bluuebunny/crossref_metadata_2025 huggingface dataset:
Competition between curvature and chemistry in a spherically expanding detonation
Authors: Bukiet B. , Jones J.
Published: July 2002

The methods of bifurcation theory and asymptotic analysis are used to study curvilinear perturbations of a plane detonation wave. An asymptotic model is derived for the small curvature limit. The nonlinear competition between curvature and chemistry is manifested by a highly degenerate critical point in the state space of the model equations. Computer simulations are presented showing agreement of this model with the reactive Euler equations in the small curvature limit. In particular, we find that the leading order correction to the wave speed due to the curvature of the detonation front depends on the order of the chemical reaction to leading order in curvature.
Investigation of stress effects on the direct current characteristics of GaAs metal-semiconductor field-effect transistors through the use of externally applied loads
Authors: McNally Patrick J. , Cooper Lisa S. , Rosenberg James J. , Jackson T. N.
Published: July 2002

This letter describes an experimental investigation of the effects of mechanical stress on the direct current (dc) electrical parameters in GaAs metal-semiconductor field-effect transistors, with the aim of separating out those effects which are direct manifestations of residual stresses in the completed device (e.g., piezoelectric effects) from stress effects which manifest themselves during the device processing (e.g., stress-enhanced diffusion during implant annealing). This study extends previous work by examining individual transistors under both tensile and compressive loads. Systematic yet nonmonotomic variations in the dc parameters observed in several devices also suggest the possibility of defect generation in the device structures, even at relatively low bulk stresses.
Structural phase transformation in the superconducting YLaxBa2−xCu3O7 compounds
Authors: Huang T. C. , Tokura Y. , Torrance J. B. , Nazzal A. I. , Karimi R.
Published: July 2002

An orthorhombic-to-tetragonal phase transformation in YLaxBa2−xCu3O7 has been observed as a function of x, but with the same oxygen concentration, from x-ray powder diffraction data. Compounds with x≤0.37 have Tc in the 93–53 K range and are orthorhombic, while compounds with x≥0.45 have Tc≤42 K and are tetragonal. An analysis of the YLa0.6Ba1.4Cu3O6.99 diffraction pattern suggests that the tetragonal structure is closely related to those of the orthorhombic YBa2Cu3O7 and the tetragonal YBa2Cu3O6 with a partial substitution of La3+ ion on the Ba2+ site and a disorder of oxygen atoms at the midpoints of both a and b unit cell edges.
Fabrication of the Bi1Sr1Ca1Cu2Ox high T
c superconductor
Authors: Garland M. M.
Published: July 2002

The high Tc superconductor Bi1Sr1Ca1Cu2Ox has been fabricated from a solid-state reaction of SrCO3, CaCO3, Cu2O, and Bi. The onset temperature is found to be 120 K. A second phase is present with an onset of 90 K. Both phases are observed in ac susceptibility and resistance data. This compound appears to be more stable than Y1Ba2Cu3O7 in the presence of water.
Fabrication of multifilamentary Y-Ba-Cu-O oxide superconductors
Authors: Sekine H. , Inoue K. , Maeda H. , Numata K. , Mori K. , Yamamoto H.
Published: July 2002

Workability of Y-Ba-Cu-O composite wires with a Ag sheath has been studied and multifilamentary Y-Ba-Cu-O superconductors with a Ag matrix have been successfully fabricated. Observations of the wires with a scanning electron microscope reveal that as the areal reduction ratio R increases, the average Y-Ba-Cu-O grain size decreases. The study on the workability of the composite wires reveals that composite wires consisting of the Y-Ba-Cu-O powder and a Ag matrix can be rolled or drawn to any extent by a cold-work process with intermediate annealings at 150 °C for R=∼10. Based on this study, we have succeeded in fabricating a 252 filament Y-Ba-Cu-O wire which shows a Tc (onset) of ∼95 K.
Short interspersed transposable elements (SINEs) are excluded from imprinted regions in the human genome
Authors: Greally John M.
Published: July 2002

To test whether regions undergoing genomic imprinting have
unique genomic characteristics, imprinted and nonimprinted human loci
were compared for nucleotide and retroelement composition. Maternally
and paternally expressed subgroups of imprinted genes were found to
differ in terms of guanine and cytosine, CpG, and retroelement
content, indicating a segregation into distinct genomic compartments.
Imprinted regions have been normally permissive to L1 long interspersed
transposable element retroposition during mammalian evolution but
universally and significantly lack short interspersed transposable
elements (SINEs). The primate-specific Alu SINEs, as well as the more
ancient mammalian-wide interspersed repeat SINEs, are found at
significantly low densities in imprinted regions. The latter
paleogenomic signature indicates that the sequence characteristics of
currently imprinted regions existed before the mammalian radiation.
Transitions from imprinted to nonimprinted genomic regions in cis are
characterized by a sharp inflection in SINE content, demonstrating that
this genomic characteristic can help predict the presence and extent of
regions undergoing imprinting. During primate evolution, SINE
accumulation in imprinted regions occurred at a decreased rate compared
with control loci. The constraint on SINE accumulation in imprinted
regions may be mediated by an active selection process. This selection
could be because of SINEs attracting and spreading methylation, as has
been found at other loci. Methylation-induced silencing could lead to
deleterious consequences at imprinted loci, where inactivation of one
allele is already established, and expression is often essential for
embryonic growth and survival.
Sequence analysis of mouse vomeronasal receptor gene clusters reveals common promoter motifs and a history of recent expansion
Authors: Lane Robert P. , Cutforth Tyler , Axel Richard , Hood Leroy , Trask Barbara J.
Published: July 2002

