IUBio

methyltransferase specificity

Markus Winter m.winter at auckland.ac.nz
Wed Mar 31 15:59:19 EST 1999


Hi Neil,

you are probably right in suspecting a methyltransferase with overlapping
specificity.

The first step would be to to a lign-up of all the Hae III sequences (+/- 15 nts)
in your vector that are not interfered with. They do not contain a recognition
site for the unknown Restriction/Modification-System.

Run them through a program that does restriction (like GeneJockey, DNA strider,
DNA* etc.) to see which enzymes do not recognise it. Then take your cut sequence
and check out which enzymes do recognise (and methylate or restrict) it.

Have fun!

Markus


> I guess my original message wasn't too clear. Basically when I transform a
> particular strain with a plasmid, one (out of many) HaeIII sites is not
> cleaved. If this DNA is then retransformed back into E. coli that HaeIII
> site becomes cleavable again. A likely explanation for this is that the
> first cytosine in  that particular HaeIII site is methylated by the strain,
> inhibiting the action of the RE. If there is a MTase around then it doesn't
> recognise just the HaeIII site because the others aren't affected. I've
> looked for REs that might recognise the region around that HaeIII site but
> none of the others, but without luck.
>
> Just curious as to what caused me some days of concern wondering why the
> plasmid I got out was different to the one I put in.
>
> Not sure if I want to chase this fellow up, but if anyone has some pointers
> to quick and easy protocols for isolating RE / MTases then please pass them
> on.
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