Glutamine Synthetase cDNA Research Articles

Molecular Cloning of Glutamine Synthetase cDNA from Lactuca sativa: Sequence Analysis and Gene Expression during Storage

Molecular Cloning of Glutamine Synthetase cDNA from Lactuca sativa: Sequence Analysis and Gene Expression during Storage

Research note: Characterization of a cDNA encoding glutamine synthetase II from Gelidium crinale (Rhodophyta)

SUMMARY A cDNA encoding glutamine synthetase (GS) was characterized from the red alga, Gelidium crinale (Turner) Gaillon, using reverse-transcriptase polymerase chain reaction and the 5′- and 3′-rapid amplification of cDNA ends. Sequence analysis of a 1231-bp GS cDNA transcript included both 5′ and 3′ untranslated regions and a 1056-bp open reading frame encoding a 352 amino acid polypeptide. Comparison with GS sequences from other organisms revealed that the G. crinale cDNA encodes a type-II GS, and the absence of a N-terminal plastid signal sequence suggests that it is a cytosolic isoenzyme. Phylogenetic analyses of GSII amino acid sequences supports the multiple origin of cytosolic and plastid isoenzymes during eukaryotic evolution.

Immunoassay for Serum Glutamine Synthetase in Serum: Development, Reference Values, and Preliminary Study in Dementias

Glutamine synthetase (GS) is a ubiquitous enzyme present at high concentrations in liver, muscle, kidney, and brain (1). GS in the brain is astrocyte specific, is involved in the detoxification of ammonia and glutamate, and is overexpressed after brain injury (2). Monomeric GS protein was found in 38 of 39 cerebrospinal fluid samples obtained from Alzheimer disease (AD) patients (3), and the concentration of GS in the lumbar cerebral spinal fluid of AD patients was increased significantly but nonspecifically (4). GS has also been reported in serum (4). Markers for AD have poor sensitivity and specificity for overt or preclinical AD (5)(6). We have developed an ELISA that is rapid, highly sensitive, and GS specific. This ELISA, together with the S100B and neuron-specific enolase (NSE) ELISAs, was used to retrospectively assess serum GS in healthy controls, AD patients, and other dementia patients. Recombinant human GS (rhGS) cDNA was purchased from ATCC (ATCC No. 409071). The full-length rhGS open reading frame was obtained by PCR and subcloning in pET28a ( Nde l/ Xho I). The construct included a polyhistidine tag at the N-terminal domain of the rhGS open reading frame and no extra sequence at the C-terminal domains. The protein was expressed in E. coli BL21 (DE3) (7). We prepared extracts and solubilized products as described (8). Affinity purification was performed by nickel-nitrilotriacetic acid chromatography. Female Balb/c mice (7–8 weeks of age) were immunized by subcutaneous injection of 50 μg of rhGS emulsified in complete Freund’s adjuvant. An intraperitoneal booster injection was given 3 weeks later (50 μg in incomplete Freund’s adjuvant) and then at 2- to 3-week intervals (three times or more) with a final injection of 50 μg of antigen in buffer via intraperitoneal route 3 days before cell fusion. Hybridoma cultures were screened by ELISA, and …

Characterization and sequencing of glutamine synthetase cDNA from liver of the ureotelic gulf toadfish ( Opsanus beta)

The hepatic enzyme, glutamine synthetase (GSase) is a pivotal protein in the regulation of urea synthesis in fish. The sequence of the DNA encoding for GSase from liver of the ureotelic gulf toadfish ( Opsanus beta) was analyzed through a suite of molecular techniques (including cDNA cloning, RACE PCR, and genomic PCR). An open reading frame (ORF) was identified in the cDNA sequence which codes for a protein of 394 amino acids with high identity (86%) to dogfish shark GSase. In the course of generating a suitable probe, a partial sequence was also obtained for horned shark GSase which also had high identity with the dogfish shark gene (93%). Like the dogfish shark GSase, the toadfish gene has two methionine translation initiation sites; the downstream site apparently codes for a cytoplasmic isozyme, while the upstream site adds an N-terminal peptide leader sequence of 23 amino acids to the ‘cytoplasmic’ protein. This leader sequence has characteristics consistent with a mitochondrial targeting peptide, including a cleavage recognition motif (Arg-X-Phe) and the apparent ability to form an amphiphathic helix. Northern analysis revealed that there is a single predominant transcript of ∼2 kb in size. These results are consistent with the interpretation that in the gulf toadfish GSase cytoplasmic and mitochondrial isozymes are coded for by a single gene and mRNA transcript which is differentially translated at either initiation site. These results are discussed in the context of prior results for enzyme kinetic characteristics and urea synthesis/excretion physiology.

