bluuebunny/crossref_metadata_2025 huggingface dataset:
Dynamical symmetry group and quantum splittings for a free particle on the group manifold AdS3
Authors: Chenaghlou A. , Fakhri H.
Published: July 2005

It is shown that the set of all quantum states corresponding to the motion of a free particle on the group manifold AdS3 as the bases with two different labels, constitute a Hilbert space. The second label is bounded by the first one however, the first label is semibounded. The Casimir operator corresponding to the simultaneous and agreeable shifting generators of both labels along with the Cartan subalgebra generator describe the Hamiltonian of a free particle on AdS3 with dynamical symmetry group U(1,1) and infinite-fold degeneracy for the energy spectrum. The Hilbert space for the Lie algebra of the dynamical symmetry group is a reducible representation space. But the Hilbert subspaces constructed by all the bases which have a given constant value for the difference of two their labels, constitute an irreducible representation for it. It is also shown that the irreducible representation subspaces of the Lie algebras u(1,1) and u(2) are separately spanned by the bases which have the same value for the second and first labels, respectively. These two bunches of Hilbert subspaces present two different types of quantum splittings on the Hilbert space.
Fabrication, morphology, structure, and photoluminescence of ZnS and CdS nanoribbons
Authors: Zhou X. T. , Kim P.-S. G. , Sham T. K. , Lee S. T.
Published: July 2005

One-dimensional semiconductor nanoribbons of hexagonal wurtzite sulfides (ZnS and CdS) have been prepared in bulk quantity by a thermal evaporation technique using thiol-capped gold nanoparticles as catalysts. Compared to their starting materials, ZnS and CdS powders, the band-gap photoluminescence excited by ultraviolet light from ZnS and CdS nanoribbons at room temperature was significantly enhanced. X-ray-excited optical luminescence at the S K edge confirms the near-band-gap and the defect origin of the luminescence.
Compressibility effect on magnetic-shear-localized ideal magnetohydrodynamic interchange instability
Authors: Gupta Sangeeta , Callen J. D. , Hegna C. C.
Published: July 2005

Eigenmode analysis of a magnetic-shear-localized ideal magnetohydrodynamic interchange instability in the presence of plasma compressibility indicates the marginal stability criterion (DI=1∕4) is not affected by the compressibility effects. Above the marginal stability criterion, plasma compressibility causes a significant reduction in the growth rate of an ideal interchange instability.
Role of step edges in oxygen vacancy transport into SrTiO3(001)
Authors: Zhu X. D. , Fei Y. Y. , Lu H. B. , Yang G. Z.
Published: July 2005

Using a combination of oblique-incidence optical reflectivity difference and specular reflection high energy electron diffraction techniques, we studied vacuum annealing of pulsed-laser deposited Nb-doped SrTiO3 monolayers on SrTiO3(100) near 730°C. In oxygen-free ambient, the as-grown monolayers are oxygen deficient. The excess oxygen vacancies in the monolayers are reduced by vacancy diffusion into the bulk. We found that the reduction rate is characterized by an activation energy E=1.0eV. More interestingly, the pre-exponential factor decreases significantly as the annealing time interval increases between two successive monolayer depositions. We propose that the indiffusion of excess surface oxygen vacancies takes place at step edges, and the Oswald ripening governs the density of the latter during vacuum annealing.
Tactile microsensor elements prepared from arrayed superelastic carbon microcoils
Authors: Chen Xiuqin , Yang S. , Hasegawa M , Kawabe K. , Motojima S.
Published: July 2005

Superelastic carbon microcoils (SCMCs) with high elasticity and coiling chirality were prepared by the Ni-catalyzed pyrolysis of acetylene, and novel tactile microsensor elements using the SCMCs as the sensor material were prepared. The sensor elements with a very small size of 80×80×80μm3 showed a very high sensitivity of 0.3mgf (1Pa). It was found that the SCMC arraying in the matrix and the placement method on the electrodes dramatically affected the sensing properties.
Docking kinetics and equilibrium of a GAAA tetraloop-receptor motif probed by single-molecule FRET
Authors: Hodak Jose H. , Fiore Julie L. , Nesbitt David J. , Downey Christopher D. , Pardi Arthur
Published: July 2005

Docking kinetics and equilibrium of fluorescently labeled RNA molecules are studied with single-molecule FRET methods. Time-resolved FRET is used to monitor docking/undocking transitions for RNAs containing a single GAAA tetraloop-receptor tertiary interaction connected by a flexible single-stranded linker. The rate constants for docking and undocking are measured as a function of Mg
2+
, revealing a complex dependence on metal ion concentration. Despite the simplicity of this model system, conformational heterogeneity similar to that noted in more complex RNA systems is observed; relatively rapid docking/undocking transitions are detected for approximately two-thirds of the RNA molecules, with significant subpopulations exhibiting few or no transitions on the 10- to 30-s time scale for photobleaching. The rate constants are determined from analysis of probability densities, which allows a much wider range of time scales to be analyzed than standard histogram procedures. The data for the GAAA tetraloop receptor are compared with kinetic and equilibrium data for other RNA tertiary interactions.
An archaeal antioxidant: Characterization of a Dps-like protein from
Sulfolobus solfataricus
Authors: Wiedenheft Blake , Mosolf Jesse , Willits Deborah , Yeager Mark , Dryden Kelly A. , Young Mark , Douglas Trevor
Published: July 2005

