Abstract
To better understand the regulation of insulin-like growth factor binding protein-5 (IGFBP-5) expression, we cloned the IGFBP-5 gene from human genomic libraries and identified a region in the 5' flanking sequence which functions as a promoter. The human IGFBP-5 gene is divided into four exons which, primarily due to a first intron of approximately 25 kilobases, span approximately 33 kilobases of DNA. Southern analysis identified a single copy of the IGFBP-5 gene in the haploid human genome, and several independent mapping strategies found this gene tightly linked with, and in opposite transcriptional orientation to, the IGFBP-2 gene at chromosomal region 2q33-34. Primer extension studies identified the IGFBP-5 mRNA cap site 772 base pairs (bp) 5' to the first nucleotide of the translation start codon. Analysis of the 5'-flanking sequence identified a potential TATA element beginning 33 bp 5' to the mRNA cap site. When a DNA fragment containing this cap site and 461 bp of upstream sequence was placed 5' to the chloramphenicol acetyltransferase reporter gene and transfected into MDA-MB-468 human breast cancer cells, it directed chloramphenicol acetyltransferase expression in an orientation-specific manner, suggesting that this region contains elements essential for IGFBP-5 promoter activity.
Highlights
To better understand the regulation of insulin-like other five members of the IGFBPfamily [1,4].Additional studgrowth factor binding protein-5 (IGFBP-5) expression, ies found that IGFBP-5 was expressed by a wide variety of we cloned theIGFBP-5 gene from human genomic libratirs-sues [1,4]
The human IGFBPg-e6ne ably dependingon post-translational modifications of IGFBP-5 is divided into four exons which, primarily due to a afnirdston the specific cell targets of IGF action
Primer Extensionof mRNA Expressed by p46lCAT-Total RNA was RESULTS Complete Sequence of hZGFBP-5cDNA-Much of the 3'-unisolated from MDA-MB-468 cells harvested 22 h after transfection with translated region of hIGFBP-5 cDNA clone BP-5.12 was not p461CAT
Summary
Complete Sequence of hZGFBP-5cDNA-Much of the 3'-unisolated from MDA-MB-468 cells harvested 22 h after transfection with translated region of hIGFBP-5 cDNA clone BP-5.12 was not p461CAT. Hybridization with oligonucleotide probes 5735 and5:PE, located 5' and 3' to theSfiI cleavage site,respectively (see Fig. 6), determined the relative order of the nineEcoRI fragments of the cosmid insert. The authenticity of the cosmid fragments was confirmed by hybridizing the 32P-labeledcosmid clone to EcoRI-cleaved total human DNA. PCR amplifications from a human-rodent cell hybrid; representative the 5"flanking region of the hIGFBP-5 gene, hybridized with analysis of PCR products generated by amplification of total genomic -16-, 1.0-, and 4.6-kb fragments of EcoRI-cleaved total human. B, fluorescence hybridizationof genomic exon-containing ment ofEcoRI-cleaved total human DNA(dantaot shown) PCR product from each primer pair was of identical length probes hybridized to different PFGE fragments than did their when either total human DNA, the cosmid subclone phBP5- respective 3' probes.
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