Drosophila polymorphism database (DPDB): a portal for nucleotide polymorphism in Drosophila

As a growing number of haplotypic sequences from resequencing studies are now accumulating for Drosophila in the main primary sequence databases, collectively they can now be used to describe the general pattern of nucleotide variation across species and genes of this genus. The Drosophila Polymorphism Database (DPDB) is a secondary database that provides a collection of all well-annotated polymorphic sequences in Drosophila together with their associated diversity measures and options for reanalysis of the data that greatly facilitate both multi-locus and multi-species diversity studies in one of the most important groups of model organisms. Here we describe the state-of-the-art of the DPDB database and provide a step-by-step guide to all its searching and analytic capabilities. Finally, we illustrate its usefulness through selected examples. DPDB is freely available at http://dpdb.uab.cat.     

From bioassays to Drosophila genetics: strategies for characterizing an essential insect neurohormone, bursicon

We describe the molecular analysis and cellular expression of the insect peptide neurohormone, bursicon. Bursicon triggers the sclerotization of the soft insect cuticle after ecdysis. Using protein elution analyses from SDS gels, we determined the molecular weight of bursicon from different insects to be approximately 30 kDa. Four partial peptide sequences of Periplaneta americana bursicon were obtained from purified nerve cord homogenates separated on two-dimensional gels. Antibodies produced against one of the sequences identified the cellular location of bursicon in different insects and showed that bursicon is co-produced with crustacean cardioactive peptide (CCAP) in the same neurons in all insects tested so far. Additionally, using the partial peptide sequences, we successfully searched the Drosophila genome project for the gene encoding bursicon. With Drosophila as a tool, we can now verify the function of the sequence using transgenic flies. Sequence comparisons also allowed us to verify that bursicon is conserved, corroborating the older data from bioassays and immunohistochemical analyses. The sequence of bursicon will enable further analysis of its function, release, and evolution.     

A strategy for mapping the heterochromatin of chromosome 2 of Drosophila melanogaster

The heterochromatin of chromosome 2 of Drosophila melanogaster has been among the best characterized models for functional studies of heterochromatin owing to its abundance of genetic markers. To determine whether it might also provide a favorable system for mapping extended regions of heterochromatin, we undertook a project to molecularly map the heterochromatin of the left arm of chromosome 2 (2Lh). In this paper, we describe a strategy that used clones and sequence information available from the Drosophila Genome Project and chromosome rearrangements to construct a map of the distal most portion of 2Lh. We also describe studies that used fluorescent in situ hybridization (FISH) to examine the resolution of this technique for cytologically resolving heterochromatic sequences on mitotic chromosomes. We discuss how these mapping studies can be extended to more proximal regions of the heterochromatin to determine the structural patterns and physical dimensions of 2Lh and the relationship of structure to function.     

Computational identification of novel chitinase-like proteins in the Drosophila melanogaster genome

MOTIVATION: Multiple chitinases as well as lectins closely related to them have been characterized previously from many insect species and the corresponding genes/cDNAs have been cloned. However, the identification of the entire assortment of genes for chitinase family proteins and their differences in biochemical properties have not been carried out in any individual insect species. The completion of the entire DNA sequence of Drosophila melanogaster (fruit fly) genome and identification of open reading frames presents an opportunity to study the structures and functions of chitinase-like proteins, and also to identify new members of this family in DROSOPHILA: We are, therefore, interested in studying the functional genomics of chitinase-like gene families in insects. METHODS: We searched the Drosophila protein sequences database using fully characterized insect chitinase sequences and BLASTP software, identified all the putative chitinase-like proteins encoded in Drosophila genome, and predicted their structures using domain analysis tools. A phylogenetic analysis of the chitinase-like proteins from Drosophila and several other insect species was carried out. The structures of these chitinases were modeled using homology modeling software. RESULTS: Our analysis revealed the presence of 18 chitinase-like proteins in the Drosophila protein database. Among these are seven novel chitinase-like proteins that contain four signature amino acid sequences of chitinases belonging to family 18 glycosylhydrolases, including both acidic and hydrophobic amino acid residues critical for enzyme activity. All the proteins contain at least one catalytic domain with one having four catalytic domains. Phylogenetic analysis of chitinase-like proteins from Drosophila and other insects revealed an evolutionary relationship among all these proteins, which indicated gene duplication and domain shuffling to generate the observed diversity in the encoded proteins. Homology modeling showed that all the Drosophila chitinase-like proteins contain one or more catalytic domains with a (alpha/beta)8 barrel-like structure. Our results suggest that insects utilize multiple family 18 chitinolytic enzymes and also non-enzymatic chitinase-like proteins for degrading/remodeling/binding to chitin in different insect anatomical extracellular structures, such as the cuticle, peritrophic membrane, trachea and mouth parts during insect development, and possibly for other roles including chitin synthesis. AVAILABILITY: Perl program and supplementary material are available at http://www.ksu.edu/bioinformatics/supplementary.htm     

FlyNets and GIF-DB, two internet databases for molecular interactions in Drosophila melanogaster.

