Functional Analysis of Human Cytosolic NADP+-dependent Malic Enzyme Gene Promoter

• Main Authors: Huang Hsin-lung, 黃新龍
• Other Authors: Chou wei-yun
• Format: Others
• Language: zh-TW
• Published: 1998
• Online Access: http://ndltd.ncl.edu.tw/handle/43857860757517208148

碩士 === 國防醫學院 === 生物化學研究所 === 86 === To investigate the structure of the promoter of human cytosolic malic enzyme (ME)(EC1.1.1.40) gene, the 5*-flanking region in the length of 7.5Kb was sequenced. It revealed no potential TATA-box or CCAAT-box, but was rich in G+C base pairs in the core promoter. A series of deletions were constructed and their promoter activities were analyzed by transient transfection of the luciferase reporter gene. Four cell lines, HepG2, Hep3B, HEK and BC-M1 were used for analysis. The construct -700/+54 had relative maximal luciferase activity in HepG2 and HEK cell lines. Whereas, the maximal luciferase activities was shifted to -567bp in BC-M1 and Hep3B. For construct of -1447/+54, a significant promoter activity was observed in the HepG2 cell line. However, no similar promoter activity was observed in other three cell lines. Putative HNF-1, C/EBP, and ElK response elements were identified in this region by computer analysis. Electrophoresis mobility shift assay (EMSA) showed two HepG2 specific shift, which were competitively displaced by ElK and HNF-1, but not by C/EBP response elements The reverse transcriptase polymerase chain reaction (RT-PCR) was used to examine the existence of these three transcription factors. No ElK PCR product was identified in all four cell lines. The PCR product of C/EBP was found predominantly in the HepG2. The largest amount of HNF-1 PCR product was found in HEK cell line. We postulated that in the malic enzyme promoter, the activation of C/EBP was facilitated by HNF-1 in a cell-type dependent manner.