Mitochondrial DNA

Cercopithecus ascanius is an African primate species encompassing five geographic types with unresolved taxonomy. Recent publications have analyzed C. ascanius genetic diversity and taxonomy; however, few publications have addressed the genetic diversity and phylogenetic relationships of C. ascanius from wild populations. My objectives for this thesis were to determine mtDNA diversity within the C. Ascanius species and investigate C. ascanius genetic structure. Results from this thesis support findings from previous studies wherein C. ascanius depicted high mitochondrial diversity and all C. ascanius subspecies form a monophyletic clade within the Cercopithecus genus. Analyzing additional samples of C. ascanius monkeys will strengthen molecular diversity estimation and clarify genetic structure within the C. ascanius lineage.

Mitochondria are inherited uniparentally in almost all eukaryotic models studied to date. The fathers mitochondria are eliminated and there have been several hypothesis as to how this occurs. One hypothesis is that the sperm mitochondria are actively targeted and destroyed. Ubiquitin has been proposed a possible candidate involved in this process. My research investigated the normal mitochondrial inheritance pattern in C. elegans. I also examined the possible role of the C34F11.1 gene in mitochondrial inheritance. This gene is sperm-specific and has ubiquitin-ligase properties. It was determined that the normal mitochondrial inheritance pattern in C. elegans is maternal and that the sperm mitochondria are eliminated. It was also concluded that the C34F11.1 gene does not have a role in normal mitochondrial inheritance.

Aging is a multifactoral biological process of progressive and deleterious changes partially attributed to a build up of oxidatively damaged biomolecules resulting from attacks by free radicals. Methionine sulfoxide reductases (Msrs) are enzymes that repair oxidized methionine (Met) residues found in proteins. Oxidized Met produces two enantiomers, Met-S-(o) and Met-R-(o), reduced by MsrA and MsrB respectively. Unlike other model organisms, our MsrA null fly mutant did not display increased sensitivity to oxidative stress or shortened lifespan, suggesting that in Drosophila, having either a functional copy of either Msr is sufficient. Here, two Msr mutant types were phenotypically assayed against isogenic controls. Results suggest that only the loss of both MsrA and MsrB produces increased sensitivity to oxidative stress and shortened lifespan, while locomotor defects became more severe with the full Msr knockout fly.

Mitochondrial disorders resulting from defects in oxidative phosphorylation are the most common form of inherited metabolic disease. Mutations in the human mitochondrial translation elongation factor GFM1 have recently been shown to cause the lethal pediatric disorder Combined Oxidative Phosphorylation Deficiency Syndrome (COXPD1). Children harboring mutations in GFM1 exhibit severe developmental, metabolic and neurological abnormalities. This work describes the identification and extensive characterization of the first known mutations in iconoclast (ico), the Drosophila orthologue of GFM1. Expression of human GFM1 can rescue ico null mutants, demonstrating functional conservation between the human and fly proteins. While point mutations in ico result in developmental defects and death during embryogenesis, animals null for ico survive until the second or third instar larval stage. These results indicate that in addition to loss-of-function consequences, point mutations in ico appear to produce toxic proteins with antimorphic or neomorphic effects. Consistent with this hypothesis, transgenic expression of a mutant ICO protein is lethal when expressed during development and inhibits growth when expressed in wing discs. In addition, animals with a single copy of an ico point mutation are more sensitive to acute hyperthermic or hypoxic stress. Removal of the positively-charged tail of the protein abolishes the toxic effects of mutant ICO, demonstrating that this domain is necessary for the harmful gain-of-function phenotypes observed in ico point mutants.

Aging is a process characterized by accumulated oxidative damage to DNA, proteins, and lipids,which leads to the gradual degeneration of cellular activity. Mitochondria play a central role in aging because they produce both cellular energy and oxidative stress. As resultof accumulated oxidative damage, mitochondria function decays, which leads to a cellular energy deficit and compromises cellular function. Iron is an essential nutrient reequired by mitodhondria to function optimally. It has been proved that iron supplementation increases the lifespan of several yeast strains, including superoxide dismutase mutants. We are interested in finding where the iron is going and what it might be doing that is beneficial to the cell. We have used Saccharomyces cerevisiae as our molecular model of aging. Our results indicate that the extra iron is being transported into the mitochoindria.

Telomerase is associated with telomere production and nDNA protection. However, studies by Santos et al. have demonstrated that human telomerase has a mitochondrial entry sequence and in the presence of hydrogen peroxide it has been found inside the mitochondrion and may cause mitochondrial DNA mutations. Saccharomyces cerevisiae contains telomerase, but it does not have the mitochondrial entry sequence. To determine if the presence of telomerase in the mitochondria can induce mutations an experiment was developed in which a mitochondrion entry sequence would be fused to the S. cerevisiae telomerase enzyme. This fusion could then be screened in S. cerevisiae with an ade2 mutation for a simple color assay of mitochondrial activity. To date, no successful transformant has been identified. The frequency of incorrect ligations has been recognized and may indicate that the desired fusion is lethal to E. coli cells.