Characterization of amplification core and esterase B1 gene responsible for insecticide resistance in Culex.

Abstract

Organophosphorus insecticide (OP) resistance in several Culex species is associated with increased esterase activity resulting from amplification of the corresponding structural gene. In Culex pipiens quinquefasciatus, high levels of OP resistance (=800 times) are due to the esterase Bi gene, which is amplified at least 250-fold. This gene has now been sequenced, and the structure of the amplification unit (amplicon) encompassing the structural gene has been partially characterized. The inferred amino acid sequence of the enzyme revealed regions of strong homology with other eukaryotic serine-esterases, such as cholinesterases, which are the target of OPs. The amplicon covers at least 30 kilobases and contains a constant and highly conserved "core" of 25 kilobases. This core carries a single copy of the esterase gene (2.8 kilobases) as well as other sequences that are present as single or low number copies in the genomes of mosquitoes lacking overproduction of the esterase Bi protein. In the amplicon, the esterase gene is framed by two DNA sequences that are repeated in other parts of the genome of resistant mosquitoes and found in the genome of susceptible mosquitoes but not near the esterase Bi gene. It is suggested that these repetitive sequences may have a role in the amplification process.Gene amplification seems to be a fundamental and widely occurring mechanism of overproduction ofproteins for counteracting environmental stress. Amplification has been shown to result in high levels ofresistance in insects that have been under selection with organophosphorus insecticides (OPs) (1-3), in alfalfa controlled with the herbicide Lphosphinothricin (4), and in Leishmania (5) and cultured mammalian cells (6) grown in the presence ofantiproliferative drugs. Populations of several species of mosquitoes of the genus Culex have become resistant to a variety of OPs due to increased production of detoxifying esterases (7). The three esterases B studied thus far-i.e., B1, B2, and B3-were shown to be amplified (1, 2). There is evidence indicating that amplification of each esterase B gene occurred as a single and independent event (2), suggesting that it may be induced by a specific mechanism. In order to explore the nature of this mechanism, we have undertaken the characterization of the amplified DNA structures. We report here the nucleotide sequencett and intron and exon organization of the esterase B1 gene, which, in Californian Culex pipiens quinquefasciatus, has been shown to be amplified at least 250-fold (1). In addition, we show that large DNA sequences are coamplified with the structural gene and characterize part of these sequences. (2)]. MATERIALS AND METHODSCloning Genomic DNA. Genomic DNA of Tem-R adults was partially digested by restriction enzyme Sau3AI. Sizeselected 15-to 22-kilobase (kb) DNA fragments thus generated were ligated to the BamHI site of the arms of phage vector A-EMBL3. After packaging of the concatenated DNA molecules, the recombinant phage library was plated on Escherichia coli C6...

Amplification and methylation of an esterase gene associated with insecticide-resistance in greenbugs, Schizaphis graminum (Rondani) (Homoptera: Aphididae)

“…Because of the shared ancestral origin, similarities between genes could have resulted in similar amplification processes in response to selective pressures from exposure to organophosphate insecticides in both insects. The Type I gene appeared to have a relatively low copy number in the resistant individuals compared with EstB1 1 of Culex mosquitoes (Mouchès et al, 1990) but similar to that estimated in M. persicae .…”

Relationship between sequence conservation and three‐dimensional structure in a large family of esterases, lipases, and related proteins

“…In glutactin, a 1,023-amino acid residue glycoprotein located in basement membranes of Drosophila, it is the N-terminal segment that is homologous to esterases. Although the sequence identity of these three nonhydrolytic proteins with other proteins in this Shimada et al, 1990Shimada et al, 1989Kawaguchi et al, 1989Longhi et al, 1992Schumacher et al, 1986Sikorav et al, 1987Rachinsky et al, 1990Doctor et al, 1990Soreq et al, 1990Hall and Spierer, 1986Hall and Malcolm, 1991Lockridge et al, 1987Rachinsky et al, 1990Jbilo and Chatonnet, 1990Robbi et al, 1990Munger et al, 1992Long et al, 1991Korza and Ozols, 1988Ozols, 1989Ovnic et al, 1991Long et al, 1988Takagi et al, 1988Oakeshott et al, 1987Collet et al, 1990Hanzlik et al, 1989Mouches et al, 1990Han et al, 1987Kissel et al, 1989 Hui andKissel, 1990;Nilsson et al, 1990;Baba et al, 1991Kyger et al, 1989Rubino et al, 1989Bomblies et al, 1990 de la Escalera et al ., 1990Olson et al, 1990Mercken et al, 1985 family is within 16-28%, there is no evolutionary pressure to maintain the geometry of the active site. One may, therefore, predict that their 3D structures will show more divergence from GCL and TcAChE than other proteins in this group.…”

“…Even within AChEs this loop varies in length by 4 residues. Only two sequences lack these cysteines: Culex esterase B1 (Mouches et al, 1990) and Heliothis juvenile hormone esterase (Hanzlik et al, 1989). Although this disulfide bridge is highly conserved, its role and importance are not apparent from the 3D structure alone.…”

Insecticide Resistance and Vector Control

“…In Diptera, they occur as a gene cluster on the same chromosome (26,27). Individual members of the gene cluster may be modified in instances of insecticide resistance, for example, by changing a single amino acid that converts the specificity of an esterase to an insecticide hydrolase (28) or by existing as multiple-gene copies that are amplified in resistant insects (the best studied examples are the B1 [29] and A2-B2 [30] amplicons in Culex pipiens and C. quinquefasciatus).…”