Glutamine Synthetase Gene Expression Research Articles

Response of transgenic poplar overexpressing cytosolic glutamine synthetase to phosphinothricin

Glutamine synthetase (GS) is the main enzyme involved in ammonia assimilation in plants and is the target of phosphinothricin (PPT), an herbicide commonly used for weed control in agriculture. As a result of the inhibition of GS, PPT also blocks photorespiration, resulting in the depletion of leaf amino acid pools leading to the plant death. Hybrid transgenic poplar ( Populus tremula × P. alba INRA clone 7171-B4) overexpressing cytosolic GS is characterized by enhanced vegetative growth [Gallardo, F., Fu, J., Cantón, F.R., García-Gutiérrez, A., Cánovas, F.M., Kirby, E.G., 1999. Expression of a conifer glutamine synthetase gene in transgenic poplar. Planta 210, 19–26; Fu, J., Sampalo, R., Gallardo, F., Cánovas, F.M., Kirby, E.G., 2003. Assembly of a cytosolic pine glutamine synthetase holoenzyme in leaves of transgenic poplar leads to enhanced vegetative growth in young plants. Plant Cell Environ. 26, 411–418; Jing, Z.P., Gallardo, F., Pascual, M.B., Sampalo, R., Romero, J., Torres de Navarra, A., Cánovas, F.M., 2004. Improved growth in a field trial of transgenic hybrid poplar overexpressing glutamine synthetase. New Phytol. 164, 137–145], increased photosynthetic and photorespiratory capacities [El-Khatib, R.T., Hamerlynck, E.P., Gallardo, F., Kirby, E.G., 2004. Transgenic poplar characterized by ectopic expression of a pine cytosolic glutamine syntetase gene exhibits enhanced tolerance to water stress. Tree Physiol. 24, 729–736], enhanced tolerance to water stress (El-Khatib et al., 2004), and enhanced nitrogen use efficiency [Man, H.-M., Boriel, R., El-Khatib, R.T., Kirby, E.G., 2005. Characterization of transgenic poplar with ectopic expression of pine cytosolic glutamine synthetase under conditions of varying nitrogen availability. New Phytol. 167, 31–39]. In vitro plantlets of GS transgenic poplar exhibited enhanced resistance to PPT when compared with non-transgenic controls. After 30 days exposure to PPT at an equivalent dose of 275 g ha −1, growth of GS transgenic poplar plantlets was 5-fold greater than controls. The response of young leaves to PPT treatment depends on physiological state as indicated by GS and Rubisco (LSU) levels. Young leaves from control plants, typically in a low differentiation state, respond to the herbicide showing up-regulation of GS and LSU. In contrast, young leaves from transgenic lines, with higher initial GS and LSU levels compared to control, display up-regulation of NADP +-isocitrate dehydrogenase. Differences between control and GS transgenics in their response to PPT are discussed in relation to their differences in photosynthetic and photorespiratory capacities ( El-Khatib et al., 2004).

Oestradiol Up‐Regulates Glutamine Synthetase mRNA and Protein Expression in the Hypothalamus and Hippocampus: Implications for a Role of Hormonally Responsive Glia in Amino Acid Neurotransmission

Rapidly emerging evidence suggests that glial cells in the central nervous system are sensitive to oestrogen actions. However, the functional consequences of the cellular mechanisms of these cells have proven difficult to study in vivo because of the intimate relationships between neurones and glia. Microarray technology offers the potential to uncover steroid hormone regulation of glial-specific genes that may play a role in hormone-dependent neuronal-glial interactions. Analysis of transcriptomes from the medial basal hypothalamus (MBH) of oestradiol and vehicle-treated adult ovariectomised mice revealed an up-regulation of several glial specific genes by oestradiol, including glutamine synthetase (GS), which facilitates the conversion of glutamate to glutamine and plays an integral role in amino acid neurotransmission. In situ hybridisation confirmed that oestradiol treatment resulted in an up-regulation of GS gene expression in the arcuate and ventromedial nuclei of the MBH, as well as the medial amygdala and hippocampus. Moreover, oestradiol increased protein expression of GS in both the MBH and hippocampus. Neurones are incapable of de novo net synthesis of glutamate from glucose and are dependent on glial-provided precursors such as glutamine to renew their amino acid transmitter pools. Thus, oestradiol induced expression of GS suggests a significant role for glial cells in hormonal modulation of glutamatergic neurotransmission important to female reproductive behaviours, neuroendocrine physiology and cognitive functions.

