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The complicated evolution of methionine adenosyltransferase in euglenids and eukaryotes in general
Szabová, Jana ; Hampl, Vladimír (advisor) ; Krajčovič, Juraj (referee) ; Sanchez-Perez, Gabino (referee)
Many eukaryotic genes do not follow vertical inheritance pattern. In the present work, we have chosen as a model the gene for methionine adenosyltransferase (MAT), in which we have decided to examine in detail the evolutionary history. MAT is a ubiquitous essential enzyme that, in eukaryotes, occurs in two relatively divergent paralogs: MAT and MATX. Both paralogs have punctate distributions across the tree of eukaryotes and, except for a few cases, they are mutually exclusive. This points to the complicated evolutionary history of this gene couple, which may be caused by either differential loss of old paralogs or the spread of one of these paralogs by horizontal gene transfer (HGT). We have focused on the evolution of this enzyme particularly within one of the best-known groups of flagellates, the euglenids, because it was hypothesized that MATX evolved in photosynthetic euglenids before it spread to other lineages. We gained 26 new sequences from 23 euglenid lineages and one prasinophyte alga Pyramimonas parkeae. MATX was found only in photoautotrophic euglenids. Both, mixotroph Rapaza viridis and the prasinophyte alga Pyramimonas parkeae, the closest known relative of the euglenid plastid ancestor, only displayed the MAT paralog. In contrast, both paralogues were found in two euglenid species...
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The use of environmental sequencing in the studies on eukaryotic diversity
Lukešová, Soňa ; Hampl, Vladimír (advisor) ; Škaloud, Pavel (referee)
The bachelor's thesis focuses on environmental sequencing method and its usage in examining the diversity of microbial eukaryotic organisms. It describes the method, its mechanism, application and the problems associated with it. The work describes the current view of the phylogeny of the major eukaryotic lineages with their closer characteristics. Particular attention is paid to newly discovered groups. The work addresses the relationships inside these groups, summarizes findings of their ecology, food resources, and morphology. A separate chapter is devoted to the occurrence of the protist groups in extreme environmental conditions. Key words: Diversity of eukaryotes, environmental DNA, sequencing, rRNA, phylogenetics.
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Morphological and phylogenetic characterization of tetratrichomonads of group A
Krížová, Kateřina ; Čepička, Ivan (advisor) ; Hampl, Vladimír (referee)
Trichomonads is a group of anaerobic single-celled eukaryotes currently classified in the kingdom Excavata (phylum Parabasalia), although does not share any of typically excavate features (the characteristic arrangement of the cytoskeleton and the ventral feeding groove). The taxonomy is based only on the molecular-phylogenetic analyses. Genus Tetratrichomonas contains probably the highest number of known species within the phylum Parabasalia, parasites and commensals of numerous vertebrate and invertebrate hosts. According to phylogenetic methods the genus has been divided into group A and B. This thesis is focused on the group A, which is split into 12 lineages (lineage 1 - 10, novel lineage 2, lineage LP). During our survey we discovered a new lineage - called sheep lineage, related to this group. Its position in group A is still unclear. Morphological differences between lineages have more or less confirmed the position of other lineages in phylogeny based taxonomy tree, which has been published previously. In this work, the differences between lineages have been described detailly. It is obvious that almost all lineages or groups of lineages are distinguishable from each other on the basis of morphological characters (size and shape of the body, the number of anterior flagella, parabasal body size,...
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Search for the remnant of plastid in the cell of Rhabdomonas sp.
