bluuebunny/crossref_metadata_2025 huggingface dataset:
Global distribution of Streptococcus pneumoniae serotypes isolated from paediatric patients during 1999–2000 and the in vitro efficacy of telithromycin and comparators
Authors: Farrell D J , Jenkins S G , Reinert R R
Published: October 2004

Few data exist on the distribution of Streptococcus pneumoniae serotypes in many countries and in non-invasive disease overall. Here, data are presented from 772 paediatric isolates from children with community-acquired respiratory tract infections isolated from the PROTEKT global surveillance study during 1999–2000. Overall, 60.0 % of isolates were covered by the 7-valent pneumococcal vaccine formulation (PCV7), with greater coverage in the USA compared with Europe (69.6 vs 55.5 %, P = 0.014). Geographically dispersed clones of serogroups 3, 11 and 15 accounted for most of the isolates outside PCV7 coverage. Overall, macrolide, penicillin and cotrimoxazole non-susceptibility rates were high; however, all isolates were susceptible to telithromycin. Although only 7.4 % of isolates were resistant to amoxycillin/clavulanate, a higher prevalence of resistance was found in isolates from the USA and South Korea. This study shows the feasibility and importance of serotyping antibiotic surveillance study isolates and the potential of telithromycin as an important option for empiric therapy.
Genetic relatedness of antibiotic-resistant pneumococci isolated during case clusters
Authors: Clarke S C , Lawrie D , Diggle M A
Published: October 2004

Multilocus sequence typing of Streptococcus pneumoniae associated with two case clusters of disease is reported here for the first time. Isolates from the first cluster were serotype 19F, resistant to penicillin and erythromycin, and were characterized as ST 320. Isolates from the second cluster were serogroup 4, resistant to ciprofloxacin, and were characterized as ST 206. Therefore, the isolates from these clusters were antibiotic-resistant, of serotypes infrequently isolated, and of uncommon sequence types.
Identification of C. elegans DAF-12-binding sites, response elements, and target genes
Authors: Shostak Yuriy , Van Gilst Marc R. , Antebi Adam , Yamamoto Keith R.
Published: October 2004

Intracellular receptor DAF-12 regulates dauer formation and developmental age and affects Caenorhabditis elegans lifespan. Genetic analyses place DAF-12 at the convergence of several signal transduction pathways; however, the downstream effectors and the molecular basis for the receptor's multiple physiological outputs are unknown. Beginning with C. elegans genomic DNA, we devised a procedure for multiple rounds of selection and amplification that yielded fragments bearing DAF-12-binding sites. These genomic fragments mediated DAF-12-dependent transcriptional regulation both in Saccharomyces cerevisiae and in C. elegans; that is, they served as functional DAF-12 response elements. We determined that most of the genomic fragments that displayed DAF-12 response element activity in yeast were linked to genes that were regulated by DAF-12 in C. elegans; indeed, the response element-containing fragments typically resided within clusters of DAF-12-regulated genes. DAF-12 target gene regulation was developmental program and stage specific, potentially predicting a fit of these targets into regulatory networks governing aspects of C. elegans reproductive development and dauer formation.
Elongation by RNA polymerase II: the short and long of it
Authors: Sims Robert J. , Belotserkovskaya Rimma , Reinberg Danny
Published: October 2004

Appreciable advances into the process of transcript elongation by RNA polymerase II (RNAP II) have identified this stage as a dynamic and highly regulated step of the transcription cycle. Here, we discuss the many factors that regulate the elongation stage of transcription. Our discussion includes the classical elongation factors that modulate the activity of RNAP II, and the more recently identified factors that facilitate elongation on chromatin templates. Additionally, we discuss the factors that associate with RNAP II, but do not modulate its catalytic activity. Elongation is highlighted as a central process that coordinates multiple stages in mRNA biogenesis and maturation.
The geminivirus nuclear shuttle protein is a virulence factor that suppresses transmembrane receptor kinase activity
Authors: Fontes Elizabeth P.B. , Santos Anesia A. , Luz Dirce F. , Waclawovsky Alessandro J. , Chory Joanne
Published: October 2004

Despite the large number of leucine-rich-repeat (LRR) receptor-like-kinases (RLKs) in plants and their conceptual relevance in signaling events, functional information is restricted to a few family members. Here we describe the characterization of new LRR-RLK family members as virulence targets of the geminivirus nuclear shuttle protein (NSP). NSP interacts specifically with three LRR-RLKs, NIK1, NIK2, and NIK3, through an 80-amino acid region that encompasses the kinase active site and A-loop. We demonstrate that these NSP-interacting kinases (NIKs) are membrane-localized proteins with biochemical properties of signaling receptors. They behave as authentic kinase proteins that undergo autophosphorylation and can also phosphorylate exogenous substrates. Autophosphorylation occurs via an intermolecular event and oligomerization precedes the activation of the kinase. Binding of NSP to NIK inhibits its kinase activity in vitro, suggesting that NIK is involved in antiviral defense response. In support of this, infectivity assays showed a positive correlation between infection rate and loss of NIK1 and NIK3 function. Our data are consistent with a model in which NSP acts as a virulence factor to suppress NIK-mediated antiviral responses.
The Arabidopsis MutS homolog AtMSH4 functions at an early step in recombination: evidence for two classes of recombination in Arabidopsis
Authors: Higgins James D. , Armstrong Susan J. , Franklin F. Christopher H. , Jones Gareth H.
Published: October 2004

