Origin and direction of replication in mitochondrial DNA molecules from Drosophila melanogaster.

Abstract

From a consideration of the various structural forms of partially replicated mitochondrial DNA (mtDNA) molecules from Drosophila melanogaster embryos observed in the electron microscope, it appears that the majority of molecules are replicated by a highly asymmetrical mode in which synthesis on one strand is up to 99% complete before synthesis on the second strand is initiated. Replication of the minority of molecules involves a more nearly symmetrical synthesis of the two complementary strands. The D. melanogaster mtDNA molecules have physical features with respect to which the origin and direction of replication could be mapped. These features are (i) a single region accounting for approximately 25% of the circular contour length and rich in adenine + thymine, and (ih) four EcoRI sites, all of which lie outside of this region.Molecules of this mtDNA were subjected to partial denaturation, EcoRI digestion, or partial denaturation after EcoRI digestion and the products were examined in the electron microscope. Complex forms interpretable as originating-from replicative intermediates were observed. The size and structure of the components of these complex forms were wholly consistent with the interpretation that, in all of these mtDNA molecules, replication originates at, or close to, the center of the adenine + thymine-rich region and proceeds unidirectionally around the molecule toward the EcoRI site lying closest to-the adenine + thymine-rich region.Circular mitochondrial DNA (mtDNA) molecules (molecular weight, 12.35 X 106) of -Drosophila melanogaster are distinguished by a region, accounting for 25% of the circular contour (genome) length, that denatures at a lower temperature than does the rest of the molecule due to a high content of adenine + thymine (A+T) (1-8). By electron microscopy we have studied replicative intermediates present in D. melanogaster mtDNA and, using the relative location of the A+T-rich region and sites sensitive to cleavage by the restriction enzyme EcoRI (5) we have located the origin and determined the direction of replication in these molecules. MATERIALS AND METHODSThe D. melanogaster strain used in these experiments was Oregon R-Utah (Oak Ridge, TN) (5). Growth of flies, collection of eggs, and preparation of mtDNA from embryonated eggs by use of preparative cesium chloride centrifugation were as described (5).mtDNA was centrifuged to equilibrium in cesium chloride/ethidium bromide (9). The regions of the gradient containing two distinct fluorescent bands and a third, more diffuse, intermediately located band were collected separately and the ethidium bromide was removed as described (10). The relative concentrations of the DNA in the three regions were determined by measuring absorbance at 260 nm.Digestion of mtDNA molecules with restriction endonuclease EcoRI was accomplished as described (5), except that incubations were carried out at 250 in order to minimize branch migration (11). Partial denaturation of circular mtDNA molecules and EcoRI-produced fragments of mtDN...

Replication Origin of Mitochondrial DNA in Insects

“…In all species examined, the free 59 ends of the minor coding strands were found in a very narrow range of several nucleotides in the A 1 T-rich region, located at the middle portion for four Drosophila species, T. castaneum, and L. migratoria and near the 12S rRNA gene for B. mori ( Figures 2B, 3B, and 4). D. yakuba and D. virilis were used in a previous mapping study using electron microscopy, and the free 59 ends were found in the middle of the A 1 T-rich region (Goddard and Wolstenholme 1980). Therefore, the results of the present study are consistent with the previous results.…”

“…If the recognition sequence for replication initiation is included in the repeated sequence, the O R 's must also be situated tandemly in D. melanogaster mtDNA. However, the O N has been found only at the central portion of the A 1 T-rich region by electron microscopy (Goddard and Wolstenholme 1978). The prominent T-stretch in the major coding strand is located at the central intervening portion between the type I and II repeats and is excluded from the repeated sequences.…”

Drosophila Mitochondrial Transcription Factor B2 Regulates Mitochondrial DNA Copy Number and Transcription in Schneider Cells

“…Relatively little is known about the mechanism of mtDNA replication in Drosophila (47), and new experimental approaches have led to a reconsideration of the long standing model proposed for mouse and man (48 -50). In the recent model, mtDNA replication is proposed to initiate bidirectionally within a broad zone and proceed symmetrically, becoming unidirectional when one fork encounters a barrier in the vicinity of the previously designated leading strand origin (51-53).…”

Genetic aspects of mitochondrial genome evolution

“…The most widely accepted "orthodox" hypothesis explaining the mode of mtDNA replication is the so called strand displacement model (Robberson et al, 1972;Goddard and Wolstenholme, 1980;Clayton, 1982). We can give here only a brief outline and refer to the reviews by Shadel and Clayton (1997) and Taanman (1999) for much more detailed description of the replication process.…”