Functional analysis of the mouse galactose-1-phosphate uridyl transferase (GALT)promoter.
Galactose-1-phosphate uridyltransferase (GALT) is expressed in most tissues, but the near total absence of catalytic activity in humans with the disease galactosemia leads to specific organ dysfunction, the pathophysiology of which remains an enigma. To characterize the transcriptional regulation of the mouse GALT gene, we isolated and sequenced over 3 kb of a 5'-flanking sequence and functionally characterized the region using in vitro transient transfection and in transgenic mice. A minimal promoter of 145 bp was found to function in both HepG2 cells and NS20Y mouse neuroblastoma cells. The minimal promoter contains regions of homology to the corresponding rat and human GALT genes. In transgenic mice expressing a luciferase transgene under control of a 1.9-kb fragment of the mGALT promoter region, reporter activity was found in most tissues, with higher than expected reporter levels in neonatal brain. To determine if high galactose levels in tissues could induce promoter activity, we bred the mGALT:luciferase transgene into a line of mice in which the GALT gene function has been eliminated by homologous recombination. High tissue levels of galactose and metabolites did not induce reporter activity above background. The studies show that GALT transcriptional regulation is complex and not directly induced by substrate levels.
Leslie
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Division of Human Genetics, Children's Hospital Medical Center, Children's Hospital Research Foundation, 3333 Burnet Avenue, Cincinnati, Ohio 45229-3039, USA. Lesln0@chmcc.org
Bai
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D000483:Alleles; D000818:Animals; D000831:Animals, Newborn; D001483:Base Sequence; D015152:Blotting, Northern; D001921:Brain / Q000378:metabolism; D002384:Catalysis; D002460:Cell Line; D018076:DNA, Complementary / Q000378:metabolism; D005091:Exons; D005690:Galactose / Q000378:metabolism / Q000493:pharmacokinetics; D017353:Gene Deletion; D006801:Humans; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008822:Mice, Transgenic; D008957:Models, Genetic; D008969:Molecular Sequence Data; D010957:Plasmids / Q000378:metabolism; D011401:Promoter Regions, Genetic; D051381:Rats; D017422:Sequence Analysis, DNA; D013997:Time Factors; D014018:Tissue Distribution; D014158:Transcription, Genetic; D014162:Transfection; D019076:Transgenes; D014407:Tumor Cells, Cultured; D005694:UTP-Hexose-1-Phosphate Uridylyltransferase / Q000096:biosynthesis* / Q000235:genetics*
D018076:DNA, Complementary; D008156:Luciferases; D005694:UTP-Hexose-1-Phosphate Uridylyltransferase; D005690:Galactose