Project description:We used Affymetrix microarrays to determine the cisplatin-induced gene expression changes in E. coli deficient in dam and mismatch repair (dam, dam mutS, and mutS mutant strains). E. coli deficient in dam are hypersensitive to cisplatin. However introducing an additional mutation in mismatch repair (i.e., a mutation in mutS or mutL) abrogates this sensitivity and essentially restores wildtype levels of resistance. Keywords: Drug-induced expression
Project description:We used Affymetrix microarrays to determine the cisplatin-induced gene expression changes in E. coli deficient in dam and mismatch repair (dam, dam mutS, and mutS mutant strains). E. coli deficient in dam are hypersensitive to cisplatin. However introducing an additional mutation in mismatch repair (i.e., a mutation in mutS or mutL) abrogates this sensitivity and essentially restores wildtype levels of resistance. Experiment Overall Design: Overnight cultures were diluted 1000-fold and grown in Luria-Bertani (LB) medium until the cells reached exponential growth as determined by OD600. The exponentially growing cells were resuspended in M9 minimal medium at a cell density of 2 X 108 cells/ml in a volume of 15 ml and treated with 150 uM cisplatin for 2 hours at 37°C. Following treatment cultures were resuspended in 15 ml LB and allowed to recover for 90 minutes at 37°C. OD600 readings were taken after the recovery period, when RNA isolation began. Total RNA was isolated from cells by extraction using the MasterPure RNA Purification Kit (Epicentre Technologies) according to the manufactureâs protocol. The isolated total RNA was quantitated by absorption at 260 nm (typical yield from a 15 ml culture was 250-500 µg of total RNA), and the purity was determined by the ratio of absorption values at 260/280nm. RNA quality was determined by formaldehyde agarose gel electrophoresis (1.2% agarose in FA Buffer pH 7.0 (20mM 3-[N-morpholino]propanesulfonic acid, 5mM sodium acetate, 1mM ethylenadiaminetetraacetic acide (EDTA)) or by analysis on an Agilent 2100 Bioanalyzer. All samples visualized by gel electrophoresis or by the bioanalyzer electropherogram showed clear distinct bands correlating to 16S and 23S ribosomal RNA, indicating that no detectable RNA degradation occurred and that RNA integrity was maintained throughout the RNA isolation procedure.
Project description:We show there is minimal genome-wide chromatin rearrangements (as measured by DNA accessibility) during tissue differentiation in C. elegans transgenic expression of E. coli DAM (DNA Adenine Methyltransferase) from tissue-specific promoters followed by mapping of methylated sites using a procedure that captures 20bp sequences flanking methylated sites for Illumina sequencing PD3994 = transgenic line expressing DAM from myo-3 promoter PD3995 = transgenic line expressing DAM from rol-6 promoter PD3997 = transgenic line expressing DAM from vit-2 promoter N2dam = wildtype (N2) line; its genomic DNA was in vitro DAM-methylated parsed = Solexa reads in which linker sequences were successfully parsed out by the authors WS170DAMtags_ALL.txt = the set of all in_silico-generated DAM tags from C. elegans genome version WS170
Project description:Heterochromatin protein HP1 is thought to play key role in chromatin structure and gene regulation. We performed a genome-wide mapping of HP1 target genes in the non-polytenic Drosophila Kc cells by using DamID. This approach is based on the ability of a chromatin protein fused to Escherichia coli DNA adenine methyltransferase (Dam) to methylate the native binding site of the chromatin protein. Dam-fusion proteins are expressed at very low levels to avoid mistargeting. Subsequently, methylated DNA fragments are isolated, labeled (using Cy3 or Cy5) and hybridized to a microarray. Methylated DNA fragments from cells transfected with Dam alone served as reference. Genomic binding sites of the protein can then be identified based on the targeted methylation pattern. For detailed background information on DamID, see: van Steensel, B., Delrow, J. & Henikoff, S. Chromatin profiling using targeted DNA adenine methyltransferase. Nat Genet 27, 304-8 (2001); van Steensel, B. & Henikoff, S. Identification of in vivo DNA targets of chromatin proteins using tethered dam methyltransferase. Nat Biotechnol 18, 424-8 (2000). We performed three independent replicates. We used for this study a cDNA array developed by the GeneCore facility in EMBL (Heidelberg, Germany), covering the DGC1 and DGC2 cDNA libraries from the Berkeley Drosophila Genome Project, which represents more than 70% of the coding Drosophila genome.
Project description:Purpose: Genome-wide DNA-binding analysis for Ttk in intestinal progenitor cells and comparing Su(H) binding sites between seq overexpressed and ttk-RNAi intestinal progenitor cells in Drosophila midgut by DNA adenine methyltransferase identification(DamID) Methods: Dam(Ctrl) , Ttk-Dam transgenes, Su(H)-Dam, Su(H)-Dam&seq and Su(H)-Dam&ttk-RNAi were expressed in intestinal progenitor cells by esg-Gal4.The guts within 2 days was used for DNA adenine methyltransferase identification (DamID), followed by deep sequencing analysis Results: Ttk suppresses neural specific Notch targets through suppressing Seq in intestinal progenitor cells
Project description:Profiling of DAF-16 binding by comparison of DNA methylation of a C. elegans control strain expressing the DNA adenine methyltransferase (DAM) and an experimental strain expressing a DAF-16::DAM. Both strains were fed daf-2 RNAi to identify DAF-16 binding associated with long lived worms.
Project description:We report the application of targeted DNA Adenine Methyltransferase identification DNA sequencing technology for high-throughput profiling of Pcl occupancy in the Gr5a cells of male Drosophila melanogaster adults. By expressing the UAS-LT3-Dam::Pcl and UAS-LT3-Dam transgene using the Gr5a-GAL4;tubulinGAL80ts driver.
Project description:We report the application of targeted DNA Adenine Methyltransferase identification DNA sequencing technology for high-throughput profiling of OGT occupancy in the Gr5a cells of male Drosophila melanogaster adults. By expressing the UAS-LT3-Dam::Pcl and UAS-LT3-Dam transgene using the Gr5a-GAL4;tubulinGAL80ts driver.
Project description:We report the application of targeted DNA Adenine Methyltransferase identification DNA sequencing technology for high-throughput profiling of Pcl occupancy in the Gr5a cells of male Drosophila melanogaster adults. By expressing the UAS-LT3-Dam::Pcl and UAS-LT3-Dam transgene using the Gr5a-GAL4;tubulinGAL80ts driver.
Project description:We report DNA adenine methyltransferase identification (DamID)-sequencing data to identify Gon4l-associated genomic regions in wild-type (WT) tailbud (TB) stage zebrafish embryos. Methylated, and therefore Gon4l-proximal, gDNA was isolated from embryos expressing a Gon4l-Dam fusion and compared to that from embryos expressing GFP-Dam control.