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The role of Prp45p in mRNA expression and maturation
Abrhámová, Kateřina ; Půta, František (advisor) ; Pichová, Alena (referee) ; Janderová, Blanka (referee)
Prp45p of Saccharomyces cerevisiae and Snw1p of Schizosaccharomyces pombe are essential proteins, which share extensive homology with the mammalian transcription regulator and splicing factor SNW/SKIP. We have analyzed the essential function of these proteins in both yeasts and found a mutation (prp45(1-169)) that exhibited temperature sensitivity. The mutant strain harboring the corresponding chromosomal deletion shows temperature sensitive phenotype and hypersensitivity to cycloheximide, hydroxyurea, calcofluor white, and to microtubule inhibitors. At 30řC, the cells are often elongated, deformed, and larger than wt. After synchronization prp45(1-169) cells stop their growth with 2N DNA content at 37řC. We found that the temperature sensitivity is not overcome and the hypersensitivity to microtubule destabilizing drugs is only partially suppressed by the excision of intron from TUB1 gene. This distinguishes prp45(1-169) from those splicing factor's mutants that cause tubulin-dependent G2/M arrest, which can be relieved by the expression of intronless tub1. We performed analysis of splicing in vitro and found that splicing of optimal substrates is not impaired. We also compared the content and stability of RNA in wt-cells and in prp45(1-169) cells at variol temperatures using microarrays. The...
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Alakali-metal-cation homeostasis in pathogenic Candida species
Elicharová, Hana ; Sychrová, Hana (advisor) ; Heidingsfeld, Olga (referee) ; Půta, František (referee)
Several tens of Candida species belong to the opportunistic human pathogens capable of inducing life-threatening infections in immunocompromised patients. Virulence of single Candida species depends among others on their resistance to the variable external conditions. The maintenance of alkali-metal-cation homeostasis, which means the ability to accumulate sufficient amount of potassium cations and on the other hand to survive under high extracellular concentrations of alkali-metal cations, is essential for growth and virulence of Candida cells. We observed the negative effect of fluconazole (FLC) on salt-tolerance of six Candida species and found that it is independent of the species level of FLC- resistance. FLC hyperpolarizes plasma membrane of Candida cells and therefore increases non-specific uptake of alkali-metal cations which results in strongly increased salt-sensitivity of Candida cells. The FLC-induced hyperpolarization also results in an increased sensitivity of Candida cells to the antifungals which are positively charged and are driven into the cells by the membrane potential. The effect of fluconazole on membrane potential and thus on the uptake of alkali- metal cations into the cell turned our attention to the homeostasis of potassium cations whose high intracellular concentration is...
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CUG Codon in Pathogenic Yeasts of the Genus Candida
Marečková, Lucie ; Půta, František (referee) ; Heidingsfeld, Olga (advisor)
2. Abstract In many Candida species the standard leucine CUG codon is translated as a serine, although not in 100% cases. This dual specifity of the CUG codon has evolved through a mechanism that required codon ambiguity mediated by a unique tRNACAG, which is in vitro aminoacylated more often by serine than by leucine. This codon ambiguity has been tolerated for more than 170 million years. The explanation at least for now is that the CUG codon reassignment could have generated genetic diversity that facilitated occurrence of new phenotypes resistant to stress. Beside this, an important step was to reduce negative impact of the codon ambiguity by crucial mutations in the structure of the ser-tRNACAG. Candida species became a valuable experimental model for elucidation of the genetic code changes. While consequences of the CUG codon reassignment have been extensively studied in Candida albicans, this topic has not yet been addressed in Candida parapsilosis. Solving the structure of C. parapsilosis secreted proteinase Sapp1p provided a tool to carry out a "case study" of possible effects of the CUG codon ambiguity. The SAPP1 gene contains one CUG codon, and the respective serine is located on the loop in the close proximity of the active site of the proteinase.
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Preparation of rat NK cell receptors using HEK293T expression system.
Celadová, Petra ; Bezouška, Karel (advisor) ; Půta, František (referee)
Natural killer cells play a significant role in the immune response against tumor and infected cells. NK cells express a wide variety of surface receptors, including NKRP1, a C-type lectin-like family of both activating and inhibitory receptors. Recently, ligands have been found for some of these previously orphan molecules, some of them lying within the same family. This is also the case of rat Clr-b as a cognitive ligand for rat NKRP1B. It has been shown that in rat, this inhibitory NKRP1B-Clr-b mutual receptor system is subverted by rat cytomegalovirus protein RCTL, a viral version of Clr-b, which serves as a decoy ligand for NK cells. The aim of my diploma thesis was cloning and production of the above mentioned C-type lectin-like proteins based on transient transfection of HEK293T cell line in a suspension culture. This expression system allows us not only to obtain proteins of our interest with a satisfactory yield but also in their native conformation, removing the need for time consuming and often fruitless refolding procedures required in case of using the E. coli expression system. Success was achieved in case of Clr-b and NKRP1B receptors from both WAG and SD strains. Proteins were purified using IMAC followed by gel filtration, identified by mass spectrometry and characterized by disulfide...
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