Cloning and characterization of the zebrafish 10-formyltetrahydrofolate dehydrogenase promoter region

• Main Authors: I-hsiao Chung, 鍾宜孝
• Other Authors: Tzu-Fun Fu
• Format: Others
• Language: zh-TW
• Published: 2007
• Online Access: http://ndltd.ncl.edu.tw/handle/08249215821525321063

碩士 === 國立成功大學 === 醫學檢驗生物技術學系 === 95 === 10-formyltetrahydrofolate dehydrogenase (FDH) converts 10-formyl- tetrahydrofolate (10-formyl-THF) to tetrahydrofolate (THF) and participates in the biosynthesis nucleotide precursor. It has been shown that FDH was down regulated during cell proliferation while over expression of FDH induced cell cycle arrest and apoptosis in cancer cells. Therefore the expression regulation of FDH is believed to tightly correlate to tumorgenesis and embryonic development. The aim of this study is to search for the possible mechanism of FDH expression regulation by cloning and characterizing the promoter region of zebrafish FDH (zFDH) gene. The 1703 bps fragment of zFDH promoter region was cloned upstream of luciferase coding sequence on pGL3-basic plasmid for transactivation activity analysis with Dual-luciferase assay in zebrafish liver epithelial cells (ZLE cell line). The 5’-UTR was cloned and the transcription starting site identified by PCR-cloning from the zebrafish full-length 5’-RACE cDNA library. Based on the results of serial deletion on promoter region and activity analysis, we found that the 164 bps (-124/+40) fragment is sufficient for substantial promoter activity. Deletion of the 40 bps 5’-UTR in exon 1 results in completely lost of promoter activity. The 164 bps fragment was DIG-labeled and incubated with nuclear extract in Electrophoresis gel Mobility Shift Assay (EMSA) and specific protein-nucleotide complexes were observed. Sequence analysis of the 164 bps fragment reveals potential binding site for Sp1 and c-Myb transcription factors. Site-directed mutagenesis to abolish these corresponding target sites results in decreased for Sp1 but no significant change for c-Myb transactivation activity. The coding sequence of zebrafish Sp1 had been cloned into the pET43.1a and pcDNA 3.1-myc/his expression vectors. We purified and characterized of the over-expressed Sp1 in E.coli and co-expressed the Sp1 in ZLE cells for promoter activity analysis.