bluuebunny/crossref_metadata_2025 huggingface dataset:
Rice Transposable Elements: A Survey of 73,000 Sequence-Tagged-Connectors
Authors: Mao Long , Wood Todd C. , Yu Yeisoo , Budiman Muhammad A. , Tomkins Jeff , Woo Sung-sick , Sasinowski Maciek , Presting Gernot , Frisch David , Goff Steve , Dean Ralph A. , Wing Rod A.
Published: July 2002

As part of an international effort to sequence the rice genome, the Clemson University Genomics Institute is developing a sequence-tagged-connector (STC) framework. This framework includes the generation of deep-coverage BAC libraries from O. sativa ssp.japonica c.v. Nipponbare and the sequencing of both ends of the genomic DNA insert of the BAC clones. Here, we report a survey of the transposable elements (TE) in >73,000 STCs. A total of 6848 STCs were found homologous to regions of known TE sequences (E<10−5) by FASTX search of STCs against a set of 1358 TE protein sequences obtained from GenBank. Of these TE-containing STCs (TE–STCs), 88% (6027) are related to retroelements and the remaining are transposase homologs. Nearly all DNA transposons known previously in plants were present in the STCs, including maize Ac/Ds,En/Spm, Mutator, and mariner-like elements. In addition, 2746 STCs were found to contain regions homologous to known miniature inverted-repeat transposable elements (MITEs). The distribution of these MITEs in regions near genes was confirmed by EST comparisons to MITE-containing STCs, and our results showed that the association of MITEs with known EST transcripts varies by MITE type. Unlike the biased distribution of retroelements in maize, we found no evidence for the presence of gene islands when we correlated TE–STCs with a physical map of the CUGI BAC library. These analyses of TEs in nearly 50 Mb of rice genomic DNA provide an interesting and informative preview of the rice genome.
Active Conservation of Noncoding Sequences Revealed by Three-Way Species Comparisons
Authors: Dubchak Inna , Brudno Michael , Loots Gabriela G. , Pachter Lior , Mayor Chris , Rubin Edward M. , Frazer Kelly A.
Published: July 2002

Human and mouse genomic sequence comparisons are being increasingly used to search for evolutionarily conserved gene regulatory elements. Large-scale human–mouse DNA comparison studies have discovered numerous conserved noncoding sequences of which only a fraction has been functionally investigated A question therefore remains as to whether most of these noncoding sequences are conserved because of functional constraints or are the result of a lack of divergence time.

[The sequence data described in this paper have been submitted to the GenBank data library under accession nos. AF276990.]
Normalization and Subtraction of Cap-Trapper-Selected cDNAs to Prepare Full-Length cDNA Libraries for Rapid Discovery of New Genes
Authors: Carninci Piero , Shibata Yuko , Hayatsu Norihito , Sugahara Yuichi , Shibata Kazuhiro , Itoh Masayoshi , Konno Hideaki , Okazaki Yasushi , Muramatsu Masami , Hayashizaki Yoshihide
Published: July 2002

In the effort to prepare the mouse full-length cDNA encyclopedia, we previously developed several techniques to prepare and select full-length cDNAs. To increase the number of different cDNAs, we introduce here a strategy to prepare normalized and subtracted cDNA libraries in a single step. The method is based on hybridization of the first-strand, full-length cDNA with several RNA drivers, including starting mRNA as the normalizing driver and run-off transcripts from minilibraries containing highly expressed genes, rearrayed clones, and previously sequenced cDNAs as subtracting drivers. Our method keeps the proportion of full-length cDNAs in the subtracted/normalized library high. Moreover, our method dramatically enhances the discovery of new genes as compared to results obtained by using standard, full-length cDNA libraries. This procedure can be extended to the preparation of full-length cDNA encyclopedias from other organisms.
Genetic Analysis of Case/Control Data Using Estimated Haplotype Frequencies: Application to APOE Locus Variation and Alzheimer's Disease
Authors: Fallin Daniele , Cohen Annick , Essioux Laurent , Chumakov Ilya , Blumenfeld Marta , Cohen Daniel , Schork Nicholas J.
Published: July 2002

There is growing debate over the utility of multiple locus association analyses in the identification of genomic regions harboring sequence variants that influence common complex traits such as hypertension and diabetes. Much of this debate concerns the manner in which one can use the genotypic information from individuals gathered in simple sampling frameworks, such as the case/control designs, to actually assess the association between alleles in a particular genomic region and a trait. In this paper we describe methods for testing associations between estimated haplotype frequencies derived from multilocus genotype data and disease endpoints assuming a simple case/control sampling design. These proposed methods overcome the lack of phase information usually associated with samples of unrelated individuals and provide a comprehensive way of assessing the relationship between sequence or multiple-site variation and traits and diseases within populations. We applied the proposed methods in a study of the relationship between polymorphisms within the APOE gene region and Alzheimer's disease. Cases and controls for this study were collected from the United States and France. Our results confirm the known association between the APOE locus and Alzheimer's disease, even when the ɛ4 polymorphism is not contained in the tested haplotypes. This suggests that, in certain situations, haplotype information and linkage disequilibrium-induced associations between polymorphic loci that neighbor loci harboring functional sequence variants can be exploited to identify disease-predisposing alleles in large, freely mixing populations via estimated haplotype frequency methods.
Microchip Module for Blood Sample Preparation and Nucleic Acid Amplification Reactions
Authors: Yuen Po Ki , Kricka Larry J. , Fortina Paolo , Panaro Nicholas J. , Sakazume Taku , Wilding Peter
Published: July 2002

