Os elementos transponíveis (TEs) constituem grande parte do material genético de diversos eucariotos, alcançando entre 50-80% do genoma de gramíneas. Os projetos genoma proporcionaram um aumento das informações disponíveis sobre estes elementos, o que evidenciou sua importância e possibilitou o desenvolvimento de novas abordagens para seu estudo. O sistema Mutator (Mu) de milho é o mais ativo e mutagênico transposon de plantas. Além do elemento autônomo, MuDR, o sistema compreende ainda um conjunto de elementos bastante heterogêneo em sua seqüência e estrutura, chamados MuLEs, que podem conter até mesmo fragmentos de genes do hospedeiro. As seqüências de transposons mais abundantemente expressas no transcriptoma de cana-de-açúcar são relacionadas a MuDR e se agrupam em quatro clados (nomeados Classes I, II, II e IV), existentes antes da divergência entre Mono e Eudicotiledôneas. O trabalho apresentado aqui teve o objetivo de aprofundar o conhecimento sobre o sistema Mutator em cana-de-açúcar a partir da análise comparativa entre seqüências dessa planta e de arroz (cujo genoma está totalmente seqüenciado). Foi possível avaliar a abundância e diversidade do sistema Mu em gramíneas, ficando evidente uma amplificação de elementos clado-específica, tendo a Classe II sofrido uma explosão no número de cópias ao longo da evolução destas plantas. Análises estruturais revelaram que, enquanto as Classes I e II compreendem elementos com características de transposons, as Classes III e IV são, na verdade, transposases domesticadas. Foram completamente seqüenciados dois clones de BAC de cana-de-açúcar, um proveniente de cada parental do híbrido (Saccharum officinarum e Saccharum spontaneum), ambos contendo elementos da Classe III. Estes elementos foram caracterizados e a seqüência genômica de cana foi comparada com sua ortóloga em arroz, revelando um acúmulo de TEs nas regiões intergênicas. / Transposable elements (TEs) constitute great part of eukaryote genetic material, in grasses, they comprise between 50-80% of the genome. Genome projects have significantly increased the amount of information about these elements, revealing their importance and allowing the development of new approaches for their study. The Mutator system (Mu) of maize is the most active and mutagenic plant transposon. Beyond the autonomous element, MuDR, the system comprises a very heterogeneous, in sequence and structure, set of elements, called MuLEs, that can contain even host gene fragments. The most abundant transposon related sequences expressed in sugarcane transcriptoma are the MuDR-like. They group into four clades (called Classes I, II, III and IV) that exist prior to the Mono and Eudicot split. The aim of this work is to gain knowledge about the Mutator system in sugarcane through the comparative analysis against rice (whose genome is completely sequenced). The results described the abundance and diversity of the Mu system in grasses, evidencing a clado-specific amplification with a burst of Class II along the evolution of this plant group. Structural analyses showed that, while Classes I and II comprise elements with transposon characteristics, Classes III and IV are domesticated transposases. One BAC clone from each sugarcane parental genotype (Saccharum officinarum and Saccharum spontaneum) have been completely sequenced, both containing Class III elements. These elements have been characterized and the sugarcane genomic sequences were compared with their orthologues in rice. The comparative analyses showed an accumulation of TEs in the intergenic regions.

The components contributing to cancer progression, especially the transition from early to invasive are unknown. Consequently, the biological reasons are unclear as to why some patients diagnosed with atypia and ductal carcinoma in situ (DCIS) never progress into invasive breast cancer. The “one gene at a time” approach does not sufficiently predict progression. To elucidate the early stage progression to invasive ductal cancer, expression signature of transcripts and transposable elements in micropunched samples of formalin-fixed, paraffin embedded (FFPE) tissue was conducted. A bioinformatics pipeline to analyze poor quality, short reads (>36 nts) from RNA-Seq data was created to compare the most common tools for alignment and differential expression. Most samples from patients prepared for RNA-seq analysis are acquired through archived FFPE tissue collections, which have low RNA quality. The pipeline analytics revealed that STAR alignment software outperformed others. Furthermore, our comparison revealed both DESeq2 and edgeR, with the estimateDisp function applied, both perform well when analyzing greater than 12 replicates. Transcriptome analysis revealed progressive diversification into known oncogenic pathways, a few novel biochemical pathways, in addition to antiviral and interferon activation. Furthermore, the transposable element (TE) signature during breast cancer progression at early stages indicated long terminal repeat (LTRs) as the most abundantly differentially expressed TEs. LTRs belong to endogenous retroviruses (ERV), a subclass of TEs. The retroviral and innate immune response activity in DCIS, which indirectly corroborates the increase in ERV expression in this pre-malignant stage. Finally, to demonstrate the potential role of TEs in the transition from pre-malignant to malignant breast cancer we used pharmacological approaches to alter global TE expression and inhibit retrotransposition activity in control and breast cancer cell lines. It was expected that dysregulation of TEs be associated with increased invasiveness and growth. However, our results indicated that DNA methyltransferase inhibitor 5-Azacytidine (AZA) consistently retarded cell migration and growth. While unexpected, these findings corroborate recent studies that AZA may induce an interferon response in cancer via increased ERV expression. This body of work illustrates the importance of understanding bioinformatics methods used in RNA-seq analysis of common clinical samples. These studies suggest the potential for TEs as biomarkers for disease progression and novel therapeutic approach to investigate in additional model systems.

