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Characterization of 5 prime flanking region of 230 kDa bullous pemphigoid antigen gene

230 kDa 大疱性类天疱疮抗原基因 5 主要侧翼区域的表征

基本信息

批准号：
05670718
负责人：
SAWAMURA Daisuke
金额：
$ 1.41万
依托单位：
HIROSAKI UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1993
资助国家：
日本
起止时间：
1993 至 1995
项目状态：
已结题

项目摘要

Bullous pemphigoid antigens (BPA) are proteins which are recognized by circulating autoantibodies in the sera of the patients with bullous pemphigoid. At least two antigens have been reported so far ; one of which is a 230 kDa protein, wereas another protein is 180 kDa collagen. This study intended to characterize the 5 prime flanking region of 230 kDa BPA gene in order to know regulation of this gene expression. First, we screened a human and a mouse genomic libraries with a human 230 kDa BPA cDNA,resulting in human and mouse positive clones. Nucleotide sequencing of these clones showed that some clones contained the 5 prime flanking regions. And the sequences of about 3 kb of 5 prime flanking regions detected several putative cis-acting elements. High sequence homology between the human and mouse 5 prime flanking regions suggested that regulation of this gene expression is critical for the function of keratinocytes. Furthermore, after PCR amplification of a human 2kb segment and of a mouse 1.2kb segment upstream from translation starting sites, we made chlolamphenicol acetyltranferase (CAT) constructs with these fragments, and transfected them to cultured cells including keratinocytes. Detection of CAT activity in only keratinocytes indicated that these fragments contained cis-elements responsible for keratinocyte specific expression. We constructed deletion mutants and transfected them to keratinocytes to examine localization of the elements. The CAT activities revieled that the elements for keratinocyte specific expression resided in -216 to -197 of the human 5 prime flanking region, and in -525 to -213 of the mouse region.We made a few studies accompanied with the study mentioned above. We showed chromosomal localization of mouse 230 kDa BPA gene using the mouse clone, and examined a role of AP-1 site in the promoter of stromelysin gene in UV-induced skin changes to clarify the function of the AP-1 site in 5 prime flanking region of 230 kDa BPA gene.

大疱性类天疱疮抗原（Bullous pemphigoid antigens，BPA）是一类能被大疱性类天疱疮患者血清中自身抗体识别的蛋白质。迄今为止已经报道了至少两种抗原;其中一种是230 kDa蛋白，另一种蛋白是180 kDa胶原。本研究旨在对230 kDa BPA基因5 ′侧翼区进行分析，以了解该基因的表达调控。首先，我们用人230 kDa BPA cDNA筛选人和小鼠基因组文库，得到人和小鼠阳性克隆。这些克隆的核苷酸测序表明，一些克隆含有5个主要侧翼区。在5个引物侧翼区约3 kb的序列中检测到了几个推测的顺式作用元件。人和小鼠5 '侧翼区之间的高序列同源性表明，该基因表达的调节对于角质形成细胞的功能是至关重要的。此外，在PCR扩增翻译起始位点上游的人2kb片段和小鼠1.2kb片段后，我们用这些片段制备氯霉素乙酰转移酶（CAT）构建体，并将它们转染到培养的细胞（包括角质形成细胞）中。仅在角质形成细胞中检测CAT活性表明，这些片段含有负责角质形成细胞特异性表达的顺式元件。我们构建了缺失突变体，并将其转染到角质形成细胞中，以检查元件的定位。CAT活性的测定结果表明，角质形成细胞特异性表达的元件位于人5 ′侧翼区的-216 ~-197和小鼠5 ′侧翼区的-525 ~-213。我们使用小鼠克隆显示了小鼠230 kDa BPA基因的染色体定位，并检查了在UV诱导的皮肤变化中基质分解素基因启动子中的AP-1位点的作用，以阐明230 kDa BPA基因5 ′侧翼区的AP-1位点的功能。