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Ad4BP gene in the frog, Rana rugosa
Gene 222 (1998) 169–176
Molecular cloning and expression of the SF-1/Ad4BP gene in the frog, Rana rugosa Ki-ichirou Kawano a, Ikuo Miura a, Ken-ichirou Morohashi b, Minoru Takase a, Masahisa Nakamura a,* a Laboratory for Amphibian Biology, Faculty of Science, Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima, Hiroshima 739-8526, Japan b National Institute for Basic Biology, Myodaiji-cho, Okazaki, Aichi 444-8585, Japan Received 14 May 1998; received in revised form 10 September 1998; accepted 12 September 1998; Received by A. Nakazawa
Abstract SF-1/Ad4BP is a transcriptional factor that was originally found to be a mammalian homologue of the Drosophila Ftz-F1 ( fushi tarazu factor 1) (Morohashi et al., 1992), and transcribed from a gene designated the Ftz-F1 gene (Nomura et al., 1995). Ftz-F1 gene-deficient mice lack adrenal glands and gonads. Besides mammals, however, the SF-1/Ad4BP cDNA has only been isolated to date in fish and birds. To understand its role(s) for adrenal and gonadal development in vertebrates, cloning of this gene in animals other than mammals is required. In this study, we succeeded to isolate frog (Rana rugosa) SF-1/Ad4BP cDNA from a testis lgt10 cDNA library. It encoded a protein of 468 amino acids, and its open reading frame (ORF ) shared 70% similarity with that of chicken OR2.1 (a SF-1/Ad4BP homologue) and 62% with bovine SF-1/Ad4BP. SF-1/Ad4BP mRNA was expressed in the testes, brains, adrenals/kidneys and spleens, but not ovaries, of adult frogs. In addition, we also cloned the 5∞-untranslated region (4.6 kb) of the SF-1/Ad4BP gene with exons I and II. Genomic structure analysis revealed that frog SF-1/Ad4BP was also transcribed from the same gene as that of mammals. However, many Ftz-F1-related proteins have been reported so far. The Ftz-F1 gene does not encode all of those Ftz-F1-related proteins. Thus, the name of Ftz-F1 is not adequate for the gene coding SF-1/Ad4BP. Here, we propose the use of SF-1/Ad4BP instead of Ftz-F1 for the gene that encodes SF-1/Ad4BP in vertebrates. © 1998 Elsevier Science B.V. All rights reserved. Keywords: cDNA; 5∞-UTR; Frog; SF-1/Ad4BP; SF-1/Ad4BP gene; Testis
1. Introduction Many transcriptional factors in the orphan nuclear receptor superfamily have recently been cloned. FTZ-F1 was originally identified in Drosophila as a transcriptional regulator of the fushi tarazu homeobox gene (Lavorgna et al., 1991). The expression of this gene is required for blastoderm and nervous-system develop* Corresponding author: Tel/Fax: +81 824 24 7483. e-mail: [email protected] Abbreviations: bp, base pair(s); DNase, deoxyribonuclease; dNTP, deoxyribonucleotide triphospate; DTT, dithiothreitol; ELP, embryonal long-terminal repeat-binding protein; 5∞-UTR, 5∞-untranslated region; FTZ-F1, fushi tarazu factor 1; kb, kilobase(s) or 1000 bp; ORF, open reading frame; RACE, rapid amplification of cDNA ends; RNase, ribonuclease; RT-PCR, reverse transcriptase-polymerase chain reaction; SDS, sodium dodecyl sulfate; SF-1, steroidogenic factor-1; SSC, 0.15 M NaCl/0.015 M Na · citrate pH 7.6. 3
ment in Drosophila. Recently, many FTZ-F1 homologues such as bovine SF-1/Ad4BP (Honda et al., 1993), mouse LRH-1 and Xenopus XFF1r (EllingerZiegelbauer et al., 1994) were reported. SF-1/Ad4BP promotes cell-specific expression of P-450scc genes in the adrenal glands (Honda et al., 1990; Lala et al., 1992; Morohashi et al., 1993; Ikeda et al., 1993; Takayama et al., 1994; Monte et al., 1998), and its mRNA is expressed in the gonads (Morohashi et al., 1992; Ikeda et al., 1994; Morohashi and Omura, 1996). Interestingly, the gene encoding SF-1/Ad4BP encodes another nuclear factor ELP (embryonal long-terminal repeat-binding protein; Tsukiyama et al., 1989). These two factors are transcribed by alternative promoter usage and splicing (Nomura et al., 1995). In Ftz-F1 gene-disrupted mice, the adrenal glands and gonads are completely lost (Luo et al., 1994; Sadovsky et al., 1995). Gonadotrope-specific markers like LH-b subunit and GnRH receptors also disappear (Ingraham et al., 1994). Thus, SF-1/Ad4BP
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is essential for adrenal and gonadal development and sexual differentiation [for reviews, see Manglesdorf et al. (1995), Nordqvist (1995) and Morohashi (1997)]. Sex steroid hormones play a pivotal role in sexual development in amphibians (Chang and Witschi, 1956; Nishioka et al., 1993), reptiles (Pieau, 1974; Jeyasuria et al., 1994; Crews, 1996) and birds ( Elbrecht and Smith, 1992; Andrews et al., 1997). The sex can be reversed by sex steroid hormones in frogs (Nishioka et al., 1993). A cloning of the cDNA encoding SF-1/Ad4BP in amphibians will, therefore, extend our understanding of (1) whether a common gene controls gonadal development in vertebrates, and (2) whether this gene has been conserved through evolution. Still, other than mammals, SF-1/Ad4BP has only been cloned in fish and birds. Here, we report molecular clonings of the frog SF1/Ad4BP cDNA and the 5∞-untranslated region (5∞-UTR) with the first and second exons. Tissues expressing SF-1/Ad4BP mRNA are also reported.
