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Iron-Sulfur cluster assembly in Monocercomonoides exilis
Vacek, Vojtěch ; Hampl, Vladimír (advisor) ; Balk, Janneke (referee) ; Tsaousis, Anastasios (referee)
In the search for the mitochondrion of oxymonads, DNA of Monocercomonoides exilis - an oxymonad isolated from the gut of Chinchilla, was isolated and its genome was sequenced. Sequencing resulted in a fairly complete genome which was extensively searched or genes for mitochondrion related proteins, but no reliable candidate for such gene was identified. Even genes for the ISC pathway, which is responsible for Fe-S cluster assembly and considered to be the only essential function of reduced mitochondrion-like organelles (MROs), were absent. Instead, we were able to detect the presence of a SUF pathway which functionally replaced the ISC pathway. Closer examination of the SUF pathway based on heterologous localisation revealed that this pathway localised in the cytosol. In silico analysis showed that SUF genes are highly conserved at the level of secondary and tertiary structure and most catalytic residues and motifs are present in their sequences. The functionality of these proteins was further indirectly confirmed by complementation experiments in Escherichia coli where SUF proteins of M. exilis were able to restore at least partially Fe-S cluster assembly of strains deficient in the SUF and ISC pathways. We also proved by bacterial adenylate cyclase two-hybrid system that SufB and SufC can form...
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Iron-Sulfur cluster assembly in Monocercomonoides exilis
Vacek, Vojtěch ; Hampl, Vladimír (advisor) ; Balk, Janneke (referee) ; Tsaousis, Anastasios (referee)
In the search for the mitochondrion of oxymonads, DNA of Monocercomonoides exilis - an oxymonad isolated from the gut of Chinchilla, was isolated and its genome was sequenced. Sequencing resulted in a fairly complete genome which was extensively searched or genes for mitochondrion related proteins, but no reliable candidate for such gene was identified. Even genes for the ISC pathway, which is responsible for Fe-S cluster assembly and considered to be the only essential function of reduced mitochondrion-like organelles (MROs), were absent. Instead, we were able to detect the presence of a SUF pathway which functionally replaced the ISC pathway. Closer examination of the SUF pathway based on heterologous localisation revealed that this pathway localised in the cytosol. In silico analysis showed that SUF genes are highly conserved at the level of secondary and tertiary structure and most catalytic residues and motifs are present in their sequences. The functionality of these proteins was further indirectly confirmed by complementation experiments in Escherichia coli where SUF proteins of M. exilis were able to restore at least partially Fe-S cluster assembly of strains deficient in the SUF and ISC pathways. We also proved by bacterial adenylate cyclase two-hybrid system that SufB and SufC can form...
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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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Genomics and cell biology of oxymonads
Treitli, Sebastian Cristian ; Hampl, Vladimír (advisor) ; Brune, Andreas (referee) ; Beneš, Vladimír (referee)
Oxymonads are a group of poorly studied protists living as intestinal endosymbionts in the gut of insects and vertebrates. In this thesis I focused on the study of phylogeny, genomics and cell biology of oxymonads. Using culture-based approaches, we uncovered the hidden diversity of small oxymonads and described one new genus and six new species. In Monocercomonoides exilis, the only oxymonad with a published genome, we investigated the genome organization using fluorescence in situ hybdridization (FISH) against the telomeric regions and single-copy genes. Our results show that the genome is most probably haploid being organized in 6-7 chromosomes. Annotation of the genome revealed that the DNA replication and repair mechanisms in M. exilis are canonical and they seem more complete than those of other metamonads whose genomes are available. Although M. exilis lacks in any traces of mitochondria, its genome annotation revealed that other cellular systems do not markedly differ from other eukaryotes. Our taxon-rich phylogenetic analyses suggested that the genus Monocercomonoides is closely related to the oxymonad Streblomastix strix, which is found exclusively in the gut of the termites. Streblomastix strix, as opposed to M. exilis, is highly adapted to harbour bacterial ectosymbionts. Since S. strix...
