Cleavage of Replicating Forms of Mitochondrial DNA by Eco RI Endonuclease

121

Mitochondrial DNA extracted from an isofemale strain of Drosophila mauritiana (subgroup melanogaster) appeared to be heterogeneous in size. A short genome [S; 18,500 base pairs (bp)] and a longer one (L; 19,000 bp) coexist in the preparation. The additional 500 bp have been located within the A+T-rich region. Hpa I digest patterns suggest that the S genome may carry a duplication of a 500-bp sequence including an Hpa I site and that the L genome may carry a triplication of the same sequence. At the 30th generation of the isofemale strain, 60 female genotypes were examined individually. Half of the flies were pure either for the S or the L DNA. The remaining 50% exhibited various degrees of heteroplasmy for the two DNA types. Among metazoan animals, this D. mauritiana strain offers an exceptional situation with regard to the number of individuals heterogeneous for mtDNA and the relative stability of heteroplasmy through generations.In metazoan animals, the lack of mitochondrial mutants is compensated by the use of restriction endonucleases to study the heredity and the evolution of mitochondrial DNA. Three general features of animal mitochondrial genetics emerge from different analyses (1). (i) mtDNA is maternally inherited: the first evidences from interspecific hybridization experiments (2, 3) have been confirmed later by intraspecific crosses. (ii) Sequence differences are frequent between individuals within the same species and even within the same population (4-8). (iii) From the existence of interindividual differences, a heteroplasmic transitory phase might be expected; however, for each individual the mtDNA always appears as a molecular clone (6-10): the mtDNA is homogeneous for a given organ, and the DNA molecules are identical from one organ to another within an individual (4, 11). This last observation suggests a rapid purification of the mitochondrial genome.Drosophila mtDNA exhibits these general properties (12-14). In the course of a sequence variability study within the melanogaster subgroup of Drosophila, we analyzed numerous isofemale strains. As For physical mapping, mitochondria were isolated either from embryos, from virgin eggs, or from adult flies. mtDNA was purified on CsCl gradients. If necessary, a second purification was carried out on CsCI/4',6-diamidino-2-phenylindol-2.HCl (DAPI) gradients. Cleavage sites of nine restriction endonucleases were mapped by analysis of partial and complete single digests and of double digests. The digestion fragments were separated by vertical electrophoresis on 1% agarose or 4.5% acrylamide slab gels. HindIII/A phage and HindII/4X174 phage DNA digests were used as molecular weight standards for calibration. After electrophoresis the gels were stained by standard procedures with ethidium bromide and then photographed under short wave UV light.For individual mitochondrial genotype comparison, it was not possible to get from a single fly enough DNA for a slab gel analysis; so we used the progeny of each female fly under study. About 30 generations...

Section: Resultsmentioning

confidence: 99%

Mitochondrial DNA heteroplasmy in Drosophila mauritiana.

Solignac¹,

Monnerot²,

Mounolou³

1983

121

“…The reaction mixture (10 ,l), containing about 1000 cpm of end-labeled 7S mtDNA in the presence of [1][2][3][4][5][6][7][8][9][10] Mg of carrier tRNA, 6 mM Tris-HCl (pH 7.5), 6 mM MgCl2, 6 mM NaCl, 6 mM 2-mercaptoethanol, and 0.5-4 units of Hae III enzyme (21,22) (New England Biolabs), was incubated at 370 for 2 hr, with a second addition of enzyme after 1 hr. The reaction was terminated by addition of EDTA to 25 mM and analyzed by electrophoresis as described above.…”

Section: Methodsmentioning

confidence: 99%

“…Such molecules have been termed displacement-loop (D-loop) mtDNA. For mouse L cells it has been shown that this primer segment is a heavy-strand sequence (1), as defined by the alkaline buoyant densities of the separate strands of mtDNA, and is located at the origin of mtDNA replication (2,3). More recent labeling data from L cells in vvo have indicated that this segment is rapidly lost and resynthesized on a substantial number of mtDNA molecules (4,5).…”

mentioning

confidence: 99%

“…We thus present sequence information on the replication origin of mtDNA, as defined by the naturally occurring 7S mtDNA. A novel feature of animal cell mitochondrial DNA (mtDNA) is the maintenance of a short primer fragment hydrogenbonded at a unique location on the closed circular genome, which results in a localized displacement of the opposite parental strand (1)(2)(3). Such molecules have been termed displacement-loop (D-loop) mtDNA.…”

mentioning

confidence: 99%

See 1 more Smart Citation

Displacement-loop replication initiation sequence in animal mitochondrial DNA exists as a family of discrete lengths

