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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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Ammonia production by colonies of mutants and aging of wrinkled colonies of Saccharomyces cerevisiae
Nedbálková, Jana ; Janderová, Blanka (advisor) ; Heidingsfeld, Olga (referee)
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 ; Mašek, Tomáš (advisor) ; Janderová, Blanka (referee)
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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The role of transport systems for potassium cations in the physiology of Saccharomyces cerevisiae yeast
Marešová, Lydie ; Sychrová, Hana (advisor) ; Pácha, Jiří (referee) ; Janderová, Blanka (referee)
[n this work, we present the characteristics of kha 1 6,. strains in the background of various multiple mutations in genes encoding alkali-metal-cation transporters. Two main phenotype manifestations of the khal deletion were growth defect on high externa\ pH and hygromycin sensitivity. The correlation between these phenotypes and the kha 1 deletion was confirmed by plasmid complementation. Fluorescence microscopy of GFP-tagged Kha 1 p showed that this antiporter is localized preferentially intracellu\arly (in contrast to the plasma-membrane Na+/H+ antiporter Nha 1 p). Based on these findings, Kha 1 p is probably not localized in plasma membrane and do es not mediate efflux of alkali metal cations from cells (as published before in RammÍrez el aJ., 1998), but is important for the regulation of intracelular cation homeostasis and optima\ pH contro l, similarly as the Nhx l p. The khal deletion phenotypes were complemented by heterologous expression of a plant antiporter AtChx 17, showing that the proteins AfChx 17 and SeKhal could have similar ťunction and that S cerevisiae khal deletion mutants could serve for heterologous expression and characterization of some plant transporters in yeast, especially those localized intracellularly. We also showed that the presence of the Tokl channel strongly infl uences...
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The role of HOG MAPK signaling pathway during osmotic stress in Saccharomyces cerevisiae
Vrbová, Michaela ; Pospíšek, Martin (advisor) ; Janderová, Blanka (referee)
THE ROLE OF HOG MAPK SIGNALING PATHWAY DURING OSMOTIC STRESS IN SACCHAROMYCES CEREVISIAE Budding yeast (Saccharomyces cerevisiae) cells utilize a conserved mitogen-activated protein kinase (MAPK) signaling cascade (the high- osmolarity glycerol or HOG pathway) during conditions of increased external osmolarity. It evokes cellular responses necessary to permit continued growth. Activation of HOG pathway with Hog1 MAP kinase results in production of glycerol to prevent dehydration and up regulation of other Hog1 dependent genes because of cell adaptation. We were trying to find difference in translation between wild-type cells and two mutants of hog1 gene before and after 0,4 M NaCl osmotic stress (2, 6, 30 min). We used deletion mutant hog1Δ and hog1-as mutant with point mutation which allows inhibition of Hog1 MAPK during presence of specific AS inhibitor. We tested AS inhibitor by plate test and have found optimal concentration of 5 μM for blocking Hog1 MAPK in hog1-as mutant. Translation profiling proves that osmotic stress decreases translation in general. Hog1Δ mutant and hog1-as AS inhibited mutant behave similarly and their translation recovers slower than the wild-type's. That confirms that HOG1 gene is important for cell recovery from the osmotic stress. Microarray analysis shows that Hog1 dependent...
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Phenotypes of Saccharomyces cerevisiae cells lacking Isw2 chromatin remodelling factor
Frýdlová, Ivana ; Hašek, Jiří (advisor) ; Janderová, Blanka (referee) ; Sychrová, Hana (referee)
8 SUMMARY nd invasive growth. We showed that this phenotype is caused by de-repression that ncentrations of pheromone. Detailed analysis however uncovered some differences birth scar, rea that is commonly prohibited for budding. Based on our microarray data and double eletion mutant analyses, we identified one gene that is responsible for this phenotype. w2Δ cells as well as rth-scar phenotype as well as CWI pathway activation. These data prove, that de-regulation of expression of the daughter cell-specific gene DSE1 is The non-essential Saccharomyces cerevisiae protein Isw2 is a subunit of the ATP- dependent chromatin remodelling complex Isw2 that regulates the structure of chromatin, and thus plays an important role in regulation of transcription. Absence of the Isw2p induces the α-mating type-specific aberrant "shmoo-like" morphology a of a-specific genes in MATα cells and production of inappropriate a-factor which in turn activates the pheromone response pathway of isw2Δ MATα cells. Our results showed isw2Δ MATα cells can express the MATa-specific genes at the level which enables them to mate with other MATα cells. Invasion of isw2Δ MATα cells depends neither on the cell surface protein Flo11, nor on the invasive growth-specific transcription factor Tec1. On the other hand it requires components of the...
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The role of HOG MAPK signaling pathway during osmotic stress in Saccharomyces cerevisiae
Vrbová, Michaela ; Pospíšek, Martin (advisor) ; Janderová, Blanka (referee)
THE ROLE OF HOG MAPK SIGNALING PATHWAY DURING OSMOTIC STRESS IN SACCHAROMYCES CEREVISIAE Budding yeast (Saccharomyces cerevisiae) cells utilize a conserved mitogen-activated protein kinase (MAPK) signaling cascade (the high- osmolarity glycerol or HOG pathway) during conditions of increased external osmolarity. It evokes cellular responses necessary to permit continued growth. Activation of HOG pathway with Hog1 MAP kinase results in production of glycerol to prevent dehydration and up regulation of other Hog1 dependent genes because of cell adaptation. We were trying to find difference in translation between wild-type cells and two mutants of hog1 gene before and after 0,4 M NaCl osmotic stress (2, 6, 30 min). We used deletion mutant hog1Δ and hog1-as mutant with point mutation which allows inhibition of Hog1 MAPK during presence of specific AS inhibitor. We tested AS inhibitor by plate test and have found optimal concentration of 5 μM for blocking Hog1 MAPK in hog1-as mutant. Translation profiling proves that osmotic stress decreases translation in general. Hog1Δ mutant and hog1-as AS inhibited mutant behave similarly and their translation recovers slower than the wild-type's. That confirms that HOG1 gene is important for cell recovery from the osmotic stress. Microarray analysis shows that Hog1 dependent...
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