bluuebunny/crossref_metadata_2025 huggingface dataset:
Interleukin (IL)-15 and IL-7 Jointly Regulate Homeostatic Proliferation of Memory Phenotype CD8+ Cells but Are Not Required for Memory Phenotype CD4+ Cells
Authors: Tan Joyce T. , Ernst Bettina , Kieper William C. , LeRoy Eric , Sprent Jonathan , Surh Charles D.
Published: July 2002

The overall size and composition of the pool of naive and memory T cells are tightly regulated by homeostatic mechanisms. Recent work has shown that homeostasis of naive T cells is controlled by two factors, self-major histocompatibility complex (MHC)/peptide ligands and a cytokine, interleukin (IL)-7. In particular, contact with these two factors is required for naive CD4+ and CD8+ cells to undergo “homeostatic” proliferation, i.e., proliferation induced as a consequence of severe T cell depletion. In contrast to naive T cells, the factors that drive memory T cells to undergo homeostatic proliferation are poorly understood. To address this issue, purified memory phenotype CD4+ and CD8+ cells from normal mice were adoptively transferred into various gene-knockout mice rendered T cell–deficient by sublethal irradiation. Three findings are reported. First, unlike naive T cells, homeostatic proliferation of memory T cells is largely MHC independent. Second, memory CD8+ cells can utilize either IL-7 or IL-15 to undergo homeostatic proliferation; however, in the absence of both IL-7 and IL-15, homeostatic proliferation fails to occur. Third, unlike memory CD8+ cells, homeostatic proliferation of memory CD4+ cells is independent of IL-7 and IL-15 (also IL-4). Thus, the homeostatic proliferation mechanisms that control memory CD8+ cells and memory CD4+ cells are quite distinct.
Chemokine Requirements for B Cell Entry to Lymph Nodes and Peyer's Patches
Authors: Okada Takaharu , Ngo Vu N. , Ekland Eric H. , Förster Reinhold , Lipp Martin , Littman Dan R. , Cyster Jason G.
Published: July 2002

B cell entry to lymph nodes and Peyer's patches depends on chemokine receptor signaling, but the principal chemokine involved has not been defined. Here we show that the homing of CXCR4−/− B cells is suppressed in CCL19 (ELC)- and CCL21 (SLC)-deficient paucity of lymph node T cells mice, but not in wild-type mice. We also find that CXCR4 can contribute to T cell homing. Using intravital microscopy, we find that B cell adhesion to high endothelial venules (HEVs) is disrupted when CCR7 and CXCR4 are predesensitized. In Peyer's patches, B cell entry is dependent on CXCR5 in addition to CCR7/CXCR4. CXCL12 (SDF1) is displayed broadly on HEVs, whereas CXCL13 (BLC) is found selectively on Peyer's patch follicular HEVs. These findings establish the principal chemokine and chemokine receptor requirements for B cell entry to lymph nodes and Peyer's patches.
Inducing Tumor Immunity through the Selective Engagement of Activating Fcγ Receptors on Dendritic Cells
Authors: Kalergis Alexis M. , Ravetch Jeffrey V.
Published: July 2002

Induction of tumor-specific immunity requires that dendritic cells (DCs) efficiently capture and present tumor antigens to result in the expansion and activation of tumor-specific cytotoxic T cells. The transition from antigen capture to T cell stimulation requires a maturation signal; in its absence tolerance, rather than immunity may develop. While immune complexes (ICs) are able to enhance antigen capture, they can be poor at inducing DC maturation, naive T cell activation and protective immunity. We now demonstrate that interfering with the inhibitory signal delivered by FcγRIIB on DCs converts ICs to potent maturation agents and results in T cell activation. Applying this approach to immunization with DCs pulsed ex-vivo with ICs, we have generated antigen-specific CD8+ T cells in vivo and achieved efficient protective immunity in a murine melanoma model. These data imply that ICs may normally function to maintain tolerance through the binding to inhibitory FcγRs on DCs, but they can be converted to potent immunogenic stimuli by selective engagement of activating FcγRs. This mechanism suggests a novel approach to the development of tumor vaccines.
Critical Roles for Interleukin 1 and Tumor Necrosis Factor α in Antibody-induced Arthritis
Authors: Ji Hong , Pettit Allison , Ohmura Koichiro , Ortiz-Lopez Adriana , Duchatelle Veronique , Degott Claude , Gravallese Ellen , Mathis Diane , Benoist Christophe
Published: July 2002

In spontaneous inflammatory arthritis of K/BxN T cell receptor transgenic mice, the effector phase of the disease is provoked by binding of immunoglobulins (Igs) to joint surfaces. Inflammatory cytokines are known to be involved in human inflammatory arthritis, in particular rheumatoid arthritis, although, overall, the pathogenetic mechanisms of the human affliction remain unclear. To explore the analogy between the K/BxN model and human patients, we assessed the role and relative importance of inflammatory cytokines in K/BxN joint inflammation by transferring arthritogenic serum into a panel of genetically deficient recipients. Interleukin (IL)-1 proved absolutely necessary. Tumor necrosis factor (TNF)–α was also required, although seemingly less critically than IL-1, because a proportion of TNF-α–deficient mice developed robust disease. There was no evidence for an important role for IL-6. Bone destruction and reconstruction were also examined. We found that all mice with strong inflammation exhibited the bone erosion and reconstruction phenomena typical of K/BxN arthritis, with no evidence of any particular requirement for TNFα for bone destruction. The variability in the requirement for TNF-α, reminiscent of that observed in treated rheumatoid arthritis patients, did not appear genetically programmed but related instead to subtle environmental changes.
Emp47p and Its Close Homolog Emp46p Have a Tyrosine-containing Endoplasmic Reticulum Exit Signal and Function in Glycoprotein Secretion inSaccharomyces cerevisiae
Authors: Sato Ken , Nakano Akihiko
Published: July 2002

