<p>Estimating substitution frequencies at sites that influence (Ka) and do not influence (Ks) the amino acid sequence is important for understanding the dynamics of molecular sequence evolution and the selective pressures that have shaped genetic variation. </p><p>The aim of this work was to gain a deeper understanding of the driving forces of substitution frequency variation in human pathogens. <i>Rickettsia prowazekii</i>, the causative agent of epidemic typhus and <i>Helicobacter pylori</i>, which has been implicated in gastric diseases were used as model systems. A specific focus was on the evolution of orphan genes in <i>Rickettsia</i>. Additionally, adaptive sequence evolution and factors influencing protein evolutionary rates in <i>H. pylori</i> were studied.</p><p>The comparative sequence analyses of orphan genes using Typhus Group (TG) and Spotted Fever Group (SFG) <i>Rickettsia</i>, indicate that orphan genes in the SFG correspond to pseudogenes in the TG and that pseudogenes in the SFG correspond to extensively degraded gene remnants in the TG. The analysis also showed that ancestral gene sequences could be reconstructed from extant gene remnants of closely related species. The studies of split genes in <i>R. conorii</i> indicate that many of the small fragmented ORFs are probably pseudogenes. Analysis of the <i>H. pylori </i>carbamoyl phosphate synthetase provided an opportunity to understand natural selection acting on a protein undergoing adaptive evolution. Factors such as network properties, protein-protein interactions, gene essentiality and chromosomal position on protein evolutionary rates in <i>H. pylori</i> were studied, of which antigenicity and gene location were identified as the strongest factors. </p><p>In conclusion, high Ka/Ks ratios in human pathogens may reflect either adaptive sequence evolution or gene deterioration. Distinguishing between the two is an important task in molecular evolution and also of great relevance for medical microbiology and functional genomics research.</p>

<p>The majority of the vertebrate genome sequence is not coding for proteins. In recent years, the evolution of this noncoding fraction of the genome has gained interest. These studies have been greatly facilitated by the availability of full genome sequences. The aim of this thesis is to study evolution of the noncoding vertebrate genome through bioinformatic analysis of large-scale genomic datasets.</p><p>In a first analysis we addressed the use of conservation of sequence between highly diverged genomes to infer function. We provided evidence for a turnover of the patterns of negative selection. Hence, measures of constraint based on comparisons of diverged genomes might underestimate the functional proportion of the genome.</p><p>In the following analyses we focused on length variation as found in small-scale insertion and deletion (indel) polymorphisms and microsatellites. For indels in chicken, replication slippage is a likely mutation mechanism, as a large proportion of the indels are parts of tandem-duplicates. Using a set of microsatellite polymorphisms in chicken, where we avoid ascertainment bias, we showed that polymorphism is positively correlated with microsatellite length and AT-content. Furthermore, interruptions in the microsatellite sequence decrease the levels of polymorphism.</p><p>We also analysed the association between microsatellite polymorphism and recombination in the human genome. Here we found increased levels of microsatellite polymorphism in human recombination hotspots and also similar increases in the frequencies of single nucleotide polymorphisms (SNPs) and indels. This points towards natural selection shaping the levels of variation. Alternatively, recombination is mutagenic for all three kinds of polymorphisms. </p><p>Finally, I present the program ILAPlot. It is a tool for visualisation, exploration and data extraction based on BLAST.</p><p>Our combined results highlight the intricate connections between evolutionary phenomena. It also emphasises the importance of length variability in genome evolution, as well as the gradual difference between indels and microsatellites.</p>

