bluuebunny/crossref_metadata_2025_split huggingface dataset:
The Chloroplast atpA Gene Cluster inChlamydomonas reinhardtii1
Authors: Drapier Dominique , Suzuki Hideki , Levy Haim , Rimbault Blandine , Kindle Karen L. , Stern David B. , Wollman Francis-André 
Published: July 2002

Abstract
Most chloroplast genes in vascular plants are organized into polycistronic transcription units, which generate a complex pattern of mono-, di-, and polycistronic transcripts. In contrast, most Chlamydomonas reinhardtii chloroplast transcripts characterized to date have been monocistronic. This paper describes the atpA gene cluster in the C. reinhardtii chloroplast genome, which includes the atpA, psbI,cemA, and atpH genes, encoding the α-subunit of the coupling-factor-1 (CF1) ATP synthase, a small photosystem II polypeptide, a chloroplast envelope membrane protein, and subunit III of the CF0 ATP synthase, respectively. We show that promoters precede the atpA,psbI, and atpH genes, but not thecemA gene, and that cemA mRNA is present only as part of di-, tri-, or tetracistronic transcripts. Deletions introduced into the gene cluster reveal, first, that CF1-α can be translated from di- or polycistronic transcripts, and, second, that substantial reductions in mRNA quantity have minimal effects on protein synthesis rates. We suggest that posttranscriptional mRNA processing is common in C. reinhardtii chloroplasts, permitting the expression of multiple genes from a single promoter.
Purification and Characterization of NAD-Isocitrate Dehydrogenase from Chlamydomonas reinhardtii1
Authors: Martı́nez-Rivas José M. , Vega JoséM. 
Published: July 2002

Abstract
NAD-isocitrate dehydrogenase (NAD-IDH) from the eukaryotic microalgaChlamydomonas reinhardtii was purified to electrophoretic homogeneity by successive chromatography steps on Phenyl-Sepharose, Blue-Sepharose, diethylaminoethyl-Sephacel, and Sephacryl S-300 (all Pharmacia Biotech). The 320-kD enzyme was found to be an octamer composed of 45-kD subunits. The presence of isocitrate plus Mn2+ protected the enzyme against thermal inactivation or inhibition by specific reagents for arginine or lysine. NADH was a competitive inhibitor (Ki, 0.14 mm) and NADPH was a noncompetitive inhibitor (Ki, 0.42 mm) with respect to NAD+. Citrate and adenine nucleotides at concentrations less than 1 mm had no effect on the activity, but 10 mm citrate, ATP, or ADP had an inhibitory effect. In addition, NAD-IDH was inhibited by inorganic monovalent anions, butl-amino acids and intermediates of glycolysis and the tricarboxylic acid cycle had no significant effect. These data support the idea that NAD-IDH from photosynthetic organisms may be a key regulatory enzyme within the tricarboxylic acid cycle.
Expression of a Soybean Gene Encoding the Tetrapyrrole-Synthesis Enzyme Glutamyl-tRNA Reductase in Symbiotic Root Nodules1
Authors: Sangwan Indu , O'Brian Mark R. 
Published: July 2002

