PROMOTER OF THE GENE ENCODING THE BOVINE CATALYTIC SUBUNIT OF CAMP-DEPENDENT PROTEIN-KINASE ISOFORM C-BETA-2

Genomic sequences flanking the 5' end of the cDNA encoding isoform C b eta 2 of the catalytic subunit of bovine cAMP-dependent protein kinase were cloned, sequenced and analyzed for promoter activity and transcr iption initiation sites. A region of 913 bp upstream the translation i nitiator ATG was amplified from genomic DNA by vectorette polymerase c hain reaction. In primer extension reactions and RNase protection assa ys, residues C (at position -91), T (-71) and G (-70) were found to se rve as transcription initiation sites of the gene. Amplification produ cts and sub-fragments thereof were ligated upstream of the reporter ge ne chloramphenicol acetyltransferase to test for promoter activity. Co nstructs were transiently transfected into a Chinese hamster ovary cel l line which was shown to express endogenous C beta 2 mRNA. The genomi c sequence upstream the C beta 2 cDNA does have promoter activity. The region from position - 51 to - 292 proved sufficient to drive efficie nt transcription of the reporter gene. The promoter is AT rich (68%), does not contain a TATA box within 50 bp upstream of the first initiat ion site and possesses putative binding sites for several transcriptio n factors such as PEA-3 and a glucocorticoid receptor.