bluuebunny/crossref_metadata_2025 huggingface dataset:
Mechanisms associated with cGMP binding and activation of cGMP-dependent protein kinase
Authors: Wall Michael E. , Francis Sharron H. , Corbin Jackie D. , Grimes Kennard , Richie-Jannetta Robyn , Kotera Jun , Macdonald Brian A. , Gibson Rowena R. , Trewhella Jill
Published: March 2003

Using small-angle x-ray scattering, we have observed the cGMP-induced elongation of an active, cGMP-dependent, monomeric deletion mutant of cGMP-dependent protein kinase (Δ
1–52
PKG-Iβ). On saturation with cGMP, the radius of gyration of Δ
1–52
PKG-Iβ increases from 29.4 ± 0.1 Å to 40.1 ± 0.7 Å, and the maximum linear dimension increases from 90 Å ± 10% to 130 Å ± 10%. The elongation is due to a change in the interaction between structured regulatory (R) and catalytic (C) domains. A model of cGMP binding to Δ
1–52
PKG-Iβ indicates that elongation of Δ
1–52
PKG-Iβ requires binding of cGMP to the low-affinity binding site of the R domain. A comparison with cAMP-dependent protein kinase suggests that both elongation and activation require cGMP binding to both sites; cGMP binding to the low-affinity site therefore seems to be a necessary, but not sufficient, condition for both elongation and activation of Δ
1–52
PKG-Iβ. We also predict that there is little or no cooperativity in cGMP binding to the two sites of Δ
1–52
PKG-Iβ under the conditions used here. Results obtained by using the Δ
1–52
PKG-Iβ monomer indicate that a previously observed elongation of PKG-Iα is consistent with a pure change in the interaction between the R domain and the C domain, without alteration of the dimerization interaction. This study has revealed important features of molecular mechanisms in the biochemical network describing PKG-Iβ activation by cGMP, yielding new insight into ligand activation of cyclic nucleotide-dependent protein kinases, a class of regulatory proteins that is key to many cellular processes.
Atomic-level observation of macromolecular crowding effects: Escape of a protein from the GroEL cage
Authors: Elcock Adrian H.
Published: March 2003

Experimental work has demonstrated that the efficient operation of the GroEL-GroES chaperonin machinery is sensitive to the presence of macromolecular crowding agents. Here, I describe atomically detailed computer simulations that provide a microscopic view of how crowding effects are exerted. Simulations were performed to compute the free energy required to extract the protein rhodanese from the central cavity of GroEL into solutions containing a range of crowder concentrations. The computed energetics allow the total yield of folded protein to be predicted; the calculated yields show a nonlinear dependence on the concentration of crowding agent identical to that observed experimentally. The close correspondence between simulation and experiment prompts the use of the former in a truly predictive setting: simulations are used to suggest that more effective crowding agents might be designed by exploiting an “agoraphobic effect.”
Ends-out, or replacement, gene targeting in
Drosophila
Authors: Gong Wei J. , Golic Kent G.
Published: March 2003

Ends-in and ends-out refer to the two arrangements of donor DNA that can be used for gene targeting. Both have been used for targeted mutagenesis, but require donors of differing design. Ends-out targeting is more frequently used in mice and yeast because it gives a straightforward route to replace or delete a target locus. Although ends-in targeting has been successful in
Drosophila
, an attempt at ends-out targeting failed. To test whether ends-out targeting could be used in
Drosophila
, we applied two strategies for ends-out gene replacement at the endogenous
yellow
(
y
) locus in
Drosophila
. First, a mutant allele was rescued by replacement with an 8-kb

y
+

DNA fragment at a rate of ≈1/800 gametes. Second, a wild-type gene was disrupted by the insertion of a marker gene in exon 1 at a rate of ≈1/380 gametes. The I-SceI endonuclease component alone is not sufficient for targeting: the FLP recombinase is also needed to generate the extrachromosomal donor. When both components are used we find that ends-out targeting can be approximately as efficient as ends-in targeting, and is likely to be generally useful for
Drosophila
gene targeting.
Identification of the coupling between skeletal muscle store-operated Ca
2+
entry and the inositol trisphosphate receptor
Authors: Launikonis Bradley S. , Barnes Melissa , Stephenson D. George
Published: March 2003

Examination of store-operated Ca
2+
entry (SOC) in single, mechanically skinned skeletal muscle cells by confocal microscopy shows that the inositol 1,4,5-trisphosphate (IP
3
) receptor acts as a sarcoplasmic reticulum [Ca
2+
] sensor and mediates SOC by physical coupling without playing a key role in Ca
2+
release from internal stores, as is the case with various cell types in which SOC was investigated previously. The results have broad implications for understanding the mechanism of SOC that is essential for cell function in general and muscle function in particular. Moreover, the study ascribes an important role to the IP
3
receptors in skeletal muscle, the role of which with respect to Ca
2+
homeostasis was ill defined until now.
Dietary restriction normalizes glucose metabolism and BDNF levels, slows disease progression, and increases survival in huntingtin mutant mice
Authors: Duan Wenzhen , Guo Zhihong , Jiang Haiyang , Ware Melvin , Li Xiao-Jiang , Mattson Mark P.
Published: March 2003

