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研究生:楊政修
研究生(外文):Cheng-hsiu Yang
論文名稱:分析LMO7在細胞有絲分裂時所扮演的角色
論文名稱(外文):Analysis for the role of LMO7 in mitosis progression
指導教授:莊育梩
指導教授(外文):Yue-Li Juang
學位類別:碩士
校院名稱:慈濟大學
系所名稱:分子生物及細胞生物研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
畢業學年度:96
語文別:中文
論文頁數:44
中文關鍵詞:紡綞體檢查點
外文關鍵詞:LMO7spindle assembly checkpointMAD1
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紡綞體檢查點主要的功能是在細胞進行有絲分裂時,確認複製後的姐妹染色體會正確的分離到兩個不同的子細胞中。人類的紡綞體檢查點包含著許多蛋白質,其中主要有MPS1、CENP-E、BUBR1、BUB3、MPS1、 MAD1、Aurora-B、NEK2、PLK1、LIS1、CLIP170、MAD2、ZW10以及ROD等。利用MAD1透過酵母菌雙雜交分析實驗從人類的肝臟基因庫中(liver cDNA library)我們找到了LMO7的C端298個胺基酸的片段,因此得知LMO7會和MAD1有交互作用。之後我們以純化後的LMO7抗體去偵測細胞內LMO7的分布,在免疫螢光染色的結果中我們發現LMO7會跑到細胞核、actin filament以及在metaphase時期時會和MAD1一起到中心體上,但是卻沒有參與細胞分裂末期cleavage furrow的形成。為了更進一步的研究LMO7的功能,我們製作了一系列將LMO7上不同功能性domain分別去除掉的基因突變株,並分別命名為LMO7bΔCH、LMO7bΔPDZ、LMO7bΔLIM、LMO7bΔMID、LMO7bΔC298、LMO7C298以及LIM domain only。將這些基因接上GFP後,放入人類子宮頸癌細胞中大量表現並觀察其在細胞中的位置與加入nocodazole後對於SAC功能的影響。我們看到在大量表現全長的LMO7時,會使得加入nocodazole所啟動的SAC機制被抑制。然而當去除LMO7上的LIM domain時,LMO7會喪失與actin filament結合的能力,並且會造成LMO7無法再去抑制nocodazole所啟動的SAC。接著我們又以轉殖LMO7 siRNA的方式去抑制細胞內LMO7蛋白質表現後,觀察到當LMO7被抑制時對於加入nocodazole所啟動的SAC機制並無影響。由上述的實驗結果我們可以推測LMO7調控SAC的功能可能與其和actin filament的結合有關。
The spindle assembly checkpoint (SAC)can ensure precise separation of paired sister-chromatids into two daughter cells during mitosis. The SAC components include MPS1, CENP-E, BUBR1, BUB3, MPS1, MAD1, Aurora-B, NEK2, PLK1, LIS1, CLIP170, MAD2, ZW10 and ROD. By yeast two-hybrid screening, we have identified that MAD1 interacted with LIM domain only-7(LMO7). Fluorescence microscopic analysis revealed that LMO7 localized to nucleus, actin filament, and seemingly the centrosome. The LIM domain of LMO7 was sufficient for its localization to the actin filament. LMO7 was not involved in the formation of cleavage furrow. We further made a series of LMO7 truncation construct to define its function in the SAC. We found that overexpression of the full-length LMO7 could significantly resulted in a defect in the SAC, and that deletion of the LIM domain could relieve the negative effect on the SAC caused by overexpression of LMO7. Besides, deletion of LIM domain made LMO7 very diffusively distributed with the cytosol in both interphase and mitotic cells. However, depletion of LMO7 by LMO7 siRNA did not affect the SAC activated by nocodazole treatment. These observations indicated that the LIM domain of LMO7 was important for its localization to actin filaments and its function toward the SAC.
第1章、序論 1
1.1 細胞週期及檢查點(cell cycle and checkpoint) 1
1.2 紡錘體檢查點(spindle assembly checkpoint) 1
1.3 MAD1 3
1.4 LIM domain only 7(LMO7) 3
1.5 LMO7與紡垂體檢查點的關係 5
第2章、實驗方法與材料 7
2.1 細胞株及菌種 7
2.2 質體 7
2.3 LMO7基因於表現載體之構築 8
2.3.1小量質體之萃取 8
2.3.2 大腸桿菌勝任細胞的製備 8
2.3.3 限制酵素處理LMO7基因及表現載體 9
2.3.4 選殖載體轉型進入勝任細胞 10
2.4 LMO72多源抗體的製備 10
2.4.1 重組蛋白質的大量表達及純化 10
2.4.2 LMO7親合性管柱的製備 11
2.4.3 LMO7抗體的純化 11
2.5 細胞免疫螢光染色 11
2.5.1 細胞繼代培養 11
2.5.2 細胞之轉染作用 12
2.5.3 免疫螢光染色 12
2.6 LMO7功能性分析 13
2.6.1 細胞的同期化 13
2.6.2 功能性分析實驗 13
2.6.3 LMO7 siRNA抑制LMO7蛋白質表現分析 13
2.6.4 西方墨點法蛋白質分析 14
2.7 酵母菌雙蛋白質雜交系統 15
2.7.1 酵母菌轉型作用(Yeast transformation) 15
2.7.2 酵母菌蛋白質萃取 15
2.7.3 蛋白質雙雜交實驗 16
第3章、實驗結果 17
3.1 LMO7抗體的製備 17
3.2 人類LMO7在細胞內的分布 17
3.3 LMO7所具有之各個functional domain對於LMO7 localization的影響 18
3.4 LMO7及LMO7 truncation mutants對於SAC功能的影響 19
3.5 Small interfering RNA(siRNA)抑制LMO7蛋白質表現後對於SAC功能的影響 20
第4章、討論 22
4.1 LMO7與MAD1之間的交互作用 22
4.2 LMO7在細胞中的分布 22
4.3 LMO7對於SAC造成的影響 23
4.4 LMO7上的功能性片段對於LMO7本身功能的影響 23
參考資料 25
附錄 42
1-1. Bacterial strains 42
1-2. PCR primers 44
1-3 LMO7 siRNA 44
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張智雲, 民96. 在細胞有絲分裂期的磷酸化蛋白質LMO7之分析/慈濟大學/分子生物及細胞生物研究所碩士論文
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