[Characteristic of the active substance of the Saccharomyces cerevisiae preparation having radioprotective properties]. / Характеристика активной субстанции препарата дрожжей Saccharomyces сerevisiae, обладающей радиопротекторными свойствами.

The paper describes some biological features of the radioprotective effect of double-stranded RNA preparation. It was found that yeast RNA preparation has a prolonged radioprotective effect after irradiation by a lethal dose of 9.4 Gy. 100 % of animals survive on the 70th day of observation when irradiated 1 hour or 4 days after 7 mg RNA preparation injection, 60 % animals survive when irradiated on day 8 or 12. Time parameters of repair of double-stranded breaks induced by gamma rays were estimated. It was found that the injection of the RNA preparation at the time of maximum number of double-stranded breaks, 1 hour after irradiation, reduces the efficacy of radioprotective action compared with the injection 1 hour before irradiation and 4 hours after irradiation. A comparison of the radioprotective effect of the standard radioprotector B-190 and the RNA preparation was made in one experiment. It has been established that the total RNA preparation is more efficacious than B-190. Survival on the 40th day after irradiation was 78 % for the group of mice treated with the RNA preparation and 67 % for those treated with B-190. In the course of analytical studies of the total yeast RNA preparation, it was found that the preparation is a mixture of single-stranded and double-stranded RNA. It was shown that only double-stranded RNA has radioprotective properties. Injection of 160 µg double-stranded RNA protects 100 % of the experimental animals from an absolutely lethal dose of gamma radiation, 9.4 Gy. It was established that the radioprotective effect of double-stranded RNA does not depend on sequence, but depends on its double-stranded form and the presence of "open" ends of the molecule. It is supposed that the radioprotective effect of double-stranded RNA is associated with the participation of RNA molecules in the correct repair of radiation-damaged chromatin in blood stem cells. The hematopoietic pluripotent cells that have survived migrate to the periphery, reach the spleen and actively proliferate. The newly formed cell population restores the hematopoietic and immune systems, which determines the survival of lethally irradiated animals.

[COMPARATIVE STUDY OF Hrs AND OTHER ENDOSOMAL MARKERS CELLULAR LOCALIZATION IN DROSOPHILA MELANOGASTER SPERMATOGENESIS BY GFP-CHIMERICAL PROTEIN APPROACH].

Acrosome is a special organelle in spermatozoids necessary for fertilizing oocyte and originates, according to various theories, either from Golgi apparatus, or from endosomes and lysosomes. One of the proteins, found at mammalian acrosome, is Hgs, a homologue of Drosophila melanogaster Hrs (Hepatocyte growth factor regulated tyrosine kinase substrate), a known marker of multivesicular bodies (MVBs). However, although Drosophila acrosome was extensively studied, it is yet unknown whether Hrs localizes at acrosome similar to Hgs and, more generally, whether the spectrum of acrosomal proteins in Drosophila is the same as in mammals. Hrs (hepatocyte growth factor regulated tyrosine kinase substrate) is the multidomain vesicular protein participating in the endosome-lysosome protein sorting. We demonstrated that two protein variants of the Drosophila Hrs are expressed in testes: a longer isoform B, and a shorter isoform A, which lacks VHS and FYVE domains that are necessary for anchoring Hrs in endosomes. We found that Hrs isoform B is concentrated at fusoma of spermatocytes in contrast to mammalian Hrs. This localization requires the C-terminus of the protein, starting from the aminoacid residue 383. In situ hybridization of hrs RNA probe showed that the gene is expressed early in spermatogenesis consistently with Hrs localization in early fusoma. Additionally, we demonstrated that Hrs is dispensable for cytokinesis. Finally, it was found that although Drosophila Hrs does not localize at acrosome, the other endosomal markers--Rab4, Rab7, and Rab11--are detected at the organelle.

[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.

[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.

[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.

[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.

[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.

[A study of expression of cell cycle genes in Drosophila using an enhancer trap and radioautography]. / Izuchenie ékspressii genov kletochnogo tsikla Drozofily s pomoshch'iu énkhansernoi lovushki i radioavtografii.

The phase of expression of genes CycB, CycE, and chb were determined in the cell cycle of neuroblasts of D. melanogaster 3rd instar larvae using the previously described radioautographic method and software. CycB was expressed at G2 phase and upon transition from G2 phase to M phase, while CycE was expressed at the end of G1 phase and upon transition from G1 phase to S phase. The phase of expression of the centrosome-associated protein was determined more precisely in G2 phase. The mean life span of reporter beta-galactosidase in neuroblasts was 4 h. The existence of more than one peak of expression of the gene in question in the cell cycle is discussed.

[Comparative study of effect of infrared, submillimeter, and millimeter electromagnetic radiation on wing somatic mutations in Drosophila melanogaster induced by gamma-irradiation]. / Sravnitel'noe issledovanie vliianiia élektromagnitnogo izlucheniia infrakrasnogo, submillimetrovogo i millimetrovogo diapazonov na indutsirovannye gamma-oblucheniem somaticheskie mutatsii kletok kryl'ev Drosophila melanogaster.

It was shown that the number of spontaneous and gamma-radiation-induced somatic mutations in wing cells of fruit flies (third instar larvae) exposed to laser irradiation of submillimeter range (lambda = 81.5 microns) was significantly lower than in control. Laser irradiation did not affect the number of recombinations. Exposure to laser radiation in the infrared range and electromagnetic waves of the millimeter range (lambda = 3.8 mm) enhanced the effect of gamma-irradiation.

[Phase-specific elements of the regulatory zone of the Drosophila melanogaster string gene]. / Fazospetsifichnye élementy reguliatornoi zony gena string Drosophila melanogaster.

The phases of the reporter gene expression controlled by different fragments of the string (stg) gene regulatory region were determined in Drosophila neuroblasts by detection of beta-galactosidase activity and radioautography. In the D10 and D22 lines carrying the constructs pstg beta-E4.9 and pstg beta-E5.3, respectively, the reporter gene activity was detected in the G1 phase of the cell cycle. In the D12 and D20 lines (pstg beta-E6.4 and pstg beta-E2.6), no periodic expression was observed. The regulatory regions of the stg from lines D10 and D22 and that of Drosophila gene cyclin D shared consensus aagaactttg, which was also expressed in the G1 phase. The phase-specific expression of the cell-cycle genes was compared in a model for the mitotic-wave cells of eye imaginal disk and neuroblasts of the nerve ganglia.

[Determination of the expression phase of chb(V40) gene in the cell cycle of Drosophila melanogaster]. / Opredelenie fazy ékspressii gena chb(V40) v kletochnom tsikle Drosophila melanogaster.

Using autoradiography, we have determined cell cycle parameters in neuroblasts of III-rd instar larvae of D. melanogaster. The overall duration of the cell cycle is 9 h, tG1 = 4 h, tS = 3.5 h, tG2 = 1 h, and tM = = 0.5 h. Using histochemical staining for beta-galactosidase activity, we have determined the stage of expression of the reporter gene of P[1ArB] element inserted into chb gene. The chb gene, which codes for a centrosomal protein, is expressed during the last third of S phase. This result illustrates the feasibility of using the enhancer trap P[1ArB] to study differential expression of cell cycle genes.