bluuebunny/crossref_metadata_2025 huggingface dataset:
Crystal structure of the receptor-binding domain of human B7-2: Insights into organization and signaling
Authors: Zhang Xuewu , Schwartz Jean-Claude D. , Almo Steven C. , Nathenson Stanley G.
Published: March 2003

B7-1 and B7-2 are homologous costimulatory ligands expressed on the surfaces of antigen-presenting cells. Their interactions with CD28/CTLA-4 receptors expressed on T cell surfaces are crucial for the proper regulation of T cell activity. B7-1 and B7-2 display distinct roles in immune regulation, although they are usually considered to have redundant functions. Here, we report the crystal structure of the receptor-binding (Ig V-type) domain of human B7-2 at 2.7-Å resolution. Structures of unliganded and liganded B7-1 and B7-2 suggest a physical–chemical basis for the observed functional similarities and differences between these two costimulatory ligands. Of particular note, whereas the majority of the residues mediating B7-1 dimerization are hydrophobic, the B7-2 dimer observed in the B7-2/CTLA-4 complex displays a very hydrophilic dimer interface. These differences provide a mechanism for preventing the formation of B7-1/B7-2 heterodimers. The divergence at the putative dimer interface is also consistent with the lower tendency of B7-2 to dimerize, as shown by the monomeric state of unliganded B7-2 both in solution and crystalline form, and may result in detailed differences in signaling mechanisms associated with B7-1 and B7-2.
Requirement for the synaptic protein interaction site for reconstitution of synaptic transmission by P/Q-type calcium channels
Authors: Mochida Sumiko , Westenbroek Ruth E. , Yokoyama Charles T. , Zhong Huijun , Myers Scott J. , Scheuer Todd , Itoh Kanako , Catterall William A.
Published: March 2003

Ca
v
2.1 channels, which conduct P/Q-type Ca
2+
currents, were expressed in superior cervical ganglion neurons in cell culture, and neurotransmission initiated by these exogenously expressed Ca
2+
channels was measured. Deletions in the synaptic protein interaction (synprint) site in the intracellular loop between domains II and III of Ca
v
2.1 channels reduced their effectiveness in synaptic transmission. Surprisingly, this effect was correlated with loss of presynaptic localization of the exogenously expressed channels. Ca
v
1.2 channels, which conduct L-type Ca
2+
currents, are ineffective in supporting synaptic transmission, but substitution of the synprint site from Ca
v
2.1 channels in Ca
v
1.2 was sufficient to establish synaptic transmission initiated by L-type Ca
2+
currents through the exogenous Ca
v
1.2 channels. Substitution of the synprint site from Ca
v
2.2 channels, which conduct N-type Ca
2+
currents, was even more effective than Ca
v
2.1. Our results show that localization and function of exogenous Ca
2+
channels in nerve terminals of superior cervical ganglion neurons require a functional synprint site and suggest that binding of soluble NSF attachment protein receptor (SNARE) proteins to the synprint site is a necessary permissive event for nerve terminal localization of presynaptic Ca
2+
channels.
Selective modulation of excitatory and inhibitory microcircuits by dopamine
Authors: Gao Wen-Jun , Goldman-Rakic Patricia S.
Published: March 2003

Dopamine plays an important role in the working memory functions of the prefrontal cortex, functions that are impacted in age-related memory decline, drug abuse, and a wide variety of disorders, including schizophrenia and Parkinson's disease. We have previously reported that dopamine depresses excitatory transmission between pyramidal neurons in the prefrontal cortex. Here, using paired recordings, we have investigated dopaminergic modulation of excitatory transmission from pyramidal neurons to fast-spiking (FS) interneurons. In contrast to its effect on recurrent excitation, dopamine was without effect on excitatory transmission to FS interneurons. However, dopamine has directly enhanced the excitability of the FS interneurons to the extent that even a single excitatory postsynaptic potential could initiate spiking with great temporal precision in some of them. These results indicate that dopamine's effects on excitatory transmission are target-specific and that the axon terminals of pyramidal neurons can be selectively regulated at the level of individual synapses. Thus, dopamine's net inhibitory effect on cortical function is remarkably constrained by the nature of the microcircuit elements on which it acts.
Molecular mechanism of recruitment of TFIIF- associating RNA polymerase C-terminal domain phosphatase (FCP1) by transcription factor IIF
Authors: Kamada Katsuhiko , Roeder Robert G. , Burley Stephen K.
Published: March 2003

After mRNA transcription termination in eukaryotes, the hyperphosphorylated form of RNA polymerase II (pol II0) must be recycled by TFIIF-associating C-terminal domain phosphatase (FCP1), the phosphatase responsible for dephosphorylating the C-terminal domain of the largest polymerase subunit. Transcription factor (TF)-IIF stimulates the activity of FCP1, and the RNA polymerase II-associating protein 74 subunit of TFIIF forms a complex with FCP1 in both human and yeast. Here, we report a cocrystal structure of the winged-helix domain of human RNA polymerase II-associating protein 74 bound to the α-helical C terminus of human FCP1 (residues 944–961). These results illustrate the molecular mechanism by which TFIIF efficiently recruits FCP1 to the pol II transcription machinery for recycling of the polymerase.
Polyamines protect
Escherichia coli
cells from the toxic effect of oxygen
Authors: Chattopadhyay Manas K. , Tabor Celia White , Tabor Herbert
Published: March 2003

