Erratum pasted onto front end-paper. / Copies of author's previously published article inserted. / Bibliography: leaves 167-180. / 180 leaves, [51] leaves of plates : ill. (chiefly col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Reanalyses the effects of the absence of CreA in the cell, raises antibodies for the detection of CreA and identifies new loci involved in carbon catabolite repression by using different genetic selection methods. / Thesis (Ph.D.)--University of Adelaide, Dept. of Genetics, 1999?

Thesis (MSc)--University of Stellenbosch, 2004. / ENGLISH ABSTRACT: This study aimed to assess the contribution of anaplerotic C prOVISIOn VIa phosphoenolpyruvate carboxylase (pEPc, EC 4.1.1.31), during Pi stress in the root and nodule components of Lupinus angustifolius. The role of PEPc in DIC metabolism in roots and nodules of phosphate-starved plants was studied. The symbioses involving leguminous plants and species of Rhizobium and Bradyrhizobium bacteria form an integral part of effective management ofN in the environment. In agricultural settings, roughly 80% of this biologically fixed N2 comes from this type of symbiotic relationship. Nitrogen-fixing bacteria in concert with legumes fix atmospheric nitrogen, which is then available to the infected plant. Worldwide, legumes are grown on approximately 250 Mha and they fix about 90 Tg (90 billion tons) of N, per year. The overall stoichiometry for nitrogen assimilation in the nodule requires one molecule of oxaloacetate to be converted to one molecule of asparagine per dinitrogen molecule fixed. One possible source for the required oxaloacetate is the reaction catalysed by PEPc. The reaction catalysed by PEPc is a major source of anaplerotic carbon for the plant and it is expected that this reaction will be even more important to plants under Pi stress, as the reaction is not ATP-dependent. Seeds of Lupinus angustifolius (cv. Wong a) were inoculated with Rhizobium sp. (Lupinus) bacteria and grown in hydroponic culture. Tanks were supplied with either 2 11MP04 (LP) or 2 mM P04 (HP) and air containing 360 ppm CO2. Roots experienced pronounced P stress with a greater decline in Pi, compared to nodules. Under P stress, PEPc activities increased in roots but not in nodules and these changes were not related to the expression of the enzyme. Root and nodular PEPc were not regulated by expression, but possibly by posttranslational control. LP roots also synthesised more pyruvate from malate than LP nodules. The role of pyruvate accumulation under Pi stress, was further highlighted by the metabolism of PEP via both the pyruvate kinase (PK, Ee 2.7.1.40) and PEPc routes. The enhanced PK activities supported these high pyruvate levels. The results show unequivocally that nodules do not experience P stress to the same extent as roots. Implications of the findings are that nodules require low P to function normally. Maintenance of phosphate levels in nodules may be at the expense of host. It can be suggested that when nodules are P-starved they can become aggressive scavengers for available P and even out-compete roots. / AFRIKAANSE OPSOMMING: Die doel van hierdie studie was om die bydrae van anaplerotiese koolstof-voorsiening via fosfo-enolpirovaatkarboksilase (pEPc, EC 4.1.1.31), tydens fosfaatstremming in die wortels en wortelknoppies van Lupinus angustifolius te bepaal. Die rol van PEPc in die metabolisme van opgeloste anorganiese koolstofdioksied in fosfaat-beperkte wortels en wortelknoppies is ondersoek. Die simbiose tussen peulplante en spesies van Rhizobium en Bradyrhizobium bakterieë vorm 'n integrale deel van die doeltreffende bestuur van stikstof in die omgewing. In die landbou word ongever 80 %van biologies-gefikseerde stikstof deur hierde simbiotiese verhouding geproduseer. Stikstotbindende bakterieë, in simbiose met peulplante, fikseer atmosferiese stikstof, wat dan beskikbaar is vir die geïnfekteerde plant. Wêreldwyd fikseer peulplante ongeveer 90 biljoen ton stikstof per jaar. Die algehele stoïgiometrie vir stikstof-fiksering in wortelknoppies vereis dat een molekule oksaalsuur na een molekule asparagien omgesit word per stikstofmolekule wat gefikseer word. Een moontlike bron vir die benodigde oksaalsuur is die reaksie wat deur PEPc gekataliseer word. Die reaksie wat deur PEPc gekataliseer word is 'n belangrike bron van anaplerotiese koolstof vir die plant en dit word vermoed dat hierdie reaksie van nog groter belang sal wees vir plante onder fosfaatstremming, omdat die reaksie nie ATP-afhanklik is nie. Sade van Lupinus angustifolius (cv. Wonga) is geïnokuleer met Rhizobium sp. (Lupinus) bakterieë en gekweek in waterkultuur. Tenke is voorsien met óf 2 !lM P04 (LP), óf 2 mM P04 (HP) en lug wat 360 ppm CO2 bevat het. Wortels het skerp fosfaatstremming ervaar, met 'n groter afname in Pi, vergelykbaar met wortelknoppies. Tydens fosfaatstremming het die aktiwiteit van PEPc toegeneem in wortels, maar nie in wortelknoppies nie en hierdie veranderinge was nie verwant aan die uitdrukking van die ensiem nie. PEPc van wortels en wortelknoppies is nie gereguleer deur uitdrukking nie, maar moontlik deur post-translasie kontrole. Wortels onder 'n lae-fosfaat voorsiening het ook meer pirodruiwesuur vanaf malaat gesintetiseer as wortelknoppies. Die rol van pirodruiwesuur-akkumulering tydens fosfaatstremming is verder beklemtoon deur die metabolisme van PEP via beide die pirovaatkinase- (PK, EC 2.7.1.40) en PEPc- roetes. Die verhoogde PK-aktiwiteite verklaar hierdie hoër vlakke van pirodruiwesuur. Die resultate toon ondubbelsinnig dat wortelknoppies me tot dieselfde mate fosfaatstremming ervaar as wortels nie. Dit impliseer dat wortelknoppies min fosfaat benodig om normal te funksioneer. Handhawing van fosfaatvlakke in wortelknoppies mag ten koste van die wortel wees. Dit is moontlik dat, wanneer wortelknoppies fosfaatbeperk is, hulle aggressiewe opruimers word vir beskikbare fosfaat en selfs beter funksioneer as die wortels.

