[Role of GAGA Factor in Drosophila Primordial Germ Cell Migration and Gonad Development].

The GAGA protein of drosophila is a factor involved in epigenetic transcription regulation of a large gene group controlling developmental processes. In this paper, the role of GAGA factor in germ cell migration is demonstrated as well as its effect on the gonad development in drosophila embryogenesis. Mutations in the Trl gene, encoding GAGA factor, prematurely induces the active migration program and relocation of the primordial cells inward the embryo before the beginning of gastrulation. The germ cells that prematurely separated from the main group migrate ectopically, lose orientation, and stay out of gonad development. Expression pattern of the Trl gene suggests its activity in epithelial cells of the embryonic blastoderm, part of which contact primordial cells. Thus, GAGA factor influences migration of these cells in an indirect manner via their somatic environment.

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.

[Main components of gene network controlling development of dorsal appendages of egg chorion in Drosophila melanogaster].

The development of dorsal appendages of the chorion (specialized structures in the D. melanogaster egg which look like elastic tubes and ensure the breathing of the developing embryo) is an attractive model for the study of genetic mechanisms of the development of organs and tissues, whose generation is based on transformation of the epithelial tissue in the tubular structures. In the present review, we present information on genes and proteins that control the development of dorsal appendages of the chorion. We demonstrated that three signal pathways (EGFR, DPP, and NOTCH), which are combined together in a single gene network through a number of components, play a major role in the development of dorsal appendages of the chorion.

[Basic types of respiratory system structure in insect egg envelopes, and genes controlling their formation].

Insects is a taxon surprisingly rich with species and varieties, and its representatives are considered as the most fitted and "evolutionary successful" living things. Insects are distinguished by diversity and abundance of adaptations to environmental conditions, representatives of this class inhabit different ecological niches, they can be found practically in every corner of the Earth and, in particular, in close adjacency to man. Among them are those who man benefits from and those who man struggles against. This determines man's interest in studying peculiarities of their development as well as adaptations formed by them in the course of evolution to become more viable. In the paper, data are presented on morphological structure of respiratory systems in insect egg envelopes that ensure respiration process of developing embryo. Variability of these systems and their dependence on environmental conditions are demonstrated for different insect species. The information about genes controlling development of respiratory systems in fruit fly eggs is brought together, and occurrence of evolutionary conservative genes participating in development of such systems in other insect species is ascertained.

Analysis and recognition of the GAGA transcription factor binding sites in Drosophila genes.

The transcription factor GAGA, encoded by the gene Trl, controls expression of many Drosophila melanogaster genes. We have compiled the presently largest sample (120 sites) of published nucleotide sequences with experimentally confirmed binding to GAGA protein. Analysis of the sample has demonstrated that despite an apparent structural diversity of the GAGA sites, they fall into four distinct groups, namely, (1) the sites containing two GAG trinucleotides with no more than one nucleotide substitution in each and separated by spacers with a length of 1 or 3 nucleotides (GAGnGAG and GAGnnnGAG); (2) the sites containing a single GAGAG motif; (3) (GA)(3-9) microsatellite repeats; and (4) the sites corresponding to three and more direct repeats of GAG trinucleotide homolog and its inverted repeats separated by spacers of various lengths. Using the software package SITECON, the methods were elaborated for recognizing the sites of GAGnGAG (method 1) and GAGnnnGAG (method 2) types in DNA sequences. Experimental verification confirmed the ability to interact with the GAGA factor for 72% of the sites predicted using method 1 and 94.5% of the sites predicted by method 2. Application of the experimentally verified methods to analyzing the localization of potential GAGA binding sites in the target genes of this transcription factor has demonstrated that the 5'-untranslated regions (5'UTRs) and first introns are enriched for these sites (two-threefold relative to the average occurrence frequency in the D. melanogaster genome) as compared with a moderate enrichment (not exceeding 1.5-fold) of promoter regions (-4000/+200 bp or -1000/+100 bp).

[Maintenance of the patterns of expression of homeotic genes in the development of Drosophila melanogaster by proteins of the polycomb, trithorax, and ETP groups].

Proteins encoded by genes of the groups Polycomb (PcG), trithorax (trxG), and the Enhancer of Trithorax and Polycomb Group (ETP) are important regulators of expression of most developmental genes. Data concerning all currently described genes assigned to these groups are summarized in the review. Genetic interactions of these genes and phenotypical manifestation of their mutations are described. Data on the PcG, trxG, and ETP proteins are systemized. Questions are considered concerning the formation of multimeric complexes containing proteins of these groups, recruitment of these complexes to regulatory elements of target genes, and the mechanisms of activation/repression of gene expression.

