Thiazole Orange as an Alternative to Antibody Binding for Detecting Triple-helical DNA in Heterochromatin of Drosophila and Rhynchosciara.

The standard method for detecting triple-stranded DNA over the last 1.5 decades has been immune detection using antibodies raised against non-canonical nucleic acid structures. Many fluorescent dyes bind differentially to nucleic acids and often exhibit distinctive staining patterns along metaphase chromosomes dependent upon features, including binding to the major and minor DNA grooves, level of chromatin compaction, nucleotide specificity, and level of dye stacking. Relatively recently, the fluorochrome Thiazole Orange (TO) was shown to preferentially bind to triplex DNA in gels. Here, we demonstrate that TO also detects triplex DNA in salivary gland chromosomes of Drosophila melanogaster and Rhynchosciara americana identical in location and specificity to observations using antibodies. This finding may enable triple-stranded DNA investigations to be carried out on a much broader and reproducible scale than hitherto possible using antibodies, where a frequently encountered problem is the difference in detection specificity and sensitivity between one antibody and another.

Visualization of the Genomic Loci That Are Bound by Specific Multiprotein Complexes by Bimolecular Fluorescence Complementation Analysis on Drosophila Polytene Chromosomes.

We have developed a procedure that enables visualization of the genomic loci that are bound by complexes formed by a specific combination of chromatin-binding proteins. This procedure is based on imaging bimolecular fluorescence complementation (BiFC) complexes on Drosophila polytene chromosomes. BiFC complexes are formed by the facilitated association of two fluorescent protein fragments that are fused to proteins that interact with, or are in close proximity to, each other. The intensity of BiFC complex fluorescence at individual genomic loci is greatly enhanced by the parallel alignment of hundreds of chromatids within the polytene chromosomes. The loci that are bound by the complexes are mapped by comparing the locations of BiFC complex fluorescence with the stereotypical banding patterns of the chromosomes. We describe strategies for the design, expression, and validation of fusion proteins for the analysis of BiFC complex binding on polytene chromosomes. We detail protocols for the preparation of polytene chromosome spreads that have been optimized for the purpose of BiFC complex visualization. Finally, we provide guidance for the interpretation of results from studies of BiFC complex binding on polytene chromosomes and for comparison of the genomic loci that are bound by BiFC complexes with those that are bound by subunits of the corresponding endogenous complexes. The visualization of BiFC complex binding on polytene chromosomes provides a unique method to visualize multiprotein complex binding at specific loci, throughout the genome, in individual cells.

The origin of in situ hybridization - A personal history.

In situ hybridization is the technique by which specific RNA or DNA molecules are detected in cytological preparations. Basically it involves formation of a hybrid molecule between an endogenous single-stranded RNA or DNA in the cell and a complementary single-stranded RNA or DNA probe. In its original form the probe was labeled with (3)H and the hybrid was detected by autoradiography. The first successful experiments in 1968 involved detection of the highly amplified ribosomal DNA in oocytes of the frog Xenopus, followed soon after by the reiterated "satellite DNA" in mouse and Drosophila chromosomes. Fluorescent probes were developed about ten years later.

A new record of Chironomus (Chironomus) acidophilus Keyl (Diptera, Chironomidae) from the Uzon volcanic caldera (Kronotsky Reserve, Kamchatka Peninsula, Russia), its karyotype, ecology and biology.

Morphology, cytology, ecology and biology of Holarctic Chironomus (Chironomus) acidophilus Keyl, 1960 (Diptera, Chironomidae) was examined from material collected in the geothermal Vosmerka Lake (pH=2.0-2.5). An illustrated redescription of C. acidophilus is given on the basis of adult males reared from field-collected pupae, and of simultaneously collected larvae. Additional larvae belonging to the pseudothummi-complex were identified as C. acidophilus on the basis of their karyotype. The karyotype of C. acidophilus (2n=8) and detailed mapping of the 4 chromosome arms A, E, D and F are provided. The population of C. acidophilus from Kamchatka was found to be karyologically monomorphic. Information on distribution and ecology of C. acidophilus from Vosmerka Lake (total mineralization 1583.5 mg/l) is also given. Chironomus acidophilus is the only species of aquatic insects recorded in this lake. Lack of competition and a richness of food resources contribute to the high abundance (35161 ind./m2) and biomass (11.342 g/m2) of the larvae of C. acidophilus in Vosmerka Lake.

