Philosophiae Doctor - PhD (Biotechnology)

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Molecular detection and genetic manipulation of the Black Queen Cell Virus
(University of the Western Cape, 2002) Benjeddou, Mongi; Davison, Sean; Dept. of Biotechnology; Faculty of Science
The South African isolate of the Black Queen-Cell Virus (BQCV), a honeybee virus, was previously found to have an 8550 nucleotide genome excluding the poly(A) tail. Its genome contained two ORFs, a 5'-proximal ORF encoding a putative replicase protein and a 3'-proximal ORF encoding a capsid polyprotein.A reverse transcriptase PCR (RT -PCR) assay was developed for the detection of BQCV and acute bee-paralysis virus (ABPV). Complete genomes sequences w ere used to design unique PCR primers within a l-kb region from the 3' end of both genomes to amplify a fragment of 70.0 bp from BQCV and 900 bp from ABPV. The combined guanidinium thiocyanate and silica membrane method was used to extract total RNA from samples of healthy and laboratory-infected bee pupae. In a blind test, RT-PCR successfully identified the samples containing BQCV and ABPV. Sensitivities were of the order of 130 genome equivalents of purified BQCV and 1600 genome equivalents of ABPV.
The molecular characterization of trichoplusia ni single nucleocapsid nucleopolyhedrovirus: a study on early regulatory features
(University of the Western Cape, 2003) Wang, Weizhou; Davison, S
With the development of biological insecticides, many research efforts have been made in baculoviruses to investigate fundamental molecular aspects of these viruses, such as the function and regulation of genes, genome organization, mode of entry, DNA replication and virus factors that determine the host range and virulence. Previously, a South African Trichoplusia ni single capsid nuclear polyhedrosis virus (TnSNPV) isolate was partially characterized as a novel baculovirus. During the process of the characterization, a few late genes of the virus were identified. This thesis describes a molecular characterization of the TnSNPV early genes to gain insight into the functional roles of these genes, their unique features and further determination of the placement of TnSNPV in baculovirus phylogeny.
Characterization of two Arabidopsis thaliana genes with roles in plant homeostasis
(University of the Western Cape, 2004) Ludidi, Ndomelele Ndiko; Gehring, C. A.
Plants are continuously exposed to varying conditions in their environment, to which they have to adapt by manipulating various cellular processes. Environmental (abiotic) and pathogen (biotic) stress are challenges against which plants have to defend themselves. Many plant responses to stress stimuli are a result of cellular processes that can be divided into three sequential steps; namely signal perception, signal transduction m1d execution of a response. Stress signal perception is, in most of these cases, facilitated by cell surface or intracellular receptors that act to recognize molecules presented to the cell. In several cases, hormones are synthesized in response to stress signals and in turn these hormones are perceived by cellular receptors that trigger signal transduction cascades. Propagation of signal transduction cascades is a complex process that results from activation of various signaling molecules within the cell. Second messengers like calcium (Ca2+) and guanosine 3', 5'-cyclic monophosphate (cGMP) play a vital role in mediating many signal transduction processes. The result of these signal transduction cascades is, in most instances, expression of genes that contribute to the plant's ability to cope with the challenges presented to it. Plant natriuretic peptides (PNPs) are novel plant hormones that regulate water and salt homeostasis via cGMP-dependent signaling pathways that involve deployment of Ca2+. The aim of this study is to partially characterize a PNP and a guanylyl cyclase, both from Arabidopsis thaliana. Guanylyl cyclases synthesize cGMP from the hydrolysis of guanosine 5' -triphosphate (GTP) in the cell. The study also aims to investigate the effect of drought and salinity on cGMP levels in plants, using sorbitol to mimic the osmolarity/dehydration effect of drought and NaCl as a source of salinity stress and thus link NaCl and sorbitol responses to both AtPNP-A and cGMP up-regulation.
Identification and characterisation of a novel gene, DWNN, isolated from promoter-trapped Chinese hamster ovary cells
(University of the Western Cape, 2005) Skepu, Amanda; Rees, D.J.G.; Dept. of Biotechnology; Faculty of Science
The process of cytotoxic T lymphocyte (CTL) killing involves the recognition and destruction of foreign antigens by cytotoxic T cells and is of crucial importance to the defence of the organism against viral infections. Defects in this process can lead to various autoimmune diseases and cancer. The aim of this study was to identify more genes involved in the cell death pathway and to link CTL killing, apoptosis and cancer.
