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FlyBase provides a centralized resource for the genetic and genomic data of Drosophila melanogaster. As FlyBase enters our fourth decade of service to the research community, we reflect on our unique aspects and look forward to our continued collaboration with the larger research and model organism communities. In this study, we emphasize the dedicated reports and tools we have constructed to meet the specialized needs of fly researchers but also to facilitate use by other research communities. We also highlight ways that we support the fly community, including an external resources page, help resources, and multiple avenues by which researchers can interact with FlyBase.

The Alliance of Genome Resources (Alliance) is a consortium of the major model organism databases and the Gene Ontology that is guided by the vision of facilitating exploration of related genes in human and well-studied model organisms by providing a highly integrated and comprehensive platform that enables researchers to leverage the extensive body of genetic and genomic studies in these organisms. Initiated in 2016, the Alliance is building a central portal (www.alliancegenome.org) for access to data for the primary model organisms along with gene ontology data and human data. All data types represented in the Alliance portal (e.g. genomic data and phenotype descriptions) have common data models and workflows for curation. All data are open and freely available via a variety of mechanisms. Long-term plans for the Alliance project include a focus on coverage of additional model organisms including those without dedicated curation communities, and the inclusion of new data types with a particular focus on providing data and tools for the non-model-organism researcher that support enhanced discovery about human health and disease. Here we review current progress and present immediate plans for this new bioinformatics resource.

Since 1992, FlyBase (flybase.org) has been an essential online resource for the Drosophila research community. Concentrating on the most extensively studied species, Drosophila melanogaster, FlyBase includes information on genes (molecular and genetic), transgenic constructs, phenotypes, genetic and physical interactions, and reagents such as stocks and cDNAs. Access to data is provided through a number of tools, reports, and bulk-data downloads. Looking to the future, FlyBase is expanding its focus to serve a broader scientific community. In this update, we describe new features, datasets, reagent collections, and data presentations that address this goal, including enhanced orthology data, Human Disease Model Reports, protein domain search and visualization, concise gene summaries, a portal for external resources, video tutorials and the FlyBase Community Advisory Group.

We report the current status of the FlyBase annotated gene set for Drosophila melanogaster and highlight improvements based on high-throughput data. The FlyBase annotated gene set consists entirely of manually annotated gene models, with the exception of some classes of small non-coding RNAs. All gene models have been reviewed using evidence from high-throughput datasets, primarily from the modENCODE project. These datasets include RNA-Seq coverage data, RNA-Seq junction data, transcription start site profiles, and translation stop-codon read-through predictions. New annotation guidelines were developed to take into account the use of the high-throughput data. We describe how this flood of new data was incorporated into thousands of new and revised annotations. FlyBase has adopted a philosophy of excluding low-confidence and low-frequency data from gene model annotations; we also do not attempt to represent all possible permutations for complex and modularly organized genes. This has allowed us to produce a high-confidence, manageable gene annotation dataset that is available at FlyBase (http://flybase.org). Interesting aspects of new annotations include new genes (coding, non-coding, and antisense), many genes with alternative transcripts with very long 3′ UTRs (up to 15–18 kb), and a stunning mismatch in the number of male-specific genes (approximately 13% of all annotated gene models) vs. female-specific genes (less than 1%). The number of identified pseudogenes and mutations in the sequenced strain also increased significantly. We discuss remaining challenges, for instance, identification of functional small polypeptides and detection of alternative translation starts.

In the context of the FlyBase annotated gene models in Drosophila melanogaster, we describe the many exceptional cases we have curated from the literature or identified in the course of FlyBase analysis. These range from atypical but common examples such as dicistronic and polycistronic transcripts, noncanonical splices, trans-spliced transcripts, noncanonical translation starts, and stop-codon readthroughs, to single exceptional cases such as ribosomal frameshifting and HAC1-type intron processing. In FlyBase, exceptional genes and transcripts are flagged with Sequence Ontology terms and/or standardized comments. Because some of the rule-benders create problems for handlers of high-throughput data, we discuss plans for flagging these cases in bulk data downloads.

