Science Collected Works - Theses
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ItemThe construction of global orthogonal curvilinear grids and their use in ocean general circulation models(University of Melbourne, 2005)
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ItemThe prediction of plantation forest products by quality and mix in South Eastern Australia(University of Melbourne, 2005)
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ItemThe 3D structural evolution of the shipwreck trough, Otway Basin, southeastern Australia : architecture of an oblique rift margin(University of Melbourne, 2005)The Otway Basin formed during Late Jurassic�Late Paleocene rifting and separation of the Australian and Antarctic continents. Rifting proceeded from west to east, and by the end of the Early Cretaceous the intra-cratonic rift stretched from the Indian Ocean in the west to the eastern side of the Gippsland Basin to the east. During the Late Cretaceous, the architecture changed in the east-central Otway Basin as the rift axis began to propagate to the south, along the western margin of Tasmania, creating a NNW-SSE trending left-lateral oblique transform margin. The change caused decreased extension and the eventual abandonment of the Bass Basin rift segment as Tasmania remained attached to the Indo-Australian Plate. Previous workers have hypothesized that the development of the oblique margin was controlled by the extensional reactivation of the N-S trending lithospheric- scale suture between the Palaeozoic Lachlan and Delamerian mobile belts. The Shipwreck Trough is located along strike of the Palaeozoic suture, at the northern tip of the Late Cretaceous oblique margin, and has been the location of numerous recent natural gas discoveries. These discoveries have driven the acquisition of high-resolution 3D seismic data sets making the Shipwreck Trough an ideal location to study the structural development of this oblique rift margin. This study has illuminated the evolution of the Shipwreck Trough through 3D structural mapping of seismic horizons, faults, and growth strata geometry and distribution. The Shipwreck Trough evolved through two rift phases. Commencing in the Late Jurassic-Barremian, the N-S trending Proto-Shipwreck Trough and NE trending Minerva Graben, Princetown Horst, and Geographe Horst were formed by the probable extensional reactivation of NE and NNW trending Palaeozoic basement structures. These early Cretaceous depocentres were filled with up to 4 km of non-marine fluvial to lacustrine sediments providing an anisotropic mechanical stratigraphy that would strongly influence Late Cretaceous structural development. In the Barremian and continuing through the Albian, many of the basement-involved structures became inactive and were buried by fluvial volcaniclastic sandstone shales and coaly sediments of the Eumeralla Formation providing the source-rock for later hydrocarbon accumulations. During the Cenomanian, Early Cretaceous depocentres to the east of the Shipwreck Trough were inverted and eroded to create the Otway Unconformity. Inversion within the Shipwreck Trough was significantly less as the contact appears primarily conformable implying a non-depositional disconformity. Rifting re-commenced in the Cenomanian-Late Paleocene creating the WNW-trending Tartwaup-Mussel Fault Zone (TMFZ) and the N-S trending Shipwreck Trough. The TMFZ was composed of individual WNW-trending, south-dipping, right-stepping en echelon fault segments which together formed the primary northern structural boundary to Late Cretaceous syn-rift deposition. The Late Cretaceous sedimentary thickness expands up to 4 km across the TMFZ. The 140� orientation of the TMFZ was oblique to the ~190� extension direction suggested by comparison of the TMFZ/Shipwreck Trough fault orientations to orientation produced by analogue models of oblique rifts. Developed synchronously and to the north of the TMFZ, the Shipwreck Trough was a north trending structural depocentre composed of W to NW trending normal faults which were bound to the east by a NNW trending left-lateral-oblique transfer fault system, the Shipwreck Fault Zone (SFZ). The WNW trending normal faults in the center of the trough were not linked to the underlying basement-involved faults, but were detached within the Late Jurassic-Early Cretaceous syn-rift fill sequence of the Proto-Shipwreck Trough. Conversely, the NNW trending SFZ was both hard and soft-linked to the faults forming the eastern margin of the underlying Proto-Shipwreck Trough providing an excellent 3D example of a basement-controlled transfer fault that shows both focused and distributed structural styles. The SFZ transferred extension from the Boggy Creek/Sherbrook Faults in the north to the Prawn/Sorell Faults in the south thus leaving the Prawn Platform to the east as a structural high. Left-lateral oblique extension on the SFZ caused synchronous compressive inversion of the Minerva Graben, creating the NE-oriented gas-bearing Minerva Anticline, and the development of "horse-tail" or wedge to rhombic-shaped extensional depocentres such as the Investigator Graben. The strike-slip component of offset on the SFZ was relatively small (<5 km). Left-lateral oblique offset on the 160� trending SFZ is compatible with a 190� Late Cretaceous extension direction. En echelon fault geometries and bi-modal to tri-modal fault orientation distributions within the Shipwreck Trough are similar to fault patterns developed in analogue models of ~30� rift obliquity. Corroborating analogue models, the primary factors that influenced the structural development of the Shipwreck Trough were: 1) reactivation of structural fabrics within the pre-rift basement, 2) the mechanical stratigraphy of the syn-rift fill sequences, and 3) the obliquity of rifting.
