Asparagine Synthetase Gene Expression Research Articles

Asparagine Synthesis During Tobacco Leaf Curing.

Senescence is a genetically controlled mechanism that modifies leaf chemistry. This involves significant changes in the accumulation of carbon- and nitrogen-containing compounds, including asparagine through the activity of asparagine synthetases. These enzymes are required for nitrogen re-assimilation and remobilization in plants; however, their mechanisms are not fully understood. Here, we report how leaf curing—a senescence-induced process that allows tobacco leaves to dry out—modifies the asparagine metabolism. We show that leaf curing strongly alters the concentration of the four main amino acids, asparagine, glutamine, aspartate, and glutamate. We demonstrate that detached tobacco leaf or stalk curing has a different impact on the expression of asparagine synthetase genes and accumulation of asparagine. Additionally, we characterize the main asparagine synthetases involved in the production of asparagine during curing. The expression of ASN1 and ASN5 genes is upregulated during curing. The ASN1-RNAi and ASN5-RNAi tobacco plant lines display significant alterations in the accumulation of asparagine, glutamine, and aspartate relative to wild-type plants. These results support the idea that ASN1 and ASN5 are key regulators of asparagine metabolism during leaf curing.

Contrasting gene expression patterns in grain of high and low asparagine wheat genotypes in response to sulphur supply

BackgroundFree asparagine is the precursor for acrylamide formation during cooking and processing of grains, tubers, beans and other crop products. In wheat grain, free asparagine, free glutamine and total free amino acids accumulate to high levels in response to sulphur deficiency. In this study, RNA-seq data were acquired for the embryo and endosperm of two genotypes of bread wheat, Spark and SR3, growing under conditions of sulphur sufficiency and deficiency, and sampled at 14 and 21 days post anthesis (dpa). The aim was to provide new knowledge and understanding of the genetic control of asparagine accumulation and breakdown in wheat grain.ResultsThere were clear differences in gene expression patterns between the genotypes. Sulphur responses were greater at 21 dpa than 14 dpa, and more evident in SR3 than Spark. TaASN2 was the most highly expressed asparagine synthetase gene in the grain, with expression in the embryo much higher than in the endosperm, and higher in Spark than SR3 during early development. There was a trend for genes encoding enzymes of nitrogen assimilation to be more highly expressed in Spark than SR3 when sulphur was supplied. TaASN2 expression in the embryo of SR3 increased in response to sulphur deficiency at 21 dpa, although this was not observed in Spark. This increase in TaASN2 expression was accompanied by an increase in glutamine synthetase gene expression and a decrease in asparaginase gene expression. Asparagine synthetase and asparaginase gene expression in the endosperm responded in the opposite way. Genes encoding regulatory protein kinases, SnRK1 and GCN2, both implicated in regulating asparagine synthetase gene expression, also responded to sulphur deficiency. Genes encoding bZIP transcription factors, including Opaque2/bZIP9, SPA/bZIP25 and BLZ1/OHP1/bZIP63, all of which contain SnRK1 target sites, were also expressed. Homeologues of many genes showed differential expression patterns and responses, including TaASN2.ConclusionsData on the genetic control of free asparagine accumulation in wheat grain and its response to sulphur supply showed grain asparagine levels to be determined in the embryo, and identified genes encoding signalling and metabolic proteins involved in asparagine metabolism that respond to sulphur availability.

Loss of asparagine synthetase suppresses the growth of human lung cancer cells by arresting cell cycle at G0/G1 phase.

The aim of this research is to determine the role of human asparagine synthetase (ASNS) in human lung cancer. In the present study, immunohistochemical staining and the Oncomine database mining showed that the expression of ASNS gene was higher in lung cancer tissues than that in the normal tissues by. In addition, western blot assay showed that ASNS was elevated in lung cancer A549 and 95D cell lines as compared with that in H1299 and H460 cells. Therefore, A549 and 95D cells were chosen for subsequent MTT and colony formation assay. It was found that knockdown of ASNS inhibited the growth and colony formation abilities of A549 and 95D cells. Flow cytometry showed that ASNS silencing arrested cell cycle progression at G0/G1 phase in A549 cells, probably through regulating the expression of cell cycle molecules such as CDK2 and Cyclin E1 as shown by quantitative real-time PCR. Taken together, our study indicates that ASNS may be an important target for lung cancer diagnosis and treatment.

