ABSTRACT
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.
Subject(s)
Databases, Genetic , Drosophila melanogaster/genetics , Genome, Insect/genetics , Genomics , Animals , Protein Domains/genetics , SoftwareABSTRACT
The transcriptome is the readout of the genome. Identifying common features in it across distant species can reveal fundamental principles. To this end, the ENCODE and modENCODE consortia have generated large amounts of matched RNA-sequencing data for human, worm and fly. Uniform processing and comprehensive annotation of these data allow comparison across metazoan phyla, extending beyond earlier within-phylum transcriptome comparisons and revealing ancient, conserved features. Specifically, we discover co-expression modules shared across animals, many of which are enriched in developmental genes. Moreover, we use expression patterns to align the stages in worm and fly development and find a novel pairing between worm embryo and fly pupae, in addition to the embryo-to-embryo and larvae-to-larvae pairings. Furthermore, we find that the extent of non-canonical, non-coding transcription is similar in each organism, per base pair. Finally, we find in all three organisms that the gene-expression levels, both coding and non-coding, can be quantitatively predicted from chromatin features at the promoter using a 'universal model' based on a single set of organism-independent parameters.
Subject(s)
Caenorhabditis elegans/genetics , Drosophila melanogaster/genetics , Gene Expression Profiling , Transcriptome/genetics , Animals , Caenorhabditis elegans/embryology , Caenorhabditis elegans/growth & development , Chromatin/genetics , Cluster Analysis , Drosophila melanogaster/growth & development , Gene Expression Regulation, Developmental/genetics , Histones/metabolism , Humans , Larva/genetics , Larva/growth & development , Models, Genetic , Molecular Sequence Annotation , Promoter Regions, Genetic/genetics , Pupa/genetics , Pupa/growth & development , RNA, Untranslated/genetics , Sequence Analysis, RNAABSTRACT
Animal transcriptomes are dynamic, with each cell type, tissue and organ system expressing an ensemble of transcript isoforms that give rise to substantial diversity. Here we have identified new genes, transcripts and proteins using poly(A)+ RNA sequencing from Drosophila melanogaster in cultured cell lines, dissected organ systems and under environmental perturbations. We found that a small set of mostly neural-specific genes has the potential to encode thousands of transcripts each through extensive alternative promoter usage and RNA splicing. The magnitudes of splicing changes are larger between tissues than between developmental stages, and most sex-specific splicing is gonad-specific. Gonads express hundreds of previously unknown coding and long non-coding RNAs (lncRNAs), some of which are antisense to protein-coding genes and produce short regulatory RNAs. Furthermore, previously identified pervasive intergenic transcription occurs primarily within newly identified introns. The fly transcriptome is substantially more complex than previously recognized, with this complexity arising from combinatorial usage of promoters, splice sites and polyadenylation sites.
Subject(s)
Drosophila melanogaster/genetics , Gene Expression Profiling , Transcriptome/genetics , Alternative Splicing/genetics , Animals , Drosophila melanogaster/anatomy & histology , Drosophila melanogaster/cytology , Female , Male , Molecular Sequence Annotation , Nerve Tissue/metabolism , Organ Specificity , Poly A/genetics , Polyadenylation , Promoter Regions, Genetic/genetics , RNA, Long Noncoding/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , Sex Characteristics , Stress, Physiological/geneticsABSTRACT
We expanded the knowledge base for Drosophila cell line transcriptomes by deeply sequencing their small RNAs. In total, we analyzed more than 1 billion raw reads from 53 libraries across 25 cell lines. We verify reproducibility of biological replicate data sets, determine common and distinct aspects of miRNA expression across cell lines, and infer the global impact of miRNAs on cell line transcriptomes. We next characterize their commonalities and differences in endo-siRNA populations. Interestingly, most cell lines exhibit enhanced TE-siRNA production relative to tissues, suggesting this as a common aspect of cell immortalization. We also broadly extend annotations of cis-NAT-siRNA loci, identifying ones with common expression across diverse cells and tissues, as well as cell-restricted loci. Finally, we characterize small RNAs in a set of ovary-derived cell lines, including somatic cells (OSS and OSC) and a mixed germline/somatic cell population (fGS/OSS) that exhibits ping-pong piRNA signatures. Collectively, the ovary data reveal new genic piRNA loci, including unusual configurations of piRNA-generating regions. Together with the companion analysis of mRNAs described in a previous study, these small RNA data provide comprehensive information on the transcriptional landscape of diverse Drosophila cell lines. These data should encourage broader usage of fly cell lines, beyond the few that are presently in common usage.
