Isolation and Characterization of Tilapia (Oreochromis mossambicus) Insulin-Like Growth Factors Gene and Proximal Promoter Region

Abstract

To understand the molecular mechanism which controls the transcription of the insulin-like growth factors (IGFs) gene, we have cloned and sequenced the cDNA for the proximal promoter region of the tilapia IGFs gene and have characterized its activity by chloramphenicol acetyltransferase (CAT) transient transfected expression assays. Tilapia (Oreochromis mossambicus) IGF-I cDNA (549 bp) was amplified by PCR from single-stranded cDNA of growth hormone (GH)-induced liver RNA using a pair of oligonucleotides specific for fish IGF-I as amplification primers. Tilapia IGF-I and IGF-II 5' termini were analyzed by rapid amplification of cDNA 5' ends (5'RACE). Analysis of the 5'RACE results revealed two transcription start sites in IGF-I and one transcription start site in IGF-II. Different fragments of the 5' flanking region were transfected into human lung adenocarcinoma cells. In the cell line, maximum promoter activity was located in the distal 657 basepairs of the IGF-I 5' flanking region and in the distal 450 basepairs of the IGF-II 5' flanking region. The in vivo actions of the IGFs promoter on developmental stage expression were investigated further in transgenic zebrafish in which an IGFs promoter-driven green fluorescent protein (GFP) encoding the cDNA transgene was microinjected into embryos. Morphologic and RT-PCR studies of the transgenic zebrafish indicated that IGF-I promoter-driven GFP transcripts appeared for the first time in the 1-K-cell stage and the IGF-II promoter-driven GFP transcripts appeared for the first time in the 32-cell stage. Fluorescent (GFP) distribution was apparent within 48 h in IGF-II-transgenic zebrafish embryos, especially in eye, muscle, corpuscle, floor plate, horizontal myoseptum, yolk sac extension, and yolk sac. These results indicate that the IGF-I and IGF-II promoters are active in tissue and in a development-specific manner. Our findings also indicate that the IGF-II promoter influences the growth of fish embryos earlier than does IGF-I, and IGF-II has higher levels of expression than does IGF-I. These results suggest that the IGF-II promoter plays a growth factor role in teleost embryo development.

Immunomodulatory effects of prolactin and growth hormone in the tilapia, Oreochromis mossambicus

“…The PCR primers were designed to amplify PRL 177 (5 -CTATAGACAGGGTTCTCGCG-3 , forward; 5 -GCAGGACAGCAGTTTGGTAA-3 , reverse), PRL 188 (5 -GTCAGATTTGATGTCCCTGG-3 , forward; 5 -GCAGGACAGCAGAAAGTTGA-3 , reverse), GH (5 -CAGCTGTCGGTTGTGTGTTT-3 , forward; 5 -CAGCAGCAAGATTCCCGTTT-3 , reverse), IGF-I (5 -ATAAACCAACAGGCTATGGC-3 , forward; 5 -TTCTGGTGGACTTCCTTGA-3 , reverse), PRL-R (5 -CAGAGATCAAATGCCGTTCTCC-3 , forward; 5 -ATTTCAGGCAGCCGTCATGATC-3 , reverse), and -actin (5 -CATGAAGTGCGACGTTGACA-3 , forward; 5 -CACATCTGCTGGAAGGTGGA-3 , reverse) based on Swennen et al (1992), Reinecke et al (1997), Chen et al (1998), Sekkali et al (1999), Shiraishi et al (1999), Takeuchi (2000) and N R Staten (personal communication). After DNase treatment, RT-PCR was performed with an Access RT-PCR System (Promega Corporation, Madison, WI, USA), BcaBEST RNA PCR Kit version 1·1 (Takara, Tokyo, Japan) and AmpliTaqGold DNA Polymerase (Roche Molecular Systems, Branchburg, NJ, USA) using a PTC-200 Peltier Thermal Cycler (MJ Research Inc., Waltham, MA, USA) and a GeneAmp 2400 PCR System (Perkin Elmer, Norwalk, CT, USA).…”

Primary cultured hepatocytes of the bony fish, Oreochromis mossambicus, the tilapia: a valid tool for physiological studies on IGF-I expression in liver

“…IGF-I sequences are highly conserved throughout evolution (Reinecke & Collet 1998). IGF-I cDNA sequences have recently been characterised in several bony fish species: Oncorhynchus kisutch (Cao et al 1989), O. mykiss (Shamblott & Chen 1992), Oreochromis mossambicus , Chen et al 1998, Cyprinus carpio (Hashimoto et al 1997), Cottus scorpius (Loffing-Cueni et al 1998) and Lates calcarifer (Kinhult-Stahlbom et al 1999). These showed about 80% sequence homology to each other, and approximately 45% to the human IGF-I sequence.…”

Molecular Cloning, Developmental Expression, and Hormonal Regulation of Zebrafish (Danio rerio) β Crystallin B1, a Member of the Superfamily of β Crystallin Proteins

“…The PCR reaction was carried out in a final volume of 100 L. The reaction consisted of the cDNA library liquids, 10 L of 10ϫ PCR buffer (HT Biotechnology, U.S.A.), 200 M of each dNTP, 1 g of the IGFBP and HDLBP forward and reverse primers, and 2.5 units of Taq DNA polymerase. The reaction and process followed those we published before (12)(13)(14)(15). The PCR product was purified by electroelution and used as a probe for isolating 1 million clones from a zebrafish heart cDNA library and a zebrafish 24-h embryo cDNA library by the plaque hybridization method, as we published previously (12)(13)(14)(15).…”