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Mutagenesis

Mutagenesis

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.

see also Ames Test; Mutagen; Mutation; Mutation Rates.

John Heddle

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/>.

Michael Smith of Canada received the 1993 Nobel Prize in physiology or medicine for invention of site-directed mutagenesis. He shared the prize with Kary Mullis, who invented the polymerase chain reaction.

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Mutagen

Mutagen

A mutagen is any substance or agent that can cause a mutation, or change in the sequence or structure of DNA. Mutagens are classified on the basis of their physical nature and the types of damage they do. A mutagen is not the same as a carcinogen. Carcinogens are agents that cause cancer. While many mutagens are carcinogens as well, many others are not. The Ames test is a widely used test to screen chemicals used in foods or medications for mutagenic potential.

Chemical Mutagens

There are many hundreds of known chemical mutagens. Some resemble the bases found in normal DNA; others alter the structures of existing bases; others insert themselves in the helix between bases; while others work indirectly, creating reactive compounds that directly damage the DNA structure.

"Base analogs" are molecules whose chemical structure is similar to one of the four DNA bases (adenine, thymine, cytosine, and guanine). Because of this similarity, they can be incorporated into the helix during DNA replication. A key feature of mutagenic base analogs is that they form base pairs with more than one other base. This can cause mutations during the next round of replication, when the replication machinery tries to pair a new base with the incorporated mutagen. For instance, 5-bromo-deoxyuridine (5BU) exists in two different forms. One mimics thymine and therefore pairs with adenine during replication, while the other mimics cytosine and therefore pairs with guanine. In its thymine-mimicking form, 5BU can be incorporated across from an adenine. If it then converts to its cytosine-like form, during the next round of replication, it will cause a gua-nine to enter the opposite strand, rather than the correct adenine.

"Base-altering mutagens" cause chemical changes in bases that are part of the DNA. For example, nitrite preservatives in food convert to the mutagen nitrous acid. Nitrous acid causes deamination, or loss of an-NH2 group, of cytosine. When this occurs, cytosine becomes uracil, a base that is not normally incorporated in DNA but that is very similar to thymine. Unless repaired, this uracil will cause an adenine to enter the opposite strand instead of a guanine. Many base-altering mutagens are complex organic molecules. These are formed in large quantities in smoke, making up the "tar" of cigarette smoke, for example. They act as alkylating agents, combining with DNA to form bulky groups that interrupt replication.

"Intercalating agents" are flat molecules that insert themselves between adjacent bases in the double helix, distorting the shape at the point of insertion. Where this occurs, DNA polymerase may add an additional base opposite the intercalating agent. If this occurs in a gene, it induces a frameshift mutation (that is, it alters the reading of the gene transcript, changing which amino acids are added to the encoded protein). Ethidium bromide is one such agent, widely used in DNA research because its dark color allows DNA to be easily visualized. This is useful in gel electrophoresis, for instance, to find the DNA bands that have been separated in a gel.

Other damaging agents include chemicals that create "free radicals" inside a cell. Free radicals are compounds in which an atom, usually an oxygen, has an unbonded electron. Free radicals are highly reactive and can cause several types of damage to DNA.

Light and Radiation

Radiation refers to two different phenomena: light and high-energy particles. Visible light represents a small slice of the electromagnetic spectrum, which includes long-wavelength (low-energy) radio waves and short-wave-length (high-energy) ultraviolet waves, plus X rays and gamma rays. These high-energy forms can directly disrupt DNA by breaking its chemical bonds. In severe cases, this can break apart chromosomes, causing chromosome aberrations. More often, they create mutagenic free radicals in the cell. X rays were first used by Hermann Muller to induce mutations in fruit flies. They continue to be used to create mutations in model organisms to study genes and their effects.

Ultraviolet light is less energetic than X rays but causes mutations nonetheless. The higher-energy form, UV-B, is more toxic than UV-A, because of its potential to cause cross-linking between adjacent thymine or cytosine bases, creating a so-called pyrimidine dimer (cytosine and thymine are chemically classified as pyrimidines). Pyrimidine dimers interrupt replication. UV-A does not cause dimer formation but can still cause mutations by creating free radicals.

Another meaning of the term "radiation" is high-energy particles released during the breakdown of radioactive elements, such as uranium. These particles are either electrons (called beta particles) or helium nuclei (called alpha particles). Their energy is sufficient to disrupt DNA structure, or to create free radicals.

