Sulfate removal from sulfomucin is believed to be a rate-limiting step in sulfomucin degradation by bacteria from the digestive tract. A novel sulfomucin-desulfating enzyme has been discovered in the anaerobic bacterium, Prevotella strain RS2, which can grow on colonic mucin as its sole energy source. The enzyme, located in the periplasm, was assayed by measuring p-nitrophenol removal from the model substrate sulfate-6-N-acetylglucosamine-1-p-nitrophenol, sulfate-6-N-acetylglucosamine being the other product. This activity differs from that of sulfatases which remove the sulfate ester group from sulfate-6-N-acetylglucosamine and its analogues substituted at the Cl position. The enzyme has been termed a sulfate-6-N-acetylglucosaminidase or sulfoglycosidase (SGL). The SGL was purified to a single protein band of 100 kDa as analyzed by SDS-PAGE. The purified SGL protein was trypsin-digested and peptide fragments were sequenced. PCR and inverse PCR were then used to amplify the entire sg/ gene from Prevotella strain RS2 genomic DNA. After inserting the gene into a suitable plasmid, active recombinant SGL was expressed using an Escherichia coli expression system. The SGL was characterized using a selection of model substrates, and shown to be an exo-enzyme that removes non-reducing terminal sulfate-6-N-acetylglucosamine residues by glycosidic bond cleavage. When tested against its putative physiological substrate, sulfomucin, the only small molecular size product detected corresponded to a sulfate-6-N-acetylglucosamine residue. Thus the SGL can catalyze a reaction, formerly thought to be performed in bacteria by the combined actions of a N-acetylglucosamine-6-sulfatase and a N-acetylglucosaminidase. Inhibition studies on the SGL were carried out. Inhibitors of the SGL and those of the sulfatases were used to confirm the presence or absence of SGL-like activity in other bacteria that inhabit environments containing sulfomucin. Four isolates, including Prevotella strain RS2, of the thirteen strains tested, appeared to have SGL-like activity. This research on the SGL with its novel catalytic activity, suggests a new mechanism by which sulfomucin desulfation can occur. The physiological importance of the enzyme is postulated to be (i) to provide energy in the form of sulfate-6-N-acetylglucosamine, for the bacterium, (ii) to remove sulfate-6-N-acetylglucosamine groups present on mucin chains, thus creating or removing sites for different adhesins, and (iii) removal of inhibitory sulfate-6-N-acetylglucosamine groups from mucin chains that limit degradation of the chain by exoglycosidases and neuraminidases.

Whole document restricted at the request of the author. / The pathways by which nutritional status is signalled to the mammary glands and the metabolic sites targeted by these pathways have not been identified. Understanding of these pathways is of particular importance in species such as rodents and ruminants in which mammary metabolism is extremely sensitive to food availability. The studies in this thesis investigated mechanisms by which nutritional state was signalled to the mammary glands using the lactating rat as an experimental model. An in vivo preparation for analysis of the effects of altered nutritional state on substrate supply to, and uptake by, the inguinal mammary glands was established, as this had not been accurately performed before in rats. This preparation was used to record a large fall in mammary glucose uptake with food deprivation and a rapid restoration of uptake after refeeding. Results demonstrated that mammary glucose uptake, and hence mammary metabolism, was not closely linked to glucose supply in lactating rats. Glucose supply is unlikely to be a key factor signalling nutritional state to the mammary glands. Experiments in which the cutaneous branch of the posterior division of the femoral nerve innervating the inguinal mammary glands was severed showed that these neural pathways did not contribute to the maintenance of mammary metabolic activity in the fed and refed states or to the suppressed activity in food deprived rats. Neural signalling is unlikely to have a direct role in controlling mammary metabolism in rats. An in vitro method for measuring the uptake of glucose by rat mammary acini was developed. Insulin administration increased glucose uptake in acini from both fed and food deprived rats. Treatment with a crude gut extract enhanced uptake of glucose in acini from food deprived, lactating rats, but not in acini from fed rats. It was concluded that insulin and/or a factor from the gut may be involved in signalling the mammary gland of the restoration of nutrient supply when food deprived rats are refed. Proteomic studies were performed to investigate the effect of food deprivation and insulin on the abundance of intracellular proteins in acini from fed and food deprived, lactating rats. Analysis of over 800 protein spots detected 7 that were regulated by food deprivation, 26 that were regulated by insulin, and 9 in which the regulation was different in acini from fed and food deprived rats. None were regulated by both food deprivation and insulin. This suggests that decreased blood insulin concentration during food deprivation is unlikely to be the only signal that results in decreased mammary metabolism. Identification of proteins affected by food deprivation and insulin has led to new insights into some of the intracellular mechanisms regulated by these factors.

