Transcription profiling of juvenile Daphnia manga following exposure to various doses of Lufenuron, a benzoylphenylurea growth inhibitor (using Message Amp II)
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ABSTRACT:
ORGANISM(S): Daphnia magna
SUBMITTER: Amanda Callaghan Christopher Hill
PROVIDER: E-TABM-797 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
Similar Datasets
Project description:Juvenile daphnia magna (<24hrs old, n=25 / test vessel) were exposed to methomyl (M) a carbamate insecticide (10.52ug/l), propanil (P), an analide herbicide (363ug/l), an independent action (IA) mixture of M (10.52ug/l and P (363ug/l) representing effect concentration 1 (EC1) for each chemical, based on standard immobility assay (OECD) or to control (untreated medium). All exposures were carried out in reconstituted water (see protocol GROWTH). Treated vessels were spiked with stock solutions of pesticides dissolved directly into reconstituted water. Test vessels were 1L glass beakers arranged in a randomised block design. Transcriptomic screening was used to assess the mechanism of toxicity of the two single pesticides and to explain (in part) the interaction of methomyl and propanil in an independent action (IA) EC1 mixture.
2010-09-15 | E-TABM-793 | biostudies-arrayexpress
Project description:Juvenile (<24hr old) Daphnia magna populations (n=250) were exposed (24hr) to Benzoylphenylurea growth inhibitor (14C labeled) lufenuron. Treatments were 0.15 ug/L, 0.33 ug/L, 0.65 ug/L lufenuron solvent control was 100 ul/L acetone carrier . Transcriptomic responses were screened using a D.magna cDNA microarray and the regulation of gene expression via log fold-change in transcript abundance, assessed. Genes involved in the synthesis of chitin were expected to respond to lufenuron, given the mode of action
2010-10-13 | E-TABM-798 | biostudies-arrayexpress
Project description:Microarray Construction The D. magna microarray was constructed using DNA fragments from three sources: i) Stress-specific cDNAs were generated using Selective Subtractive Hybridisation (SSH) on organisms exposed to the stressors shown in Table 1. SSH was performed with forward and reverse subtraction using a PCR-Select cDNA Subtraction Kit (Clontech Laboratories Inc, USA). cDNA was prepared from mRNA extracted from batches of 100 D. magna, 48 h old, using a Straight AM-CM-"M-BM-,s mRNA Isolation Kit System (Novagen Inc., UK) (2,029 clones). ii) ESTs obtained from a cDNA library from unexposed mixed aged organisms obtained from a 3-4 week old culture (Watanabe et al. 2005) (10,272 clones). iii) cDNAs isolated following an SSH, as above, between 15 adults carrying eggs and 75 juveniles (Soetaert et al. 2006) (1,143 clones). Gene fragment annotation Sequencing of the 3,172 SSH ESTs was carried out with M13-Long primers. Blast searches were performed on specific fragments that responded significantly to the exposure treatment. Sequences were annotated according to BlastX homology search against Uniprot Swissprot (http://www.ebi.ac.uk/swissprot) and Uniprot Trembl (invertebrate section, http://www.ebi.ac.uk/trembl) March 2006. Sequences were only annotated if they were found to have a Blast hit with the expect value smaller than 0.00001 and a score above 50. GeneBank/UniProt Accession number and speciesM-CM-"M-BM-, match were recorded with each annotation. We printed PCR products generated from the above, onto glass slides. All spots have been sequenced except for (iii), only a few available and some sequencing failed for various reasons. Therefore we have the sequences for around 90% of the spots. BLAST searches revealed a large proportion (nr 60%) had no significant match with existing entries in GenBank/Uniprot/Trembl. Because of low confidence we've not considered them, otherwise this would lead to huge and erroneous assumptions. All (i and ii) sequences are available either on GenBank or on our website. All the spots that came out as significant on our experiments were reviewed and only those that have been annotated, with significant BLAST match, are being published, with respective Accession numbers, species match and E-values. Fragments were therefore described as M-CM-"M-BM-,SunnanotatedM-CM-"M-BM-,M-CM-= where no significant difference was measured or M-CM-"M-BM-,Sno significant matchM-CM-"M-BM-,M-CM-= for those that were significant but no matches were found. Significant genes were annotated respectively as described above. Dose Reponse Tests were carried out using the developed microarray on cadmium, ph, lufenuron, calcium limitation and ibuprofen; details below: Cadmium: RNA was obtained from populations of 240 D. magna, 24-48 h old, exposed for 24 h to sublethal concentrations of cadmium chloride from 0, 10, 33 to 60 M-CM-^BM-BM-5g L-1 (corresponding to 0, 6.13, 20.24 and 36.79 M-CM-^BM-BM-5g L-1 Cd2+), in quadruplicates. pH: RNA was obtained from populations of 240 D. magna, 24-48 h old, exposed for 24 h to pH ranging from 8.0 (controls), 5.5, 5.3 and 5.2, in quadruplicates. pH was maintained by adding HCl on a daily basis. Lufenuron: RNA was obtained from populations of 240 D. magna, 24-48 h old, exposed for 24 h to sublethal concentrations of C14 labelled, lufenuron ranging from 0, 0.35, 0.59 to 1.00 ug L-1, in quadruplicates. Calcium: RNA was obtained from populations of 240 D. magna, 24-48 h old, exposed for 24 h to calcium chloride limitation ranging from 195.87 (culture condition), 20, 10 and 5 mg L-1 (CaCo3), in quadruplicates. Ibuprofen: RNA was obtained from populations of 300 D. magna, 24-48 h old, exposed for 24 h to ibuprofen (IB) sodium; 0 (control), 20, 40 and 80 mg L-1, in *quadruplicates. *One replicate at 80 mg L-1 was lost, thus this treatment was done in triplicate. IB was measured as the pharmacological active ingredient. Juvenile D. magna were used in order to restrict the response to genes associated with stress responses and growth, rather than genes associated with different stages of reproduction. Following exposure, daphnids were stored in RNAlater (Ambion, UK) at -80M-CM-^BM-BM-0C.
