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Title: Molecular biological characterisation of amplified esterases from organophosphate resistant and susceptible 'Culex quinquefasciatus'
Author: Vaughan, Ashley Michael
Awarding Body: London School of Hygiene & Tropical Medicine
Current Institution: London School of Hygiene and Tropical Medicine (University of London)
Date of Award: 1995
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Culex mosquitoes, as well as being vectors of filariasis and Japanese encephalitis, are a world wide biting nuisance. Organophosphorus insecticides (OPs) have been widely used to control Culex populations. Resistance to OPs has occurred and is typically mediated by the increase in non-specific esterase activity. The two esterases involved are classified as 'A' and 'B' esterases with respect to their preference for the substrates α- or β- naphthyl acetate. The commonest phenotype involves two elevated esterases, A2 and B2, which occur in complete linkage disequilibrium. The over expression of esterase B1 is due to gene amplification. Initially, in order to further study the molecular biology of OP resistance, full length cDNAs coding for both A2 and B2 esterases were isolated and sequenced from an OP resistant Sri Lankan strain of Culex quinquefasciatus, PelRR. The B2 esterase cDNA was isolated with PCR using primers sharing homology with the B1 esterase cDNA and has 97.4% homology with esterase B1 at the amino acid level. This confirmed that the B esterases belong to an allelic series. Partial genomic sequences of B2 esterase from PelRR and four other OP resistant Culex strains were identical. This suggests that the initial B2 esterase amplification has occurred only once. However, the cDNA sequence of a B1 esterase cDNA isolated from an OP resistant Cuban strain of Culex quinquefasciatus, MRES, was different to that of the previously published B1 esterase gene sequence. At the genomic level, the haplotype of the Cuban B1 esterase gene, based on EcoRI endonuclease analysis, was also different, suggesting that the initial B1 esterase gene amplification event has occurred at least twice. AB esterase cDNA from an OP susceptible strain, PelSS, has also been partially sequenced. PelSS was derived from the same origins as PelRR but its B esterase cDNA sequence and haplotype of the gene are different. Thus, the B2 esterase gene conferring OP resistance, as well as being amplified, is only found in the resistant strain, PelRR. The A2 esterase cDNA was isolated by screening a PelRR cDNA expression library with an anti-A2 antiserum. The cDNA coded for a protein of 540 amino acids (the same as B2 esterase) and shared 47% amino acid homology with B2 esterase. This strongly suggests that the two genes arose from a duplication of an ancestral counterpart. Furthermore, screening of a PelRR genomic library with A2 and B2 esterase gene probes suggests that the two esterase genes, A2 and B2 are situated in tandem within the genome. PCR was used to amplify the coding region of the PelRR A2 esterase cDNA and this was co-transfected into the baculovirus expression system. The recombinant virus expressed an active A esterase.
Supervisor: Hemingway, J. Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
Keywords: Mosquitoes; Insecticide resistance Molecular biology Cytology Genetics Biochemistry