In addition to neurological deficits, Huntington's disease (HD) patients and transgenic mice expressing mutant human huntingtin exhibit reduced levels of brain-derived neurotrophic factor, hyperglycemia, and tissue wasting. We show that the progression of neuropathological (formation of huntingtin inclusions and apoptotic protease activation), behavioral (motor dysfunction), and metabolic (glucose intolerance and tissue wasting) abnormalities in huntingtin mutant mice, an animal model of HD, are retarded when the mice are maintained on a dietary restriction (DR) feeding regimen resulting in an extension of their life span. DR increases levels of brain-derived neurotrophic factor and the protein chaperone heat-shock protein-70 in the striatum and cortex, which are depleted in HD mice fed a normal diet. The suppression of the pathogenic processes by DR in HD mice suggests that mutant huntingtin promotes neuronal degeneration by impairing cellular stress resistance, and that the body wasting in HD is driven by the neurodegenerative process. Our findings suggest a dietary intervention that may suppress the disease process and increase the life span of humans that carry the mutant huntingtin gene.
A human peripheral blood monocyte-derived subset acts as pluripotent stem cells
Authors: Zhao Yong , Glesne David , Huberman Eliezer
Published: March 2003

We have identified, cultured, characterized, and propagated adult pluripotent stem cells (PSC) from a subset of human peripheral blood monocytes. These cells, which in appearance resemble fibroblasts, expand in the presence of macrophage colony-stimulating factor and display monocytic and hematopoietic stem cell markers including CD14, CD34, and CD45. We have induced these cells to differentiate into mature macrophages by lipopolysaccharide, T lymphocytes by IL-2, epithelial cells by epidermal growth factor, endothelial cells by vascular endothelial cell growth factor, neuronal cells by nerve growth factor, and liver cells by hepatocyte growth factor. The pluripotent nature of individual PSC was further confirmed by a clonal analysis. The ability to store, expand, and differentiate these PSC from autologous peripheral blood should make them valuable candidates for transplantation therapy.
Relish-mediated immune deficiency in the transgenic mosquito
Aedes aegypti
Authors: Shin Sang Woon , Kokoza Vladimir , Lobkov Iouri , Raikhel Alexander S.
Published: March 2003

The lack of genetic means has been a serious limitation in studying mosquito immunity. We generated Relish-mediated immune deficiency (RMID) by transforming
Aedes aegypti
with the Δ
Rel
transgene driven by the
vitellogenin
(
Vg
) promoter using the pBac[3xP3-EGFP, afm] vector. A stable transformed line had a single copy of the
Vg-ΔRel
transgene. The
Vg-ΔRel
transgene expression was highly activated by blood feeding, and transgenic mosquitoes were extremely susceptible to the infection by Gram-negative bacteria. This RMID phenotype was characterized by severely reduced postinfection levels of antimicrobial peptides genes,
defensin
and
cecropin
. Crossing the RMID line with the wild-type strain produced the same RMID phenotype, indicating its dominant nature, whereas crossing with the
Vg-def
transgenic line, in which Defensin A was activated by blood feeding, restored the immunity to
Enterobacter cloacae
.
The weighted-volume derivative of a space-filling diagram
Authors: Edelsbrunner Herbert , Koehl Patrice
Published: March 2003

Computing the volume occupied by individual atoms in macromolecular structures has been the subject of research for several decades. This interest has grown in the recent years, because weighted volumes are widely used in implicit solvent models. Applications of the latter in molecular mechanics simulations require that the derivatives of these weighted volumes be known. In this article, we give a formula for the volume derivative of a molecule modeled as a space-filling diagram made up of balls in motion. The formula is given in terms of the weights, radii, and distances between the centers as well as the sizes of the facets of the power diagram restricted to the space-filling diagram. Special attention is given to the detection and treatment of singularities as well as discontinuities of the derivative.
Retroevolution of λ Cro toward a stable monomer
Authors: LeFevre Kelly R. , Cordes Matthew H. J.
Published: March 2003

The Cro protein from bacteriophage λ has a dimeric α+β fold that evolved from an ancestral all-α monomer. The sequence mutations responsible for this dramatic structural evolution are unknown. Here we use analysis of sequence alignments to show that Ala-33, a small side chain in the hydrophobic “ball-and-socket” dimer interface of λ Cro, was a much larger tryptophan side chain at a previous point in evolution. The retroevolutionary λ Cro-A33W mutant shows a 10-fold reduction in dimerization affinity relative to the wild type as well as a large increase in monomer thermal stability (Δ
T
m
> 10°C), apparently due to partial filling of the hydrophobic socket from within the same monomer. An additional mutation in the dimer interface, F58D, almost completely abolishes detectable dimerization while maintaining the high monomer stability. The secondary structure content of the monomerized versions of λ Cro is similar to that of the wild-type protein, and the tertiary structure of the monomer appears relatively well defined. These results (
i
) support a model in which the ball-and-socket dimer interface of λ Cro was created by altered volume mutations within a limited branch of the Cro lineage and (
ii
) suggest the possibility that the evolution of the α+β dimer from an all-α monomer proceeded through an α+β monomer intermediate.
Single-cell perforin and granzyme expression reveals the anatomical localization of effector CD8+T cells in influenza virus-infected mice
Authors: Johnson Barbara J. , Costelloe Elaine O. , Fitzpatrick David R. , Haanen John B. A. G. , Schumacher Ton N. M. , Brown Lorena E. , Kelso Anne
Published: March 2003

