In yeast genome we have introduced a mutation which replaces transcription factor (TF) binding site to site for restriction endonuclease. The integration marker is URA3. The correctness of marker integration and the presence of mutation in the yeast genome was verified by PCR from genome DNA followed by restriction analysis of PCR product. To excise URA3, we transformed mutant yeast with PCR product which contains regions that flank URA3 marker and does not contain the TF site and grow yeast transformants on 5-FOA. Surprisingly, we obtain colonies that mostly have URA3 marker excised, but carrying no mutation as judged by PCR with restriction analysis. We observe this with a number of colonies that had been obtained from different initial colonies carrying marker and mutation. Could it be due to a repair of a mutation or an artifact of a procedure we used to excise URA3 marker?
How did you make the first integration event? Was it a single cross over pop-in or a replacement event? Depending on the initial structure, you may be getting a pop out that leaves behind the initial wt allele.
First step is an integration via homologous recombination without generation of repeats. I use integration cassette with URA3 marker flanked by different fragments of promoter region. It leads to insertion of URA3 marker within promoter. After insertion I select strains with mutated TF binding site.
I don't think that there is any way possible to "revert" back to the wildtype promoter sequence if the original insertion went as planned. There should no longer be a wildtype sequence available. I still wonder if you initial integration event has the expected structure. When you did the PCR checks, did you use primers that are "outside" of the original URA3 integration cassette? Sometimes you can get single-sided integration events that can fool you. That is why in the "old days" Southern blot analyses were so much better -- you saw the entire structure of the locus.
Thank you very much Craig! That is very interesting. I have checked the integration event only at one side where mutation must occur supposing that the same has happened on the other side. I will check the other side too. I guess, single-sided integration is the reason for transient integrants.
Yes, the cell often gives you crazy, unexpected recombination products. We typically use primers outside (5' and 3') of the homologous regions used to target the initial event. I guess in your case, you should see a URA3 insertion if you use a pair of primers that are in the 5' part of the promoter and within the ORF?
So, we found out that it is the single-sided integration. The 3' region with mutation is OK, but no PCR product for the 5' adjacent region with primers outside of 5'-region of recombination. Something really bad happened there.
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