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Query: UNIPROT:P06889 (Mol)
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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.
Plant Mol Biol 1990 Mar
PMID:Molecular analysis of barley mutants deficient in chloroplast glutamine synthetase. 198 86

A glutamine synthetase (GS) cDNA isolated from an alfalfa cell culture cDNA library was found to represent a cytoplasmic GS. The full-length alfalfa GS1 coding sequence, in both sense and antisense orientation and under the transcriptional control of the cauliflower mosaic virus 35S promoter, was introduced into tobacco. Leaves of tobacco plants transformed with the sense construct contained greatly elevated levels of GS transcript and GS polypeptide which assembled into active enzyme. Leaves of the plants transformed with the antisense GS1 construct showed a significant decrease in the level of both GS1 and GS2 polypeptides and GS activity, but did not show any significant decrease in the level of endogenous GS mRNA. We have proposed that antisense inhibition using a heterologous antisense GS RNA occurs at the level of translation. Our results also suggest that the post-translational assembly of GS subunits into a holoenzyme requires an additional factor(s) and is under regulatory control.
Mol Gen Genet 1993 Jan
PMID:Modulation of glutamine synthetase gene expression in tobacco by the introduction of an alfalfa glutamine synthetase gene in sense and antisense orientation: molecular and biochemical analysis. 809 85

A full-length cDNA clone (pGSP114) encoding glutamine synthetase was isolated from a lambda gt11 library of the gymnosperm Pinus sylvestris. Nucleotide sequence analysis showed that pGSP114 contains an open reading frame encoding a protein of 357 amino acid residues with a calculated molecular mass of 39.5 kDa. The derived amino acid sequence was more homologous to cytosolic (GS1) (78-82%) than to chloroplastic (GS2) (71-75%) glutamine synthetase in angiosperms. The lack of N-terminal presequence and C-terminal extension which define the primary structure of GS2, also supports that the isolated cDNA encodes cytosolic GS. Southern blot analysis of genomic DNA from P. sylvestris and P. pinaster suggests that GS may be encoded by a small gene family in pine. GS mRNA was more abundant in cotyledons and stems than in roots of both Scots and maritime pines. Western blot analysis in P. sylvestris seedlings showed that only one GS polypeptide, similar in size to GS1 in P. pinaster, could be detected in several different tissues. Our results suggest that cytosolic GS is mainly responsible for glutamine biosynthesis in pine seedlings.
Plant Mol Biol 1993 Aug
PMID:Molecular characterization of a cDNA clone encoding glutamine synthetase from a gymnosperm, Pinus sylvestris. 810 6

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.
Plant Mol Biol 1993 Oct
PMID:Differential expression of six glutamine synthetase genes in Zea mays. 810 13

Glutamine synthetase (GS) catalyzes the ATP-dependent condensation of NH3 with glutamate to produce glutamine. In plants GS is an octameric enzyme and is located either in the cytoplasm (GS1) or in the chloroplast (GS2). Two distinct classes of GS1 genes with unique 3'-untranslated region (3'UTR) have been identified in alfalfa. We have demonstrated that the two classes exhibit differential expression pattern in the different plant organs suggesting different functional roles for the different isozymes. To determine the functional significance of the two classes of GS1 genes in alfalfa, we have utilized antisense gene constructs aimed specifically at the 3'UTR of the two GS1 genes and introduced them individually into alfalfa. Our data show that the gene constructs are effective in lowering the corresponding transcript level very effectively though there were organ-specific differences in the level of reduction. No transcript corresponding to the antisense gene construct was detected in any of the alfalfa transformants though they accumulated to significant levels in transgenic tobacco containing the same construct. This suggests that the antisense transcript was not stable in the presence of the homologous target sequence. Transgenic alfalfa with up to 80% reduction in the transcript level corresponding to each gene class, however, showed no reduction in GS activity or GS1 polypeptide level. The results suggest that GS1 mRNA levels are not rate-limiting for GS1 polypeptide synthesis and that GS levels are controlled both at the transcriptional and translational/post-translational level.
Plant Mol Biol 1998 Jun
PMID:Down-regulation of specific members of the glutamine synthetase gene family in alfalfa by antisense RNA technology. 961 20

The potential role of photorespiration in the protection against salt stress was examined with transgenic rice plants. Oryza sativa L. cv. Kinuhikari was transformed with a chloroplastic glutamine synthetase (GS2) gene from rice. Each transgenic rice plant line showed a different accumulation level of GS2. A transgenic plant line, G39-2, which accumulated about 1.5-fold more GS2 than the control plant, had an increased photorespiration capacity. In another line, G241-12, GS2 was almost lost and photorespiration activity could not be detected. Fluorescence quenching analysis revealed that photorespiration could prevent the over-reduction of electron transport systems. When exposed to 150 mM NaCl for 2 weeks, the control rice plants completely lost photosystem II activity, but G39-2 plants retained more than 90% activity after the 2-week treatment, whereas G241-12 plants lost these activities within one week. In the presence of isonicotinic acid hydrazide, an inhibitor of photorespiration, G39-2 showed the same salt tolerance as the control plants. The intracellular contents of NH4+ and Na+ in the stressed plants correlated well with the levels of GS2. Thus, the enhancement of photorespiration conferred resistance to salt in rice plants. Preliminary results suggest chilling tolerance in the transformant.
Plant Mol Biol 2000 May
PMID:Enhanced tolerance to salt stress in transgenic rice that overexpresses chloroplast glutamine synthetase. 1094 77

