[Obtaining the transgenic lines of finger millet Eleusine coracana (L.) Gaertn. With dinitroaniline resistance].

The current data is dedicated to the study of bioballistic and Agrobacterium-mediated transformation of finger millet with the constructs carrying the mutant alpha-tubulin gene (TUAm 1), isolated from R-biotype goosegrass (Eleusine indica L.), for the decision of problem of dinitroaniline-resistance. It was found that 10 microM of trifluralin is optimal for the selection of transgene plants of finger millet. PCR analysis of transformed lines confirmed the transgene nature of plants. The analysis of seed of T1 oftransgene lines confirmed heterozygous character of inheritance of the resistance.

[cDNA library construction from panicle meristem of finger millet].

The protocol for production of full-size cDNA using SuperScript Full-Length cDNA Library Construction Kit II (Invitrogen) was tested and high quality cDNA library from meristematic tissue of finger millet panicle (Eleusine coracana (L.) Gaertn) was created. The titer of obtained cDNA library comprised 3.01 x 10(5) CFU/ml in avarage. In average the length of cDNA insertion consisted about 1070 base pairs, the effectivity of cDNA fragment insertions--99.5%. The selective sequencing of cDNA clones from created library was performed. The sequences of cDNA clones were identified with usage of BLAST-search. The results of cDNA library analysis and selective sequencing represents prove good functionality and full length character of inserted cDNA clones. Obtained cDNA library from meristematic tissue of finger millet panicle represents good and valuable source for isolation and identification of key genes regulating metabolism and meristematic development and for mining of new molecular markers to conduct out high quality genetic investigations and molecular breeding as well.

[Creation of transgenic sugar beet lines expressing insect pest resistance genes cry1C and cry2A].

Impact of insect pests makes a significant limitation of the sugar beet crop yield. Integration of cry-genes of Bacillus thuringiensis into plant genome is one of the promising strategies to ensure plant resistance. The aim of this work was to obtain sugar beet lines (based on the MM 1/2 line) transformed with cry2A and cry1Cgenes. We have optimized transformation protocol and direct plant let regeneration protocol from leaf explants using 1 mg/l benzylaminopurine as well as 0,25 mg/l benzylaminopurine and 0,1 mg/l indole-butyric acid. Consequently, transgenic sugar beet lines transformed with vector constructs pRD400-cry1C and pRD400-cry2A have been obtained. PCR analysis revealed integration of cry2A and cry1C into genome of transgenic lines and expression of these genes in leaf tissues was shown by reverse transcription PCR.

[Establishment of in vitro culture, plant regeneration and genetic transformation of Camelina sativa].

The results on in vitro culture establishment, plantlet regeneration and rooting of Camelina sativa cultivar sample Peremozhets and cultivar Mirazh are presented. Effective concentrations of sterilizing agents and duration of plant material treatment were estimated. Phytohormone ratio, sucrose concentration in nutrient medium that induce effective formation of C. sativa shoots and NAA concentration for plantlet rooting have been established. The method of Agrobacterium-mediated transformation of Camelina by using binary vector pGH217 carrying reporter beta-glucoronidase (gus) gene driven under 35S CaMV promoter and nos-terminator, and selective marker hpt gene conferring hygromycin-resistance in transgenic plant was elaborated.

[Genetic transformation of flax (Linum usitatissimum L.) with chimeric GFP-TUA6 gene for visualisation of microtubules].

The data of Agrobacterium-mediated transformation of some Linum usitatissimum cultivars zoned on the territories of Belarus and Ukraine with the plasmid carrying chimeric GFP-TUA6 gene and nptII gene as selectable marker conferring resistance to kanamycin are presented in this study. Transformation was affected by a number of factors including optical density (OD600), time of inoculation of explants with Agrobacterium and co-culture conditions. Transgenic nature of obtained lines was confirmed by PCR analysis. Expression of GFP-TUA6 gene was detected with confocal laser scanning microscopy. The obtained transgenic lines can be used for further functional studies the role of microtubules in the processes of building the flax fibres and resistance to wind.

[Effect of inhibitors serine/threonine protein kinases and protein phosphatases on mitosis progression of synchronized tobacco by-2 cells].

