European sea bass (Dicentrarchus labrax L.) cytochrome P450arom: cDNA cloning, expression and genomic organization

Journal of Steroid Biochemistry & Molecular Biology 80 (2002) 25–34

European sea bass (Dicentrarchus labrax L.) cytochrome P450arom: cDNA cloning, expression and genomic organization L. Dalla Valle∗ , L. Lunardi, L. Colombo, P. Belvedere Department of Biology, University of Padova, Via U. Bassi 58/B, 35131 Padova, Italy Received 2 August 2001; accepted 13 September 2001

Abstract Cytochrome P450arom, a key enzyme in the hormonal steroidogenic pathway, mediates the conversion of androgens to estrogens. This work describes the molecular cloning of the cDNA encoding the European sea bass (Dicentrarchus labrax L.) cytochrome P450arom by means of reverse transcriptase and polymerase chain reaction (RT–PCR) and 5 and 3 -rapid amplification of cDNA ends (RACE) analyses. The cDNA is 1822 bp in length and encodes a putative protein of 517 amino acids. Northern blot analysis revealed that the ovary expressed a transcript of about 2.2 kb in size. Analysis of the deduced amino acid sequence indicated 62–86% identity with ovarian P450arom of other teleost fish, the highest identity being found with the Japanese flounder, Paralichthys olivaceous. Identity was lower (56–65%) with the P450arom forms first reported in teleost brain. Only 52% identity was observed with the corresponding fragment of the cartilaginous fish, Dasyatis sabina. RT–PCR revealed that the sea bass P450arom mRNA was also expressed, at low levels, in testis and brain. Between the 5 and 3 -untranslated terminal regions (UTR), the sea bass CYP19 gene contains eight introns. All introns conform to the GT/AG rule for RNA splicing and are inserted in exactly the same positions as those found in Oryzias latipes and the human CYP19 gene. © 2002 Elsevier Science Ltd. All rights reserved. Keywords: Cytochrome P450arom cDNA; European sea bass; Dicentrarchus labrax; Cloning; Gonad; Brain; Genomic organization

1. Introduction The conversion of C19 steroids to estrogens is catalyzed by the enzyme complex aromatase, which comprises a specific form of steroid cytochrome P450, named cytochrome P450arom, and a nicotinamide adenine dinucleotide phosphate (NADPH)-dependent cytochrome P450 reductase, the expression of which is considered to be ubiquitous. Estrogens play a fundamental role in the control of female sexual differentiation [1] and reproductive physiology in all vertebrates, as they regulate the estrus cycle [2], the sexual differentiation of the nervous system [3] and, in egg-laying species, vitellogenesis [4]. Until now, this cytochrome has been cloned from the ovary of a number of freshwater teleosts, such as Oncorhynchus mykiss [5], Carassius auratus [6], Oryzias latipes [7], Ictalurus punctatus [8], Oreochromis niloticus [9], Oreochromis mossambicus (accession number AF135850), Danio rerio [10]) and the seawater teleost, Paralichthys olivaceous [11]. Moreover, the aromatase has been cloned in Dasyatis sabina, a cartilaginous fish [12]. ∗

Corresponding author. Tel.: +39-49-8276188; fax: +39-49-8276199. E-mail address: [email protected] (L. Dalla Valle).

The European sea bass (Dicentrarchus labrax L., Moronidae) is a carnivorous marine fish commonly found in estuaries and lagoons of the European Atlantic coasts and Mediterranean sea. It is an important aquaculture species and the subject of both basic and applied research [13]. The aim of this work was to isolate the cDNA and to characterize the expressed transcript encoding the sea bass ovarian P450arom and to define the genomic organization of the corresponding CYP19 gene. This would allow the future study of the seasonally dependent P450arom expression during the reproductive cycle and its sex-dependent expression during the period of embryonic sex differentiation in this commercially valuable species.

