本文選題：基因沉默 + 沉默子　； 參考：《吉林大學(xué)》2011年博士論文

【摘要】：釀酒酵母染色質(zhì)的大部分端粒附近,rDNA區(qū)域以及HML和HMR區(qū)域基因是沉默不表達(dá)的,形成了類似高等真核生物的異染色質(zhì)結(jié)構(gòu)。沉默子HML-E和HML-I對(duì)其第三號(hào)染色質(zhì)的HML區(qū)域基因沉默起著決定作用,它們由復(fù)制起點(diǎn)識(shí)別復(fù)合物(Origin Recognition Complex, ORC)結(jié)合位點(diǎn), (repressor activator Protein, Raplp)結(jié)合位點(diǎn)和自主復(fù)制序列(autonomously replicating sequence binding factor, Ab.f1p)結(jié)合位點(diǎn)中的三個(gè)或者兩個(gè)結(jié)合位點(diǎn)所組成,這些結(jié)合位點(diǎn)被稱為原沉默子(protosilencer)。沉默子通過(guò)多種蛋白募集沉默信息(silent information regulator, Sir)蛋白到它上面,進(jìn)而沿著染色質(zhì)纖維傳遞,形成基因沉默的異染色質(zhì)區(qū)域。但組成沉默子的原沉默子單獨(dú)情況下對(duì)基因沉默的作用尚未研究清楚,此外常染色質(zhì)與異染色質(zhì)之間的轉(zhuǎn)化需要染色質(zhì)的重構(gòu),但任意一種Sir蛋白都沒(méi)有染色質(zhì)重構(gòu)活性,而染色質(zhì)重構(gòu)因子例如Fun30, Snf2, IswI等對(duì)基因沉默起一定作用,但是染色質(zhì)重構(gòu)因子在基因沉默以及異染色質(zhì)形成過(guò)程中的具體作用尚未研究清楚。 為研究原沉默子對(duì)基因沉默的作用,本研究采用帶有報(bào)告基因URA3的不同原沉默子替代HML區(qū)域HML-I沉默子,比較URA3的基因表達(dá)的差別,發(fā)現(xiàn)原沉默子單獨(dú)情況下沒(méi)有沉默作用,但與HML-E沉默子共同作用下能在兩者之間形成一定的基因沉默；為研究原沉默子拷貝數(shù)對(duì)基因沉默的影響,本研究利用帶有報(bào)告基因URA3的不同拷貝數(shù)的原沉默子Abflp結(jié)合位點(diǎn)替代HML-I沉默子,發(fā)現(xiàn)增加拷貝數(shù)能增強(qiáng)基因沉默作用；為研究原沉默子在不同位置對(duì)基因沉默的影響,本研究利用帶有報(bào)告基因URA3的原沉默子Raplp結(jié)合位點(diǎn)在不同位置的序列替代HML-I沉默子,發(fā)現(xiàn)Raplp在本身原來(lái)的位置有一定的基因沉默作用,在其他位置基本沒(méi)有基因沉默作用。為研究原沉默子基因沉默產(chǎn)生差異的原因,通過(guò)DNA拓?fù)浣Y(jié)構(gòu)法研究了不同條件下原沉默子對(duì)該區(qū)域染色質(zhì)DNA螺旋程度產(chǎn)生的影響,發(fā)現(xiàn)發(fā)現(xiàn)負(fù)超螺旋DNA的比例Abflp結(jié)合位點(diǎn)最高,ORC次之,Rap1結(jié)合位點(diǎn)最弱；為研究它們對(duì)對(duì)染色質(zhì)結(jié)構(gòu)產(chǎn)生的影響,通過(guò)不完全MNase降解結(jié)合Southern blot法發(fā)現(xiàn)不同的原沉默子與沉默子相互作用下原沉默子附近的核小體結(jié)構(gòu)發(fā)生較大變化,但沉默子附近的染色質(zhì)結(jié)構(gòu)沒(méi)有發(fā)生變化,通過(guò)染色質(zhì)結(jié)構(gòu)變化上不同推測(cè),可能是沉默子與原沉默子之間相互作用會(huì)隨著它們種類,數(shù)目,位置的不同而不同,從而導(dǎo)致形成的基因沉默上存在差別。 為研究重構(gòu)蛋白在異染色質(zhì)重排中的具體作用,本研究敲除了酵母的IswIp, Isw2p, Chdlp, Ino80p, Rad54p, Fun30等基因。發(fā)現(xiàn)敲除Isw2p, Chdlp, Ino80p, Rad54p基因?qū)湍傅幕虺聊瑳](méi)有影響,而敲除IswIp, Fun30基因會(huì)酵母的基因沉默產(chǎn)生影響；通過(guò)拓?fù)浣Y(jié)構(gòu)法以及MNase不完全降解結(jié)合Southern blot法發(fā)現(xiàn)敲除Fun30對(duì)異染色質(zhì)的形成影響很大而對(duì)異染色質(zhì)的維持影響不大,而敲除IswI對(duì)異染色質(zhì)的形成影響不大但對(duì)異染色質(zhì)的穩(wěn)定維持影響很大。 本研究創(chuàng)新之處：1)不同的原沉默子對(duì)插入的外源基因有不同的沉默作用,對(duì)異染色質(zhì)核小體的排列以及染色質(zhì)DNA螺旋有不同的影響；2)增加原沉默子Abfl結(jié)合位點(diǎn)拷貝數(shù)能增強(qiáng)基因沉默作用：3)改變?cè)聊拥呐帕幸矔?huì)影響基因沉默；4)染色質(zhì)重構(gòu)因子Fun30和IswI這兩種蛋白對(duì)基因沉默起增強(qiáng)作用；5)Fun30對(duì)異染色質(zhì)的形成起重要作用而對(duì)異染色質(zhì)的穩(wěn)定維持起很小作用,而IswI對(duì)異染色質(zhì)的形成起很小作用,而對(duì)異染色質(zhì)的穩(wěn)定維持起重要作用。
[Abstract]:Near the most telomere of Saccharomyces cerevisiae, the rDNA region and the HML and HMR region genes are silent and non expressed, forming a heterochromatin similar to the higher eukaryotes. The silencing HML-E and HML-I play a decisive role in the HML region gene silencing of its third chromatin, which are identified by the replication starting point (Origin Recogn). Ition Complex, ORC) binding sites (repressor activator Protein, Raplp) binding sites and three or two binding sites in the binding site (autonomously replicating sequence binding factor). These binding sites are called the original silencers. The silencers pass through a variety of eggs. The white recruitment of silent information regulator (Sir) protein is above it, and then transmitted along the chromatin fiber to form a gene silenced heterochromatin region. However, the role of the original silencer, which is composed of the silent child, has not been clearly studied, and the transformation between the outer chromatin and heterochromatin needs to be dyed. However, any kind of Sir protein has no chromatin remodeling activity, but chromatin remodeling factors such as Fun30, Snf2, and IswI play a role in gene silencing, but the specific role of chromatin remodeling factor in the process of gene silencing and heterochromatin has not been clearly studied.