Quantified effects of chromosome-nuclear envelope attachments on 3D organization of chromosomes.

We use a combined experimental and computational approach to study the effects of chromosome-nuclear envelope (Chr-NE) attachments on the 3D genome organization of Drosophila melanogaster (fruit fly) salivary gland nuclei. We consider 3 distinct models: a Null model - without specific Chr-NE attachments, a 15-attachment model - with 15 previously known Chr-NE attachments, and a 48-attachment model - with 15 original and 33 recently identified Chr-NE attachments. The radial densities of chromosomes in the models are compared to the densities observed in 100 experimental images of optically sectioned salivary gland nuclei forming "z-stacks." Most of the experimental z-stacks support the Chr-NE 48-attachment model suggesting that as many as 48 chromosome loci with appreciable affinity for the NE are necessary to reproduce the experimentally observed distribution of chromosome density in fruit fly nuclei. Next, we investigate if and how the presence and the number of Chr-NE attachments affect several key characteristics of 3D genome organization: chromosome territories and gene-gene contacts. This analysis leads to novel insight about the possible role of Chr-NE attachments in regulating the genome architecture. Specifically, we find that model nuclei with more numerous Chr-NE attachments form more distinct chromosome territories and their chromosomes intertwine less frequently. Intra-chromosome and intra-arm contacts are more common in model nuclei with Chr-NE attachments compared to the Null model (no specific attachments), while inter-chromosome and inter-arm contacts are less common in nuclei with Chr-NE attachments. We demonstrate that Chr-NE attachments increase the specificity of long-range inter-chromosome and inter-arm contacts. The predicted effects of Chr-NE attachments are rationalized by intuitive volume vs. surface accessibility arguments.

Controlled expression of Drosophila homeobox loci using the Hostile takeover system.

BACKGROUND: Hostile takeover (Hto) is a Drosophila protein trapping system that allows the investigator to both induce a gene and tag its product. The Hto transposon carries a GAL4-regulated promoter expressing an exon encoding a FLAG-mCherry tag. Upon expression, the Hto exon can splice to a downstream genomic exon, generating a fusion transcript and tagged protein. RESULTS: Using rough-eye phenotypic screens, Hto inserts were recovered at eight homeobox or Pax loci: cut, Drgx/CG34340, Pox neuro, araucan, shaven/D-Pax2, Zn finger homeodomain 2, Sex combs reduced (Scr), and the abdominal-A region. The collection yields diverse misexpression phenotypes. Ectopic Drgx was found to alter the cytoskeleton and cell adhesion in ovary follicle cells. Hto expression of cut, araucan, or shaven gives phenotypes similar to those of the corresponding UAS-cDNA constructs. The cut and Pox neuro phenotypes are suppressed by the corresponding RNAi constructs. The Scr and abdominal-A inserts do not make fusion proteins, but may act by chromatin- or RNA-based mechanisms. CONCLUSIONS: Hto can effectively express tagged homeodomain proteins from their endogenous loci; the Minos vector allows inserts to be obtained even in transposon cold-spots. Hto screens may recover homeobox genes at high rates because they are particularly sensitive to misexpression.

Specific features in linear and spatial organizations of pericentromeric heterochromatin regions in polytene chromosomes of the closely related species Drosophila virilis and D. kanekoi (Diptera: Drosophilidae).

Heterochromatin plays an important role in the spatial arrangement and evolution of the eukaryotic genetic apparatus. The closely related species Drosophila virilis (phyla virilis) and D. kanekoi (phyla montana) differ in the amount of heterochromatin along the chromosomes as well as by the presence of the metacentric chromosome 2, which emerged as a result of a pericentric inversion during speciation, in the D. kanekoi karyotype. The purpose of this study was to establish if chromosome rearrangements have any influence on the linear redistribution of centromeric heterochromatin in polytene chromosomes and the spatial organization of chromosomes in the nuclei of nurse cell. We have microdissected the chromocenter of D. virilis salivary gland polytene chromosomes; obtained a DNA library of this region (DvirIII); and hybridized (FISH) DvirIII to the salivary gland and nurse cell polytene chromosomes of D. virilis and D. kanekoi. We demonstrated that DvirIII localizes to the pericentromeric heterochromatin regions of all chromosomes and peritelomeric region of chromosome 5 in both species. Unlike D. virilis, the DvirIII signal in D. kanekoi chromosomes is detectable in the telomeric region of chromosome 2. We have also conducted a 3D FISH of DvirIII probe to the D. virilis and D. kanekoi nurse cell chromosomes. In particular, the DvirIII signal in D. virilis was observed in the local chromocenter at one pole of the nucleus, while the signal belonging to the telomeric region of chromosome 5 was detectable at the other pole. In contrast, in D. kanekoi there exist two separate DvirIII-positive regions. One of these regions belongs to the pericentromeric region of chromosome 2 and the other, to pericentromeric regions of the remaining chromosomes. These results suggest that chromosome rearrangements play an important role in the redistribution of heterochromatin DNA sequences in the genome, representing a speciation mechanism, which, in general, could also affect the chromosome orientation in the 3D nuclear space.

