Blackwell Science, LtdOxford, UKFISFisheries Science0919 92682005 Blackwell Publishing Asia Pty Ltd713465470Original ArticleOyster amino acid transporter/H Toyohara et al.
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2005; 71: 465–470
Osmo-responsive expression of oyster amino acid transporter gene and analysis of the regulatory region involved Haruhiko TOYOHARA,1* Masashi IKEDA,1a Chika GOTO,1b Hideki SAWADA,1 Masatomi HOSOI,1 Kazuaki TAKEUCHI,1c Isao HAYASHI,1 Shintaro IMAMURA2d AND Michiaki YAMASHITA2 1
Division of Applied Biosciences, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502 and National Research Institute of Fisheries Science, Yokohama 236-8648, Japan
2
ABSTRACT: To elucidate the involvement of amino acid transporter in osmotic adaptation of oysters, the expression of the amino acid transporter gene in response to environmental osmotic changes was investigated. As expected, the expression of the amino acid transporter gene was increased by hyper-osmotic stress, probably to elevate intracellular osmolality. Unexpectedly, the expression was also increased by hypo-osmotic stress, and the level of expression was higher than that induced by hyper-osmotic stress. To identify the region regulating the expression of the oyster amino acid transporter gene in response to changes in environmental osmolality, the 5¢-flanking region of approximately 2.3 kb upstream from the translation start site was cloned. Expression vectors with luciferase as a reporter gene driven by 5¢-flanking regions with different lengths were constructed and their promoter activities were compared. As a result, the osmo-responsive regulatory region responding to osmolality by both hyper- and hypo-osmolality was found within 132 bp from the transcription start site. KEY WORDS: amino acid, Crassostrea gigas, osmolality, osmolyte, oyster, promoter, transporter.
INTRODUCTION Osmoconforming marine animals have mechanisms to adjust the concentration of intracellular osmolytes in order to adapt to fluctuations in the salinity of ambient seawater.1 Cells export osmolytes to decrease the intracellular osmolality under hypo-osmotic conditions, while they import them to increase the intracellular osmolality under hyper-osmotic conditions. Free amino acids are demonstrated to contribute dominantly to the intracellular osmolality in marine bivalves.2 The giant Pacific oyster is the most important oyster species for the shellfish industry. It has been successfully introduced into various countries and accounts for approximately 80% of world oyster
*Corresponding author: Tel: 81-75-753-6446. Fax: 81-75-753-6446. Email:
[email protected] a Present address: The Ministry of Agriculture, Forestry and Fisheries of Japan, Tokyo 100-0013, Japan.bPresent address: Nippon Lever K. K, Tochigi 321-3325, Japan.cPresent address: Nippon Suisan Kaisha Ltd. Tokyo 192-0906, Japan.dPresent address: Department of Cancer Biology, Dana-Faber Cancer Institute, Harvard Medical School, 44 Bunney, Boston, MA 02115, USA. Received 30 September 2004. Accepted 5 November 2004.
production.3,4 In its natural habitat, like other species of oysters, the giant Pacific oyster has been shown to possess the ability to adapt to a wide range of osmolalities.5–7 Recently, Hosoi et al. reported that the giant Pacific oyster is able to live in a wide range of osmolalities owing to its cellular osmo-regulatory mechanism that utilizes amino acids as osmolytes.8 In mammals, membranous protein molecules that play an important role in the import and export of osmolytes have been identified and characterized. In the hypo-osmotic response, osmolytes are exported non-specifically through various channels.9 In the hyper-osmotic response, however, osmolytes are imported through osmolyte-specific transporters.10,11 In fish, cellular response to osmotic change is particularly important in the epithelial cells. We have identified taurine transporter genes that respond to hyper-osmolality in carp and tilapia and have demonstrated their taurine importing function in COS-7-expression experiments.12,13 In marine invertebrates, however, the molecular mechanism of osmolyte transport has hardly been studied. Recently, we successfully identified the taurine transporter gene in the Mediterranean blue
