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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 吳益群 | |
dc.contributor.author | Mei-Hsuan Wu | en |
dc.contributor.author | 吳美萱 | zh_TW |
dc.date.accessioned | 2021-07-10T21:34:57Z | - |
dc.date.available | 2021-07-10T21:34:57Z | - |
dc.date.copyright | 2016-11-02 | |
dc.date.issued | 2016 | |
dc.date.submitted | 2016-08-17 | |
dc.identifier.citation | Reference
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Franks, D.M., et al., C. elegans pharyngeal morphogenesis requires both de novo synthesis of pyrimidines and synthesis of heparan sulfate proteoglycans. Dev Biol, 2006. 296(2): p. 409-20. 20. Chaparian, M.G. and D.R. Evans, Intracellular location of the multidomain protein CAD in mammalian cells. FASEB J, 1988. 2(14): p. 2982-9. 21. Sigoillot, F.D., et al., Nuclear localization and mitogen-activated protein kinase phosphorylation of the multifunctional protein CAD. J Biol Chem, 2005. 280(27): p. 25611-20. 22. Ben-Sahra, I., et al., Stimulation of de novo pyrimidine synthesis by growth signaling through mTOR and S6K1. Science, 2013. 339(6125): p. 1323-8. 23. Robitaille, A.M., et al., Quantitative phosphoproteomics reveal mTORC1 activates de novo pyrimidine synthesis. Science, 2013. 339(6125): p. 1320-3. 24. Lindsey-Boltz, L.A., et al., The human Rad9 checkpoint protein stimulates the carbamoyl phosphate synthetase activity of the multifunctional protein CAD. 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Massant, J., et al., Metabolic channeling of carbamoyl phosphate, a thermolabile intermediate: evidence for physical interaction between carbamate kinase-like carbamoyl-phosphate synthetase and ornithine carbamoyltransferase from the hyperthermophile Pyrococcus furiosus. J Biol Chem, 2002. 277(21): p. 18517-22. 31. Thoden, J.B., et al., Structure of carbamoyl phosphate synthetase: a journey of 96 A from substrate to product. Biochemistry, 1997. 36(21): p. 6305-16. 32. Porter, T.N., Y. Li, and F.M. Raushel, Mechanism of the dihydroorotase reaction. Biochemistry, 2004. 43(51): p. 16285-92. 33. Buechner, M., Tubes and the single C. elegans excretory cell. Trends Cell Biol, 2002. 12(10): p. 479-84. | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/76680 | - |
dc.description.abstract | 計畫性細胞凋亡是ㄧ個受到許多基因調控的機制,且在生物的發育過程中扮演一個重要的角色。在秀麗隱桿線蟲中,要進行計畫性細胞凋亡的細胞是藉由不對稱分裂所產生的,其中比較大的細胞會繼續進行分裂、分化,然而比較小的細胞會進行計畫性細胞凋亡。我們實驗室先前已發現在計畫性細胞凋亡和不對稱分裂同時受損的線蟲中,尾巴腹側會有一個突起的表現型,我們稱之為“tail defect”,而此tail defect是由於尾部產生多餘的表皮細胞hyp8/9所造成的。我們利用此表現型,在線蟲具有不對稱分裂受損的情況下,進行正向基因篩選 (forward genetic screen),以找尋調控計畫性凋亡的新基因。我們篩選出一個在pyr-1基因上有點突變的突變株,pyr-1 基因與人類的 cad (carbamoyl phosphate synthetase、aspartate transcarbamylase 與 dihydroorotase)是同源基因,而其為一個嘧啶合成酶。我們先前已發現PYR-1具有一個促使細胞凋亡的功能,而此功能是與其原本的酵素活性無關,也證明了PYR-1在CED-3的下游去促進細胞凋亡。此論文中,我發現除了hyp8/9外,pyr-1的突變也會造成多餘的分泌細胞產生。我也找到PYR-1的第1690個氨基酸可能是CED-3的截切位置,且此氨基酸對於PYR-1促使細胞凋亡的功能是重要的。更重要的是,我也證明了PYR-1(349~1690)就已足夠去促使細胞凋亡,且PYR-1(349~ 742)和PYR-1(743~1690)需要一起作用才可以發揮此促進細胞凋亡的功能。此外,我也發現不同的PYR-1受截切的小片段具有不同的表現位置,因此我推測PYR-1 (349~1690)可能是由於表現位置的改變而具有了促進細胞凋亡的功能。總而言之,我們的研究指出嘧啶合成酵素在CED-3截切過後,擁有一個促進細胞凋亡的新功能,並推測PYR-1可能是由於表現位置的改變,來扮演了一個雙面的角色。 | zh_TW |
