Mutant Hunts To Select or to Screen Perhaps Even by Brute Force

The first step in a genetic analysis of a process it to isolate mutant individuals that are unable to carry out that process or carry it out in an aberrant way. The researcher must hypothesize what characteristics, changes in growth capabilities, morphology, etc. will be exhibited by this individual and to develop a means of identifying such individuals from among a larger group of normal individuals.

A mutation is a heritable change in the genetic material, and mutagenesis is the process of producing such changes. Spontaneous mutations are the by-product of natural processes, particularly errors resulting from DNA replication and repair and the movement of transposable elements. The spontaneous rate of mutation varies from species to species and the rate of spontaneous production of different classes of mutation (point mutation, insertion, deletion) varies. The geneticist also has available a variety of methods for enhancing the rate of mutagenesis above the spontaneous, or background, rate including treatment with various chemical agents, ultraviolet light, X-rays, and transposon-mediated events. Different mutagens produce different types of genetic change. The best choice of mutagenic agent and the best method of delivering that mutagen will depend on one's particular experimental needs and the organism under study.

Regardless of whether the mutation is spontaneous or induced and regardless of which type of mutagen is used, the researcher is unable to direct mutations to specific sequences or particular genes. To a first approximation, mutagenesis is a random process, and any particular gene has an equal chance of being damaged by a mutagen or a spontaneous error in a cellular process such as DNA replication or repair. So how does one find those individuals carrying mutations in a gene of interest, i.e. a gene involved in the process under study? In other words, how does one distinguish the interesting mutant individuals from all the other individuals in the pool of potential mutant individuals? One searches for them or carries out what geneticists sometimes call a mutant hunt. To do this, one must develop a method of identifying mutants. This is the first job of the geneticist and, in many respects, it is the most difficult job.

An individual carrying a heritable genetic change is referred to as a mutant and the process of identifying mutants of interest is called either a selection or a screen, depending upon how it is carried out. Note that one identifies mutant individuals by observing their phenotype. Phenotype is an indirect expression of genotype and is the product of the biochemical and physiological interplay among all the genes of an individual's genome. For this reason, geneticists prefer to work with isogenic strains, i.e. strains that are genetically identical at all but perhaps one or a limited number of known genes. In this way the effect of a newly induced mutation on the phenotype of the individual can clearly be associated with that specific single genetic alteration. If isogenic strains are not available, then efforts should be made to work with very closely related strains such as a congenic strain. A congenic strain is one obtained by repeated backcrosses to a common parent. The products of the tenth backcross should be better than 99.9% identical.

In the earlier yeast literature, prior to the early 1970s, researchers did not seem to pay a great deal of attention to working with genetically related strains. The influx of bacterial and phage geneticists into the yeast research community brought an awareness of the importance of working with isogenic strains where possible. In the prokaryotic systems the technology for constructing isogenic strains is rather straightforward. In yeast the ability to construct isogenic strains came with the development of Saccharomyces DNA technology. One can now change the mating type of a strain by the simple use of a plasmid expressing the HO gene.

To identify mutant individuals carrying alterations in a gene of interest, the geneticist will hypothesize the probable phenotype of these mutant individuals and develop a method of identifying them based on their phenotypic difference from the starting strain. The best method is to select for the mutant individuals. A selection method is one in which only individuals with the desired phenotype are able to grow, or move toward a light source, or carry out a function. For example, one is interested in studying the mechanism of action of an antibacterial drug, and the drug-resistant mutant bacteria capable of growing in the presence of the drug is isolated. This is a selection because millions of bacteria will be spread on the surface of a petri dish containing nutrient medium with the drug. Almost all the bacteria will die because of the action of the drug, but a few will grow to form a colony because these individuals carry a mutation that makes them resistant to the drug.

