Genome-wide mapping reveals single-origin chromosome replication in Leishmania, a eukaryotic microbe

Background DNA replication initiates on defined genome sites, termed origins. Origin usage appears to follow common rules in the eukaryotic organisms examined to date: all chromosomes are replicated from multiple origins, which display variations in firing efficiency and are selected from a larger pool of potential origins. To ask if these features of DNA replication are true of all eukaryotes, we describe genome-wide origin mapping in the parasite Leishmania. Results Origin mapping in Leishmania suggests a striking divergence in origin usage relative to characterized eukaryotes, since each chromosome appears to be replicated from a single origin. By comparing two species of Leishmania, we find evidence that such origin singularity is maintained in the face of chromosome fusion or fission events during evolution. Mapping Leishmania origins suggests that all origins fire with equal efficiency, and that the genomic sites occupied by origins differ from related non-origins sites. Finally, we provide evidence that origin location in Leishmania displays striking conservation with Trypanosoma brucei, despite the latter parasite replicating its chromosomes from multiple, variable strength origins. Conclusions The demonstration of chromosome replication for a single origin in Leishmania, a microbial eukaryote, has implications for the evolution of origin multiplicity and associated controls, and may explain the pervasive aneuploidy that characterizes Leishmania chromosome architecture

Genome cartography through domain annotation

The evolutionary history of eukaryotic proteins involves rapid sequence divergence, addition and deletion of domains, and fusion and fission of genes. Although the protein repertoires of distantly related species differ greatly, their domain repertoires do not. To account for the great diversity of domain contexts and an unexpected paucity of ortholog conservation, we must categorize the coding regions of completely sequenced genomes into domain families, as well as protein families

THoR: a tool for domain discovery and curation of multiple alignments

We describe a tool, THoR, that automatically creates and curates multiple sequence alignments representing protein domains. This exploits both PSI-BLAST and HMMER algorithms and provides an accurate and comprehensive alignment for any domain family. The entire process is designed for use via a web-browser, with simple links and cross-references to relevant information, to assist the assessment of biological significance. THoR has been benchmarked for accuracy using the SMART and pufferfish genome databases

Unsung heroes: Constituency election agents in British general elections

Despite their central role in the electoral process, constituency agents have been largely overlooked by political scientists and this article seeks to rectify the omission. It sketches the origins and development of the role of agent from the late 19th century and suggests that a serious rethink of the role took place in the 1990s. Survey-based evidence about the social characteristics of agents is presented confirming that they are largely middle-aged, middle-class, well-educated men. They are also becoming more experienced, offer realistic assessments of the impact of constituency campaigning and, arguably, many take a long-term view of how their party's support can be maximised

Zygote morphogenesis but not the establishment of cell polarity in plasmodium berghei Is controlled by the small GTPase, RAB11A

Plasmodium species are apicomplexan parasites whose zoites are polarized cells with a marked apical organisation where the organelles associated with host cell invasion and colonization reside. Plasmodium gametes mate in the mosquito midgut to form the spherical and presumed apolar zygote that morphs during the following 24 hours into a polarized, elongated and motile zoite form, the ookinete. Endocytosis-mediated protein transport is generally necessary for the establishment and maintenance of polarity in epithelial cells and neurons, and the small GTPase RAB11A is an important regulator of protein transport via recycling endosomes. PbRAB11A is essential in blood stage asexual of Plasmodium. Therefore, a promoter swap strategy was employed to down-regulate PbRAB11A expression in gametocytes and zygotes of the rodent malaria parasite, Plasmodium berghei which demonstrated the essential role of RAB11A in ookinete development. The approach revealed that lack of PbRAB11A had no effect on gamete production and fertility rates however, the zygote to ookinete transition was almost totally inhibited and transmission through the mosquito was prevented. Lack of PbRAB11A did not prevent meiosis and mitosis, nor the establishment of polarity as indicated by the correct formation and positioning of the Inner Membrane Complex (IMC) and apical complex. However, morphological maturation was prevented and parasites remained spherical and immotile and furthermore, they were impaired in the secretion and distribution of microneme cargo. The data are consistent with the previously proposed model of RAB11A endosome mediated delivery of plasma membrane in Toxoplasma gondii if not its role in IMC formation and implicate it in microneme function

