Generation of full-length cDNAs for eight putative GPCnR from the cattle tick, R. microplus using a targeted degenerate PCR and sequencing strategy

Corley, S.W., Piper, E.K. and Jonsson, N.N. (2012) Generation of full-length cDNAs for eight putative GPCnR from the cattle tick, R. microplus using a targeted degenerate PCR and sequencing strategy. PLoS ONE, 7(3), e32480. (doi:10.1371/journal.pone.0032480) (PMID:22403662) (PMCID:PMC3293813)

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Abstract

We describe here a rapid and efficient method for the targeted isolation of specific members of gene families without the need for cloning. Using this strategy we isolated full length cDNAs for eight putative G-protein coupled neurotransmitter receptors (GPCnR) from the cattle tick Rhipicephalus (Boophilus) microplus. Gene specific degenerate primers were designed using aligned amino acid sequences of similar receptor types from several insect and arachnid species. These primers were used to amplify and sequence a section of the target gene. Rapid amplification of cDNA ends (RACE) PCR was used to generate full length cDNA sequences. Phylogenetic analysis placed 7 of these sequences into Class A G-protein coupled receptors (GPCR) (Rm_α2AOR, Rm_β2AOR, Rm_Dop1R, Rm_Dop2R, Rm_INDR, Rm_5-HT7R and Rm_mAchR), and one into Class C GPCR (Rm_GABABR). Of the 7 Class A sequences, only Rm_mAchR is not a member of the biogenic amine receptor family. The isolation of these putative receptor sequences provides an opportunity to gain an understanding of acaricide resistance mechanisms such as amitraz resistance and might suggest possibilities for the development of new acaricides.

Item Type:Articles
Status:Published
Refereed:Yes
Glasgow Author(s) Enlighten ID:Jonsson, Professor Nicholas
Authors: Corley, S.W., Piper, E.K., and Jonsson, N.N.
College/School:College of Medical Veterinary and Life Sciences > Institute of Biodiversity Animal Health and Comparative Medicine
Journal Name:PLoS ONE
Publisher:Public Library of Science
ISSN:1932-6203
Published Online:05 March 2012
Copyright Holders:Copyright © 2012 The Authors
First Published:First published in PLoS ONE 7(3):e32480
Publisher Policy:Reproduced under a Creative Commons License

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