The cloning and promoter analysis of the 5' flanking region of chick acetylcholinesterase gene

Acetylcholinesterase (AChE) is a highly polymorphic enzyme that is under strict transcriptional and translational regulatory control in brain and muscle. In this study, the 5′ flanking region (∼8 kilobases) upstream to chick AChE gene was cloned by the strategies of the rapid amplification of genomic ends (RAGE) and by 5′-rapid amplification of cDNA ends (5′-RACE). Analysis of chick AChE cDNAs revealed the existence of two alternatively spliced AChE mRNA transcripts, which are named AChE-E1 and AChE-E1a mRNA. The two mRNA transcripts are different only at the first 5′-untranslated exon1. AChE-E1a mRNA was exclusively expressed in neuronal tissues whereas AChE-E1 mRNA is expressed in both the neuronal and muscle tissues. The expression of AChE mRNAs were also developmentally regulated.; Analysis of the 5′ flanking regions showed that the exon1a and exon1 are located at 6.9 kb and 3.6 kilobase upstream to ATG start codon respectively. Unlike the mammalian AChE genes which contain a single large exon2, there is evidence of alternatively spliced exon2 in the chick AChE gene.; Putative transcription factor binding sites such as AP1, AP2, avian C-type LTR CCAAT box, CREB, Ebox, EGFIC, Ets-1, Elk-1, GATA-1, muscle TATA box, myogenin, MyoD, NFκB, SP1 and v-Myb have been identified in this region. Myogenic transcription factors MyoD and myogenin are distributed close to exon 1 but not exon 1a. Promoter activity was studied by cloning various stretches of DNA upstream to a luciferase reporter gene and the promoter-driven luciferase activity was measured in transiently transfected NG108-15 and C2C12 cells. The stretch of DNA corresponding to ∼365 by upstream to ATG site resulted in 5- and 2.5-fold increase in promoter activity in NG108-15 and C2C12 cells, respectively. Although no CREB was found in this region; luciferase activity increased 2 fold after Bt₂-CAMP or forskolin stimulation in NG108-15 cells. These results strongly suggested that cAMP-mediated regulatory pathway is involved in the regulation of chick AChE gene.