91 results on '"Gilchrist, MJ"'
Search Results
2. Towards pharmacokinetic boosting of phenoxymethylpenicillin (penicillin-V) using probenecid for the treatment of bacterial infections.
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Wilson RC, Riezk A, Arkell P, Ming D, Armiger R, Latham V, Gilchrist MJ, Märtson AG, Hope WW, Holmes AH, and Rawson TM
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- Humans, Male, Adult, Female, Streptococcus pneumoniae drug effects, Young Adult, Microbial Sensitivity Tests, Middle Aged, Healthy Volunteers, Bacterial Infections drug therapy, Bacterial Infections microbiology, Probenecid pharmacokinetics, Probenecid pharmacology, Probenecid administration & dosage, Anti-Bacterial Agents pharmacokinetics, Anti-Bacterial Agents administration & dosage, Cross-Over Studies, Penicillin V pharmacokinetics, Penicillin V administration & dosage
- Abstract
In the face of increasing antimicrobial tolerance and resistance there is a global obligation to optimise oral antimicrobial dosing strategies including narrow spectrum penicillins, such as penicillin-V. We conducted a randomised, crossover study in healthy volunteers to characterise the influence of probenecid on penicillin-V pharmacokinetics and estimate the pharmacodynamics against Streptococcus pneumoniae. Twenty participants took six doses of penicillin-V (250 mg, 500 mg or 750 mg four times daily) with and without probenecid. Total and free concentrations of penicillin-V and probenecid were measured at two timepoints. A pharmacokinetic model was developed, and the probability of target attainment (PTA) calculated. The mean difference (95% CI) between penicillin-V alone and in combination with probenecid for serum total and free penicillin-V concentrations was significantly different at both timepoints (total: 45 min 4.32 (3.20-5.32) mg/L p < 0.001, 180 min 2.2 (1.58-3.25) mg/L p < 0.001; free: 45 min 1.15 (0.88-1.42) mg/L p < 0.001, 180 min 0.5 (0.35-0.76) mg/L p < 0.001). There was no difference between the timepoints in probenecid concentrations. PTA analysis shows probenecid allows a fourfold increase in MIC cover. Addition of probenecid was safe and well tolerated. The data support further research into improved dosing structures for complex outpatient therapy and might also be used to address penicillin supply shortages., (© 2024. The Author(s).)
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- 2024
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3. Working collaboratively across schools to promote oral health education through interprofessional education.
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Padilla R, Kowlowitz V, Quinonez RB, Ciarrocca K, Gilchrist MJ, Gilliland KO, Koonce TF, Lampiris L, and Beck Dallaghan GL
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- Curriculum, Humans, Oral Health, Schools, Medical, Health Education, Dental, Students, Medical
- Abstract
Purpose: The Association of American Medical Colleges and American Dental Education Association have identified oral health knowledge, skills, and attitudes shared by both medical and dental professionals. Although oral health was deemed an essential competency for medical practitioners, our state struggled to ensure learners received proper training. This training deficit resulted in conducting a needs assessment and implementing an oral health interprofessional module at our schools., Methods: First-year medical students and clinical faculty were emailed surveys in 2016 to obtain baseline information. A team of faculty and students from the Schools of Medicine and Dentistry reviewed the curriculum to determine where to augment oral health content. An oral health module to teach a basic head, neck, and oral examination to first-year medical students during their patient-centered care small-group sessions was implemented and evaluated., Results: Only 13.6% of faculty respondents were aware of national oral health competency recommendations, and <50% rated oral health important for primary care physicians (PCPs) to include in history, physical exam, or oral health counseling. On baseline, ≤25% of PCP respondents reported integrating the listed skills in their practice, and most indicated lacking expertise to teach oral health. Teaching sessions were rated helpful by students and faculty. After the teaching sessions, ratings on the importance of including oral health significantly increased from baseline., Conclusion: Collaboration between Schools of Dentistry and Medicine successfully integrated oral health into medical school curriculum and improved the tutors' attitudes of its importance., (© 2020 American Dental Education Association.)
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- 2020
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4. Genomics Methods for Xenopus Embryos and Tissues.
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Gilchrist MJ, Cho KWY, and Veenstra GJC
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- Animals, Chromatin metabolism, Chromatin Immunoprecipitation methods, DNA genetics, DNA metabolism, Embryo, Nonmammalian embryology, Genome genetics, Xenopus embryology, Chromatin genetics, Embryo, Nonmammalian metabolism, Genomics methods, High-Throughput Nucleotide Sequencing methods, Xenopus genetics
- Abstract
High-throughput sequencing methods have created exciting opportunities to explore the regulatory landscape of the entire genome. Here we introduce methods to characterize the genomic locations of bound proteins, open chromatin, and sites of DNA-DNA contact in Xenopus embryos. These methods include chromatin immunoprecipitation followed by sequencing (ChIP-seq), a combination of DNase I digestion and sequencing (DNase-seq), the assay for transposase-accessible chromatin and sequencing (ATAC-seq), and the use of proximity-based DNA ligation followed by sequencing (Hi-C)., (© 2020 Cold Spring Harbor Laboratory Press.)
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- 2020
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5. Transcriptomics and Proteomics Methods for Xenopus Embryos and Tissues.
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Gilchrist MJ, Veenstra GJC, and Cho KWY
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- Animals, Chromatography, Liquid methods, Embryo, Nonmammalian embryology, Proteome genetics, Proteome metabolism, Sequence Analysis, RNA methods, Tandem Mass Spectrometry methods, Xenopus Proteins genetics, Xenopus Proteins metabolism, Xenopus laevis embryology, Embryo, Nonmammalian metabolism, Gene Expression Profiling methods, Gene Expression Regulation, Developmental, Proteomics methods, Xenopus laevis genetics, Xenopus laevis metabolism
- Abstract
The general field of quantitative biology has advanced significantly on the back of recent improvements in both sequencing technology and proteomics methods. The development of high-throughput, short-read sequencing has revolutionized RNA-based expression studies, while improvements in proteomics methods have enabled quantitative studies to attain better resolution. Here we introduce methods to undertake global analyses of gene expression through RNA and protein quantification in Xenopus embryos and tissues., (© 2020 Cold Spring Harbor Laboratory Press.)
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- 2020
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6. An RNA-Seq Protocol for Differential Expression Analysis.
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Owens NDL, De Domenico E, and Gilchrist MJ
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- Animals, Gene Expression Regulation, Developmental, Gene Library, Specimen Handling, Xenopus embryology, Gene Expression Profiling methods, RNA-Seq methods
- Abstract
Here we consider RNA-Seq, used to measure global gene expression through RNA fragmentation, capture, sequencing, and subsequent computational analysis. Xenopus , with its large number of RNA-rich, synchronously developing, and accessible embryos, is an excellent model organism for exploiting the power of high-throughput sequencing to understand gene expression during development. Here we present a standard RNA-Seq protocol for performing two-state differential gene expression analysis (between groups of replicates of control and treated embryos) using Illumina sequencing. Samples contain multiple whole embryos, and polyadenylated mRNA is measured under relative normalization. The protocol is divided into two parts: wet-lab processes to prepare samples for sequencing and downstream computational analysis including quality control, quantification of gene expression, and differential expression., (© 2019 Cold Spring Harbor Laboratory Press.)
- Published
- 2019
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7. Planning to halve Gram-negative bloodstream infection: getting to grips with healthcare-associated Escherichia coli bloodstream infection sources.
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Otter JA, Galletly TJ, Davies F, Hitchcock J, Gilchrist MJ, Dyakova E, Mookerjee S, Holmes AH, and Brannigan ET
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- Adolescent, Adult, Aged, Aged, 80 and over, Child, Child, Preschool, Drug Resistance, Bacterial, Escherichia coli drug effects, Escherichia coli isolation & purification, Female, Hospitals, Humans, Infant, Infant, Newborn, Male, Middle Aged, Risk Factors, Young Adult, Bacteremia epidemiology, Bacteremia prevention & control, Cross Infection epidemiology, Cross Infection prevention & control, Escherichia coli Infections epidemiology, Escherichia coli Infections prevention & control, Infection Control methods
- Abstract
Background: A thorough understanding of the local sources, risks, and antibiotic resistance for Escherichia coli bloodstream infection (BSI) is required to focus prevention initiatives and therapy., Aim: To review the sources and antibiotic resistance of healthcare-associated E. coli BSI., Methods: Sources and antibiotic resistance profiles of all 250 healthcare-associated (post 48 h) E. coli BSIs that occurred within our secondary and tertiary care hospital group from April 2014 to March 2017 were reviewed. Epidemiological associations with urinary source, gastrointestinal source, and febrile neutropenia-related BSIs were analysed using univariable and multivariable binary logistic regression models., Findings: E. coli BSIs increased 9% from 4.0 to 4.4 per 10,000 admissions comparing the 2014/15 and 2016/17 financial years. Eighty-nine cases (36%) had a urinary source; 30 (34%) of these were classified as urinary catheter-associated urinary tract infections (UTIs). Forty-five (18%) were related to febrile neutropenia, and 38 (15%) had a gastrointestinal source. Cases were rarely associated with surgical procedures (11, 4%) or indwelling vascular devices (seven, 3%). Female gender (odds ratio: 2.3; 95% confidence interval: 1.2-4.6) and older age (1.02; 1.00-1.05) were significantly associated with a urinary source. No significant associations were identified for gastrointestinal source or febrile neutropenia-related BSIs. Forty-seven percent of the isolates were resistant to ciprofloxacin, 37% to third-generation cephalosporins, and 22% to gentamicin., Conclusion: The gastrointestinal tract and febrile neutropenia together accounted for one-third of E. coli BSI locally but were rare associations nationally. These sources need to be targeted locally to reduce an increasing trend of E. coli BSIs., (Copyright © 2018 The Healthcare Infection Society. Published by Elsevier Ltd. All rights reserved.)
- Published
- 2019
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8. New methods for computational decomposition of whole-mount in situ images enable effective curation of a large, highly redundant collection of Xenopus images.
