The contribution of yeast strain background, specifically auxotrophic markers, to stability and fidelity of mtDNA replication was investigated. In summary, the ade2, his3delta200, and hap1 mutations have complex effects on mitochondrial functions, the severity of which appears to depend on other components in the genetic background of the strain. These results are important as many commonly used laboratory strains are related to the respiratory hampered S288c strain and are used for studies of orthologous human mutations associated with various mitochondrial diseases. These observations have added to our understanding of fungal mtDNA replication and have informed the mitochondrial community of problematic strains that need to be considered when using this model organism. The function of the yeast mitochondrial DNA polymerase (Mip1p) carboxyl-terminal extension (CTE) was investigated both in vivo and in vitro by genetically engineering various truncations of the CTE. The respiratory competence of mip1delta175 and mip1delta205 cells, in which Mip1p lacks the C-terminal 175 and 205 residues respectively, are indistinguishable from that of wild-type. In contrast, strains harbouring Mip1pdelta351, Mip1pdelta279, Mip1pdelta241, and Mip1pdelta222 rapidly lose mtDNA. At a low frequency, mip1delta216 cells grow poorly on glycerol. Fluorescence microscopy and Southern blot analysis revealed lower levels of mtDNA in these cells, and rapid loss of mtDNA during fermentative growth. Therefore, only the polymerase-proximal segment of the Mip1p CTE is necessary for mitochondrial function. To determine more precisely the defects associated with polymerase truncation variants, these proteins were overexpressed in yeast and used in a novel non-radioactive mtDNA polymerase assay. The threonine-661 and alanine-661 variants, shown by others to be responsible for the increased mtDNA mutability of various laboratory yeast strains at increased temperature, were examined in combination with CTE-truncations. These experiments suggest that exonuclease function is not effected in the alanine-661 variant at 37 degrees Celsius whereas polymerase activity is, and this higher relative level of exonuclease activity could be a contributing factor to mtDNA instability in S288c-related strains. Lastly, isogenic CTE truncation variants all have less DNA polymerase activity than their parental wild-type. Based on these results, several possible roles for the function of the CTE in mtDNA replication are suggested. / October 2008

As one of the major preys of many important economic fish species such as swordfish and dolphinfish in waters off estern Taiwan, flyingfish belongs to low-end consumers in the food chain with the function of maintaining the stability of the Kuroshio marine ecosystem. Hirundichthys oxycephalus is the primary component of flyingfish-egg fishery captures in the northeastern waters of Taiwan, and is also one of the dominant species of flyingfish in eastern waters of Taiwan. However, the significant drop of the flyingfish and flyingfish-egg catch from 2006 to 2007 and the effects on ecosystem and fishery caused major concern from the fishery sector and academic field. In order to manage this marine resource effectively, the phylogenetic relationships and population structure needed to be characterized first. In this study, the phylogenetic relationships of Hirundichthys oxycephalus of Northwestern Pacific was characterized based on the mitochondrial COI fragment. Totally 55 samples were collected between July, 2008 and November, 2010 in waters of Keelung, Ilan, Hualian, and Green Island. In addition, 12 more samples were obtained in Sebtember, 2009 from Tanegashima Island, and Yakushima Island of Japan. The DNA sequencing results of samples from Taiwan showed a total number of 29 haplotypes. The length of partial COI sequence was found to be 657 bp while the mean genetic distance was found to be 0.6%. In phylogenetic analyses, two major groups were identified in the phylogenetic trees by neighbor-joining and maximum-likelihood methods. The majority of "Keelung inshore group" came from Keelung and Ilan waters. The main population of "Kuroshio group" came from Green Island. The variation between two groups was found to be 61.75% by amova. The DNA sequencing results of samples from Japan showed a total number of 8 haplotypes. The length of partial COI sequences was found to be 657 bp with a mean genetic distance of 0.53%. In the phylogenetic tree, the samples from Japan were found to belong to "Kuroshio group". The variation between the two major groups was found to be 60% by amova. It was inferred that the differentiation of flyingfish into the two major groups in Taiwan was due to the flow pattern difference of Kuroshio in northeast waters of Taiwan. It was also inferred that phylogenetic similarity of the samples from Japan and the Kuroshio group was due to the distribution of both groups locating on the same path of the main current of Kuroshio. However, applying different distribution assumption may result in different conclusion such as one single stock hypothesis. Further studies will be needed to confirm the stock structure of the species.

