The ascospores of Saccharomyces cerevisiae are surrounded by a complex wall that protects the spores from environmental stresses. The outermost layer of the spore wall is composed of a polymer that contains the cross-linked amino acid dityrosine. This dityrosine layer is important for stress resistance of the spore. This work reports that the dityrosine layer acts as a barrier blocking the diffusion of soluble proteins out of the spore wall into the cytoplasm of the ascus. Diffusion of a fluorescent protein out of the spore wall was used as an assay to screen for mutants affecting spore wall permeability. One of the genes identified in this screen, OSW3 ( RRT12 / YCR045c ), encodes a subtilisin-family protease localized to the spore wall. Mutation of the active site serine of Osw3 results in spores with permeable walls, indicating that the catalytic activity of Osw3 is necessary for proper construction of the dityrosine layer. These results indicate that dityrosine promotes stress resistance by acting as a protective shell around the spore. OSW3 and other OSW genes identified in this screen are strong candidates to encode enzymes involved in assembly of this protective dityrosine coat.

The profusion of high-throughput instruments and the explosion of new results in the scientific literature, particularly in molecular biomedicine, is both a blessing and a curse to the bench researcher. Even knowledgeable and experienced scientists can benefit from computational tools that help navigate this vast and rapidly evolving terrain. In this paper, we describe a novel computational approach to this challenge, a knowledge-based system that combines reading, reasoning, and reporting methods to facilitate analysis of experimental data. Reading methods extract information from external resources, either by parsing structured data or using biomedical language processing to extract information from unstructured data, and track knowledge provenance. Reasoning methods enrich the knowledge that results from reading by, for example, noting two genes that are annotated to the same ontology term or database entry. Reasoning is also used to combine all sources into a knowledge network that represents the integration of all sorts of relationships between a pair of genes, and to calculate a combined reliability score. Reporting methods combine the knowledge network with a congruent network constructed from experimental data and visualize the combined network in a tool that facilitates the knowledge-based analysis of that data. An implementation of this approach, called the Hanalyzer, is demonstrated on a large-scale gene expression array dataset relevant to craniofacial development. The use of the tool was critical in the creation of hypotheses regarding the roles of four genes never previously characterized as involved in craniofacial development; each of these hypotheses was validated by further experimental work.

DNA double-strand breaks (DSBs) can be repaired by homologous recombination (HR), which can involve Holliday junction (HJ) intermediates that are ultimately resolved by nucleolytic enzymes. An N-terminal fragment of human GEN1 has recently been shown to act as a Holliday junction resolvase, but little is known about the role of GEN-1 in vivo. Holliday junction resolution signifies the completion of DNA repair, a step that may be coupled to signaling proteins that regulate cell cycle progression in response to DNA damage. Using forward genetic approaches, we identified a Caenorhabditis elegans dual function DNA double-strand break repair and DNA damage signaling protein orthologous to the human GEN1 Holliday junction resolving enzyme. GEN-1 has biochemical activities related to the human enzyme and facilitates repair of DNA double-strand breaks, but is not essential for DNA double-strand break repair during meiotic recombination. Mutational analysis reveals that the DNA damage-signaling function of GEN-1 is separable from its role in DNA repair. GEN-1 promotes germ cell cycle arrest and apoptosis via a pathway that acts in parallel to the canonical DNA damage response pathway mediated by RPA loading, CHK1 activation, and CEP-1/p53–mediated apoptosis induction. Furthermore, GEN-1 acts redundantly with the 9-1-1 complex to ensure genome stability. Our study suggests that GEN-1 might act as a dual function Holliday junction resolvase that may coordinate DNA damage signaling with a late step in DNA double-strand break repair.

