(一) 建立試管中B型肝炎表面抗原起動子活性之測定 (二) CCAAT及CCAAT有關的motif對小鼠白蛋白基因起動子活性之影響

Abstract

(一)藉著試管中轉譯活性競爭(in vitro transcription competition)的測定方式，經加入合成的 UE, r-fibrinogen, NF-1, AP-1, HBV-E, HNF-l 寡核?酸片段，我發現在 HBsAg 轉譯控制區 (BstEⅡ2843 到 ECoRⅠ3221)，僅 HNF-l 可有效的在大鼠肝、脾臟細胞核萃取液中減低 HBV ?動子的轉譯活性．在將 HNF-1 與 HBV 序列比對後，發現在 3019 至 3032 的 13 個核?酸中(CAAGTAGGACTGG)有 9 個與 HNF-1 相同。由膠體電率轉移( Gel mobility shift assay)試驗知 HNF-1 為肝臟專有之蛋白質因數(liver specific factor)，由此似乎對 B 型肝炎病毒的肝趨性(Hepatotropism)特性給予吾人一些?示． (二) CCAAT 結合蛋白質目前已知具多樣性(multiplicity)，是以基因族(Gene family)的存在所轉譯，為了探討不同 CCAAT 結合蛋白在認知類似 CCAAT Box 序列時引起的生物活性差異，我們利用小鼠白蛋白基因構築了同質(homo logus)及異質(hetero-logus)的?動子系統作?我們研究的模式．實驗發現經接入人類乳突狀腫瘤病毒(HPV16)的一段 25bp 對稱序列及 SRE (serum response element) 片段可在活性轉譯測試中於肝臟萃取液顯出活性提高．Hela 細胞核莘取液則不會．表示如此的試管中測試模式應是日後研究此一問題的可行方向．

(1) Using Recombinant DNA techniques, we have constructed a plasmid PHBV400 which contains the transcription control region of the hepatitis B virus surface antiten gene. The plasmid directs efficient transcription by nuclear extracts from rat liver. Synthetic oligonucleotides UE, r-fibrinogen, NF-I, AP-I, HBV-E, and HNF-I were used from competition experiments in in vitro transcription reaction. Only HNF-I could compete for the transcription activity of HBV promoter. comparing the HNF-I with the HBV promoter sequences, a high degree of homology located at 3019-3032 (CAAGTAGGACTGG) was found. HNF-I is a tissue specific transcription factor, it may be involved in the regulation of the surface antigen gene and may be the element which reflects the hepatotropism of HBV. (2) The multiplicity of CCAAT binding protein has been well documented. To test the function of different CCAAT-related elements in a homologous and heterologous promoter using in vitro transcription assay is the main purpose of this study. It has been found that SRE and a plaindromic sequences derived from HPV16 could enhance the activity of mouse albumin promoter with rat liver nuclear extracts but not with HeLa nuclear extracts. Other CCAAT-related elements either have no significant effects or have detrimental effects. Therefore, an in vitro test of the enhancer function of CCAAT motif could be established based on our approaches.