The reasons of Trithorax-like expression disturbance in Trl 3609 allele of Drosophila melanogaster.

The regulatory region of the Trl gene was analyzed using the mutation Trl 3609 , resulting from the insertion of the P-element into the promoter region of the gene as well as mutations obtained on its basis. It is shown that two last transcription start sites, which are most often used in vitro in S2 cells, are almost not used in vivo. Experimental data indicate that transcription terminators in transposons play an important role in the decrease in the transcription level of the recipient gene.

A standard photomap of ovarian nurse cell chromosomes in the European malaria vector Anopheles atroparvus.

Anopheles atroparvus (Diptera: Culicidae) is one of the main malaria vectors of the Maculipennis group in Europe. Cytogenetic analysis based on salivary gland chromosomes has been used in taxonomic and population genetic studies of mosquitoes from this group. However, a high-resolution cytogenetic map that could be used in physical genome mapping in An. atroparvus is still lacking. In the present study, a high-quality photomap of the polytene chromosomes from ovarian nurse cells of An. atroparvus was developed. Using fluorescent in situ hybridization, 10 genes from the five largest genomic supercontigs on the polytene chromosome were localized and 28% of the genome was anchored to the cytogenetic map. The study established chromosome arm homology between An. atroparvus and the major African malaria vector Anopheles gambiae, suggesting a whole-arm translocation between autosomes of these two species. The standard photomap constructed for ovarian nurse cell chromosomes of An. atroparvus will be useful for routine physical mapping. This map will assist in the development of a fine-scale chromosome-based genome assembly for this species and will also facilitate comparative and evolutionary genomics studies in the genus Anopheles.

Identification of functionally significant elements in the second intron of the Drosophila melanogaster Trithorax-like gene.

It is known that a lot of genes having a distinct expression pattern require the complex system of transcription regulation. The regulatory regions of such genes can include not only the 5'-flanking regions, but also other regions, particularly their intron sequences. The Drosophila melanogaster Trithorax-like (Trl) gene, encoding the GAGA protein, is one of the genes with complex expression pattern. GAGA is one of a few transcription factors that can regulate gene expression at multiple levels. The GAGA-mediated modulation of expression seems to be linked with modifications of the chromatin structure. Nowadays, the regulatory potential of the Trl 5'-flanking region that contains multiple GAGA binding sites has been analyzed, but the presence of the functionally significant elements in other Trl regions has not been examined. We found DNase I hypersensitive sites, evolutionary-conserved sequences and numerous GAGA binding sites in the second intron of the Trl gene. Interestingly, these sequences localize in two main regions of the intron in immediate proximity to preferred regions of transposon insertions. Additionally, we revealed that deletion of the intron fragment in the Trl(1-72) mutants caused an alteration of the Trl expression pattern. These results allow us to conclude that the second intron of the Trl gene contains functionally significant elements.

[Molecular and genetic description of a new hypomorphic mutation of Trithorax-like gene and analysis of its effect on Drosophila melanogaster oogenesis].

The Trithorax-like (Trl) gene of Drosophila melanogaster encodes the multifunctional protein GAGA involved in many cellular processes. We have isolated and described a new hypomorphic mutation of the Trl gene--Trl(en82). The mutation is the insertion of a 1.4 kb P-element into the 5' untranslated region. Trl expression decreased in the ovaries of mutant flies by about 30%; however, it caused abnormalities. The Trl(en82) mutation combined with the null allele of Trl caused female sterility: the females laid a few small eggs with abnormal shape. Many egg chambers demonstrated abnormalities in the Trl(en82) mutants: the oocyte had a regular shape and intruded into the egg chamber region with nurse cells; the rapid transport of nurse cell cytoplasm into the oocyte was disturbed, which resulted in the "dumpless" phenotype of the chambers in mutants; follicular cells often did not completely cover the oocyte and concentrated on its posterior end; and the migration of centripetal cells was affected. We propose that the sterility of the Trl(en82) females is due to the abnormal functioning of follicular cells resulting from low Trl expression. This proposal is confirmed by normalizing the mutant phenotype of Trl(en82) females after the transfection of Trl cDNA. Note that even an insignificant decrease in Trl expression in such females seriously affected the somatic cell functioning, while a significant decrease in its expression in strong hypomorphic mutants affected both somatic and germline cells in the egg chambers.

[Systemic regulation of genetic and cytogenetic processes by a signal cascade of actin remodeling: locus agnostic in Drosophila].

The concept on systemic regulation of genetic and cytogenetic processes has acquired a new perspective after the completion of the Human Genome project, when the view on systemic realization of genetic activity in the dynamic spatial organization of the genome is the nucleus was generally accepted. This organization underlies plasticity of complex biological systems. Chromosome position within the nucleus determined both processes of normal development and the development of genomic diseases, i.e., changes according to the environmental requirements, current needs of the organism, and its individual experience. Nuclear actin has been envisioned as a main factor bridging three levels of the genome organization (nucleotide, structural, and spatial), due to its capability of (1) regulating transcription by activating all three classes of RNA polymerase; (2) participating in chromatin remodeling by interacting with numerous proteins; and (3) lining the nuclear membrane, determining the chromosome attachment points and regulating export from the nucleus. In view of this, the role of actin remodeling factors (LIMK1, cofilin, actin) in the development of neurodegenerative diseases, including prionic ones, and in the mechanisms of generation of genomic diseases, syndromes resulting from unequal recombination, has been intensely studied. Drosophila is a helpful model organism to determine the sequence of events in this system of hierarchical relationships. Using spontaneous and mutant variants of the agnostic locus, we have designed a model of the Williams syndrome, which also reproduces main diagnostic traits of neurodegenerative diseases.