A computer numerical control-machined plexiglas-based microchip module was designed and constructed for the integration of blood sample preparation and nucleic acid amplification reactions. The microchip module is comprised of a custom-made heater-cooler for thermal cycling, a series of 254 μm × 254 μm microchannels for transporting human whole blood and reagents in and out of an 8–9 μL dual-purpose (cell isolation and PCR) glass-silicon microchip. White blood cells were first isolated from a small volume of human whole blood (<3 μL) in an integrated cell isolation–PCR microchip containing a series of 3.5-μm feature-sized “weir-type” filters, formed by an etched silicon dam spanning the flow chamber. A genomic target, a region in the human coagulation Factor V gene (226-bp), was subsequently directly amplified by microchip-based PCR on DNA released from white blood cells isolated on the filter section of the microchip mounted onto the microchip module. The microchip module provides a convenient means to simplify nucleic acid analyses by integrating two key steps in genetic testing procedures, cell isolation and PCR and promises to be adaptable for additional types of integrated assays.
Self-Reporting PNA/DNA Primers for PCR Analysis
Authors: Fiandaca Mark J. , Hyldig-Nielsen Jens J. , Gildea Brian D. , Coull James M.
Published: July 2002

We report a new fluorogenic method for sealed-tube PCR analysis using a quencher-labeled peptide nucleic acid (Q-PNA) probe. The Q-PNA hybridizes to a complementary tag sequence located at the 5′ end of a 5′ fluorophore-labeled oligonucleotide primer, quenching the primer's fluorescence. Incorporation of the primer into a doublestranded amplicon causes displacement of the Q-PNA such that the fluorescence of the sample is a direct indication of the amplicon concentration. The Q-PNA is able to quench multiple primers bearing distinct 5′ fluorophores in a single reaction. We show realtime quantitative detection of a single-copy gene, K-ras, from human genomic DNA, as well as an endpoint multiplex assay for Chlamydia trachomatis and Neisseria gonorrhoeae targets. Because the Q-PNA may be used to quench any primer that contains the 5′ tag sequence, it is possible to inexpensively adapt an existing primer set for use in a self-reporting fluorescent assay by including the tag sequence in one of the primers.
Surveying Saccharomyces Genomes to Identify Functional Elements by Comparative DNA Sequence Analysis
Authors: Cliften Paul F. , Hillier LaDeana W. , Fulton Lucinda , Graves Tina , Miner Tracie , Gish Warren R. , Waterston Robert H. , Johnston Mark
Published: July 2002

Comparative sequence analysis has facilitated the discovery of protein coding genes and important functional sequences within proteins, but has been less useful for identifying functional sequence elements in nonprotein-coding DNA because the relatively rapid rate of change of nonprotein-coding sequences and the relative simplicity of non-coding regulatory sequence elements necessitates the comparison of sequences of relatively closely related species. We tested the use of comparative DNA sequence analysis to aid identification of promoter regulatory elements, nonprotein-coding RNA genes, and small protein-coding genes by surveying random DNA sequences of severalSaccharomyces yeast species, with the goal of learning which species are best suited for comparisons with S. cerevisiae. We also determined the DNA sequence of a few specific promoters and RNA genes of several Saccharomyces species to determine the degree of conservation of known functional elements within the genome. Our results lead us to conclude that comparative DNA sequence analysis will enable identification of functionally conserved elements within the yeast genome, and suggest a path for obtaining this information.
Assembly of the Working Draft of the Human Genome with GigAssembler
Authors: Kent W. James , Haussler David
Published: July 2002

The data for the public working draft of the human genome contains roughly 400,000 initial sequence contigs in ∼30,000 large insert clones. Many of these initial sequence contigs overlap. A program,GigAssembler, was built to merge them and to order and orient the resulting larger sequence contigs based on mRNA, paired plasmid ends, EST, BAC end pairs, and other information. This program produced the first publicly available assembly of the human genome, a working draft containing roughly 2.7 billion base pairs and covering an estimated 88% of the genome that has been used for several recent studies of the genome. Here we describe the algorithm used byGigAssembler.
An Oligonucleotide Fingerprint Normalized and Expressed Sequence Tag Characterized Zebrafish cDNA Library
Authors: Clark Matthew D. , Hennig Steffen , Herwig Ralf , Clifton Sandy W. , Marra Marco A. , Lehrach Hans , Johnson Stephen L. , Group the WU-GSC EST
Published: July 2002

The zebrafish is a powerful system for understanding the vertebrate genome, allowing the combination of genetic, molecular, and embryological analysis. Expressed sequence tags (ESTs) provide a rapid means of identifying an organism's genes for further analysis, but any EST project is limited by the availability of suitable libraries. Such cDNA libraries must be of high quality and provide a high rate of gene discovery. However, commonly used normalization and subtraction procedures tend to select for shorter, truncated, and internally primed inserts, seriously affecting library quality. An alternative procedure is to use oligonucleotide fingerprinting (OFP) to precluster clones before EST sequencing, thereby reducing the re-sequencing of common transcripts. Here, we describe the use of OFP to normalize and subtract 75,000 clones from two cDNA libraries, to a minimal set of 25,102 clones. We generated 25,788 ESTs (11,380 3′ and 14,408 5′) from over 16,000 of these clones. Clustering of 10,654 high-quality 3′ ESTs from this set identified 7232 clusters (likely genes), corresponding to a 68% gene diversity rate, comparable to what has been reported for the best normalized human cDNA libraries, and indicating that the complete set of 25,102 clones contains as many as 17,000 genes. Yet, the library quality remains high. The complete set of 25,102 clones is available for researchers as glycerol stocks, filters sets, and as individual EST clones. These resources have been used for radiation hybrid, genetic, and physical mapping of the zebrafish genome, as well as positional cloning and candidate gene identification, molecular marker, and microarray development.