Neural tube defects (NTDs) are a world health issue, affecting approximately 1 in every 1000 live births. These congenital defects arise from the improper closure of the neural tube during development, resulting in significant, life-threatening malformations of the central nervous system. Although it has been observed that supplementing women of child-bearing age with folates greatly decreases the chances of having an NTD affected baby, unfortunately these defects still occur. It is accepted that these complex disorders arise from a combination of genetic, environmental, and dietary influences. One such dietary influence is the one-carbon metabolism metabolite, homocysteine. Homocysteine is a byproduct of methylation reactions in the cell that exists in an inverse homeostasis with folate. Homocysteine can also undergo a transformation that allows it to then react with exposed lysine or cysteine residues on proteins, in a process known as N-homocysteinylation or S-homocysteinylation respectively. High levels of homocysteine have been long correlated with many disease states, including NTDs. One potential mechanism by which homocysteine confers its negative effects is through protein N-homocysteinylation. Here, a novel and high-throughput assay for N-homocysteinylation determination is described. This assay is shown to be accurate with mass spectrometry then shown to be biologically relevant using known hyperhomocysteinemia mouse models. This assay was then applied to a cohort of neural tube closure staged mouse embryos with two different genetic mutations that have previously been shown to predispose mice to NTDs. The genotypes explored here are mutations to the LRP6 gene and the Folr1 gene, both of which have been described as folate-responsive NTD mouse models. It was seen that maternal diet and embryonic genotype had the largest influence on the developmental outcome of these embryos; however, the inverse relationship between folate and homocysteine seemed to be established at this early time point, emphasizing the importance of the balance in one-carbon metabolism. One of these genes, LRP6, was then explored in a human cohort of spina bifida cases. Four novel mutations to the LRP6 gene were found and compared to the mouse model used in the previous study. One of the mutations found in the human population was seen to mimic that of the LRP6 mouse model, therefore expanding the potential of this NTD model. / text

"May 2004" / Addendum inside back page. / Bibliography: p. 99-114. / xii, 114 p. : ill. (some col.), photos (col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Describes the cloning and analysis of creD, and the characterization of the acrB gene, two components of a regulatory network controlling carbon source utilization in the filamentous fungus Aspergillus nidulans that involves ubiquitination and deubiquitination. / Thesis (Ph.D.)--University of Adelaide, School of Molecular and Biomedical Science, Discipline of Genetics, 2004