Every day more than half of the world consumes rice as a primary dietary resource. Thus, rice is one of the most important food crops in the world. Rice and its wild relatives are part of the genus Oryza. Studying the genome structure, function, and evolution of Oryza species in a comparative genomics framework is a useful approach to provide a wealth of knowledge that can significantly improve valuable agronomic traits. The Oryza genus includes 27 species, with 11 different genome types as identified by genetic and cytogenetic analyses. Six genome types, including that of domesticated rice - O. sativa and O. glaberrima, are diploid, and the remaining 5 are tetraploids. Three of the tetraploid species contain the CCDD genome types (O. grandiglumis, O. latifolia, and O. alta), which arose less than 2 million years ago. Polyploidization is one of the major contributors to evolutionary divergence and can thereby lead to adaptation to new environmental niches. An important first step in the characterization of the polyploid Oryza species is the generation of a high-quality reference genome sequence. Unfortunately, up until recently, the generation of such an important and fundamental resource from polyploid species has been challenging, primarily due to their genome complexity and repetitive sequence content. In this project, I assembled two high-quality genomes assemblies for O. grandiglumis and O. latifolia using PacBio long-read sequencing technology and an assembly pipeline that employed 3 genome assemblers (i.e., Canu/2.0, Mecat2, and Flye/2.5) and multiple rounds of sequence polishing with both Arrow and Pilon/1.23. After the primary assembly, sequence contigs were arranged into pseudomolecules, and homeologous chromosomes were assigned to their respective genome types (i.e., CC or DD). Finally, the assemblies were extensively edited manually to close as many gaps as possible. Both assemblies were then analyzed for transposable element and structural variant content between species and homoeologous chromosomes. This enabled us to study the evolutionary divergence of those two genomes, and to explore the possibility of neo-domesticating either species in future research for my PhD dissertation.

