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Robert A. Bender - One of the best experts on this subject based on the ideXlab platform.

  • isolation of a negative control mutant of the Nitrogen Assimilation control protein nac in klebsiella aerogenes
    Journal of Bacteriology, 2003
    Co-Authors: Brian K Janes, Christopher J Rosario, Robert A. Bender
    Abstract:

    A negative control mutant of the Nitrogen Assimilation control protein, NAC, has been isolated. Mutants with the leucine at position 111 changed to a nonhydrophobic residue activate transcription from hut and ure promoters, but fail to repress gdhA expression. This failure does not result from failure to bind to either of the two sites required for gdhA repression, but the binding at those sites is altered in the mutant. It appears that the NAC negative control mutants fail to form the complex structures (probably tetramers) formed by wild-type NAC at the gdhA promoter.

  • repression of glutamate dehydrogenase formation in klebsiella aerogenes requires two binding sites for the Nitrogen Assimilation control protein nac
    Journal of Bacteriology, 2002
    Co-Authors: Thomas J Goss, Brian K Janes, Robert A. Bender
    Abstract:

    In Klebsiella aerogenes, the gdhA gene codes for glutamate dehydrogenase, one of the enzymes responsible for assimilating ammonia into glutamate. Expression of a gdhAp-lacZ transcriptional fusion was strongly repressed by the Nitrogen Assimilation control protein, NAC. This strong repression (>50-fold under conditions of severe Nitrogen limitation) required the presence of two separate NAC binding sites centered at -89 and +57 relative to the start of gdhA transcription. Mutants lacking either or both of these sites lost the strong repression. The distance between the two sites was less important than the face of the helix on which they lay. Insertion or deletion of 10 bp between the sites had little effect on the strong repression, but insertion of 5 bp or deletion of either 5 or 15 bp decreased the repression significantly. We propose that the strong repression of gdhAp-lacZ expression requires an interaction between the NAC molecules bound at the two sites. A weaker repression of gdhAp-lacZ expression (about threefold) required only the NAC site centered at -89. This weaker repression appears to result from NAC's ability to prevent the action of a positive effector the target of which overlaps the NAC binding site centered at -89. Point mutations and deletions of this region result in the same threefold reduction in gdhAp-lacZ expression as the presence of NAC at this site.

  • the amino terminal 100 residues of the Nitrogen Assimilation control protein nac encode all known properties of nac from klebsiella aerogenes and escherichia coli
    Journal of Bacteriology, 1999
    Co-Authors: Wilson B Muse, Robert A. Bender
    Abstract:

    The Nitrogen Assimilation control protein (NAC) from Klebsiella aerogenes or Escherichia coli (NACK or NACE, respectively) is a transcriptional regulator that is both necessary and sufficient to activate transcription of the histidine utilization (hut) operon and to repress transcription of the glutamate dehydrogenase (gdh) operon in K. aerogenes. Truncated NAC polypeptides, generated by the introduction of stop codons within the nac open reading frame, were tested for the ability to activate hut and repress gdh in vivo. Most of the NACK and NACE fragments with 100 or more amino acids (wild-type NACK and NACE both have 305 amino acids) were functional in activating hut and repressing gdh expression in vivo. Full-length NACK and NACE were isolated as chimeric proteins with the maltose-binding protein (MBP). NACK and NACE released from such chimeras were able to activate hut transcription in a purified system in vitro, as were NACK129 and NACE100 (a NACK fragment of 129 amino acids and a NACE fragment of 100 amino acids) released from comparable chimeras. A set of NACE and NACK fragments carrying nickel-binding histidine tags (his6) at their C termini were also generated. All such constructs derived from NACE were insoluble, as was NACE itself. Of the his6-tagged constructs derived from NACK, NACK100 was inactive, but NACK120 was active. Several NAC fragments were tested for dimerization. NACK120-his6 and NACK100-his6 were dimers in solution. MBP-NACK and MBP-NACK129 were monomers in solution but dimerized when the MBP was released by cleavage with factor Xa. MBP-NACE was readily cleaved by factor Xa, but the resulting NACE was also degraded by the protease. However, MBP-NACE-his6 was completely resistant to cleavage by factor Xa, suggesting an interaction between the C and N termini of this protein.

