Glutamate Decarboxylase Gene Research Articles

Variation in acid resistance among enterohaemorrhagic Escherichia coli in a simulated gastric environment

To compare survival of enterohaemorrhagic Escherichia coli (EHEC) strains of two clonal groups in a simulated gastric environment and to quantify the effect of storage in an acidic food, apple juice, on subsequent survival of EHEC in the simulated gastric environment. To characterize acid resistance of EHEC under conditions simulating the gastric environment, survival was measured in a model stomach system (MSS) for two clonal groups of EHEC: 14 EHEC 1 strains of serotype O157:H7 and 12 EHEC 2 strains of serotypes O26:H11 and O111:H8. There were significant differences between the two EHEC groups, with the average survival rate of O157 strains in the MSS twice as great as the O26/O111 strains. Strains of the two groups also differed in the quantity of injured cells in MSS and in the transcript levels of the glutamate decarboxylase genes (measured by quantitative PCR) in stationary phase before cultures entered the MSS. The results indicate that E. coli O157:H7 strains have superior ability to survive simulated gastric acidity compared with the non-O157 EHEC. E. coli O157:H7 becomes acid resistant rapidly upon entry into stationary phase, which may underlie the low infectious dose of this pathogen.

Enhancement of γ-aminobutyric acid production in Chungkukjang by applying a Bacillus subtilis strain expressing glutamate decarboxylase from Lactobacillus brevis

For a foreign glutamate decarboxylase (GAD) to be expressed in Bacillus host system, a recombinant DNA (pLip/LbGAD) was constructed by ligating an LbGAD gene from Lactobacillus brevis OPK-3 into Escherichia coli-Bacillus shuttle vector, pLip. The pLip/LbGAD construct was then transformed into Bacillus subtilis. The culture of the transformed Bacillus strain with the pLip/LbGAD construct had higher GAD activity and gamma-aminobutyric acid (GABA) concentration than those of untransformed Bacillus counterpart. In addition, Chungkukjang, a traditional Korean fermented soybean product prepared by the transformed Bacillus subtilis, contained a significantly higher level of GABA than conventional ones. Thus, by introducing a foreign GAD gene, Bacillus strains have been genetically engineered to produce high levels of GAD and GABA.

EIL2 Transcription Factor and Glutathione Synthetase Are Required for Defense of Tobacco Against Tobacco Blue Mold

In order to identify tobacco (Nicotiana megalosiphon) genes involved in broad-spectrum resistance to tobacco blue mold (Peronospora hyoscyami f. sp. tabacina), suppression subtractive hybridization was used to generate cDNA from transcripts that are differentially expressed during an incompatible interaction. After differential screening by membrane-based hybridization, clones corresponding to 182 differentially expressed genes were selected, sequenced, and analyzed. The cDNA collection comprised a broad repertoire of genes associated with various processes. Northern blot analysis of a subset of these genes confirmed the differential expression patterns between the compatible and incompatible interaction. Subsequent virus-induced gene silencing (VIGS) of four genes that were found to be differentially induced was pursued. While VIGS of a lipid transfer protein gene or a glutamate decarboxylase gene in Nicotiana megalosiphon did not affect blue mold resistance, silencing of an EIL2 transcription factor gene and a glutathione synthetase gene was found to compromise the resistance of Nicotiana megalosiphon to P. hyoscyami f. sp. tabacina. Potentially, these genes can be used to engineer resistance in blue mold-susceptible tobacco cultivars.

Cloning, sequencing and expression of a novel glutamate decarboxylase gene from a newly isolated lactic acid bacterium, Lactobacillus brevis OPK-3

Lactobacillus brevis OPK-3, having 84.292 mg/L/h of γ-aminobutyric acid (GABA) productivity, was isolated from Kimchi, a traditional fermented food in Korea. A core fragment of glutamate decarboxylase (GAD) DNA was isolated from the L. brevis OPK-3, using primers based on two highly conserved regions of GAD. A full-length GAD ( LbGAD) clone was subsequently isolated through rapid amplification of cDNA ends (RACE) PCR. Nucleotide sequence analysis revealed that the open reading frame (ORF) consisted of 1401 bases and encoded a protein of 467 amino acid residues with a calculated molecular weight of 53.4 kDa and a pI of 5.65. The amino acid sequence deduced from LbGAD ORF showed 83%, 71%, and 60% identity to the Lactobacillus plantarum GAD, Lactococcus lactis GAD, and Listeria monocytogenes GAD sequences, respectively. The LbGAD gene was expressed in Escherichia coli strain UT481, and the extract of transformed E. coli UT481 contained an induced 53.4 kDa protein and had significantly enhanced GAD activity.

