Cloning, characterization and promoter analysis of the common carp IGF-II gene.
Insulin-like growth factors (IGFs) belong to a family of growth factors with structural homology to proinsulin. Up till now, no specific details regarding the transcriptional regulation by autocrine, paracrine or endocrine effector molecules in vivo have been described for the IGF-II gene. This is in big contrast to IGF-I gene transcription which has been studied more extensively. To better understand how the IGF-II gene is controlled at the gene transcription level, we have isolated the common carp IGF-II gene together with the 5'-flanking region by genomic library screening. The mature IGF-II protein was encoded by exon 2 and exon 3. Transient transfection of the 5'-flanking region containing a TATA box-like sequence into cultured eukaryotic cells revealed that it is a strong promoter with definitive tissue specificity. Nucleotides between -301 and -62 in the promoter are essential to drive the basal IGF-II gene expression; whereas nucleotides between -891 and -416 in the promoter are responsible for the growth hormone activation. Using electrophoretic mobility shift assay and yeast one-hybrid screening, it was demonstrated that alpha1-antitrypsin could bind specifically to the nucleotide position -301 to -262 of the gene promoter. Co-transfection studies revealed that the over-expression of alpha1-antitrypsin increased the IGF-II promoter activity by 3.4-fold, further confirming that alpha1-antitrypsin acts as a trans-acting factor on the IGF-II promoter.
Tse
Margaret C L
MC
Department of Biochemistry, The Chinese University of Hong Kong, Shatin, NT, Hong Kong, China.
Chan
K M
KM
Cheng
Christopher H K
CH
D024506:5' Flanking Region; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002347:Carps / Q000235:genetics*; D002460:Cell Line; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D015870:Gene Expression; D013006:Growth Hormone / Q000494:pharmacology; D007335:Insulin-Like Growth Factor II / Q000235:genetics*; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic / Q000187:drug effects; D017384:Sequence Deletion; D014162:Transfection; D020798:Two-Hybrid System Techniques; D000515:alpha 1-Antitrypsin / Q000378:metabolism / Q000494:pharmacology
D017931:DNA Primers; D018076:DNA, Complementary; D000515:alpha 1-Antitrypsin; D007335:Insulin-Like Growth Factor II; D013006:Growth Hormone