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研究生:鍾一全
研究生(外文):Yichuen Chong
論文名稱:大鼠懷孕特殊醣蛋白質基因受RBPJk和Notch調控之探討
論文名稱(外文):Regulation of a Rat Pregnancy-Specific Glycoprotein Gene by RBPJk and Notch
指導教授:陳宏文陳宏文引用關係
指導教授(外文):Hungwen Chen
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:生化科學研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2000
畢業學年度:88
語文別:中文
中文關鍵詞:懷孕特殊醣蛋白質啟動子轉染胎盤
外文關鍵詞:Pregnancy-specific glycoproteinpromotertransfectionplacenta
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懷孕特殊醣蛋白質 (Pregnancy-specific glycoprotein , PSG) 會專一性地表現在哺乳動物的胎盤中 , 且PSG蛋白質的表現程度會隨著胎盤的發育而大幅增加。為了進一步了解PSG基因表現的調控機制 , 我們針對其中一種齧齒類動物PSG基因 : rnCGM3 的啟動子區域進行研究探討。rnCGM3 基因的啟動子區域包含三個調控區域 : FPI , FPII 及FPIII區域。其中FPII結合因子已經證實為C/EBPbeta , 它能促進rnCGM3基因的表現。在本實驗室之前的研究 , 我們篩選得到一個FPIII結合因子 , rRBPJk-2N ( rodent Jk recombination signal sequence binding protein )。在本篇研究報告中 , 繼上述研究成果 , 我們進一步透過EMSA ( electrophoretic mobility shift assay ) 分析方法證實RBPJk 能夠結合至FPIII區域。我們也透過轉染實驗證明由FPIII-driven SV40啟動子所引發的基因表現 , 會受到rRBPJk-2N 的抑制 , 但NotchIC卻可以抵消此抑制效應。同樣地 , rnCGM3啟動子活性受C/EBPbeta促進增加的效應也會受到rRBPJk-2N 的抑制 , 但是可以受NotchIC的增進。此外 , 我們發現在FPI區域含有另一個RBPJk結合區域 , 也會受到NotchIC的影響。綜合以上實驗結果 , 顯示rnCGM3基因在胎盤中的表現 , 牽涉到 C/EBPbeta 及NotchIC的共同調節影響。

Pregnancy-specific glycoproteins (PSGs) are specifically expressed in placenta and the level of PSG increases exponentially during placental development. To understand the regulation of PSG expression, we characterized the promoter elements of a rodent PSG gene, rnCGM3. The promoter region of rnCGM3 gene contains three regulatory elements ( FPI, FPII, and FPIII ). The FPII-binding factor is shown to be C/EBPbeta, which enhances rnCGM3 gene expression. In previous study, we isolated a FPIII-binding factor, rRBPJk-2N (rodent Jk recombination signal sequence binding protein). In this study, we further revealed that RBPJk can bind to FPIII site by electrophoretic mobility shift assay. In a transient expression assay, we also demonstrated that rRBPJk-2N repressed the expression from an FPIII-driven SV40 promoter, but the active intracellular domain of Notch (NotchIC) could counteract the repressor activities of rRBPJk-2N. In the native context of rnCGM3 promoter, the promoter activity stimulated by C/EBPbeta was also repressed by rRBPJk-2N, but enhanced by NotchIC. In addition, we found that NotchIC stimulated C/EBPbeta-dependent expression was through another RBPJk site within the FPI site. Our data suggest that the placenta-specific expression of rnCGM3 may be connected to the C/EBPbeta and Notch signaling pathway.

目錄 ………………………………………………………………………… I
英文摘要 ………………………………………………………………… III
中文摘要 ………………………………………………………………… IV
縮寫表 ……………………………………………………………………… V
第一章 前言 ……………………………………………………………… 1
第一節 懷孕特殊醣蛋白質家族 Pregnancy-specific glycoproteins family (PSGs) …………………………………………………………… 1
第二節 CCAAT/Enhancer-binding Proteins (C/EBPs) 家族 ……… 2
第三節 Notch receptors受體家族 …………………………………… 3
3.1 Notch receptors家族之結構與功能 ………………………… 3
3.2 無脊椎動物 (Invertebrate) Notch 基因家族 …………… 5
3.3 脊椎動物 (Vertebrate) Notch 基因家族 ………………… 7
3.4 bHLH transcription factor 轉錄因子 …………………… 10
3.5 DSL ligand 配體蛋白質 ……………………………………… 11
3.6 Notch signalling pathway 研究進況 ……………………… 14
第四節 細胞核因子 Nuclear factor : CSL proteins ……………… 16
第二章 材料與方法 ……………………………………………………… 19
第一節 Plasmid construct 質體構築 ………………………………… 19
1.1 pCMV C/EBPb , pCMV rRBPJk-2N , pCMVmNotchIC 表現質體 19
1.2 (FPIII)6SV40CAT, (FPIIIM34)5SV40CAT , (FPIIIM56)4 SV40CAT , (FPI)6SV40CAT , (FPIM34A)6SV40CAT 報告質體 ……………………………………………………………………………… 19
1.3 pCGM3A , pCGM3A-DFPIII , pCGM3A-FPI&FPIIIM34A, pCGM3B , pCGM3B-FPIIIM34A 報告質體 ……………………………… 20
第二節 Electrophoretic mobility shift assay (EMSA) 分析方法 ……………………………………………………………………………… 23
第三節 Cell culture and transfection 細胞培養及轉染作用 ……………………………………………………………………………… 24
第四節 CAT assay (chloramphenicol acetyl transferase) 分析方法 …………………………………………………………………………… 24
第五節 Luciferase assay 分析方法 ………………………………… 25
第六節 Dynabeads M-280 streptavidin-biotinylated FPI oligonucleotide 結合分析方法 ……………………………………… 25
第七節 Western blotting 西方轉印法 ……………………………… 26
第三章 結果 ………………………………………………………… 27
第一節 在體外分析RBPJk 與FPIII之間的作用結合 ………………… 27
第二節 RBPJk 及Notch 對FPIII-報告構築質體的影響 …………… 28
第三節 Notch 與RBPJk 影響 C/EBPb 對rnCGM3啟動子的轉錄活化… 28
結果圖表 ………………………………………………………… 32
第四章 結論與討論 …………………………………………………… 37
第五章 參考文獻 ………………………………………………………… 40

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