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2u plasmids

Tue Sep 6 16:35:57 EST 1994

It is possible that only a fraction of the cells expressed the three 
proteins but I had done the expt suggested by steve Kron, I looked at 
single cells (probably 5 to 9 times) each would 
overexpress the three proteins. I also 
believe the interaction I saw ( 1 protein interacting with two other 
proteins leading to a three protein complex) since I was able to disrupt 
this complex by growing 
the cells at 37C (one of the interactor was ts) and restoring it in the 
presence of the suppressor protein (the second interactor). All the immunoprecipitation 
expt I performed with this system reproduced the results we obtained 
using the two-hybrid system for which one of the two-hybrid vector also 
carried the third gene needed for the interaction with three partners. 
The genetics data the two hybrid data and the immunoprecipitation data 
were consistent!   

On 6 Sep 1994, Steve Kron wrote:

> Wow!  Three 2 *m's in the same cell will make a lot of the 2 hybrid
> strategies sound reasonable.  However, I am concerned that only a small
> proportion of Christine Chapon's cells actually were expressing all three
> proteins!  Any interactions detected could be occurring after lysis and
> during immunoprecipitation.  I think that it would be worth looking at a
> few single colonies that grow under triple selection to make sure that all
> three plasmids or inserts are still there. Isn't the recombinogenic part of
> a 2 *m plasmid is in its 2 *m origin, or am I confused about 2 *m
> replication?

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