Fusobacterium nucleatum is a prominent member of the oral microbiota and is a common cause of human infection. F. nucleatum includes five subspecies: polymorphum , nucleatum , vincentii , fusiforme , and animalis . F. nucleatum subsp. polymorphum ATCC 10953 has been well characterized phenotypically and, in contrast to previously sequenced strains, is amenable to gene transfer. We sequenced and annotated the 2,429,698 bp genome of F. nucleatum subsp. polymorphum ATCC 10953. Plasmid pFN3 from the strain was also sequenced and analyzed. When compared to the other two available fusobacterial genomes ( F. nucleatum subsp. nucleatum , and F. nucleatum subsp. vincentii ) 627 open reading frames unique to F. nucleatum subsp. polymorphum ATCC 10953 were identified. A large percentage of these mapped within one of 28 regions or islands containing five or more genes. Seventeen percent of the clustered proteins that demonstrated similarity were most similar to proteins from the clostridia, with others being most similar to proteins from other gram-positive organisms such as Bacillus and Streptococcus . A ten kilobase region homologous to the Salmonella typhimurium propanediol utilization locus was identified, as was a prophage and integrated conjugal plasmid. The genome contains five composite ribozyme/transposons, similar to the Cd ISt IStrons described in Clostridium difficile . IStrons are not present in the other fusobacterial genomes. These findings indicate that F. nucleatum subsp. polymorphum is proficient at horizontal gene transfer and that exchange with the Firmicutes , particularly the Clostridia , is common.

Nutrient availability is an important environmental variable during development that has significant effects on the metabolism, health, and viability of an organism. To understand these interactions for the nutrient copper, we used a chemical genetic screen for zebrafish mutants sensitive to developmental copper deficiency. In this screen, we isolated two mutants that define subtleties of copper metabolism. The first contains a viable hypomorphic allele of atp7a and results in a loss of pigmentation when exposed to mild nutritional copper deficiency. This mutant displays incompletely penetrant skeletal defects affected by developmental copper availability. The second carries an inactivating mutation in the vacuolar ATPase that causes punctate melanocytes and embryonic lethality. This mutant, catastrophe , is sensitive to copper deprivation revealing overlap between ion metabolic pathways. Together, the two mutants illustrate the utility of chemical genetic screens in zebrafish to elucidate the interaction of nutrient availability and genetic polymorphisms in cellular metabolism.

Nucleotide Excision Repair (NER), which removes a variety of helix-distorting lesions from DNA, is initiated by two distinct DNA damage-sensing mechanisms. Transcription Coupled Repair (TCR) removes damage from the active strand of transcribed genes and depends on the SWI/SNF family protein CSB. Global Genome Repair (GGR) removes damage present elsewhere in the genome and depends on damage recognition by the XPC/RAD23/Centrin2 complex. Currently, it is not well understood to what extent both pathways contribute to genome maintenance and cell survival in a developing organism exposed to UV light. Here, we show that eukaryotic NER, initiated by two distinct subpathways, is well conserved in the nematode Caenorhabditis elegans . In C. elegans , involvement of TCR and GGR in the UV-induced DNA damage response changes during development. In germ cells and early embryos, we find that GGR is the major pathway contributing to normal development and survival after UV irradiation, whereas in later developmental stages TCR is predominantly engaged. Furthermore, we identify four ISWI/Cohesin and four SWI/SNF family chromatin remodeling factors that are implicated in the UV damage response in a developmental stage dependent manner. These in vivo studies strongly suggest that involvement of different repair pathways and chromatin remodeling proteins in UV-induced DNA repair depends on developmental stage of cells.

Cell-to-cell fusion is involved in multiple fundamental biological processes. Prominent examples include osteoclast and giant cell formation, fertilization and skeletal myogenesis which involve macrophage, sperm-egg and myoblast fusion, respectively. Indeed, the importance of cell fusion is underscored by the wide range of homeostatic as well as pathologic processes in which it plays a key role. Therefore, rapid and sensitive systems to trace and measure cell fusion events in various experimental systems are in demand. Here, we introduce a bipartite cell fusion monitoring system based on a genetic switch responsive to the site-specific recombinase FLP. To allow flexible deployment in both dividing as well as non-dividing cell populations, inducer and reporter modules were incorporated in lentivirus vector particles. Moreover, the recombinase-inducible transcription units were designed in such a way as to minimize basal activity and chromosomal position effects in the “off” and “on” states, respectively. The lentivirus vector-based conditional gene expression assay was validated in primary human mesenchymal stem cells and in a differentiation model based on muscle progenitor cells from a Duchenne muscular dystrophy patient using reporter genes compatible with live- and single-cell imaging and with whole population measurements. Using the skeletal muscle cell differentiation model, we showed that the new assay displays low background activity, a 2-log dynamic range, high sensitivity and is amenable to the investigation of cell fusion kinetics. The utility of the bipartite cell fusion monitoring system was underscored by a study on the impact of drug- and RNAi-mediated p38 MAPK inhibition on human myocyte differentiation. Finally, building on the capacity of lentivirus vectors to readily generate transgenic animals the present FLP-inducible system should be adaptable, alone or together with Cre/loxP-based assays, to cell lineage tracing and conditional gene manipulation studies in vivo .

