Computational Methods for Locating and Analyzing Conserved Gene Regulatory DNA Elements

This thesis presents methods for locating and analyzing cis-regulatory DNA elements involved with the regulation of gene expression in multicellular organisms. The regulation of gene expression is carried out by the combined effort of several transcription factor proteins collectively binding the DNA on the cis-regulatory elements. Only sparse knowledge of the 'genetic code' of these elements exists today. An automatic tool for discovery of putative cis-regulatory elements could help their experimental analysis, which would result in a more detailed view of the cis-regulatory element structure and function. We have developed a computational model for the evolutionary conservation of cis-regulatory elements. The elements are modeled as evolutionarily conserved clusters of sequence-specific transcription factor binding sites. We give an efficient dynamic programming algorithm that locates the putative cis-regulatory elements and scores them according to the conservation model. A notable proportion of the high-scoring DNA sequences show transcriptional enhancer activity in transgenic mouse embryos. The conservation model includes four parameters whose optimal values are estimated with simulated annealing. With good parameter values the model discriminates well between the DNA sequences with evolutionarily conserved cis-regulatory elements and the DNA sequences that have evolved neutrally. In further inquiry, the set of highest scoring putative cis-regulatory elements were found to be sensitive to small variations in the parameter values. The statistical significance of the putative cis-regulatory elements is estimated with the Two Component Extreme Value Distribution. The p-values grade the conservation of the cis-regulatory elements above the neutral expectation. The parameter values for the distribution are estimated by simulating the neutral DNA evolution. The conservation of the transcription factor binding sites can be used in the upstream analysis of regulatory interactions. This approach may provide mechanistic insight to the transcription level data from, e.g., microarray experiments. Here we give a method to predict shared transcriptional regulators for a set of co-expressed genes. The EEL (Enhancer Element Locator) software implements the method for locating putative cis-regulatory elements. The software facilitates both interactive use and distributed batch processing. We have used it to analyze the non-coding regions around all human genes with respect to the orthologous regions in various other species including mouse. The data from these genome-wide analyzes is stored in a relational database which is used in the publicly available web services for upstream analysis and visualization of the putative cis-regulatory elements in the human genome.

Essays on Illegal Activities, Institutions and Economic Growth

In public economics, two extremist views on the functions of a government compete: one emphasizes government working for the public interest to provide value for the citizens, while another regards government mainly as a workhorse for private interests. Moreover, as the sole legitimate authority, the government has the right to define the rules and laws as well as to enforce them. With respect to regulation, two extremes arise: from too little regulation to too much of it. If the government does not function or ceases to exist, the state falls into anarchy or chaos (Somalia). If it regulates too much, it will completely suffocate private activities, which might be considered extralegal (the former Soviet Union). In this thesis I scrutinize the government s interventionist policies and evaluate the question of how to best promote economic well-being. The first two essays assume that the government s policies promote illegal activity. The first paper evaluates the interaction between the government and the mafia, and pays attention to the law enforcement of underground production. We show that the revenue-maximizing government will always monitor the shadow economy, as monitoring contributes to the government s revenue. In general, both legal and illegal firms are hurt by the entry of the mafia. It is, however, plausible that legal firms might benefit by the entry of the mafia if it competes with the government. The second paper tackles the issue of the measurement of the size of the shadow economy. To formulate policies it is essential to know what drives illegal economic activity; is it the tax burden, excess regulation, corruption or a weak legal environment? In this paper we propose an additional explanation for tax evasion and shadow production, namely cultural factors as manifested by religion as determinants of tax morality. According to our findings, Catholic and Protestant countries do not differ in their tax morale. The third paper contributes to the literature discussing the role of the government in promoting economic and productivity growth. Our main result is that, given the complex relationship between economic growth and economic freedom, marketization has not necessarily been beneficial in terms of growth. The last paper builds on traditional growth literature and revisits the debate on convergence clubs arising from demographic transition. We provide new evidence against the idea that countries within a club would converge over time. Instead, we propose that since the demographic transition is a dynamic process, one can expect countries to enter the last regime of stable, modern growth in stages.

DNA Packaging and Host Cell Lysis: Late Events in Bacteriophage PRD1 Infection

The object of this study is a tailless internal membrane-containing bacteriophage PRD1. It has a dsDNA genome with covalently bound terminal proteins required for replication. The uniqueness of the structure makes this phage a desirable object of research. PRD1 has been studied for some 30 years during which time a lot of information has accumulated on its structure and life-cycle. The two least characterised steps of the PRD1 life-cycle, the genome packaging and virus release are investigated here. PRD1 shares the main principles of virion assembly (DNA packaging in particular) and host cell lysis with other dsDNA bacteriophages. However, this phage has some fascinating individual peculiarities, such as DNA packaging into a membrane vesicle inside the capsid, absence of apparent portal protein, holin inhibitor and procapsid expansion. In the course of this study we have identified the components of the DNA packaging vertex of the capsid, and determined the function of protein P6 in packaging. We managed to purify the procapsids for an in vitro packaging system, optimise the reaction and significantly increase its efficiency. We developed a new method to determine DNA translocation and were able to quantify the efficiency and the rate of packaging. A model for PRD1 DNA packaging was also proposed. Another part of this study covers the lysis of the host cell. As other dsDNA bacteriophages PRD1 has been proposed to utilise a two-component lysis system. The existence of this lysis system in PRD1 has been proven by experiments using recombinant proteins and the multi-step nature of the lysis process has been established.

