gus stability- the replies

[djackson at nature.berkeley.edu]

Thanks to those who replied to my question about GUS stability in plant
cells.  The replies are posted below.   Seems that no one is trying to
engineer a more unstable version, though I discussed this with Richard
Jefferson and he suggested a strategy to screen for ts mutants in E. coli. 
I am going to try fusing a domain of the maize homeobox gene Knotted1 to
GUS- this domain is similar to PEST domains in many rapidly turned over
animal proteins.  If I find anything I'll keep the network informed,  
David Jackson.



djackson at NATURE.BERKELEY.EDU
> I'm trying to find an accurate estimate of the half life of GUS protein in
> plant cells.  I believe it is between one and three days; I am planning
> experiments where this could cause some problems.  Is anyone trying to
> engineer a less stable form of the enzyme?
> 
> David Jackson
> USDA/UC Berkeley Plant Gene Expression Centre.

Date: Wed, 8 Dec 1993 06:24:04 -0500
From: Andrew John Millar <ajm2m at faraday.clas.virginia.edu>
X-Mailer: Mail User's Shell (7.2.3 5/22/91)
To: djackson at nature.berkeley.edu
Subject: GUS half life

Dear David,
The firefly luciferase activity has a half life of approximately
2-3 hours in planta, in the presence of its subrate, luciferin.
Try Millar et al. 1992, P.M.B. Reporter 10, 324, if you are not
locked into a GUS strategy.
Best,
Andrew Millar

To: arabidopsis at net.bio.net
From: botjlh at lure.latrobe.edu.au
Subject: Re: GUS stability
Date: 7 Dec 93 09:43:54 GMT
Sender: news at lugb.latrobe.edu.au (USENET News System)

Concerning the half life of the GUS protein in plant cells, Jefferson et al.,
reported it to be approximately 50 hours in living mesophyll protoplasts
(p3904).

Jefferson, R.A., Kavanagh, T.A. and Bevan, M.W. (1987). GUS fusions:
B-glucuronidase as a sensitive and versatile gene fusion marker in higher
plants. EMBO J. (6). pp3901-3907.

Hope this helps,
Joshua. 

Date: Tue, 30 Nov 1993 06:42:08 -0700 (PDT)
From: "Prof. David Oliver" <doliver at uidaho.edu>
Subject: Re: GUS stability
To: djackson at nature.berkeley.edu
Content-Type: TEXT/PLAIN; charset=US-ASCII

        From our experience with GUS your estimate of a half life of 
three days for the protein is rather conservative.  We have been using 
the light-induced promoter for the H-protein of glycine decarboxylase to 
drive GUS expression.  One of my goals was to place plants with a high 
level of expression in the dark to deplete the mRNA and then measure the 
kinetics of appearance upon reillumination.  This was straight forward at 
the level of mRNA, but we could not keep the plants alive long enough in 
the dark to see a significant decrease in GUS activity.  After two weeks 
there was no decrease in GUS level on a protein basis (there probably was 
some decrease in total protein).

        While there has been some discussion about designing a more 
unstable GUS protein, I am not aware of anyone that is presently working 
on the problem.

David Oliver
Department of Biochemistry and Molecular Biology
University of Idaho