Subject(s)
Drosophila/genetics , Genetic Variation , MicroRNAs/genetics , RNA, Small Interfering/genetics , Animals , Base Sequence , Cell Line , Computational Biology/methods , Gene Expression , Genetic Loci , Germ Cells , High-Throughput Nucleotide Sequencing , MicroRNAs/chemistry , Molecular Sequence Annotation , Molecular Sequence Data , Nucleic Acid Conformation , RNA, Small Interfering/chemistry , Sequence AlignmentABSTRACT
Drosophila melanogaster is one of the most well studied genetic model organisms; nonetheless, its genome still contains unannotated coding and non-coding genes, transcripts, exons and RNA editing sites. Full discovery and annotation are pre-requisites for understanding how the regulation of transcription, splicing and RNA editing directs the development of this complex organism. Here we used RNA-Seq, tiling microarrays and cDNA sequencing to explore the transcriptome in 30 distinct developmental stages. We identified 111,195 new elements, including thousands of genes, coding and non-coding transcripts, exons, splicing and editing events, and inferred protein isoforms that previously eluded discovery using established experimental, prediction and conservation-based approaches. These data substantially expand the number of known transcribed elements in the Drosophila genome and provide a high-resolution view of transcriptome dynamics throughout development.
Subject(s)
Drosophila melanogaster/growth & development , Drosophila melanogaster/genetics , Gene Expression Profiling , Gene Expression Regulation, Developmental/genetics , Transcription, Genetic/genetics , Alternative Splicing/genetics , Animals , Base Sequence , Drosophila Proteins/genetics , Drosophila melanogaster/embryology , Exons/genetics , Female , Genes, Insect/genetics , Genome, Insect/genetics , Male , MicroRNAs/genetics , Oligonucleotide Array Sequence Analysis , Protein Isoforms/genetics , RNA Editing/genetics , RNA, Messenger/analysis , RNA, Messenger/genetics , RNA, Small Untranslated/analysis , RNA, Small Untranslated/genetics , Sequence Analysis , Sex CharacteristicsABSTRACT
Core promoters are critical regions for gene regulation in higher eukaryotes. However, the boundaries of promoter regions, the relative rates of initiation at the transcription start sites (TSSs) distributed within them, and the functional significance of promoter architecture remain poorly understood. We produced a high-resolution map of promoters active in the Drosophila melanogaster embryo by integrating data from three independent and complementary methods: 21 million cap analysis of gene expression (CAGE) tags, 1.2 million RNA ligase mediated rapid amplification of cDNA ends (RLM-RACE) reads, and 50,000 cap-trapped expressed sequence tags (ESTs). We defined 12,454 promoters of 8037 genes. Our analysis indicates that, due to non-promoter-associated RNA background signal, previous studies have likely overestimated the number of promoter-associated CAGE clusters by fivefold. We show that TSS distributions form a complex continuum of shapes, and that promoters active in the embryo and adult have highly similar shapes in 95% of cases. This suggests that these distributions are generally determined by static elements such as local DNA sequence and are not modulated by dynamic signals such as histone modifications. Transcription factor binding motifs are differentially enriched as a function of promoter shape, and peaked promoter shape is correlated with both temporal and spatial regulation of gene expression. Our results contribute to the emerging view that core promoters are functionally diverse and control patterning of gene expression in Drosophila and mammals.
