Cloning of CYP1A in Atlantic salmon (Salmo salar)

Aquaculture 246 (2005) 11 – 23 www.elsevier.com/locate/aqua-online

Cloning of CYP1A in Atlantic salmon (Salmo salar)B Christopher B. Rees, Hong Wu, Weiming LiT Department of Fisheries and Wildlife, Michigan State University, 13 Natural Resources Building, East Lansing, MI 48824, USA Accepted 1 December 2004

Abstract To better understand the molecular relationship between contaminant exposure in Atlantic salmon and the cytochrome P450 system, we have cloned a CYP1A gene using reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). The cloned cDNA possesses all characteristic motifs of teleost CYP1A genes including the start codon, heme-binding region, stop codon, and poly-adenylation signal. The cloned gene is also characterized by an extended 3V untranslated region (UTR; 1025bp) containing three AUUUA sequences that are likely to be a target for rapid degradation of its mRNA. Its coding region (1569 base pairs) shares 97.4% and 96.7% sequence identity with the corresponding regions of rainbow trout CYP1A1 and CYP1A3 genes, respectively. In addition, the deduced amino acid sequence of salmon CYP1A shows 96.9% and 96.2% identity with rainbow trout P450 1A1 and P450 1A3 enzymes, respectively. Phylogenetic analysis of a sample of CYP genes confirmed that Atlantic salmon CYP1A is closely related to rainbow trout CYP1A genes as well. Northern analysis showed that CYP1A mRNA is at a significantly higher level in the liver of Atlantic salmon injected intraperitoneally with h-Naphthoflavone (BNF), suggesting that this gene is inducible. Southern analysis verified the presence of at least one copy of the CYP1A gene in the genomic DNA of Atlantic salmon. These results reveal the transcriptional and inductive properties of an Atlantic salmon CYP1A gene with an extended 3V UTR, and provide molecular tools for studying the effects of xenobiotics on Atlantic salmon populations. D 2005 Elsevier B.V. All rights reserved. Keywords: CYP1A; Cytochrome P450; Atlantic salmon; Salmo salar; Cloning

1. Introduction Atlantic salmon populations are in a steep decline across the eastern seaboard, including both the United B

Atlantic salmon GenBank Accession Number: AF361643. T Corresponding author. Tel.: +1 517 353 9837; fax: +1 517 432 1699. E-mail address: [email protected] (W. Li). 0044-8486/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2004.12.025

States and Canada, especially the southern reaches of their native range (Parrish et al., 1998). This population fall has lead to the listing of Atlantic salmon on the Endangered Species List in the state of Maine as of November 2000 (U.S. Department of Interior, 2000). Due to the rapid reduction in feral salmon populations in the last few decades, the aquaculture industry has been under heavy pressure to maintain the genetic integrity of salmon stocks. In fact, it has been reported

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that as high as 94% of the Atlantic salmon that exist today reside in aquaculture facilities (Gross, 1998). Environmental pollutants have long been suspected of negatively impacting Atlantic salmon populations (Waldichuk, 1979; Parrish et al., 1998). Recent research has focused on the possibility that endocrine-disrupting chemicals, particularly 4-nonylphenol, may be affecting the return rates of Atlantic salmon in Canada (Fairchild et al., 1999; McMaster, 2001). The problems associated with salmon exposure to environmental pollution are twofold. First, one of the most sensitive stages of stream life, smoltification (Moyle and Cech, 2000), coincides seasonally with the peak agricultural application of pesticides (Albanis et al., 1998). Secondly, many studies suggest that endocrine-disrupting chemicals may have profound effects (decreased reproductive success, lower recruitment, etc.) on teleosts at sublethal concentrations (Little et al., 1990; Jones et al., 1998; Gagne et al., 1999), which over time, may lead to population decline. Therefore, the acute and chronic effects these chemicals have on Atlantic salmon development may be subtle, emphasizing the need to study their consequences from the molecular level. Research on the P450 detoxification system is broad and recently has become an important model in how organisms cope with exposure to organic contaminants. Cytochrome P450 constitutes a ubiquitous enzyme system noted for its inducibility and diversity (Andersson and Fo¨rlin, 1992; Mansuy, 1998). These enzymes have been found in all species studied to date including plants, animals, bacteria, and other organisms (Nelson et al., 1996). P450s are involved with the metabolism and detoxification of many organic compounds (Mehmood et al., 1996; Goksøyr and Husøy, 1998), such as inhaled odorants (Nef et al., 1989; Dahl and Hadley, 1991; Lazard et al., 1991), steroids (Brittebo and Rafter, 1984; Ding and Coon, 1994), drugs (Nef et al., 1989; Schlenk et al., 1993), and carcinogens (Kawajiri and Fujii-Kuriyama, 1991; Hrelia et al., 1996). Historically, mammals have been the primary models of P450 studies. More recently, fish species have been frequently used in P450 research. In fish, the most intensively studied P450 genes are CYP1As (Nelson et al., 1996; Stegeman, 2000). Their nucleotide sequences have been determined in rainbow trout (Heilmann et al., 1988; Berndtson and Chen, 1994), brook trout (Rees and Li, 2004), lake

