A regulatory element is characterized by purine-mediated and cell-type-specific gene transcription.
Purines and purine nucleotides were found to affect transcription of the hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene in whole nuclei isolated from intestinal mucosa of adult rats fed a purine- and purine nucleotide-free diet. Nuclear run-on transcription assays, performed on whole nuclei from different tissues and cell types, identified an intestine-specific decrease in the overall incorporation of [alpha-32P]UTP in HPRT transcripts from intestinal epithelial cell nuclei when exogenous purines or purine nucleotides were omitted from either the diet or culture medium. Using a 990-base-pair genomic fragment that contains the 5'-flanking region from the HPRT gene, we generated plasmid constructs with deletions, transfected the DNA into various cell types, and assayed for chloramphenicol acetyltransferase (CAT) reporter activity in vitro. We determined that an element upstream from the putative transcriptional start site is necessary to maintain the regulatory response to purine and nucleotide levels in cultured intestinal epithelial cells. These results were tissue and cell type specific and suggest that in the absence of exogenous purines, the presence of specific factors influences transcriptional initiation of HPRT. This information provides evidence for a mechanism by which the intestinal epithelium, which has been reported to lack constitutive levels of de novo purine nucleotide biosynthetic activity, could maintain and regulate the salvage of purines and nucleotides necessary for its high rate of cell and protein turnover during fluctuating nutritional and physiological conditions. Furthermore, this information may provide more insight into regulation of the broad class of genes recognized by their lack of TATA and CCAAT box consensus sequences within the region proximal to the promoter.
Walsh
M J
MJ
Department of Pediatrics, Mount Sinai School of Medicine, New York, New York 10029.
Sanchez-Pozo
A
A
Leleiko
N S
NS
D000199:Actins / Q000235:genetics*; D000818:Animals; D001483:Base Sequence; D001921:Brain / Q000201:enzymology; D002467:Cell Nucleus / Q000378:metabolism*; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004032:Diet; D005796:Genes / Q000187:drug effects; D005809:Genes, Regulator / Q000187:drug effects; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics*; D007413:Intestinal Mucosa / Q000201:enzymology; D008099:Liver / Q000201:enzymology; D008297:Male; D051379:Mice; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D011687:Purines / Q000494:pharmacology*; D051381:Rats; D015183:Restriction Mapping; D014158:Transcription, Genetic; D014162:Transfection; D014544:Uridine Triphosphate / Q000378:metabolism
D000199:Actins; D011687:Purines; D007041:Hypoxanthine Phosphoribosyltransferase; D014544:Uridine Triphosphate