The yeast open reading frame YLR080w/EMP46 encodes a homolog of the Golgi protein Emp47p. These two proteins are 45% identical and have a single transmembrane domain in their C-terminal regions and a carbohydrate recognition domain signature in the N-terminal region. The C-terminal tail of Emp46p includes a dilysine signal. This protein is localized to Golgi membranes at steady state by subcellular fractionation and green fluorescent protein labeling. On block of forward transport in sec12-4 cells, redistribution of Emp46p from the Golgi to the endoplasmic reticulum is observed. These localization features are similar to those previously reported for Emp47p. In addition, mutagenesis of the C-terminal region identified a tyrosine-containing motif as a critical determinant of the Golgi-localization and interaction with both COPI and COPII components. Similar motifs are also observed in the C-terminal tail of Emp47p and other mammalian homologs. Disruption of Emp47p displays a growth defect at a high temperature or on Ca2+-containing medium, which is rescued by overexpression of Emp46p, suggesting a partially overlapping function between Emp46p and Emp47p. In addition, we found that the disruption of both Emp46p and Emp47p show a marked defect in the secretion of a subset of glycoproteins. Analysis of the C-terminal mutants for Ca2+sensitivity revealed that the forward transport of Emp46/47p is essential for their function, whereas the retrograde transport is not. We propose that Emp46p and Emp47p are required for the export of specific glycoprotein cargo from the endoplasmic reticulum.
Modelling biological processes using workflow and Petri Net models
Authors: Peleg Mor , Yeh Iwei , Altman Russ B.
Published: July 2002

Motivation: Biological processes can be considered at many levels of detail, ranging from atomic mechanism to general processes such as cell division, cell adhesion or cell invasion. The experimental study of protein function and gene regulation typically provides information at many levels. The representation of hierarchical process knowledge in biology is therefore a major challenge for bioinformatics. To represent high-level processes in the context of their component functions, we have developed a graphical knowledge model for biological processes that supports methods for qualitative reasoning.

Results: We assessed eleven diverse models that were developed in the fields of software engineering, business, and biology, to evaluate their suitability for representing and simulating biological processes. Based on this assessment, we combined the best aspects of two models: Workflow/Petri Net and a biological concept model. The Workflow model can represent nesting and ordering of processes, the structural components that participate in the processes, and the roles that they play. It also maps to Petri Nets, which allow verification of formal properties and qualitative simulation. The biological concept model, TAMBIS, provides a framework for describing biological entities that can be mapped to the workflow model. We tested our model by representing malaria parasites invading host erythrocytes, and composed queries, in five general classes, to discover relationships among processes and structural components. We used reachability analysis to answer queries about the dynamic aspects of the model.

Availability: The model is available at http://smi.stanford.edu/projects/helix/pubs/process-model/

Contact: peleg@smi.stanford.eduyeh@smi.stanford.eduruss.altman@stanford.edu

* To whom correspondence should be addressed.
Arrayplot for visualization and normalization of cDNA microarray data
Authors: Marc Philippe , Jacq Claude
Published: July 2002

Summary: Arrayplot is an application which allows filtering, visualization and normalization of raw cDNA microarray data.

Availability: MS-Windows binaries are freely available for non-profit use at http://www.biologie.ens.fr/yeast-publi.html

Contact: pmarc@biologie.ens.fr

Supplementary information: Scripts for online creation of Arrayplot files are available at http://transcriptome.ens.fr/lgm_bioinfo/

* To whom correspondence should be addressed.
CaGE: Cardiac gene expression knowledgebase
Authors: Bober Michael , Wiehe Kevin , Yung Christina , Onal Suzek Tugba , Lin Matthew , Baumgartner William , Winslow Raimond
Published: July 2002

Summary: CaGE is a Cardiac Gene Expression knowledgebase we have developed to facilitate the analysis of genes important to human cardiac function. CaGE integrates the functionality of the LocusLink database with data from several human cardiac expression libraries, phenotypic data from OMIM™ and data from large-scale microarray gene expression studies to create a knowledgebase of gene expression in human cardiac tissue. The knowledgebase is fully searchable via the web using several intuitive query interfaces. Results can be displayed in several concise easy to navigate formats.

Availability: CaGE is located at http://www.cage.wbmei.jhu.edu

Contact: mbbober@pol.net; rwinslow@bme.jhu.edu

* To whom correspondence should be addressed.
SNAPper: gene order predicts gene function
Authors: Kolesov Grigory , Mewes Hans-Werner , Frishman Dmitrij
Published: July 2002

Summary: SNAPper is a network service for predicting gene function based on the conservation of gene order.

Availability: The SNAPper server is available at http://pedant.gsf.de/snapper. SNAPper-based functional predictions will soon be offered as part of the PEDANT genome analysis server http://pedant.gsf.de.

