Given a mutation in a gene of interest, two genetic tests are used to search for partners: (1) identification of a second mutation that ameliorates the effects of the primary mutation and (2) identification of a second mutation that makes the phenotype more severe, often lethal. A specialized class of enhancer mutations, called synthetic lethal mutations, is particularly useful in the analysis of genetic pathways in yeast. In this case, mutations in two genes in the same pathway, if present in the same cell, even as heterozygotes, cannot be tolerated, so the cell dies. It is thought that each mutation lowers the level of production of some critical factor just a bit and that the combination of the two effectively means that the output of the pathway is insuffcient for survival. These tests can be made with existing collections of mutations by genetically crossing mutant organisms. Alternatively, one can seek new mutations created by a second round of mutagenesis. The results depend on the architecture of the particular pathway. If the products of the genes in question operate in a sequence, analysis of single and double mutants can often reveal their order in the pathway. For essential genes in haploid organisms, a conditional allele of the primary mutation simplifies the experiment. Synthetic interactions may also be discovered by overproduction of wild-type genes on a plasmid. Caution is required in interpreting suppressor and enhancer mutations, given the complexity of cellular systems and the possibility of unanticipated consequences of the mutations.

Another approach to find protein partners is called a two-hybrid assay. This assay depends on the observation that some activators of transcription have two modular domains with discrete functions: One domain binds target sites on DNA, and the other recruits the transcriptional apparatus. The target gene is expressed if both activities are present at the transcription start site, even if the activities are on two different proteins. For the two-hybrid assay, the coding sequence of the protein whose partners are to be identified is fused to the coding sequence of a yeast protein that recognizes a target DNA sequence upstream of a gene that provides the readout of the assay. This so-called bait protein is expressed constitutively in yeast cells. A plasmid library is constructed consisting of cDNA sequences of all possible interaction partners, each fused to the coding sequence of an activator domain and a nuclear localization sequence. This library of prey proteins is introduced into the bait yeast strain. The readout gene is expressed if a prey protein binds the bait protein and recruits the transcriptional apparatus. Many variations of this assay exist. One produces an enzyme that makes a colored product, so colonies of yeast with interacting proteins can be identified visually. In another version, the target gene encodes a gene essential for production of a particular amino acid, so only cells with a bait-prey interaction will grow on agar plates lacking that amino acid. Putative interactions must subsequently be tested carefully to define specificity, as false-positive results are common. Moreover, some valid interactions are missed owing to false-negative results.