Transcript Cairns et al.

The origin of mutations
Lamarck
Until 1943 there were two
hypotheses concerning the
origin of mutations:
•Beneficial mutations are
somehow induced by the
environment in which they
are useful
•Mutations are ‘directed’
Darwin
•Mutations occur
spontaneously independent
of their effects
•Mutations are random
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The basis of mutational change: How do mutations arise?
• Luria and Delbruck came up with an ingenious
experiment to test whether mutations arose:
i) randomly – mutants pre-existed in a
population before exposure to a selective
environment (e.g., an antibiotic)
Genetics 1943
ii) by direction/adaption – induced in the
population by exposure to a selective
environment
•
Distinguishing between these two possibilities is
difficult – requires knowing if an individual has a
mutation before it is exposed to a selection pressure –
once an individual is exposed to selection it is unclear
whether or not the selection pressure caused the
mutation (directed) or the mutation was already
present in the individual (random)
•E.g., think about how you would determine if a bacteria
was antibiotic resistant without exposing it to the
antibiotic
The basis of mutational change: How do mutations arise?
Each level
represents a
round of cell
division
Luria and Delbruck started
populations of bacteria from a
small number of cells…
… and allowed them to grow up to
reach a large number of cells (~10
billion)
then determined the number of
bacteriophage resistant cells:
bacteriophage
on the plate kill
sensitive cells
resistant cells live and form colonies,
sensitive cells die
but – cells can only be identified as
being resistant (having a mutation)
after exposure to the selective
environment – how does this
experiment solve our problem?
The basis of mutational change: How do mutations arise?
Directed/Adaption hypothesis
If mutations are induced in
response to selection they will
occur only in the final generation
of cells – the ones that are
exposed to phage
If mutations occur randomly they
will occur throughout population
growth – i.e., prior to selection
grow up population
without selection (e.g.
no bacteriophage)
expose the population to
selection and count the
number of mutant individuals
(colonies)
The basis of mutational change: How do mutations arise: the fluctuation test
Directed mutation/
A ‘jackpot’ – a population happened to get a
mutation early in its growth. Because descendants of
this early mutant inherit the mutation a large number
of mutants will be present
Fig. 12.1
•If mutations are directed
in response to selection,
each cell has an
independent probability
of becoming resistant –
predicts a Poisson
distribution of mutants
across populations (low
variance; variance =
mean)
•If mutations are random
cells have different
probabilities of being
resistant (it depends on
their parent) – if
mutations happen early,
a large number of
mutants are present
(high variance; variance
>> mean)
The basis of mutational change: How do mutations arise?
The random
mutation
hypothesis
predicts the
variance
should be
much higher
than the mean
– the directed
mutation
hypothesis
predicts the
variance and
mean should
be similar
A ‘jackpot’ – a
population
happened to get
a mutation early
in its growth.
Because
descendants of
this early mutant
inherit the
mutation, a large
number of
mutants are
present
Luria & Delbruck, Genetics 1943
The basis of mutational change: How do mutations arise?
Do you buy it? Do (all?) mutations occur randomly and irrespective of their usefulness?
Recap of the experiment:
•
Hypothesis: If mutations are directed, then we expect them to be Poisson distributed across replicates; If
mutations arise randomly, then we expect them to be distributed with high variance across replicates
•
Prediction: As for hypothesis but specific to phage resistance tested in this experiment (any reason that
what is true for phage may not be true for mutations generally?)
•
Test: Determine distribution of phage resistance across replicate populations
•
Interpretation: Use a statistical model to determine if observed distribution is Poisson or has higher variance
(more specifically, they falsified the completely directed mutation model, but does this necessarily mean that
the random mutation model is true?)
Luria & Delbruck, Genetics 1943
For the next 45 years it was largely accepted that the probability that a mutation
occurs is not influenced by whether or not it will be useful, then…
Cairns et al. 1988
[Luria-Delbruck (L-D) and
Lederberg and Lederberg]
Summary of
LuriaDelbruck
(L-D)
finding
Even worse…
knowing what
we now know,
the L-D
experiment
couldn’t have
detected a
signal of
directed
mutation
Pointing out that
concluding from LD and co. that ALL
mutations are
spontaneous is an
example of the
logical fallacy
affirming the
consequent
• Can you put these plots into words? How does the distribution of mutations
over independent populations differ under the directed and random mutation
models?
