Isolation and characterization of the rat gene encoding glutamate dehydrogenase

Abstract

The concentration of glutamate dehydrogenase (GDH) varies strongly between different organs and between different regions within organs. To permit further studies on the regulation of GDH expression, we isolated and characterized the rat gene encoding the GDH protein. This gene contains 13 exons and spans approximately 34 kbp. The GDH gene is present as a single, autosomally located copy in the Wistar rat genome, but shows an extensive restriction-fragment-length polymorphism for several enzymes. Promoter activity of the 5'-flanking sequence is shown by transient transfection experiments. The 5'-flanking sequence contains a TTAAAA sequence at position -29, instead of a consensus TATA box and, like many other TATA-less promoters, is characterized by a very high G + C content. In addition, consensus sequences for the binding sites of the transcription factors Spl and Zif268 are present in the G + C-rich upstream region.Glutamate dehydrogenase (GDH), a mitochondria1 matrix enzyme, catalyzes the reversible oxidative deamination of L-glutamate to 2-oxoglutarate and ammonia, using NAD' or NADP' as cofactor [l]. GDH therefore plays an important role in ammonia and amino acid metabolism. Although GDH is an ubiquitous enzyme, its concentration in different organs varies markedly, the highest concentrations being found in liver, brain, kidney and pancreas [2, 31.In liver GDH is highly expressed only in the hepatocytes. In the adult rat [4-91 and human [lo] liver the GDH concentration depends on the position of the hepatocyte along the central-portal distance, the highest activity and protein concentration being found in hepatocytes around the central veins. In rat liver two mRNAs are present which differ in the length of their 3'-untranslated region [ l l , 121. As shown by in situ hybridization, these mRNAs are also heterogeneously distributed within the adult rat liver, both showing a gradient in cellular concentration decreasing along the central-portal distance [13]. The heterogeneous mRNA distribution in the liver lobule demonstrates the presence of regional differences in the rate of pretranslational processes involved in GDH

Identification and analysis of a matrix‐attachment region 5′ of the rat glutamate‐dehydrogenase‐encoding gene

“…to -557, with the transcription initiation site numbered +1 [20]. The addition of different amounts of E. coli competitor DNA (varying from 0-SO0 pg/ml, corresponding to a 0-5000-fold excess by mass) did not significantly influence the binding, which demonstrates the specificity of the binding.…”

“…As a first approach to identify MAR sequences in the rat GDH gene, in vitro MAR assays were used. Bacteriophage A genomic clones containing the GDH gene [20] were digested with restriction endonucleases. The resulting DNA fragments were tested for binding to lithium 3,5-diiodosalicylate-extracted rat liver nu- …”

NRF-1, an activator involved in nuclear-mitochondrial interactions, utilizes a new DNA-binding domain conserved in a family of developmental regulators.

“…), 5 (Chau et al 1992), 6 (Braidotti et al 1993), Z (Kreysing et al 1990), 8(Mitsubuchi et al 1991, 9 (Forrest et al 1991), t° (Tasanen et al 1992), ~ (Giallongo et al 1990), ~2 (Das et al 1993), la (Jenkins et al 1991), 14(Katz andKahana 1988), lS (Moshier et al 1992), 16 (Wen et al 1989), lZ (Amy et al 1990), 18 (Yagawa et al 1990), W (Miyake et al 1986), 2° (Cavailles et al 1993), 21 (Miyatake et al 1985), 22 (Degen et al 1991), 2a{Zhou et al 1992), ~4 (Tewari et al 1989), 2S (Mamula and Goldfine 1992), 26 (Lutfalla et al 1992), 27 (Haendler andHofer 1990), 28{Kutt et al 1989), 29 (Nojima 1989), S° (Parmentier and Lefort 1991), al (Tsukamoto et al 1991), a2 (Lee et al 1992), a3 (Khew-Goodall et al 1991), 34 (Kurihara et al 1990), aS (Pearson et al 1991), 36{Kunze et al 1991), 37 (Baer et al 1990), aa{Biamonti et al 1989, 39 Perry 1985), 4°{Hurt et al 1989), 4~ (Sittman et al 1983), 42 (Furuno et al 1991), 43 (Ku et al 1993), 44{Adachi and Tsujimoto 1990), 45 (Park et al 1992), 46 (Luethy et al 1990), 47 (Walter et al 1989), 48 …”

FISH mapping of three ammonia metabolism genes (Glul, Cps1, Glud1) in rat, and the chromosomal localization of GLUL in human and Cps1 in mouse

“…In recent years, the rat genes encoding glutamate dehydrogenase (GLUD; Das et al 1993), glutamine synthetase (glutamateammonia ligase, GLUL; van de Zande et al 1990) and carbamoylphosphate synthetase 1 (CPS; Van den Hoff et al 1995) have been isolated. These enzymes have important functions in ammonia metabolism, and each of them is encoded by a single gene.…”

“…The rat G-band nomenclature is according to Levan (1974); for an updated recent version of the idiogram, see RATMAP database (URL http://ratmap.gen.gu.se/ratmap/WWW Nomen/RNOIdiogrRev96new.GIF). For the mapping of rat Glud1, two clones of genomic DNA (pgGDH6d, 6700 bp containing exon 1, and pgGDH2u, 5800 bp containing exons 8-12; Das et al 1993) were used. The results from each of the probes were the same, and Glud1 could be sublocalized to RNO16p16 (Fig.…”