We used the GENECONV program, the Hsu et al. (2010) method and phylogenetic analyses to analyze the gene conversions which occurred in the growth hormone, folate receptor and trypsin gene families of six primate species. Significant positive correlations were found between sequence similarity and conversion length in all but the trypsin gene family. Converted regions, when compared to non-converted ones, also displayed a significantly higher GC-content in the growth hormone and folate receptor gene families. Finally, all detected gene conversions were found to be less frequent in conserved gene regions and towards functionally important genes. This suggests that purifying selection is eliminating all gene conversions having a negative functional impact.

We used the GENECONV program, the Hsu et al. (2010) method and phylogenetic analyses to analyze the gene conversions which occurred in the growth hormone, folate receptor and trypsin gene families of six primate species. Significant positive correlations were found between sequence similarity and conversion length in all but the trypsin gene family. Converted regions, when compared to non-converted ones, also displayed a significantly higher GC-content in the growth hormone and folate receptor gene families. Finally, all detected gene conversions were found to be less frequent in conserved gene regions and towards functionally important genes. This suggests that purifying selection is eliminating all gene conversions having a negative functional impact.

Transposable elements (TEs) have had immense impact on the structure, function and evolution of eukaryotic genomes. The work in this thesis identified Kat1, a novel domesticated DNA transposase of the hAT family in the yeast Kluyveromyces lactis. Kat1 triggers a genome rearrangement that results in a switch of mating type from MATa to MATα. Furthermore, Kat1 acts on sequences that presumably are ancient remnants of a long-lost transposable element. Therefore, Kat1 provides a remarkable example of the intricate relationship between transposable elements and their hosts. We showed that Kat1 generates two DNA double strand breaks (DSBs) in MATa and that the DDE motif and several other conserved amino acid residues are important for Kat1 cleavage activity. DNA hairpins were formed on one end of the DSBs whereas the DNA between the DSBs was joined into a circle. Kat1 was transcriptionally activated by nutrient limitation through the transcription factor Mts1 and negatively regulated by translational frameshifting. In conclusion, Kat1 is a highly regulated domesticated transposase that induces sexual differentiation.  In another study, we developed an assay to measure switching rates in K. lactis and found that the switching rate was ~6x10-4 events/generation. In a genetic screen for mutations that increased mating-type switching, we found mutations in the RAS1 gene. The small GTPase Ras1 regulates cellular cyclic AMP levels and we demonstrated that Mts1 transcription is regulated by the RAS/cAMP pathway and the transcription factor Msn2. Since Ras activity is regulated by nutrient availability, these data likely explains why nutrient limitation induces mating-type switching.

The rates and patterns at which nucleotide substitutions occur vary significantly across the genome sequence of vertebrates. A prominent example is the difference in the rate of evolution of functional sequences versus nonfunctional (neutrally evolving) sequences, which is explained by the influence of natural selection on functional sequences. However, even within neutrally evolving sequences there is striking variation in the rates and patterns of nucleotide substitutions. Unraveling the underlying processes that induce this variation is necessary to understand the basic principles of variation in neutral substitution profiles, which in turn is crucial for the identification of regions in the genome where natural selection acts. This research question builds the main focus of the present thesis. I have studied the causes and consequences of variation in different patterns of nucleotide substitutions. In particular, I have investigated substitutional strand asymmetries in mammalian genes and could show that they result from the asymmetric nature of DNA replication and transcription. Comparative analysis of substitutional asymmetries then suggested that the organization of DNA replication and the level of transcription are conserved among mammals. Further, I have examined the variation in CpG mutation rate among human genes and could show that beside DNA methylation also GC content plays a decisive role in CpG mutability. In addition, I have studied the signatures of GC-biased gene conversion and its impact on the evolution of the GC isochore structure in chicken. By comparison of the results in chicken to previous results in human I found evidence that karyotype stability is critical for the evolution of GC isochores. Finally, beside the empirical studies, I have performed theoretical investigations of substitution rates in functional sequences. More precisely, I have explored the temporal dynamics of estimates of the ratio of non-synonymous to synonymous substitution rates dN/dS in a phylogentic-population genetic framework.