MSH4, a meiosis-specific member of the MutS-homolog family of genes, is required for normal levels of recombination and fertility in budding yeast, mouse, and Caenorhabditis elegans. In this paper, we report the identification and characterization of the Arabidopsis homolog of MSH4 (AtMSH4). We demonstrate that AtMSH4 expression can only be detected in floral tissues, consistent with a role in reproduction. Immunofluorescence studies indicate that its expression is limited to early meiotic prophase I, preceding the synapsis of homologous chromosomes. A T-DNA insertional mutant (Atmsh4) exhibited normal vegetative growth but a severe reduction in fertility, consistent with a meiotic defect; this was confirmed by cytological analysis of meiosis. RNAi-induced down-regulation of the MSH4 gene resulted in a similar fertility and meiotic phenotype. We demonstrate that prophase I chromosome synapsis is delayed and may be incomplete in Atmsh4, and metaphase I chiasma frequency is greatly reduced to ∼15% of wild type, leading to univalence and nondisjunction. We show that these residual chiasmata are randomly distributed among cells and chromosomes. These features of chiasma frequency and distribution in Atmsh4 show close parallels to MSH4-independent crossovers in budding yeast that have been proposed to originate by a separate pathway. Furthermore, the characteristics of the MSH4-independent chiasmata in the Atmsh4 mutant closely parallel those of second-pathway crossovers that have been postulated from Arabidopsis crossover analysis and mathematical modeling. Taken together, this evidence strongly indicates that Arabidopsis possesses two crossover pathways.
A Gateway-Compatible Yeast One-Hybrid System
Authors: Deplancke Bart , Dupuy Denis , Vidal Marc , Walhout Albertha J.M.
Published: October 2004

Since the advent of microarrays, vast amounts of gene expression data have been generated. However, these microarray data fail to reveal the transcription regulatory mechanisms that underlie differential gene expression, because the identity of the responsible transcription factors (TFs) often cannot be directly inferred from such data sets. Regulatory TFs activate or repress transcription of their target genes by binding to cis-regulatory elements that are frequently located in a gene's promoter. To understand the mechanisms underlying differential gene expression, it is necessary to identify physical interactions between regulatory TFs and their target genes. We developed a Gateway-compatible yeast one-hybrid (Y1H) system that enables the rapid, large-scale identification of protein-DNA interactions using both small (i.e., DNA elements of interest) and large (i.e., gene promoters) DNA fragments. We used four well-characterized Caenorhabditis elegans promoters as DNA baits to test the functionality of this Y1H system. We could detect ∼40% of previously reported TF-promoter interactions. By performing screens using two complementary libraries, we found novel potentially interacting TFs for each promoter. We recapitulated several of the Y1H-based protein-DNA interactions using luciferase reporter assays in mammalian cells. Taken together, the Gateway-compatible Y1H system will allow the high-throughput identification of protein-DNA interactions and may be a valuable tool to decipher transcription regulatory networks.
Feasibility of Genome-Scale Construction of Promoter::Reporter Gene Fusions for Expression in Caenorhabditis elegans Using a MultiSite Gateway Recombination System
Authors: Hope Ian A. , Stevens Jonathan , Garner Anna , Hayes Josie , Cheo David L. , Brasch Michael A. , Vidal Marc
Published: October 2004

The understanding of gene function increasingly requires the characterization of DNA segments containing promoters and their associated regulatory sequences. We describe a novel approach for linking multiple DNA segments, here applied to the generation of promoter::reporter fusions. Promoters from Caenorhabditis elegans genes were cloned using the MultiSite Gateway cloning technology. The capacity for using this system for efficient construction of chimeric genes was explored by constructing promoter::reporter gene fusions with a gfp reporter. The promoters were found to provide appropriate expression of GFP upon introduction into C. elegans, demonstrating that the short Gateway recombination site between the promoter and the reporter did not interfere with transcription or translation. The recombinational cloning involved in the Gateway system, which permits the highly efficient and precise transfer of DNA segments between plasmid vectors, makes this technology ideal for genomics research programs.
C. elegans ORFeome Version 3.1: Increasing the Coverage of ORFeome Resources With Improved Gene Predictions
Authors: Lamesch Philippe , Milstein Stuart , Hao Tong , Rosenberg Jennifer , Li Ning , Sequerra Reynaldo , Bosak Stephanie , Doucette-Stamm Lynn , Vandenhaute Jean , Hill David E. , Vidal Marc
Published: October 2004

The first version of the Caenorhabditis elegans ORFeome cloning project, based on release WS9 of Wormbase (August 1999), provided experimental verifications for ∼55% of predicted protein-encoding open reading frames (ORFs). The remaining 45% of predicted ORFs could not be cloned, possibly as a result of mispredicted gene boundaries. Since the release of WS9, gene predictions have improved continuously. To test the accuracy of evolving predictions, we attempted to PCR-amplify from a highly representative worm cDNA library and Gateway-clone ∼4200 ORFs missed earlier and for which new predictions are available in WS100 (May 2003). In this set we successfully cloned 63% of ORFs with supporting experimental data (“touched” ORFs), and 42% of ORFs with no supporting experimental evidence (“untouched” ORFs). Approximately 2000 full-length ORFs were cloned in-frame, 13% of which were corrected in their exon/intron structure relative to WS100 predictions. In total, ∼12,500 C. elegans ORFs are now available as Gateway Entry clones for various reverse proteomics (ORFeome v3.1). This work illustrates why the cloning of a complete C. elegans ORFeome, and likely the ORFeomes of other multicellular organisms, needs to be an iterative process that requires multiple rounds of experimental validation together with gradually improving gene predictions.
Toward Improving Caenorhabditis elegans Phenome Mapping With an ORFeome-Based RNAi Library
Authors: Rual Jean-François , Ceron Julian , Koreth John , Hao Tong , Nicot Anne-Sophie , Hirozane-Kishikawa Tomoko , Vandenhaute Jean , Orkin Stuart H. , Hill David E. , van den Heuvel Sander , Vidal Marc
Published: October 2004

