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.
Ectopic auxin overproduction in transgenic potato leads to enhanced productivity accompanied with concerted and occasional changes in hormonal status, and causing altered response of transformants to exogenous auxin or cytokinin. Previously, we generated potato transformants expressing Agrobacterium-derived auxin synthesis gene tms1 driven by tuber-specific patatin gene promoter (B33-promoter). Here, we studied the endogenous hormonal status and the response to exogenous phytohormones in tms1 transformants cultured in vitro. Adding indole-3-acetic acid (IAA) or kinetin to culture medium affected differently tuberization of tms1-transformed and control plants, depending also on sucrose content in the medium. Exogenous phytohormones ceased to stimulate the tuber initiation in transformants at high (5-8%) sucrose concentration, while in control plants the stimulation was observed in all experimental settings. Furthermore, exogenous auxin partly inhibited the tuber initiation, and exogenous cytokinin reduced the average tuber weight in most transformants at high sucrose content. The elevated auxin level in tubers of the transformants was accompanied with a decrease in content of cytokinin bases and their ribosides in tubers and most shoots. No concerted changes in contents of abscisic, jasmonic, salicylic acids and gibberellins in tubers were detected. The data on hormonal status indicated that the enhanced productivity of tms1 transformants was due to auxin and not mediated by other phytohormones. In addition, exogenous cytokinin was shown to upregulate the expression of genes encoding orthologs of auxin receptors. Overall, the results showed that tms1 expression and local increase in IAA level in transformants affect both the balance of endogenous cytokinins and the dynamics of tuberization in response to exogenous hormones (auxin, cytokinin), the latter reaction depending also on the carbohydrate supply. We introduce a basic model for the hormonal network controlling tuberization.
Phytohormones, auxins in particular, play an important role in plant development and productivity. Earlier data showed positive impact of exogenous auxin on potato (Solanum tuberosum L.) tuberization. The aim of this study was to generate potato plants with increased auxin level predominantly in tubers. To this end, a pBinB33-tms1 vector was constructed harboring the Agrobacterium auxin biosynthesis gene tms1 fused to tuber-specific promoter of the class I patatin gene (B33-promoter) of potato. Among numerous independently generated B33:tms1 lines, those without visible differences from control were selected for detailed studies. In the majority of transgenic lines, tms1 gene transcription was detected, mostly in tubers rather than in shoots. Indoleacetic acid (IAA) content in tubers and the auxin tuber-to-shoot ratio were increased in tms1-expressing transformants. The organ-specific increase in auxin synthesis in B33:tms1-transformants accelerated and intensified the process of tuber formation, reduced the dose of carbohydrate supply required for in vitro tuberization, and decreased the photoperiodic dependence of tuber initiation. Overall, a positive correlation was observed between tms1 expression, IAA content in tubers, and stimulation of tuber formation. The revealed properties of B33:tms1 transformants imply an important role for auxin in potato tuberization and offer prospects to magnify potato productivity by a moderate organ-specific enhancement of auxin content.Keywords: Auxin; gene expression; Solanum tuberosum; tms1; transgenic potato; tuberization; tuber yield Citation: Kolachevskaya OO, Alekseeva VV, Sergeeva LI, Rukavtsova EB, Getman IA, Vreugdenhil D, Buryanov YI, Romanov GA (2015) Expression of auxin synthesis gene tms1 under control of tuberspecific promoter enhances potato tuberization in vitro.
Electrophoretic mobility shift analysis (EMSA) is a well-characterized and widely used technique for the analysis of protein-DNA interaction and the analysis of transcription factor combinatorics. Currently implemented EMSA generally involves the time-consuming use of radiolabeled DNA and polyacrylamide gel electrophoresis. We are studying the bionanoscience of self-assembling supramolecular protein-nucleic nanostructures. We have undertaken these studies because they promise to enhance our understanding of assemblies formed during prebiotic evolution, provide tools for analysis of biological processes like DNA recombination, and may lead to the development of nanoscale biosensors designed for site-specific molecular targeting. During the course of that work, we noted that EMSA of these complex structures could be effectively implemented with microfluidics chips designed for the separation of DNA fragments. In this report we compare the two techniques and demonstrate that the microfluidics system is also capable of resolving complex mixtures produced by decorating DNA recombination intermediates with mixtures of DNA binding proteins. Moreover, the microfluidics chip system improves EMSA by permitting analysis with smaller samples, avoiding the use of radiolabeling, and reducing the time involved to a matter of minutes.
The present study deals with the binding and cleavage by EcoRII endonuclease of concatemer DNA duplexes containing EcoRII recognition sites (formula; see text) in which dT is replaced by dU or 5-bromodeoxyuridine, or 5'-terminal dC in the dT-containing strand is methylated at position 5. The enzyme molecule is found to interact with the methyl group of the dT residue of the DNA recognition site and to be at least in proximity to the H5 atom of the 5'-terminal dC residue in dT-containing strand of this site. Modification of any of these positions exerts an equal effects on the cleavage of both DNA strands. Endonuclease EcoRII was found to bind the substrate specifically. At the same time modification of the bases in recognized sequence may result in the formation of unproductive, though stable, enzyme-substrate complexes.
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