Saudi Cultural Missions Theses & Dissertations
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Item Restricted Comparative genomic analysis and evolutionary study of repetitive DNA for estimating variation and mobility(University of Leicester, 2024-09) Alwadani, Khawla; Heslop-Harrison, PatRepetitive DNA sequences, motifs repeated hundreds, thousands of times in the nuclear genome, represent typically half of the DNA and are some of the most rapidly evolving sequences. The objective of this study was to see how repetitive sequences differ and evolve between and within the wider Polygonaceae family and Malus domestica species. High volume DNA sequence reads from 12 Polygonaceae species were analysed using individual graphbased repeat clustering, revealing substantial differences in amount and composition of TEs. Many genus-specific repetitive elements were found in the comparative clustering of 21 samples, notwithstanding the close relationship of the genera. The repetitive sequence landscape was characterised among three apple cultivars. Repetitive DNA represents some 45 % of the apple genome. Graph-based sequence-read clustering showed the amount and genome composition of TEs with genome-wide dispersal was similar in various apple cultivars. LTR-retrotransposons were the most abundant, and similar Ty1-copia and Ty3-gypsy proportions. Analysis of in situ hybridisation to chromosomes was used for localisation of the various repetitive elements (including the 5S and 45S rDNA loci) and comparing abundance and organisation with published genome assemblies and unassembled raw sequence reads. In apple fruit, sectors of different colours are occasionally observed, just in the skin (peel) of the apple. The colour sectors arise from movement of TEs around the MYB transcription factor, giving rise to different expression of anthocyanin genes and hence pigmentation. Genomic DNA (c. 12-fold genome coverage) from the skin sectors was analysed. Notably, Gala is heterozygous for a key MYB gene. Insertional polymorphisms were identified in or around the MYB genes between the sectors and rest of the fruit, showing that DNA sequence movement was likely to cause the colour differences.51 0Item Restricted Timekeeper: Advancing Circadian Research with GST-Tagged Per3 Protein(The University of Edinburgh, 2024) Alanezi, Sarah Abdullah; Arribas, RaquelThe Per3 gene plays a crucial role in regulating circadian rhythms, profoundly impacting sleep-wake cycles and metabolic processes. Despite its biological significance, the expression and purification of Per3 have presented substantial challenges, often resulting in low yields and poor solubility. This study tackles these obstacles by employing innovative cloning and expression techniques, particularly utilizing a GST-Per3long to enhance solubility and purification efficiency. We designed primers incorporating homology regions complementary to restriction enzyme-digested ends and employed the In-Fusion cloning method to integrate the Per3 gene into the p3E plasmid vector with high precision. The transformation efficiencies were remarkable, with colony counts reaching 2.292 x 10^8 colonies per μg of plasmid DNA. PCR amplification confirmed the successful integration of the Per3 gene, with distinct bands observed at the expected size of 1143 bp, which was further confirmed by DNA sequencing. Protein expression trials identified 25°C as the optimal temperature, significantly improving the yield and solubility of the GST-tagged Per3 protein. Subsequent purification through GST Affinity chromatography and gel filtration chromatography yielded a highly pure protein, as confirmed by SDS-PAGE and native gel electrophoresis. Although the initial yield was modest, the high purity of the purified protein provides a robust foundation for future functional and structural studies. This study not only establishes a reliable protocol for Per3 expression and purification but also opens avenues for investigating the interactions of Per3 with other circadian proteins, such as CRY and BMAL1, to determine their mutual exclusivity and relative affinity. These interactions are crucial for understanding the biological role of Per3 in fine-tuning the circadian clock. Future work should focus on optimizing expression conditions to further increase yield and investigate the intricate biological activity of the Per3 protein within the circadian network.20 0Item Restricted Timekeeper: Advancing Circadian Research with GST-Tagged Per3 Protein(The University of Edinburgh, 2024) Alanezi, Sarah Abdullah; Arribas, RaquelThe Per3 gene plays a crucial role in regulating circadian rhythms, profoundly impacting sleep-wake cycles and metabolic processes. Despite its biological significance, the expression and purification of Per3 have presented substantial challenges, often resulting in low yields and poor solubility. This study tackles these obstacles by employing innovative cloning and expression techniques, particularly utilizing a GST-Per3long to enhance solubility and purification efficiency. We designed primers incorporating homology regions complementary to restriction enzyme-digested ends and employed the In-Fusion cloning method to integrate the Per3 gene into the p3E plasmid vector with high precision. The transformation efficiencies were remarkable, with colony counts reaching 2.292 x 108 colonies per µg of plasmid DNA. PCR amplification confirmed the successful integration of the Per3 gene, with distinct bands observed at the expected size of 1143 bp, which was further confirmed by DNA sequencing. Protein expression trials identified 25°C as the optimal temperature, significantly improving the yield and solubility of the GST-tagged Per3 protein. Subsequent purification through GST Affinity chromatography and gel filtration chromatography yielded a highly pure protein, as confirmed by SDS-PAGE and native gel electrophoresis. Although the initial yield was modest, the high purity of the purified protein provides a robust foundation for future functional and structural studies. This study not only establishes a reliable protocol for Per3 expression and purification but also opens avenues for investigating the interactions of Per3 with other circadian proteins, such as CRY and BMAL1, to determine their mutual exclusivity and relative affinity. These interactions are crucial for understanding the biological role of Per3 in fine-tuning the circadian clock. Future work should focus on optimizing expression conditions to further increase yield and investigate the intricate biological activity of the Per3 protein within the circadian network.21 0Item Restricted KsPETAse: A Potential Novel-Enzyme in The Biodegradation of PET Plastics.(Saudi Digital Library, 2023-11-14) Alsaytari, Abdulmalik Ibrahim; Ariza, AntonioPlastics have become an essential part of modern life due to their versatility, low cost and multiple uses. However, most types of synthetic plastic have a downside: they tend to last for hundreds of years before they break down and degrade, leaving behind harmful substances known as microplastics. The methods currently used to dispose of and recycle plastics have not proven to be the most ideal solutions. The lack of a highly efficient tool to solve the problem of plastic pollution and its accumulation in the ecosystem has been worrying scientists for years. The search for biodegrading organisms has emerged as a promising alternative to conventional methods of dealing with plastic waste. The discovery of the PETase enzyme system, consisting of two novel enzymes: IsPETase and IsMHETase, in the bacterium Ideonella sakaiensis, which can use PET (polyethylene terephthalate), one of the most widely produced and distributed types of plastic, as an energy source, has opened up the possibility of developing biotechnological tools that could use microorganisms to degrade and detoxify the plastic waste that has been choking the environment for years. The search for improving the functional and catalytic activities of these newly discovered classes of enzymes, as well as the search for other organisms that may have developed a mechanism for degrading plastic, is now more necessary than ever. The novel enzyme KsPETase, which holds structural similarities to IsPETase, was investigated in this study as a potential enzyme to be studied for its structural and functional properties that are expected to be resemble those of IsPETase. It was also intended for the investigation of whether it performs the catalytic functions more efficiently.66 0