The recently completed Caenorhabditis elegans genome sequence allows application of high-throughput (HT) approaches for phenotypic analyses using RNA interference (RNAi). As large phenotypic data sets become available, “phenoclustering” strategies can be used to begin understanding the complex molecular networks involved in development and other biological processes. The current HT-RNAi resources represent a great asset for phenotypic profiling but are limited by lack of flexibility. For instance, existing resources do not take advantage of the latest improvements in RNAi technology, such as inducible hairpin RNAi. Here we show that a C. elegans ORFeome resource, generated with the Gateway cloning system, can be used as a starting point to generate alternative HT-RNAi resources with enhanced flexibility. The versatility inherent to the Gateway system suggests that additional HT-RNAi libraries can now be readily generated to perform gene knockdowns under various conditions, increasing the possibilities for phenome mapping in C. elegans.
Concerted Assembly and Cloning of Multiple DNA Segments Using In Vitro Site-Specific Recombination: Functional Analysis of Multi-Segment Expression Clones
Authors: Cheo David L. , Titus Steven A. , Byrd Devon R.N. , Hartley James L. , Temple Gary F. , Brasch Michael A.
Published: October 2004

The ability to clone and manipulate DNA segments is central to molecular methods that enable expression, screening, and functional characterization of genes, proteins, and regulatory elements. We previously described the development of a novel technology that utilizes in vitro site-specific recombination to provide a robust and flexible platform for high-throughput cloning and transfer of DNA segments. By using an expanded repertoire of recombination sites with unique specificities, we have extended the technology to enable the high-efficiency in vitro assembly and concerted cloning of multiple DNA segments into a vector backbone in a predefined order, orientation, and reading frame. The efficiency and flexibility of this approach enables collections of functional elements to be generated and mixed in a combinatorial fashion for the parallel assembly of numerous multi-segment constructs. The assembled constructs can be further manipulated by directing exchange of defined segments with alternate DNA segments. In this report, we demonstrate feasibility of the technology and application to the generation of fusion proteins, the linkage of promoters to genes, and the assembly of multiple protein domains. The technology has broad implications for cell and protein engineering, the expression of multidomain proteins, and gene function analysis.
WormBase as an Integrated Platform for the C. elegans ORFeome
Authors: Chen Nansheng , Lawson Daniel , Bradnam Keith , Harris Todd W. , Stein Lincoln D.
Published: October 2004

The ORFeome project has validated and corrected a large number of predicted gene models in the nematode C. elegans, and has provided an enormous resource for proteome-scale studies. To make the resource useful to the research and teaching community, it needs to be integrated with other large-scale data sets, including the C. elegans genome, cell lineage, neurological wiring diagram, transcriptome, and gene expression map. This integration is also critical because the ORFeome data sets, like other `omics' data sets, have significant false-positive and false-negative rates, and comparison to related data is necessary to make confidence judgments in any given data point. WormBase, the central data repository for information about C. elegans and related nematodes, provides such a platform for integration. In this report, we will describe how C. elegans ORFeome data are deposited in the database, how they are used to correct gene models, how they are integrated and displayed in the context of other data sets at the WormBase Web site, and how WormBase establishes connection with the reagent-based resources at the ORFeome project Web site.
High-Throughput Computational and Experimental Techniques in Structural Genomics
Authors: Chance Mark R. , Fiser Andras , Sali Andrej , Pieper Ursula , Eswar Narayanan , Xu Guiping , Fajardo J. Eduardo , Radhakannan Thirumuruhan , Marinkovic Nebojsa
Published: October 2004

Structural genomics has as its goal the provision of structural information for all possible ORF sequences through a combination of experimental and computational approaches. The access to genome sequences and cloning resources from an ever-widening array of organisms is driving high-throughput structural studies by the New York Structural Genomics Research Consortium. In this report, we outline the progress of the Consortium in establishing its pipeline for structural genomics, and some of the experimental and bioinformatics efforts leading to structural annotation of proteins. The Consortium has established a pipeline for structural biology studies, automated modeling of ORF sequences using solved (template) structures, and a novel high-throughput approach (metallomics) to examining the metal binding to purified protein targets. The Consortium has so far produced 493 purified proteins from >1077 expression vectors. A total of 95 have resulted in crystal structures, and 81 are deposited in the Protein Data Bank (PDB). Comparative modeling of these structures has generated >40,000 structural models. We also initiated a high-throughput metal analysis of the purified proteins; this has determined that 10%-15% of the targets contain a stoichiometric structural or catalytic transition metal atom. The progress of the structural genomics centers in the U.S. and around the world suggests that the goal of providing useful structural information on most all ORF domains will be realized. This projected resource will provide structural biology information important to understanding the function of most proteins of the cell.
Versatile Gene-Specific Sequence Tags forArabidopsisFunctional Genomics: Transcript Profiling and Reverse Genetics Applications
Authors: Hilson Pierre , Allemeersch Joke , Altmann Thomas , Aubourg Sébastien , Avon Alexandra , Beynon Jim , Bhalerao Rishikesh P. , Bitton Frédérique , Caboche Michel , Cannoot Bernard , Chardakov Vasil , Cognet-Holliger Cécile , Colot Vincent , Crowe Mark , Darimont Caroline , Durinck Steffen , Eickhoff Holger , de Longevialle Andéol Falcon , Farmer Edward E. , Grant Murray , Kuiper Martin T.R. , Lehrach Hans , Léon Céline , Leyva Antonio , Lundeberg Joakim , Lurin Claire , Moreau Yves , Nietfeld Wilfried , Paz-Ares Javier , Reymond Philippe , Rouzé Pierre , Sandberg Goran , Segura Maria Dolores , Serizet Carine , Tabrett Alexandra , Taconnat Ludivine , Thareau Vincent , Van Hummelen Paul , Vercruysse Steven , Vuylsteke Marnik , Weingartner Magdalena , Weisbeek Peter J. , Wirta Valtteri , Wittink Floyd R.A. , Zabeau Marc , Small Ian
Published: October 2004