2. Materials and methods 2.1. cDNA cloning and sequence analysis A testis cDNA library in lgt10 vector was constructed from poly(A)+ RNA of 2-year-old frogs (Rana rugosa) ( Yamamoto et al., 1996). Bovine SF-1/Ad4BP cDNA (1965 bp) was labelled with [a-32P] dCTP (Amersham, Cleveland, OH ) using a random primer DNA labelling kit (Amersham) and used to screen the cDNA library by plaque hybridization. Hybridization was carried out at 62°C in 0.5% SDS, 6× SSC, 5× Denhardt’s solution and denatured salmon sperm DNA (100 mg/ml ) as previously described ( Yamamoto et al., 1996). Membranes were washed in 0.2× SSC and 0.1% SDS at 62°C, and autoradiographed. Positive cDNAs were subcloned into the pUC19 vector. The sequence analysis was performed using an ABI 373A automated DNA sequencer following the manufacturer’s instructions (Perkin Elmer, Palo Alto, CA). 2.2. Determination of the transcription initiation site of the Ad4BP gene by 5∞-RACE To determine the transcription initiation site of the Ad4BP gene, 5∞-RACE (rapid amplification of cDNA ends) was employed. First-strand cDNA was synthesized from DNase-treated total RNA (2 mg) of Rana rugosa testes using a 5∞-RACE kit (Boehringer Mannheim, Germany), and purified using a High Pure PCR product Purification kit (Boehringer Mannheim). A poly(A) tail was then added to the 3∞ end of the cDNA by incubation at 37°C with 0.2 mM dATP and 10 u of terminal transferase. Tailed cDNA was amplified by PCR using a primer (5∞-AGGAGCCCATAGTGATAACCAG-3∞ cor-
responding to nucleotide positions 301-322 of the frog SF-1/Ad4BP cDNA) with an oligo dT-anchor primer following the manufacturer’s instructions (Boehringer Mannheim). The obtained cDNA was further amplified by a second PCR using primers (5∞CCGCTTCCTATTTACACCTCACC-3∞ and 5∞-TGCAGTCCCCGTCCACCTTAGATA-3∞ corresponding to nucleotide positions 60-82 and 163-186, respectively) and the PCR anchor primer following the manufacturer’s instructions (Boehringer Mannheim). The PCR reaction consisted of 2 min at 94°C, followed by 40 cycles of 94°C (40 s), 63°C (1 min), and 72°C (2 min), ending with 15 min of extension at 72°C. The PCR product was ligated into the pCR2.1 vector for sequence analysis following the manufacturer’s instructions (Invitrogen, Carlsbad, CA). 2.3. Northern blot analysis Extraction of total RNA from various tissues of frogs was performed according to Chomczynski and Sacchi (1987). Fifteen micrograms of total RNA were electrophoresed on 1.2% denaturing formaldehyde agaroseMOPS gel and transferred on to nylon membranes ( Hybond N, Amersham). RNA was then hybridized with [a-32P] dCTP-labelled frog SF-1/Ad4BP cDNA. An image analyzer (FUJIX BAS-2000) was used to detect positive bands, since they were hardly detectable by exposure to X-ray film, even after several days. 2.4. Southern blot analysis of RT-PCR products The total RNA from different tissues of adult frogs was treated with 6 u of RNase-free DNase I (Promega, Madison, WI ) for 1 h at 37°C to avoid genomic DNA contamination, and then used as the initial templates for RT-PCR. cDNA was synthesized by incubating 2 mg of RNA in 20 ml of the first-strand buffer (Gibco-BRL, Detroit, MI ), which was supplemented with 200 u of Superscript II (Gibco-BRL), 0.5 mM dNTPs ( Takara, Kusatsu, Japan), 10 mM DTT (Gibco-BRL), and 0.4 mM oligo-dT primers at 42°C for 50 min. Samples 25 were then incubated at 70°C for 10 min to inactivate the reverse transcriptase. One-tenth of the first-strand mixture was added to a 50-ml PCR buffer containing 200 mM dNTPs, 0.2 mM, PCR primers (the forward primer; 5∞-TGTCAGCCATTGCAGCCCAG-3∞ corresponding to nucleotide positions 1258–1277 of the frog SF-1/Ad4BP cDNA and the backward primer; 5∞-CAAGTCAGTTTAGGCTCGCT-3∞ corresponding to nucleotide positions 1636–1655), and 2.5 u of Ex Taq polymerase ( Takara). The PCR reaction consisted of 3 min at 94°C, followed by 30 cycles of 94°C (40 s), 65°C (2 min), and 72°C (3 min), ending with 7 min of extension at 72°C. With this procedure, a 398-bp fragment of the SF-1/Ad4BP cDNA was amplified. The
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PCR products (0.4 kb) were then electrophoresed on 2% agarose gel and transferred to nylon membranes (GeneScreen@; NEN Research Products, Boston, MA). The DNA was hybridized with DIG-labelled frog SF-1/Ad4BP cDNA (2.1 kb) as a probe. The DIGDNA labelling kit and DIG luminescent detection kit (Boehringer Mannheim) were used for this analysis, following the manufacturer’s instructions. 2.5. Cloning of the 5∞-UTR of the SF-1/AD4BP gene and sequence analysis After genomic DNAs from frog erythrocytes were cleaved with EcoRI, they were run electrophoretically on 0.8% agarose gel and transferred to nylon membranes. The DNA was then hybridized with frog SF-1/Ad4BP cDNA labelled with [a-32P] dCTP, as explained earlier, and exposed to X-ray film ( Fuji RX ) overnight at −80°C. DNA fragments corresponding to positive bands were excised from agarose gels and extracted using a GENECLEAN kit (BIO 101, Inc., Vista, CA), following the manufacturer’s instructions. The DNA fragments were then ligated into the lgt10 vector for screening by plaque hybridization. Hybridization was carried out as described elsewhere ( Yamamoto et al., 1996). DNA clones were sequenced on both DNA strands using the ABI 373 A automated DNA sequencer following the manufacturer’s instructions (Perkin Elmer).
3. Results and discussion 3.1. Nucleotide and deduced amino acid sequence of SF-1/Ad4BP cDNA By screening 1×105 plaques for a testis cDNA library, only one positive cDNA clone was identified. This clone was subcloned and sequenced. It contained 2,126 nucleotides with a putative 1,404-bp ORF, but not a consensus polyadenylation signal or a poly(A) tail (Fig. 1). The cDNA encoded a protein of 468 amino acids with a molecular weight of 53,788. The nucleotide sequence of the ORF of frog SF-1/Ad4BP had 62% similarity with bovine SF-1/Ad4BP and 70% with chicken OR2.1. 3.2. Comparison of amino acid sequences of SF-1/Ad4BPs When full-length amino acid sequences for frog and bovine SF-1/Ad4BPs, and chicken OR2.1 were deduced from the nucleotide sequences of cDNAs, all the amino acid sequences appeared to be very similar ( Fig. 2). The amino acid sequence of frog SF-1/Ad4BP had 80% similarity with chicken OR2.1 ( Kudo and Sutou, 1997), and 68% with bovine (Honda et al., 1993) and rat
Fig. 1. Nucleotide sequence of the cDNA encoding frog SF-1/Ad4BP. The translated amino acid sequence is shown in standard one-letter code below the nucleotide sequence. The nucleotides and amino acid residues are numbered. The P box ( ESCKG) and FTZ-F1 box are boxed with solid and dotted lines, respectively. The synthetic oligonucleotides used as the primers for 5∞ RACE and Southern blot analysis are underlined and dotted-underlined, respectively. An asterisk (*) indicates a stop codon, The nucleotide sequence data will appear in the DDBJ, EMBL, and GenBank Sequence Databases under Accession No. AB017352.
