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

组氨酸脱羧酶(HDC)是哺乳动物细胞中催化组氨酸合成组胺的唯一酶。组胺仅在造血细胞中的嗜碱性和肥大细胞系中表达，因此我们对该基因的组织特异性表达机制感兴趣。我们制备了不同的报告构建体，并将它们导入多种细胞系，发现该基因的上游6kb到下游1kb之间不存在组织特异性元件。我们通过基因组DNA印迹在启动子区域发现了组织特异的DNase I超敏感部位，这表明转录因子很容易接近这个区域来表达他们的基因。此外，在HDC不表达的细胞系中，许多胞嘧啶分子在该启动子区域发生甲基化。因此，HDC基因的细胞特异性表达被认为是由于染色质结构和甲基化模式的差异，而不是反式作用因子的差异。我们对小鼠HDC基因也很感兴趣，因为我们可以研究许多人类无法解决的生理和药理学问题。我们首先构建了含有荧光素酶基因转录起始点上游1099个碱基的融合表达载体，并将其稳定地导入小鼠肥大细胞系P815。利用该质粒，我们报道了同源BDF1小鼠(J.Biol.Chem.271,28439-28444)P815细胞在腹膜腔内诱导表达hDC基因的机制。我们计划使用以前的稳定转化子来阐明赋予这种诱导性的顺式作用元件。通过将多种药物作用于该细胞系并检测其荧光素酶活性，我们可以评估这些药物对HDC基因反式激活活性的影响。我们现在正在克隆人类稳定的转化子，用于评估各种药物的活性。

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.