trout (Rees and Li, 2004), plaice (Leaver et al., 1993), Atlantic tomcod (Roy et al., 1995), toadfish and scup (Morrison et al., 1995), killifish (Morrison et al., 1998), red sea bream (Mizukami et al., 1994), and sea bass (Stien et al., 1998). The induction mechanism of these genes through binding of toxicants to the Ahreceptor has been demonstrated (in review; Goksøyr and Husøy, 1998; Hahn, 1998; Hahn et al., 1998). These experiments have provided tools to understand the structure and function of cytochrome P450 proteins in general, and the role of CYP1A in metabolism of a variety of toxicants in particular. In Atlantic salmon, the P450 1A1 enzyme system has been studied extensively through several methods. Initially, P450 1A1 enzyme activity and inducibility were examined in Atlantic salmon liver through measurement of 7-ethoxyresorufin O-deethylase (EROD) activity (Goksøyr and Larsen, 1991; Larsen et al., 1992). Later, the utility of this P450 1A1 as a biomarker for environmental monitoring and toxicological testing was examined with an ELISA (enzyme-linked immunosorbent assay) using polyclonal and monoclonal antibodies raised against a purified form of Atlantic cod (Gadus morhua) P450 1A1 (Goksøyr and Husøy, 1992). Also, effects of xenobiotics on CYP1A mRNA levels of Atlantic salmon have been demonstrated using Northern hybridization (Grosvik et al., 1997). Recently, CYP1A mRNA induction by organic contaminants has been analyzed using quantitative PCR (Miller et al., 1999; Rees et al., 2003; Rees and Li, 2004). In mammalian species, it has been shown that an important mechanism to regulate fluctuating mRNA levels of CYP genes is through rapid degradation mediated by AUUUA repeats, a pentanucleotide that recurs frequently in the 3V untranslated region (UTR; Shaw and Kamen, 1986; Binder et al., 1989). In fish species, an extended 3V UTR with the recurrence of repeated AUUUA sequences has yet to be demonstrated in CYP1A cDNAs. Approaches toward understanding Atlantic salmon P450 1A1 structure, degradation, induction, and its evolutionary relationship to other teleost CYP1A cDNAs have been limited because its sequence is not known. Evidence is emerging that P450 enzymes can have an important role in developmental processes such as pattern formation, morphogenesis, cell differentiation, growth (Stoilov, 2001), and the development of

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reproductive organs (Arukwe and Goksøyr, 1997; Arukwe et al., 2001; Navas and Segner, 2001). This is particularly intriguing in reference to the status of Atlantic salmon across many rivers of the eastern U.S. and Canada. Arukwe et al. (2001) reported reduced plasma zona radiata protein and vitellogenin levels along with induction of the CYP1A gene in Atlantic salmon treated with the antiestrogenic compound PCB-77 in combination with the estrogenic compound nonylphenol, the first time this relationship has been demonstrated in fish. Likewise, it was discovered for the first time in fish that higher levels of estradiol (E2) have suppressive effects on the levels of CYP1A gene expression in rainbow trout (Navas and Segner, 2001). This new information necessitates the need to understand the genetic relationship between CYP1A metabolic activities and the developmental and reproductive toxicity associated with exposure of Atlantic salmon to environmental contaminants. The first step to understanding these relationships is to study the structure, function, and sequence of the CYP1A gene in Atlantic salmon. In order to address these concerns, we examined the cDNA sequence, deduced amino acid sequence, and inducibility of CYP1A in Atlantic salmon liver.

2. Materials and methods 2.1. Fish Thirty Atlantic salmon (Salmo salar) approximately 20 cm in length and 50 g in weight were acquired from a fish hatchery (Lake Superior State University, Sault Ste. Marie, Michigan) and acclimated for 2 weeks at the Michigan State University Lower River Laboratory in an 800 l flow through tank (600 l hr 1, 0.75 R) at 11.5 8C. A 12 h light–dark cycle was maintained during the acclimation period. Salmon were fed Purina AquaMaxn Grower 400 (lot A-5D04; Purina Mills, Inc.; St. Louis, MO) daily at a level of 3.0% body weight. Two days prior to tissue collection, salmon were taken off of feed. 2.2. Tissue collection Fish were sacrificed by an overdose of MS-222. The liver was removed immediately, sectioned 3 times,

13

stored in at least a 10 volume of RNALater n (Ambion; Austin, Texas), and subsequently stored at 80 8C. 2.3. RNA extraction Total RNA was extracted using TRIzolR Reagent according to the manufacturer’s protocol (Life Technologies; Rockville, MD), resuspended in 50 Al diethylpyrocarbonate-treated water, and quantified on a UV spectrophotometer (Beckman DUR 7400; Fullerton, CA). RNA solution was mixed with 3 volumes of 70% ethanol and 3 M sodium acetate to a final concentration of 0.3 M and stored at 80 8C until further analysis (Sambrook et al., 1989). 2.4. First strand cDNA synthesis Total RNA (1–5 Ag) was mixed with 10 pM Al 1 Oligo d(T)18 and diluted with deionized water to a final volume of 12 Al. This mixture was heated at 70 8C for 10 min and quick chilled on ice. Next, 4 Al 5 first strand buffer, 2 Al 0.1 M dithiothreitol, and 1 Al 10 mM dNTP were added. After incubation of this mixture at 42 8C for 2 min, 200 units of MMLVRT (Life Technologies) were added making the final reaction volume 20 Al. The reaction mixture was incubated at 42 8C for 50 min and inactivated at 70 8C for 15 min. Finally, 10 units of RNAseH (Life Technologies) were added, incubated at 37 8C for 20 min, and inactivated at 94 8C for 5 min. The synthesized cDNA was stored at 20 8C until used for PCR. 2.5. Polymerase chain reaction (PCR) To select a region of the CYP1A gene for initial amplification, we first aligned three rainbow trout CYP1A sequences (Heilmann et al., 1988; Berndtson and Chen, 1994) using the CLUSTAL W algorithm. Then, using a region spanning the heme-binding domain and the helical region, we designed degenerate primers that flanked a region of approximately 580 bp in known teleost genes (Table 1). Degenerate primers were synthesized at the Macromolecular Structure, Synthesis, and Sequencing Facility, Michigan State University.