[The sequence data described in this paper have been submitted to the dbEST/GenBank data library under accession nos. AA497144–AA497369, AA542435–AA542678,AA545709–AA545724, AI384176–AI384205, AI384761–AI384796,AI396646–AI396663, AI396733–AI396777, AI396895–AI396938,AI397015–AI397130, AI397219–AI397252, AI397388–AI397484,AI415743–AI416403, AI436854–AI437493, AI444118–AI444540,AI461280–AI461395, AI476823–AI478024, AI496677–AI497576,AI522337–AI522810, AI544445–AI546083, AI558267–AI558995,AI584192–AI585023, AI585025–AI585238, AI588088–AI588836,AI601277–AI601868, AI626134–AI626875, AI629052–AI629398,AI641018–AI641780, AI657549–AI658347, AI666929–AI667197,AI667264–AI667414, AI667488–AI667567, AI721460–AI721747,AI721839–AI721978, and AI722283–AI722483.]
Shannon Information Theoretic Computation of Synonymous Codon Usage Biases in Coding Regions of Human and Mouse Genomes
Authors: Zeeberg Barry
Published: July 2002

Exonic GC of human mRNA reference sequences (RefSeqs), as well as A, C, G, and T in codon position 3 are linearly correlated with genomic GC. These observations utilize information from the completed human genome sequence and a large, high-quality set of human and mouse coding sequences, and are in accord with similar determinations published by others. A Shannon Information Theoretic measure of bias in synonymous codon usage was developed. When applied to either human or mouse RefSeqs, this measure is nonlinearly correlated with genomic, exonic, and third codon position A, C, G, and T. Information values between orthologous mouse and human RefSeqs are linearly correlated: mouse = 0.092 + 0.55 human. Mouse genes were consistently placed in genomic regions whose GC content was closer to 50% than was the GC content of the human ortholog. Since the (nonlinear) information versus percent GC curve has a minimum at 50% GC and monotonically increases with increasing distance from 50% GC, this phenomenon directly results in the low slope of 0.55. This appears to be a manifestation of an evolutionary strategy for placement of genes in regions of the genome with a GC content that relates synonymous codon bias and protein folding.

[The following individuals kindly provided reagents, samples, or unpublished information as indicated in the paper: D. Church and D. Maglott.]
A Multilocus Genotyping Assay for Candidate Markers of Cardiovascular Disease Risk
Authors: Cheng Suzanne , Grow Michael A. , Pallaud Céline , Klitz William , Erlich Henry A. , Visvikis Sophia , Chen John J. , Pullinger Clive R. , Malloy Mary J. , Siest Gérard , Kane John P.
Published: July 2002

A number of chronic diseases, including cardiovascular disease, appear to have a multifactorial genetic risk component. Consequently, techniques are needed to facilitate evaluation of complex genetic risk factors in large cohorts. We have designed a prototype assay for genotyping a panel of 35 biallelic sites that represent variation within 15 genes from biochemical pathways implicated in the development and progression of cardiovascular disease. Each DNA sample is amplified using two multiplex polymerase chain reactions, and the alleles are genotyped simultaneously using an array of immobilized, sequence-specific oligonucleotide probes. This multilocus assay was applied to two types of cohorts. Population frequencies for the markers were estimated using 496 unrelated individuals from a family-based cohort, and the observed values were consistent with previous reports. Linkage disequilibrium between consecutive pairs of markers within theapoCIII, LPL, and ELAM genes was also estimated. A preliminary analysis of single and pairwise locus associations with severity of atherosclerosis was performed using a composite cohort of 142 individuals for whom quantitative angiography data were available; evaluation of the potentially interesting associations observed will require analysis of an independent and larger cohort. This assay format provides a research tool for studies of multilocus genetic risk factors in large cardiovascular disease cohorts, and for the subsequent development of diagnostic tests.
G-protein β3 Subunit Gene Splice Variant and Body Fat Distribution in Nunavut Inuit
Authors: Hegele Robert A. , Anderson Carol , Young T. Kue , Connelly Philip W.
Published: July 2002

The GNB3 825T allele encodes a product that has enhanced activation of heterotrimeric G proteins in vitro and could play a role in adipogenesis. We therefore evaluated the possibility that theGNB3 825T allele was associated with obesity in a sample of 213 healthy Canadian Inuit. We found that body weight, body mass index, waist girth, hip girth, subscapular skinfold thickness, and triceps skinfold thickness were significantly higher in subjects with theGNB3 825T/T genotype than in subjects with other genotypes. Furthermore, two anthropometric ratios, namely that of waist to hip circumference and that of subscapular to triceps skinfold thickness, were not significantly different across genotypes. This suggested that the increased deposition of fat in subjects with the GNB3825T/T genotype was generalized and not localized to particular subregions. There was no association of this genetic variation with blood pressure. The GNB3 825T/T genotype accounted for between 1.6% and 3.3% of the total variation (≤13% of attributable variation) of the obesity-related traits. The potential for a genetic marker of obesity creates opportunities for future studies in the Inuit, not just to confirm the associations, but also to examine prospectively the influence of interventions and possible relationships between GNB3 825T and longer term complications of obesity.
Human Genome Anatomy: BACs Integrating the Genetic and Cytogenetic Maps for Bridging Genome and Biomedicine
Authors: Korenberg Julie R. , Chen Xiao-Ning , Sun Zhiguang , Shi Zheng-Yang , Ma Shaowu , Vataru Eddy , Yimlamai Dean , Weissenbach Jean S. , Shizuya Hiroaki , Simon Melvin I. , Gerety Sebastian S. , Nguyen Huy , Zemsteva Irina S. , Hui Lester , Silva James , Wu Xiaoyun , Birren Bruce W. , Hudson Thomas J.
Published: July 2002

