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.
Five forms of insulin-like growth factor-I (IGF-I) complementary DNA (cDNA) were isolated by PCR from goldfish 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 differences 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 from 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.
Kermouni
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Department of Biological Sciences, University of Calgary, Alberta, Canada.
Mahmoud
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Wang
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Moloney
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Habibi
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HR
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001667:Binding, Competitive; D015139:Blotting, Southern; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000032:analysis* / Q000235:genetics / Q000302:isolation & purification; D005260:Female; D006054:Goldfish / Q000235:genetics*; D006801:Humans; D007334:Insulin-Like Growth Factor I / Q000737:chemistry / Q000235:genetics*; D008099:Liver / Q000737:chemistry; D008969:Molecular Sequence Data; D010053:Ovary / Q000737:chemistry; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000032:analysis; D012194:RNA-Directed DNA Polymerase; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA
D018076:DNA, Complementary; D012333:RNA, Messenger; D007334:Insulin-Like Growth Factor I; D012194:RNA-Directed DNA Polymerase