Wild-type
Escherichia coli
cells grow normally in 95% O
2
/5% CO
2
. In contrast, cells that cannot make polyamines because of mutations in the biosynthetic pathway are rapidly killed by incubation in 95% O
2
/5% CO
2
. Addition of polyamines prevents the toxic effect of oxygen, permitting cell survival and optimal growth. Oxygen toxicity can also be prevented if the growth medium contains an amino acid mixture or if the polyamine-deficient cells contain a manganese-superoxide dismutase (Mn-SOD) plasmid. Partial protection is afforded by the addition of 0.4 M sucrose or 0.4 M sorbitol to the growth medium. We also report that concentrations of H
2
O
2
that are nontoxic to wild-type cells or to mutant cells pretreated with polyamines kill polyamine-deficient cells. These results show that polyamines are important in protecting cells from the toxic effects of oxygen.
ToxR regulon of
Vibrio cholerae
and its expression in vibrios shed by cholera patients
Authors: Bina James , Zhu Jun , Dziejman Michelle , Faruque Shah , Calderwood Stephen , Mekalanos John
Published: March 2003

Toxigenic
Vibrio cholerae
cause cholera, a severe diarrheal disease responsible for significant morbidity and mortality worldwide. Two determinants, cholera enterotoxin (CT) and toxin coregulated pilus (TCP) are critical factors responsible for this organism's virulence. The genes for these virulence determinants belong to a network of genes (the ToxR regulon) whose expression is modulated by transcriptional regulators encoded by the
toxRS
,
tcpPH
, and
toxT
genes. To define the ToxR regulon more fully, mutants defective in these regulatory genes were transcriptionally profiled by using
V. cholerae
genomic microarrays. This study identified 13 genes that were transcriptionally repressed by the
toxT
mutation (all involved in CT and TCP biogenesis), and 27 and 60 genes that were transcriptionally repressed by the
tcpPH
and
toxRS
mutations, respectively. During the course of this analysis, we validated the use of a genomic DNA-based reference sample as a means to standardize and normalize data obtained in different microarray experiments. This method allowed the accurate transcriptional profiling of
V. cholerae
cells present in stools from cholera patients and the comparison of these profiles to those of wild-type and mutant strains of
V. cholerae
grown under optimal conditions for CT and TCP expression. Our results suggest that vibrios present in cholera stools carry transcripts for these two virulence determinants, albeit at relatively low levels compared with optimal
in vitro
conditions. The transcriptional profile of vibrios present in cholera stools also suggests that the bacteria experienced conditions of anaerobiosis, iron limitation, and nutrient deprivation within the human gastrointestinal tract.
Autonomous T cell trafficking examined
in vivo
with intravital two-photon microscopy
Authors: Miller Mark J. , Wei Sindy H. , Cahalan Michael D. , Parker Ian
Published: March 2003

The recirculation of T cells between the blood and secondary lymphoid organs requires that T cells are motile and sensitive to tissue-specific signals. T cell motility has been studied
in vitro
, but the migratory behavior of individual T cells
in vivo
has remained enigmatic. Here, using intravital two-photon laser microscopy, we imaged the locomotion and trafficking of naïve CD4
+
T cells in the inguinal lymph nodes of anesthetized mice. Intravital recordings deep within the lymph node showed T cells flowing rapidly in the microvasculature and captured individual homing events. Within the diffuse cortex, T cells displayed robust motility with an average velocity of ≈11 μm⋅min
−1
. T cells cycled between states of low and high motility roughly every 2 min, achieving peak velocities >25 μm⋅min
−1
. An analysis of T cell migration in 3D space revealed a default trafficking program analogous to a random walk. Our results show that naïve T cells do not migrate collectively, as they might under the direction of pervasive chemokine gradients. Instead, they appear to migrate as autonomous agents, each cell taking an independent trafficking path. Our results call into question the role of chemokine gradients for basal T cell trafficking within T cell areas and suggest that antigen detection may result from a stochastic process through which a random walk facilitates contact with antigen-presenting dendritic cells.
Modulation of cardiac Ca
V
1.2 channels by dihydropyridine and phosphatase inhibitor requires Ser-1142 in the domain III pore loop
Authors: Erxleben Christian , Gomez-Alegria Claudio , Darden Thomas , Mori Yasuo , Birnbaumer Lutz , Armstrong David L.
Published: March 2003

Dihydropyridine-sensitive, voltage-activated calcium channels respond to membrane depolarization with two distinct modes of activity: short bursts of very short openings (mode 1) or repetitive openings of much longer duration (mode 2). Here we show that both the dihydropyridine, BayK8644 (BayK), and the inhibitor of Ser/Thr protein phosphatases, okadaic acid, have identical effects on the gating of the recombinant cardiac calcium channel, Ca
V
1.2 (α
1
C). Each produced identical mode 2 gating in cell-attached patches, and each prevented rundown of channel activity when the membrane patch was excised into ATP-free solutions. These effects required Ser or Thr at position 1142 in the domain III pore loop between transmembrane segments S5 and S6, where dihydropyridines bind to the channel. Mutation of Ser-1142 to Ala or Cys produced channels with very low activity that could not be modulated by either BayK or okadaic acid. A molecular model of Ca
V
1.2 indicates that Ser-1142 is unlikely to be phosphorylated, and thus we conclude that BayK binding stabilizes mode 2 gating allosterically by either protecting a phospho Ser/Thr on the α
1
C subunit or mimicking phosphorylation at that site.
Interaction of FK506-binding protein 13 with brefeldin A-inhibited guanine nucleotide-exchange protein 1 (BIG1): Effects of FK506
Authors: Padilla Philip Ian , Chang Min-ju , Pacheco-Rodriguez Gustavo , Adamik Ronald , Moss Joel , Vaughan Martha
Published: March 2003