We have analyzed the organization and sequence of 73 V1R
genes encoding putative pheromone receptors to identify
regulatory features and characterize the evolutionary history of the
V1R family. The 73 V1Rs arose from seven ancestral genes around the
time of mouse–rat speciation through large local duplications, and
this expansion may contribute to speciation events. Orthologous V1R
genes appear to have been lost during primate evolution. Exceptional
noncoding homology is observed across four V1R subfamilies at one
cluster and thus may be important for locus-specific transcriptional
regulation.
Experience-dependent changes in cerebellar contributions to motor sequence learning
Authors: Doyon Julien , Song Allen W. , Karni Avi , Lalonde François , Adams Michelle M. , Ungerleider Leslie G.
Published: July 2002

Studies in experimental animals and humans have stressed the role of the cerebellum in motor skill learning. Yet, the relative importance of the cerebellar cortex and deep nuclei, as well as the nature of the dynamic functional changes occurring between these and other motor-related structures during learning, remains in dispute. Using functional magnetic resonance imaging and a motor sequence learning paradigm in humans, we found evidence of an experience-dependent shift of activation from the cerebellar cortex to the dentate nucleus during early learning, and from a cerebellar–cortical to a striatal–cortical network with extended practice. The results indicate that intrinsic modulation within the cerebellum, in concert with activation of motor-related cortical regions, serves to set up a procedurally acquired sequence of movements that is then maintained elsewhere in the brain.
Mechanical disruption of individual nucleosomes reveals a reversible multistage release of DNA
Authors: Brower-Toland Brent D. , Smith Corey L. , Yeh Richard C. , Lis John T. , Peterson Craig L. , Wang Michelle D.
Published: July 2002

The dynamic structure of individual nucleosomes was examined by stretching nucleosomal arrays with a feedback-enhanced optical trap. Forced disassembly of each nucleosome occurred in three stages. Analysis of the data using a simple worm-like chain model yields 76 bp of DNA released from the histone core at low stretching force. Subsequently, 80 bp are released at higher forces in two stages: full extension of DNA with histones bound, followed by detachment of histones. When arrays were relaxed before the dissociated state was reached, nucleosomes were able to reassemble and to repeat the disassembly process. The kinetic parameters for nucleosome disassembly also have been determined.
The enthalpy of the alanine peptide helix measured by isothermal titration calorimetry using metal-binding to induce helix formation
Authors: Lopez Maria M. , Chin Der-Hang , Baldwin Robert L. , Makhatadze George I.
Published: July 2002

The goal of this study is to use the model system described earlier to make direct measurements of the enthalpy of helix formation at different temperatures. For this we studied model alanine peptides in which helix formation can be triggered by metal (La
3+
) binding. The heat of La
3+
interaction with the peptides at different temperatures is measured by isothermal titration calorimetry. Circular dichroism spectroscopy is used to follow helix formation. Peptides of increasing length (12-, 16-, and 19-aa residues) that contain a La
3+
-binding loop followed by helices of increasing length, are used to separate the heat of metal binding from the enthalpy of helix formation. We demonstrate that (
i
) the enthalpy of helix formation is −0.9 ± 0.1 kcal/mol; (
ii
) the enthalpy of helix formation is independent of the peptide length; (
iii
) the enthalpy of helix formation does not depend significantly on temperature in the range from 5 to 45°C, suggesting that the heat capacity change on helix formation is very small. Thus, the use of metal binding to induce helix formation has an enormous potential for measuring various thermodynamic properties of α-helices.
Biological water at the protein surface: Dynamical solvation probed directly with femtosecond resolution
Authors: Pal Samir Kumar , Peon Jorge , Zewail Ahmed H.
Published: July 2002

Biological water at the interface of proteins is critical to their equilibrium structures and enzyme function and to phenomena such as molecular recognition and protein–protein interactions. To actually probe the dynamics of water structure at the surface, we must examine the protein itself, without disrupting the native structure, and the ultrafast elementary processes of hydration. Here we report direct study, with femtosecond resolution, of the dynamics of hydration at the surface of the enzyme protein Subtilisin
Carlsberg
, whose single Trp residue (Trp-113) was used as an intrinsic biological fluorescent probe. For the protein, we observed two well separated dynamical solvation times, 0.8 ps and 38 ps, whereas in bulk water, we obtained 180 fs and 1.1 ps. We also studied a covalently bonded probe at a separation of ≈7 Å and observed the near disappearance of the 38-ps component, with solvation being practically complete in (time constant) 1.5 ps. The degree of rigidity of the probe (anisotropy decay) and of the water environment (protein vs. micelle) was also studied. These results show that hydration at the surface is a dynamical process with two general types of trajectories, those that result from weak interactions with the selected surface site, giving rise to bulk-type solvation (≈1 ps), and those that have a stronger interaction, enough to define a rigid water structure, with a solvation time of 38 ps, much slower than that of the bulk. At a distance of ≈7 Å from the surface, essentially all trajectories are bulk-type. The theoretical framework for these observations is discussed.
Climate change hastens population extinctions
Authors: McLaughlin John F. , Hellmann Jessica J. , Boggs Carol L. , Ehrlich Paul R.
Published: July 2002

