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CEN vs. 2 u plasmid expression

jbhogen at merle.acns.nwu.edu jbhogen at merle.acns.nwu.edu
Tue Oct 11 14:00:28 EST 1994


Buddies,
	Help!  We have a signalling system set up in yeast both on 2 u plasmids 
and on CEN vectors using the identical promoter and terminator (ADH).  
Basically, it's a lex fusion protein that responds to a ligand and activates 
reporter genes (HIS or LacZ or both).  I'm seeing higher background and more 
expression off my CEN vector, than off the 2 u, which I think is curious. In 
addition, the background rates for both constructs are extremely high (1-5 % 
loss of function, defined as no response to ligand).  
	This problem has plagued me for months, does anyone out there have any 
idea what could be happening?  I would like to do some saturation mutagenesis 
work on these constructs, but the basal rate is really high and variable, 
making it difficult to spot "real" mutants.
	I have done the appropriate controls, and their LOF rates are nowhere 
near what these are.  I have also tried four different strains and making the 
GAL4 fusions instead--same results.

Any ideas would be greatly appreciated,

John Hogenesch
jbhogen at merle.acns.nwu.edu



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