Genetic mutations

Genetica per Scienze Naturalia.a. 04-05 prof S. Presciuttini

Genetic mutations A A mutation mutation is a is a herheriitatableble change change of the genetic materialof the genetic material Geneticists recognize three different levels at which mutation takes Geneticists recognize three different levels at which mutation takes

place.place. In In gene mutationgene mutation, an allele of a gene changes, becoming a different allele. , an allele of a gene changes, becoming a different allele.

Because such a change takes place within a single gene and maps to one Because such a change takes place within a single gene and maps to one chromosomal locus ("point"), a gene mutation is sometimes called a point chromosomal locus ("point"), a gene mutation is sometimes called a point mutation.mutation.

In In chromosome mutationchromosome mutationss, the structure of one or more chromosome is altered., the structure of one or more chromosome is altered. Gene mutation is not necessarily a part of such a process; the effects of Gene mutation is not necessarily a part of such a process; the effects of chromosome mutation are due more to the new arrangement of chromosomes and chromosome mutation are due more to the new arrangement of chromosomes and of the genes that they contain. Nevertheless, some chromosome mutations, in of the genes that they contain. Nevertheless, some chromosome mutations, in particular those proceeding from chromosome breaks, are accompanied by gene particular those proceeding from chromosome breaks, are accompanied by gene mutations caused by the disruption at the breakpoint.mutations caused by the disruption at the breakpoint.

In In genome mutationsgenome mutations, whole chromosomes, or even entire sets of chromosomes, , whole chromosomes, or even entire sets of chromosomes, change. Duplications of entire genomes in the course of evolution are particularly change. Duplications of entire genomes in the course of evolution are particularly important as a mechanism resulting in sudden expansions in gene numberimportant as a mechanism resulting in sudden expansions in gene number


Basic terminology about gene mutation The ultimate source of genetic variation is gene mutationThe ultimate source of genetic variation is gene mutation To consider change, we must have a fixed reference point, or standard. In genetics, To consider change, we must have a fixed reference point, or standard. In genetics,

thethe wild typewild type provides the standard provides the standard ((the wild-type allele may be either the form the wild-type allele may be either the form found in nature or the form found in a standard laboratory stockfound in nature or the form found in a standard laboratory stock))..

Any change away from the wild-type allele is called Any change away from the wild-type allele is called forward mutation;forward mutation; any any change back to the wild-type allele is called change back to the wild-type allele is called reverse mutationreverse mutation..

The non-wild-type allele of a gene is The non-wild-type allele of a gene is oftenoften called a called a mutationmutation. To use the same word . To use the same word for the process and the product may seem confusing, but in practice little confusion for the process and the product may seem confusing, but in practice little confusion arises.arises.

Thus, we can speak of a Thus, we can speak of a dominant mutationdominant mutation or a or a recessive mutation recessive mutation. . Consider, Consider, however, how however, how arbitrary these arbitrary these definitionsdefinitions are; the wild type of today may have been a are; the wild type of today may have been a mutation in the evolutionary past, and vice versa.mutation in the evolutionary past, and vice versa.

Another useful term is Another useful term is mutantmutant. A mutant organism or cell is one whose changed . A mutant organism or cell is one whose changed phenotype is attributable to the possession of a mutation. Sometimes the noun is left phenotype is attributable to the possession of a mutation. Sometimes the noun is left unstated; in this case, a mutant always means an individual or cell with a phenotype unstated; in this case, a mutant always means an individual or cell with a phenotype that shows that it bears a mutation.that shows that it bears a mutation.

Two other useful terms are Two other useful terms are mutation eventmutation event, which is the actual occurrence of a , which is the actual occurrence of a mutation, and mutation, and mutation frequencymutation frequency, the proportion of mutations in a population of , the proportion of mutations in a population of cells or individual organisms.cells or individual organisms.


Somatic mutations AA somatic mutationsomatic mutation occurs in a single cell occurs in a single cell of of developing somatic tissuedeveloping somatic tissue in an in an

individual organism;individual organism; that cell that cell may becomemay become the progenitor of a population of the progenitor of a population of identical mutant cells, all of which have descended from the cell that mutatedidentical mutant cells, all of which have descended from the cell that mutated; this ; this phenomenon is particularly important in phenomenon is particularly important in cancercancer..

