Background: Cacao swollen shoot virus (CSSV), Cacao swollen shoot CD virus (CSSCDV), and Cacao swollen shoot Togo A virus (CSSTAV) cause cacao swollen shoot disease (CSSD) in West Africa. During 2000-2003, leaf and shoot-swelling symptoms and rapid tree death were observed in cacao in Cote d'Ivoire and Ghana. Molecular tests showed positive infection in only similar to 50-60% of symptomatic trees, suggesting the possible emergence of an unknown badnavirus. Methods: The DNA virome was determined from symptomatic cacao samples using Illumina-Hi Seq, and sequence accuracy was verified by Sanger sequencing. The resultant 14, and seven previously known, full-length badnaviral genomic and RT-RNase H sequences were analyzed by pairwise distance analysis to resolve species relationships, and by Maximum likelihood (ML) to reconstruct phylogenetic relationships. The viral coding and non-coding sequences, genome organization, and predicted conserved protein domains (CPDs) were identified and characterized at the species level. Results: The 21 CSSD-badnaviral genomes and RT-RNase H sequences shared 70-100% and 72-100% identity, respectively. The RT-RNase H analysis predicted four species, based on an >= 80% species cutoff. The ML genome sequence tree resolved three well-supported clades, with >= 70% bootstrap, whereas, the RT-RNase H phylogeny was poorly resolved, however, both trees grouped CSSD isolates within one large clade, including the newly discovered Cacao red vein virus (CRVV) proposed species. The genome arrangement of the four species consists of four, five, or six predicted open reading frames (ORFs), and the CPDs have similar architectures. By comparison, two New World cacao-infecting badnaviruses encode four ORFs, and harbor CPDs like the West African species. Conclusions: Three previously recognized West African cacao-infecting badnaviral species were identified, and a fourth, previously unidentified species, CRVV, is described for the first time. The CRVV is a suspect causal agent of the rapid decline phenotype, however Koch's Postulates have not been proven. To reconcile viral evolutionary with epidemiology considerations, more detailed information about CSSD-genomic variability is essential. Also, the functional basis for the multiple genome arrangements and subtly distinct CPD architectures among cacao-infecting badnaviruses is poorly understood. New knowledge about functional relationships may help explain the diverse symptomatologies observed in affected cacao trees.

京都大学 / 0048 / 新制・課程博士 / 博士(生命科学) / 甲第22135号 / 生博第422号 / 新制||生||55(附属図書館) / 京都大学大学院生命科学研究科統合生命科学専攻 / (主査)教授 藤田 尚志, 教授 朝長 啓造, 教授 永尾 雅哉 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