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Table 1 Primers used in PCR and RACE amplifications of CYP1A cDNA Application

Nucleotide sequence

Primer name

PCR

5V-CCT TGK KRA TGA ART AGC CMT TGA GG-3V 5V-CCA ACR TMA TCT SWG GMA TGT GC-3V 5V-CTG TCT TGG GCC GTT GTG TAC CTT GTG-3V 5V-TAT CCT TGA TCG TGC AGT GTG GGA TGG-3V 5V-GCT CAG TTC CTG ATG CAG TCT TTC CTG-3V 5V-CGG CTC ATT TGG CTC ATA ACG GAA GAT-3V

WML38 WML39 WML51 WML52 WML55 WML56

RACE

PCR master mix consisted of the following constituents (final reaction concentrations in parentheses): 2 Al of cDNA, dNTPs (200 AM), 5 PCR Buffer (1), MgCl2 (4 mM), WML38 sense primer (500 nM), and WML39 antisense primer (500 nM). The final 50 Al reaction was amplified in a PTC-200 MJ Research Thermocycler (1 cycle 94 8C 4 min; 30 cycles 94 8C 1 min, 56 8C 45 s, 72 8C 1 min; 1 cycle 72 8 5 min; 1 cycle 4 8C forever).

sequencing of templates greater than 1.7 kb. DNA sequence comparison and analysis were performed either with BLAST (http://www.ncbi.nlm.nih.gov/ BLAST), Sequencher 3.1 (Gene Codes Corporation; Ann Arbor, MI), MacVector 6 (Oxford Molecular Group PLC; Huntsville, MD), or DNASTAR (DNASTAR, Inc.; Madison, WI).

2.6. Molecular cloning

Gene specific primers (GSP) for 5Vand 3VRACE were designed by using the sequence of the previous PCR product. RACE was carried out utilizing the Advantage II RACE system (Clontech; Palo Alto, CA) according to the manufacturer’s protocol. One microgram of total RNA from Atlantic salmon liver was used as template for synthesis of 5Vand 3VRACE Ready cDNA. GSPs were used individually with the universal primer mix supplied in the kit for both 3Vand 5VRACE. Nested GSPs were used to enhance the efficiency of RACE amplification (Table 1). To clone a full length CYP1A cDNA, a two-step long distance RACE-PCR was utilized. Briefly, a 3VRACE GSP (WML51) was designed to extend the cDNA to the 3Vend (poly A tail) while a 5VRACE GSP (WML52) was used to extend to the 5Vend. The two RACE products produced by this method were expected to have a region of overlap of ~200 bp. Both RACE products were cloned and sequenced as previously described and analyzed using BLAST on the world-wide-web. To eliminate the possibility of producing a hybrid sequence of a full length CYP1A gene, we conducted a second RACE with a GSP designed upstream from the start codon. The expected product from this RACE would include all functional domains of a single, full-length CYP1A gene.

All PCR products were size-fractionated on a 1% agarose gel containing 0.1 Ag/ml ethidium bromide (Sambrook et al., 1989). PCR products were cleaned using the Wizard DNA clean-up system (Promega Corporation; Madison, WI), ligated into a pGEM–T Easy vector (Promega Corporation), and subsequently electroporated into DH5a competent cells (Clontech). Plasmid DNA minipreps were extracted by the Wizard DNA Miniprep Kit (Promega Corporation). 2.7. Sequencing Preliminary sequencing reactions were performed using the Sequitherm EXCELk II DNA Sequencing Kit (Epicentre Technologies; Madison, WI). Hexlabeled T7 (forward sequencing primer) and SP6 (reverse sequencing primer) primers were purchased from Integrated DNA Technologies (Coralville, IA). Once CYP1A positive inserts were identified using dideoxy sequencing and BLAST, extended sequencing was carried out by the Plant Biology DNA Sequencing Facility, Michigan State University. Primer walking was used for shorter inserts, between 1 and 1.5 kb. In vitro transposon insertion was used (Genome Priming System, New England Biolabs) for

2.8. Rapid amplification of cDNA ends (RACE)

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2.9. Phylogenetic analysis The coding region of Atlantic salmon CYP1A was aligned to the coding region of a sample of P450 genes using the CLUSTAL W algorithm. Genetic relationships and distances were generated using the Neighbor-joining method. This same analysis was also performed on CYP1A amino acid sequences as well. Genes selected for this analysis comprised mostly teleost CYP1A genes, several mammalian CYP1A genes, and two rainbow trout P450 genes from subfamilies 2 and 3 (accession numbers and references for the genes used in the analysis are given in Table 2). Initially, teleost genes were aligned after which subsequent mammalian genes were aligned. Finally, rainbow trout CYP2K1 and CYP3A27 were added for the alignment analysis. 2.10. Induction To demonstrate the inducibility of the CYP1A gene in Atlantic salmon, randomly selected individuals (n=4) were sampled and given either an intraperitoneal injection of h-Naphthoflavone (BNF,