Cloning, expression and characterization of Novel Lipase and Esterases from Burkholderia multivorans UWC10
(2005) Rashamuse, Konanani J; Cowan, Don A
An esterase and lipase producing Burkholderia multivorans strain was isolated by culture enrichment strategies. A shotgun library of Burkholderia multivorans genomic DNA (prepared in E. coli/pUC18) was screened for lipase and esterase activities. Three positive recombinant clones, pTEND5, pHOLA6 and pRASHI4, conferring esterolytic and lipolytic phenotypes respectively, were identified. Full-length sequencing of DNA inserts was performed using subeloning and "primer-walking" strategies. Nucleotide sequence analysis revealed that the pRASH14 plasmid DNA consisted of two open reading frames (ORPI and ORP2) encoding 356 and 350 amino acids, respectively. Database searches revealed that ORPI and ORP2 were homologous to lipases and chaperones from subfamily I.2. In the pTEND5 sequence, an open reading frame consisting of 978 bp, encoding 326 amino acids, was identified. Database searches revealed that this open reading frame was homologous to family Vesterases. Nucleotide sequence analysis revealed that pHOLA6, plasmid DNA consisted of 1194 bp encoding 398 amino acids and showed homology to family VIII esterases. The primary structures of LipA, EstEFH5 and EstBL from pRASHI4, pTEND5 and pHOLA6, respectively, showed a classical GxSxG motif, which is conserved in many serine hydrolases. In addition, EstBL also showed a consensus SxxK motif, the serine of which acts as a catalytic nucleophile in class C B-lactames and some peptidases.
Characterization and engineering of Bacillus megaterium AS-35, for use in biodegradation of processed olive wastewater
(University of Western Cape, 2005) Van Schalkwyk, Antoinette; Cowan, Don A
The popularization and health benefits associated with the "Mediterranean diet" saw a world wide increase in the production and consumption of processed olives and olive oil. During the brining of table olives large quantities of processed olive waste water is seasonally generated. This blackish-brown, malodours liquid is rich in organic and phenolic compounds, which cause environmental problems upon discarding. Currently, processed wastewater is discarded into large evaporation ponds where it poses serious environmental risks. The biodegradation of organic substrates present in the olive wastewater is inhibited by the high concentrations of phenolic compounds.
Structure, enzymology and genetic engineering of Bacillus sp. RAPc8 nitrile hydratase
(University of the Western Cape, 2005) Tsekoa, Tsepo L.; Cowan, Don A.; Dept. of Biotechnology; Faculty of Science
Microbial nitrile hydratases (NHases) are important industrial enzymes that catalyse the conversion of nitriles to the corresponding amides. A thermostable, cobalt-type Bacillus sp. RAPc8 NHase was previously cloned and expressed in E. coli. In this study, the primary aim was to determine the molecular structure of Bacillus sp. RAPc8 NHase. The heterotetrameric enzyme was purified to near homogeneity using heatpurification, hydrophobic interaction chromatography and ion exchange chromatography. Purified NHase was crystallised using the hanging-drop vapourdiffusion method. Crystals produced in the presence of 30% PEG 400, 0.1M MES pH 6.5 and 0.1M magnesium chloride were selected for X-ray diffraction studies. These crystals diffracted well, with diffraction spots visible beyond 2.4Å, with little mosaicity. At 2.5Å, the data were 93% complete. The crystal structure of Bacillus sp. RAPc8 NHase was solved via molecular replacement using the crystal structure of Pseudonocardia thermophila NHase as a search model. The final refined structure had good refinement statistics and geometry. The overall fold was very similar to that of previously determined NHase structures. Bacillus sp. RAPc8 NHase was most similar to Bacillus smithii NHase (0.355År.m.s.d.) and least similar to Rhodococcus sp. R312 NHase (1.191Å r.m.s.d.). One cobalt atom per heterodimer was bound to a typical NHase metal-binding motif, with post-translationally modified cysteine residues among the ligands to the metal. The substrate-binding and catalytic cavity of Bacillus sp. RAPc8 NHase was identified and described in detail. Surface representation of the structure revealed an extended, curved solvent accessible channel with access to bulk solvent from two locations in the heterodimer. The amino-acid residues forming the channel were identified and the geometric dimensions measured. Enzyme inhibition kinetics indicated that benzonitrile was a potent uncompetitive inhibitor of NHase. This information was used to aid the genetic engineering of aromatic substrate specificity into Bacillus sp. RAPc8 NHase. Site-directed mutants of NHase were prepared using the Quickchange mutagenesis procedure. Mutant W76G showed a two to three fold decrease in benzonitrile inhibition compared with the wild-type. Analysis of the substrate channel of this mutant NHase showed an 11% increase in volume and a 20% increase in inner surface area compared to that of the wild-type NHase. Due to the lack of other significant differences between the two structures (an r.m.s.d. of only 0.101Å was observed), this difference was thought to be responsible for the decrease in benzonitrile inhibition. A structure-modelling based approach for assessing the likely structural differences that may result as a result of a specific mutation was suggested and tested. This approach may be of value in future mutagenesis work.
