Patterns of <Emphasis Type="Italic"> Hermes </Emphasis>transposition in <Emphasis Type="Italic"> Drosophila melanogaster</Emphasis>

Transposable elements are being developed as tools for genomics and for the manipulation of insect genotypes for the purposes of biological control. An understanding of their transposition behavior will facilitate the use of these elements. The behavior of an autonomous Hermes transposable element from Musca domestica in the soma and germ-line of Drosophila melanogaster was investigated using the method of transposon display. In the germ-line, Hermes transposed at a rate of approximately 0.03 jumps per element per generation. Within the soma Hermes exhibited markedly non-random patterns of integration. Certain regions of the genome were distinctly preferred over others as integration targets, while other regions were underrepresented among the integration sites used. One particular site accounted for 4.4% of the transpositions recovered in this experiment, all of which were located within a 2.5-kb region of the actin5C promoter. This region was also present within the Hermes element itself, suggesting that this clustering is an example of transposable element "homing". Clusters of integration sites were also observed near the original donor sites; these represent examples of local hopping. The information content (sequence specificity) of the 8-bp target site was low, and the consensus target site resembles that determined from plasmid-based integration assays.     

Gene <Emphasis Type="Italic">dilp6</Emphasis> regulates octopamine metabolism in <Emphasis Type="Italic">Drosophila melanogaster</Emphasis>

The effect of strong hypomorphic mutation of the insulin-like protein gene (dilp6) on metabolism of octopamine (one of the main biogenic amines in insects) was studied in Drosophila melanogaster males and females. The activity of tyrosine decarboxylase (the key enzyme of octopamine synthesis) and the activity of octopamine-dependent N-acetyltransferase (the enzyme of its degradation) were measured. It was demonstrated that the activity of both studied enzymes is decreased under normal conditions in the dilp6⁴¹ mutants (as we previously demonstrated, this is correlated with an increased level of octopamine). It was also found that hypomorphic mutation of the dilp6 gene decreases the intensity of tyrosine decarboxylase response to heat stress. Thus, it was demonstrated for the first time that insulin-like DILP6 protein in drosophila influences the level of octopamine (regulating the activity of the enzyme degrading octopamine).     

Patterns of Gene Duplication in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> and <Emphasis Type="Italic">Caenorhabditis elegans</Emphasis>

In this paper we present a new method for detecting block duplications in a genome. It is more stringent than previous ones in that it requires a more rigorous definition of paralogous genes and that it requires the paralogous proteins on the two blocks to be contiguous. In addition, it provides three criterion choices: (1) the same composition (i.e., having the same paralogues in the two windows), (2) the same composition and gene order, and (3) the same composition, gene order, and gene orientation. The method is completely automated, requiring no visual inspection as in previous methods. We applied it to analyze the complete genomes of S. cerevisiae and C. elegans. In yeast we detected fewer duplicated blocks than previously reported. In C. elegans, however, we detected more block duplications than previously reported, indicating that although our method has a more stringent definition of block duplication than previous ones, it may be more sensitive in detection because it considers every possible window rather than only fixed nonoverlapping windows. Our results show that block duplication is a common phenomenon in both organisms. The patterns of block duplication in the two species are, however, markedly different. The yeast shows much more extensive block duplication than the nematode, with some chromosomes having more than 40% of the duplications derived from block duplications. Moreover, in the yeast the majority of block duplications occurred between chromosomes, while in the nematode most block duplications occurred within chromosomes.     

Egg-laying rhythm in <Emphasis Type="Italic">Drosophila melanogaster</Emphasis>

Extensive research has been carried out to understand how circadian clocks regulate various physiological processes in organisms. The discovery of clock genes and the molecular clockwork has helped researchers to understand the possible role of these genes in regulating various metabolic processes. In Drosophila melanogaster, many studies have shown that the basic architecture of circadian clocks is multi-oscillatory. In nature, different neuronal subgroups in the brain of D. melanogaster have been demonstrated to control different circadian behavioural rhythms or different aspects of the same circadian rhythm. Among the circadian phenomena that have been studied so far in Drosophila, the egg-laying rhythm is unique, and relatively less explored. Unlike most other circadian rhythms, the egg-laying rhythm is rhythmic under constant light conditions, and the endogenous or free-running period of the rhythm is greater than those of most other rhythms. Although the clock genes and neurons required for the persistence of adult emergence and activity/rest rhythms have been studied extensively, those underlying the circadian egg-laying rhythm still remain largely unknown. In this review, we discuss our current understanding of the circadian egg-laying rhythm in D. melanogaster, and the possible molecular and physiological mechanisms that control the rhythmic output of the egg-laying process.     

