Gene mutation
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The ultimate source of genetic variation is gene mutation. How, in
fact, do genetic variants arise?
A mutation is a heritable change of the genetic material.
Geneticists recognize two different levels at which mutation takes
place. In gene mutation, an allele of a gene changes, becoming a
different allele. 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 mutation.
In chromosome mutations, segments of chromosomes, whole
chromosomes, or even entire sets of chromosomes change.
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Gene mutation is not necessarily a part of such a process; the effects of
chromosome mutation are due more to the new arrangements of chromosomes
and of the genes that they contain. Nevertheless, some chromosome mutations,
in particular those proceeding from chromosome breaks, are accompanied by
gene mutations caused by the disruption at the breakpoint.
Genetica per Scienze Naturali
a.a. 03-04 prof S. Presciuttini
Basic terminology
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To consider change, we must have a fixed reference point, or standard. In genetics,
the wild type provides the standard (the wild-type allele may be either the form
found in nature or the form found in a standard laboratory stock).
Any change away from the wild-type allele is called forward mutation; any change
back to the wild-type allele is called reverse mutation.
The non-wild-type allele of a gene is often called a mutation. To use the same word
for the process and the product may seem confusing, but in practice little confusion
arises.
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Thus, we can speak of a dominant mutation or a recessive mutation. Consider,
however, how arbitrary these definitions are; the wild type of today may have been a
mutation in the evolutionary past, and vice versa.
Another useful term is mutant. A mutant organism or cell is one whose changed
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
that shows that it bears a mutation.
Two other useful terms are mutation event, which is the actual occurrence of a
mutation, and mutation frequency, the proportion of mutations in a population of
cells or individual organisms.
Genetica per Scienze Naturali
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Somatic mutations
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A somatic mutation occurs in a single cell of developing somatic tissue in an
individual organism; that cell may become the progenitor of a population of
identical mutant cells, all of which have descended from the cell that mutated; this
phenomenon is particularly important in cancer.
A population of identical cells derived asexually from one progenitor cell is called a
clone. 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
phenotypically mutant cells called a mutant sector. The earlier in development the
mutation event, the larger the mutant sector will be.
Somatic mutation in the red Delicious apple. The
mutant allele determining the golden color arose
in a flower's ovary wall, which eventually
developed into the fleshy part of the apple. The
seeds are not mutant and will give rise to redappled trees. In fact, the golden Delicious apple
originally arose as a mutant branch on a red
Delicious tree.
Genetica per Scienze Naturali
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Germinal mutations
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Somatic mutations are never passed on to progeny.
On the contrary, mutations that occurs in the germ line, special tissue
that is set aside in the course of development to form sex cells, will be
passed on to the next generation. These are called germinal
mutations.
An individual of perfectly normal phenotype and of normal ancestry
can harbor undetected mutant sex cells. These mutations can be
detected only if they are included in a zygote.
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For example, the 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.
Genetica per Scienze Naturali
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Hemophilia
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.
Genetica per Scienze Naturali
a.a. 03-04 prof S. Presciuttini
Point mutations: base substitutions
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Point mutations typically refer to alterations of single base pairs of
DNA or of a small number of adjacent base pairs.
At the DNA level, there are two main types of point mutational
changes: base substitutions and base additions or deletions.
Base substitutions are those mutations in which one base pair is
replaced by another. Base substitutions again can be divided into two
subtypes: transitions and transversions.
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To describe these subtypes, we consider how a mutation alters the sequence on
one DNA strand. A transition is the replacement of a base by the other base of
the same chemical category (purine replaced by purine: either A to G or G to A;
pyrimidine replaced by pyrimidine: either C to T or T to C). A transversion is the
opposite the replacement of a base of one chemical category by a base of the
other (pyrimidine replaced by purine: C to A, C to G, T to A, T to G; purine
replaced by pyrimidine: A to C, A to T, G to C, G to T). In describing the same
changes at the double-stranded level of DNA, we must state both members of a
base pair: an example of a transition would be G·C  A·T; that of a
transversion would be G·C  T·A.
