Transcription and transcript processing of the Acanthamoeba castellanii mitochondrial genome

Submission note: A thesis submitted in total fulfilment of the requirements for the degree of Doctor of Philosophy to the Department of Physiology, Anatomy and Microbiology, School of Life Sciences, College of Science, Health and Engineering, La Trobe University, Bundoora.

Mitochondria are essential organelles of eukaryotic cells with crucial roles in cellular processes such as energy production and respiration. Mitochondria contain their own genomes, which are extremely diverse across the eukaryotic domain. One of the highest diversities with regard to genome size, structure, gene organisation and gene expression is found in protozoan mitochondria. Studying the genetic processes in protozoan mitochondria contribute to our overall understanding of the evolution of mitochondria and their genomes. In this study, gene expression and the processing of the resulting transcripts was investigated in the mitochondria of the amoebozoan Acanthamoeba castellanii. Northern hybridisation analyses revealed the presence of six large polycistronic transcripts, all of which are processed substantially into mature mono-, di-, or tricistronic RNA molecules through mechanisms that in most cases involve the excision of tRNAs. Reverse Transcription Polymerase Chain Reactions across the intervening regions between the six major transcripts demonstrated that these transcripts derived from an even larger primary transcript. The identification of the transcription initiation site from which the primary transcript is generated was attempted in vitro capping experiments but failed to reveal the exact location. In addition, the optimal growth conditions, the grazing behaviour on various bacterial species and the sensitivity of A. castellanii to the growth inhibitor geneticin were established to form the basis for the development of a transformation system to genetically modify the organism.