A population of identical cells derived asexually from one progenitor cell is called a A population of identical cells derived asexually from one progenitor cell is called a cloneclone. Because the members of a clone tend to stay close to one another during . Because the members of a clone tend to stay close to one another during development, an observable outcome of a somatic mutation is often a patch of development, an observable outcome of a somatic mutation is often a patch of phenotypically mutant cells called a mutant sector. The earlier in development the phenotypically mutant cells called a mutant sector. The earlier in development the mutation event, the larger the mutant sector will be.mutation event, the larger the mutant sector will be.

Somatic mutation in the red Delicious apple. The Somatic mutation in the red Delicious apple. The mutant allele determining the golden color arose in a mutant allele determining the golden color arose in a flower's ovary wall, which eventually developed into flower's ovary wall, which eventually developed into the fleshy part of the apple. The seeds are not mutant the fleshy part of the apple. The seeds are not mutant and will give rise to red-appled trees. and will give rise to red-appled trees. IIn fact, the n fact, the golden Delicious apple originally arose as a mutant golden Delicious apple originally arose as a mutant branch on a red Delicious tree.branch on a red Delicious tree.


Germinal mutations SSomatic mutations omatic mutations are nare never passed on to progenyever passed on to progeny.. On the contrary, On the contrary, mutations that mutations that occurs in the germ line, special tissue occurs in the germ line, special tissue

that is set aside in the course of development to form sex cellsthat is set aside in the course of development to form sex cells, , will be will be passed on to the next generationpassed on to the next generation. These are called . These are called germinal germinal mutationmutationss. .

An individual of perfectly normal phenotype and of normal ancestry An individual of perfectly normal phenotype and of normal ancestry can harbor undetected mutant sex cells. These mutations can be can harbor undetected mutant sex cells. These mutations can be detected only if they are included in a zygotedetected only if they are included in a zygote.. For example, tFor example, the X-linked hemophilia mutation in European royal families is he X-linked hemophilia mutation in European royal families is

thought to have arisen in the germ cells of Queen Victoria or one of her parents.thought to have arisen in the germ cells of Queen Victoria or one of her parents.


Hemophilia

The original hemophilia mutation in the pedigree of the royal families of Europe arose The original hemophilia mutation in the pedigree of the royal families of Europe arose in the reproductive cells of Queen Victoria's parents or of Queen Victoria herself. in the reproductive cells of Queen Victoria's parents or of Queen Victoria herself.


Point mutations: base substitutions PPoint mutations typically refer to alterations of single base pairs of oint mutations typically refer to alterations of single base pairs of

DNA or of a small number of adjacent base pairs.DNA or of a small number of adjacent base pairs. At the DNA level, there are two main types of point mutational At the DNA level, there are two main types of point mutational

changes: changes: base substitutionsbase substitutions and and base additionsbase additions or or deletions.deletions. Base substitutionsBase substitutions are those mutations in which one base pair is are those mutations in which one base pair is

replaced by another. Base substitutions again can be divided into two replaced by another. Base substitutions again can be divided into two subtypes: subtypes: transitionstransitions and and transversionstransversions..

AdditionAddition or or deletion mutationsdeletion mutations are actually of are actually of nucleotidenucleotide pairs; pairs; nevertheless, the convention is to call them nevertheless, the convention is to call them basebase-pair additions or -pair additions or deletions. The simplest of these mutations are single-base-pair deletions. The simplest of these mutations are single-base-pair additions or single-base-pair deletions. Gene mutations may arise additions or single-base-pair deletions. Gene mutations may arise through simultaneous addition or deletion of multiple base pairs at through simultaneous addition or deletion of multiple base pairs at once.once.


