Isolation of the ibis PGHα gene

When the last male Japanese crested ibis of the Japanese origin (named Midori) died in 1995, most of his organs were preserved in liquid nitrogen (Ishii, 1999). Genomic DNA was extracted from approximately 15 mg of the preserved kidney using a GenomicPrep Cells and Tissue DNA Isolation Kit (Amersham Biosciences, NJ). The extracted DNA was digested with EcoRI and separated in aga-rose gel electrophoresis. DNA fragments of 4–10 kb long were recovered from the gel and ligated with the Lambda ZAPII vector (Stratagene, CA). An original library of 2.5×10⁵ clones was generated and amplified once to 1.3×10¹¹ clones. Approximately 2.5×10⁵ clones in the amplified library were screened by plaque hybridization. To prepare a hybridization probe, the PGHα cDNA fragment of Japanese quail (Ando and Ishii, 1994) was randomly labeled with [α-³²P]dCTP (Amersham Biosciences, UK) and used as a probe. Prehybridization was performed at 42°C for more than 2 hr with denatured salmon sperm DNA (0.2 mg/ml) in a hybridization buffer containing 6×SSC (1×SSC: 150 mM NaCl, 15 mM sodium citrate, pH7.0), Denhardt's solution (0.02% Ficoll, 0.02% bovine serum albumin, and 0.02% polyvinylpyrrolidone), and 0.1% SDS. Hybridization was carried out at 55°C overnight in the hybridization buffer containing the labeled probe. Membranes were washed once with 1×SSC containing 0.1% SDS at 60°C for 20 min and hybridization signals on the membranes were analyzed with a BAS-2000II Bio-Imaging Analyzer (Fuji Photo Film, Japan). After third round of screening, ten positive clones were isolated and the insert fragments were subcloned into the pBluescript phagemid vector. After identification of these clones by restriction enzyme digestion, a clone (pIA1) was selected for sequence analysis. Nucleotide sequence was determined with a Thermo Sequenase Cycle Sequencing Kit (USB Corporation, OH) with a DNA sequencer Model 4000L (LI-COR, NE).

Isolation of the ibis FSHβ gene

In order to obtain a nucleotide sequence of a part of the FSHβ gene, PCR was carried out with genomic DNA as a template. Primers used were as follows: FSH-F, 5′-TG(T/C)TCIGGITA(C/T)TG(C/T)T(A/T)(A/C)ACIA(A/G)(A/G)G-3′ and FSH-R, 5′-CA(A/G)TCIGT(A/G)CT(A/G)TCI(C/G)T(A/G)TC(A/G)CAI(G/T)TI(C/T)C(A/G)CA(A/G)TG(A/G)C-3′, for the sense and antisense, respectively. They were originally used for amplifying a part of the FSHβ cDNA in Japanese quail (Kikuchi et al., 1998). The PCR was performed with 30 cycles of 1 min at 95°C, 1 min at 50°C, 3 min at 72°C using a Premix Ex Taq (Takara Shuzo, Japan). The PCR products were subjected to agarose gel electrophoresis and then cloned into pCR2.1 plasmid vector (Invitrogen, CA). After sequencing, a clone (pIFPCR) was selected for further study.

Another genomic library was constructed in the Lambda EMBL3 phage vector (Stratagene, CA) with genomic DNA partially digested with BamHI. An original library of 5.0×10⁴ clones was amplified once to 8.0×10⁹ clones, and 1.0×10⁵ clones were screened by plaque hybridization. The pIFPCR insert was randomly labeled with [α−³²P]dCTP and used as a probe. Prehybridization was performed as described above, and hybridization was carried out at 60°C overnight. Membranes were washed once with 1×SSC containing 0.1% SDS at 60°C for 20 min and once with 0.1×SSC containing 0.1% SDS at 60°C for 20 min. After third round of screening, one positive clone (IF1) was isolated. The insert fragment was digested with EcoRI and subcloned into pBluescriptII phagemid vector (Stratagene, CA) for sequencing.

