[Expression of cecropin P1 gene increases resistance of Camelina sativa (L.) plants to microbial phytopathogenes].

Transgenic plants of camelina (Camelina sativa (L.) Crantz) with the synthetic gene of antimicrobial peptide cecropin P1 (cecP1) were obtained. Agrobacterium-mediated transformation is performed using the binary vector pGA482::cecP1 by vacuum infiltration of flower buds. The presence of the cecP1 gene in the genome of plants was confirmed by PCR. cecP1 gene expression in transgenic plants was shown by Western blot analysis and by antimicrobial activity of plant extracts against the bacterial phytopathogene Erwinia carotovora. The plants of F0 and F1 generations had the normal phenotype and retained the ability to form viable seeds in self-pollination. cecP1 plants exhibit enhanced resistance to bacterial and fungal phytopathogens: Erwinia carotovora and Fusarium sporotrichioides. The increased sustainability of cecropin P1-expressing plants against salt stress is shown. The possibility of the integration of the cecP1 gene into the overall protective system of plants against biotic and abiotic stresses is discussed.

[Peculiarities of 5-methylcytosine distribution in eukaryotic genome].

Most of it functions DNA methylation realizes as an integral part of the mechanism of remodeling and modification of chromatin structure. At the same time the global pattern of this complex reaction's net is still to be determined and we are just approaching to studying the mechanisms controlling epigenetic processes of histone modification and DNA methylation. Though cytosine methylation occurs predominantly at CpG sequences of eukaryotic genome, it also takes place at symmetric CpHpG and non-symmetric CpHpH sites (where H-A, T, or C). Various modification efficiency for these three site-specific DNA methylation types is observed depending on their genome localization. Different regions in eukaryotic genome are remarkable for their methylation features: CpG-islands, CpG-islands shores, differentially methylated regions of imprinted genes, and regions of non-alternative site-specific modification. Dependence of three canonical types (CpG, CpHpG, and CpHpH) of DNA methylation efficiency on their surrounding nucleotide context is noted. Existence of epigenetic code of DNA methylation, in which these context differences play specific functional role, has been supposed. The present review summarizes main up-to-date data on structural-functional features of site-specific cytosine methylation in eukaryotic genomes. Pathogenesis-related alterations of eukaryotic genome methylation pattern are considered as well.

[The use of RNA interference for the metabolic engineering of plants].

The metabolic engineering of plants is aimed at the realization of new biochemical reactions by transgenic cells. These reactions are determined by enzymes encoded by foreign or self-modified genes. Plants are considered to be the most interesting objects for metabolic engineering. Although they are characterized by the same pathways for the synthesis of basic biological compounds, plants differ by the astonishing diversity of their products: sugars, aromatic compounds, fatty acids, steroid compounds, and other biologically active substances. RNA interference aimed at modifying metabolic pathways is a powerful tool that allows for the obtainment of plants with new valuable properties. The present review discusses the main tendencies for research development directed toward the obtainment of transgenic plants with altered metabolism.

[The influence of colonizing methylobacteria on morphogenesis and resistance of sugar beet and white cabbage plants to Erwinia carotovora].

The influence of colonization of sugar beet (Beta vulgaris var. saccharifera (Alef) Krass) and white cabbage (Brassica oleracea var. capitata L.) plants by methylotrophic bacteria Methylovorus mays on the growth, rooting, and plant resistance to phytopathogen bacteria Erwinia carotovora was investigated. The colonization by methylobacteria led to their steady association with the plants which had increased growth speed, root formation and photosynthetic activity. The colonized plants had increased resistance to Erwinia carotovora phytopathogen and were better adapted to greenhouse conditions. The obtained results showed the perspectives for the practical implementation of methylobacteria in the ecologically clean microbiology substances used as the plant growth stimulators and for the plant protection from pathogens.

[Inhibition of agrobacterial oncogenes expression by means of antisense RNA].

