董桂書臺灣大學：分子與細胞生物學研究所陳雅妮Chen, Ya-NiYa-NiChen2007-11-252018-07-062007-11-252018-07-062005http://ntur.lib.ntu.edu.tw//handle/246246/49944在減數分裂中，若染色體聯會不正常或不能完成重組，粗絲期檢控點會使細胞停留在粗絲期直到缺失修復為止，目前對此檢控點的分子作用機制尚未明瞭。在酵母菌(Saccharomyces cerevisiae)中，Ndt80 與 Sum1 是兩個已知會被粗絲期檢控點調控的蛋白質。Ndt80是一個減數分裂時特定表現的轉錄因子(transcription activator)，它能夠誘導減數分裂中、後期，包括核分裂以及孢子形成相關基因的表現。在因粗絲期檢控點啟動而中止週期的細胞中，受Ndt80誘導的下游基因不會表現，若將檢控點相關基因突變，這些下游基因則回復表現。Sum1 則是在減數分裂早期擔任NDT80的轉錄抑制子 (transcription repressor) 的蛋白質。當細胞要進行核分前，Sum1蛋白會被降解。而當檢控點啟動時，Sum1蛋白持續穩定。 檢控點調控Sum1蛋白的穩定性有其中以下兩種可能的機制。檢控點直接調控Sum1蛋白的穩定性，進而降低NDT80基因的表現。另一種可能性是，粗絲期檢控點主要控制Ndt80蛋白的活性，再間接影響Sum1蛋白的穩定性。為了區分這兩種假設，我們分析Sum1蛋白在野生型，dmc1突變株，與dmc1 NDT80-bc突變株的細胞中的穩定性。NDT80-bc 是一個的特殊片段缺失突變株，它能讓細胞略過粗絲期檢控點。實驗結果顯示，與之前的研究符合，在野生型細胞中，當細胞進入減數分裂，Sum1蛋白會被降解。而當檢控點啟動時，Sum1蛋白持續穩定。有趣的是，我們發現Sum1蛋白在dmc1 NDT80-bc突變株的細胞中，即使在粗絲期檢控點被啟動的情形下，因為Ndt80-bc蛋白的存在，Sum1蛋白仍然會被降解。因此我們認為粗絲期檢控點可能不是直接調控Sum1蛋白穩定性，而是間接透過Ndt80蛋白的活性。In budding yeast, cells defective in meiotic recombination undergo checkpoint-mediated arrest at the pachytene stage. The molecular mechanism of the pachytene checkpoint machinery in controlling meiotic cell cycle is not clear. It is known that Ndt80 and Sum1 are involved in the arrest. Ndt80 is a meiosis-specific transcription activator of middle sporulation genes, and it plays a critical role in regulating the progression during meiosis. Triggering of pachytene checkpoint prevents the accumulation of active Ndt80. Sum1 is a transcription repressor which inhibits the expression of middle sporulation genes, including the NDT80, at the early stage of meiosis. The level of Sum1 protein decreases transiently in wild-type cells during meiosis. In the pachytene-arrested cells, the level of Sum1 protein is stabilized, indicating that the stability of Sum1 protein is regulated by the pachytene checkpoint. Two different models have been proposed for the regulation of Sum1 stability by the pachytene checkpoint. The pachytene checkpoint may directly control the stability of Sum1. Alternatively, the stability of Sum1 is indirectly regulated through the activity of Ndt80 protein. To distinguish between these two models, the stability of Sum1 protein was analyzed in wild-type, dmc1, and dmc1 NDT80-bc cells. NDT80-bc is a dominant allele, which can completely bypass the pachytene arrest. Consistent with the previous study, the level of Sum1 decreased as the progress of meiosis in wild-type cells. In the dmc1 mutant, the pattern of Sum1 protein level was relatively constant during meiosis. In the dmc1 NDT80-bc strain, although the pachytene checkpoint was activated, the level of Sum1 protein was still declined in the presence of active Ndt80-bc protein. These data indicated that the Sum1 protein is probably not regulated directly by the pachytene checkpoint, and its stability might be controlled by the Ndt80 activity.ABSTRACT .............................................i 中文摘要 ............................................ii TABLE OF CONTENTS ....................................iii LIST OF FIGURES.......................................v LIST OF TABLE ........................................v CHAPTER 1 INTRODUTCION ...............................1 Meiosis Overview......................................1 Meiosis in Yeast......................................2 The Pachytene Checkpoint..............................3 The NDT80 Gene........................................4 The SUM1 Gene.........................................5 The Aim of Study......................................6 CHAPTER 2. MATERIALS AND METHODS .....................7 Strains and Media.....................................7 DNA Preparation.......................................8 DNA electrophoresis...................................8 DNA purification......................................8 Plasmid preparation...................................9 Plasmid construction..................................9 Yeast Construction....................................11 Epitope tagging of Sum1...............................11 Protein Analysis......................................12 Protein extract.......................................12 Brandford assay......................................12 SDS-PAGE..........................................13 Western bolt analysis.............................13 Cytology Analysis.....................................14 Analysis of nuclear division..........................14 Chromosome spreading..................................15 Tunicamycin Treatment.................................16 CHAPTER 3. RESULTS....................................20 Analysis of Sum1 Stability............................20 There is no obvious degradation of Sum1 protein in BR2495 background..................................20 Sum1 protein degrade in SK1-derived dmc1, NDT80-bc strain ..............................................21 Sum1 shift to lower mobility form before degradation..21 Timing of MeiosisⅠ and Pachytene Stage...............22 Cells proceed to nuclear division at 5 hours after induction to meiosis..................................22 The peak of pachytene stage is at 6 hr in the wild-type cell..................................................23 Studies for post-translational modification of Sum1...23 Western analysis of in-frame deletions of Sum1........23 SUM1 is not enlarged in DNA level by PCR analysis.....24 Tunicamycin treatment of the Sum1 protein.............24 CHAPTER 4. DISCUSSION.................................26 Regulation Mechanism of Sum1 Stability................26 Pachytene checkpoint may regulate Sum1 stability through Ndt80 activity................................26 The Sum1 is degraded before the pachytene checkpoint is inactivated.............................28 The dmc1 NDT80-bc cells are delayed in entry into the pachytene stage.......................29 Slight degradation of Sum1 in BR2495 may be due to low sporulation frequency.............................29 Post-translationally modification and protein stability ......................................................30 The Constant Post-translational Modification of Sum1..31 REFERENCES............................................34359016 bytesapplication/pdfen-US酵母菌粗絲期檢控點減數分裂budding yeastpachytene checkpointmeiosisotherhttp://ntur.lib.ntu.edu.tw/bitstream/246246/49944/1/ntu-94-R92b43018-1.pdf