Current medical genetics research is dominated by a single theory that supports the Human Genome Project rationale. This thesis investigates this and several alternative hypotheses and the ethical context related to their development. Firstly, the hypotheses are discussed in detail followed by a subsection in which research evidence based on each hypothesis is cited. Secondly, these medical genetics hypotheses are situated within the contemporary medical paradigm. To conclude, the thesis examines in depth the ethical and practical implications of medical genetics research. A framework of analysis of scientific responsibility is used to explore these implications. Scientific responsibility, as presented in this thesis, is a process consisting of three steps: (1) scientific discourse; (2) the development of the nature of scientific responsibility; and, (3) effective criticism. Once scientific responsibility is defined, the term is applied specifically to the field of medical genetics research.

<p> When challenged by difficult biological samples, the forensic analyst is far more likely to obtain useful data by sequencing the human mitochondrial DNA (mtDNA). Nextgeneration sequencing (NGS) technologies are currently being evaluated by the Forensic Science Program at Western Carolina University for their ability to reliably detect lowlevel variants in mixtures of mtDNA. The sequence profiles for twenty individuals were obtained by sequencing amplified DNA derived from the mitochondrial hypervariable (HV) regions using Sanger methods. Two-person mixtures were then constructed by mixing quantified templates, simulating heteroplasmy at discrete sites and in defined ratios. Libraries of unmixed samples, artificial mixtures, and instrument controls were prepared using Illumina^&reg; Nextera^&reg; XT and deep-sequenced on the Illumina^&reg;MiSeq&trade;. Analysis of NGS data using a novel bioinformatics pipeline indicated that minor variants could be detected at the 5, 2, 1, and 0.5% levels of detection. Additional experiments which examined the occurrence of sequence variation in hair tissue demonstrates that a considerable amount of sequence variation can exist between hairs and other tissues derived from a single donor. </p>

<p> The Archaea represent a fascinating domain of life where each species comprises a hybrid of bacterial and eukaryal features. Unfortunately, there have been few investigations of the Archaea and many fundamental questions remain regarding their biochemistry, genetics, and genomics. One reason for this is that few archaea are amenable to detailed experimental analysis; however, a few halophilic archaea (haloarchaea) can be easily manipulated in the laboratory and contain a sequenced genome, allowing for bioinformatics studies, such as recording changes in the transcriptomes. Haloarchaea are found wherever seawater is concentrated above 2 M NaCl and contain a similarly high concentration of salts internally, without producing compatible solutes. They exhibit a variety of novel molecular characteristics, including acidic proteins that resist the denaturing effects of salts, and DNA repair systems that minimize the deleterious effects of desiccation and intense solar radiation. In addition, haloarchaea are metabolically versatile and respond to a wide variety of environmental signals, including extremes of radiation, salinity, temperature, heavy metals, pollutants, and pH by modulating the activity of key genes. </p><p> Of the above naturally occurring stresses, all have been previously studied but one, pH. Haloarchaea are routinely isolated from both acidic and alkaline saline lakes; however, their ability to thrive under these conditions still remains an unexplored mystery. Therefore, I have endeavored to unlock a few of these secrets by growing sequenced haloarchaeal strains at extremes of pH and cataloging the changes in their transcriptomes compared to growth at optimal pH. For my experiments, RNA was isolated at the end of logarithmic growth and labeled cDNA was generated and hybridized to custom-designed microarray slides that I designed. Changes in the transcriptomes were cataloged and compared to each other as well as to changes from previously studied bacteria. The results from my experiments showed that haloarchaea are primarily responding to extremes of pH in a manner similar to known bacterial cells, making these responses similar across two domains of life. However, there are a few differences, compared to bacteria, due to the unique nature of the Archaea.</p>

Supportive breeding is currently being used in the management of threatened populations, but the effects of this management strategy remain relatively untested. The common house fly, Musca domestica, was used as a model to assess the relative effectiveness of supportive breeding strategies for endangered species management. Captive populations were subjected to one of two captive breeding treatments, and served as the source of migrants for "wild" populations, which received either 5% or 50% of their total census size from captive migrants each generation. One "wild" population treatment served as a control and received no migrants. Measures of fitness were assessed from each population for nine generations. Within the captive populations, analyses found highly significant declines in fitness across generations, with much higher total fitness in the equalized breeding strategy as compared to the structured breeding scheme. Captive population fitness did not prove to be a good predictor of "wild" population fitness after reintroduction, but the inbreeding co-efficient of the captive populations were found to be correlated with recipient "wild" population fitness. This suggests that captive breeding programs with goals of future reintroduction to the wild should focus on overall kinship as opposed to fitness levels and make every attempt to minimize the co-efficient of inbreeding within the captive population. "Wild" populations that received low levels of ideally bred captive migrants performed significantly better than control populations, which received no outside migration. No other "wild" population treatments were found to differ significantly from the control populations, which may be due in part to the small population sizes that limited the effects of selection and migration and strengthened the influence of factors such as genetic drift and inbreeding. It is likely that the constraints of small population size led to the control populations having the lowest overall fitness. Based on this study, it does not appear that sweeping generalizations can be made about the effects of migrant breeding treatment and level of migration on wild populations. Further research is warranted before supportive breeding programs are implemented for a broader range of threatened and endangered species.

