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研究生:楊于萱
研究生(外文):Yu-Hsuan Yang
論文名稱:鑑定可補救酵母菌Δhtl1導致染色體多倍性表徵的基因
論文名稱(外文):Identification and Characterization of Genes that Rescue the Polyploidy Phenotype of Yeast htl1 Mutation
指導教授:鄭明媛
指導教授(外文):Ming-Yuan Cheng
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:生命科學暨基因體科學研究所
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:中文
論文頁數:64
中文關鍵詞:酵母菌RSC複合物染色質重組複合物HTL1基因染色體套數倍增mTn-LacZ/LEU2基因庫
外文關鍵詞:Sacchromyces cerevisiaeRSC complexchromatin remodeling complexHTL1ploidy shiftmTn/LacZ-LEU2 minitransposon library
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在Sacchromyces cerevisiae中,RSC複合物是染色質重組複合物,需靠ATP水解獲取能量執行移動核小體的功能,與酵母菌中的SWI/SNF複合物為相似物。Htl1p是RSC複合物中的一個次單元,先前研究指出,HTL1基因對於單套染色體細胞維持染色體穩定很重要。但是,HTL1如何影響細胞維持染色體穩定性目前仍不清楚。為了研究在Δhtl1突變株中,造成染色體套數改變的分子機制,我們先利用mTn-LacZ/LEU2基因庫在已帶有Δhtl1突變的菌株染色體DNA中製造隨機的插入性突變,藉此找出當產生插入性突變時,可以補救Δhtl1所造成的染色體套數倍增現象,從這些基因中找出HTL1對於染色體穩定性的可能機制。以分子遺傳篩選,找出經由標記基因周圍的酵母菌相似序列與細胞染色體DNA上的序列進行同源重組,將標記基因插入染色體DNA,以此篩選到12535個轉型株。經過染色體倍增測試,確認11株候選菌株。其中,我們發現其中一株可以補救Δhtl1導致的染色體套數增加的菌株其mTn-LacZ/LEU2插入在IRC5基因。但是,將Irc5p大量表現在該候選菌株中,無法將性狀回復成Δhtl1單一突變的狀態。此外,在Δhtl1突變株中將irc5完全剔除,此雙突變株仍然表現Δhtl1單一突變時,染色體套數倍增的性狀。若將此候選菌株與不帶有Δhtl1突變的wild-type菌株交配,產生的雙倍體再進行四分體孢子分離,測試孢子基因型及性狀,發現在Δhtl1存在的孢子中,即使有mTn-LacZ/LEU2存在,染色體倍增的現象並未下降。表示mTn-LacZ/LEU2插入的基因與Δhtl1所導致之染色體倍增現象下降沒有關連性,可能有未知突變存在部分候選菌株中。之後仍可以其他候選菌株繼續對HTL1基因與染色體套數維持的機制進行研究。
Yeast RSC complex is a homolog of SWI/SNF ATP-dependent chromatin remodeling complex, which mediates nucleosome rearrangement by ATP hydrolysis, in Sacchromyces cerevisiae. Htl1p is a subunit of RSC complex. Previous study showed that HTL1 is important for the maintenance of a single set of chromosome in haploid cells. However, how HTL1 maintains chromosome of ploidy in yeast cells is still unknown. To study the molecular mechanism that underlies ploidy change of Δhtl1 mutant, we induced genomic random mutagenesis using mTn/LacZ-LEU2 minitransposon library and screened for functional recovery of a disruption marker with an overlapping sequence in the presence of the inserted marker to isolate the genes which suppress the ploidy shift of Δhtl1 mutant. After screening of 12535 transformants, 11 mutant candidates were isolated. Among them, a mutant with the insertion in the IRC5 gene rescued the ploidy increase caused by Δhtl1 mutation. However, subsequent analysis by overexpressing IRC5 in the mutant strain or deleting IRC5 gene in Δhtl1 mutant failed to reveal the phenotype of Δhtl1 single mutant or rescue the chromosome gain of Δhtl1, respectively. After analyzing the correlation of the chromosome gain phenotype and the inserted marker of spores that generated from a cross between the candidate strains and the wild type strain, we do not find their correlation suggesting that other unknown mutations may exist in some candidate strains.
目錄
誌謝…………………………………………………………………………………i
中文摘要……………………………………………………………………………ii
英文摘要…………………………………………………………………………… iii
目錄…………………………………………………………………………………iv
緒論………………………………………………………………………………………………1
染色體倍數性………………………………………………………………………………1
染色質重塑複合物…………………………………………………………………………1
SWI/SNF複合物和RSC複合物……………………………………………………………2
RSC複合物…………………………………………………………………………………3
Htl1 …………………………………………………………………………………………3
研究目標及策略………………………………………………………………. ………………5
實驗架構………………………………………………………………………………6
材料方法………………………………………………………………………………7
大腸桿菌品系………………………………………………………………………………7
酵母菌品系…………………………………………………………………………………7
寡核甘酸……………………………………………………………………………………7
質體…………………………………………………………………………………………8
酵母菌基因庫………………………………………………………………………………9
培養基………………………………………………………………………………………9
酵母菌基因轉殖……………………………………………………………………………10
產孢作用及四分體孢子分離………………………………………………………………10
大腸桿菌基因轉殖…………………………………………………………………………10
小量抽取質體法……………………………………………………………………………10
粗萃取酵母菌染色體DNA…………………………………………………………………11
聚合酶連鎖反應……………………………………………………………………………11
mTn-LacZ/LEU2製造基因突變……………………………………………………………11
染色體倍增分析……………………………………………………………………………12
刀豆氨酸測試………………………………………………………………………………12
FACS…………………………………………………………………………………………12
恢復質體製作………………………………………………………………………………13
粗萃取酵母菌蛋白質………………………………………………………………………13
蛋白質泳動分析……………………………………………………………………………13
西方墨點法…………………………………………………………………………………14
位置搜尋聚合酶連鎖反應…………………………………………………………………14
結果……………………………………………………………………………………………16
Δhtl1/SH777四分體孢子分離……………………………………………………………16
[Ycp50]/Δhtl1/SH777及[pYJ9]/Δhtl1/SH777四分體孢子分離…………………………16
[pYES2-HTL1]/Δhtl1/SH777四分體孢子分離……………………………………………16
染色體增加測試……………………………………………………………………………17
PDH673轉型mTn-LacZ/Leu2基因庫………………………………………………………18
二次確認染色體倍增情形─刀豆氨酸測試………………………………………………18
確認候選菌株被mTn-LacZ/LEU2插入的基因……………………………………………19
確認IRC5基因與Δhtl1突變造成的染色體倍增降低之相關性…………………………20
確認候選菌株的染色體套數倍增降低是由被
mTn-LacZ/LEU2插入的基因導致 …………………………………………………… 21
以位置搜尋聚合酶連鎖反應(Site-Finding PCR)確認
mTn-LacZ/LEU2插入位置 …………………………………………………………… 21
討論……………………………………………………………………………………………23
參考文獻………………………………………………………………………………………28
圖表……………………………………………………………………………………………32
附錄………………………………………………………………........………………………55
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