[Influence of Drosophila melanogaster genotype on biological effects of endocymbiont Wolbachia (stamm wMelPop)].

Comparative analyses of symbiotic bacteria Wolbachia (stamm wMelPop reducing lifespan of flies) morphology in normal and mutant strains of Drosophila melanogaster as well as the influence of Wolbachia on the host cell ultrastructure have been done. Wolbachia infected D. melanogaster mutation strains Trithorax-like -- Trl362/TM3, Sb1 Ser y+ and Trlen82/TM3, Sb1 Ser y+ have been received by special flies crossing. Uninfected strain D. melanogaster white-1118 (w1118) have been obtained by antibiotic treatment of initially infected strain D. melanogaster [w]w1118. Complex of different methods and approaches let to investigate for the first time the morphology of cell structure before and after bacterial infection of insects and to value the bacterial presence effect on flies viability and reproduction of normal and mutant flies. Morphology af cytoplasmic compartments in early embryos and eggs layed by infected and uninfecyed females Trl362/TM3, Sb1 Ser y+ and Trlen82/TM3, Sb1 Ser y+ have been analyzed. Electron microscopy has shown that D. melanogaster embryos contain typical Wolbachia contacting with different host organelles that verify preservation of their functional activity. Atificial mitochondria and Wolbachia (wMelPop) of unusual morphology with defective bacterial membranes have been visualised in D. melanogaster [w]Trl362/TM3, Sb1 Ser y+. Wolbachia presence in ovarium cells from strains [w]Trl362/TM3, Sb1 Ser y+ and [w]Trlen82/TM3, Sb1 Ser y+ did not influence on eggs quantity layed by females. We have demonstrated for the first time that lifespan of infected and uninfected strains: D. melanogaster Trl362/TM3, Sb1 Ser y+ and Trlen82/TM3, Sb1 Ser y+ were similar. However the lifespan of imago from strain [w]w1118 was lower in comparison to those from strains Trl362/TM3, Sb1 Ser y+ and Trlen82/TM3, Sb1 Ser y+. It suggests that either chromosomal balancer TM3 or Trl mutation play an importance role in host-symbiotic relationship. Next experiments have revealed that lifespan of homozygotic flies decreased essentially and was close to lifespan of strain [w]w1118. Data obtained confirm that chromosomal balancer TM3 can affect on symbiont-host relationship.

[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.

[Basic aspects of ovarian development in Drosophila melanogaster].

Modern views of the development and structural organization of the female reproductive system in Drosophila melanogaster are reviewed. Special emphasis is placed on the generation and development of follicles in the germarium and the interactions of germline and somatic cells in the egg chamber. Detailed consideration is given to the main events that ensure and regulate the transport of mRNA, proteins, and organelles from nurse cells to the oocyte in the germarium and at later stages of egg chamber development.

[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.

[Specific DNA sequences are associated with nuclear envelopes of pseudonurse cells in Drosophila melanogaster otu 11].

Nuclei of ovarian pseudonurse cells from the mutant strain of Drosophila melanogaster otu 11 are suitable for mapping the attachment of chromosomes to the nuclear envelope (NE). Loci in contact with the NE included region 20CD of the X chromosome, region 41 of chromosome 2, the proximal end of region 81 of chromosome 3, and region 101 of chromosome 4. In situ hybridization revealed that all 4 regions contained sequences homologous to clone lambda20p1.4. DNA of clone lambda20p1.4 was previously found to bind specifically to purified D. melanogaster lamins. These results suggest that specific DNA sequences are involved in attachment of chromosomes to NE in vivo.

[Architecture of the X chromosome, expression of LIM kinase 1, and recombination in the agnostic mutants of Drosophila: a model of human Williams syndrome]. / Osobennosti arkhitektury X-khromosomy, ékspressii LIM kinazy 1 i rekombinatsii u mutantov drozofily lokusa agnostic: model' sindroma uil'iamsa cheloveka.

