Developmental and physiological responses of Brachypodium distachyon to fluctuating nitrogen availability.

The Nitrogen Use Efficiency (NUE) of grain cereals depends on nitrate (NO3-) uptake from the soil, translocation to the aerial parts, nitrogen (N) assimilation and remobilization to the grains. Brachypodium distachyon has been proposed as a model species to identify the molecular players and mechanisms that affects these processes, for the improvement of temperate C3 cereals. We report on the developmental, physiological and grain-characteristic responses of the Bd21-3 accession of Brachypodium to variations in NO3- availability. As previously described in wheat and barley, we show that vegetative growth, shoot/root ratio, tiller formation, spike development, tissue NO3- and N contents, grain number per plant, grain yield and grain N content are sensitive to pre- and/or post-anthesis NO3- supply. We subsequently described constitutive and NO3--inducible components of both High and Low Affinity Transport Systems (HATS and LATS) for root NO3- uptake, and BdNRT2/3 candidate genes potentially involved in the HATS. Taken together, our data validate Brachypodium Bd21-3 as a model to decipher cereal N nutrition. Apparent specificities such as high grain N content, strong post-anthesis NO3- uptake and efficient constitutive HATS, further identify Brachypodium as a direct source of knowledge for crop improvement.

S. pombe cdc11p, together with sid4p, provides an anchor for septation initiation network proteins on the spindle pole body.

BACKGROUND: The signal for the onset of septum formation in the fission yeast Schizosaccharomyces pombe is transduced by the septation initiation network (SIN). Many of the components of the SIN are located on the spindle pole body during mitosis, from where it is presumed that the signal for septum formation is delivered. Cdc11 mutants are defective in SIN signaling, but the role of cdc11 in the pathway has remained enigmatic. RESULTS: We have cloned the cdc11 gene by a combination of chromosome walking and transfection of cosmids into a cdc11 mutant. Cdc11p most closely resembles Saccharomyces cerevisiae Nud1p and is essential for septum formation. Cdc11p is a phosphoprotein, which becomes hyperphosphorylated during anaphase. It localizes to the spindle pole body at all stages of the cell cycle, in a sid4p-dependent manner, and cdc11p is required for the localization of all the known SIN components, except sid4p, to the SPB. Cdc11p and sid4p can be coimmunoprecipitated from cell extracts. Finally, like its S. cerevisiae ortholog Nud1p, cdc11p is involved in the proper organization of astral microtubules during mitosis. CONCLUSIONS: We propose that cdc11p acts as a bridge between sid4p and the other SIN proteins, mediating their association with the spindle pole body.

B-Cell coactivator OBF-1 exhibits unusual transcriptional properties and functions in a DNA-bound Oct-1-dependent fashion.

Eukaryotic transcriptional activators generally comprise both a DNA-binding domain that recognizes specific cis-regulatory elements in the target genes and an activation domain which is essential for transcriptional stimulation. Activation domains typically behave as structurally and functionally autonomous modules that retain their intrinsic activities when directed to a promoter by a variety of heterologous DNA-binding domains. Here we report that OBF-1, a B-cell-specific coactivator for transcription factor Oct-1, challenges this traditional view in that it contains an atypical activation domain that exhibits two unexpected functional properties when tested in the yeast Saccharomyces cerevisiae. First, OBF-1 by itself has essentially no intrinsic activation potential, yet it strongly synergizes with other activation domains such as VP16 and Gal4. Second, OBF-1 exerts its effect in association with DNA-bound Oct-1 but is inactive when attached to a heterologous DNA-binding domain. These findings suggest that activation by OBF-1 is not obtained by simple recruitment of the coactivator to the promoter but requires interaction with DNA-bound Oct-1 to stimulate a step distinct from those regulated by classical activation domains.

The role of alpha 2M receptor/LRP in chylomicron remnant metabolism.

