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Transposable elements (TEs) are interspersed repetitive sequences that are present in most genomes. Miniature inverted repeat transposable elements (MITEs) are the most numerous Class II elements in higher eukaryotes. Little is known about their origin, transposition and function. In this study, three novel MITE families (Kiddo, MDM1 and MDM2) were identified in the rice genome. They bear terminal inverted repeats (TIRs) and show target site duplications (TSDs) at the insertion sites. Each family is present in hundreds of copies with length that range from 200 bp to 400 bp. An evolutionary relationship between Mutator elements and MDM1 and MDM2 family was established. The absence of an observed transposition event, together with the mutated ancestral elements identified by in silico analysis, led to a conclusion that Kiddo and its autonomous elements are not presently active. To overcome laborious and time consuming manual analysis of MITEs on a genomic scale, MAK, a computational tool kit, was developed to automatically retrieve MITE sequences, their neighboring genes and ancestral elements from genome sequences. MAK has been functionally tested and is now available to the research community. Studies on the effect of MITE (Kiddo and MDM1) insertions into a rice ubiquitin (rubq2) promoter revealed a two-edged role of MITEs on gene regulation. While Kiddo and MDM1 contribute ~40% to rubq2 promoter activity, they also induce progressive silencing of this promoter. The evolutionary implications of the two-edged role of MITEs in gene regulation are discussed.

Ma thèse vise à caractériser l’évolution dynamique et l’organisation des génomes des différentes espèces du blé (genres Triticum et Aegilops) en relation avec la prolifération des éléments transposables (TEs) dans leur génome (>80%), les polyploïdisations récurrentes ainsi que la syntenie avec d’autres espèces de la famille des Poaceae. En constituant des sets de séquences génomiques représentatives et en étudiant la variabilité entre des haplotypes des génomes du blé, j’ai caractérisé la dynamique et la prolifération différentielle des TEs qui est la résultante de l’équilibre entre leurs insertions et aussi leurs éliminations actives. Le taux moyen de remplacement de l’espace TEs, mesurant les différences de séquences dues aux insertions et aux délétions entre deux haplotypes, a été ainsi estimé à 86% par million d’années (Ma) et dépasse celles bien documentées du maïs. Les insertions des TEs mais aussi leurs éliminations par recombinaisons illégitimes de l’ADN (pouvant atteindre plusieurs dizaines de kb) ainsi que les recombinaisons génétiques entre haplotypes divergents représentent les principaux mécanismes à la base des changements rapides de l’espace TEs. Sur une échelle d’évolution plus longue (60 Ma), j’ai analysé la conservation des gènes et l’évolution du locus (Ha) entre différentes espèces des Poaceae. J’ai pu ainsi préciser l’émergence du caractère grain tendre et des gènes Ha, comme nouveaux membres de la famille des gènes de Prolamine, dans l’ancêtre commun des Pooideae (blé et Brachypodium, de la tribu des Triticeae et des Brachypodieae) et des Ehrhartoideae (riz), après leur divergence des Panicoideae (maïs, sorgho). / My PhD aims to characterize dynamic evolution and organization of wheat genomes from différent species (Triticum and Aegilops genera) in relation to transposable element (TE) proliferation in their genomes (>80%), polyploidizations and synteny with other Poaceae species. By constituting and comparing representative genomic sequences and analyzing haplotype variability of the wheat genomes, I have characterized dynamics and differential proliferation of TEs, as resulting from the combinations of their insertions and deletions. Mean replacement rate of the TE space, which measures sequence differences due to insertion and removal of TEs between two haplotypes, was estimated to 86% per one million year (My). This is more important than the well-documented haplotype variability found in maize. It was observed that TE insertions and DNA elimination by illegitimate recombination (implicating several ‘tens’ of kb) as well as homologous recombination between divergent haplotypes represent the main molecular basis for rapid change of the TE space. At a longer evolutionary scale (60 My), I have compared gene conservation at the Ha locus region between different Poaceae species. The comparative genome analysis and evolutionary comparison with genes encoding grain reserve proteins of grasses suggest that an ancestral Ha-like gene emerged, as a new member of the Prolamin gene family, in a common ancestor of the Pooideae (wheat and Brachypodium from the Triticeae and Brachypodieae tribes) and Ehrhartoideae (rice), between 60 and 50 My, after their divergence from Panicoideae (Sorghum).

