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ever seen GFP in S cerevisiae ?

Sepp D. Kohlwein kohlwein at ftugax.tu-graz.ac.at
Wed Jun 5 09:47:18 EST 1996


Guillaume Stahl <stahl at infobiogen.fr> wrote:
>Hi GFPyeastNetters,
>
>I've integrated recently a fusion protein with wt GFP en Cterm in the 
>cerevisiae genome. After YPD culture (+/- 1 OD), directly under scope, 
>using fluoresceine filters, I've not been able to see anything (except 
>some cells, strange shape, greennish/yellowish). Same result with same 
>culture O.N. in the fridge, and same result with... negative control.
>
>My question is simple : what are the condition (media, OD, wash, fridge, 
>filters,...) you used if you ever saw GFP in yeast. Do you think a 
>single copy of 'medium expressed' GFP is sufficient. Should I try it in 
>a FACS ? Do you have good conditions/suggestions for the last point ?
>
>Thanks in advance
>
>Guillaume Stahl, Orsay, France


Dear Guillaume,
if you use wt GFP you need to use the "DAPI" filter set to get good 
results, since the excitation maximum is about 390 nm, emission 510 nm.
The medium to use and OD at harvest depend on the regulation of your 
protein (YFP). If you have "medium expression" of your protein in YPD at OD 
1 that should be ok and sufficient to see the GFP.
Wash should not be required.
Storage in the refrigerator depends on the stability/expression of your 
fusion protein. I think, a general rule cannot be given here. I would 
always analyze fresh cells.
The main problem, however, seems to be the transformants that you use. 
Endogenous fluorescence might result from material accumulating in the 
vacuole of ade1 or ade2 mutant strains - make sure that your strains are 
Ade+. GFP expressed in S.c. stays in the cytosol.
Another problem with YFP-GFP fusions is that your protein might not be 
(fully or partially) functional. If YFP is important for the cell, then a 
non fully-functional fusion may have some impact on viability/ 
morphology,etc.
In order to evaluate the localization data for a GFP-fusion it is important 
to show functionality (which is rather easy if the protein in question is 
essential and a deletion can be fully complemented by the GFP-fusion 
construct - although it might still be that 95% are mislocalized and 
detected in the wrong place and only 5% are suffucient for 
complementation...). It is also important to realize that overexpression 
may result in mis-localization.
BTW, there is a new, improved double mutant variant of GFP on the market. 
It is supposed to be >60 times brighter than the wt GFP and it can be 
detected using fluorescein filter sets...
Also, there is a GFP newsgroup on the net: 
bionet.molbio.proteins.fluorescent

Hope this helps,

Best wishes and good luck

Sepp D. Kohlwein
 



***********************************************************************
Sepp D. Kohlwein, PhD              
Genetics and Molecular Biology Group
Department of Biochemistry and Food Chemistry
Technical University Graz                   phone:  ++43 (316) 873-6456
Petersgasse 12                              fax:    ++43 (316) 873-6952
A 8010 Graz, Austria               e-mail kohlwein at ftugax.tu-graz.ac.at
***********************************************************************





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