Contact: g.kolesov@gsf.de

* To whom correspondence should be addressed.
Selecting targets for structural determination by
navigating in a graph of protein families
Authors: Portugaly Elon , Kifer Ilona , Linial Michal
Published: July 2002

Motivation: A major goal in structural genomics is to enrich the catalogue of proteins whose 3D structures are known. In an attempt to address this problem we mapped over 10 000 proteins with solved structures onto a graph of all Swissprot protein sequences (release 36, ∼73 000 proteins) provided by ProtoMap, with the goal of sorting proteins according to their likelihood of belonging to new superfamilies. We hypothesized that proteins within neighbouring clusters tend to share common structural superfamilies or folds. If true, the likelihood of finding new superfamilies increases in clusters that are distal from other solved structures within the graph.

Results: We defined an order relation between unsolved proteins according to their ‘distance’ from solved structures in the graph, and sorted ∼48 000 proteins. Our list can be partitioned into three groups: ∼35 000 proteins sharing a cluster with at least one known structure; ∼6500 proteins in clusters with no solved structure but with neighbouring clusters containing known structures; and a third group contains the rest of the proteins, ∼6100 (in 1274 clusters). We tested the quality of the order relation using thousands of recently solved structures that were not included when the order was defined. The tests show that our order is significantly better (P-value ∼105) than a random order. More interestingly, the order within the union of the second and third groups, and the order within the third group alone, perform better than random (P-values: 0.0008 and 0.15, respectively) and are better than alternative orders created using PSI-BLAST. Herein, we present a method for selecting targets to be used in structural genomics projects.

Availability: List of proteins to be used for targets selection combined with a set of biological filters for narrowing down potential targets is in http://www.protarget.cs.huji.ac.il

Contact: michall@mail.ls.huji.ac.il
Quantitative assessment of filter-based cDNA microarrays: gene expression
profiles of human T-lymphoma cell lines
Authors: Dodson J.M. , Charles P.T. , Stenger D.A. , Pancrazio J.J.
Published: July 2002

Motivation: While the use of cDNA microarrays for functional genomic analysis has become commonplace, relatively little attention has been placed on false positives, i.e. the likelihood that a change in measured radioactive or fluorescence intensity may reflect a change in gene expression when, in fact, there is none. Since cDNA arrays are being increasingly used to rapidly distinguish biomarkers for disease detection and subsequent assay development (Wellman et al., Blood, 96, 398–404, 2000), the impact of false positives can be significant. For the use of this technology, it is necessary to develop quantitative criteria for reduction of false positives with radioactively-labeled cDNA arrays.

Results: We used a single source of RNA (HuT78 T lymphoma cells) to eliminate sample variation and quantitatively examined intensity ratios using radioactively labeled cDNA microarrays. Variation in intensity ratios was reduced by processing microarrays in side-by-side (parallel mode) rather than by using the same microarray for two hybridizations (sequential mode). Based on statistical independence, calculation of the expected number of false positives as a function of threshold showed that a detection limit of |log2R| >0.65 with agreement from three replicates could be used to identify up- or down-modulated genes. Using this quantitative criteria, gene expression differences between two related T lymphoma cell lines, HuT78 and H9, were identified. The relevance of these findings to the known functional differences between these cell types is discussed.

Contact: jxp@cbmse.nrl.navy.mil

* To whom correspondence should be addressed.
Revenue‐Sharing in Movie Exhibition and the Arrival of Sound
Authors: Hanssen F. Andrew
Published: July 2002

During the silent film era, film companies rented the vast majority of their films to exhibitors for flat per‐day fees. A technology “shock” in the form of the coming of sound led to the widespread replacement of flat fees by revenue sharing. This article seeks to determine why. It finds that sound technology altered the structure of incentives in movie exhibition, significantly reducing the scope for exhibitor shirking, reducing the cost of dividing attendance revenue ex post (necessary for revenue sharing), and, initially at least, raising the difficulty of negotiating lump‐sum rental fees. As a result, percent‐of‐gross pricing became the norm. These findings allow additional light to be shed on the reasons for share contracts in general—most previous studies have been limited to an examination a cross‐section of contracts.
Gifts, Cash, and Stigma
Authors: Waldfogel Joel
Published: July 2002

Each year individuals in the United States transfer between $50 and $72 billion in resources in the form of noncash holiday gifts, despite the fact that holiday gift recipients apparently value their noncash gifts at about 10% less than the prices paid by the givers. We document that cash giving is more likely from givers who tend to give unwanted gifts, indicating that givers are concerned with the utility of their recipients. The rarity of cash gifts can be rationalized by a stigma of cash giving that we are able to parameterize and estimate using a simple structural model.
Spontaneous muscle action potentials fail to develop without fetal‐type acetylcholine receptors
Authors: Takahashi Masazumi , Kubo Tai , Mizoguchi Akira , Carlson C George , Endo Katsuaki , Ohnishi Katsunori
Published: July 2002