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ItemNature of the mineralised (ore-shoot) environment within the Magdala gold deposit, western Lachlan fold belt, Australia(University of Melbourne, 2005)Gold mineralisation within the Magdala gold deposit is contained within a series of steeply plunging ore-shoots hosted within a system of faults, fault bound laminated veins and shear zones that occur within a horizon of iron-rich altered pelites along the western flank of a doubly plunging basalt dome (Magdala Dome) In this study, the controls on the formation of the ore- shoots within the lode system are identified using a combination of structural and lithological mapping and ore-shoot scale two-dimensional numerical modelling. The study was focused around the Dukes Basalt Nose, the western most of a series of steeply southwest-dipping basalt sheets or flow lobes that comprise the Magdala Dome. Two main regions of mineralisation occur around the Dukes Nose: The Central Lode, which occurs along the upper or western boundary of the chlorite altered pelites that overly the basalt (Magdala Facies) and the Basalt Contact Ore Zone that occurs along the contact between the basalt and the altered pelites. The geometry as well as rheological contrasts between different stratigraphic units influence the partitioning of strain and the orientation of structures m the altered pelites that overlie the Dukes Nose. The shape of the nose is highly irregular along strike and down-plunge. Variations m the orientation of contacts and pinching out of smaller subsidiary noses or flows on the west flank of the basalt results in sudden changes in the thickness of overlying altered meta-sedimentary unit. The lack of evidence for early ductile deformation in the basalt and the partitioning of strain into the overlying Albion Formation early in the deformation process suggests that the variable geometry of the Dukes Basalt Nose is a product of primary volcanic processes, rather than ductile deformation. Gold mineralisation within the Central Lode is contained within a system of southwest-dipping faults, shear zones and fault bound laminated veins. Comparisons between regions of Central Lode that contain higher gold grades (ore-shoots) with those associated with low gold grades, shows that mineralised regions are those that have undergone west over east transport overprinting an earlier southwest over northeast directed event as indicated by striations and extension veins around faults. Mineralisation occurs as an overprint on pre-existing structures formed during the southwest over northeast transport event and is associated with an episode of low-displacement, west over east directed hangingwall transport. Due to the severe mis-orientation of the pre-existing faults and shear zones for reactivation in this east-west stress field, localised failure occurs on fault segments that dip at less than 65� associated with high-pore fluid pressures. The west over east event was overprinted by sinistral oblique or northwest over southeast transport on the west-dipping faults that led to further or redistribution mineralisation in some areas. Gold mineralisation within the Basalt Contact Ore Zone is hosted within faults and shear zones that occur parallel to the basalt contact and also concentrated in and around relatively flat- lying to shallow dipping quartz-carbonate-chlorite extension veins. Much of the mineralisation may have entered the region synchronous with mineralisation in Central Lode, associated with west over east transport on shallow-dipping fault segments or splays from Central Lode. Late in, or post the west over east event, strong stilpnomelane and/or silica alteration of the Magdala Facies in the region adjacent to the basalt resulted in permeability reduction. During subsequent northwest-southeast compression periodic fluid overpressuring occurred within the alteration zones and resulted in hydro-fracturing of the rock in the alteration zones and infilling of the open, fluid-filled fractures by quartz-carbonate and later by chlorite. Gold already in the area may have been remobilised following influx of new fluids and changing pressure conditions and was concentrated and precipitated in wall rock halos around the veins along with arsenopyrite and pyrite. Controls on the distribution of elevated gold grades interpreted from the results of field mapping have been further tested and refined using a series of two-dimensional numerical models constructed using the finite-difference code FLAG. Modelling was conducted using a series of ore-shoot scale cross-sections through the Dukes and Extended Basalt Noses as well as a plan section model. The section models were designed to be parallel to the interpreted transport directions during the formation and mineralisation events recognised in the Central Lode, allowing oblique movement to be modelled in two-dimensions. The results of the models show that during west over east directed transport, shearing is partitioned around low-angle contacts while tensile failure occurs in steeper sections of units with low-tensile strength and permeability. These mineralisation models show that much of the fluid flow is focused within shear zones immediately over the top of the Dukes Nose, the development of which has widespread influence on fluid flow. Isolated tensile failure has more localised effects on fluid Flow and as such mineralised tensile structures may occur in close proximity to shear zones. Ore-shoots along the flank of the Dukes Basalt can be replicated at as little as 0.18% shortening. The low-amounts of shortening leads to the development of a series of isolated shear zones in the 2D section over the top of the upper-terminations of the basalt flows where the Magdala Facies thickens.