A strong candidate for the long-searched CR receptor might be the alpha 2MR/LRP. Presently, we are overseeing a whole series of in vitro experiments from different laboratories that show that LRP expresses all the features for being such a receptor protein. LRP is localized on the liver cell surface, as well as on most other animal cells. It recognizes apo E-enriched lipoproteins as beta-VLDL and CR. There is evidence that CR contain LPL and it has been demonstrated that LPL binds with high affinity to LRP. This has been shown in cell binding experiments with subsequent cross-linking and in direct assays on purified receptor protein. HL, which is expressed in liver cells and localized at the liver cell surface, is also able to bind to LRP. Moreover, LRP is found in endosomes and can mediate the uptake of beta-VLDL and CR. Further studies are necessary to evaluate its role in vivo as well as its regulation. The interplay between the different ligands of this large multifunctional receptor protein needs to be clarified. It should be emphasized here that, by describing LPL as a new mediator of CR uptake in the liver and by providing evidence for a direct interaction between LPL and LRP, the role of LRP in the remnant catabolism has become even more likely.

Functional complementation of the mvrA mutation of Escherichia coli by plant ferredoxin-NADP+ oxidoreductase.

Escherichia coli cells carrying the mvrA mutation are unable to grow aerobically in the presence of the radical propagator methyl viologen (MV). Resistance against MV toxicity could be restored by the introduction of cloned DNA sequences encoding pea chloroplast ferredoxin-NADP+ reductase (FNR), a member of a class of flavoenzymes involved in redox pathways in bacteria, plants and animals. Complementation was strictly dependent on the accumulation of a functional transgenic FNR, since mutated reductases showing decreased enzymatic activities only partially rescued the MV-resistant phenotype. These results support recent observations suggesting that the E. coli mvrA gene encodes a ferredoxin (flavodoxin)-NADP+ reductase (V. Bianchi et al. (1993) J. Bacteriol. 175, 1590-1595). The mvrA mutant cells showed a moderate decrease in the flavodoxin-dependent activation of enzymes essential for anaerobic growth of E. coli. This effect is prevented by expression of a functional pea FNR in the mutant cells.

Translocation of the yeast dolichol-phosphate-mannose synthase into microsomal membranes.

Dolichol-phosphate-mannose synthase catalyzes the formation of Dolichol-phosphate-mannose from Dolichol-phosphate and GDP-mannose. Analysis of the primary amino acid sequence of the yeast enzyme predicts a luminal orientation of the enzyme in the endoplasmic reticulum. We analysed the translocation of the Dolichol-phosphate-mannose synthase into dog pancreatic microsomal membranes: resistance to proteolytic attack provides evidence of its luminal orientation and asks for a reevaluation of the topology of the reaction.

Ribulose-1,5-bisphosphate carboxylase-oxygenase, other Calvin-cycle enzymes, and chlorophyll decrease when glucose is supplied to mature spinach leaves via the transpiration stream.

The inhibition of photosynthesis after supplying glucose to detached leaves of spinach (Spinacia oleracea L.) was used as a model system to search for mechanisms which potentially contribute to the "sink" regulation of photosynthesis. Detached leaves were supplied with 50 mM glucose or water for 7 d through the transpiration stream, holding the leaves in low irradiance (16 µmol photons · m(-2) · s(-1)) and a cycle of 9 h light/15 h darkness to prevent any endogenous accumulation of carbohydrate. Leaves supplied with water only showed marginal changes of photosynthesis, respiration, enzyme levels or metabolites. When leaves were supplied with 50 mM glucose, photosynthesis was gradually inhibited over several days. The inhibition was most marked when photosynthesis was measured in saturating irradiance and ambient CO2, less marked in saturating irradiance and saturating CO2, and least marked in limiting irradiance. There was a gradual loss of ribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco) protein, fructose-1,6-bisphosphatase, NADP-glyceraldehyde-3-phosphate dehydrogenase and chlorophyll. The inhibition of photosynthesis was accompanied by a large decrease of glycerate-3-phosphate, an increase of triose-phosphates and fructose-1,6-bisphospate, and a small decrease of ribulose-1,5-bisphosphate. The stromal NADPH/NADP ratio increased (as indicated by increased activation of NADP-malate dehydrogenase), and the ATP/ADP ratio increased. Chlorophyll-fluorescence analysis indicated that thylakoid energisation was increased, and that the acceptor side of photosystem II was more reduced. Similar results were obtained when glucose was supplied by floating leaf discs in low irradiance on glucose solution, and when detached spinach leaves were held in high light to produce an endogenous accumulation of carbohydrate. Feeding glucose also led to an increased rate of respiration. This was not accompanied by any changes of pyruvate kinase, phosphofructokinase, or pyrophosphate: fructose-6-phosphate phosphotransferase activity. There was a decrease of phosphoenolpyruvate, glycerate-3-phosphate and glycerate-2-phosphate, an increase of pyruvate and triose-phosphates, and an increased ATP/ADP ratio. These results show (i) that accumulation of carbohydrate can inhibit photosynthesis via a long-term mechanism involving a decrease of Rubisco and other Calvin-cycle enzymes and (ii) that respiration is stimulated due to an unknown mechanism, which increases the utilisation of phosphoenolpyruvate.