As sessile organisms, plants have developed the ability to synthesize an impressive array of metabolites to meet environmental challenges. Terpenes constitute the largest group of plant natural products, yet the biological functions of most of these compounds remain unknown. One class of terpenes, the triterpenoids, are 30-carbon compounds derived from squalene that include hormones, membrane sterols, and antifungal compounds among others. Indeed, more than 100 triterpene skeletons have been identified in plants (Xu et al., 2004). Triterpenoid biosynthesis occurs through a sequence of enzymatic reactions beginning with the conversion of squalene into oxidosqualene by squalene epoxidase (SQE) enzymes. This work focuses on characterizing the squalene epoxidase family in the plant Arabidopsis thaliana. I used biochemical and molecular genetic approaches to characterize the Arabidopsis SQE family. Six putative squalene epoxidase encoding genes were identified in the Arabidopsis genome. Using heterologous expression in yeast and chemical analysis, I demonstrated that three members of the SQE family (SQE1, SQE2, and SQE3) have squalene epoxidase activity. In contrast, the other members of the SQE family (SQE4, SQE5, and SQE6) lacked SQE activity in yeast and may have adopted unique substrate preferences. I used molecular genetic approaches to survey SQE expression in plants and characterize mutations in each of the Arabidopsis SQE genes. SQE1 and SQE3 are expressed in most plant tissues throughout development, whereas expression other SQE genes is more restricted. I demonstrated that SQE1 is essential for normal plant development. sqe1-3 and sqe1-4 mutants are dwarf plants with root, stem, and hypocotyl elongation defects. In addition, sqe1-3 and sqe1-4 mutants produce inviable seed. Chemical analysis demonstrated that sqe1-3 roots and aerial tissues have &sim;10-fold increase in squalene, suggesting the triterpene pathway is blocked in this mutant. Together, this work demonstrates that members of the Arabidopsis SQE gene family have unique and overlapping roles in plant growth and development, and suggests that diversity of function at the squalene epoxidase step may contribute to triterpenoid diversity in plants.

Dictyostelium discoideum is a eukaryotic micro-organism with a unique life cycle. The amoebae live as haploid, free-living cells in the soil feeding on bacteria and dividing asexually. Under starvation conditions, the cells aggregate and undergo a process of differentiation into spores and stalk cells. We speculate that the stalk cells are sacrificed to help raise the spores above the substrate and to improve their dispersal and survival. In the case of a mix between two or more genetically different clones, a conflict may arise over which cells become spores and survive and which become stalk and die. One that differentiates more spores than its fair share in chimera is called a "cheater" and the other a "loser". Dictyostelium discoideum is a useful organism for studying the complexity of social behavior in microorganisms. Molecular tools have been developed allowing the study of genetic mechanisms that underlie this social behavior. To investigate the molecular basis of cooperation, several pools of knock-out mutants were generated using the REMI (R&barbelow;estriction E&barbelow;nzyme M&barbelow;ediated I&barbelow;ntegration) technique. To simulate evolutionary selection for cheaters, the different mutants were subjected to rounds of spore germination, growth and development in a mixed population. Only the spores were taken to the next generation. Real Time PCR confirmed that cheaters became over-represented in the evolving population because they contribute spores with a higher efficiency than the other mutants. Mutants expressing a normal phenotype were picked and isolated after 10 and 20 cycles of selection and mixed in pairwise experiments with the parental wild type. At least 35 mutants have been tested and 29 were cheaters. Analysis of the isolated genes suggested that several genetic pathways are involved in regulating or modulating the complex cooperation process in Dictyostelium discoideum. Finally we characterized one cheater mutant, called chtB, which shows apparently normal phenotype when plated clonally. The mutant is lacking in the expression of the gene chtB. In chimeras, this causes the reduction of the expression of the prespore marker cotB in the wild type strain, enabling the cheater to differentiate more spores than the parental strain.

Genetic linkage analysis entails estimating the distance between two genes on a chromosome using genotype information from a sample of individuals. For human pedigree data counting the number of meiotic crossovers or recombination events is impossible due to the lack of complete information. Consequently maximum likelihood methods are used to estimate the recombination frequency in these cases. Since the advent of high resolution genetic maps, errors in genetic linkage data have become more of a problem. Errors can introduce spurious recombinations which increase the map distance and distort linkage maps reducing the power to locate genetic diseases. A general method for detecting errors in pedigree genotype data is presented. Its performance is evaluated with power studies using Monte Carlo methods on simulated data with pedigree structures similar to the CEPH pedigrees and a larger disease pedigree used in the study of idiopathic dilated cardiomyopathy. An investigation of the effect that errors have on the power of locating a disease gene in a proposed linkage study is also presented. The study's results can be used to plan linkage studies which account for error thereby increasing their probability of success. The error detection method and power study results are important tools for performing linkage studies now and in the future which require high resolution maps.

