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標題: | 斑馬魚轉錄因數STAT5基因之選殖與性質分析 Cloning, Expression and Characterization of STAT5 gene from the Zebrafish (Danio rerio) |
作者: | Pei-Shan Li 李佩珊 |
出版年 : | 2001 |
學位: | 碩士 |
摘要: | 細胞會利用細胞膜上之受體或其他分子來接受外界傳來的訊息,分析後產生改變以調節細胞基因表現活動,而STAT訊號傳遞途徑乃是其中重要之一環,而關於魚類 STAT基因之研究報告較少。為進一步瞭解魚類 STAT 基因之作用模式,本論文利用兼併性引子進行 cDNA 選殖,配合3'-及5’-RACE,選殖得到斑馬魚 STAT5 (z-ST5)基因全長為 2844bp,其中2358bp為可轉譯區域,轉譯出含785佃肢基酸的蛋白質,與人類 STAT5A ( h-ST5a)及STAT5B ( h-ST5b)基因肢基酸序列相比,發現其相同度分別為76%及77 %,在分子結構上具有STAT家族特殊之功能性區域;利用基因演化分析顯示z-STS 與 h-ST5a、 h-ST5b、 h-ST6 和 D-ST在演化上的相關性較高,可歸類為同一群組。 為克服魚類缺乏STAT家族上游活化因數活化 STAT之難題,於是將Z-STS基因全長轉譯區域構築在pcDNA3 表現載體,形成z-STAT5-HA ;並將z-ST5 基因與鯉魚JAKI之酪臉酸磷酸激醃區域JHI組成z-STAT5-HA-JHI,此重組蛋白可進行自身酪肢酸磷酉劉匕,使 z –ST5具有活性。將 z-STAT5-HA及 z-STAT5-HA-JHI進行離體轉錄及轉譯產生其蛋白質產物,並進行SDS-PAGE 蛋白質電泳分析,分別用anti-HA 和 anti-phosphotyrosine 單株抗體進行西方墨點分析,確定此二基因表現質體所表現之蛋白與預期大小相符,且 z-STAT-HA-JHI會對自身產生酪肢酸磷酸化之作用,而 z-STAT5-HA則不會被磷酸化。 將 STAT家族中之DNA 結合序列(TTCN2-4GAA)標記32P放射性作為探針,在和活化態之 z-STAT5-HA-JHI離體轉錄轉譯產物進行EMSA 分析,結果顯示 z-STAT-HA-JHI 會與ST5、Int16 和col7這些具有h-ST5 結合序列(TTC (T/C) N (G/A)GAA)作強而有力之結合;對探針ST4 也有微弱之結合,而對探針ST1、ST3、ST6、GAS、SIE則不會作用。 將表現質體 z-STAT5-HA及 z-STAT5-HA-JHI分別與β-casein-CAT 共轉染至 COS-l 及鯉魚CF細胞株以測試表現質體 z-STAT5-HA-JHI之活化基因轉錄能力。結果顯示在COS-1細胞中,只含有β-casein-CAT基因表現質體對受質之轉換率只有4%;含有z-STAT5-HA重組質體的轉換率有5%,而含有z-STAT5-HA-JH1重組質體對β-casein-CAT質體的轉換率卻有27%, CAT基因表現量有增加之趨勢。在鯉魚CF細胞實驗中也有相似的結果,表示活化之 z-STAT5-HA-JHI可與特定DNA序列結合,進而活化基因轉錄之機制,使基因之表現量增加。 One STAT homologue from zebrafish ( Danio rerio ) has been isolated . The full-length cDNA of zebrafish STAT5 (z-ST5) contains 2,844 bp and encodes a protein of 785 amino acids . From the amino acid sequence comparison , the z-ST5 is much closer to human STAT5 with an overall identity of 77% and 76% to human-STAT5b ( h-ST5b) and human-STAT5a ( h-ST5a ). respectiVely. Since there is no cytokine , interleukins , or growth factors available , at present , we decided to construct a constitutively active variant of z-ST5 . To obtain this variant , we constructed the z-ST5, which has been demonstrated to be autophosphorylated , with the JH1 domain of carp JAKI . Using anti-phosphotyrosine mAb PY-99, z-STAT5-HA-JHI, but not z-STAT5-HA was demonstrated to be tyrosine-phosphorylated . The phophorylated z-STAT5-HA-JHI was further used to investigate the DNA binding properties. As expected , z-STAT5-HA-JHI exhibited high affinity binding activity with STAT5 consensus binding site , which was identified in the promoter of mammalian β-casein gene and with Int 16 binding site that was located in the intron 16 of puffer fish JAK 1 gene . In addition z-STAT5-HA-JH1 also exhibited moderate binding activity with ST4 probe , but no binding activity with other STI , ST3 , ST6 , GAS , and SIE probes . To determine the transactivation activity of z-STAT5-HA-JHI mammalian COS-1 cells and carp CF cells were transiently cotransfected with a reporter plasmid containing the CAT gene driven by β-casein gene promoter and activator z-STAT5- HA-JHI . z-STAT5-HA-JHI shows the moderate transcriptional activity toward β-casein gene promoter in both mammalian cells and fish cells . |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/75164 |
全文授權: | 未授權 |
顯示於系所單位: | 生化科學研究所 |
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