Human genome sequencing is accelerating rapidly. Multiple genome maps link this sequence to problems in biology and clinical medicine. Because each map represents a different aspect of the structure, content, and behavior of human chromosomes, these fundamental properties must be integrated with the genome to understand disease genes, cancer instability, and human evolution. Cytogenetic maps use 400–850 visible band landmarks and are the primary means for defining prenatal defects and novel cancer breakpoints, thereby providing simultaneous examination of the entire genome. Recent genetic, physical, and transcript maps use PCR-based landmarks called sequence-tagged sites (STSs). We have integrated these genome maps by anchoring the human cytogenetic to the STS-based genetic and physical maps with 1021 STS–BAC pairs at an average spacing of ∼1 per 3 Mb. These integration points are represented by 872 unique STSs, including 642 polymorphic markers and 957 bacterial artificial chromosomes (BACs), each of which was localized on high resolution fluorescent banded chromosomes. These BACs constitute a resource that bridges map levels and provides the tools to seamlessly translate questions raised by genomic change seen at the chromosomal level into answers based at the molecular level. We show how the BACs provide molecular links for understanding human genomic duplications, meiosis, and evolution, as well as reagents for conducting genome-wide prenatal diagnosis at the molecular level and for detecting gene candidates associated with novel cancer breakpoints.
Identification of Three Novel Ca2+ Channel γ Subunit Genes Reveals Molecular Diversification by Tandem and Chromosome Duplication
Authors: Burgess Daniel L. , Davis Caleb F. , Gefrides Lisa A. , Noebels Jeffrey L.
Published: July 2002

Gene duplication is believed to be an important evolutionary mechanism for generating functional diversity within genomes. The accumulated products of ancient duplication events can be readily observed among the genes encoding voltage-dependent Ca2+ ion channels. Ten paralogous genes have been identified that encode isoforms of the α1 subunit, four that encode β subunits, and three that encode α2δ subunits. Until recently, only a single gene encoding a muscle-specific isoform of the Ca2+ channel γ subunit (CACNG1) was known. Expression of a distantly related gene in the brain was subsequently demonstrated upon isolation of the Cacng2 gene, which is mutated in the mouse neurological mutant stargazer (stg). In this study, we sought to identify additional genes that encoded γ subunits. Because gene duplication often generates paralogs that remain in close syntenic proximity (tandem duplication) or are copied onto related daughter chromosomes (chromosome or whole-genome duplication), we hypothesized that the known positions of CACNG1 andCACNG2 could be used to predict the likely locations of additional γ subunit genes. Low-stringency genomic sequence analysis of targeted regions led to the identification of three novel Ca2+ channel γ subunit genes, CACNG3,CACNG4, and CACNG5, on chromosomes 16 and 17. These results demonstrate the value of genome evolution models for the identification of distantly related members of gene families.

[The sequence data described in this paper have been submitted to the GenBank data library under accession numbersAF142618–AF142625 and AF148220.]
Identification of a New Pea Gene, PsNlec1, Encoding a Lectin-like Glycoprotein Isolated from the Symbiosomes of Root Nodules
Authors: Kardailsky I. V. , Sherrier D. J. , Brewin N. J.
Published: July 2002

A 27-kD glycoprotein antigen recognized by monoclonal antibody MAC266 was purified from isolated symbiosomes derived from pea (Pisum sativum) root nodules containing Rhizobium. The N-terminal amino acid sequence was obtained, and the corresponding cDNA clone was isolated by a polymerase chain reaction-based strategy. The clone contained a single open reading frame, and the gene was termed PsNlec1. Phylogenetic analysis of 31 legume sequences showed that the PsNlec1 protein is related to the legume lectin family but belongs to a subgroup that is very different from pea seed lectin. Expression of the PsNlec1 transcript was much stronger in nodules than in other parts of the plant. It was found in both infected and uninfected cells in the central tissue of the nodule and in the stele of the root near the attachment point of the nodule. When uninfected pea seedlings were grown on medium containing nitrate, weak transcription of PsNlec1 was observed in the root system. The identification of PsNlec1 inside the symbiosome is consistent with the observation that legume lectins are generally vacuolar proteins that may serve as transient storage components.
Protective Mechanism of the Mexican Bean Weevil against High Levels of α--Amylase Inhibitor in the Common Bean
Authors: Ishimoto M. , Chrispeels M. J.
Published: July 2002

