©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Mosquito Carboxylesterase Est2 (A)
CLONING AND SEQUENCE OF THE FULL-LENGTH cDNA FOR A MAJOR INSECTICIDE RESISTANCE GENE WORLDWIDE IN THE MOSQUITO CULEX QUINQUEFASCIATUS(*)

Ashley Vaughan , Janet Hemingway (§)

From the (1)School of Pure and Applied Biology, University of Wales Cardiff, P.O. Box 915, Cardiff CF1 3TL, United Kingdom

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Organophosphorus insecticide resistance in Culex mosquitoes is commonly caused by increased activity of one or more esterases. The commonest phenotype involves elevation of the esterases Est2 (A) and Est2 (B). A cDNA encoding the Est2 esterase has now been isolated from a Sri Lankan insecticide-resistant mosquito (Culex quinquefasciatus, Say) expression library. In line with a recently suggested nomenclature system (Karunaratne, S. H. P. P.(1994) Characterization of Multiple Variants of Carboxylesterases Which Are Involved in Insecticide Resistance in the Mosquito Culex quinquefasciatus. Ph.D. thesis, University of London), as the first sequenced variant of this esterase, it is now referred to as Est2. The full-length cDNA of est2 codes for a 540-amino acid protein, which has high homology with other esterases and lipases and belongs to the serine or B-esterase enzyme family. The predicted secondary structure of Est2 is similar to the consensus secondary structure of proteins within the esterase/lipase family where the secondary and tertiary structures have been resolved. The level of identity (47% at the amino acid level) between the est2 and the various Culex est (B and B) cDNA alleles that have been cloned and sequenced suggests that the two esterase loci are closely related and arose originally from duplication of a common ancestral gene. The lack of a distinct hydrophobic signal sequence for Est2 and two possible N-linked glycosylation sites, both situated close to the active site serine, suggest that it is a nonglycosylated protein that is not exported from the cell. Southern and dot blot analysis of genomic DNA from various insecticide-resistant and susceptible mosquito strains show that the est2 gene, like est2, is amplified in resistant strains. The restriction fragment length polymorphism patterns, after probing Southern blots of EcoRI-digested genomic DNA with est2 cDNA, show that the amplified and nonamplified est alleles differ in the resistant and susceptible Sri Lankan mosquitoes.


INTRODUCTION

Insecticide resistance is a significant problem in many insect pests. Elevation of carboxylesterase activity is the major mechanism of resistance to the organophosphorus insecticides in a wide range of insect species(1, 2, 3, 4, 5, 6, 7) . However, the resistance mechanism has been studied in depth at the biochemical and/or molecular level in only a few species. In the mosquito Culex quinquefasciatus and the aphid Myzus persicae, amplification of at least one esterase gene underlies the increase in esterase activity, with up to 250 copies of an esterase gene per genome being recorded in Culex (8-10). The esterases involved in both species are standard B or serine esterases according to the classification of Aldridge(11) . This is a widely distributed family of enzymes, which hydrolyze carboxylester, amide, and thioester bonds in a variety of compounds.

The mosquito C. quinquefasciatus is a major biting nuisance insect worldwide, and a vector of filarial and viral disease on at least two continents. Esterase banding patterns after polyacrylamide or starch gel electrophoresis of crude homogenates of this mosquito can be complex(12, 13) . Esterases can be classified on the basis of their biochemical, molecular, immunological, and electrophoretic characteristics, although their broad substrate specificities and multi-allelic nature make classification difficult. A classification system based on the esterase's preference for the general esterase substrates - or -naphthyl acetate and their relative electrophoretic mobilities has been suggested(14) . On the basis of this classification, two predominant elevated esterase phenotypes, B and co-elevated A/B, which cause insecticide resistance, can be distinguished electrophoretically in the mosquito. The A/B phenotype is by far the most common, occurring in all continents where C. quinquefasciatus is found. The elevated A and B esterases are in complete linkage disequilibrium in mosquito populations, i.e. the two esterases are co-elevated in all mosquitoes. Amplification of the B esterase was first shown in the Californian Tem-R strain of mosquito(9) . Immunological and molecular studies have since shown that B and B were originally alleles of the same locus and that the B gene is also amplified in resistant insects(10, 15, 16, 17) . Initial reports suggested that there was no cross-reaction between the A and B esterases at either an immunological or molecular level, and it was concluded that if these genes had arisen from duplication of a common ancestral gene, this must undoubtedly have occurred a long time ago(15, 18) . However, we have recently shown that a polyclonal antiserum raised to the nondenatured A esterase does cross-react with the B, although the specificity is 50-fold lower for B than A, suggesting that the two enzymes share some common epitopes(19, 20) .