Genetica per Scienze Naturali
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Point mutations: additions/deletions
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Addition or deletion mutations are actually of nucleotide pairs;
nevertheless, the convention is to call them base-pair additions or
deletions. The simplest of these mutations are single-base-pair
additions or single-base-pair deletions. There are examples in which
mutations arise through simultaneous addition or deletion of multiple
base pairs at once.
What are the functional consequences of these different types of point
mutations?
Genetica per Scienze Naturali
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Functional consequences of point mutations (1)
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We first consider what happens when a mutation arises in a polypeptide coding part
of a gene. Depending on the consequences, single-base substitutions are classified
into:
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Silent mutations: the mutation changes one codon for an amino acid into another codon
for that same amino acid.
Missense mutations: the codon for one amino acid is replaced by a codon for another
amino acid.
Nonsense mutations: the codon for one amino acid is replaced by a translation
termination (stop) codon.
The severity of the effect of missense and nonsense mutations on the polypeptide
may differ. If a missense mutation causes the substitution of a chemically similar
amino acid (synonymous substitution), then it is likely that the alteration will have
a less-severe effect on the protein's structure and function. Alternatively, chemically
different amino acid substitutions, called nonsynonymous substitutions, are more
likely to produce severe changes in protein structure and function.
Nonsense mutations will lead to the premature termination of translation. Thus, they
have a considerable effect on protein function. Unless they occur very close to the 3’
end of the open reading frame, so that only a partly functional truncated polypeptide
is produced, nonsense mutations will produce inactive protein products.
Genetica per Scienze Naturali
a.a. 03-04 prof S. Presciuttini
Functional consequences of point mutations (2)
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On the other hand, single-base additions or deletions have
consequences on polypeptide sequence that extend far beyond the site
of the mutation itself, like nonsense mutations.
Because the sequence of mRNA is "read" by the translational
apparatus in groups of three base pairs (codons), the addition or
deletion of a single base pair of DNA will change the reading frame
starting from the location of the addition or deletion and extending
through to the carboxy terminal of the protein. Hence, these lesions
are called frameshift mutations.
These mutations cause the entire amino acid sequence translationally
downstream of the mutant site to bear no relation to the original amino
acid sequence.
Thus, frameshift mutations typically exhibit complete loss of normal
protein structure and function.
Genetica per Scienze Naturali
a.a. 03-04 prof S. Presciuttini
Examples of point mutations
Genetica per Scienze Naturali
a.a. 03-04 prof S. Presciuttini
Mutations in non-coding regions
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Now let's turn to those mutations that occur in regulatory and other non-coding
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
transcription-regulating proteins must bind. At the RNA level, there are also
important functional sequences such as the ribosome-binding sites of bacterial
mRNAs and the self-ligating sites for intron excision in eukaryote mRNAs.
The consequences of mutations in parts of a gene other that the polypeptide-coding
segments are difficult to predict. In general, the functional consequences of any
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
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
environmental cues or in certain tissues.
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.
Genetica per Scienze Naturali
a.a. 03-04 prof S. Presciuttini
Do mutations arise spontaneously?
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Since the beginning of the past century it was widely known that when a population
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 population (resistance).
In addition, the resistant phenotype was often stable and so appeared to be the
consequence of true genetic mutations.
However, it was not known if these mutants were produced spontaneously or if they
were induced by the presence of the toxic agent. For many years, most
microbiologists believed that mutations in bacteria were induced by exposure to a
particular environment. (Salvador Luria once said that "bacteriology is the last
stronghold of Lamarckism".)
The first rigorous evidence that mutations in bacteria followed the Darwinian
principle of random variation and selection came from a study by Luria and
Delbruck (1943).
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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.