(Nomura et al., 1995) SF-1/Ad4BPs. There were only minor differences in the sequences of the first 120 amino acids among frog, chicken and bovine (<7%). SF-1/Ad4BP has three distinct conserved sequences of amino acids, namely a DNA-binding domain and two putative ligand binding domains (I and II ). The amino acid sequence of the DNA-binding domain of frog SF-1/Ad4BP shared 92% identity with that of bovine SF-1/Ad4BP and 94% with chicken OR2.1. It is known that the P box of a five-amino-acid sequence (ESCKG) is present in the zinc finger motif of bovine SF-1/Ad4BP cDNA (Honda et al., 1993). This box was also found in frog SF-1/Ad4BP cDNA
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Fig. 2. Alignment of the amino acid sequences of SF-1/Ad4BP obtained by sequencing chicken OR2.1 and bovine Ad4BP. The sequence data for bovine SF-1/Ad4BP and chicken OR2.1 are from [Honda et al. (1993)], and [ Kudo and Sutou (1997)], respectively. Dots indicate positions where residues are identical among the polypeptide sequences. Amino acid residues are numbered. The DNA-binding domain and FTZ-F1 box are boxed with solid and dotted lines, respectively. The 8-proline stretch of bovine Ad4BP is underlined.
(Fig. 1). In addition, an 8-proline stretch is seen between the DNA-binding domain and ligand-binding domain I of bovine SF-1/Ad4BP (Honda et al., 1993). However, this stretch was not observed in frog SF-1/Ad4BP, or in chicken OR2.1 ( Kudo and Sutou, 1997) ( Fig. 2). Moreover, the FTZ-F1 box, which can recognize and bind to DNA as a monomer ( Ueda et al., 1992), abuts the C-terminal end of the zinc finger motif. This box (28 amino acids long) in frog SF-1/Ad4BP is 100% identical to bovine SF-1/Ad4BP (Honda et al., 1993) and chicken OR2.1 ( Kudo and Sutou, 1997) (Figs. 1 and 2). These findings support the hypothesis that SF-1/Ad4BP has been highly conserved through evolution, and that it plays an essential role(s) for gonadal development in vertebrates. Recently, two FTZ-F1-related orphan receptors were cloned from chicken embryos ( Kudo and Sutou, 1997). The nucleotide sequence of one clone (OR2.0) had a high degree of similarity with that of mouse LRH-1 and Xenopus xFFlr, whereas the other (OR2.1) was very similar to mammalian SF-1/Ad4BPs. When the amino acid sequence of frog SF-1/Ad4BP was aligned with those of other known SF-1/Ad4BP proteins and FTZF1-related orphan receptors by the UPGMA method (Felsenstein, 1993), high sequence similarities were observed (Fig. 3). The Rana rugosa sequence appeared to be closer to that of chickens than mammals, as expected. Chicken OR2.0 is the inner group of mouse LRH-1 and Xenopus xFFlr. From the phylogenetic tree shown in Fig. 3, it can be concluded that frog SF-1/Ad4BP and Drosophila FTZ-F1 evolved from the same gene, and that frog SF-1/Ad4BP and chicken OR2.1 then diverged separately.
3.3. Northern blot analysis The total RNA from different tissues of adult frogs was extracted and analyzed by Northern blot using frog SF-1/Ad4BP cDNA as a probe. As shown in Fig. 4A, a positive signal (approximately 2.4 kb) was observed only in the testis among the 10 tissues examined. The results are compatible with those of Kudo and Sutou (1997), showing that chicken OR2.1 mRNA is detected in the testis. To confirm whether the testis is the only tissue that expresses SF-1/Ad4BP mRNA among various tissues of adult frogs, RT-PCR was employed to amplify the transcripts for detection by Southern blot analysis. As shown in Fig. 4C, SF-1/Ad4BP mRNA was expressed in the brains, spleens and adrenals/kidneys in addition to the testes. However, SF-1/Ad4BP mRNA could not be detected in frog ovaries, as opposed to the results of other studies, which show that the ovaries express Ad4BP mRNA in rats, mice and chickens (Honda et al., 1993; Kudo and Sutou, 1997; Liu et al., 1997). We have no explanation for this discrepancy at the present time. It should also be noted that the expression of SF-1/Ad4BP mRNA is very low in adult frog tissues. According to Honda et al. (1993), Ad4BP mRNA was detectable only in adrenals of bovines by Northern blot analysis, but in testes, ovaries, brains and spleens by Southern blot analysis of RT-PCR products. It would be interesting to compare the level of SF-1/Ad4BP mRNA in different tissues of frogs. However, its level in frog tissues was not determined precisely because of the low quantity. In this study, the SF-1/Ad4BP mRNA was detected in frog brains. The cytochrome P-450scc gene is
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Fig. 3. Phylogenetic tree of frog SF-1/Ad4BP and other known FTZ-F1-related orphan receptors in vertebrates. The database source and accession number are given in parentheses. The phylogenetic tree was constructed by the UPGMA method ([Felsenstein, 1993]).
expressed in murine brains (Le Goascogne et al., 1987; Ikeda et al., 1994). SF-1/Ad4BP regulates the activity of steroidogenic enzymes in mouse adrenal cells, and bovine luteal cells (Morohashi et al., 1993; Takayama et al., 1994; Liu and Simpson, 1997). Therefore, SF-1/Ad4BP may also control steroidogenesis in brains of frogs. It should also be mentioned that frog
adrenals/kidneys expressed SF-1/Ad4BP mRNA. Since adrenals adhere strongly to the midventral surface of each kidney in frogs (Gilbert, 1972), they cannot be completely separated from the kidneys surgically. Thus, both the adrenals and kidneys were excised in the present studies. As no expression of SF-1/Ad4BP gene was observed in bovine and chicken kidneys (Honda et al.,
Fig. 4. (A) Northern blot analysis of SF-1/Ad4BP mRNA in frog tissues. The RNA size markers were 28 and 18S ribosomal RNAs, and their positions are indicated to the right of the panel. (B) Total RNA stained with ethidium bromide. (C ) Southern blot analysis of RT-PCR products. Fifteen microliters of RT-PCR products were electrophoresed on 2% agarose gel, followed by Southern blot analysis, as described in Section 2.
