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Ryazanova A.Y.,Moscow State University | Kubareva E.A.,Moscow State University | Grman I.,Comenius University | Lavrova N.V.,Gamaleya Institute of Epidemiology and Microbiology | And 3 more authors.

The interaction of (cytosine-5)-DNA methyltransferase SsoII (M.SsoII) with double-stranded DNA was studied by means of thickness shear mode acoustic method (TSM) and gel electrophoresis. M.SsoII recognizes in double-stranded DNA the methylation site 5′-CCNGG-3′ (N = A, C, G, T) and methylates the inner cytosine residue. M.SsoII also acts as a transcription factor via binding to the regulatory site 5′-AGGACAAATTGTCCT-3′ in the promoter region of SsoII restriction-modification system. We designed three 60-mer biotinylated DNA duplexes: with the methylation site (60met), with the regulatory site (60reg), and without a specific binding site (60oct). A strong binding of M.SsoII with each one of the studied DNA immobilized on the TSM transducer has been shown. The equilibrium dissociation constants, KD, of the M.SsoII-DNA complexes decreased in the order 60oct > 60reg > 60met, suggesting a higher stability of M.SsoII-60met complex in comparison with the others. The association rate constant, ka, was also higher for 60met, while similar values were obtained for 60reg and 60oct. The difference in the kinetic parameters for 60met and 60reg suggested a possible way of coordination between the two M.SsoII functions in a cell. © 2011 The Royal Society of Chemistry. Source

Kalle E.,Swedish University of Agricultural Sciences | Gulevich A.,Institute of Agricultural Biotechnology | Rensing C.,Copenhagen University
Journal of Microbiological Methods

In a mixed template, the presence of homologous target DNA sequences creates environments that almost inevitably give rise to artifacts and biases during PCR. Heteroduplexes, chimeras, and skewed template-to-product ratios are the exclusive attributes of mixed template PCR and never occur in a single template assay. Yet, multi-template PCR has been used without appropriate attention to quality control and assay validation, in spite of the fact that such practice diminishes the reliability of results. External and internal amplification controls became obligatory elements of good laboratory practice in different PCR assays. We propose the inclusion of an analogous approach as a quality control system for multi-template PCR applications. The amplification controls must take into account the characteristics of multi-template PCR and be able to effectively monitor particular assay performance. This study demonstrated the efficiency of a model mixed template as an adequate external amplification control for a particular PCR application. The conditions of multi-template PCR do not allow implementation of a classic internal control; therefore we developed a convenient semi-internal control as an acceptable alternative. In order to evaluate the effects of inhibitors, a model multi-template mix was amplified in a mixture with DNAse-treated sample. Semi-internal control allowed establishment of intervals for robust PCR performance for different samples, thus enabling correct comparison of the samples. The complexity of the external and semi-internal amplification controls must be comparable with the assumed complexity of the samples. We also emphasize that amplification controls should be applied in multi-template PCR regardless of the post-assay method used to analyze products. © 2013 Elsevier B.V. Source

Globa A.G.,Institute of Agricultural Biotechnology | Alekseev Y.I.,Institute of Agricultural Biotechnology | Arzumanyan V.G.,Russian Academy of Medical Sciences | Zaborova V.A.,Moscow State University | Guridov A.A.,Russian Academy of Medical Sciences
Biomeditsinskaya Khimiya

A test system has been developed for determination of propionic bacterial species residing on human skin. This system developed in the real time PCR format is applicable for quantitative determination and also detection of genomes of the following Pmpionibacterium spccics: P. acnes, P. g)-anulosim and P. avidum. This system was used for analysis of wash samples from the skin of 17 pentathlon sportsmen and 16 students. All three species of propionic bacteria were found in all skin wash samples. However, contamination with P. acnes was two times higher in control group than in the group of pentathlon sportsmen. Source

Drobyazina P.E.,Institute of Agricultural Biotechnology | Khavkin E.E.,Institute of Agricultural Biotechnology

Day length controls development in many plants. In Arabidopsis thaliana, the CONSTANS (CO) gene has been firmly established as a key component in the photoperiodic pathway of floral transition; less is known about CONSTANS-LIKE1 (COL1) orthologues of this gene in Arabidopsis and several other species. The CONSTANS protein comprises two B-box-type zinc fingers, CCT domain, and a variable middle region (MR) which corresponds to exon 2 in the COL1 genes of Solanum species. Solanum COL1 proteins are over 85% identical within the genus and about 50% similar to Arabidopsis CO. Comparative COL1 analysis in several cultivated and wild Solanum species discerned two gene variants, which differed in the structures of exon 2 and introns 1 and 2. In exon 2, two variants were primarily discerned by the numbers of AAC/AAT and CAA/CAG repeats coding for polyasparagine and polyglutamine tracts in MR; therefore two variants were dubbed short and long COL1 genes (sCOL1 and lCOL1). However, intron 1 in lCOL1 was shorter than in sCOL1 due to three indels, whereas intron 2 in available COL1 sequences was represented by three different variants. The temporal profiles of sCOL1 and lCOL1 expression in tuberosum potato dramatically differed under short and long day, and the level of sCOL1 expression exceeded that of lSOL1 by an order of magnitude. Both sCOL1 and lCOL1 were found in each Solanum genome under study and in each individual plant, and the ratio of their copy numbers was not related to plant ploidy and photoperiodic response. Evidently the evolution of two COL1 genes preceded Solanum speciation, and the day-length response of diverse Solanum genotypes does not stem from the primary COL1 structure. © 2010 Elsevier B.V. Source

Pankin A.A.,Institute of Agricultural Biotechnology | Khavkin E.E.,Institute of Agricultural Biotechnology
American Journal of Botany

Premise of the study: Traditional taxonomy and nomenclature of Brassiceae (Brassicaceae) species do not reflect their phylogeny. Revision of the species and generic limits supported by extensive molecular data seems crucial. Methods and Results: Genome-specific polymorphisms extracted from non-coding and coding sequences were used to develop 14 sequencec haracterized a mplified r egion (SCAR) markers specific for the Brassica B genome. These SCARs were verified against 77 accessions of six U-triangle Brassica species and used to screen 23 accessions of seven wild Brassiceae species to test for their cross-species amplification. SCARs were found in all B-genome Brassica species and also in Sinapisarvensis. Conclusions: SCAR markers can be employed for discerning B-genome chromosomes in Brassica species and S. arvensis to reliably identify B-genome species and their natural hybrids. The combined molecular evidence supports the suggestion to revise the generic limits of Brassica and Sinapis. © 2011 Botanical Society of America. Source

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