Zoological Studies

Vol. 37 No. 3, 1998

Cloning and Characterization of Insulin-like Growth Factor I cDNA from Black Seabream (Acanthopagrus schlegeli)

Mark Hung-Chih Chen1,2, Gen-Hwa Lin1, Hong-Yi Gong1, Chiou-Yueh Lee1, Chi-Yao Chang1, Thomas T. Chen3 and Jen-Leih Wu1,*

1Laboratory of Marine Molecular Biology and Biotechnology, Institute of Zoology, Academia Sinica, Taipei, Taiwan 115
2Institute of Marine Biology, National Taiwan Ocean University, Taipei, Taiwan 202
3Department of Molecular and Cell Biology and Biotechnology Center, University of Connecticut, Storrs, Connecticut 06269, USA

Mark Hung-Chih Chen, Gen-Hwa Lin, Hong-Yi Gong, Chiou-Yueh Lee, Chi-Yao Chang, Thomas T. Chen and Jen-Leih Wu (1998) A cDNA library was constructed in Uni-ZAP XR using mRNA from the liver of black seabream, Acanthopagrus schlegeli. In this study, we designed 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 seabream by reverse transcription/polymerase chain reaction (RT/PCR). Using the internal probe to screen the cDNA library, we obtained 16 positive clones. Subsequent restriction enzyme map analysis suggested that about 6 forms ranging in size from 1.8 kilo-base pair (kb) to 2.3 kb of IGF-I were present. We took 2 longer cDNA clones to read the full sequence. Sequences of 2 clones of IGF-I cDNAs were found to be 2238 base pairs (bp) and 2299 bp in length. Except for a sequence of 61 nucleotides missing in the 5'-untranslated region (5'-UTR) and 6 nucleotides being different in the 3'-untranslated region (3'-UTR), the other nucleotides of these 2 clones are identical. This showed that the black seabream IGF-I gene contains polymorphism or gene duplication. Both cDNAs containing an open reading frame (ORF) encode 185 amino acids, including a 44-amino acids leader peptide, the 67-amino acids mature peptide in the B, C, A, and D domains, and a 74-amino acids extended carboxyl-terminal 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 mature 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 compared 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 encode a particular subtype of IGF-I from liver, IGF-I Ea- 4. Furthermore, this IGF-I is phylogenetically most closely related to that of Sparidae.

Key words: IGF-I, Sparidae, cDNA.

*Correspondence: Tel: 886-2-27899568. Fax: 886-2-27858059. E-mail: zojlwu@ccvax.sinica.edu.tw