- Author
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Patrushev I, James-Zorn C, Ciau-Uitz A, Patient R, and Gilchrist MJ
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- Animals, Embryo, Nonmammalian metabolism, Gene Expression, Gene Expression Profiling methods, Gene Expression Regulation, Developmental, In Situ Hybridization methods, In Situ Hybridization, Fluorescence methods, Software, Transcriptome, Xenopus laevis embryology, Computational Biology methods, Data Curation methods, Image Processing, Computer-Assisted methods
- Abstract
The precise anatomical location of gene expression is an essential component of the study of gene function. For most model organisms this task is usually undertaken via visual inspection of gene expression images by interested researchers. Computational analysis of gene expression has been developed in several model organisms, notably in Drosophila which exhibits a uniform shape and outline in the early stages of development. Here we address the challenge of computational analysis of gene expression in Xenopus, where the range of developmental stages of interest encompasses a wide range of embryo size and shape. Embryos may have different orientation across images, and, in addition, embryos have a pigmented epidermis that can mask or confuse underlying gene expression. Here we report the development of a set of computational tools capable of processing large image sets with variable characteristics. These tools efficiently separate the Xenopus embryo from the background, separately identify both histochemically stained and naturally pigmented regions within the embryo, and can sort images from the same gene and developmental stage according to similarity of gene expression patterns without information about relative orientation. We tested these methods on a large, but highly redundant, collection of 33,289 in situ hybridization images, allowing us to select representative images of expression patterns at different embryo orientations. This has allowed us to put a much smaller subset of these images into the public domain in an effective manner. The 'isimage' module and the scripts developed are implemented in Python and freely available on https://pypi.python.org/pypi/isimage/., Competing Interests: The authors have declared that no competing interests exist.
- Published
- 2018
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9. Developmentally regulated long non-coding RNAs in Xenopus tropicalis.
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Forouzmand E, Owens NDL, Blitz IL, Paraiso KD, Khokha MK, Gilchrist MJ, Xie X, and Cho KWY
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- Animals, Embryo, Nonmammalian metabolism, Exons genetics, Gene Expression Profiling, Gene Expression Regulation, Developmental, Models, Genetic, RNA, Long Noncoding biosynthesis, RNA, Long Noncoding isolation & purification, Transcriptome, Xenopus embryology, RNA, Long Noncoding genetics, Xenopus genetics
- Abstract
Advances in RNA sequencing technologies have led to the surprising discovery that a vast number of transcripts emanate from regions of the genome that are not part of coding genes. Although some of the smaller ncRNAs such as microRNAs have well-characterized functions, the majority of long ncRNA (lncRNA) functions remain poorly understood. Understanding the significance of lncRNAs is an important challenge facing biology today. A powerful approach to uncovering the function of lncRNAs is to explore temporal and spatial expression profiling. This may be particularly useful for classes of lncRNAs that have developmentally important roles as the expression of such lncRNAs will be expected to be both spatially and temporally regulated during development. Here, we take advantage of our ultra-high frequency (temporal) sampling of Xenopus embryos to analyze gene expression trajectories of lncRNA transcripts over the first 3 days of development. We computationally identify 5689 potential single- and multi-exon lncRNAs. These lncRNAs demonstrate clear dynamic expression patterns. A subset of them displays highly correlative temporal expression profiles with respect to those of the neighboring genes. We also identified spatially localized lncRNAs in the gastrula stage embryo. These results suggest that lncRNAs have regulatory roles during early embryonic development., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)
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- 2017
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10. Editorial: The Xenopus laevis genome.
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Harland RM and Gilchrist MJ
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- Animals, Cell Cycle, Transcriptome, Developmental Biology, Genome, Xenopus Proteins genetics, Xenopus laevis genetics, Xenopus laevis physiology
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- 2017
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11. A catalog of Xenopus tropicalis transcription factors and their regional expression in the early gastrula stage embryo.
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Blitz IL, Paraiso KD, Patrushev I, Chiu WTY, Cho KWY, and Gilchrist MJ
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- Animals, Base Sequence, Embryo, Nonmammalian metabolism, Humans, Mice, Species Specificity, Transcription Factors genetics, Xenopus embryology, Xenopus genetics, Xenopus Proteins genetics, Gastrula metabolism, Gene Expression Regulation, Developmental, Transcription Factors biosynthesis, Xenopus metabolism, Xenopus Proteins biosynthesis
- Abstract
Gene regulatory networks (GRNs) involve highly combinatorial interactions between transcription factors and short sequence motifs in cis-regulatory modules of target genes to control cellular phenotypes. The GRNs specifying most cell types are largely unknown and are the subject of wide interest. A catalog of transcription factors is a valuable tool toward obtaining a deeper understanding of the role of these critical effectors in any biological setting. Here we present a comprehensive catalog of the transcription factors for the diploid frog Xenopus tropicalis. We identify 1235 genes encoding DNA-binding transcription factors, comparable to the numbers found in typical mammalian species. In detail, the repertoire of X. tropicalis transcription factor genes is nearly identical to human and mouse, with the exception of zinc finger family members, and a small number of species/lineage-specific gene duplications and losses relative to the mammalian repertoires. We applied this resource to the identification of transcription factors differentially expressed in the early gastrula stage embryo. We find transcription factor enrichment in Spemann's organizer, the ventral mesoderm, ectoderm and endoderm, and report 218 TFs that show regionalized expression patterns at this stage. Many of these have not been previously reported as expressed in the early embryo, suggesting thus far unappreciated roles for many transcription factors in the GRNs regulating early development. We expect our transcription factor catalog will facilitate myriad studies using Xenopus as a model system to understand basic biology and human disease., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2017
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12. Antimicrobial stewardship: are we failing in cross-specialty clinical engagement?
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Rawson TM, Moore LS, Gilchrist MJ, and Holmes AH
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- Cross-Sectional Studies, Humans, United Kingdom, Anti-Bacterial Agents therapeutic use, Drug Resistance, Bacterial, Drug Utilization standards
- Abstract
Background: Antimicrobial resistance (AMR) is a public health priority and leading patient safety issue. Globally, antimicrobial stewardship (AMS) has been integral in promoting therapeutic optimization whilst minimizing harmful antimicrobial use. A cross-sectional, observational study was undertaken to investigate the coverage of AMS and antibacterial resistance across clinical scientific conferences in 2014, as a surrogate marker for current awareness and attributed importance., Methods: Clinical specialties were identified, and the largest corresponding clinical scientific/research conferences in 2014 determined (i) within the UK and (ii) internationally. Conference characteristics and abstracts were interrogated and analysed to determine those related to AMS and AMR. Inter-specialty variation was assessed using χ(2) or Fisher's exact statistical analysis., Results: In total, 45 conferences from 23 specialties were analysed representing 59,682 accepted abstracts. The UK had a significantly greater proportion of AMS-AMR-related abstracts compared with international conferences [2.8% (n = 221/7843) compared with 1.8% (n = 942/51,839); P < 0.001]. Infection conferences contained the greatest proportion of AMS-AMR abstracts, representing 20% (732/3669) of all abstracts [UK 66% (80/121) and international 18% (652/3548); P < 0.0001]. AMS-AMR coverage across all general specialties was poor [intensive care 9% (116/1287), surgical 1% (8/757) and medical specialties 0.64% (332/51,497)] despite high usage of antimicrobials across all., Conclusions: Despite current AMS-AMR strategies being advocated by infection specialists and discussed by national and international policy makers, AMS-AMR coverage remained limited across clinical specialty scientific conferences in 2014. We call for further intervention to ensure specialty engagement with AMS programmes and promote the AMR agenda across clinical practice., (© The Author 2015. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)
- Published
- 2016
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13. Measuring Absolute RNA Copy Numbers at High Temporal Resolution Reveals Transcriptome Kinetics in Development.
- Author
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Owens NDL, Blitz IL, Lane MA, Patrushev I, Overton JD, Gilchrist MJ, Cho KWY, and Khokha MK
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- Animals, Bayes Theorem, Embryo, Nonmammalian metabolism, Expressed Sequence Tags, Gene Dosage, Kinetics, MicroRNAs metabolism, Xenopus growth & development, Xenopus metabolism, RNA metabolism, Transcriptome
- Abstract
Transcript regulation is essential for cell function, and misregulation can lead to disease. Despite technologies to survey the transcriptome, we lack a comprehensive understanding of transcript kinetics, which limits quantitative biology. This is an acute challenge in embryonic development, where rapid changes in gene expression dictate cell fate decisions. By ultra-high-frequency sampling of Xenopus embryos and absolute normalization of sequence reads, we present smooth gene expression trajectories in absolute transcript numbers. During a developmental period approximating the first 8 weeks of human gestation, transcript kinetics vary by eight orders of magnitude. Ordering genes by expression dynamics, we find that "temporal synexpression" predicts common gene function. Remarkably, a single parameter, the characteristic timescale, can classify transcript kinetics globally and distinguish genes regulating development from those involved in cellular metabolism. Overall, our analysis provides unprecedented insight into the reorganization of maternal and embryonic transcripts and redefines our ability to perform quantitative biology., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)
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- 2016
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14. The Xenopus ORFeome: A resource that enables functional genomics.
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Grant IM, Balcha D, Hao T, Shen Y, Trivedi P, Patrushev I, Fortriede JD, Karpinka JB, Liu L, Zorn AM, Stukenberg PT, Hill DE, and Gilchrist MJ
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- Animals, Base Sequence, Cloning, Molecular, DNA, Complementary genetics, Databases, Genetic, Disease genetics, Genomics, Humans, Models, Genetic, Molecular Sequence Data, Sequence Homology, Nucleic Acid, Species Specificity, Xenopus Proteins genetics, Xenopus laevis genetics, Open Reading Frames, Xenopus genetics
- Abstract
Functional characterisation of proteins and large-scale, systems-level studies are enabled by extensive sets of cloned open reading frames (ORFs) in an easily-accessible format that enables many different applications. Here we report the release of the first stage of the Xenopus ORFeome, which contains 8673 ORFs from the Xenopus Gene Collection (XGC) for Xenopus laevis, cloned into a Gateway® donor vector enabling rapid in-frame transfer of the ORFs to expression vectors. This resource represents an estimated 7871 unique genes, approximately 40% of the non-redundant X. laevis gene complement, and includes 2724 genes where the human ortholog has an association with disease. Transfer into the Gateway system was validated by 5' and 3' end sequencing of the entire collection and protein expression of a set of test clones. In a parallel process, the underlying ORF predictions from the original XGC collection were re-analysed to verify quality and full-length status, identifying those proteins likely to exhibit truncations when translated. These data are integrated into Xenbase, the Xenopus community database, which associates genomic, expression, function and human disease model metadata to each ORF, enabling end-users to search for ORFeome clones with links to commercial distributors of the collection. When coupled with the experimental advantages of Xenopus eggs and embryos, the ORFeome collection represents a valuable resource for functional genomics and disease modelling., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2015
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15. Molecular asymmetry in the 8-cell stage Xenopus tropicalis embryo described by single blastomere transcript sequencing.