Development of Transfection and High-Producer Screening Protocols for the CHOK1SV Cell System

To date, the FDA has approved 18 monoclonal antibody (MAb) therapeutic drugs with targets ranging from asthma and rheumatoid arthritis to leukemia. Many of these approved products are produced in Chinese hamster ovary cells (CHO) making CHO a significant and relevant host system. We studied the applicability of CHOK1SV cells as a potential host cell line for MAb production in terms of timelines, achievable titers, transfectant stability, and reproducibility. CHOK1SV, developed by Lonza Biologics, is a suspension, protein-free-adapted CHOK1-derivative utilizing the glutamine synthetase (GS) gene expression system. CHOK1SV expresses the GS enzyme endogenously; thus, positive transfectants were obtained under the dual selection of methionine sulfoximine (MSX) and glutamine-free media. We examined outgrowth efficiencies, specific productivities, and achievable batch titers of three different IgG MAbs transfected into CHOK1SV. Reducing the MSX concentration in the initial selection medium resulted in a decreased incubation time required for transfectant colonies to appear. Specific productivities of "high-producers" ranged between 11 and 49 pg/c/d with batch titers ranging from 105 to 519 mg/L. Transfectant stability and the effects of MSX also were investigated, which indicated that the addition of MSX was necessary to maintain stable MAb production. Cell growth was stable regardless of MSX concentration.

Toward More Efficient Protein Expression: Keep the Message Simple

Optimization of gene coding-sequence, including preferred codon usage and removal of cryptic splice sites and mRNA-destabilizing motifs, has been shown to improve recombinant protein production of different proteins. Here, we present data to show that gene optimization can also be used to improve the production of a complex macromolecule, namely an antibody. When applied to the heavy and light chain genes of our model antibody, we found that greater numbers of high-producing transfectants as well as increased levels of protein production were observed (approximately 1.5-fold). In this test model, production was improved even though the antibody has previously been demonstrated to give high expression in stably transfected cells (up to 5 g/L in bioreactors). Because the parental heavy chain sequence contained introns, and the process of gene optimization is most efficiently performed on sequences without introns, we demonstrated that removal of introns in the coding sequence had no effect on the quantity of antibody produced. All constructs were evaluated using Lonza's glutamine synthetase gene expression vectors in Chinese hamster ovary cells. Our findings suggest that significant improvements in product yields can be achieved by gene optimization, which may facilitate the processing and translation of gene transcripts.

Inherent characteristics of gene expression for buffering environmental changes without the corresponding transcriptional regulations.

Gene expression patterning is crucial for environmental nutritional responses such as the nitrogen response in Escherichia coli. The nitrogen response is primarily regulated by the expression of glutamine synthetase (GS), which catalyzes the sole reaction of glutamine formation, by cis-logic regulatory circuits. Here, by removing the entire corresponding operator and promoter regions required for the control of GS, we constructed an E. coli strain that enables the detection of the basal GS gene expression, which is expressed from a plain promoter unrelated to the nitrogen response, and measured by co-transcribed GFP expression, an indicator of GS expression. Using strain cultures, we found that the GS expression level was able to shift inversely against the change of the environmental glutamine concentration. As a control experiment, we repeated similar experiments with another strain in which the GS regulatory region remained intact and the GFP gene following the plain promoter was introduced into a different chromosomal site. For this strain, we found that the GFP expression level did not shift in accordance with the environmental glutamine concentration. These results showed that GS expression from the plain promoter exhibited a responsive ability to buffer environmental changes, whereas the GS expression shift did not correlate with the specific characteristics of the plain promoter and GFP expression. This study identifies the inherent characteristics of basal gene expression in response to environmental changes, facilitating a deeper understanding of cellular design principles.

