Novel messenger RNA and alternative promoter for murine acetylcholinesterase

Abstract

A portion of the 5'-flanking region of murine acetylcholinesterase was cloned from genomic DNA by 5'-rapid amplification of genomic ends, identified in a mouse genomic library, and sequenced. Multiple potential binding sites for universal and tissue-specific transcription factors were suggestive of a promoter region within this DNA sequence. Potential promoter activity was confirmed by coupling the new sequence to the open reading frame of a luciferase reporter gene in transient expression experiments with nerve and muscle cells. 5'-Rapid amplification of cDNA ends with templates from multiple sources revealed a novel transcription start site (at position - 626, relative to translation start), located 32 bases downstream from a TATAA sequence. This start site appeared to mark a novel exon (1a) comprising 291 base pairs between positions -335 and -626, relative to the translation start. Supporting this conclusion, polymerase chain reactions with cDNA from mouse brain, heart, and other tissues, consistently amplified a transcript containing the exon 1a sequence fused to the invariant sequence beginning at position -22 in exon 2, but lacking exon 1. Northern blot analyses confirmed the in vivo expression of exon 1a-containing transcripts, especially in heart, brain, liver, and kidney. These results indicate that the murine acetylcholinesterase gene has a functioning alternative promoter that may influence expression of acetylcholinesterase in certain tissues.