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PubMed2000

A whole-genome assembly of Drosophila.

We report on the quality of a whole-genome assembly of Drosophila melanogaster and the nature of the computer algorithms that accomplished it. Three independent external data sources essentially agree with and support the assembly's sequence and ordering of contigs across the euchromatic portion of the genome. In addition, there are isolated contigs that we believe represent nonrepetitive pockets within the heterochromatin of the centromeres. Comparison with a previously sequenced 2.9- megabase region indicates that sequencing accuracy within nonrepetitive segments is greater than 99. 99% without manual curation. As such, this initial reconstruction of the Drosophila sequence should be of substantial value to the scientific community.

Algorithms↗

PubMed2006

The nicotinic acetylcholine receptor Dalpha7 is required for an escape behavior in Drosophila.

Acetylcholine is the major excitatory neurotransmitter in the central nervous system of insects. Mutant analysis of the Dalpha7 nicotinic acetylcholine receptor (nAChR) of Drosophila shows that it is required for the giant fiber-mediated escape behavior. The Dalpha7 protein is enriched in the dendrites of the giant fiber, and electrophysiological analysis of the giant fiber circuit showed that sensory input to the giant fiber is disrupted, as is transmission at an identified cholinergic synapse between the peripherally synapsing interneuron and the dorsal lateral muscle motor neuron. Moreover, we found that gfA1, a mutation identified in a screen for giant fiber defects more than twenty years ago, is an allele of Dalpha7. Therefore, a combination of behavioral, electrophysiological, anatomical, and genetic data indicate an essential role for the Dalpha7 nAChR in giant fiber-mediated escape in Drosophila.

Alleles↗

PubMedDate not supplied

Developmental instability of the Drosophila wing as an index of genomic perturbation and altered cell proliferation.

We experimentally induced different levels of instability affecting the development of specific wing regions of Drosophila melanogaster using the UAS-GAL4 system. A common index of developmental instability is fluctuating asymmetry (FA), that is, random differences between body sides of single individuals. We studied the FA in transgenic strains carrying random genomic insertions (UAS strains), as well as insertions in the regulatory region of genes involved in the organization of wing development (GAL4 strains). In addition, the expression of genes that increase (dp110 and 3622) or decrease (dPTEN) cell proliferation was ectopically induced. Our results are related to different levels of perturbation. Through the first kind of perturbation, genome integrity was compromised by the insertion of foreign DNA. In all cases, we observed a general increase in FA, although it was rarely found significant. The second kind of perturbation involved a modification of genes controlling wing development through the insertion of a GAL4 sequence in their promoter region. The third kind involved the ectopic expression of genes controlling cell proliferation. Our results show that (i) the level of FA is connected with the level of morphological perturbation induced, (ii) FA increase was higher in the wing regions that were the target of the genetic perturbation, and (iii) developmental instability was also observed in regions that were not directly addressed by the perturbation. The results were discussed on the basis of the running models about Drosophila wing development.

Analysis of Variance↗

PubMed1996

Genetic and molecular analysis of a set of unstable white mutants in Drosophila melanogaster.

Two related unstable mutants at the white locus of Drosophila melanogaster show different interactions with the zeste1 mutant: one mutated white gene becomes repressed in males, whereas the other is unaffected by z1. By use of Southern blot techniques and by constructing genomic lambda-libraries, molecular analyses of the white regions of these two strains were performed. The results showed a single difference at a site 2.5 kb (kilobases) downstream of the white transcription unit. In both strains, FB (foldback) elements were integrated at this site, but the repressed strain also harboured a 4 kb NOF (Nofretete) element. No other restriction site polymorphisms between the two strains were observed within a 120 kb region surrounding the white gene. The extent of twelve white deletions and twelve white transpositions deriving from these unstable strains was analysed by in situ hybridisation and Southern blot techniques. The results revealed that the distal breakpoint of all aberrations coincided with the insertion site of the mobile elements, but that the centromere proximal breakpoints varied. The mechanisms for the instability and the interaction with the zeste1 mutant are discussed.

Animals↗

PubMed2006

Testing chromosomal phylogenies and inversion breakpoint reuse in Drosophila.

