GFP Splice Site Responses

[Mark Guiltinan]

Here are the most important comments I got on the GFP cryptic splice site. 
Apparently it is only used by some species, Arabidopsis seems to have a
particulary bad time with it.  The mutated form is available as cited below. 
I anticipate that new spliceless versions and others based on the more
efficient mutants will be available in plant expression vectors soon, either
commercially or through the generosity of our collegues.  I also heard through
the grapevine (thanks Simon) that GFP splicing only seems to be a problem with
stable transformation, it works fine in transient assays (only a rumor?).

Mark Guiltinan
Penn State University
Department of Horticulture
Biotechnology Institute
mjg at psuen.psu.edu




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Mark,

The information that I have received form Clontech states that the 
cryptic intron (84 bp) in the GFP is:

    AG|GUAUUGA --------UCAUGGCAG|AC
    
According to Paul Kitts at Clontch, Jim Hasseloff changed the 5' 
splice site and elevated the CG content of theis region.  This 
greatly improved the fluorescence in Arabidopsis under the 35S 
promoter.  Recently Heim et al. (Nature 373, page 664) have used 
point mutations to increase fluorescence with blue light.

I am very interested in trying GFP with our barley micropsore 
cultures to monitor our transformation frequencies.  Clontech seems 
very slow in making available improved constructs suitable for work 
with plants.  If you do engineer an improved construct I would be 
very interested in trying/buying it for use with our system.  In the 
meantime I am interested in trying the available GFP constructs in 
barley.  If I could use one of your constructs for that purpose I'd 
be very grateful.

The flourescent protein newsgroup is up and running apparently.  That 
should be a useful forum for information.  Hope you can engineer a 
sequence useful in plants - Joan


Joan Krochko
Crop Science Department
University of Guelph
Guelph, Ontario 
N1G 2W1  CANADA
TEL: (519) 824-4120 ext. 8185
FAX: (519) 763-8933
email: jkrochko at crop.uoguelph.ca

________________________________________________

Hi  contact Jim Haseloff for mGFP with altered splice site.

his e-mail adress:  jph at mrc-lmb.cam.ac.uk (Jim Haseloff)


S.P. Dinesh-Kumar
Plant Gene Expression Center/UC Berkeley
Albany, CA 94710