Repairing the Damage

DNA is constantly being damaged, and it is constantly being repaired as well. It is only when the damage is not repaired that a mutation can lead to cancer or cell death. The DNA repair enzymes can recognize damaged nucleotides and remove and replace them. The human liver contains a large number of enzymes whose role is to detoxify toxic compounds, mutagenic or otherwise, by chemically reacting them. However, in some cases these enzymes (called cytochrome P450s) actually create mutagens during the course of these reactions. Such "bioactivation" may be a significant source of mutagens.

see also Carcinogens; Chromosomal Aberrations; DNA Repair; Muller, Hermann; Mutagenesis; Mutation.

Richard Robinson

Bibliography

Philp, Richard B. Ecosystems and Human Health: Toxicology and Environmental Hazards, 2nd ed. Boca Raton, FL: Lewis Publishers, 2001.

Brusick, David. Principles of Genetic Toxicology, 2nd ed. New York: Plenum Press, 1987.

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Mutagen

Mutagen


Mutagens are chemical agents that cause changes in the genetic code which are then passed on to future generations of an organism. Mutations are usually chemical in nature and often carcinogenic, but may also be caused by physical damage produced by x rays or other causes. A mutation changes the activity of a gene. Mutations are frequent in lower forms of life and may help these organisms adapt to changes in their environments.

Proteins are composed of chains of amino acids. In the genetic code of deoxyribonucleic acid (DNA ), a codon or three-base sequence codes for the placement of each amino acid; for example, the codon UUU places phenylalanine at that location in the protein and replacement of the third base with adenine results in the placement of leucine instead of phenylalanine. If a portion of the original code read UUUACG , deleting one of the uridine bases would cause that portion of the code to read UUACG ; the sequence UUA would then specify leucine. A point mutation changing one base might result in the formation of a different protein.

Mutations can occur by several mechanisms, such as replacing one nucleotide base with another or by adding or removing a base; they can also develop when a carcinogenic agent such as an aromatic hydrocarbon molecule is inserted between the strands of DNA, causing the code to be misread. Some chemical mutagens such as nitrites change one base into another, resulting in a new sequence of amino acids and the synthesis of a new protein. The modified protein might function normally or might not be useful at all, but it could be dangerous.

Many mutagenic agents are also carcinogenic, and the Ames test provides a quick method for screening foods and other substances for potential cancer-causing agents.

HOW DOES THE AMES TEST WORK?

The Ames test is a method for screening potential mutagens. The test uses auxotrophs (strains that have lost the ability to synthesize a needed substance) of Salmonella typhimurium that carry mutant genes, making them unable to synthesize histidine. They can live on media containing histidine, but die when the amino acid is depleted. The bacteria are especially sensitive to back mutations that reactivate the gene for the synthesis of histidine; exposure to mutagenic substances allows the bacteria to grow rapidly, developing large and numerous colonies.

see also Carcinogen; Codon.

Dan M. Sullivan

Bibliography

Devlin, Thomas M., ed. (2002). Textbook of Biochemistry: With Clinical Correlations, 5th edition. New York: Wiley-Liss.

Voet, D.; Voet, J. G.; and Pratt, C. W. (2003). Biochemistry, 3rd edition. New York: Wiley.

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mutagen

mu·ta·gen / ˈmyoōtəjən/ • n. an agent, such as radiation or a chemical substance, that causes genetic mutation. DERIVATIVES: mu·ta·gen·e·sis / ˌmyoōtəˈjenəsəs/ n. mu·ta·gen·ic / ˌmyoōtəˈjenik/ adj.

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mutagen

mutagen An agent that causes an increase in the number of mutants (see mutation) in a population. Mutagens operate either by causing changes in the DNA of the genes, so interfering with the coding system, or by causing chromosome damage. Various chemicals (e.g. colchicine) and forms of radiation (e.g. X-rays) have been identified as mutagens.

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mutagen

mutagen An agent that increases the mutation rate within an organism. Examples of mutagens are X-rays, gamma rays, neutrons, and certain chemicals, such as carcinogens.

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mutagen

mutagen An agent that increases the mutation rate within an organism. Examples of mutagens are X-rays, gamma rays, neutrons, and certain chemicals, such as carcinogens.

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mutagen

mutagen An agent that increases the mutation rate within an organism. Examples of mutagens are X-rays, gamma rays, neutrons, and certain chemicals such as carcinogens.

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mutagen

mutagen Compound that causes mutations and may be carcinogenic; see also Ames test.

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mutagen

mutagen: see mutation.

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