BIG1 and BIG2 are brefeldin A-inhibited guanine nucleotide-exchange proteins that activate ADP-ribosylation factors (ARFs), critical components of vesicular trafficking pathways. These proteins can exist in macromolecular complexes and move between Golgi membranes and cytosol. In the BIG1 molecule, a centrally located Sec7 domain is responsible for ARF activation, but functions of other regions are largely unknown. Yeast two-hybrid screens of a human placenta cDNA library with BIG1 cDNA constructs revealed specific interaction of the N-terminal region (amino acids 1–331) with FK506-binding protein 13 (FKBP13). The association was confirmed by immunoprecipitation of both endogenous BIG1 and FKBP13 from Jurkat T cells with antibodies against either one. Binding of BIG1, BIG2, and ARF to cell membranes
in vitro
was increased by guanosine 5′-[γ-thio]triphosphate, and further increases were induced by FK506. Incubation of Jurkat T cells with FK506 increased binding of BIG1, BIG2, and ARF to Golgi and other membranes in a time- and concentration-dependent manner, without effects on clathrin or γ-adaptin binding. Binding of BIG1, BIG2, and ARF to membranes was also increased by L-732,531, an agonist structurally related to FK506, but was not increased by a related antagonist, L-685,818, nor by cyclosporin A or rapamycin. These findings are consistent with a role for FKBP13 and FK506 in vesicular trafficking, influencing ARF activity through their guanine nucleotide-exchange proteins.
Tumor necrosis factor-related apoptosis-inducing ligand induces neuronal death in a murine model of HIV central nervous system infection
Authors: Miura Yoshiharu , Misawa Naoko , Kawano Yuji , Okada Hiroshi , Inagaki Yoshio , Yamamoto Naoki , Ito Mamoru , Yagita Hideo , Okumura Ko , Mizusawa Hidehiro , Koyanagi Yoshio
Published: March 2003

HIV-1 infection in the brain induces neuronal apoptosis leading to HIV-associated dementia. To explore the underlying mechanism, we developed a murine model by using human peripheral blood mononuclear cell (PBMC)–transplanted nonobese diabetic (NOD)–severe combined immunodeficiency (SCID) (hu-PBMC-NOD-SCID) mice. Administration of lipopolysaccharide (LPS) to HIV-1-infected hu-PBMC-NOD-SCID mice induced infiltration of HIV-1-infected human cells into the perivascular region of the brain and neuronal apoptosis was found in macrophage (M)-tropic but not T cell (T)-tropic HIV-1-infected brains. The apoptotic neurons were frequently colocalized with the HIV-1-infected macrophages that expressed tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL). Administration of a neutralizing antibody against human TRAIL but not human TNF-α or Fas ligand (FasL) blocked the neuronal apoptosis in the HIV-1-infected brain. These results strongly suggest a critical contribution of TRAIL expressed on HIV-1-infected macrophages to neuronal apoptosis.
The C-terminal domain phosphatase and transcription elongation activities of FCP1 are regulated by phosphorylation
Authors: Friedl Erika M. , Lane William S. , Erdjument-Bromage Hediye , Tempst Paul , Reinberg Danny
Published: March 2003

The C-terminal domain (CTD) of the largest subunit of RNA polymerase II (RNAPII) is heavily phosphorylated during the transition from transcription initiation to the establishment of an elongation-competent transcription complex. FCP1 is the only phosphatase known to be specific for the CTD of the largest subunit of RNAPII, and its activity is believed to be required to reactivate RNAPII, so that RNAPII can enter another round of transcription. We demonstrate that FCP1 is a phosphoprotein, and that phosphorylation regulates FCP1 activities. FCP1 is phosphorylated at multiple sites
in vivo
. The CTD phosphatase activity of phosphorylated FCP1 is stimulated by TFIIF, whereas dephosphorylated FCP1 is not. In addition to its role in the recycling of RNAPII, FCP1 also affects transcription elongation. Phosphorylated FCP1 is more active in stimulating transcription elongation than the dephosphorylated form of FCP1. We found that only phosphorylated FCP1 can physically interact with TFIIF. We set out to purify an FCP1 kinase from HeLa cells and identified casein kinase 2, which, surprisingly, displayed a negative effect on FCP1-associated activities.
Plasma fibronectin promotes thrombus growth and stability in injured arterioles
Authors: Ni Heyu , Yuen Peter S. T. , Papalia Jessie M. , Trevithick Jane E. , Sakai Takao , Fässler Reinhard , Hynes Richard O. , Wagner Denisa D.
Published: March 2003

