Bio- ja ympäristötieteellinen tiedekunta


Recent Submissions

  • Bulanova, Daria (Helsingin yliopisto, 2016)
    Breast cancer is the most common malignancy in women. Familial history and inherited genetic mutations are the most important risk factors for the development of this type of neoplasia. However, according to current estimations, known highly penetrant germline mutations (e.g. in BRCA1 and BRCA2 genes) explain the origin of less than 30% of familial breast cancer cases 2 5. It urges for identification of the novel genetic determinants of breast carcinogenesis. In the presented work, we performed a whole exome sequencing-based search for new breast cancer-predisposing genetic mutations. We identified a germline inactivating mutation c.183delG [p. Arg61fs] in an orphan G protein-coupled receptor-encoding gene GPRC5A as a novel genetic determinant of breast cancer, which is highly prevalent in BRCA1-associated familial tumors. Following this finding, we revealed that GPRC5A modulates BRCA1 expression and function in homologous recombination-mediated DNA repair, suggesting that GPRC5A may act as a genetic modifier of BRCA1-mediated breast cancer progression. GPRC5A has been implicated in the epithelial carcinogenesis, but so far its function remains controversial. Exploring the effect of GPRC5A deficiency or excessive expression on different aspects of cancer progression, we discovered that GPRC5A plays a role in cancer cell-matrix interactions, and found that it functions as a positive modulator of integrin β1-mediated adhesion. The data suggest that GPRC5A may play a role in progression of particular types of epithelial tumors. We found that GPRC5A associated with two more matrix interaction regulators, Ephrin receptor A2 (EphA2) and hyaluronic acid receptor RHAMM; the effect of GPRC5A on RHAMM expression was BRCA1-dependent. Finally, we revealed that GPRC5A expression in mammary glands is estrogen-dependent and correlates with proliferative capacity of estrogen receptor-positive (ER+) mammary tumors, proposing GPRC5A as a biomarker of ER+ tumors progression. We propose that GPRC5A is a determinant of breast carcinogenesis, whose function depends on the context of the genetic background (presence of BRCA1 mutation) and the hormonal status of the tumor.
  • Siligato, Riccardo (2016)
    Plants possess the rare capability to shape the own architecture according to biotic and abiotic stimuli received from the environment. Spatially defined groups of cells, called meristems, contribute to the division and differentiation processes continuously occurring inside the organism. Meristems can be classified as primary meristems, if they are specified during embryogenesis, or secondary meristems, if they form from undifferentiated, quiescent cells outside the primary meristems. Primary meristems, like the Root Apical Meristem (RAM) and the Shoot Apical Meristem (SAM), coordinate the apical growth of the plant in opposite directions, while secondary meristems shape the radial architecture, regulating the thickness and branching of the primary root and shoot. Cambium is a secondary meristem which produces the vascular tissues xylem and phloem. Xylem transports water and minerals from the root to the photosynthetic tissues; it comprises lignified dead conducting cells called tracheary elements, living parenchyma cells, and lignified dead cells, called fibres, which confer mechanical support and strength. Phloem distributes glucose, RNA, viruses, and proteins from the photosynthetic sources to the sink cells; it consists of empty living sieve elements, supporting companion cells, and parenchyma cells. In order to investigate the regulation of primary and secondary growth, we developed a new chemically inducible system to control the timing and location of the induction of an effector or gene of interest. This enables us to avoid deleterious effects such as seed lethality or sterility when studying the role of a gene in a particular cell type. For example, the meristem cambium is difficult to access through normal techniques, since mutations affecting cambial cell divisions often inhibit the primary growth, too. We developed the inducible system by combining the Multi-Site Gateway cloning technology with the already extant XVE inducible system. This system was used to perform part of the research presented in the thesis. Phytohormones are involved in virtually every aspect of plant life, from development to stress response. They are small molecules which act cellautonomously or non-cell-autonomously to mediate the majority of developmental and environmental responses and, consequently, the activity of the meristems throughout the plant life cycle. Auxin and cytokinins, which were among the first phytohormones discovered, regulate almost every aspect of plant life, such as the division and differentiation processes occurring continuously in the RAM and SAM. The two phytohormones have long been known to interact, and recent studies have uncovered significant crosstalk on the level of biosynthesis, transport, signalling and degradation. We investigated the dynamic role of auxin in maintaining the balance between division, elongation, differentiation in the RAM of the model organism Arabidopsis thaliana. Our results confirm that an optimal level of auxin response is required for division and elongation, while differentiation mechanisms require just a minimal concentration of auxin to proceed normally. We discovered that auxin and cytokinin responses interact synergistically to specify the stem cells and to regulate the timing of divisions in the cambium of Arabidopsis thaliana. The auxin and cytokinin signalling pathways both have a positive role in triggering secondary growth, but the hierarchy of the crosstalk between them is still unclear. Finally, auxin transported via the AUX1/LAX auxin influx carriers regulates the differentiation of vessel elements in the later stages of root cambium development. In summary, we confirm that auxin and cytokinins behave as master regulators of meristematic activities throughout the root, as the signalling pathways associated with both phytohormones heavily influence primary and secondary growth.
