In order to overcome the inherent limitations of enzyme assays, we utilized a heterologous DNA probe specific foratp6 in maize orBrassica tournefortii to detect mitochondria. The samples were treated with SDS, proteinase K, and RNase A followed by agarose gel electrophoresis, and blotting. The immobilized DNA was detected with ³²P]-labelled probes, and quantified using a phosphor imager. The assay is specific, sensitive, and independent of species, cell type, and developmental stage, thus circumventing the need for expressed protein to assay enzyme activity.