We here report the original cloning and cDNA sequence of the A mosquito esterase from an insecticide-resistant strain of C. quinquefasciatus from Peliyagoda, Sri Lanka(21) . The cDNA sequence is compared with the other mosquito and aphid esterases that are involved in insecticide resistance. We also show that the underlying mechanism of A elevation in the Pel RR insecticide-resistant strain is gene amplification.


EXPERIMENTAL PROCEDURES

Mosquito Esterase Nomenclature

The Culex esterase nomenclature is currently out of line with that used for other organisms, and there is confusion between the earlier general esterase classification (11) and that subsequently proposed for mosquitoes(14) . Karunaratne (22) has proposed that the Culex esterases should be renamed using and rather than the A and B of Raymond et al.(14) , with numerical superscripts denoting sequence variants with the same electrophoretic mobility. Hence the two amplified Bs, which differ in their inferred amino acid sequence but are electrophoretically identical, become Est1 and Est1, from the Californian and Cuban strains of Culex, respectively, and the nonelevated B from the Sri Lankan Pel SS strain, again with the same electrophoretic mobility, becomes Est1. This system ensures that esterases can still be named initially on the basis of their electrophoretic mobilities, but distinct DNA sequence variants can also be indicated as this level of data becomes available. As this nomenclature system will be adopted in this paper, a summary of the past and proposed nomenclature for mosquito strains where esterase DNA sequence data is available is given in . On the basis of this classification(22) , the Pel RR A esterase now becomes Est2, as the first available sequence of this esterase.

Mosquito Strains

A heterogeneous population (Pel) of mosquito was collected from Peliyagoda, Sri Lanka, in 1986. It was selected to give an insecticide-susceptible strain, Pel SS, and a resistant strain, Pel RR (23, 24). Pel RR was 31-fold more resistant to the organophosphorus insecticide temephos than Pel SS(25) .

The Pel SS strain was obtained by multiple single family selection from the Pel strain(23) . The Pel RR strain was obtained by mass selection with temephos(24) . Since then, insecticide resistance in this strain has been maintained by exposing fourth instar larvae every third generation to the LD concentration of temephos. The only organophosphate resistance mechanism in this strain is co-elevation of the Est2 and Est2 esterases.

Production of a Polyclonal Antiserum

A polyclonal antiserum was prepared by injecting a New Zealand White rabbit with purified nondenatured Est2 from Pel RR. The esterase was administered after mixing with reconstituted Ribi adjuvant to give a final enzyme concentration of 200 µg/ml. Four injections of 1 ml of antigen were given at 2-week intervals. Each injection was split between intramuscular, intradermal, and subcutaneous sites(26) . The resultant antiserum had a high level of specificity to Est2 and much lower (50-fold) levels of sensitivity to the Est2 and Est1 esterases and the organophosphate target site acetylcholinesterase(19, 20) . This antiserum was used to screen a Pel RR cDNA expression library.

Isolation of a cDNA Clone for Esterase Est2

Pel RR cDNA Library Construction

The cDNA for library construction was synthesized from 5 µg of mRNA from fourth instar Pel RR larvae by a method previously used(17) . The cDNA was blunted and size-selected (>400 base pairs) on a Sephacryl S-400 column (Pharmacia Biotech Inc.). After EcoRI linker addition (Promega) and subsequent phosphorylation, the cDNA was ligated into Lambda ZAP II (Stratagene). This vector allows plasmid containing cDNAs to be directly excised from the phage library. The bacteriophage arms were supplied digested with EcoRI and dephosphorylated. 5 µg of arms were ligated to 125 ng of cDNA and packaged with Packagene extracts (Promega). Approximately 200,000 recombinant clones were obtained.