The genus Capricornis Ogilby, 1837, is divided into three species and widely distributed in sourthern China, Tibet, Myanmar, IndoChinese peninsula, Malaysia peninsula, Sumatra, Japanese archipelagos and Taiwan. Using complete cytochrome b sequences (1140 bp) analyzes the genetic variation and phylogeny of genus Capricornis from Taiwan, Japan and Mainland China. Constructed by both distance and maximum parsimony methods, the phyloenetic tree distinguish the Capricornis to three clades: Formosan serow, Japanese serow, and Sumatran serow from mainland China. Formosan serow is more familiar with Sumatran serow than Japanese serow. Local populations of Formosan serow of Taiwan island and Japanese serow of the Japanese archipelagos are already differentiated. Serow and goral are apparently distinguishable. The results of Principal Component Analysis and Discriminant Analysis show that serows from Taiwan, Japan and mainland China and goral are apparently distinguishable at morphological characters. The variation of morphological analysis may be a good tool to identify serow and goral. From the paleogeology and fossil records of serow of Quaternary, we could infer that ancestors of serow from southwestern mountain of mainland China migrated to Taiwan island and Japanese archipelagos through the land bridge of east Asian islands to mainland China in the early Pleistocene caused by the glaciation of Quaternary, then separated from mainland of east Asia and speciation of serow occured in Taiwan island and Japanese archipelagos after the end of the glaciation of Quaternary.

Genetic analyses of two endangered species of mammals in the Lower Keys of Florida (Lower Keys marsh rabbit, LKMR, Sylvilagus palustris hefneri; silver rice rat, SRR, Oryzomys palustris natator) were performed to evaluate the genetic structure of their populations. Mitochondrial sequence data (control region; 763 base pairs (bp), LKMR; 788 bp, SRR) were used to explore patterns of genetic variation within and among island populations in both species. Analysis of the SRR also included 8 polymorphic nuclear microsatellite loci (9 to 16 alleles). Phylogenetic analyses of mitochondrial sequence data for both species revealed two main lineages corresponding to eastern and western localities, with high levels of genetic structuring (LKMR FST = 0.982, SRR ΦST = 0.916). The two species differed in the level of sequence divergence between eastern and western populations (LKMR, 19 bp; SRR 4 bp). In addition to an overall similar pattern of genetic subdivision, populations of both species possessed low levels of mtDNA variation (haplotypic diversity in the LKMR = 66.1%, SRR = 58.6%). Microsatellite analyses of the SRR revealed subdivision between eastern and western regions. Although less pronounced than the structure observed in mtDNA, the overall pattern was still apparent. Additional examination of divergence between mainland and Lower Keys rice rats revealed a genetic division that indicated a lack of recent gene exchange between the regions (i.e. no shared haplotypes, the presence of private alleles, and distinctive separation in numerous analyses). Although this degree of division does not warrant species designation, the levels and patterns of divergence, both morphological and genetic, do suggest genetic isolation of mainland and island forms. This fact, along with restricted gene flow between the Lower Keys and the Everglades, suggests that the SRR is on an evolutionary trajectory separate from its mainland counterparts and validates its identification as a separate subspecies, Oryzomys palustris natator. Finally, the genetic division between eastern and western populations of the SRR and LKMR suggests that populations of both species in these two regions of the Lower Keys should be treated as separate management units, especially when considering the enhancement of populations via translocations.

