GBE 212E – GENETICS Assist. Prof. Muhammet AY Lecture 10 OCCURRENCE AND CAUSES OF MUTATION OCCURRENCE AND CAUSES OF MUTATION Geneticists categorize the cause of mutation in one of two ways. Spontaneous mutations are changes in DNA structure that result from abnormalities in biological processes, whereas induced mutations are caused by environmental agents (Table 16.4 ). Overall, a distinguishing feature of spontaneous mutations is that their underlying cause originates within the cell. By comparison, the cause of induced mutations originates outside the cell. Induced mutations are produced by environmental agents, either chemical or physical, that enter the cell and lead to changes in DNA structure. Agents known to alter the structure of DNA which lead to mutations are called mutagens. Spontaneous Mutations Are Random Events Salvadore Luria and Max Delbrück were interested in the ability of bacteria to become resistant to infection by a bacteriophage called T1. When a population of E. coli cells is exposed to T1, a small percentage of bacteria are found to be resistant to T1 infection and pass this trait to their progeny. Luria and Delbrück were interested in whether such resistance, called tonr (T one resistance), is due to the occurrence of random mutations or whether it is a physiological adaptation that occurs at a low rate within the bacterial population. According to the physiological adaptation hypothesis, the rate of mutation should be a relatively constant value and depend on exposure to the bacteriophage. Therefore, when comparing different populations of bacteria, the number of tonr bacteria should be an essentially constant proportion of the total population. Spontaneous Mutations Are Random Events In contrast, the random mutation hypothesis depends on the timing of mutation. If a tonr mutation occurs early within the proliferation of a bacterial population, many tonr bacteria will be found within that population because mutant bacteria will have time to grow and multiply. However, if it occurs much later in population growth, fewer tonr bacteria will be observed. In general, a random mutation hypothesis predicts a much greater fluctuation in the number of tonr bacteria among different populations. To distinguish between the physiological adaptation and random mutation hypotheses, Luria and Delbrück inoculated one large flask and 20 individual tubes with E. coli cells and grew them in the absence of T1 phage. Spontaneous Mutations Are Random Events The flask was grown to produce a very large population of cells, while each individual culture was grown to a smaller population of approximately 20 million cells. They then plated the individual cultures onto media containing T1 phage. Likewise, 10 subsamples, each consisting of 20 million bacteria, were removed from the large flask and plated onto media with T1 phage. The results of the Luria and Delbrück experiment are shown in Figure 16.6. Within the smaller individual cultures, a great fluctuation was observed in the number of tonr mutants. This test, therefore, has become known as the fluctuation test. Spontaneous Mutations Are Random Events Which hypothesis do these results support? The data are consistent with a random mutation hypothesis in which the timing of a mutation during the growth of a culture greatly affects the number of mutant cells. For example, in tube 14, many tonr bacteria were observed. Luria and Delbrück reasoned that a mutation occurred randomly in one bacterium at an early stage of the population growth, before the bacteria were exposed to T1 on plates. This mutant bacterium then divided to produce many daughter cells that inherited the tonr trait. In other tubes, such as 1 and 3, this spontaneous mutation did not occur, so none of the bacteria had a tonr phenotype. Spontaneous Mutations Are Random Events By comparison, the cells plated from the large flask tended toward a relatively constant and intermediate number of tonr bacteria. Because the large flask had so many cells, several independent tonr mutations were likely to have occurred during different stages of its growth. In a single flask, however, these independent events would be mixed together to give an average value of tonr cells. Several years later, Joshua and Esther Lederberg were also interested in the relationship between mutation and the environmental conditions that select for mutation. To distinguish between the physiological adaptation and random mutation hypotheses, they developed a technique known as replica plating in the 1950s. As shown in Figure 16.7 , they plated a large number of bacteria onto a master plate that did not contain any selective agent (namely, the T1 phage). Spontaneous Mutations Are Random Events A sterile piece of velvet cloth was lightly touched to this plate in order to pick up a few bacterial cells from each colony. This replica was then transferred to two secondary plates that contained an agent that selected for the growth of bacterial cells with a particular genotype. In the example shown in Figure 16.7, the secondary plates contained T1 bacteriophages. On these