  • Mönttinen, Heli (2015)
    The right-hand-shaped polymerases comprising the DNA/RNA polymerase superfamily represent at least six different protein families containing replicases, transcriptases and repair proteins from all three domains of life as well as from their viruses. All of these polymerases have at least three subdomains: fingers, palm and thumb, which form together a structure resembling a right-hand. The catalytic site is located in the palm subdomain, in which polymerization process is catalysed by two Mg2+ ions. There can also be additional ions such as the non-catalytic ion in the RNA-dependent RNA polymerase of phage φ6, which is located approximately 6 Ångströms from the catalytic site. The phylogeny and common regions of the right-hand-shaped polymerases have been previously studied mainly using amino acid sequence alignments. However, the sequence similarity between polymerases belonging into different protein families is low and therefore, the structure alignment provides a potential alternative because protein structures can retain similarity longer in evolution than amino acid sequences. In this thesis, the common structural features between and within the families of the right-hand-shaped polymerases are described, and based on the structural cores the phylogenetic trees are deduced. In addition, the phylogenetic relationships between the right-hand-shaped polymerases and other structurally related proteins are described. As results, it is shown that a phylogenetic tree following the established boundaries of protein families is possible to construct based on structural core sharing no sequence identity. This tree, illustrating long distance phylogenetic relationships suggests that the known right-hand-shaped polymerase families are not the closest relatives to each other. The phylogenies within polymerase families suggest that the relationships among the polymerases do not always follow the evolution of the corresponding organism, which implies horizontal gene transfer between cells, and cells and viruses. The phylogeny of RNA virus RNA polymerases seem to be dependent on the priming mechanism and it does not follow the virion architecture or the Baltimore classification. In addition, the viral RNA-dependent RNA polymerases seem to share a third ion binding site in the proximity of catalytic site.
  • Jaakkola, Salla (Helsingin yliopisto, 2014)
    Extremely saline environments include salt lakes, evaporation ponds, and terrestrial environments, such as salt deserts and underground halite deposits. They are inhabited by halophilic microbes that require salt for living. Cell densities in hypersaline waters can be as high as 107-108 cfu/ml, and most of the cells are archaeal. The number of viruses can be ten times higher than that of the cells. In buried halite, the cell counts are generally low, but viable bacteria and archaea have been isolated from samples up to Permian in age (250-280 million years). Icosahedral tailless virus types seem to be common in hypersaline waters, based on microscopic studies. However, only few such viruses have been isolated and studied. In this thesis Haloarcula hispanica icosahedral virus 2 (HHIV-2) was studied using virological, biochemical, sequencing, lipidomic, and cryo-electron microscopy methods. HHIV-2 infects a halophilic euryarchaeal host and is virulent. It is icosahedral, tailless, and contains an inner membrane. The properties of HHIV-2 were compared to two structurally related viruses: haloarchaeal virus SH1 and thermophilic phage P23-77. The comparison revealed the evolutionary stability of the virion capsid structure, in contrast to the host-interacting structures of viruses. It was also established that different virus capsid assembly pathways can lead to identical capsid architecture. Drill core samples from deeply buried halite deposits were used for isolating halophilic microbes. Nine novel unique archaeal strains belonging to Halobacterium and Halolamina were obtained. No bacteria or viruses could be isolated. Three archaeal isolates from 40 million years old halite were found to be polyploid. Polyploidy is connected to higher mutation resistance, which might positively affect the survival of cells inside halite deposits. One unique isolate was obtained from 123 million years old halite. The complete genomic sequence of this isolate was resolved. Based on sequence data and DNA-DNA hybridization, the isolate represented a novel species, and was named Halobacterium hubeiense. The isolate was found to be closely related to halophilic archaea residing in surface habitats.
