4 Genetic Pathways
Walter P. Suza; Philip W. Becraft; Faizo Kasule; and Madan K. Bhattacharyya
Introduction
Genes do not function in isolation but in groups of matching sets that perform their biological functions. When different genes function in different sequential steps of a biological process, it is known as a genetic pathway. Perhaps the most conceptually intuitive type of pathway is a biosynthetic pathway, where a precursor molecule is chemically modified through a series of enzymatically catalyzed intermediate steps to produce a bioactive product. Since enzymes are proteins, the genes that encode these enzymes are considered a genetic pathway. In a regulatory pathway, some stimulus leads to a change in the expression or activity of a particular gene product, which in turn acts to alter the expression or activity of another gene product or products, which in turn could regulate yet another level of activity. Ultimately, these regulatory changes in expression or activity result in a response to the stimulus, typically involving changes in gene expression. Again, the genes that encode the various types of regulatory molecules, mainly proteins, constitute a genetic pathway. Pathways are often targets in the biotechnological manipulation of traits, which requires an understanding of the behavior of those pathways. Also, many biosynthetic and regulatory systems are better described as networks because pathways often branch, converge, and interact with other systems. However, for simplicity, such complex networks will not be discussed here.
Biosynthetic Pathways
Genes Encode Enzymes that Catalyze Steps in the Synthesis of a Compound
A biosynthetic pathway describes a process of converting a precursor substrate into a product. In addition to the precursor substrate and product, the biosynthetic pathway includes the enzymes, the chemical reactions catalyzed by the enzymes, and all the intermediate compounds. A pathway consists of a series of steps where a precursor molecule acts as a substrate for an enzyme, which catalyzes a chemical reaction to produce a product, which then serves as a substrate for a subsequent step.
We have already discussed the one gene, one enzyme hypothesis, which was an important development in the discovery of how genes encode proteins. This analysis also provides a nice example of the concept of a biosynthetic pathway. This hypothesis was discovered by analyzing auxotrophic Neurospora mutants that were defective in the production of amino acids. That is, the biosynthetic pathways producing specific amino acids were disrupted by mutations. Figure 1 shows the arginine pathway. Mutants that disrupt this pathway cannot grow on a medium lacking arginine but can grow if arginine is supplied. The same fundamental pathway also produces arginine in plants.
Mutations that disrupt the function of specific enzymes in the pathway block the progression of the pathway at that corresponding step. For example, a mutation in the argF gene would disrupt the enzyme ornithine carbamoyltransferase. This would block step 6 in the pathway (Figure 1), the conversion of L-Ornithine to L-Citrulline. As a result, the intermediate compound L-Ornithine will likely accumulate, whereas all the compounds that occur later in the pathway (i.e., L-Citrulline, L-arginosuccinate, and L-arginine) will be deficient.
The Anthocyanin Pathway
The anthocyanin pathway is slightly more complex, although it is still simple regarding genetic pathways. It is of significant interest to horticulturists because anthocyanin pigments are responsible for many plant colors, particularly in flowers and fruits (think red wine). Anthocyanin pigments range from orange to red, purple, and blue. For example, the range of pigments seen in the petunia assortment below reflects variations in the anthocyanin pathway (Figure 2). In addition to the attractive colors anthocyanins provide, there is also considerable recent interest in anthocyanins for their dietary value, acting as strong antioxidants and possibly anti-cancer agents.
The anthocyanin biosynthetic pathway is shown in Figure 3. The enzyme chalcone synthase catalyzes the first committed step in the pathway. Subsequent enzymatic steps catalyze the synthesis of intermediate compounds, resulting in the production of anthocyanidins. Chemical modifications, such as the 3′ or 3′,5′ hydroxylation of the B ring, result in the production of different classes of anthocyanidins with varying characteristics of color. Anthocyanidins are subsequently glucosylated and transported into the cell vacuole, where they accumulate and are stored as anthocyanins. Intermediate compounds are colorless until the production of anthocyanidins. pH greatly affects the color properties, and the acidic pH of the vacuole is required for the colors normally observed for anthocyanin pigments.
Not all plants produce all these compounds, and some produce yet more varieties. For example, neither the rose nor maize genomes encode a flavanone 3′,5′-hydroxylase enzyme, so these species do not normally produce delphinidin.
