In this thesis work, I have used a comparative genomics approach to study a fungal model organism, Neurospora tetrasperma. My specific focus has been on genomic introgression, intron evolution, chromosomal structural rearrangements and codon usage. All of the studies are based on large-scale dataset generated by next-generation sequencing technology (NGS), combined with other techniques, such as Optical Mapping. In the introgression study, we detected large-scale introgression tracts in three N. tetrasperma lineages, and the introgression showed allele-specific and chromosomal-specific pattern. In the study of introns, we found indications of mRNA mediated intron loss and non-homologous end joining (NHEJ) mediated intron gains in N. tetrasperma. We found that selection is involved in shaping intron gains and losses, and associated with intron position, intron phase and GC content. In the study of chromosomal structural rearrangements, we found a lineage specific chromosomal inversion pattern in N. tetrasperma, which indicates that inversions are unlikely to associate with the origin of the suppressed recombination and the mating system transition in N. tetrasperma. The result suggests inversions are the consequences, rather than the causes, of suppressed recombination on the mating-type chromosome of N. tetrasperma. In the final study, analyses of codon usage indicated that the region of suppressed recombination in N. tetrasperma is subjected to genomic degeneration, and selection efficiency has been much reduced in this region.

This thesis aims to address issues relating to genome architecture and base composition. The first part of this thesis addresses questions relating to codon usage. Initially I will investigate thousands of bacterial species using a detailed analysis of strengths of selection acting upon codons usage while also investigating patterns of optimal codon changes with respect to genomic base composition and tRNA abundance. I report that selection on codon usage increases throughout the length of highly expressed genes, in particular, the first quarter of genes have significantly lower selection. Further, it is clear that factors affecting genomic base composition can eventually lead to changes in optimal codons if the change in base composition is strong enough, however these patterns differ substantially between amino acids. The debate over translational efficiency vs. accuracy was addressed by comparing sites of differing conservation. Differing conservation were defined using a phylogenetic method, allowing sites to change in their extent of conservation throughout the tree. The results show that translational accuracy acts strongly on the top 10% of conserved sites, however is relatively weak when compared to the efficiency for other sites. Also detected is a reduction in apparent selection on codon usage on the bottom 10% of conserved sites which is likely to be caused by conflicting positive selection on amino acids. Finally, although differences in patterns are observed between amino acids, the general relationship to conservation is similar. As much of the variation in codon usage is determined by variation in base composition, this aspect of base composition is investigated in the second part of the thesis. The observed variation in intragenomic base composition in bacteria was found to be far higher than expected for GC-rich bacteria. The non-core part of the genome contributes to this variation to a greater extent than the core part, suggesting that processes such as AT-rich horizontal gene transfer may be involved. Secondly, base composition is modelled under Brownian motion and as an extension, the Ornstein- Uhlenbeck process, which allows for multiple optima throughout the tree. The model including optima fits the data better than standard Brownian motion or Brownian motion with multiple diffusion coefficients. Finally, I investigate a case where a previous codon usage analysis has been seriously confounded by an unusual genome architecture of abnormal regional base composition in two species of eukaryotic parasites in the genus Theileria. In both species, the background G+C content is 37% at most, out of the four syntenic chromosomes. In many orthologous regions however, T.annulata has a decreased G+C content of 28% while T.parva has an increased G+C content of 41%. Various factors coincide with this remarkable divergence: increased divergence at all types of site, recombination hot spots in T.parva, an increased frequency of tandem repeats and DNA sequence motifs in both species. The evolutionary origins of these unusual patterns will be discussed.

The genetic code is redundant, with most amino acids coded by multiple codons. In many organisms, codon usage is biased towards particular codons. A variety of adaptive and non-adaptive explanations have been proposed to explain these patterns of codon usage bias. Using mechanistic models of protein translation and population genetics, I explore the relative importance of various evolutionary forces in shaping these patterns. This work challenges one of the fundamental assumptions made in over 30 years of research: codons with higher tRNA abundances leads to lower error rates. I show that observed patterns of codon usage are inconsistent with selection for translation accuracy. I also show that almost all the variation in patterns of codon usage in S. cerevisiae can be explained by a model taking into account the effects of mutational biases and selection for efficient ribosome usage. In addition, by sampling suboptimal mRNA secondary structures at various temperatures, I show that melting of ribosomal binding sites in a special class of mRNAs known as RNA thermometers is a more general phenomenon.