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De Domenico E, Owens ND, Grant IM, Gomes-Faria R, and Gilchrist MJ
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- Animals, Body Patterning genetics, Female, Gene Knockdown Techniques, Humans, Male, Models, Animal, Monosaccharide Transport Proteins antagonists & inhibitors, Monosaccharide Transport Proteins genetics, Mutation, RNA, Messenger genetics, RNA, Messenger metabolism, Transcription, Genetic, Xenopus metabolism, Xenopus Proteins antagonists & inhibitors, Xenopus Proteins genetics, Blastomeres metabolism, Xenopus embryology, Xenopus genetics
- Abstract
Correct development of the vertebrate body plan requires the early definition of two asymmetric, perpendicular axes. The first axis is established during oocyte maturation, and the second is established by symmetry breaking shortly after fertilization. The physical processes generating the second asymmetric, or dorsal-ventral, axis are well understood, but the specific molecular determinants, presumed to be maternal gene products, are poorly characterized. Whilst enrichment of maternal mRNAs at the animal and vegetal poles in both the oocyte and the early embryo has been studied, little is known about the distribution of maternal mRNAs along either the dorsal-ventral or left-right axes during the early cleavage stages. Here we report an unbiased analysis of the distribution of maternal mRNA on all axes of the Xenopus tropicalis 8-cell stage embryo, based on sequencing of single blastomeres whose positions within the embryo are known. Analysis of pooled data from complete sets of blastomeres from four embryos has identified 908 mRNAs enriched in either the animal or vegetal blastomeres, of which 793 are not previously reported as enriched. In contrast, we find no evidence for asymmetric distribution along either the dorsal-ventral or left-right axes. We confirm that animal pole enrichment is on average distinctly lower than vegetal pole enrichment, and that considerable variation is found between reported enrichment levels in different studies. We use publicly available data to show that there is a significant association between genes with human disease annotation and enrichment at the animal pole. Mutations in the human ortholog of the most animally enriched novel gene, Slc35d1, are causative for Schneckenbecken dysplasia, and we show that a similar phenotype is produced by depletion of the orthologous protein in Xenopus embryos., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2015
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16. A Database of microRNA Expression Patterns in Xenopus laevis.
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Ahmed A, Ward NJ, Moxon S, Lopez-Gomollon S, Viaut C, Tomlinson ML, Patrushev I, Gilchrist MJ, Dalmay T, Dotlic D, Münsterberg AE, and Wheeler GN
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- Animals, Embryo, Nonmammalian, Gene Expression Regulation, Developmental, MicroRNAs classification, MicroRNAs genetics, RNA, Messenger genetics, Sequence Analysis, RNA, Xenopus laevis growth & development, Embryonic Development genetics, MicroRNAs biosynthesis, RNA, Messenger biosynthesis, Xenopus laevis genetics
- Abstract
MicroRNAs (miRNAs) are short, non-coding RNAs around 22 nucleotides long. They inhibit gene expression either by translational repression or by causing the degradation of the mRNAs they bind to. Many are highly conserved amongst diverse organisms and have restricted spatio-temporal expression patterns during embryonic development where they are thought to be involved in generating accuracy of developmental timing and in supporting cell fate decisions and tissue identity. We determined the expression patterns of 180 miRNAs in Xenopus laevis embryos using LNA oligonucleotides. In addition we carried out small RNA-seq on different stages of early Xenopus development, identified 44 miRNAs belonging to 29 new families and characterized the expression of 5 of these. Our analyses identified miRNA expression in many organs of the developing embryo. In particular a large number were expressed in neural tissue and in the somites. Surprisingly none of the miRNAs we have looked at show expression in the heart. Our results have been made freely available as a resource in both XenMARK and Xenbase.
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- 2015
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17. A pipeline for the systematic identification of non-redundant full-ORF cDNAs for polymorphic and evolutionary divergent genomes: Application to the ascidian Ciona intestinalis.
- Author
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Gilchrist MJ, Sobral D, Khoueiry P, Daian F, Laporte B, Patrushev I, Matsumoto J, Dewar K, Hastings KE, Satou Y, Lemaire P, and Rothbächer U
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- Algorithms, Animals, Base Sequence, Biological Evolution, Evolution, Molecular, Gene Expression Profiling, Humans, Multigene Family genetics, Open Reading Frames genetics, Sequence Alignment, Sequence Analysis, DNA, Ciona intestinalis genetics, Gene Regulatory Networks genetics, Genetic Predisposition to Disease
- Abstract
Genome-wide resources, such as collections of cDNA clones encoding for complete proteins (full-ORF clones), are crucial tools for studying the evolution of gene function and genetic interactions. Non-model organisms, in particular marine organisms, provide a rich source of functional diversity. Marine organism genomes are, however, frequently highly polymorphic and encode proteins that diverge significantly from those of well-annotated model genomes. The construction of full-ORF clone collections from non-model organisms is hindered by the difficulty of predicting accurately the N-terminal ends of proteins, and distinguishing recent paralogs from highly polymorphic alleles. We report a computational strategy that overcomes these difficulties, and allows for accurate gene level clustering of transcript data followed by the automated identification of full-ORFs with correct 5'- and 3'-ends. It is robust to polymorphism, includes paralog calling and does not require evolutionary proximity to well annotated model organisms. We developed this pipeline for the ascidian Ciona intestinalis, a highly polymorphic member of the divergent sister group of the vertebrates, emerging as a powerful model organism to study chordate gene function, Gene Regulatory Networks and molecular mechanisms underlying human pathologies. Using this pipeline we have generated the first full-ORF collection for a highly polymorphic marine invertebrate. It contains 19,163 full-ORF cDNA clones covering 60% of Ciona coding genes, and full-ORF orthologs for approximately half of curated human disease-associated genes., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2015
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18. A New Nomenclature of Xenopus laevis Chromosomes Based on the Phylogenetic Relationship to Silurana/Xenopus tropicalis.
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Matsuda Y, Uno Y, Kondo M, Gilchrist MJ, Zorn AM, Rokhsar DS, Schmid M, and Taira M
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- Animals, Chromosome Banding, Chromosomes genetics, Chromosomes ultrastructure, Female, Hybridization, Genetic genetics, In Situ Hybridization, Fluorescence, Metaphase, Phylogeny, Species Specificity, Tetraploidy, Xenopus classification, Xenopus laevis classification, Chromosomes classification, Terminology as Topic, Xenopus laevis genetics
- Abstract
Xenopus laevis (XLA) is an allotetraploid species which appears to have undergone whole-genome duplication after the interspecific hybridization of 2 diploid species closely related to Silurana/Xenopus tropicalis (XTR). Previous cDNA fluorescence in situ hybridization (FISH) experiments have identified 9 sets of homoeologous chromosomes in X. laevis, in which 8 sets correspond to chromosomes 1-8 of X. tropicalis (XTR1-XTR8), and the last set corresponds to a fusion of XTR9 and XTR10. In addition, recent X. laevis genome sequencing and BAC-FISH experiments support this physiological relationship and show no gross chromosome translocation in the X. laevis karyotype. Therefore, for the benefit of both comparative cytogenetics and genome research, we here propose a new chromosome nomenclature for X. laevis based on the phylogenetic relationship and chromosome length, i.e. XLA1L, XLA1S, XLA2L, XLA2S, and so on, in which the numbering of XLA chromosomes corresponds to that in X. tropicalis and the postfixes 'L' and 'S' stand for 'long' and 'short' chromosomes in the homoeologous pairs, which can be distinguished cytologically by their relative size. The last chromosome set is named XLA9L and XLA9S, in which XLA9 corresponds to both XTR9 and XTR10, and hence, to emphasize the phylogenetic relationship to X. tropicalis, XLA9_10L and XLA9_10S are also used as synonyms.
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- 2015
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19. Homogeneity of antimicrobial policy, yet heterogeneity of antimicrobial resistance: antimicrobial non-susceptibility among 108,717 clinical isolates from primary, secondary and tertiary care patients in London.
- Author
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Moore LS, Freeman R, Gilchrist MJ, Gharbi M, Brannigan ET, Donaldson H, Livermore DM, and Holmes AH
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- Adolescent, Adult, Aged, Aged, 80 and over, Bacterial Infections epidemiology, Cohort Studies, Drug Therapy standards, Female, Health Policy, Hospitals, Teaching, Humans, London epidemiology, Male, Middle Aged, Primary Health Care methods, Secondary Care methods, Tertiary Healthcare methods, Young Adult, Anti-Bacterial Agents therapeutic use, Bacteria drug effects, Bacterial Infections microbiology, Drug Resistance, Bacterial
- Abstract
Objectives: We examined the 4 year trend in antimicrobial susceptibilities and prescribing across levels of care at two London teaching hospitals and their multisite renal unit, and for the surrounding community., Methods: Laboratory and pharmacy information management systems were interrogated, with antimicrobial use and susceptibilities analysed between hospitals, within hospitals and over time., Results: A total of 108,717 isolates from 71,687 patients were identified, with significant differences (at P < 0.05) in antimicrobial susceptibility between and within hospitals. Across the 4 years, rates of ESBL-/AmpC-producing Enterobacteriaceae ranged from 6.4% to 10.7% among community isolates, 17.8% to 26.9% at ward level and 25.2% to 52.5% in critical care. Significant variations were also demonstrated in glycopeptide-resistant enterococci (ward level 6.2%-17.4%; critical care 21.9%-56.3%), MRSA (ward level 18.5%-38.2%; critical care 12.5%-47.9%) and carbapenem-resistant Pseudomonas spp. (ward level 8.3%-16.9%; critical care 19.9%-53.7%). Few instances of persistently higher resistance were seen between the hospitals in equivalent cohorts, despite persistently higher antimicrobial use in Hospital 1 than Hospital 2. We found significant fluctuations in non-susceptibility year on year across the cohorts, but with few persistent trends., Conclusions: The marked heterogeneity of antimicrobial susceptibilities between hospitals, within hospitals and over time demands detailed, standardized surveillance and appropriate benchmarking to identify possible drivers and effective interventions. Homogeneous antimicrobial policies are unlikely to continue to be suitable as individual hospitals join hospital networks, and policies should be tailored to local resistance rates, at least at the hospital level, and possibly with finer resolution, particularly for critical care., (© The Author 2014. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy.)
- Published
- 2014
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20. Activation of the aryl hydrocarbon receptor dampens the severity of inflammatory skin conditions.
