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Characteristics of expression vectors for Corynebacterium glutamicum and their use for studies of sigma factors of RNA polymerase
Dvořáková, Pavla ; Pátek, Miroslav (advisor) ; Konopásek, Ivo (referee)
The aim of the thesis was to characterize chosen expression vectors used in biotechnologically important bacterial species, Corynebacterium glutamicum, and to test their use in studies of promoter activity control by sigma factors of RNA polymerase. Different properties of these vectors (level of expression of the cloned gene, leaky expression without inducer, dependence of expression level on inducer concentration and cell population homogeneity) were found by determination of expression level of the model gfpuv gene by fluorescence intensity assay of the produced protein and by gfpuv-expressing C. glutamicum cell population analysis using flow cytometry. The vector pEC-XT99A was chosen for testing the bi-plasmid system for assignment of a sigma factor to the chosen promoter. Although the level of expression provided by pEC-XT99A was not high, the vector showed no leaky expression, expression from the vector was comparable for a wide range of IPTG concentrations and the cell population was homogenous concerning the gene expression. Using pEC-XT99A from which individual stress sig genes were expressed, the σD factor was clearly assigned to the up-to-now unknown Pcg0420 promoter. Another vector for isolation and purification of C. glutamicum proteins was used to express the C. glutamicum sigM gene and to...
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The role of alternative sigma factors of RNA polymerase in regulation of gene expression in Corynebacterium glutamicum
Šilar, Radoslav ; Nešvera, Jan (advisor) ; Branny, Pavel (referee) ; Lichá, Irena (referee)
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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Functions of sigma factors of RNA polymerase in Corynebacterium glutamicum
Dvořáková, Pavla ; Pátek, Miroslav (advisor) ; Dvořáček, Lukáš (referee)
The aim of this thesis was to characterize the function of sigma factors of the bacterium Corynebacterium glutamicum and to analyze the promoter sequences which are recognized by individual sigma factors. Sigma (σ) factors are the subunits of RNA polymerase, which allow recognizing the sequences of specific promoter regions of the gene and initiating its transcription. The C. glutamicum genome carries genes encoding the primary sigma factor σA and six alternative sigma factors, σB , σC , σD , σE , σH and σM , whose expression is changed depending on the growth conditions and in response to the stimuli from the surrounding environment. Regulation of gene expression at the level of transcription is one of the mechanisms of the adaptation of cells to changes of living conditions. At the conclusion of this work, a model of the regulatory network of sigma factors, which is a core of the complex regulatory network controlling all processes in the cell, is proposed. Key words: sigma factor (SF), RNA polymerase, Corynebacterium glutamicum, transcription, promoter
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Role of promoter in the regulation of alternative splicing
Kozáková, Eva ; Staněk, David (advisor) ; Půta, František (referee) ; Blažek, Dalibor (referee)
It was shown that 95 % of human multi-exon genes are alternatively spliced and the regulation of alternative splicing is extremely complex. Most pre-mRNA splicing events occur co- transcriptionally and there is increasing body of evidence, that chromatin modifications play an important role in the regulation of alternative splicing. Here we showed that inhibition of histone deacetylases (HDACs) modulates alternative splicing of ~700 genes via induction of histone H4 acetylation and increase of Pol II elongation rate along alternative region. We identified HDAC1 the catalytic activity of which is responsible for changes in alternative splicing. Then, we analyzed whether acetylhistone binding protein Brd2 regulates alternative splicing and showed that Brd2 occupies promoter regions of targeted genes and controls alternative splicing of ~300 genes. Later we showed that knockdown of histone acetyltransferase p300 promotes inclusion of the alternative fibronectin (FN1) EDB exon. p300 associates with CRE sites in the promoter via the CREB transcription factor. We created mini-gene reporters driven by an artificial promoter containing CRE sites. Both deletion and mutation of the CRE site affected EDB alternative splicing in the same manner as the p300 knockdown. Next we showed that p300 controls histone...
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Regulation of gene expression in anaerobic parasitic protist and practical application of knowledge
Brzoň, Ondřej ; Zubáčová, Zuzana (advisor) ; Horváthová, Lenka (referee)
Because of its importance for every single cell, regulation of gene expression is often studied at all levels today. On the other hand, rather sparse amount of information is available for protists and our understanding of their gene expression is nearly limited to several model organisms. Three anaerobic human parasites have been studied in details: Trichomonas vaginalis, Entamoeba histolytica and Giardia intestinalis. Most recently, some others anaerobic protists (e.g. Entamoeba invadens or some species of Spironucleus genus) have been researched as well. This work is focused on the structure of promoter regions in protein coding genes and application of this knowledge in construction of transfection systems as one of the most effective and powerful tools in molecular and cell biology.