dc.description.abstract | Programmed cell death (PCD) is a tightly regulated suicide program and plays an important role during animal development. In Caenorhabditis elegans (C. elegans), cells died by PCD are generated through asymmetric cell divisions (ACDs) that produce a large cell and a small one which will undergo programmed cell deaths. Previously, our lab found that blockage of both PCD and ACD leads to the generation of extra cells in specific cell lineages, such as hyp8/9 hypodermal cells, and caused a bulge behind the anus, a phenotype we called tail defect. Using this phenotype, a forward genetic screen was conducted in the ACD-defective background to identify new PCD genes. One of these mutants was identified to have a mutation on pyr-1, the C. elegans homolog of cad (carbamoyl phosphate synthetase, aspartate transcarbamylase, and dihydroorotase), which is a pyrimidine synthetase required for the de novo pyrimidine synthesis pathway. PYR-1 has a novel pro-apoptotic function independent of its enzymatic activity and acts downstream of CED-3 to promote programmed cell deaths. In this thesis, I find that, in addition to hyp8/9, the pyr-1 mutation also causes extra excretory cell(s). I also identify the 1690th aspartic acid (D1690), which is a potential CED-3 cleavage site, as a critical site for the pro-apoptotic function of PYR-1. More importantly, I also demonstrate that PYR-1(349~1690) is sufficient to elicit the pro-apoptotic function of PYR-1. Moreover, PYR-1(349~742) and PYR-1(743~1690) need each other to exert the pro-apoptotic function. In addition, truncated PYR-1 fragments after the CED-3 cleavages have different expression patterns and localizations, suggesting that the translocalization of PYR-1(349~1690) may account for the novel pro-apoptotic function of PYR-1. Taken together, our studies reveal that pyrimidine synthetase possesses a novel pro-apoptotic function after CED-3 cleavages in specific PCDs and suggest that PYR-1 has a dual role in PCD, depending on its localization. | en |
dc.description.provenance | Made available in DSpace on 2021-07-10T21:34:57Z (GMT). No. of bitstreams: 1 ntu-105-R03b43005-1.pdf: 11213997 bytes, checksum: da032550703744093a7ac03239bb33a8 (MD5) Previous issue date: 2016 | en |
dc.description.tableofcontents | 致謝 i
中文摘要 ii Abstract iv Table of content vi Introduction 1 Materials and Methods 11 Strains and culture conditions 11 Plasmid constructions 11 Transgenic animals 16 Excretory cell and RMEV neuron analysis 16 Cell corpse analysis 16 Prediction of CED-3 cleavage sites 16 GFP expression and localization analysis 17 Statistical analysis and histograms 17 Results 18 pyr-1(tp12); grp-1(gm350) mutants had extra excretory cell(s) 18 pyr-1(tp12); grp-1(gm350) mutants had no extra RMEV neuron 19 pyr-1 is required for a specific set of cell deaths in C. elegans 21 Overexpression of pyr-1 rescued the decreased number of cell corpses but failed to induce ectopic cell deaths 22 The 348th, 742th, and 1690th aspartic acid (D348, D742, and D1690) are potential CED-3 cleavage sites of PYR-1 23 The potential CED-3 cleavage site D1690 is important for the pro-apoptotic activity of PYR-1 25 The N-terminal fragment of PYR-1 (N1690) possesses a pro-apoptotic function 26 PYR-1(349~1690) possessed a pro-apoptotic activity 27 Different PYR-1 fragments have different localizations and expression patterns 29 Discussion 31 PYR-1 possesses two opposite functions- pyrimidine synthetase and pro-apoptotic activity 31 The 561th 1602th and 1690th amino acids are important for the pro-apoptotic