Regulation of pre-mRNA splicing is a key process for most if not all eukaryotes. The process can, in the abstract, be considered as a series of trans-acting factors that interact with cis-motifs in the RNA to enable the removal of introns and joining of exons. As the cis factors need not only be the splice sites themselves, but also motifs in the exons, the splicing process has the potential to impose selective constraint on exonic sequence in addition to the normal selection on the amino acid content of the protein. To understand this more clearly, in this thesis, I mainly focus on a type of important and widely investigated cis-motifs, exonic splicing enhancers (ESEs), which bind with SR proteins to re-enforce the splice sites and so ensure splicing correctly. First, I explore splice-related cis-motif usage of the Ectocarpus genome, which is a species phylogenetically very distant from vertebrates but, like vertebrates in having abundant large introns. A deep phylogenetic conservation of exonic splice-related constraints is observed (Chapter II). Then I extend the analysis across taxa in a phylogenetically explicit framework. In this section stronger selection on exon end synonymous sites can be detected within humans when the exons are flanked by larger introns. Additionally I report evidence that reduced Ne might lead to larger introns and weakened splice sites. Thus I suggest an unusual circumstance in which selection (for cis-motifs to control error-prone splicing) might be stronger when population sizes are smaller; this is unexpected and would be a necessary complement to nearly-neutral theory (Chapter III). Third, I ask whether what we know about biases in the usage of ESEs and splicing control elements allows us to understand where in human genes pathogenic mutations tend to occur (Chapter IV). By examining the relationship between determinants of the usage of splice-associated cis-motifs and the distribution of human pathogenic SNPs, I found certain exons are vulnerable to splice disruption owing to low ESE density and a “fragile” exon model we proposed could describe and explain this phenomenon (Chapter IV). Finally I perform preliminary analysis, with a view to biotechnological optimization of transgenes, to address whether there might be such a thing as a tissue specific ESE. To this end I examine ESE usage in tissue specific genes. I find some preliminary evidence for tissue specific biased usage of certain ESEs.

Synonymous mutations have traditionally been thought to have no significant effect on fitness. However, a growing body of recent research has shown that this is not always the case. In an experimentally evolved population of Pseudomonas fluorescens grown in minimal glucose media, synonymous mutations arose in a glucose transport gene that resulted in beneficial fitness effects comparable to those of non-synonymous mutations. We found that the increase in fitness was a direct result of increased gene expression; however, the precise mechanism was unclear. Synonymous mutations have been shown to affect gene expression on transcriptional and translational levels through changes in mRNA secondary structure and codon usage. Our study investigates the underlying mechanisms in which these evolved synonymous mutations lead to increased gene expression. In addition to the evolved mutations, we have a library of 42 strains with single synonymous mutations within the glucose transport gene and found a positive correlation between fitness and gene expression. To determine whether these mutations affect transcript levels, translational efficiency or a combination of both, we systematically incorporated transcriptional and translational fusions of a yellow fluorescent protein within the glucose transport operon. We found that the evolved mutations predominantly act on the level of transcription and have strong polar downstream effects. Additionally, through manipulation of the local genetic sequence, we investigated the specific molecular requirements necessary for the increased expression. We found that for one of our evolved synonymous mutants, mRNA secondary structure does not play an essential role, but we speculate that the mutation may strengthen a weak internal promoter sequence to confer its increased expression. Our study provides evidence of the adaptive mechanisms of beneficial synonymous mutations in an experimentally evolved setting.

Protein-coding sequences have long been assumed to evolve under selection, but the quantification of the process at the nucleotide sequence level only started when a simple null model, the neutral theory of molecular evolution, was formulated by Kimura. Several methods were developed, which were based on the assumption that synonymous sites (nucleotides at third codon positions which do not change the encoded amino acid) evolve close to neutrally, and could be used as local neutral standards. Most of our current knowledge on the direction and strength of selection still depends on this simple assumption. One method, notably the non-synonymous to synonymous substitution rate ratio (dN/dS) has gained prevalence and is still widely used, in spite of the growing body of evidence that synonymous sites evolve under selection. In this thesis, I quantify the strength of selection in different sequence compartments of mammalian genomes, in order to obtain estimates of their functional importance from comparative genomics analyses. I quantify the fraction of mutations that have been selectively eliminated since the divergence of the species pairs examined, the so called genome wide selective constraint. This in turn is used to approximate the genomic deleterious mutation rate, which is an important parameter for several evolutionary problems. As estimates of selection depend on a large extent on the chosen neutral standard, here I use orthologous transposable elements, so called ancestral repeats, as these have been found to be evolving at a largely neutral fashion, and contain the least number of constrained sites in mammalian genomes. This enables me to quantify the level of selection even at synonymous sites, and the results suggest that these sites indeed evolve under constraint, the consequences of which I discuss. The selective constraint estimates enable me to test some simple hypotheses, such as Ohta's nearly neutral theory of molecular evolution, which suggests that selection is more efficient in species with larger effective population sizes. Beside the choice of neutral standards, there are several additional factors which are known to affect the selective constraint estimates. Here I also test the consequences of one of these, notably when sequences are not at compositional equilibrium (i.e. their GC content is away from the equilibrium GC content), which predicts that sequences with different GC content should evolve with different rates. This can cause bias in the estimates of level of selection or can even imitate selection in sequences which evolve completely neutrally. This effect is quantified here, and a simple correction is discussed.

