Let's use the example of arginine biosynthesis. There are several enzymes involved in this process, and we want to generate mutants in all of them. So, we mutagenize bacteria and look for colonies that cannot grow unless arginine is supplied in the medium. These bacteria are auxotrophic for arginine. If we generate a lot of mutants and cross them all with each other, we can discover a series of complementation groups; that is, mutants whose mutations do not complement each other and are thus in the same gene. The following chart illustrates such an experiment:
Mutants 1 and 4 can complement each other. They are therefore in different genes. Mutants 1 and 2 do not complement each other. They are in the same gene and in the same complementation group.
1 ----> 2 ----> 3 ----> arginineAnd we want to find out which of our three mutants corresponds to the enzyme that catalyzes each step. We can give our mutants various intermediates and test for growth:
The mutant that can use the least number of intermediates to grow is mutated the latest in the pathway. The mutant that can use many intermediates to grow is mutated the earliest in the pathway. The later in a pathway a mutant is blocked, the less can be done to rescue it. Thus we can establish that:
1 ----> 2 ----> 3 ----> arginine Arg1 Arg2 Arg3
We can use this phenomenon to order elements of a pathway. By generating double mutants, and discovering which phenotype is masked, we can order the genes in a pathway. For example:
In this case, the later a mutant is in a pathway, the more early mutants it can repress (that is, mask their phenotype by exhibiting its own). Thus, we get the same result as before:
1 ----> 2 ----> 3 ----> arginine Arg1 Arg2 Arg3We can use the concept of epistasis to order genes in regulatory as well as biosynthetic pathways, but we will look at those examples in terms of eukaryotic gene regulation.