Microbial diversity and gene mining in Antarctic Dry Valley mineral soils
(University of the Western Cape, 2006) Smith, Jacques J.; Cowan, Don A.; Dept. of Biotechnology; Faculty of Science
Soil communities are regarded as among the most complex and diverse assemblages of microorganisms with estimated bacterial numbers in the order of 109-1 cells.g. Studies on extreme soils however, have reported lower cell densities, supporting the perception that the so-called extreme environments exhibit low species diversity. To assess the extent of microbial diversity within an extreme environment, the mineral soils of the Dry Valleys, Ross Dependency, Eastern Antarctica were investigated using 16S rDNA analysis. Three mineral soils designated MVG, PENP and BIS were analysed, each differing with respect to altitude, protein, lipid, water and DNA content. The mid-altitude sample, MVG, yielded the highest levels of DNA and the low altitude BIS soil contained the highest levels of protein, lipid and water. 16S clone libraries were constructed and 60 unique clones were identified and sequenced. BLASTn analysis revealed eight phylogenetic groups with Cyanobacteria, Actinobacteria and Acidobacteria representing the majority. The Cyanobacterial phylotypes were unique to the desiccated high-altitude soils of the PENP sample, suggesting a soil-borne Cyanobacterial population. 21% of the phylotypes identified were assigned as ‘uncultured’. DNA isolated from the Antarctic mineral soils was also used to construct a metagenomic clone library consisting of 90700 clones with an average insert size of 3.5 kb, representing an estimated 3.4% of the available metagenome. Activity-based screening of the library for genes conferring lipolytic activity yielded no positive clones. It is suggested that the failure to produce positive clones might be a result of insufficient nucleotide coverage of the metagenomic DNA. The metagenomic DNA extracted from the Dry Valley mineral soils was further analyzed using PCR. Two sets of degenerate primers based on conserved regions within lipolytic genes were used to target lipase and esterase genes. One set of primers was selected from a previous study. A second primer set was designed manually from amino acid alignments of true lipase genes from family I, sub-families I-VI. PCR analysis resulted in nine partial gene fragments varying between 240 bp and 300 bp. Bioinformatic analysis revealed that all nine partial gene fragments harboured α/β-hydrolase motifs, putatively identifying two esterases and three lipases from both bacterial and fungal origin.
Studies on the population genetics of Euphausiids: a comparison of patterns in plagic taxa displaying different distributions and life-histories
(University of the Western Cape, 2006) Harkins, Gordon W.; D’Amato, M.E.; Gibbons, M.J.; Faculty of Science
The systematic and population genetic relationships were characterised for three ecologically related euphausiid species: Euphausia lucens, E. recurva and E. vallentini. These species have different geographical distributions and life histories. All three species have a circumpolar distribution in the Southern Hemisphere while E. recurva is also distributed in the North Pacific. DNA sequence variation was determined for three regions of mitochondrial DNA and a single nuclear gene. It was conclusively demonstrated that both E. lucens and E. vallentini represent valid taxonomic species with fixed differences observed in both the nuclear and mitochondrial genes and that the low divergences previously reported for these species with 16SrRNA and CO1 resulted from a species misidentification. It was also shown that previous attempts to date the divergence between Antarctic and Sub-Antarctic euphausiid species based on 16SrRNA distances suffer from a large overestimation due to a calculation error.
