CLONING AND CHARACTERIZATION OF THE HUMAN OSTEOPONTIN GENE AND ITS PROMOTER
CLONING AND CHARACTERIZATION OF THE HUMAN OSTEOPONTIN GENE AND ITS PROMOTER
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DOI：
10.1042/bj3030255
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发表时间：
1994-10-01
影响因子：
4.1
通讯作者：
YAMAMOTO, S
中科院分区：
文献类型：
--
作者：
HIJIYA, N;SETOGUCHI, M;YAMAMOTO, S
我们分离了人骨桥蛋白(HOP)基因及其5‘上游区域，并分析了其外显子-内含子结构和启动子区域的潜在调控序列，并与小鼠和猪的基因进行了比较。编码序列被分成7个外显子，与小鼠基因相似，尽管HOP基因比小鼠基因长。长度上的差异主要是由于内含子3的变异，内含子3的长度类似于小鼠OP基因的2.7倍。HOP的5‘上游区域高度保守，最高可达250个核苷酸，包含许多潜在的顺式调控共识序列。检测了一系列5‘端缺失的嵌合克隆对氯霉素乙酰转移酶(CAT)的激活能力。初步CAT分析表明，在人单核细胞系SCC-3中，-474到-270、-124到-80和-55到-39位置的核苷酸含有顺式作用增强序列，尽管-124到-80区域比其他区域更活跃。-474和-270之间的序列的缺失使该顺式区域定位于-439到-410的序列，而在-124到-80之间的缺失使区域定位到-124到-115和-94到-80。凝胶位移分析显示，分别在-124~-115和-94~-80处合成了10和15个碱基对的双链DNA，每个探针与SCC-3细胞制备的核蛋白形成了一条主带。
We isolated the human osteopontin (hOP) gene and the 5' upstream region, and analysed its exon-intron structure and potential regulatory sequences of the promoter region in comparison with those of the mouse and porcine gene. The coding sequence is split into 7 exons which are similar to those of the mouse gene, although the hOP gene is longer than the mouse gene. The difference in length is mainly due to variations in intron 3, which is similar to 2.7-fold longer than that of the mouse OP gene. The 5' upstream region of the hOP, which is highly conserved up to nucleotide -250, contains a number of potential cis regulatory consensus sequences. A series of sequentially 5'-deleted chimeric clones was tested for the ability to stimulate chloramphenicol acetyltransferase (CAT). Initial CAT analysis demonstrated that nucleotides at positions -474 to -270, -124 to -80, and -55 to -39 contained cis-acting enhancing sequences in a human monocyte cell line, SCC-3, although the -124 to -80 region was much more active than other regions. Deletion of the sequences between -474 and -270 localized this cis region to the sequence at positions -439 to -410, whereas the deletion between -124 to -80 localized the regions to -124 to -115, and -94 to -80. Gel-shift analysis using as probes synthesized double-stranded DNA corresponding to the 10 and 15 bp region at positions -124 to -115 and -94 to -80 respectively revealed that each probe formed a major band complexed with nuclear proteins prepared from SCC-3 cells.