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.     

Analysis of the chromocenter DNA composition in polytene chromosomes of <Emphasis Type=Italic>Drosophila orena</Emphasis> ovarian nurse cells

Chromocenter DNA fragments of polytene chromosomes of Drosophila orena ovarian nurse cells were cloned from a region-specific library (Dore1) in a plasmid vector to yield 133 clones. A total of 76 clones were selected and sequenced. The total length of the sequenced fragments was 23940 bp. Analysis with several software packages revealed various repetitive sequences among the fragments of the Dore1 library, including mobile genetic elements (25 fragments homologous to various LTR retrotransposons, five fragments homologous to LINEs, three fragments homologous to Helitrons, one fragment homologous to Polinton, and one fragment homologous to the mini-me non-LTR retrotransposon), four minisatellites, a satellite (SAR_DM), the (TATATG)n simple sequence repeat, and a low-complexity T-rich repeat. Sequences homologous to protein-coding genes were also found in the Dore1 library. Various repetitive DNA sequences and gene homologs were identified as conserved sequences of pericentric heterochromatin of polytene chromosomes of ovarian nurse cells in nine species of the melanogaster species subgroup.     

Study of the organization of polytene chromosomes in the nuclei of Drosophila melanogaster pseudonurse cells with the use of the DNA library of the Drosophila orena chromocenter

The location of the Drosophila orena chromocenter in polytene chromosomes of pseudonurse cells of the D. melanogaster ovaries (the otu11 mutation) and salivary glands has been studied. Numerous sites of location of the D. orena chromocenter DNA have been found throughout the length of D. melanogaster chromosomes. The specific distribution of the binding sites for the DNA probe has made it possible to identify chromosomes and analyze their mutual positions in the three-dimensional space of the nuclei of pseudonurse cells. The mutual positions of chromosomes have been found to vary, the pericentromeric regions of different chromosomes differing from one another in associative ratios.     

Three-dimensional organization of telomeres in nuclei of <Emphasis Type="Italic">Drosophila melanogaster</Emphasis> salivary glands

3D-FISH method was employed to investigate the telomere topology in polytene nuclei of salivary glands in Drosophila melanogaster. The majority of telomeres in polytene nuclei of salivary glands in Drosophila strain y^2-717 is localized in the nuclear central area and have no contacts with nuclear membrane. In the females ectopic contacts between telomeres occur in frequency 25% higher than in males. HeT-A DNA in y^2-717alk3-2 strain, which is a derivative of the y^2-717 stain carrying an inversion between 1B and 13C bands, is found in the region 13 of X chromosome. The frequency of ectopic contacts of telomeres in y^2-717alk3-2 males is 10% higher than in the y^2-717 strain. The number of ectopic contacts can be differ significantly in independent experiments, possibly indicating the role of random factors in the contact formation.     

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.     

Molecular and genetic organization of <Emphasis Type="Italic">Drosophila melanogaster</Emphasis>polytene chromosomes: evidence for two types of interband regions

The 3A and 60E regions of Drosophila melanogaster polytene chromosomes containing inserted copies of the P{lArB} transposon have been subjected to an electron microscopic (EM) analysis. We show that both inserts led to formation of new bands within the interband regions 3A4/A6 and 60E8-9/E10. This allowed us to clone DNA of these interbands. Their sequences, as well as those of DNA from other four interbands described earlier, have been analyzed. We have found that, with the exception of 60E8-9/E10 interband, all other five regions under study corresponded to 5' or 3' ends of genes. We have further obtained the evidence for 60E8-9/E10 interband to harbor the 'housekeeping' RpL19 gene, which is transcribed in many tissues, including salivary glands. Based upon the genetic heterogeneity of the interbands observed a revised model of polytene chromosome organization is discussed.     

Distorted heterochromatin replication in <Emphasis Type="Italic">Drosophila melanogaster</Emphasis> polytene chromosomes as a result of euchromatin-heterochromatin rearrangements

Studies of the position effect resulting from chromosome rearrangements in Drosophila melanogaster have shown that replication distortions in polytene chromosomes correlate with heritable gene silencing in mitotic cells. Earlier studies mostly focused on the effects of euchromatin-heterochromatin rearrangements on replication and silencing of euchromatic regions adjacent to the heterochromatin breakpoint. This review is based on published original data and considers the effect of rearrangements on heterochromatin: heterochromatin blocks that are normally underrepresented or underreplicated in polytene chromosomes are restored. Euchromatin proved to affect heterochromatin, preventing its underreplication. The effect is opposite to the known inactivation effect, which extends from heterochromatin to euchromatin. The trans-action of heterochromatin blocks on replication of heterochromatin placed within euchromatin is discussed. Distortions of heterochromatin replication in polytene chromosomes are considered to be an important characteristic associated with the functional role of the corresponding genome regions.     