In order to investigate the role of various serine/ threonine protein kinases and protein phosphatases in the regulation of mitosis progression in plant cells the influence of cyclin-dependent (olomoucine) and Ca2+ -calmodulin-dependent (W7) protein kinases inhibitors, as well as protein kinase C inhibitors (H7 and staurosporine) and protein phosphatases inhibitor (okadaic acid) on mitosis progression in synchronized tobacco BY-2 cells has been studied. It was found that BY-2 culture treatment with inhibitors of cyclin dependent protein kinases and protein kinase C causes prophase delay, reduces the mitotic index and displaces of mitotic peak as compare with control cells. Inhibition of Ca2+ -calmodulin dependent protein kinases enhances the cell entry into prophase and delays their exit from mitosis. Meanwhile inhibition of serine/threonine protein phosphatases insignificantly enhances of synchronized BY-2 cells entering into all phases of mitosis.

[Morphological and histochemical analysis of mucous membrane transformation of the artificial urinary bladder].

The performed morphologic study of transformation mechanisms of the intestinal reservoir mucous membrane using modern immunochemical and histochemical methods revealed three morphologic phases of enteric epithelium adaptation; reactive-inflammatory, compensatory-protective and atrophic. Enteric urinary reservoir preserves the cambial apparatus typical for the enteric phenotype with an inherent apoptosis activity and proliferation which confirms a low level of tumor transformation risk.

[Developing transgenic barley lines producing human lactoferrin using mutant alfa-tubulin gene as selective marker gene].

Method of biolistic transformation was used for genetic improvement of commercial barley cultivars (Oksamitoviy, Vodogray and Getman). The plasmid pHLFTuBA was used for particle bombardment that consists of the hLF gene under the control of the barley glutelin B-1 promoter and a selectable marker gene, alpha-tubulin conferring resistance to trifluralin (dinitroanilinr herbicide). Preliminary screening of different trifluralin concentration range from 0,1 to 30 microM was tested for determination of effective selective agent concentration. Two transgenic barley line of genotype Oksamitiviy and transgenic callus line of cultivar Getman were obtained after selection on 10 microM of trifluralin. To confirm the transgenic nature of regenerated plants, the PCR analysis was carried out. The 734bp length fragment of hLFgene was amplified from both regenerated plants.

[Peculiarities of urinary bladder cancer tumor cells apoptosis response on neoadjuvant chemotherapy].

Induced apoptosis in urinary bladder cancer tumor cells of patients was studied using TUNEL reaction. It was shown that increase in induced apoptosis value had a definite correlation between corresponding features of tumor reaction as a response on Gemcitabine-Cisplatin neoadjuvant chemotherapy application. It was found that evaluation of induced apoptosis in urinary bladder cancer tumor cells using TUNEL method allows forecasting the effectiveness of chemotherapy on the cellular level in patients with this type of cancer.

[The effect of inhibitors of serinethreonine protein kinases on Arabidopsis thaliana root morphology and microtubules organization in its cells].

The effect of different types of serine/thereonine protein kinases inhibitors (cyclin-dependent, Ca2+-calmodulin-dependent and protein kinase C) on microtubules organization in cells of Arabidopsis thaliana main primary root zones were investigated in vivo. The microtubules in epidermal and cortex cells in the transition and elongation zones as well as microtubules in trichoblasts and atrichoblasts in the differentiation zone showed the greatest sensitivity to protein kinases inhibitors studied. It was established that microtubules in these cell types modified their initial transverse/oblique orientation to a chaotic or longitudinal relative to the major axis of primary root as a result of serinethereonine protein kinases inhibition. The microtubules in cells in root meristematic zone as well as in root hairs were less sensitive to influence of protein kinases inhibitors tested. Alterations of microtubules orientation in the cells in primary root zones under the influence of serinethereonine protein kinases inhibitors led to further disturbances in growth and differentiation processes. It was assumed that phosphorylation of microtubules proteins, especially tubulin, might be involved in the regulation of these processes.

[MAST2-like protein kinase from grape vine Vitis vinifera: cloning of catalytic domain cDNA].

