Native Drosophila Research Articles

DNA binding dynamics and target location mechanism of pioneer factor GAF.

The chromatin landscape regulates transcription by localizing and regulating the activity of sequence-specific DNA binding proteins, chromatin remodeling enzymes and transcription machinery. Pioneer transcription factors can target DNA motifs on closed chromatin and trigger local chromatin remodeling to increase chromatin accessibility. Despite this knowledge, how pioneer factors search for their cognate motif on chromatin remains poorly understood. Here, we use multicolor single-molecule fluorescence resonance energy transfer (smFRET) to investigate how a prototypical Drosophila pioneer factor GAF locates its cognate motif on chromatin. To investigate how GAF searches for its cognate motif on DNA and nucleosomes, we strategically placed fluorophores on the DNA binding domain of GAF (GAF-DBD) and DNA motifs, allowing distinction of motif-specific binding and nonspecific DNA binding by FRET. We observed GAF-DBD dwells on a DNA molecule containing one GAF binding motif for 3-4 seconds before dissociation. Interestingly, on a DNA sequence with two GAF binding motifs, GAF-DBD can slide between two motifs while staying bound to the DNA. This suggests GAF monomers use one-dimensional (1D) sliding to search for its motif on nucleosome-free DNA. We next investigated the scenario where one of the two motifs is protected by a nucleosome. If the motif is located at the edge of a 601 nucleosome (SHL7 location), GAF-DBD more frequently lands on the other motif on linker DNA before it slides to the nucleosomal motif. Nucleosomes effectively block 1D sliding when the motif is located more internally on the nucleosome, at SHL5 or SHL3. However, on a nucleosome reconstituted on a native Drosophila DNA sequence, GAF-DBD can directly target internal motifs by three-dimensional diffusion, instead of 1D sliding. Our findings reveal different search modes that can be used by pioneer factors to locate their cognate motifs on chromatin.

Host searching and host preference of resident pupal parasitoids of Drosophila suzukii in the invaded regions

In its invaded regions, Drosophila suzukii (Matsumura) is a novel host for the community of resident parasitoids of Drosophila. To attain a high parasitization rate on the novel host, the parasitoids have to locate it and accept it in the presence of other Drosophila hosts. We conducted a laboratory choice experiment and a semifield trial to investigate host searching and host preference of the three pupal parasitoid species Trichopria drosophilae (Perkins), Pachycrepoideus vindemmiae (Rondani) and Spalangia erythromera Förster. All three parasitoid species preferred D. suzukii over two common native hosts in the choice experiment. In field cages, most parasitoid offspring emerged from D. suzukii hosts. While P. vindemmiae mainly parasitized hosts in the foliage, most T. drosophilae offspring emerged from pupae presented on the ground. Both P. vindemmiae and T. drosophilae have the potential to find and parasitize D. suzukii in the field. If released early in the season, possible nontarget effects on native Drosophila should be minimal.

A simple and versatile system for the ATP-dependent assembly of chromatin

Chromatin is the natural form of DNA in the eukaryotic nucleus and is the substrate for diverse biological phenomena. The functional analysis of these processes ideally would be carried out with nucleosomal templates that are assembled with customized core histones, DNA sequences, and chromosomal proteins. Here we report a simple, reliable, and versatile method for the ATP-dependent assembly of evenly spaced nucleosome arrays. This minimal chromatin assembly system comprises the Drosophila nucleoplasmin-like protein (dNLP) histone chaperone, the imitation switch (ISWI) ATP-driven motor protein, core histones, template DNA, and ATP. The dNLP and ISWI components were synthesized in bacteria, and each protein could be purified in a single step by affinity chromatography. We show that the dNLP-ISWI system can be used with different DNA sequences, linear or circular DNA, bulk genomic DNA, recombinant or native Drosophila core histones, native human histones, the linker histone H1, the non-histone chromosomal protein HMGN2, and the core histone variants H3.3 and H2A.V. The dNLP-ISWI system should be accessible to a wide range of researchers and enable the assembly of customized chromatin with specifically desired DNA sequences, core histones, and other chromosomal proteins.

Ectotherms in Variable Thermal Landscapes: A Physiological Evaluation of the Invasive Potential of Fruit Flies Species.

