Web-enabled knowledge-based analysis of genetic data

We present a web-based implementation of GenePath, an intelligent assistant tool for data analysis in functional genomics. GenePath considers mutant data and uses expert-defined patterns to find gene-to-gene or gene-to-outcome relations. It presents the results of analysis as genetic networks, wherein a set of genes has various influence on one another and on a biological outcome. In the paper, we particularly focus on its web-based interface and explanation mechanisms

Gene prioritization by compressive data fusion and chaining

Data integration procedures combine heterogeneous data sets into predictive models, but they are limited to data explicitly related to the target object type, such as genes. Collage is a new data fusion approach to gene prioritization. It considers data sets of various association levels with the prediction task, utilizes collective matrix factorization to compress the data, and chaining to relate different object types contained in a data compendium. Collage prioritizes genes based on their similarity to several seed genes. We tested Collage by prioritizing bacterial response genes in Dictyostelium as a novel model system for prokaryote-eukaryote interactions. Using 4 seed genes and 14 data sets, only one of which was directly related to the bacterial response, Collage proposed 8 candidate genes that were readily validated as necessary for the response of Dictyostelium to Gram-negative bacteria. These findings establish Collage as a method for inferring biological knowledge from the integration of heterogeneous and coarsely related data sets

A new social gene in Dictyostelium discoideum, chtB

Background: Competitive social interactions are ubiquitous in nature, but their genetic basis is difficult to determine. Much can be learned from single gene knockouts in a eukaryote microbe. The mutants can be competed with the parent to discern the social impact of that specific gene. Dictyostelium discoideum is a social amoeba that exhibits cooperative behavior in the construction of a multicellular fruiting body. It is a good model organism to study the genetic basis of cooperation since it has a sequenced genome and it is amenable to genetic manipulation. When two strains of D. discoideum are mixed, a cheater strain can exploit its social partner by differentiating more spore than its fair share relative to stalk cells. Cheater strains can be generated in the lab or found in the wild and genetic analyses have shown that cheating behavior can be achieved through many pathways. Results: We have characterized the knockout mutant chtB, which was isolated from a screen for cheater mutants that were also able to form normal fruiting bodies on their own. When mixed in equal proportions with parental strain cells, chtB mutants contributed almost 60% of the total number of spores. To do so, chtB cells inhibit wild type cells from becoming spores, as indicated by counts and by the wild type cells’ reduced expression of the prespore gene, cotB. We found no obvious fitness costs (morphology, doubling time in liquid medium, spore production, and germination efficiency) associated with the cheating ability of the chtB knockout. Conclusions: In this study we describe a new gene in D. discoideum, chtB, which when knocked out inhibits the parental strain from producing spores. Moreover, under lab conditions, we did not detect any fitness costs associated with this behavior

Polymorphic members of the lag gene family mediate kin discrimination in Dictyostelium

Self and kin discrimination are observed in most kingdoms of life and are mediated by highly polymorphic plasma membrane proteins. Sequence polymorphism, which is essential for effective recognition, is maintained by balancing selection. Dictyostelium discoideum are social amoebas that propagate as unicellular organisms but aggregate upon starvation and form fruiting bodies with viable spores and dead stalk cells. Aggregative development exposes Dictyostelium to the perils of chimerism, including cheating, which raises questions about how the victims survive in nature and how social cooperation persists. Dictyostelids can minimize the cost of chimerism by preferential cooperation with kin, but the mechanisms of kin discrimination are largely unknown. Dictyostelium lag genes encode transmembrane proteins with multiple immunoglobulin (Ig) repeats that participate in cell adhesion and signaling. Here, we describe their role in kin discrimination. We show that lagB1 and lagC1 are highly polymorphic in natural populations and that their sequence dissimilarity correlates well with wild-strain segregation. Deleting lagB1 and lagC1 results in strain segregation in chimeras with wild-type cells, whereas elimination of the nearly invariant homolog lagD1 has no such consequences. These findings reveal an early evolutionary origin of kin discrimination and provide insight into the mechanism of social recognition and immunity

Cell–Cell Adhesion Prevents Mutant Cells Lacking Myosin II from Penetrating Aggregation Streams ofDictyostelium