Traditional medicinal uses and biological activities of some plant extracts of African Combretum Loefl., Terminalia L. and Pteleopsis Engl. species

In Africa various species of Combretum, Terminalia and Pteleopsis are used in traditional medicine. Despite of this, some species of these genera have still not been studied for their biological effects to validate their traditional uses. The aim of this work has been to document the ethnomedicinal uses of several species of Combretum and Terminalia in Mbeya region, south-western Tanzania, and to use this information for finding species with good antimicrobial and cytotoxic potential. During a five weeks expedition to Tanzania in spring 1999 sixteen different species of Combretum and Terminalia, as well as Pteleopsis myrtifolia were collected from various locations in the districts of Mbeya, Iringa and Dar-es-Salaam. Traditional healers in seven different villages in the Mbeya region were interviewed in Swahili and Nyakyusa on the medicinal uses of Combretum and Terminalia species shown to them. A questionnaire was used during the interviews. The results of the interviews correlated well between different villages, the same species being used in similar ways in different villages. Of the ten species shown to the healers six were frequently used for treatment of skin diseases, bacterial infections, diarrhea, oedema and wounds. The dried plants were most commonly prepared into hot water decoctions or mixed into maize porridge, Ugali. Infusions made from dried or fresh plant material were also common. Wounds and topical infections were treated with ointments made from the dried plant material mixed with sheep fat. Twenty-one extracts of six species of Combretum and four of Terminalia, collected from Tanzania, were screened for their antibacterial effects against two gram-negative and five gram-positive bacteria, as well as the yeast, Candida albicans, using an agar diffusion method. Most of the screened plants showed substantial antimicrobial activity. A methanolic root extract of T. sambesiaca showed the most potent antibacterial effects of all the plant species screened, and gave a MIC value of 0.9 mg/ml against Enterobacter aerogenes. Also root extracts of T. sericea and T. kaiserana gave excellent antimicrobial effects, and notably a hot water extract of T. sericea was as potent as extracts of this species made from EtOH and MeOH. Thus, the traditional way of preparing T. sericea into hot water decoctions seems to extract antimicrobial compounds. Thirty-five extracts of five species of Terminalia, ten of Combretum and Pteleopsis myrtifolia were screened for their antifungal effects against five species of yeast (Candida spp.) and Cryptococcus neoformans. The species differed from each other to their antifungal effects, some being very effective whereas others showed no antifungal effects. The most effective extracts showed antifungal effects comparable to the standard antibiotics itraconazol and amphotericin B. Species of Terminalia gave in general stronger antifungal effects than those of Combretum. The best effects were obtained with methanolic root extracts of T. sambesiaca, T. sericea and T. kaiserana, and this investigation indicates that decoctions of these species might be used for treatment of HIV-related fungal infections. Twenty-seven crude extracts of eight species of Combretum, five of Terminalia and Pteleopsis myrtifolia were evaluated for their cytotoxic effects against human cancer cell lines (HeLa, cervical carcinoma; MCF 7, breast carcinoma, T 24 bladder carcinoma) and one endothelial cell line (BBCE, bovine brain capillary endothelial cells). The most outstanding effects were obtained with a leaf extract of Combretum fragrans, which nearly totally inhibited the proliferation of T 24 and HeLa cells at a concentration of 25 ug/ml and inhibited 60 % of the growth of the HeLa cells at a concentration of 4.3 ug/ml. The species of Terminalia were less cytotoxically potent than the Combretum species, although T. sericea and T. sambesiaca gave good cytotoxic effects (< 30 % proliferation). In summary this study indicates that some of the species of Terminalia, Combretum and Pteleopsis, used in Tanzanian traditional medicine, are powerful inhibitors of both microbial and cancer cell growth. In depth studies would be needed to find the active compounds behind these biological activities.

