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Chan Y.-S.,University of Cambridge | Takeuchi R.,Fred Hutchinson Cancer Research Center | Jarjour J.,Pregenen Inc. | Huen D.S.,University of Cambridge | And 4 more authors.
PLoS ONE | Year: 2013

The homing endonuclease gene (HEG) drive system, a promising genetic approach for controlling arthropod populations, utilises engineered nucleases to spread deleterious mutations that inactivate individual genes throughout a target population. Previous work with a naturally occurring LAGLIDADG homing endonuclease (I-SceI) demonstrated its feasibility in both Drosophila and Anopheles. Here we report on the next stage of this strategy: the redesign of HEGs with customized specificity in order to drive HEG-induced 'homing' in vivo via break-induced homologous recombination. Variants targeting a sequence within the Anopheles AGAP004734 gene were created from the recently characterized I-OnuI endonuclease, and tested for cleavage activity and frequency of homing using a model Drosophila HEG drive system. We observed cleavage and homing at an integrated reporter for all endonuclease variants tested, demonstrating for the first time that engineered HEGs can cleave their target site in insect germline cells, promoting targeted mutagenesis and homing. However, in comparison to our previously reported work with I-SceI, the engineered I-OnuI variants mediated homing with a reduced frequency, suggesting that site-specific cleavage activity is insufficient by itself to ensure efficient homing. Taken together, our experiments take a further step towards the development of a viable HEG-based population control strategy for insects. © 2013 Chan et al.


Chan Y.-S.,University of Cambridge | Huen D.S.,University of Cambridge | Huen D.S.,University of Wolverhampton | Glauert R.,University of Cambridge | And 4 more authors.
PLoS ONE | Year: 2013

Homing endonuclease gene (HEG) drive is a promising insect population control technique that employs meganucleases to impair the fitness of pest populations. Our previous studies showed that HEG drive was more difficult to achieve in Drosophila melanogaster than Anopheles gambiae and we therefore investigated ways of improving homing performance in Drosophila. We show that homing in Drosophila responds to increased expression of HEGs specifically during the spermatogonia stage and this could be achieved through improved construct design. We found that 3′-UTR choice was important to maximise expression levels, with HEG activity increasing as we employed Hsp70, SV40, vasa and βTub56D derived UTRs. We also searched for spermatogonium-specific promoters and found that the Rcd-1r promoter was able to drive specific expression at this stage. Since Rcd-1 is a regulator of differentiation in other species, it suggests that Rcd-1r may serve a similar role during spermatogonial differentiation in Drosophila. Contrary to expectations, a fragment containing the entire region between the TBPH gene and the bgcn translational start drove strong HEG expression only during late spermatogenesis rather than in the germline stem cells and spermatogonia as expected. We also observed that the fraction of targets undergoing homing was temperature-sensitive, falling nearly four-fold when the temperature was lowered to 18°C. Taken together, this study demonstrates how a few simple measures can lead to substantial improvements in the HEG-based gene drive strategy and reinforce the idea that the HEG approach may be widely applicable to a variety of insect control programs. © 2013 Chan et al.


Kolesnikov N.,European Bioinformatics Institute | Hastings E.,European Bioinformatics Institute | Keays M.,European Bioinformatics Institute | Melnichuk O.,European Bioinformatics Institute | And 15 more authors.
Nucleic Acids Research | Year: 2015

The ArrayExpress Archive of Functional Genomics Data (http://www.ebi.ac.uk/arrayexpress) is an international functional genomics database at the European Bioinformatics Institute (EMBL-EBI) recommended by most journals as a repository for data supporting peer-reviewed publications. It contains data from over 7000 public sequencing and 42 000 array-based studies comprising over 1.5 million assays in total. The proportion of sequencing-based submissions has grown significantly over the last few years and has doubled in the last 18 months, whilst the rate of microarray submissions is growing slightly. All data in ArrayExpress are available in the MAGE-TAB format, which allows robust linking to data analysis and visualization tools and standardized analysis. The main development over the last two years has been the release of a new data submission tool Annotare, which has reduced the average submission time almost 3-fold. In the near future, Annotare will become the only submission route into ArrayExpress, alongside MAGE-TAB format-based pipelines. ArrayExpress is a stable and highly accessed resource. Our future tasks include automation of data flows and further integration with other EMBL-EBI resources for the representation of multiomics data. © The Author(s) 2014.


