Philosophiae Doctor - PhD / The plasticity of single-stranded viral genomes permits the formation of secondary structures through complementary base-pairing of their component nucleotides. Such structures have been shown to regulate a number of biological processes during the viral life-cycle including, replication, translation, transcription, post-transcriptional editing and genome packaging. However, even randomly generated single-stranded nucleotide sequences have the capacity to form stable secondary structures and therefore, amongst the numerous secondary structures formed in large viral genomes only a few of these elements will likely be biologically relevant. While it is possible to identify functional elements through series of laboratory experiments, this is both excessively resource- and time-intensive, and therefore not always feasible. A more efficient approach involves the use of computational comparative analyses methods to study the signals of molecular evolution that are consistent with selection acting to preserve particular structural elements. In this study, I systematically deploy a collection of computationally-based molecular evolution detection methods to analyse the genomes of viruses belonging to a number of ssRNA viral families (Alphaflexiviridae, Arteriviridae, Caliciviridae, Closteroviridae, Coronavirinae, Flaviviridae, Luteoviridae, Picornaviridae, Potyviridae, Togaviridae and Virgaviridae), for evidence of selectively stabilised secondary structures. To identify potentially important structural elements the approach incorporates structure prediction data with signals of natural selection, sequence co-evolution and genetic recombination. In addition, auxiliary computational tools were used to; 1) quantitatively rank the identified structures in order of their likely biological importance, 2) plot co-ordinates of structures onto viral genome maps, and 3) visualise individual structures, overlaid with estimates from the molecular evolution analyses. I show that in many of these viruses purifying selection tends to be stronger at sites that are predicted to be base-paired within secondary structures, in addition to strong associations between base-paired sites and those that are complementarily co-evolving. Lastly, I show that in recombinant genomes breakpoint locations are weakly associated with co-ordinates of secondary structures. Collectively, these findings suggest that natural selection acting to maintain potentially functional secondary structures has been a major theme during the evolution of these ssRNA viruses.

Please read the abstract in the section 00front of this document / Dissertation (MSc (Microbiology))--University of Pretoria, 2006. / Microbiology and Plant Pathology / unrestricted

Ceratocystis fimbriata is the type species for the genus Ceratocystis and was first described as the causal agent of black rot in sweet potatoes. However, evidence from DNA sequence data suggests that C. fimbriata is in fact a species complex (C. fimbriata sensu lato) consisting of many morphologically similar cryptic species. Species in this complex are pathogens of important root and fruit crops and trees in the forestry industry world-wide. Population studies on some of these species have mainly relied on microsatellite markers. However, nothing is known regarding the microsatellite structure within Ceratocystis species or any species in the order Microascales in which Ceratocystis resides. The need for a more robust identification tool is also required to differentiate between species in this complex. The first chapter of this thesis provides a review of the literature on microsatellite markers, particularly in fungi. It also discusses the history of microsatellites, mechanisms of microsatellite evolution and functional importance in selected fungal examples. In addition, isolation methodologies are compared and contrasted to newly developed techniques that include bioinformatic searches of genome sequences. Opportunities to use and develop microsatellite markers in Ceratocystis species is also discussed with an emphasis on the possibilities that more microsatellites markers would provide. Microsatellites are abundant in eukaryotic genomes, and fungi are no exception. Analyses of microsatellite content in eukaryotic and fungal genomes have shown that fungi contain fewer microsatellites and that each organism shows preference for particular motifs. In Chapter 2 of this thesis, the abundance and distribution of microsatellites in the recently sequenced C. fimbriata genome is investigated. Comparisons to other fungi and eukaryotes show that C. fimbriata follows the general pattern of microsatellite structure, however it is unique in its preference for certain motifs. The C. fimbriata sensu lato species complex contains morphologically indistinct species. Microsatellite markers previously developed for a population study could differentiate between some of the cryptic species based on their geographic location and host-specificity. In Chapter 3 a subset of microsatellite markers identified in gene regions in Chapter 2 are used to develop a diagnostic test to differentiate between species in the complex. Microsatellite markers that are polymorphic between species but monomorphic within species were selected for this purpose. However, not all species could be distinguished using this diagnostic test. This thesis is presented as a series of chapters in which Chapters 3 2 and 3 are in manuscript format. Consequently each chapter represents an independent article and repetition between these chapters has been unavoidable. / Dissertation (MSc)--University of Pretoria, 2014. / Genetics / Unrestricted