AbstractWhen a small number of fluorescently labeled myosin II mutant cells (mhcA−) are mixed with wild-type cells and development of the chimeras is observed by confocal microscopy, the mutant cells are localized to the edges of aggregation streams and mounds. Moreover, the mutant cells stick to wild-type cells and become distorted (Shelden and Knecht, 1995). Two independent adhesion mechanisms, Contact Sites A and Contact Sites B, function during the aggregation stage and either one or both might be responsible for excluding the myosin II null cells. We have mixedmhcA−cells with cells in which the appearance of Contact Sites B is delayed (strain TL72) as well as cells which lack Contact Sites A (strain GT10) and double mutants in which both adhesion mechanisms are affected (strain TL73). In all chimeras, themhcA−cells were distorted by interactions with the adhesion mutant cells, indicating that it does not require significant adhesive interaction to distort the flaccid cortex ofmhcA−cells.mhcA−cells were excluded from streams composed of cells lacking either Contact Sites A or Contact Sites B but mixed randomly with cells lacking both adhesion systems. By 10 hr of development, cells of strain TL73 acquire Contact Sites B adhesion. If cells of this strain were mixed with labeledmhcA−cells, allowed to develop for 9 hr, and then dissociated before replating, the myosin II null cells were seen to be distorted and excluded from the reaggregates. Thus the exclusion ofmhcA−cells from streams can be accomplished by either Contact Sites A or B. When chimeras of labeled TL73 and wild-type cells were made, the TL73 cells were found to be randomly mixed into aggregation streams. This result indicates that adhesive sorting does not function during aggregation and so cannot account for the exclusion ofmhcA−cells from streams. We hypothesize that the flaccid cortex ofmhcA−cells cannot generate sufficient protrusive force to break the contacts between adhered cells in aggregation streams but can enter streams where the cells are weakly adherent

Xpf and Not the Fanconi Anaemia Proteins or Rev3 Accounts for the Extreme Resistance to Cisplatin in Dictyostelium discoideum

Organisms like Dictyostelium discoideum, often referred to as DNA damage “extremophiles”, can survive exposure to extremely high doses of radiation and DNA crosslinking agents. These agents form highly toxic DNA crosslinks that cause extensive DNA damage. However, little is known about how Dictyostelium and the other “extremophiles” can tolerate and repair such large numbers of DNA crosslinks. Here we describe a comprehensive genetic analysis of crosslink repair in Dictyostelium discoideum. We analyse three gene groups that are crucial for a replication-coupled repair process that removes DNA crosslinks in higher eukarya: The Fanconi anaemia pathway (FA), translesion synthesis (TLS), and nucleotide excision repair. Gene disruption studies unexpectedly reveal that the FA genes and the TLS enzyme Rev3 play minor roles in tolerance to crosslinks in Dictyostelium. However, disruption of the Xpf nuclease subcomponent results in striking hypersensitivity to crosslinks. Genetic interaction studies reveal that although Xpf functions with FA and TLS gene products, most Xpf mediated repair is independent of these two gene groups. These results suggest that Dictyostelium utilises a distinct Xpf nuclease-mediated repair process to remove crosslinked DNA. Other DNA damage–resistant organisms and chemoresistant cancer cells might adopt a similar strategy to develop resistance to DNA crosslinking agents

Genomic Signatures of Cooperation and Conflict in the Social Amoeba

Molecular evolution analyses reveal the history of social conflict Genes that mediate social conflict show signatures of frequency-dependent selection Balanced polymorphisms suggest that cheating may be stable and endemic Cooperative systems are susceptible to invasion by selfish individuals that profit from receiving the social benefits but fail to contribute. These so-called cheaters can have a fitness advantage in the laboratory, but it is unclear whether cheating provides an important selective advantage in nature. We used a population genomic approach to examine the history of genes involved in cheating behaviors in the social amoeba Dictyostelium discoideum, testing whether these genes experience rapid evolutionary change as a result of conflict over spore-stalk fate. Candidate genes and surrounding regions showed elevated polymorphism, unusual patterns of linkage disequilibrium, and lower levels of population differentiation, but they did not show greater between-species divergence. The signatures were most consistent with frequency-dependent selection acting to maintain multiple alleles, suggesting that conflict may lead to stalemate rather than an escalating arms race. Our results reveal the evolutionary dynamics of cooperation and cheating and underscore how sequence-based approaches can be used to elucidate the history of conflicts that are difficult to observe directly

WASP family proteins and formins compete in pseudopod- and bleb-based migration

Actin pseudopods induced by SCAR/WAVE drive normal migration and chemotaxis in eukaryotic cells. Cells can also migrate using blebs, in which the edge is driven forward by hydrostatic pressure instead of actin. In Dictyostelium discoideum, loss of SCAR is compensated by WASP moving to the leading edge to generate morphologically normal pseudopods. Here we use an inducible double knockout to show that cells lacking both SCAR and WASP are unable to grow, make pseudopods or, unexpectedly, migrate using blebs. Remarkably, amounts and dynamics of actin polymerization are normal. Pseudopods are replaced in double SCAR/WASP mutants by aberrant filopods, induced by the formin dDia2. Further disruption of the gene for dDia2 restores cells’ ability to initiate blebs and thus migrate, though pseudopods are still lost. Triple knockout cells still contain near-normal F-actin levels. This work shows that SCAR, WASP, and dDia2 compete for actin. Loss of SCAR and WASP causes excessive dDia2 activity, maintaining F-actin levels but blocking pseudopod and bleb formation and migration