Thesis (MSc (Genetics. Institute for Plant Biotechnology (IPB)))--Stellenbosch University, 2008. / Dextran is a high value glucose polymer used in medicine and an array of laboratory techniques. It is synthesised by lactic-acid bacteria from sucrose but has also reportedly been produced by Gluconobacter oxydans (G. oxydans) from a range of maltooligosaccharides (MOS) via the action of dextrin dextranase (DDase). In this study the presence of DDase is investigated in two G. oxydans strains (ATCC 621H and ATCC 19357) and shown to be present in the ATCC 19357 strain, but not in the ATCC 621H strain. The enzyme was partially purified from the ATCC 19357 strain, and its kinetic properties investigated. The partially purified protein was also digested with trypsin, and de novo peptide sequences obtained from it. Several attempts were made to obtain the gene coding for the DDase. These include amplifying an open reading frame from the G. oxydans genome coding for a glycosyltransferase with the approximate molecular weight of the DDase, using the peptide sequences obtained from the partially purified protein to design degenerate PCR primers and the production of a genomic DNA library for functional screening in E. coli. None of these approaches led to the successful isolation of the extracellular DDase sequence.

Thesis (MSc)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: See item for full abstract / AFRIKAANS OPSOMMING: Sien item vir volteks / IPB, National Research Foundation (NRF) and SASRI for funding

Thesis (M.A.)--Eastern Illinois University, 2010. / Includes bibliographical references (leaves 61-65).

Thesis (M.S.)--University of Missouri-Columbia, 2004. / Typescript. Includes bibliographical references (leaves 84-87). Also available on the Internet.

Thesis (PhD (Plant Biotechnology))--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: Virus Induced Gene Silencing (VIGS) was optimized to allow for the study of starch metabolism. The plastidial inorganic pyrophosphatase gene, for which a mutant has never been identified, was studied using VIGS and it was found to have a broad role in this subcellular compartment. The accumulation of inorganic pyrophosphate limited the production of starch, carotenoids, chlorophyll, and increased the plants susceptibility to drought stress. These effects highlight the importance of this enzyme in maintaining a low intraplastidial concentration of PPi providing an environment which facilitates these anabolic processes. Several genes involved in starch synthesis and degradation were also targeted with the aim of establishing a system of multiple gene silencing for the study of metabolic pathways. One, two and three genes were successfully silenced using this system which was validated based on previously published data. Interestingly, simultaneous silencing of the two isoforms of disproportionating enzyme led to a novel phenotype as a large reduction in starch instead of the expected increase was observed. / No Afrikaans abstract available

Thesisa (MSc (Genetics. Plant Biotechnology))--University of Stellenbosch, 2005. / The study had two main objectives: 1) to investigate specific enzyme activity profiles at various developmental stages and to determine possible implications for sucrose metabolism, 2) to incorporate enzyme activity data of different internodes to obtain a detailed model of every stage in the tissue maturation process. The most significant findings of the regulation of sucrose accumulation in this study are centred on three main point controls in sucrose metabolism pathway. Firstly, the maturation of sugarcane internodes coincided with an increase of SPS in most genotypes, and this underlines the key role of this enzyme in sucrose accumulation. Secondly, SuSy activity (cleavage reaction) correlated negatively with sucrose concentration and hence with tissue maturation process, in most of the varieties. This finding indicates that SuSy could well be implicated in sucrose metabolism. Thirdly, in vitro PFP activity was found to be negatively correlated to sucrose content in sugarcane varieties differing in amount of sucrose.

