In an attempt to clone
ace-3, a sequence (clone 48) was obtained by RT-PCR on total RNAs from C. elegans
ace-1 null mutants. This clone hybridized to YACs Y48B6 and Y59C8 indicating a site on chromosome II near
unc-52, the expected location of
ace-3. Blast examination of genomic sequences covering this region in C. briggsae showed that two sequences on chromosome II in this species were related to clone 48. One was the homologous of clone 48 and the other had also the characteristics of an ace gene. Those two sequences were located in very close proximity on chromosome II with only 372 bp between the stop of the upstream ORF and the ATG of the downstream ORF. A similar tandem organization of these two ace genes was also found in C. elegans by PCR (with 356 bp between the two ORFs and 200 bp separating the polyadenylation site of the 5' gene and the trans-splicing site of the 3' gene). Due to the close proximity of the two genes on chromosome II it was not possible to decide which sequence corresponded to
ace-3 (defined as encoding AChE of class C) and those genes were provisionally named ace-x (upstream ORF) and ace-y (downstream ORF). Using RT-PCR, both genes were shown to be transcribed in both species. In both C.elegans and C. briggsae, ace-x contains 16 introns and ace-y 7 introns. We found FGQSAG (ace-x) and VGESAG (ace-y) for sequences around the active serine: both sequences suggest a low catalytic activity. The C-terminal sequences are of the H type suggesting that ACE-X and ACE-Y are glycolipid-anchored membrane proteins. We then sequenced ace-x and ace-y in the mutant
ace-3 (strain
dc2) in order to identify
ace-3. We found that the only change was a deletion (580 nt-long) covering the non coding region between ace-x and ace-y as well as two small portions of coding sequence including the stop codon of ace-x and the initiator ATG of ace-y. The deletion did not shift the reading frame, so that a single long ORF was present in the mutant
dc2. A single large mRNA was found by RT-PCR in
dc2 instead of two distinct mRNAs in N2 strain. It is likely that the folding of the large encoded protein prevents catalytic activity of ace-x and ace-y. Thus the
dc2 mutant did not allow the identification of ace-x or ace-y to
ace-3.