ADH1 Promoter Enhancement?

mmelan at marcie.wellesley.edu mmelan at marcie.wellesley.edu
Wed Nov 17 21:19:31 EST 1993


Dear Yeast-netters,

        Forgive me if this falls under the FAQ category but I'm not a frequent
reader of this board.....
        I am working with the Two-hybrid system of Fields and Bartel and am
having what seem to be low expression problems on my positively-interacting
clones.  The cause of this low expression of the GAL1-LacZ fusion could be that
one of the test fusions is a cytoskeleton-binding protein and is probably
binding to microtubules out in the cytoplasm and not making it into the nucleus
to interact and induce the GAL1-LacZ expression.  My reason for thinking this
is that I *eventually* get blue colonies when I lift and react with X-Gal but
it takes about a week for color development.  The activation and binding
domain plasmids are driven by the ADH1 promoter and I seem to recall reading
somewhere that expression off of this promoter is increased if glucose in the
media becomes limited.  I am currently growing my colonies on plates with 2%
glucose.  Would lowering the amount of glucose increase the expression off of
the plasmids and give a stronger signal (i.e. swamping the binding of my
proteins to the yeast microtubules and letting more of the protein get to the
nucleus)?  Is there a better/more sensitive assay for GAL1-LacZ expression
than X-Gal colony lifts?
        Any help would be greatly appreciated.  Thank you.
================================================================================Melissa Melan                                   mmelan at lucy.wellelsey.edu
Department of Biological Sciences
Wellelsey College
Wellesley, MA  02181
(617) 283-3137
================================================================================



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