( om the herpes virus VP16 protein. 10 I...)New Mammalian Two-Hybrid System Detects Protein-Protein Interactions

om the herpes virus VP16 protein.¹⁰ In addition, whereas overexpression of transcription factors containing the activation domain from VP16 can be toxic,¹¹ overexpression of the activation domain from NF-kB p65 has not been reported to be toxic.^10,12

It was demonstrated that the transcriptional-activation domain of NF-kB p65 is located in its carboxyl terminus.⁹ To determine the region of p65 that has the highest transcription activity, various segments of the mouse NF-kB p65 protein were PCR amplified. The PCR products of the NF-kB p65 segments (amino acids 283 to 550, 364 to 550, and 519 to 550) were subcloned to make pGAL4 BD-NF-kB 478, pGAL4 BD-NF-kB 476, and pGAL4 BD-NF-kB 479, respectively (Figure 3, Panel A).

Fig.3

To map domains required for maximum transactivation, we constructed chimaeric p65 proteins that contain at their N-termini the DNA binding domain of the GAL4 protein (Figure 3, Panel A). The transactivating potential of the GAL4 BD-p65 fusion proteins was tested by cotransfection with a luciferase reporter construct that contains the luciferase gene downstream of five GAL4 binding sites (pFR-Luc: PathDetect reporter plasmid). Our results indicated that sequences of p65 between amino acids 364 and 550 gave the highest luciferase activity and were even higher than the activity obtained with the GAL4 BD-VP16-fusion protein (Figure 3, Panel B). Therefore, this segment (amino acids 364
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