Abstract
Heme and chlorophyll accumulate to high levels in legume root nodules and in photosynthetic tissues, respectively, and they are both derived from the universal tetrapyrrole precursor δ-aminolevulinic acid (ALA). The first committed step in ALA and tetrapyrrole synthesis is catalyzed by glutamyl-tRNA reductase (GTR) in plants. A soybean (Glycine max) root-nodule cDNA encoding GTR was isolated by complementation of anEscherichia coli GTR-defective mutant for restoration of ALA prototrophy. Gtr mRNA was very low in uninfected roots but accumulated to high levels in root nodules. The induction ofGtr mRNA in developing nodules was subsequent to that of the gene Enod2 (early nodule) and coincided with leghemoglobin mRNA accumulation. Genomic analysis revealed two Gtr genes, Gtr1 and a 3′ portion of Gtr2, which were isolated from the soybean genome. RNase-protection analysis using probes specific toGtr1 and Gtr2 showed that both genes were expressed, but Gtr1 mRNA accumulated to significantly higher levels. In addition, the qualitative patterns of expression ofGtr1 and Gtr2 were similar to each other and to total Gtr mRNA in leaves and nodules of mature plants and etiolated plantlets. The data indicate thatGtr1 is universal for tetrapyrrole synthesis and that aGtr gene specific for a tissue or tetrapyrrole is unlikely. We suggest that ALA synthesis in specialized root nodules involves an altered spatial expression of genes that are otherwise induced strongly only in photosynthetic tissues of uninfected plants.
An Arabidopsis GSK3/shaggy-Like Gene That Complements Yeast Salt Stress-Sensitive Mutants Is Induced by NaCl and Abscisic Acid
Authors: Piao Hai Lan , Pih Kyeong Tae , Lim Jeong Hwa , Kang Shin Gene , Jin Jing Bo , Kim Sung Hee , Hwang Inhwan 
Published: July 2002

Abstract
GSK3/shaggy-like genes encode kinases that are involved in a variety of biological processes. By functional complementation of the yeast calcineurin mutant strain DHT22-1a with a NaCl stress-sensitive phenotype, we isolated the Arabidopsis cDNA AtGSK1, which encodes a GSK3/shaggy-like protein kinase. AtGSK1 rescued the yeast calcineurin mutant cells from the effects of high NaCl. Also, theAtGSK1 gene turned on the transcription of the NaCl stress-inducible PMR2A gene in the calcineurin mutant cells under NaCl stress. To further define the role of AtGSK1 in the yeast cells we introduced a deletion mutation at theMCK1 gene, a yeast homolog of GSK3, and examined the phenotype of the mutant. The mck1 mutant exhibited a NaCl stress-sensitive phenotype that was rescued by AtGSK1. Also, constitutive expression of MCK1 complemented the NaCl-sensitive phenotype of the calcineurin mutants. Therefore, these results suggest that Mck1p is involved in the NaCl stress signaling in yeast and that AtGSK1 may functionally replace Mck1p in the NaCl stress response in the calcineurin mutant. To investigate the biological function of AtGSK1 in Arabidopsis we examined the expression ofAtGSK1. Northern-blot analysis revealed that the expression is differentially regulated in various tissues with a high level expression in flower tissues. In addition, theAtGSK1 expression was induced by NaCl and exogenously applied ABA but not by KCl. Taken together, these results suggest that AtGSK1 is involved in the osmotic stress response in Arabidopsis.
Autophosphorylation-Dependent Activation of a Calcium-Dependent Protein Kinase from Groundnut1
Authors: Chaudhuri Subho , Seal Anindita , Gupta Maitrayee Das 
Published: July 2002

Abstract
Ca2+-dependent protein kinases (CDPKs) containing a calmodulin-like domain integrated in their primary sequence are present primarily in plants. A member of this family was characterized from the groundnut (Arachis hypogea) plant and called GnCDPK (M. DasGupta [1994] Plant Physiol 104: 961–969). GnCDPK specifically uses the myosin light chain synthetic peptide (MLCpep), which is the phosphate-accepting domain of smooth muscle myosin light chains (KKRPQRATSNVFS), as an exogenous substrate under in vitro experimental conditions. In this report we show that GnCDPK undergoes intramolecular autophosphorylation. This self-phosphorylation occurs in threonine residues in a Ca2+-dependent (K0.5 = 0.5 μm) and calmodulin-independent manner. The kinase activity toward MLCpep and its sensitivity to Ca2+ were unaffected by prior autophosphorylation when measured under saturating ATP concentrations. The role of autophosphorylation in the exogenous substrate MLCpep phosphorylation reaction was reinvestigated at low ATP concentrations. A pronounced lag time of 1 to 2 min, followed by a linear increase of activity for 7.5 min, was seen in the initial rate of MLCpep phosphorylation under such suboptimal conditions. Prior autophosphorylation completely abolished this lag phase, and a sharp rise of exogenous substrate phosphorylation was seen from the 1st min. Our results suggest that autophosphorylation is a prerequisite for the activation of GnCDPK.
Interaction of Root Gravitropism and Phototropism in Arabidopsis Wild-Type and Starchless Mutants
Authors: Vitha Stanislav , Zhao Liming , Sack Fred David 
Published: July 2002