Because IF1 was found to cover only a 5′ part of the FSHβ gene, screening was repeated to obtain the remaining 3′ part. The pIFPCR insert was digested with BamHI and then a fragment of approximately 180 bp was recovered to exclude a part overlapping with IF1. This fragment was randomly labeled with [α-³²P]dCTP and used as a probe. Since the genomic library did not give any positive signal, we constructed other genomic library with the Lambda EMBL3 vector and BamHI-digested genomic DNA. The resultant library was 1.6×10⁵ clones and was screened without amplification. Hybridization was carried out at 50°C overnight, and membranes were washed once with 3×SSC containing 0.1% SDS at 50°C for 20 min. Consequently, three positive clones were isolated. Based on identification by restriction enzyme digestion, a clone (IF4) was selected and a region of this insert was subcloned into the pBluescriptII phagemid vector. Nucleotide sequence was determined with a BigDye Terminator v3.0 Ready Reaction Cycle Sequencing Kit and an ABI PRISM 377 DNA Sequencing System (Applied Biosystems, CA).

Analysis of the second intron in the ibis FSHβ gene in two individuals

Nucleotide sequences in the second intron of the FSHβ gene were determined in two individuals. One was Midori of Japanese origin, and the other was Long-Long which had been lent from China to Japan and died in Japan in 1994. The genomic DNA of Long-Long was extracted from the cryo-preserved kidney (Ishii, 1999). Genomic DNA extraction, PCR using the primers FSH-F and FSH-R, cloning and sequencing were performed as described above. After sequencing, the PCR products were digested with EcoRI and subjected to electrophoresis.

Isolation and nucleotide sequence of the ibis PGHα gene

Ten positive clones were isolated from the genomic library and nucleotide sequence of a clone (pIA1) was determined (Fig. 1). The determined sequence of 5026 bp was compared with PGHα cDNA sequences of turkey (Foster and Foster, 1991), chicken (Foster et al., 1992), Japanese quail (Ando and Ishii, 1994) and two species of duck (Hsieh et al., 2001). Comparison results showed remarkable sequence similarities which enabled us to infer the exonintron structure (Fig. 2). We could recognize three putative exons containing the whole coding region and 3′ untranslated region within this sequence, but we were unable to find a sequence corresponding to most of 5′ untranslated region. Exon-intron junctions were determined according to the GTAG rule (Breathnach and Chambon, 1981). Then, amino acid sequence was deduced and aligned with those of PGHα of turkey (Foster and Foster, 1991), chicken (Foster et al., 1992), Japanese quail (Ando and Ishii, 1994), ostrich (Koide et al., 1996), and two species of duck (Hsieh et al., 2001) (Fig. 3). Three amino acid residues in the putative ibis PGHα precursor were different from those in each of the other birds over a signal peptide of 24 residues, while 96 residues in a mature protein were completely identical to those of the other birds except for ostrich (ostrich mature protein differed in three amino acid residues from those of the other birds). Ten cysteine residues forming disulfide bonds and two putative N-linked glycosylation sites were conserved among all these birds. These findings assured that the acquired nucleotide sequence encoded the ibis PGHα precursor molecule.

Fig. 1

Structure and sequencing strategy of a part of the gene for the PGHα in the Japanese crested ibis. Solid and open boxes indicate the coding and the untranslated region of exons, respectively. Lines represent introns and 3′ flanking region. Arrows indicate the direction and extent of sequencing. Restriction sites for EcoRI are indicated with E.

Fig. 2

Nucleotide sequence of the part of the PGHα gene in the Japanese crested ibis. Exons are shown in capital letters, and introns and flanking regions are shown in lowercase letters. The polyadenylation signal, AATAAA, is underlined. Deduced amino acid residues are represented with three letter codes below the nucleotide sequence and numbered sequentially from the N-terminus. The nucleotide sequence data in this figure is available in the EMBL/GenBank/DDBJ Data Bank with Accession No.  AB089503.

Fig. 3

Alignment of amino acid sequences of signal peptides (A) and mature proteins (B) of the PGHα precursor molecules of the Japanese crested ibis, duck (Hsieh et al., 2001), chicken (Foster et al., 1992), Japanese quail (Ando and Ishii, 1994), turkey (Foster and Foster, 1991) and ostrich (Koide et al., 1996). Residues identical to those in the Japanese crested ibis are indicated with dots. Ten conserved cysteine residues and two putative N-linked glycosylation sites are denoted by • and ▾, respectively.

