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http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/75320完整後設資料紀錄
| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.author | Hsin-Yi Chen | en |
| dc.contributor.author | 陳心怡 | zh_TW |
| dc.date.accessioned | 2021-07-01T08:12:40Z | - |
| dc.date.available | 2021-07-01T08:12:40Z | - |
| dc.date.issued | 2002 | |
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J Cell Biol. 68(3):620-8. 133．朱蔡啟。（1997）鯉魚c-mos基因表現蛋白性狀及功能之探討。國立台灣大學動物學研究所碩士論文。 | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/75320 | - |
| dc.description.abstract | 細胞進行有絲分裂時，染色體必須先複製、濃縮，再經由分離染色體的母機—紡錘體，將之分到兩個子細胞中。進行減數分裂的細胞也必須歷經以上過程，然而由於染色體組態及紡錘體行為的差異，減數分裂必須有所修飾或特化。研究顯示，Nek2和Ran與染色體濃縮皆有直接的關聯，Ran更為紡錘體形成機制的一員，而減數分裂特有蛋白Mos可能透過作用到相關元件，影響減數分裂染色體組態及紡錘體的形成、變動與維持。 細胞分裂時染色體、紡錘體狀態的控制元件彼此之間勢必需要直接的相互作用或間接的溝通，為了探究減數分裂細胞染色體組態變化及紡錘體行為特殊風貌的分子機制，瞭解相關的元件為第一步，因此本實驗自鯉魚的卵巢選殖了Nek2、Ran基因，於細菌表現Nek2重組蛋白，並製作了anti-Nek2抗血清，加上先前實驗室已選殖、表現的Mos基因，希望以此做為建立in vitro研究系統的基礎。由於Mos表現蛋白出現自我聚集的現象，為了探討此現象是否為內生性性狀，本實驗也於細菌表現了失去激?活性及C端truncated突變種Mos蛋白，希望能夠藉此瞭解Mos的性質並獲得有活性的表現蛋白，建立in vitro assay系統，釐清Nek2、Ran及Mos蛋白在減數分裂的相互關係。 | zh_TW |
| dc.description.abstract | Chromosome condensation and spindle formation are the principle phenomena of mitosis. Compared with mitosis, meiosis has the particular characters in chromosome configuration and spindle behavior. However, chromosome condensation and spindle formation are still necessary meiotic process. Because of the different between mitosis and meiosis, the meiotic machine must be modified. Nek2 and Ran had be shown to involve in chromosome condensation. Furthermore, Ran participates in spindle formation. On the other hand, Mos knockout data implied that the vertebrate meiosis protein is related to chromosome status and microtubule dynamics. For the sake of completing the separation, cell chromosome condensation components have to coordinate, via either direct interaction or indirect communication. To investigate the characteristic of meiosis molecular mechanism, the cDNA of Nek2 and Ran genes had been cloned from the carp ovary. The carp Nek2 recombinant protein expressed in bacteria was used to produce Nek2 antiserum. Combining with the previous cloned Mos, it will be the basis of in vitro assay system. When expressing Mos recombinant protein, we observed that the recombinant protein tended to aggregation. To inquiry if this is Mos intrinsic characteristic, kinase-dead and C-terminal truncated mos mutant were also expressed in bacteria. In the expect of grasping Mos property, obtaining active expression protein and establishing a in vitro assay system to deliberate the connection between Mos, Nek2 and Ran in the future. | en |
| dc.description.provenance | Made available in DSpace on 2021-07-01T08:12:40Z (GMT). No. of bitstreams: 0 Previous issue date: 2002 | en |
