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Updated: Oct 2, 2026
Protocol for Production of a Genetic Cross of the Rodent Malaria Parasites
Published on: January 3, 2011
Topology and replication of a nuclear episomal plasmid in the rodent malaria Plasmodium berghei
Donald H Williamson1, Chris J Janse, Peter W Moore
1Division of Parasitology, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK. dwillia@nimr.mrc.ac.uk
Abstract:
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.
Insights
Genetic transformation of malaria parasites Plasmodium berghei involves episomes converting into large, non-rearranged circular concatamers within two weeks. These aggregates form through a rolling circle mechanism and internal recombination in asexual blood stages.
Area of Science:
- Molecular Parasitology
- Genetics of Malaria Parasites
- Plasmodium berghei Research
Background:
- Plasmodium berghei, a rodent malaria model, is genetically transformable via exogenous DNA uptake in blood-stage parasites.
- Episomal DNA with selectable markers replicates and is maintained under selection post-transformation.
Purpose of the Study:
- Investigate the fate of episomal DNA in dividing asexual blood-stage Plasmodium berghei.
- Characterize the structure and formation of episomal concatamers during parasite replication.
Main Methods:
- Pulsed field gel electrophoresis (PFGE) to analyze large DNA molecules.
- Ionizing radiation to probe DNA aggregate structure.
- 2D gel electrophoresis to identify replication origins.
Main Results:
- Episomes are converted into non-rearranged circular concatamers (9-15 monomer copies) within 2 weeks post-infection.
- These concatamers form slow-moving aggregates linked by radiation-sensitive, single-stranded DNA components.
- Cairns-type replication origins were absent; rolling circle replication and internal recombination are proposed mechanisms.
Conclusions:
- Episomal DNA in Plasmodium berghei undergoes significant structural changes during asexual blood-stage replication.
- A model involving rolling circle replication and recombination explains concatemer and aggregate formation.
- Understanding these mechanisms is crucial for genetic manipulation of malaria parasites.
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