hi,

use of MMS is very toxic and other than MMS you can try with THF (tetra hydro folic acid) or SAM (S- Adenosyl methionine) as these two are good for methylation process. you can try out your work with methonine amino acid too in MS medium and check for the results. i have not tried this for my tissue culture work you try out with this three agents. i have attached one file for your reference.

all the best.

Refer this article and you get some idea to carry out the rest of your work.

Peter Nunn University of Portsmouth UK. Glycine betaine is a common plant methylating compound, which may be helpful - and it is cheap to buy. Many plant betaines are known (see numerous papers by Gerald Blunden), but few ae commercially available. Some stabilised forms of S-adenosylmethionine (SAM) are available as SAM itself is very unstable.

Dear all, thanks so much for the answers; I've got some good clues :)

Dear Rowshon,

I do not think you can alter methylation levels of cultures and their cognate genomic DNAs simply by the addition of methylating agents in the media! There are many barriers between medium agent and DNA methylation such as culture's ability to take up, receptor's sensitivity, proper ionic form of the agent, inter and intracelluar transport of the agent, pH and other cytoplasmic requirements and various transport factors and induction of series of factors needed to methylate DNA. There is no gross mechanism of addition of methylating agent into the medium and its immediate result of DNA methylation!!

What are you trying to do, produce non-physiolgical DNA methylations that require repair or are mutagenic (that's what you'll get with MMS (Methyl methanesulfonate)) or influence normal methylations involved in regulation etc???

I've produced a transgenic line overexpressing a gene that is involved in demethylation; I was planning a simple study to demonstrate the functionally active overexpression of the gene by growing WT and transgenic lines on growth medium containing a methylating agent; my point was to see if the ratio of methylated DNA Vs total DNA is changed in the transgenic line compared to the WT. I guess I can do it in normal medium too; but having the data on normal as well as methylated medium would complement each other; this is a rough idea; I welcome any feedback :)

If your 'demethyase' is concerned with the conversion of 5-Methylcytosine to cytosine you are unlikely to get good results using a chemical methylator as they generate little 5-Methylcytosine when they interact with DNA. If it is a DNA-dioxygenases which oxidatively demethylate N1-methyladenine and N3-methylcytosine in DNA ie a repair protein for methylated/damaged normal bases a chemical methylating agent could be of value in your studies. All methylating agents of this type are potentially mutagenic and carcinogenic .

Dear Philip, yes, my demethylase nicks 5-methylcytosine; in such case what would you advice?

I 'm not really a molecular biologist but I would look for the increased expression of an endogenous (or introduced) gene, the product of which is known to be silenced (or diminished) by promoter hypermethylation.

I post a very similar reply a few minutes ago but didn't see it listed if this is posted twice I apologize.

Thanks so much Philip; I can do that; I had to do qPCR to show the overexpression anyway; now I just need to do it for one more gene; that reduces my workload :) thanks again :)