the origin of mutations lamarck darwin ...
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The origin of mutations
Lamarck
Darwin
www.princessleia.com/Lamarck.phpfreethinkersasylum.com/2010/03/freethinkers-book-club-darwins-sacred-cause/
• Beneficial mutations are somehow induced by the
environment in which they are useful
• Mutations are ‘directed’
• Mutations occur spontaneously independent
of their effects
• Mutations are random
Until 1943 there were two hypotheses concerning the
origin of mutations:
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)
ii) by direction/adaption – induced in the population by exposure to a selective
environment
Genetics 1943
• 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?
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:
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?
Each level represents a round of cell
division
bacteriophage on the plate kill sensitive cells
The basis of mutational change: How do mutations arise?
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)
Directed/Adaption hypothesis
The basis of mutational change: How do mutations arise: the fluctuation test
• 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)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
Directed mutation/
Fig. 12.1
The basis of mutational change: How do mutations arise?
Luria & Delbruck, Genetics 1943
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
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
The basis of mutational change: How do mutations arise?
Luria & Delbruck, Genetics 1943
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?)
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…
Summary of Luria-Delbruck
(L-D) finding
Pointing out that concluding from L-D and co. that ALL mutations
are spontaneous is an example of
the logical fallacy affirming the consequent
Even worse… knowing what we now know,
the L-D experiment
couldn’t have detected a signal of directed mutation
Cairns et al. 1988
[Luria-Delbruck (L-D) and Lederberg and Lederberg]
• 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
Directed mutation
pro
babili
ty o
f havin
g X
or
more
m
uta
nts
number of mutants X
1
1
Mean number of mutation events per
culture
Mean number of mutation events on the
plate
• Just what distribution of mutants should you expect if both directed and random mutations are present?
Random mutation
Directed mutation
pro
babili
ty o
f havin
g X
or
more
m
uta
nts
number of mutants X
0.5 1 2 4 8 16
1 2 5 10
20
30
Mean number of mutation events per
culture
Mean number of mutation events on the
plate
• Just what distribution of mutants should you expect if both directed and random mutations are present?
Random mutation
Directed mutation
Directed and random mutation
pro
babili
ty o
f havin
g X
or
more
m
uta
nts
number of mutants X
Mean number of mutation events per
culture
0.5 1 2 4 8 16
Mean number of mutation events on the
plate
1 2 5 10
20
30
1 25
10
20
30
0
1 random mutation + X directed mutations
What do you take away from this plot?
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+)
Experimental design
lac- lac+ (independent 1 bp deletion mutations restoring the proper reading frame are shown)
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
start codon
selection for
growth on
lactose
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?)
Experimental design
add lac- cells and lactose immediately
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?
time
num
ber
of
lac+
muta
nts ~20 colonies
on day 2 and ~8 cells per day thereafter
count lac+ mutant colonies arising over time (only lac+ can form colonies on minimal lac medium)
Experimental design
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)?
add lac- cells immediately and:
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?)
Cairns et al. 1988
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
If lac+ mutants occur randomly on the plate – independent of the presence of lactose, then mutants will be
formed prior to lactose being added
time
nu
mb
er
of
lac+
mu
tan
ts
0 2 46 8 10lac- cells
added at t=0
lactose addedexperiment 1
lactose addedexperiment 2
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?
• 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.
Assignment 3