In order to study the effect of the original silencer on gene silencing, this study uses a different original silencer with a reporter gene URA3 to replace the HML-I silencer in the HML region, and compares the difference in the gene expression of the URA3. It is found that the original silencer has no silence in a single case, but it can form a certain gene sink between the two and the HML-E silencers. In order to study the effect of the original silencing number on gene silencing, this study uses the Abflp binding site of the original silencer with different copies of the reporter gene URA3 to replace the HML-I silencer. It is found that the increase of the number of copies can enhance the gene silencing effect. The original silencer Raplp binding site of the reporter gene URA3 replaced the HML-I silencer in different locations. It was found that Raplp had a certain gene silencing effect in its original position, and there was no gene silencing in other locations. The reason for the difference of the silence of the original silencing subgene was studied by the DNA topological structure method. Under the same condition, the effect of the original silencer on the degree of helicity of chromatin DNA in the region was found. It was found that the proportion of Abflp binding sites in the negative super spiral DNA was the highest, ORC time and the Rap1 binding site were the weakest, and the effect of them on the chromatin structure was studied, and the different original silencers were found by the incomplete MNase reduction and Southern blot method. The nucleosome structure in the vicinity of the original silencer changes greatly with the interaction of the silencer, but the chromatin structure near the silencer does not change. It is presumed that the interaction between the silencer and the original silencer may be different with the species, the number and the position of the original silencers. There is a difference in the formation of gene silencing.
In order to study the specific role of recombinant protein in heterochromatin rearrangement, this study knocked out the genes of yeast IswIp, Isw2p, Chdlp, Ino80p, Rad54p, Fun30. It was found that the knockout of Isw2p, Chdlp, Ino80p, Rad54p genes had no effect on the gene silencing of yeast, and the Fun30 gene would affect the gene silencing of yeast; Papping structure method and MNase incomplete degradation combined with Southern blot method found that knockout Fun30 has a great influence on the formation of heterochromatin, but it has little effect on the maintenance of heterochromatin, but the knock off of IswI has little effect on the formation of heterochromatin but has a great influence on the stability of heterochromatin.
The innovation of this study: 1) different original silencers have different silencing effects on the inserted foreign genes, and have different effects on the arrangement of heterochromatin nucleosomes and chromatin DNA helix; 2) increasing the copy number of the binding site of the original silencer can enhance the gene silencing effect: 3) change the arrangement of the original silencer and affect the gene sink. 4) two proteins, chromatin remodeling factors Fun30 and IswI, enhance the gene silencing; 5) Fun30 plays an important role in the formation of heterochromatin and has a small effect on the stability of heterochromatin, while IswI plays a small role in the formation of heterochromatin, and it plays an important role in maintaining the stability of heterochromatin.

【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2011
【分類號(hào)】：R346

【共引文獻(xiàn)】

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本文編號(hào)：1854109