Note that the word “pathway” does not adequately describe anthocyanin biosynthesis. There is not a single pathway but rather a series of intersecting pathways. For example, the intermediate compound naringenin can be subject to 3 different chemical reactions, leading to different intermediate products and ultimately to any of the four anthocyanidins shown (Figure 3). And one of the products, dihydrokaempferol, can again be subject to 3 different chemical reactions, leading to 3 different anthocyanidins. Many biosynthetic systems are far more complex, and multiple pathways often intersect. For example, carbohydrate metabolism and amino acid metabolism interconnect at multiple points. For this reason, biosynthetic systems are often referred to as biosynthetic or metabolic “networks.”
For more information, please see The Genetics and Biochemistry of Floral Pigments (Grotewold, 2006).
Effects of Mutations at Different Steps in Anthocyanin Biosynthesis
Of course, each enzyme in the anthocyanin pathway is encoded by a gene. As such, mutations can generate variation or disrupt the pathway. The maize anthocyanin pathway and examples of mutants are shown in Figure 4. The a2 mutant causes a deficiency in the anthocyanidin synthase enzyme, blocking the production of any pigmented compounds. The a1 and c2 mutants are similarly colorless (not shown). The bz2 mutant is deficient in an enzyme related to glutathione-S-transferase, which is required to transport anthocyanidins into the vacuole, resulting in the bronze-colored pigment due to the more alkaline environment of the cytosol. The bz1 mutant (not shown) produces a similar phenotype. Mutation of the pr1 gene eliminates just one class of anthocyanins. The pr1 gene encodes flavanone 3′-hydroxylase, required to produce cyanidin, which generates purple anthocyanin. In the mutant, only pelargonidin is synthesized, producing a red pigment.
Learning Activity
To answer the following questions, refer to the pathways illustrated in Figs 3 and 4. They are labeled slightly differently, but a1 encodes DFR, which catalyzes the production of leucoanthocyanidins, and a2 encodes AS or LDOX/ANS, which subsequently catalyzes the production of anthocyanidins. Assume that normal, wild-type maize kernels are the deep purple/black color as in Figure 4.
What would you predict to happen to the levels of leucoanthocyanidins in an a2 mutant compared to normal?
- no change
- increase
- The expectation would be B, that leucoanthocyanidins would increase. Because the pathway is blocked at the subsequent step, leucoanthocyanidins cannot be converted to anthocyanidins. But since all the preceding enzymes remain functional, those reactions would be expected to continue, resulting in the accumulation of leucoanthocyanidins.
- decrease
- can’t predict
- D is also a correct response. Biosynthetic pathways do not always behave as expected. For example, there could be feedback inhibition of one or more enzymes by intermediate compounds. Bottom line, we won’t know for certain until the levels are measured experimentally.
What would you predict to happen to the levels of pelargonidin in a pr1 mutant compared to normal?
- no change
- increase
- Once more, the expectation would be B, that pelargonidin would increase. Normally, some fractions of the intermediate compounds are channeled through the pathway branch leading to cyaniding. Because the pathway leading to cyanidin is blocked, it might be expected that all the intermediates would be converted to pelargonidin, causing an increased accumulation.
- decrease
- can’t predict
- D is also a correct response. As discussed in problem 1, biosynthetic pathways do not always behave as expected. Feedback inhibition could be a factor, or it may be possible that the enzymatic capacity of the pelargonidin branch of the pathway is already saturated. To increase flux through the pathway, it could be required to increase the levels or activities of one or more rate-limiting enzymes. Again, we won’t know for certain until the levels are measured experimentally.
The anthocyanin biosynthetic pathway has been subjected to extensive biotechnological manipulation. Some examples are reviewed in the accompanying article (Tanaka et al, 2008). One striking example is the production of lavender roses by the addition of a pansy gene encoding flavanone 3′,5′-hydroxylase. This enzyme is normally not encoded by the rose genome, so the delphinidin pigment produced in these transgenic roses produces beautiful flower colors not naturally found in roses (Figure 5). Similar strategies can be applied to other biosynthetic pathways to produce novel products for enhanced nutrition, pest resistance, or industrial applications.
For more information, please see Seeing is believing: engineering anthocyanin and carotenoid biosynthetic pathways (Tanaka and Ohmiya, 2008).
Regulatory Pathways
Introduction
As mentioned, a regulatory pathway is a situation where one gene or gene product controls the expression or activity of another gene or gene product, which may also perform a regulatory function. As we discussed, gene function can be regulated at many points, from transcriptional to post-translational regulation. We will examine a couple of specific examples of regulatory pathways, but first, let us consider some of the general features of regulatory pathways. Understanding the logic of regulatory pathways will become important later when we seek to use biotechnology to manipulate traits by altering their regulation.
