SadA, a novel adhesion receptor in Dictyostelium

Little is known about cell–substrate adhesion and how motile and adhesive forces work together in moving cells. The ability to rapidly screen a large number of insertional mutants prompted us to perform a genetic screen in Dictyostelium to isolate adhesion-deficient mutants. The resulting substrate adhesion–deficient (sad) mutants grew in plastic dishes without attaching to the substrate. The cells were often larger than their wild-type parents and displayed a rough surface with many apparent blebs. One of these mutants, sadA−, completely lacked substrate adhesion in growth medium. The sadA− mutant also showed slightly impaired cytokinesis, an aberrant F-actin organization, and a phagocytosis defect. Deletion of the sadA gene by homologous recombination recreated the original mutant phenotype. Expression of sadA–GFP in sadA-null cells restored the wild-type phenotype. In sadA–GFP-rescued mutant cells, sadA–GFP localized to the cell surface, appropriate for an adhesion molecule. SadA contains nine putative transmembrane domains and three conserved EGF-like repeats in a predicted extracellular domain. The EGF repeats are similar to corresponding regions in proteins known to be involved in adhesion, such as tenascins and integrins. Our data combined suggest that sadA is the first substrate adhesion receptor to be identified in Dictyostelium

A fluorescent resonant energy transfer–based biosensor reveals transient and regional myosin light chain kinase activation in lamella and cleavage furrows

Approaches with high spatial and temporal resolution are required to understand the regulation of nonmuscle myosin II in vivo. Using fluorescence resonance energy transfer we have produced a novel biosensor allowing simultaneous determination of myosin light chain kinase (MLCK) localization and its [Ca2+]4/calmodulin-binding state in living cells. We observe transient recruitment of diffuse MLCK to stress fibers and its in situ activation before contraction. MLCK is highly active in the lamella of migrating cells, but not at the retracting tail. This unexpected result highlights a potential role for MLCK-mediated myosin contractility in the lamella as a driving force for migration. During cytokinesis, MLCK was enriched at the spindle equator during late metaphase, and was maximally activated just before cleavage furrow constriction. As furrow contraction was completed, active MLCK was redistributed to the poles of the daughter cells. These results show MLCK is a myosin regulator in the lamella and contractile ring, and pinpoints sites where myosin function may be mediated by other kinases

dictyBase, the model organism database for Dictyostelium discoideum

dictyBase () is the model organism database (MOD) for the social amoeba Dictyostelium discoideum. The unique biology and phylogenetic position of Dictyostelium offer a great opportunity to gain knowledge of processes not characterized in other organisms. The recent completion of the 34 MB genome sequence, together with the sizable scientific literature using Dictyostelium as a research organism, provided the necessary tools to create a well-annotated genome. dictyBase has leveraged software developed by the Saccharomyces Genome Database and the Generic Model Organism Database project. This has reduced the time required to develop a full-featured MOD and greatly facilitated our ability to focus on annotation and providing new functionality. We hope that manual curation of the Dictyostelium genome will facilitate the annotation of other genomes

Xanthusbase: adapting wikipedia principles to a model organism database

xanthusBase () is the official model organism database (MOD) for the social bacterium Myxococcus xanthus. In many respects, M.xanthus represents the pioneer model organism (MO) for studying the genetic, biochemical, and mechanistic basis of prokaryotic multicellularity, a topic that has garnered considerable attention due to the significance of biofilms in both basic and applied microbiology research. To facilitate its utility, the design of xanthusBase incorporates open-source software, leveraging the cumulative experience made available through the Generic Model Organism Database (GMOD) project, MediaWiki (), and dictyBase (), to create a MOD that is both highly useful and easily navigable. In addition, we have incorporated a unique Wikipedia-style curation model which exploits the internet's inherent interactivity, thus enabling M.xanthus and other myxobacterial researchers to contribute directly toward the ongoing genome annotation

Annotating the human genome with Disease Ontology

<p>Abstract</p> <p>Background</p> <p>The human genome has been extensively annotated with Gene Ontology for biological functions, but minimally computationally annotated for diseases.</p> <p>Results</p> <p>We used the Unified Medical Language System (UMLS) MetaMap Transfer tool (MMTx) to discover gene-disease relationships from the GeneRIF database. We utilized a comprehensive subset of UMLS, which is disease-focused and structured as a directed acyclic graph (the Disease Ontology), to filter and interpret results from MMTx. The results were validated against the Homayouni gene collection using recall and precision measurements. We compared our results with the widely used Online Mendelian Inheritance in Man (OMIM) annotations.</p> <p>Conclusion</p> <p>The validation data set suggests a 91% recall rate and 97% precision rate of disease annotation using GeneRIF, in contrast with a 22% recall and 98% precision using OMIM. Our thesaurus-based approach allows for comparisons to be made between disease containing databases and allows for increased accuracy in disease identification through synonym matching. The much higher recall rate of our approach demonstrates that annotating human genome with Disease Ontology and GeneRIF for diseases dramatically increases the coverage of the disease annotation of human genome.</p