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mussel Mytilus galloprovincialis (accession number AB190909) and the amino acid transporter gene in the Pacific oyster (accession number AB185408). The deduced amino acid sequence of mussel taurine transporter consists of 666 amino acids and it transports taurine, while the Pacific oyster amino acid transporter consists of 692 amino acids and it transports leucine, phenylalanine, threonine and valine. Details as to structure and function of these transporters will be published elsewhere. In the present study, we examined the expression of the oyster amino acid transporter gene in response to osmotic changes and attempted to identify the promoter region that regulates its expression in response to osmotic stresses. MATERIALS AND METHODS Materials Giant Pacific oysters Crassostrea gigas used in the present study were purchased alive from a commercial supplier. All other materials were of the highest quality commercially available. Exposure of oysters to hyper-osmotic and hypo-osmotic stresses The Pacific oysters were acclimatized in artificial seawater (ASW; Marine Merit, Matsuda, Japan) of 3.0% salinity (100% ASW) for at least 1 week without feeding before exposure to osmotic stresses. About 20 oysters were maintained in a 50-L tank with aeration. Water temperature was kept at 15∞C. To expose oysters to hypo-osmotic stress, oysters acclimatized to 100% ASW were transferred to 50% ASW corresponding to 1.5% salinity. To expose them to hyper-osmotic stress, oysters acclimatized to 100% ASW were transferred to 150% ASW corresponding to 4.5% seawater. A part of the shell edge (about 10 mm long and 5 mm wide) of each specimen was chipped away in order to ensure the free exchange of seawater between the inside and outside of the shell. Oysters were taken at 0, 2, 8 and 24 h after exposure to the osmotic changes and used for Northern blot analysis. Northern blot analysis Routine molecular cloning techniques used in the present study were carried out according to standard procedures.14 Total RNA was isolated from the gill and adductor muscle by the guanidine isothio-
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cyanate/cesium chloride method. Northern blot analysis was performed on total RNA (10 mg) to evaluate the expression of oyster amino acid transporter mRNA by using a polymerase chain reaction (PCR) fragment as a probe. PCR was performed for 30 cycles (95∞C for 30 s, 42∞C for 1 min, and 72∞C for 2 min) in a Program Temp Control System PC-700 (Astec, Fukuoka, Japan). Amplifications were performed in 20 mL of reaction mixture containing 2.0 U Ex-taq polymerase (Takara, Tokyo, Japan), 10 mM dATP, dCTP, dGTP and dTTP, 1 ¥ reaction buffer, 0.5 mM primers and oyster DNA (100 ng). Primers used were 5¢-CGGAATTCGGNAAYGTNTGGAGRTTYCC-3¢ and 5¢-CGGAATTCGTNCCNGCRTCNATCCANAC-3¢, where N and Y are defined according to IUPAC nomenclature. A pBluescript carrying a full-length oyster amino acid transporter gene was used as a template for the preparation of a probe. Hybridization was carried out at 42∞C in a solution containing 5 ¥ standard saline citrate (SSC; 1 ¥ SSC: 150 mM NaCl, 15 mM sodium citrate, pH 7.0) containing 0.1% sodium dodecylsulfate (SDS), 50% formamide, 100 mg/mL denatured salmon sperm DNA, 0.6% bovine serum albumin, and a 32P-labeled probe prepared from the PCR fragment obtained above with a Megaprime DNA labeling system (Amersham Biosciences Corp., Piscataway, NJ, USA). After hybridization, filters were rinsed four times with 2 ¥ SSC at room temperature and three times with 2 ¥ SSC containing 0.5% SDS at 68∞C prior to autoradiography. Promoter cloning Oyster DNA was prepared from the gill according to the standard method including proteinase K treatment, phenol-chloroform extraction, and ethanol precipitation.14 The 5¢-flanking region of the oyster amino acid transporter gene was cloned by using a Genome Walker Kit (Clontech, Palo Alto, CA, USA) according to the manufacturer’s protocol. Briefly, oyster DNA was cleaved with EcoR V, Dra I, Pvu II and Ssp I (Takara, Tokyo, Japan). The adapter supplied by the manufacturer was ligated to the 5¢end of the cleaved products and PCR was carried out with a sense primer supplied by the manufacturer and antisense primers designed according to the nucleotide sequence of the oyster amino acid transporter gene according to the manufacturer’s protocol. Amplified products were subcloned into pGEMT Easy Vector (Promega, Woods Hollow Road, Madison, WI, USA), and nucleotide sequences were determined with a Dye Terminator Cycle Sequencing Kit (Beckman Coulter, Fullerton, CA, USA). The
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nucleotides were analyzed using TFSEARCH (http://mbs.cbrc.jp/research/db/TFSEARCHJ.ht) to identify putative transcriptional motifs. Primer extension To identify the transcription start site, primer extension was performed with a Primer Extension System-AMV Reverse Transcriptase Kit (Promega) according to the manufacturer’s protocol. Gill mRNA was used after purification with oligotexdT30 (Takara, Tokyo, Japan). The primer used was 5¢-TTCTGGAGATTAATGTAGTCAAATAC-3¢, which corresponds to the reverse strand sequence of the 1–26 bp upstream region of the translation start site. Assay of promoter activity in response to osmotic changes
