Structure of the germline genome of Tetrahymena thermophila and relationship to the massively rearranged somatic genome

The germline genome of the binucleated ciliate Tetrahymena thermophila undergoes programmed chromosome breakage and massive DNA elimination to generate the somatic genome. Here, we present a complete sequence assembly of the germline genome and analyze multiple features of its structure and its relationship to the somatic genome, shedding light on the mechanisms of genome rearrangement as well as the evolutionary history of this remarkable germline/soma differentiation. Our results strengthen the notion that a complex, dynamic, and ongoing interplay between mobile DNA elements and the host genome have shaped Tetrahymena chromosome structure, locally and globally. Non-standard outcomes of rearrangement events, including the generation of short-lived somatic chromosomes and excision of DNA interrupting protein-coding regions, may represent novel forms of developmental gene regulation. We also compare Tetrahymenas germline/soma differentiation to that of other characterized ciliates, illustrating the wide diversity of adaptations that have occurred within this phylum.</p

Models of natural computation : gene assembly and membrane systems

This thesis is concerned with two research areas in natural computing: the computational nature of gene assembly and membrane computing. Gene assembly is a process occurring in unicellular organisms called ciliates. During this process genes are transformed through cut-and-paste operations. We study this process from a theoretical point of view. More specifically, we relate the theory of gene assembly to sorting by reversal, which is another well-known theory of DNA transformation. In this way we obtain a novel graph-theoretical representation that provides new insights into the nature of gene assembly. Membrane computing is a computational model inspired by the functioning of membranes in cells. Membrane systems compute in a parallel fashion by moving objects, through membranes, between compartments. We study the computational power of various classes of membrane systems, and also relate them to other well-known models of computation.Netherlands Organisation for Scientific Research (NWO), Institute for Programming research and Algorithmics (IPA)UBL - phd migration 201

Life cycle studies of the red tide dinoflagellate species complex Alexandrium tamarense

Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy at the Massachusetts Institute of Technology and the Woods Hole Oceanographic Institution February 2011Blooms of toxic species within the algal dinoflagellate species complex Alexandrium tamarense may cause Paralytic Shellfish Poisoning, a significant and growing environmental threat worldwide. However, blooms of closely related nontoxic A. tamarense also occur, sometimes in close geographical proximity to toxic blooms. This thesis explores the interactions between toxic and nontoxic blooms by examining sexual crosses between each of five ribosomal clades within the A. tamarense complex (termed Groups I-V). Several lines of evidence argue that these clades represent separate species. Particular emphasis was given to interactions between toxic Group I and nontoxic Group III species because they are among the most closely related A. tamarense clades and because they share a natural range boundary in several parts of the world. Interspecies hybridization appeared widespread between different clades and between geographically dispersed isolates. However, subsequent germination studies of hypnozygotes produced from combinations of Group I and Group III clones failed to yield new vegetative cultures in multiple trials. The possibility that these hypnozygotes were actually inbred (i.e. the result of pairs of only Group I or only Group III gametes) was considered and rejected using a nested PCR assay that was developed to assess the parentage of individual cysts. The assay was also suitable for analyzing cysts collected from the field and was applied to individual cysts collected from Belfast Lough, an area where both Group I and Group III blooms were known to occur. Two Group I/Group III hybrids were detected in fourteen successful assays from the Belfast sample, showing that hybridization does occur in nature. These findings have several important implications. First, the failure of Group I/Group III hybrids to produce new vegetative cultures serves as a proof that the A. tamarense clades represent cryptic species because they are unable to produce genetic intermediates. Second, the presence of hybrid cysts in Belfast Lough indicates ongoing displacement of a nontoxic population by a toxic one (or vice versa) in that region. Third, the inviability of toxic/nontoxic hybrids suggests a remediation strategy whereby the recurrence of toxic A. tamarense blooms might be combated through the introduction of nontoxic cells.research support from NSF (grants OCE-0402707 and OCE- 9808173 awarded to Don Anderson), the Woods Hole Center for Oceans and Human Health (NSF Grant no. OCE-0430724 and NIEHS Grant no. P50ES012742-0), an EPA STAR graduate fellowship (FP-91688601), and the Coastal Ocean Institute at WHOI. I’ve also benefitted greatly from conference travel funds provided by the NOAA Center for Sponsored Coastal Ocean Research, the MIT Student Assistance Fund, and the Academic Programs Office at WHOI

