[Cytophotometric determination of genome size in two species of Cyclops Lake Baikal (Crustacea: Copepoda: Cyclopoina) in ontogenetic development].

Genome size of Cyclops in cells at early stages of cleavage (up to the 5th division) and in somatic cells were estimated by a static digital Feulgen cytophotometry in order to study the quantitative changes in DNA content during chromatin diminution. Our realization of the cytophotometric method was approbeted on five different digital-imaging systems in blood cells of four vertebrate species. In all cases, we observed a direct correlation of the obtained and known from the literature data on the genome size and a high reproducibility, which allows to use these systems in future work. We also optimized the conditions for DNA hydrolysis of both blood smears, and for two species of Cyclops from the Moscow population, as 30 min in 5 N HCl at 24 degrees C. Here we first revealed chromatin diminution in two endemic Baikal species of Cyclopoida: Acanthocyclops incolotaenia and Diacyclops galbinus estimated the extent ofchromatin diminution in Diacyclops galbinus as 95.5-96.2 %. Cytometric analysis of the third species, Mesocyclops leuckarti, did not reveal obvious chromatin diminution. We also optimized the conditions for DNA hydrolysis of both blood smear preparations, and for two species of copepods from the Moscow population, as 30 min in 5N HCl at 24 degrees C.

[Drosophila melanogaster imaginal discs mitotic anomalies induced by tumor-supressor dlg silencing construction].

Mitosis, cytokinesis and nuclear texture of wing imaginal discs cells silenced by UAS-RNAi-dlg construct induced by 1096-Ga 14 driver were studied. The silencing construct contains coding region of dlg gene and the complementary region. Further, this RNA hairpin (Dietzl et al., 2007) is processed by endogenous protein Dicer and the resulting RNA fragments silence mRNA dlg. Tumor suppressor gene dlg is encoding for 21 transcripts. The construct UAS-RNAi-dlg inactivates 14 transcripts--RE, RH, RQ, RS, RG, RD, RL, RB, RK, RR, RT, RN, RA, RP--and does not silenced the other 7 (RO, RF, RI, RU, RJ, RC, RM). This permits to study functions of proteins containg guanilate-kinase domain IPR008145 at C-end of the protein. The most important consequences of the silencing are abnormal mitotic exit and the formation of binuclear cells. Quantitative fluorescence measurements of anti-H3-p histone and DAPI signals showed phase-specific changes in nuclear texture. The inactivation of cellular cortex polarization is the most likely target of dlg inactivation in mitosis.

[Role of the Drosophila melanogaster hrs gene in wing formation].

The Hrs (hepatocyte growth factor receptor tyrosine kinase substrate) protein is an endosomal protein whose function is to transport receptor tyrosine kinases from early endosomes to lysosomes. Since receptor tyrosine kinases are involved in various signaling pathways, HSR defects lead to various malformations. A study of the role of the hrs gene in wing development in Drosophila confirmed that the gene is involved in the formation of the D/V boundary of the wing imaginal disk and suggested a new role in wing vein refinement for the gene. Structural analysis of the hrs gene transcripts indicated that transcript B is responsible for vein refinement.

[Nucleus DNA content measurement methods features].

We suggest a new theoretical method of the flow cytometry DNA histograms and apply it for Drosophila melanogaster imaginal discs cells. The model gives a possibility to determine the proportions of cells in G1, G2 (M) and S cell cycle phases. We show that the precision of G1 and G2 (M) DNA content measurements is limited by the precision of device zero signal arrangement. The usage of calculated device zero and dividing cells as the DNA content standards may improve the precision of DNA content measurements. We also compared the precisions of different DNA content methods and draw the conclusion that the current precisions of different methods are similar and lie within 2-6% interval.

[The influence of morphogene Wg on the formation of an ectopic eye in Drosophila melanogaster].

