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Recombinant preparation of DNA binding domain of transcription factor TEAD1
Kúdelová, Veronika ; Novák, Petr (advisor) ; Dračínská, Helena (referee)
TEAD proteins belong to a significant family of transcription factors that contribute to the regulation of organism growth and cell differentiation during its development by activating the expression of a wide variety of genes. This family shares two highly conserved sites, the TEA DNA binding domain, after which the proteins have been named, and the domain by which transcription factors bind other coactivators. Because TEAD proteins are not able to activate transcription themselves, they interact with a number of coactivators. These coactivators allow the transcription of the gene of interest to be regulated. Failure of TEAD protein activity regulation can lead to cancer. Therefore, TEAD family proteins nowadays play an important role in the development of new anticancer drugs. One way of inhibiting these proteins is to block the active site in their DNA binding domain, thus, to block their binding to DNA. This bachelor thesis deals with recombinant expression of said DNA binding domain of transcription factor TEAD1, which is extended by amino acids in unstructured regions. After finding suitable conditions of protein production, we proceeded to large volume production which was followed by purification and protein identity verification. Finally, the ability of the produced protein to interact...
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Study of effect of Bacillus subtilis yxkO gene on motility during stress response to osmotic upshift.
Streitová, Eliška ; Lichá, Irena (advisor) ; Krásný, Libor (referee)
Bacillus subtilis is gram-positive soil bacteria. In its natural environment it is constantly exposed to changes of chemical and physical conditons, including changes of osmolality. It responds to high osmolality by transporting of potassium ions and afterthat transporting and/or synthetising of compatible solutes. In last years the mutant strain Bacillus subtilis L-42 was isolated with non-specific insertional mutagenesis (mini Tn10) in our laboratory. This strain displays limited growth and inability to cope with hyperosmotic shock in a defined medium with potassium concentration of < 1 mmol/l. Insertion of transposon was located in yxkO gene which encodes a protein of unknown biological function. Some other data also indicate a possible role of disruption of yxkO gene in regulation of expression of hag gene, which encodes flagelin - a pivotal protein of bacterial flagellum. The goal of this thesis was to clarify if the disruption of yxkO gene influences motility and whether is affected the transcription of hag gene. With integrative vector pMUTIN4 a mutant strain with specific mutation of yxkO gene was prepared. Vector was pasted into chromosome of Bacillus subtilis strain 1A839 - genotype of this strain allows to extrude the known transcriptional regulation of hag gene. Cell's motility was...
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Transcriptional regulation of miR-17-92 microRNA cluster during macrophage differentiation.
Rybářová, Jana ; Stopka, Tomáš (advisor) ; Pospíšek, Martin (referee)
miR-17-92 cluster (Oncomir1) encodes seven microRNAs (miRNA, miR) regulating many biological processes including proliferation, differentiation or apoptosis. Overexpression of microRNAs encoded by miR-17-92 cluster is found in a number of tumors including acute and chronic myeloid leukemias (Dixon-McIver et al., 2008; Li et al., 2008; Venturini et al., 2007). Myeloid progenitors express miR-17-92 cluster at a high level, while macrophage differentiation associates with its downregulation. Our laboratory found, that miR-17-92 cluster is repressed by transcription factor Early growth response 2 (Egr2) upon differentiation of primary myeloid PUER progenitors, induced with transcription factor PU.1. Aim of this thesis is to further test the abovementioned data by preparing a reporter vectors set, carrying various fragments of miR-17-92 putative promoter, which enables us to study regulation of transcription of miR-17-92 cluster. This task complicated by presence of increased GC content of the miR-17-92 promoter was successfully accomplished resulting in amplification of eight fragments containing the various parts of miR-17-92 promoter including region -3.3 to 0 kb relative to the start of miR-17-5p sequence, that were inserted into pGL3 reporter vector. Transfection of pGL3 reporter vector carrying...
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Molecular mechanisms in diabetic embryopathy
Čerychová, Radka ; Pavlínková, Gabriela (advisor) ; Kolář, František (referee)
Diabetic embryopathy is one of many serious complications associated with diabetes. It is known that maternal diabetes increases the frequency of congenital defects up to ten times. The most common defects are cardiovascular and neural tube defects. Molecular mechanisms of diabetic embryopathy are still not known. This work contributes to elucidation of molecular processes leading to development of cardiovascular defects in diabetic embryopathy. This study is based on observation that maternal diabetes affects transcriptional regulation of hypoxia-inducible factor 1 (HIF-1) in developing embryo. To study the influence of maternal diabetes on HIF-1 signaling pathway, we used mouse model heterozygous for "knock-out" of Hif1α gene. Our analyses showed the negative combinational effects of maternal diabetes and Hif1α+/- genotype on embryonic development and increased risk of diabetic embryopathy. Histological analysis demonstrated the increased incidence of cardiovascular defects, particularly defects of interventricular septum and hypoplastic compact left ventricular wall in embryonic day (E) 14.5 Hif1α+/- embryos compared to wt littermates from the diabetic pregnancy. Using qPCR, we analyzed gene expression changes in the embryonic hearts at E9.5 and E10.5. We selected genes important for the...
