Next-generation sequencing (NGS) has revolutionized the field of genetics by providing a means for fast and relatively affordable sequencing. With the advancement of NGS, whole- genome sequencing (WGS) has become more commonplace. However, sequencing an entire genome is still not cost effective or even beneficial in all cases. In studies that do not require a whole-genome survey, WGS yields lower sequencing depth and sequencing of uninformative loci. Targeted sequencing utilizes the speed and low cost of NGS while providing deeper coverage for desired loci. This thesis applies targeted sequencing to the genomes of two different, non-model plants, Artemisia tridentate (sagebrush) and Lupinus luteus (yellow lupine). We first targeted the transcriptomes of three species of sagebrush (Artemisia) using RNA-seq. By targeting the transcriptome of sagebrush we have built a resource of transcripts previously unmatched in sagebrush and identify transcripts related to terpenes. Terpenes are of growing interest in sagebrush because of their ability to identify certain species of sagebrush and because they play a role in the feeding habits of the threatened sage-grouse. Lastly, using paralogs with synonymous mutations we reconstructed an evolutionary time line of ancient genome duplications. Second, we targeted the flanking loci of recognition sites of two endorestriction enzymes in genome of L. luteus genome through genotyping-by-sequencing (GBS). GBS of yellow lupine provided enough single-nucleotide polymorphic loci for the construction of a genetic map of yellow lupine. Additionally we compare GBS strategies for plant species without a reference genome sequence.

In eukaryotes, sequences that code for the amino acid structure of proteins represent a small fraction of the total sequence space in the genome. These are referred to as coding sequences, whereas the remaining majority of the genome is designated as noncoding. Studies of translation, the process in which a ribosome decodes a coding sequence to synthesize proteins, have primarily focused on coding sequences, mainly due to the belief that translation outside of canonical coding sequences occurs rarely and with little impact on a cell. However, recently developed techniques such as ribosome profiling have revealed pervasive translation in a diverse set of noncoding sequences, including long noncoding RNAs (lncRNAs), introns, and both the 5’ and 3’ UTRs of mRNAs. Although proteins with amino acid sequences derived partially or entirely from noncoding regions may be functional, they will often be nonfunctional or toxic to the cell and therefore need to be removed. Translation outside of canonical coding regions may further expose the noncoding genome to selective pressure at the protein level, leading to the generation of novel functional proteins over evolutionary timescales. Despite the potentially significant impact of these processes on the cell, the cellular mechanisms that function to detect and triage translation in diverse noncoding regions, as well as how peptides that escape triage may evolve into novel functional proteins, remain poorly understood.This thesis will describe novel findings that offer new insight into the process of noncoding translation mitigation revealed by a combination of high-throughput systems-based approaches and validated by biochemical and genetic approaches. Chapter 1 will discuss general concepts in the translation of noncoding sequences and the relevant cellular systems and impacts on human health. Chapter 2 will discuss the results of a high-throughput reporter assay investigating translation in thousands of noncoding sequences from diverse sources. The results discussed in this chapter revealed two factors involved in the mitigation of proteins derived from noncoding sequences: C-terminal hydrophobicity and proteasomal degradation. Chapter 3 will build on Chapter 2 and discuss the results of a genome-wide CRISPR/Cas9 knockout screen that identified the BAG6/TRC35/RNF126 membrane protein chaperone complex as a key cellular pathway in the detection and degradation of proteins with translated noncoding sequences. Having identified the BAG6 complex as targeting a specific reporter of translation of the 3’ UTR in the AMD1 gene, a series of knockout cell lines validated these results and demonstrated the participation of two additional genes, SGTA and UBL4A. Through coimmunoprecipitation western blots and rescue assays with flow cytometry as a readout, we confirmed physical interaction between BAG6 and the 3’ UTR of AMD1, and a similar experiment confirmed interaction between BAG6 and a readthrough mutant of the SMAD4 tumor suppressor gene. Finally, by combining our high-throughput reporter library with our BAG6 knockout cell line, we demonstrated that BAG6 targets hydrophobic C-terminal tails in many noncoding sequences of diverse origin. Finally, Chapter 4 will discuss the evolutionary perspective of noncoding translation through analyses of the sequence content of human and mouse genomes. The findings of this chapter demonstrate a significant trend for increased uracil content in noncoding regions of the genome, which frequently results in the translation of hydrophobic amino acids. We also find that many functional translated noncoding peptides localize to membranes, providing a theoretical link between the shuttling of translated noncoding sequences to a protein complex involved in membrane protein quality control and the emergence of newly evolving proteins from the noncoding genome.

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.