Understanding the mechanism of recombination has important implications for genome evolution and genomic variability. The work presented in this thesis studies the properties of recombination by investigating the effects it has on genome evolution in humans and dogs. Using alignments of human genes with chimpanzee and macaque orthologues we studied substitution patterns along the human lineage and scanned for evidence of positive selection. The properties mirror the situation in human non-coding sequences with the fixation bias ‘GC-biased gene conversion’ (gBGC) as a driving force in the most rapidly evolving regions. By assigning candidate genes to distinct classes of evolutionary forces we quantified the extent of those genes affected by gBGC to 20%. This suggests that human-specific characters can be prompted by the fixation bias of gBGC, which can be mistaken for selection. The gene PRDM9 controls recombination in most mammals, but is lacking in dogs. Using whole-genome alignments of dog with related species we examined the effects of PRDM9 inactivation. Additionally, we analyzed genomic variation in the genomes of several dog breeds. We identified that non-allelic homologous recombination (NAHR) via sequence identity, often GC-rich, creates structural variants of genomic regions. We show that these regions, which are also found in dog recombination hotspots, are a subset of unmethylated CpG-islands (CGIs). We inferred that CGIs have experienced a drastic increase in biased substitution rates, concurrent with a shift of recombination to target these regions. This enables recurrent episodes of gBGC to shape their distribution. The work presented in this thesis demonstrates the importance of meiotic recombination on patterns of molecular evolution and genomic variability in humans and dogs. Bioinformatic analyses identified mechanisms that regulate genome composition. gBGC is presented as an alternative to positive selection and is revealed as a major factor affecting allele configuration and the emergence of accelerated evolution on the human lineage. Characterization of recombination-induced sequence patterns highlights the potential of non-methylation and establishes unmethylated CGIs as targets of meiotic recombination in dogs. These observations describe recombination as an interesting process in genome evolution and provide further insights into the mechanisms of genomic variability.

We used the GENECONV program, the Hsu et al. (2010) method and phylogenetic analyses to analyze the gene conversions which occurred in the growth hormone, folate receptor and trypsin gene families of six primate species. Significant positive correlations were found between sequence similarity and conversion length in all but the trypsin gene family. Converted regions, when compared to non-converted ones, also displayed a significantly higher GC-content in the growth hormone and folate receptor gene families. Finally, all detected gene conversions were found to be less frequent in conserved gene regions and towards functionally important genes. This suggests that purifying selection is eliminating all gene conversions having a negative functional impact.

The concept of species in bacteria is a matter of contention. The current definition is based on DNA-DNA hybridisation and does not account for evolutionary forces that are important in demarcating species. In this thesis we investigate two evolutionary forces that are important in speciation in bacteria, propose novel statistical models for them and infer parameters of interest. We present the first attempt at inferring the bias in the recombination process from whole bacterial genomes. Despite empirical evidence that recombination is biased and theoretical results that this bias is important in speciation, it is usually ignored. We propose a coalescent based model that accounts for the bias in the recombination process. We use approximate Bayesian computation for inference and describe an efficient method for simulating from the model. We show that our method performs well on simulated datasets and is robust to slight misspecification of the history of the samples. Application of our method to a Bacillus cereus dataset shows that it contain evidence that the recombination process depends on the evolutionary distance between donors and recipients. We demonstrate that the rate of bias in the recombination process for this dataset is far lower than what theoretical studies require for the spontaneous generation of populations that can be called species under neutral model. Next we propose a model for occurrence of adaptive events on a phylogenetic tree. We use the model to infer the boundaries of clusters on a phylogenetic tree that correspond to ecologically distinct lineages. we characterise our method using simulated datasets and show that it is conservative in estimating the number of adaptive events. Finally we apply our method to two bacterial datasets of Salmonella enterica and Vibrionaceae. We show that there is decisive evidence that isolates in these datasets partition into numerous ecologically distinct lineages and use our method to delineate the boundaries of these lineages.

We used the GENECONV program, the Hsu et al. (2010) method and phylogenetic analyses to analyze the gene conversions which occurred in the growth hormone, folate receptor and trypsin gene families of six primate species. Significant positive correlations were found between sequence similarity and conversion length in all but the trypsin gene family. Converted regions, when compared to non-converted ones, also displayed a significantly higher GC-content in the growth hormone and folate receptor gene families. Finally, all detected gene conversions were found to be less frequent in conserved gene regions and towards functionally important genes. This suggests that purifying selection is eliminating all gene conversions having a negative functional impact.