A combination of cytogenetic and bioinformatic procedures was used to test the chromosomal phylogeny relating Drosophila buzzatii with D. repleta. Chromosomes X and 2, harboring most of the inversions fixed between these two species, were analyzed. First, chromosomal segments conserved during the divergence of the two species were identified by comparative in situ hybridization to the D. repleta chromosomes of 180 BAC clones from a BAC-based physical map of the D. buzzatii genome. These conserved segments were precisely delimited with the aid of clones containing inversion breakpoints. Then GRIMM software was used to estimate the minimum number of rearrangements necessary to transform one genome into the other and identify all possible rearrangement scenarios. Finally, the most plausible inversion trajectory was tested by hybridizing 12 breakpoint-bearing BAC clones to the chromosomes of seven other species in the repleta group. The results show that chromosomes X and 2 of D. buzzatii and D. repleta differ by 12 paracentric inversions. Nine of them are fixed in chromosome 2 and entail two breakpoint reuses. Our results also show that the cytological relationship between D. repleta and D. mercatorum is closer than that between D. repleta and D. peninsularis, and we propose that the phylogenetic relationships in this lineage of the repleta group be reconsidered. We also estimated the rate of rearrangement between D. repleta and D. buzzatii and conclude that rates within the genus Drosophila vary substantially between lineages, even within a single species group.

Animals↗

PubMed2006

Identification and comparative analysis of accessory gland proteins in Orthoptera.

Accessory reproductive gland proteins (Acps) in Drosophila evolve quickly and appear to play an important role in ensuring the fertilization success of males. Moreover, Acps are thought to be involved in establishing barriers to fertilization between closely related species. While accessory glands are known to occur in the males of many insect groups, the proteins that are passed on to females by males during mating have not been well characterized outside of Drosophila. To gain a better understanding of these proteins, we characterized ESTs from the accessory glands of two cricket species, Allonemobius fasciatus and Gryllus firmus. Using an expressed sequence tag (EST) approach, followed by bioinformatic and evolutionary analyses, we found that many proteins are secreted and, therefore, available for transfer to the female during mating. Further, we found that most ESTs are novel, showing little sequence similarity between taxa. Evolutionary analyses suggest that cricket proteins are subject to diversifying selection and indicate that Allonemobius is much less polymorphic than Gryllus. Despite rapid nucleotide sequence divergence, there appears to be functional conservation of protein classes among Drosophila and cricket taxa.

Amino Acid Sequence↗

PubMed1998

Isolation and characterization of a pufferfish MLL (mixed lineage leukemia)-like gene (fMll) reveals evolutionary conservation in vertebrate genes related to Drosophila trithorax.

The MLL gene is interrupted and fused to a number of partner genes as a result of chromosomal translocations in human leukemias. MLL is a very large protein with a unique domain structure and large regions of homology to Drosophila trx. To define the key structural and functional domains of the MLL protein in vertebrates, we have cloned the genomic region encoding an MLL-like gene in the compact model vertebrate genome of Fugu rubripes. While the similarity between the mouse and human MLL proteins is very high, a lower overall similarity is present between the Fugu and mammalian proteins. Several new highly conserved regions were identified in the portion of the protein included in the MLL leukemia-associated fusion proteins. The conserved nature of regions of similarity between vertebrate forms of MLL and the Drosophila TRX proteins, as well as other domains previously suggested to have a functional role in MLL (including the AT hooks and the DNA methyltransferase domain), was also observed. Therefore, strong evolutionary constraints limited sequence divergence within these domains. The information derived from this comparative analysis will form the basis for the functional study of the MLL protein, particularly as it relates to human leukemogenesis.

Amino Acid Sequence↗

PubMed1998

A novel class of supercoil-independent nuclease hypersensitive site is comprised of alternative DNA structures that flank eukaryotic genes.

