3. Electronic Theses and Dissertations (ETDs) - All submissions
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Item The recombinant expression and structural characterization of movement protein BC1 from South African cassava mosaic virus(2018) Nankoo, NikitaSouth African cassava mosaic virus (SACMV) is a circular ssDNA bipartite begomovirus, whose genome comprises of DNA A (encodes six genes) and DNA B (encodes BC1 (cell-to-cell movement protein) and BV1 (nuclear shuttle protein)). Begomoviruses cause cassava mosaic disease (CMD) resulting in substantial root yield losses impacting farmers and potential starch yields for industrial purposes. The structural and physiochemical characteristics of begomoviruses have not been elucidated to date. Additionally, it is crucial to identify protein-protein interactions between SACMV BC1 and cassava host proteins that facilitate SACMV movement. In this study, in silico characterization studies for BC1 were undertaken. The FASTA amino acid sequence of BC1 was uploaded onto a webserver (Predict Protein (https://www.predictprotein.org)) and resulted in the predicted secondary structure of BC1 as well as predicted protein binding sites. BC1 was cloned into a pET-28a(+) expression vector and transformed into host cells E.coli BL21 (DE3). The optimal conditions for the expression of BC1 protein were found to be induction with 0.25 mM IPTG at an OD600 of ~0.6 expressed at 37 °C for four hours. The protein was refolded using stepwise dialysis. The molecular weight of BC1 was 35 kDa (SDS-PAGE). The secondary structure of BC1 was confirmed to be predominantly β-strands (CD). An ANS (1-anilino-8-naphthalene sulphonate) binding assay confirmed that BC1 possesses hydrophobic pockets with the ability to bind ligands such as ATP. A 2’ (3’)-N-methylanthraniloyl-ATP (MANT-ATP) assay showed binding of MANTATP to BC1. Intrinsic tryptophan fluorescence studies indicated significant conformational change in the denatured form of BC1 in the presence of ATP compared to in the absence of ATP. Literature and the PredictProtein webserver were employed to in silico predict suitable cassava host prey proteins for the yeast-two-hybrid (Y2H) system. Cloning of host proteins (HSP70 and Histone H3) into pDEST™22 plasmids was done at GenScript USA. The in silico study and structural characterization of BC1 provide insights to the structural characteristics of BC1 and further explains its functionality. Completion of Y2H assays will confirm or refute that cassava host proteins Histone H3 and HSP70 interact with the cell-to-cell movement protein of SACMVItem Characterisation of the replication-associated protein of South African cassava mosaic virus and elucidation of protein binding partners in cassava(2018) Ayres, Frances MargaretGeminiviruses are a large family of plant-infecting viruses that have severely affected a variety of economically important crops, such as cassava. Cassava is a perennial crop that plays a vital economic role in the lives of subsistence farmers of the developing world. Cassava is a salient crop in developing countries and is favoured due to its high starch content and drought tolerance. Africa has emerged as the leading producer of cassava; however, yields of cassava tuberous roots and leaves have been negatively impacted by the emergence of viral pathogens. Geminiviruses such as South African cassava mosaic virus (SACMV) introduce cassava mosaic disease which negatively impacts upon crop yields. The geminivirus-encoded replication-associated protein (Rep) is a highly conserved protein that is crucial for viral replication, and modulates a number of virus-host interactions. The aims of this study included the expression and characterisation of SACMV Rep and determining host protein binding partners of the viral protein. Expression of a soluble recombinant protein was successfully carried out. Structural analyses were undertaken by studying the secondary and tertiary structural features of the protein using circular dichroism (CD), and intrinsic fluorescence spectroscopy. Secondary structure studies were inconclusive, with intrinsic fluorescence revealing that adenosine triphosphate (ATP) binding did not induce significant protein conformational changes. Binding of 8-Anilino-1-naphthalenesulfonic acid (ANS) to SACMV Rep was observed, with the interaction not disrupted in the presence of ATP. This confirmed the lack of a persistent SACMV Rep-ATP interaction. Functional characterisation was undertaken using a DNA binding and cleavage assay. A comparison between results obtained in this study and previous geminivirus Rep research suggests cleavage of the nucleotide probe, and binding to the cleaved 5ʹ-end. The formation of a protein-DNA complex confirmed that the expressed recombinant protein was functionally competent. Interaction of cassava proteins with SACMV Rep were probed using a yeast two-hybrid assay (YTH). Putative cassava protein binding partners of SACMV Rep were selected based on previous research and geminivirus related literature. The putative binding partners, histone H3, cyclin D3;2 and cyclin D4;2, were used as the prey proteins with bait protein SACMV Rep utilised as the bait. Results obtained from the X-gal filter lift assay suggested that there was no direct interaction of SACMV Rep with histone H3, cyclin D3;2 or cyclin D4;2. This study presents the first investigation into the elucidation of structural and functional features of SACMV Rep, and the first effort into identifying host protein binding partners of SACMV RepItem Gene expression studies towards the elucidation of host responses to South African cassava mosaic virus(2014-04-22) Allie, FarhahnaUnable to load abstract.