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1993; Kudo and Sutou, 1997), it is conceivable that the adrenals, but not the kidneys, express SF-1/Ad4BP mRNA in frogs. 3.4. Isolation of the 5∞-UTR of the frog SF-1/Ad4BP gene To understand the function(s) of SF-1/Ad4BP for gonadal development in vertebrates, it is important to determine the genomic structure, in particular the promoter region of this gene, in animals other than mammals. Fig. 5 shows exons I and II in a genomic EcoRIcleaved DNA fragment of frogs. This is the first report showing the 5∞-UTR of the SF-1/Ad4BP gene in vertebrates except for murine and human. The amino acid sequence in exon II of the SF-1/Ad4BP gene was identical among three animals (frog, chicken and bovine)
(see Figs. 2 and 5). Exon 1 is probably not translated in frogs as it is in mammalian species (Nomura et al., 1995; Oba et al., 1996). Next, the 5∞-RACE was employed in order to identify the transcription initiation site of the frog SF-1/Ad4BP gene. By this method, we could extend the 5∞-end of the SF-1/Ad4BP cDNA by 6 nt (5∞-AATGTC ) (see Fig. 5). The consensus sequence for the CCAAT box was observed at the nucleotide positions of 874 and 1,111 bp in the 5∞-UTR (Fig. 5). Therefore, the transcription initiation site is probably the nucleotide A at the nucleotide positions of 1,175. The E box sequence (5∞-CACGTG) in the promoter region is required for the expression of the murine SF-1/Ad4BP gene (Nomura et al., 1995; Woodson et al., 1997). According to Nomura et al. (1995), the E box is located at −82 to −77 bp in the 5∞-UTR of the rat SF-1/Ad4BP gene. In addition, only a 90-bp proximal promoter fragment is sufficient to direct mouse SF-1/Ad4BP gene expression, and three elements (the E box, CAAT box, and Sp1 site) are required for activity of the SF-1/Ad4BP promoter ( Woodson et al., 1997). In this study, the E box-like sequence (5∞-CANNTG) was observed at positions 181, 193, 316 and 594 in the 5∞-UTR of the frog SF-1/Ad4BP gene (see Fig. 5), but their locations were further from the region than that found in the murine SF-1/Ad4BP gene, leading us to conclude that they may not function as the cis-element. However, further studies will be required to clarify whether this is true. Furthermore, there was neither a recognizable TATA element nor an Sp1 site in the 5∞-end of exon I. They are unlikely to function as a TATA box. In view of these findings, we can conclude that the nucleotide sequence of the 5∞-UTR in the frog SF-1/Ad4BP gene has a low similarity with that of mouse and human. We have no explanation at present for which element(s) is required to direct the expression of the SF-1/Ad4BP gene in frogs. 3.5. Structure of the nucleotide sequences around the translation initiation site among frog, mouse and human SF-1/Ad4BP genes
Fig. 5. DNA sequence of the 5∞-UTR of the frog SF-1/Ad4BP gene. Exons I and II are boxed. Nucleotides are numbered as the first nucleotide of +1. The amino acid sequence deduced from a nucleotide sequence is shown in exon II. The CCAAT box, E box, and TA- and TTA-rich elements are boxed. The first stop codon ( TGA) before exon II is underlined. An arrow indicates the translation initiation site determined by 5∞ RACE. The nucleotide sequences (1561-2220 and 2521-4644) are not shown because of space. The nucleotide sequence data will appear in the DDBJ, EMBL, and GenBank Sequence Databases under Accession No. AB017353.
As mentioned earlier, the Ftz-F1 single gene encodes SF-1/Ad4BP and ELP in murines. Murine FLP is transcribed within SF-1/Ad4BP intron I, and 77 amino acid residues are translated before the first methionine of SF1/Ad4BP (Honda et al., 1993). In contrast to murines, ELP is unlikely to be present in humans (Oba et al., 1996). Thus, it is of interest to know whether ELP is present in amphibians. Based on the nucleotide sequence shown in Fig. 5, 36 amino acids could be translated proceeding the first methionine within exon II of the SF-1/Ad4BP gene. However, no ELP initiation methionine was observed in those 36 amino acids before a stop codon ( TGA) at positions 2,306–2,308 (Fig. 5). ELP is,
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Fig. 6. Genomic structures of frog, mouse and human SF-1/Ad4BP genes around exon II. The boxes indicate exons I–III. Methionine in exon II indicates the translation initiation site. The deduced amino acid sequence is shown in standard one-letter code below the nucleotide sequence. Nucleotide sequences for human and mouse are given by the DDBJ sequence databases under Accession Nos. D84207 and D49682, respectively.
therefore, unlikely to be present in frogs, at least for Rana rugosa. As shown in Fig. 6, finally, a gene encoding SF-1/Ad4BP is designated the Ftz-F1 gene in mammals. We recently isolated a FTZ-F1 homologue from an embryo cDNA library of the frog, Rana rugosa. The genomic structure of the Rana rugosa FTZ-F1 homologue was different from that of SF-1/Ad4BP (to be published elsewhere). The Ftz-F1 gene encodes F-1/Ad4BP and ELP in mice, but only SF-1/Ad4BP in humans and frogs (Fig. 6). Judging from the nucleotide sequences of the 5∞-UTR of the frog SF-1/Ad4BP and FTZ-F1 homologue genes, both proteins are unlikely to be generated from a single gene. Besides, the Ftz-F1 gene does not encode all proteins such as SF-1/Ad4BP, ELP, and estrogen and androgen receptors in the FTZF1-related orphan nuclear receptor superfamily. Thus, we propose here the use of SF-1/Ad4BP for the gene encoding SF-1/Ad4BP instead of Ftz-F1.
4. Conclusion (1) Frog SF-1/Ad4BP cDNA, which shares a high degree of similarity with mammalian SF-1/Ad4BP and chicken OR2.1, was cloned. (2) SF-1/Ad4BP mRNA is expressed in the testes, brains adrenals/kidneys and spleens, but not ovaries, of adult frogs. (3) The 5∞-UTR with exons I and II of the frog SF-1/Ad4BP gene was cloned from a genomic DNA library. The sequence analysis revealed that ELP is unlikely to be present in the frog, Rana rugosa.
(4) It is proposed that SF-1/Ad4BP, not Ftz-F1, should be used as a name for the gene encoding SF-1/Ad4BP in vertebrates.
Acknowledgement This work was supported in part by Grant-in-Aids from the Ministry of Education, Science and Culture, Japan to M.N.