Functional consequences of base changes We fWe first consider what happens when a mutation arises in a polypeptideirst consider what happens when a mutation arises in a polypeptide coding part coding part

of a gene.of a gene. Depending on the consequences, Depending on the consequences, single-base substitutionssingle-base substitutions are classified are classified into:into:

Silent or synonymous Silent or synonymous mutationsmutations:: the mutation changes one codon for an amino acid the mutation changes one codon for an amino acid into another codon for that same amino acid.into another codon for that same amino acid.

Missense mutationsMissense mutations:: the codon for one amino acid is replaced by a codon for another the codon for one amino acid is replaced by a codon for another amino acid.amino acid.

Nonsense mutationsNonsense mutations:: the codon for one amino acid is replaced by a translation the codon for one amino acid is replaced by a translation termination (stop) codon.termination (stop) codon.

The severity of the effect of missense and nonsense mutations on the polypeptide The severity of the effect of missense and nonsense mutations on the polypeptide may may differ. differ. IIf a missense mutation causes the substitution of a chemically similar f a missense mutation causes the substitution of a chemically similar amino acidamino acid ( (conservative substitutionconservative substitution),), then it is likely that the alteration will have then it is likely that the alteration will have a less-severe effect on the protein's structure and function. Alternatively, chemically a less-severe effect on the protein's structure and function. Alternatively, chemically different amino acid substitutions, called different amino acid substitutions, called nonconservative substitutionsnonconservative substitutions,, are more are more likely to produce severe changes in protein structure and function.likely to produce severe changes in protein structure and function.

Nonsense mutations will lead to the premature termination of translation. Thus, they Nonsense mutations will lead to the premature termination of translation. Thus, they have a considerable effect on protein function.have a considerable effect on protein function. U Unless they occur very close to the nless they occur very close to the 33’’ end of the open reading frame, so that only a partly functional truncated end of the open reading frame, so that only a partly functional truncated polypeptide is produced, nonsense mutations will produce inactive protein products.polypeptide is produced, nonsense mutations will produce inactive protein products.


Functional consequences of frameshift mutations On the other hand,On the other hand, single-base additions or deletionssingle-base additions or deletions have have

consequences on polypeptide sequence that extend far beyond the site consequences on polypeptide sequence that extend far beyond the site of the mutation itselfof the mutation itself,, llike nonsense mutations.ike nonsense mutations.

Because the sequence of mRNA is "read" by the translational Because the sequence of mRNA is "read" by the translational apparatus in groups of three base pairs (codons), the addition or apparatus in groups of three base pairs (codons), the addition or deletion of a single base pair of DNA will change the reading frame deletion of a single base pair of DNA will change the reading frame starting from the location of the addition or deletion and extending starting from the location of the addition or deletion and extending through to the carboxy terminal of the protein. Hence, these lesions through to the carboxy terminal of the protein. Hence, these lesions are called are called frameshiftframeshift mutations.mutations.

These mutations cause the entire amino acid sequence translationally These mutations cause the entire amino acid sequence translationally downstream of the mutant site to bear no relation to the original downstream of the mutant site to bear no relation to the original amino acid sequence.amino acid sequence.

Thus, frameshift mutations typically exhibit complete loss of normal Thus, frameshift mutations typically exhibit complete loss of normal protein structure and function.protein structure and function.


Examples of point mutations


Mutations in non-coding regions Now let's turn to those mutations that occur in regulatory and other nonNow let's turn to those mutations that occur in regulatory and other non--coding coding

sequences. Those parts of a gene that are not protein coding contain a variety of sequences. Those parts of a gene that are not protein coding contain a variety of crucial functional sites. At the DNA level, there are sites to which specific crucial functional sites. At the DNA level, there are sites to which specific transcription-regulating proteins must bind. At the RNA level, there are also transcription-regulating proteins must bind. At the RNA level, there are also important functional sequences such as the ribosome-binding sites of bacterial important functional sequences such as the ribosome-binding sites of bacterial mRNAs and the self-ligating sites for intron excision in eukaryote mRNAs.mRNAs and the self-ligating sites for intron excision in eukaryote mRNAs.