Expression of <Emphasis Type="Italic">Ciona intestinalis</Emphasis> AOX causes male reproductive defects in <Emphasis Type="Italic">Drosophila melanogaster</Emphasis>

Background

Mitochondrial alternative respiratory-chain enzymes are phylogenetically widespread, and buffer stresses affecting oxidative phosphorylation in species that possess them. However, they have been lost in the evolutionary lineages leading to vertebrates and arthropods, raising the question as to what survival or reproductive disadvantages they confer. Recent interest in using them in therapy lends a biomedical dimension to this question.

Methods

Here, we examined the impact of the expression of Ciona intestinalis alternative oxidase, AOX, on the reproductive success of Drosophila melanogaster males. Sperm-competition assays were performed between flies carrying three copies of a ubiquitously expressed AOX construct, driven by the α-tubulin promoter, and wild-type males of the same genetic background.

Results

In sperm-competition assays, AOX conferred a substantial disadvantage, associated with decreased production of mature sperm. Sperm differentiation appeared to proceed until the last stages, but was spatially deranged, with spermatozoids retained in the testis instead of being released to the seminal vesicle. High AOX expression was detected in the outermost cell-layer of the testis sheath, which we hypothesize may disrupt a signal required for sperm maturation.

Conclusions

AOX expression in Drosophila thus has effects that are deleterious to male reproductive function. Our results imply that AOX therapy must be developed with caution.

     

<Emphasis Type="Italic">Wolbachia</Emphasis> infection increases recapture rate of field-released <Emphasis Type="Italic">Drosophila melanogaster</Emphasis>

Wolbachia pipientis is a commonly occurring endosymbiont with well-characterised effects on host reproductive biology associated with its infection of the gonads. Wolbachia infections are also widespread in somatic tissues and consequently they have the potential to play a much broader role in host biology. Recently, Wolbachia was shown to alter the locomotion of Drosophila melanogaster in response to food cues in the laboratory. To determine whether this laboratory-based phenotype might translate to real differences for insects in the field, we performed a simple mark-release-recapture experiment with Wolbachia-infected D. melanogaster in a forested habitat. We demonstrate that infected flies are recaptured at twice the rate of uninfected flies, although infection does not affect the distance traveled by those flies recaptured. The differences in recapture could be explained by infection-induced changes in physiology or behavior. If generalizable, such changes may affect the interpretation of behavioral studies for Wolbachia-infected insects and have potential implications for the dynamics of Wolbachia infections in natural populations, including situations where Wolbachia-infected insects are being released for biological control.     

The endosymbiont <Emphasis Type="Italic">Wolbachia</Emphasis> in Eurasian populations of <Emphasis Type="Italic">Drosophila melanogaster</Emphasis>

The endosymbiotic α-proteobacteria Wolbachia is widely spread among arthropods and Filariidae nematodes. This bacterium is transmitted vertically via a transovarian route. Wolbachia is a cause of several reproductive abnormalities in the host species. We analyzed the isofemale lines created using flies collected from Drosophila melanogaster natural populations for infection with the endosymbiont Wolbachia. Wolbachia were genotyped according to five variable markers: the presence of insertion sequence IS5 in two loci, the copy number of two minisatellite repeats, and an inversion. Overall, 665 isofemale lines isolated from the populations of D. melanogaster from Ukraine, Belarus, Moldova, Caucasus, Central Asia, Ural, Udmurtia, Altai, West and East Siberia, and Far East in 1974 through 2005 were used in the work. The samples from Ukrainian, Altaian, and Middle Asian populations were largest. The infection rate of D. melanogaster populations from Middle Asia, Altaian, and Eastern Europe (Ukraine, Moldavia, and Belarus) with Wolbachia amounted to 64, 56, and 39%, respectively. The D. melanogaster population from the Caucasus displayed heterogeneity in the genotypes of this cytoplasmic infection. The Wolbachia genotype wMel, detected in all the populations studied, was the most abundant. The genotype wMelCS2 was always present in the populations from Middle Asia and Altai and was among the rare variants in the D. melanogaster populations from the Eastern Europe. Single instances of the Wolbachia genotype wMelCS occurred in a few flies from the Central Asian and Altai populations, but was not found this genotype in the other regions.     

