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v, 100 leaves : ill., graphs, tables ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.1976) from the Dept. of Genetics, University of Adelaide

Thesis (Ph.D. 1976) from the Department of Genetics, University of Adelaide.

Barramundi (Lates calcarifer) is a centropomid teleost with a wide distribution across the Indo Pacific. In Australia, barramundi are native to the tropical zone from Exmouth Gulf in Western Australia, across the northern part of the continent, to the Mary River in Queensland. Barramundi are protandrous hermaphrodites, and are euryhaline, with a catadromous life history. Barramundi are a valuable Australian resource, with important commercial and recreational fisheries and aquaculture production to the value of $11 million dollars per year. Recent declines in the availability of the fish in some rivers has led to an interest in the possibility of restocking rivers with barramundi from other areas. Determining the genetic structure of barramundi populations in Australia is important for understanding biogeographic history, and appropriate management practices for both aquaculture and recreational and commercial fishing. Previous studies have concentrated on the east coast of Australia, and have largely ignored the western populations. In this study, I obtained DNA data from barramundi populations across the Australian range of the species, as well as populations from Papua New Guinea and Indonesia. The aims of this study were to use the genetic data to determine: 1. if populations in Western Australia show genetic differences between geographic regions 2. if these populations show an ancestral split from populations in the east of Australia and 3. the ancestral origins of Australian barramundi. Previous studies of DNA data from barramundi have discovered an east/west split occurring at the Torres Strait that was assumed to be caused by the closing of the strait during lowered sea levels. However, these studies suffered from a bias in sampling area, concentrating either on the eastern half of the range of barramundi, or on the western tip of the range. Data from these studies were combined and reanalyzed. Two major clades were discovered, with considerable biogeographic structuring, but their geographic locations did not coincide with the reported vicariance event at the Torres Strait. Instead, historical divisions among freshwater drainage systems appeared to have driven the evolutionary history of barramundi in Australia. In order to investigate these historical divisions further, a 290 bp section of the mitochondrial DNA control region was sequenced in 284 barramundi from seven populations across the Australian geographic range of the species and from one population in Papua New Guinea and one population in Indonesia. Analyses of molecular variance within and among populations showed significant geographic structuring, based on biogeographical provinces and drainage divisions. Nested clade analyses indicated that these geographical associations were the result of restricted gene flow, range expansion, and past fragmentation events. I hypothesise that the Ord River area in the west of the continent was the ancestral source population for the rest of the species’ range across Australia, with Indonesia being the most likely origin of this source. Populations of barramundi from the Pilbara region are genetically distinct and geographically isolated, with strong evidence of an ancestral divide along geographical barriers to dispersal. There is a strong association between Papua New Guinea and Australia, although further investigations using the cytochrome b region of mitochondrial DNA indicated a more ancestral divide between the two than is currently evident, which could reflect an ancient geographical divide between the two, or could be evidence of a secondary migration route to Australia. For a more detailed study of evolutionary processes acting on populations of barramundi in Western Australia, allelic diversity was examined at five microsatellite loci. All loci were polymorphic and genotypic frequencies conformed to Hardy-Weinberg expectations, with no significant linkage between loci evident in any population. Measures of within population diversity were significantly related to latitude, suggesting southerly migration from a northern source population. The Ord River was the most genetically diverse population, and the most likely ancestral migration source to the area, with diversity decreasing down the west coast. Although there were significant differences among populations, the nuclear microsatellite data do not indicate the same degree of genetic structuring as is evident in the mitochondrial data. This may be a consequence of rapid evolutionary change at microsatellite loci, with past separations or population differences masked by recombination and back mutation of the microsatellite alleles. However, the nature of nuclear and mitochondrial inheritance may also indicate life history differences between the sexes, where significant genetic contribution to gene flow by males and limited female gene flow may lead to preservation of maternally inherited population substructure. The principal findings from this study are: • There is no genetic evidence for an east/west division of barramundi populations in Australia, as suggested by previous research. • Despite barramundi’s catadromous life history, and ability to disperse through marine waters, the present genetic structure indicates a division principally among river drainages. From a population genetic viewpoint, the species can be regarded as freshwater, rather than marine. • The most likely origin of barramundi in Australia is the Ord River region, with Indonesia as the route of migration. • Differences in the population structure demonstrated by nuclear and mitochondrial data indicate possible life history differences between the sexes. • Barramundi populations in different biogeographical provinces may have been substantially isolated over a long period of time, and may therefore represent independently evolving populations. This has important implications for fishery management and translocation issues for restocking rivers.

