Expression of a human acetylcholinesterase promoter-reporter construct in developing neuromuscular junctions of Xenopus embryos.
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.
Ben Aziz-Aloya
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R
Department of Biological Chemistry, Hebrew University of Jerusalem, Israel.
Seidman
S
S
Timberg
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R
Sternfeld
M
M
Zakut
H
H
Soreq
H
H
D000110:Acetylcholinesterase / Q000032:analysis / Q000235:genetics* / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D004247:DNA / Q000235:genetics; D004625:Embryo, Nonmammalian / Q000502:physiology*; D005314:Embryonic and Fetal Development; D005091:Exons; D006801:Humans; D007438:Introns; D008969:Molecular Sequence Data; D009469:Neuromuscular Junction / Q000502:physiology*; D009838:Oligodeoxyribonucleotides; D009865:Oocytes / Q000502:physiology*; D016133:Polymerase Chain Reaction / Q000379:methods; D011401:Promoter Regions, Genetic; D012326:RNA Splicing; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic; D014982:Xenopus laevis
D009838:Oligodeoxyribonucleotides; D012333:RNA, Messenger; D004247:DNA; D000110:Acetylcholinesterase