Food safety: Structure and expression of the asparagine synthetase gene family of wheat

Asparagine is an important nitrogen storage and transport molecule, but its accumulation as a free amino acid in crops has implications for food safety because free asparagine is a precursor for acrylamide formation during cooking and processing. Asparagine synthesis occurs by the amidation of aspartate, catalysed by asparagine synthetase, and this study concerned the expression of asparagine synthetase (TaASN) genes in wheat. The expression of three genes, TaASN1-3, was studied in different tissues and in response to nitrogen and sulphur supply. The expression of TaASN2 in the embryo and endosperm during mid to late grain development was the highest of any of the genes in any tissue. Both TaASN1 and TaASN2 increased in expression through grain development, and in the grain of field-grown plants during mid-development in response to sulphur deprivation. However, only TaASN1 was affected by nitrogen or sulphur supply in pot-based experiments, showing complex tissue-specific and developmentally-changing responses. A putative N-motif or GCN4-like regulatory motif was found in the promoter of TaASN1 genes from several cereal species. As the study was completed, a fourth gene, TaASN4, was identified from recently available genome data. Phylogenetic analysis showed that other cereal species have similar asparagine synthetase gene families to wheat.

Analysis of gene profiles involved in the enhancement of all-trans retinoic acid-induced HL-60 cell differentiation by sesquiterpene lactones identifies asparagine synthetase as a novel target for differentiation-inducing therapy

All-trans retinoic acid (ATRA) is one of the most useful drugs in the treatment for acute promyelocytic leukemia (APL), but its adverse effects, which include drug resistance and hypercalcemia are obstacles to achieving complete remission. Our previous study showed that some sesquiterpene lactones (STLs), i.e., helenalin (HE) and parthenolide (PA) but not sclareolide (SC), enhance ATRA-induced differentiation of HL-60 APL cells with no unexpected effects, but the precise mechanism on underlying this synergism is not yet fully understood. In this study, we investigated the distinctive transcriptional profile of cells treated with effective STL compounds, which were identified by comparing the profile with that of cells treated with SC. Genome-wide approaches using cDNA microarrays showed that co-treatment with the differentiation-enhancing STLs HE and PA maximized the transcriptional variation regulated by the suboptimal concentration of ATRA in HL-60 cells. Of the genes of interest, asparagine synthetase was remarkably downregulated by ATRA co-treated with either HE or PA, but not with SC. In an additional analysis for the role of asparagine synthetase, ATRA-mediated HL-60 cell differentiation was enhanced when asparagine in the culture media was depleted by an addition of L-asparaginase, indicating that downregulation of asparagine synthetase gene expression may be involved in the enhanced cell differentiation by STL compounds. These results provide useful insight into differentiation-inducing therapy in the treatment of leukemia.

Low expression of asparagine synthetase in lymphoid blasts precludes its role in sensitivity to L-asparaginase

Childhood acute lymphoblastic leukemia (ALL) is treated with combined chemotherapy, including L-asparaginase (L-asp). Recent studies question the traditional view that the level of asparagine synthetase (ASNS), an enzyme producing the intracellular asparagine, correlates with the response to L-asp treatment. However, the importance of ASNS in response to L-asp has neither been confirmed nor refuted so far. In this study, we wanted to elucidate the effect of ASNS expression level on the sensitivity of ALL cells to L-asp treatment. We used four ALL cell lines (NALM-6, RS4;11, REH, and UOCB6) and 30 diagnostic bone marrow samples of ALL patients to study the relationship between ASNS expression and sensitivity to L-asp using MTS proliferation assay. RNA interference was used to study the effect of a range of ASNS levels on the response to L-asp treatment. Using a cell line model with a gradually knocked-down ASNS gene, we defined a cutoff level below which ASNS gene expression does not correlate with sensitivity to L-asp. Importantly, ASNS gene expression in patients' ALL blasts is below this level. We confirmed that there was no correlation between ASNS gene expression and sensitivity to L-asp in ALL blasts. In addition, we show that cells with low ASNS expression level do not respond to asparagine deprivation by upregulation of ASNS gene expression. In conclusion, the ASNS expression level does not predict sensitivity to L-asp in leukemic blasts. Moreover, cell lines with high basal expression of ASNS cannot serve as a valid model for studies on the relationship between the ASNS and L-asp cytotoxic effect.

