Zhang, Fukai (2022). Investigation of condensin-related proteins and purification of the DNA excision machinery in Paramecium tetraurelia. (Thesis). Universität Bern, Bern
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Abstract
The spatial organization of chromosomes is vital to life at the cellular level. DNA loops formed by condensin are the crucial structure of the meiotic and mitotic genomes which organize chromatin in interphase and metaphase. The structural maintenance of chromosomes (SMCs) protein family, which is now thought to serve as a DNA motor, is central to this organization. Paramecium tetraurelia is a ciliate that performs RNA-mediated widely genome rearrangement, mainly Internal Elimination Sequence (IES) excision, during sexual reproduction. The scanning model, which includes two sets of small RNAs (scanRNA and iesRNA), is widely used to explain this process. While the exact mechanism by which interphase and metaphase genome structure interact with IES excision is unknown to date. Given the importance of SMCs to chromosome structure and the poorly reported condensin in Paramecium tetraurelia, we explored the function of condensin SMCs in IES elimination and investigated whether their cooperation displayed different effects on this progress. One of the SMC4s in Paramecium tetraurelia, SMC4-2, is a novel player of IES elimination. Knockdown of SMC4-2 is lethal and causes the retention of almost all IESs, while the knockdown of SMC2s and SMC4-1 exhibit lethality to progeny without any sign of IES retention. Most interestingly, combining SMC4-2 RNAi with any of the other SMCs mentioned above abolished the retention in SMC4-2 single silencing. The SMC4-1 and SMC4-2 Co-immunoprecipitation and mass spectrometry analysis indicates a possibility that the interactions between SMC4-1 and SMC4-2 are decided by their concentration ratio of them. Additionally, we developed and optimized a purification method for IES-specific in vivo DNA binding complexes. The exact players of IES excision, especial the complexes that perform recognizing, binding and cleaving, are still unclear. This method allows us to purify specific-IES binding excision machinery from the new developing MAC where the old MAC contamination can be excluded. After that, we also tried to analyse the PiggyMac (PGM) interacting proteins by using Flag-HA tagged PGM 3A mutant. We were unable to enrich the PGM 3A-Flag-HA on anti-HA beads. However, we still provide some hints which could be helpful to further study.
| Item Type: | Thesis |
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| Granting Institution: | Faculty of Medicine, University of Bern |
| Dissertation Type: | Cumulative |
| Date of Defense: | 15 November 2022 |
| Subjects: | 500 Science > 570 Life sciences; biology |
| Institute / Center: | 08 Faculty of Science > Department of Biology > Institute of Cell Biology > Ciliate |
| Depositing User: | Fukai Zhang |
| Date Deposited: | 03 Jan 2024 09:44 |
| Last Modified: | 03 Jan 2024 23:25 |
| URI: | https://boristheses.unibe.ch/id/eprint/2153 |
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