bluuebunny/crossref_metadata_2025 huggingface dataset:
Panmixia in the European eel: a matter of time…
Authors: Dannewitz Johan , Maes Gregory E , Johansson Leif , Wickström Håkan , Volckaert Filip A.M , Järvi Torbjörn
Published: June 2005

The European eel (Anguilla anguillaL.) has been a prime example of the panmixia paradigm because of its extraordinary adaptation to the North Atlantic gyral system, semelparous spawning in the Sargasso Sea and long trans-oceanic migration. Recently, this view was challenged by the suggestion of a genetic structure characterized by an isolation-by-distance (IBD) pattern. This is only likely if spawning subpopulations are spatially and/or temporally separated, followed by non-random larval dispersal. A limitation of previous genetic work on eels is the lack of replication over time to test for temporal stability of genetic structure. Here, we hypothesize that temporal genetic variation plays a significant role in explaining the spatial structure reported earlier for this species. We tested this by increasing the texture of geographical sampling and by including temporal replicates. Overall genetic differentiation among samples was low, highly significant and comparable with earlier studies (FST=0.0014;p<0.01). On the other hand, and in sharp contrast with current understandings, hierarchical analyses revealed no significant inter-location genetic heterogeneity and hence no IBD. Instead, genetic variation among temporal samples within sites clearly exceeded the geographical component. Our results provide support for the panmixia hypothesis and emphasize the importance of temporal replication when assessing population structure of marine fish species.
Footer: A quantitative comparative genomics method for efficient recognition ofcis-regulatory elements
Authors: Corcoran David L. , Feingold Eleanor , Dominick Jessica , Wright Marietta , Harnaha Jo , Trucco Massimo , Giannoukakis Nick , Benos Panayiotis V.
Published: June 2005

The search for mammalian DNA regulatory regions poses a challenging problem in computational biology. The short length of the DNA patterns compared with the size of the promoter regions and the degeneracy of the patterns makes their identification difficult. One way to overcome this problem is to use evolutionary information to reduce the number of false-positive predictions. We developed a novel method for pattern identification that compares a pair of putative binding sites in two species (e.g., human and mouse) and assigns two probability scores based on the relative position of the sites in the promoter and their agreement with a known model of binding preferences. We tested the algorithm's ability to predict known binding sites on various promoters. Overall, it exhibited 83%sensitivityand thespecificitywas 72%, which is a clear improvement over existing methods. Our algorithm also successfully predicted two novel NF-κB binding sites in the promoter region of the mouse autotaxin gene (ATX, ENPP2), which we were able to verify by using chromatin immunoprecipitation assay coupled with quantitative real-time PCR.
A BAC-based physical map of theDrosophila buzzatiigenome
Authors: González Josefa , Nefedov Michael , Bosdet Ian , Casals Ferran , Calvete Oriol , Delprat Alejandra , Shin Heesun , Chiu Readman , Mathewson Carrie , Wye Natasja , Hoskins Roger A. , Schein Jacqueline E. , de Jong Pieter , Ruiz Alfredo
Published: June 2005

Large-insert genomic libraries facilitate cloning of large genomic regions, allow the construction of clone-based physical maps, and provide useful resources for sequencing entire genomes.Drosophila buzzatiiis a representative species of therepletagroup in theDrosophilasubgenus, which is being widely used as a model in studies of genome evolution, ecological adaptation, and speciation. We constructed a Bacterial Artificial Chromosome (BAC) genomic library ofD. buzzatiiusing the shuttle vector pTARBAC2.1. The library comprises 18,353 clones with an average insert size of 152 kb and an ∼18× expected representation of theD. buzzatiieuchromatic genome. We screened the entire library with six euchromatic gene probes and estimated the actual genome representation to be ∼23×. In addition, we fingerprinted by restriction digestion and agarose gel electrophoresis a sample of 9555 clones, and assembled them using FingerPrint Contigs (FPC) software and manual editing into 345 contigs (mean of 26 clones per contig) and 670 singletons. Finally, we anchored 181 large contigs (containing 7788 clones) to theD. buzzatiisalivary gland polytene chromosomes by in situ hybridization of 427 representative clones. The BAC library and a database with all the information regarding the high coverage BAC-based physical map described in this paper are available to the research community.
Genome-wide prediction of imprinted murine genes
Authors: Luedi Philippe P. , Hartemink Alexander J. , Jirtle Randy L.
Published: June 2005

Imprinted genes are epigenetically modified genes whose expression is determined according to their parent of origin. They are involved in embryonic development, and imprinting dysregulation is linked to cancer, obesity, diabetes, and behavioral disorders such as autism and bipolar disease. Herein, we train a statistical model based on DNA sequence characteristics that not only identifies potentially imprinted genes, but also predicts the parental allele from which they are expressed. Of 23,788 annotated autosomal mouse genes, our model identifies 600 (2.5%) to be potentially imprinted, 64% of which are predicted to exhibit maternal expression. These predictions allowed for the identification of putative candidate genes for complex conditions where parent-of-origin effects are involved, including Alzheimer disease, autism, bipolar disorder, diabetes, male sexual orientation, obesity, and schizophrenia. We observe that the number, type, and relative orientation of repeated elements flanking a gene are particularly important in predicting whether a gene is imprinted.
Genome-scale analysis of Streptomyces coelicolor A3(2) metabolism
Authors: Borodina Irina , Krabben Preben , Nielsen Jens
Published: June 2005