Estradiol modulation of astrocytic form and function: Implications for hormonal control of synaptic communication

There is a growing appreciation for the importance of glial cells to overall brain function. For decades, glial cells have been considered relatively passive supporters of nerve cell function, providing only structural and metabolic support to the communicating neurons. Now, rapidly emerging evidence demonstrates that glial cells are active participants in the processes of synaptic patterning and synaptic transmission. Like their neuronal neighbors residing in steroid sensitive brain regions, glial cells demonstrate a responsiveness to gonadal steroids that has been best characterized by physical changes in their morphology. However, because of their intimate relationship, the nature of neuronal–glial interactions has been challenging to study in vivo and until recently, the functional relevance of steroid-induced changes in glial morphology to neuroendocrine functions could only be implied from anatomical and in vitro studies. The advent of microarray technology offers the potential to uncover steroid regulation of glial-specific genes that may play a role in hormone-dependent neuronal–glial interactions. Our microarray analysis of the rodent hypothalamus has revealed that estradiol increases the expression of a number of glial-specific genes, including glutamine synthetase, an enzyme that inactivates glutamate through its conversion to glutamine. Given that glutamine is the predominant precursor for releasable pools of glutamate, our observation that estradiol increases glutamine synthetase gene and protein expression suggests that hormonal regulation of glutamate neurotransmission involves hormonally responsive glia. Thus, hormonally responsive glia may play a pivotal role in estradiol-mediated synaptic transmission underlying neuroendocrine function.

Expression and amplification of glutamine synthetase gene endows HepG2 cells with ammonia-metabolizing activity for bioartificial liver support system

To establish the ammonia-metabolizing cell lines for a bioartificial liver support system, CHO-K1 and HepG2 were transformed with pBK-CMV-GS vector that contains glutamine synthetase ( gs) gene. The recombinant cell lines were selected under the various concentrations of glutamine synthetase inhibitor, methionine sulfoximine (MSX). The host CHO-K1 and HepG2 cell lines produces ammonia, but the both MSX tolerable CHO (GS-CHO) and HepG2 (GS-HepG2) cell lines endowed with the high GS activity could metabolize the ammonium from medium. The ammonia-metabolizing activity of CHO and HepG2 cell was about one-fourth of that of primary hepatocyte.

Transgenic poplar characterized by ectopic expression of a pine cytosolic glutamine synthetase gene exhibits enhanced tolerance to water stress.

Physiological responses to water stress in hybrid poplar (INRA 7171-B4, Populus tremula L. x P. alba L.) lines transformed to overexpress a pine cytosolic glutamine synthetase (GS1) gene were compared with those of non-transgenic plants. Before, during and after a drought treatment, net photosynthetic rates (Anet) were higher in transgenic than in non-transgenic plants. Stomatal conductance (gs) was higher in transgenic than in non-transgenic plants before, but not after exposure to drought. Before drought treatment, a sudden reduction in photosynthetic photon flux caused a greater burst of CO2 efflux in transgenic than non-transgenic plants, indicating greater photorespiratory activity. Drought caused greater reductions in photochemical quenching, photosystem II (PSII) antennae transfer efficiency (Fv'/Fm') and light-adapted PSII yield (PhiPSII) in non-transgenic than in transgenic plants, especially at low irradiances. Antennae-based thermal dissipation was higher in transgenic plants than in non-transgenic plants both during the imposition of drought and 1 or 3 days after the relief of drought. Under severe water stress and subsequently, transgenic plants maintained a higher expression of glutamine synthetase, glutamate synthase and Rubisco and higher concentrations of chlorophyll and glycine than non-transgenic plants. These findings indicate that overexpression of pine cytosolic GS1 enhanced sustained photosynthetic electron transport capacity during severe stomatal limitation. The data also suggest that ectopic expression of cytosolic GS increases photorespiratory activity, and that this serves as a protective sink for electrons from photosynthetic reaction centers.

Changes in Glutamine Synthetase Activity and Gene Expression During Storage of Moso Bamboo Shoot

Changes in Glutamine Synthetase Activity and Gene Expression During Storage of Moso Bamboo Shoot

Cloning and expression of mycobacterial glutamine synthetase gene in Escherichia coli

Extracellular glutamine synthetase (GS) is one of the prominent proteins secreted by pathogenic mycobacteria such as Mycobacterium tuberculosis and Mycobacterium bovis. Non-pathogenic species like Mycobacterium smegmatis and Mycobacterium phlei do not secrete this protein. GS has been proposed to play an indispensable role in intracellular survival of pathogenic mycobacteria. In this study, the structural gene for extracellular glutamine synthetase of M. tuberculosis was PCR amplified and expressed as fusion protein with hexahistidine residues in Escherichia coli. Expression of GS in E. coli under transcriptional regulation of T5 promoter yielded an insoluble protein aggregating to form inclusion bodies. The inclusion bodies were solubilized in presence of 8 M urea and the enzyme was purified to homogeneity under denaturing conditions using nitrilotriacetic acid (Ni–NTA) affinity chromatography. The denatured protein was renatured by gradual removal of the urea while immobilized on (Ni–NTA) column. The yield of purified recombinant glutamine synthetase was 40 mg/L. The purified recombinant enzyme was obtained in highly active state having specific activity of 200 U/mg protein. This is the first report describing cloning and expression of mycobacterial glutamine synthetase gene in E. coli.