Influenza virus infection activates cytolytic T lymphocytes (CTL) that contribute to viral clearance by releasing perforin and granzymes from cytoplasmic granules. Virus-specific, perforin-dependent CD8+CTL were detected in freshly isolated cells from the mouse lung parenchyma but not from the mediastinal lymph nodes (MLN), where they are primed, or from the spleen during primary influenza virus infection. To determine whether this difference was due to the low frequency or incomplete maturation of effector CTL in MLN, we measured expression of perforin, granzymes A, B, and C, and IFN-γ mRNAs in CD8+populations and single cells immediately after isolation from virus-infected mice. Quantitative PCR revealed significant expression of perforin, granzyme A, granzyme B, and IFN-γ in activated CD8+cells from MLN, spleen, and lung parenchyma. Granzyme C expression was not detected. Individual activated or nucleoprotein peptide/class I tetramer-binding CD8+cells from the three tissues expressed diverse combinations of perforin, granzyme, and IFN-γ mRNAs. Although cells from lung expressed granzymes A and B at higher frequency, each of the tissues contained cells that coexpressed perforin with granzymes A and/or B. The main difference between MLN and lung was the elevated frequency of activated CD8+T cells in the lung, rather than their perforin/granzyme expression profile. The data suggest that some CTL mature into perforin/granzyme-expressing effector cells in MLN but reach detectable frequencies only when they accumulate in the infected lung.
Structural basis of two-stage voltage-dependent activation in K+channels
Authors: Silverman William R. , Roux Benoît , Papazian Diane M.
Published: March 2003

The structure of the voltage sensor and the detailed physical basis of voltage-dependent activation in ion channels have not been determined. We now have identified conserved molecular rearrangements underlying two major voltage-dependent conformational changes during activation of divergent K+channels, ether-à-go-go (eag) and Shaker. Two conserved arginines of the S4 voltage sensor move sequentially into an extracellular gating pocket, where they interact with an acidic residue in S2. In eag, these transitions are modulated by a divalent ion that binds in the gating pocket. Conservation of key molecular details in the activation mechanism confirms that voltage sensors in divergent K+channels share a common structure. Molecular modeling reveals that structural constraints derived from eag and Shaker specify the unique packing arrangement of transmembrane segments S2, S3, and S4 within the voltage sensor.
Extraordinarily slow binding of guanosine to the
Tetrahymena
group I ribozyme: Implications for RNA preorganization and function
Authors: Karbstein Katrin , Herschlag Daniel
Published: March 2003

The
Tetrahymena
ribozyme derived from the self-splicing group I intron binds a 5′-splice site analog (S) and guanosine (G), catalyzing their conversion to a 5′-exon analog (P) and GA. Herein, we show that binding of guanosine is exceptionally slow, limiting the reaction at near neutral pH. Our results implicate a conformational rearrangement on guanosine binding, likely because the binding site is not prearranged in the absence of ligand. The fast accommodation of guanosine (10
2
to 10
3
⋅s
−1
) and prior structural data suggest local rather than global rearrangements, raising the possibility that folding of this and perhaps other large RNAs is not fully cooperative. Guanosine binding is accelerated by addition of residues that form helices, referred to as P9.0 and P10, immediately 5′ and 3′ to the guanosine. These rate enhancements provide evidence for binding intermediates that have the adjacent helices formed before accommodation of guanosine into its binding site. Because the ability to form the P9.0 and P10 helices distinguishes the guanosine at the correct 3′-splice site from other guanosine residues, the faster binding of the correct guanosine can enhance specificity of 3′-splice site selection. Thus, paradoxically, the absence of a preformed binding site and the resulting slow guanosine binding can contribute to splicing specificity by providing an opportunity for the adjacent helices to increase the rate of binding of the guanosine specifying the 3′-splice site.
Domestication quantitative trait loci inTriticum dicoccoides, the progenitor of wheat
Authors: Peng Junhua , Ronin Yefim , Fahima Tzion , Röder Marion S. , Li Youchun , Nevo Eviatar , Korol Abraham
Published: March 2003

Wild emmer wheat,Triticum dicoccoides, is the progenitor of modern tetraploid and hexaploid cultivated wheats. Our objective was to map domestication-related quantitative trait loci (QTL) inT. dicoccoides. The studied traits include brittle rachis, heading date, plant height, grain size, yield, and yield components. Our mapping population was derived from a cross betweenT. dicoccoidesandTriticum durum. Approximately 70 domestication QTL effects were detected, nonrandomly distributed among and along chromosomes. Seven domestication syndrome factors were proposed, each affecting 5–11 traits. We showed: (i) clustering and strong effects of some QTLs; (ii) remarkable genomic association of strong domestication-related QTLs with gene-rich regions; and (iii) unexpected predominance of QTL effects in the A genome. The A genome of wheat may have played a more important role than the B genome during domestication evolution. The cryptic beneficial alleles at specific QTLs derived fromT. dicoccoidesmay contribute to wheat and cereal improvement.
Activation of presynaptic NMDA receptors coupled to NaV1.8-resistant sodium channel C-fibers causes retrograde mechanical nociceptor sensitization
Authors: Parada Carlos Amílcar , Vivancos Gustavo Gameiro , Tambeli Claudia Herrera , de Queiróz Cunha Fernando , Ferreira Sérgio Henrique
Published: March 2003

