The Nature of Mutation
E3: Lecture 3
The Return
of Black Box
BLACK
BOX 1
BLACK
BOX 2
BLACK
BOX 3
BLACK
BOX 4
Deniz-Somer-Clementine Hypothesis
O1=
Closest prime greater than or equal to I, if I is odd
Closest prime greater than I, otherwise
O2=
0, if I is odd
1, otherwise
Neem’s Hypothesis
Y, if I=O1
N, otherwise
O3=
Should we input 3 or 7?
O1=
I+1, if I<5
I, otherwise
O2=
I-1, if I<5
0, otherwise
O3=
N, if I<5
Y, otherwise
Deniz-Toni-Somer Hypothesis
BLACK
BOX 5
BLACK
BOX 7
BLACK
BOX 6
BLACK
BOX 8
DATA
I
O1
O2
O3
2
3
1
NO
1
2
0
NO
5
5
0
YES
O1=
Random in {I-1, I, I+1}
O2=
O1 modulo I
O3=
Y, if I>4
N, otherwise
Natural Selection
•
Richard Lewontin (1970) laid out Darwin’s postulates:
1. There is variation between individuals
2. There is heritability across generations
3. There is differential survival/reproduction
Richard Lewontin
•
Thus conceived, natural selection destroys variation by weeding out unfit individuals.
•
Regarding the first condition, there must be a process that generates variation or
else selection grinds to a halt.
•
This process is mutational change and is conceptually separable from selection.
Natural Selection and Mutation
• In The Origin of Species, Darwin noted the following:
“Some [writers] have even imagined that natural selection
induces variability, whereas it implies only the preservation
of such variations as arise and are beneficial to the being
under its conditions of life.”
• Darwin conceptually separated the process of variation
generation from natural selection.
• In the Vignieri et al. study on dorsal coat coloration in
mice, in order for selection to favor different heritable
colors under different substrates, there must have been
mutational changes that affected coat color.
mainland mouse colonizes beach
several generations
The “sieve” of natural selection
light mutant
arises
selection
favors light
mutant
M
The “generator” of mutation
The Nature of Mutation
Lecture Outline
• Introduction to genetic mutation
• The Luria-Delbrück experiment
• A mutation controversy
• Selection for mutation rate
• Summary
The Nature of Mutation
Lecture Outline
• Introduction to genetic mutation
• The Luria-Delbrück experiment
• A mutation controversy
• Selection for mutation rate
• Summary
The Central Dogma
5’
3’
•
•
The central dogma suggests that information
flows from nucleic acid sequences (DNA to
RNA) to protein, but not in the reverse direction.
Crick (1970) stated:
“Although the details of the classification proposed
here are plausible, our knowledge of molecular
biology, even in one cell, let alone for all the
organisms in nature, is still far too incomplete to allow
us to assert dogmatically that it is correct.”
G G A
C T C
G
T T
A A
G A G
C C T
C
DNA
transcription
A A
G A G
C C U
C
mRNA
translation
Mutations are changes to the genetic material
(DNA or RNA) due to:
-
Errors during replication
Exposure to radiation
Exposure to mutagens
protein
An example of a mutation
3’
If mutations remain unrepaired, then the
change can be inherited.
5’
parental strand
G G A
•
Glutamic
Acid
•
Proline
- Prions (protein mediated change of proteins)
Glutamine
- Reverse transcription (RNA to DNA)
C T C
A A G
C C T
new strand
G
T T
Types of Mutation
•
Classifying mutation by effect on structure
5’
3’
- Point mutation
• Synonomous (codes for the same aa)
• Non-synonomous
- Insertion (of bases or longer stretches)
- Deletion (of bases or longer stretches)
-
•
G G G
C C C
Classifying mutation by effect on function
-
G
T T
A A
G A G
C C T
C
- Missense (codes for new aa)
- Nonsense (codes for stop)
•
C T C
G G A
C T C
G A G
G
C
T T
A A
CCC still
gives Proline
Loss-of-function mutation (due to missense,
nonsense, or frameshift mutations)
Gain-of-function mutation
Lethal mutation
G
C
T
A
C T C
G
T T
A T G A G
A A
C
Other classifications (e.g., coding vs. regulatory)
wild-type
sickle-cell
CAU now
gives Histidine
wildtype
whiteeye
G G A A
insertion
point mutation
nonmotile
wildtype
deletion
C C T T
T C
A G
G
C
UAG is
stop codon
T T
A A
Fitness Effects of Mutation
Most mutations are either
neutral or deleterious.