Bacteria have small, streamlined genomes and evolve rapidly. Their large population sizes allow selection to be the main driver of evolution. With advances in sequencing technologies and precise methods for genetic engineering, many bacteria are excellent models for studying elementary questions in evolutionary biology. The work in this thesis has broadly been devoted to adaptive evolution and fitness effects of different types of mutations. In Paper I we experimentally tested the fitness constrains of horizontal gene transfer (HGT), which could be used to predict how the fixation of HGT events are affected by selection and fitness effects. We found that the majority of the examined HGT inserts were indistinguishable from neutral, implying that extra DNA transferred by HGT, even though it does not confer an immediate selective advantage, could be maintained at transfer-selection balance and serve as a reservoir for the evolution of novel beneficial functions. Paper II examined why four synonymous mutations in rpsT (encoding ribosomal protein S20) reduced fitness, and how this cost could be genetically compensated. We found that the cause for the fitness reduction was low S20 levels and that this lead to a defective subpopulation of 30S subunits lacking S20. In an adaptive evolution experiment, these impairments were compensated by up-regulation of S20 though various types of mutations. In Paper III we continued the studies of how the deleterious rpsT mutations could be compensated. The mutations either down-regulated the global regulator Fis or altered a subunit of the RNA polymerase (rpoA). We found that the decreased S20 levels in the cells causes an assembly defect of the 30S particles and that the fis and rpoA mutations restored the skewed S20:ribosome ratio by both increasing S20 levels and decreasing other ribosomal components. Paper IV examined adaptation of two bacterial species to different growth media. A total of 142 different adaptive mutations were identified and 112 mutants were characterized in terms of fitness. We found that the experimental variation in fitness measurements could be reduced 10-fold by introducing some adaptive mutations prior to the experiment, allowing measurements of fitness differences as small as 0.04%.

<p>Various types of genetic constructs are widely used as diagnostic, prophylactic, and therapeutic tools for human diseases. They are also the workhorse in biotech and pharmaceutical industry for production of therapeutic antibodies and proteins. Since the majority of the genetic constructs encode protein products, it is therefore of tremendous value to human health and the society that we could find a way to fine-tune and optimize genetic constructs and hence protein expression for achieving maximal potency or long-lasting effects in therapeutics or for obtaining highest yields in pharmaceutical protein production. However, for protein-coding genes to be expressed in a heterologous host, the coding sequences need to be optimized by using synonymous codons to achieve reasonable levels of expression, if at all. Since codon optimization is done in a protein-by-protein basis with respect to specific host organisms, tissue/cell types, even health conditions, and there is no set of standard rules to follow, this process is still very unpredictable and time-consuming.</p><p>This thesis presents the development of a feasible platform for solving the problem of optimizing regular and long DNA constructs for academic or industrial purposes through the development of a novel cloning method for complex gene libraries, and based on the library expression system constructed in such manner, a platform for high-throughput screening of codon-optimized and error-corrected proteins, and a novel protocol for screening long gene constructs which could be extremely difficult to achieve by using regular screening methods. This multi-step platform has the potential for studying the natural systems: how codon bias correlates to protein expression efficiency, for generating improved pharmaceutical proteins and enhanced DNA vaccines and for constructing improved genome libraries.</p> / Dissertation

