CHARACTERIZATION OF THE CHROMOSOMAL GENE AND PROMOTER FOR HUMAN INSULIN-LIKE GROWTH-FACTOR BINDING PROTEIN-5

To better understand the regulation of insulin-like growth factor bind ing protein-5 (IGFBP-5) expression, we cloned the IGFBP-5 gene from hu man genomic libraries and identified a region in the 5' flanking seque nce which functions as a promoter. The human IGFBP-5 gene is divided i nto four exons which, primarily due to a first intron of similar to 25 kilobases, span similar to 33 kilobases of DNA. Southern analysis ide ntified a single copy of the IGFBP-5 gene in the haploid human genome, and several independent mapping strategies found this gene tightly li nked with, and in opposite transcriptional orientation to, the IGFBP-2 gene at chromosomal region 2q33-34. Primer extension studies identifi ed the IGFBP-5 mRNA cap site 772 base pairs (bp) 5' to the first nucle otide of the translation start codon. Analysis of the 5'-flanking sequ ence identified a potential TATA element beginning 33 bp 5' to the mRN A cap site. When a DNA fragment containing this cap site and 461 bp of upstream sequence was placed 5' to the chloramphenicol acetyltransfer ase 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 elem ents essential for IGFBP-5 promoter activity.