12 research outputs found

    Ciliate Gene Unscrambling with Fewer Templates

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    One of the theoretical models proposed for the mechanism of gene unscrambling in some species of ciliates is the template-guided recombination (TGR) system by Prescott, Ehrenfeucht and Rozenberg which has been generalized by Daley and McQuillan from a formal language theory perspective. In this paper, we propose a refinement of this model that generates regular languages using the iterated TGR system with a finite initial language and a finite set of templates, using fewer templates and a smaller alphabet compared to that of the Daley-McQuillan model. To achieve Turing completeness using only finite components, i.e., a finite initial language and a finite set of templates, we also propose an extension of the contextual template-guided recombination system (CTGR system) by Daley and McQuillan, by adding an extra control called permitting contexts on the usage of templates.Comment: In Proceedings DCFS 2010, arXiv:1008.127

    Analysis of DIE5 and LIA5 reveals the importance of DNA repair in programmed DNA rearrangement of Tetrahymena thermophila

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    During its somatic nuclear differentiation, the single cell eukaryote Tetrahymena thermophila undergoes genome-wide programmed DNA rearrangement to eliminate transposon-like elements from its future soma. This process involves small RNA-directed heterochromatin formation followed by extensive nuclear reorganization to form subnuclear domains. While more has been known about small RNAs and heterochromatin, the mechanisms and players involved in the process of nuclear reorganization and the subsequent removal of transposon-like elements from the somatic genome are just starting to unravel. My thesis work centers on the study of two novel nuclear proteins Die5p: Chapter 2) and Lia5p: Chapter 3) and their roles in DNA rearrangement. These essential proteins function downstream of small RNA targeted heterochromatin establishment. While Lia5p is essential for nuclear reorganization to form distinct subnuclear structures, Die5p is a protein conserved across ciliate species and appears to be important for the integrity of the differentiating genome. Maintaining genome integrity during somatic nuclear differentiation has proven to be an active process. Similar to V(D)J recombination during mammalian B and T cell maturation, programmed DNA rearrangement in Tetrahymena induces global DNA damage that requires proper response and repair. Through the study of LIA5 and DIE5, we show that nuclear reorganization during Tetrahymena DNA rearrangement is intimately associated with the response to DNA damage. Furthermore, we implicate a chromodomain protein Pdd1 as a component of the DNA damage response system, thus providing evidence to support the link between heterochromatin and DNA repair during the reprogramming of Tetrahymena somatic genome

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

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    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 ChromatinĂ€nderungen 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 fĂŒr die Genregulation und Polymerase II AktivitĂ€t notwendig ist. Histonmodifikationen zeigen breite Verteilungen in Genen, wĂ€hrend intergenische Regionen Nukleosomen-frei sind. Ein Stilllegen von Genen scheint ohne die Rekrutierung von Nukleosomen zu erfolgen, womit die Genregulation in Paramecium dem aktuellen VerstĂ€ndnis 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 vielfĂ€ltige Ptiwi-Funktionen offenbart. Die Studien zeigen epigenetische Mechanismen zur Genregulation in einem kompakten Genom, wobei Ptiwis zur Transkriptom- und Chromatindynamik beitragen

    The Role of dsRNA in Nuclear Differentiation and Remodeling in the Ciliate, Tetrahymena thermophila

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    The ciliate, Tetrahymena thermophila, like a handful of other eukaryotes, engages in massive genome reorganization known collectively as chromatin diminution. Part of this process involves large-scale DNA excision known as DNA elimination. Recent data has shown DNA elimination to be dependent on RNA interference: RNAi). Using T. thermophila, I have sought to determine the role of non-coding RNA: ncRNA) in RNAi-dependent DNA elimination through studies of DNA sequences that are to be eliminated called internal eliminated sequences: IESs) and through a conjugation-specific Dicer protein and its putative tandem dsRNA-binding motif: DSRM) protein partners. Studies of the R IES revealed the requirement of IES DNA for production of long, bidirectional ncRNA early in conjugation. This ncRNA is essential for IES excision in zygotic nuclei later in conjugation. The conjugation-specific Dicer homologue, DCL1, was shown to be required for production of a species of sRNA called scnRNAs from the long, bidirectional ncRNA from IESs. Knockouts of DCL1 displayed a loss of these scnRNAs as well as an increase in the long, bidirectional ncRNA precursors. A deficiency in these scnRNAs was sufficient to block modification of chromatin associated with IESs and prevent their rearrangement later in conjugation. Failure of DNA elimination caused DCL1 knockout cells to arrest before completion of conjugation. Further studies of the tandem DSRM-containing proteins, DRB2 and DRB1, revealed that neither are solely partners for DCL1 or any other Dicer protein but play other important roles during conjugation. Zygotic expression of DRB2 was shown to be essential for DNA elimination and completion of conjugation. Interaction with the chromo-domain containing protein, Pdd1p, by Drb2p implicates ncRNA or sRNA in later stages of conjugation after scnRNA production. Knockouts of the tandem DSRM-containing DRB1 caused higher numbers of cells to abort conjugation and therefore produce fewer progeny. Localization of this protein to the crescent micronucleus during prophase of meiosis I links DRB1 to a probable role in ensuring proper recombination during meiosis for haploid gamete production. All these studies suggest that ncRNA has many roles in conjugation-specific processes including RNAi-directed DNA elimination