La tesi esta basada en l'adaptació de l'ús de codons a genomes procariotes, especialment l'adaptació de l'ús de codons a una alta expressió. Hi ha un grup de genomes procariotes, els quals estan sota una selecció traduccional, que tenen un grup de gens amb un ús de codons esbiaixat de la resta de gens del genoma i adaptats a l'abundància dels tRNA. Hem desenvolupat un nou algoritme per a avaluar si un genoma esta sota selecció traduccional i predir els gens altament expressat de tots els genomes sota selecció traduccional. Aquestes prediccions són públiques a la base de dades HEG-DB (http://genomes.urv.cat/HEG-DB), la qual s'ha publicat a la revista Nucleic Acids Research. Les prediccions de gens altament expressats s'han fet servir com a filtre en les prediccions de gens adquirits per transferència horitzontal, ja que els gens altament expressats molts cops son predits com a falsos positius en la predicció de gens adquirits. Amb les dades de la predicció de gens altament expressats, també hem desenvolupat una nova eina Bioinformàtica, anomenada OPTIMIZER (http://genomes.urv.cat/OPTIMIZER) i publicada al Nucleic Acids Research, per tal d'optimitzar l'ús de codons d'un gen per a incrementar la seva expressió en experiments d'expressió heteròloga de proteïnes. També hem estudiat un cas particular d'adaptació de l'ús de codons. El cas de l' 'amelioration', que és l'adaptació de l'ús de codons que pateix un gen inserit en un genoma hoste. Aquest cas l'hem estudiat amb els gens mitocondrials que varen saltar al genoma nuclear i varen haver d'adaptar el seu us de codons mitocondrial a l'ús de codons del genoma nuclear. Per tal d'estudiar l''amelioration', hem desenvolupat un nou índex anomenat CAI esperat (eCAI) i una nova eina Bioinformàtica anomenada CAIcal (http://genomes.urv.cat/CAIcal), que està en procés de revisió a la revista BMC Bioinformatics. Analitzant l'anàlisi de l'ús de codons dels genomes completament sequenciats vàrem realitzar una troballa que s'aparta una mica del tema central de la tesi. Vàrem veure que els genomes que estan adaptats a la (hiper)termofília tenen un patró de l'ús de codons i d'aminoàcids diferent a la resta de genomes (mesòfils). Aquest fet ens ha permès descobrir casos de guany i pèrdua (recents i antics) de la capacitat d'adaptació termofílica en genomes procariotes. Aquests resultats han donat lloc a una publicació a la revista Trends in Genetics. Durant la tesi he realitzat una estada de 4 mesos (Febrer - Juny, 2006) en el laboratori de bioinformàtica del departament de biologia de la universitat nacional d'Irlanda a Maynooth sota la supervisió del Dr James McInerney on vaig desenvolupar un nou programa per a la comparació d'arbres filogenètics anomenat TOPD/FMTS (http://genomes.urv.cat/topd) el qual està publicat a la revista Bioinformatics. / This thesis is based in codon usage adaptation in prokaryotic genomes, especially the codon usage adaptation to a high expression. In genomes under translational selection, the group of highly expressed genes has a codon usage adapted to the most abundant tRNA species. We have developed a new iterative algorithm which predicts a group of highly expressed genes in genomes under translational selection by using the Codon Adaptation Index and the group of ribosomal protein genes as a seed. We have developed a new genomic database, called HEG-DB, to store genes that are predicted as highly expressed in prokaryotic complete genomes under strong translational selection. The database is freely available at http://genomes.urv.cat/HEG-DB and it has been published in Nucleic Acids Research. The predicted highly expressed genes are used as an initial filter to reduce the number of false positives of the Horizontal Gene Transfer Database, due to highly expressed genes are usually false positive in predictions of acquired genes. We have developed a new web sever, called OPTIMIZER (http://genomes.urv.cat/OPTIMIZER), which has been published in Nucleic Acids Research, to optimize the codon usage of DNA or RNA sequences. This new web server can be used to predict and optimize the level expression of a gene in heterologous gene expression or to express new genes that confer new metabolic capabilities in a given species. We have also analyzed an especial case of codon usage adaptation, which is called 'amelioration'. The 'amelioration' is the adaptation of foreign genes to a new genome. This is the case of mitochondrial genes encoded in the human nuclear genome and originally encoded in the proto-mitochondria. To test the 'amelioration' process we have developed an expected value of CAI (eCAI) to find out whether the differences in the CAI are statistically significant or whether they are the product of biased nucleotide and/or amino acid composition and a new bioinformatics tool called CAIcal (http://genomes.urv.cat/CAIcal). We have also analyzed the evolution of thermophilic adaptation in prokaryotes and we suggest that the amino acid composition signature in thermophilic organisms is a consequence of or an adaptation to living at high temperatures, not its cause. Our findings suggest that there have been several cases where the capacity for thermophilic adaptation has been gained or lost throughout the evolution of prokaryotes. These results have been published in Trends in Genetics. During my thesis I have worked for four months in the Bioinformatics Laboratory of the Biology Department at the National University of Ireland under the supervision of Dr James O. McInerney where I developed a new software program to compare phylogenetic trees called TOPD/FMTS (http://genomes.urv.cat/topd), that has been published in Bioinformatics.