Mice lacking both of the best-known platelet ligands, von Willebrand factor and fibrinogen, can still form occlusive thrombi in injured arterioles. The platelets of these animals accumulate excessive amounts of fibronectin (FN). These observations led us to examine the contribution of plasma FN (pFN) to thrombus formation. Inactivation of the
FN
gene in FN conditional knockout mice reduced pFN levels to <2% and platelet FN to ≈20% of the levels in similarly treated control mice. The mice were then observed in a model of arterial injury to evaluate their capacity to form thrombi. The deficiency of pFN did not affect the initial platelet adhesion, but a delay of several minutes in thrombus formation was observed in the arterioles of pFN-deficient mice as compared with control mice. The thrombi that formed in the absence of pFN were stably anchored to the vessel wall but continuously shed platelets or small platelet clumps, thus slowing their growth significantly; the platelet/platelet cohesion was apparently diminished. Consequently the occlusion of pFN-deficient vessels was delayed, with the majority of vessels remaining patent at the end of the 40-min observation period. We conclude that, in addition to von Willebrand factor and fibrinogen, FN plays a significant role in thrombus initiation, growth, and stability at arterial shear rates and that deficiency in each of the three platelet ligands has its own specific impact on platelet plug formation.
Tyrosine-phosphorylated and nonphosphorylated isoforms of α-dystrobrevin
Authors: Grady R. Mark , Akaaboune Mohammed , Cohen Alexander L. , Maimone Margaret M. , Lichtman Jeff W. , Sanes Joshua R.
Published: March 2003

α-Dystrobrevin (DB), a cytoplasmic component of the dystrophin–glycoprotein complex, is found throughout the sarcolemma of muscle cells. Mice lacking αDB exhibit muscular dystrophy, defects in maturation of neuromuscular junctions (NMJs) and, as shown here, abnormal myotendinous junctions (MTJs). In normal muscle, alternative splicing produces two main αDB isoforms, αDB1 and αDB2, with common NH2-terminal but distinct COOH-terminal domains. αDB1, whose COOH-terminal extension can be tyrosine phosphorylated, is concentrated at the NMJs and MTJs. αDB2, which is not tyrosine phosphorylated, is the predominant isoform in extrajunctional regions, and is also present at NMJs and MTJs. Transgenic expression of either isoform in αDB−/− mice prevented muscle fiber degeneration; however, only αDB1 completely corrected defects at the NMJs (abnormal acetylcholine receptor patterning, rapid turnover, and low density) and MTJs (shortened junctional folds). Site-directed mutagenesis revealed that the effectiveness of αDB1 in stabilizing the NMJ depends in part on its ability to serve as a tyrosine kinase substrate. Thus, αDB1 phosphorylation may be a key regulatory point for synaptic remodeling. More generally, αDB may play multiple roles in muscle by means of differential distribution of isoforms with distinct signaling or structural properties.
AP-1 binding to sorting signals and release from clathrin-coated vesicles is regulated by phosphorylation
Authors: Ghosh Pradipta , Kornfeld Stuart
Published: March 2003

The adaptor protein complex-1 (AP-1) sorts and packages membrane proteins into clathrin-coated vesicles (CCVs) at the TGN and endosomes. Here we show that this process is highly regulated by phosphorylation of AP-1 subunits. Cell fractionation studies revealed that membrane-associated AP-1 differs from cytosolic AP-1 in the phosphorylation status of its β1 and μ1 subunits. AP-1 recruitment onto the membrane is associated with protein phosphatase 2A (PP2A)–mediated dephosphorylation of its β1 subunit, which enables clathrin assembly. This Golgi-associated isoform of PP2A exhibits specificity for phosphorylated β1 compared with phosphorylated μ1. Once on the membrane, the μ1 subunit undergoes phosphorylation, which results in a conformation change, as revealed by increased sensitivity to trypsin. This conformational change is associated with increased binding to sorting signals on the cytoplasmic tails of cargo molecules. Dephosphorylation of μ1 (and μ2) by another PP2A-like phosphatase reversed the effect and resulted in adaptor release from CCVs. Immunodepletion and okadaic acid inhibition studies demonstrate that PP2A is the cytosolic cofactor for Hsc-70–mediated adaptor uncoating. A model is proposed where cyclical phosphorylation/dephosphorylation of the subunits of AP-1 regulate its function from membrane recruitment until its release into cytosol.
Chromosome order in HeLa cells changes during mitosis and early G1, but is stably maintained during subsequent interphase stages
Authors: Walter Joachim , Schermelleh Lothar , Cremer Marion , Tashiro Satoshi , Cremer Thomas
Published: March 2003

Whether chromosomes maintain their nuclear positions during interphase and from one cell cycle to the next has been controversially discussed. To address this question, we performed long-term live-cell studies using a HeLa cell line with GFP-tagged chromatin. Positional changes of the intensity gravity centers of fluorescently labeled chromosome territories (CTs) on the order of several μm were observed in early G1, suggesting a role of CT mobility in establishing interphase nuclear architecture. Thereafter, the positions were highly constrained within a range of ∼1 μm until the end of G2. To analyze possible changes of chromosome arrangements from one cell cycle to the next, nuclei were photobleached in G2 maintaining a contiguous zone of unbleached chromatin at one nuclear pole. This zone was stably preserved until the onset of prophase, whereas the contiguity of unbleached chromosome segments was lost to a variable extent, when the metaphase plate was formed. Accordingly, chromatin patterns observed in daughter nuclei differed significantly from the mother cell nucleus. We conclude that CT arrangements were stably maintained from mid G1 to late G2/early prophase, whereas major changes of CT neighborhoods occurred from one cell cycle to the next. The variability of CT neighborhoods during clonal growth was further confirmed by chromosome painting experiments.
Virtual crystals and fermionic formulas of type 𝐷_{𝑛+1}⁽²⁾, 𝐴_{2𝑛}⁽²⁾, and 𝐶_{𝑛}⁽¹⁾
Authors: Okado Masato , Schilling Anne , Shimozono Mark
Published: March 2003

We introduce “virtual” crystals of the affine types

g

=

D

n
+
1

(
2
)

