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Modern Genetic Analysis Chapter 6. Gene Interaction
Gene Interaction Leads to Modified Dihybrid Ratios

Genetic analysis can identify the genes that interact in the determination of a particular biological property. The key diagnostic is that two interacting genes produce modified dihybrid ratios. There are various types of interactions, and these lead to a range of different modifications. An important distinction is between interacting genes in different biological pathways and those in the same pathway.

Interacting Genes in Different Pathways

A simple, yet striking, example of gene interaction is the inheritance of skin coloration in corn snakes. The natural color is a repeating black-and-orange camouflage pattern, as shown in Figure 6-11a. The phenotype is produced by two separate pigments, both of which are under genetic control. One gene determines the orange pigment, and the alleles we shall consider are o+ (presence of orange pigment) and o (absence of orange pigment). Another gene determines the black pigment, with alleles b+ (presence of black pigment) and b (absence of black pigment). These two genes are unlinked. The natural pattern is produced by the genotype o+/ ; b+/. A snake that is o/o ; b+/is black because it lacks orange pigment (Figure 6-11b), and a snake that is o+/ ; b/b is orange because it lacks the black pigment (Figure 6-11c). The double homozygous recessive o/o ; b/b is albino, as shown in Figure 6-11d. Notice, however, that the faint pink color of the albino is from yet another pigment, the hemoglobin of the blood that is visible through this snake's skin when the other pigments are absent. The albino snake also shows clearly that there is another element to the skin pigmentation pattern in addition to pigmentthe repeating motif in and around which pigment is deposited. Since there are two genes in this system, we obtain a typical dihybrid inheritance pattern, and the four unique phenotypes form a 9:3:3:1 ratio in the F2. A typical analysis might be as follows:

In summary, the 9:3:3:1 dihybrid ratio is produced because the mutations are in two parallel biochemical pathways:

When the mutations are in one pathway, different ratios are seen, as in the next examples.

Interacting Genes in the Same Pathway

Here we need only return to the example of petal color in harebells, discussed under complementation. The pathway terminated in a blue pigment, and the intermediates were all colorless. Two different white-petaled homozygous lines of harebells were crossed, and the F1 was blue-flowered, showing complementation. What will the F2 resulting from crossing the F1 plants be like? The F2 shows both blue and white plants in a ratio of 9:7. How can these results be explained? The 9:7 ratio is clearly a modification of the dihybrid 9:3:3:1 ratio, with the 3:3:1 combined to make 7. The cross of the two white lines and subsequent generations can be represented as follows:

The results show that homozygosity for the recessive mutant allele of either or both genes causes a plant to have white petals. To have the blue phenotype, a plant must have at least one dominant allele of both genes.

If one or more intermediates in the pathway are colored, then a different F2 ratio is produced. In the following example, taken from the plant blue-eyed Mary (Collinsia parviflora), the pathway is

The w and m genes are not linked. If homozygous white and magenta plants are crossed, the F1 and F2 are as follows:

Overall a 9:3:4 phenotypic ratio is produced. This kind of interaction is called epistasis, which literally means "standing on"; in other words, an allele of one gene masks expression of alleles of another gene. In this example the w allele is epistatic on m+ and m. Conversely, m+ and m can be expressed only in the presence of w+. Because a recessive allele is epistatic, this is a case of recessive epistasis.

MESSAGE
Epistasis is inferred when an allele of one gene eliminates expression of alleles of another gene and expresses its own phenotype instead.

In general, every time one gene is higher, or "upstream," in some biochemical pathway, we would expect there to be an epistatic effect of a defective allele on alleles of genes later in the sequence. Therefore, finding a case of epistasis (for example, the 9:4:3 modified dihybrid ratio) can provide insight about the sequence in which genes act. This principle can be useful in piecing together biochemical pathways.

Another case of recessive epistasis well known to most of us is the yellow coat color of Labrador retriever dogs. Two alleles B and b stand for black and brown coats, respectively, but the allele e of another gene is epistatic on these, giving a yellow coat (Figure 6-12). Therefore the genotypes B/ ; e/e and b/b ; e/e are both of yellow phenotype whereas B/ ; E/ and b/b ; E/ are black and brown, respectively. This case of epistasis is not caused by an upstream block in a pathway leading to dark pigment. Yellow dogs can make black or brown pigment, as can be seen in their nose and lips. The action of the allele e is to prevent deposition of the pigment in hairs. In this case, the epistatic gene is developmentally downstream; it represents a kind of developmental target that has to be in the E state before pigment can be deposited.