Rapidly accumulating genomic data as a result of high-throughput sequencing has necessitated development of efficient computational methods to decode the biological information underlying these data. DNA composition varies across structurally or functionally different regions of a genome as well as those of distinct evolutionary origins. We adapted an integrative framework that combines a top-down, recursive segmentation algorithm with a bottom-up, agglomerative clustering algorithm to decipher compositionally distinct regions in genomes. The recursive segmentation procedure entails fragmenting a genome into compositionally distinct segments within a statistical hypothesis testing framework. This is followed by an agglomerative clustering procedure to group compositionally similar segments within the same framework. One of our main objectives was to decipher distinctive evolutionary patterns in sex chromosomes via unraveling the underlying compositional heterogeneity. Application of this approach to the human X-chromosome provided novel insights into the stratification of the X chromosome as a consequence of punctuated recombination suppressions between the X and Y from the distal long arm to the distal short arm. Novel "evolutionary strata" were identified particularly in the X conserved region (XCR) that is not amenable to the X-Y comparative analysis due to massive loss of the Y gametologs following recombination cessation. Our compositional based approach could circumvent the limitations of the current methods that depend on X-Y (or Z-W for ZW sex determination system) comparisons by deciphering the stratification even if only the sequence of sex chromosome in the homogametic sex (i.e. X or Z chromosome) is available. These studies were extended to the plant sex chromosomes which are known to have a number of evolutionary strata that formed at the initial stage of their evolution, presenting an opportunity to examine the onset of stratum formation on the sex chromosomes. Further applications included detection of horizontally acquired DNAs in extremophilic eukaryote, Galdieria sulphuraria, which encode variety of potentially adaptive functions, and in the taxonomic profiling of metagenomic sequences. Finally, we discussed how the Markovian segmentation and clustering method can be made more sensitive and robust for further applications in biological and biomedical sciences in future.

Bacteria live under constant assault by bacteriophages and have evolved a diverse array of defense strategies. CRISPR-Cas systems are prokaryotic adaptive immune systems that rely on RNA-guided binding for the recognition and degradation of invading nucleic acids. Intriguingly, some bacteria also encode divergent CRISPR-Cas systems that can bind to — but cannot degrade — target nucleic acids. In this dissertation, I describe the study of nuclease-deficient CRISPR-Cas systems alongside the evolutionary pressures that led to their persistence in bacterial genomes. I present experimental data for the existence of CRISPR-associated transposons (CASTs) that utilize the RNA-guided DNA binding ability of Type I-F CRISPR-Cas systems to direct transposition to new target sites in a heterologous Escherichia coli host. This RNA-guided DNA integration pathway can tolerate large cargos of up to 10 kilo-base pairs in size, and is highly specific for the programmed target site, as determined by deep sequencing experiments. We further reveal the physical link between the CRISPR-Cas and transposition machineries through biochemical experiments and by determining cryo-EM structures of the transposition protein TniQ in complex with the CRISPR-Cas effector. After bioinformatic analyses and experimental validation we established an array of twenty diverse CAST systems for which a subset works completely orthogonally. This dataset revealed the modular nature of CASTs by showcasing the horizontal acquisition of targeting modules and by characterizing a system that encodes both a programmable, RNA-dependent pathway, and a fixed, RNA-independent pathway. Further analysis of the transposon-encoded cargo genes uncovered the striking presence of anti-phage defense systems, suggesting a role in transmitting innate immunity between bacteria. Finally, we exploit high-throughput screening assays to determine the specific sequence and spacing requirements of the transposon ends, and use this knowledge to develop a CAST-mediated endogenous gene-tagging approach. Intriguingly, our experiments uncover the involvement of a previously unknown cellular protein, integration host factor (IHF), which is critical for transposition of VchCAST, but not other homologous systems. Collectively, the work presented in this dissertation describes the discovery of RNA-guided DNA integration employed by CASTs, substantially advances our biological understanding of these systems, and expands the suite of RNA-guided transposases for programmable, large-scale genome engineering.

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No description available.