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A common trans-acting factor, Ad4-binding protein, to the promoters of steroidogenic P-450s. J. Biol. Chem. 267, 17913–17919. Morohashi, K., 1997. The ontogenesis of the steroidogenic tissues. Genes Cells 2, 95–106. Morohashi, K.-I., Zanger, U.M., Honda, S.-I., Hara, M., Watermasn, M.R., Omura, T., 1993. Activation of CYP11A and CYP11B gene promoters by the steroidogenic cell-specific transcription factor, Ad4BP. Mol. Endocrinol. 7, 1196–1204. Morohashi, K.-I., Omura, T., 1996. Ad4BP/SF-1, a transcription factor essential for the transcription of steroidogenic cytochrome P450 genes and for the establishment of the reproductive function. FASEB J. 10, 1569–1577. Nishioka, M., Miura, I., Saitoh, K., 1993. Sex chromosomes of Rana rugosa with special reference to local differences in sex-determining mechanism. Sci. Rep. Lab. Amphib. Biol. Hiroshima Univ. 12, 55–81. Nomura, M., Bartsch, S., Nawata, H., Omura, T., Morohashi, K., 1995. An E box element is required for the expression of the ad4bp gene, a mammalian homologue of ftz-f1 gene, which is essential for adrenal and gonadal development. J. Biol. Chem. 270, 7453–7461. Nordqvist, K., 1995. Sex differentiation—gonadogenesis and novel genes. Int. J. Dev. Biol. 39, 727–736. Oba, K., Yanase, T., Nomura, M., Morohashi, K., Takayanagi, R., Nawata, H., 1996. Structural characterization of human Ad4bp (SF1) gene. Biochem. Biophys. Res. Commun. 226, 261–267. Pieau, C., 1974. Differentiation du sexe en fonction de la temperature chez les embryons d’Emys orcbicularis L. (Chelonien); effects des hormones sexuelles. Ann. Embryol. Morphol. 7, 365–394. Sadovsky, Y., Crawford, P.A., Woodsan, K.G., Polish, J.A., Clements, M.A., Tourtellotte, L.A., Simburger, K., Milbrandt, J., Mice deficient in the orphan receptor steroidogenic factor 1 lack adrenal glands and gonads but express P450 side-chain-cleavage enzyme in the placenta and have normal embryonic serum levels of corticosteroids. 1995. Proc. Natl. Acad. Sci. USA 92, 10939–10943. Takayama, K., Morohashi, K.-I., Honda, S.-I., Hara, N., Omura, T., 1994. Contribution of Ad4BP, a steroidogenic cell-specific transcription factor, to regulation of the human CYP11A and bovine CYP11B genes through their distal promoters. J. Biochem. 116, 193–203. Tsukiyama, T., Niwa, O., Yokora, K., 1989. Mechanism of suppression of the long terminal repeat of Moloney leukaemia virus in mouse embryonal carcinoma cells. Mol. Cell. Biol. 9, 4670–4676. Ueda, H., Sun, G.-C., Murata, T., Hirose, S., 1992. A novel DNAbinding motif abuts the zinc finger domain of insect nuclear hormone receptor FTZ-F1 and mouse embryonal long terminal repeat-binding protein. Mol. Cell. Biol. 12, 5667–5672. Woodson, K.G., Crawford, P.A., Sadovsky, Y., Milbrandt, J., 1997. Characterization of the promoter of SF-1, an orphan nuclear receptor required for adrenal and gonadal development. Mol. Endocrinol. 11, 117–126. Yamamoto, S., Kondo, Y., Hanada, H., Nakamura, M., 1996. Strong expression of the calreticulin gene in the liver of Rana rugosa tadpoles, but not adult frogs. J. Exp. Zool. 275, 431–443.
Molecular cloning and expression of the SF-1/Ad4BP gene in the frog, Rana rugosa Ki-ichirou Kawano a, Ikuo Miura a, Ken-ichirou Morohashi b, Minoru Takase a, Masahisa Nakamura a,* a Laboratory for Amphibian Biology, Faculty of Science, Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima, Hiroshima 739-8526, Japan b National Institute for Basic Biology, Myodaiji-cho, Okazaki, Aichi 444-8585, Japan Received 14 May 1998; received in revised form 10 September 1998; accepted 12 September 1998; Received by A. Nakazawa
Abstract SF-1/Ad4BP is a transcriptional factor that was originally found to be a mammalian homologue of the Drosophila Ftz-F1 ( fushi tarazu factor 1) (Morohashi et al., 1992), and transcribed from a gene designated the Ftz-F1 gene (Nomura et al., 1995). Ftz-F1 gene-deficient mice lack adrenal glands and gonads. Besides mammals, however, the SF-1/Ad4BP cDNA has only been isolated to date in fish and birds. To understand its role(s) for adrenal and gonadal development in vertebrates, cloning of this gene in animals other than mammals is required. In this study, we succeeded to isolate frog (Rana rugosa) SF-1/Ad4BP cDNA from a testis lgt10 cDNA library. It encoded a protein of 468 amino acids, and its open reading frame (ORF ) shared 70% similarity with that of chicken OR2.1 (a SF-1/Ad4BP homologue) and 62% with bovine SF-1/Ad4BP. SF-1/Ad4BP mRNA was expressed in the testes, brains, adrenals/kidneys and spleens, but not ovaries, of adult frogs. In addition, we also cloned the 5∞-untranslated region (4.6 kb) of the SF-1/Ad4BP gene with exons I and II. Genomic structure analysis revealed that frog SF-1/Ad4BP was also transcribed from the same gene as that of mammals. However, many Ftz-F1-related proteins have been reported so far. The Ftz-F1 gene does not encode all of those Ftz-F1-related proteins. Thus, the name of Ftz-F1 is not adequate for the gene coding SF-1/Ad4BP. Here, we propose the use of SF-1/Ad4BP instead of Ftz-F1 for the gene that encodes SF-1/Ad4BP in vertebrates. © 1998 Elsevier Science B.V. All rights reserved. Keywords: cDNA; 5∞-UTR; Frog; SF-1/Ad4BP; SF-1/Ad4BP gene; Testis
1. Introduction Many transcriptional factors in the orphan nuclear receptor superfamily have recently been cloned. FTZ-F1 was originally identified in Drosophila as a transcriptional regulator of the fushi tarazu homeobox gene (Lavorgna et al., 1991). The expression of this gene is required for blastoderm and nervous-system develop* Corresponding author: Tel/Fax: +81 824 24 7483. e-mail: [email protected] Abbreviations: bp, base pair(s); DNase, deoxyribonuclease; dNTP, deoxyribonucleotide triphospate; DTT, dithiothreitol; ELP, embryonal long-terminal repeat-binding protein; 5∞-UTR, 5∞-untranslated region; FTZ-F1, fushi tarazu factor 1; kb, kilobase(s) or 1000 bp; ORF, open reading frame; RACE, rapid amplification of cDNA ends; RNase, ribonuclease; RT-PCR, reverse transcriptase-polymerase chain reaction; SDS, sodium dodecyl sulfate; SF-1, steroidogenic factor-1; SSC, 0.15 M NaCl/0.015 M Na · citrate pH 7.6. 3
ment in Drosophila. Recently, many FTZ-F1 homologues such as bovine SF-1/Ad4BP (Honda et al., 1993), mouse LRH-1 and Xenopus XFF1r (EllingerZiegelbauer et al., 1994) were reported. SF-1/Ad4BP promotes cell-specific expression of P-450scc genes in the adrenal glands (Honda et al., 1990; Lala et al., 1992; Morohashi et al., 1993; Ikeda et al., 1993; Takayama et al., 1994; Monte et al., 1998), and its mRNA is expressed in the gonads (Morohashi et al., 1992; Ikeda et al., 1994; Morohashi and Omura, 1996). Interestingly, the gene encoding SF-1/Ad4BP encodes another nuclear factor ELP (embryonal long-terminal repeat-binding protein; Tsukiyama et al., 1989). These two factors are transcribed by alternative promoter usage and splicing (Nomura et al., 1995). In Ftz-F1 gene-disrupted mice, the adrenal glands and gonads are completely lost (Luo et al., 1994; Sadovsky et al., 1995). Gonadotrope-specific markers like LH-b subunit and GnRH receptors also disappear (Ingraham et al., 1994). Thus, SF-1/Ad4BP
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is essential for adrenal and gonadal development and sexual differentiation [for reviews, see Manglesdorf et al. (1995), Nordqvist (1995) and Morohashi (1997)]. Sex steroid hormones play a pivotal role in sexual development in amphibians (Chang and Witschi, 1956; Nishioka et al., 1993), reptiles (Pieau, 1974; Jeyasuria et al., 1994; Crews, 1996) and birds ( Elbrecht and Smith, 1992; Andrews et al., 1997). The sex can be reversed by sex steroid hormones in frogs (Nishioka et al., 1993). A cloning of the cDNA encoding SF-1/Ad4BP in amphibians will, therefore, extend our understanding of (1) whether a common gene controls gonadal development in vertebrates, and (2) whether this gene has been conserved through evolution. Still, other than mammals, SF-1/Ad4BP has only been cloned in fish and birds. Here, we report molecular clonings of the frog SF1/Ad4BP cDNA and the 5∞-untranslated region (5∞-UTR) with the first and second exons. Tissues expressing SF-1/Ad4BP mRNA are also reported.
