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Title: | 結合NP-KB和RBPJK之複合單元調控大鼠懷孕特殊醣蛋白質, rnCGM3 基因之?動子活性 A composite element for NP-KB and RBPJK mediates the promoter activity of the rat pregnancy specific glycoprotein gene,rnGGM3 |
Authors: | Chorng-Der Wang 王崇德 |
Publication Year : | 2000 |
Degree: | 碩士 |
Abstract: | 懷孕特殊醣蛋白(pregnancy-specific glycoproteins,PSGs) ,是在哺乳類動物的懷孕過程中,胎盤所表現之主要蛋白質。為了瞭解此類蛋白質的基因表現和生理機制,囑齒類之PSG 基因,mCGM3,及其cDNA 序列已被分離並鑑識。由DNA序列之分析發現mCGM3 之全長為2761 bp ,可轉譯出475 個氨基酸。由和另一高相似性之基因mCGM6 之比較發現轉錄之起始位均在轉譯起始位上游之197 個核?酸位置,其功能區段具高度保留性。
進一步研究其基因之?動子區域,具有三個DNA 結合之單元:FPI ,FPII,和FPⅢ。我們發現,FP I 和FP Ⅲ 和RBPJK 有結合作用,而FPII 則和C/EBPβ 有結合作用。利用酵母菌之雜交系統(yeast one-hybrid system) 我們找出另一重要之轉錄因數:NF-KB和ppm 有結合作用;其中NF-KB 之二主要次單位和FPⅢ之結合作用中, p65 的結合較弱;而p50之結合較強。故而於此我們欲進一步研究兩種不同之因數:NF-KB 和RBPJK,於FPⅢ之結合效應對於基因之表現所產生之影響。 由EMSA (electrophoretic mobility shift assay) 和報導基因(reporter gene expression assay) 之分析,我們發現NF-KB 與其不同次單位p50, p65的或其異元二聚體均有結合作用,並且其於FPⅢ 所認知之核心序列變異會減弱結合作用。報導基因之分析發現,經轉染293 細胞株暫時性表現NF-KB 具活化作用之p65 次單位可刺激報導基因之表現;而p50 則不具有活化效應。同時NF-KB 之異元二聚體嵌合蛋白質亦具有活化基因表現之功能。RBPJK 可和FPⅢ產生結合作用,經由EMSA 之分析可知其和NF-KB 與FPⅢ可形成不同之DNA-蛋白質複合物。並且以NF-KB 所刺激報導基因之表現可被RBPJK 所抑制。我們的結果顯示藉由NF-KB與RBPJK 作用的訊息傳遞途徑可能影響mCGM3 基因的表現。 Pregnancy-specific glycoproteins (PSGs) are primarily expressed in the placenta and become the major glycoproteins at term. To understand the function and regulation of PSG regulation in pregnancy ,the promoter elements and cDNA sequences of a rodent PSG gene have been characterized. The cDNA of the rat pregnancy gene ,mCGM3 ,is 276lbp in length and contains an open reading frame that encodes a 475 amino acid polypeptide. The transcription initiation site of mCGM3 is located at nucleotide -197 upstream of the translation start site. Three nuclear protein binding sites: FP I ,FP II and FPⅢ in 5'-flanking region of mCGM3 gene have been characterized. The FP II binding factor was shown to be C/EBPβ.RBPJK was found to be the factor binds to both FP I and FPⅢ. We also found another important transcription factor ,the p65 subunit of NF-KB,which binds to the FPⅢelement in the yeast one-hybrid system. In this study ,we showed that both pSG and p65 subunits of NF-KB bind to the FPill element in the electrophoretic mobility shift assay (EMSA). A core sequence ,5’-GGGAAA-3' ,on the FPⅢ element for NF-KB was defined by mutagenesis analysis. Based on EMSA with NF-KB-enriched nuclear extracts from 293 cells transfected with different subunits of NF-KB,we found that both mono- and heterodimer of NF-KB bind to FPⅢ. We further studied the effect of two different transcription factors ,NF-KB and RBPJK,on the regulation of mCGM3 gene expression. By transient expression analyses ,we found the expression of CAT reporter gene could be activated by p65 and this stimulatory effect of p65 could be repressed by RBPJK. Our results suggest that mCGM3 gene expression may be mediated by the NF-KB and the Notch signaling pathway via RBPJK. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/75086 |
Fulltext Rights: | 未授權 |
Appears in Collections: | 生化科學研究所 |
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