15

a potent inducer of CYP1A; 50 mg kg 1 body weight) dissolved in corn oil (10 mg BNF ml 1 corn oil) or corn oil alone. Individual salmon were then placed for 48 h in a 40 l flow-through aquarium (20 l h 1; 0.5 R), sacrificed with an overdose of MS-222, and dissected for liver tissues which were immediately stored in RNALatern (Ambion). 2.11. Northern blotting Total RNA (10 Ag) from control and induced samples was separated on 1% agarose-formaldehyde gels and transferred to positively charged nylon membranes (Ambion). The P450 full sequence clone (2.626 kb) was used as a probe and was P32-labeled using the Random Primed DNA Labeling System (Life Technologies). Blots were hybridized and washed according to the manufacturer’s protocol. Probes for actin mRNA were produced by the same labeling technique as above from a 400 bp partial Atlantic salmon hactin cDNA (Rogers et al., 1999, unpublished, GenBank Accession AF012125) fragment confirmed by sequencing.

Table 2 CYP genes and accession numbers used in the phylogenetic analysis Name in analysis (general name)

Reference

GenBank Accession Number

Fundulus heteroclitus CYP1A (Killifish) Liza saliens CYP1A (Mullet) Dicentrarchus labrax CYP1A (Seabass) Limanda limanda CYP1A (Sand dab) Stenotomus chrysops CYP1A (Scup) Chaetodon capistratus CYP1A (Butterflyfish) Opsanus tau CYP1A (Toadfish) Microgadus tomcod CYP1A (Tomcod) Oncorhyncus mykiss CYP1A1V1 (Rainbow trout) Oncorhyncus mykiss CYP1A1V3 Oncorhyncus mykiss CYP1A1V2 Oncorhyncus mykiss CYP1A3 Salmo salar CYP1A (Atlantic salmon) Anguilla japonica CYP1A (Japanese eel) Homo sapiens CYP1A1 (Human) Macaca iris CYP1A1 (Crab eating monkey) Mus musculus CYP1A1 (Mouse) Homo sapiens CYP1B1 Rattus norvegicus CYP1B1 (Rat) Oncorhyncus mykiss CYP2K1 Oncorhyncus mykiss CYP3A27

Morrison et al., 1998 Sen et al., 1999 (unpublished) Stien et al., 1998 Robertson, 1997 (unpublished) Morrison et al., 1995 Vrolijk et al., 1995 (unpublished) Morrison et al., 1995 Roy et al., 1995 Berndtson and Chen, 1994 Bailey et al., 1997 (unpublished) Bailey et al., 1997 (unpublished) Berndtson and Chen, 1994 Rees et al. (this paper) Mitsuo et al., 1999 (unpublished) Jaiswal et al., 1985 Ohmachi et al., 1993 (unpublished) Kimura et al., 1984 Sutter et al., 1994 Bhattacharyya et al., 1995 Buhler et al., 1994 Lee et al., 1998

AF026800 AF072899 U78316 AJ001724 U14162 U19855 U14161 L41886 S69278 U62797 U62796 S69277 AF361643 AB020414 K03191 D17575 Y00071 U03688 X83867 L11528 U96077

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2.12. Southern blotting

3. Results

Southern blots were carried out by first extracting Atlantic salmon genomic DNA using the WIZARD Genomic Preps (Promega Inc.). Genomic DNA (5 Ag) was digested using HindIII, EcoR1, and BamH1 restriction endonucleases and loaded into a 0.7% agarose gel. The membrane was probed with the same P32-labeled full-length CYP1A cDNA as in the Northern analysis.

3.1. Atlantic salmon CYP1A Initial PCR amplification of CYP1A from Atlantic salmon liver cDNA produced a fragment approximately 580 bp long. Molecular cloning and sequencing of this PCR product established that it was homologous to rainbow trout CYP1A and exactly 576 bp long. The 3V and 5V RACE experiments with gene specific