In mammals, two combinations of muscle nicotinic acetylcholine receptors (AChRs) are used: α2βγδ (γ‐AChR) or α2βϵδ (ϵ‐AChR). After birth, γ‐AChRs are replaced by ϵ‐AChRs (γ/ϵ‐switch). The two receptors have different conductances and open times. During perinatal period, the long open time γ‐AChRs generate random myofiber action potentials from uniquantal miniature end‐plate potentials (mEPPs). ϵ‐AChRs are suitable for strong adult muscle activities. Since the effect of the γ/ϵ‐switch on neuromuscular development was unclear, despite the many differences in channel characteristics, we carried out this study to generate γ‐subunit‐deficient mice. Homozygotes born alive survived for 2 days in a stable condition, and were able to move their forelimbs. Endplate AChRs included ϵ‐subunits, and muscle fibers had multiple neuromuscular junctions. Both pre‐ and postsynapses were abnormal and spontaneous action potentials generated from mEPPs were totally absent. Results suggest a requirement for γ‐AChRs in mediating synaptically‐induced action potential activity critical for neuromuscular development.
Negative regulation of transcription by the type II arginine methyltransferase PRMT5
Authors: Fabbrizio Eric , Messaoudi Selma El , Polanowska Jolanta , Paul Conception , Cook Jeffry R , Lee Jin‐Hyung , Nègre Vincent , Rousset Mathieu , Pestka Sidney , Le Cam Alphonse , Sardet Claude
Published: July 2002

We have identified previously a repressor element in the transcription start site region of the cyclin E1 promoter that periodically associates with an atypical, high molecular weight E2F complex, termed CERC. Purification of native CERC reveals the presence of the type II arginine methyltransferase PRMT5, which can mono‐ or symetrically dimethylate arginine residues in proteins. Chromatin immunoprecipitations (ChIPs) show that PRMT5 is associated specifically with the transcription start site region of the cyclin E1 promoter. ChIP analyses also show that this correlates with the presence on the same promoter region of arginine‐methylated proteins including histone H4, an in vitro substrate of PRMT5. Consistent with its presence within the repressor complex, forced expression of PRMT5 negatively affects cyclin E1 promoter activity and cellular proliferation, effects that require its methyltransferase activity. These data provide the first direct experimental evidence that a type II arginine methylase is involved in the control of transcription and proliferation.
Dynamic Ideal Point Estimation via Markov Chain Monte Carlo for the U.S. Supreme Court, 1953–1999
Authors: Martin Andrew D. , Quinn Kevin M.
Published: July 2002

At the heart of attitudinal and strategic explanations of judicial behavior is the assumption that justices have policy preferences. In this paper we employ Markov chain Monte Carlo methods to fit a Bayesian measurement model of ideal points for all justices serving on the U.S. Supreme Court from 1953 through 1999. We are particularly interested in determining to what extent ideal points of justices change throughout their tenure on the Court. This is important because judicial politics scholars oftentimes invoke preference measures that are time invariant. To investigate preference change, we posit a dynamic item response model that allows ideal points to change systematically over time. Additionally, we introduce Bayesian methods for fitting multivariate dynamic linear models to political scientists. Our results suggest that many justices do not have temporally constant ideal points. Moreover, our ideal point estimates outperform existing measures and explain judicial behavior quite well across civil rights, civil liberties, economics, and federalism cases.
Insects and low temperatures: from molecular biology to distributions and abundance
Authors: Bale J. S.
Published: July 2002

Insects are the most diverse fauna on earth, with different species occupying a range of terrestrial and aquatic habitats from the tropics to the poles. Species inhabiting extreme low–temperature environments must either tolerate or avoid freezing to survive. While much is now known about the synthesis, biochemistry and function of the main groups of cryoprotectants involved in the seasonal processes of acclimatization and winter cold hardiness (ice–nucleating agents, polyols and antifreeze proteins), studies on the structural biology of these compounds have been more limited.

The recent discovery of rapid cold–hardening, ice–interface desiccation and the daily resetting of critical thermal thresholds affecting mortality and mobility have emphasized the role of temperature as the most important abiotic factor, acting through physiological processes to determine ecological outcomes. These relationships are seen in key areas such as species responses to climate warming, forecasting systems for pest outbreaks and the establishment potential of alien species in new environments.
Molecular genetic analysis of cold–regulated gene transcription
Authors: Viswanathan C. , Zhu Jian-Kang
Published: July 2002

Chilling and freezing temperatures adversely affect the productivity and quality of crops. Hence improving the cold hardiness of crop plants is an important goal in agriculture, which demands a clear understanding of cold stress signal perception and transduction. Pharmacological and biochemical evidence shows that membrane rigidification followed by cytoskeleton rearrangement, Ca
2+
influx and Ca
2+
–dependent phosphorylation are involved in cold stress signal transduction. Cold–responsive genes are regulated through C–repeat/dehydration–responsive elements (CRT/DRE) and abscisic acid (ABA)–responsive element cis elements by transacting factors C–repeat binding factors/dehydration–responsive element binding proteins (CBFs/DREBs) and basic leucine zippers (bZIPs) (SGBF1), respectively. We have carried out a forward genetic analysis using chemically mutagenized
Arabidopsis
plants expressing cold–responsive RD29A promoter–driven luciferase to dissect cold signal transduction. We have isolated the
fiery1
(
fry1
) mutant and cloned the
FRY1
gene, which encodes an inositol polyphosphate 1–phosphatase. The
fry1
plants showed enhanced induction of stress genes in response to cold, ABA, salt and dehydration due to higher accumulation of the second messenger, inositol (1,4,5)– triphosphate (IP
3
). Thus our study provides genetic evidence suggesting that cold signal is transduced through changes in IP
3
levels. We have also identified the
hos1
mutation, which showed super induction of cold–responsive genes and their transcriptional activators. Molecular cloning and characterization revealed that
HOS1
encodes a ring finger protein, which has been implicated as an E3 ubiquitin conjugating enzyme. HOS1 is present in the cytoplasm at normal growth temperatures but accumulates in the nucleus upon cold stress. HOS1 appears to regulate temperature sensing by the cell as cold–responsive gene expression occurs in the
hos1
mutant at relatively warm temperatures. Thus HOS1 is a negative regulator, which may be functionally linked to cellular thermosensors to modulate cold–responsive gene transcription.
Low–temperature sensors in bacteria
Authors: Eriksson Sofia , Hurme Reini , Rhen Mikael
Published: July 2002

