MUTAGENESIS in promoters: "ethical issues"

Heidi Moss hmmoss at MAIL.MED.CORNELL.EDU
Tue Jul 8 16:23:24 EST 1997


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




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