Ciliates are important model organisms that have been used to study many aspects of cellular biology, including telomeres, histone modifications, and ribozymes. These unicellular eukaryotes house both a germline genome and a somatic genome in distinct nuclear structures within a single cell. One of their most unique features is their ability to undergo complex programmed genome rearrangements, during which their germline genome is fragmented and rearranged to form a new somatic genome. This rearrangement process results in a highly specialized somatic genome with many polyploid short chromosomes that are rich with genes. While all ciliates can undergo this developmental process, Oxytricha trifallax experiences particularly complex rearrangements that result in a more radically unconventional structure in its somatic genome. Much of the previous work studying Oxytricha has been focused on the complex rearrangements that it undergoes during sexual development and the mechanisms that allow it to perform these genome rearrangements events at the level of accuracy required for proper somatic function afterwards. Due to this particular focus on Oxytricha sexual development, the rest of Oxytricha’s unique biology has not been studied to the same degree. For my thesis I examined two aspects of Oxytricha biology that have not been well understood. In Chapter 1 I report the results of a proteomic survey of both types of nuclei found within the vegetative cell, the somatic macronucleus and the germline micronucleus. We performed mass spectrometry on enriched samples of both nuclear types and analyzed the enrichment of proteins between the two. Despite some mitochondrial contamination, we found that many categories of functional proteins were enriched in one of the two nuclei. We validated the appropriate nuclear localization of specific proteins from each subcategory through imaging Our results confirmed many previously predicted aspects of the two nuclei and provide a valuable resource for further studies on nuclear proteins in Oxytricha. In Chapter 2 I describe various features of a comparative analysis between the somatic genomes of multiple strains of Oxytricha trifallax. Previous work from the lab has focused primarily on the reference strains JRB310 and JRB510, which are most commonly used due to their ability to mate. We generated four new draft assemblies of the somatic genomes of strains JRB27, JRB39, SLC89, and SLC92. Many metrics demonstrate that these new assemblies are largely complete. Our analyses of these new strains revealed that there are numerous strain-specific chromosomes in Oxytricha that can encode genes. While they do not seem to encode core genes that would be missing otherwise, they are prime candidates for further examination to identify mating type-related genes.

Ciliates are model organisms for studying programmed genome rearrangement because each cell houses two distinct genomes. During postzygotic development, the somatic genome rearranges from a copy of the germline genome via extensive genome remodeling, including DNA elimination, religation and sometimes translocation or inversion of genomic regions. Previous studies of this process were restricted to a few model ciliates including Tetrahymena thermophila, Paramecium tetraurelia and Oxytricha trifallax. Oxytricha diverged from Tetrahymena and Paramecium over one billion years ago, and it possesses a massively fragmented and scrambled germline genome. My thesis compares Oxytricha to more closely related ciliates to address the evolutionary origin of genome complexity. Chapter 1 provides a general introduction to genome architecture, comparison of well-studied ciliate genomes and challenges of studying genome rearrangement in non-model ciliates. Chapter 2 describes a computational pipeline, SIGAR (Split-read Inference of Genome Architecture and Rearrangements), which infers genome rearrangement features without a germline genome assembly. We validated the pipeline using a published Oxytricha dataset, and also applied it to six diverse ciliate species including Ichthyophthirius multifiliis, a fish pathogen. This pipeline enables pilot surveys or exploration of chromosomal rearrangement in ciliates with limited germline DNA access, thereby providing new insights into the evolution of DNA rearrangement. Chapter 3 presents a comparative genomic study of three ciliate species including Oxytricha trifallax, Tetmemena sp. and Euplotes woodruffi. Collaborating with my colleagues, I assembled and annotated germline genomes in Tetmemena and E. woodruffi, as well as E. woodruffi’s somatic genome. We identified scrambled genes in all three species, especially the earlier-diverged E. woodruffi, though at a lower level (7.3% of gene loci) compared to Oxytricha (15.6%) and Tetmemena (13.6%). E. woodruffi may therefore represent an intermediate between the nonscrambled ancestral genome and more massively scrambled genomes as can be seen in Oxytricha and Tetmemena. We also found that scrambled genes tend to have more paralogs or have partial MDS duplications, suggesting that local duplications might play a role in the evolutionary origin of scrambled genes. Chapter 4 reports a new genetic code identified in a basal spirotrich ciliate, Licnophora macfarlandi. Ciliates have been a hot spot for the evolution of alternative genetic codes. All variant genetic codes in ciliates reassign canonical stop codons to amino acids, and in most cases the UAA and UAG are reassigned to the same amino acid, or are both used as stop codons. The codon usage analysis in Licnophora revealed an unprecedented genetic code that translates the UAA to glutamic acid and the UAG to glutamine. We also detected candidate tRNAs from the somatic genome which can recognize the UAA and UAG. Chapter 5 describes possible future directions to understand the genome complexity of ciliates.