[Effect of hypomorphic mutation in Trithorax-like gene on Drosophila melanogaster oogenesis].

We generated and characterized a new hypomorphic mutation of Drosophila melanogaster Trithorax-like (Trl) gene named Trl362. The Trl362 homozygous females are sterile and lay a small number of eggs; most embryos die at the early developmental stages. The transcriptional Trl level of adult Trl362 females was markedly lowered. Little or no GAGA protein, encoded by Trl@, was detected in the nurse cell nuclei. The ovaries of Trl362 females showed impairments, such considerable changes in the structure of both ovarioles and individual egg chambers. We believe that the observed ovarian defects in Trl362 mutants are mostly due to a decreased amount of GAGA protein in the germline cells. An increase of GAGA-519 protein caused by introduction of hsp83:GAGA-519 transgene against Trl362 background rescued partially the female fertility. It may well be that a decrease of GAGA protein in Trl362 germline cells leads to a defective expression of the genes regulated by transcription factor GAGA, whose products are essential for normal Drosophila oogenesis.

[Generation and analysis of novel mutations of the trithorax-like gene in Drosophila melanogaster].

The Trithorax-like (Trl) gene of Drosophila melanogaster encodes the multifunctional GAGA factor. The expression of Trl is known to depend on numerous factors, such as the organ, the tissue, the ontogenetic stage, and the ambient temperature. Apparently, this expression is controlled by a complex system of regulatory elements, which so far has been scarcely studied. Our preliminary results indicate that the second intron of the Trl gene bears functionally significant elements. To test this assumption, we generated 23 novel alleles of the gene via P-induced male recombination and analyzed them cytogenetically. Of these mutations, 13 (recessive lethals) are deletions, disrupting the coding gene region. Ten mutations (seven deletions and three duplications) remove parts of the second Trl intron only. Some of these mutant stocks exhibit lower viability at different temperatures. These results suggest that the second intron region harbors functionally significant elements. The deletion mapping results verified the localization of the Trl gene in the 70F1-2 region.

[Characterization and comparative analysis of DNA from the pericentric heterochromatin of chromosome 2 of Anopheles atroparvus V. Tiel (Culicidae, Diptera)]. / Kharakteristika i sravnitel'nyi analiz DNK iz pritsentromernogo geterokhromatina khromosomy 2 Anopheles atroparvus V Teil (Culicidae, Diptera)

The minilibrary containing DNA sequences from the diffuse pericentric heterochromatin from the right arm of Anopheles atroparvus V. Tiel (Culicidae, Diptera) chromosome 2 (2R) was generated by use of chromosome microdissection technique. Southern-blot hybridization of the minilibrary fragments with the labeled genomic DNA of A. atroparvus and analysis of their primary structure showed that this heterochromatin region contained repeated DNA sequences differed by their primary structure and the number of copies. These were mostly AT-rich sequences harboring the features characteristic of the S/MAR regions. Based on the clones homology to the sequences from the An. gambiae and Drosophila melanogaster genomes, it was demonstrated that the pericentric heterochromatin from the right arm of An. atroparvus chromosome 2 contained gypsy-like transposable elements, as well as the sequences homologous to the structural genes. In situ hybridization with the chromosomes of A. atroparvus and of the two representatives of the Anopheles maculipennis species complex, A. messeae and A. beklemishevi, showed that pericentric regions of all these chromosomes contained DNA sequences homologous to the sequences from the region-specific minilibrary. Cloned fragments of conserved repetitive DNA revealed upon interspecific Southern-blot hybridization of the clones with the labeled genomic DNA of A. messeae can be utilized in further investigations of evolutionary rearrangements of the pericentric heterochromatin within the Anopheles maculipennis species complex.

[Molecular-cytogenetic characteristics of B-chromosomes in chironomid (Diptera, Chironomidae)]. / Molekuliarno-tsitogeneticheskaia kharakteristika B-khromosom khironomid (Diptera, Chironomidae).

Morphological and molecular study of B-chromosomes of three Chironomus species (siblings Ch. borokensis and Ch. phumosus from plumosus group, and Ch. heterodentatus from obtusidens group) was carried out. Morphological similarity of B-chromosome banding pattern and telomer-centromeric region banding pattern of chromosome IV in Ch. borokensis was shown. Polytene B-chromosomes of Ch. borokensis and Ch. heterodentatus were microdissected, and their DNA was amplified using degenerate oligonucleotide primer polymerase chain reaction. Comparative analysis of the localization of homologous B-chromosome DNA sequences of A- and B-polytene chromosomes was made using in situ fluorescence hybridization. It has been shown that B-chromosomes in the studied species are composed mainly of repetitive DNA sequences homologous to sequences of centromeric and telomeric DNA of A-chromosomes, and also these of the mobile element NLRCthl. The B-chromosome DNA, homologous to sequences of DNA mobile element, was scattered on A-chromosomes (more than 100 sites). No ribosomal DNA repeats were identified in B-chromosome. Heterologous FISH of B-chromosome DNA to polytene A-chromosomes of Ch. thummi, a species lacking B-chromosomes, enabled us to reveal the presence of numerous sites homologous to DNA of B-chromosomes. These are mainly mobile element sites. An origin of B-chromosomes and peculiarities of their organization in chironomids are discussed.