  • Dopie, Joseph (2014)
    Actin controls numerous nuclear events including transcription factor activity, chromatin remodeling and RNA polymerase activity. As a component of the cytoskeleton in the cytoplasm, actin traditionally influences cell motility, cell division, cell shape and intracellular transport. In the cytoplasm, actin-binding proteins (ABPs) regulate the dynamic interplay between actin polymerization into filaments and depolymerization into monomers, a process that is central to the cytoplasmic functions of actin. Details of the nuclear functions of actin are unclear and it remains unknown whether actin polymerization and depolymerization in the nucleus is directly linked to the nuclear functions of actin. Many cytoplasmic ABPs have also been identified in the nucleus and shown to influence gene expression, yet their nuclear function in relation to actin is not clear. Actin utilizes an active mechanism to exit the nucleus, however, the nuclear import mechanism for actin has not been characterized. This work provides evidence to support an active nuclear shuttling mechanism for actin and identify novel regulators of nuclear actin. Our live cell imaging data show that actin actively and constantly shuttles between the nucleus and the cytoplasm. Using RNA interference (RNAi) mediated loss-of-function analysis, we show that unphosphorylated cofilin, an ABP, and importin 9, a member of the karyopherin β family of transport receptors, are required for nuclear localization of actin. Protein interaction experiments show that importin 9, cofilin and actin form an import complex that mediates nuclear localization of actin to promote efficient transcriptional activity. Our genome-wide RNAi screens have identified novel and conserved regulators of nucleocytoplasmic transport of actin. Notably, we identified cell division cycle (CDC)73, also known as parafibromin, a component of the RNA polymerase II associated factor homolog (PAF)1 complex and cyclin-dependent kinase 13 (CDK13), a protein that controls cell fate, as regulators of nuclear export of actin. On the other hand our data implicate protein kinase activated gamma subunit 1 (PRKAG1), a regulatory subunit of the AMP-activated protein kinase (AMPK) and RAB18, a member of the ras-related protein family, as factors that promote nuclear import of actin. Also, we identify novel regulators of cofilin phosphorylation that influence nuclear localization of actin. These include; Capping protein B (CPB), an actin filament barbed end capping protein; shibire (SHI)/dynamin, involved in endocytosis; BTB and CNC homology 2 (BACH2), a Pox virus and Zinc finger domain-containing transcriptional regulator; receptor for protein kinase C 1 (RACK1) and structure-specific recognition protein (SSRP), a member of the facilitates chromatin transcription (FACT) complex. BACH2 promotes cofilin dephosphorylation via repression of Lim kinase (LIMK) and testis-specific kinase (TESK) expression, while CPB promotes cofilin dephosphorylation via activation of the expression of slingshot. This study has thus provided essential insights into the regulatory mechanism of nuclear actin and identified several routes through which nuclear actin levels could be regulated.