plates, only those mutant cells that are tonr could grow. On the secondary plates, a few colonies were observed. Strikingly, they occupied the same location on each plate. These results indicated that the mutations conferring tonr occurred randomly while the cells were growing on the nonselective master plate. The presence of the T1 phage in the secondary plates simply selected for the growth of previously occurring tonr mutants. These results supported the random mutation hypothesis. Spontaneous Mutations Are Random Events In contrast, the physiological adaptation hypothesis would have predicted that tonr bacterial mutants would occur after exposure to the selective agent. If that had been the case, the colonies would not be expected to arise in identical locations on different secondary plates but rather in random patterns. Taken together, the results of Luria and Delbrück and those of the Lederbergs supported the random mutation hypothesis, now known as the random mutation theory. According to this theory, mutations are a random process—they can occur in any gene and do not involve exposure of an organism to a particular condition that causes specific types of mutations to happen. In some cases, a random mutation may provide a mutant organism with an advantage, such as resistance to T1 phage. Although such mutations occur as a matter of random chance, growth conditions may select for organisms that happen to carry them. Spontaneous Mutations Are Random Events As researchers have learned more about mutation at the molecular level, the view that mutations are a totally random process has required some modification. Within the same individual, some genes mutate at a much higher rate than other genes. Why does this happen? Some genes are larger than others, which provides a greater chance for mutation. Also, the relative locations of genes within a chromosome may cause some genes to be more susceptible to mutation than others. Even within a single gene, hot spots are usually found—certain regions of a gene that are more likely to mutate than other regions. Mutation Rates and Frequencies Are Ways to Quantitatively Assess Mutation in a Population Because mutations occur spontaneously among populations of living organisms, geneticists are greatly interested in learning how prevalent they are. The term mutation rate is the likelihood that a gene will be altered by a new mutation. This rate is commonly expressed as the number of new mutations in a given gene per cell generation. In general, the spontaneous mutation rate for a particular gene is in the range of 1 in 100,000 to 1 in 1 billion, or 10–5 to 10–9 per cell generation. An average person will carry about 100 to 200 new mutations in their entire genome compared with their parents. Most of these are single nucleotide changes. With a human genome size of approximately 3,200,000,000 bp, these numbers tell us that a mutation is a relatively infrequent event. However, the mutation rate is not a constant number. The presence of certain environmental agents, such as X-rays, can increase the rate of induced mutations to a much higher value than the spontaneous mutation rate. Mutation Rates and Frequencies Are Ways to Quantitatively Assess Mutation in a Population The rate of new mutation is different from the concept of mutation frequency. The mutation frequency for a gene is the number of mutant genes divided by the total number of genes within a population. If 1 million bacteria were plated and 10 were found to be mutant, the mutation frequency would be 1 in 100,000, or 10–5. Luria and Delbrück showed that among the bacteria in the 20 tubes, the timing of mutations influenced the mutation frequency within any particular tube. Some tubes had a high frequency of mutation, but others did not. Therefore, the mutation frequency depends not only on the mutation rate but also on the timing of mutation, and on the likelihood that a mutation will be passed to future generations. The mutation frequency is an important genetic concept, particularly in the field of population genetics. Spontaneous Mutations Can Arise by Depurination, Deamination, and Tautomeric Shifts The most common type of chemical change that occurs naturally is depurination, which involves the removal of a purine (adenine or guanine) from the DNA. The covalent bond between deoxyribose and a purine base is somewhat unstable and occasionally undergoes a spontaneous reaction with water that releases the base from the sugar, thereby creating an apurinic site (Figure 16.8a ). In a typical mammalian cell, approximately 10,000 purines are lost from the DNA in a 24-hour period at 37°C. The rate of loss is higher if the DNA is exposed to agents that cause certain types of base modifications such as the attachment of alkyl groups (methyl or ethyl groups). Fortunately, apurinic sites are recognized by DNA base excision repair enzymes that repair the site. If the repair system fails, however, a mutation may result during subsequent rounds of DNA replication. Spontaneous Mutations Can Arise by Depurination, Deamination, and Tautomeric Shifts What happens at