\mathfrak {g}=D_{n+1}^{(2)}

,

A

2
n

(
2
)

A_{2n}^{(2)}

and

C
n

(
1
)

C_n^{(1)}

by naturally extending embeddings of crystals of types

B
n

B_n

and

C
n

C_n

into crystals of type

A

2
n
−
1

A_{2n-1}

. Conjecturally, these virtual crystals are the crystal bases of finite dimensional

U
q
′

(

g

)

U_q’(\mathfrak {g})

-modules associated with multiples of fundamental weights. We provide evidence and in some cases proofs of this conjecture. Recently, fermionic formulas for the one-dimensional configuration sums associated with tensor products of the finite dimensional

U
q
′

(

g

)

U_q’(\mathfrak {g})

-modules were conjectured by Hatayama et al. We provide proofs of these conjectures in specific cases by exploiting duality properties of crystals and rigged configuration techniques. For type

A

2
n

(
2
)

A_{2n}^{(2)}

we also conjecture a new fermionic formula coming from a different labeling of the Dynkin diagram.
The Phylogenetic Extent of Metabolic Enzymes and Pathways
Authors: Peregrin-Alvarez José Manuel , Tsoka Sophia , Ouzounis Christos A.
Published: March 2003

The evolution of metabolic enzymes and pathways has been a subject of intense study for more than half a century. Yet, so far, previous studies have focused on a small number of enzyme families or biochemical pathways. Here, we examine the phylogenetic distribution of the full-known metabolic complement of Escherichia coli, using sequence comparison against taxa-specific databases. Half of the metabolic enzymes have homologs in all domains of life, representing families involved in some of the most fundamental cellular processes. We thus show for the first time and in a comprehensive way that metabolism is conserved at the enzyme level. In addition, our analysis suggests that despite the sequence conservation and the extensive phylogenetic distribution of metabolic enzymes, their groupings into biochemical pathways are much more variable than previously thought.
SLAM: Cross-Species Gene Finding and Alignment with a Generalized Pair Hidden Markov Model
Authors: Alexandersson Marina , Cawley Simon , Pachter Lior
Published: March 2003

Comparative-based gene recognition is driven by the principle that conserved regions between related organisms are more likely than divergent regions to be coding. We describe a probabilistic framework for gene structure and alignment that can be used to simultaneously find both the gene structure and alignment of two syntenic genomic regions. A key feature of the method is the ability to enhance gene predictions by finding the best alignment between two syntenic sequences, while at the same time finding biologically meaningful alignments that preserve the correspondence between coding exons. Our probabilistic framework is the generalized pair hidden Markov model, a hybrid of (1) generalized hidden Markov models, which have been used previously for gene finding, and (2) pair hidden Markov models, which have applications to sequence alignment. We have built a gene finding and alignment program called SLAM, which aligns and identifies complete exon/intron structures of genes in two related but unannotated sequences of DNA. SLAM is able to reliably predict gene structures for any suitably related pair of organisms, most notably with fewer false-positive predictions compared to previous methods (examples are provided for Homo sapiens/Mus musculus andPlasmodium falciparum/Plasmodium vivax comparisons). Accuracy is obtained by distinguishing conserved noncoding sequence (CNS) from conserved coding sequence. CNS annotation is a novel feature of SLAM and may be useful for the annotation of UTRs, regulatory elements, and other noncoding features.
Gene Expression Analyses of Arabidopsis Chromosome 2 Using a Genomic DNA Amplicon Microarray
Authors: Kim Heenam , Snesrud Erik C. , Haas Brian , Cheung Foo , Town Christopher D. , Quackenbush John
Published: March 2003

The gene predictions and accompanying functional assignments resulting from the sequencing and annotation of a genome represent hypotheses that can be tested and used to develop a more complete understanding of the organism and its biology. In the model plantArabidopsis thaliana, we developed a novel approach to constructing whole-genome microarrays based on PCR amplification of the 3′ ends of each predicted gene from genomic DNA, and constructed an array representing more than 94% of the predicted genes and pseudogenes on chromosome 2. With this array, we examined various tissues and physiological conditions, providing expression-based validation for 84% of the gene predictions and providing clues as to the functions of many predicted genes. Further, by examining the distribution of expression along the physical chromosome, we were able to identify a region of repressed transcription that may represent a previously undescribed heterochromatic region.

[The sequence data from this study have been submitted to ArrayExpress under accession nos.: For the Array Design, A-TIGR-2. For the three subgroups of experiments: AbioticStress, E-TIGR-2; BioticStress, E-TIGR-3; Tissues, E-TIGR-4.]
Genomic DNA Insertions and Deletions Occur Frequently Between Humans and Nonhuman Primates
Authors: Frazer Kelly A. , Chen Xiyin , Hinds David A. , Pant P.V. Krishna , Patil Nila , Cox David R.
Published: March 2003

Comparative DNA sequence studies between humans and nonhuman primates will be important for understanding the genetic basis of the phenotypic differences between these species. Here we compare ∼27 Mb of human chromosome 21 with chimpanzee DNA sequences identifying 57 genomic rearrangements (deletions and insertions ranging in size from 0.2 to 8.0 kb) between the two species. These rearrangements are distributed along the entire length of chromosome 21, with ∼35% found in genomic intervals encoding genes (genic intervals), and have occurred in the genomes of both humans and chimpanzees. Comparison of ∼9 Mb of human chromosome 21 with orangutan, rhesus macaque, and woolly monkey DNA sequences identified a combined total of 114 genomic rearrangements between humans and nonhuman primates. Analysis of these rearrangements revealed that they are randomly distributed with respect to genic and nongenic intervals and identified one deletion that has likely resulted in the inactivation of a gene (β1,3-galactosyltransferase) in the woolly monkey. Our data show that genomic rearrangements have occurred frequently during primate genome evolution and significantly contribute to the DNA differences between these species. These DNA rearrangements are commonly found in genic intervals, and thus provide natural starting points for focused investigations of qualitative and quantitative gene expression differences between humans and other primates.