Metagenomic approaches to gene discovery
(University of the Western Cape, 2006) Meyer, Quinton Christian; Cowan, Don A.; Dept. of Biotechnology; Faculty of Science
The classical approach to gene discovery has been to culture micro-organisms demonstrating a specific enzyme activity and then to recover the gene of interest through shotgun cloning. The realization that these standard microbiological methods provide limited access to the true microbial biodiversity and therefore the available microbial genetic diversity (collectively termed the Metagenome) has resulted in the development of environmental nucleic acid extraction technologies designed to access this wealth of genetic information, thereby avoiding the limitations of culture dependent genetic exploitation. In this work several gene discovery technologies was employed in an attempt to recover novel bacterial laccase genes (EC 1.10.3.2), a group of enzymes in which considerable biotechnological interest has been expressed. Metagenomic DNA extracted from two organic rich environmental samples was used as the source material for the construction of two genomic DNA libraries. The small insert plasmid based library derived from compost DNA consisted of approximately 106 clones at an average insert size of 2.7Kbp, equivalent to 2.6 Gbp of cloned environmental DNA. A Fosmid based large insert library derived from grape waste DNA consisted of approximately 44000 cfu at an average insert size of 25Kbp (1.1 Gbp cloned DNA). Both libraries were screened for laccase activity but failed to produce novel laccase genes. As an alternative approach, a multicopper oxidase specific PCR detection assay was developed using a laccase positive Streptomyces strain as a model organism. The newly designed primers were used to detect the presence of bacterial multicopper oxidases in environmental samples. This resulted in the identification of nine novel gene fragments showing identity ranging from 37 to 94% to published putative bacterial multicopper oxidase gene sequences. Three clones pMCO6, pMCO8 and pMCO9 were significantly smaller than those typically reported for bacterial laccases and were assigned to a recently described clade of Streptomyces bacterial multicopper oxidases. Two PCR based techniques were employed to attempt the recovery of flanking regions for two of these genes (pMCO7 and pMCO8). The use of TAIL-PCR resulted in the recovery of 90% of the pMCO7 ORF. As an alternative approach the Vectorette™ system was employed to recover the 3’ downstream region of pMCO8. The complexity of the DNA sample proved to be a considerable technical challenge for the implementation of both these techniques. The feasibility of both these approaches were however demonstrated in principle. Finally, in an attempt to expedite the recovery of fulllength copies of these genes a subtractive hybridization magnetic bead capture technique was adapted and employed to recover a full – length putative multicopper oxidase gene from a Streptomyces strain in a proof of concept experiment. The StrepA06pMCO gene fragment was used as a ‘driver’ against fragmented Streptomyces genomic DNA (‘tester’) and resulted in the recovery of a 1215 bp open reading frame. Unexpectedly, this ORF showed only 80% identity to the StrepA06pMCO gene sequence at nucleotide level, and 48% amino acid identity to a putative mco gene derived from a Norcardioides sp JS614.
Mining a Chinese hyperthermophilic metagenome
(University of the Western Cape, 2007) Du Plessis, Morne Graham; Cowan, Don A.; Arieff, Zainu; Dept. of Biotechnology; Faculty of Science
Metagenomic sequencing of environmental samples provide direct access to genomic information of organisms within the respective environments. This sequence information represents a significant resource for the identification and subsequent characterization of potentially novel genes, or known genes with acquired novel characteristics. Within this context, the thermophilic environments are of particular interest due to its potential for deriving novel thermostable enzymes with biotechnological and industrial applications. In this work metagenomic library construction, random sequencing and sequence analysis strategies were employed to enhance identification and characterisation of potentially novel genes, from a thermophilic soil sample. High molecular weight metagenomic DNA was extracted from two Chinese hydrothermal soil samples. This was used as source material for the construction of four genomic DNA libraries. The combined libraries were estimated to contain in the order of 1.3 million genes, which provides a rich resource for gene identification. Approximately 70 kbp of sequence data was generated from one of the libraries as a resource for sequence-based analysis. Initial BLAST analysis predicted the presence of 53 ORFs/partial ORFs. The BLAST similarity scores for the investigated ORFs were sufficiently high (>40%) to infer homology with database proteins while also being indicative of novel sequence variants of these database matches. In an attempt to enhance the potential for deriving more full length ORFs a novel strategy, based on WGA technology, was employed. This resulted in the recovery of the near complete sequence of partial ORF5, directly from the WGA DNA of the environmental sample. While the full length ORF5 could not be recovered, the feasibility of this novel approach, for enhanced metagenomic sequence recovery was proved in principle. The implementation of multiple insilico strategies resulted in the identification of two ORFs, classified as homologs of the DUF29 and Usp protein families respectively. The functional inference obtained from the integrated in-silico predictions was furthermore highly suggestive of a putative nucleotide binding/interaction role for both ORFs. A putative novel DNA polymerase gene (denoted TC11pol) was identified from the sequence data. Expression and characterization of the full length TC11pol did however not result in detectable polymerase activity. The implementation of a homology modeling approach proved succesfull for deriving a structural model of the polymerase that was used for: (i) deriving functional inferences of the potential activities of the polymerase and (ii) deriving a 5’ exonuclease deletion mutant for functional analysis. Expression and subsequent functional characterization of the putative 5’exo- TC11pol mutant resulted in detectable polymerase and 3’-5’ exonuclease activity at 37 and 45 oC, following a heat denaturation step at 55 oC for 1 hour. It was, therefore concluded that the putative 5’exo- TC11pol mutant was functionally equivalent to the Klenow fragment of E. coli, while exhibiting increased thermostability.
Identification of ruantitative trait loci controlling the requirement for chilling in vegetative budbreak in apple (Malus x domestica Borkh.)
