Project description:This RNA-Seq analysis compares gene expression of the RNA polymerase II CTD-mutants contrasting to the WT RNA polymerase II CTD (with isogenic 3' selection marker) in the fission yeast S. pombe
Project description:A pool of 3633 tagged heterozygous transposon disruption mutants underwent haploinsufficiency profiling in the presence of different synthetic compounds to identify their cellular targets
Project description:The structural integrity of the nucleosome is central to regulation of DNA metabolism and transcription. We describe a library of 486 systematic histone H3 and H4 substitution and deletion mutants in Saccharomyces cerevisiae that probe the contribution of each residue to nucleosome function and can be episomal or genomically integrated. We tagged each mutant histone gene with unique molecular barcodes, facilitating identification of mutant pools through barcode amplification, labeling, and microarray hybridization. We probed fitness contributions of each residue to chemical perturbagens of chromosome integrity and transcription, mapping global patterns of chemical sensitivities and requirements for three forms of transcriptional silencing onto the nucleosome surface. Lethal mutants were surprisingly rare and of distinct types; one set of mutations mapped precisely to the DNA interaction surface. The barcode microarrays were useful for scoring complex phenotypes such as competitive fitness in a chemostat, proficiency of DNA repair, and synthetic genetic interactions. Keywords: genetic modification These nine datasets characterize a microarray platform repurposed for profiling a barcoded comprehensive library of synthetic histone mutants. The first two datasets establish the wide dynamic range and high sensitivity and specificity of data from this platform. The other seven datasets demonstrate the usefulness of this technology for scoring subtle and complex phenotypes of the histone H3 and H4 alleles in this library.
Project description:The structural integrity of the nucleosome is central to regulation of DNA metabolism and transcription. We describe a library of 486 systematic histone H3 and H4 substitution and deletion mutants in Saccharomyces cerevisiae that probe the contribution of each residue to nucleosome function and can be episomal or genomically integrated. We tagged each mutant histone gene with unique molecular barcodes, facilitating identification of mutant pools through barcode amplification, labeling, and microarray hybridization. We probed fitness contributions of each residue to chemical perturbagens of chromosome integrity and transcription, mapping global patterns of chemical sensitivities and requirements for three forms of transcriptional silencing onto the nucleosome surface. Lethal mutants were surprisingly rare and of distinct types; one set of mutations mapped precisely to the DNA interaction surface. The barcode microarrays were useful for scoring complex phenotypes such as competitive fitness in a chemostat, proficiency of DNA repair, and synthetic genetic interactions. Keywords: genetic modification
Project description:The construction of synthetic gene circuits relies on our ability to engineer regulatory architectures that are orthogonal to the host's native regulatory pathways. However, as synthetic gene circuits become larger and more complicated, we are limited by the small number of parts, especially transcription factors, that work well in the context of the circuit. The current repertoire of transcription factors consists of a limited selection of activators and repressors, making the implementation of transcriptional logic a complicated and component-intensive process. To address this, we modified bacteriophage T7 RNA polymerase (T7 RNAP) to create a library of transcriptional AND gates for use in Escherichia coli by first splitting the protein and then mutating the DNA recognition domain of the C-terminal fragment to alter its promoter specificity. We first demonstrate that split T7 RNAP is active in vivo and compare it with full-length enzyme. We then create a library of mutant split T7 RNAPs that have a range of activities when used in combination with a complimentary set of altered T7-specific promoters. Finally, we assay the two-input function of both wild-type and mutant split T7 RNAPs and find that regulated expression of the N- and C-terminal fragments of the split T7 RNAPs creates AND logic in each case. This work demonstrates that mutant split T7 RNAP can be used as a transcriptional AND gate and introduces a unique library of components for use in synthetic gene circuits.
Project description:RNA Polymerase II was mapped over 4% of the yeast genome by ChIP, in wild-type and a handful of mutants in transcriptional termination factors. Keywords: ChIP-chip, transcription termination
