|
|
Construction and characterization of recombinant systems for stress response analysis in pollutant-degrading rhodococci
Křenková, Lucie ; Štěpánek, Václav (advisor) ; Marešová, Helena (referee)
Genus Rhodococcus is represented by Gram-positive, mycolate-containing non- sporulating actinobacteria that excel in their ability to withstand effects of various physical and chemical stressors. Their enormous metabolic capacity allows them to efficiently degrade a wide range of toxic compounds. To benefit from these capabilities, it is essential to know the molecular basis of their response to the stresses, the regulation of which is primarily controlled by σ factors of RNA polymerase. These σ factors are still poorly described in rhodococci. This thesis aims to explore the potential of Rhodococcus erythropolis CCM2595 as a model organism for studying stress responses in rhodococci. R. erythropolis ΔsigH mutant and single-plasmid strains have been constructed which allow studying promoter activities of genes responding to osmotic, heat, and oxidative stress by measurement of GFP fluorescence intensity, in the cells of R. erythropolis. Further, homologous two- plasmid strains, which support induced overproduction of s factors, were also constructed. Comparison with analogous Corynebacterium glutamicum host-based systems showed that the use of rhodococci themselves as hosts for plasmid systems is still very limited, probably due to the plasmid instability in rhodococcal cells. Thus, for routine...
|
|
|
Analysis of promoters of genes encoding sigma factors in pathogenic Corynebacteria using Corynebacterium glutamicum model
Kučera, Tomáš ; Pátek, Miroslav (advisor) ; Mikušová, Gabriela (referee)
The aim of this thesis was to idetify potential promoters of genes encoding sigma factors in pathogenic corynebacteria using the model bacterium Corynebacterium glutamicum and to assess the effect of amino acid sequences of sigma proteins on promoter recognition. Sigma factors are essential for transcription and regulation of gene expression and play a key role in controlling promoter activity. Understanding the regulation of genes encoding sigma factors is esential because they contribute to virulence and pathogenicity and regulate the host immune response. In this study, the promoter regions of genes encoding sigma factors in Corynebacterium ulcerans and Corynebacterium diphtheriae were analyzed. First, potential promoter sequences in the genomes of C. ulcerans and C. diphtheriae were identified based on knowledge of consensus sequences of confirmed promoters in the model organism. The already tested two-plasmid system established in C. glutamicum was used to evaluate the activity of the proposed promoter regions. A potentional promoter region was inserted into the pEPR1 vector upstream of the gfpuv reporter gene, and sigma factor genes were inserted downstream of the inducible promoter in the pEC-XT99A vector. Using fluorescence, the activity of the reporter gene in response to induced sigma...
|
|
|
Expression, localisation, and interactome of the RefZ protein during sporulation in Bacillus subtilis.
Paliesková, Anna Mária ; Krásný, Libor (advisor) ; Nešvera, Jan (referee)
Bacillus subtilis is a gram-positive sporulating bacterium. Under unfavorable conditions, it initiates the sporulation process that results in a resistant spore. The transcription factor Spo0A is a master regulator of sporulation initiation. The hallmark of sporulation is the formation of an asymmetric septum near a cell pole, which divides the cell into the larger mother cell and smaller prespore. The asymmetric septum is localized at 1/6 of the cell length relative to the nearer pole. One of the players involved in this localization is the RefZ protein, referred to as the FtsZ regulatory protein, which forms the Z-ring. The Z-ring is important for the formation of both the vegetative (mid-cell) and asymmetric septa. RefZ facilitates the relocalization of the Z-ring from midcell to the poles at an early stage of sporulation. RefZ also binds DNA (RefZ binding motifs [RBMs]) near the ori site of the chromosome, thereby promoting precise positioning of the chromosome arms during sporulation. The entire sporulation process is controlled by a cascade of compartment-specific sporulation σ factors that recognize specific consensus sequences in the promoter regions of genes, thereby allowing RNA polymerase to initiate transcription of sporulation-specific genes. These σ factors ensure spatially and...
