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Identification of non-coding RNAs of Clostridium beijerinckii NRRL B-598 using RNA-Seq data
Pomykalová, Barbora ; Sedlář, Karel (referee) ; Jurečková, Kateřina (advisor)
This bachelor thesis contains short introduction into bacterial small non-coding RNA problematic. It is oriented on their features and functions in organisms, especially in bacteria Clostridium beijerinckii NRRL B-598. Bachelor thesis also contains description of various laboratory methods for gene expression determination and suggests a detection method for small non-coding RNA in bacteria Clostridium beijerinckii NRRL B-598. Suggested method works with data, which were obtained by RNA-Seq technology. Within the framework of the bachelor thesis was suggested method implemented in programming and numeric computing platform MATLAB and its results were discussed.
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Identification of non-coding RNAs of Clostridium beijerinckii NRRL B-598 using RNA-Seq data
Pomykalová, Barbora ; Sedlář, Karel (referee) ; Jurečková, Kateřina (advisor)
This bachelor thesis contains short introduction into bacterial small non-coding RNA problematic. It is oriented on their features and functions in organisms, especially in bacteria Clostridium beijerinckii NRRL B-598. Bachelor thesis also contains description of various laboratory methods for gene expression determination and suggests a detection method for small non-coding RNA in bacteria Clostridium beijerinckii NRRL B-598. Suggested method works with data, which were obtained by RNA-Seq technology. Within the framework of the bachelor thesis was suggested method implemented in programming and numeric computing platform MATLAB and its results were discussed.
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Intracellular and intercellular regulation of gene expression in Gram-positive bacteria.
Pospíšil, Jiří ; Krásný, Libor (advisor) ; Lichá, Irena (referee) ; Malínský, Jan (referee)
Bacteria, the most dominant organisms on Earth, are an everyday presence in our lives. Symbiotic bacteria, which are present in the digestive tract of animals, usually have a beneficial effect on the body. On the opposite side of the spectrum are pathogenic species that cause more or less serious diseases around the world. In order to fight pathogens effectively, it is necessary to learn as much as possible about the molecular mechanisms by which bacteria respond to their environment, and also about the types of communication within bacterial populations that allow them to react to environmental changes as "multicellular" organisms. This Thesis consists of two main parts. In the first part, selected aspects of bacterial gene expression are characterized, using Bacillus subtilis and Mycobacterium smegmatis as model organisms. DNA-dependent RNA polymerase (RNAP) is the enzyme that is responsible for transcription of DNA into RNA, and thus plays a key role in gene expression. This Thesis deals with the structure of bacterial RNAP and important auxiliary factors (proteins and RNA) that associate with this enzyme and modulate its function. In the second part, the focus is on cell-to-cell communication, revealing which factors/mechanisms, including gene expression, affect this process in B. subtilis....
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The effect of selected endogenous and exogenous factors on bacterial growth
Šiková, Michaela ; Krásný, Libor (advisor) ; Valášek, Leoš (referee) ; Vopálenský, Václav (referee)
The growth of bacteria by binary division is a key characteristic of these organisms. This growth depends on two types of factors: endogenous and exogenous. Endogenous factors make up the molecular apparatus of cells. Among important endogenous factors belong also those involved in gene expression and its regulation. Exogenous factors are external conditions such as nutrient availability, temperature, pH, various stresses or the presence of antibacterial agents. The main aim of my Thesis was to study the effects of selected endogenous and exogenous factors on bacterial growth. As endogenous factors I studied RNase J1 in Bacillus subtilis and a small RNA called Ms1 in Mycobacterium smegmatis, which are involved in regulation of gene expression at the transcriptional level. I showed that RNase J1 can, besides its role in RNA degradation, play a role in genome integrity by removing stalled RNA polymerase (RNAP) complexes from DNA. I further showed that Ms1 binds to the RNAP core and affects the level of RNAP in the cell. The results revealed new mechanistic aspects of the transcription apparatus and show how individual components or their combinations affect bacterial growth. As exogenous factors I studied the recently discovered antibacterial compounds, called lipophosphonoxins, their interaction...
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Study of RNAi mechanisms in tobacco BY-2 cell line and potato plants
Tyč, Dimitrij ; Fischer, Lukáš (advisor) ; Kovařík, Aleš (referee) ; Moravec, Tomáš (referee)
Knowledge of the processes of RNA interference, the regulation of gene expression by small RNAs (sRNAs), has grown at an unprecedented rate over the last 30 years. Some of the findings were literally revolutionary, as they revealed events that overturned many long-held notions. Many phenomena have been shown to be highly conserved and common to organisms of different species, but others are specific to certain lineages or have not yet been fully explored. There is also a lack of knowledge about the interconnection of numerous pathways - for example between silencing at the transcriptional (TGS, leading to the promoter methylation) and post-transcriptional levels (PTGS, affecting mRNA stability or translation). The present work summarizes the findings of two published and two unpublished works and attempts to describe some of the less known sites of RNA interference using various plant model organisms. Research on Solanum tuberosum transgenic lines has revealed the ability of 5-azacytidine to restore the expression of transcriptionally silenced transgenes at the whole plant level. De novo regeneration from leaves of such plants can lead to re-silencing of reactivated transgenes and thus serves as a selection method to exclude lines prone to spontaneous silencing. The nature of changes in the...