Climate change is expected to alter the distribution and abundance of many species. Predictions of climate-induced population extinctions are supported by geographic range shifts that correspond to climatic warming, but few extinctions have been linked mechanistically to climate change. Here we show that extinctions of two populations of a checkerspot butterfly were hastened by increasing variability in precipitation, a phenomenon predicted by global climate models. We model checkerspot populations to show that changes in precipitation amplified population fluctuations, leading to rapid extinctions. As populations of checkerspots and other species become further isolated by habitat loss, climate change is likely to cause more extinctions, threatening both species diversity and critical ecosystem services.
Natural β-sheet proteins use negative design to avoid edge-to-edge aggregation
Authors: Richardson Jane S. , Richardson David C.
Published: July 2002

The fact that natural β-sheet proteins are usually soluble but that fragments or designs of β structure usually aggregate suggests that natural β proteins must somehow be designed to avoid this problem. Regular β-sheet edges are dangerous, because they are already in the right conformation to interact with any other β strand they encounter. We surveyed edge strands in a large sample of all-β proteins to tabulate features that could protect against further β-sheet interactions. β-barrels, of course, avoid edges altogether by continuous H-bonding around the barrel cylinder. Parallel β-helix proteins protect their β-sheet ends by covering them with loops of other structure. β-propeller and single-sheet proteins use a combination of β-bulges, prolines, strategically placed charges, very short edge strands, and loop coverage. β-sandwich proteins favor placing an inward-pointing charged side chain on one of the edge strands where it would be buried by dimerization; they also use bulges, prolines, and other mechanisms. One recent β-hairpin design has a constrained twist too great for accommodation into a larger β-sheet, whereas some β-sheet edges are protected by the bend and reverse twist produced by an Lβ glycine. All free edge strands were seen to be protected, usually by several redundant mechanisms. In contrast, edge strands that natively form β H-bonded dimers or rings have long, regular stretches without such protection. These results are relevant to understanding how proteins may assemble into β-sheet amyloid fibers, and they are especially applicable to the
de novo
design of β structure. Many edge-protection strategies used by natural proteins are beyond our current abilities to constrain by design, but one possibility stands out as especially useful: a single charged side chain near the middle of what would ordinarily be the hydrophobic side of the edge β strand. This minimal negative-design strategy changes only one residue, requires no backbone distortion, and is easy to design. The accompanying paper [Wang, W. & Hecht, M. H. (2002)
Proc. Natl. Acad. Sci. USA
99, 2760–2765] makes use of the inward-pointing charge strategy with great success, turning highly aggregated β-sandwich designs into soluble monomers.
In vitrocartilage formation by human adult stem cells from bone marrow stroma defines the sequence of cellular and molecular events during chondrogenesis
Authors: Sekiya Ichiro , Vuoristo Jussi T. , Larson Benjamin L. , Prockop Darwin J.
Published: July 2002

One approach to resolving the complexities of chondrogenesis is to examine simplified systemsin vitro. We analyzed cartilage differentiation by human adult stem cells from bone marrow stroma. Marrow stromal cells were cultured as micromass pellets for 21 days in serum-free medium containing transforming growth factor (TGF)-β3, dexamethasone, and bone morphogenetic protein (BMP)-6. Assays for pulse-labeled [3H]DNA and for total DNA indicated that there was little proliferation and a progressive loss of cells in the pellets. There were continuous increases in mRNAs for cartilage matrix (proteoglycans and COL2, -9, -10, and -11), receptors [fibroblast growth factor 2 (FGFR2) and parathyroid hormone-related peptide receptor (PTHrP-R)], and transcription factors (SOX5, -6, and -9) as demonstrated by histochemical and microarray assays. Reverse transcription–PCR assays for 11 mRNAs confirmed the microarray data. SOX4, vascular endothelial growth factor (VEGF), and matrix metalloproteinase 14 (MMP14) increased at day 1 and decreased thereafter, suggesting roles early in chondrogenesis. Also, forkhead, CD10, and MMP13 increased up to day 7 and decreased thereafter, suggesting roles in an intermediate stage of chondrogenesis. In addition, two collagens (COL3A1 and COL16A1), a signaling molecule (WNT11), a homeobox homolog (BAPX1), a receptor (IL-1R1), an IGFs modulator (IGFBP5), and a mettaloproteinase (MMP16) increased progressively up to about day 14, suggesting roles later in chondrogenesis. Our results indicate that the simplicity of the system makes it possible to define in detail the cellular and molecular events during chondrogenesis.
Toward a quantum-mechanical description of metal-assisted phosphoryl transfer in pyrophosphatase
Authors: Heikinheimo P. , Tuominen V. , Ahonen A.-K. , Teplyakov A. , Cooperman B. S. , Baykov A. A. , Lahti R. , Goldman A.
Published: July 2002

The wealth of kinetic and structural information makes inorganic
pyrophosphatases (PPases) a good model system to study the details of
enzymatic phosphoryl transfer. The enzyme accelerates metal-complexed
phosphoryl transfer 10
10
-fold: but how? Our structures of
the yeast PPase product complex at 1.15 Å and fluoride-inhibited
complex at 1.9 Å visualize the active site in three different states:
substrate-bound, immediate product bound, and relaxed product bound.
These span the steps around chemical catalysis and provide strong
evidence that a water molecule (O
nu
) directly attacks PPi
with a p
K
a
vastly lowered by coordination to
two metal ions and D117. They also suggest that a low-barrier hydrogen
bond (LBHB) forms between D117 and O
nu
, in part because of
steric crowding by W100 and N116. Direct visualization of the double
bonds on the phosphates appears possible. The flexible side chains at
the top of the active site absorb the motion involved in the reaction,
which may help accelerate catalysis. Relaxation of the product allows a
new nucleophile to be generated and creates symmetry in the elementary
catalytic steps on the enzyme. We are thus moving closer to
understanding phosphoryl transfer in PPases at the quantum mechanical
level. Ultra-high resolution structures can thus tease out overlapping
complexes and so are as relevant to discussion of enzyme mechanism as
structures produced by time-resolved crystallography.
ARGONAUTE1
is required for efficient RNA interference in
Drosophila
embryos
Authors: Williams Robert W. , Rubin Gerald M.
Published: July 2002

