Developmentally Regulated Expression of the Gene Family for Cytosolic Glutamine Synthetase in Pisum sativum

In Pisum sativum, two classes of genes encode distinct isoforms of cytosolic glutamine synthetase (GS). The first class comprises two nearly identical or “twin” GS genes (GS341 and GS132), while the second comprises a single GS gene (GS299) distinct in both coding and noncoding regions from the “twin” GS genes. Gene-specific analyses were used to monitor the individual contribution of each gene for cytosolic GS during root nodule development and in cotyledons during germination, two contexts where large amounts of ammonia must be assimilated by GS for nitrogen transport. mRNAs corresponding to all three genes for cytosolic GS were shown to accumulate coordinately during a time course of nodule development. All the GS mRNAs also accumulate to wild-type levels in mutant nodules formed by a nifD⁻ strain of Rhizobium leguminosarum indicating that induced GS expression in pea root nodules does not depend on the production of ammonia. Distinct patterns of expression for the two classes of GS genes were observed in certain mutant root nodules and most dramatically in cotyledons of germinating seedlings. The different patterns of expression between the two classes of genes for cytosolic GS suggests that their distinct gene products may serve nonoverlapping functions during pea development.     

Specific Insulin-Mediated Regulation of Glutamine Synthetase in Cultured Chick Astroglial Cells

The expression of glutamine synthetase (GS; L-glutamate ammonia ligase; EC 6.3.1.2) in primary cultures of chick astroglial cells and neurons grown in a chemically defined medium, with and without insulin added, was investigated. An inhibitory effect of insulin toward GS activity, and specific to chick astroglial cells, was observed. Neurons in culture were not sensitive to the hormone effect. Modulation of the activating effect of hydrocortisone on glial GS by insulin was also observed. The data suggest that insulin contributes to the regulation of the metabolism of amino acid neurotransmitters via its effect on GS.     

Glucocorticoid Stimulation of Glutamine Synthetase Production in Cultured Rat Glioma Cells

Abstract: A sensitive radioisotopic assay has been used to examine the kinetic properties and regulation of biosynthesis of glutamine synthetase in C-6 glioma cultures. The K_m values for glutamate, MgATP, and ammonium ion were 5mM, 14 mM, and 0.042 mM, respectively, when measured at the pH optimum of 7.2. There was an absolute requirement for a divalent metal ion, with 15 mM- Mg²⁺ being the preferred ion at pH 7.2. Activity was completely inhibited after 30 min with 8 mM-L-methionhe sulfoximine. The addition of 1 μM-cortisol to C-6 cultures caused a two to threefold increase in glutamine synthetase specific activity over a 96-h period, while dexamethasone at the same concentration elevated the level some 7-10-fold. This was specific for glucocorticoids, as other steroid hormones or catecholamines did not significantly affect glutamine synthetase specific activity. Cycloheximide (30 μM) or actinomycin D (0.01 μg/ml) blocked the hormone response. The continued presence of hormone was required in order to maintain an elevated enzyme level. The results suggest that glucocorticoids act to induce glutamine synthetase by stimulating new enzyme synthesis.     

Derepression of the Glutamine Synthetase in Neuroblastoma Cells at Low Concentrations of Glutamine

Abstract: Regulation of the biosynthesis of glutamine synthetase was studied in neuroblastoma cells (Neuro-2A) by use of a recently developed, sensitive radioisotopic assay. The removal of glutamine from the culture medium of these cells for 24 h resulted in a 10-fold increase in glutamine synthetase specific activity (15-fold after 2 weeks) compared with the basal level found in cells grown in the presence of 2 m M glutamine. Following the growth of these cells for 2 weeks in the presence of various concentrations of glutamine, a negative linear correlation was observed between the specific activity of glutamine synthetase (from 1.7 to 0.14 unit/mg) and the concentration of glutamine in the growth medium (from 0.5 to 2 m M ). Cycloheximide or actinomycin D blocked the increase in glutamine synthetase activity observed in the absence of glutamine. These results suggest that the removal of glutamine led to the induction of glutamine synthetase by stimulating new enzyme synthesis. The enzyme was not degraded, but only diluted, by growth upon readdition of glutamine to the medium. The influence of glutamine depletion is also reported for C-6 glioma cells and glial cells in primary cultures.     

