Abstract
The purine metabolic gene adenosine deaminase (ADA) is expressed along a defined spatiotemporal pattern in the developing mammalian small intestine, where high-level expression is limited to the villous epithelium of the duodenum. This activation is observed in rodents as the intestine completes the final maturation resulting in adult crypt-villus structures at 2-3 weeks postpartum. A regulatory module responsible for this pattern of expression has been identified in the second intron of the human ADA gene. Of the multiple duodenal proteins that can interact with this small duodenal enhancer region, the studies contained in this work describe the identification of five of these proteins as the dispersed homeobox protein PDX-1. This transcription factor exhibits a profile of expression in the small intestine similar to that observed for ADA, making it an ideal candidate factor for the duodenum-specific ADA enhancer. Loss of PDX-1 binding, via a PDX-1 mutated enhancer transgenic construction, resulted in complete loss of high-level activation in the duodenum, demonstrating the absolute requirement for this factor in vivo. However, co-transfection experiments suggest that other proteins that bind the enhancer are also required for enhancer function because PDX-1 alone was incapable of significant transactivation.
Highlights
Many molecular events and interactions are required in the complex process of organogenesis of the gut
The purine metabolic gene adenosine deaminase (ADA) is expressed along a defined spatiotemporal pattern in the developing mammalian small intestine, where high-level expression is limited to the villous epithelium of the duodenum
Each differentiated cell type is produced along the length of the intestine, a particular cell type can manufacture widely differing gene products that result in functional demarcations that correspond to the physical demarcations
Summary
Preparation of Intestinal Nuclear Extracts for EMSA Experiments— All materials, rotors, solutions, etc. are at 4 °C unless otherwise stated. An aliquot was diluted to 10 pmol/l and served as 100ϫ competitor in gel shift experiments Another aliquot was purified through 4% Biogel (Qbiogene, Carlsbad, CA) and the DNA isolated using the Mermaid kit (Qbiogene). Binding reactions contained 5 l of 5ϫ buffer E (125 mM Tris, pH 8.0, 32.5 mM MgCl2, 2.5 mM dithiothreitol, 2.5 mM EDTA, 250 mM KCl, and 0.6 g/l bovine serum albumin), 0.5 l of poly(dI1⁄7dC)/poly(dA1⁄7dT) (2 g/l), 10 l of nuclear extract or extract diluted with buffer D (as in nuclei preparation), and competitor oligonucleotide and/or antibody as needed plus water to a final volume of 24 l. CAT assays, protein determination, and copy number analysis were performed as described previously [13]. The cell extract was assayed for CAT and protein concentration as described previously [17]
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