CRISPR/Cas9 technology is currently one of the most important tools of genome engineering. This technology allows a precise site-specific gene editing and such ability was applied to study the role of TALE (TALE - three amino acids loop extension) homeodomain transcription factors during neural crest cells development. The main genes of interest, belonging to sub-family of TALE proteins, are Meis1 transcription factors that are present in the zebrafish genome as two paralogous genes, meis1a and meis1b. Their function was assessed by mutating their DNA-binding domain - homeodomain to abrogate the ability of transcription factor to bind DNA and by that disturb regulatory network, in which Meis1 proteins operate in. Phenotype analysis of mutant fish would reveal a potential role of Meis1 proteins in regulation of neural crest cells development and outline the functional significance of the homeodomain in regulatory operations. To determine the regulatory relationship between meis1a and meis1b genes morpholino-based knock-down of the genes was performed. Preliminary results suggest a dominant role of Meis1b in neural crest cells regulation and importance of its homeodomain. Furthermore, knock-down of Meis1a indicates its contribution to regulation of craniofacial development. However, a detailed...

Several techniques have recently been described for precise mutagenesis of selected target sites in the genome. This thesis establishes the method of gene targeting by CRISPR/Cas system in D. melanogaster and compares it with gene targeting using TALENs. To test the mutagenesis systems, we choose an endogenous gene encoding concentrative nucleoside transporter gene (CNT1). We have received two mutants containing large deletions affecting the N-terminal part of the CNT1 gene. We show that CRISPR/Cas is useful tool for targeted gene disruption in D. melanogaster.

Phosphoglucosamine mutase (GlmM) is an enzyme of bacterial cell wall biosynthesis. The main aim of this thesis was to find out, whether gene glmM is essential for viability of Streptococcus pneumoniae. Therefore, we prepared merodiploid strain containing two copies of glmM; the genomic gene and ectopic copy under control of zinc inducible promoter. Subsequently, depletion strain was prepared by deletion of genomic copy of glmM. This strain was further used for analysis of viability and phenotype features in the medium containing various concentrations of zinc ions, an inducer of ectopic glmM expression. We found out, that the viability of this strain was strictly dependent on the concentration of inducer and further, that depletion of GlmM resulted in remarkable morphological defects. The rescue of mutant strain was observed after addition of inducer up to the level of the control sample. These results have provided the evidence of glmM essentiality for S. pneumoniae viability. Furthermore, we analyzed, whether phosphorylation of key amino acid residues, S99 and S101, is essential for GlmM functionality. Four different strains were prepared by means of site-directed mutagenesis expressing glmM with substitutions of key serine residues for alanine or glutamic acid. Since deletion of chromosomal locus in...

4 Abstract Purinergic P2X receptors are membrane ion channels activated by extracellular ATP. There are seven isoforms of mammalian P2X receptors designated as P2X1-7, which according to their structure represent a specific family of ligand gated ionic channels, with extraordinary structural/functional properties. The P2X receptor consists of three subunits and each subunit has two transmembrane domains. Crystalographic data demonstrate that ionic channel pore is situated between the second transmembrane domains. Crystal structure of P2X4 receptor from the zebrafish (Danio rerio) is available in both open and closed state of the channel and the exact structure of ATP binding site is solved. The aim of this thesis was to study the structure-function relationships in a model of recombinant P2X4 receptor of the rat. By employing the point mutagenesis and electrophysiological recording, the functional importance of conserved cysteine residues in the ectodomain and amino acid residues which form the extracellular vestibule was investigated. All ten cysteins were substituted one by one with alanine or threonine and ATP-induced currents were measured from HEK293T cells expressing wild type (WT) and mutated P2X4 receptors. The results indicate that C116A, C126A, C149A and C165A mutations disrupt two disulfide bonds...