Immunoscreening

This was carried out with the Protoblot Immunoscreening System (Promega). The 200,000 unamplified recombinant clones were screened with the polyclonal anti-Est2 esterase antisera at a dilution of 1:10,000.

In Vivo Excision and Sequencing

Positive clones were purified by successive rounds of screening and in vivo excised to give recombinant pBluescript plasmids. Sequencing was carried out with Sequenase Version 2.0 (Amersham Corp.) using universal primers complemented with the ExoIII/Mung Bean Nuclease deletion method (Stratagene) and with primers specific to the est2 clone. This allowed the sequencing of both strands of the insert cDNA from the plasmid.

Modified 5`-Rapid Amplification of cDNA Ends (RACE)

A modified 5`-RACE procedure as performed previously (17) was used to isolate the full-length est2 cDNA. The reverse primer used in the PCR was 5`-ACCGTACATCTCCACTCC-3` and was close to the 5`-end of the cDNA isolated from the cDNA library. The PCR product was subcloned into the pBluescript T vector(17, 27) . Two separate PCR products were sequenced in both directions.

Genomic DNA Studies

A Pel RR est2 cDNA fragment was used as a probe to determine the haplotype of the Est esterases from the Pel RR and Pel SS strains. Genomic DNA was isolated from fourth instar larvae as described previously(17) . 10 µg of genomic DNA was digested to completion with EcoRI and separated by gel electrophoresis through 0.8% (w/v) agarose. The DNA was transferred to charged nylon membranes (Amersham Corp.) and hybridized with a P-labeled Est2 cDNA probe (specific activity > 2 10 cpm/µg) at 65 °C for 16 h in hybridization buffer (5 Denhardt's solution, 6 SSC, 0.1% (w/v) SDS, 0.1% (w/v) sodium pyrophosphate, 5% (w/v) PEG 8000, and 100 µg/ml boiled, sheared herring sperm DNA). The final washes were at 65 °C in 0.1 SSC and 0.1% (w/v) SDS for 20 min.

Sequence Analysis

Similarity searches of protein data bases with the the est2 cDNA sequence were undertaken using the B17049 and MPsearch programs available through NCBI (National Institutes of Health) and EMBL (Heidelberg), respectively. Family structure determination was undertaken using the Prosite section of the Motif finder program through the Motif E-mail server on Genome. Prediction of secondary structure was undertaken using the PHD program(28) . Sequence alignments were undertaken using the MegAlign program of the LASERGENE package (DNASTAR). Similarity indexes were calculated using the FASTA algorithm, via the GeneMan program of LASERGENE.


RESULTS

Four positive plaques were obtained from the initial screening of the 200,000 recombinant clones of the unamplified Pel RR cDNA library. The number of positives obtained from this screening suggested that the target sequence was initially present at a higher frequency than that expected for a single copy gene. The four plaques were purified, in vivo excised, and partially sequenced with M13 forward and reverse primers. The sequence for all four clones over 400 nucleotides was identical. The insert from one of the plasmids (pBlueAV.A2) was completely sequenced in both directions.

