I have been diligently using the Fields two-hybrid screen with a yeast gene
as the probe. Only now have I realized that there might be a fatal flaw in
the method when applied to yeast genes. The GAL4 gene, which of course is
present in the pGAD library, will give a positive signal regardless of the
presence or absence of the Gal4-binding-domain plasmid, thereby circumventing
the need for an interaction between two hybrids. Since the GAL4 will express
a positive signal in any reading frame in either orientation, it will provide
the majority, if not the entirety, of the cloned plasmids selected by the
screen. Screens with non-yeast libraries, which obviously do not have the
yeast GAL4 gene, would not present this difficulty.
My lab isolated 78 candidates in an exhaustive screen (of ~460,000
transformants, and checked every one of them, obly to learn that each and
every one of them was GAL4. This of course leads to extreme depression
among lab personnel.
My question to yeast netters: Is there a way around this background problem?
For instance, is there a yeast library made from a GAL4 minus strain,
preferably a delta-GAL4 strain, in the pGAD line of vectors? Such a library
would prevent the cloning of GAL4? If such a library is not yet made, is
somebody planning to make one? If such a library is not available, are there
other ways around the problem, for instance, a method for detection and
elimination of GAL4 positive clones early in the course of the screen?
Any worthwhile suggestions would be gratefully appreciated.
Michael Cusick MEC at BioVax.TAMU.Edu
Dept. of Medical Biochemistry & GEnetics
TExas A&M College of Medicine
College Station, TX 77843-1114