TGTGCAGAAGCCACAAAAAAAAATACATAAAAATGGTTCTCATGATACTACCCATTATTGGCTCAGTCTCTGTGTCTGAGGGGCTGGTGGCCATGG M V L M I L P I I G S V S V S E G L V A M TAACACTATGCCTGGTGTACATGTTCATGAAGTACAAGCACACAGAGATCCCAGAGGGACTGAAACGGCTCCCAGGACCAAAGCCCCTGCCCATCA V T L C L V Y M F M K Y K H T E I P E G L K R L P G P K P L P I TCGGGAATGTGCTGGAGGTGCACAACAACCCTCACCTCAGCCTGACTGCCATGAGTGAGCGCTACGGCTCAGTCTTCCAGATCCAGATAGGGATGC I G N V L E V H N N P H L S L T A M S E R Y G S V F Q I Q I G M GGCCTGTGGTTGTTCTGAGTGGCAGCGAGACAGTCCGCCAGGCTCTTATCAAGCAAGGGGAAGACTTCGCCGGGAGGCCCGATCTATACAGCTTCA R P V V V L S G S E T V R Q A L I K Q G E D F A G R P D L Y S F AGTTCATCAACGACGGCAAGAGCTTGGCCTTCAGCACCGACAAGGCTGGGGTATGGCGCGCCCGCCGCAAGCTAGCTATGAGCGCCCTCCGCTCTT K F I N D G K S L A F S T D K A G V W R A R R K L A M S A L R S TCGCCACCCTGGAGGGATCGACCCCAGAGTACTCCTGTGCCCTGGAGGAGCACGTCTGCAAGGAGGGAGAGTACCTGGTAAAACAGCTGACCTCCG F A T L E G S T P E Y S C A L E E H V C K E G E Y L V K Q L T S TCATGGATGTCAGTGGCAGCTTTGACCCCTTCCGCCATATTGTCGTATCGGTGGCCAACGTCATCTGTGGAATGTGCTTCGGCCGGCGCTACAGCC V M D V S G S F D P F R H I V V S V A N V I C G M C F G R R Y S ATGATGACCAGGAGCTGTTGAGCTTGGTGAACTTGAGTGATGAGTTTGGGCAGGTGGTGGGCAGCGGCAACCCTGCAGACTTCATTCCCATCCTTC H D D Q E L L S L V N L S D E F G Q V V G S G N P A D F I P I L GTTACCTACCAAACCGCACCATGAAGAGGTTTATGGATATCAATGACCGTTTCAACGCCTTTGTGCAGAAGATTGTCAGTGAGCACTATGAAAGCT R Y L P N R T M K R F M D I N D R F N A F V Q K I V S E H Y E S ATGACAAGGACAACATCCGTGACATCACTGACTCCCTCATTGACCACTGTGAGGACAGGAAACTAGATGAGAATGCCAACATCCAGGTTTCTGATG Y D K D N I R D I T D S L I D H C E D R K L D E N A N I Q V S D AGAAGATTGTGGGCATTGTCAATGATCTGTTTGGGGCAGGTTTTGACACTATCAGCACAGCTCTGTCTTGGGCTGTTGTGTACCTTGTGGCTTACC E K I V G I V N D L F G A G F D T I S T A L S W A V V Y L V A Y CAGAGATCCAGGAAAGACTGCATCAGGAACTGACGGAAAAGGTGGGATTGAATCGCACTCCCCGTCTCTCAGACAAAACCAACTTACCTCTGCTGG P E I Q E R L H Q E L T E K V G L N R T P R L S D K T N L P L L AAGCCTTCATCCTGGAGATCTTCCGGCACTCTTCCTTCCTGCCGTTCACCATCCCACACTGCACGATCAAGGATACATCCCTCAATGGCTACTTCA E A F I L E I F R H S S F L P F T I P H C T I K D T S L N G Y F TTCCCAAGGACACCTGTGTCTTCATCAACCAGTGGCAGGTCAACCATGACCCGGAGCTGTGGAAGGAGCCTTCTTTATTCAACCCTGACCGTTTCC I P K D T C V F I N Q W Q V N H D P E L W K E P S L F N P D R F TGAGTGCTGATGGCACAGAACTCAACAAGCTGGAGGGGGAGAAGGTGCTCGTATTCGGCATGGGCAAGCGCCGCTGCATCGGTGAGGCCATCGGAC L S A D G T E L N K L E G E K V L V F G M G K R R C I G E A I G GCAACGAGGTCTACCTCTTCTTGGCCATCCTGCTCCAAAGGCTGCGCTTCCAGGAGAAACCTGGGCACCCGCTGGACATGACCCCAGAGTACGGCC R N E V Y L F L A I L L Q R L R F Q E K P G H P L D M T P E Y G TCACTATGAAGCACAAGCGCTGTCAGCTGAAGGCTAGCCTGCGGCCATGGGGGCAGGAGGAGTGAAGGACATGGTCACATTTATGATTCTCAACAT L T M K H K R C Q L K A S L R P W G Q E E CACTATAACTGATTTATAGTTAGCCCTACATACTTGATGGCATGAGATGAGTTCAGATTTAAAAGCCAGAGGGTACATTTTCTCTCTAGGAAAGCC CGGGCTTGACAACTCTCTTGATTCATAGGAGAATTAACAGTGAGGGAAAACTGAGTCAAATTGTAGACAATTTGAAATCGATAAAATTATAATCTG AAATGGTATGTAAGGGGATTACAAACAAGTGTTGTATTGGATAAAGGACCTTCATGACCAGCATACCACAGGGCCTACTAGAGTTTGTCAGGTGTT GCTTCAGAAGATATCAGGAGAGACTATTGCTGTTTAGCTGCCTTTTTGCCTACATCATCGTGTTTTACTGCTCCTCTTGCCTTGACCACAGCTCAT ATACTGTATCAGCTCGCTTTAAAGAGGATCAATCTATAAAACATACACATAAACCCATAGTGGGGTTTTAAAGAGCATTGTTTTTTAGTTCCATGT TTGGGTATAATTTTTTGCTACAGAATTTTTTGAGGCAAAAATGTTACAACCTACATGATAAAGTGCCTGTTCTCGTACTTTTGAGTTATTGTTCAG GTGGTCACAGACAGAGTTGAATGGATGTATCCTCCCAATTCTAAGCTAGATATTTTTATTCCACTGAATAAATCAGTCAAATGTGGACACAACACA CTGAAGCTATGTTTGTATCCCACCAACATGTGATTGATTGTTTGAGTACATTTTTAAGCCTAGATGTATTTTGAAAAGTCCTTTTTTGAAATGTGC CTAAAATGTGTATATATTGTGGTGGCTTTGTATGACTTTATCAGGATGCCAAAATACGTATGTATTACTGACTGTGTTATAAATGTCAACATTTTA TAATGTAACGAAGGACTCTTGTAAACTAAAACCCCAACTGTATACACTATGCATTGTGTTGTTGGTAGCTATCTAGGTAGCTACTGAAATAAAAGC TGAAAAGCAAAAAAAAAAAAAAAAAAAAAAAAAA

