Komplikovaná evoluce methionin adenosyltransferázy u eukaryot se zvláštním zaměřením na euglenidy

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...Velká část eukaryotických genů se v evoluci nepřenášela výhradně vertikálně z rodičů na potomstvo. V této disertační práci jsme si vybrali jeden z takových genů, a to gen pro methionin adenosyltransferázu (MAT), a pokusili se podrobně zmapovat jeho evoluci. MAT je všudypřítomný esenciální enzym, který se u eukaryot nachází ve formě dvou paralogů: MAT a MATX. Oba paralogy jsou mezi eukaryoty nerovnoměrně rozšířeny a s výjimkou několika málo případů se u daného organismu vyskytuje jen jeden z nich. To ukazuje na komplikovanou evoluční historii tohoto genu, která může zahrnovat takové evoluční procesy jako genové duplikace a následné ztráty nebo horizontální genový přenos (HGT). My jsme se zaměřili zejména na výskyt obou forem tohoto genu u jedné z nejznámějších skupin bičíkovců, skupiny Euglenida. Předpokládalo se totiž, že by tato skupina mohla být kolébkou paralogu MATX, ze které se tento gen následně šířil do dalších eukaryotických linií. Podařilo se nám získat 26 nových sekvencí z 23 linií euglenidů a jedné prasinofytní řasy Pyramimonas parkeae, která představuje nejbližšího známého příbuzného euglenidího plastidu. MATX byl zjištěn pouze u fotoautotrofních euglenidů, přičemž mixotrof Rapaza viridis a P. parkeae vykazovali přítomnost pouze paralogu MAT. Oba typy paralogů byly nalezeny u dvou druhů...Department of ParasitologyKatedra parazitologieFaculty of SciencePřírodovědecká fakult

Komplikovaná evoluce methionin adenosyltransferázy u eukaryot se zvláštním zaměřením na euglenidy

Velká část eukaryotických genů se v evoluci nepřenášela výhradně vertikálně z rodičů na potomstvo. V této disertační práci jsme si vybrali jeden z takových genů, a to gen pro methionin adenosyltransferázu (MAT), a pokusili se podrobně zmapovat jeho evoluci. MAT je všudypřítomný esenciální enzym, který se u eukaryot nachází ve formě dvou paralogů: MAT a MATX. Oba paralogy jsou mezi eukaryoty nerovnoměrně rozšířeny a s výjimkou několika málo případů se u daného organismu vyskytuje jen jeden z nich. To ukazuje na komplikovanou evoluční historii tohoto genu, která může zahrnovat takové evoluční procesy jako genové duplikace a následné ztráty nebo horizontální genový přenos (HGT). My jsme se zaměřili zejména na výskyt obou forem tohoto genu u jedné z nejznámějších skupin bičíkovců, skupiny Euglenida. Předpokládalo se totiž, že by tato skupina mohla být kolébkou paralogu MATX, ze které se tento gen následně šířil do dalších eukaryotických linií. Podařilo se nám získat 26 nových sekvencí z 23 linií euglenidů a jedné prasinofytní řasy Pyramimonas parkeae, která představuje nejbližšího známého příbuzného euglenidího plastidu. MATX byl zjištěn pouze u fotoautotrofních euglenidů, přičemž mixotrof Rapaza viridis a P. parkeae vykazovali přítomnost pouze paralogu MAT. Oba typy paralogů byly nalezeny u dvou druhů...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...Katedra parazitologieDepartment of ParasitologyFaculty of SciencePřírodovědecká fakult

Evoluce proteomu plastidu euglenidů

Vznik plastidů endosymbiózou a jejich horizontální šíření je široce rozšířený evoluční jev a jedna z významných hnacích sil evoluce eukaryot. Integrace nové organely je doprovázena změnami v její struktuře, genovém obsahu, biogenezi a importu proteinů a propojením jejích metabolických drah s drahami hostitele. Studium těchto procesů v různých skupinách sekundárních řas a srovnávání mezi nimi je důležité pro porozumění obecným principům evoluce plastidů. Krásnoočka (Euglenophyta) získala své plastidy od zelených řas po poměrně dlouhém období heterotrofie. V této práci jsem se podílela na analýze nově vygenerovaných sekvenčních datasetů: transkriptomů Euglena gracilis a Euglena longa a plastidového proteomu E. gracilis determinovaného pomocí hmotnostní spektrometrie, a to s ohledem na potenciální inovace související se získáním a integrací plastidu. Ve výsledných publikacích jsme se zaměřili zvláště na složení a evoluci systému pro targeting a import jaderně kódovaných proteinů do plastidu a zjistili, že plastidy krásnooček obsahují extrémně redukovaný TIC a zcela postrádají TOC komplex. Na základě plastidového proteomu jsme identifikovali několik nových potenciálních translokáz odvozených od proteinů endomembránového systému a popsali některé dříve nepovšimnuté vlastnosti N-terminálních...Endosymbiotic gain and transfer of plastids is a widespread evolutionary phenomenon and a major driving force of eukaryotic evolution. The integration of a new organelle is accompanied by changes in its structure, gene content, molecular mechanisms for biogenesis and transport, and re-wiring of the host and organelle metabolic pathways. To understand the course and underlying mechanisms of plastid evolution, it is important to study these processes in variety of secondary algae and notice their differences and similarities. Euglenophytes gained their plastids from green eukaryotic algae after a long history of heterotrophic lifestyle. In my thesis, I participated in analyses of newly generated sequence datasets: transcriptomes of Euglena gracilis and Euglena longa and mass spectrometry-determined proteome of E. gracilis plastid with especial regard to the potential novelties associated with plastid gain and incorporation. In the resulting publications we particularly focus on plastid protein import machinery and targeting signals and report extremely reduced TIC and completely absent TOC in euglenophyte plastid. Using the proteomic dataset, we predict potential novel plastid protein translocases recruited from ER/Golgi and re-analyze plastid signal domains, characterizing previously overlooked...Katedra parazitologieDepartment of ParasitologyPřírodovědecká fakultaFaculty of Scienc