  • two roles for the dna recognition site of the klebsiella aerogenes Nitrogen Assimilation control protein
    Journal of Bacteriology, 1998
    Co-Authors: Pablo J Pomposiello, Brian K Janes, Robert A. Bender
    Abstract:

    The Nitrogen Assimilation control protein (NAC) binds to a site within the promoter region of the histidine utilization operon (hutUH) of Klebsiella aerogenes, and NAC bound at this site activates transcription of hutUH. This NAC-binding site was characterized by a combination of random and directed DNA mutagenesis. Mutations that abolished or diminished in vivo transcriptional activation by NAC were found to lie within a 15-bp region contained within the 26-bp region protected by NAC from DNase I digestion. This 15-bp core has the palindromic ends ATA and TAT, and it matches the consensus for LysR family transcriptional regulators. Protein-binding experiments showed that transcriptional activation in vivo decreased with decreasing binding in vitro. In contrast to the NAC-binding site from hutUH, the NAC-binding site from the gdhA promoter failed to activate transcription from a semisynthetic promoter, and this failure was not due to weak binding or greatly distorted protein-DNA structure. Mutations in the promoter-proximal half-site of the NAC-binding site from gdhA allowed this site to activate transcription. Similar studies using the NAC-binding site from hut showed that two mutations in the promoter proximal half-site increased binding but abolished transcriptional activation. Interestingly, for symmetric mutations in the promoter-distal half-site, loss of transcriptional activation was always correlated with a decrease in binding. We conclude from these observations that if the binding in vitro reflects the binding in vivo, then binding of NAC to DNA is not sufficient for transcriptional activation and that the NAC-binding site can be functionally divided in two half-sites, with related but different functions.

  • alanine catabolism in klebsiella aerogenes molecular characterization of the dadab operon and its regulation by the Nitrogen Assimilation control protein
    Journal of Bacteriology, 1998
    Co-Authors: Brian K Janes, Robert A. Bender
    Abstract:

    Klebsiella aerogenes strains with reduced levels of d-amino acid dehydrogenase not only fail to use alanine as a growth substrate but also become sensitive to alanine in minimal media supplemented with glucose and ammonium. The inability of these mutant strains to catabolize the alanine provided in the medium interferes with both pathways of glutamate production. Alanine derepresses the Nitrogen regulatory system (Ntr), which in turn represses glutamate dehydrogenase, one pathway of glutamate production. Alanine also inhibits the enzyme glutamine synthetase, the first enzyme in the other pathway of glutamate production. Therefore, in the presence of alanine, strains with mutations in dadA (the gene that codes for a subunit of the dehydrogenase) exhibit a glutamate auxotrophy when ammonium is the sole source of Nitrogen. The alanine catabolic operon of Klebsiella aerogenes, dadAB, was cloned, and its DNA sequence was determined. The clone complemented the alanine defects of dadA strains. The operon has a high similarity to the dadAB operon of Salmonella typhimurium and the dadAX operon of Escherichia coli, each of which codes for the smaller subunit of d-amino acid dehydrogenase and the catabolic alanine racemase. Unlike the cases for E. coli and S. typhimurium, the dad operon of K. aerogenes is activated by the Ntr system, mediated in this case by the Nitrogen Assimilation control protein (NAC). A sequence matching the DNA consensus for NAC-binding sites is located centered at position −44 with respect to the start of transcription. The promoter of this operon also contains consensus binding sites for the catabolite activator protein and the leucine-responsive regulatory protein.

Brian K Janes - One of the best experts on this subject based on the ideXlab platform.

