DNA nanotweezers studied with a coarse-grained model of DNA

We introduce a coarse-grained rigid nucleotide model of DNA that reproduces the basic thermodynamics of short strands: duplex hybridization, single-stranded stacking and hairpin formation, and also captures the essential structural properties of DNA: the helical pitch, persistence length and torsional stiffness of double-stranded molecules, as well as the comparative flexibility of unstacked single strands. We apply the model to calculate the detailed free-energy landscape of one full cycle of DNA 'tweezers', a simple machine driven by hybridization and strand displacement.Comment: 4 pages, 5 figure

Structural, mechanical and thermodynamic properties of a coarse-grained DNA model

We explore in detail the structural, mechanical and thermodynamic properties of a coarse-grained model of DNA similar to that introduced in Thomas E. Ouldridge, Ard A. Louis, Jonathan P.K. Doye, Phys. Rev. Lett. 104 178101 (2010). Effective interactions are used to represent chain connectivity, excluded volume, base stacking and hydrogen bonding, naturally reproducing a range of DNA behaviour. We quantify the relation to experiment of the thermodynamics of single-stranded stacking, duplex hybridization and hairpin formation, as well as structural properties such as the persistence length of single strands and duplexes, and the torsional and stretching stiffness of double helices. We also explore the model's representation of more complex motifs involving dangling ends, bulged bases and internal loops, and the effect of stacking and fraying on the thermodynamics of the duplex formation transition.Comment: 25 pages, 16 figure

Plasmodium berghei calcium-dependent protein kinase 3 is required for ookinete gliding motility and mosquito midgut invasion

Apicomplexan parasites critically depend on a unique form of gliding motility to colonize their hosts and to invade cells. Gliding requires different stage and species-specific transmembrane adhesins, which interact with an intracellular motor complex shared across parasite stages and species. How gliding is regulated by extracellular factors and intracellular signalling mechanisms is largely unknown, but current evidence suggests an important role for cytosolic calcium as a second messenger. Studying a Plasmodium berghei gene deletion mutant, we here provide evidence that a calcium-dependent protein kinase, CDPK3, has an important function in regulating motility of the ookinete in the mosquito midgut. We show that a cdpk3(–) parasite clone produces morphologically normal ookinetes, which fail to engage the midgut epithelium, due to a marked reduction in their ability to glide productively, resulting in marked reduction in malaria transmission to the mosquito. The mutant was successfully complemented with an episomally maintained cdpk3 gene, restoring mosquito transmission to wild-type level. cdpk3(–) ookinetes maintain their full genetic differentiation potential when microinjected into the mosquito haemocoel and cdpk3(–) sporozoites produced in this way are motile and infectious, suggesting an ookinete-limited essential function for CDPK3

Attempted isolation of the gene encoding the 21 Kd Plasmodium berghei ookinete transmission blocking antigen from Plasmodium yoelli and Plasmodium vivax

The 21kD ookinete antigen of Plasmodium berghei (Pbs 21) has been shown to elicit an effective and long lasting transmission blocking immune response in mice. Having cloned and sequenced this antigen (Paton et al. 1993) the sequence was compared to the genes of the same family previously identified in P. falciparum, P. gallinaceum (Kaslow et al. 1989) and P. reichenowi (Lal et al. 1990). Four conserved areas were identified in this comparison, to which degenerate oligonucleotides were designed. PCR amplification and screening of genomic libraries was then carried out using these oligonucleotides. The P. yoelii gene was successfully cloned and a number of novel P. vivax genes identified but the P. vivax homologue of Pbs21 remains elusive

Triplet repeat DNA structures and human genetic disease: dynamic mutations from dynamic DNA.

Fourteen genetic neurodegenerative diseases and three fragile sites have been associated with the expansion of (CTG)n (CAG)n, (CGG)n (CCG)n, or (GAA)n (TTC)n repeat tracts. Different models have been proposed for the expansion of triplet repeats, most of which presume the formation of alternative DNA structures in repeat tracts. One of the most likely structures, slipped strand DNA, may stably and reproducibly form within triplet repeat sequences. The propensity to form slipped strand DNA is proportional to the length and homogeneity of the repeat tract. The remarkable stability of slipped strand DNA may, in part, be due to loop-loop interactions facilitated by the sequence complementarity of the loops and the dynamic structure of three-way junctions formed at the loop-outs

DNA cruciform arms nucleate through a correlated but non-synchronous cooperative mechanism

