Comparative analysis of multiple genomes in a phylogenetic framework dramatically improves the precision and sensitivity of evolutionary inference, producing more robust results than single-genome analyses can provide. The genomes of 12 Drosophila species, ten of which are presented here for the first time (sechellia, simulans, yakuba, erecta, ananassae, persimilis, willistoni, mojavensis, virilis and grimshawi), illustrate how rates and patterns of sequence divergence across taxa can illuminate evolutionary processes on a genomic scale. These genome sequences augment the formidable genetic tools that have made Drosophila melanogaster a pre-eminent model for animal genetics, and will further catalyse fundamental research on mechanisms of development, cell biology, genetics, disease, neurobiology, behaviour, physiology and evolution. Despite remarkable similarities among these Drosophila species, we identified many putatively non-neutral changes in protein-coding genes, non-coding RNA genes, and cis-regulatory regions. These may prove to underlie differences in the ecology and behaviour of these diverse species.

FlyBase (flybase.org) is a knowledge base that supports the community of researchers that use the fruit fly, Drosophila melanogaster, as a model organism. The FlyBase team curates and organizes a diverse array of genetic, molecular, genomic, and developmental information about Drosophila. At the beginning of 2018, ‘FlyBase 2.0’ was released with a significantly improved user interface and new tools. Among these important changes are a new organization of search results into interactive lists or tables (hitlists), enhanced reference lists, and new protein domain graphics. An important new data class called ‘experimental tools’ consolidates information on useful fly strains and other resources related to a specific gene, which significantly enhances the ability of the Drosophila researcher to design and carry out experiments. With the release of FlyBase 2.0, there has also been a restructuring of backend architecture and a continued development of application programming interfaces (APIs) for programmatic access to FlyBase data. In this review, we describe these major new features and functionalities of the FlyBase 2.0 site and how they support the use of Drosophila as a model organism for biological discovery and translational research.

Over-replication of two clusters of chorion genes in Drosophila ovarian follicle cells is essential for rapid eggshell biosynthesis. The relationship of this amplification to the follicle cell cycles has remained unclear. To investigate the regulation of amplification, we developed a technique to detect amplifying chorion genes in individual follicle cells using BrdU incorporation and FISH. Amplification occurs in two developmental phases. One of the gene clusters begins to amplify periodically during S phases of follicle cell endocycles. Subsequently, after endocycles have ceased, both clusters amplify continuously during the remainder of oogenesis. In contrast to the early phase, late amplification commences synchronously among follicle cells. The pattern of Cyclin E expression mirrors these two phases. We present evidence that Cyclin E is required positively for amplification. We suggest that Cyclin E also acts negatively to inhibit refiring of most origins within a cycle, and that specific factors at chorion origins allow them to escape this negative rereplication control. Our findings suggest that chorion amplification is a model for understanding metazoan replicons and the controls that restrict replication to once per cell cycle.[Key Words: Drosophila; oogenesis; chorion; amplification; replication; cyclin E] Received November 10, 1997; revised version accepted January 15, 1998.To maintain euploid gene balance, DNA sequences must be replicated every cell cycle but not more than once. Recent evidence indicates that cell cycle control of DNA replication is effected by a two-step mechanism (for review, see Diffley 1996). Origins first become competent to replicate by assembling proteins comprising prereplication complexes onto chromatin in G 1 , and then later, during S, those origins initiate replication (Diffley et al. 1994). Replication from or through an origin dissociates functional prereplication complexes. Once destroyed, these complexes cannot reassemble until the subsequent G 1 , thereby precluding refiring of an origin in a single cycle. Several lines of evidence suggest that cyclin dependent kinases (CDKs), in addition to being required positively for cell cycle progression, act negatively and are responsible for blocking reassembly of replication complexes in S, G 2 , and M (Broek et al. 1991;Hayles et al. 1994;Moreno and Nurse 1994;Dahmann et al. 1995;Sauer 1995;Hua et al. 1997;Jallepalli et al. 1997;Tanaka et al. 1997). It is only after passage through mitosis, during a period in G 1 when kinase levels are low, that complexes can reassemble onto chromatin. This two-step mechanism of assembly and firing linked to kinase levels ensures that each region of the genome is replicated only once per cycle.In Drosophila melanogaster, as in many multicellular eukaryotes including humans, certain tissues become polyploid by entering an endocycle characterized by alternating S and G phases without intervening mitoses (for review, see Carminati and Orr-Weaver 1996). As in other cycles, Cyclin E (CycE), with...

