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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.

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.

The lark gene was initially identified in a behavioral screen for flies that displayed circadian rhythm defects (Newby and Jackson, 1993, 1996). It encodes an RNAbinding protein of the RNA Recognition Motif (RRM) class. Whereas lark heterozygotes exhibit a rhythm defect, homozygotes die during embryonic development because of a zygotic requirement for the gene product. Subsequently, it was shown that there is also a maternal requirement for lark function during oogenesis and early embryonic development (McNeil et al., 1999). Currently, several fly labs are pursuing studies of LARK function as related to oogenesis and embryonic development. LARK protein contains three potential RNA binding domains, two RNA recognition motifs, and a single retroviral type zinc finger, that have been shown to be important for LARK function during development but appear to play functionally redundant roles in the circadian regulation of eclosion (Newby and Jackson, 1996; McNeil et al., 2000). The LARK protein is widely expressed in most if not all tissues and throughout development (Zhang et al., 2000). In the majority of cells that express LARK, the protein has a nuclear localization. A notable exception occurs in a subset of neurosecretory cells that express the neuropeptide crustacean cardioactive peptide (CCAP), in which LARK is cytoplasmic. Of interest for the rhythm phenotype, CCAP cells exhibit circadian changes in LARK abundance (Zhang et al., 2000). In order to further characterize LARK protein function we generated pP{UAS-lark T:Ivir }(UAS-lark-3HA, Fig. 1, panel a). To distinguish GAL4 driven expression of the UAS-lark transgene from endogenous LARK expression, sequences encoding a triple hemagglutinin (HA) epitope were incorporated into the construct immediately prior to the LARK stop codon. Seven independent UAS-lark3HA transgenic lines were generated and here we report the preliminary characterization of three of these lines. We employed one line carrying a second chromosome insertion (w; P{w mC UAS-lark }94A) and two lines with independent third chromosome insertions (w; P{w mC UAS-lark }9A and (w; P{w mC UAS-lark }23A). All three lines are homozygous viable and fertile. To examine the effect of LARK overexpression, we first used the strong ubiquitous Act5C-GAL4 driver to drive expression of UAS-lark-3HA transgenes. Flies carrying an Act5C-GAL4 third chromosome insert (balanced over TM6b, Tb) were crossed to flies homozygous for a UAS-lark-3HA transgene. Adults carrying both the Act5C-GAL4 driver and any of the three UAS-lark-3HA transgenes were never observed; i.e., the only adult progeny that resulted from these crosses carried the TM6b, Tb chromosome (Table 1). In the case of two of the UAS-lark-3HA lines, 94A and 9A, some of the LARK overexpressing flies were able to develop to the early pupal stage (as indicated by a few Tb pupae in the vials; data not shown). However, these individuals ceased to develop soon after pupal formation and no brown or black Tb pupae were observed in these crosses. In the case of crosses with the 23A UAS-lark-3HA line, Tb pupae were never observed, suggesting that ubiquitous overexpression of LARK from this insertion is lethal prior to pupation. To determine if the observed lethality was caused by increased LARK protein expression in the nervous system, we used an elav-GAL4 driver (c155) to express UAS-lark-3HA transgenes in all differentiated neurons. Such a pan-neural overexpression of LARK caused lethality, the severity of which was dependent on the particular UAS-lark-3HA line employed in crosses. For example, crosses of males bearing the X-linked elav-GAL4 driver to females homozygous for the 23A UAS-lark-3HA transgene failed to produce any female offspring (Table 1). In similar crosses with elav-GAL4 and either the 94A or 9A UAS-lark-3HA transgene, GAL4/UAS flies were observed to eclose at a reduced frequency compared to UAS sibling controls (Table 1). Of the GAL4/UAS flies that do eclose, all display a cuticle-tanning defect characterized by a significant delay in cuticle hardening and pigmentation (not seen in control flies). In addition, the GAL4/UAS flies do not show normal wing expansion (Fig. 1, panel b). With the 94A UAS-lark-3HA transgene, none of the GAL4/UAS flies ever expanded their wings. Approximately 2% of the GAL4/UAS flies displayed nor

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.

Genetic analysis in Drosophila melanogaster has identified molecules important for the function of insect circadian clocks, and this has resulted in the elaboration of explicit biochemical models of the clock mechanism. Comparable molecular genetic analysis coupled with neuroanatomical approaches has also delineated cellular elements of the circadian pacemaker controlling insect activity rhythms. However, not much is known about the transfer of temporal information from clock cells in the insect brain to downstream neural elements or other target cells that are regulated by the clock (i.e. clock output pathways). In this review, we focus on the insect literature, with special reference to the fruitfly D. melanogaster and the hawkmoth Manduca sexta, to discuss the candidate molecules, biochemical mechanisms and cell types implicated in the clock control of behavior.

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.

Past studies have implicated the Drosophila LARK protein in the circadian control of adult eclosion behavior. LARK has a broad tissue pattern of distribution, and is pan-neuronal in the differentiated brain. In certain peptidergic neurons, LARK abundance changes in a circadian manner. However, the precise cellular requirement for LARK, with respect to circadian behavior, is still not known. To explore this issue, we employed the GAL4/UAS binary expression system to increase LARK abundance in defined neuronal cell types. Interestingly, LARK expression in Crustacean Cardioactive Peptide (CCAP) neurons caused an early-eclosion phenotype, whereas a similar perturbation in the Eclosion Hormone (EH) cells resulted in abnormally late peaks of eclosion. Surprisingly, LARK expression in Pigment Dispersing Factor (PDF)- or TIMELESS (TIM)-containing clock neurons caused behavioral arrhythmicity, even though clock protein cycling was found to be normal in these flies. Although the observed effects of LARK expression mirrored those seen with genetic ablation of the relevant peptidergic populations, there was no evidence of defective cell development or morphology. This suggests that an alteration of cell function rather than cell death is the cause of the aberrant phenotypes. Diminished PDF immunoreactivity in flies expressing LARK in the PDF neurons suggests that an effect on neuropeptide synthesis, transport, or release may contribute to the observed arrhythmicity. Importantly, the expression of LARK in several other cell populations did not have detectable effects on development, viability or behavior, indicating a specificity of action within certain cell types.

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.