The overall goal of this project is to dissect the process of mitochondrial biogenesis that occurs in this lower eukaryotic parasitic cell in molecular terms, both in terms of the mitochondrial genome and the nuclear genome. In addition we would like to understand the unusual molecular differentiation of this mitochondrial DNA in a structural and functional sense.
Specific aims are as follows: 1. Completion of entire sequence of L. tarentolae maxicircle. 2. Identification of 3' and 5' ends of all transcripts. 3. Identification of primary transcripts. 4. Isolation and sequencing of tRNAs. 5. Transcription of specific maxicircle genes under different physiological conditions. 6. Transcription of maxicircle genes in life cycle of T. brucei. 7. Isolation of mitochondrial RNA polymerase. Identification of promoters of maxicircle genes. 8. Comparative maxicircle gene organization in different species. 9. Sequencing of 9 and 12s genes from several species. 10. Isolation of cytochrome oxidase or FOF1 ATPase from L. tarentolae. 11. Cloning of nuclear genes for cytochrome oxidase subunits 4-7 of FOF1 ATPase subunits. 12. Cloning nuclear genes for mitochondrial proteins. 13. Analysis of developmentally regulated transcription of the BhR sequence in T. brucei. 14. Analysis of possible minicircle transcription.
