Insects form close partnerships, or symbioses, with bacteria. These partnerships allow the insects to use resources that would be unavailable to them otherwise. Certain insects, hemipterans, are able to feed on nutrient-poor plant-sap because these bacteria supplement their diets. While this association is beneficial for both the insect and bacteria, it also comes with consequences: the genomes of bacterial symbionts typically undergo extreme degradation, becoming small and lacking many genes necessary for typical bacterial functioning. In the Hemiptera, aphids, mealybugs, cicadas, true bugs,and their relatives, these bacterial partnerships tend to be stable over millions of years. However, adelgids (Aphidoidea: Adelgidae) are highly unusual in that their symbiotic bacteria have been frequently replaced. These replacements offer a unique opportunity to explore the effects of symbiont role and age on symbiont genome degradation. My dissertation uses the pattern of adelgid symbiont gains and losses to understand the process of symbiont replacements and co-symbiont gain. I accomplished this by sequencing and annotating the genomes of adelgid symbionts from across the family, first focusing on the symbionts from a pest species, then expanding to representatives from across the family, and finally conducting an in-depth exploration of how the genomes of a symbiont found in two branches of the adelgids varies between species. Through this work I demonstrate that adelgid symbionts are nutritional providers, they have a unique pattern for distributing the work of providing nutrients between the symbiont pairs, and that a symbiont’s precedence, whether it was there first or whether it joined another symbiont, has an impact on genome degradation.

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The brain is incredibly complex, with billions of diverse cells performing a variety of necessary functions. It is fascinating then, that a small group of progenitor cells are capable of generating all of the neural cell types. During development, robust and stable expression of identity factors is necessary for diverse cell fate determination, but progenitor cells must also be flexible to quickly change expression programs in response to developmental cues. The metazoan genome is non-randomly organized, and this organization is thought to underlie cell type specific gene expression programs. However, the process by which genome organization is stabilized, and then reorganized, is not well-understood. A Drosophila neuroblast nuclear factor, Distal antenna (Dan), was previously identified as a key regulator of this process. Downregulation of Dan is necessary for a developmentally-timed genome reorganization in neural progenitors that terminates their competence to specify early-born cell types. Maintaining Dan expression prevents genome reorganization, extending the early competence window, and implicating Dan in the stabilization of the early competence state. The mechanisms through which Dan functions to stabilize the genome architecture is not known. In this work, we take advantage of the Drosophila embryonic ventral nerve cord model system to study Dan and its role in regulating neuroblast competence. We find that Dan, a DNA- binding protein that localizes throughout the nucleus in distinct puncta, coalesces into large, liquid condensates that relocalize to the nuclear periphery when DNA-binding is inhibited. The size of the droplets increases as impairment to the DNA-binding domain increases, suggesting that Da normally exists in a competitive tug-of-war between genome binding and protein condensation at the nuclear periphery. We further find that while Dan is a highly intrinsically disordered protein, formation of the large droplets requires a LARKS domain – a glycine-rich, structural motif that forms kinked beta-sheets associated with labile interactions that underlie phase-separation. In embryos, Dan’s ability to maintain neural progenitor early competence requires both its Pipsqueak motif DNA-binding domain and phase separation properties. Finally, we find that Dan interacts with proteins of the nuclear pore complex. In particular, we find that Elys, a core scaffold protein which has been shown to bind DNA and regulate nuclear architecture, is required for termination of the early competence window. Together, we propose a mechanism by which a single protein can exert opposing forces between DNA binding and self- association to organize progenitor genome architecture and regulate neuronal diversification.