Plant's infection with soil bacteria Agrobacterium tumefaciens lead to tumour formation, so called crown galls. The reason of tumorigenesis is integration of agrobacterial genes for phytohormone synthesis auxins and cytokinins in plant genome, the most important of them are iaaM and ipt. Obtaining of transgenic plants able to inhibit these genes expression, creates conditions for producing of plants resistant to crown gall disease. With this purpose single and double transformants of tobacco plants with antisense copies of iaaM and ipt genes under the control of single and double promoters of 35S RNA of cauliflower mosaic virus (CaMV 35S and CaMV 35SS) were produced. Infection with virulentA. tumefaciens strains C58 (pTiC58) and A6 (pTiA6) of all types transgenic plants with antisense oncogenes copies showed essential but incomplete inhibition of these genes expression. After agrobacterial transformations of transgenic plants only "weakened" tumours of various morphology, able to regenerate the whole plants, were formed. The analysis data of inhibition of iaaM and ipt genes expression in formed tumour cells were presented. The results indicate perspective RNA-interference strategy for producing of plants resistant to agrobacterial crown gall disease.

[Enhanced resistance to phytopathogenic bacteria in transgenic tobacco plants with synthetic gene of antimicrobial peptide cecropin P1].

Plasmids with a synthetic gene of the mammalian antimicrobial peptide cecropin P1 (cecP1) controlled by the constitutive promoter 35S RNA of cauliflower mosaic virus were constructed. Agrobacterial transformation of tobacco plants was conducted using the obtained recombinant binary vector. The presence of gene cecP1 in the plant genome was confirmed by PCR. The expression of gene cecP1 in transgenic plants was shown by Northern blot analysis. The obtained transgenic plants exhibit enhanced resistance to phytopathogenic bacteria Pseudomonas syringae, P. marginata, and Erwinia carotovora. The ability of transgenic plants to express cecropin P1 was transmitted to the progeny. F1 and F2 plants had the normal phenotype (except for a changed coloration of flowers) and retained the ability to produce normal viable seeds upon self-pollination. Lines of F1 plants with Mendelian segregation of transgenic traits were selected.

[Biosynthesis of mini-antibodies to human ferritin in plant and bacterial producers]. / Biosintez mini-antitel k ferritinu cheloveka v rastitel'nykh i bakterial'nykh produtsentakh.

A recombinant scFv antibody against human spleen ferritin was expressed as a barstar-fused protein in Escherichia coli and in Nicotiana tabacum plants and suspension cell cultures. As demonstrated by immunoblotting with antibarstar antibodies, direction of the recombinant protein to the endomembrane system of plant cells ensured its stability and solubility. Production of the recombinant protein did not differ between parental transgenic plants and their first-generation progeny. Fusion with barstar allowed not only immunochemical detection of the recombinant scFv antibody, but also their purification from the plant material by affinity chromatography with barnase-His6 immobilized on a metal-affinity carrier.

[Analysis of transgenic tobacco plants carrying the gene for the surface antigen of the hepatitis B virus]. / Analiz transgennykh rastenii tabaka, soderzhashchikh gen poverkhnostnogo antigena virusa gepatita B.

The plasmids carrying the gene encoding the hepatitis B surface antigen (HBsAg) under the control of 35S RNA single or dual promoters of the cauliflower mosaic virus CaMV 35S were constructed. These constructions were used for obtaining transgenic tobacco plants that synthesize the HBS antigen. The presence of HBsAg in tobacco plant extracts was confirmed by the enzyme-linked immunoassay using antibodies against the native HBs antigen. The antigen amount in plants carrying the HbsAg gene under a single 35 S promoter was 0.0001-0.001 of the total soluble protein whereas the use of a dual 35S promoter increased the antigen synthesis to 0.002-0.05% of the protein. The antigen-synthesizing ability was inherited by the offspring. In the F1 plants, the antigen expression varied in different lines comprising 0.001 to 0.03% of the total soluble protein, which corresponded to the antigen amount in the F0 plants.