[Supplemental material is available online atwww.genome.org.]
Comparative Analysis of Superintegrons: Engineering Extensive Genetic Diversity in the Vibrionaceae
Authors: Rowe-Magnus Dean A. , Guerout Anne-Marie , Biskri Latefa , Bouige Philippe , Mazel Didier
Published: March 2003

Integrons are natural tools for bacterial evolution and innovation. Their involvement in the capture and dissemination of antibiotic-resistance genes among Gram-negative bacteria is well documented. Recently, massive ancestral versions, the superintegrons (SIs), were discovered in the genomes of diverse proteobacterial species. SI gene cassettes with an identifiable activity encode proteins related to simple adaptive functions, including resistance, virulence, and metabolic activities, and their recruitment was interpreted as providing the host with an adaptive advantage. Here, we present extensive comparative analysis of SIs identified among the Vibrionaceae. Each was at least 100 kb in size, reaffirming the participation of SIs in the genome plasticity and heterogeneity of these species. Phylogenetic and localization data supported the sedentary nature of the functional integron platform and its coevolution with the host genome. Conversely, comparative analysis of the SI cassettes was indicative of both a wide range of origin for the entrapped genes and of an active cassette assembly process in these bacterial species. The signature attC sites of each species displayed conserved structural characteristics indicating that symmetry rather than sequence was important in the recognition of such a varied collection of target recombination sequences by a single site-specific recombinase. Our discovery of various addiction module cassettes within each of the different SIs indicates a possible role for them in the overall stability of large integron cassette arrays.

[Supplemental material is available online atwww.genome.org. The sequence data from this study have been submitted to GenBank under accession nos. listed in Table 1.]
Genome Duplication, a Trait Shared by 22,000 Species of Ray-Finned Fish
Authors: Taylor John S. , Braasch Ingo , Frickey Tancred , Meyer Axel , Van de Peer Yves
Published: March 2003

Through phylogeny reconstruction we identified 49 genes with a single copy in man, mouse, and chicken, one or two copies in the tetraploid frog Xenopus laevis, and two copies in zebrafish (Danio rerio). For 22 of these genes, both zebrafish duplicates had orthologs in the pufferfish (Takifugu rubripes). For another 20 of these genes, we found only one pufferfish ortholog but in each case it was more closely related to one of the zebrafish duplicates than to the other. Forty-three pairs of duplicated genes map to 24 of the 25 zebrafish linkage groups but they are not randomly distributed; we identified 10 duplicated regions of the zebrafish genome that each contain between two and five sets of paralogous genes. These phylogeny and synteny data suggest that the common ancestor of zebrafish and pufferfish, a fish that gave rise to ∼22,000 species, experienced a large-scale gene or complete genome duplication event and that the pufferfish has lost many duplicates that the zebrafish has retained.

[Supplemental material is available online at www.genome.org.]
The Genetic Core of the Universal Ancestor
Authors: Harris J. Kirk , Kelley Scott T. , Spiegelman George B. , Pace Norman R.
Published: March 2003

Molecular analysis of conserved sequences in the ribosomal RNAs of modern organisms reveals a three-domain phylogeny that converges in a universal ancestor for all life. We used the Clusters of Orthologous Groups database and information from published genomes to search for other universally conserved genes that have the same phylogenetic pattern as ribosomal RNA, and therefore constitute the ancestral genetic core of cells. Our analyses identified a small set of genes that can be traced back to the universal ancestor and have coevolved since that time. As indicated by earlier studies, almost all of these genes are involved with the transfer of genetic information, and most of them directly interact with the ribosome. Other universal genes have either undergone lateral transfer in the past, or have diverged so much in sequence that their distant past could not be resolved. The nature of the conserved genes suggests innovations that may have been essential to the divergence of the three domains of life. The analysis also identified several genes of unknown function with phylogenies that track with the ribosomal RNA genes. The products of these genes are likely to play fundamental roles in cellular processes.
Gene Discovery in the Apicomplexa as Revealed by EST Sequencing and Assembly of a Comparative Gene Database
Authors: Li Li , Brunk Brian P. , Kissinger Jessica C. , Pape Deana , Tang Keliang , Cole Robert H. , Martin John , Wylie Todd , Dante Mike , Fogarty Steven J. , Howe Daniel K. , Liberator Paul , Diaz Carmen , Anderson Jennifer , White Michael , Jerome Maria E. , Johnson Emily A. , Radke Jay A. , Stoeckert Christian J. , Waterston Robert H. , Clifton Sandra W. , Roos David S. , Sibley L. David
Published: March 2003

Large-scale EST sequencing projects for several important parasites within the phylum Apicomplexa were undertaken for the purpose of gene discovery. Included were several parasites of medical importance (Plasmodium falciparum, Toxoplasma gondii) and others of veterinary importance (Eimeria tenella, Sarcocystis neurona, and Neospora caninum). A total of 55,192 ESTs, deposited into dbEST/GenBank, were included in the analyses. The resulting sequences have been clustered into nonredundant gene assemblies and deposited into a relational database that supports a variety of sequence and text searches. This database has been used to compare the gene assemblies using BLAST similarity comparisons to the public protein databases to identify putative genes. Of these new entries, ∼15%–20% represent putative homologs with a conservative cutoff of p < 10−9, thus identifying many conserved genes that are likely to share common functions with other well-studied organisms. Gene assemblies were also used to identify strain polymorphisms, examine stage-specific expression, and identify gene families. An interesting class of genes that are confined to members of this phylum and not shared by plants, animals, or fungi, was identified. These genes likely mediate the novel biological features of members of the Apicomplexa and hence offer great potential for biological investigation and as possible therapeutic targets.