[On the possibility of spontaneous interspecific hybridization in the nature of representatives of sibling-Species Chironomus riparius Kieffer and Chironomus piger Strenzke (Diptera, Chironomidae) from Armenia].

F2 hybrid (Chironomus riparius x Chironomus piger) x C. piger was found in mixed community of C. riparius and C. piger. Mechanism of its origin and problem of hybrid viability is considered.

A simple approach for multicolor immunofluorescence staining in different Drosophila cell types.

Multicolor immunostaining analysis is often a desirable tool in cell biology for most researchers. Nonetheless, this is not an easy task and often not affordable by many laboratories as it might require expensive instrumentation and sophisticated analysis software. Here, we describe a simple protocol for performing sequential immunostainings on two different Drosophila specimens. Our strategy relies on an efficient and reproducible method for removal primary antibodies and/or fluorophore-conjugated secondary antibodies that does not affect antigene integrity. We show that alternation of multiple rounds of antibody incubation and removal on the same slide, followed by registration of the same DAPI-stained image, provides a simple framework for the sequential detection of several antigens in the same cell. Given that the sample fixation procedures used for Drosophila tissues are compatible with most specimen processing protocols, we can envisage that the multicolor immunostaining strategy presented here can be also adapted to different samples including mammalian tissues and/or cells.

[Species-specific localization of DNA from pericentromeric heterochromatin on polytene chromosomes in the salivary gland cells and 3D-nuclear organization nurse cells in Drosophila virilis, and Drosophila kanekoi (Diptera: Drosophilidae)].

Microdissection of the chromocenter of D. virilis salivary gland polytene chromosomes has been carried out and the region-specific. DNA library (DvirIII) has been obtained. FISH was used for DvirIII hybridization with salivary gland polytene chromosomes and ovarian nurse cells of D. virilis and D. kanekoi. Localization of DvirIII in the pericentromeric regions of chromosomes and in the telomeric region of chromosome 5 was observed in both species. Moreover, species specificity in the localization of DNA sequences of DvirIII in some chromosomal regions was detected. In order to study the three-dimensional organization of pericentromeric heterochromatin region of polytene chromosomes of ovarian nurse cells of D. virilis and D. kanekoi, 3D FISH DvirIII was performed with nurse cells of these species. As a result, species specificity in the distribution of DvirIII signals in the nuclear space was revealed. Namely, the signal was detected in the local chromocenter at one pole of the nucleus in D. virilis, while the signal from the telomeric region of chromosome 5 was detected on another pole. At the same time, DvirIII signals in D. kanekoi are localized in two separate areas in the nucleus: the first belongs to the pericentromeric region of chromosome 2 and another to pericentromeric regions of the remaining chromosomes.

[Structural features of chromatin organization of 3C6/C7 interband in Drosophila melanogaster polytene chromosomes].