The aim of our work is the identification of protein kinases phosphorylating microtubule proteins in plant cells. Using bioinformatic approach, we found genes of putative homologues of microtubule-associated mammalian protein kinase MAST2 in higher plant genomes. The gene of closest MAST2 homologue, putative protein, named GMLK (Grape MAST2-Like Kinase, A7NTE9_VITVI), was found in grape Vitis vinifera. We report here the cloning of cDNA of GMLK (A7NTE9) from Pinot Noir grape vine leaves.

[Antimitotic activity of new 2,6-dinitroaniline derivatives and their synergistic activity in compositions with graminicides].

Shown antimicrotrubules activity of new 2,6-dinitroaniline compounds. Investigated their ability on apoptotic processes in a plant cell when used as a composition with inhibitors of acetyl-CoA carboxylase.

[Agrobacterium-mediated transformation of flax with a mutant tubulin gene responsible for resistance to dinitroaniline herbicides].

Agrobacterium tumefaciens was used to transform fiber flax with the pBITUBA8 plasmid carrying the mutant alpha-tubulin gene imparting resistance to dinitroaniline herbicides and the nptII selective marker gene imparting resistance to canamycin. The transformants were selected in parallel on media containing canamycin and trifluralin (a dinitroaniline herbicide). The transgenic nature of the resultant regenerants resistant to dinitroaniline herbicides was confirmed by means of Southern blotting and polymerase chain reaction (PCR) analysis using specific probes for the ntpll gene and the gene of alpha-tubulin.

[Estimation of the efficiency of callus formation and regeneration in barley spring varieties zoned in Ukraine].

The data on in vitro culture establishment and on estimation of callus formation and plant regeneration for eight barley Hordeum vulgare varieties (Getman, Tabora, Adagio, Galactic, Europrestige, Korona, Nevada and Stalker) zoned in Ukraine is represented. Mature embryos of these genotypes were used to study the callus induction and plant regeneration. Using of this approach can rather simplify and intensify the work. Medium, that consists of MS salts supplemented with caseine hydrolysate (1 g/L), L-proline (690 mg/L), thiamine-HCl (1 mg/L), maltose (30 g/L), 2,4-D (2 mg/L), CuSO4 (12.5 mg/L), myoinositol (250 mg/l), gerlite (3.5 g/L) pH 5.6-5.8 was used for both callus formation and plant regeneration. Formation of callus from mature embryos was observed in all studied varieties and showed high frequency (from 65 +/- 3.4% to 100%). It was found that cultivars Korona (88 +/- 2.8%), Europrestige (89 +/- 6.5%), Tabora (93 +/- 3.4%), Getman (99 +/- 0.8%) and Nevada (100%) had the highest regeneration potential. Organogenesis and somatic embryogenesis were the two ways of plant regeneration from callus tissues in barley. The highest general regeneration potential of cultivar Getman (50 +/- 5%) was observed. This cultivar was selected for the further work for development of effective genetic transformation protocol.

[The effect of okadaic acid on root morphology of Arabidopsis thaliana and microtubule organization in its cells].

To investigate the functional role of protein hyperphosphorylation in plant cells the general morphology of Arabidopsis thaliana primary roots and structural-functional property of cortical microtubules were studied after treatment with okadaic acid, specific inhibitor of protein phosphatases PPI and PP2A. It has been estimated that okadaic acid affects microtubule organization in a different manner depending from the type of the cells and functional zones of the primary root. It was found that the microtubules in epidermis and cortex cells of distal elongation zone which depolymerized after inhibitor treatment were the most sensitive to 0, 1, 1 and 10 nM okadaic acid concentrations. In trichoblasts, antichoblasts and cortex cells of differentiation zone treatment with okadaic acid caused the microtubules stabilization. Okadaic acid influences were particularly evident in root hair morphology, root hairs swelling and branching as a result of abnormal microtubules orientation observed. According to the data obtained, we can suggest that induction of protein hyperphosphorylation as a result of protein phosphatase inhibition plays crucial key in plant cell microtubule organization.

[Reorganization of microtubules as a response to realization of NO (II) signal pathways in plant cell].

