The Candida Genome Database (CGD): incorporation of Assembly 22, systematic identifiers and visualization of high throughput sequencing data.

The Candida Genome Database (CGD, http://www.candidagenome.org/) is a freely available online resource that provides gene, protein and sequence information for multiple Candida species, along with web-based tools for accessing, analyzing and exploring these data. The mission of CGD is to facilitate and accelerate research into Candida pathogenesis and biology, by curating the scientific literature in real time, and connecting literature-derived annotations to the latest version of the genomic sequence and its annotations. Here, we report the incorporation into CGD of Assembly 22, the first chromosome-level, phased diploid assembly of the C. albicans genome, coupled with improvements that we have made to the assembly using additional available sequence data. We also report the creation of systematic identifiers for C. albicans genes and sequence features using a system similar to that adopted by the yeast community over two decades ago. Finally, we describe the incorporation of JBrowse into CGD, which allows online browsing of mapped high throughput sequencing data, and its implementation for several RNA-Seq data sets, as well as the whole genome sequencing data that was used in the construction of Assembly 22.

The Candida Genome Database: the new homology information page highlights protein similarity and phylogeny.

The Candida Genome Database (CGD, http://www.candidagenome.org/) is a freely available online resource that provides gene, protein and sequence information for multiple Candida species, along with web-based tools for accessing, analyzing and exploring these data. The goal of CGD is to facilitate and accelerate research into Candida pathogenesis and biology. The CGD Web site is organized around Locus pages, which display information collected about individual genes. Locus pages have multiple tabs for accessing different types of information; the default Summary tab provides an overview of the gene name, aliases, phenotype and Gene Ontology curation, whereas other tabs display more in-depth information, including protein product details for coding genes, notes on changes to the sequence or structure of the gene and a comprehensive reference list. Here, in this update to previous NAR Database articles featuring CGD, we describe a new tab that we have added to the Locus page, entitled the Homology Information tab, which displays phylogeny and gene similarity information for each locus.

The Aspergillus Genome Database: multispecies curation and incorporation of RNA-Seq data to improve structural gene annotations.

The Aspergillus Genome Database (AspGD; http://www.aspgd.org) is a freely available web-based resource that was designed for Aspergillus researchers and is also a valuable source of information for the entire fungal research community. In addition to being a repository and central point of access to genome, transcriptome and polymorphism data, AspGD hosts a comprehensive comparative genomics toolbox that facilitates the exploration of precomputed orthologs among the 20 currently available Aspergillus genomes. AspGD curators perform gene product annotation based on review of the literature for four key Aspergillus species: Aspergillus nidulans, Aspergillus oryzae, Aspergillus fumigatus and Aspergillus niger. We have iteratively improved the structural annotation of Aspergillus genomes through the analysis of publicly available transcription data, mostly expressed sequenced tags, as described in a previous NAR Database article (Arnaud et al. 2012). In this update, we report substantive structural annotation improvements for A. nidulans, A. oryzae and A. fumigatus genomes based on recently available RNA-Seq data. Over 26 000 loci were updated across these species; although those primarily comprise the addition and extension of untranslated regions (UTRs), the new analysis also enabled over 1000 modifications affecting the coding sequence of genes in each target genome.

Improved gene ontology annotation for biofilm formation, filamentous growth, and phenotypic switching in Candida albicans.

The opportunistic fungal pathogen Candida albicans is a significant medical threat, especially for immunocompromised patients. Experimental research has focused on specific areas of C. albicans biology, with the goal of understanding the multiple factors that contribute to its pathogenic potential. Some of these factors include cell adhesion, invasive or filamentous growth, and the formation of drug-resistant biofilms. The Gene Ontology (GO) (www.geneontology.org) is a standardized vocabulary that the Candida Genome Database (CGD) (www.candidagenome.org) and other groups use to describe the functions of gene products. To improve the breadth and accuracy of pathogenicity-related gene product descriptions and to facilitate the description of as yet uncharacterized but potentially pathogenicity-related genes in Candida species, CGD undertook a three-part project: first, the addition of terms to the biological process branch of the GO to improve the description of fungus-related processes; second, manual recuration of gene product annotations in CGD to use the improved GO vocabulary; and third, computational ortholog-based transfer of GO annotations from experimentally characterized gene products, using these new terms, to uncharacterized orthologs in other Candida species. Through genome annotation and analysis, we identified candidate pathogenicity genes in seven non-C. albicans Candida species and in one additional C. albicans strain, WO-1. We also defined a set of C. albicans genes at the intersection of biofilm formation, filamentous growth, pathogenesis, and phenotypic switching of this opportunistic fungal pathogen, which provides a compelling list of candidates for further experimentation.