Biochemical analysis of a blood meal-induced Aedes aegypti glutamine synthetase gene

Glutamine synthetase (GS) in the mosquito, Aedes aegypti, is induced in the midgut following a blood meal. Mosquito GS message is detected as soon as 1 h post-blood feeding and remains stable for 18 h. Using a PCR product encoding mosquito GS, a λgt10 adult female mosquito cDNA library was screened. A cDNA clone, pCl5A2, encoding the full translation product of mosquito GS was isolated and sequence analyses performed. Mosquito GS cDNA is 2.5 kb in length and its putative translation product shares all the conserved regions characteristic of the GS gene family, including the presumed ATP biding site. Glutamine synthetase activity in the mosquito midgut is highest at 18 h post-blood feeding. Activity can be detected over a broad pH range, from 6.0 to 7.5. Unlike other cellular GS enzymes, mosquito GS is not active in the presence of ATP. Very low dosages (0.05 mM) of L-methionine S-sulfoximine are sufficient to partially inhibit mosquito GS activity. Inhibition of GS disrupts the normal formation of the midgut peritrophic matrix, suggesting that GS enzyme might be involved in the initial pathway of chitin synthesis. The unique expression pattern and inducible nature of the mosquito GS gene make it an interesting candidate for studying promoter function. Additionally, the blood meal activation of the GS gene makes this a potentially valuable tool in mosquito transformation studies.

Quick Identification of the Members of the Glutamine Synthetase Gene Family from Sunflower by Simultaneous Amplification of cDNA with Degenerate Primers

A single degenerate glutamine synthetase (GS)-specific primer was used to amplify the 3′ end of cDNAs derived from different GS genes that are expressed in leaves and roots of sunflower (Helianthus annuus L. cv. Peredovic). Four types of GS cDNA (I, II, III and IV) were simultaneously amplified from leaves and five types (I, II, V, VI, VII) from roots with a minimum investment of time and experimental work. cDNAs II, III and IV encode chloroplastic isoforms as deduced by the presence of chloroplastic GS-specific features in their sequences. The rest of cDNAs codifies cytosolic isoforms. Using cDNA-specific probes and primers, homologous sequences to all GS cDNAs amplified from cv. Peredovic, except to cDNAs III and IV, were detected in the inbred line R41. This result strongly suggests that the three cDNAs for chloroplastic isoform are allelic sequences from the same locus, and since cDNA type IV contains sequences derived from cDNAs II and III, it indicates a recombinational origin. The results presented are consistent with the existence of a GS gene family in sunflower with at least five members. Four of them, named ggs1.1 to ggs1.4, codify for the cytosolic isoforms (cDNAs I, V, VI and VII). A fifth member, named ggs2, from which three allelic sequences (cDNAs II, III and IV) have been cloned, encodes the chloroplastic isoform.

Cross‐species amplification of glutamine synthetase cDNA by polymerase chain reaction with degenerate primers

