bluuebunny/crossref_metadata_2025 huggingface dataset:
Integrative Analysis of Cell Cycle Control in Budding Yeast
Authors: Chen Katherine C. , Calzone Laurence , Csikasz-Nagy Attila , Cross Frederick R. , Novak Bela , Tyson John J.
Published: June 2004

The adaptive responses of a living cell to internal and external signals are controlled by networks of proteins whose interactions are so complex that the functional integration of the network cannot be comprehended by intuitive reasoning alone. Mathematical modeling, based on biochemical rate equations, provides a rigorous and reliable tool for unraveling the complexities of molecular regulatory networks. The budding yeast cell cycle is a challenging test case for this approach, because the control system is known in exquisite detail and its function is constrained by the phenotypic properties of >100 genetically engineered strains. We show that a mathematical model built on a consensus picture of this control system is largely successful in explaining the phenotypes of mutants described so far. A few inconsistencies between the model and experiments indicate aspects of the mechanism that require revision. In addition, the model allows one to frame and critique hypotheses about how the division cycle is regulated in wild-type and mutant cells, to predict the phenotypes of new mutant combinations, and to estimate the effective values of biochemical rate constants that are difficult to measure directly in vivo.
Lipid Raft-dependent Glucagon-like Peptide-2 Receptor Trafficking Occurs Independently of Agonist-induced Desensitization
Authors: Estall Jennifer L. , Yusta Bernardo , Drucker Daniel J.
Published: June 2004

The intestinotrophic and cytoprotective actions of glucagon-like peptide-2 (GLP-2) are mediated by the GLP-2 receptor (GLP-2R), a member of the class II glucagon-secretin G protein-coupled receptor superfamily. Although native GLP-2 exhibits a short circulating half-life, long-acting degradation-resistant GLP-2 analogues are being evaluated for therapeutic use in human subjects. Accordingly, we examined the mechanisms regulating signaling, internalization, and trafficking of the GLP-2R to identify determinants of receptor activation and desensitization. Heterologous cells expressing the transfected rat or human GLP-2R exhibited a rapid, dose-dependent, and prolonged desensitization of the GLP-2–stimulated cAMP response and a sustained GLP-2–induced decrease in levels of cell surface receptor. Surprisingly, inhibitors of clathrin-dependent endocytosis failed to significantly decrease GLP-2R internalization, whereas cholesterol sequestration inhibited ligand-induced receptor internalization and potentiated homologous desensitization. The hGLP-2R localized to both Triton X-100–soluble and –insoluble (lipid raft) cellular fractions and colocalized transiently with the lipid raft marker caveolin-1. Although GLP-2R endocytosis was dependent on lipid raft integrity, the receptor transiently associated with green fluorescent protein tagged-early endosome antigen 1–positive vesicles and inhibitors of endosomal acidification attenuated the reappearance of the GLP-2R on the cell surface. Our data demonstrate that GLP-2R desensitization and raft-dependent trafficking represent distinct and independent cellular mechanisms and provide new evidence implicating the importance of a clathrin- and dynamin-independent, lipid raft-dependent pathway for homologous G protein-coupled receptor internalization.
Cdc42, Rac1, and Rac2 Display Distinct Patterns of Activation during Phagocytosis
Authors: Hoppe Adam D. , Swanson Joel A.
Published: June 2004

The small G proteins Cdc42, Rac1, and Rac2 regulate the rearrangements of actin and membrane necessary for Fcγ receptor-mediated phagocytosis by macrophages. Activated, GTP-bound Cdc42, Rac1, and Rac2 bind to the p21-binding domain (PBD) of PAK1, and this interaction provided a basis for microscopic methods to localize activation of these G proteins inside cells. Fluorescence resonance energy transfer-based stoichiometry of fluorescent chimeras of actin, PBD, Cdc42, Rac1, and Rac2 was used to quantify G protein activation relative to actin movements during phagocytosis of IgG-opsonized erythrocytes. The activation dynamics of endogenous G proteins, localized using yellow fluorescent protein-labeled PBD, was restricted to phagocytic cups, with a prominent spike of activation over an actin-poor region at the base of the cup. Refinements of fluorescence resonance energy transfer stoichiometry allowed calculation of the fractions of activated GTPases in forming phagosomes. Cdc42 activation was restricted to the leading margin of the cell, whereas Rac1 was active throughout the phagocytic cup. During phagosome closure, activation of Rac1 and Rac2 increased uniformly and transiently in the actin-poor region of phagosomal membrane. These distinct roles for Cdc42, Rac1, and Rac2 in the component activities of phagocytosis indicate mechanisms by which their differential regulation coordinates rearrangements of actin and membranes.
Essential Role for the Myotubularin-related Phosphatase Ymr1p and the Synaptojanin-like Phosphatases Sjl2p and Sjl3p in Regulation of Phosphatidylinositol 3-Phosphate in Yeast
Authors: Parrish William R. , Stefan Christopher J. , Emr Scott D.
Published: June 2004

