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 (CLO NTECH) was screened using P-32-labeled rat p67 cDNA. A clone containin g a segment of 5'-upstream region of p67 genomic DNA was obtained. The DNA (about 1.7 kilobase pairs) was isolated and characterized. Sequen ce analysis of this DNA fragment showed that the 898 base pairs at the 5'-end of the upstream region was identical to several long intersper sed nucleotide sequences. One hundred forty eight base pairs at the 3' -end contained the beginning of the first exon including the ATG initi ator codon. The remaining 652 base pairs in between contained two AT-r ich regions and several regulatory sequences. The mRNA initiation site was identified at 89 base pairs upstream from the translation start c odon. The DNA fragment was also analyzed by transient transfection. Wh en linked to a firefly luciferase reporter gene, this fragment enhance d transcription in a rat hepatoma cell line (KRC-7). Using a series of deletions in the DNA, the minimum essential promoter region (from -17 7 to -60) was identified. The promoter activity was also enhanced by t reatment 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 s equences significantly reduced PMA enhancement, Deletion of both of th ese sequences almost completely eliminated PMA enhancement.