Giant coronary aneurysms

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A novel repressor of nif and glnA expression in the methanogenic archaeon Methanococcus maripaludis.

Nitrogen assimilation in the methanogenic archaeon Methanococcus maripaludis is regulated by transcriptional repression involving a palindromic 'nitrogen operator' repressor binding sequence. Here we report the isolation of the nitrogen repressor, NrpR, from M. maripaludis using DNA affinity purification. Deletion of the nrpR gene resulted in loss of nitrogen operator binding activity in cell extracts and loss of repression of nif (nitrogen-fixation) and glnA (glutamine synthetase) gene expression in vivo. Genetic complementation of the nrpR mutation restored all functions. NrpR contained a putative N-terminal winged helix-turn-helix motif followed by two mutually homologous domains of unknown function. Comparison of the migration of NrpR in gel-filtration chromatography with its subunit molecular weight (60 kDa) suggested that NrpR was a tetramer. Several lines of evidence suggested that the level of NrpR itself is not regulated, and the binding affinity of NrpR to the nitrogen operator is controlled by an unknown mechanism. Homologues of NrpR were found only in certain species in the kingdom Euryarchaeota. Full length homologues were found in Methanocaldococcus jannaschii and Methanothermobacter thermoautotrophicus, and homologues lacking one or more of the three polypeptide domains were found in Archaeoglobus fulgidus, Methanopyrus kandleri, Methanosarcina acetivorans, and Methanosarcina mazei. NrpR represents a new family of regulators unique to the Euryarchaeota.

CAAT/Enhancer Binding Protein Activates an Enhancer in the Glutamine Synthetase Distal 5′-Flanking Sequence

The glutamine synthetase (GS) gene is expressed at high levels in several cell types, including astrocytes, pericentral hepatocytes, and adipocytes. During hormone-mediated adipocyte differentiation of 3T3-L1 cells, GS gene expression increases several hundred fold. We previously reported that elements in the distal 5′-flanking sequence and intron-1 participate in establishing the temporal pattern of GS transcription during adipocyte differentiation. To examine the role of the distal 5′-flanking region in regulating adipocyte-specific GS expression, GS–CAT fusion genes were constructed and analyzed in transiently transfected 3T3-L1 cells. In this way, adipocyte differentiation-responsive enhancer activity was localized to a 422-bp sequence that occurs about 3.5 kb upstream from the transcription start site. This sequence includes several putative C/EBP binding sites and is activated by ectopic expression of C/EBPα in NIH-3T3 cells. Thus, our data indicate that C/EBPα has the capacity to activate functional C/EBP sites in the GS gene distal 5′-flanking region.

Glutamine synthetase gene expression at elevated hydrostatic pressure in a deep-sea piezophilic Shewanella violacea

A glutamine synthetase gene ( glnA) was isolated from a deep-sea piezophilic bacterium, Shewanella violacea strain DSS12. A 7.5-kb SacI fragment containing the complete glnA gene was cloned and sequenced. The glnA gene was found to encode a protein consisting of 469 amino acid residues, showing 75.0% identity to the glutamine synthetase of Escherichia coli. Primer extension analyses revealed two transcription initiation sites in glnA and expression from each site was positively regulated by pressure. Putative promoters recognized by σ 70 and σ 54 were identified in the region upstream of glnA. An electrophoretic mobility shift assay demonstrated that S. violacea σ 54 specifically binds to the promoter region of glnA, suggesting that σ 54 may play an important role in pressure-regulated transcription in this piezophilic bacterium.

