mutagenesis

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Formation or development of a mutation.

[MUTA(TION) + –GENESIS.]

The induction or occurrence of a genetic mutation.

Mutagenesis is the process of inducing mutations. Mutations may occur due to exposure to natural mutagens such as ultraviolet (UV) light, to industrial or environmental mutagens such as benzene or asbestos, or by deliberate mutagenesis for purposes of genetic research. For geneticists, the study of mutagenesis is important because mutants reveal the genetic mechanisms underlying heredity and gene expression. Mutations are also important for studying protein function: Often the importance of a protein cannot be characterized unless a mutant can be made in which that protein is absent.

Noninduced Mutagenic Agents

Environmental agents can influence the mutation rate not only by increasing it, but also by decreasing it. For example, antioxidants, which are found commonly in fruits and vegetables, are thought by many to protect against mutagens that are generated by normal cellular respiration. In addition to protective agents, however, many plants also contain deleterious mutagens known as carcinogens. Many chemical mutagens exist both naturally in the environment and as a result of human activity. Benzo(a)pyrene, for example, is produced by any incomplete burning, whether of tobacco in a cigarette or of wood in forest fires.

Spontaneous (noninduced) mutations are very rare, and finding them is difficult because most are recessive. The recessive nature of most mutations means that they will not be evident in most of the individuals who inherit them, for they will be hidden by the presence of the dominant allele. The rarity of mutations means that many individuals must be examined to find a mutant, whether they are people, other organisms, or even cells in culture.

Creating Mutations

To overcome the problem of the rarity of mutations, researchers induce mutations with a variety of agents. Hermann Muller was the first to do this when, in 1927, he used X rays on fruit flies (Drosophila) to increase the mutation rate by more than 100-fold. Other high-energy forms of radiation can also be used to create mutations.

The first chemical to be recognized as a mutagen was mustard gas, which had been developed during World War I, but not tested until World War II by Auerbach and Robson, at the University of Edinburgh. Since then a wide variety of chemicals have been discovered that are also mutagenic. Some induce mutations at any point in the cell cycle, by disrupting DNA structure. Others only act during DNA replication. Called base analogs, these latter chemicals have structures similar to the bases found in DNA, and are incorporated instead of the normal base.

Transposable genetic elements (also called transposons, or "jumping genes") can also induce mutations. These elements insert randomly into the genome, and may disrupt gene function if inserted into a gene or its promoter. Finding the organism with a disrupted gene is made easier if the transposon carries with it a reporter gene whose product can be identified, or a selectable marker that allows the transformed cells to live while non-transformed ones die. (The use of reporter genes and selectable markers are techniques used in genetic analysis in the laboratory.) The transposon sequence itself serves as a molecular tag. Thus, if the target gene (the gene being studied) is interrupted, finding the transposon allows the researcher to find the gene.

All of the above methods disrupt genes randomly. However, specific genes can also be targeted, for "site-directed mutagenesis," if their sequence is known. Using the known sequence, a matching DNA sequence is inserted into a single-stranded vector. Short, complementary, partial sequences containing the desired mutation are then synthesized. These are allowed to pair up, and DNA polymerase is then used to complete the complementary strand. Further replication amplifies he number of copies of the mutant. In bacteria, the mutant gene can be placed on a plasmid for transformation of the bacteria. The bacteria make the mutant protein, and the effect of the mutation can then be studied. This is a key tool in studying how amino acid sequences affect protein structure, since individual amino acids can be changed, one at a time.

In eukaryotes, the mutant gene can be inserted into the chromosome of an experimental organism by "homologous recombination," a system in which the mutant gene switches places with the normal chromosomal gene. Such techniques can "knock out" and "knock in" genes bearing the desired mutations.

The First Mutagenesis Assay

Before DNA sequencing became widespread, most mutations could only be detected by their effects on the phenotype of the organism. Many mutations are recessive, however, and do not affect the phenotype if present in only one allele. Hermann Muller, who pioneered the study of mutations, overcame this problem by focusing on the X chromosome in his studies of the genetics of Drosophila, the fruit fly. While females have two X chromosomes, males have only one, so any mutated gene carried on the X chromosome is expressed in males, even if it is recessive. Hence recessive lethal mutations on the X chromosome kill any male inheriting them, but would not kill a female. Muller's method examined all of the genes on the X chromosome that could mutate to give a recessive lethal mutation. Muller used X rays to generate mutants. X rays are a very high-energy form of radiation, and break the DNA at numerous points. The method is shown in Figure 1.