•
This can be shown by
performing a mutation
accumulation experiment:
-
-
As an analogy, think of
two versions of the game
“telephone”
I know class
481 is fun !
Aye, no gas
for 81 infants!
I know glass
for 81 is fun!
“not-for-parties” version
standard version
•
Experimental treatment: A
random offspring individual is
picked to establish the next
generation
Control treatment: Several
individuals continually
establish the next generation.
treatment
control
I know glass
for 81 is fun!
I know classes
for 81 infants!
Experimental Results
control
I know class
481 is fun !
I know class
481 is fun!
*$#%
&%#@%
fitness
•
treatment
generation
The Nature of Mutation
Lecture Outline
• Introduction to genetic mutation
• The Luria-Delbrück experiment
• A mutation controversy
• Selection for mutation rate
• Summary
The Mutational Trigger
•
Zoologists and botanists are often able to see mutants arise
that do not bring fitness benefits to their bearers.
•
Early microbiologists were less fortunate: mutants of the
microorganisms they studied could often be revealed only
under selective conditions in which the mutation allowed
survival.
•
Consider antibiotic resistance (something we’re interested in
from a medical point of view):
mutant fruit fly
- Let’s say you have a growing culture of bacteria in a test tube.
-
•
You then add an antibiotic to this tube.
Your culture quickly clears (due to the death of a large number
of cells)
However, after further incubation, the culture recovers (due to
the growth of drug-resistant cells).
But what was the trigger of these drug-resistant mutants?
-
Did the presence of the antibiotic stimulate the resistance
mutations?
Did the mutations occur randomly (e.g., even before the
application of the antibiotic)?
?
mutant pea plant
wild type
wild type
Two Hypotheses
•
Imagine a single cell placed in a test tube with
growth media. This cell doubles and then its
offspring double and so on… This can be
represented as an inverted tree.
•
At a certain point in the population growth, the
selective pressure (an antibiotic) is applied
(represented by the red dashed line).
•
Extreme versions of our hypotheses:
-
Directed Mutation: Mutations occur only after
application of the selective pressure
Random Mutation: Mutations occur
spontaneously at any doubling event.
•
The problem is that resistant cells and sensitive
cells don’t look different– we can only tell them
apart by imparting the selective pressure (e.g.,
after a “red line”)
•
The stakes are big– are mutation and selection
linked or independent?
•
This question was addressed in 1943 by
Salvador Luria and Max Delbruck.
Salvador Luria
Max Delbrück
The Luria-Delbruck Experiment
•
The key insight that Luria and Delbruck had, was to look at several replicates to see
how mutant numbers were distributed across replicates.
•
The Directed Mutation hypothesis predicts a fairly even distribution of mutants
across replicates.
•
The Random Mutation hypothesis predicts an uneven distribution of mutants across
replicates, with a few replicates having unusually high numbers of mutants (these are
called jackpots).
A Signal to Distinguish Hypotheses
•
After growing up several replicate populations and then placing each population
under selective conditions, Luria and Delbruck could calculate the average number of
mutants and the variance in the number of mutants.
1 n
m  m2  m3  ...  mn
average  m   mr  1
n r 1
n
1 n
(m1 - m )2  (m2 - m )2  ...  (mn - m )2
2
variance    (mr - m ) 
n r 1
n
2
m
m
 m2 
2  3 1 4  2  2
 2.33
6
(2 - 2.33) 2  (3 - 2.33) 2  (1 - 2.33) 2  (4 - 2.33) 2  (2 - 2.33) 2  (2 - 2.33) 2
 1.07
6
m
 m2 
2 1 2  2  8  2
 2.83
6
(2 - 2.83) 2  (1 - 2.83) 2  (2 - 2.83) 2  (2 - 2.83) 2  (8 - 2.83) 2  (2 - 2.83) 2
 6.57
6
•
Relative to the mean, jackpots tend to inflate the variance across replicates in the
number of mutants.
