Cloning of the rat insulin-like growth factor-binding protein-2 gene and identification of a functional promoter lacking a TATA box.
We have isolated clones encoding the rat insulin-like growth factor-binding protein-2 (IGFBP-2) gene and determined its organization and nucleotide sequence. The rat IGFBP-2 gene spans at least 8 kilobases and consists of four exons, each of which contains protein-coding sequences. The amino acid sequences of exons 1, 3, and 4 are 32-50% identical to the corresponding exons of human IGFBP-1 and IGFBP-3, and 87-91% identical to those of human IGFBP-2. The 18 cysteines in the mature binding proteins are conserved. Exon 2 shows negligible homology. Primer-extended reverse transcription indicated that the 5' end of IGFBP-2 mRNA is 151 nucleotides up-stream from the translation start site [designated nucleotide (nt) -151]. Consistent with this result, IGFBP-2 mRNA protected a genomic fragment terminating at approximately nt -148, as well as smaller fragments. A 1260 nt fragment containing 1144 nt of 5' flanking region had promoter activity when inserted in the correct orientation into a plasmid containing a promoterless luciferase reporter gene and transiently transfected into BRL-3A rat liver cells, which express IGFBP-2, but not when transfected into H4-II-E cells, which do not express IGFBP-2. The IGFBP-2 gene lacks a TATA box immediately up-stream from the transcription initiation site. It is GC rich (66% between nt -270 and +385) and contains GC boxes that might be recognized by transcription factors Sp1 or ETF. The promoter region contains multiple direct and indirect repeats. One direct repeat contains a variant Sp1 site (-158 to -150) near the consensus Sp1 site at nt -138 to -130. The 5' flanking region also contains motifs that might be recognized by transcription factors AP-1 (Jun/Fos), AP-2, and liver factor B1. The role of these sites in basal and regulated expression of the IGFBP-2 gene remains to be determined.
Brown
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Growth and Development Section, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892.
Rechler
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D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics*; D003001:Cloning, Molecular; D005091:Exons; D018971:Insulin-Like Growth Factor Binding Protein 2; D018969:Insulin-Like Growth Factor Binding Proteins; D007438:Introns; D008156:Luciferases / Q000235:genetics; D008969:Molecular Sequence Data; D010957:Plasmids; D011401:Promoter Regions, Genetic / Q000235:genetics*; D051381:Rats; D012091:Repetitive Sequences, Nucleic Acid; D015183:Restriction Mapping; D012260:Ribonucleases; D012689:Sequence Homology, Nucleic Acid; D016385:TATA Box; D014158:Transcription, Genetic; D014162:Transfection
D002352:Carrier Proteins; D018971:Insulin-Like Growth Factor Binding Protein 2; D018969:Insulin-Like Growth Factor Binding Proteins; D008156:Luciferases; D012260:Ribonucleases