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Di Meglio P, Duarte JH, Ahlfors H, Owens ND, Li Y, Villanova F, Tosi I, Hirota K, Nestle FO, Mrowietz U, Gilchrist MJ, and Stockinger B
- Subjects
- Adjuvants, Immunologic pharmacology, Aminoquinolines pharmacology, Animals, Aryl Hydrocarbon Hydroxylases biosynthesis, Azo Compounds pharmacology, Basic Helix-Loop-Helix Transcription Factors agonists, Basic Helix-Loop-Helix Transcription Factors genetics, Carbazoles pharmacology, Cytochrome P-450 CYP1A1 biosynthesis, Cytochrome P-450 CYP1B1, Cytokines pharmacology, Environmental Exposure, Humans, Imiquimod, Keratinocytes immunology, Mice, Mice, Knockout, Psoriasis pathology, Pyrazoles pharmacology, Receptors, Aryl Hydrocarbon agonists, Receptors, Aryl Hydrocarbon genetics, Signal Transduction immunology, Skin immunology, Skin metabolism, Transcription Factors biosynthesis, Up-Regulation, Basic Helix-Loop-Helix Transcription Factors immunology, Inflammation immunology, Psoriasis immunology, Receptors, Aryl Hydrocarbon immunology
- Abstract
Environmental stimuli are known to contribute to psoriasis pathogenesis and that of other autoimmune diseases, but the mechanisms are largely unknown. Here we show that the aryl hydrocarbon receptor (AhR), a transcription factor that senses environmental stimuli, modulates pathology in psoriasis. AhR-activating ligands reduced inflammation in the lesional skin of psoriasis patients, whereas AhR antagonists increased inflammation. Similarly, AhR signaling via the endogenous ligand FICZ reduced the inflammatory response in the imiquimod-induced model of skin inflammation and AhR-deficient mice exhibited a substantial exacerbation of the disease, compared to AhR-sufficient controls. Nonhematopoietic cells, in particular keratinocytes, were responsible for this hyperinflammatory response, which involved upregulation of AP-1 family members of transcription factors. Thus, our data suggest a critical role for AhR in the regulation of inflammatory responses and open the possibility for novel therapeutic strategies in chronic inflammatory disorders., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2014
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21. High-resolution analysis of gene activity during the Xenopus mid-blastula transition.
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Collart C, Owens ND, Bhaw-Rosun L, Cooper B, De Domenico E, Patrushev I, Sesay AK, Smith JN, Smith JC, and Gilchrist MJ
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- Animals, Embryo, Nonmammalian metabolism, Gene Expression Profiling, Molecular Sequence Annotation, Poly A metabolism, Polyadenylation genetics, RNA Stability genetics, RNA, Messenger, Stored genetics, RNA, Messenger, Stored metabolism, Reproducibility of Results, Transcription Factors metabolism, Transcription, Genetic, Xenopus Proteins genetics, Xenopus Proteins metabolism, Zebrafish genetics, Blastula metabolism, Gene Expression Regulation, Developmental, Xenopus embryology, Xenopus genetics
- Abstract
The Xenopus mid-blastula transition (MBT) marks the onset of large-scale zygotic transcription, as well as an increase in cell cycle length and a loss of synchronous cell divisions. Little is known about what triggers the activation of transcription or how newly expressed genes interact with each other. Here, we use high-resolution expression profiling to identify three waves of gene activity: a post-fertilisation wave involving polyadenylation of maternal transcripts; a broad wave of zygotic transcription detectable as early as the seventh cleavage and extending beyond the MBT at the twelfth cleavage; and a shorter post-MBT wave of transcription that becomes apparent as development proceeds. Our studies have also allowed us to define a set of maternal mRNAs that are deadenylated shortly after fertilisation, and are likely to be degraded thereafter. Experimental analysis indicates that the polyadenylation of maternal transcripts is necessary for the establishment of proper levels of zygotic transcription at the MBT, and that genes activated in the second wave of expression, including Brachyury and Mixer, contribute to the regulation of genes expressed in the third. Together, our high-resolution time series and experimental studies have yielded a deeper understanding of the temporal organisation of gene regulatory networks in the early Xenopus embryo.
- Published
- 2014
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22. Transcriptional regulation of Caenorhabditis elegans FOXO/DAF-16 modulates lifespan.
- Author
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Bansal A, Kwon ES, Conte D Jr, Liu H, Gilchrist MJ, MacNeil LT, and Tissenbaum HA
- Abstract
Background: Insulin/IGF-1 signaling plays a central role in longevity across phylogeny. In C. elegans, the forkhead box O (FOXO) transcription factor, DAF-16, is the primary target of insulin/IGF-1 signaling, and multiple isoforms of DAF-16 (a, b, and d/f) modulate lifespan, metabolism, dauer formation, and stress resistance. Thus far, across phylogeny modulation of mammalian FOXOs and DAF-16 have focused on post-translational regulation with little focus on transcriptional regulation. In C. elegans, we have previously shown that DAF-16d/f cooperates with DAF-16a to promote longevity. In this study, we generated transgenic strains expressing near-endogenous levels of either daf-16a or daf-16d/f, and examined temporal expression of the isoforms to further define how these isoforms contribute to lifespan regulation., Results: Here, we show that DAF-16a is sensitive both to changes in gene dosage and to alterations in the level of insulin/IGF-1 signaling. Interestingly, we find that as worms age, the intestinal expression of daf-16d/f but not daf-16a is dramatically upregulated at the level of transcription. Preventing this transcriptional upregulation shortens lifespan, indicating that transcriptional regulation of daf-16d/f promotes longevity. In an RNAi screen of transcriptional regulators, we identify elt-2 (GATA transcription factor) and swsn-1 (core subunit of SWI/SNF complex) as key modulators of daf-16d/f gene expression. ELT-2 and another GATA factor, ELT-4, promote longevity via both DAF-16a and DAF-16d/f while the components of SWI/SNF complex promote longevity specifically via DAF-16d/f., Conclusions: Our findings indicate that transcriptional control of C. elegans FOXO/daf-16 is an essential regulatory event. Considering the conservation of FOXO across species, our findings identify a new layer of FOXO regulation as a potential determinant of mammalian longevity and age-related diseases such as cancer and diabetes.
- Published
- 2014
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23. In vivo T-box transcription factor profiling reveals joint regulation of embryonic neuromesodermal bipotency.
- Author
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Gentsch GE, Owens ND, Martin SR, Piccinelli P, Faial T, Trotter MW, Gilchrist MJ, and Smith JC
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- Animals, DNA genetics, DNA metabolism, Embryonic Stem Cells cytology, Embryonic Stem Cells metabolism, Mesoderm cytology, Mesoderm metabolism, Neural Tube cytology, Neural Tube metabolism, Neurons cytology, Neurons metabolism, T-Box Domain Proteins genetics, Xenopus, Embryonic Development physiology, T-Box Domain Proteins metabolism
- Abstract
The design of effective cell replacement therapies requires detailed knowledge of how embryonic stem cells form primary tissues, such as mesoderm or neurectoderm that later become skeletal muscle or nervous system. Members of the T-box transcription factor family are key in the formation of these primary tissues, but their underlying molecular activities are poorly understood. Here, we define in vivo genome-wide regulatory inputs of the T-box proteins Brachyury, Eomesodermin, and VegT, which together maintain neuromesodermal stem cells and determine their bipotential fates in frog embryos. These T-box proteins are all recruited to the same genomic recognition sites, from where they activate genes involved in stem cell maintenance and mesoderm formation while repressing neurogenic genes. Consequently, their loss causes embryos to form an oversized neural tube with no mesodermal derivatives. This collaboration between T-box family members thus ensures the continuous formation of correctly proportioned neural and mesodermal tissues in vertebrate embryos during axial elongation., (Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2013
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24. Efficient high-throughput sequencing of a laser microdissected chromosome arm.
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Seifertova E, Zimmerman LB, Gilchrist MJ, Macha J, Kubickova S, Cernohorska H, Zarsky V, Owens ND, Sesay AK, Tlapakova T, and Krylov V
- Subjects
- Animals, Chromosome Mapping, Genomics, Nucleic Acid Amplification Techniques, Nucleic Acid Hybridization, Xenopus genetics, Chromosomes genetics, High-Throughput Nucleotide Sequencing methods, Lasers, Microdissection, Sequence Analysis, DNA methods
- Abstract
Background: Genomic sequence assemblies are key tools for a broad range of gene function and evolutionary studies. The diploid amphibian Xenopus tropicalis plays a pivotal role in these fields due to its combination of experimental flexibility, diploid genome, and early-branching tetrapod taxonomic position, having diverged from the amniote lineage ~360 million years ago. A genome assembly and a genetic linkage map have recently been made available. Unfortunately, large gaps in the linkage map attenuate long-range integrity of the genome assembly., Results: We laser dissected the short arm of X. tropicalis chromosome 7 for next generation sequencing and computational mapping to the reference genome. This arm is of particular interest as it encodes the sex determination locus, but its genetic map contains large gaps which undermine available genome assemblies. Whole genome amplification of 15 laser-microdissected 7p arms followed by next generation sequencing yielded ~35 million reads, over four million of which uniquely mapped to the X. tropicalis genome. Our analysis placed more than 200 previously unmapped scaffolds on the analyzed chromosome arm, providing valuable low-resolution physical map information for de novo genome assembly., Conclusion: We present a new approach for improving and validating genetic maps and sequence assemblies. Whole genome amplification of 15 microdissected chromosome arms provided sufficient high-quality material for localizing previously unmapped scaffolds and genes as well as recognizing mislocalized scaffolds.
- Published
- 2013
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25. A large pseudoautosomal region on the sex chromosomes of the frog Silurana tropicalis.
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Bewick AJ, Chain FJ, Zimmerman LB, Sesay A, Gilchrist MJ, Owens ND, Seifertova E, Krylov V, Macha J, Tlapakova T, Kubickova S, Cernohorska H, Zarsky V, and Evans BJ
- Subjects
- Animals, Female, Gene Expression, Genotype, Male, Evolution, Molecular, Sex Chromosomes genetics, Xenopus genetics
- Abstract
Sex chromosome divergence has been documented across phylogenetically diverse species, with amphibians typically having cytologically nondiverged ("homomorphic") sex chromosomes. With an aim of further characterizing sex chromosome divergence of an amphibian, we used "RAD-tags" and Sanger sequencing to examine sex specificity and heterozygosity in the Western clawed frog Silurana tropicalis (also known as Xenopus tropicalis). Our findings based on approximately 20 million genotype calls and approximately 200 polymerase chain reaction-amplified regions across multiple male and female genomes failed to identify a substantially sized genomic region with genotypic hallmarks of sex chromosome divergence, including in regions known to be tightly linked to the sex-determining region. We also found that expression and molecular evolution of genes linked to the sex-determining region did not differ substantially from genes in other parts of the genome. This suggests that the pseudoautosomal region, where recombination occurs, comprises a large portion of the sex chromosomes of S. tropicalis. These results may in part explain why African clawed frogs have such a high incidence of polyploidization, shed light on why amphibians have a high rate of sex chromosome turnover, and raise questions about why homomorphic sex chromosomes are so prevalent in amphibians.
- Published
- 2013
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26. Exon capture and bulk segregant analysis: rapid discovery of causative mutations using high-throughput sequencing.