The Candida genome database incorporates multiple Candida species: multispecies search and analysis tools with curated gene and protein information for Candida albicans and Candida glabrata.

The Candida Genome Database (CGD, http://www.candidagenome.org/) is an internet-based resource that provides centralized access to genomic sequence data and manually curated functional information about genes and proteins of the fungal pathogen Candida albicans and other Candida species. As the scope of Candida research, and the number of sequenced strains and related species, has grown in recent years, the need for expanded genomic resources has also grown. To answer this need, CGD has expanded beyond storing data solely for C. albicans, now integrating data from multiple species. Herein we describe the incorporation of this multispecies information, which includes curated gene information and the reference sequence for C. glabrata, as well as orthology relationships that interconnect Locus Summary pages, allowing easy navigation between genes of C. albicans and C. glabrata. These orthology relationships are also used to predict GO annotations of their products. We have also added protein information pages that display domains, structural information and physicochemical properties; bibliographic pages highlighting important topic areas in Candida biology; and a laboratory strain lineage page that describes the lineage of commonly used laboratory strains. All of these data are freely available at http://www.candidagenome.org/. We welcome feedback from the research community at candida-curator@lists.stanford.edu.

The Aspergillus Genome Database (AspGD): recent developments in comprehensive multispecies curation, comparative genomics and community resources.

The Aspergillus Genome Database (AspGD; http://www.aspgd.org) is a freely available, web-based resource for researchers studying fungi of the genus Aspergillus, which includes organisms of clinical, agricultural and industrial importance. AspGD curators have now completed comprehensive review of the entire published literature about Aspergillus nidulans and Aspergillus fumigatus, and this annotation is provided with streamlined, ortholog-based navigation of the multispecies information. AspGD facilitates comparative genomics by providing a full-featured genomics viewer, as well as matched and standardized sets of genomic information for the sequenced aspergilli. AspGD also provides resources to foster interaction and dissemination of community information and resources. We welcome and encourage feedback at aspergillus-curator@lists.stanford.edu.

ProPhylER: a curated online resource for protein function and structure based on evolutionary constraint analyses.

ProPhylER (Protein Phylogeny and Evolutionary Rates) is a next-generation curated proteome resource that uses comparative sequence analysis to predict constraint and mutation impact for eukaryotic proteins. Its purpose is to inform any research program for which protein function and structure are relevant, by the predictive power of evolutionary constraint analyses. ProPhylER currently has nearly 9000 clusters of related proteins, including more than 200,000 sequences. It serves data via two interfaces. The "ProPhylER Interface" displays predictive analyses in sequence space; the "CrystalPainter" maps evolutionary constraints onto solved protein structures. Here we summarize ProPhylER's data content and analysis pipeline, demonstrate the use of ProPhylER's interfaces, and evaluate ProPhylER's unique regional analysis of evolutionary constraint. The high accuracy of ProPhylER's regional analysis complements the high resolution of its single-site analysis to effectively guide and inform structure-function investigations and predict the impact of polymorphisms.

Comprehensive annotation of secondary metabolite biosynthetic genes and gene clusters of Aspergillus nidulans, A. fumigatus, A. niger and A. oryzae.

BACKGROUND: Secondary metabolite production, a hallmark of filamentous fungi, is an expanding area of research for the Aspergilli. These compounds are potent chemicals, ranging from deadly toxins to therapeutic antibiotics to potential anti-cancer drugs. The genome sequences for multiple Aspergilli have been determined, and provide a wealth of predictive information about secondary metabolite production. Sequence analysis and gene overexpression strategies have enabled the discovery of novel secondary metabolites and the genes involved in their biosynthesis. The Aspergillus Genome Database (AspGD) provides a central repository for gene annotation and protein information for Aspergillus species. These annotations include Gene Ontology (GO) terms, phenotype data, gene names and descriptions and they are crucial for interpreting both small- and large-scale data and for aiding in the design of new experiments that further Aspergillus research. RESULTS: We have manually curated Biological Process GO annotations for all genes in AspGD with recorded functions in secondary metabolite production, adding new GO terms that specifically describe each secondary metabolite. We then leveraged these new annotations to predict roles in secondary metabolism for genes lacking experimental characterization. As a starting point for manually annotating Aspergillus secondary metabolite gene clusters, we used antiSMASH (antibiotics and Secondary Metabolite Analysis SHell) and SMURF (Secondary Metabolite Unknown Regions Finder) algorithms to identify potential clusters in A. nidulans, A. fumigatus, A. niger and A. oryzae, which we subsequently refined through manual curation. CONCLUSIONS: This set of 266 manually curated secondary metabolite gene clusters will facilitate the investigation of novel Aspergillus secondary metabolites.

