CLONING AND CHARACTERIZATION OF THE HUMAN OSTEOPONTIN GENE AND ITS PROMOTER

We isolated the human osteopontin (hOP) gene and the 5' upstream regio n, and analysed its exon-intron structure and potential regulatory seq uences of the promoter region in comparison with those of the mouse an d porcine gene. The coding sequence is split into 7 exons which are si milar 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 mous e OP gene. The 5' upstream region of the hOP, which is highly conserve d up to nucleotide -250, contains a number of potential cis regulatory consensus sequences. A series of sequentially 5'-deleted chimeric clo nes was tested for the ability to stimulate chloramphenicol acetyltran sferase (CAT). Initial CAT analysis demonstrated that nucleotides at p ositions -474 to -270, -124 to -80, and -55 to -39 contained cis-actin g enhancing sequences in a human monocyte cell line, SCC-3, although t he -124 to -80 region was much more active than other regions. Deletio n 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 corresp onding 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 complex ed with nuclear proteins prepared from SCC-3 cells.