Evolution of an oxygenic atmosphere required primordial life to accommodate the toxicity associated with reactive oxygen species. We have characterized an archaeal antioxidant from the hyperthermophilic acidophile
Sulfolobus solfataricus
. The amino acid sequence of this ≈22-kDa protein shares little sequence similarity with proteins with known function. However, the protein shares high sequence similarity with hypothetical proteins in other archaeal and bacterial genomes. Nine of these hypothetical proteins form a monophyletic cluster within the broad superfamily of ferritin-like diiron-carboxylate proteins. Higher order structural predictions and image reconstructions indicate that the
S. solfataricus
protein is structurally related to a class of
D
NA-binding
p
rotein from
s
tarved cells (Dps). The recombinant protein self assembles into a hollow dodecameric protein cage having tetrahedral symmetry (
Ss
Dps). The outer shell diameter is ≈10 nm, and the interior diameter is ≈5 nm. Dps proteins have been shown to protect nucleic acids by physically shielding DNA against oxidative damage and by consuming constituents involved in Fenton chemistry.
In vitro
, the assembled archaeal protein efficiently uses H
2
O
2
to oxidize Fe(II) to Fe(III) and stores the oxide as a mineral core on the interior surface of the protein cage. The
ssdps
gene is up-regulated in
S. solfataricus
cultures grown in iron-depleted media and upon H
2
O
2
stress, but is not induced by other stresses.
Ss
Dps-mediated reduction of hydrogen peroxide and possible DNA-binding capabilities of this archaeal Dps protein are mechanisms by which
S. solfataricus
mitigates oxidative damage.
Nephrogenic diabetes insipidus in mice lacking all nitric oxide synthase isoforms
Authors: Morishita Tsuyoshi , Tsutsui Masato , Shimokawa Hiroaki , Sabanai Ken , Tasaki Hiromi , Suda Osamu , Nakata Sei , Tanimoto Akihide , Wang Ke-Yong , Ueta Yoichi , Sasaguri Yasuyuki , Nakashima Yasuhide , Yanagihara Nobuyuki
Published: July 2005

Nitric oxide (NO) is produced in almost all tissues and organs, exerting a variety of biological actions under physiological and pathological conditions. NO is synthesized by three different isoforms of NO synthase (NOS), including neuronal, inducible, and endothelial NOSs. Because there are substantial compensatory interactions among the NOS isoforms, the ultimate roles of endogenous NO in our body still remain to be fully elucidated. Here, we have successfully developed mice in which all three NOS genes are completely deleted by crossbreeding singly NOS-/-mice. NOS expression and activities were totally absent in the triply NOS-/-mice before and after treatment with lipopolysaccharide. Although the triply NOS-/-mice were viable and appeared normal, their survival and fertility rates were markedly reduced as compared with the wild-type mice. Furthermore, these mice exhibited marked hypotonic polyuria, polydipsia, and renal unresponsiveness to an antidiuretic hormone, vasopressin, all of which are characteristics consistent with nephrogenic diabetes insipidus. In the kidney of the triply NOS-/-mice, vasopressin-induced cAMP production and membranous aquaporin-2 water channel expression were reduced associated with tubuloglomerular lesion formation. These results provide evidence that the NOS system plays a critical role in maintaining homeostasis, especially in the kidney.
Thickness of ventromedial prefrontal cortex in humans is correlated with extinction memory
Authors: Milad Mohammed R. , Quinn Brian T. , Pitman Roger K. , Orr Scott P. , Fischl Bruce , Rauch Scott L.
Published: July 2005

The ventromedial prefrontal cortex (vmPFC) has been implicated in fear extinction [Phelps, E. A., Delgado, M. R., Nearing, K. I. & Ledoux, J. E. (2004)
Neuron
43, 897-905; Herry, C. & Garcia, R. (2003)
Behav. Brain Res
. 146, 89-96]. Here, we test the hypothesis that the cortical thickness of vmPFC regions is associated with how well healthy humans retain their extinction memory a day after having been conditioned and then extinguished. Fourteen participants underwent a 2-day fear conditioning and extinction protocol. The conditioned stimuli (CSs) were pictures of virtual lights, and the unconditioned stimulus (US) was an electric shock. On day 1, participants received 5 CS+US pairings (conditioning), followed by 10 CS trials with no US (extinction). On day 2, the CS was presented alone to test for extinction memory. Skin conductance response (SCR) was the behavioral index of conditioning and extinction. Participants underwent MRI scans to obtain structural images, from which cortical thickness was measured. We performed a vertex-based analysis across the entire cortical surface and a region-of-interest analysis of
a priori
hypothesized territories to measure cortical thickness and map correlations between this measure and SCR. We found significant, direct correlation between thickness of the vmPFC, specifically medial orbitofrontal cortex, and extinction retention. That is, thicker medial orbitofrontal cortex was associated with lower SCR to the conditioned stimulus during extinction recall (i.e., greater extinction memory). These results suggest that the size of the vmPFC might explain individual differences in the ability to modulate fear among humans.
Loss of the
mtr
operon in
Methanosarcina
blocks growth on methanol, but not methanogenesis, and reveals an unknown methanogenic pathway
Authors: Welander Paula V. , Metcalf William W.
Published: July 2005

