Materials and Methods
The eye samples and RT-PCR amplification:
The eyes of African elephant (L. africana) and Asian elephant (E. maximus) were sampled from necropsied females of 24 and 57 years old, respectively. Their total retinal RNAs were isolated using the procedures of YOKOYAMA et al. (1995). The entire coding regions of all different types of opsin cDNAs of the African elephant have been cloned in two steps: (1) cloning of the internal segments by using RT-PCR and (2) completion of the cloning by using 5'- and 3'-rapid amplification of cDNA ends (RACE; see the next section). To characterize internal segments, we first cloned the segment between codon positions 248 and 300 of all opsin cDNAs by using the forward and reverse degenerate primers designed by CARLETON and KOCHER (2001) (Figure 1A, all internal), where the amino acid site numbers are standardized by those of bovine RH1 pigment (GenBank accession nos. K00502, K00503, K00504, K00505, K00506). Note that these degenerate primers have been designed to clone all RH1, RH2, SWS1, SWS2, and M/LWS opsin genes. Using these primers, cDNA was reverse transcribed at 42° for 1 hr and 95° for 5 min and then PCR amplification was carried out for 30 cycles at 94° for 45 sec, 55° for 1.5 min, and 72° for 2 min. The PCR products were gel isolated and subcloned into the T-tailed EcoRV-digested Bluescript plasmid vector with T-overhang attached to 3' ends (HADJEB and BERKOWITZ 1996). Nucleotide sequences of these cDNA clones were determined by cycle-sequencing reactions using the Sequitherm Excel II long-read kits (Epicentre Technologies, Madison, WI) with dye-labeled M13 forward and reverse primers. Reactions were run on a LI-COR (Lincoln, NE) 4200LD automated DNA sequencer. To clone the longer internal fragments, we then designed additional sets of the RH1, SWS1, and M/LWS gene-specific degenerate primers. The forward primers (internal forward) for the RH1, SWS1, and LWS opsin cDNAs are located around codon sites 51, 70, and 51, respectively, while the corresponding reverse primers (internal reverse) are located between codon sites 260 and 280, making the total lengths of RT-PCR products between 573 and 691 nucleotide sites (Figure 1A).
RACE:
To determine the rest of the cDNA sequences, we have constructed additional gene-specific primers (GSPs) (Figure 1A) and conducted 5'- and 3'-RACE analyses according to the manufacturer's protocol (GIBCO BRL, Gaithersburg, MD). In short, for the 3'-RACE, the first-strand cDNA was made using the oligo(dT)-containing adapter primer (AP) provided by the manufacturer and the original mRNA was degraded by RNase H. Then, two sequential PCR amplifications were performed, applying two sets of GSPs and universal adapter primers (UAPs) to these cDNAs, first using GSP1 with UAP and then the nested GSP2 with abridged UAP (Figure 1A), where the UAPs were supplied by the manufacturer. For the 5'-RACE, the cDNAs were first synthesized from total RNA using GSP1 and the mRNA was degraded by RNase H. The cDNA was purified using a spin column and then a poly(C) tail was added to the 3' end of the cDNAs using dCTP and terminal transferase. Then, the entire coding region was obtained by two sequential PCR amplifications, first using nested GSP2 with abridged AP and then nested GSP3 and abridged UAP (Figure 1A), where the UAPs were again supplied by the manufacturer. The nucleotide sequences of the resulting cDNA products were again determined by using a LI-COR 4200LD automated DNA sequencer.
Expression and spectral analyses of pigments (in vitro assay):
The contiguous full-length opsin cDNAs were then obtained by RT-PCR using primers based on the nucleotide sequences of the 5' and 3' end cDNA clones (Figure 1B). The opsin cDNAs of full length were subcloned into the EcoRI and SalI restriction sites of the expression vector pMT5 (KHORANA et al. 1988). All of these DNA fragments were sequenced to rule out spurious mutations. These plasmids were expressed in COS1 cells by transient transfection. The pigments were regenerated by incubating the opsins with 11-cis-retinal (Storm Eye Institute, Medical University of South Carolina) and purified using immobilized 1D4 (The Culture Center, Minneapolis) in buffer W1 [50 mM N-(2-hydroxyethyl) piperazine-N'-2-ethanesulfonic acid (HEPES) (pH 6.6), 140 mM NaCl, 3 mM MgCl2, 20% (w/v) glycerol, and 0.1% dodecyl maltoside] (for more details, see YOKOYAMA 2000b). UV-visible spectra were recorded at 20° using a Hitachi U-3000 dual beam spectrophotometer. Visual pigments were bleached for 3 min using a 60-W standard light bulb equipped with a Kodak Wratten no. 3 filter at a distance of 20 cm. Data were analyzed using Sigmaplot software (Jandel Scientific, San Rafael, CA).
Site-directed mutagenesis:
Mutant opsins were generated by using the QuickChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). All DNA fragments that were subjected to mutagenesis were sequenced to rule out spurious mutations.
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