Subject(s)
Computational Biology , Drosophila melanogaster/genetics , Genome, Insect/genetics , Promoter Regions, Genetic , 3' Untranslated Regions/genetics , Animals , Chromosome Mapping , Drosophila melanogaster/embryology , Expressed Sequence Tags , Gene Expression Profiling , Gene Expression Regulation/genetics , Genome-Wide Association Study , Transcription Initiation SiteABSTRACT
Drosophila melanogaster cell lines are important resources for cell biologists. Here, we catalog the expression of exons, genes, and unannotated transcriptional signals for 25 lines. Unannotated transcription is substantial (typically 19% of euchromatic signal). Conservatively, we identify 1405 novel transcribed regions; 684 of these appear to be new exons of neighboring, often distant, genes. Sixty-four percent of genes are expressed detectably in at least one line, but only 21% are detected in all lines. Each cell line expresses, on average, 5885 genes, including a common set of 3109. Expression levels vary over several orders of magnitude. Major signaling pathways are well represented: most differentiation pathways are "off" and survival/growth pathways "on." Roughly 50% of the genes expressed by each line are not part of the common set, and these show considerable individuality. Thirty-one percent are expressed at a higher level in at least one cell line than in any single developmental stage, suggesting that each line is enriched for genes characteristic of small sets of cells. Most remarkable is that imaginal disc-derived lines can generally be assigned, on the basis of expression, to small territories within developing discs. These mappings reveal unexpected stability of even fine-grained spatial determination. No two cell lines show identical transcription factor expression. We conclude that each line has retained features of an individual founder cell superimposed on a common "cell line" gene expression pattern.
Subject(s)
Drosophila melanogaster/genetics , Genetic Variation , Transcription, Genetic , Animals , Cell Line , Cluster Analysis , Exons , Female , Gene Expression Profiling , Male , Molecular Sequence Data , Signal Transduction/genetics , Transcription Factors/geneticsABSTRACT
Sequencing of multiple related species followed by comparative genomics analysis constitutes a powerful approach for the systematic understanding of any genome. Here, we use the genomes of 12 Drosophila species for the de novo discovery of functional elements in the fly. Each type of functional element shows characteristic patterns of change, or 'evolutionary signatures', dictated by its precise selective constraints. Such signatures enable recognition of new protein-coding genes and exons, spurious and incorrect gene annotations, and numerous unusual gene structures, including abundant stop-codon readthrough. Similarly, we predict non-protein-coding RNA genes and structures, and new microRNA (miRNA) genes. We provide evidence of miRNA processing and functionality from both hairpin arms and both DNA strands. We identify several classes of pre- and post-transcriptional regulatory motifs, and predict individual motif instances with high confidence. We also study how discovery power scales with the divergence and number of species compared, and we provide general guidelines for comparative studies.
Subject(s)
Drosophila/classification , Drosophila/genetics , Evolution, Molecular , Genome, Insect/genetics , Genomics , Animals , Base Sequence , Binding Sites , Conserved Sequence , Drosophila Proteins/genetics , Exons/genetics , Gene Expression Regulation/genetics , Genes, Insect/genetics , MicroRNAs/genetics , Molecular Sequence Data , Organ Specificity , Phylogeny , Untranslated Regions/geneticsABSTRACT
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.
Subject(s)
Drosophila/classification , Drosophila/genetics , Evolution, Molecular , Genes, Insect/genetics , Genome, Insect/genetics , Genomics , Phylogeny , Animals , Codon/genetics , DNA Transposable Elements/genetics , Drosophila/immunology , Drosophila/metabolism , Drosophila Proteins/genetics , Gene Order/genetics , Genome, Mitochondrial/genetics , Immunity/genetics , Multigene Family/genetics , RNA, Untranslated/genetics , Reproduction/genetics , Sequence Alignment , Sequence Analysis, DNA , Synteny/geneticsABSTRACT
In fruit fly research, chromosomal deletions are indispensable tools for mapping mutations, characterizing alleles and identifying interacting loci. Most widely used deletions were generated by irradiation or chemical mutagenesis. These methods are labor-intensive, generate random breakpoints and result in unwanted secondary mutations that can confound phenotypic analyses. Most of the existing deletions are large, have molecularly undefined endpoints and are maintained in genetically complex stocks. Furthermore, the existence of haplolethal or haplosterile loci makes the recovery of deletions of certain regions exceedingly difficult by traditional methods, resulting in gaps in coverage. Here we describe two methods that address these problems by providing for the systematic isolation of targeted deletions in the D. melanogaster genome. The first strategy used a P element-based technique to generate deletions that closely flank haploinsufficient genes and minimize undeleted regions. This deletion set has increased overall genomic coverage by 5-7%. The second strategy used FLP recombinase and the large array of FRT-bearing insertions described in the accompanying paper to generate 519 isogenic deletions with molecularly defined endpoints. This second deletion collection provides 56% genome coverage so far. The latter methodology enables the generation of small custom deletions with predictable endpoints throughout the genome and should make their isolation a simple and routine task.
