Animals and embryos

Mature adults of the orange-red variety of the medaka fish O. latipes were purchased from a dealer. Embryos of O. latipes Hd-rR inbred strain were kindly provided by Professor Akihiro Shima, University of Tokyo. The embryos were kept in indoor tanks under artificial reproductive conditions (10 hr dark and 14 hr light at 27°C) and were fed on Otohime B2 (Nisshin Seifun Group Inc., Tokyo, Japan).

Preparation of RNA

Total RNA was isolated from various adult organs of the orange-red variety of O. latipes by the acid guanidinium thiocyanatephenol-chloroform extraction method (Chomczynski et al., 1987). Poly(A)⁺RNA was prepared using Oligotex-dT30 (Roche, Basel, Switzerland), according to the manufacturer's protocol.

Amplification of cDNA fragments for membrane GCs by RTPCR

Three degenerate oligonucleotide primers (P2, 5′-GAYATHGTNGGNTTYAC-3′; P6, GTRTTNACNTRTCNCC-3′; P7, 5′-ARRCARTANCKNGGAT-3′) were synthesized based on the amino acid sequences of 3 conserved regions (DIVGFT, MPRYCL, GDTVNT) in known membrane GCs and these primers were used to amplify membrane GC cDNA fragments from cDNA reverse-transcribed total RNA of the medaka fish ovary, as described previously (Seimiya et al., 1997). The product was purified, subcloned into the plasmid vector pBluescript II KS(–) (Stratagene, La Jolla, CA, USA), and sequenced.

5′- and 3′-Rapid Amplification of cDNA Ends (5′- and 3′-RACE)

To obtain a full-length cDNA sequence of a new membrane GC clone referred to as OlGC8, the 5′- or 3′-portion of the cDNA was amplified by the 5′- or 3′-RACE method (Frohman et al., 1988), respectively, using the 5′-RACE System for Rapid Amplification of cDNA Ends, Ver 2.0 (Gibco BRL, Groningen, The Netherlands) or the 3′-Full RACE Core Set (Takara Shuzo Co., Ltd. Tokyo, Japan), as described previously (Yamagami et al., 2001).

Total RNA (2 μg) isolated from the adult medaka fish ovary was reverse-transcribed with gene-specific antisense oligonucleotide primers (GSP1, GSP4, GSP7, and GSP10) and used as a template for 5′-RACE with the Abridged Anchor Primer (Life Technologies, Groningen, The Netherlands) and other gene-specific antisense oligonucleotide primers (GSP2, GSP5, GSP8, and GSP11). Amplification in the 5′-RACE was performed as follows: denaturation at 94°C for 5 min followed by 30 cycles of denaturation for 30 sec at 96°C, annealing for 1 min at 58°C, 61°C, 63°C, and 58°C, respectively, and extension for 90 sec at 72°C, and the final extension was carried out at 72°C for 10 min. To enrich the 5′-RACE products, a one-fifteenth volume of the primary products was reamplified by PCR using the Abridged Anchor Primer and nested primers (GSP3, GSP6, GSP9, and GSP12). PCR was performed under the same conditions as those used for GSP2, except for the amplification cycle (25 cycles) and the annealing temperature (58°C, 61°C, 62°C, and 55°C, respectively). On the other hand, 3 μg of total RNA was reverse-transcribed with an Oligo dT-3′ sites Adaptor Primer (Takara Shuzo) for the 3′-RACE method. The cDNA was amplified by PCR with the 3′ sites Adaptor Primer (Takara Shuzo) and another gene-specific oligonucleotide primer (GSP13). A one-fifteenth volume of the PCR product was amplified by PCR with 3′ sites Adaptor Primer and another gene-specific oligonucleotide primer (GSP14). Amplification was performed under the same conditions as those used for GSP2, except for the amplification cycle and the annealing temperature (30 cycles at 63°C and 25 cycles at 62°C, respectively). The gene-specific primers used were complementary to the following nucleotide positions: 4281–4301 (GSP1), 3735–3754 (GSP2), 2568–2589 (GSP3), 1421–1440 (GSP4), 4252– 4272 (GSP5), 3534–3555 (GSP6), 2538–2559 (GSP7), 1359–1377 (GSP8), 4147–4166 (GSP9), 3450–3471 (GSP10), 2511–2531 (GSP11), 1268–1286 (GSP12), 3643–3661 (GSP13), and 3809–3830 (GSP14). The RACE products overlapped at 53–655 bp with the 5′ or 3′ end of the isolated cDNA clone.