  • isolation of a negative control mutant of the Nitrogen Assimilation control protein nac in klebsiella aerogenes
    Journal of Bacteriology, 2003
    Co-Authors: Brian K Janes, Christopher J Rosario, Robert A. Bender
    Abstract:

    A negative control mutant of the Nitrogen Assimilation control protein, NAC, has been isolated. Mutants with the leucine at position 111 changed to a nonhydrophobic residue activate transcription from hut and ure promoters, but fail to repress gdhA expression. This failure does not result from failure to bind to either of the two sites required for gdhA repression, but the binding at those sites is altered in the mutant. It appears that the NAC negative control mutants fail to form the complex structures (probably tetramers) formed by wild-type NAC at the gdhA promoter.

  • repression of glutamate dehydrogenase formation in klebsiella aerogenes requires two binding sites for the Nitrogen Assimilation control protein nac
    Journal of Bacteriology, 2002
    Co-Authors: Thomas J Goss, Brian K Janes, Robert A. Bender
    Abstract:

    In Klebsiella aerogenes, the gdhA gene codes for glutamate dehydrogenase, one of the enzymes responsible for assimilating ammonia into glutamate. Expression of a gdhAp-lacZ transcriptional fusion was strongly repressed by the Nitrogen Assimilation control protein, NAC. This strong repression (>50-fold under conditions of severe Nitrogen limitation) required the presence of two separate NAC binding sites centered at -89 and +57 relative to the start of gdhA transcription. Mutants lacking either or both of these sites lost the strong repression. The distance between the two sites was less important than the face of the helix on which they lay. Insertion or deletion of 10 bp between the sites had little effect on the strong repression, but insertion of 5 bp or deletion of either 5 or 15 bp decreased the repression significantly. We propose that the strong repression of gdhAp-lacZ expression requires an interaction between the NAC molecules bound at the two sites. A weaker repression of gdhAp-lacZ expression (about threefold) required only the NAC site centered at -89. This weaker repression appears to result from NAC's ability to prevent the action of a positive effector the target of which overlaps the NAC binding site centered at -89. Point mutations and deletions of this region result in the same threefold reduction in gdhAp-lacZ expression as the presence of NAC at this site.

  • two roles for the dna recognition site of the klebsiella aerogenes Nitrogen Assimilation control protein
    Journal of Bacteriology, 1998
    Co-Authors: Pablo J Pomposiello, Brian K Janes, Robert A. Bender
    Abstract:

    The Nitrogen Assimilation control protein (NAC) binds to a site within the promoter region of the histidine utilization operon (hutUH) of Klebsiella aerogenes, and NAC bound at this site activates transcription of hutUH. This NAC-binding site was characterized by a combination of random and directed DNA mutagenesis. Mutations that abolished or diminished in vivo transcriptional activation by NAC were found to lie within a 15-bp region contained within the 26-bp region protected by NAC from DNase I digestion. This 15-bp core has the palindromic ends ATA and TAT, and it matches the consensus for LysR family transcriptional regulators. Protein-binding experiments showed that transcriptional activation in vivo decreased with decreasing binding in vitro. In contrast to the NAC-binding site from hutUH, the NAC-binding site from the gdhA promoter failed to activate transcription from a semisynthetic promoter, and this failure was not due to weak binding or greatly distorted protein-DNA structure. Mutations in the promoter-proximal half-site of the NAC-binding site from gdhA allowed this site to activate transcription. Similar studies using the NAC-binding site from hut showed that two mutations in the promoter proximal half-site increased binding but abolished transcriptional activation. Interestingly, for symmetric mutations in the promoter-distal half-site, loss of transcriptional activation was always correlated with a decrease in binding. We conclude from these observations that if the binding in vitro reflects the binding in vivo, then binding of NAC to DNA is not sufficient for transcriptional activation and that the NAC-binding site can be functionally divided in two half-sites, with related but different functions.

