Movement-associated proteins of Potato virus A: attachment to virus particles and phosphorylation

The particles of Potato virus A (PVA; genus Potyvirus) are helically constructed filaments that contain multiple copies of a single type of coat-protein (CP) subunit and a single copy of genome-linked protein (VPg), attached to one end of the virion. Examination of negatively-stained virions by electron microscopy revealed flexuous, rod-shaped particles with no obvious terminal structures. It is known that particles of several filamentous plant viruses incorporate additional minor protein components, forming stable complexes that mediate particle disassembly, movement or transmission by insect vectors. The first objective of this work was to study the interaction of PVA movement-associated proteins with virus particles and how these interactions contribute to the morphology and function of the virus particles. Purified particles of PVA were examined by atomic force microscopy (AFM) and immuno-gold electron microscopy. A protrusion was found at one end of some of the potyvirus particles, associated with the 5' end of the viral RNA. The tip contained two virus-encoded proteins, the genome-linked protein (VPg) and the helper-component proteinase (HC-Pro). Both are required for cell-to-cell movement of the virus. Biochemical and electron microscopy studies of purified PVA samples also revealed the presence of another protein required for cell-to-cell movement the cylindrical inclusion protein (CI), which is also an RNA helicase/ATPase. Centrifugation through a 5-40% sucrose gradient separated virus particles with no detectable CI to a fraction that remained in the gradient, from the CI-associated particles that went to the pellet. Both types of particles were infectious. AFM and translation experiments demonstrated that when the viral CI was not present in the sample, PVA virions had a beads-on-a-string phenotype, and RNA within the virus particles was more accessible to translation. The second objective of this work was to study phosphorylation of PVA movement-associated and structural proteins (CP and VPg) in vitro and, if possible, in vivo. PVA virion structural protein CP is necessary for virus cell-to-cell movement. The tobacco protein kinase CK2 was identified as a kinase phosphorylating PVA CP. A major site of CK2 phosphorylation in PVA CP was identified as a single threonine within a CK2 consensus sequence. Amino acid substitutions affecting the CK2 consensus sequence in CP resulted in viruses that were defective in cell-to-cell and long-distance movement. The CK2 regulation of virion assembly and cell-to-cell movement by phosphorylation of CP was possibly due to the inhibition of CP binding to viral RNA. Four putative phosphorylation sites were identified from an in vitro phosphorylated recombinant VPg. All four were mutated and the spread of mutant viruses in two different host plants was studied. Two putative phosphorylation site mutants (Thr45 and Thr49) had phenotypes identical to that of a wild type (WT) virus infection in both Nicotiana benthamiana and N. tabacum plants. The other two mutant viruses (Thr132/Ser133 and Thr168) showed different phenotypes with increased or decreased accumulation rates, respectively, in inoculated and the first two systemically infected leaves of N. benthamiana. The same mutants were occasionally restricted to single cells in N. tabacum plants, suggesting the importance of these amino acids in the PVA infection cycle in N. tabacum.

The life cycle and genetic structure of the red alga Furcellaria lumbricalis on a salinity gradient

The life cycle and genetic diversity of the red alga Furcellaria lumbricalis (Hudson) Lamouroux were investigated in 15 populations in northern Europe. The occurrence of different life cycle phases and seasonality of reproduction were studied in four brackish populations in the northern Baltic Sea. Furthermore, a new method, based on genome screening with ISSR markers combined with a restriction-ligation method, was developed to discover microsatellite markers for population genetic analyses. The mitochondrial DNA cox2-3 spacer sequence and four microsatellite markers were used to examine the genetic diversity and differentiation of red algal populations in northern Europe. In addition, clonality and small-scale genetic structure of one Irish and four Baltic Sea populations were studied with microsatellite markers. It was discovered that at the low salinities of the northern Baltic Sea, only tetrasporophytes and males were present in the populations of F. lumbricalis and that winter was the main season for tetrasporangial production. Furthermore, the population occurring at the lowest salinity (3.6 practical salinity units, psu) did not produce spores. The size of the tetraspores was smaller in the Baltic Sea populations than that in the Irish population, and there were more deformed spores in the Baltic Sea populations than in the Irish populations. Studies with microsatellite markers indicated that clonality is a common phenomenon in the Baltic Sea populations of F. lumbricalis, although the proportion of clonal individuals varied among populations. Some genetic divergence occurred within locations both in Ireland and in the northern Baltic Sea. Even though no carpogonia were detected in the field samples during the study, the microsatellite data indicated that sexual reproduction occurs at least occasionally in the northern Baltic Sea. The genetic diversity of F. lumbricalis was highest in Brittany, France. Since no variation was discovered in the mtDNA cox2-3 spacer sequence, which is generally regarded as an informative phylogeographic marker in red algae, it can be assumed that the studied populations probably share the same origin.

On the force potential of strategy texts: a critical discourse analysis of a strategic plan and its power effects in a city organization

Despite increasing interest in the discursive aspects of strategy, few studies have examined strategy texts and their power effects. We draw from Critical Discourse Analysis to better understand the power of strategic plans as a directive genre. In our empirical analysis, we examined the creation of the official strategic plan of the City of Lahti in Finland. As a result of our inductive analysis, we identified five central discursive features of this plan: self-authorization, special terminology, discursive innovation, forced consensus and deonticity. We argue that these features can, with due caution, be generalized and conceived as distinctive features of the strategy genre. We maintain that these discursive features are not trivial characteristics; they have important implications for the textual agency of strategic plans, their performative effects, impact on power relations and ideological implications.

Dual-trap optical tweezers with real-time force clamp control

Single molecule force clamp experiments are widely used to investigate how enzymes, molecular motors, and other molecular mechanisms work. We developed a dual-trap optical tweezers instrument with real-time (200 kHz update rate) force clamp control that can exert 0–100 pN forces on trapped beads. A model for force clamp experiments in the dumbbell-geometry is presented. We observe good agreement between predicted and observed power spectra of bead position and force fluctuations. The model can be used to predict and optimize the dynamics of real-time force clamp optical tweezers instruments. The results from a proof-of-principle experiment in which lambda exonuclease converts a double-stranded DNA tether, held at constant tension, into its single-stranded form, show that the developed instrument is suitable for experiments in single molecule biology.