Insulin-like growth factor binding protein-3. Organization of the human chromosomal gene and demonstration of promoter activity.

Insulin-like growth factor binding protein-3 (IGFBP-3) can modulate the mitogenic and metabolic effects of the insulin-like growth factors (IGFs). IGFBP-3 protein levels are developmentally regulated and influenced by a number of hormonal stimuli both in vitro and in vivo. As a first step toward understanding how hormonal and developmental factors regulate IGFBP-3 production, we are characterizing the human IGFBP-3 chromosomal gene and promoter. Southern analysis demonstrates a single copy of the IGFBP-3 gene in the human genome. This gene spans 8.9 kilobases; the protein-coding region is divided into four exons while a fifth exon contains the 3'-untranslated region. Primer extension studies locate the IGFBP-3 mRNA cap site 132 base pairs 5' to the ATG translation initiation codon. On the chromosomal gene, this cap site is located 30 base pairs 3' to the start of a TATA box and 97 base pairs 3' to a consensus GC upstream promoter element, an organization common to many eukaryotic promoters. When this potential IGFBP-3 promoter region is placed upstream to the chloramphenicol acetyltransferase reporter gene, it directs high-level production of chloramphenicol acetyltransferase in transfected COS-1 cells. These observations suggest an uncomplicated organization for the IGFBP-3 chromosomal gene and promoter in the human genome.     

Identification of a cyclic adenosine monophosphate-responsive element in the rat corticotropin-releasing hormone gene

The molecular mechanisms involved in the regulation of expression of the rat CRH gene have been examined in rat pheochromocytoma (PC-12) cells transiently transfected with a chimeric gene containing 1.4 kilobases of rat CRH 5'-flanking DNA fused to the bacterial reporter gene encoding chloramphenicol acetyltransferase. Cyclic AMP analogs and activators of adenylate cyclase positively regulate the expression of this chimeric gene in PC-12 cells, inducing chloramphenicol acetyltransferase activity more than 15-fold. The DNA sequence required for this response to cAMP has been localized to a 59 base pair region located between 238 and 180 base pairs 5' to the putative CRH mRNA cap site. This sequence can confer cAMP-responsiveness on a heterologous promoter in an orientation independent fashion and has homology to cAMP regulatory regions from a number of other eukaryotic genes.