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Financial education mechanism of handicapped pupils at secondary schools
Filip, Josef ; Krutská, Dagmar (advisor) ; Svoboda, Petr (referee)
The thesis deals with problems of disabled pupils'education financing at secondary schools. Its aim is to create the integral survey about the disabled pupils'schooling, to compare and analyse normative financing of the most expanded educational specialization in accordance with regions. Theoretical part includes national educational concepts and programs directed at handicapped pupils. Besides detailed characterization of the health troubles'single types there are also described institutial schooling and normative financing of handicapped pupils. There are the analysed reports of MŠMT (Ministry of education and youth's psychical education) about handicapped pupils at the research part. Charts and graphs contain datas of pupils in the Czech republic according to health trouble's type, number of pupils at special classrooms and of integrated pupils during 2007-2012. The inseparable component of the research there is survey of pupils'schooling in accordance with the schools'promoters. Conclusion is devoted to detailed comparison of normative financing of the most expanded educational specialization 65-51-E/01 Stravovací a ubytovací služby (The board and accommodational service) in the first place for mental disabled pupils and pupils suffering with developmental disorders of learning and behaviour.
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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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Characterization of promoter regions of HGSNAT and GBA genes, and a contribution to the study of pathogenesis of MPS IIIC and Gaucher disease
Richtrová, Eva ; Hřebíček, Martin (advisor) ; Macek, Milan (referee) ; Adam, Tomáš (referee)
Pathogenesis of mucopolysaccharidosis type IIIC (MPS IIIC) and Gaucher disease has not been yet fully clarified, and the causes of phenotypical variability between the patients with the same genotype in Gaucher disease remain obscure. Because the variants in the regulatory regions of genes can cause phenotypical differences mentioned above, I have studied promoter regions of HGSNAT and GBA genes mutated in these lysosomal disorders. I have shown that there is an alternative promoter of GBA (P2). Additional studies were aimed to elucidate possible physiological functions of P2, and its possible role in the pathogenesis of Gaucher disease. I have found that P2 is not tissue specific, and that its variants do not influence the variability of phenotype in Gaucher patients with the same genotype. P2 is used differentially neither during the differentiation of monocytes to macrophages nor in macrophages from controls and Gaucher patients, in whom there is a prominent storage only in cells of macrophage origin. We have thus not found any changes that would suggest a role for P2 in the pathogenesis of Gaucher disease. I have characterized the promoter region of HGSNAT and shown that the binding of Sp1 transcription factor is important for its expression. Sequence variants found in HGSNAT promoter in...
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Regulation of transcription in mycobacteria.
Páleníková, Petra ; Krásný, Libor (advisor) ; Mrvová, Silvia (referee)
The bacterial cell has to be able to cope with environmental changes. Adaptation to these changes is achieved by changes in gene expression. Gene expression is regulated mostly at the level or transcription initiation. Transcription initiation depends on the sequence of promoters and is regulated by alternative sigma factors and many transcription factors acting either as activators or repressors. This work describes various ways of transcription regulation in the bacterial genus Mycobacterium that includes deathly pathogens such as M. tuberculosis and M. leprae. The typical characteristics of this genus are poorly conserved promoters, a high number of sigma and transcription factors, the presence of two-component systems and a lot of small RNAs that have not been characterized in detail so far.
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Expression of genes for the conversion of nitriles and amides in Rhodococcus erythropolis
Kracík, Martin ; Pátek, Miroslav (advisor) ; Mikušová, Gabriela (referee)
The strain Rhodococcus erythropolis A4 is a source of enzymes nitrilhydratase and amidase, that catalyse conversion of nitriles and amides. These enzymes are used in industrial biotransformation and bioremediation. Since it was difficult to carry out genetic manipulations aimed at increasing the production of these enzymes in the strain A4, the corresponding genes (ami and nha1 + nha2) of a related strain R. erythropolis CCM2595, in which both plasmid and chromosome manipulations can be routinely performed, were identified and analyzed in this diploma theses. The ami and nha1 + nha2 genes from the strain R. erythropolis CCM2595 were isolated and sequenced together with the flanking regions (5.5 kb in total). The organization of these genes and the expected regulatory genes was described in the strain CCM2595 and mechanisms of regulation of expression of these genes were studied. For the analysis of transcription of amidase and nitrilhydratase genes from both strains of R. erythropolis, the promoter-probe vector pEPR1 replicating in Escherichia coli and R. erythropolis was used. Transcriptional fusion of Pami promoters of the strains A4 and CCM2595 and the reporter gfp gene were constructed. The activity of the Pami promoter was measured by means of fluorescence of gfp gene product (green fluorescent...
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