Sequence requirement for transcription in vivo of the human preproenkephalin A gene

The nucleotide sequence of the 5'-flanking region of the cloned human preproenkephalin A gene, extending to 949 bp upstream of the capping site, has been determined. The preproenkephalin A gene, when joined with an SV40 vector and introduced into COS monkey cells, is efficiently transcribed from its own promoter. To assess the DNA sequence required for promoter function, we have constructed a series of 5'-deletion mutants of a fusion gene that consists of the 949-bp 5'-flanking sequence and capping site of the preproenkephalin A gene and the structural sequence of the herpes simplex virus thymidine kinase gene. The deletions up to 757-172 bp upstream of the capping site exert essentially no effect on the expression of the fusion gene, whereas the deletions up to 145, 111, 81 and 67 bp upstream of the capping site result in a gradual decrease in the transcriptional efficiency. No detectable amount of the fusion gene transcript is produced with the mutants having deletions up to 67, 43 and 28 bp upstream of the capping site. These results indicate that a functional promoter of the preproenkephalin A gene lies between 67 and 171 bp upstream of the capping site. This promoter region corresponds to a highly GC-rich segment with short repeated sequences and palindromes.