Constitutive expression of the gene for the cell-specific p48 DNA-binding subunit of pancreas transcription factor 1 in cultured cells is under control of binding sites for transcription factors Sp1 and alphaCbf.

We have cloned and characterized the rat gene that encodes the p48 DNA-binding subunit of pancreas transcription factor 1 (Ptf1), a cell-specific basic region helix-loop-helix (bHLH) protein. The ptf1-p48 gene measures 1.8 kilobases in size and occurs as a single copy in the haploid genome. Run-on transcription assays suggest that this gene is subject to transcriptional control since no activity of its promoter is detected in nonproducing cells. The gene specifies two mRNAs that encode the same protein and originate from transcription initiation at alternative sites. Expression analysis of hybrid genes bearing deletions of the gene's 5'-flanking region fused to a reporter gene defines a promoter region within the gene-proximal 260 base pairs of DNA. The cis-acting elements that control promoter activity include binding sites for transcription factors Sp1 and alphaCbf, a 60-kDa CCAAT box-binding protein. The gene promoter, however, functions not only in exocrine pancreatic cells but also in cells of other origin. No cell-specific transcriptional control element was detected in as much as 10 kilobases of 5'-flanking region. We discuss models of how the cell-specific expression of the endogenous ptf1-p48 gene might be established during development of the animal.

Methylammonium-resistant mutants of Nicotiana plumbaginifolia are affected in nitrate transport.

This work reports the isolation and preliminary characterization of Nicotiana plumbaginifolia mutants resistant to methylammonium. Nicotiana plumbaginifolia plants cannot grow on low levels of nitrate in the presence of methylammonium. Methylammonium is not used as a nitrogen source, although it can be efficiently taken up by Nicotiana plumbaginifolia cells and converted into methylglutamine, an analog of glutamine. Glutamine is known to repress the expression of the enzymes that mediate the first two steps in the nitrate assimilatory pathway, nitrate reductase (NR) and nitrite reductase (NiR). Methylammonium has therefore been used, in combination with low concentrations of nitrate, as a selective agent in order to screen for mutants in which the nitrate pathway is de-repressed. Eleven semi-dominant mutants, all belonging to the same complementation group, were identified. The mutant showing the highest resistance to methylammonium was not affected either in the utilization of ammonium, accumulation of methylammonium or in glutamine synthase activity. A series of experiments showed that utilization of nitrite by the wild-type and the mutant was comparable, in the presence or the absence of methylammonium, thus suggesting that the mutation specifically affected nitrate transport or reduction. Although NR mRNA levels were less repressed by methylammonium treatment of the wild-type than the mutant, NR activities of the mutant remained comparable with or without methylammonium, leading to the hypothesis that modified expression of NR is probably not responsible for resistance to methylammonium. Methylammonium inhibited nitrate uptake in the wild-type but had only a limited effect in the mutant. The implications of these results are discussed.