Due to the ectopic expression of the ey gene in the wing imaginal disc under the action of the 1096-Gal4 driver, a part of the wing disc cells change their fate and become eye cells. Ectopic eyes are induced in definite regions of the wing disc and form a stable pattern on the wing of an adult fly. Here, we have shown that the ectopic expression of Wg inhibits the formation of ectopic eyes, and conversely the expression of Wg is reduced in the sites of ectopic Ey expression. Experiments with overexpression of the vesicular traffic protein H rs capable of inhibiting the Wg signaling agree with the notion on antagonism of Wg and Ey in ectopic eyes. Our results confirm that the processes of formation of normal and ectopic eyes are principally similar with regard to genetic control.

[Duration of the cell cycle phases in mutants for the tumor suppressor Merlin in Drosophila melanogaster].

The cell cycle duration was estimated in Drosophila melanogaster mutants for the tumor suppressor Merlin with the use of different approaches. Experiments on induction of mosaic clones in tissues of the larval wing imaginal disc showed that the cell cycle in mutant discs is shorter than that in control. Flow fluorescence cytometry revealed no differences between mutant and normal animals in the relative duration of the cell cycle phases, which suggests proportional shortening of the cell cycle phases. The study with pulse-labeled mitoses confirmed these results and showed that the length of the cell cycle is 7 h (S phase duration 3 h) in control individuals and 5 h (S phase duration 2 h) in Merlin gene mutants.

[DNA contents in nuclei of Cyclops kolensis and C. insignis (Crustacea, Copepoda)].

Chromatin diminution (CD) in two Cyclopoida species, Cyclops kolensis and C. insignis, was studied by static digital Feulgen cytophotometry. DNA content (pg/cell) was evaluated by standard curves builded up using blood cells of five organisms with known DNA content, which ranged from 1.25 to 14.70 pg. According to data obtained, diploid genome of C. kolensis has about 40 pg DNA before CD and 1.8-2.0 pg DNA after CD. These values are similar for both Moscow and Baikal populations of C. kolensis and 6-10 times exceed estimates made earlier (Grishanin, 2008), Our data confirm that CD in C. kolensis is 94-96% of DNA. In mitotic dividing cells of C. insignis, DNA content was about 7.5 pg both in early and late embryos, and CD was not revealed for this species. The data obtained show that, among Cyclopoida studied, the genome of C. kolensis before CD has a maximum content of DNA.

[Fluorometric identification of chromatin packing level laying out of the light microscopy resolution].

Confocal microscopy permits to perform quantitative analysis of the fluorescent signals of the nuclei. We determined the level of DAPI and phosphorylated histone-H3 fluorescence, the volume of DAPI and phosphorylated histone-H3 fluorescence in normal (Hikone AW) and colchicine treated third instar Drosophila melanogaster wing imaginal disc mitotic cells. Our analysis permitted to indentify two unknown levels of chromatin package; one in the prometaphase and another at the end of metaphase. These levels disappeared in colchicine treated mitoses. We conclude that quantitative analysis of confocal images is able to detect the differences in chromatin package laying over the resolution of light microscopy.

[Study of the functional role of mutation in the guinea pig-adapted Ebola virus genome on a Drosophila melanogaster model].

Ebola virus virulence in guinea pigs, which appears through virus adaptation to this animal host, correlates with substitutions in the gene encoding vp24 protein. In particular, the substitution His-->Tyr186 was found when obtaining strain 8 ms. An attempt was made to clarify the functional role of this substitution in a transgenic fruit fly model. Using the drosophila transformation technique provided transgenic strains that contained genomic insertions of wild-type Ebola virus vp24 gene and the mutant gene with the His-->Tyr substitution at the above position. Thus, the drosophila strains carrying the sequences encoding for the vp24 proteins of Ebola virus Zaire and 8 ms in pUAST vector were obtained. This makes it possible to study the expression of transgenic constructs in various D. melanogaster organs and tissues.

[Nuclear texture in mitotic cells].

Changes of nuclear texture in mitotic cells of Drosophila melanogaster imaginal discs were studied. The distribution of voxels DAPI fluorescence intensities was used as the quantitative measure of the nuclear texture. The integral characteristics such as the portion of voxels with a given fluorescent signal level and autocorrelation of pixel intensities were used. We showed the nuclear texture has specific changes at different mitotic stages and this can be used for more precise staging of mitosis. Colchicines treatment pathologies, connected to abnormal mitoses, by nuclear-texture approach.