Retroviruses are viruses which are able to integrate to genome of host cell. Nonrandom integration of provirus near or inside some cellular genes may result in their deregulation, activation or silencing. This can later lead to cell transformation and tumor formation. This thesis discusses identification of viral integration sites (VIS) and common integration sites (CIS) in tumors originating from different organs (mostly kidneys, lungs and liver) with using mostly avian retroviruses subgroup J, specifically first natural isolate HPRS-103 and laboratory made virus MAV-J, which was made by replacing gene envB by envJ. Infection was made in ovo using chicken breeds Brown Leghorn and White Leghorn and tumors were isolated from 8 to 28 weeks after infection. For molecular analyses was used inverse PCR method and sequencing. From 74 molecularly analyzed tumors there was detected 373 VIS and 6 CIS with statistical significance over 2.10-2 . Gene with the highest number of hits was FRK (14 times), then TERT (5 times), CTDSPL (5 times), EGFR/ERBB1 (3 times), MYB (3 times) and MYC (3 times). Except 6 CIS there were other genes found, which had smaller statistical significance. Keywords: retrovirus, insertional mutagenesis, subgroup J, oncogenesis, oncogenes, MAV-J, HPRS-103, proviral integration sites, tumors

Phytohormone auxin and its directional distribution plays an essential role in the regulation of numerous processes during vegetative and reproductive plant development. Regulation of the expression, localization and activity of the PIN-FORMED (PIN) proteins is important for proper polar auxin transport in plant tissues. PIN proteins have been described as the major auxin efflux carriers regulating auxin's directional flow to build up gradients that provide information for the coordination of plant development. PIN protein structure topology prediction through bioinformatic analysis is still insufficient to understand their transport mechanism. Experimental analysis of PIN protein domains can provide valuable insight into understanding their role in mediating auxin transport. In this study, the C-terminal part of PINs have been modified by gradual trimming to determine the existence of relevant functional domains, which could be important for auxin transport. Seven modified PIN proteins from Arabidopsis thaliana and Nicotiana tabacum were prepared. Transiently transformed tobacco cell line Bright Yellow-2 (BY-2) was used to monitor differences in PIN transport activity. This approach allowed indirect monitoring of intracellular auxin levels using the DR5 reporter system. Transiently expressed...

This master thesis is focused on the molecular characterization of the eight red yeasts species. For molecular characterisation, the most variable rDNA regions ITS1, 5,8S ITS2 and the region encoding the large ribosomal subunit (26S) were amplified. This long region of the yeasts DNA was sequenced and compared by NCBI database for identification. The red yeasts identification was confirmed by data from DGGE method. Another aim of this thesis was to select the best yeasts producer of carotenoids and triacylglycerols. Rhodosporidium toruloides was found as the best producer and, thus, this strain was subjected to random mutagenesis by UV irradiation. The results of the production of metabolites by R. toruloides were compared with mutant strains, which were also adapted to the glycerol and waste whey substrates. The mutant strain G33 was found as the best producer of total carotenoids with a yield of 7.14 mg.g-1 of biomass. The highest production of ergosterol was demonstrated by the mutant strain Y34, the ergosterol yield was 47.72 mg.g-1 of biomass. The wild type of R. toruloides was able to produce the highest amount of both carotene (2.42 mg.g-1 of biomass) and TAG (76.32 mg.g-1 of biomass) on glucose medium.

Předložená diplomová práce se zabývá produkcí polyhydroxyalkanoátů (PHA) bakterií Cupriavidus necator H16. Cílem práce byla příprava, selekce a charakterizace mutantních kmenů schopných vyšší produkce PHA. V teoretické části byla zpracována literární rešerše zabývající se nejdůležitějšími typy PHA, bakterií Cupriavidus necator a způsoby indukce mutageneze. V experimentální části byly připraveny mutantní kmeny pomocí fyzikální a chemické mutageneze. Mutantní kmeny schopné nadprodukce PHA byly selektovány pomocí kultivace na minerálním médium s olejem. Pro další studium byly vybrány 4 mutantní kmeny schopné nadprodukce PHA. Tyto mutantní kmeny byly dále podrobeny biochemické charakterizaci. Byly naměřeny specifické aktivity vybraných intracelulárních enzymů včetně enzymů podílejících se na biosyntéze PHA. Také byla naměřena resistence mutantů vůči oxidačnímu stresu. Bylo zjištěno, že mutantní kmeny schopné nadprodukce PHA mají vyšší aktivity enzymů produkujících NADPH. NADPH je jeden z klíčových substrátů ovlivňujících směr toku acetyl-CoA metabolizmem. Vyšší intracelulární koncentrace NADPH parciálně inhibuje Krebsův cyklus a aktivuje akumulaci PHA. Aktivity acetoacetyl-CoA reduktázy a PHA syntázy, enzymů zapojených do syntézy PHA, těchto mutantů proto byly také vyšší stejně jako molekulová hmotnost připravených polymerů. Aplikace fyzikálních a chemických mutagenů je způsob, kterým lze připravit biotechnologicky perspektivní mutantní kmeny schopné nadprodukce PHA.

