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 acetylcholinesteras e classes (A, B, and C) in the nematode Caenorhabditis elegans. A frag ment 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 posi tion that perfectly matched the location of ace-1 previously determine d by genetic methods. Comparison of genomic and cDNA sequences showed that the open reading frame was interrupted by eight introns. The prod uct of ace-1 (ACE-1, 620 amino acids) presented 42% identity with Torp edo and human acetylcholinesterases, 41% with human butyrylcholinester ase, and 35% with Drosophila acetylcholinesterase. The overall structu re of cholinesterases was conserved in ACE-1 as indicated by the conse rved 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 cDN A. The secreted enzyme was active and existed as hydrophilic 5 and 11. 5 S molecular forms. It hydrolyzed both acetylthiocholine and butyrylt hiocholine and was inhibited by acetylthiocholine above 10 mM.