Diss. (sammanfattning). Uppsala : Sveriges lantbruksuniv. / Härtill 4 uppsatser.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniv., 2009. / Härtill 4 uppsatser.

Mitochondria are essential cellular organelles and the location of the electron transport chain, the site of the majority of energy production in the cell. Mitochondria contain their own circular genome approximately 16,000 base pairs in length. The mitochondrial genome (mtDNA) encodes 11 protein-coding genes essential for the electron transport chain, 22 tRNA genes, and two rRNA genes. Mitochondrial malfunction occurs in many diseases, and changes in the mitochondrial genome lead to numerous disorders. Multiple mitochondrial haplotypes and sequence features are associated with Alzheimer's disease. In this dissertation we utilized TreeScanning, an evolutionary-based haplotype approach to identify haplotypes and sequence variation associated with specific phenotypes: Alzheimer's disease case-control status, mitochondrial copy number, and 16 neuroimaging phenotypes related to Alzheimer's disease neurodegeneration. In the first two studies we utilized 1007 complete mitochondrial genomes from participants in the Cache County Study on Memory Health and Aging. First, individuals with mitochondrial haplotypes H6A1A and H6A1B showed a reduced risk of AD. Our study is the largest to date and the only study with complete mtDNA genome sequence data. Next, each cell contains multiple mitochondria, and each mitochondrion contains multiple copies of its own circular genome. The ratio of mitochondrial genomes to nuclear genomes is referred to as mitochondrial copy number. Decreases in mitochondrial copy number are known to occur in many tissues as people age, and in certain diseases. Three variants belonging to mitochondrial haplogroups U5A1 and T2 were significantly associated with higher mitochondrial copy number in our dataset. Each of these three variants was associated with higher mitochondrial copy number and we suggest several hypotheses for how these variants influence mitochondrial copy number by interacting with known regulators of mitochondrial copy number. Our results are the first to report sequence variation in the mitochondrial genome that lead to changes in mitochondrial copy number. The identification of these variants that increase mtDNA copy number has important implications in understanding the pathological processes that underlie these phenotypes. Lastly, we used an endophenotype-based approach to further characterize mitochondrial genetic variation and its relationship to risk markers for Alzheimer's disease. We analyzed longitudinal data from non-demented, mild cognitive impairment, and late onset Alzheimer's disease participants in the Alzheimer's Disease Neuroimaging Initiative with genetic, brain imaging, and behavioral data. Four clades were associated with three different endophenotypes: whole brain volume, percent change in temporal pole thickness, and left hippocampal atrophy over two years. This was the first study of its kind to identify mitochondrial variation associated with brain imaging endophenotypes of Alzheimer's disease. Together, these projects provide evidence of mtDNA involvement in the risk and physiological changes of Alzheimer's disease.

No / Evolutionary biologists are increasingly relying on ancient DNA from archaeological animal bones to study processes such as domestication and population dispersals. As many animal bones found on archaeological sites are likely to have been cooked, the potential for DNA preservation must be carefully considered to maximise the chance of amplification success. Here, we assess the preservation of mitochondrial DNA in a medieval cattle bone assemblage from Coppergate, York, UK. These bones have variable degrees of thermal alterations to bone collagen fibrils, indicative of cooking. Our results show that DNA preservation is not reliant on the presence of intact collagen fibrils. In fact, a greater number of template molecules could be extracted from bones with damaged collagen. We conclude that moderate heating of bone may enhance the retention of DNA fragments. Our results also indicate that ancient DNA preservation is highly variable, even within a relatively recent assemblage from contexts conducive to organic preservation, and that diagenetic parameters based on protein diagenesis are not always useful for predicting ancient DNA survival.