Effects of exogenic NO donor, sodium nitroprusside, on orientation and organization of cortical microtubules in Arabidopsis thaliana root cells that express GFP-MAP4 were studied in vivo. It was found that sodium nitroprusside treatment (10-500 microM, 24 h) caused the acceleration of primary root growth and enhanced initiation of root hairs in differentiation zone. The influence of sodium nitroprusside revealed in alterations of cortical: microtubules orientation and organization in different types of cells of A. thaliana root. The most sensitive to sodium nitroprusside exposure were microtubules in epidermal cells of elongation zone where native transverse orientation of cortical microtubules turned into random, oblique or longitudinal relatively to primary root axis. We suppose that NO as one of the intracellular secondary messengers triggers cells differentiation by cortical microtubules reorientation possibly via tubulin nitrotyrosination.

[Structural-biological characteristics of tubulin interaction with dinitroanilines].

The interaction of dinitroaniline compounds with tubulin molecules is characterized by extraordinary selectivity--these matters effectively associate with plant as well as protozoan tubulin and practically don't interact with fungal and animal tubulin in spite of extraordinarily high level of similarity of their sequences. Structural features and mechanisms of this interaction are generalized and in detail analysed in this research. In particular, the regularities of dinitroaniline binding sites' structure and localization on surfaces of tubulin different subunits and tubulins of different origin are characterized. Dinitroaniline binding sites are disposed on the surfaces of longitudinal contacts between tubulin subunits, contain residues of diamine amino acids (lysine or arginine) coupling al nitrile group (s) of dinitroanilines. Binding site location on the surfaces of the same subunit of different origin (for example, plant and protozoan alpha-tubulins) is coincided, however site localisation on surface of alpha- and beta-subunits is distinct. The described sites potentially can be the binding areas for another antimicrotubular compounds, in particular, cyanoacrilates.

[Screening of new 2,4- and 2,6-dinitroaniline derivates for phytotoxicity and antimitotic activity].

The results of Allium-test screening of new 2,4- and 2,6-dinitroaniline derivates on antimitotic activity and phytotoxicity are presented in the work. It is revealed that all studied compounds which are derivates of 2,4-dinitroaniline, 2,6-dinitro-(4-fluoromethyl)-aniline as well as (methylsulfonyl) nitrobenzol, can evoke a change in mitotic index value, an appearance of cytogenetic damages and also have phytotoxic effect on Allium cepa seedling roots. On data obtained the continuation of investigation the action of N-(2,4-dinitrophenyl)-orto-aminobenzoic acid, N,N-diethyl-2,6-dinitro-4-(trifluoromethyl)-aniline and 1-methylsulfonyl-3-nitrobenzol as potential herbicides is proposed.

[Molecular and structural-biological analysis of Nicotiana plumbaginifolia mutants for identification of the site of beta-tubulins interaction with dinitroanilines and phosphorotioamidates].

The identification of point mutation locations on beta-tubulin molecules of amiprophosmethyl- and trifluralin-resistant Nicotiana plumbaginifolia lines have described in the work. It was shown that in the first case this mutation is connected with the substitution ofserine residue on proline in position 248; in the second case--with the substitution of phenilalanine on serine in position 317 of beta-tubulin amino acid sequence. Three-dimensional models of beta-tubulin molecule from Chlamydomonas with well-known location of mutations conferring dinitroaniline- and phosphorotioamidate resistance (substitution of lysine residue to methionine on position 350), and beta-tubulin from Nicotiana plumbaginifolia have been reconstructed. On the basis of analysis of site of interaction with dinitroanilines and phosphorotioamides on Chlamydomonas beta-tubulin molecule it was concluded that the revealed mutations on Nicotiana plumbaginifolia beta-tubulin affect amino acid residues participating in formation of this site.

[Bioinformatic search for plant homologs of Ste20-like serine/threonine protein kinases].

Eleven plant homologs of animal and yeast Ste20-like protein kinases were identified. It was shown that the nearest plant homologs of the Ste20-like protein kinases are the unknown proteins A9RVK0 from Physcomitrella patens ssp. patens and A7P2E2 from Vitis vinifera. Cladistic analysis showed a protein kinase dstl from Dictyostelium discoideum as the closest protein to the newly found plant homologs. A predicted spatial structure of the A9RVK0 from P. patens ssp. patens catalytic domain is presented.