The present study investigated whether activation of presynaptic
N
-methyl-
d
-aspartate (NMDA) receptors in the spinal cord produces a retrograde nociceptor sensitization (hypernociception) to mechanical nonnoxious stimulus. By using an electronic version of the von Frey hair test (pressure meter), s.c. intraplantar administration of prostaglandin E
2
(PGE
2
) (50–400 ng per paw) evoked a dose-related ipsilateral paw hypernociception. In contrast, intrathecal (i.t.) administration of NMDA (5–80 ng) and PGE
2
(15–150 ng) evoked dose-related bilateral paw hypernociception. The s.c. intraplantar administration of dipyrone (80–320 μg per paw) or morphine (3 and 9 μg per paw), usually used to antagonize peripheral PGE
2
(100 ng per paw), induced hypernociception and also antagonized the ipsilateral (without affecting the contralateral) paw hypernociception induced by i.t. injections of NMDA (40 ng) or PGE
2
(50 ng). These doses of drugs did not modify the basal mechanical sensitivity of control paws. This result shows that intraspinal NMDA or PGE
2
produces sensitization of the primary sensory neuron in response to mechanical stimulation. In a second series of experiments it was shown that the i.t. treatment with NaV1.8 (SNS/PN3) sodium channel antisense oligodeoxynucleotides, but not mismatch oligodeoxynucleotides, decreased the mRNA expression of sodium tetrodotoxin-resistant channels on the dorsal root ganglia and abolished the mechanical hypernociception induced by i.t. administration of NMDA. Thus, our results support the suggestion that glutamate release in the spinal cord during inflammation causes retrograde hypernociception of nociceptors associated with sodium tetrodotoxin-resistant channels in primary nociceptive sensory neurons.
Integrin β cytoplasmic domain interactions with phosphotyrosine-binding domains: A structural prototype for diversity in integrin signaling
Authors: Calderwood David A. , Fujioka Yosuke , de Pereda Jose M. , García-Alvarez Begoña , Nakamoto Tetsuya , Margolis Ben , McGlade C. Jane , Liddington Robert C. , Ginsberg Mark H.
Published: March 2003

The cytoplasmic domains (tails) of heterodimeric integrin adhesion receptors mediate integrins' biological functions by binding to cytoplasmic proteins. Most integrin β tails contain one or two NPXY/F motifs that can form β turns. These motifs are part of a canonical recognition sequence for phosphotyrosine-binding (PTB) domains, protein modules that are present in a wide variety of signaling and cytoskeletal proteins. Indeed, talin and ICAP1-α bind to integrin β tails by means of a PTB domain–NPXY ligand interaction. To assess the generality of this interaction we examined the binding of a series of recombinant PTB domains to a panel of short integrin β tails. In addition to the known integrin-binding proteins, we found that Numb (a negative regulator of Notch signaling) and Dok-1 (a signaling adaptor involved in cell migration) and their isolated PTB domain bound to integrin tails. Furthermore, Dok-1 physically associated with integrin αIIbβ3. Mutations of the integrin β tails confirmed that these interactions are canonical PTB domain–ligand interactions. First, the interactions were blocked by mutation of an NPXY motif in the integrin tail. Second, integrin class-specific interactions were observed with the PTB domains of Dab, EPS8, and tensin. We used this specificity, and a molecular model of an integrin β tail–PTB domain interaction to predict critical interacting residues. The importance of these residues was confirmed by generation of gain- and loss-of-function mutations in β7 and β3 tails. These data establish that short integrin β tails interact with a large number of PTB domain-containing proteins through a structurally conserved mechanism.
Stochastic gene expression as a many-body problem
Authors: Sasai Masaki , Wolynes Peter G.
Published: March 2003

Gene expression has a stochastic component because of the single-molecule nature of the gene and the small number of copies of individual DNA-binding proteins in the cell. We show how the statistics of such systems can be mapped onto quantum many-body problems. The dynamics of a single gene switch resembles the spin-boson model of a two-site polaron or an electron transfer reaction. Networks of switches can be approximately described as quantum spin systems by using an appropriate variational principle. In this way, the concept of frustration for magnetic systems can be taken over into gene networks. The landscape of stable attractors depends on the degree and style of frustration, much as for neural networks. We show the number of attractors, which may represent cell types, is much smaller for appropriately designed weakly frustrated stochastic networks than for randomly connected networks.
Hypothermia rescues hippocampal CA1 neurons and attenuates down-regulation of the AMPA receptor GluR2 subunit after forebrain ischemia
Authors: Colbourne Frederick , Grooms Sonja Y. , Zukin R. Suzanne , Buchan Alastair M. , Bennett Michael V. L.
Published: March 2003

Brief forebrain ischemia in rodents induces selective and delayed neuronal death, particularly of hippocampal CA1 pyramidal neurons. Neuronal death is preceded by down-regulation specific to CA1 of GluR2, the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunit that limits Ca
2+
influx. This alteration is hypothesized to cause neurodegeneration by permitting a lethal influx of Ca
2+
and/or Zn
2+
through newly formed GluR2-lacking AMPA receptors. Two days of mild hypothermia induced 1 h after ischemia potently and lastingly protects against ischemic injury. We examined molecular mechanisms underlying hypothermia-induced neuroprotection. We report that hypothermia rescues most hippocampal CA1 neurons from ischemia-induced cell death and attenuates ischemia-induced down-regulation of mRNA encoding the AMPA receptor subunit GluR2. Ischemia induced a marked down-regulation of GluR2 mRNA and a small down-regulation of GluR1 mRNA in CA1 at 2 days, as assessed by quantitative
in situ
hybridization. The ischemia-induced changes in gene expression were cell-specific in that GluR2 was not significantly altered in CA3 or dentate gyrus. After ischemia treated by hypothermia GluR2 mRNA expression was modestly reduced at 2 days and exhibited complete recovery to control levels at 7 days. Hypothermia prevented ischemia induced changes in GluR1 mRNA expression. These findings suggest that intervention at the level of transcriptional regulation of the GluR2 gene may be a mechanism by which prolonged postischemic cooling rescues CA1 neurons otherwise “destined to die.”
Recent advances in our understanding of risk-sensitive foraging preferences
Authors: Bateson Melissa
Published: March 2003