Maintenance of genomic integrity and stability is of prime importance for the survival of an organism. Upon exposure to different damaging agents, DNA acquires various lesions such as base modifications, single-strand breaks (SSBs), and double-strand breaks (DSBs). Organisms have evolved specific repair pathways in order to efficiently correct such DNA damages. Among various types of DNA damages, DSBs are the most serious when present inside cells. Unrepaired or misrepaired DSBs account for some of the genetic instabilities that lead to secondary chromosomal rearrangements, such as deletions, inversions, and translocations and consequently to cancer predisposition. Nonhomologous DNA end joining (NHEJ) is one of the major DSB repair pathways in higher organisms. Mitochondrial DNA (mtDNA) deletions identified in humans are flanked by short directly-repeated sequences, however, the mechanism by which these deletions arise are unknown. mtDNA deletions are associated with various types of mitochondrial disorders related to cancer, aging, diabetes, deafness, neurodegenerative disorders, sporadic and inherited diseases. Compared to nuclear DNA (nDNA), mtDNA is highly exposed to oxidative stress due to its proximity to the respiratory chain and the lack of protective histones. DSBs generated by reactive oxygen species, replication stalling or radiation represents a highly dangerous form of damage to both nDNA and mtDNA. However, the repair of DSBs in mitochondria and the proteins involved in this repair are still elusive. Animals deficient for any one of the known Classical-NHEJ factors are immunodeficient. However, DSB repair (DSBR) is not eliminated entirely in these animals suggesting evidence of alternative mechanism, ‘alternative NHEJ’ (A-NHEJ/A-EJ). Several lines of evidence also suggest that alternative and less well-defined backup NHEJ (B-NHEJ) pathways play an important role in physiological and pathological DSBR. We studied NHEJ in different tissue mitochondrial protein extracts using oligomeric DNA substrates which mimics various endogenous DSBs. Results showed A-EJ, as the predominant pathway in mitochondria. Interestingly, immunoprecipitation (IP) studies and specific inhibitor assays suggested, mitochondrial end joining (EJ) was dependent on A-EJ proteins and independent of C-NHEJ proteins. Further, colocalization studies showed A-EJ proteins localize into mitochondria in HeLa cells. More importantly knockdown experiments showed the involvement of DNA LIGASE III in mitochondrial A-EJ. These observations highlight the central role of A-EJ in maintenance of the mammalian mitochondrial genome. By using oligomeric DNA substrates mimicking various endogenous DSBs, NHEJ in different cancer cell lines were studied. We found that the efficiency of NHEJ varies among cancer cells; however, there was no remarkable difference in the mechanism and expression of NHEJ proteins. Interestingly, cancer cells with lower levels of BCL2 possessed efficient NHEJ and vice versa. Removal of BCL2 by immunoprecipitation and protein fractionation using size exclusion column chromatography showed elevated levels of EJ. Most importantly, the overexpression of BCL2 in vivo or the addition of purified BCL2 in vitro led to the downregulation of NHEJ in cancer cells. Further, we found that BCL2 interacts with KU proteins both in vitro and in vivo using immunoprecipitation and immunofluorescence, respectively. Hence, NHEJ in cancer cells is negatively regulated by the anti-apoptotic protein, BCL2, and this may contribute towards increased chromosomal abnormalities in cancer. In summary, our study showed that the efficiency of EJ in cancers could be regulated by the antiapoptotic protein BCL2. However, it may not affect the mechanistic aspect of EJ. BCL2 instead may interfere with EJ by sequestering KU and preventing it from binding to DNA ends. This may help in better understanding towards increased chromosomal abnormalities in cancer. Study of mitochondrial DSBR in mammalian system highlights the central role of microhomology-mediated A-EJ in the maintenance of the mammalian mitochondrial genome and this knowledge will helpful for the development of future therapeutic strategies against variety of mitochondria associated diseases.