In the methanogenic archaeon
Methanosarcina barkeri
Fusaro, the
N
5
-methyl-tetrahydrosarcinapterin (CH
3
-H
4
SPT):coenzyme M (CoM) methyltransferase, encoded by the
mtr
operon, catalyzes the energy-conserving (sodium-pumping) methyl transfer from CH
3
-H
4
SPT to CoM during growth on H
2
/CO
2
or acetate. However, in the disproportionation of C-1 compounds, such as methanol, to methane and carbon dioxide, it catalyzes the reverse, endergonic transfer from methyl-CoM to H
4
SPT, which is driven by sodium uptake. It has been proposed that a bypass for this energy-consuming reaction may occur via a direct methyl transfer from methanol to H
4
SPT. To test this, an
mtr
deletion mutant was constructed and characterized in
M. barkeri
Fusaro. The mutant is unable to grow on methanol, acetate or H
2
/CO
2
, but can grow on methanol with H
2
/CO
2
and, surprisingly, methanol with acetate.
13
C labeling experiments show that growth on acetate with methanol involves a previously unknown methanogenic pathway, in which oxidation of acetate to a mixture of CO
2
and formic acid is coupled to methanol reduction. Interestingly, although the mutant is unable to grow on methanol alone, it remains capable of producing methane from this substrate. Thus, the proposed Mtr bypass does exist, but is unable to support growth of the organism.
Slc7a11
gene controls production of pheomelanin pigment and proliferation of cultured cells
Authors: Chintala Sreenivasulu , Li Wei , Lamoreux M. Lynn , Ito Shosuke , Wakamatsu Kazumasa , Sviderskaya Elena V. , Bennett Dorothy C. , Park Young-Mee , Gahl William A. , Huizing Marjan , Spritz Richard A. , Ben Songtao , Novak Edward K. , Tan Jian , Swank Richard T.
Published: July 2005

In mammals, >100 genes regulate pigmentation by means of a wide variety of developmental, cellular, and enzymatic mechanisms. Nevertheless, genes that directly regulate pheomelanin production have not been described. Here, we demonstrate that the subtle gray (
sut
) mouse pigmentation mutant arose by means of a mutation in the
Slc7a11
gene, encoding the plasma membrane cystine/glutamate exchanger xCT [Kanai, Y. & Endou, H. (2001)
Curr. Drug Metab.
2, 339-354]. A resulting low rate of extracellular cystine transport into
sut
melanocytes reduces pheomelanin production. We show that
Slc7a11
is a major genetic regulator of pheomelanin pigment in hair and melanocytes, with minimal or no effects on eumelanin. Furthermore, transport of cystine by xCT is critical for normal proliferation, glutathione production, and protection from oxidative stress in cultured cells. Thus, we have found that the
Slc7a11
gene controls the production of pheomelanin pigment directly. Cells from
sut
mice provide a model for oxidative stress-related diseases and their therapies.
Characterization of complex apurinic/apyrimidinic-site clustering associated with an authentic site-specific radiation-induced DNA double-strand break
Authors: Datta Kamal , Neumann Ronald D. , Winters Thomas A.
Published: July 2005

Radiation lethality is largely attributed to radiation-induced DNA double-strand breaks (DSBs). A range of structural complexity is predicted for radiation-induced DSBs. However, this lesion has never been analyzed in isolation at the molecular level. To address this problem, we have created authentic site-specific radiation-induced DSBs in plasmid DNA by triplex-forming oligonucleotide-targeted
125
I decay. No significant difference in DSB yield was observed after irradiation in the presence or absence of the radical scavenger DMSO, suggesting that DSB formation is a result of the direct effect of the radiation. A restriction fragment terminated by the DSB was isolated and probed with the
Escherichia coli
DNA repair enzyme endonuclease IV (endo IV), which recognizes apurinic/apyrimidinic (AP) sites. Enzymatic probing demonstrated clustering of AP sites within 10 bases of the
125
I-targeted base in the DNA duplex. Our results suggest scavengeable radicals may not play a large role in the generation of AP sites associated with DSB formation, because at least 30% of all fragments have endo IV-sensitive sites, regardless of irradiation conditions. An internal control fragment recovered from the
125
I linearized plasmid did not exhibit endo IV sensitivity in excess of that observed for a similar fragment recovered from an undamaged plasmid. Thus, AP site clustering proximal to the DSB resulted from the
125
I decays responsible for DSB formation and was not due to untargeted background irradiation.
Proliferation and invasion: Plasticity in tumor cells
Authors: Gao Chong-Feng , Xie Qian , Su Yan-Li , Koeman Julie , Khoo Sok Kean , Gustafson Margaret , Knudsen Beatrice S. , Hay Rick , Shinomiya Nariyoshi , Woude George F. Vande
Published: July 2005

Invasive and proliferative phenotypes are fundamental components of malignant disease, yet basic questions persist about whether tumor cells can express both phenotypes simultaneously and, if so, what are their properties. Suitable
in vitro
models that allow characterization of cells that are purely invasive are limited because proliferation is required for cell maintenance. Here, we describe glioblastoma cells that are highly invasive in response to hepatocyte growth factor/scatter factor (HGF/SF). From this cell population, we selected subclones that were highly proliferative or displayed both invasive and proliferative phenotypes. The biological activities of invasion, migration, urokinase-type plasminogen activation, and branching morphogenesis exclusively partitioned with the highly invasive cells, whereas the highly proliferative subcloned cells uniquely displayed anchorage independent growth in soft agar and were highly tumorigenic as xenografts in immune-compromised mice. In response to HGF/SF, the highly invasive cells signal through the MAPK pathway, whereas the selection of the highly proliferative cells coselected for signaling through Myc. Moreover, in subcloned cells displaying both invasive and proliferative phenotypes, both signaling pathways are activated by HGF/SF. These results show how the mitogen-activated protein kinase and Myc pathways can cooperate to confer both invasive and proliferative phenotypes on tumor cells and provide a system for studying how transitions between invasion and proliferation can contribute to malignant progression.
Regulation of human methylenetetrahydrofolate reductase by phosphorylation
Authors: Yamada Kazuhiro , Strahler John R. , Andrews Philip C. , Matthews Rowena G.
Published: July 2005