The requirement of Vps34p, the sole phosphatidylinositol (PI) 3-kinase in Saccharomyces cerevisiae, for protein sorting to the vacuole in yeast has exemplified the essential role for phosphoinositides, phosphorylated derivatives of PI, in membrane trafficking. To better understand mechanisms that regulate PI 3-phosphate [PI(3)P]-mediated signaling, the role of the yeast myotubularin-related PI(3)P phosphatase Ymr1p was investigated. We found that Ymr1p and the synaptojanin-like phosphatase Sjl3p function as key regulators of the localization and levels of PI(3)P. Our data indicated that the ymr1Δ sjl3Δ double mutant aberrantly accumulated PI(3)P and demonstrated a steady-state redistribution of this lipid that leads to enrichment on the vacuolar membrane. This resulted in vacuole protein sorting defects, vacuolar fragmentation, and the misregulation of PI(3)P-specific effectors. Triple deletion of YMR1, SJL2, and SJL3 was lethal, suggesting an essential requirement for phosphatase-mediated PI(3)P regulation. Consistent with this, growth was restored to a ymr1Δ sjl2Δ sjl3Δ triple mutant by a PI(3)P-targeted Sac1p domain chimera (GFP-Sac1ΔC-FYVEEEA1) that returned PI(3)P to levels comparable with wild-type cells. Together, this study demonstrated that Ymr1p, a myotubularin phosphatase family member, functions in the control of PI(3)P-dependent signaling and the maintenance of endosomal system integrity. In addition, this work defined an essential overlapping role for lipid phosphatases in the regulation of 3′ phosphoinositides in yeast.
Modulation of Telomere Length Dynamics by the Subtelomeric Region ofTetrahymenaTelomeres
Authors: Jacob Naduparambil K. , Stout Angela R. , Price Carolyn M.
Published: June 2004

Tetrahymena telomeres usually consist of ∼250 base pairs of T2G4repeats, but they can grow to reach a new length set point of up to 900 base pairs when kept in log culture at 30°C. We have examined the growth profile of individual macronuclear telomeres and have found that the rate and extent of telomere growth are affected by the subtelomeric region. When the sequence of the rDNA subtelomeric region was altered, we observed a decrease in telomere growth regardless of whether the GC content was increased or decreased. In both cases, the ordered structure of the subtelomeric chromatin was disrupted, but the effect on the telomeric complex was relatively minor. Examination of the telomeres from non-rDNA chromosomes showed that each telomere exhibited a unique and characteristic growth profile. The subtelomeric regions from individual chromosome ends did not share common sequence elements, and they each had a different chromatin structure. Thus, telomere growth is likely to be regulated by the organization of the subtelomeric chromatin rather than by a specific DNA element. Our findings suggest that at each telomere the telomeric complex and subtelomeric chromatin cooperate to form a unique higher order chromatin structure that controls telomere length.
Proteomic Analysis of Interchromatin Granule Clusters
Authors: Saitoh Noriko , Spahr Chris S. , Patterson Scott D. , Bubulya Paula , Neuwald Andrew F. , Spector David L.
Published: June 2004

A variety of proteins involved in gene expression have been localized within mammalian cell nuclei in a speckled distribution that predominantly corresponds to interchromatin granule clusters (IGCs). We have applied a mass spectrometry strategy to identify the protein composition of this nuclear organelle purified from mouse liver nuclei. Using this approach, we have identified 146 proteins, many of which had already been shown to be localized to IGCs, or their functions are common to other already identified IGC proteins. In addition, we identified 32 proteins for which only sequence information is available and thus these represent novel IGC protein candidates. We find that 54% of the identified IGC proteins have known functions in pre-mRNA splicing. In combination with proteins involved in other steps of pre-mRNA processing, 81% of the identified IGC proteins are associated with RNA metabolism. In addition, proteins involved in transcription, as well as several other cellular functions, have been identified in the IGC fraction. However, the predominance of pre-mRNA processing factors supports the proposed role of IGCs as assembly, modification, and/or storage sites for proteins involved in pre-mRNA processing.
Correcting the loss of cell-cycle synchrony in clustering analysis of microarray data using weights
Authors: Duan Fenghai , Zhang Heping
Published: June 2004

Motivation: Due to the existence of the loss of synchrony in cell-cycle data sets, standard clustering methods (e.g. k-means), which group open reading frames (ORFs) based on similar expression levels, are deficient unless the temporal pattern of the expression levels of the ORFs is taken into account.

Methods: We propose to improve the performance of the k-means method by assigning a decreasing weight on its variable level and evaluating the ‘weighted k-means’ on a yeast cell-cycle data set. Protein complexes from a public website are used as biological benchmarks. To compare the k-means clusters with the structures of the protein complexes, we measure the agreement between these two ways of clustering via the adjusted Rand index.

Results: Our results show the time-decreasing weight function—exp[−(1/2)(t2/C2)—]which we assign to the variable level of k-means, generally increases the agreement between protein complexes and k-means clusters when C is near the length of two cell cycles.
DBRF–MEGN method: an algorithm for deducing minimum equivalent gene networks from large-scale gene expression profiles of gene deletion mutants
Authors: Kyoda Koji , Baba Kotaro , Onami Shuichi , Kitano Hiroaki
Published: June 2004

Motivation: Large-scale gene expression profiles measured in gene deletion mutants are invaluable sources for identifying gene regulatory networks. Signed directed graph (SDG) is the most common representation of gene networks in genetics and cell biology. However, no practical procedure that deduces SDGs consistent with such profiles has been developed.