The pullulanase gene from Caldocellum saccharolyticum, an obligate thermophilic anaerobe, was sequenced and expressed in E. coli. Expression and substrate induction studies in E. coli showed that while gene expression was substrate inducible and the enzyme was exported into the growth medium in C. saccharolyticum, expression was non-inducible in E. coli and the enzyme remained in the cytoplasm. The nucleotide sequence of the pulA gene was shown to be 2478 basepairs (bp) in length, coding for a protein of 96 kDa. The proposed promoter sequences showed homology to both the standard E. coli sequences and the consensus sequences obtained from other C. saccharolyticum genes. The enzyme from the native organism was purified from the growth medium and shown to have a molecular mass of approximately 120 kDa. Periodic acid-Schiffs staining showed that this enzyme was glycosylated and substrate characterisation revealed that the enzyme debranched pullulan to produce only maltotriose, but hydrolysed amylopectin, amylose and β-limit dextran to produce a number of smaller oligosaccharides. The enzyme was expressed in E. coli from its own promoters and was purified from the cytoplasmic fraction. Substrate characterisation revealed that the enzyme debranched pullulan to produce only maltotriose, but had only limited activity on β-limit dextran and amylopectin, and no activity on amylose. The pullulanase gene was also expressed under the control of a heat-inducible overexpression system in E. coli and a copper-inducible expression system in yeast. Amino acid homology comparisons of the pullulanase to other pullulanase sequences and related enzymes revealed a high degree of homology, particularly around three highly conserved regions. In α-amylases amino acids in these regions are involved in catalytic activity, substrate binding and metal ion binding.

Prumnopitys taxifolia (Banks et Sol. ex D. Don) de Laub. (matai) and P. ferruginea (D. Don) de Laub. (miro) are two New Zealand podocarps with morphologically very different reproductive structures. In P. taxifolia the pollen cones are sessile on spicate fertile branches, whereas in P. ferruginea they are solitary on short scaly peduncles. Likewise, in P. taxifolia the ovulate strobilus is an attenuated spicate structure bearing globose seeds, whereas in P. ferruginea the reduced ovulate strobilus bears a dorsi-ventrally flattened, usually solitary seed on a slender scaly peduncle. Such differences throw doubt on the validity of the current delimitations of the genus Prumnopitys. A comparative embryological study of these two species was done in parallel with a morphological and anatomical study of the reproductive structures to fill gaps in their known reproductive life cycles, and to determine whether there are any differences in the male and female cones or in their embryogeny that are of taxonomic significance at the generic level. In addition the hydrodynamic properties of their saccate pollen grains were compared with that of the pollen of several other conifers and the spores of three ferns and one lycopod. Morphology and Embryology Embryologically these two species are shown to be more similar to each other and to the Chilean Prumnopitys andina than to other podocarp genera. For instance, in both species: the long tapered archegonia have prominent neck cells with a thickened cap overlying them; five free nuclear mitoses occur before cell formation in the proembryo; meiosis can result in both linear and non-linear tetrads of megaspores; the microgametophyte typically forms 7 or 8 prothallial cells before dispersal. However, new morphological and anatomical differences have emerged from this study. In the pollen cones of P. taxifolia, the microsporangia are free with a transverse stomium located close to the cone axis; in P. ferruginea they are fused with the transverse stomium located basally. In P. taxifolia ovules, a tanniniferous layer develops in the epimatium and not in the integument, and fusion of epimatium/integument/nucellus is greatest dorsi-ventrally; in P. ferruginea ovules a prominent tanniniferous layer develops only in the integument, and fusion of epimatium/integument/nucellus is greatest laterally. Furthermore, observations of insect predation point to biochemical differences between P. taxifolia and P. ferruginea. An undescribed species of gall midge appears to be specialized exclusively for the P. taxifolia life cycle, and several other insect larvae prey on the developing and mature seeds, whereas the ovules/seeds of P. ferruginea appear to have almost no insect predators. Morphologically, anatomically and biochemically, P. taxifolia and P. ferruginea may therefore be sufficiently different to warrant being two separate genera; comparable differences have been used to distinguish other genera in the Podocarpaceae. Since embryogeny is likely to be under less evolutionary pressure than morphology and