Soukal, Petr ; Hampl, Vladimír (advisor) ; Oborník, Miroslav (referee)
Phylum Euglenozoa contsists of four groups - Euglenoidea, Kinetoplastea, Symbiontida and Diplonemea. Phototrophic euglenids, which constitute a clade, possess green plastid acquired via secondary endosymbiosis from green alga related to recent genus Pyramimonas. According to generally accepted plastid-late hypothesis, the endo- symbiosis took place after split between phototrophic euglenids and all other euglenozo- ans. Endosymbiotic event is always associated with gene transfer from endosymbiont to nuclei of host. Even if the endosymbiont is completely lost we should be able to observe enrichment of the host genome with the genes derived from endosymbiont. Some recent phylogenetic analyses uncovered genes related to green algae in trypanosomas (Kineto- plastea: Trypanosomatida). Based on this observation, authors postulated a hypothesis that the plastid was present already in common ancestor of kinetoplastids and euglenids and was lost in kinetoplastids and some euglenids including osmotrophic Rhabdomonas costata. During analysis of transcriptome of R. costata we found 63 genes, which could originated from green (24 genes) or other (49 genes) algae. In phylogenetic trees only one was robustly related to green and four were robustly related to other algae. Since the number of genes related to...
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Mitochondrion of Trimastix pyriformis
Novák, Lukáš ; Hampl, Vladimír (advisor) ; Kolísko, Martin (referee)
2013 DIPLOMOVÁ PRÁCE Lukáš Novák Abstract Free-living microaerophilic protist Trimastix pyriformis is closely related to oxymonads which are the largest eukaryotic group without any known mitochondrion. In contrast to oxymonads, an enigmatic reduced mitochondrion has been found in the cell of T. pyriformis. In EST data of T. pyriformis, a number of genes has been identified whose products are putatively localized in the mitochondrion. Among these are genes for all the components of the glycine cleavage system, [FeFe]hydrogenases and the mitochondrial marker Cpn60. We performed experiments in order to determine the cellular localization of these proteins. Our results show that the glycine cleavage system is localized in the mitochondrion. Results of the experiments carried out in order to localize two hydrogenases suggest also the mitochondrial localization but are not fully convincing. The attempt to localize Cpn60 has failed. We have also identified a set of new genes in transcripts of T. pyriformis and Monocercomonoides sp. (Oxymonadida). These genes code for some components of the SUF system of FeS cluster synthesis and a peroxidase rubrerythrin. Key words: Trimastix, Monocercomonoides, mitochondrion, hydrogenosome, mitosome, hydrogenase, glycine cleavage system, SUF system.
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Transfer of genetic information between parasite and its host
Soukal, Petr ; Hampl, Vladimír (advisor) ; Votýpka, Jan (referee)
Horizontal gene transfer (HGT) is considered a rare evolutionary event. It can take place between unrelated organisms that coexist in an intimate symbiotic relationship. Such relationship have some parasites with its host. HGT between eukaryotic parasite and its host was identified in some holoparazitic and hemiparazitic plants, the most important human protozoan parasites, microsporidias, human blood-flukes, parasitoids and fruit flies.
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Mitochondrion of oxymonads
Vacek, Vojtěch ; Hampl, Vladimír (advisor) ; Eliáš, Marek (referee)
Oxymonads are among the last larger eukaryotic groups in which mitochondrion-like organelles were not detected. Among almost one million transcriptome sequences of oxymonad Monocercomonoides we found sequences for only three proteins commonly associated with mitochondrion-like organelles - pyruvate:ferredoxin oxidoreductase (PFO), [FeFe]hydrogenase and pyridine:nucleotide transhydrogenase (PNT). Out of these tree proteins PNT is most closely associated with mitochondrion-like organelles. Although the transcriptome data set is quite large we did not detect any other mitochondrial proteins including proteins for synthesis of FeS clusters and proteins associated with mitochondrial protein import. In the phylogenetic tree, PNT from Monocercomonoides formed a clade with sequences of other eukaryotes. This suggests that PNT of Monocercomonoides is likely of mitochondrial origin; however, the possibility that Monocercomonoides acquired PNT by lateral gene transfer cannot be excluded. We tried to localize PNT in the cell of Monocercomonoides by immunofluorescent microscopy but the results were difficult to interpret. We could not prove the presence of mitochondrion-like organelle in Monocercomonoides. If there is any such organelle, it is highly probable that this organelle is very reduced because we did not find...
