Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Expression analysis of Sphingomonas paucimobilis sp. EPA505 during biodegradation kinetics of various PAHs and detection of gene in a contaminated soil S3

ABSTRACT: Studying eight enzymes involved in the degradation of polycyclic aromatic hydrocarbons from the model strain Sphingomonas paucimobilis sp. EPA505 using a designed microarray of 8,048 probes. During the biodegradation kinetics with phenanthrene, fluoranthene or a mix of both pollutants, we identified the targeted set of genes induced by these pollutants, compared to basal expression detected with glucose. Hybridizing total DNA extracted from S3, we show the efficiency of our probe design to study a complex environment. Despite the relative small size of our probes (23-mers), their sensitivity is reliable as we can detect the presence of genes in this complex mixture. Obtained results are further described in Sébastien Terrat, Eric Peyretaillade, Olivier Gonçalves, Eric Dugat-Bony, Fabrice Gravelat, and Pierre Peyret. 2010 - Studying the ‘Unkown’ with Metabolic Design, a new probe design software for explorative functional microarrays development. Nucleic Acids Research (submited). A 17 chip study was realized using total RNA recovered from separate cultures of Sphingomonas paucimobilis sp. EPA505 with phenanthrene, fluoranthene or a mix of these both pollutants as sole carbon and energy source. A negative kinetic expermient was realized with glucose as sole carbon and energy source. Each chip measures the expression level of 8 genes from Sphingomonas paucimobilis sp. EPA505 with 23-mer probes (a total of 8,048 probes) using a new design approach. We also assess metabolic capacities of microbial communities in an aromatic hydrocarbons contaminated soil named S3. Each probe was spotted in triplicate, and a total of 8,863 random probes was used to determine the background noise.

ORGANISM(S): Sphingomonas paucimobilis

SUBMITTER: TERRAT Sebastien

PROVIDER: E-GEOD-21402 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Detecting variants with Metabolic Design, a new software tool to design probes for explorative functional DNA microarray development.

Terrat Sébastien S Peyretaillade Eric E Gonçalves Olivier O Dugat-Bony Eric E Gravelat Fabrice F Moné Anne A Biderre-Petit Corinne C Boucher Delphine D Troquet Julien J Peyret Pierre P

BMC bioinformatics 20100923

<h4>Background</h4>Microorganisms display vast diversity, and each one has its own set of genes, cell components and metabolic reactions. To assess their huge unexploited metabolic potential in different ecosystems, we need high throughput tools, such as functional microarrays, that allow the simultaneous analysis of thousands of genes. However, most classical functional microarrays use specific probes that monitor only known sequences, and so fail to cover the full microbial gene diversity pres ...[more]

PMID: 20860850

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Project description:CD4+ ‘helper’ T cells (TH) are pivotal for the generation and maintenance of CD8+ T cell responses. ‘Helped’ CD8+ T cells receive signals during priming that prevent the induction of the pro-apoptotic molecule TRAIL during reactivation, thereby enabling robust secondary expansion. Conversely, ‘helpless’ CD8+ T cells primed in the absence of TH induce TRAIL expression upon restimulation and undergo activation-induced cell death (AICD). We have investigated the molecular basis for the differential regulation of TRAIL in helped versus helpless CD8+ T cells by comparing their transcriptional profiles, and have identified a transcriptional corepressor, NGFI-A binding protein 2 (Nab2) that is selectively induced in helped CD8+ T cells. Enforced expression of Nab2 prevents TRAIL induction upon restimulation of primary helpless CD8+ T cells, and expression of a dominant-negative form of Nab2 in helped CD8+ T cells impairs their secondary proliferative response that is reversible by TRAIL blockade. Finally, we observe that the CD8+ T cell autocrine growth factor interleukin-2 (IL-2) coordinately increases Nab2 expression and decreases TRAIL expression. These findings identify Nab2 as a mediator of TH-dependent CD8+ T cell memory responses through regulation of TRAIL and promotion of secondary expansion, and suggest a mechanism through which this operates. Helped and helpless E1B192-200-specific CD8+ T cells were generated as previously described16. Briefly, C57BL/6J mice were treated with 100 ug GK1.5 intraperitoneally (helpless) or left untreated (helped) prior to subcutaneous immunization with 1x107 irradiated (3000 rad) TAP-/-Ad5E1-MEC. 3 days post immunization, all mice were treated with 100 ug GK1.5. Spleens and draining lymph nodes were harvested 7 days post immunization, and CD8+ T cells were purified with the CD8 negative isolation kit (Miltenyi) according to the manufacturer’s protocol. Purity was 88-95%. Microarray and data analysis Purified CD8+ T cells from helped and helpless mice were sorted on CD44hi expression by flow cytometry. Cells were restimulated for 4h with 2ug/ml E1B192-200 peptide in the presence of 20uM qVD-OPh (R&D Systems). RNA was extracted using TriZol (Gibco BRL) according to the manufacturer's instructions. 5ug of total RNA was used to generate cDNA with RT Superscript III (Invitrogen). cDNA was indirectly labeled using Cyanine 3 and Cyanine 5 (Amersham). Labeled samples were purified and spotted in an equimolar ratio on the Mouse Exonic Evidence Based Oligonucleotide (MEEBO) array. Images were preview scanned on an Axon 4000B and channels were balanced until an overall Cy5/Cy3 ratio of 1 was achieved before a final data scan at 5µm was performed in GenePix. The images were manually gridded, and then saved for analysis. Data were analysed using Spotfire Decision Site. Gene Ontology terms were downloaded from BioMart <http://www.biomart.org> version NCBI37, 09 April 2009. Genes with Biological Process term "regulation of transcription" (GO:0045449) were assumed to encode transcription factors. Of 276 probes with an absolute log2 ratio >2, 231 probes were present in BioMart; the remaining cDNA probes did not match currently known genes.

Dataset Information

Expression analysis of Sphingomonas paucimobilis sp. EPA505 during biodegradation kinetics of various PAHs and detection of gene in a contaminated soil S3

Publications

Detecting variants with Metabolic Design, a new software tool to design probes for explorative functional DNA microarray development.

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