Animals and cDNA library of an octopus hemisected eye

Living Japanese common octopuses (Octopus vulgaris; local common name ‘madako’) were purchased from a local fishery (Tokusui Shoji, Akashi, Japan). Total RNA was prepared from one hemisected eye by standard CsCl-ultracentrifugation methods (Maniatis et al., 1982). cDNA was synthesized from the total RNA with a random 9-mer as a primer with Moloney murine leukemia virus reverse transcriptase (Super Script RT, Life Technologies, Rockville, USA) and used as a template for polymerase chain reaction (PCR). A cDNA library of the octopus eye was then constructed with a λZap-II system (Stratagene, La Jolla, USA) and in vitro-packaged by Giga Pack Gold III (Stratagene, La Jolla, USA).

Polymerase chain reaction

Degenerated PCR primers were designed according to the conserved amino acid motifs of the α subunit of G protein. A forward primer corresponded to amino acid sequence KQM(K/R)IIH, and a reverse primer corresponded to amino acid sequence (K/R)KWI(H/Q)CF (F1 and R1, respectively; Fig. 1 and shaded boxes in Fig. 2). PCRs were performed with random-primed cDNA as a template, and the PCR products were then analyzed by agarose gel electrophoresis, recovered from the agarose gel (Band prep kit, Amersham Pharmacia Biotech, Uppsala, Sweden) and subcloned in a pT7blue vector (Stratagene, La Jolla, USA). The nucleotide sequence was determined in both directions from several independent clones, using the primer-labeled cycle sequence method according to the manufacturer's protocol (Amersham Pharmacia Biotech, Uppsala, Sweden). Sequences were analyzed with an automatic DNA sequencer (SQ3000, Hitachi Electronics Engineering, Tokyo, Japan or Shimadzu DSQ 1000L, Shimadzu, Kyoto, Japan). RACE-PCR, with a combination of a cDNA fragment specific primer (3′-RACE 1, 2, 5′-RACE 1 or 2 in Fig. 1) and a vector primer (P8, SK, P7 or T7 in Fig. 1), was performed to obtain a 3′- and 5′-sequence using 1μl aliquots of the O. vulgaris eye cDNA library as a template. Finally, the whole open reading frame (ORF) was PCR-amplified with a combination of specific primers based on the 3′ and 5′ non-coding sequence of each G protein α subunit (Gα -N1, -N2, -C1, or -C2 in Fig. 1).

Fig. 1

Cloning strategy for octopus G protein α subunit genes. Degenerate oligonucleotides (F1 and R1) corresponding to conserved amino acid sequences were synthesized and used to amplify cDNA fragments of octopus G protein α subunits (PCR 1). Gene-specific primers were then synthesized (3′-RACE-1 and 2, and 5′-RACE-1 and 2) and used with vector-specific primers (T7, P7, P8 and SK) to amplify 5′ or 3′-cDNA fragments (PCR 2 and 3). Finally, gene-specific primers were made to amplify the whole coding sequence (Gα-N1 and 2, and Gα-C1 and 2; PCR 4).

Fig. 2

Comparison of the amino acid sequences of octopus G protein α subunits. Dots represent identical amino acids; dashes are alignment gaps; asterisks mark conserved amino acid residues. Shaded boxes show the amino acid sequence used to design PCR primers (F1 and R1 in Fig. 1) to amplify a cDNA fragment of G protein α subunit. Open boxes represent A-, C- and G-boxes (in order from the N-terminal), which are conserved within all G protein α subunits. Sequence data used in the figure were taken from GenBank, EMBL and DDBJ data bases: Hs (Homo sapiens) Gi3 (M20604), HsGo (M60162), HsGq (M69013), HsGs (X04408) and HsGt (X15088). Sequence data for octopus are results of the present study, with accession numbers as follows: Ov (Octopus vulgaris) Gi (AB025780), OvGo (AB025781), OvGq (AB025782), and OvGs (AB025783).