Thus, when E. coli is spread on a plate with 1010 phage T1, most of the cells are killed.
However, rare T1 resistant (TonR) colonies can arise due to mutations in E. coli that alter
the T1 receptor in the cell wall.
Genetica per Scienze Naturali
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Spontaneous vs induced mutations
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Luria noted that the two theories of mutation (spontaneous vs
induced) made different statistical predictions.
If the TonR mutations were induced by exposure to phage T1, then
every population of cells would be expected to have an equal
probability of developing resistance and hence a nearly equal number
of TonR colonies would be produced from different cultures.
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For example, if there was a 10-8 probability that exposure to phage T1 would
induce a TonR mutant, then approximately 10 colonies would arise on each plate
spread with 109 bacteria.
In contrast, if TonR mutations were due to random, spontaneous
mutations that occured sometime during the growth of the culture
prior to exposure to phage T1, then the number of TonR colonies
would vary widely between each different culture.
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For example, although there is an equal probability (say 10-8) that a TonR
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 first
cell divisions or one of the last cell divisions.
Genetica per Scienze Naturali
a.a. 03-04 prof S. Presciuttini
Different predictions
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The figure shows a cartoon of the two
alternative predictions. TonS cells are
indicated in white and TonR cells are
indicated in black. The shaded area
indicates when the cells were exposed to
phage T1.
In either of these two cases, if multiple
samples from a single culture of bacteria
were plated on phage T1, each of the
resulting plates should yield approximately
the same number of colonies. However,
the two possibilities can be distinguished
mathematically by comparing the mean
and variance of the number of the number
of mutants in each culture.
Genetica per Scienze Naturali
a.a. 03-04 prof S. Presciuttini
The “fluctuation test”
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Luria and Delbruck designed their "fluctuation test" as follows.
They inoculated 20 small cultures (0.2 ml), each with a few cells, and incubated
them until there were 108 cells per ml. 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 10 aliquots of the same size from the large culture were
plated in the presence of phage.
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 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
phage.
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
roughly the same rate and little variation from culture to culture would be expected.
Genetica per Scienze Naturali
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Results of the
fluctuation test
Variation from plate to plate was indeed
observed in the individual 0.2-ml cultures
but not in the samples from the bulk
culture (which represent a kind of control
experiment). This situation cannot be
explained by physiological change,
because all the samples spread had the
same approximate number of cells. The
simplest explanation is random
mutation, occurring early in the
incubation of the 0.2-ml cultures
(producing a large number of resistant
cells and, therefore, a large number of
colonies) or late (producing few resistant
cells and colonies) or not at all (producing
no resistant cells).
Genetica per Scienze Naturali
a.a. 03-04 prof S. Presciuttini
Measuring the mutation rate
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Luria and Delbrück's fluctuation test provides a way of measuring the mutation rate
towards acquisition of resistance to phage T1.
The 20 cultures that were tested for phage T1 resistance can be divided among those
in which no mutation had occurred (11 of the 20 = 0.55) and those in which a
mutation had occurred once or more (we cannot distinguish the cultures in which
one mutation only had occurred from the others). This situation is well described by
the Poisson distribution, in which the probability of zero occurrences of an event
(P0) is given by P0 = e-m, where m is the probability that an event will occur.
We can estimate m from data by letting 0.55 = e-m and solving for m (m = -ln 0.55);
this gives m = 0.6 mutations/tube.
We can now estimate the number of cell duplications (n) that occurred in each tube.
If n is very large, and if the original number of cells was very small, then a
sufficiently accurate estimate of the number of cell divisions is given by n itself.
Thus, since n = 0.2 × 108, we arrive at an estimate of the mutation rate (m) of m =
0.6/ 0.2 × 108 = 3 × 10-8.
Note that m is expressed as number of mutations per gene per DNA replication.
Genetica per Scienze Naturali
a.a. 03-04 prof S. Presciuttini