<p>Although low temperature plays an important role in determining agricultural yield, little is known about the effect on the underlying biochemical and physiological processes that influence plant growth. Photosynthesis and respiration are central to plant growth and both processes are heavily affected by temperature. However, many plants have the ability to cope with low temperature and resume growth by cold acclimating.</p><p>We have shown that enhancement of carbon fixation, an increased flux of carbon into sucrose and the recovery of diurnal export is crucial for the recovery of functional carbon metabolism at low temperature in Arabidopsis thaliana. The recovery of efflux is governed by increased expression of sucrose transporters along with changes in vascularisation. We also demonstrate the importance of controlling the flux of metabolites between the chloroplast and the cytosol by regulating the expression of AtTPT.</p><p>We further investigated the difference in response between leaves developed at low temperature but originating from warm grown Arabidopsis and leaves from plants grown from seed at low temperature. We were able to distinguish factors that respond specifically to low temperature from those that are connected to the actual stress. Substantial difference could be seen in the different metabolomes. One conclusion drawn is that the increase in sucrose reported at low temperature is an essential feature for life in the cold. </p><p>In an extended study we were able to transfer some of the key factor of cold acclimation in Arabidopsis to other species. The study included forbs, grasses and evergreen trees/shrubs showed that there are striking similarities in the extent and biochemical changes that underpin acclimation among the different functional groups.</p><p>Low temperature does not only influence growth of the leaves, perennial organs such as the corm of the ornamental plant Crocus vernus is also affected. However in these plants low temperature has a positive effect on the final size of the corm. We were able to show that this enhanced growth was an affect of increased cell size and thus increased sink capacity, which ultimately delays leaf senescence</p>

Although low temperature plays an important role in determining agricultural yield, little is known about the effect on the underlying biochemical and physiological processes that influence plant growth. Photosynthesis and respiration are central to plant growth and both processes are heavily affected by temperature. However, many plants have the ability to cope with low temperature and resume growth by cold acclimating. We have shown that enhancement of carbon fixation, an increased flux of carbon into sucrose and the recovery of diurnal export is crucial for the recovery of functional carbon metabolism at low temperature in Arabidopsis thaliana. The recovery of efflux is governed by increased expression of sucrose transporters along with changes in vascularisation. We also demonstrate the importance of controlling the flux of metabolites between the chloroplast and the cytosol by regulating the expression of AtTPT. We further investigated the difference in response between leaves developed at low temperature but originating from warm grown Arabidopsis and leaves from plants grown from seed at low temperature. We were able to distinguish factors that respond specifically to low temperature from those that are connected to the actual stress. Substantial difference could be seen in the different metabolomes. One conclusion drawn is that the increase in sucrose reported at low temperature is an essential feature for life in the cold. In an extended study we were able to transfer some of the key factor of cold acclimation in Arabidopsis to other species. The study included forbs, grasses and evergreen trees/shrubs showed that there are striking similarities in the extent and biochemical changes that underpin acclimation among the different functional groups. Low temperature does not only influence growth of the leaves, perennial organs such as the corm of the ornamental plant Crocus vernus is also affected. However in these plants low temperature has a positive effect on the final size of the corm. We were able to show that this enhanced growth was an affect of increased cell size and thus increased sink capacity, which ultimately delays leaf senescence