[An intergral method for quantitative cell DNA content measurements by digital microphotography].

Quantitative nuclei DNA content measurement based on Feulgen reaction and the analysis of CCD images was tested. The measurements were performed in monochorome CCD option (650 X 514 pixels) with the wavelength 551 nm. The linear dependence of photomatrix elements signals on the falling light was shown with the use of multigraded light absorption filter. The optimal microscope and camera setting and an approach for elimination of the optic blur were found. We have shown that the proper Feulgen fluorescence does not affect our measurements. Densitometric DNA content measurements of blood cells of four vertebrate species (Gallus domesticus, Danio rerio, Homo sapiens and Rana arvalis) showed good consistence to the literature data (http://www.genomesize.com). The precision of our approach is comparable to the other known methods. Current improvement of CCD technical parameters and wide usage of CCD cameras in biological applications gives perspectives for the suggested approach.

[Drosophila melanogaster gene Merlin interacts with the clathrin adaptor protein gene lap].

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.

[Dynamics of the spatial organization of the chromosome set in cells of Drosophila melanogaster imaginal disks normally and under the action of the tumor-inducing mutation Merlin].

Fluorescence of H3-p histone and DAPI was studied at different stages of interphase and mitosis in cells of imaginal disks of third-instar Drosophila melanogaster larvae. Three stages differing in the spatial organization of the chromosome set in mitosis were revealed. At the first stage (prophase, prometaphase), the histone 3 phosphorylation level rises, and the volume occupied by the chromosome set in the nucleus increases. The distinctive features of the second stage (metaphase) are a gradual decrease in the histone 3 phosphorylation (the density ofphosphorylation remaining constant) and a reduction of the volume occupied by the chromosome set. At the third stage (anaphase, telophase), the intensity and density of the signal from H3-p histone decrease, and the volume occupied by the chromosome set reduces. At this stage, in Mer4 larvae, in contrast to the control strain, the cells prematurely pass from anaphase into telophase. In addition, a subpopulation of cells with an abnormally large volume of nuclear DNA during the G1 period was revealed in Mer4 larvae. The cells of this subpopulation do not enter into the DNA synthesis and quit the cycle.

[Effect of mutations in Drosophila melanogaster tumor suppressor Merlin on proliferation and differentiation of wing cells].

Experiments on transplantation of wing imaginal discs homozygous for a mutation in the tumor suppressor gene Merlin have demonstrated that this mutation does not induce malignant tumors. Marking of the wing disc compartment borders by specific antibodies showed the absence of essential compartment border defects in case of the Merlin mutation. Drosophila melanogaster cells mutant for Merlin have shorter cell cycle than normal cells. Proliferation of imaginal discs lasts longer in case of the mutation. It is known that beginning from some moment of development, wing veins serve as clonal restriction lines that cannot be crossed by growing mosaic clones. We showed that the Merlin mutation leads to depression of vein clonal restriction property. This means that this gene is involved not only in the control of cell proliferation, but also in the control of cell mobility and adhesion.

[The role of the functional sites of the Merlin tumor suppressor in Drosophila spermatogenesis].

The Merlin gene of Drosophila is homologous to the human Neurofibromatosis 2 (NF2) gene an important regulator of proliferation and endocytosis of cell receptors. It was earlier shown that the Thr5 residue of the Drosophila Merlin protein was homologous to Ser518 of the human protein (which was already known to undergo phosphorylation); hence, it was assumed that Thr559 of Drosophila also was a substrate of phosphorylation. The mutant Merlin proteins MerT559D (an analog of the phosphorylated form) and MerT559A (a nonphosphorylated form) were constructed and tested, under the conditions of ectopic expression for the ability to correct the spermatogenesis defects induced by the Mer4 mutation. The mutant form MerT559D was demonstrated to restore the abnormal nebenkern phenotype induced by this mutation, whereas the MerT559A substituted form did not restore this phenotype. Ectopic expression o the wild-type Merlin protein, MerT559A mutant form, and mycMer345-635 truncated protein in a normal genotype resulted in the abnormal nebenkern phenotype, whereas this phenotype was not observed in the case ofectopic expression of the MerT559D analog of the phosphorylated form. Ectopic expression of the mycMer3, mycMerABB, and mycMer-379 truncate variants led to disturbance of meiotic cytokinesis.