anatomy, and since P. taxifolia (along with P. andina) is regarded by some as basal in the conifers, with P. ferruginea less so, their embryological similarity does not necessarily indicate close relationship, only that they are closer to each other than to the other podocarps. Although their morphological and anatomical differences may be of taxonomic significance, a better understanding of the other species currently in Prumnopitys is necessary before the relationships within the genus can be clarified. Pollen Hydrodynamics Despite the morphological differences in their ovulate cones, P. taxifolia and P. ferruginea have very similar pollination mechanisms involving an inverted micropyle, a pollination drop and saccate pollen. Saccate grains have sometimes been referred to as 'non-wettable' due to their buoyant properties, while non-saccate pollen grains have been described as 'wettable'. The hydrodynamic properties of saccate pollen grains of seven podocarp species in five genera, Dacrydium Sol. ex G. Forst., Dacrycarpus (Endl.) de Laub., Manoao Molloy, Podocarpus L'Hér. ex pers. and Prumnopitys Phil. have been tested in water, together with saccate and non-saccate pollen of four other conifer genera, Cedrus Trew (Pinaceae), Cephalotaxus Siebold & Zucc. ex Endl. (Cephalotaxaceae), Cupressus L. (Cupressaceae) and Phyllocladus Rich. ex Mirb. (Phyllocladaceae), and spores of three fern species and one lycopod species. All four spore types studied were non-wettable, whereas the bisaccate and trisaccate pollen types, like all the other conifer pollen types, were wettable, enabling the grains to cross the surface tension barrier of water. Once past this barrier, grain behaviour was governed by presence or absence of sacci. Non-saccate and vestigially saccate grains sank, whereas saccate grains behaved like air bubbles, floating up to the highest point. In addition, the grains were observed to float in water with sacci uppermost, consistent with the suggestion that distally-placed sacci serve to orientate the germinal furrow of the pollen grain towards the nucellus of an inverted ovule. Observations of pollen grains in the pollen chambers of naturally pollinated Prumnopitys ovules confirmed this. The combination of buoyancy and wettability in saccate pollen has implications for the efficiency of the typical podocarp pollination mechanism. Since saccate pollen, inverted ovules and pollination drops are also characteristic of the Pinaceae, the similarities in the pollination mechanisms and the pollen grains of P. taxifolia and P. ferruginea have no taxonomic significance at the generic level.

Potyvirus is the largest and one of the most economically important of the virus genera infecting plants. The complexities of potyvirus identification resulting from many different species, mixed infections, emerging new viruses, new hosts, and new vectors, etc., often requires the use of multiple detection methods which is time consumable and costly. Therefore an assay that can test for a range of potyviruses simultaneously, with good specificity and sensitivity, is desirable. This study looked at the feasibility of producing an oligonucleotide microarray for detection and identification of potyviruses at both species and strain level. Thirty plant samples with suspected potyvirus infections were collected from field and research laboratories in New Zealand and partial NIb gene, complete CP gene and 3΄UTR were sequenced. Twelve definitive potyviruses, one tentative potyvirus, one non-potyvirus, and one novel potyviruslike sequence were identified, six of which were first records for New Zealand. Sequence analysis showed that NIb and CP genes and the 3΄UTR contained both conserved and variable sequences which were used to design both species and strain level specific probes. Four Potyvirus species were chosen for a “proof of concept” study and probes were designed using two different software programs (ROSO and CAG software). A total of eighty five probes including 33 perfect-match and 52 mismatch probes were selected to represent the four targeted potyviruses. Each probe was synthesized with spacers of either 6 or 12 poly-cytosine or poly-thymine at the 5΄ terminus. Arrays showed high specificity to the targets when tested using nineteen different geographically diverse potyvirus isolates representing the four target species, four distinct but closely related New Zealand potyviruses, and four healthy plant species. Factors affecting the hybridization efficiency, e.g. the size of the target fragments, secondary structure of probes and targets, label type, strandedness, Tm and GC content of probes, were also investigated. The approaches and protocols developed in this study should form a useful basis for developing other potyviruses arrays and the results also provide useful insights into issues of generic interest for the development of arrays for detecting other pathogens.