Streptomyces are filamentous soil bacteria that produce more than half of the known microbial antibiotics. We present the first genome-scale metabolic model of a representative of this group—Streptomyces coelicolor A3(2). The metabolism reconstruction was based on annotated genes, physiological and biochemical information. The stoichiometric model includes 819 biochemical conversions and 152 transport reactions, accounting for a total of 971 reactions. Of the reactions in the network, 700 are unique, while the rest are iso-reactions. The network comprises 500 metabolites. A total of 711 open reading frames (ORFs) were included in the model, which corresponds to 13% of the ORFs with assigned function in the S. coelicolor A3(2) genome. In a comparative analysis with the Streptomyces avermitilis genome, we showed that the metabolic genes are highly conserved between these species and therefore the model is suitable for use with other Streptomycetes. Flux balance analysis was applied for studies of the reconstructed metabolic network and to assess its metabolic capabilities for growth and polyketides production. The model predictions of wild-type and mutants' growth on different carbon and nitrogen sources agreed with the experimental data in most cases. We estimated the impact of each reaction knockout on the growth of the in silico strain on 62 carbon sources and two nitrogen sources, thereby identifying the “core” of the essential reactions. We also illustrated how reconstruction of a metabolic network at the genome level can be used to fill gaps in genome annotation.
Operon formation is driven by co-regulation and not by horizontal gene transfer
Authors: Price Morgan N. , Huang Katherine H. , Arkin Adam P. , Alm Eric J.
Published: June 2005

The organization of bacterial genes into operons was originally ascribed to the benefits of co-regulation. More recently, the “selfish operon” model, in which operons are formed by repeated gain and loss of genes, was proposed. Indeed, operons are often subject to horizontal gene transfer (HGT). On the other hand, non-HGT genes are particularly likely to be in operons. To clarify whether HGT is involved in operon formation, we identified recently formed operons in Escherichia coli K12. We show that genes that have homologs in distantly related bacteria but not in close relatives of E. coli—indicating HGT—form new operons at about the same rates as native genes. Furthermore, genes in new operons are no more likely than other genes to have phylogenetic trees that are inconsistent with the species tree. In contrast, essential genes and ubiquitous genes without paralogs—genes believed to undergo HGT rarely—often form new operons. We conclude that HGT is not a cause of operon formation but instead promotes the prevalence of pre-existing operons. To explain operon formation, we propose that new operons reduce the amount of regulatory information required to specify optimal expression patterns and infer that operons should be more likely to evolve than independent promoters when regulation is complex. Consistent with this hypothesis, operons have greater amounts of conserved regulatory sequences than do individually transcribed genes.
Multilocus patterns of nucleotide variability and the demographic and selection history of Drosophila melanogaster populations
Authors: Haddrill Penelope R. , Thornton Kevin R. , Charlesworth Brian , Andolfatto Peter
Published: June 2005

Uncertainty about the demographic history of populations can hamper genome-wide scans for selection based on population genetic models. To obtain a portrait of the effects of demographic history on genome variability patterns in Drosophila melanogaster populations, we surveyed noncoding DNA polymorphism at 10 X-linked loci in large samples from three African and two non-African populations. All five populations show significant departures from expectations under the standard neutral model. We detect weak but significant differentiation between East (Kenya and Zimbabwe) and West/Central sub-Saharan (Gabon) African populations. A skew toward high-frequency-derived polymorphisms, elevated levels of linkage disequilibrium (LD) and significant heterogeneity in levels of polymorphism and divergence in the Gabon sample suggest that this population is further from mutation-drift equilibrium than the two Eastern African populations. Both non-African populations harbor significantly higher levels of LD, a large excess of high-frequency-derived mutations and extreme heterogeneity among loci in levels of polymorphism and divergence. Rejections of the neutral model in D. melanogaster populations using these and similar features have been interpreted as evidence for an important role for natural selection in shaping genome variability patterns. Based on simulations, we conclude that simple bottleneck models are sufficient to account for most, if not all, polymorphism features of both African and non-African populations. In contrast, we show that a steady-state recurrent hitchhiking model fails to account for several aspects of the data. Demographic departures from equilibrium expectations in both ancestral and derived populations thus represent a serious challenge to detecting positive selection in genome-wide scans using current methodologies.
Discovering regulatory binding-site modules using rule-based learning
Authors: Hvidsten Torgeir R. , Wilczyński Bartosz , Kryshtafovych Andriy , Tiuryn Jerzy , Komorowski Jan , Fidelis Krzysztof
Published: June 2005