We have developed and optimized a consistent polymerase chain reaction (PCR)‐based strategy to quickly obtain specific sequence information on novel plant glutamine synthetase (GS, EC 6.3.1.2) cDNAs. Two sets of degenerate primer pairs were designed to discriminate regions conserved in either any kind of GS messenger or exclusively in those for the chloroplastic GS. Novel GS cDNA sequences were successfully amplified from total RNA obtained from 14 different monocotyledonous and dicotyledonous plants. The procedure, coupled with a further restriction analysis, allowed us to uncover the presence of GS cDNA polymorphism, which most likely stems from the different GS gene family members within a single species. Contrary to previously reported strategies in other systems, GS cDNA oligonucleotide primers were designed keeping the degeneracy level to a minimum, together with a high melting temperature. This approach proved to be particularly effective, generating high yields of the expected products without requiring extra nested amplification steps or time‐consuming optimization steps for each species GS cDNA amplification. Different clones containing sequence information from either the coding or the 3′‐untranslated regions were further sequenced and characterized, confirming the high sequence identity and size uniformity the of GS cDNAs across higher plant species. Therefore, this approach is proposed as a stand‐alone procedure to quickly determine the sequence of unknown GS cDNAs, as well as to speed up and complement classical molecular cloning methodologies.

Cross-species amplification of glutamine synthetase cDNA by polymerase chain reaction with degenerate primers

Cross-species amplification of glutamine synthetase cDNA by polymerase chain reaction with degenerate primers

Chromosomal Mapping of a Family of Human Glutamine Synthetase Genes: Functional Gene (GLUL) on 1q25, Pseudogene (GLULP) on 9p13, and Three Related Genes (GLULL1, GLULL2, GLULL3) on 5q33, 11p15, and 11q24

Glutamine synthetase (GS) is a ubiquitous enzyme that catalyzes the ATP-dependent conversion of glutamate to glutamine using ammonia as the nitrogen source. Using human GS cDNA as a probe, a bacterial artificial chromosome (BAC) library consisting of twofold coverage of the human genome was screened, and 18 clones were obtained. The restriction analysis of the human insert DNAs provided a basis to divide these 18 BAC clones into five groups, suggesting the existence of a GS gene family in the human genome. PCR analysis using three sets of primers designed from the reported sequences for GS cDNA and a processed pseudogene identified the corresponding BAC clones. Fluorescencein situhybridization analysis revealed the chromosomal localization of these five genes: the GS gene to 1q25 (GLUL), the processed pseudogene to 9p13 (GLULP), and three related genes to 5q33 (GLULL1), 11p15 (GLULL2), and 11q24 (GLULL3), respectively.

Genetic engineering of hybridoma glutamine metabolism

The murine hybridoma PQXB1/2 cannot be adapted to grow in culture media containing <0.5 m m glutamine. Transformants selected following electroporation of PQXB1/2 cells with vectors containing a Chinese hamster glutamine synthetase (GS) cDNA under the control of the SV40 early promoter also failed to grow in the absence of glutamine in the culture medium. PQXB1/2 cells have, however, been transformed to glutamine independence following electroporation with a vector containing this glutamine synthetase cDNA under the control of the human cytomegalovirus immediate early promoter. In these cells, sufficient active glutamine synthetase was expressed from one vector per cell to enable growth in glutamine-free media. The specific activity of glutamine synthetase in two transformed cell lines producing parental levels of antibody was increased by 128 and 152%, respectively (0.57 and 0.63 μmol min −1 per 10 6 cells in transformants compared with parental levels of 0.25 μmol min −1 per 10 6 cells). This reprogramming of glutamine synthetase expression and glutamine metabolism is important for developing strategies to deal with ammonia toxicity and the production of cell lines with improved metabolic processes.

Detection of a functional promoter/enhancer in an intron-less human gene encoding a glutamine synthetase-like enzyme

A human genomic clone, ΨGS, containing an intron-less glutamine synthetase (GS)-encoding pseudogene, was isolated by screening a human library. A sequence of 3004 bp, containing the GS coding region and both the 5' and 3' flanking sequences, was identified that exhibits all the characteristics of a processed pseudogene. The coding region shows 93% identity with the human GS cDNA (h GS) sequence and contains two frame-shifts and two termination codons. The coding sequence is flanked by a 9-bp AT repeat and a putative polyadenylation site, AATAAA, at the 3' end. Primer extension analysis and S1 nuclease mapping showed a transcription start point ( tsp) 62 bp upstream from the start codon indicating a shorter untranslated region than h GS. Transfection of HeLa cells with cat constructs containing portions of the 5' flanking sequence showed the presence of a functional promoter/enhancer within 200 bp of the tsp, independent of its orientation.