Molecular phylogenetic analysis

The deduced amino acid sequence of OlGC8 was compared with those of various membrane GCs using the Clustal W program (Thompson et al., 1994) and the sequence editor SeqPub (Gilbert, Indiana University, Bloomington, IN, USA). The unrooted phylo-genetic tree was constructed using the aligned sequences by the neighbor-joining algorithms in the PROTRAS program of PHYLIP ver. 3.572 (Felsenstein, 1989) and the Clustal W program (Saitou and Nei, 1987). For the neighbor-joining analysis, the evolutionary distance was estimated using Kimura's empirical method for protein distances (Kimura, 1983). The following GenBank/EMBL/DDBJ accession numbers for the sequences were used:  X14773 (rat GCA);  M26896 (rat GC-B);  M55636 (rat GC-C);  L37203 (rat GC-D);  L36029 (rat GC-E);  L36030 (rat GC-F);  AF024622 (rat GC-G);  AB 004921 (OlGC1);  AB030274 (OlGC2);  AB000899 (OlGC3);  AB000900 (OlGC4);  AB000901 (OlGC5);  AB007192 (OlGC6);  AB023489 (OlGC7).

Expression of OlGC8 or medaka fish natriuretic peptide receptors in COS-7 cells

The whole protein-coding region of the OlGC8 cDNA (nucleotides 2151-4865) or the cDNA (nucleotides 3827-4865) corresponding to the catalytic domain of OlGC8 (CAT-8) was amplified by RT-PCR using the medaka kidney RNA. The primers used for the amplification were 5′-TTCATCGTCCGTCCATCAGTCC-3′ (sense) and 5′-CGTCAAACACGTGTGACGCTG-3′ (antisense) for the whole protein, and those for the CAT-8 amplification were 5′-TCGGTGCCGTTCTCATAACCC-3′ (sense) and 5′-CGTCAAACACGTGTGACGCTG-3′ (antisense). A medaka fish natriuretic peptide receptor/membrane GC (e.g., OlGC1, OlGC2 or OlGC7) was also amplified by RT-PCR using the following primers: 5′-AGAATTCAAGACGGTCGAGTGTCCTCTCC-3′ and 5′-CCGCTCGAGCGGGCGACTCAGATAACGTAC-3′ for OlGC1, and 5′-CAGAAGCTTCACCTGCTGGAAGTGGACC3′ and 5′-CATCTAGACCTCCTGTCATCTCATTGGC-3′ for OlGC2, and 5′-GGAATTCTGGCTTATCATCATGGC-3′ and 5′-CCTCTAGAGTCCTTCATCCGTTTGAGTTATCC-3′ for OlGC7. The amplified cDNA was subcloned into the expression vectors pCR®3.1 using the Eukaryotic TA Cloning® Kit (Invitrogen).

COS-7 cells were maintained in Dulbecco's modified Eagle medium (DMEM) plus 10% heat-inactivated fetal bovine serum (FBS) (HyClone®, Logan, UT, USA), 100 U/ml penicillin, 100 U/ml streptomycin, and 292 μg/ml glutamine (GIBCO^TM) under 5% CO_2-95% air at 37°C in a humidified atmosphere. One day before transfection, cells were plated at 4×10⁵ cells per well in a six-well plate (for the intracellular cGMP assay; Fig. 3C), at 2×10⁶ cells per 10-cm dish (for the assay of the GC activity in the membrane fraction; Fig. 3D), or at 8×10⁴ cells per well in a 24-well plate (for the intra-cellular cGMP assay; Fig. 4). Cells were transfected by lipofection with the plasmid DNA (2 μg of DNA/7.5 μl of LIPOFECTAMINE 2000 (LF2000) Reagent per well in a six-well plate; 6 μg of DNA/22.5 μl of LF2000 Reagent per 10-cm dish; 0.8 μg of DNA/2 μl of LF2000 Reagent per well in a 24-well plate) according to the manufacturer's protocol (Life Technologies, Inc.). As a control, pCR3.1 vector was transfected alone. Forty-eight hr after the addition of the respective construct DNA, the intracellular cGMP concentrations and the GC activity in the membrane fractions were determined.

Preparation of membranes and assay of membrane GC activity

For the preparation of the membrane fractions, COS-7 cells were plated in three 10-cm dishes and harvested 48 hr after transfection. Cells expressing OlGC8 were washed twice with 5 ml of ice-cold phosphate-buffered saline (PBS) and then were scraped into a 0.5 ml dish containing an ice-cold solution consisting of the homogenization buffer and 1:1000 dilution of a Protease Inhibitors Mixture and DMSO solution for Mammalian Cell and Tissue Extracts (Wako Pure Chemical Industries, Ltd. Osaka, Japan). Cells were homogenized with a glass homogenizer and centrifuged for 1 hr at 2°C at 100,000×g. The resulting membrane pellet was washed twice with 500 μl of the homogenization buffer. The membranes were resuspended in 200 μl of the homogenization buffer and used for assaying the membrane GC activity or for the Western blot analysis.

Assays of membrane GC activity were performed in a 100 μl reaction mixture consisting of 50 mM NaCl, 0.25 mM IBMX, 0.1% BSA, 5 mM creatine phosphate, creatine phosphokinase (10 U), 1 mM GTP, 1 mM ATP, 5 mM MgCl₂/3 mM MnCl₂, and 25 mM HEPES, pH 7.4, as well as 0.1% Triton X-100, as indicated. The reaction was started by the addition of a membrane fraction to the reaction mixture and continued for 10 min at 37°C and stopped by addition of a 10% solution of trichloroacetic acid (TCA) to give a final concentration of 5%. TCA in the reaction solution was extracted with diethyl ether, and then the cGMP concentration was determined by PROTOCOL 1 of the cGMP enzyme immunoassay (EIA) system (Amersham Pharmacia Biotech., Buckinghamshire, UK) according to the manufacturer's protocol.