2. Material and methods 2.1. Tissue preparation and RNA extraction Samples of ovary, brain and posterior kidney were collected from 4-year-old females reared at a marine fish farm near Venice, Italy, during the reproductive period, whereas testes were collected from both normal and triploid males. At the time of tissue collection, the fish were sedated in an

0960-0760/02/$ – see front matter © 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 9 6 0 - 0 7 6 0 ( 0 1 ) 0 0 1 7 0 - 4

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ice bath and sacrificed by decapitation. Tissues were immediately removed by dissection, frozen in liquid nitrogen and stored at −80 ◦ C until analyzed. Total RNA was extracted using the commercial product RNAzol B (Celbio, Milan, Italy) according to the manufacturer’s instructions. The RNA samples were kept at −80 ◦ C until use. The RNA was enriched in polyadenylated mRNA utilizing the NucleoTrap mRNA Kit (M-Medical, Florence, Italy). 2.2. Oligonucleotides The oligonucleotides used as polymerase chain reaction (PCR) primers are listed in Table 1. The non-specific primers, ns-Sb-1, ns-Sb-2, ns-Sb-3 and ns-Sb-4, were designed on highly conserved regions of the P450arom cDNAs from O. niloticus (U72071), O. latipes (D82968), O. mykiss [5], D. rerio (AF004521), I. punctatus (S75715) and P. olivaceous (AB017182). Specific primers were then suited to our new sequence. The set of primer BT1 and BT2 is specific for rainbow trout ␤-actin (AF157514), but can also amplify the ␤-actin cDNA of sea bass. 2.3. Cloning of sea bass ovarian P450arom cDNA The sea bass P450arom cDNA was cloned and sequenced in five fragments by reverse transcriptase and PCR (RT–PCR) amplification of the coding region and rapid amplification of cDNA ends (RACE) analyses. The cloning of the coding region was performed with three reactions of RT–PCR: in the first reaction, we used a pair of non-specific primers (ns-Sb-1 and ns-Sb-2) selected on highly conserved regions of fish P450arom cDNAs; in the second reaction, we used a 5 -primer (s-Sb-1) specific for the sea bass sequence and a non-specific 3 -primer (ns-Sb-3); finally in

Table 1 Primers used in RT–PCR and 5 and 3 -RACE analyses for sequencing and expression analysis of sea bass cytochrome P450arom Primer

Sequence

Nucleotide position

ns-Sb-1 ns-Sb-2 ns-Sb-3 ns-Sb-4 ns-Sb-5 s-Sb-1 s-Sb-2 s-Sb-3 s-Sb-4 s-Sb-5 s-Sb-6 s-Sb-7 s-Sb-8 BT-1 BT-2

5 -CCTCCACACAGACTCACCT-3

+583 → +601 +986 → +970 +1522 → +503 +348 → +366 +303 → +286 +933 → +950 +651 → +632 +1006 → +1022 +1403→ +1420 +471 → +453 +419→ +402 +33 → +52 +1758 → +1737 +396 → +416a +873 → +853a

5 -TGCGATCACCATCTCCA-3 5 -TGGTGCTCTACAGGCTGCTG-3 5 -TGGATCGATGGAGAGGAGA-3 5 -CTGTGCCTATACCAGTCC-3 5 -GGCGAACTGACTGCTGAG-3 5 -AGCGCAGCAAACTGAGGA-3 5 -TCAGCCTCTTCTTTATG-3 5 -GATGATGAAGTCCATCCT-3 5 -CATACATGCCCATGCAGCT-3 5 -ATGTCCATTCTTCAGTAC-3 5 -TAAATGGATCTGATCTCTGC-3 5 -CGTACATTTCACATAATAGCTC-3 5 -CAGGGAGAAGATGACCCAGAT-3 5 -GATACCGCAAGACTCCATACC-3

a Positions are relative to the trout ␤-actin cDNA sequence (accession number AF157514).