Random mutation
Mean number of
mutation events per
culture
1
probability of having
X or more mutants
Directed mutation
1
Mean number of mutation
events on the plate
number of mutants X
• Just what distribution of mutants should you expect if both directed and
random mutations are present?
Random mutation
2
1
0.5
Mean number of
mutation events per
culture16
8
4
1
2
5
10
20 30
Mean number of mutation
events on the plate
probability of having
X or more mutants
Directed mutation
number of mutants X
• Just what distribution of mutants should you expect if both directed and
random mutations are present?
Random mutation
2
1
0.5
Mean number of
mutation events per
culture16
8
4
Directed and random mutation
0
1
2
1 random
mutation + X
directed
mutations
5
10
20 30
1
2
5
10
20 30
Mean number of mutation
events on the plate
probability of having
X or more mutants
Directed mutation
number of mutants X
What do you take
away from this
plot?
Experimental design
An experiment that aims to identify random and directed mutations should use a
“marker” that is expressed immediately after a mutational change  e.g.,
resumption of the ability to grow on the sugar lactose (lac- to lac+)
Addition of a ‘C’ to lacZ creates a
frameshift mutation that reveals an
ochre stop codon (taa) and prevents
functional LacZ being formed – without
LacZ, a cell can’t grow on lactose
selection
for growth
on lactose
start
codon
lac-
lac+ (independent 1 bp deletion
mutations restoring the proper
reading frame are shown)
Experimental design
add lac- cells and
lactose immediately
count lac+ mutant
colonies arising over
time (only lac+ can
form colonies on
minimal lac medium)
number of lac+
mutants
Observation: (i) distribution of lac+ mutants on plates suggests influence of
random and directed mutations (ii) lac- cells plated on minimal lactose medium
continue to produce lac+ colonies at a constant rate – suggests that mutations
occur during selection on plates (directed?)
~20 colonies on
day 2 and ~8
cells per day
thereafter
time
Mutant colonies rise at an approx. constant rate over time
• Is this consistent with the new mutations being directed (as suggested by
Cairns et al.)?
• Is there any other explanation? If so, what?
• How could you test for the influence of directed mutations (in addition to
random mutations) in this experiment?
Experimental design
Observation: (i) distribution of lac+ mutants on plates suggests influence of
random and directed mutations (ii) lac- cells plated on minimal lactose medium
continue to produce lac+ colonies at a constant rate – suggests that mutations
occur during selection on plates (directed?)
add lac- cells immediately and:
add lactose
immediately
wait 3 days before
adding lactose
Count colonies arising after addition of lactose (nothing to grow on before it is
added)
• What would you predict if mutants arose before lactose addition (random)?
• What would you predict if mutants only arose after lactose addition
(directed)?
Cairns et al. 1988
If lac+ mutants occur
randomly on the plate –
independent of the
presence of lactose, then
mutants will be formed
prior to lactose being
added
number of lac+ mutants
Predictions:
If lac+ mutants are being
induced by lactose – so
only happen in its
presence, then mutants
will not be formed prior to
lactose being added
0
2
4
lac- cells
added at
t=0
lactose added
experiment 1
6
time
lactose added
experiment 2
8
10
Cairns et al. 1988
Bjedov et al.
Read by next Tuesday (March 3) and come to class with a list of points you did not understand. You’ll
discuss and attempt to resolve each others concerns in a small group. The aim is for us to discuss the
paper on Thursday (March 5) and evaluate experiments and interpretations. To do this, you need to have
a working knowledge of the experimental design and analysis.
E.g.
Bjedov et al.
Read by next Tuesday (March 3) and come to class with a list of points you did not understand. You’ll
discuss and attempt to resolve each others concerns in a small group. The aim is for us to discuss the
paper on Thursday (March 5) and evaluate experiments and interpretations. To do this, you need to have
a working knowledge of the experimental design and analysis.
E.g. In the third column, what’s the meaning of values above vs. below 1? Why choose the genes listed
in the first column? What was done with them?
Assignment 3
• An independent research project
• Each group (3-4 people, unless something else makes more sense in a particular
instance) should meet to come up with some research topics of interest.
• Research topics can be: experimental, bioinformatic/computational, theoretical,
conceptual…
• The week of March 3 I will meet with each group to refine and develop a project.
Before this meeting each group should do some research on their ideas to evaluate
what is feasible.