BACKGROUND:Next-generation DNA sequencing (NGS) technologies have made huge impacts in many fields of biological research, but especially in evolutionary biology. One area where NGS has shown potential is for high-throughput sequencing of complete mtDNA genomes (of humans and other animals). Despite the increasing use of NGS technologies and a better appreciation of their importance in answering biological questions, there remain significant obstacles to the successful implementation of NGS-based projects, especially for new users.RESULTS:Here we present an 'A to Z' protocol for obtaining complete human mitochondrial (mtDNA) genomes - from DNA extraction to consensus sequence. Although designed for use on humans, this protocol could also be used to sequence small, organellar genomes from other species, and also nuclear loci. This protocol includes DNA extraction, PCR amplification, fragmentation of PCR products, barcoding of fragments, sequencing using the 454 GS FLX platform, and a complete bioinformatics pipeline (primer removal, reference-based mapping, output of coverage plots and SNP calling).CONCLUSIONS:All steps in this protocol are designed to be straightforward to implement, especially for researchers who are undertaking next-generation sequencing for the first time. The molecular steps are scalable to large numbers (hundreds) of individuals and all steps post-DNA extraction can be carried out in 96-well plate format. Also, the protocol has been assembled so that individual 'modules' can be swapped out to suit available resources.

BACKGROUND: Uruguay exhibits one of the highest rates of breast cancer in Latin America, similar to those of developed nations, the reasons for which are not completely understood. In this study we investigated the effect that ancestral background has on breast cancer susceptibility among Uruguayan women. METHODS: We carried out a case-control study of 328 (164 cases, 164 controls) women enrolled in public hospitals and private clinics across the country. We estimated ancestral proportions using a panel of nuclear and mitochondrial ancestry informative markers (AIMs) and tested their association with breast cancer risk. RESULTS: Nuclear individual ancestry in cases was (mean ± SD) 9.8 ± 7.6% African, 13.2 ± 10.2% Native American and 77.1 ± 13.1% European, and in controls 9.1 ± 7.5% African, 14.7 ± 11.2% Native American and 76.2 ± 14.2% European. There was no evidence of a difference in nuclear or mitochondrial ancestry between cases and controls. However, European mitochondrial haplogroup H was associated with breast cancer (OR = 2.0; 95% CI 1.1, 3.5). CONCLUSIONS: We have not found evidence that overall genetic ancestry differs between breast cancer patients and controls in Uruguay but we detected an association of the disease with a European mitochondrial lineage, which warrants further investigation.