A simplistic but useful way to help understand the logic of regulatory pathways is to consider them as a series of switches with on and off states.
Positive vs. Negative Regulators
A key feature of regulatory pathways is that regulators might function positively or negatively. A positive regulator functions to activate the next component in the pathway, whereas a negative regulator would inhibit the next component. For example, a transcription factor that activated the transcription of a gene would be considered a positive regulator. In contrast, one that repressed the transcription of a gene would be a negative regulator. Another common type of regulator is a protein kinase. Protein kinases are proteins that add a phosphate group to another protein. Phosphorylation is a common type of post-translational modification that can regulate the activity of proteins. Sometimes the phosphorylated protein becomes activated, and sometimes it is repressed.
Example 1:
A positive regulatory step is represented with an arrow. For example, “protein A activates protein B” would be represented like:
Example 2:
A negative regulatory step is represented by a bar. Therefore, “Protein C represses protein D” would be represented like:
Active vs. Inactive States of Regulators
A second point to consider in the logic of regulatory pathways is the activity state of each component. A helpful way to think about this is that each component can have an “on” or an “off” state. Of course, many proteins can have intermediate activity levels, but for simplicity’s sake, we will consider the “on” and “off” states.
The effect of a regulator being in the “on” or “off” state depends on whether it is a positive or negative regulator.
If a positive regulator is in the “on” state, it will function to activate the next factor. In example #1, if A is in the “on” or active state, it will function to switch B to the “on” or active state.
If a negative regulator is in the “on” state, it will function to switch the next factor to the “off” or inactive state. For the example #2, if C is in the “on” or active state, it will switch D to the “off” or inactive state.
Steps of Regulatory Pathways
Regulatory pathways sometimes contain many, or sometimes few, steps. They can also contain a mixture of positive and negative regulators. Let us explore a few hypothetical examples to see how the pieces fit together to regulate plant responses to stimuli.
When a pathway is depicted, all the steps are shown. For the sake of simplicity, it is assumed that the default activity state of any given component is such that the preceding step functions to change it. We will begin with a simple example containing several positive regulatory steps that activate a cellular activity, Activity 1, in response to Stimulus 1. In the pathway shown below, Stimulus 1 activates A. Therefore, we assume that in the absence of this stimulus, A is in the inactive or off state. Since A is inactive, it is not functioning to activate B, which is therefore also in the inactive state, and likewise for C. Ultimately Activity 1 does not occur in the absence of Stimulus 1 but occurs in response to the stimulus.
We can use color coding to help visualize this. Light blue represents the OFF state, and red represents the ON state.
Or we can make a table to help keep track of the states of each component in the presence or absence of a stimulus.
Now let’s look at another example containing both positive and negative regulators.
In the preceding example, components D and E would be inactive in the absence of a stimulus because they would not have been activated. But E functions as a negative regulator of F. Since E is inactive, it is not functioning to inhibit F, which then remains in the active state to promote cellular Activity 2. When Stimulus 2 is present, D and E become activated, and E functions to inactivate F. Since F is off, Activity 2 does not occur. Thus, the net response to Stimulus 2 is the repression of Activity 2.
OR
Learning Activity
Try to complete each of the following tables to determine whether the hypothetical Activity will be activated or repressed in response to each Stimulus.
activated
| Stimulus 3 → | D ⊣ | E ⊣ | F → | Activity 3 |
|---|---|---|---|---|
repressed
| Stimulus 4 → | D ⊣ | E ⊣ | F ⊣ | Activity 4 |
|---|---|---|---|---|
Click to reveal
activated
| Stimulus 3 → | D ⊣ | E ⊣ | F → | Activity 3 |
|---|---|---|---|---|
| no | off | on | off | no |
| yes | on | off | on | yes |
repressed
| Stimulus 4 → | D ⊣ | E ⊣ | F ⊣ | Activity 4 |
|---|---|---|---|---|
| no | off | on | off | yes |
| yes | on | off | on | no |
Now that we have considered regulatory pathways from a hypothetical perspective, let us consider a couple of real ones. As mentioned, regulatory pathways can take on many forms. They can consist of a series of transcription factors that regulate the expression of one another’s genes, ultimately resulting in the regulation of genes that effect some biological response. Here, we will look at a signal transduction pathway consisting of a series of post-translational modifications that occur in response to a hormone stimulus and culminate in gene expression changes to effect a response.