  • Yuan, Qiang (Qiang Yuan, 2012)
    SNARE Complex Regulation in Membrane Fusion SNAREs are membrane associated proteins essential for intracellular protein trafficking and membrane fusion. There are at least 24 SNAREs in the yeast Saccharomyces cerevisiae and more than 35 SNAREs in mammalian cells. Syntaxin1, SNAP25 and VAMP in mammalian cells can form a SNARE complex and function for the synaptic vesicle docking and fusion at neuronal synapses. The yeast cells contain two syntaxin homologous SNAREs, Sso1p and Sso2p that together with Sec9p and Snc1/2pmediate membrane fusion during exocytosis at the plasma membrane. Based on new phosphorylation mass spectrometry data, the contribution of phosphorylation on Sso protein in vivo function was assessed. Basal or overexpression of phosphomimicking or putative non-phosphorylated Sso1p or Sso2p mutants resulted in no obvious growth phenotype. Sso1p is specifically required for de novo formation of the prospore membrane during meiosis in sporulating cells. Cells expressing only mutant versions of Sso1p, however, did not display any detectable sporulation defects. In addition to sporulation, pseudohyphal and invasive growth modes are regulated by the availability of nutrients. Deletion of SSO1 or SSO2, or expression of the phospho-mutant versions of SSO1 or SSO2 as the sole copies of SSO genes caused no defects in haploid or diploid pseudohyphal and invasive growth. The results indicate that the tested phosphorylation sites in Sso1p or Sso2p are not functional in vivo. In addition to the SNAREs, the Sec1/Munc18 (SM) protein family and other proteins also function in the membrane fusion by facilitating the SNARE complex assembly. However, the molecular interactions for these proteins are not well understood. This study shows that Mso1p, a yeast Sec1p binding protein, interacts with cellular membranes through two biochemically distinct sites. The N-terminal region can insert into the lipid bilayer whereas the C-terminal region of the protein binds phospholipids mainly through electrostatic interactions and may associate with secretory vesicles. Mso1p lipid binding is essential for the plasma membrane localization of the Mso1p-Sec1p complex and for the function of Mso1p in membrane fusion in vivo. The results suggest the existence of a conserved mode of molecular interactions between SM protein binding proteins, Rab GTPases and lipid binding in SNARE mediated membrane fusion. We used a novel Sec1p mutant to study how Sec1p interacts with other proteins involved in SNARE assembly. The results indicate that Sec9p displays a novel type of interaction mode with Sec1p. In addition, the results suggest that Sec1p is required for the plasma membrane interaction of Sec9p-Sec4p-Sro7p complexes. Collectively, the results in the thesis suggest novel interaction modes and previously unknown regulation for the SNARE complex formation for exocytosis in yeast.
  • Sarin, Peter (2010)
    Double-stranded RNA (dsRNA) viruses encode only a single protein species that contains RNA-dependent RNA polymerase (RdRP) motifs. This protein is a central component in the life cycle of a dsRNA virus, carrying out both RNA transcription and replication. The architecture of viral RdRPs resembles that of a 'cupped right hand' with fingers, palm and thumb domains. Those applying de novo initiation have additional structural features, including a flexible C-terminal domain that constitutes the priming platform. Moreover, viral RdRPs must be able to interact with the incoming 3'-terminus of the template and position it so that a productive binary complex is formed. Bacteriophage phi6 of the Cystoviridae family is to date one of the best studied dsRNA viruses. The purified recombinant phi6 RdRP is highly active in vitro and possesses both RNA replication and transcription activities. The extensive biochemical observations and the atomic level crystal structure of the phi6 RdRP provides an excellent platform for in-depth studies of RNA replication in vitro. In this thesis, targeted structure-based mutagenesis, enzymatic assays and molecular mapping of phi6 RdRP and its RNA were used to elucidate the formation of productive RNA-polymerase binary complexes. The positively charged rim of the template tunnel was shown to have a significant role in the engagement of highly structured ssRNA molecules, whereas specific interactions further down in the template tunnel promote ssRNA entry to the catalytic site. This work demonstrated that by aiding the formation of a stable binary complex with optimized RNA templates, the overall polymerization activity of the phi6 RdRP can be greatly enhanced. Furthermore, proteolyzed phi6 RdRPs that possess a nick in the polypeptide chain at the hinge region, which is part of the extended loop, were better suited for catalysis at higher temperatures whilst favouring back-primed initiation. The clipped C-terminus remains associated with the main body of the polymerase and the hinge region, although structurally disordered, is involved in the control of C-terminal domain displacement. The accumulated knowhow on bacteriophage phi6 was utilized in the development of two technologies for the production of dsRNA: (i) an in vitro system that combines the T7 RNA polymerase and the phi6 RdRP to generate dsRNA molecules of practically unlimited length, and (ii) an in vivo RNA replication system based on restricted infection with phi6 polymerase complexes in bacterial cells to produce virtually unlimited amounts of dsRNA. The pools of small interfering RNAs derived from dsRNA produced by these systems were validated and shown to efficiently decrease the expression of both exogenous and endogenous targets.