Cloning and Expression of a Full-Length Glutamate Decarboxylase Gene from Lactobacillus plantarum

In order to investigate the molecular mechanism of γ-aminobutyric acid (GABA) production in lactic acid bacteria, we cloned a glutamate decarboxylase (GAD) gene from Lactobacillus plantarum using polymerase chain reaction (PCR). One PCR product DNA was obtained and inserted into a TA cloning vector with a T7 promoter. The recombinant plasmid was used to transform E. coli. The insertion of the product was confirmed by EcoRI digestion of the plasmid purified from the transformed E. coli. Nucleotide sequence analysis showed that the insert is a full-length Lactobacillus plantarum GAD and that the sequence is 100% and 72% identical to the regions of Lactobacillus plantarum GAD and Lactococcus lactis GAD sequences deposited in GenBank, accession nos: NP786643 and NP267446, respectively. The amino acid sequence deduced from the cloned Lactobacillus plantarum GAD gene showed 100% and 68% identities to the GAD sequences deduced from the genes of the NP786643 and NP267446, respectively. To express the GAD protein in E. coli, an expression vector with the GAD gene (pkk/GAD) was constructed and used to transform the UT481 E. coli strain and the expression was confirmed by analyzing the enzyme activity. The Lactobacillus plantarum GAD gene obtained may facilitate the study of the molecular mechanisms regulating GABA metabolism in lactic acid bacteria.

The root-specific glutamate decarboxylase (GAD1) is essential for sustaining GABA levels in Arabidopsis

In plants, as in most eukaryotes, glutamate decarboxylase catalyses the synthesis of GABA. The Arabidopsis genome contains five glutamate decarboxylase genes and one of these genes (glutamate decarboxylase1; i.e. GAD1 ) is expressed specifically in roots. By isolating and analyzing three gad1 T-DNA insertion alleles, derived from two ecotypes, we investigated the potential role of GAD1 in GABA production. We also analyzed a promoter region of the GAD1 gene and show that it confers root-specific expression when fused to reporter genes. Phenotypic analysis of the gad1 insertion mutants revealed that GABA levels in roots were drastically reduced compared with those in the wild type. The roots of the wild type contained about sevenfold more GABA than roots of the mutants. Disruption of the GAD1 gene also prevented the accumulation of GABA in roots in response to heat stress. Our results show that the root-specific calcium/calmodulin-regulated GAD1 plays a major role in GABA synthesis in plants under normal growth conditions and in response to stress.

Polymorphisms in glutamate decarboxylase genes: analysis in schizophrenia.

The decrease of glutamic acid decarboxylase (GAD) has been reported as an important neurochemical alteration of the inhibitory GABAergic interneurons in schizophrenia. To our knowledge no studies have investigated the genetic variants influencing GAD expression. To search for markers contributing to the genetic susceptibility of schizophrenia, we typed two polymorphisms by polymerase chain reaction-restriction fragment length polymorphism in both GAD1 and GAD2 genes in 112 triad families and 46 case-controls. We used the Transmission Disequilibrium Test to perform the qualitative family-based analyses and found negative results (GAD1, chi2 = 0.273, 1 degree of freedom, P = 0.60; GAD2, chi2 = 0, 1 degree of freedom, P = 1). In addition there were no associations with GAD1 and GAD2 and quantitative measures of suicide behaviour in this sample. Although our results are negative, this was the first study to investigate GAD genes in schizophrenia, and further studies of these genes, particularly with schizophrenia subtypes, may prove valuable.

PH-dependent catabolic protein expression during anaerobic growth of Escherichia coli K-12.

