Genomic organization and transcriptional analysis of the human l-glutaminase gene.
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. 18 kb 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.
Pérez-Gómez
Cristina
C
Departamento de Biología Molecular y Bioquímica, Laboratorio de Química de Proteínas, Facultad de Ciencias, Universidad de Málaga, 29071 Málaga, Spain.
Matés
José M
JM
Gómez-Fabre
Pedro M
PM
del Castillo-Olivares
Antonio
A
Alonso
Francisco J
FJ
Márquez
Javier
J
D024506:5' Flanking Region; D000595:Amino Acid Sequence; D001483:Base Sequence; D050976:Basic-Leucine Zipper Transcription Factors; D001665:Binding Sites; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D017930:Genes, Reporter; D015894:Genome, Human; D005972:Glutaminase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D051537:Hepatocyte Nuclear Factor 1; D051538:Hepatocyte Nuclear Factor 1-alpha; D051539:Hepatocyte Nuclear Factor 1-beta; D006801:Humans; D008099:Liver / Q000201:enzymology; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009687:Nuclear Proteins; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D016415:Sequence Alignment; D016329:Sp1 Transcription Factor / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured
D050976:Basic-Leucine Zipper Transcription Factors; D004268:DNA-Binding Proteins; C490671:HLF protein, human; C496487:HNF1A protein, human; C496495:HNF1B protein, human; D051538:Hepatocyte Nuclear Factor 1-alpha; D009687:Nuclear Proteins; D016329:Sp1 Transcription Factor; D014157:Transcription Factors; D051537:Hepatocyte Nuclear Factor 1; D051539:Hepatocyte Nuclear Factor 1-beta; D005972:Glutaminase