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Two-hybrid problems

Michael D. Hebert mhebert at emory.edu
Wed Apr 2 18:47:49 EST 1997

I was wondering if anyone out there has had this problem when doing a
two-hybrid screen.  I am using the yeast strain Y190, a bait in pAS2, and
a cDNA library in pACT.  I have obtained -HLT colonies and found them to
have lacZ activity.  Subsequently, I have isolated the prey vector and
transformed this vector back into Y190 with bait.  This is where my
problem starts.  The resulting transformants are LT+ but are unable to
grow on -HLT and show no lacZ.  My current thinking is that the original
colony (which could grow on -HLT and was lacZ +) was the result of a
mutation somewhere on the chromosome- possibly causing transcription from
the UASg promoter without the need for interacting bait and prey
proteins.  This would explain my results with the transformation of the
prey into a new Y190 + bait strain.
Does this make any sense and is this a viable hypothesis?
Thank you in advance for helpful comments.

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