Immunological methods

A polyclonal antibody against OlGC8 was created in rabbits using a synthetic peptide, YMTTESICGPWIPDDGKIF, corresponding to the amino acid sequence of a part of the extracellular domain (residues 114–132). The Western blot analysis was carried out essentially by the method of Towbin et al. (1979). The proteins of the membrane fractions prepared from COS-7 cells transfected with the OlGC8 plasmid were separated on a 6% sodium dodecyl sulfate (SDS)-polyacrylamide gel and transferred electrophoretically to an Immobilon^TM PVDF membrane (Millipore, Bedford, MA, USA). After incubation with Block Ace (Dainippon Pharmaceutical Co. Ltd., Osaka, Japan) at 4°C overnight, the membrane was washed with PBST (137 mM NaCl, 2.7 mM KCl, 8.1 mM Na₂HPO₄, 1.5 mM KH₂PO₄, and 0.1% Tween 20) and incubated in a 1:1000 dilution of anti-OlGC8 rabbit anti-serum for 1 hr at room temperature. The membrane was washed three times with PBST and then reacted with a 1:3000 dilution of a horseradish peroxidase-linked anti-rabbit antibody (Amersham Pharmacia Biotech.) in PBST. After washing the membrane three times with PBST, the antibody-reacted protein bands were visualized using an enhanced chemiluminescence (ECL) detection kit (Amersham Pharmacia Biotech.).

Tissue extraction

Brain (402.1 mg), heart (96.7 mg), kidney (257.4 mg), testis (117.1 mg), and ovary (352.6 mg) tissues were obtained from 100 mature adult individuals of the orange-red variety of O. latipes and were homogenized in a five-fold weight/volume homogenization buffer containing 10% glycerol, 100 mM NaCl, 1 mM EDTA, and 50mM HEPES, pH 7.4. The homogenate was then boiled for 10 min and cooled before centrifugation at 10,000 × g for 30 min at 4°C. The resulting supernatant sample (500 μl) was used for further experiments.

Intracellular cyclic GMP formation in intact COS-7 cells

The intracellular cGMP concentrations increased by transiently expressed membrane GC in intact COS-7 cells were determined by the method described by Koller et al. (1991). For the determination of cGMP accumulation in COS-7 cells expressing the appropriate plasmid DNA, the medium was exchanged with DMEM (without additives) containing 0.1 mM 3-isobutyl-1-methylxanthine (IBMX) at 37°C. After 10 min, the medium was exchanged with DMEM containing 0.1 mM IBMX without additives (Fig. 3C, or controls in Fig. 4) or with 1 μM each the following rat natriuretic peptide: ANP (28 amino acids), BNP-32, CNP (32-53), and C-ANF (4-23) (des [Gln¹⁸, Ser¹⁹, Gln²⁰, Leu²¹, Gly²²] ANF (4-23)-NH₂) (Peninsula Laboratories, Inc., San Carlos, CA, USA) or the medium was exchanged with 20 μl medaka fish tissue extract, and incubated for an additional 10 min. The reaction was stopped by replacing the medium with lysis reagent 1 working solution (cGMP EIA system) and the plate was shaken on a microplate shaker for 10 min at room temperature. The cGMP concentration was estimated by PROTOCOL 3 of the cGMP EIA system according to the manufacturer's protocol.

Southern blot hybridization

The Southern blot was carried out with the same membrane as that used in the previous study (Yamagami et al., 2001). The membrane was reprobed with boiled 0.5% SDS solution before prehybridization. A 319-bp OlGC8 genomic DNA fragment (nucleotides 3552–3780) was labeled with [α-³²P] dCTP using the Random Primer DNA Labeling kit Version 2 (Takara Shuzo) and was used as a probe. The membrane was washed twice with 2×SSC containing 0.1% SDS and once with 1×SSC containing 0.1% SDS at 50°C for 15 min.

Isolation and characterization of a genomic DNA clone for OlGC8 from a medaka fish bacterial artificial chromosome (BAC) library

High-density replica membranes of an O. latipes Hd-rR inbred strain genomic BAC library were uesd for the screening (Matsuda et al., 2001). The membrane preparation and the screening method were essentially performed as described previously (Yamagami et al., 2001). To isolate the BAC clone containing the OlGC8 gene, the hybridization was carried out using a probe made by PCR using the O. latipes Hd-rR inbred strain genomic DNA as a template and the following primers: the 5′-C1 primer (5′-CAGGTGGTGGACTTCCTCAAC-3′) and the 3′-5 primer (5′-CAACGCTAGCTGGAGACGT-3′). Genomic DNA was isolated from one individual of O. latipes Hd-rR inbred strain, as described previously (Yamagami et al., 2001).

