Complementation Tests Determine Whether Different Recessive Mutations Are in the Same Gene
Researchers using the classical genetic approach to studying a particular cellular process often isolate multiple recessive mutations that produce the same phenotype. A common test for determining whether these mutations are in the same gene or in different genes exploits the phenomenon of genetic complementation; that is, restoration of the wild-type phenotype by mating two different mutants. If two recessive mutations, a and b, are in the same gene, then a diploid organism carrying one a allele and one b allele will exhibit the mutant phenotype because neither allele provides a functional copy of the gene. In contrast, if mutations a and b are in separate genes, then heterozygotes carrying a single copy of each mutant allele will not exhibit the mutant phenotype because a wild-type allele of each gene is also present. In this case, the mutations are said to complement each other. Complementation analysis cannot be performed on dominant mutations because the phenotype conferred by the mutant allele is displayed even in the presence of a wild-type allele of the gene.
Complementation analysis of a set of mutants exhibiting the same phenotype can distinguish the individual genes in a set of functionally related genes, all of which must function to produce a given phenotypic trait. For example, the screen for cdc mutations in Saccharomyces described previously yielded many recessive temperature-sensitive mutants that appeared to be arrested at the same cell cycle stage. To determine how many genes were affected by these mutations, Hartwell and his colleagues performed complementation tests on all of the pair-wise combinations of their cdc mutants, following the general protocol outlined in Figure 6-7. These tests organized more than 100 cdc mutations into about 20 different CDC genes. The subsequent molecular characterization of the CDC genes and their encoded proteins, as described in detail in Chapter 19, has provided a framework for understanding how cell division is regulated in organisms ranging from yeast to humans.
EXPERIMENTAL FIGURE 6-7 Complementation analysis determines whether recessive mutations are in the same or different genes. Complementation tests in yeast are performed by mating haploid a and α cells carrying different recessive mutations to produce diploid cells. In the analysis of cdc mutations, pairs of different haploid temperature-sensitive cdc strains were systematically mated and the resulting diploids tested for growth at the permissive and nonpermissive temperatures. In this hypothetical example, the cdcX and cdcY mutants complement each other, and thus have mutations in different genes, whereas the cdcX and cdcZ mutants have mutations in the same gene.