Bacteria are ubiquitous colonizers of various environments and host organisms, and they are therefore often subjected to drastic temperature alterations. Temperature alterations set demands on these colonizers, in that the bacteria need to readjust their biochemical constitution and physiology in order to survive and resume growth at the new temperature. Furthermore, temperature alteration is also a main factor determining the expression or repression of bacterial virulence functions. To cope with temperature variation, bacteria have devices for sensing temperature alterations and a means of translating this sensory event into a pragmatic gene response. While such regulatory cascades may ultimately be complicated, it appears that they contain primary sensor machinery at the top of the cascade. The functional core of such machinery is usually that of a temperature–induced conformational or physico–chemical change in the central constituents of the cell. In a sense, a bacterium can use structural alterations in its biomolecules as the primary thermometers or thermostats.
Plant D–type cyclins and the control of G1 progression
Authors: Oakenfull E. Ann , Riou-Khamlichi Catherine , Murray A. H.
Published: July 2002

The basic pattern of controls that operate during the G1 phase of the plant cell cycle shows much closer similarity to animals than to the yeasts and other fungi. The activity of D–type cyclin (CycD) kinases is induced in response to stimulatory signals, and these phosphorylate the plant homologue of the retinoblastoma tumour susceptibility (Rb) protein. It is likely that Rb phosphorylation results in the activation of genes under the control of E2F transcription factors, including those required for S phase entry. As the initial triggers of the cascade, attention has focused on the CycDs, and a family of 10 genes is present inArabidopsis, divided into three major and three minor groups. Analysis to date suggests that these groups are functionally distinct.
Concluding remarks
Authors: Bale Jeff
Published: July 2002

There are clearly a substantial number of people who work on low temperature. Some of you might want to put a label on your collar to say that you are a taxonomically based lowtemperature biologist: a botanist, zoologist or microbiologist. Or some of you may describe yourself by the level of organization at which you work: a structural biologist, biochemist, physiologist, or ecologist. But all of you have one common interest; that is low temperature. One success of this meeting therefore, has been to bring together people who ordinarily might not otherwise have met. It is clear that many of us do not know what each other does, because from a literature search on some of the speakers and their papers, it was apparent that we do not cross–cite each other very much. In other words, entomologists do not really know what botanists are doing, who do not really know what bacteriologists are doing and so on. So, a second success of this meeting is that we have actually come together and, at least by knowing each other's names, we might become more familiar in future with each other's work. By that approach, I am sure that we will start to see some analogies and common ground across our different experimental systems. That objective was one of the main bene. ts that Dai Rees, Peter Lillford, Ian Shanks and Diana Bowles wanted to emerge from this meeting, so on your behalf, I will also pass our thanks to them for their advice and support.
The Arabidopsis kinase-associated protein phosphatase controls internalization of the somatic embryogenesis receptor kinase 1
Authors: Shah Khalid , Russinova Eugenia , Gadella Theodorus W.J. , Willemse Joost , de Vries Sacco C.
Published: July 2002

The AtSERK1 protein is a plasma membrane-located LRR receptor-like serine threonine kinase that is transiently expressed during plant embryogenesis. Our results show that AtSERK1 interacts with the kinase-associated protein phosphatase (KAPP) in vitro. The kinase interaction (KI) domain of KAPP does not interact with a catalytically inactive kinase mutant. Using mutantAtSERK1 proteins in which Thr 462, Thr 463, and Thr 468 in the A-loop of the AtSERK1 kinase domain were replaced by alanines, we show that phosphorylation status of the receptor is involved in interaction with KAPP. KAPP and AtSERK1 cDNAs were fused to two different variants of green fluorescent protein (GFP), the yellow fluorescent protein (YFP) or the cyan fluorescent protein (CFP). Both KAPP and AtSERK1 proteins are found at the plasma membrane. Our results show that AtSERK1-CFP becomes sequestered into intracellular vesicles when transiently coexpressed with KAPP-YFP proteins. AtSERK1T463A-CFP andAtSERK13T→A-CFP proteins were partially sequestered intracellularly in the absence of KAPP-YFP protein, suggesting an active role for KAPP dephosphorylation of threonine residues in theAtSERK1 A-loop in receptor internalization. The interaction between the KAPP-CFP/YFP and AtSERK1-CFP/YFP fusion proteins was investigated with fluorescence spectral imaging microscopy (FSPIM). Our results show that AtSERK1-CFP and KAPP-YFP proteins are colocalized at the plasma membrane but only show fluorescence energy transfer (FRET) indicative of physical interaction in intracellular vesicles. These results suggest that KAPP is an integral part of theAtSERK1 endocytosis mechanism.
The barrier function of an insulator couples high histone acetylation levels with specific protection of promoter DNA from methylation
Authors: Mutskov Vesco J. , Farrell Catherine M. , Wade Paul A. , Wolffe Alan P. , Felsenfeld Gary
Published: July 2002

