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Function of stress sigma factors of RNA polymerase SigD, SigE, SigH and SigM in transcription regulation network of Corynebacterium glutamicum
Dostálová, Hana ; Pátek, Miroslav (advisor) ; Bobek, Jan (referee) ; Halgašová, Nora (referee)
Grampositive bacterium Corynebacterium glutamicum is an important industrial producer of amido acids and other metabolites. Its genome encodes 7 sigma (σ) subunits of RNA polymerase: primary factor σA , primary-like σB and five alternative sigma factors, σC , σD , σE , σH and σM (sigma factors with extracytoplasmic function). This study is focused on revealing so far unknown regulons of stress sigma factors or closer description of regulons whose genes are controled by σD , σE , σH and σM . These factors were partially described for their activity during surface (σD and σE ), heat (σE , σH and σM ) and oxidative (σH and σM ) stress response. We assumed that the genes of each regulon are transcribed from promoters of a single class. For the purpose of detailed promoter analysis, it was necessary to develop methods which can quickly and reliably assign sigma factor to particular promoters and, thus, respective genes. For this purpose, a combination of in vivo (two-plasmid system) and in vitro (in vitro transcription) techniques was developed that allow to specify this assignment. We identified 9 σH /σE - promiscuous promoters (PamtR, Pcg0378, Pcg1121, Pcg3309, Pcg3344, PclgR, PdnaJ, PdnaK and PsigB), 7 σD /σH - promiscuous promoters (Pcg0607, Pcg2047, Pcmt2, PfadD2, Plpd, PlppS and PrsdA) a 9 σH /σM...
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Clinical classification of sequence variants in non-coding regulatory regions in breast cancer susceptibility genes.
Bubáková, Eliška ; Ševčík, Jan (advisor) ; Vodička, Pavel (referee)
Inactivation of tumor supressor gene BRCA1 causes a life-long risk of breast carcinoma development. Genetic screenings of indicated individuals from high-risk families help to identify large number of sequence variants in known predisposing genes. Majority of discovered variants doesn't have clinical significance yet which causes a big problem for diagnostics. Some of these variants are found within regulatory non-coding regions of gene. A part of the clinical classification of variants is their functional characterization. The goal of this thesis was to create a model system for functional characterization of variants in non-coding regions and to verify its function. Model system was based on targeted gene manipulation by co-transfecting CRISPR-Cas9 construct and donor construct that contained a portion of BRCA1 gene sequence with analyzed modifications, into U2 OS cells. The cells have stably integrated DR-GFP system which allows the activity of homologous recombination (HR) to be determined. Monoallelic modifications were induced into U2 OS cells. These modifications were in a Kozak sequence region of BRCA1 gene. Expression level of BRCA1 mRNA was determined by qRT-PCR, which showed the same levels of mRNA in all cells with analyzed alterations. Next, expression level of BRCA1 protein was...
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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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Factors interacting with bacterial RNA polymerase and their effect on the regulation of transcription initiation
Ramaniuk, Volha
(ENGLISH) The bacterial cell needs to regulate its gene expression in response to changing environmental conditions. RNA polymerase (RNAP) is the pivotal enzyme of this process and its activity is controlled by a number of auxiliary factors. Here I focus on RNAP-associating factors involved in regulation of transcription in G+ bacteria: factors, initiating nucleoside triphosphates (iNTPs), HelD, δ and small RNA Ms1. The main emphasis is on σ factors from Bacillus subtilis. σ factors allow RNAP to specifically recognize promoter DNA. In my first project I set up in vitro transcription systems with purified alternative σ factors, σB , σD , σH , σI from B. subtilis. Using these systems, I studied the effect of initiating NTP concentration ([iNTP]) on transcription initiation. I showed that promoters of alternative factors are often regulated by [iNTP]. In the next project I comprehensively characterized one of the least explored alternative factors from B. subtilis, I . I identified ~130 genes affected by I , though only 16 of them were directly affected. Moreover, I discovered that I is involved in iron metabolism. Finally, I showed that I binding requires not only the conserved -35 and -10 hexamers, but also extended -35 and -10 elements located in the spacer region. In collaboration with...
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Factors interacting with bacterial RNA polymerase and their effect on the regulation of transcription initiation
Ramaniuk, Volha ; Krásný, Libor (advisor) ; Lichá, Irena (referee) ; Valášek, Leoš (referee)
(ENGLISH) The bacterial cell needs to regulate its gene expression in response to changing environmental conditions. RNA polymerase (RNAP) is the pivotal enzyme of this process and its activity is controlled by a number of auxiliary factors. Here I focus on RNAP-associating factors involved in regulation of transcription in G+ bacteria: factors, initiating nucleoside triphosphates (iNTPs), HelD, δ and small RNA Ms1. The main emphasis is on σ factors from Bacillus subtilis. σ factors allow RNAP to specifically recognize promoter DNA. In my first project I set up in vitro transcription systems with purified alternative σ factors, σB , σD , σH , σI from B. subtilis. Using these systems, I studied the effect of initiating NTP concentration ([iNTP]) on transcription initiation. I showed that promoters of alternative factors are often regulated by [iNTP]. In the next project I comprehensively characterized one of the least explored alternative factors from B. subtilis, I . I identified ~130 genes affected by I , though only 16 of them were directly affected. Moreover, I discovered that I is involved in iron metabolism. Finally, I showed that I binding requires not only the conserved -35 and -10 hexamers, but also extended -35 and -10 elements located in the spacer region. In collaboration with...