The cell makes a fundamental distinction between genes and non-gene sequences, which mechanistically underlies the process of gene regulation. Here, we describe the properties of a novel class of genetic sites that reproducibly flank and delineate the coding regions of the eukaryotic genes tested. Defined in vitro reaction conditions that include altered solvation and elevated temperature rendered the sites hypersensitive to nuclease cleavage. Consequently, the complete coding regions of the Drosophila genes tested were quantitatively excised from genomic DNA or genomic clones by this treatment. Identical reaction products were generated from linear or supercoiled DNA substrates. Chemical modification and fine-structure analysis of several cleavage sites flanking Drosophila genes showed that the cleavage sites were stable nucleic acid structures that contained specific arrangements of paired and unpaired nucleotides. The locations and properties of the cleavage sites did not correspond to previously known nuclease hypersensitive sites nor to known alternative DNA structures. Thus, they appear to represent a new class of genetic site. In a deletion analysis, the minimal sequence information necessary to direct in vitro nuclease cleavage 3' to the Drosophila GART gene co-localized with the signal required for termination of transcription in vivo. The data suggest that a novel class of DNA site with distinct structural properties encodes biological information by marking the boundaries of at least some gene expression units in organisms as diverse as Plasmodium and Drosophila.

Animals↗

PubMed2005

AnoEST: toward A. gambiae functional genomics.

Here, we present an analysis of 215,634 EST and cDNA sequences of a major vector of human malaria Anopheles gambiae structured into the AnoEST database. The expressed sequences are grouped into clusters using genomic sequence as template and associated with inferred functional annotation, including the following: corresponding Ensembl gene prediction, putative orthologous genes in other species, homology to known proteins, protein domains, associated Gene Ontology terms, and corresponding classification into broad GO-slim functional groups. AnoEST is a vital resource for interpretation of expression profiles derived using recently developed A. gambiae cDNA microarrays. Using these cDNA microarrays, we have experimentally confirmed the expression of 7961 clusters during mosquito development. Of these, 3100 are not associated with currently predicted genes. Moreover, we found that clusters with confirmed expression are nonbiased with respect to the current gene annotation or homology to known proteins. Consequently, we expect that many as yet unconfirmed clusters are likely to be actual A. gambiae genes. [AnoEST is publicly available at http://komar.embl.de, and is also accessible as a Distributed Annotation Service (DAS).].

Animals↗

PubMed2006

Genome-wide analyses identify transcription factors required for proper morphogenesis of Drosophila sensory neuron dendrites.

Dendrite arborization patterns are critical determinants of neuronal function. To explore the basis of transcriptional regulation in dendrite pattern formation, we used RNA interference (RNAi) to screen 730 transcriptional regulators and identified 78 genes involved in patterning the stereotyped dendritic arbors of class I da neurons in Drosophila. Most of these transcriptional regulators affect dendrite morphology without altering the number of class I dendrite arborization (da) neurons and fall primarily into three groups. Group A genes control both primary dendrite extension and lateral branching, hence the overall dendritic field. Nineteen genes within group A act to increase arborization, whereas 20 other genes restrict dendritic coverage. Group B genes appear to balance dendritic outgrowth and branching. Nineteen group B genes function to promote branching rather than outgrowth, and two others have the opposite effects. Finally, 10 group C genes are critical for the routing of the dendritic arbors of individual class I da neurons. Thus, multiple genetic programs operate to calibrate dendritic coverage, to coordinate the elaboration of primary versus secondary branches, and to lay out these dendritic branches in the proper orientation.

Animals↗

PubMed1999

A novel mechanism for P element homing in Drosophila.

P element insertion is essentially random at the scale of the genome. However, P elements containing regulatory sequences from Drosophila engrailed and polyhomeotic genes and from the Bithorax and Antennapedia complexes show some insertional specificity by frequently inserting near the parent gene (homing) and/or near genes containing Polycomb group response elements (preferential insertion). This phenomenon is thought to be mediated by Polycomb group proteins. In this report, we describe a case of homing of P elements containing regulatory sequences of the linotte gene. This homing occurs with high frequency (up to 20% of the lines) and high precision (inserted into a region of <1 kilobase). We present evidence showing that it is not mediated by Polycomb group proteins but by a new, as yet unknown, mechanism. We also suggest that P element homing could be a more frequent phenomenon than generally assumed and that it could become a powerful tool of Drosophila reverse genetics, for which there is no other described gene targeting technique.

Amino Acid Sequence↗

PubMed2004

An active transposable element, Herves, from the African malaria mosquito Anopheles gambiae.

