Publications by year
2020
Sidda JD, Song L, Parker JL, Studholme DJ, Sambles C, Grant M (2020). Diversity of secoiridoid glycosides in leaves of UK and Danish ash provide new insight for ash dieback management.
Scientific Reports,
10(1).
Abstract:
Diversity of secoiridoid glycosides in leaves of UK and Danish ash provide new insight for ash dieback management
© 2020, the Author(s). Secoiridoid glycosides are anti-feeding deterrents of the Oleaceae family recently highlighted as potential biomarkers in Danish ash trees to differentiate between those tolerant and susceptible to the fungal disease ash dieback. With the knowledge that emerald ash borer has recently entered Europe from Russia, and that extensive selection trials are ongoing in Europe for ash dieback tolerant European ash (Fraxinus excelsior), we undertook comprehensive screening of secoiridoid glycosides in leaf extracts of trees tolerant and susceptible to ash dieback sampled from sites in the UK and Denmark. Here we report an unexpected diversity of secoiridoid glycosides in UK trees and higher levels of secoiridoid glycosides in the UK sample group. While it is unlikely that secoiridoid glycosides generally can serve as reliable markers for ash dieback susceptibility, there are differences between tolerant and susceptible groups for specific secoiridoids. We predict that the high levels—and structural diversity—of secoiridoids present in the UK group may provide a robust reservoir of anti-feeding deterrents to mitigate future herbivore threats such as the Emerald ash borer.
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Guo Y, Dupont P-Y, Mesarich CH, Yang B, McDougal RL, Panda P, Dijkwel P, Studholme DJ, Sambles C, Win J, et al (2020). Functional analysis of RXLR effectors from the New Zealand kauri dieback pathogen Phytophthora agathidicida.
Mol Plant Pathol,
21(9), 1131-1148.
Abstract:
Functional analysis of RXLR effectors from the New Zealand kauri dieback pathogen Phytophthora agathidicida.
New Zealand kauri is an ancient, iconic, gymnosperm tree species that is under threat from a lethal dieback disease caused by the oomycete Phytophthora agathidicida. To gain insight into this pathogen, we determined whether proteinaceous effectors of P. agathidicida interact with the immune system of a model angiosperm, Nicotiana, as previously shown for Phytophthora pathogens of angiosperms. From the P. agathidicida genome, we defined and analysed a set of RXLR effectors, a class of proteins that typically have important roles in suppressing or activating the plant immune system. RXLRs were screened for their ability to activate or suppress the Nicotiana plant immune system using Agrobacterium tumefaciens transient transformation assays. Nine P. agathidicida RXLRs triggered cell death or suppressed plant immunity in Nicotiana, of which three were expressed in kauri. For the most highly expressed, P. agathidicida (Pa) RXLR24, candidate cognate immune receptors associated with cell death were identified in Nicotiana benthamiana using RNA silencing-based approaches. Our results show that RXLRs of a pathogen of gymnosperms can interact with the immune system of an angiosperm species. This study provides an important foundation for studying the molecular basis of plant-pathogen interactions in gymnosperm forest trees, including kauri.
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Sambles C, Venkatesan L, Shittu OM, Harrison J, Moore K, Tripathi L, Grant M, Warmington R, Studholme DJ (2020). Genome sequencing data for wild and cultivated bananas, plantains and abacá.
Data in Brief,
33Abstract:
Genome sequencing data for wild and cultivated bananas, plantains and abacá
© 2020 the Author(s) We performed shotgun genome sequencing on a total of 19 different Musa genotypes including representatives of wild banana species Musa acuminata and M. balibisiana, allopolyploid bananas and plantains, Fe'i banana, pink banana (also known as hairy banana) and abacá (also known as hemp banana). We aligned sequence reads against a previously sequenced reference genome and assessed ploidy and, in the case of allopolyploids, the contributions of the a and B genomes; this provides important quality-assurance data about the taxonomic identities of the sequenced plant material. These data will be useful for phylogenetics, crop improvement, studies of the complex story of intergenomic recombination in AAB and ABB allotriploid bananas and plantains and can be integrated into resources such as the Banana Genome Hub.
