CLONING AND CHARACTERIZATION OF INSULIN-LIKE-GROWTH-FACTOR-I CDNA FROM BLACK SEABREAM (ACANTHOPAGRUS-SCHLEGELI)

A cDNA library was constructed in Uni-ZAP XR using mRNA from the liver of black seabream, Acanthopagrus schlegeli. In this study, we designe d a pair of primers from the C and E domains of trout IGF-I cDNA, and synthesized an internal probe of IGF-I from the liver of black seabrea m by reverse transcription/polymerase chain reaction (RT/PCR). Using t he internal probe to screen the cDNA library, we obtained 16 positive clones. Subsequent restriction enzyme map analysis suggested that abou t 6 forms ranging in size from 1.8 kilo-base pair (kb) to 2.3 kb of IG F-I were present. We took 2 longer cDNA clones to read the full sequen ce. Sequences of 2 clones of IGF-I cDNAs were found to be 2238 base pa irs (bp) and 2299 bp in length. Except for a sequence of 61 nucleotide s missing in the 5'-untranslated region (5'-UTR) and 6 nucleotides bei ng different in the 3'-untranslated region (3'-UTR), the other nucleot ides of these 2 clones are identical. This showed that the black seabr eam IGF-I gene contains polymorphism or gene duplication. Both cDNAs c ontaining an open reading frame (ORF) encode 185 amino acids, includin g a 44-amino acids leader peptide, the 67-amino acids mature peptide i n the B, C, A, and D domains, and a 74-amino acids extended carboxyl-t erminal peptide in the E domain. The predicted propeptide of IGF-I can be divided into B, C, A, D, and E domains. Owing to the conserved mat ure peptide of IGF-I, we compared IGF-I of other teleosts with that of black seabream. The comparisons showed 100%, 91%, 91%, 87%, 87%, 58%, and 48% amino acid identity, respectively, with the IGF-I of Sparus, salmon, trout, carp, catfish, hagfish, and amphioxus. Moreover, we com pared the E domain of IGF-I of Salmonidae with that of black seabream. The comparisons revealed that the E domain of black seabream belongs to the largest Ea form. These data imply that black seabream cDNAs enc ode a particular subtype of IGF-I from liver, IGF-I Ea-4. Furthermore, this IGF-I is phylogenetically most closely related to that of Sparid ae.