function of PYR-1 33 CED-3 just cleaves PYR-1 at GLN and DHO domains 35 Different expression patterns and locations of PYR-1 and truncated PYR-1 36 pyr-1 has a novel apoptotic function and acts synergistically with grp-1 to promote cell deaths 37 Both PYR-1 and CAD may have a moderate pro-apoptotic function after CED-3/Caspase-3 cleavages 38 The formation of cysts and vacuoles in pyr-1(tp12); grp-1(gm350) mutants was not caused by the extra excretory cells 40 Reference 41 Figures 44 Figure 1. The schemes of the PCD process in C.elegans. 44 Figure 2. grp-1; ced-3 mutants have extra cells. 45 Figure 3. PYR-1 is a pyrimidine synthetase and contains five domains involved in the de novo pyrimidine synthesis pathway. 46 Figure 4. The cell corpses of the aunt cells of hyp8/9 and excretory cell ere missing in pyr-1(tp12) and pyr-1(tp12); grp-1(gm350) mutants. 47 Figure 5. pyr-1(tp12); grp-1(gm350) mutants had extra excretory cell(s). 49 Figure 6. The pyr-1 (tp12) mutation did not cause extra RMEV neuron(s) in the grp-1(gm350) background. 50 Figure 7. The proposed lineages of the excretory cell and RMEV neuron in different genotypes 52 Figure 8. PYR-1 acts a downstream of CED-3 and is a direct substrate of CED-3 and acts downstream of ced-3 to promote programmed cell deaths....54 Figure 9. D348, D742 and D1690 are potential CED-3 cleavage sites in PYR-1. 56 Figure 10. The 1690th aspartic acid (D) is critical for the pro-apoptotic function of PYR-1. 58 Figure 11. There are six potential truncated PYR-1 fragments after CED-3 cleavages. 59 Figure 12. N1690 fragment can rescue the tail defect of pyr-1(tp12); grp-1(gm350) mutants. 60 Figure 13. PYR-1 1~348, PYR-1 349~742 and PYR-1 743~1690 fragments failed to rescue the tail defect of pyr-1(tp12); grp-1(gm350) mutants. 61 Figure 14. PYR-1 349~1690 fragment can rescue the tail defect of pyr-1(tp12); grp-1(gm350) mutants. 62 Figure 15. Different PYR-1 fragments have different localizations and expression patterns. 63 Figure 16. PYR-1(1~348) and PYR-1(349~742) did not co-localize with mitochondria. 64 Figure 17. PYR-1(349~1690) and PYR-1(743~1690) may co-localize with mitochondria. 66 Figure 18. N1690-GFP is too weak to be observed despite of a long exposure time. 68 Tables 69 Table 1. The reduced cell corpses of pyr-1(tp12) mutants was rescued by pyr-1 gene overexpression to a wild-type level. 69 Supplementary Figures 70 Figure S1. CED-3 just cleaves PYR-1 at GLN and DHO domains. 70 Figure S2. The cysts and vacuoles in pyr-1(tp12); grp-1(gm350) mutants were not caused by the extra excretory cells. 71 | |
dc.language.iso | en | |
dc.title | 線蟲CED-3對嘧啶合成酶之截切促進特定的計畫性細胞凋亡 | zh_TW |
dc.title | Cleavage of pyrimidine synthetase by CED-3 promotes specific programmed cell deaths in Caenorhabditis elegans | en |
dc.type | Thesis | |
dc.date.schoolyear | 104-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 吳瑞菁,江皓森 | |
dc.subject.keyword | 計畫性細胞凋亡,嘧啶合成?,CED-3,線蟲, | zh_TW |
dc.subject.keyword | programmed cell death,pyrimidine synthetase,CED-3,Caenorhabditis elegans, | en |
dc.relation.page | 71 | |
dc.identifier.doi | 10.6342/NTU201602800 | |
dc.rights.note | 未授權 | |
dc.date.accepted | 2016-08-18 | |
dc.contributor.author-college | 生命科學院 | zh_TW |
dc.contributor.author-dept | 分子與細胞生物學研究所 | zh_TW |
顯示於系所單位: | 分子與細胞生物學研究所 |
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