PEOPLE
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Post Docs
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Baker, Bienyameen Dr.
Synopsis of Research:
tbc
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PhD
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Garwe, Dahlia Ms.
Synopsis of Research:
tbc
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Govender, Kershini Ms.
Synopsis of Research:
Gene: Trx2 (Peroxiredoxin type 2)
Peroxiredoxins (Prxs) are enzymes that reduce hydrogen peroxide and alkyl hydroperoxides. Prxs are part of the antioxidant defense. These antioxidants therefore protects the plants from harmful ROS and is said to also play a role in signalling. These prxs are grouped in four classes:
a) 2-Cys Prx
b) PrxQ
c) PrxII
d) 1-Cys Prx
A full length cDNA cold stressed library of Xerophyta viscosa was synthesised. Sixty genes were selected based on size considerations and sequence information. Sequence alignment and blast searches revealed their identity based on homology to characterised genes. Eight genes were selected for further analyses. Five genes were probed against mRNA obtained from stressed [cold (4°C), dehydration/rehydration, abscissic acid (100 mM) and salt (150 mM)] and non stressed X. viscosa plants to determine expression patterns during the respective stresses. Consequently, XVTrx2 was selected as it displayed characteristic stress inducible expression.
Northern blot analyses [viz. low temperature (4°C), dehydration/rehydration, abscissic acid (ABA, 100mM), NaCl (150 mM), heat stress (42°C) and high light (1500 mmol/m2/s)] proved that this gene is differentially expressed depending on the type of stress.
Southern blot analyses has confirmed the presence of XVTrx2 in the X. viscosa genome.
The following review article has already been published:
Mundree, S.G., B. Baker, S. Mowla, S. Peters, S. Marais, C. Vander Willigen, K. Govender, A. Maredza, S. Muyanga, J.M. Farrant and J.A. Thomson. 2002. Physiological and Molecular Insights into Drought Tolerance. Afri. J. Biotech. 1(2):28-38.
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Maredza, Alice Ms
Synopsis of Research:
BSc Honours in Biochemistry (University of Zimbabwe), MSc in Biotechnology (University of Zimbabwe)
Phd student
Research interests
Aldose reductase (AR) is one of the genes that were identified from a Xerophyta viscosa dehydration library using the strategy of 'complementation by functional sufficiency'. Sorbitol mutants of E. coli that contained ALDRXV4, the gene coding for AR in X. viscosa, were able to grow on media supplemented with sorbitol showing that the gene is involved in osmotic stress tolerance. AR converts glucose to sorbitol, a known osmoprotectant. Northern and western blot analyses showed that the AR gene is expressed and the enzyme is synthesised in X. viscosa under dehydration conditions. The possible functions of AR during dessication and its contribution to drought stress tolerance will be studied. The overall objective of the project is to determine whether genes coding for single stress-protective proteins can confer the same tolerance characteristics if cloned into susceptible plants such as Arabidopsis thaliana and Digitaria sanguinalis.
Objectives
a) To transform A. thaliana and D. sanguinalis with ALDRXV4, the gene coding for the AR,
b) To determine whether ALDRXV4 confers osmotic stress tolerance to transgenic A. thaliana and D. sanguinalis,
c) To characterise the transgenic plants at the physiological, molecular and biochemical levels.
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Mowla, Shaheen Ms.
Synopsis of Research:
Joined UCT in 1997. Graduated with BSc (Microbiology & Biochemistry) in 1999.
Graduated with BSc Hons in Microbiology in 2000.
Currently doing her final year of PhD (Plant Biotechnology).
Project title:
Molecular characterization of a novel stress-inducible antioxidant gene, XvPer1, from the resurrection plant Xerophyta viscosa Baker.
Environmental stresses such as drought, heat and freezing occur in various parts of the world, especially in response to global climate change. As a result crop plant productivity is negatively affected globally, but more so in sub-Saharan Africa which, despite the Green Revolution, is still struggling to feed its people (Thomson, 2002). Nearly 200 million of the 600 million people who live there are chronically undernourished.
