Project description:We use Illumina sequencing to monitor mutations in the bacteriophage T7 genome in the presence of T7 DNA polymerase that has an altered exonuclease active site. These alterations include mutation of key residues in the exonuclease active site.
Project description:Transcription, the copying of genetic information into RNA, is accomplished by multi-subunit RNA polymerases (msRNAPs) in all living organisms. msRNAPs are highly conserved in evolution and invariantly share a ~400 kDa five-subunit catalytic core1,2. A group of hypothetical single-chain proteins of unknown function, present in various bacteria and bacteriophages, was predicted to be distantly related to msRNAPs, having diverged from them before the Last Universal Common Ancestor3. Here, we studied a ~100 kDa protein, YonO, from this group encoded by the bacteriophage SPβ of Bacillus subtilis. We show that despite homology to only several amino acids of msRNAP, and the absence of most of the conserved domains, YonO is a highly processive and fast DNA-dependent RNA polymerase. Unlike msRNAPs, YonO can start specific transcription on double-stranded DNA without additional factors. We show that YonO is a bona fide RNAP of SPβ bacteriophage that transcribes its late genes. This new class of RNAPs may represent an intermediate step in the evolution of an ancestor of all msRNAPs.
Project description:We performed the whole transcriptome analysis in Zscan4 positive ES cells (Em+) and Zscan4 negative ES cells (Em-) by using FACS-sorted MC1-ZE7 ES cells.
Project description:We analyzed the genome-wide chromatin states in Zscan4 positive ES cells (Em+) and Zscan4 negative ES cells (Em-) by using FACS-sorted MC1-ZE7 ES cells. H3K27 hyperacetylation and DNA demethylation were detected in heterochromatic regions of Em+ cells. These results suggested that the heterochromatin is activated in Zscan4 positive state.
Project description:By using FACS-sorted MC1-ZE7 ES cells, we analyzed the genome-wide distribution of H3K27ac and DNA methylation in Zscan4 positive ES cells (Em+) and Zscan4 negative ES cells (Em-). H3K27 hyperacetylation and DNA demethylation were detected in heterochromatic regions of Em+ cells, but not Em- cells. These results suggested that the Zscan4 state takes "open" chromatin conformation in ES cells.
Project description:Linear amplification of RNA by T7 bacteriophage polymerase is widely used in molecular biology. We performed 5’RACE-Seq to identify T7 promoter variants with enhanced transcriptional activity that generate up to five-fold higher RNA output in large scale synthesis reactions. In single-cell RNA-Sequencing, optimized T7 promoters facilitate library preparation, and substantially increase library complexity and the number of expressed genes detected per cell, highlighting a particular value for bioanalytical applications
