By using an expression cloning strategy, we isolated a single positive clone encoding a tilapia prolactin (PRL) receptor. Tilapia PRL188 was used to screen a freshwater tilapia kidney expression library transfected in COS cells. The tilapia PRL receptor is a mature protein of 606 amino acids. The extracellular domain is devoid of the tandem repeat units present in birds and has two pairs of cysteine residues, a Trp-Ser-Xaa-Trp-Ser motif, and two potential N-glycosylation sites. The cytoplasmic domain contains 372 amino acids, including box 1, a sequence previously shown to be important for signal transduction in mammalian species. Thus, the general structure is similar to the long form of mammalian PRL receptors; however, amino acid comparisons reveal a rather low identity (-37%). Northern blot analysis shows the existence of a single transcript in osmoregulatory tissues and reproductive organs. This localization is in agreement with known functions of PRL in teleosts.Prolactin (PRL) is a pituitary polypeptide hormone that is implicated in many physiological actions in vertebrates including fish (1). Since the pioneering work by Pickford and Phillips in 1959 (2) demonstrating that hypophysectomized Fundulus heteroclitus require PRL for survival in freshwater, numerous studies have confirmed that PRL is one of the major hormones regulating the maintenance of water and electrolyte homeostasis on osmoregulatory surfaces (3-5). In tilapia, two distinct PRL forms have been well characterized (tiPRL188 or tiPRL, and tiPRL177 or tiPRLII), which share only 69% identity (6-8).These two tilapia PRLs (tiPRLs) were shown to be differentially regulated during adaptation to a hyperosmotic environment (9-11) and to exhibit both common and distinct biological effects and potencies (6, 10, 12). These effects are mediated by a specific cell membrane receptor. In tilapia, initial studies using ovine PRL (oPRL) as a ligand revealed the presence of PRL receptors in various tissues (13-15). Using bioactive recombinant tiPRL forms (16), high specific binding of PRL (up to 45% with tiPRL188) has recently been shown in gill and kidney, involving only one class of receptors, which binds tiPRL188 with higher affinity than tiPRL177 (17).PRL receptor cDNAs have been cloned from several mammalian species and sources (18)(19)(20) and two avian species (21, 22). These receptors belong to a superfamily including the receptors for growth hormone (GH), cytokines, and erythropoietin (18). This superfamily has several common structural features (23) including two pairs of conserved extracellular cysteine residues, a single transmembrane domain, and an intracellular proline-rich region (24). In lower vertebrates, however, no PRL receptor cDNA has been identified.We report in this paper the isolation of a tiPRL receptor cDNAI by an expression cloning approach (25). The characterized tiPRL receptor is a mature protein of 606 amino acids with a single extracellular unit and a long cytoplasmic domain. Moreover, tissue distribution studies indica...

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Metamorphosis in amphibians is marked by dramatic thyroid hormone-induced changes that include tail regression. To examine thyroid hormone effects on gene transcription during the early stages of tail resorption, we i jected exogenous genes directly into the caudal skeletal muscle of Xenopus tadpoles and followed their expression in vivo. Gene expression was both strong and reproducible, and it correlated with the amount of DNA injected. Moreover, expression continued as long as the animals were blocked in prometamorphosis by antithyroid drugs (for up to 4 months). Thyroid hormone-dependent effects on transcription were examined by using a palindromic thyroid hormone response element linked to a chloramphenicol acetyltransferase reporter gene. Reporter gene expressions were normalized for transfection efficiency by using a constitutively expressed luciferase construct. Physiological concentrations of 3,5,3' trllodo-L-thyronine (1 nM), applied for 120 hr, produced a 5-fold increase in transcription (P <0.05) from the thyroid hormone response element but did not modify transcription from constitutive viral promoters. This study thus demonstrates that by directly expressing genes in Xenopus tadpole muscle in vivo, one can exploit the powerful experimental advantages of gene transfer systems in an intact, physiologically normal animal.