During aerobic growth of Escherichia coli, expression of catabolic enzymes and envelope and periplasmic proteins is regulated by pH. Additional modes of pH regulation were revealed under anaerobiosis. E. coli K-12 strain W3110 was cultured anaerobically in broth medium buffered at pH 5.5 or 8.5 for protein identification on proteomic two-dimensional gels. A total of 32 proteins from anaerobic cultures show pH-dependent expression, and only four of these proteins (DsbA, TnaA, GatY, and HdeA) showed pH regulation in aerated cultures. The levels of 19 proteins were elevated at the high pH; these proteins included metabolic enzymes (DhaKLM, GapA, TnaA, HisC, and HisD), periplasmic proteins (ProX, OppA, DegQ, MalB, and MglB), and stress proteins (DsbA, Tig, and UspA). High-pH induction of the glycolytic enzymes DhaKLM and GapA suggested that there was increased fermentation to acids, which helped neutralize alkalinity. Reporter lac fusion constructs showed base induction of sdaA encoding serine deaminase under anaerobiosis; in addition, the glutamate decarboxylase genes gadA and gadB were induced at the high pH anaerobically but not with aeration. This result is consistent with the hypothesis that there is a connection between the gad system and GabT metabolism of 4-aminobutanoate. On the other hand, 13 other proteins were induced by acid; these proteins included metabolic enzymes (GatY and AckA), periplasmic proteins (TolC, HdeA, and OmpA), and redox enzymes (GuaB, HmpA, and Lpd). The acid induction of NikA (nickel transporter) is of interest because E. coli requires nickel for anaerobic fermentation. The position of the NikA spot coincided with the position of a small unidentified spot whose induction in aerobic cultures was reported previously; thus, NikA appeared to be induced slightly by acid during aeration but showed stronger induction under anaerobic conditions. Overall, anaerobic growth revealed several more pH-regulated proteins; in particular, anaerobiosis enabled induction of several additional catabolic enzymes and sugar transporters at the high pH, at which production of fermentation acids may be advantageous for the cell.

Normalization of glutamate decarboxylase gene expression in the entopeduncular nucleus of rats with a unilateral 6-hydroxydopamine lesion correlates with increased gabaergic input following intermittent but not continuous levodopa

The expression of mRNA encoding for the 67 kilodalton isoform of glutamate decarboxylase (GAD67) was examined by in situ hybridization histochemistry in the entopeduncular nucleus (EP) of adult rats with a 6-hydroxydopamine unilaterally lesion of dopamine neurons. Our results provide original evidence that continuous or intermittent levodopa administration is equally effective at reversing the lesion-induced increase in GAD67 mRNA expression in the EP when compared with vehicle controls. To characterize the GABAergic interactions that may mediate levodopa-induced alterations in the EP, double-labeling in situ hybridization was conducted with a combination of GAD67 radioactive and preproenkephalin or preprotachykinin digoxigenin-labeled complementary RNA probes in the striatum. Levels of GAD67 mRNA labeling were significantly increased by intermittent, but not continuous levodopa. Analysis at the cellular level in a dorsal sector of the striatum revealed that GAD67 mRNA levels increased predominantly in preproenkephalin-unlabeled neuronal profiles, presumably striatal/EP neurons (+99.3%). Saturation analyses of 3H-flunitrazepam binding to GABA A receptors in the EP showed that the increase in GAD67 mRNA in preproenkephalin-unlabeled neurons by intermittent levodopa paralleled a significant decrease in number of GABA A receptors (Bmax) in the EP ipsilateral to the lesion. Continuous levodopa failed to alter striatal GAD67 mRNA levels, or the number or affinity of GABA A receptors when compared with vehicle-treated controls. These results suggest the normalization of GAD gene expression in the EP by intermittent levodopa involves an increase in GABAergic inhibition by striatonigral/EP neurons of the direct pathway. Conversely, the effects of continuous levodopa on GAD mRNA levels in the EP do not appear to be mediated by GABA.

GadE (YhiE) activates glutamate decarboxylase-dependent acid resistance in Escherichia coli K-12.