  • alanine catabolism in klebsiella aerogenes molecular characterization of the dadab operon and its regulation by the Nitrogen Assimilation control protein
    Journal of Bacteriology, 1998
    Co-Authors: Brian K Janes, Robert A. Bender
    Abstract:

    Klebsiella aerogenes strains with reduced levels of d-amino acid dehydrogenase not only fail to use alanine as a growth substrate but also become sensitive to alanine in minimal media supplemented with glucose and ammonium. The inability of these mutant strains to catabolize the alanine provided in the medium interferes with both pathways of glutamate production. Alanine derepresses the Nitrogen regulatory system (Ntr), which in turn represses glutamate dehydrogenase, one pathway of glutamate production. Alanine also inhibits the enzyme glutamine synthetase, the first enzyme in the other pathway of glutamate production. Therefore, in the presence of alanine, strains with mutations in dadA (the gene that codes for a subunit of the dehydrogenase) exhibit a glutamate auxotrophy when ammonium is the sole source of Nitrogen. The alanine catabolic operon of Klebsiella aerogenes, dadAB, was cloned, and its DNA sequence was determined. The clone complemented the alanine defects of dadA strains. The operon has a high similarity to the dadAB operon of Salmonella typhimurium and the dadAX operon of Escherichia coli, each of which codes for the smaller subunit of d-amino acid dehydrogenase and the catabolic alanine racemase. Unlike the cases for E. coli and S. typhimurium, the dad operon of K. aerogenes is activated by the Ntr system, mediated in this case by the Nitrogen Assimilation control protein (NAC). A sequence matching the DNA consensus for NAC-binding sites is located centered at position −44 with respect to the start of transcription. The promoter of this operon also contains consensus binding sites for the catabolite activator protein and the leucine-responsive regulatory protein.

  • alanine catabolism in klebsiella aerogenes molecular characterization of the dadab operon and its regulation by the Nitrogen Assimilation control protein
    Journal of Bacteriology, 1998
    Co-Authors: Brian K Janes, Robert A. Bender
    Abstract:

    Klebsiella aerogenes strains with reduced levels of d-amino acid dehydrogenase not only fail to use alanine as a growth substrate but also become sensitive to alanine in minimal media supplemented with glucose and ammonium. The inability of these mutant strains to catabolize the alanine provided in the medium interferes with both pathways of glutamate production. Alanine derepresses the Nitrogen regulatory system (Ntr), which in turn represses glutamate dehydrogenase, one pathway of glutamate production. Alanine also inhibits the enzyme glutamine synthetase, the first enzyme in the other pathway of glutamate production. Therefore, in the presence of alanine, strains with mutations in dadA (the gene that codes for a subunit of the dehydrogenase) exhibit a glutamate auxotrophy when ammonium is the sole source of Nitrogen. The alanine catabolic operon of Klebsiella aerogenes, dadAB, was cloned, and its DNA sequence was determined. The clone complemented the alanine defects of dadA strains. The operon has a high similarity to the dadAB operon of Salmonella typhimurium and the dadAX operon of Escherichia coli, each of which codes for the smaller subunit of d-amino acid dehydrogenase and the catabolic alanine racemase. Unlike the cases for E. coli and S. typhimurium, the dad operon of K. aerogenes is activated by the Ntr system, mediated in this case by the Nitrogen Assimilation control protein (NAC). A sequence matching the DNA consensus for NAC-binding sites is located centered at position −44 with respect to the start of transcription. The promoter of this operon also contains consensus binding sites for the catabolite activator protein and the leucine-responsive regulatory protein.

Alexander J Ninfa - One of the best experts on this subject based on the ideXlab platform.