All the PGHα genes previously reported in mammals and teleosts consist of four exons and three introns (Fiddes and Goodman, 1981; Goodwin et al., 1983; Burnside et al., 1988; Gordon et al., 1988; Kato et al., 1990; Golos et al., 1991; Huang et al., 1992; Suzuki et al., 1995). The exonintron structure in the ibis PGHα gene was compared with that of carp (Huang et al., 1992), mouse (Gordon et al., 1988), bovine (Goodwin et al., 1983) and human (Fiddes and Goodman, 1981) (Fig. 4). The result indicated that the clone we obtained lacked a 5′ part corresponding to the first exon of other vertebrates. By aligning amino acid sequences, locations of the introns in the coding region were compared. The location of the first intron in the part of the ibis PGHα gene was identical with that of the corresponding intron (the second intron) in mammals, but different from that in teleosts with an amino acid residue (Fig. 5), while the location of the second intron (the third in mammals and teleosts) was conserved through the ibis, mammals and teleosts.

Fig. 4

Comparison of the exon-intron structure of the part of the PGHα gene in the Japanese crested ibis with those of PGHα gene of carp (Huang et al., 1992), mouse (Gordon et al., 1988), bovine (Goodwin et al., 1983) and human (Fiddes and Goodman, 1981). Solid and open boxes represent the coding and the untranslated regions of exons, respectively. Solid lines indicate introns and flanking regions. Thin broken lines between boxes link corresponding exons. A thick broken line in the Japanese crested ibis represents part whose nucleotide sequence is not available at present.

Fig. 5

Alignment of a part of amino acid sequences of PGHα for comparison of location of the second intron. Locations of the intron are shaded. Numbers at the top represent positions of amino acid residues in the PGHα in the Japanese crested ibis. References are as follows; carp (Huang et al., 1992), salmon (Suzuki et al., 1995), rat (Burnside et al., 1988), mouse (Gordon et al., 1988), bovine (Goodwin et al., 1983), porcine (Kato et al., 1990), rhesus (Golos et al., 1991), and human (Fiddes and Goodman, 1981).

Isolation and nucleotide sequence of the ibis FSHβ gene

Strategy to obtain the ibis FSHβ gene is illustrated in Fig. 6. The PCR gave a single band of approximately 800 bp and the determined 780 bp sequence was compared with FSHβ cDNA sequences of Japanese quail (Kikuchi et al., 1998) and chicken (Shen and Yu, 2002). This sequence was found to contain an intron of 582 bp flanked by partial exons of 23 and 175 bp of the putative FSHβ gene. A clone (IF1), which was isolated from the genomic library, contained an insert of approximately 10 kb, of which the nucleotide sequence of 4059 bp around the 3′ part was determined. Comparison of the sequence with the FSHβ cDNA sequences of the two bird species revealed that IF1 contained a 5′ part of the FSHβ gene, but lacked its 3′ part. Then, three clones were isolated by repeated screening of the other genomic library. A partial nucleotide sequence of a clone (IF4), 3580 bp in size, was compared with the FSHβ cDNA sequences of the two bird species. IF4 was shown to contain a 3′ part of the FSHβ gene which was missing in IF1.

Fig. 6

Structure and sequencing strategy of the FSHβ gene in the Japanese crested ibis. Insert fragments of pIFPCR, IF1 and IF4 are shown with arrows which indicate the direction and extent of sequencing. At the bottom, the structure of ibis FSHβ gene is shown with exons indicated as boxes. Solid and open boxes represent coding and untranslated regions, respectively. Restriction sites for BamHI are indicated with B.

By collecting the results from the two phage clones (IF1 and IF4) and the PCR fragment (pIFPCR), a sequence of 7633 bp was acquired (Fig. 7). Exon-intron structure was inferred by sequence similarities with the FSHβ cDNA in Japanese quail and chicken and exon-intron junctions were determined based on the GT-AG rule (Breathnach and Chambon, 1981). Particularly, we inferred the putative transcriptional start site and the polyadenylation site referring to the 5′ and 3′ end of the chicken FSHβ cDNA determined by rapid amplification of cDNA end (RACE) method. Finally, the assembled nucleotide sequence was found to contain 2820 bp of the 5′ flanking region followed by three exons of 34, 160 and 2549 bp, separated by two introns of 458 and 582 bp. An amino acid sequence was deduced and aligned with those of Japanese quail (Kikuchi et al., 1998), chicken (Shen and Yu, 2002) and ostrich (Koide et al., 1996) (Fig. 8). The ibis amino acid sequence showed remarkable similarities to those of FSHβ of Japanese quail (94.7%) and chicken (95.4%). High similarity (93.4%) was also observed with the ostrich FSHβ except for a deletion of five residues in the C-terminus. Twelve cysteine residues and two putative N-linked glycosylation sites were conserved through these four bird species. These results enabled us to conclude that the nucleotide sequence we obtained encoded the FSHβ gene of the Japanese crested ibis.