Subject(s)
DNA Transposable Elements , Drosophila melanogaster/genetics , Sequence Deletion , Animals , Genome , Mutagenesis, InsertionalABSTRACT
Human industries generate hundreds of thousands of chemicals, many of which have not been adequately studied for environmental safety or effects on human health. This deficit of chemical safety information is exacerbated by current testing methods in mammals that are expensive, labor-intensive, and time-consuming. Recently, scientists and regulators have been working to develop new approach methodologies (NAMs) for chemical safety testing that are cheaper, more rapid, and reduce animal suffering. One of the key NAMs to emerge is the use of invertebrate organisms as replacements for mammalian models to elucidate conserved chemical modes of action across distantly related species, including humans. To advance these efforts, here, we describe a method that uses the fruit fly, Drosophila melanogaster, to assess chemical safety. The protocol describes a simple, rapid, and inexpensive procedure to measure the viability and feeding behavior of exposed adult flies. In addition, the protocol can be easily adapted to generate samples for genomic and metabolomic approaches. Overall, the protocol represents an important step forward in establishing Drosophila as a standard model for use in precision toxicology.
Subject(s)
Drosophila melanogaster , Drosophila , Animals , Adult , Humans , Genomics , Feeding Behavior , Risk Assessment , MammalsABSTRACT
A global isotopic labeling strategy combined with multidimensional liquid chromatographies and tandem mass spectrometry was used for quantitative proteome analysis of a presymptomatic A53T alpha-synuclein Drosophila model of Parkinson disease (PD). Multiple internal standard proteins at different concentration ratios were spiked into samples from PD-like and control animals to assess quantification accuracy. Two biological replicates isotopically labeled in forward and reverse directions were analyzed. A total of 253 proteins were quantified with a minimum of two identified peptide sequences (for each protein); 180 ( approximately 71%) proteins were detected in both forward and reverse labeling measurements. Twenty-four proteins were differentially expressed in A53T alpha-synuclein Drosophila; up-regulation of troponin T and down-regulation of fat body protein 1 were confirmed by Western blot analysis. Elevated expressions of heat shock protein 70 cognate 3 and ATP synthase are known to be directly involved in A53T alpha-synuclein-mediated toxicity and PD; three up-regulated proteins (muscle LIM protein at 60A, manganese-superoxide dismutase, and troponin T) and two down-regulated proteins (chaoptin and retinal degeneration A) have literature-supported associations with cellular malfunctions. That these variations were observed in presymptomatic animals may shed light on the etiology of PD. Protein interaction network analysis indicated that seven proteins belong to a single network, which may provide insight into molecular pathways underlying PD. Gene Ontology analysis indicated that the dysregulated proteins are primarily associated with membrane, endoplasmic reticulum, actin cytoskeleton, mitochondria, and ribosome. These associations support prior findings in studies of the A30P alpha-synuclein Drosophila model (Xun, Z. Y., Sowell, R. A., Kaufman, T. C., and Clemmer, D. E. (2007) Protein expression in a Drosophila model of Parkinson's disease. J. Proteome Res. 6, 348-357; Xun, Z. Y., Sowell, R. A., Kaufman, T. C., and Clemmer, D. E. (2007) Lifetime proteomic profiling of an A30P alpha-synuclein Drosophila model of Parkinson's disease. J. Proteome Res. 6, 3729-3738) that defects in cellular components such as actin cytoskeleton and mitochondria may contribute to the development of later symptoms.