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The role of alternative sigma factors of RNA polymerase in regulation of gene expression in Corynebacterium glutamicum
Šilar, Radoslav
Abstract Regulation of transcription by extracytoplasmic-function (ECF) sigma factors of RNA polymerase is an efficient way of cell adaptation to diverse environmental stresses. Amino acid- producing gram-positive bacterium Corynebacterium glutamicum codes for seven sigma factors: the primary sigma factor SigA, the primary-like sigma factor SigB and five ECF stress- responsive sigma factors (SigC, SigD, SigE, SigH and SigM). The sigH gene encoding SigH sigma factor is located in a gene cluster together with the rshA gene, encoding the anti-sigma factor of SigH. Anti-sigma factors bind to their cognate sigma factors and inhibit their transcriptional activity. Under the stress conditions the binding is released allowing the sigma factors to bind to the RNAP core enzyme. In this thesis, regulation of expression of genes encoding the most important ECF sigma factor SigH and its anti-sigma factor RshA as well as genes belonging to the SigH-regulon were mainly studied. The transcriptional analysis of the sigH-rshA operon revealed four housekeeping promoters of the sigH gene and one SigH-dependent promoter of the rshA gene. For testing the role of the complex SigH-RshA in gene expression, the C. glutamicum ΔrshA strain was used for genome-wide transcription profiling with DNA Microarrays technique under...
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Effect of promoter sequence on utilization of NAD+ as a substrate for transcription initiation by RNA polymerase
Pinkas, Daniel ; Krásný, Libor (advisor) ; Fišer, Radovan (referee)
For a long time, 5' cap has been thought to be privilege only for eukaryotic organisms in form of 7-methylguanosine cap at the end of mRNA. This was changed only a few years ago. By using methods liquid chromatography and mass spectrometry a new molecule associated with RNA of Escherichia coli has been found. This molecule turned out to be nicotinamide adenine dinucleotide (NAD+ ) attached to 5' end of some small regulatory RNAs (sRNA). Later it has been shown, that RNA polymerase can attach NAD+ at 5' of RNA ab initio, meaning that RNA polymerase can utilize NAD+ as a substrate for transcription initiation. To some extent substrate for transcription initiation is chosen based on promoter sequence. Crucial requirement is presence of adenine at +1 position of DNA coding strand. This thesis focuses on promoter sequence requirements for transcription initiation with NAD+ . As a template for transcription four promoters with different modifications and their chimeras are used: RNA1, Pveg, lac UV5 and rrnB P1. Also, I tried to compare RNA polymerase from E. coli and B. subtilis in terms of transcription initiation substrate usage. Lastly, I describe here isolation of NudC, enzyme that cleaves NAD+ to nicotinamide mononucleotide (NMN) and adenosine monophosphate (AMP). NudC will be used for upcoming...
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Inducible promoters and their use in yeast cell manipulation
Přibáňová, Gabriela ; Palková, Zdena (advisor) ; Vopálenský, Václav (referee)
Promoters which can be regulated by different chemical or physical factors are often used in cell manipulations. This thesis focuses predominantly on promoter systems which use light as an inductor. There are two main approaches to controlling a promoter by light. The first one uses so-called "caged molecules", chemical inducers whose inducing activity is "masked" by a photolabile protecting group. The second approach includes optogenetic systems, which can regulate transcription in cells. These systems are encoded in the DNA of the organism, and light is the only external regulatory stimulus. Photoreceptors that need a specific cofactor (chromophore) are the main components of optogenetic systems. There are several groups of photoreceptors classified by the type of chromophore and photoactivation mechanism. This thesis gives an overview of optogenetic systems used for transcription regulation and focuses on different photoreceptors and induction mechanism used. The systems using photocaged molecules are described as well. Furthermore, the thesis deals with light- systems in yeast as a model organism as well as organism used for biotechnological purposes. Finally, some limitations of light inducible promoters are discussed, including the chromophore type, the wavelength of the light, and the...
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Regulatory mechanisms of CD47 surface expression
Jakubec, Martin ; Drbal, Karel (advisor) ; Dibus, Michal (referee)
CD47 glycoprotein can be found on the surface of all healthy cells in our body. The interaction of CD47 with inhibitory receptor SIRPα on the macrophage leads to the inhibition of phagocytosis. This makes CD47 irreplaceable for the safe recognition of own cells and removal of aged or apoptotic cells. Apart from this, CD47 plays a major role in several essential signalling pathways, such as cell adhesion and motility or calcium homeostasis. The level of CD47 expression and its presence on the cell membrane depends not only on the type of tissue, but also on the age of a cell. An increased expression of CD47 protein has also been observed in the cells undergoing tumorigenic transformation, allowing them to escape from tumour immunosurveillance. Spontaneous regulation of the CD47 gene expression is achieved via regulatory transcription factors, such as NF-κB or HIF-1. Another mechanism of CD47 regulation includes the 3'UTR of CD47 mRNA, which serves as a binding site for either regulatory proteins, such as HuR, or miRNAs. CD47 expression can thus be regulated on both transcriptional, as well as translational level. However, appropriate topological CD47 localization within the cell and on the cell surface has also an important effect of its physiological function. Our in depth understanding of key regulatory...
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