Detalle de: cDNA cloning and sequence of European sea bass (Dicentrarchus labrax) somatolactin

Three cDNA clones encoding for European sea bass somatolactin (SL) were obtained by RT-PCR and 3' RACE of RNA of pituitary origin. Clone 1 was 582 bp in length, and included a part of the signal peptide and the 5' end of the mature protein. Clone 2 (1075 bp) included a fragment of the coding sequence and the 3' untranslated region, which was 888 bp in length and contained two putative polyadenylation signals (AATAAA) at 12-17, and 202-207 nucleotides upstream of the poly (A) tail. Clone 3 was 624 bp in length and its nucleotide sequence encoding for the entire mature protein confirmed the sequence already determined from the first two clones. The size of SL mRNA transcripts was estimated by Northern blot analysis and a single band of approximately 1.6 kb was observed with pituitary RNAs. No band was found with RNAs of brain and liver origin. Alignment of the deduced amino acid sequence revealed that European sea bass SL shared 90-84% identity with perciform, pleuronectiform and scorpaeniform fish SLs, and 77-57% with other SLs of more distant fish orders, with a strict conservation of Cys residues and the N-glycosylation site (Asn-Lys-Thr) at 121-123 amino acid positions. The reconstruction of the phylogenetic tree based on SL nucleotide sequences. and analyzed by maximum likelihood distances, showed the same clustering as the present hierarchy of fish. When comparisons were made among SL, prolactin and growth hormone of European sea bass, the overall amino identity was relatively low (22-23%). However, a high degree of amino acid homology was found at the C-terminus, which contains three of the four Cys residues strictly conserved in all the members of GH/PRL family.