the third reaction, we used a specific 3 -primer (s-Sb-2) and a non-specific 5 -primer (ns-Sb-4). For these analyses, we utilized 2 ␮g of total RNA of sea bass ovary and the SuperScript One-Step RT–PCR System (Gibco-BRL, Milan, Italy), changing the conditions according to the annealing temperature of the sets of oligonucleotides. For the 3 -RACE, the cDNA was synthesized by incubating 2 ␮g of total RNA of sea bass ovary in 20 ␮l of the first-strand buffer which was supplemented with 200 U of SuperScript II reverse transcriptase (RT) (Gibco-BRL), 0.5 mM of dNTPs, 10 mM DTT and 0.5 ␮M dT17 primer (5 -GAC TCG AGT CGA CAT CGA TTT TTT TTT TTT TTT TT-3 ) at 50 ◦ C for 1 h. The first-strand mixture was diluted to 200 ␮l with water and 2 ␮l were added to 50 ␮l of the PCR buffer containing 200 ␮M of dNTP, 0.2 ␮M of the anchor primer (5 -CTG GTT CGG CCC AGA CTC GAG TCG ACA TCG-3 ), 2.5 U of Taq polymerase (Celbio), and the specific primer s-Sb-3. The cDNA obtained was further amplified by a second PCR using the primer s-Sb-4 and the PCR anchor primer. The amplification procedure consisted of 2 min at 95 ◦ C followed by 10 cycles at 95 ◦ C for 45 s (DNA denaturation), 58 ◦ C for 45 s (annealing), and 72 ◦ C for 2 min (extension) and 25 cycles at 95 ◦ C (45 s), 58 ◦ C (45 s), and 72 ◦ C (2 min) with 2 s of time increment per cycle in each extension phase. The extension phase of the last cycle was prolonged by 10 min. The fragment amplified by 3 -RACE was purified and sequenced. The 5 -RACE was carried out using the 5 -RACE System (Gibco-BRL) following the manufacturer’s instructions. Briefly, the cDNA was synthesized by incubating 2 ␮g of total RNA of sea bass ovary in 25 ␮l of the first-strand buffer, which was supplemented with 200 U of SuperScript II RT, 0.4 mM of dNTPs, 10 mM DTT and 0.16 ␮M of the specific primer s-Sb-5, at 50 ◦ C for 1 h. Terminal transferase was used to add a homopolymeric C-tail to the 3 end of the first-strand cDNA purified with a GlassMAX DNA isolation Spin Cartridge Purification kit (Gibco-BRL). The tailed cDNA was then amplified by PCR using the specific primer s-Sb-6 and the oligo dG-anchor primer (5 -GGC CAC GCG TCG ACT AGT ACG GGI IGG GII GGG IIG-3 ). As the amplification product was scarce, a second PCR was conducted using an aliquot of the first PCR reaction as a template with the internal non-specific primer ns-Sb-5 and the dG-anchor primer. The amplification procedure consisted of a touch-down PCR reaction with annealing temperatures decreasing from 62 to 55 ◦ C over 16 cycles and the final 24 cycles maintained at 54 ◦ C. The extension phase of the last cycle was prolonged by 10 min. The resultant amplicon was purified from the sliced gel band and directly sequenced. 2.4. Northern blot analysis We prepared a cRNA probe corresponding to the coding region of sea bass P450arom cDNA (nucleotides 33 to 1758), which was amplified by RT–PCR from total ovarian RNA using a pair of specific primers (s-Sb-7 and s-Sb-8; Table 1).

