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Heat-induced stress granules of Saccharomyces cerevisiae.
Groušl, Tomáš ; Hašek, Jiří (advisor) ; Janderová, Blanka (referee) ; Sychrová, Hana (referee)
(English) In response to environmental stresses, cells try to adapt to changed living conditions. Regulation of translation process provides fast-responding and versatile system enabling execution of stress-induced expression program. Messenger ribonucleoprotein complexes (mRNPs) engaged in translation and mRNA turnover are remodelled and may accumulate into higher-order assemblies, in connection to stress-induced translational changes. Stress granules (SGs) and processing bodies (PBs) are examples of such assemblies. Through them, further fate of mRNA molecules and certain translation machinery components is determined. In an effort to better understand the entire role of SGs in cellular metabolism, we performed the analysis of heat-induced SGs in model organism Saccharomyces cerevisiae. We contributed to the finding that SGs phenomenon is evolutionary conserved in eukaryotic kingdom proving that SGs are formed also in unicellular yeast S. cerevisiae under robust heat stress. The SGs reassemble their counterparts from higher eukaryotes in core composition and proposed functions. However, they possess also unique nature, which seems to be specific to the yeast. We further extended the data about heat-induced SGs, with a focus on additional composition, dynamics, associated proteins and a relation...
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Biochemical and molecular basis of specific properties of non-conventional osmotolerant yeast
Dušková, Michala ; Sychrová, Hana (advisor) ; Malcová, Ivana (referee) ; Janderová, Blanka (referee)
Unicellular organisms such as yeasts are permanently exposed to environmental changes, especially to changes of the concentration of osmotically active substances. Yeast species, which are able to cope with these changes properly, are called osmotolerant (Zygosaccharomyces rouxii, Pichia sorbitophila, Debaryomyces hansenii, etc.). The osmotolerance of yeasts depends on many physiological parameters but the most important is the efficient metabolism of internal osmolytes. This role is played by the small molecule of glycerol in most yeast species. Yeasts have to release the surplus of glycerol during the hypoosmotic shock. On the other hand, the hyperosmotic conditions require synthesis and accumulation of this small compound in a high quantity. The model yeast Saccharomyces cerevisiae has two systems transporting glycerol. First of them is the ScFps1 channel which is important mainly for releasing of glycerol during hypoosmotic shock. The second transporter, called ScStl1, is employed in accumulation of glycerol in case of hyperosmotic conditions. Detailed study of physiological properties of the three above mentioned species was performed within this work. Although they do not differ in basic morphological parameters from S. cerevisiae, their difference lies in the ability to survive desiccation,...
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Role of translational elongation factors in dynamics of stress granules.
Hlaváček, Adam ; Hašek, Jiří (advisor) ; Janderová, Blanka (referee)
eIF5A seems to be involved in both, translation initiation and elongation. It was also reported to affect assembly of P-bodies. Given similarities of P-bodies with stress granules (SGs) we decided to test the role of eIF5A in dynamics of heat-induced SGs and its implications for the cell recovery. For the evaluation of eIF5A function in SGs formation was used the temperature- sensitive (ts) mutant eIF5A-3 (C39Y/G118D) cultivated under permissive temperature 25řC and Rpg1-GFP fusion protein as a marker of SGs. The cells were exposed to robust heat shock at 46řC for 10 minutes. The ability of the mutant cells to recover was tested by propidium iodine staining and colony forming units plating. We found that the eIF5A-3 mutant forms heat-induced SGs more loosely aggregated, indicating that the fully functional eIF5A is necessary for SGs assembly. However, it does not seem to affect the rate of SGs dissolution. Survival tests indicate that eIF5A-3 mutant cells are susceptible to dying in a similar way as WT cells; nevertheless, their ability to resume proliferation is significantly better. We also observed a loss of the ts phenotype of the eIF5A-3 mutant. This loss cannot be explained by reversion of mutated eIF5A sequence into normal. Probable cause lies in the adaptive evolution. Our results indicate role of...
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Function and localization of the SUN family of proteins in yeast populations
Kuznetsov, Evgeny ; Palková, Zdena (advisor) ; Janderová, Blanka (referee) ; Malcová, Ivana (referee)
The SUN family of proteins (Uth1p, Sun4p, Sim1p, Nca3p) is a group of fungal proteins similar to cell wall glucanases and highly homologous in their 256 long C- terminal amino acid domain. Our previous studies on yeast colony development revealed that members of the SUN family of proteins may be involved in the aging process and may play important role for survival during the development and grow of multicellular yeast populations. Our lab implemented a microarray analysis of expression changes in Saccharomyces cerevisiae colonies, which showed significant changes in the expression level of the SUN family member - UTH1. In addition, a strain with a disrupted UTH1 gene displayed a poorer grow and rate of survival in yeast colonies in comparison to the wild type. However, in this work, we focused on identifying and better understanding the functions of particular SUN proteins and determitation of their exact localization. Interestingly, some SUN family proteins have dual localization (Uth1p, Sun4p) to the mitochondria and cell wall and may thus be involved in mitochondrial and cell wall function. In this thesis, the "Results and discussion" section is divided into two parts as follows: the first part addresses questions concerning localization, oxygen-depended regulation and the possible involvement...