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2019
Studholme DJ, Panda P, Sanfuentes Von Stowasser E, González M, Hill R, Sambles C, Grant M, Williams NM, McDougal RL (2019). Genome sequencing of oomycete isolates from Chile supports the New Zealand origin of Phytophthora kernoviae and makes available the first Nothophytophthora sp. genome.
Mol Plant Pathol,
20(3), 423-431.
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Genome sequencing of oomycete isolates from Chile supports the New Zealand origin of Phytophthora kernoviae and makes available the first Nothophytophthora sp. genome.
Genome sequences were generated for six oomycete isolates collected from forests in Valdivia, Chile. Three of the isolates were identified morphologically as Phytophthora kernoviae, whereas two were similar to other clade 10 Phytophthora species. One isolate was tentatively identified as Nothophytophthora valdiviana based on nucleotide sequence similarity in the cytochrome oxidase 1 gene. This is the first genome sequence for this recently described genus. The genome assembly was more fragmented and contained many duplicated genes when compared with the other Phytophthora sequences. Comparative analyses were performed with genomic sequences of the P. kernoviae isolates from the UK and New Zealand. Although the potential New Zealand origin of P. kernoviae has been suggested, new isolations from Chile had cast doubt on this hypothesis. We present evidence supporting P. kernoviae as having originated in New Zealand. However, investigation of the diversity of oomycete species in Chile has been limited and warrants further exploration. We demonstrate the expediency of genomic analyses in determining phylogenetic relationships between isolates within new and often scantly represented taxonomic groups, such as Phytophthora clade 10 and Nothophytophthora. Data are available on GenBank via BioProject accession number PRJNA352331.
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Tennant RK, Lux T, Sambles C, Kuhn N, Petticrew E, Oldfield R, Parker D, Hatton J, Moore K, Lee R, et al (2019). Palaeogenomics of the Hydrocarbon Producing Microalga Botryococcus braunii.
Scientific Reports,
9 Full text.
2017
Sambles CM, Salmon DL, Florance H, Howard TP, Smirnoff N, Nielsen LR, McKinney LV, Kjær ED, Buggs RJA, Studholme DJ, et al (2017). Ash leaf metabolomes reveal differences between trees tolerant and susceptible to ash dieback disease.
Sci Data,
4Abstract:
Ash leaf metabolomes reveal differences between trees tolerant and susceptible to ash dieback disease.
European common ash, Fraxinus excelsior, is currently threatened by Ash dieback (ADB) caused by the fungus, Hymenoscyphus fraxineus. To detect and identify metabolites that may be products of pathways important in contributing to resistance against H. fraxineus, we performed untargeted metabolomic profiling on leaves from five high-susceptibility and five low-susceptibility F. excelsior individuals identified during Danish field trials. We describe in this study, two datasets. The first is untargeted LC-MS metabolomics raw data from ash leaves with high-susceptibility and low-susceptibility to ADB in positive and negative mode. These data allow the application of peak picking, alignment, gap-filling and retention-time correlation analyses to be performed in alternative ways. The second, a processed dataset containing abundances of aligned features across all samples enables further mining of the data. Here we illustrate the utility of this dataset which has previously been used to identify putative iridoid glycosides, well known anti-herbivory terpenoid derivatives, and show differential abundance in tolerant and susceptible ash samples.
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Sambles C (2017). Genome annotation of the oleaginous yeast Rhodotorula toruloides strain CGMCC 2.1609.
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Genome annotation of the oleaginous yeast Rhodotorula toruloides strain CGMCC 2.1609
Genome annotation of the oleaginous yeast Rhodotorula toruloides strain CGMCC 2.1609 as a GFF file. Genome sequence and further information is available on NCBI.