Expression of asparagine synthetase genes in sunflower ( Helianthus annuus) under various environmental stresses

In sunflower, asparagine synthetase (AS; EC 6.3.5.4) is encoded by a small family of three genes ( HAS1, HAS1.1 and HAS2) that are differentially regulated by light, carbon and nitrogen availability. In this study, the response of each gene to various stress conditions was examined by Northern analysis with gene-specific probes in leaves and roots. The expression of HAS1 and HAS1.1 genes was induced by osmotic stress (300 mM mannitol), salt stress (150 mM NaCl), and heavy-metal stress (20 μM CuSO 4), more in roots than in leaves. The expression of HAS2 was not significantly altered by stress treatments. The positive response of HAS1 and HAS1.1 genes to osmotic and salt stresses occurred in the light, in contrast to that previously found in unstressed plants. Measurements of sucrose and total free amino acid contents in leaves and roots indicate that the expression of root HAS1 and HAS1.1 genes in stressed plants is not under metabolic control by the intracellular C/N ratio, suggesting the involvement of some specific stress factor(s). Growth of plants at 40 °C for 12 h negatively affected the expression of HAS1 and HAS1.1 but not that of HAS2.

Role of Glutamine Depletion in Directing Tissue-specific Nutrient Stress Responses to L-Asparaginase

Role of Glutamine Depletion in Directing Tissue-specific Nutrient Stress Responses to L-Asparaginase

Contrasted responses to carbohydrate limitation in tomato fruit at two stages of development

AbstractThe metabolic consequences of long‐term carbohydrate depletion have been well documented in many sink organs but not extensively in fruit. Therefore, in the present study the response to sugar limitation in tomato fruit (Lycopersicon esculentum Mill.) was investigated at two developmental stages; during the cell division and cell expansion phases. First, the response in excised fruit cultured in vitro was characterized. Sugar depletion caused an arrest of growth and an exhaustion of carbon reserves. The proteins that were degraded and the nitrogen released was transiently stored as asparagine and glutamine in both developmental stages and also as γ ‐aminobutyric acid (GABA) in expanding fruit. Fruit at the cell division stage appeared to be more sensitive to sugar limitation. The response to sugar depletion was then characterized in fruit from plants submitted to extended darkness. In planta, the effects of sugar‐limitation were similar to those described in vitro but much more attenuated, especially in expanding fruit, which still accumulated dry matter. The expression of cell cycle genes, sugar‐ and nitrogen‐related genes was reduced by darkness. Only asparagine synthetase gene expression was induced in both dark‐treated fruit. Together the present data revealed that the effects of the carbon limitation are more pronounced in the youngest fruits as it is probably controlled by the relative sink strength of the fruit.

Molecular cloning and characterization of two cDNAs encoding asparagine synthetase from Astragalus sinicus nodules

Two cDNAs that encode asparagine synthetase were cloned from root nodules of Astragalus sinicus cv. Japan (Renge-sou). The expression of the transcripts was nodule-enhanced. The expression of both genes was reduced in nodules when (NH 4) 2SO 4 was added to the culture medium. This is the first report of the inhibition of asparagine synthetase gene expression by ammonium in root nodules.

