Genetics - Theses
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ItemCharacterisation of genes involved in L-Arabinose metabolism in Arabidopsis(University of Melbourne, 2003)Glycosyl hydrolases are important mediators of plant cell wall modification during plant development. These enzymes catalyse the hydrolytic release of specific sugars, such as L-arabinose, from the polysaccharide-rich cell wall matrix. Here, the cloning and expression analysis of two genes, AtASD1 and AtASD1, encoding putative ?-L-arabinofuranosidases, is reported in Arabidopsis thaliana. AtASD1 and AtASD2 identities were assigned on the basis of homology to plant and microbial family 51 glycoside hydrolases. Despite numerous attempts, no enzyme activity was demonstrated for either protein. Over-expression of AtASD1 in the cell wall mutant, mur1, resulted in a genetic interaction that caused extreme dwarfism in plants. An AtASD2 T-DNA knockout mutant presented no visible phenotypes. Using the experimental approaches of RT-PCR, RNA gel blot analysis and GUS reporter gene expression analysis, AtASD1 and AtASD2 were shown to have different developmental expression profiles. High levels of AtASD1 promoter activity are present in multiple tissues during vegetative and reproductive growth. In comparison, AtASD2 expression is limited to the vasculature of older root tissue and to some floral organs and floral abscission zones. These findings implicate multiple and complex developmental roles for L-arabinosidases in addition to a simple role in carbon homeostasis. A previously described Arabidopsis mutant, ara1, exhibits a visible sensitivity to exogenous L-arabinose and encodes a defective L-arabinose kinase gene allele. The molecular characteristics of an ARA1 homologue, denoted ARA2, are reported. Expression studies revealed ARA1 is expressed during early vegetative growth, in floral organs and in conjunction with ARA2, developing pollen. Attempts to identify an ARA2 T-DNA mutant were made. Thus, a total of four genes that are involved in L-arabinose metabolism in Arabidopsis, are described. In summary, these findings have contributed significant insights into an intricate and highly regulated mechanism, whereby the concerted action of many enzymes mediate development-related cell wall remodelling that ultimately influences plant form and function.
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ItemFunctional analysis of conserved domains in the Drosophila TRPL channel(University of Melbourne, 2003)The Drosophila Transient Receptor Potential (TRP) and Transient Receptor Potential Like (TRPL) proteins form non selective cation channels whose activity depolarize the photoreceptor neurons in response to light. The initial steps that trigger the photoreceptor response are well characterized. However, the molecular mechanisms that directly activate and regulate the channels remain unknown. The elucidation of critical domains and their contribution to channel activation might constitute a starting point to understand the activating mechanism of TRPL and other TRP channels. The function of conserved regions amongst TRP channels was explored by creating mutations in the TRPL channel (truncations and point mutations) in three regions of the protein with high conservation through human and Drosophila TRP channels. The first conserved domains studied were the Ankyrin Repeats (AR) motifs that are present in the Amino-terminal region of the TRPC and TRPV subfamilies of TRP channels. These motifs were deleted in the TRPL channel, expressed in HEK cells and studied by patch-clamp whole cell recordings. Two truncated channels lacking all or only the 3rd and 4th AR showed activation by GTP-y-S included in the patch pipette, indicating that these conserved motifs do not play a key role in TRPL channel activation. Within the transmembrane domains region, the loop linking the S4 and S5 transmembrane domains, which shows significant degree of conservation, was studied. The substitution of Leucine 542 and Serine 545 for Alanines (mutant LS) did not affect TRPL activation, whereas changing Glycine 540 and Glutamine 543 for Alanines (mutant GO) eliminated the channel response to GTP-?-S. The possible role of this region was also investigated by dialysis of a peptide derived from this S4-S5 loop into HEK cells expressing wild type TRPL. This treatment elicited currents in cells expressing TRPL as well as in cells expressing the human homologue TRPC6. In overlay assays this peptide bound a Drosophila head protein of approximately 200 KDa. In conjunction these results indicate that the region is necessary for normal channel activation and that it probably regulates TRPL channel activity by interacting with other proteins. The C-terminal region was the third region investigated. The deletion of aminoacids 682-1025 (mutant ?C1), located immediately C-terminal to the last transmembrane domain, abolished TRPL channel responses to both GTP-?-S and Linolenic acid (LNA). The deletion of the highly conserved region 682-698, which is present in all TRPC channels, significantly reduced the response to GTP-?-S and prevented LNA activation of the channel. These results provide evidence of the regions within the channel that are required for its normal activation by GTP-?-S or by lipids. Although, the actual role that these identified regions is playing in channel functioning can not be established, different possible scenarios are discussed.
