Evolution in a glass
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Experimental work with bacteria, eukaryotic micro-organisms and
very small animals can tell us much about the occurrence and
properties of mutations, including beneficial mutations. Over the last
fifty years or so beneficial mutations have been observed to occur in a
number of studies
Most of these experiments were done in a continuous culture system
called a chemostat.
A chemostat consists of a bottle in which the organisms grow. Growth
medium (i.e. food) is continuously pumped into the bottle and waste
products, residual medium and organisms flow out. The contents of
the bottle are well mixed so that each critter in the chemostat has an
equal chance of getting at each bit of food. Factors that affect the
growth of the organisms such as temperature are controlled,
sometimes quite rigourously. Several variations of chemostats have
been developed.
Genetica per Scienze Naturali
a.a. 05-06 prof S. Presciuttini
The chemostat
Continuous culture, in a device called a chemostat, can be used to maintain a bacterial population
at a constant density, a situation that is, in many ways, more similar to bacterial growth in natural
environments. In a chemostat, the growth chamber is connected to a reservoir of sterile medium.
Once growth is initiated, fresh medium is continuously supplied from the reservoir. The volume
of fluid in the growth chamber is maintained at a constant level by some sort of overflow drain.
Fresh medium is allowed to enter into the growth chamber at a rate that limits the growth of the
bacteria. The bacteria grow (cells are formed) at the same rate that bacterial cells (and spent
medium) are removed by the overflow. The rate of addition of the fresh medium determines the
rate of growth because the fresh medium always contains a limiting amount of an essential
nutrient.
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Schematic diagram of a chemostat, a device for the
continuous culture of bacteria. The chemostat relieves the
environmental conditions that restrict growth by
continuously supplying nutrients to cells and removing
waste substances and spent cells from the culture medium
Genetica per Scienze Naturali
a.a. 05-06 prof S. Presciuttini
Fluctuations of mutant strains
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In an early study, resistance to a phage was used as a marker to follow the
appearance of some mutations in a chemostat culture.
Novick and Szilard grew E. coli in a chemostat at a steady-state density of about 3 ×
108 cells per ml. Periodically they assayed cells sampled from the chemostat for
resistance to infection by bacteriophage T5 and calculated the density of T5 resistant
cells in the culture.
At no time was phage T5 present in the chemostat nor had the cells in the chemostat
been exposed to phage T5. They found that there was always a fraction of cells in
the culture that was resistant to T5.
The density of resistant cells fluctuated betweeen 102 and 103 per ml.
The increases and decreases reflect the occurrence of mutations within strains in the
chemostat. The initial increase in the frequency of resistant cells occurs because a
mutation occurs within a T5 resistant strain that makes it (and its descendents) the
fastest growing cells in the culture. As long as this strain remains the fastest growing
one its representation in the population will increase. Eventually different favorable
mutation occurs in a cell that is sensitive to T5 that makes it (and its descendents)
the fastest growing cells in the culture. This causes the frequency of T5 resistance to
decline. Later a different mutation occurs in a T5 resistant strain that makes it the
fastest growing strain. Its frequency increases, and so on.
Genetica per Scienze Naturali
a.a. 05-06 prof S. Presciuttini
Neutral mutations
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It is important to note that in this environment sensitivity and resistance to infection
by T5 is a neutral trait here. Because there is no T5 in the environment, resistance
does not provide an advantage.
But it doesn't seem to provide much disadvantage either. If it provided a
disadvantage, the resistant cells would washout of the chemostat. In this
environment, it is selectively neutral.
Mutations in other genes cause some cells to have a higher growth rate. It is just a
matter of whether these mutations occur first in resistant or sensitive cells that
determines whether the frequency of T5 resistant cells increases or decreases.
It's a hitchhiking effect - the T5 resistance gene just goes along for the ride with the
genes causing the fluctuations.
Bacteria carrying neutral mutations constitute a fluctuating proportion of growing
cultures. The fluctuations are attributed to periodic selection of fitter clones, with
each successive sweep replacing less fit members of the population, including those
with neutral mutations. The frequency of neutral mutations can also change in clonal
populations as a consequence of hitchhiking with favorable mutations.
Genetica per Scienze Naturali
a.a. 05-06 prof S. Presciuttini
An example with yeast
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Frequency of canavanine resistant cells
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Paquin & Adams (1983) studied haploid and diploid populations of yeast to estimate
the relative rate that beneficial mutations would arise in an asexual population of
each type.
Populations were kept in a chemostat (a fairly constant environment) at a population
size of about 5 billion. Initially, the population was started from a single clone (one
genotype).
A neutral marker, canavanine resistance then increased in frequency due to mutation
pressure alone (amino acid mutation rate = 10-7), although the mutations always
remained low in frequency (< 10-5) during the hundreds of generations of the
experiment.