Abstract
Root gravitropism in wild-type Arabidopsis and in two starchless mutants,pgm1-1 and adg1-1, was evaluated as a function of light position to determine the relative strengths of negative phototropism and of gravitropism and how much phototropism affects gravitropic measurements. Gravitropism was stronger than phototropism in some but not all light positions in wild-type roots grown for an extended period, indicating that the relationship between the two tropisms is more complex than previously reported. Root phototropism significantly influenced the time course of gravitropic curvature and the two measures of sensitivity. Light from above during horizontal exposure overestimated all three parameters for all three genotypes except the wild-type perception time. At the irradiance used (80 μmol m−2 s−1), the shortest periods of illumination found to exaggerate gravitropism were 45 min of continuous illumination and 2-min doses of intermittent illumination. By growing roots in circumlateral light or by gravistimulating in the dark, corrected values were obtained for each gravitropic parameter. Roots of both starchless mutants were determined to be about three times less sensitive than prior estimates. This study demonstrates the importance of accounting for phototropism in the design of root gravitropism experiments in Arabidopsis.
Limited Correlation between Expansin Gene Expression and Elongation Growth Rate
Authors: Caderas Doina , Muster Matthias , Vogler Hannes , Mandel Therese , Rose Jocelyn K.C. , McQueen-Mason Simon , Kuhlemeier Cris 
Published: July 2002

Abstract
The aim of this work was to study the role of the cell wall protein expansin in elongation growth. Expansins increase cell wall extensibility in vitro and are thought to be involved in cell elongation. Here, we studied the regulation of two tomato (Lycopersicon esculentum cv Moneymaker) expansin genes,LeExp2 and LeExp18, in rapidly expanding tissues. LeExp2 was strongly expressed in the elongation zone of hypocotyls and in the faster growing stem part during gravitropic stimulation. LeExp18 expression did not correlate with elongation growth. Exogenous application of hormones showed a substantial auxin-stimulation of LeExp2 mRNA in etiolated hypocotyls and a weaker auxin-stimulation ofLeExp18 mRNA in stem tissue. Analysis of transcript accumulation revealed higher levels of LeExp2 andLeExp18 in light-treated, slow-growing tissue than in dark-treated, rapidly elongating tissue. Expansin protein levels and cell wall extension activities were similar in light- and dark-grown hypocotyl extracts. The results show a strong correlation between expansin gene expression and growth rate, but this correlation is not absolute. We conclude that elongation growth is likely to be controlled by expansin acting in concert with other factors that may limit growth under some physiological conditions.
Redistribution of Golgi Stacks and Other Organelles during Mitosis and Cytokinesis in Plant Cells,
Authors: Nebenführ Andreas , Frohlick Jennifer A. , Staehelin L. Andrew 
Published: July 2002