Thesis (MSc (Genetics. Plant Biotechnology)--University of Stellenbosch, 2009. / ENGLISH ABSTRACT: Glycogen was isolated from E. coli and analysed for the amount of phosphate present within it. It was confirmed that a significant proportion of the glucose residues were phosphorylated at the C6 position. This glycogen phosphate was found also in both glgb- (glycogen branching enzyme) and glgp- (glycogen phosphorylase enzyme) mutants, demonstrating that a mechanism for phosphate incorporation that does not involve GlgP alone, and which is capable of incorporating phosphate into linear glucans could exist. The degree of phosphorylation depended on the amount of phosphate present in the media, which less being incorporated in media where phosphate was reduced. Screening for glycogen phosphorylating genes using a E. coli genomic library in a functional expression system identified the malP gene as a possible candidate for incorporation of the phosphate at the C6 position. There was no difference, however, between the glycogen phosphate content of the mutant and wild type. Efforts were made to construct a malp-/glgp- double mutant, but these were unsuccessful. In addition the influence of plants and human proteins on yeast glycogen metabolism was also investigated. These proteins have been demonstrated to have an effect on starch or glycogen in humans, plant and E. coli, but the data from this study indicated that this was not the case in yeast. / AFRIKAANSE OPSOMMING: Glikogeen, wat geisoleer was uit E.coli was geanaliseer vir fosfaat inhoud daarin. Daar was gevind dat `n beduidende proporsie van die glukose residue gefosforileerd was op die C6 posisie. Hierdie gefosforileerde glikogeen was ook gevind in glg- (glikogeen vertakkingsensieme) en glgp- (glikogeen fosforileringsensieme) mutante wat daarop dui dat `n meganisme vir fosforilering bestaan was nie slegs aangewese is op die aktiwiteit van GlgP nie, en om fosfaat te inkorporeer in linêre glukane. Die graad van fosforilering was ook afhanklik van die hoeveelheid fosfaat teenwoordig in die medium, met gevolglik minder wat geinkorporeer kan word in medium waar fosfaat verminderd was. Seleksie-gebaseerde ondersoeking vir fosforileringsensieme van glikogeen deur gebruik te maak van E. coli genomiese biblioteke in `n funksionele uitdrukkingssisteem het die malP geen geidentifiseer as een van die moontlike kandidate wat verantwoordelik kan wees vir inkorporering van fosfaat in the C6 posisie. Daar was egter geen verskil in die fosfaat inhoud van glikogeen tussen die wilde tipe en die mutante. Pogings wat aangewend is om `n malp-/glgpdubbel mutant te konstrueer was onsuksesvol. Verder is die invloed van plant en mens proteine op gis glikogeen ook bestudeer. Vroeër is aangetoon dat hierdie proteine `n invloed op stysel en glikogeen het in mense, plante en E. coli, maar data van hierdie studie toon aan dat dit nie die geval in gis is nie.