Microarray transcript profiling and RNA interference are two new technologies crucial for large-scale gene function studies in multicellular eukaryotes. Both rely on sequence-specific hybridization between complementary nucleic acid strands, inciting us to create a collection of gene-specific sequence tags (GSTs) representing at least 21,500Arabidopsisgenes and which are compatible with both approaches. The GSTs were carefully selected to ensure that each of them shared no significant similarity with any other region in theArabidopsisgenome. They were synthesized by PCR amplification from genomic DNA. Spotted microarrays fabricated from the GSTs show good dynamic range, specificity, and sensitivity in transcript profiling experiments. The GSTs have also been transferred to bacterial plasmid vectors via recombinational cloning protocols. These cloned GSTs constitute the ideal starting point for a variety of functional approaches, including reverse genetics. We have subcloned GSTs on a large scale into vectors designed for gene silencing in plant cells. We show that in planta expression of GST hairpin RNA results in the expected phenotypes in silencedArabidopsislines. These versatile GST resources provide novel and powerful tools for functional genomics.
From ORFeome to Biology: A Functional Genomics Pipeline
Authors: Wiemann Stefan , Arlt Dorit , Huber Wolfgang , Wellenreuther Ruth , Schleeger Simone , Mehrle Alexander , Bechtel Stephanie , Sauermann Mamatha , Korf Ulrike , Pepperkok Rainer , Sültmann Holger , Poustka Annemarie
Published: October 2004

As several model genomes have been sequenced, the elucidation of protein function is the next challenge toward the understanding of biological processes in health and disease. We have generated a human ORFeome resource and established a functional genomics and proteomics analysis pipeline to address the major topics in the post-genome-sequencing era: the identification of human genes and splice forms, and the determination of protein localization, activity, and interaction. Combined with the understanding of when and where gene products are expressed in normal and diseased conditions, we create information that is essential for understanding the interplay of genes and proteins in the complex biological network. We have implemented bioinformatics tools and databases that are suitable to store, analyze, and integrate the different types of data from high-throughput experiments and to include further annotation that is based on external information. All information is presented in a Web database (http://www.dkfz.de/LIFEdb). It is exploited for the identification of disease-relevant genes and proteins for diagnosis and therapy.
1274 Full-Open Reading Frames of Transcripts Expressed in the Developing Mouse Nervous System
Authors: Bonaldo Maria F. , Bair Thomas B. , Scheetz Todd E. , Snir Einat , Akabogu Ike , Bair Jennifer L. , Berger Brian , Crouch Keith , Davis Aja , Eyestone Mari E. , Keppel Catherine , Kucaba Tamara A. , Lebeck Mark , Lin Jenny L. , de Melo Anna I.R. , Rehmann Joshua , Reiter Rebecca S. , Schaefer Kelly , Smith Christina , Tack Dylan , Trout Kurtis , Sheffield Val C. , Lin Jim J-C. , Casavant Thomas L. , Soares Marcelo B.
Published: October 2004

As part of the trans-National Institutes of Health (NIH) Mouse Brain Molecular Anatomy Project (BMAP), and in close coordination with the NIH Mammalian Gene Collection Program (MGC), we initiated a large-scale project to clone, identify, and sequence the complete open reading frame (ORF) of transcripts expressed in the developing mouse nervous system. Here we report the analysis of the ORF sequence of 1274 cDNAs, obtained from 47 full-length-enriched cDNA libraries, constructed by using a novel approach, herein described. cDNA libraries were derived from size-fractionated cytoplasmic mRNA isolated from brain and eye tissues obtained at several embryonic stages and postnatal days. Altogether, including the full-ORF MGC sequences derived from these libraries by the MGC sequencing team, NIH_BMAP full-ORF sequences correspond to ∼20% of all transcripts currently represented in mouse MGC. We show that NIH_BMAP clones comprise 68% of mouse MGC cDNAs ≥5 kb, and 54% of those ≥4 kb, as of March 15, 2004. Importantly, we identified transcripts, among the 1274 full-ORF sequences, that are exclusively or predominantly expressed in brain and eye tissues, many of which encode yet uncharacterized proteins.
ORFeome Cloning and Systems Biology: Standardized Mass Production of the Parts From the Parts-List
Authors: Brasch Michael A. , Hartley James L. , Vidal Marc
Published: October 2004

