In this example, the wild-type protein has a specific cell function denoted by the red rays. Mutations that eliminate this function or inactivate it at higher temperatures are shown. The conditional mutant protein carries an amino acid substitution (red) that prevents its proper folding at 37ºC, but allows the protein to fold and function normally at 25ºC. Such temperature-sensitive conditional mutations are especially useful for studying essential genes; the organism can be grown under the permissive condition and then be moved to the nonpermissive condition to study the consequences of losing the gene product.
of function. It is easy to determine if a mutation is dominant or recessive. One simply mates a mutant with a wild type to obtain diploid cells or organisms. The progeny from the mating will be heterozygous for the mutation. If the mutant phenotype is no longer observed, one can conclude that the mutation is recessive and is very likely to be a loss-of-function mutation (see Panel 8–2).
Complementation Tests Reveal Whether Two Mutations Are in the Same Gene or Different Genes
A large-scale genetic screen can turn up many different mutations that show the same phenotype. These defects might lie in different genes that function in the same process, or they might represent different mutations in the same gene. Alternative forms of the same gene are known as alleles. The most common difference between alleles is a substitution of a single nucleotide pair, but different alleles can also bear deletions, substitutions, and duplications. How can we tell, then, whether two mutations that produce the same phenotype occur in the same gene or in different genes? If the mutations are recessive—if, for example, they represent a loss of function of a particular gene—a complementation test can be used to ascertain whether the mutations fall in the same gene or in different genes. To test complementation in a diploid organism, an individual that is homozygous for one mutation—that is, it possesses two identical alleles of the mutant gene in question—is mated with an individual that is homozygous for the other mutation. If the two mutations are in the same gene, the offspring show the mutant phenotype, because they still will have no normal copies of the gene in question (Figure 8–48). If, in contrast, the mutations fall in different genes, the resulting offspring show a normal phenotype, because they retain one normal copy (and one mutant copy) of each gene; the mutations thereby complement one another and restore a normal phenotype. Complementation testing of mutants identified during genetic screens has revealed, for example, that 5 different genes are required for yeast to digest the sugar galactose, 20 genes are needed for E. coli to build a functional flagellum, 48 genes are involved in assembling bacteriophage T4 viral particles, and hundreds of genes are involved in the development of an adult nematode worm from a fertilized egg.
Once a set of genes involved in a particular biological process has been identified, the next step is often to determine in which order the genes function. Gene order is perhaps easiest to explain for metabolic pathways, where, for example, enzyme A is necessary to produce the substrate for enzyme B. In this case, we would say that the gene encoding enzyme A acts before (upstream of) the gene encoding enzyme B in the pathway. Similarly, where one protein regulates the activity of another protein, we would say that the former gene acts before the latter. Gene order can, in many cases, be determined purely by genetic analysis without any knowledge of the mechanism of action of the gene products involved.
Suppose we have a biosynthetic process consisting of a sequence of steps, such that performance of step B is conditional on completion of the preceding step A; and suppose gene A is required for step A, and gene B is required for step