This article presents an analysis of the causes of chromatin decompaction of interbands in Drosophila polytene chromosomes. On the example of interband 3C6/C7 of X chromosome, we investigate the ability of different DNA segments from the region to form an interband in a new genetic environment. Site-specific FLP recombination between two transposons with FRT-sites allows introducing the DNA fragments from the inter-band 3C6/C7 into plCon(dv) transposon located in cytologically well-characterized 84F region of chromosome 3 followed by electron microscopic analysis of changes in the region, caused by insertion of the DNA fragments into the transposon. Thus, it has shown that the insertion of DNA fragment 276 bp in length from 3C6/C7 region into the plCon(dv) transposon leads to the formation of a new interband between two thin bands which are represented by material of the transposon. To date, the DNA fragment is minimal known sequence that is necessary and sufficient for interband formation. In addition, the sequence containing three tandemly repeated copies of DNA fragment 0.9 kb including a fragment of 276 bp from the interband 3C6/C7 was integrated in the transposon. The presence of additional copies of the DNA fragment did not change the morphology of the resulting interband. It was shown that sites of hypersensitivity to DNase I persist in interbands formed in the new genetic environment. The data obtained allow us to start analysis of the specific factors (proteins, DNA motifs, etc.) that determine the formation of decompacted chromatin state in sertain interband region and, as a whole, chromometric organization of interphase chromosomes in Drosophila.

Organization and maintenance of Drosophila telomeres: the roles of terminin and non-terminin proteins.

Drosophila telomeres are elongated by occasional transposition of specialized retroelements rather than telomerase activity, and are assembled independently of the sequence of the DNA termini. Drosophila telomeres are capped by terminin, a complex formed by the HOAP, Moi, Ver and HipHop proteins that localize exclusively at telomeres and protect them from fusion events. Other proteins required to prevent end-to-end fusion include HP 1 Eff/UbcD 1, ATM, the components of the Mrel 1-Rad50-Nbs (MRN) complex, and the Woc transcription factor. The terminin proteins are encoded by fast-evolving genes and are not evolutionarily conserved outside the Drosophila species. In contrast, the non-terminin telomere capping proteins are not fast-evolving, do not localize only at telomeres and are conserved from yeasts to mammals. We propose that following telomerase loss, Drosophila rapidly evolved terminin to bind chromosome ends in a sequence-independent manner, and that non-terminin proteins did not evolve as rapidly as terminin because of the functional constraints imposed by their involvement in diverse cellular processes. This hypothesis suggests that the Drosophila non-terminin proteins might correspond to ancestral telomere-associated proteins with homologues in other organisms including humans.

First description of polytene chromosomes in biting midges (Diptera: Ceratopogonidae).

Polytene chromosomes are described from secretory cells in larvae of Forcipomyia nigra (Winnertz). They are present in large glandular-trichogen cells at the bases of secretory setae and in midgut cells that were observed by transmission electron microscopy and light microscopy. Polytene chromosomes, isolated from the glandular-trichogen cells using aceto-orcein squash technique, measure 50-200 microm, have braid-like strands of chromatin and no bands, features that are unique within the Culicomorpha.

Differential occurrence of chromosome inversion polymorphisms among Muller's elements in three species of the tripunctata group of Drosophila, including a species with fast chromosomal evolution.

Detailed chromosome maps with reliable homologies among chromosomes of different species are the first step to study the evolution of the genetic architecture in any set of species. Here, we present detailed photo maps of the polytene chromosomes of three closely related species of the tripunctata group (subgenus Drosophila): Drosophila mediopunctata, D. roehrae, and D. unipunctata. We identified Muller's elements in each species, using FISH, establishing reliable chromosome homologies among species and D. melanogaster. The simultaneous analysis of chromosome inversions revealed a distribution pattern for the inversion polymorphisms among Muller's elements in the three species. Element E is the most polymorphic, with many inversions in each species. Element C follows; while the least polymorphic elements are B and D. While interesting, it remains to be determined how general this pattern is among species of the tripunctata group. Despite previous studies showing that D. mediopunctata and D. unipunctata are phylogenetically closer to each other than to D. roehrae, D. unipunctata shows rare karyotypic changes. It has two chromosome fusions: an additional heterochromatic chromosome pair and a pericentric inversion in the X chromosome. This especial conformation suggests a fast chromosomal evolution that deserves further study.

Chironomus polonicus sp. n. (Diptera: Chironomidae) from southern Poland.

The paper describes larval, pupal and adult morphology as well as the karyotype of Chironomus polonicus sp. n. from southern Poland. The material has been obtained from reared egg masses collected in Boleslaw pool, near Kraków. The species belongs to the pseudothummi cytocomplex with 2n = 8 and chromosome arm combinations AE, BF, CD, G. Several homozygous inversions distinguish arm A of the new species from that of C. pseudothummi Strenzke. Arm F is similar to that of C. aprilinus Meigen and differs from it by few steps of homozygous inversions. Few morphological differences in the pupa and adult are also presented.