Functional analyses of variants reveal a significant role for dominant negative and common alleles in oligogenic Bardet-Biedl syndrome.

Technological advances hold the promise of rapidly catalyzing the discovery of pathogenic variants for genetic disease. However, this possibility is tempered by limitations in interpreting the functional consequences of genetic variation at candidate loci. Here, we present a systematic approach, grounded on physiologically relevant assays, to evaluate the mutational content (125 alleles) of the 14 genes associated with Bardet-Biedl syndrome (BBS). A combination of in vivo assays with subsequent in vitro validation suggests that a significant fraction of BBS-associated mutations have a dominant-negative mode of action. Moreover, we find that a subset of common alleles, previously considered to be benign, are, in fact, detrimental to protein function and can interact with strong rare alleles to modulate disease presentation. These data represent a comprehensive evaluation of genetic load in a multilocus disease. Importantly, superimposition of these results to human genetics data suggests a previously underappreciated complexity in disease architecture that might be shared among diverse clinical phenotypes.

How to Use the Candida Genome Database.

The Candida Genome Database provides access to biological information about genes and proteins of several medically important Candida species. The website is organized into easily navigable pages that enable data retrieval and analysis. This chapter shows how to explore the CGD Home page and Locus Summary pages, which are the main access points to the database. It also provides a description of how to use the GO analysis tools, GO Term Finder, and GO Slim Mapper and how to browse large-scale datasets using the JBrowse genome browser. Finally, it shows how to search and retrieve data for user-defined sets of genes using the Advanced Search and Batch Download tools.

Identification of the Otopetrin Domain, a conserved domain in vertebrate otopetrins and invertebrate otopetrin-like family members.

BACKGROUND: Otopetrin 1 (Otop1) encodes a multi-transmembrane domain protein with no homology to known transporters, channels, exchangers, or receptors. Otop1 is necessary for the formation of otoconia and otoliths, calcium carbonate biominerals within the inner ear of mammals and teleost fish that are required for the detection of linear acceleration and gravity. Vertebrate Otop1 and its paralogues Otop2 and Otop3 define a new gene family with homology to the invertebrate Domain of Unknown Function 270 genes (DUF270; pfam03189). RESULTS: Multi-species comparison of the predicted primary sequences and predicted secondary structures of 62 vertebrate otopetrin, and arthropod and nematode DUF270 proteins, has established that the genes encoding these proteins constitute a single family that we renamed the Otopetrin Domain Protein (ODP) gene family. Signature features of ODP proteins are three "Otopetrin Domains" that are highly conserved between vertebrates, arthropods and nematodes, and a highly constrained predicted loop structure. CONCLUSION: Our studies suggest a refined topologic model for ODP insertion into the lipid bilayer of 12 transmembrane domains, and highlight conserved amino-acid residues that will aid in the biochemical examination of ODP family function. The high degree of sequence and structural similarity of the ODP proteins may suggest a conserved role in the intracellular trafficking of calcium and the formation of biominerals.

Using the Candida Genome Database.

Studying Candida biology requires access to genomic sequence data in conjunction with experimental information that together provide functional context to genes and proteins, and aid in interpreting newly generated experimental data. The Candida Genome Database (CGD) curates the Candida literature, and integrates functional information about Candida genes and their products with a set of analysis tools that facilitate searching for sets of genes and exploring their biological roles. This chapter describes how the various types of information available at CGD can be searched, retrieved, and analyzed. Starting with the guided tour of the CGD Home page and Locus Summary page, this unit shows how to navigate the various assemblies of the C. albicans genome, how to use Gene Ontology tools to make sense of large-scale data, and how to access the microarray data archived at CGD, as well as visualize high-throughput sequencing data through the use of JBrowse.

Structural and molecular evolutionary analysis of Agouti and Agouti-related proteins.