2. Materials and methods 2.1. cDNA cloning and sequence analysis A testis cDNA library in lgt10 vector was constructed from poly(A)+ RNA of 2-year-old frogs (Rana rugosa) ( Yamamoto et al., 1996). Bovine SF-1/Ad4BP cDNA (1965 bp) was labelled with [a-32P] dCTP (Amersham, Cleveland, OH ) using a random primer DNA labelling kit (Amersham) and used to screen the cDNA library by plaque hybridization. Hybridization was carried out at 62°C in 0.5% SDS, 6× SSC, 5× Denhardt’s solution and denatured salmon sperm DNA (100 mg/ml ) as previously described ( Yamamoto et al., 1996). Membranes were washed in 0.2× SSC and 0.1% SDS at 62°C, and autoradiographed. Positive cDNAs were subcloned into the pUC19 vector. The sequence analysis was performed using an ABI 373A automated DNA sequencer following the manufacturer’s instructions (Perkin Elmer, Palo Alto, CA). 2.2. Determination of the transcription initiation site of the Ad4BP gene by 5∞-RACE To determine the transcription initiation site of the Ad4BP gene, 5∞-RACE (rapid amplification of cDNA ends) was employed. First-strand cDNA was synthesized from DNase-treated total RNA (2 mg) of Rana rugosa testes using a 5∞-RACE kit (Boehringer Mannheim, Germany), and purified using a High Pure PCR product Purification kit (Boehringer Mannheim). A poly(A) tail was then added to the 3∞ end of the cDNA by incubation at 37°C with 0.2 mM dATP and 10 u of terminal transferase. Tailed cDNA was amplified by PCR using a primer (5∞-AGGAGCCCATAGTGATAACCAG-3∞ cor-
responding to nucleotide positions 301-322 of the frog SF-1/Ad4BP cDNA) with an oligo dT-anchor primer following the manufacturer’s instructions (Boehringer Mannheim). The obtained cDNA was further amplified by a second PCR using primers (5∞CCGCTTCCTATTTACACCTCACC-3∞ and 5∞-TGCAGTCCCCGTCCACCTTAGATA-3∞ corresponding to nucleotide positions 60-82 and 163-186, respectively) and the PCR anchor primer following the manufacturer’s instructions (Boehringer Mannheim). The PCR reaction consisted of 2 min at 94°C, followed by 40 cycles of 94°C (40 s), 63°C (1 min), and 72°C (2 min), ending with 15 min of extension at 72°C. The PCR product was ligated into the pCR2.1 vector for sequence analysis following the manufacturer’s instructions (Invitrogen, Carlsbad, CA). 2.3. Northern blot analysis Extraction of total RNA from various tissues of frogs was performed according to Chomczynski and Sacchi (1987). Fifteen micrograms of total RNA were electrophoresed on 1.2% denaturing formaldehyde agaroseMOPS gel and transferred on to nylon membranes ( Hybond N, Amersham). RNA was then hybridized with [a-32P] dCTP-labelled frog SF-1/Ad4BP cDNA. An image analyzer (FUJIX BAS-2000) was used to detect positive bands, since they were hardly detectable by exposure to X-ray film, even after several days. 2.4. Southern blot analysis of RT-PCR products The total RNA from different tissues of adult frogs was treated with 6 u of RNase-free DNase I (Promega, Madison, WI ) for 1 h at 37°C to avoid genomic DNA contamination, and then used as the initial templates for RT-PCR. cDNA was synthesized by incubating 2 mg of RNA in 20 ml of the first-strand buffer (Gibco-BRL, Detroit, MI ), which was supplemented with 200 u of Superscript II (Gibco-BRL), 0.5 mM dNTPs ( Takara, Kusatsu, Japan), 10 mM DTT (Gibco-BRL), and 0.4 mM oligo-dT primers at 42°C for 50 min. Samples 25 were then incubated at 70°C for 10 min to inactivate the reverse transcriptase. One-tenth of the first-strand mixture was added to a 50-ml PCR buffer containing 200 mM dNTPs, 0.2 mM, PCR primers (the forward primer; 5∞-TGTCAGCCATTGCAGCCCAG-3∞ corresponding to nucleotide positions 1258–1277 of the frog SF-1/Ad4BP cDNA and the backward primer; 5∞-CAAGTCAGTTTAGGCTCGCT-3∞ corresponding to nucleotide positions 1636–1655), and 2.5 u of Ex Taq polymerase ( Takara). The PCR reaction consisted of 3 min at 94°C, followed by 30 cycles of 94°C (40 s), 65°C (2 min), and 72°C (3 min), ending with 7 min of extension at 72°C. With this procedure, a 398-bp fragment of the SF-1/Ad4BP cDNA was amplified. The
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PCR products (0.4 kb) were then electrophoresed on 2% agarose gel and transferred to nylon membranes (GeneScreen@; NEN Research Products, Boston, MA). The DNA was hybridized with DIG-labelled frog SF-1/Ad4BP cDNA (2.1 kb) as a probe. The DIGDNA labelling kit and DIG luminescent detection kit (Boehringer Mannheim) were used for this analysis, following the manufacturer’s instructions. 2.5. Cloning of the 5∞-UTR of the SF-1/AD4BP gene and sequence analysis After genomic DNAs from frog erythrocytes were cleaved with EcoRI, they were run electrophoretically on 0.8% agarose gel and transferred to nylon membranes. The DNA was then hybridized with frog SF-1/Ad4BP cDNA labelled with [a-32P] dCTP, as explained earlier, and exposed to X-ray film ( Fuji RX ) overnight at −80°C. DNA fragments corresponding to positive bands were excised from agarose gels and extracted using a GENECLEAN kit (BIO 101, Inc., Vista, CA), following the manufacturer’s instructions. The DNA fragments were then ligated into the lgt10 vector for screening by plaque hybridization. Hybridization was carried out as described elsewhere ( Yamamoto et al., 1996). DNA clones were sequenced on both DNA strands using the ABI 373 A automated DNA sequencer following the manufacturer’s instructions (Perkin Elmer).