α--Amylase inhibitor (α-AI) protects seeds of the common bean (Phaseolus vulgaris) against predation by certain species of bruchids such as the cowpea weevil (Callosobruchus maculatus) and the azuki bean weevil (Callosobruchus chinensis), but not against predation by the bean weevil (Acanthoscelides obtectus) or the Mexican bean weevil (Zabrotes subfasciatus), insects that are common in the Americas. We characterized the interaction of α-AI-1 present in seeds of the common bean, of a different isoform, α-AI-2, present in seeds of wild common bean accessions, and of two homologs, α-AI-Pa present in seeds of the tepary bean (Phaseolus acutifolius) and (α-AI-Pc in seeds of the scarlet runner bean (Phaseolus coccineus), with the midgut extracts of several bruchids. The extract of the Z. subfasciatus larvae rapidly digests and inactivates α-AI-1 and α-AI-Pc, but not α-AI-2 or [alphha]AI-Pa. The digestion is caused by a serine protease. A single proteolytic cleavage in the [beta] subunit of α-AI-1 occurs at the active site of the protein. When degradation is prevented, α-AI-1 and α-AI-Pc do not inhibit the α--amylase of Z. subfasciatus, although they are effective against the α--amylase of C. chinensis. α-AI-2 and α-AI-Pa, on the other hand, do inhibit the α--amylase of Z. subfasciatus, suggesting that they are good candidates for genetic engineering to achieve resistance to Z. subfasciatus.
Energy Sources for HCO3− and CO2 Transport in Air-Grown Cells ofSynechococcus UTEX 6251
Authors: Li Qinglin , Canvin David T.
Published: July 2002

Light-dependent inorganic C (Ci) transport and accumulation in air-grown cells ofSynechococcus UTEX 625 were examined with a mass spectrometer in the presence of inhibitors or artificial electron acceptors of photosynthesis in an attempt to drive CO2 or HCO3− uptake separately by the cyclic or linear electron transport chains. In the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea, the cells were able to accumulate an intracellular Ci pool of 20 mm, even though CO2 fixation was completely inhibited, indicating that cyclic electron flow was involved in the Ci-concentrating mechanism. When 200 μmN,N-dimethyl-p-nitrosoaniline was used to drain electrons from ferredoxin, a similar Ci accumulation was observed, suggesting that linear electron flow could support the transport of Ci. When carbonic anhydrase was not present, initial CO2 uptake was greatly reduced and the extracellular [CO2] eventually increased to a level higher than equilibrium, strongly suggesting that CO2transport was inhibited and that Ci accumulation was the result of active HCO3− transport. With 3-(3,4-dichlorophenyl)-1,1-dimethylurea-treated cells, Citransport and accumulation were inhibited by inhibitors of CO2 transport, such as COS and Na2S, whereas Li+, an HCO3−-transport inhibitor, had little effect. In the presence ofN,N-dimethyl-p-nitrosoaniline, Ci transport and accumulation were not inhibited by COS and Na2S but were inhibited by Li+. These results suggest that CO2 transport is supported by cyclic electron transport and that HCO3− transport is supported by linear electron transport.
Rearrangement of Actin Microfilaments in Plant Root Hairs Responding to Rhizobium etli Nodulation Signals1
Authors: Cárdenas Luis , Vidali Luis , Domı́nguez Jimena , Pérez Héctor , Sánchez Federico , Hepler Peter K. , Quinto Carmen
Published: July 2002

The response of the actin cytoskeleton to nodulation (Nod) factors secreted by Rhizobium etli has been studied in living root hairs of bean (Phaseolus vulgaris) that were microinjected with fluorescein isothiocyanate-phalloidin. In untreated control cells or cells treated with the inactive chitin oligomer, the actin cytoskeleton was organized into long bundles that were oriented parallel to the long axis of the root hair and extended into the apical zone. Upon exposure to R. etli Nod factors, the filamentous actin became fragmented, as indicated by the appearance of prominent masses of diffuse fluorescence in the apical region of the root hair. These changes in the actin cytoskeleton were rapid, observed as soon as 5 to 10 min after application of the Nod factors. It was interesting that the filamentous actin partially recovered in the continued presence of the Nod factor: by 1 h, long bundles had reformed. However, these cells still contained a significant amount of diffuse fluorescence in the apical zone and in the nuclear area, presumably indicating the presence of short actin filaments. These results indicate that Nod factors alter the organization of actin microfilaments in root hair cells, and this could be a prelude for the formation of infection threads.
Characterization of Cadmium Binding, Uptake, and Translocation in Intact Seedlings of Bread and Durum Wheat Cultivars
Authors: Hart Jonathan J. , Welch Ross M. , Norvell Wendell A. , Sullivan Lori A. , Kochian Leon V.
Published: July 2002

High Cd content in durum wheat (Triticum turgidum L. var durum) grain grown in the United States and Canada presents potential health and economic problems for consumers and growers. In an effort to understand the biological processes that result in excess Cd accumulation, root Cd uptake and xylem translocation to shoots in seedlings of bread wheat (Triticum aestivum L.) and durum wheat cultivars were studied. Whole-plant Cd accumulation was somewhat greater in the bread wheat cultivar, but this was probably because of increased apoplastic Cd binding. Concentration-dependent109Cd2+-influx kinetics in both cultivars were characterized by smooth, nonsaturating curves that could be dissected into linear and saturable components. The saturable component likely represented carrier-mediated Cd influx across root-cell plasma membranes (Michaelis constant, 20–40 nm; maximum initial velocity, 26–29 nmol g−1 fresh weight h−1), whereas linear Cd uptake represented cell wall binding of109Cd. Cd translocation to shoots was greater in the bread wheat cultivar than in the durum cultivar because a larger proportion of root-absorbed Cd moved to shoots. Our results indicate that excess Cd accumulation in durum wheat grain is not correlated with seedling-root influx rates or root-to-shoot translocation, but may be related to phloem-mediated Cd transport to the grain.
Ultraviolet-B Radiation Effects on Water Relations, Leaf Development, and Photosynthesis in Droughted Pea Plants1
Authors: Nogués Salvador , Allen Damian J. , Morison James I.L. , Baker Neil R.
Published: July 2002