Yeasts are like other organisms constantly exposed to environmental influences. Their survival depends on the skills to adapt to environmental changes, including the ability to use various alternative sources of nutrients. In presented PhD thesis carotenogenic yeast belonging to the genera Rhodotorula, Sporobolomyces and Cystofilobasidium were tested for ability to use of selected waste substrates, and also subjected to several types of exogenous stress effects and mutations in order to increase the production of microbial biomass enriched with specific metabolites. As alternative nutrient sources derived from waste substrates from agricultural and farm production apple peel, pulp, corn germ and more were tested. Yeasts were also exposed to osmotic, oxidative and combined stress (benefits of various concentrations of NaCl and H2O2 to the culture media), followed by metal ions of selenium and chromium in concentrations of 0.01 mM, 0.1 mM and 1 mM. The effect of mutagen methanesulfonic acid ethyl ester was tested too. In all experiments the adaptivity of cells, morphological changes, color pigments produced by the media while some important fungal metabolites production and changes in chromosomal DNA fragmentation were analyzed. In order to evaluate potential changes in the yeast genome after treatment with mutagen and stress factors methods for isolation of intact chromosomal DNA and DNA analysis by pulsed field gel electrophoresis was optimized. The amount of produced metabolites was mainly analyzed by RP-HPLC with UV/VIS and MS detection. The work has been shown that most strains are able to use waste substrates and produced selected target metabolites. Biomass, for example, in R. aurantiaca on apple fiber was about 7 g/l and in C. capitatum cultivated on modified whey reached to 9 g/l. Amount of produced carotenoids by R. aurantiaca cultivated on wheat germ and maize after enzymatic hydrolysis by F. solani was 1.01 mg/g and S. roseus on pasta 4.3 mg/g. The values of ergosterol synthesis in R. aurantiaca are on the apple shells around 4.8 mg/g, in S. roseus on pasta with the enzymatic hydrolysis of P. chrysosporium 8.9 mg/g. The best substrate for biomass production and induction of carotenoids are waste substartes containing a mixture of simple and complex carbohydrates enriched with the addition of nitrogen compounds. Potential cytotoxic effect of stress factors of low concentrations was demonstrated. Red yeast genome was able to distribute by optimized PFGE, the karyotype of tested yeasts contain 11 or more chromosomes with visible differences between yeast species and genera. During exchange internship the ability of recombinant yeast S. cerevisiae to convert xylose to xylitol, which would be achieved by increasing the production of bioethanol as alternative fuel sources was studied. It turned out that both ligninocellulose materials to bioethanol production, as well as various waste substrates for microbial synthesis of carotenoids would reduce costs for industrial production of yeast metabolites, as well as to reduce the negative burden on the environment.

The described experimental model of clonal tumors induced through the insertional mutagenesis with MAV-2 proved to be a valid and rich source of information describing the process of transformation of normal into tumor cell. We have mapped more than 2000 individual clonal VISs from several hundreds of tumor tissue samples. We have analyzed five tumor types of different histology and tissue of origin along with their derivative tissue cultures. Furthermore, we have discovered the industasis phenomenon and described it during the course of the study. The goal of my study was to uncover common reasons for neoplastic transformation of the cell. The results of my study led me to the paradoxical conclusion that the significance of genetic changes as the primary cause of induction of neoplastic transformation is being overestimated. Although studying the functions of individual genes and search for new tumor markers and therapeutical targets are still beneficial, I believe that the traditional perception of tumor formation as a function/result of mutation accumulation and selection is becoming a serious drawback in further investigations. These conclusions are further discussed in the last section of the presented Ph.D. thesis.