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ItemOoidal ironstones in the cretaceous and early tertiary sediments of the Otway Basin, Southeastern Australia(University of Melbourne, 2005)Research on ooidal/oolitic ironstones has generally focused on those in the northern latitudes. This study presents three southern latitude examples of ooidal ironstones in the Otway Basin, southeastern Australia: the Late Cretaceous Banoon Member and Nullawarre Greensand, and the early Tertiary Pebble Point Formation. Three types of iron-rich ooids are present in these units: berthierine-dominated ooids, Fe oxide/oxyhydroxide-dominated ooids and siderite�calcite�phosphate�pyrite-dominated ooids. Microprobe analysis, X-ray diffraction and scanning electron microscopy has identified the green clay mineral berthierine to be present in each unit along with varying proportions of iron-rich ooids, glauconite, phosphate, siderite, pyrite, and calcite. Synsedimentary marine cerium- rich rhabdophane was also identified in the Nullawarre Greensand as fibrous inclusions along cracks in altered glauconite grains, as laminae in berthierine-dominated ooid cortices, and as minute crystals in berthierine cement. Glauconite grains are interpreted as reworked. Precipitation of berthierine is interpreted to occur in the zone of suboxic diagenesis below the sediment-water interface. Berthierine builds up as concentrically laminated ooid cortices in the zone of reworking along with rhabdophane. Below the zone of reworking, but still in the suboxic zone, berthierine continues to precipitate as an isopachous cement on mineral grains and co-precipitates with rhabdophane. After the precipitation of isopachous berthierine cement, but still in the zone of suboxic diagenesis, phosphate (carbonate fluor-apatite) and siderite precipitate. Later stage non-isopachous berthierine cement precipitated after carbonate fluor-apatite. Deeper in the sulphate reduction zone, pyrite precipitates. Much later in the burial history of the sediment calcite precipitates and replaces the primary fabric of the sediment. The presence of rhabdophane in the Nullawarre Greensand is of significance as rhabdophane has not previously been identified in sandstone sediments of any age in Australia. Its co-precipitation with berthierine has led to the conclusion that this REE-phosphate mineral precipitates in an early-diagenetic marine environment. The Banoon Member, Nullawarre Greensand and Pebble Point Formation were deposited during transgressive and regressive periods in varying depositional environments including: back-beach lagoonal (Banoon Member), delta front sands in an abandoned delta or delta lobe (Nullawarre Greensand), and estuarine (Pebble Point Formation). The mineralogy of the Banoon Member, Nullawarre Greensand and Pebble Point Formation has led to a greater understanding of the environment in which these units were deposited in and in turn has contributed to the body of knowledge surrounding the formation of ooidal/oolitic ironstones.
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ItemFunctional analysis of the barley cellulose synthase like (CSL)D and CSLF gene families(University of Melbourne, 2005)Plant cells are encased by walls that provide structure and support for the plant, and are crucial to growth and development. The cell wall is a complex and dynamic network that must be rigid to provide support, but also flexible to allow growth. The assembly and maintenance of the wall has been proposed to involve several thousand genes (Carpita et al., 2001). Polysaccharides make up the bulk (>90%) of the primary cell wall. Typically, microfibrils of cellulose form a rigid framework that is embedded in a matrix of non-cellulosic and pectic polysaccharides. The enzymes responsible for polysaccharide synthesis are largely unknown, but the CESA superfamily (containing the CESA gene family and eight cellulose synthase-like (CSLA-H) gene families) are thought to encode enzymes that catalyse processive sugar linkages to make the backbone of many of these polysaccharides. Determining the functions) of the CSL genes is the goal of many researchers investigating the process of cell wall synthesis. This goal has proved challenging, even with the vast resources available for the model dicot plant, Arabidopsis, suggesting that study of other biological systems may be necessary. As a case in point, the initial breakthrough in the discovery of a function for members of the CSLA family of genes in mannan synthesis was achieved through the analysis of tissues rich in mannan, the endosperm of guar seeds (Dhugga et al., 2004). The difficulties in proving functions for the CSL genes hint at an elaborate system of regulation for the synthesis of cell wall polysaccharides. The purpose of this PhD was to add to the existing knowledge of CSLs by identifying and characterising CSLD genes of barley (Hordeum vulgare L.), with the aim of elucidating the function of these genes by comparing and contrasting barley CSLDs with those of dicots. Soon after commencing, the scope was widened to include the CSLF genes, which are very similar in sequence to the CSLDs and CESA s, but so far have only been found in cereal genomes. They are therefore good candidates to encode the synthases of mixed-linkage ?