The cDNA sequence had an open reading frame at its 5`-end, and terminated in a stop codon, 3`-untranslated region, and a poly(A) tail. There was no start methionine codon (AUG), so a modified 5`-RACE procedure (17) was used to isolate the 5`-end of the cDNA. The sequence of the two subcloned 5`-RACE PCR products analyzed was identical in both directions and also overlapped exactly with the previously sequenced partial length cDNA clone. The full-length cDNA (made up of the insert from pBlueAV.A2 and the 5`-RACE PCR product) had an open reading frame of 1623 base pairs complete with an AUG start codon and coded for a protein of 540 amino acids. This is in the expected range, since the purified monomeric protein has an estimated molecular mass of 58-67 kDa estimated by SDS-PAGE, native PAGE, and Sephacryl S200 chromatography(29) . Fig. 1shows the full-length est2 cDNA nucleotide sequence and the proposed amino acid sequence. Fig. 2shows the predicted amino acid sequence of Est2 and its alignment with the Culex Est2 (Pel RR B) and aphid E4 amplified esterases. The Est2 sequence, like the Est2, contained nine cysteine residues, but only three of these are conserved between the two esterases. The triad of precisely located active site amino acids, Ser, Glu, and His, of the serine esterase family were present in the Est2 sequence at positions 190, 324, and 445, respectively.


Figure 1: The nucleotide and predicted amino acid sequence of the cDNA for carboxylesterase Est2 (A) from Pel RR, an OP-resistant strain of Culex quinquefasciatus. The cDNA was isolated from a cDNA expression library by immunoscreening with antibody against Est2 and by a modified RACE procedure. The nucleotide and amino acid sequences are numbered from the ATG start codon. An in-frame stop codon (TGA) upstream of the ATG start codon is underlined. The start (ATG, amino acid M) and stop (TAG, represented by X) codons are underlined, as is the putative polyadenylation signal (AATAAA) beginning at nucleotide 1727. The amino acid residues thought to make up the active site triad (Ser, Glu, and His) are doubleunderlined, and the start of the poly(A) tail is represented by $.




Figure 2: Alignment of the amino acid sequence of Culex mosquito serine Est2 and Est2 esterases with the aphid E4 esterase involved in insecticide resistance. Possible glycosylation sites are underlined. Conserved amino acids in a large range of esterases (30) are in boldface.



The est2 had a similarity index of 49.2 with est2, which was the highest similarity in the protein database. The top 30 alignments of est2 undertaken with both the B17049 and MPsearch programs were all esterases or lipases. The protein was also unambiguously assigned to the carboxylesterase B-1 family of B type serine esterases using the Motif program.

Possible N-linked glycosylation sites, conforming to the sequence NXT or NXS, where X is not proline are underlined in Fig. 2. There are only two possible glycosylation sites in est2; both are very close to the active site serine and are not shared by other esterases in the alignment. Since the est2 is not proceeded by a signal sequence, and as the purified mature protein is not retained by Con A chromatography(22) , it is probable that the protein is not glycosylated.

The predicted secondary structure of Est2 obtained using the PHD program (28) is given in Fig. 3compared with the known consensus secondary structure of the Torpedo californica acetylcholinesterase and Geotrichum candidum lipase(30) . The predicted secondary structures of the Est2 and Est2 proteins, determined independently, were identical with the exception of the final -helix, which was not predicted in Est2. The predicted secondary structure is remarkably similar to that of the known consensus sequence for serine esterases and lipases, differing only in three -helices. One of these, the helix between -sheets 8 and 9 is also present in the G. candidum lipase structure but is not present in T. californica acetylcholinesterase (30).


Figure 3: Predicted secondary structure of Est2 compared with the known consensus secondary structure of T. californica acetylcholinesterase and G. candidum lipase. The predicted structure of Est2 differs only in the final -helix. Arrows indicate -sheets, and boxes indicate -helices. A, consensus secondary structure of T. californica acetylcholinesterase and G. candidum lipase. B, predicted secondary structure of the Est2Culex esterase.