Heather L.C.,University of Oxford | Wang X.,Cambridge Systems Biology Center | Wang X.,MRC Human Nutrition Research | West J.A.,Cambridge Systems Biology Center | And 3 more authors.
Journal of Molecular and Cellular Cardiology | Year: 2013

Metabolomics has refreshed interest in metabolism across biology and medicine, particularly in the areas of functional genomics and biomarker discovery. In this review we will discuss the experimental techniques and challenges involved in metabolomic profiling and how these technologies have been applied to cardiovascular disease. Open profiling and targeted approaches to metabolomics are compared, focusing on high resolution NMR spectroscopy and mass spectrometry, as well as discussing how to analyse the large amounts of data generated using multivariate statistics. Finally, the current literature on metabolomic profiling in human cardiovascular disease is reviewed to illustrate the diversity of approaches, and discuss some of the key metabolites and pathways found to be perturbed in plasma, urine and tissue from patients with these diseases. This includes studies of coronary artery disease, myocardial infarction, and ischemic heart disease. These studies demonstrate the potential of the technology for biomarker discovery and elucidating metabolic mechanisms associated with given pathologies, but also in some cases provide a warning of the pitfalls of poor study design. This article is part of a Special Issue entitled "Focus on Cardiac Metabolism". © 2012 Elsevier Ltd.


Boschetti C.,University of Cambridge | Carr A.,University of Cambridge | Carr A.,Cambridge Systems Biology Center | Crisp A.,University of Cambridge | And 8 more authors.
PLoS Genetics | Year: 2012

Bdelloid rotifers are microinvertebrates with unique characteristics: they have survived tens of millions of years without sexual reproduction; they withstand extreme desiccation by undergoing anhydrobiosis; and they tolerate very high levels of ionizing radiation. Recent evidence suggests that subtelomeric regions of the bdelloid genome contain sequences originating from other organisms by horizontal gene transfer (HGT), of which some are known to be transcribed. However, the extent to which foreign gene expression plays a role in bdelloid physiology is unknown. We address this in the first large scale analysis of the transcriptome of the bdelloid Adineta ricciae: cDNA libraries from hydrated and desiccated bdelloids were subjected to massively parallel sequencing and assembled transcripts compared against the UniProtKB database by blastx to identify their putative products. Of ~29,000 matched transcripts, ~10% were inferred from blastx matches to be horizontally acquired, mainly from eubacteria but also from fungi, protists, and algae. After allowing for possible sources of error, the rate of HGT is at least 8%-9%, a level significantly higher than other invertebrates. We verified their foreign nature by phylogenetic analysis and by demonstrating linkage of foreign genes with metazoan genes in the bdelloid genome. Approximately 80% of horizontally acquired genes expressed in bdelloids code for enzymes, and these represent 39% of enzymes in identified pathways. Many enzymes encoded by foreign genes enhance biochemistry in bdelloids compared to other metazoans, for example, by potentiating toxin degradation or generation of antioxidants and key metabolites. They also supplement, and occasionally potentially replace, existing metazoan functions. Bdelloid rotifers therefore express horizontally acquired genes on a scale unprecedented in animals, and foreign genes make a profound contribution to their metabolism. This represents a potential mechanism for ancient asexuals to adapt rapidly to changing environments and thereby persist over long evolutionary time periods in the absence of sex. © 2012 Boschetti et al.


Chan Y.-S.,University of Cambridge | Naujoks D.A.,University of Cambridge | Naujoks D.A.,Imperial College London | Huen D.S.,University of Cambridge | And 2 more authors.
Genetics | Year: 2011

Insects play a major role as vectors of human disease as well as causing significant agricultural losses. Harnessing the activity of customized homing endonuclease genes (HEGs) has been proposed as a method for spreading deleterious mutations through populations with a view to controlling disease vectors. Here, we demonstrate the feasibility of this method in Drosophila melanogaster, utilizing the well-characterized HEG, I-SceI. In particular, we show that high rates of homing can be achieved within spermatogonia and in the female germline. We show that homed constructs continue to exhibit HEG activity in the subsequent generation and that the ectopic homing events required for initiating the strategy occur at an acceptable rate. We conclude that the requirements for successful deployment of a HEG-based gene drive strategy can be satisfied in a model dipteran and that there is a reasonable prospect of the method working in other dipterans. In characterizing the system we measured repair outcomes at the spermatogonial, spermatocyte, and spermatid stages of spermatogenesis. We show that homologous recombination is restricted to sper-matogonia and that it immediately ceases when they become primary spermatocytes, indicating that the choice of DNA repair pathway in the Drosophila testis can switch abruptly during differentiation.


