Mitochondrial DNA of the malarial parasite Plasmodium falciparum comprises approximately 20 copies per cell of a 6 kb genome, arranged mainly as polydisperse linear concatemers. In synchronous blood cultures, initiation of mtDNA replication coincides with the start of the 4–5 doublings in nuclear DNA that mark the reproductive phase of the erythrocytic cycle. We show that mtDNA replication coincides with a recombination process reminiscent of the replication mechanism used by certain bacteriophages and plasmids. The few circular forms of mtDNA which are also present do not replicate by a theta mechanism, but are themselves the product of recombination, and we propose they undergo rolling circle activity to generate the linear concatemers.

SummaryIn common with other apicomplexan parasites, Plasmodium falciparum, a causative organism of human malaria, harbours a residual plastid derived from an ancient secondary endosymbiotic acquisition of an alga. The function of the 35 kb plastid genome is unknown, but its evolutionary origin and genetic content make it a likely target for chemotherapy. Pulsed field gel electrophoresis and ionizing radiation have shown that essentially all the plastid DNA comprises covalently closed circular monomers, together with a tiny minority of linear 35 kb molecules. Using two-dimensional gels and electron microscopy, two replication mechanisms have been revealed. One, sensitive to the topoisomerase inhibitor ciprofloxacin, appears to initiate at twin D-loops located in a large inverted repeat carrying duplicated rRNA and tRNA genes, whereas the second, less drug sensitive, probably involves rolling circles that initiate outside the inverted repeat. IntroductionLike many other members of the phylum Apicomplexa, malaria parasites harbour a residual highly derived plastid resulting from secondary endosymbiosis by a progenitor that also gave rise to dinoflagellates (Fast et al., 2001). Electron microscopic examination of this organelle's DNA from four malarial species has revealed covalently closed circular molecules (Wilson and Williamson, 1997), and sequencing of the Plasmodium falciparum version has shown that it has a size of nearly 35 kb and is packed with about 67 genes, almost all of which are involved in its own expression al., 1993;Lu et al., 1996), twin D-loops have been reported in Chlamydomonas (Waddell et al., 1984), but only a single origin has been detected in Euglena (Koller and Delius, 1982). Although often positioned near genes for rRNA, there is limited conservation of origin sequences between different plastid genomes, whether of the D-loop or Cairns variety.The evidence reported here, based on two-dimensional analysis and PFGE supported by electron microscopy, shows that essentially all the plastid DNA from P. falciparum is circular in form, although a minor proportion of linear 35 kb molecules is also present. Two replication mechanisms have been revealed: one seems to initiate at twin D-loops located in a large inverted repeat (IR) carrying duplicated rRNA and tRNA genes and is relatively sensitive to the topoisomerase inhibitor ciprofloxacin. The second mechanism, somewhat less sensitive to the inhibitor, probably involves rolling circles, which initiate replication elsewhere on the molecule. ResultsA partial restriction map of the plastid molecule is shown in Fig. 1, which records all the restriction sites and cloned fragments we used as probes. The latter were chosen to allow us to target sectors, sometimes overlapping and almost invariably generated in double digests, spanning the entire molecule. Figure 2 shows the gross time course of replication of nuclear and plastid DNA. Replication of the plastid DNA was initiated in late trophozoites slightly before that of chromosomal DNA and continued ...

Transfection of the human malaria parasite Plasmodium falciparum is currently performed with circularised plasmids that are maintained episomally in parasites under drug selection but which are rapidly lost when selection pressure is removed. In this paper, we show that in instances where gene targeting is not favoured, transfected plasmids can change to stably replicating forms (SRFs) that are maintained episomally in the absence of drug selection. SRF DNA is a large concatamer of the parental plasmid comprising at least nine plasmids arranged in a head-to-tail array. We show as well that the original unstable replicating forms (URFs) are also present as head-to-tail concatamers, but only comprise three plasmids. Limited digestion and gamma irradiation experiments revealed that while URF concatamers are primarily circular, as expected, SRF concatamers form a more complex structure that includes extensive single-stranded DNA. No evidence of sequence rearrangement or additional sequence was detected in SRF DNA, including in transient replication experiments designed to select for more efficiently replicating plasmids. Surprisingly, these experiments revealed that the bacterial plasmid alone can replicate in parasites. Together, these results imply that transfected plasmids are required to form head-to-tail concatamers to be maintained in parasites and implicate both rolling-circle and recombination-dependent mechanisms in their replication.

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