  • escherichia coli pii signal transduction protein controlling Nitrogen Assimilation acts as a sensor of adenylate energy charge in vitro
    Biochemistry, 2007
    Co-Authors: Peng Jiang, Alexander J Ninfa
    Abstract:

    PII signal transduction proteins are among the most widely distributed signaling proteins in nature, controlling Nitrogen Assimilation in organisms ranging from bacteria to higher plants. PII proteins integrate signals of cellular metabolic status and interact with and regulate receptors that are signal transduction enzymes or key metabolic enzymes. Prior work with Escherichia coli PII showed that all signal transduction functions of PII required ATP binding to PII and that ATP binding was synergistic with the binding of alpha-ketoglutarate to PII. Furthermore, alpha-ketoglutarate, a cellular signal of Nitrogen and carbon status, was observed to strongly regulate PII functions. Here, we show that in reconstituted signal transduction systems, ADP had a dramatic effect on PII regulation of two E. coli PII receptors, ATase, and NRII (NtrB), and on PII uridylylation by the signal transducing UTase/UR. ADP acted antagonistically to alpha-ketoglutarate, that is, low adenylylate energy charge acted to diminish signaling of Nitrogen limitation. By individually studying the interactions that occur in the reconstituted signal transduction systems, we observed that essentially all PII and PII-UMP interactions were influenced by ADP. Our experiments also suggest that under certain conditions, the three nucleotide binding sites of the PII trimer may be occupied by combinations of ATP and ADP. In the aggregate, our results show that PII proteins, in addition to serving as sensors of alpha-ketoglutarate, have the capacity to serve as direct sensors of the adenylylate energy charge.

  • enzymological characterization of the signal transducing uridylyltransferase uridylyl removing enzyme ec 2 7 7 59 of escherichia coli and its interaction with the pii protein
    Biochemistry, 1998
    Co-Authors: Peng Jiang, James A Peliska, Alexander J Ninfa
    Abstract:

    The uridylyltransferase/uridylyl-removing enzyme (UTase/UR) of Escherichia coli plays an important role in the regulation of Nitrogen Assimilation by controlling the uridylylation state of the PII ...

  • role of the glnk signal transduction protein in the regulation of Nitrogen Assimilation in escherichia coli
    Molecular Microbiology, 1998
    Co-Authors: Mariette R Atkinson, Alexander J Ninfa
    Abstract:

    Summary Two structurally similar but functionally distinct PIIlike proteins, PII and GlnK, regulate Nitrogen Assimilation in Escherichia coli. Studies with cells indicated that both PII (the glnB product) and GlnK (the glnK product) acted through the kinase/phosphatase NRII [NtrB, the glnL (ntrB ) product] to reduce transcription initiation from Ntr promoters, apparently by regulating the phosphorylation state of the transcriptional activator NRI, P( NtrC ,P, the phosphorylated form of the glnG (ntrC ) product). Both GlnK and PII also acted through adenylyltransferase (ATase, the glnE product) to regulate the adenylylation state of glutamine synthetase (GS). The activity of both GlnK and PII was regulated by the signal-transducing uridylyltransferase/ uridylyl-removing enzyme (UTase/UR, glnD product). Our experiments indicate that either PII or GlnK could effectively regulate ATase, but that PII was required for the efficient regulation of NRII required to prevent expression of glnA, which encodes GS. Yet, GlnK also participated in regulation of NRII. Although cells that lack either PII or GlnK grew well, cells lacking both of these proteins were defective for growth on Nitrogen-rich minimal media. This defect was alleviated by the loss of NRII, and was apparently due to unregulated expression of the Ntr regulon. Also, mutations in glnK, designated glnK *, were obtained as suppressors of the Ntr π phenotype of a double mutant lacking PII and the UTase/UR. These suppressors appeared to reduce, but not eliminate, the ability of GlnK to prevent Ntr gene expression by acting through NRII. We hypothesize that one role of GlnK is to regulate the expression of the level of NRI,P during conditions of severe Nitrogen starvation, and by so doing to contribute to the regulation of certain Ntr genes.

Pablo J Pomposiello - One of the best experts on this subject based on the ideXlab platform.