New Zealand is a land with a unique and very diversified flora which, along with the great range of habitats to be found throughout the country, has given rise to some remarkable plant communities. As a result of a considerable volume of ecological research, mainly by the efforts of Cockayne, our plant associations are well known and delimited. There have, however, been few detailed analyses of their structure and of the factors operating in any of them, Cockayne's work being primarily descriptive. From the developmental point of view the general life-history of the Kauri forest has been known for many years, but nothing more detailed is known about the changes occuring. It is for the above reason that an attempt has been made in the present research to try and elucidate some of these factors while there are still considerable areas of Kauri forest in a more or less untouched state.

Annual reproductive cycles and the related changes in the pyloric caeca of the New Zealand starfish Stichaster australis and Coscinasterias calamaria (Echinodermata, Asteroidea) were determined at Maori Bay on the west coast of Auckland. Both species were shown to have clearly defined reproductive and pyloric caeca cycles, closely repeated from year to year. Pyloric caeca cycles showed an approximate although slightly displaced inverse relationship to gonad cycles. Changes in the gonad shown by histological sections confirmed these cycles. In addition to sexual reproduction C. calamaria may reproduce asexually by autotomy. Frequency of asexual reproduction did not vary seasonally and may be related to physiological stress or mechanical damage. Oocytes of S. australis and C. calamaria were fertilized in the laboratory and the resulting larvae reared through to metamorphosed starfish. The methods used in larval culture and embryological and larval development are described. Larvae of S. australis and C. calamaria closely resemble the larvae of Asterias rubens, described by Gemmill (1914), and of other planktotrophic asteroid larvae. Abnormal larvae found in some cultures are thought to result from, unsuitable culture conditions. It was found that oocytes of both species could be cross fertilized with sperm of the other. The settling behaviour was described and substrate preferences of advanced brachiolaria larvae of S. australis and C. calamaria were determined with larvae reared in the laboratory. These results were correlated with field observations of habitat preferences of juvenile starfish. C. calamaria larvae settled on almost any hard substratum provided it was coated with a primary film. Recently metamorphosed C. calamaria could not be located at Maori Bay and it is inferred that there is low recruitment from the plankton to the Maori Bay population. S. australis larvae would only settle, metamorphose and feed on the encrusting coralline alga Mesophyllum insigne. Nursery areas of offshore boulders covered with this alga were located at Maori Bay and these were found to support populations of juvenile starfish. Mesophyllum insigne appears to provide a stable and abundant food source for juvenile S. australis. The structure of the brachiolar arms and adhesive disc of S. australis and C. calamaria was determined from light microscopy and from scanning and transmission electron microscopy. The structure of these organs was very similar in S. australis and C. calamaria. The brachiolar arms are comprised of a stem region terminating in a crown of adhesive papillae. Adhesive papillae are made up of a variety of secretory cell types. Principal among these are elongated cells producing very electron dense secretory particles. These are released at the free cell surface attached to cilia. secretory particles appear to be important in temporary attachment of the brachiolar arms to the substratum. Ciliary sense cells, possibly used in the recognition of specific substrates are located at the tip of adhesive papillae. The adhesive disc is comprised of large cells packed with secretory droplets and elongated intracellular fibres. In the attached adhesive disc secretory droplets have been lost, having formed the cement attaching the disc to the substratum. The intracellular fibres are the principal anchoring structures running from the disc surface microvilli (locked into the attachment cement) to the underlying connective tissue of the attachment stalk. S. australis juveniles reared in the laboratory were maintained on Mesophyllum insigne substrata and the growth rates and feeding behaviour of individual starfish was determined. Growth of yearly settlement cohorts on a nursery site on the shore used in conjunction with laboratory results gave a fairly clear picture of growth after settlement. Growth rates followed a typical logistic curve, growth being slow initially and becoming more rapid later. The numbers of juveniles recruited to nursery areas vary from year to year but mortality following settlement appears to be low. It was found that juvenile S. australis predate M. insigne exclusively until they reach approximately 0.8cm in diameter (7-8 months old) when they may occasionally predate juvenile perna canaliculus. The incidence of carnivorous feeding gradually increases until juveniles are approximately 2.0-2.5cm in diameter (15-18 months old) by which time they are exclusively carnivorous on small P. canaliculus. As growth continues juvenile starfish gradually migrate from nursery areas to adjacent reefs where dense beds of adult P. canaliculus occur. Starfish of this species become sexually mature when they reach approximately 8.0cm in diameter. Juvenile C. calamaria reared in the laboratory feed on the primary film coating the substratum. Field observations at Leigh showed juveniles of 0.8-0.9cm diameter to be feeding on small barnacles on the undersides of boulders. Juveniles could not be reared to this size in the laboratory and the point at which juveniles change from a herbivorous or microphagous to a carnivorous diet is still unknown. Because of frequent autotomy it was not possible to determine growth rates of C. calamaria in the laboratory. Starfish of this species become sexually mature they reach 5.0-10.0cm in diameter. / Whole document restricted, but available by request, use the feedback form to request access.

