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Figure 1 | BMC Biotechnology

Figure 1

From: Development of a Premature Stop Codon-detection method based on a bacterial two-hybrid system

Figure 1

Principles for the design of pREAL. A: Principle of E.coli two-hybrid system based on functional complementation of adenylate cyclase domains of Bordetella pertussis. The two spheres represent the T25 and T18 domains corresponding to amino acids 1–224 and 225–339 of the cya protein. In a, the full-length catalytic domain, when expressed in the cya- BTH102 strain exhibits a basal activity that results in cAMP synthesis. In b, the two independent fragments are unable to interact and no cAMP is synthesized. In c, the two fragments, fused to two interacting proteins -X and Y- are brought into close proximity, resulting in functional complementation followed by cAMP production. B: Principle of the design of the pREAL plasmid. Both adenylate cyclase fragments (T25 and T18) are subcloned into the same plasmid, leaving a multiple cloning site in between to insert a fragment of a human gene of interest. In a, T25 and T18 are linked by a human polypeptide chain and are functionally active when a PSC-free human gene is introduced in pREAL. In b, any PSC in the human gene inhibits the complete translation of both fragments as a single protein. In case of an in-frame translation re-initiation event post PSC, T18 will be present but unable to interact with T25; hence no cAMP will be synthesized.

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