Le paludisme est une des maladies tropicales les plus dévastatrices au monde causée par des parasites protozoaires intracellulaires du genre Plasmodium. Cinq espèces de plasmodies sont responsables du paludisme chez l'homme et causent 600 000 décès par an principalement chez les enfants de moins de 5 ans et les femmes enceintes vivant dans les régions les plus pauvres du globe. Les parasites ont généré une résistance contre les chimiothérapies existantes et aucun vaccin efficace n'est encore disponible. Il est donc impératif d'identifier et de valider de nouvelles cibles qui peuvent être exploitées pour la découverte de nouveaux médicaments.Cette étude a porté sur la caractérisation d'un enzyme, la phosphoénolpyruvate carboxylase (PEPC), produit d'un gène spécifique au parasite et absent chez l'hôte humain, ce qui constitue l'un des pré-requis d'une cible potentielle pour la découverte de médicaments. Le gène avait été montré comme essentiel pour des parasites seulement en absence de malate ou de fumarate, suggérant un rôle de la protéine dans le métabolisme du carbone intermédiaire des parasites.Mes études de thèse avaient pour but de caractériser le rôle de la PEPC en utilisant la métabolomique. J'ai d'abord établi et normalisé une méthodologie d'analyses métabolomiques des globules rouges infectés par Plasmodium et optimisé l'analyse des métabolites hydrophiles présents dans le parasite intracellulaire et sa cellule hôte. Nous nous sommes concentrés sur les métabolites du métabolisme du carbone intermédiaire, où la PEPC pouvait jouer un rôle déterminant par analogie avec les plantes et les bactéries. Des analyses ciblées utilisant un marquage isotopique du métabolome à partir de 13C-U-glucose, 13C-bicarbonate et 13C, 15N-glutamine ont aussi été réalisées permettant de mieux appréhender les conséquences d'un KO de l'enzyme PEPC sur le métabolisme du parasite.Les données montrent que l'enzyme PEPC permet une fixation du bicarbonate et catalyse une réaction anaplérotique conduisant à du malate qui est introduit dans le cycle de l'acide tricarboxylique mitochondrial, transférant ainsi des équivalents réducteurs du cytoplasme à la mitochondrie et fournissant aussi un point d'entrée du squelette carboné dans le cycle. Les résultats montrent surtout que les parasites possèdent un cycle complet et de type oxydatif de l'acide tricarboxylique mitochondrial. Il parait y avoir trois points d'entrée: 1. l'acétyl CoA résultant du pyruvate généré par la glycolyse et décarboxylé dans la mitochondrie; 2. l'acide alpha-cétoglutarique provenant du glutamate, qui lui-même résulte de la désamination de la glutamine essentiellement fournie par l'environnement externe; 3. le malate, produit en aval de la malate déshydrogénase qui réduit l'oxaloacétate produit par la PEPC. En aval de la PEPC, la biosynthèse des pyrimidines opère grâce à l'activité de l'aspartate aminotransférase agissant sur oxaloacétate.En dehors du malate, le fumarate est le seul autre métabolite qui permet de s'opposer au défaut de croissance des parasites déficients en PEPC, ce qui a conduit à évaluer le rôle de la fumarase. À cette fin, l'étiquetage du gène endogène fumarase avec une étiquette HA, a permis de montrer que la protéine est exprimée dans les stades intra-érythrocytaires de P. falciparum et de montrer que la protéine se trouve à la fois dans la mitochondrie et le cytoplasme. La protéine recombinante a été exprimée avec succès et partiellement caractérisée biochimiquement. De nombreuses tentatives visant à générer des mutants de délétion génétique de P. falciparum n'ont pas abouti, laissant en suspens la question du caractère essentiel du gène pour les parasites. Cependant, il est possible de cibler le locus du gène via un marquage C-terminal. Ceci suggère que l'enzyme peut être essentielle pour la survie du parasite et donc une cible exploitable pour la découverte d'un type nouveau de médicament antipaludique. / Malaria is one of the world's most devastating tropical diseases caused by obligate intracellular protozoan parasites of the genus Plasmodium. Five species of these parasites cause malaria in humans and infection results in ~600,000 deaths annually primarily in children under the age of 5 and pregnant women living in the poorest areas of the globe. The parasites have an outstanding ability to generate resistance against existing chemotherapies and an efficacious vaccine is not available yet. Therefore it is imperative that attempts are being made to identify and validate new targets that can be exploited for future drug discovery.This study focused on the validation and elucidation of a parasite-specific gene product namely phosphoenolpyruvate carboxylase (PEPC), which is not present in the human host and thus has one of the pre-requisites of a potential drug target. The gene had been previously genetically validated and it was demonstrated that mutant parasites lacking pepc were only viable in the presence of malate or fumarate, suggesting a role of the protein in intermediary carbon metabolism of the parasites.My studies had the goal to assess the role of PEPC using a metabolomics approach. Initially the methodologies to perform metabolomics analyses of Plasmodium-infected RBCs were established and standardised and it was assessed how to best analyse the hydrophilic metabolites present in the intracellular parasites and its host cell. We focused on metabolites of intermediary carbon metabolism, as it is likely that PEPC is important for metabolic functions linked to this in the parasites, in analogy to plants and bacteria. While global metabolomics analyses were appealing, it was decided to apply a targeted metabolomics and comparative approach using stable isotope labelling of the parasite metabolomes with 13C-U-glucose, 13C-bicarbonate and 13C-,15N-glutamine to assess the consequences of the pepc knockout on parasite metabolism.The data demonstrated that PEPC has an anaplerotic function fixing bicarbonate and leading to generation of malate that is fed into the mitochondrial tricarboxylic acid cycle and so transfers reducing equivalents from cytoplasm to mitochondrion as well as providing an entry point of carbon skeleton into the cycle. The most important findings with respect to parasite mitochondrial metabolism were that the parasites possess a complete and oxidative tricarboxylic acid cycle, which appears to have three entry points: 1. Acetyl CoA resulting from glycolytically generated pyruvate that is decarboxylated in the mitochondrion; 2. α-ketoglutarate from the reaction of glutamate dehydrogenase and 3. malate, which is a downstream product of malate dehydrogenase that reduces oxaloacetate the reaction product of PEPC. Other downstream reactions supported by PEPC activity are pyrimidine biosynthesis through the activity of aspartate aminotransferase also acting on the PEPC-derived oxaloacetate.Apart from malate, fumarate was the only other metabolite that reversed the growth defect of pepc mutant parasites. Hence the role of fumarase in the parasites was also assessed. To this end the endogenous fumarase gene of P. falciparum was tagged with an HA-tag, which showed that the protein is expressed in the intra-erythrocytic stages of P. falciparum and demonstrated that the protein is located in both mitochondrion and cytoplasm. In addition, the recombinant protein was produced and partially biochemically characterised. Numerous independent attempts to generate genetic deletion mutants of P. falciparum were unsuccessful, leaving the question whether the gene is essential for the parasites unanswered. However, it was possible to manipulate the locus by C-terminal tagging of the fumarase gene suggesting that fumarase might be indeed essential for parasite survival and therefore possibly suitable for future drug design and discovery.