Many experiments have shown that foraging animals are sensitive to the riskiness, or variance, associated with alternative food sources. For example, when offered a choice of a constant feeding option that always offers three seeds, and a risky option that offers either no seeds or six seeds with equal probability, most animals tested will be either risk-averse or risk-prone, preferring either the fixed or variable option respectively. Whether animals are risk-averse or risk-prone appears to depend on a range of factors, including the energetic status of the forager, the type of variance associated with the feeding options and even the number of feeding options between which the animal is choosing. These behavioural phenomena have attracted much theoretical interest, and a range of different explanations have been suggested, some based on a consideration of the psychological mechanisms involved in decision making, and others on a consideration of the Darwinian fitness consequences of risk-averse or risk-prone behaviour for the forager. A brief review of the recent literature on risk-sensitive foraging will be presented, focusing on results from the two experimental systems with which I have been involved: wild rufous hummingbirds (Selasphorus rufus) foraging on artificial flowers; European starlings (Sturnus vulgaris) foraging in operant boxes in the laboratory. It will be argued that to understand the foraging decisions of animals account needs to be taken of both the psychological mechanisms underlying decision-making and the fitness consequences of different decisions for the forager.
Minus-end capture of preformed kinetochore fibers contributes to spindle morphogenesis
Authors: Khodjakov Alexey , Copenagle Lily , Gordon Michael B. , Compton Duane A. , Kapoor Tarun M.
Published: March 2003

Near-simultaneous three-dimensional fluorescence/differential interference contrast microscopy was used to follow the behavior of microtubules and chromosomes in living α-tubulin/GFP-expressing cells after inhibition of the mitotic kinesin Eg5 with monastrol. Kinetochore fibers (K-fibers) were frequently observed forming in association with chromosomes both during monastrol treatment and after monastrol removal. Surprisingly, these K-fibers were oriented away from, and not directly connected to, centrosomes and incorporated into the spindle by the sliding of their distal ends toward centrosomes via a NuMA-dependent mechanism. Similar preformed K-fibers were also observed during spindle formation in untreated cells. In addition, upon monastrol removal, centrosomes established a transient chromosome-free bipolar array whose orientation specified the axis along which chromosomes segregated. We propose that the capture and incorporation of preformed K-fibers complements the microtubule plus-end capture mechanism and contributes to spindle formation in vertebrates.
A role of topoisomerase II in linking DNA replication to chromosome condensation
Authors: Cuvier Olivier , Hirano Tatsuya
Published: March 2003

The condensin complex and topoisomerase II (topo II) have different biochemical activities in vitro, and both are required for mitotic chromosome condensation. We have used Xenopus egg extracts to investigate the functional interplay between condensin and topo II in chromosome condensation. When unreplicated chromatin is directly converted into chromosomes with single chromatids, the two proteins must function together, although they are independently targeted to chromosomes. In contrast, the requirement for topo II is temporarily separable from that of condensin when chromosome assembly is induced after DNA replication. This experimental setting allows us to find that, in the absence of condensin, topo II becomes enriched in an axial structure within uncondensed chromatin. Subsequent addition of condensin converts this structure into mitotic chromosomes in an ATP hydrolysis–dependent manner. Strikingly, preventing DNA replication by the addition of geminin or aphidicolin disturbs the formation of topo II–containing axes and alters the binding property of topo II with chromatin. Our results suggest that topo II plays an important role in an early stage of chromosome condensation, and that this function of topo II is tightly coupled with prior DNA replication.
A mechanism of coupling RCC1 mobility to RanGTP production on the chromatin in vivo
Authors: Li Hoi Yeung , Wirtz Denis , Zheng Yixian
Published: March 2003

The RanGTP gradient across the interphase nuclear envelope and on the condensed mitotic chromosomes is essential for many cellular processes, including nucleocytoplasmic transport and spindle assembly. Although the chromosome-associated enzyme RCC1 is responsible for RanGTP production, the mechanism of generating and maintaining the RanGTP gradient in vivo remains unknown. Here, we report that regulator of chromosome condensation (RCC1) rapidly associates and dissociates with both interphase and mitotic chromosomes in living cells, and that this mobility is regulated during the cell cycle. Our kinetic modeling suggests that RCC1 couples its catalytic activity to chromosome binding to generate a RanGTP gradient. Indeed, we have demonstrated experimentally that the interaction of RCC1 with the chromatin is coupled to the nucleotide exchange on Ran in vivo. The coupling is due to the stable binding of the binary complex of RCC1–Ran to chromatin. Successful nucleotide exchange dissociates the binary complex, permitting the release of RCC1 and RanGTP from the chromatin and the production of RanGTP on the chromatin surface.
Identification of a tight junction–associated guanine nucleotide exchange factor that activates Rho and regulates paracellular permeability
Authors: Benais-Pont Gaëlle , Punn Anu , Flores-Maldonado Catalina , Eckert Judith , Raposo Graça , Fleming Tom P. , Cereijido Marcelino , Balda Maria S. , Matter Karl
Published: March 2003

