Identification of 5 ' regulatory region of pyruvate kinase gene from Drosophila melanogaster

• Main Authors: Pi-Feng Hsiao, 蕭碧鳳
• Other Authors: Yi-Chih Chien
• Format: Others
• Language: zh-TW
• Published: 2001
• Online Access: http://ndltd.ncl.edu.tw/handle/54842910248087559177

碩士 === 國立彰化師範大學 === 生物學系 === 89 === Pyruvate kinase (PK, EC 2,7,1,40) is a key glycolytic enzyme of Drosophila melanogaster. It catalyzes the conversion of phosphoenolpyruvate into pyruvate with the transfer of a phosphate group to ADP to form ATP. “ATP” provides energy for cell growth and metabolism, and “pyruvate” participates in many metabolic reactions. Therefore PK plays an important role in cell metabolism. Southern blot analysis and PCR were used to determine the content of a Drosophila genomic Pyk, lPK61. The result indicated that the insert of lPK61 comprised 8,330 bp upstream of and 7,186 bp downstream of TSP of Pyk gene. In other words, the size of the insert of lPK61 was 15,516 bp in total, which contained 6 genes including Pyk. Deletion mapping method was applied to identify the promoter region and cis-acting elements of 5’ of PyK from D. melanogaster. Ten serial deletions produced by PCR were inserted into upstream of a reporter gene (LacZ) to form recombinant plasmids, which then were transfected into Drosophila S2 cells. The results revealed that the regions of —1475~-1033 and —1033~-534 bp of 5’ end of PyK possessed positive regulatory function for PyK gene expression, i.e. raise PyK gene expression. We found that there were 53 possible cis-acting elements including EcRE (Ecdysone Response Element) and E74A and BRCZ (Broad Complex Zinc finger) binding sites. E74A and BRCZ belong to the “early” gene regulated by ecdysone. This result suggested that Pyk might be regulated by ecdysone, directly or indirectly. In addition, we found that in these regions there were many cis-acting elements in relation to egg and embryo development. Both regions of —258~-254 and —167~-163 bp comprise of CAAT box, and deletion of these regions resulted in decrease of reporter gene expression. Therefore, it was suggested that both CAAT boxes be functional, and the promoter of Pyk might be located in the region of —258~+109 bp. No TATA box and DPE (Downstream Promoter Element) were identified around TSP of Pyk. The 5’ region contains high ratio of C and G nucleotides. Besides, PyK might share the regulatory region with neighboring unknown gene. It was concluded that Pyk fit the characteristics of a housekeeping gene.