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Application of molecular and cellular biology methods in research of protozoa Eimeria
Vrba, Vladimír ; Škvor, Jiří (advisor) ; Čepička, Ivan (referee) ; Hampl, Vladimír (referee)
Eimeria is an apicomplexan parasite causing disease coccidiosis that is most prominent in poultry farming industry. This thesis is aimed to develop new molecular tools and resolve issues that would be a valuable contribution in the field from both research and industry perspective. Because immunity to Eimeria is strictly species- specific, it is important to know and recognize correctly all species that parasitize the host. Traditional diagnostic approaches rely on classical methods such as oocyst morphology determination under the microscope, measurement of prepatent period or in-vivo assessment of lesions caused by this parasite. However, diagnostics of individual species using these methods is very time-consuming and it is often unreliable, especially when mixture of multiple species whose parameters overlap is analyzed. Methods utilizing conventional PCR to distinguish species already exist, however, they lack advantages offered by quantitative real-time PCR (qPCR). The first aim of this thesis was to develop qPCR assays for detection and quantification of seven Eimeria species which infect chicken utilizing single-copy non-polymorphic targets in order to ensure maximal specifity and coverage of all strains of each species. Usefulness of this method was demonstrated by analysis of field...
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Evolution of the genetic code and classification of oxymonads
Šrámová, Eliška ; Hampl, Vladimír (advisor) ; Čepička, Ivan (referee)
Oxymonads are a group of heterotrophic flagellates living in low oxygen environment. These protists inhabit mainly the gut of xylophagous insects (cockroaches, termites), with an exception of the genus Monocercomonoides, which was described from the intestinal contents of many vertebrates. On the basis of molecular data, Oxymonadida are classified into the supergroup Excavata (Cavalier-Smith, 2002; Simpson et al., 2006, Hampl et al. 2009). This thesis was focused on the diversity of genus Monocecomonoides from the morphologically simplest family Polymastigidae. The main goal of our work was to gather sequence data from strains isolated from a wide spectrum of hosts. We have obtained 26 partial sequences of the gene for the SSU rDNA in total, of which two belonged to another oxymonad, apparently genus Oxymonas. Our phylogenetic analysis indicated that the representatives of the genus Monocercomonoides form one group, however with a low bootstrap support. On the basis of published data about the presence of non-canonical genetic code in some oxymonads (Keeling and Leander, 2003; de Koning et al., 2008), we decided to explore this rare phenomenon in representatives of the genus Monocercomonoides. For this part of the study we gathered 9 partial sequences of α-tubulin gene. In these sequences we have not...
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Protein import into mitochondria and peroxisomes of parasitic protists
Žárský, Vojtěch ; Tachezy, Jan (advisor) ; Hampl, Vladimír (referee)
The presented thesis includes three related projects, that are linked by a common interest in the evolution of eukaryotic organelles and machineries that import proteins into these compartments. The first project considers the possibility of peroxisomes (eukaryotic organelles known in aerobic organisms) being conserved in two related anaerobic protists: a free-living amoeba Mastigamoeba balamuthi and a parasite Entamoeba histolytica. The most important hint for the presence of peroxisomes was the discovery of proteins that are homologous to known components of the peroxisomal protein import machinery. The second project aims to characterize the unknown protein translocase of the inner membrane (TIM) in the mitosomes (extremely reduced mitochondria) of an anaerobic protozoan Giardia intestinalis. We have discovered an important subunit of the mitosomal translocase (Tim44), which usually tethers the Hsp70/PAM (presequence translocase-associated motor) complex to the TIM translocon. The last project shows that the protein translocase of the outer mitochondrial membrane in trypanosomatids is related to a typical eukaryotic channel Tom40. This finding is important because the absence of Tom40 was previously considered an ancestral feature of trypanosomatids.
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