The effects of ultraviolet-B (UV-B) radiation on water relations, leaf development, and gas-exchange characteristics in pea (Pisum sativum L. cv Meteor) plants subjected to drought were investigated. Plants grown throughout their development under a high irradiance of UV-B radiation (0.63 W m−2) were compared with those grown without UV-B radiation, and after 12 d one-half of the plants were subjected to 24 d of drought that resulted in mild water stress. UV-B radiation resulted in a decrease of adaxial stomatal conductance by approximately 65%, increasing stomatal limitation of CO2 uptake by 10 to 15%. However, there was no loss of mesophyll light-saturated photosynthetic activity. Growth in UV-B radiation resulted in large reductions of leaf area and plant biomass, which were associated with a decline in leaf cell numbers and cell division. UV-B radiation also inhibited epidermal cell expansion of the exposed surface of leaves. There was an interaction between UV-B radiation and drought treatments: UV-B radiation both delayed and reduced the severity of drought stress through reductions in plant water-loss rates, stomatal conductance, and leaf area.
Changes in Growth CO2 Result in Rapid Adjustments of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase Small Subunit Gene Expression in Expanding and Mature Leaves of Rice
Authors: Gesch Russ W. , Boote Kenneth J. , Vu Joseph C.V. , Hartwell Allen L. , Bowes George
Published: July 2002

The accumulation of soluble carbohydrates resulting from growth under elevated CO2 may potentially signal the repression of gene activity for the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (rbcS). To test this hypothesis we grew rice (Oryza sativa L.) under ambient (350 μL L−1) and high (700 μL L−1) CO2in outdoor, sunlit, environment-controlled chambers and performed a cross-switching of growth CO2 concentration at the late-vegetative phase. Within 24 h, plants switched to high CO2 showed a 15% and 23% decrease in rbcSmRNA, whereas plants switched to ambient CO2 increased 27% and 11% in expanding and mature leaves, respectively. Ribulose-1,5-bisphosphate carboxylase/oxygenase total activity and protein content 8 d after the switch increased up to 27% and 20%, respectively, in plants switched to ambient CO2, but changed very little in plants switched to high CO2. Plants maintained at high CO2 showed greater carbohydrate pool sizes and lower rbcS transcript levels than plants kept at ambient CO2. However, after switching growth CO2 concentration, there was not a simple correlation between carbohydrate and rbcS transcript levels. We conclude that although carbohydrates may be important in the regulation of rbcS expression, changes in total pool size alone could not predict the rapid changes in expression that we observed.
Superoxide Dismutase in Arabidopsis: An Eclectic Enzyme Family with Disparate Regulation and Protein Localization
Authors: Kliebenstein Daniel J. , Monde Rita-Ann , Last Robert L.
Published: July 2002

A number of environmental stresses can lead to enhanced production of superoxide within plant tissues, and plants are believed to rely on the enzyme superoxide dismutase (SOD) to detoxify this reactive oxygen species. We have identified seven cDNAs and genes for SOD in Arabidopsis. These consist of three CuZnSODs (CSD1,CSD2, and CSD3), three FeSODs (FSD1, FSD2, and FSD3), and one MnSOD (MSD1). The chromosomal location of these seven SOD genes has been established. To study this enzyme family, antibodies were generated against five proteins: CSD1, CSD2, CSD3, FSD1, and MSD1. Using these antisera and nondenaturing-polyacrylamide gel electrophoresis enzyme assays, we identified protein and activity for two CuZnSODs and for FeSOD and MnSOD in Arabidopsis rosette tissue. Additionally, subcellular fractionation studies revealed the presence of CSD2 and FeSOD protein within Arabidopsis chloroplasts. The seven SOD mRNAs and the four proteins identified were differentially regulated in response to various light regimes, ozone fumigation, and ultraviolet-B irradiation. To our knowledge, this is the first report of a large-scale analysis of the regulation of multiple SOD proteins in a plant species.
Turgor Regulation via Cell Wall Adjustment in White Spruce1
Authors: Marshall John G. , Dumbroff Erwin B.
Published: July 2002

Turgor regulation at reduced water contents was closely associated with changes in the elastic quality of the cell walls of individual needles and shoots of naturally drought-resistant seedlings of white spruce (Picea glauca [Moench] Voss.) and of seedlings of intermediate resistance that had been pretreated with paclobutrazol, a stress-protecting, synthetic plant-growth regulator. Paclobutrazol-treated seedlings showed marked increases in drought resistance, and pressure-volume analysis combined with Chardakov measurements confirmed observations that water stress was ameliorated during prolonged drought. Turgor was maintained in the paclobutrazol-treated and in the naturally resistant drought-stressed seedlings despite water contents near or below the turgor-loss volumes of well-watered controls. The maintenance of turgor in these seedlings was in large part a function of the dynamic process of cell wall adjustment, as reflected by marked reductions in both the saturated and turgor-loss volumes and by large increases in the elastic coefficients of the tissues. Shear and Young's moduli, calculated from pressure-volume curves and the radii and wall thicknesses of mesophyll cells, also confirmed observed changes in the elastic qualities of the cell walls. Elastic coefficients of well-watered, paclobutrazol-treated seedlings were consistently larger than those in well-watered controls and several times larger than the values in untreated plants, which succumbed rapidly to drought. In contrast, untreated seedlings that withstood prolonged drought without wilting displayed elastic coefficients similar to those in seedlings that had been treated with paclobutrazol but that had not been exposed to drought.
Cell-Specific Production and Antimicrobial Activity of Naphthoquinones in Roots of Lithospermum erythrorhizon1
Authors: Brigham Lindy A. , Michaels Paula J. , Flores Hector E.
Published: July 2002

