Gene mutations can arise spontaneously or they can be induced. Spontaneous mutations are naturally occurring mutations and arise in all cells. Induced mutations arise through the action of certain agents called mutagens that increase the rate at which mutations occur. In this section, we consider the nature of spontaneous mutations.

The origin of spontaneous hereditary change has always been a topic of considerable interest. Among the first questions asked by geneticists was, Do spontaneous mutations occur in response to the selecting agent or are variants present at a low frequency in most populations? An ideal experimental system to address this important question was the analysis of mutations in bacteria that confer resistance to specific environmental agents not normally tolerated by wild-type cells.

One experiment by Salvador Luria and Max Delbrück in 1943 was particularly influential in shaping our understanding of the nature of mutation, not only in bacteria, but in organisms generally. It was known at the time that, if E. coli bacteria are spread on a plate of nutrient medium in the presence of phage T1, the phages soon infect and kill the bacteria. Rarely, but regularly, colonies were seen that were resistant to phage attack; these colonies were stable and so appeared to be genuine mutants. However, whether these mutants were produced spontaneously but randomly in time or the presence of the phage induced a physiological change that caused resistance was not known.

587

Luria reasoned that, if mutations occurred spontaneously, then the mutations might be expected to occur at different times in different cultures. In this case, the numbers of resistant colonies per culture should show high variation (or “fluctuation” in his word). He later claimed that the idea came to him as he watched the fluctuating returns obtained by colleagues gambling on a slot machine at a faculty dance in a local country club; hence the origin of the term “jackpot” mutation.

Luria and Delbrück designed their “fluctuation test” as follows. They inoculated 20 small cultures, each with a few cells, and incubated them until there were 10⁸ cells per milliliter. At the same time, a much larger culture also was inoculated and incubated until there were 10⁸ cells per milliliter. The 20 individual cultures and 20 samples of the same size from the large culture were plated in the presence of phage. The 20 individual cultures showed high variation in the number of resistant colonies: 11 plates had 0 resistant colonies, and the remainder had 1, 1, 3, 5, 5, 6, 35, 64, and 107 per plate (Figure 16-5a). The 20 samples from the large culture showed much less variation from plate to plate, all in the range of 14 to 26. If the phage were inducing mutations, there was no reason why fluctuation should be higher on the individual cultures because all were exposed to phage similarly. The best explanation was that mutation was occurring randomly in time: the early mutations gave the higher numbers of resistant cells because the mutant cells had time to produce many resistant descendants. The later mutations produced fewer resistant cells (Figure 16-5b). This result led to the reigning “paradigm” of mutation; that is, whether in viruses, bacteria, or eukaryotes, mutations can occur in any cell at any time and their occurrence is random. For this and other work, Luria and Delbrück were awarded the Nobel Prize in Physiology or Medicine in 1969. Interestingly, this was after Luria’s first graduate student, James Watson, won his Nobel Prize (with Francis Crick in 1964) for the discovery of the DNA double-helix structure.

588

This elegant analysis suggests that the resistant cells are selected by the environmental agent (here, phage) rather than produced by it. Can the existence of mutants in a population before selection be demonstrated directly? This demonstration was made possible by the use of a technique called replica plating, developed largely by Esther Lederberg in 1952. A population of bacteria was plated on nonselective medium—that is, medium containing no phage—and from each cell a colony grew. This plate was called the master plate. A sterile piece of velvet was pressed down lightly on the surface of the master plate, and the velvet picked up cells wherever there was a colony (Figure 16-6). In this way, the velvet picked up a colony “imprint” from the whole plate. The velvet was then touched to replica plates containing selective medium (that is, containing T1 phage). On touching velvet to plates, cells clinging to the velvet are inoculated onto the replica plates in the same relative positions as those of the colonies on the original master plate. As expected, rare resistant mutant colonies were found on the replica plates, but the multiple replica plates showed identical patterns of resistant colonies (Figure 16-7). If the mutations had occurred after exposure to the selective agents, the patterns for each plate would have been as random as the mutations themselves. The mutation events must have occurred before exposure to the selective agent. Again, these results confirm that mutation is occurring randomly all the time, rather than in response to a selective agent.