The est2 cDNA was used as a probe for Southern blot analysis of EcoRI restriction digests of equal amounts of genomic DNA from the insecticide-susceptible (Pel SS) and resistant (Pel RR) mosquito strains. After hybridization and high stringency washing, the probe bound to a single 7.4-kb band of Pel SS DNA (Fig. 4), which demonstrates the existence of an est gene with high homology to est2 in this strain. A 7.5-kb band was found in the resistant strain at an equally low intensity, suggesting that the resistant strain still carries a nonamplified est allele. The Pel RR strain contained a 5.8-kb band with a high signal intensity, which was not present in the Pel SS strain. The higher signal intensity in the resistant strain compared with the susceptible, implies that gene amplification is the underlying mechanism of the Est2-associated resistance. Further proof of this was obtained by undertaking dot blots with the est2 cDNA probe, using genomic DNA from four other resistant strains of C. quinquefasciatus from different geographical locations with elevated Est2 activity. All the resistant strains gave a much higher intensity of signal than the susceptible strain (Pel SS), and the signals obtained were similar when the blots were reprobed with an est2 cDNA. This suggests that the amplification levels of the est2 and est2 genes are similar in the resistant strains.


Figure 4: Southern blot of EcoRI restriction digests of equal amounts of Pel RR (lane 1) and Pel SS (lane 2) genomic DNA hybridized with a Pel RR Est2 cDNA probe.




DISCUSSION

In this paper a new Culex esterase nomenclature system (17) has been adopted, as the molecular data now accumulating is making the old system unworkable. For example, a nonamplified B esterase from a susceptible Culex strain, which has an identical electrophoretic mobility to B, has been shown to be distinct from the Bs from TemR and MRES, which are in turn distinct from each other, and from esterase B at both a kinetic and amino acid level(17) .()Thus on the basis of restriction fragment length polymorphism (RFLP) patterns, DNA sequences, inferred amino acid sequences, and kinetic interactions of these enzymes at least one nonamplified and two amplified ``B'' esterases occur(17) .()This level of variability of electrophoretically identical esterases makes application of the earlier classification difficult, and the problem has been compounded by electrophoretically similar esterases being given different numerical values on the basis of their distinct RFLP patterns(31) . The nomenclature system we have used allows preliminary assignment of the esterase by electrophoresis, with an extension when sequence data is available, while the old system means esterases may need complete reclassification once sequence or RFLP data is available. With the new classification the esterase that we have now cloned, which was previously referred to as A, is classified as Est2 on the basis of its electrophoretic mobility and sequence.

The est2 esterase cDNA from the insecticide-resistant Pel RR strain of the C. quinquefasciatus mosquito has now been cloned and sequenced. The high number of positive clones obtained from the initial screening of the cDNA library suggested either that there was increased transcription of the gene or that gene amplification was the underlying mechanism of increased esterase activity. Southern blot analysis showed that the resistant strain had a unique, amplified 5.8-kb EcoRI RFLP when compared with the susceptible strain, which had a single unamplified band at 7.4 kb. Amplification of the est2 gene was also shown in other C. quinquefasciatus strains with elevated Est2 and Est2 activity, using dot blot analysis of genomic DNA. Since amplification of est2 is already well documented(17) , the underlying genetic mechanism for organophosphorus insecticide resistance in mosquitoes with the elevated Est2/Est2 activity phenotype is therefore amplification of both esterase genes.

The different RFLP pattern for est in the Pel RR and Pel SS strains demonstrates the existence of two distinct est alleles. The amplified and nonamplified Est and Est esterases have been purified and characterized physically and kinetically from a range of insecticide-resistant strains and a susceptible strain of mosquito(28, 32, 33, 34) . The Est enzymes from the susceptible (Pel SS) and resistant (Pel RR) strains are kinetically and electrophoretically distinct from each other, suggesting that they are different alleles, which is supported by the different RFLP patterns seen in the two strains. The amplified Est2, Est1, and Est2 esterases all have similar sizes (60 kDa) and PI values(29, 33) . We now know that all three esterases are coded for by cDNAs with the same length open reading frame. The amplified esterases E4 and FE4 in insecticide-resistant aphids are comparable in function and size with the mosquito esterases, but unlike the Culex esterases these enzymes confer resistance to both organophosphates and pyrethroids(35, 36, 37) . The E4 esterase and the Est2 and various Est Culex esterases all produce resistance to the organophosphates by sequestration, although the aphid and mosquito Est and Est esterases share only 21.8 and 22.9% similarity, respectively, while the similarity of the mosquito esterases is 49.2%. Hence the same kinetic properties can clearly be conferred by a large number of different sequences. This is perhaps not surprising when the predicted secondary structures of these esterases are considered. The predicted secondary structures for the Est2 and Est2 esterases differ only in the final element, and the structure bears a striking resemblance to actual secondary structures already resolved for two members of the serine esterase/lipase family(30) .

