PRIMARY SEQUENCE AND FUNCTIONAL-ANALYSIS OF THE BOVINE GALANIN GENE PROMOTER IN HUMAN NEUROBLASTOMA-CELLS

Galanin (GAL) is a biologically active neuropeptide that has been sugg ested to play a role in stress-induced inhibition of insulin secretion , in dementia of the Alzheimer's type, and in the regulation of growth hormone secretion. We report here the isolation of a bovine genomic c lone containing more than 5-kb 5'-flanking sequences. Partial sequence analysis of the genomic clone revealed an atypical TATA-box in the pr omoter (ATAAATA) and several consensus sequences that typically bind t ranscription factors, including those that bind NF kappa B, Sp1, and A P-2. Primer extension and RNase protection analyses revealed that tran scription is initiated at two sites, 28 and 31 bp, respectively, downs tream from the TATA-box. To locate functionally active regulatory elem ents on the GAL gene, we first identified a neural crest-derived human neuroblastoma cell line, SK-N-SH subclone SH-SY5Y, that expressed eas ily detectable levels of endogenous GAL mRNA. We then constructed plas mids containing various lengths of bovine GAL 5'-flanking sequences an d the first exon fused to a reporter plasmid encoding luciferase. Tran sfection of these plasmids into the SH-SY5Y cells and analysis by tran sient expression indicated that 131 bp of 5' gene sequence was suffici ent to obtain maximal basal expression. Further, expression was suppre sed 16-fold when 5 kb were included, suggesting the presence of a dist al repressor element(s). In another set of experiments, we found that GAL mRNA levels could be induced more than 10-fold by 20-hr treatment with phorbol 12-myristate 13-acetate (PMA). In cells transfected with the same plasmids, luciferase activity was also induced by PMA, but th e degree of induction did not significantly differ among the deletion constructions (varying from six- to eight-fold), suggesting that eleme nts conferring PMA induction and/or RNA stabilization may be located w ithin 131 bp of the transcriptional start site, in the first exon, or on gene sequences not studied here.