Species recognition is an integral component of mate selection and must occur in all sexually reproducing organisms to avoid costly hybridisation. Species recognition abilities may be comprised of both innate components and experience during ontogeny through the learning of visual, acoustic, and other sensory species-specific cues. But how greatly is the ability to recognise one‟s own species (conspecifics) over others (heterospecifics) dependent on the phylogeographic relationship of the array of potential species as social partners and to what extent is the discriminatory behaviour modulated by subject ontogeny versus species identity? Using a model system, which is widely studied in all disciplines of avian research, the zebra finch (Taeniopygia guttata castanotis), I aimed to investigate the visual and acoustic cues involved in conspecific recognition by both female and male individuals of this species. I used an array of previously untested phylogeographically relevant estrildid heterospecifics as my stimuli and tested subjects of diverse experimental ontogenetic treatments. By scoring a wide-selection of measured behavioural responses my research indicates that female and male zebra finches prefer live conspecifics over live phylogeographically relevant heterospecific stimuli and this preference is more consistent by females than males. Female zebra finches rely on both visual and acoustic features of potential social partners for accurate species discrimination; in this regard video playbacks or the diverse colour morphs of domesticated zebra finches may be useful tools for further experimentation. Additionally, females display significant individuality in their behavioural responses which may be relevant for pair bonding decisions made by both sexes. I further documented that normally-reared zebra finches will prefer song playbacks of their own species but that both rearing in an indoor restricted acoustic environment of conspecifics or cross-fostering to another species will reduce discrimination preferences, although the results may depend on the behavioural metrics analysed. This dissertation is presented as a general overview with details of my specific contributions towards the work included in this thesis, followed by discrete review and data chapters, and a final general discussion.

Anthocyanin concentration is an important determinant of the colour of many fruits. In apple (Malus x domestica), centuries of breeding have produced numerous varieties in which levels of anthocyanin pigment vary widely and change differently in response to environmental and developmental stimuli. The apple fruit cortex is usually colourless, although germplasm does exist where the cortex is highly pigmented due to the accumulation of anthocyanins, carotenoids or chlorophylls. From studies in a diverse array of plants species, it is apparent that anthocyanin biosynthesis is controlled at the level of transcription. In this thesis the transcript levels of the anthocyanin biosynthetic genes in a red-fleshed apple are compared with a white-fleshed cultivar. A MYB transcription factor (TF), MdMYB10, was isolated and is shown to be similar in sequence to known anthocyanin regulators in other species. Further, this TF is shown to induce anthocyanin accumulation in both heterologous and homologous systems, generating pigmented patches in transient assays in tobacco leaves and highly pigmented apple plants following stable transformation with constitutively expressed MdMYB10. Efficient induction of anthocyanin biosynthesis in transient assays by MdMYB10 was dependent on the co-expression of two bHLH proteins from apple, MdbHLH3 and MdbHLH33. A strong correlation between expression of MdMYB10 and apple anthocyanin levels during fruit development suggests that this TF is responsible for controlling anthocyanin biosynthesis in apple fruit; in the red-fleshed cultivar and in the skin of other varieties, there is an induction of MdMYB10 expression concurrent with colour formation during development. Mutations in the genes encoding for either the biosynthetic or transcriptional regulation of the anthocyanin pathway have been linked to colour phenotypes. Generally this is a loss of function resulting in a reduction or a change in the distribution of anthocyanin. The upstream