In article <*-2805971659100001 at j.keeling.sbs.auckland.ac.nz>,
> I require some information regarding the autofluorescence of Saccharomyces
> cerevisiae. I am trying to get GFP expression in my yeast and I was
> wondering how much background fluorescence I should expect. Also do the
> yeast fluoresce more when they are old or dying??
>> Any tips on optimizing GFP expression would be greatly appreciated, we are
> using Clontech EGFP.
> Leigh Bradbury
> School of Biological Sciences
> University of Auckland
> New Zealand.
Autofluorescence in yeast does not seem to be a problem as long as you are
using Ade+ strains. ade1 and ade2 mutants accumulate a fluorescent pigment
in the vacuole (strongly increasing in "old" cells) that is excited at the
same wavelength as GFP (488 nm), and shows up to some extent using an FITC
band pass filter.
"Optimization" means that you optimize the signal (increse expression
levels) without affecting localization and function of the tagged protein.
There are no general rules for optimization. In many cases expression from
a GAL-controlled, episomal plasmid will give you a functional and properly
targetted protein that is bright enough to be detected. In other cases, a
single integrated copy of the fusion under its native promoter will not be
functional (so any GFP-fluorescence that you might detect would not tell
you too much). You also have to consider N- or C-terminal fusions,
depending on potential localization/ targetting signals on your protein
Hope this helps,
Sepp D. Kohlwein