Masters of Science / Viral genomes consist of either deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). The viral RNA molecules are responsible for two functions, firstly, their sequences contain the genetic code, which encodes the viral proteins, and secondly, they may form structural elements important in the regulation of the viral life-cycle. Using a host of computational and bioinformatics techniques we investigated how predicted secondary structure may influence the evolutionary dynamics of a group of single-stranded RNA viruses from the Picornaviridae family. We detected significant and marginally significant correlations between regions predicted to be structured and synonymous substitution constraints in these regions, suggesting that selection may be acting on those sites to maintain the integrity of certain structures. Additionally, coevolution analysis showed that nucleotides predicted to be base paired, tended to co-evolve with one another in a complimentary fashion in four out of the eleven species examined. Our analyses were then focused on individual structural elements within the genome-wide predicted structures. We ranked the predicted secondary structural elements according to their degree of evolutionary conservation, their associated synonymous substitution rates and the degree to which nucleotides predicted to be base paired coevolved with one another. Top ranking structures coincided with well characterized secondary structures that have been previously described in the literature. We also assessed the impact that genomic secondary structures had on the recombinational dynamics of picornavirus genomes, observing a strong tendency for recombination breakpoints to occur in non-coding regions. However, convincing evidence for the association between the distribution of predicted RNA structural elements and breakpoint clustering was not detected.

Longtemps utilisés en palynologie pour l’étude des paléo-environnements, les coprolithes, excréments fossilisés, sont également d’importantes sources d’information pour des espèces disparues, producteurs ou proies. Grâce à de nombreux échantillons archéologiques provenant d’une dizaine de grottes, comme la Grotte Chauvet-Pont d’Arc, deux espèces ont pu être étudiées : le loup, Canis lupus, et l’hyène des cavernes, Crocuta crocuta spelaea.À partir d’un coprolithe de canidé, daté à 34 500 ans, un génome mitochondrial complet de Canis lupus a pu être reconstitué. Les analyses phylogénétiques ont montré que ce spécimen se situe en dehors de la diversité des chiens et loups actuels. Puis, les analyses menées sur des gènes nucléaires ont montré que le spécimen de Chauvet ne présente pas de traces évidentes de domestication. L’étude du coprolithe met en évidence un régime carné, avec un bol alimentaire comportant des traces d’ADN d’ours des cavernes, Ursus spelaeus.Dans un second temps, grâce à l’étude de nombreux coprolithes d’hyène des cavernes, une alimentation variée composée d’animaux de grande comme de petite taille a été mise en évidence pour ce carnivore. L’analyse plus fine des séquences d’ADN contenues dans l’un des échantillons a permis de reconstituer un génome mitochondrial complet pour une espèce aujourd’hui éteinte : le bison des forêts, Bison schoetensacki. En parallèle, grâce à l’étude d’un ossement de bison des steppes, Bison priscus, un génome mitochondrial complet a été obtenu pour cette espèce éteinte. L’ajout de ces deux nouvelles séquences mitochondriales à la phylogénie des bovidés a permis d’apporter des éclaircissements à cette dernière. / Coprolites have long been used in palynology for paleoenvironments reconstruction. They also are an important source of information on the DNA of the producing species and its diet. Using numerous archeological samples from several caves, including the Chauvet-Pont d’Arc cave, we studied coprolites for two species: the wolf, Canis lupus and the cave hyena, Crocuta crocuta spelaea.Using a canid coprolite from the Chauvet cave, dated back to 34 500 years, we obtained a complete mitochondrial genome sequence. Phylogenetic analyses highlight a maternal lineage that positions outside the diversity of extant dogs and wolfs. Then, analyzes conducted on the nuclear genes showed that the Chauvet canis lupus specimen does not display obvious indication of domestication. Analysing the coprolite for other species to indicate the diet of this specimen, we detected cave bear (Ursus spelaeus) DNA sequences.Second, using many cave hyena coprolites, a flexible diet consisting of large as well as small animals was demonstrated for this extinct carnivore. Focusing the analysis on a coprolite samples that contained large amounts of bovine DNA, we obtained for the first time a complete mitochondrial genome sequence for the extinct European forest bison, Bison schoetensacki. In parallel, a bone sample for the extinct steppe bison provided the first complete mitochondrial genome sequence for Bison priscus. These two genome sequences shed new lights on the phylogeny of Bovinae.

