Frequent recombination events generate diversity within the multi-copy variant antigen gene families of Plasmodium falciparum

The human malaria parasite Plasmodium falciparum utilises a mechanism of antigenic variation to avoid the antibody response of its human host and thereby generates a long-term, persistent infection. This process predominantly results from systematic changes in expression of the primary erythrocyte surface antigen, a parasite-produced protein called PfEMP1 that is encoded by a repertoire of over 60 var genes in the P. falciparum genome. var genes exhibit extensive sequence diversity, both within a single parasite's genome as well as between different parasite isolates, and thus provide a large repertoire of antigenic determinants to be alternately displayed over the course of an infection. Whilst significant work has recently been published documenting the extreme level of diversity displayed by var genes found in natural parasite populations, little work has been done regarding the mechanisms that lead to sequence diversification and heterogeneity within var genes. In the course of producing transgenic lines from the original NF54 parasite isolate, we cloned and characterised a parasite line, termed E5, which is closely related to but distinct from 3D7, the parasite used for the P. falciparum genome nucleotide sequencing project. Analysis of the E5 var gene repertoire, as well as that of the surrounding rif and stevor multi-copy gene families, identified examples of frequent recombination events within these gene families, including an example of a duplicative transposition which indicates that recombination events play a significant role in the generation of diversity within the antigen encoding genes of P. falciparum.     

Petunia hybrida CAROTENOID CLEAVAGE DIOXYGENASE7 Is Involved in the Production of Negative and Positive Branching Signals in Petunia