Identical functional organization of nonpolytene and polytene chromosomes in Drosophila melanogaster.

Salivary gland polytene chromosomes demonstrate banding pattern, genetic meaning of which is an enigma for decades. Till now it is not known how to mark the band/interband borders on physical map of DNA and structures of polytene chromosomes are not characterized in molecular and genetic terms. It is not known either similar banding pattern exists in chromosomes of regular diploid mitotically dividing nonpolytene cells. Using the newly developed approach permitting to identify the interband material and localization data of interband-specific proteins from modENCODE and other genome-wide projects, we identify physical limits of bands and interbands in small cytological region 9F13-10B3 of the X chromosome in D. melanogaster, as well as characterize their general molecular features. Our results suggests that the polytene and interphase cell line chromosomes have practically the same patterns of bands and interbands reflecting, probably, the basic principle of interphase chromosome organization. Two types of bands have been described in chromosomes, early and late-replicating, which differ in many aspects of their protein and genetic content. As appeared, origin recognition complexes are located almost totally in the interbands of chromosomes.

[Localization of the Dras1 sequence to polytene chromosomes in sibling species of the Drosophila virilis group].

Drasl gene was mapped by in situ hybridization to polytene chromosomes of several sibling species of the Drosophila virilis group and hybrids between them. A 1037 bp fragment of the Drasl gene of the D. virilis genome was used as a probe. The gene sequence is localized to the region of the disk 25 A-B on the chromosome 2 of the polytene chromosome map of D. virilis.

Induced decondensation of heterochromatin in Drosophila melanogaster polytene chromosomes under condition of ectopic expression of the Supressor of underreplication gene.

Overexpression of Suppressor of Underreplication protein (SUUR) induces giant reversible swellings in intercalary and pericentric heterochromatin of salivary gland polytene chromosomes. Here, we demonstrate that morphology and extent of swellings are highly dependent on the fixation conditions used: upon glutaraldehyde fixation, we observed moderate decondensation of heterochromatic regions, which was significantly more pronounced upon acetic-acid fixation. Swellings are formed in a PARP-independent fashion. Together with data on inactive transcription in them, this indicates that the swelling-forming regions fail to acquire any features of puffs, the regions typically forming locally decondensed chromatin. Large swellings display striking re-localization of histones and SUUR protein, which are now found at the periphery of the swellings, in contrast to the DNA that fills the entirety of the swelling. We show that swelling-embedded DNA is capable of undergoing replication, however SUUR overexpression drastically alters replication timing in salivary gland cells. We speculate that swelling formation results from SUUR tipping the balance against other proteins that contribute to the organization of repressed chromatin regions.

[Non-histone skeleton of mitotic chromosome in situ].

Studying giant nuclei of Chironomus plumosus in situ (Makarov, Chentsov, 2010), we concluded that polythene chromosome structure appears after 2 M NaCl and DNase treatment in presence of 2 mM CuCl2. Cu2+ -ions may stabilize bonds between specific non-histone components, arranged into non-histone matrix of polythene chromosome. Here, we investigated the non-histone matrix of pig embryo mitotic chromosomes in situ, using 2 mM CuCl2-stabilization method. In 2 mM CuCl2-stabilized cells the residual chromosome body (non-histone matrix) could be visualized in every stage of mitosis. Mitotic chromosome non-histone matrix had the same reaction on preliminary hypotonic treatment as normal chromosome: different decondensation of non-histone material was observed. Topoisomerase IIalpha and SMC 1 had uniform localization inside chromosomal body and did not form any axial structures.

[Karyofunds of Chironomus plumosus (L.) (Diptera, Chironomidae). V. terminal and interstitial populations].

The study of polytene chromosomes of Chironomus plumosus from Rybinsk reservoir, its inflows and surrounding water bodies allow to mark out two type's of population. Terminal populations--presented by karyoforms with concrete limited composition of genotype combinations and zygotic arm combinations, which adapted to specific complex of the habitat factors. The immediate exchange of genes between different terminal populations is absent (even they placed in nearness) from allochronie of life cycles and impossibility of larvae-immigrants to finish ontogenesis in unfit for they habitat conditions. Interstitial populations inhabit the biotopes, where the karyoforms of two or more terminal populations can to finish ontogenesis and to reproduce. The genetic exchange between terminal population implement through interstitial populations.