FlyBase (flybase.org) is the primary online database of genetic, genomic, and functional information about Drosophila species, with a major focus on the model organism Drosophila melanogaster. The long and rich history of Drosophila research, combined with recent surges in genomic-scale and high-throughput technologies, mean that FlyBase now houses a huge quantity of data. Researchers need to be able to rapidly and intuitively query these data, and the QuickSearch tool has been designed to meet these needs. This tool is conveniently located on the FlyBase homepage and is organized into a series of simple tabbed interfaces that cover the major data and annotation classes within the database. This unit describes the functionality of all aspects of the QuickSearch tool. With this knowledge, FlyBase users will be equipped to take full advantage of all QuickSearch features and thereby gain improved access to data relevant to their research. © 2016 by John Wiley & Sons, Inc.

Whole genome sequencing of the model organisms has created increased demand for efficient tools to facilitate the genome annotation efforts. Accordingly, we report the further implementations and analyses stemming from our publicly available P{wHy} library for Drosophila melanogaster. A two-step regime—large scale transposon mutagenesis followed by hobo-induced nested deletions—allows mutation saturation and provides significant enhancements to existing genomic coverage. We previously showed that, for a given starting insert, deletion saturation is readily obtained over a 60-kb interval; here, we perform a breakdown analysis of efficiency to identify rate-limiting steps in the process. Transrecombination, the hobo-induced recombination between two P{wHy} half molecules, was shown to further expand the P{wHy} mutational range, pointing to a potent, iterative process of transrecombination–reconstitution–transrecombination for alternating between very large and very fine-grained deletions in a self-contained manner. A number of strains also showed partial or complete repression of P{wHy} markers, depending on chromosome location, whereby asymmetric marker silencing allowed continuous phenotypic detection, indicating that P{wHy}-based saturational mutagenesis should be useful for the study of heterochromatin/positional effects.

We have sequenced the genome of a second Drosophila species, Drosophila pseudoobscura, and compared this to the genome sequence of Drosophila melanogaster, a primary model organism. Throughout evolution the vast majority of Drosophila genes have remained on the same chromosome arm, but within each arm gene order has been extensively reshuffled, leading to a minimum of 921 syntenic blocks shared between the species. A repetitive sequence is found in the D. pseudoobscura genome at many junctions between adjacent syntenic blocks. Analysis of this novel repetitive element family suggests that recombination between offset elements may have given rise to many paracentric inversions, thereby contributing to the shuffling of gene order in the D. pseudoobscura lineage. Based on sequence similarity and synteny, 10,516 putative orthologs have been identified as a core gene set conserved over 25–55 million years (Myr) since the pseudoobscura/melanogaster divergence. Genes expressed in the testes had higher amino acid sequence divergence than the genome-wide average, consistent with the rapid evolution of sex-specific proteins. Cis-regulatory sequences are more conserved than random and nearby sequences between the species—but the difference is slight, suggesting that the evolution of cis-regulatory elements is flexible. Overall, a pattern of repeat-mediated chromosomal rearrangement, and high coadaptation of both male genes and cis-regulatory sequences emerges as important themes of genome divergence between these species of Drosophila.

With the available eukaryotic genome sequences, there are predictions of thousands of previously uncharacterized genes without known function or available mutational variant. Thus, there is an urgent need for efficient genetic tools for genomewide phenotypic analysis. Here we describe such a tool: a deletion-generator technology that exploits properties of a double transposable element to produce molecularly defined deletions at high density and with high efficiency. This double element, called P { wHy }, is composed of a “deleter” element hobo , bracketed by two genetic markers and inserted into a “carrier” P element. We have used this P { wHy } element in Drosophila melanogaster to generate sets of nested deletions of sufficient coverage to discriminate among every transcription unit within 60 kb of the starting insertion site. Because these two types of mobile elements, carrier and deleter, can be found in other species, our strategy should be applicable to phenotypic analysis in a variety of model organisms.

