Characterization of the 5'-flanking region of the rat phospholipase C β3 gene

Abstract

As a first step toward understanding regulation of the phosphatidylinositol - specific phospholipase C(PLC) isoform genes, the 5`` flanking region of the Rat PLC-$\beta$3 gene was sequenced and characterized. Sequence analysis of the 5`` flanking region revealed that it contains a CCAAT sequence at position -289 and a extremely high GC- rich region(-305 to -16bp: 78\%). But, TATA box was not found in this region. This 5`` region also contained binding sites for the transcription factors Sp1, AP-1, AP-2, and PEA3. When the putative promoter was ligated into a promoterless CAT vector and transfected into C$_6$Bu1 cells, CAT activity was expressed, indicating the promoter region was located in this fragment. To define sequences that influence transcription of the PLC-$\beta$3 gene, transcription activities of various 5`` deletion mutants of its upstream region, fused to the CAT gene, were examined in terms of CAT expression after transfection into C$_6$Bu1 cells. Deletion mutational analysis revealed that the PLC-$\beta$3 promoter contains at least three positive elements; -1827 to -1543 bp, -1232 to -1072 bp, and -587 to -489 bp. In addition to these positive elements, there are two negative elements; -1543 to -1393 bp and -643 to -587 bp. Gel mobility shift assays revealed that these elements form multiple protein- DNA complexes with nuclear proteins in C$_6$Bu1 cells. These results suggest the presence of multiple trans - acting factors that act on these positive and negative cis - acting elements and regulate the transcription of the PLC-$\beta$3 gene.