bluuebunny/crossref_metadata_2025 huggingface dataset:
Loss of Heterozygosity Assay for Molecular Detection of Cancer Using Energy-transfer Primers and Capillary Array Electrophoresis
Authors: Medintz Igor L. , Lee Chyi-Chia Richard , Wong Wendy W. , Pirkola Kristin , Sidransky David , Mathies Richard A.
Published: July 2002

Microsatellite DNA loci are useful markers for the detection of loss of heterozygosity (LOH) and microsatellite instability (MI) associated with primary cancers. To carry out large-scale studies of LOH and MI in cancer progression, high-throughput instrumentation and assays with high accuracy and sensitivity need to be validated. DNA was extracted from 26 renal tumor and paired lymphocyte samples and amplified with two-color energy-transfer (ET) fluorescent primers specific for loci associated with cancer-induced chromosomal changes. PCR amplicons were separated on the MegaBACE-1000 96 capillary array electrophoresis (CAE) instrument and analyzed with MegaBACE Genetic Profiler v.1.0 software. Ninety-six separations were achieved in parallel in 75 minutes. Loss of heterozygosity was easily detected in tumor samples as was the gain/loss of microsatellite core repeats. Allelic ratios were determined with a precision of  ± 10% or better. Prior analysis of these samples with slab gel electrophoresis and radioisotope labeling had not detected these changes with as much sensitivity or precision. This study establishes the validity of this assay and the MegaBACE instrument for large-scale, high-throughput studies of the molecular genetic changes associated with cancer.
Efficiency of Database Search for Identification of Mutated and Modified Proteins via Mass Spectrometry
Authors: Pevzner Pavel A. , Mulyukov Zufar , Dancik Vlado , Tang Chris L
Published: July 2002

Although protein identification by matching tandem mass spectra (MS/MS) against protein databases is a widespread tool in mass spectrometry, the question about reliability of such searches remains open. Absence of rigorous significance scores in MS/MS database search makes it difficult to discard random database hits and may lead to erroneous protein identification, particularly in the case of mutated or post-translationally modified peptides. This problem is especially important for high-throughput MS/MS projects when the possibility of expert analysis is limited. Thus, algorithms that sort out reliable database hits from unreliable ones and identify mutated and modified peptides are sought. Most MS/MS database search algorithms rely on variations of the Shared Peaks Count approach that scores pairs of spectra by the peaks (masses) they have in common. Although this approach proved to be useful, it has a high error rate in identification of mutated and modified peptides. We describe new MS/MS database search tools, MS-CONVOLUTION andMS-ALIGNMENT, which implement the spectral convolution and spectral alignment approaches to peptide identification. We further analyze these approaches to identification of modified peptides and demonstrate their advantages over the Shared Peaks Count. We also use the spectral alignment approach as a filter in a new database search algorithm that reliably identifies peptides differing by up to two mutations/modifications from a peptide in a database.
Sequence-Based Design of Single-Copy Genomic DNA Probes for Fluorescence In Situ Hybridization
Authors: Rogan Peter K. , Cazcarro Patricia M. , Knoll Joan H.M.
Published: July 2002

Chromosomal rearrangements are frequently monitored by fluorescence in situ hybridization (FISH) using large, recombinant DNA probes consisting of contiguous genomic intervals that are often distant from disease loci. We developed smaller, targeted, single-copy probes directly from the human genome sequence. These single-copy FISH (scFISH) probes were designed by computational sequence analysis of ∼100-kb genomic sequences. ScFISH probes are produced by long PCR, then purified, labeled, and hybridized individually or in combination to human chromosomes. Preannealing or blocking with unlabeled, repetitive DNA is unnecessary, as scFISH probes lack repetitive DNA sequences. The hybridization results are analogous to conventional FISH, except that shorter probes can be readily visualized. Combinations of probes from the same region gave single hybridization signals on metaphase chromosomes. ScFISH probes are produced directly from genomic DNA, and thus more quickly than by recombinant DNA techniques. We developed single-copy probes for three chromosomal regions—the CDC2L1 (chromosome 1p36), MAGEL2(chromosome 15q11.2), and HIRA (chromosome 22q11.2) genes—and show their utility for FISH. The smallest probe tested was 2290 bp in length. To assess the potential utility of scFISH for high-resolution analysis, we determined chromosomal distributions of such probes. Single-copy intervals of this length or greater are separated by an average of 29.2 and 22.3 kb on chromosomes 21 and 22, respectively. This indicates that abnormalities seen on metaphase chromosomes could be characterized with scFISH probes at a resolution greater than previously possible.
Physical Mapping of a 670-kb Region of Chromosomes XVI and XVII from the Human Protozoan Parasite Trypanosoma cruzi Encompassing the Genes for Two Immunodominant Antigens
Authors: Santos Marcia R.M. , Lorenzi Hernan , Porcile Patricio , Carmo Mirian S. do , Schijman Alejandro , Brandão Adeilton , Araya Jorge E. , Gomes Henrique B.M. , Chiurillo Miguel A. , Ramirez José L. , Degrave Wim M. , Levin Mariano J. , da Silveira José Franco
Published: July 2002