Gene Expression : From Microarrays to Functional Genomics

The time of the large sequencing projects has enabled unprecedented possibilities of investigating more complex aspects of living organisms. Among the high-throughput technologies based on the genomic sequences, the DNA microarrays are widely used for many purposes, including the measurement of the relative quantity of the messenger RNAs. However, the reliability of microarrays has been strongly doubted as robust analysis of the complex microarray output data has been developed only after the technology had already been spread in the community. An objective of this study consisted of increasing the performance of microarrays, and was measured by the successful validation of the results by independent techniques. To this end, emphasis has been given to the possibility of selecting candidate genes with remarkable biological significance within specific experimental design. Along with literature evidence, the re-annotation of the probes and model-based normalization algorithms were found to be beneficial when analyzing Affymetrix GeneChip data. Typically, the analysis of microarrays aims at selecting genes whose expression is significantly different in different conditions followed by grouping them in functional categories, enabling a biological interpretation of the results. Another approach investigates the global differences in the expression of functionally related groups of genes. Here, this technique has been effective in discovering patterns related to temporal changes during infection of human cells. Another aspect explored in this thesis is related to the possibility of combining independent gene expression data for creating a catalog of genes that are selectively expressed in healthy human tissues. Not all the genes present in human cells are active; some involved in basic activities (named housekeeping genes) are expressed ubiquitously. Other genes (named tissue-selective genes) provide more specific functions and they are expressed preferably in certain cell types or tissues. Defining the tissue-selective genes is also important as these genes can cause disease with phenotype in the tissues where they are expressed. The hypothesis that gene expression could be used as a measure of the relatedness of the tissues has been also proved. Microarray experiments provide long lists of candidate genes that are often difficult to interpret and prioritize. Extending the power of microarray results is possible by inferring the relationships of genes under certain conditions. Gene transcription is constantly regulated by the coordinated binding of proteins, named transcription factors, to specific portions of the its promoter sequence. In this study, the analysis of promoters from groups of candidate genes has been utilized for predicting gene networks and highlighting modules of transcription factors playing a central role in the regulation of their transcription. Specific modules have been found regulating the expression of genes selectively expressed in the hippocampus, an area of the brain having a central role in the Major Depression Disorder. Similarly, gene networks derived from microarray results have elucidated aspects of the development of the mesencephalon, another region of the brain involved in Parkinson Disease.

Microbial communities in Pb contaminated boreal forest soil

Lead contamination in the environment is of particular concern, as it is a known toxin. Until recently, however, much less attention has been given to the local contamination caused by activities at shooting ranges compared to large-scale industrial contamination. In Finland, more than 500 tons of Pb is produced each year for shotgun ammunition. The contaminant threatens various organisms, ground water and the health of human populations. However, the forest at shooting ranges usually shows no visible sign of stress compared to nearby clean environments. The aboveground biota normally reflects the belowground ecosystem. Thus, the soil microbial communities appear to bear strong resistance to contamination, despite the influence of lead. The studies forming this thesis investigated a shooting range site at Hälvälä in Southern Finland, which is heavily contaminated by lead pellets. Previously it was experimentally shown that the growth of grasses and degradation of litter are retarded. Measurements of acute toxicity of the contaminated soil or soil extracts gave conflicting results, as enchytraeid worms used as toxicity reporters were strongly affected, while reporter bacteria showed no or very minor decreases in viability. Measurements using sensitive inducible luminescent reporter bacteria suggested that the bioavailability of lead in the soil is indeed low, and this notion was supported by the very low water extractability of the lead. Nevertheless, the frequency of lead-resistant cultivable bacteria was elevated based on the isolation of cultivable strains. The bacterial and fungal diversity in heavily lead contaminated shooting sectors were compared with those of pristine sections of the shooting range area. The bacterial 16S rRNA gene and fungal ITS rRNA gene were amplified, cloned and sequenced using total DNA extracted from the soil humus layer as the template. Altogether, 917 sequenced bacterial clones and 649 sequenced fungal clones revealed a high soil microbial diversity. No effect of lead contamination was found on bacterial richness or diversity, while fungal richness and diversity significantly differed between lead contaminated and clean control areas. However, even in the case of fungi, genera that were deemed sensitive were not totally absent from the contaminated area: only their relative frequency was significantly reduced. Some operational taxonomic units (OTUs) assigned to Basidiomycota were clearly affected, and were much rarer in the lead contaminated areas. The studies of this thesis surveyed EcM sporocarps, analyzed morphotyped EcM root tips by direct sequencing, and 454-pyrosequenced fungal communities in in-growth bags. A total of 32 EcM fungi that formed conspicuous sporocarps, 27 EcM fungal OTUs from 294 root tips, and 116 EcM fungal OTUs from a total of 8 194 ITS2 454 sequences were recorded. The ordination analyses by non-parametric multidimensional scaling (NMS) indicated that Pb enrichment induced a shift in the EcM community composition. This was visible as indicative trends in the sporocarp and root tip datasets, but explicitly clear in the communities observed in the in-growth bags. The compositional shift in the EcM community was mainly attributable to an increase in the frequencies of OTUs assigned to the genus Thelephora, and to a decrease in the OTUs assigned to Pseudotomentella, Suillus and Tylospora in Pb-contaminated areas when compared to the control. The enrichment of Thelephora in contaminated areas was also observed when examining the total fungal communities in soil using DNA cloning and sequencing technology. While the compositional shifts are clear, their functional consequences for the dominant trees or soil ecosystem remain undetermined. The results indicate that at the Hälvälä shooting range, lead influences the fungal communities but not the bacterial communities. The forest ecosystem shows apparent functional redundancy, since no significant effects were seen on forest trees. Recently, by means of 454 pyrosequencing , the amount of sequences in a single analysis run can be up to one million. It has been applied in microbial ecology studies to characterize microbial communities. The handling of sequence data with traditional programs is becoming difficult and exceedingly time consuming, and novel tools are needed to handle the vast amounts of data being generated. The field of microbial ecology has recently benefited from the availability of a number of tools for describing and comparing microbial communities using robust statistical methods. However, although these programs provide methods for rapid calculation, it has become necessary to make them more amenable to larger datasets and numbers of samples from pyrosequencing. As part of this thesis, a new program was developed, MuSSA (Multi-Sample Sequence Analyser), to handle sequence data from novel high-throughput sequencing approaches in microbial community analyses. The greatest advantage of the program is that large volumes of sequence data can be manipulated, and general OTU series with a frequency value can be calculated among a large number of samples.