-(1?3),(1?4)-D-glucans (MLG), a polysaccharide specific to the walls of some commelinoid monocots (Smith and Harris, 1999). Initially, it was necessary to clone barley members of each of these families, as there was little barley sequence available when this thesis commenced. The identification of barley CSLD and CSLF genes, using a combination of bioinformatics and molecular techniques, is outlined in Chapter 2. There were three barley CSLDs identified (HvCSLD1-HvCSLD3), and the full-length sequence was determined for two of these. Phylogenetic trees of CSLD genes from a number of different species confirmed previous observations that orthologs are generally more similar than paralogs (Holland et al., 2000; Richmond and Somerville, 2000); however one clade of the tree indicated a possible division between dicot and monocot sequences. The number of CSLD genes in Arabidopsis and cereals such as rice suggested that there are more than three CSLD sequences in barley, a prediction that is supported by the identification of a single EST for a third CSLD gene not previously detected in screens for CSLD family members. Three barley CSLF genes were also identified. The full-length sequence is known for two of these, and the third is a contig of 6 ESTs. The final number of barley CSLFs is more difficult to predict, as these genes have so far only been found in cereal genomes and some of the more similar rice sequences may be the result of recent duplications. However, from analysis of wheat and rice sequences (representing at least 4 wheat CSLFs and 8 rice CSLFs), it seems likely that there are more CSLFs yet to be identified from barley. These may have been missed because the probe used to screen for CSLF sequences was derived from one of the more divergent members of this family. Five of the genes were mapped and correlations sought between their genetic location and QTLs for wall-related traits. The HvCSLD1 and HvCSLD2 genes were on chromosomes 7H and 1H, respectively, and the HvCSLF1, HvCSLF2 and HvCSLF3 genes were on chromosomes 7H, 2H and 1H respectively. None of the five genes were close to each other. HvCSLD1 and HvCSLF1 were in genomic regions where a number of QTLs had already been mapped. Interestingly, HvCSLF1 was in an area containing QTLs for several malting quality traits. An accurate map location for HvCSLF2 could not be determined, but this gene is potentially located in a major QTL for barley MLG content. In Chapter 3 the expression of the available barley CSLD and CSLF genes was analysed, with the exception of HvCSLD3, as too little sequence was available for the design of primers for quantitative Real Time RT-PCR (Q-PCR). HvCSLD1 and HvCSLD2 showed different expression profiles. HvCSLD1 was expressed in all tissues examined and had strongest expression in suspension culture cells and developing endosperm, while expression of HvCSLD2 was mostly in root tips, with little or no significant expression in other tissues. The expression of both CSLD genes suggests that they are important in young tissues and may have a role in polysaccharide synthesis during cell division. Their expression is consistent with an involvement in cellulose synthesis, especially for HvCSLD1 in developing endosperm, but it was not possible to correlate the expression of either CSLD gene with the accumulation of a particular polysaccharide. EST analysis of CSLDs from other species supported the prediction that there were likely to be more barley CSLDs, as a clade of genes with pollen-specific expression was identified for which there was no barley ortholog. Mutations in the Arabidopsis gene AtCSLD3 result in the kojak phenotype, which is characterised by the absence of hairs on the root. The ortholog of the kojak/AtCSLD3 gene was not obvious among the barley CSLD genes, because both HvCSLD1 and HvCSLD2 grouped in the same clade as AtCSLD3 and were expressed in tissues where root hairs are forming. However, as both HvCSLD1 and AtCSLD3 are expressed in many plant tissues, HvCSLD1 was considered more likely to be the barley ortholog. For the CSLF genes, HvCSLF1 had high levels of expression in all tissues examined except leaf tips, with the strongest expression being in coleoptiles and elongating stems. HvCSLF2 and HvCSLF3 had their highest expression in coleoptiles and root tips. The observation that all three genes were strongly expressed in coleoptiles was significant, as there are high amounts of MLG synthesised in elongating coleoptiles. This finding suggested that CSLFs could be involved in MLG synthesis. Experiments aimed at determining the subcellular location of the barley CSLD proteins are presented in Chapter 4. The subcellular location of polysaccharide synthases has implications for their function, as cellulose and callose are the only wall polysaccharides known to be made at the plasma membrane (PM). All other wall polysaccharides are thought to be synthesised in the Golgi apparatus (GA) before being transported to the wall. Antibodies against the predicted cytoplasmic domain of HvCSLD1 were developed and used to label protein blots from membrane fractions enriched for different subcellular compartments. The CSLD1 antibody did not detect any proteins in barley PM-enriched fractions or rice GA-enriched fractions. However, the CSLD1 antibody did detect a protein of approximately 70 kDa in the crude homogenate and mixed membrane fractions of both barley and rice. As a protein of similar size was also detected in rice by an anti-CESA antibody, the possibility that this ~70 kDa protein is related to CESAs or CSLDs is worth investigating further. Another approach used to determine subcellular location was to tag CSLDs with GFP and express them in tobacco protoplasts. GFP- tagged HvCSLD2 was located within intracellular vesicles that did not appear to be associated with either the endoplasmic reticulum (ER), GA or PM. Chapter 5 outlines results of functional analyses by either gene suppression, via RNAi, or heterologous expression by transformation of Arabidopsis. Stable transformation of barley with RNAi constructs against HvCSLD1 and HvCSLF1 did not lead to the production of transgenic plants, although plants transformed with the backbone vector could be regenerated. This result suggested that HvCSLD1 and HvCSLF1 could be important at some stage in the development of transgenic shoots from callus. Transient expression of HvCSLD1 RNAi constructs in barley coleoptiles did not give a detectable phenotype, perhaps because the expression of HvCSLD1 and HvCSLD2 is relatively low in coleoptile tissues. Transient expression of a HvCSLF1 RNAi construct in coleoptiles may