QIAGEN plasmid midi and maxi kits (QIAGEN, Hilden, Germany) were used for BAC DNA isolation from the bacterial culture. Based on the nucleotide sequence of the OlGC8 cDNA, various specific oligonucleotide primers were designed to amplify the intron regions of positive BAC clones and to effectively carry out sequencing (Table 1). Long and accurate polymerase chain reaction (LAPCR) was performed using a combination of these primers under the following conditions: after an initial denaturing step of 5 min at 94°C, the PCR was carried out with 30 cycles of denaturation for 20 sec at 98°C, followed by annealing and extension for 20 min at 59–68°C (depending on the primers), and a final extension at 72°C for 10 min. Primers positioned in the exon were used to determine the intron size by PCR of the positive BAC clone DNA used as a template. The PCR product was also subcloned into the plasmid vector pBluescript II KS(–) or KS(+) (Stratagene) and sequenced.

Table 1

Primers used in LA-PCR analysis of the OlGC8 gene

Primer extension analysis

In order to identify the transcription start site, a primer extension experiment was carried out as described previously (Yamagami et al., 2001). A ³²P-end-labeled oligonucleotide primer (5′-TGTCAGGAATGTTTGGACGC-3′; 5×10⁴cpm) was hybridized with 5 μg of the medaka fish ovary poly(A)⁺RNA under the following conditions: hybridization took place in a total of 20 μl of 10 mM Tris-HCl (pH 8.3) containing 1 mM EDTA and 0.25 M KCl kept at 65°C for 90 min and then at room temperature for 90 min or in a 10 μl solution containing 2 M NaCl and 50 mM PIPES (pH 6.4) kept at 85°C for 2 min and then at 56°C for overnight.

Northern blot hybridization

Northern blot analysis was performed using poly(A)⁺RNA (7.5 μg) isolated from the adult medaka fish brain, gill, kidney, testis, and ovary, and a nucleotide fragment corresponding to the OlGC8 cDNA (nucleotides 1607–2115 or 2151–2937) was used as a probe according to a previously described procedure (Yamagami et al., 2001). The membrane was washed twice with 2×SSC containing 0.1% SDS at 42°C for 15 min and with 0.1×SSC containing 0.1% SDS for 10 min at 50°C.

RNase protection analysis

An RNase protection experiment was carried out using the total RNA (10 μg) prepared from various adult medaka fish organs and an antisense cRNA probe transcribed from the 5′-noncoding region corresponding to nucleotides 2029-2336 of the OlGC8 cDNA, as described previously (Yamagami et al., 2001). A plasmid containing a cDNA fragment of the 3′-noncoding region (1741-1840) of medaka fish cytoplasmic actin gene OlCA1 (Kusakabe et al., 1999) was used as a template for the synthesis of a cRNA probe.

RNA Ligase-Mediated Rapid Amplification of cDNA Ends (RLM-RACE), and Riverse Transcription-Polymerase Chain Reaction (RT-PCR)

To confirm the sequence of the 3.3 kb-OlGC8 mRNA, the 5′-portion of the cDNA was amplified by the RLM-RACE method (Maruyama and Sugano, 1994) using the First Choice^TM RLM-RACE Kit (Ambion, Austin, TX, USA). This experiment was carried out using the total RNA (5 μg) isolated from the adult medaka fish brain and the 5′ RLM-RACE kit according to the manufacturer's standard protocol. After the reaction of reverse transcription, PCR was performed as follows: denaturation at 94°C for 3 min followed by 30 cycles of denaturation for 30 sec at 96°C, annealing for 30 sec at 60°C, and extension for 2.5 min at 72°C, and the final extension was carried out at 72°C for 10 min. The 5′-RACE Outer Primer and gene-specific antisense oligonucleotide primer 5′-23 (nucleotides 2511–2532) were used for Outer 5′ RLM-RACE PCR. A 1:25 volume of the primary products was reamplified by PCR using the same conditions as those given above; Inner 5′-RACE Inner Primer was used for this experiment, and gene specific primer 5′-63 (nucleotides 2313–2333) was used for Inner 5′ RLM-RACE PCR.

For RT-PCR to detect that no excision of introns had taken place, 5 μg of total RNA from various adult medaka fish organs was used as a template to synthesize the first-strand cDNA using an oligo(dT) primer according to the manufacturer's protocol (Super Script Preamplification System for First Strand cDNA Synthesis). The cDNA fragment containing the catalytic domain of OlGC8 was amplified by PCR from the of medaka fish brain. The primer pair used was as follows: 5′-TCGGTGCCGTTCTCATAACCC-3′ (identical to nucleotides 3828-3848) and 5′-CGTCAAACACGTGTGACGCTG-3′ (complementary to nucleotides 4846–4866).

To determine the sequences of several spliced variants from the testis, we carried out RT-PCR using the testis and kidney first strand cDNA of medaka fish. The primer pair used was as follows: 5′-CCTGTGATCTCCTGCACGCTC-3′ (identical to nucleotides 1288– 1308 in the genome) and 5′-AGCAGTTCTCTCCGTTGGCGTC-3′ (complementary to nucleotides 3849–3870 in the genome). The RACE and RT-PCR products were purified and subcloned into the plasmid vector pBluescript II KS(–) (Stratagene).

