The amplification of carboxylesterase structural genes followed by their overexpression is the most common mechanism of resistance to organophosphorus insecticides in Culex mosquitoes. Most resistant Culex quinquefasciatus mosquitoes have co-amplified estα21 and estβ21 genes. Recently, Southern, DNA dot-blot analysis and phosphorimaging technology were used to quantify the est gene copy number in aphids and mosquitoes. Although more accurate than autoradiography, this method relies on probe hybridization, which can be variable. We have directly measured gene and mRNA copy number by using real-time quantitative PCRs in mosquitoes. The acquisition of fluorescence from incorporation of the double-strand-specific dye SYBR GreenI into a PCR product once per cycle is used to provide an absolute quantification of the initial template copy number. Thus it has been possible to show that estα21 and estβ21 are co-amplified approx. 80-fold in the genome of the resistant PelRR strain of C. quinquefasciatus. The two genes, although co-amplified in a 1:1 ratio, are differentially transcribed: the estβ21 gene from this amplicon has greater transcription than estα21 in all individual mosquito larvae tested, with an average ratio of 10:1. Purified esterases from mosquito homogenates were found in a ratio of 3:1, which, combined with the quantitative mRNA data, suggests the operation of both transcriptional and translational control mechanisms to regulate the expression of the amplified genes in C. quinquefasciatus insecticide-resistant mosquitoes.
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Research Article|
February 08 2000
Quantitative analysis of gene amplification in insecticide-resistant Culex mosquitoes
Michael G. PATON;
Michael G. PATON
1School of Biosciences, University of Wales Cardiff, P.O. Box 915, Cardiff CF1 3TL, U.K.
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S. H. P. Parakrama KARUNARATNE;
S. H. P. Parakrama KARUNARATNE
1School of Biosciences, University of Wales Cardiff, P.O. Box 915, Cardiff CF1 3TL, U.K.
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Elsa GIAKOUMAKI;
Elsa GIAKOUMAKI
1School of Biosciences, University of Wales Cardiff, P.O. Box 915, Cardiff CF1 3TL, U.K.
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Neil ROBERTS;
Neil ROBERTS
1School of Biosciences, University of Wales Cardiff, P.O. Box 915, Cardiff CF1 3TL, U.K.
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Janet HEMINGWAY
Janet HEMINGWAY
1
1To whom correspondence should be addressed (e-mail sabjh@;cardiff.ac.uk).
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Publisher: Portland Press Ltd
Received:
August 09 1999
Revision Received:
November 01 1999
Accepted:
November 25 1999
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London © 2000
2000
Biochem J (2000) 346 (1): 17–24.
Article history
Received:
August 09 1999
Revision Received:
November 01 1999
Accepted:
November 25 1999
Citation
Michael G. PATON, S. H. P. Parakrama KARUNARATNE, Elsa GIAKOUMAKI, Neil ROBERTS, Janet HEMINGWAY; Quantitative analysis of gene amplification in insecticide-resistant Culex mosquitoes. Biochem J 15 February 2000; 346 (1): 17–24. doi: https://doi.org/10.1042/bj3460017
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