Commensal and pathogenic strains of Escherichia coli possess three inducible acid resistance systems that collaboratively protect cells against acid stress to pH 2 or below. The most effective system requires glutamate in the acid challenge media and relies on two glutamate decarboxylases (GadA and B) combined with a putative glutamate:gamma-aminobutyric acid antiporter (GadC). A complex network of regulators mediates induction of this system in response to various media, pH and growth phase signals. We report that the LuxR-like regulator GadE (formerly YhiE) is required for expression of gadA and gadBC regardless of media or growth conditions. This protein binds directly to the 20 bp GAD box sequence found in the control regions of both loci. Two previously identified AraC-like regulators, GadX and GadW, are only needed for gadA/BC expression under some circumstances. Overexpression of GadX or GadW will not overcome a need for GadE. However, overexpression of GadE can supplant a requirement for GadX and W. Data provided also indicate that GadX and GadE can simultaneously bind the area around the GAD box region and probably form a complex. The gadA, gadBC and gadE genes are all induced by low pH in exponential phase cells grown in minimal glucose media. The acid induction of gadA/BC results primarily from the acid induction of gadE. Constitutive expression of GadE removes most pH control over the glutamate decarboxylase and antiporter genes. The small amount of remaining pH control is governed by GadX and W. The finding that gadE mutations also diminish the effectiveness of the other two acid resistance systems suggests that GadE influences the expression of additional acid resistance components. The number of regulatory proteins (five), sigma factors (two) and regulatory feedback loops focused on gadA/BC expression make this one of the most intensively regulated systems in E. coli.

Evaluation of the glutamate decarboxylase genes Gad1 and Gad2 as candidate genes for acute ethanol withdrawal severity in mice

Previous studies in crosses between the C57BL/6J (B6) and the DBA/2J (D2) mice have implicated a role of the genes encoding for the 67- and 65-kDa isoforms of the glutamate decarboxylase (Gad1 and Gad2) in the manifestation and severity of multiple ethanol-related traits such as acute ethanol withdrawal severity [Buck, K.J., Metten, P., Belknap, J.K., Crabbe, J.C., 1997. Quantitative trait loci involved in genetic predisposition to acute alcohol withdrawal in mice. J. Neurosci. 17, 3946–3955], ethanol preference [Phillips, T.J., Belknap, J.K., Buck, K.J., Cunningham, C.L., 1998. Genes on mouse chromosomes 2 and 9 determine variation in ethanol consumption. Mamm. Genome 9, 936–941] and ethanol-induced locomotion [Demarest, K., McCaughran Jr., J., Mahjubi, E., Cipp, L., Hitzemann, R., 1999. Identification of an acute ethanol response quantitative trait locus on mouse chromosome 2. J. Neurosci. 19, 549–561]. Strain-specific sequencing experiments as well as gene expression studies in drug-naı̈ve and ethanol-treated D2 and B6 mice were carried out. The Gad1 sequence was similar, the Gad2 cDNA carried only a silent polymorphism (1017 G>C) between both strains. In addition, no significant GAD65 or GAD67 expression differences were detected in either drug-naı̈ve or acute ethanol withdrawn animals by Western blot experiments. Therefore, these results do not support the hypothesis of an involvement of Gad1 or Gad2 in the pathophysiology of acute ethanol withdrawal severity and the other ethanol related traits.

Acid tolerance and gad mRNA levels of Escherichia coli O157:H7 grown in foods

We examined the acid tolerance and gad mRNA levels of Escherichia coli O157:H7 (three strains) and nonpathogenic E. coli (strains K12, W1485, and B) grown in foods. The E. coli cells (approximately 30,000 cells) were inoculated on the surface of 10 g of solid food samples (asparagus, broccoli, carrot, celery, cucumber, eggplant, ginger, green pepper, onion, potato, radish, tomato and beef) and in 10 ml of cow's milk, cultured statically at 10–25 °C for 1–14 days, and subjected to an acid challenge at 37 °C for 1 h in LB medium (pH 3.0). When grown at 20 and 25 °C in all foods, except for tomato and ginger, the strains showed a stationary-phase specific acid tolerance. The acid tolerance of the O157 strains changed depending on the types of foods (3–10% survival), but was clearly lower than that of the cells grown in EC medium (more than 90% survival). Tomato and ginger induced relatively high acid tolerances (10–30% survival) in the O157 strains irrespective of the growth phase, probably because of their acidity. No remarkable difference was observed in the acid tolerance between the O157 and nonpathogenic strains grown in all foods. When grown at 10 and 15 °C in the foods and EC medium, none of the strains showed the stationary-phase specific acid tolerance. In beef, broccoli, celery, potato and radish, the acid tolerance showed a tendency to decrease with the prolonged cultivation time. In other foods, the acid tolerance was almost constant (about 0.1% survival) irrespective of the growth stage. The mRNA level of glutamate decarboxylase genes ( gadA and gadB) correlated to the acid tolerance level when the E. coli cells were grown at 25 °C, but was very low even in the stationary phase when the E. coli cells were grown at 15 °C or below.