No description available.

AgaR, a protein in <i>Escherichia coli</i> thought to control the metabolism of N-acetylgalactosamine, is a member of the DeoR family of transcriptional regulators. Three transcriptional promoters within a cluster of genes containing the gene for AgaR were identified, specific for <i>agaR, agaZ</i> and <i>agaS</i>, and the transcription start sites mapped. Transcription from these promoters was specifically induced by N-acetylgalactosamine or galactosamine, though K-12 strains lacked the ability to utilize these as sole sources of carbon. The activity of these promoters was constitutively elevated in a strain in which <i>agaR</i> had been disrupted confirming that the promoters are subject to negative regulation by AgaR. AgaR-His6, purified using immobilized metal affinity chromatography, was used for DNase I footprint analysis of the promoter regions. Four operator sites bound by AgaR were identified. A putative consensus binding sequence for AgaR was proposed based on these four sites. <i>In vivo</i> and <i>in vitro</i> analysis of the <i>agaZ</i> promoter indicated that this promoter was activated by the cAMP-cAMP receptor protein (CRP). Expression from the <i>aga</i> promoters was less sensitive to catabolite repression in revertants capable of <i>N</i>-acetylgalactosamine utilization, suggesting that these revertants have mutation(s) that result in an elevated level of inducer for AgaR. A cluster of genes at minute 87.7 of the <i>E. coli</i> genome contains a gene that encodes another member of the DeoR family of transcriptional regulators. This protein, YihW, is more similar to GlpR, transcriptional regulator of <i>sn</i>-glycerol 3-phosphate metabolism in <i>E. coli</i>, than other members of the DeoR family. Despite the high degree of similarity, YihW lacked the ability to repress P_glpK, a promoter known to be controlled by GlpR. A variant of YihW containing substitutions in the putative recognition helix to more closely match the recognition helix of GlpR was also unable to repress P_glpK. Transcriptional promoters identified in this cluster of genes were negatively regulated by YihW. Regulation of genes involved in the metabolism of <i>sn</i>-glycerol 3-phosphate in <i>E. coli</i> by GlpR has been well characterized. However, the function of a protein (GlpE) encoded by a gene cotranscribed with that for GlpR was unknown prior to this work. GlpE was identified as a single-domain, 12-kDa rhodanese (thiosulfate:cyanide sulfurtransferase). The enzyme was purified to near homogeneity and characterized. As shown for other characterized rhodaneses, kinetic analysis revealed that catalysis occurs via an enzyme-sulfur intermediate utilizing a double-displacement mechanism requiring an active-site cysteine. K_m (SSO₃²⁻) and K_m (CN⁻) were determined to be 78 mM and 17 mM, respectively. The native molecular mass of GlpE was 22.5 kDa indicating that GlpE functions as a dimer. GlpE exhibited a kcat of 230 s-1. Thioredoxin, a small multifunctional dithiol protein, served as sulfur-acceptor substrate for GlpE with an apparent K_m of 34 mM when thiosulfate was near its K_m, suggesting thioredoxin may be a physiological substrate. / Ph. D.

No description available.