cDNA sequence, gene structure, and in vitro expression of ace-1, the gene encoding acetylcholinesterase of class A in the nematode Caenorhabditis elegans.
Three genes, ace-1, ace-2, and ace-3, encode three acetylcholinesterase classes (A, B, and C) in the nematode Caenorhabditis elegans. A fragment of genomic DNA was amplified by a polymerase chain reaction (PCR) using degenerate oligonucleotides based on sequences conserved in the cholinesterase family. This fragment mapped to chromosome X at a position that perfectly matched the location of ace-1 previously determined by genetic methods. Comparison of genomic and cDNA sequences showed that the open reading frame was interrupted by eight introns. The product of ace-1 (ACE-1, 620 amino acids) presented 42% identity with Torpedo and human acetylcholinesterases, 41% with human butyrylcholinesterase, and 35% with Drosophila acetylcholinesterase. The overall structure of cholinesterases was conserved in ACE-1 as indicated by the conserved sequence positions of Ser-216, His-468, and Glu-346 (S200, H440, E327 in Torpedo (AChE) as components of the catalytic triad, of the six cysteines which form three intrachain disulfide bonds, and of Trp-99(84), a critical side chain in the choline binding site. Spodoptera Sf9 cells were infected by a recombinant baculovirus containing ace-1 cDNA. The secreted enzyme was active and existed as hydrophilic 5 and 11.5 S molecular forms. It hydrolyzed both acetylthiocholine and butyrylthiocholine and was inhibited by acetylthiocholine above 10 mM.
Arpagaus
M
M
INRA, Montpellier, France.
Fedon
Y
Y
Cousin
X
X
Chatonnet
A
A
Bergé
J B
JB
Fournier
D
D
Toutant
J P
JP
D000110:Acetylcholinesterase / Q000096:biosynthesis* / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D017173:Caenorhabditis elegans / Q000201:enzymology* / Q000235:genetics* / Q000254:growth & development; D002460:Cell Line; D017124:Conserved Sequence; D017931:DNA Primers; D018076:DNA, Complementary / Q000032:analysis; D004330:Drosophila / Q000201:enzymology / Q000235:genetics; D015870:Gene Expression; D006801:Humans; D007438:Introns; D007527:Isoenzymes / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008322:Mammals / Q000235:genetics; D008969:Molecular Sequence Data; D009036:Moths; D016133:Polymerase Chain Reaction; D012326:RNA Splicing; D015183:Restriction Mapping; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid; D014162:Transfection; D014714:Vertebrates / Q000235:genetics
D017931:DNA Primers; D018076:DNA, Complementary; D007527:Isoenzymes; D000110:Acetylcholinesterase