Human parvovirus B19: tissue persistence and prevalence of prototypic and new variants

Human parvovirus B19 is a minute ssDNA virus causing a wide variety of diseases, including erythema infectiosum, arthropathy, anemias, and fetal death. After primary infection, genomic DNA of B19 has been shown to persist in solid tissues of not only symptomatic but also of constitutionally healthy, immunocompetent individuals. In this thesis, the viral DNA was shown to persist as an apparently intact molecule of full length, and without persistence-specific mutations. Thus, although the mere presence of B19 DNA in tissue can not be used as a diagnostic criterion, a possible role in the pathogenesis of diseases e.g. through mRNA or protein production can not be excluded. The molecular mechanism, the host-cell type and the possible clinical significance of B19 DNA tissue persistence are yet to be elucidated. In the beginning of this work, the B19 genomic sequence was considered highly conserved. However, new variants were found: V9 was detected in 1998 in France, in serum of a child with aplastic crisis. This variant differed from the prototypic B19 sequences by ~10 %. In 2002 we found, persisting in skin of constitutionally healthy humans, DNA of another novel B19 variant, LaLi. Genetically this variant differed from both the prototypic sequences and the variant V9 also by ~10%. Simultaneously, B19 isolates with DNA sequences similar to LaLi were introduced by two other groups, in the USA and France. Based on phylogeny, a classification scheme based on three genotypes (B19 types 1-3) was proposed. Although the B19 virus is mainly transmitted via the respiratory route, blood and plasma-derived products contaminated with high levels of B19 DNA have also been shown to be infectious. The European Pharmacopoeia stipulates that, in Europe, from the beginning of 2004, plasma pools for manufacture must contain less than 104 IU/ml of B19 DNA. Quantitative PCR screening is therefore a prerequisite for restriction of the B19 DNA load and obtaining of safe plasma products. Due to the DNA sequence variation among the three B19 genotypes, however, B19 PCR methods might fail to detect the new variants. We therefore examined the suitability of the two commercially available quantitative B19 PCR tests, LightCycler-Parvovirus B19 quantification kit (Roche Diagnostics) and RealArt Parvo B19 LC PCR (Artus), for detection, quantification and differentiation of the three B19 types known, including B19 types 2 and 3. The former method was highly sensitive for detection of the B19 prototype but was not suitable for detection of types 2 and 3. The latter method detected and differentiated all three B19 virus types. However, one of the two type-3 strains was detected at a lower sensitivity. Then, we assessed the prevalence of the three B19 virus types among Finnish blood donors, by screening pooled plasma samples derived from >140 000 blood-donor units: none of the pools contained detectable levels of B19 virus types 2 or 3. According to the results of other groups, B19 type 2 was absent also among Danish blood-donors, and extremely rare among symptomatic European patients. B19 type 3 has been encountered endemically in Ghana and (apparently) in Brazil, and sporadical cases have been detected in France and the UK. We next examined the biological characteristics of these virus types. The p6 promoter regions of virus types 1-3 were cloned in front of a reporter gene, the constructs were transfected into different cell lines, and the promoter activities were measured. As a result, we found that the activities of the three p6 promoters, although differing in sequence by >20%, were of equal strength, and most active in B19-permissive cells. Furthermore, the infectivity of the three B19 types was examined in two B19-permissive cell lines. RT-PCR revealed synthesis of spliced B19 mRNAs, and immunofluorescence verified the production of NS1 and VP proteins in the infected cells. These experiments suggested similar host-cell tropism and showed that the three virus types are strains of the same species, i.e. human parvovirus B19. Last but not least, the sera from subjects infected in the past either with B19 type 1 or type 2 (as evidenced by tissue persistence of the respective DNAs), revealed in VP1/2- and VP2-EIAs a 100 % cross-reactivity between virus types 1 and 2. These results, together with similar studies by others, indicate that the three B19 genotypes constitute a single serotype.

