GFP in yeasts

Michael Moser moser at U.WASHINGTON.EDU
Thu Mar 23 14:09:02 EST 1995


Dear Yeasties,
	I thought that I would elaborate on our successes with GFP in S. 
cerevisiae and S. pombe.  I made a construct that allows fusion of GFP to
the N-terminus of any protein of interest via an NcoI site.  Another thing
that I did was to create a 10 amino acid Gly-Ala flexible spacer at the
fusion junction.  I also incorporated the "6X GFP" mutation into my
constucts.  All three fusion proteins we have made so far complement a
chromosomal delete (1 in pombe and 2 in cerevisiae).  I would say the
mutant form of the protein allows a significant increase in fluorescence
within the yeast.  There was a range of brightness between the 3 fusions. 
The fusion to S. pombe calmodulin gave reasonable fluorescence, the
cerevisiae NUF1 fusion a bit less and the cerevisiae MYO2 fusion was quite
faint.  All the fusions could be visualized much easier using confocal
microscopy.  One problem with the 6X mutant was that it seems to bleach a
bit faster on the confocal than the wt GFP, althouguh it is not supposed
to.  Another important point I found is that the cells have to be freshly
grown log phase cells for optimal results.  I believe the spacer that I
engineered may allow the chromophore domain to be cleaved from the fusion
over time in stationary phase cells.  I also saw that overexpression of
the fusion protein in S. pombe caused significant mislocalization in that
the entire cytoplasm was fluorescent in those cells.  In pombe I had to
integrate my construct for optimal results.  I want to thank people out
there for sharing their unpublished results on this topic and I hope
others will come forward to share their results on this exciting topic.
Sincerely,

Mike Moser                                             Tel: 206-543-5354
Department of Biochemistry  SJ-70                      FAX: 206-685-1792
University of Washington                          moser at u.washington.edu
Seattle, WA  98195                          Make peace my beast is yeast




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