  • Kolari, Pasi (2010)
    The forest vegetation takes up atmospheric carbon dioxide (CO2) in photosynthesis. Part of the fixed carbon is released back into the atmosphere during plant respiration but a substantial part is stored as plant biomass, especially in the stems of trees. Carbon also accumulates in the soil as litter and via the roots. CO2 is released into the atmosphere from these carbon stocks in the decomposition of dead biomass. Carbon balance of a forest stand is the difference between the CO2 uptake and CO2 efflux. This study quantifies and analyses the dynamics of carbon balance and component CO2 fluxes in four Southern Finnish Scots pine stands that covered the typical economic rotation time of 80 years. The study was based on direct flux measurements with chambers and eddy covariance (EC), and modelling of component CO2 fluxes. The net CO2 exchange of the stand was partitioned into component fluxes: photosynthesis of trees and ground vegetation, respiration of tree foliage and stems, and CO2 efflux from the soil. The relationships between the component fluxes and the environmental factors (light, temperature, atmospheric CO2, air humidity and soil moisture) were studied with mathematical modelling. The annual CO2 balance varied from a source of about 400 g C/m2 at a recently clearcut site to net CO2 uptake of 200 300 g C/m2 in a middle-aged (40-year-old) and a mature (75-year-old) stand. A 12-year-old sapling site was at the turning point from source to a sink of CO2. In the middle-aged stand, photosynthetic production was dominated by trees. Under closed pine canopies, ground vegetation accounted for 10 20% of stand photosynthesis whereas at the open sites the proportion and also the absolute photosynthesis of ground vegetation was much higher. The aboveground respiration was dominated by tree foliage which accounted for one third of the ecosystem respiration. Rate of wood respiration was in the order of 10% of total ecosystem respiration. CO2 efflux from the soil dominated the ecosystem respiratory fluxes in all phases of stand development. Instantaneous and delayed responses to the environmental driving factors could predict well within-year variability in photosynthetic production: In the short term and during the growing season photosynthesis follows primarily light while the seasonal variation is more strongly connected to temperature. The temperature relationship of the annual cycle of photosynthesis was found to be almost equal in the southern boreal zone and at the timberline in the northern boreal zone. The respiratory fluxes showed instantaneous and seasonal temperature relationships but they could also be connected to photosynthesis at an annual timescale.