Together with metabolites, proteins and RNAs form complex biological systems through highly intricate networks of physical and functional interactions. Large-scale studies aimed at a molecular understanding of the structure, function, and dynamics of proteins and RNAs in the context of cellular networks require novel approaches and technologies. This Special Issue of Genome Research features strategies for the high-throughput construction and manipulation of complete sets of protein-encoding open reading frames (ORFeome), gene promoters (promoterome), and noncoding RNAs, as predicted from genome and transcriptome sequences. Here we discuss the use of a recombinational cloning system that allows efficiency, adaptability, and compatibility in the generation of ORFeome, promoterome, and other resources.
Evidence for Serine/Threonine and Histidine Kinase Activity in the Tobacco Ethylene Receptor Protein NTHK2
Authors: Zhang Zhi-Gang , Zhou Hua-Lin , Chen Tao , Gong Yan , Cao Wan-Hong , Wang Yu-Jun , Zhang Jin-Song , Chen Shou-Yi
Published: October 2004

Ethylene plays important roles in plant growth, development, and stress responses. Two ethylene receptors, ETR1 from Arabidopsis and NTHK1 from tobacco (Nicotiana tabacum), have been found to have His kinase (HK) activity and Ser/Thr kinase activity, respectively, although both show similarity to bacterial two-component HK. Here, we report the characterization of another ethylene receptor homolog gene, NTHK2, from tobacco. This gene also encodes a HK-like protein and is induced by dehydration and CaCl2 but not significantly affected by NaCl and abscisic acid treatments. The biochemical properties of the yeast (Schizosaccharomyces pombe)-expressed NTHK2 domains were further characterized. We found that NTHK2 possessed Ser/Thr kinase activity in the presence of Mn2+ and had HK activity in the presence of Ca2+. Several lines of evidence supported this conclusion, including hydrolytic stability, phosphoamino acid analysis, mutation, deletion, and substrate analysis. These properties have implications in elucidation of the complexity of the ethylene signal transduction pathway and understanding of ethylene functions in plants.
Boron Tolerance in Barley Is Mediated by Efflux of Boron from the Roots
Authors: Hayes Julie E. , Reid Robert J.
Published: October 2004

Many plants are known to reduce the toxic effects of high soil boron (B) by reducing uptake of B, but no mechanism for limiting uptake has previously been identified. The B-tolerant cultivar of barley (Hordeum vulgare L.), Sahara, was shown to be able to maintain root B concentrations up to 50% lower than in the B-sensitive cultivar, Schooner. This translated into xylem concentrations that were approximately 64% lower and leaf concentrations 73% lower in the tolerant cultivar. In both cultivars, B accumulation was rapid and reached a steady-state concentration in roots within 3 h. In Schooner, this concentration was similar to the external medium, whereas in Sahara, the root concentration was maintained at a lower concentration. For this to occur, B must be actively extruded from the root in Sahara, and this is presumed to be the basis for B tolerance in barley. The extrusion mechanism was inhibited by sodium azide but not by treatment at low temperature. Several anion channel inhibitors were also effective in limiting extrusion, but it was not clear whether they acted directly or via metabolic inhibition. The ability of Sahara to maintain lower root B concentrations was constitutive and occurred across a wide range of B concentrations. This ability was lost at high pH, and both Schooner and Sahara then had similar root B concentrations. A predictive model that is consistent with the empirical results and explains the tolerance mechanism based on the presence of a borate anion efflux transporter in Sahara is presented.
Measurement and Interpretation of the Oxygen Isotope Composition of Carbon Dioxide Respired by Leaves in the Dark
Authors: Cernusak Lucas A. , Farquhar Graham D. , Wong S. Chin , Stuart-Williams Hilary
Published: October 2004

We measured the oxygen isotope composition (δ18O) of CO2 respired by Ricinus communis leaves in the dark. Experiments were conducted at low CO2 partial pressure and at normal atmospheric CO2 partial pressure. Across both experiments, the δ18O of dark-respired CO2 (δR) ranged from 44‰ to 324‰ (Vienna Standard Mean Ocean Water scale). This seemingly implausible range of values reflects the large flux of CO2 that diffuses into leaves, equilibrates with leaf water via the catalytic activity of carbonic anhydrase, then diffuses out of the leaf, leaving the net CO2 efflux rate unaltered. The impact of this process on δR is modulated by the δ18O difference between CO2 inside the leaf and in the air, and by variation in the CO2 partial pressure inside the leaf relative to that in the air. We developed theoretical equations to calculate δ18O of CO2 in leaf chloroplasts (δc), the assumed location of carbonic anhydrase activity, during dark respiration. Their application led to sensible estimates of δc, suggesting that the theory adequately accounted for the labeling of CO2 by leaf water in excess of that expected from the net CO2 efflux. The δc values were strongly correlated with δ18O of water at the evaporative sites within leaves. We estimated that approximately 80% of CO2 in chloroplasts had completely exchanged oxygen atoms with chloroplast water during dark respiration, whereas approximately 100% had exchanged during photosynthesis. Incorporation of the δ18O of leaf dark respiration into ecosystem and global scale models of C18OO dynamics could affect model outputs and their interpretation.
Galactinol synthase1. A Novel Heat Shock Factor Target Gene Responsible for Heat-Induced Synthesis of Raffinose Family Oligosaccharides in Arabidopsis
Authors: Panikulangara Tressa Jacob , Eggers-Schumacher Gabriele , Wunderlich Markus , Stransky Harald , Schöffl Fritz
Published: October 2004

