LW rhodopsin
The bee long-wavelength rhodopsin is one of a family of three rhodopsin genes in bees: long wavelength (green), blue, and UV rhodopsin (Towson et al. 1998, Chang et al. 1996). Rhodopsins are G-protein-coupled receptor proteins that perform the first steps in visual transduction in most organisms. Long-wavelength rhodopsins have been characterized and sequenced in ants (Popp et al. 1996), mantids (Towner & Gärtner 1994), butterflies (Briscoe 1999) and flies (Drosophila, Huber et al. 1997). The long-wavelength rhodopsin (hereafter LW opsin) has been used for phylogenetic analysis of bees (Mardulyn & Cameron 1999, Cameron & Mardulyn 2001, Ascher et al. 2001, Cameron & Williams 2003), Heliconius butterflies (Hsu et al. 2001), cynipid wasps (Rokas et al. 2002, Nylander et al. 2004), and aphids (Ortiz-Rivas et al. 2003). Previous phylogenetic analyses of bees (Mardulyn & Cameron 1999, Cameron & Mardulyn 2001, Ascher et al. 2001, Cameron & Williams 2003) used opsin primers LWRhFor, and LWRhRev, which produce an approximately 700 bp PCR product consisting of three exons and two introns (primers are listed in the attached pdf file below).
We expanded the opsin data set beyond the range of previous studies using 3'-RACE, as described below. Poly-A mRNAs were isolated from heads of recently collected, frozen bees (Agapostemon virescens, Andrena carlini, Augochlorella striata, Lasioglossum leucozonium, Colletes inaequalis, Halictus ligatus, and Lasioglossum (Dialictus) zephyrum; all collected in Ithaca, NY) using Oligotex Direct mRNA Micro Kit (Qiagen, Chatsworth, CA). The mRNA were then used for reverse-transcription to generate cDNA. PCR of cDNA fragments using opsin-specific primers and poly-T reverse primers were used to generate PCR products, which were then ligated into a pGEM vector (Promega, Madison, WI), and transformed into DH5-alpha library efficiency competent cells (Gibco, Grand Island, NY). The transformed cells were plated out on LB medium. Plasmids containing opsin inserts were isolated and sequenced using an ABI 373A automated sequencer. We aligned the sequences obtained with the three different opsin copies in Apis (Chang et. al 1996) to confirm that the cloned fragments matched the LW gene family. All sequences spanned the 3' end of the gene and all included the stop codon plus the 3' non-coding region. Based on these sequences we developed two reverse primers (Opsin Rev4, Opsin Rev4a; see pdf file below) to amplify the 3' end of the gene (including one intron).
Our opsin data set spans positions 434 to 1146 of the coding region in the Apis mellifera LW opsin paralog (Chang et al. 1996), and includes four exons and three introns (see attached pdf file with the map of the gene). The region we sequenced spans transmembrane regions III to VII plus the intracellular loop downstream of transmembrane region VII. The locations of introns coincide with the junction of transmembrane and extra-membrane regions, suggesting that the introns flank functional domains of the protein.
The honey bee, Apis mellifera, complete LW opsin sequence is available on Genbank as accession number U26026 (Chang et al. 1996).
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Gene Information: |
Map of Opsin gene |
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