Rapid PCR-based method which can determine both phenotype and genotype of Lactococcus lactis subspecies.

A highly efficient, rapid, and reliable PCR-based method for distinguishing Lactococcus lactis subspecies (L. lactis subsp. lactis and L. lactis subsp. cremoris) is described. Primers complementary to positions in the glutamate decarboxylase gene have been constructed. PCR analysis with extracted DNA or with cells of different L. lactis strains resulted in specific fragments. The length polymorphism of the PCR fragments allowed a clear distinction of the L. lactis subspecies. The amplified fragment length polymorphism with the primers and the restriction fragment length polymorphism of the amplified products agreed perfectly with the identification based on genotypic and phenotypic analyses, respectively. Isolates from cheese starters were investigated by this method, and amplified fragments of genetic variants were found to be approximately 40 bp shorter than the typical L. lactis subsp. cremoris fragments.

Conserved Function ofCaenorhabditis elegansUNC-30 and Mouse Pitx2 in Controlling GABAergic Neuron Differentiation

We are taking a cross-species approach to identify genes that are required for mammalian GABAergic neuron differentiation. On the basis of homeodomain similarity, the vertebrate Pitx genes appear to be orthologs of unc-30, a Caenorhabditis elegans gene necessary for differentiation of the GABAergic phenotype of type D neurons. One of the Pitx genes, Pitx2, is expressed in regions of GABAergic neurogenesis in the mammalian brain. These observations led us to test the functional conservation of the mouse Pitx2 and worm unc-30 genes using a rescue assay. Pitx2 rescues the GABAergic differentiation defect and partially rescues the axon guidance and behavioral phenotypes of unc-30 mutants, indicating a high degree of functional conservation between these evolutionarily related genes. Previous studies show that UNC-30 directly regulates the unc-25/glutamate decarboxylase gene that encodes the enzyme for GABA synthesis. We find that the promoter regions of the mouse and human genes coding for the 67 kDa glutamate decarboxylase (Gad1) also contain binding sites matching the UNC-30/Pitx2 consensus binding site sequence. We show that these sites specifically bind to Pitx2 protein in vitro and that in transfected neuroblastoma cells, the Pitx2 binding sites contribute to the basal activity of the Gad1 promoter. Furthermore, in cotransfection experiments, we find that Pitx2 strongly activates the Gad1 promoter. These results indicate that Pitx2 may regulate Gad1 expression in mammals, suggesting a new role for this key developmental transcription factor as a regulator of GABAergic differentiation during mammalian neural development. Our results suggest that some of the mechanisms regulating GABAergic differentiation are evolutionarily conserved.

An activator of glutamate decarboxylase genes regulates the expression of enteropathogenic Escherichia coli virulence genes through control of the plasmid-encoded regulator, Per.