Thesis (PhD (Food Sc)--Stellenbosch University, 2011. / ENGLISH ABSTRACT: Consumers have the right to accurate information on the fish products they purchase to enable them to make educated seafood selections that will not endanger their own wellbeing or the wellbeing of the environment. Unfortunately, marine resource scarcity, financial incentives and inadequate or poorly enforced regulations have all promoted the mislabelling of fish species on global markets, the results of which may hold economic, conservation and health consequences. The primary aims of this study were to determine the most commonly available fish species on the South African market, to establish and compare DNA-based methods for the unambiguous identification of these species and to utilise the most applicable methods to evaluate the extent of mislabelling on the local fisheries market. The results from surveys of n = 215 restaurants and n = 200 retail outlets in four South African provinces (Western Cape, Kwa-Zulu Natal, Eastern Cape and Gauteng) indicated that 34 and 70 nominal fish types were available in restaurants and retail outlets, respectively, the most common of which were kingklip, salmon and hake. Over 30% of the fish species being sold were of conservation concern, while several outlets marketed specially-protected, illegal-to-sell species in South Africa. Fish purveyors were poorly equipped to provide information on the identity, origin, production method (farmed/wild) and sustainability of the fish they were selling and the labelling of many packaged fish products was in contravention with South African regulations. Data were published for the first time comparing the efficiency of five methods (urea-SDS-proteinase K, phenol-chloroform, salt extraction, SureFood PREP kit and Wizard Genomic DNA Purification kit) for the extraction of DNA from the muscle tissue of fish species available in South Africa. The SureFood kit was identified as the most suitable method for DNA extraction from fish muscle, extracting significantly (P < 0.05) higher DNA yields than all other methods evaluated and being simple and safe to use. A comprehensive reference library of genetic information was compiled for the first time that contains sufficient DNA sequence data from different mitochondrial DNA loci (16S ribosomal RNA (rRNA), 12S rRNA and cytochrome c oxidase I (COI) genes, as well as the control region) to allow the explicit identification of 53 fish species in South Africa. Although 16S and 12S rRNA gene sequencing allowed the identification of most fish to the genus level, the discrimination of closely-related, congeneric species was problematic when based on these gene regions. Conversely, the vast majority (98%) of fish examined could be readily differentiated by their COI sequences, with only members of the genus Thunnus requiring supplementary control region sequencing for species confirmation. Lastly, sequencing of the COI region was used to show that 9% of fish samples collected from local seafood wholesalers and 31% of samples from retail outlets were mislabelled. This study has established that fish mislabelling is a reality on the South African market and that DNA-based methods should be applied by both industry and regulatory bodies to deter illegal activities and to promote transparency on the domestic fisheries market. / AFRIKAANSE OPSOMMING: Verbruikers het die reg tot akkurate informasie rakende die visprodukte wat hulle aankoop. Hierdie inligting sal hulle bemagtig om ingeligte seekos keuses te maak wat voordelig sal wees vir beide die verbruiker se eie, sowel as die omgewing, se voortbestaan. Ongelukkig het 'n gebrek aan seelewebronne, geldelike aansporings en onvanpaste of swak geïmplimenteerde regulasies gelei tot die verkeerde etikettering van visspesies op die wêreldmarkte. Dit mag ekonomiese-, bewarings- en gesondheidsgevolge inhou. Die primêre doelwitte van hierdie studie was om te bepaal watter visspesies die algemeenste beskikbaar is in die Suid-Afrikaanse mark, om DNS-gebaseerde metodes vir die duidelike identifisering van hierdie spesies te vind en te vergelyk, en om die mees gepaste metodes te gebruik om die omvang van verkeerde etikettering in die plaaslike vismarkte te evalueer. Die resultate van opnames van n = 215 restaurante en n = 200 winkels in vier Suid-Afrikaanse provinsies (Wes-Kaap, Kwa-Zulu Natal, Oos-Kaap en Gauteng) het gewys dat 34 en 70 nominale visspesies in onderskeidelik restaurante en kleinhandelaars beskikbaar was. Koningklip, salm en stokvis was die mees algemene spesies. Meer as 30% van die visspesies wat te koop was is van bewaringsbelang, terwyl verskeie winkels spesiaal-beskermde, onwettig-om-te-verkoop spesies in Suid-Afrika bemark het. Visverkopers was swak bemagtig om informasie oor die identiteit, oorsprong, produksiemetode (teel/wild) en volhoubaarheid van die vis wat hulle verkoop het te kon gee. Verder was die etikettering van baie verpakte visprodukte in stryd met Suid-Afrikaanse regulasies. Vir die eerste keer is data gepubliseer wat vyf metodes (ureum-SDS-proteïenase K, fenolchloroform, sout-ekstraksie, SureFood PREP stel en Wizard Genomic DNS suiwering stel) vergelyk in hul doeltreffendheid om DNS vanuit die spierweefsel van visspesies wat in Suid-Afrika beskikbaar is te ekstraheer. Die SureFood stel is as die mees geskikte metode vir DNS ekstraksie vanuit visweefsel geïdentifiseer aangesien die DNS opbrengs betekenisvol (P < 0.05) hoër was met hierdie metode, en dit ook 'n eenvoudige en veilige metode is om te gebruik. 'n Omvattende verwysingsbiblioteek van genetiese informasie wat voldoende DNS volgordebepalingsdata van verskillende mitokondriale DNS lokusse (16S ribosomale RNS (rRNS), 12S rRNS en sitochroom c oksidase I (COI) gene, sowel as die kontrolegebiede) bevat, is vir die eerste keer opgestel om die besliste identifisering van 53 visspesies in Suid-Afrika toe te laat. Alhoewel 16S en 12S rRNS geenvolgordebepaling die identifisering van meeste visse op genusvlak toegelaat het, was die diskriminasie van naby-verwante, gelyksoorting spesies problematies wanneer hierdie geengebiede gebruik is. Die oorgrote meerderheid (98%) vis wat ondersoek is geredelik onderskei op grond van hul COI volgordebepalings, met slegs lede van die genus Thunnus wat addisionele kontrolegebied volgordebepaling vir spesies bevestiging vereis het. Laastens, is volgordebepaling van die COI-gebied gebruik om te wys dat 9% van die vismonsters van plaaslike seekosgroothandelaars en 31% van die monsters van kleinhandelaars verkeerd geëtiketteer is. Hierdie studie het bevestig dat die verkeerde etikettering van vis in Suid-Afrika 'n realiteit is, en dat DNS-gebaseerde metodes gebruik moet word deur die industrie sowel as die regulerende liggame om onwettige aktiwiteite teen te werk en om deursigtigheid in plaaslike vismarkte te bevorder.

