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Zusammenfassung:
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.
