B. Then a null mutation (a mutation that abolishes function) in gene A will arrest the process at step A, regardless of whether gene B is functional or not, whereas a null mutation in gene B will cause arrest at step B only if gene A is still active. In such a case, gene A is said to be epistatic to gene B. By comparing the phenotypes of the different combinations of mutations, we can therefore discover the order in which the genes act. This type of analysis is called epistasis analysis. As an example, the pathway of protein secretion in yeast has been analyzed in this way. Different mutations in this pathway cause proteins to accumulate aberrantly in the endoplasmic reticulum (ER) or in the Golgi apparatus. When a yeast cell is engineered to carry both a mutation that blocks protein processing in the ER and a mutation that blocks processing in the Golgi apparatus, proteins accumulate in
Figure 8–48 A complementation test can reveal that mutations in two different genes are responsible for the same abnormal phenotype. When an albino (white) bird from one strain is bred with an albino from a different strain, the resulting offspring (bottom) have normal coloration. This restoration of the wild-type plumage indicates that the two white breeds lack color because of recessive mutations in different genes. (From W. Bateson, Mendel’s Principles of Heredity, 1st ed. Cambridge, UK: Cambridge University Press, 1913.) the ER. This indicates that proteins must pass through the ER before being sent to the Golgi before secretion (Figure 8–49). Strictly speaking, an epistasis analysis can only provide information about gene order in a pathway when both mutations are null alleles. When the mutations retain partial function, their epistasis interactions can be difficult to interpret.
Sometimes, a double mutant will show a new or more severe phenotype than either single mutant alone. This type of genetic interaction is called a synthetic phenotype, and if the phenotype is death of the organism, it is called synthetic lethality. In most cases, a synthetic phenotype indicates that the two genes act in two different parallel pathways, either of which is capable of mediating the same cell process. Thus, when both pathways are disrupted in the double mutant, the process fails altogether, and the synthetic phenotype is observed.
Once a collection of mutant organisms with interesting phenotypes has been obtained, the next task is to identify the gene or genes responsible for the altered phenotype. If the phenotype has been produced by insertional mutagenesis, locating the disrupted gene is fairly simple. DNA fragments containing the insertion (a transposon or a retrovirus, for example) are amplified by PCR, and the nucleotide sequence of the flanking DNA is determined. The gene affected by the insertion can then be identified by a computer-aided search of the complete genome sequence of the organism.
If a DNA-damaging chemical was used to generate the mutations, identifying the inactivated gene is often more laborious, but there are several powerful strategies available. If the genome size of the organism is small (for example, for bacteria or simple eukaryotes), it is possible to simply determine the genome sequence of the mutant organism and identify the affected gene by comparison with the wild-type sequence. Because of the continuous accumulation of neutral mutations, there will probably be differences between the two genome sequences in addition to the mutation responsible for the phenotype. One way of proving that a mutation is causative is to introduce the putative mutation back into a normal organism and determine whether or not it causes the mutant phenotype. We will discuss how this is accomplished later in the chapter.
Genetic screens in model experimental organisms have been spectacularly successful in identifying genes and relating them to various phenotypes, including many that are conserved between these organisms and humans. But how can we study humans directly? They do not reproduce rapidly, cannot be treated with mutagens, and, if they have a defect in an essential process such as DNA replication, would die long before birth.
Despite their limitations compared to model organisms, humans are becoming increasingly attractive subjects for genetic studies. Because the human