Multifunctional RNA silencing pathway polymerase QDE-1 of Neurospora crassa

Double-stranded RNA and associated proteins are known to regulate the gene expression of most eukaryotic organisms. These regulation pathways have different components, outcomes and distinct nomenclature depending on the model system, and often they are referred to collectively as RNA silencing. In many cases, RNA-dependent RNA polymerases (RdRPs) are found to be involved in the RNA silencing, but their targets, activities, interaction partners and reaction products remain enigmatic. In the filamentous fungus Neurospora crassa, the RdRP QDE-1 is critical for silencing of transgenes a phenomenon known as quelling. In this thesis the structure, biochemical activities and biological functions of QDE-1 were extensively studied. This dimeric RdRP was shown to possess five distinct catalytic in vitro activities that could be dissected by mutagenesis and by altering reaction conditions. The biochemical characterization implied that QDE-1 is actually an active DNA-dependent RNA polymerase that has additional RdRP activity. It also provided a structural explanation for the dimerization and suggested a biological framework for the functions of QDE-1 in vivo. (I) QDE-1 was also studied in a broader context along with the other components of the quelling pathway. It was shown that DNA damage in Neurospora causes a dramatic increase in the expression level of the Argonaute protein QDE-2 as well as the synthesis of a novel class of small RNAs known as qiRNAs. The accumulation of qiRNAs was shown to be dependent on several quelling components, and particularly to be derived from an aberrant ssRNA (aRNA) molecule that is synthesized by QDE-1 in the nucleus. The genomic distribution of qiRNA targets was analyzed and the possible biological significance of qiRNAs was studied. Importantly, qiRNAs are the first class of small RNAs that are induced by DNA damage. (II) After establishing that QDE-1 is a multifunctional RNA polymerase with several activities, template specificities and subcellular locations, the focus was turned onto its interaction partners. It had been previously known that QDE-1 associates with Replication Protein A (RPA), but the RecQ helicase QDE-3 was now shown to regulate this interaction. RPA was also observed to promote QDE-1 dependent dsRNA synthesis in vitro. By characterizing the interplay between QDE-1, QDE-3 and RPA, a working model of quelling and qiRNA pathways in Neurospora was presented. (III) This work sheds light on the complexity of the various RNA silencing pathways of a fungal model system. It shows how an RdRP can regulate gene expression on many levels, and suggests novel lines of research in other eukaryotic organisms.

Molecular details of phage phi6 RNA-dependent RNA synthesis

For most RNA viruses RNA-dependent RNA polymerases (RdRPs) encoded by the virus are responsible for the entire RNA metabolism. Thus, RdRPs are critical components in the viral life cycle. However, it is not fully understood how these important enzymes function during viral replication. Double-stranded RNA (dsRNA) viruses perform the synthesis of their RNA genome within a proteinacous viral particle containing an RdRP as a minor constituent. The phi6 bacteriophage is the best-studied dsRNA virus, providing an excellent background for studies of its RNA synthesis. The purified recombinant phi6 RdRP is highly active in vitro and it possesses both RNA replication and transcription activities. The crystal structure of the phi6 polymerase, solved in complex with a number of ligands, provides a working model for detailed in vitro studies of RNA-dependent RNA polymerization. In this thesis, the primer-independent initiation of the phi6 RdRP was studied in vitro using biochemical and structural methods. A C-terminal, four-amino-acid-long loop protruding into the central cavity of the phi6 RdRP has been suggested to stabilize the incoming nucleotides of the initiation complex formation through stacking interactions. A similar structural element has been found from several other viral RdRPs. In this thesis, this so-called initiation platform loop was subjected to site-directed mutagenesis to address its role in the initiation. It was found that the initiation mode of the mutants is primer-dependent, requiring either an oligonucleotide primer or a back-priming initiation mechanism for the RNA synthesis. The crystal structure of a mutant RdRP with altered initiation platform revealed a set of contacts important for primer-independent initiation. Since phi6 RdRP is structurally and functionally homologous to several viral RdRPs, among them the hepatitis C virus RdRP, these results provide further general insight to understand primer-independent initiation. In this study it is demonstrated that manganese phasing could be used as a practical tool for solving structures of large proteins with a bound manganese ion. The phi6 RdRP was used as a case study to obtain phases for crystallographic analysis. Manganese ions are naturally bound to the phi6 RdRP at the palm domain of the enzyme. In a crystallographic experiment, X-ray diffraction data from a phi6 RdRP crystal were collected at a wavelength of 1.89 Å, which is the K edge of manganese. With this data an automatically built model of the core region of the protein could be obtained. Finally, in this work terminal nucleotidyl transferase (TNTase) activity of the phi6 RdRP was documented in the isolated polymerase as well as in the viral particle. This is the first time that such an activity has been reported in a polymerase of a dsRNA virus. The phi6 RdRP used uridine triphosphates as the sole substrate in a TNTase reaction but could accept several heterologous templates. The RdRP was able to add one or a few non-templated nucleotides to the 3' end of the single- or double-stranded RNA substrate. Based on the results on particle-mediated TNTase activity and previous structural information of the polymerase, a model for termination of the RNA-dependent RNA synthesis is suggested in this thesis.