Double-stranded RNA (dsRNA) triggers homology-dependent posttranscriptional gene interference (RNAi) in a diverse range of eukaryotic organisms, in a process mechanistically related to viral and transgene-mediated cosuppression. RNAi is characterized by the conversion of long dsRNA into ≈21–25-nt small interfering RNAs (siRNA) that guide the degradation of homologous mRNA. Many of the genes required for siRNA production and target mRNA degradation are widely conserved. Notably, members of the
Argonaute-
like gene family from
Arabidopsis
,
Caenorhabditis
elegans
,
Drosophila
, and
Neurospora
have been genetically and/or biochemically identified as components of the RNAi/cosuppression pathway. We show here that mutations in the
Drosophila Argonaute1
(
AGO1
) gene suppress RNAi in embryos. This defect corresponds to a reduced ability to degrade mRNA in response to dsRNA
in vitro
. Furthermore,
AGO1
is not required for siRNA production
in vitro
nor can the introduction of siRNA bypass
AGO1
mutants
in vivo
. These data suggest that
AGO1
functions downstream of siRNA production.
Investigation of the role of ansocalcin in the biomineralization in goose eggshell matrix
Authors: Lakshminarayanan Rajamani , Kini R. Manjunatha , Valiyaveettil Suresh
Published: July 2002

The role of proteins in biomineralization and the mechanism of eggshell formation are not well understood. We have isolated and purified the major protein, ansocalcin from goose eggshell matrix. The amino acid sequence study indicates that ansocalcin is homologous to the chicken eggshell protein, ovocleidin 17, and C-type lectins. Ansocalcin nucleates polycrystalline aggregates of calcite crystals in
in vitro
mineralization experiments. The polycrystalline aggregates obtained at higher concentration of ansocalcin appears to be similar to the crystals observed at the mamillary layer of the eggshell.
Maximum likelihood estimation of a migration matrix and effective population sizes innsubpopulations by using a coalescent approach
Authors: Beerli Peter , Felsenstein Joseph
Published: July 2002

A maximum likelihood estimator based on the coalescent for unequal migration rates and different subpopulation sizes is developed. The method uses a Markov chain Monte Carlo approach to investigate possible genealogies with branch lengths and with migration events. Properties of the new method are shown by using simulated data from a four-populationn-island model and a source–sink population model. Our estimation method as coded inmigrateis tested againstgenetree; both programs deliver a very similar likelihood surface. The algorithm converges to the estimates fairly quickly, even when the Markov chain is started from unfavorable parameters. The method was used to estimate gene flow in the Nile valley by using mtDNA data from three human populations.
In vivo
restoration of laminin 5 β3 expression and function in junctional epidermolysis bullosa
Authors: Robbins Paul B. , Lin Qun , Goodnough Julia B. , Tian Hongsheng , Chen Xinjian , Khavari Paul A.
Published: July 2002

The blistering disorder, lethal junctional epidermolysis bullosa
(JEB), can result from mutations in the
LAMB3
gene,
which encodes laminin 5 β3 (β3). Appropriate expression of LAMβ3
in JEB skin tissue could potentially ameliorate the symptoms of the
underlying disease. To explore the utility of this therapeutic
approach, primary keratinocytes from six unrelated JEB patients were
transduced with a retroviral vector encoding β3 and used to
regenerate human skin on severe combined immunodeficient (SCID) mice.
Tissue regenerated from β3-transduced JEB keratinocytes produced
phenotypically normal skin characterized by sustained β3 expression
and the formation of hemidesmosomes. Additionally, β3 gene transfer
corrected the distribution of a number of important basement membrane
zone proteins including BPAG2, integrins β4/β1, and
laminins α3/γ2. Skin produced from β3-negative (β3[−]) JEB
cells mimicked the hallmarks of the disease state and did not exhibit
any of the aforementioned traits. Therefore, by effecting therapeutic
gene transfer to β3-deficient primary keratinocytes, it is possible
to produce healthy, normal skin tissue
in vivo
. These
data support the utility of gene therapy for JEB and highlight the
potential for gene delivery in the treatment of human genetic skin
disease.
Cyclooxygenase-2 expression is induced during human megakaryopoiesis and characterizes newly formed platelets
Authors: Rocca Bianca , Secchiero Paola , Ciabattoni Giovanni , Ranelletti Franco O. , Catani Lucia , Guidotti Lia , Melloni Elisabetta , Maggiano Nicola , Zauli Giorgio , Patrono Carlo
Published: July 2002

