Rat hepatic glutaminase: identification of the full coding sequence and characterization of a functional promoter.
Glutamine catabolism in mammalian liver is catalysed by a unique isoenzyme of phosphate-activated glutaminase. The full coding and 5' untranslated sequence for rat hepatic glutaminase was isolated by screening lambda ZAP cDNA libraries and a Charon 4a rat genomic library. The sequence produces a mRNA 2225 nt in length, encoding a polypeptide of 535 amino acid residues with a calculated molecular mass of 59.2 kDa. The deduced amino acid sequence of rat liver glutaminase shows 86% similarity to that of rat kidney glutaminase and 65% similarity to a putative glutaminase from Caenorhabditis elegans. A genomic clone to rat liver glutaminase was isolated that contains 3.5 kb of the gene and 7.5 kb of the 5' flanking region. The 1 kb immediately upstream of the hepatic glutaminase gene (from -1022 to +48) showed functional promoter activity in HepG2 hepatoma cells. This promoter region did not respond to treatment with cAMP, but was highly responsive (10-fold stimulation) to the synthetic glucocorticoid dexamethasone. Subsequent 5' deletion analysis indicated that the promoter region between -103 and +48 was sufficient for basal promoter activity. This region does not contain an identifiable TATA element, indicating that transcription of the glutaminase gene is driven by a TATA-less promoter. The region responsive to glucocorticoids was mapped to -252 to -103 relative to the transcription start site.
Chung-Bok
M I
MI
Department of Nutritional Sciences, Thompson Hall, Cook College, Rutgers, The State University of New Jersey, New Brunswick, NJ 08903, USA.
Vincent
N
N
Jhala
U
U
Watford
M
M
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D017173:Caenorhabditis elegans / Q000201:enzymology; D015500:Chloramphenicol O-Acetyltransferase / Q000096:biosynthesis; D018076:DNA, Complementary; D005091:Exons; D015698:Genomic Library; D005938:Glucocorticoids / Q000494:pharmacology; D005972:Glutaminase / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D007668:Kidney / Q000201:enzymology; D008099:Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000096:biosynthesis; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D017386:Sequence Homology, Amino Acid; D014158:Transcription, Genetic / Q000187:drug effects
D018076:DNA, Complementary; D005938:Glucocorticoids; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D015500:Chloramphenicol O-Acetyltransferase; D005972:Glutaminase