DNA double strand breaks (DSB) are cytotoxic lesions that can lead to genome rearrangements and genomic instability, which are hallmarks of cancer. The two main DSB repair pathways are non-homologous end joining and homologous recombination (HR). While HR is generally highly accurate, it has the potential for gross chromosomal rearrangements (GCRs) that occur directly or through intermediates generated during the repair process. Whole genome sequencing of cancers has revealed numerous types of structural rearrangement signatures that are often indicative of repair mediated by sequence homology. However, it can be challenging to delineate repair mechanisms from sequence analysis of rearrangement end products from cancer genomes, or even model systems, because the same rearrangements can be generated by different pathways. Numerous studies have provided insights into the types of spontaneous GCRs that can occur in various Saccharomyces cerevisiae mutants. However, understanding the mechanism and frequency of formation of these GCR without knowledge of the initiating lesions is limited. Here, we focus on DSB-induced repair pathways that lead to GCRs. Inverted duplications occur at a surprisingly high frequency when a DSB is formed near short inverted repeats in cells deficient for the nuclease activity of Mre11. Similar to previously proposed models, the inverted duplications occur through intra-strand foldback annealing at resected inverted repeats to form a hairpin-capped chromosome that is a precursor to dicentric chromosomes. Surprisingly, we found that DNA polymerase δ proof-reading activity but not the Rad1-Rad10 nuclease is required for inverted duplication formation, suggesting a role for Pol δ in the removal of the heterologous tails formed during foldback annealing. Contrary to previous work on spontaneous inverted duplications, we find that DSB-induced inverted duplications require the Pol δ processivity subunit Pol32 and that RPA plays little role in their inhibition, suggesting that spontaneous inverted duplications arise differently than those induced by DSBs. We show that stabilization of dicentric chromosomes after breakage involves telomere capture through a strand-invasion step mediated by repeat sequences and requires Rad51. Previous work on spontaneous inverted duplications suggested that Tel1, but not Mre11-Sae2, inhibits inverted duplications that initiate from inverted repeats separated by long spacers (> 12 bp). However, we do not find evidence for this requirement. Cells with Tel1 deletion can still resolve hairpins containing loops up to 30 nt long. Furthermore, deletion of Sae2, but not Tel1, increases the frequency of inverted duplications when a DSB is induced near an inverted repeat separated by a 20 bp-long spacer. This highlights another difference between spontaneous and DSB-induced GCRs. Finally, we find that the sequence context of a DSB affects the type of GCR outcome. Inverted repeats are required for the formation of inverted duplications, as the deletion of a DSB-proximal inverted repeat significantly reduces the incidence of this type of rearrangement. Furthermore, introduction of a DSB near short telomere-like sequence is required for chromosome truncations stabilized by de novo telomere addition. The effect of the sequence context can partly explain how repair pathways can be channeled to different mutagenic outcomes. Our results highlight the importance of considering how the initiating lesion can affect the type of resulting GCRs and the mechanisms by which they occur.

京都大学 / 0048 / 新制・課程博士 / 博士(生命科学) / 甲第20782号 / 生博第388号 / 新制||生||51(附属図書館) / 京都大学大学院生命科学研究科統合生命科学専攻 / (主査)教授 藤田 尚志, 教授 朝長 啓造, 教授 永尾 雅哉 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

No description available.

A strain of Trichomonas vaginalis infected with a double-stranded RNA virus showed pronounced cytopathology in the form of giant syncytia generated by the recruitment of single cells. The giant cells ultimately lysed, releasing virus into the culture medium. In the infected cells, clusters of electron-dense particles resembling viral structures were found in the cytoplasm. In addition, distinctive inclusions composed of similar particles were present in the nuclei of some cells. Double-stranded viral RNA of 5.5 kbp was demonstrated in both cytoplasmic and nuclear fractions from these cells. Viral particles collected from the cell-free culture supernatant were of the same shape and size as the RNA virus isolated from a strain of T. vaginalis described previously (Wang and Wang, Journal of Biological Chemistry, 260: 3697-3702, 1985; Wang and Wang, Proceedings of the National Academy of Sciences of the U.S.A. 83: 7956-7986) which does not show this cytopathology.

No description available.

Double-stranded RNA (dsRNA) is a key molecule that initiates the immune response to viral infection, but increasingly endogenous (self) dsRNA has been found to be central to the pathology of diverse non-infectious diseases, from neurodegenerative disease to autoimmunity to cancers. Therefore, it is critical to understand the mechanisms that regulate endogenous dsRNA and the pattern recognition receptors that sense dsRNA. In this dissertation, I address three main questions pertaining to this. First, why is the brain so prone to dsRNA-mediated non-infectious disease, especially considering that dsRNA sensors are expressed in almost all tissues. Using stem cell differentiation, gene expression manipulation, and microscopy, I determined that neurons are a special cell type that express high levels of endogenous dsRNA. This high dsRNA burden in neurons is driven by global lengthening of 3`untranslated regions (3`UTRs) and induces tonic inflammation. Second, I examined the mechanism through which the dsRNA regulator ADAR1 controls endogenous dsRNA levels. I employed heavy use of tissue culture and visualization of dsRNA by confocal microscopy to determine that both the dsRNA-binding and dsRNA-editing activities of ADAR1 are required to suppress global endogenous dsRNA levels. Third, after identifying the existence of transcript isoforms of the key dsRNA sensor PKR, I explored their regulatory potential on the PKR protein itself. By genetically altering human cells, I identify that 3`UTR isoforms of PKR regulate transcript localization, translation efficiency, and PKR protein activatability. Overall, the studies described herein demonstrate novel regulatory roles of endogenous dsRNA and underscore the importance of dsRNA in neurological disease.

