For some proteins,this approach offers additional advantages over screening a library using a traditional two-hybrid scheme.Proteins that activate transcription when fused to LexA or another DNA-binding domain can be difficult to use in conventional interactor hunts.Though methods are available to reduce the sensitivity of the reporter genes (Durfee et al.,1993; Estojak et al.,1995; Chapter 2,3,4)it is not always possible to reduce the reporter sensitivity below the threshold of activation for some baits.Moreover,reduction in reporter sensitivity carries with it the risk that the reporters will not detect weakly interacting proteins.Furthermore,spontaneously occurring yeast mutations,for example those that increase the copy number of the bait plasmid,can increase the activating potential of weakly activating baits (R.L.F.,R.B.,A.Mendelsohn,unpublished data); such mutations are typically scored as positive in the early stages of an interactor hunt,and they are not readily detected in schemes where the specificity test is performed by removing the bait plasmid from the strain containing the prey and mating the strain with other bait strains.Thus,an alternative for proteins that activate transcription as baits,is to use them as preys to screen existing panels of baits,or even libraries of baits.Interaction mating approaches also have clear advantages for proteins that are somewhat toxic to yeast; the prey vector allows conditional expression of toxic proteins in the presence of a bait,and often the interaction can be observed as the reporters are activated even if the cells are inviable.An example of the use of interaction mating together with a large panel of bait strains to characterize a protein that both activates transcription and is toxic to yeast,Drosophila Cyclin E (Finley,Zavitz,Thomas,Richardson,Zipursky,and Brent,in prep),is discussed in Section 7.
Figure 1.Mating assay for interactions between a prey and 96 baits
Figure 1.
Top.The plate on the left holds 96 different yeast strains in patches (or colonies)that each express a different bait protein.The plate on the right holds 96 patches,each of the same yeast strain (prey strain)that expresses a protein fused to an activation domain (prey).The plate of bait strains and the plate of prey strains are each pressed to the same replica velvet and the impression is lifted with a plate containing YPD medium.After one day of growth on the YPD plate,during which time the two strains mate to form diploids,the YPD plate is pressed to a new replica velvet and the impression is lifted with a plate containing diploid selection medium and an indicator like X-Gal.Blue patches (dark spots)on the X-Gal plate indicate that the lacZ reporter is transcribed,suggesting that the prey interacts with the bait at that location.
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Protocol 2.Collecting bait (and prey)strains
Materials
Freezing media: 1:1 solution of minimal glucose media lacking appropriate amino acids (e.g.-u-h Glu for bait strains): sterile glycerol solution (65% (v/v)glycerol,0.1 M MgSO4,25 mM Tris-HCl pH 7.4)
1.0 to 1.5 ml cryotubes
Yeast strains freshly streaked to minimal glucose plates
Sterile wooden applicator strips
Methods
1.Streak bait strains to -u-h Glu plates,or prey strains to -w Glu plates,and incubate at 30℃ for 24 to 48 hours.Yeast should be taken from the plates and frozen no more than 4 days after being streaked.
2.With a sterile wooden applicator stick,grab a dollop of yeast from the plates and inoculate 0.5 ml of freezing solution in a cryotube.Vortex lightly.This solution should have an OD600 over 3.0.
3.Alternatively,inoculate 0.5 ml of -u-h Glu liquid media to an OD600 less than 0.2,incubate at 30℃ with shaking until OD600 = 1.5 to 2.0 (log phase),and add 0.25 ml of this culture to 0.25 ml of sterile glycerol solution in a cryotube.
4.Freeze by placing cryotubes in -80℃ freezer.Most strains can be recovered after up to at least two years by scraping the surface of the ice and streaking to minimal glucose plates.Avoid allowing entire contents of cryotube to thaw.