Abstract
We have followed the redistribution of Golgi stacks during mitosis and cytokinesis in living tobacco BY-2 suspension culture cells by means of a green fluorescent protein-tagged soybean α-1,2 mannosidase, and correlated the findings to cytoskeletal rearrangements and to the redistribution of endoplasmic reticulum, mitochondria, and plastids. In preparation for cell division, when the general streaming of Golgi stacks stops, about one-third of the peripheral Golgi stacks redistributes to the perinuclear cytoplasm, the phragmosome, thereby reversing the ratio of interior to cortical Golgi from 2:3 to 3:2. During metaphase, approximately 20% of all Golgi stacks aggregate in the immediate vicinity of the mitotic spindle and a similar number becomes concentrated in an equatorial region under the plasma membrane. This latter localization, the “Golgi belt,” accurately predicts the future site of cell division, and thus forms a novel marker for this region after the disassembly of the preprophase band. During telophase and cytokinesis, many Golgi stacks redistribute around the phragmoplast where the cell plate is formed. At the end of cytokinesis, the daughter cells have very similar Golgi stack densities. The sites of preferential Golgi stack localization are specific for this organelle and largely exclude mitochondria and plastids, although some mitochondria can approach the phragmoplast. This segregation of organelles is first observed in metaphase and persists until completion of cytokinesis. Maintenance of the distinct localizations does not depend on intact actin filaments or microtubules, although the mitotic spindle appears to play a major role in organizing the organelle distribution patterns. The redistribution of Golgi stacks during mitosis and cytokinesis is consistent with the hypothesis that Golgi stacks are repositioned to ensure equal partitioning between daughter cells as well as rapid cell plate assembly.
Abscisic Acid Stimulation of Phospholipase D in the Barley Aleurone Is G-Protein-Mediated and Localized to the Plasma Membrane
Authors: Ritchie Sian , Gilroy Simon 
Published: July 2002

Abstract
We have previously determined that phospholipase D (PLD) is activated by abscisic acid (ABA), and this activation is required for the ABA response of the cereal aleurone cell. In this study, ABA-stimulated PLD activity was reconstituted in vitro in microsomal membranes prepared from aleurone protoplasts. The transient nature (20 min) and degree (1.5- to 2-fold) of activation in vitro were similar to that measured in vivo. Stimulation by ABA was only apparent in the membrane fraction and was associated with a fraction enriched in plasma membrane. These results suggest that an ABA receptor system and elements linking it to PLD activation are associated with the aleurone plasma membrane. The activation of PLD in vitro by ABA was dependent on the presence of GTP. Addition of GTPγS transiently stimulated PLD in an ABA-independent manner, whereas treatment with GDPβS or pertussis toxin blocked the PLD activation by ABA. Application of pertussis toxin to intact aleurone protoplasts inhibited the ability of ABA to activate PLD as well as antagonizing the ability of ABA to down-regulate gibberellic acid-stimulated α-amylase production. All of these data support the hypothesis that ABA stimulation of PLD activity occurs at the plasma membrane and is mediated by G-protein activity.
Identification of the Maize Amyloplast Stromal 112-kD Protein as a Plastidic Starch Phosphorylase,
Authors: Yu Ying , Mu Helen He , Wasserman Bruce P. , Carman George M. 
Published: July 2002

Abstract
Amyloplast is the site of starch synthesis in the storage tissue of maize (Zea mays). The amyloplast stroma contains an enriched group of proteins when compared with the whole endosperm. Proteins with molecular masses of 76 and 85 kD have been identified as starch synthase I and starch branching enzyme IIb, respectively. A 112-kD protein was isolated from the stromal fraction by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subjected to tryptic digestion and amino acid sequence analysis. Three peptide sequences showed high identity to plastidic forms of starch phosphorylase (SP) from sweet potato, potato, and spinach. SP activity was identified in the amyloplast stromal fraction and was enriched 4-fold when compared with the activity in the whole endosperm fraction. Native and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses showed that SP activity was associated with the amyloplast stromal 112-kD protein. In addition, antibodies raised against the potato plastidic SP recognized the amyloplast stromal 112-kD protein. The amyloplast stromal 112-kD SP was expressed in whole endosperm isolated from maize harvested 9 to 24 d after pollination. Results of affinity electrophoresis and enzyme kinetic analyses showed that the amyloplast stromal 112-kD SP preferred amylopectin over glycogen as a substrate in the synthetic reaction. The maize shrunken-4 mutant had reduced SP activity due to a decrease of the amyloplast stromal 112-kD enzyme.
Maintaining Methylation Activities during Salt Stress. The Involvement of Adenosine Kinase
Authors: Weretilnyk Elizabeth A. , Alexander Kristin J. , Drebenstedt Martina , Snider Jamie D. , Summers Peter S. , Moffatt Barbara A. 
Published: July 2002