As part of the Trypanosoma cruzi Genome Initiative, we have mapped a large portion of the chromosomal bands XVI (2.3 Mb) and XVII (2.6 Mb) containing the highly repetitive and immunodominant antigenic gene families h49 and jl8. Restriction mapping of the isolated chromosomal bands and hybridization with chromosome specific gene probes showed that genes h49 and jl8 are located in a pair of size-polymorphic homologous chromosomes. To construct the integrated map of the chromosomes harboring the h49 andjl8 loci, we used YAC, cosmid, and λ phage overlapping clones, and long range restriction analysis using a variety of probes (i.e., known gene sequences, ESTs, polymorphic repetitive sequences, anonymous sequences, STSs generated from the YAC ends). The total length covered by the YAC contig was approximately 670 kb, and its map agreed and was complementary to the one obtained by long-range restriction fragment analysis. Average genetic marker spacing in a 105 kb region around h49 and jl8 genes was estimated to be 6.2 kb/marker. We have detected some polymorphism in the H49/JL8 antigens-encoding chromosomes, affecting also the coding regions. The physical map of this region, together with the isolation of specific chromosome markers, will contribute in the global effort to sequence the nuclear genome of this parasite.
Automated Assessment of Conditioning Parameters for Context and Cued Fear in Mice
Authors: Contarino Angelo , Baca Leonardo , Kennelly Arthur , Gold Lisa H.
Published: July 2002

A behavioral technique often used to evaluate the cognitive performance of rats and mice is the fear conditioning paradigm. During conditioned fear experiments, freezing responses shown by rodents after exposure to environmental stimuli previously paired to an aversive experience provide a behavioral index of the animal's associative abilities. The present study examined the ability of a computer-controlled automated Freeze Monitor system for recording immobility behavior in mice. The sensitivity of the automated procedure to detect group differences caused by the application of various training protocols was also evaluated. Statistical analyses revealed significant positive correlations between immobility scores obtained with the automated apparatus and hand-scored data collected by a continuous or a time-sampling method. Behavioral patterns recorded by the computerized system were very similar to those obtained by the hand-scoring methods adopted. In particular, during context testing, exposure to environmental stimuli previously paired with a mild foot shock (unconditioned stimulus [US]) evoked increased immobility behavior in mice conditioned with the US compared with levels of immobility displayed by mice previously confined to the same contextual stimuli without receiving the US. Moreover, although during conditioned stimulus (CS) testing, mice previously exposed to the US displayed high levels of immobility when confined to environmental cues much different from those paired with the US (contextual fear generalization), both hand-scored and automated results revealed the effect of CS–US pairing (increased immobility) only in mice trained to associate the two stimuli (paired group) but not in mice exposed to both CS and US separated by a 40-sec time interval (unpaired group) or in mice receiving only the US (US group) during conditioning sessions. Overall, the results show associative conditioning measured in an automated apparatus and highlight the utility of obtaining both latency as well as beam interruption parameters.
Polysynaptic Potentiation at Different Levels of Rat Olfactory Pathways Following Learning
Authors: Mouly Anne Marie , Gervais Rémi
Published: July 2002