It is widely believed that DNA replication in multicellular animals (metazoa) begins at specific origins to which a pre-replicative complex (pre-RC) binds. Nevertheless, a consensus sequence for origins has yet to be identified in metazoa. Origin identity can change during development, suggesting that there are epigenetic influences. A notable example of developmental specificity occurs in Drosophila, where somatic follicle cells of the ovary transition from genomic replication to exclusive re-replication at origins that control amplification of the eggshell (chorion) protein genes. Here we show that chromatin acetylation is critical for this developmental transition in origin specificity. We find that histones at the active origins are hyperacetylated, coincident with binding of the origin recognition complex (ORC). Mutation of the histone deacetylase (HDAC) Rpd3 induced genome-wide hyperacetylation, genomic replication and a redistribution of the origin-binding protein ORC2 in amplification-stage cells, independent of effects on transcription. Tethering Rpd3 or Polycomb proteins to the origin decreased its activity, whereas tethering the Chameau acetyltransferase increased origin activity. These results suggest that nucleosome acetylation and other epigenetic changes are important modulators of origin activity in metazoa.

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Initiation of DNA replication at origins more than once per cell cycle results in rereplication and has been implicated in cancer. Here we use Drosophila to examine the checkpoint responses to rereplication in a developmental context. We find that increased Double-parked (Dup), the Drosophila ortholog of Cdt1, results in rereplication and DNA damage. In most cells, this rereplication triggers caspase activation and apoptotic cell death mediated by both p53-dependent and -independent pathways. Elevated Dup also caused DNA damage in endocycling cells, which switch to a G/S cycle during normal development, indicating that rereplication and the endocycling DNA reduplication program are distinct processes. Unexpectedly, however, endocycling cells do not apoptose regardless of tissue type. Our combined evidence suggests that endocycling apoptosis is repressed in part because proapoptotic gene promoters are silenced. Normal endocycling cells had DNA lesions near heterochromatin, which increased after rereplication, explaining why endocycling cells must constantly repress the genotoxic apoptotic response. Our results reveal a novel regulation of apoptosis in development and new insights into the little-understood endocycle. Similar mechanisms may operate during vertebrate development, with implications for cancer predisposition in certain tissues.[Keywords: DNA replication; DNA damage; endocycle; checkpoint; apoptosis] Supplemental material is available at http://www.genesdev.org. The timely duplication of the genome during S phase of every cell division cycle requires that DNA replication initiate from thousands of origins. If too few origins initiate, replication forks can collapse, resulting in DNA damage and incomplete replication of the genome. Initiation of DNA replication from origins more than once per cell cycle, however, results in "rereplication" and subsequent DNA damage (Arias and Walter 2007). In recent years, it has become increasingly apparent that problems with DNA replication are common in premalignant cells, with subsequent checkpoint defects leading to genome instability and cancer (Dutta 2007). It remains unclear, however, whether all cells in development are equivalent with respect to their regulation of DNA replication and checkpoint responses. Here, we use Drosophila to investigate the checkpoint responses to rereplication in a developmental context. Two important steps in the cell cycle regulation of DNA replication are the assembly and activation of a prereplicative complex (pre-RC) (Sivaprasad et al. 2006). The pre-RC assembles onto origins in early G1 and is subsequently activated in S phase. During pre-RC assembly, the hexameric origin recognition complex (ORC) serves as a scaffold for origin association of Cdc6 and Cdt1, which are both required to load the hexameric minichromosome maintenance complex (MCM), the replicative helicase (Randell et al. 2006;Sivaprasad et al. 2006). Once the MCM complex is tightly bound, the origins are considered to be "licensed" and competent to initiate replic...

