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標題: | Ndt80 蛋白質磷酸化與其功能之關係 Relationship between Ndt80 phosporylation and its function |
作者: | Kuei-Shu Tung 董桂書 |
出版年 : | 2001 |
學位: | 碩士 |
摘要: | Ndt80蛋白為酵母菌Saccharomyces cerevisiae減數分裂特定表現的轉錄因數,能夠誘導減數分裂中、後期基因的表現。Ndt80的多重磷酸化與其活性有密切的相關,可能是決定其活性的關鍵,而Ndt80的磷酸化則可能是粗絲期檢控機制的重要作用點。本論文主要探討Ndi80的功能調控與磷酸化之問的關係。首先利用定點突變的方式將Ndt80上可能的磷酸化胺基酸改變,構築一系列的ndt80特定突變株後,進一步對這些突變株進行產孢效能、孢子存活率以及蛋白質磷酸化的分析。我們發現Ndt80-T318A與Ndt80-Y216A蛋白呈現磷酸化嚴重缺失而且完全不會產生孢子,推測T318和Y216為Ndt80的兩個主要之磷酸化位置。而在其他蛋白質已經證實以負電荷的胺基酸取代磷酸化胺基酸具有模擬磷酸化的效應,令人意外的是T318D與T318E並不能持續表現Ndt80的活性;不過,由免疫螢光分析的結果發現,Ndt80-T318D與Ndt80-T318E蛋白位於細胞核內,但是Ndt80-T318A則和粗絲期中止細胞中之未磷酸化正常的Ndt80蛋白一樣,分佈在細胞質中。由此結果推測T318的磷酸化可能控制了Ndi80的細胞核輸入。另外,我們過量表現ndt80突變株,分析Ndt80的磷酸化與隱抑粗絲期中止突變株zipl缺失能力之問的關係,整體而言,ndt80突變株產孢功能與隱抑zipl缺失的功能呈現正相關的關係。此外,我們亦構築ndt80片段缺失突變株,希望藉此定出Ndt80上的功能區域,作為日後進一步研究Ndt80功能的基礎工作。 In yeast, Saccharomyces cerevisiae, the NDT80 gene encodes a meiosis-specific transcriptional activator that regulates the expression of middle- and late-sporulation genes. Ndt8O is phosphorylated and the phosphorylation may be a major target of the pachytene checkpoint. This thesis is to further study the functions of Ndt80 and the regulation of Ndt80 activity by phosphorylation. We generated a series of point mutations at potential phosphorylation sites on Ndt80 and tested for their effects on sporulation, spore viability and phosphorylation. We found that residue T318 and Y216 are two important phosphorylation sites on Ndt80. The ndt80-T318A and ndt80-Y216A mutations display phosphorylation defects and eliminate the ability of sporulation. In several other proteins, it has been shown that substitutions of phosphorylated residues with negatively charged amino acids could mimic the phosphorylation state of the protein. Unexpectedly, changes of T318→D and T318→E cause sporulation defect instead of making Ndt80 constitutively active. However, Ndt80-T318D and Ndt80-T318E protein localize to nuclei, while Ndt80-T318A, as well as Ndt80 in pachytene-arrested cells, stay in the cytoplasm. These results suggest that phosphorylation at T318 might control nuclear import of the Ndt80 protein. We also tested the ability of suppressing zipl defects in sporulation by overexpression of these ndt80 mutant alleles. In general, the suppression of zipl in sporulation is correlated with the sporulation ability of each ndt80 mutants. In addition, we constructed several in-frame-deletion mutations and tried to define the functional domains of Ndt80. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/75188 |
全文授權: | 未授權 |
顯示於系所單位: | 植物科學研究所 |
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