Hairy roots of Brassica napus: II. Glutamine synthetase overexpression alters ammonia assimilation and the response to phosphinothricin

Hairy roots of Brassica napus (rape cv. Giant) have been produced that contain the cytosolic glutamine synthetase (GS) gene from Glycine max (soybean). Leaf explants were cocultivated with Agrobacterium rhizogenes strain A4T harbouring the binary vector pLN16. This vector was constructed by inserting a soybean cytosolic GS cDNA into the multiple cloning site of pGA643, placing it under the control of the CaMV promoter. In addition, the T-DNA region of pLN16 contained a NPTII gene for selection of transformed cells. Transgenic hairy roots grew prolifically on hormone-free media containing a selective level of kanamycin. Southern and northern analyses confirmed the presence of soybean GS DNA and transcripts, respectively. These transformed hairy roots also have a greater abundance of the GS polypeptide, approximately 3-6 fold greater GS activity and lower levels of endogenous ammonia. Hairy roots provide a useful system for studying responses to phosphinothricin (PPT). Hairy roots grown in media containing PPT had lower GS activity, greater ammonia accumulation and slower growth than controls. The presence of the soybean GS gene in the hairy roots reduced these PPT-induced effects and resulted in higher GS activity, lower ammonia levels and faster growth than in PPT-treated controls. Greater tolerance of PPT was also seen in shoots regenerated from the hairy roots displaying elevated levels of GS activity.

Effects of modafinil-induced wakefulness on glutamine synthetase regulation in the rat brain

Changes in the level of glutamine synthetase (GS), an enzyme chiefly found in glial cells, were investigated in the brains of rats treated with modafinil, an awakening drug interfering with central catecholamine function. Two hours (waking period) and 7 h (recovery period) after intra-peritoneal injection of 128 mg/kg modafinil, a significant increase in the level of GS protein was observed by immunotitration in both the locus coeruleus (+30%) and in the frontoparietal cortex (+50%). No changes were observed with 64 mg/kg of modafinil. Gs mRNA was quantified in the entire cortex by Northern blot hybridization using an oligonucleotidic GS cDNA probe. A significant increase in the GS-mRNA level (+70%) was observed in the CX of rats 2 h after injection of 128 mg/kg modafinil; the level tended to return to control values 7 h later during the recovery period. The level of glial acid fibrillary protein (GFAP), an astroglial marker, was unchanged after modafinil treatment. These changes in GS levels after modafinil treatment are discussed in terms of neuron-glia interactions in the regulation of brain metabolism during pharmacologically induced wakefulness, excluding possible stress effects.

Dexamethasone increases jejunal glutamine synthetase expression via translational regulation

Glutamine provides energy and precursors for nucleotide biosynthesis for the gut mucosa, and it is essential for intestinal metabolism and function. During stress states, glutamine uptake of circulating and luminal glutamine may be diminished, but the ability of the gut mucosa to synthesize glutamine de novo in response to this decreased delivery remains undefined. Since the glucocorticoids play an important role in regulating interorgan glutamine metabolism during catabolic states, we hypothesized that these hormones induce the expression of gut mucosal glutamine synthetase (GS), the enzyme that catalyzes the intracellular biosynthesis of glutamine. Adult rats were treated with dexamethasone (DEX, 0.5 mg/kg intraperitoneally) or saline (controls). At various times after treatment (4, 12, 24, 48, and 72 hours), jejunal mucosal GS-specific activity was assayed, and total RNA was extracted. GS transcripts were detected by Northern blot analysis, using a radiolabeled rat GS cDNA probe. Transcripts were quantitated by phospho-imaging and normalized to β-actin. An anti-GS polyclonal antibody was used to quantitate GS protein concentrations by Western blot analysis. The relative quantities of GS translated were measured using a cell-free protein-synthesizing system (reticulocyte lysate assay). Data were analyzed using analysis of variance and were considered statistically significant for p < 0.05. DEX increased GS activity by 45% 12 hours after administration. Western blot analysis revealed an increase in the concentration of the GS protein in the jejunum of DEX-treated animals. Northern blot analysis demonstrated no significant change in GS mRNA levels after DEX treatment, indicating the possibility of posttranscriptional regulation. In vitro translational experiments demonstrated that the quantity of GS translated was increased by 25% after the administration of DEX. These data suggest that glucocorticoids may increase jejunal mucosal GS levels by accelerating protein translation. This adaptive response could provide ghitamine for the gut mucosa during stress, when exogenous glutamine supplies may be rate limiting.