Northern blot analysis

Total RNA from several tissues of O. vulgaris was prepared by the standard CsCl-ultracentrifugation method. This RNA (10 μg) was then separated on a 0.8% agarose-formamide gel and transferred to nylon membrane (Hybond N⁺, Amersham Pharmacia Biotech, Uppsala, Sweden) and cross-linked with a UV cross-linker (CL-1000 Ultra Violet Cross Linker, UVP, San Gabriel, USA). Hybridization was carried out with random primed ³²P-labeled probes for each class of G protein α subunit (full length of ORF) (Megaprime DNA labeling system, Amersham Pharmacia Biotech, Uppsala, Sweden) in 50% formamide, 6 × SSC (0.9 M NaCl, 90 mM sodium citrate), 1% SDS, 5 × Denhardt's solution and 20 μg/ml denatured salmon sperm DNA at 42°C overnight. The filters were then washed twice in 2 × SSPE (360 mM NaCl, 20 mM sodium phosphate, 2 mM EDTA, pH 7.7) and 0.1% SDS for 10 minutes at room temperature, and twice in 1 × SSPE and 0.1% SDS for 15 minutes at 65°C. The washed membranes were then analyzed with an imaging plate (BAS2000, Fuji film, Tokyo, Japan).

In situ hybridization

Digoxigenin-labeled anti-sense and sense riboprobes for each G protein α subunit message were made according to the manufacturer's protocol (DIG RNA labeling Kit, Boeringer-Mannheim, Mannheim, Germany). Octopus retinas were fixed with PLP (4% paraformaldehyde, 75 mM lysin, 37.5 mM phosphate buffer, pH 7.4, 10 mM sodium periodate) and washed in PBS (10 mM sodium phosphate, pH 7.5, 154 mM NaCl). The fixed tissues were then incubated in PBS containing 20% sucrose and 5% glycerol and embedded in OCT compound (Miles Inc., Indiana, USA). Cryostat sections (about 10 μm thickness) were dried in air, fixed again in 4% formaldehyde in PBS for 20 min at room temperature and washed in PBS (10 min, 3 times), followed by distilled water (1 min). The sections were then acetylated, briefly washed in 2x SSC, dehydrated in a graded EtOH series (50 –100%) and dried in air. They were then hybridized with each digoxigenin-labeled riboprobe at 50°C overnight. Unhybridized riboprobes were then digested with RNase at 37°C for 30 min, and washed in 2x SSC for 1 hr at room temperature, followed by 0.1x SSC for 1 hr at 60°C. The sections were then incubated with anti-DIG antibody - alkaline phosphatase conjugates at room temperature for 1 hr and the color was developed according to the manufacturer's protocol (Boehringer-Mannheim, Germany), for 4 hr - overnight at room temperature. After a wash with TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH7.6), the sections were fixed with 1% glutaraldehyde in distilled water followed by a wash with distilled water. They were then dehydrated through EtOH, cleared in xylene, and embedded in Permount (Fisher Scientific, New Jersey, USA).

Molecular structure of cDNA clones of octopus G protein α subunits

Four different classes of cDNA clones were obtained by PCR amplification of octopus eye cDNA with primers designed for the conserved amino acid sequence of G protein α sub-unit. Full length cDNA clones were then obtained from a cDNA library of a hemisected eye of O. vulgaris with a series of runs of PCR with vector-specific and cDNA-specific primers. The nucleotide sequences will appear in the DDBJ, EMBL and GenBank Nucleotide Sequence Databases under Accession Nos. AB025780 – AB025783. The cDNA sequences contained a single open reading frame and the deduced amino acid sequences from the clones were 353 or 354 amino acids in length, except one of 377 amino acids. Based on comparison of the deduced amino acid sequences with those of human G protein α subunits (Table 1 and Fig. 2), they are classified as O. vulgaris G protein α subunits (OvGα_i, OvGα_o, OvGα_q and OvGα_s). They show high similarity (74 – 84%) to the corresponding subclasses of human G protein α subunit (Table 1, numbers in bold type). The well conserved amino acid motifs of G protein α subunits, domains A, C and G, were found in all the deduced sequences (open boxes in Fig. 2). Since these domains play an important role in the binding and catalysis of a guanine nucleotide, the octopus G protein α subunits obtained appear to be functional proteins in octopus eye.