[Hypermethylation of the human calcitonin gene as a molecular marker in acute lymphoid leukemia]. / Gipermetilirovanie gena kal'tsitonina cheloveka kak molekuliarnyi marker ostroi limfoidnoi leikemii.

HpaII/MspI blot-hybridization analysis of the 5'-end region of the calcitonin (CT) gene methylation in cells of bone marrow and peripheral blood of patients with acute lymphoblastic leukemias (ALL) has been carried out. ALLs are accompanied by hypermethylation of the inner cytosine in the CCGG sequences of this region of the CT gene. The level of hypermethylation of the CT gene corresponded to the degree of disease progression and malignancy. At a long-term remission, hypermethylation of the CT gene is not observed. In case of primary resistance or if the complete remission has not been achieved the CT gene remained hypermethylated. It has been shown that in relapse the normal CT gene methylation pattern reversed to hypermethylation. This phenomenon was detected 1-8 months before the obvious clinical and laboratory signs of the disease progression (relapse). The large size of abnormal HpaII-fragments of the 5'-end region of the calcitonin gene had a direct correlation with the malignancy status of ALL.

[Phenotypic changes in transgenic tobacco plants with an antisense form of the hmg1 gene]. / Fenotipicheskie izmeneniia transgennykh rastenii tabaka s antismyslovoi formoi gena hmg1.

Tobacco plants were genetically transformed with the Arabidopsis thaliana heterologous hmg1 gene encoding 3-hydroxy-3-methylglutaryl-CoA reductase, a key enzyme involved in the metabolism of terpenoid compounds. The hmg1 gene was inserted under the control of the 35S RNA double promoter from the cauliflower mosaic virus (CaMV 35S) both in direct and reverse orientation relative to the promoter. DNA analysis by polymerase chain reaction (PCR) and Southern blotting confirmed the transgenic nature of the tobacco plants obtained. DNA-RNA hybridization revealed expression of the hmg1 gene in these tobacco plants. The plants transformed with the antisense copy of the hmg1 gene differed from the control plants in delayed development and in flower color and shape.

[The lack of the CpNpG methylation at the 5'-terminal region of the human calcitonin gene in norm and in leukemia []. / Otsutstvie CpNpG-metilirovaniia v 5'-kontsevoi oblasti gena kal'tsi- tonina cheloveka v norme i pri leikozakh [(letter)].

The inner cytosine methylation was analyzed in the CCWGG sequences of the 5'-terminal region of the human calcitonin gene from peripheral blood and bone marrow cells in various forms of leukemia. Since these sequences remain nonmethylated both in norm and in various leukemia forms, the CpG dinucleotide hypermethylation of the 5'-terminus of the human calcitonin gene, characteristic for the development of leukemias, does not spread over adjacent CpNpG sequences.

[DNA methyltransferases for detection of the level of methylation of cytosine in the DNA CCWGG sequence]. / DNK-metiltransferazy dlia opredeleniia urovnia metilirovaniia tsitozina v posledovatel'nosti CCWGG DNK.

An assay for the cytosine methylation level in the eukaryotic DNA CCWGG sequence is proposed. The method is based on the ability of DNA methylase BstNI to methylate DNA containing in a CCWGG site a nonmodified or 5-methylated cytosine to yield N4-methyl- or N4,5'-dimethylcytosine, respectively.

[Formation of deletional derivatives of the Ti-plasmid pGV3850 in a conjugative transfer from Agrobacterium tumefaciens to Escherichia coli]. / Obrazovanie deletsionnykh proizvodnykh Ti-plasmidy pGV3850 pri kon"iugatsionnom perenose iz Agrobacterium tumefaciens v Escherichia coli.