Methylenetetrahydrofolate reductase (MTHFR) catalyzes the reduction of methylenetetrahydrofolate to methyltetrahydrofolate, the methyl donor for the conversion of homocysteine to methionine. Regulation of MTHFR activity is crucial for maintaining cellular concentrations of methionine and
S
-adenosylmethionine (AdoMet). Purified recombinant human MTHFR expressed in insect cells is multiply phosphorylated on an N-terminal extension of the protein that contains a highly conserved serine-rich region. Treatment by alkaline phosphatase removes seven phosphoryl groups from the enzyme. Thr-34 was identified as one of the seven phosphorylation sites by using a monoclonal antibody directed toward
p
Thr-Pro. Mutation of Thr-34 to Ala completely blocks modification as judged by mass spectrometric analysis, suggesting that Thr-34 is the priming phosphorylation site. The Thr34Ala mutant was expressed in baculovirus-infected insect cells, and its enzymic properties were compared with wild-type enzyme. The mutant enzyme and alkaline phosphatase-treated wild-type enzyme are more active than untreated wild-type enzyme and less sensitive to inhibition by saturating AdoMet, indicating that phosphorylation at Thr-34 is critical for allosteric regulation of human MTHFR activity by AdoMet. The absence of methionine and the presence of adenosine in the cell culture medium, which lead to a low intracellular AdoMet/
S
-adenosylhomocysteine ratio, are associated with faster electrophoretic mobility of MTHFR, presumably because of less or no phosphorylation. Because the faster-mobility MTHFR is associated with the more active form of MTHFR, this response is likely to increase methionine production. Those observations suggest that AdoMet functions not only as an allosteric inhibitor but also to control phosphorylation of human MTHFR.
Effective function annotation through catalytic residue conservation
Authors: George Richard A. , Spriggs Ruth V. , Bartlett Gail J. , Gutteridge Alex , MacArthur Malcolm W. , Porter Craig T. , Al-Lazikani Bissan , Thornton Janet M. , Swindells Mark B.
Published: July 2005

Because of the extreme impact of genome sequencing projects, protein sequences without accompanying experimental data now dominate public databases. Homology searches, by providing an opportunity to transfer functional information between related proteins, have become the de facto way to address this. Although a single, well annotated, close relationship will often facilitate sufficient annotation, this situation is not always the case, particularly if mutations are present in important functional residues. When only distant relationships are available, the transfer of function information is more tenuous, and the likelihood of encountering several well annotated proteins with different functions is increased. The consequence for a researcher is a range of candidate functions with little way of knowing which, if any, are correct. Here, we address the problem directly by introducing a computational approach to accurately identify and segregate related proteins into those with a functional similarity and those where function differs. This approach should find a wide range of applications, including the interpretation of genomics/proteomics data and the prioritization of targets for high-throughput structure determination. The method is generic, but here we concentrate on enzymes and apply high-quality catalytic site data. In addition to providing a series of comprehensive benchmarks to show the overall performance of our approach, we illustrate its utility with specific examples that include the correct identification of haptoglobin as a nonenzymatic relative of trypsin, discrimination of acid-d-amino acid ligases from a much larger ligase pool, and the successful annotation of BioH, a structural genomics target.
Yeast prions [URE3] and [PSI
+
] are diseases
Authors: Nakayashiki Toru , Kurtzman Cletus P. , Edskes Herman K. , Wickner Reed B.
Published: July 2005

Viruses, plasmids, and prions can spread in nature despite being a burden to their hosts. Because a prion arises
de novo
in more than one in 10
6
yeast cells and spreads to all offspring in meiosis, its absence in wild strains would imply that it has a net deleterious effect on its host. Among 70 wild
Saccharomyces
strains, we found the [
PIN
+
] prion in 11 strains, but the [URE3] and [
PSI
+
] prions were uniformly absent. In contrast, the “selfish” 2μ DNA was in 38 wild strains and the selfish RNA replicons L-BC, 20S, and 23S were found in 8, 14, and 1 strains, respectively. The absence of [URE3] and [
PSI
+
] in wild strains indicates that each prion has a net deleterious effect on its host.
Towards performance control on the Grid
Authors: Mayes K.R , Luján M , Riley G.D , Chin J , Coveney P.V , Gurd J.R
Published: July 2005

Advances in computational Grid technologies are enabling the development of simulations of complex biological and physical systems. Such simulations can be assembled from separate components—separately deployable computation units of well-defined functionality. Such an assemblage can represent an application composed of interacting simulations or might comprise multiple instances of a simulation executing together, each running with different simulation parameters. However, such assemblages need the ability to cope with heterogeneous and dynamically changing execution environments, particularly where such changes can affect performance.