Optical Diagnostics on Helical Flux Compression Generators

Explosively driven magnetic flux compression (MFC) has been object of research for more than three decades. Actual interest in the basic physical picture of flux compression has been heightened by a newly started Department of Defense (DoD) Multi-University Research Initiative. The emphasis is on helical flux compression generators comprising a hollow cylindrical metal liner filled with high explosives and at least one helical coil surrounding the liner. After the application of a seed current, magnetic flux is trapped and high current is generated by moving, i.e., expanding, the liner explosively along the winding of the helical coil. Several key factors involved in the temporal development can be addresses by optical diagnostics. 1) The uniformity of liner expansion is captured by framing camera photography and supplemented by laser illuminated high spatial and temporal resolution imaging. Also, X-ray flash photography is insensitive to possible image blur by shockwaves coming from the exploding liner. 2) The thermodynamic state of the shocked gas is assessed by spatially and temporally resolved emission spectroscopy. 3) The moving liner-coil contact point is a possible source of high electric losses and is preferentially monitored also by emission spectroscopy. Since optical access to the region between liner and coil is not always guaranteed, optical fibers can he used to extract light from the generator. The information so gained will give, together with detailed electrical diagnostics, more insight in the physical loss mechanisms involved in MFC

'Transcriptional differentiation of Trypanosoma brucei during in vitro acquisition of resistance to acoziborole

Subspecies of the protozoan parasite Trypanosoma brucei are the causative agents of Human African Trypanosomiasis (HAT), a debilitating neglected tropical disease prevalent across sub-Saharan Africa. HAT case numbers have steadily decreased since the start of the century, and sustainable elimination of one form of the disease is in sight. However, key to this is the development of novel drugs to combat the disease. Acoziborole is a recently developed benzoxaborole, currently in advanced clinical trials, for treatment of stage 1 and stage 2 HAT. Importantly, acoziborole is orally bioavailable, and curative with one dose. Recent studies have made significant progress in determining the molecular mode of action of acoziborole. However, less is known about the potential mechanisms leading to acoziborole resistance in trypanosomes. In this study, an in vitro-derived acoziborole-resistant cell line was generated and characterised. The Aco(R) line exhibited significant cross-resistance with the methyltransferase inhibitor sinefungin as well as hypersensitisation to known trypanocides. Interestingly, transcriptomics analysis of Aco(R) cells indicated the parasites had obtained a procyclic- or stumpy-like transcriptome profile, with upregulation of procyclin surface proteins as well as differential regulation of key metabolic genes known to be expressed in a life cycle-specific manner, even in the absence of major morphological changes. However, no changes were observed in transcripts encoding CPSF3, the recently identified protein target of acoziborole. The results suggest that generation of resistance to this novel compound in vitro can be accompanied by transcriptomic switches resembling a procyclic- or stumpy-type phenotype

Trypanosoma brucei ATR Links DNA Damage Signaling during Antigenic Variation with Regulation of RNA Polymerase I-Transcribed Surface Antigen

Trypanosoma brucei evades mammalian immunity by using recombination to switch its surface expressed Variant Surface Glycoprotein (VSG), whilst ensuring only one of many subtelomeric multigene VSG expression sites are transcribed at a time. DNA repair activities have been implicated only in catalysis of VSG switching by recombination, not transcriptional control. How VSG switching is signalled to guide the appropriate reaction, or to integrate switching into parasite growth, is unknown. Here we show that loss of ATR, a DNA damage signalling protein kinase, is lethal, causing nuclear genome instability and increased VSG switching through VSG-localised damage. Furthermore, ATR loss leads to increased transcription of silent VSG expression sites and expression of mixed VSGs on the cell surface, effects that are associated with altered localisation of RNA Polymerase I and VEX1. This work therefore reveals that ATR acts in antigenic variation both through DNA damage signalling and surface antigen expression control

Heritability and Tissue Specificity of Expression Quantitative Trait Loci

Variation in gene expression is heritable and has been mapped to the genome in humans and model organisms as expression quantitative trait loci (eQTLs). We applied integrated genome-wide expression profiling and linkage analysis to the regulation of gene expression in fat, kidney, adrenal, and heart tissues using the BXH/HXB panel of rat recombinant inbred strains. Here, we report the influence of heritability and allelic effect of the quantitative trait locus on detection of cis- and trans-acting eQTLs and discuss how these factors operate in a tissue-specific context. We identified several hundred major eQTLs in each tissue and found that cis-acting eQTLs are highly heritable and easier to detect than trans-eQTLs. The proportion of heritable expression traits was similar in all tissues; however, heritability alone was not a reliable predictor of whether an eQTL will be detected. We empirically show how the use of heritability as a filter reduces the ability to discover trans-eQTLs, particularly for eQTLs with small effects. Only 3% of cis- and trans-eQTLs exhibited large allelic effects, explaining more than 40% of the phenotypic variance, suggestive of a highly polygenic control of gene expression. Power calculations indicated that, across tissues, minor differences in genetic effects are expected to have a significant impact on detection of trans-eQTLs. Trans-eQTLs generally show smaller effects than cis-eQTLs and have a higher false discovery rate, particularly in more heterogeneous tissues, suggesting that small biological variability, likely relating to tissue composition, may influence detection of trans-eQTLs in this system. We delineate the effects of genetic architecture on variation in gene expression and show the sensitivity of this experimental design to tissue sampling variability in large-scale eQTL studies