Transcription factors regulate expression by binding selectively to sequence sites incis-regulatory regions of genes. It is therefore reasonable to assume that genes regulated by the same transcription factors should all contain the corresponding binding sites in their regulatory regions and exhibit similar expression profiles as measured by, for example, microarray technology. We have used this assumption to analyze genome-wide yeast binding-site and microarray expression data to reveal the combinatorial nature of gene regulation. We obtained IF-THEN rules linking binding-site combinations (binding-site modules) to genes with particular expression profiles, and thereby provided testable hypotheses on the combinatorial coregulation of gene expression. We showed that genes associated with such rules have a significantly higher probability of being bound by the same transcription factors, as indicated by a genome-wide location analysis, than genes associated with only common binding sites or similar expression. Furthermore, we also found that such genes were significantly more often biologically related in terms of Gene Ontology annotations than genes only associated with common binding sites or similar expression. We analyzed expression data collected under different sets of stress conditions and found many binding-site modules that are conserved over several of these condition sets, as well as modules that are specific to particular biological responses. Our results on the reoccurrence of binding sites in different modules provide specific data on how binding sites may be combined to allow a large number of expression outcomes using relatively few transcription factors.
The L-type calcium channel blocker nifedipine impairs extinction, but not reduced contingency effects, in mice
Authors: Cain Christopher K. , Godsil Bill P. , Jami Shekib , Barad Mark
Published: June 2005

We recently reported that fear extinction, a form of inhibitory learning, is selectively blocked by systemic administration of L-type voltage-gated calcium channel (LVGCC) antagonists, including nifedipine, in mice. We here replicate this finding and examine three reduced contingency effects after vehicle or nifedipine (40 mg/kg) administration. In the first experiment, contingency reduction was achieved by adding USs to the training protocol (degraded contingency), a phenomenon thought to be independent of behavioral inhibition. In the second experiment, contingency reduction was achieved by varying the percentage of CS-US pairing, a phenomenon thought to be weakly dependent on behavioral inhibition. In the third and fourth experiments, contingency reduction was achieved by adding CSs to the training protocol (partial reinforcement), a phenomenon thought to be completely dependent on behavioral inhibition. We found that none of these reduced contingency effects was impaired by nifedipine. In a final experiment, we found that extinction conducted 1 or 3 h post-acquisition, but not immediately, was LVGCC-dependent. Taken together, the results suggest that reduced contingency effects and extinction depend on different molecular mechanisms and that LVGCC dependence of behavioral inhibition develops with time after associative CS-US learning.
Mapping neuronal activation and the influence of adrenergic signaling during contextual memory retrieval
Authors: Zhang Wei-Ping , Guzowski John F. , Thomas Steven A.
Published: June 2005

We recently described a critical role for adrenergic signaling in the hippocampus during contextual and spatial memory retrieval. To determine which neurons are activated by contextual memory retrieval and its sequelae in the presence and absence of adrenergic signaling, transcriptional imaging for the immediate-early geneArcwas used in control and mutant mice lacking norepinephrine and epinephrine. This imaging approach permits the identification of neuronal genomic activation specific to one of two behavioral epochs in the same animal. Analysis revealed several brain regions that were more greatly activated by re-exposure to a salient versus neutral context 1 d after training in control mice (e.g., hippocampal CA3 and CA1, the amygdala, the dorsolateral caudate/putamen, the primary motor cortex, and parts of the rhinal cortices). In mice lacking norepinephrine and epinephrine, many of these regions exhibited significantly reduced activation (e.g., hippocampal CA1), while other regions did not (e.g., hippocampal CA3). In consideration with previous results, the current findings suggest a hypothesis in which adrenergic signaling may be critical for the transfer of retrieved contextual information from CA3 to CA1, where it would be compared to online sensory information coming directly from the cortex.
Involvement of the rat anterior cingulate cortex in control of instrumental responses guided by reward expectancy
Authors: Schweimer Judith , Hauber Wolfgang
Published: June 2005

The anterior cingulate cortex (ACC) plays a critical role in
stimulus-reinforcement learning and reward-guided selection of actions. Here
we conducted a series of experiments to further elucidate the role of the ACC
in instrumental behavior involving effort-based decision-making and
instrumental learning guided by reward-predictive stimuli. In Experiment 1,
rats were trained on a cost-benefit T-maze task in which they could either
choose to climb a barrier to obtain a high reward (four pellets) in one arm or
a low reward (two pellets) in the other with no barrier present. In line with
previous studies, our data reveal that rats with quinolinic acid lesions of
the ACC selected the response involving less work and smaller reward.
Experiment 2 demonstrates that breaking points of instrumental performance
under a progressive ratio schedule were similar in sham-lesioned and
ACC-lesioned rats. Thus, lesions of the ACC did not interfere with the effort
a rat is willing to expend to obtain a specific reward in this test. In a
subsequent task, we examined effort-based decision-making in a lever-press
task where rats had the choice between pressing a lever to receive preferred
food pellets under a progressive ratio schedule, or free feeding on a less
preferred food, i.e. lab chow. Results show that sham- and ACC-lesioned
animals had similar breaking points and ingested comparable amounts of
less-preferred food. Together, the results of Experiment 1 and 2 suggest that
the ACC plays a role in evaluating how much effort to expend for reward;
however, the ACC is not necessary in all situations requiring an assessment of
costs and benefits. In Experiment 3 we investigated learning and reversal
learning of instrumental responses guided by reward predictive stimuli. A
reaction time (RT) task demanding conditioned lever release was used in which
the upcoming reward magnitude (five vs. one food pellet) was signalled in
advance by discriminative visual stimuli. Results revealed that rats with ACC
lesions were able to discriminate reward magnitude-predictive stimuli and to
adapt instrumental behavior to reversed stimulus-reward magnitude
contingencies. Thus, in a simple discrimination task as used here, the ACC
appears not to be required to discriminate reward magnitude-predictive stimuli
and to use the learned significance of the stimuli to guide instrumental
behavior.
Pontine stimulation overcomes developmental limitations in the neural mechanisms of eyeblink conditioning
Authors: Freeman John H. , Rabinak Christine A. , Campolattaro Matthew M.
Published: June 2005