A screen is where the individuals of a population are observed one by one following growth in a particular condition and individuals with a particular phenotype are chosen. For example, one is interested in tryptophan synthesis, and needs to isolate mutant strains unable to synthesize tryptophan. Such mutants would be unable to grow in the absence of added tryptophan in the medium because tryptophan is an essential amino acid. Colonies (clones of cells) are grown on the surface of a solid (agar-containing medium in a petri dish or petri plate) medium containing tryptophan. Clones are genetically identical individuals. Each clone is tested by a simple test called replica plating to see if it grows on the same medium only lacking tryptophan. (Replica plating is a method of making duplicate copies of the pattern of colonies growing on the surface of solid media in a petri dish. Colonies are grown on the media in a petri dish; these colonies are transferred to a sterile velvet cloth by pressing the cloth to the surface of the plate. Then the colonies are transferred to other plates by pressing the cloth to the surface of new plates.) Colonies grown on a medium plus tryptophan are replica plated to plates containing the same medium with and without tryptophan. Those colonies that do not grow on the plate lacking tryptophan but do grow on the plate containing tryptophan have a tryptophan minus phenotype.

Only when all else fails will the geneticist resort to a brute force screen in which individuals are screened for a complex phenotypic trait by a difficult procedure. These are few and far between, and the geneticist would have to be desperate. Before undertaking a brute force screen, the geneticist will attempt to enrich for mutants of the desired type. Penicillin enrichment for E. coli mutants is an example. Penicillin kills only growing cells because it blocks cell wall growth causing the expanding cytoplasm to burst the cell. Mutagenized cells asked to grow in penicillin-

containing medium lacking histidine should be enriched for histidine minus mutants because these would have been unable to grow in the absence of histidine and therefore would have survived penicillin treatment.

Geneticists refer to the original strain isolated from nature and prior to any mutagenesis as the wild-type strain and the allele of each gene in this strain as the wild-type allele. Any alteration in a gene, whether associated with an altered phenotype or not, is a mutant allele. Most often, the strain used in a selection or screen will carry one or more known mutations that are for the use of the geneticist. These mutant genes are often referred to as genetic markers or marker genes because they mark the existence and position of the gene in the genome and are used to expedite the genetic analysis. Marker genes with easily identifiable phenotypes are generally chosen, such as nutritional mutations or drug-resistant alleles. Thus, because the original strain used to isolate new mutation is not truly wild-type, it is usually referred to as the parental strain.

A mutant allele may be dominant or recessive to the wild-type, and this can only be determined by making the heterozygous diploid individual and determining its phenotype. If the mutant phenotype is exhibited by the heterozygous diploid, then the mutation is dominant. If the wild-type phenotype is exhibited by the heterozygous diploid, then the mutation is recessive, i.e. the wild-type allele dominates the phenotype. A recessive mutation is a loss of function mutation; that is, the gene either no longer produces a product or the product produced is so abnormal that it is unable to carry out the function. A dominant mutation is a change of function mutation; that is, the product of the gene has retained all or part of its original functional characteristics but also has acquired new functions or carries out its usual functions differently.

The more rare a particular type of mutation, the more aggressively one must look. Selection is more aggressive than screening because more individuals can be tested. This statement does not contradict the fact that mutagenesis is a random process. What it does say is that the genetic alteration needed to produce a certain phenotype can be more or less difficult to achieve. For example, to knock out all function of a gene one can delete it, insert a DNA fragment into it, or change its sequence in any of a variety of ways. One does not care because no function need be retained. But if one wants a mutant with a slightly changed phenotype, such as resistance to a drug, then the product of the altered gene must be able to carry out its normal function but with different characteristics. Alterations capable of achieving this are few and far between and only certain select changes will do the trick. In other words, it takes a scalpel to make a dominant mutation but you can use a grenade to make a recessive mutation. A priori, the geneticist does not know how rare the searched-for defects will be so, ideally speaking, one tries to devise a selection. This is not always feasible, and so a screen may have to be used. The geneticist will often repeat a mutant search using subtle variations in the selection/ screening method. The particular method used undoubtedly biases the types of mutation isolated. By varying the isolation method, it is hoped that the additional genes involved in the process will be identified or that different alleles of an already identified gene with subtly different phenotypes will be obtained.