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The directed search of genes for secondary metabolites in streptomycetes.
Bakal, Tomáš ; Najmanová, Lucie (advisor) ; Janderová, Blanka (referee)
Discoveries of new natural antibiotics are now relatively rare, therefore the construction of strains producing hybrid substances seems to be a very promising opportunity to gain new interesting biologically active compounds. This work is part of a larger project focused on the preparation of new biologically active substances derived from the antibiotic lincomycin. Lincomycin is composed of saccharide (MTL) and amino acid (propylhygric acid) moieties condensed by amide bond. Various modifications of amino acid moiety, especially of the side alkyl chain, are known to improve the antibiotic properties of final molecule. The bottleneck of biosynthesis of such modified compounds is the condensing enzyme NDL-synthetase, and especially its A-domain, which, similarly to nonribosomal peptide synthetases (NRPS), specifically recognizes and activates the amino acid precursor. In this work a set of degenerate primers for PCR searching of NRPS A-domains was proposed and the conditions of PCR reaction were optimized. In the first step a collection approximately 800 isolates of soil actinomycetes will serve as a source of genetic information for search of interesting NRPS A-domains, applicable for the construction of hybrid biosynthetic clusters. The isolates of this collection have been also characterized taxonomically...
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Preparation and characterization of LmbX protein involved in lincomycin biosynthesis
Jiráčková, Petra ; Janata, Jiří (advisor) ; Janderová, Blanka (referee)
Lincomycin is an antibiotic used in clinical praxis. It is produced by Streptomyces lincolnensis. Lincomycin is composed of an amino-sugar and an amino-acid moiety linked by an amide bond. The amino-acid precursor is propylproline (PPL), whose biosynthesis undergoes the pathway derived from tyrosine. The modified PPL biosynthesis pathway was also discovered in pyrrolobenzodiazepines (PBD) and hormaomycin. In the biosynthesis of PBD the PPL precursor is further modified by reactions catalysed by specific enzymes missing in the biosynthesis of lincomycin. The genes encoding these enzymes could be transferred to the lincomycin biosynthetic gene cluster. In this way we could get producers of hybrid antibiotics with better properties and even antimalaric effects. Six enzymes participate in PPL biosynthesis, which are encoded in the lincomycin biosynthetic gene cluster. The first two reactions of PPL biosynthesis pathway are proven, therefore, this work focuses on the third reaction that is supposed to be catalysed by protein LmbX according to literature. The proposed function of LmbX is a hydrolysis of C-C bond. However, LmbX belongs to the protein family of isomerases by sequence homology. The protein LmbX was overproduced in this work and its activity was tested in the presence of the expected...
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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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Ammonia production by colonies of mutants and aging of wrinkled colonies of Saccharomyces cerevisiae
Nedbálková, Jana ; Heidingsfeld, Olga (referee) ; Janderová, Blanka (advisor)
Production of ammonia by the colonies of mutants and aging of wrinkled colonies of Saccharomyces cerevisiae The aim of this diploma thesis is to observe the development, respectively the aging of cells in yeast colonies Saccharomyces cerevisiae. Yeast cells S. cerevisiea form multicellular organized structures on a solid substrate, i.e. colonies, which the intercellular interactions occur in. These interactions influence forming, morphology and aging of yeast colonies. This diploma thesis is focused partly on the changes in ammonia production by giant colonies of deletion mutants and partly on the aging of colonies with the wrinkled morphology. I characterized mutant strains of S. cerevisiae with the deletion in RTG1, RTG2, RTG3, FIS1, CIT2 genes. Their products play an important role in the colony development. The transcription of these genes changes during the transition from the acidic to alkali phase during developmental process of the colonies. I have found out that the ammonium production rate was in accordance with the results of the alkalization in giant colonies surroundings and mentioned mutants derived from the BY strain has been producing ammonia since the 15th day. The rate of the ammonia production by rtg3∆ strain was comparable to the parental strain. Compared to parental strain, lower...
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Yeast as a tool in development of new antivirals
Dubská, Jitka ; Janderová, Blanka (referee) ; Mašek, Tomáš (advisor)
The increase of infectious chronic diseases is the main health problem of modern civilization. These diseases, which are spread both in developing as well as industrial countries, cause more than 59% of all life ends. Statistically, the most frequent infection is hepatitis C virus (HCV) infection. Incurable hepatic illnesses such as cirrhosis and hepatocellular carcinoma are the outcomes of the HCV infection and the liver transplantation is the only successful therapeutic option for the patients in industrial countries. It is estimated that about 170 million people worldwide are chronically infected with this virus. HCV is a small coated virus with single stranded RNA genome of positive polarity. Lots of research has been done since it was discovered twenty years ago but the efficient drug is still missing. There is a strong tendency to focus the research on searching for translation initiation inhibitors in last few years, because the whole life cycle of HCV, including RNA replication, new virions assembly etc., is dependent on translation products. Nowadays there is a small group of human proteins (so called ITAFs) known for up- or down-regulate HCV and other viruses translation initiation. It is believed, that this group is much more comprehensive. The aim of this diploma work was to construct yeast...
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