In mammals, glutaminase (GA) is expressed in most tissues, but the regulation of organ-specific expression is largely unknown. Therefore, as an essential step towards studying the regulation of GA expression, the human liver-type GA (hLGA) gene has been characterized. LGA genomic sequences were isolated using the genome walking technique. Analysis and comparison of these sequences with two LGA cDNA clones and the Human Genome Project database, allowed the determination of the genomic organization of the LGA gene. The gene has 18 exons and is approx. 18kb long. All exon/intron junction sequences conform to the GT/AG rule. Progressive deletion analysis of LGA promoter—luciferase constructs indicated that the core promoter is located between nt −141 and +410, with several potential regulatory elements: CAAT, GC, TATA-like, Ras-responsive element binding protein and specificity protein 1 (Sp1) sites. The minimal promoter was mapped within +107 and +410, where only an Sp1 binding site is present. Mutation experiments suggested that two CAAT recognition elements near the transcription-initiation site (-138 and −87), play a crucial role for optimal promoter activity. Electrophoretic mobility-shift assays confirmed the importance of CAAT- and TATA-like boxes to enhance basal transcription, and demonstrated that HNF-1 motif is a significant distal element for transcriptional regulation of the hLGA gene.
Abbreviations used: C/EBP, CAAT-enhancer binding protein; CREB, cAMP-responsive element binding protein; EMSA, electrophoretic mobility-shift assay; ER, oestrogen receptor; GA, phosphate-activated glutaminase; HLF, hepatic leukaemia factor; KGA, kidney-type glutaminase; hKGA, human kidney-type glutaminase; rKGA, rat kidney-type glutaminase; LGA, liver-type glutaminase; hLGA, human liver-type glutaminase; rLGA, rat liver-type glutaminase; PDZ, PSD95/Dlg/ZO1 domains; RREB, Ras-responsive element binding protein; RT, reverse transcription; Sp1, specificity protein 1; UTR, untranslated region.
This paper is dedicated to Professor I. Núñez de Castro on the occasion of his retirement. We are indebted to him for his example as a scientist and as a colleague.
Both authors contributed equally to this work.
The nucleotide sequence data reported in this paper have been submitted to the DDBJ, EMBL, GenBank® and GSDB Nucleotide Sequence Databases under the accession number AF348119.