Pontine neuronal activation during auditory stimuli increases
ontogenetically between postnatal days (P) P17 and P24 in rats. Pontine
neurons are an essential component of the conditioned stimulus (CS) pathway
for eyeblink conditioning, providing mossy fiber input to the cerebellum. Here
we examined whether the developmental limitation in pontine responsiveness to
a CS in P17 rats could be overcome by direct stimulation of the CS pathway.
Eyeblink conditioning was established in infant rats on P17-P18 and P24-P25
using pontine stimulation as a CS. There were no significant age-related
differences in the rate or level of conditioning. Eyeblink conditioned
responses established with the stimulation CS were abolished by inactivation
of the ipsilateral cerebellar nuclei and overlying cortex in both age groups.
The findings suggest that developmental changes in the CS pathway play an
important role in the ontogeny of eyeblink conditioning.
Distinct prefrontal molecular mechanisms for information storage lasting seconds versus minutes
Authors: Runyan Jason D. , Dash Pramod K.
Published: June 2005

The prefrontal cortex (PFC) is known to actively hold information
“online” for a period of seconds in working memory for guiding
goal-directed behavior. It has been proposed that relevant information is
stored in other brain regions, which is retrieved and held in working memory
for subsequent assimilation by the PFC in order to guide behavior. It is
uncertain whether PFC stores information outside the temporal limits of
working memory. Here, we demonstrate that although enhanced cAMP-dependent
protein kinase A (PKA) activity in the PFC is detrimental to working memory,
it is required for performance in tasks involving conflicting representations
when memory storage is needed for minutes. This study indicates that distinct
molecular mechanisms within the PFC underlie information storage for seconds
(working memory) and for minutes (short-term memory). In addition, our results
demonstrate that short-term memory storage within the prefrontal cortex is
required for guiding behavior in tasks with conflicts and provides a plausible
mechanism by which the prefrontal cortex executes cognitive control.
Reinforcement in an in vitro analog of appetitive classical conditioning
of feeding behavior in Aplysia: Blockade by a dopamine
antagonist
Authors: Reyes Fredy D. , Mozzachiodi Riccardo , Baxter Douglas A. , Byrne John H.
Published: June 2005

In a recently developed in vitro analog of appetitive classical
conditioning of feeding in Aplysia, the unconditioned stimulus (US)
was electrical stimulation of the esophageal nerve (En). This nerve is rich in
dopamine (DA)-containing processes, which suggests that DA mediates
reinforcement during appetitive conditioning. To test this possibility,
methylergonovine was used to antagonize DA receptors. Methylergonovine (1 nM)
blocked the pairing-specific increase in fictive feeding that is usually
induced by in vitro classical conditioning. The present results and previous
observation that methylergonovine also blocks the effects of contingent
reinforcement in an in vitro analog of appetitive operant conditioning suggest
that DA mediates reinforcement for appetitive associative conditioning of
feeding in Aplysia.
AMPA/kainate, NMDA, and dopamine D1 receptor function in the nucleus accumbens core: A context-limited role in the encoding and consolidation of instrumental memory
Authors: Hernandez Pepe J. , Andrzejewski Matthew E. , Sadeghian Kenneth , Panksepp Jules B. , Kelley Ann E.
Published: June 2005

Neural integration of glutamate- and dopamine-coded signals within the nucleus accumbens (NAc) is a fundamental process governing cellular plasticity underlying reward-related learning. Intra-NAc core blockade of NMDA or D1 receptors in rats impairs instrumental learning (lever-pressing for sugar pellets), but it is not known during which phase of learning (acquisition or consolidation) these receptors are recruited, nor is it known what role AMPA/kainate receptors have in these processes. Here we show that pre-trial intra-NAc core administration of the NMDA, AMPA/KA, and D1 receptor antagonists AP-5 (1 μg/0.5 μL), LY293558 (0.01 or 0.1 μg/0.5 μL), and SCH23390 (1 μg/0.5 μL), respectively, impaired acquisition of a lever-pressing response, whereas post-trial administration left memory consolidation unaffected. An analysis of the microstructure of behavior while rats were under the influence of these drugs revealed that glutamatergic and dopaminergic signals contribute differentially to critical aspects of the initial, randomly emitted behaviors that enable reinforcement learning. Thus, glutamate and dopamine receptors are activated in a time-limited fashion—only being required while the animals are actively engaged in the learning context.
Crystallization and preliminary X-ray analysis of native and selenomethionyl vinorine synthase from Rauvolfia serpentina
Authors: Ma Xueyan , Koepke Juergen , Bayer Anja , Fritzsch Günter , Michel Hartmut , Stöckigt Joachim
Published: June 2005