Enteropathogenic Escherichia coli (EPEC) is a major cause of infantile diarrhoea in a number of developing countries and is the prototype of pathogenic bacteria that cause attaching and effacing (A/E) intestinal lesions. A chromosomal pathogenicity island, termed the locus of enterocyte effacement (LEE), contains all the genes necessary for the A/E phenotype as well as genes for a type III secretion system and intimate adhesion. Genes in the LEE and genes involved in the synthesis of bundle-forming pili (BFP) are positively regulated by the plasmid-encoded regulator (Per) and comprise the per regulon. In order to identify factors that control the per regulon, we screened an EPEC genomic library for clones that modulate the expression of per. A plasmid clone that decreased the expression of per was isolated using a lacZ reporter gene fused to the per promoter. Subcloning revealed that YhiX, a putative AraC/XylR family transcriptional regulator, was the effector of per repression. Through downregulation of per, a plasmid overproducing YhiX reduced the synthesis of intimin, BfpA, Tir, and CesT, factors important for EPEC virulence. yhiX is located downstream of gadA, which encodes glutamate decarboxylase, an enzyme involved in acid resistance of E. coli. YhiX was found to be an activator of gadA, and the cloned yhiX gene increased production of glutamate decarboxylases (GAD) and activated the transcription of the gadA and gadB promoters. Therefore, yhiX was renamed gadX. Analysis of a gadX mutant grown in the different culture media with acidic and alkaline pH showed that regulation of perA, gadA and gadB by GadX was altered by the external pH and the culture media condition. Under conditions in which EPEC infects cultured epithelial cells, GadX negatively regulated perA expression, and the derepression in the gadX mutant increased translocation of Tir into epithelial cells relative to wild-type EPEC. DNA mobility shift experiments showed that purified GadX protein bound to the perA, gadA and gadB promoter regions in vitro, indicating that GadX is a transcriptional regulator of these genes. On the basis of these results, we propose that GadX may be involved in the appropriate expression of genes required for acid resistance and virulence of EPEC. Our data are consistent with a model in which environmental changes resulting from passage from the stomach to the proximal small intestine induce the functional effect of GadX on per and GAD expression in order to prevent inappropriate expression of the products of these two systems.

Glutamate decarboxylase genes as a prescreening marker for detection of pathogenic Escherichia coli groups.

The enzyme glutamate decarboxylase (GAD) is prevalent in Escherichia coli but few strains in the various pathogenic E. coli groups have been tested for GAD. Using PCR primers that amplify a 670-bp segment from the gadA and gadB genes encoding GAD, we examined the distribution of the gadAB genes among enteric bacteria. Analysis of 173 pathogenic E. coli strains, including 125 enterohemorrhagic E. coli isolates of the O157:H7 serotype and its phenotypic variants and 48 isolates of enteropathogenic E. coli, enterotoxigenic E. coli, enteroinvasive E. coli, and other Shiga toxin-producing E. coli (STEC) serotypes, showed that gadAB genes were present in all these strains. Among the 22 non-E. coli isolates tested, only the 6 Shigella spp. carried gadAB. Analysis of naturally contaminated water and food samples using a gadAB-specific DNA probe that was labeled with digoxigenin showed that a gadAB-based assay is as reliable as standard methods that enumerate E. coli organisms on the basis of lactose fermentation. The presence of few E. coli cells initially seeded into produce rinsates could be detected by PCR to gadA/B genes after overnight enrichment. A multiplex PCR assay using the gadAB primers in combination with primers to Shiga toxin (Stx) genes stx(1) and stx(2) was effective in detecting STEC from the enrichment medium after seeding produce rinsate samples with as few as 2 CFU. The gadAB primers may be multiplexed with primers to other trait virulence markers to specifically identify other pathogenic E. coli groups.

Neuronal Development of Embryonic Stem Cells: A Model of GABAergic Neuron Differentiation

Neural cultures derived from differentiating embryonic stem (ES) cells are a potentially powerful in vitro model of neural development. We show that neural cells derived from mouse ES cells express mRNAs characteristic of GABAergic neurons. The glutamate decarboxylase genes (Gad1 and Gad2), required for GABA synthesis and the vesicular inhibitory amino acid transporter (Viaat) gene, required for GABA vesicular packaging are activated in the ES-derived cultures. Nearly half of the ES-derived neurons express the GAD67 protein, the product of the Gad1 gene. Building on these results we show that Gad1-lacZ “knockin” reporter ES cell lines can be used to easily monitor Gad1 expression patterns and expression levels during ES differentiation. We also demonstrate that the ES-derived neural progenitors can be infected with retroviruses or transfected with plasmids via lipofection. These experiments outline the basic strategies and methods required for studies of GABAergic gene expression and regulation in ES-derived neuronal cultures.