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del Viso F, Bhattacharya D, Kong Y, Gilchrist MJ, and Khokha MK
- Subjects
- Animals, Base Sequence, Chromosome Mapping, Clone Cells, Genetic Linkage, Genotype, High-Throughput Nucleotide Sequencing, Meiosis genetics, Molecular Sequence Data, Phenotype, Sequence Analysis, DNA, Exome, Exons, Mutation, Polymorphism, Single Nucleotide, Xenopus genetics, Xenopus Proteins genetics
- Abstract
Background: Exome sequencing has transformed human genetic analysis and may do the same for other vertebrate model systems. However, a major challenge is sifting through the large number of sequence variants to identify the causative mutation for a given phenotype. In models like Xenopus tropicalis, an incomplete and occasionally incorrect genome assembly compounds this problem. To facilitate cloning of X. tropicalis mutants identified in forward genetic screens, we sought to combine bulk segregant analysis and exome sequencing into a single step., Results: Here we report the first use of exon capture sequencing to identify mutations in a non-mammalian, vertebrate model. We demonstrate that bulk segregant analysis coupled with exon capture sequencing is not only able to identify causative mutations but can also generate linkage information, facilitate the assembly of scaffolds, identify misassembles, and discover thousands of SNPs for fine mapping., Conclusion: Exon capture sequencing and bulk segregant analysis is a rapid, inexpensive method to clone mutants identified in forward genetic screens. With sufficient meioses, this method can be generalized to any model system with a genome assembly, polished or unpolished, and in the latter case, it also provides many critical genomic resources.
- Published
- 2012
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27. Rsx is a metatherian RNA with Xist-like properties in X-chromosome inactivation.
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Grant J, Mahadevaiah SK, Khil P, Sangrithi MN, Royo H, Duckworth J, McCarrey JR, VandeBerg JL, Renfree MB, Taylor W, Elgar G, Camerini-Otero RD, Gilchrist MJ, and Turner JM
- Subjects
- Animals, Female, Gene Expression Regulation, Gene Silencing, Mice, Transgenes, Monodelphis genetics, Monodelphis metabolism, RNA genetics, RNA metabolism, X Chromosome genetics, X Chromosome metabolism, X Chromosome Inactivation
- Abstract
In female (XX) mammals, one of the two X chromosomes is inactivated to ensure an equal dose of X-linked genes with males (XY). X-chromosome inactivation in eutherian mammals is mediated by the non-coding RNA Xist. Xist is not found in metatherians (marsupials), and how X-chromosome inactivation is initiated in these mammals has been the subject of speculation for decades. Using the marsupial Monodelphis domestica, here we identify Rsx (RNA-on-the-silent X), an RNA that has properties consistent with a role in X-chromosome inactivation. Rsx is a large, repeat-rich RNA that is expressed only in females and is transcribed from, and coats, the inactive X chromosome. In female germ cells, in which both X chromosomes are active, Rsx is silenced, linking Rsx expression to X-chromosome inactivation and reactivation. Integration of an Rsx transgene on an autosome in mouse embryonic stem cells leads to gene silencing in cis. Our findings permit comparative studies of X-chromosome inactivation in mammals and pose questions about the mechanisms by which X-chromosome inactivation is achieved in eutherians.
- Published
- 2012
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28. A large scale screen for neural stem cell markers in Xenopus retina.
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Parain K, Mazurier N, Bronchain O, Borday C, Cabochette P, Chesneau A, Colozza G, El Yakoubi W, Hamdache J, Locker M, Gilchrist MJ, Pollet N, and Perron M
- Subjects
- Animals, Base Sequence, Biomarkers metabolism, In Situ Hybridization, Molecular Sequence Data, Neural Stem Cells metabolism, Polymerase Chain Reaction, Retina metabolism, Xenopus, Biomarkers analysis, Databases, Genetic, Gene Expression Profiling, Neural Stem Cells cytology, Retina cytology
- Abstract
Neural stem cell research suffers from a lack of molecular markers to specifically assess stem or progenitor cell properties. The organization of the Xenopus ciliary marginal zone (CMZ) in the retina allows the spatial distinction of these two cell types: stem cells are confined to the most peripheral region, while progenitors are more central. Despite this clear advantage, very few genes specifically expressed in retinal stem cells have been discovered so far in this model. To gain insight into the molecular signature of these cells, we performed a large-scale expression screen in the Xenopus CMZ, establishing it as a model system for stem cell gene profiling. Eighteen genes expressed specifically in the CMZ stem cell compartment were retrieved and are discussed here. These encode various types of proteins, including factors associated with proliferation, mitotic spindle organization, DNA/RNA processing, and cell adhesion. In addition, the publication of this work in a special issue on Xenopus prompted us to give a more general illustration of the value of large-scale screens in this model species. Thus, beyond neural stem cell specific genes, we give a broader highlight of our screen outcome, describing in particular other retinal cell markers that we found. Finally, we present how these can all be easily retrieved through a novel module we developed in the web-based annotation tool XenMARK, and illustrate the potential of this powerful searchable database in the context of the retina., (Copyright © 2011 Wiley Periodicals, Inc.)
- Published
- 2012
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29. From expression cloning to gene modeling: the development of Xenopus gene sequence resources.
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Gilchrist MJ
- Subjects
- Animals, Cloning, Molecular, DNA, Complementary chemistry, Expressed Sequence Tags, Genome, RNA, Messenger chemistry, Sequence Analysis, Xenopus laevis genetics, Models, Genetic, Transcriptome, Xenopus genetics, Xenopus Proteins genetics
- Abstract
The Xenopus community has made concerted efforts over the last 10-12 years systematically to improve the available sequence information for this amphibian model organism ideally suited to the study of early development in vertebrates. Here I review progress in the collection of both sequence data and physical clone reagents for protein coding genes. I conclude that we have cDNA sequences for around 50% and full-length clones for about 35% of the genes in Xenopus tropicalis, and similar numbers but a smaller proportion for Xenopus laevis. In addition, I demonstrate that the gaps in the current genome assembly create problems for the computational elucidation of gene sequences, and suggest some ways to ameliorate the effects of this., (Copyright © 2012 Wiley Periodicals, Inc.)
- Published
- 2012
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30. Widespread transcription in an amphibian oocyte relates to its reprogramming activity on transplanted somatic nuclei.
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Simeoni I, Gilchrist MJ, Garrett N, Armisen J, and Gurdon JB
- Subjects
- Animals, Cell Nucleus metabolism, Female, Gene Expression Profiling, High-Throughput Nucleotide Sequencing, RNA Splicing, RNA, Messenger metabolism, Stem Cells cytology, Stem Cells metabolism, Transcriptional Activation, Xenopus, Xenopus Proteins metabolism, Cell Nucleus genetics, Nuclear Transfer Techniques, Oocytes cytology, Oocytes metabolism, RNA, Messenger genetics, Transcriptome genetics, Xenopus Proteins genetics
- Abstract
Amphibian oocytes have the special ability to directly induce the transcription of pluripotency and other genes in transplanted somatic nuclei. To this extent, oocytes induce a stem cell-like pattern of transcription in somatic cell nuclei. We ask whether the induced transcription in transplanted nuclei reflects the normal transcriptional activity of oocyte genes. We describe here the transcript content of a wide range of genes in Xenopus tropicalis oocytes. Using accurate quantitation, we find that each mature oocyte has accumulated several hundred transcripts of cell-type specific genes. This value is several orders of magnitude greater than the "leakage" level found in most somatic cells and about the same level found in somatic cells where these genes are fully expressed. Illumina sequencing confirms the high transcript content of a mature Xenopus oocyte. Most of the transcripts from these highly expressed genes in oocytes are correctly and efficiently spliced. Our results contribute a more quantitative view of certain amphibian oocyte transcripts than previously available. Our results also show that transplanted somatic nuclei conform, with respect to the genes analyzed, to the transcriptional characteristics of the recipient oocytes.
- Published
- 2012
- Full Text
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31. Databases of gene expression in Xenopus development.
- Author
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Gilchrist MJ and Pollet N
- Subjects
- Animals, Arabidopsis Proteins genetics, Arabidopsis Proteins metabolism, Data Mining, Databases, Genetic, Embryo, Nonmammalian metabolism, Expressed Sequence Tags, Intramolecular Transferases genetics, Intramolecular Transferases metabolism, Organ Specificity, Search Engine, Software, Transcriptome, Xenopus growth & development, Xenopus Proteins metabolism, Gene Expression Regulation, Developmental, Xenopus genetics, Xenopus Proteins genetics
- Abstract
Gene expression data for Xenopus are collected and curated in diverse forms and locations. The intention of this chapter is to give the reader a guide to the publicly accessible databases where these data can be found and an idea of the current scope and limitations of the data in these resources. Instructions are given on how to access and interpret the data provided by the NCBI Gene database, Xenbase, and the Xenopus full-length EST, quickImage, and Xenmark databases.
- Published
- 2012
- Full Text
- View/download PDF
32. Genomic targets of Brachyury (T) in differentiating mouse embryonic stem cells.
- Author
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Evans AL, Faial T, Gilchrist MJ, Down T, Vallier L, Pedersen RA, Wardle FC, and Smith JC
- Subjects
- Animals, Axin Protein genetics, Axin Protein metabolism, Base Sequence, Binding Sites genetics, Blotting, Western, Cell Line, Cells, Cultured, Embryo, Mammalian cytology, Embryo, Mammalian embryology, Embryo, Mammalian metabolism, Embryonic Stem Cells cytology, Fetal Proteins genetics, Fibroblast Growth Factor 8 genetics, Fibroblast Growth Factor 8 metabolism, Gene Expression Profiling, Gene Expression Regulation, Developmental, Humans, In Situ Hybridization, Mice, Mice, 129 Strain, Mice, Knockout, Promoter Regions, Genetic genetics, Protein Binding, Proteins genetics, Reverse Transcriptase Polymerase Chain Reaction, T-Box Domain Proteins genetics, Time Factors, Wnt3A Protein genetics, Wnt3A Protein metabolism, Cell Differentiation, Embryonic Stem Cells metabolism, Fetal Proteins metabolism, Proteins metabolism, T-Box Domain Proteins metabolism
- Abstract
Background: The T-box transcription factor Brachyury (T) is essential for formation of the posterior mesoderm and the notochord in vertebrate embryos. Work in the frog and the zebrafish has identified some direct genomic targets of Brachyury, but little is known about Brachyury targets in the mouse., Methodology/principal Findings: Here we use chromatin immunoprecipitation and mouse promoter microarrays to identify targets of Brachyury in embryoid bodies formed from differentiating mouse ES cells. The targets we identify are enriched for sequence-specific DNA binding proteins and include components of signal transduction pathways that direct cell fate in the primitive streak and tailbud of the early embryo. Expression of some of these targets, such as Axin2, Fgf8 and Wnt3a, is down regulated in Brachyury mutant embryos and we demonstrate that they are also Brachyury targets in the human. Surprisingly, we do not observe enrichment of the canonical T-domain DNA binding sequence 5'-TCACACCT-3' in the vicinity of most Brachyury target genes. Rather, we have identified an (AC)(n) repeat sequence, which is conserved in the rat but not in human, zebrafish or Xenopus. We do not understand the significance of this sequence, but speculate that it enhances transcription factor binding in the regulatory regions of Brachyury target genes in rodents., Conclusions/significance: Our work identifies the genomic targets of a key regulator of mesoderm formation in the early mouse embryo, thereby providing insights into the Brachyury-driven genetic regulatory network and allowing us to compare the function of Brachyury in different species.