Climate change and biological invasions pose one of the greatest threats to biodiversity. Most analyses of the potential biological impacts have focused on changes in mean temperature, but changes in thermal variance may also impact native and invasive organisms, although differentially. We assessed the combined effects of the mean and the variance of temperature on the expression of heat shock protein (hsp90) in adults of the invasive fruit fly Drosophila melanogaster and the native Drosophila gaucha in Mediterranean habitats of central Chile. We observed that, under these experimental conditions, hsp90 mRNA expression was higher in the invasive species but absent in the native one. Apparently, the biogeographic origin and niche conservatisms are playing a role in the heat shock response of these species under different putative scenarios of climate change. We suggest that in order to develop more realistic predictions about the biological impact of climate change and biological invasions, one must consider the interactions between the mean and variance of climatic variables, as well as the evolutionary original conditions of the native and invasive species.

Prevalence of exotic frugivorous Drosophila species, D. simulans and D. immigrans (Diptera: Drosophilidae), and its effects on local parasitoids in Sapporo, northern Japan

The prevalence of exotic species has a variety of effects on native biological communities. To understand the effect of exotic species on a local Drosophila–parasitoid community, I investigated associations of native and exotic frugivorous Drosophila species (Diptera: Drosophilidae) with local parasitoids in Sapporo, northern Japan. In this survey, two exotic species, D. simulans Sturtevant and D. immigrans Sturtevant, accounted for approximately 88 % of Drosophila individuals that emerged from banana bait; two native species, D. auraria Peng and D. biauraria Bock & Wheeler, accounted for approximately 10 %. Seven larval parasitoid species were recorded as emerging from Drosophila larvae occurring in banana bait; six of these were examined for host use in laboratory experiments. Drosophila simulans was favored as host by four larval parasitoids, but D. immigrans was not favored by most of the larval parasitoids. Nevertheless, more than one-third of parasitoid individuals emerged from D. immigrans in the field study, probably because of its high abundance. Thus, exotic species are assumed to affect the abundance and distributions of native parasitoid species. In this study it was not clear whether the parasitoid species mediate the effects of exotic Drosophila species on native Drosophila species, because the incidence of parasitism was usually low.

The History of the Drosophila TRP Channel: The Birth of a New Channel Superfamily

: Transient receptor potential (TRP) channels are polymodal cellular sensors involved in a wide variety of cellular processes, mainly by changing membrane voltage and increasing cellular Ca2+. This review outlines in detail the history of the founding member of the TRP family, the Drosophila TRP channel. The field began with a spontaneous mutation in the trp gene that led to a blind mutant during prolonged intense light. It was this mutant that allowed for the discovery of the first TRP channels. A combination of electrophysiological, biochemical, Ca2+ measurements, and genetic studies in flies and in other invertebrates pointed to TRP as a novel phosphoinositide-regulated and Ca2+-permeable channel. The cloning and sequencing of the trp gene provided its molecular identity. These seminal findings led to the isolation of the first mammalian homologues of the Drosophila TRP channels. We now know that TRP channel proteins are conserved through evolution and are found in most organisms, tissues, and cell-types. The TRP channel superfamily is classified into seven related subfamilies: TRPC, TRPM, TRPV, TRPA, TRPP, TRPML, and TRPN. A great deal is known today about participation of TRP channels in many biological processes, including initiation of pain, thermoregulation, salivary fluid secretion, inflammation, cardiovascular regulation, smooth muscle tone, pressure regulation, Ca2+ and Mg2+ homeostasis, and lysosomal function. The native Drosophila photoreceptor cells, where the founding member of the TRP channels superfamily was found, is still a useful preparation to study basic features of this remarkable channel.

Electron microscopy and three-dimensional reconstruction of native thin filaments reveal species-specific differences in regulatory strand densities

Throughout the animal kingdom striated muscle contraction is regulated by the thin filament troponin–tropomyosin complex. Homologous regulatory components are shared among vertebrate and arthropod muscles; however, unique protein extensions and/or components characterize the latter. The Troponin T (TnT) isoforms of Drosophila indirect flight and tarantula femur muscle for example contain distinct C-terminal extensions and are ∼20% larger overall than their vertebrate counterpart. Using electron microscopy and three-dimensional helical reconstruction of native Drosophila, tarantula and frog muscle thin filaments we have identified species-specific differences in tropomyosin regulatory strand densities. The strands on the arthropod thin filaments were significantly larger in diameter than those from vertebrates, although not significantly different from each other. These findings reflect differences in the regulatory troponin–tropomyosin complex, which are likely due to the larger TnT molecules aligning and extending along much of the tropomyosin strands’ length. Such an arrangement potentially alters the physical properties of the regulatory strands and may help establish contractile characteristics unique to certain arthropod muscles.