The biology of South African Bryde’s whales (Balaenoptera brydei/edeni), with a focus on the inshore form, was investigated through estimates of abundance and survival rate, seasonality of occurrence and variation in mitochondrial and nuclear DNA. Photographs, sightings data and biopsy samples were collected in Plettenberg Bay, on the south-east coast of South Africa. Additional genetic material was obtained from the Iziko South African Museum, Marine and Coastal Management, and the Port Elizabeth Museum. Mark-recapture methods applied to photo-identification data were used to estimate abundance and survival rate. Estimates of abundance ranged from 130 to 250 (CV = 0.07 - 0.38) and the estimated annual survival rate was 0.93 (CV = 0.047, 95% CI = 0.852 - 1.0). Seasonal increases in the encounter rate and number of individual whales were observed during summer and autumn, with a peak in April, which corresponded to increased feeding activity and larger average aggregation sizes. Chlorophyll-a, sea surface temperature and wind speed were all significant factors in explaining the variability in the occurrence of whales. No seasonality in the occurrence of calves was detected. Mitochondrial DNA control region sequences (685bp) were compared to published sequences. This confirmed the offshore form as Balaenoptera brydei and the inshore form as closely related to B.brydei, possibly at the sub-specific level, but excluded it as B.edeni. Phylogenetic analyses support complete separation between the two forms. The use of 10 polymorphic microsatellite loci revealed no population structure among the inshore samples (FST = 0.006). Pairwise estimates of relatedness found most individuals to be unrelated, with only a few distant relatives detected.

The prototype uncoupling protein 1 (UCP1) mediates proton leak-dependent thermogenesis in mammals, but the physiological functions of the novel UCP2-5 are unclear. Nematodes only express one uncoupling protein that is most similar to UCP4 in the human brain, which is believed to be the most evolutionarily conserved of the uncoupling proteins. Consistent with reported UCP functions in mammals, we observed that ceUCP4-null nematodes had decreased metabolic rates and increased adiposity compared to wild type. Surprisingly, these phenotypes corresponded to decreased succinate-mediated mitochondrial respiration without apparent changes in mitochondrial uncoupling. ceUCP4-null mitochondria exhibited normal electron transport chain functions, but had a decreased capacity for succinate import. Supporting the functional importance of ceUCP4-dependent complex II regulation in vivo, ceUCP4 deficiency was demonstrated to result in a selectively lethal response to genetic and pharmacological inhibition of Complex I. Similarly, ceUCP4-deficiency significantly prolonged lifespan in the short-lived mev-1 mutant that generates deleterious complex II-derived reactive oxidants. These results define a new physiological function for the ancestral ceUCP4 in the regulation of complex II-mediated oxidative phosphorylation through an unexpected effect on mitochondrial succinate transport. The data described in this dissertation also describe a novel mechanism by which uncoupling proteins mediate mitochondrial bioenergetics. / text

Alzheimer's disease (AD) is a progressive, irreversible, neurodegenerative disease. The key to understanding AD is to elucidate the pathogenesis of neuron degeneration in specific brain regions.We hypothesize that there is increased DNA oxidation in AD brain compared to age-matched control subjects, especially in mitochondrial DNA (mtDNA), and that the changes in DNA bases will affect protein expression in mitochondria and contribute to neurodegeneration in AD. To test this hypothesis:1) We quantified multiple oxidized bases in nuclear DNA (nDNA) and mtDNA of frontal, parietal, and temporal lobes and cerebellum from late-stage AD (LAD), mild cognitive impairment (MCI), and age-matched control subjects using gas chromatography/mass spectrometry with selective ion monitoring (GC/MS-SIM). Also, we quantified oxidized DNA bases in cortex of APP/PS1 transgenic mice. (a) nDNA and mtDNA were extracted from eight LAD and eight control subjects. We found levels of multiple oxidized bases were significantly higher in frontal, parietal, and temporal lobes and that mtDNA had approximately 10-fold higher levels of oxidized bases than nDNA. Eight-hydroxyguanine was approximately 10-fold higher than other oxidized base adducts in both LAD and control subjects. These results suggest that oxidative damage to mtDNA may contribute to the neurodegeneration of AD. (b) Mild Cognitive Impairment (MCI), the phase between normal aging and early dementia, is a common problem in the elderly with many subjects going on to develop AD. Results from eight amnestic MCI and six control subjects suggest oxidative damage to DNA occurs in the earliest detectable phase of AD. (c) Analysis of nDNA from the cortex of four groups (3m, 6m, 9m, 12m) of APP/PS1 and wild type mice showed elevations of 8-hydroxyguanine in 12 month old APP/PS1 mice.2) To analyze mitochondrial protein changes in LAD, 2D gels were run to separate proteins and MALDI-TOF mass spectrometry was used to identify proteins.Five mitochondrial proteins were significantly decreased in LAD. This proteomic study provides a proteome map of mitochondria in LAD brain and an insight into the pathogenesis of neuron degeneration in Alzheimer's disease.