One of the key factors that defines plant form is the regulation of when and where branches develop. The diversity of form observed in nature results, in part, from variation in the regulation of branching between species. Two CAROTENOID CLEAVAGE DIOXYGENASE (CCD) genes, CCD7 and CCD8, are required for the production of a branch-suppressing plant hormone. Here, we report that the decreased apical dominance3 (dad3) mutant of petunia (Petunia hybrida) results from the mutation of the PhCCD7 gene and has a less severe branching phenotype than mutation of PhCCD8 (dad1). An analysis of the expression of this gene in wild-type, mutant, and grafted petunia suggests that in petunia, CCD7 and CCD8 are coordinately regulated. In contrast to observations in Arabidopsis (Arabidopsis thaliana), ccd7ccd8 double mutants in petunia show an additive phenotype. An analysis using dad3 or dad1 mutant scions grafted to wild-type rootstocks showed that when these plants produce adventitious mutant roots, branching is increased above that seen in plants where the mutant roots are removed. The results presented here indicate that mutation of either CCD7 or CCD8 in petunia results in both the loss of an inhibitor of branching and an increase in a promoter of branching.The dynamic process that leads to a plant''s architecture is regulated by developmental factors and by environmental conditions. Whether or not axillary meristems grow to form branches is one key component of plant architecture. Plants with altered architecture have been important in agronomy since the earliest selections were made by humans. More recent examples are vital to the productivity of our current farming systems. The domestication of maize (Zea mays) and the dwarfing of wheat (Triticum aestivum) and rice (Oryza sativa; as part of the Green Revolution) involved alterations to plant height and branch number that dramatically improved productivity (for review, see Sakamoto and Matsuoka, 2004).Arabidopsis (Arabidopsis thaliana), rice, pea (Pisum sativum), and petunia (Petunia hybrida) are important model plants in which axillary branching has been studied. The growth habits of these plants show differences when grown under standard floral inductive conditions. This is due, in part, to the differing developmental programs controlling the outgrowth of axillary branches. Petunia (inbred genetic stock V26) produces basal axillary branches between nodes two and eight that begin their growth during the vegetative growth phase (Snowden and Napoli, 2003). Axillary branches may also form in the nodes immediately below the first flower after the floral transition (Napoli et al., 1999). Arabidopsis generally produces axillary branches after flowering, releasing axillary meristems in the rosette and also from cauline leaves (Hempel and Feldman, 1994). Wild-type, tall pea cultivars such as Parvus are very unlikely to produce basal axillary branches at any stage of growth but do branch at the nodes immediately below the first flower (Stafstrom, 1995). Cultivated rice produces basal axillary branches, called tillers, during vegetative growth. The tillers formed early in plant development will produce panicles (flowering branches), and the remainder will senesce (Hanada, 1993). How these differences in development arise is yet to be understood.Although the overall architecture of plants varies considerably, the genes so far identified that control branching are frequently conserved between species. In particular, two CAROTENOID CLEAVAGE DIOXYGENASE (CCD) genes, CCD7 and CCD8, appear to be well conserved among the plant species studied. Mutations in these two genes result in increased branching phenotypes in every species studied to date (Sorefan et al., 2003; Booker et al., 2004; Snowden et al., 2005; Zou et al., 2005; Johnson et al., 2006; Arite et al., 2007). One interesting line of enquiry is to consider whether differences in the regulation or activity of these two genes are involved in the diversity of architecture seen in plants.Grafting experiments have provided insight into the control of axillary branching, in particular the discovery that signals move from roots to shoots. In petunia, Arabidopsis, and pea, some of the increased branching mutants (ccd7 and ccd8 mutants in particular) can be reverted to a wild-type phenotype by grafting mutant scions onto wild-type rootstocks (for review, see Drummond et al., 2009). Additionally, ccd8 mutant plant lines have been reverted to the wild type by the insertion of a small piece (approximately 2 mm) of wild-type hypocotyl into the hypocotyls of mutant petunia or by insertion of a small piece of epicotyl into the epicotyl of mutant pea (Napoli, 1996; Foo et al., 2001). In Arabidopsis, the ccd7 mutant has been similarly reverted using hypocotyl interstock grafts (Booker et al., 2004). Together, these results suggest the presence of a mobile branch inhibitor produced in wild-type tissue. However, an observation by Napoli (1996) suggested that decreased apical dominance1 (dad1) mutant roots may also have a branch-inducing effect in certain circumstances. A similar result was observed for pea in Parvus by Foo et al. (2001). The discussion presented by Napoli (1996) did not exclude either a branch-inducing or a branch-suppressing signal, although current models generally only consider the presence of a branch inhibitor, and recent efforts have focused on the identification of inhibitors of branching.Strigolactones have recently been identified as signaling molecules that inhibit axillary branch outgrowth in plants (Gomez-Roldan et al., 2008; Umehara et al., 2008). Strigolactones were previously identified as signal molecules secreted from roots. When arbuscular mycorrhizae detect strigolactones, they undergo a preinfection hyperbranching response that is thought to aid fungal colonization of the roots, frequently leading to improved nutrient uptake by the plant (Akiyama et al., 2005). The seeds of the parasitic plants Orobanche species and Striga species are also induced to germinate upon detection of strigolactones in the soil, resulting in significant yield losses for some crops (Cook et al., 1966; Siame et al., 1993; Yokota et al., 1998). The production of strigolactones in rice and pea has been shown to require the action of both CCD7 and CCD8 (Gomez-Roldan et al., 2008; Umehara et al., 2008). The discovery that strigolactones can alter branching confirmed a new layer of regulatory complexity in the control of branching that has long been hidden beneath the global plant growth regulators of auxin and cytokinin.In this study, we have focused on the role of the CCD7 gene in the control of branching in petunia. We have isolated a petunia CCD7 ortholog (PhCCD7) and show that the increased branching phenotype of the dad3 mutant is caused by a lesion in this gene. The phenotype of the dad3 mutant is less severe than that of the petunia ccd8 mutant (dad1), and the double ccd7ccd8 mutant is shown to be additive. These observations are contrasted with what has been observed for other plant species. We show that the regulation of PhCCD7 is similar to that of the PhCCD8 gene, with expression predominantly in root and stem tissue (although at a reduced level) and up-regulation of expression in plants with increased numbers of branches. We also provide evidence for the presence of a branch-promoting signal in mutant roots of petunia. These results suggest that there is an added layer of complexity to the control of branching that is not fully described by current models and indicate that the CCD7 gene may have a role in the diversity of plant architecture.     

An Unusual Recent Expansion of the C-Terminal Domain of RNA Polymerase II in Primate Malaria Parasites Features a Motif Otherwise Found Only in Mammalian Polymerases

The tail of the enzyme RNA polymerase II is responsible for integrating the diverse events of gene expression in eukaryotes and is indispensable for life in yeast, fruit flies, and mice. The tail features a C-terminal domain (CTD), which is comprised of tandemly repeated Y₁-S₂-P₃-T₄-S₅-P₆-S₇ amino acid heptads that are highly conserved across evolutionary lineages, with all mammalian polymerases featuring 52 identical heptad repeats. However, the composition and function of protozoan CTDs remain less well understood. We find that malaria parasites (genus Plasmodium) display an unprecedented plasticity within the length and composition of their CTDs. The CTD in malaria parasites which infect human and nonhuman primates has expanded compared to closely related species that infect rodents or birds. In addition, this variability extends to different isolates within a single species, such as isolates of the human malaria parasite, Plasmodium falciparum. Our results indicate that expanded CTD heptads in malaria parasites correlates with parasitism of primates and provide the first demonstration of polymorphism of the RNA polymerase II CTD within a single species. The expanded set of CTD heptads feature lysine in the seventh position (Y₁-S₂-P₃-T₄-S₅-P₆-K₇), a sequence only seen otherwise in the distal portion of mammalian polymerases. These observations raise new questions for the radiation of malaria parasites into diverse hosts and for the molecular evolution of RNA polymerase II.     

