Functional Analysis of Human Cytosolic NADP+-dependent Malic Enzyme Gene Promoter
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 p...
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ndltd-TW-086NDMC01070042015-10-13T11:06:17Z http://ndltd.ncl.edu.tw/handle/43857860757517208148 Functional Analysis of Human Cytosolic NADP+-dependent Malic Enzyme Gene Promoter 人類胞漿蘋果酸基因啟動子活性之分析 Huang Hsin-lung 黃新龍 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. Chou wei-yun 周慰遠 1998 學位論文 ; thesis 52 zh-TW |
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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.
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author2 |
Chou wei-yun |
author_facet |
Chou wei-yun Huang Hsin-lung 黃新龍 |
author |
Huang Hsin-lung 黃新龍 |
spellingShingle |
Huang Hsin-lung 黃新龍 Functional Analysis of Human Cytosolic NADP+-dependent Malic Enzyme Gene Promoter |
author_sort |
Huang Hsin-lung |
title |
Functional Analysis of Human Cytosolic NADP+-dependent Malic Enzyme Gene Promoter |
title_short |
Functional Analysis of Human Cytosolic NADP+-dependent Malic Enzyme Gene Promoter |
title_full |
Functional Analysis of Human Cytosolic NADP+-dependent Malic Enzyme Gene Promoter |
title_fullStr |
Functional Analysis of Human Cytosolic NADP+-dependent Malic Enzyme Gene Promoter |
title_full_unstemmed |
Functional Analysis of Human Cytosolic NADP+-dependent Malic Enzyme Gene Promoter |
title_sort |
functional analysis of human cytosolic nadp+-dependent malic enzyme gene promoter |
publishDate |
1998 |
url |
http://ndltd.ncl.edu.tw/handle/43857860757517208148 |
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