Heat shock factors (HSFs) are transcriptional regulators of the heat shock response. The major target of HSFs are the genes encoding heat shock proteins (HSPs), which are known to have a protective function that counteracts cytotoxic effects. To identify other HSF target genes, which may be important determinants for the generation of stress tolerance in Arabidopsis, we screened a library enriched for genes that are up-regulated in HSF3 (AtHsfA1b)-overexpressing transgenic plants (TPs). Galactinol synthase1 (GolS1) is one of the genes that is heat-inducible in wild type, but shows constitutive mRNA levels in HSF3 TPs. The generation and analysis of TPs containing GolS1-promoter::β-glucuronidase-reporter gene constructs showed that, upon heat stress, the expression is transcriptionally controlled and occurs in all vegetative tissues. Functional consequences of GolS1 expression were investigated by the quantification of raffinose, stachyose, and galactinol contents in wild type, HSF3 TPs, and two different GolS1 knockout mutants (gols1-1 and gols1-2). This analysis demonstrates that (1) raffinose content in leaves increases upon heat stress in wild-type but not in the GolS1 mutant plants; and (2) the level of raffinose is enhanced and stachyose is present at normal temperature in HSF3 TPs. These data provide evidence that GolS1 is a novel HSF target gene, which is responsible for heat stress-dependent synthesis of raffinose, a member of the raffinose family oligosaccharides. The biological function of this osmoprotective substance and the role of HSF-dependent genes in this biochemical pathway are discussed.
Maximizing the Efficacy of SAGE Analysis Identifies Novel Transcripts in Arabidopsis
Authors: Robinson Stephen J. , Cram Dustin J. , Lewis Christopher T. , Parkin Isobel A.P.
Published: October 2004

The efficacy of using Serial Analysis of Gene Expression (SAGE) to analyze the transcriptome of the model dicotyledonous plant Arabidopsis was assessed. We describe an iterative tag-to-gene matching process that exploits the availability of the whole genome sequence of Arabidopsis. The expression patterns of 98% of the annotated Arabidopsis genes could theoretically be evaluated through SAGE and using an iterative matching process 79% could be identified by a tag found at a unique site in the genome. A total of 145,170 reliable experimental tags from two Arabidopsis leaf tissue SAGE libraries were analyzed, of which 29,632 were distinct. The majority (93%) of the 12,988 experimental tags observed greater than once could be matched within the Arabidopsis genome. However, only 78% were matched to a single locus within the genome, reflecting the complexities associated with working in a highly duplicated genome. In addition to a comprehensive assessment of gene expression in Arabidopsis leaf tissue, we describe evidence of transcription from pseudo-genes as well as evidence of alternative mRNA processing and anti-sense transcription. This collection of experimental SAGE tags could be exploited to assist in the on-going annotation of the Arabidopsis genome.
The Mechanics of Surface Expansion Anisotropy in Medicago truncatula Root Hairs
Authors: Dumais Jacques , Long Sharon R. , Shaw Sidney L.
Published: October 2004

Wall expansion in tip-growing cells shows variations according to position and direction. In Medicago truncatula root hairs, wall expansion exhibits a strong meridional gradient with a maximum near the pole of the cell. Root hair cells also show a striking expansion anisotropy, i.e. over most of the dome surface the rate of circumferential wall expansion exceeds the rate of meridional expansion. Concomitant measurements of expansion rates and wall stresses reveal that the extensibility of the cell wall must vary abruptly along the meridian of the cell to maintain the gradient of wall expansion. To determine the mechanical basis of expansion anisotropy, we compared measurements of wall expansion with expansion patterns predicted from wall structural models that were either fully isotropic, transversely isotropic, or fully anisotropic. Our results indicate that a model based on a transversely isotropic wall structure can provide a good fit of the data although a fully anisotropic model offers the best fit overall. We discuss how such mechanical properties could be controlled at the microstructural level.
Bacteroid Proline Catabolism Affects N2 Fixation Rate of Drought-Stressed Soybeans
Authors: Curtis Jason , Shearer Georgia , Kohl Daniel H.
Published: October 2004

In prior work, we observed that soybean (Glycine max L. cv Merr.) seeds inoculated with a mutant Bradyrhizobium japonicum strain unable to catabolize Pro (Pro dehydrogenase− [ProDH−]) resulted in plants that, when forced to depend on N2 fixation as the sole source of nitrogen and subjected to mild drought stress, suffered twice as large a loss in seed yield as did plants inoculated with the parental strain. Here, we used a continuous gas flow system to measure H2 evolution as a function of time and leaf water potential (ΨL). Since one H2 is produced for every N2 fixed as an obligate part of the mechanism of N2 fixation, these measurements serve as the basis for continuous monitoring of the N2 fixation rate. In five replicate experiments, the slope of the decline in N2 fixation rate in response to water stress was always greater for plants inoculated with the mutant strain unable to catabolize Pro or take up H2 (ProDH−, hup−) than it was for plants inoculated with the parental strain (ProDH+, hup−). In aggregate, the probability that this difference occurred by chance alone was 0.005. In combination with the earlier result, this is consistent with bacteroid catabolism of Pro synthesized in response to mild drought stress having a positive impact on N2 fixation rate and seed yield.
Arsenic Hyperaccumulation in Gametophytes ofPteris vittata. A New Model System for Analysis of Arsenic Hyperaccumulation
Authors: Gumaelius Luke , Lahner Brett , Salt David E. , Banks Jo Ann
Published: October 2004

