The human mitochondrial DNA polymerase (Pol γ) catalyzes mitochondrial DNA synthesis, and thus is essential for the integrity of the organelle. Mutations of Pol γ have been implicated in more than 150 human diseases. Reduced Pol γ activity caused by inhibition of anti-HIV drugs targeted to HIV reverse transcriptase confers major drug toxicity. To illustrate the structural basis for mtDNA replication and facilitate rational design of antiviral drugs, I have determined the crystal structure of human Pol γ holoenzyme. The structure reveals heterotrimer architecture of Pol γ holoenzyme with a monomeric catalytic subunit Pol γA, and a dimeric processivity factor Pol γB. While the polymerase and exonuclease domains in Pol γA present high structural homology with the other members of the DNA Pol I family, the spacer between the two functional domains shows a unique fold, and constitutes the subunit interface. The structure suggests a novel mechanism for Pol γ’s high processivity of DNA replication. Furthermore, the structure reveals dissimilarity in the active sites between Pol γ and HIV RT, thereby indicating an exploitable space for design of less toxic anti-HIV drugs. Interestingly, the structure shows an asymmetric subunit interaction, that is, one monomer of dimeric Pol γB primarily participates in interactions with Pol γA. To understand the roles of each Pol γB monomer, I generated a monomeric human Pol γB variant by disrupting the dimeric interface of the subunit. Comparative studies of this variant and dimeric wild-type Pol γB reveal that each monomer in the dimeric Pol γB makes a distinct contribution to processivity: one monomer (proximal to Pol γA) increases DNA binding affinity whereas the other monomer (distal to Pol γA) enhances the rate of polymerization. The pol γ holoenzyme structure also gives a rationale to establish the genotypic-phenotypic relationship of many disease-implicated mutations, especially for those located outside of the conserved pol or exo domains. Using the structure as a guide, I characterized a substitution of Pol γA residue R232 that is located at the subunit interface but far from either active sites. Kinetic analyses reveal that the mutation has no effect on intrinsic Pol γA activity, but shows functional defects in the holoenzyme, including decreased polymerase activity and increased exonuclease activity, as well as reduced discrimination between mismatched and corrected base pair. Results provide a molecular rationale for the Pol γA-R232 substitution mediated mitochondrial diseases. / text

Biogeographers have long searched for common processes responsible for driving diversification in the Holarctic region. Although terrestrial flora and fauna have been well studied, much of the marine biogeographic work addresses patterns and processes occurring over a relatively recent timescale. A prerequisite to comparative biogeographic analysis requires well-resolved phylogenies of similarly distributed taxa that diverged over a similar timeframe. The overall aim of my Ph.D. thesis was to address fundamental questions in the systematics and biogeography of a family of Holarctic fish (Osmeridae) and place these results in a broad comparative biogeographic framework. With eight conflicting morphological hypotheses, the northern hemisphere smelts have long been the subjects of systematic disagreement. In addition to the uncertainty in the interrelationships within this family, the relationship of the Osmeridae to several other families remains unclear. Using DNA sequence data from three mitochondrial and three nuclear genes from multiple individuals per species, I reconstructed the phylogenetic relationships among the 6 genera and 15 osmerid species. Phylogenetic reconstruction and divergence dating yielded a well-resolved phylogeny of the osmerid genera and revealed several interesting evolutionary patterns within the family: (1) Hypomesus chishimaensis and H. nipponensis individuals are not reciprocally monophyletic, suggesting that they are conspecific and H. chishimaensis is a recently evolved freshwater ecotype that invaded the Kuril Islands following the last glaciation, (2) The trans-Pacific sister relationships in Hypomesus based on lateral line scale counts are not supported, implying that this phenotype evolved in parallel on each side of the North Pacific Ocean, (3) The Plecoglossidae are the Osmeridae sister group, (4) Over half of the characters from previous studies show evidence of parallel evolution; however, 27 traits reflect ancestral relationships, (5) Multiple divergences within the Osmeridae date to both the mid-Miocene cooling period and the Pliocene Bering Seaway opening, suggesting these events were important in the evolution of these fishes, and (6) Divergences in many marine taxa for which dated phylogenies are available are also correlated with these time periods. Future research should target additional Holarctic marine taxa for further comparative analysis.

The objective - To identify Zemaitukai mtDNA D-loop region and genetic relationships between Zemaitukai and some other breeds based on mtDNA sequence variation. Methods - To perform a phylogenetic analysis of 10 Zemaitukai, mtDNA D-loop 49 horses in the 20 different horse breeds sequence were included from GenBank (http://www.ncbi.nlm.nih.gov/GenBank). DNA was extracted from hair roots using the DNEasy® Tissue Kit (Qiagen). 1,280 bp fragment of mtDNA D-loop were amplify out in PTC-100™ termocycler. Amplified products were sequenced on a LI-COR® 4200S-2 automated sequencer. Sequencher v 4.1.4. software package was used to generate the actual DNA sequence for each of the animals. Multiple alignments of sequences were performed with CLUSTAL X 1.8 (Thompson et al., 1997). The Neighbor-joining tree (Saitou and Nei, 1987) of mtDNA sequences was constructed from Jukes-Cantor distances, performed on the pairwise deletion using the MEGA software (Kumar et al., 1993). Results –Within Zemaitukai breed 19 polymorphic sites were detected and 6 haplotypes. At the individual level 4 zemaitukai haplotypes were private to one individuals, 1 haplotype-between 2 individuals and 1 haplotype was shared between 4 individuals. On the Neighbor-joining tree showed one Zemaitukai haplotype closer genetic relationships to the Icelandic and to the Norwegian Fiord horse haplotypes. Zemaitukai breed formed a separate branch with the ancient DNA haplotype. The phylogenetic analysis reflects the presence... [to full text]