DNA damage is a constant threat to our genomes which drives genome instability and contributes to cancer progression. DNA damage interferes with important DNA transactions such as transcription and replication. DNA lesions are removed by repair pathways that ensure genome stability during transcription and replication. Here, we identify and characterize distinct roles for the ultra violet stimulated scaffold protein A (UVSSA) in the maintenance of genome stability during transcription in human cells. First, we unravel a novel function for UVSSA in transcription-coupled repair of DNA interstrand crosslinks (ICLs), genotoxic adducts that covalently bind opposing strands of the DNA and block transcription and replication. UVSSA knockout cells are sensitive to ICL inducing drugs, and UVSSA is specifically required for transcription-coupled repair of ICLs in a fluorescence-based reporter assay. Based on analysis of the UVSSA protein interactome in crosslinker treated cells we propose a model for transcription-coupled ICL repair (TC-ICR) that is initiated by stalling of transcribing RNA polymerase II (Pol II) at an ICL. Stalled Pol II is first bound by CSA and CSB, followed by UVSSA which recruits TFIIH to initiate downstream lesion removal steps. Second, we establish that UVSSA counteracts MYC dependent transcription stress to promote genome stability in cells aberrantly expressing the cMYC oncogene. UVSSA knockdown sensitizes cells to MYC expression, resulting in synthetic sickness and increased doubling time. UVSSA knockdown impacts Pol II dynamics in MYC activated cells. We conclude that UVSSA is required for regulation of Pol II during MYC induced transcription to prevent transcription stress. Together, these studies expand our understanding of UVSSA’s role in genome stability during transcription and elucidates the poorly understood transcription-coupled ICL repair pathway.

Genomic instability is a hallmark of cancer that can be caused by various forms of DNA replication stress. Collision of the replication fork with obstacles ahead of the replication machinery can result in replication fork stalling and collapse. To overcome or bypass these obstacles and resume proper replication fork progression, the cell has various replication restart mechanisms involving homologous recombination (HR) that help preserve genome integrity and ensure cell survival. However, HR can also lead to genome rearrangements, particularly when recombination occurs between repetitive DNA sequences. The tandem duplicator phenotype found in breast and ovarian cancer is an example of a genome-wide instability configuration that has been associated with pathways related to homologous recombination and replication stress, but the exact mechanism of how these duplicated sequences are formed is not fully understood. To examine the molecular mechanisms regulating genome instability in response to replication stress, we have established a genetic system in Saccharomyces cerevisiae to detect recombination events that result in tandem duplications (TDs) and deletions. Using this system, we investigated the mechanisms of recombination upon site-specific replication fork stalling initiated by a protein-induced replication fork barrier. We have found that a Tus/Ter-induced fork block downstream of direct repeats results in an induction in recombination events resulting in TDs and deletions compared to spontaneous frequencies, and that these recombination events have specific genetic requirements. Mainly focusing on the recombination mechanisms generating Tus/Ter-induced TDs, we determined that formation of these TDs is dependent on Rad52, Rad51, the Mph1 translocase, and structure-selective endonucleases, and that these events appear to be enhanced by disruption of the MRX complex and sister chromatid cohesion. We also found that genetic requirements for recombination in response to fork stalling by a protein-DNA barrier are distinct from those involved in fork collapse at a nick. Taken together, these studies give insight into the mechanisms governing copy number variation in the context of replication fork stalling, which may ultimately provide a better understanding of how replication stress contributes to cancer and other diseases characterized by genome instability.

