Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Comparison of gene expression profiles of E. coli W3110 normal colonies vs. L-form colonies

ABSTRACT: Expression data comparing typical E.coli colonies grown on BHI and BHI+Sucrose+MgSO4 to L-form colonies grown on BHI+Sucrose+MgSO4+Penicillin G. Keywords: one time point DNA microarray analysis of E.coli colonies grown on BHI and BHI+Sucrose+MgSO4 (controls) with L-form colonies grown on BHI+Sucrose+MgSO4+Penicillin G (experimental). An isolated colony of fresh W3110 was grown for 5hrs to log phase, dilutions of the inoculmn were spread onto three media: BHI, BHI+Sucrose+MgSO4, and BHI+Sucrose+MgSO4+Penicillin G to yield isolated colonies. Isolated control colonies which grow overnight were harvested the next day while isolated L-form colonies which do not grow until 48-72 hrs were harvested after 72hrs. All colonies were harvest with a PBS moistened sterile swab and dipped into a sterile microcentrifuge tube containing 500ul of PBS. RNA was isolated from pelleted cells using MasterPure RNA Purification Kit and reverse transcribed for making probes for array hybridization on E.coli Genome 2.0 Affymetrix microarrays.

ORGANISM(S): Escherichia coli str. K-12 substr. W3110

SUBMITTER: Yanqin Yang

PROVIDER: E-GEOD-14796 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:We investigated the influence of genome position on propensity to amplify. First, we integrated a mutant form of DHFR into different positions in the human genome, challenged cells with methotrexate and then studied the genomic alterations arising in drug resistant cells. We observed site specific differences in methotrexate sensitivity, organization of amplicons and amplification frequency. One site was uniquely associated with a significantly enhanced propensity to amplify and recurrent amplicon boundaries, possibly implicating a rare folate sensitive fragile site in initiating amplification. Hierarchical clustering of gene expression patterns and subsequent gene enrichment analysis revealed two clusters differing significantly in expression of MYC target genes independent of integration site. We introduced a mutant form of DHFR (L22F), which confers greater resistance to methotrexate than the wild type (endogenous) gene into HCT116+chr3 cells and isolated independent clones containing DHFR* at different positions in the genome and identified genome sequences flanking the integration site of DHFR* using inverse PCR. For further analysis, we selected only clones, which were considered to have a single insertion of DHFR* by inverse PCR (13 independent insertion sites). The individual insertion site clones were further characterized with respect to genome copy number profiles. To select methotrexate resistant colonies, we exposed cells to a concentration of methotrexate that was three to four times the IC-50 for each integration site. Genomic copy number profiles were obtained for isolated resistant colonies (GSE6262) by using UCSF HumArray platform (GPL4421). Twelve methotrexate resistant colonies (four different integration sites) were selected for microarray analysis of gene expression at the mRNA level. The untreated integration site clone was used as the reference (Cy5 labeled cDNA) for each of the hybridizations with its respective resistant colonies (Cy3 labeled cDNA). Hybridizations were carried out on arrays of printed long oligonucleotides (70mers) containing 21,000 elements (Operon V2.0, printed in J. David Gladstone Institutes, Genomics Core Laboratory).

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Comparison of gene expression profiles of E. coli W3110 normal colonies vs. L-form colonies

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