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,
jbhogen at merle.acns.nwu.edu