The The consequencesconsequences of mutations in parts of a gene other that the polypeptide-coding of mutations in parts of a gene other that the polypeptide-coding segments are segments are difficultdifficult to predict. In general, the functional consequences of any to predict. In general, the functional consequences of any point mutation (substitution or addition or deletion) in such a region depend on its point mutation (substitution or addition or deletion) in such a region depend on its location and on whether it disrupts a functional site. Mutations that disrupt these location and on whether it disrupts a functional site. Mutations that disrupt these sites have the potential to change the expression pattern of a gene in terms of the sites have the potential to change the expression pattern of a gene in terms of the amount of product expressed at a certain time or in response to certain amount of product expressed at a certain time or in response to certain environmental cues or in certain tissues. environmental cues or in certain tissues.

It is important to realize that such regulatory mutations will affect the amount of It is important to realize that such regulatory mutations will affect the amount of the protein product of a gene, but they will not alter the structure of the protein.the protein product of a gene, but they will not alter the structure of the protein.


Do mutations arise spontaneously? Since the beginning of theSince the beginning of the past past century it was widely known that when a population century it was widely known that when a population

of bacteria is exposed to a toxic environment, some rare cells may acquire the ability of bacteria is exposed to a toxic environment, some rare cells may acquire the ability to grow much better than most of the other cells in the populationto grow much better than most of the other cells in the population (resistance) (resistance)..

In addition, the resistant phenotypeIn addition, the resistant phenotype w was oftenas often stable and so appeared to be stable and so appeared to be the the consequence of true consequence of true gengenetic mutationsetic mutations..

However, it was not known if these mutants were produced spontaneously or if they However, it was not known if these mutants were produced spontaneously or if they were induced by the presence of the were induced by the presence of the toxic agenttoxic agent.. For many years, most For many years, most microbiologists believed that mutations in bacteria were induced by exposure to a microbiologists believed that mutations in bacteria were induced by exposure to a particular environment. (particular environment. (SSalvador Luria alvador Luria once once said that "bacteriology is the last said that "bacteriology is the last stronghold of Lamarckism".) stronghold of Lamarckism".)

The first rigorous evidence that mutations in bacteria followed the Darwinian The first rigorous evidence that mutations in bacteria followed the Darwinian principleprinciple of random variation of random variation and selectionand selection came from a study by Luria and came from a study by Luria and DelbruckDelbruck (1943) (1943)..

They studied mutations that made E. coli resistant to phage T1. Phage T1 interacts with They studied mutations that made E. coli resistant to phage T1. Phage T1 interacts with specific receptors on the surface of E. coli, enters the cell, and subsequently kills the cell. specific receptors on the surface of E. coli, enters the cell, and subsequently kills the cell. Thus, when E. coli is spread on a plate with 10Thus, when E. coli is spread on a plate with 101010 phage T1, most of the cells are killed. phage T1, most of the cells are killed. However, rare T1 resistant (TonHowever, rare T1 resistant (TonRR) colonies can arise due to mutations in E. coli that alter ) colonies can arise due to mutations in E. coli that alter the T1 receptor in the cell wall.the T1 receptor in the cell wall.


Spontaneous vs induced mutations Luria noted that the two theories of mutation Luria noted that the two theories of mutation (spontaneous vs (spontaneous vs

induced) induced) made different statistical predictions.made different statistical predictions. If the TonIf the TonRR mutations were mutations were inducedinduced by exposure to phage T1, then by exposure to phage T1, then

every population of cells would be expected to have an equal every population of cells would be expected to have an equal probability of developing resistance and hence a nearly equal number probability of developing resistance and hence a nearly equal number of Tonof TonRR colonies would be produced from different cultures. colonies would be produced from different cultures. For example, if there was a 10For example, if there was a 10-8-8 probability that exposure to phage T1 would probability that exposure to phage T1 would

induce a Toninduce a TonRR mutant, then approximately 10 colonies would arise on each plate mutant, then approximately 10 colonies would arise on each plate spread with 10spread with 1099 bacteria. bacteria.

