No description available.

No description available.

Glutamine synthetases are one of the most ancient functioning enzymes in existence and these large oligomeric complexes are found in all extant forms of life where they play a critical role in nitrogen metabolism. Over the past five decades, extensive biochemical studies together with structural investigations have helped build a picture of the mechanism of functioning and regulation in the GSI and GSII families. The most divergent GSIII family, however, is poorly characterized and has only recently been recognized. Structural studies, using both cryo-EM and X-ray crystallography, were undertaken on the type III GS, GlnN, from the opportunistic human pathogen, Bacteroides fragilis, with a view to better understanding the GSIII family in the light of the known structure functionrelationships of the other GS enzymes, and to investigate the potential for the design of selective inhibitors against the divergent family. A low-resolution (16 Ã) reconstruction of GlnN was first determined by single particle cryo-EM and image processing. This structure revealed that GlnN was a double-ringed dodecamer with D6 symmetry and the arrangement of active sites within the hexameric rings closely matched the GSI structure. Following the design of a rapid purification protocol and improvements to the stability and solubility of GlnN, conditions were discovered for the production of diffraction quality inhibitor-bound crystals. A second better diffracting crystal form was also produced following proteolytic processing. The crystal structure of GlnN was solved to near atomic resolution (3.0 Ã) following phase extension of low-resolution SAD phases, taking into account the cryo-EM structure. The higher resolution of the crystal structure revealed that, surprisingly, the orientation of the hexameric rings in GlnN is inverted in comparison to other families. These results have raised interesting questions surrounding the mechanism and driving forces responsible for the evolution of quaternary structure in the GS enzymes and have suggested that the GSI and GSII structure arose following truncation of a large GSIII-like ancestor. Despite the differences in higher order assembly, the GlnN monomer displayed a high degree of similarity with the GSI and GSII structures in the core active site region, thus, suggesting a conservation of reaction mechanism. Structure-based multiple sequence alignment showed that the residues forming the nucleotide binding pocket are the least conserved in the GS superfamily, and several residue positions, which represent altered modes of ligand binding, were suggested as potential avenues for the design of selective inhibitors against GlnN.

Modelling the Evolution of HIV-1 Protein-Coding Sequences with Particular Focus on the Early Stages of Infection Natasha Thandi Wood, February 2010 The evolution of the viral genome sequence over the course of HIV-1 infection is of interest for vaccine and drug design, and for the development of effective treatment strategies. Characteristics of the transmitted viral genome that could render the virus more sensitive to host immune responses, are of particular interest for vaccine studies. However, sequence samples from the earliest phase of HIV infection are scarce, and inferences about the nature of the infecting virus and its evolution during the course of early infection are often made from samples isolated from later stages, or from chronic infections. To establish in detail the adaptive changes that occur in early infection, an investigation was carried out on a large dataset consisting of sequences isolated from individuals in early infection. The majority of these infections were inferred to have resulted from transmission of a single virion or virally infected cell, which permitted a detailed investigation of HIV-1 diversification in early infection for the first time. Comparing viral diversification across multiple patients, it was possible to identify specific evolutionary patterns in the HIV-1 genome that occur frequently during the earliest stages of infection. The analyses revealed that APOBEC-mediated hypermutation has an important role in early viral diversification and may enable rapid escape from the first wave of host immune responses. Several mutations in early infection that were likely to result in immune escape were identified, some of which have subsequently been confirmed experimentally. In general, experimental verification of model-based inferences is necessary, but can be expensive and time-consuming. To reduce the costs involved, it is essential that the evolutionary methods produce accurate results. Simulation results presented in this thesis show that inferences made about viral evolution can be subject to bias when key aspects of viral biology are not accounted for by the models used. In particular, some previous comparisons between sequence groups that share genealogical histories, positive selection studies that fail to account for recombination, and research on HIV covariation, may need to be revisited, using more accurate evolutionary models. The results presented in this thesis demonstrate the importance of accurate evolutionary models to understand the selection pressures acting on the virus during various stages of infection. Furthermore, using a phylogenetic model it was possible to identify sites in the HIV genome that were evolving adaptively and are implicated in CTL immune escape during early infection. Characterising escape mutations in the transmitted virus may lead to novel approaches to develop vaccines and antiviral drugs.

By differential screening of a cDNA library of the resurrection plant Xerophyta viscosa, a cDNA termed XvPer I was isolated for its over-expression under dehydration stress. Analysis of the cDNA sequence indicated a hydrophilic protein of a predicted Mw of 24.2 kDa with high identity to plant l-Cys peroxiredoxins, a novel antioxidant enzyme. Southern blot analysis revealed that XvPer I was most probably a single copy gene. The polypeptide sequence had significant identities (-70%) with other recently identified plant I-Cys peroxiredoxins with an absolutely conserved active site (PVCTTE). The protein sequence also had a putative bipartite nuclear localization signal.

