We have determined by electron microscopy the molecular weights of circular mitochondrial DNA (mtDNA) molecules from 39 species representing 13 groups of five subgenera of the genus Drosophila. mtDNA molecules of all species examined, other than members of the melanogaster group, had, with one exception, molecular weights in the rather narrow range 9.90 X 106 to 10.34 X 106. The one exception was D. robusta, which had a molecular weight of 10.61 X 106. In contrast, mtDNA molecules from species within the melanogaster group had molecular weights covering the considerably greater range 9.92 X 106 to 12.35 X 108. Applying the electron microscope denaturation mapping technique of Inman to mtDNA molecules of eight species of the melanogaster group, we found each of them to contain a region [presumably rich in adenine and thymine (A+T)J which denatured at a specific temperature (400) at which most of the remainder of the molecule remained undenatured. The size of the A+T-rich region was constant for mtDNA molecules of a species, but varied from 0.62 X 106 to 3.41 X 106 for mtDNA molecules of different species. It was demonstrated that the differences in molecular weights of the A+T-rich regions can almost completely account for the differences in total molecular weights of the mtDNA molecules from species of the melanogaster group. Mitochondrial DNA (mtDNA) of Drosophila melanogaster is in the form of circular molecules with contour lengths of about 6.2 ,um (molecular weight = 12.35 X 106) (1-4). The findings of triphasic (1, 4) and biphasic (2) hyperchromic changes in this DNA upon heating indicated that a portion of it contained a distinctly higher content of adenine and thymine than the remainder. Using Mitochondria were prepared from eggs and DNA was prepared from the mitochondria and analyzed by neutral and alkaline CsCI equilibrium density gradient centrifugation and electron microscopy as described elsewhere (9, 10).Ovaries were hand dissected from anesthetized flies in 0.15 M NaCl, 0.1 M EDTA, 0.05 M Tris-HCI (pH 8.0) and immediately frozen on solid CO2. Ovaries from approximately 300 flies of a single species were thawed and lysed by addition of sodium dodecyl sulfate to 2%, and the lysate was subjected to preparative CsCI equilibrium density gradient centrifugation (10). The buoyant densities of individual fractions of the resuiting gradient were determined from their refractive indices, and the configurations of the DNA molecules each fraction contained were determined by electron microscopy.DNA samples comprising 50-80% open circular molecules (each containing at least one phosphodiester bond break) used for thermal denaturation studies were obtained as the upper fluorescent band in a CsCI/ethidium bromide equilibrium density gradient (10, 11) of pooled circular-molecule-containing fractions obtained by CsCl centrifugation of ovary lysates.
3623Abbreviations: mtDNA, mitochondrial DNA; Mr, molecular weight.