Abstract
We cloned and sequenced the acetylcholinesterase gene and cDNA of zebrafish, Danio rerio. We found a single gene (ache) located on linkage group LG7. The relative organization of ache, eng2, and shh genes is conserved between zebrafish and mammals and defines a synteny. Restriction fragment length polymorphism analysis was allowed to identify several allelic variations. We also identified two transposable elements in non-coding regions of the gene. Compared with other vertebrate acetylcholinesterase genes, ache gene contains no alternative splicing at 5' or 3' ends where only a T exon is present. The translated sequence is 60-80% identical to acetylcholinesterases of the vertebrates and exhibits an extra loop specific to teleosts. Analysis of molecular forms showed a transition, at the time of hatching, from the globular G4 form to asymmetric A12 form that becomes prominent in adults. In situ hybridization and enzymatic activity detection on whole embryos confirmed early expression of the acetylcholinesterase gene in nervous and muscular tissues. We found no butyrylcholinesterase gene or activity in Danio. These findings make zebrafish a promising model to study function of acetylcholinesterase during development and regulation of molecular forms assembly in vivo.
Highlights
We cloned and sequenced the acetylcholinesterase gene and cDNA of zebrafish, Danio rerio
A Single Acetylcholinesterase in Zebrafish—Cholinesterase activity was first detected, in whole embryos, using a histochemical reaction adapted from Karnovsky and Roots [15]
AChE Is Encoded by a Unique Gene—The insertional polymorphism observed in intron 4 allowed us to locate ache gene on zebrafish genome by segregation on MOP crosshaploid mapping panel [13]
Summary
Materials—D. rerio adults and embryos were from our facility. Fish are maintained at 28 °C on a 13-h light/11-h dark cycle. Expression of Recombinant AChE in Drosophila S2 Cells and in Zebrafish Embryos—A mini-gene containing the entire coding sequence of AChE was assembled from three genomic fragments and cloned in the expression vector pMT/V5-His (Invitrogen). The final construct was totally sequenced before transfection in Drosophila S2 cells This mini-gene was introduced in pcDNA3 vector for in vivo overexpression in zebrafish embryos. Results were identical for recombinant or native AChE, with a standard error less than 10%
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