have resulted in reduced elongation of coleoptiles, and, in severe cases, the development of a split along the length of the coleoptile. However, Q-PCR analysis did not show a link between the appearance of this phenotype and a reduction in HvCSLF1 transcript. The barley CSLDs were further analysed by heterologous expression in Arabidopsis mutant kojak (kjk) plants to determine if they were capable of complementing the lack of root hair phenotype. HvCSLD1 was able to complement the phenotype, thus confirming the prediction that HvCSLD1 is the ortholog of AtCSLD3. The demonstration that the product of a barley gene is capable of performing the same function in Arabidopsis means that further predictions about the function of cereal CESAs and CSLs based on findings in Arabidopsis can be made with some confidence. Although HvCSLD2 was not capable of complementing the kjk phenotype, expression of HvCSLD2 did result in a change in phenotype in both kjk and WT Arabidopsis plants. Plants transformed with HvCSLD2 germinated and established more rapidly than their untransformed counterparts. A difference in the speed of germination and establishment between WT Columbia and kjk-3 plants was also observed, suggesting that the expression of AtCSLD3 also has an effect at this early stage of development, even though HvCSLD2 and AtCSLD3 do not appear to be orthologs. This difference in seedling development has not been previously reported, and strengthens the suggestion from the expression analysis that CSLDs are important at early growth stages, and perhaps have a role in cell division. It is also possible that they are involved in the production of mucilage around the seed coat or at the root tip. In conclusion, this study has contributed to current knowledge of the CSL family by the identification and characterisation of new CSLD and CSLF genes of barley. Complementation experiments enabled the identification of a barley ortholog to the dicot gene, AtCSLD3, and fluorescent tagging of CSLD proteins suggests they are located in post-Golgi vesicles and may have some association with the PM. This location is similar to the reported location of KOR, an endo-1,4-?-glucanase involved in cellulose synthesis (Robert & H�fte, pers. comm.). If CSLDs are located in post-Golgi vesicles or the PM, it is unlikely that they are involved in the synthesis of non-cellulosic polysaccharides such as glucuronarabinoxylan, xyloglucan or MLG, as these polysaccharides are believed to be synthesised within the GA before their secretion, via vesicles, to the cell wall. If the CSLDs are involved in the synthesis of cellulose, then the similarities between CSLD and KOR location studies raise the possibility that these proteins are both involved at the same stage of cellulose synthesis. Read and Bacic (2002) propose the existence of different CESA polypeptides responsible for the initiation or the elongation of cellulose microfibrils. It is plausible that CSLD proteins could initiate cellulose synthesis by producing ?-glucan �primers� that are then transferred to the CESA rosette complex after cleavage by KOR. Obviously, this and other possibilities need to be explored further, and much more work is needed to determine the exact functions of CSLDs or CSLFs. Determining the functions of these proteins will, in turn, be just one step in unravelling and understanding the elaborate processes involved in the assembly and maintenance of a plant cell wall.
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ItemThe role of NaGSL1 in callose synthesis in the pollen tubes of nicotiana alata(University of Melbourne, 2005)Plant cell walls are dynamic structures that play a key role in plant growth and development and in the responses of plants to stress. Callose, a 1,3-?-glucan, is found in the cell wall at specialized locations throughout plant development, and is deposited in response to wounding and pathogen challenge. The enzyme callose synthase (UDP-Glc: 1,3-?-D-glucan 3-?-D-glucosyl transferase; EC 2.4.1.34 ), or CalS, is responsible for the production of callose. Until quite recently, CalS was unique among plant polysaccharide synthases for being both active and stable in vitro. Despite these biochemical advantages, the proteins that comprise this enzyme in plants have not been definitively identified. A family of genes, the glucan-synthase-like (GSL) genes, has been proposed to encode plant CalS enzymes based on the similarity of these genes to genes believed to encode the fungal 1,3-?-glucan synthase (Saxena and Brown, 2000; Cui et al., 2001; Doblin et al., 2001; Hong et al., 2001a). A number of studies have provided evidence to support a role for GSL genes in callose synthesis at both the biochemical (Cui et al., 2001; Hong et al., 2001a; Li et al., 2003) and molecular (Cui et al., 2001; Doblin et al., 2001; Hong et al., 2001a; Jacobs et al., 2003; Nishimura et al., 2003) levels; however, definitive proof of function for the GSL genes has not yet been shown. In this thesis, pollen tubes of Nicotiana alata Link et Otto are used as an experimental system to study callose synthesis. Pollen tubes form upon germination of pollen grains, and usually grow through the style to deliver the sperm to the ovaries. N. alata pollen tubes can also be grown in culture. The wall of N. alata pollen tubes is made predominantly (86%) of callose (Li et al., 1999), and a highly active, developmentally regulated CalS enzyme has been biochemically characterized from pollen tubes (Schl�pmann et al., 1993; Li et al., 1997; Turner et al., 1998; Li et al., 1999). An N. alata GSL gene, NaGSLl, is abundantly expressed specifically in pollen grains and tubes and is therefore a candidate to encode the pollen-tube CalS (Doblin et al., 2001). This thesis aims to determine if the polypeptide encoded by the NaGSL1 gene, NaGSL1, is the pollen-tube CalS and, following a positive identification, to investigate the regulation of NaGSL1 and thus of callose synthesis in