<p> Phylogenetic analysis of gene order data is a developing area, and many questions on gene orders are still unresolved. In this project, we started from the OGRe database, where we obtained the mitochondrial genome information (after some corrections), designed a logarithm correction for breakpoint distance, applied distance matrix methods to both breakpoint distance and the logarithm of breakpoint distance for gene orders, and then focused on Arthropoda phylogeny We tried many phylogenetic methods to infer Arthropod phylogeny; however, no method yielded a satisfying result. We constructed an Arthropod phylogenetic tree based on both molecular and morphological evidences. After we estimated the phylogenetic tree, we used maximum likelihood methods to estimate branch lengths for tRNAs and proteins, calculated the breakpoint numbers and inversion numbers for gene orders, and calculated the correlations among these four measures. We found that: when gene order rearrangements and mutations on sequences are small, the changes are independent, and, when the rearrangements and mutations are large, the changes seem to be correlated. The branch lengths in the tRNA and protein trees are highly correlated in low mutation situations and less correlated when mutation rates are larger. </p> / Thesis / Master of Science (MSc)

Transcription-associated RNA surveillance is vital to the well-being of the cell by processing both coding and noncoding RNA (ncRNA). When left unregulated, RNA transcripts can cause genomic instability by forming structures called R-loops that leave a single strand of DNA exposed to potentially harmful events like nicks and mutations. The RNA exosome is the 3’ exoribonuclease that plays an important role in degrading both coding and ncRNA, and its role in processing ncRNA transcripts has been demonstrated extensively in the recent past. The role of ncRNA transcription and the RNA exosome is best exemplified in B cell programmed DNA recombination, namely class switch recombination (CSR). In CSR, ncRNA transcription and RNA exosome recruitment to switch sequences allows for targeting of the activation-induced cytidine deaminase (AID) enzyme and ultimate formation of DNA double strand breaks, which results in recombination between the appropriate switch regions. While the mechanism of the RNA exosome in processing this transcribed ncRNA and regulating CSR is well-established, the mechanism of RNA exosome regulation and recruitment remains unknown. RNA base modifications are another facet of RNA surveillance that can have profound effects on RNA biology. While some modifications are fixed, reversible base modifications such as methylation are particularly influential due to their dynamic nature. Among the most widely studied of reversible modifications is that of N⁶-methyladenosine (m⁶A). M⁶A has been identified for its role in regulation of gene expression and cell differentiation via its effect on regulating mRNA levels and downstream function. Although it has been shown to be present on some ncRNA, the role of m6A in regulating ncRNA turnover and genome dynamics is largely unclear. We interrogated the hypothesis that m6A plays a role in regulating RNA exosome recruitment in B cell programmed DNA recombination. We initially assessed the role of the RNA exosome cofactor MPP6 in CSR using CRISPR/Cas9-mediated knockout mouse B cell hybridoma CH12F3 cells (CH12 cells) and established a connection between the RNA exosome and m6A by demonstrating an interaction between MPP6 and m6A reader protein YTHDC1. We then utilized mouse models to knockout METTL3, a m6A methyltransferase, in primary B cells and assessed the efficiency of CSR. We performed RNA sequencing to assess the levels of ncRNA in B cells upon METTL3 loss and find that primarily G-rich RNA transcripts are accumulated upon METTL3 loss, exemplified by switch region transcript SμGLT. In order to specifically show that methylation of SμGLT is required for efficient CSR, we used fusion proteins of catalytically dead Cas13b fused to either m6A demethylase ALKBH5 or FTO and targeted SμGLT with a guide RNA specific for the transcript. We found that methylation of SμGLT specifically is required for efficient CSR, with targeted demethylation resulting in a decrease in CSR efficiency. We also interrogated the role of m6A reader protein YTHDC1 in CSR via CRISPR/Cas9-mediated knockout CH12 cells to assess the mechanism of m6A-mediated downstream function. We found that YTHDC1 is also required for efficient CSR, likely via interaction with MPP6 and the RNA exosome to degrade SμGLT. We also assessed the role of MPP6, METTL3, and YTHDC1 in facilitating the association between AID and the RNA exosome via 3D stochastic optical reconstruction microscopy (3D-STORM) and found that all three components are required for nucleation of AID and the RNA exosome (for which helicase MTR4 was used as a proxy). We then wanted to assess whether m⁶A loss promotes genomic instability in B cell programmed DNA breaks. We performed linear amplification-mediated high-throughput genome-wide sequencing (LAM-HTGTS) to assess the levels of on- versus off-target rearrangements in METTL3 knockout primary B cells. We found that upon METTL3 loss there is an increase in off-target, non-immunoglobulin translocations as well as an increase in microhomology-mediated end joining, indicative of defects in on-target recombination and the canonical nonhomologous end joining pathway. These data suggest that m⁶A is required for targeted programmed DNA rearrangements and protection of nonhomologous end joining in B cells. Taken together, these findings have elucidated a novel mechanism by which m6A regulates ncRNA turnover, RNA exosome targeting, and genome dynamics in B cell programmed DNA rearrangements.

3D genome organization recapitulates genome function, typically at the level of transcriptional state or chromatin state. Ciliates like Oxytricha trifallax possess highly specialized germline genomes, which play a role in sexual development but are largely transcriptionally silent. During Oxytricha development, the germline undergoes genome-wide rearrangement, transforming into a structurally distinct, transcriptionally active somatic genome. This wholesale rearrangement is programmed by transgenerational RNA copies of the somatic genome, which guide the retention of a small portion of the germline. Retained and eliminated sequences must be distinguished from each other and processed separately, but how this dichotomy is effected spatially or by chromatin organization is unknown. The breadth of Oxytricha genome dynamics presents the possibility of a complex relationship between chromatin architecture and genome function. We present new tools for studying the Oxytricha germline and apply them to map the 3D organization of the genome’s vegetative and developmental states using Hi-C. We find that the vegetative conformation primes the germline for development, while a massive spatial reorganization of the genome during development differentiates between retained and eliminated regions and precedes rearrangement of DNA. Further experiments suggest a role for RNA-DNA interactions and chromatin remodeling in this reorganization. This evidence supports a critical role for 3D architecture in programmed genome rearrangement.

Thesis (Ph. D.) -- University of Texas Southwestern Medical Center at Dallas, 2003. / Vita. Bibliography: 127-154.

Thesis (Ph. D.)--University of Oklahoma. / Bibliography: leaves 182-199.

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.

リポジトリの登録にあたっては、Peer reviewされた最終版のみ可能であり、その際には下記の出版社のウェブサイトのアドレスを記載することが求められる。当該論文は2014年6月の出版であり、12ヶ月を経過していることから、公開には差し支えはない。http://clincancerres.aacrjournals.org/content/20/12/3087.full / 京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19617号 / 医博第4124号 / 新制||医||1015(附属図書館) / 32653 / 京都大学大学院医学研究科医学専攻 / (主査)教授 小川 誠司, 教授 野田 亮, 教授 伊達 洋至 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Thesis (Ph. D.)--University of Washington, 2000. / Vita. Includes bibliographical references (leaves 106-115).

Thesis (Ph.D.)--University of Alabama at Birmingham, 2009. / Title from PDF title page (viewed on July 1, 2010). Includes bibliographical references.

Thesis (Ph. D.)--University of Washington, 2000. / Vita. Includes bibliographical references (leaves 139-193).