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Epistasis points to interaction of genes in some biochemical or developmental sequence.

Suppressors

Another important type of gene interaction is suppression. A suppressor is an allele that reverses the effect of a mutation of another gene, resulting in the normal (wild-type) phenotype. For example, assume that an allele a+ produces the normal phenotype, whereas a recessive mutant allele a results in abnormality. A recessive mutant allele s at another gene suppresses the effect of a, so that the genotype a/a . s/s will have wild-type (a+-like) phenotype. Suppressor alleles often have no effect on the wild-type allele of the target gene, so in this example the phenotype of a+/a+ . s/s would be wild-type. Suppressors also result in modified dihybrid ratios. Let's look at a real example from Drosophila, using a recessive suppressor su of the unlinked recessive purple eye-color allele pd. A homozygous purple-eyed fly is crossed to a homozygous red-eyed stock carrying the suppressor.

The overall ratio in the F2 is 13 red:3 purple. This ratio is characteristic of a recessive suppressor acting on a recessive mutation. Both recessive and dominant suppressors are found.

Suppression is sometimes confused with epistasis. However, note that because a suppressor cancels the expression of a mutant allele and restores the corresponding wild-type phenotype, the modified dihybrid F2 ratio can involve only two phenotypes (normal and abnormal), whereas in the case of epistasis the epistatic allele introduces a third phenotype into the ratio.

How do suppressors work at the molecular level? There are many possible mechanisms. A well-researched type is nonsense suppressors, which act on mutations caused by stop ("nonsense") codons in the middle of the protein-coding sequence. Nonsense mutants show premature amino acid chain termination. However, a mutation in a tRNA anticodon that allows the tRNA to insert an amino acid at a nonsense codon will suppress the effect of the mutation by allowing protein synthesis to proceed past the site of the mutation in the mRNA. Since there is often repetition of tRNA genes, this suppressor will be perfectly viable. Another type of suppression is possible in cases of protein-protein interactions. If two proteins normally fit together to provide some type of cellular function, when a mutation causes a shape change in one protein, no bonding, and hence no function, occurs. However, a compensatory shape change by mutation in the second protein can act as a suppressor to restore normal binding.

Finally, in situations in which a mutation causes a block in a metabolic pathway, the suppressor finds some way of circumventing the block, for example, by channeling in substances beyond the block from related pathways.

Because of the demonstrable interaction of a suppressor with its target gene, geneticists deliberately seek suppressors as another way of piecing together a set of interacting genes that affect one biological process or structure. The approach is relatively easyperform a large-scale mutation-induction experiment starting with a mutant line (say, genotype m), and simply look for rare individuals that are wild-type. Most of these will be m+ reverse mutations, but some will be suppressed (m . su), and these can be distinguished by the dihybrid ratios produced on crossing. This procedure can be very easily applied in haploid organisms. For example, if large numbers of cells of an argininerequiring mutant (arg) are spread on a plate of growth medium lacking arginine, most cells will not grow; however, reverse mutations to the true wild-type allele (arg+) and suppressed mutations (arg . su) will grow and announce their presence by forming visible colonies. The suppressed colonies can be distinguished by crossing to wild type because arginine-requiring progeny will be produced:

MESSAGE
Suppressors cancel the expression of a mutant allele of another gene, resulting in normal wild-type phenotype.

Coat Color in a Mammalian Model, the Mouse

The analysis of coat color in mammals is a beautiful example of how different genes cooperate in the determination of overall coat appearance. The mouse is a good mammal for genetic studies because it is small and thus easy to maintain in the laboratory, and because its reproductive cycle is short. It is the best-studied mammal with regard to the genetic determination of coat color. The genetic determination of coat color in other mammals closely parallels that of mice, and for this reason the mouse acts as a model system. We shall look at examples from other mammals as our discussion proceeds. At least five major genes interact to determine the coat color of mice: the genes are A,B, C, D, and S.

The A gene.

