charrof at ircm.umontreal.ca
Thu Sep 26 13:20:49 EST 1996
I am doing structure-function studies of a mammalian transcription factor
using pGBT9 (Clontech) fusions. It is a 2u ori, TRP1, GAL4-DBD expression
vector driven by the ADH1 promoter. I use SFY526 yeast strain, which harbors
a lacZ driven by the GAL1 promoter, integrated in URA3.
When I transform freshly grown SFY526 (using LiOAc technique) with a
GAL4-DBD/(my transcription factor (full length)) fusion, I see around 90%
blue colonies after on-plate b-gal assay (shortly, I permeabilize yeast
colonies on-plate using chloroform, 5 min, then decant the chlofoform, dry
inverted at 30C, 5 min, and overlay with 1% agarose, 100 mM KPO4, pH 7.0, 1
mg/ml X-Gal. I then incubate at 30C O/N (Trends Genet. (1996) 12, 340).
However, using various deletions of my transcription factor, I get 10% blue
colonies of different intensites, but the majority (90%) of them appear
white. My question is, why isn't that uniform?
I don't have much experience with yeast, but I know that 2u plasmids copy
number can vary a lot from cell to cell. However, a colony would be a
population of cells containing different number of plasmid, right? So I would
expect each colony to be about the same intensity. I also oberve colonies of
different size, but this doesn't seem to affect the color.
What else could affect this heterogeneity? I thought about mutations, but
this would be a rather high rate (10%).
Thanks for any help,
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