Intercalary heterochromatin in polytene chromosomes of <Emphasis Type="Italic">Drosophila melanogaster</Emphasis>

Intercalary heterochromatin consists of extended chromosomal domains which are interspersed throughout the euchromatin and contain silent genetic material. These domains comprise either clusters of functionally unrelated genes or tandem gene duplications and possibly stretches of noncoding sequences. Strong repression of genetic activity means that intercalary heterochromatin displays properties that are normally attributable to classic pericentric heterochromatin: high compaction, late replication and underreplication in polytene chromosomes, and the presence of heterochromatin-specific proteins. Late replication and underreplication occurs when the suppressor of underreplication protein is present in intercalary heterochromatic regions. Intercalary heterochromatin underreplication in polytene chromosomes results in free double-stranded ends of DNA molecules; ligation of these free ends is the most likely mechanism for ectopic pairing between intercalary heterochromatic and pericentric heterochromatic regions. No support has been found for the view that the frequency of chromosome aberrations is elevated in intercalary heterochromatin.     

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.     

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.     

DNA content of mitotically-active condensed chromosomes of <Emphasis Type="Italic">Drosophila melanogaster</Emphasis>

Two-wavelength Feulgen microspectrophotometry was used to determine the DNA content of mitotically-active ganglionic cells of first-and thirdinstar larvae of Drosophila melanogaster. The measurements revealed that the DNA values differ, on the average, by a factor of approximately two, with the metaphase cells of the first-instar larvae having about four times the haploid amount of the spermatozoon, and the metaphase cells of the third-instar larvae having about eight times the haploid amount. The increase from 4C to 8C in the course of development without any pronounced modification of the heterochromatic—euchromatic ratio is interpreted as evidence of an increase in the number of chromosomal strands. It is suggested, accordingly, that these mitotically-active chromosomes are multistranded or polynemie.     

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.     

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).     

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.     

A simple and rapid method for constructing ring-<Emphasis Type="Italic">X</Emphasis> chromosomes in <Emphasis Type="Italic">Drosophila melanogaster</Emphasis>

Ring chromosomes are of basic interest to the geneticist and cell biologist who study their behavior in meiotic and mitotic divisions. In addition, the mitotic instability associated with some ring-X chromosomes has proven useful in Drosophila as a means to produce gynandromorphs for developmental studies. We describe a method to construct ring-X chromosomes in Drosophila via I-CreI-mediated exchange in rDNA, and then rapidly diagnose the recovery of ring chromosomes via FLP-mediated sister chromatid exchange within the ring. The method we describe provides a ready means to tailor the genetic content of ring-X chromosomes, making it suited to produce ring-X chromosomes for a variety of experimental purposes.     

Deleterious mutations in various <Emphasis Type="Italic">Drosophila melanogaster</Emphasis> strains carrying meiotic mutation <Emphasis Type="Italic">c(3)G</Emphasis>

In the absence of meiotic recombination, deleterious mutations, decreasing the viability, are accumulated and fixed in small Drosophila populations. Study of the viability of hybrid progenies of three laboratory Drosophila melanogaster strains carrying meiotic mutation c(3)G ¹⁷ has suggested that the deleterious mutations are negatively synergistic in their interaction. The deleterious mutations localized to the pericentromeric region of chromosome 3 are threefold more efficient as compared with the mutations located in distal regions. Substitution of a new chromosome for the balancer chromosome in a strain with meiotic mutation c(3)G ¹⁷ partially restores (by ～20%) the viability of homozygotes c(3)G ¹⁷ /c(3)G ¹⁷ over the first 20–30 generations. Further cultivation for 30 generations with the same balancer again decreases the viability to the initial level. An epigenetic nature of deleterious mutations is discussed.     

<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.     

<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.     

<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">Ty</Emphasis>1<Emphasis Type="Italic">/copia</Emphasis>- and <Emphasis Type="Italic">Ty</Emphasis>3<Emphasis Type="Italic">/gypsy</Emphasis>-like DNA sequences in <Emphasis Type="Italic">Helianthus </Emphasis>species

Two repeated DNA sequences isolated from a partial genomic DNA library of Helianthus annuus, p HaS13 and p HaS211, were shown to represent portions of the int gene of a Ty3 /gypsy retroelement and of the RNase-Hgene of a Ty1 /copia retroelement, respectively. Southern blotting patterns obtained by hybridizing the two probes to BglII- or DraI-digested genomic DNA from different Helianthus species showed p HaS13 and p HaS211 were parts of dispersed repeats at least 8 and 7 kb in length, respectively, that were conserved in all species studied. Comparable hybridization patterns were obtained in all species with p HaS13. By contrast, the patterns obtained by hybridizing p HaS211 clearly differentiated annual species from perennials. The frequencies of p HaS13- and p HaS211-related sequences in different species were 4.3x10(4)-1.3x10(5) copies and 9.9x10(2)-8.1x10(3) copies per picogram of DNA, respectively. The frequency of p HaS13-related sequences varied widely within annual species, while no significant difference was observed among perennial species. Conversely, the frequency variation of p HaS211-related sequences was as large within annual species as within perennials. Sequences of both families were found to be dispersed along the length of all chromosomes in all species studied. However, Ty3 /gypsy-like sequences were localized preferentially at the centromeric regions, whereas Ty1/ copia-like sequences were less represented or absent around the centromeres and plentiful at the chromosome ends. These findings suggest that the two sequence families played a role in Helianthusgenome evolution and species divergence, evolved independently in the same genomic backgrounds and in annual or perennial species, and acquired different possible functions in the host genomes.     

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.     

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.     

In vivo analysis of <Emphasis Type="Italic">Drosophila</Emphasis> SU(<Emphasis Type="Italic">Z</Emphasis>)12 function

Polycomb group (PcG) proteins are required to maintain a stable repression of the homeotic genes during Drosophila development. Mutants in the PcG gene Supressor of zeste 12 (Su(z)12) exhibit strong homeotic transformations caused by widespread misexpression of several homeotic genes in embryos and larvae. Su(z)12 has also been suggested to be involved in position effect variegation and in regulation of the white gene expression in combination with zeste. To elucidate whether SU(Z)12 has any such direct functions we investigated the binding pattern to polytene chromosomes and compared the localization to other proteins. We found that SU(Z)12 binds to about 90 specific eukaryotic sites, however, not the white locus. We also find staining at the chromocenter and the nucleolus. The binding along chromosome arms is mostly in interbands and these sites correlate precisely with those of Enhancer-of-zeste and other components of the PRC2 silencing complex. This implies that SU(Z)12 mainly exists in complex with PRC2. Comparisons with other PcG protein-binding patterns reveal extensive overlap. However, SU(Z)12 binding sites and histone 3 trimethylated lysine 27 residues (3meK27 H3) do not correlate that well. Still, we show that Su(z)12 is essential for tri-methylation of the lysine 27 residue of histone H3 in vivo, and that overexpression of SU(Z)12 in somatic clones results in higher levels of histone methylation, indicating that SU(Z)12 is rate limiting for the enzymatic activity of PRC2. In addition, we analyzed the binding pattern of Heterochromatin Protein 1 (HP1) and found that SU(Z)12 and HP1 do not co-localize.