Browsing by Author "Govender, Kershini"

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    Characterisation of XvPrx2 : a type II peroxiredoxin isolated from the resurrection plant Xerophyta viscosa (Baker)
    (2006) Govender, Kershini; Mundree, Sagadevan G; Thomson, Jennifer Ann
    Knowledge of the biochemical and molecular mechanisms by which plants tolerate environmental stresses is necessary for genetic engineering approaches to improve crop performance. A unique feature of resurrection plants, such as Xerophyta viscosa, is their ability to cope with severe water loss of greater than 90%. A full-length cDNA library was synthesised from a cold stressed X viscosa plant. Sequencing and BLAST analysis revealed the identity of sixty genes. A type 2 peroxiredoxin (XvPrx2) was selected for further analyses as it was observed, by northern analyses, to be stress-inducible. The XvPrx2 protein was confirmed to be involved in the stress response by Western analyses. The XvPrx2 gene, which displays highest identity to a rice orthologue, has an open reading frame of 162 amino acids, and codes for a hydrophilic polypeptide of 162 residues with a predicted molecular weight of 17.5 kDa. The XvPrx2 polypeptide displays significant identity with other plant type II Prxs, with an absolutely conserved amino acid sequence proposed to constitute the active site of the enzyme (PGAFTPTCS). The XvPrx2 protein has a single cataly1ic cysteine residue at position 51 similar to Prxs from Oryza sativa and Candida boidinii. A mutated protein (XvV76C) was generated by converting the valine at position 76 to a cysteine resulting in a conformational change as determined by limited proteolysis. An in vitro DNA protection assay showed that, in the presence of either XvPrx2 or XvV76C, DNA protection occurred. In addition, an in vivo assay showed that increased protection was conferred on cell lines over-expressing either XvPrx2 or XvV76C. Several upstream promoter regions were identified for the XvPrx2 gene using the splinkerette method. Southern and two dimensional gel analyses revealed that multiple XvPrx2 homologues exist within the X viscosa genome. These homologues have similar pI values to Arabidopsis orthologues. Immuno-cytochemical data revealed that XvPrx2 is localised to the chloroplast, however, this could be attributed to cross reactivity with a chloroplastic homologue. Using YFP technology, the protein was observed to be expressed in the cytosol, and this location is supported by the absence of an upstream targeting signal in the XvPrx2 sequence. The XvPrx2 activity was maximal with DTT as electron donor and HzOz as substrate with t-BOOH being the next preferred. Using Trx£. coli a 2-15 fold lower enzyme activity was observed. The XvPrx2 activity with GSH was significantly lower and Grx had no measurable effect on this reaction. The XvV76C protein displayed significantly lower activity compared to XvPrx2 for all substrates assessed. Enzymatic kinetic parameter values determined for XvPrx2 using DTT as electron donor and HzOz as substrate were: Km = 45 IlM, V max = 278 Ilmol min-I.mg-I protein, kcat 6.173 x 103 s-1 and kcaJKm = 0.136 X 103 IlM-1.s-l. Based on knowledge-based models of XvPrx2 and XvV76C no structural differences were observed between the two molecules.
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    Population genetic studies of economically important Gracilaria and Gracilariopsis (Rhodophyta) in the south western Cape
    (2001) Govender, Kershini; Bolton, John J; Coyne, Vernon
    Gracilaria (Rhodophyta) encompasses some of the world's most valuable agarophytes and has accordingly been the subject of much research in recent decades. Gracilaria, however, has presented a problem to taxonomists over the years in that extreme phenotypic variability in certain entities, and the occurrence of similar morphologies among different taxa (in both Gracilaria and Gracilariopsis), combine to impede species recognition. Gracilaria and Gracilariopsis occur naturally from the Eastern Cape Province, South Africa to northern Namibia and the genetic variation within this widespread species or species complex is unknown. In this study, samples were connected from various locations within the Langebaan Lagoon-Saldanha Bay system and the nearby St. Helena Bay. Their positions were accurately mapped with the aid of a Global Positioning System (GPS). Reproductive structures are of great taxonomic importance within the Gracilariaceae. The local utility of reproductive features is questionable however, as south western Cape material is very seldom fertile which presents a problem for identification. External morphology lacks taxonomic significance due to the isomorphic nature of terete taxa. It was found that one cannot differentiate between Gracilaria gracilis and Gracilariopsis longissima based on external morphology. Adding to this, it was found that under varying ecological conditions different external morphologies occur. Although these specimens appear different externally, molecular studies show that they belong to the same species. Various molecular methods have proven successful in differentiating within and between populations, species and genera in the Gracilariaceae. Sequencing results of a 299 bp variable region of the ISS rRNA gene reveal that G. gracilis predominates in the Langebaan Lagoon-Saldanha Bay system, whereas Gp. Longissima predominates in the St. Helena Bay system. From the AFLP data one can infer that Saldanha Bay isolates display moderate divergence, probably due to favourable ecological conditions in this locality.