Results: We developed the DBRF–MEGN (difference-based regulation finding–minimum equivalent gene network) method in which an algorithm deduces the most parsimonious SDGs consistent with expression profiles of gene deletion mutants. Positive (or negative) directed edges representing positive (or negative) gene regulations are deduced by comparing the gene expression level between the wild-type and mutant. The most parsimonious SDGs are deduced using graph theoretical procedures. Compensation for excess removal of edges by restoring a minimum number of edges makes the method applicable to cyclic gene networks. Use of independent groups of edges greatly reduces the computational cost, thus making the method applicable to large-scale expression profiles. We confirmed the applicability of our method by applying it to the gene expression profiles of 265 Saccharomyces cerevisiae deletion mutants, and we confirmed our method's validity by comparing the pheromone response pathway, general amino acid control system, and copper and iron homeostasis system deduced by our method with those reported in the literature. Interpretation of the gene network deduced from the S. cerevisiae expression profiles by using our method led to the prediction of 132 transcriptional targets and modulators of transcriptional activity of 18 transcriptional regulators.

Availability: The software is available on request.

Supplementary information:  http://www.so.bio.keio.ac.jp/dbrf-megn/
Mining gene expression data based on template theory
Authors: Yang Zheng Rong
Published: June 2004

Motivation: It is understood that clustering genes are useful for exploring scientific knowledge from DNA microarray gene expression data. The explored knowledge can be finally used for annotating biological function for novel genes. Representing the explored knowledge in an efficient manner is then closely related to the classification accuracy. However, this issue has not yet been paid the attention it deserves.

Result: A novel method based on template theory in cognitive psychology and pattern recognition is developed in this study for representing knowledge extracted from cluster analysis effectively. The basic principle is to represent knowledge according to the relationship between genes and a found cluster structure. Based on this novel knowledge representation method, a pattern recognition algorithm (the decision tree algorithm C4.5) is then used to construct a classifier for annotating biological functions of novel genes. The experiments on five published datasets show that this method has improved the classification performance compared with the conventional method. The statistical tests indicate that this improvement is significant.

Availability: The software package can be obtained upon request from the author.
CoPS: Comprehensive Peptide Signature Database
Authors: Prakash Tulika , Khandelwal Mamta , Dasgupta Dipayan , Dash Debasis , Brahmachari Samir K.
Published: June 2004

Summary: We present the development of a Comprehensive database of 12 076 invariant Peptide Signatures (CoPS) derived from 52 bacterial genomes with a minimum occurrence in at least seven organisms. These peptides were observed in functionally similar proteins and are distributed over nearly 1250 different functional proteins. The database provides function, structure and occurrence in biochemical pathways of the proteins containing these signature peptides. It houses additional information on the signature peptides, such as identical match in other motif/pattern (e.g. PROSITE, BLOCKS, PRINTS and Pfam) databases and the database of interacting proteins, human proteome and mutation effect on these signature peptides. There is a wide applicability of this database in the identification of critical functional residues in proteins. The database also facilitates the identification of folding nucleus/structural determinants in proteins and functional assignment to yet unknown proteins. We demonstrate functional assignment to 2605 hypothetical proteins in bacterial genomes and 112 unknown proteins in human using this database.

Availability: The database can be freely accessed through the following URL: http://203.195.151.46/copsv2/index.html or http://203.90.127.70/copsv2/index.html

Supplementary information:  http://203.195.151.46/copsv2/aboutcops.html and http://203.90.127.70/copsv2/aboutcops.html
ICBS: a database of interactions between protein chains mediated by β-sheet formation
Authors: Dou Yimeng , Baisnée Pierre-François , Pollastri Gianluca , Pécout Yann , Nowick James , Baldi Pierre
Published: June 2004

Motivation: Interchain β-sheet (ICBS) interactions occur widely in protein quaternary structures, interactions between proteins and protein aggregation. These interactions play a central role in many biological processes and in diseases ranging from AIDS and cancer to anthrax and Alzheimer's.

Results: We have created a comprehensive database of ICBS interactions that is updated on a weekly basis and allows entries to be sorted and searched by relevance and other criteria through a simple Web interface. We derive a simple ICBS index to quantify the relative contributions of the β-ladders in the overall interchain interaction and compute first- and second-order statistics regarding amino acid composition and pairing at different relative positions in the β-strands. Analysis of the database reveals a 15.8% prevalence of significant ICBS interactions, the majority of which involve the formation of antiparallel β-sheets and many of which involve the formation of dimers and oligomers. The frequencies of amino acids in ICBS interfaces are similar to those in intrachain β-sheet interfaces. A full range of non-covalent interactions between side chains complement the hydrogen-bonding interactions between the main chains. Polar amino acids pair preferentially with polar amino acids and non-polar amino acids pair preferentially with non-polar amino acids among antiparallel (i, j) pairs. We anticipate that the statistics and insights gained from the database will guide the development of agents that control interchain β-sheet interactions and that the database will help identify new protein interactions and targets for these agents.

