Subtle mutagenesis by ends-in recombination in malaria parasites

The recent advent of gene-targeting techniques in malaria (Plasmodium) para sites provides the means for introducing subtle mutations into their genome . Here, we used the TRAP gene of Plasmodium berghei as a target to test whe ther an ends-in strategy, i.e., targeting plasmids of the insertion type, m ay be suitable for subtle mutagenesis, We analyzed the recombinant loci gen erated by insertion of linear plasmids containing either basepair substitut ions, insertions, or deletions in their targeting sequence. We show that pl asmid integration occurs via a double-strand gap repair mechanism, Although sequence heterologies located close (less than 450 bp) to the initial doub le-strand break (DSB) were often lost during plasmid integration, mutations located 600 bp and farther from the DSB were frequently maintained in the recombinant loci. The short lengths of gene conversion tracts associated wi th plasmid integration into TRIP suggests that an ends-in strategy may be w idely applicable to modify plasmodial genes and perform structure-function analyses of their important products.