A metagenome originated from a shotgun sequencing of a microbial community is a heterogeneous mixture of rather short sequences. A vast majority of microbial species in a given community (99%) are likely to be non-cultivable. Many protein-coding regions in a new metagenome are likely to code for barely detectable homologs of already known proteins. Therefore, an ab initio method that would accurately identify the new genes is a vitally important tool of metagenomic sequence analysis. However, a heuristic model method for finding genes in short prokaryotic sequences with anonymous origin was proposed in 1999 prior to the advent of metagenomics. With hundreds of new prokaryotic genomes available it is now possible to enhance the original approach and to utilize direct polynomial and logistic approximations of oligonucleotide frequencies. The idea was to bypass traditional ways of parameter estimation such as supervised training on a set of validated genes or unsupervised training on an anonymous sequence supposed to contain a large enough number of genes. The codon frequencies, critical for the model parameterization, could be derived from frequencies of nucleotides observed in the short sequence. This method could be further applied for initializing the algorithms for iterative parameters estimation for prokaryotic as well as eukaryotic gene finders.

Thesis (PhD)--Macquarie University, Division of Environmental & Life Sciences, Department of Biological Sciences, 2008. / Bibliography: p. 257-274. / Literature review -- General materials and methods -- Characterisation of strain collection -- Distribution of integrons and gene cassettes in pseudomonas -- Genomic context of pseudomonas integrons -- Evolutionary analysis of pseudomonas spp. integrons 199 -- Final discussion -- Appendix -- References. / Integrons associated with mobile genetic elements have played a central role in the emergence and spread of multiple antibiotic resistance in many pathogenic bacteria. However, the discovery of integrons in the chromosomes of diverse, non-pathogenic bacteria suggests that integrons have a broader role in bacterial evolution. The Pseudomonas stutzeri species complex is a well studied model for bacterial diversity. Members of the complex are genetically closely related, but sub-taxa are not able to be defined by exclusively shared sets of phenotypic characters. Rather, on the basis of total DNA:DNA similarity, Ps. stutzeri strains have been divided into 17 different groups (termed genomovars). Two Ps. stutzeri strains have been found to contain Chromosomal Integrons (CIs). This thesis involved exploration of the hypothesis that a CI was present in the common ancestor of the Ps. stutzeri species complex and assessed the impact of integrons on diversity across all Pseudomonads. The history and significance of integrons is discussed in Chapter 1 as part of a literature review, and general materials and methods are provided in Chapter 2. Chapters 3 - 6 comprise the sections in which data generated during my PhD project are presented. A comprehensive analysis of the relationships between the strains being analysed is presented in Chapter 3. In Chapter 4, results of PCR and hybridisation screening for integrons across the strain collection are presented. In Chapter 5 the recovery of additional integrons and in depth sequence analysis of the recovered integrons are described. Finally, Chapter 6 contains statistical analyses of integron-associated genes and Chapter 7 contains a final discussion the most significant findings. Twenty-three Pseudomonas spp. strains were screened for the presence of integrons. All but three were found to contain integron-like sequences; however, most integron sequences recovered contained inactivated core integrons. viii Despite having a chromosomal locus, integrons in Pseudomonas were found to have properties indicative of frequent horizontal transfer. Evidence was also obtained which suggests that integrons have been acquired at the same locus on multiple independent occasions. This has not been observed in other families of chromosomal integrons and suggests that the loci at which integrons in Pseudomonas are found are hotspots for recombination. / Mode of access: World Wide Web. / xiii, 274 p. ill

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.