This paper describes the design and implementation of a prototype performance control system (PerCo), which is capable of monitoring the progress of simulations and redeploying them so as to optimize performance. The ability to control performance by redeployment is demonstrated using an assemblage of lattice Boltzmann simulations running with and without control policies. The cost of using PerCo is evaluated and it is shown that PerCo is able to reduce overall execution time.
Grid-based steered thermodynamic integration accelerates the calculation of binding free energies
Authors: Fowler Philip W , Jha Shantenu , Coveney Peter V
Published: July 2005

The calculation of binding free energies is important in many condensed matter problems. Although formally exact computational methods have the potential to complement, add to, and even compete with experimental approaches, they are difficult to use and extremely time consuming. We describe a Grid-based approach for the calculation of relative binding free energies, which we call Steered Thermodynamic Integration calculations using Molecular Dynamics (STIMD), and its application to Src homology 2 (SH2) protein cell signalling domains. We show that the time taken to compute free energy differences using thermodynamic integration can be significantly reduced: potentially from weeks or months to days of wall-clock time. To be able to perform such accelerated calculations requires the ability to both run concurrently and control in realtime several parallel simulations on a computational Grid. We describe how the RealityGrid computational steering system, in conjunction with a scalable classical MD code, can be used to dramatically reduce the time to achieve a result. This is necessary to improve the adoption of this technique and further allows more detailed investigations into the accuracy and precision of thermodynamic integration. Initial results for the Src SH2 system are presented and compared to a reported experimental value. Finally, we discuss the significance of our approach.
Evaluation of regulatory potential and conservation scores for detecting cis-regulatory modules in aligned mammalian genome sequences
Authors: King David C. , Taylor James , Elnitski Laura , Chiaromonte Francesca , Miller Webb , Hardison Ross C.
Published: July 2005

Techniques of comparative genomics are being used to identify candidate functional DNA sequences, and objective evaluations are needed to assess their effectiveness. Different analytical methods score distinctive features of whole-genome alignments among human, mouse, and rat to predict functional regions. We evaluated three of these methods for their ability to identify the positions of known regulatory regions in the well-studied HBB gene complex. Two methods, multispecies conserved sequences and phastCons, quantify levels of conservation to estimate a likelihood that aligned DNA sequences are under purifying selection. A third function, regulatory potential (RP), measures the similarity of patterns in the alignments to those in known regulatory regions. The methods can correctly identify 50%–60% of noncoding positions in the HBB gene complex as regulatory or nonregulatory, with RP performing better than do other methods. When evaluated by the ability to discriminate genomic intervals, RP reaches a sensitivity of 0.78 and a true discovery rate of ∼0.6. The performance is better on other reference sets; both phastCons and RP scores can capture almost all regulatory elements in those sets along with ∼7% of the human genome.
Gene-breaking: A new paradigm for human retrotransposon-mediated gene evolution
Authors: Wheelan Sarah J. , Aizawa Yasunori , Han Jeffrey S. , Boeke Jef D.
Published: July 2005

The L1 retrotransposon is the most highly successful autonomous retrotransposon in mammals. This prolific genome parasite may on occasion benefit its host through genome rearrangements or adjustments of host gene expression. In examining possible effects of L1 elements on host gene expression, we investigated whether a full-length L1 element inserted in the antisense orientation into an intron of a cellular gene may actually split the gene's transcript into two smaller transcripts: (1) a transcript containing the upstream exons and terminating in the major antisense polyadenylation site (MAPS) of the L1, and (2) a transcript derived from the L1 antisense promoter (ASP) that includes the downstream exons of the gene. Bioinformatic analysis and experimental follow-up provide evidence for this L1 “gene-breaking” hypothesis. We identified three human genes apparently “broken” by L1 elements, as well as 12 more candidate genes. Most of the inserted L1 elements in our 15 candidate genes predate the human/chimp divergence. If indeed split, the transcripts of these genes may in at least one case encode potentially interacting proteins, and in another case may encode novel proteins. Gene-breaking represents a new mechanism through which L1 elements remodel mammalian genomes.
Evolutionarily conserved elements in vertebrate, insect, worm, and yeast genomes
Authors: Siepel Adam , Bejerano Gill , Pedersen Jakob S. , Hinrichs Angie S. , Hou Minmei , Rosenbloom Kate , Clawson Hiram , Spieth John , Hillier LaDeana W. , Richards Stephen , Weinstock George M. , Wilson Richard K. , Gibbs Richard A. , Kent W. James , Miller Webb , Haussler David
Published: July 2005

We have conducted a comprehensive search for conserved elements in vertebrate genomes, using genome-wide multiple alignments of five vertebrate species (human, mouse, rat, chicken, and Fugu rubripes). Parallel searches have been performed with multiple alignments of four insect species (three species of Drosophila and Anopheles gambiae), two species of Caenorhabditis, and seven species of Saccharomyces. Conserved elements were identified with a computer program called phastCons, which is based on a two-state phylogenetic hidden Markov model (phylo-HMM). PhastCons works by fitting a phylo-HMM to the data by maximum likelihood, subject to constraints designed to calibrate the model across species groups, and then predicting conserved elements based on this model. The predicted elements cover roughly 3%–8% of the human genome (depending on the details of the calibration procedure) and substantially higher fractions of the more compact Drosophila melanogaster (37%–53%), Caenorhabditis elegans (18%–37%), and Saccharaomyces cerevisiae (47%–68%) genomes. From yeasts to vertebrates, in order of increasing genome size and general biological complexity, increasing fractions of conserved bases are found to lie outside of the exons of known protein-coding genes. In all groups, the most highly conserved elements (HCEs), by log-odds score, are hundreds or thousands of bases long. These elements share certain properties with ultraconserved elements, but they tend to be longer and less perfectly conserved, and they overlap genes of somewhat different functional categories. In vertebrates, HCEs are associated with the 3′ UTRs of regulatory genes, stable gene deserts, and megabase-sized regions rich in moderately conserved noncoding sequences. Noncoding HCEs also show strong statistical evidence of an enrichment for RNA secondary structure.
Random mutagenesis of proximal mouse chromosome 5 uncovers predominantly embryonic lethal mutations
Authors: Wilson Lawriston , Ching Yung-Hao , Farias Michael , Hartford Suzanne A. , Howell Gareth , Shao Hongguang , Bucan Maja , Schimenti John C.
Published: July 2005

