Textbook Assignment: Chapter 14, pages 389-406. Material between pages 406 and 409, listed in the syllabus as part of this lecture, will be included in Lecture 26.
Major concepts
Introduction: These notes are unusually long, at least in part because they bring together into one place many aspects of mutation and mutagenesis that have already been introduced at least briefly in previous lectures. As you read through the familiar parts, please do not overlook the substantial amount of new material that has been blended in to provide a more thorough understanding of the overall concepts.
Mutation: A mutation is any change in genetic information relative to a reference "wild-type" genome, including changes that affect expression of genes without altering their coding sequences and changes that do not cause any detectable phenotypic difference (silent mutations). In a complex organism, mutation can occur at many different structural levels and can be classified in many different ways:
Point mutations were originally defined as those involving a chromosomal region that was too small for the change to be detected cytologically (particularly in the giant polytene chromosomes of Drosophila larval salivary glands). In current usage, point mutations are usually understood to involve only one base pair, but to include both substitutions (transitions and transversions), and the addition or deletion of a single base pair. A point mutation can result in missense (amino acid substitution), nonsense (insertion of a stop codon), or frameshift (either positive or negative).
Gene mutations are defined as those that occur entirely within one gene (and its upstream regulatory sequences) and may be either point mutations or other small disruptions of normal chromosomal structure that occur entirely within one gene.
Chromosomal mutations are defined as those that involve deletion, inversion, duplication, or other changes of a chromosomal region that is large enough so the change can be detected cytologically. By definition, chromosomal mutations are limited entirely to a single chromosome, although there could be more than one chromosomal mutation within a genome.
Genomic mutations are defined as those that involve loss or gain of whole chromosomes, translocation from one chromosome to another or other gross chromosomal rearrangements. Note that both chromosomal and genomic mutations can cause aneuploidy.
The importance of mutation: Genes are stable repositories of the information needed for synthesis of all of the RNA and proteins in a living organism. Survival and stability of each species is dependent on faithful replication of genetic information for use by each new generation. However, a low level of mutational change is highly desirable. Over an extended period of time, mutational changes provide the ability for species to adapt to changing conditions and challenges, and thus serve as the raw material for selective survival and the evolution of more advanced and efficient species, as well as the development of biological diversity.
Somatic and germ-line mutations: The mutations that we normally deal with in genetics are those that occur in the germ-line and are thus passed on to subsequent generations. However, mutations can also occur in somatic cells. Those mutations affect only the immediate progeny of the cells they occur in and are not inherited. Colored spots in Indian corn are caused by back mutation of a relatively unstable mutation that is responsible for loss of pigmentation. Cancer is caused by somatic mutations that alter normal cellular growth regulatory mechanisms in a single cell and its direct progeny.
Morphological mutations: Classical genetics was based almost exclusively on the study of mutations that caused affected progeny to be visibly altered. Mendel's original work was done with inbred strains of peas that were true-breeding for particular traits. However, in the years following the rediscovery of Mendel's laws, mutations were generated in wild type stocks by exposure to X-rays or other mutagenic treatments. In order to be detected and studied, the mutations had to be visibly different from the wild-type parental strains. Although the term "morphological" normally refers to structural properties, the term "morphological mutation" is often used more broadly to refer to any visible change, including changes in coloration.
Naming of mutations: When working with classical morphological mutations, it is important to remember that the names given to induced mutations usually describe the recessive phenotype. Thus, a gene named for white eyes codes for a gene product needed for normal synthesis of pigments in wild-type eyes. Similarly, a gene called brown codes for a step in the synthesis of vermillion pigment, which, when absent, leaves the eyes with a brown color. This nomenclature can be thoroughly confusing when one begins to analyze the molecular and biochemical mechanisms responsible for classical mutations. It is important to remember that for most classical recessive mutations, the wild-type allele codes for the protein that must be present and functional to prevent expression of the mutant phenotype.