The high level of amino acid identity of Est2 and Est2 suggests that they originated from a common ancestor. The two genes probably arose through gene duplication and subsequently diversified. The Culex esterase cDNAs each code for proteins of 540 amino acids, and they have a higher sequence homology with each other than for any other sequence within the data banks. The two esterases also share a number of common features. For example, many carboxylesterases contain a short hydrophobic leader sequence, which initially directs protein sorting down a secretory pathway. All sequenced human liver carboxylesterases and the aphid E4 carboxylesterase contain signal sequences of between 17 and 23 amino acids(38, 39) . The mosquito est2 sequence and the est2 sequence are relatively unusual in that they contain no signal sequence, suggesting that neither esterase is exported from the cell. Neither of these purified esterases bind to Con A chromatography columns(22) , which suggests that the esterases are not glycosylated, in contrast to the aphid and some human esterases(39, 40) . This is further supported by the sequence data, since there are only two possible N-linked glycosylation sites on the Est2 esterase, which are situated only 4 and 31 amino acids from the active site serine.

In many soluble carboxylesterases that are retained by the endoplasmic reticulum, the tetrapeptide KDEL (Lys-Asp-Glu-Leu) occurs at the carboxyl terminus(41) . Variants of the KDEL sequence that direct intracellular retention of proteins have since been identified, although it appears that the Glu-Leu is a major requirement(42) . The four Culex est cDNAs sequenced to date all end in the sequence NDELF. However, the carboxyl terminus of Est2 is KDKLY.

Twenty-three amino acids are conserved through a series of 29 related proteins, and it was argued that these amino acids are essential for the structure (salt bridges, packing, and disulfide bridges) and function (active site) of the proteins(30) . Only 21 of these are conserved in the Est2. A cysteine is one of the nonconserved residues, and it is notable that both the Est2 and Est2 have nine cysteine residues, the majority of which are not conserved in any other serine esterase. The missing cysteine, at position 65 (Cys and Cys in T. californica AChE and G. candidum lipase, respectively) in the Pel RR Est2 sequence, forms a disulfide bridge in T. californica acetylcholinesterase and G. candidum lipase with a cysteine at position 84 (Cys and Cys), which is a serine in the Est2 and various Est Culex esterases. In the alignment of Cygler et al.(30) , the only esterase in which the latter cysteine was not conserved was the Culex TemR Est1. The reason for the large number of cysteines in the Culex esterases is unknown, but it has been suggested that five of these may not be involved in disulfide bond formation, leaving them free to oxidize. This results in the development of evenly spaced satellite bands after native polyacrylamide gel electrophoresis of purified native esterases under buffer conditions that do not protect the thiol groups(43) .

Having shown that the mechanism of elevation of the Est2 esterase is gene amplification, we now intend to determine the genomic structure of this esterase. The almost complete linkage disequilibrium in which these two elevated esterases occur, coupled with their sequence similarities, indicating that they arose through gene duplication, may also suggest that the two esterases are situated on the same amplification unit. This has, however, been contradicted by some classical genetic data on the inheritance patterns of these esterases,(12, 44) , and the final determination of the physical location of the two esterases in relation to each other awaits further genomic studies.

  
Table: Suggested nomenclatures for the esterases of the mosquito Culex quinquefasciatus



FOOTNOTES

*
This work was funded by the Wellcome Trust, The Royal Society, and Zeneca Public Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank/EMBL Data Bank with accession number(s) Z47988.

§
To whom correspondence should be addressed: E-mail: sabjh@cardiff.ac.uk.

The abbreviations used are: RACE, rapid amplification of cDNA ends; PCR, polymerase chain reaction; kb, kilobase.

