title: Using two-hybrid interactions to identify separation-of-function mutations.
abstract: Protein functions within cells frequently require they interact physically with a number of partner proteins to coordinate the appropriate biochemical processes. Mutational analysis has been quite useful for analyzing how the loss of a gene or protein impacts cell function or more specifically particular pathways. However, the genetics approach to studying gene function can be limited by not having the right mutations; for example because the mutant allele ablates all function, as is the case for deletion (null) alleles and most temperature-sensitive alleles. To dissect the relative contributions of a protein's interactions, the researcher needs mutations that specifically affect one but not all of the protein's interactions. In genetics parlance such mutations are called separation-of-function mutations. The yeast two-hybrid system has been exploited for two decades to identify protein-binding partners. Here we describe a fairly simple protocol, within reach of laboratories with molecular biology experience, for using the two-hybrid system to identify separation-of-function mutations.
authors.affiliation: Department of Biochemistry and Molecular Biology, SUNY Upstate Medical University, 750 E. Adams St., Syracuse, NY, 13210, USA.
authors.forename: Brian
authors.initials: B
authors.lastname: Haarer
authors.forename: David C
authors.initials: DC
authors.lastname: Amberg
mesh_terms: D000483:Alleles; D016296:Mutagenesis; D009154:Mutation; D011485:Protein Binding; D060066:Protein Interaction Maps; D011506:Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D020798:Two-Hybrid System Techniques
chemical_list: D011506:Proteins