<Emphasis Type="Italic">Drosophila melanogaster</Emphasis> gene <Emphasis Type="Italic">Merlin</Emphasis> interacts with the clathrin adaptor protein gene <Emphasis Type="Italic">lap</Emphasis>

The protein Merlin is involved in the regulation of cell proliferation and differentiation in the eyes and wings of Drosophila and is a homolog of the human protein encoded by the Neurofibromatosis 2 (NF2) gene whose mutations cause auricular nerve tumors. Recent studies show that Merlin and Expanded cooperatively regulate the recycling of membrane receptors, such as the epidermal growth factor receptor (EGFR). By performing a search for potential genetic interactions between Merlin (Mer) and the genes important for vesicular trafficking, we found that ectopic expression in the wing pouch of the clathrin adapter protein Lap involved in clathrin-mediated receptor endocytosis resulted in the formation of extra vein materials. On the one hand, coexpression of wild-type Merlin and lap in the wing pouch restored normal venation, while overexpression of a dominant-negative mutant Mer ^DBB together with lap enhanced ectopic vein formation. Using various constructs with Merlin truncated copies, we showed the C-terminal portion of the Merlin protein to be responsible for the Merlin-lap genetic interaction. Furthermore, we showed that the Merlin and Lap proteins colocalized at the cortex of the wing imaginal disc cells.     

<Emphasis Type="Italic">Drosophila melanogaster</Emphasis> as a polymicrobial infection model for <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> and <Emphasis Type="Italic">Staphylococcus aureus</Emphasis>

Non-mammalian infection models have been developed over the last two decades, which is a historic milestone to understand the molecular basis of bacterial pathogenesis. They also provide small-scale research platforms for identification of virulence factors, screening for antibacterial hits, and evaluation of antibacterial efficacy. The fruit fly, Drosophila melanogaster is one of the model hosts for a variety of bacterial pathogens, in that the innate immunity pathways and tissue physiology are highly similar to those in mammals. We here present a relatively simple protocol to assess the key aspects of the polymicrobial interaction in vivo between the human opportunistic pathogens, Pseudomonas aeruginosa and Staphylococcus aureus, which is based on the systemic infection by needle pricking at the dorsal thorax of the flies. After infection, fly survival and bacteremia over time for both P. aeruginosa and S. aureus within the infected flies can be monitored as a measure of polymicrobial virulence potential. The infection takes ~24 h including bacterial cultivation. Fly survival and bacteremia are assessed using the infected flies that are monitored up to ~60 h post-infection. These methods can be used to identify presumable as well as unexpected phenotypes during polymicrobial interaction between P. aeruginosa and S. aureus mutants, regarding bacterial pathogenesis and host immunity.     

Molecular characterization of the <Emphasis Type="Italic">singed wings</Emphasis> locus of <Emphasis Type="Italic">Drosophila melanogaster</Emphasis>

Background  

Hormones frequently guide animal development via the induction of cascades of gene activities, whose products further amplify an initial hormonal stimulus. In Drosophila the transformation of the larva into the pupa and the subsequent metamorphosis to the adult stage is triggered by changes in the titer of the steroid hormone 20-hydroxyecdysone. singed wings (swi) is the only gene known in Drosophila melanogaster for which mutations specifically interrupt the transmission of the regulatory signal from early to late ecdysone inducible genes.     

Functional conservation of the<Emphasis Type="Italic"> Sex-lethal</Emphasis> sex determining promoter,<Emphasis Type="Italic"> Sxl-Pe</Emphasis>, in<Emphasis Type="Italic"> Drosophila virilis</Emphasis>

The primary sex determination signal in Drosophila melanogaster, the ratio of X chromosomes to autosomes, sets the activity state of the switch gene, Sex-lethal ( Sxl), by regulating the establishment promoter, m-Sxl-Pe. We have identified and characterized the establishment promoter, v-Sxl-Pe, of the distantly related species Drosophila virilis. Like melanogaster, the virilis Sxl-Pe is organized into four sub-domains: the Sxl-Pe mRNA leader and exon E1 of Sxl protein, the core promoter, the sex-specific element and the augmentation element. The core promoter and sex-specific element of v-Sxl-Pe show considerable sequence similarity to m-Sxl-Pe and contain target sites for components of the X/A signaling system. While the augmentation element of v-Sxl-Pe also has sequence motifs that could function as target sites for the X/A signaling system, it shows little similarity to the melanogaster augmentation element. Functional studies reveal that v-Sxl-Pe drives sex-specific expression in D. melanogaster embryos and that the activity of the virilis promoter is controlled by known components of the melanogaster X/A counting system. Although v-Sxl-Pe responds appropriately to the melanogaster sex determination signal, it is less active than Sxl-Pe from melanogaster. Unexpectedly, the reduced activity is due to differences in the activity of the conserved core promoter, while the non-conserved augmentation element functions effectively. These findings suggest that low-affinity target sites for the X/A counting system are critical for the functioning of Sxl-Pe.     

