suspicious yeast - troubles with transformation during two hybrid assay (Jan/30/2007 )

Hi,

This message is to anybody working with yeast cells, especially in conjunction with yeast two hybrid assays. I was busy with the two-hybrid system for years, but I’m really running out of ideas how to address the enigmatic troubles stated below.

In our lab we are using the Matchmaker GAL4 Two-Hybrid System 3 from Clontech. Recently, we encounter the following issue during two-hybrid assays: When we performed double transformations with selection on SD/-Leu-Trp we obtained colonies of obviously diverse phenotypes. The colonies varied conspicuously in size and color. Moreover, the colonies exhibited weird shapes and surfaces. LacZ overlay assay revealed differing activation of the reporter gene. Taken together, we observed at least two distinct phenotypes on the same selection pate, regarding size, shape, color, and activation of reporter genes. Until this point the obvious solution to the problem, seems to be that some contaminating foreign yeast strains screwed up our experiments. But the issue is more complicated.

First, the problems can’t be circumvented by replacing all transformation solutions (PEG, LiAC, ssDNA, H2O). Moreover, single transformation revealed that the problems might be cause by the plasmid coding for the GAL4 activation domain (pGAD10, pACT2, pGADT7), since transformation with pGBKT7 and selection on SD/-trp does not lead to odd colony phenotypes. In contrary, single transformation of pGADT7 fusion constructs and selection on SD/-leu results in the same confusing pattern of yeast phenotypes as described for the double transformations. The effect is independent of whether we used DNA originated from Minipreps or Maxipreps. Furthermore, it seems to be irrelevant whether the plasmid codes for a protein supposed to display interaction or an unrelated inert protein. All DNAs were verified by restriction digest and sequencing, thus ensuring that we did not apply mixtures of DNA. Frequently, the DNA preps for pGBKT7 and pGADT7 constructs were done at the same time and with the same kits. We also could not fix the troubles by replacing the yeast strain. Both, AH109 and Y190 demonstrated the same strange behavior. Finally, we organized a yeast strain (AH109) from an independent source, but the outcome of our transformations was the same.

Maybe somehow the dropout medium became permissive for growth of non transformed yeast cells. However, this seems unlikely because the effect is the same for two independent dropout media (SD/-leu and SD/-leu-trp). I speculated that in a small percentage of yeast clones the LEU2 selection marker coded by plasmids such as pGADT7 integrated into the yeast genome; thereby conferring the ability for growth on dropout medium while at the same time loosing fusion protein expression. I was searching the literature for some solution on the issue of distinct yeast phenotypes resulting from a single transformation, but could not succeed. It is really frustrating during two hybrid assays, because the final results depend on the arbitrary selected clones. From a single plate we are able to pick a clone that activates the LacZ reporter gene and one that does not; a fact that consequently diminishes the reliability of the assay. We are currently avoiding the problem by plating the entire transformation mixture on SD/-Leu-Trp and at the same time on SD/-Ade-His-Leu-Trp. The trouble with divers phenotypes remains on the double selection, however seems to disappear when additional selection for reporter gene activation is applied. The phenotypes on SD/-Ade-His-Leu-Trp are uniformly and unsuspicious. Still, we obtained ambiguous results for some candidate proteins, because the numbers of transformants on SD/-Leu-Trp and SD/-Ade-His-Leu-Trp differed considerable. These, results are hardly to interpret and may be again due to the annoying multiple yeast phenotypes.

If anybody has encountered the same or similar problems please let me know. I’d really appreciate any proposals.

Hi,
I had some problems with Y2H too. When I co-transform pGADT7 and pGBKT7 on SD/-Leu-Trp. They are giving me the colonies. Infact more than the testing interactions. Can you please tell me the amount you are plating on SD/-Ade-His-Leu-Trp plates? Can we do direct plating on SD/-Ade-His-Leu-Trp plates? I will be very happy if u can send me the protocol for this.
Regards,
Niraj

I was just wondering what the "weird" phenotypes of the yeast are. I have done several yeast two hybrids and haven't seen strange colonies but i have a potential suggestion. If you have variable activation of reporter genes in your strains (which can happen, even with proteins that interact, depending on expression levels etc) then this can lead to different phenotypes. If yeast lack adenine they they turn pink - is this the strange thing about yours? Also, it's fairly typical, in my experience, to get fewer transformants on -lei-trp-his-ade than on -leu-trp because of slightly variable expression of proteins, the quadruple dropout media is much more stringent than the double. Some proteins that interact grow poorly on the quadruple.