an apurinic site during DNA replication? Because a complementary base is not present to specify the incoming base for the new strand, any of the four bases are added to the new strand in the region that is opposite the apurinic site (Figure 16.8b). This may lead to a new mutation. A second spontaneous lesion that may occur in DNA is the deamination of cytosines. The other bases are not readily deaminated. As shown in Figure 16.9a, deamination involves the removal of an amino group from the cytosine base. This produces uracil. DNA repair enzymes can recognize uracil as an inappropriate base within DNA and subsequently remove it. However, if such repair does not take place, a mutation may result because uracil hydrogen bonds with adenine during DNA replication. Spontaneous Mutations Can Arise by Depurination, Deamination, and Tautomeric Shifts Figure 16.9b shows the deamination of 5-methylcytosine. If 5methylcytosine is deaminated, the resulting base is thymine, which is a normal constituent of DNA. Therefore, this poses a problem for DNA repair because DNA repair proteins cannot distinguish which is the incorrect base—the thymine that was produced by deamination or the guanine in the opposite strand that originally base-paired with the methylated cytosine. For this reason, methylated cytosine bases tend to produce hot spots for mutation. As an example, researchers analyzed 55 spontaneous mutations that occurred within the lacI gene of E. coli and determined that 44 of them involved changes at sites that were originally occupied by a methylated cytosine base. Spontaneous Mutations Can Arise by Depurination, Deamination, and Tautomeric Shifts A third way that mutations may arise spontaneously involves a temporary change in base structure called a tautomeric shift. In this case, the tautomers are bases, which exist in keto and enol or amino and imino forms. These forms can interconvert by a chemical reaction that involves the migration of a hydrogen atom and a switch of a single bond and an adjacent double bond. The common, stable form of guanine and thymine is the keto form; the common form of adenine and cytosine is the amino form (Figure 16.10a ). At a low rate, G and T can interconvert to an enol form, and A and C can change to an imino form. Though the relative amounts of the enol and imino forms of these bases are relatively small, they can cause a mutation because these rare forms of the bases do not conform to the AT/GC rule of base pairing. Spontaneous Mutations Can Arise by Depurination, Deamination, and Tautomeric Shifts How does a tautomeric shift cause a mutation? The answer is that it must occur immediately prior to DNA replication. When DNA is in a double-stranded condition, the base pairing usually holds the bases in their more stable forms. After the strands unwind, however, a tautomeric shift may occur. In the example shown in Figure 16.10c, a thymine base in the template strand has undergone a tautomeric shift just prior to the replication of the complementary daughter strand. During replication, the daughter strand incorporates a guanine opposite this thymine, creating a base mismatch. This mismatch could be repaired via the proofreading function of DNA polymerase or via a mismatch repair system. Oxidative Stress May Also Lead to DNA Damage and Mutation Aerobic organisms use oxygen as a terminal acceptor of their electron transport chains. Reactive oxygen species (ROS), such as hydrogen peroxide, superoxide, and hydroxyl radical, are products of oxygen metabolism in all aerobic organisms. In eukaryotes, ROS are naturally produced as unwanted by-products of energy production in mitochondria. They may also be produced during certain types of immune responses and from a variety of detoxification reactions in the cell. If ROS accumulate, they can damage cellular molecules, including DNA, proteins, and lipids. To prevent this from happening, cells use a variety of enzymes, such as superoxide dismutase and catalase, to prevent the buildup of ROS. Oxidative Stress May Also Lead to DNA Damage and Mutation Oxidative stress refers to an imbalance between the production of ROS and an organism’s ability to break them down. If ROS overaccumulate, one particularly harmful consequence is oxidative DNA damage, which refers to changes in DNA structure that are caused by ROS. DNA bases are very susceptible to oxidation. Guanine bases are particularly vulnerable to oxidation, which can lead to several different oxidized guanine products. The most thoroughly studied guanine oxidation product is 7,8-dihydro-8-oxoguanine, which is commonly known as 8-oxoguanine (8-oxoG) (Figure 16.11 ). Researchers often measure the amount of 8-oxoG in a sample of DNA to determine the extent of oxidative stress. Although oxidative DNA damage can occur spontaneously, it also results from environmental agents, such as ultraviolet light, X-rays, and many chemicals including those found in cigarette smoke. Oxidative Stress May Also Lead to DNA Damage and Mutation DNA Sequences Known as Trinucleotide Repeats Are Hotspots for Mutation Researchers have discovered that several