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Following PCR, the amplified cDNA was resolved on 1.2% agarose gel and the gel-purified fragment was ligated into a pGEM-T vector using a pGEM-T Vector System I according to the supplier’s recommendations (Promega, Milan, Italy). The insert was sequenced from two different plasmids in order to verify probe specificity, orientation and sequence. A recombinant plasmid was then linearized by restriction cleavage and used as a template for generating the cRNA probe. The cRNA transcript was digoxigenin-labeled by in vitro transcription using a DIG RNA Labeling kit (Roche Diagnostics, Milan, Italy). Poly(A)+ -enriched RNAs were extracted from sea bass ovary and brain. The poly(A)+ RNAs (2.5 ␮g) were electrophoresed with the High Range RNA Ladder (MBI Fermentas, DASIT, Milan, Italy) through 1.1% formaldehyde-denaturing gel, blotted onto a nylon membrane (Hybond-N, Amersham, Milan, Italy) and baked under vacuum at 80 ◦ C for 2 h. The remaining 28S and 18S rRNAs in each poly(A)+ -enriched RNA preparation were visualized by methylene blue staining to check RNA loading and integrity. The membrane was incubated overnight at 68 ◦ C with the DIG-labeled cRNA probe in 5 × SSC containing 50% formamide, 0.02% SDS, 0.1% lauroylsarcosine, 1% blocking reagent and 100 ␮g/ml of transfer RNA. After incubation with an anti-DIG antibody, signals were detected using a DIG nucleotide detection kit (Roche) according to the manufacturer’s instructions. The signal was revealed by exposing the membrane to an X-ray film for 2 h. 2.5. Tissue-specific expression of P450arom Total RNA was isolated from the ovary (four samples), brain (three samples) and posterior kidney (one sample) of 4-year-old sea bass females, and from the testes of normal male (one sample) and triploid males (two samples), as described above. Reverse transcription of sea bass P450arom cDNA (primers ns-SB-1 and ns-Sb-2) and ␤-actin cDNA (primers BT1 and BT2) was carried out with 1 ␮g of total RNA and the SuperScript One-Step RT–PCR System. The primers were selected so that the resultant amplicon would span three exon/intron boundaries of the sea bass CYP19 gene, thus eliminating amplification of genomic DNA. In preliminary experiments, each primer set was used to amplify equal amounts of cDNA derived from the samples with the higher levels of specific messenger for 10–40 cycles, and based on this analysis, a predetermined number of cycles was chosen for each primer set to maintain product accumulation in the linear range. The concentration of MgSO4 was adjusted to 1.5 mM and primers were added to a final concentration of 0.2 ␮M. The cycling conditions for RT–PCR were: one incubation at 54 ◦ C for 30 min followed by 2 min at 94 ◦ C to inactivate the RT; 95 ◦ C for 30 s, 58 ◦ C for 30 s, and 72 ◦ C for 30 s for 32 cycles (cytochrome P450arom) or 20 cycles (␤-actin). The extension phase of the last cycle was prolonged by 10 min. PCR products were resolved on 1.2% agarose gel and stained with ethidium bromide.

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2.6. Genomic organization of the sea bass P450arom Sea bass genomic DNA was extracted using a Genomic DNA Purification Kit (MBI Fermentas). Amplification of genomic CYP19 gene fragments was performed with the sets of primers, s-Sb-7 and ns-Sb-2, ns-Sb-1 and ns-Sb-2, s-Sb-1 and s-Sb-8, using 200 ng of sea bass genomic DNA as template. Amplification was performed changing the temperature conditions according to the annealing temperature of each set of oligonucleotides. The products from genomic DNA were larger than those obtained as cDNA, indicating that the sequences were interrupted by one or more introns. Direct sequencing of the PCR genomic products was performed using the oligonucleotides listed in Table 1, and the exon–intron boundaries as well as the intron sequences were determined by comparing the genomic sequence with the cDNA sequence. 2.7. Nucleotide sequencing Sequencing was performed on double-stranded DNA using the ABI PRISM Dye Terminator Cycle Sequencing Core Kit (Applied Biosystems, Monza, Italy). Electrophoresis of sequencing reactions was completed in the ABI PRISM model 377, version 2.1.1, automated sequencer. The homology searches were carried out using the Basic Blast program, version 2.0, at http://www.ncbi.nlm.nih.gov/ BLAST/, whereas sequence alignment was performed using the ClustalW program at http://www2.ebi.ac.uk/clustalw/. Transmembrane prediction analysis was carried out with the ExPASy Molecular Biology Server (http://www.expasy. ch/) using the Prediction of Transmembrane Regions SOSUI software. The protein was also scanned for the occurrence of patterns stored in the PROSITE database (http://www. expasy.ch/prosite/). The molecular weight was obtained using the ProtParam tool program (http://www.expasy.ch/). 3. Results 3.1. Isolation of sea bass P450arom cDNA The sea bass P450arom cDNA sequence was obtained by sequencing five overlapping PCR fragments. Internal fragments were obtained by RT–PCR reactions using primers selected on highly conserved regions of fish P450arom cDNAs available from GeneBank and total RNA extracted from ovaries of 4-year-old females. To ensure that the ovaries were expressing the P450arom, the same tissues were analyzed by means of an aromatase assay and found to be able to convert tritiated androstenedione into 17␤-estradiol (data not shown). Hence, a 404-bp fragment was amplified with non-specific oligonucleotide primers. A database search revealed a high similarity of this PCR product to other teleost fish P450arom cDNAs. Specific primers were then designed based on our sequence and

