: Malaria, the world's most important parasitic disease, is caused by intraerythrocytic protozoan parasites of the genus Plasmodium. Plasmodium falciparum causes the most severe form of the disease and contributes to 1.5 to 2.7 million deaths annually. The P. falciparum genome project that began in earnest in May 1996 is now completed. This progress has led to a rapid accumulation of genes, many of which have no homologs in other databases and a very large proportion have unknown functions. The available technology to characterize the function of these genes by gene interruption or in vivo tagging is at an early stage of development. Several months are needed to generate a single knockout of a non-essential gene, and study of essential genes by complementation and plasmid shuffling can take much longer. Therefore, only a handful of genes could be studied using this technology, very likely resulting in less than optimal usage of the collected sequence data. The proposed research project seeks to develop transposition as a tool for large-scale functional analysis of the P. falciparum genome. The premise is that this tool will help to dissect the function of multiple genes in a single-step and in a short period of time. Here, we propose to develop mariner-based transposable elements as genetic tools for generating systematic disruptions of P. falciparum genes. A transposon, pmariner-BSD, that can shuttle between P. falciparum and E. coli, has been designed in our laboratory and shown to move efficiently and randomly in vitro and allows easy recovery of transposition events by transposon-rescue in bacteria. Moreover, transgenic blastieidin-refractory parasites harboring pmariner-BSD in an episomal form have been obtained.
In Specific Aim One, we will express the Drosophila mariner (MOS1) transposase in P. falciparum clones harboring pmariner-BSD and attempt to recover all the events of pmariner-BSD transposition into the genome by rescue of the plasmid and the genomic flanking regions in E. coli. The identities of the targeted genes will be determined by sequencing DNA adjacent to the transposon and the efficiency of transposition will be determined.
In Specific Aim Two, we will perform large-scale limiting dilution cloning to isolate individual parasites harboring each single integration event. The studies outlined above are designed to generate a valuable library of information that could lead to better understanding of the basic biology of P. falciparum development and its interaction with its host. Information and clones generated from these studies will be accessible to the general scientific community, providing valuable resources for advancing our understanding of the biology of this important parasite. ? ?
