Isolation and characterization of tilapia ( XXXX ) insulin-like growth factors gene and proximal promoter region.

@entity1 @entity22820 @entity13864 @entity2755 @entity2220 @entity3392 @entity22819
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 @entity2755 acetyltransferase (CAT) transient transfected expression assays. Tilapia ( @entity13864 ) @entity22819 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 @entity22819 as amplification primers. Tilapia @entity22819 and @entity22820 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 @entity22819 and one transcription start site in @entity22820 . Different fragments of the 5' flanking region were transfected into @entity1 @entity2220 cells. In the cell line, maximum promoter activity was located in the distal 657 basepairs of the @entity22819 5' flanking region and in the distal 450 basepairs of the @entity22820 5' flanking region. The in vivo actions of the IGFs promoter on developmental stage expression were investigated further in transgenic @entity3392 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 @entity3392 indicated that @entity22819 promoter-driven GFP transcripts appeared for the first time in the 1-K-cell stage and the @entity22820 promoter-driven GFP transcripts appeared for the first time in the 32-cell stage. Fluorescent (GFP) distribution was apparent within 48 h in @entity22820 -transgenic @entity3392 embryos, especially in eye, muscle, corpuscle, floor plate, horizontal myoseptum, yolk sac extension, and yolk sac. These results indicate that the @entity22819 and @entity22820 promoters are active in tissue and in a development-specific manner. Our findings also indicate that the @entity22820 promoter influences the growth of fish embryos earlier than does @entity22819 , and @entity22820 has higher levels of expression than does @entity22819 . These results suggest that the @entity22820 promoter plays a growth factor role in teleost embryo development.