Availability: The database is available at: http://www.igb.uci.edu/servers/icbs/

Supplementary Information:  http://www.igb.uci.edu/servers/icbs/
Faster cyclic loess: normalizing RNA arrays via linear models
Authors: Ballman Karla V. , Grill Diane E. , Oberg Ann L. , Therneau Terry M.
Published: June 2004

Motivation: Our goal was to develop a normalization technique that yields results similar to cyclic loess normalization and with speed comparable to quantile normalization.

Results: Fastlo yields normalized values similar to cyclic loess and quantile normalization and is fast; it is at least an order of magnitude faster than cyclic loess and approaches the speed of quantile normalization. Furthermore, fastlo is more versatile than both cyclic loess and quantile normalization because it is model-based.

Availability: The Splus/R function for fastlo normalization is available from the authors.
Command line tool for calculating theoretical MS spectra for given sequences
Authors: Boehm Andreas M. , Grosse-Coosmann Florian , Sickmann Albert
Published: June 2004

Summary: Scientists usually want to verify the ion matching process of algorithms that look up peptide sequences in DNA or protein databases. The verification step is often done numerically or visually. Not all search algorithms present the appropriate theoretical spectrum information within their results. Thus, the theoretical spectrum for each result should be calculated from the sequence of the matched peptide. We present an operating-system-independent command line tool for this purpose that can be integrated easily into complex as well as existing environments, and can be used to present the theoretical spectrum to the user in either graphical or tabular format by third party products.

Availability: The code is available via the website http://www.protein-ms.de.
BacTregulators: a database of transcriptional regulators in bacteria and archaea
Authors: Martínez-Bueno Manuel , Molina-Henares Antonio J. , Pareja Eduardo , Ramos Juan L. , Tobes Raquel
Published: June 2004

Motivation: The BacTregulators database is intended to collect and to integrate information on proteins belonging to defined families of transcriptional regulators in prokaryotes.

Results: The BacTregulators database currently contains data on two families of transcriptional regulators: AraC-XylS and TetR. The proteins included in the BacTregulators database have been identified by screening 123 genomes from archaea and bacteria and the SWISS-PROT and TrEMBL databases with profiles defining each family. As the result of an integration process, we have included 1326 different protein sequences from the AraC-XylS family and 1487 different protein sequences from the TetR family. The definition of an entry in BacTregulators is based on protein sequence, source organism, genome element and position in this genome element. The BacTregulators site allows the user to retrieve protein sequences, functional features and experimental evidence supporting the functions, references and the three-dimensional structure of the regulator when available. BacTregulators supplies an innovative tool that allows the researcher to obtain an integrated report that shows the data corresponding to other entries which are related by sequence similarity to the query entry. BacTregulators detects and classifies the regulators belonging to AraC-XylS and TetR families present in prokaryotic genomes, and thus contributes to a more accurate annotation of regulators in genomes. The information collected on each protein in the family can be useful to characterize a new regulator or compile information on the biological properties of a known regulator.

Availability: The BacTregulators is available at www.bactregulators.org

Supplementary information:  www.bactregulators.org/supplementary/
Qxpak: a versatile mixed model application for genetical genomics and QTL analyses
Authors: Pérez-Enciso M. , Misztal I.
Published: June 2004

Motivation: Current methodology and software for quantitative trait loci (QTL) analyses do not use all available information and are inadequate to deal with the huge amount of QTL analyses to be needed in forecoming genetical genomics' studies.

Results: We show that a mixed model statistical framework provides a very flexible tool for QTL modeling in a variety of populations, be it a cross between inbred lines, a within population study, or experiments involving a mixture of populations or crosses. The software allows multitrait and multiQTL analyses, inclusion of infinitesimal genetic value and a batch multitrait option suitable for genetical genomics studies. It also allows massive association studies between single nucleotide polymorphisms and the trait(s) of interest.

Availability: A software (Qxpak), together with a manual and example files, is freely available for research purposes. So far, the compiled program is available for linux systems, the windows version will follow soon. See http://www.icrea.es/pag.asp?id=Miguel.Perez
Data mining techniques to study the disulfide-bonding state in proteins: signal peptide is a strong descriptor
Authors: Tessier Dominique , Bardiaux Benjamin , Larré Colette , Popineau Yves
Published: June 2004

In the eucaryotic cell, the formation of disulfide bonds takes place in general inside the endoplasmic reticulum which provides a unique folding environment. The DisulfideDB database gathers information about this biological process with structural, evolutionary and neighborhood information on cysteines in proteins. Mining this information with an association rule discovery program permits to extract some strong rules for the prediction of the disulfide-bonding state of cysteines.