Vinorine synthase (VS) is a central enzyme of the biosynthesis of the antiarrhythmic drug ajmaline and is a member of the BAHD superfamily of acyltransferases. So far, no three-dimensional structure with significant sequence homology with VS is known. Crystals of VS and selenomethionyl-labelled VS from the medicinal plant Rauvolfia serpentina have been obtained by the hanging-drop technique at 305 K with ammonium sulfate and PEG 400 as precipitants. VS crystals diffract to 2.8 Å and belong to space group P212121, with unit-cell parameters a = 82.3, b = 89.6, c = 136.2 Å. The selenomethionyl VS crystal was nearly isomorphous with the VS crystal.
The incorporation of large impurities into virus crystals
Authors: Kuznetsov Yu. G. , Makino Debora L. , Malkin Alexander J. , McPherson Alexander
Published: June 2005

Virus crystals can incorporate a wide range of unusual impurities, not possible for conventional crystals, or even most protein crystals because of the large size of their constituent particles. These impurities include anomalous virions, satellite viruses and biological fibers. Examples of several of these unusual impurities are presented here, along with some of the consequences for the crystal lattices. The high solvent content, the forgiving character of the lattice and the plasticity of the virions allow these incorporations to be possible.
Anaerobic purification and crystallization to improve the crystal quality: ferredoxin II from Desulfovibrio gigas
Authors: Hsieh Ying-Cheng , Liu Ming-Yih , Le Gall Jean , Chen Chun-Jung
Published: June 2005

Sulfate-reducing bacteria (SRB), which are strict anaerobes, contain an electron-transfer chain from pyridine nucleotides to molecular oxygen. This unique enzymatic equipment allows the bacterium to produce ATP when exposed to air from the degradation of internal reserves of polyglucose. Ferredoxin II (Fd II) is a small electron-transfer protein isolated from the strict anaerobic sulfate-reducing bacterium Desulfovibrio gigas. The protein contains 58 amino acids and an iron–sulfur cluster. The cluster [3Fe–4S] spontaneously undergoes conversion to [4Fe–4S] when it is used as an electron mediator in the phosphoroclastic reaction. The iron–sulfur geometries and interconversion mechanism appear to have physiological significance between the oxidized and reduced states. Crystallization of Fd II in an anaerobic environment was achieved at a higher resolution of 1.37 Å and the differences between the anaerobic and aerobic structures will reveal the unique iron-storage function and electron-transfer mechanism of ferredoxin II from D. gigas.
HalX: an open-source LIMS (Laboratory Information Management System) for small- to large-scale laboratories
Authors: Prilusky Jaime , Oueillet Eric , Ulryck Nathalie , Pajon Anne , Bernauer Julie , Krimm Isabelle , Quevillon-Cheruel Sophie , Leulliot Nicolas , Graille Marc , Liger Dominique , Trésaugues Lionel , Sussman Joel L. , Janin Joël , van Tilbeurgh Herman , Poupon Anne
Published: June 2005

Structural genomics aims at the establishment of a universal protein-fold dictionary through systematic structure determination either by NMR or X-ray crystallography. In order to catch up with the explosive amount of protein sequence data, the structural biology laboratories are spurred to increase the speed of the structure-determination process. To achieve this goal, high-throughput robotic approaches are increasingly used in all the steps leading from cloning to data collection and even structure interpretation is becoming more and more automatic. The progress made in these areas has begun to have a significant impact on the more `classical' structural biology laboratories, dramatically increasing the number of individual experiments. This automation creates the need for efficient data management. Here, a new piece of software, HalX, designed as an `electronic lab book' that aims at (i) storage and (ii) easy access and use of all experimental data is presented. This should lead to much improved management and tracking of structural genomics experimental data.
Protein crystal perfection and its application
Authors: Helliwell John R.
Published: June 2005

Knowledge of protein crystal perfection in theory and practice is reviewed. X-ray methods of assessing perfection such as mosaicity, topography and reciprocal-space mapping are described. X-ray diffraction physics applications of protein crystals such as in Laue geometry, the large-angle oscillation technique and forming polychromatic profiles across diffraction spots are covered. Cryo- and room-temperature cases are discussed including in X-ray and neutron protein crystallography. Experience of freezing very large crystals, which are commonly used today in neutron protein crystallography, is highlighted.
Reproducible growth of well diffracting ribosomal crystals
Authors: Auerbach-Nevo Tamar , Zarivach Raz , Peretz Moshe , Yonath Ada
Published: June 2005

The crystallization of ribosomal particles is associated with extraordinary challenging demands. This originates mainly from the ribosome's natural tendency to deteriorate and from its multi-conformational heterogeneity, both of which stem from its functional flexibility. To increase the level of homogeneity of ribosomal preparations, systematic searches for conditions yielding populations of fully defined chemical compositions were employed and the variables essential for high functional activity were analyzed and optimized. These include temperature, cell-growth duration and media, the cell-harvesting stage, ribosomal purification and storage. The functional state that is most suitable to yield quality crystals was identified as that of the polysome and it was found that this fraction reproducibly yielded crystals of superior properties.
Measurable characteristics of lysozyme crystal growth
Authors: Gorti Sridhar , Forsythe Elizabeth L. , Pusey Marc L.
Published: June 2005

