Malarial parasites have two organelle genomes, a 35 kb circular DNA with chloroplast-like characteristics and a 6 kb tandemly repeated linear element which is the mitochondrial DNA. Compounds which inhibit the activity of gene products encoded by these DNAs have anti-malarial effects, arguing that expression of both organelles is critical to parasite survival. All genes identified from the 35 kb DNA encode components of the transcriptional/translational machinery, thus the function(s) required for survival must be in the 40% of that molecule which remains unsequenced. Evidence suggests that the mitochondrial respiratory system is differentially expressed between erythrocytic and insect stages of Plasmodium falciparum and genes from both organelle DNAs have been shown to vary in transcript abundance among erythrocytic stages. This project is designed to gain an understanding of the organization and expression of the organelle genomes during the life cycle of P. falciparum and to evaluate their importance to parasite survival. The remaining sequence of the 35 kb DNA will be determined and its genes analyzed. Transcripts from both organelles will be mapped and analyzed for processing events, which commonly regulate organelle gene expression. Transcript and protein expression of selected genes from the 35 kb DNA and the 6 kb element will be evaluated in erythrocytic stages, gametocytes, and sporozoites to provide an integrated picture of organelle gene expression during the life cycle. Cytochrome oxidase II is normally mitochondrial but is not encoded by the 6 kb element and may have been transferred to the nucleus; its gene will be isolated and its transcript abundance during the life cycle characterized to ascertain if it is coordinately regulated with the mitochondrially encoded cytochromes. The proposed experiments will clarify the role of the 35 kb DNA, provide an integrated picture of gene expression for these extrachromosomal DNAs, and allow assessment of nuclear/organellar coordination. Insights into the basic biochemistry of malarial parasites will be obtained and novel mechanisms for gene regulation may be found. Furthermore, the anti-malarial activity of compounds which interfere with organelle genome expression indicate that a more complete understanding of organelle expression may suggest new approaches for chemotherapeutics.