96 21 192 53 288 85 384 117 480 149 576 181 672 213 768 245 864 277 960 309 1056 341 1152 373 1248 405 1344 437 1440 469 1536 501 1632 522 1728 1824 1920 2016 2112 2208 2304 2400 2496 2592 2626

Fig. 1. cDNA and deduced amino acid residue sequence of Atlantic salmon CYP1A. The start codon, arginine residue critical to enzymatic function (position 246), heme-binding cysteine codon (position 463), stop codon (position 523), ATTTA (AUUUA) sequences, and putative poly-adenylation signal are all underlined and boldfaced.

C.B. Rees et al. / Aquaculture 246 (2005) 11–23 F G MG K R R C I G

Butterflyfish 1A

Vrolijk et al. 1995

F G L G R R R C I G

Killifish 1A

Morrison et al. 1998

F G MG K R R C I G

Mullet 1A

Sen et al. 1999

F G MG K R R C I G

Salmon 1A

Rees et al. (this paper)

F G MG K R R C I G

Sand dab 1A

Robertson 1997

F G MG K R R C I G

Scup 1A

Morrison et al. 1995

F G L G KR R C I G

Seabass 1A

Stien et al. 1998

F G L G KR R C I G

Toadfish 1A

Morrison et al. 1995

F G MG K R R C I G

Tomcod 1A

Roy et al. 1995

F G MD K R R C I G

Trout 1A1V1

Berndtson and Chen 1994

F G MG K R R C I G

Trout 1A1V2

Bailey et al. 1997

F G MG K R R C I G

Trout 1A1V3

Bailey et al. 1997

F G MG K R R C I G

Trout 1A3

Berndtson and Chen 1994

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Fig. 2. Alignment and comparison of the heme-binding region (amino acid residues 456–465) from representative teleost P450 1A enzymes. Boxed areas show amino acid residue differences from salmon P450 1A.

primers obtained from this preliminary sequence resulted in CYP1A fragments of 1669 bp and 1394 bp, respectively. Both 3V and 5V RACE fragments were roughly 95% homologous to both rainbow trout

CYP1A1 and CYP1A3 cDNA molecules, and were found sharing a region of overlap of 205 bp. To verify that the two overlapping products encode a single CYP1A gene, a long distance 3V RACE

Table 3 Phylogenetic analysis of cytochrome P450 genes

Dicentrarchus labrax CYP1A Chaetodon capistratus CYP1A Stenotomus chrysops CYP1A Limanda limanda CYP1A Fundulus heteroclitus CYP1A Liza saliens CYP1A Opsanus tau CYP1A Microgadus tomcod CYP1A Oncorhyncus mykiss CYP1A1V1 Oncorhyncus mykiss CYP1A1V3 Oncorhyncus mykiss CYP1A1V2 Oncorhyncus mykiss CYP1A3 Salmo salar CYP1A Anguilla japonica CYP1A Homo sapiens CYP1A1 Macaca iris CYP1A1 Mus musculus CYP1A1 Homo sapiens CYP1B1 Rattus norvegicus CYP1B1 Oncorhyncus mykiss CYP2K1 Oncorhyncus mykiss CYP3A27

65.5 60

50

40

30

20

10

0

Gene accession numbers and references of sequences used for this analysis are given in Table 2. Multiple sequence alignment was carried out using the CLUSTAL W algorithm (1515 nucleotides of the coding region). The phylogenetic tree and genetic distances were determined using the Neighbor-joining method. Cytochrome P450 nomenclature followed that of Morrison et al. (1998).

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Table 4 Phylogenetic analysis of cytochrome P450 proteins

Fundulus heteroclitus CYP1A Liza saliens CYP1A Dicentrarchus labrax CYP1A Limanda limanda CYP1A Stenotomus chrysops CYP1A Chaetodon capistratus CYP1A Opsanus tau CYP1A Microgadus tomcod CYP1A Oncorhyncus mykiss CYP1A1V1 Oncorhyncus mykiss CYP1A1V3 Oncorhyncus mykiss CYP1A1V2 Oncorhyncus mykiss CYP1A3 Salmo salar CYP1A Anguilla japonica CYP1A Homo sapiens CYP1A1 Macaca iris CYP1A1 Mus musculus CYP1A1 Homo sapiens CYP1B1 Rattus norvegicus CYP1B1 Oncorhyncus mykiss CYP2K1 Oncorhyncus mykiss CYP3A27

120.5 120

100

80

60

40

20

0

Analysis was carried out as described in Table 3 utilizing 505 amino acid residues of each enzyme. Accession numbers and references for the genes encoding these enzymes are given in Table 2.