Abstract
Synthesis of the compatible osmolyte Gly betaine is increased in salt-stressed spinach (Spinacia oleracea). Gly betaine arises by oxidation of choline from phosphocholine. Phosphocholine is synthesized in the cytosol by three successiveS-adenosyl-Met-dependent N-methylations of phosphoethanolamine. With each transmethylation, a molecule ofS-adenosylhomo-Cys (SAH) is produced, a potent inhibitor of S-adenosyl-Met-dependent methyltransferases. We examined two enzymes involved in SAH metabolism: SAH hydrolase (SAHH) catabolizes SAH to adenosine plus homo-Cys and adenosine kinase (ADK) converts adenosine to adenosine monophosphate. In vitro SAHH and ADK activities increased incrementally in extracts from leaves of spinach plants subjected to successively higher levels of salt stress and these changes reflected increased levels of SAHH and ADK protein and transcripts. Another Gly betaine accumulator, sugar beet (Beta vulgaris), also showed salt-responsive increases in SAHH and ADK activities and protein whereas tobacco (Nicotiana tabacum) and canola (Brassica napus), which do not accumulate Gly betaine, did not show comparable changes in these enzymes. In spinach, subcellular localization positions SAHH and ADK in the cytosol with the phospho-base N-methyltransferase activities. Because SAHH activity is inhibited by its products, we propose that ADK is not a stress-responsive enzyme per se, but plays a pivotal role in sustaining transmethylation reactions in general by serving as a coarse metabolic control to reduce the cellular concentration of free adenosine. In support of this model, we grew Arabidopsis under a short-day photoperiod that promotes secondary cell wall development and found both ADK activity and transcript levels to increase severalfold.
Temperature-Sensitive Alleles of RSW2 Link the KORRIGAN Endo-1,4-β-Glucanase to Cellulose Synthesis and Cytokinesis in Arabidopsis
Authors: Lane Diana R. , Wiedemeier Allison , Peng Liangcai , Höfte Herman , Vernhettes Samantha , Desprez Thierry , Hocart Charles H. , Birch Rosemary J. , Baskin Tobias I. , Burn Joanne E. , Arioli Tony , Betzner Andreas S. , Williamson Richard E. 
Published: July 2002

Abstract
An 8.5-kb cosmid containing the KORRIGAN gene complements the cellulose-deficient rsw2-1 mutant of Arabidopsis. Three temperature-sensitive alleles of rsw2show single amino acid mutations in the putative endo-1,4-β-glucanase encoded by KOR. The F1 from crosses betweenkor-1 and rsw2 alleles shows a weak, temperature-sensitive root phenotype. The shoots ofrsw2-1 seedlings produce less cellulose and accumulate a short chain, readily extractable glucan resembling that reported forrsw1 (which is defective in a putative glycosyltransferase required for cellulose synthesis). The double mutant (rsw2-1 rsw1) shows further reductions in cellulose production relative to both single mutants, constitutively slow root growth, and enhanced temperature-sensitive responses that are typically more severe than in either single mutant. Abnormal cytokinesis and severely reduced birefringent retardation in elongating root cell walls of rsw2 link the enzyme to cellulose production for primary cell walls and probably cell plates. The Rsw2− phenotype generally resembles the Kor−and cellulose-deficient Rsw1− phenotypes, but anther dehiscence is impaired in Rsw2-1−. The findings link a second putative enzyme activity to cellulose synthesis in primary cell walls of Arabidopsis and further increases the parallels to cellulose synthesis in Agrobacterium tumefaciens where thecelA and celC genes are required and encode a putative glycosyltransferase and an endo-1,4-β-glucanase related to RSW1 and KOR, respectively.
Conformational and thermodynamic properties of peptide binding to the human S100P protein
Authors: Gribenko Alexey V. , Guzmán‐Casado Mercedes , Lopez Maria M. , Makhatadze George I. 
Published: July 2002