One-step purification of plant ferredoxin-NADP+ oxidoreductase expressed in Escherichia coli as fusion with glutathione S-transferase.

Complementary DNA sequences encoding the mature form of pea ferredoxin-NADP+ reductase were cloned in-frame at the 3' end of the Schistosoma japonicum glutathione S-transferase gene in the expression vector pGEX-3X (Smith and Johnson, Gene 67, 31-40, 1988). A spacer sequence linking the two genes was modified to provide a proteolytic site just before the first amino acid residue of mature pea reductase. When introduced into competent Escherichia coli cells and induced, the resulting plasmid (pGF205) directed the expression of a 60-kDa immunoreactive peptide that results from the fusion between glutathione S-transferase and ferredoxin-NADP+ reductase sequences. The fused protein could be purified in a single step by selective absorption onto glutathione-agarose beads, followed by elution with free glutathione. It showed both transferase and reductase activities. Removal of the transferase portion by cleavage with the restriction protease Xa rendered ferredoxin-NADP+ reductase electrophoretically homogeneous. The purified transgenic enzyme showed kinetic and spectroscopic properties that were similar to those reported for the plant flavoprotein, indicating that, even when fused to the 27-kDa transferase portion, the reductase was still able to assemble FAD and to acquire an active conformation in the bacterial host. The expression-purification protocol employed here allows the isolation of up to 1 mg of active ferredoxin-NADP+ reductase/g of transformed cells. The system is potentially useful for the purification of activity-impaired forms of the flavoprotein.

Expression, assembly, and processing of an active plant ferredoxin-NADP+ oxidoreductase and its precursor protein in Escherichia coli.

The flavoprotein ferredoxin-NADP+ reductase (FNR) catalyzes the final step of the photosynthetic electron transport chain, i.e. the reduction of NADP+ by ferredoxin. A cloned FNR cDNA from a pea library (Newman, B., and Gray, J. (1988) Plant Mol. Biol. 10, 511-520) was used to construct plasmids which express the apoenzyme in Escherichia coli. Two recombinant vectors were prepared, one containing the sequence corresponding to the mature enzyme and another including, in addition, the sequence of the transit peptide that directs FNR to the chloroplast. These proteins were expressed as fusion products to the NH2-terminal portion of beta-galactosidase. In both cases, a 35-kDa immunoreactive polypeptide was the major product, suggesting that the proteins were processed in vivo. NH2-terminal sequence determination of the purified recombinant proteins indicate cleavage at positions -1/-2 with respect to the normal processing site in chloroplasts. The processed enzymes showed enzymatic activities and spectral properties that were similar or identical to those of native plant FNR. When a La protease-deficient E. coli strain was used as a host, the expressed FNR precursor was found to be poorly processed, associated to bacterial pellets, and showed no detectable FNR activity. The overall results indicate that acquisition of the native enzyme conformation and assembly of the prosthetic group takes place in the bacterial host, generating an enzyme that is, as far as studied, indistinguishable from plant FNR.

The p48 DNA-binding subunit of transcription factor PTF1 is a new exocrine pancreas-specific basic helix-loop-helix protein.

We report the isolation of cDNA for the p48 DNA-binding subunit of the heterooligomeric transcription factor PTF1. A sequence analysis of the cDNA demonstrates that p48 is a new member of the family of basic helix-loop-helix (bHLH) transcription factors. The p48 bHLH domain shows striking amino acid sequence similarity with the bHLH domain of proteins that act as developmental regulators, including the twist gene product, myogenic factors and proteins involved in hematopoietic differentiation. We show that reduced p48 synthesis correlates with a diminished expression of genes encoding exocrine pancreas-specific functions. The synthesis of p48 mRNAs, and therefore also the protein, is restricted to cells of the exocrine pancreas in the adult and to the pancreatic primordium in the embryo. Thus the pancreas-specific DNA-binding activity of PTF1 originates from the synthesis of at least one cell-specific component rather than from a cell-specific assembly of more widely distributed proteins.