The process of the transfer of the Ti-plasmid vector pGV3850 with the plasmid pBR322 inserted into the T-DNA region from Agrobacterium tumefaciens to a non-plasmid strain of Escherichia coli was studied. The transferred Ti-plasmid was found to be exposed to deletions and formed a wide range of derivatives with a size ranging from 3-4 kb to 50 kb, maintained in E. coli due to ColE1-replicon. The Ti-plasmid is also inserted into the chromosome of the recipient bacterium with at least a 100-fold lower frequency than the formation of deletional derivatives. It was shown that the induction of vir genes controlling the transfer of T-DNA into plants has no appreciable effect on the efficiency of obtaining transconjugates in mating with E. coli. The deletion of the genetic material of megaplasmids with the inserted functional site OriV ColE1, as a result of the conjugative transfer from cells of different bacteria to the cells of E. coli, was proposed for molecular cloning.

[Formation of two types of enzyme-substrate complexes during the interaction of EcoRII restriction enzyme with synthetic DNA-duplexes]. / Obrazovanie dvukh tipov ferment-substratnykh kompleksov pri vzaimodeistvii restriktazy EcoRII s sinteticheskimi DNA-dupleksami.

Binding of EcoRII restriction endonuclease to synthetic oligodeoxyribonucleotide substrates of 11-30 base pairs long was investigated by polyacrylamide gel electrophoresis under nondenaturing conditions in the absence of Mg2+ ions. Irrespective of the length of a substrate, two types of specific DNA-protein complexes were shown to be formed. Their mobility in gel was close to that of the monomer (45 kDa) and dimer (90 kDa) of marker protein, ovalbumin. The ratio of these complexes in solution depended on that of the molar concentrations of EcoRII restriction endonuclease and DNA duplexes. The possible structure of the complexes is discussed.

[Interaction of EcoRII restriction and modification enzymes with synthetic DNA fragments. Determination of the size of EcoRII binding site]. / Vzaimodeistvie fermentov restriktsii i modifikatsii EcoRII s sinteticheskimi fragmentami DNK. XI. Opredelenie velichiny mesta posadki éndonukleazy EcoRII na substrat.

EcoRII restriction endonuclease binding site has been determined on the basis of comparison of the binding parameters of the enzyme with synthetic DNA-duplexes of concatemer type containing a different number of EcoRII recognition sites. It has been shown that it consists of 21 +/- 1 base pairs.

[UV- and CD-spectra of restriction endonuclease EcoRII and DNA-methylase EcoRII]. / UF- i KD-spektry restriktsionnoi éndonukleazy EcoRII i DNK-metilazy EcoRII.

UV- and CD-spectra of homogeneous enzymes have been measured. Extinction coefficients estimated from the UV-spectra are 0.97 for restriction endonuclease EcoRII at 279.5 nm and 1.17 for DNA-methylase EcoRII at 279 nm. As it follows from the CD spectra, both enzymes have a well developed tertiary structure and a highly ordered secondary structure, which consists of 22% alpha-helices, 64% beta-structure and 9% bends for REcoRII and of 44% alpha-helices, 48% beta-structure and 4% bends for MEcoRII. Restriction endonuclease denatures at 50 degrees C, while DNA-methylase denatures at 45 degrees C, with partial reversibility upon cooling.

[Isolation, purification and properties of restrictase and methylase BstN1 from Bacillus stearothermophilus]. / Vydelenie, ochistka i nekotorye svoistva restriktazy i metilazy BstN1 iz Bacillus stearothermophilus.

Restriction-methylation enzymes BstN1 from Bacillus stearothermophilus were isolated and purified. These enzymes are related to a new class of restriction-methylation enzymes of the second type, whose modifying component is N4-cytosine-DNA-methylase. Both enzymes recognize the DNA sequence CC(A/T)GG. Restrictase BstN1 is a protein made up of one subunit with a molecular mass of 25 kDa. The molecular mass of native DNA-methylase BstN1 is about 55 kDa. The temperature optima for restrictase and methylase BstN1 are around 60 degrees C. Possible uses of BstN1 restriction-methylation enzymes for the analysis of cytosine methylation in bacterial and higher plant DNA are discussed.