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

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?

• 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