Polymorphism of <Emphasis Type="Italic">yellow</Emphasis> locus in <Emphasis Type="Italic">Drosophila melanogaster</Emphasis> mutants from natural populations

We have studied the molecular characteristics of the yellow locus (y; 1–0.0), which determines the body color of phenotypically wild-type and mutant alleles isolated in different years from geographically distant populations of Drosophila melanogaster. According to the Southern blot, data restriction maps of the yellow locus of all examined strains differ from one another, as well as from Oregon stock. FISH analysis shows that, in the neighborhood of the yellow locus in the X chromosome, neither P nor hobo elements are found in y^1–775 stock, while only hobo is found in these region in y^1–859 and y^1–866 stocks, only the P element is found in y⁺sn⁸⁴⁹ stock, and both elements are found in y^1–719 stock. Thus, all yellow mutants studied are of independent origin. Locus yellow located on the end of X chromosome (region 1A5–8 on the cytologic map) carries significantly more transposon than retrotransposon induced mutations compared to the white locus (region 3C2). It is possible that, at the ends of Drosophila melanogaster chromosomes, transposons are more active than retrotransposons.     

Prevalence of full-size <Emphasis Type="Italic">P</Emphasis> and <Emphasis Type="Italic">KP</Emphasis> elements in North American populations of <Emphasis Type="Italic">Drosophila melanogaster</Emphasis>

The P transposable element invaded the Drosophila melanogaster genome in the middle of the twentieth century, probably from D. willistoni in the Caribbean or southeastern North America. P elements then spread rapidly and became ubiquitous worldwide in wild populations of D. melanogaster by 1980. To study the dynamics and long-term fate of transposable genetic elements, we examined the molecular profile of genomic P elements and the phenotype in the P-M system of the current North American natural populations collected in 2001-2003. We found that full-size P and KP elements were the two major size classes of P elements present in the genomes of all populations ("FP + KP predominance") and that the P-related phenotypes had largely not changed since the 1980s. Both FP + KP predominance and phenotypic stability were also seen in other populations from other continents. As North American populations did not show many KP elements in earlier samples, we hypothesize that KP elements have spread and multiplied in the last 20 years in North America. We suggest that this may be due to a transpositional advantage of KP elements, rather than to a role in P-element regulation.     

Mutagenesis testing using the <Emphasis Type="Italic">LacZ</Emphasis> reporter activity of the reparation gene <Emphasis Type="Italic">mus209</Emphasis> in <Emphasis Type="Italic">Drosophila melanogaster</Emphasis>

We studied a set of Drosophila melanogaster strains that could be potentially suitable for testing a variety of mutagenic factors. Their genomes contained insertions of the enhancer trap P {lacW}-in which the activity of the LacZ reporter is under the control of the reparation genes’ regulatory region. We demonstrated that the beta-galactosidase reporter, which is encoded by insertion of P {lacW} element in the gene mus209, is induced by irradiation in the cells of the salivary glands and wing imaginal discs. Despite the fact that the reporting coloration is not associated with the dose of radiation treatment, we found that the induction threshold of the reporter is different for these tissues. Thus, coloration in salivary glands is detectable after the dose of 200 rad and above, whereas the imaginal discs get colored with 500 rads and above. Thereby, multiple thresholds for induction of the reporter in the various tissues allow approximating the received dose.     

The regulation of <Emphasis Type="Italic">yp3</Emphasis> expression in the <Emphasis Type="Italic">Drosophila melanogaster</Emphasis> fat body

The regulation of the Drosophila melanogaster yolk protein genes 1 and 2 have been well characterised. Cis-acting DNA elements and trans-acting factors regulating ovarian fat body and sex-specific expression have been identified. In this paper we have analysed the regulation of yolk protein 3, which is separated from the other two genes on the X-chromosome. We have separated sex-specific control from fat body control in some constructs in transgenic flies. We propose that the organisation of the regulatory elements in yp3 differs from yp1 and yp2 for control of fat body expression and that it closely resembles the regulation of a reporter gene using Musca and Calliphora yp promoter enhancer sequences in transgenic Drosophila.     

Structural comparison of the <Emphasis Type="Italic">hsp70</Emphasis> gene cluster in the <Emphasis Type="Italic">Drosophila virilis</Emphasis> species group

One of the Drosophila montana hsp70 genes was cloned and sequenced. Its 3′-flanking sequence proved to harbor a fragment of the SGM mobile element. The element was also found in the hsp70 3′-flanking region of and other species of the species group. A reorganization of the cluster with the involvement of full-length SGM was found in a strain. It was assumed that the presence of SGM in the cluster is conserved among species of the group and that SGM played a role in evolutionarily rearrangements of the cluster.