期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：1993
卷号：90
期号：6
页码：2471-2475
DOI：10.1073/pnas.90.6.2471
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：We have employed Xenopus embryos to express human acetylcholinesterase (AcChoEase; EC 3.1.1.7 ) in developing synapses. Transcription of AcChoEase mRNA was driven by a 2.2-kb sequence upstream from the initiator AUG in the ACHE gene encoding AcChoEase, with multiple potential sites for binding universal and tissue-specific transcription factors. These included clustered MyoD elements, E-box, SP1, EGR1, AP-2, and the development-related GAGA motif. A DNA construct composed of this sequence linked to a 2.1-kb sequence encoding human AcChoEase was designated human AcChoEase promoter-reporter (HpACHE). HpACHE but none of its several 5'-truncated derivatives was transcriptionally active in developing Xenopus embryos. Furthermore, PCR analysis using chimeric PCR primers revealed usage of the same 1.5-kb intron and 74-bp exon within the HpACHE sequence in microinjected embryos and various human tissues. Cytochemical staining revealed conspicuous accumulation of overexpressed AcChoEase in neuromuscular junctions and within muscle fibers of apparently normal 2-day Xenopus embryos injected with HpACHE. The same reporter driven by the cytomegalovirus promoter was similarly efficient in directing the heterologous human enzyme toward neuromuscular junctions, attributing the evolutionary conservation of AcChoEase targeting to the coding sequence. Our findings demonstrate that a short DNA sequence is sufficient to promote the exogenous transcription and faithful splicing of human AcChoEase mRNA in developing Xenopus embryos and foreshadow their use for integrative studies of cholinergic signaling and synapse formation.