If several genes are expected to be involved in a particular process or pathway, large numbers of mutant individuals must be obtained so as to ensure that mutations in all of these genes are isolated. This is referred to as saturating the system. Mutations in different genes involved in the same process may have the same or slightly different phenotypes. Sometimes different mutations in the same gene may have different phenotypes. A great deal may be learned about the normal function of a gene by carrying out a careful phenotypic analysis of the different mutant alleles obtained.

A forward mutation is a genetic alteration of a wild-type gene. It produces a mutant strain with a mutant phenotype and mutant genotype. A reverse mutation, also called a reversion, is an alteration that produces a revertant strain with a wildtype or pseudo wild-type phenotype. If the reverse mutation exactly reproduces the original wild-type genetic sequence, then the revertant strain is a true revertant and has the wild-type phenotype and genotype. As we will see in Chapter 8, it is possible for a mutation at a different site, even in a different gene, to produce a revertant strain with what appears to be a wild-type phenotype even though the genotype is not wild-type but is actually a double mutant. In this situation the second mutation interacts with the original mutation to suppress its mutant phenotype, and the second mutation is called a suppressor mutation. Suppression is an important, interesting, and very valuable tool for the molecular geneticist, and we will explore it in great detail in future chapters.

A null mutation is one in which the alteration completely eliminates gene function. Null mutations result from deletions of the gene that include large sections of the ORF or the 5' end of the gene, nonsense mutations near the 5' end of the ORF, transposon insertions, or other changes that block gene expression or production of the gene product. Null mutations are always recessive.

A strain carrying a conditional mutation is one that exhibits the mutant phenotype under some conditions and the wild-type phenotype under other conditions. The strain is a mutant strain but whether the strain exhibits the mutant phenotype or the wild-type phenotype depends on the growth conditions. The condition that is most often varied is the growth temperature. Most organisms prefer to grow at a particular temperature. For example, the ideal growth temperature of Saccharomyces is 30°C, but wild-type strains are able to grow at temperatures from 14°C to 42°C. Mutant strains that exhibit the mutant phenotype at temperatures above the preferred growth temperature are called temperature sensitive, and the mutant gene is referred to as a temperature-sensitive or ts allele. Mutant strains that exhibit the mutant phenotype at temperatures below the preferred growth temperature are said to be cold sensitive, and the mutant gene is referred to as a cold-sensitive or cs allele. Since there is a condition where the mutant gene functions somewhat normally, conditional alleles are not null alleles and most often are changes of a single amino acid residue in the encoded protein. The growth condition at which the mutant phenotype is exhibited is called the nonpermissive or restrictive condition. The permissive or nonrestrictive condition is the one in which the wild-type phenotype is expressed.

Researchers interested in essential cellular processes like cell division or transcription must isolate conditional mutations. An essential gene encodes a product required for normal growth and cell division and its function cannot be by-passed by nutrient supplementation of the medium. A cell carrying a mutation in a gene encoding a necessary component of RNA polymerase II cannot survive if the mutant exhibits the mutant phenotype under all conditions. There is no way of adding a nutrient or drug to the medium or altering the growth conditions that will enable this cell to grow. Such a mutation in an essential gene is a lethal mutation. A conditional mutation in an essential gene allows the researcher to culture the mutant strain under the permissive condition and to observe the mutant phenotype under the nonpermissive condition. This type of mutation is referred to as a conditional lethal mutation. The use of conditional mutations is a very powerful tool of the geneticist and its many uses will become obvious as we read the literature.

Genetic Techniques for Biological Research Corinne A. Michels Copyright © 2002 John Wiley & Sons, Ltd ISBNs: 0-471-89921-6 (Hardback); 0-470-84662-3 (Electronic)

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