The world of precision medicine was revolutionized by the discovery of CRISPR-Cas systems. In particular, the capabilities of the programmable nuclease Cas9 and its derivatives have unlocked a world in which applied genome engineering to cure human disease is a reality being pursued in patient clinical trials. Gene editing via the induction of programmable, site-specific double strand breaks (DSBs) has been revolutionary for the precision medicine field. However, there are many safety concerns centered on the induction of DSBs causing potential undesirable on- and off-target consequences, particularly for in vivo CRISPR applications. To circumvent these warranted concerns, many groups have attempted to repurpose recombinases or engineer new fusion systems to perform programmable genome engineering without the induction of DSBs. This dissertation will first highlight the development of recombinases for programmable DNA insertions over the course of decades, including efforts to evolve novel DNA recognition sequences, efforts to tether recombinases to programmable DNA-binding proteins, and the recent discovery of naturally occurring RNA-guided DNA transposition systems. This dissertation will then highlight the development of CRISPR-associated transposases (CASTs) as DSB-independent programmable mammalian gene editing tools capable of integrating large DNA cargos, as well as the future directions that may further enhance CAST activity in human cells. The works in this dissertation detail the initial efforts to engineer and optimize a new class of genome manipulation tools that were previously absent from the gene editing toolkit.

DNA double-strand breaks (DSBs) are one of the most dangerous lesions cells encounter, given that DSBs can lead to genomic instability and cell death if not repaired properly. Cells have two primary pathways to repair DSBs: Homologous recombination (HR) and nonhomologous end-joining (NHEJ). HR is the high-fidelity branch of the DSB repair pathway since it employs a process of homology search and synthesis from a homologous template. The homology search is carried out by ssDNA that is generated on either side of the DSB by end resection. End resection occurs via a two-step mechanism involving resection initiation, followed by long-range resection. Previous work has revealed that long-range resection is dispensable for some cases of HR; however, it is currently unclear why the requirement for long-range resection is context- dependent. Furthermore, it is not completely clear how the mechanisms of HR, including requirements for long-range resection, apply to single-ended DSBs (seDSBs) arising during replication. Therefore, we defined the role of long-range resection in two-ended DSB repair in different chromosomal contexts. We also established a Cas9 nickase (Cas9n) system to study seDSB repair and defined genetic requirements for repair. To study the requirement for long-range resection in HR, we employed inter- and intrachromosomal genetic recombination assays in haploid yeast. We found that long-range resection is required for interchromosomal HR, but not for intrachromosomal HR. This difference is linked to the observation that the DNA damage checkpoint, which is deficient in the absence of long-range resection, is activated in interchromosomal HR, but not intrachromosomal HR. The DNA damage checkpoint has also previously been implicated in promoting chromosome mobility. Therefore, we reason that the requirement for long-range resection in interchromosomal repair is due to a need to activate the DNA damage checkpoint and chromosome mobility, specifically during slower repair events. To study seDSB repair, we implemented Cas9n, which creates nicks that can cause replication fork collapse. We demonstrated that expression of Cas9n with an efficient gRNA can induce replication fork collapse and that repair of these seDSBs breaks is dependent on the HR machinery. A genome-wide screen using Cas9n revealed a requirement for replication-coupled nucleosome assembly (RCNA) in repair of seDSBs, specifically in replication origin-deplete regions of the genome. Consistent with the model of seDSB repair, we found that Cas9n-induced seDSBs preferentially undergo sister chromatid recombination. This preference was altered in the absence of Mre11, which we hypothesize is due to a role of MRX in sister chromatid tethering. Altogether, the results presented in this thesis offer a different perspective on the role of long-range resection in two-ended DSB repair and establish a Cas9n-based system to better study single-ended DSB repair.