Supplementary information: The web supplement to this paper, including the UML diagram of the database and some procedures used with the association rule discovery tool, may be found at http://www.nantes.inra.fr/centre/unites-recherche/urpvi/bioinformatique/publi.html.
Genomic Regionality in Rates of Evolution Is Not Explained by Clustering of Genes of Comparable Expression Profile
Authors: Lercher Martin J. , Chamary Jean-Vincent , Hurst Laurence D.
Published: June 2004

In mammalian genomes, linked genes show similar rates of evolution, both at fourfold degenerate synonymous sites (K4) and at nonsynonymous sites (KA). Although it has been suggested that the local similarity in the synonymous substitution rate is an artifact caused by the inclusion of disparately evolving gene pairs, we demonstrate here that this is not the case: after removal of disparately evolving genes, both (1) linked genes and (2) introns from the same gene have more similar silent substitution rates than expected by chance. What causes the local similarity in both synonymous and nonsynonymous substitution rates? One class of hypotheses argues that both may be related to the observed clustering of genes of comparable expression profile. We investigate these hypotheses using substitution rates from both human–mouse and mouse–rat comparisons, and employing three different methods to assay expression parameters. Although we confirm a negative correlation of expression breadth with both K4 and KA, we find no evidence that clustering of similarly expressed genes explains the clustering of genes of comparable substitution rates. If gene expression is not responsible, what about other causes? At least in the human–mouse comparison, the local similarity in KA can be explained by the covariation of KA and K4. As regards K4, our results appear consistent with the notion that local similarity is due to processes associated with meiotic recombination.
DNA Mapping Using Microfluidic Stretching and Single-Molecule Detection of Fluorescent Site-Specific Tags
Authors: Chan Eugene Y. , Goncalves Nuno M. , Haeusler Rebecca A. , Hatch Amie J. , Larson Jonathan W. , Maletta Anthony M. , Yantz Gregory R. , Carstea Eugene D. , Fuchs Martin , Wong Gordon G. , Gullans Steven R. , Gilmanshin Rudolf
Published: June 2004

We have developed a rapid molecular mapping technology—Direct Linear Analysis (DLA)—on the basis of the analysis of individual DNA molecules bound with sequence-specific fluorescent tags. The apparatus includes a microfluidic device for stretching DNA molecules in elongational flow that is coupled to a multicolor detection system capable of single-fluorophore sensitivity. Double-stranded DNA molecules were tagged at sequence-specific motif sites with fluorescent bisPNA (Peptide Nucleic Acid) tags. The DNA molecules were then stretched in the microfluidic device and driven in a flow stream past confocal fluorescence detectors. DLA provided the spatial locations of multiple specific sequence motifs along individual DNA molecules, and thousands of individual molecules could be analyzed per minute. We validated this technology using the 48.5 kb λ phage genome with different 8-base and 7-base sequence motif tags. The distance between the sequence motifs was determined with an accuracy of ±0.8 kb, and these tags could be localized on the DNA with an accuracy of ±2 kb. Thus, DLA is a rapid mapping technology, suitable for analysis of long DNA molecules.
eShadow: A Tool for Comparing Closely Related Sequences
Authors: Ovcharenko Ivan , Boffelli Dario , Loots Gabriela G.
Published: June 2004

Primate sequence comparisons are difficult to interpret due to the high degree of sequence similarity shared between such closely related species. Recently, a novel method, phylogenetic shadowing, has been pioneered for predicting functional elements in the human genome through the analysis of multiple primate sequence alignments. We have expanded this theoretical approach to create a computational tool, eShadow, for the identification of elements under selective pressure in multiple sequence alignments of closely related genomes, such as in comparisons of human-to-primate or mouse-to-rat DNA. This tool integrates two different statistical methods and allows for the dynamic visualization of the resulting conservation profile. eShadow also includes a versatile optimization module capable of training the underlying Hidden Markov Model to differentially predict functional sequences. This module grants the tool high flexibility in the analysis of multiple sequence alignments and in comparing sequences with different divergence rates. Here, we describe the eShadow comparative tool and its potential uses for analyzing both multiple nucleotide and protein alignments to predict putative functional elements.
Annotation Transfer Between Genomes: Protein–Protein Interologs and Protein–DNA Regulogs
Authors: Yu Haiyuan , Luscombe Nicholas M. , Lu Hao Xin , Zhu Xiaowei , Xia Yu , Han Jing-Dong J. , Bertin Nicolas , Chung Sambath , Vidal Marc , Gerstein Mark
Published: June 2004

Proteins function mainly through interactions, especially with DNA and other proteins. While some large-scale interaction networks are now available for a number of model organisms, their experimental generation remains difficult. Consequently, interolog mapping—the transfer of interaction annotation from one organism to another using comparative genomics—is of significant value. Here we quantitatively assess the degree to which interologs can be reliably transferred between species as a function of the sequence similarity of the corresponding interacting proteins. Using interaction information from Saccharomyces cerevisiae, Caenorhabditis elegans, Drosophila melanogaster, and Helicobacter pylori, we find that protein–protein interactions can be transferred when a pair of proteins has a joint sequence identity >80% or a joint E-value <10–70. (These “joint” quantities are the geometric means of the identities or E-values for the two pairs of interacting proteins.) We generalize our interolog analysis to protein–DNA binding, finding such interactions are conserved at specific thresholds between 30% and 60% sequence identity depending on the protein family. Furthermore, we introduce the concept of a “regulog”—a conserved regulatory relationship between proteins across different species. We map interologs and regulogs from yeast to a number of genomes with limited experimental annotation (e.g., Arabidopsis thaliana) and make these available through an online database at http://interolog.gersteinlab.org. Specifically, we are able to transfer ∼90,000 potential protein–protein interactions to the worm. We test a number of these in two-hybrid experiments and are able to verify 45 overlaps, which we show to be statistically significant.
Recombinatorial Cloning Using Heterologous Lox Sites
Authors: Siegel Robert W. , Velappan Nileena , Pavlik Peter , Chasteen Leslie , Bradbury Andrew
Published: June 2004

