gain of function mutagenesis, how to do?

Frederik Börnke ricky_boernke at gmx.net
Wed Jan 19 09:20:11 EST 2000



"A.F. Simpson" schrieb:
> 
> Frederik Börnke wrote:
> 
> > I need some advice on the following experiment:
> > Protein X from tobacco interacts with isoform 1 and 2 of protein Y
> > in the yeast-two hybrid system. Protein X from spinach interacts
> > with isoform 1 of protein Y only. X from tobacco and spinach share
> > about 70% homology. Now I'd like to find out the structural basis
> > of the different isoform specific interaction of the two proteins.
> 
> <snip choice between constructing chimeras and randomly mutating the
> spinach protein>
> 
> Would there be any advantage of an inital set of experiments mutating
> the tobacco protein X to look of sites which give _loss_ of function
> (ie. produce a protein which interacts with isoform Y1 only)?
> 
> This might narrow down the important areas and allow you to target the
> construction of chimeras to certain regions.

Actually, I did some deletion constructs. However, if I delete only
small
portions from either C or N terminus interaction is completely
abolished.
What does that tell me wiht respect to the isoform speceficity of inter
action?
 
> (I don't have much experience in this kind of experiment, so this is
> more of a question than a suggestion.)
> 
For more practical reasons it would be easier to assay for gain of
function since the two hybrid system is based on a positive selection.
And if using the random mutagenesis approach you would never know
whether
loss of function is due to aa exchanges or tbe introduction of stop
codons.
If you have any ideas to circumvent this, I would be pleased to hear.




See you
Ricky

> 
> love
> Anna

-- 
*******************************************************************

Frederik Boernke
Research Group of  Molecular Plant Physiology
Institute for Plant Genetics and Crop Plant Research (IPK)
Corrensstr. 3
06466 Gatersleben
Tel.  039482 -5 321
Fax. 039482 -5 515
http://www.ipk-gatersleben.de




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