Africa needs to improve crop productivity. Most technologies developed to boost production and economic growth during the Green Revolution were not applied in Africa (Conway and Sechler, 2000). Scientists are now investigating the development and use of GM crops geared specifically for Africa.
XvPer1 was isolated and identified as a stress-inducible gene (Ndima et al. 2001). The gene codes for a 1-Cys peroxiredoxin. Peroxiredoxins are one of the most recently discovered types of enzymatic antioxidants (Chae et al. 1994); being active on substrates such as hydroperoxides and alkyl hydroperoxides. A consequence of many environmental stresses is oxidative stress within the plant, i.e. the accumulation of reactive oxygen species (ROS) within cells, damaging cellular structures (Foyer et al. 1994).
The cDNA named XvPer1, was isolated from X. viscosa by differential screening of a cDNA library. Characterization of the cDNA showed that XvPer1 has an ORF of 849 bp encoding a polypeptide of 210 residues. The XvPer1 cDNA contains a putative polyadenylation site and codes for a putative bipartite nuclear localization signal (NLS) near the 3'-end. The cDNA corresponds to 1-Cys peroxiredoxin, an evolutionary conserved thiol-specific antioxidant enzyme. XvPer1 polypeptide shows significant sequence identity (~70%) to other recently identified plant 1-Cys peroxiredoxins. The amino acid sequence proposed to constitute the active site of the enzyme (PVCTTE) is highly conserved in XvPer1. Southern blot analysis revealed a single copy of XvPer1 in the genome. Analysis of the gene expression using northern blot indicated that the transcript is absent in fully hydrated X. viscosa tissues but is induced under abiotic stresses such as dehydration, heat (42oC),high light (1500 mmol m-2 s-1) and when treated with abscisic acid (100 mM) and sodium chloride (150 mM). Western blot data correlated with the patterns of expression of XvPer1. Preliminary localization studies using immunofluorescence revealed that XvPer1 is localized in the nucleus of dehydrated X. viscosa leaf cells. These results suggest that the stress-inducible gene Xvper1 may function to protect nucleic acids within the nucleus against oxidative injury.
Current experiments:
XvPer1 has been cloned into the plant expression vector pA53 and transformed (biolistics) into the monocot model plant Digitaria sanguinalis (crabplant). At this point in time, putative transgenics are being analyzed.
XvPer1 was also cloned into the yeast expression vector pYES2 and transformed into yeast (Saccharomyces cerevisiae) oxidative mutants TSA1, TSA2 and YBL064C. The aim is to be able to investigate the oxidative capability of the XvPer1 protein.
Electron microscopy studies are also to be undertaken by immuno-gold localization in order to localize the exact location of XvPer1 in the X. viscosa vegetative tissue, as well as seeds and roots.
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MSc
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Brocklehurst, David Mr.
Synopsis of Research:
My MSc project is focused on the cloning and characterisation of a G-box
binding protein from a resurrection grass species, Eragrostis nindensis.
The G-box is a plant DNA cis-acting element involved in the regulation
of gene expression in response to a range of environmental signals
including anaerobiosis, dehydration and light as well as by abscisic
acid (ABA). Basic leucine zipper (bZIP) transcription factors have been
shown to specifically bind and activate transcription from G-boxes in a
dimerized form. I have cloned a 1.4 kb cDNA for a bZIP class
transcription factor, designated EnGBF1, by degenerate RT-PCR. EnGBF1
shows greatest homology to GBF1 from maize which is induced during
flooding stress as well as high homology to OSBZ8 from rice which is
induced by ABA treatment. Southern blot analysis indicates that EnGBF1
is present as a single copy gene in the E. nindensis genome. Further
characterisation of this gene will include studying the expression
profile of EnGBF1 in response to dehydration and ABA treatment as well
as elucidating the DNA sequence-binding specificity of the recombinant
protein.