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.     

Structural organization characteristics of the <Emphasis Type="Italic">DIP1</Emphasis> gene in <Emphasis Type="Italic">Drosophila melanogaster</Emphasis> strains mutant for the <Emphasis Type="Italic">flamenco</Emphasis> gene

Molecular cloning of the DIP1 gene located in the 20A4-5 region has been performed from the following strains with the flamenco phenotype: flam ^SS (SS) and flam ^MS (MS) characterized by a high transposition rate of retrotransposon gypsy (mdg4), flam ^{py + (P)} carrying the insertion of a construction based on the P element into the region of the flamenco gene, and flamenco ⁺. The results of restriction analysis and sequencing cloned DNA fragments has shown that strains flam ^SS , flam ^MS considerably differ from flam ^{py + (P)}, and flamenco ⁺ in the structure of DIP1. Strains flam ^SS and flam ^MS have no DraI restriction site at position 1765 in the coding region of the gene, specifically, in the domain determining the signal of the nuclear localization of the DIP1 protein. This mutation has been found to consist in a nucleotide substitution in the recognition site of DraI restriction endonuclease, which is transformed from TTTAAA into TTTAAG and, hence, is not recognized by the enzyme. This substitution changes codon AAA into AAG and is translationally insignificant, because both triplets encode the same amino acid, lysine. The DIP1 gene of strains flam ^SS and flam ^MS has been found to contain a 182-bp insertion denoted IdSS (insertion in DIP1 strain SS); it is located in the second intron of the gene. The IdSS sequence is part of the open reading frame encoding the putative transposase of the mobile genetic element HB1 belonging to the Tc1/mariner family. This insertion is presumed to disturb the conformations of DNA and the chromosome, in particular, by forming loops, which alters the expression of DIP1 and, probably, neighboring genes. In strains flamenco ⁺ and flam ^{py + (P)}, the IdSS insertion within the HB1 sequence is deleted. The deletion encompasses five C-terminal amino acid residues of the conserved domain and the entire C-terminal region of the putative HB1 transposase. The obtained data suggest that DIP1 is involved in the control of gypsy transpositions either directly or through interaction with other elements of the genome.     

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).     

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.     

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.     

The polytene dot chromosome of <Emphasis Type="Italic">Drosophila</Emphasis>: <Emphasis Type="Italic">D. melanogaster</Emphasis> and <Emphasis Type="Italic">D. subobscura</Emphasis>

The chromosome arms are assumed to be homologous within the genus Drosophila. Homology at the level of the polytene chromosome banding pattern between non-sibling species is, however, almost impossible to establish as different processes such as inversion, transposition and unequal crossing over, have disturbed it. Even though the band sequences cannot be followed, we may ask whether there is a correlation in the total number of bands between species. The polytene dot chromosome is an excellent starting point for such an approach. Here we present the detailed cytology of polytene chromosome 4 of D. melanogasterand the polytene dot chromosome of D. subobscura using electron microscopy. The results show that the number of bands is about the same, around 30, in both species. We predict that by using thin sections and electron microscopy for the longer polytene chromosome arms, both species will turn out to have approximately equal band numbers.     

<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.     

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.     

<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.     

Localization of <Emphasis Type="Italic">Dras1</Emphasis> gene in polytene chromosomes of sibling species of <Emphasis Type="Italic">Drosophila virilis</Emphasis> group

The Dras1 gene was mapped by in situ hybridization to polytene chromosomes of several sibling species of the Drosophila virilis group and their hybrids. A 1037-bp fragment of Dras1 gene from the D. virilis genome was used as the probe. The gene sequence was localized in the region of a 25 A-B disk in chromosome 2 (in accordance with the D. virilis polytene chromosome map (Gubenko and Evgen’ev, 1984).     

<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.