  • two roles for the dna recognition site of the klebsiella aerogenes Nitrogen Assimilation control protein
    Journal of Bacteriology, 1998
    Co-Authors: Pablo J Pomposiello, Brian K Janes, Robert A. Bender
    Abstract:

    The Nitrogen Assimilation control protein (NAC) binds to a site within the promoter region of the histidine utilization operon (hutUH) of Klebsiella aerogenes, and NAC bound at this site activates transcription of hutUH. This NAC-binding site was characterized by a combination of random and directed DNA mutagenesis. Mutations that abolished or diminished in vivo transcriptional activation by NAC were found to lie within a 15-bp region contained within the 26-bp region protected by NAC from DNase I digestion. This 15-bp core has the palindromic ends ATA and TAT, and it matches the consensus for LysR family transcriptional regulators. Protein-binding experiments showed that transcriptional activation in vivo decreased with decreasing binding in vitro. In contrast to the NAC-binding site from hutUH, the NAC-binding site from the gdhA promoter failed to activate transcription from a semisynthetic promoter, and this failure was not due to weak binding or greatly distorted protein-DNA structure. Mutations in the promoter-proximal half-site of the NAC-binding site from gdhA allowed this site to activate transcription. Similar studies using the NAC-binding site from hut showed that two mutations in the promoter proximal half-site increased binding but abolished transcriptional activation. Interestingly, for symmetric mutations in the promoter-distal half-site, loss of transcriptional activation was always correlated with a decrease in binding. We conclude from these observations that if the binding in vitro reflects the binding in vivo, then binding of NAC to DNA is not sufficient for transcriptional activation and that the NAC-binding site can be functionally divided in two half-sites, with related but different functions.

  • activation of the escherichia coli lacz promoter by the klebsiella aerogenes Nitrogen Assimilation control protein nac a lysr family transcription factor
    Journal of Bacteriology, 1995
    Co-Authors: Pablo J Pomposiello, Robert A. Bender
    Abstract:

    A chimeric promoter with the Nitrogen Assimilation control protein binding site from hutUp of Klebsiella aerogenes fused to the lacZ core promoter from Escherichia coli was built and cloned in a lacZ reporter plasmid. This construct showed a 14-fold increase of beta-galactosidase activity upon Nitrogen limitation. Primer extension experiments showed that the Nitrogen Assimilation control protein activates lacZp1 in a position-dependent manner.

Gloria M Coruzzi - One of the best experts on this subject based on the ideXlab platform.

  • arabidopsis gls mutants and distinct fd gogat genes implications for photorespiration and primary Nitrogen Assimilation
    The Plant Cell, 1998
    Co-Authors: Karen Coschigano, Rosana Melooliveira, Gloria M Coruzzi
    Abstract:

    Ferredoxin-dependent glutamate synthase (Fd-GOGAT) plays a major role in photorespiration in Arabidopsis, as has been determined by the characterization of mutants deficient in Fd-GOGAT enzyme activity (gls). Despite genetic evidence for a single Fd-GOGAT locus and gene, we discovered that Arabidopsis contains two expressed genes for Fd-GOGAT (GLU1 and GLU2). Physical and genetic mapping of the gls1 locus and GLU genes indicates that GLU1 is linked to the gls1 locus, whereas GLU2 maps to a different chromosome. Contrasting patterns of GLU1 and GLU2 expression explain why a mutation in only one of the two genes for Fd-GOGAT leads to a photorespiratory phenotype in the gls1 mutants. GLU1 mRNA was expressed at the highest levels in leaves, and its mRNA levels were specifically induced by light or sucrose. In contrast, GLU2 mRNA was expressed at lower constitutive levels in leaves and preferentially accumulated in roots. Although these results suggest a major role for GLU1 in photorespiration, the sucrose induction of GLU1 mRNA in leaves also suggests a role in primary Nitrogen Assimilation. This possibility is supported by the finding that chlorophyll levels of a gls mutant are significantly lower than those of the wild type when grown under conditions that suppress photorespiration. Both the mutant analysis and gene regulation studies suggest that GLU1 plays a major role in photorespiration and also plays a role in primary Nitrogen Assimilation in leaves, whereas the GLU2 gene may play a major role in primary Nitrogen Assimilation in roots.

