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Title: | 圓斑河魨轉錄因數STAT基因之選殖與表現 Cloning and expression of the STAT (signal transducers and activators of transcription) genes in the round spotted pufferfish, Tetraodon fluviatilis |
Authors: | Ting-Jia Fan 範廷佳 |
Publication Year : | 1998 |
Degree: | 碩士 |
Abstract: | 為了探究魚類的JAK-STAT信號傳遞途徑,我們進行圓斑河魨轉錄因數STAT家族的基因選殖。利用5’端及3’端RACE的方法,得到STAT 3A、STAT 3B和STAT 5B完整的cDNA序列。STAT 3A全長有3.3kb,可轉譯成含764個胺基酸的蛋白質,推演分子質量為87411 Da。經胺基酸序列比對,與人及鱒魚STAT 3序列相同性分別?86.5%和92.8%。STAT 3B全長2.6kb,可轉譯758個胺基酸,推演分子質量為87506 Da,與人及鱒魚STAT 1胺基酸序列相同性分別為67.3%和77.9%。STAT 5B全長2.5kb,可轉譯785個胺基酸,推演分子質量為89798 Da,與人的STAT 5A及STAT 5B胺基酸序列相同性分別為78.0%和78.9%。 進一步將STAT 3A、STAT 3B及STAT 5B之全長cDNA構築於適當的表現載體,利用離體轉錄轉譯系統,可獲得如預期大小的蛋白質產物。若將這些表現載體轉染至哺乳類細胞株,進行暫時性基因表現分析,結果並無法檢測到STAT基因產物之表現。 另一方面,為暸解圓斑河魨STAT家族的基因結構,我們針對圓斑河魨STAT 3A、STAT 3B和STAT 5B的基因結構與啟動子進行選殖。結果顯示圓斑河魨STAT 3A基因是由23個外顯子與22個內隱子所組成,STAT 3B基因是由25個外顯子與24個內隱子所組成,STAT 5B基因是由19個外顯子與18內隱子所組成。STAT 3A基因由5’端RACE的終點,其上游1.5kb啟動子區域之核酸序列經電腦比對,顯示此區域具有c-Myb、CREB、CCAAT、GATA、AP1、Ets-1和Myc等轉錄因數的調控序列;而SSTAT 3B基因上游1.5kb之啟動子區域,則具有Sp1、GATA、Myc、E2F、AP1和c-Myb等轉錄因數的調控序列。為進一步分析圓斑河魨STAT3B基因啟動子區域之活性,我們將STAT 3A基因1.3kb的啟動子區域,以及STAT 3B基因1.5kb的啟動子區域,分別進行報告基因氯黴素乙醯轉換?(CAT),重組質體經轉染至鯉魚CF細胞,制備細胞萃取物後,測定氯黴素乙醯轉換?活性。結果發現pST3AP-CAT(-1360/+127)以及pST3B-CAT(-1512/+65)的CAT活性較高,分別校正對照組pRSV-CAT之活性增加1.3倍及0.8倍,顯示STAT 3A基因自-1360到+127的區域,以及STAT 3B基因自-1512到+65的區域,在鯉魚CF細胞中?具有功能的啟動子區域。 Signal transducers and activators of transcription (STATs) are cytoplasmic transcription factors that translocate to the nucleus and regulate gene expression in response to cytokine and growth factor stimulation. In this study, by using RT-PCR. 5'- and 3'-RACE, we have isolated the full-length cDNAs encoding the round-spotted pufferfish STAT 3A, STAT 3B and STAT 5B. The pufferfish STAT 3A cDNA encodes a protein of 764 amino acids whereas the STAT 3B cDNA encodes another protein of 758 amino acids. The deduced amino acid sequence of the pufferfish STAT 3A shows higher identity of 86.5 % and 92.8% to that of human STAT 3 and rainbow trout STAT 3, respectively. However, the amino acid sequence identity between the pufferfish STAT 3B and the human STAT 1 and the rainbow trout STAT l is only 67.3% and 77.9%, respectively. The pufferfish STAT 5B cDNA encodes a protein of 785 amino acids whose amino acid sequence shows 78% and 78.9% identity to that of human STAT 5A and STAT 5B. Using in vitro transcription and translation, the molecular mass of the gene products of these three pufferfish STAT cDNAs was the same as that calculated from the deduced amino acid sequence. However, when the mammalian expression vector containg these three pufferfish STAT cDNAs were transfected into 293T cell line, the putative gene products could not detected in the cell lysates. Three pufferfish STAT genes were also isolated from a round-spotted pufferfish genomic library and characterized. The pufferfish STAT 3A consists of 23 exons and 22 introns spanning about 8 kb whereas the STAT 3B consists of 25 exons and 24 introns spanning about 5.5 kb. However, the STAT 5B is composed of only 19 exons and 18 introns spanning at least 10 kb of genomic DNA. Examination of 1.5 kb of 5'- flanking sequence of the STAT 3A gene revealed potential binding sites for a variety of transcription factors such as c-Myb. CREB, GATA, AP1. Ets-1. and Myc. The sequence of the 1.5 kb region upstream of the first exon of the STAT3B gene contains numerous potential binding sites for transcription factors including Sp1. GATA, Myc. E2F. Ap1 and c-Myb. When the 1.3 kb DNA fragment (-1360 to +127) of the STAT 3A gene and the 1.5 kb DNA fragment (-1512 to +65) of the STAT 3B gene were placed upstream of the chloramphenicol acetyltransferase (CAT) reporter gene respectively, and then transfected into a carp CF cell line. The former and the latter construct could drive the synthesis of CAT enzyme 1.3 and 0.8 times than could the RSV promoter. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/76336 |
Fulltext Rights: | 未授權 |
Appears in Collections: | 生化科學研究所 |
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