(2008) Labuschagnè, Iwan; Rees, D.J.G.
The domesticated apple (Malus x domestica Borkh.) has been distributed into diverse climatic conditions worldwide for commercial production of fruit. Apple trees need exposure to cold temperatures, referred to as chill unit (CU) accumulation during winter, in order for budbreak to occur promptly and uniformly after winter. In warmer production areas the application of dormancy breaking chemicals has enabled successful production of high chilling requiring apple cultivars in suboptimal environmental conditions. In the Western Cape region of South Africa it is common orchard practice to apply dormancy breaking chemicals after winter in order to stimulate vegetative growth. If this is not done prolonged dormancy symptoms (PDS) are experienced which include extended rest, less synchronised breaking of buds and reduced branching. An increasing awareness of both global warming and the negative effects associated with the use of chemical sprays (for both pest and disease resistance and growth regulation) has resulted in the need to breed cultivars better adapted to current and future environmental conditions. The breeding of new cultivars using conventional breeding methods is a time consuming process, especially in perennial tree species with a long juvenile phase such as apple. The implementation of marker-assistedbreeding (MAB) and selection (MAS) will enable the selection of favourable genotypes at a very early seedling stage. Although markers linked to genes involved in disease resistance for a variety of known apple pathogens have been identified and are already in use in breeding programs, the genetic determinants of dormancy related characteristics residing within the bud itself (endodormancy) are poorly understood. This hampers the genetic improvement of such characters. Although this study focused on time of initial vegetative budbreak IVB, there are various other characteristics that can be associated with dormancy, such as position and number of budbreak and budbreak duration. The unravelling of the genetic basis of complex traits such as dormancy, can be done through the construction of a genetic linkage map followed by the identification of genomic regions, known as quantitative trait loci (QTL), that can be ssociated with the trait of interest. This study involved the construction of genetic linkage maps for two mapping pedigrees where the low chilling requiring cultivar ‘Anna’ was used as common male parent in crosses with the higher chilling requiring ‘Golden Delicious’ and ‘Sharpe’s Early’. A third mapping pedigree, with ‘Golden Delicious’ as female parent and ‘Prima’ as male parent, was also included. Maps consisted of transferable SSR markers only, facilitating the alignment with the proposed apple reference map (Silfverberg-Dilworth et al., 2006) and adherence to the common LG numbering system now being used for apple genetic linkage maps (Maliepaard et al., 1998). A number of newly developed EST-SSR markers are reported, some of which are candidates for filling large gaps between adjacent SSR markers on the apple reference map. An interactive database was developed to successfully manage the large amount of data generated during this investigation. A selective mapping, or bin mapping strategy (Vision et al., 2000) was developed for two of the three mapping populations in order to facilitate the incorporation and positioning of newly developed markers onto existing genetic linkage maps. This involves the screening of new markers on a small subset of the population, drastically reducing the cost and time involved. Genetic linkage maps constructed allowed for the detection of 18 putative QTLs affecting the time of IVB. Four of these QTLs co-localize with previously identified QTLs. A QTL identified on LG 8 confirms a previously identified QTL (Segura et al., 2007), while one of the QTLs identified on LG 9 might coincide with a QTL identified on the corresponding LG 3 of the genetic linkage map constructed by Conner et al. (1998). Two QTLs identified on LG 10 might coincide with markers found to co-segregate with time of budbreak in an earlier study conducted by Lawson et al. (1995). An additional 14 QTLs involved in time of IVB have been identified. We proposed the testing of four markers in a validation study conducted on a second mapping pedigree derived from a cross between ‘Anna’ and ‘Golden Delicious’. These markers are CH04a12, CH04c06y, CH01h01 and A267. Not only do these markers show significant levels of association with the time of IVB, but segregation of parental alleles from the cultivar ‘Anna’ for two of these markers, CH04c06y and CH01h01, were found to be associated with the time of IVB in different genetic backgrounds. The identification of markers closely associated with time of IVB will facilitate the implementation of MAS in breeding programs in order to breed cultivars that are better adapted to local climatic conditions.