•
By looking at the ratio of the variance over the average, we can distinguish these
hypotheses. This ratio is expected to be one for the directed mutation hypothesis
and is expected to be greater than one for the spontaneous mutation hypothesis.
An Exercise
•
We’re going to simulate the Luria-Delbruck
experiment in class. You will receive a piece of
paper with two inverted trees on it and a die.
Random Mutation Simulation
2
•
In the “Random Mutation Simulation” start at the top
of the tree and make your way down always doing
the following:
- Record the roll of your die in the box
- If the roll was a 1, then fill in all boxes and circles
to the bottom right of the box.
- If the roll was not a 1, then do nothing.
- As you move down the tree, skip all filled in
boxes.
•
In the “Directed Mutation Simulation” simply roll your
die 8 times and record the numbers in the 8 boxes. If
the roll was a 1, fill in the circle to the bottom right of
the relevant box.
•
Circles represent bacterial cells, boxes represent
replication events, and filled circles represent
mutants.
6
1
4
3
1
5
1
3
4
6
Directed Mutation Simulation
3
4
2
2
1
4
6
1
Their Data
•
In all of their replicated experiments, Luria & Delbruck found a significantly higher
variance than average– consistent with spontaneous mutation.
An Independent Check
•
In 1952, Joshua and Esther Lederberg provided
independent support for the Random Mutation hypothesis.
•
They grew up bacteria on an agar-filled Petri dish until it
formed a confluent lawn.
•
Then they replica plated this population onto several new
agar plates with selective media (e.g., the presence of an
antibiotic).
replica plating tool
Random
Mutation
some same locations
of mutant colonies
velvet
Esther
Lederberg
different locations
of mutant colonies
Directed
Mutation
Joshua
Lederberg
The Nature of Mutation
Lecture Outline
• Introduction to genetic mutation
• The Luria-Delbrück experiment
• A mutation controversy
• Selection for mutation rate
• Summary
Directed Mutation Strikes Back
•
John Cairns, Julie Overbaugh and others suggested that for
some executions of the Luria-Delbruck protocol (considering
other mutations), the variance was too low to be accounted
for solely by spontaneous mutation.
•
Indeed, they argued that the distribution of mutants in
replicated populations looked like a combination of the
distributions predicted from the spontaneous mutation and
directed mutation processes.
•
Further, they claimed to provide independent evidence that
directed mutation played at least some role:
-
-
•
They saw E. coli unable to use the sugar lactose (Lac-) mutate
into two types of Lac+ bacteria (with different colony shapes).
The first type (fast colony former) had a high variance for its
mean across replicates.
The second type (slow colony former) had a variance similar to
its mean across replicates.
The authors claimed the second mutant was the result of
directed mutation (mutation to Lac+ in the presence of lactose)
The mechanism? “[Perhaps] the cell could produce a highly
variable set of mRNA molecules and then reverse-transcribe
the one that made the best protein.”
John
Cairns
Julie
Overbaugh
replicate 1
replicate 2
replicate 3
replicate 4
replicate 5
replicate 6
DNA
DNA
mRNA
mRNA
protein
protein
central dogma
Cairnsian
revision?
Data Consistent with Directed Mutation
•
An E. coli strain was studied by Spencer Benson that lacked
the ability to take up large maltodextrins (Dex-).
•
Two point mutations can restore this ability in E. coli (either in
the OmpC locus or OmpF locus).
•
Benson (1988) found Dex+ mutants via OmpF are generated
at a much higher frequency than Dex+ mutants via OmpC.
•
Benson suggested that this bias might be due to directed
mutation that causes the OmpF change (under appropriate
selective conditions), but not the OmpC change.
Spencer Benson
Take 5 minutes to talk to your group about the following:
1. Is Benson’s suggestion the only plausible process to explain the pattern?
2. If not, what other hypotheses would you suggest?
3. How might you experimentally test your hypotheses?
The Return of Random Mutation
•
The Luria-Delbruck random mutation hypothesis makes an important
assumption: The mutant (whenever generated) duplicates at the same
rate as its ancestor.
•
Benson et al. (1991) checked the growth rates of OmpF and OmpC
Dex+ mutants and found OmpF mutants to have a higher growth rate.
