Acetylcholinesterase (AChE) is a key enzyme at cholinergic synapses, where it hydrolyses the neurotransmitter acetylcholine ending its synaptic action. It was reported previously that three genes,
ace-1,
ace-2 and
ace-3 encode three pharmacological classes of AChE in the nematode Caenorhabditis elegans [1]. The first to be cloned and characterized was
ace-1 [2] but sequences of three other ace genes
ace-2, ace-x and ace-y were recently reported [3]. We are interested in the function of each AChE class and in the regulation of their expression during development.
ace-1. The 5' flanking region of
ace-1 was cloned in C. elegans and in C. briggsae. Several conserved regions were identified. For the functional study of this region, phenotype rescue of null mutants was obtained by microinjection of DNA and GFP reporter constructs were used to analyze the developmental and tissue expression pattern of
ace-1 in live animals. The
ace-1 promoter drives GFP expression mainly in muscle cells (body-wall muscles, pharynx muscle cells
pm5, anal sphincter and four vulval muscle cells) and in only a few neurons (4 neurons of the head ganglia, 2 neurons of the retrovesicular ganglion and 2-3 neurones in the body). Deletion analysis suggests that the
ace-1 promoter is built of a short basal promoter region modulated by several separate tissue-specific activator cis-elements.
ace-2. A partial cDNA clone (#40), obtained by PCR using degenerate primers, was mapped to YACs Y44E3 and Y52G11 on a region of chromosome I compatible with the genetic localization of
ace-2. It presents all characteristics of an AChE gene and is trans-spliced to SL1. To confirm that it was indeed
ace-2, we sequenced the corresponding cDNA in the null mutant
ace-2- (allele
gp72) [4]. A point mutation (G441E) adjacent to H440 of the catalytic triad was found. The localization of
ace-2 expression was studied using injection of GFP reporter constructs. The expression pattern is limited exclusively to nerve cells in head ganglia and tail ganglia. ace-x and ace-y. Another clone (#48) obtained by PCR, hybridized to YACs Y48B6 and Y59C8 on chromosome II near
unc-52, the expected location of
ace-3. Blast examination of genomic sequences released from the genome sequencing program of C. briggsae, showed that two sequences on chromosome II in this species were related to clone 48 of C. elegans. One was the homologue of clone 48 in C. briggsae; the other also possessed all the characteristics of an acetylcholinesterase gene. These two sequences were located in very close proximity on chromosome II with only 360 bp between the stop of the upstream ORF and the ATG of the downstream ORF. A similar tandem organization was also found in the genome of C. elegans. We checked by PCR that these two ace genes were also transcribed in vivo, in C elegans. Due to their close proximity on chromosome II it is not possible, at the moment, to decide which sequence corresponds to
ace-3 (defined in the literature as encoding a class C type AChE [1]). Thus these third and fourth ace genes are provisionally named ace-x (upstream ORF) and ace-y (downstream) [3]. [1] Johnson, C.J. (1991) In Cholinesterases. ASC Conference series, 136-140. [2] Arpagaus, M., Fedon, Y., Cousin, X., Chatonnet, A., Berg!, J.-B., Fournier, D. and Toutant, J.-P. (1994) J. Biol. Chem. 269, 9957-9965. [3] Grauso, M., Culetto, E., Combes, D., Fedon, Y., Toutant, J.-P. and Arpagaus, M. (1998) FEBS Lett. 424, 279-284. [4] Culotti, J.G., Von Ehrenstein, G., Culotti, M.R. and Russell, R.L. (1981) Genetics 97, 281-305.