Promoter Elements of the Rat Choline Acetyltransferase Gene Allowing Nerve Growth Factor Inducibility in Transfected Primary Cultured Cells

Abstract

Choline acetyltransferase, the enzyme responsible for the synthesis of acetylcholine, provides a convenient index for cholinergic neurons. Using a previously identified rat cDNA clone, we have isolated several corresponding genomic clones and have characterized a 1,902-bp fragment that contains part of the first noncoding exon as well as promoter sequences. The promoter activity of this fragment was tested, taking advantage of the recently developed lipopolyamine-mediated DNA transfer method, which allows transfection of primary neurons. The 1,902-bp sequence drives the expression of the bacterial chloramphenicol acetyltransferase (CAT) reporter gene in a culture of dissociated cells prepared from the septal area of fetal (embryonic day 17) rats, a structure rich in cholinergic neurons. Moreover, addition of nerve growth factor to the culture increases CAT expression by approximately 56-fold, indicating that our DNA fragment contains sequences required for NGF induction. In addition, it contains consensus sequences for various transcription factors, including those of the basic helix-loop-helix family. Finally, experiments to characterize the transcription start site are presented.