Genomic diversity is shaped by the interplay between mutation, genetic drift, recombination, and natural selection. A major goal of evolutionary biology is to understand the relative contribution of these different microevolutionary forces to patterns of genetic variation both within and across species. The advent of massive parallel sequencing technologies opened new avenues to investigate the extent to which alternative evolutionary mechanisms impact the genome and the footprints they leave. We can leverage genomic information to, for example, trace back the demographic trajectory of populations and to identify genomic regions underlying adaptive traits. In this dissertation, I employ genomic data to explore the role of demography and natural selection in two New World bird systems distributed along steep environmental gradients: the Altamira Ori-ole (Icterus gularis), a Mesoamerican bird that exhibits large variation in body size across its range, and the Hairy and Downy woodpecker (Dryobates villosus and D. pubescens), two sympatric species whose phenotypes vary extensively in response to environments in North America. In Chapter 1, I combine ecological niche model, phenotypic and ddRAD sequencing data from several individuals of I. gularis to investigate which spatial processes best explain geographic variation in phenotypes and alleles: (i) isolation by distance, (ii) isolation by history or (iii) isolation by environment. I find that the pronounced genetic and phenotypic variation in I. gularis are only partially correlated and differ regarding spatial predictors. Whereas genomic variation is largely explained by historical barriers to gene flow (IBH), variation in body size can be best predicted by contemporary environmental heterogeneity (IBE), which is consistent with a pattern produced by either natural selection or environmental plasticity. In Chapter 2, I conduct whole genome resequencing on 140 individuals of Downy and Hairy Woodpecker from across North America to more explicitly elucidate the impact of demography and natural selection on the genome. I find that despite spatial congruence in allele frequencies, population structure in these two species has been produced at different temporal scales. Whereas Hairy Woodpeckers were isolated into two east-west glacial refugia, Downy woodpecker populations seem to have expanded from a single ancestral refugium. Demographic analyses suggest large variation in Ne over the past one million years in both Hairy and Downy Woodpeckers, with repeated episodes of bottleneck followed by population expansion, consistent with the onset of the climatic oscillations of the Pleistocene. Nucleotide diversity in both species was positively correlated with recombination rate and negatively correlated with gene density, suggesting the effect of linked selection. The magnitude of this effect, however, seems to have been modulated by the individual demographic trajectory of populations and species. Nevertheless, patterns of nucleotide diversity along the genome are highly correlated between Hairy and Downy Woodpecker, which may be attributed to pervasive selection acting on a conserved genomic landscape of recombination. Finally, in Chapter 3, I use a suite of statistical methods to scan the genome of Hairy and Downy Woodpecker for signatures of natural selection associated with population-specific environmental differences. I test whether climatic adaptation was achieved through selection on the same loci in both species, which would indicate parallel genetic mechanisms for adaptation. I find limited evidence of genomic parallelism at the SNP level, but large parallelism at the gene level. Candidate genes were involved in a broad range of biological processes, including immune response, nutritional metabolism, mitochondrial respiration, and embryonic development. Lastly, I identify potential candidates for key phenotypic traits in Downy and Hairy Woodpecker, such as genes in the IGF signaling pathway, putatively linked to differences in body size, and the melanoregulin gene (MREG), potentially involved in plumage variation. Together, these findings highlight the significant role of demography and natural selection in shaping genomic variation.

Gene expression regulation is a set of critical biological processes that give rise to the diversity of cell types across tissues and development stages. Noncoding regions of the genome (intergenic + intronic, >98% of genome) play an important role in these processes, with noncoding genetic variation quantitatively affecting transcriptional activity, splicing of pre-mRNA, and localization, stability, and translational control of mRNA transcripts. Previous genetic studies of human disease have implicated numerous common noncoding loci with small but significant effect in common conditions. Recently, we and others have reported evidence supporting a role of rare noncoding variants with larger effect in early onset conditions such as birth defects and neurodevelopmental disorders. These early onset conditions are quite common in aggregate, affecting over 3% of young children. A better understanding of the functional impact of rare regulatory noncoding variants will enable novel genetic discovery, give insights of disease mechanisms, and ultimately improve diagnosis, treatment, and clinical care. In this thesis dissertation, I describe three related projects. First, we used a combinatorial multi-testing framework to find excess burden of noncoding de novo mutations in congenital heart disease (impacting both transcriptional and post-transcriptional regulatory stages). This finding was central to the rest of my work, motivating the development of new computational approaches to predict genetic effect of noncoding variants through the lens of post-transcriptional regulation. Second, we used convolutional neural networks to model and understand sequence specific RBP binding processes. Finally, we designed a graphical neural network model capable of integrating cause and consequence to predict genetic effect of rare noncoding variants. In summary, we developed new machine learning methods to analyze multimodal human genome sequencing data, uncover deeper insights into post-transcriptional gene regulatory processes, and advance genomic medicine.

