Cloning and Characterization of Rainbow Trout (Oncorhynchus mykiss) Somatolactin cDNA and Its Expression in Pituitary and Nonpituitary Tissues

Abstract

A cDNA clone encoding rainbow trout (Oncorhynchus mykiss) somatolactin (rtSL) has been isolated from a rainbow trout pituitary cDNA library. This 2329-bp cDNA clone includes a very short 7-bp 5′-untranslated region, a coding region of 702 bp, and a long 3′-untranslated region of 1620 bp. The deduced amino acid sequence of rtSL shows a polypeptide of 233 amino acid residues which consists of a 24-amino-acid putative signal peptide followed by a 209-amino-acid mature polypeptide. This mature polypeptide has a molecular weight of 24 kDa. The rtSL shares 99% amino acid identity with chum salmon SL (csSL) and approximately 53–77% amino acid identity with SLs in other fishes, including the 7 conserved cysteine residues. Although a glycosylation site has been identified in SL of other fish species, none is observed in rtSL polypeptide. The level of rtSL mRNA in a single pituitary gland was determined by RNA blot hybridization. Results showed that levels of SL mRNA in pituitary glands of 2-year-old fish were 4- to 7-fold higher than those of 1-year-old fish. The tissue distribution of SL gene expression in adult fish was determined by reverse transcription–polymerase chain reaction (RT-PCR) and DNA blot hybridization. In addition to the pituitary gland, SL mRNA was detected in all tissues examined including brain, gill, heart, kidney, liver, skeleton muscle, spleen, ovary, testis, and immature oocytes. The extrapituitary expression of the SL gene was also detected in embryos and fry. The PCR products which contained the region coding for mature SL from heart and kidney were cloned and characterized. Nucleotide sequence analysis showed that the SL mRNAs in heart and kidney were identical to that in the pituitary gland. These results suggest that, although the pituitary gland is the predominant tissue for producing SL, it is not the only tissue that SL gene is expressed in, and the extrapituitary expression of rtSL gene starts from very early developmental stages.