This study was aimed at investigating the consequences of learning on late polysynaptic components of evoked field potential signals recorded in parallel at different levels of the olfactory pathways. For this, evoked field potentials induced by electrical stimulation of the olfactory bulb were recorded simultaneously in the anterior piriform cortex, the posterior piriform cortex, the lateral entorhinal cortex, and the dentate gyrus. The different parameters of late components were measured in each site before and after completion of associative learning in anesthetized rats. In the learning task, rats were trained to associate electrical stimulation of one olfactory bulb electrode with the delivery of sucrose (positive reward) and stimulation of a second olfactory bulb electrode with the delivery of quinine (negative reward). In this way, stimulation of the same olfactory bulb electrodes used for inducing field potentials served as a discriminative cue in the learning paradigm. The data confirmed previous observation that learning was associated with a lowering in late-component-1 intensity of induction in the posterior piriform cortex. The use of simultaneous recording allowed us to further specify the consequences of learning on late-component distribution in the studied network. Indeed the data showed that whereas before learning, late component 1 was rather uniformly distributed among the recorded sites; following learning, its expression was facilitated preferentially in the posterior piriform cortex and lateral entorhinal cortex. Furthermore, learning was accompanied by the emergence of a new late component (late component 2), which occurred simultaneously in the four recording sites. The possible involvement of potentiation of polysynaptic components in recognition and/or consolidation processes will be discussed.
Learning Motor Synergies Makes Use of Information on Muscular Load
Authors: Fernández-Ruiz Juan , Hall-Haro Cynthia , Díaz Rosalinda , Mischner Jacqueline , Vergara Patricia , Lopez-Garcia Juan Carlos
Published: July 2002

Prism adaptation, a form of procedural learning, requires the integration of visual and motor information for its proper acquisition. Although the role of the visual feedback has begun to be understood, the nature of the motor information necessary for the development of the adaptation remains unknown. In this work we have tested the idea that modifying the arm load at different stages of the adaptation process, and the ensuing change of motor information perceived by the subjects, would modify the final properties of the adaptation. We trained a set of subjects to throw balls to a target while wearing prism glasses and varied the weight of their arms at different time points during the task. We observed that the acquisition of the adaptation was not affected by the change in load. However, its persistence (i.e., the aftereffect) was reduced when tested under a weight condition different from the training trials. Furthermore, when the training weight conditions were restored later during testing, a second, late aftereffect was unmasked, suggesting that the missing aftereffect did not disappear but had remained latent. Our results show that the internal representation of a motor memory incorporates information about load conditions and that the memory stored under a specific weight condition can be fully retrieved only when the original training condition is restored.
Ancestral Multipartite Units in Light-Responsive Plant Promoters Have Structural Features Correlating with Specific Phototransduction Pathways
Authors: Arguello-Astorga G. R. , Herrera-Estrella L. R.
Published: July 2002

Regulation of plant gene transcription by light is mediated by multipartite cis-regulatory units. Previous attempts to identify structural features that are common to all light-responsive elements (LREs) have been unsuccessful. To address the question of what is needed to confer photoresponsiveness to a promoter, the upstream sequences from more than 110 light-regulated plant genes were analyzed by a new, phylogenetic-structural method. As a result, 30 distinct conserved DNA module arrays (CMAs) associated with light-responsive promoter regions were identified. Several of these CMAs have remained invariant throughout the evolutionary radiation of angiosperms and are conserved between homologous genes as well as between members of different gene families. The identified CMAs share a gene superfamily-specific core that correlates with the particular phytochrome-dependent transduction pathway that controls their expression, i.e. ACCTA(A/C)C(A/C) for the cGMP-dependent phenylpropanoid metabolism-associated genes, and GATA(A/T)GR for the Ca2+/calmodulin-dependent photosynthesis-associated nuclear genes. In addition to suggesting a general model for the functional and structural organization of LREs, the data obtained in this study indicate that angiosperm LREs probably evolved from complex cis-acting elements involved in regulatory processes other than photoregulation in gymnosperms.
Sugar Regulates mRNA Abundance of H+-ATPase Gene Family Members in Tomato
Authors: Mito N. , Wimmers L. E. , Bennett A. B.
Published: July 2002

