FIGURE 6-8 Analysis of double mutants can often order the steps in biosynthetic or signaling pathways. When mutations in two different genes affect the same cellular process but produce distinctly different phenotypes, the phenotype of the double mutant can often reveal the order in which the two genes must function. (a) In the case of mutations that affect the same biosynthetic pathway, a double mutant will accumulate the intermediate immediately preceding the step catalyzed by the protein that acts earlier in the wild-type organism. (b) Double-mutant analysis of a signaling pathway is possible if two mutations have opposite effects on expression of a reporter gene. In this case, the observed phenotype of the double mutant provides information about the order in which the proteins act and whether they are positive or negative regulators.