- Published
- 2012
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33. Genome-wide analysis of gene expression during Xenopus tropicalis tadpole tail regeneration.
- Author
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Love NR, Chen Y, Bonev B, Gilchrist MJ, Fairclough L, Lea R, Mohun TJ, Paredes R, Zeef LA, and Amaya E
- Subjects
- Animals, Larva genetics, NADP genetics, Regeneration, Tail physiology, Xenopus genetics, Gene Expression Regulation, Developmental, Genome, Larva physiology, Xenopus physiology
- Abstract
Background: The molecular mechanisms governing vertebrate appendage regeneration remain poorly understood. Uncovering these mechanisms may lead to novel therapies aimed at alleviating human disfigurement and visible loss of function following injury. Here, we explore tadpole tail regeneration in Xenopus tropicalis, a diploid frog with a sequenced genome., Results: We found that, like the traditionally used Xenopus laevis, the Xenopus tropicalis tadpole has the capacity to regenerate its tail following amputation, including its spinal cord, muscle, and major blood vessels. We examined gene expression using the Xenopus tropicalis Affymetrix genome array during three phases of regeneration, uncovering more than 1,000 genes that are significantly modulated during tail regeneration. Target validation, using RT-qPCR followed by gene ontology (GO) analysis, revealed a dynamic regulation of genes involved in the inflammatory response, intracellular metabolism, and energy regulation. Meta-analyses of the array data and validation by RT-qPCR and in situ hybridization uncovered a subset of genes upregulated during the early and intermediate phases of regeneration that are involved in the generation of NADP/H, suggesting that these pathways may be important for proper tail regeneration., Conclusions: The Xenopus tropicalis tadpole is a powerful model to elucidate the genetic mechanisms of vertebrate appendage regeneration. We have produced a novel and substantial microarray data set examining gene expression during vertebrate appendage regeneration.
- Published
- 2011
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34. The ANISEED database: digital representation, formalization, and elucidation of a chordate developmental program.
- Author
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Tassy O, Dauga D, Daian F, Sobral D, Robin F, Khoueiry P, Salgado D, Fox V, Caillol D, Schiappa R, Laporte B, Rios A, Luxardi G, Kusakabe T, Joly JS, Darras S, Christiaen L, Contensin M, Auger H, Lamy C, Hudson C, Rothbächer U, Gilchrist MJ, Makabe KW, Hotta K, Fujiwara S, Satoh N, Satou Y, and Lemaire P
- Subjects
- Animals, Chordata embryology, Chordata genetics, Chordata growth & development, Computational Biology methods, Databases, Factual, Developmental Biology methods, Gene Expression Regulation, Developmental, Image Processing, Computer-Assisted methods, Internet, Urochordata embryology, Urochordata genetics, Urochordata growth & development
- Abstract
Developmental biology aims to understand how the dynamics of embryonic shapes and organ functions are encoded in linear DNA molecules. Thanks to recent progress in genomics and imaging technologies, systemic approaches are now used in parallel with small-scale studies to establish links between genomic information and phenotypes, often described at the subcellular level. Current model organism databases, however, do not integrate heterogeneous data sets at different scales into a global view of the developmental program. Here, we present a novel, generic digital system, NISEED, and its implementation, ANISEED, to ascidians, which are invertebrate chordates suitable for developmental systems biology approaches. ANISEED hosts an unprecedented combination of anatomical and molecular data on ascidian development. This includes the first detailed anatomical ontologies for these embryos, and quantitative geometrical descriptions of developing cells obtained from reconstructed three-dimensional (3D) embryos up to the gastrula stages. Fully annotated gene model sets are linked to 30,000 high-resolution spatial gene expression patterns in wild-type and experimentally manipulated conditions and to 528 experimentally validated cis-regulatory regions imported from specialized databases or extracted from 160 literature articles. This highly structured data set can be explored via a Developmental Browser, a Genome Browser, and a 3D Virtual Embryo module. We show how integration of heterogeneous data in ANISEED can provide a system-level understanding of the developmental program through the automatic inference of gene regulatory interactions, the identification of inducing signals, and the discovery and explanation of novel asymmetric divisions.
- Published
- 2010
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35. A study of alternative splicing in the pig.
- Author
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Nygard AB, Cirera S, Gilchrist MJ, Gorodkin J, Jørgensen CB, and Fredholm M
- Abstract
Background: Since at least half of the genes in mammalian genomes are subjected to alternative splicing, alternative pre-mRNA splicing plays an important contribution to the complexity of the mammalian proteome. Expressed sequence tags (ESTs) provide evidence of a great number of possible alternative isoforms. With the EST resource for the domestic pig now containing more than one million porcine ESTs, it is possible to identify alternative splice forms of the individual transcripts in this species from the EST data with some confidence., Results: The pig EST data generated by the Sino-Danish Pig Genome project has been assembled with publicly available ESTs and made available in the PigEST database. Using the Distiller package 2,515 EST clusters with candidate alternative isoforms were identified in the EST data with high confidence. In agreement with general observations in human and mouse, we find putative splice variants in about 30% of the contigs with more than 50 ESTs. Based on the criteria that a minimum of two EST sequences confirmed each splice event, a list of 100 genes with the most distinct tissue-specific alternative splice events was generated from the list of candidates. To confirm the tissue specificity of the splice events, 10 genes with functional annotation were randomly selected from which 16 individual splice events were chosen for experimental verification by quantitative PCR (qPCR). Six genes were shown to have tissue specific alternatively spliced transcripts with expression patterns matching those of the EST data. The remaining four genes had tissue-restricted expression of alternative spliced transcripts. Five out of the 16 splice events that were experimentally verified were found to be putative pig specific., Conclusions: In accordance with human and rodent studies we estimate that approximately 30% of the porcine genes undergo alternative splicing. We found a good correlation between EST predicted tissue-specificity and experimentally validated splice events in different porcine tissue. This study indicates that a cluster size of around 50 ESTs is optimal for in silico detection of alternative splicing. Although based on a limited number of splice events, the study supports the notion that alternative splicing could have an important impact on species differentiation since 31% of the splice events studied appears to be species specific.
- Published
- 2010
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36. The genome of the Western clawed frog Xenopus tropicalis.
- Author
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Hellsten U, Harland RM, Gilchrist MJ, Hendrix D, Jurka J, Kapitonov V, Ovcharenko I, Putnam NH, Shu S, Taher L, Blitz IL, Blumberg B, Dichmann DS, Dubchak I, Amaya E, Detter JC, Fletcher R, Gerhard DS, Goodstein D, Graves T, Grigoriev IV, Grimwood J, Kawashima T, Lindquist E, Lucas SM, Mead PE, Mitros T, Ogino H, Ohta Y, Poliakov AV, Pollet N, Robert J, Salamov A, Sater AK, Schmutz J, Terry A, Vize PD, Warren WC, Wells D, Wills A, Wilson RK, Zimmerman LB, Zorn AM, Grainger R, Grammer T, Khokha MK, Richardson PM, and Rokhsar DS
- Subjects
- Animals, Chickens genetics, Chromosome Mapping, Chromosomes genetics, Computational Biology, Conserved Sequence, DNA Transposable Elements, DNA, Complementary, Embryo, Nonmammalian metabolism, Evolution, Molecular, Expressed Sequence Tags, Gene Duplication, Genes, Humans, Phylogeny, Synteny, Vertebrates genetics, Xenopus embryology, Xenopus Proteins genetics, Genome, Sequence Analysis, DNA, Xenopus genetics
- Abstract
The western clawed frog Xenopus tropicalis is an important model for vertebrate development that combines experimental advantages of the African clawed frog Xenopus laevis with more tractable genetics. Here we present a draft genome sequence assembly of X. tropicalis. This genome encodes more than 20,000 protein-coding genes, including orthologs of at least 1700 human disease genes. Over 1 million expressed sequence tags validated the annotation. More than one-third of the genome consists of transposable elements, with unusually prevalent DNA transposons. Like that of other tetrapods, the genome of X. tropicalis contains gene deserts enriched for conserved noncoding elements. The genome exhibits substantial shared synteny with human and chicken over major parts of large chromosomes, broken by lineage-specific chromosome fusions and fissions, mainly in the mammalian lineage.
- Published
- 2010
- Full Text
- View/download PDF
37. Factors associated with rabid animals since the introduction of raccoon rabies variant in Massachusetts, 1992-2007.
- Author
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Wang X, Smole S, DeMaria A, and Gilchrist MJ
- Subjects
- Animals, Humans, Logistic Models, Massachusetts epidemiology, Rabies pathology, Raccoons virology, Risk Factors, Animals, Domestic virology, Animals, Wild virology, Rabies diagnosis, Rabies epidemiology
- Abstract
Rabies virus has been identified in 26 animal species since the introduction of the raccoon rabies variant (RRV) into the Commonwealth of Massachusetts in 1992. This study used data from 47,162 testable specimens, including 4538 (9.62%) rabid animals, to produce a multi-categorical logistic regression model to identify factors associated with a positive rabies laboratory test. The model was adjusted by the animal type and animal species, using the least tested and the least found rabid animal species pooled as a reference group. The c-statistic for the final model was 0.94, and a receiver operator characteristic curve plot shows the increased sensitivity and the decreased false-positive proportion of the model. Introduction of RRV into the county where the animal was found (OR = 17.3), not up-to-date on vaccination (OR = 3.88), exposure of multiple humans, or pets, or human and pet (OR = 1.88), reason for rabies testing (using human exposure only as the reference group, the odds ratio for both human and animal exposure is 2.14; for pet/companion exposure only is 2.96; and for undefined reasons/sick animal is 1.49), reported syndromes/observation of aggression (OR = 4.13), ataxia (OR = 1.36), disorientation (OR = 1.67), paralysis ( OR = 1.37), and the presence of unexplained wounds (OR = 1.27) were all significantly associated with a positive rabies testing result at the alpha = 0.05 level.
