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研究生:桂泰如
研究生(外文):Gui, Tai-Ru
論文名稱:人類精胺基琥珀酸鹽合成酉每基因加強子之研究
論文名稱(外文):Characterization of enhancer elements of human argininosuccinate synthetase gene
指導教授:蘇宗笙
指導教授(外文):Su, Tsung-Sheng
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:1995
畢業學年度:83
語文別:中文
中文關鍵詞:遺傳精胺基琥珀酸鹽
外文關鍵詞:HEREDITY
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人類精胺基琥珀酸鹽合成酵素(argininosuccinate synthetase,AS)是尿素循環中的一個酵素,其催化的反應是將瓜胺酸(citrulline)和天門冬胺酸(aspartate)合成為精胺基琥珀酸鹽(argininosuccinate)。由於尿素循環在肝臟中進行,因此肝細胞中精胺基珀酸鹽合成酵素表現量最高,而且會受到升糖素 (glulcagon),糖皮質激素(gluucocorticoid),及cAMP等之誘導而增加在肝中的表現量。
利用forskolin會刺激adenylate cyclase以增加細胞內cAMP的含量之特性,處理11種人類肝癌細胞株和人類表皮細胞株RPMI2650及其變異株Canrl,結果顯示Canrl以及肝癌細胞株HuH-7,Hep3B-T2,HuH-6 clone5等4種細胞株中,精胺基琥珀酸鹽合成酵素mRNA明顯地增加。所以精胺基琥珀酸鹽合成酵素在有些人類肝癌細胞株中及Canrl細胞中的確可以受到。cAMP的誘導而增加mRNA含量。而利用一種人工合成的糖皮質激素類似物(agonist)-dexamethasone,同樣處理肝癌細胞株,及人類表皮細胞株RPMI2650和Canrl,結果在所有的細胞株中均未出現精胺基琥珀酸鹽合成酵素mRNA受dexamethasone誘導而增加的現象。
為瞭解DNA甲基化如何調控人類精胺基琥珀酸鹽合成酵素基因的表現,以NotI限制酵素尋找“CpG island”所在位置,於轉錄起始點下游1kb(第一個內子中)及上游10kb處各發現一個NotI切點;進一步分析第一個內子中NotI切點附近的序列,發現此處可能就是所謂的“CpG island”;經由序列比對發現在此區域有一個“加強子核心序列”(enhancer core sequence);另外發現一些transcription factor的結合序列,如GC box,AP2蛋白質結合序列等。
為明瞭精胺基琥珀酸鹽合成酵素在肝臟中大量表現的組織專一性調控 (tissue-specific regulation),吾等利用DNase I核酸酵素高敏感區定位法(DNase I hypersenstive site mapping,DNase I HSsmapping)尋找cis-acting regulatory element的所在位置,並對這些區域做功能分析 (functinal assay)。利用人類肝癌細胞株HuH-7和表皮細胞株RPMI2650及其變異株Canrl,在人類精胺基琥珀酸鹽合成酵素基因5'端找到4個DNaseI HSs,分別是位於距轉錄起始點-10kb,-6.3kb,-0.2kb,及+1kb的位置,並命名為HS-10,HS-6.3,HS-0.2及HS+1。HS-0.2即是精胺基琥珀酸鹽合成酵素基因的起動子所在位置;而HS+1與HS-10正是2個NotI限制酵素切點的位置。利用以thmidine kinase(tk)起動子的Chloramphenical acetyltransferase(CAT)的表現質體,把含有HS+1,HS-6.3,及HS-10的片段正向或反向分別接在tk起動子之前,短暫轉染至HuH-7細胞株後,發現HS+1有約增強CAT表現15-20倍之效應,而HS-6.3及HS-10各約增強5倍。而若將AS起動子前的HSS做各式的刪除,也發現其CAT的表現下降,顯然這些HSs確實為加強子之所在,其存在能增加tk及AS起動子的活性
本研究中也發現,HS-10片段對forskolin的誘導有反應,推斷其可能含有cAMP response element(CRE)。而HS+1只在HuH-7細胞中明顯存在,當將帶有HS+1片段的質體送入HeLa細胞時,其轉錄增強效應相當得差;另外帶有HS-6.3片段的質體也有類似情形發生,顯示不同細胞類型會影響HS+1及HS-6.3之增強轉錄作用;所以其中可能存在有肝細胞專一性的加強子(liver-specific enhancer)。
Argininosuccinate synthetase catalyzes the conversion of citrulline and aspartate to argininosuccinate, which is further converted to arginine by argininosuccinate lyase. Argininosuccinate synthetase is present in all tissues and cultured cells studied, but the highest enzyme activity is in the liver where the enzyme functions in the urea cycle to eliminate ammonia.
The mechanism of liver-specific enhancement of argininosuccinate synthetase is known to under the regulation of a tissue-specific extinguisher, i.e. Tse-1, which encoding a regulatory subunit R1α of protein kinase A. The action of Tse-1 is at the transcriptional level, thus, the liver-specific enhancement of argininosuccinate synthetase is at least in part controlled at the transcription initiation. To define the cis-regulatory elements, we used an indirect end-labeling method to locate DNase I hypersensitive sites (DNase I HSs) in the 5' region of the human argininosuccinate synthetase gene. Four DNase I hypersensitive sites, i.e. HS-10, HS-6.3, HS-0.2 and HS+1, at -10 kb, -6.3 kb, -0.2 kb and +1 kb in respect to the transcription start site are located. HS-0.2 is at promoter region while HS+1 is mainly present in liver cells. In addition, using Not I restriction enzyme digestion, two CpG islands that coresponding to HS-10 and HS+1 are located.
To study the function of these DNase I hypersensitive sites, the restriction enzyme fragment containing an individual site was inserted at the 5' end of an expression plasmid, i.e. tk-CAT, in either direct or reverse orientation. A 15-20 fold enhancement of CAT expression was observed when the expression plasmid with HS+1 was transiently expressed in HuH-7 cells. And a 5 fold enhancement was also observed when constructs with HS-6.3 or HS-10 was analyzed. In addition, if DNase I hypersensitive sites were deleted singly or in combination, the CAT expression under argininosucinate synthetase promoter regulation is also reduced. These results suggest that enhancer elements are located at these DNase I hypersensitive sites and trans-factors bound on these sites regulate cooperatively the transcription of the human argininosuccinate gene. We also found that when transfecting HS10-tk-CAT into HuH-7 cells, the CAT activity is subjected to forskolin induction, suggesting that cAMP response element may locate on the HS-10 region. In addition, considerable reduction in CAT expression was observed when HS+1-tk-CAT or HS-6.3-tk-CAT construct was transfected into HeLa cells when comparing to that in HuH-7 cells. The data imply that liver-specific enhancers may locate on HS+1 and HS-6.3 sequences. Thus, liver-specific transcriptional activators may bind on these enhancers to contribute the liver-specific enhancement of argininosuccinate synthetase expression.



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