Mitochondrial DNA genetics and the basis of maternal inheritance

In most Eukaryotes, the mitochondrial DNA (mtDNA) is an essential cytoplasmic chromosome encoding several vital components of the electron transport chain and mitochondrial ATPase - enzymes required for the ATP-generating process of aerobic respiration. In humans, mtDNA mutations are associated with a number of maternally inherited metabolic diseases, yet we lack animal models to study these diseases and the basis for their inheritance. Previously, genetic analysis of mtDNA was confounded by its polyploidy and relaxed segregation during cell division. However, we developed an efficient selection for homoplasmic mtDNA mutant fly lines based on mitochondria-targeted restriction enzymes. We isolated a homoplasmic mtDNA mutant with age-onset phenotypes resembling human mtDNA disease, arguing that the delayed onset of human mtDNA diseases is not necessarily caused by the gradual accumulation of mutant genomes. We also isolated a male sterile mtDNA mutant that we used to study how organisms cope with mtDNA mutations causing male-specific defects. Lastly, we isolated a mtDNA deletion that allowed us to discriminate different mtDNA genotypes in a mixed population, and follow the fate of paternal mtDNA in a cross. This work advances flies as an animal model for mtDNA disease. We additionally characterized the mechanisms that ensure maternal mtDNA inheritance. We attempted to follow paternal mtDNA through a cross, but discovered that fertilized embryos lacked paternal mtDNA. Furthermore, we determined that mature sperm also lacked mtDNA, despite containing giant mitochondria. Next, we documented two distinct processes that eliminate mtDNA from developing sperm mitochondria. A primary mechanism digests mtDNA during sperm elongation while a backup mechanism collects and discards residual mtDNA in conjunction with cytoplasmic trimming. Using targeted genetic screens, we determined that two mitochondrial nucleases, EndoG and Tamas, are required to eliminate mtDNA during sperm elongation. We propose that EndoG nicks mtDNA, allowing Tamas, an exonuclease, to initiate mtDNA digestion. When we removed both EndoG and Tamas, we robustly inhibited mtDNA elimination. However, these mt-DNA containing sperm were dysfunctional, suggesting that mtDNA elimination is vital to sperm function. We argue that these early acting barriers to paternal mtDNA transmission may have evolved to restrict the spread of malicious mtDNA to progeny