Table 1

Percentage amino acid identity between octopus and human G protein α subunits.

Tissue distribution of G proteins

To determine the tissue distribution of messages corresponding to octopus G protein α subunits, Northern blot hybridization was performed with RNA purified from a variety of octopus tissues (Fig. 3). Two messages of different size were observed for OvGα_i in optic lobe, heart and epidermis; and three for OvGα_q in eye, intestine and epidermis. They were evident also in other RNA preparations, suggesting that they are not the products of a single degraded message.

Fig. 3

Northern blot analysis of octopus G protein α subunit message in different tissues of Octopus vulgaris. Total RNA (10 μg) from each indicated tissue was analyzed with a specific probe for each class of octopus G protein α subunit message containing for entire coding and 3′-noncoding region. Arrows at right are size markers.

As shown in Fig. 3, OvGα_i was expressed in the all tissues, with a weaker signal in the eye and liver than in the other tissues. The expression of OvGα_o was restricted to the neural tissues (brain and optic lobe) and was not observed in the eye. Although the cDNA clone of OvGα_o was obtained from the cDNA library of a hemisected eye, the absence of a signal indicates that the amount of OvGα_o in the eye is very low. These results agree well with our biochemical data, in that the amount of G_o class G protein α subunit was less than 10% of that of G_q class in the eye (unpublished results). The expression of OvGα_q was high in the eye, lower in the optic lobe, brain, pancreas, heart, intestine and epidermis, and negligible in the kidney, resembling the eye-specific expression of vertebrate transducin and photoreceptor protein (opsin). OvGα_s was present as a single band in the all tissues except the eye, with an intensity different for each tissue.

Distribution of OvGα_q in octopus retina

The spatial distribution of G protein messages in octopus photoreceptor cells was investigated by in situ hybridization of retinal sections. Particular attention was paid to the expression of OvGα_q and OvGα_o in the photoreceptor cells, since both classes of G protein have been reported to couple with photo-activated rhodopsin in invertebrates (Lee et al., 1990; Suzuki et al., 1995; Kikkawa et al., 1996; Kojima et al., 1997). Unfortunately, no signal was obtained with the anti-sense RNA probe for OvGα_o, presumably because the amount of OvGα_o is very low in the eye, as indicated by the Northern blot analysis (Fig. 3).

The anti-sense probe for OvGα_q showed a signal in the inner segment layer around the nuclei of the photoreceptor cells (Fig. 4A), while the sense-probe did not (Fig. 4B). These results indicate that OvGα_q is expressed in the photoreceptor cells not in the supporting cells, because the nuclei of the supporting cells form a layer just between the inner segment and the outer segment (arrowhead in Fig 4A).

Fig. 4

In situ hybridization of octopus retina with digoxigenin-labeled probes for OvGα_q or octopus opsin message. A, Anti-sense probe for OvGα_q. The positive signal is observed in the nuclear layer of the photoreceptor cells. B, Sense probe for OvGα_q. C, Anti-sense probe for octopus opsin. D, Anti-sense probe for OvGα_q (cf. A), for comparison with C. Square brackets and arrowheads to the right of each figure indicate nuclear layers of the photoreceptor cells and supporting cells, respectively. Scale bars,100 μm.

Opsin, the apoprotein of rhodopsin, is clearly expressed in photoreceptor cells (square bracket in Fig. 4C): all the photoreceptor cells showed an intense signal not only around the nuclei but also in the cytoplasm. This is consistent with the fact that only one opsin gene has been found in the octopus to date and that the photoreceptor cells of the octopus are morphologically similar. Under higher magnification, the expression of OvGα_q was observed around the photoreceptor nuclei (Fig. 4D) but with a signal intensity weaker than that of opsin (cf. Fig. 4C). Again almost all the photoreceptor cells expressed OvGα_q, suggesting that photoreceptor cells commonly co-express opsin and OvGα_q.