Gillum

Clayton

1978

Self Cite

The single-stranded mitochondrial DNA (mtDNA) displacement-loop initiation sequence (7S mtDNA) is hydrogen-bonded at the origin of replication in animal cell mtDNA. Analysis of 7S mtDNA from several cell sources indicates that this initiation sequence exists as a family of fragments of relatively discrete lengths. mtDNA from both mouse L cells and mouse liver has four major sizes of 7S mtDNA fragments, ranging from 500 to 580 nucleotides in length. The 5'-end region

“…Construction of heteroduplex molecules failed to reveal evidence of gross nucleotide sequence changes associated with differences in EcoRI sensitivity. Circular mitochondrial DNA (mtDNA) molecules of various metozoans are sensitive to cleavage by the Escherichia coli restriction endonuclease EcoRI (1)(2)(3)(4)(5). The mtDNA molecules from an individual organism are usually homogeneous in regard to the EcoRI-sensitive sites they contain.…”

mentioning

confidence: 99%

EcoRI cleavage site variants of mitochondrial DNA molecules from rats.

Buzzo¹,

Fouts²,

Wolstenholme³

1978

The mitochondrial DNA (mtDNA) molecules of some rats obtained from the SASCO colony contain six EcoRI-sensitive sites while the mtDNA molecules of other rats obtained from the same source contain only four EcoRI sites. By mapping the positions of the EcoRI sites on the molecules relative to the D-loop it was determined that mtDNA molecules from all rats have four EcoRI sites in common, but all the mtDNA molecules of some rats have two extra EcoRI sites. EcoRI sensitivity of mtDNA molecules was shown to be maternally inherited. Construction of heteroduplex molecules failed to reveal evidence of gross nucleotide sequence changes associated with differences in EcoRI sensitivity. Mitochondria were isolated from rat livers as described (7), except that mitochondria were pelleted by centrifugation at 15,000 X g, and DNA was prepared from mitochondria and separated into covalently closed circular molecules and open (nicked) circular molecules as described in Wolstenholme and Fauron (4). Fragments resulting fr i either EcoRI or HindIII digestion (4, 9) were circularized as follows. The restriction enzyme was destroyed by heating to 630 for 5 min and the DNA fragment-containing solution was dialyzed againsts 33 mM Tris-HCI (pH 7.6). MgCl2 was added to 6.6 mM, ATP to 0.06 mM, and dithiothreitol to 10 mM (10). One unit of DNA ligase isolated from T4-infected E. coil cells (Miles Laboratories) was added to 100 ,1 of solution containing DNA at a concentration of less than 10 ,gg/ml, and the mixture was incubated for 48 hr at 40 and prepared for electron microscopy at room temperature.Heteroduplexes were constructed as follows. A 1:4 mixture of nicked circular mtDNA molecules containing six EcoRI sites and fragments resulting from complete EcoRI digestion of mtDNA molecules containing four EcoRI sites were dialyzed against 95% formamide/10 mM EDTA (pH 8.2) for 1 hr to denature the DNA, then against 50% formamide/100 mM Tris. HCI (pH 8.2)/10 mM EDTA for 1 hr to achieve approximately 50% renaturation, and prepared directly for electron microscopy with a hypophase containing 20% formamide/10 mM Tris-HCI (pH 8.2)/i mM EDTA (11). The lengths (as percentage circular genome) of double and single-stranded segments of heteroduplex molecules were determined by comparison with the mean lengths of totally double-stranded and totally single-stranded circular molecules contained in the same preparation.All of the confidence limits given (+) are standard deviations, and the number of observations in each case is given by n. RESULTSA preparation of mtDNA obtained from the pooled livers of six rats was shown by electron microscopy to contain approximately 95% circular molecules. The molecular weights of these circular molecules had a unimodal distribution with a mean of 10.34 + 0.26 X 106 (n = 30). Analysis of this mtDNA (or of separated covalently closed and nicked circular molecules) by agarose gel (1.6%) electrophoresis after EcoRI digestion revealed eight bands at positions expected for fragments with mean lengths equivalent to a...