A region-specific ENU mutagenesis screen was conducted to elucidate the functional content of proximal mouse Chr 5. We used the visibly marked, recessive, lethal inversion Rump White (Rw) as a balancer in a three-generation breeding scheme to identify recessive mutations within the ∼50 megabases spanned by Rw. A total of 1003 pedigrees were produced, representing the largest inversion screen performed in mice. Test-class animals, homozygous for the ENU-mutagenized proximal Chr 5 and visibly distinguishable from nonhomozygous littermates, were screened for fertility, hearing, vestibular function, DNA repair, behavior, and dysmorphology. Lethals were identifiable by failure to derive test-class animals within a pedigree. Embryonic lethal mutations (total of 34) were overwhelmingly the largest class of mutants recovered. We characterized them with respect to the time of embryonic death, revealing that most act at midgestation (8.5–10.5) or sooner. To position the mutations within the Rw region and to guide allelism tests, we performed complementation analyses with a set of new and existing chromosomal deletions, as well as standard recombinational mapping on a subset of the mutations. By pooling the data from this and other region-specific mutagenesis projects, we calculate that the mouse genome contains ∼3479–4825 embryonic lethal genes, or about 13.7%–19% of all genes.
Identification of novel mammalian growth regulatory factors by genome-scale quantitative image analysis
Authors: Harada Josephine N. , Bower Kristen E. , Orth Anthony P. , Callaway Scott , Nelson Christian G. , Laris Casey , Hogenesch John B. , Vogt Peter K. , Chanda Sumit K.
Published: July 2005

Functional profiling technologies using arrayed collections of genome-scale siRNA and cDNA arrayed libraries enable the comprehensive global analysis of gene function. However, the current repertoire of high-throughput detection methodologies has limited the scope of cellular phenotypes that can be studied. In this report, we describe the systematic identification of mammalian growth-regulatory factors achieved through the integration of automated microscopy, pattern recognition analysis, and cell-based functional genomics. The effects of 7364 human and mouse proteins, encoded by individually arrayed cDNAs, upon proliferation and viability in U2OS osteosarcoma cells were evaluated in a live-cell, kinetic assay using quantitative image analysis. Overexpression of more than 86 cDNAs (1.15%) conferred dramatic increases in the proliferation, as determined cell enumeration. These included several known growth regulators, as well as previously uncharacterized ones (LRRK1, Ankrd25). In addition, novel functional roles for two genes (5033414D02Rik, 2810429O05Rik), now termed Gatp1 and Gatp2, respectively, were identified. Further analysis demonstrated that these encoded proteins promoted cellular proliferation and transformation in primary cells. Conversely, cells depleted for Gatp1 underwent apoptosis upon serum reduction, suggesting that Gatp1 is essential for cell survival under growth-factor-restricted conditions. Taken together, our findings offer new insight into the regulation of cellular growth and proliferation, and demonstrate the value and feasibility of assessing cellular phenotypes through genome-level computational image analysis.
The scale of mutational variation in the murid genome
Authors: Gaffney Daniel J. , Keightley Peter D.
Published: July 2005

Mutation rates vary across mammalian genomes, but little is known about the scale over which this variation occurs. Knowledge of the magnitude and scale of mutational variation is required to understand the processes that drive mutation, and is essential in formulating a robust null hypothesis for comparative genomics studies. Here we estimate the scale of mutational variation in the murid genome by calculating the spatial autocorrelation of nucleotide substitution rates in ancestral repeats. Such transposable elements are good candidates for neutrally evolving sequence and therefore well suited for the study of mutation rate variation. We find that the autocorrelation coefficient decays to a value close to zero by ∼15 Mb, with little apparent variation in mutation rate under 100 kb. We conclude that the primary scale over which mutation rates vary is subchromosomal. Furthermore, our analysis shows that within-chromosome mutational variability exceeds variation among chromosomes by approximately one order of magnitude. Thus, differences in mutation rate between different regions of the same chromosome frequently exceed differences both between whole autosomes and between autosomes and the X-chromosome. Our results indicate that factors other than the time spent in the male germ line are important in driving mutation rates. This raises questions about the biological mechanism(s) that produce new mutations and has implications for the study of male-driven evolution.
Human ribosomal RNA gene arrays display a broad range of palindromic structures
Authors: Caburet Sandrine , Conti Chiara , Schurra Catherine , Lebofsky Ronald , Edelstein Stuart J. , Bensimon Aaron
Published: July 2005