    Chromosome Descrambling Order Analysis in ciliates

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    Ciliates are a type of unicellular eukaryotic organism that has two types of nuclei within each cell; one is called the macronucleus (MAC) and the other is known as the micronucleus (MIC). During mating, ciliates exchange their MIC, destroy their own MAC, and create a new MAC from the genetic material of their new MIC. The process of developing a new MAC from the exchanged new MIC is known as gene assembly in ciliates, and it consists of a massive amount of DNA excision from the micronucleus, and the rearrangement of the rest of the DNA sequences. During the gene assembly process, the DNA segments that get eliminated are known as internal eliminated segments (IESs), and the remaining DNA segments that are rearranged in an order that is correct for creating proteins, are called macronuclear destined segments (MDSs). A topic of interest is to predict the correct order to descramble a gene or chromosomal segment. A prediction can be made based on the principle of parsimony, whereby the smallest sequence of operations is likely close to the actual number of operations that occurred. Interestingly, the order of MDSs in the newly assembled 22,354 Oxytricha trifallax MIC chromosome fragments provides evidence that multiple parallel recombinations occur, where the structure of the chromosomes allows for interleaving between two sections of the developing macronuclear chromosome in a manner that can be captured with a common string operation called the shuffle operation (the shuffle operation on two strings results in a new string by weaving together the first two, while preserving the order within each string). Thus, we studied four similar systems involving applications of shuffle to see how the minimum number of operations needed to assemble differs between the types. Two algorithms for each of the first two systems have been implemented that are both shown to be optimal. And, for the third and fourth systems, four and two heuristic algorithms, respectively, have been implemented. The results from these algorithms revealed that, in most cases, the third system gives the minimum number of applications of shuffle to descramble, but whether the best implemented algorithm for the third system is optimal or not remains an open question. The best implemented algorithm for the third system showed that 96.63% of the scrambled micronuclear chromosome fragments of Oxytricha trifallax can be descrambled by only 1 or 2 applications of shuffle. This small number of steps lends theoretical evidence that some structural component is enforcing an alignment of segments in a shuffle-like fashion, and then parallel recombination is taking place to enable MDS rearrangement and IES elimination. Another problem of interest is to classify segments of the MIC into MDSs and IESs; this is the second topic of the thesis, and is a matter of determining the right "class label", i.e. MDS or IES, on each nucleotide. Thus, training data of labelled input sequences was used with hidden Markov models (HMMs), which is a well-known supervised machine learning classification algorithm. HMMs of first-, second-, third-, fourth-, and fifth-order have been implemented. The accuracy of the classification was verified through 10-fold cross validation. Results from this work show that an HMM is more likely to fail to accurately classify micronuclear chromosomes without having some additional knowledge