<p> Two studies are presented in this thesis. First part is about the origin of tRNA genes in Trypanosoma and Leishmania. These organisms have special mitochondrial DNA, termed kinetoplast DNA (kDNA), which is unique in its structure and function. kDNA is a massive network which is composed of thousands of connected DNA circles. Unlike most other mitochondrial genomes, there is no gene encoding tRNAs in their kDNAs. So all the tRNAs used in mitochondria must be encoded on nuclear genes and transported from the cytoplasm into the mitochondria. So our question of interest is where the tRNA genes in their nucleus come from. We carry out phylogenetic analysis of these genes and the corresponding ones in bacteria, mitochondria and eukaryotic nuclei. There is no evidence indicating gene transfer from mitochondria to nucleus on the basis of this analysis. These results are consistent with the simplest hypothesis, i.e. that all tRNA genes of Trypanosoma and Leishmania have the same origin as nuclear genes of other eukaryotes.</p> <p> The second part is about the comparison of eukaryote phylogenies obtained from mitochondrial rRNA and protein sequences. We carried out phylogenetic analysis for the species which have complete mitochondrial genomes by using both concatenated mitochondrial rRNA and protein sequences. We got phylogenies for three groups, fungi/metazoan, plant/algae and stramenopile/alveolate group. The analysis is useful for the further study of position of the genetic code changes and the mechanisms involved.</p> / Thesis / Master of Science (MSc)

Genome evolution is not always shaped by a Darwinian-fashion of vertical inheritance from ancestral lineages. The historical gene content of a species contains many atypical gene sequences showing high similarity to those of distantly related taxa. This evolutionary phenomenon is referred to as lateral gene transfer (LGT). Lateral gene transfer permits the exchange of genetic material across lineages, completely ignoring any concept of taxonomic boundary. The rapid acquisition of foreign genes into bacterial genomes has greatly obscured the historical phylogeny of prokaryotes. In this thesis we calculate the rate of LGT on a Bacillaceae phylogeny, to determine the extent to which it controls species evolution. First, we examined the evolution of the phylogeny according to a simple model of maximum likelihood. We assume equal rates of gene insertion and deletion on the phylogeny and show high rates of evolution in the genomes of B. anthracis, B. cereus, and B. thuringiensis (Bc group), representative of adaptive evolution. We then improved the model to account for differential rates of gene insertion and deletion, thus offering a more realistic model of gene evolution. Again, we demonstrate that members of the Bc group are rapidly evolving, with the rate of gene insertion being significantly higher than the rated of gene deletion. Finally, we evaluate the sole effect of LGT on the phylogeny in a simple birth-death analysis with immigration. We show that LGT is the main vehicle of gene acquisition when the number of gene families substantially increases from external taxa to members of the Bc group. Collectively, our findings suggest that the Bacillaceae genome is rapidly expanding, and that laterally transferred genes may facilitate adaptive evolution and subsistence in a new niche. / Thesis / Master of Science (MSc)

Much of our understanding of eukaryotic life has come from studying mitochondrial DNA, giving rise to leading hypotheses in evolution. To enable these studies, efficient algorithms are needed to interpret, analyze, and draw relevant conclusions from the available mitochondrial sequence data. The central theme of this work is to provide such algorithms for two biological problems in mitogenomes. The key element of both methods is the de Bruijn graph. Small sequence segments of length k, called k-mers, of the genomes represented in the graph form the vertices. Two vertices are connected if the suffix of length (k-1) of the first vertex is equal to the prefix of length (k-1) of the second vertex. The edges are thus specified by the (k+1)-mer consisting of the k-prefix of the first vertex and the last character of the second vertex. The first problem is the automated accurate annotation of genes in complete mitochondrial sequences. For this purpose, a new method, called DeGeCI, is presented. The method uses a large collection of mitogenomes whose sequence data is represented as an annotated de Bruijn graph. To annotate an input genome sequence, initially, a subgraph induced by all (k+1)-mers of the sequence is constructed. Unmapped parts of the sequence result in disconnected components in this subgraph, which are bridged in the next step. For this purpose, alternative trails with a high sequence similarity to the respective unmapped subsequences of the input genome are identified in the database graph and added to the subgraph. Using a clustering approach, DeGeCI aggregates annotations contained in the resulting subgraph to obtain gene predictions for the input sequence. The thesis also presents the follow-up version of DeGeCI, which offers additional features and, in contrast to DeGeCI, can be used via a web server front-end. Genome rearrangements, which change the arrangement of the genes in the genome, are particularly common in mitogenomes. The locations in the genomes where the gene order differs are called breakpoints. The second objective of this thesis is to localize these breakpoints in the nucleotide sequences of complete mitochondrial genomes, taking into account possible high substitution rates. A novel method, DeBBI, is presented to address this task. The method constructs a colored de Bruijn graph of the input sequences, where each color is associated with one of the sequences. This graph is searched for certain structures that can be associated with the breakpoint locations. These so-called breakpoint bulges are common paths that branch into two separate paths and rejoin again at another location. One of the branches is short and of a single color, while the other branch is long and color-alternating. Sequence dissimilarities distort these structures by introducing additional branches. To identify the bulges despite these distortions, DeBBI uses a heuristic algorithm.