The standard model of eukaryotic ribosomal RNA (rRNA) genes involves tandem arrays with hundreds of units in clusters, the nucleolus organizer regions (NORs). A first genomic overview for human cells is reported here for these regions, which have never been sequenced in their totality, by using molecular combing. The rRNA-coding regions are examined by fluorescence on single molecules of DNA with two specific probes that cover their entire length. The standard organization assumed for rDNA units is a transcribed region followed by a nontranscribed spacer. While we confirmed this arrangement in many cases, unorthodox patterns were also observed in normal individuals, with one-third of the rDNA units rearranged to form apparently palindromic structures (noncanonical units) independent of the age of the donors. In cells from individuals with a deficiency in the WRN RecQ helicase (Werner syndrome), the proportion of palindromes increased to one-half. These findings, supported by Southern blot analyses, show that rRNA genes are a mosaic of canonical and (presumably nonfunctional) palindromic units that may be altered by factors associated with genomic instability and pathology.
A functional survey of the enhancer activity of conserved non-coding sequences from vertebrateIroquoiscluster gene deserts
Authors: de la Calle-Mustienes Elisa , Feijóo Cármen Gloria , Manzanares Miguel , Tena Juan J. , Rodríguez-Seguel Elisa , Letizia Annalisa , Allende Miguel L. , Gómez-Skarmeta José Luis
Published: July 2005

Recent studies of the genome architecture of vertebrates have uncovered two unforeseen aspects of its organization. First, large regions of the genome, called gene deserts, are devoid of protein-coding sequences and have no obvious biological role. Second, comparative genomics has highlighted the existence of an array of highly conserved non-coding regions (HCNRs) in all vertebrates. Most surprisingly, these structural features are strongly associated with genes that have essential functions during development. Among these, the vertebrateIroquois(Irx) genes stand out on both fronts. MammalianIrxgenes are organized in two clusters (IrxAandIrxB) that span >1 Mb each with no other genes interspersed. Additionally, a large number of HCNRs exist withinIrxclusters. We have systematically examined the enhancer activity of HCNRs from theIrxBcluster using transgenicXenopusand zebrafish embryos. Most of these HCNRs are active in subdomains of endogenousIrxexpression, and some are candidates to contain shared enhancers of neighboring genes, which could explain the evolutionary conservation ofIrxclusters. Furthermore, HCNRs present in tetrapodIrxBbut not in fish may be responsible for novelIrxexpression domains that appeared after their divergence. Finally, we have performed a more detailed analysis on twoIrxBultraconserved non-coding regions (UCRs) duplicated inIrxAclusters in similar relative positions. These four regions share a core region highly conserved among all of them and drive expression in similar domains. However, inter-species conserved sequences surrounding the core, specific for each of these UCRs, are able to modulate their expression.
TaVRT-2, a Member of the StMADS-11 Clade of Flowering Repressors, Is Regulated by Vernalization and Photoperiod in Wheat
Authors: Kane Ndjido A. , Danyluk Jean , Tardif Guylaine , Ouellet François , Laliberté Jean-François , Limin Allen E. , Fowler D. Brian , Sarhan Fathey
Published: July 2005

The initiation of the reproductive phase in winter cereals is delayed during winter until favorable growth conditions resume in the spring. This delay is modulated by low temperature through the process of vernalization. The molecular and genetic bases of the interaction between environmental factors and the floral transition in these species are still unknown. However, the recent identification of the wheat (Triticum aestivum L.) TaVRT-1 gene provides an opportunity to decipher the molecular basis of the flowering-time regulation in cereals. Here, we describe the characterization of another gene, named TaVRT-2, possibly involved in the flowering pathway in wheat. Molecular and phylogenetic analyses indicate that the gene encodes a member of the MADS-box transcription factor family that belongs to a clade responsible for flowering repression in several species. Expression profiling of TaVRT-2 in near-isogenic lines and different genotypes with natural variation in their response to vernalization and photoperiod showed a strong relationship with floral transition. Its expression is up-regulated in the winter genotypes during the vegetative phase and in photoperiod-sensitive genotypes during short days, and is repressed by vernalization to a level that allows the transition to the reproductive phase. Protein-protein interaction studies revealed that TaVRT-2 interacts with proteins encoded by two important vernalization genes (TaVRT-1/VRN-1 and VRN-2) in wheat. These results support the hypothesis that TaVRT-2 is a putative repressor of the floral transition in wheat.
Development of a Virus-Induced Gene-Silencing System for Hexaploid Wheat and Its Use in Functional Analysis of the Lr21-Mediated Leaf Rust Resistance Pathway
Authors: Scofield Steven R. , Huang Li , Brandt Amanda S. , Gill Bikram S.
Published: July 2005

Virus-induced gene silencing (VIGS) is an important tool for the analysis of gene function in plants. In VIGS, viruses engineered to carry sequences derived from plant gene transcripts activate the host's sequence-specific RNA degradation system. This mechanism targets the RNAs of the viral genome for degradation, and as the virus contains transcribed plant sequence, homologous host mRNAs are also targeted for destruction. While routinely used in some dicots, no VIGS system was known for monocot plants until the recent report of silencing in barley (Hordeum vulgare) by barley stripe mosaic virus (BSMV). Here, we report development of protocols for use of BSMV to efficiently silence genes in hexaploid wheat (Triticum aestivum). The VIGS system was first optimized in studies silencing phytoene desaturase expression. Next, we used it to assay genes functioning in leaf rust resistance mediated by Lr21, which encodes a nucleotide binding site-leucine-rich repeat class resistance gene product. We demonstrated that infection with BSMV constructs carrying a 150-bp fragment of Lr21 caused conversion of incompatible interactions to compatible, whereas infection with a control construct or one that silences phytoene desaturase had no effect on resistance or susceptibility. Additionally, silencing the RAR1, SGT1, and HSP90 genes, known to be required in many but not all nucleotide binding site-leucine-rich repeat resistance pathways in diverse plant species, resulted in conversion to compatibility, indicating that these genes are essential in Lr21-mediated resistance. These studies indicate that BSMV-VIGS is a powerful tool for dissecting the genetic pathways of disease resistance in hexaploid wheat.
SERK and APOSTART. Candidate Genes for Apomixis in Poa pratensis
Authors: Albertini Emidio , Marconi Gianpiero , Reale Lara , Barcaccia Gianni , Porceddu Andrea , Ferranti Francesco , Falcinelli Mario
Published: July 2005

