Analysis of the human liver/bone/kidney alkaline phosphatase promoterin vivoandin vitro

Abstract

We have carried out an analysis of the promoter for the human liver/bone/kidney alkaline phosphatase (LBK AP) gene. Using transient transfection assays, the intact promoter directs equal expression of a linked cat gene in Saos-2 cells (osteoblast-derived cells which express very high levels of endogenous LBK AP mRNA) and in HeLa and HepG2 cells (which express low levels of endogenous message). The activity of the transfected promoter apparentty mimics the true in vivo situation since nuclear run-on assays employing Saos-2 and HeLa cells indicate that the endogenous gene is transcribed at approximately the same rate In these two cell types. Transfections of a series of 5' deletion mutants indicate that promoter activity is dependent on multiple motifs, which possibly include several putative Spi binding sites and a TATA box. The LBK AP promoter also directs accurate transcription initiation in HeLa whole cell extracts and in vitro activities of the 5' deletion mutants also suggest that the promoter utilizes multiple motifs.