A highly condensed genome without heterochromatin : orchestration of gene expression and epigenomics in Paramecium tetraurelia

Epigenetic regulation in unicellular ciliates can be as complex as in metazoans and is well described regarding small RNA (sRNA) mediated effects. The ciliate Paramecium harbors several copies of sRNA-biogenesis related proteins involved in genome rearrangements resulting in chromatin alterations. The global chromatin organization thereby is poorly understood, and unusual characteristics of the somatic nucleus, like high polyploidy, high genome coding density, and absence of heterochromatin, ought to call for complex regulation to orchestrate gene expression. The present study characterized the nucleosomal organization required for gene regulation and proper Polymerase II activity. Histone marks reveal broad domains in gene bodies, whereas intergenic regions are nucleosome free. Low occupancy in silent genes suggests that gene inactivation does not involve nucleosome recruitment. Thus, Paramecium gene regulation counteracts the current understanding of chromatin biology. Apart from global nucleosome studies, two sRNA binding proteins (Ptiwis) classically associated with transposon silencing were investigated in the background of transgene-induced silencing. Surprisingly, both Ptiwis also load sRNAs from endogenous loci in vegetative growth, revealing a broad diversity of Ptiwi functions. Together, the studies enlighten epigenetic mechanisms that regulate gene expression in a condensed genome, with Ptiwis contributing to transcriptome and chromatin dynamics.Epigenetische Regulation kann in einzelligen Ciliaten so komplex sein wie in Vielzellern und wurde umfassend angesichts kleiner RNA (sRNA)-vermittelter Effekte untersucht. Der Ciliat Paramecium besitzt mehrere Kopien sRNA-Biogenese assoziierter Proteine, die an Genomprozessierungen und resultierenden Chromatinänderungen beteiligt sind. Die globale Organisation des Chromatins ist dabei kaum verstanden und obskure Eigenschaften des somatischen Kerns, wie hohe Polyploidie, Kodierungsdichte und Fehlen von Heterochromatin, sollten eine komplexe Regulation zur Steuerung der Genexpression erfordern. Die vorliegende Studie charakterisiert die Chromatinorganisation, die für die Genregulation und Polymerase II Aktivität notwendig ist. Histonmodifikationen zeigen breite Verteilungen in Genen, während intergenische Regionen Nukleosomen-frei sind. Ein Stilllegen von Genen scheint ohne die Rekrutierung von Nukleosomen zu erfolgen, womit die Genregulation in Paramecium dem aktuellen Verständnis der Chromatinbiologie widerspricht. Neben Nukleosomenstudien wurden zwei sRNA-bindende Proteine (Ptiwis), die klassisch mit Transposon-Silencing assoziiert sind, im Hintergrund des Transgeninduzierten Silencings untersucht. Überraschenderweise laden Ptiwis sRNAs von endogenen Loci im vegetativen Wachstum, was vielfältige Ptiwi-Funktionen offenbart. Die Studien zeigen epigenetische Mechanismen zur Genregulation in einem kompakten Genom, wobei Ptiwis zur Transkriptom- und Chromatindynamik beitragen