Glutamine synthetase gene expression at elevated hydrostatic pressure in a deep-sea piezophilic Shewanella violacea

A glutamine synthetase gene (glnA) was isolated from a deep-sea piezophilic bacterium, Shewanella violacea strain DSS12. A 7.5-kb SacI fragment containing the complete glnA gene was cloned and sequenced. The glnA gene was found to encode a protein consisting of 469 amino acid residues, showing 75.0% identity to the glutamine synthetase of Escherichia coli. Primer extension analyses revealed two transcription initiation sites in glnA and expression from each site was positively regulated by pressure. Putative promoters recognized by sigma(70) and sigma(54) were identified in the region upstream of glnA. An electrophoretic mobility shift assay demonstrated that S. violacea sigma(54) specifically binds to the promoter region of glnA, suggesting that sigma(54) may play an important role in pressure-regulated transcription in this piezophilic bacterium.

Expression of a conifer glutamine synthetase gene in transgenic poplar

The assimilation of ammonium into organic nitrogen catalyzed by the enzyme glutamine synthetase (GS; EC 6.3.1.2) has been suggested to be the limiting step for plant nitrogen utilization (H-M. Lam et al. 1995, Plant Cell 7: 887-898). We have developed a molecular approach to increase glutamine production in transgenic poplar by the overexpression of a conifer GS gene. A chimeric construct consisting of the cauliflower mosaic virus 35S promoter fused to pine cytosolic GS cDNA and nopaline synthetase polyadenylation region was transferred into pBin19 for transformation of a hybrid poplar clone (INRA 7171-B4, Populus tremula x P. alba) via Agrobacterium tumefaciens. Transformed poplar lines were selected by their ability to grow on selective medium containing kanamycin. The presence of the introduced gene in the poplar genome was verified by Southern blotting and polymerase chain reaction analysis. Transgene expression was detected in all selected poplar lines at the mRNA level. The detection of the corresponding polypeptide (41 kDa) and increased GS activity in the transgenics suggest that pine transcripts are correctly processed by the angiosperm translational machinery and that GS1 subunits are assembled in functional holoenzymes. Expression of the pine GS1 gene in poplar was associated with an increase in the levels of total soluble protein and an increase in chlorophyll content in leaves of transformed trees. Furthermore, the mean net growth in height of GS-overexpressing clones was significantly greater than that of non-transformed controls, ranging from a 76% increase in height at 2 months to a 21.3% increase at 6 months. Our results suggest that the efficiency of nitrogen utilization may be engineered in trees by genetic manipulation of glutamine biosynthesis.

Effect of portacaval anastomosis on glutamine synthetase protein and gene expression in brain, liver and skeletal muscle.

The effects of chronic liver insufficiency resulting from end-to-side portacaval anastomosis (PCA) on glutamine synthetase (GS) activities, protein and gene expression were studied in brain, liver and skeletal muscle of male adult rats. Four weeks following PCA, activities of GS in cerebral cortex and cerebellum were reduced by 32% and 37% (p<0.05) respectively whereas GS activities in muscle were increased by 52% (p<0.05). GS activities in liver were decreased by up to 90% (p<0.01), a finding which undoubtedly reflects the loss of GS-rich perivenous hepatocytes following portal-systemic shunting. Immunoblotting techniques revealed no change in GS protein content of brain regions or muscle but a significant loss in liver of PCA rats. GS mRNA determined by semi-quantitative RT-PCR was also significantly decreased in the livers of PCA rats compared to sham-operated controls. These findings demonstrate that PCA results in a loss of GS gene expression in the liver and that brain does not show a compensatory induction of enzyme activity, rendering it particularly sensitive to increases in ammonia in chronic liver failure. The finding of a post-translational increase of GS in muscle following portacaval shunting suggests that, in chronic liver failure, muscle becomes the major organ responsible for the removal of excess blood-borne ammonia.

Expression analysis of a cytosolic glutamine synthetase gene in cotyledons of Scots pine seedlings: developmental, light regulation and spatial distribution of specific transcripts.