Cloning and characterization of a nodule-enhanced glutamine synthetase-encoding gene from Lupinus luteus

Glutamine synthetase (GS)-encoding genes in Lupinus luteus constitute a small family of genes showing different expression patterns [Boron et al., Acta Biochim. Polon. 36 (1989) 295–301]. One member of this family, the LINGS1 gene, is strongly induced in root nodules close to the onset of nitrogen fixation and is referred to as a nodule-enhanced GS gene. We present here the structure of the nodule-enhanced LINGS1 gene, the first gene of this class which has been sequenced. LINGS1 is composed of twelve exons and shows structural similarity to the GS gene from Medicago sativa, indicating structure conservation of GS genes in legumes. Comparison of protein coding regions, as well as 5'-untranslated regions derived from LINGS1 and a Lupinus angustifolius pGS5 GS cDNA clone [Grant et al., Plant Mol. Biol. 13 (1989) 481–490], revealed a high degree of shared identity between both genes, indicating that they are orthologous. The sequence of the LINGS1 5'-flanking region (2.3 kb) contains several elements implicated in regulation of nodulin genes, as well as other characteristic DNA motifs. RNA blot hybridization analysis carried out using a probe corresponding to the LINGS1 3'-untranslated region revealed that this gene is also transcribed in leaves, but at a barely detectable level.

Differential expression of six glutamine synthetase genes in Zea mays

The maize genome has been shown to contain six glutamine synthetase (GS) genes with at least four different expression patterns. Noncoding 3' gene-specific probes were constructed from all six GS cDNA clones and used to examine transcript levels in selected organs by RNA gel blot hybridization experiments. The transcript of the single putative chloroplastic GS2 gene was found to accumulate primarily in green tissues, whereas the transcripts of the five putative GS1 genes were shown to accumulate preferentially in roots. The specific patterns of transcript accumulation were quite distinct for the five GS1 genes, with the exception of two closely related genes.

Dexamethasone regulates glutamine synthetase expression in rat lung

Since the lungs play a central role in maintaining glutamine homeostasis in normal and catabolic disease states, we studied the regulation of glutamine synthetase (GS) expression by dexamethasone in rat lung. Adult rats received saline (controls) or dexamethasone (0.5 mg/kg). Lung total RNA was extracted for Northern hybridization and labeled with an α- 32P rat GS cDNA probe. The mRNA of the constitutively expressed gene β-actin was the control for RNA loading. GS transcripts were measured by laser densitometry and normalized to actin, and GS specific activity was also determined. Following a single injection of dexamethasone (0.5 mg/kg), lung GS activity increased by 40% at 4 hours and by 75% at 8 hours. The dexamethasone-mediated increase in GS activity was associated with a marked increase in GS mRNA levels, which preceded the increase in enzyme activity by approximately 2 hours. Serial daily dexamethasone administration for 3 and 6 days caused an even greater increase in GS mRNA levels and specific activity. No effect was seen on β-actin levels, demonstrating that the expression of GS was not part of a global response to steroids. Therefore, glucocorticoids stimulate GS expression in rat lung. This regulation appears to be one mechanism by which lung glutamine release is augmented during critical illness.