human genetic diseases are caused by an unusual form of mutation known as trinucleotide repeat expansion (TNRE). The term refers to the phenomenon in which a repeated sequence of three nucleotides can readily increase in number from one generation to the next. In humans and other species, certain genes and chromosomal locations contain regions where trinucleotide sequences are repeated in tandem. These sequences are usually transmitted normally from parent to offspring without mutation. However, in persons with TNRE disorders, the length of a trinucleotide repeat has increased above a certain critical size and thereby causes disease symptoms. DNA Sequences Known as Trinucleotide Repeats Are Hotspots for Mutation Table 16.5 describes several human diseases that involve these types of expansions, including spinal and bulbar muscular atrophy (SBMA), Huntington disease (HD), spinocerebellar ataxia (SCA1), fragile X syndromes (FRAXA and FRAXE), and myotonic muscular dystrophy (DM). In some cases, the expansion is within the coding sequence of the gene. Typically, such an expansion is a CAG repeat. Because CAG encodes a glutamine codon, these repeats cause the encoded proteins to contain long tracts of glutamine. The presence of glutamine tracts causes the proteins to aggregate. This aggregation of proteins or protein fragments carrying glutamine repeats is correlated with the progression of the disease. DNA Sequences Known as Trinucleotide Repeats Are Hotspots for Mutation Mutagens Alter DNA Structure in Different Ways Mutagenic agents are usually classified as chemical or physical mutagens. Examples of both types of agents are listed in Table 16.6. In some cases, chemicals that are not mutagenic can be altered to a mutagenically active form after being ingested. Cellular enzymes such as oxidases have been shown to activate some mutagens. Mutagens can alter the structure of DNA in various ways. Some mutagens act by covalently modifying the structure of bases. For example, nitrous acid (HNO2) replaces amino groups with keto groups ( “NH2 to “O), a process called deamination. This can change cytosine to uracil and adenine to hypoxanthine. When this altered DNA replicates, the modified bases do not pair with the appropriate bases in the newly made strand. Instead, uracil pairs with adenine, and hypoxanthine pairs with cytosine (Figure 16.15 ). Testing Methods Can Determine If an Agent Is a Mutagen To determine if an agent is mutagenic, researchers use testing methods that can monitor whether or not an agent increases the rate of mutation. Many different kinds of tests have been used to evaluate mutagenicity. One common method is the Ames test, which was developed by Bruce Ames. This test uses strains of a bacterium, Salmonella typhimurium, that cannot synthesize the amino acid histidine. These strains contain a point mutation within a gene that encodes an enzyme required for histidine biosynthesis. The mutation renders the enzyme inactive. Therefore, the bacteria cannot grow on petri plates unless histidine has been added to the growth medium. However, a second mutation—a reversion— may occur that restores the ability to synthesize histidine. In other words, a second mutation can cause a reversion back to the wild-type condition. The Ames test monitors the rate at which this second mutation occurs, thereby indicating whether an agent increases the mutation rate above the spontaneous rate. Testing Methods Can Determine If an Agent Is a Mutagen Figure 16.18 outlines the steps in the Ames test. The suspected mutagen is mixed with a rat liver extract and a strain of Salmonella that cannot synthesize histidine. A mutagen may require activation by cellular enzymes, which are provided by the rat liver extract. This step improves the ability of the test to identify agents that may cause mutation in mammals. After the incubation period, a large number of bacteria are then plated on a growth medium that does not contain histidine. The Salmonella strain is not expected to grow on these plates. However, if a mutation has occurred that allows the strain to synthesize histidine, it can grow on these plates to form a visible bacterial colony. To estimate the mutation rate, the colonies that grow on the media are counted and compared with the total number of bacterial cells that were originally streaked on the plate. Testing Methods Can Determine If an Agent Is a Mutagen For example, if 10,000,000 bacteria were plated and 10 growing colonies were observed, the rate of mutation is 10 out of 10,000,000; this equals 1 in 106, or simply 10–6. As a control, bacteria that have not been exposed to the mutagen are also tested, because a low level of spontaneous mutations is expected to occur. How do we judge if an agent is a mutagen? Researchers compare the mutation rate in the presence and absence of the suspected mutagen. The experimental approach shown in Figure 16.18 is conducted several times. If statistics reveal that the mutation rates in the experimental and control samples are significantly different, researchers may tentatively conclude that the agent may be a mutagen.
