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研究生:張純瑜
研究生(外文):Chun-Yu Chang
論文名稱:粗絲期檢控點調控Ndt80活性之機制
論文名稱(外文):The control mechanisms of Ndt80 activity by the pachytene checkpoint
指導教授:董桂書
指導教授(外文):Kuei-Shu Tung
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:2005
畢業學年度:93
語文別:英文
論文頁數:60
中文關鍵詞:酵母菌減數分裂Ndt80粗絲期檢控點
外文關鍵詞:budding yeastmeiosisNdt80pachytene checkpoint
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在減數分裂過程中,染色體配對、聯會以及重組發生異常時,粗絲期檢控點會阻止細胞進入下一時期,直到缺失修復為止。在酵母菌(Saccharomyces cerevisiae)中,Ndt80是一個受到粗絲期檢控點調控的轉錄因子(transcription activator),它能促使減數分裂中後期有關細胞核分裂與孢子生成基因的表現,缺少Ndt80的細胞會停留在粗絲期,因此在細胞從粗絲期進入第一次減數分裂的過程中,Ndt80扮演重要的角色。
之前本實驗室發現一個特殊的NDT80突變株,NDT80-bc。它可以使細胞不受粗絲期檢控點的調控,在染色體聯會發生異常的情況下(例如zip1突變株)完成減數分裂並產生孢子。有趣的是,zip1 NDT80/NDT80-bc細胞進入核分裂以及孢子生成的時間有延遲現象,推測是失去活性的Ndt80存在並干擾Ndt80-bc的能力。為了探討粗絲期檢控點調控Ndt80活性的機制,我們利用不同的抗原決定基分別標定Ndt80以及Ndt80-bc,經過初步產孢效能與蛋白質分析,我們決定利用NDT80-6XHA/NDT80-bc-6XMYC來同時觀察Ndt80及Ndt80-bc在野生型與zip1突變株中的表現。在zip1 NDT80-6XHA/NDT80-bc-6XMYC細胞中,核分裂與孢子生成仍然有延遲的現象。在蛋白質表現上,Ndt80與Ndt80-bc蛋白開始表現的時間相似,另一方面,Ndt80與Ndt80-bc表現量沒有明顯的差異。透過免疫螢光染色,我們發現在zip1 NDT80-6XHA/NDT80-bc-6XMYC細胞中,Ndt80與Ndt80-bc蛋白細進入細胞核的效率有明顯的差異,推測Ndt80蛋白進入細胞核的過程受到粗絲期檢控點的監控,進而調節Ndt80的活性。
Mutants that confer defects in meiotic recombination (e.g., zip1) will trigger the checkpoint mechanism to arrest cells at the pachytene stage of meiosis. One of the control targets of the pachytene checkpoint is Ndt80. Ndt80 is a meiosis-specific protein that activates transcription of the middle sporulation genes, including genes required for nuclear divisions and spore formation. The Ndt80 activity is regulated by the pachytene checkpoint. Therefore, NDT80 is a critical factor which controls the progression from the pachytene into meiosis I.
Previous studies have isolated NDT80-bc, a dominant NDT80 mutant, which is not regulated by the pachytene checkpoint. Interestingly, the zip1 NDT80/NDT80-bc cells displayed a delay in nuclear division and spore formation, suggesting that inactive Ndt80 might exist and interfere with the activity of Ndt80-bc. In order to explore the regulatory mechanism of the pachytene checkpoint on Ndt80, we tried to use different epitopes to label Ndt80 and Ndt80-bc, respectively. Among all of these tagged Ndt80 and Ndt80-bc, NDT80-6XHA/NDT80-bc-6XMYC heterozygous in wild-type and zip1 were used to monitor the difference between Ndt80 and Ndt80-bc simultaneously. The zip1 NDT80-6XHA/NDT80-bc-6XMYC cells also displayed a delay in the kinetics of nuclear division and spore formation. We found that there is no significant difference in kinetics of protein expression between Ndt80 and Ndt80-bc in the zip1 NDT80-6XHA/NDT80-bc-6XMYC cells. Cytological analyses showed that the kinetics of nuclear import between Ndt80 and Ndt80-bc were different in zip1 NDT80-6XHA/NDT80-bc-6XMYC. Our results suggest that the Ndt80 activity is probably controlled by the pachytene checkpoint at the step of nuclear import.
ABSTRACT .................................................i
中文摘要 ................................................ii
TABLE OF CONTENTS ......................................iii
LIST OF TABLES .......................................vi
LIST OF FIGURES ......................................vii

CHAPTER 1. INTRODUCTION ...............................1
Meiosis Overview ........................................1
Meiotic Checkpoint ...............................2
The Pachytene Checkpoint ...............................3
NDT80 .................................................4
NDT80-bc .................................................6

CHAPTER 2. MATERIALS AND METHODS ......................8
Media and Genetic Methods ...............................8
DNA Preparation, Transformation, and Molecular Biology Methods .................................................8
Plasmids Construction .....................................9
Yeast Strains ............................................11
Kinetics of the Meiotic Cells ............................12
Protein Extraction and Western Blot Analysis ............12
Immunofluorescence Staining of Yeast Cells ............13
Quantification of Subcellular Localization ............14

CHAPTER 3. RESULTS ..............................16
The Effects of Epitopes on Ndt80 and Ndt80-bc ............16
Epitope-tagged version of Ndt80 and Ndt80-bc ............16
The N-terminal 3XMYC affects the function of Ndt80-bc.....16
The effect of C-terminal epitopes on Ndt80 ............18
The Regulation of Ndt80 by the Pachytene Checkpoint.......19
Construction of NDT80-6XHA/NDT80-bc-6XMYC ............19
Time course analysis of zip1 NDT80-6XHA/NDT80-bc-6XMYC ...20
There is no significant difference in protein expression between Ndt80 and Ndt80-bc in zip1 .......................20
Nuclear import of Ndt80 and Ndt80-bc are different .......22

CHAPTER 4. DISCUSSION ....................................25
The Regulation of Ndt80 Activity by the Pachytene Checkpoint ...............................................25
The expression of Ndt80 is similar to Ndt80-bc ...........25
The phosphorylation of Ndt80 is delayed in
zip1 NDT80-6XHA/NDT80-bc-6XMYC ...........................25
The nuclear import of Ndt80 might be
regulated by the pachytene checkpoint ....................26
The phosphorylation and nuclear import of Ndt80 ..........28
The Effects of Epitopes on Ndt80 and Ndt80-bc ............29
The positional effect of 3XMYC on the mobility of Ndt80 ..29
General discussion for 3XMYC-Ndt80-bc ....................30
The effect of C-terminal epitopes on Ndt80 ...............31
REFERENCES ...............................................33
APPENDIX ................................................59
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