Ecological processes have the potential to influence evolution through their effects on selection. This thesis explores the effects of two ecological factors - competition and ecological opportunity. Intraspecific (within-species) competition is often expected to drive adaptation and diversification by increasing selection for the use of novel resources, thereby alleviating the detrimental effects of competition. However, this is not always the expected outcome; theory suggests that intraspecific competition can also drive convergent evolution. On the other hand, interspecific (between-species) competition is usually expected to impede adaptation and diversification because competitor species occupy potential available niches, preventing the focal species from diversifying to do so. In this thesis, I review previous experimental studies exploring the effects of competition on adaptive diversification, and then directly test these effects using experimental evolution of the bacterium Pseudomonas fluorescens. I confirm that intraspecific competition drives adaptive diversification, while the effects of interspecific competition are varied. Strong interspecific competition impedes adaptation and diversification, while the presence of weak, non-diversifying interspecific competitors drives diversification through increased resource competition. The presence of ecological opportunity is essential for adaptation and diversification, and so variation in attributes of those opportunities is expected to have important effects on the dynamics of adaptive evolution. In another evolution experiment with P. fluorescens, I tested the effects of variation in ecological opportunity on adaptive evolution and found that the type and arrangement of ecological opportunities drives adaptation but, in this system, not diversification. I also show that ecological opportunity drives differences in the degree of parallel evolution at the phenotypic and genotypic level. Finally, I explore some unexpected genetic changes identified in one of these evolved populations - two synonymous mutations that conferred fitness benefits, and show that the observed fitness improvements are the result of increased gene expression. I have shown that ecological processes can play an important role in shaping the evolutionary trajectories taken by populations. Understanding the interactions between ecological and evolutionary processes is vital for our understanding of evolutionary dynamics as a whole, and the studies laid out in this thesis represent valuable contributions to this field of study.

The human genome is highly heterogeneous in its GC composition. How codon usage affects translation rates has been extensively studied and exploited to increase protein expression. Although effects on virtually all other steps in gene expression have been reported as well, so far no systematic approach has been taken to quantitatively measure the contribution of each to overall protein levels in human cells. Here, I utilise a library of several hundred synonymous variants of the Green fluorescent protein (GFP) to characterise the influence of codon usage on gene expression in human cells. In an initial small-scale screen, I show that protein levels are largely correlated with codon-usage and particularly GC-content. Additionally, I demonstrate that these changes can already be seen on the RNA level, confirming more broadly previously published data from our lab (Kudla et al., 2006). In order to assess the consequences of randomised codon usage on a larger scale, I established and validated a high-throughput approach for the phenotypic profiling of reporter genes. Using a pool of cells stably expressing >200 GFP variants, I measured multiple parameters simultaneously, such as protein levels, translational state, RNA levels, stability and export. Data from these experiments confirm a strong relationship between GC-content, protein levels, as well as RNA export, reproducibly in two cell lines. Low expression of especially GC-poor variants could not be rescued by splicing, but increased nuclear-to-cytoplasmic RNA ratio, suggesting further mechanisms important for efficient gene expression. These effects are even more pronounced when the distribution of GC is spread evenly along the coding sequence. Interestingly, our data also suggests that high GC within the first 200nt is more predictive of efficient gene expression, contrasting studies performed on bacteria, in which strong secondary folding near the ribosomal binding site was shown to be non-permissive for translation (Kudla et al., 2009). By relating experimentally derived parameters to sequence features known to inhibit expression, I demonstrate that cryptic splicing is a major factor leading to decreased levels of particularly GC-poor GFP variants. An attempt to quantitatively assess the relative contribution of several sequence features (e.g. tAI, GC3, CpG) using multiple regression analysis lead to inconclusive results, leaving the requirement for the exploration of alternative approaches in order to dissect the role of individual parameters, as well as to identify novel determinants of gene expression.

Ecological processes have the potential to influence evolution through their effects on selection. This thesis explores the effects of two ecological factors - competition and ecological opportunity. Intraspecific (within-species) competition is often expected to drive adaptation and diversification by increasing selection for the use of novel resources, thereby alleviating the detrimental effects of competition. However, this is not always the expected outcome; theory suggests that intraspecific competition can also drive convergent evolution. On the other hand, interspecific (between-species) competition is usually expected to impede adaptation and diversification because competitor species occupy potential available niches, preventing the focal species from diversifying to do so. In this thesis, I review previous experimental studies exploring the effects of competition on adaptive diversification, and then directly test these effects using experimental evolution of the bacterium Pseudomonas fluorescens. I confirm that intraspecific competition drives adaptive diversification, while the effects of interspecific competition are varied. Strong interspecific competition impedes adaptation and diversification, while the presence of weak, non-diversifying interspecific competitors drives diversification through increased resource competition. The presence of ecological opportunity is essential for adaptation and diversification, and so variation in attributes of those opportunities is expected to have important effects on the dynamics of adaptive evolution. In another evolution experiment with P. fluorescens, I tested the effects of variation in ecological opportunity on adaptive evolution and found that the type and arrangement of ecological opportunities drives adaptation but, in this system, not diversification. I also show that ecological opportunity drives differences in the degree of parallel evolution at the phenotypic and genotypic level. Finally, I explore some unexpected genetic changes identified in one of these evolved populations - two synonymous mutations that conferred fitness benefits, and show that the observed fitness improvements are the result of increased gene expression. I have shown that ecological processes can play an important role in shaping the evolutionary trajectories taken by populations. Understanding the interactions between ecological and evolutionary processes is vital for our understanding of evolutionary dynamics as a whole, and the studies laid out in this thesis represent valuable contributions to this field of study.

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