The transcriptional apparatus of Chlamydomonas chloroplasts

The transcriptional apparatus of higher plant chloroplasts is well characterised and consists of a plastid-encoded polymerase (PEP) and a nuclear encoded polymerase (NEP). PEP is dispensable to cell viability. The situation in green algal species, however, is less clear. Chloroplast genes encoding subunits of the PEP have been cloned and sequenced in the green alga Chlamydomonas reinhardtii and preliminary reverse-genetic studies suggest that PEP is essential to cell viability, which is in contrast to the situation in higher plants. To investigate this further a series of gene knockouts were constructed using the chloroplast gene rpoC2, encoding the " subunit of PEP. Results indicate that PEP is essential to C. reinhardtii cell viability. In addition, inhibitors of PEP have been used in an in vivo transcription assay to try to identify a second RNA polymerase activity in C. reinhardtii chloroplasts. In all higher plant and red algal species so far studied the PEP factor is encoded in the nuclear genome. A C. reinhardtii nuclear gene (rpoD) encoding a putative PEP factor has been cloned and partially sequenced. This is the first factor cloned from a green algal species. A transcript of ~2.9 kb was detected for the rpoD gene by northern analysis. Finally, epitope tagging technology was developed for chloroplast and bacterial gene products. The rpoC2 gene of C. reinhardtii was modified to produce a 6x-histidine tagged polypeptide and an attempt was made to purify this polypeptide from C. reinhardtii cells using IMAC. In addition, a 3x haemagglutinin (HA) epitope tag was codon optimised for use in C. reinhardtii chloroplasts and this epitope was used to tag -galactosidase in E. coli. The protein was detected in a western blot using anti-HA monoclonal antibodies. This epitope will prove useful as a tool to tag C. reinhardtii chloroplast proteins

Kinetoplastid Genomics and Beyond

This book includes a collection of eight original research articles and three reviews covering a wide range of topics in the field of kinetoplastids. In addition, readers can find a compendium of molecular biology procedures and bioinformatics tools

An exploration of phosphorylases for the synthesis of carbohydrate polymers

Phosphorylases are interesting enzymes with regard to both their role in metabolism and their use in the in vitro synthesis of carbohydrates. The disaccharide phosphorylases have attracted attention because of their strict stereo- and regiospecificity and their tractability. The polymerising phosphorylases have received less attention due to heterogeneous product formation, requiring more complex analyses. In this work three polymerising carbohydrate phosphorylases have been studied. The plant α-1,4-glucan phosphorylase PHS2 is closely related to the well characterised mammalian glycogen phosphorylase. We present the first crystal structures of the plant enzyme which reveals a unique surface binding site. PHS2 allowed the production of novel starch like surface, both in two and three dimensions, which show some of the same properties as a native starch granule. This can now be used to study starch-active enzymes on an insoluble glucan surface which is analogous to the native starch granule. The bacterial β-1,4-glucan phosphorylase CDP is involved in degradation of cellulose. In the reverse direction this enzyme allows the rapid synthesis of cellulose polymers in solution and also allows the synthesis of hemicellulose-like materials. The substrate specificity can in part be probed in the crystal structure presented here, which represents the first structure of a polymerising, inverting phosphorylase. Together these data provide the foundation for further work with this enzyme in the synthesis of plant cell wall related glycans. The third enzyme studied was the β-1,3-glucan from the unsequenced alga Euglena gracilis, which was used for the facile enzymatic synthesis of β-glucosyl glycerols. In order to identify the sequence of this enzyme we obtained de novo transcriptome sequencing data from this alga, which has revealed unexpected metabolic diversity. Aside from complex carbohydrate metabolism, there are also many surprising features, including novel enzyme architectures, antioxidants only previously noted in human parasites and complex natural product synthases

Mechanism of protein biosynthesis in mammalian mitochondria

Protein synthesis in mammalian mitochondria produces 13 proteins that are essential subunits of the oxidative phosphorylation complexes. This review provides a detailed outline of each phase of mitochondrial translation including initiation, elongation, termination, and ribosome recycling. The roles of essential proteins involved in each phase are described. All of the products of mitochondrial protein synthesis in mammals are inserted into the inner membrane. Several proteins that may help bind ribosomes to the membrane during translation are described, although much remains to be learned about this process. Mutations in mitochondrial or nuclear genes encoding components of the translation system often lead to severe deficiencies in oxidative phosphorylation, and a summary of these mutations is provided