Cyclooxygenase (COX)-1 or -2 and prostaglandin (PG) synthases catalyze the formation of various PGs and thromboxane (TX) A
2
. We have investigated the expression and activity of COX-1 and -2 during human megakaryocytopoiesis. We analyzed megakaryocytes from bone marrow biopsies and derived from thrombopoietin-treated CD34
+
hemopoietic progenitor cells in culture. Platelets were obtained from healthy donors and patients with high platelet regeneration because of immune thrombocytopenia or peripheral blood stem cell transplantation. By immunocytochemistry, COX-1 was observed in CD34
+
cells and in megakaryocytes at each stage of maturation, whereas COX-2 was induced after 6 days of culture, and remained detectable in mature megakaryocytes. CD34
+
cells synthesized more PGE
2
than TXB
2
(214 ± 50 vs. 30 ± 10 pg/10
6
cells), whereas the reverse was true in mature megakaryocytes (TXB
2
8,440 ± 2,500 vs. PGE
2
906 ± 161 pg/10
6
cells). By immunostaining, COX-2 was observed in <10% of circulating platelets from healthy controls, whereas up to 60% of COX-2-positive platelets were found in patients. A selective COX-2 inhibitor reduced platelet production of both PGE
2
and TXB
2
to a significantly greater extent in patients than in healthy subjects. Finally, we found that COX-2 and the inducible PGE-synthase were coexpressed in mature megakaryocytes and in platelets. We conclude that both COX-isoforms contribute to prostanoid formation during human megakaryocytopoiesis and that COX-2-derived PGE
2
and TXA
2
may play an unrecognized role in inflammatory and hemostatic responses in clinical syndromes associated with high platelet turnover.
Unpaired terminal nucleotides and 5′ monophosphorylation govern 3′ polyadenylation by
Escherichia coli
poly(A) polymerase I
Authors: Feng Yanan , Cohen Stanley N.
Published: July 2002

In bacteria, most mRNAs and certain regulatory RNAs are rapidly turned over, whereas mature tRNA and ribosomal RNA are highly stable. The selective susceptibility of unstable
Escherichia coli
RNAs to 3′ polyadenylation by the
pcnB
gene product, poly(A) polymerase I (PAP I),
in vivo
is a key factor in their rapid degradation by 3′ to 5′ exonucleases. Using highly purified His-tagged recombinant PAP I, we show that differential adenylation of RNA substrates by PAP I occurs
in vitro
and that this capability resides in PAP I itself rather than in any ancillary protein(s). Surprisingly, the efficiency of 3′ polyadenylation is affected by substrate structure at both termini; single-strand segments at either the 5′ or 3′ end of RNA molecules and monophosphorylation at an unpaired 5′ terminus dramatically increase the rate and length of 3′ poly(A) tail additions by PAP I. Our results provide a mechanistic basis for the susceptibility of certain RNAs to 3′ polyadenylation. They also suggest a model of “programmed” RNA decay in which endonucleolytically generated RNA fragments containing single-stranded monophosphorylated 5′ termini are targeted for poly(A) addition and further degradation.
Assembly of RecA-like recombinases: Distinct roles for mediator proteins in mitosis and meiosis
Authors: Gasior Stephen L. , Olivares Heidi , Ear Uy , Hari Danielle M. , Weichselbaum Ralph , Bishop Douglas K.
Published: July 2002

Members of the RecA family of recombinases from bacteriophage T4,
Escherichia coli
, yeast, and higher eukaryotes function in recombination as higher-order oligomers assembled on tracts of single-strand DNA (ssDNA). Biochemical studies have shown that assembly of recombinase involves accessory factors. These studies have identified a class of proteins, called recombination mediator proteins, that act by promoting assembly of recombinase on ssDNA tracts that are bound by ssDNA-binding protein (ssb). In the absence of mediators, ssb inhibits recombination reactions by competing with recombinase for DNA-binding sites. Here we briefly review mediated recombinase assembly and present results of new
in vivo
experiments. Immuno-double-staining experiments in
Saccharomyces cerevisiae
suggest that Rad51, the eukaryotic recombinase, can assemble at or near sites containing ssb (replication protein A, RPA) during the response to DNA damage, consistent with a need for mediator activity. Correspondingly, mediator gene mutants display defects in Rad51 assembly after DNA damage and during meiosis, although the requirements for assembly are distinct in the two cases. In meiosis, both Rad52 and Rad55/57 are required, whereas either Rad52 or Rad55/57 is sufficient to promote assembly of Rad51 in irradiated mitotic cells. Rad52 promotes normal amounts of Rad51 assembly in the absence of Rad55 at 30°C but not 20°C, accounting for the cold sensitivity of
rad55
null mutants. Finally, we show that assembly of Rad51 is induced by radiation during S phase but not during G
1
, consistent with the role of Rad51 in repairing the spontaneous damage that occurs during DNA replication.
Short tandem repeat profiling provides an international reference standard for human cell lines
Authors: Masters John R. , Thomson Jim A. , Daly-Burns Bernadette , Reid Yvonne A. , Dirks Wilhelm G. , Packer Phil , Toji Lorraine H. , Ohno Tadao , Tanabe Hideyuki , Arlett Colin F. , Kelland Lloyd R. , Harrison Maureen , Virmani Arvind , Ward Timothy H. , Ayres Karen L. , Debenham Paul G.
Published: July 2002

Cross-contamination between cell lines is a longstanding and frequent cause of scientific misrepresentation. Estimates from national testing services indicate that up to 36% of cell lines are of a different origin or species to that claimed. To test a standard method of cell line authentication, 253 human cell lines from banks and research institutes worldwide were analyzed by short tandem repeat profiling. The short tandem repeat profile is a simple numerical code that is reproducible between laboratories, is inexpensive, and can provide an international reference standard for every cell line. If DNA profiling of cell lines is accepted and demanded internationally, scientific misrepresentation because of cross-contamination can be largely eliminated.
The evolutionary history of methicillin-resistant
Staphylococcus aureus
(MRSA)
Authors: Enright Mark C. , Robinson D. Ashley , Randle Gaynor , Feil Edward J. , Grundmann Hajo , Spratt Brian G.
Published: July 2002

