Genetics - Theses
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ItemA genetic investigation of congenital defects in alpacas( 2013)The aim of this PhD project was to understand the genetic mechanisms contributing to congenital defects in alpacas. Alpaca veterinarians report a prevalence of congenital defects much higher than any other livestock species. A reduction in genetic diversity due to mating between closely related individuals can cause congenital defects. In this study, inbreeding coefficients estimated from genomic data were compared between individuals with congenital defects and healthy individuals. Australian alpacas with congenital defects did not show significantly higher levels of inbreeding than alpacas without diagnosed defects. Therefore, high levels of inbreeding cannot explain the prevalence of congenital defects in Australian alpacas. One common congenital defect is the blue-eyed white phenotype which is characterized by solid white fleece, two blue eyes and often deafness. A genetic investigation of the blue-eyed white phenotype and the mapping of this trait form the second aim of this project. Case-control association analyses were performed and KIT was identified as the gene likely to be responsible for this trait. Two haplotypes were present in BEW (blue-eyed white) individuals and this suggested that two mutations contribute to this phenotype. Next-generation sequencing was used to identify possible causative mutations. Single nucleotide polymorphism analysis was used to refine the region which contains the mutations responsible for this trait and to examine the linkage disequilibrium in this region. The experimental results from this thesis were used to formulate a model for the genetic inheritance of the BEW phenotype in alpacas. The genetic markers examined in the study have the potential to provide a useful genetic test for breeders who view the BEW phenotype as a congenital defect which should be culled from the population. Together the aspects of this project aim to provide breeders with information on the genetic diversity of Australian alpaca stock as well as provide a genetic tool to enable the elimination of a deleterious phenotypic trait.
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ItemSelection and characterisation of Arabidopsis mutants resistant to a glutathione biosynthesis inhibitor( 2003)This thesis describes the investigation of GSH regulation and signaling in Arabidopsis thaliana by a genetic approach. A procedure is described to select for mutants resistant to a chemical inhibitor of glutathione (GSH) biosynthesis, L-buthionine-SR-sulfoximine (BSO). Fifteen mutants were selected and preliminary genetic examinations indicated that BSO-resistance phenotypes are often highly variable. Consequently five mutants were chosen for further genetic, molecular and biochemical characterisation. All five of these mutants were shown to have altered GSH levels. Moreover, root growth among these mutants varied indicating that different mechanisms may be conferring BSO-resistance. Molecular mapping techniques were used to assign the BSO-resistance loci of four of the mutants to chromosome regions. Two mutants (a T-DNA insertion mutant and an EMS-derived mutant) were found to map to the same position between the markers CHS1 and R89998 on chromosome 5. Subsequently, complementation tests confirmed that these mutants define a single locus. The mutated locus in the T-DNA insertion mutant was determined using molecular techniques. The gene that contained the insert encodes a putative transport protein and was designated the BSO Resistance Transporter1 (BRT1). No transcript was detected in the T-DNA insertion mutant (brt1-1) which suggests that brt1-1 is likely to be a null allele. The EMS-derived mutant (brt1-2) contained an intron splice-site consensus sequence mutation which resulted in transcripts that included an unspliced intron. The function of the BRT1 gene was investigated by phenotype testing and despite the brt1 mutants having 2-fold GSH levels they were not resistant to known oxidising agents. Northern analysis of GCS and the use of a GCS-GUS reporter line indicated that the increase in GSH levels in these mutants was not accompanied by increases in GCS transcription and translation. This suggests that BRT1 is a novel component influencing GSH metabolism. BRT1 defines a novel family of three genes in Arabidopsis. Preliminary experiments indicate that these three genes are expressed in the same tissues providing for possible a level of redundancy between them. Supporting this is a newly identified insertion mutant in a second BRT gene (brt2-1) which is also resistant to BS0. Moreover, the double mutant, brt1-1;brt2-1, exhibits increased BSO-resistance suggesting an additive effect of the mutations. Database searches revealed that there were homologues in other organisms including one from the malaria parasite (PfCRT). Mutations in PfCRT confer resistance to a widely used antimalarial, chloroquine (CQ). However the function of PfCRT is not known. The BRT1 mutant was shown to have a level of functional conservation with a malaria parasite homologue, PfCRT, and therefore further characterisation of these proteins may provide insights into GSH metabolism and perhaps CQ-resistance in the malaria parasite.
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ItemThe regulation and function of the Drosophila melanogaster Cytochrome P450 gene, Cyp12d1( 2011)Cytochrome P450s are an important family of monooxygenase enzymes implicated in numerous xenobiotic detoxification events as well as in essential endogenous functions. The vinegar fly Drosophila melanogaster has 85 P450 genes; however, the large majority of them remain uncharacterised in terms of their function and regulation. Cyp12d1 is arguably the most xenobiotic inducible P450 gene in the D. melanogaster genome. It has been suggested that Cyp12d1 is an excellent candidate gene to study Drosophila xenobiotic induction pathways as it responds to a wide range of chemical inducers, indicating that it contains most if not all of the cis-regulatory elements needed for xenobiotic induction in Drosophila. Hence, the transcriptional regulation of Cyp12d1 was investigated to identify novel P450 induction pathways in D. melanogaster. Cyp12d1 basal transcriptional regulators were found in Cyp12d1 upstream and downstream regulatory regions, while enhancers for Phenobarbital and caffeine induction were located upstream. Site-directed mutagenesis experiments identified GATA family transcription factors as important Cyp12d1 midgut expression regulatory proteins. However, their role in xenobiotic induction remains unclear. Biochemical sequencing of electomobility-shift assay protein bands, and genetic RNAi screens of genes encoding other candidate transcription factors, failed to identify any other potential xenobiotic regulatory proteins. Cyp12d1 function was also investigated in this study. Cyp12d1 overexpression has been shown to confer resistance to the insecticides DDT and dicyclanil, but other functions have not been identified prior to this study. Adult Cyp12d1 functions were investigated through Cyp12d1 RNAi and overexpression studies. Cyp12d1 was found to be involved in adult longevity and oxidative stress resistance, suggesting other potential functions in addition to known detoxification functions. Cyp12d1 has been tandemly duplicated in D. melanogaster, and this duplication exists as a polymorphism in field populations. The geographical distribution of the Cyp12d1 duplication was examined in flies collected along the eastern coastline of Australia. The frequency of the duplicated Cyp12d1 gene was found to vary spatially, with flies in lower latitudes being more likely to possess the Cyp12d1 duplication and flies in higher latitudes being less likely. Cyp12d1 tissue-specific embryonic expression and mRNA transcript length was different in Cyp12d1-duplicated lines when compared to non-Cyp12d1 duplicated lines. These results indicate the Cyp12d1 duplication confers changes in Cyp12d1 expression patterns and suggest that Cyp12d1 may be involved in local adaptation to the microenvironment.