Signal Transduction Pathway for Hormone Signaling
Brassinosteroids (BRs) are a class of plant steroid hormones that control many aspects of plant physiology. They are most noted for their role in promoting plant growth, and mutants deficient in BR biosynthesis or signaling show dwarfism. BRs also promote a range of other responses, including yield and increased stress resistance. Figure 6 shows examples of several rice mutants with varying levels of BR deficiencies.
BR hormones, of which brassinolide (BL) is considered the most active, are recognized by a receptor called BRI1. BRI1 is a type of receptor known as a receptor kinase, which spans the cell membrane. On the outside of the cell is a receptor domain, which specifically recognizes and binds to BR. There is also a membrane-spanning domain and a protein kinase domain inside the cell. Binding to BR outside the cell then activates the protein kinase domain inside the cell. Protein kinases are enzymes that function to add phosphate groups to other proteins. As previously discussed, phosphorylation can alter the activity of a protein. Activation of the BRI1 receptor kinase then results in the activation of a signaling pathway that ultimately regulates the activity of transcription factors inside the nucleus, changing the expression of genes that regulate growth and stress resistance.
The BR signal transduction pathway is well studied and is summarized in Figure 7. Do not be concerned with memorizing the names of all these factors but focus on understanding the regulatory logic. The overall regulatory relationships of the pathway are depicted in panel a. The main target of regulation is a pair of closely related transcription factors called BZR1 and BZR2. As shown in panel a, BZR1/2 are negatively regulated by a protein called BIN2 in the absence of BR. BIN2 is a protein kinase that phosphorylates BZR1/2, which results in BZR1/2’s exclusion from the nucleus and their proteolytic degradation. In the absence of BR, BIN2 contains a phosphate group required for its activity.
As shown in Figure 7b, binding of BR to the BRI1 receptor domain outside the cell results in the activation of the kinase domain inside the cell. Activated BRI1 then phosphorylates a protein called BSK. Phosphorylated BSK binds another protein called BSU1, a protein phosphatase. Protein phosphatases are enzymes that remove phosphate groups from other proteins. BSK binding activates BSU1, which then catalyzes the removal of the phosphate from BIN2, thereby inactivating BIN2. Thus, BSU1 is a negative regulator of BIN2. When BIN2 is inactivated, that allows BZR1/2 to accumulate, enter the nucleus, and regulate the expression of genes that effect the BR response, including promoting plant growth and yield.
For more information, please see Erect leaves caused by brassinosteroid deficiency increase biomass production and grain yield in rice (Sakamoto, 2006).
Effects of Mutation on Different Steps
Mutations can affect regulatory pathways in multiple ways. Most mutations are loss-of-function mutations where the function of the gene or gene product is partly or completely impaired. Considering the BR signaling pathway, the mutation phenotypes will differ depending on whether the mutation affects the pathway’s positive or negative regulator. A loss-of-function mutation in the bri1 gene will block the perception of the hormone. Therefore, the pathway will not be activated, resulting in a dwarf phenotype. On the other hand, a loss-of-function in the bin2 gene will release BZR1/2 from regulation. The result will be constant BZR1/2 activity regardless of whether BR hormone is present. This might be expected to generate giant plants, but the effects of deregulating hormone responses are more complex. Let us call it unregulated growth.
Mutations can also be gain-of-function, where the gene product assumes a form that is constantly in the active state. Such mutations are typically dominant and are much less common than loss-of-function. Again, the outcome of a gain-of-function mutation on the BR signaling pathway will vary depending on whether it affects the pathway’s positive or negative regulator. A gain-of-function mutation in a positive regulator would cause deregulated hormone responses, whereas such a mutation in a negative regulator would permanently shut down the pathway and cause dwarfism.
Complementary Gene Action and Genetic Epistasis in the Context of Pathways
Several genetic principles make more sense considering that biological processes are controlled by multiple genes acting together. First, it becomes clear how mutations in different genes can produce the same phenotypic effects. Since multiple genes are often required for a single biological process, disruption of different genes in the process might have the same net effect. From this follows the concept of complementary gene action. Mutant plants with the same phenotype are crossed together, and the offspring are normal-looking. The mutations in the parent plants likely affected different genes in the same pathway. Finally, the concept of epistasis can be best understood in the context of pathways. Note that the term epistasis is used differently by different geneticists. In quantitative genetics, epistasis refers to any gene interaction. Here, the term epistasis refers to a specific type of gene interaction where the action of one gene is masked by the action of another gene. In other words, if two mutations are combined in an individual, only one of the phenotypes is apparent. The mutant whose phenotype is apparent is said to be epistatic to the one that is masked. Let us consider these concepts in detail within the contexts of biosynthetic and regulatory pathways.