Activation of the Unfolded Protein Response Pathway Induces Human Asparagine Synthetase Gene Expression

The gene for the amino acid biosynthetic activity asparagine synthetase (AS) is induced by both amino acid and glucose deprivation of cells. The data reported here document that the human AS gene is induced following activation of the Unfolded Response Pathway (UPR), also known as the Endoplasmic Reticulum Stress Response (ERSR) in mammals. Increased AS transcription occurs in response to glucose deprivation, tunicamycin, or azetidine-2-carboxylate, all known to activate the UPR/ERSR pathway. Previously identified ERSR target genes contain multiple copies of a single highly conserved cis-element. In contrast, the human AS gene does not contain the ERSR element, as it has been described for other responsive genes. Instead, AS induction requires an Sp1-like sequence, a sequence previously shown to be associated with amino acid control of transcription, and possibly, a third region containing no consensus sequences for known transcription factors. Oligonucleotides covering each of these regions form DNA-protein complexes in vitro, and for some the amount of these complexes is greater when nuclear extracts from glucose-starved cells are tested. These results document that a wider range of metabolic activities are activated by the UPR/ERSR pathway than previously recognized and that genomic elements other than those already described can serve to enhance transcription of specific target genes.

Metabolic regulation of asparagine synthetase gene expression in maize (Zea mays L.) root tips

Differential hybridization of a cDNA library constructed with poly(A)+ mRNA from 24 h starved maize (Zea mays L.) root tips, resulted in the isolation of a cDNA (called pZSS1) that was highly induced during glucose deprivation. The nucleotide sequence analysis of the full-length cDNA allowed its identification by comparison with sequence data bases. The 586 amino acid sequence encoded by pZSS1 was shown to be about 60% identical to sequences of asparagine synthetases (EC 6.3.5.4) from Asparagus officinalis, Pisum sativum, Arabidopsis thaliana and Brassica oleracea. Southern blot analysis of maize genomic DNA showed that asparagine synthetase may be encoded by at least two genes. The use of a specific probe for the 3' untranslated region of pZSS1 in Northern blot experiments, revealed that the isolated AS gene was essentially expressed in roots of maize seedlings. Time course analysis revealed that maximal expression of the gene corresponding to pZSS1 occurs between 18 and 24 h after the onset of the starvation treatment. The steady-state levels of transcripts in maize root tips were found to change under various incubation conditions. Exogenous supply of metabolizable sugars downregulated the gene expression, while carbohydrate deprivation or feeding with non-metabolizable sugars resulted in the induction of gene expression. In addition to carbohydrate deprivation, the effects of nitrogen metabolite supply and stress conditions indicate that gene expression might be under metabolic control in maize root tips. The intracellular nitrogen to carbon ratio might be an important factor for the regulation of asparagine synthetase gene expression.

Cis- and trans-acting elements involved in amino acid regulation of asparagine synthetase gene expression.

We have previously shown that asparagine synthetase (AS) mRNA expression can be dramatically up-regulated by asparagine deprivation in ts11 cells, mutants of BHK hamster cells which encode a temperature-sensitive AS. The expression of AS mRNA was also induced upon starvation for one of several essential amino acids in HeLa cells. We also showed that regulation of AS mRNA expression by amino acid concentration has both transcriptional and posttranscriptional components. Here we report the analysis of the elements in the human AS promoter region important for its basal activity and activation by amino acid starvation. Our results indicate that a DNA fragment spanning from nucleotides -164 to +44 of the AS promoter is sufficient for uninduced and induced gene expression. Mutations in a region located 15 to 30 bp downstream from the major transcription start site that shows good homology to a sequence in the first exon of c-fos implicated as a negative regulatory element resulted in a significant increase in basal gene expression but did not affect regulation. Interestingly, this region binds single-stranded-DNA-binding proteins that are specific for the AS coding strand. Mutations in either one of two putative binding sites for transcription factor Sp1, in a region of approximately 60 bp where many minor RNA start sites are located, or at the major transcription start site decreased promoter activity, but significant induction by amino acid starvation was still observed. Strikingly, mutations centered around nucleotide -68 not only decreased the basal promoter activity but also abolished amino acid regulation. This DNA region contains the sequence 5'-CATGATG-3', which we call the amino acid response element (AARE), that can bind a factor(s) present in HeLa cells nuclear extracts that is not capable of binding to an AS promoter with mutations or deletions of the AARE. This finding is in line with the hypothesis that transcriptional activation of AS gene expression is mediated through the binding of a positive regulatory element. We did not detect changes in the level of binding of this factor to the AARE by using nuclear extracts from HeLa cells grown under starved conditions, suggesting that activation of this factor(s) results from posttranslational modification or complexing with other proteins that do not affect its DNA-binding properties.

