Gal1 promoter construct of mine

Marc Lamphier lamphier at po.infosphere.or.jp
Sun Nov 26 22:35:17 EST 1995


In article <vvsvetlov-2111950922530001 at tcooper2.utmem.edu>,
vvsvetlov at utmem1.utmem.edu (killer yeast) wrote:

> In article <*-2111951143180001 at 130.216.20.102>, *@sbsu1.auckland.ac.nz
> (*@sbsu1) wrote:
> 
> Gal1 in pYES2 appears to leak on glucose. "tight" regulation of Gal1
> expression (which is not that tight) is achieved through multiple paths
> working simultaneosly - down regulation of Gal4 expression, negative
> regulation via Gal80p-Gal4p interactions, repression by Mig1/Ssn6/Tup1
> complex etc. I would not be surprised (but too lazy to check) if
> Gal1-derived promoter in pYES2 lacks Mig1p-binding sites. After all the
> idea was to get overexpression vector... 
> Regards, 
> Vladmeister
> 
> -- 

A number of derivatives of the GAL1 promoter do show low level expression
in
glucose -- you should check whether the promoter is intact or not. Also
expression from an episomal
plasmd (is pYES a 2um replicator?) is often leaky. There is little you can
do about it
as far as I know -- some of the leakiness is GAL4-independent (still occurs
in a GAL4- strain) so 
increasing expression of GAL80 might not help. Get an intact GAL1 promoter
and a lower-copy
or integrate the plasmid if it is practical. There are many varieties of
the GAL promoter around
so it shouldn't be hard to find something better. 

M. Lamphier
Univ. of Tokyo



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