Vector control is an essential and effective approach for controlling transmission of vector-borne diseases. However, increasing resistance to insecticide and drugs suggests that new strategies to control vector-borne diseases are needed. One possible strategy involves replacing mosquito populations with disease-resistant transgenic mosquitoes. Transposable elements (TEs) are an important component in this new strategy due to their ability to integrate exogenous DNA into chromosomes. They could potentially be useful tools in assisting the spread of disease-resistant genes in mosquito populations. This research focuses on two related subjects, TEs and their regulation. The first subject is on a Long Terminal Repeat (LTR) retrotransposon in the African malaria mosquito, Anopheles gambiae, namely Belly. The second subject focuses on the characterization of piwi genes in the dengue and yellow fever mosquito, Aedes aegypti. For the first subject we characterized Belly by identifying the two identical LTRs and one intact open reading frame. We also defined the target site duplications and boundaries of the full-length Belly element. This novel retrotransposon has nine full-length copies in the An. gambiae sequenced genome and their nucleotide similarity suggests that there has been fairly recent retrotransposon. We have shown that Belly is transcribed and translated in An. gambiae. Single LTR circles were recovered from An. gambiae cells, which is consistent with active transposition of Belly. The second subject focuses on the piwi genes of Ae. aegypti. We found nine potential piwi genes in Ae. aegypti and two in An. gambiae. Phylogenetic analysis suggests that these piwis formed two subgroups and gene duplication within each group occurred after the divergence between the two mosquito species. RT-PCR and transcriptome analysis showed Ago3 as well as all the seven tested piwi genes were expressed either in germline tissues or developing embryos. Differential expression patterns were observed. While most piwis were transcribed in the ovaries, testis, and embryos, two piwis appear to have a zygotic expression. Three piwi genes (piwi 3, piwi 4, and Ago3) were also detected in adult somatic tissues of Ae. aegypti. The expansion of the number of piwi genes in Ae. aegypti compared to An. gambiae and D. melanogaster may be correlated with a larger genome size and greater amount of TEs. The finding of piwi expression in adult somatic tissues is intriguing. It is possible that these piwi genes were expressed in the adult stem cells. It is also possible that they may be involved with anti-viral defense. Both of these hypotheses require further testing. / Master of Science

Doctor of Philosophy / Division of Biology / Mark C. Ungerer / Nuclear genome size varies immensely across flowering plants, spanning nearly 2400-fold. The causes and consequences of this vast amount of variation have intrigued biologists since it became clear that nuclear DNA amount did not reflect organismal complexity (the so-called C-value paradox). In my dissertation I utilize wild sunflower species in the genus Helianthus to examine the role of transposable elements (TEs), and in particular, long terminal repeat (LTR) retrotransposons, in generating genome size variation and whether variation in genome size influences aspects of plant growth and development across multiple organizational levels. The genus Helianthus provides an excellent system for studying these questions given four-fold variation in nuclear DNA content among diploid species and well-resolved phylogenetic relationships. Utilizing short-read Illumina data and sequence information from a diverse panel of Helianthus annuus (common sunflower) full-length LTR retrotransposons, I found that nuclear genome size in Helianthus species is positively correlated with repetitive DNA, and LTR retrotransposon subtypes generally show similar patterns in genomic abundance across taxa. Helianthus species with the largest genomes, however, exhibit large-scale amplification of a small number of LTR retrotransposon subtypes. Measuring aspects of plant growth and development at cell-, organ- and whole plant-levels in a panel of diploid Helianthus species that vary 4-fold in nuclear genome size, I found that genome size is negatively correlated with cell production rate, but that this negative correlation does not persist at higher organizational levels. Taken together, these results provide insights into the mechanisms contributing to genome size evolution in plants and the organizational level at which genome size may impact growth patterns and developmental rates. Genome expansion in wild sunflowers is influenced most significantly by amplification of a small number of TEs and not necessarily by a greater diversity of TEs. Genome size is strongly negatively correlated with cell production rate but this relationship weakens at higher organizational levels, such as that of organ and whole-plant development.

