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Modern Genetic Analysis Chapter 7. Gene Mutations
Mutational Analysis

Understanding the molecular basis of mutations lays the groundwork for the mutational analysis of biochemical, cellular, and developmental pathways. The usual situation is that the investigator is interested in some particular biological process and wishes to use the genetic approach to dissect this process. The goal and the challenge are to identify all of the genes that contribute to the process and then to understand the nature of the gene products (usually proteins) and how they contribute and interact in this process or pathway. Genes are identified through their mutant alleles; therefore the genetic approach begins with mutants. Generally, the following steps are followed to genetically dissect a process:

1. Design an effective mutation-detection system.
2. Use a mutagen to induce a large collection of mutants that show variations in the wild-type process.
3. Group the mutations into genes by using complementation tests.
4. Map the genes to their chromosomal loci.
5. Isolate the genes by using DNA technology.
6. Characterize the structure and function of the genes.
7. From analysis of gene and protein interaction, piece together an integrated picture of how the biological process under study works.

Detailed examples of the application of these ideas to developmental genetics will be given in Chapters 15 and 16. For the present, we need to consider the general approaches.

There is a large chasm between the expectation of the kinds of mutations that could arise in a gene and the actual recovery of mutations after mutagenesis. Several factors are crucial in the practical recovery of mutants. The choice of mutagen is important, because different mutagens will produce a different mutational array. The phenotypes that are used to identify mutant-bearing individuals also are important, because only a subset of the huge array of mutations that can arise in a given gene might lead to the production of the relevant mutant phenotype. Thus, in any given mutagenesis procedure, every gene will have its own "target size." It is possible that a gene can present a relatively large target in one kind of mutagenesis but a relatively small one in another. The challenge for the experimental geneticist is then to figure out how to design mutagenesis that will "saturate" the system; that is, identify every component in the biological process being studied.

Somatic versus Germinal Mutation

One question in the design of mutational analysis is the type of tissue or cells to examine. In multicellular organisms, genes can mutate in either somatic or germinal tissue, and these changes are called somatic mutations and germinal mutations, respectively. These two different types are shown diagrammatically in Figure 7-29. If a somatic mutation occurs in a single cell in developing somatic tissue, that cell is the pro-genitor of a population of identical mutant cells, all of which are descended from the cell that mutated. A population of identical cells derived asexually from one progenitor cell is called a clone. Because the members of a clone tend to stay close to one another during development, an observable outcome of a somatic mutation is often a patch of phenotypically mutant cells called a mutant sector. The earlier in development the mutation event, the larger the mutant sector will be (Figure 7-30). Mutant sectors can be identified visually only if their phenotype contrasts with the phenotype of the surrounding wild-type cells (Figure 7-31).

In diploids, any dominant somatic mutation is expected to show up in the phenotype of the cell or clone of cells containing it. On the other hand, a recessive mutation will not be expressed, because it is masked by a wild-type allele that is by definition dominant to the recessive mutation. A second mutation could create a homozygous recessive mutation, but this event would be extremely rare.

What would be the consequences of a somatic mutation in a cell of a fully developed organism? If the mutation is in tissue in which the cells are still dividing, then there is the possibility of a mutant clone arising. If the mutation is in a postmitotic cellthat is, one that is no longer dividingthen the effect on phenotype is likely to be negligible. Even when dominant mutations result in a cell that is either dead or defective, this loss of function will be compensated for by other normal cells in that tissue. However, mutations that give rise to cancer are a special case. Mechanisms of cancer will be considered in Chapter 15.

Are somatic mutations ever passed on to progeny? This is impossible, because somatic cells by definition are those that are never transmitted to progeny. However, note that, if we take a plant cutting from a stem or leaf that includes a mutant somatic sector, the plant that grows from the mutant cutting may develop mutant germinal tissue. Put another way, a branch bearing flowers (that is, germinal tissue) can grow out of the mutant somatic sector. Hence, what arose as a somatic mutation can be transmitted sexually. An example is shown in Figure 7-32.