Fig. 7

Nucleotide sequence of the FSHβ gene and its flanking region in the Japanese crested ibis. Exons are shown in capital letters, and introns and flanking regions are shown in lowercase letters. The two potential TATAAA sequences and the five potential polyadenylation signals, AATAAA, are underlined. The bent arrow indicates the putative transcriptional start site deduced from 5′ end of chicken FSHβ cDNA (Shen and Yu, 2002). Eight ATTTA motifs in the putative 3′ flanking region are boxed. Deduced amino acid residues are represented with three letter codes below the nucleotide sequence and numbered sequentially from the N-terminus. The nucleotide sequence data in this figure is available in the EMBL/GenBank/DDBJ Data Bank with Accession No.  AB089502.

Fig. 8

Alignment of the amino acid sequences of signal peptides (A) and mature proteins (B) of FSHβ precursor molecules of the Japanese crested ibis, chicken (Shen and Yu, 2002), Japanese quail (Kikuchi et al., 1998), and ostrich (Koide et al., 1996). Residues identical to those in the Japanese crested ibis are indicated with dots. Twelve conserved cysteines and two putative N-linked glycosylation sites are denoted by • and ▾, respectively.

The ibis FSHβ gene showed a structure with three exons and two introns. The similar exon-intron structure has been previously reported in mammals and teleosts (Jameson et al., 1988; Kim et al., 1988; Gharib et al., 1989; Hirai et al., 1990; Guzman et al., 1991; Kumar et al., 1995; Sohn et al., 1998; Rosenfeld et al., 2001). Locations of the first intron in the 5′ untranslated region and the second intron between amino acid residue 33 and 34 of mature protein in the ibis FSHβ gene were also similar to locations reported for mammalian and teleostean counterparts. Comparison of the nucleotide sequence in the 5′ flanking region between the ibis and non-avian species (teleosts and mammals) revealed that the ibis sequence could be well aligned with the sequences in mammals within the promoter region (Fig. 9).

Fig. 9

Comparison of nucleotide sequences of the promoter region of FSHβ genes of the Japanese crested ibis, ovine (Guzman et al., 1991), bovine (Kim et al., 1988), porcine (Hirai et al., 1990), rat (Gharib et al., 1989) and human (Jameson et al., 1988). The nucleotide sequence is numbered from the putative transcriptional start site in the ibis FSHβ gene. A progesterone responsive element (PRE)-like sequence (Webster et al., 1995) and two AP-1 responsive element-like sequences (Huang et al., 2001) reported in ovine FSHβ gene are boxed with solid line with consensus sequence attached in parenthesis. Parts in which TATAAA sequences are observed in the ibis are shaded.

Analysis of the second intron of the ibis FSHβ gene in two individuals

Results of sequencing the PCR fragments of Midori and Long-Long showed that two types of sequences were present in the second intron of the FSHβ gene (Fig. 10). The difference between the two types was the 32 bp sequence in the 5′ portion. Ten and five clones were sequenced for Midori and Long-Long, respectively. Only the shorter type was observed in Midori, whereas both the shorter (in two clones) and longer (in three clones) types were found in Long-Long. In addition to the length difference, three nucleotide substitutions were observed. A substitution, which was located in the most 3′ position, changed the sequence “GAATTC” (the recognition site of EcoRI) in the shorter type into “CAATTC” in the longer type. Utilizing this sequence difference, we could differentiate the two types by EcoRI digestion of the PCR products. Namely, the PCR product containing only the shorter type (approximately 780 bp) was divided into two fragments of 550 and 230 bp, while the PCR product of the longer type (approximately 810 bp), was not cleaved. The digestion results showed that only two fragments of 550 and 230 bp were observed for Midori and three fragment of 810, 550 and 230 bp for Long-Long, indicating that Midori had only the shorter type and Long-Long had both types.

Fig. 10

Nucleotide sequences of the second intron of the FSHβ gene in the Japanese crested ibis, (L) the longer type and (S) the shorter type. Dashes indicate a gap inserted to maximize identity. Three nucleotide mismatches are marked with a reverse triangle. Part recognized by EcoRI in the short type is boxed.