In contrast, if TonIn contrast, if TonRR mutations were due to random, spontaneous mutations were due to random, spontaneous mutations that occured sometime during the growth of the culture mutations that occured sometime during the growth of the culture priorprior to exposure to phage T1, then the number of Ton to exposure to phage T1, then the number of TonRR colonies colonies would vary widely between each different culture.would vary widely between each different culture. For example, although there is an equal probability (say 10For example, although there is an equal probability (say 10 -8-8) that a Ton) that a TonRR

mutant would arise per cell division, the number of resistant bacteria in each mutant would arise per cell division, the number of resistant bacteria in each culture would depend upon whether the mutation occurred during one of the culture would depend upon whether the mutation occurred during one of the first cell divisions or one of the last cell divisions.first cell divisions or one of the last cell divisions.


Different predictions The figure shows a cartoon of theThe figure shows a cartoon of the two two

alternative predictions. Tonalternative predictions. TonSS cells are cells are indicated in white and Tonindicated in white and TonRR cells are cells are indicated in black. The shaded area indicated in black. The shaded area indicates when the cells were exposed to indicates when the cells were exposed to phage T1.phage T1.

In either of these two cases, if multiple In either of these two cases, if multiple samples from a single culture of bacteria samples from a single culture of bacteria were plated on phage T1, each of the were plated on phage T1, each of the resulting plates should yield resulting plates should yield approximately the same number of approximately the same number of colonies. However, the two possibilities colonies. However, the two possibilities can be distinguished mathematically by can be distinguished mathematically by comparing the mean and variance of the comparing the mean and variance of the number of the number of mutants in each number of the number of mutants in each culturculture.e.


The “fluctuation test” Luria and Delbruck designed their "fluctuation test" as follows.Luria and Delbruck designed their "fluctuation test" as follows. They inoculated 20 small culturesThey inoculated 20 small cultures (0.2 ml) (0.2 ml), each with a few cells, and incubated , each with a few cells, and incubated

them until there were 10them until there were 1088 cells per m cells per mll.. At the same time, a much larger culture also At the same time, a much larger culture also was inoculated and incubated until there were 10was inoculated and incubated until there were 1088 cells per milliliter. cells per milliliter. The 20 The 20 individual cultures and individual cultures and 110 aliquots of the same size from the large culture were 0 aliquots of the same size from the large culture were plated in the presence of phage.plated in the presence of phage.

If resistance were due to random mutation during the incubation period, each If resistance were due to random mutation during the incubation period, each culture tube would produce a different number of resistant cells; the number would culture tube would produce a different number of resistant cells; the number would vary depending on how early in the cascade of growing cells the mutation occurred. vary depending on how early in the cascade of growing cells the mutation occurred. These resistant cells would each produce a separate colony when plated with the T1 These resistant cells would each produce a separate colony when plated with the T1 phage.phage.

If resistance were due to a physiological adaptation occurring after exposure of the If resistance were due to a physiological adaptation occurring after exposure of the cells on the T1 phage, all cultures would be expected to generate resistant cells at cells on the T1 phage, all cultures would be expected to generate resistant cells at roughly the same rate and little variation from culture to culture would be expected.roughly the same rate and little variation from culture to culture would be expected.


Results of thefluctuation test

Variation from plate to plate was indeed Variation from plate to plate was indeed observed in the individual 0.2-ml cultures observed in the individual 0.2-ml cultures but not in the samples from the bulk but not in the samples from the bulk culture (which represent a kind of control culture (which represent a kind of control experiment). This situation cannot be experiment). This situation cannot be explained by physiological change, explained by physiological change, because all the samples spread had the because all the samples spread had the same approximate number of cells. The same approximate number of cells. The simplest explanation is simplest explanation is random random mutationmutation, occurring early in the , occurring early in the incubation of the 0.2-ml cultures incubation of the 0.2-ml cultures (producing a large number of resistant (producing a large number of resistant cells and, therefore, a large number of cells and, therefore, a large number of colonies) or late (producing few resistant colonies) or late (producing few resistant cells and colonies) or not at all (producing cells and colonies) or not at all (producing no resistant cells).no resistant cells).

Genetic mutations

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