Isolating novel phages using Streptomyces azureus, which produces antibiotic thiostrepton, as a host, and characterizing the genomes may help us to find new tools that could be used to develop antibiotics in addition to contribute to the databases of phages and specifically, Streptomyces phages. Streptomyces phages Alsaber, Omar, Attoomi, Rowa, and ZamZam were isolated using during this study. They were isolated from enriched soil and sequenced by Illumina sequencing method. They were isolated from three different geographical regions. They are siphoviridae phages that create small clear plaques with a diameter of approximately 0.5-1 mm, except for Rowa which has cloudy plaques, and they have varied sizes of their heads and tails. ZamZam was not characterized at this time. The sequencing shows that they are circular genome with 3' sticky overhang and various genomes' sizes with high percentage of GC content with the average of 66%. Alsaber was classified under sub-cluster BD3, while Omar was categorized under sub-cluster BD2. They share the same cluster of Cluster BD. Rowa was placed in Cluster BL and Attoomi is currently a singleton that does not fit into an established cluster. Alsaber yields 76 putative genes with no tRNA, Omar 81 putative genes with 1 tRNA. Attoomi 53 putative genes with no tRNA, and Rowa with 61 orfs and 7 tRNA. Rowa also was a putative temperate phage due to its lysogenic activity, and Row was not able to reinfect the lysogenic strain, S. azureus (Rowa). All of the isolated phages infected S. indigocolor, while only Attoomi and Rowa were able to infect S. tricolor. Upon completion of this project, we acquired more data and understanding of S. azureus phages and Actinobacteriophage in general, which will expand the scale of future research of Streptomyces bacteriophages.

Next-generation sequencing has generated enormous amount of DNA and RNA sequences that potentially carry volumes of genetic information, e.g. protein-coding genes. The thesis is divided into three main parts describing i) GeneMarkS-2, ii) GeneMarkS-T, and iii) MetaGeneTack. In prokaryotic genomes, ab initio gene finders can predict genes with high accuracy. However, the error rate is not negligible and largely species-specific. Most errors in gene prediction are made in genes located in genomic regions with atypical GC composition, e.g. genes in pathogenicity islands. We describe a new algorithm GeneMarkS-2 that uses local GC-specific heuristic models for scoring individual ORFs in the first step of analysis. Predicted atypical genes are retained and serve as ‘external’ evidence in subsequent runs of self-training. GeneMarkS-2 also controls the quality of training process by effectively selecting optimal orders of the Markov chain models as well as duration parameters in the hidden semi-Markov model. GeneMarkS-2 has shown significantly improved accuracy compared with other state-of-the-art gene prediction tools. Massive parallel sequencing of RNA transcripts by the next generation technology (RNA-Seq) provides large amount of RNA reads that can be assembled to full transcriptome. We have developed a new tool, GeneMarkS-T, for ab initio identification of protein-coding regions in RNA transcripts. Unsupervised estimation of parameters of the algorithm makes unnecessary several steps in the conventional gene prediction protocols, most importantly the manually curated preparation of training sets. We have demonstrated that the GeneMarkS-T self-training is robust with respect to the presence of errors in assembled transcripts and the accuracy of GeneMarkS-T in identifying protein-coding regions and, particularly, in predicting gene starts compares favorably to other existing methods. Frameshift prediction (FS) is important for analysis and biological interpretation of metagenomic sequences. Reads in metagenomic samples are prone to sequencing errors. Insertion and deletion errors that change the coding frame impair the accurate identification of protein coding genes. Accurate frameshift prediction requires sufficient amount of data to estimate parameters of species-specific statistical models of protein-coding and non-coding regions. However, this data is not available; all we have is metagenomic sequences of unknown origin. The challenge of ab initio FS detection is, therefore, twofold: (i) to find a way to infer necessary model parameters and (ii) to identify positions of frameshifts (if any). We describe a new tool, MetaGeneTack, which uses a heuristic method to estimate parameters of sequence models used in the FS detection algorithm. It was shown on several test sets that the performance of MetaGeneTack FS detection is comparable or better than the one of earlier developed program FragGeneScan.

