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Cloning and expression analysis of Aedes aegypti opsin: adaptation of an in situ hybridization protocol for mosquitoes
Author(s) -
Graf R.,
Godknecht A.,
Nakano M.,
LJ X.,
Ackermann U.,
Helbiing P.
Publication year - 1996
Publication title -
insect molecular biology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.955
H-Index - 93
eISSN - 1365-2583
pISSN - 0962-1075
DOI - 10.1111/j.1365-2583.1996.tb00051.x
Subject(s) - biology , opsin , in situ hybridization , complementary dna , microbiology and biotechnology , cloning (programming) , rhodopsin , aedes aegypti , cdna library , genetics , gene , gene expression , biochemistry , botany , retinal , larva , computer science , programming language
Opsin is a G protein coupled photoreceptor that actlvates a signal transduction cascade in the ommatidia. Its primary and secondary structure Is conserved both In insects and vertebrates as exemplified by the Drosophiia opslns. Through serendipitous cloning of a PCR fragment, we have Identified an opsln cDNA. The latter was used to clone full length cDNAs from a mosquito head library. the main purpose of cloning was to have a positive control probe to establish an In situ hybridization protocol for less abundant probes. Opsin‐mRNA is localized specifically to the visual receptor cells in the ommatida. No other cells in the brain or the remainder of the body are positive. This is confirmed by Northern blot analysis. The sequence of the receptor, of which we have found two different transcripts, confirms Its typical topology, including the seven transmembrane spanning regions and the intracellular carboxy terminus that has potential phosphorylatlon sites. Our in situ hybridization protocol combines several procedures: the most important points are: (a) the immediate processing of sections after cutting, and (b) the sections are never allowed to dry out once the procedure was started. Our protocol has a much higher sensitivity, using approximately 50times lower concentrations of probe compared to published protocols. In addition to the detection of opsin‐mRNA, It has been successfully applied to the detection of the low abundant Insulin receptor homologue. Furthermore, Aedes aegypti probes were visualizing a similar tissue specificity when applied to the malaria mosquito Anopheles albimanus .