DNA end-resection and nuclear actin-based movements orchestrate clustering of double-strandbreaks (DSBs) into homology-directed repair (HDR) domains. Using genomic approaches, we analyze how actin nucleation by ARP2/3 affects damage-dependent and -independent 3D genome reorganization and facilitates pathologic repair. Chromosome conformation capture techniques (Hi-C) reveal multi-scale alterations in genome organization following damage, including changes in chromatin insulation and compartmentalization. Nuclear actin polymerization promotes interactions between DSBs, which in turn facilitates aberrant intra- and inter-chromosomal rearrangements as visualized by high-throughput translocation assays (HTGTS). Notably, BRCA1 deficiency, which decreases end-resection, DSB mobility, and subsequent HDR, nearly abrogates recurrent translocations between AsiSI DSBs. In contrast, loss of functional BRCA1 yields unique translocations genome-wide, reflecting a critical role in preventing spontaneous genome instability and subsequent rearrangements. Our work establishes that the assembly of DSB repair domains is coordinated with multiscale alterations in genome architecture that enable HDR despite increased risk of translocations with pathologic potential.

Maintenance of genomic integrity is essential for the survival of an organism and its ability to pass genetic information to its progeny. However, DNA is constantly exposed to exogenous and endogenous sources of damage, which demands cells to possess DNA repair mechanisms. Of the many forms of DNA damage, double-strand breaks (DSBs) are particularly cytotoxic DNA lesions that cause genome instability and cell lethality, but also provide opportunities to manipulate the genome via repair. One of the major DSB repair pathways shared between single-celled yeast and humans is homologous recombination (HR). HR is initiated by the evolutionarily conserved Mre11-Rad50-Xrs2/Nbs1 (MRX in yeast, MRN in mammals) complex. The MRX complex has a multitude of functions such as damage sensing, adduct removal from DSB ends, and end tethering – a process to maintain the two ends of a DSB in close proximity. The role of the MRX complex has been uncovered by studying the repair of DSBs generated from meganucleases such as HO and I-SceI. However, it is unclear if this knowledge translates to the repair of DSBs from genome editing nucleases such as TALEN and CRISPR-Cas9 (Cas9), as these nucleases create DSBs with different end polarities. While the repair efficiencies and outcomes of TALEN and Cas9 are actively studied, less is known about the earlier stages of repair. The objective of this thesis is to examine the role of the MRX complex in repair processes at both ends of a DSB after cleavage with I-SceI, TALEN, and Cas9 in vivo using the model organism Saccharomyces cerevisiae. In Chapter 1, I describe the importance of DSB repair, a summary of HR and its sub-pathways, the functions of the MRX complex, and properties of I-SceI, TALEN, and Cas9. The materials and methods used in this thesis are detailed in Chapter 2. The work described in Chapter 3 focuses on end tethering and recruitment of downstream repair proteins in haploid cells. I find that DSB ends from the three nucleases all depend on the MRX complex for end tethering, and that initial end polarity does not affect tethering. DSBs created by Cas9 show greater dependence on the Mre11 nuclease of the MRX complex for Rad52 recruitment compared to DSBs from I-SceI and TALEN. Despite Mre11-dependent end processing and Rad52 recruitment at Cas9-induced DSBs, Cas9 stays bound to one DNA end after cleavage, irrespective of the MRX complex. These results suggest that Mre11 exonuclease activity required for adduct removal from DSB ends is not critical for Rad52 recruitment, and that Mre11 endonuclease activity may be driving processing of Cas9-bound DSBs. I also find that MRX tethers DSB ends even after Rad52 recruitment, and unexpectedly, untethered ends are processed asymmetrically in the absence of MRX for all three nucleases. In Chapter 4, I explore the interaction of DSB ends with their repair template, the intact homologous chromosome, in diploid cells. The primary goal is to monitor interhomolog contact in real time from homology search to completion of HR. Although technical limitations make it difficult to capture the entire HR program from DSB formation to repair, I show that untethered ends interact with the homolog separately in the absence of the MRX complex. Similar to haploids, diploid cells display defects in end tethering and end processing without the MRX complex. Repair outcomes of WT cells show an even distribution of G2 crossovers and non-crossovers, while pre-replication crossovers and break-induced replication are undetected. Overall, the results in this thesis provide insight into the functions of the MRX complex in repairing different DSB ends created by I-SceI, TALEN, and Cas9. In Chapter 5, I summarize all of these findings and discuss the motivation for future cell biology studies of HR.

No description available.