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Sollars ESA, Harper AL, Kelly LJ, Sambles CM, Ramirez-Gonzalez RH, Swarbreck D, Kaithakottil G, Cooper ED, Uauy C, Havlickova L, et al (2017). Genome sequence and genetic diversity of European ash trees.
Nature,
541(7636), 212-216.
Abstract:
Genome sequence and genetic diversity of European ash trees.
Ash trees (genus Fraxinus, family Oleaceae) are widespread throughout the Northern Hemisphere, but are being devastated in Europe by the fungus Hymenoscyphus fraxineus, causing ash dieback, and in North America by the herbivorous beetle Agrilus planipennis. Here we sequence the genome of a low-heterozygosity Fraxinus excelsior tree from Gloucestershire, UK, annotating 38,852 protein-coding genes of which 25% appear ash specific when compared with the genomes of ten other plant species. Analyses of paralogous genes suggest a whole-genome duplication shared with olive (Olea europaea, Oleaceae). We also re-sequence 37 F. excelsior trees from Europe, finding evidence for apparent long-term decline in effective population size. Using our reference sequence, we re-analyse association transcriptomic data, yielding improved markers for reduced susceptibility to ash dieback. Surveys of these markers in British populations suggest that reduced susceptibility to ash dieback may be more widespread in Great Britain than in Denmark. We also present evidence that susceptibility of trees to H. fraxineus is associated with their iridoid glycoside levels. This rapid, integrated, multidisciplinary research response to an emerging health threat in a non-model organism opens the way for mitigation of the epidemic.
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Sambles C, Middelhaufe S, Soanes D, Kolak D, Lux T, Moore K, Matoušková P, Parker D, Lee R, Love J, et al (2017). Genome sequence of the oleaginous yeast Rhodotorula toruloides strain CGMCC 2.1609.
Genomics Data,
13, 1-2.
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Tennant RK, Sambles CM, Diffey GE, Moore KA, Love J (2017). Metagenomic Analysis of Silage.
Journal of Visualized Experiments,
119, e54936-e54936.
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Sambles C, Moore K, Lux TM, Jones K, Littlejohn GR, Gouveia JD, Aves SJ, Studholme DJ, Lee R, Love J, et al (2017). Metagenomic analysis of the complex microbial consortium associated with cultures of the oil-rich alga. Botryococcus braunii.
MicrobiologyOpen, e00482-e00482.
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2016
Jones KJ, Moore K, Sambles C, Love J, Studholme DJ, Aves SJ (2016). Draft Genome Sequences of Achromobacter piechaudii GCS2, Agrobacterium sp. Strain SUL3, Microbacterium sp. Strain GCS4, Shinella sp. Strain GWS1, and Shinella sp. Strain SUS2 Isolated from Consortium with the Hydrocarbon-Producing Alga Botryococcus braunii.
Genome Announc,
4(1).
Abstract:
Draft Genome Sequences of Achromobacter piechaudii GCS2, Agrobacterium sp. Strain SUL3, Microbacterium sp. Strain GCS4, Shinella sp. Strain GWS1, and Shinella sp. Strain SUS2 Isolated from Consortium with the Hydrocarbon-Producing Alga Botryococcus braunii.
A variety of bacteria associate with the hydrocarbon-producing microalga Botryococcus braunii, some of which may influence its growth. We report here the genome sequences for Achromobacter piechaudii GCS2, Agrobacterium sp. strain SUL3, Microbacterium sp. strain GCS4, and Shinella sp. strains GWS1 and SUS2, isolated from a laboratory culture of B. braunii, race B, strain Guadeloupe.
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Studholme DJ, McDougal RL, Sambles C, Hansen E, Hardy G, Grant M, Ganley RJ, Williams NM (2016). Genome sequences of six Phytophthora species associated with forests in New Zealand.
Genomics Data,
7, 54-56.
