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Structural flexibility of regulatory DNA element
Řezáčová, Barbora ; Štěpánek, Josef (advisor) ; Rosenberg, Ivan (referee)
Transcription of the genetic code is controlled by numerous proteins. Some of them, so-called transcription factors, bind to certain DNA regions possessing anomalous properties due to their specific and highly conservative base sequence. One of these DNA segments is SRE (Serum Response Element). The diploma work is linked to results of the previous study indicating that the structural flexibility of this segment, related to the CArG box sequence, plays a key role in its physiological activity. Our work is devoted to study of this DNA segment and its model sequential variants. The approach of accurate measurements of temperature dependent UV absorption spectra and their consecutive analysis by means of factor analysis techniques and difference spectra construction, the main part of which was also developed within the framework of this work, was employed. The results indicate that separate chains of SRE with various length form surprisingly stable hairpins, which in some cases prevail even in mixtures of both chains of the SRE segment over duplexes. By using a fitting to thermodynamic equilibrium equations, thermodynamic characteristics of temperature induced association and dissociation transitions of complexes, both intermolecular (duplexes) and intramolecular (hairpins), were obtained.
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Thermodynamic Stability of Hybrid Nucleic Acid Complexes: Microcalorimetric Study
Ottová, Pavla ; Štěpánek, Josef (advisor) ; Vaisocherová, Hana (referee)
This work deals with implementation of differential scanning kalorimetry (DSC) into research of potential oligonucleotide therapeutics. Capabilities and limits of the DSC device have been determined and optimal conditions for measurement of nucleic acids complexes specified. Four types of modified oligonucleotides with isopolar, non-isosteric internucleotide linkage, consisting in insertion of a methylene group into the phosphodiester linkage between the phosphorus and one of the two ester oxygens (the methylene group inserted or at 3' or at 5' side of the linkage, the modified linkages alternate with the natural ones or all linkages are modified) were investigated. We determined thermodynamics characteristics of hybrid duplexes composed of modified deoxythymidine pentadecamers and complementary RNA polymer chain, polyadenylic acid. The oligonucleotide with the methylene group at the 3' side, the all linkages of which are modified, does not form complexes with polyadenylic acid. Other oligonucleotides form complexes and their stability increase in succession (3' end, alternating) ~ (5' end, all) < natural (dT)15 < (5' end, alternating). High cooperativity of the oligonucleotide to the polymer binding has been found out for all complexes. RNA hairpin of HIV-1 TAR element and its R06 aptamer have been also...
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Comprehensive characterization of cellular suspensions by means of elastic light scattering
Macháň, Radek ; Štěpánek, Josef (advisor) ; Plášek, Jaromír (referee)
Studies of yeast Saccharomyces cerevisiae - a model eukaryot organism - often require determining the number of cells in a suspension. One of the methods often used for studying cell suspensions is the measuring of elastic scattering of light passing through the suspensions. Usual output of those measurements is the angular dependency of the intensity of scattered light, which bears information about the size of scatterers. This thesis studies time-dependencies of the intensity of scattered light and relationship of their statistical properties to the number of scatterers in a unit volume. For this purpose a simple model was proposed, which describes the connection between this quantity for a monodisperse suspension and the dependency of standard deviation of time-behaviour of the scattered intensity on the mean value of the same time-behaviour. An apparatus for computer controlled measurements of angular and timedependencies of scattered light intensity was completed for carrying out the experiments. Elastic scattering was measured on suspensions of yeast Saccharomyces cerevisiae and a model system (suspensions of 8m polymer particles). Experimental data were interpreted within the proposed model. Although yeast suspensions cannot be regarded as monodisperse, experimental data were in a good agreement with...
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