Double Mutants Are Useful in Assessing the Order in Which Proteins Function

Based on careful analysis of mutant phenotypes associated with a particular cellular process, researchers often can deduce the order in which a set of genes and their protein products function. Two general types of processes are amenable to such analysis: (a) biosynthetic pathways in which a precursor material is converted via one or more intermediates to a final product and (b) 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 pathway to the next. In E. coli, the genes encoding these enzymes lie adjacent to one another in the genome, constituting the

(a) Analysis of a biosynthetic pathway

A mutation in A accumulates intermediate 1. A mutation in B accumulates intermediate 2.

PHENOTYPE OF DOUBLE MUTANT:

INTERPRETATION:

A double mutation in A and B accumulates intermediate 1.

The reaction catalyzed by A precedes the reaction catalyzed by B.

clusive ordering of the steps. Double mutants defective in two steps in the pathway are particularly useful in ordering such pathways (Figure 9-8a).

In Chapter 17 we discuss the classic use of the doublemutant 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.

(b) Analysis of a signaling pathway

A mutation in A gives repressed reporter expression. A mutation in B gives constitutive reporter expression.

A mutation in A gives repressed reporter expression. A mutation in B gives constitutive reporter expression.

PHENOTYPE OF DOUBLE MUTANT:

INTERPRETATION:

A double mutation in A and B gives constitutive reporter expression.

B negatively regulates reporter expression and is negatively regulated by A.

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