A physical gene map of the bacteriophage P22 late region: Genetic analysis of cloned fragments of P22 DNA

A physical gene map of the late region of the P22 chromosome has been constructed by genetic analysis of restriction enzyme fragments of P22 DNA cloned in a plasmid vector. Cleavage sites for restriction endonucleases SalI, SstI, SmaI, Xhoi, and BglI were mapped on P22 DNA to provide physical reference points in addition to the EcoRI, HindIII, and BamHI cleavage sites previously mapped. Restriction enzymes KpnI, BglII, and Xbal were found to have no cleavage sites on P22 DNA. Fragments of P22 DNA produced by cleavage with EcoRI, BamHI, or EcoRI plus BamHI were cloned in Escherichia coli using the plasmid vector pBR322, and the resulting recombinant plasmids were introduced into Salmonella typhimurium. The genes present on a cloned fragment were identified by the ability of the hybrid plasmid to complement or recombine with P22 amber mutations in known genes when mutant phage were used to infect S. typhimurium strains carrying the recombinant plasmids. These experiments place all phage genes required for P22 head morphogenesis except gene 3 on the physical map between coordinates 0.000 and 0.318. The coding capacity of this interval is in close agreement with the molecular weights of the proteins assigned to it. The single gene for the P22 base plate protein is placed between coordinates 0.376 and 0.420 on the physical map. These results also show that distances on the recombination frequency map are significantly distorted relative to the physical gene map of the late region. The recombination frequency map is expanded in the region of the physical gene map where terminally redundant ends of the circularly permuted mature chromosomes fall.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/23263/1/0000197.pd

dictyBase—a Dictyostelium bioinformatics resource update

dictyBase (http://dictybase.org) is the model organism database for Dictyostelium discoideum. It houses the complete genome sequence, ESTs and the entire body of literature relevant to Dictyostelium. This information is curated to provide accurate gene models and functional annotations, with the goal of fully annotating the genome. This dictyBase update describes the annotations and features implemented since 2006, including improved strain and phenotype representation, integration of predicted transcriptional regulatory elements, protein domain information, biochemical pathways, improved searching and a wiki tool that allows members of the research community to provide annotations

The genetic architecture underlying prey-dependent performance in a microbial predator

Natural selection should favour generalist predators that outperform specialists across all prey types. Two genetic solutions could explain why intraspecific variation in predatory performance is, nonetheless, widespread: mutations beneficial on one prey type are costly on another (antagonistic pleiotropy), or mutational effects are prey-specific, which weakens selection, allowing variation to persist (relaxed selection). To understand the relative importance of these alternatives, we characterised natural variation in predatory performance in the microbial predator Dictyostelium discoideum. We found widespread nontransitive differences among strains in predatory success across different bacterial prey, which can facilitate stain coexistence in multi-prey environments. To understand the genetic basis, we developed methods for high throughput experimental evolution on different prey (REMI-seq). Most mutations (~77%) had prey-specific effects, with very few (~4%) showing antagonistic pleiotropy. This highlights the potential for prey-specific effects to dilute selection, which would inhibit the purging of variation and prevent the emergence of an optimal generalist predator

Mutant resources for functional genomics in Dictyostelium discoideum using REMI-seq technology

Background Genomes can be sequenced with relative ease, but ascribing gene function remains a major challenge. Genetically tractable model systems are crucial to meet this challenge. One powerful model is the social amoeba Dictyostelium discoideum, a eukaryotic microbe widely used to study diverse questions in the cell, developmental and evolutionary biology. Results We describe REMI-seq, an adaptation of Tn-seq, which allows high throughput, en masse, and quantitative identification of the genomic site of insertion of a drug resistance marker after restriction enzyme-mediated integration. We use REMI-seq to develop tools which greatly enhance the efficiency with which the sequence, transcriptome or proteome variation can be linked to phenotype in D. discoideum. These comprise (1) a near genome-wide resource of individual mutants and (2) a defined pool of ‘barcoded’ mutants to allow large-scale parallel phenotypic analyses. These resources are freely available and easily accessible through the REMI-seq website that also provides comprehensive guidance and pipelines for data analysis. We demonstrate that integrating these resources allows novel regulators of cell migration, phagocytosis and macropinocytosis to be rapidly identified. Conclusions We present methods and resources, generated using REMI-seq, for high throughput gene function analysis in a key model system