Erythrocytic stages of mammalian malarial parasites contain acristate mitochondria whose functions are not well understood. Moreover, little is known about the genome of these organelles. We have previously reported that all species of malarial parasites examined carry highly conserved, tandemly arrayed DNA with a unit length of 6 kb that is transcribed into discrete RNA molecules in erythrocytic stages. The entire 6 kb repeating unit from a rodent parasite, Plasmodium yoelii, has been sequenced in our laboratory. The 6 kb sequence has similarities with cytochrome oxidase I and cytochrome b genes as well as with portions of rRNA genes. Sequence analyses and several other pieces of evidence strongly suggest that the 6 kb tandem arrays are mitochondria-like organelle DNA molecules. Considering many features of their organization, sequence, and size, these tandem arrays appear to be a very unusual form of mitochondrial DNA (mtDNA). Because of the physiological significance of these genes and their products, we propose to carry out an extensive investigation of organelle genes of malarial parasites. Transcripts from the 6 kb DNA will be characterized by primer extension, RNA sequencing, and cDNA cloning. Possible RNA processing events will be revealed by these experiments. Developmental regulation of transcription from these sequences will be studied by examining various vertebrate and insect stages of the parasites. We shall also examine mitochondrial fractions for the presence of any companion DNA molecules that are different from the 6 kb tandem arrays. Immunological probes will be developed to assess the expression proteins predictively encoded by the 6 kb sequence and to see what other proteins are associated with these predicted peptides. Biochemical and biophysical properties of these proteins will be studied. Significance of small RNAs with peptidyl transferase domain-like structure will be investigated. Evolutionary relationship between 6 kb DNAs from three species of plasmodia will be examined through DNA sequence comparison. With these investigations, we hope to fill a gap in our knowledge of plasmodial metabolism.
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