Release 6, the latest reference genome assembly of the fruit fly Drosophila melanogaster, was released by the Berkeley Drosophila Genome Project in 2014; it replaces their previous Release 5 genome assembly, which had been the reference genome assembly for over 7 years. With the enormous amount of information now attached to the D. melanogaster genome in public repositories and individual laboratories, the replacement of the previous assembly by the new one is a major event requiring careful migration of annotations and genome-anchored data to the new, improved assembly. In this report, we describe the attributes of the new Release 6 reference genome assembly, the migration of FlyBase genome annotations to this new assembly, how genome features on this new assembly can be viewed in FlyBase (http://flybase.org) and how users can convert coordinates for their own data to the corresponding Release 6 coordinates.

The genes of the trithorax group (trxG) in Drosophila melanogaster are required to maintain the pattern of homeotic gene expression that is established early in embryogenesis by the transient expression of the segmentation genes. The precise role of each of the diverse trxG members and the functional relationships among them are not well understood. Here, we report on the isolation of the trxG gene moira (mor) and its molecular characterization. mor encodes a fruit fly homolog of the human and yeast chromatin-remodeling factors BAF170, BAF155, and SWI3. mor is widely expressed throughout development, and its 170-kDa protein product is present in many embryonic tissues. In vitro, MOR can bind to itself and it interacts with Brahma (BRM), an SWI2-SNF2 homolog, with which it is associated in embryonic nuclear extracts. The leucine zipper motif of MOR is likely to participate in self-oligomerization; the equally conserved SANT domain, for which no function is known, may be required for optimal binding to BRM. MOR thus joins BRM and Snf5-related 1 (SNR1), two known Drosophila SWI-SNF subunits that act as positive regulators of the homeotic genes. These observations provide a molecular explanation for the phenotypic and genetic relationships among several of the trxG genes by suggesting that they encode evolutionarily conserved components of a chromatin-remodeling complex.

FlyBase is a database of genetic and molecular data concerning Drosophila. FlyBase is maintained as a relational database (in Sybase) and is made available as html documents and flat files. The scope of FlyBase includes: genes, alleles (and phenotypes), aberrations, transposons, pointers to sequence data, clones, stock lists, Drosophila workers and bibliographic references. The Encyclopedia of Drosophila is a joint effort between FlyBase and the Berkeley Drosophila Genome Project which integrates FlyBase data with those from the BDGP.

The availability of sequenced genomes from 12 Drosophila species has enabled the use of comparative genomics for the systematic discovery of functional elements conserved within this genus. We have developed quantitative metrics for the evolutionary signatures specific to protein-coding regions and applied them genome-wide, resulting in 1193 candidate new protein-coding exons in the D. melanogaster genome. We have reviewed these predictions by manual curation and validated a subset by directed cDNA screening and sequencing, revealing both new genes and new alternative splice forms of known genes. We also used these evolutionary signatures to evaluate existing gene annotations, resulting in the validation of 87% of genes lacking descriptive names and identifying 414 poorly conserved genes that are likely to be spurious predictions, noncoding, or species-specific genes. Furthermore, our methods suggest a variety of refinements to hundreds of existing gene models, such as modifications to translation start codons and exon splice boundaries. Finally, we performed directed genome-wide searches for unusual protein-coding structures, discovering 149 possible examples of stop codon readthrough, 125 new candidate ORFs of polycistronic mRNAs, and several candidate translational frameshifts. These results affect >10% of annotated fly genes and demonstrate the power of comparative genomics to enhance our understanding of genome organization, even in a model organism as intensively studied as Drosophila melanogaster.

FlyBase (http://flybase.org/) is the primary database of genetic and genomic data for the insect family Drosophilidae. Historically, Drosophila melanogaster has been the most extensively studied species in this family, but recent determination of the genomic sequences of an additional 11 Drosophila species opens up new avenues of research for other Drosophila species. This extensive sequence resource, encompassing species with well-defined phylogenetic relationships, provides a model system for comparative genomic analyses. FlyBase has developed tools to facilitate access to and navigation through this invaluable new data collection.