Other methods

SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was carried out essentially as described by Laemmli (1970). The protein concentration was determined by the method of Schacterle and Pollack (1973). The nucleotide sequence was determined by the dideoxy chain termination method (Sanger et al., 1977) with an Applied Biosystems 377 sequencer or a 3100 Genetic Analyzer, and analyzed with DNASIS software (Hitachi Software Engineering Co., Yokohama, Japan) and GENETY X-MAC/version 7.2.0. (Software Development Co., Ltd. Tokyo, Japan).

Alignment of the amino acid sequences was carried out using ClustalW available on WWW Service at the European Bioinformatics Institute (URL;  http://www.ebi.ac.uk/clustalw) (Thompson et al., 1994). Transcription-factor binding sequences within the enhancer element were scanned using the TFSEARCH program from GenomeNet ( http://www.rwcp.or.jp/papia/) (Heinemeyer et al., 1998). Genome sequences held on the Ensembl Genome Browser ( http://www.ensembl.org/) and the Fugu database (UK HGMP-RC;  http://fugu.hgmp.mrc.ac.uk) were analyzed using the BLAST or TBLASTN search programs available at the site.

Isolation and characterization of cDNA clone encoding OlGC8

The comparison of the nucleotide and deduced amino acid sequences of a 341 bp-cDNA fragment obtained from the medaka fish ovary by RT-PCR with those of known vertebrate and invertebrate membrane GCs indicated that the cDNA fragment is similar but not identical to those of other known membrane GCs (Fig. 1). We obtained the full-length cDNA clone (referred to as OlGC8) by repeated 5′-RACE (4 times) and 3′-RACE, the products of which overlapped at 46-1150 bp with the end of the clone that had been isolated. The complete OlGC8 cDNA was 4958 bp in length and consisted of a 2196-bp 5′-untranslated region (UTR), a 2577-bp open reading frame (ORF), and a 186-bp 3′-UTR. Termination codons occurred in all three frames upstream of the putative initiation codon (ATG) and the nucleotides around the putative initiation codon fit the preferred sequence context for the initiation of protein synthesis in eukaryotic mRNAs (Kozak, 1983). The ORF of OlGC8 cDNA predicted a protein of 859 amino acids and contains an amino-terminal signal sequence of 21 amino acids (Kyte and Doolittle, 1982; von Heijne, 1983). The mature protein with 838 amino acids was composed of a rather small extracellular domain (residues 1–214), a transmembrane segment (residues 215–233), an intracellular protein kinase-like domain (residues 266–549), and a cyclase catalytic domain (residues 569–796). The comparison of the amino acid sequence of OlGC8 with those of other known membrane GCs indicated a very low degree of similarity in the extracellular domain. There were 14 Cys residues in the extracellular domain of OlGC8, and only one potential N-linked carbohydrate binding site was identified in the domain (Fig. 2). The amino acid sequence identity in the catalytic domain of OlGC8 to that of rat GC-G, rat GC-A, rat GC-B, and medaka fish OlGC1 was 56%, 61%, 61%, and 62%, respectively.

Fig. 1

Molecular phylogenetic analysis of the amino acid sequences of medaka fish and rat membrane GCs. The amino acid sequences of the catalytic domains of GCs were subjected to phylogenetic analysis (see “Materials and Methods”).

Fig. 2

The nucleotide and deduced amino acid sequences of OlGC8 cDNA. The deduced amino acid sequence is indicated by the single-letter code. The signal peptide sequence is indicated by lower-case letters. The amino acids are numbered relative to the predicted signal cleavage site (+1). A potential N-linked carbohydrate binding site is underlined. Open boxes denote cysteine residues in the extracellular domain. A stop codon is indicated by an asterisk. Open boxes (I) ∼ (IV) indicate the extracellular, transmembrane, kinase-like, and cyclase catalytic domains, respectively. Open box (A) indicates the region used for the probe in the RNase protection analysis. The first and second transcription initiation sites are indicated by an open circle. The upstream-pointing arrow inside the 5′-UTR designates the site used to form the primers for the primer extension analysis (see Fig. 7). The downstream-pointing arrows inside the 5′-UTR designate the site which was deleted by splicing in the testis tissue samples (see Fig. 9A). The four arrowheads point out the sites of introns 1, 2, 17, and 18.

Expression of OlGC8 activity

To confirm whether or not the OlGC8 gene encodes an active GC, the whole cDNA or the cDNA fragment for the catalytic domain was expressed in COS-7 cells and the GC activity was measured (Fig. 3A). As shown in Fig. 3B, Western blot analysis using the antibody specific to the extra-cellular domain of OlGC8 demonstrated that a 95 kDa protein was the only protein that specifically reacted with the antibody when the membrane proteins prepared from COS-7 cells transfected with the OlGC8 construct for the whole protein were used. The molecular mass of the former protein corresponded to a molecular weight of 93,780, as calculated based on the amino acid composition of OlGC8. As shown in Fig. 3C, the cGMP concentrations in COS-7 cells transfected with the OlGC8 construct for the whole protein or with a 283 amino acid fragment (the putative catalytic domain) were significantly higher than those with vector (pCR3.1) alone. The membrane fractions prepared from COS-7 cells transfected with the OlGC8 construct for the whole protein exhibited higher GC activity in the presence of Mn²⁺/Triton X-100 than that in the presence of Mn²⁺ alone (Fig. 3D).

