Multicopy suppressor screen
Lab/Group: Wolf Frommer lab (Carnegie Institution, Stanford)
Related Journal & Article Information
Journal: Nature
Introduction
To identify mutations that restore the activity of a defective ammonium transporter, natural suppressors or supprssors generated by mutagenesis can be identified.
Materials
Reagents
Equipment
Procedure
1) Select suppressor mutants from the 31019b yeast strain(1) carrying the high copy number plasmid pDR-AtAMT1;1Thr460Ala by growing at 28ºC for 8 days on solid YNB medium supplemented with 3% glucose and 2 mM ammonium chloride.
2) Pick suppressors and amplify in liquid YNB medium supplemented with 3% glucose and 2 mM ammonium.
3) Isolate plasmid from yeast by CTAB extraction and use for E. coli transformation.
4) Amplify plasmids in E. coli, isolate and sequence.
Troubleshooting
Critical Steps
Anticipated Results
When an inactive transporter Trans is subjected to a multi-copy suppressor screen, point mutations (single base changes, primarily) may occur spontaneously in the sequence of Trans. Such a spontaneous mutation occurs first in a single plasmid molecule, yielding a single Trans-Mut sequence. If Trans-Mut does not contribute to transport and growth, the cell will die and the sequence will be lost. If Trans-Mut does improve transport, it will initially function in (Trans)2(Trans-Mut)1 complexes, as Trans is present in vast excess. If activity is sufficient to lead to growth, Trans-Mut will replicate sufficiently to be observed by plasmid DNA sequencing. Reversion to the wild-type sequence in a single plasmid molecule would yield (Trans)2(wt)1 and (Trans)1(wt)2 complexes at the outset (wt3 would be a sufficiently small portion of the population that it would not contribute to growth). The absence of wild-type revertants from the suppressor screens suggests that these complexes are inactive.
References
(1) Marini, A. M., Soussi-Boudekou, S., Vissers, S. & André, B. A family of ammonium transporters in Saccharomyces cerevisiae. Mol. Cell. Biol. 17, 4282-4293 (1997).
Acknowledgements
Keywords
Yeast, Random mutagenesis, Yeast complementation