- Published
- 2010
- Full Text
- View/download PDF
38. Abundant and dynamically expressed miRNAs, piRNAs, and other small RNAs in the vertebrate Xenopus tropicalis.
- Author
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Armisen J, Gilchrist MJ, Wilczynska A, Standart N, and Miska EA
- Subjects
- Animals, Female, Gene Expression Profiling, Gene Expression Regulation, Developmental, Germ Cells metabolism, MicroRNAs metabolism, RNA, Small Interfering metabolism, RNA, Small Nuclear metabolism, Signal Transduction genetics, Vertebrates genetics, Vertebrates metabolism, Xenopus metabolism, MicroRNAs genetics, RNA, Small Interfering genetics, RNA, Small Nuclear genetics, Xenopus genetics
- Abstract
Small regulatory RNAs have recently emerged as key regulators of eukaryotic gene expression. Here we used high-throughput sequencing to determine small RNA populations in the germline and soma of the African clawed frog Xenopus tropicalis. We identified a number of miRNAs that were expressed in the female germline. miRNA expression profiling revealed that miR-202-5p is an oocyte-enriched miRNA. We identified two novel miRNAs that were expressed in the soma. In addition, we sequenced large numbers of Piwi-associated RNAs (piRNAs) and other endogenous small RNAs, likely representing endogenous siRNAs (endo-siRNAs). Of these, only piRNAs were restricted to the germline, suggesting that endo-siRNAs are an abundant class of small RNAs in the vertebrate soma. In the germline, both endogenous small RNAs and piRNAs mapped to many high copy number loci. Furthermore, endogenous small RNAs mapped to the same specific subsets of repetitive elements in both the soma and the germline, suggesting that these RNAs might act to silence repetitive elements in both compartments. Data presented here suggest a conserved role for miRNAs in the vertebrate germline. Furthermore, this study provides a basis for the functional analysis of small regulatory RNAs in an important vertebrate model system.
- Published
- 2009
- Full Text
- View/download PDF
39. Database of queryable gene expression patterns for Xenopus.
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Gilchrist MJ, Christensen MB, Bronchain O, Brunet F, Chesneau A, Fenger U, Geach TJ, Ironfield HV, Kaya F, Kricha S, Lea R, Massé K, Néant I, Paillard E, Parain K, Perron M, Sinzelle L, Souopgui J, Thuret R, Ymlahi-Ouazzani Q, and Pollet N
- Subjects
- Animals, Humans, Software, Xenopus laevis anatomy & histology, Databases, Genetic, Gene Expression, Gene Expression Regulation, Developmental, Xenopus laevis embryology, Xenopus laevis genetics
- Abstract
The precise localization of gene expression within the developing embryo, and how it changes over time, is one of the most important sources of information for elucidating gene function. As a searchable resource, this information has up until now been largely inaccessible to the Xenopus community. Here, we present a new database of Xenopus gene expression patterns, queryable by specific location or region in the embryo. Pattern matching can be driven either from an existing in situ image, or from a user-defined pattern based on development stage schematic diagrams. The data are derived from the work of a group of 21 Xenopus researchers over a period of 4 days. We used a novel, rapid manual annotation tool, XenMARK, which exploits the ability of the human brain to make the necessary distortions in transferring data from the in situ images to the standard schematic geometry. Developmental Dynamics 238:1379-1388, 2009. (c) 2009 Wiley-Liss, Inc.
- Published
- 2009
- Full Text
- View/download PDF
40. Sequence assembly.
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Scheibye-Alsing K, Hoffmann S, Frankel A, Jensen P, Stadler PF, Mang Y, Tommerup N, Gilchrist MJ, Nygård AB, Cirera S, Jørgensen CB, Fredholm M, and Gorodkin J
- Subjects
- Computational Biology methods, DNA chemistry, Expressed Sequence Tags, Genome, Polymorphism, Single Nucleotide, Repetitive Sequences, Nucleic Acid, Genomics methods
- Abstract
Despite the rapidly increasing number of sequenced and re-sequenced genomes, many issues regarding the computational assembly of large-scale sequencing data have remain unresolved. Computational assembly is crucial in large genome projects as well for the evolving high-throughput technologies and plays an important role in processing the information generated by these methods. Here, we provide a comprehensive overview of the current publicly available sequence assembly programs. We describe the basic principles of computational assembly along with the main concerns, such as repetitive sequences in genomic DNA, highly expressed genes and alternative transcripts in EST sequences. We summarize existing comparisons of different assemblers and provide a detailed descriptions and directions for download of assembly programs at: http://genome.ku.dk/resources/assembly/methods.html.
- Published
- 2009
- Full Text
- View/download PDF
41. A gene regulatory network directed by zebrafish No tail accounts for its roles in mesoderm formation.
- Author
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Morley RH, Lachani K, Keefe D, Gilchrist MJ, Flicek P, Smith JC, and Wardle FC
- Subjects
- Animals, Base Sequence, Binding Sites, Body Patterning genetics, Cell Lineage, Conserved Sequence, Fetal Proteins genetics, Gastrulation genetics, Gene Expression Regulation, Developmental, Homeodomain Proteins genetics, Homeodomain Proteins metabolism, Molecular Sequence Data, Muscles cytology, Protein Binding, T-Box Domain Proteins genetics, Transcription Factors genetics, Transcription Factors metabolism, Transcription, Genetic, Zebrafish Proteins genetics, Zebrafish Proteins metabolism, Fetal Proteins metabolism, Gene Regulatory Networks, Mesoderm embryology, T-Box Domain Proteins metabolism, Zebrafish embryology, Zebrafish genetics
- Abstract
Using chromatin immunoprecipitation combined with genomic microarrays we have identified targets of No tail (Ntl), a zebrafish Brachyury ortholog that plays a central role in mesoderm formation. We show that Ntl regulates a downstream network of other transcription factors and identify an in vivo Ntl binding site that resembles the consensus T-box binding site (TBS) previously identified by in vitro studies. We show that the notochord-expressed gene floating head (flh) is a direct transcriptional target of Ntl and that a combination of TBSs in the flh upstream region are required for Ntl-directed expression. Using our genome-scale data we have assembled a preliminary gene regulatory network that begins to describe mesoderm formation and patterning in the early zebrafish embryo.
- Published
- 2009
- Full Text
- View/download PDF
42. Identification of direct T-box target genes in the developing zebrafish mesoderm.
- Author
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Garnett AT, Han TM, Gilchrist MJ, Smith JC, Eisen MB, Wardle FC, and Amacher SL
- Subjects
- Animals, Animals, Genetically Modified, Binding Sites genetics, DNA genetics, DNA metabolism, Fetal Proteins, Gene Expression Regulation, Developmental, Mesoderm embryology, Mesoderm metabolism, Mice, Molecular Sequence Data, Mutation, Nerve Tissue Proteins genetics, RNA, Messenger genetics, RNA, Messenger metabolism, Recombinant Fusion Proteins genetics, Recombinant Fusion Proteins metabolism, Regulatory Elements, Transcriptional, Sequence Homology, Nucleic Acid, Signal Transduction, T-Box Domain Proteins metabolism, Tail embryology, Tail metabolism, Wnt Proteins genetics, Wnt Proteins metabolism, Zebrafish metabolism, Zebrafish Proteins metabolism, T-Box Domain Proteins genetics, Zebrafish embryology, Zebrafish genetics, Zebrafish Proteins genetics
- Abstract
The zebrafish genes spadetail (spt) and no tail (ntl) encode T-box transcription factors that are important for early mesoderm development. Although much has been done to characterize these genes, the identity and location of target regulatory elements remain largely unknown. Here, we survey the genome for downstream target genes of the Spt and Ntl T-box transcription factors. We find evidence for extensive additive interactions towards gene activation and limited evidence for combinatorial and antagonistic interactions between the two factors. Using in vitro binding selection assays to define Spt- and Ntl-binding motifs, we searched for target regulatory sequence via a combination of binding motif searches and comparative genomics. We identified regulatory elements for tbx6 and deltaD, and, using chromatin immunoprecipitation, in vitro DNA binding assays and transgenic methods, we provide evidence that both are directly regulated by T-box transcription factors. We also find that deltaD is directly activated by T-box factors in the tail bud, where it has been implicated in starting the segmentation clock, suggesting that spt and ntl act upstream of this process.
- Published
- 2009
- Full Text
- View/download PDF
43. Loss of REEP4 causes paralysis of the Xenopus embryo.
- Author
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Argasinska J, Rana AA, Gilchrist MJ, Lachani K, Young A, and Smith JC
- Subjects
- Amino Acid Sequence, Amphibian Proteins chemistry, Amphibian Proteins genetics, Animals, Base Sequence, Biomarkers, Conserved Sequence, Down-Regulation, Embryo, Nonmammalian abnormalities, Embryo, Nonmammalian metabolism, Gene Expression Regulation, Developmental, Membrane Transport Proteins chemistry, Membrane Transport Proteins genetics, Molecular Sequence Data, Muscle Development, Paralysis genetics, Phylogeny, Sequence Alignment, Xenopus genetics, Amphibian Proteins metabolism, Membrane Transport Proteins metabolism, Paralysis embryology, Paralysis metabolism, Xenopus abnormalities, Xenopus metabolism
- Abstract
Members of the REEP (Receptor expression enhancing protein) family contain a TB2/DP1, HVA22 domain that is involved in intracellular trafficking and secretion. Consistent with the presence of this domain, REEP1 and REEP3 enhance the expression of odorant and taste receptors in mammals, while mutation of these genes causes defects in neural development. REEP4 was identified in the course of a functional antisense morpholino oligonucleotide screen searching for genes involved in the early development of Xenopus tropicalis: although over-expression of the gene causes no phenotype, embryos lacking REEP4 develop a slightly kinked body axis and are paralysed. At tailbud stages of development, REEP4 is expressed in the somites and neural tube. The paralysis observed in embryos lacking REEP4 might therefore be caused by defects in the nervous system or in muscle. To address this point, we examined the expression of various neural and muscle markers and found that although all are expressed normally at early stages of development, many are down regulated by the tailbud stage. This suggests that REEP4 plays a role in the maintenance of both the nervous system and the musculature.
- Published
- 2009
- Full Text
- View/download PDF
44. Evading the annotation bottleneck: using sequence similarity to search non-sequence gene data.
- Author
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Gilchrist MJ, Christensen MB, Harland R, Pollet N, Smith JC, Ueno N, and Papalopulu N
- Subjects
- Animals, Base Sequence, Conserved Sequence, Humans, Xenopus, Databases, Genetic, Information Storage and Retrieval methods
- Abstract
Background: Non-sequence gene data (images, literature, etc.) can be found in many different public databases. Access to these data is mostly by text based methods using gene names; however, gene annotation is neither complete, nor fully systematic between organisms, and is also not generally stable over time. This provides some challenges for text based access, especially for cross-species searches. We propose a method for non-sequence data retrieval based on sequence similarity, which removes dependence on annotation and text searches. This work was motivated by the need to provide better access to large numbers of in situ images, and the observation that such image data were usually associated with a specific gene sequence. Sequence similarity searches are found in existing gene oriented databases, but mostly give indirect access to non-sequence data via navigational links., Results: Three applications were built to explore the proposed method: accessing image data, literature and gene names. Searches are initiated with the sequence of the user's gene of interest, which is searched against a database of sequences associated with the target data. The matching (non-sequence) target data are returned directly to the user's browser, organised by sequence similarity. The method worked well for the intended application in image data management. Comparison with text based searches of the image data set showed the accuracy of the method. Applied to literature searches it facilitated retrieval of mostly high relevance references. Applied to gene name data it provided a useful analysis of name variation of related genes within and between species., Conclusion: This method makes a powerful and useful addition to existing methods for searching gene data based on text retrieval or curated gene lists. In particular the method facilitates cross-species comparisons, and enables the handling of novel or otherwise un-annotated genes. Applications using the method are quick and easy to build, and the data require little maintenance. This approach largely circumvents the need for annotation, which can be a major obstacle to the development of genomic scale data resources.