B cell development in chicken takes place in a specific primary lymphoid organ, the bursa of Fabricius. The bursa is considered to provide a microenvironment that promotes B lymphocyte survival and maturation. The most important maturation step in the bursa is the immunoglobulin (Ig) gene conversion, a process that is responsible for immunoglobulin repertoire in avian species. The Ig-gene conversion is strictly regulated, and only progenitors that are able to initiate the process will develop into fully functional B lymphocytes. In this study the late embryonic B lymphocyte stages are investigated, the bursal stem cell stage and the onset of Ig-gene conversion stage. Previous studies identified functional and phenotypic differences between the two stages, showing high rate of proliferation at both, but a significant increase in apoptotic activity at the onset of gene conversion stage. The molecular basis behind the initiation of Ig-gene conversion is not well understood. Here two approaches are presented to provide information on the B lymphocyte developmental process. In chapter II proteomic analysis of the two cell stages was performed. The proteins were sorted into functional groups and signal transductions pathways were identified that are associated with proliferation, differentiation, cell adhesion and apoptosis. The project identified differences in protein profiles that might explain the changes in B lymphocyte physiology and bursal microenvironment at the initiation of Ig-gene conversion. In chapter III the antigen recognized by a bursal secretory dendritic cell specific monoclonal antibody, GIIF3 was identified and cloned. The antigen was shown to be expressed by bursal secretory dendritic cells only during the late embryonic period. The antigen was identified as smooth muscle gamma actin. Futher work will investigate what role the gene plays in dendritic cell funtion.

Understanding how genetic changes ultimately affect morphology and physiology is essential for understanding and predicting how organisms will adapt to environmental changes. Although most traits are complex and involve the interplay of many different genetic loci, some exceptions exist. These include the convergent evolution of tetrodotoxin resistance in snakes, which has a simple genetic basis and can be used as a model system to investigate the genetic basis of adaptive evolution. Tetrodotoxin is a potent neurotoxin used as a chemical defense by various animals, including toxic newts. Snakes have evolved resistance through mutations in voltage-gated sodium channels, the protein targets of tetrodotoxin, sparking an evolutionary arms race between predator and prey. In this dissertation, I describe how genomic rearrangements have led to sex-linkage of four of the voltage-gated sodium channel genes in snakes and compare allele frequencies across populations and sexes to make inferences about how sex linkage has influenced the evolution of resistance in garter snakes. By measuring gene expression in different snake tissues, I show that three of these sex-linked sodium channel genes are dosage compensated in embryos, adult muscle, and adult brain. In contrast, two channels show sexual dimorphism in their expression levels in the heart, which may indicate differences in dosage compensation among tissues. I then use comparative genomics to track the evolutionary history of tetrodotoxin resistance across all nine sodium channel genes in squamate reptiles and show how historical changes have paved the way for full-body resistance in certain snakes. Finally, I use targeted sequence capture to obtain the sodium channel sequences of salamanders and show evidence that tetrodotoxin self-resistance in toxic newts was likely accelerated through gene conversion between resistant and non-resistant sodium channel paralogs. Together, these results illustrate parallelism in evolutionary mechanisms and processes contributing to the appearance of an extreme and complex trait that arose independently in two distinct taxa separated by hundreds of millions of years. / Doctor of Philosophy / Western North America is the site of an ongoing battle between highly toxic species of salamanders (toxic newts) and their garter snake predators. In certain regions, garter snakes have countered newt defenses by evolving resistance to their toxins, and the newts have become more toxic in response. This interaction has been the focus of scientists for decades because it teaches us about the ways in which animals can respond to changes in their environment. In living organisms, DNA is used a blueprint to determine the ultimate traits that are expressed (e.g., whether an organism will have five fingers or four, or whether it will be resistant or sensitive to a toxin). By comparing DNA sequences of different life forms, we are beginning to understand the rules that determine how these blueprints are read and how they can change over time. Because life is built upon the same basic building blocks (DNA, mRNA, and proteins), information about this snake-newt system can be used to understand the way that other systems, such as humans and pathogens, might interact. In my dissertation, I compare DNA sequences from snakes and lizards to identify the history of changes leading to the extreme toxin resistance in the garter snakes. I show that toxin resistance began hundreds of millions of years ago, with all lizards having a low baseline level of resistance, and that resistance built up slowly in the lineages leading to garter snakes. I also show that because of DNA rearrangements, female snakes have fewer copies of some of the genes involved in resistance, and this may have led to differences among the sexes. Lastly, I compare DNA sequences among salamanders, revealing a similar pattern to that in snakes and lizards. Specifically, newts have evolved self-resistance to their own toxin, and this has happened gradually over hundreds of millions of years, with all salamanders having some toxin resistance. I also show that an unusual process occurred within the DNA of toxic newts, resulting in a rapid change from toxin sensitivity to toxin resistance in some genes. Taken together, this work helps advance our understanding of the processes and limitations that determine how organisms can function and change over time.