The long-term goals of these studies are to examine the different mechanisms by which Plasmodium falciparum diversifies its genome and identify the molecular factors and genes involved in genetic recombination during meiosis in mosquito. It is well established that point mutation confers resistance of parasite to pyrimethamine and sulfadoxine and also that antigen polymorphisms hamper the development of malarial vaccines. These studies will give us opportunities under conditions of natural transmission of malaria to address whether point mutations (nucleotide substitution) or spontaneous deletions of sub-telomeric genes are likely to occur during mitosis among P. falciparum parasites in persistently infected subjects in Mali. These will be performed using a sub-cohort of 80 subjects between 2 and 60 years of age. They will be followed during 28 days (D0, D3, D7, D11, D14, D28) to detect subjects with persistent infection with a single parasite allotype. In addition, we will examine the mechanisms of chromosomal exchange between parasites with the same allotypes and also with different allotypes during meiosis when the diploid zygote is formed in the mosquito. A sub-cohort of 40 infected subjects with gametocytes obtained from the first sub-cohort will be used to infect F1 generation An. gambiae. Oocyst DNA obtained from these mosquitoes will be amplified by Nested PCR and compared to parent gametocyte allotypes. The technique of Differential Display-PCR and microarray will be used to screen genes, which are differentially expressed in P. falciparum at the time of zygote formation during meiosis. The basic information to be obtained in these studies will clarify the mechanisms responsible for genetic diversity in malaria parasites and should therefore help clarify the obstacles confronting successful development of new antimalarial drugs and vaccines.