Recombination systems based on λ and Cre/loxPhave been described to facilitate gene transfer from one vector to another in a high-throughput fashion, avoiding the bottlenecks associated with traditional cloning. However, no system described to date is suitable for the cloning of affinity reagents selected from display libraries, which requires that the recombination signals flanking the affinity reagent are translated with a minimum impact on functionality. As affinity reagents will be essential tools in the functional characterization of proteomes, and display technologies represent the most effective means to generate such affinity reagents on a genomic scale, we developed a Cre/loxP-based system, using mutually exclusive heterologousloxPsites placed 5′ (Lox 2372) and 3′ (Lox WT) of an affinity reagent (scFv). The translated lox sites have minimal impact on scFv expression or functionality, and, in association with a conditionally lethal gene (SacB) permit efficient, high-fidelity transfer to destination vectors. This approach will considerably facilitate the high-throughput downstream use of affinity reagents selected by display technologies, as well as being widely applicable to general recombinatorial cloning for genomic purposes.
Coexpression Analysis of Human Genes Across Many Microarray Data Sets
Authors: Lee Homin K. , Hsu Amy K. , Sajdak Jon , Qin Jie , Pavlidis Paul
Published: June 2004

We present a large-scale analysis of mRNA coexpression based on 60 large human data sets containing a total of 3924 microarrays. We sought pairs of genes that were reliably coexpressed (based on the correlation of their expression profiles) in multiple data sets, establishing a high-confidence network of 8805 genes connected by 220,649 “coexpression links” that are observed in at least three data sets. Confirmed positive correlations between genes were much more common than confirmed negative correlations. We show that confirmation of coexpression in multiple data sets is correlated with functional relatedness, and show how cluster analysis of the network can reveal functionally coherent groups of genes. Our findings demonstrate how the large body of accumulated microarray data can be exploited to increase the reliability of inferences about gene function.
Comparative Genomic Hybridization Provides New Insights Into the Molecular Taxonomy of the Saccharomyces Sensu Stricto Complex
Authors: Edwards-Ingram Laura C. , Gent Manda E. , Hoyle David C. , Hayes Andrew , Stateva Lubomira I. , Oliver Stephen G.
Published: June 2004

The science of taxonomy is constantly improving as new techniques are developed. Current practice is to construct phylogenetic trees based on the analysis of the DNA sequence of single genes, or parts of single genes. However, this approach has recently been brought into question as several tree topologies may be produced for the same clade when the sequences for various different genes are used. The availability of complete genome sequences for several organisms has seen the adoption of microarray technology to construct molecular phylogenies of bacteria, based on all of the genes. Similar techniques have been used to reveal the relationships between different strains of the yeast Saccharomyces cerevisiae. We have exploited microarray technology to construct a molecular phylogeny for the Saccharomyces sensu stricto complex of yeast species, which is based on all of the protein-encoding genes revealed by the complete genome sequence of the paradigmatic species, S. cerevisiae. We also analyze different strains of S. cerevisiae itself, as well as the putative species S. boulardii. We show that in addition to the phylogeny produced, we can identify and analyze individual ORF traits and interpret the results to give a detailed explanation of evolutionary events underlying the phylogeny.
Coexpression of Neighboring Genes in the Genome of Arabidopsis thaliana
Authors: Williams Elizabeth J.B. , Bowles Dianna J.
Published: June 2004

Large-scale analyses of expression data of eukaryotic organisms are now becoming increasingly routine. The data sets are revealing interesting and novel patterns of genomic organization, which provide insight both into molecular evolution and how structure and function of a genome interrelate. Our study investigates, for the first time, how genome organization affects expression of a gene in the Arabidopsis genome. The analyses show that neighboring genes are coexpressed. This pattern has been found for all eukaryotic genomes studied so far, but as yet, it remains unclear whether it is due to selective or nonselective influences. We have investigated reasons for coexpression of neighboring genes in Arabidopsis, and our evidence suggests that orientation of gene pairs plays a significant role, with potential sharing of regulatory elements in divergently transcribed genes. Using the data available in the KEGG database, we find evidence that genes in the same pathway are coexpressed, although this is not a major cause for the coexpression of neighboring genes.
Building Genomic Profiles for Uncovering Segmental Homology in the Twilight Zone
Authors: Simillion Cedric , Vandepoele Klaas , Saeys Yvan , Van de Peer Yves
Published: June 2004