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Analyses of Monocercomonoides genome sizes, ploidies and karyotypes
Kornalíková, Martina ; Hampl, Vladimír (advisor) ; Krylov, Vladimír (referee)
Oxymonads are a group of flagellate protists living in low oxygen environments - mainly the guts of insects and vertebrates. In this study, we focus on the analysis of ploidy and karyotype of various species of oxymonads using Fluorescence In Situ Hybridization (FISH) with probes against single copy genes and telomeric repeats as well as estimating the DNA content in the nuclei of these oxymonads using flow cytometry. Using specific FISH probes against SufDSU gene, which is present in a single copy in the haploid genome, we showed that all studied strains are probably haploid. From the genome of Monocercomonoides exilis strain PA203 we know that oxymonads have the ancestral type of telomeric repeat (TTAGGG). Using a probe against these repeats we tried to label chromosome ends and estimate the number of chromosomes for seven strains (five species) of Monocercomonoides. With a single exception, the average number of signals per nucleus was below 20 indicating number of chromosomes below 10. In the strains of M. mercovicensis, we observed much higher number of signals suggesting that the cells have much higher number of chromosomes. Finally, we established the DNA content for several strains using flow cytometry. We used as a standard M. exilis strain PA203 knowing that the haploid genome size is...
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Losses of mitochondria and plastids in the evolution of eukaryotes
Trokšiar, David ; Hampl, Vladimír (advisor) ; Hadariová, Lucia (referee)
- 5 - Abstract: Mitochondria and plastids were acquired by endosymbiotic event, where prokaryotic organism was engulfed by ancestors of extant eukaryotes. There are more known endosymbiotic events in plastid evolution. In primary endosymbiosis cyanobacterium cell was engulfed by heterotrophic eukaryotic organism. In following secondary, tertiary and quaternary endosymbiotic events eukaryotic cell was engulfed by another eukaryote. Mitochondria originated by engulfment of α-proteobacteria. In the evolution of eukaryotes, reduction of mitochondria occurred in many lineages, making living under anaerobic conditions possible. The least reduced form is anaerobic mitochondria, which together with aerobic mitochondria and hydrogen producing mitochondria, possess genome. Hydrogenosomes and the most reduced form mitosomes, does not possess genome. Plastid reductions led to loss of photosynthetic ability. In last years, more examples of organisms that lost entirely their semi- autonomous organelle, are coming. Loss occur at two parasitic representatives of the Alveolata group, and one endobiotic oxymonad. Parasites Cryptosporidium parvum and Hematodinium lost nonphotosyntetic plastid, whereas Monocercomoides lost its mitochondria. Semi-autonomous organelles were dispensable, because all representatives have access to...
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Analysis of gene regulatory regions in the genome of oxymonad Monocercomonoides
Brzoň, Ondřej ; Hampl, Vladimír (advisor) ; Vopálenský, Václav (referee)
iv Abstract Regulation of gene expression is a key ability of every single cell in its development, differentiation and homeostasis. On the other hand, rather sparse amount of information is available for protists and our understanding of regulation of gene expression in eukaryotes is limited to a few model organisms. Our research is aimed at oxymonads, poorly studied group of anaerobic protists, which inhabit digestive tract of some animals. In this study we focus on the genus Monocercomonoides. Gene expression is modulated at multiple levels by many mechanisms. This thesis is focused on structure of promoter regions, 5' untranslated regions and basal transcription and translation initiation factors. Our results are compared to the closest studied relatives of Monocercomonoides - Trichomonas vaginalis and Giardia intestinalis. We have identified several conserved motifs in promoter regions of Monocercomonoides, including TATA box and TATA-like motif. These motifs potentially play a role in the transcription regulation. 5' untranslated regions are relatively short (typically 20 - 30 nucleotides) and GC content in these regions is low compared to model organisms. In selected genes, the quality of the automatic prediction of UTR was verified by RACE. We have annotated sets of basic transcription (23 proteins)...
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