Xerophyta viscosa Baker (family Velloziaceae) can survive extremes of dehydration (desiccation), down to 5% relative water content (RWC) and resumes full physiological activity within 80 h of rehydration. A thorough understanding of this phenomenon may provide further insight into possible mechanisms for improving drought tolerance in other plants. In this respect a comprehensive analysis of the nuclear proteome of this plant and its response to dehydration stress at 35% RWC was carried out. The RWC at 35% represents a distinct phase of the dehydration process where induction of late protection mechanisms is initiated and is a characteristic of desiccation tolerant species. We optimized nuclei isolation and nuclear protein extraction protocols and successfully employed these protocols to isolate highly purified nuclei and subsequently nuclear proteins from fully hydrated and dehydrated X. viscosa leaf samples. The integrity of the purified nuclei was confirmed with light and fluorescent microscopy. The nuclei were uniform spheres, approximately 5 μm in size. The purity and enrichment of the nuclear proteins were confirmed by chlorophyll assay and Western blot analysis. The nuclear proteins were investigated using two-dimensional (2D) and isobaric tags for relative and absolute quantitation (iTRAQ) technologies. Using the 2DE approach, a total of 438 proteins spots were reproducibly detected and analysed of which 18 protein spots were shown to be up-regulated in response to dehydration. These proteins contained both regulatory and functional proteins. The largest category comprised five novel protein factors and two proteins with unassigned functions. The second category comprised proteins involved in gene regulation and signal transduction. The third category comprised stress responsive proteins with chaperone type activities. Other categories include proteins involved in energy metabolism, protein degradation and translation. These results demonstrate that dehydration was controlled by multiple genes within the plant nucleus and X. viscosa may possess its own specific nuclear proteins that are involved in desiccation stress. In addition we comprehensively analyzed the nuclear proteome of X. viscosa using iTRAQ with two-dimensional liquid chromatography and tandem mass spectrometry to complement the data obtained from the 2DE approach. Using iTRAQ, we reproducibly University of Cape Town identified 128 proteins with confidence ¥ 95% (Ï < 0.05). Sixty six percent of the identified proteins showed consistent expression levels. The remaining 34% proteins showed significant changes in expressions. Of the latter, 23% were shown to be up regulated in response to dehydration stress. The remaining 11% were shown to be down regulated. The nuclear proteins of X. viscosa up-regulated in response to dehydration stress showed a coordinated response involving both regulatory and functional proteins and were implicated in diverse cellular functions. The characteristic feature of the X. viscosa nuclear proteins is the high level of stress molecules among the dehydration responsive proteins with evident functions in defense mechanisms compared to down regulated proteins and proteins showing consistent expression levels. These results demonstrate that enhanced defense capacity is crucial to desiccation tolerance and strongly support the notion that late dehydration responsive proteins are involved in protection of the cellular structures during dehydration. Proteins showing consistent expression levels during dehydration most likely maintain the minimum viability in cells under all conditions or may be indirectly associated with desiccation tolerance. Down-regulated proteins are likely important for plant survival under normal growth conditions. The proteins up-regulated in response to dehydration stress were assumed to be associated directly with the acquisition of desiccation tolerance. The up-regulated proteins were further categorized into nine functional groups to gain more insight into their roles in desiccation tolerance. The largest group was shown to be involved in gene regulation and signal transduction (36%), which reflects the role of the nucleus in gene expression and regulation. The second group included stress responsive molecules such as antioxidants, molecular chaperones and compatible solutes (33%). This reflects the importance of strong defense systems in preventing lipid peroxidation, protein aggregation, membrane leakage and maintaining the integrity of cellular structures during dehydration and in the dried state. The third group contained proteins involved in nucleocytoplasmic transport (10%). This might reflect the capacity of this plant to control the movement of molecules to and from the nucleus during dehydration and the importance of this process in adaptation to dehydration stress. The fourth group contained proteins involved in protein translation (7%). Proteins categorized to other functions, include proteins with miscellaneous and unknown functions. Proteins with unknown functions were considered to be X. viscosa nuclear-specific proteins. There was good correlation between the up-regulated proteins identified by 2-DE and iTRAQ approaches. In conclusion, this study revealed that X. viscosa nuclear proteome was responsive to dehydration stress and desiccation tolerance is University of Cape Town genetically encoded. Secondly, X. viscosa relies on readily inducible protection to combat desiccation and desiccation tolerance is controlled by multiple genes within the plant nucleus. Thirdly, the protective mechanisms of desiccation tolerance utilized by X. viscosa appear to involve signal perception genes and modulating gene expression of appropriate genes encoding protective molecules including antioxidants, molecular chaperones, compatible solutes, proteins of translation and degradation machinery, proteins with miscellaneous functions and novel protein factors. Lastly, proteins are crucial to desiccation tolerance allowing X. viscosa to possess a unique stress tolerance with versatile and coordinated actions to provide protection for its cellular structures during desiccation and in the dried state. To our best knowledge this is the first study to provide insight into the nuclear (organellar) proteome of a desiccation tolerant plant.