The plant plasma membrane H+-ATPase energizes the secondary uptake of nutrients and may facilitate cell expansion by acidifying the cell wall. In yeast, Glc stimulates the accumulation of H+-ATPase mRNA, and the growth rate supported by various sugars is correlated with H+-ATPase protein abundance. Expression of three H+-ATPase genes, LHA1, LHA2, and LHA4, was previously detected in tomato (Lycopersicon esculentum). We have characterized the sequence of the LHA4 gene and examined the expression of these three tomato H+-ATPase genes in growing tissues and in response to exogenous sugars. LHA4 is a member of the H+-ATPase subfamily, including the Arabidopsis thaliana genes AHA1, AHA2, and AHA3. The 5[prime] untranslated region of the deduced LHA4 cDNA contains a short, open reading frame very similar to that in the Nicotiana plumbaginifolia gene PMA1. LHA4 transcript abundance in seedlings is correlated with cell growth, being 2.5 times greater in hypocotyls of dark- versus light-grown plants. The accumulation of both LHA4 and LHA2 mRNAs is induced by the addition of exogenous sugars and this induction appears to be dependent on sugar uptake and metabolism, because mannitol and 3–O-methylglucose do not stimulate mRNA acumulation. These results suggest that the induction of expression of H+-ATPase genes by metabolizable sugars may be part of a generalized cellular response to increased cell growth and metabolism promoted by the availability of an abundant carbon source.
Evidence for Transcriptional Regulation of Plastid Photosynthesis Genes in Arabidopsis thaliana Roots
Authors: Isono K. , Niwa Y. , Satoh K. , Kobayashi H.
Published: July 2002

Mechanisms underlying suppressed levels of transcripts for plastid photosynthesis genes in nongreen tissues such as roots and calli were analyzed in Arabidopsis thaliana, a plant suitable for further genetic dissection. A region encoding promoters of rbcL, the gene encoding the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase, and the atpB/E operon for the [beta] and [epsilon] subunits of coupling factor one were cloned and sequenced. Transcripts for rbcL, atpB/E, and psbA, the gene for the D1 protein in the photosystem II reaction center, were barely detectable in roots of A. thaliana, whereas 16S rRNA was detected at a low level. The run-on transcription experiment revealed that expression of rbcL, atpB/E, and psbA was regulated at transcription. The copy number of plastid DNA in roots was one-fifth that in green leaves on the basis of total cellular DNA, suggesting that in the latter the DNA copy-number regulation also exists in plastid gene expression. Digestion of DNA with methyl-sensitive and -insensitive isoschizomeric endonucleases and subsequent polymerase chain reaction, as well as in vitro transcription of plastid DNAs with Escherichia coli RNA polymerase, resulted in no evidence of regulation by DNA modification. In spite of predominant suppression of expression of rbcL, atpB/E, and psbA at transcription in roots and calli, 16S rRNA levels were decreased because of low RNA stability.
Cytoplasmic Free Ca2+ in Arabidopsis Roots Changes in Response to Touch but Not Gravity
Authors: Legue V. , Blancaflor E. , Wymer C. , Perbal G. , Fantin D. , Gilroy S.
Published: July 2002

Changes in cytoplasmic Ca2+ concentration ([Ca2+]i) have been proposed to be involved in signal transduction pathways in response to a number of stimuli, including gravity and touch. The current hypothesis proposes that the development of gravitropic bending is correlated with a redistribution of [Ca2+]i in gravistimulated roots. However, no study has demonstrated clearly the development of an asymmetry of this ion during root curvature. We tested this hypothesis by quantifying the temporal and spatial changes in [Ca2+]i in roots of living Arabidopsis seedlings using ultraviolet-confocal Ca2+-ratio imaging and vertical stage fluorescence microscopy to visualize root [Ca2+]i. We observed no changes in [Ca2+]i associated with the graviresponse whether monitored at the whole organ level or in individual cells in different regions of the root for up to 12 h after gravistimulation. However, touch stimulation led to transient increases in [Ca2+]i in all cell types monitored. The increases induced in the cap cells were larger and longer-lived than in cells in the meristematic or elongation zone. One millimolar La3+ and 100 [mu]M verapamil did not prevent these responses, whereas 5 mM EGTA or 50 [mu]M ruthenium red inhibited the transients, indicating an intracellular origin of the Ca2+ increase. These results suggest that, although touch responses of roots may be mediated through a Ca2+-dependent pathway, the gravitropic response is not associated with detectable changes in [Ca2+]i.
Promoter Analysis and Expression of a Phospholipase D Gene from Castor Bean
Authors: Xu L. , Zheng S. , Zheng L. , Wang X.
Published: July 2002

