By careful analysis of mutant phenotypes associated with a particular cellular process, researchers can often deduce the order in which a set of genes and their protein products function. Two general types of processes are amenable to such analysis: (1) biosynthetic pathways in which a precursor material is converted via one or more intermediates to a final product, and (2) signaling pathways that regulate other processes and involve the flow of information rather than chemical intermediates.

Ordering of Biosynthetic Pathways A simple example of the first type of process is the biosynthesis of a metabolite such as the amino acid tryptophan in bacteria. In this case, each of the enzymes required for synthesis of tryptophan catalyzes the conversion of one of the intermediates in the biosynthetic pathway to the next. In E. coli, the genes encoding these enzymes lie adjacent to one another in the genome, constituting the trp operon (see Figure 5-13). The order of action of the different genes for these enzymes, and hence the order of the biochemical reactions in the pathway, was initially deduced from the types of intermediate compounds that accumulated in each mutant. In the case of complex synthetic pathways, however, phenotypic analysis of mutants defective in a single step may give ambiguous results that do not permit conclusive ordering of the steps. Double mutants defective in two steps in the pathway are particularly useful in ordering such pathways (Figure 6-8a).

In Chapter 14, we discuss the classic use of the double-mutant strategy to help elucidate the secretory pathway. In this pathway, proteins to be secreted from the cell move from their site of synthesis on the rough endoplasmic reticulum (ER) to the Golgi complex, then to secretory vesicles, and finally to the cell surface.

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Ordering of Signaling Pathways As we will learn in later chapters, the expression of many eukaryotic genes is regulated by signaling pathways that are initiated by extracellular hormones, growth factors, or other signals. Such signaling pathways may include numerous components, and double-mutant analysis can often provide insight into the functions and interactions of these components. The only prerequisite for obtaining useful information from this type of analysis is that the two mutations must have very different, or even opposite, effects on the output of the same regulated pathway as measured by expression of a reporter gene. Most commonly, one mutation represses expression of a particular reporter gene even when the signal is present, while another mutation results in reporter gene expression even when the signal is absent (i.e., constitutive expression). As illustrated in Figure 6-8b, two simple regulatory mechanisms are consistent with such single mutants, but the double-mutant phenotype can distinguish between them. This general approach has enabled geneticists to delineate many of the key steps in a variety of different regulatory pathways, setting the stage for more specific biochemical assays.

Note that this technique differs from the complementation analysis just described in that both dominant and recessive mutants can be subjected to double-mutant analysis. When two recessive mutations are tested, the double mutant created must be homozygous for both mutations.