The role of lipases and LRP in the catabolism of triglyceride-rich lipoproteins.

A strong candidate for the long searched CR receptor might be the a2MR/LRP. We oversee a whole series of in vitro experiments from different laboratories today which show that LRP expresses all features for being such a receptor protein. LRP is localized on the liver cell surface, as well as on most other animal cells. It recognizes apo E enriched lipoproteins, as beta-VLDL and CR. There is evidence that CR contains LPL and it has been demonstrated that LPL binds with high affinity to LRP. This has been shown in cell binding experiments with subsequent cross-linking and in direct binding assays on purified receptor protein. HL which is expressed in liver cells and localized at the liver cell surface is also able to bind to LRP. LRP is moreover found in endosomes and can mediate the uptake of beta-VLDL and CR. Further studies are necessary to evaluate its role in vivo as well as its regulation. The interplay between the different ligands of this large multifunctional receptor protein needs to be clarified. It should be emphasized here that by describing LPL as a new mediator of CR untake in the liver and providing evidence for an interaction between LPL and LRP the role of LRP in the remnant catabolism has become even more likely.

The role of ferredoxin-NADP+ reductase in the concerted cell defense against oxidative damage -- studies using Escherichia coli mutants and cloned plant genes.

Ferredoxin-NADP+ reductases (FNR) participate in cellular defense against oxidative damage. Escherichia coli mutants deficient in FNR are abnormally sensitive to methyl viologen and hydrogen peroxide. Tolerance to these oxidants was regained by expression of plant FNR, superoxide dismutase, or catalase genes in the mutant cells. FNR contribution to the concerted defense against viologen toxicity under redox-cycling conditions was similar to that of the two major E. coli superoxide dismutases together, as judged by the phenotypes displayed by relevant mutant strains. However, FNR expression in sodA sodB strains failed to increase their tolerance to viologens, indicating that the FNR target is not the superoxide radical. Sensitivity of FNR-deficient cells to oxidants is related to extensive DNA damage. Incubation of the mutant bacteria with iron chelators or hydroxyl radical scavengers provided significant protection against viologens or peroxide, suggesting that oxidative injury in FNR-deficient cells was mediated by intracellular iron through the formation of hydroxyl radicals in situ. The NADP(H)-dependent activities of the reductase were necessary and sufficient for detoxification, without participation of either ferredoxin or flavodoxin in the process. Possible mechanisms by which FNR may exert its protective role are discussed.

Growth of tobacco in short-day conditions leads to high starch, low sugars, altered diurnal changes in the Nia transcript and low nitrate reductase activity, and inhibition of amino acid synthesis.

