臺灣博碩士論文加值系統

目前已知RSC複合物對於基因和細胞週期調控上扮演著舉足輕重的角色。而越來越多的研究資料顯示，HTL1基因和RSC複合物的構型、種類和功能有密切的關係。在本論文中利用了離子交換層析法、蔗糖梯度分離、高拷貝抑制基因策略篩選和蛋白剪切酵素的處理，希望能更深入地了解HTL1的功能以及它和RSC複合物之間的交互作用。
在離子交換層析法和蔗糖梯度分離實驗中得知，在野生型菌株和htl1刪除株中，RSC複合物的成員是否有被DNase處理過所純化出來以及在蔗糖梯度溶液中的分佈不同。除此之外，也利用了蛋白剪切酵素的處理觀察到缺乏Htl1p時，Rsc8p對蛋白剪切酵素endopeptidase Glu-C較敏感，這可能就是缺乏Htl1p會影響到RSC複合物穩定性的原因。為了更進一步探討HTL1基因的特性，利用高拷貝抑制基因策略篩選抑制htl1刪除株對benomyl過度敏感的基因，結果沒有挑到抑制者。
目前大家認為htl1刪除是直接造成酵母菌高溫致死的原因。根據我們實驗室先前利用蔗糖濃度梯度的方式來分析比較在37℃下生長的野生型菌株 (SEY6211) 和htl1刪除 (CKLa2)，發現缺乏Htl1p會影響RSC複合物的穩定性，使Rsc8p蛋白質和Sth1p蛋白質間的交互作用變的不穩定，然而，可能RSC複合物中的組成蛋白質間的交互作用不穩定才是造成酵母菌高溫致死的直接因素。為了更進一步觀察是否Rsc8p蛋白質和Sth1p蛋白質間的交互作用的穩定度是造成高溫致死現象的重要關鍵，因此設計了一個融合蛋白的實驗。我們將所建構出的STH1-LINKER-RSC8 (SLR) DNA序列轉殖入htl1刪除株中。目前觀察到有孢子可以生長在30℃和 37℃ SD-U培養基上。

RSC complex, an essential chromatin-remodeling complex in Saccharomyces cerevisiae, performs central roles in transcriptional regulation and cell cycle progression. RSC complex has been found to interact with Htl1p. To study the relationship between Htl1p and RSC complex, a series of analysis, including ion exchange column chromatography, sucrose gradient assay, multiple copy suppressor and partial protease, were performed in this thesis. By ion exchange column chromatography and sucrose gradient assay, the eluted protein and the distribution of subunits of RSC complex with or without DNase treatment were different between htl1 mutant and wild type cells. According to the analysis of sucrose gradient distribution in wild type and htl1 deletion strains at 37℃, we found Htl1p may play a role in maintaining stability of RSC complex, especially in the interaction between Rsc8p and Sth1p. Therefore, the instability of RSC complexes might be the main reason that causes temperature sensitivity. To further characterize if the interaction of Sth1p and Rsc8p is crucial for the temperature sensitive phenotype. A fusion protein experiment was designed. We constructed STH1-LINKER-RSC8 (SLR) fusion gene and inserted it into the strain without HTL1. So far that Ura+ spore can grow at 30℃ and 37℃. Partial protease digestion demonstrated that Rsc8p exhibits endopeptidase Glu-C sensitivity in htl1 deletion mutant. In order to further characterize HTL1 gene, we screened suppressors of Benomyl sensitivity of htl1 deletion strain by multicopy suppressor scheme. However, no suppressor of htl1 deletion strain was obtained.

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