Cloning and characterization of the promoter region of a gene encoding a 67-kDa glycoprotein

A rat genomic library constructed in lambda-EMBL3 (SP6/T7) vector (CLONTECH) was screened using P-32-labeled rat p67 cDNA. A clone containing a segment of 5'-upstream region of p67 genomic DNA was obtained. The DNA (about 1.7 kilobase pairs) was isolated and characterized. Sequence analysis of this DNA fragment showed that the 898 base pairs at the 5'-end of the upstream region was identical to several long interspersed nucleotide sequences. One hundred forty eight base pairs at the 3'-end contained the beginning of the first exon including the ATG initiator codon. The remaining 652 base pairs in between contained two AT-rich regions and several regulatory sequences. The mRNA initiation site was identified at 89 base pairs upstream from the translation start codon. The DNA fragment was also analyzed by transient transfection. When linked to a firefly luciferase reporter gene, this fragment enhanced transcription in a rat hepatoma cell line (KRC-7). Using a series of deletions in the DNA, the minimum essential promoter region (from -177 to -60) was identified. The promoter activity was also enhanced by treatment with phorbol 13-myristate 12-acetate (PMA). This enhancement required an AP-1 sequence (-298 to -292; 5'-TGACTCA-3') and a similar sequence (-97 to -88; 5'-ATGACATCAT-3'), Deletion of either of these sequences significantly reduced PMA enhancement, Deletion of both of these sequences almost completely eliminated PMA enhancement.