La maturation des ARN implique un grand nombre d’évènements post-transcriptionnels, parmi lesquels la polyadénylation qui constitue une étape clé. Chez Arabidopsis, la présence de l’hétérochromatine au niveau des introns de certains gènes peut influencer considérablement la polyadénylation de leur transcrits. INCREASED IN BONSAI METHYLATION2 (IBM2) est une protéinequi contrôle cette catégorie de gènes en reconnaissant l’hétérochromatine au niveau des introns via son domaine BOMO-ADJACENT HOMOLOGY (BAH). IBM2 se lie à l’ARNm par son motif RNA RECOGNOTION (RRM), afin d’assurer la transcription complète de ces gènes cibles en favorisant l’utilisation d’un site distal de polyadénylation. Par conséquent, en mutant IBM2, des plus transcrits courts sont synthétisés suite à une polyadénylation précoce au niveau de la régionhétérochromatique. Durant ma thèse, j’ai cherché à comprendre les mécanismes moléculaires sous-jacents de cette régulation tout en étudiant le rôle du complexe protéique IBM2. Nous avons identifié des protéines partenaires d’IBM2 déjà étudiées telle que ENHANCED DOWNY MILDEW2 (EDM2) et ASI-IMMUNOPRECIPITATED PROTEIN1 (AIPP1), ainsi qu’une nouvelle protéine interagissant physiquement avec IBM2 et d’autres protéines. La mutation du gène correspondant à cette protéine conduit à une réduction de l’expression globale des cibles d’IBM2testées, accompagnée d’un niveau réduit de transcrits longs fonctionnels. Moyennant un crible génétique des suppresseurs de la mutation ibm2, nous avons identifié plusieurs facteurs agissant en amont de la voie IBM2, notamment la protéine FLOWERING TIME CONTROL (FPA). FPA est une protéine capable de s’associer à l’ARN pour favoriser l’utilisation de sites proximaux de polyadénylation de plusieurs gènes cibles, avec parmi eux des gènes contrôlés par IBM2, ce qui suggère que la transcription complète de ces gènes dépend étroitement des actions antagonistes entre IBM2 et FPA. Nos résultats ont montré que le choix du site de polyadénylation de gènes contenant de l’hétérochromatine dépend de plusieurs protéines agissant en différents complexes ainsi que l’interconnexion avec d’autres voies. / RNA maturation implies numerous post-transcriptional modifications in whichpolyadenylation is a key step. In Arabidopsis, the heterochromatin found within introns(intronic-HC) can impact transcripts polyadenylation of host genes. INCREASED IN BONSAI METHYLATION2 (IBM2), an RNA-binding protein containing a bromo-adjacent homology (BAH) domain, interacts with intronic-HC to produce functional full-length transcripts by promoting distal polyadenylation. Loss of IBM2 function triggers short transcripts production due to premature polyadenylation from the heterochromatic region. During my thesis, I investigated the role of proteins that may belong to different sub-complexes in the regulation of intronic-HC containing genes. We identified IBM2 partners, including ENHANCED DOWNY MILDEW 2 (EDM2) and ASI-IMMUNOPRECIPITATED PROTEIN1 (AIPP1), and a novel partner that interacts directly with IBM2 and other proteins. Mutating the corresponding gene of the novel partner results in decreased expression of tested IBM2-targets such as IBM1 encoding an H3K9demethylase and the disease resistance gene RECOGNITION OF PERONOSPORA PARASITICA 7 (RPP7), accompanied with compromised use of their distal polyadenylation sites. By conducting a genetic screen of ibm2 mutation suppressors, we identified factors belonging to different pathways that act upstream of IBM2, among them the FLOWERING TIME CONTROL PROTEIN (FPA). FPA is an RNA-binding protein that promotes the use of proximal polyadenylation sitesof several genes such as IBM1. Our data bring evidence that antagonistic actions of FPA and IBM2 regulates polyadenylation sites choice at intronic-HC containing genes. These results provide new insights to understand the interplay between heterochromatin and RNA processing.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第18526号 / 農博第2083号 / 新制||農||1026(附属図書館) / 学位論文||H26||N4870(農学部図書室) / 31412 / 京都大学大学院農学研究科農学専攻 / (主査)教授 奥本 裕, 教授 米森 敬三, 教授 冨永 達 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

No description available.

Whole genome sequencing (WGS) projects for model mammalian organisms exposed the magnitude to which transposable elements (TEs) have contributed to DNA content, but led to inferences on repeat composition and activity patterns which do not capture the true diversity within Mammalia. Understanding of the evolutionary importance of TEs and the development of TEs for biological applications are hindered when considering only the data gathered from a limited sampling of model organisms. The research presented here begins with analysis of a mouse lemur WGS dataset, revealing an exception in the primate lineage to the generalizations garnered from model organisms. A recently active TE was uncovered with evidence of horizontal transfer involving a primate and another mammal; this discovery may lead to a useful transfection vector with known efficacy in mammalian cells. Furthermore, an opportunity to observe atypical patterns of TE diversification within a mammalian family was achieved through survey sequencing and comparative analyses of five vespertilionid taxa and a phyllostomid outgroup. A potential inverse relationship between the two classes of TEs was tentatively described, and further explored by an in-depth analysis of the dominant TE family from each class in a species with WGS data available. The interplay of the two classes could not previously be investigated in mammals, due to ~40 million years of Class II TE inactivity in model organisms. Through exploration of activity patterns of both classes, this study provides insight on the relationship between TEs and their host.

This research focuses on non-Long Terminal Repeat (non-LTR) retrotransposons in the African malaria mosquito, Anopheles gambiae and other mosquito species. An unprecedented diversity of non-LTRs was discovered by genome analysis of the An. gambiae genome assembly. One hundred and four families were found by a reiterative and comprehensive search using the conserved reverse transcriptase domains of known non-LTRs from a number of organisms as the starting queries. These families range in copy number from a few to approximately 2000 and occupy at least 3% of the genome. An. gambiae non-LTRs represent 8 of the 15 previously defined clades, plus two novel clades, Loner and Outcast, raising the total number of known clades to 17. The first invertebrate L1 clade representatives were also found. All clades except one have families with sequence characteristics suggesting recent activity. Juan, a non-LTR of the Jockey clade originally discovered in the mosquito Culex pipiens quinquefasciatus (Mouches et al. 1991), has been implicated in horizontal transfer in three non-sibling species of the Aedes genus (Mouches, Bensaadi, and Salvado 1992). PCR was used to obtain sequences from 18 mosquito species of six genera. Phylogenetic analysis demonstrates predominant vertical inheritance of Juan elements among these species. There is strong evidence from sequence analysis supporting the recent activity of Juan in several divergent species. We hypothesize that the sustained activity (versus quick inactivation) of non-LTRs in mosquitoes may contribute to the diversity we observe in the An. gambiae genome today. Promoter and transcriptional analyses were performed for several families previously identified as potentially active elements based on sequence analysis. RT-PCR results indicate that transcripts are present in An. gambiae cell lines that contain sequences corresponding to 13 of 15 tested non-LTR families. The 5' UTRs of An. gambiae non-LTRs from the I, Jockey, and L1 clades support basal transcription in divergent mosquito cell lines from 3 species. The Jen-1 5'UTR did not support transcription in Ae. aegypti and had low activity in Ae. albopictus. In summary, this research shows that Non-LTRs have been highly successful genomic elements that have flourished in many divergent mosquito species. / Ph. D.