regulatory region of MdMYB10 was investigated and found to contain a rearrangement. This modification is responsible for increasing the level of anthocyanin throughout the plant to produce a striking phenotype that includes red foliage and red fruit flesh. It consists of a series of multiple repeats, forming a minisatellite-like structure that comprises five direct tandem repeats of a 23 base pair sequence. This MdMYB10 rearrangement is present in all the red foliage apple varieties and species tested, but in none of the white fleshed varieties. Transient assays demonstrated that the 23 bp sequence motif is a target of the MdMYB10 protein itself, and the number of repeat units correlates with an increase in transactivation by MdMYB10 protein. The repeat motif is capable of binding MdMYB10 protein in electrophoretic mobility shift assays. Taken together, these results indicate that a rearrangement in the promoter of MdMYB10 has generated an autoregulatory locus and this autoregulation is sufficient to account for the increase in MdMYB10 transcript levels and subsequent ectopic accumulation of anthocyanins throughout the plant. Characterisation of MdMYB10 and the rearrangement in the promoter region has implications for the development of new varieties through classical breeding or a biotechnological approach. Understanding whether this mutation is simply an allele of other recently published apple MYB TFs, or if this is the only R2R3 MYB TF involved in apple anthocyanin response, is a challenge for future research.

The cell cycle is one of the fundamental processes in nature, and is primarily concerned with the faithful replication of cellular contents, followed by even division to produce two identical daughter cells. It is made up of five discrete biochemical steps, comprising the interphase (G1, S and G2) and the mitotic phase (mitosis and cytokinesis), with two major regulatory checkpoints at G1 and G2. The focus of this research is the G2 checkpoint, which ensures the successful achievement of DNA replication, prior to the initiation of mitosis. Arrest or progression is principally mediated by the CDK1/cyclin B1 complex; phosphorylation of CDK1 by wee1 kinase prevents progression to mitosis, and subsequent dephosphorylation by the CDC25 phosphatases, initially the B isoform, leads to mitotic onset. The aim of this research was the biophysical and/or biochemical characterisation of the molecular complexes that form at the G2 checkpoint to regulate entry into mitosis. CDK1 and cyclin B1 were separately expressed and purified from baculovirus-infected Sf9 cells. The wee1/14-3-3β complex was also expressed and purified, incorporating either full length wee1 or a truncated version from which the N-terminal domain of wee1 was deleted. Both exhibited wee1 kinase activity, at equivalent levels, p<0.001, with a 2.4 fold increase in kinase activity when wee1 is bound by 14-3-3β, p>0.001. Tryptic digestion of the complex indicated that its architecture was likely to be flexible and open, particularly within the N-terminal domain of wee1. CD analyses indicated that the wee1/14-3-3β complex was folded, with 30-40% α-helical content and 10-20% β-sheet content. Dissociation experiments were unsuccessful, however, indicating a high strength of interaction between wee1 and 14-3-3β. The empirical stoichiometry of the complex was determined as 1:1; subsequent native molecular weight determination suggested that the minimal functional unit is likely to be a 2:2 wee1/14-3-3β arrangement. It was proposed that the structural architecture of this complex may be similar to the serotonin N-acetyltransferase/14-3-3ζ complex. Experiments to determine the structure experimentally, using either TEM or x-ray crystallography, were unsuccessful, as the complex appeared to exhibit a high degree of flexibility in solution. CDC25B was also expressed and purified, and was found to co-purify with a putative Sf9 14-3-3 protein. Consequently, it was re-cloned to co-express with 14-3-3β, and subsequent analysis of the resulting CDC25B/14-3-3β complex indicated that the empirical stoichiometry was 1:1, with the functional organization likely to be a 2:2 arrangement. It was proposed that the structural arrangement of this complex is most likely to be similar to that of the wee1/14-3-3β complex.