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RNA directed DNA methylation in Arabidopsis thaliana
Motylová, Šárka ; Fischer, Lukáš (advisor) ; Moravec, Tomáš (referee)
The differential transcriptional activity of the genome is provided by epigenetic modifications, which include DNA methylation, alteration of histone N-terminal amino acids and changes in histone variants. RNA interference is a regulatory process, in which transcriptional or post-transcriptional silencing of exogenous or endogenous sequences is mediated by the action of small RNAs derived from these sequences. The 24-nucleotide siRNAs, forming a fraction of small RNAs, direct de novo DNA methylation and participate in the maintenance of DNA methylation (RNA-directed DNA methylation; RdDM), which facilitates transcriptional silencing of heterochromatin and transposable elements representing a large part of plant genomes. The presence of two RNA polymerases involved in this pathway is characteristic for flowering plants, which were discovered for the first time in the genome of Arabidopsis thaliana, which has also become the main plant model for the study of RdDM. Polymerase IV transcribes siRNA precursors; siRNAs are subsequently associated with AGO4 proteins and guide methylation enzymes to the target sequences via complementarity with polymerase V transcripts.
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Reporter expression system for study of silencing of provirus integrated inside transcriptionally active gene
Slavková, Martina ; Hejnar, Jiří (advisor) ; Španielová, Hana (referee)
Retroviral vectors are used as mighty tools for an introduction of recombinant genes into the recipient genome in gene therapy trials. In the vector design, great emphasis is put on safety and efficiency. In spite of a great progress in retroviral vector design with the purpose to stabilize its expression, e.g. introduction of protective elements into the viral regulatory sequences, the current approaches are still not sufficiently effective and the majority of proviruses is transcriptionally silenced. The understanding of the silencing mechanism is of special importance to the optimization of the vector design and handling. In this master thesis, I have designed and constructed an expression system for study of the mechanism involved in the silencing of retroviruses integrated inside gene bodies. This artificial system will be utilized for testing of hypothesis that retroviruses integrated into gene bodies are silenced by DNMT-dependent mechanism and this process is triggered by transcriptional read-through of the provirus from nearby host promoter. I have obtained preliminary results suggesting the validity of the suggested hypothesis; however the verification of general validity of this hypothesis for various retroviruses and elements will be a matter of further studies in our laboratory. Powered by...
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Studies of S. cerevisiae BR-S strain with deletion of SIR2 gene
Novotná, Pavla ; Kuthan, Martin (advisor) ; Pichová, Iva (referee)
Yeasts are unicellular eukaryotic microorganisms, capable of forming of organised multicellular communities, the colonies. Many yeast strains possess a characteristic colony morphology under defined living conditions. Another feature typical for many feral and pathogenic yeast strains is the ability to switch their morphotype. This phenomenon, called the phenotypic switching, contributes to a rapid adaptation to the changing harmful environment and is often connected with changes of the stress resistance or with the changes of virulence of pathogenic yeasts. Phenotypic switching can be observed even in non-pathogenic yeast Saccharomyces cerevisiae. The strain BR-F, isolated from nature, switches under laboratory conditions from fluffy to smooth morphology of the strain BR-S. This phenotypic switch is accompanied by broad changes in the phenotype. Transcriptome analyses of the strains BR-F and BR-S have shown, among others, changes in expression of the subtelomeric genes that are under control of the histone acetylases and deacetylases. My work was aimed to the histone deacetylase Sir2p, which could influence the phenotypic switching in Saccharomyces cerevisiae. The sir2 deletion mutant of the strain BR-S, prepared in our laboratory, was used for my studies. The results show, that the strain BR-S...
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RNA directed DNA methylation in Arabidopsis thaliana
Motylová, Šárka ; Fischer, Lukáš (advisor) ; Moravec, Tomáš (referee)
The differential transcriptional activity of the genome is provided by epigenetic modifications, which include DNA methylation, alteration of histone N-terminal amino acids and changes in histone variants. RNA interference is a regulatory process, in which transcriptional or post-transcriptional silencing of exogenous or endogenous sequences is mediated by the action of small RNAs derived from these sequences. The 24-nucleotide siRNAs, forming a fraction of small RNAs, direct de novo DNA methylation and participate in the maintenance of DNA methylation (RNA-directed DNA methylation; RdDM), which facilitates transcriptional silencing of heterochromatin and transposable elements representing a large part of plant genomes. The presence of two RNA polymerases involved in this pathway is characteristic for flowering plants, which were discovered for the first time in the genome of Arabidopsis thaliana, which has also become the main plant model for the study of RdDM. Polymerase IV transcribes siRNA precursors; siRNAs are subsequently associated with AGO4 proteins and guide methylation enzymes to the target sequences via complementarity with polymerase V transcripts.
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Study of the function of selected genes in the colonies of wild yeast strains
Tarabová, Eva ; Kuthan, Martin (advisor) ; Heidingsfeld, Olga (referee)
Saccharomyces cerevisiae strains isolated from the wild are able to exhibit multicellular social behaviour and to form complex structured colonies resembling in many properties highly resistant biofilms of pathogenic yeasts. The capability of phenotypic variability, i.e. high frequency transition between two or more different phenotypes, is another feature typical for the wild yeast strains. Such phenotypic changes are in case of pathogenic yeast often connected with changes in virulence and resistance to stress and antifungal treatment. Long-term cultivation of the wild yeast strains under laboratory conditions leads to their domestication, i.e. transition to smooth colonies and loss of some features typical for structured colonies. This process is, similarly to phenotypic switching, accompanied by significant changes in gene expression and global change of colony lifestyle. Mechanisms underlying yeast phenotypic transitions are ascribed to epigenetic regulation of gene expression via transcriptional silencing conferred by histone deacetylases. This work deals with the study of such mechanisms using knock-outs of selected genes with putative function in formation of structured colonies in wild and domesticated strains. The achieved results show, that NAD+-dependent histone deacetylase Sir2p influences...
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