Agouti (ASIP) and Agouti-related protein (AgRP) are endogenous antagonists of melanocortin receptors that play critical roles in the regulation of pigmentation and energy balance, respectively, and which arose from a common ancestral gene early in vertebrate evolution. The N-terminal domain of ASIP facilitates antagonism by binding to an accessory receptor, but here we show that the N-terminal domain of AgRP has the opposite effect and acts as a prodomain that negatively regulates antagonist function. Computational analysis reveals similar patterns of evolutionary constraint in the ASIP and AgRP C-terminal domains, but fundamental differences between the N-terminal domains. These studies shed light on the relationships between regulation of pigmentation and body weight, and they illustrate how evolutionary structure function analysis can reveal both unique and common mechanisms of action for paralogous gene products.

A novel His158Arg mutation in TIMP3 causes a late-onset form of Sorsby fundus dystrophy.

PURPOSE: To describe the phenotype and genotype of a family with suspected Sorsby fundus dystrophy (SFD). DESIGN: Case reports and results of deoxyribonucleic acid (DNA) analysis. METHODS: Clinical features were determined by complete ophthalmologic examination or by review of medical records. Mutational analysis of the tissue inhibitor of metalloproteinase (TIMP)3 gene was performed by DNA resequencing. Biochemical properties of the mutant TIMP3 protein were studied, and phylogenetic and molecular modeling analyses of TIMP proteins were performed. RESULTS: Fundi of four affected family members demonstrated active or regressed bilateral choroidal neovascularization, whereas another affected individual displayed severe diffuse pigmentary degeneration associated with nyctalopia characteristic of SFD. Onset of disease occurred in the fifth to seventh decades of life. A heterozygous His158Arg mutation was found in seven affected family members and was absent from an unaffected member and 98 unrelated controls. Bioinformatic analyses indicate that histidine 158 is an evolutionarily conserved residue in most vertebrate TIMP homologs and predict that substitution by arginine disrupts TIMP3 function. The mutant protein appears to be expressed by fibroblasts from an affected family member. Molecular modeling suggests that TIMP3 residue 158 may be part of a protein-protein interaction interface. CONCLUSION: A novel mutation in TIMP3 causes a late-onset form of SFD in this family. His158Arg is the first reported TIMP3 SFD coding sequence mutation that does not create an unpaired cysteine. Further study of this unusual mutation may provide insight into the mechanism of SFD pathogenesis.

How to Use the Candida Genome Database.

Studying Candida biology requires access to genomic sequence data in conjunction with experimental information that provides functional context to genes and proteins. The Candida Genome Database (CGD) integrates functional information about Candida genes and their products with a set of analysis tools that facilitate searching for sets of genes and exploring their biological roles. This chapter describes how the various types of information available at CGD can be searched, retrieved, and analyzed. Starting with the guided tour of the CGD Home page and Locus Summary page, this unit shows how to navigate the various assemblies of the C. albicans genome, how to use Gene Ontology tools to make sense of large-scale data, and how to access the microarray data archived at CGD.

A cell cycle phosphoproteome of the yeast centrosome.

Centrosomes organize the bipolar mitotic spindle, and centrosomal defects cause chromosome instability. Protein phosphorylation modulates centrosome function, and we provide a comprehensive map of phosphorylation on intact yeast centrosomes (18 proteins). Mass spectrometry was used to identify 297 phosphorylation sites on centrosomes from different cell cycle stages. We observed different modes of phosphoregulation via specific protein kinases, phosphorylation site clustering, and conserved phosphorylated residues. Mutating all eight cyclin-dependent kinase (Cdk)-directed sites within the core component, Spc42, resulted in lethality and reduced centrosomal assembly. Alternatively, mutation of one conserved Cdk site within γ-tubulin (Tub4-S360D) caused mitotic delay and aberrant anaphase spindle elongation. Our work establishes the extent and complexity of this prominent posttranslational modification in centrosome biology and provides specific examples of phosphorylation control in centrosome function.

The integrity of a cholesterol-binding pocket in Niemann-Pick C2 protein is necessary to control lysosome cholesterol levels.

The neurodegenerative disease Niemann-Pick Type C2 (NPC2) results from mutations in the NPC2 (HE1) gene that cause abnormally high cholesterol accumulation in cells. We find that purified NPC2, a secreted soluble protein, binds cholesterol specifically with a much higher affinity (K(d) = 30-50 nM) than previously reported. Genetic and biochemical studies identified single amino acid changes that prevent both cholesterol binding and the restoration of normal cholesterol levels in mutant cells. The amino acids that affect cholesterol binding surround a hydrophobic pocket in the NPC2 protein structure, identifying a candidate sterol-binding location. On the basis of evolutionary analysis and mutagenesis, three other regions of the NPC2 protein emerged as important, including one required for efficient secretion.