Mass production of translationally optimized bacteriophages (hereafter referred to as phages) is the need of the hour in the application of phages to therapy. Understanding translational efficiency of phages is the major preliminary step for mass producing efficient phages. The objective of this thesis is to understand factors affecting translational efficiency of phages. In chapter two, we hypothesized that weak translation initiation efficiency is responsible for weak codon concordance of Escherichia coli lambdoid phages with that of their hosts. We measured the strength of translation initiation using two indices namely minimum folding energy (MFE) and proportion of Shine-Dalgarno sequence (PSD). Empirical results substantiate our hypothesis suggesting lack of strong selection for improving codon adaptation in these phages is due to their weak translation initiation. In chapter three, we measured codon usage concordance between GC-rich and GC-poor Aeromonas phages with their GC-rich host Aeromonas salmonicida. We found low codon usage concordance in the GC-poor Aeromonas phages. We were interested in testing for the role of tRNAs in the GC-poor phages. We observed that the GC-poor phages carry tRNAs for codons that are overused by the phages and underused by the host. These findings suggest that the GC-poor Aeromonas phages carry their own tRNAs for compensating for the compositional difference between their genomes and that of their host. Previously several studies have reported observed avoidance of stable secondary structures in start site of mRNA in a wide range of species. We probed the genomes of 422 phage species and measured their secondary structure stability using MFE. We observed strong patterns of secondary structure avoidance (less negative MFE values) in the translation initiation region (TIR) and translation termination region (TTR) of all analyzed phages. These findings imply selection is operating at these translationally important sites to control stable secondary structures in order to maintain efficient translation.

In this study we have focused on the codon usage bias in E. coli. In chapter 3, we use the population genetics model and the data available on the protein and mRNA levels of the E. coli genes to understand the pattern of codon usage in different genes with different expression levels and see which measure best explains the codon usage pattern. Besides codon bias, by testing for the over-parametrization of the model, we are able to test for the existence of context dependent mutation. We have also fitted the model for the codon usage patter in the Yeast and also tested for the context dependent mutation in this organism. In chapter 4, we focus on the first 10-15 codons in the genes of E. coli. Motivated by the fact that in this region we observe two phenomena, reduction in translation efficiency and suppression of mRNA secondary structures, we investigate whether the former is a side effect of selection for the latter. For this matter we have generated a set of synonymous randomized sequences, and then by selecting the ones which show weak secondary structures in the mentioned region, we would be able to test the theory. We will also look at the frequencies of the amino acids in E. coli genes and see whether the selection for weak secondary structures in the translation initiation region could be strong enough to not only affect the codon usage, but also the choice of amino acids. We would also provide information on the correlation between the strength of the mRNA secondary structure in the first 13 codons and the overall translation efficiency of the genes. / Thesis / Master of Science (MSc)

The human genome comprises 8% sequences of retroviral origin, so-called human endogenous retroviruses (HERVs). Most of these proviral sequences are defective, but some possess open reading frames. They can lead to the formation of viral transcripts, when activated by intrinsic and extrinsic factors. HERVs are thought to play a pathological role in inflammatory diseases and cancer. Since the consequences of activated proviral sequences in the human body are largely unexplored, selected envelope proteins of human endogenous retroviruses associated with inflammatory diseases, namely HERV-K18, HERV-K113, and HERV-Fc1, were investigated in the present study. A formation of glycosylated envelope proteins was demonstrated in different mammalian cell lines. Nevertheless, protein maturation seemed to be incomplete as no transport to the plasma membrane was observed. Instead, the proteins remained in the ER where they induced the expression of genes involved in unfolded protein response, such as HSPA5 and sXBP1. Furthermore, low expression levels of native envelope proteins were increased by codon optimization. Cell-free expression systems showed that both the transcriptional and translational level is affected. By generating different codon-optimized variants of HERV-K113 envelope, the influence of single rare t-RNA pools in certain cell lines was demonstrated. The mRNA secondary structure also appears to play an important role in the translation of the tested viral envelope proteins. In summary, the formation of certain HERV proteins is basically possible. However, their complete maturation and thus full biologic activity seems to depend on additional factors that might be disease-specific and await elucidation in the future.