Analysis of the somatic and germline genomes of the ciliate Blepharisma stoltei

Ciliaten sind prototypische, üblicherweise einzellige Eukaryoten mit getrennten Keimbahn- und somatischen Zellkernen. Das somatische Genom entsteht aus dem Keimbahngenom durch einen Prozess der Transposase-vermittelten DNA-Eliminierung und Genom-Neuordnung während der sexuellen Fortpflanzung. Aktuelle Modelle für die Reorganisation des Genoms bei Wimpertierchen gehen davon aus, dass kleine RNAs während der sexuellen Fortpflanzung in den sich entwickelnden somatischen Kern transportiert werden und Transposasen dabei helfen, keimlinienspezifische Sequenzen zu identifizieren und auszuschneiden. Diese Sequenzen, die so genannten intern eliminierten Sequenzen (IES), und ihre Exzisasen werden von einer Maschinerie begleitet, die ihre Entfernung durchführt. Dazu gehören Dicer-ähnliche und Piwi/Argonaute-Proteine, die kleine RNAs erzeugen und transportieren, sowie Proteine, die das Chromatin verändern und die DNA für die Exzision zugänglich machen. Blepharisma gehört zu einer früh divergiereden Klasse von Ciliaten, die als Heterotrichea bekannt sind. Obwohl die Reorganisation des Genoms bei später divergierenden Ciliaten wie den oligohymenophoren Ciliaten Tetrahymena und Paramecium und den spirotrichen Oxytricha untersucht wurde, gibt es deutliche Unterschiede in der Art und Weise, wie sie dies tun. Die Untersuchung dieses Prozesses in einem früh divergiereden Ciliaten wie Blepharisma ist ein wichtiger Beitrag zum Verständnis, wie konserviert die verschiedenen Elemente der Genom- Reorganisationsmaschinerie unter Ciliaten sind. Diese Arbeit bietet den ersten Blick aus genomischer Sicht auf die verschiedenen Teilnehmer und mutmaßlichen Mechanismen der Genomreorganisation in Blepharisma. Mittels Long-Read-Sequenzierung und Annotationsmethoden, die auf die atypischen Genomeigenschaften von Blepharisma zugeschnitten sind, wurden annotierte Referenzgenome für die somatischen und Keimbahnkerne von Blepharisma stoltei (Stamm ATCC 30299) erstellt. Das somatische Genom von B. stoltei ist kompakt (41 Mb), gen-dicht (25710 Gene) und enthält kurze, 15-16 Nukleotide umfassende spliceosomale Introns. Wir haben Schlüsselkomponenten identifiziert, die an der Reorganisation des Genoms im somatischen Genom von Blepharisma beteiligt sind, und sie mit denen der Modell-Ciliaten Paramecium, Tetrahymena und Oxytricha verglichen. Es wurden vier Transposase-Familien gefunden, die in den somatischen und Keimbahn-Genomen kodiert sind, nämlich die PiggyBac-,Tc1/Mariner-, Mutator- und Merlin-Familien. Es ist bekannt, dass PiggyBac-Transposasen die wichtigsten Transposasen sind, die in den Modell-Ciliaten Paramecium und Tetrahymena an der Reorganisation des Genoms beteiligt sind, während sie in Oxytricha, wo vermutlich eine Transposase aus einer anderen Familie verwendet wird, gänzlich fehlen. In Paramecium koordinieren sechs somatisch kodierte PiggyBacs, die nicht zur Katalyse fähig sind, sowie ein katalytisch vollständiges Homolog, namens PiggyMac, die DNA-Exzision. Dies ähnelt der Situation in Blepharisma, wo es dreizehn Homologe der PiggyBac-Transposase gibt, von denen nur eine eine vollständige katalytische Triade besitzt und daher wahrscheinlich die primäre Exzisase ist. Die keimbahnbegrenzten genomischen Regionen von Blepharisma wurden ebenfalls charakterisiert. Die IES von Blepharisma haben zwei wesentliche Merkmale mit den IES von Paramecium gemeinsam, nämlich eine periodische Längenverteilung für kurze IES und überwiegend durch TA-Dinukleotide abgegrenzte IES-Grenzen. Wir haben auch eine Klasse von kleinen RNAs („small RNAs“ ) mit 24 Nukleotiden identifiziert, die mit fortschreitender Entwicklung in Blepharisma zunehmend den IESs zugeordnet werden. Diese Tendenzen ähneln denen, die in Paramecium und Tetrahymena beobachtet wurden, weshalb wir vorschlagen, dass es sich auch hier um so genannte "Scan"-RNAs (scnRNAs) handelt, die die IES-Exzision steuern. Die phylogenetische Analyse der PiggyBac-Homologe von Blepharisma hat gezeigt, dass sie einen gemeinsamen Ursprung mit den PiggyBac-Homologen von Paramecium und Tetrahymena haben, wobei letztere evolutionär stärker divergieren als Blepharisma und auf jüngeren Zweigen des phylogenetischen Stammbaums der Ciliaten zu finden sind. Mehrere Indizien aus diesen Studien deuten daher darauf hin, dass eine PiggyBac-Transposase höchstwahrscheinlich die wichtigste IES-Exzisase in Blepharisma ist und dass der letzte gemeinsame Vorfahre der Ciliaten ebenfalls diesen Transposasetyp besaß.Ciliates are prototypical, conventionally unicellular eukaryotes with separate germline and somatic nuclei. The somatic genome arises from the germline genome through a process of transposase-mediated DNA elimination and genome rearrangement during sexual reproduction. Current models for genome reorganization in ciliates posit that small RNAs are transported to the developing somatic nucleus during sexual reproduction, aiding transposases in identifying and excising germline-specific sequences. Accompanying these sequences, known as Internally Eliminated Sequences (IESs), and their excisases is the machinery to carry out their removal. This includes Dicer-like and Piwi/Argonaute proteins, which generate and transport small RNAs, as well as proteins that alter chromatin, and make DNA accessible for excision. The ciliate Blepharisma belongs to an early diverging class of ciliates known as the Heterotrichea. Though genome reorganization has been studied in later diverging ciliates such the oligohymenophorean ciliates Tetrahymena and Paramecium and the spirotrich Oxytricha there are pronounced differences in how they do so. Studying this process in an early diverging ciliate like Blepharisma is an important contribution to the understanding of how conserved the different elements of the genome reorganization machinery among ciliates are. This thesis provides the first look, from a genomic perspective, at the various participants and putative mechanisms of genome reorganization in Blepharisma. Annotated reference genomes for the somatic and germline nuclei of Blepharisma stoltei (strain ATCC 30299) were generated using long-read sequencing and annotation methods tailored to the atypical genome properties of Blepharisma. The B. stoltei somatic genome is compact (41 Mb), gene-dense (25710 genes) and contains short, 15-16 nucleotide spliceosomal introns. We identified key components involved in genome reorganization in the Blepharisma somatic genome and compared them with those of the model ciliates Paramecium, Tetrahymena and Oxytricha. Four transposase families were found encoded in the somatic and germline genomes, namely the PiggyBac, Tc1/Mariner, Mutator and Merlin families. PiggyBac transposases are known to be the main transposases involved in genome reorganization in the model ciliates Paramecium and Tetrahymena, but are entirely absent in Oxytricha, which is thought to use a transposase from another family. In Paramecium, six somatically encoded PiggyBacs incapable of catalysis, plus one catalytically complete homolog called the PiggyMac, coordinate DNA excision. This resembles the situation in Blepharisma, which has thirteen homologs of the PiggyBac transposase, only one of which has a complete catalytic triad and is hence likely to be the primary excisase. The germline-limited genomic regions of Blepharisma were also characterized. Blepharisma IESs share two key features with the IESs of Paramecium, namely a periodic length distribution for short IESs and predominantly TA-dinucleotide delineated IES boundaries. We also identified a class of 24-nucleotide small RNAs that increasingly map to IESs as development progresses in Blepharisma. These trends are similar to those observed in Paramecium and Tetrahymena, hence we propose that they are also so-called “scan” RNAs (scnRNAs) that guide IES excision. Phylogenetic analysis of the Blepharisma PiggyBac homologs showed that they share common ancestry with the PiggyBac homologs of Paramecium and Tetrahymena, where the latter are evolutionarily more divergent than Blepharisma and are located on more recently diverging branches of the ciliate phylogenetic tree. Several lines of evidence from these studies therefore indicate that a PiggyBac transposase is the most likely the main IES excisase in Blepharisma and that the last ciliate common ancestor also possessed this type of transposase

