國立臺灣大學分子與細胞生物學研究所董桂書2006-07-252018-07-062006-07-252018-07-062005http://ntur.lib.ntu.edu.tw//handle/246246/5048減數分裂在高等生物有性繁殖中扮演關鍵性的角色，一次完整的減數分裂包含了一次DNA 複製，卻有二次染色體分離，因此而能平衡子代染色體的數目。減數分裂過程中，藉由同源染色體的配對、互換，而使染色體得以正常分離。為確認配對和互換在第一次染色體分離前完成，減數分裂細胞週期中從粗絲期進入到第一次核分裂期的轉換受到粗絲期檢控點的嚴密監控。此檢控點的分子作用機制是我們研究的主要目標。 酵母菌在減數分裂時特定表現的轉錄活化因子Ndt80 是驅動細胞進入第一次染色體分離期的必需蛋白。而Ndt80 的多重磷酸化與減數分裂週期的正常進行又有直接的關聯，因此Ndt80 蛋白質本身或是其磷酸化可能正是粗絲期檢控機制的直接作用點。但亦有研究報告認為粗絲期檢控點對Ndt80 活性的檢控是透過Sum1 轉錄抑制因子對於NDT80 基因表現的調控而間接作用。我們在界定Ndt80 功能區的研究中發現了一個顯性的片段缺失突變型，NDT80-bc。此一片段缺失使得Ndt80-bc 蛋白似乎不再受粗絲期檢控點的控制而維持恆常活性。Ndt80-bc 提供了研究粗絲期檢控點的重要方向。 本研究計畫即針對Ndt80-bc 與粗絲期檢控點間的作用關係，做進一步的分析，以確認Ndt80-bc 的作用確實是一般性的不受粗絲期檢控點控制，而非只針對特殊突變體（如zip1）。我們的實驗結果也充分支持Ndt80 蛋白本身即是粗絲期檢控點的作用標的。但磷酸化與Ndt80 功能之間的關係，目前尚未有明確的結論。此外，利用此缺失片段篩選與Ndt80 蛋白質直接作用的檢控點蛋白質也已開始進行。這些實驗結果除了可以增進我們對減數分裂的了解，同時也能幫助我們進一步探討細胞週期，更可以應用在許多人類遺傳疾病和癌症的研究上。Meiosis is essential for sexual reproduction. To ensure the success of meiosis, meiotic checkpoints operate to coordinate the proper order of meiotic events. In particular, the pachytene checkpoint prevents exit from the pachytene stage of meiotic prophase when meiotic recombination and chromosome synapsis are incomplete. Our research is planned to study the molecular mechanism of the pachytene checkpoint in detail. In budding yeast Saccharomyces cerevisiae, the NDT80 gene encodes a meiosis-specific transcription activator that is required for progression from pachytene into meiosis I. Multiple phosphorylation of Ndt80 occur in wild-type cells, but it is inhibited in cells arrested at the pachytene stage by the pachytene checkpoint. Based on these results, we suggested that Ndt80 is a direct target of the pachytene checkpoint. On the other hand, other reports proposed that Ndt80 activity is regulated indirectly at a transcriptional level by the pachytene checkpoint through the Sum1 transcription repressor. In our studies on defining Ndt80 functional domains, we have isolated a dominant deletion mutation, NDT80-bc. In this study, we have confirmed that the Ndt80-bc protein is resistant to the control of the pachytene checkpoint. It bypassed all the pachytene-arrested mutants tested, and it did not restore spore viability and crossover frequency. We proposed that the Ndt80 protein itself is a direct target of the pachytene checkpoint. The deleted region from Ndt80-bc might be the interacting site with an unidentified checkpoint protein(s). The Ndt80-bc provides a good evidence for our hypothesis and points out an important direction for the studies on pachytene checkpoint. The information learned from this project will provide valuable information for the understanding of meiotic cell-cycle control. It is also useful and applicable to researches on mitotic cell cycle, human genetic diseases, and cancers.application/pdf41658 bytesapplication/pdfzh-TW國立臺灣大學分子與細胞生物學研究所減數分裂細胞週期粗絲期檢控點酵母菌meiosiscell cyclepachytene checkpointbudding yeast[SDGs]SDG3reporthttp://ntur.lib.ntu.edu.tw/bitstream/246246/5048/1/922311B002102.pdf