Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology
Cloning of the Atlantic salmon (Salmo salar) estrogen receptor-α gene
Introduction
Estrogens exert their genomic effects via specific receptor proteins, the estrogen receptors (ERs), which are members of the nuclear receptor superfamily (Evans et al., 1988, O’Malley, 1990, Manglesdorf et al., 1995). This superfamily also includes the receptors for the steroids, thyroid hormones, vitamin D3 and retinoids as well as many so-called orphan receptors for which ligands have yet to be identified. The members of this superfamily have a number of common features and their proteins can be divided into several distinct domains (Evans et al., 1988, Tsai et al., 1994). The DNA-binding domain (C domain) and the ligand-binding domain (E domain) are highly conserved between species. There are also variable regions at the N and C termini and between the DNA-binding and ligand binding domains (A/B, F and D domains, respectively). Two isoforms of ER, designated ER-α and ER-β, have been described in vertebrates (Nilsson et al., 1998). The two isoforms are not functionally redundant — they have distinct expression profiles and the phenotype of transgenic mice lacking ER-α (Rissman et al., 1995, Ogawa et al., 1997, Nilsson et al., 1998) is markedly different to that of transgenic mice lacking ER-β (Krege et al., 1998).
Estrogens are the major female sex hormones and probably have many target genes associated with this role. For example, the vitellogenin gene of oviparous animals, which encodes a major precursor of egg yolk proteins, is strongly upregulated by estrogens (Shapiro et al., 1990). In mammals, estrogens are also needed for development of the male reproductive tract and have a variety of other roles in both males and females, particularly in the cardiovascular system and in the development of brain, skeletal and soft tissues (Smith et al., 1994, Rissman et al., 1995, Nilsson et al., 1998).
In salmon, estrogens have been implicated in the control of smoltification — the transformation from freshwater dwelling parr to saltwater tolerant smolt. Smoltification involves a number of co-ordinated changes in morphology, physiology and biochemistry (Hoar, 1998). In comparison to parr, smolts are silvery, more streamlined, synthesise different haemoglobin isoforms, show increased activity of the gill Na+/K+-ATPase and greater hypo-osmoregulatory ability. The role of estrogens in smoltification is unclear, since some reports describe a rise in the plasma levels of estradiol (E2) during smoltification of the coho salmon (Oncorhynchus kisutch) (Sower et al., 1984, Sower et al., 1992), but other workers have not found this increase in either coho salmon (Patiño et al., 1986) or amago salmon (Oncorhynchus rhodurus) (Nagahama et al., 1982). Treatment of Atlantic salmon with E2 has been reported to inhibit smoltification (Madsen et al., 1997). The E2 treated fish show decreased gill Na+/K+-ATPase activity, lower gill chloride cell density and reduced hypo-osmoregulatory ability in comparison to controls. To further investigate the role of estrogens in the salmon life cycle, we isolated clones of the ER-α gene of salmon and used reverse transcriptase-polymerase chain reaction (RT-PCR) to determine the tissue distribution of ER-α mRNA both in male and female parr.
Section snippets
Isolation of a fragment of Atlantic salmon ER-α by RT-PCR
mRNA was prepared from Atlantic salmon tissues using Tri- reagent (Sigma). A homologous probe for salmon ER was isolated by RT-PCR from female adult liver total RNA using degenerate primers (5′ CAT/CATGATCGCCTGGGC 3′ and 5′ GATGTAA/GAGGTGCTCCAT 3′) derived from rainbow trout (Oncorhynchus mykiss) (Pakdel et al., 1989) and Xenopus laevis (Weiler et al., 1987) ER sequences. Amplification was performed for 35 cycles (95°C for 1 min, 56°C for 1 min, 72°C for 2 min) in buffer containing 2 mM MgCl2.
Cloning and sequencing of the Atlantic salmon ER-α gene
In order to obtain an Atlantic salmon ER probe, a 500 bp fragment of an Atlantic salmon ER gene was obtained by RT-PCR from liver RNA from an adult female. This fragment was used to screen a cDNA library constructed using poly (A)+ RNA from the liver of an adult female salmon and a cDNA clone of a salmon ER was subsequently isolated and sequenced. The sequence of this clone (nucleotides 197–3135 in Fig. 1) spans 2938 nucleotides (nt) including a large open reading frame (nt 197–1798) and a 3′
Discussion
We have determined the complete coding sequence of the ER-α gene from the Atlantic salmon. As expected, the salmon sequence shows greatest similarity to the ER-α gene of rainbow trout. Surprisingly however, some regions in the salmon ER-α amino acid sequence differ from the rainbow trout sequence more than from ER-α genes of less closely related fish. For example, within the amino terminal region (A/B domain) of the salmon ER-α gene, the sequence phe 15-pro 46 shows only 17% aa identity with
Acknowledgements
We thank Dr A. Youngson and Mr J. MacLean (Scottish Office Agriculture, Environment and Fisheries Department) for their help in obtaining tissue samples and Dr R. Male for providing a salmon genomic DNA library. SA Rogers was funded by a BBSRC studentship.
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