A germinal mutation arises in the germ line, special tissue that is set aside during development to form gametes. If a mutant gamete participates in fertilization, then the mutation will be passed on to the next generation. However, an individual of normal phenotype and of normal ancestry can harbor undetected mutant gametes. These mutations can be detected only if they are included in a zygote (Figures 7-33 and 7-34). Recall from Chapter 4 that the X-linked hemophilia mutation in the European royal families is thought to have arisen in the germ cells of Queen Victoria or one of her parents. The mutation was expressed only in her male descendants.

Detection systems in haploids are often quite straightforward: because there is only one set of chromosomes, a mutation will automatically express itself as a mutant phenotype. Therefore, if we are interested (for example) in the pathway of the synthesis of the orange carotenoid pigment normally found in the cells of the fungus Neurospora, any mutation that inactivates that pathway will result either in colorless colonies or in colonies with a different pigment that might be an intermediate in the synthetic pathway. Such colonies are easily distinguishable with the naked eye. Scanning a large number of individuals in the search for a mutant is called a screen. Screening should be differentiated from a selection experiment, in which the mutant phenotype survives some selective treatment whereas wild types do not. For example, if we are interested in the genes that code for actin filaments (part of the cytoskeleton) in a fungus, we can select mutants by treating cells with cytochalasin, an inhibitor that binds to wild-type actin and inactivates it, preventing cell proliferation. Mutants that grow on cytochalasin will have mutations in genes that code for actin and related proteins. One possibility is that the mutation causes a shape change in actin that results in its inability to be bound by cytochalasin.

One problem in haploid genetics is that recessive lethal mutations (an abundant phenotypic class) cannot be recovered without special methods. In diploids, recessive lethal mutations are viable in heterozygotes, but then the problem is detection.

In diploids, detecting any recessive mutations is a challenge because, if a mutation arises, it will be masked by the dominant wild-type allele. One way around this problem is to start with heterozygotes A/a in which one allele is already a recessive mutation (a). Then any new recessive mutations, a*, arising from the conversion A a* can be detected:

This protocol for mutational detection is called a specific-locus test. Examples of specific-locus tests in germinal and somatic tissues are shown in Figures 7-35 and 7-36.

The next stage (grouping the mutations into genes) uses the complementation test (Chapter 6). If mutations fail to complement, they are in the same gene. Mapping of the mutant locus is based on the recombination-based techniques of Chapter 5 and on molecular protocols (Chapter 12). Interaction studies use the techniques of Chapter 6. The subsequent stages of analysis (molecular characterization of the genes concerned) will be discussed in Chapters 10 and 11.

Forward and Reverse Mutation

The process leading to any change away from the wild-type allele is called forward mutation; the process leading to any change back toward the wild-type allele is reverse mutation (also called reversion or back mutation). For example,

In most mutational analyses, forward mutations are sought. However, for certain studies, revertants are useful. For example, if the purpose of the study is to detect chemicals that might be mutagens, then simple reversion of auxotrophic alleles in a haploid fungus or bacterium is convenient because large numbers of cells can be plated and the prototrophic revertants will announce themselves as colonies (in other words, there is a built-in selection system). For example, to detect revertants of an auxotrophic mutation at the leu-3 locus of yeast, large numbers of cells are plated on leucine-free medium on which only the prototrophic leu-3+ revertants can grow. Reversion analysis is also useful in seeking suppressor mutations that might represent genes whose products interact with those of the suppressed gene. These mutations can be distinguished from true revertants because, upon crossing, the original mutation will appear in some descendants, having segregated away from the suppressor.

Mutant Phenotypes

In any mutational analysis, a crucial question is, What mutant phenotype should be sought? This is where knowledge of the nature of mutation and of the biology of the process under study are of paramount significance, and it is here that some of the most inspired pieces of analytical design are seen.