Nutritional and biochemical mutations: For microorganisms that can be grown on defined (or semi-defined) culture media, it is possible to select for auxotrophic mutations that require nutrients that the wild type organisms can make for themselves. Wild type organisms that are able to multiply in a medium lacking such a nutrient are called prototrophs. There are also many biochemical mutations that affect proteins other than those involved in synthesis of nutrients.
Lethal mutations: Any mutation that disrupts an essential function needed for survival will be lethal when homozygous. In many cases, heterozygotes can function reasonably normally, and they may be virtually indistinguishable from wild type. In other cases, the heterozygote may have a distinctive phenotype, as in the tailless Manx cat. In such cases, the gene is described as a dominant lethal, as discussed in earlier lectures. (Note that a more precise description would refer to the phenotype of the heterozygote as dominant and the lethality as recessive )
Molecular nature of point mutations: Point mutations can occur in a variety of ways (including frameshift mutations, which are discussed separately below). A change in a single base pair that alters a codon and causes an amino acid substitution in the coded protein is called a missense mutation. If one purine is replaced by another purine or if one pryimidine is replaced by another pyrimidine in the sense strand base sequence (with complementary changes in the antisense strand), the substitution is called a transition. If the substitution involves replacement of a purine with a pyrimidine or a pyrimidine with a purine, it is called a transversion.
Missense mutations: Most base pair substitutions change the amino acid specified by the codon in which they occur. Such mutations are described as missense mutations because they cause an amino acid substitution in the coded protein. Depending on the nature of the amino acid substitution and its location within the protein, missense mutations may have a variety of effects, ranging from complete loss of biological activity to reduced activity or temperature-sensitive activity or no funtional effect at all.
Nonsense mutations: Base pair mutations that generate a translation stop codon (TAA, TAG or TGA in the DNA sense strand, transcribed as UAA, UAG or UGA in the mRNA) cause premature termination of translation of the coded protein and are referred to as nonsense mutations. In some cases, the effects of nonsense mutations can be suppressed by modified tRNA molecules that insert an amino acid with a low efficiency when a stop codon is encountered. Bacterial strains that contain such tRNAs are referred to as suppressor strains.
Silent mutations: In some cases, base pair substitutions generate a different codon for the same amino acid, with no biological effect whatsoever. This is most likely to happen in the third position (wobble base) of redundant codons for the same amino acid. Such changes are considered to be mutations because they alter the genetic code. However, because they have no phenotypic effect, even at the level of protein amino acid sequence, they are called silent mutations.
Frameshift mutations: The genetic code is translated three nucleotide bases (one codon) at a time, with no punctuation between the codons. Addition or deletion of a single base pair in the middle of a coding sequence will result in out-of-frame translation of all of the downstream codons, and thus result in a completely different amino acid sequence, which is often prematurely truncated by stop codons (UAG,UAA,UGA) generated by reading the coding sequence out-of-frame. Such mutations, which are a special subclass of point mutations, are referred to as frameshift mutations. Deletion of a single base pair results in moving ahead one base in all of the codons, and is often referred to as a positive frameshift. Addition of one base pair (or loss of two base pairs) shifts the reading frame behind by one base, and is often referred to as a negative frameshift. Note that deletion or addition of three base pairs (or multiples of threes) does not cause a frameshift, but instead results in deletion or addition of one or more amino acids in the coded protein.
Conditional mutations: Some types of mutations exert their phenotypic effects only under certain environmental conditions. Such mutations are called conditional mutations.
Temperature-sensitive (ts) mutations are missense mutations that do not seriously affect the biological activity of the coded proteins, but cause them to have a reduced thermal stability. Such proteins become denatured at temperatures that do not affect the corresponding wild-type proteins. However, when the mutant strains are maintained at a lower temperature, the proteins are still able to function reasonably well, and no mutant phenotype is observed. Temperature-sensitive mutations are particularly useful for studying vital functions, such as progression through the cell division cycle. In order to maintain stock cultures of organisms carrying such mutations, it is necessary to be able to expand populations under conditions where the mutations are not expressed phenotypically. Growth at low temperature and analysis of the mutant phenotype at a higher temperature provides such a system.