Fig. 3

Guanylyl cyclase activity in COS-7 cells overexpressing OlGC8. (A) The schematic diagram of OlGC8 and GC8-CAT. OlGC8 contained an extracellular domain, a transmembrane domain, and intracellularly, a kinase-like domain and a catalytic domain. GC8-CAT encoded only the catalytic domain. (B) Immunoprecipitation of OlGC8 expressed in COS-7 cells. The COS-7 cells transfected with OlGC8 or the expression vector (pCR3.1) were homogenized and immunoprecipitated with the polyclonal antibody specific to the extracellular domain of OlGC8 and the cells were analyzed on a 6% polyacrylamide gel, as described in Materials and Methods. The arrowhead indicates a 95 kDa-protein which was the only protein to specifically react with the antibody. (C) The cGMP concentrations in COS-7 cells transfected with OlGC8, GC8-CAT, and pCR3.1 expression vector in a 6-well plate. Cells were incubated twice for 10 min at 37°C in the presence of 0.1 M IBMX. The transfection and cGMP assay were carried out four times independently, and the data are expressed as means ± S.D. (D) Membranes of COS-7 cells expressing OlGC8 in the 10-cm dish were assayed for GC activity. Membrane fractions were incubated in a reaction mixture in the presence of 5 mM Mg²⁺, 5 mM Mg²⁺ and 0.1% Triton X-100, 5 mM Mn²⁺, or 5 mM Mn²⁺ and 0.1% Triton X-100 for 10 min at 37°C and the reaction was stopped by the addition of TCA (at a final concentration of 5%).

To examine whether or not adult medaka fish tissues contain specific ligands for OlGC8 in addition to the already isolated and characterized putative medaka fish natriuretic peptide receptors (OlGC1, OlGC2, and OlGC7), we tested the effects of various medaka fish tissue extracts on COS-7 cells transfected with the OlGC8 construct as well as the OlGC1, OlGC2 or OlGC7 construct. As shown in Fig. 4A, the cGMP concentrations in COS-7 cells transfected with the OlGC8 construct retained the basal level in the presence as well as in the absence of tissue extract. However, the cGMP concentrations in COS-7 cells transfected with the OlGC1 construct were increased by addition of the brain extracts and those transfected with the OlGC2 construct were increased by heart extract. Both the brain and heart extracts increased the cGMP concentrations in COS-7 cells transfected with the OlGC7 construct. As shown in Fig. 4B, rat BNP and CNP activated OlGC1, which was expressed in COS-7 cells; rat ANP activated both OlGC1 and OlGC2, although rat C-ANF did not stimulate the activity of OlGC1 and OlGC2 expressed in COS-7 cells. Moreover, all of the rat natriuretic peptides (ANP, BNP, CNP, and C-ANF) stimulated the GC activity of OlGC7 expressed in COS-7 cells much more effectively than they stimulated the GC activity of OlGC1 and OlGC2, which were expressed similarly in the COS-7 cells; however, OlGC8 remained at a constant low level of activity with or without tissue extract or rat natriuretic peptides.

Fig. 4

GC activity of OlGC8 and three medaka natriuretic peptide receptors with various medaka fish tissue extracts or rat natriuretic pep-tides. COS-7 cells expressing the appropriate receptor (OlGC1, OlGC2, OlGC7, or OlGC8) or empty expression vector (pCR3.1) in a 24-well plate were treated with the medaka fish tissue extract (A), with 1 μM rat natriuretic peptides (B), or with medium alone, and then the intra-cellular cGMP concentrations were determined. Transfection and cGMP assays were performed four times independently, and the resulting values are expressed as means ± S.D. (A) medaka fish tissue extracts: 1, brain; 2, heart; 3, kidney; 4, testis; 5, ovary; 6, control (medium alone). (B) rat natriuretic peptides: 1, ANP; 2, BNP; 3, CNP; 4, C-ANF; 5, control (medium alone).

The comparison of the deduced amino acid sequence of OlGC8 with those of three putative medaka fish natriuretic peptide receptors, OlGC1, OlGC2 and OlGC7 is shown in Fig. 5. The positions and the number of Cys residues in the extracellular domain differ between OlGC8 and the other three OlGCs. The His-Trp residues, which are considered to be the ligand-binding site and are conserved in the three putative medaka fish natriuretic peptide receptors, were not seen in OlGC8 in the corresponding positions.