Subject(s)
Disease Models, Animal , Drosophila melanogaster/metabolism , Parkinson Disease/metabolism , Proteomics/methods , alpha-Synuclein/genetics , Algorithms , Animals , Animals, Genetically Modified , Cluster Analysis , Drosophila melanogaster/genetics , Drosophila melanogaster/physiology , Female , Humans , Male , Mutation, Missense , Parkinson Disease/genetics , Parkinson Disease/pathology , Peptide Fragments/analysis , Proteome/analysisABSTRACT
Parkinson's disease (PD) is characterized by loss of dopaminergic neurons in the substantia nigra and formation of intracytoplasmic Lewy bodies (LBs). Loss-of-function mutations in parkin which encodes an E3 ubiquitin protein ligase contribute to a predominant cause of a familial form of PD termed autosomal recessive juvenile Parkinsonism (AR-JP). Drosophila parkin null mutants display muscle degeneration and mitochondrial dysfunction, providing an animal model to study Parkin-associated molecular pathways in PD. To define protein alterations involved in Parkin pathogenesis, we performed quantitative proteomic analyses of Drosophila parkin null mutants and age-matched controls utilizing both global internal standard technology (GIST) and extracted ion chromatogram peak area (XICPA) label-free approaches. A total of 375 proteins were quantified with a minimum of two peptide identifications from the combination of the XICPA and GIST measurements applied to two independent biological replicates. Sixteen proteins exhibited significant alteration. Seven of the dysregulated proteins are involved in energy metabolism, of which six were down-regulated. All five proteins involved in transporter activity exhibited higher levels, of which larval serum protein 1alpha, larval serum protein 1beta, larval serum protein 1gamma, and fat body protein 1 showed >10-fold up-regulation and substantially higher level of fat body protein 1 was confirmed by Western blot analysis. These findings suggest that abnormalities in energy metabolism and protein transporter activity pathways may be associated with the pathogenesis of Parkin-associated AR-JP.
Subject(s)
Drosophila Proteins , Isotope Labeling/methods , Proteome , Proteomics/methods , Animals , Animals, Genetically Modified , Blotting, Western , Drosophila/metabolism , Drosophila Proteins/analysis , Drosophila Proteins/genetics , Drosophila Proteins/metabolism , Energy Metabolism , Female , Hemoglobins/analysis , Male , Peptide Fragments/analysis , Protein Transport , Proteome/analysis , Proteome/metabolism , Reference Standards , Reproducibility of Results , Research Design , Ubiquitin-Protein Ligases , alpha-Synuclein/genetics , alpha-Synuclein/metabolismABSTRACT
The sequencing of the 12 genomes of members of the genus Drosophila was taken as an opportunity to reevaluate the genetic and physical maps for 11 of the species, in part to aid in the mapping of assembled scaffolds. Here, we present an overview of the importance of cytogenetic maps to Drosophila biology and to the concepts of chromosomal evolution. Physical and genetic markers were used to anchor the genome assembly scaffolds to the polytene chromosomal maps for each species. In addition, a computational approach was used to anchor smaller scaffolds on the basis of the analysis of syntenic blocks. We present the chromosomal map data from each of the 11 sequenced non-Drosophila melanogaster species as a series of sections. Each section reviews the history of the polytene chromosome maps for each species, presents the new polytene chromosome maps, and anchors the genomic scaffolds to the cytological maps using genetic and physical markers. The mapping data agree with Muller's idea that the majority of Drosophila genes are syntenic. Despite the conservation of genes within homologous chromosome arms across species, the karyotypes of these species have changed through the fusion of chromosomal arms followed by subsequent rearrangement events.
Subject(s)
Chromosomes/genetics , Drosophila/genetics , Genome, Insect/genetics , Physical Chromosome Mapping , Animals , Genetic Markers , Karyotyping , Sequence Alignment , SyntenyABSTRACT
Model organism databases (MODs) have been collecting and integrating biomedical research data for 30 years and were designed to meet specific needs of each model organism research community. The contributions of model organism research to understanding biological systems would be hard to overstate. Modern molecular biology methods and cost reductions in nucleotide sequencing have opened avenues for direct application of model organism research to elucidating mechanisms of human diseases. Thus, the mandate for model organism research and databases has now grown to include facilitating use of these data in translational applications. Challenges in meeting this opportunity include the distribution of research data across many databases and websites, a lack of data format standards for some data types, and sustainability of scale and cost for genomic database resources like MODs. The issues of widely distributed data and application of data standards are some of the challenges addressed by FAIR (Findable, Accessible, Interoperable, and Re-usable) data principles. The Alliance of Genome Resources is now moving to address these challenges by bringing together expertly curated research data from fly, mouse, rat, worm, yeast, zebrafish, and the Gene Ontology consortium. Centralized multi-species data access, integration, and format standardization will lower the data utilization barrier in comparative genomics and translational applications and will provide a framework in which sustainable scale and cost can be addressed. This article presents a brief historical perspective on how the Alliance model organisms are complementary and how they have already contributed to understanding the etiology of human diseases. In addition, we discuss four challenges for using data from MODs in translational applications and how the Alliance is working to address them, in part by applying FAIR data principles. Ultimately, combined data from these animal models are more powerful than the sum of the parts.