Methicillin-resistant
Staphylococcus aureus
(MRSA) is a major cause of hospital-acquired infections that are becoming increasingly difficult to combat because of emerging resistance to all current antibiotic classes. The evolutionary origins of MRSA are poorly understood, no rational nomenclature exists, and there is no consensus on the number of major MRSA clones or the relatedness of clones described from different countries. We resolve all of these issues and provide a more thorough and precise analysis of the evolution of MRSA clones than has previously been possible. Using multilocus sequence typing and an algorithm,
burst
, we analyzed an international collection of 912 MRSA and methicillin-susceptible
S. aureus
(MSSA) isolates. We identified 11 major MRSA clones within five groups of related genotypes. The putative ancestral genotype of each group and the most parsimonious patterns of descent of isolates from each ancestor were inferred by using
burst
, which, together with analysis of the methicillin resistance genes, established the likely evolutionary origins of each major MRSA clone, the genotype of the original MRSA clone and its MSSA progenitor, and the extent of acquisition and horizontal movement of the methicillin resistance genes. Major MRSA clones have arisen repeatedly from successful epidemic MSSA strains, and isolates with decreased susceptibility to vancomycin, the antibiotic of last resort, are arising from some of these major MRSA clones, highlighting a depressing progression of increasing drug resistance within a small number of ecologically successful
S. aureus
genotypes.
Enhancement of induced disease resistance by simultaneous activation of salicylate- and jasmonate-dependent defense pathways in
Arabidopsis
thaliana
Authors: van Wees Saskia C. M. , de Swart Erik A. M. , van Pelt Johan A. , van Loon Leendert C. , Pieterse Corné M. J.
Published: July 2002

The plant-signaling molecules salicylic acid (SA) and jasmonic acid (JA) play an important role in induced disease resistance pathways. Cross-talk between SA- and JA-dependent pathways can result in inhibition of JA-mediated defense responses. We investigated possible antagonistic interactions between the SA-dependent systemic acquired resistance (SAR) pathway, which is induced upon pathogen infection, and the JA-dependent induced systemic resistance (ISR) pathway, which is triggered by nonpathogenic
Pseudomonas
rhizobacteria. In
Arabidopsis thaliana
, SAR and ISR are effective against a broad spectrum of pathogens, including the foliar pathogen
Pseudomonas syringae
pv.
tomato
(
Pst
). Simultaneous activation of SAR and ISR resulted in an additive effect on the level of induced protection against
Pst
. In
Arabidopsis
genotypes that are blocked in either SAR or ISR, this additive effect was not evident. Moreover, induction of ISR did not affect the expression of the SAR marker gene
PR-1
in plants expressing SAR. Together, these observations demonstrate that the SAR and the ISR pathway are compatible and that there is no significant cross-talk between these pathways. SAR and ISR both require the key regulatory protein NPR1. Plants expressing both types of induced resistance did not show elevated
Npr1
transcript levels, indicating that the constitutive level of NPR1 is sufficient to facilitate simultaneous expression of SAR and ISR. These results suggest that the enhanced level of protection is established through parallel activation of complementary, NPR1-dependent defense responses that are both active against
Pst
. Therefore, combining SAR and ISR provides an attractive tool for the improvement of disease control.
An ethylene-induced cDNA encoding a lipase expressed at the onset of senescence
Authors: Hong Yuwen , Wang Tzann-Wei , Hudak Katalin A. , Schade Frank , Froese Carol D. , Thompson John E.
Published: July 2002

A cDNA clone encoding a lipase (lipolytic acyl hydrolase) expressed at the onset of petal senescence has been isolated by screening a cDNA expression library prepared from carnation flowers (
Dianthus caryophyllus
). The cDNA contains the lipase consensus sequence, ITFAGHSLGA, and encodes a 447-amino acid polypeptide with a calculated molecular mass of 50.2 kDa that appears to be a cytosolic protein. Over-expression of the clone in
Escherichia coli
yielded a protein of the expected molecular weight that proved capable of deesterifying fatty acids from
p
-nitrophenylpalmitate, tri-linolein, soybean phospholipid, and Tween in both
in vitro
and
in situ
assays of enzyme activity. The abundance of the lipase mRNA increases just as carnation flowers begin to senesce, and expression of the gene is also induced by treatment with ethylene. Southern blot analyses of carnation genomic DNA have indicated that the lipase is a single copy gene. The lipase gene is also expressed in carnation leaves and is up-regulated when the leaves are treated with ethylene. Deesterification of membrane lipids and ensuing loss of membrane structural integrity are well established early events of plant senescence, and the expression pattern of this lipase gene together with the lipolytic activity of its cognate protein indicate that it plays a fundamentally central role in mediating the onset of senescence.
TheArabidopsis dnd1“defense, no death” gene encodes a mutated cyclic nucleotide-gated ion channel
Authors: Clough Steven J. , Fengler Kevin A. , Yu I-ching , Lippok Bernadette , Smith Roger K. , Bent Andrew F.
Published: July 2002