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After 6 h of osmotic stress, cells were harvested and luciferase activity was evaluated with a DualGlo Luciferase Assay System (Promega) according to the manufacturer’s protocol by using a luminometer, 1420 ARVOTM SX-FL (Wallac, Turku, Finland). Statistical analysis was performed by t-test using Excel X (Microsoft, Redmond, WA, USA), and P < 0.05 was used as the level of significance. Nucleotide sequence The nucleotide sequence of the 5¢-flanking region reported in the present study has been deposited to DDBJ/EMBL/GenBank Nucleotide Sequence Database under the accession number AB185409. RESULTS
Promoter activity was analyzed by using luciferase as a reporter gene. Promoter regions with various lengths (Proms. I to VI) were amplified by PCR and ligated into pGL3-Basic Vector (Promega). These vectors were transfected with Lipofectamine (Invitrogen, Carlsbad, CA, USA) into HEK-293T cells, which were derived from human embryo kidney. HEK-293T cells were cultured in Minimal Essential Medium (Nissui Pharmaceutical, Tokyo, Japan) buffered with HEPES containing 10% fetal bovine serum at 37∞C for 24 h. The medium was then replaced with hypo-osmotic, control (isotonic) or hyper-osmotic medium. The osmolalities of the hypo- and hyper-osmotic media were, respectively, adjusted to 200 mOsm/ kg and 500 mOsm/kg by the addition of NaCl, while that of the isotonic medium was adjusted to 300 mOsm/kg. Osmolality was measured by freezing point depression using a Knauer semimicro osmometer (Knauer, Berlin, Germany).
Expression of oyster amino acid transporter gene in response to hypo-osmotic and hyper-osmotic stresses As shown in Fig. 1, the amounts of oyster amino acid transporter mRNA in the gill and adductor muscle were increased in response to the environmental osmotic changes. The marked induction was observed after 24 h of the hyper-osmotic stress in the gill and adductor muscle, while it was observed after 8 h of the hypo-osmotic stress in the same tissues. Interestingly, hypo-osmotic stress induced a more intense and acute increase than hyper-osmotic stress especially in the gill. Expression of amino acid transporter in response to osmotic stresses suggested that the transporter gene contains the osmo-regulatory region for both hypo- and hyper-osmotic stresses. Thus, we attempted to clone the 5¢-flanking region of oyster amino acid transporter gene to specify the regulatory region for the osmotic stresses.
Hyper-osmotic stress
Hypo-osmotic stress
Gill Fig. 1 Northern blot analysis of the expression of the oyster amino acid transporter in response to osmotic changes. Experimental conditions are detailed in the text.
Adductor muscle 0
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+1 +165 TSP
-2,141 Prom. VI Prom. V
-1,670 -1,177
Prom. IV Prom. III
-718 -356
Prom. II
-132 Prom. I
Fig. 2 Results of promoter sequence motif search and schema of deletion mutants of the 5’-flanking region of the oyster amino acid transporter gene. Deletion mutants (Proms. I–VI) were amplified by polymerase chain reaction and ligated in pGL-3 Basic Vector and used for the experiment shown in Fig. 3. TSP, transcription start site. The translation start site is located 165 bp downstream of TSP. , CCAAT box; , cyclic AMP responsive element binding motif; , GC box; , heat shock factor-binding motif; , hypoxia inducible factor-binding motif.
Cloning of the 5¢¢-flanking region of the oyster amino acid transporter gene
Identification of the osmo-responsive promoter region
The 5¢-flanking region of the oyster amino acid gene was cloned up to 2306 bp from the translation start site. Primer extension analysis demonstrated that the transcription start site is located 165 bp upstream from the translation start site. Thus, we cloned the region of 2141 bp upstream from the transcription start site. A sequence motif search revealed that CCAAT box, cyclic AMP responsive element binding motif, and GC box are localized at 39 bp, 216 bp and 288 bp upstream from the transcription start site, respectively, as shown in Fig. 2. Heat shock factorbinding motifs (NGAANNTTCN)15 are found at 537 bp, 905 bp and 939 bp upstream from the transcription start site, and hypoxia inducible factor-binding motifs (A/GCGTG)16,17 are found at 1657 bp, 1670 bp and 1784 bp upstream from the same site. These findings suggest that expression of the oyster amino acid transporter gene would be induced by a variety of stresses besides osmotic stress. A hyper-osmolality-responsive element called the tonicity-responsive element (TonE, TGGAAAAGTCCA) was identified in Mardin– Darby canine kidney cells (MDCK) by investigating the 5¢-flanking region of the betaine transporter gene.18 TonE has been shown to regulate the expression of aldose reductase and sodium/myoinositol cotransporter gene.19–21 A TonE sequence, however, was not found in the 5¢flanking region of the oyster amino acid transporter gene we cloned.
