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gain of function mutagenesis, how to do?

A.F. Simpson AFS7 at le.ac.uk
Wed Jan 19 20:01:41 EST 2000

Frederik Börnke wrote:

> > 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)?

> 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?

Well, I suppose it tells you deletion mutants are a bit of a dodgy idea
sometimes :-)

Did you try any internal deletions?
> 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.

I was thinking of screening mutant tobacco protein Xs in parallel
against Y1 and Y2, looking for tobacco mutants which interact with Y1
but don't interact with Y2.  This would presumably exclude the stop
codons.  However, I have only a vague idea about the protocol for yeast
two-hybrid experiments, so this might be a silly idea.

How about making chimeras of the spinach and tobacco X proteins?

> See you
> Ricky


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