Rho family GTPases are important regulators of epithelial tight junctions (TJs); however, little is known about how the GTPases themselves are controlled during TJ assembly and function. We have identified and cloned a canine guanine nucleotide exchange factor (GEF) of the Dbl family of proto-oncogenes that activates Rho and associates with TJs. Based on sequence similarity searches and immunological and functional data, this protein is the canine homologue of human GEF-H1 and mouse Lfc, two previously identified Rho-specific exchange factors known to associate with microtubules in nonpolarized cells. In agreement with these observations, immunofluorescence of proliferating MDCK cells revealed that the endogenous canine GEF-H1/Lfc associates with mitotic spindles. Functional analysis based on overexpression and RNA interference in polarized MDCK cells revealed that this exchange factor for Rho regulates paracellular permeability of small hydrophilic tracers. Although overexpression resulted in increased size-selective paracellular permeability, such cell lines exhibited a normal overall morphology and formed fully assembled TJs as determined by measuring transepithelial resistance and by immunofluorescence and freeze-fracture analysis. These data indicate that GEF-H1/Lfc is a component of TJs and functions in the regulation of epithelial permeability.
Quantum loop modules
Authors: Chari Vyjayanthi , Greenstein Jacob
Published: March 2003

We classify the simple infinite-dimensional integrable modules with finite-dimensional weight spaces over the quantized enveloping algebra of an untwisted affine algebra. We prove that these are either highest (lowest) weight integrable modules or simple submodules of a loop module of a finite-dimensional simple integrable module and describe the latter class. Their characters and crystal basis theory are discussed in a special case.
A Classification-Based Machine Learning Approach for the Analysis of Genome-Wide Expression Data
Authors: Lyons-Weiler James , Patel Satish , Bhattacharya Soumyaroop
Published: March 2003

Three important areas of data analysis for global gene expression analysis are class discovery, class prediction, and finding dysregulated genes (biomarkers). The clinical application of microarray data will require marker genes whose expression patterns are sufficiently well understood to allow accurate predictions on disease subclass membership. Commonly used methods of analysis include hierarchical clustering algorithms, t-, F-, and Z-tests, and machine learning approaches. We describe an approach called the maximum difference subset (MDSS) algorithm that combines classification algorithms, classical statistics, and elements of machine learning and provides a coherent framework. By integrating prediction accuracy, the MDSS algorithm learns the critical threshold of statistical significance (the α orP-value), eliminating the arbitrariness of setting a threshold of statistical significance and minimizing the effect of the normality assumptions. To reduce the false positive rate and to increase external validity of the predictive gene set, a jackknife step is used. This step identifies and removes genes in the initial MDSS with low combined predictive utility. The overall MDSS provides a prediction that is less dependent on an arbitrary study design (sample inclusion or exclusion) and should thus have high external validity. We demonstrate that this approach, unlike other published methods, identifies biomarkers capable of predicting the outcome of anthracycline-cytarabine chemotherapy in cases of acute myeloid leukemia. By incorporating two criteria—statistical significance and predictive utility—the approach learns the significance level relevant for a given data set. The MDSS approach can be used with any test and classifier operator pair.
Screening of Gene-Associated Polymorphisms by Use of In-Gel Competitive Reassociation and EST (cDNA) Array Hybridization
Authors: Gotoh Koshichi , Oishi Michio
Published: March 2003

In-gel competitive reassociation (IGCR) is a method of differential subtraction to enrich polymorphic DNA restriction fragments between two DNA samples without probes or specific sequence information. Here, we show that by combining IGCR and expressed sequence tags (EST) array hybridization, polymorphic DNA fragments associated with genes in complex higher organisms (Arabidopsis thaliana) can be effectively screened, demonstrating that this procedure offers a simple and efficient way to obtain gene-associated polymorphic DNA markers.

[The following individuals kindly provided reagents, samples, or unpublished information as indicated in the paper: N. Sakurai, D. Shibata, and C. Kuwata.]
Segmental Duplications in Euchromatic Regions of Human Chromosome 5: A Source of Evolutionary Instability and Transcriptional Innovation
Authors: Courseaux Anouk , Richard Florence , Grosgeorge Josiane , Ortola Christine , Viale Agnes , Turc-Carel Claude , Dutrillaux Bernard , Gaudray Patrick , Nahon Jean-Louis
Published: March 2003

Recent analyses of the structure of pericentromeric and subtelomeric regions have revealed that these particular regions of human chromosomes are often composed of blocks of duplicated genomic segments that have been associated with rapid evolutionary turnover among the genomes of closely related primates. In the present study, we show that euchromatic regions of human chromosome 5—5p14, 5p13, 5q13, 5q15–5q21—also display such an accumulation of segmental duplications. The structure, organization and evolution of those primate-specific sequences were studied in detail by combining in silico and comparative FISH analyses on human, chimpanzee, gorilla, orangutang, macaca, and capuchin chromosomes. Our results lend support to a two-step model of transposition duplication in the euchromatic regions, with a founder insertional event at the time of divergence betweenPlatyrrhiniandCatarrhini(25–35 million years ago) and an apparent burst of inter- and intrachromosomal duplications in theHominidaelineage. Furthermore, phylogenetic analysis suggests that the chronology and, likely, molecular mechanisms, differ regarding the region of primary insertion—euchromatic versus pericentromeric regions. Lastly, we show that as their counterparts located near the heterochromatic region, the euchromatic segmental duplications have consistently reshaped their region of insertion during primate evolution, creating putative mosaic genes, and they are obvious candidates for causing ectopic rearrangements that have contributed to evolutionary/genomic instability.