  • Mattsson, Tuija (2010)
    The terrestrial export of dissolved organic matter (DOM) is associated with climate, vegetation and land use, and thus is under the influence of climatic variability and human interference with terrestrial ecosystems, their soils and hydrological cycles. The present study provides an assessment of spatial variation of DOM concentrations and export, and interactions between DOM, catchment characteristics, land use and climatic factors in boreal catchments. The influence of catchment characteristics, land use and climatic drivers on the concentrations and export of total organic carbon (TOC), total organic nitrogen (TON) and dissolved organic phosphorus (DOP) was estimated using stream water quality, forest inventory and climatic data from 42 Finnish pristine forested headwater catchments, and water quality monitoring, GIS land use, forest inventory and climatic data from the 36 main Finnish rivers (and their sub-catchments) flowing to the Baltic Sea. Moreover, the export of DOM in relation to land use along a European climatic gradient was studied using river water quality and land use data from four European areas. Additionally, the role of organic and minerogenic acidity in controlling pH levels in Finnish rivers and pristine streams was studied by measuring organic anion, sulphate (SO4) and base cation (Ca, Mg, K and Na) concentrations. In all study catchments, TOC was a major fraction of DOM, with much lower proportions of TON and DOP. Moreover, most of TOC and TON was in a dissolved form. The correlation between TOC and TON concentrations was strong and TOC concentrations explained 78% of the variation in TON concentrations in pristine headwater streams. In a subgroup of 20 headwater catchments with similar climatic conditions and low N deposition in eastern Finland, the proportion of peatlands in the catchment and the proportion of Norway spruce (Picea abies Karsten) of the tree stand had the strongest correlation with the TOC and TON concentrations and export. In Finnish river basins, TOC export increased with the increasing proportion of peatland in the catchment, whereas TON export increased with increasing extent of agricultural land. The highest DOP concentrations and export were recorded in river basins with a high extent of agricultural land and urban areas, reflecting the influence of human impact on DOP loads. However, the most important predictor for TOC, TON and DOP export in Finnish rivers was the proportion of upstream lakes in the catchment. The higher the upstream lake percentage, the lower the export indicating organic matter retention in lakes. Molar TOC:TON ratio decreased from headwater catchments covered by forests and peatlands to the large river basins with mixed land use, emphasising the effect of the land use gradient on the stoichiometry of rivers. This study also demonstrated that the land use of the catchments is related to both organic and minerogenic acidity in rivers and pristine headwater streams. Organic anion dominated in rivers and streams situated in northern Finland, reflecting the higher extent of peatlands in these areas, whereas SO4 dominated in southern Finland and on western coastal areas, where the extent of fertile areas, agricultural land, urban areas, acid sulphate soils, and sulphate deposition is highest. High TOC concentrations decreased pH values in the stream and river water, whereas no correlation between SO4 concentrations and pH was observed. This underlines the importance of organic acids in controlling pH levels in Finnish pristine headwater streams and main rivers. High SO4 concentrations were associated with high base cation concentrations and fertile areas, which buffered the effects of SO4 on pH.
  • Sirkiä, Saija (Helsingin yliopisto, 2010)
    The Capercaillie (Tetrao urogallus L.) is often used as a focal species for landscape ecological studies: the minimum size for its lekking area is 300 ha, and the annual home range for an individual may cover 30 80 km2. In Finland, Capercaillie populations have decreased by approximately 40 85%, with the declines likely to have started in the 1940s. Although the declines have partly stabilized from the 1990s onwards, it is obvious that the negative population trend was at least partly caused by changes in human land use. The aim of this thesis was to study the connections between human land use and Capercaillie populations in Finland, using several spatial and temporal scales. First, the effect of forest age structure on Capercaillie population trends was studied in 18 forestry board districts in Finland, during 1965 1988. Second, the abundances of Capercaillie and Moose (Alces alces L.) were compared in terms of several land-use variables on a scale of 50 × 50 km grids and in five regions in Finland. Third, the effects of forest cover and fine-grain forest fragmentation on Capercaillie lekking area persistence were studied in three study locations in Finland, on 1000 and 3000 m spatial scales surrounding the leks. The analyses considering lekking areas were performed with two definitions for forest: > 60 and > 152 m3ha 1 of timber volume. The results show that patterns and processes at large spatial scales strongly influence Capercaillie in Finland. In particular, in southwestern and eastern Finland, high forest cover and low human impact were found to be beneficial for this species. Forest cover (> 60 m3ha 1 of timber) surrounding the lekking sites positively affected lekking area persistence only at the larger landscape scale (3000 m radius). The effects of older forest classes were hard to assess due to scarcity of older forests in several study areas. Young and middle-aged forest classes were common in the vicinity of areas with high Capercaillie abundances especially in northern Finland. The increase in the amount of younger forest classes did not provide a good explanation for Capercaillie population decline in 1965 1988. In addition, there was no significant connection between mature forests (> 152 m3ha 1 of timber) and lekking area persistence in Finland. It seems that in present-day Finnish landscapes, area covered with old forest is either too scarce to efficiently explain the abundance of Capercaillie and the persistence of the lekking areas, or the effect of forest age is only important when considering smaller spatial scales than the ones studied in this thesis. In conclusion, larger spatial scales should be considered for assessing the future Capercaillie management. According to the proposed multi-level planning, the first priority should be to secure the large, regional-scale forest cover, and the second priority should be to maintain fine-grained, heterogeneous structure within the separate forest patches. A management unit covering hundreds of hectares, or even tens or hundreds of square kilometers, should be covered, which requires regional-level land-use planning and co-operation between forest owners.