The glutamine synthetase gene family of Arabidopsis thaliana light-regulation and differential expression in leaves, roots and seeds

Glutamine synthetase (GS) plays an important role in the assimilation of nitrogen by higher plants. We present here a molecular analysis of the GS polypeptides, mRNAs, and genes of Arabidopsis thaliana. Western blot analysis of leaf and root protein extracts revealed at least two distinct GS polypeptides; 43 kDa and 39 kDa GS polypeptides were present in leaves, while only a 39 kDa GS was detected in roots. The 43 kDa GS polypeptide is light-inducible. In etiolated seedlings only the 39 kDa GS was detected. However, upon greening the 43 kDa GS increased to levels comparable to those observed in light-grown plants. Four distinct GS cDNA clones, lambda Atgsl1, lambda Atgsr1, lambda Atgsr2 and lambda Atkb6 were isolated and characterized. Their complete nucleotide and deduced amino acid sequences are presented. The coding sequences of the four clones are 70-88% similar while their 5' and 3' untranslated regions exhibit less than 50% similarity. Northern blots of leaf, root and germinated seed RNA revealed that the four cDNAs hybridize to mRNAs which are differentially expressed in the organs of Arabidopsis thaliana. lambda Atgsl1 is leaf-specific and hybridizes to a 1.6 kb mRNA. Both lambda Atgsr1 and lambda Atgskb6 hybridize to 1.4 kb mRNAs which are expressed in both roots and germinated seeds. lambda Atgsr2 hybridizes to a 1.4 kb mRNA, which is primarily expressed in roots with low levels of expression in seeds and leaves. lambda Atgsl1, which represents the leaf-specific mRNA, is induced by light. lambda Atgsl1 mRNA levels increase during the greening of etiolated seedlings while lambda Atgsr1 levels remain constant. Southern blot analysis indicated that the Arabidopsis genome contains at least four and possibly five distinct GS genes.

Expression of three plant glutamine synthetase cDNA in Escherichia coli

Three cDNA clones encoding the closely related glutamine synthetase (GS) alpha, beta and gamma polypeptides of Phaseolus vulgaris (French bean) were recombinantly expressed in Escherichia coli. The GS expression plasmids correctly synthesised the recombinant alpha, beta and gamma polypeptides which then assembled into catalytically active homo-octameric isoenzymes. These isoenzymes behaved similarly to their native homologues on ion-exchange and gel-filtration chromatography. Furthermore, the alpha and gamma isoenzymes complemented a GS(glnA)-deficient mutant, thus demonstrating their physiological activity in E. coli. Differences were observed between the three recombinant GS plasmids in their quantitative expression of the GS polypeptides and their ability to complement the E. coli mutant. These differences were correlated to the degree of solubility of the polypeptide, which was observed to be dependent on the temperature of expression. The production of active GS isoenzymes in E. coli facilitates the isolation and characterisation of the individual P. vulgaris homo-octameric GS isoenzymes.

Molecular analysis of barley mutants deficient in chloroplast glutamine synthetase

A barley leaf cDNA library has been screened with two oligonucleotide probes designed to hybridize to conserved sequences in glutamine synthetase (GS) genes from higher plants. Two GS cDNA clones were identified as hybridizing strongly to one or both probes. The larger clone (pcHvGS6) contained a 1.6 kb insert which was shown by primer extension analysis to be an almost full-length cDNA. Both clones were more closely related to cDNAs for the chloroplast form of GS (GS2) from pea and Phaseolus vulgaris than to cDNAs for the cytosolic form (GS1). A sequence identical to an N-terminal sequence determined from a purified preparation of the mature GS2 polypeptide (NH2-XLGPETTGVIQRMQQ) was found in the pcHvGS6-encoded polypeptide at residues 46-61, indicating a pre-sequence of at least 45 amino acids. The pre-sequence has only limited sequence homology to the pre-sequences of pea and P. vulgaris GS2 subunits, but is similarly rich in basic residues and possesses some of the structural features common to the targeting sequences of other chloroplast proteins. The molecular lesions responsible for the GS2-deficient phenotypes of eight photorespiratory mutants of barley were investigated using a gene-specific probe from pcHvGS6 to assay for GS2 mRNA, and an anti-GS antiserum to assay for GS2 protein. Three classes of mutants were identified: class I, in which absence of cross-reacting material was correlated with low or undetectable levels of GS2 mRNA; class II, which had normal or increased levels of GS2 mRNA but very little GS2 protein; and class III, which had significant amounts of GS2 protein but little or no GS2 activity.