TI  - Genetic characterization of a mammalian protein-protein interaction domain
      by using a yeast reverse two-hybrid system.
PG  - 10321-6
AB  - Many biological processes rely upon protein-protein interactions. Hence,
      detailed analysis of these interactions is critical for their
      understanding. Due to the complexities involved, genetic approaches are
      often needed. In yeast and phage, genetic characterizations of protein
      complexes are possible. However, in multicellular organisms, such
      characterizations are limited by the lack of powerful selection systems.
      Herein we describe genetic selections that allow single amino acid changes
      that disrupt protein-protein interactions to be selected from large
      libraries of randomly generated mutant alleles. The strategy, based on a
      yeast reverse two-hybrid system, involves a first-step negative selection
      for mutations that affect interaction, followed by a second-step positive
      selection for a subset of these mutations that maintain expression of
      full-length protein (two-step selection). We have selected such mutations
      in the transcription factor  <prot>E2F1</prot>  that affect its ability to heterodimerize
      with  <prot>DP1</prot> . The mutations obtained identified a putative helix in the marked
      box, a region conserved among E2F family members, as an important
      determinant for interaction. This two-step selection procedure can be used
      to characterize any interaction domain that can be tested in the
      two-hybrid system.
AD  - Massachusetts General Hospital Cancer Center, Charlestown 02129, USA.