N. alata pollen tubes. This required the production of a full-length NaGSL1 cDNA and antibodies against a bacterially expressed region of NaGSL1, and the successful production of these molecular tools is described in Chapter 2. A biochemical link between NaGSL and the pollen-tube CalS is established in Chapter 3. The pollen-tube CalS was enriched several hundred fold using continuous-density-gradient centrifugation and product entrapment, essentially as described by Turner et al. (1998). Polypeptides from each stage of the enrichment were analysed by SDS-PAGE. The most abundant polypeptide in the product-entrapped material had a molecular weight of 220 kDa and was identified as NaGSL1 from tryptic peptides using both peptide mass fingerprinting (PMF) by MALDI-TOF MS, and MS/MS. Labelling with the anti-GSL antibodies showed that NaGSL1 is enriched through the CalS-enrichment process and abundant in the product-entrapped material, with the relative abundance of NaGSL1 correlating with CalS activity. This data thus provided a strong biochemical link between NaGSL1 and CalS activity, leading to the conclusion that NaGSL1 is the pollen-tube CalS. PMF was also used to investigate the identity of other polypeptides in the product-entrapped material. The only other plant polypeptides identified, a 103-kDa plasma membrane H+-ATPase and a 60-kDa ? subunit of the mitochondrial ATPase, were deduced to be contaminants. All other polypeptides were present in low abundance, and so could not be identified. Therefore, it appears that NaGSL1 does not require the presence of another protein for CalS activity. Once it was established that NaGSL1 is the pollen-tube CalS (Chapter 3), the regulation of NaGSL1 was investigated in Chapter 4 by tracking the abundance and location of the NaGSLl polypeptide through pollen-tube growth using the anti-GSL antibodies, and then relating these data to levels of CalS activity. Because the pollen- tube CalS can be activated in vitro by trypsin (Schl�pmann et al., 1993; Li et al., 1997, 1999), CalS activity assays were conducted without trypsin (measuring the amount of active enzyme) and with trypsin (measuring the total amount of enzyme). NaGSL1 was present in low abundance 30 min after pollen-grain hydration, the same time at which CalS activity is first detected. As the NaGSL1 mRNA is present in mature pollen grains well before germination, the production of NaGSLl appears to be regulated at translation. The amount of the NaGSL1 protein increased over the first 16 h of pollen-tube growth, and paralleled the increases in the amount of CalS over this time. After continuous-gradient-density centrifugation of membranes from 4-h and 16-h pollen tubes, NaGSL1 was present in fractions enriched for intracellular membranes and in fractions enriched for plasma membrane (PM). Without trypsin activation, CalS was predominantly in the PM-enriched fractions. Trypsin treatment increased the CalS activity in the PM-enriched fractions and also revealed a previously inactive CalS in the fractions enriched for intracellular membranes. The relative abundance of NaGSL1 in different fractions detected with the anti-GSL antibodies correlated with the level of CalS activity after trypsin treatment, that is, with the total CalS activity. The subcellular location of NaGSL1 were determined by immuno-electron-microscopy (immunoEM). Both the results from membrane fractionation and from immunoEM show that NaGSL1 is located predominantly in the ER and Golgi during the early stages of pollen-tube growth, and is located predominantly in a population of vesicles and the PM during the later stages of pollen-tube growth. Western blot analysis and CalS activity assays were used to analyse the mechanism of trypsin activation and determine if this occurred via the removal of a sizeable autoinhibitory domain from NaGSL1. The results are described in Chapter 5 and show that there was no consistent correlation between the production by trypsin of lower-molecular-weight forms of NaGSL1 and a high level of CalS activity. Activation of NaGSL1 was concluded not to be due to cleavage of a sizeable autoinhibitory domain. Whilst it is possible that trypsin activation is due to the removal of a di- or tri-peptide from the C-terminus of NaGSL1, it seems more likely that trypsin is acting upon a separate, inhibitory protein and causing its dissociation from NaGSL1. This inhibitory protein would not be present in a fully activated, product-entrapped CalS preparation, in agreement with the results of Chapter 3. Heterologous expression was used to investigate the function and properties of NaGSL1, and this is described in Chapter 6. Yeast was selected as the heterologous host as it contains homologous genes, the FKS genes, believed to encode a 1,3-?-glucan synthase (Douglas et al., 1994a). When the full-length NaGSL1 cDNA was transformed into a range of yeast fks mutants, neither full nor partial complementation were observed, even though the full-length NaGSL1 polypeptide was expressed in yeast and targeted (at least partially) to the PM. The heterologously expressed NaGSL1 did not display in vitro CalS activity, and the addition of trypsin or pollen- tube homogenate did not stimulate CalS activity. Instead a low-molecular-weight, non-proteinaceous component of the yeast lysate appeared to inhibit the pollen-tube CalS. The failure of NaGSL1 to complement fks mutants and to display in vitro CalS activity when expressed in yeast may be due to the yeast biosynthetic machinery being unable to correctly fold, post-translationally modify and/or regulate the plant protein. The lack of fks complementation may therefore relate to differences in the production and/or regulation of the plant GSL and fungal FKS proteins, even though they appear to have the same catalytic function. Heterologous expression of NaGSL1 in plant cells other than pollen tubes may be required to investigate the molecular mechanism of NaGSL1 and its activation.