The identification of homologous regions within and between genomes is an essential prerequisite for studying genome structure and evolution. Different methods already exist that allow detecting homologous regions in an automated manner. These methods are based either on finding sequence similarities at the DNA level or on identifying chromosomal regions showing conservation of gene order and content. Especially the latter approach has proven useful for detecting homology between highly divergent chromosomal regions. However, until now, such map-based approaches required that candidate homologous regions show significant collinearity with other segments to be considered as being homologous. Here, we present a novel method that creates profiles combining the gene order and content information of multiple mutually homologous genomic segments. These profiles can be used to scan one or more genomes to detect segments that show significant collinearity with the entire profile but not necessarily with individual segments. When applying this new method to the combined genomes of Arabidopsis and rice, we find additional evidence for ancient duplication events in the rice genome.
The Small Chromosomes of Trypanosoma brucei Involved in Antigenic Variation Are Constructed Around Repetitive Palindromes
Authors: Wickstead Bill , Ersfeld Klaus , Gull Keith
Published: June 2004

Most eukaryotic genomes contain large regions of satellite DNA. These arrays are often associated with essential chromosomal functions, but remain largely absent from genome projects because of difficulties in cloning and sequence assembly. The numerous small chromosomes of the parasite Trypanosoma brucei fall into this category, yet are critical to understanding the genome because of their role in antigenic variation. Their relatively small size, however, makes them particularly amenable to physical mapping. We have produced fine-resolution maps of 17 complete minichromosomes and partial maps of two larger intermediate-sized chromosomes. This revealed a canonical structure shared by both chromosomal classes based around a large central core of 177-bp repeats. Around the core are variable-length genic regions, the lengths of which define chromosomal class. We show the core region to be a repetitive palindrome with a single inversion point common to all the chromosomes of both classes, suggesting a mechanism of genesis for these chromosomes. Moreover, palindromy appears to be a feature of (peri)centromeres in other species that can be easily overlooked. We propose that sequence inversion is one of the higher-order sequence motifs that confer chromosomal stability.
Bacterial Genomes as New Gene Homes: The Genealogy of ORFans in E. coli
Authors: Daubin Vincent , Ochman Howard
Published: June 2004

Differences in gene repertoire among bacterial genomes are usually ascribed to gene loss or to lateral gene transfer from unrelated cellular organisms. However, most bacteria contain large numbers of ORFans, that is, annotated genes that are restricted to a particular genome and that possess no known homologs. The uniqueness of ORFans within a genome has precluded the use of a comparative approach to examine their function and evolution. However, by identifying sequences unique to monophyletic groups at increasing phylogenetic depths, we can make direct comparisons of the characteristics of ORFans of different ages in the Escherichia coli genome, and establish their functional status and evolutionary rates. Relative to the genes ancestral to γ-Proteobacteria and to those genes distributed sporadically in other prokaryotic species, ORFans in the E. coli lineage are short, A+T rich, and evolve quickly. Moreover, most encode functional proteins. Based on these features, ORFans are not attributable to errors in gene annotation, limitations of current databases, or to failure of methods for detecting homology. Rather, ORFans in the genomes of free-living microorganisms apparently derive from bacteriophage and occasionally become established by assuming roles in key cellular functions.
Automatic Identification of Subcellular Phenotypes on Human Cell Arrays
Authors: Conrad Christian , Erfle Holger , Warnat Patrick , Daigle Nathalie , Lörch Thomas , Ellenberg Jan , Pepperkok Rainer , Eils Roland
Published: June 2004

Light microscopic analysis of cell morphology provides a high-content readout of cell function and protein localization. Cell arrays and microwell transfection assays on cultured cells have made cell phenotype analysis accessible to high-throughput experiments. Both the localization of each protein in the proteome and the effect of RNAi knock-down of individual genes on cell morphology can be assayed by manual inspection of microscopic images. However, the use of morphological readouts for functional genomics requires fast and automatic identification of complex cellular phenotypes. Here, we present a fully automated platform for high-throughput cell phenotype screening combining human live cell arrays, screening microscopy, and machine-learning-based classification methods. Efficiency of this platform is demonstrated by classification of eleven subcellular patterns marked by GFP-tagged proteins. Our classification method can be adapted to virtually any microscopic assay based on cell morphology, opening a wide range of applications including large-scale RNAi screening in human cells.
Differential Alu Mobilization and Polymorphism Among the Human and Chimpanzee Lineages
Authors: Hedges Dale J. , Callinan Pauline A. , Cordaux Richard , Xing Jinchuan , Barnes Erin , Batzer Mark A.
Published: June 2004

Alu elements are primate-specific members of the SINE (short interspersed element) retroposon family, which comprise ∼10% of the human genome. Here we report the first chromosomal-level comparison examining the Alu retroposition dynamics following the divergence of humans and chimpanzees. We find a twofold increase in Alu insertions in humans in comparison to the common chimpanzee (Pan troglodytes). The genomic diversity (polymorphism for presence or absence of the Alu insertion) associated with these inserts indicates that, analogous to recent nucleotide diversity studies, the level of chimpanzee Alu diversity is ∼1.7 times higher than that of humans. Evolutionarily recent Alu subfamily structure differs markedly between the human and chimpanzee lineages, with the major human subfamilies remaining largely inactive in the chimpanzee lineage. We propose a population-based model to account for the observed fluctuation in Alu retroposition rates across primate taxa.
WebLogo: A Sequence Logo Generator: Figure 1
Authors: Crooks Gavin E. , Hon Gary , Chandonia John-Marc , Brenner Steven E.
Published: June 2004

