Reducibility of Gene Patterns in Ciliates using the Breakpoint Graph

Gene assembly in ciliates is one of the most involved DNA processings going on in any organism. This process transforms one nucleus (the micronucleus) into another functionally different nucleus (the macronucleus). We continue the development of the theoretical models of gene assembly, and in particular we demonstrate the use of the concept of the breakpoint graph, known from another branch of DNA transformation research. More specifically: (1) we characterize the intermediate gene patterns that can occur during the transformation of a given micronuclear gene pattern to its macronuclear form; (2) we determine the number of applications of the loop recombination operation (the most basic of the three molecular operations that accomplish gene assembly) needed in this transformation; (3) we generalize previous results (and give elegant alternatives for some proofs) concerning characterizations of the micronuclear gene patterns that can be assembled using a specific subset of the three molecular operations.Comment: 30 pages, 13 figure

Genome Structure Drives Patterns of Gene Family Evolution in Ciliates, a Case Study Using \u3ci\u3eChilodonella uncinata\u3c/i\u3e (Protista, Ciliophora, Phyllopharyngea)

In most lineages, diversity among gene family members results from gene duplication followed by sequence divergence. Because of the genome rearrangements during the development of somatic nuclei, gene family evolution in ciliates involves more complex processes. Previous work on the ciliate Chilodonella uncinata revealed that macronuclear β-tubulin gene family members are generated by alternative processing, in which germline regions are alternatively used in multiple macronuclear chromosomes. To further study genome evolution in this ciliate, we analyzed its transcriptome and found that (1) alternative processing is extensive among gene families; and (2) such gene families are likely to be C. uncinata specific. We characterized additional macronuclear and micronuclear copies of one candidate alternatively processed gene family-a protein kinase domain containing protein (PKc)-from two C. uncinata strains. Analysis of the PKc sequences reveals that (1) multiple PKc gene family members in the macronucleus share some identical regions flanked by divergent regions; and (2) the shared identical regions are processed from a single micronuclear chromosome. We discuss analogous processes in lineages across the eukaryotic tree of life to provide further insights on the impact of genome structure on gene family evolution in eukaryotes

Twisted Tales: Insights into Genome Diversity of Ciliates Using Single-Cell 'Omics.

The emergence of robust single-cell 'omics techniques enables studies of uncultivable species, allowing for the (re)discovery of diverse genomic features. In this study, we combine single-cell genomics and transcriptomics to explore genome evolution in ciliates (a > 1 Gy old clade). Analysis of the data resulting from these single-cell 'omics approaches show: 1) the description of the ciliates in the class Karyorelictea as "primitive" is inaccurate because their somatic macronuclei contain loci of varying copy number (i.e., they have been processed by genome rearrangements from the zygotic nucleus); 2) gene-sized somatic chromosomes exist in the class Litostomatea, consistent with Balbiani's (1890) observation of giant chromosomes in this lineage; and 3) gene scrambling exists in the underexplored Postciliodesmatophora (the classes Heterotrichea and Karyorelictea, abbreviated here as the Po-clade), one of two major clades of ciliates. Together these data highlight the complex evolutionary patterns underlying germline genome architectures in ciliates and provide a basis for further exploration of principles of genome evolution in diverse microbial lineages

IMPACTS OF GENOME AND NUCLEAR ARCHITECTURE ON MOLECULAR EVOLUTION IN EUKARYOTES

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

Sorting Permutations: Games, Genomes, and Cycles

Permutation sorting, one of the fundamental steps in pre-processing data for the efficient application of other algorithms, has a long history in mathematical research literature and has numerous applications. Two special-purpose sorting operations are considered in this paper: context directed swap, abbreviated cds, and context directed reversal, abbreviated cdr. These are special cases of sorting operations that were studied in prior work on permutation sorting. Moreover, cds and cdr have been postulated to model molecular sorting events that occur in the genome maintenance program of certain species of single-celled organisms called ciliates. This paper investigates mathematical aspects of these two sorting operations. The main result of this paper is a generalization of previously discovered characterizations of cds-sortability of a permutation. The combinatorial structure underlying this generalization suggests natural combinatorial two-player games. These games are the main mathematical innovation of this paper.Comment: to appear in Discrete Mathematics, Algorithms and Application

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

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

Twisted Tales: Insights into Genome Diversity of Ciliates Using Single-Cell ‘Omics

The emergence of robust single-cell ‘omics techniques enables studies of uncultivable species, allowing for the (re)discovery of diverse genomic features. In this study, we combine single-cell genomics and transcriptomics to explore genome evolution in ciliates (a \u3e 1 Gy old clade). Analysis of the data resulting from these single-cell ‘omics approaches show: 1) the description of the ciliates in the class Karyorelictea as “primitive”is inaccurate because their somatic macronuclei contain loci of varying copy number (i.e., they have been processed by genome rearrangements from the zygotic nucleus); 2) gene-sized somatic chromosomes exist in the class Litostomatea, consistent with Balbiani’s (1890) observation of giant chromosomes in this lineage; and 3) gene scrambling exists in the underexplored Postciliodesmatophora (the classes Heterotrichea and Karyorelictea, abbreviated here as the Po-clade), one of two major clades of ciliates. Together these data highlight the complex evolutionary patterns underlying germline genome architectures in ciliates and provide a basis for further exploration of principles of genome evolution in diverse microbial lineages

Spliced DNA sequences in the Paramecium germline: their properties and evolutionary potential

Despite playing a crucial role in germline-soma differentiation, the evolutionary significance of developmentally regulated genome rearrangements (DRGRs) has received scant attention. An example of DRGR is DNA splicing, a process that removes segments of DNA interrupting genic and/or intergenic sequences. Perhaps, best known for shaping immune-system genes in vertebrates, DNA splicing plays a central role in the life of ciliated protozoa, where thousands of germline DNA segments are eliminated after sexual reproduction to regenerate a functional somatic genome. Here, we identify and chronicle the properties of 5,286 sequences that putatively undergo DNA splicing (i.e., internal eliminated sequences [IESs]) across the genomes of three closely related species of the ciliate Paramecium (P. tetraurelia, P. biaurelia, and P. sexaurelia). The study reveals that these putative IESs share several physical characteristics. Although our results are consistent with excision events being largely conserved between species, episodes of differential IES retention/excision occur, may have a recent origin, and frequently involve coding regions. Our findings indicate interconversion between somatic—often coding—DNA sequences and noncoding IESs, and provide insights into the role of DNA splicing in creating potentially functional genetic innovation