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28 • "PROTIST—2016
mologues were probably replaced by eukaryotic specific homologue or in the secondarily amito-chondriate groups where it is not necessary anymore. In the other hand, recA genes were subject to further duplication events in green plants, where they present several forms. The reconstruction of the RecA phylogeny with its EGT events retells the very evolutionary history of the euka-ryotes and also bacteria and enables a further understanding of endosymbiosis. We showed a group-wide presence of mitochondrial recA genes in Amoebozoa, Oomycetes, green plants and minor groups giving support to an ancestral EGT acquisition ofmitochondrial recA prior to eukaryotic diversification, as well as chloroplastic recA in all major photosynthesizing groups.
UNVEILING SSU RDNA INTRAGENOMIC POLYMORPHISM IN DEEP-SEA FORAMI-NIFERA USING SINGLE-CELL HIGH THROUGHPUT SEQUENCING Holzmann Maria1, Apothelot-Perret-Gentil Laure1, Voltsky Ivan12, Lejzerowicz Franck1
1 - University of Geneva
2 - Ben Gurion University of the Negev [email protected]
The SSU rRNA genes are commonly used for the assessment of protist diversity. It is assumed that the rDNA copies are identical within the individual cells. However, previous studies, using cloning and Sanger sequencing approach showed a high level of intra-individual polymorphism in shallow water benthic foraminifera. Here, we present the results of single cell high-throughput analysis of SSU rDNA in about 200 specimens of abyssal foraminifera. Our data confirm the presence of intragenomic polymorphism in foraminifera but levels of intra-individual divergence are different among taxono-mic groups. Our approach further allows us a quick sorting of foraminifera for barcoding purposes by distinguishing unknown foraminiferal sequences from those that are already present in our database. In the case of unknown sequences the longer SSU fragment is Sanger sequenced afterwards to phylogenetically characterize the putative new species.
ANCIENT MITOCHONDRIAL PROTEIN SECRETION
Horvathova L.1, Zarsky V.1, Derrelle R.2, Krupickova A.1, Klapst'ova V.1, Voleman L.1, Petru M.1, Elias M.3, Panek T.3, Cepicka I.4, Huysmans G.5, Chami M.6, Francetic O.7, Dolezal P.1 1 - Dept. of Parasitology, Charles University in Prague
2 - Dept. de Biologie, Université Paris-Sud 11
3 - Dept. ofBiology and Ecology, University ofOstrava
4 - Dept. of Zoology, Charles University in Prague
5 - Weill Cornell Medical College, Cornell University
6 - Biozentrum der Universität Basel
7 - Institut Pasteur [email protected].
The bacterial origin of mitochondria has been evidenced by a number of shared features with current bacteria, including some of the protein transport components. However, most of the original bacterial protein transport pathways have been lost from the mitochondria and replaced by the protein import apparatus. To some detail, mitochondria of Discoba represent an evolutionary intermediate stage as they carry the largest mitochondrial genomes encoding bacterial SecY and TAT trans-locases. By a multi-phylome approach we have analyzed eukaryotic proteomes for nuclear encoded genes, which are exclusive to Discoba. We show that their nuclei encode for about forty genes not found in other eukaryotic lineages. These include eight components of bacterial type II secretion system (T2SS). We show that mitochondria of Discoba express minimalist T2SS, which includes the pore forming secretin in the outer mitochondrial membrane and pseudopilin in the intermembrane space. Using the bacterial and yeast two hybrid assays, we are currently looking for the putative substrate of the ancient mitochondrial protein secretion pathway.
OCHER-COLORED GRANULES IN THE CILIATE CYCLOTRICHIUM SP. ARE EVIDENTLY DIATOM CHLOROPLASTS Hoshina Ryo1, Suzaki Toshinobu2, Kusuoka Yasushi3
1 - Nagahama Institute of Bioscience and Technology
2 - Kobe University
3 - Lake Biwa Museum [email protected]
As a crucial part of our exploration ofthe diversity of animal protists associated with algal endosymbionts, we have examined the ultrastructure and molecular characteristics of a ciliate ofthe genus Cyclotrichium collected from Lake Biwa, the largest and oldest lake in Japan. The ciliates, which are nearly spherical and about 100 ^m in diameter, are filled with hundreds of 2—3 ^m ocher-colored granules that impart their color to the whole cell. The internal structure of these granules is indistinct by light microscopy, but examination by SEM shows them to be chloroplasts consisting ofthree-layered thylakoid membranes. Many of the chloroplasts seem to be at different phases of digestion in food vacuoles (from
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intact-looking to half-digested), but there are also some naked chloroplasts with three surrounding membranes in the ciliate cytoplasm. To identify the origin of these chloroplasts, we attempted to analyze PCR products obtained from isolated whole ciliate cells using various specific primers. The diatom-targeted primers yielded a probable nuclear SSU rDNA, and phylogenetic analyses based on Neighbor Joining showed that this sequence pertains to the centric diatom genus Discostella (Stephanodiscaceae). It was especially close to that of the diatom symbiont of the dinoflagellate Peridiniopsis penardii. We are now carrying out a detailed study of the ciliate and its symbiont from taxonomic and life history points of view.