Stably integrated transgenes flanked by the chicken β-globin HS4 insulator are protected against chromosomal position effects and gradual extinction of expression during long-term propagation in culture. To investigate the mechanism of action of this insulator, we used bisulfite genomic sequencing to examine the methylation of individual CpG sites within insulated transgenes, and compared this with patterns of histone acetylation. Surprisingly, although the histones of the entire insulated transgene are highly acetylated, only a specific region in the promoter, containing binding sites for erythroid-specific transcription factors, is highly protected from DNA methylation. This critical region is methylated in noninsulated and inactive lines. MBD3 and Mi-2, subunits of the Mi-2/NuRD repressor complex, are bound in vivo to these silenced noninsulated transgenes. In contrast, insulated cell lines do not show any enrichment of Mi-2/NuRD proteins very late in culture. In addition to the high levels of histone acetylation observed across the entire insulated transgene, significant peaks of H3 acetylation are present over the HS4 insulator elements. Targeted histone acetylation by the chicken β-globin insulator occurs independently of gene transcription and does not require the presence of a functional enhancer. We suggest that this acetylation is in turn responsible for the maintenance of a region of unmethylated DNA over the promoter. Whereas DNA methylation often leads to histone deacetylation, here acetylation appears to prevent methylation.
Genome Size Reduction through Illegitimate Recombination Counteracts Genome Expansion in Arabidopsis
Authors: Devos Katrien M. , Brown James K.M. , Bennetzen Jeffrey L.
Published: July 2002

Genome size varies greatly across angiosperms. It is well documented that, in addition to polyploidization, retrotransposon amplification has been a major cause of genome expansion. The lack of evidence for counterbalancing mechanisms that curtail unlimited genome growth has made many of us wonder whether angiosperms have a “one-way ticket to genomic obesity.” We have therefore investigated an angiosperm with a well-characterized and notably small genome, Arabidopsis thaliana, for evidence of genomic DNA loss. Our results indicate that illegitimate recombination is the driving force behind genome size decrease in Arabidopsis, removing at least fivefold more DNA than unequal homologous recombination. The presence of highly degraded retroelements also suggests that retrotransposon amplification has not been confined to the last 4 million years, as is indicated by the dating of intact retroelements.
Lymphopenia in the BB Rat Model of Type 1 Diabetes is Due to a Mutation in a Novel Immune-Associated Nucleotide (Ian)-Related Gene
Authors: MacMurray Armand J. , Moralejo Daniel H. , Kwitek Anne E. , Rutledge Elizabeth A. , Van Yserloo Brian , Gohlke Paul , Speros Sara J. , Snyder Ben , Schaefer Jonathan , Bieg Sabine , Jiang Jianjie , Ettinger Ruth A. , Fuller Jessica , Daniels Terri L. , Pettersson Anna , Orlebeke Kimberly , Birren Bruce , Jacob Howard J. , Lander Eric S. , Lernmark Åke
Published: July 2002

The BB (BioBreeding) rat is one of the best models of spontaneous autoimmune diabetes and is used to study non-MHC loci contributing to Type 1 diabetes. Type 1 diabetes in the diabetes-prone BB (BBDP) rat is polygenic, dependent upon mutations at several loci.Iddm1, on chromosome 4, is responsible for a lymphopenia (lyp) phenotype and is essential to diabetes. In this study, we report the positional cloning of theIddm1/lyplocus. We show that lymphopenia is due to a frameshift deletion in a novel member (Ian5) of the Immune-Associated Nucleotide (IAN)-related gene family, resulting in truncation of a significant portion of the protein. This mutation was absent in 37 other inbred rat strains that are nonlymphopenic and nondiabetic. The IAN gene family, lying within a tight cluster on rat chromosome 4, mouse chromosome 6, and human chromosome 7, is poorly characterized. Some members of the family have been shown to be expressed in mature T cells and switched on during thymic T-cell development, suggesting thatIan5may be a key factor in T-cell development. The lymphopenia mutation may thus be useful not only to elucidate Type 1 diabetes, but also in the function of theIangene family as a whole.

[Sequence data reported in this paper has been deposited in GenBank and assigned the following accession nos:AF517674,AF517675,AF517676, andAF517677. Supplemental material is available online athttp://depts.washington.edu/rhwlab/and http:www.genome.org. ] The following individuals and institutions kindly provided reagents, samples, or unpublished information as indicated in the paper: K. Matsumoto and the Sir Frederick Banting Research Centre.
Potentiation of Developmentally Regulated Plant Defense Response by AtWRKY18, a Pathogen-Induced Arabidopsis Transcription Factor
Authors: Chen Chunhong , Chen Zhixiang
Published: July 2002

AtWRKY18 is a pathogen- and salicylic acid-induced Arabidopsis transcription factor containing the plant-specific WRKY zinc finger DNA-binding motif. In the present study, we have transformed Arabidopsis plants with AtWRKY18under control of the cauliflower mosaic virus 35S promoter. Surprisingly, transgenic plants expressing high levels ofAtWRKY18 were stunted in growth. When expressed at moderate levels, AtWRKY18 potentiated developmentally regulated defense responses in transgenic plants without causing substantial negative effects on plant growth. As they grew from seedling to mature stages, transgenic AtWRKY18 plant showed marked increase in the expression of pathogenesis-related genes and resistance to the bacterial pathogen Pseudomonas syringae, whereas wild-type plants exhibited little enhancement in these defense responses. Potentiation of developmentally regulated defense responses by AtWRKY18 was not associated with enhanced biosynthesis of salicylic acid but required the disease resistance regulatory protein NPR1/NIM1. Thus, AtWRKY18 can positively modulate defense-related gene expression and disease resistance. To study the regulated expression ofAtWRKY18, we have identified a cluster of WRKY binding sites in the promoter of the gene and demonstrated that they acted as negative regulatory elements for the inducible expression ofAtWRKY18. These negative cis-acting elements may prevent overexpression of AtWRKY18 during the activation of plant defense responses that could be detrimental to plant growth as inferred from the transgenic plants ectopically expressing the transgene.
Regulation of CLV3 Expression by Two Homeobox Genes in Arabidopsis
Authors: Brand Ulrike , Grünewald Margit , Hobe Martin , Simon Rüdiger
Published: July 2002