Diurnal changes in carbohydrates and nitrate reductase (NR) activity were compared in tobacco (Nicotiana tabacum. L.cv. Gatersleben) plants growing in a long (18 h light/6 h dark) and a short (6 h light/18 h dark) day growth regime, or after short-term changes in the light regime. In long-day-grown plants, source leaves contained high levels of sugars throughout the light and dark periods. In short-day-grown plants, levels of sucrose and reducing sugars were very low at the end of the night and, although they rose during the light period, remained much lower than in long days and declined to very low levels again by the middle of the night. Starch accumulated more rapidly in short-day-than long-day-grown plants. Starch was completely remobilised during the night in short days, but not in long days. A single short day/long night cycle sufficed to stimulate starch accumulation during the following light period. In long-day-grown plants, the Nia transcript level was high at the end of the night, decreased during the day, and recovered gradually during the night. In short-day-grown plants, the Nia transcript level was relatively low at the end of the night, decreased to very low levels at the end of the light period, increased to a marked maximum in the middle of the night, and decreased during the last 5 h of the dark period. In long-day-grown plants, NR activity in source leaves rose by 2- to 3-fold in the first part of the light period and decreased in the second part of the light period. In short-day-grown plants, NR activity was low at the end of the night, and only increased slightly after illumination. Dark inactivation of source-leaf NR was partially reversed in long-day-grown plants, but not in short day-grown plants. In both growth regimes, mutants with one instead of four functional copies of the Nia gene had a 60% reduction in maximum NR activity in the source leaves, compared to wild-type plants. The diurnal changes in NR activity were almost completely suppressed in the mutants in long days, whereas the mutants showed similar or slightly larger diurnal changes than wild-type plants in short days. When short-day-grown plants were transferred to long-day conditions for 3 d, NR activity and the diurnal changes in NR activity resembled those in long-day-grown plants. Phloem export from source leaves of short-day-grown plants was partially inhibited by applying a cold-girdle for one light and dark cycle. The resulting increase in leaf sugar was accompanied by an marked increase in the Nia transcript level and a 2-fold increase in NR activity at the end of the dark period. When wild-type plants were subjected to a single short day/long night cycle of increasing severity, NR activity in source leaves at the end of the night decreased when the endogenous sugars declined below about 3 mumol hexose (g FW)-1. In sink leaves in short-day conditions, sugars were higher and the light-induced rise in NR activity was much larger than in source leaves on the same plants. The source leaves of wild-type plants in short-day conditions contained very high levels of nitrate, very low levels of glutamine, low levels of total amino acids, and lower protein and chlorophyll, compared to long-day-grown plants. Plants grown in short days had relatively high levels of glutamate and aspartate, and extremely low levels of most of the minor amino acids in their source leaves at the end of the night. Illumination led to a decrease in glutamate and an increase in the minor amino acids. A single short day/long night cycle led to an increase in glutamate, and a large decrease in the minor acids at the end of the dark period, and reillumination led to a decrease in glutamate and an increase in the minor amino acids. It is proposed that sugar-mediated control of Nia expression and NR activity overrides regulation by nitrogenous compounds when sugars are in short supply, resulting in a severe inhibition of nitrate assimilation. It is also proposed that su

Conformational requirements of a recombinant ferredoxin-NADP+ reductase precursor for efficient binding to and import into isolated chloroplasts.

The cytosolic precursor of the chloroplast flavoprotein ferredoxin-NADP+ reductase was expressed in Escherichia coli rendering a soluble protein that contained bound FAD and could be imported by isolated chloroplasts. The mechanism of plastid translocation was studied under defined conditions using this recombinant precursor holoprotein and intact pea chloroplasts. The first step in the import pathway, namely, binding of the reductase precursor to isolated chloroplasts, was saturable at about 2000 molecules/plastid, and showed a high-affinity interaction with a dissociation constant Kd of approximately 5 nM. Binding was not affected by the addition of soluble leaf extracts or by prior denaturation of the precursor with urea. Analysis of the initial import rates at different precursor concentrations indicated the existence of a single translocation system for this protein. Inclusion of leaf extracts in the assay resulted in a three-fold increase of the maximal import rates to 14,000 molecules . min-(1).chloroplast-(1), with a concomitant decrease in the apparent Km for the recombinant precursor, from 1 microM to 100-150 nM. Comparison of Km and Kd values under various conditions indicated that the binding step of the translocation process is largely irreversible, favouring import and processing. In the absence of extract, a denatured precursor obtained by incubation with urea was a better substrate for plastid import than the holoprotein. Treatment of the precursor with either extract or urea resulted in similar increases in import efficiency (V/Km), suggesting that stimulation by leaf extracts is probably related to unfolding of the precursor prior to translocation.

Methicillin resistance study in clinical isolates of coagulase-negative staphylococci and determination of their susceptibility to alternative antimicrobial agents.

AIMS: To achieve reliable detection of methicillin resistance in clinical isolates of coagulase-negative staphylococci. METHODS AND RESULTS: Strains (105) were evaluated by normatized antimicrobial susceptibility methods, and for the presence of the methicillin resistance-determining mecA gene, using the polymerase chain reaction. Correlation between phenotypic and genotypic methods was obtained in 87.6% of the samples. Six strains, classified as methicillin-susceptible by phenotypic assays, revealed the presence of the mecA gene, indicating that methicillin resistance expression was probably repressed. Another seven isolates failed to show mecA amplification after displaying methicillin resistance in phenotypic evaluations. The susceptibility of the methicillin-resistant isolates to other antimicrobial agents was variable. CONCLUSION: Genotypic determination of the mecA gene proved to be the most reliable method for detection of methicillin resistance. SIGNIFICANCE AND IMPACT OF THE STUDY: Correct assessment of methicillin resistance, such as that attained through genotyping, is essential for defining therapeutic strategies, particularly when treating severely compromised patients.