The sporophyte of the fern Pteris vittata is known to hyperaccumulate arsenic (As) in its fronds to >1% of its dry weight. Hyperaccumulation of As by plants has been identified as a valuable trait for the development of a practical phytoremediation processes for removal of this potentially toxic trace element from the environment. However, because the sporophyte of P. vittata is a slow growing perennial plant, with a large genome and no developed genetics tools, it is not ideal for investigations into the basic mechanisms underlying As hyperaccumulation in plants. However, like other homosporous ferns, P. vittata produces and releases abundant haploid spores from the parent sporophyte plant which upon germination develop as free-living, autotrophic haploid gametophyte consisting of a small (<1 mm) single-layered sheet of cells. Its small size, rapid growth rate, ease of culture, and haploid genome make the gametophyte a potentially ideal system for the application of both forward and reverse genetics for the study of As hyperaccumulation. Here we report that gametophytes of P. vittata hyperaccumulate As in a similar manner to that previously observed in the sporophyte. Gametophytes are able to grow normally in medium containing 20 mm arsenate and accumulate >2.5% of their dry weight as As. This contrasts with gametophytes of the related nonaccumulating fern Ceratopteris richardii, which die at even low (0.1 mm) As concentrations. Interestingly, gametophytes of the related As accumulator Pityrogramma calomelanos appear to tolerate and accumulate As to intermediate levels compared to P. vittata and C. richardii. Analysis of gametophyte populations from 40 different P. vittata sporophyte plants collected at different sites in Florida also revealed the existence of natural variability in As tolerance but not accumulation. Such observations should open the door to the application of new and powerful genetic tools for the dissection of the molecular mechanisms involved in As hyperaccumulation in P. vittata using gametophytes as an easily manipulated model system.
Starch Division and Partitioning. A Mechanism for Granule Propagation and Maintenance in the Picophytoplanktonic Green Alga Ostreococcus tauri
Authors: Ral Jean-Philippe , Derelle Evelyne , Ferraz Conchita , Wattebled Fabrice , Farinas Benoit , Corellou Florence , Buléon Alain , Slomianny Marie-Christine , Delvalle David , d'Hulst Christophe , Rombauts Stephane , Moreau Hervé , Ball Steven
Published: October 2004

Whereas Glc is stored in small-sized hydrosoluble glycogen particles in archaea, eubacteria, fungi, and animal cells, photosynthetic eukaryotes have resorted to building starch, which is composed of several distinct polysaccharide fractions packed into a highly organized semicrystalline granule. In plants, both the initiation of polysaccharide synthesis and the nucleation mechanism leading to formation of new starch granules are currently not understood. Ostreococcus tauri, a unicellular green alga of the Prasinophyceae family, defines the tiniest eukaryote with one of the smallest genomes. We show that it accumulates a single starch granule at the chloroplast center by using the same pathway as higher plants. At the time of plastid division, we observe elongation of the starch and division into two daughter structures that are partitioned in each newly formed chloroplast. These observations suggest that in this system the information required to initiate crystalline polysaccharide growth of a new granule is contained within the preexisting polysaccharide structure and the design of the plastid division machinery.
Metabolic Profiling of the Sink-to-Source Transition in Developing Leaves of Quaking Aspen
Authors: Jeong Mijeong Lee , Jiang Hongying , Chen Huann-Sheng , Tsai Chung-Jui , Harding Scott A.
Published: October 2004

Profiles of small polar metabolites from aspen (Populus tremuloides Michx.) leaves spanning the sink-to-source transition zone were compared. Approximately 25% of 250 to 300 routinely resolved peaks were identified, with carbohydrates, organic acids, and amino acids being most abundant. Two-thirds of identified metabolites exhibited greater than 4-fold changes in abundance during leaf ontogeny. In the context of photosynthetic and respiratory measurements, profile data yielded information consistent with expected developmental trends in carbon-heterotrophic and carbon-autotrophic metabolism. Suc concentration increased throughout leaf expansion, while hexose sugar concentrations peaked at mid-expansion and decreased sharply thereafter. Amino acid contents generally decreased during leaf expansion, but an early increase in Phe and a later one in Gly and Ser reflected growing commitments to secondary metabolism and photorespiration, respectively. The assimilation of nitrate and utilization of stored Asn appeared to be marked by sequential changes in malate concentration and Asn transaminase activity. Principal component and hierarchical clustering analysis facilitated the grouping of cell wall maturation (pectins, hemicelluloses, and oxalate) and membrane biogenesis markers in relation to developmental changes in carbon and nitrogen assimilation. Metabolite profiling will facilitate investigation of nitrogen use and cellular development in Populus sp. varying widely in their growth and pattern of carbon allocation during sink-to-source development and in response to stress.
Interactions between Ethylene and Gibberellins in Phytochrome-Mediated Shade Avoidance Responses in Tobacco
Authors: Pierik Ronald , Cuppens Mieke L.C. , Voesenek Laurentius A.C.J. , Visser Eric J.W.
Published: October 2004