Subject(s)
Animals, Laboratory , Databases as Topic , Translational Research, Biomedical/methods , Animals , Models, AnimalABSTRACT
The purpose of this chapter in FlyBook is to acquaint the reader with the Drosophila genome and the ways in which it can be altered by mutation. Much of what follows will be familiar to the experienced Fly Pusher but hopefully will be useful to those just entering the field and are thus unfamiliar with the genome, the history of how it has been and can be altered, and the consequences of those alterations. I will begin with the structure, content, and organization of the genome, followed by the kinds of structural alterations (karyotypic aberrations), how they affect the behavior of chromosomes in meiotic cell division, and how that behavior can be used. Finally, screens for mutations as they have been performed will be discussed. There are several excellent sources of detailed information on Drosophila husbandry and screening that are recommended for those interested in further expanding their familiarity with Drosophila as a research tool and model organism. These are a book by Ralph Greenspan and a review article by John Roote and Andreas Prokop, which should be required reading for any new student entering a fly lab for the first time.
Subject(s)
Drosophila melanogaster/genetics , Genome, Insect/genetics , Mutation/genetics , Science/history , Animals , Chromosome Aberrations , History, 20th Century , History, 21st Century , Miosis/geneticsABSTRACT
In Drosophila melanogaster a functional pericentriolar matrix (PCM) at mitotic centrosomes requires Centrosomin-Long Form (Cnn-LF) proteins. Moreover, tissue culture cells have shown that the centrosomal localization of both Cnn-LF and Polo kinase are co-dependent, suggesting a direct interaction. Our recent study found Cnn potentially binds to and is phosphorylated by Polo kinase at 2 residues encoded by Exon1A, the initiating exon of a subset of Cnn isoforms. These interactions are required for the centrosomal localization of Cnn-LF in syncytial embryos and a mutation of either phosphorylation site is sufficient to block localization of both mutant and wild-type Cnn when they are co-expressed. Immunoprecipitation experiments show that Cnn-LF interacts directly with mitotically activated Polo kinase and requires the 2 phosphorylation sites in Exon1A. These IP experiments also show that Cnn-LF proteins form multimers. Depending on the stoichiometry between functional and mutant peptides, heteromultimers exhibit dominant negative or positive trans-complementation (rescue) effects on mitosis. Additionally, following the completion of meiosis, Cnn-Short Form (Cnn-SF) proteins are required for polar body formation in embryos, a process previously shown to require Polo kinase. These findings, when combined with previous work, clearly demonstrate the complexity of cnn and show that a view of cnn as encoding a single peptide is too simplistic.
Subject(s)
Centrosome/metabolism , Drosophila Proteins/metabolism , Drosophila melanogaster/metabolism , Homeodomain Proteins/metabolism , Protein Serine-Threonine Kinases/metabolism , Amino Acid Sequence , Animals , Drosophila Proteins/chemistry , Drosophila melanogaster/cytology , Drosophila melanogaster/growth & development , Embryo, Nonmammalian/cytology , Embryo, Nonmammalian/metabolism , Homeodomain Proteins/chemistry , Mitosis , PhosphorylationABSTRACT
During embryonic development of the Drosophila brain, the Hox gene labial is required for the regionalized specification of the tritocerebral neuromere. In order to gain further insight into the mechanisms of Hox gene action in the CNS, we have studied the molecular and genetic basis of cross-regulatory interactions between labial and other more posterior Hox genes using the GAL4/UAS system for targeted misexpression. Misexpression of posterior Hox genes in the embryonic neuroectoderm results in a labial loss-of function phenotype and a corresponding lack of Labial protein expression in the tritocerebrum. This is due to repression of labial gene transcription in the embryonic brain. Enhancer analysis suggests that this transcriptional repression operates on a 3.65 kb brain-specific labial-enhancer element. A functional analysis of Antennapedia and Ultrabithorax protein domains shows that the transcriptional repression of labial requires homeodomain-DNA interactions but is not dependent on a functional hexapeptide. The repressive activity of a Hox protein on labial expression in the tritocerebrum can, however, be abolished by concomitant misexpression of a Hox protein and the cofactors Homothorax and nuclear-targeted Extradenticle. Taken together, these results provide novel and detailed insight into the cross-regulatory interactions of Hox genes in embryonic brain development and suggest that specification of tritocerebral neuronal identity requires equilibrated levels of a Hox protein and Hth and n-Exd cofactors.
