An alteration in concatameric structure is associated with efficient segregation of plasmids in transfected Plasmodium falciparum parasites

Transfection of the human malaria parasite Plasmodium falciparum is current ly performed with circularised plasmids that are maintained episomally in p arasites under drug selection but which are rapidly lost when selection pre ssure is removed. In this paper, we show that in instances where gene targe ting is not favoured, transfected plasmids can change to stably replicating forms (SRFs) that are maintained episomally in the absence of drug selecti on. SRF DNA is a large concatamer of the parental plasmid comprising at lea st 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-tai l concatamers, but only comprise three plasmids, Limited digestion and gamm a 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 rearrangeme nt or additional sequence was detected in SRF DNA, including in transient r eplication experiments designed to select for more efficiently replicating plasmids, Surprisingly, these experiments revealed that the bacterial plasm id alone can replicate in parasites. Together, these results imply that tra nsfected plasmids are required to form head-to-tail concatamers to be maint ained in parasites and implicate both rolling-circle and recombination-depe ndent mechanisms in their replication.