Nonsense suppression: Another approach to conditional mutation that is used extensively in studies on bacterial viruses is to generate nonsense mutations involving the amber codon (UAG). Viruses bearing such mutations can often be maintained in amber suppressor strains of bacteria and then transferred to regular strains to study the phenotypic effects of the mutations. The amber suppressor strains contain an altered transfer RNA that inefficiently reads the UAG codon as coding for an amino acid. If the protein is able to function with that particular amino acid inserted at the location of the amber mutation, the virus is able to replicate, although often with reduced efficiency, in the amber suppressor strain (see page 333 of the textbook).
Permissive and nonpermissive conditions: The conditions that allow growth or function without phenotypic expression of conditional mutations are referred to as permissive. The conditions that cause phenotypic expression to occur are referred to as nonpermissive. This nomenclature refers primarily to conditions that permit growth or do not permit growth, but can also be used for other types of conditional mutations, such as loss of pigmentation at higher temperatures in Siamese cats and Himalayan rabbits (Figure 7.3 in the textbook). Permissive conditions allow the non-mutant phenotype to be expressed.
Conditional lethal mutations: Conditional mutations that do not allow survival of the organism under nonpermissive conditions are referred to as conditional lethal mutations. Note that many other conditional mutations cause expression of mutant phenotypes at non-permissive temperatures without being lethal. Bleaching of coat color on warmer parts of the bodies of Siamese cats is an example of this.
Historical considerations: The textbook devotes several pages (pp. 390-392) to early studies that pointed the way toward modern genetic concepts of mutation prior to the availability of DNA sequence analysis as a method for determining the exact structure of genes. Some of the key points are summarized briefly below.
Delbruck and Luria fluctuation test: This test was designed to determine whether bacterial mutations were induced by stress conditions, such as bacteriophage infection, or whether they occurred spontaneously and were present prior to exposure to the stress conditions. Delbruck and Luria argued that if mutation was spontaneous, cultures started from small populations and grown up to large numbers in the absence of bacteriophage T1 should exhibit major variability in numbers of phage-resistant cells that they contain, depending on when during the culture history the mutations to phage resistance occurred, This was in fact what they found when the cultures were inoculated onto plates that contained large numbers of phage (Table 14.1). This was one of the final steps in disproving the Lamarkian view that genetic change was induced in response to environmental conditions.
Mechanisms of mutation: This portion of the lecture deals primarily with the mechanisms responsible for point mutations and their reversion or suppression. Strictly speaking, the term reversion should be used only to describe an exact reversal of the original mutational change. Many other secondary changes, either within the same gene, or in other genes can suppress the effects of a mutation. Such changes are called intragenic suppression and intergenic suppression, respectively.
Tautomerization: Spontaneous mutations that involve base pair substitutions are caused primarily by configurational changes within the individual bases that result in mispairing. These changes, which are called tautomeric shifts, involve momentary expression of rare alternative molecular configurations that exist in equilibrium with the more common forms. Specifically, proton shifts can convert the amino groups in adenine and cytosine to imino groups, and the keto groups in guanine and thymine to enol groups (Figures 14.7, 14.8)
Transitions: A tautomeric shift in any of the four DNA bases can lead to mispairing of A to C or G to T. The tautomeric state can occur either in the template base or the incoming base. During the next round of DNA synthesis, the mispaired base pairs with its normal partner, resulting in a transition, in which an AT base pair replaces a GC or a GC replaces an AT, with no change in the purine:pyrimidine polarity of the base pair (Figure 14.9). Transitions are the most common type of mutation resulting from spontaneous mispairing due to tautomerization.
Transversions: To achieve a transversion, in which the positions of purine and pyrimidine are reversed in the DNA double helix, two events are thought to be involved, tautomerization of one of the bases and rotation of the other to yield a purine:purine pairing. Based on information from the previous textbook for this course, the frequency of spontaneous transversions, which is lower than that of transitions, appears to be consistent with this interpretation. However, that book also warns that recent studies suggest that the overall picture may be more complex. Our current text does not discuss transversions in much detail. A second possible mechanism for transversions is the formation of an apurinic site (described below), which can result in replacement of the original purine with any of the four bases.
