Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Tiling path analysis of a library of transposon mutants generated in Salmonella enterica serovar Typhimurium strain SL1344 before and after infection in mice to identify mutants attenuated in vivo

ABSTRACT: A library of transposon mutants was generated in Salmonella enterica serovar Typhimurium strain SL1344 using custom Tn5 and Mu transposons with outward-facing T7 and SP6 promoters. The library was grown up in vitro, and used as the input pool for mouse infection experiments. An in vivo output pool was obtained from the mouse livers 2 days post-infection.

Genomic DNA was extracted from the input and output pools and digested with the restriction enzyme RsaI. Cy5-labelled RNA run-offs were produced seperately from the T7 and SP6 promoters and hybridised to a tiling microarray along with the Cy3-labelled in vitro transcription products from an untransposed SL1344 wild type control. Analysis of the microarray data allows the location of the transposon inserts to be determined. Mutants that are present in the input pool but that are absent or less prevalent in the output pools are inferred to be attenuated in vivo.

ORGANISM(S): Salmonella typhimurium

SUBMITTER: Roy Chaudhuri

PROVIDER: E-MEXP-2076 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Cellular responses to signalling pathways are often highly dynamic, however most analyses of developmental signalling pathways focus on a single endpoint. We have analyzed the temporal changes in transcription following a short Notch activation treatment and related these to the recruitment of the Notch pathway transcription factor, CSL [Suppressor of Hairless, Su(H), in Drosophila], and to the state of RNA Polymerase II (Pol II) binding. A total of 154 genes showed significant differential expression over time and their expression profiles stratified into 14 clusters based on temporal and quantitative differences in their responses. These differences were partially reflected in the profiles of Pol II and Su(H) binding. However, neither could fully account for the different response profiles. Furthermore, the timing of the different responses was unaffected by more prolonged Notch activation. Instead our data suggest that regulatory relationships between genes that segregate into different response clusters can partially account for the stratification. Thus, feed-forward repression, where products of early responding Enhancer of split bHLH genes (E(spl)bHLH) inhibit expression of endogenous repressors, is one mechanism that explains the profile of genes that exhibit delayed up-regulation. E(spl)bHLH genes may therefore be responsible for co-ordinating the Notch response of a wide spectrum of other targets, explaining their critical functions in many developmental and disease contexts. DmD8 cells were collected at 7 time points (0M-bM-^@M-^Y, 10M-bM-^@M-^Y, 20M-bM-^@M-^Y, 30M-bM-^@M-^Y, 40M-bM-^@M-^Y, 60M-bM-^@M-^Y and 100M-bM-^@M-^Y) after a 5 minute Notch stimulation as 3 independent replicates. Immunoprecipitation was performed with Su(H) antibody and compared to the total input DNA. Samples from replicates #1 and #2 were labelled with Cy5, and replicate #3 was labelled with Cy3 as a dye-swap. For time point 10M-bM-^@M-^Y, only 2 biological replicates were obtained.

Dataset Information

Tiling path analysis of a library of transposon mutants generated in Salmonella enterica serovar Typhimurium strain SL1344 before and after infection in mice to identify mutants attenuated in vivo

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