Re-two hybrid false? positi

David Stillman david.stillman at qmserver.genetics.utah.edu
Tue Apr 28 10:39:03 EST 1998


Subject:  Re:two hybrid false? positives

In article <2.2.32.19980421210206.00674cf0 at mail.cc.umanitoba.ca> Deb Court,
dcourt at cc.UManitoba.CA writes:
>Subject: two hybrid false? positives
>From: Deb Court, dcourt at cc.UManitoba.CA
>Date: 21 Apr 1998 14:51:11 -0700
>>Hi! Does anyone have any experience with two hybrid "positives" in which
>only a few (<10) aa have been fused to the activation domain? The clones
>seem to have passed the genetic tests for false positives.
>Thanks,
>Deb Court
>

Hi, 

We have had similar experiences in a two hybrid screen with a yeast genomic
library, and the following discussion pertains only to such a genomic library,
but not to a cDNA library. 

As described in Mol. Gen. Genet. 26:376 (1997), we identified a number of two
hybrid positives that had only a few amino acids fused to the activation
domain. The inserts in these clones were 5 - 10 kb, and we found that another
open reading frame in the insert was responsible for activation of the two
hybrid promoter. These genes do not require fusion to the Gal4 activation
domain (GAD), and we referred to these as clones as GAD-independent. Thus when
these GAD-independent genes are overexpressed from a simple multicopy yeast
plasmid they can, with the bait, activate the two hybrid promoter. This
suggests that these proteins contain their own activation domains. 

Several other labs have identified GAD-independent two hybrid activation, as
listed in the references below. Interestingly, most of these screens involved
transcriptional regulators. 

There are several ways to quickly characterize a clone as GAD-independent. We
used fusions to LexA as bait, and there is a unique MluI site within the lexA
coding region. One can digest the clone with MluI, fill out with Klenow, and
religate. (This assumes that the insert lacks a MluI site; otherwise a MluI
partial digest is required.) This destroys the reading frame of lexA, as well
as that of any lexA fusion. If the resulting "MluI blowout" clone retains the
ability to activate the two hybrid promoter one can conclude that it is
GAD-independent activation. For Gal4 DNA-binding domain fusions, there is an
XhoI site that can be used similarly.  
A second way to characterize a clone as GAD-independent is to subclone the
insert into a standard YEp vector, without any activation domain fusion. 

Hope this helps!

David Stillman
stillman at genetics.utah.edu

References

Kasten, M. M., and D. J. Stillman. 1997. Identification of the Saccharomyces
cerevisiae STB1 - STB5 genes encoding Sin3p binding proteins. Mol. Gen. Genet.
256:376-386.

Lesage, P., X. Yang, and M. Carlson. 1996. Yeast SNF1 protein kinase interacts
with SIP4, a C6 zinc cluster transcriptional activator: a new role for SNF1 in
the glucose response. Mol. Cell. Biol. 16:1921-1928.


Page, N., J. Sheraton, J. L. Brown, R. C. Stewart, and H. Bussey. 1996.
Identification of ASK10 as a multicopy activator of Skn7p-dependent
transcription of a HIS3 reporter gene. Yeast 12:267-272.

Wotton, D., K. Freeman, and D. Shore. 1996. Multimerization of Hsp42p, a novel
heat shock protein of Saccharomyces cerevisiae, is dependent on a conserved
carboxyl-terminal sequence. J. Biol. Chem. 271:2717-2723.







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