The behavior of protein crystal growth is estimated from measurements performed at both the microscopic and molecular levels. In the absence of solutal flow, it was determined that a model that balances the macromolecular flux toward the crystal surface with the flux of the crystal surface well characterizes crystal growth observed using microscopic methods. Namely, it was determined that the model provides accurate estimates for the crystal-growth velocities upon evaluation of crystal-growth measurements obtained in time. Growth velocities thus determined as a function of solution supersaturation were further interpreted using established deterministic models. From analyses of crystal-growth velocities, it was found that the mode of crystal growth varies with respect to increasing solution supersaturation, possibly owing to kinetic roughening. To verify further the hypothesis of kinetic roughening, crystal growth at the molecular level was examined using atomic force microscopy (AFM). From the AFM measurements, it was found that the magnitude of surface-height fluctuations, h(x), increases with increasing solution supersaturation. In contrast, the estimated characteristic length, ξ, decreases rapidly upon increasing solution supersaturation. It was conjectured that the magnitude of both h(x) and ξ could possibly determine the mode of crystal growth. Although the data precede any exact theory, the non-critical divergence of h(x) and ξ with respect to increasing solution supersaturation was nevertheless preliminarily established. Moreover, approximate models to account for behavior of both h(x) and ξ are also presented.
Comparison of lysozyme structures derived from thin-film-based and classical crystals
Authors: Pechkova Eugenia , Sivozhelezov Victor , Tropiano Giuseppe , Fiordoro Stefano , Nicolini Claudio
Published: June 2005

The present report is dedicated to a systematic comparison of crystal structures produced by the nanobiofilm template method and by the classical hanging-drop vapour-diffusion method. Crystals grown by the innovative nanostructured template method appear indeed radiation-resistant even in the presence of a third-generation highly focused beam at the European Synchrotron Radiation Facility. The implications of this finding for protein crystallography are discussed here in terms of water redistribution and of the detailed atomic resolution comparative studies of the two crystal structures with or without nanobiofilm template, as emerging also from circular-dichroism and thermal denaturation studies.
Crystallization of canavalin as a function of pH
Authors: Forsythe Elizabeth L. , Gorti Sridhar , Pusey Marc L.
Published: June 2005

The question was posed of what happens to a protein that is known to grow as an n-mer when it is placed in solution conditions where it is monomeric. The trypsin-treated or cut form of the protein canavalin (CCAN) has been shown to nucleate and grow crystals as a trimer from neutral to slightly acidic solutions. Under these conditions the solution is composed almost wholly of trimers. The crystalline protein can be readily dissolved by weakly basic solution, which has been proposed to result in a solution that is monomeric. Results are presented for crystallization experiments of CCAN over the pH 6.4–9.6 range. CCAN was found to crystallize in the canonical rhombohedral form at all pH values. Light scattering and size-exclusion chromatography data gave no indication of monomeric CCAN. The light-scattering data also showed that trimeric CCAN slowly self-associates to form larger species. Results are presented for crystallization experiments of CCAN over the pH 6.4–9.6 range. Fluorescence anisotropy, coupled with light-scattering and gel-filtration experiments, show that the solutions are primarily trimers, with association to form larger species occurring as a function of protein concentration. These results indicate that CCAN crystal nucleation occurs by the self-association of trimers to form progressively larger species in solution, which eventually become critically sized crystal nuclei.
Supersaturated lysozyme solution structure studied by chemical cross-linking
Authors: Hall Clayton L. , Clemens John R. , Brown Amanda M. , Wilson Lori J.
Published: June 2005

Glutaraldehyde cross-linking followed by separation has been used to detect aggregates of chicken egg-white lysozyme (CEWL) in supersaturated solutions. In solutions of varying NaCl content, the number of aggregates was found to be related to the ionic strength of the solution. Separation by SDS–PAGE showed that percentage of dimer in solution ranged from 25.3% for no NaCl to 27.1% at 15% NaCl, and the aggregates larger than dimer increased from 1.9% for no NaCl to 36.8% at 15% NaCl. Conversely, the percentage of monomers decreased from 72.8% without NaCl to 36.1% at 15% NaCl. Molecular weights by capillary electrophoresis (SDS–CE) were found to be multiples of the monomer molecular weights, with the exception of trimer, which indicates a very compact structure. Native separation was accomplished using size-exclusion chromatography (SEC) and gave a lower monomer concentration and higher aggregate concentration than SDS–CE, which is a denaturing separation method. Most noticeably, trimers were absent in the SEC separation. The number of aggregates did not change with increased time between addition of NaCl and addition of cross-linking agent when separated by gel electrophoresis (SDS–PAGE). The results suggest that high ionic strength CEWL solutions are highly aggregated and that denaturing separation methods disrupt cross-linked products.
Crystal quality and differential crystal-growth behaviour of three proteins crystallized in gel at high hydrostatic pressure
Authors: Kadri A. , Lorber B. , Charron C. , Robert M.-C. , Capelle B. , Damak M. , Jenner G. , Giegé R.
Published: June 2005