There are two classes of transposable elements: DNA transposons and retrotransposons. DNA transposons spread in the genome by “cut and paste” mechanism. In contrast, retrotransposons use copy and paste strategy involving RNA and retrotranscriptase mediated mechanism; these include long interspersed nuclear elements-1 (LINE-1, L1) and short interspersed nuclear elements (SINE). In mammals, in order to maintain genome integrity both types of transposons are tightly repressed. However, some copies of retrotransposons are still active in germ cells contributing to natural variation. Surprisingly, recent reports indicate that also somatic cells support L1 reactivation in early development, in particular in the brain leading to mosaicism. However, whether L1 retrotransposition is a part of other cell lineage developmental programs and its functional significance in the context of cell differentiation remain to be elucidated. To address this question, I investigated whether L1 retrotransposition was occurring during in vitro osteogenic and adipogenic differentiation of bone marrow derived human mesenchymal stem cells (hMSCs). Interestingly, clinical observations have revealed loss of bone density in HIV-infected individuals treated with nucleoside analogs that inhibit HIV retrotranscriptase, as well as the endogenous one encoded by L1s. This observation made us to hypothesize that transposable elements played a positive role in post-natal bone homeostasis. I found that while adipogenesis is “retrotransposition free”, osteogenic differentiation is a “retrotransposition-prone” process and its inhibition blocks its genetic program. Indeed, L1 DNA content does not change during adipogenic differentiation and that of retrotranscriptase does not have any effect on the acquisition of a terminally differentiated phenotype. In contrast, soon after MSCs commitment into pre-osteoblasts, L1 retrotransposable elements increase their expression and actively transpose. Inhibition of retrotransposition and knock down of L1 mRNA strongly impairs matrix deposition. Moreover, I forced L1 expression in in vitro adipogenesis, by directly delivering L1 mRNA to the cells. Interestingly, overexpression of L1 elements was detrimental for in vitro adipogenesis. Then, I performed loss of function experiments in osteogenesis by directly targeting and degrading the L1 endogenous transcript. This experiment confirmed the positive role of L1 reactivation in the osteogenic context, suggesting also a possible role for L1 RNA, distinct from retrotransposition.

Many factors may explain why certain transposable elements (TEs) spread in some species and not others. On the one hand, they include processes that affect the rate of transposition, such as differences in the regulation of expression; on the other hand, they include characteristics of a genome that affect the consequences of transposition. In particular genome size may have an effect: a genome that is large due to non-essential repetitive DNA may be permissive for TE movement, as insertion events are less likely to be deleterious. Genome size may also help explain the pattern of TE distribution between species of mosquitoes, including the important vectors of arboviruses, Aedes aegypti and Culex pipiens sensu lato. These species have genomes 3- 5 times larger than a third genus, the Anopheles mosquitoes, which includes the malaria vectors. While all mosquitoes carry a diverse range of TEs, only culicines have the super abundant retroposon, Juan which can contribute up to 3% of the genome. The genome sequences of various insect species were compared and the mosquitoes show a significant trend of increase in genome size, which can be attributed to the increase in retroposon sequences. Two variants of Juan are reported, and new information is added regarding these elements. Previous publication of these elements contained errors in their sequences. A unique triple repeat of a cysteine rich region with a CCHC motif is present in the open reading frame. This sequence is a zinc-knuckle domain, important for the replication mechanism of these elements. In comparison, a third recently active but very low copy number retroposon, termed Pip1, is also described. The results show that Pip1 is related to the Juan elements and also possess the triple CCHC motif. The PCR results also supports previous findings of polymorphism in insertion sites of this element, suggesting that Pip1 was active after the establishment of the different strains. Pip1 copies can be grouped into three distinct groups based on nucleotide differences. Pip1 could also be using an alternative start codon to initiate transcription. Full length intact copies of the three TEs in this study were been cloned into a germline transformation vector based on piggyBac and used for germline transformation in Drosophila melanogaster. Drosophila melanogaster has no Juan or Pip1 elements and an even smaller genome than anophelines mosquitoes, so insertion events from unregulated TE movement should be more detectable. We found that the elements have been successfully introduced into the Drosophila lines. The lines were inbred to obtain a homozygous population. A range of transformed lines were monitored. No effects of hybrid dysgenesis was found. Flies with black spotted eyes were identified in a Pip1 line but this phenotype was not heritable. Whole genome sequencing was carried out on the flies using next generation sequencing (NGS) technology. Retroposon sequences was detected at a high frequency. Insertion junctions were not detected but this result does not eliminate the possibility that a junction is present but the sequencing was not sensitive enough. A possible explanation is the retroposon is present as extrachromosomal plasmid DNA.

