Purely FYI -
Here is a synopsis of the responses I received to my plant-GFP posting
(together with my personal thanks to all involved for the timely and useful
suggestions).
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Original Posting:
Is anyone aware of a GFP clone that:
1 - has plant-optimized DNA sequence
2 - has a emission wavelength shifted from the original green
and
3 - is available for research purposes
We would like to use GFP as a screenable marker for plant genetic
transformation but have encountered considerable background fluorescence at
the 'wildtype' GFP emission wavelength.
You can reply to my email address (jvelten at mail.csrl.ars.usda.gov) and I will
post a synopsis of all responses.
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Response 1:
Seth Davis' comments (see below [#2] for Seth's own response) as forwarded by:
Mike Moser Tel: 206-616-7391
UW Department of Pathology FAX: 206-543-3644
Box 357470 moser at u.washington.edu
Seattle, WA 98195 http://weber.u.washington.edu/~moser
These GFPs are Haseloff's mGFP4 (cryptic splice corrected) with three
mutations that improve GFP solubility (F99S, M153T, V163A) and may contain
spectral changes (S65T or Y66H). I published these clones in Weeds World,
a web-based journal, and am completing a second report to be sent to Plant
Molecular Biology. These clones were donated to the Arabidopsis Stock
Center for public distribution and are starting to see wide use throughout
the plant community. If your interested, they work in human cells as well
(you work on humans, right?), so you can try them for free (see below).
smGFP refers to soluble-modified GFP. "Soluble" means the solublility
mutations were introduced, while "modified" refers to the codon changes to
avoid missplicing of the mRNA.
The soluble-modified FP collection is publicly available at the Arabidopsis
Biological Resource Center at the Ohio State University. You can "order"
them through their web page. The order location for these stocks is
http://aims.cps.msu.edu/aims/menu/search2.html. The three stocks are:
CD3-326 soluble-modified GFP
CD3-327 soluble-modifed Red-shifted GFP
CD3-328 soluble-modified Blue FP
============================================================
Response 2:
From: Seth J. Davis (6/19/97)
I've made two that have shifted spectrum: soluble-modified red-shifted GFP
and soluble-modified blue FP. For your application, I recommend smRS-GFP.
These clones were donated to the arabidopsis stock center (AIMS). You can
read about them in WeedsWorld 3(2).
========================================================
Response 3:
From: Kirby Siemering (6/20/97)
Have you tried mgfp5-ER (see http://brindabella.mrc-lmb.cam.ac.uk/ and
references stated therein for details)? We have engineered this version so
that it can be efficiently excited with either UV or blue light. One of
the reasons for this is that autofluorescence that looks like GFP usually
only occurs under one or the other of the exciting wavelengths. Therefore,
if you toggle between filter blocks set up for UV and blue light
excitation, you can usually tell real GFP fluorescence from
autofluorescence. My experience is from work with Arabidopsis.
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Response 4:
From: Dr. George McNamara (6/19/97)
The Haseloff lab has this. See http://brindabella.mrc-lmb.cam.ac.uk for
details. Also their papers: Haseloff et al (1997) PNAS 94: 2122-2127 and
K.R. Siemering, R. Golbik, R. Sever, J. Haseloff (1996) Mutations that
suppress the thermostability of green fluorescent protein. Current Biology
6(12):1653-1663.
Another example is: Stewart at http://www.uncg.edu/~cnstewar/by960003.htm
You might also try EGFP (490/520 nm) or GFPuv (400/520 nm) from Clontech,
or a BFP from Tsien or Quantum Biotechnologies or one of the spectral
variants from G.J. Palm, A. Zdanov, G.A. Gaitanaris, R. Stauber, G.N.
Pavlakis, A. Wlodawer (1997) The structural basis for spectral variation in
green fluorescent protein. Nature Biotechnology 4: 361-365.
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Response 5:
From: David Micklem (6/20/97)
Jeff - I strongly recommend getting in touch with Jim Haseloff (sp?) at the
Laboratory of Molecular Biology, MRC, in Cambridge UK.
I'm sure he's got plant-codon'd GFP and I think its possible he may have
colour-shifted variants too - or he would probably know if they exist.
Try Proc Natl Acad Sci 1997, 94, 2122-and Current Biology 1996, 6, 1653-for
relevant papers.
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Response 5:
From: Friedrich Bischoff (6/26/97)
Using the Haseloff-construct, we got expression levels that were high
enough to distinguish transformed calli from those with background
fluorescence. Same is possible for particle bombardement.
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Over and out!
\\\\/
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