Inverted repeat (IR) sequences in DNA can form non-canonical cruciform structures to relieve torsional stress. We use Monte Carlo simulations of a recently developed coarse-grained model of DNA to demonstrate that the nucleation of a cruciform can proceed through a cooperative mechanism. Firstly, a twist-induced denaturation bubble must diffuse so that its midpoint is near the centre of symmetry of the IR sequence. Secondly, bubble fluctuations must be large enough to allow one of the arms to form a small number of hairpin bonds. Once the first arm is partially formed, the second arm can rapidly grow to a similar size. Because bubbles can twist back on themselves, they need considerably fewer bases to resolve torsional stress than the final cruciform state does. The initially stabilised cruciform therefore continues to grow, which typically proceeds synchronously, reminiscent of the S-type mechanism of cruciform formation. By using umbrella sampling techniques we calculate, for different temperatures and superhelical densities, the free energy as a function of the number of bonds in each cruciform along the correlated but non-synchronous nucleation pathways we observed in direct simulations.Comment: 12 pages main paper + 11 pages supplementary dat

Jak-STAT regulation of cyst stem cell development in the Drosophila testis

Establishment and maintenance of functional stem cells is critical for organ development and tissue homeostasis. Little is known about the mechanisms underlying stem establishment during organogenesis. Drosophila testes are among the most thoroughly characterized systems for studying stem cell behavior, with germline stem cells (GSCs) and somatic cyst stem cells (CySCs) cohabiting a discrete stem cell niche at the testis apex. GSCs and CySCs are arrayed around hub cells that also comprise the niche and communication between hub cells, GSCs, and CySCs regulates the balance between stem cell maintenance and differentiation. Recent data has shown that functional, asymmetrically dividing GSCs are first established at similar to 23 h after egg laying during Drosophila testis morphogenesis (Sheng et al., 2009). This process correlates with coalescence of the hub, but development of CySCs from somatic gonadal precursors (SGPs) was not examined. Here, we show that functional CySCs are present at the time of GSC establishment, and that Jak-STAT signaling is necessary and sufficient for CySC maintenance shortly thereafter. Furthermore, hyper-activation of Jak in CySCs promotes expansion of the GSC population, while ectopic Jak activation in the germline induces GSC gene expression in GSC daughter cells but does not prevent spermatogenic differentiation. Together, these observations indicate that, similar to adult testes, Jak-STAT signaling from the hub acts on both GSCs and CySC to regulate their development and differentiation, and that additional signaling from CySCs to the GSCs play a dominant role in controlling GSC maintenance during niche formation. (c) 2012 Elsevier Inc. All rights reserved

Proteomic analysis of Plasmodium in the mosquito: progress and pitfalls

Here we discuss proteomic analyses of whole cell preparations of the mosquito stages of malaria parasite development (i.e. gametocytes, microgamete, ookinete, oocyst and sporozoite) of Plasmodium berghei. We also include critiques of the proteomes of two cell fractions from the purified ookinete, namely the micronemes and cell surface. Whereas we summarise key biological interpretations of the data, we also try to identify key methodological constraints we have met, only some of which we were able to resolve. Recognising the need to translate the potential of current genome sequencing into functional understanding, we report our efforts to develop more powerful combinations of methods for the in silico prediction of protein function and location. We have applied this analysis to the proteome of the male gamete, a cell whose very simple structural organisation facilitated interpretation of data. Some of the in silico predictions made have now been supported by ongoing protein tagging and genetic knockout studies. We hope this discussion may assist future studie

Proteomic analysis of the Plasmodium male gamete reveals the key role for glycolysis in flagellar motility.

BACKGROUND: Gametogenesis and fertilization play crucial roles in malaria transmission. While male gametes are thought to be amongst the simplest eukaryotic cells and are proven targets of transmission blocking immunity, little is known about their molecular organization. For example, the pathway of energy metabolism that power motility, a feature that facilitates gamete encounter and fertilization, is unknown. METHODS: Plasmodium berghei microgametes were purified and analysed by whole-cell proteomic analysis for the first time. Data are available via ProteomeXchange with identifier PXD001163. RESULTS: 615 proteins were recovered, they included all male gamete proteins described thus far. Amongst them were the 11 enzymes of the glycolytic pathway. The hexose transporter was localized to the gamete plasma membrane and it was shown that microgamete motility can be suppressed effectively by inhibitors of this transporter and of the glycolytic pathway. CONCLUSIONS: This study describes the first whole-cell proteomic analysis of the malaria male gamete. It identifies glycolysis as the likely exclusive source of energy for flagellar beat, and provides new insights in original features of Plasmodium flagellar organization