Spontaneous mutations arise from a variety of sources. One source is the DNA-replication process. Although DNA replication is a remarkably accurate process, mistakes are made in the copying of the millions, even billions, of base pairs in a genome. Spontaneous mutations also arise in part because DNA is a very labile molecule and the cellular environment itself can damage it. As described in Chapter 15, mutations can even be caused by the insertion of a transposable element from elsewhere in the genome. In this chapter, we focus on mutations that are not caused by transposable elements.

Errors in DNA replication An error in DNA replication can result when an illegitimate nucleotide pair (say, A–C) forms in DNA synthesis, leading to a base substitution that may be either a transition or a transversion. Other errors may add or subtract base pairs such that a frameshift mutation is created.

Transitions You saw in Chapter 7 that each of the bases in DNA can appear in one of several tautomeric forms that can pair to the wrong base. Mismatches can also result when one of the bases becomes ionized. This type of mismatch may occur more frequently than mismatches due to tautomerization. These errors are frequently corrected by the proofreading (editing) function of bacterial DNA pol III (see Figure 7-18). If proofreading does not occur, all the mismatches described so far lead to transition mutations, in which a purine substitutes for a purine or a pyrimidine for a pyrimidine (see Figure 16-2). Other repair systems (described later in this chapter) correct many of the mismatched bases that escape correction by the polymerase editing function.

589

Transversions In transversion mutations, a pyrimidine substitutes for a purine, or vice versa (see Figure 16-2). The creation of a transversion by a replication error would require, at some point in the course of replication, the mispairing of a purine with a purine or a pyrimidine with a pyrimidine. Although the dimensions of the DNA double helix render such mismatches energetically unfavorable, we now know from X-ray diffraction studies that G–A pairs, as well as other purine–purine pairs, can form.

Frameshift mutations Replication errors can also lead to frameshift mutations. Recall from Chapter 9 that such mutations result in greatly altered proteins.

Certain kinds of replication errors can lead to indel mutations—that is, insertions or deletions of one or more base pairs. These insertions or deletions produce frameshift mutations when they add or subtract a number of bases not divisible by three (the size of a codon) in the protein-coding regions. The prevailing model (Figure 16-8) proposes that indels arise when loops in single-stranded regions are stabilized by the “slipped mispairing” of repeated sequences in the course of replication. This mechanism is sometimes called replication slippage.

Spontaneous lesions In addition to replication errors, spontaneous lesions, naturally occurring damage to the DNA, can generate mutations. Two of the most frequent spontaneous lesions result from depurination and deamination.

Depurination, the more common of the two, is the loss of a purine base. Depurination consists of the interruption of the glycosidic bond between the base and deoxyribose and the subsequent loss of a guanine or an adenine residue from the DNA. The DNA backbone remains intact.

A mammalian cell spontaneously loses about 10,000 purines from its DNA in a 20-hour cell-cycle period at 37°C. If these lesions were to persist, they would result in significant genetic damage because, in replication, the resulting apurinic sites cannot specify a base complementary to the original purine. However, as we will see later in the chapter, efficient repair systems remove apurinic sites. Under certain conditions (to be described later), a base can be inserted across from an apurinic site; this insertion will frequently result in a mutation.

590

The deamination of cytosine yields uracil.

Unrepaired uracil residues will pair with adenine in replication, resulting in the conversion of a G · C pair into an A · T pair (a G · C → A · T transition).

Oxidatively damaged bases represent a third type of spontaneous lesion that generates mutations. Active oxygen species, such as superoxide radicals (O₂ · ^–), hydrogen peroxide (H₂O₂), and hydroxyl radicals (· OH), are produced as by-products of normal aerobic metabolism. They can cause oxidative damage to DNA, as well as to precursors of DNA (such as GTP), resulting in mutation. Mutations from oxidative damage have been implicated in a number of human diseases. Figure 16-9 shows two products of oxidative damage. The 8-oxo-7-hydrodeoxyguanosine (8-oxo dG, or GO) product frequently mispairs with A, resulting in a high level of G · T transversions. The thymidine glycol product blocks DNA replication if unrepaired.