The chemotherapeutic doxorubicin (DOX) induces DNA double-strand break (DSB) damage. In order to identify conserved genes that mediate DOX resistance, we screened the Saccharomyces cerevisiae diploid deletion collection and identified 376 deletion strains in which exposure to DOX was lethal or severely reduced growth fitness. This diploid screen identified 5-fold more DOX resistance genes than a comparable screen using the isogenic haploid derivative. Since DSB damage is repaired primarily by homologous recombination in yeast, and haploid cells lack an available DNA homolog in G1 and early S phase, this suggests that our diploid screen may have detected the loss of repair functions in G1 or early S phase prior to complete DNA replication. To test this, we compared the relative DOX sensitivity of 30 diploid deletion mutants identified under our screening conditions to their isogenic haploid counterpart, most of which (n = 26) were not detected in the haploid screen. For six mutants ( bem1Δ, ctf4Δ, ctk1Δ, hfi1Δ,nup133Δ, tho2Δ) DOX-induced lethality was absent or greatly reduced in the haploid as compared to the isogenic diploid derivative. Moreover, unlike WT, all six diploid mutants displayed severe G1/S phase cell cycle progression defects when exposed to DOX and some were significantly enhanced ( ctk1Δ and hfi1Δ ) or deficient ( tho2Δ ) for recombination. Using these and other “THO2-like” hypo-recombinogenic, diploid-specific DOX sensitive mutants ( mft1Δ, thp1Δ, thp2Δ ) we utilized known genetic/proteomic interactions to construct an interactive functional genomic network which predicted additional DOX resistance genes not detected in the primary screen. Most (76%) of the DOX resistance genes detected in this diploid yeast screen are evolutionarily conserved suggesting the human orthologs are candidates for mediating DOX resistance by impacting on checkpoint and recombination functions in G1 and/or early S phases.

The histiocytoses are a heterogeneous group of disorders characterised by an excessive number of histiocytes. In most cases the pathophysiology is unclear and treatment is nonspecific. Faisalabad histiocytosis (FHC) (MIM 602782) has been classed as an autosomal recessively inherited form of histiocytosis with similarities to Rosai-Dorfman disease (RDD) (also known as sinus histiocytosis with massive lymphadenopathy (SHML)). To elucidate the molecular basis of FHC, we performed autozygosity mapping studies in a large consanguineous family and identified a novel locus at chromosome 10q22.1. Mutation analysis of candidate genes within the target interval identified biallelic germline mutations in SLC29A3 in the FHC kindred and in two families reported to have familial RDD. Analysis of SLC29A3 expression during mouse embryogenesis revealed widespread expression by e14.5 with prominent expression in the central nervous system, eye, inner ear, and epithelial tissues including the gastrointestinal tract. SLC29A3 encodes an intracellular equilibrative nucleoside transporter (hENT3) with affinity for adenosine. Recently germline mutations in SLC29A3 were also described in two rare autosomal recessive disorders with overlapping phenotypes: (a) H syndrome (MIM 612391) that is characterised by cutaneous hyperpigmentation and hypertrichosis, hepatomegaly, heart anomalies, hearing loss, and hypogonadism; and (b) PHID (pigmented hypertrichosis with insulin-dependent diabetes mellitus) syndrome. Our findings suggest that a variety of clinical diagnoses (H and PHID syndromes, FHC, and familial RDD) can be included in a new diagnostic category of SLC29A3 spectrum disorder.