Thesis (MSc (Genetics. Plant Biotechnology)) --University of Stellenbosch, 2009. / ENGLISH ABSTRACT: This project examined the role of proteins in starch phosphate metabolism. The first part was aimed at the functional characterization of the SEX4, LSF1 and LSF2 genes in both plants and bacteria. Constructs were produced to allow for expression of the three proteins in E. coli with the SEX4 and LSF2 proteins being successfully purified and used to produce antibodies. Immunoblot analysis indicated that the antibodies recognised the repective proteins in extracts, but it was not clear if they actually recognised the proteins or the GST tags they were fused to. Virus induced gene silencing constructs were also produced to allow repression of these three genes in Nicotiana benthamiana. This resulted in a starch excess phenotype being observed in the leaves of silenced plants which is consistent with the known or presumed roles for the genes. The antibodies produced were not specific enough to confirm that the respective protein were actually repressed, but it is likely that this was the case as plants infiltrated at the same time with a VIGS vector designed to repress phytoene desaturase exhibited a chlorophyll bleaching phenotype. These data confirm that SEX4 and LSF1 probable play the same role in N. benthamiana as in Arabidopsis, and provide evidence that LSF2 is also necessary for starch degradation. It was also attempted to characterise these proteins with respect to their substrate utilization by setting up a glyco-array experiment. Various potato starches from genetically modified plants were subjected to hydrolytic attack by starch degrading enzymes and fractionated by anion exchange chromatography to produce a multitude of glucans. These will be spotted onto glass filters and probed with the purified proteins to see if they bind to specific starch breakdown products preferentially. iv The project also involved investigating the effect the SEX4 protein has on E. coli glycogen contents. SEX4 was expressed in wild type and glgX mutant E. coli strains as it has been shown that this stops glycogen accumulation in the wild type, but not the glgX mutant. The cells were grown in liquid culture and glycogen contents measured. In liquid cultures SEX4 had no effect on glycogen contents in the wild type, possible because of problems with plasmid stability in the strain used. This final part of the project investigated the effect that a gwd mutation has on carbohydrate metabolism in leaves and fruits of the Micro-tom tomato cultivar. Starch and soluble sugar contents were measured in leaves and ripening fruits. A starch excess phenotype was found in the leaves, but no change in starch contents was determined in either the placenta or pericarp of the fruit. Soluble sugar contents were reduced in the fruit tissues, although the reason for this in unclear. / AFRIKAANSE OPSOMMING: Hierdie projek het die rol van proteine in stysel-fosfaat metabolisme ondersoek. Die eerste deel handel oor die funksionele karaktiseering van die SEX4, LSF1 en LSF2 gene in beide plante en bakteriee. Vektore is gekonstrueer om die uitdrukking van die drie proteine in E.coli toe te laat terwyl die SEX4 en LSF2 proteine suksesvol gesuiwer is vir die gebruik vir teenliggaam produksie. Immunoklad analises het getoon dat die teenligame die spesifieke proteine in die ekstrak herken het, maar dit was nie duidelik of dit die onderskeie proteine was of die GST-verklikker waaraan die onderskeie proteine verbind was nie. Virus geindiseerde geen onderdrukking konstrukte is ook geproduseer om toe te laat vir die onderdrukking van hierdie drie gene in Nicotiana benthamiana. Dit het ‘n stysel oorskot fenotipe tot gevolg gehad in die blare van onderdrukte plante wat konstant is met die bekende of voorgestelde rolle van die gene. Die teenliggame wat geproduseer is was nie spesifiek genoeg om te bewys dat die onderskeie proteine wel onderdrukis nie. Dit kon wel die geval gewees het want plante geinfiltreer op dieselfde tyd met ‘n VIGS vektor wat ontwerp is om phytoene desaturase te onderdruk het ‘n chlorofil bleikings fenotipe getoon. Hierdie data bevestig dus dat SEX4 en LSF1 moontlik dieselfde rol speel in N. benthamiana as in Arabidopsis, en toon bewyse dat LSF2 ook nodig is vir stysel afbreek. Karakterisasie van die onderskeie proteine met respek tot hul substraat gebruik is ondersoek deur ‘n gliko-array eksperiment. Verskillende aartappel stysels van genetiese gemodifiseerde plante was geonderwerp aan hydrolitiese afbreek deur stysel afbrekende ensieme en geskei deur anioon uitruilings chromotografie om veelvuldige glukans te vi vervaardig. Dit is geplaas op glas filters en is ondersoek saam met die gesuiwerde proteine om te sien of dit mag bind aan spesifieke stysel afbreek produkte. ‘n Verdere ondersoek is onderneem na die effek van die SEX4 protein op E. coli glikogeen inhoud. SEX4 was uitgedruk in die E .coli wildetipe en glgX mutant omdat dit reeds bewys is dat SEX4 glikogeen ophoping veroorsaak in die wildetipe maar nie in die glgX mutant. Die selle is opgegroei in vloeibare media en glikogeen inhoud is gemeet. In vloeibare media het SEX4 geen effek op die wildetipe se glikogeen inhoud nie wat moontlik kan wees as gevolg van plasmied stabiliteit in die E. coli ras wat gebruik is. Die finale deel van die projek was om die effek van ‘n gwd mutasie op koolhidraat metabolisme in blare en vrugte van die Micro-tom tamatie kultivar te ondersoek. Stysel en oplosbare suikers is gemeet in blare en rypwordende vrugte. ‘n Oortollige stysel fenotipe is in die blare gevind maar geen verandering in stysel inhoud is waargeneem in die plasenta of perikarp van die vrug nie. Oplosbare suiker inhoud het afgeneem in die vrugweefsel dog is die rede hiervoor nie te verstane.