[High frequency induction of small mosaic cell clones in the Drosophila wings under the influence of gamma-irradiation].

As it was shown earlier in Gonzalez-Gaitan et al., one-cell and two-cells clones (tailing clones) are induced in the Drosophila wings after irradiation and represent a significant portion of clones detected with the use of mwh genetic marker. Our experiments shown that gamma-irradiation occur to be more efficient inductor of such small clones. Earlier small clones were considered as a result of the induced chromosomal aneuploidy of those low proliferating cells. Our data suggest that the small clones descend from the low proliferative cells of non-imaginal disc origin that migrate to the wing imaginal disc at some developmental point.

[The role of Gilgamesh protein kinase in Drosophila melanogaster spermatogenesis].

The cellular function of the gilgamesh mutation (89B9-12) of casein kinase gene in Drosophila spermatogenesis was studied. It was demonstrated that the sterility resulting from this mutation is connected with the abnormalities in spermatid individualization. A phylogenetic study of the protein sequences of casein kinases 1 from various organisms was conducted. The Gilgamesh protein was shown to be phylogenetically closer to the cytoplasmic casein kinase family, represented by the YCK3, YCK2, and YCK1 proteins of Saccharomyces cerevisiae and animal gamma-casein kinases. It is known that these yeast casein kinases are involved in vesicular trafficking, which, in turn, is related in its genetic control to the cell membrane remodeling during spermatid individualization. Thus, the data of phylogenetic analysis fit well the results obtained by studying the mutation phenotype.

[Specifics of anaphase chromatid disjunction in Drosophila melanogaster mitotic mutants].

Anaphase chromatid behavior defects (CBDs) were quantitatively and qualitatively studied in nerve ganglion cells of third-instar larvae of several control wild-type Drosophila melanogaster strains and four strains with mutations of the aar(v158), ff3, mast(v40), and CycB(2g) cell-cycle genes. A linear specificity was observed for the CBD frequency, type, determination, and correction probability. The probability of anaphase CBD correction was close to unity in the control strains and lower in the mutant strains. The lower correction probability in the mutant strains was explained in the context of two findings, that the mutations induced the CBDs that were atypical of the wild-type strains and were potentially uncorrectable in anaphase and that the mutations negatively affected the relative anaphase time in mitosis.

[Chromosome nondisjunction in Drosophila strains mutant for tumor suppressor Merlin].

The effect of mutation for gene Merlin on chromosome disjunction in Drosophila during meiosis was genetically studied. Chromosome nondisjunction was not registered in females heterozygous for this mutation and containing structurally normal X chromosomes. In cases when these females additionally contained inversion in one of chromosomes X, a tendency toward the appearance of nondisjunction events was observed in individuals containing mutation in the heterozygote. The genetic construct was obtained allowing the overexpression of protein corresponding to a sterile allele Mer3 in the germ cell line. This construct relieves the lethal effect of Mer4 mutation. The ectopic expression of this mutant protein leads to chromosome nondisjunction in male meiosis.

[Role of the porcupine gene in the development of the wing imaginal disk of Drosophila melanogaster].

A search for the genes interacting with the Merlin tumor suppressor gene revealed a Merlin-porcupine interaction during wing morphogenesis. Ectopic expression of the porcupine gene in the wing imaginal disk reduced the adult wing, while addition of an UAS construct with a full-length or truncated copy of the Merlin gene partly restored the wing phenotype. The highest restoration level was observed upon adding the fragments coding for the C end of the Merlin protein. In addition, the porcupine gene was shown to mediate the wingless gene autoregulation, which occurs at two ontogenetic stages, segmentation during embryo development and determination of the wg expression band at the boundary between the dorsal and ventral compartments of the wing imaginal disk.