|
|
|
Evolution of the gene regulatory network underlying the formation of the gastrula organizer
Macháčová, Simona ; Kozmikova, Iryna (advisor) ; Krylov, Vladimír (referee) ; Buchtová, Marcela (referee)
During gastrulation, the vertebrate embryo is organized from the clump of cells into a bilaterally symmetric body. This organization process is driven by the gastrula organizer. Its establishment is induced by maternal Wnt/β-catenin signaling and Nodal/Activin signaling localized in the presumptive dorsal region of the embryo. The regulative environment then triggers the expression of the organizer-specific genes which create morphogen gradients in the embryonic body and therefore give each cell positional information. However, the evolution of vertebrate organizer establishment remains vague. Here we aim to test the compatibility of the invertebrate cis-regulatory modules with the vertebrate gene regulatory network (GRN). We introduced fluorescent reporter genes under the control of the invertebrate regulatory sequence of organizer-specific genes into a vertebrate model to observe their behavior in the context of the vertebrate GRN. We found and functionally verified a 500 bp-long amphioxus sequence (an enhancer) that is necessary and sufficient to drive a correct Chordin gene expression in the gastrula organizer in zebrafish. Chordin is a prominent organizer-specific gene antagonizing Bone Morphogenetic Protein (BMP) signaling. We tested also other invertebrate genes for their compatibility with...
|
|
|
Porovnání promotorů pro využití v klíštěcím (\kur{Ixodes}) expresním systému
MUSTACOVÁ, Johana
Gene manipulation can be a convenient tool for tick control, however functional procedure for gene manipulation of ticks was not determined yet. The creation of plasmid vectors with high ability of gene expression driven by functional promoters is crucial for genetically modified ticks. To investigate effective tools for tick genes manipulation within the scope of this master thesis, transfection procedures for various types of Ixodes tick cell lines were optimized. As well as expression vectors for use in tick cells were tested. For this purpose, expression plasmid vectors containing luciferase reporter genes driven by eukaryotic and viral promoters were used.
|
|
|
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...
|
|
|
Preparation of the constructs for analysis of expression of nuclear receptor nhr-97 by using transgenic techniques in the model system Caenorhabditis elegans
Boušová, Kristýna ; Stiborová, Marie (advisor) ; Hudeček, Jiří (referee)
The aim of this work was to prepare two constructs of the promoter of a gene coding for nuclear hormone receptor nhr-97 in C. elegans. Nuclear receptors belong to a large group of genes sharing homologous sequences in some vertebrate nuclear receptors. The first part of the work describes the structure of nuclear hormone receptors, their function and significance in the nematodes C. elegans. The model organism C. elegans, its anatomy, life cycle and genome were also described. The work also discusses the structure and use of green fluorescent protein (GFP), which serves to localize the expression of the nhr-97 gene in C. elegans. In the practical part of the work, the preparation of two constructs of the promoter is described. Isolation of genomic DNA of C. elegans, PCR amplification of the promoters and their subsequent cloning into vector pPD95.67 containing a gene coding for green fluorescent protein were performed. To verify the successful cloning of the promoter constructs, sequencing DNA was performed. Cloned promoters of nhr-97 will be used for microinjetions to C. elegans gonads and the expression of this gene regulated from particular promoters will be subsequently monitored using expression of green fluorescent protein in progeny.
|
|
|
Cis and trans elements in plant response to drought (with a particular focus on photosynthetic genes)
Panchártek, Daniel ; Holá, Dana (advisor) ; Feketová, Zuzana (referee)
Abiotic stresses, such as drought, high salinity and cold can strongly affect plant development and growth. Cis and trans elements are one of the options how plants regulate their own metabolism in those cases. That's the way how the expression of many target genes is induced. The products of these genes function not only in stress tolerance but also in general stress response. Many transcription factors and regulatory proteins (trans elements) are involved in these adaptions; those often interact with specific sequences in gene promoters (cis elements). Recenly, a progress has been made in analyzing the signal paths and complex cascades of gene expression regulation, although a little is still known about this regulation during drought conditions. Some of these target genes code products participating in photosyntesis and the regulation of their expression can significantly affect this process. This essay briefly describes main cis and trans elements of plant response to drought (with a particular focus on the regulation of photosyntetic genes). Keywords: drought, gene expression, cis-element, transcription factor, abcisic acid, photosyntesis, promoter.
|
|
|
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.
|
|
|
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.
|
guest ::
