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Influence of a composition of hybridization mixtures on fluorescence intensity during the in-situ hybridization
Janíček, Tomáš ; Kejík, Lukáš (oponent) ; Vojtěch,, Hudzieczek (vedoucí práce)
Fluorescence in situ hybridization (FISH) is widely used method for detection of certain DNA sequence on the chromosomes. The goal of this thesis is a comparison of three different chemical compounds (formamide, ethylene carbonate and sodium cations) used in mixtures for in situ hybridization. All three mentioned compoundsi nfluence renaturation of DNA and it is interesting to compare their physical properties. This thesis is split into two major parts. First part deals with question of thermodynamic parameters used for FISH experiments, such as melting temperature. Determining enthalpy of helix-coil transition give us insight about newly established bounds between bases and each mixture composition. In addition, entropy values determine the order inside mixture – DNA system. Second part compares optimized probe melting temperatures used for in situ hybridization and obtained mean fluorescence signal intensity. As a probe was used sub-telomeric X43.1 repeat, which is located on the Y-chromosome of plant model organism Silene latifolia. The mixture containing formamide has the best performance during longer hybridization procedure, whereas ethylene carbonate yielded higher signal intensity and therefore it is more suitable for fast FISH protocols.
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Influence of a composition of hybridization mixtures on fluorescence intensity during the in-situ hybridization
Janíček, Tomáš ; Kejík, Lukáš (oponent) ; Vojtěch,, Hudzieczek (vedoucí práce)
Fluorescence in situ hybridization (FISH) is widely used method for detection of certain DNA sequence on the chromosomes. The goal of this thesis is a comparison of three different chemical compounds (formamide, ethylene carbonate and sodium cations) used in mixtures for in situ hybridization. All three mentioned compoundsi nfluence renaturation of DNA and it is interesting to compare their physical properties. This thesis is split into two major parts. First part deals with question of thermodynamic parameters used for FISH experiments, such as melting temperature. Determining enthalpy of helix-coil transition give us insight about newly established bounds between bases and each mixture composition. In addition, entropy values determine the order inside mixture – DNA system. Second part compares optimized probe melting temperatures used for in situ hybridization and obtained mean fluorescence signal intensity. As a probe was used sub-telomeric X43.1 repeat, which is located on the Y-chromosome of plant model organism Silene latifolia. The mixture containing formamide has the best performance during longer hybridization procedure, whereas ethylene carbonate yielded higher signal intensity and therefore it is more suitable for fast FISH protocols.
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Structure and evolution of plant sex chromosomes: The use of BAC library
Kejnovský, Eduard ; Lengerová, Martina ; Hobza, Roman ; Široký, Jiří ; Vyskot, Boris
Sex chromosomes of plant species Silene latifolia are the ideal model for evolutionary biology studies because they represent early stages of X and Y chromosomes history. We used fluorescence in situ hybridization (FISH) with bacterial artificial chromosome (BAC) clones in studies of chromosome structure to demonstrate that a majority of genes are located in subtelomeric regions of all chromosomes. Our results also indicate the absence of accumulation of main classes of repetitive sequences - retroelements and centromeric tandem repeats - in non-recombining parts of the sex chromosomes.
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Genetic and epigenetic acpects of sex determination
Janoušek, Bohuslav ; Hodurková, Jaromíra ; Žlůvová, Jitka ; Lengerová, Martina ; Hobza, Roman ; Nix, Tomáš ; Vyskot, Boris
The article concentrates on the latest knowledge concerning the role of epigenetic mechanisms in sex determination in model dioecious species Silene latifolia. Results suggest that the locus suppressing development of female organs in male plants is hypersensitive to the action of agents influencing level of DNA methylation (5-azacytidine) and histone acetylation (trichostatin A). According to the results obtained via inter-specific hybridisation and the ability of the smut Ustilago violacea to activate development of male organs in female plants, it can be deduced that there is a locus suppressing male organ development in standard female plants. Function of this locus is not influenced by the drugs mentioned above.
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