Thesis (MSc (Genetics. Plant Biotechnology))--University of Stellenbosch, 2007. / Carbon partitioning was investigated in sugarcane (Saccharum spp. hybrids) that was genetically modified with sucrose: sucrose 1-fructosyltransferase (1-SST; EC 2.4.1.99) from Cynara scolymus. This enzyme catalyses the transfer of a fructosyl moiety from one sucrose molecule to another to produce the trisaccharide 1-kestose. Molecular characterisation of four sugarcane lines, regenerated after transformation, confirmed that two lines (2153 and 2121) were transgenic, with at least one intact copy of 1-SST present in line 2153, and a minimum of five copies (or portions thereof) present in line 2121. The novel gene was successfully transcribed and translated in both lines, as confirmed by cDNA gel blot hybridisation and HPLC analysis respectively. Kestose production was stable under field resembling conditions and levels of this trisaccharide progressively increased with increasing internodal maturity from 7.94 ± 2.96 nmol.g-1 fresh mass (fm) in internode 6 to 112.01 ± 17.42 nmol.g-1 fm in internode 16 of 2153, and by 1.05 ± 0.93 nmol.g-1 fm from the youngest to the oldest internode in line 2121. Sugarcane line 2153 contained 100 times more 1-kestose than 2121 in the oldest sampled internode hence the lines were referred to as high- and low-1-kestose producers. The production of 1-kestose did not reduce sucrose levels in the transgenics, instead they contained significantly higher levels of sucrose than the control line NCo310 (p<0.01, N=72). The production of this alternative sugar in addition to elevated sucrose levels significantly increased the total sugar content in the transgenic lines (p<0.01, N=72). Moreover, the high-1-kestose producer had statistically more total sugar than the low-1-kestose producer (p<0.01, N=72). Soluble acid invertase (SAI) and neutral invertase (NI, β-fructofuranosidase EC 3.2.1.26) from non-transgenic sugarcane internodal tissues were separated and partially purified. Kinetic analysis of the purified invertases revealed two isoforms of SAI eluting at approximately 100 mM KCl in a linear gradient while NI eluted at approximately 500 mM KCl. The final specific activities of SAI and NI were 88.57 pkat.mg-1 protein and 92.31 pkat.mg-1 protein, respectively. This implied a 16- fold purification of SAI, and 4- fold purification of NI. The pH optimum for NI was 7.0 and that for soluble acid invertase less than 5.0. Due to the broad pH activities of the invertases, activities significantly overlapped between pH 4.5 and 7.0. The affinity of these invertases for 1-kestose hydrolysis was tested. The invertases displayed hyperbolic saturation kinetics for sucrose, and had low affinities for 1-kestose with Km values ranging from 50 - 247 mM. Furthermore, the presence of 200 mM 1-kestose had an inhibitory effect on SAI-mediated sucrose hydrolysis reducing activity to 51 % and 54 % for isoform 1 and 2 respectively. To determine whether carbon allocation had been altered by the expression and activity of 1-SST, 14C whole-plant radiolabelling experiments were conducted. Radiolabelled CO2 was fed to the leaf subtending internode 5 and the allocation of carbon to different parts of the culm was assessed. There was no significant difference in the distribution of total radiolabel down the culm of the three sugarcane lines (p>0.05, N=72). However, the percentage of total radiolabel in the water-soluble fraction per internode in the high-1-kestose producer was significantly higher than the other two lines (p<0.01, N=72). As a result, the percentage radiolabel in the waterinsoluble fraction in this transgenic was concomitantly lower than in the other lines. Carbon was therefore redirected from the water-insoluble fraction to the water-soluble fraction to account for the additive production of 1-kestose. The expression of 1-SST in sugarcane therefore established an additional carbohydrate sink by the flow of carbon from the sucrose pool into 1-kestose. This did not lead to a depletion of the sucrose pool, but rather stimulated carbon channelling into this pathway, thereby increasing the non-structural carbohydrate content of the plant in one of the transgenics. The work described in this study is the first to report on carbon partitioning in 1- kestose-producing sugarcane grown under field resembling conditions. It contributes significantly to an improved understanding of carbon partitioning in the culm, and demonstrates that an alternative sugar can be produced in sugarcane under field resembling conditions.

The propagation of wild-type virus and a transgenic viral vector was examined in cultured plant cells to identify factors affecting viral infection of and accumulation in cultured cells and to determine if viral vectors could be used to facilitate the expression of heterologous proteins in vitro. Tobacco mosaic virus (TMV) accumulation was examined in Nicotiana tabacum and N. benthamiana suspension and hairy root cultures. TMV accumulation was superior in N. benthamiana hairy roots. Hairy roots were infected by adding TMV to the liquid culture medium at the same time as root inoculation. Hairy root growth was unaffected by virus infection. The distribution of virus within root mats from shake-flask grown cultures was non-uniform and the concentration of virus accumulated in replicate cultures was variable. When N. benthamiana hairy roots were infected using 1.5 μg mL-1 TMV, the average maximum concentration of virus accumulated in the biomass was 1.6 ?? 0.25 mg g-1 dry weight, or 15-fold lower than in TMV infected N. tabacum leaves. Virus coat protein accumulated to a level of (26 ?? 10)% total soluble protein in the hairy roots. Inoculum virus concentration and the medium in which infection was performed affected the virus yield and the percentage of inoculated cultures that accumulated virus. When cultures were inoculated using 9.0 μg mL-1 TMV, virus accumulated in the biomass to 4.2 ?? 0.60 mg g-1 dry weight. Proportional scale-up of hairy root infection in shake flasks did not result in constant virus concentrations in the scaled cultures. TMV accumulation in bioreactor-infected and -grown hairy roots was poor. N. benthamiana hairy roots were infected with a TMV-based vector (30B-GFPC3) that encoded Cycle 3 green fluorescent protein (GFP). TMV-GFPC3 was (260 ?? 140)-fold less infectious than TMV as measured by local lesion assays. Propagation of TMV-GFPC3 could not be confirmed using mass balance. GFP was not detected in the infected hairy roots or the culture medium. Hairy roots represent a potentially viable culture-based system for the in vitro production of virus and virus products when field-grown agricultural systems do not adequately address issues of containment or product safety.