[Supplemental material is available online atwww.genome.org. The following individuals kindly provided reagents, samples, or unpublished information as indicated in the paper: D. Le Paslier, A. McKenzie, J. Melki, C. Sargent, J. Scharf and S. Selig.]
Large-Scale Identification of Single-Feature Polymorphisms in Complex Genomes
Authors: Borevitz Justin O. , Liang David , Plouffe David , Chang Hur-Song , Zhu Tong , Weigel Detlef , Berry Charles C. , Winzeler Elizabeth , Chory Joanne
Published: March 2003

We have developed a high-throughput genotyping platform by hybridizing genomic DNA from Arabidopsis thaliana accessions to an RNA expression GeneChip (AtGenome1). Using newly developed analytical tools, a large number of single-feature polymorphisms (SFPs) were identified. A comparison of two accessions, the reference strain Columbia (Col) and the strain Landsberg erecta (Ler), identified nearly 4000 SFPs, which could be reliably scored at a 5% error rate. Ler sequence was used to confirm 117 of 121 SFPs and to determine the sensitivity of array hybridization. Features containing sequence repeats, as well as those from high copy genes, showed greater polymorphism rates. A linear clustering algorithm was developed to identify clusters of SFPs representing potential deletions in 111 genes at a 5% false discovery rate (FDR). Among the potential deletions were transposons, disease resistance genes, and genes involved in secondary metabolism. The applicability of this technique was demonstrated by genotyping a recombinant inbred line. Recombination break points could be clearly defined, and in one case delimited to an interval of 29 kb. We further demonstrate that array hybridization can be combined with bulk segregant analysis to quickly map mutations. The extension of these tools to organisms with complex genomes, such as Arabidopsis, will greatly increase our ability to map and clone quantitative trait loci (QTL).

[Supplemental material is available online atwww.genome.org.]
Large-Scale Analysis of the Meningococcus Genome by Gene Disruption: Resistance to Complement-Mediated Lysis
Authors: Geoffroy Marie-Claude , Floquet Stéphanie , Métais Arnaud , Nassif Xavier , Pelicic Vladimir
Published: March 2003

The biologic role of a majority of the Neisseria meningitidis 2100 predicted coding regions is still to be assigned or experimentally confirmed. Determining the phenotypic effect of gene disruption being a fundamental approach to understanding gene function, we used high-density signature-tagged transposon mutagenesis, followed by a large-scale sequencing of the transposon insertion sites, to construct a genome-wide collection of mutants. The sequencing results for the first half of the 4548 mutants composing the library suggested that we have mutations in 80%–90% of N. meningitidisnonessential genes. This was confirmed by a whole-genome identification of the genes required for resistance to complement-mediated lysis, a key to meningococcal virulence. We show that all the genes we identified, including four previously uncharacterized, were important for the synthesis of the polysialic acid capsule or the lipooligosaccharide (LOS), suggesting that these are likely to be the only meningococcal attributes necessary for serum resistance. Our work provides a valuable and lasting resource that may lead to a global map of gene function in N. meningitidis.

[Supplemental material is available online at www.genome.org. The following individuals kindly provided reagents, samples, or unpublished information as indicated in the paper: E. Carbonnelle, M. Joussemet, D. Caugant, M.-J. Quentin-Millet.]
The Development of a Highly Informative Mouse Simple Sequence Length Polymorphism (SSLP) Marker Set and Construction of a Mouse Family Tree Using Parsimony Analysis
Authors: Witmer Philip D. , Doheny Kimberly F. , Adams Marcia K. , Boehm Corinne D. , Dizon Jane S. , Goldstein Janet L. , Templeton Tira M. , Wheaton Ariana M. , Dong Penny N. , Pugh Elizabeth W. , Nussbaum Robert L. , Hunter Kent , Kelmenson Jennifer A. , Rowe Lucy B. , Brownstein Michael J.
Published: March 2003

To identify highly informative markers for a large number of commonly employed murine crosses, we selected a subset of the extant mouse simple sequence length polymorphism (SSLP) marker set for further development. Primer pairs for 314 SSLP markers were designed and typed against 54 inbred mouse strains. We designed new PCR primer sequences for the markers selected for multiplexing using the fluorescent dyes FAM, VIC, NED, and ROX. The number of informative markers for C57BL/6J × DBA/2J is 217, with an average spacing of 6.8 centiMorgans (cM). For all other pairs of strains, the mean number of informative markers per cross is 197.0 (SD 37.8) with a mean distance between markers of 6.8 cM (SD 1.1). To confirm map positions of the 224 markers in our set that are polymorphic betweenMus musculusandMus spretus, we used The Jackson Laboratory (TJL) interspecific backcross mapping panel (TJL BSS); 168 (75%) of these markers had not been previously mapped in this cross by other investigators, adding new information to this community map resource. With this large data set, we sought to reconstruct a phylogenetic history of the laboratory mouse using Wagner parsimony analysis. Our results are largely congruent with the known history of inbred mouse strains.

