This project proposes to develop methods for transformation of the mitochondria of kinetoplastid parasites. The kinetoplastid protozoa are the causative agents of devastating diseases in tropical and sub-tropical parts of the world and include visceral and cutaneous leishmaniasis, sleeping sickness and Chagas disease. The mitochondrial DNA (kinetoplast DNA or kDNA) of these parasites consists of thousands of small DNAs termed minicircles and 20-30 maxicircles, which encode proteins involved in oxidative metabolism and genes for mitochondrial ribosomal RNAs. Replication of kDNA and expression of the maxicircle genes involves essential sequences such as replication origins for initiation of minicircle and maxicircle replication, and promoter sequences for protein expression, ribosomal RNA synthesis and for synthesis of guide RNAs required for editing of maxicircle transcripts. To analyze the function of such DNA elements it is necessary to be able to manipulate these sequences in vitro and to be able to introduce the sequences back into the mitochondria. Transformation of the nucleus of kinetoplastid parasites by electroporation of DNA into the nucleus of the cell has been enormously successful in analyzing the regulation and expression of chromosomal genes. The inability to transform the cell's mitochondria by this method has been an impediment to similar studies of mitochondrial genes in other eukaryotes as well as in kinetoplastids. Two recent developments offer a promising approach to transforming mitochondria of kinetoplastids. First, the development of a particle gun method for introducing DNA into the mitochondria of yeast and Chlamydomonas, a unicellular alga, should be applicable to transformation of the mitochondria of kinetoplastids. Second, a gene in the maxicircle of Leishmania tarentolae that encodes a mutant form of cytochrome c has been shown to confer resistance to the drug antimycin A. This mutation was selected by prolonged growth of L. tarentolae in the presence of the drug. We are proposing to develop plasmids and/or linear DNA fragments containing this mutant gene for transformation of wild- type L. tarentolae by particle bombardment with tungsten spheres coated with the DNA. DNA constructs would be created for replication as a mitochondrial episome using a minicircle or maxicircle replication origin or as an integrated DNA segment by re-combinational insertion into the maxicircle. The level of expression of the gene conferring antimycin resistance would be increased by the inclusion of other promoter sequences including the strong promoter for the 12S ribosomal RNA. The proposed research will develop methods for altering the DNA sequence of the mitochondrial DNA in parasites commonly found in tropical and sub-tropical areas of the world. The mitochondrial DNA carries the genetic information for the parasite's machinery for metabolism and energy production. An understanding of the molecular and biochemical basis for energy production in these parasites will aid in the development of novel drugs for the treatment of parasitic diseases. ? ? ?
