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Overlaps of regulons of stress sigma factors of RNA polymerase in corynebacteria and rhodococci
Blumenstein, Jan ; Pátek, Miroslav (advisor) ; Hnilicová, Jarmila (referee) ; Ulrych, Aleš (referee)
Bacteria of the genera Rhodococcus and Corynebacterium are biotechnologically important microorganisms. This dissertation thesis focuses on the role of sigma (σ) factors of RNA polymerase in the regulation of gene expression in these bacteria. The C. glutamicum genome harbors 7 genes encoding σ factors, namely σA , σB , σC , σD , σE , σH and σM . With the exception of σM , these factors and their functions have been well described. The R. erythropolis genome encodes 21 putative genes for σ factors, but none of them has been analyzed in detail. The same holds for another biotechnologically useful bacterium, R. opacus. A group of genes whose expression is mediated by a single σ factor is called σ regulon. Overlaps of σ regulons are mainly caused by overlaps of recognition specificity of σ factors, i.e. when two or more σ factors recognize the same promoter. This thesis is focused on σ factors of group 4 (ECF) and their regulons: σD , σE , σH (C. glutamicum, R. erythropolis, R. opacus) and σM (C. glutamicum). We used RNA-sequencing, in vivo, in vitro and in silico methods (homology modeling and molecular dynamics simulations) to achieve the intended aims. The overlap of σD and σH regulons was demonstrated in the natural hybrid promoter Pcg0441. Using the combination of in vivo and in silico methods,...
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Hybrid sigma factors of RNA polymerase in Corynebacterium glutamicum
Blumenstein, Jan ; Štěpánek, Václav (advisor) ; Krásný, Libor (referee)
Corynebacterium glutamicum is a Gram-positive non-sporulating soil bacterium which is used in biotechnology as a producer of amino acids, nucleotides, biofuels and alcohols. The aim of this thesis was to create a hybrid σ factor of RNA polymerase which would be able to recognize a matching hybrid promoter without effect on expression of the host genes. Based on the σD and σH amino acid sequence, two types of hybrid factors, σDH and σHD , were designed by the sequence combination of sigD and sigH. As an alternative approach, based on the in silico homology modeling, mutations of wild-type σH in the region recognizing the -35 promoter element of the σH -dependent promoter were introduced. Hybrid promoters were constructed by combining the -35 and -10 promoter regions that were derived from the σD - and σH - dependent promoters. Promoter activity was determined by using gfpuv reporter gene under the control of hybrid promoter. The expression of gfpuv in strains with hybrid sigma factors σDH / σHD and hybrid promoters was rather low compared to strains that carried wild-type σ factor and the respective promoter. The aim of the thesis was achieved by using one of the mutant σH factor (σmutH_6A ) with alterations in the region recognizing the -35 element of the σH -dependent promoter. This mutant σ...
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The role of methylation of histone 3 on lysine 4 (H3K4) during mouse spermatogenesis
Blumenstein, Jan ; Trachtulec, Zdeněk (advisor) ; Král, Jiří (referee)
Epige eti ké odifika e hro ati u hrají z ač ou roli v regula i přístup osti DNA. odifika í je etyla e histo ů. V H a lysi u ěhe sper atoge eze yši do á í a to zej é a ve Běhe eioti ké profáze I do hází k hro ozo ů, a k eioti ké reko i a i. Tyto pro esy jsou kriti ký ode tvor y pohlav í h u ěk a jeji h selhá í vede k eioti ké zástavě a tí páde k poruše tvor y haploid í h u ěk. Histo ová etyltra sferáza PRDM9 hraje klíčovou roli v eioti ké reko i a e, e oť je zodpověd á za urče í progra ova ý h dvouřetěz ový h zlo ů v íste h zva ý h hotspoty. Ni é ě ge je důležitý i ve spe ia i, a to řed i tví fe o é u hy rid í sterility. Další i protei y, které se podílí a etyla i histo u H a lysi u ěhe sper atoge eze, jsou MLL KMT D , MLL KMT E a yší h varlate h ovše az ačují, že regula i této odifika e hro ati u podílí ví e ge ů. Avšak ejsou z á é ko krét í fu k e, které ají tyto z ývají í ge y ěhe vývoje sa čí h pohlav í h u ěk. Další zkou á í tě hto ge ů ohou ýt užiteč á pro o jas ě í částeč é e o úpl é eplod os Klíčová slova: eioti ká reko i a e, dvouřetěz ové zlo y, hy rid í sterilita, sper atoge eze, etyla e histo ů
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