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Interaction of nucleic acids with RNA polymerase
Janoušková, Martina ; Krásný, Libor (advisor) ; Vopálenský, Václav (referee) ; Knejzlík, Zdeněk (referee)
Regulation of gene expression by RNA polymerase (RNAP) is an essential ability of living organisms, required for their adaption to a changing environment and ultimately enabling their survival. Interaction of RNAP with ribonucleic acids (DNA or RNA) is crucial for transcription and its regulation. This Doctoral Thesis contains two projects addressing interactions of RNAP with nucleic acids: (i) Transcription of modified DNA templates and (ii) Ms1, a small RNA (sRNA) from M. smegmatis. (i) We investigated the influence of modifications in the major groove of DNA on bacterial transcription in vitro. We found out that transcription of modified DNA templates is influenced on the transcription initiation level and that the promoter sequence is important for the effect of the modifications. Furthermore, we successfully performed transcription switch ON and OFF in vitro by bioorthogonal reactions. This regulation of transcription by artificial DNA modifications has a future in biotechnologies and/or medical therapy. (ii) Regulators of transcription are also small non-coding RNAs. These molecules have an important role in gene expression regulation among prokaryotes and eukaryotes. Ms1 is an sRNA found in mycobacteria. It makes a complex with the RNAP core and it is abundant in stationary phase (in amounts...
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Regulation of expression of Ms1, a sRNA from Mycobacterium smegmatis
Páleníková, Petra ; Krásný, Libor (advisor) ; Lichá, Irena (referee)
Bacteria are exposed to various environmental conditions during their growth. They have to cope with rapid changes in temperature, lack of nutrition, etc. To survive, bacteria alter their gene expression. One type of regulation of gene expression is regulation by small RNAs (sRNAs). In bacteria, a well-studied sRNA is 6S RNA that binds to the RNA polymerase holoenzyme. However, 6S RNA has not been identified in several bacterial species. Mycobacteria are a genus that probably does not have 6S RNA. Instead, Mycobacterium smegmatis possess another sRNA - Ms1. Ms1 structurally resembles 6S RNA and indeed it was first identified as a 6S RNA structural homologue. However, Ms1 binds to RNAP devoid of any sigma factor, and, therefore, is significantly distinct from 6S RNA. This work describes regulation of expression of Ms1. DNA fragments of different length from the region upstream of the Ms1 gene were prepared. These fragments were fused to the lacZ reporter gene and their activity was tested in different growth phases and under stress. This allowed identification and characterization of the core promoter sequence and regulatory sequences that might interact with transcription factor(s). Promoter activity increased with increased length of the promoter fragment and after transition into stationary...
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Characterization of Ms1, a newly identified small RNA from Mycobacterium smegmatis
Pospíšil, Jiří ; Krásný, Libor (advisor) ; Lichá, Irena (referee)
Introduction: In recent years, there has been growing interest in regulation of gene expression by small non-coding RNA (sRNA). The first sRNA discovered in 1960s was 6S RNA from E. coli (length ~184 nt). It took ~ 30 years to obtain meaningful insights into its function. 6S RNA binds during stationary phase to RNA polymerase (RNAP) containing sigma factor 70 (primary sigma factor), thereby preventing transcription from σ70 - dependent promoters. In our laboratory we discovered a small RNA (length ~300 nt) in stationary phase of growht in Mycobacterium smegmatis. This sRNA was named Ms 1. The function of Ms 1 is uknown and preliminary experiments indicated that Ms 1may bind to RNAP that lacks σ factor (σA ). Goals: The aim of this Diploma project is to contribute to the characterization of Ms 1. Approaches: First, by molecular cloning, affinity chromatography and in vitro transcription I prepared the tools for subsequent experiments in vitro: RNAP, σA , Ms 1 and its mutated variants. Next, these tools were used for binding experiments on native gels and for transcription experiments. Results: RNAP, σA , Ms 1 and its variants were prepared. In vitro binding assays showed that wt Ms 1 but not a mutated variant of Ms 1 binds to RNAP. Using this assays were identified areas of Ms 1 that are important...
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Trichurids in ruminants from Czech Republic.
Antošová, Tereza ; Langrová, Iva (advisor) ; Ivana, Ivana (referee)
The goal of this paper was to determine rate of presence of whipworms of genus
Trichuris in bodies of selected ruminants (sheep, roe deer) in certain areas and to morphologically state different species of whipworms using molecular revision and professional literature on samples found during helmitological dissections of selected ruminants. Two hypotheses were stated: H1: species that are found in highest volume in case of roe deer and sheep are whipworms Trichuris discolor and Trichuris ovis H2: these whipworms can not be positively distinguished when using morphometrical methods.
Material needed for the study, i.e. the intestines of examined ruminants, was recovered in different areas of Czech Republic. Later were the intestines dissected in a laboratory using standardized procedure and hereby collected samples were analysed.
Based on selected methods it was determined that in roe deer the rate of occurence of Trichuris discolor is much higher compared to that of Trichuris ovis. With sheep the difference between rates of presence is smaller. These results confirm the first hypothesis by showing high rate of presence of whipworms in these hosts. Collected females of genus Trichurids were morphometrically differentiated by their sex and in 4 morphotypes. Following this differentiation, the most present were the females of morphotype M2, those with a vulval opening without an everted vagina. The second hypothesis was also confirmed.
Multihosting species Trichuris discolor and Trichuris ovis are prevalent in the bodies of roe deer and sheep. Thus we can say the roe deer are a potential source of whipworm contamination to sheep breeding. It can not be excluded that sheep are infected by roe deer and vice versa. Molecular determination is a necessary tool for correct assessment of whipworm species, considering the fact that morphological methods may lead to incorrect results.
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