The protozoan parasite Giardia intestinalis (syn. G. lamblia, G. duodenalis) is one of the most common causes of diarrheal disease throughout the world, where an estimated 500 million people are infected annually. Despite efforts in trying to elucidate factors associated with virulence in G. intestinalis little is currently known. The disease outcome is highly variable in Giardia infected individuals, ranging from asymptomatic carriers to severe disease. The reasons behind the differences in disease outcome are vaguely understood and studies trying to link infectivity to different Giardia assemblages or sub-assemblages have rendered conflicting results. Prior to this study, little was known about the prevalence and genetic diversity of different G. intestinalis assemblages across the world. In this thesis, molecular characterization of clinical G. intestinalis samples from Eastern Africa and Central America, has been performed, enabling a better understanding of the prevalence of different Giardia genotypes in endemic areas (Papers I and II). A correlation between Giardia colonization and the presence of Helicobacter pylori in the human host was established. We found that the currently available genotyping tools provide low resolution when used to characterize assemblage A Giardia. Also, genotyping of assemblage B isolates at these loci is troublesome due to the polymorphic substitutions frequently found in the sequencing chromatograms. This ambiguity was investigated by using micromanipulation to isolate single assemblage B Giardia cells (Paper III). Both cultured trophozoites and cysts from giardiasis patients were analyzed. The data showed that allelic sequence heterozygosity (ASH) does occur at the single cell level, but also that multiple sub-assemblage infections appear to be common in human giardiasis patients. Furthermore, genome-wide sequencing followed by comparative genomics was performed in order to better characterize differences between and within different Giardia assemblages. The genome of a non-human infecting, assemblage E isolate (Paper IV) was sequenced.  The genomes of two freshly isolated human infecting assemblage AII isolates were also sequenced (Paper V). Subsequent, comparative analyses were performed and included the genomes of two human infecting isolates, WB (AI) and GS/M (B). Several important differences were found between assemblages A, B and E, but also within assemblage A; including unique gene repertoires for each isolate, observed differences in the variable gene families and an overall difference in ASH between the different isolates. Also, a new multi-locus genotyping (MLG) strategy for genotyping of assemblage A Giardia has been established and evaluated on clinical samples from human giardiasis patients.

Estimating substitution frequencies at sites that influence (Ka) and do not influence (Ks) the amino acid sequence is important for understanding the dynamics of molecular sequence evolution and the selective pressures that have shaped genetic variation. The aim of this work was to gain a deeper understanding of the driving forces of substitution frequency variation in human pathogens. Rickettsia prowazekii, the causative agent of epidemic typhus and Helicobacter pylori, which has been implicated in gastric diseases were used as model systems. A specific focus was on the evolution of orphan genes in Rickettsia. Additionally, adaptive sequence evolution and factors influencing protein evolutionary rates in H. pylori were studied. The comparative sequence analyses of orphan genes using Typhus Group (TG) and Spotted Fever Group (SFG) Rickettsia, indicate that orphan genes in the SFG correspond to pseudogenes in the TG and that pseudogenes in the SFG correspond to extensively degraded gene remnants in the TG. The analysis also showed that ancestral gene sequences could be reconstructed from extant gene remnants of closely related species. The studies of split genes in R. conorii indicate that many of the small fragmented ORFs are probably pseudogenes. Analysis of the H. pylori carbamoyl phosphate synthetase provided an opportunity to understand natural selection acting on a protein undergoing adaptive evolution. Factors such as network properties, protein-protein interactions, gene essentiality and chromosomal position on protein evolutionary rates in H. pylori were studied, of which antigenicity and gene location were identified as the strongest factors. In conclusion, high Ka/Ks ratios in human pathogens may reflect either adaptive sequence evolution or gene deterioration. Distinguishing between the two is an important task in molecular evolution and also of great relevance for medical microbiology and functional genomics research.

The majority of the vertebrate genome sequence is not coding for proteins. In recent years, the evolution of this noncoding fraction of the genome has gained interest. These studies have been greatly facilitated by the availability of full genome sequences. The aim of this thesis is to study evolution of the noncoding vertebrate genome through bioinformatic analysis of large-scale genomic datasets. In a first analysis we addressed the use of conservation of sequence between highly diverged genomes to infer function. We provided evidence for a turnover of the patterns of negative selection. Hence, measures of constraint based on comparisons of diverged genomes might underestimate the functional proportion of the genome. In the following analyses we focused on length variation as found in small-scale insertion and deletion (indel) polymorphisms and microsatellites. For indels in chicken, replication slippage is a likely mutation mechanism, as a large proportion of the indels are parts of tandem-duplicates. Using a set of microsatellite polymorphisms in chicken, where we avoid ascertainment bias, we showed that polymorphism is positively correlated with microsatellite length and AT-content. Furthermore, interruptions in the microsatellite sequence decrease the levels of polymorphism. We also analysed the association between microsatellite polymorphism and recombination in the human genome. Here we found increased levels of microsatellite polymorphism in human recombination hotspots and also similar increases in the frequencies of single nucleotide polymorphisms (SNPs) and indels. This points towards natural selection shaping the levels of variation. Alternatively, recombination is mutagenic for all three kinds of polymorphisms. Finally, I present the program ILAPlot. It is a tool for visualisation, exploration and data extraction based on BLAST. Our combined results highlight the intricate connections between evolutionary phenomena. It also emphasises the importance of length variability in genome evolution, as well as the gradual difference between indels and microsatellites.

