MUTAGENESIS in promoters: "ethical issues"

[Eric Anderson]

In article <Pine.A32.3.91.970708162646.6386A-100000 at mail.med.cornell.edu>,
hmmoss at MAIL.MED.CORNELL.EDU (Heidi Moss) wrote:

>greetings all...
>
>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
>        fragment:
>
>        -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 
>possible.
>
>Thanks in advance!!!

heidi,

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

-- 
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