This gene determines the distribution of pigment in the hair. The wild-type allele A produces a phenotype called agouti. Agouti is an overall grayish color with a brindled, or "salt-and-pepper," appearance. It is a common color of mammals in nature. The effect is caused by a band of yellow on the otherwise dark hair shaft. In the nonagouti phenotype (determined by the allele a), the yellow band is absent, so there is solid dark pigment throughout (Figure 6-13). The lethal allele AY, discussed in an earlier section, is another allele of this gene; it makes the entire shaft yellow. Still another allele at results in a "black-and-tan" effect, a yellow belly with dark pigmentation elsewhere.

The B gene.

This gene determines the color of pigment. There are two major alleles, B coding for black pigment and b for brown. The allele B gives the normal agouti color in combination with A but gives solid black with a/a. The genotype A/ ; b/b gives a streaked brown color called cinnamon, and a/a ; b/b gives solid brown. In horses, the breeding of domestic lines seems to have eliminated the A allele that determines the agouti phenotype, although certain wild relatives of the horse do have this allele. The color we have called brown in mice is called chestnut in horses, and this phenotype also is recessive to black. Note that we have already encountered these same alleles in the determination of coat color of Labrador retrievers.

The C gene.

The wild-type allele C permits color expression, and the allele c prevents color expression. The c/c constitution is epistatic to the other color genes. The c/c animals are of course albinos, which we have already discussed in the section on human autosomal recessive disorders. Albinos are common in many mammalian species and have also been reported among birds, snakes, and fish (Figure 6-14). In most cases, the gene codes for the melanin-producing enzyme tyrosinase. In rabbits an allele of this gene, the ch (Himalayan) allele, determines that pigment will be deposited only at the body extremities. In mice the same allele also produces the phenotype called Himalayan, and in cats the same allele produces the phenotype called Siamese (Figure 6-15). The allele ch can be considered a version of the c allele with heat-sensitive expression. It is only at the colder body extremities that ch is functional and can make pigment. In warm parts of the body it is expressed just like the albino allele c. This allele shows clearly how the expression of an allele depends on the environment.

The D gene.

The D gene controls the intensity of pigment specified by the other coat color genes. The genotypes D/D and D/d permit full expression of color in mice, but d/d "dilutes" the color, making it look milky. The effect is due to an uneven distribution of pigment in the hair shaft. Dilute agouti, dilute cinnamon, dilute brown, and dilute black coats all are possible. A gene with such an effect is called a modifier gene. In horses, the D allele shows incomplete dominance. Figure 6-16 shows how dilution affects the appearance of chestnut and bay horses. Cases of dilution in the coats of house cats also are commonly seen.

Modifier gene action can be based on many different molecular mechanisms. One case involves regulatory genes that bind to the upstream region of the gene near the promoter and affect the level of transcription. Positive regulators increase ("up-regulate") transcription rates, and negative regulators decrease ("down-regulate") transcription rates. As an example, consider the regulation of a gene G. G is the normal allele coding for active protein, whereas g is a null allele (caused by a base-pair substitution) that codes for inactive protein. At an unlinked locus, R codes for a regulatory protein that causes high levels of transcription at the G locus, whereas r yields protein that allows only a basal level. If a dihybrid G/g ; R/r is selfed, a 9:3:4 ratio of protein activity is produced, as follows:

The S gene.

The S gene controls the presence or absence of spots by controlling the migration of clumps of melanocytes (pigment-producing cells) across the surface of the developing embryo. The genotype S/ results in no spots, and s/s produces a spotting pattern called piebald in both mice and horses. This pattern can be superimposed on any of the coat colors discussed earlier, with the exception of albino, of course. Piebald mutations are also known in humans.

We see that the normal coat appearance in wild mice is produced by a complex set of interacting genes determining pigment type, pigment distribution in the individual hairs, pigment distribution on the animal's body, and the presence or absence of pigment. Such interactions are deduced from modified ratios in dihybrid crosses. Figure 6-17 illustrates some of the pigment patterns in mice. Interacting genes such as these determine most characters in any organism.

MESSAGE
Different kinds of modified dihybrid ratios point to different ways in which genes can interact with one another to determine phenotype.

© 1999 by W. H. Freeman and Company. All rights reserved.