The ability of meristems to continuously produce new organs depends on the activity of their stem cell populations, which are located at the meristem tip. In Arabidopsis, the size of the stem cell domain is regulated by two antagonistic activities. TheWUS (WUSCHEL) gene, encoding a homeodomain protein, promotes the formation and maintenance of stem cells. These stem cells express CLV3(CLAVATA3), and signaling of CLV3 through the CLV1/CLV2 receptor complex restricts WUS activity. Homeostasis of the stem cell population may be achieved through feedback regulation, whereby changes in stem cell number result in corresponding changes inCLV3 expression levels, and adjustment ofWUS expression via the CLV signal transduction pathway. We have analyzed whether expression ofCLV3 is controlled by the activity of WUSor another homeobox gene, STM (SHOOT MERISTEMLESS), which is required for stem cell maintenance. We found that expression of CLV3 depends onWUS function only in the embryonic shoot meristem. At later developmental stages, WUS promotes the level ofCLV3 expression, together with STM. Within a meristem, competence to respond to WUS activity by expressing CLV3 is restricted to the meristem apex.
Cold-Regulated Cereal Chloroplast Late Embryogenesis Abundant-Like Proteins. Molecular Characterization and Functional Analyses
Authors: NDong Christian , Danyluk Jean , Wilson Kenneth E. , Pocock Tessa , Huner Norman P.A. , Sarhan Fathey
Published: July 2002

Cold acclimation and freezing tolerance are the result of complex interaction between low temperature, light, and photosystem II (PSII) excitation pressure. Previous results have shown that expression of the Wcs19 gene is correlated with PSII excitation pressure measured in vivo as the relative reduction state of PSII. Using cDNA library screening and data mining, we have identified three different groups of proteins, late embryogenesis abundant (LEA) 3-L1, LEA3-L2, and LEA3-L3, sharing identities with WCS19. These groups represent a new class of proteins in cereals related to group 3 LEA proteins. They share important characteristics such as a sorting signal that is predicted to target them to either the chloroplast or mitochondria and a C-terminal sequence that may be involved in oligomerization. The results of subcellular fractionation, immunolocalization by electron microscopy and the analyses of target sequences within the Wcs19 gene are consistent with the localization of WCS19 within the chloroplast stroma of wheat (Triticum aestivum) and rye (Secale cereale). Western analysis showed that the accumulation of chloroplastic LEA3-L2 proteins is correlated with the capacity of different wheat and rye cultivars to develop freezing tolerance. Arabidopsis was transformed with the Wcs19 gene and the transgenic plants showed a significant increase in their freezing tolerance. This increase was only evident in cold-acclimated plants. The putative function of this protein in the enhancement of freezing tolerance is discussed.
Single and Double Knockouts of the Genes for Photosystem I Subunits G, K, and H of Arabidopsis. Effects on Photosystem I Composition, Photosynthetic Electron Flow, and State Transitions
Authors: Varotto Claudio , Pesaresi Paolo , Jahns Peter , Leßnick Angela , Tizzano Marco , Schiavon Fabio , Salamini Francesco , Leister Dario
Published: July 2002

Photosystem I (PSI) of higher plants contains 18 subunits. Using Arabidopsis En insertion lines, we have isolated knockout alleles of the genes psaG,psaH2, and psaK, which code for PSI-G, -H, and -K. In the mutants psak-1 andpsag-1.4, complete loss of PSI-K and -G, respectively, was confirmed, whereas the residual H level in psah2-1.4is due to a second gene encoding PSI-H, psaH1. Double mutants, lacking PSI-G, and also -K, or a fraction of -H, together with the three single mutants were characterized for their growth phenotypes and PSI polypeptide composition. In general, the loss of each subunit has secondary, in some cases additive, effects on the abundance of other PSI polypeptides, such as D, E, H, L, N, and the light-harvesting complex I proteins Lhca2 and 3. In the G-less mutantpsag-1.4, the variation in PSI composition suggests that PSI-G stabilizes the PSI-core. Levels of light-harvesting complex I proteins in plants, which lack simultaneously PSI-G and -K, indicate that PSI subunits other than G and K can also bind Lhca2 and 3. In the same single and double mutants, psag-1.4,psak-1, psah2-1.4,psag-1.4/psah2-1.4, andpsag-1.4/psak-1 photosynthetic electron flow and excitation energy quenching were analyzed to address the roles of the various subunits in P700 reduction (mediated by PSI-F and -N) and oxidation (PSI-E), and state transitions (PSI-H). Based on the results, we also suggest for PSI-K a role in state transitions.
The Physiology and Biophysics of an Aluminum Tolerance Mechanism Based on Root Citrate Exudation in Maize
Authors: Piñeros Miguel A. , Magalhaes Jurandir V. , Carvalho Alves Vera M. , Kochian Leon V.
Published: July 2002

