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Study of the function of the hybrid proline-rich protein family
Dvořáková, Lenka ; Fischer, Lukáš (advisor) ; Vágner, Martin (referee) ; Havel, Ladislav (referee)
Lenka Dvořáková SUI'^,ÍARYoF TřE PH.D. TFDSIS 4. CONCLUSIONS Ancestral HyPRP evolved probably from a lipid transfer protein (LTP) relative that had acquired a sequence encoding a long proline-rich N-terminal domain. The origin of HyPRPs may be one of the evolutionary innovationsof seedplants. Specific sequence is not probably important for formation of the functional 8 CM domain of HyPRPs with exception of eight conserved cysteine residues.which stabilize the tertiary structureof the domain. HyPRPs with long proline-rich N-terminal domains are relatively well-conserved and probably evolutionary more original. Proteins with aýpical N-terminal domains (very short. glycine-rich) evolved apparently relatively recently and independentlyin differentplant speciespossibly by means of shortening, loss or re€uTangements of the ancestral longer proline-rich domains. Glycine-rich domains could originate from proline-rich ones by way of inversion in the coding sequence.This mechanismcould have greatimpact in evolution of cell wall proteins in general, becausethey are often rich eitherin prolineor glycine. N-terminal domains of angiosperm C-type HyPRPs remained relatively well conserved (long and hydrophobic), while the rest of angiosperm HyPRPs has been undergoing rapid and continuous diversification. However, in the...
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Function of PsbO isoforms
Duchoslav, Miloš ; Fischer, Lukáš (advisor) ; Špunda, Vladimír (referee) ; Sobotka, Roman (referee)
(English version) Oxygenic photosynthesis is crucial for most forms of the life on the Earth. The splitting of water and evolution of oxygen is conducted by photosystem II (PSII), a multi-subunit pigment- protein complex embedded in the thylakoid membrane. PsbO is an indispensable subunit of PSII, bound to its transmembrane subunits from the luminal side. The main function of PsbO is to stabilise and protect Mn4CaO5 cluster where the water splitting occurs. However, it has probably also some auxiliary functions. These additional functions might be different for isoforms of PsbO proteins, as suggested for Arabidopsis thaliana, which expresses two genes encoding protein isoforms PsbO1 and PsbO2. This thesis studies auxiliary functions of PsbO with a focus on functional differences between PsbO isoforms. We found that besides Arabidopsis thaliana, also many other plant species express two psbO genes. Interestingly, the duplication of psbO gene occurred many times independently, generally at the roots of modern angiosperm families. In spite of this, the PsbO isoforms differ at similar sites in the protein structure, suggesting that similar subfunctionalisation of PsbO isoforms occurred parallelly in various lineages. Biochemical characterisation of PsbO from green alga Chlamydomonas reinhardtii and...
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The influence of RDR6 activity and mode of RNAi induction on dynamics and mechanism of silencing of the reporter GFP gene in tobacco cell line BY-2
Motylová, Šárka ; Fischer, Lukáš (advisor) ; Kovařík, Aleš (referee)
RNA interference (RNAi) is a process mediated by small RNAs (sRNA), which is significantly involved in the regulation of gene expression in plants. Diverse RNAi pathways can be divided into two basic mechanisms, which are post-transcriptional and transcriptional gene silencing (PTGS and TGS). Production of sRNAs is dependent on the presence of a double-stranded RNA molecule (dsRNA), which is cleaved by one of DCL proteins to produce sRNAs usually of 21-24 nt in length. One strand of the sRNA is subsequently loaded onto AGO protein. During PTGS, the AGO-sRNA complex interacts with the target RNA based on its sequence complementarity to the sRNA and cleaves it or blocks its translation. In the case of TGS, AGO interacts with plant-specific RNA Pol V and its transcripts, which are again complementary to the sRNA. This interaction allows assembling of a protein complex facilitating DNA and histone methylation inhibiting RNA Pol II transcription. There are numerous ways the dsRNA can arise. A significant part of dsRNA cell production is dependent on synthesising the complementary strand of the dsRNA by RDR6 (RNA-dependent RNA polymerase 6). RDR6 is also involved in the process of the secondary sRNA formation. The significance of RDR6 during PTGS was examined using a GFP reporter gene either during...
