User:Torsten Waldminghaus/Notebook/Methylation array

Idea

• Analyse the methylation of GATC sites genomewide in E. coli.

Notes

• Løbner-Olesen et al, 2003 [1] used a aroK17::cat strain to have more hemimethylation in the cell, since a polar effect on dam leads to a reduced Dam content in the cell (only 30% of wt [2]). Could be used as control and for interesting analysis.
• methylation in different strains could be interesting:
  • dam-overproduction
  • seqA deletion, over-production, under-production
  • synchronized cells
  • does introduction of a GATC cluster alter methylation of surrounding sites?

• As independent method and first step one could analyse cutting by methylation sensitive REN with qPCR
  • on whole chromosome
  • clusters and isolated GATCs
  • coding regions and intergenic regions

Possible restriction enzymes that are Dam methylation sensitive:

Enzyme	Site	Notes	Link
HphI	GGTGA	No star activity; reaction at 37°C; heatinactivation at 20 min 65°C	http://www.fermentas.com/catalog/re/hphi.htm
MboII	GAAGA	Star activity; reaction at 37°C; heatinactivation at 20 min 65°C	http://www.fermentas.com/catalog/re/mboii.htm
TaqI	TCGA	No star activity; reaction at 65 °C; no heatinactivation after 20 min 80°C	http://www.fermentas.com/catalog/re/taqi.htm

• HphI seems to be a goog choice since it can be heatinactivated and has no star activity (does not cleave unspecific when DNA is overdigested).

1. Løbner-Olesen A, Marinus MG, and Hansen FG. Role of SeqA and Dam in Escherichia coli gene expression: a global/microarray analysis. Proc Natl Acad Sci U S A. 2003 Apr 15;100(8):4672-7. DOI:10.1073/pnas.0538053100 |
2. Løbner-Olesen A, Boye E, and Marinus MG. Expression of the Escherichia coli dam gene. Mol Microbiol. 1992 Jul;6(13):1841-51. DOI:10.1111/j.1365-2958.1992.tb01356.x |
3. Braun RE and Wright A. DNA methylation differentially enhances the expression of one of the two E. coli dnaA promoters in vivo and in vitro. Mol Gen Genet. 1986 Feb;202(2):246-50. DOI:10.1007/BF00331644 |
4. Kücherer C, Lother H, Kölling R, Schauzu MA, and Messer W. Regulation of transcription of the chromosomal dnaA gene of Escherichia coli. Mol Gen Genet. 1986 Oct;205(1):115-21. DOI:10.1007/BF02428040 |
5. Campbell JL and Kleckner N. E. coli oriC and the dnaA gene promoter are sequestered from dam methyltransferase following the passage of the chromosomal replication fork. Cell. 1990 Sep 7;62(5):967-79. DOI:10.1016/0092-8674(90)90271-f |
6. Pfister S, Schlaeger C, Mendrzyk F, Wittmann A, Benner A, Kulozik A, Scheurlen W, Radlwimmer B, and Lichter P. Array-based profiling of reference-independent methylation status (aPRIMES) identifies frequent promoter methylation and consecutive downregulation of ZIC2 in pediatric medulloblastoma. Nucleic Acids Res. 2007;35(7):e51. DOI:10.1093/nar/gkm094 |