Frameshifts: Spontaneous frameshift mutations are believed to arise primarily from mispairing within long runs of the same base in a coding sequence. Such regions are believed to be one of the causes of mutational "hot spots" that have been observed during fine-structure genetic mapping.
Deamination: Our current textbook discusses deamination of cytosine primarily in terms of mutagenesis by nitric oxide (page 405), but spontaneous deamination also has an important role, particularly in methylated regions of DNA. If a cytosine undergoes oxidative deamination, it becomes uracil, which is capable of pairing with adenine (as in RNA synthesis), but is detected as an anomoly in DNA and may trigger repair mechanisms. However, if 5-methylcytosine is deaminated, it forms thymine, which is a normal DNA base that is not detected by repair systems (other than proofreading of GT mispairing during DNA synthesis). Because of selective methylation of CG sequences in many DNAs, there is a tendancy for all non-essential CG sequences to be converted to TG sequences over time. Methylated CG sequences are thus hot spots for mutation, such that in DNA in general, CG sequences tend to be far less frequent that TG sequences. (Remember that a sequence is always described in 5' to 3' terms, such that CG means 5'-CG-3').
Spontaneous mutation rate: For single-celled organisms ranging from bacteria to cultured mammalian cells, mutation rate is usually measured as the probability of mutation within a specific gene per cell division. For higher animals, the rate is measured in terms of the probability per gamete per generation (remember that each new individual contains the contributions from two separate gametes). Bacterial rates are typically in the range of 10-8 to 10-6 per generation. Mammalian (including human) rates for individual easily observed mutations tend to be on the order of 10-5 per generation (See Table 14.2).
Chemical mutagenesis: A variety of chemical mutagens have been discovered that act in several distinctly different ways. Many chemicals that are used in modern industry and technology are potentially mutagenic, which includes their ability to cause cancer as a result of somatic mutation. Page 409 of the textbook contains a description of the Ames test for carcinogens, which is based on mutagenicity in specially engineered strains of bacterial cells that have been stripped of most of their repair mechanisms, and that must undergo back mutation in a gene for histidine synthesis to be able to form colonies on a selective medium. In some cases, a liver extract is added to simulate metabolic conversion of potential carcinogens into active carcinogens in the human body. The test has been further refined through the use of strains that respond to different types of mutagenic activity (base substitution vs. frameshift).
Base analogues: One of the more popular approaches to experimental mutagenesis is the use of base analogues. These are substances that are sufficiently similar to naturally occurring DNA bases so that their deoxyribonucleotide triphosphates will incorporate into DNA in place of the normal bases. However, they also have anomolous base-pairing properties, leading to an increased rate of mutagenesis. For example, 5-bromouracil (Fig. 14.10) pairs like thymine (5-methyluracil), but undergoes more enol tautomerization, leading to more frequent mispairing with guanine. Similarly, 2-aminopurine normally pairs with thymine, but can also pair with cytosine (see Insights and Solutions #2, page 423). These mispairings lead to an increase in the frequency of transition mutagenesis.
Nitrous acid: Treatment of DNA with nitrous acid leads to deamination of cytosine and adenine, again resulting in transitions, as described above for spontaneous deamination (Figure 14.13).
Alkylating agents: Certain alkylating agents, such as ethyl methane sulfonate (EMS) and ethyl ethane sulfonate (EES) add alkyl groups to purines, which can cause mispairing (Fig.14.11), and also destabilize the bond between the purine and deoxyribose, leaving apurinic sites. The absence of a base-pairing partner allows any base to be inserted during the next round of DNA synthesis. This frequently leads to transversions (as well as transitions).
Intercalation: Certain flat aromatic molecules, such as acridine orange and proflavin become inserted between base pairs in DNA, which can lead to misalignment during replication and the occurence of frameshift mutation (fig 14.12).
