Genomic Structure and Promoter Region of c-fos Gene of Round-spotted Pufferfish, Tetraodon nigroviridis (Syn. T. fluviatilis)

圓斑河豚（Tetraodon nigroviridis [Syn. T. fluviatilis]）c-fos基因結構與起動子活性分析

張茂山(Mau-Sun Chang) ； 呂健宏(Jiann-Horng Leu) ； 姚振文(Chen-Wen Yao) ； 黃火鍊(Fore-Lien Huang) ； 黃銓珍(Chang-Jen Huang)

《Zoological Studies》 36卷3期 (1997/07) Pp. 159-169

摘要

圓斑河豚屬於豚形目四齒豚科，廣泛分佈於東南亞及東印度的河口區域，在臺灣容易自水族館購得，其基因體大小約為380 Mb，只有人類基因體大小的八分之一，適於從事基因體結構分析與比較。在鯉魚蛋白激酶JAK1基因的啓動子區域有兩處轉錄因子AP1結合的核酸序列，為了研究AP1如何調控JAK1基因的轉錄，我們便著手魚類c-fos基因的分子選殖。在本報告中，我們自圓斑河豚染色基因體基因庫篩選得到c-fos基因並定其基因結構。實驗的結果顯示圓斑河豚c-fos基因共包含4個外顯子和3個內隱子，可轉譯一個含374個胺基酸的蛋白質，其分子量約為42 kd，其胺基酸序列與虎河豚（Fugu rubripes）、人類、小白鼠與雞的相同度分別為90%、58%、58%與53%。我們利用引子延展分析決定了兩個可能的轉錄起始點，並且在圓斑河豚c-fos基因0.75 kb的啓動子區域中也發現了CREB、E2A、Ets-1、GATA-1、HNF-5、c-Myb及Sp1等轉錄因子的核酸結合序列。另外在氯徽素乙醯轉換酵素活性分析的實驗中，我們發現在圓斑河豚c-fos基因-746到+153的啓動子區域具有較對照組pRSV-CAT高出2.7倍的氯徽素乙醯轉換酵素活性，而此啓動子區域在5%胎牛血清的刺激下，其酵素活性亦明顯增加，證明圓斑河豚c-fos基因-746到+153的區域是一有功能的啓動子區域。

關鍵字

基因結構 ； 起動子 ； 圓斑河豚（Tetraodon nigroviridis）

並列摘要

The round-spotted pufferfish c-fos gene was isolated from a liver genomic library. Complete DNA sequences, including 0.75-kb of the promoter region were determined. Our data indicate that this gene is composed of 4 exons and 3 introns spanning about 3 kb. This gene encodes a protein of 374 amino acids with a molecular mass of 42 262 Da. The amino acid sequence identities between this puffer fish c-fos protein and fugu (another pufferfish species), human, mouse, and chicken c-fos protein are 90%, 58%, 58%, and 53%, respectively, Two potential transcription initiation sites were located by primer extension analysis, Examination of 0.75-kb of a 5'-flanking sequence revealed potential binding sites for a variety of transcription factors such as CREB, E2A, Ets-1, GATA-1, HNF-5, c-Myb, and Sp1. When the O.9-kb DNA fragment (-746 to +153) was placed upstream of the chloramphenicol acetyltransferase (CAT) reporter gene and transfected into a carp CF cell line, it could drive the synthesis of CAT enzyme 2.7 times more efficiently than could the RSV promoter. Although lacking the known serum-response element, the CAT activity of this region still could be induced by serum.

並列關鍵字

Genomic DNA sequence ； c-fos ； Round-spotted pufterfish Tetraodon nigroviridis ； Chloramphenicol acetyltransferase (CAT)