Characterization of genomic diversity in extraintestinal pathogenic Escherichia coli (ExPEC) and development of a diagnostic DNA microarray for the differentiation of ExPEC isolates causing urinary tract infections

Extraintestinal pathogenic Escherichia coli (ExPEC) represent a diverse group of strains of E. coli, which infect extraintestinal sites, such as the urinary tract, the bloodstream, the meninges, the peritoneal cavity, and the lungs. Urinary tract infections (UTIs) caused by uropathogenic E. coli (UPEC), the major subgroup of ExPEC, are among the most prevalent microbial diseases world wide and a substantial burden for public health care systems. UTIs are responsible for serious morbidity and mortality in the elderly, in young children, and in immune-compromised and hospitalized patients. ExPEC strains are different, both from genetic and clinical perspectives, from commensal E. coli strains belonging to the normal intestinal flora and from intestinal pathogenic E. coli strains causing diarrhea. ExPEC strains are characterized by a broad range of alternate virulence factors, such as adhesins, toxins, and iron accumulation systems. Unlike diarrheagenic E. coli, whose distinctive virulence determinants evoke characteristic diarrheagenic symptoms and signs, ExPEC strains are exceedingly heterogeneous and are known to possess no specific virulence factors or a set of factors, which are obligatory for the infection of a certain extraintestinal site (e. g. the urinary tract). The ExPEC genomes are highly diverse mosaic structures in permanent flux. These strains have obtained a significant amount of DNA (predictably up to 25% of the genomes) through acquisition of foreign DNA from diverse related or non-related donor species by lateral transfer of mobile genetic elements, including pathogenicity islands (PAIs), plasmids, phages, transposons, and insertion elements. The ability of ExPEC strains to cause disease is mainly derived from this horizontally acquired gene pool; the extragenous DNA facilitates rapid adaptation of the pathogen to changing conditions and hence the extent of the spectrum of sites that can be infected. However, neither the amount of unique DNA in different ExPEC strains (or UPEC strains) nor the mechanisms lying behind the observed genomic mobility are known. Due to this extreme heterogeneity of the UPEC and ExPEC populations in general, the routine surveillance of ExPEC is exceedingly difficult. In this project, we presented a novel virulence gene algorithm (VGA) for the estimation of the extraintestinal virulence potential (VP, pathogenicity risk) of clinically relevant ExPECs and fecal E. coli isolates. The VGA was based on a DNA microarray specific for the ExPEC phenotype (ExPEC pathoarray). This array contained 77 DNA probes homologous with known (e.g. adhesion factors, iron accumulation systems, and toxins) and putative (e.g. genes predictably involved in adhesion, iron uptake, or in metabolic functions) ExPEC virulence determinants. In total, 25 of DNA probes homologous with known virulence factors and 36 of DNA probes representing putative extraintestinal virulence determinants were found at significantly higher frequency in virulent ExPEC isolates than in commensal E. coli strains. We showed that the ExPEC pathoarray and the VGA could be readily used for the differentiation of highly virulent ExPECs both from less virulent ExPEC clones and from commensal E. coli strains as well. Implementing the VGA in a group of unknown ExPECs (n=53) and fecal E. coli isolates (n=37), 83% of strains were correctly identified as extraintestinal virulent or commensal E. coli. Conversely, 15% of clinical ExPECs and 19% of fecal E. coli strains failed to raster into their respective pathogenic and non-pathogenic groups. Clinical data and virulence gene profiles of these strains warranted the estimated VPs; UPEC strains with atypically low risk-ratios were largely isolated from patients with certain medical history, including diabetes mellitus or catheterization, or from elderly patients. In addition, fecal E. coli strains with VPs characteristic for ExPEC were shown to represent the diagnostically important fraction of resident strains of the gut flora with a high potential of causing extraintestinal infections. Interestingly, a large fraction of DNA probes associated with the ExPEC phenotype corresponded to novel DNA sequences without any known function in UTIs and thus represented new genetic markers for the extraintestinal virulence. These DNA probes included unknown DNA sequences originating from the genomic subtractions of four clinical ExPEC isolates as well as from five novel cosmid sequences identified in the UPEC strains HE300 and JS299. The characterized cosmid sequences (pJS332, pJS448, pJS666, pJS700, and pJS706) revealed complex modular DNA structures with known and unknown DNA fragments arranged in a puzzle-like manner and integrated into the common E. coli genomic backbone. Furthermore, cosmid pJS332 of the UPEC strain HE300, which carried a chromosomal virulence gene cluster (iroBCDEN) encoding the salmochelin siderophore system, was shown to be part of a transmissible plasmid of Salmonella enterica. Taken together, the results of this project pointed towards the assumptions that first, (i) homologous recombination, even within coding genes, contributes to the observed mosaicism of ExPEC genomes and secondly, (ii) besides en block transfer of large DNA regions (e.g. chromosomal PAIs) also rearrangements of small DNA modules provide a means of genomic plasticity. The data presented in this project supplemented previous whole genome sequencing projects of E. coli and indicated that each E. coli genome displays a unique assemblage of individual mosaic structures, which enable these strains to successfully colonize and infect different anatomical sites.