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When a beneficial mutation
occurred, it was most likely to arise
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in a canavanine sensitive cell.
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The beneficial mutation would then
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sweep through the population.
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Canavanine sensitivity would "hitch3
hike" along, driving back down the
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frequency of canavanine resistance.
1
0
0
50
100
150
Generations
200
250
Genetica per Scienze Naturali
a.a. 05-06 prof S. Presciuttini
Interpreting mutant fluctuations
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This chart is an explanation of what happens in the chemostat
Genetica per Scienze Naturali
a.a. 05-06 prof S. Presciuttini
Genetic drift
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Fluctuations of mutant-clone frequencies in the chemostat are
examples of the process known as genetic drift.
If a population is finite in size (as all populations are) and if a given
pair of parents of a diploid species has only a small number of
offspring, then, even in the absence of all selective forces, the
frequency of a gene will not be exactly reproduced in the next
generation, because of sampling error.
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If, in a population of 1000 individuals, the frequency of a is 0.5 in one
generation, then it may by chance be 0.493 or 0.505 in the next generation
because of the chance production of slightly more or slightly fewer progeny of
each genotype. In the second generation, there is another sampling error based
on the new gene frequency, so the frequency of a may go from 0.505 to 0.511 or
back to 0.498. This process of random fluctuation continues generation after
generation, with no force pushing the frequency back to its initial state, because
the population has no "genetic memory" of its state many generations ago. Each
generation is an independent event.
Genetica per Scienze Naturali
a.a. 05-06 prof S. Presciuttini
Extinction of genetic variability by genetic drift
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The final result of this random change in allelic frequency is that the population
eventually drifts to p = 1 or p = 0. After this point, no further change is possible; the
population has become homozygous. A different population, isolated from the first,
also undergoes this random genetic drift, but it may become homozygous for allele
A, whereas the first population has become homozygous for allele a. As time goes
on, isolated populations diverge from each other, each losing heterozygosity. The
variation originally present within populations now appears as variation among
populations.
Computer simulation of genetic
drift. The frequency of an allele
(e.g., A in a system with A and a) is
shown for five replicate populations
over the course of 100 generations,
with a population size (N) of 20. The
effect of drift is inversely
proportional to population size, a
fundamental driving force in many
evolutionary divergences
Genetica per Scienze Naturali
a.a. 05-06 prof S. Presciuttini
Genetic drift over evolutionary time
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The appearance, loss, and eventual incorporation of neutral mutations in the life of a
population. If random genetic drift does not cause the loss of a new mutation, then it
must eventually cause the entire population to become homozygous for the
mutation.
At that point, the mutation has been fixed.
In the figure, 10 mutations have arisen, of which 9 (light red at bottom of graph)
increased slightly in frequency and then died out. Only the fourth mutation
eventually spread into the population.
Therefore, a steady substitution
of one allele for another is
expected to occur due to genetic
drift alone
Genetica per Scienze Naturali
a.a. 05-06 prof S. Presciuttini
The probability of fixation of a neutral allele
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It has been proven by matematical analysis (and it is quite intuitive)
that the probability of fixation u of any neutral allele a is equal to its
frequency in the population:
u = pa
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In a finite diploid population, pa takes discrete values only, starting
from 1/(2N) in diploid species (when one copy only of allele a is
present in the population), and incrementing at steps of 1/(2N).
In other words, the initial frequency of a mutant allele is, by
definition, pa = 1/(2N)
Thus, the probability of ultimate fixation of any new neutral mutation
is equal to the reciprocal of twice the population size.
Genetica per Scienze Naturali
a.a. 05-06 prof S. Presciuttini
The concept of gene substitution
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It is important to distinguish between "Mutation" and "Substitution" with respect to
individuals and populations:
The rate of gene substitution (K) is defined as the number of mutants reaching
fixation per unit time.
If neutral mutations occur at a rate of μ per gene per generation, then the number of
mutants arising in a diploid population of size N is 2N μ mutant alleles per
generation. Since the probability of fixation for each of these mutations is 1/(2N),
we obtain the result that
K = μ.
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Thus theoretically, if the mutation rate μ is constant over time, neutral alleles
accumulate at a fixed rate independently of population size, and their rate of
accumulation can be used as an evolutionary clock to measure divergence times.
This is one of the fundamental tenants of molecular evolution.
This result can be intuitively understood by noting that, in a large population, the
number of mutations arising every generation is high but the fixation probability of
each mutation is low. In comparison, in a small population, the number of mutations
arising every generation is low, but the fixation probability of each mutation is high.
As a consequence, the rate of substitution for neutral mutations is independent of
population size.
Genetica per Scienze Naturali
a.a. 05-06 prof S. Presciuttini