The resilience of alternative community states driven by priority effects: a microcosm investigation

A thesis submitted to the University of Bedfordshire, in partial fulfilment of the requirements for the degree of Master of Science by Research.Within an ecosystem, there are a variety of interactions between species which affect the overall community. One of the strongest influences of community structure within a habitat is the order in which species arrive and establish; resulting in populations either coexisting or excluding one another to extinction. This can either be invading species excluding residents (competitive exclusion) or residents excluding invaders (priority effects), often due to freely depleting any shared resources before invaders arrive. Priority effects are predicted to be weaker when the invasion occurs simultaneously with warming towards and above the thermal tolerance of one species as the pressure put on the species can be too much to allow a population to grow or establish to survive. This experiment investigated whether an 8°C temperature range altered protist ability to invade or be invaded in simple aquatic microcosms, where the order of invasion of Colpidium and Tetrahymena was varied. I measured the changes to population density of both species over time, to identify changes in maximum population density and time to extinction. Results showed very strong priority effects between the two species, but this was never affected by temperature. In all treatments, resident Tetrahymena could never be invaded by Colpidium. However, Tetrahymena can invade resident Colpidium and populations can coexist for weeks, but Colpidium always eventually exclude Tetrahymena to extinction. The only factors temperature affected were maximum population density and time to extinction in single species microcosms, with earlier extinction and lower maximum population densities at warmer temperatures. This study suggests that arrival of species into an environment is vital in determining the final habitat composition. Although temperature does not affect priority effects, it does alter the duration species may be able to survive and coexist, which could be fundamental in conservation work in a world with changing habitats and climates