Substantial concentrations of NH4+ are found in the apoplast of the leaves of Brassica napus. Physiological studies on isolated mesophyll protoplasts with 15NH4+ revealed the presence of a high-affinity ammonium transporter that shared physiological similarity to the high-affinity NH4+ transporters in Arabidopsis thaliana (AtAMT1;3). PCR techniques were used to isolate a full-length clone of a B. napus homologue of AMT1 from shoot mRNA which showed 97% similarity to AtAMT1;3. The full-length cDNA when cloned into the yeast expression vector pFL61 was able to complement a yeast mutant unable to grow on media with NH4+ as the sole nitrogen source. Regulatory studies with detached leaves revealed a stimulation of both NH4+ uptake and expression of mRNA when the leaves were supplied with increasing concentrations of NH4+. Withdrawal of NH4+ supply for up to 96 h had little effect on mRNA expression or NH4+ uptake; however, plants grown continuously at high NH4+ levels exhibited decreased mRNA expression. BnAMT1;2 mRNA expression was highest when NH4+ was supplied directly to the leaf and lowest when either glutamine or glutamate was supplied to the leaves, which directly paralleled chloroplastic glutamine synthetase (GS2) activity in the same leaves. These results provide tentative evidence that BnAMT1;2 may be regulated by similar mechanisms to GS2 in leaves.
Plant Mol Biol 2002 Jul
PMID:Regulation of the high-affinity ammonium transporter (BnAMT1;2) in the leaves of Brassica napus by nitrogen status. 1209 Jun 24

In this study, the in vitro and in vivo transfection capacity of novel pH-sensitive sugar-based gemini surfactants was investigated. In an aqueous environment at physiological pH, these compounds form bilayer vesicles, but they undergo a lamellar-to-micellar phase transition in the endosomal pH range as a consequence of an increased protonation state. In the same way, lipoplexes made with these amphiphiles exhibit a lamellar morphology at physiological pH and a non-lamellar phase at acidic pH. In this study, we confirm that the gemini surfactants are able to form complexes with plasmid DNA at physiological pH and are able to transfect efficiently CHO cells in vitro. Out of the five compounds tested here, two of these amphiphiles, GS1 and GS2, led to 70% of transfected cells with a good cell survival. These two compounds were tested further for in vivo applications. Because of their lamellar organisation, these lipoplexes exhibited a good colloidal stability in salt and in serum at physiological pH compatible with a prolonged stability in vivo. Indeed, when injected intravenously to mice, these stable lipoplexes apparently did not substantially accumulate, as inferred from the observation that transfection of the lungs was not detectable, as examined by in vivo bioluminescence. This potential of avoiding 'preliminary capture' in the lungs may, thus, be further exploited in developing devices for specific targeting of gemini lipoplexes.
J Mol Med (Berl) 2006 Sep
PMID:Transfection mediated by pH-sensitive sugar-based gemini surfactants; potential for in vivo gene therapy applications. 1676 Nov 43

We present the first cloning and study of glutamine synthetase (GS) genes in wheat (Triticum aestivum L.). Based on sequence analysis, phylogenetic studies and mapping data, ten GS sequences were classified into four sub-families: GS2 (a, b and c), GS1 (a, b and c), GSr (1 and 2) and GSe (1 and 2). Phylogenetic analysis showed that the wheat GS sub-families together with the GS genes from other monocotyledonous species form four distinct clades. Immunolocalisation studies in leaves, stems and rachis in plants at flowering showed GS protein to be present in parenchyma, phloem companion and perifascicular sheath cells. In situ localisation confirmed that GS1 transcripts were present in the perifascicular sheath cells whilst those for GSr were confined to the vascular cells. Studies of the expression and protein profiles showed that all GS sub-families were differentially expressed in the leaves, peduncle, glumes and roots. Expression of GS genes in leaves was developmentally regulated, with both GS2 and GS1 assimilating or recycling ammonia in leaves during the period of grain development and filling. During leaf senescence the cytosolic isozymes, GS1 and GSr, were the predominant forms, suggesting major roles in assimilating ammonia during the critical phases of remobilisation of nitrogen to the grain. A preliminary analysis of three different wheat genotypes showed that the ratio of leaf GS2 protein to GS1 protein was variable. Use of this genetic variation should inform future efforts to modulate this enzyme for pre-breeding efforts to improve nitrogen use in wheat.
Plant Mol Biol 2008 May
PMID:Gene expression, cellular localisation and function of glutamine synthetase isozymes in wheat (Triticum aestivum L.). 1828 74

GS2 (PNPLA4; iPLAeta) is the smallest member of the patatin-like family of phospholipases (PNPLA). It was initially identified by its ability to hydrolyze retinylesters (RE) in cell homogenates, and was later found to esterify retinol using a variety of acyl donors. In the present study we set out to determine its cellular function and examined its impact on RE status in 293T cells transfected with GS2, GS2-M1 (a non-translatable mutant of GS2) and empty vector, in fibroblasts isolated from normal and GS2-null donors and in SCC12b and in a somatic cell knock-out of GS2 (SCC12b-GS2(neo/-)), that we generated by homologous recombination. At 50nM medium retinol, GS2 had no significant impact on RE accumulation. However, at 2muM retinol, GS2 promoted a 1.6- to 5-fold increase in RE accumulation. To verify role of GS2 as a catalyst, RE levels were measured in 293T transfected wild type GS2, catalytic dyad mutants devoid of enzymatic activity, or alanine substitution mutants spanning the entire GS2 sequence. Surprisingly, every GS2 mutant promoted RE accumulation. This activity was also observed in the GS2 paralogues and rat orthologue. The data demonstrate that within the context of the cell GS2 promotes RE accumulation and may do so either as a catalyst or as a regulatory protein that enhances RE formation catalyzed by other acyl transferases.
Mol Genet Metab 2009 Apr
PMID:GS2 as a retinol transacylase and as a catalytic dyad independent regulator of retinylester accretion. 1918 55


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