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Chopera, Denis Mr
Synopsis of Research:
Graduated with a BSc Honours in Biochemistry (first class) from the University of Zimbabwe (2002). Worked on the Galactinol Synthase (GolS) gene. The gene encodes the enzyme that catalyses the committed step in the biosynthesis of raffinose family oligosaccharides (RFOs). The RFOs play an important role in the physiology of plants which include acting as osmoprotectants during various osmotic stresses. They (RFOs) are, however, a limitation to the utilisation of legumes as protein sources in human diets. Monogastrics lack the enzyme, a-galactosidase which cleaves the a-galactosidic linkages in RFOs. When ingested, the RFOs pass into the large intestine where they are anaerobically fermented by microflora to produce gases such as methane and hydrogen resulting in the acidification of the gut. The long term goal of the project was to produce transgenic legumes that express low levels of RFOs using RNA interference (RNAi).
At present, I am working on the molecular and biochemical characterization of XvIno1, a myo-inositol-1-phosphate synthase gene from Xerophyta viscosa. Myo-inositol-1-phosphate synthase catalyses the conversion of glucose-6-phosphate to myo-inositol-1-phosphate which is subsequently dephosphorylated to myo-inositol. Myo-inositol is a precursor for a number of important metabolites which include membrane components, storage molecules, phytohormones and a variety of osmoprotectants. The gene has been shown to be up-regulated during various stresses. If there is sufficient evidence that XvIno1 is involved in abiotic stress response in X. viscosa, the gene will be overexpressed in crop plants in combination with other stress-responsive genes in order to develop stress-tolerant crops.
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Conrad, Nailah Ms
Synopsis of Research:
Characterisation of stress responsive calcium signalling through investigation of XvCaM, a gene encoding the calcium-binding protein calmodulin.
In order for stress tolerance mechanisms to be initiated by a plant, the stress has to be perceived and this message has to be conveyed to parts of the plant where the necessary physiological changes can be brought about. Because of the ubiquitous use of Ca2+ in cell signalling, it is important to study the role of calcium signalling proteins in response to various stress conditions.
XvCaM was isolated from a cDNA library constructed from low temperature stressed Xerophyta viscosa. Initial molecular characterisation will include Southern blot analysis on genomic DNA to predict the copy number of the gene and confirm its presence in the genome. Northern blot analysis will be performed on low and high temperature stress, ABA, dehydrated/rehydrated and NaCl treated plant material to test the expression profile of the gene under various stress conditions.
XvCaM will be cloned into a high expression vector to isolate the recombinant protein. This will be used to raise antibodies which will be used in western blot analysis and immunolocalistion. The purified protein will be used to conduct a calcium-binding assay to demonstrate the binding capacity of the EF-hands in XVCAM.
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Davis, Emily Ms.
Synopsis of Research:
Characterisation of ALDRXV4, an aldose reductase homologue
Mundree et al. (2000) showed ALDRXV4 to be upregulated in response to desiccation stress.
Other abiotic stresses, such as high salinity and high and low temperature stress commonly cause a water deficit stress on the cell. Therefore I am looking at changes in expression levels of ALDRXV4 in response to these above-mentioned stresses.
Expression of ALDRXV4 in response to application of ABA will also be studied in order to determine whether or not this gene is regulated by an ABA-dependant or independent pathway.
ALDRXV4 protein expression and enzyme activity assays ascertain whether or not the functional protein is induced under these stress conditions. Aldose reductase is an enzyme that reduces nonphosphorylated sugars to sugar alcohols, specifically glucose to sorbitol. Therefore changes in cellular sugar content in response to these stresses will be determined in order to try to elucidate the effect of ALDRXV4 upregulation.