fragment of 2626 bp was obtained. Sequence analysis indicated this product included 32 bp of the 5V untranslated region (UTR), a 1569 bp coding region, and a 1025 bp 3V UTR containing 3 AUUUA sequences. It encodes a protein of 523 amino acid residues. The obtained sequence also possesses all major functional domains and characteristics of previously discovered CYP1A molecules including the heme-binding cysteine (position 463), arginine codon (position 246) integral to enzymatic function, stop codon (position 523), and polyadenylation signal (Fig. 1).

rainbow trout P450 1A1 and 96.2% amino acid similarity with rainbow trout P450 1A3. Likewise, the salmon CYP1A protein demonstrates between 68 and 83% amino acid homology with other teleost CYP1A proteins. The heme-binding region of Atlantic salmon CYP1A, encompassing the amino acid sequence FGMGKRRCIG (positions 456–465), is identical to the same region of many fish CYP1A, or differs by only 1 or two amino acid residues (Fig. 2).

1

2

3.2. Phylogenetic analysis 2.7 kb

This cloned RACE cDNA shares 97.4% sequence identity with the corresponding rainbow trout CYP1A1 gene and 96.7% sequence identity with rainbow trout CYP1A3. In addition, this gene shares between 70 and 76% nucleotide homology with other teleost CYP1A genes. The deduced amino acid sequence shows 96.9% amino acid identity with

Fig. 3. Northern analysis of CYP1A total RNA from Atlantic salmon liver. Ten micrograms of total RNA from each individual 2 d after exposure was applied to each lane. Lane 1: untreated salmon liver; Lane 2: salmon liver treated with h-Naphthoflavone. The 2.7 kb CYP1A mRNA was hybridized with a P32-labeled CYP1A fulllength cDNA probe.

C.B. Rees et al. / Aquaculture 246 (2005) 11–23

1

2

3

19

4. Discussion 6 kb

5.1 kb

Fig. 4. Southern analysis of CYP1A genomic DNA from Atlantic salmon muscle. Lane 1: EcoR1 digested DNA; Lane 2: HindIII digested DNA; Lane 3: BamH1 digested DNA. The membrane was probed with a P32-labeled 2.7 kb full-length cDNA probe.

Multiple sequence alignment using the CLUSTAL W algorithm followed by construction of a phylogenetic tree using the Neighbor-joining method suggested that Atlantic salmon CYP1A is most highly related to rainbow trout CYP1A of the genes compared (Table 3). The phylogenetic tree also showed that Atlantic salmon CYP1A is related to representative teleost CYP1A genes, followed by mammalian CYP1A cDNAs, and finally members of rainbow trout CYP2 and CYP3 genes. The same analysis based on predicted amino acid sequences demonstrated a similar relationship (Table 4). The phylogenetic methods carried out here showed that Atlantic salmon CYP1A is a close relative of rainbow trout CYP1A genes. 3.3. Induction of CYP1A In the Northern analysis, the CYP1A probe hybridized with an mRNA approximately 2.7 kb in length from salmon treated with h-Naphthoflavone as well as salmon injected with corn oil only (Fig. 3). In addition, a probe designed from an amplified PCR product of the Atlantic salmon h-actin gene hybridized with mRNA of approximately 1.2 kb from both control and induced samples with both bands showing roughly the same density. 3.4. Southern analysis Southern blots demonstrated the presence at least one copy of a CYP1A gene in each of the digests (HindIII, EcoR1, BamH1). The full-length Atlantic salmon CYP1A probe hybridized to genomic fragments in the range of 5.1–6.0 kb. Refer to Fig. 4 for a picture of these results.

There are several lines of evidence that suggest the cDNA we have cloned is a full-length CYP1A molecule from Atlantic salmon. It contains all of the positional characteristics of a full-length coding cDNA including a start codon and a stop codon followed by a poly A tail. This cDNA also carries characteristics of many teleost CYP1A genes such as the heme-binding domain, arginine codon integral to enzymatic function, and a rather large 3V UTR (1025 bp). The coding region (1569 bp), which encodes a protein of 523 amino acid residues, is the same size as the rainbow trout P450 1A protein. This gene shows 97.4% and 96.7% sequence identity with rainbow trout CYP1A1 and CYP1A3, respectively. The deduced amino acid sequence of this salmon gene is highly homologous to rainbow trout CYP1A1 and CYP1A3 proteins (96.9% and 96.2%, respectively). Furthermore, this gene was demonstrated to be inducible by BNF, an important characteristic of CYP1A genes. Whether Atlantic salmon has two CYP1A genes needs to be explored. Although our Southern and Northern blots verify the presence of one CYP1A gene, the results are inconclusive in determining the potential for multiple CYP1A genes in the Atlantic salmon genome. In most fish species, only one copy of the CYP1A gene has been confirmed (Stegeman, 2000). It has been demonstrated that rainbow trout has two separately functioning CYP1A genes, CYP1A1 and CYP1A3 (Berndtson and Chen, 1994). It would not be surprising if species of Salmo, which is the sister genus of Oncorhyncus (Allendorf and Waples, 1996), are found to possess two CYP1A molecules with highly homologous sequences. This raises two issues. The first issue is whether the isolated cDNA is from a single CYP1A gene. During initial PCR amplification, the primer sequences utilized in these experiments showed 100% homology to both rainbow trout CYP1A genes. In addition, the product amplified also showed less than a 1% difference in sequence identity between both CYP1A genes in rainbow trout. Therefore, the RACE primers initially utilized also were bnon-distinguishingQ between the sequences of the possible CYP1A genes in Atlantic salmon liver. The first CYP1A gene published in rainbow trout was named CYP1A1 (Heilmann et al., 1988). Recently, it has been reported

