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GAL4 Nuclear Localization Signal

Neigeborn, Lenore neigeborn at OCELOT.RUTGERS.EDU
Mon Oct 24 17:17:28 EST 1994

Hi folks,
     This is in response to George's concern that his protein may not be
functional when fused to the GAL4-DNA Binding Domain.  He wants to do a
complementation test with his fusion protein to confirm that his protein is
still functional (and presumably retains the ability to associate with its
normal partners).  Right, George?  

     My question is this:  Have you tried the complementation test yet?  I know
that you're worried because your protein is normally cytoplasmic and you think
it will not complement because it is targetted to the nucleus.  We've found,
however, that our fusion protein is able to fully complement our mutation
despite nuclear targetting (we don't know whether our WT protein is
nuclear-localized or not).  I've known several people who are doing this with
cytoplasmic proteins and they have the same complementation success.  My
feeling is that the ADH promoter that is driving the 2-hybrid fusions is
overexpressing enough product to permit some "mis-localization" of the fusion
protein, so that it doesn't all go to (or stay in) the nucleus.  Of course this
phenomenon will be variable, depending on the protein you're studying, but it
seems worth a try.

Lenore Neigeborn

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