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Transgene silencing in potato plants and tobacco cell line BY-2
Tyč, Dimitrij ; Fischer, Lukáš (advisor) ; Honys, David (referee)
Accidental discovery of RNA interference in plants a few decades ago and its description by Fire et al. (1998) brought a real revolution in molecular biology and in many other branches of biology. Researchers have uncovered a large and unexpected mechanism of regulation of gene expression at many levels in almost all studied eukaryotes. The first studies have revealed silencing of both exogenous and endogenous genes in plants. So, it soon became clear that this process serves to organisms not only in the defense against viruses, but also against mobile genetic elements as well as in modulation of expression own genes. The key role in all RNAi mechanisms is played by dsRNA that is processed into small RNAs. In association with a number of other proteins, small RNAs can direct either cleavage of complementary or nearly complementary mRNAs or block their translation in the process of posttranscriptional gene silencing (PTGS). Additionaly, sRNAs can affect gene expression at transcriptional level via modifying DNA in a process called RNA dependent DNA methylation (RdDM). In plant transformation, RNAi cause variability amongst transgene lines and instability in the expression of introduced genes, which complicates the use of transgenic lines, because many biotechnological approaches require a defined...
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Dynamics of de novo DNA methylation and its impact on transgene expression and CRISPR/Cas9 mutagenesis
Přibylová, Adéla ; Fischer, Lukáš (advisor) ; Pečinka, Aleš (referee) ; Fajkus, Jiří (referee)
Genetic information must be protected, maintained and copied from cell to daughter cells, from generation to generation. In plants, most of the cells contain complete genetic information, and many of these cells can regenerate to a whole new plant. Such a feature leads to the need for precise control of which genes will be active and which not because in growth and differentiation, only the activity of specific genes for the individual cells, tissues, organs are required. One of the mechanisms controlling the gene activity is RNA interference (RNAi), which down- regulates or blocks the expression of specific genes at the transcriptional or post-transcriptional level. The crucial part of the RNAi is guiding the RNAi machinery to the target. It is mediated via sequence complementarity of the target with a small RNA (sRNA), which is diced from a double- stranded RNA (dsRNA) precursor. The molecular mechanism of dsRNA and sRNA formation and also the target origin predestinates the subsequent silencing pathway. In transcriptional gene silencing (TGS), the gene expression is regulated through chromatin epigenetic modifications. One of the epigenetic marks is cytosine methylation, which is established mainly by RNA-directed DNA-methylation (RdDM) pathway. Although the protein machinery was relatively...
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Dynamics and variability of induced transgene silencing in tobacco cell line BY-2
Čermák, Vojtěch ; Fischer, Lukáš (advisor) ; Pečinka, Aleš (referee) ; Lafon Placette, Clément (referee)
RNA interference (RNAi) is an important mechanism regulating gene expression. In plants, RNAi is triggered by double-stranded RNA (dsRNA) which is processed into small RNAs (sRNAs), usually 21-24 nt long. The sRNAs are loaded into Argonaut (AGO) protein and recognize the target based on sequence complementarity. When the target is mRNA, they can slice it or block translation leading to posttranscriptional gene silencing (PTGS). When the target is DNA, they can induce DNA methylation and chromatin changes, which when present in the promoter can lead to transcriptional gene silencing (TGS). The individual components of RNAi are well described, but less is known about the impact of different types of dsRNA precursors on the dynamics of RNAi. To study these aspects of RNAi, we used tobacco BY-2 cell line expressing GFP reporter and inducible silencers. The silencers used different ways of triggering the dsRNA formation by transcripts from antisense (AS), unterminated sense (UT) and inverted repeat (IR) GFP sequence to initiate PTGS. Additionally, one IR silencer based on the CaMV 35S promoter initiated TGS. This allowed us to study RNAi from the beginning throughout the steady state level and till the recovery phase, all in the highly homogeneous system. Using this system, we described several features...