Background: The recent completion of the Drosophila melanogaster genomic sequence to high quality and the availability of a greatly expanded set of Drosophila cDNA sequences, aligning to 78% of the predicted euchromatic genes, afforded FlyBase the opportunity to significantly improve genomic annotations. We made the annotation process more rigorous by inspecting each gene visually, utilizing a comprehensive set of curation rules, requiring traceable evidence for each gene model, and comparing each predicted peptide to SWISS-PROT and TrEMBL sequences. Results: Although the number of predicted protein-coding genes in Drosophila remains essentially unchanged, the revised annotation significantly improves gene models, resulting in structural changes to 85% of the transcripts and 45% of the predicted proteins. We annotated transposable elements and non-protein-coding RNAs as new features, and extended the annotation of untranslated (UTR) sequences and alternative transcripts to include more than 70% and 20% of genes, respectively. Finally, cDNA sequence provided evidence for dicistronic transcripts, neighboring genes with overlapping UTRs on the same DNA sequence strand, alternatively spliced genes that encode distinct, non-overlapping peptides, and numerous nested genes. Conclusions: Identification of so many unusual gene models not only suggests that some mechanisms for gene regulation are more prevalent than previously believed, but also underscores the complex challenges of eukaryotic gene prediction. At present, experimental data and human curation remain essential to generate high-quality genome annotations.

The well-established inaccuracy of purely computational methods for annotating genome sequences necessitates an interactive tool to allow biological experts to refine these approximations by viewing and independently evaluating the data supporting each annotation. Apollo was developed to meet this need, enabling curators to inspect genome annotations closely and edit them. FlyBase biologists successfully used Apollo to annotate the Drosophila melanogaster genome and it is increasingly being used as a starting point for the development of customized annotation editing tools for other genome projects.

We have conducted a genetic analysis of the region flanking the 68C glue gene cluster in Drosophila melanogaster by isolating lethal and semilethal mutations uncovered by deficiencies which span this region. Three different mutagens were used: ethyl methanesulfonate (EMS), ethyl nitrosourea (ENU) and diepoxybutane (DEB). In the region from 68A3 to 68C11, 64 lethal, semilethal, and visible mutations were recovered. These include alleles of 13 new lethal complementation groups, as well as new alleles of rotated, low xanthine dehydrogenase, lethal(3)517 and lethal(3)B76. Six new visible mutations from within this region were recovered on the basis of their reduced viability; all proved to be semiviable alleles of lethal complementation groups. No significant differences were observed in the distributions of lethals recovered using the three different mutagens. Each lethal was mapped on the basis of complementation with overlapping deficiencies; mutations that mapped within the same interval were tested for complementation, and the relative order of the lethal groups within each interval was determined by recombination. The cytological distribution of genes within the 68A3-68C11 region is not uniform: the region from 68A2,3 to 68B1,3 (seven to ten polytene chromosome bands) contains at least 13 lethal complementation groups and the mutation low xanthine dehydrogenase; the adjoining region from 68B1,3 to 68C5,6 (six to nine bands) includes the 68C glue gene cluster, but no known lethal or visible complementation groups; and the interval from 68C5,6 to 68C10,11 (three to five bands) contains at least three lethal complementation groups and the visible mutation rotated. The developmental stage at which lethality is observed was determined for a representative allele from each lethal complementation group.

The 68C intermolt puff of Drosophila melanogaster contains a cluster of three glue protein genes, Sgs-3, Sgs-7, and Sgs-8. By analysis of chromosomal rearrangements which break near the glue gene cluster, we have established that a region of no more than 20 kb is required for normal expression of the glue genes and for formation of the 68C puff. Using P element-mediated transformation, we have introduced defined segments of the 68C region into the fly genome and assayed the expression of the Sgs-3 gene. Based on the criteria of correct tissue- and stage-specific expression, transcription of an RNA of appropriate size and abundance, and production of an sgs-3 protein, the correctly regulated expression of the Sgs-3 gene requires less than 3.4 kb of total flanking sequences, approximately 2.3 kb 5' and 1.1 kb 3'. Formation of a new intermolt puff at the site of insertion is not observed for all transformants which produce high levels of Sgs-3 RNA. Only transformants in which the introduced DNA from 68C also contains the Sgs-7 and Sgs-8 genes cause a new intermolt puff at the chromosomal location of the insert.