Pressure is a non-invasive physical parameter that can be used to control and influence protein crystallization. It is also found that protein crystals of superior quality can be produced in gel. Here, a novel crystallization strategy combining hydrostatic pressure and agarose gel is described. Comparative experiments were conducted on hen and turkey egg-white lysozymes and the plant protein thaumatin. Crystals could be produced under up to 75–100 MPa (lysozymes) and 250 MPa (thaumatin). Several pressure-dependent parameters were determined, which included solubility and supersaturation of the proteins, number, size and morphology of the crystals, and the crystallization volume. Exploration of three-dimensional phase diagrams in which pH and pressure varied identified growth conditions where crystals had largest size and best morphology. As a general trend, nucleation and crystal-growth kinetics are altered and nucleation is always enhanced under pressure. Further, solubility of the lysozymes increases with pressure while that of thaumatin decreases. Likewise, changes in crystallization volumes at high and atmospheric pressure are opposite, being positive for the lysozymes and negative for thaumatin. Crystal quality was estimated by analysis of Bragg reflection profiles and X-ray topographs. While the quality of lysozyme crystals deteriorates as pressure increases, that of thaumatin crystals improves, with more homogeneous crystal morphology suggesting that pressure selectively dissociates ill-formed nuclei. Analysis of the thaumatin structure reveals a less hydrated solvent shell around the protein when pressure increases, with ∼20% less ordered water molecules in crystals grown at 150 MPa when compared with those grown at atmospheric pressure (0.1 MPa). Noticeably, the altered water distribution is seen in depressurized crystals, indicating that pressure triggers a stable structural alteration on the protein surface while its polypeptide backbone remains essentially unaltered.
Comparison of the crystallization and crystal packing of two Fab single-site mutant protein L complexes
Authors: Granata Vincenzo , Housden Nicholas G. , Harrison Stephen , Jolivet-Reynaud Colette , Gore Michael G. , Stura Enrico Adriano
Published: June 2005

Protein L from Peptostreptococcus magnus (PpL) is a multidomain protein composed of four or five immunoglobulin-binding domains that target the κ light chain of a large repertoire of human and murine antibodies. Thus, a single domain of this protein can be used to aid the crystallization of Fab, free or complexed to their antigen when it is not possible to obtain crystals without it. Each wild-type PpL domain has two light-chain binding sites that target the same region of the light chain and can thus bring together two Fab–antigen complexes within the crystal lattice. In this context the small PpL domain is sandwiched between two Fab and cannot participate in crystal contacts, thus mutants are unlikely to increase the chances of crystallizing a particular complex. However, it is possible to design mutants that can bind at only one site by making use of the crystal structures obtained so far. Such mutants will have a free surface that can participate in crystal contacts and that can be modified to improve its crystal contact-forming properties. Here, a comparison of two single-site mutants that differ at three different positions is reported. In both mutants two different tryptophan residues participate in crystal-packing interactions, suggesting that this residue may be particularly interesting for enhancing crystal-contact formation.
Effects of macromolecular impurities and of crystallization method on the quality of eubacterial aspartyl-tRNA synthetase crystals
Authors: Moreno A. , Théobald-Dietrich A. , Lorber B. , Sauter C. , Giegé R.
Published: June 2005

Although macromolecular purity is thought to be essential for the growth of flawless protein crystals, only a few studies have investigated how contaminants alter the crystallization process and crystal quality. Likewise, the outcome of a crystallization process may vary with the crystallization method. Here, it is reported how these two variables affect the crystallogenesis of aspartyl-tRNA synthetase from the eubacterium Thermus thermophilus. This homodimeric enzyme (M
r = 130 000) possesses a multi-domain architecture and crystallizes either in a monoclinic or an orthorhombic habit. Minute amounts of protein impurities alter to a different extent the growth of each crystal form. The best synthetase crystals are only obtained when the crystallizing solution is either enclosed in capillaries or immobilized in agarose gel. In these two environments convection is reduced with regard to that existing in an unconstrained solution.
Development of a novel ampholyte buffer for isoelectric focusing: electric charge-separation of protein samples for X-ray crystallography using free-flow isoelectric focusing
Authors: Kim Seong-Hoon , Miyatake Hideyuki , Ueno Tomoyuki , Nagao Takuya , Miki Kunio
Published: June 2005

The purification of biological macromolecules has remained a severe bottleneck in three-dimensional structure determination in the field of protein crystallography. By the use of the Free Flow Electrophoresis (ProTeam FFE; TECAN Group Ltd) apparatus, target protein samples can be purified in a high yield, while a preparative separation is performed in solution without using solid matrices such as polyacrylamide gel. A novel ampholyte buffer suitable for crystallization in the use of the ProTeam FFE apparatus has been developed. This buffer is able to generate pH gradients owing to the use of low-molecular-weight electrolytes. The effect of the flow rate on the pH gradient using the novel ampholyte buffer has been elucidated. When the flow rate was lowered to 78 ml h−1, the pH gradient in the electrophoretic chambers was generated in an almost linear fashion.
Structure determination of GGA-GAE and γ1-ear in complex with peptides: crystallization of low-affinity complexes in membrane traffic
Authors: Yamada Yusuke , Inoue Michio , Shiba Tomoo , Kawasaki Masato , Kato Ryuichi , Nakayama Kazuhisa , Wakatsuki Soichi
Published: June 2005