Fig. 5

Alignment of the amino acid sequence of OlGC8 with that of three medaka fish natriuretic peptide receptor/membrane GC homologs. The signal peptide sequence is indicated by lowercase letters. The identical amino acid residues among four proteins are indicated by asterisks below the sequence. Gaps in the sequence are indicated by dashes (–). The locations of introns are indicated by vertical bold lines. Open boxes indicate the transmembrane, kinase-like, and cyclase catalytic domains. Open circles indicate the conserved Cys and His-Trp residues. The open circle by the striped line in catalytic domain is represented by E, was reported in the mutagenesis analysis (Wedel et al., 1997).

Genomic Southern analysis

Southern blot hybridization using the medaka fish (HdrR strain) genomic DNA demonstrated that the OlGC8 probe produced only one positive signal in each of the three lanes (Fig. 6). The size of the band in each lane was consistent with that of the DNA fragment obtained from digestion of the genomic DNA by the restriction enzymes, suggesting that a single OlGC8 gene exists in the medaka fish genome.

Fig. 6

Genomic Southern hybridization analysis. Genomic DNA (10 μg) from a single individual of the O. latipes Hd-rR inbred strain was digested with BamHI, EcoRI, or HindIII. The blot was hybridized with a ³²P-labeled OlGC8 cDNA probe.

Characterization of a genomic DNA clone for OlGC8

A genomic BAC library of the O. latipes Hd-rR strain was screened with a genomic DNA fragment of OlGC8 (nucleotides 3644–4866, including exons/introns 14–24) used as a probe. By sequencing a positive BAC clone, we obtained a complete nucleotide sequence of the OlGC8 gene with 14.3 kbp consisting of 24 exons and a promoter region (Fig. 7). The nucleotide sequences of all the splice junctions were in good agreement with the GT/AG rule (Mount, 1982). The exon-intron organization and the exon sizes were completely different from those of mammalian GC-A or medaka fish natriuretic peptide receptor/membrane GCs (Fig. 5). The 5′-UTR of the OlGC8 cDNA was rather big and there was an intron 1 at the end of the 5′-UTR.

Fig. 7

Genomic structure and schematic diagram of cDNA of OlGC8 and identification of the transcription initiation site by primer extension analysis of the OlGC8 gene. The open boxes indicate 5′- and 3′-untranslated regions, and the solid boxes show protein coding regions in the genomic structures. Introns are indicated by lines. Two schematic diagrams of cDNAs are presented under the genomic structure. Exon numbers are shown in the cDNA structure. ECD, extracellular domain; TM, transmembrane domain; KLD, kinase-like domain; CYC, cyclase catalytic domain. The numbers inside the open boxes indicate the nucleotide position in the cDNA. The regions of the probe using Northern blot analysis are shown as two bars under the structure of the cDNA. A lack of excision of introns 17 and 18 is indicated by two striped boxes inside the 3261 bp-cDNA. The box at the lower left indicates the results of the primer extension analysis. A radiolabeled antisense oligonucleotide primer corresponding to the 20 bp (183–202; indicated by an arrow in Fig. 2) for OlGC8 was hybridized with 5 μg of poly(A)⁺RNA isolated from the medaka fish ovary. In order to produce the sequence ladder, a dideoxy DNA sequencing reaction of OlGC8 genomic DNA was performed using the same antisense oligonucleotide primer. The corresponding nucleotide in the sequencing ladder is marked by an asterisk (*). The hybridization was carried out at 65°C for 90 min and then was continued at room temperature (RT) for 90 min, and the other hybridization was performed at 85°C for 2 min and then was maintained at 56°C overnight.

Identification of the 5′-end nucleotide of the OlGC8 tran-scripts by primer extension analysis

As shown in Fig. 7 (box at the lower left), a primer extension analysis demonstrated that the 5′-end nucleotide was at the nucleotide position 2196 bp (G) upstream of the putative initiation codon, as shown by using poly(A)⁺ RNA from the ovary; this finding indicated that the complete OlGC8 cDNA is 4958 bp in length and possesses a 2196-bp 5′-untranslated region (UTR). There was no typical TATA box in the 5′-flanking region upstream of the first putative transcription initiation site of the OlGC8 gene.

Tissue-specific expression of different sizes of the OlGC8 transcripts

As shown in Fig. 8A, Northern blot hybridization using poly(A)⁺ RNA from the adult medaka fish tissues and using a cDNA fragment corresponding to the 5′-UTR as a probe (Fig. 7, probe A) demonstrated the existence of a 5 kb-OlGC8 transcript in the kidney and testis. However, when a cDNA fragment corresponding to exon 2 to exon 8 encoding the extracellular domain of OlGC8 (Fig. 7, probe B) was used as a probe for hybridization using the same membrane, another signal at a position corresponding to 3.3 kb was detected with the brain poly(A)⁺RNA, whereas a signal at 5 kb was also detected with poly(A)⁺RNA from the kidney and testis (Fig. 8B). To clarify how the 3.3 kb-mRNA was produced in the brain, an RNase protection assay was carried out using a probe complementary to the 306 bases of the 5 kb-mRNA (Fig. 9A, striped arrow). As shown in Fig. 9B (arrowhead A), a strong signal at 306 bp due to the 5 kb-OlGC8 transcript was detected with poly(A)⁺RNA from the kidney and testis, and a weak signal at 306 bp due to the 5 kb-OlGC8 transcript was detected with that from the ovary and brain. Another band migrated between 154 and 201 bp, which was thought to be the result of the protection of the last 168 bp of the probe (Fig. 9A, arrow C; Fig. 9B, arrowhead C); this latter band was detected only in the case of the brain poly(A)⁺RNA. On the other hand, several different bands with weak intensity were detected in the poly(A)⁺RNA of the kidney and testis; these bands were located somewhat under the 220 bp marker, a finding which was attributed to the protection of the last 210 bp of the probe (Fig. 9A, arrow B; Fig. 9B, arrowhead B). Moreover, three weak bands migrated between 75 bp and 134 bp, and were detected in the testis samples (Figs. 9A and 9B, arrow d, e, f).