GIF-DB and FlyNets are two WWW databases describing molecular (protein-DNA, protein-RNA and protein-protein) interactions occuring in the fly Drosophila melanogaster (http://gifts.univ-mrs.fr/GIFTS_home_page.html ). GIF-DB is a specialised database which focuses on molecular interactions involved in the process of embryonic pattern formation, whereas FlyNets is a new and more general database, the long-term goal of which is to report on any published molecular interaction occuring in the fly. The information content of both databases is distributed in specific lines arranged into an EMBL- (or GenBank-) like format. These databases achieve a high level of integration with other databases such as FlyBase, EMBL, GenBank and SWISS-PROT through numerous hyperlinks. In addition, we also describe SOS-DGDB, a new collection of annotated Drosophila gene sequences, in which binding sites for regulatory proteins are directly visible on the DNA primary sequence and hyperlinked both to GIF-DB and TRANSFAC database entries.     

Molecular genetics of Drosophila vision

The fruitfly, Drosophila melanogaster, is an excellent organism for dissecting the components of vision genetically. Many mutations have been generated that affect a diversity of processes important in vision. Through a combined application of molecular and genetic approaches many of the genes important in Drosophila vision are now being identified.     

MTV sings jubilation for telomere biology in Drosophila

Telomere protects the ends of linear chromosomes. Telomere dysfunction fuels genome instability that can lead to diseases such as cancer. For over 30 years, Drosophila has fascinated the field as the only major model organism that does not rely on the conserved telomerase enzyme for end protection. Instead of short DNA repeats at chromosome ends, Drosophila has domesticated retrotransposons. In addition, telomere protection can be entirely sequence-independent under normal laboratory conditions, again dissimilar to what has been established for telomerase-maintained systems. Despite these major differences, recent studies from us and others have revealed remarkable similarities between the 2 systems. In particular, with the identification of the MTV complex as an ssDNA binding complex essential for telomere integrity in Drosophila (Zhang et al. 2016 Plos Genetics), we have now established several universal principles that are intrinsic to chromosome extremities but independent of the underlying DNA sequences or the telomerase enzyme. Telomere studies in Drosophila will continue to yield fundamental insights that are instrumental to the understanding of the evolution of telomere and telomeric functions.     

Polymorphism and Locus-Specific Effects on Polymorphism at Microsatellite Loci in Natural Drosophila Melanogaster Populations

We have studied the natural variation at microsatellite loci in two African and five non-African populations of Drosophila melanogaster. Ten dinucleotide simple sequence loci were cloned from chromosomally mapped P1 clones and typed for single individuals from isofemale lines of the respective populations. We find that the African populations harbor the largest degree of diversity, while the non-African populations show a lower diversity. This supports previous results that D. melanogaster originated in Africa and spread across the rest of the world in historic times. Using genetic distance measures, we find also a distinct population subdivision between the non-African populations. Most interestingly, we find for some loci in some populations a strongly reduced variability, which cannot be explained by bottleneck effects. Employing a conservative test based on the variance in repeat number, we find that at least one locus in one population deviates significantly from the expectations of mutation-drift equilibrium. We suggest that this may be due to a recent selective sweep in this chromosomal region that may have been caused by a linked locus that was involved in local adaptation of the population.     

Genome, evolution, Drosophila and beyond: the new dimensions

A century ago a little fly with red eyes was first used for genetic studies. That insignificant fly, called at that time Drosophila ampelophila, revolutionized biology while becoming the model we know today under the name of Drosophila melanogaster. Since then its study has never ceased, but the field of interest has somewhat changed during the century. To caricature a little, today we essentially learn from Drosophila meetings that the fly has a brain! It is true that the fly is a tremendous model organism for neurobiology. But this fly is, in fact, an appropriate and recognized model for the whole of biology. Indeed, Drosophila meetings are exceptional opportunities to gather biologists of diverse backgrounds together. There we not only learn about the latest improvements in our field of interest, but surely appreciate learning another bit of biology. From this biological melting pot has emerged a culture very specific to the fly community. Thus besides neurobiology, cell biology and development, a diversity of other research fields exist; they all have their own place in the cultural and historical dimension of the "drosophila" model. Several communications from those diverse research fields were presented at the 8th Japanese Drosophila Research Conference (JDRC8) and are briefly covered here. We believe it more judicious to call the model "drosophila" without a capital initial, as the model has never really been limited to only the Drosophila genus. The vernacular name "drosophila" is currently used to designate any fly of the Drosophilidae family and we believe the term more appropriate than "small fruit fly" or "vinegar fly" to better include the species and ecological diversity of the model.     