Regulation of asparagine synthetase gene expression by amino acid starvation.

We have studied the regulation of expression of the asparagine synthetase (AS) gene in ts11 cells, a mutant of BHK hamster cells which encodes a temperature-sensitive AS and therefore does not produce endogenous asparagine at 39.5 degrees C. Incubation of ts11 cells at the nonpermissive temperature drastically increases the level of AS mRNA, and the stimulation of AS mRNA expression is effectively suppressed by the addition of asparagine to the medium. We show here that regulation of AS gene expression involves cis-acting elements which are contained in the mRNA as well as in the 5' genomic region. When a plasmid containing the human AS cDNA under the control of the human AS promoter region was stably transfected into ts11 cells, the expression of human AS RNAs was regulated as that of the endogenous hamster transcripts, indicating that this construct contained all cis elements necessary for regulation. Expression of the AS cDNA in ts11 cells under the control of a constitutive foreign promoter was also regulated by the concentration of asparagine, and this regulation required translation. When we introduced by mutagenesis a number of stop codons in the AS cDNA, the mutant mRNAs with short open reading frames were expressed at low levels that were not increased by asparagine deprivation. Inhibition of protein and RNA synthesis also prevented down-regulation of AS mRNA levels by high concentrations of asparagine. In a parallel series of experiments, we showed that an AS DNA fragment including the promoter and first exon can also regulate RNA expression in response to asparagine concentration. Furthermore, similar increases in the levels of AS RNAs are produced not only by asparagine deprivation in ts11 cells but also by deprivation of human and wild-type BHK cells of leucine, isoleucine, or glutamine. Thus, regulation of AS gene expression is a response to amino acid starvation through mechanisms which appear to involve both changes in RNA stability and change in the rates of transcription initiation or elongation.

Regulation of Asparagine Synthetase Gene Expression by Amino Acid Starvation

We have studied the regulation of expression of the asparagine synthetase (AS) gene in ts11 cells, a mutant of BHK hamster cells which encodes a temperature-sensitive AS and therefore does not produce endogenous asparagine at 39.5 degrees C. Incubation of ts11 cells at the nonpermissive temperature drastically increases the level of AS mRNA, and the stimulation of AS mRNA expression is effectively suppressed by the addition of asparagine to the medium. We show here that regulation of AS gene expression involves cis-acting elements which are contained in the mRNA as well as in the 5' genomic region. When a plasmid containing the human AS cDNA under the control of the human AS promoter region was stably transfected into ts11 cells, the expression of human AS RNAs was regulated as that of the endogenous hamster transcripts, indicating that this construct contained all cis elements necessary for regulation. Expression of the AS cDNA in ts11 cells under the control of a constitutive foreign promoter was also regulated by the concentration of asparagine, and this regulation required translation. When we introduced by mutagenesis a number of stop codons in the AS cDNA, the mutant mRNAs with short open reading frames were expressed at low levels that were not increased by asparagine deprivation. Inhibition of protein and RNA synthesis also prevented down-regulation of AS mRNA levels by high concentrations of asparagine. In a parallel series of experiments, we showed that an AS DNA fragment including the promoter and first exon can also regulate RNA expression in response to asparagine concentration. Furthermore, similar increases in the levels of AS RNAs are produced not only by asparagine deprivation in ts11 cells but also by deprivation of human and wild-type BHK cells of leucine, isoleucine, or glutamine. Thus, regulation of AS gene expression is a response to amino acid starvation through mechanisms which appear to involve both changes in RNA stability and change in the rates of transcription initiation or elongation.