| dc.description.tableofcontents | 目錄……………………………………………………i 圖目錄……………………………………………………iii 簡（縮）寫表……………………………………………………iv 中文摘要……………………………………………………vii 英文摘要……………………………………………………viii 引言……………………………………………………1 壹、有絲分裂與減數分裂的分子機制……………………………………………………2 一、染色體濃縮與紡錘體組裝為分裂共同現象……………………………………………………2 1．染色體濃縮……………………………………………………2 2．紡錘體組裝……………………………………………………3 二、減數分裂分子機制必須特化……………………………………………………4 貳、NIMA與染色體濃縮……………………………………………………5 參、Ran為紡錘體形成的重要分子……………………………………………………7 肆、Mos為減數分裂特有的蛋白……………………………………………………8 一、Mos在卵母細胞成熟的角色……………………………………………………9 1．MPF自我放大迴路……………………………………………………9 2．GVBD……………………………………………………10 3．MI/MII transition……………………………………………………11 4．CSF……………………………………………………12 二、Mos的分解……………………………………………………13 三、Mos與染色體濃縮及紡錘體形成的關係……………………………………………………13 伍、Mos活化機制……………………………………………………14 一、激?的活化機制……………………………………………………14 二、Mos的活化……………………………………………………15 陸、實驗目的……………………………………………………16 一、Mos、Nek2、Ran間的交互作用……………………………………………………16 二、表現蛋白的聚集……………………………………………………17 材料與方法……………………………………………………19 壹、基因選殖……………………………………………………19 一、獲取基因片段序列……………………………………………………19 二、以RACE法選殖全長基因……………………………………………………19 貳、蛋白質表現……………………………………………………20 一、質體構築……………………………………………………20 二、蛋白質表現誘發……………………………………………………20 1．一般蛋白質表現……………………………………………………21 2．毒性蛋白表現……………………………………………………21 三、蛋白質萃取……………………………………………………21 四、蛋白質純化……………………………………………………22 1．denature純化……………………………………………………22 2．native純化……………………………………………………22 參、鯉魚卵萃液製備……………………………………………………23 一、超高速離心法……………………………………………………23 二、研磨法……………………………………………………23 肆、Glycerol gradient離心法測定蛋白質分子量……………………………………………………24 伍、蛋白質refolding……………………………………………………24 一、透析法……………………………………………………24 二、快速稀釋法……………………………………………………25 結果……………………………………………………26 壹、鯉魚卵巢Nek2基因……………………………………………………26 一、基因全長序列……………………………………………………26 二、蛋白質表現、純化與抗體製造……………………………………………………26 三、於鯉魚卵萃取液中偵測Nek2蛋白……………………………………………………27 貳、鯉魚卵巢Ran基因……………………………………………………28 參、鯉魚Mos表現蛋白的性狀探討……………………………………………………28 一、分離選殖無激?活性及C端truncated Mos基因……………………………………………………28 二、Anti-Mos抗體製造……………………………………………………28 三、以glycerol gradient離心法測定Mos蛋白複合體分子量……………………………………………………29 四、表現突變種Mos蛋白並與野生型比較溶解度……………………………………………………29 五、鯉魚Mos表現蛋白refolding……………………………………………………30 討論……………………………………………………44 壹、Nek2的調控……………………………………………………44 貳、Ran序列分析……………………………………………………46 參、激?間協調與transport系統……………………………………………………46 肆、Ran系統與transport及MPF活化……………………………………………………48 伍、Nek2和RCC1-Ran共同協調染色體組態……………………………………………………50 陸、核膜破裂前後Ran的角色定位及與Nek2、Mos的作用……………………………………………………51 柒、Mos聚集現象……………………………………………………52 引用文獻……………………………………………………54 | |
| dc.language.iso | zh-TW | |
| dc.title | 鯉魚減數分裂染色體濃縮相關基因Nek2、Ran之選殖 | zh_TW |
| dc.title | Molecular cloning of chromosome condensation related genes, Nek2 and Ran, in the carp oocytes | en |
| dc.date.schoolyear | 90-2 | |
| dc.description.degree | 碩士 | |
| dc.relation.page | 65 | |
| dc.rights.note | 未授權 | |
| dc.contributor.author-dept | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 動物學研究所 | zh_TW |
| 顯示於系所單位： | 動物學研究所 | |
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