Cloning and characterization of a chitin synthase cDNA from the mosquito Aedes aegypti

Characterization of the enzymes involved in the chitin biosynthetic pathway in mosquitoes is critical due to the importance of chitin in the formation of the peritrophic matrix [Phl] and its potential impact on vector compete nce. Chitin is the homopolymer of the amino sugar N-acetyl-D glucosamine [G lcNAc]. The final step of incorporation of GlcNAc into the chitin polymer i s catalyzed by the enzyme chitin synthase [CS]. CS is a membrane bound enzy me, but the mechanism of its action in the biosynthesis of the PM is not un derstood. We have isolated and sequenced a CS-encoding cDNA clone from the mosquito Aedes aegypti, compared its sequence with CS from other organisms and studied its RNA expression. The cDNA is 3.5 kb in length with an open r eading frame of 2.6 kb that encodes a protein of 865 amino acids with a pre dicted molecular mass of 99.5 kDa. The putative translation product shares 90% similarity to two CS proteins from Caenorhabditis elegans and 50% simil arity to Saccharomyces cerevisiae in the catalytic domain of CS enzymes. Da ta suggest that CS is a single copy gene. RT-PCR analysis shows CS message in whole non-blood-fed females, whole blood-fed females, non-blood-fed midg uts and in midguts dissected at different time points post-blood-feeding. I n situ hybridization studies of midgut samples revealed that CS mRNA increa ses following a bloodmeal and is localized to the periphery of the epitheli al cells facing the midgut lumen. (C) 2000 Elsevier Science Ltd. All rights reserved.