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ItemInvestigating the structure and function of lymphocyte perforin(University of Melbourne, 2003)Cytotoxic T Lymphocytes (CTL) and Natural Killer (NK) cells are involved in the elimination of virus-infected and malignant cells. Perforin, a key cytotoxin secreted by cytotoxic lymphocytes acts synergistically with the co-secreted serine proteases (granzymes) to kill the target cell. The devastating effects of perforin deficiency are mirrored in perforin-deficient mice and children diagnosed with familial haemophagocytic lymphohistiocytosis (FHL), a lethal immune deficiency requiring bone marrow transplantation as the only successful therapy. Perforin�s pivotal role in killer cell function makes it an attractive target for therapeutic intervention. Currently however, a large gap exists in our understanding of how perform operates at the molecular level, principally due to a lack of expression systems capable of synthesising this cytotoxic protein. This thesis describes a novel retroviral expression system that was successfully used to express wild type perforin, allowing the first ever mutagenic analysis of the molecule. Using this technology, perforin was expressed in Rat Basophilic Leukemia (RBL) cells, which can synthesis and store the protein in secretory granules. Degranulation and perforin release were achieved through the use of an anti-trinitrophenyl (TNP) IgE antibody to crosslink the Fee receptor on RBL cells with TNP-labelled EL-4 target cells. This resulted in death of the EL-4 cells, however RBL cells transduced with empty viral vector did not induce cell death. Using the same methodology, two mutations identified in FHL (P5 mutation: G429E and P6 mutation: P345L) were expressed in the RBL cells and shown to be associated with complete loss of cytotoxic function. Both mutated perforin molecules were correctly targeted to the secretory granules, and released upon Fee receptor crosslinking. This suggested that in each case, the defect in perforin function mapped downstream of release from cytotoxic lymphocytes. Retroviral transduction was also used to investigate the role of putative calcium-binding aspartate residues located in perforin�s carboxy terminus C2-like domain. The negatively charged aspartate side chains have been predicted to cluster and bind calcium ions, which is an obligate requirement for perform function. Single and joint mutation of two of the five aspartate residues conserved in rat, mouse and human perforins caused a complete loss of perforin-mediated cytotoxicity, suggesting that aspartate residues 484 and 486 are both indispensable for perforin function.
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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 role of cytochrome P450s in pyrethroid resistance in the AN02 strain of Helicoverpa armigera( 2003)Helicoverpa armigera is a lepidopteran moth species that is one of the largest agricultural pests in the world. In Australia, it is especially damaging to the cotton industry. Previous research on pyrethroid resistance in Helicoverpa armigera has suggested several resistance mechanisms present in the field. Research on a specific strain of H. armigera, AN02, suggested that resistance to pyrethroids in this particular strain is due to metabolism of the insecticide by cytochrome P450. This thesis describes work undertaken to gain further knowledge of the resistance mechanism present in this strain. Chapter 2 describes expression testing and elimination of the CYP6B cluster of P450s as candidates for resistance in the AN02, which were the most likely candidates available at the time. Chapter 3 describes construction, probing and sequence analysis of a midgut cDNA library of H.armigera to clone more P450 sequences and analyse the general transcriptome of one of the tissues where detoxification is likely to occur. Chapter 4 describes expression testing of new P450 sequences obtained from different sources to determine whether any of them have increased expression that is associated with the resistance genotype. Several new P450 genes were found to have increased expression and are candidates for further study.