Expression, Enzymatic Activities and Subcellular Localization of Hepatitis E Virus and Semliki Forest Virus Replicase Proteins

Viruses are submicroscopic, infectious agents that are obligate intracellular parasites. They adopt various types of strategies for their parasitic replication and proliferation in infected cells. The nucleic acid genome of a virus contains information that redirects molecular machinery of the cell to the replication and production of new virions. Viruses that replicate in the cytoplasm and are unable to use the nuclear transcription machinery of the host cell have developed their own transcription and capping systems. This thesis describes replication strategies of two distantly related viruses, hepatitis E virus (HEV) and Semliki Forest virus (SFV), which belong to the alphavirus-like superfamily of positive-strand RNA viruses. We have demonstrated that HEV and SFV share a unique cap formation pathway specific for alphavirus-like superfamily. The capping enzyme first acts as a methyltransferase, catalyzing the transfer of a methyl group from S-adenosylmethionine to GTP to yield m7GTP. It then transfers the methylated guanosine to the end of viral mRNA. Both reactions are virus-specific and differ from those described for the host cell. Therefore, these capping reactions offer attractive targets for the development of antiviral drugs. Additionally, it has been shown that replication of SFV and HEV takes place in association with cellular membranes. The origin of these membranes and the intracellular localization of the components of the replication complex were studied by modern microscopy techniques. It was demonstrated that SFV replicates in cytoplasmic membranes that are derived from endosomes and lysosomes. According to our studies, site for HEV replication seems to be the intermediate compartment which mediates the traffic between endoplasmic reticulum and the Golgi complex. As a result of this work, a unique mechanism of cap formation for hepatitis E virus replicase has been characterized. It represents a novel target for the development of specific inhibitors against viral replication.

Novel Molecular Mechanisms of Arabidopsis Disease Resistance

Plants are capable of recognizing phytopathogens through the perception of pathogen-derived molecules or plant cell-wall degradation products due to the activities of pathogen-secreted enzymes. Such elicitor recognition events trigger an array of inducible defense responses involving signal transduction networks and massive transcriptional re-programming. The outcome of a pathogen infection relies on the balance between different signaling pathways, which are integrated by regulatory proteins. This thesis characterized two key regulatory components: a damage control enzyme, chlorophyllase 1 (AtCHL1), and a transcription factor, WRKY70. Their roles in defense signaling were then investigated. The Erwinia-derived elicitors rapidly activated the expression of AtCLH1 and WRKY70 through different signaling pathways. The expression of the AtCHL1 gene was up-regulated by jasmonic acid (JA) but down-regulated by salicylic acid (SA), whereas WRKY70 was activated by SA and repressed by JA. In order to elucidate the functions of AtCLH1 and WRKY70 in plant defense, stable transgenic lines were produced where these genes were overexpressed or silenced. Additionally, independent knockout lines were also characterized. Bacterial and fungal pathogens were then used to assess the contribution of these genes to the Arabidopsis disease resistance. The transcriptional modulation of AtCLH1 by either the constitutive over-expression or RNAi silencing caused alterations in the chlorophyll-to-chlorophyllide ratio, supporting the claim that chlorophyllase 1 has a role in the chlorophyll degradation pathway. Silencing of this gene led to light-dependent over-accumulation of the reactive oxygen species (ROS) in response to infection by Erwinia carotovora subsp. carotovora SCC1. This was followed by an enhanced induction of SA-dependent defense genes and an increased resistance to this pathogen. Interestingly, little effect on the pathogen-induced SA accumulation at the early infection was observed, suggesting that action of ROS might potentiate SA signaling. In contrast, the pathogen-induced JA production was significantly reduced in the RNAi silenced plants. Moreover, JA signaling and resistance to Alternaria brassicicola were impaired. These observations provide support for the argument that the ROS generated in chloroplasts might have a negative impact on JA signaling. The over-expression of WRKY70 resulted in an enhanced resistance to E. carotovora subsp. carotovora SCC1, Pseudomonas syringae pv. tomato DC3000 and Erysiphe cichoracearum UCSC1, whilst an antisense suppression or an insertional inactivation of WRKY70 led to a compromised resistance to E. carotovora subsp. carotovora SCC1 and to E. cichoracearum UCSC1 but not to P. syringae pv. tomato DC3000. Gene expression analysis revealed that WRKY70 activated many known defense-related genes associated with the SAR response but suppressed a subset of the JA-responsive genes. In particular, I was able to show that both the basal and the induced expression of AtCLH1 was enhanced by the antisense silencing or the insertional inactivation of WRKY70, whereas a reduction in AtCLH1 expression was observed in the WRKY70 over-expressors following an MeJA application or an A. brassicicola infection. Moreover, the SA-induced suppression of AtCLH1 was relieved in wrky70 mutants. These results indicate that WRKY70 down-regulates AtCLH1. An epistasis analysis suggested that WRKY70 functions downstream of the NPR1 in an SA-dependent signaling pathway. When challenged with A. brassicicola, WRKY70 over-expressing plants exhibited a compromised disease resistance while wrky70 mutants had the opposite effect. These results confirmed the WRKY70-mediated inhibitory effects on JA signaling. Furthermore, the WRKY70-controlled suppression of A. brassicicola resistance was mainly through an NPR1-dependent mechanism. Taking all the data together, I suggest that the pathogen-responsive transcription factor WRKY70 is a common component in both SA- and JA-dependent pathways and plays a crucial role in the SA-mediated suppression of JA signaling.