S. H. P. P. Karunaratne, K. G. I. Jayawardena, A. Vaughan, and J. Hemingway, submitted for publication.

G. J. Small, S. H. P. P. Karunaratne, and J. Hemingway, submitted for publication.


REFERENCES
  1. Chang, C. K., and Jordan, T. W. (1983) Pestic. Biochem. Physiol.19, 190-195
  2. Herath, P. R. J., Hemingway, J., Weerasinghe, I. S., and Jayawardena, K. G. I. (1987) Pestic. Biochem. Physiol.29, 157-162
  3. Kao, L. R., Motoyama, N., and Dauterman, W. C. (1985) Pestic. Biochem. Physiol.23, 228-239
  4. Malkenson, N. C. D., Wood, E. J., and Zerba, E. N. (1984) Insect Biochem.4, 481-486
  5. Matsumura, F., and Sakai, K. (1968) J. Econ. Entomol.61, 598-605
  6. Motoyama, N., Kao, L. R., Lin, P. T., and Dauterman, W. C. (1984) Pestic. Biochem. Physiol.21, 139-147
  7. Parker, A. G., Russell, R. J., Delves, A. C., and Oakeshott, J. G. (1991) Pestic. Biochem. Physiol.41, 305-318
  8. Field, L. M., Devonshire, A. L., and Forde, B. G. (1988) Biochem. J.251, 309-312 [Medline] [Order article via Infotrieve]
  9. Mouches, C., Pasteur, N., Berge, J. B., Hyrien, O., Raymond, M., De Saint Vincent, B. R., De Silvestri, M., and Georghiou, G. P. (1986) Science233, 778-780 [Medline] [Order article via Infotrieve]
  10. Raymond, M., Beyssat-Arnaouty, V., Sivasubramanian, N., Mouches, C., Georghiou, G. P., and Pasteur, N. (1989) Biochem. Genet.27, 417-423 [Medline] [Order article via Infotrieve]
  11. Aldridge, W. N. (1953) Biochem. J.53, 110-117
  12. Callaghan, A., Hemingway, J., and Malcolm, C. A. (1993) Biochem. Genet.31, 459-472 [Medline] [Order article via Infotrieve]
  13. Georghiou, G. P. and Pasteur, N. (1978) J. Econ. Entomol.71, 201-205 [Medline] [Order article via Infotrieve]
  14. Raymond, M., Pasteur, N., Georghiou, G. P., Mellon, R. B., Wirth, M. C., and Hawley, M. (1987) J. Med. Entomol.24, 24-27 [Medline] [Order article via Infotrieve]
  15. Mouches, C., Magnin, M., Berge, J. B., De Silvestri, M., Beyssat, V., Pasteur, N., and Georghiou, G. P. (1987) Proc. Natl. Acad. Sci. U. S. A.84, 2113-2116 [Abstract]
  16. Mouches, C., Pauplin, Y., Agarwal, M., Lemieux, L., Herzog, M., Abadon, M., Beyssat-Arnaouty, V., Hyrien, O., De Saint Vincent, B. R., Georghiou, G. P., and Pasteur, N. (1990) Proc. Natl. Acad. Sci. U. S. A.87, 2574-2578 [Abstract]
  17. Vaughan, A., Rodriguez, M., and Hemingway, J. (1995) Biochem. J.305, 651-658 [Medline] [Order article via Infotrieve]
  18. Fournier, D., Bride, J.-M., Mouches, C., Raymond, M., Magnin, M., Berge, J. -B., Pasteur, N., and Georghiou, G. P. (1987) Pestic. Biochem. Physiol.27, 211-217
  19. Karunaratne, S. H. P. P., Jayawardena, K. G. I., Hemingway, J., and Ketterman, A. J. (1993) Biochem. Soc. Trans.22, 127S
  20. Karunaratne, S. H. P. P., Jayawardena, K. G. I., and Hemingway, J. (1995) Pestic. Biochem. Physiol.51, in press
  21. Peiris, H. T. R., and Hemingway, J. (1993) Bull. Entomol. Res.83, 127-132
