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標題: | 鑑定梨形鞭毛蟲的Pax同源蛋白質對於cyst wall protein 1 和 2基因的轉錄調控之影響 Characterization of a novel Pax homologue involved in transcriptional regulation of cyst wall protein 1 and 2 genes in Giardia lamblia |
作者: | Yi-Ting Wang 王怡婷 |
指導教授: | 孫錦虹 |
關鍵字: | 梨形鞭毛蟲,cyst wall protein 1(Cwp1),cyst wall protein 2(Cwp2),gPax1,paired domain,nuclear localization signal(NLS), Giardia lamblia,cyst wall protein 1(Cwp1),cyst wall protein 2(Cwp2),gPax1,paired domain,nuclear localization signal(NLS), |
出版年 : | 2009 |
學位: | 碩士 |
摘要: | Pax蛋白質被發現於脊椎動物,果蠅和線蟲,功能上被認為是一個轉錄因子,參與細胞增生與發育;而在植物,真菌,以及原蟲中仍未被發現。我們發現在梨形鞭毛蟲中有類似pax的基因(gpax1),經由序列分析以及SMART軟體分析,發現梨形鞭毛蟲的gPax1蛋白質具有一個在C端的paired domain。在滋養體時期和囊體時期,我們發現內生性gPax1基因在mRNA層級上表現量相似。我們將AU1標記接到gPax1轉染到梨形鞭毛蟲,利用免疫螢光分析法發現,在滋養體時期以及囊體化時期,gPax1皆表現在細胞核,蛋白質層級表現量在此二時期也都差不多。我們利用cyst wall protein 1(cwp1)和cwp2基因的啟動子作為探針,電泳遷移率分析結果顯示gPax1蛋白質可以和cwp1和cwp2啟動子結合,推測其為一個轉錄因子,並可能影響cwp1和cwp2基因轉錄的結果。突變序列分析的結果也顯示,gPax1喜歡結合在AT-rih的序列上。我們從RNA表現層次和蛋白質表現層次皆可觀察到,當大量表現gPax1會促使cwp1和cwp2的RNA表現量和Cwp1的蛋白質表現量增加。將gPax1的paired domain刪除,稱之為gPax1del,發現gPax1del和cwp1以及cwp2基因的啟動子結合能力消失,gPax1del表現位於細胞質和細胞核中,且當大量表現gPax1del的細胞株相較於大量表現wild type gPax1的細胞株的cwp1和cwp2的RNA減少,且降低Cwp1蛋白質的表現量。推測gPax1的paired domain對gPax1的調控能力有很重要的影響。我們也發現在paired domain中有兩段正價胺基酸集中區域可能為nuclear localization signal (NLS)片段,分別將其序列上的正價性胺基酸作突變,分別命名為gPaxm1和gPax1m2,發現gPaxm1和gPax1m2都失去和cwp1以及cwp2基因的啟動子結合的能力。其中gPax1m1表現在細胞核中,且大量表現gPax1m1的細胞株相較於大量表現wild type gPax1的細胞株會使cwp1和cwp2的RNA表現減少,且Cwp1蛋白質表現量也減少。gPax1m2以囊泡型式,散布在細胞質中,大量表現gPax1m2的細胞株相較於大量表現wild type gPax1的細胞株的cwp1和cwp2的RNA量減少,且Cwp1蛋白質表現量也減少。由以上結果,我們認為只找到一段gPax1的NLS並且存在於paired domain (由gPax1m2的結果得知),此NLS的突變造成gPax1減少入核以及減低其調控能力。另一段存在於paired domain的突變序列雖然不會影響gPax1的表現位置,但減低其DNA結合能力及轉錄活化能力 (由gPax1m1的結果得知),故我們推測,gPax1蛋白質為一個特異性的轉錄因子,和cwp1和cwp2基因轉錄之轉錄活化有關,且其功能與paired domian的完整性有很重要的關係。 Pax proteins with paired domains have been identified in Drosophila, nematodes, and vertebrates. Pax proteins have been demonstrated as specialized transcription factors involved in cell proliferation and development. To date, pax-like gene has not been found in plant, protozoa and fungi. We found one Pax-like open reading frame from Giardia lamblia genome data base and we named it gPax1. gPax1 has a paired domain near its C terminus as predicted by SMART program. During vegetative growth and encystation stage, endogenous gPax1 gene was expressed at similar levels of mRNA. To understand the function of gPax1, we transfected a construct which expressed AU1-tagged gpax1 gene into G. lamblia. Inmunofluorescence assay and Western blot analysis showed that the AU1-tagged gPax1 was localized to neclei and expressed at similar protein levels during vegetative growth and encystation stages. Using Electrophoretic Mobility Shift assays (EMSA), we found that gPax1 can bind to the promoter of cyst wall protein 1 (cwp1) and cwp2 genes, suggesting that gPax1 could be a transcription factor and involved in transcriptional regulation of the cwp1 and cwp2 genes. Mutation analysis revealed that gPax1 prefered to bind to AT-rich sequence. Overexpression of gPax1 resulted in a significant increase of levels of cwp1 and cwp2 mRNA and of the Cwp1 protein. A gPax1 mutant, gPax1del, that lacks the paired domain was analyzed. We found that gPax1del lost the binding ability to the promoter of the cwp1 and cwp2 genes. gPax1del was localized to both nuclei and cytoplasm. We also found that the levels of the cwp1 and cwp2 mRNA and Cwp1 protein in the gPax1del overexpressing cell line decreased significantly relative to the levels in the gPax1 overexpressing cell line, suggesting that the paired domain of gPax1 could be very important to the regulation ability of gPax1. Two stretches of basic amino acids were found in the paired domain of gPax1. We tried to understand whether these two regions are nuclear localization signal (NLS). When we mutated the basic amino acids in these two regions to neural amino acids (gPax1m1 and gPax1m2 mutants), the binding ability to the promoter of the cwp1 and cwp2 genes was lost. gPax1m1 was localized to nuclei. We found that the levels of the cwp1 and cwp2 mRNA and Cwp1 protein in the gPax1m1 overexpressing cell line decreased relative to the levels in the gPax1 overexpressing cell line. gPax1m2 was localized to some vesicles in cytoplasm. The levels of the cwp1 and cwp2 mRNA and Cwp1 protein in the gPax1m2 overexpressing cell line decreased significantly relative to the levels in the gPax1 overexpressing cell line. One stretch of basic amino acids in the paired domain could be NLS as shown by the results from the gPax1m2 mutant. Mutation of this potential NLS resulted in a decrease of nuclear localization and the regulation ability of gPax1. Mutation of another stretch of basic amino acids in the paired domain did not change the localization of gPax1 to the nuclei, but this mutation still decreased the DNA binding and transactivation ability of gPax1 (gPax1m1). We suggested that gPax1 may be an important transcriptional activator in regulation of the cwp1 and cwp2 genes, and the paired domain may contribute to the function of gPax1. |
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