Biochemical characterization of Drosophilagamma-glutamyl carboxylase and its role in fly development

To investigate structure-function relationships in gamma-glutamyl carboxylases, the enzyme from Drosophila melanogaster was characterized. Four cysteine residues were shown to be important determinants for enzymatic activity. Native Drosophila substrates have not yet been identified, but propeptides of human prothrombin and factor IX are recognized by the Drosophila enzyme. The presence of the propeptide region increased apparent affinity by approximately 200-fold, and mutation of a hydrophobic residue of factor IX propeptide (F-16A) decreased carboxylation by 90%, as in the human enzyme. Substrate recognition appears to be highly conserved between the human and Drosophila gamma-glutamyl carboxylases. Inactivation of Drosophila gamma-glutamyl carboxylase by non-sense mutations or insertional mutagenesis by P-element insertion have no apparent effects on growth and fertility under laboratory conditions.

Pharmacological profiles of recombinant and native insect nicotinic acetylcholine receptors

Nicotinic acetylcholine receptors (nAChRs) are targets for insect-selective neonicotinoid insecticides exemplified by imidacloprid (IMI) and mammalian-selective nicotinoids including nicotine and epibatidine (EPI). Despite their importance, insect nAChRs are poorly understood compared with their vertebrate counterparts. This study characterizes the [ 3H]IMI, [ 3H]EPI, and [ 3H] α-bungarotoxin ( α-BGT) binding sites in hybrid nAChRs consisting of Drosophila melanogaster (fruit fly) or Myzus persicae (peach–potato aphid) α2 coassembled with rat β2 subunits (D α2/R β2 and Mp α2/R β2) and compares them with native insect and vertebrate α4 β2 nAChRs. [ 3H]IMI and [ 3H]EPI bind to D α2/R β2 and Mp α2/R β2 hybrids but [ 3H] α-BGT does not. In native Drosophila receptors, [ 3H]EPI has a single high-affinity binding site that is independent from that for [ 3H]IMI and, interestingly, overlaps the [ 3H] α-BGT site. In the Mp α2/R β2 hybrid, [ 3H]IMI and [ 3H]EPI bind to the same site and have similar pharmacological profiles. On considering both neonicotinoids and nicotinoids, the D α2/R β2 and Mp α2/R β2 receptors display intermediate pharmacological profiles between those of native insect and vertebrate α4 β2 receptors, limiting the use of these hybrid receptors for predictive toxicology. These findings are consistent with the agonist binding site being located at the nAChR subunit interface and indicate that both α and β subunits influence the pharmacological properties of insect nAChRs.

Isolation and genetic characterisation of the Drosophila homologue of (SCE)REV3, encoding the catalytic subunit of DNA polymerase ζ

In Drosophila, about 30 mutants are known that show hypersensitivity to the methylating agent methyl methane sulfonate (MMS). Addition of this agent to the medium results in an increased larval mortality of the mutants. Using a P-insertion mutagenesis screen, three MMS-sensitive mutants on chromosome II were isolated. One of these is allelic to the known EMS-induced mus205 (mutagen sensitive) mutant. In the newly isolated mutant, a P-element is detected in region 43E by in situ hybridisation. The localisation of mus205 to this region was confirmed by deficiency mapping. The gene was cloned and shows strong homology to the Saccharomyces cerevisiae REV3 gene. The REV3 gene encodes the catalytic subunit of DNA polymerase ζ, involved in translesion synthesis. The P-element is inserted in the first exon of the mus205 gene resulting in an aberrant mRNA, encoding a putative truncated protein containing only the first 13 of the 2130 aa native Drosophila protein. The mus205 mutant is hypersensitive to alkylating agents and UV, but not to ionising radiation. In contrast to reported data, in germ cells, the mutant has no effect on mutability by X-rays, NQO and alkylating agents. In somatic cells, the mutant shows no effect on MMS-induced mutations and recombinations. This phenotype of the Drosophila mus205 mutant is strikingly different from the phenotype of the yeast rev3 mutant, which is hypomutable after UV, X-rays, NQO and alkylating agents.

Novel neonicotinoid-agarose affinity column for Drosophila and Musca nicotinic acetylcholine receptors.