We constructed deletion mutants of the putative promoter region and inserted them in the pGL3Basic Vector. These vectors were then transfected into HEK-293T cells. Prior to the experiment, we confirmed that pGL3-Basic Vector without an insert demonstrated no detectable luciferase activity with or without osmotic stress (data not shown). When we examined the promoter activity responding to osmotic changes, the vectors carrying Proms. I–VI demonstrated significantly higher luciferase activity than the corresponding controls in isotonic medium as shown in Fig. 3. Because the shortest mutant of Prom. I containing the nucleotide sequence of 132 bp upstream from the transcription start site exhibited significantly higher luciferase activities than the control in response to hypo- and hyper-osmotic stresses, it is suggested that the regulatory element(s) responding to osmotic stresses are localized within this region. DISCUSSION It has been reported that HEK-293T cells, which were derived from human embryonic kidney cells, expressed a potassium channel gene in response to osmotic changes,22 suggesting that HEK-293T cells are equipped with osmo-responsive signal transduction cascade that finally activates transcriptional factors involved in novel expression of the gene required for the osmotic adaptation. Because promoter regions cloned from oyster functioned in
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Oyster amino acid transporter
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VI
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V Fig. 3 Promoter activity of the deletion mutants in response to osmotic changes. Vectors were transfected into HEK-293T cells, which were then exposed to hypo- and hyper-osmotic stresses by changing the medium. Promoter activity was evaluated by measuring luciferase activity. One unit of the activity is defined as the activity of Prom. VI without osmotic stress. Horizontal bars show the standard errors. *, significant differences (P < 0.05) from the values of each control.
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IV
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III II
Control
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Hypo-osmolality
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response to osmotic changes in HEK-293T cells (Fig. 3), transcriptional factors in HEK-293T cells involved in osmotic regulation could bind to the promoter region of the oyster amino acid transporter gene. Therefore, it seems probable that transcriptional factors involved in the regulation of the osmotic responsive genes are well conserved among vertebrates and invertebrates. Animals living in water are affected by environmental osmotic conditions.23 In particular, most marine invertebrates lack an endocrine system to regulate the osmolality of their body fluid and must cope with environmental osmotic stress at the cellular level.1 In adapting to hyper-osmotic stress, cells import and accumulate small organic solutes called osmolytes by means of transporters specific for each osmolyte,10,11 while they export osmolytes through non-specific channels in adapting to hypo-osmotic stress.9 In this connection, various fish cells and mammalian kidney cells express osmolyte transporter genes only in response to hyper-osmotic stress.10–13 In this study, we demonstrated that in contrast to vertebrate transporters, the oyster amino acid transporter gene was expressed in response to hypo-osmotic stress as well as hyper-osmotic stress. In the previous study, we have already observed the induction of the taurine transporter gene in response to hypo- and hyper-osmotic stresses in the Mediterranean Blue Mussel Mytilus galloprovincialis.24 Thus, responsiveness to hypoosmotic stress may be a common feature of osmolyte transporters in marine bivalves living in brackish water. To identify the osmo-responsive element(s) in the oyster amino acid transporter gene, we cloned the 5¢-flanking region of the gene
Hyper-osmolality
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and found that the element(s) are localized within 132 bp from the transcription start site. In an attempt to identify the sequence responding to these osmotic stresses, we performed DNase footprint analysis, but this was unsuccessful. It will be necessary to make shorter deletion mutants to identify the nucleotide sequence of osmo-responsive regulatory element(s) in the oyster amino acid transporter gene. ACKNOWLEDGMENTS The authors wish to thank Professor P. Hawkes, Osaka International University, for critical reading of the manuscript. This study was partly supported by a grant from The Salt Science Research Foundation (No. 9943) and a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (No. 11660204). REFERENCES 1. Yancy PH, Clark ME, Steven CH, Bowlus RD, Somero GN. Living with water stress: Evolution of osmolyte systems. Science 1982; 217: 1214–1222. 2. Somero GN, Bowlus RD. Osmolyte and metabolic end products of mollusks: the design of compatible solute systems. In: Hochachka PW (ed). Mollusca, Vol. 2. Academic Press, New York. 1983; 77–100. 3. Chew KK. Global bivalve shellfish introductions. World Aquacult. 1990; 21: 9–22. 4. Shatkin G, Shumway SE, Hawes R. Considerations regarding the possible introduction of the Pacific oyster (Crassostrea gigas) to the Gulf of Maine: a review of global experience. J. Shellfish. Res. 1997; 16: 463–478.
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