    DNA elimination in the ciliate Tetrahymena

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    WĂ€hrend der sexuellen Fortpflanzung finden im neu entstehenden Makronukleus von Tetrahymena weitreichende Umgestaltungen des Genoms statt, bei denen mehr als 30 % des Genoms eliminiert werden. Dieser Prozess beinhaltet die spezifische Erkennung interner eliminierter Sequenzen durch einen Argonaut-scanRNA Komplex und die darauf folgende Heterochromatinbildung. Diese fĂŒhrt zur Akkumulierung der methylierten Lysine 9 und 27 des Histons H3 woraufhin das ChromodomĂ€nen Protein Pdd1p rekrutiert wird. Im Anschluss daran bilden sich sichtbar abgegrenzte Heterochromatinkörperchen von denen man davon ausgeht, dass in ihnen die DNS Eliminierung stattfindet. In meiner Doktorarbeit konnte ich zeigen, dass die piggyBac-Ă€hnliche Transposase Tpb2p in diese Heterochromatinkörperchen rekrutiert wird und dass sie fĂŒr die DNS Eliminierung unerlĂ€sslich ist. Zudem erkennt und schneidet das rekombinant in Bakterien hergestellte Tpb2p die Grenzen verschiedener interner eliminierter Sequenzen in vitro, wenn diese in der Mitte einer kĂŒnstlich ausgearbeiteten und synthetisierten oligo DNS vorhanden sind. Daher fĂŒhrt Tpb2p wahrscheinlich auch den initialen Doppelstrangbruch wĂ€hrend des Ausschneidens der internen eliminierten Sequenzen aus. Um einen Einblick zu erhalten wie die PrĂ€zission wĂ€hrend des Ausschneidens erreicht wird, habe ich die Nuklease AktivitĂ€t des rekombinanten Tpb2p genauer untersucht. DafĂŒr verwendete ich synthetisierte oligo DNS in der jede Position der linken Grenze des gut untersuchten R-Elements (Sequenz AGTGAT) individuell mutiert wurde. Ich konnte zeigen, dass die Positionen drei und vier wichtig fĂŒr ein effektives Schneiden der Sequenz durch Tpb2p sind. Des Weiteren konnte ich veranschaulichen, dass diese Positionen auch fĂŒr die prĂ€zise Eliminierung des R-Elements wichtig sind. Daher trĂ€gt die PrĂ€ferenz von Tpb2p fĂŒr bestimmte DNS Sequenzen sicherlich zur genauen Eliminierung der internen eliminierten Sequenzen bei. Andererseits lokalisiert Tpb2p in den Heterochromatinkörperchen und ist fĂŒr deren Ausbildung essentiell. Daher ist es auch möglich, dass Tpb2p direkt mit dem Heterochromatin interagiert, und dass diese Interaktion die prĂ€zise Eliminierung ermöglicht. Tpb2p hat eine Endonuklease DomĂ€ne und eine Zink Finger DomĂ€ne. Ich habe herausgefunden, dass die Zink Finger DomĂ€ne, allerdings nicht die Endonuklease DomĂ€ne essentiell fĂŒr die Ausbildung der Heterochromatinkörperchen ist. Außerdem konnte ich zeigen, dass die Zink Finger DomĂ€ne in vitro an Peptide des Histons H3 bindet, wenn diese an Lysin 9 oder 27 tri-methyliert sind. Dies lĂ€sst die Schlussfolgerung zu, dass diese Modifikationen, die spezifisch sind fĂŒr interne eliminierte Sequenzen – zusammen mit der Sequenz PrĂ€ferenz von Tpb2p – das prĂ€zise Ausschneiden der internen eliminierten Sequenzen vermittelt.During sexual reproduction the new developing macronucleus of Tetrahymena undergoes massive programmed DNA rearrangement, where over 30 % of the genome is eliminated. This process involves the sequence specific recognition of internal eliminated sequences by an Argonaute-scan RNA complex followed by heterochromatin formation including the accumulations of methylated histone H3 at lysine 9 and lysine 27 and the chromodomain protein Pdd1p. This heterochromatin formation eventually leads to the formation of distinct heterochromatin bodies in which DNA elimination is believed to occur. In my thesis I demonstrated that the piggyBac-like transposase Tpb2p is recruited to the heterochromatin bodies and that it is essential for DNA elimination. Furthermore, the recombinantly expressed Tpb2p from bacteria can recognize and cut boundaries of different internal eliminated sequences in vitro when they are placed in the middle of an artificially designed and synthesized oligo. Thus Tpb2p probably introduces the initial double strand break during DNA excision. To get insight into how the precision of excision is achieved, I first analyzed the nuclease activity of recombinant Tpb2p in more detail. Using synthesized oligo DNAs where every position of the reported left boundary of the well studied R element (sequence AGTGAT) was individually mutated, I found that the third and fourth positions in the boundary sequence are important for efficient cleavage by Tpb2p. Furthermore, an in vivo study confirmed that these two positions were crucial for the precise elimination of the R IES element. Therefore, some DNA sequence preference of Tpb2p clearly contributes to the precise elimination of internal eliminated sequences. On the other hand, because Tpb2p is a component of heterochromatin and is required for heterochromatin body formation, heterochromatin interaction with Tpb2p might also be involved in the precise DNA elimination. Tpb2p has an endonuclease domain and a zinc finger domain. I found that the zinc finger domain, but not the endonuclease domain, was essential for heterochromatin body formation. I could show in vitro that the zinc finger domain binds to histone H3 peptides that are tri-methylated at lysines 9 or 27 suggesting that these modifications specific to internal eliminated sequences are - together with the sequence preference of Tpb2p- specifying the precise IES excision

    Hox3 duplication and divergence in the Lepidoptera

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    Using the Speckled Wood Butterfly Pararge aegeria as the model species, this thesis presents the possible evolutionary significance of a set of duplications found in the Hox cluster of the Lepidoptera, called the Special Homeobox genes. An annotation of this duplicated cluster across a wide number of Lepidoptera was performed in order to assess patterns of duplication and loss across the order. The sequences recovered revealed a large amount of variation associated with the duplicate genes, indicating these are evolving very rapidly in different lineages. Patterns of sequence variation were examined to ascertain whether the observed variation was maintained due to selection at three separate levels of divergence: within the Ditrysia, within the more recently diverged Heliconius genus, and at the intraspecific level by quantifying nucleotide polymorphism within Pararge aegeria. Selective pressures were found to be operating between paralogous and orthologous genes, suggesting these have evolved, in part, under positive selection. The potential function of the duplicates was examined by means of CRISPR/Cas9 geneome editing, but revealed inconclusive results. Genome editing, however, was shown to be largely applicable to P. aegeria, and resulted in consistent mutations associated with wing patterning genes. The potential significance of the duplications for Lepidopeteran biology are discussed, as well as future applications for genome editing techniques in P. aegeria
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