The plastid DNA of the malaria parasite Plasmodium falciparum is replicated by two mechanisms

Abstract

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 ...

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“…PfGyrA expression could be detected only during the trophozoite and schizont stages. It is interesting that apicoplast DNA replication overlaps with nuclear DNA replication (5,28,38,56). Since nuclear DNA replication takes place during the late trophozoite and schizont stages (24), it can be suggested that the expression patterns of PfGyrA and PfGyrB coincide with the time of DNA replication.…”

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“…In P. falciparum, the mtDNA and the apDNA are replicated at about the same time as the nuclear genome, just before the onset of schizogony , Smeijsters et al 1994, Williamson et al 2002. In the early stage of schizogony, the mitochondrion changes its morphology from the simple rod-like shape to an amorphous extended shape with many branches (van Dooren et al 2005).…”

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