Complementation and Biochemical Genetics

Complementation

When we study prokaryotic genetics, we often randomly mutagenize bacteria and look for mutants that are deficient in the process that we're studying. If we generate many mutants, how do we tell how many of them are mutated in the same gene, and how many different genes there are? One method is to do a complementation test. If two bacteria are crossed to each other, and each is mutated in the same gene, function will not be restored. On the other hand, if the mutations are in different genes, then the good copy of each gene in the other bacterium will fulfill the function of the mutated gene, or complement it. How do we cross haploid, asexually reproducing bacteria? We use some of the techniques described on the previous page to generate pseudo-diploid offspring, so that we have two copies of the same strand of DNA to compare.

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.

Elucidating Biochemical Pathways: Feeding Intermediates

Now that we have a bunch of different mutants in arginine biosynthesis, we want to find the order of the genes in the pathway for arginine biosynthesis. We know the following intermediates in the arginine biosynthesis pathway:

         1 ----> 2 ----> 3 ----> arginine

And 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

Elucidating Biochemical Pathways: Epistasis

In the above example, if we generated a bacterium that was deficient in both genes Arg1 and Arg3, the phenotype would be like that of Arg3 mutants: the bacteria would not be able to grow on intermediate 2 as can Arg1 mutants, because it is also lacking enzyme Arg3. Thus, mutants in Arg3 are epistatic to mutants in Arg1: the phenotype of Arg3 masks that of Arg1.

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    Arg3

We 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.

hyperbio@mit.edu