Glucocorticoids mediate their effects by binding to the glucocorticoid receptor (GR), resulting in modulation of transcription of target genes via direct binding to DNA or tethering via proteinprotein interactions. A central question is what determines the rank order of ligand-selective transcription with different GR ligands for the same gene in the same cell. Using a panel of twelve GR ligands, including agonists, partial agonists and antagonists, the relationship between the extent of GR phosphorylation at S226, GR turnover and transcriptional response, was investigated using a variety of biochemical approaches. Using a phospho-S226-specific GR antibody, ligand-selective S226 phosphorylation was shown to occur in both COS-1 and U2OS cells, while GR phosphorylation at S226 was shown to inhibit maximal transactivation and transrepression efficacy. Attempts to identify the kinases responsible for this interaction were inconclusive but suggested a combination of kinases is responsible for the in vivo phosphorylation of the hGR in these cells. Similarly the rate of GR degradation was different for the different ligands. Interestingly, both ligand-selective GR phosphorylation and half-life were found to correlate with efficacy for transactivation and transrepression of model synthetic reporter genes, where agonists resulted in the greatest extent of phosphorylation and the fastest vii rate of GR turnover, suggesting a link between these functions. Furthermore experiments where transcription was blocked suggest that GR turnover does not require transcription. However, using a S226A GR mutant, as well as in experiments where GR turnover was blocked, it was established that neither phosphorylation of the GR at S226 nor GR degradation rate determines the rank order of ligand-selective GR transactivation. The mechanisms whereby GR phosphorylation influence GR-mediated transcription was further investigated using a triple phosphorylation deficient mutant. It was shown that phosphorylation at one or more of residues S203/S211/S226 is required for transactivation of a MMTV promoter but does not affect unliganded or agonist-induced GR degradation and acetylation. Additionally, it was shown that phosphorylation at S203/S211/S226 is not the sole determinant of co-activator p300 recruitment to the GR. Interestingly, GR-mediated transrepression via AP-1 is less sensitive to GR phosphorylation than GR-mediated transactivation, indicating different mechanisms in the role of GR phosphorylation on transactivation vs. transrepression. Pull-down and chromatin immunoprecipitation assays showed that phosphorylation of the GR at one or more of these residues are required for interaction of the GR with the co-activator GRIP-1 in vitro and for maximal recruitment of GR and GRIP-1 to the MMTV promoter in intact cells. Cellular fractionation showed that phosphorylation at these residues is not however required for GR nuclear localisation. Taken together these results support the conclusion that phosphorylation at one or more of S203/S211/S226 of the hGR is required for maximal transactivation response to enable GRIP-1 recruitment to the hGR.

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Includes abstract / Includes bibliographical references / Gracilaria gracilis that grows naturally at Saldanha Bay, South Africa is economically important as a source of agar. The Gracilaria yields from natural beds at Saldanha Bay are however unreliable, and consequently the South African Gracilaria industry has experienced a number of setbacks over the years. The only way a consistent supply can be assured is by mariculture to supplement the natural harvests. In 1993 the Seaweed Research Institute (SRU) found that mariculture of G. gracilis in Saldanha Bay is feasible but that there is potential to improve yields by technical research and development (Anderson et al.1996a). Jaffray and Coyne (1996) developed a pathogenicity assay that demonstrated that agarolytic bacteria isolated from Saldanha Bay Gracilaria induced disease symptoms such as thallus bleaching, while non-agarolytic isolates did not. It is thought that unfavorable environmental conditions such as elevated water temperature and nutrient depletion, which occur during the summer months in the surface layers of the water column in Saldanha Bay, induce the onset of agarase production or result in changes in the bacterial community structure in which agarase-producers become more dominant. By using the pathogenicity assay, Jaffray and Coyne (1996) identified the highly agarolytic Gracilaria gracilis pathogen, Pseudoalteromonas sp. LS2i. The aim of this study was to characterize the bacterial pathogen, Pseudoalteromonas sp. LS2i to further our understanding of virulence regulation and specifically, the role of the agarase enzymes in the process of seaweed-pathogen interaction. Two agarolytic clones, pEB1 and pJB1, were obtained after constructing and screening a Pseudoalteromonas sp. LS2i genomic library in Esherichia coli. Restriction enzyme mapping suggested that both clones contain the same agarase gene. Southern hybridization studies confirmed the origin of the cloned DNA and sequencing studies revealed the 1062 bp ORF, putative promoter region, putative ribosome binding site and putative transcriptional start point of the cloned agarase gene. The ORF showed sequence identity to several other β-agarases and was identified as a member of the GH-16 family of glycoside hydrolases. The agarase was purified from the E. coli JM109 (pEB3) transformant. The molecular weight was estimated to be 39 kDa by SDS-PAGE. Zymogram analysis confirmed that the purified protein is agarolytic and TLC analysis revealed that the predominant end-products of agar hydrolysis are neoagarohexaose and neoagarobiose, which indicates the same mode of action as that observed for the agarase produced extracellularly by Pseudoalteromonas sp. LS2i.

Word processed copy. / Includes bibliographical references (p. 141-165). / The marine bacterium Vibrio cholerae produces pyomelanin through the catabolism of L-tyrosine to homogentisic acid. Various types of melanins are used by microbes as defense mechanisms against a variety of environmental stresses. This thesis investigated the defensive role of V. cholerae pyomelanin against exogenous stresses, particularly hydrogen-peroxide (H2O2).