Transposable elements have proven to be invaluable tools for genetically manipulating a wide variety of plants, animals, and microbes. Some have suggested that they could be used to spread desirable genes, such as refractoriness to Plasmodium infection, through target populations of Anopheles gambiae, thereby disabling the mosquito's ability to transmit malaria. To achieve this, a transposon must remain mobile and intact after the initial introduction into the genome. Endogenous, active class II transposable elements from An. gambiae have not been exploited as gene vectors/drivers because none have been isolated. We report the discovery of an active class II transposable element, Herves, from the mosquito An. gambiae. Herves is a member of a distinct subfamily of hAT elements that includes the hopper-we element from Bactrocera dorsalis and B. cucurbitae. Herves was transpositionally active in mobility assays performed in Drosophila melanogaster S2 cells and developing embryos and was used as a germ-line transformation vector in D. melanogaster. Herves displays an altered target-site preference from the distantly related hAT elements, Hermes and hobo. Herves is also present in An. arabiensis and An. merus with copy numbers similar to that found in An. gambiae. Preliminary data from an East African population are consistent with the element being transpositionally active in mosquitoes.

Africa↗

PubMed2003

Evolutionary genomics of inversions in Drosophila pseudoobscura: evidence for epistasis.

Drosophila pseudoobscura harbors a rich polymorphism for paracentric inversions on the third chromosome, and the clines in the inversion frequencies across the southwestern United States indicate that strong natural selection operates on them. Isogenic inversion strains were made from isofemale lines collected from four localities, and eight molecular markers were mapped on the third chromosome. Nucleotide diversity was measured for these loci and formed the basis of an evolutionary genomic analysis. The loci were differentiated among inversions. The inversions did not show significant differences among populations, however, likely the result of extensive gene flow among populations. Some loci had significant reductions in nucleotide diversity within inversions compared with interspecies divergence, suggesting that these loci are near inversion breakpoints or are near targets of directional selection. Linkage disequilibrium (LD) levels tended to decrease with distance between loci, indicating that some genetic exchange occurs among gene arrangements despite the presence of inversions. In some cases, however, adjacent genes had low levels of interlocus LD and loosely linked genes had high levels of interlocus LD, suggesting strong epistatic selection. Our results support the hypothesis that the inversions of D. pseudoobscura have emerged as suppressors of recombination to maintain positive epistatic relationships among loci within gene arrangements that developed as the species adapted to a heterogeneous environment.

Animals↗

PubMed2006

Functional genomics of cactus host shifts in Drosophila mojavensis.

Understanding the genetic basis of adaptation to novel environments remains one of the major challenges confronting evolutionary biologists. While newly developed genomic approaches hold considerable promise for addressing this overall question, the relevant tools have not often been available in the most ecologically interesting organisms. Our study organism, Drosophila mojavensis, is a cactophilic Sonoran Desert endemic utilizing four different cactus hosts across its geographical range. Its well-known ecology makes it an attractive system in which to study the evolution of gene expression during adaptation. As a cactophile, D. mojavensis oviposits in the necrotic tissues of cacti, therefore exposing larvae and even adults to the varied and toxic compounds of rotting cacti. We have developed a cDNA microarray of D. mojavensis to examine gene expression associated with cactus host use. Using a population from the Baja California population we examined gene expression differences of third instar larvae when reared in two chemically distinct cactus hosts, agria (Stenocereus gummosus, native host) vs. organpipe (Stenocereus thurberi, alternative host). We have observed differential gene expression associated with cactus host use in genes involved in metabolism and detoxification.

Animals↗

PubMed1998

Isolation and chronobiological analysis of a neuropeptide pigment-dispersing factor gene in Drosophila melanogaster.

In this article, the authors isolate a gene encoding a neuropeptide in Drosophila melanogaster. The substance is called pigment-dispersing factor (PDF), based on one of the roles it plays in crustaceans (the arthropods in which this factor was initially discovered). The PDF-encoding Drosophila gene (pdf) is intronless and present in a single copy per haploid genome. The cytological location of pdf is 97B on the third chromosome. The putative 102-amino-acid precursor (prepro-PDF) consists of a signal peptide and a PDF-associated peptide, followed by the mature PDF. The PDF-associated peptide region of the precursor is highly diverged from those of the crustacean precursors, whereas the primary structure of the mature PDF is conserved in other members of the pigment-dispersing hormone family. A single pdf transcript (ca. 0.8 kb) is expressed predominantly in the head; the expression levels of pdf mRNA are consistently higher in males than in females. Putative pdf homologous transcripts are present in other Drosophila species, which exhibit similar sexual dimorphic expression patterns. Cyclic expression of pdf over the course of the day and night was assessed, but the mRNA exhibited at best very gentle cycling. The pdf expression in two behaviorally arrhythmic mutants were examined; the expression was intact in a period0 mutant but absent in the disconnected mutant.