Life cycle studies of the red tide dinoflagellate species complex Alexandrium tamarense

Thesis (Ph. D.)--Joint Program in Oceanography/Applied Ocean Science and Engineering (Massachusetts Institute of Technology, Dept. of Biology; and the Woods Hole Oceanographic Institution), 2011.Cataloged from PDF version of thesis.Includes bibliographical references.Blooms of toxic species within the algal dinoflagellate species complex Alexandrium tamarense may cause Paralytic Shellfish Poisoning, a significant and growing environmental threat worldwide. However, blooms of closely related nontoxic A. tamarense also occur, sometimes in close geographical proximity to toxic blooms. This thesis explores the interactions between toxic and nontoxic blooms by examining sexual crosses between each of five ribosomal clades within the A. tamarense complex (termed Groups I-V). Several lines of evidence argue that these clades represent separate species. Particular emphasis was given to interactions between toxic Group I and nontoxic Group III species because they are among the most closely related A. tamarense clades and because they share a natural range boundary in several parts of the world. Interspecies hybridization appeared widespread between different clades and between geographically dispersed isolates. However, subsequent germination studies of hypnozygotes produced from combinations of Group I and Group III clones failed to yield new vegetative cultures in multiple trials. The possibility that these hypnozygotes were actually inbred (i.e. the result of pairs of only Group I or only Group III gametes) was considered and rejected using a nested PCR assay that was developed to assess the parentage of individual cysts. The assay was also suitable for analyzing cysts collected from the field and was applied to individual cysts collected from Belfast Lough, an area where both Group I and Group III blooms were known to occur. Two Group I/Group III hybrids were detected in fourteen successful assays from the Belfast sample, showing that hybridization does occur in nature. These findings have several important implications. First, the failure of Group I/Group III hybrids to produce new vegetative cultures serves as a proof that the A. tamarense clades represent cryptic species because they are unable to produce genetic intermediates. Second, the presence of hybrid cysts in Belfast Lough indicates ongoing displacement of a nontoxic population by a toxic one (or vice versa) in that region. Third, the inviability of toxic/nontoxic hybrids suggests a remediation strategy whereby the recurrence of toxic A. tamarense blooms might be combated through the introduction of nontoxic cells. The results from these experiments also highlighted several shortfalls in our understanding of the mechanisms governing sexual compatibility between clones and also our ability to replicate these organisms' sexual cycle in the laboratory. Two initiatives were begun with the ultimate goal of better characterizing sexual processes in natural populations. The first initiative was the application of an imaging flow cytometer to detect sexual events in natural blooms. An existing instrument, the Imaging FlowCytobot, was adapted to positively identify A. tamarense Group I cells in mixed species assemblages and measure cell DNA content. A collection of four samples were analyzed, three from the development and decline of a local Group I bloom and one from a Group I red tide that occurred near Portsmouth, NH and led to a major deposition of new cysts in the southern Gulf of Maine. Several unanticipated patterns were revealed including the discovery of a persistent layer of cells with 2c DNA content near the surface and disproportionately high rates of infection by an Amoebophrya sp. parasite in large A. tamarense planozygotes. The second initiative was the application of high throughput Illumina sequencing to define the transcriptomes of toxic Group I, nontoxic Group III, and toxic Group IV clones. Additional steps were taken to isolate RNA and prepare a cDNA library from a natural sample of Group I hypnozygotes. The applications for these data are expected to be extensive and include the discovery of sexual biomarkers and further characterization of the differences between toxic and nontoxic A. tamarense species. Preliminary results from the sequencing of these libraries and their initial assembly are described.by Michael L. Brosnahan.Ph.D