Abstract:
Genome sequences of six Phytophthora species associated with forests in New Zealand
© 2015 the Authors. In New Zealand there has been a long association of Phytophthora diseases in forests, nurseries, remnant plantings and horticultural crops. However, new Phytophthora diseases of trees have recently emerged. Genome sequencing has been performed for 12 Phytophthora isolates, from six species: Phytophthora pluvialis, Phytophthora kernoviae, Phytophthora cinnamomi, Phytophthora agathidicida, Phytophthora multivora and Phytophthora taxon Totara. These sequences will enable comparative analyses to identify potential virulence strategies and ultimately facilitate better control strategies. This Whole Genome Shotgun data have been deposited in DDBJ/ENA/GenBank under the accession numbers LGTT00000000, LGTU00000000, JPWV00000000, JPWU00000000, LGSK00000000, LGSJ00000000, LGTR00000000, LGTS00000000, LGSM00000000, LGSL00000000, LGSO00000000, and LGSN00000000.
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Pirbadian S, Barchinger SE, Subramanian P, Sambles CM, Baker CS, Burroughs NJ, Jensen GJ, Golbeck JH, El-Naggar MY (2016). Multiheme Cytochromes and the Bacterial Nanowires of Shewanella oneidensis MR-1: Regulation, Structure, and Extracellular Electron Transport Mechanisms.
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Barchinger SE, Pirbadian S, Sambles C, Baker CS, Leung KM, Burroughs NJ, El-Naggar MY, Golbeck JH (2016). Regulation of Gene Expression in Shewanella oneidensis MR-1 during Electron Acceptor Limitation and Bacterial Nanowire Formation.
Appl Environ Microbiol,
82(17), 5428-5443.
Abstract:
Regulation of Gene Expression in Shewanella oneidensis MR-1 during Electron Acceptor Limitation and Bacterial Nanowire Formation.
UNLABELLED: in limiting oxygen as an electron acceptor, the dissimilatory metal-reducing bacterium Shewanella oneidensis MR-1 rapidly forms nanowires, extensions of its outer membrane containing the cytochromes MtrC and OmcA needed for extracellular electron transfer. RNA sequencing (RNA-Seq) analysis was employed to determine differential gene expression over time from triplicate chemostat cultures that were limited for oxygen. We identified 465 genes with decreased expression and 677 genes with increased expression. The coordinated increased expression of heme biosynthesis, cytochrome maturation, and transport pathways indicates that S. oneidensis MR-1 increases cytochrome production, including the transcription of genes encoding MtrA, MtrC, and OmcA, and transports these decaheme cytochromes across the cytoplasmic membrane during electron acceptor limitation and nanowire formation. In contrast, the expression of the mtrA and mtrC homologs mtrF and mtrD either remains unaffected or decreases under these conditions. The ompW gene, encoding a small outer membrane porin, has 40-fold higher expression during oxygen limitation, and it is proposed that OmpW plays a role in cation transport to maintain electrical neutrality during electron transfer. The genes encoding the anaerobic respiration regulator cyclic AMP receptor protein (CRP) and the extracytoplasmic function sigma factor RpoE are among the transcription factor genes with increased expression. RpoE might function by signaling the initial response to oxygen limitation. Our results show that RpoE activates transcription from promoters upstream of mtrC and omcA the transcriptome and mutant analyses of S. oneidensis MR-1 nanowire production are consistent with independent regulatory mechanisms for extending the outer membrane into tubular structures and for ensuring the electron transfer function of the nanowires. IMPORTANCE: Shewanella oneidensis MR-1 has the capacity to transfer electrons to its external surface using extensions of the outer membrane called bacterial nanowires. These bacterial nanowires link the cell's respiratory chain to external surfaces, including oxidized metals important in bioremediation, and explain why S. oneidensis can be utilized as a component of microbial fuel cells, a form of renewable energy. In this work, we use differential gene expression analysis to focus on which genes function to produce the nanowires and promote extracellular electron transfer during oxygen limitation. Among the genes that are expressed at high levels are those encoding cytochrome proteins necessary for electron transfer. Shewanella coordinates the increased expression of regulators, metabolic pathways, and transport pathways to ensure that cytochromes efficiently transfer electrons along the nanowires.