Proteases play key roles in plants, maintaining strict protein quality control and degrading specific sets of proteins in response to diverse environmental and developmental stimuli. Similarities and differences between the proteases expressed in different species may give valuable insights into their physiological roles and evolution. Systematic comparative analysis of the available sequenced genomes of two model organisms led to the identification of an increasing number of protease genes, giving insights about protein sequences that are conserved in the different species, and thus are likely to have common functions in them and the acquisition of new genes, elucidate issues concerning non-functionalization, neofunctionalization and subfunctionalization. The involvement of proteases in senescence and PCD was investigated. While PCD in woody tissues shows the importance of vacuole proteases in the process, the senescence in leaves demonstrate to be a slower and more ordered mechanism starting in the chloroplast where the proteases there localized become important. The light-harvesting complex of Photosystem II is very susceptible to protease attack during leaf senescence. We were able to show that a metallo-protease belonging to the FtsH family is involved on the process in vitro. Arabidopsis knockout mutants confirmed the function of FtsH6 in vivo.

In order to identify expressologs (orthologs exhibiting the highest expression profile ranking) among a variety of plant species, bioinformatic methods were used in order to first identify sequence orthologs and subsequently to rank these orthologs based on expression profile similarity. Analyses conducted on these data suggested that expressologs exhibited greater functional equivalency. A comparison of drought response in A. thaliana and Populus showed that expressologs exhibited a higher correlation when computed using stress data as opposed to developmental data. This suggested that the use of condition-specific data sets is more appropriate when examining specific conditions. Analysis was conducted in order to investigate the hypothesis that neutral evolution was a predominant factor in gene expression divergence. Some evidence was found for selection acting on expression pattern maintenance. Further analysis will be required in order to confirm the type of selection acting to maintain expression patterns across species.

In order to identify expressologs (orthologs exhibiting the highest expression profile ranking) among a variety of plant species, bioinformatic methods were used in order to first identify sequence orthologs and subsequently to rank these orthologs based on expression profile similarity. Analyses conducted on these data suggested that expressologs exhibited greater functional equivalency. A comparison of drought response in A. thaliana and Populus showed that expressologs exhibited a higher correlation when computed using stress data as opposed to developmental data. This suggested that the use of condition-specific data sets is more appropriate when examining specific conditions. Analysis was conducted in order to investigate the hypothesis that neutral evolution was a predominant factor in gene expression divergence. Some evidence was found for selection acting on expression pattern maintenance. Further analysis will be required in order to confirm the type of selection acting to maintain expression patterns across species.

