replies: GFP for nuclear localization

[Iris meier]

Dear netters:

I am posting the replies I got about the use of GFP for nuclear 
localization experiments. Thank you all for your help.

Iris Meier


Rick Vierstra wrote:

 
Check the latest WEEDS WORLD for a series of improved GFPs we have made.  
They
will be available in the OHIO STATE STOCK CENTER shortly.
 
RICK VIERSTRA
University of WIsconsin.


Brian Pickett wrote:


I know that it has worked well in yeast, talk to Jasper Rine or one of 
the
folks in his lab at Berkeley.

I sent just this suggestion to Vivian Irish a few months ago, you might 
also
check with her to see if she has decided to use this approach.

Good luck,

Bryan Pickett, fpicket at orion.it.luc.edu


Bernd Mueller-Roeber wrote:


we have used GFP for stable transformation of potato and tobacco plants. 
In
these plants GFP goes preferentially into the nucleus without the 
addition
of any targeting sequence, just the plain GFP does it. Other people have
also seen this. What I heard from people doing transient expression 
studies,
GFP mainly stays in the cytosol. Nevertheless, there might be a portion 
of
the protein that still enters the nucleus. However, assuming that a
targeting sequence is attached, you might expect to see most of the GFP, 
if
not all, in the nucleus. 

************************************************************************
**
Dr. Bernd Mueller-Roeber
Max-Planck-Institute of Molecular Plant Physiology (MPI-MOPP)
- Junior Research Group -
Karl-Liebknecht-Str. 25
Haus 20
D-14476 Golm / Potsdam
Germany

Tel.: + 49 - 331 - 977 2787
Fax:  + 49 - 331 - 977 2301


Tony Schaeffer, Munich, also warned that GFP tends to localize in the 
nucleus. He referred to Jen Scheen and Brian Seed for obtaining 
constructs.

*   email: schaeff at lmb.uni-muenchen.de



Albrecht von Arnim wrote:


GFP as a tag gives excellent subcellular resolution and is much more
reliable and less finicky than GUS as a tag.

I have constructed vectors for expressing C- and N-terminal fusion 
proteins
to GFP in plant cells.  These vectors carry the 35S promoter and are 
based
on the modified GFP sequence (mGFP) distributed by Jim Haseloff.  There 
is
a limted number of cloning sites (NcoI for N-terminus; BglII for
C-terminus).  I have both wild type and S65T mutant versions.  The
sensitivity of detection of GFP is comparable to that of a strongly
expressed GUS construct (which requires short staining times).

But:

Using GFP for studying nuclear localization requires caution because 
even
unfused GFP will accumulate in the nucleus of plant cells.  The reason 
for
this is not known.  Under certain circumstances GFP might be useful,
though.

vonarnim at utkux.utcc.utk.edu