利用酵母菌雙雜合系統篩選與Ndt80作用的蛋白質

The yeast two-hybrid screen for Ndt80-interacting proteins

何家瑋(Chia-Wei Ho)

指導教授 ： 董桂書

國立臺灣大學/生命科學院/分子與細胞生物學研究所/碩士(2006年)

https://doi.org/10.6342/NTU.2006.10518

摘要

在減數分裂時，若染色體聯會不正常或是不能完成重組，粗絲期檢控點會使細胞停滯在粗絲期，直到缺失修復為止。目前對於粗絲期檢控點作用的分子機制尚未完全明瞭。在酵母菌(Saccharomyces cerevisiae)中，Ndt80是一個減數分裂時期特定表現的轉錄因子(transcription factor)，它能誘導減數分裂中期、後期的基因表現，這些基因中，包含核分裂以及孢子形成相關的基因。目前已知Ndt80會受粗絲期檢控點控制，可能是透過transcription以及post-translation的兩個層次，但詳細的分子機制仍是未知的。在本實驗室之前的研究中發現，當Ndt80在346~402a.a這個區域缺失時(Ndt80-bc)，會不受粗絲期檢控點所控制，使粗絲期停滯突變株可以通過粗絲期檢控點，所以推測Ndt80受到粗絲期檢控點控制可能是透過蛋白質和蛋白質之間的交互作用來達成，而346~402a.a這個區域，正是粗絲期檢控點控制Ndt80活性的區域。
本篇論文的目的是要找出跟Ndt80有交互作用的蛋白質，並在這些有交互作用的蛋白質中，找出和粗絲期檢控點有關的蛋白質，藉此了解和粗絲期檢控點調控Ndt80活性的機制。利用酵母菌雙雜合系統搜尋和Ndt80有交互作用的蛋白質，我們發現Akr2和Ndt80有交互作用。同時，在zip1的情形下同時過量表現Ndt80和Akr2會使得Ndt80使突變株通過粗絲期檢控點的能力下降，我們推測，Akr2可能參與在抑制Ndt80活性的機制中。而且，Akr2也和Ndt80-bc有交互作用，所以我們推測，造成Ndt80-bc和Ndt80在活性上的不同，並不是透過Akr2，可能是其他因素造成的現象。但zip1akr2本身仍然無法通過粗絲期檢控點，顯示Akr2並不是粗絲期檢控點抑制Ndt80活性的主要機制，可能還有其他分子參與調控的過程。

關鍵字

粗絲期檢控點 ； 孢子 ； 酵母菌雙雜合系統 ； 減數分裂

並列摘要

In budding yeast Saccharomyces cerevisiae, cells defective in meiotic recombination and chromosome synapsis undergo checkpoint-mediated arrest at the pachytene stage. Previous study suggests that this checkpoint-mediated arrest is through Ndt80. Ndt80 is a meiosis-specific transcription factor that activates the expression of middle and late sporulation genes, including genes required for nuclear division and spore formation. Ndt80 is regulated by pachytene checkpoint at transcriptional and post-translational level. However, the detail mechanism is not clear. In our previous study, we found a dominant allele of Ndt80, Ndt80-bc. The deletion region of Ndt80-bc is in 346~402 a.a. We hypothesized that this region is the binding target of pachytene checkpoint to Ndt80. We proposed that the pachytene checkpoint regulates the activity of Ndt80 through protein-protein interaction.
In this study, we perform yeast two-hybrid screen to identify potential Ndt80-interacting proteins. We find that Akr2 has the potential to interact with Ndt80. Moreover, overproduction of Ndt80 and Akr2 in zip1 mutant decreases the suppression effect of Ndt80 overproduction in zip1 mutant. We suppose that Akr2 may negatively regulate Ndt80 activity. However, zip1akr2 mutant is still arrest at pachytene stage, suggesting that Akr2 may not be the most critical factor that negatively regulates Ndt80 activity. Furthermore, Akr2 also interacts with Ndt80-bc, suggesting that the different activity of Ndt80 and Ndt80-bc is not caused by Akr2, and there should be other reason that leads to the difference. We suggest that there would be other factors involved in the control mechanism.

並列關鍵字

pachytene checkpoint ； yeast two-hybrid screen ； meiosis ； Ndt80 ； sporulation

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