Neonicotinoids such as the insecticide imidacloprid (IMI) act as agonists at the insect nicotinic acetylcholine receptor (nAChR). Head membranes of Drosophila melanogaster and Musca domestica have a single high-affinity binding site for [3H]IMI with KD values of 1-2 nM and Bmax values of 560-850 fmol/mg of protein. Locusta and Periplaneta nAChRs isolated with an alpha-bungarotoxin (alpha-BGT)-agarose affinity column are known to be alpha-subunit homooligomers. This study uses 1-[N-(6-chloro-3-pyridylmethyl)-N-ethyl]amino-1-amino-2-nitroethene++ + (which inhibits [3H]IMI binding to Drosophila and Musca head membranes at 2-3 nM) to develop a neonicotinoid-agarose affinity column. The procedure-introduction of Triton-solubilized Drosophila or Musca head membranes into this neonicotinoid-based column, elution with IMI, and analysis by lithium dodecyl sulfate-polyacrylamicle gel electrophoresis-gives only three proteins (69, 66, and 61 kDa) tentatively assigned as putative subunits of the nAChR; the same three proteins are obtained with Musca using the alpha-BGT-agarose affinity column. Photoaffinity labeling of the Drosophila and Musca putative subunits from the neonicotinoid column with 125I-alpha-BGT-4-azidosalicylic acid gives a labeled derivative of 66-69 kDa. The yield is 2-5 micrograms of receptor protein from 1 g of Drosophila or Musca heads. Neonicotinoid affinity chromatography to isolate native Drosophila and Musca receptors will facilitate studies on the structure and function of insect nAChRs.

Bacterial expression, characterization and DNA binding studies on Drosophila melanogaster c-Myb DNA-binding protein.

The Drosophila Myb homologue retains an evolutionarily conserved typical sequence of three imperfect tandem tryptophan repeat units (R1-R2-R3) of 51-53 amino acids towards its N-terminus as its presumptive DNA binding domain. Using PCR amplification and the T7 expression vector pET 11d, we have overproduced this tryptophan repeat domain of Drosophila Myb in Escherichia coli and the protein has been purified. Circular dichroic measurements indicate that the protein has a high helical component (58.6%) in its overall structure. The protein is found to recognize the same cognate target sequence TAACGG, as recognized by the vertebrate proteins. The DNA binding properties of the protein have been investigated in detail by fluorescence spectroscopy taking advantage of the large number of tryptophan residues present in the protein. The fluorescence of the native Drosophila R123 was quenched when synthetic duplex DNA oligomers were added to the protein. The oligomers containing specific Myb target sites quenched the protein fluorescence to a greater extent than the non-specific DNA. Binding constants of the protein to the targets were also length dependent for smaller oligomers. Experiments with the collisional quencher acrylamide and cysteine modification reagent indicated that the specific and non-specific target sequences interact with the protein differently. In the former case both the buried and the exposed tryptophan residues were affected by DNA binding whereas in the latter only the solvent-exposed residues were involved.

K + channel expression in primary cell cultures mediated by vaccinia virus

A recombinant vaccinia virus (VV) was used to express functional Drosophila Shaker H4 K + channels in primary cell cultures from rat heart (atrial and ventricular myocytes, fibroblasts), autonomic ganglia (SCG neurons) and CNS (hippocampal neurons, cerebral astroglia). In most cells the expressed currents possessed the typical characteristics of the native Drosophila muscle A currents; a few cells showed evidence of hetero-oligomers with new properties. The maximum current density corresponded to a channel density of 2–3/μm 3. Voltage recordings in heart cells showed altered action potential waveforms after successful infection. VV vectors thus are useful for studying altered excitability and cell-specific processing of ion channel proteins.

Field Dispersal and Population Sizes of Native Drosophila from North Carolina

Native Drosophila were periodically captured, dusted with micronized fluorescent dust and released during the summer of 1977 at two locations near Raleigh, North Carolina. Two or three days after each release, flies were collected in baited traps placed at regular intervals along a transect (800 m or 1,200 m long) centered on the point of release. Recaptured data provided estimates of the shape of the distribution of release flies as well as the dispersal rate of Drosophila, in particular D. melanogaster and D. simulans. Simultaneously collected unmarked flies provided estimates of natural population densities and effective sizes during the summer and early fall. Movement of marked flies averaged 150 m per day with a variance of around 20,000 m2. The observed distribution of a large sample of marked flies appeared to fit a Brownian motion model, in spite of a tendency for Drosophila to aggregate in moist, shady spots. The density of flies varied over the 4 mo tested, ranging from near zero to roughly 2-3 flies per 100 m2 for both D. melanogaster and D. simulans at the two experimental sites. Wright's formulas give estimates of effective population size in the range of 1,000-10,000 individuals during the peak summer populations, while the neighborhood (breeding) radius varied between 200-400 m. Dispersion and population size may be underestimated since evidence suggested dispersion of marked flies beyond the experimental area.