Wolbachia Infections in Drosophila Melanogaster and D. Simulans: Polymorphism and Levels of Cytoplasmic Incompatibility

Wolbachia are endosymbiotic bacteria, widespread in terrestrial Arthropods. They are mainly transmitted vertically, from mothers to offspring and induce various alterations of their hosts' sexuality and reproduction, the most commonly reported phenomenon being Cytoplasmic Incompatibility (CI), observed in Drosophila melanogaster and D. simulans. Basically, CI results in a more or less intense embryonic mortality, occurring in crosses between males infected by Wolbachia and uninfected females. In D. simulans, Wolbachia and CI were observed in 1986. Since then, this host species has become a model system for investigating the polymorphism of Wolbachia infections and CI. In this review we describe the different Wolbachia infections currently known to occur in D. melanogaster and D. simulans. The two species are highly contrasting with regard to symbiotic diversity: while five Wolbachia variants have been described in D. simulans natural populations, D. melanogaster seems to harbor one Wolbachia variant only. Another marked difference between these two Drosophila species is their permissiveness with regard to CI, which seems to be fully expressed in D. simulans but partially or totally repressed in D. melanogaster, demonstrating the involvement of host factors in the control of CI levels. The potential of the two host species regarding the understanding of CI and its evolution is also discussed.     

Unusual pattern of single nucleotide polymorphism at the exuperantia2 locus of Drosophila pseudoobscura

We have investigated the pattern of DNA sequence variation at the exuperantia2 locus in Drosophila pseudoobscura. This adds to the increasing dataset of genetic variation in D. pseudoobscura, a useful model species for evolutionary genetic studies. The level of silent site nucleotide diversity and the divergence from an outgroup Drosophila miranda are comparable with those for other X-linked loci. One peculiar pattern at the exu2 locus of D. pseudoobscura is a complete linkage disequilibrium between two SNPs, one of which is a replacement site. As a result, there are two distinct haplotype groups in our dataset. Based upon the comparisons with the outgroup sequences from D. miranda and Drosophila persimilis, we show that the newly derived haplotype group has lower diversity than the ancestral haplotype group. The pattern of protein evolution at exu2 shows some deviation from the neutral model. Together, these and other characteristics of the exu2 locus suggest the action of selection on the pattern of SNP variation, consistent with a partial selective sweep associated with the newly derived haplotype.     

Molecular evolution of phosphoprotein phosphatases in Drosophila

Phosphoprotein phosphatases (PPP), these ancient and important regulatory enzymes are present in all eukaryotic organisms. Based on the genome sequences of 12 Drosophila species we traced the evolution of the PPP catalytic subunits and noted a substantial expansion of the gene family. We concluded that the 18-22 PPP genes of Drosophilidae were generated from a core set of 8 indispensable phosphatases that are present in most of the insects. Retropositons followed by tandem gene duplications extended the phosphatase repertoire, and sporadic gene losses contributed to the species specific variations in the PPP complement. During the course of these studies we identified 5, up till now uncharacterized phosphatase retrogenes: PpY+, PpD5+, PpD6+, Pp4+, and Pp6+ which are found only in some ancient Drosophila. We demonstrated that all of these new PPP genes exhibit a distinct male specific expression. In addition to the changes in gene numbers, the intron-exon structure and the chromosomal localization of several PPP genes was also altered during evolution. The G-C content of the coding regions decreased when a gene moved into the heterochromatic region of chromosome Y. Thus the PPP enzymes exemplify the various types of dynamic rearrangements that accompany the molecular evolution of a gene family in Drosophilidae.     

Fungal diversity associated with hawaiian Drosophila host plants

Hawaiian Drosophila depend primarily, sometimes exclusively, on specific host plants for oviposition and larval development, and most specialize further on a particular decomposing part of that plant. Differences in fungal community between host plants and substrate types may establish the basis for host specificity in Hawaiian Drosophila. Fungi mediate decomposition, releasing plant micronutrients and volatiles that can indicate high quality substrates and serve as cues to stimulate oviposition. This study addresses major gaps in our knowledge by providing the first culture-free, DNA-based survey of fungal diversity associated with four ecologically important tree genera in the Hawaiian Islands. Three genera, Cheirodendron, Clermontia, and Pisonia, are important host plants for Drosophila. The fourth, Acacia, is not an important drosophilid host but is a dominant forest tree. We sampled fresh and rotting leaves from all four taxa, plus rotting stems from Clermontia and Pisonia. Based on sequences from the D1/D2 domain of the 26S rDNA gene, we identified by BLAST search representatives from 113 genera in 13 fungal classes. A total of 160 operational taxonomic units, defined on the basis of ≥97% genetic similarity, were identified in these samples, but sampling curves show this is an underestimate of the total fungal diversity present on these substrates. Shannon diversity indices ranged from 2.0 to 3.5 among the Hawaiian samples, a slight reduction compared to continental surveys. We detected very little sharing of fungal taxa among the substrates, and tests of community composition confirmed that the structure of the fungal community differed significantly among the substrates and host plants. Based on these results, we hypothesize that fungal community structure plays a central role in the establishment of host preference in the Hawaiian Drosophila radiation.