  • the molecular genetics of Nitrogen Assimilation into amino acids in higher plants
    Annual Review of Plant Physiology and Plant Molecular Biology, 1996
    Co-Authors: Honming Lam, Karen Coschigano, Igor C Oliveira, Rosana Melooliveira, Gloria M Coruzzi
    Abstract:

    Nitrogen Assimilation is a vital process controlling plant growth and development. Inorganic Nitrogen is assimilated into the amino acids glutamine, glutamate, asparagine, and aspartate, which serve as important Nitrogen carriers in plants. The enzymes glutamine synthetase (GS), glutamate synthase (GOGAT), glutamate dehydrogenase (GDH), aspartate aminotransferase (AspAT), and asparagine synthetase (AS) are responsible for the biosynthesis of these Nitrogen-carrying amino acids. Biochemical studies have revealed the existence of multiple isoenzymes for each of these enzymes. Recent molecular analyses demonstrate that each enzyme is encoded by a gene family wherein individual members encode distinct isoenzymes that are differentially regulated by environmental stimuli, metabolic control, developmental control, and tissue/cell-type specificity. We review the recent progress in using molecular-genetic approaches to delineate the regulatory mechanisms controlling Nitrogen Assimilation into amino acids and to define the physiological role of each isoenzyme involved in this metabolic pathway.

  • use of arabidopsis mutants and genes to study amide amino acid biosynthesis
    The Plant Cell, 1995
    Co-Authors: Honming Lam, Karen Coschigano, Igor C Oliveira, Rosana Melooliveira, Carolyn J Schultz, Gabrielle Tjaden, Nora Ngai, Minghsiun Hsieh, Gloria M Coruzzi
    Abstract:

    Studies of enzymes involved in Nitrogen Assimilation in higher plants have an impact on both basic and applied plant research. First, basic research in this area should uncover the mechanisms by which plants regulate genes involved in a metabolic pathway. Second, because Nitrogen is a rate-limiting element in plant growth (Hageman and Lambert, 1988), it may be possible to increase the yield or improve the quality of crop plants by the molecular or genetic manipulation of genes involved in Nitrogen Assimilation. Research on Nitrogen Assimilation into amino acids has been complicated by the fact that some of these reactions are catalyzed by multiple isoenzymes located in distinct subcellular compartments. With traditional biochemical approaches, it has been impossible to sort out the function of each isoenzyme in plant Nitrogen metabolism. The discovery that genes for chloroplastic and cytosolic isoenzymes of glutamine synthetase (GS) are expressed in distinct cell types (Edwards et al., 1990; Carvalhoet al., 1992; Kamachi et al., 1992)suggeststhat traditional biochemical studies, which begin with tissue disruption, artificially mix isoenzymes that may not coexist in the same cell type in vivo. Thus, in vitro biochemical methods commonly used to define the rate-limiting enzyme in a pathway in unicellular microorganisms may lead to erroneous interpretations when employed to study plant metabolic pathways. An alternative way to define the in vivo function of a particular isoenzyme or to define a rate-limiting enzyme in a pathway is by mutant analysis, as shown by studies of Escherichia coli and yeast. Plant mutants defective in particular isoenzymes of GS or ferredoxin-dependent glutamate synthase (Fd-GOGAT) have been identified in screens for photorespiratory mutants in Arabidopsis and barley (Somerville and Ogren, 1980,1982; Wallsgrove et al., 1987). More recently, Arabidopsis mutants with alterations in the activity of additional enzymes of Nitrogen Assimilation have been identified using a screening method that does not depend on a growth phenotype (Schultz and Coruzzi, 1995). The in vivo role of the mutated isoenzyme