  • Jaatinen, Silja (Silja Jaatinen, 2009)
    In this thesis three icosahedral lipid-containing double-stranded (ds) deoxyribonucleic acid (DNA) bacteriophages have been studied: PRD1, Bam35 and P23-77. The work focuses on the entry, exit and structure of the viruses. PRD1 is the type member of the Tectiviridae family, infecting a variety of Gram-negative bacteria. The PRD1 receptor binding complex, consisting of the penton protein P31, the spike protein P5 and the receptor binding protein P2 recognizes a specific receptor on the host surface. In this study we found that the transmembrane protein P16 has an important stabilization function as the fourth member of the receptor binding complex and protein P16 may have a role in the formation of a tubular membrane structure, which is needed in the ejection of the genome into the cell. Phage Bam35 (Tectiviridae), which infects Gram-positive hosts, has been earlier found to resemble PRD1 in morphology and genome organization The uncharacterized early and late events in the Bam35 life cycle were studied by electrochemical methods. Physiological changes in the beginning of the infection were found to be similar in both lysogenic and nonlysogenic cell lines, Bam35 inducing a temporal decrease of membrane voltage and K+ efflux. At the end of the infection cycle physiological changes were observed only in the nonlysogenic cell line. The strong K+ efflux 40 min after infection and the induced premature cell lysis propose that Bam35 has a similar holin-endolysin lysis system to that of PRD1. Thermophilic icosahedral dsDNA Thermus phages P23-65H, P23-72 and P23-77 have been proposed to belong to the Tectiviridae family. In this study these phages were compared to each other. Analysis of structural protein patterns and stability revealed these phages to be very similar but not identical. The most stable of the studied viruses, P23-77, was further analyzed in more detail. Cryo-electron microscopy and three-dimensional image reconstruction was used to determine the structure of virus to 14 Å resolution. Results of thin layer chromatography for neutral lipids together with analysis of the three dimensional reconstruction of P23-77 virus particle revealed the presence of an internal lipid membrane. The overall capsid architecture of P23-77 is similar to PRD1 and Bam35, but most closely it resembles the structure of the capsid of archaeal virus SH1. This complicates the classification of dsDNA, internal lipid-containing icosahedral viruses.
  • Koivunen, Minni (Helsingin yliopisto, 2006)
    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.