Gene-for-gene disease resistance typically includes a programmed cell death response known as the hypersensitive response (HR). TheArabidopsis thaliana dnd1mutant was previously isolated as a line that failed to produce the HR in response to avirulentPseudomonas syringaepathogens; plants homozygous for the recessivednd1-1mutation still carry out effective gene-for-gene resistance. Thednd1-1mutation also causes constitutive systemic resistance and elevated levels of salicylic acid. In the present study, a positional cloning approach was used to isolateDND1. DND1encodes the same protein as AtCNGC2, a cyclic nucleotide-gated ion channel of previously unknown organismal function that can allow passage of Ca2+, K+and other cations [Leng, Q., Mercier, R. W., Yao, W. & Berkowitz, G. A. (1999)Plant Physiol.121, 753–761]. By using a nahG transgene, we found that salicylic acid is required for the elevated resistance caused by thednd1mutation but that removal of salicylic acid did not completely eliminate the dwarf and loss-of-HR phenotypes of mutantdnd1plants. A stop codon that would severely truncate theDND1gene product was identified in thednd1-1allele. This demonstrates that broad-spectrum disease resistance and inhibition of the HR can be activated in plants by disruption of a cyclic nucleotide-gated ion channel.
Sir3-dependent assembly of supramolecular chromatin structuresin vitro
Authors: Georgel Philippe T. , Palacios DeBeer Madeleine A. , Pietz Gregory , Fox Catherine A. , Hansen Jeffrey C.
Published: July 2002

Baculovirus-expressed recombinant Sir3p (rSir3p) has been purified to near homogeneity, and its binding to naked DNA, mononucleosomes, and nucleosomal arrays has been characterizedin vitro. At stoichiometric levels rSir3p interacts with intact nucleosomal arrays, mononucleosomes, and naked DNA, as evidenced by formation of supershifted species on native agarose gels. Proteolytic removal of the core histone tail domains inhibits but does not completely abolish rSir3p binding to nucleosomal arrays. The linker DNA in the supershifted complexes remains freely accessible to restriction endonuclease digestion, suggesting that both the tail domains and nucleosomal DNA contribute to rSir3p–chromatin interactions. Together these data indicate that rSir3p cross-links individual nucleosomal arrays into supramolecular assemblies whose physical properties transcend those of typical 10-nm and 30-nm fibers. Based on these data we hypothesize that Sir3p functions, at least in part, by mediating reorganization of the canonical chromatin fiber into functionally specialized higher order chromosomal domains.
Evaluation of two-dimensional gel electrophoresis-based proteome analysis technology
Authors: Gygi Steven P. , Corthals Garry L. , Zhang Yanni , Rochon Yvan , Aebersold Ruedi
Published: July 2002

Proteome analysis is most commonly accomplished by a combination of two-dimensional gel electrophoresis (2DE) to separate and visualize proteins and mass spectrometry (MS) for protein identification. Although this technique is powerful, mature, and sensitive, questions remain concerning its ability to characterize all of the elements of a proteome. In the current study, more than 1,500 features were visualized by silver staining a narrow pH range (4.9–5.7) 2D gel in which 0.5 mg of total soluble yeast protein was separated. Fifty spots migrating to a region of 4 cm
2
were subjected to MS protein identification. Despite the high sample load and extended electrophoretic separation, proteins from genes with codon bias values of <0.1 (lower abundance proteins) were not found, even though fully one-half of all yeast genes fall into that range. Proteins from genes with codon bias values of <0.1 were found, however, if protein amounts exceeding the capacity of 2DE were fractionated and analyzed. We conclude that the large range of protein expression levels limits the ability of the 2DE-MS approach to analyze proteins of medium to low abundance, and thus the potential of this technique for proteome analysis is likewise limited.
TOM1
, an
Arabidopsis
gene required for efficient multiplication of a tobamovirus, encodes a putative transmembrane protein
Authors: Yamanaka Takuya , Ohta Takehiro , Takahashi Miki , Meshi Tetsuo , Schmidt Renate , Dean Caroline , Naito Satoshi , Ishikawa Masayuki
Published: July 2002

Host-encoded factors play an important role in virus multiplication, acting in concert with virus-encoded factors. However, information regarding the host factors involved in this process is limited. Here we report the map-based cloning of an
Arabidopsis thaliana
gene,
TOM1
, which is necessary for the efficient multiplication of tobamoviruses, positive-strand RNA viruses infecting a wide variety of plants. The
TOM1
mRNA is suggested to encode a 291-aa polypeptide that is predicted to be a multipass transmembrane protein. The Sos recruitment assay supported the hypothesis that TOM1 is associated with membranes, and in addition, that TOM1 interacts with the helicase domain of tobamovirus-encoded replication proteins. Taken into account that the tobamovirus replication complex is associated with membranes, we propose that TOM1 participates in the
in vivo
formation of the replication complex by serving as a membrane anchor.
Critical roles of glycosylphosphatidylinositol for
Trypanosoma brucei
Authors: Nagamune Kisaburo , Nozaki Tomoyoshi , Maeda Yusuke , Ohishi Kazuhito , Fukuma Toshihide , Hara Tatsuru , Schwarz Ralph T. , Sütterlin Christine , Brun Reto , Riezman Howard , Kinoshita Taroh
Published: July 2002

Trypanosoma brucei
, the protozoan parasite responsible for sleeping sickness, evades the immune response of mammalian hosts and digestion in the gut of the insect vector by means of its coat proteins tethered to the cell surface via glycosylphosphatidylinositol (GPI) anchors. To evaluate the importance of GPI for parasite survival, we cloned and disrupted a trypanosomal gene,
TbGPI10
, involved in biosynthesis of GPI.
TbGPI10
encodes a protein of 558 amino acids having 25% and 23% sequence identity to human PIG-B and
Saccharomyces cerevisiae
Gpi10p, respectively.
TbGPI10
restored biosynthesis of GPI in a mouse mutant cell line defective in mouse
Pig-b
gene.
TbGPI10
also rescued the inviability of
GPI10
-disrupted
S. cerevisiae
, indicating that
TbGPI10
is the orthologue of
PIG-B
/
GPI10
that is involved in the transfer of the third mannose to GPI. The bloodstream form of
T. brucei
could not lose
TbGPI10
; therefore, GPI synthesis is essential for growth of mammalian stage parasites. Procyclic form cells (insect stage parasites) lacking the surface coat proteins because of disruption of
TbGPI10
are viable and grow slower than normal, provided that they are cultured in nonadherent flasks. In regular flasks, they adhered to the plastic surface and died. Infectivity to tsetse flies is partially impaired, particularly in the early stage. Therefore, parasitespecific inhibition of GPI biosynthesis should be an effective chemotherapy target against African trypanosomiasis.
Adaptive evolution of a candidate gene for aging inDrosophila
Authors: Schmidt Paul S. , Duvernell David D. , Eanes Walter F.
Published: July 2002