Reversion: As indicated earlier, the term reversion should only be used to describe an exact reversal of a mutation. For a base-substitution mutation (missense or nonsense), this would mean replacement of the substituted base with the original base. For a frameshift, this would mean removal of the inserted base pair or replacement of the deleted base pair. The net result of reversion is to restore the original genetic sequence exactly. Note that complete failure to revert usually indicates that a mutation is the result of a major change, such as a deletion that is incapable of being reversed.
Intragenic suppression: Intragenic suppression refers to a second mutation within the same coding unit that reverses the effect of the first mutation without actually correcting it. For example, if correct protein folding depended on interaction of a positive charge with a negative charge and the positive was mutated to negative, function could be restored by mutating the original negative to positive so that there was once again a positive-negative pair to guide the folding. Similarly, a frameshift might be reversed by a nearby second frameshift in the opposite direction, such that only a few non-essential amino acids were altered.
Intergenic suppression: In some cases, a second mutation in another gene can reverse the effects of a mutation. For example, if heterodimer formation is required for function, a complementary change in the second protein could allow proper pairing to occur once again. This is also the presumed mechanism for the intracistronic complementation that is sometimes observed, although in this case, the two changes are are in the same protein, making it intragenic suppression. Another example is suppression of a nonsense mutation by an altered tRNA that reads the stop codon as an amino acid specifying codon (Page 333).
Sickle-cell anemia as an example of a missense mutation: Sickle-cell anemia was identified in 1957 as being caused by a missense mutation resulting in a single amino acid substitution in the beta-globulin subunit of the hemoglobin tetramer (2 alpha + 2 beta subunits). A transversion causes the codon GAG to be changed to GUG (GTG in the DNA). This replaces a glutamic acid with a valine as the sixth amino acid (counting from the N-terminus) in the mature beta-globulin molecule. That substitution causes the hemoglobin to precipitate into fibrous aggregates that distort the shapes of red blood cells under low-oxygen conditions, resulting both in blockage of capillary circulation and breakage of the red blood cells. (Described on pages 371-373 of our textbook)
Heterozygote advantage: One obvious question is why a genetic disease as severe as sickle-cell anemia is present at such a high level in African-American populations. The reason is that in regions of Africa with a high incidence of malaria, individuals who are heterozygous for the altered beta-globulin have a better survival rate due to malaria resistance than individuals who are homozygous for unaltered beta-globulin. Thus, the heterozygotes had enough selective advantage so that the sickle-cell gene became well established in the population even though homozygotes were severely unhealthy and usually experienced early death. Unfortunately, this genetic legacy will persist for many generations, even in the absence of the selective effect of malaria.
Molecular basis for dominance and recessiveness: As we have gained a better understanding of the molecular nature of different types of mutaitons, we have also begun to understand what makes a particular mutation recessive or dominant.
Recessive mutations usually result from partial or complete loss of a wild type function. Amorphic alleles are those that have completely lost the function. An example would be a mutation in which production of pigment is completely lost in the homozygous state, causing albinism. Hypomorphic alleles are those in which function is reduced, but not completely lost. An example would be a mutation that causes a partial loss of pigmentation, giving a lighter color when homozygous.
Dominance can be caused in a wider variety of ways. There are three classes of so called gain-of-function alleles. Hypermorphic alleles are those that cause excess product to be produced. Antimorphic alleles are those that produce an altered gene product that "poisons" or disrupts the function of the normal gene product. Neomorphic alleles cause the gene product to be expressed in the wrong types of cells, and can have drastic effects, such as that of the antennapedia gene that coverts the antennae of flies into legs.
Another type of dominance is haplo-insufficiency. In this case, loss of a gene product causes a recognizably different phenotype in the heterozygote. This is considered to be a dominant mutation because the presence of one copy of the mutant allele in combination with one copy of the wild-type allele causes an altered phenotype. In many cases, the homozygote is lethal, as in the case of the Manx cat. In cases where the mutation is not lethal when homozygous, haplo-insufficiency is more likely to be called partial dominance, as in the formation of a pink flower by a heterozygote containing one red allele and one white allele (Pages 80-81, Figure 4.1).