Complementary Gene Action in Biosynthetic Pathways
As mentioned, one simple means by which complementary gene action can occur is when mutations disrupt genes required for different steps in a pathway. Recall that gene complementation can only occur with recessive mutations, which, except in rare instances, would be loss-of-function mutations. Refer to the maize anthocyanin pathway shown in Figure 4A. Recessive loss-of-function mutations in the c2 gene produce a “colorless” phenotype virtually identical to mutations in the a2 gene. Now consider the following cross:
Parents: A2+/A2+; c2/c2 (colorless) X a2/a2; C2+/C2+ (colorless)
↓
F 1 : A2+/a2; c2/C2+ (purple pigmented)
The first parent has a “colorless” mutant phenotype because it is homozygous for two nonfunctional copies of the c2 gene. As such, it does not encode a functional chalcone synthase enzyme; therefore, it cannot produce any pigmented products of the anthocyanin pathway. The second parent carries two non-functional copies of the a2 gene, does not encode a functional anthocyanidin synthase enzyme, and cannot synthesize anthocyanin pigments. The F1 progeny received one functional copy of A2+ and one functional copy of C2+ from the parents; therefore, they encode functional anthocyanidin synthase and chalcone synthase enzymes. Hence, the progeny can produce purple anthocyanin pigments, and these two mutants complement.
It can also happen that two or more genes are required for a single enzymatic step. This occurs in the case of heterodimeric enzymes, enzymes composed of two different protein subunits, with each subunit encoded by a different gene. An example is ADP-glucose pyrophosphorylase, a key enzyme in starch biosynthesis (Hannah, 2005). Maize mutants disrupted in this enzyme are defective in starch production and accumulate high levels of sugars, starch precursors. This is the basis for some types of commercial sweet corn. ADP-glucose pyrophosphorylase is a heterodimeric enzyme requiring proteins encoded by two different genes, Sh2 and Bt2. Mutations in either gene disrupt the function of the ADP-glucose pyrophosphorylase enzyme, therefore, are phenotypically very similar. As discussed in the previous paragraph, if a sh2 mutant is crossed to a bt2 mutant, the F1 progeny inherits a functional copy of each gene and can produce a functional enzyme.
Epistasis in a Biosynthetic Pathway
The concept of epistasis can be readily explained in the context of a biosynthetic pathway. Consider again the anthocyanin pathway (Figure 3 and Figure 4) and the a2 and bz2 maize mutations depicted in Figure 4. Suppose an individual was homozygous for both mutations; what would be the phenotype of this double mutant? Because the a2 mutation blocks the anthocyanidin synthase step, the pathway would stop with the production of leucoanthocyanidin intermediate compounds, which are colorless. Whether any subsequent steps, including those catalyzed by the Bz2 gene product, are functional makes no difference since no substrates are produced for them to act on. Thus, the double mutant phenotype would be colorless like a2. The bz2 phenotype would be masked, and a2 would be epistatic to bz2.
Learning Activity
Predict the phenotype of an a2; pr1 double mutant
- Predict the phenotype of an a2; pr1 double mutant
- Can′t tell
- Red like pr1; pr1 would be epistatic to a2
- They would all be light red; both genes would be partially epistatic
- Colorless like a2; a2 would be epistatic to pr1
- The correct answer is D) colorless like a2; a2 would be epistatic to pr1. Because of the pr1 mutation, the left-hand branch of the pathway in Figure 4.4A would be blocked, and all the flux would be through the right-hand branch. However, this branch would be blocked by the a2 mutation, preventing the conversion of the colorless leucopelargonidin to the pigmented pelargonidin. Thus, the double mutant would be colorless, and a2 would be epistatic to pr1.
In a strictly linear pathway, such as the one shown for arginine biosynthesis (Figure 1), the rule of thumb is that the “upstream” gene (the one earlier in the pathway) is epistatic to the more “downstream” gene. However, as we see, this rule breaks down in branched pathways.
Complementary Gene Action in a Regulatory Pathway
The concept of complementary gene action is no different in a regulatory pathway than in a biosynthetic pathway. Mutations in different genes with similar roles in the pathway (e.g., both positive regulators of the pathway) would be expected to cause similar mutant phenotypes. If such mutants in different genes were crossed together, the F1 progeny would be normal because both genes would be heterozygous and therefore a functional copy of both factors would be present.
