薬剤スクリーニングの簡素化-ヒト・ヒスチジン脱炭酸酵素

点击翻译按钮获取中文摘要

Histidine decarboxylase (HDC) is the unique enzyme which catalyze the synthetic reaction of histamine from histidine in mammalian cells. Histamine is expressed exclusively in basophilic and mast cell lineage in hematopoietic cells, therefore we are interested in the mechanism of tissue specific expression of this gene. We prepared various reporter constructs and transfected them into verious cell lines and clarified no tissue specific element exists from 6 Kb upstream to 1 Kb downstream of this gene. We found tissue specific DNase I hypersensitive sites in the promoter region by genomic DNA blotting, suggesting that this region is easily approached by transcription factors for their gene expression. Moreover, many cytosine molecules were methylated in this promoter region in HDC-non-expressing cell lines. Therefore, the cell specific expression of HDC gene is thought to be confirred to the differences of chromatin structure and methylation pattern rather than those of trans-acting factors.We also are interested in mouse HDC gene, because we can approach many physiological and pharmacological problem which could not be approached in human. We, first, constructed a plasmid that contains 1099 bases upstream of the transcription initiation site fused with luciferase gene and transfected this plaamid stably into P815 mouse mast cell line. With this plasmid, we reported the mechanism of how P815 cells was induced to express HDC gene in peritoneal cavity of syngenic BDF1 mouse (J.Biol.Chem.271,28439-28444). We are planning to elucidate the cis-acting element confering this inducibility using former stable transformant. By applying many kinds of medicine to this cell line and measuring the luciferase activity, we can assess the effect of these medicine on the transactivational activity of HDC gene. We are now under cloning of human stable transformant to be used for the assessment of activity of various medicine.