Gene and cDNA Structures of Flounder Insulin-Like Growth Factor-I (IGF-I): Multiple mRNA Species Encode a Single Short Mature IGF-I

Abstract

To understand the comprehensive mechanisms of gene expression and processing for insulin-like growth factor-I (IGF-I) in vertebrates, we have investigated the gene organization, promoter and transcriptional initiation sites, alternative splicing and polyadenylating sites, and the cDNA structures of this gene in the Japanese flounder, Paralichthys olivaceus. The flounder IGF-I gene was found to be composed of five exons and four introns spanning 17.5 kb. By Northern blot analysis, two major mRNA classes of 4.7 kb and 2.9 kb were found in the liver. cDNA cloning and reverse transcription polymerase chain reaction (RT-PCR) analysis indicated that these two mRNA classes result from two different-sized 3'-noncoding regions generated by alternative usage of two polyadenylating signals. Further analysis by RT-PCR and sequencing revealed that these mRNA classes both contain two subclasses of mRNA encoding two forms of IGF-I prepropeptide, preproIGF-I-1 and preproIGF-I-2. The two forms of preproIGF-I share the identical signal peptide and mature IGF-I domain but contain different E domains as a result of alternative splicing in exon 3. The mature form of flounder IGF-I was found to comprise 68 amino acid residues, showing a small molecular weight, 7486. In the 5'-flanking region, one major and four minor transcription start sites have been identified by ribonuclease protection assay between -230 and -130 from the translation initiation codon, but no canonical TATA box or GC box was detected in their upstream regions up to -724. The results suggest that some unknown transcription initiation factors are functioning in the promotion of IGF-I gene expression.

Molecular cloning and expression of insulin-like growth factor-I (IGF-I) in Eurasian perch (Perca fluviatilis): lack of responsiveness to growth hormone treatment

“…The utilizing of an alternative 5 0 splice site resulted in the deletion of an 81-nt region encoding 27 residues of the E domain. The identified perch IGF-Ia and IGF-Ib correspond to the two IGF-I transcripts isolated from Japanese flounder (Tanaka et al 1998) and barramundi (Lates calcarifer) (Sta˚hlbom et al 1999). Correspondingly, two alternative spliced transcripts were also identified in each of the duplicated IGF-I genes in salmonids (Wallis and Devlin 1993;Duan et al 1994;Duguay et al 1994).…”

“…Multiple long IGF-I transcripts have previously been identified in human and several fish species by Northern blot (Cao et al 1989;Sussenbach et al 1992;Lund 1994;Schmid et al 1999;Sta˚hlbom et al 1999). In Japanese flounder, two large IGF-I cDNA of 4691 and 2877 nt with different sized 3 0 UTR were isolated (Tanaka et al 1998) that corresponds to the 4.9 and 3.0 kb perch IGF-I transcripts identified by Northern blot analysis. Even the shortest transcript of about 1.5 kb is considerable longer than the isolated IGF-I cDNA sequence.…”

Molecular cloning, identification and characterization of four distinct receptor subtypes for insulin and IGF-I in Japanese flounder, Paralichthys olivaceus

“…1 legend), these four receptors may be the products of four different genes. On the other hand, molecules of insulin (Andoh & Nagasawa 1998) and IGF-I (Tanaka et al 1998) have been reported to be produced by a single gene in Japanese flounder. The amino acid sequences of fIR-1 and fIR-2 are more homologous to that of human IR than to that of human IGF-IR, and those of fIGF-IR-1 and fIGF-IR-2 are more similar to that of human IGF-IR than human IR.…”

Characterization of structure and expression of the growth hormone receptor gene of the Japanese flounder (Paralichtys olivaceus)

“…An RNase protection assay was performed by the previously described method (Tanaka et al 1998). Briefly, Figure 1 The nucleotide sequence of the flounder GHR gene.…”