The Drosophila maternal effect mutant, bicaudal (bic), affects the anterior-posterior axis of the embryo. It is incompletely penetrant, producing a variety of lethal embryonic phenotypes, the most severe of which is replacement of the anterior half with a mirror image duplication of the posterior half. Genetic mapping has placed this locus within a region defined by the overlap of two chromosomal deficiencies on the second chromosome. Indirect genetic evidence has suggested that a lethal mutation that maps to the same region, $vr22\sp{P3}$ may represent a more severe allele of the bicaudal locus. A chromosome walk was performed in this region and over 65 kb of overlapping clones isolated. The location of the P-element insertion responsible for the $vr22\sp{P3}$ mutation was localized in the walk, and cDNAs corresponding to two adjacent transcription units isolated. These clones have been analyzed and sequenced, and one shows at least a 65% identity with eukaryotic release factor. Recombination work with bicaudal mutant stocks has improved penetrance of the mutation to a level allowing genetic analysis. These stocks were used in genetic rescue experiments to determine the locations of bic and $vr22\sp{P3}.$ The results of genetic rescue and polymorphism mapping provide good evidence that $vr22\sp{P3}$ and bicaudal are in fact alleles at the same locus, and the gene codes for a protein with strong homology to release factor. The bic protein most likely plays a role in negative regulation of translation of downstream maternal genes such as nanos.

Shutoff of host biosynthesis is one of the earliest and most dramatic events occurring in viral infection and requires the expression of viral early genes. To understand the mechanisms of bacteriophage SPO1 induced host-shutoff, two SPO1 early genes, e3 and e22, were cloned and sequenced, and the roles of e3 in host-shutoff and in phage growth were studied. Both e3 and e22 are novel genes, and are actively expressed during the first few minutes of infection before being promptly shut off. Expression of a plasmid-borne e3 gene, in either B. subtilis or E. coli, caused the inhibition of host DNA, RNA and protein synthesis, and ultimately led to cell death. To identify the primary target of e3-induced shutoff, an e3-resistant E. coli mutant was isolated and analyzed. Plasmid libraries of this mutant's genomic DNA, when screened for genes that could protect wild-type E. coli against e3, yielded the rpoB and dksA genes, which specify the RNA polymerase $\beta$ subunit and a suppressor for DnaK, respectively. The wild-type dksA gene, but not the wild-type rpoB gene, was able to protect against e3, suggesting that the primary e3-resistant mutation was in the rpoB gene and that protection by the dksA gene depended upon overexpression from the plasmid. I suggest that e3 acts by distorting the structure of the host RNA polymerase, thus preventing host transcription, and that this distortion can be prevented or reversed by a chaperonin-like activity specified by dksA. The host-shutoff still occurred normally during infection by an SPO1 mutant which lacked e3 activity, and it occurred much more rapidly than that caused by expression of e3 in uninfected cells. Thus, the e3 product must be only one component of the host-shutoff machinery, which must include elements whose function is redundant to that of e3. At high multiplicities of infection, the mutant SPO1 produced more phage progeny than the wild-type SPO1, suggesting that high concentrations of e3 can be inhibitory to phage growth as well as to host function. Perhaps for that reason, expression of both e3 and e22 is shut off after a brief period of high activity.

In order for cells to grow and survive, they must be able to overcome changes in their external environment. For example, stresses such as high or low temperatures and nutrient deprivation have been shown to elicit a response pathway which eventually results in the transcriptional or translational regulation of various genes. This study focuses on the ability of yeast cells to respond to and recover from the environmental stress of a change in external osmolarity. Because of its unicellular nature, it is vital that the sudden changes in internal turgor pressure caused by osmotic stress are compensated for, usually through the accumulation of glycerol inside the cell. Recently, it was found that strains with mutations in the single essential yeast actin gene, ACT1, were not able to grow at high temperatures or high osmolarity. This phenotype suggested that an underlying physiological process affecting cytoskeletal actin was involved in the response to osmotic stress. In order to understand this process, wild type yeast cells were analyzed at the morphological, genetic and molecular level. Results from this characterization led to the hypothesis that the response of actin to osmotic stress is regulated by interactions with other actin binding proteins. Potential candidates for these interactions were discovered by generating second site suppressors of an actin mutant and analysing them genetically. A dominant suppressor, RAH3, was found with four alleles, each having different phenotypes. Cloning of the recessive single mutant revealed that the gene product was the yeast homolog to fimbrin, a protein thought to regulate stress responses. Sequence analysis of the different alleles indicates that several sites dispersed throughout the fimbrin gene are important in regulating the cytoskeletal response to osmotic stress.