[The following individuals kindly provided reagents, samples, or unpublished information as indicated in the paper: E. Eicher, T. Golovkina, J. Cheverud, S. Cropp, P. Denny, and A. Southwell.]
A Highly Efficient Recombineering-Based Method for Generating Conditional Knockout Mutations
Authors: Liu Pentao , Jenkins Nancy A. , Copeland Neal G.
Published: March 2003

Phage-based Escherichia coli homologous recombination systems have recently been developed that now make it possible to subclone or modify DNA cloned into plasmids, BACs, or PACs without the need for restriction enzymes or DNA ligases. This new form of chromosome engineering, termed recombineering, has many different uses for functional genomic studies. Here we describe a new recombineering-based method for generating conditional mouse knockout (cko) mutations. This method uses homologous recombination mediated by the λ phage Red proteins, to subclone DNA from BACs into high-copy plasmids by gap repair, and together with Cre or Flpe recombinases, to introduce loxP or FRT sites into the subcloned DNA. Unlike other methods that use short 45–55-bp regions of homology for recombineering, our method uses much longer regions of homology. We also make use of several new E. coli strains, in which the proteins required for recombination are expressed from a defective temperature-sensitive λ prophage, and the Cre or Flpe recombinases from an arabinose-inducible promoter. We also describe two newNeo selection cassettes that work well in both E. coli and mouse ES cells. Our method is fast, efficient, and reliable and makes it possible to generate cko-targeting vectors in less than 2 wk. This method should also facilitate the generation of knock-in mutations and transgene constructs, as well as expedite the analysis of regulatory elements and functional domains in or near genes.
15,000 Unique Zebrafish EST Clusters and Their Future Use in Microarray for Profiling Gene Expression Patterns During Embryogenesis
Authors: Lo Jane , Lee Sorcheng , Xu Min , Liu Feng , Ruan Hua , Eun Alvin , He Yawen , Ma Weiping , Wang Weefuen , Wen Zilong , Peng Jinrong
Published: March 2003

A total of 15,590 unique zebrafish EST clusters from two cDNA libraries have been identified. Most significantly, only 22% (3437) of the 15,590 unique clusters matched 2805 (of 15,200) clusters in theDanio rerio UniGene database, indicating that our EST set is complementary to the existing ESTs in the public database and will be invaluable in assisting the annotation of genes based on the upcoming zebrafish genome sequence. Blast search showed that 7824 of our unique clusters matched 6710 known or predicted proteins in the nonredundant database. A cDNA microarray representing ∼3100 unique zebrafish cDNA clusters has been generated and used to profile the gene expression patterns across six different embryonic stages (cleavage, blastula, gastrula, segmentation, pharyngula, and hatching). Analysis of expression data using K-means clustering revealed that genes coding for muscle-specific proteins displayed similar expression patterns, confirming that the coordinate gene expression is important for myogenesis. Our results demonstrate that the combination of microarray technology with the zebrafish model system can provide useful information on how genes are coordinated in a genetic network to control zebrafish embryogenesis and can help to identify novel genes that are important for organogenesis.

[Supplemental material is available online at www.genome.org. The sequence data from this study have been submitted to EMBL under accession nos.AL901610–AL928536.]
Analysis of Primate Genomic Variation Reveals a Repeat-Driven Expansion of the Human Genome
Authors: Liu Ge , Program NISC Comparative Sequencing , Zhao Shaying , Bailey Jeffrey A. , Sahinalp S. Cenk , Alkan Can , Tuzun Eray , Green Eric D. , Eichler Evan E.
Published: March 2003

We performed a detailed analysis of both single-nucleotide and large insertion/deletion events based on large-scale comparison of 10.6 Mb of genomic sequence from lemur, baboon, and chimpanzee to human. Using a human genomic reference, optimal global alignments were constructed from large (>50-kb) genomic sequence clones. These alignments were examined for the pattern, frequency, and nature of mutational events. Whereas rates of single-nucleotide substitution remain relatively constant (1–2 × 10−9 substitutions/site/year), rates of retrotransposition vary radically among different primate lineages. These differences have lead to a 15%–20% expansion of human genome size over the last 50 million years of primate evolution, 90% of it due to new retroposon insertions. Orthologous comparisons with the chimpanzee suggest that the human genome continues to significantly expand due to shifts in retrotransposition activity. Assuming that the primate genome sequence we have sampled is representative, we estimate that human euchromatin has expanded 30 Mb and 550 Mb compared to the primate genomes of chimpanzee and lemur, respectively.

[Supplemental material is available online at www.genome.org.]
Laser-Capture Microdissection, a Tool for the Global Analysis of Gene Expression in Specific Plant Cell Types: Identification of Genes Expressed Differentially in Epidermal Cells or Vascular Tissues of Maize[W]
Authors: Nakazono Mikio , Qiu Fang , Borsuk Lisa A. , Schnable Patrick S.
Published: March 2003

Laser-capture microdissection (LCM) allows for the one-step procurement of large homogeneous populations of cells from tissue sections. In mammals, LCM has been used to conduct cDNA microarray and proteomics studies on specific cell types. However, LCM has not been applied to plant cells, most likely because plant cell walls make it difficult to separate target cells from surrounding cells and because ice crystals can form in the air spaces between cells when preparing frozen sections. By fixing tissues, using a cryoprotectant before freezing, and using an adhesive-coated slide system, it was possible to capture large numbers (>10,000) of epidermal cells and vascular tissues (vascular bundles and bundle sheath cells) from ethanol:acetic acid–fixed coleoptiles of maize. RNA extracted from these cells was amplified with T7 RNA polymerase and used to hybridize a microarray containing ∼8800 maize cDNAs. Approximately 250 of these were expressed preferentially in epidermal cells or vascular tissues. These results demonstrate that the combination of LCM and microarrays makes it feasible to conduct high-resolution global gene expression analyses of plants. This approach has the potential to enhance our understanding of diverse plant cell type–specific biological processes.