Pigmented naphthoquinone derivatives of shikonin are produced at specific times and in specific cells of Lithospermum erythrorhizon roots. Normal pigment development is limited to root hairs and root border cells in hairy roots grown on “noninducing” medium, whereas induction of additional pigment production by abiotic (CuSO4) or biotic (fungal elicitor) factors increases the amount of total pigment, changes the ratios of derivatives produced, and initiates production of pigment de novo in epidermal cells. When the biological activity of these compounds was tested against soil-borne bacteria and fungi, a wide range of sensitivity was recorded. Acetyl-shikonin and β-hydroxyisovaleryl-shikonin, the two most abundant derivatives in both Agrobacterium rhizogenes-transformed “hairy-root” cultures and greenhouse-grown plant roots, were the most biologically active of the seven compounds tested. Hyphae of the pathogenic fungiRhizoctonia solani, Pythium aphanidermatum, and Nectria hematococca induced localized pigment production upon contact with the roots. Challenge byR. solani crude elicitor increased shikonin derivative production 30-fold. We have studied the regulation of this suite of related, differentially produced, differentially active compounds to understand their role(s) in plant defense at the cellular level in the rhizosphere.
Water Deficit and Spatial Pattern of Leaf Development. Variability in Responses Can Be Simulated Using a Simple Model of Leaf Development1
Authors: Granier Christine , Tardieu François
Published: July 2002

We analyzed the effect of short-term water deficits at different periods of sunflower (Helianthus annuus L.) leaf development on the spatial and temporal patterns of tissue expansion and epidermal cell division. Six water-deficit periods were imposed with similar and constant values of soil water content, predawn leaf water potential and [ABA] in the xylem sap, and with negligible reduction of the rate of photosynthesis. Water deficit did not affect the duration of expansion and division. Regardless of their timing, deficits reduced relative expansion rate by 36% and relative cell division rate by 39% (cells blocked at the G0-G1 phase) in all positions within the leaf. However, reductions in final leaf area and cell number in a given zone of the leaf largely differed with the timing of deficit, with a maximum effect for earliest deficits. Individual cell area was only affected during the periods when division slowed down. These behaviors could be simulated in all leaf zones and for all timings by assuming that water deficit affects relative cell division rate and relative expansion rate independently, and that leaf development in each zone follows a stable three-phase pattern in which duration of each phase is stable if expressed in thermal time (C. Granier and F. Tardieu [1998b] Plant Cell Environ 21: 695–703).
Nicotianamine Chelates Both FeIII and FeII. Implications for Metal Transport in Plants1
Authors: von Wirén Nicolaus , Klair Sukhbinder , Bansal Suhkibar , Briat Jean-Francois , Khodr Hicham , Shioiri Takayuki , Leigh Roger A. , Hider Robert C.
Published: July 2002

Nicotianamine (NA) occurs in all plants and chelates metal cations, including FeII, but reportedly not FeIII. However, a comparison of the FeII and ZnII affinity constants of NA and various FeIII-chelating aminocarboxylates suggested that NA should chelate FeIII. High-voltage electrophoresis of the FeNA complex formed in the presence of FeIII showed that the complex had a net charge of 0, consistent with the hexadentate chelation of FeIII. Measurement of the affinity constant for FeIII yielded a value of 1020.6, which is greater than that for the association of NA with FeII (1012.8). However, capillary electrophoresis showed that in the presence of FeII and FeIII, NA preferentially chelates FeII, indicating that the FeIINA complex is kinetically stable under aerobic conditions. Furthermore, Fe complexes of NA are relatively poor Fenton reagents, as measured by their ability to mediate H2O2-dependent oxidation of deoxyribose. This suggests that NA will have an important role in scavenging Fe and protecting the cell from oxidative damage. The pH dependence of metal ion chelation by NA and a typical phytosiderophore, 2′-deoxymugineic acid, indicated that although both have the ability to chelate Fe, when both are present, 2′-deoxymugineic acid dominates the chelation process at acidic pH values, whereas NA dominates at alkaline pH values. The consequences for the role of NA in the long-distance transport of metals in the xylem and phloem are discussed.
Decrease in Phosphoribulokinase Activity by Antisense RNA in Transgenic Tobacco. Relationship between Photosynthesis, Growth, and Allocation at Different Nitrogen Levels1
Authors: Banks Fiona M. , Driscoll Simon P. , Parry Martin A.J. , Lawlor David W. , Knight Jacqui S. , Gray John C. , Paul Matthew J.
Published: July 2002

