VP16/HLF1-265 +

VP16/HLF 1-265 C25S VP16

GalDBD +

Fig. 2. Cys-25 in the basic region of HLF is critical for Ref-1 interaction. COS-1 cells were cotransfected with 300 ng of Gal4-luciferase reporter G5Elb-Luc, 50 ng of expression vector chimeric for the activation domain of VP16 and HLF 1-265 or the HLF cysteine-to-serine point mutant HLF 1-265 C25S, and 50 ng of GalDBD/Ref-1 or GalDBD as indicated. After 24 hr, cells were harvested and luciferase activity was normalized relative to cotransfected pRL-TK control reporter. Results are from two independent experiments performed in triplicate and are represented as fold induction relative to reporter gene activity of nonchimeric VP16 with GalDBD. Error bars represent the standard deviation.

activity by reducing Cys-25 located in the basic DNA-binding region of HLF.8 As shown in Fig. 2, when HLF Cys-25 is mutated to a serine residue (VP16/HLF 1-265 C25S) interaction with GalDBD/Ref-1 is abolished. This suggests that the interaction between Ref-1 and HLF may depend on the formation of transient disulfide linkages, and that the two-hybrid system is capable of detecting this type of weak interaction.

A possible caveat of the mammalian two-hybrid system is that artifactual interactions between protein fragments may be invoked by spurious structures created by the chimeric fusion proteins. Although such false-positive interactions are not commonly observed, they can be guarded against by creating reciprocal chimeras to verify interactions in a new protein context. Thus, to demonstrate that the interaction of Ref-1 with HLF observed in Fig. 2 was not peculiar to the GalDBD and VP16 constructs utilized, we tested (Fig. 3) reciprocal chimeras in which

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