Muller treated adult males with X rays and mated them to females who carried one copy of a specially prepared X chromosome, called ClB. This chromosome had a gene to prevent crossing over (C), which kept the chromosome intact; a lethal recessive gene (l) to kill any males that inherit it; and a dominant "bar eye" gene (B) that resulted in a distinctive phenotypic change, making it easy to find female flies that inherited it.

Muller mated X-ray treated males with ClB-carrying females. All female offspring from this crossbreeding received one treated X chromosome from the male (which might or might not have carried a lethal recessive gene). They also received one X from the female, either normal or ClB. He selected only the bar-eyed females for further mating. To determine which of these females carried an X-ray induced lethal recessive, he separated each female into a separate jar, and examined their offspring.

Three types of males were created in this cross, depending on what type of X chromosome they inherited. Males inheriting the ClB chromosome died, due to the presence of the l gene. Males inheriting an X-ray-treated X chromosome with a lethal recessive died. Males inheriting an X-ray-treated X chromosome without a lethal recessive lived. Therefore, any jar with live males indicated that the mother did not carry a lethal recessive. Any jars with no males indicates the mother carried a lethal recessive, originally induced in the X-ray-treated male. The analysis was rapid because an experienced person could examine a bottle of flies and see at a glance if there were males present. Subsequent studies showed that this method tested almost 1,000 genes simultaneously, thus making it practical to use when detecting rare mutations. Unfortunately the breeding takes quite a lot of time, so this assay has now fallen largely into disuse, despite its historical importance.

Detecting Mutations

Today the mutagenic potential of chemicals is considered in evaluating the mutagenic risks posed by chemical exposure. Many new methods have been developed to determine if chemicals to which people will be exposed, such as new drugs, food additives, and pesticides, are mutagens. Since mutations can occur in any organism, and because there are many different kinds of mutations, there are a correspondingly wide variety of tests to detect them. No one test detects them all.

The Ames test was the first and remains the only test to be almost universally required by regulatory agencies as a minimum standard for determining if a chemical is mutagenic. The test is conducted in Salmonella bacteria. Since bacteria have only one chromosome, recessive mutations can be detected readily. Rare mutations are easily detected because mutants can be selected very simply. Several variants have been added to the original test, allowing for detection of many types of mutations. In an effort to make the test more relevant to human risk, one variant uses an extract of liver to mimic the biochemical modifications of chemicals that occur in the human liver.

Assays for Chromosome Aberrations

Chromosomal aberrations can be detected by examining cells in mitosis or meiosis for changes (see Figure 2). Typically, bone marrow cells of mice or rats are examined for in vivo tests. Any cells can be used for tests of cells in culture, but Chinese hamster cells or human fibroblasts are most commonly used. Another test, called the micronucleus test, is also commonly used. Micronuclei are small nuclei that arise from pieces of chromosomes or whole chromosomes that have been lost during cell division. They are conveniently detected in mouse red blood cells, which have no normal nucleus but which often retain micronuclei. The micronucleus assay is also widely used in cultured cells.

Assays for Somatic Mutations

Recessive mutations can be detected more readily on a mammalian X chromosome than on the other chromosomes, because only one X chromosome is active. Therefore, detection of the mutagenic potential of a substance in mammals can be most efficiently performed by analyzing the X-linked mutations. A system using the X-linked gene hprt has been widely used because the enzyme is not essential and because the addition of the drug thioguanine kills all cells except mutants. A count of the cells that can be cultured in the presence of thioguanine is a count of hprt mutants.

Bibliography

Griffiths, Anthony J. F., et al. An Introduction to Genetic Analysis. New York: W. H.Freeman, 2000.

Muller, Hermann J. "Artificial Transmutation of the Gene." Science 66 (1927): 84-87.

Rubin, G. M., and A. C. Spradling. "Genetic Transformation of Drosophila withTransposable Element Vectors." Science 218 (1982): 348-353.

Internet Resource

United Nations Scientific Committee on the Effects of Atomic Radiation. http://www.unscear.org/.

—John Heddle

The process of inducing genetic mutation.

• site directed m. — a set of methods used to experimentally create specific changes in the nucleotide sequence of a gene.