Amino Acid Sequence↗

PubMed2005

Sequence signature analysis of chromosome identity in three Drosophila species.

BACKGROUND: All eukaryotic organisms need to distinguish each of their chromosomes. A few protein complexes have been described that recognise entire, specific chromosomes, for instance dosage compensation complexes and the recently discovered autosome-specific Painting of Fourth (POF) protein in Drosophila. However, no sequences have been found that are chromosome-specific and distributed over the entire length of the respective chromosome. Here, we present a new, unbiased, exhaustive computational method that was used to probe three Drosophila genomes for chromosome-specific sequences. RESULTS: By combining genome annotations and cytological data with multivariate statistics related to three Drosophila genomes we found sequence signatures that distinguish Muller's F-elements (chromosome 4 in D. melanogaster) from all other chromosomes in Drosophila that are not attributable to differences in nucleotide composition, simple sequence repeats or repeated elements. Based on these signatures we identified complex motifs that are strongly overrepresented in the F-elements and found indications that the D. melanogaster motif may be involved in POF-binding to the F-element. In addition, the X-chromosomes of D. melanogaster and D. yakuba can be distinguished from the other chromosomes, albeit to a lesser extent. Surprisingly, the conservation of the F-element sequence signatures extends not only between species separated by approximately 55 Myr, but also linearly along the sequenced part of the F-elements. CONCLUSION: Our results suggest that chromosome-distinguishing features are not exclusive to the sex chromosomes, but are also present on at least one autosome (the F-element) in Drosophila.

Animals↗

PubMed2005

Whole-genome analysis reveals a strong positional bias of conserved dMyc-dependent E-boxes.

Myc is a transcription factor with diverse biological effects ranging from the control of cellular proliferation and growth to the induction of apoptosis. Here we present a comprehensive analysis of the transcriptional targets of the sole Myc ortholog in Drosophila melanogaster, dMyc. We show that the genes that are down-regulated in response to dmyc inhibition are largely identical to those that are up-regulated after dMyc overexpression and that many of them play a role in growth control. The promoter regions of these targets are characterized by the presence of the E-box sequence CACGTG, a known dMyc binding site. Surprisingly, a large subgroup of (functionally related) dMyc targets contains a single E-box located within the first 100 nucleotides after the transcription start site. The relevance of this E-box and its position was confirmed by a mutational analysis of a selected dMyc target and by the observation of its evolutionary conservation in a different Drosophila species, Drosophila pseudoobscura. These observations raise the possibility that a subset of Myc targets share a distinct regulatory mechanism.

Animals↗

PubMed1998

The organization of the alpha-tubulin gene family in the Drosophila montium subgroup of the melanogaster species group.

The alpha 1-, alpha 2-, alpha 3-, and alpha 4-tubulin genes have been mapped by in situ hybridization to the polytene chromosomes of five species representative of the Drosophila montium subgroup geographical distribution. A lambda phage clone containing alpha 1-tubulin specific sequences was isolated from a genomic DNA library of Drosophila auraria and its restriction endonuclease pattern is presented. Both well-characterized heterologous and homologous probes were used to assess orthogonality of gene members between species groups. The in situ hybridization pattern observed in all species studied is consistent with that of Drosophila melanogaster, since alpha 1-, alpha 2-, and alpha 3-tubulin genes are located on the same polytene arm, and the alpha 4-tubulin gene is found on a different arm. Cross-hybridization was observed among alpha 1-, alpha 2-, and alpha 3-tubulin specific sequences in all species studied, using either heterologous or homologous probes. However, unlike D. melanogaster, in all montium species studied, both alpha 1- and alpha 3-tubulin specific probes hybridize to the same polytene band, indicating a clustered organization of the above genes. The chromosomal organization of this gene family would suggest that taxa within the montium subgroup are closer to their common ancestors than are the taxa in the melanogaster species group. A mode of evolution for this gene family in Drosophila is proposed.

Animals↗