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ItemExpression of recombinant proteins in model plant systems(University of Melbourne, 2005)The production of recombinant proteins in plants and algae has led to the development of important plant produced proteins including biopharmaceuticals such as cytokines, hormones, monoclonal antibodies, enzymes and vaccines (Miele 1997; Le�n- Banares et al. 2004). This thesis looked at the expression of proteins in two different systems, a novel red algal system and the model plant system Arabidopsis thaliana (from now, known as Arabidopsis). The potential of the red algae Galdieria sulphuraria as a protein expression system was evaluated. This microalgae, with growth characteristics of microorganisms, provides a novel eukaryotic system that, in addition to the usual features (e.g. folding and glycosylation) grows optimally at low pH (<2.0) and high temperatures (40-70�C). Therefore there should be minimal problems with contamination making this red algae an ideal system for expression of recombinant proteins. A reporter gene construct containing green fluorescent protein (GFP) was introduced into G. sulphuraria using a particle inflow gun (P.I.G.). Transient expression of GFP was obtained using the cauliflower mosaic virus 35S promoter (CaMV35S) which is commonly used in plant transformation. We tested the suitability of a wide range of antibiotics and herbicides as selective agents for the development of a stable transformation system in G. sulphuraria. Attempts at stable transformation resulted in no transformants. The remainder of the thesis describes protein expression in Arabidopsis. The development of plant-derived vaccines has become a new strategy for combating diseases, particularly in developing countries. Two antigens from the tapeworm Taenia solium were selected for expression in the model plant system Arabidopsis. Arabidopsis was selected as it is easy to transform, has a fast growth rate, and plant cell suspension cultures can be produced rapidly. Two constructs were designed for the expression and secretion of the T. solium antigens called TSOL-18 and TSOL-45. Transformation was successful and resulted in both TSOL-18 and TSOL-45 transgenic plants. Plant cell suspension cultures were developed from single lines. Analysis of whole plants expressing recombinant TSOL-18 protein showed that the recombinant protein was truncated and expressed at low levels. Although truncated, some antigenicity was retained as evidenced by TSOL-18 specific antibody binding. Full length TSOL-45 recombinant protein was obtained from the appropriate transformants at higher levels than TSOL-18. Plant cell suspension cultures were analysed for the presence of the recombinant protein in both the suspension cells and the media. Analysis of the suspension cells detected TSOL-45 protein in the cell suspension but no recombinant proteins were detected in TSOL-18 cell suspension. Despite the proteins being targeted to the cell surface using endoplasmic reticulum signals, no protein could be recovered from the culture media suggesting that the recombinant proteins may be degraded. Due to the low viability of the plant cell cultures, it was decided to focus on whole plants for further analysis. To investigate whether either of the recombinant proteins were subjected to specific plant glycosylation and/or other modifications during plant cell secretion, proteins were purified for protein sequencing and mass spectrometry. Both nickel purification and immunopurification were used to isolate the recombinant protein. High performance liquid chromatography (HPLC) was used to resolve the proteins further. Although insufficient material was obtained in this study for sequencing, methods have been developed that with further modifications, will permit immunogenicity testing in the future. This project has evaluated the potential of two tapeworm antigens as plant- derived vaccines. It has been shown that T. solium antigens can be expressed in a plant system such as Arabidopsis. Proteins can be resolved using various purification strategies. The work described here will aid in the future evaluation of these proteins as plant-derived vaccines to treat T. solium infections in animals.
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ItemGenome evolution in Australian marsupials(University of Melbourne, 2005)In this study genome evolution has been studied in marsupials at three different comparative levels; the chromosome level, the gene arrangement level and the DNA sequence level. The karyotypes of different marsupial species comprise different chromosome numbers and morphologies, but G-banding has revealed conservation of large chromosome blocks. In this study, I have explored the rearrangements of large chromosome blocks and individual genes within them, between two distantly related Australian marsupials, the tammar wallaby (Macropus eugenii) and the stipe faced dunnart (Sminthopsis macroura), and used these results for comparative analysis with other marsupials, other mammals and other vertebrate species. I used �chromosome painting� to investigate the degree of conservation between the chromosomes of the tammar wallaby (chromosome number 2n=16) and dunnart (2n=14). I hybridized DNA from flow sorted tammar wallaby chromosomes to the chromosomes of the dunnart and discovered a high degree of conservation, even though the two marsupial species are in different orders and last shared a common ancestor 66-72 MYA (million years ago; Springer et al., 1997). Painting of the tammar chromosomes to the wombat karyotype (also a 2n=14 Australian marsupial) revealed chromosome blocks almost completely conserved with those of the dunnart. These results, together with results from previous chromosome painting in other Diprotodont marsupials and earlier G-banding studies, mean a 2n=14 ancestral karyotype can be deduced for Australian marsupials. In order to take the genome comparison, between the tammar wallaby and the dunnart, to the next level of resolution two different sets of genes were chosen for mapping and sequencing in both species. The first set of genes chosen were the hemoglobin genes, of which homologues have previously been found in species ranging from bacteria to mammals. The second set of genes were the milk protein genes Early Lactation Protein (ELP) and Whey Acidic Protein (WAP), both specific to mammals. I isolated large genomic insert BAG clones containing the ? (alpha) hemoglobin genes, the ? (beta) hemoglobin genes, the ELP gene and the WAP gene. Mapping of BACs containing hemoglobin and milk protein genes by Fluorescence In Situ Hybridization confirmed and extended the painting results by providing specific chromosome reference points within the conserved chromosome blocks between the tammar wallaby and dunnart. In addition, previous inconsistent mapping positions of the hemoglobin genes in marsupials by the older technique of Radioactive In Situ Hybridization were resolved. Sequencing the hemoglobin clusters of the dunnart revealed both genes and regulatory regions, many of which are shared with eutherians. This study found that the dunnart p globin cluster contained only two genes in the following order 5�-?