Thesis (Ph.D.)--East Carolina University, 2009. / Presented to the faculty of the Department of Exercise and Sports Science. Advisor: Joseph A. Houmard. Title from PDF t.p. (viewed Apr. 30, 2010). Includes bibliographical references.

published_or_final_version / abstract / Obstetrics and Gynaecology / Doctoral / Doctor of Philosophy

Mitochondrial DNA (mtDNA) was isolated from 8 different natural populations of the rodent species Otomys irroratus (Muridae: Otomyinae) and from one population of the species 0. angoniensis occurring in South Africa. MtDNA samples were cleaved with five different restriction endonucleases, end-labelled with phosphorous-32, separated by electrophoresis on horizontal 1 % agarose gels and the resulting fragment bands were detected by autoradiography. The individual-specific fragment banding patterns were analysed and compared among the various populations. The percent sequence divergence among and between the populations was calculated using the formula of Nei (1979). A matrix of sequence divergence values for all intergenomic pairwise comparisons was subjected to a clustering analysis by the unweighted pair group method with arithmetic means (UPGMA, Sneath and Sokal, 1973), using the computer programme NTSYS (Rohlf: 1988). The results of these analyses allowed for a preliminary identification of phenetic groupings in the data set. A matrix generated by scoring the restriction endonuclease fragments as present or absent was used to generate a phylogenetic dendogram using the BIOSYS (Swofford and Selander, 1989) programme. The overall restriction fragment variation uncovered in this study revealed 15 different mtDNA haplotypes within the 20 individuals examined. This corresponded to a high degree of polymorphism in the populations where more than one specimen was available, as well as within the species 0. irroratus. There were no clones that were shared between any of the populations sampled. The intrapopulation sequence divergence values uncovered in this study were high (range 0.35 % to 5.08 %), but also consistent with some other reports in the literature for intrapopulation variation. The outgroup, 0. angoniensis revealed the highest divergence values when compared to the mtDNA clones found in 0. irroratus. The phenetic and cladistic cluster diagrams revealed overall similarity with one another. There appeared to be little correlation between the topology of the mtDNA haplotype phenograms and the geographic distance of the sample localities. There was, however, a marked congruence between the distribution of mtDNA haplotypes and the distribution of three distinct cytotypes occurring over the species range. A possible polyphyletic evolution of populations of 0. irroratus was inferred from the cladistic analysis. / Thesis (M.Sc.)-University of Natal, 1994.

An interpopulation study was done on the rodent species Otamys irroratus (Muridae:Otomyinae) using restriction fragment length Polymorphisms to examine the mitochondrial DNA (mtDNA) of 30 vlei rats (Otamys irroratus) from three South African locations and 12 Angoni vlei rats (O.angoniensis) from two locations which were included as an outgroup. The three O.irroratus Populations originated from Karkloof and Kamberg in the Natal midlands and from Rietvlei in the Southern Transvaal. Mitochondrial DNA was extracted and purified by cesium-chloride/ethidium-bromide ultracentrifugation and digested with 19 class 11 restriction endonucleases. The fragments were end-labelled with 32P-dCTP, separated by electrophoresis on horizontal 1% agarose gels and the bands detected by autoradiography. The resultant individual-specific fragment patterns were analysed using the Restsite analysis program (v 1.1; Nei and Miller, 1990) to obtain a measure of the percent sequence divergences between and within the 3 POpulations of O.irroratus as well as between this species and the outgroup. The 19 endonucleases detected 19 distinct O.irroratus mtDNA maternal lineages and 3 O.angoniensis lineages. The O.irroratus lineages were clearly geographical ly structured and most closely reflected the Avise et al. (1987) category I (phylogenetic discontinuity with spatial separation). The only exception was a possible ancestral lineage represented by single individuals from Kamberg and Karkloof. Phylogenetic affinities between the most diverse lineages found at Kamberg and most Karkloof clones appear to be consistent with the finding of Pillay et al. (1993) and Contrafatto et al. (1992b) that Kamberg O.irroratus is an incipient sibling species of Karkloof O.irroratus. The mtDNA data indicates that the O.irroratus Populations at Karkloof and Kamberg last shared a common ancestor approximately 365 000 years ago. By contrast, O.angoniensis showed no evidence of geographic mtDNA structuring and is best described by the Avise et al. (1987) category Ill, which reflects phylogenetic continuity with spatial separation. These classifications must be regarded with caution given the limited distributional range of each species covered by this investigation. The interspecific mtDNA sequence divergence between O.irroratus and O.angoniensis of 11.57% substaniates morphological, karyotypic and allozymic evidence that these two sympatric species are also sibling species and they appear to have last shared a common ancestor between 1.2 and 2.4 million years ago. / Thesis (M.Sc.)-University of Natal, Durban, 1993.