AbstractS100P is a member of the S100 subfamily of calcium‐binding proteins that are believed to be associated with various diseases, and in particular deregulation of S100P expression has been documented for prostate and breast cancer. Previously, we characterized the effects of metal binding on the conformational properties of S100P and proposed that S100P could function as a Ca2+ conformational switch. In this study we used fluorescence and CD spectroscopies and isothermal titration calorimetry to characterize the target‐recognition properties of S100P using a model peptide, melittin. Based on these experimental data we show that S100P and melittin can interact in a Ca2+‐dependent and ‐independent manner. Ca2+‐independent binding occurs with low affinity (Kd ≈ 0.2 mM), has a stoichiometry of four melittin molecules per S100P dimer and is presumably driven by favorable electrostatic interactions between the acidic protein and the basic peptide. In contrast, Ca2+‐dependent binding of melittin to S100P occurs with high affinity (Kd ≈ 5 μM) has a stoichiometry of two molecules of melittin per S100P dimer, appears to have positive cooperativity, and is driven by hydrophobic interactions. Furthermore, Ca2+‐dependent S100P‐melittin complex formation is accompanied by significant conformational changes: Melittin, otherwise unstructured in solution, adopts a helical conformation upon interaction with Ca2+‐S100P. These results support a model for the Ca2+‐dependent conformational switch in S100P for functional target recognition.
A general method for the quantitative analysis of functional chimeras: Applications from site‐directed mutagenesis and macromolecular association
Authors: Luong Tinh N. , Kirsch Jack F. 
Published: July 2002

AbstractTwo new parameters, I and C, are introduced for the quantitative evaluation of functional chimeras: I (impact) and C (context dependence) are the free energy difference and sum, respectively, of the effects on a given property measured in forward and retro chimeras. The forward chimera is made by substitution of a part “a” from ensemble A into the analogous position of homologous ensemble B (SB → A). The C value is a measure of the interaction of the interrogated position with its surroundings, whereas I is an expression of the quantitative importance of the probed position. Both I and C vary with the evaluated property, for example, kinetics, binding, thermostability, and so forth. The retro chimera is the reverse substitution of the analogous part “b” from B into A, SA → B. The I and C values derived from original data for forward and retro mutations in aspartate and tyrosine aminotransferase, from literature data for quasi domain exchange in oncomodulin and for the interaction of Tat with bovine and human TAR are evaluated. The most salient derived conclusions are, first, that Thr 109 (AATase) or Ser 109 (TATase) is an important discriminator for dicarboxylic acid selectivity by these two enzymes (I < −2.9 kcal/mol). The T109S mutation in AATase produces a nearly equal and opposite effect to S109T in TATase (C < 0.4 kcal/mol). Second, an I value of 5.5 kcal/mol describes the effects of mirror mutations D94S (site 1) and S55D (site 2) in the Ca2+ binding sites of oncomodulin on Ca2+ affinity. The second mirror set, G98D (site 1) and D59G (site 2), yields a smaller impact (I = −3.4 kcal/mol) on Ca2+ binding; however, the effect is significantly more nearly context independent (C = −0.6 versus C = −2.7 kcal/mol). Third, the stem and loop regions of HIV and BIV TAR are predominantly responsible for the species specific interaction with BIV Tat65–81 (I = −1.5 to −1.6 kcal/mol), whereas I = 0.1 kcal/mol for bulge TAR chimeras. The C values are from −0.3 to −1.2 kcal/mol. The analysis described should have important applications to protein design.