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Bechtold U, Penfold CA, Jenkins DJ, Legaie R, Moore JD, Lawson T, Matthews JSA, Vialet-Chabrand SRM, Baxter L, Subramaniam S, et al (2016). Time-Series Transcriptomics Reveals That AGAMOUS-LIKE22 Affects Primary Metabolism and Developmental Processes in Drought-Stressed Arabidopsis.
Plant Cell,
28(2), 345-366.
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Time-Series Transcriptomics Reveals That AGAMOUS-LIKE22 Affects Primary Metabolism and Developmental Processes in Drought-Stressed Arabidopsis.
In Arabidopsis thaliana, changes in metabolism and gene expression drive increased drought tolerance and initiate diverse drought avoidance and escape responses. To address regulatory processes that link these responses, we set out to identify genes that govern early responses to drought. To do this, a high-resolution time series transcriptomics data set was produced, coupled with detailed physiological and metabolic analyses of plants subjected to a slow transition from well-watered to drought conditions. A total of 1815 drought-responsive differentially expressed genes were identified. The early changes in gene expression coincided with a drop in carbon assimilation, and only in the late stages with an increase in foliar abscisic acid content. To identify gene regulatory networks (GRNs) mediating the transition between the early and late stages of drought, we used Bayesian network modeling of differentially expressed transcription factor (TF) genes. This approach identified AGAMOUS-LIKE22 (AGL22), as key hub gene in a TF GRN. It has previously been shown that AGL22 is involved in the transition from vegetative state to flowering but here we show that AGL22 expression influences steady state photosynthetic rates and lifetime water use. This suggests that AGL22 uniquely regulates a transcriptional network during drought stress, linking changes in primary metabolism and the initiation of stress responses.
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2015
Sambles C, Schlenzig A, O'Neill P, Grant M, Studholme DJ (2015). Draft genome sequences of Phytophthora kernoviae and Phytophthora ramorum lineage EU2 from Scotland.
Genomics Data,
6, 193-194.
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Sambles CM, White DA (2015). Genome Sequence of Rhodococcus sp. Strain PML026, a Trehalolipid Biosurfactant Producer and Biodegrader of Oil and Alkanes.
Genome Announc,
3(3).
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Genome Sequence of Rhodococcus sp. Strain PML026, a Trehalolipid Biosurfactant Producer and Biodegrader of Oil and Alkanes.
Rhodococcus sp. strain PML026 produces an array of trehalolipid biosurfactant compounds in order to utilize hydrophobic carbon sources, such as oils and alkanes. Here, we report the high-quality draft genome sequence of this strain, which has a total length of 5,168,404 bp containing 4,835 protein-coding sequences, 12 rRNAs, and 45 tRNAs.
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Sharma R, Xia X, Cano LM, Evangelisti E, Kemen E, Judelson H, Oome S, Sambles C, van den Hoogen DJ, Kitner M, et al (2015). Genome analyses of the sunflower pathogen Plasmopara halstedii provide insights into effector evolution in downy mildews and Phytophthora.
BMC Genomics,
16Abstract:
Genome analyses of the sunflower pathogen Plasmopara halstedii provide insights into effector evolution in downy mildews and Phytophthora.