Considerable variation exists both within and between species in nuclear DNA content. Despite there being no obvious functional role for much of this DNA, many studies have reported phenotypic correlations with genome size at various taxonomic levels. This suggests that DNA plays a functional role beyond the traditionally understood mechanisms. One such example of a phenotypic correlation with DNA content is present in the genus Silene, where a negative correlation between DNA content and flower size exists within and between species. This relationship is consistent with the direction of sexual dimorphism in DNA content (caused by heteromorphic sex-chromosomes) and flower size in the most studied species in the genus: S. latifolia. This thesis takes a comparative approach between two closely related species in the genus (S. latifolia and S. marizii), which differ markedly in their nuclear DNA content, in order to investigate the nature and phenotypic impacts of variation in DNA content. A phenotypic survey from a number of S. marizii populations reveals that the pattern of DNA content variation in this species is very different to that in S. latifolia. In particular, phenotypic correlations with DNA content appear be much weaker, whilst sexual dimorphism in DNA content, when present, appears to occur in either direction. A survey of interspecific hybrids suggests that this may be due to an enlarged S. marizii X-chromosome and that DNA content in hybrids may be biased with regard to their parents. Repetitive elements may be significant constituents of plant genomes. A study of Ty1-copia class retrotransposons in the two species reveals that they are present as a large and highly heterogeneous population. Phylogenetic analysis of these elements suggests a substantial degree of genetic isolation between the two species. Finally, an assessment of the flow-cytometric method, used to estimate DNA content, reveals substantial error associated with the method, but only limited evidence for stoichiometric effects.

Significant alterations in the environmental conditions can have pronounced effects on plant genome stability. Recent evidence argues for a global involvement of the components of epigenetic modules in the regulation of genome homeostasis both immediately after stress exposure and long after environmental cues were acquired. The last observation is of particular interest as the memory of imposing stress can be maintained at the molecular level throughout plant ontogenesis and may be faithfully propagated into the following generation. Our study provides evidence that epigenetic repercussions exerted by stress exposure of parental plants manifest themselves in untreated progeny at all three levels of the epigenetic module: DNA methylation, histone posttranslational modifications and small RNA metabolism. Additionally, the results of our study shed new light on the engagement of the epigenetic machinery in the maintenance of plant genome integrity by counteracting the activity of invading nucleic acids. / xv, 280 leaves : ill. ; 29 cm

L’inférence de génomes ancestraux est une étape essentielle pour l’étude de l’évolution des génomes. Connaissant les génomes d’espèces éteintes, on peut proposer des mécanismes biologiques expliquant les divergences entre les génomes des espèces modernes. Diverses méthodes visant à résoudre ce problème existent, se classant parmis deux grandes catégories : les méthodes de distance et les méthodes de synténie. L’état de l’art des distances génomiques ne permettant qu’un certain répertoire de réarrangements pour le moment, les méthodes de synténie sont donc plus appropriées en pratique. Nous proposons une méthode de synténie pour la reconstruction de génomes ancestraux basée sur une définition relaxée d’adjacences de gènes, permettant un contenu en gène inégal dans les génomes modernes causé par des pertes de gènes de même que des duplications de génomes entiers (DGE). Des simulations sont effectuées, démontrant une capacité de former une solution assemblée en un nombre réduit de régions ancestrales contigües par rapport à d’autres méthodes tout en gardant une bonne fiabilité. Des applications sur des données de levures et de plantes céréalières montrent des résultats en accord avec d’autres publications, notamment la présence de fusion imbriquée de chromosomes pendant l’évolution des céréales. / Ancestral genome inference is a decisive step for studying genome evolution. Knowing genomes from extinct species, one can propose biological mecanisms explaining divergences between extant species genomes. Various methods classified in two categories have been developped : distance based methods and synteny based methods. The state of the art of distance based methods only permit a certain repertoire of genomic rearrangements, thus synteny based methods are more appropriate in practice for the time being. We propose a synteny method for ancestral genome reconstruction based on a relaxed defenition of gene adjacencies, permitting unequal gene content in extant genomes caused by gene losses and whole genome duplications (WGD). Simulations results demonstrate our method’s ability to form a more assembled solution rather than a collection of contiguous ancestral regions (CAR) with respect to other methods, while maintaining a good reliability. Applications on data sets from yeasts and cereal species show results agreeing with other publications, notably the existence of nested chromosome fusion during the evolution of cereals.