-?-3�, which contrasts the human p globin cluster which contains five functional genes in the following order 5�-? -A?-G?-?-?-3'. Phylogenetic footprinting of the dunnart p cluster sequence 5� to the e gene revealed a region similar to the eutherian locus control region (LCR) containing sequences and transcription factor binding sites with homology to the eutherian hypersensitive sites 1-5. This demonstrates that the LCR in the dunnart is equally as complex as that of eutherians, and supports the theory that the LCR acts as an integral unit to regulate the ? globin genes. A previous finding of a ?-like gene ? (omega) in the a cluster in the tammar wallaby (Wheeler et al., 2001) suggested that the ? and ? clusters evolved via genome duplication and loss rather than tandem duplication. This study has found the dunnart ? gene lies next to the ? gene, implying that the ?-? juxtaposition is likely to be conserved in all marsupials. In addition, the LUC7L gene was found 3� of the dunnart ? locus, a gene order shared with humans, but not mouse, chicken or fish. Sequencing of the dunnart WAP gene revealed a five exon gene, with the four disulphide core (4DSC) a protein motif present in the middle three exons. This contrasts the eutherian milk WAPs which have four exons with the 4DSC located in the middle two exons. Expression studies of genes in lactating mammary tissue in Sminthopsis crassicaudata (also known as the fat tailed dunnart and closely related to the stripe faced dunnart) revealed that ELP is expressed at only the earlier phase of lactation and WAP is expressed only at the later phase of lactation. This finding of asynchronous expression during the lactation cycle in a dunnart species is a similar pattern of expression to that which has previously been identified in the distantly related tammar wallaby, and shows that this pattern of asynchronous expression is likely to be ancestral, at least to Australian marsupials. The aims of this study are to examine genome evolution in marsupials at three different comparative levels; the chromosome level, the gene arrangement level and the DNA sequence level. The techniques employed to achieve these aims are chromosome painting, DNA sequencing and gene mapping.
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ItemBehavioural ecology of the Bobuck (Trichosurus cunninghami)(University of Melbourne, 2005)This thesis demonstrates the profound influence that patterns of resource distribution and abundance can have on the behavioural ecology of a marsupial. I studied two neighbouring populations of bobucks, Trichosurus cunninghami, within a fragmented forest landscape in the Strathbogie Ranges, south-eastern Australia. There were numerous marked differences between the 'forest population', which inhabited a forest patch and the 'roadside population' which occurred in linear roadside vegetation. The forest population was socially monogamous; the adult sex ratio was at parity and there were no significant differences in the number of den-trees used by adult females and males or in their home range sizes. Pair-members had strongly overlapping home ranges, shared den-trees and remained close to one another during their nocturnal activity period. Pair-bonds ended only as a result of the death of one pair-member. Two-thirds of young were sired by the female's social partner and most males sired one young per year. There was a significantly male-biased offspring sex ratio and all males that were horn at this site and survived to three years of age dispersed; all females were philopatric. Females inherited some of their mothers' den-trees and, as adults, established home ranges that overlapped substantially with their mothers' ranges. Patterns of genetic relatedness indicated that all adult males in the forest population were immigrants. In contrast, the roadside population was polygynous; the adult sex ratio was female-biased and male home ranges were significantly larger than those of females. Each male's home range overlapped with those of two or three females, and males used significantly more den-trees than females. There was no bias in offspring sex ratio; genetic data revealed that there was no sex-bias in dispersal, and that males sired multiple young per year. Both of the key resources for bobucks (den-trees and silver wattle. Acacia dealbata, the main dietary item) occurred at significantly higher density at the roadside site, and were located in close proximity to one another. Conversely, these resources were widely separated at the forest site. There were no significant differences in the number of den-trees or silver wattle trees located within the home ranges of females at the two sites, but females at the forest site had home ranges approximately three times the size of those of roadside females. At the forest site, patterns of den-use and den-sharing, and the relative location of den-trees and preferred foraging areas, suggested that suitable den-trees located close to food trees were limiting. To test this hypothesis, I artificially supplemented hollow availability by installing nestboxes within the core foraging range of each adult female in the forest population. In contrast to the findings of other studies, the nestboxes were rapidly colonised by bobucks, indicating that if natural hollows were available closer to food resources, as they were at the roadside site, they would almost certainly be used. My study establishes that bobucks have a variable mating system, governed by the availability and distribution of key resources. Specifically, where resources were sparse and widely distributed, females had large home ranges, and males were constrained to social monogamy by the spatial distribution of females.