35S Promoter Methylation in Kanamycin-Resistant Kalanchoe (Kalanchoe pinnata L.) Plants Expressing the Antimicrobial Peptide Cecropin P1 Transgene.

Transgenic kalanchoe plants (Kalanchoe pinnata L.) expressing the antimicrobial peptide cecropin P1 gene (cecP1) under the control of the 35S cauliflower mosaic virus 35S RNA promoter and the selective neomycin phosphotransferase II (nptII) gene under the control of the nopaline synthase gene promoter were studied. The 35S promoter methylation and the cecropin P1 biosynthesis levels were compared in plants growing on media with and without kanamycin. The low level of active 35S promoter methylation further decreases upon cultivation on kanamycin-containing medium, while cecropin P1 synthesis increases.

[The obtainment and characteristics of Kalanchoe pinnata L. plants expressing the artificial gene of the cecropin P1 antimicrobial peptide].

Kalanchoe pinnata L. plants bearing an artificial CP1 gene encoding the cecropin P1 antimicrobial peptide have been obtained. The presence of the CP1 gene in the plant genome has been confirmed by PCR. Cecropin P1 synthesis in transgenic plants has been shown by MALDI mass spectrometry and Western blotting. The obtained plants have been highly resistant to bacterial and fungal phytopathogens, and their extracts have demonstrated antimicrobial activity towards human and animal pathogens. It has been shown that transgenic plants bearing the CP1 gene can be colonized by the beneficial associative microorganisms Methylovorus mays.

Expression of exogenous DNA methyltransferases: application in molecular and cell biology.

DNA methyltransferases might be used as powerful tools for studies in molecular and cell biology due to their ability to recognize and modify nitrogen bases in specific sequences of the genome. Methylation of the eukaryotic genome using exogenous DNA methyltransferases appears to be a promising approach for studies on chromatin structure. Currently, the development of new methods for targeted methylation of specific genetic loci using DNA methyltransferases fused with DNA-binding proteins is especially interesting. In the present review, expression of exogenous DNA methyltransferase for purposes of in vivo analysis of the functional chromatin structure along with investigation of the functional role of DNA methylation in cell processes are discussed, as well as future prospects for application of DNA methyltransferases in epigenetic therapy and in plant selection.

[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.

Effect of hypermethylation of CCWGG sequences in DNA of Mesembryanthemum crystallinum plants on their adaptation to salt stress.

Under salt stress conditions, the level of CpNpG-methylation (N is any nucleoside) of the nuclear genome of the facultative halophyte Mesembryanthemum crystallinum in the CCWGG sequences (W = A or T) increases two-fold and is coupled with hypermethylation of satellite DNA on switching-over of C3-photosynthesis to the crassulacean acid metabolism (CAM) pathway of carbon dioxide assimilation. The methylation pattern of the CCWGG sequences is not changed in both the 5'-promoter region of the gene of phosphoenolpyruvate carboxylase, the key enzyme of C4-photosynthesis and CAM, and in the nuclear ribosomal DNA. Thus, a specific CpNpG-hypermethylation of satellite DNA has been found under conditions of expression of a new metabolic program. The functional role of the CpNpG-hypermethylation of satellite DNA is probably associated with formation of a specialized chromatin structure simultaneously regulating expression of a large number of genes in the cells of M. crystallinum plants on their adaptation to salt stress and switching-over to CAM metabolism.

DNA methyltransferases and structural-functional specificity of eukaryotic DNA modification.

Properties of the main families of mammalian, plant, and fungal DNA methyltransferases are considered. Structural-functional specificity of eukaryotic genome sequences methylated by DNA methyltransferases is characterized. The total methylation of cytosine in DNA sequences is described, as well as its relation with RNA interference. Mechanisms of regulation of expression and modulation of DNA methyltransferase activity in the eukaryotic cell are discussed.

Hypermethylation of 5'-region of the human calcitonin gene in leukemias: structural features and diagnostic significance.

Methylation of the 5'-region of the calcitonin gene was investigated in bone marrow and peripheral blood cells of 27 healthy volunteers and 25 leukemic patients. In all patients suffering from various forms of myeloid and lymphoid leukemia, hypermethylation of CpG sequences was observed in this region of the calcitonin gene. Cytosine hypermethylation in the CpG sequence did not involve cytosines of adjacent CpNpG sequences (where N is any nucleoside). The 5'-region of the calcitonin gene lacked CpNpG methylation both in healthy controls and in leukemic patients; this apparently represents specific "non-alternative" type of CpG methylation in the extended DNA sequence. Methylation of the calcitonin gene was monitored in 18 leukemic patients during malignant progression and medical treatment. Hypermethylation of the calcitonin gene was not observed on long-term clinical hematological remission. In ten patients characterized by unstable (or incomplete) remission hypermethylation of the calcitonin gene persisted through the whole period of observation. In relapses, hypermethylation of the calcitonin gene appeared again and in six patients, this "molecular relapse" being registered 1-8 months before onset of clinical and laboratory signs of disease progression. The leukemia-specific hypermethylation of CpG sequences of the 5'-region of the calcitonin gene is a promising prognostic and diagnostic marker of leukemias and might be useful for monitoring of this disease.

[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.

[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.

Effect of the M-EcoRII methyltransferase-encoding gene on the phenotype of Nicotiana tabacum transgenic cells.

The EcoRII DNA methyltransferase (M-EcoRII; MTase) modifies a cytosine in the DNA sequence CCWGG which contains a CNG methylation motif characteristic of plant DNA. The gene (ecoRIIM) encoding this MTase has been cloned into the T-DNA of the wild-type Agrobacterium Ti-plasmid pTiC58 downstream from the plant expression nopaline synthase-encoding gene promoter. Nicotiana tabacum cells have been transformed with Agrobacterium tumefaciens harbouring this recombinant Ti-plasmid. The primary transformed tabacco tissue line has given rise to novel stable lines which are morphologically distinctive. Southern hybridization analysis of all transformed tissue lines has shown the presence, in each of them, of ecoRIIM. The tissue studied differed in morphology in callus culture, dependence on phytohormones and the ability to synthesize nopaline.

[The rate and time indices of mitral and tricuspid valve movement during the cardiac cycle in congenital heart defects]. / Skorostnye i vremennýe pokazateli dvizheniia mitral'nogo i trikuspidal'nogo klapanov vo vremia serdechnogo tsikla pri nekotorykh vrozhdennykh porokakh serdtsa.

Ultrasonic Doppler echotachocardiogram of the mitral and tricuspid valves together with polycardiogram were recorded in 44 patients with interatrial septal defect and in 18 patients with interventricular septal defect. The patients with interatrial septal defect manifested an increase of the velocity of the movement of the mitral valve during its opening, deceleration of the movement velocity in the atrial systole, a tendency towards movement deceleration during the closure, and a rise of the time of the mitral valve during its closure and opening. In interventricular septal defect, there was a tendency towards deceleration of the movement velocity of the mitral valve in the atrial systole. The velocity and time parameters of the movement of the tricuspid valve remained unchanged in patients with congenital defects under study.