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J. Biol. Chem., Vol. 266, Issue 2, 752-758, Jan, 1991

Transcriptional analysis of the human ornithine aminotransferase promoter

JF Engelhardt, G Steel and D Valle
Howard Hughes Medical Institute, Department of Pediatrics, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.

The regulation of ornithine delta-aminotransferase (OAT) expression is poorly characterized in humans, but in rat there are tissue-specific responses to nutritional and hormonal stimuli. We analyzed 1.3 kilobases of 5'-flanking sequence and part of intron 1 of the human OAT gene and found several candidate regulatory sequences including four copies of a motif also present in the promoters of three other urea cycle-related enzymes (urea cycle element). We transfected a series of promoter deletion constructs into HepG2 (human hepatoma), H4 (rat hepatoma), and HEK (human embryonic kidney) cells and obtained maximal expression with 134 base pairs (bp) of 5'-flanking DNA. One of the urea cycle elements and the 3' end of exon 1 had positive effects on expression in all cell lines. However, mutations in a 22-bp element which overlaps the transcriptional start site decreased activity 4-fold in H4 cells only. cAMP increased endogenous OAT mRNA 4-fold in HepG2 cells and the expression of constructs containing as little as -85 bp of 5'-flanking DNA 2- 5-fold in HepG2 and H4 cells. DNase I protection analysis of the first 134 bp of 5'-flanking sequence with nuclear extracts from rat liver, rat kidney, HEK, and HepG2 cells showed two patterns of protection over one of the urea cycle elements. These studies provide the foundation for the understanding of tissue-specific regulation of OAT.
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