[The sequence data from this study have been submitted to dbEST division of GenBank under accession nos.:Toxoplasma gondii: BG657138–BG661027,BI921045–BI921090, BI946571–BI946588, BM003839–BM004582,BM039066– BM040645, BM131233–BM133172, BM174962–BM176879,BM188953–BM189923, BM271559–BM271694. Plasmodium falciparum:BI670521–BI670830, BI813842–BI816393, BI936022–BI936312,BM273300–BM276553. Sarcocystis neurona: BE574328, BE574347,BE574384, BE574386, BE574409, BE574465, BE574508, BE574543, BE574561,BE574633, BE574689, BE574694, BE574723, BE635418–BE636244,BE574288–BE574724, BF323572–BF324064, BM252128–BM253024,BM303125–BM305293. Eimeria tenella: AI755306–AI758088,AI759179–AI759181, AI759254–AI759304, AI759182–AI759253, AI759305–AI759387, AI759463–AI759546,AI759388–AI759462, AI759547–AI759621, BE027133– BE028807,BF023640–BF023711, BF023609–BF023639, BG235514–BG235880,BG413067–BG413336, BG466192–BG467045, BG515959–BG517044,BG560819–BG562379, BG724474–BG725148, BI895002–BI896127,BM305294–BM306971, BM321464–BM322026. Neospora caninum: BF248514–BF249435, BF716421–BF717094, BF823742,BF823805– BF823813, BF823743–BF824633, BG235070–BG235513.]
Large-Scale Discovery of Induced Point Mutations With High-Throughput TILLING
Authors: Till Bradley J. , Reynolds Steven H. , Greene Elizabeth A. , Codomo Christine A. , Enns Linda C. , Johnson Jessica E. , Burtner Chris , Odden Anthony R. , Young Kim , Taylor Nicholas E. , Henikoff Jorja G. , Comai Luca , Henikoff Steven
Published: March 2003

TILLING (Targeting Induced LocalLesions inGenomes) is a general reverse-genetic strategy that provides an allelic series of induced point mutations in genes of interest. High-throughput TILLING allows the rapid and low-cost discovery of induced point mutations in populations of chemically mutagenized individuals. As chemical mutagenesis is widely applicable and mutation detection for TILLING is dependent only on sufficient yield of PCR products, TILLING can be applied to most organisms. We have developed TILLING as a service to the Arabidopsis community known as the ArabidopsisTILLING Project (ATP). Our goal is to rapidly deliver allelic series of ethylmethanesulfonate-induced mutations in target 1-kb loci requested by the international research community. In the first year of public operation, ATP has discovered, sequenced, and delivered >1000 mutations in >100 genes ordered by Arabidopsis researchers. The tools and methodologies described here can be adapted to create similar facilities for other organisms.
Alteration of Microtubule Dynamic Instability during Preprophase Band Formation Revealed by Yellow Fluorescent Protein–CLIP170 Microtubule Plus-End Labeling[W]
Authors: Dhonukshe Pankaj , Gadella Theodorus W. J.
Published: March 2003

At the onset of mitosis, plant cells form a microtubular preprophase band that defines the plane of cell division, but the mechanism of its formation remains a mystery. Here, we describe the use of mammalian yellow fluorescent protein–tagged CLIP170 to visualize the dynamic plus ends of plant microtubules in transfected cowpea protoplasts and in stably transformed and dividing tobacco Bright Yellow 2 cells. Using plus-end labeling, we observed dynamic instability in different microtubular conformations in live plant cells. The interphase plant microtubules grow at 5 μm/min, shrink at 20 μm/min, and display catastrophe and rescue frequencies of 0.02 and 0.08 events/s, respectively, exhibiting faster turnover than their mammalian counterparts. Strikingly, during preprophase band formation, the growth rate and catastrophe frequency of plant microtubules double, whereas the shrinkage rate and rescue frequency remain unchanged, making microtubules shorter and more dynamic. Using these novel insights and four-dimensional time-lapse imaging data, we propose a model that can explain the mechanism by which changes in microtubule dynamic instability drive the dramatic rearrangements of microtubules during preprophase band and spindle formation in plant cells.
Characterization of the Last Subunit of the Arabidopsis COP9 Signalosome: Implications for the Overall Structure and Origin of the Complex[W]
Authors: Serino Giovanna , Su Hongwen , Peng Zhaohua , Tsuge Tomohiko , Wei Ning , Gu Hongya , Deng Xing Wang
Published: March 2003

