Identification of the promoter region of the rat protein phosphatase 2Aα gene

Abstract

We have cloned a genomic fragment containing the promoter region of the rat protein phosphatase 2A alpha gene (PP-2A alpha). A 1.6 kb fragment of the 5' flanking region was sequenced. Three major transcriptional initiation sites were identified by the primer extension method using rat liver mRNA and found to be located 225, 222 and 220 bases upstream of the translational initiation site, respectively. Bacterial chloramphenicol acetyltransferase (CAT) assay revealed that a 503 bp SmaI fragment containing the transcriptional initiation sites had promoter activity, which was stronger than that of the SV40 early promoter on the pSV2CAT plasmid when introduced into NIH3T3 cells. Deletion of a 119 bp SacII fragment decreased its promoter activity considerably. The promoter region has an extremely high GC content and does not contain either a 'TATA box' or a 'CAAT box' suggesting that this promoter can be classified as that of a 'house keeping' gene, although there is only one typical GC-box (GGGCGG) immediately preceding the transcriptional initiation sites. There is a 10 base pair palindrome, 5'-GTGACGTCAC-3', 26 base pairs upstream of the +1 transcriptional initiation site, which is highly conserved in many other genes, whose expression is regulated by cAMP. The promoter activity was shown to be increased by forskolin treatment (10 microM) in NIH3T3 cells.