The relationship between structure and thermostability of a nitrile hydratase from Goebacillus pallidus RAPc8
(University of the Western Cape, 2008) Van Wyk, Jennifer Caroline; Sayed, M.F.; Cowan, Don A.; Dept. of Biotechnology; Faculty of Science
Nitrile hydratases (NHases) are very important biocatalysts for the enzymatic conversion of nitriles to industrially important amides such as acrylamide and nicotinamide. An “ideal” NHase should fulfil several essential criteria including, high substrate conversion rates, being able to tolerate high substrate and product concentrations as well as being highly thermostable. The NHase used in the present study was isolated from Geobacillus pallidus RAPc8, a moderate thermophile. The primary aims of this study were to use random mutagenesis to engineer the G. pallidus RAPc8 NHase towards improved thermostability and then to use X-ray crystallography to investigate the molecular mechanism(s) involved in the enhanced thermostability. Two randomly mutated libraries were constructed using MnCl2 mediated errorprone PCR. The PCR reaction was performed using 0.05 mM and 0.10 mM MnCl2 and a biased dNTP concentration. The hydroxamic acid assay was used to screen the randomly mutated libraries for NHase mutants with enhanced thermostability. Six mutants that exhibited thermostability-enhancing mutations were isolated from the randomly mutated libraries. The thermostabilised mutants contained between 3 and 7 nucleotide changes per NHase operon. The wild-type and four thermostabilised mutant NHases (7D, 8C, 9C, 9E) were over-expressed, purified, crystallised and subjected to X-ray crystallography. The resolution of the diffraction data for the all the mutant NHases were better than the 2.4Å previously obtained for the wild-type G. pallidus NHase. The best quality data was collected for mutant 9E, which diffracted to a resolution of 1.15Å. The high quality crystal structures allowed each thermostability-enhancing mutation to be viewed in detail. As most of the NHase mutants contained multiple mutations, the crystal structures were important in correlating the observed thermostabilisation with the structural effect of the mutations. Analysis of the X-ray crystal structures illustrated the importance of electrostatic interactions, particularly salt bridges and hydrogen bonds in enhancing the thermostability of the mutant NHases. The difference in the free energy of activation of thermal unfolding (DDG) was used to compare the wild-type and mutant NHases thermostability. The most improved NHase, mutant 9C, was stabilised by both a buried inter-subunit salt bridge between aR169 and bD218 and an inter-helical hydrogen bond between bK43 and bK50. The stabilisation provided by these electrostatic interactions was 7.62 kJ/mol. Mutant 8C was primarily stabilised by the introduction an electrostatic network consisting of a salt bridge between bE96 and aR28 and a hydrogen bond between bE96 and bE92. Also, an intra-helical salt bridge between aE192 and aK195 stabilised the helix consisting of a190-196 in mutant 8C by shielding the helix backbone from solvation and preventing co-operative unfolding of the a helix. However, mutant 8C was also destabilised by a mutation that disrupted a water-mediated hydrogen bond between bD167 and bK168 at the heterotetramer interface of the enzyme. Consequently, the net stabilisation energy provided as a result of stabilising and destabilising interactions was 6.16 kJ/mol. Mutant 7D was the only NHase mutant with only one possible thermostabilising mutation. This mutant was stabilised by 3.40 kJ/mol as the result of a water-mediated hydrogen bond between aS47 and bE33. Similarly, a water-mediated hydrogen bond between aS23 and bS103 provided a stabilisation energy of 4.27 kJ/mol to mutant 9E. This project has shown that moderate-frequency randomly mutated libraries can yield mutants with multiple thermostabilising interactions. Also, the importance of utilising X-ray crystallography to investigate structure-function relationships in proteins has been illustrated.
Comparison of actinobacterial diversity in Marion Island terrestrial habitats
(University of the Western Cape, 2008) Sanyika, Walter Tendai; Cowan, Donald A.; Faculty of Arts
The major aim of this study is to determine and compare the distribution of bacteria and actinobacteria in Marion Island terrestrial habitats.
Saturation sequencing, characterisation and mapping of the NBS-LRR resistance gene family in apple, Malus x domestica (Borkh)
(University of the Western Cape, 2008) Mafofo, Joseph; Rees, D.J.G.; Faculty of Science
To date five classes of resistance proteins have been identified in plants and these include the intracellular protein kinases, receptor-like protein kinases with extracellular leucinerich repeat (LRR) domain, LRR proteins that encode membrane bound extracellular proteins, toxin reductase and intracellular LRR proteins with a nucleotide-binding site (NBS). These proteins recognise "invading pathogen" and in turn trigger defence response systems that act to protect plants from invading pathogens. The NBS-LRR genes which constitutes the major class encode a family of resistance proteins that are made up of a centrally located nucleotide binding site domain and a C-terminal leucine rich repeat receptor. This class of genes constitute the largest family of resistance genes identified in plants to date. They make up the majority of proteins involved in the plant basal and inducible defence systems against pathogen infection.