In the hours and days following acute CNS injury, a secondary wave of events is initiated that exacerbate spinal tissue damage and neuronal cell death. A potential mechanism driving these secondary events is opening of the mitochondrial permeability transition pore (mPTP) and subsequent release of several cell death proteins. Previous studies have shown that inhibition of cyclophilin D(CypD), the key regulating component in mPTP opening, was protective against insults that induce necrotic cell death. We therefore hypothesized that CypD-null mice would show improved functional and pathological outcomes following spinal cord injury (SCI) and traumatic brain injury (TBI). Moderate and severe spinal contusion was produced in wild-type (WT) and CypD-null mice at the T-10 level using the Infinite Horizon impactor. Changes in locomotor function were evaluated using the Basso Mouse Scale (BMS) at 3 days post-injury followed by weekly testing for 4 weeks. Histological assessment of tissue sparing and lesion volume was performed 4 weeks post SCI. Calpain activity, measured by calpain-mediated spectrin degradation, was assessed in moderate injury only by western blot 24 hours post SCI. Results showed that following moderate SCI, CypD-null mice had no significant improvement in locomotor recovery or tissue sparing compared to wild-type mice. Following severe SCI, CypD-null mice showed significantly lower locomotor recovery and decreased tissue sparing compared to WT mice. Calpain-mediated spectrin degradation was not significantly reduced in CypD-null mice compared to WT mice 24h post moderate SCI. The lack of protective effects in CypD-null mice suggests that more dominant mechanisms are involved in the pathology of SCI. In addition, CypD may have a pro survival role that is dependent on the severity of the spinal cord injury.

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.

The Leptonycteris yerbabuenae is found in southern Arizona, Mexico, Guatemala, and El Salvador. Some females are migratory, mating in southern Mexico, and migrating to maternity roosts in northern Mexico and southern Arizona to give birth. Twelve microsatellite loci markers and the Mitochondrial DNA Control Region (CR) were amplified to examine population structure and phylogenetic relationships among roosts. Twelve polymorphic microsatellite loci were isolated from L. yerbabuenae. A total of sixteen localities in AZ and Mexico were sampled. The mtDNA CR fragment resulted in 102 haplotypes. The phylogenetic analyses resulted in two clades, but no observable geographic structuring. The average FST value across all loci and all sampled localities was 0.022. Program STRUCTURE analyses indicate one population (K=1) throughout the sampling area. These results suggest movement between maternity colonies and transient roosts in Arizona, Sonora, and Chamela, Management recommendations based on these results would be to manage as a single population.

The phylo-geography and population genetics of three Crotalus species in Southern Arizona (C. atrox, C. cerastes, and C. scutulatus) were examined using mitochondrial DNA genes and nuclear microsatellite DNA markers. My focus was twofold: (1) the phylo-geography and population structure in Southern Arizona and (2) possible genetic signatures of population fragmentation by linear barriers on rattlesnakes populations at Picacho Peak. My results show genetic signatures of geneflow restrictions in one species (C. atrox) which coincide with Interstate 10. I did not observe similar genetic effects in C. cerastes or scutulatus, possibly caused by smaller sample sizes and marker numbers. I found limited phylo-geographic and population genetic structure for all three species in Southern Arizona indicating large interconnected populations. This study provides wildlife management with a powerful genetic toolset and provides important baseline data for future assessment and monitoring efforts of important predators and their populations in the Sonoran desert habitat.