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Analysis of single-cell genomic data of Saccinobaculus sp.
Gajdošová, Petra ; Treitli, Sebastian Cristian (advisor) ; Kolísko, Martin (referee)
The progress of single-cell genomics and metagenomics techniques allows us to explore the uncultured organisms in greater depth. In this work, we focused on the genome assembly and genetic code of the oxymonad Sacinobaculus ambloaxostylus, which inhabits the hindgut of wood-eating cockroaches from the genus Cryptocercus. Using single cell picking and whole genome amplification we obtained sequencing data from three cells of S. ambloaxostylus. The obtained data was used to generate a single-cell genome assembly using SPAdes. The assembly was further binned and decontaminated to remove any potential bacterial or eukaryotic contaminants. After decontamination and re-assembly, we obtained a genome assembly with a total length of ∼417Mbp distributed to ∼185 thousand scaffolds. Despite its low completeness of 13.71%, we attempted to determine the genetic code used by S. ambloaxostylus. For this we manually modeled 33 genes from various metabolic pathways. Aligning these gene models with homologs from closely related oxymonads, we calculated the usage of stop codons as sense codons with focus on conserved positions. Our results suggest that the stop codons UAA and UAG act as sense codons and most probably they encode glutamine. Our 18S rRNA phylogeny was unable to answer the question if the ciliate...
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Morphological, ultrastructural, and molecular characterization of novel isolates of Anaeramoeba (Metamonada: Anaeramoebae)
Pavlátová, Magdaléna ; Čepička, Ivan (advisor) ; Kolísko, Martin (referee)
Anaeramoebae is a phylum of anaerobic marine amoebae and amoeboflagellates that belongs to the Metamonada supergroup. To date, this phylum has been represented by only six free- living species belonging to a single genus, Anaeramoeba. In this study five isolates with amoeboid stage of the genus Anaeramoeba were morphologically characterized, one on which also contained a flagellate stage. The ultrastructure of three isolates was investigated. Prokaryotes are in symbiotic relationships with all known members of the genus Anaeramoeba and our results show that these symbionts occur in three different arrangements within host cells. Furthermore, the distance between the nucleus and the acentriolar centrosome was studied and found to vary between species. The 18S rRNA gene sequences were obtained from three isolates and were included with previously sequenced strains in a phylogenetic analysis. In the remaining two isolates 18S rRNA gene sequencing was unsuccessful, but morphological and ultrastructural features support a relationship of these isolates to A. ignava. Phylogenetic analyse revealed that one of the isolates based on its sequence belongs to the A. flamelloides complex and two of the of the isolates represent a new species of the genus Anaeramoeba having the smallest cells of all known...
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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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Genome Assembly and Annotation of Biparental Bee Ceratina nigrolabiata
Fraňková, Tereza ; Straka, Jakub (advisor) ; Kolísko, Martin (referee)
Biparental care in Hymenoptera is a little studied behaviour. This kind of parental care was discovered and the ethological aspect described in the bee Ceratina nigrolabiata from the Czech Republic and is well understood on the ethological level. However, biparental care is not a common behaviour and the lack of genomic studies of this behaviour complicates the understanding of the origin of the biparental care and its underlying pathways on the genomic and physiological level. This master's thesis presents the genome analyses of a biparental bee Ceratina nigrolabiata. It consists of a brief summary of the known parental behaviour across insects, characteristics of the genus Ceratina, summary of the used genomic methods and presents a candidate genes for the transcriptomic study of the biparental behaviour of Ceratina nigrolabiata. Key words: biparental care, genomics, Hymenoptera, Insecta, Ceratina, small carpenter bees
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Phylogenetic Position of Genus Polymastix and Its Prokaryotic Symbionts
Kubánková, Aneta ; Hampl, Vladimír (advisor) ; Kolísko, Martin (referee)
Polymastigidae is one of the five families of oxymonads (Metamonada, Preaxostyla). It includes small flagellates inhabiting the digestive tract of insects. Recently obtained molecular phylogenetic data have questioned the monophyly of this group. The type genus of the family Polymastigidae is Polymastix. The phylogenetic position of this genus has not been studied yet, although this information could significantly help to clarify the relationships within this group. This work provides the first sequence data of this genus, which we have obtained using a single- cell genome amplification and subsequent whole genome sequencing on Illumina HiSeq X Ten (Macrogene) from three individuals of P. melolonthae inhabiting the posterior hindgut of crane fly larvae. Phylogenetic analysis based on the gene for 18S rRNA, EF-1α and HSP90 yielded several roughly equally probable hypotheses about the position of this genus within Oxymonadida. The first of them places the genus Polymastix in a close proximity to the genus Streblomastix, according to the second hypothesis, Polymastix is sister to Termitimonas travisae. A characteristic feature of this protist is ectosymbiotic bacteria attached to its surface. These were necessarily amplified and sequenced together with their hosts, which allowed us to obtain sequence...
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Diversity and host specificity of intestinal protists of cockroaches and termites (Blattodea)
Soviš, Matyáš ; Čepička, Ivan (advisor) ; Kolísko, Martin (referee)
The cockroaches (Blattodea) are morphologically and ecologically diverse group of insects living in manifold habitats. Some of them including many termites (Isoptera) feed on wood in various stages of decay. This is however difficult to metabolise for the vast majority of cockroaches and termites and this is why they harbour in their hindguts symbiotic protists belonging mostly to the excavate lineages Parabasalia and Preaxostyla. In this thesis I sum up the knowledge on the diversity of organisms living in the hindgut of cockroaches and termites and on the host specificity of the most important groups of symbiotic protists. Keywords: Blattodea, Isoptera, hindgut, host specificity, diversity, protist, Parabasalia, Oxymonadida
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Mitochondrion-related organelles in diplomonads
Stoklasa, Michal ; Tachezy, Jan (advisor) ; Kolísko, Martin (referee)
Order Diplomonadida includes parasitic and free-living species that adapted to the oxygen-poor environment. They possess reduced form of mitochondria (hydrogenosome or mitosome). These organelles lack Krebs cycle and membrane electron-transport chain. ATP synthesis by oxidative phosphorylation and other mitochondrial metabolic pathways are modified or entirely absent. Main difference between hydrogenosome and mitosome is synthesis of hydrogen using the enzyme hydrogenase and ATP synthesis by substrate level phosphorylation in hydrogenosomes that are absent in mitosomes. The most studied diplomonads are a human pathogen Giardia intestinalis possessing the mitosomes and a salmon parasite Spironucleus salmonicida with hydrogenosomes. This thesis was focused on determining the type of mitochondrial organelles in angelfish parasite Spironucleus vortens and free living Hexamita sp. It has not been described whether they possess the hydrogenosomes or the mitosomes so far. In both protists transmission electron microscopy revealed presence of double membrane vesicles, possibly their mitochondrial organelles. Homologous S. vortens anti- hydrogenase and anti-HydE antibodies were produced and tested in order to determine their cellular localization. Using the western blot analysis and immunofluorescence...
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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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