Characterisation of microbial communities associated with hypolithic environments in Antarctic Dry Valley soils
(University of the Western Cape, 2008) Khan, Nuraan; Cowan, Don A.; Dept. of Biotechnology; Faculty of Science
The Eastern Antarctic Dry Valley region is a polar desert, where conditions of extreme aridity, high temperature fluctuations and high irradiation levels make it one of the most extreme environments on earth. Despite the harsh environment, the soils in this region yield a wide range of bacterial and eukaryotic phylotypes in greater abundance than previously believed. In the Dry Valleys, highly localized niche communities colonise the underside of translucent quartz rocks and present macroscopic growth.
Identification of bacterial pathogenic gene classes subject to diversifying selection
(University of the Western Cape, 2009) Panji, Sumir; Hide, Winston; Bajic, Vladimir; Faculty of Science
Availability of genome sequences for numerous bacterial species comprising of different bacterial strains allows elucidation of species and strain specific adaptations that facilitate their survival in widely fluctuating micro-environments and enhance their pathogenic potential. Different bacterial species use different strategies in their pathogenesis and the pathogenic potential of a bacterial species is dependent on its genomic complement of virulence factors. A bacterial virulence factor, within the context of this study, is defined as any endogenous protein product encoded by a gene that aids in the adhesion, invasion, colonization, persistence and pathogenesis of a bacterium within a host. Anecdotal evidence suggests that bacterial virulence genes are undergoing diversifying evolution to counteract the rapid adaptability of its host’s immune defences. Genome sequences of pathogenic bacterial species and strains provide unique opportunities to study the action of diversifying selection operating on different classes of bacterial genes.
Plant natriuretic peptides - elucidation of the mechanisms of action
(University of the Western Cape, 2009) Ruzvidzo, Oziniel; Gehring, Chris; NULL; Faculty of Science
Several lines of cellular and physiological evidence have suggested the presence of a novel class of systemically mobile plant molecules that are recognized by antibodies generated against vertebrate atrial natriuretic peptides (ANPs). Functional characterization of these immunoanalogues, referred to as immunoreactive plant natriuretic peptides (irPNPs) or plant natriuretic peptides (PNPs), has shown that they play important roles in a number of cellular processes crucial for plant growth and maintenance of cellular homeostasis. Although the various biological roles of PNPs in plants are known, their exact mode of action remains elusive. To elucidate the mechanisms of action for these immunoanalogues, we have prepared a biologically active recombinant PNP from Arabidopsis thaliana (AtPNP-A) and the biological activity was demonstrated by showing its ability to induce water uptake into Arabidopsis thaliana protoplasts. In addition, the molecule was shown to downregulate photosynthesis while at the same time up-regulating respiration, transpiration as well as net water uptake and retention capacities in the sage Plectranthus ecklonii. Further analysis of the recombinant AtPNP-A indicated that the peptide can induce systemic response signalling though the phloem. A recombinant Arabidopsis wall associated kinase-like protein (AtWAKL10) that has a domain organization resembling that of vertebrate natriuretic peptide (NP) receptors was also partially characterized as a possible receptor for the recombinant AtPNP-A. Vertebrate NP receptors contain an extracellular ligand-binding domain and an intracellular guanylate cyclase (GC)/kinase domain and signal through the activity of their GC domain that is capable of generating intracellular cGMP from GTP. The structural resemblance of AtWAKL10 to vertebrate NP receptors could suggest a functional homology with receptor molecules and it is conceivable that such a receptor may recognize PNPs as ligands. The characterization of the recombinant AtWAKL10 showed that the molecule functions as both a GC and a kinase in vitro. This strengthened the suggestion that AtWAKL10 could be a possible AtPNP-A receptor especially considering the fact that AtPNP-A applications to plant cells also trigger cGMP transients. Furthermore, a bioinformatic analysis of the functions of AtPNP-A and AtWAKL10 has inferred both molecules in plant pathogen responses and defense mechanisms, thus indirectly functionally linking the two proteins.