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Marais, Saberi Mr
Synopsis of Research:
Vacuolar ATPases have been implicated in response to salinity stress. V-ATPase response has been studied at the transcript, protein and enzymatic levels. Subunit c proteolipid is integral to V-ATPase function, and its c-DNAs have been identified and characterised in a range of angiosperm species such as Avena sativa, Mesembryanthemum crystallinum, Daucus carota, Beta vulgaris, Arabidopsis thaliana and Tortula ruralis. Generally, Subunit c's steady-state transcript levels display a 2 to 4-fold increase in response to salinity stress. As a result, the abundance of V-ATPase subunits increases and assembles to form functional V-ATPase heteromultimers. The resultant V-ATPases acidify intracellular compartments, including the vacuolar lumen, and contributes to a proton motive force capable of driving the secondary transport of ions and metabolites across membranes. Transcript of a subunit c homologue was found to be upregulated in X. viscosa in response to dehydration stress. My project deals with the molecular characterisation of this subunit c homologue, VATP1XV.
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Peters, Shaun Mr
Synopsis of Research:
Of the genes identified thus far in X. viscosa XVGols, a full length cDNA encoding for a galactinol synthase, may be an important component in compatible solute biosynthesis. XVGols was found to be up-regulated in the leaves of X. viscosa during drought stress. GolS enzymes represent the first step in the synthesis of Raffinose Family Oligosaccharides (RFOs), major soluble carbohydrates occurring in the seeds and other vegetative tissues of plants. Galactinol, an *-galactoside of myo-inositol, is an unusual molecule found exclusively in plants. Biosynthesis of galactinol is catalysed by galactosyltransferase and galactinol synthase, which utilises myo-inositol and UDP-galactose as substrates.
RFOs are derivatives of sucrose to which galactosyl units are added to the glucose moiety of sucrose, via *-(1,6) linkages. The most common are the trisaccharide raffinose, the tetrasaccharide stachyose and the pentasaccahride verbascose. The first step in the biosynthesis of RFOs is the reversible transfer of the galactosyl residue from the donor molecule, galactinol, to sucrose. This reaction results in the formation of raffinose, the first member in the family, which serves as the acceptor molecule for the transfer of another galactosyl residue from galactinol, to form stachyose. The reactions are catalysed by the enzymes raffinose synthase and stachyose synthase respectively. RFOs have been extensively characterised as principle agents in carbon translocation in plants but have also been observed to accumulate under cold, drought or salinity stress, implying a role for RFOs in stress adaptation.
The project presently encompasses characterising XvGolS expression (transcription and translation) under simulated environmental stress (drought, low and high temperature, high salinity) using Southern, northern, and western blot hybridisations. In-silico bioinformatic analyses on XvGolS indicated that the cDNA encoded a polypeptide with an ORF of 1014 bp corresponding to a MW of 38.04 kD. The cDNA showed highest identity to galactinol synthases from Brassica napus, Arabodpisis thaliana and Oryza sativa in the order of 72%, 72% and 70% respectively. A conserved functional domain belonging to the galactosyl tranferases, family 8, was present in all sequences analysed. We have shown that the locus is significantly induced 1h after exposure to low temperature shock (-20°).
Long term goals include generating transgenic Digitaria sanguinalis plants which over-express XvGolS and immuno-localisation .studies to determine intra-cellular location of XvGolS.
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Technician
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Bezuidenhout, Marion Mrs
Synopsis of Research:
Marion is the lab plant tissue culture technician.
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Alumni
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Bali, Nosi Ms.
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Bohms, Andreas Mr.
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Chigorimbo, Nyaradzo Ms.
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Ekmekci, Yasemin Ms.
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Ewer, Jonathan Mr.
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Msagati, Titus Mr.
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Muanga, Samson Dr.
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Oberle, Bettina Ms.
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Tawokam, Jean Bernard Mr.
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Vander Willigen, Clare Dr.
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Walford, Sally Ms.
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