Study of the genetic relationships and evolutionary histories of pupfish populations (Cyprinodontidae: Cyprinodon spp.) from the remnant aquatic habitats of Death Valley was approached by exploring the genetic structure and divergence within and among populations using mitochondrial and nuclear DNA markers. The findings of these studies illustrate the influences of population size and isolation time in the divergence of small, fragmented populations largely via genetic drift. The information revealed in this study has implications for assessing priorities in the conservation of the unique evolutionary heritage among populations of the Death Valley pupfishes. A new retrotransposable element family was identified and characterized. This family of genetic elements was uncovered during a search of the pupfish genome for transposable elements to be used as molecular markers for population analyses. The description of this element family, named "Swimmer 1" (SW1), provides new insights into the evolution of long interspersed nuclear elements (LINEs) in vertebrates. Therefore, a full characterization of the SW1 element family was undertaken in the Japanese medaka (Oryzias latipes) as well as in the pupfish genome. The Japanese medaka is a model organism widely used for genetic and developmental biology studies. / Ph. D.

Strawberry and potato are both important crop species in the world providing various nutritional values. The cultivated strawberry, Fragaria ananassa, is a fruit crop with a complex genome (2n=8x=56) whereas the diploid woodland strawberry, Fragaria vesca, has a smaller genome (2n=2x=14, 240 Mb) and lots of other qualities that make it a good model for genetic and genomic study, such as high yield of seeds and efficient transformation. Potato (Solanum tuberosum, 2n=4x=48) is an important vegetable crop in the world and is highly heterozygous. The successful sequencing of the homozygous doubled monoploid clone of potato provides good insight into the study of important genes in this species in improving the pest resistance and improving yield. One approach to characterize gene function in a model system is having large populations of T-DNA insertional or transposon tagged mutants. The idea of using AcDs construct to create transposon tagged mutant populations has also been applied in many species. Here we transformed two species, Fragaria vesca and a monoploid potato, Solanum phureja 1-3-516, which is the progenitor of the sequenced doubled monoploid clone, with the same AcDs construct, Ac-DsATag-Bar_gosGFP, to generate mutant collection, compare the marker gene performance and transposition efficiency, as well as characterizing phenotypic mutants with genes of interest. Transposants were found to reinsert to unlinked sites from the launch pad site in the strawberry genome, whereas in potato transposants tended to locate locally from the launch pad position when using the same construct. One transposon based activation tagging strawberry mutant, with its insertion in the promoter region of gene of interest in strawberry from the Ac-DsATag-Bar_gosGFP population was studied. In a segregating T2 population, expression level of the candidate gene, epidermis-specific secreted glycoprotein EP1 precursor, was 670 fold higher in petioles of homozygotes than in wild type plants, suggesting the function of this gene involved in maintaining mechanical strength of petioles. Since the often-used transposase gene was cloned from the monocot species maize, the efficiency of obtaining germinal transposants was many times lower than expected in order to saturate the genome for diploid species. In order to improve the chance of getting unique transposants, we attempted to codon optimize the transposase gene, as well as switching to microspore specific promoters that had been well characterized to control timing of expression of the transposase gene. Transposants were found in both T0 primary regenerates and anther culture derived potatoes using both the pAcDs-AtSCP and pAcDs-AmDEFH125 constructs. Sequencing of the empty donor site revealed that excision occurred in different cells during anther culture. A strawberry mutant with sugar transport deficiency due to T-DNA insertion near a sucrose transporter-2 gene showing stunted phenotype with increased level of anthocyanin was also characterized. The concentrations of sucrose, glucose, and fructose were significantly greater in source leaves of the mutant than wild type plants, suggesting these compounds might be substrates of this gene in transporting to sink leaves and roots. / Ph. D.