In a mutant hunt, what type of altered function can be expected, and what type would be preferred? A wild-type allele has some type of active function specific to the biological role of that particular gene. Mutations are generally destructive, so the most common type of mutation can be predicted to be a loss-of-function mutation, either a null or a leaky mutation. In cases of haplo-sufficiency (Chapter 3), loss-of-function mutations are recessive, but, in cases of haplo-insufficiency, they are dominant. The action of recessive loss-of-function mutations is represented diagrammatically in Figure 7-37a and b.

Gain-of-function mutations, which produce new phenotypes, will probably be dominant and should be expressed in a heterozygote for the mutant and the wild-type alleles. Expression of gain-of-function mutations is represented in Figure 7-37c. Some specific mechanisms of gain-of-function dominant mutations are discussed in Chapters 15 and 16.

MESSAGE
Dominant mutations can be caused by haploinsufficiency of loss-of-function mutations or by a gain-of-function mutation.

Some general types of mutant phenotypes are those due to morphological, lethal, biochemical, or conditional mutations. These phenotypes are useful in thinking about the ways in which mutant alleles might show up. This classification is not meant to be complete. Furthermore, a given mutation may fall into more than one of these categories.

Morphological mutations.

Morph means "form." Morphological mutations affect the outwardly visible properties of an organism, such as shape, color, or size. Albino ascospores in Neurospora, curly wings in Drosophila, and dwarf stature in peas are morphological mutations. Additional examples are shown in Figure 7-38.

Lethal mutations.

A new lethal mutant allele is recognized by its effects on the survival of the organism. Sometimes a primary cause of death from a lethal mutation is easy to identify (for example, in certain blood abnormalities). But often the cause of death is hidden, and the mutant allele is recognizable only by its effects on viability. An example of a morphological mutation that would be lethal in nature is shown in Figure 7-39. The analysis of lethal mutations is of considerable importance in the genetic dissection of development (Chapter 16).

Biochemical mutations.

Microbial cultures are convenient material for the study of biochemical mutations. Wild-type microorganisms are prototrophic, existing on a substrate of simple inorganic salts and an energy source. From these simple raw materials, the microorganisms synthesize all necessary compounds by using their many biochemical pathways. In contrast, biochemical mutants are often auxotrophic and must be supplied certain additional nutrients if they are to grow. The chemicals that will restore growth are those presumed to be missing in the mutant cells. Therefore analyzing the precise set of chemicals that restores growth is instructive in piecing together the relevant biochemical pathway. The practical method of testing for the auxotrophic or protoptrophic phenotype is shown in Figure 7-40.

Although microbial cultures are used for the experimental induction of biochemical mutations, we should note that many human hereditary diseases are biochemical mutations defective in some step of cellular chemistry. The expression inborn errors of metabolism is sometimes used to describe such biochemical disorders. Phenylketonuria and alkaptonuria are two examples.

Conditional mutations.

Not all mutations reliably produce a mutant phenotype, regardless of environmental conditions. Indeed, conditional mutations have been a gold mine for genetic analysis. A conditional mutant allele causes a mutant phenotype only in a certain environment, called the restrictive condition, but produces a wild-type phenotype in some different environment, called the permissive condition. Geneticists have studied many temperature-conditional mutations. For example, certain Drosophila mutations are known as "dominant heat-sensitive lethals." Heterozygotes (say, H+/H) are wild type at 20°C (the permissive condition) but die if the temperature is raised to 30°C (the restrictive condition).

Many mutant organisms are less vigorous than their normal counterparts and thus more troublesome as experimental subjects. For this reason, conditional mutants are useful because they can be grown under permissive conditions and then shifted to restrictive conditions for study. Another advantage of conditional mutations is that they allow the determination of a developmental sensitive period at which specific time the gene acts. In these studies, organisms carrying some specific conditional mutation are shifted from permissive to restrictive conditions at different times in the course of development. Some shifts will lead to mutants, others to wild types, and, from these results, the sensitive period of gene action is assessed.

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