Baker D.A.,University of Cambridge | Nolan T.,Imperial College London | Fischer B.,Cambridge Systems Biology Center | Pinder A.,Imperial College London | And 3 more authors.
BMC Genomics | Year: 2011

Background: The mosquito, Anopheles gambiae, is the primary vector of human malaria, a disease responsible for millions of deaths each year. To improve strategies for controlling transmission of the causative parasite, Plasmodium falciparum, we require a thorough understanding of the developmental mechanisms, physiological processes and evolutionary pressures affecting life-history traits in the mosquito. Identifying genes expressed in particular tissues or involved in specific biological processes is an essential part of this process.Results: In this study, we present transcription profiles for ~82% of annotated Anopheles genes in dissected adult male and female tissues. The sensitivity afforded by examining dissected tissues found gene activity in an additional 20% of the genome that is undetected when using whole-animal samples. The somatic and reproductive tissues we examined each displayed patterns of sexually dimorphic and tissue-specific expression. By comparing expression profiles with Drosophila melanogaster we also assessed which genes are well conserved within the Diptera versus those that are more recently evolved.Conclusions: Our expression atlas and associated publicly available database, the MozAtlas (http://www.tissue-atlas.org), provides information on the relative strength and specificity of gene expression in several somatic and reproductive tissues, isolated from a single strain grown under uniform conditions. The data will serve as a reference for other mosquito researchers by providing a simple method for identifying where genes are expressed in the adult, however, in addition our resource will also provide insights into the evolutionary diversity associated with gene expression levels among species. © 2011 Baker et al; licensee BioMed Central Ltd.


Kruse K.,University of Cambridge | Sewitz S.,University of Cambridge | Sewitz S.,Cambridge Systems Biology Center | Madan Babu M.,University of Cambridge
Nucleic Acids Research | Year: 2013

Experimental techniques for the investigation of three-dimensional (3D) genome organization are being developed at a fast pace. Currently, the associated computational methods are mostly specific to the individual experimental approach. Here we present a general statistical framework that is widely applicable to the analysis of genomic contact maps, irrespective of the data acquisition and normalization processes. Within this framework DNA-DNA contact data are represented as a complex network, for which a broad number of directly applicable methods already exist. In such a network representation, DNA segments and contacts between them are denoted as nodes and edges, respectively. Furthermore, we present a robust method for generating randomized contact networks that explicitly take into account the inherent 3D nature of the genome and serve as realistic null-models for unbiased statistical analyses. By integrating a variety of large-scale genome-wide datasets we demonstrate that meiotic crossover sites display enriched genomic contacts and that cohesin-bound genes are significantly colocalized in the yeast nucleus. We anticipate that the complex network framework in conjunction with the randomization of DNA-DNA contact networks will become a widely used tool in the study of nuclear architecture. © 2012 The Author(s).


Huen D.S.,University of Cambridge | Russell S.,University of Cambridge | Russell S.,Cambridge Systems Biology Center
BMC Bioinformatics | Year: 2010

Background: In eukaryotes, most DNA-binding proteins exert their action as members of large effector complexes. The presence of these complexes are revealed in high-throughput genome-wide assays by the co-occurrence of the binding sites of different complex components. Resampling tests are one route by which the statistical significance of apparent co-occurrence can be assessed.Results: We have investigated two resampling approaches for evaluating the statistical significance of binding-site co-occurrence. The permutation test approach was found to yield overly favourable p-values while the independent resampling approach had the opposite effect and is of little use in practical terms. We have developed a new, pragmatically-devised hybrid approach that, when applied to the experimental results of an Polycomb/Trithorax study, yielded p-values consistent with the findings of that study. We extended our investigations to the FL method developed by Haiminen et al, which derives its null distribution from all binding sites within a dataset, and show that the p-value computed for a pair of factors by this method can depend on which other factors are included in that dataset. Both our hybrid method and the FL method appeared to yield plausible estimates of the statistical significance of co-occurrences although our hybrid method was more conservative when applied to the Polycomb/Trithorax dataset.A high-performance parallelized implementation of the hybrid method is available.Conclusions: We propose a new resampling-based co-occurrence significance test and demonstrate that it performs as well as or better than existing methods on a large experimentally-derived dataset. We believe it can be usefully applied to data from high-throughput genome-wide techniques such as ChIP-chip or DamID. The Cooccur package, which implements our approach, accompanies this paper. © 2010 Huen and Russell; licensee BioMed Central Ltd.


Baker D.A.,University of Cambridge | Russell S.,University of Cambridge | Russell S.,Cambridge Systems Biology Center
Genetics | Year: 2011

Gene expression in Anopheles gambiae shows a deficiency of testis-expressed genes on the X chromosome associated with an excessive movement of retrogene duplication. We suggest that the degeneration of sex chromosomes in this monandrous species is likely the result of pressures from X inactivation, dosage compensation, and sexual antagonism. © 2011 by the Genetics Society of America.

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