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研究生:楊惠如
研究生(外文):Hui-Ju Yang
論文名稱:功能分析tlk-1,一個作用在線蟲染色體分離的基因
論文名稱(外文):Characterization of tlk-1 function in mitosis in C. elegans
指導教授:吳益群
指導教授(外文):Yi-Chun Wu
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:分子與細胞生物學研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:英文
論文頁數:69
中文關鍵詞:後期促進複合體擬混亂激酶有絲分裂線蟲染色體分離
外文關鍵詞:TLK-1AIR-2aneuploidymitosisanaphase-promoting complexchromosome segregation
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在有絲分裂時(mitosis),細胞必須完成遺傳物質均等的分配到子細胞中,而蛋白質的週期性降解(proteolysis)為一個主要的機制用以調控此一事件能有條不紊的進行。我們的研究顯示擬混亂激酶(Tousled-Like Kinase-1; TLK-1)參與細胞週期的調控機制而其本身也遭受週期性降解。利用一種斷絕基因表現的方法─核醣核酸干擾(RNA interference),我們發現tlk-1對於線蟲的存活是絕對需要的,因此是一個必須基因(essential gene)。在tlk-1核醣核酸干擾突變株中,線蟲的胚胎(embryos)都死在早期的細胞時期並伴隨著脹大的核(enlarged nuclei),除此,我們也觀察到DNA橋接(DNA bridges)和非整倍體(aneuploidy)的現象,因此我們推測TLK-1作用於染色體分離(chromosome segregation)。藉由免疫染色(immunostaining),我們發現TLK-1的表現受細胞週期所調控,TLK-1在有絲分裂末期(telophase),歷經間期(interphase)和有絲分裂前期(prometaphase)持續表現,但是在有絲分裂的中期(metaphase)和後期(anaphase)則會消失。後期促進複合體(anaphase-promoting complex, APC)為多單元的泛素連接酶(multimeric ubiquitin ligase),其會催化泛素(ubiquitin)與帶有摧毀訊息(destruction box)的蛋白質形成複合物;被泛素(ubiquitin)標誌的蛋白質將被蛋白酵體(prteosome)降解。摧毀訊息(destruction box)也被發現在TLK-1的蛋白質C端。進一步檢視在後期促進複合體(anaphase-promoting complex)的突變株下,發現TLK-1會不正常的環繞於中期(metaphase)的染色體;因此推測TLK-1是受APC調節而降解。在線蟲中,極光相關激酶(AIR-2)在染色體分離的進程中扮演了重要的角色。我們發現在極光相關激酶(AIR-2)的突變株下,TLK-1也會不正常的環繞於中期(metaphase)的染色體。除了弁鈳鄍?loss-of-function)的tlk-1會導致線蟲胚胎死亡外,過量表現TLK-1也會造成線蟲胚胎的死亡率提高;這樣的結果顯示TLK-1的表現量受到嚴密的調控。在線蟲中外源性表現TLK-1在人類的同源蛋白可以部分救援tlk-1核醣核酸干擾突變株所造成的死亡率,證明tlk-1的弁鄔呇b不同物種間具有高度保守性。本篇論文提供了tlk-1的表現受細胞週期調節以及可能的相關分子機制,進而為細胞週期的研究提供一個方向。
Faithful chromosome segregation during mitosis assure the transmission of genetic information and this event is much dependent on protein proteolysis. This thesis shows that TLK-1, a protein encodes serine/threonine kinase, plays key roles on mitosis and regulated by degradation. The tlk-1(RNAi) mutants arrested at about 50-cell stage showing the phenotype of aneupolidy and DNA bridge. TLK-1 protein was expressed in a cell-cycle-dependent manner. It is detected in interphase and prophase nuclei but not in mitotic cells from metaphase through anaphase. As TLK-1 harbors two D-box motifs, which are anaphase-promoting complex (APC)-dependent proteolysis signals, we investigated if these motifs are important for the dramatic disappearance of TLK-1 at metaphase. Mutations in D-box motifs caused the persistent presence of TLK-1 at metaphase, indicating that D box motifs are required for the degradation of TLK-1 at metaphase. The C. elegans mat-2 and emb-30 encodes subunits of APC. When any of these genes is mutated, TLK-1 protein is abnormally present at metaphase. The C. elegans AIR-2, Aurora/Ipl1-related Kinases, associates with chromosome and midbody microtubules is required for chromosome segregation. We further examined TLK-1 localization under air-2(RNAi) background; intriguingly, TLK-1 was found associated with metaphase chromosome. In addition to tlk-1 loss-of–function causing dead embryos, we found tlk-1 overexpression can also lead to embryonic lethal. Supposedly, the amount of TLK-1 in normal cells is tightly controlled in balance. The human homologue of TLK-1 can partially rescue the tlk-1(RNAi) mutant embryos, indicating that the function of tlk-1 is evolutionary functional conserved.
Abstract 2
中文摘要 3
Introduction 5
Experimental Procedure 12
C. elegans Strains and Alleles 12
Molecular Constructs 12
Transgenic Animals 14
Heat Shock Experiments 14
Bacteria-mediated RNA Interference 14
Embryonic Lethality Assay 15
Antibodies 16
Immunostaining 17
Microscopy 18
Results 19
Characterization of tlk-1 19
tlk-1(RNAi) mutants arrest at early embryogenesis showing the defects of chromosome segregation 20
Cell-cycle dependent of TLK-1 expression and localization 21
tlk-1(RNAi) mutants show an increased number of mitotic cells 22
Overexpression of tlk-1 increases the embryonic lethality 23
Mutations in D-box motifs cause the embryonic lethality and the persistent presence of TLK-1 at metaphase 24
TLK-1 protein is abnormally present at metaphase after APC genes are mutated 25
Depletion of AIR-2/Aurora B results in TLK-1 unnatural presence at metaphase 26
Mutation of CSC-1, a subunit of AIR-2/Aurora B kinase complex, caused TLK-1 to be present in metaphase 27
FZY-1 is normally localized to prophase and metaphase chromosome in tlk-1(RNAi) embryos 28
Depletion of asf-1 or caf-1, the potential targets of TLK-1, by bacteria-mediated RNAi does not cause embryonic lethality 29
Overexpression of human Tlk2 under the control of heat-shock promoters rescues the embryonic lethality caused by tlk-1(RNAi) 30
Discussion 32
Degradation of TLK-1 is mediated by anaphase-promoting complex pathway 32
The role of tlk-1 in histone H3 phosphorylation 32
TLK-1 and chromosomal passengers 33
tlk-1 is required for proper chromosome segregation 35
Figures and tables 39
References 65
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