臺灣博碩士論文加值系統

蛋白質與纏繞成染色質絲的DNA的交互作用很可能會被核小體列或是更高級的結構所阻擋。為了解決此問題，在酵母菌Sacchromyces cerevisiae中的三磷酸腺苷依賴之染色質重塑複合物 (ATP-dependent chromatin remodeling complex)，如SWI/SNF和其相似物RSC，被證實涉及干擾核小體的結構。RSC8譯碼出558個胺基酸的蛋白質並且與SWI/SNF複合物中的Swi3p有30%的相同性及52%的相似性。不同於Swi3p的是，Rsc8p對於細胞存活的進行是必需的 (essential)。Swi3p的SWIRM主域被證實是一個能與DNA結合並且有可能與蛋白質結合的主域。為了想找出是否Rsc8的SWIRM主域也能結合DNA及影響到複合物的完整性，在本篇論文中，利用位置導向突變技術 (site-directed mutagenesis)，產生Rsc8p上分別相對於Swi3p SWIRM主域中K383及K387的A158及K162的置換突變，來觀察SWIRM主域對於Rsc8結合DNA的性質以及RSC複合物完整性的影響。並且透過觀察比較野生型菌株與突變株的表型，檢驗是否Rsc8p的A158及K162的置換突變在功能上有重要影響。在本篇論文中，結果指出與野生型菌株比較下，(1) A158D、A158K、K162A、K162D與A158/K162的雙重突變，皆沒有顯著的生長情形缺失，(2)與染色質絲的連結情形也沒有明顯改變，(3)RSC複合物完整性也不受影響。

The interaction of proteins with DNA packaged into chromatin may be blocked by the nucleosome arrays and the higher order structure of chromatin. To solve this problem, in yeast Sacchromyces cerevisiae, ATP-dependent chromatin remodeling complex, such as SWI/SNF and its homolog RSC, have been proven to be involved in the perturbation of nucleosome structure. RSC8 encodes a polypeptide of 558 amino acids residues and is 30% identical and 52% similar to that of Swi3p in the SWI/SNF complex. Unlike Swi3p, Rsc8p is essential for cell viability. The SWIRM domain of Swi3p has been shown to be a DNA- and possibly a protein-binding domain. To investigate if SWIRM domain of Rsc8 could bind DNA and affects complex integrity, the influence of SWIRM domain on DNA-binding property of Rsc8 and the RSC complex integrity are evaluated , using the site-directed mutagenesis to generate substitution mutations on Rsc8p A158 and K162 relative to Swi3p K383 and K387 on the SWIRM domain. To examine if the substitution mutations on A158 and K162 of Rsc8p affect its function, the phenotypes of the Rsc8p mutants are examined and were compared with that of the wild type cells. The results demonstrated that, (1) A158D, A158K, K162A, K162D and the double mutants on A158/K162 showed no significant growth defect, (2) the chromatin association have not changed and (3) the RSC complex integrity are not affected.

中文摘要 I
英文摘要 II

緒論 1
核小體 (nucleosome) 的組成及功能 1
三磷酸腺苷依賴之染色質重塑複合物 (ATP-dependent chromatin
remodeling complex) 2
SWI/SNF複合物與RSC複合物之關係 3
RSC複合物之組成及功能 4
RSC8與SWIRM domain 5

實驗材料 7
一、酵母菌品系 (Saccharomyces cerevisiae strains) 7
二、大腸桿菌品系 (Escherichia coli strains) 7
三、寡核甘酸引子 (Oligonucleotide primers) 7
四、質體 (Plasmids) 8
五、培養基及培養液 (Media and broth) 11

實驗方法 12
1. 大腸桿菌質體轉殖 (Escherichia coli transformation) 12
2. 小量製備質體DNA (Mini-preparation of plasmid DNA) 12
3. 聚合酶連鎖反應 (Polymerase chain reaction; PCR) 13
4. 位置導向突變技術 (Site-directed mutagenesis) 13
5. 酵母菌質體轉殖 (Yeast transformation) 13
6. Tet-Off技術 (Tet-Off technique) 14
7. 點菌分析 (Spot assay) 14
8. 染色質連結分析 (Chromatin association assay) 14
9. 成孢作用及四分體孢子分析 (Sporulation and tetrad analysis) 15
10. 共同免疫沉澱 (Co-immunoprecipitation) 15
11. SDS-PAGE電泳分析 (Sodium dodecyl-sulfate polyacrylamide
gel electrophoresis) 16
12. 西方漬墨法 (Western blotting) 17

實驗結果 18
1. rsc8胺基酸置換突變株相較於野生型菌株生長情形未改變 18
2. rsc8胺基酸置換突變株與染色質絲連結情形未改變 21
3. rsc8胺基酸置換突變株複合物完整性沒有被影響 22

討論 23
rsc8胺基酸置換突變株對於酵母菌生長情形的影響 23
rsc8胺基酸置換突變株與染色質絲的連結 25
rsc8胺基酸置換突變株中RSC複合物完整性 27
Rsc8p SWIRM主域在RSC複合物中可能扮演的角色 29

參考文獻 32

結果圖表 40

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