Fig. 8

Northern blot analysis of the OlGC8 transcripts in various adult medaka fish organs. (A) and (B) Northern blot analysis was carried out using poly(A)⁺RNA (7.5 μg) from the brain, gill, kidney, testis, and ovary. The radioactive bands are indicated by arrowheads. The positions and sizes of the RNA markers are shown on the left. The same blot was hybridized with a ³²P-labeled cDNA probe for a portion of the 5′-UTR (A) or the extracellular domain (B) of OlGC8 (see Fig. 7).

Fig. 9

Schematic diagram of OlGC8 cDNA expressed in kidney, brain, or testis, and the position of the probe used for RNase protection analysis and depiction of the regions of several protected bands, and RNase protection analysis of OlGC8 transcripts. (A) The upper schematic diagram denotes cDNA expressed in the kidney and testis. The second schematic diagram indicates cDNA expressed in the brain. The lower four diagrams denote spliced variants of cDNA expressed in the testis. The grey bar indicate probe using RNase protection analysis, and the protected bands are shown as black bars (A, B, C, d, e, f) pointed at by black or white arrows. The black arrows indicate that the sequence of the protected band represented by the bar was confirmed by sequencing, and the white arrows indicate that the sequence of the protected band indicated by the bar remains unknown. (B) RNase protection analysis of the OlGC8 transcripts in various adult medaka fish organs. Total RNA (10 μg) obtained from various medaka fish organs was hybridized with an antisense cRNA probe (172 nucleotides) for OlGC8. An anti-sense cRNA probe for OlCA1 (100 nucleotides) was used as an internal control. After digestion with RNase, the protected fragments were separated by electrophoresis on a 6% polyacrylamide/7 M urea gel and autoradiographed.

A variety of sizes of OlGC8 transcripts were detected in the brain samples, and were also detected in the samples from the kidney and testis; the presence of these different transcripts in the kidney and testis were confirmed by RTPCR (Fig. 8). A band protected at about 210 bp was due to an mRNA which was thought to be spliced starting at 1010 bp (Fig. 9A, broken line and arrow in 5′-UTR) to 2126 bp of the cDNA nucleotides (Fig. 9A, arrow B). The nucleotide position at 2126 bp was located at the first intron, and the nucleotide position at 2227 bp was located at the second intron. As seen in the RT-PCR products, we detected several different sizes of OlGC8 transcripts in the testis, most likely due to a deletion from somewhere between the nucleotide position at 1010 bp to the first or second intron site (Fig. 9A, arrow B, d), or else due to deletion of a nucleotide near intron 2 (Fig. 9A, arrow e, f). This finding was in good agreement with the RNase protection analysis, which revealed that a fragment of approximately 80-90 bp cDNA was protected between 75 bp to 134 bp (Fig. 9B, asterisk*) when the testis sample was examined.

Identification of the 5′-end nucleotide of the 3.3 kbmRNA by RNA Ligase-Mediated Rapid Amplification of cDNA Ends (RLM-RACE) and Riverse Transcription-Polymerase Chain Reaction (RT-PCR)

Since Northern blot and RNase protection analyses indicated that a 3.3 kb-mRNA is a major OlGC8 transcript in the adult medaka fish brain, it became important to assign the 5′-end nucleotide of the 3.3 kb-mRNA in order to clarify how this mRNA was formed. We employed the RLM-RACE method for the assignment of the 5′-end nucleotide. The nucleotide was detected at the nucleotide position at 2168 bp (A) downstream of the first transcription site using the adult medaka fish brain total RNA; this site was referred to as the second putative transcription initiation site. The size of an mRNA transcribed from the second putative transcription initiation site was estimated to be 2790 b. To determine whether or not the second transcription initiation site was for the 3.3 kb-mRNA, we amplified a partial OlGC8 mRNA expressed in the medaka fish brain by RT-PCR using OlGC8 specific primers, and we obtained two cDNA fragments of different sizes that encoded the catalytic domain. Subsequent sequencing of these RT-PCR products revealed that the shorter cDNA fragment (1039 bp) corresponded to the normal OlGC8 cDNA and the longer cDNA fragment (1510 bp) corresponded to a OlGC8 cDNA with intron 17 (178 bp) and intron 18 (293 bp) (Fig. 7, striped boxes in the schematic cDNA structure depicted at the bottom).