MUTAGENESIS in promoters: "ethical issues"
e-anderson at ski.mskcc.org
Wed Jul 9 09:22:55 EST 1997
In article <Pine.A220.127.116.110708162646.6386A-100000 at mail.med.cornell.edu>,
hmmoss at MAIL.MED.CORNELL.EDU (Heidi Moss) wrote:
>I have a question regarding site-specific mutagenesis in a given promoter.
>Specifically, I am wondering if disruption of SPACING (ie. adding or
>subtracting nucleotides) can have an impact NOT ONLY ON THE SITE OF
>INTEREST, but perhaps on OTHER transcription factor binding sites that
>might be affected by such an addition or deletion (in other words, the
>alteration of 'promoter context').
>OKAY, the specific issue at hand is this:
>THE PROMOTER in QUESTION contains two ~30bp stretches, 80bp apart, that
>are binding sites for tandem transcription factors of interest (STATs).
>--"person A" decided on this approach to analyze the friendly 500bp promoter
> -replace each of the 30bp stretches with a 6bp restriction site
> (the promoter is thus disrupted by a 48bp deletion)
>--"person B" decided to point mutate 2nt that are known to completely
>inhibit STAT binding. The same number of bp remains, thus the only change
>in is within the STAT binding site specifically.
>MY QUESTION IS: if person A obtains a completely negative result, can
>that be SOLEY attributed to the mutation in the STATs, or could such a
>large deletion affect the context of other transcription factors in the
>neighborhood? What if person B's result is not completely negative?
>I do not know much about such things (promoter contexts and all) but my
>opinion is that person B's approach is more solid. But could different
>results be obtained from the two approaches? I do not know.
>Any imput is greatly appreciated. Please respond to my e-mail address, if
>Thanks in advance!!!
i would think that both approaches have their pluses and minuses. one way
to see if person A's approach (assuming that s/he actually gets a
completely negative result) is legitimate would be to do gel shifts with
other transcription factors known to bind the promoter. the best way to
do this would be to use a large region encompassing the 2 30bp regions,
the 80bp spacer and the relevant flanking sequences on either side. (gel
shifts with this size target are a little more difficult than using a 30bp
ds oligo but are done pretty routinely in this lab (albeit not by me).
if the binding of these factors is not interrupted by the deletions made
by person A then all is well.
one thing that must be considered in both situations though is cooperative
binding between various sites, esp. the 2 STAT sites.
just a thought. good luck,
Eric C. Anderson
Sloan-Kettering Institute for Cancer Research
Dept. of Cell Biology and Genetics
Lab: (212) 639-2977
Fax: (212) 717-3298
e-anderson at ski.mskcc.org
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