<p>Sexual reproduction in fungi is governed by a specialized genomic region called the mating-type locus (MAT). The ascomycetes, the largest phylum of fungi, primarily possess a bipolar mating system while the basidiomycetes, the second largest group, are mostly tetrapolar. The human fungal pathogen and basidiomycetous yeast Cryptococcus neoformans has evolved a bipolar mating system that encodes homeodomain (HD) and pheromone/receptor (P/R) genes. The MAT locus of C. neoformans is unusually large, spans greater than 100 kb, and encodes more than 20 genes. To understand how the pathogenic Cryptococcus species complex evolved this unique bipolar mating system, we investigated the evolution of MAT in closely and distantly related species and discovered an extant sexual cycle in Cryptococcus amylolentus. </p><p>Phylogenetic analysis using a six-gene multi-locus sequencing (MLS) approach identified the most closely related species to the pathogenic Cryptococcus species complex that are currently known. The two non-pathogenic sibling species, Tsuchiyaea wingfieldii and Cryptococcus amylolentus, and the more distantly related species Filobasidiella depauperata define the Filobasidiella clade. We also resolved the phylogeny of the species located in the sister clade, Kwoniella. A comprehensive tree dendrogram revealed that the 15 Tremellales species examined suggests a common saprobic ancestor. Moreover, the pathogenic Cryptococcus species have a saprobic origin but later emerged as pathogens. We further characterized the mating-type locus for T. wingfieldii and C. amylolentus by cloning and sequencing two unlinked genomic loci encoding the HD and P/R genes. Interestingly, linked and likely divergently transcribed homologs for SXI1 and SXI2 are present in T. wingfieldii and C. amylolentus, while the P/R alleles contain many genes also found in the MAT locus of the pathogenic Cryptococcus species. Also, hypothetical genes present in C. neoformans MAT are also MAT-linked in both species and indicate a possible translocation event between chromosomes 4 and 5 of C. neoformans. Our analysis of MAT in the sibling species indicates that T. wingfieldii is likely tetrapolar, and the C. amylolentus sequence comparison of the dimorphic SXI1 and SXI2 region and the pheromone receptor, STE3, suggests that C. amylolentus is also tetrapolar. The examination of MAT in these sibling species confirms the model for MAT evolution previously proposed in which this structure in C. neoformans and C. gattii evolved from an ancestral tetrapolar mating system. Moreover, the organization of MAT in these sibling species mirrors key aspects of the proposed intermediates in the evolution of MAT in the pathogenic Cryptococcus species, and for sex chromosomes in plants, animals, and alga in general. </p><p>We discovered an extant sexual cycle for C. amylolentus, a species previously thought to be asexual. Matings between two strains of opposite mating-types produce dikaryotic hyphae with fused clamp connections and uni- and bi-nucleate basidiospores. Genotyping of basidiospores using markers linked and unlinked to MAT revealed that genetic exchange (recombination) occurs during the sexual cycle of C. amylolentus, and it is likely that either aneuploids are generated during sex or more than one meiosis event occurs within each basidium. This is in contrast to C. neoformans, where only one meiotic event per basidium has been observed. Uniparental mitochondrial inheritance has also been observed in C. amylolentus progeny; similar to the pathogenic Cryptococcus species, mtDNA is inherited from the C. amylolentus MATa parent. Analysis of sex in C. amylolentus has provided insight into the mechanisms that phylogenetically related fungi employ in orchestrating sexual reproduction. </p><p>We also extended our analysis to include the distantly related tetrapolar basidiomycete Tremella mesenterica. We completed comparisons of MAT-specific genes between five strains of T. mesenterica and identified the regions that define its mating-type system. The HD locus is limited to the SXI1- and SXI2-like genes while the P/R locus is defined by STE3, STE12, STE20, and the pheromone gene, tremerogen a-13. Interestingly, many of the genes associated with the MAT locus of the pathogenic Cryptococcus species flank the HD and P/R locus and are not incorporated in MAT in T. mesenterica. The MAT region includes transposons and C. neoformans hypothetical genes also present in T. wingfieldii and C. amylolentus. The mating-type system in T. mesenterica reflects an ancestral intermediate in the evolution of the MAT locus in the pathogenic Cryptococcus species. In conclusion, this study provides an in-depth analysis on the structure, function, and evolution of an unusual mating-type locus with broader implications for the transitions in modes of sexual reproduction in fungi that impact gene flow in populations.</p> / Dissertation

High-throughput genome processing, sophisticated protein sequence analysis, programming, and information management were used to achieve two major advances in the comparative genomics of microbial signal transduction. First, an integrated and flexible bioinformatics platform and the Microbial Signal Transduction database (MiST) were developed, which facilitated the genome-wide analysis of bacterial signal transduction. This platform was used successfully for the high-throughput identification and classification of signal transduction proteins in more than 300 archaeal and bacterial organisms. Second, analysis of information encoded in prokaryotic genomes revealed that the majority of signal transduction systems consist of one-component systems a single protein containing both input and output domains but lacking phosphotransfer domains typical of two-component systems. The prevalence of one-component systems is a paradigm-shifting discovery because two-component systems are currently viewed as the primary mode of signal transduction in prokaryotes. One-component systems are more widely distributed among bacteria and archaea and display a greater diversity of domains than two-component systems. Additionally, in-depth bioinformatic analyses were performed that further characterized the function of two, input, signaling domains. In summary, this systematic, high-throughput delineation of microbial signal transduction is another step forward in our understanding of the genomic basis of life.