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Role of sequence context in DNA methylation
Polák, Jan ; Fischer, Lukáš (advisor) ; Širl, Marek (referee)
Cytosine methylation of DNA is a pivotal epigenetic mark, which contributes to the regulation of the gene expresion, silencing of transposable elements, and co-defines chromatine state. There are three cytosine contexts: CG, CHG and CHH (where H stands for C, A, or T). Arabidopsis thaliana (and plants in general) has an arsenal of molecular mechanisms capable of cytosine methylation in all of its contexts. That said, there are two tasks at hand: maintaining of pre-existing methylation and if need be, creating new methylated spots. The actual process of maintaining of the methylation depends on the cytosine context. Methylation of symmetrical contexts of CG and CHG can utilize the information about the methylation pattern from the second DNA strand. The aymmetrical context of CHH, and also CHG need to look for this information elsewhere: in the methylation of the lysine 9 of H3 histone. This creates a self-reinforcing loop and a crosstalk between two epigenetic mechanisms. Maintaince of methylation of CHH is also navigated by small RNA complementary to the locus in question. This mechanism of enzyme navigating by RNA is also used in establishing a new methylated site for all of the contexts. CG methylation is most prevalent in both heterochromatine and euchromatine. It also has a special functions...
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Characterization of N-demethyllincomycin-methyltransferase.
Poľan, Marek ; Najmanová, Lucie (advisor) ; Petříčková, Kateřina (referee)
Lincomycin is a naturally occurring member of a lincosamide group of antibiotics. The cluster of lincomycin biosynthetic gene was already decribed and the function of many of genes has been clarified. This work, "Characterization of N-demethyllincomycin-methyltransferase", is focused on the study of the final step of lincomycin biosynthetic pathway - the methylation of nitrogen atom from the pyrollo ring of the propylproline unit of the N-demethyllicomycin (NDL). The aim of this work was the characterization of the protein LmbJ, catalysing this final biosynthetic step. All the experiments were provided for the enzyme LmbJ with N-terminal histidine tag, which had been prepared by the heterologous expression in E.coli cells. The pH and temperature optimum was determined as well as the Michaelis constants for both substrates of the reaction - N-demethyllincomycin and S-adenosyl methionine (SAM - a methyl group donor). With the exception of the pH optimum, all specified parameters have markedly differed from the data published for the enzyme isolated from the natural source. Based on the comparison of electron microscopy, blue native gel electrophoresis and gel filtration results, the hypothetical model of the LmbJ quarternary structure was created. Majority of methyltranserases, so far described occure in...
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