Project description:To determine the transcription start site and the abundance of dam mRNA, we conducted mRNA-specific RNA-Seq experiments. Essentially two strategies were tested. First strategy: We obtained the cDNA with a specific RT-PCR in order to get the mRNA from our dam construct (see DNA-seq of DamID reporter cassette). An enrichment, in which we used a Biotinylated dam oligo and Streptavidin magnetic beads, was needed. Then, similar to SHAPE-seq, the ssDNA ligation was performed with CircLigase. The last step was the PCR reaction in order to prepare the cDNA libraries to be sequenced. Second strategy: Similar to the first one, but the specificity was introduced at the PCR, not at the RT. Thereby, busk cDNA was prepared with random hexamers. The enrichment step was not needed.
Project description:To get a global view of PT modification’s function in ∆dam mutant, we did an RNA-seq to compare the gene expression change after dam knocks out and test whether PT could restore the changes.
Project description:DamID, in which a protein of interest is fused to Dam methylase, enables mapping of protein-DNA binding through readout of adenine methylation in genomic DNA. DamID offers a compelling alternative to chromatin immunoprecipitation sequencing (ChIP-Seq), particularly in cases where cell number or antibody availability are limiting. This comes at a cost, however, of high non-specific signal and a lowered spatial resolution of several kb, limiting its application to transcription factor-DNA binding. Here we show that mutations in Dam, when fused to the transcription factor Tcf7l2, greatly reduce non-specific methylation. Combined with a simplified DamID sequencing protocol, we find that these Dam mutants allow for accurate detection of transcription factor binding at a sensitivity and spatial resolution closely matching that seen in ChIP-seq.
Project description:We determined the global gene expression profiles of wildtype, dam, dam mutS, and mutS mutant E. coli strains. Keywords: Basal gene expression comparison
Project description:Both chromatin access, and binding of AID in B cells are of major scientific interest. Therefore DamID was setup in B cells. AID specific binding could not be identified. In contrast to binding, DAM profiling provide a upmost useful tool in defining chromatin access in vivo.
Project description:PacBio SMRT-seq of wild-type, ∆metJ, and ∆dam Salmonella enterica serovar Typhimurium grown under SPI-1-inducing and SPI-2-inducing conditions.
Project description:Dam, the most described bacterial DNA-methyltransferase, is widespread in gamma-proteobacteria. Dam DNA methylation can play a role in various genes expression and is involved in pathogenicity of several bacterial species. In the entomopathogenic bacterium Photorhabdus luminescens, a dam ortholog was identified. Overexpression of dam in P. luminescens did not impair growth ability in vitro. In contrast, compared to a control strain harboring an empty plasmid, a significant decrease in motility was observed in the dam-overexpressing strain. In addition, the dam-overexpressing P. luminescens strain showed a delayed virulence compared to that of the control strain after injection in larvae of the lepidopteran Spodoptera littoralis. These results reveal that Dam plays a major role during P. luminescens insect infection.
