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研究生:許惇偉
研究生(外文):Hsu, Duen-Wei
論文名稱:人類DNA甲基轉移酵素活體外功能研究
論文名稱(外文):Study of human DNA (cytosine-5)-methyltransferase function in vitro
指導教授:沈哲鯤沈哲鯤引用關係
指導教授(外文):Shen, Che-Kun
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:遺傳學研究所
學門:生命科學學門
學類:生物訊息學類
論文種類:學術論文
論文出版年:1997
畢業學年度:85
語文別:英文
論文頁數:71
中文關鍵詞:遺傳DNA
外文關鍵詞:HEREDITY
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DNA甲基轉移酵素(DNA(cytosine-5)-methyltransferase or DNA MTase)廣泛存於脊椎動物,此酵素可將甲基轉至特定區域CpG位置的胞喀咬(cytosine)上,藉以調控胚胎發育、基因活性等重要功能。該酵素主要由碳端約500胺基酸長度的酵素活性區位(domain)和被認為具活性調節功能的胺端調節區位構成。
本實驗利用聚合酵素連鎖反應(PCR)技術自人類細胞K-567分離完整(full length)及胺端缺少兩個表現子(exons)的偽完整(pseudo full length)甲基轉移酵素基因,分別命名為FMT(full length MTase,4.86kb)與MT(4.5kb);而碳端酵素活性區位已證實具新生甲基化(denovo methylation)作用,為了更了解此區位的功能,3'MT-1(2.3kb)與3'MT-2(1.5kb)兩不同長度的碳端區位被分離出。雖3'MT-1胺端有小部份由調節區位而來,但該片段蛋白卻能型新生甲基化作用。
而後再取組織蛋白(histone H1)、轉錄因子Sp 1和第一型拓撲異構酵素(topoisomnerase I)的DNA結合區位(binding domnain)接於3'MT-1的胺端以取代原有的調節區位,構築HM(Histone 3'MT-1)、SM(Sp1-3'MT-1)、TM(Topo-3'Mt-1)三種混合(hybrid)蛋白質。按著嘗試利用pKK2223-3、pET與pGEX等不同的原核生物蛋白質誘導系統大量表現這七種構築蛋白,研究它們6活體外(in vitro)的酵素活性,冀望找到適當的表現系統,以觀察FMT、MT、3'MT-1與3'MT-2甲基轉移的活性;並藉以分析具不同DNA結合區位的混合蛋白質(HM、SM與TM)能否只在特定DNA序列上行甲基轉移功能。
研究發現,SM可生成於pKK223-3系統裡,並可能有新生(de novo)甲基化作用;HM、SM與TM會表現在pET系統,但都沒有酵素活性;3'MT-1、3'MT-2、HM和SM皆可在pGEX系統中合成,唯3'MT-1才具酵素活性,且傾向新生甲基化功能。在每種表現狀態下,各蛋白質都有被嚴重分解的現象,並難以被純化,不易再做更進一步的酵素功能分析。故推測原核生物蛋白質表現系統不適於進行此研究。
DNA (cytosine-5)-methyltransferase, a DNA modifying enzyme,generally expressed in vertebrates, can transfer a methyl group to cytosine in CpG site of specific region to regulate some important function like embryogenesis, gene activity etc. This enzyme is composed an enzymatic domain at the C-terminal and a regulatory domain at the N- terminal.
PCR was used to amplify the total full length (including the newly identified two exons) and pseudo full length (lacking the two exons at the N-terminal) and were named as FMT (4.86 kb) and MT (4.5 kb) respectively. The C-terminal enzymatic domain was reported to have de novo methylation function. To understand this, two C-terminal fragments were cloned and named them as 3'MT-1 (2.3 kb) and 3'MT-2 (1.5 kb). The 3'MT-1 has some regions from the N-terminal regulatory domain and for some unknown reasons this construct failed to perform methylase function. However, 3'MT-1 have the enzymatic function and favor de novo methylation.
The primary aim of this thesis is to study the enzymatic function upon replacing the regulatory N-terminal region with some of the known DNA binding domains of the DNA binding proteins. For this reason the DNA binding domains of Histone H1, transcription factor Sp 1 and topoisomerase I were isolated and fused at the N-terminal region of 3'MT-1. Three hybrid constructs HM (Histone-3'MT-1), SM (Sp 1- 3'MT-1) and TM (Topo-3'MT-1) were made. Three different expression systems were used to express these hybrid constructs. After several experimental manipulations, it was found that SM can be expressed in pKK223-3 system, and had de novo methylation activity.
HM, SM and TM can be expressed using pET system but all the expressed proteins lost their enzymatic activity. Finally using pGEX system, 3'MT-1, 3'MT-2, HM and SM can be syntliesized, but only 3'MT-1 had the de novo enzymatic activity. As a common feature all the constructs expressed in any of the expression system have undergone serious degradation which hampered further purification or characterization of the enzyme.



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