Phylogeography of the common vampire bat (Desmodus rotundus): Marked population structure, Neotropical Pleistocene vicariance and incongruence between nuclear and mtDNA markers

Background  

The common vampire bat Desmodus rotundus is an excellent model organism for studying ecological vicariance in the Neotropics due to its broad geographic range and its preference for forested areas as roosting sites. With the objective of testing for Pleistocene ecological vicariance, we sequenced a mitocondrial DNA (mtDNA) marker and two nuclear markers (RAG2 and DRB) to try to understand how Pleistocene glaciations affected the distribution of intraspecific lineages in this bat.     

Purification of reversibly oxidized proteins (PROP) reveals a redox switch controlling p38 MAP kinase activity

Oxidation of cysteine residues of proteins is emerging as an important means of regulation of signal transduction, particularly of protein kinase function. Tools to detect and quantify cysteine oxidation of proteins have been a limiting factor in understanding the role of cysteine oxidation in signal transduction. As an example, the p38 MAP kinase is activated by several stress-related stimuli that are often accompanied by in vitro generation of hydrogen peroxide. We noted that hydrogen peroxide inhibited p38 activity despite paradoxically increasing the activating phosphorylation of p38. To address the possibility that cysteine oxidation may provide a negative regulatory effect on p38 activity, we developed a biochemical assay to detect reversible cysteine oxidation in intact cells. This procedure, PROP, demonstrated in vivo oxidation of p38 in response to hydrogen peroxide and also to the natural inflammatory lipid prostaglandin J2. Mutagenesis of the potential target cysteines showed that oxidation occurred preferentially on residues near the surface of the p38 molecule. Cysteine oxidation thus controls a functional redox switch regulating the intensity or duration of p38 activity that would not be revealed by immunodetection of phosphoprotein commonly interpreted as reflective of p38 activity.     

Focus on Water: Biodesalination: A Case Study for Applications of Photosynthetic Bacteria in Water Treatment

Shortage of freshwater is a serious problem in many regions worldwide, and is expected to become even more urgent over the next decades as a result of increased demand for food production and adverse effects of climate change. Vast water resources in the oceans can only be tapped into if sustainable, energy-efficient technologies for desalination are developed. Energization of desalination by sunlight through photosynthetic organisms offers a potential opportunity to exploit biological processes for this purpose. Cyanobacterial cultures in particular can generate a large biomass in brackish and seawater, thereby forming a low-salt reservoir within the saline water. The latter could be used as an ion exchanger through manipulation of transport proteins in the cell membrane. In this article, we use the example of biodesalination as a vehicle to review the availability of tools and methods for the exploitation of cyanobacteria in water biotechnology. Issues discussed relate to strain selection, environmental factors, genetic manipulation, ion transport, cell-water separation, process design, safety, and public acceptance.Bacteria are commonly employed for the purification of municipal and industrial wastewater but until now, established water treatment technologies have not taken advantage of photosynthetic bacteria (i.e. cyanobacteria). The ability of cyanobacterial cultures to grow at high cell densities with minimal nutritional requirements (e.g. sunlight, carbon dioxide, and minerals) opens up many future avenues for sustainable water treatment applications.Water security is an urgent global issue, especially because many regions of the world are experiencing, or are predicted to experience, water shortage conditions: More than one in six people globally are water stressed, in that they do not have access to safe drinking water (United Nations, 2006). Ninety-seven percent of the Earth’s water is in the oceans; consequently, there are many efforts to develop efficient methods for converting saltwater into freshwater. Various processes using synthetic membranes, such as reverse osmosis, are successfully used for large-scale desalination. However, the high energy consumption of these technologies has limited their application predominantly to countries with both relatively limited freshwater resources and high availability of energy, for example, in the form of oil reserves.The development of an innovative, low-energy biological desalination process, using biological membranes of cyanobacteria, would thus be both attractive and pertinent. The core of the proposed biodesalination process (Fig. 1) is a low-salt biological reservoir within seawater that can serve as an ion exchanger. Its development can be separated into several complementary steps. The first step comprises the selection of a cyanobacterial strain that can be grown to high cell densities in seawater with minimal requirement for energy sources other than those that are naturally available. The environmental conditions during growth can be manipulated to enhance natural extrusion of sodium (Na⁺) by cyanobacteria. In the second step, cyanobacterial ion transport mechanisms must be manipulated to generate cells in which sodium export is replaced with intracellular sodium accumulation. This will involve inhibition of endogenous Na⁺ export and expression of synthetic molecular units that facilitate light-driven sodium flux into the cells. A robust control system built from biological switches will be required to achieve precisely timed expression of the salt-accumulating molecular units. The third step consists of engineering efficient separation of the cyanobacterial cells from the desalinated water, using knowledge of physicochemical properties of the cell surface and their natural ability to produce extracellular polymeric substances (EPSs), which aid cell separation while preserving cell integrity. The fourth step integrates the first three steps into a manageable and scalable engineering process. The fifth and final step assesses potential risks and public acceptance issues linked to the new technology.Open in a separate windowFigure 1.Proposed usage of cyanobacterial cultures for water treatment. A, Hypothetical water treatment station. Situated in basins next to the water source, sun-powered cell cultures remove unwanted elements from the water. The clean water is separated from the cells for human uses. The produced biomass is available for other industries. The proposed biodesalination process is based on the following steps. B, Photoautotrophic cells divide to generate high-density cultures. C, The combined cell volume is low in salt as a result of transport proteins in the cell membrane that export sodium using photosynthetically generated energy. D, Through environmental and genetic manipulation, salt export is inhibited and replaced with transport modules that accumulate salt inside the cells. This process is again fueled by light energy. E, Manipulation of cell surface properties separates the salt-enriched cells from the desalinated water.In this review, we outline the state of knowledge and available technology for each of the steps, as well as summarize the current knowledge gaps and technical limitations in employing a large-scale water treatment process using cyanobacteria. Before discussing these issues, we provide some background information on the usage of cyanobacteria in biotechnology and the impact of sodium on cellular functions of cyanobacteria. The example of biodesalination provides a good vehicle to discuss the suitability of photosynthetic bacteria for water treatment more generally. The issues addressed in this review are relevant for a wide range of biotechnological applications of cyanobacteria, including bioremediation and biodegradation as well as the generation of biofuels, natural medicines, or cosmetics.     

