[Yeast] Thiamine autofluorescence ???

Isabelle Jourdain I.Jourdain at massey.ac.nz
Mon Sep 12 00:00:46 EST 2005


Hi,

I am just starting to work with S.pombe and facing some problems with 
the expression of my genes fused GFP, using the nmt1 promotor: in 
thiamine -, my cells are so green that I cannot see anything. But when 
I add thiamine, the signal localises to mitochondria, whatever my 
genes, and even with the empty GFP-vector.

I made a series of tests to try to understand where the problem was 
from:
- I grew some WT cells in EMM + thiamine, and not transformed with any 
DNA. I read somewhere that thiamine can autofluoresce if converted into 
another chemical form.
- I made some fresh thiamine and made sure to keep it in the dark. It 
was ordered from Sigma (T4625) and autoclaved.
- Between T+ and T- conditions, I washed the cells at least 3 times 
with EMM + supp
- I tried to grow the cells in 5ml to saturation first, and then in 
50-100ml
- I transformed pREP41Ngfp that I received from 2 different labs, just 
in case the one that I used for my constructions had a mutation in the 
nmt1 promoter.
- I used a range of thiamine concentrations from 0.015 to 15uM
...

And each time, I got the same mitochondrial signal (MitoTracker 
co-localises)... unfortunately, I've got no other gfp-construct to use 
as positive control that the one that  made myself. I assume this must 
be a background signal due to thiamine rather that GFP, but despite 
spending days on Medline, I could not find any paper describing this in 
yeast. I am sure that I am doing something very silly without realising 
but I am running out of ideas and get desperate for some help.
Has anyone ever got this type of mitochondrial signal? And if this is a 
background, why am I not seeing the real GFP signal anyway?
Thank you for any information you could give me,
Isabelle


Isabelle Jourdain, PhD
Post-doctoral fellow
Private bag 11222
Massey University
Palmerston North
New Zealand
Tel: (00-64)-350-5518
Internal: -2208



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