The contribution of yeast strain background, specifically auxotrophic markers, to stability and fidelity of mtDNA replication was investigated. In summary, the ade2, his3delta200, and hap1 mutations have complex effects on mitochondrial functions, the severity of which appears to depend on other components in the genetic background of the strain. These results are important as many commonly used laboratory strains are related to the respiratory hampered S288c strain and are used for studies of orthologous human mutations associated with various mitochondrial diseases. These observations have added to our understanding of fungal mtDNA replication and have informed the mitochondrial community of problematic strains that need to be considered when using this model organism. The function of the yeast mitochondrial DNA polymerase (Mip1p) carboxyl-terminal extension (CTE) was investigated both in vivo and in vitro by genetically engineering various truncations of the CTE. The respiratory competence of mip1delta175 and mip1delta205 cells, in which Mip1p lacks the C-terminal 175 and 205 residues respectively, are indistinguishable from that of wild-type. In contrast, strains harbouring Mip1pdelta351, Mip1pdelta279, Mip1pdelta241, and Mip1pdelta222 rapidly lose mtDNA. At a low frequency, mip1delta216 cells grow poorly on glycerol. Fluorescence microscopy and Southern blot analysis revealed lower levels of mtDNA in these cells, and rapid loss of mtDNA during fermentative growth. Therefore, only the polymerase-proximal segment of the Mip1p CTE is necessary for mitochondrial function. To determine more precisely the defects associated with polymerase truncation variants, these proteins were overexpressed in yeast and used in a novel non-radioactive mtDNA polymerase assay. The threonine-661 and alanine-661 variants, shown by others to be responsible for the increased mtDNA mutability of various laboratory yeast strains at increased temperature, were examined in combination with CTE-truncations. These experiments suggest that exonuclease function is not effected in the alanine-661 variant at 37 degrees Celsius whereas polymerase activity is, and this higher relative level of exonuclease activity could be a contributing factor to mtDNA instability in S288c-related strains. Lastly, isogenic CTE truncation variants all have less DNA polymerase activity than their parental wild-type. Based on these results, several possible roles for the function of the CTE in mtDNA replication are suggested.

In order to identify organizational differences between sterile Polima (pol) and fertile Campestris (cam) mitochondrial genomes that could be linked to cytoplasmic male sterility (CMS), the physical map of the pol mitochondrial genome was constructed and compared to the physical map of the cam mitochondrial genome. The only structural differences between the two genomes are confined to a region encompassed by a 4.5 kb segment, present in pol mtDNA but absent in cam mtDNA. This 4.5 kb CMS-associated pol segment contains a chimeric gene called orf224 that is cotranscribed with atpG and comprises the single mtDNA region expressed differently in fertile, sterile and fertility restored plants which makes it a good candidate for specifying the sterility trait. Sequence analysis of the pol 4.5 kb segment has shown that orf224 was the only significant open reading frame (ORF) within the segment that gives rise to abundant transcripts, strengthening the view that the orf224/atp6 gene region is conferring pol male sterility. The pol 4.5 kb segment is also present and similarly organized in the common Brassica napus nap mtDNA but the sequences flanking the two segments are unrelated. Thus, the 4.5 kb segment appears to have transposed during the evolution of the pol and nap mitochondrial genomes and appears to have been lost in the cam mitochondrial genome. Sequence analysis of the nap segment revealed the presence of an ORF related to but divergent from orf224. This open reading frame (orf222) potentially encodes a protein of 222 amino-acids with 79% homology to the predicted product of orf224. orf222 is co-transcribed with the third exon of the trans-spliced gene, nad5, and another ORF of unknown function. Expression of the orf222 gene region is tightly associated with nap CMS since the levels of orf222 transcripts are significantly reduced upon restoration while the expression of 22 other mitochondrial genes do not consistently correlate with nap CMS. Antibodies were rai