BACKGROUND: Downy mildews are the most speciose group of oomycetes and affect crops of great economic importance. So far, there is only a single deeply-sequenced downy mildew genome available, from Hyaloperonospora arabidopsidis. Further genomic resources for downy mildews are required to study their evolution, including pathogenicity effector proteins, such as RxLR effectors. Plasmopara halstedii is a devastating pathogen of sunflower and a potential pathosystem model to study downy mildews, as several Avr-genes and R-genes have been predicted and unlike Arabidopsis downy mildew, large quantities of almost contamination-free material can be obtained easily. RESULTS: Here a high-quality draft genome of Plasmopara halstedii is reported and analysed with respect to various aspects, including genome organisation, secondary metabolism, effector proteins and comparative genomics with other sequenced oomycetes. Interestingly, the present analyses revealed further variation of the RxLR motif, suggesting an important role of the conservation of the dEER-motif. Orthology analyses revealed the conservation of 28 RxLR-like core effectors among Phytophthora species. Only six putative RxLR-like effectors were shared by the two sequenced downy mildews, highlighting the fast and largely independent evolution of two of the three major downy mildew lineages. This is seemingly supported by phylogenomic results, in which downy mildews did not appear to be monophyletic. CONCLUSIONS: the genome resource will be useful for developing markers for monitoring the pathogen population and might provide the basis for new approaches to fight Phytophthora and downy mildew pathogens by targeting core pathogenicity effectors.
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2014
Airs RL, Temperton B, Sambles C, Farnham G, Skill SC, Llewellyn CA (2014). Chlorophyll f and chlorophyll d are produced in the cyanobacterium Chlorogloeopsis fritschii when cultured under natural light and near-infrared radiation.
FEBS Letters, ---.
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Chlorophyll f and chlorophyll d are produced in the cyanobacterium Chlorogloeopsis fritschii when cultured under natural light and near-infrared radiation
Abstract We report production of chlorophyll f and chlorophyll d in the cyanobacterium Chlorogloeopsis fritschii cultured under near-infrared and natural light conditions. C. fritschii produced chlorophyll f and chlorophyll d when cultured under natural light to a high culture density in a 20 L bubble column photobioreactor. In the laboratory, the ratio of chlorophyll f to chlorophyll a changed from 1:15 under near-infrared, to an undetectable level of chlorophyll f under artificial white light. The results provide support that chlorophylls f and d are both red-light inducible chlorophylls in C. fritschii.
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Wagley S, Newcombe J, Laing E, Yusuf E, Sambles CM, Studholme DJ, La Ragione RM, Titball RW, Champion OL (2014). Differences in carbon source utilisation distinguish Campylobacter jejuni from Campylobacter coli.
BMC Microbiol,
14Abstract:
Differences in carbon source utilisation distinguish Campylobacter jejuni from Campylobacter coli.
BACKGROUND: Campylobacter jejuni and C. coli are human intestinal pathogens that are the most frequent causes of bacterial foodborne gastroenteritis in humans in the UK. In this study, we aimed to characterise the metabolic diversity of both C. jejuni and C. coli using a diverse panel of clinical strains isolated from the UK, Pakistan and Thailand, thereby representing both the developed and developing world. Our aim was to apply multi genome analysis and Biolog phenotyping to determine differences in carbon source utilisation by C. jejuni and C. coli strains. RESULTS: We have identified a core set of carbon sources (utilised by all strains tested) and a set that are differentially utilised for a diverse panel of thirteen C. jejuni and two C. coli strains. This study used multi genome analysis to show that propionic acid is utilised only by C. coli strains tested. A broader PCR screen of 16 C. coli strains and 42 C. jejuni confirmed the absence of the genes needed for propanoate metabolism. CONCLUSIONS: from our analysis we have identified a phenotypic method and two genotypic methods based on propionic utilisation that might be applicable for distinguishing between C. jejuni and C. coli.
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2011
Jameson E, Mann NH, Joint I, Sambles C, Mühling M (2011). The diversity of cyanomyovirus populations along a North-South Atlantic Ocean transect.
ISME J,
5(11), 1713-1721.
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The diversity of cyanomyovirus populations along a North-South Atlantic Ocean transect.