Mutations as molecular tools : The metabolic-rate dependent molecular clock and DNA barcoding of allied species

Mutation and recombination are the fundamental processes leading to genetic variation in natural populations. This variation forms the raw material for evolution through natural selection and drift. Therefore, studying mutation rates may reveal information about evolutionary histories as well as phylogenetic interrelationships of organisms. In this thesis two molecular tools, DNA barcoding and the molecular clock were examined. In the first part, the efficiency of mutations to delineate closely related species was tested and the implications for conservation practices were assessed. The second part investigated the proposition that a constant mutation rate exists within invertebrates, in form of a metabolic-rate dependent molecular clock, which can be applied to accurately date speciation events. DNA barcoding aspires to be an efficient technique to not only distinguish between species but also reveal population-level variation solely relying on mutations found on a short stretch of a single gene. In this thesis barcoding was applied to discriminate between Hylochares populations from Russian Karelia and new Hylochares findings from the greater Helsinki region in Finland. Although barcoding failed to delineate the two reproductively isolated groups, their distinct morphological features and differing life-history traits led to their classification as two closely related, although separate species. The lack of genetic differentiation appears to be due to a recent divergence event not yet reflected in the beetles molecular make-up. Thus, the Russian Hylochares was described as a new species. The Finnish species, previously considered as locally extinct, was recognized as endangered. Even if, due to their identical genetic make-up, the populations had been regarded as conspecific, conservation strategies based on prior knowledge from Russia would not have guaranteed the survival of the Finnish beetle. Therefore, new conservation actions based on detailed studies of the biology and life-history of the Finnish Hylochares were conducted to protect this endemic rarity in Finland. The idea behind the strict molecular clock is that mutation rates are constant over evolutionary time and may thus be used to infer species divergence dates. However, one of the most recent theories argues that a strict clock does not tick per unit of time but that it has a constant substitution rate per unit of mass-specific metabolic energy. Therefore, according to this hypothesis, molecular clocks have to be recalibrated taking body size and temperature into account. This thesis tested the temperature effect on mutation rates in equally sized invertebrates. For the first dataset (family Eucnemidae, Coleoptera) the phylogenetic interrelationships and evolutionary history of the genus Arrhipis had to be inferred before the influence of temperature on substitution rates could be studied. Further, a second, larger invertebrate dataset (family Syrphidae, Diptera) was employed. Several methodological approaches, a number of genes and multiple molecular clock models revealed that there was no consistent relationship between temperature and mutation rate for the taxa under study. Thus, the body size effect, observed in vertebrates but controversial for invertebrates, rather than temperature may be the underlying driving force behind the metabolic-rate dependent molecular clock. Therefore, the metabolic-rate dependent molecular clock does not hold for the here studied invertebrate groups. This thesis emphasizes that molecular techniques relying on mutation rates have to be applied with caution. Whereas they may work satisfactorily under certain conditions for specific taxa, they may fail for others. The molecular clock as well as DNA barcoding should incorporate all the information and data available to obtain comprehensive estimations of the existing biodiversity and its evolutionary history.