Detection of a Cytosolic Glutamine Synthetase in Leaves of Nicotiana tabacum L. by Immunocytochemical Methods

Two glutamine synthetase (GS) polypeptides (44 and 39 kD) were immunodetected on western blots of leaf extracts from tobacco (Nicotiana tabacum L.), a plant that has been reported to contain only chloroplast GS in the leaves. By immunocytochemical methods, we confirmed the localization of GS in the cytosol of cells in the vascular tissue and in the chloroplasts of mesophyll cells.     

Glutamine Synthetase in Higher Plants Regulation of Gene and Protein Expression from the Organ to the Cell

Compared to other enzymatic systems, the regulation of GS isoenzymeexpression shows a unique diversity. Considering that GS isone of the oldest existing and functioning genes found in allextant life forms, we can hypothesise that the evolution ofmetabolic pathways from primitive pre-procaryotes to lower andthen higher plants might have gradually refined the functionof GS to provide reduced nitrogen forms for the rest of themetabolism (Kumada et al. 1993). This refinement might explainthe genetic and biological diversity encountered in the variousmodes of expression and regulation of higher plant GS isoenzymesboth at the cellular and intracellular levels (Fig. 1). Althoughmodel plants are valuable sources of information helping todecipher fine regulatory control mechanisms (Lam et al. 1996),the study of this genetic diversity appears to be one of themost promising areas of research, necessary to better understandammonia assimilation in plants and more generally improve nitrogenuse efficiency. (Received September 6, 1999; Accepted October 15, 1999)     

The Promoter of the Gene for Glutamine Synthetase from Rice Shows Organ-Specific and Substrate-Induced Expression in Transgenic Tobacco Plants

A 2-kb fragment from the 5'-flanking region of the RGS-28 gene,which encodes the cytosolic glutamine synthetase in Oryza sativaL., was fused to a ß-glucuronidase (GUS) reportergene and introduced into Nicotiana tabacum by Agrobacterium-mediatedtransformation. The promoter was predominantly active in theleaves of transgenic plants, as it is in authentic rice plants.The promoter also responded to externally applied ammonium ions.It is suggested that the cis-acting regulatory elements responsiblefor the recognition of the leaf as a site of synthesis and ofammonia, a substrate for glutamine synthetase, are located withina 2-kb region of the promoter. (Received October 15, 1990; Accepted January 11, 1991)     

Inhibition of Gene Expression by Peptide Nucleic Acids in Cultured Cells

Abstract

We have tested in cultured cells the capacity of antisense and antigene PNAs to inhibit, in a sequence specific manner, the expression of oncogenes in leukaemia and pancreatic carcinoma cells. The results observed appeared promising and suggest that PNA may play in the future an important role in targeting disease-related genes.     

Accumulated Expression Level of Cytosolic Glutamine Synthetase 1 Gene (OsGS1;1 or OsGS1;2) Alter Plant Development and the Carbon-Nitrogen Metabolic Status in Rice

Maintaining an appropriate balance of carbon to nitrogen metabolism is essential for rice growth and yield. Glutamine synthetase is a key enzyme for ammonium assimilation. In this study, we systematically analyzed the growth phenotype, carbon-nitrogen metabolic status and gene expression profiles in GS1;1-, GS1;2-overexpressing rice and wildtype plants. Our results revealed that the GS1;1-, GS1;2-overexpressing plants exhibited a poor plant growth phenotype and yield and decreased carbon/nitrogen ratio in the stem caused by the accumulation of nitrogen in the stem. In addition, the leaf SPAD value and photosynthetic parameters, soluble proteins and carbohydrates varied greatly in the GS1;1-, GS1;2-overexpressing plants. Furthermore, metabolite profile and gene expression analysis demonstrated significant changes in individual sugars, organic acids and free amino acids, and gene expression patterns in GS1;1-, GS1;2-overexpressing plants, which also indicated the distinct roles that these two GS1 genes played in rice nitrogen metabolism, particularly when sufficient nitrogen was applied in the environment. Thus, the unbalanced carbon-nitrogen metabolic status and poor ability of nitrogen transportation from stem to leaf in GS1;1-, GS1;2-overexpressing plants may explain the poor growth and yield.     