The expression of a cytosolic glutamine synthetase (GS1; EC 6.3.1.2) gene was examined in cotyledons of Scots pine seedlings. Light strongly stimulated GS1 mRNA accumulation during development. Similarly, steady-state levels of GS1 transcripts increased in dark-grown seedlings transferred to light and decreased in dark-adapted seedlings. Light/dark adaptation affected rbcS and lhcb2 mRNA levels and chlorophyll contents in the same manner. Light-grown seedlings in the presence of the herbicide norflurazon showed a drastic decrease in mRNA for GS and photosynthetic proteins, whereas the effect of the herbicide on mitochondrial beta-ATP synthase mRNA was limited. These results indicate that factors associated with developing chloroplasts could be required for maximal GS1 gene expression during seedling development. The level of GS polypeptide, determined by immunoblot, was up-regulated during seedling development in the light or dark. However, the levels of the polypeptide detected were unaltered by the light/dark adaptation treatments. The analysis of GS1 mRNA association with polysomes indicated that the discrepancies between GS protein and mRNA levels are not a result of a differential translational rate of the transcript in darkness relative to light. Two GS isoproteins with different isoelectric point were resolved by two-dimensional PAGE in light- and dark-germinated plants. The relative abundance of one of them was markedly affected by light and correlated with the observed changes in GS mRNA, suggesting that the other form is not a product derived from the detected transcript. In situ hybridization of cotyledon sections showed the presence of GS1 mRNAs in mesophyll and phloem cells confirming gene expression in photosynthetic tissues. High levels of transcript were detected also in meristematic cells of apical primordia. These data suggest a dual role for the GS1 gene associated with chloroplast development/activity and glutamine biosynthesis for nitrogen mobilization during early growth of Scots pine.

Glutamine synthetase gene expression in the lungs of endotoxin-treated and adrenalectomized rats.

During sepsis, the lung responds by exporting increased amounts of the amino acid glutamine. This response is accompanied by increased enzymatic activity of glutamine synthetase (GS), which catalyzes the synthesis of glutamine from glutamate and ammonia. It is also known that GS expression in the rat lung can be induced by glucocorticoid hormones. To determine whether the septic response and the response to glucocorticoids are related, we have characterized the induction of GS expression during lipopolysaccharide (LPS)-induced endotoxemia in normal, neutropenic, and adrenalectomized rats. Normal rats exhibited a time- and dose-dependent induction of GS mRNA levels after a single intraperitoneal dose of LPS. Responses to LPS were maximal at doses of 0.1 mg/kg body wt and above. A single 10 mg/kg body wt dose of LPS led to a rapid, transient sevenfold increase in GS mRNA (P < or = 0.1) and a twofold increase in GS protein level 8 h postinjection. Induction of lung GS mRNA 4 h after LPS injection was approximately fivefold in neutropenic (P < or = 0.1) and fourfold in nonneutropenic control rats (P < or = 0.1), suggesting that infiltrating neutrophils or neutrophil-derived factors are not required for GS induction. In response to high-dose, short-term endotoxemia, adrenalectomized rat lung GS mRNA increased twofold (P < or = 0.02) compared with sixfold in sham-operated control rats (P < or = 0.02). However, in response to low-dose, long-term endotoxemia, adrenalectomized rat lung GS mRNA increased threefold (P < or = 0.02) compared with fourfold in sham-operated control rats (P < or = 0.02). Adrenalectomy did not affect the elevation of lung GS mRNA levels in response to dexamethasone. In addition, GS mRNA was induced four- and sixfold in rat microvascular pulmonary endothelial cells exposed to plasma from control and septic rats, respectively. The addition of a glucocorticoid antagonist, RU-38486, completely blocked GS gene induction in the presence of control plasma but only attenuated the response to plasma from septic animals by 30%. These results suggest that GS gene induction during sepsis is only partially mediated by adrenal-derived glucocorticoid hormones.

Role of the 5′ enhancer of the glutamine synthetase gene in its organ-specific expression

In mammals, glutamine synthetase (GS) is expressed in a large number of organs, but the precise regulation of its expression is still obscure. Therefore a detailed analysis of the activity of the upstream regulatory element of the GS gene in the transcriptional regulation of its expression was carried out in transgenic mice carrying the chloramphenicol acetyltransferase (CAT) gene under the control of the upstream regulatory region of the GS gene. CAT and GS mRNA expression were compared in liver, epididymis, lung, adipocytes, testis, kidney, skeletal muscle and gastrointestinal tract, both quantitatively by ribonuclease-protection analysis and topographically by in situ hybridization. It was found that the upstream regulatory region is active with respect both to the level and to the topography of GS gene expression in liver, epididymis, gastrointestinal tract (stomach, small intestine and colon) and skeletal muscle. On the other hand, in the kidney, brain, adipocytes, spleen, lung and testis, GS gene expression is not or only partly regulated by the 5' enhancer. A second enhancer, identified within the first intron, may regulate GS expression in the latter organs. Furthermore, CAT expression in the brain did not co-localize with that of GS, showing that the 5' regulatory region of the GS gene does not direct its expression to the astrocytes.