Fig. 3

Development of techniques to examine the replication of the Plasmodium genome. a Bioinformatic analysis of conserved sequences at and surrounding replication origins in the S. cerevisiae genome has led to identification of common motifs for searching the Plasmodium genome [30]. Origins in S. cerevisiae consist of compact autonomously replicating sequences (ARS) with ‘A domain’ motifs (orange) and surrounding ‘B domains’ (green). The Plasmodium genome has a high concentration of individual A and B domains (~ every 2500 bp) but a much lower concentration when the requirement for closely associated domains is imposed (grey boxes). Bioinformatics approaches can only identify putative origin sites and may fail to identify true origins (*) or identify sequences which are incapable of functioning as origins. b Chromatin immuno-precipitation (ChIP) of the proteins required before and during replication, such as members of the Origin Recognition Complex (ORC), allows experimental characterisation of origin sequences [30]. Following reversible DNA-protein cross linking, the genome is fragmented and the proteins of interest are purified along with the associated DNA fragments, which are then sequenced. This may include origins that would never be activated, and may miss those where the protein complex has dissociated from the chromosome. c Synthetic nucleoside labelling and DNA combing techniques allow the labelling and fluorescent immunodetection of de novo DNA synthesis [31]. Parasites expressing viral thymidine kinase can incorporate the synthetic nucleosides IdU (red) and CldU (green) which can be visualised in individual nuclei or on combed DNA fibres, allowing the calculation of inter-origin distances and replication rates. The synthetic nucleosides will only be incorporated around active origins (*) while inactive origins will remain unlabelled and therefore undetected