The COP9 signalosome (CSN) is an evolutionarily conserved protein complex that resembles the lid subcomplex of proteasomes. Through its ability to regulate specific proteasome-mediated protein degradation events, CSN controls multiple aspects of development. Here, we report the cloning and characterization of AtCSN2, the last uncharacterized CSN subunit from Arabidopsis. We show that the AtCSN2 gene corresponds to the previously identified FUS12 locus and that AtCSN2 copurifies with CSN, confirming that AtCSN2 is an integral component of CSN. AtCSN2 is not only able to interact with the SCFTIR1 subunit AtCUL1, which is partially responsible for the regulatory interaction between CSN and SCFTIR1, but also interacts with AtCUL3, suggesting that CSN is able to regulate the activity of other cullin-based E3 ligases through conserved interactions. Phylogenetic analysis indicated that the duplication and subsequent divergence events that led to the genes that encode CSN and lid subunits occurred before the divergence of unicellular and multicellular eukaryotic organisms and that the CSN subunits were more conserved than the lid subunits during evolution. Comparative analyses of the subunit interaction of CSN revealed a set of conserved subunit contacts and resulted in a model of CSN subunit topology, some aspects of which were substantiated by in vivo cross-link tests.
What is reference for?
Authors: Janes Joseph
Published: March 2003

Much discussion has taken place about the future of reference work and its place within the library and the emerging information environment. It seems reasonable, then, to reflect briefly on the purposes and motivations of reference – past, present and future – to see if those do not help us get a better handle on what we should be doing in the years to come.
Scenario planning for the future of reference: five white papers posit the future and raise the bar for us all
Authors: Barbara Watstein Sarah
Published: March 2003

Provides an examination of conference papers as illustrations of scenario planning in action. Includes a summary of each author’s understanding of whether or not reference service has a future, and presents highlights of each author’s future vision for reference services. Acknowledges that white paper authors have succeeded on two levels – in looking beyond their current models of reality and in considering a future that is radically different. Suggests that this is a challenge that confronts all librarians involved in planning for, administering, delivering, and otherwise supporting reference services.
Chat: from the desk of a subject librarian
Authors: Porter Susan
Published: March 2003

Off‐campus students at La Trobe University are a growing group who do not always receive assistance through information literacy classes, or at their time of need. Previous efforts by the subject librarian to answer e‐mail queries from off‐campus nursing students have sometimes been less than satisfactory for both the student and the librarian, requiring that a telephone call be made in order to conduct a reference interview. A chat reference service was offered to off‐campus nursing students as an alternative method of contacting the subject librarian or the library. There was a trial in March and April 2002 using LivePerson, and involving three health sciences librarians. The trial would determine: if chat increased the number of students who sought/received help; if a reference interview can be conducted properly during a chat; and the infrastructure required to continue a chat service, particularly whether using subject librarians removes the need to roster staff on the service.
Information literacy and Writing across the Curriculum: sharing the vision
Authors: Elmborg James K.
Published: March 2003

Points out the similarities and differences between library instruction and writing instruction in the higher education curriculum. Notes that information literacy librarians can learn from the experiences of composition instructors regarding curricular revision and reform. Suggests that one of the keys to information literacy reaching its potential is to find common ground with programs like Writing across the Curriculum.
Integration of information literacy skills into business courses
Authors: Feast Vicki
Published: March 2003

Using content analysis and staff interviews, this study evaluated the impact of an action plan that aimed to assist in integrating information literacy skills into teaching and learning practices of eight first‐year core business courses in the Division of Business and Enterprise (BUE) at the University of South Australia (UniSA). The action plan involved a series of professional development meetings with these eight staff, library liaison staff and learning advisers. In order to evaluate the success of this action plan, comparisons were made between two groups of academic staff: those eight first‐year BUE core course coordinators who had experienced the action plan and another eight BUE first‐year staff who had not. An analysis found that there were no statistically significant differences to changes in information literacy teaching and learning practices over the 12 months between the two groups. It was therefore concluded that the action plan had not delivered the intended outcomes. This finding was partly attributed to a large number of inhibiting factors as well as to the short time over which the action plan was conducted.
Roles of the Enantioselective Glutathione
S
-Transferases in Cleavage of β-Aryl Ether
Authors: Masai Eiji , Ichimura Atsushi , Sato Yusuke , Miyauchi Keisuke , Katayama Yoshihiro , Fukuda Masao
Published: March 2003

Cleavage of the β-aryl ether linkage is the most important process in lignin degradation. Here we characterize the three tandemly located glutathione
S
-transferase (GST) genes,
ligF
,
ligE
, and
ligG
, from low-molecular-weight lignin-degrading
Sphingomonas paucimobilis
SYK-6, and we describe the actual roles of these genes in the β-aryl ether cleavage. Based on the identification of the reaction product by electrospray ionization-mass spectrometry, a model compound of β-aryl ether, α-(2-methoxyphenoxy)-β-hydroxypropiovanillone (MPHPV), was transformed by LigF or LigE to guaiacol and α-glutathionyl-β-hydroxypropiovanillone (GS-HPV). This result suggested that LigF and LigE catalyze the nucleophilic attack of glutathione on the carbon atom at the β position of MPHPV. High-pressure liquid chromatography-circular dichroism analysis indicated that LigF and LigE each attacked a different enantiomer of the racemic MPHPV preparation. The
ligG
gene product specifically catalyzed the elimination of glutathione from GS-HPV generated by the action of LigF. This reaction then produces an achiral compound, β-hydroxypropiovanillone, which is further degraded by this strain. Disruption of the
ligF
,
ligE
, and
ligG
genes in SYK-6 showed that
ligF
is essential to the degradation of one of the MPHPV enantiomers, and the alternative activities which metabolize the substrates of LigE and LigG are present in this strain.