methylation and promoter analysis

Saroj Mathupala mathu at WELCHLINK.WELCH.JHU.EDU
Sat May 21 17:52:25 EST 1994

Hi Bio-netters, we are setting up promoter analysis experiments using
vectors /kits from a company which recommends the use of E. coli JM109 for
isolation of promoter-reporter gene constructs for transfection into
mammalian cells. JM 109, and practically every other E. coli strain used
for these studies, methylates it's DNA (dam and dcm type methylation,
and/or EcoK methylase activity). In eukaryotes promoters are switched on
and off depending on the demethylated/methylated state. My question is
this-does that type of methylation interfere with promoter activity once
the reporter plasmids are transfected into mammalian cells? And, if so,
how long should one wait for the 'demethylase/s' of eukaryotic cells to
fully deprotect a transfected plasmid (i.e. After transfection, how long
should one wait before starting expression assays)? For a fully (in vitro)
methylated plasmid, it takes from 48 to 102 hours for complete
demethylation, according to available literature. Any insight/suggestions
into this matter will be greatly appreciated. Thanks in advance, Mathu. 

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