Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Characterization of transcriptional and fitness effects of a loss of function mutation in rho

ABSTRACT: In order to study the effects of a mutation to the transcriptional termination regulator Rho (referred to as rho*), we made use of expression microarrays to observe the direct and indirect effects of rho* on gene expression. In addition, we used arrays to map the fitness of strains from transposon mutagenized libraries under four conditions, showing that in each case the majority of genes with significant fitness effects were dependent on the genotype at rho. For expression arrays, we performed two-color microarrays comparing transcript levels in rho* and wild type cells during exponential growth in glucose minimal media. For selection experiments, transposon insertions were mapped through selective amplification of genomic regions adjacent to them. We then measured the fitness effects of insertions throughout the genome using two-color microarrays, comparing amplified DNA from a population grown under a selective condition of interest to an isogenic control population grown under a reference condition (glucose minimal media). All arrays were performed in duplicate, and the source material for the duplicates came from separate biological replicates.

ORGANISM(S): Escherichia coli str. K-12 substr. MG1655

SUBMITTER: Peter Freddolino

PROVIDER: E-GEOD-32022 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Somatic transposon mutagenesis in mice is an efficient strategy to investigate the genetic mechanisms of tumorigenesis. The identification of tumor driving transposon insertions traditionally requires the generation of large tumor cohorts to obtain information about common insertion sites. Tumor driving insertions are also characterized by their clonal expansion in tumor tissue, a phenomenon that is facilitated by the slow and evolving transformation process of transposon mutagenesis. We describe here an improved approach for the detection of tumor driving insertions that assesses the clonal expansion of insertions by quantifying the relative proportion of sequence reads obtained in individual tumors. To this end, we have developed a protocol for insertion site sequencing that utilizes acoustic shearing of tumor DNA and Illumina sequencing. We analyzed various solid tumors generated by PiggyBac mutagenesis and for each tumor >10^6 reads corresponding to >10^4 insertion sites were obtained. In each tumor, 9 to 25 insertions stood out by their enriched sequence read frequencies when compared to frequencies obtained from tail DNA controls. These enriched insertions are potential clonally expanded tumor driving insertions, and thus identify candidate cancer genes. The candidate cancer genes of our study comprised many established cancer genes, but also novel candidate genes such as Mastermind-like1 (Mamld1) and Diacylglycerolkinase delta (Dgkd). We show that clonal expansion analysis by high-throughput sequencing is a robust approach for the identification of candidate cancer genes in insertional mutagenesis screens on the level of individual tumors. Solid tumors in mice were generated by somatic transposon mutagenesis with a PiggyBac transposon system. Insertion sites of transposons in 11 tumors and 6 non-cancerous tail controls were determined by Illumina high-throughput sequencing. Insertions were determined both on 5' and 3' sides of the transposon (PB5 and PB3, respectively). Quantitative analysis of read numbers revealed enrichment of certain insertions in tumors, but not in controls, and these enriched insertions identify candidate cancer genes.

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Characterization of transcriptional and fitness effects of a loss of function mutation in rho

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