To study the direct effects of photosynthesis on allocation of biomass by altering photosynthesis without altering leaf N or nitrate content, phosphoribulokinase (PRK) activity was decreased in transgenic tobacco (Nicotiana tabacum L.) with an inverted tobacco PRK cDNA and plants were grown at different N levels (0.4 and 5 mmNH4NO3). The activation state of PRK increased as the amount of enzyme was decreased genetically at both levels of N. At high N a 94% decrease in PRK activity had only a small effect (20%) on photosynthesis and growth. At low N a 94% decrease in PRK activity had a greater effect on leaf photosynthesis (decreased by up to 50%) and whole-plant photosynthesis (decreased by up to 35%) than at high N. These plants were up to 35% smaller than plants with higher PRK activities because they had less structural dry matter and less starch, which was decreased by 3- to 4-fold, but still accumulated to 24% to 31% of dry weight; young leaves contained more starch than older leaves in older plants. Leaves had a higher ion and water content, and specific leaf area was higher, but allocation between shoot and root was unaltered. In conclusion, low N in addition to a 94% decrease in PRK by antisense reduces the activity of PRK sufficient to diminish photosynthesis, which limits biomass production under conditions normally considered sink limited.
Involvement of the Octadecanoid Pathway and Protein Phosphorylation in Fungal Elicitor-Induced Expression of Terpenoid Indole Alkaloid Biosynthetic Genes in Catharanthus roseus
Authors: Menke Frank L.H. , Parchmann Stefanie , Mueller Martin J. , Kijne Jan W. , Memelink Johan
Published: July 2002

Two key genes in terpenoid indole alkaloid biosynthesis,Tdc and Str, encoding tryptophan decarboxylase and strictosidine synthase, respectively, are coordinately induced by fungal elicitors in suspension-culturedCatharanthus roseus cells. We have studied the roles of the jasmonate biosynthetic pathway and of protein phosphorylation in signal transduction initiated by a partially purified elicitor from yeast extract. In addition to activating Tdc andStr gene expression, the elicitor also induced the biosynthesis of jasmonic acid. The jasmonate precursor α-linolenic acid or methyl jasmonate (MeJA) itself induced Tdc andStr gene expression when added exogenously . Diethyldithiocarbamic acid, an inhibitor of jasmonate biosynthesis, blocked both the elicitor-induced formation of jasmonic acid and the activation of terpenoid indole alkaloid biosynthetic genes. The protein kinase inhibitor K-252a abolished both elicitor-induced jasmonate biosynthesis and MeJA-induced Tdc and Strgene expression. Analysis of the expression of Strpromoter/gusA fusions in transgenic C. roseus cells showed that the elicitor and MeJA act at the transcriptional level. These results demonstrate that the jasmonate biosynthetic pathway is an integral part of the elicitor-triggered signal transduction pathway that results in the coordinate expression of the Tdc and Str genes and that protein kinases act both upstream and downstream of jasmonates.
Extracellular Carbonic Anhydrase Facilitates Carbon Dioxide Availability for Photosynthesis in the Marine DinoflagellateProrocentrum micans
Authors: Nimer Nabil A. , Brownlee Colin , Merrett Michael J.
Published: July 2002

This study investigated inorganic carbon accumulation in relation to photosynthesis in the marine dinoflagellate Prorocentrum micans. Measurement of the internal inorganic carbon pool showed a 10-fold accumulation in relation to external dissolved inorganic carbon (DIC). Dextran-bound sulfonamide (DBS), which inhibited extracellular carbonic anhydrase, caused more than 95% inhibition of DIC accumulation and photosynthesis. We used real-time imaging of living cells with confocal laser scanning microscopy and a fluorescent pH indicator dye to measure transient pH changes in relation to inorganic carbon availability. When steady-state photosynthesizing cells were DIC limited, the chloroplast pH decreased from 8.3 to 6.9 and cytosolic pH decreased from 7.7 to 7.1. Re-addition of HCO3− led to a rapid re-establishment of the steady-state pH values abolished by DBS. The addition of DBS to photosynthesizing cells under steady-state conditions resulted in a transient increase in intracellular pH, with photosynthesis maintained for 6 s, the amount of time needed for depletion of the intracellular inorganic carbon pool. These results demonstrate the key role of extracellular carbonic anhydrase in facilitating the availability of CO2 at the exofacial surface of the plasma membrane necessary to maintain the photosynthetic rate. The need for a CO2-concentrating mechanism at ambient CO2 concentrations may reflect the difference in the specificity factor of ribulose-1,5 bisphosphate carboxylase/oxygenase in dinoflagellates compared with other algal phyla.
Identification of the Soluble Starch Synthase Activities of Maize Endosperm1
Authors: Cao Heping , Imparl-Radosevich Jennifer , Guan Hanping , Keeling Peter L. , James Martha G. , Myers Alan M.
Published: July 2002

This study identified the complement of soluble starch synthases (SSs) present in developing maize (Zea mays) endosperm. The product of thedu1 gene, DU1, was shown to be one of the two major soluble SSs. The C-terminal 450 residues of DU1 comprise eight sequence blocks conserved in 28 known or predicted glucan synthases. This region of DU1 was expressed in Escherichia coli and shown to possess SS activity. DU1-specific antisera detected a soluble endosperm protein of more than 200 kD that was lacking in du1- mutants. These antisera eliminated 20% to 30% of the soluble SS activity from kernel extracts. Antiserum against the isozyme zSSI eliminated approximately 60% of the total soluble SS, and immunodepletion of du1- mutant extracts with this antiserum nearly eliminated SS activity. Two soluble SS activities were identified by electrophoretic fractionation, each of which correlated specifically with zSSI or DU1. Thus, DU1 and zSSI accounted for the great majority of soluble SS activity present in developing endosperm. The relative activity of the two isozymes did not change significantly during the starch biosynthetic period. DU1 and zSSI may be interdependent, because mutant extracts lacking DU1 exhibited a significant stimulation of the remaining SS activity.