WebLogo generates sequence logos, graphical representations of the patterns within a multiple sequence alignment. Sequence logos provide a richer and more precise description of sequence similarity than consensus sequences and can rapidly reveal significant features of the alignment otherwise difficult to perceive. Each logo consists of stacks of letters, one stack for each position in the sequence. The overall height of each stack indicates the sequence conservation at that position (measured in bits), whereas the height of symbols within the stack reflects the relative frequency of the corresponding amino or nucleic acid at that position. WebLogo has been enhanced recently with additional features and options, to provide a convenient and highly configurable sequence logo generator. A command line interface and the complete, open WebLogo source code are available for local installation and customization.
Affinity transfer by CDR grafting on a nonimmunoglobulin scaffold
Authors: Nicaise Magali , Valerio‐Lepiniec Marielle , Minard Philippe , Desmadril Michel
Published: June 2004

Neocarzinostatin (NCS) is a small “all β” protein displaying the same overall fold as immunoglobulins. This protein possesses a well‐defined hydrophobic core and two loops structurally equivalent to the CDR1 and CDR3 of immunoglobulins. NCS is the most studied member of the enediynechromoprotein family, and is clinically used as an antitumoral agent. NCS has promise as a drug delivery vehicle if new binding specificities could be conferred on its protein scaffold. Previous studies have shown that the binding specificity of the crevasse can be extended to compounds completely unrelated to the natural enediyne chromophore family. We show here that it is possible to introduce new interaction capacities to obtain a protein useful for drug targeting by modifying the immunoglobulin CDR‐like loops. We transferred the CDR3 of the VHH chain of camel antilysozyme immunoglobulin to the equivalent site in the corresponding loop of neocarzinostatin. We then evaluated the stability of the resulting structure and its affinity for lysozyme. The engineered NCS‐CDR3 presents a structure similar to that of the wild‐type NCS, and is stable and efficiently produced. ELISA, ITC, and SPR measurements demonstrated that the new NCS‐CDR3 specifically bound lysozyme.
Crystal structure of rat α‐parvalbumin at 1.05 Å resolution
Authors: Bottoms Christopher A. , Schuermann Jonathan P. , Agah Sayeh , Henzl Michael T. , Tanner John J.
Published: June 2004

The crystal structure of rat α‐parvalbumin has been determined at 1.05 Å resolution, using synchrotron data collected at Advanced Photon Source beamline 19‐ID. After refinement with SHELX, employing anisotropic displacement parameters and riding hydrogen atoms,R= 0.132 andRfree= 0.162. The average coordinate estimated standard deviations are 0.021 Å and 0.038 Å for backbone atoms and side‐chain atoms, respectively. Besides providing a more precise view of the α‐isoform than previously available, these data permit comparison with the 0.91 Å structure determined for pike β‐parvalbumin. Visualization of the anisotropic displacement parameters as thermal ellipsoids yields insight into the atomic motion within the Ca2+‐binding sites. The asymmetric unit includes three parvalbumin (PV) molecules. Interestingly, the EF site in one displays uncharacteristic flexibility. The ellipsoids for Asp‐92 are particularly large and non‐spherical, and the shape of the Ca2+ellipsoid implies significant vibrational motion perpendicular to the plane defined by the fouryandzligands. The relative dearth of crystal‐packing interactions in this site suggests that the heightened flexibility may be the result of diminished intermolecular contacts. The implication is that, by impeding conformational mobility, crystal‐packing forces may cause serious overestimation of EF‐hand rigidity. The high quality of the data permitted 11 residues to be modeled in alternative side‐chain conformations, including the two core residues, Ile‐97 and Leu‐105. The discrete disorder observed for Ile‐97 may have functional ramifications, providing a mechanism for communicating binding status between the CD and EF binding loops and between the PV metal ion‐binding domain and the N‐terminal AB region.
Insulin forms amyloid in a strain‐dependent manner: An FT‐IR spectroscopic study
Authors: Dzwolak Wojciech , Smirnovas Vytautas , Jansen Ralf , Winter Roland
Published: June 2004

The presence of 20% (v/v) ethanol triggers growth of insulin amyloid with distinct infrared spectroscopic features, compared with the fibrils obtained under ambient conditions. Here we report that the two insulin amyloid types behave in the prion strain‐like manner regarding seeding specificity and ability of the self‐propagating conformational template to overrule unfavorable environmental factors and maintain the initial folding pattern. The type of the original seed has been shown to prevail over cosolvent effects and determines spectral position and width of the amide I′ infrared band of the heterogeneously seeded amyloid. These findings imply that “strains” may be a common generic trait of amyloids.