Escherichia coli acid resistance: cAMP receptor protein and a 20 bp cis-acting sequence control pH and stationary phase expression of the gadA and gadBC glutamate decarboxylase genes

Acid resistance is an important feature of both pathogenic and non-pathogenic Escherichia coli. It enables survival in the acidic regions of mammalian gastrointestinal tracts and is largely responsible for the small number of bacteria required for infection/colonization. Three systems of acid resistance have been identified, the most efficient of which requires glutamic acid during pH 2 acid challenge. Three proteins associated with glutamate-dependent acid resistance have been identified. They are glutamate decarboxylase (encompassing two isozymes encoded by gadA and gadB) and a putative glutamate:gamma-amino butyric acid antiporter (encoded by gadC). The results confirm that the GadA and GadB proteins increase in response to stationary phase and low environmental pH. The levels of these proteins correspond to concomitant changes in gadA and gadBC mRNA levels. Fusions between lacZ and the gadA and gadBC operons indicate that this control occurs at the transcriptional level. Western blot, Northern blot and fusion analyses reveal that regulation of these genes is complex. Expression in rich media is restricted to stationary phase. However, in minimal media, acid pH alone can trigger induction in exponential or stationary phase cells. Despite this differential control, there is only one transcriptional start site for each gene. Expression in rich media is largely dependent on the alternate sigma factor sigma(S) and is repressed by the cAMP receptor protein (CRP). In contrast, sigma(S) has only a minor role in gad transcription in cells grown in minimal media. Deletions of the regulatory region upstream of gadA provided evidence that a 20 bp conserved region located 50 bp from the transcriptional start of both operons is required for expression.

Dynamic expression of a glutamate decarboxylase gene in multiple non-neural tissues during mouse development.

BackgroundGlutamate decarboxylase (GAD) is the biosynthetic enzyme for the neurotransmitter γ-aminobutyric acid (GABA). Mouse embryos lacking the 67-kDa isoform of GAD (encoded by the Gad1 gene) develop a complete cleft of the secondary palate. This phenotype suggests that this gene may be involved in the normal development of tissues outside of the CNS. Although Gad1 expression in adult non-CNS tissues has been noted previously, no systematic analysis of its embryonic expression outside of the nervous system has been performed. The objective of this study was to define additional structures outside of the central nervous system that express Gad1, indicating those structures that may require its function for normal development.ResultsOur analysis detected the localized expression of Gad1 transcripts in several developing tissues in the mouse embryo from E9.0-E14.5. Tissues expressing Gad1 included the tail bud mesenchyme, the pharyngeal pouches and arches, the ectodermal placodes of the developing vibrissae, and the apical ectodermal ridge (AER), mesenchyme and ectoderm of the limb buds.ConclusionsSome of the sites of Gad1 expression are tissues that emit signals required for patterning and differentiation (AER, vibrissal placodes). Other sites correspond to proliferating stem cell populations that give rise to multiple differentiated tissues (tail bud mesenchyme, pharyngeal endoderm and mesenchyme). The dynamic expression of Gad1 in such tissues suggests a wider role for GABA signaling in development than was previously appreciated.

Expression of a Glutamate Decarboxylase Homologue Is Required for Normal Oxidative Stress Tolerance in Saccharomyces cerevisiae

The action of gamma-aminobutyrate (GABA) as an intercellular signaling molecule has been intensively studied, but the role of this amino acid metabolite in intracellular metabolism is poorly understood. In this work, we identify a Saccharomyces cerevisiae homologue of the GABA-producing enzyme glutamate decarboxylase (GAD) that is required for normal oxidative stress tolerance. A high copy number plasmid bearing the glutamate decarboxylase gene (GAD1) increases resistance to two different oxidants, H(2)O(2) and diamide, in cells that contain an intact glutamate catabolic pathway. Structural similarity of the S. cerevisiae GAD to previously studied plant enzymes was demonstrated by the cross-reaction of the yeast enzyme to a antiserum directed against the plant GAD. The yeast GAD also bound to calmodulin as did the plant enzyme, suggesting a conservation of calcium regulation of this protein. Loss of either gene encoding the downstream steps in the conversion of glutamate to succinate reduced oxidative stress tolerance in normal cells and was epistatic to high copy number GAD1. The gene encoding succinate semialdehyde dehydrogenase (UGA5) was identified and found to be induced by H(2)O(2) exposure. Together, these data strongly suggest that increases in activity of the glutamate catabolic pathway can act to buffer redox changes in the cell.