Blue and Red Light-dependent Alterations in the Ratio of two Forms of Glutamine Synthetase in Chlorella kessleri

Two forms of glutamine synthetase (EC 6.3.1.2) can be separated in crude extracts of Chlorella kessleri on the basis of their different surface charges. The two enzyme forms (GS1 and GS2) respond differently upon transferring the cells from darkness to autotrophic growth in white light: the activity of GS2 increases, that of GS1 remains unchanged. The increase in GS2 activity is only brought about by blue light; in red light GS2 activity appears to be uninfluenced, while that of GS1 increases. There are no indications of wavelength-dependent oligomerization processes as a cause for the observed activity alterations. There is however, a strong influence of inhibitors of protein biosynthesis. Cycloheximide and lincomycin both affect the blue light-dependent increase in activity of GS2, cycloheximide preventing that of GS1 in red lgiht completely. Since literature data point to localization of GS2 in the chloroplast, and GS1 in the cytosol, the data are discussed in view of two different photoreceptors involved in the regulation of the amounts of GS1 and GS2 in different compartments of the Chlorella cell.     

Control by Glutamine of the Synthesis of Nitrate Reductase in Cultured Spinach Cells

Immunoblotting, using antibodies raised against electrophoreticallypure nitrate reductase, was used to study the regulation ofsynthesis of nitrate reductase in cultured spinach cells. Theextent of the loss of nitrate reductase activity that occurredwhen cultures were transferred to a glutamine-containing mediumwas correlated with the decrease in the level of cross-reactingmaterial (repression). Removal of exogenous glutamine resultedin the appearance of nitrate reductase activity as well as ofimmunoreactive protein (derepression). The activity of nitratereductase in spinach cells under "repressing" or "derepressing"conditions appears to be regulated by changes in the amountof the enzyme protein rather than by inactivation and activationof preexisting protein. (Received August 22, 1991; Accepted May 28, 1992)     

The Protective Effects of Riluzole on Manganese-Induced Disruption of Glutamate Transporters and Glutamine Synthetase in the Cultured Astrocytes

Chronic exposure to excessive manganese (Mn) can lead to manganism, a type of neurotoxicity accomplished with extracellular glutamate (Glu) accumulation. To investigate this accumulation, this study focused on the role of astrocyte glutamate transporters (GluTs) and glutamine synthetase (GS), which have roles in Glu transport and metabolism, respectively. And the possible protective effects of riluzole (a glutamatergic modulator) were studied in relation to Mn exposure. At first, the astrocytes were exposed to 0, 125, 250, and 500 μM MnCl(2) for 24 h, and 100 μM riluzole was pretreated to astrocytes for 6 h before 500 μM MnCl(2) exposure. Then, [(3)H]-glutamate uptake was measured by liquid scintillation counting; Na(+)-K(+) ATPase and GS activities were determined by a colorimetric method; glutamate/aspartate transporter (GLAST), glutamate transporter-1 (GLT-1), and GS mRNA expression were determined by RT-PCR and protein levels were measured by western blotting. The results showed that Mn inhibited Glu uptake, Na(+)-K(+) ATPase and GS activities, GLAST, GLT-1, and GS mRNA, and protein in a concentration-dependent manner. And they were significantly higher for astrocytes pretreated with 100 μM riluzole than the group exposed to 500 μM MnCl(2). The results suggested that Mn disrupted Glu transport and metabolism by inhibiting GluTs and GS. Riluzole activated protective effects on enhancing GluTs and GS to reverse Glu accumulation. In conclusion, Mn exposure results in the disruption of GLAST, GLT-1, and GS expression and function. Furthermore, riluzole attenuates this Mn toxicity.