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Fig. 1. Nucleotide sequence of sea bass CYP19 gene and its deduced amino acid sequence. The capital letters correspond to the exon sequence. The intron sequence is in lower-case letters. The intron dinucleotide acceptor and donor sites for RNA splicing are indicated by lower case, bold letters. One-letter symbols of encoded amino acids are shown below the DNA sequence. The numbers refer to the nucleotide positions of DNA at the end of each line. The two in-frame translation start codons as well as the stop codon are given in bold type. The positions of putative poly (A)+ signals are underlined. The cDNA sequence and the genomic DNA sequence have been submitted to the GeneBank under the accession number AJ311177 and AJ318516, respectively.

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Fig. 2. Comparison of the amino acid sequences of sea bass P450arom with those of Japanese flounder (accession number BAA74777), medaka (Q92087), rainbow trout (228574), Nile tilapia (P70091), zebrafish (AAK00643), goldfish (073686), channel catfish (Q92111), Atlantic stingray (AAF04617), African clawed frog (BAA90529), chicken (P19098), bovine (P46194), rat (P22443), human (P11511), and with the P450arom brain forms of zebrafish (AAK00642), goldfish (P79690), Nile tilapia (AAG18458), and rainbow trout (AJ311937). Identical and similar amino acid residues are marked by asterisks and dots, respectively. The numbers refer to the amino acid position at the end of each line. Roman numerals indicate high homology regions such as the I-helix (I), an aromatase-specific conserved region (II), and the heme-binding region (III) [16].

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Fig. 2. (Continued ).

utilized with non-specific external primers to obtain two fragments covering almost entirely the coding region of the P450arom cDNA. The full-length cDNA was obtained by 5 and 3 -RACE analyses using primers designed on the cDNA partial sequence of the coding region. To define more precisely the amino acid sequence of sea bass P450arom and to prepare a cRNA probe for the next experiments, the cDNA was amplified with primers flanking the open reading frame (ORF) (s-Sb-7 and s-Sb-8). The PCR products were cloned and two independent clones were sequenced. The final P450arom cDNA is 1822 bp-long (accession number AJ311177) and contains an ORF of 1551 b, starting from a putative initiation methionine which is 35 b downstream from the 5 -end; the 3 -untranslated terminal regions (UTR)is 236 b-long. The ORF encodes a putative protein of 517 amino acids with a calculated molecular weight of 58.5 kDa. Similar to other reported ovarian aromatases in teleosts, such as goldfish, [6], medaka [7], rainbow trout [5], and ice goby [14], the sea bass P450arom cDNA presents a second potential initiation site 30 b downstream from the first one, but neither is supported by a perfect consensus Kozak sequence for initiation of translation [15]. Actually, it is still unknown which ATG is the true initial codon in teleosts [14]. The 5 -UTR before the first ATG initiation codon is relatively short (35 bp), which is consistent with all