Thesis (Ph. D.)--Cornell University,1989. / Vita. Includes bibliographical references.

While the study of transposable element evolution has been conducted in several model insect organisms such as Anopheles gambiae, Drosophila melanogaster, and Bombyx mori, little investigation has been conducted into the transposable element (TE) evolution within less commonly examined model and non-model taxa within Diptera. In this work we contributed two analyses to close this gap. First, TEs in the lepidopteran, Heliconius melpomene, were characterized, and it was determined that 25% of the genome is composed of TEs. Second, TEs in oestroid and muscid flies were characterized using survey sequencing rather than whole genomes. Comparative analyses were performed on Haematobia irritans, Sarcophaga crassipalpis, Phormia regina, and Cochliomyia hominivorax. TE proportions were 5.95%, 10.00%, 22.43%, and 30.67%, for C. hominivorax, P. regina, S. crassipalpis and H. irritans, respectively. These studies provide new insights into the diversity of TEs in Insecta and suggest that in general, TE diversity is high among insects.

Transposable elements (TEs), also known as jumping genes, are DNA sequences capable of mobilizing and replicating within the genome. In mammals, it is not uncommon for 50% of the genome to be derived from TEs, yet they remain an underutilized tool for tracking evolutionary change. With the increasing number of publicly funded genome projects and affordable access to next-generation sequencing platforms, it is important to demonstrate the role TEs may play in helping us understand evolutionary patterns. The research presented herein utilizes TEs to investigate such patterns at the genomic, specific, and generic levels in three distinct ways. First at the genomic level, an analysis of the historical TE activity within the thirteen-lined ground squirrel (Spermophilus tridecemlineatus) shows that non-LTR retrotransposon activity has been declining for the past ~26 million years and appears to have ceased ~5 million years ago. Since most mammals, and all other rodents studied to date, have active TEs the extinction event in S. tridecemlineatus makes it a valuable model for understanding the factors driving TE activity and extinction. Second, we examined TEs as factors impacting genomic and species diversity. We found that DNA transposon insertions in Eptesicus fuscus, appear to have been exapted as miRNAs. When placed within a phylogenetic context a burst of transposon-driven, miRNA origination and the vespertilionid species radiation occurred simultaneously ~30 million years ago. This observation implies that lineage specific TEs could generate lineage specific regulatory pathways, and consequently lineage specific phenotypic differences. Finally, we utilized TEs to investigate their phylogenetic potential at the level of genus. In particular a method was developed that identified, over 670 thousand Ves SINE insertions in seven species of Myotis for use in future phylogenetic studies. Our method was able to accurately identify insertions in taxa for which no reference genome was available and was confirmed using traditional PCR and Sanger sequencing methods. By identifying polymorphic Ves insertions, it may be possible to resolve the phylogeny of one of the largest species radiations in mammals.