Al-induced release of Al-chelating ligands (primarily organic acids) into the rhizosphere from the root apex has been identified as a major Al tolerance mechanism in a number of plant species. In the present study, we conducted physiological investigations to study the spatial and temporal characteristics of Al-activated root organic acid exudation, as well as changes in root organic acid content and Al accumulation, in an Al-tolerant maize (Zea mays) single cross (SLP 181/71 × Cateto Colombia 96/71). These investigations were integrated with biophysical studies using the patch-clamp technique to examine Al-activated anion channel activity in protoplasts isolated from different regions of the maize root. Exposure to Al nearly instantaneously activated a concentration-dependent citrate release, which saturated at rates close to 0.5 nmol citrate h−1 root−1, with the half-maximal rates of citrate release occurring at about 20 μm Al3+ activity. Comparison of citrate exudation rates between decapped and capped roots indicated the root cap does not play a major role in perceiving the Al signal or in the exudation process. Spatial analysis indicated that the predominant citrate exudation is not confined to the root apex, but could be found as far as 5 cm beyond the root cap, involving cortex and stelar cells. Patch clamp recordings obtained in whole-cell and outside-out patches confirmed the presence of an Al-inducible plasma membrane anion channel in protoplasts isolated from stelar or cortical tissues. The unitary conductance of this channel was 23 to 55 pS. Our results suggest that this transporter mediates the Al-induced citrate release observed in the intact tissue. In addition to the rapid Al activation of citrate release, a slower, Al-inducible increase in root citrate content was also observed. These findings led us to speculate that in addition to the Al exclusion mechanism based on root citrate exudation, a second internal Al tolerance mechanism may be operating based on Al-inducible changes in organic acid synthesis and compartmentation. We discuss our findings in terms of recent genetic studies of Al tolerance in maize, which suggest that Al tolerance in maize is a complex trait.
Proteomics of Arabidopsis Seed Germination. A Comparative Study of Wild-Type and Gibberellin-Deficient Seeds
Authors: Gallardo Karine , Job Claudette , Groot Steven P.C. , Puype Magda , Demol Hans , Vandekerckhove Joël , Job Dominique
Published: July 2002

We examined the role of gibberellins (GAs) in germination of Arabidopsis seeds by a proteomic approach. For that purpose, we used two systems. The first system consisted of seeds of the GA-deficient ga1 mutant, and the second corresponded to wild-type seeds incubated in paclobutrazol, a specific GA biosynthesis inhibitor. With both systems, radicle protrusion was strictly dependent on exogenous GAs. The proteomic analysis indicated that GAs do not participate in many processes involved in germination sensu stricto (prior to radicle protrusion), as, for example, the initial mobilization of seed protein and lipid reserves. Out of 46 protein changes detected during germination sensu stricto (1 d of incubation on water), only one, corresponding to the cytoskeleton component α-2,4 tubulin, appeared to depend on the action of GAs. An increase in this protein spot was noted for the wild-type seeds but not for thega1 seeds incubated for 1 d on water. In contrast, GAs appeared to be involved, directly or indirectly, in controlling the abundance of several proteins associated with radicle protrusion. This is the case for two isoforms of S-adenosyl-methionine (Ado-Met) synthetase, which catalyzes the formation of Ado-Met from Met and ATP. Owing to the housekeeping functions of Ado-Met, this event is presumably required for germination and seedling establishment, and might represent a major metabolic control of seedling establishment. GAs can also play a role in controlling the abundance of a β-glucosidase, which might be involved in the embryo cell wall loosening needed for cell elongation and radicle extension.
The Complement of Protein Phosphatase Catalytic Subunits Encoded in the Genome of Arabidopsis
Authors: Kerk David , Bulgrien Joshua , Smith Douglas W. , Barsam Brooke , Veretnik Stella , Gribskov Michael
Published: July 2002

Reversible protein phosphorylation is critically important in the modulation of a wide variety of cellular functions. Several families of protein phosphatases remove phosphate groups placed on key cellular proteins by protein kinases. The complete genomic sequence of the model plant Arabidopsis permits a comprehensive survey of the phosphatases encoded by this organism. Several errors in the sequencing project gene models were found via analysis of predicted phosphatase coding sequences. Structural sequence probes from aligned and unaligned sequence models, and all-against-all BLAST searches, were used to identify 112 phosphatase catalytic subunit sequences, distributed among the serine (Ser)/threonine (Thr) phosphatases (STs) of the protein phosphatase P (PPP) family, STs of the protein phosphatase M (PPM) family (protein phosphatases 2C [PP2Cs] subfamily), protein tyrosine (Tyr) phosphatases (PTPs), low-M  r protein Tyr phosphatases, and dual-specificity (Tyr and Ser/Thr) phosphatases (DSPs). The Arabidopsis genome contains an abundance of PP2Cs (69) and a dearth of PTPs (one). Eight sequences were identified as new protein phosphatase candidates: five dual-specificity phosphatases and three PP2Cs. We used phylogenetic analyses to infer clustering patterns reflecting sequence similarity and evolutionary ancestry. These clusters, particularly for the largely unexplored PP2C set, will be a rich source of material for plant biologists, allowing the systematic sampling of protein function by genetic and biochemical means.