Viruses that infect the marine cyanobacterium Prochlorococcus have the potential to impact the growth, productivity, diversity and abundance of their hosts. In this study, changes in the microdiversity of cyanomyoviruses were investigated in 10 environmental samples taken along a North-South Atlantic Ocean transect using a myoviral-specific PCR-sequencing approach. Phylogenetic analyses of 630 viral g20 clones from this study, with 786 published g20 sequences, revealed that myoviral populations in the Atlantic Ocean had higher diversity than previously reported, with several novel putative g20 clades. Some of these clades were detected throughout the Atlantic Ocean. Multivariate statistical analyses did not reveal any significant correlations between myoviral diversity and environmental parameters, although myoviral diversity appeared to be lowest in samples collected from the north and south of the transect where Prochlorococcus diversity was also lowest. The results were correlated to the abundance and diversity of the co-occurring Prochlorococcus and Synechococcus populations, but revealed no significant correlations to either of the two potential host genera. This study provides evidence that cyanophages have extremely high and variable diversity and are distributed over large areas of the Atlantic Ocean.
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2008
Ford AT, Sambles C, Kille P (2008). Intersexuality in crustaceans: genetic, individual and population level effects.
Mar Environ Res,
66(1), 146-148.
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Intersexuality in crustaceans: genetic, individual and population level effects.
Scientists investigating toxicants such as endocrine disrupting chemicals (EDCs) at the cellular at the sub-cellular level are often faced with criticisms as to how these effects can be extrapolated to the level of individuals and their populations. This report aims to provide an overview of the studies undertaken on crustacean model, Echinogammarus marinus LEACH (AMPHIPODA), and intersex phenotypes, at the individual and population levels, and provide additional emergent data at the genomic level. These, normal and intersex, males and females have been investigated by cross-hybridisation microarray analysis and specific sexually dimorphic genes and corresponding properties identified between each sexual phenotype. The morphology, physiology and histology of these intersexes have been investigated in detail and a number of reproductive costs have been identified including reduced fecundity and fertility. These costs have been incorporated into a population model and simulated over a ten-year period to ascertain how different levels of intersexuality affect the stability of populations. Based on the information gained through study of intersex models (with known endocrine dysfunction) together with the substantial quantity of historical data relating to effects of chemicals on amphipod fecundity, growth and mortality, the development of appropriate biomarkers is nearer to being assessed from the level of genes to that of the population.
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2003
Blagg SL, Stewart M, Sambles C, Insall RH (2003). PIR121 regulates pseudopod dynamics and SCAR activity in Dictyostelium.
Curr Biol,
13(17), 1480-1487.
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PIR121 regulates pseudopod dynamics and SCAR activity in Dictyostelium.
BACKGROUND: the WASP/SCAR family of adaptor proteins coordinates actin reorganization by coupling different signaling molecules, including Rho-family GTPases, to the activation of the Arp2/3 complex. WASP binds directly to Cdc42 through its GTPase binding domain (GBD), but SCAR does not contain a GBD, and no direct binding has been found. However, SCAR has recently been found to copurify with four other proteins in a complex. One of these, PIR121, binds directly to Rac. RESULTS: We have identified four of the members of this complex in Dictyostelium and disrupted the pirA gene, which encodes PIR121. The resulting mutant cells are unusually large, maintain an excessive proportion of their actin in a polymerized state and display severe defects in movement and chemotaxis. They also continually extend new pseudopods by widening and splitting existing leading edges rather than by initiating new pseudopods. Comparing these cells to scar null mutants shows behavior that is broadly consistent with overactivation of SCAR. Deletion of the pirA gene in a scar(-) mutant resulted in cells resembling their scar(-) parents with no obvious changes, confirming that PIR121 mainly acts through SCAR in vivo. Surprisingly given their hyperactive phenotype, we find that pirA(-) mutants contain very little intact SCAR protein despite normal levels of mRNA, suggesting a posttranscriptional downregulation of activated SCAR. CONCLUSIONS: Our results demonstrate a genetic connection between the pirA and scar genes. PIR121 appears to inhibit the activity of SCAR in the absence of activating signals. The location of the newly formed protrusions indicates that unregulated SCAR is acting at the edges of existing pseudopods, not elsewhere in the cell. We suggest that active SCAR protein released from the inhibitory complex is rapidly removed and that this is an important and novel mechanism for controlling actin dynamics.
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