P450arom cDNAs that have been isolated from fish ovaries so far. Although a poly-A tail was attached to the 3 -end of the cDNA sequence, the canonical poly-A signal, AATAAA, characteristic of the eukaryotic mRNA, was not found in the 3 -UTR. Nevertheless, two putative poly-A signals were detected at nucleotide positions 1716–1721 and 1779–1784 of the 3 -UTR. The last signal falls within the expected range of 10-25 b upstream of the poly-A site (Fig. 1). During the course of this work, a sea bass ovary-derived P450arom sequence was deposited in GeneBank database (accession number AJ298290). Our sequence differs from the latter for a 30 nt longer 5 -UTR, three different nucleotides in the coding region determining the change of two amino acids, and two different nucleotides and two deletions in the 3 -UTR. 3.2. Comparison of amino acid sequences of P450arom The amino acid sequence deduced from the ORF of the sea bass P450arom cDNA was compared with the known amino acid sequences of P450arom from 17 other species. The highest identity (86%) was found with another seawater fish, the flounder P. olivaceous, whereas the identity with other freshwater teleost fishes, such as medaka, rainbow trout, Nile tilapia, goldfish and channel catfish, ranged between 82 and 62%. The identity was only 52% with the

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aromatase sequence of the cartilaginous fish, D. sabina. Identity was also lower (56–65%) with the P450arom forms reported in teleost brains (zebrafish, goldfish, Nile tilapia and trout). Amino acid identities of the sea bass sequence with those of the African clawed frog, chicken, bovine, rat and human P450arom were 52, 51, 51, 49 and 50%, respectively. In Fig. 2, we show the alignment between the amino acid sequences reported above. The region that can be described as hydrophobic lies between the amino acids 35 and 57, and comprises the membrane spanning region. Some regions of the P450arom sequences are conserved among different species, including sea bass; these regions include the I-helix, an aromatasespecific conserved region, and the heme-binding region [16]. The identity of the sequence presented in this work is supported by a good overall homology (up to 86% in the coding region) with already known fish sequences. As reported for other teleost ovarian P450arom amino acid sequences, the N-terminal region of sea bass aromatase is longer than that of mammals, chicken, frog and stingray ovarian aromatases. The homology of this additional region among fish species is low. It is not hydrophobic in sea bass, unlike rainbow trout in which it is assumed to function as an additional membrane insertion domain [5]. Moreover, if the downstream initiation site (+65) is utilized, this results in a 507-amino acid protein, a length similar to the aromatase of higher vertebrates. 3.3. Northern blot analysis of sea bass P450arom The size and number of P450arom transcripts were examined by Northern blot analysis. Using the sea bass P450arom cRNA probe, a transcript of about 2.2 kb was detected in the ovarian poly(A)+ -enriched RNA samples, whereas the transcript abundance was presumably too low to be seen in the corresponding brain samples (Fig. 3). The transcript was larger than the PCR-derived cDNA: about 2.2 kb against 1.8 kb, probably due to the transcript polyadenylated tail that is normally 200–400 b long. 3.4. P450arom mRNA expression in various sea bass tissues Total RNAs were prepared from various tissues in adult sea bass (ovary, brain, testis and kidney) and aromatase expression was determined by semiquantitative RT–PCR (Fig. 4, panel A). Agarose gel of total RNA was used to verify the RNA loading, whereas integrity of RNA was analysed with ␤-actin amplification (Fig. 4, panel B). Aromatase was markedly expressed in the ovarian samples, whereas expression levels were extremely low and barely detectable in the brain. The amplification levels in the testis were higher than in the brain, but lower than in the ovary. For this analysis, we used two samples of RNA extracted from triploid males and one sample from a diploid male. As can be seen in Fig. 4, the amplification level was higher in diploid than triploid males, but the number of