As the Human Genome and Drosophila Genome Projects were completed, it became clear that functions of human disease-associated genes may be elucidated by studying the phenotypic expression of mutations affecting their structural or functional homologs in Drosophila. Genomic diseases were identified as a new class of human disorders. Their cause is recombination, which takes place at gene-flanking duplicons to generate chromosome aberrations such as deletions, duplications, inversions, and translocations. The resulting imbalance of the dosage of developmentally important genes arises at a frequency of 10(-3) (higher than the mutation rate of individual genes) and leads to syndromes with multiple manifestations, including cognitive defects. Genomic DNA fragments were cloned from the Drosophila melanogaster agnostic locus, whose mutations impair learning ability and memory. As a result, the locus was exactly localized in X-chromosome region 11A containing the LIM kinase 1 (LIMK1) gene (CG1848), which is conserved among many species. Hemizygosity for the LIMK1 gene, which is caused by recombination at neighboring extended repeats, underlies cognitive disorders in human Williams syndrome. LIMK1 is a component of the integrin signaling cascade, which regulates the functions of the actin cytoskeleton, synaptogenesis, and morphogenesis in the developing brain. Immunofluorescence analysis revealed LIMK1 in all subdomains of the central complex and the visual system of Drosophila melanogaster. Like in the human genome, the D. melanogaster region is flanked by numerous repeats, which were detected by molecular genetic methods and analysis of ectopic chromosome pairing. The repeats determined a higher rate of spontaneous and induced recombination. including unequal crossing over, in the agnostic gene region. Hence, the agnostic locus was considered as the first D. melanogaster model suitable for studying the genetic defect associated with Williams syndrome in human.

[Molecular genetic analysis of Thrithorax-like gene encoded transcriptional factor GAGA in Drosophila melanogaster]. / Molekuliarno-geneticheskii analiz gena Trithorax-like, kodiruiushchego transkriptsionnyi faktor GAGA Drosophila melanogaster.

The Drosophila melanogaster Trithorax-like (Trl) gene is classed with the trx-G genes and codes for several isoforms of the GAGA transcription factor (GAF) which regulates expression of homeotic and numerous other genes. GAF acts as a transcriptional antirepressor, i.e., its interaction with nucleosomal DNA results in the open chromatin conformation in promoter gene regions. The regions thereby become accessible to other transcription factors. As mutations of the Trl gene enhance position effect variegation and disturb chromosome segregation in mitosis and meiosis, GAF is thought to play another, more significant role in determining the chromatin structure. To study the molecular basis of its pleiotropic effect, the Trl gene was subjected to a structural analysis. The genomic Trl gene was sequenced, the sizes of its exons and introns was established, and a complex structure of the 5' and 3' gene regions was demonstrated. The Trl13C, Trl62, DfTrlR67, and DfTrlR85 mutations were exactly mapped. In addition, four insertions of the P element were identified as Trl alleles (Trll(3)s2325, TrlEP(3)3184, TrlEP(3)3191, and TrlEP(3)3609). The viability at various developmental stages was studied in homozygotes for the Trl mutations and in interallelic compounds. The following lethality stages were established: hatching, (Trl13C, DfTrlR85, TrlEP(3)3609), larval molts (Trll(3)s2325), pupation, metamorphosis (DfTrlR67, Trl62), and eclosion (several compounds).

[The effect of some mutations in the Trl gene on mitosis in embryonal and larval tissues and egg chamber morphology in Drosophila melanogaster]. / Vliianie nekotorykh mutatsii po genu Trl na mitoz v émbrional'noi i lichinochnykh tkaniakh i morfologiiu iaitsevykh kamer u Drosophila melanogaster.

A study was made of three insertional mutations (Trl13C, Trls2325, and TrlEP(3)3184) located in the second intron of the Trithorax-like (Trl) gene for the GAGA transcription factor (GAF). Their cytological effects were analyzed in oogenesis, early embryonic development, and in larval development (96-108 h) in cells of nervous ganglia and imaginal disks. Notwithstanding an interallelic difference in expression, all three P-element insertions proved to be dominant as far as the examined parameters were concerned. The most substantial defects were the formation of "granular" chromatin during the interphase and mitosis and high proportions of cells with hypercondensed chromatin (which were arrested at the G2/M boundary) and cells with abnormal chromosome segregation. A higher frequency of egg chambers with trophocytes defective in number and in chromatin condensation was observed in females carrying the mutant Trl gene. The defects were assumed to result from poor coordination of the chromosome and cell cycles and, including, the nuclear and centrosomal cycles in embryonic development and the cycles of chromosome condensation and spindle formation in cells of larval imaginal disks and nervous ganglia.