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Study of expression of the nuclear receptor nhr-97 in Caenorhabditis elegans
Boušová, Kristýna ; Stiborová, Marie (advisor) ; Vaněk, Ondřej (referee)
Nuclear hormone receptors (NHR) are important transcription factors that regulate development and metabolism in the large group of animals. Caenorhabditis elegans contains 284 nuclear receptors, which is unusually large amount compared to receptors of Drosophila melanogaster (18) and humans (48). 15 receptors of the C. elegans have homologous receptor structure with receptors of D. melanogaster and mammals. The remaining 269 NHR are specific to nematodes and belong to the group of supplementary nuclear receptors (SupNRs), the evolutionary precursor of the HNF4 - an important transcription factor in humans. In this work we describe the nuclear hormone receptor nhr-97 C. elegans, whose expression and function have not yet been studied. The gene is encoded in the genome of C. elegans and is among SupNRs. Nhr-97 consists of two isoforms A and B, whose expression in C. elegans tissues is different. Localization of gene expression in vivo was determined using lines expressing nhr-97:: GFP. For the A isoform expression of nhr-97::GFP was localized in neurons in the pharynx and the tail, in the intestine and hypodermis, in isoform B in the pharynx, in neurons around the corpus of pharynx, the head mesodermal cell and in anal sphincter. Nhr-97 expression during development of C. elegans was determined by...
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Preparation and crystallization of a metabolic repressor LutR from Bacillus subtilis
Soldánová, Anna ; Maloy Řezáčová, Pavlína (advisor) ; Kutá-Smatanová, Ivana (referee)
Metabolic transcriptional repressors are proteins controlling transcription of specific genes involved in bacterial metabolism. These proteins typically consist of two domains: N-terminal DNA-binding domain (DBD) and C-terminal effector-binding domain. When an effector (usually a metabolite molecule) binds to the protein, the conformation of the protein is changed. This causes a change in affinity to its DNA operator and that subsequently modulates the transcription of genes of the specific metabolic pathway. LutR belongs to the GntR family of bacterial transcriptional regulators. In undomesticated strain RO-NN-1 of Bacillus subtilis, LutR regulates transcription of genes required for L-lactate utilization. Interestingly, LutR from laboratory strains PY79 and 168 has a different function. Due to a mutation, it lacks the first 21 amino acids and this alters its DNA recognition specificity. This LutR variant acts as a global regulator and regulates many genes associated with transition from exponential growth to stationary phase of bacterial population. Knowledge of 3D structure of LutR with DNA could elucidate the impact of this short deletion in LutR DBD on the mechanism of DNA recognition. In this work, the DBD of LutR from undomesticated strains of B. subtilis was prepared by heterologous...
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Regulatory mechanisms of ornithin transcarbamylase and beta-glucocerebrosidase gene expression and their relevance to diagnostics
Lukšan, Ondřej ; Jirsa, Milan (advisor) ; Kožich, Viktor (referee) ; Kříž, Vítězslav (referee)
5 Abstract Definitive diagnosis of inherited metabolic disorders commonly depends on the measurement of enzyme activity (which is often complicated) and/or molecular genetic testing. Yet even the standard mutation analysis can bring false negative results in the case of gross chromosomal rearrangements or incorrect regulation of gene expression due to the mutations in regulatory regions. In the present study I focused on characterization of complex mutations affecting the gene encoding ornithin transcarbamylase (OTC) followed by studies of regulatory regions of OTC and GBA (the gene encoding β-glucocerebrosidase). In the first study we identified 14 novel mutations including three large deletions in a cohort of 37 patients with OTC deficiency (OTCD). Subsequently we evaluated clinical significance of all these mutations. We also found a heterozygote carrying a hypomorphic mutation and manifesting OTCD most likely due to unfavorable X-inactivation which was observed independently in three different peripheral tissues. In order to evaluate the clinical significance of a promoter variation c.-366A>G found in a family with mild OTCD we identified three alternative transcription start sites (TSSs) of human OTC and delimited the promoter. We also found a distal enhancer and performed functional analysis of both...
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Recombinant preparation of DNA binding domain of transcription factor TEAD4
Zákopčaník, Marek ; Novák, Petr (advisor) ; Šulc, Miroslav (referee)
6 Abstract Transcription factors play a key role in the management of cell growth and differ- entiation and their deregulation is associated with many cancers. TEAD proteins utilise highly conserved DNA binding domain to recognise specific DNA sequences. This domain could facilitate new drug design and development. The goal of this master thesis includes recombinant preparation of DNA binding domain of transcriptional factor TEAD4 extended by a part of an unstruc- tured variable sequence, which connects this domain with transactivation domain. Purification steps include affinity chromatography followed by size exclusion chro- matography. The characterization of produced protein was performed by mass spectrometry and finally, native gel electrophoresis was used to prove the ability of the produced protein to bind DNA. During purification steps, a fragmentation from C-terminus was observed. Based on analysis of the mass spectra, three most represented forms of produced protein were described all of which were fragmented. The most abundant form (55%) consisted of amino acids 30-131 from TEAD4 protein. Second most abun- dant form (18%) consisted of amino acids 30-144 and the third form consisted of amino acids 30-81. Native gel electrophoresis verified the ability to bind DNA, the efficiency was however lower...
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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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