Cyclin E/Cdk2 is necessary for replication-dependent histone mRNA biosynthesis, but how it controls this process in early development is unknown. We show that in Drosophila embryos the MPM-2 monoclonal antibody, raised against a phosphoepitope from human mitotic cells, detects Cyclin E/Cdk2-dependent nuclear foci that colocalize with nascent histone transcripts. These foci are coincident with the histone locus body (HLB), a Cajal body-like nuclear structure associated with the histone locus and enriched in histone pre-mRNA processing factors such as Lsm11, a core component of the U7 small nuclear ribonucleoprotein. Using MPM-2 and anti-Lsm11 antibodies, we demonstrate that the HLB is absent in the early embryo and occurs when zygotic histone transcription begins during nuclear cycle 11. Whereas the HLB is found in all cells after its formation, MPM-2 labels the HLB only in cells with active Cyclin E/Cdk2. MPM-2 and Lsm11 foci are present in embryos lacking the histone locus, and MPM-2 foci are present in U7 mutants, which cannot correctly process histone pre-mRNA. These data indicate that MPM-2 recognizes a Cdk2-regulated protein that assembles into the HLB independently of histone mRNA biosynthesis. HLB foci are present in histone deletion embryos, although the MPM-2 foci are smaller, and some Lsm11 foci are not associated with MPM-2 foci, suggesting that the histone locus is important for HLB integrity. INTRODUCTIONCell cycle-regulated histone protein biosynthesis is controlled primarily through the regulation of histone mRNA abundance, which in cultured mammalian cells increases 35-fold at the G1-S transition (Breindl and Gallwitz, 1973;Borun et al., 1975;Detke et al., 1979;Parker and Fitschen, 1980;DeLisle et al., 1983;Heintz et al., 1983;Harris et al., 1991). This rapid rise in mRNA is achieved by increases in the rate of transcription initiation and pre-mRNA processing as cells enter S phase, followed by rapid degradation of histone mRNA at the end of S phase . How these various aspects of histone mRNA metabolism are linked to other events that drive progression through the cell cycle by regulating the activity of the cyclindependent kinases (Cdks) remains incompletely understood.In animal cells Cyclin E/Cdk2 promotes the G1-to-S transition in part by phosphorylating proteins that mediate changes in gene expression associated with the onset of DNA replication (e.g., pRb; Du and Pogoriler, 2006). These include proteins that regulate histone expression. For example, human NPAT and human HIRA are Cyclin E/Cdk2 substrates that act to stimulate and repress, respectively, histone gene transcription in cell culture experiments (Ma et al., 2000;Zhao et al., 2000;Hall et al., 2001;Nelson et al., 2002;Miele et al., 2005). How the activity of such factors is modulated by Cyclin E/Cdk2 and integrated into cell cycle-regulated histone gene expression in vivo is not known.Cyclin E/Cdk2 may also regulate features of histone mRNA biosynthesis other than transcription, such as premRNA processing. Rather than being polyaden...

It is important that chromosomes are duplicated only once per cell cycle. Over-replication is prevented by multiple mechanisms that block the reformation of a pre-replicative complex (pre-RC) onto origins in S and G2 phase. We have investigated the developmental regulation of Double-parked (Dup) protein, the Drosophila ortholog of Cdt1, a conserved and essential pre-RC component found in human and other organisms. We find that phosphorylation and degradation of Dup protein at G1/S requires cyclin E/CDK2. The N terminus of Dup, which contains ten potential CDK phosphorylation sites, is necessary and sufficient for Dup degradation during S phase of mitotic cycles and endocycles. Mutation of these ten phosphorylation sites, however, only partially stabilizes the protein,suggesting that multiple mechanisms ensure Dup degradation. This regulation is important because increased Dup protein is sufficient to induce profound rereplication and death of developing cells. Mis-expression has different effects on genomic replication than on developmental amplification from chorion origins. The C terminus alone has no effect on genomic replication,but it is better than full-length protein at stimulating amplification. Mutation of the Dup CDK sites increases genomic re-replication, but is dominant negative for amplification. These two results suggest that phosphorylation regulates Dup activity differently during these developmentally specific types of DNA replication. Moreover, the ability of the CDK site mutant to rapidly inhibit BrdU incorporation suggests that Dup is required for fork elongation during amplification. In the context of findings from human and other cells, our results indicate that stringent regulation of Dup protein is critical to protect genome integrity.