- Published
- 2008
- Full Text
- View/download PDF
45. Maternal Argonaute 2 is essential for early mouse development at the maternal-zygotic transition.
- Author
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Lykke-Andersen K, Gilchrist MJ, Grabarek JB, Das P, Miska E, and Zernicka-Goetz M
- Subjects
- Animals, Argonaute Proteins, Eukaryotic Initiation Factor-2 metabolism, Green Fluorescent Proteins metabolism, Mice, Mice, Inbred C57BL, Mice, Inbred CBA, Nucleic Acid Hybridization, RNA Interference, RNA, Double-Stranded metabolism, RNA, Messenger, Stored genetics, RNA, Small Interfering metabolism, RNA-Induced Silencing Complex genetics, Eukaryotic Initiation Factor-2 genetics, Gene Expression Regulation, Developmental, Gene Silencing, Zygote metabolism
- Abstract
Activation of zygotic gene expression in the two-cell mouse embryo is associated with destruction of maternally inherited transcripts, an important process for embryogenesis about which little is understood. We asked whether the Argonaute (Ago)/RNA-induced silencing complex, providing the mRNA "slicer" activity in gene silencing, might contribute to this process. Here we show that Ago2, 3, and 4 transcripts are contributed to the embryo maternally. By systematic knockdown of maternal Ago2, 3, and 4, individually and in combination, we find that only Ago2 is required for development beyond the two-cell stage. Knockdown of Ago2 stabilizes one set of maternal mRNAs and reduces zygotic transcripts of another set of genes. Ago2 is localized in mRNA-degradation P-bodies analogous to those that function in RNAi-like mechanisms in other systems. Profiling the expression of microRNAs throughout preimplantation development identified several candidates that could potentially work with Ago2 to mediate degradation of specific mRNAs. However, their low abundance raises the possibility that other endogenous siRNAs may also participate. Together, our results demonstrate that maternal expression of Ago2 is essential for the earliest stages of mouse embryogenesis and are compatible with the notion that degradation of a proportion of maternal messages involves the RNAi-machinery.
- Published
- 2008
- Full Text
- View/download PDF
46. In vivo imaging of oskar mRNA transport reveals the mechanism of posterior localization.
- Author
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Zimyanin VL, Belaya K, Pecreaux J, Gilchrist MJ, Clark A, Davis I, and St Johnston D
- Subjects
- Animals, Body Patterning, Drosophila Proteins metabolism, Drosophila melanogaster embryology, Drosophila melanogaster metabolism, Microtubules metabolism, Nuclear Proteins genetics, Nuclear Proteins metabolism, Oocytes chemistry, RNA, Messenger analysis, RNA-Binding Proteins genetics, RNA-Binding Proteins metabolism, Ribonucleoproteins analysis, Ribonucleoproteins metabolism, Tropomyosin genetics, Tropomyosin metabolism, Drosophila Proteins genetics, Drosophila melanogaster genetics, RNA Transport, RNA, Messenger metabolism
- Abstract
oskar mRNA localization to the posterior of the Drosophila oocyte defines where the abdomen and germ cells form in the embryo. Although this localization requires microtubules and the plus end-directed motor, kinesin, its mechanism is controversial and has been proposed to involve active transport to the posterior, diffusion and trapping, or exclusion from the anterior and lateral cortex. By following oskar mRNA particles in living oocytes, we show that the mRNA is actively transported along microtubules in all directions, with a slight bias toward the posterior. This bias is sufficient to localize the mRNA and is reversed in mago, barentsz, and Tropomyosin II mutants, which mislocalize the mRNA anteriorly. Since almost all transport is mediated by kinesin, oskar mRNA localizes by a biased random walk along a weakly polarized cytoskeleton. We also show that each component of the oskar mRNA complex plays a distinct role in particle formation and transport.
- Published
- 2008
- Full Text
- View/download PDF
47. Piwi and piRNAs act upstream of an endogenous siRNA pathway to suppress Tc3 transposon mobility in the Caenorhabditis elegans germline.
- Author
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Das PP, Bagijn MP, Goldstein LD, Woolford JR, Lehrbach NJ, Sapetschnig A, Buhecha HR, Gilchrist MJ, Howe KL, Stark R, Matthews N, Berezikov E, Ketting RF, Tavaré S, and Miska EA
- Subjects
- Animals, Argonaute Proteins, Caenorhabditis elegans genetics, Drosophila Proteins, Female, Gene Silencing, Genes, Helminth, Germ Cells growth & development, Male, Proteins genetics, RNA, Helminth metabolism, RNA-Induced Silencing Complex, Transposases metabolism, Caenorhabditis elegans metabolism, Caenorhabditis elegans Proteins metabolism, DNA Transposable Elements genetics, Germ Cells metabolism, Proteins metabolism, RNA, Small Interfering metabolism
- Abstract
The Piwi proteins of the Argonaute superfamily are required for normal germline development in Drosophila, zebrafish, and mice and associate with 24-30 nucleotide RNAs termed piRNAs. We identify a class of 21 nucleotide RNAs, previously named 21U-RNAs, as the piRNAs of C. elegans. Piwi and piRNA expression is restricted to the male and female germline and independent of many proteins in other small-RNA pathways, including DCR-1. We show that Piwi is specifically required to silence Tc3, but not other Tc/mariner DNA transposons. Tc3 excision rates in the germline are increased at least 100-fold in piwi mutants as compared to wild-type. We find no evidence for a Ping-Pong model for piRNA amplification in C. elegans. Instead, we demonstrate that Piwi acts upstream of an endogenous siRNA pathway in Tc3 silencing. These data might suggest a link between piRNA and siRNA function.
- Published
- 2008
- Full Text
- View/download PDF
48. Prdm1- and Sox6-mediated transcriptional repression specifies muscle fibre type in the zebrafish embryo.
- Author
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von Hofsten J, Elworthy S, Gilchrist MJ, Smith JC, Wardle FC, and Ingham PW
- Subjects
- Animals, Binding Sites, Cardiac Myosins genetics, Cell Differentiation, Chromatin Immunoprecipitation, Gene Expression Regulation, Developmental, Muscle Fibers, Fast-Twitch cytology, Muscle Fibers, Skeletal cytology, Muscle Fibers, Slow-Twitch cytology, Mutation genetics, Myosin Light Chains genetics, Organ Specificity, Positive Regulatory Domain I-Binding Factor 1, Promoter Regions, Genetic genetics, Up-Regulation genetics, Zebrafish genetics, DNA-Binding Proteins metabolism, Embryo, Nonmammalian metabolism, Muscle Fibers, Skeletal metabolism, Nuclear Proteins metabolism, Repressor Proteins metabolism, Transcription, Genetic, Zebrafish embryology, Zebrafish Proteins metabolism
- Abstract
The zebrafish u-boot (ubo) gene encodes the transcription factor Prdm1, which is essential for the specification of the primary slow-twitch muscle fibres that derive from adaxial cells. Here, we show that Prdm1 functions by acting as a transcriptional repressor and that slow-twitch-specific muscle gene expression is activated by Prdm1-mediated repression of the transcriptional repressor Sox6. Genes encoding fast-specific isoforms of sarcomeric proteins are ectopically expressed in the adaxial cells of ubo(tp39) mutant embryos. By using chromatin immunoprecipitation, we show that these are direct targets of Prdm1. Thus, Prdm1 promotes slow-twitch fibre differentiation by acting as a global repressor of fast-fibre-specific genes, as well as by abrogating the repression of slow-fibre-specific genes.
- Published
- 2008
- Full Text
- View/download PDF
49. Modelling and measuring single cell RNA expression levels find considerable transcriptional differences among phenotypically identical cells.
- Author
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Subkhankulova T, Gilchrist MJ, and Livesey FJ
- Subjects
- Animals, Mice, Neurons metabolism, Oligonucleotide Array Sequence Analysis, Polymerase Chain Reaction, RNA analysis, RNA genetics, RNA metabolism, RNA, Messenger genetics, RNA, Messenger metabolism, Reproducibility of Results, Gene Expression Profiling methods, Models, Genetic, Neurons cytology, Phenotype, Stem Cells metabolism, Transcription, Genetic
- Abstract
Background: Phenotypically identical cells demonstrate predictable, robust behaviours. However, there is uncertainty as to whether phenotypically identical cells are equally similar at the underlying transcriptional level or if cellular systems are inherently noisy. To answer this question, it is essential to distinguish between technical noise and true variation in transcript levels. A critical issue is the contribution of sampling effects, introduced by the requirement to globally amplify the single cell mRNA population, to observed measurements of relative transcript abundance., Results: We used single cell microarray data to develop simple mathematical models, ran Monte Carlo simulations of the impact of technical and sampling effects on single cell expression data, and compared these with experimental microarray data generated from single embryonic neural stem cells in vivo. We show that the actual distribution of measured gene expression ratios for pairs of neural stem cells is much broader than that predicted from our sampling effect model., Conclusion: Our results confirm that significant differences in gene expression levels exist between phenotypically identical cells in vivo, and that these differences exceed any noise contribution from global mRNA amplification.
- Published
- 2008
- Full Text
- View/download PDF
50. SNP-finding in pig mitochondrial ESTs.
- Author
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Scheibye-Alsing K, Cirera S, Gilchrist MJ, Fredholm M, and Gorodkin J
- Subjects
- Animals, Confidence Intervals, Gene Frequency, Genome, Humans, Species Specificity, Expressed Sequence Tags, Mitochondria genetics, Polymorphism, Single Nucleotide, Swine genetics
- Abstract
The Sino-Danish pig genome project produced 685 851 ESTs (Gorodkin et al. 2007), of which 41 499 originated from the mitochondrial genome. In this study, the mitochondrial ESTs were assembled, and 374 putative SNPs were found. Chromatograms for the ESTs containing SNPs were manually inspected, and 112 total (52 non-synonymous) SNPs were found to be of high confidence (five of them are close to disease-causing SNPs in humans). Nine of the high-confidence SNPs were tested experimentally, and eight were confirmed. The SNPs can be accessed online at http://pigest.ku.dk/more/mito.
- Published
- 2008
- Full Text
- View/download PDF
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