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Fig. 3. Northern blot analysis of the mRNA for P450arom performed with 2.5 ␮g of poly(A)+ -enriched RNAs from samples of ovary and brain extracted from 4-year-old sea bass females. The remaining ribosomal RNAs were detected by staining with methylene blue.

samples was too low for any hypothesis to be framed. The identity of the cDNAs obtained with brain and testis total RNAs was confirmed by sequencing the amplificates after a non-quantitative RT–PCR (40 cycles). 3.5. Genomic organization of sea bass P450arom The exon/intron organization of sea bass P450arom was determined by sequential amplification of overlapping gene fragments and compared with that of medaka and human CYP19 genes. The gene encoding human aromatase has been found to contain 10 exons, including an untranslated exon 1 [17]. The sea bass CYP19 gene contains nine exons, the nucleotide sequence of which coincides exactly with the cDNA sequence, except for position 1642 (AJ311177), in which C (cDNA) is replaced by T (genomic DNA) (AJ318516). As for medaka, there was no additional exon, contrary to what has been found in the 5 non-coding region of mammalian P450arom [18]. Between the 5 and 3 -UTR

Fig. 4. Semiquantitative RT–PCR analysis of the tissue distribution of P450arom and ␤-actin mRNAs in sea bass. Ovarian and brain samples were obtained from 4-year-old females; testis from triploid males and a normal male (last sample) of sea bass.

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Fig. 5. Genomic structure of the sea bass CYP19 gene. (A) Schematic diagram illustrating the relative positions of introns and exons. Exon sizes in base pairs are indicated above the scheme; intron lengths are denoted below. Exons are depicted as shaded boxes. Introns are indicated as a horizontal line. (B) Splice junction sequences of sea bass, medaka and human CYP19 genes. The nucleotide sequences of exons (upper case) and introns (lower case) at the splice junctions are shown. The corresponding amino acid residues are shown below the nucleotide sequence of exons. The consensus 5 donor (gt) and 3 acceptor (ag) sites are in bold. Intron lengths are denoted on the right.

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regions, the sea bass CYP19 gene contains eight introns (Fig. 5). All donor and acceptor sites of these introns are GT and AG, respectively, agreeing with consensus sequences [19]. The introns are inserted at exactly the same positions as those found in O. latipes and human genes. In sea bass as in medaka, the size of the introns is smaller than in humans (89–408 and 73–213 bp, respectively, against 1.25–10 kbp in humans) (see Fig. 5); all the sea bass introns, except number 7, are longer than those of the medaka. All introns interrupt the reading phase inside the boundary codon triplet, except for introns 6 and 8 in sea bass and medaka (introns 7 and 9 in humans).

4. Discussion In the present study, we report the cloning of sea bass P450arom cDNA and the analysis of the genomic organization of the corresponding CYP19 gene. The 1822-bp cDNA contains a 517 amino acid-long ORF and shares high amino acid sequence identity with that of the other teleost ovarian P450arom cDNAs. The identity is lower with the teleostean brain P450arom forms sequenced up to now. The longest ORF starts with an ATG initiation codon at nucleotide 35. There is no canonical AATAAA poly-A signal in the 3 -UTR. Although this signal is highly conserved, natural variants, as those found in sea bass cDNA, do occur [20]. Mutations in the AATAAA sequence decrease the rate of cleavage and polyadenylation to different extents. The sequence ATTAAA corresponds to the mildest mutation and the most common natural variant, whereas AGTAAA, which could also be processed efficiently, is not commonly found in natural mRNAs [20]. Semiquantitative RT–PCR analysis using primers that hybridize to the coding region revealed that the sea bass P450arom transcript is expressed in gonadal tissues (ovary and testis) and brain but not in the non-steroidogenic kidney. The aromatase expression in the sea bass testis agrees with the results reported in the dogfish, Squalus acanthias [21], channel catfish, I. punctatus [22], rainbow trout, O. mykiss [23] and Atlantic stingray, D. sabina [12]. The estrogen effects in this tissue are probably local (as in mammals), as the levels of circulating estrogens are normally low or undetectable in the plasma of males [13]. In all vertebrates, including teleost fish, P450arom is expressed in the brain. Whereas extra-gonadal expression of P450arom in mammals is obtained by the use of alternatively spliced 5 -untranslated exons and tissue-specific promoters [24], the brain form of P450arom expressed in the goldfish [6], zebrafish [10] and rainbow trout [25] originates from a second P450arom gene. The presence of different genes encoding different forms of aromatase has been demonstrated also in pig [26]. The teleostean brain and ovarian variants of P450arom were shown by means of RT–PCR to be coexpressed in neural tissues [6,10,25]. To date, it is not known if a second form of P450arom, specific for the brain, is present in the sea bass.