Plants respond to proximate neighbors with a suite of responses that comprise the shade avoidance syndrome. These phytochrome-mediated responses include hyponasty (i.e. a more vertical orientation of leaves) and enhanced stem and petiole elongation. We showed recently that ethylene-insensitive tobacco (Nicotiana tabacum) plants (Tetr) have reduced responses to neighbors, showing an important role for this gaseous plant hormone in shade avoidance. Here, we investigate interactions between phytochrome signaling and ethylene action in shade avoidance responses. Furthermore, we investigate if ethylene acts in these responses through an interaction with the GA class of hormones. Low red to far-red light ratios (R:FR) enhanced ethylene production in wild-type tobacco, resulting in shade avoidance responses, whereas ethylene-insensitive plants showed reduced shade avoidance responses. Plants with inhibited GA production showed hardly any shade avoidance responses at all to either a low R:FR or increased ethylene concentrations. Furthermore, low R:FR enhanced the responsiveness of hyponasty and stem elongation in both wild-type and Tetr plants to applied GA3, with the stem elongation process being more responsive to GA3 in the wild type than in Tetr. We conclude that phytochrome-mediated shade avoidance responses involve ethylene action, at least partly by modulating GA action.
Patellin1, a Novel Sec14-Like Protein, Localizes to the Cell Plate and Binds Phosphoinositides
Authors: Peterman T. Kaye , Ohol Yamini M. , McReynolds Lisa J. , Luna Elizabeth J.
Published: October 2004

Membrane trafficking is central to construction of the cell plate during plant cytokinesis. Consequently, a detailed understanding of the process depends on the characterization of molecules that function in the formation, transport, targeting, and fusion of membrane vesicles to the developing plate, as well as those that participate in its consolidation and maturation into a fully functional partition. Here we report the initial biochemical and functional characterization of patellin1 (PATL1), a novel cell-plate-associated protein that is related in sequence to proteins involved in membrane trafficking in other eukaryotes. Analysis of the Arabidopsis genome indicated that PATL1 is one of a small family of Arabidopsis proteins, characterized by a variable N-terminal domain followed by two domains found in other membrane-trafficking proteins (Sec14 and Golgi dynamics domains). Results from immunolocalization and biochemical fractionation studies suggested that PATL1 is recruited from the cytoplasm to the expanding and maturing cell plate. In vesicle-binding assays, PATL1 bound to specific phosphoinositides, important regulators of membrane trafficking, with a preference for phosphatidylinositol(5)P, phosphatidylinositol(4,5)P2, and phosphatidylinositol(3)P. Taken together, these findings suggest a role for PATL1 in membrane-trafficking events associated with cell-plate expansion or maturation and point to the involvement of phosphoinositides in cell-plate biogenesis.
Heavy Metal Stress. Activation of Distinct Mitogen-Activated Protein Kinase Pathways by Copper and Cadmium
Authors: Jonak Claudia , Nakagami Hirofumi , Hirt Heribert
Published: October 2004

Excessive amounts of heavy metals adversely affect plant growth and development. Whereas some regions naturally contain high levels of heavy metals, anthropogenic release of heavy metals into the environment continuously increases soil contamination. The presence of elevated levels of heavy metal ions triggers a wide range of cellular responses including changes in gene expression and synthesis of metal-detoxifying peptides. To elucidate signal transduction events leading to the cellular response to heavy metal stress we analyzed protein phosphorylation induced by elevated levels of copper and cadmium ions as examples for heavy metals with different physiochemical properties and functions. Exposure of alfalfa (Medicago sativa) seedlings to excess copper or cadmium ions activated four distinct mitogen-activated protein kinases (MAPKs): SIMK, MMK2, MMK3, and SAMK. Comparison of the kinetics of MAPK activation revealed that SIMK, MMK2, MMK3, and SAMK are very rapidly activated by copper ions, while cadmium ions induced delayed MAPK activation. In protoplasts, the MAPK kinase SIMKK specifically mediated activation of SIMK and SAMK but not of MMK2 and MMK3. Moreover, SIMKK only conveyed MAPK activation by CuCl2 but not by CdCl2. These results suggest that plants respond to heavy metal stress by induction of several distinct MAPK pathways and that excess amounts of copper and cadmium ions induce different cellular signaling mechanisms in roots.
Biochemical Characterization of the Tobacco 42-kD Protein Kinase Activated by Osmotic Stress
Authors: Kelner Anna , Pękala Izabela , Kaczanowski Szymon , Muszyńska Grażyna , Hardie D. Grahame , Dobrowolska Grażyna
Published: October 2004

In tobacco (Nicotiana tabacum), hyperosmotic stress induces rapid activation of a 42-kD protein kinase, referred to as Nicotiana tabacum osmotic stress-activated protein kinase (NtOSAK). cDNA encoding the kinase was cloned and, based on the predicted amino acid sequence, the enzyme was assigned to the SNF1-related protein kinase type 2 (SnRK2) family. The identity of the enzyme was confirmed by immunoprecipitation of the active kinase from tobacco cells subjected to osmotic stress using antibodies raised against a peptide corresponding to the C-terminal sequence of the kinase predicted from the cloned cDNA. A detailed biochemical characterization of NtOSAK purified from stressed tobacco cells was performed. Our results show that NtOSAK is a calcium-independent Ser/Thr protein kinase. The sequence of putative phosphorylation sites recognized by NtOSAK, predicted by the computer program PREDIKIN, resembled the substrate consensus sequence defined for animal and yeast (Saccharomyces cerevisiae) AMPK/SNF1 kinases. Our experimental data confirmed these results, as various targets for AMPK/SNF1 kinases were also efficiently phosphorylated by NtOSAK. A range of protein kinase inhibitors was tested as potential modulators of NtOSAK, but only staurosporine, a rather nonspecific protein kinase inhibitor, was found to abolish the enzyme activity. In phosphorylation reactions, NtOSAK exhibited a preference for Mg2+ over Mn2+ ions and an inability to use GTP instead of ATP as a phosphate donor. The enzyme activity was not modulated by 5′-AMP. To our knowledge, these results represent the first detailed biochemical characterization of a kinase of the SnRK2 family.