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that this gene actually was a chimeric sequence incorporating both CYP1A1 and CYP1A3 character (Rabergh et al., 2000). For that reason, even though overlapping 5Vand 3VRACE CYP1A products were produced, we were not sure whether this was a chimeric sequence. Since CYP1A genes in rainbow trout differ mostly in their 5VUTR, we designed another RACE primer in a nonhomologous 5Vregion upstream from the start codon, and with this primer conducted a long distance RACE to obtain a product consisting entirely of coding region and 3VUTR. Therefore, the cDNA described here is likely a single CYP1A gene from Atlantic salmon liver. The second issue dealt with is the naming of this gene. As discussed in previous teleost CYP1A cloning papers (Nelson et al., 1996; Morrison et al., 1998), careful considerations are essential to correctly assign nomenclature to each new CYP1A gene published. In recent years, functional properties as well as extended sequence analyses have become necessities in distinguishing families and subfamilies of CYP1A genes. One of the major ways to distinguish between CYP1A1 and CYP1A3 in rainbow trout is by looking for presence or absence of xenobiotic regulatory elements (XRE) in the 5V flanking region of each gene. CYP1A1 contains no XREs while CYP1A3 contains two XREs (Berndtson and Chen, 1994). Unfortunately, the 5VUTR of this salmon gene is not large enough to verify whether or not it contains XREs. Another possible way to assign a name to the CYP1A gene from Atlantic salmon is by comparison to rainbow trout CYP1A1and CYP1A3. However, rainbow trout CYP1A genes show 96% homology suggesting a more recent gene duplication event, and thus making comparisons in this manner problematic. Conservatively, we have assigned this Atlantic salmon P450 gene as CYP1A. However, based on strict sequence comparison, it is likely this cDNA is a CYP1A1 molecule. It is interesting that this gene has a 3VUTR as long as 1025 bp. The RNA sequence AUUUA recurs frequently in the 3VUTR of many CYP1A genes. In most mammalian species, CYP1A1 genes generally have 1 or 2 AUUUA, yet in hamster, it has been shown there are four AUUUA repeats (Sagami et al., 1991). This sequence has been implicated in RNA degradation (Fukuhara et al., 1989; Shaw and Kamen, 1986; Binder et al., 1989). Gene expression can be controlled by a change in mRNA stability (Alberts et al., 1994). Most

RNAs in eukaryotic cells are stable lasting for hours at a time; however, some mRNAs, whose levels vacillate frequently, harbor sequence elements which stimulate their own degradation (Alberts et al., 1994). In most cases the pentanucleotide AUUUA repeated several times in the 3VUTR serves as the consensus sequence for the binding of an endonuclease to target first for deadenylation of the mRNA and ultimately degradation (Jackson and Standart, 1990; Sachs, 1993; Ross, 1995; Jacobson and Peltz, 1996; Lewin, 1997). It is likely that the three AUUUA elements in Atlantic salmon CYP1A may also serve as the targets for the rapid removal of the CYP1A mRNA in cells. The kinetics behind this targeted degradation will likely reveal the mechanisms behind acute and chronic toxicological responses of Atlantic salmon to organic contaminants. A 3VUTR of this length with three AUUUA sequences has not been reported or discussed in teleost CYP1As previously. Further studies are needed to characterize the family of cytochrome P450 genes in Atlantic salmon and to understand their complex diversity and evolutionary relationships to the same genes of other teleosts and mammals. It is apparent that P450 genes have been genetically modified in many ways to account for and metabolize the many compounds with which fish interact throughout their life history. As Atlantic salmon populations continue to suffer, it becomes more important to understand the complex molecular relationships between environmental pollution and the detoxification system of this species. This same line of thinking also applies to maintaining the genetic diversity of this species from an aquaculture standpoint. New research has begun to reveal the links between environmental contaminant exposure, CYP1A expression, and reproductive health in Atlantic salmon (Stoilov, 2001; Navas and Segner, 2001; Arukwe et al., 2001). The future of Atlantic salmon may therefore reside in the hands of the aquaculture of this species, with an important emphasis on preserving its genetic makeup. In conclusion, we have cloned a single, fulllength CYP1A cDNA from Atlantic salmon. This cDNA contains all functional domains of teleost CYP1A genes, is most closely related to rainbow trout CYP1A1 through sequence homology comparisons and phylogenetic analysis, and is BNF inducible. This gene appears to be targeted for rapid degradation due to the presence of AUUUA sequen-

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ces in its 3VUTR. These studies along with the results in this paper lay a foundation for studying the molecular relationships between environmental contaminant exposure, CYP1A expression, and reproductive behavior in Atlantic salmon.

Acknowledgments Roger Greil and Dr. Trent Sutton of Lake Superior State University Fish Hatchery donated Atlantic salmon for this study. Sequencing expertise was provided by Dr. Tom Newman of the MSU DNA Sequencing Facility. The lab of Dr. Paul Coussens provided space and assistance during Northern and Southern blotting experiments. Marla Sanborn assisted with RACE and molecular cloning. Funding for this project was provided by the Jaqua Foundation and Michigan State University.

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