591

DNA sequence analysis has revealed the gene mutations contributing to numerous human hereditary diseases. Many are of the expected base-substitution or single-base-pair indel type. However, some mutations are more complex. A number of these human disorders are due to duplications of short repeated sequences.

A common mechanism responsible for a number of genetic diseases is the expansion of a three-base-pair repeat. For this reason, they are termed trinucleotide repeat diseases. An example is the human disease called fragile X syndrome. This disease is the most common form of inherited mental impairment, occurring in close to 1 of 1500 males and 1 of 2500 females. It is manifested cytologically by a fragile site in the X chromosome that results in breaks in vitro (but this does not lead to the disease phenotype). Fragile X syndrome results from changes in the number of a (CGG)_n repeat in a region of the FMR-1 gene that is transcribed but not translated (Figure 16-10a).

How does repeat number correlate with the disease phenotype? Humans normally show considerable variation in the number of CGG repeats in the FMR-1 gene, ranging from 6 to 54, with the most frequent allele containing 29 repeats. Sometimes, unaffected parents and grandparents give rise to several offspring with fragile X syndrome. The offspring with the symptoms of the disease have enormous repeat numbers, ranging from 200 to 1300 (Figure 16-10b). The unaffected parents and grandparents also have been found to contain increased copy numbers of the repeat, but ranging from only 50 to 200. For this reason, these ancestors have been said to carry premutations. The repeats in these premutation alleles are not sufficient to cause the disease phenotype, but they are much more unstable (that is, readily expanded) than normal alleles, and so they lead to even greater expansion in their offspring. (In general, the more expanded the repeat number, the greater the instability appears to be.)

592

The proposed mechanism for the generation of these repeats is replication slippage that occurs in the course of DNA synthesis (Figure 16-11). However, the extraordinarily high frequency of mutation at the trinucleotide repeats in fragile X syndrome suggests that in human cells, after a threshold level of about 50 repeats, the replication machinery cannot faithfully replicate the correct sequence and large variations in repeat numbers result.

Other diseases, such as Huntington disease (see Chapter 2), also have been associated with the expansion of trinucleotide repeats in a gene or its regulatory regions. Several general themes apply to these diseases. In Huntington disease, for example, the wild-type HD gene includes a repeated sequence, often within the protein-coding region, and mutation correlates with a considerable expansion of this repeat region. The severity of the disease correlates with the number of repeat copies.

Huntington disease and Kennedy disease (also called X-linked spinal and bulbar muscular atrophy) result from the amplification of a three-base-pair repeat, CAG. Unaffected persons have an average of 19 to 21 CAG repeats, whereas affected patients have an average of about 46. In Kennedy disease, which is characterized by progressive muscle weakness and atrophy, the expansion of the trinucleotide repeat is in the gene that encodes the androgen receptor.

Properties common to some trinucleotide-repeat diseases suggest a common mechanism by which the abnormal phenotypes are produced. First, many of these diseases seem to include neurodegeneration—that is, cell death within the nervous system. Second, in such diseases, the trinucleotide repeats fall within the open reading frames of the transcripts of the mutated gene, leading to expansions or contractions of the number of repeats of a single amino acid in the polypeptide (for example, CAG repeats encode a polyglutamine repeat). Thus, it is easy to understand why these diseases result from expansions of codon-size units three base pairs in length.

However, this explanation cannot hold for all trinucleotide-repeat diseases. After all, in fragile X syndrome, the trinucleotide expansion is near the 5′ end of the FMR-1 mRNA, before the translation start site. Thus, we cannot ascribe the phenotypic abnormalities of the FMR-1 mutations to an effect on protein structure. One clue to the problem with the mutant FMR-1 genes is that they, unlike the normal gene, are hypermethylated, a feature associated with transcriptionally silenced genes (see Figure 16-10b). On the basis of these findings, repeat expansion is hypothesized to lead to changes in chromatin structure that silence the transcription of the mutant gene (see Chapter 12). In support of this model is the finding that the FMR-1 gene is deleted in some patients with fragile X syndrome. These observations support a loss-of-function mutation.

593