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Specific roles of Argonaute family proteins in Arabidopsis thaliana
Teznerová, Kateřina ; Fischer, Lukáš (advisor) ; Hála, Michal (referee)
Argonaut proteins are RNA binding proteins with RNase activity. They can bind short non-coding RNAs, which are usually 21-24 nt in length. These small RNAs are formed in plants from double-stranded RNAs and are involved in the regulation of gene expression, in the protection of the plant against viruses, transposons, or other invasive genetic elements, and affect the state of chromatin. Using small RNAs, Argonaut proteins participate in a mechanism called RNA interference, a complex process used to regulate gene expression, at the transcriptional or post-transcriptional level. In the plant species Arabidopsis thaliana, the Argonaut family of proteins has 10 genes, while in other plant species they have their functional homologues (orthologs). Individual proteins differ in their preference for different types of small RNAs depending on their length, the 5 'terminal nucleotide and the way in which the source double-stranded RNA is formed. At the same time, the involvement of individual types of Argonaut in the partial processes of RNA interference and in connection with their cellular localization also differs. This work is focused on the interaction of small RNAs with Argonaut proteins, describes the processes of their functioning and further deals with individual paralogs of Argonaut proteins in...
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Use of transgenic plants in functional analysis of plant genes
Klincová, Martina ; Fischer, Lukáš (advisor) ; Sabol, Peter (referee)
Use of transgenic plants in functional analysis of plant genes Functional genomics makes it possible to detect the functions of uncharacterized genes. It allows you to understand not only the functions of individual genes, but also the relationships between them. The acquired knowledge can also be used in improving the properties of various cultivated plants. With decreasing cost and increasing sequencing efficiency, many plant genomes are sequenced, but without the associated function of individual genes. Reverse genetics approaches are used to characterize these genes. In the first place, these include approaches that include the analysis of insertional mutants or the use of RNA interference that inactivates or reduces the expression of the individual genes studied. We can also inactivate whether to specifically modify the function of a particular selected gene using site-specific endonucleases, which include meganucleases, zinc finger nucleases, TALEN (transcription activator-like effector nucleases) and CRISPR (clustered regularly interspaced short paindromic repeats). In contrast to inactivation approaches, there are approaches such as activating mutagenesis and ectopic expression, through which we increase the expression. In addition, gene expression and protein localization can be analyzed...
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Functional characterization of Alba-family genes in Arabidopsis thaliana
Kočová, Helena ; Honys, David (advisor) ; Fischer, Lukáš (referee)
(anglicky) Alba-family proteins were identified in Archaea and Eucarya and are classified among the oldest and the most conserved nucleic acid-binding proteins. The binding preferences and roles differ among certain evolution clades. In Crenarchaea they represent chromatin-binding proteins, while their role in RNA metabolism is suggested in Euryarchaea and Eukaryotes. ALBA proteins are well characterized in human, where they play a role in the RNAse P/MRP complex and in unicellular parasites, such as Plasmodium and Trypanosoma, where an involvement in the life cycle regulation is confirmed. In plants, their role is not yet well understood. The aim of this thesis is to increase a knowledge about the Alba-family proteins in the model plant Arabidopsis thaliana. Based on a minimal changes to development and reproduction in single mutants and high sequence similarity, a functional redundancy of the proteins was assumed. For better understanding of the ALBA proteins function, three smaller members of the family were edited by the same metod. The obtained triple mutant showed delay in flowering. ALBA dimer formation was confirmed in many organisms. BiFC method was used to determine Arabidopsis ALBA homodimerization. The data analysis showed potential homodimerization in most of them.
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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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