Examination of the phenotypic effects of specific mutations has been extensively used to identify candidate genes affecting traits of interest. However, such analyses do not reveal anything about the evolutionary forces acting at these loci, or whether standing allelic variation contributes to phenotypic variance in natural populations. TheDrosophilagenemethuselah(mth) has been proposed as having major effects on organismal stress response and longevity phenotype. Here, we examine patterns of polymorphism and divergence atmthin population level samples ofDrosophila melanogaster,D. simulans, andD. yakuba. Mthhas experienced an unusually high level of adaptive amino acid divergence concentrated in the intra- and extracellular loop domains of the receptor protein, suggesting the historical action of positive selection on those regions of the molecule that modulate signal transduction. Further analysis of single nucleotide polymorphisms (SNPs) inD. melanogasterprovided evidence for contemporary and spatially variable selection at themthlocus. In ten surveyed populations, the most commonmthhaplotype exhibited a 40% cline in frequency that coincided with population level differences in multiple life-history traits including lifespan. This clinal pattern was not associated with any particular SNP in the coding region, indicating that selection is operating at a closely linked site that may be involved in gene expression. Together, these consistently nonneutral patterns of inter- and intraspecific variation suggest adaptive evolution of a signal transduction pathway that may modulate lifespan in nature.
Behavioral screening for cocaine sensitivity in mutagenized zebrafish
Authors: Darland Tristan , Dowling John E.
Published: July 2002

Understanding the molecular basis of addiction could be greatly
aided by using forward genetic manipulation to lengthen the list of
candidate genes involved in this complex process. Here, we report that
zebrafish exhibit cocaine-induced conditioned place preference. In a
pilot screen of 18 F
2
generation families of mutagenized
fish, we found three with abnormally low responses to cocaine. This
behavior was inherited by the F
3
generation in a manner
that suggests the abnormalities were because of dominant mutations in
single genes. Performance profiles in secondary behavioral screens
measuring visual dark-adaptation and learning suggest that the defects
were the result of mutations in distinct genes that affect dopaminergic
signaling in the retina and brain.
Quantitative transcript imaging in normal and heat-shocked
Drosophila
embryos by using high-density oligonucleotide arrays
Authors: Leemans Ronny , Egger Boris , Loop Thomas , Kammermeier Lars , He Haiqiong , Hartmann Beate , Certa Ulrich , Hirth Frank , Reichert Heinrich
Published: July 2002

Embryonic development in
Drosophila
is characterized
by an early phase during which a cellular blastoderm is formed and
gastrulation takes place, and by a later postgastrulation phase in
which key morphogenetic processes such as segmentation and
organogenesis occur. We have focused on this later phase in
embryogenesis with the goal of obtaining a comprehensive analysis of
the zygotic gene expression that occurs during development under normal
and altered environmental conditions. For this, a functional genomic
approach to embryogenesis has been developed that uses high-density
oligonucleotide arrays for large-scale detection and quantification of
gene expression. These oligonucleotide arrays were used for
quantitative transcript imaging of embryonically expressed genes under
standard conditions and in response to heat shock. In embryos raised
under standard conditions, transcripts were detected for 37% of the
1,519 identified genes represented on the arrays, and highly
reproducible quantification of gene expression was achieved in all
cases. Analysis of differential gene expression after heat shock
revealed substantial expression level changes for known heat-shock
genes and identified numerous heat shock-inducible genes. These results
demonstrate that high-density oligonucleotide arrays are sensitive,
efficient, and quantitative instruments for the analysis of large scale
gene expression in
Drosophila
embryos.
Translation of chloroplast
psbA
mRNA is regulated by signals initiated by both photosystems II and I
Authors: Trebitsh Tova , Danon Avihai
Published: July 2002

Light controls the translation of several mRNAs in fully developed chloroplasts via at least two regulatory pathways. In the first, the light signal is transduced as a thiol-mediated signal that modulates translation in parallel to light intensity. The second light-controlled pathway, termed priming, is a prerequisite to the thiol-mediated regulatory pathway. Light regulation is rapid and requires intrachloroplast photoreceptor(s). To delineate the signaling pathways controlling each of these regulatory events, we assayed the effect of photosynthetic inhibitors and electron donors on the translation of chloroplastic
psbA
mRNA. We show that the thiol-mediated signal is generated by photosystem I and transduced by vicinal dithiol-containing proteins. We also found that the priming signal probably initiates on reduction of plastoquinone. These findings suggest that translation of chloroplast
psbA
mRNA is controlled by both linear photosynthetic electron transport, exerted by the reduction of the ferredoxin–thioredoxin system, and the relative activities of photosystems I and II, signaled by the redox state of the plastoquinone pool. These data underscore the function of the light-capturing reactions of photosynthesis as chloroplast photoreceptors.