Isolation of Sparus auratus prolactin gene and activity of the cis-acting regulatory elements Antonio Astola, a Manuela Ortiz, a Josep A. Calduch-Giner, b Jaume P erez-S anchez, b and Manuel M. Valdivia a, * a Departamento de Bioqu  ımica y Biolog  ıa Molecular, Facultad de Ciencias, Universidad de C adiz, Puerto Real, C adiz 11510, Spain b Instituto de Acuicultura Torre de la Sal, C.S.I.C. 12595 Ribera de Cabanes, Castell on, Spain Accepted 23 May 2003 Abstract A sea bream prolactin (sbPRL) gene was isolated using a prolactin cDNA fragment, generated by PCR as a probe. The gene analyzed comprises 3.5 kb of DNA containing five exons as described previously for other fish PRL genes. Analysis of 1.0 kb of the proximal promoter sequence reveals a consensus TATAA box, up to seven (A/T) 3 NCAT consensus motifs for binding of the pituitary-specific factor Pit-1 and putative CREB and GATA binding sites. CHO culture cells co-transfected with a sbPRL promoter sequence and a sea bream Pit-1 cDNA expression plasmid showed expression of a linked luciferase reporter gene. Transient expression experiments with 5 0 -delection mutants reveals at least three regulatory regions on the sbPRL gene, two with a stimulatory effect on transcription and one with apparent inhibitory effect. From a comparative point of view, this study of PRL gene in Sparus auratus, correlates well with those previously published on tilapia and rainbow trout. The molecular data reported will be useful for comparative analysis of gene regulation in the GH/PRL gene family in teleosts. Ó 2003 Elsevier Science (USA). All rights reserved. Keywords: Prolactin; Gilthead sea bream; Gene promoter; cis-acting sequences; Sparus auratus 1. Introduction Growth hormone (GH), prolactin (PRL), and somatolactin (SL) belong to a family of structurally re- lated polypeptide hormones in fishes. On the basis of nucleotide and amino acid sequences, it has been sug- gested that the members of this gene family have evolved by divergence from a common ancestral gene (Miller and Eberhardt, 1983; Niall et al., 1971; Nicoll et al., 1986; Wallis, 2001). In vertebrates, PRL is a versatile hormone, involved in many physiological processes such as growth, differentiation, metabolism, osmoregulation, and reproduction (Hirano, 1986). In teleosts, the major function of PRL seems to be osmoregulation in fresh- water to prevent the loss of Na þ and electrolyte ho- meostasis (Ayson et al., 1994; Manzon, 2002). The protein structure of PRL is believed to be composed of four a-helices arranged in an anti-parallel four-helical bundle as described similarly for GH (Barsh et al., 1983). In fish, several PRL cDNAs have been charac- terized (Chan et al., 1996; Chen et al., 1991; Santos et al., 1999; Swennen et al., 1992; Xiong et al., 1992), and two PRL genes in tilapia have been reported (Rentier-Delrue et al., 1989). Also, regulatory elements of chinook salmon, tilapia, and rainbow trout prolactin genes have been studied previously (Argenton et al., 1996; Poncelet et al., 1996; Xiong et al., 1992). In the present study we have isolated and sequenced a Sparus auratus PRL gene and analyzed the transcription activity of the cis-elements under the control of the sea bream Pit-1 factor (Mart  ınez-Barber a et al., 1997). 2. Methods 2.1. Cloning of sea bream prolactin gene First, a sbPRL probe was generated by PCR using a forward primer designed within the 5 0 end of the cDNA General and Comparative Endocrinology 134 (2003) 57–61 www.elsevier.com/locate/ygcen GENERAL AND COMPARATIVE ENDOCRINOLOGY * Corresponding author. Fax: +34-956-016288. E-mail address: manuel.valdivia@uca.es (M.M. Valdivia). 0016-6480/$ - see front matter Ó 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S0016-6480(03)00214-4