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Nucleic Acids Research, 2002, Vol. 30, No. 3 726-731
© 2002 Oxford University Press

Topology and replication of a nuclear episomal plasmid in the rodent malaria Plasmodium berghei

Donald H. Williamson*, Chris J. Janse1, Peter W. Moore, Andrew P. Waters1 and Peter R. Preiser

Division of Parasitology, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK and 1Department of Parasitology, Leiden University Medical Centre, PO Box 9600, 2300 RC Leiden, The Netherlands

The rodent malaria Plasmodium berghei is one of a small number of species of Plasmodium that can currently be genetically transformed through experimentally controlled uptake of exogenous DNA by bloodstage parasites. Circular DNA containing a selectable marker replicates and is maintained under selection pressure in a randomly segregating episomal form during the first weeks after transformation. In this study, using pulsed field gel electrophoresis and ionising radiation, we show that in dividing asexual blood stage parasites the episomes are completely converted, within 2 weeks post-infection, into non-rearranged circular concatamers ranging in size between about 9 and 15 copies of the monomer. These occur as slow-moving aggregates held together by radiation-sensitive linkers consisting partly of single-stranded DNA. The process generating these complexes is not clear but 2D gel analysis showed that Cairns-type replication origins were absent and it seems most likely that the initial concatamerisation takes place using a rolling circle mechanism followed by circularisation through internal recombination. We propose a model in which continued rolling circle replication of the large circular concatamers and the recombinational activity of the tails of the rolling circles could lead to the formation of the large aggregates.

* To whom correspondence should be addressed. Tel: +44 208 959 3666; Fax: +44 208 913 8593; Email: dwillia{at}nimr.mrc.ac.uk


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