Molecular cloning and characterization of a greenbug (Schizaphis graminum) cDNA encoding acetylcholinesterase possibly evolved from a duplicate gene lineage.
An acetylcholinesterase (AChE, EC 3.1.1.7) cDNA was cloned and characterized from a greenbug (Schizaphis graminum (Rondani)) cDNA library. The complete cDNA (3283 bp) contains a 2028-bp open reading frame encoding 676 amino acid residues. The putative AChE preproenzyme has a 17 amino acid signal peptide, a 78 amino acid activation peptide and a mature enzyme of 581 amino acid residues. The first nine amino acid residues (YTSDDPLII) that were determined by sequencing the N-terminus of a 72-kDa AChE purified from the greenbug matched the nine residues deduced from the cDNA. The key amino acid residues, including the three residues Ser206 (200 in Torpedo), Glu332 (327) and His446 (440) forming a catalytic triad, three pairs of cysteine putatively forming intrachain disulfide bonds, and 10 out of the 14 aromatic residues lining the active site gorge of the Torpedo AChE, are conserved. However, Ser336 (Phe331) in the greenbug substituted an aromatic amino acid residue that is conserved in all other known AChEs. Northern blot analysis of mRNA revealed a 3.7-kb transcript, and Southern blot analysis suggested a single copy of this gene in the greenbug. The deduced amino acid sequence is most similar to AChE1 of the nematodes Caenorhabditis briggsae and C. elegans with 43% identity. Phylogenetic analysis showed that the greenbug AChE formed a cluster with those of nematodes, a squid and ticks, and grouped out of the insect cluster. This result suggests that the cloned gene evolved from a different duplicate gene lineage of insect AChEs.
Gao
J-R
JR
Department of Entomology, 123 Waters Hall, Kansas State University, Manhattan, KS 66506-4004, USA. kzhu@ksu.edu
Kambhampati
S
S
Zhu
K Y
KY
D000110:Acetylcholinesterase / Q000145:classification / Q000235:genetics*; D000595:Amino Acid Sequence; D000818:Animals; D001042:Aphids / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary; D019143:Evolution, Molecular; D008969:Molecular Sequence Data; D010802:Phylogeny; D017386:Sequence Homology, Amino Acid
D018076:DNA, Complementary; D000110:Acetylcholinesterase