Epistasis in a Regulatory Pathway
Epistasis can be quite complex, especially in regulatory pathways. Unlike complementary gene action, epistasis is not restricted to recessive loss-of-function mutations. The situation is further complicated by the presence of positive and negative regulators in a pathway. In contrast to a biosynthetic pathway, the rule of thumb for a regulatory pathway is that the more “downstream” gene is epistatic to the more “upstream” one. Let us revert to the BR signaling pathway and look at a couple of examples to understand why.
First, consider loss-of-function mutations in the bri1 and bin2 genes, which in single mutants cause a dwarf phenotype and an unregulated growth phenotype, respectively. What do we expect if the two mutations are combined into a double mutant plant? The bri1 mutations disrupt the receptor function required to initiate signaling through the pathway. However, BIN2 functions to inhibit the activity of the pathway by repressing the function of BZR1/2. If BIN2 is rendered non-functional, then BZR1/2 will be active and promote the growth response. This will be true even if the bri1 mutation blocks previous steps. So, the double mutant would show the bin2 unregulated growth phenotype, making bin2 epistatic to bri1.
What about a gain-of-function mutation in BZR1 or BZR2 combined with a loss-of-function mutation in bri1? A gain-of-function mutation would render BZR1/2 constitutively active, regardless of the activity state of BIN2, resulting in the unregulated growth phenotype. Since BIN2 activity would be irrelevant, upstream activities would also be irrelevant. Thus, the double mutant would show unregulated growth, and BZR1/2 would be epistatic to bri1.
Learning Activity
Predict the phenotype of a BSK gain-of-function.
A gain-of-function mutation in the BZR2 gene
- Unregulated growth
- The correct answer is A) unregulated growth. BSK is a positive regulator of the pathway that functions to activate BSU1. Therefore, a gain-of-function would cause it to positively activate the pathway, even in the absence of BR hormone, causing unregulated growth.
- No effect
- Can’t predict
- dwarf
- The correct answer is D) dwarf. The gain-of-function BIN2 mutation locks BIN2 in the ON state, where it would function to inhibit the activity of BZR1/2. It would be impervious to regulation by the upstream BSU1, so even if BSU1 were continually active due to the gain-of-function BSK, the BIN2 mutant would inhibit growth and therefore be epistatic.
Now predict the phenotype of a double mutant between the BSK gain-of-function and a BIN2 gain-of-function.
- Can’t predict
- No effect
- Dwarf
- The correct answer is C) dwarf. The gain-of-function BIN2 mutation locks BIN2 in the ON state, where it would function to inhibit the activity of BZR1/2. It would be impervious to regulation by the upstream BSU1, so even if BSU1 were continually active due to the gain-of-function BSK, the BIN2 mutant would inhibit growth and therefore be epistatic.
- Unregulated growth
Chapter Summary
Genes do not act alone but function in concert with other genes to perform various biological functions, such as the biosynthesis of a compound or the regulation of a response to a stimulus. When genes control a sequential series of steps, that is called a pathway. But pathways typically branch and intersect with other pathways to form networks. Mutations affect the activity of pathways in various ways, depending on the nature of the mutation (loss vs. gain-of-function) and the function of the gene product (positive vs. negative effector).
When mutations affect different steps in a pathway, they can often have a similar overall effect on the pathway, resulting in similar phenotypic consequences. Such mutations will complement one another even though their phenotypes are identical. It is also possible that mutations in different steps of a pathway can cause different phenotypes. When such mutations are combined into a double mutant individual, the phenotype of one is often masked by the phenotype of the other epistatic mutation. When pathways are linear, the “upstream” mutation is generally epistatic in biosynthetic pathways, and in regulatory pathways, it is generally the “downstream” mutation that is epistatic. In branched pathways or networks, it is more difficult to generalize.
Complementary gene action is important for geneticists trying to identify all the steps in a pathway. This is the basis of the classic complementation test used to determine if mutations are allelic or affect independent genes. Epistasis is also useful to geneticists who want to understand whether mutations with different phenotypes affect the same pathway, and if so, to determine the order of gene action.
When biotechnology is used to manipulate a trait, it is the activity of a pathway or network being targeted. The same genetic principles outlined in this section are used to design biotechnological strategies to alter pathway activities to produce the desired outcome on the trait of interest.
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