The expression of a castor bean (Ricinus communis L.) phospholipase D (PLD; EC 3.1.4.4) gene has been studied by examining its promoter activity in transgenic tobacco (Nicotiana tabacum) carrying a PLD promoter-glucuronidase transgene and by monitoring the levels of PLD mRNA in castor bean. Sequence and the 5[prime] truncation analyses revealed that the 5[prime] flanking region from nucleotide -1200 to -730 is required for the regulation and basal function of the PLD promoter. The PLD promoter in vegetative tissues is highly active in the rapidly growing regions such as the shoot apex and the secondary meristem producing axillary buds and vascular tissues of young leaves and stems. The PLD promoter activity in floral tissues was high in stigma, ovary, and pollen grains, but low in petals, sepals, the epidermis of anthers, styles, and filaments. The PLD promoter activity was enhanced by abscisic acid. Northern-blot analysis of PLD in castor bean showed that the PLD mRNA levels were high in young and metabolically more active tissues such as expanding leaves, hypocotyl hooks, developing seeds, and young seedlings, and they decreased in mature tissues such as fully expanded leaves and developed seeds. These patterns of expression suggest a role of PLD in rapid cell growth, proliferation, and reproduction.
Tobamovirus and Potyvirus Accumulation in Minor Veins of Inoculated Leaves from Representatives of the Solanaceae and Fabaceae1
Authors: Shun Ding Xin , Carter Shelly A. , Michael Deom C. , Nelson Richard S.
Published: July 2002

Virus invasion of minor veins in inoculated leaves of a host is the likely prelude to systemic movement of the pathogen and to subsequent yield reduction and quality loss. In this study we have analyzed the cell number and arrangement in minor veins within mature leaves of various members of the Solanaceae and Fabaceae families. We then monitored the accumulation pattern of several tobamoviruses and potyviruses in these veins at the time of rapid, phloem-mediated movement of viruses. Vascular parenchyma cells were the predominant and sometimes only cells to become visibly infected among the cells surrounding the sieve elements in minor veins containing 9 to 12 cells. In no instance did we observe a companion cell infected without a vascular parenchyma cell also being infected in the same vein. This suggests that the viruses used in this study first enter the vascular parenchyma cells and then the companion cells during invasion. The lack of detectable infection of smooth-walled companion or transfer cells, respectively, from inoculated leaves of bean (Phaseolus vulgaris) and pea (Pisum sativum) during a period of known rapid, phloem-mediated movement suggests that some viruses may be able to circumvent these cells in establishing phloem-mediated infection. The cause of the barrier to virus accumulation in the companion or transfer cells, the relationship of this barrier to previously identified barriers for virus or photoassimilate transport, and the relevance of these findings to photoassimilate transport models are discussed.
Extending the Microtubule/Microfibril Paradigm1
Authors: Fisher Deborah D. , Cyr Richard J.
Published: July 2002

The cortical microtubule array provides spatial information to the cellulose-synthesizing machinery within the plasma membrane of elongating cells. Until now data indicated that information is transferred from organized cortical microtubules to the cellulose-synthesizing complex, which results in the deposition of ordered cellulosic walls. How cortical microtubules become aligned is unclear. The literature indicates that biophysical forces, transmitted by the organized cellulose component of the cell wall, provide a spatial cue to orient cortical microtubules. This hypothesis was tested on tobacco (Nicotiana tabacum L.) protoplasts and suspension-cultured cells treated with the cellulose synthesis inhibitor isoxaben. Isoxaben (0.25–2.5 μm) inhibited the synthesis of cellulose microfibrils (detected by staining with 1 μg mL−1 fluorescent dye and polarized birefringence), the cells failed to elongate, and the cortical microtubules failed to become organized. The affects of isoxaben were reversible, and after its removal microtubules reorganized and cells elongated. Isoxaben did not depolymerize microtubules in vivo or inhibit the polymerization of tubulin in vitro. These data are consistent with the hypothesis that cellulose microfibrils, and hence cell elongation, are involved in providing spatial cues for cortical microtubule organization. These results compel us to extend the microtubule/microfibril paradigm to include the bidirectional flow of information.
Intracellular pH in Arbuscular Mycorrhizal Fungi1
Authors: Jolicoeur Mario , Germette Sophie , Gaudette Martin , Perrier Michel , Bécard Guillaume
Published: July 2002