  22. Karunaratne, S. H. P. P. (1994) Characterization of Multiple Variants of Carboxylesterases Which Are Involved in Insecticide Resistance in the Mosquito Culex quinquefasciatus. Ph.D. thesis, University of London
  23. Amin, A. M., and Peiris, H. T. R. (1990) Med. Vet. Entomol.4, 269-273 [Medline] [Order article via Infotrieve]
  24. Peiris, H. T. R., and Hemingway, J. (1990) Bull. Entomol. Res.80, 453-457
  25. Peiris, H. T. R., and Hemingway, J. (1990) Bull. Entomol. Res.80, 49-55
  26. Jayawardena, K. G. I. (1992) Purification and Characterization of Two Carboxylesterases from Organophosphate Resistant Culex quinquefasciatus from Sri Lanka. Ph.D. thesis, University of London
  27. Marchuk, D., Drumm, M., Saulino, A., and Collins, F. S. (1991) Nucleic Acids Res.19, 1154 [Medline] [Order article via Infotrieve]
  28. Rost, B., and Sander, C. (1993) Proc. Natl. Acad. Sci. U. S. A.90, 7558-7562 [Abstract/Free Full Text]
  29. Ketterman, A. J., Jayawardena, K. G. I., and Hemingway, J. (1992) Biochem. J.287, 355-360 [Medline] [Order article via Infotrieve]
  30. Cygler, M., Schrag, J. D., Sussman, J. L., Harel, M., Silman, I., Gentry, M. K., and Doctor, B. P. (1993) Protein Sci.2, 366-382 [Abstract/Free Full Text]
  31. Poire, M., Raymond, M., and Pasteur, N. (1992) Biochem. Genet.30, 13-26 [CrossRef][Medline] [Order article via Infotrieve]
  32. Jayawardena, K. G. I., Karunaratne, S. H. P. P., Ketterman, A. J., and Hemingway, J. (1994) Bull. Entomol. Res.84, 39-44
  33. Karunaratne, S. H. P. P., Jayawardena, K. G. I., Hemingway, J., and Ketterman, A. J. (1993) Biochem. J.294, 575-579 [Medline] [Order article via Infotrieve]
  34. Ketterman, A. J., Karunaratne, S. H. P. P., Jayawardena, K. G. I., and Hemingway, J. (1993) Pestic. Biochem. Physiol.47, 142-148 [CrossRef]
  35. Devonshire, A. L. (1977) Biochem. J.167, 675-683 [Medline] [Order article via Infotrieve]
  36. Devonshire, A. L., and Moores, G. D. (1982) Pestic. Biochem. Physiol.18, 235-246
  37. Devonshire, A. L., and Moores, G. D. (1989) in Enzymes Hydrolysing Organophosphorus Compounds (Reiner, E., Aldridge, W. N., and Hoskin, F. C. G., eds) pp. 181-192, Ellis Horwood Chichester, UK
  38. Field, L. M., Williamson, M. S., Moores, G. D., and Devonshire, A. L. (1993) Biochem. J.294, 569-574 [Medline] [Order article via Infotrieve]
  39. Kroetz, D. L., McBride, O. W., and Gonzalez, F. J. (1993) Biochemistry32, 11606-11617 [Medline] [Order article via Infotrieve]
  40. Devonshire, A. L., Searle, L. M., and Moores, G. D. (1986) Insect Biochem.16, 659-665
  41. Pelham, H. R. B. (1990) Trends Biochem. Sci.15, 483-486 [CrossRef][Medline] [Order article via Infotrieve]
  42. Robbi, M., and Beaufay, H. (1992) Biochem. Biophys. Res. Commun.183, 836-841 [Medline] [Order article via Infotrieve]
  43. Jayawardena, K. G. I., and Hemingway, J. (1995) Med. Vet. Entomol.9,
  44. Wirth, M. C., Marquine, M., Georghiou, G. P., and Pasteur, N. (1990) J. Med. Entomol.27, 202-206 [Medline] [Order article via Infotrieve]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.