ISOLATION AND CHARACTERIZATION OF MITOCHONDRIAL DNA FROM DROSOPHILA MELANOGASTER

Abstract

A B S T R A C TMitochondrial DNA (MtDNA) with a neutral buoyant density of 1.681 g/cm a has been isolated from unfertilized eggs of Drosophila mdanogaster. This DNA is a circular molecule with an average length of 5.3 /~m; it reassociates with a low Coil~2 after denaturation, and in alkaline isopycnic centrifugation it separates into strands differing in density by 0.005 g/cm 3 MtDNA isolated from purified mitochondria of unfertilized eggs or from total larval DNA melts with three distinct thermal transitions. The three melting temperature values suggest that the molecule may have three regions differing in average base composition. DNA isolated from unfertilized eggs of D. melanogaster contains approximately equal amounts of MtDNA and another DNA with a buoyant density of 1.697 g/cm a, slightly less dense than main peak DNA. The possibility that the heavier DNA fraction consists of amplified ribosomal DNA was excluded by hybridization experiments, but otherwise nothing is known of its origin or function. I N T R O D U C T I O NMitochondrial DNA (MtDNA) 1 from several higher organisms renatures rapidly (Corneo et al., 1966;Borst and Ruttenberg, 1966;Nass, 1966;Kroon et al., 1966; Dawid and Wolstenholme, 1968), is circular (Van Bruggen et al., 1966;Sinclair and Stevens, 1966; Wolstenholmc and Dawid, 1967), and shows strand separation in alkaline isopycnic centrifugation (Dawid and Wolstenholme, 1967; Leftter et al., 1970;Corneo et al., 1968). We report here the isolation of Mt-DNA from Drosophila melanogaster and its charac1Abbreviations used in this paper: EBr, ethidium bromide; EDTA, ethylenediaminetetraacetate; MtDNA, mitochondrial DNA; rDNA, the DNA coding for ribosomal RNA; rRNA, ribosomal RNA; SSC, 0.15 M NaCI, 0.015 M Na-citrate, pH 7.0; T,,, melting temperature.terization by isopycnic centrifugation, thermal denaturation, and electron microscopy.MtDNA from D. melanogaster is a circular molecule, renatures rapidly, and separates into two strands in alkaline CsC1. However, it has unusual thermal denaturation properties. MtDNA from Rana pipiens, Xenopus laevis (Dawid, 1965), and sheep heart shows only one transition on thermal denaturation. Leffier et al. (1970) have reported a biphasic melting curve for rat liver MtDNA and correlated the transitions with sequential denaturation of nicked and intact circles. Renatured rat liver MtDNA melted with a single transition. Wolstenholme et al. (1972) have observed the same phenomenon and, using the electron microscope, have identified at least three specific regions of rat liver MtDNA which melt 1°-2°C below the rest of the circle. We have ob-$80

Replicating DNA molecules from eggs ofDrosophila melanogaster

“…Evidence has been presented that circular molecules of approximately this size represent the mitochondriM DNA (mtDNA) of D. melanogaster (Polan et al, 1973 ;Bultmann and Laird, 1973). The abundance of these circular molecules in the present preparations and their interpretation as mtDNA is in agreement with the finding that approximately one-hMf of the DNA of unfertilized eggs of D. melanogaster is mitochondrial (Polun et al, 1973).…”

A partial map of the circular mitochondrial genome of Drosophila melanogaster. Location of EcoRI-sensitive sites and the adenine-thymine-rich region.

“…melanogaster mtDNA reported previously (3,23,24,32). When these mtDNA preparations were centrifuged to equilibrium in cesium chloride-ethidium bromide gradients, two visible bands were formed.…”

“…Mitochondrial DNA (mtDNA) of Drosophila melanogaster is in the form of circular molecules with a similar contour length of about 6.2 tzm (mol wt = 12.35 x 106 daltons) (3,9,10,23,24,32). The findings of triphasic (24) and biphasic (3) hyperchromic changes in this DNA upon heating indicated that a portion of it contained a distinctly higher content of adeninethymine than the remainder.…”

“…From a consideration of the lengths of fragments resulting from incomplete Eco RI digestion, it was determined that the arrangement of the fragments in the circular genome was A-C-B-D. By electron microscope examination of partially denatured EcoRI fragments, the A-T-rich region was shown to be located in the A fragment closer to one end than to the other. By similar partialdenaturation studies of fragments resulting from incomplete EcoRI digestion, it was determined that, in the circular genome, of the two EcoRI sites which define the limits of the A fragment, the site between the A and D fragment lies nearest to the A-T-rich region.Mitochondrial DNA (mtDNA) of Drosophila melanogaster is in the form of circular molecules with a similar contour length of about 6.2 tzm (mol wt = 12.35 x 106 daltons) (3,9,10,23,24,32). The findings of triphasic (24) and biphasic (3) hyperchromic changes in this DNA upon heating indicated that a portion of it contained a distinctly higher content of adeninethymine than the remainder.…”

Determination ofWolbachiaGenome Size by Pulsed-Field Gel Electrophoresis

“…In this report, the methods used to purify mitochondria from Drosophila (28,35) were modified to prepare DNA from Wolbachia in quantities that could be visualized by ethidium bromide staining of agarose gels. Around 5 ml of adult flies (about 1,000) was collected and then homogenized in buffer as previously described (9), except without Lubrol (90 mM KCl, 55 mM CaCl 2 , 15 mM MgSO 4 , 30 mM NaCl, 250 mM sucrose) using a Dounce tissue grinder (Wheaton, Millville, N.J.).…”