The hydrophobin EAS is largely unstructured in solution and functions by forming amyloid-like structures

BACKGROUND: Fungal hydrophobin proteins have the remarkable ability to self-assemble into polymeric, amphipathic monolayers on the surface of aerial structures such as spores and fruiting bodies. These monolayers are extremely resistant to degradation and as such offer the possibility of a range of biotechnological applications involving the reversal of surface polarity. The molecular details underlying the formation of these monolayers, however, have been elusive. We have studied EAS, the hydrophobin from the ascomycete Neurospora crassa, in an effort to understand the structural aspects of hydrophobin polymerization. RESULTS: We have purified both wild-type and uniformly 15N-labeled EAS from N. crassa conidia, and used a range of physical methods including multidimensional NMR spectroscopy to provide the first high resolution structural information on a member of the hydrophobin family. We have found that EAS is monomeric but mostly unstructured in solution, except for a small region of antiparallel beta sheet that is probably stabilized by four intramolecular disulfide bonds. Polymerised EAS appears to contain substantially higher amounts of beta sheet structure, and shares many properties with amyloid fibers, including a characteristic gold-green birefringence under polarized light in the presence of the dye Congo Red. CONCLUSIONS: EAS joins an increasing number of proteins that undergo a disorder-->order transition in carrying out their normal function. This report is one of the few examples where an amyloid-like state represents the wild-type functional form. Thus the mechanism of amyloid formation, now thought to be a general property of polypeptide chains, has actually been applied in nature to form these remarkable structures.     

Isocitrate dehydrogenase 1 R132C mutation occurs exclusively in microsatellite stable colorectal cancers with the CpG island methylator phenotype

The CpG Island Methylator Phenotype (CIMP) is fundamental to an important subset of colorectal cancer; however, its cause is unknown. CIMP is associated with microsatellite instability but is also found in BRAF mutant microsatellite stable cancers that are associated with poor prognosis. The isocitrate dehydrogenase 1 (IDH1) gene causes CIMP in glioma due to an activating mutation that produces the 2-hydroxyglutarate oncometabolite. We therefore examined IDH1 alteration as a potential cause of CIMP in colorectal cancer. The IDH1 mutational hotspot was screened in 86 CIMP-positive and 80 CIMP-negative cancers. The entire coding sequence was examined in 81 CIMP-positive colorectal cancers. Forty-seven cancers varying by CIMP-status and IDH1 mutation status were examined using Illumina 450K DNA methylation microarrays. The R132C IDH1 mutation was detected in 4/166 cancers. All IDH1 mutations were in CIMP cancers that were BRAF mutant and microsatellite stable (4/45, 8.9%). Unsupervised hierarchical cluster analysis identified an IDH1 mutation-like methylation signature in approximately half of the CIMP-positive cancers. IDH1 mutation appears to cause CIMP in a small proportion of BRAF mutant, microsatellite stable colorectal cancers. This study provides a precedent that a single gene mutation may cause CIMP in colorectal cancer, and that this will be associated with a specific epigenetic signature and clinicopathological features.