Subject(s)
Brain/embryology , Drosophila Proteins/physiology , Gene Expression Regulation, Developmental , Genes, Homeobox , Homeodomain Proteins/genetics , Homeodomain Proteins/physiology , Nuclear Proteins/physiology , Transcription Factors/physiology , Animals , Antennapedia Homeodomain Protein , Brain/metabolism , Central Nervous System , Crosses, Genetic , DNA-Binding Proteins/physiology , Drosophila Proteins/biosynthesis , Drosophila melanogaster , Embryonic Development/physiology , Enhancer Elements, Genetic , Homeodomain Proteins/biosynthesis , Immunohistochemistry , Peptides/chemistry , Phenotype , Protein Binding , Time Factors , Tissue Distribution , Transcription Factors/biosynthesis , Transcription, GeneticABSTRACT
Here we describe how to generate customized microinjection needles from glass capillary tubes. Controls demonstrate the range of variables and effects on needle tip shape using a standard Flaming/Brown micropipet needle puller. Needles generated with two-cycle pulls provide a wider range of needle shapes in a predictable fashion. We used the needle puller's ramp function for multiple-cycle programs to determine the useful range of heat settings inherent to the glass capillary tube. This articlefocuses primarily on the preparation of injection needles utilized for P-element-mediated germ-line transformation in Drosophila melanogaster that do not require the dechorionation of the egg. However, these types of needles can be usefulfor numerous other types of injections, such as RNA interference, homologous recombination mutagenesis, morpholinos, transient gene regulation, drug delivery, and the transfer of cytoplasmic factors that are useful in a wide range of biological systems ranging from plants to vertebrates. Using our standard needle, we correlate the survival of injected D. melanogaster embryos with transformation efficiencies and plasmid construct characteristics.
Subject(s)
DNA Transposable Elements/genetics , Drosophila melanogaster/genetics , Gene Transfer Techniques/instrumentation , Glass , Microinjections/instrumentation , Needles , Air Pressure , Animals , Drosophila melanogaster/embryology , Equipment Design , Germ Cells , Hot Temperature , Microinjections/methods , Particle Size , Plasmids , Quality Control , Recombination, Genetic , Sensitivity and Specificity , Transformation, Genetic , ViscosityABSTRACT
The rapid evolution of essential developmental genes and their protein products is both intriguing and problematic. The rapid evolution of gene products with simple protein folds and a lack of well-characterized functional domains typically result in a low discovery rate of orthologous genes. Additionally, in the absence of orthologs it is difficult to study the processes and mechanisms underlying rapid evolution. In this study, we have investigated the rapid evolution of centrosomin (cnn), an essential gene encoding centrosomal protein isoforms required during syncytial development in Drosophila melanogaster. Until recently the rapid divergence of cnn made identification of orthologs difficult and questionable because Cnn violates many of the assumptions underlying models for protein evolution. To overcome these limitations, we have identified a group of insect orthologs and present conserved features likely to be required for the functions attributed to cnn in D. melanogaster. We also show that the rapid divergence of Cnn isoforms is apparently due to frequent coding sequence indels and an accelerated rate of intronic additions and eliminations. These changes appear to be buffered by multi-exon and multi-reading frame maximum potential ORFs, simple protein folds, and the splicing machinery. These buffering features also occur in other genes in Drosophila and may help prevent potentially deleterious mutations due to indels in genes with large coding exons and exon-dense regions separated by small introns. This work promises to be useful for future investigations of cnn and potentially other rapidly evolving genes and proteins.