EVOLUTION OF THE MITOCHONDRIAL PROTEOME, FROM THE LARGE-SCALE PATTERNS TO THE NITTY-GRITTY DETAILS Huynen M.A., Duarte I., Szklarczyk R., Elurbe D. CMBI, Radboud University Medical Centre, Nijme-gen, Netherlands [email protected]
I will review what has been inferred about the changes at the level of the proteome that accompanied the evolution of the mitochondrion from an Alphaproteobacterium, quantifying the amount of gene loss, gene replacement and gene gain. Most of the loss and replacements that separate current day mitochondria from Alphaproteobacteria took place before the radiation ofthe eukaryotes. Detailed analyses of the evolution of the mitochondrial complexes like the ribosome and oxidative phos-phorylation show that also the acquisition of new proteins occurred mainly before the radiation of the eukaryotes, supporting an early acquisition of mitochondria in eukaryotic evolution. Secondly I will analyze the accumulation ofnew supernumerary subunits and assembly factors from pre-existing protein families for one protein complex in detail: complex I. There is a spectrum of protein function conservation between the complex I representatives and their non-complex I homologs. In general, the new complex I proteins appear to have been recruited from proteins that are active in mitochondria: proteins one expects to be expressed when and where complex I is active. Within complex I and its assembly there are many cases of neo-functionalization after gene duplication, one case of sub-functionalization, and one case in which a complex I protein itself appears to have been the source of the evolution of a new protein in another complex. Complex I and its assembly can therewith be regarded as a treasure trove for pathway evolution.
CONDITIONAL EXPRESSION SYSTEM IS NOT SUITABLE FOR DEVELOPMENTAL STUDIES IN LEISHMANIA Ishemgulova A.1, Kraeva N.1, Faktorova D.2, Podesvova L.1, Lukes J.234, Yurchenko V.125
1 - Life Science Research Centre, Faculty ofScience, University ofOstrava, 71000Ostrava, Czech Republic
2 - Czech Academy of Sciences, 370 05 Ceske Budejovice (Budweis), Czech Republic
3 - Faculty of Sciences, University ofSouth Bohemia, 370 05Cesku Budejovice (Budweis), Czech Republic
4 - Canadian Institute for Advanced Research, Toronto, ONM5G1Z8, Canada
5 - Department of Pathology, Albert Einstein College of Medicine, Bronx, NY, 10461, USA [email protected]
The genus Leishmania unites parasitic protists of the family Trypanosomatidae causing leishmaniases, several closely related diseases that affect human and animal populations mainly in the tropical and subtropical regions. The clinical manifestations vary from spontaneously healing skin lesions to progressive and potentially fatal visceral infections. Leishmaniases represent a global health problem with over 350 million people at risk and an annual incidence rate of 2—10 million worldwide. Conventional and conditional systems allow for a controlled activation or repression of gene expression in time and space. Such systems are nowadays widely used to analyse a variety of cellular processes in numerous parasites including Leishmania. A T7-driven, tetracycline-inducible system for protein expression was established in a human pathogen Leishmania mexicana. The gene expression in this strain is tightly regulated and dose- and time dependent. We believe that it can be widely used by the parasitology community to analyse effects of genes of interest on biology, physiology and virulence of parasites causing cutaneous leishmaniases. This system was used to analyse gene expression profiles during L. mexicana differentiation (procyclics, metacyclics, and amastigotes). The transcription/translation of the gene of interest was severely decreased upon Leishmania differentiation into metacyclic and amastigotes. However, the same expression profile was documented for the T7 polymerase. The expression was demonstrated to be not locus-specific but dependent on untranslated regions flanking open reading frames of studied genes. We concluded that the previously established conventional gene expression systems might have certain limitations in their common applications.