  • Pahlberg, Johan (2007)
    In the present thesis, questions of spectral tuning, the relation of spectral and thermal properties of visual pigments, and evolutionary adaptation to different light environments were addressed using a group of small crustaceans of the genus Mysis as a model. The study was based on microspectrophotometric measurements of visual pigment absorbance spectra, electrophysiological measurements of spectral sensitivities of dark-adapted eyes, and sequencing of the opsin gene retrieved through PCR. The spectral properties were related to the spectral transmission of the respective light environments, as well as to the phylogentic histories of the species. The photoactivation energy (Ea) was estimated from temperature effects on spectral sensitivity in the long-wavelength range, and calculations were made for optimal quantum catch and optimal signal-to-noise ratio in the different light environments. The opsin amino acid sequences of spectrally characterized individuals were compared to find candidate residues for spectral tuning. The general purpose was to clarify to what extent and on what time scale adaptive evolution has driven the functional properties of (mysid) visual pigments towards optimal performance in different light environments. An ultimate goal was to find the molecular mechanisms underlying the spectral tuning and to understand the balance between evolutionary adaptation and molecular constraints. The totally consistent segregation of absorption maxima (λmax) into (shorter-wavelength) marine and (longer-wavelength) freshwater populations suggests that truly adaptive evolution is involved in tuning the visual pigment for optimal performance, driven by selection for high absolute visual sensitivity. On the other hand, the similarity in λmax and opsin sequence between several populations of freshwater M. relicta in spectrally different lakes highlights the limits to adaptation set by evolutionary history and time. A strong inverse correlation between Ea and λmax was found among all visual pigments studied in these respects, including those of M. relicta and 10 species of vertebrate pigments, and this was used to infer thermal noise. The conceptual signal-to-noise ratios thus calculated for pigments with different λmax in the Baltic Sea and Lake Pääjärvi light environments supported the notion that spectral adaptation works towards maximizing the signal-to-noise ratio rather than quantum catch as such. Judged by the shape of absorbance spectra, the visual pigments of all populations of M. relicta and M. salemaai used exclusively the A2 chromophore (3, 4-dehydroretinal). A comparison of amino acid substitutions between M. relicta and M. salemaai indicated that mysid shrimps have a small number of readily available tuning sites to shift between a shorter - and a longer -wavelength opsin. However, phylogenetic history seems to have prevented marine M. relicta from converting back to the (presumably) ancestral opsin form, and thus the more recent reinvention of marine spectral sensitivity has been accomplished by some other novel mechanism, yet to be found
  • Raateoja, Mika (Merentutkimuslaitos, 2006)
    Phytoplankton ecology and productivity is one of the main branches of contemporary oceanographic research. Research groups in this branch have increasingly started to utilise bio-optical applications. My main research objective was to critically investigate the advantages and deficiencies of the fast repetition rate (FRR) fluorometry for studies of productivity of phytoplankton, and the responses of phytoplankton towards varying environmental stress. Second, I aimed to clarify the applicability of the FRR system to the optical environment of the Baltic Sea. The FRR system offers a highly dynamic tool for studies of phytoplankton photophysiology and productivity both in the field and in a controlled environment. The FRR metrics obtain high-frequency in situ determinations of the light-acclimative and photosynthetic parameters of intact phytoplankton communities. The measurement protocol is relatively easy to use without phases requiring analytical determinations. The most notable application of the FRR system lies in its potential for making primary productivity (PP) estimations. However, the realisation of this scheme is not straightforward. The FRR-PP, based on the photosynthetic electron flow (PEF) rate, are linearly related to the photosynthetic gas exchange (fixation of 14C) PP only in environments where the photosynthesis is light-limited. If the light limitation is not present, as is usually the case in the near-surface layers of the water column, the two PP approaches will deviate. The prompt response of the PEF rate to the short-term variability in the natural light field makes the field comparisons between the PEF-PP and the 14C-PP difficult to interpret, because this variability is averaged out in the 14C-incubations. Furthermore, the FRR based PP models are tuned to closely follow the vertical pattern of the underwater irradiance. Due to the photoacclimational plasticity of phytoplankton, this easily leads to overestimates of water column PP, if precautionary measures are not taken. Natural phytoplankton is subject to broad-waveband light. Active non-spectral bio-optical instruments, like the FRR fluorometer, emit light in a relatively narrow waveband, which by its nature does not represent the in situ light field. Thus, the spectrally-dependent parameters provided by the FRR system need to be spectrally scaled to the natural light field of the Baltic Sea. In general, the requirement of spectral scaling in the water bodies under terrestrial impact concerns all light-adaptive parameters provided by any active non-spectral bio-optical technique. The FRR system can be adopted to studies of all phytoplankton that possess efficient light harvesting in the waveband matching the bluish FRR excitation. Although these taxa cover the large bulk of all the phytoplankton taxa, one exception with a pronounced ecological significance is found in the Baltic Sea. The FRR system cannot be used to monitor the photophysiology of the cyanobacterial taxa harvesting light in the yellow-red waveband. These taxa include the ecologically-significant bloom-forming cyanobacterial taxa in the Baltic Sea.