The homeotic mutations in the right half of the bithorax complex of Drosophila cause segmental transformations in the second through the eighth segments of the fly. A chromosomal walk in the bithorax complex has now been extended 215 kb through the right half of the complex, and lesions for over 40 mutations have been located on the DNA map. The mutations can be grouped in a series of phenotypic classes, one for each abdominal segment, although each mutation typically affects more than one segment. The mutant lesions of each class are clustered, and they are aligned on the chromosome in the order of the body segments that they affect. Complementation tests suggest interactions between widely spaced DNA regions; indeed, the right half cannot be split anywhere without some loss of function.

The Abdominal-B (Abd-B) gene of Drosophila controls the specification of segment identities in the posterior abdomen. We describe here the spatial and temporal distribution of Abd-B transcripts. At least five RNA species are detected on Northern blots with probes within the region of the Abd-B homeobox. We have identified probes specific for subsets of these transcripts and have used these probes to study the distribution of each subset by in situ hybridization to embryonic tissue sections. The transcripts can be divided into two groups: one group is expressed maximally in parasegment (PS)13 and extends anteriorly to PS10; the other is expressed only in PS14 and 15. These two different patterns of expression correspond to the anatomical domains defined by two classes of mutations in Abd-B. These results help explain the complex genetic interactions and phenotypes of mutations within this gene.

The Drosophila melanogaster 68C chromosomal locus is the site of a prominent polytene chromosome puff that harbors the genes Sgs-3, Sgs-7 and Sgs-8. These genes code for proteins that are part of the salivary glue that Drosophila larvae secrete as a means of fixing themselves to an external substrate for the duration of the pre-pupal and pupal period. The 68C glue genes are regulated by the steroid hormone ecdysterone, with the hormone required for both initiation and cessation of gene expression during the third larval instar. Previous work has defined sequences sufficient for expression of abundant levels of Sgs-3 mRNA at the correct time and in the correct tissue. We show here that sequences sufficient for normal tissue- and stage-specific accumulation of Sgs-3 RNA, but adequate only for low levels of expression, lie within 130 bp of the 5' end of the gene, or within the gene.

Publisher Summary This chapter discusses steroid-regulated vertebrate genes. The most extensively studied steroid-responsive gene is the mouse mammary tumor virus (MMTV) provirus. In a variety of cell types, chromosomally inserted proviruses give rise to a single transcript, initiating within the left long terminal repeat (LTR). This transcript is present at very low levels until the cells are exposed to a glucocorticoid hormone. Within minutes following hormone treatment, the titre of the MMTV transcript rises, reaching a level that is at least 50-fold above the basal level within 2 hours. Glucocorticoid receptor binding sites are located within the MMTV LTR, and the same region of the LTR is both necessary and sufficient to confer glucocorticoid inducibility. Receptor binding sites are clustered between 80 and 300 base pairs upstream of the transcriptional initiation sites, and additional sites are present at several positions internal to the transcript. The chapter discusses ecdysteroid-responsive genes from drosophila and glue genes.

Drosophila melanogaster begins its life as a fertilized egg, which undergoes embryogenesis within an egg shell for about a day and then hatches from the egg as a first instar larva. The wormlike larva eats yeast for a day, then sheds its first instar skin and crawls out as a second instar larva. One day later another molt occurs, resulting in a third instar larva. The third instar stage lasts approximately 40 hours at 25 °C toward the end of this stage the larva crawls out of its medium and onto a dry surface. After a few hours of wandering, it stops, contracts, and secretes a protein glue from its salivary glands that hardens and attaches the larva to its substrate (Fraenkel and Brookes 1953). The affixed larva then tans its third larval instar cuticle into a puparial case, and some hours later pupates within this case. Complete metamorphosis follows pupation, and after several days the pupal case is opened and a new adult emerges.