A method was developed to perform real-time analysis of cytosolic pH of arbuscular mycorrhizal fungi in culture using dye and ratiometric measurements (490/450 nm excitations). The study was mainly performed using photometric analysis, although some data were confirmed using image analysis. The use of nigericin allowed an in vivo calibration. Experimental parameters such as loading time and concentration of the dye were determined so that pH measurements could be made for a steady-state period on viable cells. A characteristic pH profile was observed along hyphae. For Gigaspora margarita, the pH of the tip (0–2 μm) was typically 6.7, increased sharply to 7.0 behind this region (9.5 μm), and decreased over the next 250 μm to a constant value of 6.6. A similar pattern was obtained for Glomus intraradices. The pH profile of G. margaritagerm tubes was higher when cultured in the presence of carrot (Daucus carota) hairy roots (nonmycorrhizal). Similarly, extraradical hyphae of G. intraradices had a higher apical pH than the germ tubes. The use of a paper layer to prevent the mycorrhizal roots from being in direct contact with the medium selected hyphae with an even higher cytosolic pH. Results suggest that this method could be useful as a bioassay for studying signal perception and/or H+ cotransport of nutrients by arbuscular mycorrhizal hyphae.
The Phytochrome Response of the Lemna gibba NPR1 Gene Is Mediated Primarily through Changes in Abscisic Acid Levels1
Authors: Weatherwax Sharlene C. , Williams Shirley A. , Tingay Sonia , Tobin Elaine M.
Published: July 2002

Two important signaling systems involved in the growth and development of plants, those triggered by the photoreceptor phytochrome and the hormone abscisic acid (ABA), are involved in the regulation of expression of the NPR1 gene of Lemna gibba. We previously demonstrated that phytochrome action mediates changes in ABA levels in L. gibba, correlating with changes in gene expression evoked by stimulation of the phytochrome system. We have now further characterized phytochrome- and ABA-mediated regulation of L. gibba NPR1 gene expression using a transient particle bombardment assay, demonstrating that regulatory elements controlling responses to both stimuli reside within 156 nucleotides upstream of the transcription start. Linker scan (LS) analysis of the region from −156 to −70 was used to identify two specific requisite and nonredundant cis-acting promoter elements between −143 to −135 (LS2) and −113 to −101 (LS5). Mutation of either of these elements resulted in a coordinate loss of regulation by phytochrome and ABA. This suggests that, unlike theL. gibba Lhcb2*1 promoter, in which phytochrome and ABA regulatory elements are separable, the phytochrome response of theL. gibba NPR1 gene can be attributed to alterations in ABA levels.
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

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

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

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

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

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

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

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

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

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

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

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

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.
The active magnetic control algorithm for HITSAT‐1
Authors: Sun Zhaowei , Cheng Yanping , Geng Yunhai , Cao Xibin
Published: July 2002

The HITSAT‐1 is the first small satellite developed by Harbin Institute of Technology (HIT) whose mission objective is to test several pivotal techniques. In the initial orbit period, the satellite is likely to tumble as the result of separating from the rocket. How to capture it promptly with finite magnetic torque is an important problem. In this paper, considering the restrictive conditions of the magnetic field, the variable structure control theory is used to cope with the magnetic torque. Because of using the reaction wheels and magnetorquers as the control actuators, the combination control algorithm has been adopted in the initial orbit period. The results of the computer simulation indicated that the algorithm has excellent robustness and can be designed and realized easily.