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A series of petite mutants of Saccharomyces cerevisiae , generated after treatment for various times with ethidium bromide, was isolated, and the mitochondrial deoxyribonucleic acid size for each member was estimated. It was found that, as the treatment time with ethidium bromide was increased, the mitochondrial deoxyribonucleic acid isolated from the petite series was increasingly reduced in size.

DNA from purified mitochondria of Drosophila melanogaster can be isolated as supercoiled molecules which when nicked have a contour length of 5.9 ~m. Partial denaturation mapping shows regional heterogeneity of base composition with one early denaturing region, with a calculated GC content close to zero, extending over 20% of the genome. DNA isolated from unfertilized eggs shows nuclear and mitochondrial DNA in equal proportions; we found no evidence of other cytoplasmic species.Confusion as to which of the satellite DNA peaks of Drosophila are cytoplasmic (4, 12) has been resolved in an analysis (17) which has shown that two (1.688 and 1.672 g/cm a) of the three distinct light satellites are of nuclear origin and that the remaining one (1.680 g/cm a) is of cytoplasmic origin. Polan et al. (18) and Bultmann and Laird (6) identified this 1.680 g/cm a peak as mitochondrial DNA. An additional DNA species of cytoplasmic origin (1.697 g/cm 3) has been claimed to be present in unfertilized eggs (18), and Travaglini et al. (20) report a major DNA species in unfertilized eggs to be a satellite of density 1.669 g/cm a.In this paper, we report additional characterization of the mitochondrial DNA molecule by denaturation mapping. We have failed to detect other cytoplasmic DNA species in unfertilized eggs (or early embryos). MATERIALS AND METHODS Preparation of Mitochondrial DNAD. melanogaster (Canberra wild type) eggs or embryos were harvested in NaCI (0.7%), Triton X-100 (0.015%) and dechorionated with sodium hypochlorite (5% available chlorine) for 2 min. The eggs were homogenized (Dounce) in buffer (0.25 M sucrose, 0.03 M "Iris, 1 mM EDTA, 2.5 mM CaCI2, pH 7.5) and the debris was removed by filtering through nylon mesh.Nuclei were sedimented by several cycles of centrifugation at 1,000 g and the mitochondria were collected by centrifugation at 16,000 g for 20 min. After washing, mitochondria were incubated with pancreatic deoxyribonuclease (25 /zg/ml) for 30 min at room temperature in 0.02 M Mg 2 § After incubation, EDTA (pH 8) was added to a final concentration of 0.01 M and the mitochondria were washed three times with 5 mM EDTA, 0.25 M sucrose, 0.15% bovine serum albumin (pH 8).Mitochondria were lysed in 0.5 M NaCI, 0.01 M EDTA, pH 8, containing sarkosyl (1%). The lysate was then centrifuged to equilibrium in CsCI (,o = 1.70 g/cm 3) at 35,000 rpm in a Spinco no. 40 rotor (Beckman Instruments, Inc., Spinco Div., Palo Alto, Calif.) for 60 h. Total DNA ExtractionTotal DNA was extracted as described previously (17). Base CompositionA ~P-labeled DNA copy was synthesized using Escherichia coli DNA polymerase I as described before (17).The DNA was hydrolyzed enzymatically and the bases were separated and counted. Ethidium Bromide GradientsThe mitochondrial pellet was lysed into a CsCl-Ethidium bromide gradient by the method of Clark-Walker (8). Ethidium bromide was removed by extraction with CsCl-water-saturated isopropanol.