3. Results and discussion 3.1. Nucleotide and deduced amino acid sequence of SF-1/Ad4BP cDNA By screening 1×105 plaques for a testis cDNA library, only one positive cDNA clone was identified. This clone was subcloned and sequenced. It contained 2,126 nucleotides with a putative 1,404-bp ORF, but not a consensus polyadenylation signal or a poly(A) tail (Fig. 1). The cDNA encoded a protein of 468 amino acids with a molecular weight of 53,788. The nucleotide sequence of the ORF of frog SF-1/Ad4BP had 62% similarity with bovine SF-1/Ad4BP and 70% with chicken OR2.1. 3.2. Comparison of amino acid sequences of SF-1/Ad4BPs When full-length amino acid sequences for frog and bovine SF-1/Ad4BPs, and chicken OR2.1 were deduced from the nucleotide sequences of cDNAs, all the amino acid sequences appeared to be very similar ( Fig. 2). The amino acid sequence of frog SF-1/Ad4BP had 80% similarity with chicken OR2.1 ( Kudo and Sutou, 1997), and 68% with bovine (Honda et al., 1993) and rat
Fig. 1. Nucleotide sequence of the cDNA encoding frog SF-1/Ad4BP. The translated amino acid sequence is shown in standard one-letter code below the nucleotide sequence. The nucleotides and amino acid residues are numbered. The P box ( ESCKG) and FTZ-F1 box are boxed with solid and dotted lines, respectively. The synthetic oligonucleotides used as the primers for 5∞ RACE and Southern blot analysis are underlined and dotted-underlined, respectively. An asterisk (*) indicates a stop codon, The nucleotide sequence data will appear in the DDBJ, EMBL, and GenBank Sequence Databases under Accession No. AB017352.
(Nomura et al., 1995) SF-1/Ad4BPs. There were only minor differences in the sequences of the first 120 amino acids among frog, chicken and bovine (<7%). SF-1/Ad4BP has three distinct conserved sequences of amino acids, namely a DNA-binding domain and two putative ligand binding domains (I and II ). The amino acid sequence of the DNA-binding domain of frog SF-1/Ad4BP shared 92% identity with that of bovine SF-1/Ad4BP and 94% with chicken OR2.1. It is known that the P box of a five-amino-acid sequence (ESCKG) is present in the zinc finger motif of bovine SF-1/Ad4BP cDNA (Honda et al., 1993). This box was also found in frog SF-1/Ad4BP cDNA
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Fig. 2. Alignment of the amino acid sequences of SF-1/Ad4BP obtained by sequencing chicken OR2.1 and bovine Ad4BP. The sequence data for bovine SF-1/Ad4BP and chicken OR2.1 are from [Honda et al. (1993)], and [ Kudo and Sutou (1997)], respectively. Dots indicate positions where residues are identical among the polypeptide sequences. Amino acid residues are numbered. The DNA-binding domain and FTZ-F1 box are boxed with solid and dotted lines, respectively. The 8-proline stretch of bovine Ad4BP is underlined.
(Fig. 1). In addition, an 8-proline stretch is seen between the DNA-binding domain and ligand-binding domain I of bovine SF-1/Ad4BP (Honda et al., 1993). However, this stretch was not observed in frog SF-1/Ad4BP, or in chicken OR2.1 ( Kudo and Sutou, 1997) ( Fig. 2). Moreover, the FTZ-F1 box, which can recognize and bind to DNA as a monomer ( Ueda et al., 1992), abuts the C-terminal end of the zinc finger motif. This box (28 amino acids long) in frog SF-1/Ad4BP is 100% identical to bovine SF-1/Ad4BP (Honda et al., 1993) and chicken OR2.1 ( Kudo and Sutou, 1997) (Figs. 1 and 2). These findings support the hypothesis that SF-1/Ad4BP has been highly conserved through evolution, and that it plays an essential role(s) for gonadal development in vertebrates. Recently, two FTZ-F1-related orphan receptors were cloned from chicken embryos ( Kudo and Sutou, 1997). The nucleotide sequence of one clone (OR2.0) had a high degree of similarity with that of mouse LRH-1 and Xenopus xFFlr, whereas the other (OR2.1) was very similar to mammalian SF-1/Ad4BPs. When the amino acid sequence of frog SF-1/Ad4BP was aligned with those of other known SF-1/Ad4BP proteins and FTZF1-related orphan receptors by the UPGMA method (Felsenstein, 1993), high sequence similarities were observed (Fig. 3). The Rana rugosa sequence appeared to be closer to that of chickens than mammals, as expected. Chicken OR2.0 is the inner group of mouse LRH-1 and Xenopus xFFlr. From the phylogenetic tree shown in Fig. 3, it can be concluded that frog SF-1/Ad4BP and Drosophila FTZ-F1 evolved from the same gene, and that frog SF-1/Ad4BP and chicken OR2.1 then diverged separately.
3.3. Northern blot analysis The total RNA from different tissues of adult frogs was extracted and analyzed by Northern blot using frog SF-1/Ad4BP cDNA as a probe. As shown in Fig. 4A, a positive signal (approximately 2.4 kb) was observed only in the testis among the 10 tissues examined. The results are compatible with those of Kudo and Sutou (1997), showing that chicken OR2.1 mRNA is detected in the testis. To confirm whether the testis is the only tissue that expresses SF-1/Ad4BP mRNA among various tissues of adult frogs, RT-PCR was employed to amplify the transcripts for detection by Southern blot analysis. As shown in Fig. 4C, SF-1/Ad4BP mRNA was expressed in the brains, spleens and adrenals/kidneys in addition to the testes. However, SF-1/Ad4BP mRNA could not be detected in frog ovaries, as opposed to the results of other studies, which show that the ovaries express Ad4BP mRNA in rats, mice and chickens (Honda et al., 1993; Kudo and Sutou, 1997; Liu et al., 1997). We have no explanation for this discrepancy at the present time. It should also be noted that the expression of SF-1/Ad4BP mRNA is very low in adult frog tissues. According to Honda et al. (1993), Ad4BP mRNA was detectable only in adrenals of bovines by Northern blot analysis, but in testes, ovaries, brains and spleens by Southern blot analysis of RT-PCR products. It would be interesting to compare the level of SF-1/Ad4BP mRNA in different tissues of frogs. However, its level in frog tissues was not determined precisely because of the low quantity. In this study, the SF-1/Ad4BP mRNA was detected in frog brains. The cytochrome P-450scc gene is
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Fig. 3. Phylogenetic tree of frog SF-1/Ad4BP and other known FTZ-F1-related orphan receptors in vertebrates. The database source and accession number are given in parentheses. The phylogenetic tree was constructed by the UPGMA method ([Felsenstein, 1993]).
expressed in murine brains (Le Goascogne et al., 1987; Ikeda et al., 1994). SF-1/Ad4BP regulates the activity of steroidogenic enzymes in mouse adrenal cells, and bovine luteal cells (Morohashi et al., 1993; Takayama et al., 1994; Liu and Simpson, 1997). Therefore, SF-1/Ad4BP may also control steroidogenesis in brains of frogs. It should also be mentioned that frog
adrenals/kidneys expressed SF-1/Ad4BP mRNA. Since adrenals adhere strongly to the midventral surface of each kidney in frogs (Gilbert, 1972), they cannot be completely separated from the kidneys surgically. Thus, both the adrenals and kidneys were excised in the present studies. As no expression of SF-1/Ad4BP gene was observed in bovine and chicken kidneys (Honda et al.,
Fig. 4. (A) Northern blot analysis of SF-1/Ad4BP mRNA in frog tissues. The RNA size markers were 28 and 18S ribosomal RNAs, and their positions are indicated to the right of the panel. (B) Total RNA stained with ethidium bromide. (C ) Southern blot analysis of RT-PCR products. Fifteen microliters of RT-PCR products were electrophoresed on 2% agarose gel, followed by Southern blot analysis, as described in Section 2.
