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標題: | Ndt80蛋白抑制DNA複製之機制 Mechanisms for repressing DNA replication by Ndt80 |
作者: | Feng-Hsuan Teng 鄧鳳璇 |
指導教授: | 董桂書(Kuei-Shu Tung) |
關鍵字: | Ndt80,DNA複製,酵母菌,減數分裂,細胞週期, Ndt80,DNA replication,budding yeast,meiosis,cell cycle, |
出版年 : | 2005 |
學位: | 碩士 |
摘要: | 在出芽酵母菌(Saccharomyces cerevisiae)中,Ndt80是一個在減數分裂時特定表現的轉錄因子(transcriptional activator),它能夠誘導減數分裂中期基因的表現。有趣的是,在營養細胞中異位表現 (ectopic expression) Ndt80會造成細胞生長週期停滯在G1/S時期。由這樣的結果可推測,或許Ndt80能夠抑制第一次減數分裂(meiosis I)和第二次減數分裂(meiosis II)中間發生另一次DNA複製,使得細胞能夠成功地產生單倍體的孢子。Ndt80抑制DNA複製也許是透過誘導B-type cyclins基因或其他調控細胞週期進行的基因的表現,或者是經由Ndt80本身直接地結合在DNA的複製起始點上。
為了區別這兩種可能性,我們構築一系列的Ndt80片段缺失突變株(in-frame deletions),其缺失的區域分別在DNA結合區 (DNA-binding domain)或活化轉錄區 (transcription-activation domain)。將這些突變株於營養細胞中進行異位表現,測試這些突變是否會影響NDT80在營養細胞表現時造成的細胞生長停滯。結果發現Ndt80的DNA結合區對於造成G1/S時期的生長停滯是必要的;另一方面,Ndt80的活化轉錄區對於造成生長停滯並不是絕對地需要。此外,在B-type cyclins缺失的細胞中異位表現 NDT80仍然能夠造成細胞生長週期停滯在G1/S時期,顯示抑制DNA複製的機制似乎不是透過誘導Ndt80調控的基因。然而,利用染色質免疫沉澱分析(chromatin immunoprecipitation assay)無法提供證據證明Ndt80能夠直接地與DNA複製起始點結合。 另外,在這些片段缺失突變株中,我們發現一個有趣的突變,ndt80∆404-503。利用減數分裂時間曲線的分析以及更進一步的片段缺失分析,我們推測ndt80∆404-503是一個特別且功能可區別的NDT80突變株,它喪失了抑制DNA複製的功能,但仍保有部分活化轉錄的功能。 In Saccharomyces cerevisiae, Ndt80 is a meiosis-specific transcriptional activator that binds to the promoter element termed MSE (middle sporulation element) and induces expression of middle sporulation genes. Interestingly, ectopic expression of NDT80 in vegetative cells causes cell cycle arrest at the G1/S phase. It is possible that Ndt80 may repress another round of DNA replication between meiosis I and meiosis II, thus to successfully produce haploid spores. Repression of DNA replication by Ndt80 could be due to the induction of B-type cyclins or other unidentified genes that regulate cell cycle progression. Alternatively, it could be directly due to the binding of Ndt80 itself to the origins of DNA replication. To distinguish between these two possibilities, we have constructed a series of in-frame deletions in the DNA-binding domain or the transcription-activation domain of Ndt80 and tested for their effects on cell cycle arrest. The results showed that the DNA-binding domain is essential for the repression at G1/S. On the other hand, the transcription-activation domain is not absolutely required for the cell cycle arrest. Furthermore, ectopic expression of NDT80 in null mutants of B-type cyclins still causes G1/S arrest, suggesting that the induction of Ndt80-regulated genes is unlikely to be the repression mechanism. However, the chromatin immunoprecipitation assays did not provide evidence for physical associations between Ndt80 and the origins of DNA replication for the direct mechanism. Additionally, among these deletion mutations, we have isolated an interesting one, ndt80∆404-503. Based on meiotic time course analyses and further deletion analyses, we suggest that ndt80∆404-503 is a special separation-of-function mutation of NDT80 that loses the ability in repression of DNA replication but retains partial function in transcription activation. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/24332 |
全文授權: | 未授權 |
顯示於系所單位: | 分子與細胞生物學研究所 |
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