•
Could slow growth explain Cairnes’ “two-variance” observation?
Random Mutation where Mutant Grows Slowly
Directed Mutation
•
If the mutant grows slower, then the number of mutants decreases and the variance drops (looks
more similar to the pattern predicted under directed mutation).
•
Interestingly, the colonies producing an even distribution of mutants in the Cairns experiment
were slow growing colonies…
Some Lessons from the Mutation Controversy
Experimentally:
•
It is extremely important to remember (and test for) any underlying assumptions of
your hypotheses.
•
Running appropriate controls is important:
-
The directed mutation hypothesis suggests that mutation occurs specifically at loci where
changes will improve the fitness of the organism under current selective conditions.
In order to test whether the mutation under consideration is directed, it becomes important
to consider mutations at other loci that are not under selection (think how you would do
that…)
Biologically:
•
Given no molecular mechanism for “trying out” mutations within the lifetime of an
organism and some experimental problems from the data supporting the directed
mutation hypothesis, the random mutation hypothesis is the current working model.
•
Are there many different mutations that could produce a phenotype that would
prosper under selective conditions? Do these different classes of mutants differ in
their growth rates (fitness) under non-selective conditions? Under selective
conditions?
•
Do stressful conditions raise the mutation rate of organisms? Is this an adaptive
response or simply a by-product of poor organismal condition?
The Nature of Mutation
Lecture Outline
• Introduction to genetic mutation
• The Luria-Delbrück experiment
• A mutation controversy
• Selection for mutation rate
• Summary
Variation in Mutation Rate
•
Mutation rate varies between and within species. For
instance, mutation rate can be sensitive to environmental
conditions:
-
Some organisms experience a higher mutation rate under
stressful/starvation conditions
Chemical mutagens can increase the rate of mutation
Genetic changes can affect mutation rate
•
Surveys of natural populations of bacteria suggest that
more than 1% of isolates are mutator strains. These
strains are often deficient in some DNA repair component.
•
Given heritable variation in mutation rate, it is reasonable
to consider the possibility that mutation rate could evolve
through natural selection…
mitomycin C
caffeine
E. coli adenine glycosylase
Take 5 minutes to talk to your group about the following:
1. Under what circumstances would you expect a higher mutation rate to be adaptive?
2. What are the costs to being a mutator?
3. How might you experimentally test your hypotheses?
Experimental Test for Adaptive Mutation
•
Arjan de Visser, Richard Lenski and colleagues
(1999) explored the success of mutator strains under
novel selective conditions.
•
Controlling for genetic background, they constructed
three strains, a wild-type, a mutY mutator, and a mutS
mutator.
Arjan de
Visser
Richard
Lenski
•
They grew these three strains up separately under
conditions to which the strains were not adapted.
wild-type
•
They found that both mutator strains evolved a higher
fitness after 1000 generations than the wild-type.
mutS
•
The same result was not found under conditions to
which all strains were adapted.
This experiment gives some evidence that there may
be short-term advantages to higher mutation under
novel selective conditions.
1.3
fitness
•
mutY
1.2
1.1
1.0
generations
1000
800
600
400
200
Potential trade-offs include long-term deleterious
effects of a higher mutational load.
0
•
The Results of Your Simulation…
The Nature of Mutation
Lecture Outline
• Introduction to genetic mutation
• The Luria-Delbrück experiment
• A mutation controversy
• Selection for mutation rate
• Summary
Summary
• The Neo-Darwinian perspective separates the process of mutation
from the process of selection—that is, mutations occur without respect
to their selective consequences.
• Most mutations have either neutral or slightly deleterious effects, but
rarely mutants are selectively favored.
• Luria and Delbruck designed a classic experiment using the variance
in mutants between replicate populations to distinguish between the
random mutation hypothesis (neo-Darwinian perspective) and the
directed mutation hypothesis (Cairnsian perspective).
• Luria and Delbruck (& others) found support for spontaneous mutation.
• The evidence presented by some scientists in support of directed
mutation illustrates the importance of proper controls and checking the
assumptions of your hypotheses.
• However, the directed mutation controversy has led to greater
exploration of the (potentially adaptive) changes in mutation rate.