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1993; Kudo and Sutou, 1997), it is conceivable that the adrenals, but not the kidneys, express SF-1/Ad4BP mRNA in frogs. 3.4. Isolation of the 5∞-UTR of the frog SF-1/Ad4BP gene To understand the function(s) of SF-1/Ad4BP for gonadal development in vertebrates, it is important to determine the genomic structure, in particular the promoter region of this gene, in animals other than mammals. Fig. 5 shows exons I and II in a genomic EcoRIcleaved DNA fragment of frogs. This is the first report showing the 5∞-UTR of the SF-1/Ad4BP gene in vertebrates except for murine and human. The amino acid sequence in exon II of the SF-1/Ad4BP gene was identical among three animals (frog, chicken and bovine)
(see Figs. 2 and 5). Exon 1 is probably not translated in frogs as it is in mammalian species (Nomura et al., 1995; Oba et al., 1996). Next, the 5∞-RACE was employed in order to identify the transcription initiation site of the frog SF-1/Ad4BP gene. By this method, we could extend the 5∞-end of the SF-1/Ad4BP cDNA by 6 nt (5∞-AATGTC ) (see Fig. 5). The consensus sequence for the CCAAT box was observed at the nucleotide positions of 874 and 1,111 bp in the 5∞-UTR (Fig. 5). Therefore, the transcription initiation site is probably the nucleotide A at the nucleotide positions of 1,175. The E box sequence (5∞-CACGTG) in the promoter region is required for the expression of the murine SF-1/Ad4BP gene (Nomura et al., 1995; Woodson et al., 1997). According to Nomura et al. (1995), the E box is located at −82 to −77 bp in the 5∞-UTR of the rat SF-1/Ad4BP gene. In addition, only a 90-bp proximal promoter fragment is sufficient to direct mouse SF-1/Ad4BP gene expression, and three elements (the E box, CAAT box, and Sp1 site) are required for activity of the SF-1/Ad4BP promoter ( Woodson et al., 1997). In this study, the E box-like sequence (5∞-CANNTG) was observed at positions 181, 193, 316 and 594 in the 5∞-UTR of the frog SF-1/Ad4BP gene (see Fig. 5), but their locations were further from the region than that found in the murine SF-1/Ad4BP gene, leading us to conclude that they may not function as the cis-element. However, further studies will be required to clarify whether this is true. Furthermore, there was neither a recognizable TATA element nor an Sp1 site in the 5∞-end of exon I. They are unlikely to function as a TATA box. In view of these findings, we can conclude that the nucleotide sequence of the 5∞-UTR in the frog SF-1/Ad4BP gene has a low similarity with that of mouse and human. We have no explanation at present for which element(s) is required to direct the expression of the SF-1/Ad4BP gene in frogs. 3.5. Structure of the nucleotide sequences around the translation initiation site among frog, mouse and human SF-1/Ad4BP genes
Fig. 5. DNA sequence of the 5∞-UTR of the frog SF-1/Ad4BP gene. Exons I and II are boxed. Nucleotides are numbered as the first nucleotide of +1. The amino acid sequence deduced from a nucleotide sequence is shown in exon II. The CCAAT box, E box, and TA- and TTA-rich elements are boxed. The first stop codon ( TGA) before exon II is underlined. An arrow indicates the translation initiation site determined by 5∞ RACE. The nucleotide sequences (1561-2220 and 2521-4644) are not shown because of space. The nucleotide sequence data will appear in the DDBJ, EMBL, and GenBank Sequence Databases under Accession No. AB017353.
As mentioned earlier, the Ftz-F1 single gene encodes SF-1/Ad4BP and ELP in murines. Murine FLP is transcribed within SF-1/Ad4BP intron I, and 77 amino acid residues are translated before the first methionine of SF1/Ad4BP (Honda et al., 1993). In contrast to murines, ELP is unlikely to be present in humans (Oba et al., 1996). Thus, it is of interest to know whether ELP is present in amphibians. Based on the nucleotide sequence shown in Fig. 5, 36 amino acids could be translated proceeding the first methionine within exon II of the SF-1/Ad4BP gene. However, no ELP initiation methionine was observed in those 36 amino acids before a stop codon ( TGA) at positions 2,306–2,308 (Fig. 5). ELP is,
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Fig. 6. Genomic structures of frog, mouse and human SF-1/Ad4BP genes around exon II. The boxes indicate exons I–III. Methionine in exon II indicates the translation initiation site. The deduced amino acid sequence is shown in standard one-letter code below the nucleotide sequence. Nucleotide sequences for human and mouse are given by the DDBJ sequence databases under Accession Nos. D84207 and D49682, respectively.
therefore, unlikely to be present in frogs, at least for Rana rugosa. As shown in Fig. 6, finally, a gene encoding SF-1/Ad4BP is designated the Ftz-F1 gene in mammals. We recently isolated a FTZ-F1 homologue from an embryo cDNA library of the frog, Rana rugosa. The genomic structure of the Rana rugosa FTZ-F1 homologue was different from that of SF-1/Ad4BP (to be published elsewhere). The Ftz-F1 gene encodes F-1/Ad4BP and ELP in mice, but only SF-1/Ad4BP in humans and frogs (Fig. 6). Judging from the nucleotide sequences of the 5∞-UTR of the frog SF-1/Ad4BP and FTZ-F1 homologue genes, both proteins are unlikely to be generated from a single gene. Besides, the Ftz-F1 gene does not encode all proteins such as SF-1/Ad4BP, ELP, and estrogen and androgen receptors in the FTZF1-related orphan nuclear receptor superfamily. Thus, we propose here the use of SF-1/Ad4BP for the gene encoding SF-1/Ad4BP instead of Ftz-F1.
4. Conclusion (1) Frog SF-1/Ad4BP cDNA, which shares a high degree of similarity with mammalian SF-1/Ad4BP and chicken OR2.1, was cloned. (2) SF-1/Ad4BP mRNA is expressed in the testes, brains adrenals/kidneys and spleens, but not ovaries, of adult frogs. (3) The 5∞-UTR with exons I and II of the frog SF-1/Ad4BP gene was cloned from a genomic DNA library. The sequence analysis revealed that ELP is unlikely to be present in the frog, Rana rugosa.
(4) It is proposed that SF-1/Ad4BP, not Ftz-F1, should be used as a name for the gene encoding SF-1/Ad4BP in vertebrates.
Acknowledgement This work was supported in part by Grant-in-Aids from the Ministry of Education, Science and Culture, Japan to M.N.
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