Cloning of a Full-Length Insulin-like Growth Factor-I Complementary DNA in the Goldfish Liver and Ovary and Development of a Quantitative PCR Method for Its Measurement A. Kermouni, S. S. Mahmoud, S. Wang, M. Moloney, and H. R. Habibi Department of Biological Sciences, University of Calgary, Calgary, Alberta, Canada T2N 1N4 Accepted March 25, 1998 Five forms of insulin-like growth factor-I (IGF-I) comple- mentary DNA (cDNA) were isolated by PCR from gold- fish liver and ovary, using primers based on common carp IGF-I sequence. In the goldfish liver, we cloned and sequenced three IGF-I forms (1, 2, and 3), and elucidated the full-length cDNA sequence using the 5 '- and 3 '- RACE. Two IGF-I forms (1 and 2) were cloned from the goldfish ovary and were found to have diffences with respect to both size and nucleotide sequence compared to liver IGF-I. The entire liver IGF-I form 1 sequence was found to be 833 nucleotides long, containing a 483- nucleotide open reading frame encoding 161 amino acids. The deduced amino acid sequence of the mature peptide was compared to IGF-I sequences of other vertebrates, and found to have 97 and 93%similarity to carp and salmon IGF-I, respectively. In this study we also developed a competitive quantitative PCR method and demonstrated an increase in IGF-I expression following treatments with growth hormone or gonadotropin- releasing hormone in the goldfish liver.  1998 Academic Press Key Words: goldfish; insulin-like growth factor-I; liver, ovary; PCR; cDNA library; nucleic acid; messenger. The insulin-like growth factors (IGFs) are polypep- tides and are important regulators of growth and differentiation (Jones et al., 1995; Froesch et al., 1985). The mature IGF-I peptide has a B-, a C-, and an A-domain and is highly conserved among vertebrates. The C-terminal of the IGF-I contains a D-domain and an E-domain. The E-domain is proteolytically removed as a posttranslational modification to yield the mature peptide (Daughaday and Rotwein, 1989; Lund et al., 1994). Although IGFs are predominantly synthesized by the liver and released into serum (Schwander et al., 1983), other tissues in the body also produce and release IGF-I and IGF-II to induce cell replication and differentiation in paracrine/endocrine manner (D’Erocole et al., 1984; Froesch and Hussain, 1993; Daughaday and Rotwein, 1989; Lund 1994). The amino acid and nucleotide sequence of IGF-I has been deter- mined in a number of mammalian and nonmamma- lian species. The IGF-I sequences in mammals have been determined in human, rat, mouse, pig, cow, sheep (Rinderknecht and Humbel, 1978; Roberts et al., 1987; Bell et al., 1986; Francis et al., 1988, 1989a,b), dog, deer, goat, guinea pig, kangaroo, and horse (Delafontaine et al., 1993; Moore et al., 1993; Mikawa et al., 1995; Bell et al., 1990; Yandell et al., 1996; Otte et al., 1996). In nonmammalian vertebrates, the IGF-I sequences were elucidated in frog, chicken, salmon, trout, hagfish, sea bream, shark, carp, and catfish (Kajimoto and Rotwein, 1989, 1990; Cao et al., 1989; Shamblott and Chen, 1992; Nagamatsu et al., 1991; Duguay et al., 1995, 1996; Liang et al., 1996; McRory and Sherwood, 1994). In mammals, IGF-I production is stimulated by growth hormone (GH), and there is evidence that IGF-I plays an impor- tant paracrine and autocrine role in a number of tissues General and Comparative Endocrinology 111, 51–60 (1998) Article No. GC987085 51 0016-6480/98 $25.00 Copyright  1998 by Academic Press All rights of reproduction in any form reserved.