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However, the low level of ovarian P450arom expression in the brain is not in agreement with the high level of aromatase activity present in this tissue [13], suggesting the possible presence, in this species too, of a second form of P450arom. Finally, aromatase expression has been demonstrated, by means of RT–PCR, in the non-steroidogenic kidney of rainbow trout [23] and the Atlantic stingray [12], whereas negative results were obtained with the channel catfish [22]. The lack of aromatase expression in sea bass kidney suggests that expression in this tissue could be species-regulated. With regard to the genomic organization, the suggestion that the CYP19 gene is phylogenetically conserved throughout the vertebrates on the basis of a high degree of conservation of coding sequences and gene organization between human and medaka genes [18] is confirmed by the results obtained in the sea bass. On the other hand, the sea bass CYP19 gene, as found for medaka, is much smaller (3.1 kb in length) than the human gene (larger than 50 kb) [17] due to the small introns present. In summary, we cloned and sequenced the sea bass cDNA for the cytochrome P450arom. This cDNA will be useful for the future investigation of the molecular mechanism controlling the aromatase expression, at the transcriptional level, throughout the reproductive season in the sea bass.

Acknowledgements Research was aided by Grant no. 5C 117 from the Ministry for Agricultural Policies of Italy, in the purview of the Fifth Triennial Plan for Fisheries and Aquaculture in Marine and Brackish Waters. References [1] F. Piferrer, Endocrine sex control strategies for the feminization of teleost fish, Aquaculture 197 (1-4) (2001) 229–281. [2] M.A. Cowley, A. Rao, P.J. Wright, N. Illing, R.P. Millar, I.J. Clarke, Evidence for differential regulation of multiple transcripts of the gonadotropin releasing hormone receptor in the ovine pituitary gland; effect of estrogen, Mol. Cell. Endocrinol. 146 (1-2) (1998) 141–149. [3] E.D. Lephart, A review of brain aromatase cytochrome P450, Brain Res. Rev. 22 (1) (1996) 1–26. [4] J.-M. Nicolas, Vitellogenesis in fish and the effects of polycyclic aromatic hydrocarbon contaminants, Aquat. Toxicol. 45 (2-3) (1996) 77–90. [5] M. Tanaka, T.M. Telecky, S. Fukada, S. Adachi, S. Chen, Y. Nagahama, Cloning and sequence analysis of the cDNA encoding P-450 aromatase (P450arom) from a rainbow trout (Oncorhynchus mykiss) ovary; relationship between the amount of P450arom mRNA and the production of oestradiol-17ß in the ovary, J. Mol. Endocrinol. 8 (1) (1992) 53–61. [6] A. Tchoudakova, G.V. Callard, Identification of multiple CYP19 genes encoding different cytochrome P450 aromatase isozymes in brain and ovary, Endocrinology 139 (4) (1998) 2179–2189. [7] S. Fukada, M. Tanaka, M. Matsuyama, D. Kobayashi, Y. Nagahama, Isolation, characterization, and expression of cDNAs encoding the medaka (Oryzias latipes) ovarian follicle cytochrome P-450 aromatase, Mol. Reprod. Dev. 45 (3) (1996) 285–290.

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