Confirmation of the ZFN-mediated protein identification system.

<p>Upon the interaction of well-known proteins fused with BCRZFP and FokI, ZFNs were functional and successfully cleaved DNA at target sites. (A) Transformants with the reporter vector only cannot survive on plates lacking histidine and adenine. In contrast, colonies were observed on plates with histidine, adenine, and G418. (B) Co-transformants pRS-ZFP and pYE-FokI empty expression vectors cannot survive on plates lacking histidine and adenine. (C) Transformants of the ZFP-SV40LT fusion protein and FokI-p53 fusion protein. Upon the interaction of p53 and SV40LT, ZFNs were reconstituted and bound the target binding site. Subsequently, colonies grew on plates lacking histidine and adenine upon the restoration of functional Gal4. (D) Co-transformation of BCRZFP-orfA and FokI-E2F5 expression vectors into reporter yeast cells. Transformants survived on plates lacking histidine and adenine. (E) Co-transformation of ZFP-SV40LT and FokI-E2F5 expression vectors into reporter yeast cells. (F) Co-transformation of ZFP-orfA and p53 expression vectors into reporter yeast cells. No colonies survived on selective plates when yeast co-expression of non-cognate proteins. No cross reactivity was observed between either SV40LT/E2F5 or orfA/p53. L, leucine; T, tryptophan; H, histidine; A, adenine; and G, G418.