Genetic diversity is a fundamental attribute that contributes to a species evolutionary survival. In recent times, conservation managers have recognized the need to preserve genetic diversity of declining species, and have also acknowledged the utility of genetic markers for describing genetic and ecological relationships within and among populations. Information obtained from genetic studies can be used in conjunction with information on population demography, land use patterns and habitat distribution to develop effective management strategies for the conservation of species in decline. The wallum froglet, Crinia tinnula, is one of Australia's smallest habitat specialist anurans. In recent years there has been a dramatic decrease in population numbers of this species. The habitat to which C.tinnula is endemic ('wallum' habitat) is restricted to low coastal plains along the southeast Queensland and northern New South Wales coastline. As human populations in this region expanded, the coastal areas have undergone significant development and large areas of wallum habitat have been cleared. The effect has been to convert once largely continuous patches of coastal heathland in to a matrix of small habitat patches within an area undergoing rapid urban expansion. This study aimed to document levels and patterns of genetic diversity and to define the population structure of C.tinnula populations within southeast Queensland, with the objective of defining possible conservation management units for this species. Results from 12S and COI mitochondrial markers clearly showed that two distinct evolutionary lineages of C.tinnula are present within southeast Queensland. The high level of divergence between lineages and strict geographic partitioning suggests long term isolation of C.tinnula populations. It is hypothesized that ancestral C.tinnula populations were once confined to wallum habitat refugia during the Pliocene resulting in phylogeographic delineation of 'northern' and 'southern' C.tinnula clades. Populations within each geographic region show evidence of range contraction and expansion, with subsequent restricted gene flow. Levels of genetic diversity appear, largely, to be the product of historical associations rather than contemporary gene flow. A revision of the current systematics of C.tinnula is required to ensure that discrete population groups are recognized as distinct evolutionary lineages and will therefore be protected accordingly.

Mitochondria are the main sites for adenosine triphosphate (ATP) generation within most cells. Structural and functional alterations of mitochondria due to genetic abnormalities of mitochondria can cause respiratory chain dysfunction. In this study, the important role of mitochondria in energy metabolism was determined by comparing the effect of mitochondrial DNA (mtDNA) mutations on growth patterns and oxidative phosphorylation (OXPHOS) enzyme activities of six isolated clones (B5, B12, D4, D9, E1 and E8); as well as the effect of ATP supplement to culture using the slowest growing clone. The isolated clones had shown distinct growth pattern and morphology. The difference in proliferation rates among the clones was ascertained by the doubling times (B5=26.4h. B12=43.2h. D4=25.7h. D9=33.6h. E1=26.9h and E8=28.8h). The clone's slow growth rate was likely the result of mitochondrial mutations in the 16S rRNA gene, ND1, ND4, ND6 and COX III. Five heteroplasmic mutations were found in clone B12 (G2480T, C2513G, A2520T, C9527T and C14263G), one heteroplasmic mutation in clone D9 (A4137G) and one homoplasmic mutation in clone D4 (C11496). The mutations in clone B12 appeared to be deleterious to the cell by disrupting mitochondrial OXPHOS activities and reducing energy output. Additionally, extracellular ATP supplement to OXPHOS deficient clone B12 facilitated cell growth and enhances the gene expression. Increased expression of mtDNA-encoded respiratory chain complexes observed in clone B12 compared to clone D4 may reflect mitochondrial genomic adaptation to perturbations in cellular energy requirements. The stimulation of mitochondrial biogenesis may be a cellular response in compensation for defects in OXPHOS associated with mtDNA mutations. My data support the hypothesis that the variability in functional manifestations of mtDNA is attributed to the nature of the mutation, number of mutation and the gene specifically affected. These results will help to further our understanding of the relationship between mitochondrial mutation and cellular function.

Thesis (Ph.D.)--McMaster University, 2006. / Supervisor: Jianping Xu). Includes bibliographical references.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.

Thesis (M.Sc.) - Carleton University, 2005. / Includes bibliographical references (p.103-107 ). Also available in electronic format on the Internet.

Thesis (M.S.)--Brigham Young University. Dept. of Integrative Biology, 2005. / Includes bibliographical references (leaves 21-27).

Honors Project--Smith College, Northampton, Mass., 2010. / Includes bibliographical references (p. 18-21, 54-60).