Transcription regulation and candidate diagnostic markers of esophageal cancer
(University of the Western Cape, 2009) Essack, Magbubah; Bajic, Vladimir; South African National Bioinformatics; Faculty of Science
Esophageal cancer (EC) ranks among the ten most frequent cancers worldwide. Mortality rates associated with EC are very similar to the incidence rates due to the relatively late stage of diagnosis and the poor efficacy of treatment. The aim of this study was to enhance our insights of putative transcriptional circuitry of EC genes, thereby potentially positively impacting our knowledge of therapeutic targets, providing indications as to more appropriate lines of treatment, and additionally allowing for the determination of putative candidate diagnostic markers for the early stage detection of EC. This thesis reports on the development of a novel comprehensive database (Dragon Database of Genes Implicated in Esophageal Cancer, DDEC) as an integrated knowledge database aimed at representing a gateway to esophageal cancer related data. More importantly, it illustrates how the biocurated genes in the database may represent a reliable starting point for divulging transcriptional regulation, diagnostic markers and the biology related to esophageal cancer. DDEC contains known and novel information for 529 differentially expressed EC genes compiled using scientific publications from PubMed and is freely accessible for academic and non-profit users at http://apps.sanbi.ac.za/ddec/. The novel information provided to users of the DDEC is the lists of putative transcription factors that potentially control the 529 manually curated genes. The value of the information accessible through the database was further refined by providing precompiled text-mined and data-mined reports about each of these genes to allow for easy exploration of information about associations of EC-implicated genes with other human genes and proteins, metabolites and enzymes, toxins, chemicals with pharmacological effects, disease concepts and human anatomy. This feature has the capacity to display potential associations that are rarely reported and thus difficult to identify, and it enables the inspection of potentially new ‘association hypotheses’ generated based on the precompiled reports. This study further illustrates how the biocurated esophageal squamous cell carcinoma (ESCC) genes in the database may represent a reliable starting point for exploring beyond current knowledge of the transcriptional circuitry of estrogen related hormone therapy. The genes were used to develop a method that identified 44 combinations of transcription factors (TFs) that characterize the promoter sequence of estrogen responsive genes implicated in ESCC. These significantly over-represented combinations of TFs were then used to increase confidence in the 47 novel putative estrogen response genes that may be related to ESCC too. Coincidently, two of the novel putative estrogen response genes were verified by current (2009), experimental publications.
A proteomic analysis of drought and salt stress responsive proteins of different sorghum varieties
(University of the Western Cape, 2009) Ngara, Rudo; Ndimba, Bongani K.
Sorghum (Sorghum bicolorï, a drought tolerant cereal crop, is not only an important food source in the semi arid/arid regions but also a potential model for studying and gaining a better understanding of the molecular mechanisms of drought and salt stress tolerance in cereals. This study reports on a proteomic analysis of sorghum proteomes in response to salt and hyperosmotie stresses. Two-dimensional gel electrophoresis (2DE) in combination with mass spectrometry (MS) was used to separate, visualise and identify sorghum proteins using both sorghum cell suspension cultures and whole plants. The sorghum cell suspension culture system was used as a source of culture filtrate (CF) proteins. Of the 25 visualised CBB stained CF spots, 15 abundant and well-resolved spots were selected for identification using a combination of MALDI- TOF and MALDI- TOFTOF MS, and database searching. Of these spots, 14 were positively identified as peroxidases, germ in proteins, oxalate oxidases and alpha-galactosidases with known functions in signalling processes, defense mechanisms and cell wall metabolism. Following 200 mM NaCl and 400 mM sorbitol stress treatments, the expression/abundance of a protein spot similar to a rice wall-associated protein kinase was upregulated in the sorghum secretome in response to both stresses. Amino acid sequence alignment of the matching peptides between these two proteins showed that the sorghum CF spot possesses a protein kinase domain. Therefore, this protein could possibly participate in cell signalling functions, which link the external environment with the cell's cytoplasm. Using whole plant systems, a comparative study of leaf protein expression between two sorghum varieties, AS6 (salt sensitive) and MN1618 (salt tolerant) was conducted. Forty well resolved spots of varying abundances were picked for MS analysis. Of these, 28 were positively identified, representing proteins with functions in carbohydrate metabolism (60.7%), proton transport (17.9%), protein synthesis (7.1%), hydrolytic functions (7.1%), nucleotide metabolism (3.6%) and detoxification (3.6%). Using PDQuest™ Advanced 2D Analysis Software version 8.0.1 (BIO-RAD), a comparative analysis of leaf proteome expression patterns between the two sorghum varieties was conducted. The results indicated proteins with similar expression patterns as well as qualitative and quantitative differences between the two leaf proteomes. The effect of 100 mM NaCI on leaf proteome expression between the two sorghum varieties was also studied. Western blotting analysis of leaf, sheath and root tissues using Hsp70 antibodies showed that this treatment induced Hsp70 expression, a known stress protein, in both varieties. Thereafter, the partially annotated leaf proteome map was used to landmark other salt responsive proteins. Examples of differential expression patterns included glutathione S transferase and hydroxynitrile lyase proteins whose abundances were upregulated in both varieties, while the large subunit of RuBisCo was downregulated in AS6 but upregulated in MN1618. Qualitative spot expression differences in response to salt stress were also observed between the two sorghum varieties but these remained unidentified after both MALDI-TOF and MALDI-TOF-TOF MS, possibly indicating novel and previously uncharacterised sorghum proteins. The results of this study can be used as reference tools by proteomics researchers worldwide as well as a foundation for future studies.

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