2007-11-05 | E-MAXD-20 | biostudies-arrayexpress
Project description:Regulation of milk synthesis and secretion is controlled mostly through local (intra-mammary) mechanisms. To gain insight into the molecular pathways comprising this response, an analysis of mammary gene expression was conducted in 12 lactating cows shifted from twice daily to once daily milking. Tissues were sampled by biopsy from adjacent mammary quarters of these animals during the two milking frequencies, allowing changes in gene expression to be assessed within each animal. Using bovine-specific, oligonucleotide arrays representing 21,495 unique transcripts, a range of differentially expressed genes were found as a result of less frequent milk removal. A total of 12 microarrays were used in this study. Each array was hybridized with RNA derived from a mammary sample taken at each biopsy time point, such that the twice-a-day milked sample and once-a-day milked sample were contained on the same slide for each animal (12 animals). Six of the microarrays represented animals previously shown to demonstrate a âlargeâ reduction in milk yield, whereas the other six arrays represented animals that previously displayed a âsmallâ reduction in milk yield when milked once per day. Half of each yield loss group (three animals) was represented by once-a-day milked/twice-daily-milked samples for Cy5/Cy3 labelling, with the other half labelled with the opposite dye configuration. This meant that six of 12 arrays were 'dye swapped', but with biological replicates as opposed to identical replicates.
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Project description:The insulin-like signaling (ILS) pathway regulates metabolism and is known to modulate adult lifespan in C. elegans. Altered stress responses and resistance to a wide range of stressors are also associated with changes in ILS and contribute to enhanced longevity. The transcription factors DAF-16 and HSF-1 are key effectors of the longevity phenotype. We demonstrate that increased intrinsic thermotolerance, due to lower ILS, is not dependent on stress induced HSF-1 transcriptional responses but instead requires active protein translation. Translation profiling experiments reveal genes that are post-transcriptionally regulated in response to altered ILS during heat shock in a DAF-16-dependent manner. Furthermore, several novel proteins are specifically required for ILS effects on thermotolerance. We propose that lowered-ILS results in DAF-16-induced metabolic and physiological changes that precondition a translational response to modulated survival under acute stress. 72 experimental samples were analyzed using custom oligo microarrays. A wild type sample pool was used as the Cy3 reference/control for all experimetal samples. All extracted RNA prior to array analysis was fractioned (via a sucrose gradient) based on ribosomal loading and pooled into ribosomal and free RNA (F1), light polysomes (F2) and heavy polysomes (F3) as described in the experimental procedures. The control RNAi is ‘empty’ vector L4440 RNAi feeding vector plasmid (1999 Firelab vector kit) transformed HT115(DE3), which was obtained from the Caenorhabditis Genetics Center (University of Wisconsin).
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Project description:Reducing protein synthesis slows growth and development but can increase adult lifespan. We demonstrate that knock-down of eukaryotic translation initiation factor 4G (eIF4G), which is down-regulated during starvation, results in differential translation of genes important for growth and longevity in C. elegans. Genome-wide mRNA translation state analysis showed that inhibition of IFG-1, the C. elegans ortholog of eIF4G, results in a relative increase in ribosomal loading and translation of stress response genes. Some of these genes are required for lifespan extension when IFG-1 is inhibited and are new determinants of longevity. Furthermore, enhanced ribosomal loading of certain mRNAs upon IFG-1 inhibition was correlated with increased mRNA length. This association was supported by changes in the proteome assayed via quantitative mass spectrometry. Our results support a role for IFG-1 in mediating the antagonistic effects on growth and somatic maintenance by modulating translation of a specific class of mRNA based on transcript length. 24 experimental samples were analyzed using custom oligo microarrays. A wild type sample pool was used as the Cy3 reference/control for all experimetal samples. All extracted RNA prior to array analysis was fractioned (via a sucrose gradient) based on ribosomal loading and pooled into ribosomal and free RNA (Fraction1), light polysomes (Fraction2) and heavy polysomes (Fraction3) as described in the experimental procedures. The control RNAi is ‘empty’ vector L4440 RNAi feeding vector plasmid (1999 Firelab vector kit) transformed HT115(DE3), which was obtained from the Caenorhabditis Genetics Center (University of Wisconsin).
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