Seed production generally requires the mating of opposite sex gametes. Apomixis, an asexual mode of reproduction, avoids both meiotic reduction and egg fertilization. The essential feature of apomixis is that an embryo is formed autonomously by parthenogenesis from an unreduced egg of an embryo sac generated through apomeiosis. If apomixis were well understood and harnessed, it could be exploited to indefinitely propagate superior hybrids or specific genotypes bearing complex gene sets. A more profound knowledge of the mechanisms that regulate reproductive events would contribute fundamentally to understanding the genetic control of the apomictic pathway. In Poa pratensis, we isolated and characterized two genes, PpSERK (SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE) and APOSTART. These full-length genes were recovered by rapid amplification of cDNA ends and their temporal and spatial expression patterns were assessed by reverse transcription-polymerase chain reaction and in situ hybridization, respectively. The expression of PpSERK and APOSTART differed in apomictic and sexual genotypes. Their putative role in cell-signaling transduction cascades and trafficking events required during sporogenesis, gametogenesis, and embryogenesis in plants is reported and discussed. We propose that, in nucellar cells of apomictic genotypes, PpSERK is the switch that channels embryo sac development and that it may also redirect signaling gene products to compartments other than their typical ones. The involvement of APOSTART in meiosis and programmed cell death is also discussed.
A New Substrate Cycle in Plants. Evidence for a High Glucose-Phosphate-to-Glucose Turnover from in Vivo Steady-State and Pulse-Labeling Experiments with [13C]Glucose and [14C]Glucose
Authors: Alonso Ana Paula , Vigeolas Hélène , Raymond Philippe , Rolin Dominique , Dieuaide-Noubhani Martine
Published: July 2005

Substrate (futile) cycling involving carbohydrate turnover has been widely reported in plant tissues, although its extent, mechanisms, and functions are not well known. In this study, two complementary approaches, short and steady-state labeling experiments, were used to analyze glucose metabolism in maize (Zea mays) root tips. Unidirectional rates of synthesis for storage compounds (starch, Suc, and cell wall polysaccharides) were determined by short labeling experiments using [U-14C]glucose and compared with net synthesis fluxes to determine the rate of glucose production from these storage compounds. Steady-state labeling with [1-13C]glucose and [U-13C]glucose showed that the redistribution of label between carbon C-1 and C-6 in glucose is close to that in cytosolic hexose-P. These results indicate a high resynthesis flux of glucose from hexose-P that is not accounted for by glucose recycling from storage compounds, thus suggesting the occurrence of a direct glucose-P-to-glucose conversion. An enzyme assay confirmed the presence of substantial glucose-6-phosphatase activity in maize root tips. This new glucose-P-to-glucose cycle was shown to consume around 40% of the ATP generated in the cell, whereas Suc cycling consumes at most 3% to 6% of the ATP produced. The rate of glucose-P cycling differs by a factor of 3 between a maize W22 line and the hybrid maize cv Dea, and is significantly decreased by a carbohydrate starvation pretreatment.
Rapid Phosphorylation of a Syntaxin during the Avr9/Cf-9-Race-Specific Signaling Pathway
Authors: Heese Antje , Ludwig Andrea A. , Jones Jonathan D.G.
Published: July 2005

The tomato (Lycopersicon esculentum) resistance (R) gene Cf-9 is required for resistance to races of the fungal pathogen Cladosporium fulvum expressing the elicitor Avr9 and also confers responsiveness to Avr9 in Cf-9-containing transgenic tobacco (Nicotiana tabacum; Cf9 tobacco). Although protein phosphorylation is required for many early Avr9/Cf-9-signaling events, so far the only phosphorylation targets known in this race-specific signaling pathway are three kinases: the two mitogen-activated protein kinases, wound-induced protein kinase and salicylic acid-induced protein kinase, and the calcium-dependent protein kinase NtCDPK2. Here, we provide evidence that a tobacco syntaxin is rapidly and transiently phosphorylated after Avr9 elicitation. The syntaxin was detected with an antibody against NtSyp121, a plasma membrane-localized syntaxin implicated in abscisic acid responses and secretion. Consistent with the gene-for-gene hypothesis, syntaxin phosphorylation required the presence of both Avr9 and Cf-9. This phosphorylation event occurred either upstream of the pathway leading to reactive oxygen species production or in a parallel pathway. Interestingly, rapid syntaxin phosphorylation was triggered by the race-specific elicitor Avr9 but not by flg22P.aer, a general elicitor capable of inducing other defense-related signaling events in Cf9 tobacco such as reactive oxygen species production, mitogen-activated protein kinase activation, and PR5 transcript up-regulation. Furthermore, NtSyp121 transcript levels were increased at 24 h after elicitation with Avr9 but not with flg22P.aer. Because most other previously described Avr9- and flg22P.aer-elicited responses are similar, syntaxin phosphorylation and NtSyp121 transcript up-regulation may serve as novel early biochemical and late molecular markers, respectively, to elucidate further differences in the signaling responses between these two elicitors.