Crystallization of protein–protein complexes is an important step in the studies of biological functions of proteins. However, weak and transient, even though specific, interactions often present difficulties in crystallization of protein complexes due to the heterogeneity of the sample mixture. For example, the γ1-ear domain of the AP-1 complex and the GAE domain of GGA1, responsible for the interaction with accessory proteins involved in vesicular transport, are known to interact with target proteins with affinities of the order of 1–100 µM. Such low affinities have hampered crystallization trials of the complexes. To overcome this problem, the γ1-ear and GAE domains were first co-crystallized with excess amounts of the peptides. Co-crystals of both domains were obtained and the complex structures were determined at 2.5–2.9 Å resolution. Based on the crystal packing of γ1-ear and the cognate peptide, γ1-ear fused with a peptide tag at the N-terminus was prepared. The peptide-tagged γ1-ear readily crystallized and the crystal diffracted far better, 1.9–2.2 Å resolution, compared with the co-crystallized complex, giving significantly more details without affecting the overall complex structure.
The high-throughput protein-to-structure pipeline at SECSG
Authors: Liu Zhi-Jie , Tempel Wolfram , Ng Joseph D. , Lin Dawei , Shah Ashit K. , Chen Lirong , Horanyi Peter S. , Habel Jeff E. , Kataeva Irina A. , Xu Hao , Yang Hua , Chang Jessie C. , Huang Lei , Chang Shu-Huey , Zhou Weihong , Lee Doowon , Praissman Jeremy L. , Zhang Hua , Newton M. Gary , Rose John P. , Richardson Jane S. , Richardson David C. , Wang Bi-Cheng
Published: June 2005

Using a high degree of automation, the crystallography core at the Southeast Collaboratory for Structural Genomics (SECSG) has developed a high-throughput protein-to-structure pipeline. Various robots and automation procedures have been adopted and integrated into a pipeline that is capable of screening 40 proteins for crystallization and solving four protein structures per week. This pipeline is composed of three major units: crystallization, structure determination/validation and crystallomics. Coupled with the protein-production cores at SECSG, the protein-to-structure pipeline provides a two-tiered approach for protein production at SECSG. In tier 1, all protein samples supplied by the protein-production cores pass through the pipeline using standard crystallization screening and optimization procedures. The protein targets that failed to yield diffraction-quality crystals (resolution better than 3.0 Å) become tier 2 or salvaging targets. The goal of tier 2 target salvaging, carried out by the crystallomics core, is to produce the target proteins with increased purity and homogeneity, which would render them more likely to yield well diffracting crystals. This is performed by alternative purification procedures and/or the introduction of chemical modifications to the proteins (such as tag removal, methylation, surface mutagenesis, selenomethionine labelling etc.). Details of the various procedures in the pipeline for protein crystallization, target salvaging, data collection/processing and high-throughput structure determination/validation, as well as some examples, are described.
Solution stirring initiates nucleation and improves the quality of adenosine deaminase crystals
Authors: Adachi Hiroaki , Takano Kazufumi , Niino Ai , Matsumura Hiroyoshi , Kinoshita Takayoshi , Warizaya Masaichi , Inoue Tsuyoshi , Mori Yusuke , Sasaki Takatomo
Published: June 2005

Crystals of bovine adenosine deaminase (ADA) grown over a two-week period in the presence of an inhibitor (ADA complex) were found to be of low quality for X-ray diffraction analysis. Furthermore, ADA incubated in the absence of an inhibitor (ADA native) did not form any crystals using conventional crystallization methods. A solution-stirring technique was used to obtain high-quality ADA complex and ADA native crystals. The crystals obtained using this technique were found to be of high quality and were shown to have high structural resolution for X-ray diffraction analyses. The results reported here indicate that the solution-stirring technique promotes nucleation and improves the quality of protein crystals.
An attempt to increase the efficiency of protein crystal screening: a simplified screen and experiments
Authors: Gao Wei , Li Shu-xing , Bi Ru-chang
Published: June 2005

Macromolecular crystallization remains a bottleneck in structure determination by X-ray diffraction. Based on the data reflecting success rates of crystallization conditions in different screens and the information derived from the BMCD and other related studies, a simplified screen has been designed to increase the success rate of traditional screening and to save samples, time and cost. The screen has been tested with six protein samples which had been crystallized before and its comparison with Crystal Screen (Hampton Research) was also performed with lysozyme crystallization. The experimental results show that for obtaining crystal leads, the success rate of the simplified screen is reasonably higher. In addition, it has been successful in crystallizing two new proteins from snake venom using the simplified screen that had failed with Crystal Screen. These results indicate that the simplified screen, which assembles and optimizes the efficient crystallization conditions from distinct screens, extends the region of crystallization and improves the success rate of screening. Based on information of newly published efficient crystallization conditions, the simplified screen could be developed and optimized continuously in the future.