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Document Type

Open Access Dissertation

Degree Name

Doctor of Philosophy (PhD)

Degree Program

Organismic and Evolutionary Biology

Year Degree Awarded

2018

Month Degree Awarded

February

First Advisor

Laura A. Katz

Second Advisor

Courtney Babbitt

Third Advisor

Robert L. Dorit

Fourth Advisor

Michael Hood

Subject Categories

Bioinformatics | Evolution | Genetics | Genomics | Other Microbiology

Abstract

The traditional view of genomes suggests that they are static entities changing slowly in sequence and structure through time (e.g. evolving over geological time-scales). This outdated view has been challenged as our understanding of the dynamic nature of genomes has increased. Changes in DNA content (i.e. polyploidy) are common to specific life-cycle stages in a variety of eukaryotes, as are changes in genome content itself. These dramatic genomic changes include chromosomal deletions (i.e. paternal chromosome deletion in insects; Goday and Esteban 2001; Ross, et al. 2010), developmentally regulated genome rearrangements (e.g. the V(D)J system in adaptive immunity in mammals; Schatz and Swanson 2011) and the specialization of a distinct somatic genome through epigenetically regulate DNA elimination during development (found in protists and some animals; Coyne, et al. 2012; Prescott 1994; Wang and Davis 2014; Wyngaard, et al. 2011).

What likely allows genomes to be highly flexible is the separation of germline (i.e. ‘heritable’) and somatic (i.e. ‘functional’) material, even in the context of a single nucleus. Germline-soma distinctions have been best described (and most easily seen) in lineages of multicellular eukaryotes (e.g. plants, animals and fungi) due to obvious sexual structures. Germline genomes of these taxa are restricted to specialized cells (e.g. gametes; for example, pollen grains, eggs and spores) and remain undifferentiated (and often transcriptionally inactive), whereas the somatic cells (e.g. skin, leaves, hyphae) provide the basis for ensuring organismal survival to reproductive life-stages. Sequestered germline and somatic genomes are not restricted to these well-known multi-cellular lineages but are also well-described among ciliates (the focus of this dissertation) and some foraminifera. However, in these protists, germline and somatic genomes are not isolated into distinct cells and tissues but rather are isolated into distinct nuclei that share a common cytoplasm.

Ciliates are a diverse and ancient clade of eukaryotes (~1-1.2 GYA old) and their study has led to the discovery of broad uniting eukaryotic features such as telomeres (Blackburn and Gall 1978) and self-splicing RNAs (Kruger, et al. 1982). As in the “macrobial” eukaryotes, the somatic genome (macronucleus; MAC) is transcriptionally active, transcribing all the genes necessary to maintain the cell, while the germline genome (micronucleus; MIC) remains transcriptionally inactive during the asexual portions of the life cycle. While the germline chromosomes in ciliates are physically similar to other ‘traditional’ eukaryotic chromosomes (e.g. being multi-Mbp with centromeres), the physical structure of the somatic chromosomes is highly variable. For example, in the model ciliate Tetrahymena thermophila, the somatic genome is composed of 225 unique chromosomes (most of them being ~200-400Kbp), with each at approximately 45 copies, whereas Oxytricha trifallax’s somatic genome is composed of ~16,000 gene-sized chromosomes (~2-3Kbp) with each chromosome at its own independent copy number (average copy number ~2,000).

Despite dramatic differences in somatic genome architecture in ciliates, the development of a new somatic genome involves. For all ciliates studied to date, this metamorphosis from ‘traditional’ germline chromosomal architecture to the incredibly variable somatic genome architecture includes large-scale genome rearrangements and DNA elimination. This transformation involves the epigenetically-guided retention of somatically destined DNA from the background germline genome. While genomic rearrangements in most other eukaryotes are often fatal and are symptoms of well-known diseases (e.g. some cancers), this traditionally ‘catastrophic’ event is a fundamental part of ciliate life-cycles.

Although studies of ciliate germline genomes have largely been restricted to only a few genera, there appear to be broad similarities in gene organization that may be phylogenetically conserved. Ciliate germline genome architecture has been categorized as either non-scrambled or scrambled, where non-scrambled architectures are often defined as possessing macronuclear destined sequences (MDSs; soma) that are separated by germline-limited DNA and remain in consecutive order (e.g. 1-2-3-4; Figure 3.1A and Figure 4.4A). Scrambled germline architectures are highly variable, but are broadly defined as MDSs being maintained in non-consecutive order (e.g. 1-3-4-2) and/or on opposing strands of DNA (Figure 3.1 B-D and Figure 4.4B). The germline genomes of Chilodonella uncinata (the main focus of this dissertation) possess a combination of scrambled and non-scrambled architectures. Before my thesis work, only those ciliates with gene-sized chromosomes have been demonstrated to have scrambled germline loci. Interestingly, previous work has implicated somatic genome architecture impacting the observable accelerated rates of protein evolution in ciliates, where the proteins of those ciliates possessing ‘gene-sized’ chromosomes experience the greatest evolutionary rates. These observations highlight the need for further work exploring the evolutionary impacts of different germline genome architectures, as the germline structure itself has direct impact on the development of the somatic genome.

While this dissertation aims to elucidate some aspects of the evolution of germline-soma distinctions and the impact of genome and nuclear architecture (Chapters 2-4), there remain several fundamental questions that we can start addressing. For instance, in this work we observe that the most expanded gene families in Chilodonella uncinata are composed of genes that are disproportionately found at scrambled germline loci (Chapter 3). A major step future step will be to explore the functional implications of this increased paralog diversity through forward and reverse genetics techniques. Similarly, it will be incredibly valuable to better understand the nuclear architecture of the differing genomic contents of the three distinct nuclei present during ciliate development (i.e. the degrading parental MAC, the ‘new’ MIC, and the developing MAC). There may be observable compartmentalization that is exploitable or critical to the accurate rearrangement of the germline genome into a functional somatic genome. Finally, with the increasingly apparent utility of single-cell ‘omics techniques (which we use in Chapters 3 and 4), there is opportunity to probe into taxonomic groups where physical germline-soma separations exist, which will provide a far more expansive understanding of the evolutionary and functional impacts of harboring multiple distinct genomes inside of a single cell/organism.

Available for download on Wednesday, August 01, 2018

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