The precursor of pea ferredoxin-NADP+ reductase synthesized in Escherichia coli contains bound FAD and is transported into chloroplasts.

The precursor of the chloroplast flavoprotein ferredoxin-NADP+ reductase from pea was expressed in Escherichia coli as a carboxyl-terminal fusion to glutathione S-transferase. The fused protein was soluble, and the precursor could be purified in a few steps involving affinity chromatography on glutathione-agarose, cleavage of the transferase portion by protease Xa, and ion exchange chromatography on DEAE-cellulose. The purified prereductase contained bound FAD but displayed marginally low levels of activity. Removal of the transit peptide by limited proteolysis rendered a functional protease-resistant core exhibiting enzymatic activity. The FAD-containing precursor expressed in E. coli was readily transported into isolated pea chloroplasts and was processed to the mature size, both inside the plastid and by incubation with stromal extracts in a plastid-free reaction. Import was dependent on the presence of ATP and was stimulated severalfold by the addition of plant leaf extracts.

Assembly of plant ferredoxin-NADP+ oxidoreductase in Escherichia coli requires GroE molecular chaperones.

We have recently reported the expression in Escherichia coli of an enzymatically competent ferredoxin-NADP+ oxidoreductase from cloned pea genes encoding either the mature enzyme or its precursor protein (Ceccarelli, E. A., Viale, A. M., Krapp, A. R., and Carrillo, N. (1991) J. Biol. Chem. 266, 14283-14287). Processing to the mature form by bacterial protease(s) and FAD assembly occurred in the bacterial cytosol. Expression of ferredoxin-NADP+ reductase in chaperonin-deficient (groE-) mutants of E. coli resulted in partial reductase assembly at permissive growth temperatures (i.e. 30 degrees C), and in total breakdown of holoenzyme assembly, and accumulation as aggregated inclusion bodies at non-permissive temperatures (i.e. 42 degrees C). Coexpression in these mutants of a cloned groESL operon from the phototrophic bacterium Chromatium vinosum resulted in partial or total recoveries of ferredoxin-NADP+ reductase assembly. The overall results indicate that bacterial chaperonins are required for the productive folding/assembly of eucaryotic ferredoxin-NADP+ reductase expressed in E. coli.

PCR-identification of a Nicotiana plumbaginifolia cDNA homologous to the high-affinity nitrate transporters of the crnA family.

A family of high-affinity nitrate transporters has been identified in Aspergillus nidulans and Chlamydomonas reinhardtii, and recently homologues of this family have been cloned from a higher plant (barley). Based on six of the peptide sequences most strongly conserved between the barley and C. reinhardtii polypeptides, a set of degenerate primers was designed to permit amplification of the corresponding genes from other plant species. The utility of these primers was demonstrated by RT-PCR with cDNA made from poly(A)+ RNA from barley, C. reinhardtii and Nicotiana plumbaginifolia. A PCR fragment amplified from N. plumbaginifolia was used as probe to isolate a full-length cDNA clone which encodes a protein, NRT2;1Np, that is closely related to the previously isolated crnA homologue from barley. Genomic Southern blots indicated that there are only 1 or 2 members of the Nrt2 gene family in N. plumbaginifolia. Northern blotting showed that the Nrt2 transcripts are most strongly expressed in roots. The effects of external treatments with different N sources showed that the regulation of the Nrt2 gene(s) is very similar to that reported for nitrate reductase and nitrite reductase genes: their expression was strongly induced by nitrate but was repressed when reduced forms of N were supplied to the roots.