國立臺灣大學分子與細胞生物學研究所董桂書2006-07-252018-07-062006-07-252018-07-062006http://ntur.lib.ntu.edu.tw//handle/246246/5052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，卻有二次細胞核分裂使得染色體數目減半，因此能平衡受精作用，維持子代染色體數目的恆定。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本研究的主要目標。 酵母菌在減數分裂時特定表現的轉錄活化因子Ndt80 是細胞進入第一次染色體分離期的必需蛋白。而Ndt80 的活化與減數分裂週期的正常進行有直接的關聯，因此Ndt80 蛋白質本身活性的調控可能正是粗絲期檢控機制的直接作用點。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 蛋白不再受粗絲期檢控點的控制而維持恆常活性。Ndt80-bc 提供了重要的證據以及進一步研究粗絲期檢控點的有利工具。 本研究計畫主要針對Ndt80-bc 與粗絲期檢控點間的調控關係加以分析，以了解粗絲期檢控點的分子作用機制。同時，亦將利用其缺失片段篩選與Ndt80 蛋白質直接作用的檢控點蛋白質。我們發現在粗絲期檢控點被啟動的突變體中（如zip1），正常的Ndt80 蛋白不能進入細胞核，但Ndt80-bc 卻不受控制而能進入細胞核並活化其它基因的表現。因此，我們推論粗絲期檢控點藉由阻止Ndt80 進入細胞核而使其不能發揮轉錄因子的功用，進而控制減數分裂的進行。此一重大發現已撰寫論文，經最後修飾後，即將投稿。同時我們也以two-hybrid 的方法去篩選與Ndt80 直接作用的粗絲期檢控點蛋白，目前也找到了可能的基 因。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。Meiosis is essential for sexual reproduction. Single round of DNA replication with two successive nuclear divisions produce haploid gametes; and therefore, organisms maintain a constant genetic content from generation to generation after fertilization of two gametes. 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. We have proposed that Ndt80 is a direct target of the pachytene checkpoint. In our previous studies on defining Ndt80 functional domains, we have isolated a dominant deletion mutation, NDT80-bc. This Ndt80-bc protein is resistant to the control of the pachytene checkpoint and becomes constitutively active. The NDT80-bc mutant provides a strong evidence for our hypothesis and a powerful tool for the studies on the mechanism of the pachytene checkpoint. This research project is to study the molecular interaction between Ndt80-bc and the pachytene checkpoint. The Ndt80-interacting protein of the pachytene checkpoint machinery will be isolated. Our data showed that Ndt80 was retained in cytoplasm in the zip1 mutant, while Ndt80-bc was localized into the nucleus of the same cell. These results suggest that nuclear localization of Ndt80 is regulated by the pachytene checkpoint. In the pachytene-arrest cells, the Ndt80 protein is retained in the cytoplasm by an inhibitor interacting with the bc target domain of Ndt80.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/pdf50254 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/246246/5052/1/932311B002028.pdf